MtDiCAL LiliK^'^> by - UCL Discovery€¦ · MtDiCAL LiliK^'^> ROYAL FREE nUSMlAL HAMPSTEAD. JAMIE YANCEY JEREMY Department of Chemical Pathology and Human Metabolism, The Royal Free

2807712935

VASCULAR AND PLATELET EICOSANOIDS IN THE AETIOLOGY AND

TREATMENT OF CARDIOVASCULAR DISEASE

A th e s is p resen ted to the U n iv e r s i t y o f London in p a r t

f u l f i l m e n t o f the re q u ire m e n t f o r the degree o f

DOCTOR OF PHILOSOPHY

byMtDiCAL LiliK^'^>

RO YAL FREE n U S M lA L

HAMPSTEAD.

JAMIE YANCEY JEREMY

Department o f Chemical Patho logy and Human M etabo lism ,

The Royal Free H o s p ita l and School o f M e d ic in e ,

Pond S t r e e t ,

LONDON, UK

MARCH 1993

ProQuest Number: U057204

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ABSTRACT

Methods fo r the study o f receptor-linked prostacyclin (PGI2 )

synthesis in blood vessels (from the ra t , rabb it and man) and

thromboxane A2 (TXA2 ) synthesis in human p la te le ts were developed.

The ro le o f calcium, G proteins and protein kinase C in mediating

eicosanoid synthesis in these tissues was also studied. These

methods were applied in the investiga tion o f : 1) PGI2 synthesis

fo llow ing hypothermic storage o f blood vessels in preservation

solutions used in organ transp lanta tion , 2 ) the s i te o f action o f

drugs known to modulate eicosanoid synthesis; fo r example,

phosphodiesterase in h ib ito rs (PDEIs), non-steroidal a n t i

inflammatory drugs (NSAIDs) and copper chelators, 3) the mechanisms

underlying a ltered synthesis o f vascular PGI2 in diabetes m e ll i tus

(DM) and hepatic portal hypertension (HPH) in the r a t . There were

marked varia tions in the c h a ra c te r is i t ic s o f receptor-linked PGI2

release between species (human, ra t and ra b b it ) whereas receptor-

linked PGI2 release in d i f fe re n t a r te r ies and veins o f the same

species was remarkably s im ila r . Hypothermic storage o f blood vessels

( ra t , rabb it and human) in preservation solutions exerted minimal

e ffec ts on PGI2 synthesis, a find ing o f relevance to transplant

surgery. Apart from th e ir accepted s ites o f action, NSAIDs

(cyclooxygenase in h ib i to rs ) and PDEIs (cAMP elevators) were also

found to in h ib i t eicosanoid synthesis a t the signal transduction

le ve l. Copper chelators inh ib ited eicosanoid synthesis a t the level

o f cyclooxygenase and lipoxygenase. In ra ts with DM, there were

marked d i f fe re n t ia l a lte ra t ions o f receptor-linked PGI2 synthesis in

d i f fe re n t vessels and in other non-vascular tissues (gastro-

- in te s t in a l t ra c t , urinary bladder, trachea). In ra ts with HPH,

there were marked changes in receptor-linked PGI2 release by the

aorta and mesenteric vasculature, ind ica ting an adaptive series of

events involv ing PGI2 . The systems developed in th is thesis are a

convenient means o f investiga ting not only eicosanoid synthesis but

also drug action and the aetiology o f vascular disease.

LIST OF CONTENTS

PAGE

Abstract............................................................................................................ 2

L is t o f contents............................................................................................ 4

L is t o f f ig u re s .............................................................................................. 9

L is t o f tab les ................................................................................................ 13

Abbreviations................................................................................................... 14

Acknowledgements.............................................................................................. 15

CHAPTER 1. INTRODUCTION .............................................................................. 17

1.1 A h is to r ic a l overview o f eicosanoid research.............................. 17

1.2 Receptor-linked eicosanoid synthesis; the ro le o f Ca2+ . . . . 22

1.3 The phosphoinositide cycle and eicosanoid synthesis.................29

1.4 GTP (G) proteins ...................................................................... 31

1.5 Vascular PGI2 and p la te le t TXA2 in the aetiology o f CVD.... 33

1.6 Measurement and assessment o f PGI2 and TXA2 synthesis in

vivo and in v i t r o in re la t io n to cardiovascular and

thromboembolic disease....................................................................... 36

1.7 Eicosanoids and mechanisms o f atherogenesis................................ 39

1.8. PGI2 and TXA2 ischaemic heart disease (IHD) and diseases

predisposing to IHD............................................................................... 42

a) Atherosclerosis ............................................................................. 42

b) Diabetes m e l l i t u s . . . . ..................................................................... 45

c) Hypertension ..................................................................................... 48

d) Cigarette smoking ................. 49

e) Gender .............................................................................................. 50

1.8 Eicosanoids and drug ac tion ............................................................. 51

LIST OF CONTENTS (cont)

AIMS OF THE THESIS ....................................................................................... 54

CHAPTER 2. MATERIALS AND METHODS............................................................. 55

2.1 M ateria ls ................................................................................................ 55

2.2 Instruments............................................................................................ 58

2.3 Buffer compositions ........................................................................... 58

2.4 Preparation and incubation o f vascular (and other) tissues

fo r assessment o f PG synthesis....................................................... 59

2.5 E ffec t o f agonists, antagonists and other modulators o f

in t ra c e l lu la r second messengers on vascular PG syn th es is .. . 61

2.6 E ffect o f d rugs /inh ib ito rs on stimulated PGI2 s y n th e s is . . . . 61

2.7 Preparation o f washed p la te le ts ..................................................... 63

2.8 P la te le t TXA2 synthesis by unstirred washed p la te le ts 64

2.8a E ffect o f p la te le t a c t iva to rs ............................................................64

2.8b E ffect o f drugs on stimulated TXA2 synthesis .......................... 64

2.9 Measurement o f TXB2 , 6-oxo-PGFi^, PGE2 and PGp2o f ...................65

2.10 Validation o f radioimmunoassays..................................................... 65

2.11 Measurement o f p la te le t cAMP phosphodiesterase a c t i v i t y . . . . 66

2.12 Prostanoid-stimulated cAMP synthesis............................................ 68

2.13 P la te le t lipoxygenase a c t i v i t y ....................................................... 71

2.14 PGI2 synthesis by cultured human umbilical endothelial

c e l ls (HUVECs) and human omental tissue microvascular

endothelial c e l l s ........................................... 72

2.15 Ex vivo experiments on ra ts w ith diabets (pa ir fed and fed

ad l ib itu m or hepatic portal hypertension.................................... 75

2.15a Diabetes M e ll i tus (Rat) model ....................................................... 75


2.15b Hepatic portal hypertension (Rat) model..................................... 76

2.16. S ta t is t ic s .............................................................................................. 77

CHAPTER 3 : STUDIES ON PGIg AND OTHER PROSTANOIDS RELEASED

BY BLOOD VESSELS FROM THE RAT, RABBIT AND MAN AND ON TXAg RELEASE

BY WASHED HUMAN PLATELETS............................................................................ 78

3 .1 INTRODUCTION............................................................................................ 78

3.2 RESULTS...................................................................................................... 81

3.2a E ffect o f endothelium removal on receptor-linked PGI2 release

by the ra t ao rta .................................................................................... 81

3.2b Comparative studies on PGI2 release by a rte r ies and

veins from the ra t and ra b b i t ............................................................. 82

3.2c Studies on PGI2 release by human vessels and cultured human

umbilical vein and omental fa t endothelial c e l l s ...........................83

3.2d Effect o f hypothermic storage o f ra t , rabb it and human

vessels In organ preservation solutions on PGI2 re le a s e . . . . 84

3.2e Basic studies on p la te le t TXA2 synthesis.................................. 85

3 .3 DISCUSSION ............................................................................................ 104

3.3a. Basic studies on blood vessels..................................................... 104

3.3b. Studies on hypothermic storage In organ preservation

s o lu t io n s . . ......................... 109

3.3c. Basic p la te le t s tud ies..................................................................... 114

CHAPTER 4 . DRUG EFFECTS ON VASCULAR AND PLATELET EICOSANOID

SYNTHESIS................................................................................... 118

4 .1 . INTRODUCTION........................................................................................ 118


4.1a Non steroidal antiinflammatory drugs........................................... 118

4.1b Milrinone and IBMX (PDE in h ib i to r s ) ............................................. 119

4.1c Copper chelators ................................................................................. 121

4.2 RESULTS.................................................................................................. 123

4.2a Nonsteroidal antiinflammatory drugs............................................ 123

4.2b Effect o f phosphodiesterase in h ib i to rs on TXA2 syn thes is .. . 127

4.2c Milrinone e ffec ts on phosphodiesterase a c t iv i t y and

iloprost-s t im u la tesd cAMP synthesis............................................. 127

4.2d E ffect o f copper chelators on AA-stimulated TXA2 synthesis

and lipoxygenase a c t iv i t y ............................................................... 132

4.3 DISCUSSION ............................................................................................. 137

4.3a Nonsteroidal antiinflammatory drugs............................................. 137

4.3b Milrinone ............................................................................................... 139

4.3c Copper chelators ................................................................................. 143

CHAPTER 5. STUDIES ON P6 I 2 RELEASE BY VASCULAR AND OTHER TISSUES

FROM RATS WITH EXPERIMENTAL DIABETES MELLITUS AND HEPATIC PORTAL

HYPERTENSION..................................................................................................... 147

5.1 INTRODUCTION........................................................................................... 147

5.1a Diabetes M e ll i tu s ............................................................................... 147

5.1b Hepatic porta l hypertension............................................................. 149

5.2. RESULTS.................................................................................................. 151

5.2a Diabetes M e ll i tus ............................................................................... 151

5.2b Hepatic porta l hypertension............................................................. 153

5.3 DISCUSSION ............................................................................................. 154

8


5.3a. Diabetes Mellitus .......................................................................... 154

5.3b. Portal hypertension ....................................................................... 179

CHAPTER 6 . GENERAL DISCUSSION AND CONCLUDING REMARKS........................... 184

PUBLICATIONS DERIVED FROM WORK PRESENTED IN THE THESIS..................... 190

REFERENCES........................................................................................................ 193

LIST OF FIGURES

Figure Page

1.1 Fatty acid precursors o f prostaglandins ..................................... 19

1.2 Biochemical pathways involved in eicosanoid synthesis 23

1.3 In t ra c e l lu la r mechanisms co n tro l l in g vascular PG%2

s y n th e s is . . . . ......................................................................... 27

1.4 In t ra c e l lu la r mechanisms co n tro l l in g p la te le t TXA2

synthesis.................................................................................................. 28

1.5 Schematic representation o f p la te le t a c t iv a t io n ..........................35

1.6 Possible ro les fo r PGI2 in l i p id sequestration

in vascular t is s u e ................................................................................... 43

1.7 Model fo r involvement o f PGs in vascular ce l l p ro l i fe ra t io n . 44

1.8 Hypothetical model o f mechanisms underlying atherosclerosis. 47

2.1 Pattern of PGI2 release from ra t aorta, in v i t r o ........................ 62

2.2 Validation o f 6-oxo-PGF^q, and TXB2 measurements.......................... 67

2.3. Stimulation o f cAMP in p la te le ts by i lo p ro s t and PGE 69

2.4a Thin layer chromatography separation o f

arachidonic acid metabolites (p la te le ts ) ........................................ 73

2.4b In h ib i t io n o f lipoxygenase a c t i v i t y ............................................... 73

3.1 E ffec t o f endothelium removal on PGI2 synthesis by the ra t

ao rta .............................................................................................................86

3.2 E ffec t o f endothelium removal on in h ib i t io n of PGI2

synthesis by the ra t aorta ............................................................... 87

3.3 E ffec t o f various agonists on PGI2 synthesis by

d i f fe re n t blood vessels o f the ra t ................................................ 88

3.4 E ffec t o f a range o f vasoactive substances on PGI2 release

by rabb it aorta (with and without endothelium).............................89

1 0

LIST OF FIGURES (cont.)

3.5 E ffec t o f various stimulators on PGI2 release by

d i f fe re n t blood vessels o f the ra b b i t ..............................................90

3.6 E ffec t o f various agonists on PGI2 synthesis by

human i l i a c a rtery from s ix in d iv id u a ls ........................................ 91

3.7. E ffec t o f a range o f vasoactive substances on PGI2

release by human blood vessels ....................................................... 92

3.8. E ffec t o f a range o f second messenger activa tors

on PGI2 release by human i l i a c a rte ry and vein ......................... 93

3.9 E ffec t o f various stimualtors o f PGI2 synthesis

by HUVECSs and HOTMECs.........................................................................94

3.10 E ffec t o f preservation solutions on ra t ao rt ic

PGI2 synthesis........................................................................................ 96

3.11 E ffec t o f cold storage o f ra t a o r t ic t issue in

preservation solutions on noradrenaline stimulated

PGI2 synthesis........................................................................................ 97

3.12 E ffec t o f long term cold storage o f ra t ao rt ic

tissue in preservation solutions on noradrenaline

stimulated PGI2 synthesis...................................................................98

3.13 E ffec t o f d i f fe re n t stimulators o f p la te le t

aggregation on TXA2 synthesis by washed p la te le ts ...................... 101

3.14 TX&2 release in réponse to various stimulators by A)

d i f fe re n t numbers o f p la te le ts and B) at d i f fe re n t

times over 10 min................................................................................... 102

3.15 E ffec t o f calcium channel blockers and staurosporine on TXA2

synthesis by p la te le ts ......................................................................... 103

3.16 Scheme o f s ites o f action o f d i f fe re n t stimualtors on PGI2

synthesis by iso la ted vascular t is s u e ........................................... 117

11


4.1 E ffect o f indomethacin ibuprofen and t iap ro fen ic acid on

PGI2 synthesis by isolated ra t ao rta ............................................. 124

4.2 E ffect o f indomethacin ibuprofen and t iap ro fen ic acid on

PGI2 synthesis by isolated urinary bladder.................................. 125

4.3 E ffect o f m ilrinone on p la te le t TXA2 synthesis............................128

4.4. E ffect o f d i f fe re n t concentrations o f milrinone on phorbol

ester concentrati on-TXA2 response curve.......................................... 129

4.5. E ffect o f IBMX on p la te le t TXA2 synthesis......................................130

4.6. E ffect o f m ilrinone and IBMX on conversion o f cAMP to AMP

by washed human p la te le ts ................................................................... 131

4.7 E ffect o f copper chelators on A) p la te le t TXA2 synthesis and

B) p la te le t lipoxygenase a c t i v i t y ................................................... 133

4.8 E ffect o f dimethyldithiocarbamic acid on TXA2 by washed

human p la te le ts ...................................................................................... 134

4.9 E ffect o f various metal sa lts on A) DMDTCA - inh ib ited TXA2

synthesis and B) DMDTCA-inhibited lipoxygenase a c t i v i t y 135

5.1 Adrenaline-stimulated PGI2 release by aortae from d iabetic

and control ra ts ....................................... 155

5.2 Angiotensin I l-s t im u la ted PGI2 release by aortae from

diabetic and control r a ts ......................................................................156

5.3 Phorbol es te r—stimulated PGI2 release by aortae from diabetic

and control ra ts .................................................................................... 157

5.4 Ca2+ ionophore A23187-stimulated PGI2 release by aortae from

diabetic and control r a ts ................................................................... 158

5.5 Thapsigargin-stimulated PGI2 release by aortae from d iabetic

and control r a ts ..................................................................................... 159

12


5.6 Arachidonate-stimulated PGI2 release by aortae from diabetic

and control ra ts .................................................................................... 160

5.7 Acetycholine-stimulated PGI2 release by urinary bladders

from diabetic and control r a ts ......................................................... 161

5.8 Phorbol ester dibutyrate-stimulated PGI2 release by urinary

bladders from d iabetic and control ra ts ....................................... 162

5.9 Ca2+ ionophore A23187-stimulated PGI2 release by urinary

bladders from diabetic and control r a ts ....................................... 163

5.10 Thapsigargin-stimulated PGI2 release by urinary bladders

from d iabetic and control ra ts ......................................................... 164

5.11 Acetycholine-stimulated PGI2 release by the trachea from

d iabetic and control r a ts ................................................................... 165

5.12 PGI2 synthesis by d i f fe re n t vascular tissues o f the

d iabetic ra t ............................... 166

5.13 Adrenaline-stimulated PG%2 release by a) aortae and b)

mesenteric vasculature from portal vein constricted r a t s . . . . 169

5.14 Phorbol ester-stimulated PG%2 release by a) aortae and b)


5.15 Ca2+ ionophore A23187-stimulated PGI2 release by a) aortae

and b) mesenteric vasculature from portal vein constric ted

ra ts ............................................................................................................ 171

5.16 Thapsigargin-stimulated PGI2 release by a) aortae and b)

mesenteric vasculature from portal vein constric ted r a t s . . . . 172

5.17 Arachidonate-stimulated PG%2 release by a) aortae and b)


13

LIST OF TABLES

Table page

2.1 Cross re a c t iv i t ie s o f some major PGs with anti sera ...................... 70

2.2 Inter-assay and intra-assay co e ff ic ie n ts o f va r ia t ion o f

eicosanoids measured by RIA................................................................... 71

3.1 E ffec t o f endothelium removal on PG%2 synthesis by human

i l i a c artery e l ic i te d by a range o f stimulators ......................... 95

3.2 E ffect o f hypothermic storage o f ra b b it a o rt ic r ings in

preservation solutions on P6 I 2 synthesis.............................................99

3.3 E ffect o f hypothermic storage o f human i l i a c a rtery in

preservation solutions on PGI2 synthesis......................................... 100

4.1 E ffect o f median in h ib i to ry concentrations o f indomethacin

on ra t a o rt ic prostanoid synthesis.................................................... 126

4.2 E ffec t o f d i f fe re n t copper chelators on TXA2 synthesis by

washed p la te le ts ........................................................................................ 136

5.1 PGE2 , PGF2 and TXA2 synthesis by vascular t issue from diabetic

(fed ad l ib i tu m , pa ir fed, in s u lin treated) and control ra ts

e ight weeks a f te r induction o f diabets with s tre p to zo to c in .. . 167

5.2 PGE2 , PGF2 and TXA2 synthesis by gas tro in tes tina l tissue from

diabetic (fed ad l ib i tu m , pa ir fed, in su lin treated) and

control ra ts e igh t weeks a f te r induction o f diabets with

s trep tozotoc in ............................................................................................ 168

14

ABBREVIATIONS

AA arachidonic acidACE(I) angiotensin converting enzyme ( in h ib i to r )Ach acetyl choline

ADP adenosine diphosphateAng angiotensin[Ca2+]g e x tra ce l lu la r calcium

[Ca2+ ] j in t ra c e l lu la r calcium

cAMP adenosine-3'5 ' cyc l ic monophosphatecGMP guanosine-3'5' cyc l ic monophosphateCVD cardiovascular diseaseDAG (DG) diacyl glycerolDEDTCA diethyldith iocarbamic acidDHLA dihomo - T - l in o le n ic acidDM diabetes m e ll i tusDMDTCA dimethyldithiocarbamic acidEDRF endothelium-derived re lax ing fac torEDTA ethylene glycol te traa ce t ic acidEFA essential fa t ty acidEGTA ethylenetetraacetic acidGC MS gas chromatography mass spectrometryG protein GTP binding protein

GTP guanosine triphosphateHDL high density l ipop ro te inHETE hydroxyeicosatetraenoic acidHOTMECs human omental tissue microvascular

endothelial c e l ls .HPETE hydroperoxyeicosateteraenoic acidHPH hepatic portal hypertension5-HT 5-hydroxytryptamine

H7 isoquino linylsu lfonylm ethyl pi p e r iz ine.HUVECs human umbilical vein endothelial c e l ls .

IBMX isobutyl methyl xanthineIHD ischaemic heart diseaseIL in te r le u k in

15

ABBREVIATIONS (continued)

IP3 myo-inositol 1,4,5-trisphosphate

KPS kidney preservation so lu tion

KRB Kreb's Ringer bicarbonate bu ffe r

LDL low density l ipop ro te in

LT leukotriene

MEM Minimum Essential MediumNaF sodium f lu o r id e

NDGA nordihydroguaretic acid

NSAID non-steroidal antiinflammatory drug

PDBU phorbol 12,13-dibutyratePEI polyethylimine ce llu losePDE(I) phosphodiesterase ( in h ib i to r )PI phoshatidyl in o s ito lPG prostaglandinPK protein kinase

PLAg phospholipase A2

PLC phospholipase CPMA phorbol myristate acetatePRP p la te le t r ich plasmaPVD peripheral vascular diseaseRIA radioimmunoassaySOD superoxide dismutaseTETD tr ie th y lth iu ra m disulphideTLC th in layer chromatographyTX thromboxaneUW University o f Wisconsin SolutionVSMC vascular smooth muscle c e l ls

16

ACKNOWLEDGEMENTS

I thank Dr D im i t r i M ik h a i l id is fo r h is p a t ie n t su p e rv is io n ,

and support. Special thanks go to Mr Manuel Barradas, whose

f r ie n d s h ip has helped me immeasurably. Professor Anthony Winder

deserves p a r t i c u la r thanks s ince he was the one most ins trum enta l

in encouraging me to undertake and complete t h is th e s is . For her

help and kindness, I am g ra te fu l to Mrs Pat Wisner. Special

mention must be made o f o ther c lose colleagues w ith whom I have

enjoyed an everyday and happy ra p p o r t ; Ms Oasvinder G i l l , Dr Cecil

Thompson, Ms An ita Jagroop, Ms Pamela Dale, Dr D a lv i r G i l l , Dr

An il Coumar, Dr Lauren Ozin, Ms Shiobahn O'Donaghue, and Mrs

Mol l i e Cohen.

With regard to the c o l la b o ra t iv e work c a r r ie d out in th is

th e s is I would l i k e to thank Dr Cecil Thompson fo r s e t t in g up the

d ia b e t ic r a t model, Ms Oasvinder G i l l and Mr Manuel Barradas fo r

t h e i r e x p e r t ise in preparing and assessing p la te le t s , Mr Gerard

Stansby, Mr Keith Roll es and Mr George Hamilton (Department o f

Surgery) fo r supp ly ing me w ith human blood vessels and c u ltu re d

e n d o th e l ia l c e l l s and Dr Ai den McCormick, Dr S te l io s Karatapanis

and Dr David Harry (Department o f Medicine) fo r s e t t in g up the

hepa tic p o r ta l hypertens ion r a t model. Although too numerous to

mention by name, I would l i k e to thank a l l o ther members o f the

Department o f Chemical Pathology and Human Metabolism.

The fo l lo w in g Pharmaceutical companies are g r a te f u l l y

acknowledged fo r t h e i r f in a n c ia l support: S te r l in g W inthrop, Smith

K line and Beecham, S co t ia , 3M R iker and LaFon.

17

CHAPTER 1. INTRODUCTION

1.1. Historical overview of eicosanoid research

In the 1930s, seminal f lu id was found to be a potent in v i t r o

stimulator o f mammalian smooth muscle c o n t r a c t i l i t y and to markedly

reduce blood pressure, in vivo (Kurzok and Lieb, 1930; Goldb la tt,

1933, 1935; von Euler, 1935). Since von Euler (1937) also found tha t

extracts o f prostate glands had s im ila r pharmacological e ffec ts as

seminal f lu id and were l i p id in nature, these bioactive substance(s)

were christened prostaglandins (PGs). I t is i ro n ic tha t von Euler

also studied extracts o f seminal vesicles which he found to be

b ioactive and as such termed vesiglandins (von Euler, 1937). Thus, i f

i t had been decided to opt fo r th is la t t e r term, we would now be

using such terms as vesanoids and ves icyc lin ! In 1940, Kell away and

Trethewie, reported the existence o f a substance tha t was released in

response to anaphylaxis and contracted airways tissue. This

substance, termed "slow reacting substance o f anaphylaxis", we now

know to be leukotrienes (LTs; Samuelsson, 1982). In the same decade

Burr and Burr (1930) established the existence o f essential fa t t y

acids (EFA), which over 30 years la te r were to be recognised as

precursors fo r a l l eicosanoids.

A fte r th e i r discovery, l i t t l e work was carried out on PGs u n t i l

the la te 1950s when Bergstrom (1957) is o la te d , in a pure c r y ta l l in e

form, two primary PGs: PGE and PGP ,. In the 1960s, chemical

characterisation of PGs was made possible by two major methodological

advances: gas-liqu id chromatography (GLC) and mass-spectrometry (MS).

Thus, i t was established tha t a l l PGs contain 20 carbon atoms, a 5-

membered r ing and have the same basic carbon skeleton (Bergstrom et

a l . , 1968; f i g . 1 .1 . ) . In 1964, Van Dorp e t a l . and Bergstrom e t a l.

described the biosynthesis o f PGs from dihomo-7 - l in o le n ic acid

18

(DGLA) and arachidonic acid (AA) when incubated with homogenates of

sheep vesicular glands. The tranformations involved peroxidation, the

incorporation o f molecular oxygen and cyc l isa t ion o f the fa t ty acids

to produce PGE and PGE2 . These unique transformations led to the

term cyclooxygenase to describe the enzyme tha t catalysed these

reactions. I t was also determined th a t PGs can be o f the 1, 2 or 3

series depending on the fa t ty acid from which they are derived:

monoenoic (1 se r ies), dienoic (2 series) and t r ie n o ic (3 series) PGs

are derived from DGLA, AA, and eicosapentaenoic acid (EPA),

respective ly (Bergstrom et a l . , 1968; f i g . 1 .1 .) . The PGs o f the 1,2

and 3 series possess markedly d i f fe re n t properties which has led to

therapeutic attempts to a l te r PG synthesis by the manipulation of

d ie tary intake o f o i ls (e.g. f is h o i l , evening primrose o i l ) .

Throughout the 1960's a large number o f d i f fe re n t PGs were

characterised (Bergstrom e t a l . , 1968). I t was also found tha t PGs

are potent modulators o f smooth muscle a c t iv i t y , a l te r metabolic

functions ( inc luding l i p id and carbohydrate metabolism) and exert

e ffec ts on neurotransmission (Bergstrom e t a l . , 1968). Since then,

PGs have been shown to be involved in the a c t iv i ty o f v i r t u a l l y every

mammalian ce ll type.

In the 1970's, there were considerable breakthroughs in

eicosanoid research. Although th e o re t ic a l ly postulated to e x is t by

Samuel sson in 1965, the endoperoxides PGG and PGH were ac tua lly

iso la ted in 1973 (Hamberg and Samuelsson 1973, Nugteren and Hazelhof,

1973; Fig. 1 .2 . ) . These endoperoxides are the immediate precursors o f

both the thromboxanes (TXs) and PGs (Hamberg e t a l . , 1975; f i g . 1 .2 .) .

Cyclooxygenase was also isolated and purifed by several d i f fe re n t

groups (Miyamoto e t a l . , 1976; Hemler e t a l . , 1976). I t was also

1 9

COOH

l ino l e i c a c i d

C O O H

di—h o m o - y - l i n o l e n i c a c i d P G ^

COOH

/v \ /a r a c h i d o n i c a c i d

COOH

COOH

oc — l i no lon ic a c i d

COOH

e i c o s a p e n t a e n o i c a c i d P G 3 OH

COOH

Fig. 1.1. Di-homo-7 - I in o le n ic acid and arachidonic acid are converted in to PGs of the 1 series (one double bond) and 2

series (two double bonds), respective ly . These fa t ty acidsand th e ir precursor, l in o le ic acid, are members o f the family of n-6 fa t ty acids, characterised by an end segment o f 6 carbons (a t the opposite end from the - COOH). Eicosa- -pentaenoic acid, coming from a -1 ino le n ic acid (n-3 family) is converted to 3-series PGs (three double bonds). The cha rac te r is t ic end segments o f n-6 and n-3 fam ilies arerepresented in the f igure by th ick l in e s .

2 0

found th a t haem (and the iron i t contains) is required fo r

cyclooxygenase a c t i v i t y (Hemler and Lands, 1976). Thus, v ia iron -

mediated redox reac tions , cyclooxygenase e l i c i t s the abstract ion o f

hydrogen atoms from the fa t t y acid forming rad ica l s truc tures

followed by the incorpora tion o f two oxygen molecules ( f i g . 1.2. Deby,

1988). A c y c l ic molecule is produced, PGG, which under the

peroxidasic a c t i v i t y o f cyclooxygenase is reduced to an endoperoxide,

PGH, the immediate precursor o f PGs and thromboxanes, depending on

the synthase/isomerase present in any given t issue ( f i g . 1 . 2 . ) .

Lipoxygenase was then discovered in p la te le ts and leucocytes

(Nugteren, 1975; Borgeat and Samuelsson, 1979). Lipoxygenase is also

a haem-containing enzyme, which abstracts a hydrogen atom and

incorporates an oxygen molecule in to fa t t y acids a t the C l2, 05 or

015 p o s it io n , producing hydroperoxyeicosatetraenoic acid (HPETE;

f i g . 1.2. Deby, 1988). HPETE can e i th e r be converted to leukotrienes

(LT) by LT synthases (p r in c ip a l ly in leucocytes) or spontaneously

degrade to hydroxyeicosatetraenoic acid (HETE), l ib e ra t in g a t the

same time a superoxide ion (Deby, 1988). Apart from iro n , copper has

been proposed as being a co - fa c to r o f cyclooxygenase and lipoxygenase

(Fernandez-Madrid, 1989). One o f the ob jec tives o f th is thes is was to

explore the ro le o f copper in mediating the a c t iv i t y o f these enzymes

in p la te le ts (see in tro d u c t io n o f chapter 4 fo r a more comprehensive

ra t io n a le fo r these s tu d ie s ) .

In the mid-1970s, p ros tacyc lin (PGI2 ) was discovered. In a now

c la ss ic series o f experiments, i t was found th a t blood vessels, in

v i t r o , released a substance th a t was a vaso d ila to r , a potent

in h ib i to r o f p la te le t aggregation, had a very short h a l f - l i f e and

whose synthesis was in h ib i te d by a s p ir in (a cyclooxygenase in h ib i t o r ;

2 1

Moncada e t a l . , 1977; Bunting e t a l . , 1976; Gryglewski e t a l . , 1977).

This h i th e rto "undiscovered" PG (termed PGX in i t s neonatal period)

was subsequently characterised and ca lled prostacyclin (PGI2 [now

epoprostenol] ) due to i t s unique molecular structure (see f i g . 1 . 2 . ) .

In fu r the r studies i t was suggested th a t vascular endothelial c e l ls

are the main source of PGI2 in blood vessels (Moncada et a l . , 1977;

Boeynaems e t a l . , 1985). Around the same time i t was established tha t

p la te le ts synthesise thromboxane A2 (TXA2 ; Hamberg e t a l . , 1975;

Hammarstrom and Falardeau, 1977). TXA2 was found to have a short

h a l f - l i f e , to be a potent stim u la tor o f p la te le t aggregation and to

be vasoconstrictor (Moncada and Vane, 1979). Since blood vessels and

p la te le ts synthesise large quantit ies o f eicosanoids with

d iam etr ica lly opposite actions, an intense era o f research was

in i t ia te d , aimed p r im ari ly a t evaluating the in te rac tion of PGI2 and

TXA2 in the aetio logy o f cardiovascular disease (see section 1 .5 .) .

Although i t has long been recognised tha t phospholipase A2 (PLA2 )

is a key enzyme tha t l im i ts PG formation (Vogt e t a l . , 1969), i t was

not u n t i l the la te 1970s tha t the importance of PLA2 was f u l l y

recognised (Flower and Blackwell, 1976). AA is stored, incorporated

as an ester at the sn-2 position ( f ig . 1 .3 .) o f phosholipids (PLs):

phosphatidyl serine, phosphatidyl choline, phosphatidyl ethanol ami ne

or phosphatidyl in o s ito l in b io log ica l membranes ( I rv in e , 1982;

Waite, 1987). Under the influence o f sp e c if ic stim u la tion and

in t ra c e l lu la r regu la tion (see sections 1.2 and 1.3) AA is l ibera ted ,

p r in c ip a l ly by the hyd ro ly t ic action o f PLA2 ( I rv in e , 1982; Hong and

Deykin, 1982). I t has also been suggested tha t AA is derived from

diacyl glycerol (DG) by the action o f DG lipase (McKean e t a l . , 1981)

as well as a PLA^-lysophospholipase pathway (Martin and Wysolmerski,

2 2

1987). "Liberated" AA then undergoes bio-transformations to generate

eicosanoids as described above. Several groups established tha t

cort icoste ro ids in h ib i t eicosanoid formation (Greaves and MacDonald-

Gibson, 1972; Kantrowitz e t a l . , 1975; Gryglewski e t a l . , 1975). I t

was established tha t th is e f fe c t o f cort icoste ro ids requires receptor

occupancy and de novo protein synthesis (Feldman e t a l . , 1972;

Thompson and Lippman, 1974; Flower and Blackwell, 1979; Russo-Marie

and Duval, 1979). This protein (macrocortin or l ip o c o r t in ) was

iso lated and characterised soon a f te r (Blackwell e t a l . , 1980; Hi rata

e t a l . , 1980; Rothhut e t a l . , 1983; Di Rosa e t a l . , 1984; Flower,

1988).

1.2. Receptor-linked eicosanoid synthesis and the role of calcium

During the la s t decade a tten tion has focussed on the in t r a

c e l lu la r mechanisms th a t govern eicosanoid synthesis. I t has long

been recognised tha t agonists which influence the function o f any

given tissue (e .g. vascular c o n t r a c t i l i t y ; p la te le t aggregation) also

modulate the local synthesis o f eicosanoids. A plethora o f vasoactive

agents (e .g . histamine, bradykinin, serotonin [5-hydroxytryptamine;

5-HT], LTs, angiotensin I I , noradrenaline, purinomimetics and TXA2

analogues) stimulate the release o f PGI2 from in ta c t blood vessels as

well as cultured endothelial and vascular smooth muscle c e l ls ( fo r

review see Jeremy e t a l . , 1988a). Since the re la t iv e potency o f these

agonists on PG release in blood vessels was s im ila r to the re la t iv e

potencies on c o n t r a c t i l i t y / re laxation [Godfraind et a l . , 1982]), i t

was postulated tha t there was a signal transduction pathway common to

both vascular contraction and endogenous PGI2 synthesis (Jeremy e t

a l . , 1985a).

2 3

2 4

The above re la t ionsh ip appears to hold true in other smooth

muscle types. For example, in the ra t u r inary bladder and penis,

parasympathomimetics stimulate the synthesis of PGI2 , PGE2 and PGF20,

(Jeremy e t a l . , 1986a,b), an action blocked by muscarine-receptor

antagonists (a trop ine, gal lamine and ipratropium bromide; Jeremy e t

a l . , 1986a,b). Again, the re la t iv e potency o f agonists and

antagonists on both c o n t ra c t i l i t y / re laxation and eicosanoid

synthesis was s im ila r , consolidating the concept o f a d ire c t

re la tionsh ip (or common pathway) between the two events in smooth

muscle, per se. Furthermore, the fa c t tha t a l l three PGs studied were

stimulated to an equal extent pointed to the ac tiva tion o f a phospho-

- l ipa se , ra ther than o f cyclooxygenase or o f ind iv idual synthases or

isomerases (Jeremy e t a l . , 1986a,b).

Despite the above apparent general re la t ionsh ip between

contractors o f smooth muscle and PGI2 synthesis in the ra t aorta,

there appears to be considerable in ter-species va r ia tion with regard

to the receptor type linked to PGI2 synthesis in blood vessels. For

example, acetylcholine (Ach) has no e f fe c t on PGI2 synthesis in the

ra t aorta (Jeremy e t a l . , 1985a) but has been reported to be a potent

s tim u la to r in the ra b b it aorta (Boeynaems e t a l . , 1985). S im ila r ly ,

histamine is a potent s tim u la tor o f PGI2 synthesis in cu ltu red

endothelial (bovine and human) c e l ls (Baezinger e t a l . , 1981) whereas

th is agonist has no e f fe c t on PGI2 release by the ra t aorta (Jeremy

e t a l . , 1988a). In th is context, i t is often assumed tha t agonist-

stimulated PGI2 release from whole vessels re f le c ts production by the

endothelium. This la t t e r assumption was based on the study o f Moncada

e t a l . (1977) and la te r by Boeynaems e t a l . (1985), who showed a

marked reduction o f PGI2 output by a o r t ic r ings denuded o f

2 5

endothelium. The aorta, the most widely used vessel fo r the study of

agonist - PGIg re la t ionsh ips , is also a large conduit vessel and as

such may vary considerably in i t s reactive properties from smaller

vessels, which in turn may possess d i f fe re n t receptor populations.

One o f the objectives o f th is thes is , therefore , was to investigate

responses to agonists on PGI2 synthesis in d i f fe re n t vessels from the

same species and between species ( inc lud ing man). The ro le o f the

endothelium in mediating vascular PGI2 release was also investigated

in th is thes is . A more complete ra t iona le fo r these studies is

provided in the in troduction o f chapter 3.

Since Ca2+ m obilisa tion is involved in both ac t iva tion o f PLA2

( I r v in e , 1982; Hong and Deykin, 1982; Martin and Wysolmerski, 1987)

and exc ita tion -con trac tion coupling (Somlyo and Somlyo, 1968), i t was

deemed possible th a t Ca^^ was the common ac t iva to r in the two

processes ( i . e . vasoconstriction and PG%2 synthesis; Jeremy e t a l . ,

1985a,b). Thus, i t has been shown th a t in the ra t aorta, ra t t a i l

a r te ry , urinary bladder and penis th a t receptor-linked prostanoid

synthesis is in h ib ite d by the removal o f e x tra ce l lu la r Ca2+ with Ca2+

chelators, by Ca2+ channel blockers such as verapamil, n ifed ip ine and

d ie th y ls t i lb e s t ro l and by lanthanum, a Ca^+.binding antagonist

(Stewart e t a l . , 1983; Golub e t a l . , 1985; Jeremy e t a l . ,1985a,b ) .

However, in these la t t e r studies, the amounts o f Ca2+ blockers

required to in h ib i t PG synthesis were greater than those required to

in h ib i t receptor-linked contraction in these tissues. For example,

Valloton e t a l . (1990) found tha t Ca^^ channel blockers at

concentrations th a t in h ib i t vascular contraction did not in h ib i t PGI2

release from cultured vascular smooth muscle c e l ls (VSMCs) in

response to angiontensin I I (ANG I I ) . More recently , several groups

2 6

have measured, concomitantly, PGI2 release and in t ra c e l lu la r Ca2+ in

cultured endothelial c e l ls , using Ca^+.gensitive dyes such as Quin-2

and Fura-2 (Hallam et a l . , 1989; Carter e t a l . , 1989). These groups

concluded tha t the in t ra c e l lu la r release o f Ca2+ is the p r in c ip a l, i f

not the only, source o f Ca2+ fo r PLA2 ac t iva t ion and therefore PGI2

release. Furthermore, in cultured bovine endothelial c e l ls , i t has

been demonstrated tha t the release o f AA is mediated p r in c ip a l ly by

phospholipase A^-lysophospholipase, the a c t iv i t y o f which is not only

independent o f Ca2+, but is ac tua lly enhanced by the presence o f EDTA

(Martin and Wysolmerski, 1987).

In the p la te le t , aggregation is associated with the concomitant

release of TXA2 (A r i ta e t a l . , 1989). However, the d ire c t

re la t ionsh ip between receptors and TXA2 release is complicated by the

fac t tha t aggregation, per se, causes the synthesis and release o f

TXA2 (A rita e t a l . , 1989), making i t d i f f i c u l t to d issect out the

mechanisms involved in receptor-TXA2 synthesis coupling. This is

exemplified by the fa c t tha t Ca2+ channel blockers (again a t high

concentrations) in h ib i t p la te le t aggregation and concomitant TXA2

release (Jeremy e t a l . , 1985b). However, several studies have

demonstrated tha t TXAg is synthesised by p la te le ts via Ca^^-mediated

PLA2 ac tiva tion ( I rv in e , 1982; Si ess e t a l . , 1983). In an elegant

study. Brune and U l l r ic h (1991) demonstrated th a t the Ca2+ linked to

TXA2 synthesis is derived p r in c ip a l ly from in t ra c e l lu la r stores. The

involvement o f Ca2+ and eicosanoid synthesis is represented

diagramatica lly in f ig s . 1.3. and 1.4. One objective o f th is thesis

therefore was to investigate the release o f TXA2 by washed unstirred

p la te le ts (see chapter 3 fo r a more de ta iled ra t io n a le ) .

vasoconstrictor vasodilator

GLYCOCALYX

calciumchannel

PLASMA

G p ii-

adenylatecyclase

arachidonate

cAMP

calcium stores

VASOCONSTRICTION

F i g . 1 . 3 . S c h e m a t i c r e p r e s e n t a t i o n o f t h e m e c h a n i s m s c o n t r o l l i n g v a s c u l a r P G I 2 s y n t h e s i s . V i a G p r o t e i n s ( G p ) , v a s o c o n s t r i c t o r a g o n i s t s ( e . g . n o r a d r e n a l i n e , TXAg,) a c t i v a t e p h o s p h o l i p a s e C ( P L C ) w h i c h g e n e r a t e s d i a c y l g l y c e r o l (DAG) a nd i n o s i t o l t r i s p h o s p h a t e ( I P 3 ) f r o m p h o s p h a t i d y l i n o s i t o i 4 , 5 - b i s p h o s p h a t e ( P I ) .DAG a c t i v a t e s p r o t e i n k i n a s e C (PKC) w h i c h may a c t i v a t e CaZ+ c h a n n e l s and d i r e c t l y s t i m u l a t e c o n s t r i c t i o n . I P 3 e l i c i t s t h e r e l e a s e o f Ca2+ ( [ C a 2 + ] j ) f r o m i n t r a c e l l u l a r s t o r e s . A n e t i n c r e a s e i n [ C a ^ + l j en h a n c e s c o n s t r i c t i o n ( a s w e l l as PKC a c t i v i t y ) a nd a c t i v a t e s PLA 2 ( r e l e a s e s a r a c h i d o n a t e f r o m p h o s p h o l i p i d s t o r e s t h e r e b y g e n e r a t i n g P G I 2 ) . P G I 2 s t i m u l a t e s cAMP s y n t h e s i s w h i c h a c t i v a t e s p r o t e i n k i n a s e A ( P K A ) , an e v e n t a s s o c i a t e d w i t h v a s o d i l a t i o n . PKC a l s o d o w n - r e g u l a t e s e x c i t a t o r y r e c e p t o r s . U n d e r s i m i l a r c o n t r o l , P G I 2

r e l e a s e f r o m e n d o t h e l i a l c e l l s i n h i b i t s p l a t e l e t and l e u c o c y t e a c t i v i t y . How t h i s m e c h a n i s m r e l a t e s t o c e l l p r o l i f e r a t i o n a nd l i p i d s e q u e s t r a t i o n i s g i v e n i n f i g s . 1 . 6 . , 1 . 7 . and 1 . 8 . ro

agonist

CaMK

mPLC

o u ts id e

M E M B R A N E

ins ide

aggregation / secretion

PKC\ d t s

C a ^ “ /PLA.

AA

F i g . 1 . 4 . S c h e m a t i c r e p r e s e n t a t i o n o f t h e m e c h a n i s m s c o n t r o l l i n g p l a t e l e t TXA 2 s y n t h e s i s . V i a G p r o t e i n s ( G p ) , a g o n i s t s ( e . g . c o l l a g e n , TXA 2 ) a c t i v a t e m e m b r a n e - a s s o c i a t e d PLC (mPLC) w h i c h g e n e r a t e s d i a c y l g l y c e r o l (DG) and i n o s i t o l t r i s p h o s p h a t e ( I P 3 ) f r o m p h o s p h a t i d y l i n o s i t o l 4 , 5 - b i s p h o s p h a t e ( P I P 2 ) . DG a c t i v a t e s p r o t e i n k i n a s e C (PKC) w h i c h i n t u r n e n h a n c e s s e c r e t i o n , a g g r e g a t i o n and PLA 2 a c t i v i t y . I P 3 e l i c i t s t h e r e l e a s e o f i n t r a c e l l u l a r Ca^+ ( [ C a ^ + J ^ ) f r o m t h e d e n s e t u b u l a r s y s t e m (D TS ) w h i c h i n t u r n a c t i v a t e s PLA 2 t o g e n e r a t e TXA 2 and a c t i v a t e s c a l m o d u l i n k i n a s e ( C a M K ) . TXA 2 r e l e a s e d by t h e p l a t e l e t c a n t h e n f u r t h e r e n h a n c e a g g r e g a t i o n o f n e i g h b o u r i n g p l a t e l e t s v i a t h e same m e c h a n i s m s . E l e v a t e d [ C a ^ + l ^ c a n a l s o f u r t h e r e n h a n c e DG and I P 3 g e n e r a t i o n f r o m p h o s h a t i d y l i n o s i t o l ( P I ) v i a a c t i v a t i o n o f c y t o s o l i c PLC ( c P L C ) t h e r e b y f u r t h e r e n h a n c i n g t h e

a c t i v a t i o n o f p l a t e l e t s .

fo00

2 9

1.3. The phosholnositlde cycle and eicosanoid synthesis.

In v i r t u a l ly every ce ll type h ith e r to investigated receptor-

ac t iva t ion resu lts in the hydrolysis o f phosphatidyl in o s ito l (P I),

an event widely termed 'PI tu rnove r '. I t has been long recognised

tha t the in i t i a t io n o f PI turnover is dependent on phospholipase C

(PLC) a c t iv i t y (Hokin and Hokin, 1960). PLC hydrolyses PI to generate

two in t r a c e l lu la r second messengers; 1,2-diacyl glycerol (DG) and

in o s ito l 1 ,4 ,5-trisphosphate (IP3 ) . PLC a c t iv i t y requires very low

(0.1uM) or no Ca2+ (Jackowski e t a l . , 1986). I t is therefore ^

generally accepted tha t Ca2+ response fo llow ing in i t i a t io n o f the PI

cycle is secondary ra ther than being causative. DG, which remains in

the plasmalemma, activates protein kinase C (PKC; Nishizuka, 1984,

1988). Essential co-factors fo r th is ac t iva t ion are Ca2+ and PS which

combine with DAG and PKC to form a quaternary complex w ith in the

plasmalemma (Nishizuka, 1988). PKC then acts by phosphorylating

(thereby ac tiva t ing and/or deactivating) other in t ra c e l lu la r

prote ins. PKC also down regulates receptors via phosphorylation

(Iwamoto e t a l . , 1992).

Experimentally, the DG-PKC system has la rge ly been investigated

using the DG-mimetics, phorbol esters (Nishizuka, 1984,1988; Berridge

and Irv ine , 1988). In vascular t issue , phorbol esters e l i c i t

contraction in vascular smooth muscle (Danthaluri and Deth, 1984;

Rasmussen e t a l . , 1984), an e f fe c t tha t is Ca^^-dependent and

potentiated by A23187. In tu rn , i t was found tha t phorbol ester

stimulates in v i t r o PGI2 synthesis by the ra t aorta, an e f fe c t

potentiated by noradrenaline and Ca2+ ionophore A23187 (Jeremy and

Dandona, 1987). This action o f phorbol esters was in h ib ite d by Ca2+

channel blockers and by the PKC in h ib i to r , isoqu ino liny lsu lfony l

3 0

methyl pi peri zi ne (H7; Jeremy and Dandona, 1987) which also inh ib ited

adrenoceptor-linked PGI2 synthesis (Jeremy and Dandona, 1987). These

experiments suggested tha t adrenoceptor-PGl2 synthesis coupling, at

leas t in the ra t aorta, is mediated by PKC and also tha t PKC e l i c i t s

Ca%+ m obilisation associated with eicosanoid synthesis possibly via

ac tiva t ion o f Ca^+ channels (Jeremy and Dandona, 1987). However,

other workers have provided evidence tha t PKC in i t ia te s vascular PGI2

synthesis through d ire c t ac tiva t ion o f PLA (DeMolle and Boeynaems,

1988). Phorbol esters e xh ib it dual e ffec ts on p la te le t a c t iv i t y

(Nakano e t a l , 1989): an i n i t i a l enhancement o f p la te le t responses

(aggregation and secretion) followed by in h ib i t io n o f a c t iv i t y .

Phorbol esters cause p la te le t amine storage granule secretion and

hence aggregation without increasing cy toso lic Ca%+ (Rink e t a l . ,

1983). This e f fe c t appears to be linked to phosphorylation o f P47,

the protein responsible fo r in i t i a t in g the secretory response

(Daniel, 1990). At the outset o f th is thesis l i t t l e work had been

done on PKC and TXA2 release by p la te le ts .

IP3 binds to spe c if ic s ites on the endoplasmic re ticu lum and

e l i c i t s the release and elevation o f in t r a c e l lu la r Ca^+ ([Ca^+ lj)

leve ls (Berridge and Irv in e , 1988), which in tu rn activates Ca^+ -

dependent processes, including contraction (Somlyo e t a l . , 1985) and

p la te le t aggregation (Lapetina e t a l . , 1984; Brass e t a l . , 1985).

With regard to blood vessels, receptor- linked PI turnover has been

demonstrated in response to vasoactive agents including noradrenaline

(Legan e t a l . , 1985; Rapaport, 1987; V illa lobos-Molina e t a l . , 1982;

Berta e t a l . , 1986) angiotensin I I (G ried ling e t a l . , 1986),

serotonin (Roth e t a l . , 1984), histamine (Resink e t a l . , 1987) and

p la te le t ac t iva t ing fac to r (PAF; Test and Bang, 1981). Recent

31

experiments have indicated tha t IPg-stimulated elevation o f [Ca2+]^

is the princ ipa l mechanism by which PLA2 is activated in vascular

tissue (Carter e t a l . , 1988; Hal lam e t a l . , 1989).

Considerable a tten tion has been paid to the PI cycle, Ca^+

mobilisation and TXA2 synthesis in p la te le ts . P la te le t activa tors

which have been shown to induce PI turnover include thrombin,

collagen, p la te le t ac t iva t ing fac to r (PAF), adenosine diphosphate

(ADP), vasopressin and serotonin (Daniel, 1990). I t has also been

c le a r ly demonstrated tha t IP3 e l i c i t s an increase in in t ra c e l lu la r

Ca^+ and f u l l response in p la te le ts , including TXA2 synthesis

(Lapetina e t a l . , 1984; Brass and Joseph, 1985; Brune and U l l r ic h ,

1991). The involvement o f DG and IP3 and eicosanoid synthesis is

represented diagramatica lly in f ig s . 1.3. and 1.4.

1.4. GTP binding (6) proteins

G proteins are regulatory guanosine triphosphate (GTP)-binding

proteins l in k in g receptor ac t iva t ion to mediators o f ce l l s igna ll ing

(v iz . PLC and adenylate cyclase; Nagata and Nozawa, 1990; Johnson and

Dhanasekaran, 1989). G proteins contain three subunits ( a andY ),

bind guanine nucelotides with high a f f in i t y and possess GTPase

a c t iv i t y and are modulated by toxins (cholera and pertussis;Johnson

and Dhanasekaran, 1989). However, between-tissue responses to these

tox ins can vary markedly and as such caution should be exercised when

in te rp re t ing to x in -e f fe c ts . Although well characterised in other

t issues, the ro le o f G proteins in mediating vascular and p la te le t

eicosanoid synthesis is not c lea r. However, in other t issues, i t has

been f irm ly established tha t G proteins are involved in ion channel

regu la tion , exocytotic secretion and PLA2 ac tiva tion (Nagata and

32

Nozawa, 1990; Johnson and Dhanasekaran, 1989). Although not proven

d e f in i t iv e ly , there is substantial evidence th a t a G prote in l inks

receptor ac t iva t ion to PLC (Cockcroft, 1987). Haslam and Davidson

(1984) showed th a t GTP and i t s non-hydrolysable analogue, GTPys in

e le c t r ic a l ly permeabilised human p la te le ts enhances thrombin-induced

DG formation, ind ica ting tha t G proteins couple receptors to PLC.

Other studies have also indicated tha t there is a GTP-dependent

ac tiva t ion o f cytoplasmic PLC in p la te le ts (Baldassare, 1986; Deckmyn

e t a l . , 1986). In tu rn , PKC has been postulated to act a t the level

o f G proteins and has been presumed to couple the thrombin receptor

with PLC (Halenda e t a l . , 1986). Several studies have indicated tha t

PLA2 a c t iv i t y is regulated by G proteins (Nakashima e t a l . , 1987).

Thrombin-induced AA release was completely in h ib ite d by pertussis

tox in treatment whereas PLC a c t iv i ty was decreased by only 20-40% in

tox in -trea ted p la te le ts (Nakashima e t a l . , 1987). These resu lts

ind ica te tha t AA release by PLAg is independent of PLC ac tiva t ion and

a pertussis tox in -sen s it ive GTP-binding prote in l in ks receptor

ac t iva t ion to PLA^. cAMP in p la te le ts appears to be regulated by

stimulatory (Gs) and in h ib i to ry G proteins (Gi) (Jakobs e t a l . , 1987;

Katada e t a l . , 1984).

In vascular t issue , rather than in h ib i t in g PGI2 release, cholera

tox in potentiated agonist-stimulated PGI2 release from cultured

bovine a o rt ic smooth muscle c e l ls , whereas pertussis tox in was

inac tive (Demolle and Boeynaems, 1989). Sodium f lu o r id e (NaF) has

also been used as an ac t iva to r o f G proteins (Gilman, 1987). NaF

mimics GTP by forming a fluoro-aluminate complex which then binds to

and activates G proteins (Bigay e t a l . , 1985). NaF has been shown to

stimulate PGI2 synthesis by the ra t aorta and cultured endothelial

3 3

c e l ls (Jeremy and Dandona, 1988; Stansby e t a l . , 1991), an e f fe c t

blocked by PKC in h ib i t io n , Ca^^ channel blockade and cholera tox in

(Jeremy and Dandona, 1988; Magnusson e t a l . , 1989; Garcia e t a l . ,

1991). These data ind icate tha t G proteins l inked to vascular PGI2

synthesis involves the ac tiva tion of PLC and Ca^+ m obilisa tion .

L i t t l e is known of G proteins in the control o f PGIg-linked adenylate

cyclase in vascular t issue . The possible involvement o f G proteins in

eicosanoid synthesis is represented d iagram atica lly in f ig s . 1.3. and

1.4. Some aspects o f G proteins and eicosanoid synthesis in vascular

t issue and p la te le ts is fu r the r explored in th is thes is .

1.5. Vascular PGI2 and platelet TXA2 in the aetiology of CVD.

Given the d iam etr ica lly opposing e ffec ts o f PGI2 and TXA2 and

tha t these eicosanoids are synthesised by the vascular endothelium

and p la te le t respective ly , i t was qu ick ly perceived th a t the re la t iv e

balance between these two eicosanoids may cons titu te a fundamental

haemostatic mechanism (Moncada and Vane, 1979). P rio r to discussing

th is balance i t is pertinent to describe the basic physiology and

biochemistry o f p la te le ts and the vascular endothelium as they re la te

to haemostasis.

The physiological task o f c irc u la t in g p la te le ts is to a rrest the

loss o f blood when a blood vessel is damaged. This process involves:

a) rapid adhesion to exposed subendothelium, b) p la te le t to p la te le t

adherence (aggregation) and c) formation o f p la te le t plug (see f ig .

1 .5 .) . In v i t r o , p la te le ts are activated by a wide range o f

substances (thrombin, collagen, ADP, adrenaline, serotonin,

r is to c e t in , Ca2+ ionophore, AA and TXA2 analogues; Crawford and

Scrutton, 1987). The p la te le t response to these substances is

3 4

characterised by: a) shape change b) aggregation and c) the release

o f vaso- and thrombo-active substances from the beta and dense

granules and d) generation o f TXA2 (Crawford and Scrutton, 1987). The

dense granules, less numerous and o f greater electron density than

beta granules, contain nucleotides (v iz . ADP), serotonin, Ca2+ and

pyrophosphates. Alpha-granules release p la te le t derived growth fac to r

(PDGF; mediates tissue repa ir and wound healing), p la te le t fac to r 4

(PF4), f ib rone c t in (forms covalent linkages with collagen and f ib r in

and subendothelium), beta-thromboglobulin, thromospondin, fac tor V

and von W ill lebrand 's fac to r ( fac to r V I I I ) . The p la te le t also

synthesises p la te le t ac t iva t ing factor (PAF; fo r review see

Mikhail d is and Peplow, 1990). A diagrammatic representation o f

p la te le t ac t iva t ion sequence is given in f i g . 1.5.

The vascular endothelium generates both a n t i - and pro-thrombotic

factors (Sixma 1987; Pearson and Petty, 1989). Endothelium l ines the

luminal surface o f large vessels with a monolayer o f polygonal oblong

c e l ls stre tch ing th e i r long diameter along the d irec t ion o f blood

flow. In small vessels th is "cobblestone" pattern disappears as

ind iv idua l c e l ls fo ld th e ir cytoplasm to enc irc le the en tire

c a p i l la ry lumen. Prevention o f p la te le t adhesion and aggregation to

the healthy vascular endothelium is effected by a physico-chemical

in te rac t io n o f p la te le ts with endothelial surface glycoproteins and

the secretion by the endothelium o f substances which in h ib i t p la te le t

a c t iv i t y . These a n t i -p la te le t substances include PGI2 (Moncada and

Vane, 1979), ADPase (Lieberman et a l . , 1982) and endothelium-derived

relaxing fac to r (EDRF; Furchgott, 1983), now thought to be n i t r i c

oxide (NO; Palmer e t a l . , 1987). Apart from synthesising

predominantly, PGI2 , the endothelium posesses the capacity to

36

THROMBUS

RESTING PLATELET

erythrocytes

FIBRIN

serotonin

SHAPE CHANGE

a g g r e g a t i o n

/PDGF

degranulatlon

ADHESION

Fig. 1.5. Principal events in p late let activation, in vivo. Circulating pla te lets, sensing damage to the endothelium or via stimulation by plate let activating substances, undergo shape change. Platelets then adhere to sites of vascular damage ( i .e . where endothelium removal exposes the subendothelium) and release growth factors (e.g. PDGF) which brings about endothelial repair.I f vascular damage is more severe, platelets aggregate aggressively in large numbers and form a plug, in and around which f ib r in and erythrocytes accumulate, thereby forming a thrombus at the s ite of vascular in ju ry . Platelets undergo degranulation and release a number of prothrombotic and proaggregatory factors which enhance thrombus formation. These release substances are l ike ly to 'prime' other c ircu la ting platelets.

36

generate other PGs (e.g. PGE2 , PGp2c and TXA2 [Stansby e t a l . ,

1991]).

In contrast to these a n t i -p la te le t fac to rs , p la te le t adhesion to

subendothelium requires the presence o f von Willebrand's fac to r, a

g lycoprotein synthesised by endothelial c e l ls and stored in

organelles known as Wei be l-Palade bodies (Wagner e t a l . , 1982). The

endothelium also synthesises angiotensin-converting enzyme (ACE;

Caldwell e t a l , 1976), which cleaves angiotensin I to l ibe ra te

angiotensin I I (ANG I I ; a vasconstric tor, promoter o f p la te le t

a c t iv i t y and a mitogen; Taubman et a l . , 1989). More recently , the

endothelium has been shown to synthesise endothelin-1 , the most

potent vascoconstrictor known (Yanagisawa e t a l . , 1988). Endothelium

also produces thrombomodulin (ac t iva tion o f protein C anticoagulant)

thromboplastin, t issue plasminogen ac tiva ting factor (t-PA) and t-PA

in h ib ito r -1 (Sixma, 1987; Petty and Pearson, 1989). Vascular

endothelium also possesses the enzyme, l ipop ro te in l ipase, which

cleaves fa t ty acids from l ipop ro te ins , the fa t ty acids then being

taken up fo r sequestration by vascular tissues (Eckel, 1989; see

section 1. 8 . and f i g . 1 . 6 ).

1.6. Measurement and assessment of PGI2 and TXA2 synthesis, in vivo

and in vitro, in relation to CVD and thromboembolic disease

Both PGI2 and TXA2 break down spontaneously to the stable

hydrolysates, 6-oxo-PGF-|q, and TXB2 , respective ly (Moncada and Vane,

1979). These la t te r hydrolysates are fu r the r converted by the l iv e r

to various metabolites predominantly, 2 ,3-d inor derivatives

(Rosencrantz e t a l . , 1980; FitzGerald e t a l . , 1981; Barrow and

Taylor, 1987; Barrow and R it te r ; 1988, FitzGerald et a l . , 1985).

37

Radioimmunoassay (RIA) o f TXB2 and G-oxo-PGP^în plasma has been

employed, but th is method has serious flaws; p r in c ip a l ly interference

by other plasma components (Barrow and R it te r , 1988). Apart from

s p e c i f ic i t y and s e n s i t iv i t y there are other problems with assessing

c i rc u la t in g eicosanoid metabolites. At venepuncture ac tiva t ion o f

ju s t a small number o f p la te le ts (there are several 100s o f m il l ions

in 1ml blood) may a r te fa c tu a lly a l te r the amount o f TXB2 in a blood

sample. S im ila r ly , venepuncture has been shown to e l i c i t a local

large 'trauma-stimulated' release of PGI2 from blood vessels (Jeremy

e t a l . , 1984a), p o te n t ia l ly a l te r in g the amount of PGI2 in a blood

sample. The use o f gas chromatography-mass spectrometry (GC-MS) has

obviated some o f these problems (but not o f eicosanoid release at

time o f sampling in the case o f measurement in blood) and is now

considered the d e f in i t iv e method fo r asessement o f measurement o f

c irc u la t in g metabolites o f PGI2 and TXA2 (Barrow et a l . , 1982,

FitzGerald e t a l . , 1985; Barrow and Taylor, 1987; Barrow and R it te r ,

1988).

I t has been established tha t the actual c irc u la t in g leve ls o f

TXB2 , 6-oxo-PGF-|qj and th e ir hepatic metabolites are in the ng to sub-

ng/1 range (Barrow and R i t te r , 1988) which produces profound problems

o f s e n s i t iv i t y . These s e n s i t iv i ty problems have been la rge ly obviated

by measuring d i-nor metabolites in the urine, again using GC/MS

(Catella and FitzGerald, 1987; Barrow e t a l . , 1987; Barrow and

R it te r , 1988). This approach is advantageous since: a) there are

greater concentrations o f these metabolites in the urine than in

blood, b) urine co l le c t io n is non-invasive and c) urine is devoid o f

b io log ica l material th a t can generate these eicosanoids (except, o f

course, in kidney disease, where blood ce l ls appear in the u r in e ).

3 8

The assumption, however, tha t urinary metabolites re f le c t vascular

and/or p la te le t - generated eicosanoids must be approached with

caution. We know, fo r instance, tha t the bladder also possesses the

capacity to synthesise PGs (including PGI2 and TXA2 ) in large

quan tit ies (Jeremy et a l . , 1984b; M ikha il id is e t a l . , 1987b). I t was

also found th a t variables such as distension and osmolality o f

luminal f lu id as well as disease states (e.g diabetes m e ll i tus [DM])

can profoundly a l te r bladder PG synthesis (Jeremy e t a l . , 1986d).

Thus, i t was suggested tha t where a disease state may a l te r bladder

PG synthesis (which, in tu rn , may a l te r urinary PG metabolite

p ro f i le s ) , then th is con tr ibu tion may d is to r t in te rp re ta t io n o f

re s u lts , i f i t were assumed tha t urinary PGs are so le ly o f vascular

or p la te le t o r ig in (M ikhaildis e t a l . , 1987b). In a recent paper,

th is hypothesis was consolidated when i t was demonstrated th a t the

ur inary bladder contributes as much as 50% o f the to ta l PGs present

in urine (Reyes and Klahr, 1990).

TXA2 synthesis by p la te le ts has also been widely studied by

measuring the release o f th is eicosanoid, ex v ivo. Problems o f

s e n s i t iv i ty and s p e c i f ic i t y are avoided since, p la te le ts possess the

capacity to synthesise large quantit ies o f TXA2 (1ml PRP maximally

generates microgram amounts o f TXA2 ; M ikha il id is e t a l . , 1983,

1985a,1985b). Thus, TXB2 has been measured (by RIA) in blood samples

which have been allowed to c lo t (V iin ikka and Y likorka la , 1980) and

in samples o f PRP fo llow ing aggregation in an aggregometer

(M ikh a il id is e t a l . , 1983, 1985a,1985b). One ob jective o f th is thesis

was to study the release o f TXA2 in washed, unstirred p la te le ts ( to

obviate aggregation; see in troduction o f chapter 3 fo r a more

de ta iled ra tiona le )

C learly, the study o f vascular PGI2 by vessels obtained from

3 9

volunteers or patients is not feas ib le . Thus, most o f our knowledge

on vascular PGI2 has been derived from experiments using vessels

obtained from laboratory animals. In th is approach, vessels are

excised, cut in to r ings , placed and incubated in physiological

buffers and PGI2 release assessed by RIA o f 6-oxo-PGF-|q, . In early

experiments, only the spontaneous release o f PGI2 was measured,

sometimes by bioassay ( in h ib i t io n o f p la te le t aggregation, in v i t r o ) .

However, as was discussed above, PGI2 synthesis is con tro lled by

complex in t ra c e l lu la r mechanisms. Thus, PGI2 stimulated by d i f fe re n t

substances can reveal s ites at which defects in disease states may

occur and also a t which s ites drugs may exert th e i r action (see

section 1 .9 .) . Without doubt fo r most studies on PGI2 release

vascular t issue has been obtained from cultured vascular ce l ls

(endothelial and smooth muscle).

1.7. Eicosanoids and mechanisms of atherogenesis

The presence o f advanced a therosc lero tic plaques is invariab ly

the common denominator in ind iv idua ls who develop and die of

ischaemic heart disease (IHD) or stroke (Davies and Thomas, 1985;

Meade, 1987; Woolf, 1989). In tu rn , r is k factors tha t predispose to

the development o f atherosclerosis include diabetes m e ll i tus (DM;

Kannel and McGee, 1979; Krowlewski e t a l . , 1985; Pyorala e t a l . ,

1987; Betteridge, 1990), hypertension (Hollenberg, 1991; Rau, 1991),

c iga re tte smoking (Meade, 1987; McGill, 1990; Oeremy and M ikh a il id is ,

1990), gender ( i . e . males > females; Kannel and McGee, 1979) and

hyperlipidaemia (cho les te ro l, low density l ip o ro te in [LDL] and

tr ig ly c e r id e s ; Smith, 1987; Betteridge, 1989a,b; Krauss, 1991).

Key events in the i n t i t i a t i o n and growth o f the a therosc lero tic

40

plaque, are the p ro l i fe ra t io n o f a r te r ia l smooth muscle c e l ls ,

deposition o f l i p i d and accumulation o f collagen, e la s t ic f ib res and

proteoglycans (Haust e t a l . , 1960). I t has been proposed tha t

atherogenesis is in i t ia te d by endothelial ce l l in ju ry which, in turn

leads to adherence o f p la te le ts and subsequent local release of

p la te le t substances (TXA2 , serotonin, PAF, histamine and PDGF) a l l o f

which are pro-mitogenic, promote fu r th e r adhesion and aggregation of

p la te le ts and a t t ra c t monocytes and neutrophils (Ross and Glomset,

1976; Spagnuolo e t a l . , 1988). More recen tly , invasion in to vascular

tissue o f monocytes and th e i r transformation in to macrophages has

been suggested as the progenitor o f the a therosc lero tic plaque (Ross

e t a l . , 1986; Mitchinson and B a ll , 1987). Macrophages, in tu rn ,

transform in to 'foam c e l ls ' which appear a t the edges o f most

advanced a therosc lero tic lesions and accumulate l ip id s , in

p a r t ic u la r , cholesterol (G errity 1981). Macrophages also release a

plethora o f mitogenic and inflammatory substances (histamine,

serotonin, leukotrienes, in te r leuk ins ( I t s ) , tumour necrosing factor

(TNF), PGs, TXA2 , PDGF-like substance, to x ic free rad ica ls , and

neutral proteases; Mitchinson and B a l l , 1987; Woolf, 1989).

As part o f th e i r k i l l i n g (o f bacteria) mechanism, neutrophils

generate large ammounts o f superoxide. Apart from the destruction of

bacteria , superoxide e l i c i t s l i p id peroxidation o f fa t ty acids both

in the plasma membrane and in LDL, enhancing the atherogenic ity of

th is l ipop ro te in (Witzum and Steinberg, 1991). L ip id peroxides also

in h ib i t PGI2 synthesis a t the endoperoxide synthase level (Warso and

Lands, 1983). In tu rn , PGI2 has been shown to in h ib i t the

in f i l t r a t i o n o f monocytes in to blood vessels, the adhesion o f

41

neutrophils to in ju red vascular endothelium and the release o f pro-

atherogenic substances from these c e l ls (Ham e t a l . , 1983; Oones and

Hurley, 1984; Weksler and Goldstein, 1980; Belch e t a l . , 1986;

Gryglewski e t a l . , 1987).

Notwithstanding the mechanisms th a t i n t i t i a t e atherogenesis,

p la te le ts ce r ta in ly play a ro le in the end-stage pathological events

th a t lead to thrombus formation. At end stage, the a therosc lero tic

lesion becomes c a lc i f ie d and denuded o f endothelium and the plaque

often ruptures. The vascular lumen is also markedly narrowed

(s tenosis), which leads to abnormalities in blood flow (Karino and

Goldsmith, 1987). C learly, such a pa tho log ica lly damaged s i te

constitu tes a prime ta rge t fo r p la te le t adhesion, aggregation and

c lo t formation. Shear stress caused by stenosis has also been widely

implicated as a component in p la te le t ac t iva t ion in atherosclerosis

(Karino and Goldsmith, 1987).

Apart from in h ib i t in g p la te le t and leukocyte adhesion and

aggregation, PGI2 and other PGs has been shown to in h ib i t in v i t ro

VSMC and f ib ro b la s t p ro l i fe ra t io n (Huttner e t a l . , 1977; Nilsson and

Olsson, 1984; Owen, 1985; Pomerantz and Hajja r, 1989). In an elegant

study, Libby e t a l . (1988) demonstrated tha t the cytokine,

in terleukin-1B (IL-1B), stimulated VSMC and f ib ro b la s t p ro l i fe ra t io n ,

an e f fe c t blocked by PGE and PGE2 . In tu rn , both p la te le t and

leucocyte release substances have been shown to stimulate the release

o f PGI2 and other PGs from endothelial c e l ls , VSMCs and whole vessels

(Coughlin e t a l . , 1980, 1981; Baezinger e t a l . , 1981; Van Coevorden

and Boeynaems, 1984; A llbrightson e t a l . , 1985; Jeremy e t a l . , 1985c,

1988a; Rossi e t a l . , 1985; Ristimaki and V iin ika , 1992) ind ica ting

tha t there is a complex communication between these c e l ls , in which

42

release substances o f opposing actions are the means by which ce ll

p ro l i fe ra t io n may be con tro lled in blood vessels (see f i g . 1. 6 . ) .

PGs have other properties which perta in to the aetio logy o f

atherosclerosis. For example, PGI2 has been shown to stimulate (via

cAMP; Dembinska-Kiec e t a l . , 1980) the a c t iv i t y o f cholesteryl ester

hydrolase (Hajjar e t a l . , 1982; Pomerantz e t a l . , 1989). In the

vascular c e l l , th is la t t e r enzyme converts cholesterol esters

(impermeable to ce l l membranes) to free cholesterol (permeable to

c e l l membranes), which is then libe ra ted in to the c irc u la t io n (see

f i g . 1 .7 . ) . Impairment o f cholesteryl ester hydrolase may (possibly

invo lv ing impairment o f PGI2 synthesis) lead to accumulation of

cholesteryl esters, an accepted con tr ibu ting fac tor in atherogenesis

(Krauss, 1991). Lipoproteins (both high and low density) also

stimulate PGI2 synthesis in vascular t issue , an e f fe c t ascribed to

the release o f AA fo llow ing the hyd ro ly t ic action o f l ipop ro te in

lipase (Pomerantz e t a l . , 1984b, 1989). A hypothetical model fo r the

ro le of PGI2 in l i p i d sequestration in vascular t issue is given in

f i g . 1.7.

1.8. PGI2 and TXA2 in IHD and disease predisposing to IHD

a) Atherosclerosis

In a r te r ie s , heart, lung and kidney o f rabbits w ith experimental

atherosclerosis, there is a decrease in the ex vivo release o f PGI2

(Dembinska-Kiec e t a l , , 1977, Gryglewski e t a l . , 1978). Reduced PGI2

synthesis by a therosc lero tic human blood vessels, obtained post

mortem, has been reported (Sinzinger e t a l . , 1979). Larrue e t a l . .

PHASE

T Y R O S IN E P H O SP H O R YLA TIO N

c -fo sMITOGENS PKC c - myc

PDGF

C Y T O K IN E S5 - H T

PGFzot

d a gPRO TEINRc -G p

P H O S P H O R Y L A TIO Nphosphoiipase C

caM-PK

c •■SA N T I-M IT O G E N S PGsPGE;PGJPGDPGI.

-4cAMPR c—Gp adenyl c y c la s e

F i g . 1 . 6 . P o s s ib l e i n v o l v e m e n t o f PGs in th e c o n t r o l o f v a s c u la r smooth muscle c e l l p r o l i f e r a t i o n ( t h e pathognomonic l e s i o n o f a t h e r o s c l e r o s i s ) . Mi togens e l i c i t seve ra l e a r l y e v e n ts , i n p a r t i c u l a r , p h o s p h a t id y l i n o s i t o l 4 , 5 - b i s - -phospha te (P IP 2 ) h y d r o l y s i s which i n t u r n r e s u l t s i n p r o t e i n p h o s p h o r y l a t i o n by k inase C and c a l c i u m /c a lm o d u l i n - -dependen t k inases (caM-PK) . These events induce ex p re s s io n o f two genes: c - f o s and c-myc, th u s i n i t i a t i n g DNA s y n th e s i s and c e l l d i v i s i o n . E l e v a t i o n o f cAMP i s a s s o c ia te d w i t h i n h i b i t i o n o f m i t o s i s . cAMP a c t i v a t e s PKA which p h o s p ho ry la te s p r o t e i n s t h a t c o u n t e r t h e e f f e c t s o f m i togens . Thus, the a n t i m i t o g e n ic e f f e c t o f c e r t a i n endogenous PGs, i s mediated by a c t i v a t i o n o f a d e n y la te c y c la s e . CO

BLOODVASCULAR CELL

LDL

CELDLdrop le t

CEHatherosclerosisendocytosis

e s té r i f ica t ionlipoprotein lipasecAMP

arachidonicacid

egress

M E M B R A N E

/ LDL \ f cholesteryl

V ester

L D L -R c

PGI

fre ech o le s te ro l

HDL

Fig. 1.7. P o s s ib l e invo lvem en t o f PGI2 i n l i p i d s e q u e s t r a t i o n by v a s c u la r c e l l s . Low d e n s i t y l i p o p r o t e i n (LDL) i s taken up by e n d o t h e l i a l a n d /o r v a s c u la r c e l l s where a r a c h i d o n i c a c id (AA) i s l i b e r a t e d by l i p o p r o t e i n l i p a s e f rom t r i g l y c e r i d e s c o n ta in ed i n t h e LDL. AA i s then conve r ted t o PGI2 which s t i m u l a t e s cAMP s y n th e s i s which a c t i v a t e s c h o l e s t e r y l e s t e r h y d ro la s e (CEH). In t u r n , CEH h y d ro lyses e s t e r i f i e d c h o l e s t e r o l ( impermeable t o c e l l membranes) t o f r e e c h o l e s t e r o l which can then be removed f rom th e c e l l ( p o s s i b l y f o l l o w i n g i n c o r p o r a t i o n i n t o h igh d e n s i t y l i p o p r o t e i n HDL). A t h e r o s c l e r o t i c l e s i o n s a re a s s o c ia te d w i t h an accumula t ion o f e s t e r i f i e d c h o l e s t e r o l ( i n d r o p l e t s ) . Thus, any r e d u c t i o n o f the a c t i v i t y o f t he enzymes i n v o l v e d w i t h PGI2 s y n th e s i s ( e . g . by excess c e l l u l a r c h o l e s t e r o l ) may i m p a i r t h e normal egress o f c h o l e s t e r o l f rom c e l l s and as such c o n t r i b u t e t o a th e ro g e n e s is .

4 5

(1980) also reported diminished PGI2 release by cultured VSMCs

derived from atherosc lero tic ra bb it aortae. P late le ts also adhere to

a therosc lero tic vessels more read ily than normal vessels (Dembinska-

Kiec e t a l , 1977). TXA2 production by p la te le ts from patients with

atherosclerosis and peripheral vascular disease (PVD) is increased

(FitzGerald e t a l . , 1986; M ikha il id is e t a l . , 1985a). Patients with

severe atheromatous disease excrete greater amounts o f 2,3-d inor

metabolites o f G-oxo-PGP^# and TXB2 than healthy controls (F itzgera ld

e t a l . , 1984). As was mentioned e a r l ie r , damage to vessels causes

enhanced release o f PGI2 , which may explain these increased leve ls of

PGI2 metabolites. P la te le ts , which are probably activated in vivo by

damaged atherosc lero tic vessels would also explain the increased

leve ls o f TXA2 metabolites in patients with severe atheromatous

disease. However, R it te r e t a l . (1986), found no elevation o f PGI2

metabolites in plasma from patients with severe atheromatous disease.

This, may be explicable by an 'exhaustion' o f PGI2 substrate due to

prolonged ac tiva t ion o f output in diseased vessels.

b) Diabetes Mellitus (DM)

Patients with diabetes m e ll i tus (DM) are a t greater r is k than

the healthy population o f developing cardiovascular disease (CVD;

Banga and Sixma, 1986; M ikha il id is e t a l . , 1988; Betteridge, 1989a,

1990). Of the many ae tio log ica l factors associating CVD with DM, a

marked attenuation o f prostacyclin (PGI2 ) synthesis by blood vessels

from d iabetic laboratory animals (Harrison e t a l . , 1978; Rogers et

a l . , 1981; Jeremy e t a l . , 1987a) and from vascular biopsies obtained

from diabetic patients (Johnson et a l . , 1979; Silberbauer e t a l . ,

1979) has been reported. The precise mechanisms tha t determine

4 6

reduced vascular P6 I 2 synthesis in diabetes m e ll i tu s (DM) are not

c le a r ly defined, although a reduction in substrate (arachidonate)

leve ls in blood vessels has been suggested (Holman e t a l , 1983;

Jeremy e t a l , 1987a, Dang e t a l . , 1988). As was discussed e a r l ie r ,

vascular PGIg synthesis is stimulated by vasoactive agonists, which

in tu rn is mediated by G pro te ins , PKC and IP3 (G riend ling e t a l . ,

1986; Hal lam e t a l . , 1988; Jeremy e t a l . , 1988a). The net re s u l t o f

a c t iv a t io n o f these systems is transmembrane Ca2+ in f lu x and an

increase in in t r a c e l lu la r Ca2+ ([Ca2+].) which then ac tiva tes PLA2 . A

major ob jec t ive o f th is thes is was to fu r th e r e luc ida te the

mechanisms th a t determine diminished vascular PGI2 synthesis in DM,

using a range o f s t im u la to rs th a t act a t d i f fe re n t s i te s in the

receptor ac tiva t ion -P G l2 synthesis pathway in the s trep to zo toc in -

induced d ia b e t ic ra t ( fo r a more complete ra t io n a le see the

in trodu c t ion o f chapter 5 ) .

In general, disturbances o f p la te le t function are seen only in

d ia be t ic pa tien ts where micro- or macroangiopathy is present (Hendra

and Betteridge, 1989). Possibly the area o f greatest disagreement is

th a t o f TXA2 release by p la te le ts from d ia be t ic p a t ie n ts . Increased,

decreased an unchanged re lease o f TXA2 by p la te le ts from d ia b e t ic

pa tien ts has been reported (Hendra and Betteridge, 1989; M ik h a i l id is

e t a l . , 1988). These d is p a r i t ie s may be methodological. For example,

when one assesses TXA2 re lease fo l lo w in g aggregation, i t must be

remembered th a t the more pronounced the aggregation the greater the

re lease o f TXA2 (A r i ta e t a l . , 1988). I f one looks a t re lease in non

aggregated p la te le ts ( e l i c i t e d by freeze f ra c tu r in g and/or

so n ica t io n ), then one a c tu a l ly sees a reduction in TXA2 re lease from

p la te le ts taken from d iabe tics when compared to con tro ls (Jeremy e t

47SMOOTH MUSCLE ENDOTHELIUM BLOOD

Ach _

NOnoradcGMP

vasodilation cAMP

PGI

PGI

constriction TXA

PDGFaggregate5 -H T

THROMBUSPGE

cell proliferation’CYTOKINES

MONOCYTE

MACROPHAGE

FOAM CELL NEUTROPHIL

o rA.S.

PLAQUE-peroxidation

cholesterol

F i g . 1 . 8 . P o s s ib le i n t e r r e l a t i o n s h i p s between b lood v e s s e l s , p l a t e l e t s and le u co cy te s i n a th e ro g e n s i s . Damage t o th e endo the l ium r e s u l t s i n p l a t e l e t and n e u t r o p h i l adhes ion and monocyte i n f i l t r a t i o n where they t r a n s f o r m i n t o macro- -phages ( p r o g e n i t o r s o f foam c e l l s ) . A l l these c e l l s r e l e a s e substances which are m i t o g e n i c , i n c re a s e v a s c u la r p e r m e a b i l i t y and f u r t h e r enhance le u c o c y te and p l a t e l e t ac c u m u la t i o n . However, these r e l e a s e substance a l s o s t i m u l a t e v a s c u la r PG sy n th e s i s wh ich c o u n t e r a c t these e f f e c t ( i . e . d i m in i s h p l a t e l e t and le u c o c y te adhes ion, r e l e a s e r e a c t i o n s and i n h i b i t c e l l p r o l i f e r a t i o n ) . PG r e l e a s e thus c o n s t i t u t e s a defence mechanism by which a th e ro g e n ic events a re endogenously coun te rac ted by b lood v e s s e l s . In t u r n , any impai rment o f normal PG re l e a s e may render a b lood vessel s u s c e p t i b l e t o these a t h e ro g e n ic ev e n ts .

4 8

a l . , 1988b). This la t t e r point may also ind icate tha t p la te le t

dysfunction in DM is not due to an increased a c t iv i t y o f the TXA2

synthesising pathway but is a consequence o f increased p la te le t

a c t iv i t y .

In contrast to DM, there is v i r t u a l l y universal agreement tha t in

patients with peripheral vascular disease (PVD), there is a marked

increase in p la te le t a c t iv i t y . Aggregation ( in p la te le t r ic h plasma

and in whole blood) and concomitant TXA2 release is markedly enhanced

in p la te le ts from patients with PVD (M ikhaildis e t a l . , 1985a). The

general consensus is tha t increased p la te le t a c t iv i ty fo llows rather

than precedes PVD. Certa in ly diseased and damaged vessels are l i k e ly

to cause an exc ita t ion o f c irc u la t in g p la te le ts , since th e i r innate

function is to recognise and be activated by vascular damage.

c) Hypertension

Intravenous in fusion of PGI2 decreases blood pressure in

normotensive and hypertensive patients (Papanikolaou, 1988),

ind ica ting tha t PGI2 plays a ro le in co n tro l l in g vascular tone.

Although i t may be expected tha t vascular PGI2 would be reduced in

hypertension, the m ajority o f studies ind ica te an increase in PGI2

output by vessels from hypertensive animals (Pace-Asciak, 1978; Botha

e t a l . , 1979; Rioux e t a l . , 1985). In experimental portal

hypertension, PGI2 synthesis by the hepatic portal vessel is markedly

enhanced (Hamilton e t a l . , 1981,1982; Guarner and Soraino, 1993).

Furthermore, c irc u la t in g PGI2 metabolites are increased in patients

with portal hypertension (Sitzmann and L i , 1991). Thus, i t has been

suggested tha t increased PGI2 release may constitu te an adaptive

response aimed a t diminishing hypertension (Hamilton e t a l . , 1982).

4 9

One objective o f th is thesis was to investigate changes o f

receptor-l inked PGI2 synthesis in both the aorta and mesenteric

vasculature o f the ra t with experimental portal hypertension and

possibly to p inpoin t s ites a t which a lte ra t io ns o f mechanisms common

to both PGI2 synthesis and vasoactiv ity may occur. A more complete

ra t iona le is given in the in troduction o f chapter 5.

Given tha t TXA2 is a vasoconstrictor, i t has been suggested that

p la te le t hyperac tiv ity may play a ro le in hypertension (Nyrop and

Zwe if le r, 1988). As in DM, changes in p la te le t function from

hypertensive patients are equivocal. I t appears tha t the median

a r te r ia l pressure must be > 120 mm Hg before increases in p la te le t

a c t iv i t y are manifest (Nyrop and Zw e if le r, 1988). In te re s t in g ly , i t

has been demonstrated tha t there is increased adhesion and migration

o f monocytes (which also produce TXA2 ; Davies, 1986) in to blood

vessels o f hypertensive ra ts (Haudenschild, 1979).

d) Cigarette smoking

Cigarette smokers are a t fa r greater r is k o f developing

atherosclerosis than non-smokers (Kannel, 1981; Wilhelmsen, 1988;

McGill, 1990). The e f fe c t o f c iga re tte smoking on p la te le t a c t iv i t y ,

TXA2 synthesis and vascular PGI2 is an area o f considerable

d ifference o f opinion. That p la te le t aggregation and / or release is

increased, decreased or unchanged in smokers has been reported by

d i f fe re n t groups (K u t t i , 1990). An increase in 2,3-dinor metabolites

o f TXA2 the urine o f smokers has been reported (Murray e t a l . ,

1990; Barrow e t a l . , 1989). In a recent study the author reported

tha t c iga re tte smoke condenstates are potent in h ib ito rs o f TXA2

release, in v i t r o (Jeremy and M ikh a i l id is , 1990). There are reports

60

tha t n ico tine in h ib i ts vascular PGI2 synthesis, in v i t r o (Wennmalm

and A ls te r, 1983), whereas other studies have disputed th is (Oeremy

e t a l . , 1985e). More recently , c iga re tte smoke extracts have been

shown to produce a biphasic e f fe c t on vascular PGI2 synthesis, in

v i t r o : s tim u la tion at low concentrations and in h ib i t io n at higher

concentrations o f ex trac t (Jeremy and Mikhail ids, 1990). These

find ings ind icate th a t another component(s) o f tobacco smoke (other

than n ico tine) is responsible fo r a lte red vascular PGI2 synthesis, at

leas t in v i t r o . I t is , however, unequivocal tha t plasma fibrinogen

concentrations are elevated in smokers and tha t th is coagulation

fac to r is a f irm pred ic to r o f pending IHD (Meade, 1987b). Smoking

also causes profound morphological damage to the endothelium ( P i t t i l o

e t a l , 1982, 1984, 1990; Bull e t a l . , 1988) as well as enhancing

p la te le t adhesion to the endothelium ( P i t t i l o e t a l . , 1984). Thus,

smoking does not appear to increase the r is k o f developing IHD

through a chronic ac t iva t ion o f p la te le ts but possibly via damage to

the endothelium. This la t te r s i tu a t io n may eventually re s u lt in

hyperactive p la te le ts in response to vascular damage, as in PVD (a

condition tha t has a well established association with smoking).

e) Gender

IHD is more prevalent in age-matched men than pre-menopausal

women, but in post-menopausal women th is decreased incidence o f IHD

returns to tha t o f age-matched men (C a s te l l i , 1988). I t has been

suggested tha t estrogens confer th is pro tect ive e f fe c t to women

(Jeremy and M ikh a i l id is , 1991). In con trast, women who use the

contraceptive p i l l (containing synthetic estrogens / progestagens)

51

are more l ik e ly to develop thrombosis than non-users (Upton, 1990).

Data are scant on the e ffec ts o f estrogens and androgens on vascular

PGI2 synthesis. However, androgens have been shown to in h ib i t

vascular PGI2 synthesis (Chang et a l . , 1982), whereas estrogens (and

progesterone) appear to have no e f fe c t (Jeremy and Dandona, 1985).

With regard to p la te le t TXA2 synthesis, few studies have been carried

out to elucidate the ro le o f the sex hormones on p la te le t a c t iv i t y .

This is suprising since CVD is ove rt ly gender-linked.

1.9. Eicosanoids and drug action

Apart from the ro les played by PGs and TXs in normal c e l lu la r

function and in the pathophysiology o f CVD, these eicosanoids are

important mediators o f drug action. This mediation can e ith e r be by a

d ire c t action on eicosanoid synthesising enzymes or by an in d ire c t

e f fe c t on systems tha t also control eicosanoid synthesis. The c lass ic

example o f d ire c t acting drugs are non-steroidal antiinflammatory

drugs (NSAIDs) such as asp ir in and indomethacin, which in h ib i t

cyclooxygenase a c t iv i t y (Vane, 1971; Flower, 1974). In h ib ito rs o f 5-

and 12-lipoxygenases and drugs which act at the synthase level have

also been developed (most notably r id o g re l, a combined TXA2 synthase

in h ib i to r and TXA2 receptor antagonist; Rainsford, 1988; Sanner,

1988). PLA2 in h ib i to rs include non-steroidal drugs such as mepacrine

and quinacrine as well as cort icoste ro ids (via s tim ulation of

l ip o c o r t in ) , such as dexamethasone, betamethasone prednisolone

(Flower, 1978; Rainsford, 1988). Eicosanoid receptor antagonists,

p a r t ic u la r ly against LTs and TXA2 , have also been developed and have

met with some therapeutic success (Sanner, 1988).

Apart from d ire c t ly acting drugs, other classes o f drugs have

52

been shown t o m o d u la t e e i c o s a n o i d s y n t h e s i s a t s i t e s o t h e r t h a n

enzymes (PLA2 , s y n t h a s e s , c y c l o o x y g e n a s e , l i p o x y g e n a s e a n d / o r

r e c e p t o r s ; Boeynaems, 1 9 8 8 ) . P u t a n o t h e r way, any g i v e n d ru g may a c t

a t d i f f e r e n t s i t e s i n t h e e i c o s a n o i d s y n t h e s i s i n g p a th w a y . For

e x am p le , a l t h o u g h a - a d r e n o c e p t o r a n t a g o n i s t s ( e . g . p r a z o s i n ,

y o h i m b i n e , r a u w o l s e i n e ) i n h i b i t n o r a d r e n a l i n e o r a d r e n a l i n e -

s t i m u l a t e d PGI2 r e l e a s e f r o m b l o o d v e s s e l s , t h e y have no e f f e c t on

A23187- o r AA- s t i m u l a t e d PGI2 s y n t h e s i s ( Jeremy e t a l . , 1 9 8 5 a ) . T h i s

l a t t e r e f f e c t i s r e a d i l y e x p l i c a b l e by a s p e c i f i c a c t i o n on

a d r e n o c e p t o r s w h i c h w o u l d , i p s o f a c t o , b l o c k a d r e n a l i n e - s t i m u l a t e d

PGI2 s y n t h e s i s b u t n o t t h e a c t i v i t y o f o t h e r sys tems i n v o l v e d i n

a d r e n o c e p t o r - PGI2 s y n t h e s i s c o u p l i n g . I n c o n t r a s t , Ca^^ channe l

b l o c k e r s such as n i f e d i p i n e and n i m o d i p i n e , i n h i b i t r a t a o r t i c PGI2

s y n t h e s i s when s t i m u l a t e d by # - a d r e n o c e p t o r a g o n i s t s ( S t e w a r t e t

a l . , 1984; Golub e t a l . , 1985, Jeremy e t a l . , 1985a, 1 9 8 6 c ) , A23187

( Jeremy e t a l . , 1 9 8 6 c ) , p h o rb o l e s t e r ( Je remy and Dandona, 1987) and

NaF (Jeremy and Dandona, 1988) b u t n o t a r a c h i d o n a te o r t r a u m a -

s t i m u l a t e d PGI2 r e l e a s e (Jeremy e t a l . , 1 9 8 6 c ) . T h i s l a t t e r e f f e c t i s

e x p l i c a b l e s i n c e PLA2 i s C a ^ ^ - a c t i v a t e d ( I r v i n e , 1982; Hong and

D e y k i n , 1982) and r e c e p t o r a c t i v a t i o n , PKC and G p r o t e i n s p re ce d e s

t h e PLA2 s t e p whereas A A - s t i m u l a t e d and t r a u m a s t i m u l a t e d s y n t h e s i s

b y -p a s s t h e Ca^^ m o b i l i s i n g s t e p . I n t h i s c o n t e x t , t h e a n g i o t e n s i n

c o n v e r t i n g enzyme i n h i b i t o r (A C E I ) , c a p t o p r i l , was shown t o i n h i b i t

r a t a o r t i c PGI2 s y n t h e s i s when s t i m u l a t e d by a d r e n a l i n e , U46619 and

A23187 b u t n o t by AA o r t raum a (Je remy e t a l . , 1 9 8 8 e ) . I t was

c o n c lu d e d t h a t ACE I s may t h e r e f o r e e x e r t t h e i r h y p e r t e n s i v e e f f e c t

n o t o n l y v i a i n h i b i t i o n o f ANG I I g e n e r a t i o n , b u t a l s o v i a i n h i b i t i o n

o f Ca2+ m o b i l i s a t i o n ( e s s e n t i a l f o r v a s o c o n s t r i c t i o n ; Somlyo and

53

S omlyo , 1 9 6 8 ) . T hu s , t h e a c c e p te d mode o f a c t i o n o f any one c l a s s o f

d ru g may n o t be i t s o n l y s i t e o f a c t i o n . F u r t h e r m o r e , s i n c e PGI2 and

TXA2 s y n t h e s i s i n v o l v e s a number i n t e r m e d i o r y s t e p s , i t i s t h e r e f o r e

p o s s i b l e t o d e t e c t a l t e r n a t i v e s i t e s o f a c t i o n o f any g i v e n d ru g

u s i n g d i f f e r e n t s t i m u l a t o r s w h ich a c t a t s e p a r a t e s i t e s i n t h e

r e c e p t o r - e i c o s a n o i d s y n t h e s i s c o u p l i n g p a th w a y . A m a j o r o b j e c t i v e o f

t h i s t h e s i s , t h e r e f o r e , was t o a p p l y t h i s p r i n c i p a l i n t h e

e x p l o r a t i o n o f a l t e r n a t i v e s i t e s o f a c t i o n o f some common d ru g s used

i n t h e t r e a t m e n t o f CVD. A more c o m p l e t e r a t i o n a l e i s g i v e n i n t h e

i n t r o d u c t i o n o f c h a p t e r 4 .

64

AIMS OF THE THESIS

1) To fu r the r investigate receptor-linked PGI2 synthesis in blood

vessels o f the ra t , ra b b it and man, with p a rt icu la r reference to

receptor agonists and the mediatory ro le o f calcium, G proteins and

protein kinase C.

2) To investigate the va r ia t ion o f receptor-linked PGI2 release

between species and between d i f fe re n t blood vessels (a r te r ie s and

veins) in the same species. Studying the ro le o f the endothelium in

mediating PGI2 release by blood vessels was also an ob jective .

3) To study the e f fe c t o f hypothermic storage o f blood vessels in

organ preservation solutions in re la t io n to the complications

encountered in organ transp lanta tion .

4) To develop a method fo r the study o f p la te le t TXA2 synthesis in a

s ta t ic (non-aggregating) system and to study the ro le o f calcium, G

proteins and PKC in mediating TXA2 synthesis.

5) To investigate the s i te o f action o f drugs (m ilr inone, non

steroidal antiinflammatory drugs, copper chelators) using the

techniques developed in above, in blood vessels and p la te le ts

6 ) To apply the systems developed in (1) above to c la r i f y the

mechanisms tha t lead to the altered synthesis o f vascular PGI2 in

experimental diabetes m e ll i tus and hepatic portal hypertension.

6 6

CHAPTER 2 . MATERIALS AND METHODS

2.1. MATERIALS

The fo llowing chemicals and drugs were obtained from:

a) Sigma Chemical Co. (Poole, Dorset, UK):

acetylcholine ch lo ride ,

adenosine c yc l ic 3'5'-mono phosphate,adenosine diphosphate,adrenaline b i ta r t ra te ,angiotensin I I ,apyrase,arachidonic acid (99% pure), bovine serum albumin, calcium ionophore A23187, carbachol ch loride, collagen type I I I , d ie th y ls t i lb e s t ro l ,Dulbecco's minimum essential medium (MEM),fe ta l c a l f serum,histamineN-2-Hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES),ibuprofenindomethacin,i sobutylmethylxanthi neisoquino linylsu lfonylm ethyl pi p e r iz ine (H7),medium 199,noradrenaline b i ta r t ra te , nordihydroguaretic acid,DL-penicillamine, p e n ic i l l in 6 , phenylephrine ch lo r ide , phorbol ester d ibutyra te ,

phorbol ester myristate acetate,PGs (PGE- j , PGEg, * 6 - o x o - P G F ^ ^ )

serotoni n (5-hydroxytryptamine), sodium f lu o r id e .

6 6

sodium nitroprusside,staurosporine,streptomycinstreptozotocin,substance P,thapsigargin,

thromboxane A2 ,T r is (hydroxymethyl) - méthylammonium chloride (Tris hydrochloride),

U46619 (thromboxane A2 analogue) verapamil.

b) A ldrich Chemical Co. (Gillingham, Dorset, UK):

dimethyldithiocarbamic acid, te traethylth iouram d is u lf id e t r ie n t in etr ie thy ld ith iocarbam ic acid phenanthroline

c) BDH (Poole, Dorset, UK):

absolute ethanol (A r is ta r grade)chioroformdie thyl ether

ethyl acetateg lac ia l acetic acidmethanolpetroleum ether

Unless otherwise stated, a l l bu ffer components and metal sa lts were

purchased from BDH (see 2 .3 . ) . BDH also supplied p la s t ic plates fo r

th in layer chromatography (TLC), precoated with s i l i c a gel (Kieselgel

60; w ith fluorescent ind ica to r) or precoated with polyethylim i ne

(PEI) ce llu lose .

67

The fo llowing radiochemicals were obtained from New England Nuclear

(Dreieich, Germany):

[^H ]-6-oxo-PGF-|q, ( 120 Ci/mmol),

[ 3h]-TXB2 (120Ci / mmol),

[ 3h]-PGE2 (120 01 / mmol),

[ 3h]-PGF20! (120 01 / mmol),

[ ' ^ 0 ]-arach1don1c acid (20 01 / mmol),

[^H]-adenos1ne c y c l ic monophosphate (120 01 / mmol).

Protein binding assay k i ts fo r the measurement o f cAMP were obtained

from Amersham Radiochemicals (Amersham, UK).

Polyclonal antisera against 6-oxo-PGF^q, ,1X82, PGE2 and PGF2Q, of

high serological s p e c i f ic i ty were purchased from Capell Laboratories

(West Chester, PA, USA) and Cayman Chemical Co. (Ann Arbor, MI, USA).

Polypropylene tubes (2.5 ml) fo r incubation o f tissues and

p la te le ts as well as fo r radioimmunoassays were purchased from

Luckham Ltd. (Middlesex, UK), micropipettes and automatic l iq u id

dispensers from Gilson (Anachem L td ., Luton, Beds, UK), l iq u id

s c in t i l la t io n v ia ls from G & G Chemicals (Berks, UK), l iq u id

s c in t i l la t io n f lu id from National Diagnostics (Sussex, UK), M u lt is t ix

from Ames Division (Miles Laboratories L td ., Stoke Poges, Slough,

England) and Percoll from Pharmacia (Uppsala, Sweden).

Pharmaceutical companies supplied the following drugs: milrinone

(S ter ling Winthrop, Guildford, UK), benzydamine (3M Riker,

Loughborough, UK), i lo p ro s t (Sobering, Burgess H i l l , UK), n ifed ip ine

(Bayer UK L td ., Newbury, UK), t ia p ro fe n ic acid (Rousell, Uxbridge,

UK), nabumetone and 6-MNA (Smith Kline and Beecham, Welwyn Garden

C ity , UK), BW 755C (Wellcome Pharmaceuticals, Beckenham, Kent, UK).

6 8

2.2. INSTRUMENTS

a) bench centrifuge (Centra 7R) and sonicator (Soniprep 150): MSE

Instruments (Sussex, UK).

b) shaking water bath: Grant Instruments (Cambridge, UK)

c) vortex mixer, vacuum chamber evaporator, magnetic s t i r r e r :

Gallenkamp Ltd. (Middlesex, UK)

d) pH meter: Corning Science Products, (Middlesex, UK).

e) d ig i ta l balance: Oertling (Maidstone, Kent, UK).

f ) beta p a r t ic le counter (Rakbeta 1210): LKB Wallac (Turku,Finland).

g) p la te le t counter; model ZM (Coulter Electronics L td ., Luton,

Beds, UK).

2.3. BUFFER COMPOSITIONS (in mmol / 1, unless otherwise stated)

a) HEPES buffer A: 140 NaCl ; 2.7 KCl, 1 EDTA, 0.001 PGE (or 100

un its / ml apyrase), 3.8 HEPES, 0.1% glucose, 0.1% bovine serum

albumin; adjusted to pH 7.6 with I r i s base).

b) Ca2+ - free HEPES buffer B : 140 NaCI, 2.7 KCl, 8 NagHPO*, 1.5

KH2PO4 , 0.1 MgCl2 , 1 glucose; adjusted to pH 7.6 with 1M Tris base.

c) Tris-gelatin (RIA): 50 Tris HCl, 1g / 1 porcine g e la t in , adjusted

to pH 7.4 with HCl.

d) Dextran coated charcoal (RIA): In T r is ge la tin bu ffe r , 6g . l"^

Norit activated charcoal and 100 mg / 1 dextran (M.W. 5,000).

d) Kreb's Ringer bicarbonate buffer (KRB): 118 NaCl; 4.8 KCl; 1.2

MgS04. 7H20 , 1.2 KH2PO4 , 2.5 CaCl2 . 2H20; 11 glucose; 24 NaHCOg.

6 9

e) University of Wisoconsin preservation solution: 125 KCl; 25 NaCl;

5 MgCl2 , 100 1actobionate, 25 NaHgPO , 5 NaSO , 1 a l lo p u r in o l , 3

reduced glu tath ione, 30 ra ff inose , 5 adenosine, pH 7.4,

f ) Kidney Preservation solution: 80 KCl, 80 NaCl, 40 MgCl2 , 40 NaSO ,

pH 7.4.

g) Minimum essential medium (MEM): pre weighed dissolved In 1 l i t r e

double d is t i l le d water, NaHCOg added to 24 mmol / 1; gassed with 95%

O2 : 5% C02»

h) Medium 199: pre weighed preparations dissolved In 1 l i t r e double

d is t i l l e d water, containing 24 mmol / 1 NaHCOg, lOmg / 1

streptomycin sulphate, 10 mg / 1 actlnomycin D, 10% fe ta l c a l f serum;

gassed with 95% O2 : 5% CO .

1) Phosphate buffer (for cAMP assay): 8 Na2HP04, 5 KH2PO4 , adjusted

to pH 7.6 with HCl and/or NaOH.

2.4. Preparation and incubation of vascular (and other) tissues for

assessment of PG synthesis.

Blood vessels were obtained from laboratory ra ts , rabb its or

from humans who were donors fo r l i v e r transp lan ta tion . In the

m ajority o f animal experiments, aortae were used. In some animal

experiments, mesenteric vasculature, hepatic portal vein, vena cava,

femoral and caro tid a r te r ie s were also used (see chapter 3 ). The

pr inc ipa l human vessels Investigated were the I l ia c vein and I l i a c

a r te ry . These vessels were rou tine ly harvested from donors fo r l i v e r

transp la ta t lon , since In some cases they may have been required fo r

anastomosis In the Implantation procedure. In the m ajority o f cases.

6 0

however, they were not required and were therefore used in the

present study. On excision from l i v e r donors, vessels were placed in

the University o f Wisconsin so lu tion (UW; a preservation so lu tion ;

see chapter 3) and stored on ice . Since i n i t i a l studies revealed an

unpredictable va r ia t ion in the release o f PGs by human vessels, basic

studies on the e f fe c t o f UW and hypothermic storage on prostanoid

release were carried out in vessels from animals, as well as human

blood vessels.

In animal experiments, fo llow ing excis ion, adventit ia and fa t ty

t issue were removed and the vessels cut in to 2 mm rings with a

scalpel blade on a te f lo n block. Human vessels were opened

lo n g itu d in a l ly with scissors, cut la te ra l ly in to 2mm s tr ip s and

fu r the r in to 2mm squares with a scalpel blade on a te f lo n block, care

being taken to avoid touching the endothelium. Human blood vessel

segments were then processed in the same way as fo r animal vessels.

Where the e f fe c t o f endothelium removal was investigated, endothelium

was removed e ith e r by rubbing an opened vessel with a tissue or in

in ta c t vessels w ith a pipe-cleaner.

In a l l experiments invo lv ing stim u la tion o f PG synthesis with

agonists, vascular t issue was pooled and placed in Dulbecco’ s minimum

essential medium (MEM) fo r up to 9 h to allow trauma - stimulated

release o f prostanoids to subside (Jeremy e t a l . , 1984a, 1985a,

1985b, 1985c). In some comparative experiments, urinary bladders and

tracheal rings (chapters 4 and 5) were also investigated. The

bladders were cut lo n g itu d in a l ly in to four s tr ip s and each s t r ip in to

four fu r the r segments and whole trachea in to 4 mm r ings . Tissues

were then pooled and randomised in MEM and incubated fo r up to 9h in

MEM, at 37°C, w ith changes o f medium every 30 min, again to allow

61

prostanoid release e l ic i te d by preparative handling to subside

(Jeremy et a l . , 1984a, 1985a; see f i g . 2.1. fo r explanatory notes).

2.5. Effect of agonists, antagonists and other modulators of

intracellular second messengers on vascular PG synthesis

Following pre-incubation o f vessels as described above, vessel

segments or rings were placed in 2.5ml polypropylene tubes containing

MEM. Direct s timulatory e ffec ts o f any given agent were tested by

adding the agents over a concentration range and the tissues further

incubated fo r 30 min to 1h, at 37^C. Following incubation, aliquots

o f supernatant were taken and stored in a freezer at -70°C p r io r to

radioimmunoassay (RIA; see section 2 .9 .) o f PGs. An example o f PG

release over 6h pre-incubation and fo llow ing stimulation with

noradrenaline and antagonism with yohimibine (an alpha adrenoceptor

antagonist) is shown in f ig . 2.1.

2.6. Effect of drugs on PGI2 synthesis

For studies on the e f fe c t o f drugs on PG synthesis, tissues

(fo llow ing pre-incubation) were placed in 1 ml MEM containing varying

concentrations o f drugs ( in t r ip l i c a te fo r each concentration). The

tissues were then incubated fo r up to 1 h, at 37°C, to allow

e q u i l ib ra t io n o f tissues with drugs. The media was then aspirated and

replaced with fresh media containing the same concentrations of

drugs. Prostanoid synthesis was then e l ic i te d by the addition o f

known stimulators (see re s u lt section o f chapter 3 fo r l i s t o f

stimulators used). The tissues were then fu r the r incubated fo r 30 min

or 1 h, at 37°C, in a shaking water bath and a liquots o f supernatant

taken fo r estimation of PG concentrations by radioimmunoassay (RIA;

6 2

cÊ

(D

(D3cow

O)r

lL~0Q.IOX01

CD

U)CL

160

p+0yohimbine120

-8+ 1080

- 7—G

10 M NA //

40

//

- 5

//

time ( h )

F ig . 2 . 1 . Pattern o f PGI2 (as 6-oxo-PGF^qjmeasured by RIA) release from ra t a o r t ic rings incubated in MEM at 37°C, i l l u s t r a t in g the pr inc ipa l of the method employed to study vascular PGI2 release. The figu re shows the gradual subsidance o f PGI2 release i n i t i a l l y caused by the 'trauma' of preparation. Thus, when PGI2 has reached a basal output a stim ula tor is added; ( in th is case noradrenaline [NA]) which stimulates PGI2 release l in e a r ly over a period o f 1h. In tu rn , th is e f fe c t is antagonised in a dose-dependent fashion by the addition o f increasing concentrations of yohimbine (an alpha-blocker). This response is evidence fo r a receptor-

linked process.

6 3

see section 2 .9 . ) . Trauma-stimulated PG synthesis was e l ic i te d by

both freeze fra c tu r in g and sonication o f tissues. Tissues were

sequestered in to tubes and pre-equ il ib ra ted with drugs as above.

Freeze thawing: the media was then aspirated and tubes placed in a

freezer at -70°C fo r 3 h, a f te r which time medium containing the same

concentrations o f drugs were added and the tissues incubated fo r 1 h,

at 37°C. 6- oxo-PGF-iq, (and/or other PG) concentrations were measured

in the supernatant, as above. Sonication: fo llowing preincubation

w ith drugs and replacement o f bu ffer containing drugs, a sonicator

probe was placed in the MEM containing the vascular t issue . The

tissues were then subjected to 3 x 10 sec bursts o f sonication (20

microns) and the tissues incubated as above and samples o f MEM taken

fo r RIA of PGs.

2.7. Preparation of washed platelets

Blood was drawn from volunteers or patients who had not ingested

any drugs fo r at leas t 7 days p r io r to phlebotomy. One volume o f

trisodium c i t ra te (3.8%) was added to 9 volumes o f blood and

p la te le t - r ic h plasma (PRP) prepared by cen tr ifugation at x 160g fo r

15 min. PRP was collected and used in some experiments. Where washed

p la te le ts were used, p la te le t pe l le ts were f i r s t prepared by

cen tr ifuga tion at 550 g fo r 10 min and washed with HEPES buffer A.

The washed p la te le ts were resuspended in Ca2+ - free HEPES buffer B

and adjusted with HEPES buffer to a f in a l count o f 2 x 10^1 p la te le ts

/ 1 .

6 4

2.8. Platelet TXA2 synthesis by unstirred washed platelets.

a) Effect of platelet activators

Aliquots (100ju l ) o f PRP or washed p la te le ts were placed in

polypropylene tubes and 100 ul o f various concentrations o f

stimulators o f p la te le t a c t iv i t y (collagen, calcium ionophore A23187,

NaF, phorbol ester myristate acetate, thapsigargin, arachidonic acid,

ADP, adrenaline, U46619) added. Following gentle ag ita t ion , the tubes

were incubated at 37°C fo r 30 min and the reaction stopped with 400

ul absolute u ltra -pure ethanol. Preliminary experiments detected no

change in p la te le t count a f te r the incubation with s tim u la tors . I t

was therefore deduced tha t s ig n if ic a n t aggregation (a s t i r r in g -

dependent event) had not taken place. Following vortex-mixing and

cen tr i fug a t io n , a liquots o f ethanolic supernantant were placed in to

polypropylene RIA tubes and evaporated in a vacuum chamber. Residues

were reconstitu ted in T r is assay bu ffer and TXB2 concentrations

measured by radiommunoassay. In studies on the spontaneous release of

TXB2 , p la te le ts were incubated fo r 2h a t 37°C without addition of

stimulatory agents. Reactions were stopped with ethanol and p la te le ts

processed fo r the measurement o f TXB2 as described in section 2.9.

For studies on e f fe c t o f stimulators on PRP, the only d ifference in

procedure was th a t PRP was used instead o f washed p la te le ts .

b) Effect of drugs on stimulated TXA2 synthesis

Where the e f fe c t o f drugs and / or modulators o f TXA2 synthesis

were investigated washed p la te le ts (or PRP) were pre-incubated with

drugs fo r 10 min, at 37^C. Stimulators, a t pre-determined doses were

added in 10^1 a liquo ts . The tubes were processed fo r assessment o f

TXA2 release as described above in 2.8a.

6 6

2.9. Measurement of TXB2 , 6-keto-PGF-|Q, , PGE2 and PGp2 #by RIA

In a l l assay procedures described in th is section, T r is HCl -

ge la t in bu ffer was used. P la te le t extracts were reconstitu ted with

100 p] TRIS-RIA bu ffe r . Aliquots o f supernatant from vascular

incubates (usually 5-20 p i ) was adjusted to 100pi with T r is -

ge la t in assay bu ffe r . Standard curves comprised o f 100 pi of known

concentrations o f eicosanoids over a range o f 0-1 ng. To these 100j i ]

volumes, containing known and unknown quantit i tes o f eicosanoid, 200

ul d ilu ted anti sera containing 25 nCi [^Hj-eicosanoid was added. The

tubes were incubated at 4°C overnight. Bound and unbound eicosanoid

was separated by the addition o f 0.5 ml dextran coated charcoal in

Tr is bu ffer to each tube, followed by 10 min incubation at 4°C. Tubes

were then centrifuged and supernatants decanted in to l iq u id

s c in t i l la t io n v ia ls . Liquid s c in t i l l a t io n f lu id ( 10 ml ) was added

to each v ia l and counted fo r ra d io a c t iv i ty . Standard curves were

compiled and unknown eicosanoid concentrations calculated by

extrapo lation .

2.10. Validation of radioimmunoassays.

In the RIA o f eicosanoids in the present studies, no extraction or

p u r i f ic a t io n steps were carried out. This is possible in the present

in v i t r o systems as eicosanoids are generated in large quantit ies by

p la te le ts and vessels (unlike the minute quantit ies o f eicosanoid

metabolites found in urine or plasma). Furthermore, there are minimal

in te r fe r in g substances (un like the s itu a t io n in urine or plasma)

present in the incubation bu ffe rs . Nevertheless, a series of

va lida tion procedures were carried out. F i r s t ly , anti sera were

checked fo r cross re a c t iv i t y . I t was found tha t there was

6 6

considerable v a r ia t io n (as one might expect fo r polyclonal

antibod ies) between anti sera purchased from d i f fe re n t companies and

between-batch v a r ia t io n o f anti sera from the same company. In order

to obviate in te r-b a tch v a r t ia t io n , la rge amounts o f anti sera o f the

same batch were purchased. Cross r e a c t iv i t ie s o f various PGs w ith the

an ti sera employed are given in tab le 2 .1 . In te r - and in t r a - assay

c o e f f ic ie n ts o f v a r ia t io n are given in tab le 2 .2 . In order to

es tab lish fu r th e r the accuracy and possib le in te rfe rence o f other

endogenous substances w ith in the incubation systems employed, known

amounts o f 5-oxo-PGF-| q, and TXB2 were added to supernatants from

vessel and p la te le t incubations, which were then measured by RIA (see

f i g . 2 .2 . fo r fu r th e r d e ta i ls ) . These experiments consolidated the

high s p e c i f ic i t y and r e l i a b i l i t y o f the incubation and RIA methods

employed.

2.11. Measurement of p late let cAMP phosphodiesterase activ ity

P la te le t phosphodiesterase a c t i v i t y was assayed as previously

described (Jeremy e t a l , 1988). A liquo ts (10(^1) o f washed p la te le ts

were pre-incubated w ith various concentrations o f drug fo r 10 min, at

37°C. Unlabelled cAMP (lOyumoles [1 0 / j1 cAMP so lu t ion in HEPES in 90

ul washed p la te le ts ] ) was also added so as to give approximately 50%

conversion o f cAMP to AMP under the present incubation cond it ions .

100 nCi [^H]-cAMP was then added, the p la te le ts ra p id ly ag ita ted on a

vortex mixer and incubated fo r 5 min a t 37^0. The reac tion was

stopped by the ad d it ion o f 400 ul ice cold absolute ethanol,

conta in ing excess concentrations o f cAMP and AMP (both 100yuM).

Following vortex-m ix ing, son ication fo r 2 x 10 sec and

c e n tr i fu g a t io n , the supernantant was evaporated, in vacuo. Residues

6 7

D)C

T30)u.D(0(Q0)E;o’5c(Q(0OOÔ)

100

0.1

10 10010.10

lOOr

0.1

0 0.1 10 100

eicosanoid added ( ng / ml, final conc. )

Fig . 2 .2 . V a lida tion o f RIA methods: A= p la te le t TXB2 and B = ra t a o r t ic 6-oxo-PGFiQ,»3 a o r t ic r ings and 1 ml washed p la te le ts were incubated fo r 30 min w ith 10 uM A23187. CO= indomethacin - t rea ted ; untreated ] . Following incubation and c e n tr i fu g a t io n , supernatants Were taken o f f and div ided in to 100 ul a l iq u o ts . To each a l iq u o t was added known q u a n t it ie s o f

TXB2 or 6-oxo-PGFi(% and fu r th e r a liquo ts taken fo r RIA. In the samples not trea ted w ith drugs, zero values were subtracted from the values obtained

with added eicosanoid. As can be seen the values measured equate p rec is ley with the ammounts added. Thus, i t is concluded th a t : 1) there are no in te r fe r in g substances released by p la te le t or vessels th a t may d is to r t measurements by RIA and 2) there is l in e a r i t y in immunoreactive TXB2 and 6- oxo-PGF-joj over a wide concentration range.

6 8

were resuspended in ethanol/water (50 / 50; v /v) and cAMP and AMP

separated by th in layer chromatography (TLC) on po lye thy lim ine (PEI)

ce llu lose p la tes, developed in 50 mM KCl (Jeremy e t a l . , 1988c).

Unchanged cAMP and AMP were detected under UV l ig h t and each band

removed from the p la te and placed in a l iq u id s c in t i l la t io n v ia l . KCl

(1 ml; 100 mmol / 1 ) was added and l e f t overnight in a shaking water

bath at 40°C to ex trac t the nucleotides from the PEI ce llu lose .

Liquid s c in t i l la t io n f lu id was added to each v ia l and ra d io a c t iv ity

counted from which the % in h ib i t io n o f conversion o f cAMP to AMP was

ca lcu lated.

2.12. Prostanoid - stimulated cAMP synthesis.

Aliquots o f washed p la te le ts were pre-incubated with various

concentrations o f drug and/or modulator fo r 10 min, at 37°C. PGE or

i lo p ro s t was then added over a pre-established stimulatory

concentration range (see f i g . 2.3. fo r dose-response curves) and

incubated fo r 10 min, at 37°C. The reaction was stopped by the

addition o f 400 ul ice cold absolute ethanol. Following vortex-

mixing, sonication fo r 2 x 10 sec and cen tr ifuga tion , a liquots o f

supernantant were evaporated in vacuo. Residues were resuspended in

cAMP assay bu ffer and cAMP concentrations measured by cAMP binding

prote in radioassay (Amersham International p ic ) , as previously

described (Jeremy e t a l . , 1988c).

6 9

cE

(/)CDCD

-*—»

i?Q .

00OT—

CL

<O

OEQ .

0 0.03 0.3

prostaglancdin (^ m o l 'T ' )

F ig . 2 .3 . E ffe c t o f PGE ( Q ) and i lo p ro s t ( Q ] ) on cAMP synthesis by

washed human p la te le ts . Each po in t = mean i . SD, n =7.

7 0

Table 2 .1 . Cross r e a c t iv i t ie s o f some major PGs w ith anti sera

employed in th is th e s is .

cross mass o f PG required to d isp lace 50% [^H-PG]

re a c t iv i t y mass o f PG required to disp lace 50% [^H-PG]

PG = PG against which anti serum has been raised (e .g . 6-oxo-PGFi#)

and PGy = other PG being tested fo r cross re a c t iv i t y (e .g . PGE )

% Cross re a c t iv i t y

an ti sera

eicosanoid 6-oxo -P G F^^ TXB2 PGE2

6-oxo-PGF-j 0 100 0.001 0.01 0 .03

TXBg 0.01 100 0 .03 0.01

6-oxo-PGE^q 0 .02 0 .005 0.01 0 .02

PGFgcx 0.01 0 .002 0 .02 100

PGEg 0 .003 0 .005 100 0.1

PGD2 0.01 0 .003 0.1 0 .003

PG&j 0.001 0.01 8 .0 0 .05

PGBg 0.01 0 .003 0 .05 0.003

PGA2 0 .02 0 .005 0 .03 0.002

arachidonic acid < 0.001 < 0.001 0.01 0.001

l in o le n ic acid < 0.0001 < 0.0001 0.001 <0.0001

71

Table 2.2 . Inter-assay and in tra-assay c o e f f ic ie n ts o f v a r ia t io n (CV)

o f eicosanoids measured by RIA. CVs were obtained from 10

measurements o f the same sample in the same assay ( in tra -assay CV

[%]) and in 10 separate assays ( in te r-assay CV [% ]).

Eicosanoid in tra-assay CV in te r-assay CV

6-oxo-PGF-jQ, 2 3

TXBg 2 5

PGEg 3 4

PGFg 2 2

2.13. P latelet lipoxygenase activ ity

P la te le ts contain the enzyme lipoxygenase th a t converts AA to 12-

HETE. Thus, the lipoxygenase assay is based on the incubation o f

p la te le ts w ith ra d io lab e lled AA ([^^C-AA]) followed by separation and

is o la t io n o f 12-HETE by TLC. A liquots ( 100 ^1 ) o f PRP were

cen tr ifuged a t 2,500 rpm fo r 10 min. The plasma supernatants were

discarded and the p e l le ts washed w ith HEPES bu ffe r B, to remove any

residual plasma. HEPES B ( 100^1 ) conta in ing varying concentrations

o f drug / modulator was added to the p e l le ts ( in t r i p l i c a t e fo r each

concentration o f drug) and ag ita ted v igorously to resuspend the

p la te le ts . The tubes were incubated fo r 15 min a t 37®C to a llow

e q u i l ib ra t io n o f the drug w ith the p la te le ts . To each tube, 100 nCi

o f [^^C]-arachidonic acid was added to the tubes which were then

vortexed and incubated fo r a fu r th e r 30 min a t 37°C. The incubates

were then trans fe rred to glass tubes and 500 ul d ie thy l e ther: hexane

7 2

(50:50, v /v ) added. The tubes were vortexed v igorously , neutral

l ip id s (v iz . AA and 12-HETE) being extracted in to the organic phase.

Tubes were cen tr ifuged and supernatants removed and placed in glass

tubes. The remaining aqueous phase was then extracted w ith 500 ul

Folch reagent (chloroform / methanol; 3 / 2 , v /v ) and vigorously

vortexed and cen tr i fuged . The subnatant (Folch is denser than aqueous

HEPES) was removed and added to the ether / hexane e x tra c t . The

pooled extracts were then evaporated in a vacuum chamber. Residues

were then redissolved in 50 ^1 Folch so lu t io n and spotted onto the

o r ig in o f precoated (w ith s i l i c a ge l) p la s t ic TLC p la tes . The p la tes

were then placed in chromatography tanks contain ing 100 ml d ie thy l

e ther: hexane: ace tic acid (50 : 50 : 1; v /v ) . The solvent moved up

the p la te and when a t the top was removed and dried in a i r . The plate

was then be div ided la te r a l l y in to 1 cm bands w ith a pencil and these

bands cut sequen tia lly from the o r ig in upward, placed in v ia ls and

l iq u id s c in t i l l a t i o n f l u id added. The v ia ls were then ag ita ted

v igorously and ra d io a c t iv i t y counted in a b e ta -p a r t ic le counter. The

separation p r o f i le were then compiled (see f i g 2 .4 . ) . Both the

lipoxygenase in h ib i to r s , NOGA and BW755c (but not indomethacin)

in h ib i te d conversion o f AA to HETE ( f i g . 2 .4 . ) , conso lida ting th a t

the present method re f le c ts lipoxygenase and not cyclooxygenase

a c t i v i t y .

2.14. PGI2 synthesis by cultured human umbilical endothelial cells

(HUVECS) human omental tissue microvascular endothelial cells

(HOTMECs)

HUVECs were prepared by Ms Neelima Shukla o f the Department o f

Surgery (Royal Free H osp ita l) , using the method o f Oaffe e t a l .

7 3

(D

Aarachldonlc acid

12-HETE

phospholip ids

n -T Lb

origin

0

solvent front

(OCOCCDD)>XOÛ.

O r

50co

s!cc

100

10 100

inhibitor ( uM )

F ig . 2 .4 . A. Running pos it ions o f phospholip ids, 12-HETE and arachidonic

acid (AA) on TLC pla tes coated w ith s i l i c a gel G and developed in solvent

mix: hexane / d ie thy l ether / ace tic acid (50 / 50 / 1 ; v / v / v ) .

B. E ffe c t o f NOGA (Q ;lipoxygenase in h ib i t o r ) , BW755c ( 0 ;1 ipoxygenase

in h ib i to r ) and indomethacin (jh^cyclooxygenase in h ib i to r ) on conversion o f

[l^c-AA ] to [^^C-12-HETE] by washed p la te le ts (each po in t = mean 1 S.D.)^,n — 6

7 4

(1973). Umbilical cords were obtained from women who de livered normal

healthy babies spontaneously and who experienced no complications

during pregnancy. Following f lush ing o f the um bilica l vein w ith MEM,

the cord was clamped a t one end and MEM contain ing 1% collagenase

inserted in to the venous lumen and the cord clamped a t the other end.

The cord was then immersed in KRB and incubated a t 37°C, fo r 30 min.

The clamp was removed and the f lu id conta in ing endothe lia l c e l ls

decanted in to a f la s k . Cells were washed with MEM and cen tr ifuged at

1,000 rpm and the supernatant discarded. This procedure was ca rr ied

out three times. The washed c e l ls were then placed in a 100ml cu ltu re

f la sk conta in ing 5 ml medium 199. Following i n i t i a l c u ltu re in medium

199 (conta in ing 20% fe ta l c a l f serum [FCS]) fo r 5 days, c e l ls were

trea ted w ith MEM conta in ing t ry p s in (10 Units / ml) which dislodged

the c e l ls which adhere to the f la sk bottom. The dislodged c e l ls were

seeded in to 96-well t issue c u ltu re p la tes and fu r th e r grown in medium

199 conta in ing 20% FCS. A m od if ica tion o f the method o f Kern e t a l . ,

(1983) was used to prepare HOTMECs fo r c u l tu re . Omental fa t (20-75g)

was obtained from pa tien ts undergoing ro u t ine surgery. The fa t was

incubated in 1% collagenase fo r 30 min a t 37°C. the d igest was

f i l t e r e d , allowed to s e t t le and adipocytes removed. The remaining

so lu t ion r ic h in vessel fragments were cen tr ifuged in 45% Perco l1 fo r

20 min, the vessel fragments forming a laye r near the top o f the

density g rad ien t. This endothe lia l r ic h layer was then removed and

placed in Ml99 conta in ing 20% FCS and cu ltu red as above fo r HUVECs.

When confluen t (2-5 days), the c e l ls were washed w ith pre-warmed MEM

and MEM conta in ing s t im u la to rs ( Î drugs ) added. The p la tes were

then incubated a t 37°C in an incubator and the supernatants from each

7 6

well removed and stored a t -70°C fo r radioimmunoassay o f 6-oxo-PGFiQ,

and other PGs as described above.

2.15. Ex vivo experiments on rats with diabetes (pair fed and fed ad

libitum) or hepatic portal hypertension.

2.15a. Diabetes

A ll experiments were ca rr ied out using male Sprague Dawley ra ts

( i n i t i a l body weight o f 250 g ) . Non-ketonuric, hyperglycaemic

diabetes was induced by in je c t in g s trep tozo toc in in travenously ( t a i l

vein; 65 mg / kg body w t . ) . These ra ts developed g lycosuria but not

ketonuria or haematuria. In some experiments, d ia b e t ic ra ts were fed

e i th e r ad l ib i tu m or p a ir fed w ith D iet 41B (Grain Harvester, Kent,

UK) and allowed free access to water. In the p a ir fed experiments,

a l l animals were given 25g ra t chow per day. This is the average food

intake o f normal age-matched ra ts (Thompson e t a l . , 1990). In su l in

(10 i . u . , tw ice d a i ly ) was administered to one group o f d ia b e t ic ra ts

fed ad l ib i tu m . Seven o f each group o f ra ts were s a c r if ic e d a f te r 8

weeks o f diabetes and the re s u lts compared w ith seven age matched

co n tro ls . Rat urine was monitored fo r glucose, ketone bodies and

pro te ins w ith M u l t is t ix (Ames Diagnostics, Stoke Poges). At the time

o f s a c r i f ic e , blood was co llec ted by cardiac puncture fo r measurement

o f blood glucose.

A f te r 8 weeks ra ts were anaesthetised w ith pentobarbitone (90 mg

/ kg in t ra p e r i to n e a l ly ; Sagata l: May and Baker L td . , Dagenham, UK). A

m id line in c is io n was made in to the abdominal ca v ity and the hepatic

porta l ve in , aortae, ca ro t id a r te r ie s , g a s tro in te s t in a l (GI) t r a c t ,

u r inary bladders and trachea were ra p id ly excised and placed in MEM.

Given the small s ize o f the vasculature in the mesenteric bed and the

7 6

presence o f large amounts o f adipose t is su e , venous and a r te r ia l

mesenteric vessels were not dissected from each o ther. The in te s t in a l

segments were opened by lo n g itu d in a l in c is io n and rinsed free o f

residual food. Mucosa was separated from the muscular po rt ion o f

duodenal, je juna l and i le a l t issue w ith a glass s l id e (Debnam and

Levin, 1975) and placed in MEM. Stomach mucosa and muscularis were

separated as prev ious ly described (Jeremy e t a l . , 1987). The muscular

po rt ion was cu t in to 2mm wide s t r ip w ith a scalpel blade and then

in to 2mm squares; 4 squares in dup lica te fo r each animal were placed

in polypropylene tubes conta in ing MEM and incubated in a shaking

water bath fo r 30 min. Mucosal scrapes were washed three times in MEM

followed by c e n tr i fu g a t io n . The mucosal scrape from each in te s t in a l

po rt ion and the g a s tr ic mucosal discs ( in dup lica te fo r each animal)

were placed in 1ml MEM and incubated and processed as fo r muscularis

t is s u e . Following incubation the tubes were centr ifuged a t 200 rpm,

fo r 10 min and a l iq u o ts o f supernatant stored a t -70*^0 p r io r to

measurement o f G-oxo-PGFi# , PGE2 , PGF2 (^ and TXB2 by RIA.

Mesenteric vessels were dissected away from adherent adipose

t issue and cut in to 1mm lengths o f vessel fo r each animal. Hepatic

po rta l vein was cu t in to approximately 1mm squares. Aortae and

c a ro t id a r te r ie s were cu t in to 1mm r in g s . Approximately 10 mg

vascular t issue ( in dup lica te fo r each animal) were incubated in 1ml

MEM fo r 1h a t 37°C. A liquo ts o f supernatant were stored a t -70°C fo r

measurement o f 6-oxo-PGF^q, , PGE2 , PGF20 ; and TXB2 by RIA.

2.15b. Hepatic portal hypertension.

Male Sprague Dawley ra ts o f body weight 250g were used. Portal

hypertension was induced s u rg ic a l ly by p a r t ia l l ig a t io n o f the portal

7 7

vein (Cummings e t a l . , 1986). These procedures were ca rr ied out by Dr

Ai den McCormick o f the Department o f Medicine, Royal Free H osp ita l.

This form o f experimental porta l hypertension d i f fe r s from other

means o f e l i c i t i n g th is s ta te (e .g . by adm in is tra tion o f carbon

te t ra c h lo r id e ) in th a t hypertension develops w ithout s ig n i f ic a n t

l i v e r dysfunction. Control ra ts comprised o f sham-operated animals.

A fte r 7 days, animals were anaesthetised w ith pentobarbitone (90 mg /

kg, i . p . ) and po rta l blood pressure measured fo l low ing cannulation o f

the superior mesenteric ve in . Vascular t issue was not co l lec ted from

s ite s o f cannulation or l ig a t io n . Thoracic aortae and mesenteric

vasculature were excised and processed fo r assessment o f PGI2 release

as described above.

2.16. Statistics

Throughout th is study, s t a t is t ic a l s ign if icances between mean

vaules was determined by paired or unpaired Student’ s tw o - ta i le d t

t e s t . Values are expressed as mean 2 SEM or SD.

7 8

CHAPTER 3

STUDIES ON PGIg AND OTHER PROSTANOIDS RELEASED BY BLOOD VESSELS FROM

THE RAT, RABBIT AND MAN AND ON TXA2 RELEASE BY HUMAN PLATELETS.

3 . 1 . INTRODUCTION

As ou tl ined in the general In t ro d u c t io n , the re lease o f PGI2

from vascular t issue is e l i c i t e d by a range o f vasoactive agonists.

This process, in tu rn , is mediated by in t r a c e l lu la r 2nd messengers,

inc lud ing G p ro te in s , PKC, and Ca2+. However, few systematic studies

between species and between d i f fe re n t vessels o f the same species

have been ca rr ied ou t. This is o f importance since data obtained

from any one given vessel may not be representa tive o f other vessels

in the same species. This p o in t is exemplified by the o ften -ra ised

ob jec tion against using the aorta from labora tory animals as a

representa tive t issue fo r studies on CVD or drug action (e .g .

Tomlinson e t a l . , 1992). I t has been suggested th a t as a conduit

vessel, the aorta lacks the s e n s i t iv i t y o f other smaller vessels

(v iz . a r te r io le s and microvascular beds; Tomlinson e t a l . , 1992).

Secondly, receptors and l inked mechanisms c o n tro l l in g PGI2 synthesis

in a vessel from any given species may vary considerably from other

species. This is o f importance since the mechanisms underly ing a

given b io func t ion in a s ing le species o ften d i f f e r when compared to

the general p ic tu re obtained from other species. Furthermore, since

the re s u lts o f experiments in animal models/tissues (a t le a s t in

c l in ic a l research) are o ften extrapolated to man, i t is c le a r ly

important th a t the properties o f a 'model' vessel should in fa c t ,

resemble th a t o f man. In order to examine these areas fu r th e r , PGI2

re lease, in response to a range o f receptor agonists and

7 9

i n t r a c e l lu la r mediators by d i f fe re n t blood vessels from the r a t ,

ra b b i t and man were investiga ted . The ro le o f the endothelium in

mediating vascular PGI2 release in these vessels was also

investiga ted since there is some confusion as to the re la t iv e

c o n tr ib u t io n o f the endothelium to ‘ to ta l output* o f PGI2 .

The studies on human vessels were complicated by several

fa c to rs . F i r s t l y , since blood vessels were obtained from donors fo r

l i v e r tra n sp la n ta t io n , the donors were in va r ia b ly the v ic t im s o f

t r a f f i c accidents (head in ju r ie s ) or cerebrovascular events

(aneurysm). Thus, these unfortunate v ic t im s had been sustained on

l i f e support systems fo r va r iab le times and had received a va r iab le

range o f drugs. Secondly, the vessels, fo l low ing exc is ion , were

placed in preservation so lu tions (U n ive rs ity o f Wisconsin; see

below) and stored on ice fo r up to 18h p r io r to experimentation. In

order to determine the e f fe c ts o f hypothermic storage in

preservation so lu t ions on vascular PGI2 re lease, systematic studies

on the e f fe c t o f cold preservation o f blood vessels ( r a t and ra b b it )

were also ca rr ied ou t. These studies are also o f fundamental

in te re s t to tra nsp la n t surgery fo r several reasons. Primary g ra f t

dysfunction, fo l lo w in g o r tho top ic l i v e r t ra n sp la n ta t io n , occurs in

about 10% o f cases (Maddrey e t a l . , 1988; Starzl e t a l . , 1989;

B u su tt i l e t a l . , 1987; Krom e t a l . , 1989; Quiroga e t a l . , 1991) and

is a major cause o f m orb id ity and m o r ta l i ty . The cause o f primary

g r a f t dysfunction is m u lt i fa c to r ia l but "storage in ju r y " may be an

important co n tr ib u to ry fa c to r in a subset o f pa tien ts (Kazikoe e t

a l . , 1990). Studies using r a t l iv e rs have demonstrated th a t

s ig n i f ic a n t morphological damage to endothe lia l and Kupffer c e l ls

occurs fo llow ing reperfusion a f te r hypothermic storage o f the organs

80

in Euro-Col l in s so lu t io n and to a lesser degree in U n ive rs ity o f

Wisonsin (UW) so lu t ion (Caldwel1-Kenkel e t a l . , 1989; 1990). The

functiona l s ign if ica nce o f these morphological changes is unknown.

However, the vascular endothelium influences haemostasis and

v a so a c t iv i ty , in p a r t , through the secre tion o f PGI2 , as well as

endothelium derived re la x ing fa c to r (EDRF; is ob lig a to ry fo r

re la xa t io n and also in h ib i t s p la te le t a c t i v i t y ; Furchgott, 1983;

Ignarro, 1989; Rubanyi, 1990). Damage to the endothelium and

d is ru p t io n o f EDRF and PGI2 re lease may the re fo re re s u l t in

vasoconstr ic t ion or thrombosis, which in tu rn would endanger g ra f t

fu n c t io n . Indeed there is a considerable body o f evidence th a t

ischaemic in ju r y is a major cause o f ea r ly g r a f t fa i lu re s (Maddrey

e t a l . , 1988; S tarzl e t a l . , 1989; B u su t t i l e t a l . , 1987; Krom e t

a l . , 1989; Quiroga e t a l . , 1991).

TXA2 synthesis by p la te le ts obtained from pa t ie n ts , has la rg e ly

been investiga ted e i th e r by: a) a llow ing blood to c lo t and measuring

TXA2 released in to the serum by radioimmunoassay (V i in ikka and

Y l ik o rk a la , 1981) or b) fo l lo w in g in v i t r o aggregation (ca rr ie d out

in plasma) e l i c i t e d by known aggregating agents (e .g . adrenaline,

ADP, collagen; D iczfa lusy e t a l . , 1977; M ik h a i l id is e t a l . , 1983,

1985a, 1985b). In terms o f determining the a e t io lo g ica l ro le o f TXA2

in any given d iso rder, both o f these methods have drawbacks.

F i r s t l y , components o f blood, (e .g . f a t t y acids, glucose,

l ip o p ro te in s , e t c . ) , have been shown to in fluence aggregation and

the re fo re TXA2 re lease (Hendra and Bette ridge, 1988). Although the

e f fe c t o f blood components on aggregation and or TXA2 synthesis and

re lease is in i t s e l f o f importance one is s t i l l confronted by the

question as to whether TXA2 re lease is primary or secondary to

81

p la te le t a c t iv a t io n . With regard to the study o f TXA2 released in

response to aggregating agents, we have recen t ly established th a t in

p la te le ts from pa tien ts w ith PVD, which are known to be

hyperaggregable and which l ib e ra te more TXA2 than con tro ls

(M ik h a i l id is e t a l . , 1985a), th a t TXA2 synthesising capacity is

a c tu a l ly diminished (Jeremy e t a l . , 1988b). th is l a t t e r f in d in g

implies th a t TXA2 not play an a e t io lo g ic a l ro le in

hyperaggregability o f p la te le ts but ra the r is secondary to the

enhanced aggregation.

In order to d issec t out TXA2 synthesis from aggregation and to

obviate the va r iab le o f e f fe c ts o f plasma/serum fa c to rs , a method

fo r the study o f TXA2 synthesis in u n s t i r re d , washed p la te le ts was

developed and the fo l lo w in g studies ca rr ied out to in ve s t ig a te : 1)

the e f fe c t o f pro-aggregatory agonis ts , ADP, adrenaline, co llagen,

arachidonic acid and A23187 on TXAg synthesis, 2) the ro le o f Ca2+

in mediating s t im u la t ion o f TXA2 synthesis using Ca2+ che la to rs ,

Ca2+ channel b lockers, high potassium, and thapsigarg in (mobilises

in t r a c e l lu la r Ca^*; Thastrup e t a l . , 1990), and 3) the ro le o f PKC

using phorbol es te r and staurosporine. The methods described in

chapter 2 were app lied .

3.2 . RESULTS

3.2a. Effect of endothelium removal on receptor-linked PGI2 release

by the ra t aorta.

At 3h post-endothelium removal, concentration-PGl2 response

curves o f the s t im u la to ry agents investiga ted (adrenaline,

noradrenaline, PDBU, NaF) were sh i f te d markedly to the l e f t (F ig .

3 .1 . ) . At 6h post-endothelium removal, however, there were no

82

d iffe rences between these dose-response curves from in ta c t aortae or

from aortae w ith endothelium removed (F ig . 3 .1 . ) . Substance P,

ace ty lcho line and n itrop russ ide were w ithou t s ig n i f ic a n t e f fe c t on

de novo ra t a o r t ic PGI2 synthesis, i r re s p e c t iv e o f incubation time

or the presence o f the endothelium (data not shown). At 3h post

endothelium removal, the in h ib i to ry action o f yohimbine, prazosin

and n ife d ip in e on adrenaline- on noradrenaline- stimulated PGI2

synthesis were markedly diminished ( f i g . 3 .2 .) w h i ls t those o f

con tro l aortae were s im i la r to prev ious ly described data (Jeremy e t

a l . , 1985a). At 6h post-endothelium removal the in h ib i to ry action of

yohimbine, prazosin and n ife d ip in e were p a r t ia l l y restored, although

s t i l l s ig n i f ic a n t ly diminished, w h i ls t a t 9h post-endothelium

removal, i t was completely restored in aortae w ithout endothelium

( f i g . 3 .2 . ) . Relaxors o f r a t a o r t ic t is su e ; substance P, Ach,

carbachol and n itrop russ ide ( a l l up to 3 x 10"^ M) were w ithout

s ig n i f ic a n t e f fe c t on noradrenaline-stimulated PGI2 synthesis in

aortae with and w ithout endothelium (data not shown).

3.2b. Comparative studies on PGI2 release by arteries and veins from

the ra t and rabbit.

In a l l blood vessels (o ther than the aorta) inves tiga ted in the

ra t (ca ro t id and hepatic po rta l ve in ) , PGI2 synthesis was stimulated

by adrenaline, PDBU, NaF, A23187 and thapsigarg in ( f i g . 3 .3 . ) ,

whereas histamine, Ach, carbachol, 5-HT, n itrop russ ide and KOI were

a l l w ithout s ig n i f ic a n t e f fe c t (data not shown). In the ra b b i t ,

phenylephrine (an - adrenoceptor agon is t) , Ach, A23187, AA, NaF

and PDBU a l l s timulated PGI2 release by the aorta ( f i g . 3 .4 . ) . In

the ca ro t id a r te ry , femoral a r te ry and hepatic porta l vein o f the

8 3

ra b b i t , PGI2 synthesis was stimulated by PDBU, phenylephrine,

adrenaline, Ach, A23187, NaF, carbachol and thapsigargin ( f i g .

3 .5 . ) . Histamine and 5-HT were w ithou t e f fe c t in a l l ra b b i t vessels.

Removal o f the endothelium from the ra b b i t aorta markedly reduced

the release o f PGI2 in response to phenylephrine, Ach, A23187, AA,

NaF, PDBU ( f i g . 3 .4 . ) .

3.2c. Studies on PGI2 release by human vessels, HUVECs and HOTMECs

Responses in the human vessels ( i l i a c a r te ry and v e in ) , as one

might expect, were v a r ia b le . In some samples, c lea r cu t dose-

response curves were obtained fo r the various s tim u la tors

inves tiga ted , whereas in vessels from other in d iv id u a ls , responses

were v i r t u a l l y non-ex is tent or displayed excessive background

release o f PGI2 (as one encounters in damaged vessels [e .g . by

freeze-thaw ing ]). This in te r - in d iv id u a l v a r ia t io n is exem plif ied by

the responses obtained w ith the same s t im u la to rs in i l i a c a r te r ie s

from s ix consecutive donors (the f i r s t s ix studied; f i g . 3 .6 . ) . In

concomitant experiments on co n tra c t io n -re la xa t io n in an organ bath

using segments o f the same human vessels examined fo r PG release,

there were also marked in te r - in d iv id u a l va r ia t io n s ranging from

reasonable dose-reponse curves to excessive spontaneous a c t i v i t y ,

spasmogenic responses to agonists and absolu te ly no a c t i v i t y a t a l l

(Karatapanis and Oeremy, unpublished observations). Therefore, fo r

the compilation o f dose-response curves, data from in d iv id u a ls where

there was a zero response or where responses resembled vessels where

membrane func tion had been d isrupted, were excluded. Thus, in both

i l i a c a r te ry and vein , PGI2 synthesis was stimulated by:

phenylephrine, adrenaline, Ach, carbachol, histamine, 5-HT

8 4

( f i g . 3 .7 ) , PDBU, NaF, thaps igarg in , A23187 and AA ( f i g . 3.8) in

dose-dependent manners. Using s ing le maximal doses o f selected

s t im u la to rs , removal o f endothelium in human i l i a c a r te ry and vein

had minimal e f fe c t on PGI2 synthesis ( ta b le 3 .1 . ) . In cu ltu red

HUVECs, PG%2 synthesis was stimulated w ith A23187, AA, NaF, PDBU,

histamine and thrombin but not 5-HT, carbachol, or noradrenaline

( f i g . 3.9a) and in HOTMECs w ith A23187, AA, NaF, PDBU but not by

histamine, thrombin, 5-HT, carbachol or noradrenaline ( f i g . 3 .9b).

3.2d. Effect on PGI2 release following hypothermic storage of aortae

(rats and rabbits) and human lia c artery in organ preservation

soluti ons.

Incubation o f r a t aorta in e i th e r UW or KPS completely in h ib i te d

the release o f PGI2 e l i c i t e d by noradrenaline, PDBU, A23187 and NaF

( f i g . 3 .10). In con tras t, AA-stimulated PGI2 release was not

s ig n i f ic a n t ly d i f fe re n t from con tro ls ( f i g . 3.10). Following cold

storage o f a o r t ic t issue in MEM, UW, or KPS fo r 12h, t ra n s fe r ra l o f

a l l t issues to MEM and immediate incubation in the presence o f

noradrenaline, a t 37^0 fo r 1h, there were marked enhancements o f

both basal and noradrenaline-stim ulated PGI2 release ( f i g . 3 .11.)

These enhanced responses reversed to zero time ( i . e . w ithou t cold

storage) responses ( f i g . 3 .1 1 .) . Iden tica l patterns o f enhancement

and reversal were also found fo l low ing storage fo r 24h and 48h (data

not shown). Following cold storage in UW, kidney preservation

so lu t io n or MEM fo r 24h and 48h a f te r 1h incubation in MEM,

responses to noradrenaline were s im i la r to those in fresh t issue

( f i g . 3 .1 2 .) . Following cold storage fo r 72h (and a f te r 1h

incubation in MEM) the response to noradrenaline was s ig n i f ic a n t ly

8 6

diminished when compared to the zero time dose-response curve fo r

a l l 3 so lu tions ( f i g . 3 .1 2 .) . Following cold storage o f in ta c t

ra b b i t a o r t ic r ings in UW, MEM or KPS fo r 24 h and 48h, PGI2 release

in response to Ach, phenylephrine, PDBU, NaF, A23187 and AA was

unaffected by storage in UW, MEM or KPS (tab le 3 .2 . ) . Following cold

storage o f in ta c t human i l i a c a r te ry and vein segments in UW, MEM or

KPS fo r 24 h and 48h, PGIg release in response to ace ty lcho line ,

PDBU, NaF, A23187 and AA was unaffected by storage in UW, MEM or KPS

(ta b le 3 .3 . ) .

3.2e. Basic studies on p la te le t TXAg synthesis.

TXA2 synthesis by washed, u n s t ir re d p la te le ts was stim u la ted in

dose-dependent manners by collagen, NaF, A23187, phorbol ester

m yris ta te acetate (PMA), thapsigarg in and AA but not by adrenaline,

ADP, PAF, U46619 or 5-HT ( f i g . 3 .13). Response to s tim u la to rs was

rap id and reached maximal a t 10 min a f te r add it ion o f s t im u la to rs

( f i g . 3.14b). TXA2 synthesis was d i r e c t ly proportional to the

number o f p la te le ts present, i r re s p e c t iv e o f the s t im u la to r used

( f i g . 3.14a). The Ca2+ channel blockers n ife d ip in e (3.15a) and

d ie th y ls t i Ib e s t ro l (3.15b) in h ib i te d p la te le t TXA2 synthesis when

stimulated by NaF, PMA and A23187 but not when stimulated by AA or

freeze f ra c tu r in g . The PKC in h ib i t o r , staurosporine in h ib i te d

p la te le t TXA% synthesis when stim u la ted by NaF and PMA but to a

lesser degree when stimulated by A23187 but not w ith AA or freeze

f ra c tu r in g ( f i g . 3 .15c).

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9 6

T a b le 3.1. E ffe c t o f endothelium removal on PGI2 synthesis

(pg 6-oxo-PGF-iQj / mg t issue / 30 min; mean + SD, n = 8 ) by human

i l i a c a r te ry e l i c i t e d by a range o f s t im u la to rs .

s t im u la to r In ta c t vessel w ith endothelium removed

Phenylephrine (10^M) 138 + 37 124 1 22

Acety lcho line (10 ^M) 128 + 38 118 i 35

Histamine (10 pM) 75 i 19 65 ± 16

5-HT (10 |jM) 45 i 9 40 1 14

NaF (10 mM) 150 ± 60 135 1 28

PDBU

A23187

AA

(1 |iM) 179 ± 26 166 1 28

(10 |iM) 168 1 36 150 1 22

(10 yjM) 175 i 19 170 1 24

thapsigarg in (3 |iM) 73 ± 18 68 i 16

9 6

2 0 0

cE

0)DCO

,Ç0V-*O)E

u_0Û-1oX01

CD

1 0 0

0

tI#iII

i%m%%I I

NA

%I#II

èi1

#I

I##NaF A23187 PE

f

AA

MEM UW KPS

Fig. 3.10. Effect o f incubation o f ra t a o rt ic rings in minimum essential medium (MEM), Univers ity of Wisconsin solution (UW), kidney preservation solu tion (KPS) on PGI2 synthesis when stimulated with nor-adrenaline (NA; 10^M ), NaF (10 mM), phorbol ester d ibutyrate (PE; 1 j jM ) , Ca2+ ionophore A23187 (A23187; 3^M ), arachidonic acid (AA; 3 juM).Each histogram represents mean i S.D., n = 6 .

2 0 0 r2 0 02 0 0

cE

(DZJCOco

D)

^ 100 100100

0Q.1oX01

CDO)a

1

- lo g | n o ra d re na i in ^ ( M )

F ig . 3 .1 1 . E f f e c t o f c o ld s to ra g e in MEM ( # ) , KPS ( ▼ ) , o r UW (Él ) f o r 12h fo l lo w e d by in c u b a t io n o f r a t a o r t i c r i n g s in MEM ( f o r i ] ze ro t im e , i i ] 30 min and i i i ] 1 h) p r i o r

t o s t im u la t i o n o f PGI2 w i th n o ra d re n a l in e (N A ). Each p o in t r e p re s e n ts mean o f 4

d e te r m in a t io n s . ( Q ) re p re s e n ts th e NA-PGI2 do se -response c u rv e o b ta in e d in f re s h ( n o t c o ld - s t o r e d ) a o r ta .

CON

200 200200

O)

1001 00 100

LL—

CD

D)a

0 L

- lo g [n o ra d re n a l in ^ ( M )F ig . 3 .1 2 . E f f e c t o f c o ld s to ra g e in MEM ( # ) , KPS « ^ ) o r UW ( A ) f o r : i ) 24 h, 11) 48h and 111) 72h fo l lo w e d by In c u b a t io n o f r a t a o r t i c r in g s In MEM f o r 1h p r i o r t o s t im u la t io n o f PGI2 w i th NA. Each p o in t re p re s e n ts mean o f 4 d e te r m in a t io n s . ( Q ) re p re s e n ts the NA- PGI2 do se -respo nse c u rve o b ta in e d In f re s h (n o t c o ld - s to r e d ) a o r t a .

CO00

9 9

Table 3 .2 . E ffe c t o f hypothermic storage o f ra b b it a o r t ic r ings in

preservation so lu t ions (U n ive rs ity o f Wisconsin's [UW] and kidney

preservation so lu t io n [KPS]) and c u l tu re medium (MEM) fo r 24 h and

48 h on PGI2 synthesis (pg 6 - oxo-PGF^q , / mg t issue / min; mean +

S.D., n = 8 ) e l i c i t e d by a range o f s t im u la to rs (phenylephrine [PE],

ace ty lcho line [Ach], NaF, PDBU, A23187 and AA).

stimul ator________MEM__________________ UW_________________ KPS_______

0 time 24h 48 h 24h 48h 24h 48h

PE 44+7 42t8 36tl1 50Ï8 49+9 38fl1 39±10

Ach 38+8 33+6 31i8 39+9 27 il2 28±5 29±6

NaF 120+21 115+14 108tl4 129±15 100+20 110±15 114+22

PDBU 84+9 76+8 78+12 74+7 64+12 90+12 78+22

A23187 118+26 120+22 126+28 115+24 140t32 144+29 128±24

AA 135+16 145+25 148+22 144+19 141+27 131±24 120+26

1 00

Table 3.3. E ffe c t o f hypothermic storage o f human i l i a c a r te ry in

preservation so lu t ions (U n ive rs ity o f Wisconsin's [UW] and kidney

preservation so lu t io n [KPS]) and cu l tu re medium (MEM) fo r 24 h and

48 h on PGI2 synthesis (pg 6-oxo-PGF^(^ / mg t issue / 30 min; mean 1

SD, n= 12) e l i c i t e d by a range o f s t im u la to rs (phenylephrine [PE],

ace ty lcho line [Ach], NaF, PDBU, A23187 and AA.

stimul ator_____________ MEM_____________ UW________________ KPS

0 time 24h 48 h 24h 48h 24h 48h

PE 128Î27 122Î32 110Ï11 135128 125±29 108131 119120

Ach 118+28 123+26 11 i l l 8 129ll9 112128 118t30 109124

NaF 130141 125+18 104124 129l25 109128 124135 124±29

PDBU 184129 176138 178126 174132 164124 144126 168142

A23187 198+46 180+32 176128 185144 160l32 174149 158134

AA 185126 I 75I 25 168132 174129 16H37 14ll24 130l46

101

cÊ

to•+-»(DÜ)jgD.

0 .5

00oT—D) c

*— %

X I—

OL

0 0.03 0.3 3[agent] (^g / ml )

30

0 5

[NqfJ ( mM )

10

F ig . 3 .1 3 . E f f e c t o f d i f f e r e n t s t im u la t o r s o f p l a t e l e t a g g re g a t io n

on TXA2 (m easured as TXB2 ) g e n e r a t io n by washed human p l a t e l e t s :

A23187 ( A ) , p h o rb o l e s te r m yr i s t a t e a c e ta te ( # ) , AA ( ^ ) ,

c o l la g e n (A), NaF ( < ^ ) , t h a p s ig a r g in ( Q ) » a d r e n a l in e ( Q ) ,ADP ( Q ) , U4661 9 ( D ) , 5-HT ( O ) and RAF ( [ ] ) .Each p o in t = mean 1 S i E . M . , n = 7 h e a l th y v o lu n t e e r s .

2

en

1

0

0

0.8

0.4

2 4 86 100

plate let count time (min)

F ig . 3.14. TXA2 (as TXB2 ) release in response to various stimulators by: A) d i f fe re n t numbers o f p la te le ts and B) by same number o f p la te le ts over 10 min. Each po in t = mean + S.D., n 1 jjM Phorbol ester ( □ ) , 3^M A23187 (<0>), 3yiM AA ( • ) , 10yjM thapsigargin ( O ) , 10 mM NaP ( 4 \ ) .

= 6

oro

1 0 3

wCOCD

ICJ;II

100

Hog [ d r u ^ ( M )

or

JD

100

log Qimg] ( M )

F ig . 3 .1 5 . E ffe c t o f a) n ife d ip in e , b) d ie th y ls t i lb e s t ro l and c)

staurosporine on TXA2 synthesis stim ulated by phorbol es te r m yris ta te

acetate ( • ) , NaF (<3>), A23187 ( □ ) , arachidonate (O) and freeze-

f ra c tu r in g . Each po in t = mean iS . E . M . , n = 6 .

( A )

1 0 4

3.3. DISCUSSION

3.3a. Basic studies on blood vessels

The f i r s t po in t to emerge from th is study is the remarkable

s im i la r i t y o f responses o f d i f fe re n t vessels w ith in the same

species. Thus, although absolute output o f PGI2 var ied, almost

id e n t ica l patterns o f response to the same substances were obtained

fo r the aorta , c a ro t id and hepatic po rta l vein o f the ra t and

ra b b i t . Also in the human vessels, responses were b a s ic a l ly s im i la r

in both the i l i a c a r te ry and i l i a c ve in . Thus, although i t has often

been suggested th a t the aorta is an inappropria te vessel to use as a

model o f the vasculature (Tomlinson e t a l . , 1992), the present data

ind ica te th a t the aorta con s t itu te s an adequate representa tive model

o f a t lea s t other la rg e r vessels w ith in the same species. I t is

conceded, however, th a t such may not be the case in smaller vessels,

inc lud ing a r te r io le s , venules and c a p i l la r ie s ( i . e . the micro-

vascu la ture). From an in v e s t ig a t iv e p o in t o f view, the study o f

microvasculature is fraught w ith methodological p i t f a l l s . Perfusion

o f organs w ith dense microvasculature (e .g . lung) and measurement o f

PGs in the perfusate fo l lo w in g add it ion o f vasoactive agents has

been used to assess PGIg output by microvascular beds (e .g . Stevens

e t a l . , 1992). However, i t is impossible to exclude the co n tr ib u t io n

o f surrounding t issue to the PG%2 measured in a perfusate. For

example, the lung consists o f large amounts o f bronchial smooth

muscle. In tu rn , airways t issue releases large amounts o f PGI2 in

response to agonists which do not e l i c i t PGI2 release in blood

vessels. For example, in the trachea o f the r a t , para-

sympathomimetics (but not sympathomimetics) s tim u la te PGI2 re lease.

1 0 6

(Oeremy e t a l . , 1989) whereas the opposite Is the case fo r vascular

t issue ( th is th e s is ) . This po in t is fu r th e r considered in the

analys is o f PGI2 re lease from vasculature, trachea and lungs o f

d ia b e t ic ra ts in chapter 5. Perhaps in order to f u l l y c l a r i f y the

in tra -spec ies v a r ia t io n o f PGIg release by d i f fe re n t vessels,

studies on la rg e r animals, where s u f f ic ie n t amounts o f small vessels

are ava ila b le , would fu r th e r c l a r i f y th is question.

The second po in t to emerge from the present study was the

in te r-spec ies d if fe rences in responses. The most notable va r ia t io n s

were seen w ith d i f fe r e n t receptor agonis ts . Although a l l species,

inc lud ing man, responded to adrenergic s t im u la t io n , the ra t and

ra b b it fa i le d to release PGI2 in response to histamine or 5-HT. In

con tras t , histamine and 5-HT were potent s tim u la to rs o f PG%2 in

vessels from the human vessels. The most s t r ik in g f in d in g was th a t

Ach, a re laxo r o f blood vessels (Furchgott, 1983) had no e f fe c t on

PGI2 in r a t vessels and ye t was a potent s t im u la to r o f PG%2 release

by ra b b i t and human vessels. I t had prev ious ly been suggested th a t

only a c t iva to rs o f c o n s tr ic t io n (noradrenaline, histamine, 5-HT,

angiotensin I I ) cause the release o f PGI2 by blood vessels (Jeremy

e t a l . , 1985a; 1998a). The ra b b it and human data however, confound

th is proposal since Ach is a c la ss ic re la xo r o f vascular t is s u e . In

con tras t , other re laxors (n it ro p ru ss id e , substance P) were w ithout

e f fe c t on PGI2 re lease in these vessels which would seem to exclude

a cons is tent re la t io n s h ip between vasod ila to rs and PGI2 re lease.

Further studies are c u r re n t ly underway by the author to es tab lish

which mechanisms are involved in ACh-stimulated PGI2 re lease from

vessels but due to the constra in ts o f time cannot be included in

th is th e s is .

1 0 6

In con tras t to the vasoactive agonists there was universal

s t im u la t ion o f PGI2 release w ith phorbol ester (ac tiva tes PKC;

Nishizuka, 1984), NaF (a non-spec if ic G p ro te in a c t iv a to r ; Gilman,

1987), A23187 (creates a r t i f i c i a l Ca2+ channels; Reed and Lardy,

1972), thapsigarg in (mobilises in t r a c e l lu la r Ca^^; Tharstrup, 1990)

and AA (PG%2 substra te ; Bergstrom e t a l . , 1968) by a l l vessels (as

well as in c e l l cu ltu re s ) inves tiga ted . Thus, the mechanism derived

from the ra t ao rta : th a t PGI2 synthesis is mediated sequen tia lly by

G p ro te ins , PKC, Ca2+ m o b il isa t ion , PLA2 a c t iv a t io n , AA release and

conversion o f AA to PGI2 (see f i g . 1.3. in chapter 1) appears to be

u n ive rsa lly v a l id fo r a l l vascular t is s u e . This is o f importance

since in any one study (e .g . in d issec ting out a lte ra t io n s o f

mechanisms in a disease model, ex v ivo or in v i t r o or in the study

o f s i te s o f drug ac tion ) i t is reasonable to extrapo la te data

obtained using these modulators in a vessel from a labora tory animal

model to man.

The th i r d p o in t to emerge from the present study was the ro le o f

the endothelium in mediating PGI2 re lease by whole vessels. In the

ra t aorta , the endothelium con tr ibu tes n e g l ig ib le amounts o f PGI2 to

the to ta l output o f th is prostanoid, a t le a s t in response to the

s t im u la to ry agents inves tiga ted . The main source o f PGI2 in th is

vessel appears, the re fo re , to be o f medial and/or in t im a i o r ig in .

PGI2 synthesis was observed in response to adrenaline,

noradrenaline, U46619, phorbol es te r and f lu o r id e in aortae w ithout

endothelium compared to vessels w ith in ta c t endothelium. S im i la r ly ,

a t 3h post- endothelium removal there was a marked reduction in the

in h ib i t io n o f noradrenaline-stim ula ted PGI2 synthesis by yohimbine

and prazosin and by Ca2+ channel blockade. Previous studies have

1 0 7

shown th a t removal o f the endothelium re s u lts in diminished

responsiveness to vasod ila to rs (Furchgott, 1983) and increased

response to vasoconstr ic tors (Egleme e t a l . , 1984) which has led to

the concept th a t vascular r e a c t iv i t y is co n tro l le d by EDRF

(Furchgott, 1983). The data a t 3h post- endothelium removal would

the re fo re seem to ind ica te th a t the s t im u la t ion o f r a t a o r t ic P6 I 2

synthesis and in h ib i t io n o f PGI2 by s p e c if ic alpha-adrenergic

antagonism and Ca2+ channel blockade are dependent on the secretion

o f EDRF. However, p ro tracted incubation ( 6h and 9h) o f the aorta

w ithou t endothelium normalised these changes o f response to

s tim u la to rs and in h ib i to rs o f PGI2 synthesis, despite continued

absence o f the endothelium (as assessed by scanning EM). These

f ind ings the re fo re suggest th a t removal o f the endothelium per se

ra the r than the secre tion o f EDRF, determines these i n i t i a l (a t 3h)

changes. Since endothelium removal co n s t itu te s a severe trauma to

the vessel, i t is possib le th a t th is mechanical in ju ry sens it ises

the vessel, thereby enhancing and d im in ish ing responsiveness to

s t im u la to rs and in h ib i to r s , re sp e c t ive ly . The author is aware o f no

systematic study on mechanical in ju r y and vascular reponsiveness in

v i t r o , but i t is known th a t trauma (sustained by wounded so ld ie rs in

the 1st World War) causes prolonged spasm in human a r te r ie s

(Kinmonth e t a l , 1962), although th is would seem to warrant fu r th e r

in v e s t ig a t io n .

In complete con tras t to the r a t ao rta , removal o f the

endothelium in the ra b b i t aorta abolished PGI2 release in response

to both receptor agonists (phenylephrine, Ach), in t r a c e l lu la r

modulators (NaF, PDBU, A23187) and substrate (AA). These data

in d ica te th a t in the ra b b i t the endothelium is the major source o f

1 0 8

PGl2 . That th is re lease is mediated by EDRF (as is re la xa t io n o f

th is vessel; Furchgott, 1983; Ignarro e t a l . , 1990) is u n l ik e ly

since in in ta c t vessels n itrop russ ide (degrades to form NO which in

tu rn is considered to be EDRF; Palmer e t a l . , 1989) was w ithout

e f fe c t on PGI2 re lease. In con tras t , responses in the human i l i a c

a r te ry were r e la t i v e ly unimpaired by removal o f endothelium. Thus,

o f the species investiga ted the ra b b i t appears to be anomolous v is a

v is the c o n tr ib u t io n o f the endothelium to the to ta l output o f PGI2

by conduit a r te r ie s . I t should be stressed th a t the author is not

proposing th a t endothe lia l PGI2 is not b io lo g ic a l ly re le van t.

Rather, th a t when assessing output o f PGI2 by blood vessels,

p a r t ic u la r ly in response to agonists and/ or other s t im u la to rs , the

PGI2 measured should not be so le ly ascribed to the endothelium but

ra the r to the smooth muscle component o f the vessels (w ith the

exception o f the r a b b i t ) . This po in t is exemplified by the data

obtained from human endothe lia l c e l l cu ltu res (HUVECs and HOTMECs).

In HUVECs, PGI2 re lease was stimulated by thrombin, histamine,

phorbol es te r , NaF, A23187 and AA and in HOTMECS by phorbol es te r,

NaF, A23187 and AA but not by histamine, thrombin, carbachol,

adrenaline or 5-HT. In con tras t , adrenaline, carbachol and 5-HT a l l

e l i c i t e d PGI2 re lease by whole human vessels. This d i f f e r e n t ia l may

r e f le c t the separate functions o f the two vascular zones:

haemostasis a t the endothe lia l-b lood in te r fa ce and c o n t r a c t i l i t y in

the smooth muscle component. Put another way, i t is important to

re a l is e th a t when assessing PGI2 re lease by whole vessels in any

given experimental scenario (e .g . ex v ivo PGI2 release in ra ts w ith

a r t i f i c ia l l y - in d u c e d pa tho log ies), th a t the PGI2 measured mainly

r e f le c ts th a t derived from the smooth muscle component ra the r than

1 0 9

the endothelium. However, because in the ra b b i t aorta the

endothelium is the major source o f PGI2 , then th is species could be

used to inve s tiga te changes o f PGI2 a t the endothelium. This facet

o f the ra b b it aorta has been exp lo ited in the studies on hypothermic

storage o f vessels in preservation so lu t ions (below).

3.3b Studies on hypothermic storage in organ preservation solutions

Apart from the mechanistic and lo g is t i c aspects discussed

above, the ra t io n a le derived from these experiments was applied to

the study o f the e f fe c t o f hypothermic storage in preservation

so lu tions on vascular v i a b i l i t y . Th is, in tu rn , is o f relevance to

t ra n sp la ta t io n as ou tl ined in the in t ro d u c t io n .

The f i r s t f in d in g was th a t incubation o f r a t aorta in both

organ preservation so lu tions inves tiga ted (UW and KPS) resu lted in a

potent in h ib i t io n o f r a t a o r t ic PGI2 re lease a f te r s t im u la t ion w ith

noradrenaline, NaF, A23187 or phorbol e s te r , whereas AA-stimulated

PGI2 was unaffected. Since noradrenaline, NaF and phorbol e s te r-

stimulated PGI2 synthesis are in h ib i te d by Ca2+ channel blockade

(Jeremy e t a l . , 1988a), these data in d ica te th a t these so lu t ions are

acting on the Ca2+ pools associated w ith PGI2 synthesis (v ia

a c t iv a t io n o f PLA2 ) . AA, however, acts d i r e c t ly as substrate fo r the

cyclooxygenase holoenzyme, in d ic a t in g th a t the in h ib i to ry e f fe c t o f

preservation so lu t ions is not cyclooxygenase-mediated. Since KPS

contains c i t r a t e , a Ca2+ che la tor (Lages e t a l . , 1977), th is may

exp la in the in h ib i to r y e f fe c t o f th is s o lu t io n . UW contains

lac to b ion ic ac id , a l lo p u r in o l , ra f f in o s e pentahydrate and reduced

g lu ta th ione . Which o f these la t t e r components exerts a d i re c t

in h ib i to ry e f fe c t on Ca^^-dependent PGI2 re lease w i l l requ ire

1 1 0

studies on e f fe c ts o f these in d iv id u a l components.

In con tras t, i t was demonstrated th a t washing the vessels w ith

MEM normalised the in h ib i t io n o f PG%2 fo l low ing hypothermic storage.

Thus, in vessels from the r a t and ra b b i t , storage in UW or KPS

e l i c i t e d no appreciable reduction in PGI2 in response to any o f the

s t im u la to rs . These data suggest th a t preservation so lu t ions exert

minimal e f fe c ts on smooth muscle PGI2 synthesis ( r a t aorta) or

endothe lia l PGI2 synthesis ( r a b b i t ) . The in te g r i t y o f endogenous

PGI2 synthesis is o f relevance to g r a f t func tion since th is

eicosanoid is a vasod ila to r (Moncada and Vane, 1979), exerts a

cy top ro tec tive e f fe c t (a lb e i t poorly understood; Lefer e t a l . ,

1978), reduces leucocyte sequestration w ith in damaged tissue (Jones

and Hurley, 1984) and in h ib i t s p la te le t adhesion and aggregation

(Moncada and Vane, 1979). Given these d iverse, yet re levant

p rope rt ies , i lo p ro s t (a stab le PGI2 analogue) has also been

incorporated as an ad d it ive to preservation f lush so lu tions

(Klepetko e t a l . , 1989; Tamaki e t a l . , 1988). The s t im u la to rs o f

PGI2 employed in the present study are also modulators o f other

vascular functions (NaF, phorbol es te r and A23187). Thus,

hypothermic storage in UW or KPS appear to exe rt minimal e f fe c ts on

in t r a c e l lu la r signal transduction systems which mediate not only

PGI2 synthesis but also vascular c o n t r a c t i l i t y (Danthaluri and Deth,

1984). This concept was consolidated by in v i t r o experiments on ra t

and ra b b i t ao rta , in which hypothermic storage o f blood vessels

resu lted in minimal e f fe c ts on co n tra c t io n /re la x a t io n patterns even

a t up to 72 h storage (Karatapanis e t a l . , 1990). Since con tract ion

/ re la xa t io n is e x q u is i t iv e ly se n s it ive to endothe lia l damage, these

l a t t e r data consolida te th a t hypothermic storage o f blood vessels in

1 1 1

organ preservation so lu tions e l i c i t s minimal de le ter ious e f fe c ts to

the endothelium, per se.

The present re s u lts are o f relevance to l i v e r tra nsp la n ta t ion

in several respects. A recent study reported th a t the f i r s t post

operative month con s t itu te s the period o f greatest r is k fo r g r a f t

fa i lu r e a f te r tra nsp la n ta t ion (Quiroga e t a l . , 1991). Major

con tr ibu to ry fac to rs to ea rly g r a f t fa i lu r e were vascular

complications, inc lud ing thrombosis and ischaemia (Quiroga e t a l ,

1991). The mechanisms underlying these complications are unknown but

endothe lia l in ju r y induced by preservation so lu tions and cold

storage have been suggested as playing a ro le (Kazikoe e t a l . ,

1990; Caldwel1-Kenkel e t a l , 1989; 1990; B lankensteijn and Terpstra,

1991). However, the re s u lts o f the present study would seem to

ind ica te th a t cold storage in organ preservation so lu t ions re su lts

in minimal e f fe c ts on endothelia l i n t e g r i t y . However, Caldwell-

Kenkel e t a l . (1989, 1990), found th a t hypothermic storage o f ra t

l i v e r s flushed w ith Euro C o ll ins so lu t ion (s im i la r in composition to

KPS) followed by reperfusion o f the organ w ith KRB resu lted in loss

o f v i a b i l i t y and denudation o f s inusoidal endothe lia l c e l ls as well

as a c t iv a t io n o f Kupffer c e l ls . UW s o lu t io n , however, caused less

damage than Euro C o ll ins so lu t ion (Caldwell-Kenkel, 1989; 1990).

Thus, i t is possib le th a t the vascular endothelium a t d i f fe re n t

s i te s (e .g . in tra -h e p a t ic as opposed to large a r te r ie s ) may be

d i f f e r e n t ia l l y susceptib le to these so lu t io n s . A l te rn a t iv e ly , since

the present experiments did not include a re -pe rfus ion component, i t

is also possible th a t reperfusion (e .g . v ia shear forces and/or

anchorage o f endothe lia l c e l ls to the subendothelium), in

combination w ith cold storage, e l i c i t s endothe lia l in ju r y and

1 1 2

denudation. Notwithstanding these p o s s ib i l i t ie s , i t is

unquestionable th a t hypothermic storage and reperfusion of

transplanted organs causes damage to the vascular endothelium.

C e rta in ly , disturbances o f other endothe lia l functions and

in te ra c t io n s (e .g . w ith coagulation fa c to rs , cytokines, neutrophil

adhesion and release o f reac tive oxygen species) fo l low ing

hypothermic storage and reperfusion have been demonstrated

(B lankenste ijn and Terpstra, 1991). What the present data there fore

ind ica te is th a t EDRF and PGI2 release are r e la t iv e ly re s is ta n t to

d is rup tion by hypothermic storage whereas other key endothe lia l

functions may be more vulnerable.

I t is also apparent th a t UW and KPS exerted no c lea r advantage

over the more conventional phys io log ica l so lu t io n , MEM, in terms o f

e f fe c ts on c o n t r a c t i l i t y and PGI2 re lease. The question there fore

arises as to why specia lised f lu id s such as UW are pre ferred as

perfusates in co ld -s to red organs fo r tra n sp la n ta t io n . Many studies

have demonstrated th a t UW enhances v i a b i l i t y / in te g r i t y o f o ther,

perhaps more susceptib le biosystems, than those determining

c o n t r a c t i l i t y and re la xa t io n (Jamieson e t a l , 1988; Marsh e t a l . ,

1991; Guyomard, 1990). UW was designed o r ig in a l ly to prevent c e l l

sw elling and free rad ica l in ju ry in organs inc lud ing the l i v e r ,

kidney and pancreas (Wahlberg, 1987). Rigorous studies have shown

th a t UW, compared to cu ltu re media and so lu tions l ik e KRB produces

less de le te r ious e f fe c ts on c e l ls inc lud ing hepatocytes (Onte ll e t

a l . , 1988; Jamieson e t a l . , 1988; Marsh e t a l . , 1991; Guyomard,

1990). Thus, the s im i la r i t y o f e f fe c ts o f UW with MEM reported here

does not bring in to question the usefulness o f preservation

so lu tions such as UW.

1 1 3

In conclusion, cold storage o f blood vessels in preservation

flush so lu tions used in l i v e r t ra n sp la n ta t io n re su lts in minimal

e f fe c ts on PG%2 release by the r a t and ra b b i t ao rta . Experiments on

the human vessels consolidate th a t hypothermic storage and/or

preservation so lu t ions cannot exp la in the va r iab le responses o f

blood vessels taken from human donors. I t is notable th a t the

concomitant assessment o f c o n t r a c t i l i t y / re la x a t io n o f these human

vessels also displayed marked v a r ia b i l i t y : ranging from spasmogenic

and zero responses to reasonable responses obtained in approximately

the same number o f samples as fo r PGI2 release (Karatapanis e t a l . ,

unpublished observations). Thus, the v a r ia t io n can be ascribed to :

a) in te r - in d iv id u a l (e .g . age, sex) d if fe rences , b) the drugs

administered to the donors, c) nature o f the in ju r ie s and / or cause

o f death o f donors, d) in fe c t io n (known to markedly a l te r eicosanoid

synthesis; Feuerstein and Ramwell, 1981) and e) length o f time

maintained on l i fe -s u p p o r t systems. A re tropsec tive analys is o f

these variab les in re la t io n to the responses obtained was

lo g is t i c a l l y impossible and a t the very best would be anecdotal.

Nevertheless, given the importance o f vascular patency in the

success o f t ra n sp la n t surgery (as ou t l ined above), the present data,

in conduction w ith the organ bath experiments, ind ica te th a t fu r th e r

consideration o f var iab les encountered in donors warrants

in v e s t ig a t io n . Such a study could comprise o f the assessment o f

c i r c u la t in g fac to rs (e .g . p la te le t and white c e l l a c t i v i t y and th e i r

release substances) as well as the other variab les o u t l in e d above

and to re la te these parameters to the outcome o f t ra n sp la ta t io n

success as well as to basic vascular func tions .

1 1 4

3.3c. Basic p la te le t studies

With regard to p la te le ts , the present study f i r s t l y

demonstrates th a t o f the pro-aggregatory substances inves tiga ted ,

PMA, NaF, co llagen, AA, thapsigarg in and A23187, but not ADP,

adrenaline, 5-HT, PAP or U46619, stimulated TXA2 synthesis by

u n s t ir re d p la te le ts . Thus, i t would appear th a t TXA2 is secondary to

aggregation ( i . e . is a consequence o f) ra the r than being a primary

event fo l low ing challenge w ith adrenaline, ADP, 5-HT, U46619 and

PAP. I t is notable th a t even when TXA2 re lease is assessed fo llow ing

s t im u la t ion o f aggregation these agonists generate small amounts o f

TXA2 (Barradas, Jeremy and M ik h a i l id is , unpublished observations)

whereas agents such as A23187 generate large q u a n t it ie s o f TXA2 .

A c tiva to rs o f p la te le ts can be considered as e ith e r 's tro n g ' or

'weak' (Crawford and Scrutton, 1988). The d i f fe r e n t ia l e f fe c ts

observed here may the re fo re be explained by th is v a r ia t io n in

's tre n g th ' o f agon is t.

With regard to the ro le o f Ca2+ in c o n t ro l l in g the synthesis o f

p la te le t TXA2 , the channel b lockers, n i fe d ip in e and

d ie th y ls t i lb e s t ro l in h ib i te d TXA2 synthesis when stimulated with

co llagen, in d ic a t in g , by convention, th a t e x t ra c e l lu la r Ca2+ plays a

ro le in the coupling o f these agonists to TXA2 synthesis. This

conclusion is supported by the in h ib i t io n o f TXA2 synthesis by the

Ca2+ che la to r , EGTA, which is purported not to enter c e l ls (Baker,

1972). Furthermore, A23817, which creates a r t i f i c i a l Ca '*’ channels

was a potent s t im u la to r o f TXA2 synthesis . I t has also been reported

th a t in t r a c e l lu la r Ca2+, released by IP3 , contro l TXA2 synthesis in

p la te le ts (Knezevic e t a l . , 1992). These la t t e r authors

demonstrated th a t fo l low ing in s e r t io n o f IP3 (by sap on if ica t ion )

116

in to p la te le ts , an Increase in In t r a c e l lu la r Ca2+ and TXAg synthesis

preceded secretion o f ADP and aggregation. The s t im u la t io n o f TXA2

synthesis by thaps iga rg in , a m o b il ise r o f in t r a c e l lu la r Ca2+

(Tharstrup, 1990), supports th is l a t t e r view. However, using Ca2+-

se n s it ive dyes, several studies have shown th a t the agonists th a t

d id not s tim u la te TXAg synthesis in th is study (ADP, 5-HT, PAF, 5-

HT) a c tu a l ly e levate in t r a c e l lu la r Ca^* (Rink and Sage, 1990) and

would seem to m it ig a te against a ro le fo r the e levation o f i n t r a

c e l lu la r Ca2+ in mediating TXA2 synthesis. The nature o f Ca2+

channels in p la te le ts is obscure (Rink and Sage, 1990). C erta in ly

high concentrations o f blockers are required to i n h ib i t p la te le t

aggregation (Jeremy e t a l . 1985b) as well as TXA2 synthesis.

Furthermore, high concentrations o f K'*' ( c la s s ic a l ly ac t iva te

voltage-dependent channels in smooth muscle, cardiac and neural

t is s u e ] ) f a i l to e l i c i t both aggregation and TXA^ synthesis in

p la te le ts (Barradas, Jeremy, M ik h a i l id is , unpublished observations).

Thus, i t has been suggested th a t p la te le ts do not possess Ca2+

channels s im i la r to those found in other t issues (Rink and Sage,

1990). I t is c e r ta in ly possib le th a t Ca2+ blockers are ac ting at

other Ca2+ - re q u ir in g steps (e .g . PKC and PLA^). Others have

suggested th a t Ca2+ channel blockers in h ib i t p la te le t a c t i v i t y via

non - Ca2+ channel mediated mechanisms. I t is also possib le th a t

there are s u f f ic ie n t Ca2+ stores located in the plasmalemma and

glycocalyx which can supply the required Ca2+ fo r PLA^ a c t iv a t io n .

Such a p o s s ib i l i t y warrants fu r th e r in ve s t ig a t io n .

With regard to signal transduction , both NaF (a non -spec if ic G

p ro te in a c t iv a to r ; Gilman, 1987) and PMA (a PKC a c t iv a to r ;

Nishizuka, 1984) stimulated TXA2 synthes is . In tu rn , both these

116

stim u la tors were in h ib i te d by Ca2+ channel blockers and by

staurosporine (a PKC in h ib i t o r ) . Thus, the sequence o f events

postulated to occur fo r recep to r- l inke d PGI2 in vascular t issue

appears to hold t ru e fo r TXA2 synthesis in the p la te le t .

Using the data obtained from the above experiments, a ra t io n a le

fo r studying drug action and changes in vascular t issue in selected

pathologies using a range o f s t im u la to rs was developed (see f i g .

3 .1 6 .) . The background and data obtained from th is approach are

presented in the ensuing chapters.

phorbol ester A23187 arachidonate

PKC

DG

PLA cyciooxagonist Rc bGprf PLC AA

intNaF

thapsigargin

F ig . 3 .1 6 . Scheme o f s i t e s o f a c t io n o f d i f f e r e n t s t im u la t o r s o f PGI2 s y n th e s is by i s o la t e d v a s c u la r t i s s u e . The same p r i n c i p l e a p p l ie s to TXA2 re le a s e by p l a t e l e t s . R e ce p to r (Rc) a g o n is ts ( e . g . a d r e n a l in e in th e r a t , Ach in th e r a b b i t , th ro m b in in th e p l a t e l e t ) a c t v ia a c t i v a t i o n o f Res. NaF a c t i v a t e s 6 p r o te in s (Gp) wh ich in t u r n a c t i v a t e s p h o s p h o l ip a s e C, th e re b y g e n e ra t in g d ia c y l g ly c e r o l and i n o s i t o l t r is p h o s p h a te ( I P 3 ) . Phorbol e s te r s a c t i v a t e p r o t e i n k in a s e C (PKC). A23187 c re a te s a r t i f i c i a l Ca2+ c h a n n e ls , th e re b y e l i c i t i n g i n f l u x o f e x t r a c e l l u l a r Ca2+ whereas t h a p s ig a r g in m o b i l is e s i n t r a c e l l u l a r C a^*. The ne t in c re a s e in Ca2+ a c t i v a t e s PLA2 which

h y d ro ly s e s and l i b e r a t e s AA from endogenous p h o s p h o l ip id s to r e s . AA i s c o n v e r te d to e ic o s a n o id by c y c lo o x y g e n a s e . T h is scheme is a p p l ie d to th e s tu d y o f s i t e s o f a c t i o n o f d rugs (c h a p te r 4) and assessment o f e ic o s a n o id s y n th e s is in s e le c te d p a th o lo g ie s (C h a p te r 5 ) .

1 1 8

CHAPTER 4 .

DRUG ACTION: STUDIES ON VASCULAR PROSTACYCLIN AND THROMBOXANE Ag

SYNTHESIS BY HUMAN PLATELETS

4.1 . Introduction.

The general background to drugs and eicosanoid synthesis has

been discussed e a r l ie r (1 .8 . ) . The o b je c t ive o f the ensuing studies

was to ascerta in the s i te ( s ) o f action ( i f any) o f some important

drugs used not only in the treatment o f CVD but also in other

eicosanoid mediated disorders ( v iz . in flam m ation). This ob jec t ive was

achieved by employing a range o f s t im u la to rs which act a t d i f fe re n t

s i te s in the receptor-e icosanoid synthesis pathway. The ra t io n a le o f

t h is approach was derived from data obtained and discussed in the

previous chapter (a lso f i g . 3 .16). Although the author has

investiga ted several d i f fe re n t drugs during the course o f th is thesis

(e .g . , benzydamine, r ido g re l and nabumetone), the re s u lts obtained

from three disparate drug types are presented in th is th e s is : NSAIDs,

PDE in h ib i to rs (PDEIs) and copper che la to rs , as these i l l u s t r a t e the

a p p l ic a b i l i t y o f the methods developed fo r the study o f drug ac tion .

Apart from eicosanoid synthesis, other aspects o f drug e f fe c ts were

also assessed (e .g . phosphodiesterase [PDE], adenylate cyclase and

lipoxygenase a c t i v i t y ; as described in Chapter 2 ) . For the sake o f

c l a r i t y , the background fo r each drug type is described seperate ly.

4 .1 a . NSAIDs

Non-steroidal antiin flam matory drugs (NSAIDs) are the p r in c ip a l

drugs used in the treatment o f o s te o a r th r i t is , rheumatoid a r t h r i t i s ,

ankylosing s p o n d y l i t is , gout, pain and thrombotic disease (Rainsford,

1 1 9

1988). However, NSAIDs also e l i c i t de le te r ious side e f fe c ts ,

inc lud ing g a s tr ic erosion (Atwater e t a l . , 1965; Roth and Bennett,

1987), nephro tox ic ity (L ip s e t t and Goldman, 1954; Swainson, 1984) and

hypertensive e f fe c ts (Durao e t a l . , 1977; Brown e t a l . , 1986). Since

NSAIDs reduce prostanoid synthesis through in h ib i t io n o f

cyclooxygenase a c t i v i t y (Vane, 1971; Flower, 1974), t h e i r actions and

si de -e ffec ts are la rg e ly in te rp re ted as being prostanoid-mediated.

However, there are many reports th a t NSAIDs exert e f fe c ts on other

biosystems, inc lud ing PDE (Weinryb e t a l . , 1972), Ca2+ binding a t the

plasmalemma (Northover, 1971) and phospholipase C (Bomalaski, 1986).

NSAIDs have also been shown to i n h ib i t tumour growth (Hial e t a l . ,

1976; 1977) and c e l l p r o l i fe ra t io n through a non-prostanoid mediated

mechanism (De Mello e t a l . , 1980). In tu rn , the phase o f c e l l

d iv is io n (signal transduction phase th a t precedes DNA synthesis [S

phase]) involves Ca2+ m o b il isa t ion , PKC, G prote ins and

phospholipases (Rosengurt, 1984; Wickremasinghe, 1988). Since, as has

been expounded in th is th e s is , PG synthesis is mediated by these

aforementioned mechanisms, possib le e f fe c ts o f NSAIDs (indomethacin,

t ia p ro fe n ic acid and ibuprofen) on these systems in the r a t aorta was

investiga ted using a range o f s t im u la to rs (see f i g . 3.16). In order

to te s t " t issue s p e c i f i c i t y " , e f fe c ts o f NSAIDs on PGI2 re lease by

the ra t u r ina ry bladder was also inves tiga ted .

4.1b. Milrinone and IBMX (PDE inhibitors)

M ilrinone [2 -m e th y l-5 -cyano -(3 ,4 '-b ip y r id in )-6 (1H )-o ne ] is a

non-glycoside b ip y r id in e d e r iva t ive th a t has p o s it ive in o t ro p ic and

vasod ila t ing a c t i v i t y (Alousi e t a l , 1983; Jaski e t a l , 1985; Borrow

e t a l , 1985). M ilr inone has proved e f fe c t iv e in the treatment o f

1 2 0

congestive cardiac fa i lu re (DiBianco e t a l . , 1989). Milrinone

probably exerts i t s benefic ia l action p r in c ip a l ly via in h ib i t io n o f

c y c l ic adenosine-3'5' monophosphate (cAMP) phosphodiesterase (PDE),

thereby elevating in t ra c e l lu la r cAMP concentrations and reducing

in t ra c e l lu la r Ca2+ concentrations (Earl e t a l . , 1986; Olson e t a l . ,

1987; Mylotte e t a l . , 1985). cAMP-PDE in h ib i to rs (PDEIs) have also

been shown to in h ib i t p la te le t aggregation (Lindgren e t a l . , 1990;

Simpson e t a l . , 1988; Tang e t a l . , 1980). In th is context, amrinone,

a s im ila r drug to m ilr inone, in h ib i ts p la te le t aggregation o f rabb it

and human p la te le ts (Lippton e t a l . , 1985; Patti son e t a l . , 1986). I t

is c lear tha t the in h ib i t io n o f p la te le t a c t iv i t y in patients with

congestive cardiac fa i lu re by milrinone would constitu te a desirable

additional property since: a) p la te le ts are key components in

thrombus formation (Chesterman and Berndt, 1984; Meade, 1985) and

patients with congestive heart fa i lu re have an increased r is k o f

developing thrombotic episodes i f ischaemic heart disease (IHD) is

involved (Trip e t a l . , 1990; M ikha il id is e t a l . , 1990) and b)

p la te le t release substances (v iz . TXA2 ) are potent vasoconstrictors

(as well as being proaggregatory) and as such have been widely

implicated in the aetio logy o f CVD (Meade, 1985; M ikha il id is e t a l . ,

1990).

In l i g h t o f the above, the e ffec ts and properties o f milrinone

on various aspects o f human p la te le t function , in v i t r o were

investigated:

1) cAMP PDE a c t iv i t y , as assessed by the conversion o f [^H]-cAMP to

[^H]-AMP and i lo p ro s t (a PGI2 analogue)- stimulated cAMP synthesis.

2 ) TXA2 synthesis in unstirred p la te le ts (spontaneous and when

stimulated by collagen, Ca '*' ionophore A23187, NaF, PMA, AA and

121

f re e ze - fra c tu r in g ) and 3) given th a t vascular P6 I 2 is also mediated

by fundamentally the same mechanisms, the e f fe c t o f th is drug on ra t

a o r t ic , endothe lia l c e l l cu ltu re was also inves tiga ted .

4.1c. Copper chelators

Copper imbalance is associated w ith both cardiovascular

(Klevay, 1975; Cunnane e t a l , 1979; S w if t e t a l . , 1978) and

inflammatory disease (Denko, 1989). However, there is some

ambivalence as to the precise ro le o f copper in these diseases. For

example, i t has been demonstrated th a t copper and the copper binding

p ro te in , caeruloplasmin, is elevated in both the serum and synovial

f l u id o f pa tien ts w ith rheumatoid a r t h r i t i s (Youssef e t a l . , 1983;

Scudder e t a l . , 1978a,b ) . S im i la r ly , copper che lators such as DL-

p e n ic i l lamine and cupralene have been used to t re a t inflammatory

disease (Fernandez-Madrid, 1989). In co n tra s t, complexing o f drugs

w ith copper has also been shown to markedly improve the e f f ic a c y o f

antiin flam matory drugs, inc lud ing a s p ir in (Sorenson, 1989). In CVD,

copper de fic iency ra th e r than copper excess is associated w ith

cardiovascular d isorders (Klevay, 1975). However, copper catalyses

l i p i d perox ida tion , which in tu rn is regarded as a major con tr ibu to ry

fa c to r in the ae tio logy o f both atherogenesis (Steinberg e t a l . ,

1989) and inflammation (Cuthbert e t a l . , 1989).

I t has been widely suggested th a t p la te le ts and t h e i r release

substances (v iz . TXA2 ) play a ro le in the pathophysiology o f

inflammatory (Lewis, 1984) and vascular disease (as has been

discussed p re v io u s ly ) . In tu rn , copper has been shown to modulate

eicosanoid synthesis in macrophages ( E l l i o t e t a l . , 1987), blood

vessels (M itche ll e t a l . , 1985), cardiac t issue (Cunnane e t a l . .

1 2 2

1988) and seminal ves ic le homogenates (Maddox, 1973). However, l i t t l e

Is known o f the ro le o f copper In mediating p la te le t cyclooxygenase

and lipoxygenase a c t i v i t y . In order to Inves tiga te th is area fu r th e r ,

the e f fe c t o f copper che lators on p la te le t TXA2 synthesis and

lipoxygenase a c t i v i t y was Investiga ted using systems employed

prev ious ly to Inves tiga te the ro le o f Iron w ith Iron che la tors

(Jeremy e t a l . , 1988e; Barradas e t a l . , 1989). The che la tors

Investiga ted were: dimethyld ith locarbam ic acid (DMDCA),

d ie thy ld ith loca rbam ic acid (DEDCA), te t ra e th y lth iu ra m d isu lph ide

(TETD), DL-penlcI11 amine, phenanthrolIne and t r ie n t ln e . D if fe re n t

s t im u la to rs o f TXA2 synthesis (NaF, PDBU, AA, Ca2+ Ionophore A23187

and f re e z e - fra c tu r in g ) were employed In order to es ta b lish s i te s o f

action o f the che la to rs . In te rac tions o f che lators w ith metal sa lts

(Cu2+, Cu+, Zn+, MnZ+, Fe^*, Fe+, A l^ * , Sn2+), superoxide dismutase

(SOD) and catalase were also Inves tiga ted .

1 2 3

4 .2 . RESULTS

4.2a NSAIDS

Indomethacin Inh ib ited equipotently the synthesis o f a o rt ic PGI2

synthesis when stimulated with noradrenaline, PDBU and A23187 ( f ig .

4 .1 .; fo r IC5QS see figu re legend). In contrast, there was a marked

r ig h t s h i f t (30 fo ld ) in the in h ib i to ry potency o f indomethacin when

PGI2 synthesis was stimulated with AA, sonication or freeze-

frac tu r ing ( f ig . 4 .1 .) The same pattern was found fo r ibuprofen ( f ig .

4 .1 .) and t ia p ro fe n ic acid ( f ig . 4 .1 . ) . In the aorta, in h ib i t io n o f

PGE2 , PGF2 and TXA2 synthesis by median doses o f indomethacin

(derived from NSAID ICggS in PGI2 synthesis above) were iden tica l to

the degree o f in h ib i t io n of PGI2 , when stimulated with noradrenaline,

A23187, AA or sonication (table 4 .1 . ) . S im ilar resu lts were obtained

fo r ibuprofen and t iap ro fe n ic acid. Since there were no differences

in the degree of in h ib i t io n o f each PG when stimulated with AA and

since AA-induced PG release is an index o f cyclooxygenase and / or PG

synthase/isomerase a c t iv i t y , the data ind ica te tha t the NSAIDs

studied are not acting d i f fe r e n t ia l ly on ind iv idual PG/TX synthases /

isomerases. In the urinary bladder, carbachol- and A23187-stimulated

PGI2 synthesis was in h ib ite d by indomethacin, ibuprofen and

t iap ro fe n ic acid ( f i g . 4. 2.) in a s im ila r manner to the aorta. Again,

there was a marked r ig h t s h i f t in the in h ib i to ry dose-response curves

when PGI2 synthesis was e l ic i te d by sonication or AA ( f i g . 4 .2 . ) .

Or 0 r

20

25

40

505060

n

756075

100L100100 0.01 I 'U

îndomethacir^ ( ^m ol / ( ) 0.01

[jbaprofenic acid] ( pmol/ I )

0.1 1.0 101000.01Qbuprofen] ( p m o l / l )

1.0

F ig . 4 . 1 . E f f e c t o f in d o m e th a c in , ib u p ro fe n and t i a p r o f e n i c a c id on PGI2 s y n th e s is by the i s o la t e d r a t a o r ta when s t im u la te d w i t h ; n o ra d re n a l in e ( | ) , phorbo l e s te r d ib u t y r a t e (JL )

A231 87 ( 0 ) , a ra c h i donate ( Q ) , f re e z e f r a c t u r i n g (A) and soni c a t io n ( Q ) . Each p o in t

r e p re s e n ts mean 1 SEN, n = 6 .

roA

0

202 5

2 5

4 0

5 05 0

n

7 58 0 75

100100 L

1.00.10.01

[jndomethacir^ ( pmol / 1 )0.1 1.0 10

[Tbuprofen] ( pmol/ f )100

1.00.10.01

[tiaprofenic acid] ( pm ol/ | )

F ig . 4 . 2 . E f f e c t o f in d o m e th a c in , Ib u p ro fe n and t i a p r o f e n i c a c id on PGI2 s y n th e s is by

th e i s o la t e d r a t u r in a r y b la d d e r when s t im u la te d w i t h : ca rb a ch o l ( # ) , A231 87 (A)» a ra c h i dona te (Q) and f re e z e f r a c t u r i n g ( A ) . Each p o in t re p re s e n ts mean 1 SEM, n = 6 .

roOl

1 2 6

Table 4 .1 . E ffe c t o f median In h ib i to r y concentrations o f indomethacin

(INDO) on ra t a o r t ic PGI2 (as 6-oxo-PGF.|(^ ) , PGEg, TXB2

(pg / mg tissue / rain [% in h ib i t io n ] ) when stim ulated w ith

noradrenline (NA), A23187, arachidonate (AA) and son ica tion (trauma).

_____________________ NA________A23187_________AA_________TRAUMA

6-oxo-PGF%% zero 160 (0%) 70 (0%) 190 (0%) 140 (0%)

INDO 84 (48%) 33 (33%) 80 (58%) 65 (54%)

PGE2 zero 88 (0%) 44 (0%) 95 (0%) 70 (0%)

INDO 43 (51%) 24 (45%) 40 (58%) 33 (47%)

PGF20 : zero 32 (0%) 18 (0%) 34 (0%) 26 (0%)

INDO 13 (51%) 9 (45%) 16 (58%) 12 (47%)

TXBg zero 16 (0%) 7 (0%) 18 (0%) 14 (0%)

INDO 8 (50%) 4 (53%) 9 (50%) 7 (50%)

127

stim u la to ry doses o f collagen (ICgQ: 1 x 10” ^ M; f i g . 4 .3 ) , NaF

( I C c n : 3 X 10-7 M; f i g . 4.3) and PMA (ICrn: 2.2 x 10“ 7 M; f i g . 4 .3 ) .

4.2b. Effect of PDE inhibitors on TXA2 synthesis.

M ilrinone was a potent in h ib i to r o f spontaneous TXA2 synthesis by

in ta c t p la te le ts (ICgg = 3 x IQ-7 M; f i g . 4 .3 ) . M ilr inone was also a

potent in h ib i to r o f p la te le t TXA2 synthesis when stimulated by median

'50

'gQ: o A iw ■ n ; i i y . H.of ai iu PMm i i v g Q :

In con tras t, a t s t im u la to ry doses o f A23187 and AA there was a marked

decrease in the potency o f m ilr inone in in h ib i t in g TXA2 synthesis

( f i g . 4 .3 ) . M ilr inone had a weaker e f fe c t on TXA2 synthesis when

e l i c i t e d by fre e ze - fra c tu r in g ( f i g . 4 .3 ) . Increasing concentrations

o f m ilr inone e l i c i t e d a p a ra l le l r ig h t s h i f t in the PMA concentration

- TXA2 response curve ( f i g . 4 .4 ) , in d ic a t in g a competitive in h ib i t io n

o f p ro te in kinase C by m ilr inone . In a l l experiments IBMX was a

weaker in h ib i to r o f TXA2 synthesis ( f i g . 4 .5 ) .

4.2c. Milrinone effects on phosphodiesterase activ ity and iloprost -

stimulated cAMP synthesis

Both m ilr inone and IBMX in h ib i te d p la te le t cAMP-PDE a c t i v i t y in

concentration-dependent manners when assessed by the conversion o f

[^Hl-cAMP to [^Hl-AMP ( f i g . 4 .6a ) . IC^qS were: m ilr inone; 2 x 10"^ M

and IBMX; 4.6 x 10“ ^ M. In tu rn , m ilr inone and IBMX increased cAMP

le ve ls in p la te le ts when e l i c i t e d by a median s tim u la to ry

concentration o f i lo p ro s t ( f i g . 4 .6b). ECggS (concentration o f drug

a t which i lo p ro s t-s t im u la te d cAMP synthesis was stimulated to 50% o f

maximal) were: m ilr inone , 5.6 x 10“ ^ M; IBMX, 3 x 10“ ^ M. Neither

m ilr inone or IBMX (up to 1 x 10“ ^ M) e l i c i t e d a measurable increase

in p la te le t cAMP concentrations (data not shown).

1 2 8

CO’SCDSIc>CO

CM<XH

60co4—'

5lec

1 0 0

-log Q niirinon^ ( M )

F ig . 4 . 3 . E f f e c t o f m i l r i n o n e on p l a t e l e t TXA2 (m easured as

TXBg) s y n th e s is when s t im u la t e d by ( f i n a l c o n c e n t r a t i o n s ) :

1 0 ^ 9 / ml c o l la g e n ( Q ) » 1 JuM PMA (<(>), 5 mM NaF ( ^ ) ,

1 A23187 ( 9 ) , 1 AA ( A ) , f r e e z e - f r a c t u r i n g ( A )and spo n ta n e o u s r e le a s e ( Q ) .

Each p o in t = mean t S .E .M . , n = 6 .

1 2 9

cE

CO4—'

(D0

•+ -»

_cga

00o

h-

D)C

1

0

“ log l^phorbo! ester] ( M )

F ig . 4 .4 . E f f e c t o f d i f f e r e n t c o n c e n t r a t io n s o f m i l r in o n e

on pho rbo l e s te r c o n c e n t r a t io n - TXA2 (as TXB2 ) response cu rve ( # ) + ze ro m i l r i n o n e , ( A ) + 3 x 10"? M m i l r i n o n e ;( ^ ) + 1 X 10 "6 M m i l r i n o n e ; ( B ) + 3 x 10” ® M m i l r i n o n e . Each p o in t = mean; n = 4.

1 3 0

O r

.2’co(Dsz+ -»c>>CO

cvj< X I—

5 0

co

-C■ c

100

-log [ j B M x ] ( M )

F ig . 4 . 5 . E f f e c t o f IBMX on p l a t e l e t TXA2 (m easured as TXB2 ) s y n th e s is when s t im u la t e d by ( f i n a l c o n c e n t r a t i o n s ) :

10 / ml c o l la g e n (O), 1 jJM p h o rb o l e s t e r « ^ ) , 5 mM NaF( □ ) , 1 JiM A23187 (A), 1 /iM AA ( # ) , f r e e z e - f r a c t u r i n g (A). Each p o in t = mean t S .E .M . , n = 6 .

or>>ü(Q

LUO0_Co 5 0

jec

100

c£

coo

<ooEa

- log [PDE inhlbitc^ ( M )

F ig . 4 . 6 . a . E f f e c t o f m i l r in o n e ( # ) and IBMX (A) on th e c o n v e rs io n o f [^H ]-cAM P to

C^H]-AMP by washed human p l a t e l e t s . Each p o in t = mean 1 S .E .M ., n=6 .

b . E f f e c t o f m i l r i n o n e ( Q ) and IBMX ( ) on i l o p r o s t ( 1^ M ) - s t im u la t e d cAMP

s y n th e s is by washed human p l a t e l e t s . Each p o in t = mean i S .E .M ., n=6 .

CO

1 3 2

4.2d. Effect of copper chelators on AA-stimulated TXAg synthesis and

lipoxygenase activ ity

The chelators studied In h ib ite d conversion o f AA to TXA2 ( f i g .

4.7a) in th is rank order o f potency: DMDCA > TETD > DEDCA > t r ie n t in e

> phenanthroline >> DL-penic illam ine. These chelators also in h ib i te d

lipoxygenase a c t i v i t y , the rank order o f potency being s im i la r to

th a t o f TXA2 synthesis ( f i g . 4 .7b).

The degree o f in h ib i t io n by DMDCA o f TXA2 synthesis when

stim ulated w ith A23187, NaF, phorbol es te r and AA were s im i la r fo r

a l l s t im u la to rs ( f i g . 4.8; ICggS fo r various s t im u la to rs : AA, 33uM;

A23187, 35uM; NaF, 34uM; phorbol e s te r , 30uM; freeze f ra c tu r in g ,

28uM). The s im i la r i t y in the ICgQg fo r each s t im u la to r is in d ic a t iv e

o f an e f fe c t o f che la to r on cyclooxygenase, ra ther than on other

enzymes or systems (PLA2 , p ro te in kinase C, Ca2+ m o b i l is a t io n ) . This

was confirmed using maximal in h ib i to ry doses o f the other chelators

( ta b le 4 .2 . ) . The in h ib i to ry e f fe c t o f a l l copper che la tors on both

TXA2 synthesis and lipoxygenase a c t i v i t y was reversed by the add ition

o f approximately equimolar concentrations o f Cu2+ and to a lesser

extent by Cu"*" and Zn2+ but not by Fe^^, Fe^*, A l^^, Mn^^ or Sn^^

( f ig s . 4.9a and 4 .9b). The add it ion o f ne ither SOD nor catalase to

incubates a ffec ted c h e la to r - in h ib i te d p la te le t cyclooxygenase or

lipoxygenase a c t i v i t y .

Or

20CO

CO

0

C

CO 4 0

6 0

co

J 3 8 0

100

1010.10 .01

>ÜCO0)COCOc0O)>Xoa

co

S Ic

0

20

40

60

80

100

0.01

log che lator ( uM )

0.1 10

Fig . 4 .7 . E ffe c t o f copper chelators on A) p la te le t TXA2 synthesis (stimulated with 10jjM arachidonic acid)

and B) lipoxygenase a c t iv i t y (conversion o f ^^C-AA to ^^C-HETE). Each point = mean 1 S.E.M., n = 7. dimethlydithiocarbamic acid (O), te trae thy lth iu ram disulphide ( • ) , diethyldiothiocarbamic acid (A)^ phenanthrol ine ( Q ) , t r ie n t in e DL-penici 11 ami ne ( \ ^ ) .

COCO

1 3 4

COCOLUXHZ>CO

<XH

ZOt 6 0COXz

80

100

10.10

log [chelato^ ( mM )

F ig . 4 . 8 . E f f e c t o f d im e t h y ld i t h io c a r b a m ic a c id on TXA2

s y n th e s is by washed human p l a t e l e t s when s t im u la t e d w i t h :

5 mM NaF ( 0 ) f 1 PMA ( V ) » 1 JuM a r c h id o n a te ( < ^ ) , 1 ^ M

Ca2+ lo n o p h o re A23187 ( Z \ ) , f r e e z e f r a c t u r i n g ( □ ) .

Each p o in t = mean 1 SEM, n = 8

wtn<üs:c«

20

CM<Xh-

4 0

co

!cc 6 0

8 0

100

0 .1 2 5 0 .2 5 0.5

>

oco<uwg0)O)>Xo.g.

20

4 0

co

6 0

8 0

100_l

0 .1 2 5 0 .2 5 0 .5

|metal sait| ( mM

Fig. 4 .9 . E ffec t o f various metal (ch lo r ide ) sa lts on (a) DMDTCA (100jjM )- in h ib ite d TXA2 synthesis

(stimulated with 1 0 AA) and (b) DMDTCA (100jiM) - in h ib i te d lipoxygenase a c t iv i t y (conversion o f ^^C-AA to ^^C-HETE by washed human p la te le ts . Each po in t = mean 1 S.D., n = 6 .

CuClg ( • ) , CuCl (A), ZnCl ( ■ ) , F e d 2 (A), FeClg « > ) , MnCl2 (Q), AICI3 ( + ) .CUCI2 alone ( ^ ) , NaCl alone (\7 )* CO

CJI

136

Table 4 .2 . E ffe c t o f d i f fe re n t copper che la tors on TXA2 synthesis

(% in h ib i t io n ; mean 1 SD; n = 6 ) e l i c i t e d by a range o f d i f fe re n t

s t im u la to rs .

Chelator NaF PMA AA spontaneous(cone) (10 mM) (1 ^M) (3 juM)

DMDCA 93 ± 8 94 ± 8 9 2 + 7 9 6 + 5(100 |iM)

TETD 90 ± 6 89 ± 8 88 i 10 88 ± 13(100 |iM)

DEDCA 60 ± 7 59 ± 8 63 ± 8 63 i 7(100 ^M)

phenanthrol ine 80 ± 7 83 + 6 82 + 8 80 +.7(2mM)

t r ie n t in e 7 5 + 6 7 3 + 7 7 4 + 8 7 5 + 8(5 mM)

DL-penici 11 ami ne 3 i 0.3 5.1 0.4 6 t 0.5 4 i 0.2(10 mM)

1 3 7

4.3. Discussion.

4 .3 .a NSAIDS

This study demonstrates th a t there is a marked d is p a r i ty in the

in h ib i to r y potencies o f NSAIDs on vascular PGI2 synthesis when

e l i c i t e d by s t im u la to rs which act a t d i f fe re n t s i te s in the receptor

a c t iv a t io n - PGI2 synthesis sequence. The s im i la r i t y o f e f fe c t

between the aorta and bladder ind ica tes th a t th is in h ib i t io n is not

confined to vascular smooth muscle and/or adrenoceptors. The IC^qS o f

the NSAIDs on trauma- and AA-stimulated PGI2 synthesis were ide n t ica l

but there were marked l e f t s h i f t s in the in h ib i to ry potencies when PG

synthesis was stimulated with NA, NaF, PDBU and A23187. The ICgQS

obtained from these s tim u la to rs also being id e n t ica l fo r each NSAID.

As discussed e a r l ie r , i t has been established th a t receptor agonist,

NaF and A23187 s t im u la t ion o f PG%2 synthesis in the ra t aortae is

in h ib i te d by conventional Ca2+ channel b lockers, such as verapamil

and n ife d ip in e (Jeremy e t a l . , 1985a, 1988a). In con tras t, AA-or

trauma-stimulated PGI2 synthesis are unaffected by Ca2+ channel

blockers (Jeremy e t a l , 1986c). Trauma e l i c i t s PGI2 synthesis in

smooth muscle through exogenous Ca2+ a c t iv a t io n o f PLA ( i . e . Ca2+

contained in incubation media), probably due to des truc t ion o f c e l l

membrane in te g r i t y thereby by-passing Ca2+ channels (Jeremy e t a l . ,

1986c). Since AA is the substrate fo r PG synthesis, s t im u la t io n o f

PGI2 synthesis w ith exogenous AA, as w ith trauma, also bypasssed the

Ca2+ mobi1isation-PLA2 step th a t l ib e ra te s endogenous AA. The present

data the re fo re po in ts to NSAIDs exering an e f fe c t on Ca2+

m ob il isa t ion linked to exogenous AA release as well as an e f fe c t on

cyclooxygenase.

Such a proposal is not w ithout precedent. I t has been suggested

1 3 8

th a t a major source o f ac tiva to r Ca2+ linked to both contraction and

PG syntheis is derived, in pa rt, from Ca2+ stores located in the

plasma membrane and glycocalyx (V il la m il e t a l , 1973; Loutzenheiser

and van Breemen, 1983; Jeremy e t a l , 1988a). In th is context,

Northover (1971) demonstrated th a t NSAIDs in h ib i t the uptake and

binding o f Ca2+ by plasma membranes o f vascular endothelial c e l ls and

tha t ibuprofen and indomethacin in h ib i ts [^^Ca^'*’ ] uptake by human

p la te le ts (G i l l e t a l . , 1990). NSAIDs also in h ib i t other membrane-

associated processes, including tha t o f superoxide and anion

generation by the c e l l - f r e e NADPH oxidase system o f neutrophils

(Biemond e t al,, 1986), mononuclear ce ll PLC a c t iv i ty (Bomalaski et

a t, 1986) and 12-HETE peroxidase o f the lipoxygenase pathway in

p la te le ts (Siegel e t a l . , 1986). In th is context, Abramson and

Weissman (1989) demonstrated tha t the NSAIDs, asp ir in , indomethacin

and piroxicam are also potent in h ib i to rs o f the early steps o f

receptor-activated neutrophil function, including the uptake and

m obilisation o f Ca2+ at the plasma membrane o f these c e l ls .

D ispar it ies o f the in h ib i to ry potencies o f NSAIDs on PG synthesis

have also been reported in other systems. For example, NSAIDs

( inc lud ing indomethacin and ibuprofen) are potent in h ib i to rs of

phorbol ester-stimulated PG synthesis by macrophages at

concentrations fa r less (up to 100 fo ld ) than are required to in h ib i t

PG synthesis by iso la ted seminal vesic le microsomes (Brune e t a l . ,

1981). The ICggS o f indomethacin and ibuprofen on PG synthesis by

seminal vesicles are almost identica l to the IC^qS reported here fo r

trauma- and AA- stimulated PGI2 synthesis whereas the IC^qS o f the

NSAIDs on PG synthesis by macrophages are s im ila r to IC^qS derived

here fo r agonist-, NaF-, PDBU- and A23187- stimulated PGI2 data.

139

Thus, notw ithstanding the mechanistic im p lica tions o f the present

study, i t is c lea r th a t the in h ib i to ry potencies o f NSAIDs on PG

synthesis depend on the methods used and awareness o f th is may be o f

importance in eva luating and comparing NSAID e f f ic a c y .

The present f ind ings may be o f relevance not only to the

e f f ic a c y o f NSAIDs as in h ib i to rs o f inflammation but also to th e i r

side e f fe c ts . F i r s t l y , the reduction o f inflammation by NSAIDs may be

due to in h ib i t io n o f Ca '*" m ob il isa t ion l inked not only to PG

synthesis but also to other Ca2+- re q u ir in g pro-inflammatory systems

(Westwick and P o l l , 1986). I t is also o f in te re s t th a t NSAIDs exert

s ig n i f ic a n t e f fe c ts on the metabolism o f bone and c a r t i la g e th a t are

probably not dependent on cyclooxygenase (Famaey e t a l , 1975;

Arumugham and Bose, 1982). The p o s s ib l i l i t y th a t the side e f fe c ts o f

NSAIDs (g a s tr ic erosion and u lce ra t io n , nephro tox ic ity and

hypertension), are mediated not only by the in h ib i t io n o f PG

synthesis but also through Ca^^-associated signal transduction

warrants fu r th e r in v e s t ig a t io n .

4 .3 .0 MILRINONE

The concentrations o f m ilr inone th a t in h ib i te d PDE a c t i v i t y in

the present study were approximately 30 times greater than those

which in h ib i t in v i t r o p la te le t aggregation (Barradas e t a l , 1992).

In con tras t , the concentrations o f m ilr inone th a t in h ib i te d TXAg

synthesis in the present study were s im i la r to those th a t in h ib ite d

in v i t r o p la te le t aggregation (Barradas e t a l , 1992). Furthermore,

the amounts o f m ilr inone required to i n h ib i t PDE a c t i v i t y in the

present study are somewhat greater than the accepted therapeutic

concentrations (Benotti and Hood, 1984). I t is u n l ik e ly , the re fo re .

1 4 0

th a t the in h ib i t io n o f PDE a c t i v i t y mediates the anti-aggregatory

e f fe c t o f m ilr inone . Ex v ivo , re s t in g in t r a p la te le t concentrations o f

cAMP are v i r t u a l l y undetectable and in v ivo are probably not

to n ic a l l y s tim ula ted, since i t is now accepted th a t PGI2 is not

c o n t in u a l ly secreted by blood vessels (B la i r e t a l . , 1982; Barrow and

R i t te r , 1988). Thus, e f fe c ts o f m ilr inone on PDE a c t i v i t y , and

the re fo re in t r a p la te le t cAMP le v e ls , are u n l ik e ly to come in to play

in the c i r c u la t in g , non-activated p la te le t . However, a t the s i t e o f a

mural thrombus or p la te le t aggregate, the s i tu a t io n may be qu ite

d i f fe r e n t . On adhesion and aggregation, p la te le ts release a number o f

b io ac t ive substances (TXA2 , seroton in , p la te le t derived growth

fa c to r ) a l l o f which have been shown to be potent s t im u la to rs o f PGI2

synthesis by vascular t issues (Jeremy e t a l , 1988a). I t was suggested

th a t th is p la te le t-s t im u la te d release o f PGI2 may co n s t i tu te a

haemostatic mechanism by which the size o f a thrombus is l im i te d at

the s i te o f vascular in ju ry (R i t te r e t a l . , 1983; Jeremy e t a l ,

1985a; 1985c). In th is l a t t e r scenario, lo c a l ly high concentrations

o f PGI2 w i l l e l i c i t e leva tion o f in t r a p la te le t cAMP concentrations.

Thus, m ilr inone may l i m i t thrombus s ize /e longation v ia enhancement o f

PGl2“ Stimulated cAMP in the p la te le ts involved in thrombus formation.

In con trast to PDE in h ib i t io n and cAMP modulation, m ilr inone was

a potent in h ib i to r o f TXA2 re lease when stimulated w ith co llagen, NaF

and PDBU and to a lesser extent when stim ulated with A23187,

exogenous arachidonate or f re e z e - f ra c tu r in g . This d i f f e r e n t ia l

potency o f in h ib i t io n o f TXA2 synthesis may be in d ic a t iv e o f several

possib le s i te s o f action o f m ilr inone since the s t im u la to rs used to

e l i c i t TXA2 synthesis act a t d i f fe re n t s i te s in the sequence leading

from surface receptor a c t iv a t io n to TXA2 synthesis. Thus, given the

141

d is p a r i t ie s in the in h ib i to ry potencies to d i f fe re n t s tim u la tors, the

present data indicates tha t m ilrinone is exerting i t s most potent

e f fe c t on the signal transduction mechanism l in k in g receptor

ac tiva t ion with TXA2 synthesis in the p la te le t . Since milrinone

inh ib ited [45ca2+] uptake at concentrations s im ila r to those tha t

inh ib ited PMA-, NaF- and collagen -stimulated as well as spontaneous

TXA2 synthesis (Jeremy e t a l . , 1993), the in h ib i to ry e f fe c t o f

m ilrinone on TXA synthesis may occur at any o f the Ca^* - dependent

steps: v iz . ac t iva t ion o f PKC (Nishizuka, 1988) and PLA ( I rv in e ,

1982). A s im ila r conclusion was reached by Block e t a l . , (1990), who

suggested tha t amrinone in h ib i ts p la te le t PLAg a c t iv i t y . The greater

potency o f m ilrinone on phorbol ester-stimulated TXA2 and the

p a ra lle l r ig h t s h i f t o f the phorbol ester dose-response curve,

e l ic i te d by m ilr inone, is ind ica tive o f a competitive in h ib i t io n o f

PKC a c t iv i t y . Since PKC a c t iv i t y requires Ca2+ (Nishizuka 1988), the

present data may re f le c t a d isruption o f the binding / association o f

Ca2+ to PKC. In tu rn , we have previously suggested tha t the uptake o f

[45ca2+] in response to agonists in the p la te le t re f le c ts Ca^+

sequestration a t the membrane and not changes in in t ra c e l lu la r Ca^+

(G i l l e t a l . , 1991, 1992a). Since PKC, when activated, moves in to the

plasma membrane (Nishizuka, 1988; Iwamoto, 1992), the present e ffec ts

o f m ilrinone may re f le c t loca lised events a t the ce l l surface. Such a

proposal is not w ithout precedent. A recent study demonstrated tha t

m ilrinone exerts a potent 'membrane e f fe c t ' on p la te le ts as

demonstrated by i t s in h ib i to ry action on the binding o f monoclonal

antibodies to p la te le t membrane glycoprotein Ib (Jackson e t a l . ,

1989). Milrinone has been shown to also in h ib i t Ca^+ - ATPase (a

Ca2+ - stimulated enzyme) in iso la ted heart plasma membranes

1 4 2

(Mylotte e t a l , 1987). Such e ffec ts on p la te le t membranes may also

explain the in h ib i to ry e f fe c t o f m ilrinone on p la te le t aggregation,

since membrane associated Ca2+ is essential fo r c e l l - c e l l stick iness

( i . e . aggregation; Gerrard e t a l , 1982; Rink and Sage, 1990).

I t is notable tha t IBMX, although less potent, e l i c i t s a s im ila r

pattern o f response as m ilrinone. S tru c tu ra l ly , IBMX is not

d iss im ila r to m ilr inone. I t is also o f in te re s t tha t the c lass ic

in h ib i to r o f Ca2+ in f lu x , n ifed ip ine , posesses s truc tu ra l

s im i la r i t ie s to m ilrinone (milrinone is a d ipyrid ine ; n ifed ip ine is a

monopyridine). Whether milrinone (and s t ru c tu ra l ly s im ila r PDE

in h ib i to rs ) also act via d isruption o f Ca '*' binding to e ffe c to r

proteins and what s truc tu ra l component determines these properties

warrants fu r the r inves tiga tion .

Apart from the in h ib i t io n o f p la te le t aggregation, the present

f ind ings may be o f relevance to the e f fe c t o f milrinone on other

ta rge t t issues, v iz . blood vessels and the heart. I t has been

established tha t m ilrinone is a potent vasodilator and increases

a t r ia l tension and beat rate (Alousi e t a l . , 1983; Grant e t a l . ,

1985). Both vascular re a c t iv i ty and heart function are dependent on

the mobilisation and resequestration o f Ca^^ and recent studies have

also demonstrated the ob ligatory ro le o f the G proteins, PLC and PKC

in these functions (Edes and Kranias, 1990; Danthaluri and Deth,

1984; Rasmussen a t a l , 1984). I t is also o f in te re s t tha t m ilrinone

is synerg is tic with captopril on cardiac performance and complements

e ffec ts o f captopril on the peripheral c irc u la t io n (LeOemtel e t a l ,

1985). In tu rn , i t has been demonstrated tha t captopril also in h ib i ts

adrenaline-stimulated [^^Ca^'*'] uptake by human p la te le ts in v i t r o and

ex vivo (G i l l e t a l . , 1989; G i l l e t a l . , 1992b). The synerg is tic /

1 4 3

complementary re la t io n s h ip between cap top r i l and m ilr inone po ints to

a common s i te o f ac t ion , possibly Ca^^ m ob il isa tion / t ra nspo rt at

the plasma membrane. The p o s s ib i l i t y th a t m ilr inone exerts i t s

vasod ila to r and in o tro p ic actions v ia the mechanisms postulated to

occur in the p la te le t in th is study warrants fu r th e r in ve s t ig a t io n .

4 .3c Copper chelators

The present study demonstrates th a t copper che la tors , in

p a r t ic u la r DMDCA, DEDCA and TETD in h ib i t s p la te le t TXA2 synthesis and

lipoxygenase a c t i v i t y . Both systems were notably in h ib i te d in a

s im i la r rank order o f potency, in d ica t in g a s im ila r mechanism o f

a c t ion . The in h ib i t io n o f TXAg synthesis appears to be a t the

cyclooxygenase and/or TXA2 synthase leve l ra ther th a t a t other s ites

since the in h ib i to ry potencies o f the che lators was s im i la r fo r a l l

s t im u la to rs o f TXA2 synthesis (NaF, A23187, AA and phorbol es te r.

The in h ib i to ry action o f the che lators was reversed by the presence

o f Cu2+ and to a lesser extent by Zn^* and Cu'*' but not Fe^*, Fe^^,

MnZ+ or A l^^. Since, Zn2+ has only one valence sta te and such is

u n l ik e ly to exert e f fe c ts on cyclooxygenase or lipoxygenase a c t iv i t y

i t would appear th a t the present e f fe c ts are mediated p r in c ip a l ly via

in te rac t io ns w ith Cu '*'. The reversal o f the e f fe c t may also be due to

negation o f drug a c t i v i t y since ac tive s i te s would be e f fe c t iv e ly

abolished by che la tion w ith metals.

M echan is tica lly , cyclooxygenase catalyses the oxygenation and

peroxidation o f AA to y ie ld the endoperoxide, PGG2 , which is then

converted to PGH2 by peroxidase action (Deby, 1988). Since Cu '*’

catalyses peroxidation (Cuthbert e t a l . , 1989) and the present data

demonstates a c lea r in h ib i t io n o f AA perox idation , i t is possible

1 4 4

th a t the copper che la tors act by in h ib i t in g the peroxidase a c t iv i t y

o f cyclooxygenase. A l te rn a t iv e ly , i t has been proposed th a t TXA2

synthase catalyses a dismutase-type reaction (Anderson e t a l , 1978).

Given th a t copper is an essentia l c o - fa c to r fo r SOD a c t iv i t y

(Marklund, 1982), i t is possible th a t the e f fe c ts o f copper che lators

on TXAg synthesis are mediated by e f fe c ts on copper-mediated

dismutation o f PGH2 to TXA2 . Furthermore, copper-chelator complexes

possess dismutative a c t i v i t y in t h e i r own r ig h t (Lengfelder and

E ls tner, 1978), which may con tr ibu te to the e f fe c t o f che la tors seen

here. However, the lack o f e f fe c t o f SOD on c h e la to r - in h ib i te d TXA2

synthesis does not support th is p o s s ib i l i t y .

Although i t is well established th a t cyclooxygenase holoenzyme

contains iron (Kulmacs and Lands, 1984), there is no evidence th a t

cyclooxygenase is a copper-containing enzyme. However, several

studies have demonstrated th a t copper mediates eicosanoid synthesis

in other t issue s . Maddox (1973) demonstrated th a t the presence o f

d iva le n t copper in homogenates enhances the conversion o f AA to PGF2

but in h ib i t s concomitant PGE2 generation and concluded th a t Cu2+

exerts d i f f e r e n t ia l e f fe c ts on d i f fe r e n t synthases. I t i s also

notable th a t although Cu2+ che la to rs , benzydroxamic acid and 8-

hydroxyquinoline reversed these e f fe c ts o f Cu2+, whereas DL-

penici 11 amine had an opposite e f fe c t (Maddox, 1973). In the present

study, i t was also found th a t DL-penic i 1 lamine has l i t t l e e f fe c t on

p la te le t TXA2 synthesis or lipoxygenase a c t i v i t y synthes is . I t is

d i f f i c u l t to ra t io n a l is e why pe n ic il lam ine d i f fe r s from other

che la to rs , since i t posesses high a f f i n i t y binding capacity fo r

copper. However, i t has been demonstrated th a t pen ic il lam ine augments

p la te le t aggregation (M ik h a i l id is e t a l . , 1988b) whereas other copper

1 4 6

chelators i n h ib i t p la te le t aggregation (V arga ft ig e t a l . , 1974). I t

would appear the re fo re th a t pen ic il lam ine is anomalous in the present

con text. Other extensive studies ca rr ied out by V arga ft ig e t a l .

(1974) consolidate the present f ind ings since a range o f copper

che la tors were found to i n h ib i t p la te le t aggregation, an e f fe c t also

reversed by the presence o f copper and z in c . I t is notable th a t

V arga ft ig e t a l . (1974) predicted th a t these chelators were acting

v ia in h ib i t io n o f eicosanoid synthesis.

Apart from p la te le ts , copper has been shown to markedly in fluence

eicosanoid synthesis in other t is s u e s /c e l ls . Copper de fic iency in

ra ts has been shown to reduce by about 50% PGI2 synthesis by a o r t ic

r ings compared to those fo r copper adequate animals, im p lica t in g an

ob lig a to ry ro le fo r copper in mediating the synthesis o f th is

eicosanoid (M itche ll e t a l . , 1988). I t is notable th a t a o r t ic SOD

a c t i v i t y was also reduced by approximately 50% in copper d e f ic ie n t

compared to copper adequate animals (M itche ll e t a l . , 1988). However,

in the present study, ne ithe r SOD nor catalase in fluenced ch e la to r-

in h ib i te d TXA2 synthesis. In another study, hearts from ra ts fed a

low copper or a copper supplemented d ie t were perfused and secreted

less 6-keto-PGFi0 than hearts from the copper-supplemented group

(Cunnane e t a l . , 1988). I t was suggested th a t copper de fic iency

allows increased l i p i d hydroperoxide production which is s u f f ic ie n t

to in h ib i t PGI2 synthase a c t i v i t y and hence reduce PGI2 formation.

E l l i o t e t a l . (1987) demonstrated th a t copper stimulated the release

o f both TXA2 and ^^C-AA release from macrophages and concluded th a t

copper acts on PLA ra the r than on cyclooxygenase in these c e l ls . The

present data, however, do not concur w ith e f fe c ts o f copper che lators

on PLA2 .

1 4 6

C l in ic a l ly , l i t t l e is known o f the re la t io n s h ip between copper

imbalance, eicosanoid synthesis and the ae tio logy o f disease.

Wilson's disease (a cond it ion characterised by copper overload) is

associated w ith premature o s te o a r th r i t is w ith biopsies o f symptomatic

jo in t s showing chronic inflammatory c e l l i n f i l t r a t e s (Denko, 1989).

Copper and the copper-binding p ro te in , caeruloplasmin has also been

shown to be elevated in serum and in the synovial f l u id o f pa tien ts

w ith rheumatoid a r t h r i t i s (Youssef e t a l . , 1983; Scudder e t a l . ,

1978a,b). In tu rn , eicosanoids are key mediators o f inflammation,

inc lud ing rheumatoid a r t h r i t i s (Rainsford, 1988; Brune, 1988).

Furthermore, copper che la tors have been used to t re a t inflammatory

disease (Fernandez-Madrid, 1989). However, th is drug type e l i c i t s a

number o f severe side e f fe c ts and as such have been la rg e ly re jec ted

as a major therapy fo r inflammatory disease (Fernandez-Madrid, 1989).

Nevertheless, copper che la tors may prove inva luable to o ls in

e lu c id a t in g the in te r re la t io n s h ip between copper, eicosanoid

synthesis and l i p i d peroxidation in the pathophysiology o f disease.

Development o f less to x ic che lators may also prove e f fe c t iv e as

therapeutic agents.

1 4 7

CHAPTER 5 .

STUDIES ON PGIg RELEASE BY VASCULAR AND OTHER TISSUES FROM RATS

WITH EXPERIMENTAL DIABETES MELLITUS OR HEPATIC PORTAL HYPERTENSION.

5.1 . Introduction

5.1a Diabetes Mellltus

As was discussed in the general in tro d u c t io n , DM is

characterised by a reduction in vascular PGIg synthesis (Harrison e t

a l . , 1978; Rogers e t a l . , 1981; Jeremy e t a l . , 1987a). The precise

mechanisms th a t determine reduced vascular PG%2 synthesis in DM are

not c le a r ly defined, although a reduction in substrate

(arachidonate) leve ls in blood vessels has been suggested (Holman e t

a l . , 1983; Jeremy e t a l . , 1987a, Dang e t a l . , 1988). As was

discussed e a r l ie r , vascular PG%2 synthesis is s timulated by

vasoactive agonists, which in tu rn is mediated by G p ro te ins , PKC

and IP3 (G r ied ling e t a l . , 1986; Hal lam e t a l . , 1988; Jeremy e t a l . ,

1988a). The net re s u l t o f a c t iv a t io n o f these systems is

transmembrane Ca2+ in f lu x and an increase in in t r a c e l lu la r Ca2+

([Ca2+]^) which then ac tiva tes PLA^, l ib e ra t in g AA from phospholipid

stores ( I r v in e , 1982). In order to fu r th e r e luc ida te the mechanisms

th a t determine diminished vascular PGI2 synthesis, the e f fe c t o f a

range o f s t im u la to rs th a t act a t d i f fe r e n t s i te s in the receptor

ac t iva t ion -P G l2 synthesis pathway in the s treptozotocin-induced

d ia b e t ic r a t were inves tiga ted . The s t im u la to rs employed were:

adrenaline (mediated by heterogenous cx-adrenoceptors; Jeremy e t

a l . , 1985a) angiotensin I I (Ang I I ; Hassid and W illiams, 1983;

Stevens e t a l . , 1992), PDBU (a PKC a c t iv a to r ) , thapsigarg in

(e levates in t r a c e l lu la r Ca^+; Thastrup e t a l . , 1990; Levine and

1 4 8

Watanabe, 1991) Ca2+ ionophore A23187 (creates a r t i f i c i a l Ca2+

channels; Reed and Lardy, 1972) and arachidonic acid (AA; substrate

fo r PGI2 synthesis; Hamberg, 1968).

Reports th a t vascular PGI2 synthesis is diminished in DM are

not un ive rsa l. F u j i i e t a l . , (1986) reported th a t in perfused ra t

mesenteric bed taken from d ia be t ic ra ts there was a marked increase

in the output o f PGI2 compared w ith c o n tro ls . Indeed, increased

blood flow in g a s tro in te s t in a l (GI) vessels is also associated with

the ea rly stages o f DM (Tooke e t a l . , 1986). A e t io lo g ic a l ly , these

d isorders have been la rg e ly ascribed to d ia be tic autonomic

neuropathy (L inco ln e t a l . , 1984a,b). However, i t is also well

established th a t the pathophysiology o f various GI disorders in non

d iabe tics involves endogenous PGs (Branski e t a l . , 1986). With

regard to the GI t r a c t in DM, the author is aware o f no systematic

studies on PG synthesis in man or in experimental animal models.

However, i t has been demonstrated th a t c a lo r ic depriva tion re su lts

in d i f fe r e n t ia l changes in PGI2 and TXA2 synthesis by the small

in te s t in e and mesenteric vasculature o f the ra t (Jeremy e t a l . ,

1987c). Since these PGs in fluence mesenteric blood flow and gut

m o t i l i t y (Mailman, 1982), i t was suggested th a t these changes in PG

synthesis may play a ro le in the adaptation o f the small gut in

response to fa s t in g through modulation o f gut m o t i l i t y and

mesenteric blood flow (Jeremy e t a l . , 1987c). I t is also well

established th a t in experimental DM, the GI t r a c t and associated

vasculature o f the ra t undergoes marked adaptive changes o f

s tru c tu re (mucosal and muscular hyperp lasia; Je rv is and Levin, 1966;

Schedl e t a l . , 1982) and function (increased n u tr ie n t uptake;

Lorenz-Meyer e t a l . , 1977).

1 4 9

In order to inve s t ig a te a possib le ro le fo r PGs in the

adaptation and pathology o f DM in the splanchnic vascu lature, the

re lease o f PG%2 , TXA2 , PGE2 , the mesenteric vessels and

hepatic porta l vein was inves tiga ted . PG synthesis by the aorta and

ca ro t id a r te ry o f the same animals were also measured to assess

vascular PG synthesis in 'po s t-h e p a tic ' vessels. Since d ia b e t ic ra ts

are markedly hyperphagic we also examined the e f fe c t o f p a ir feeding

(amount o f food given to d ia be t ic ra ts per day adjusted to the

average d a i ly in take o f age-matched co n tro ls ) on PG synthesis by the

above t issues . The e f fe c t o f in s u l in adm in is tra tion to d ia b e t ic ra ts

was also inve s t iga te d . Since gut m o t i l i t y a f fe c ts blood flow (Winne,

1980) and fa s t in g has been shown to markedly a l te r PG synthesis by

the GI t r a c t (Jeremy e t a l . , 1987c), PG synthesis by the mucosal and

muscular portions o f the GI t r a c t were also assessed.

In order to examine whether o ther non-vascular smooth muscle

tissues are a ffec ted in a s im i la r or d isparate manner as the aorta

and splanchnic vasculature, PGI2 synthesis by the u r ina ry bladder

and trachea was inves tiga ted . I t is also notable th a t the u r ina ry

bladder o f the d ia b e t ic r a t releases more PGI2 per u n i t weight than

con tro ls (Jeremy e t a l . , 1986a). Bladder PGI2 synthesis was

stim ulated w ith Ach (PGI2 synthesis in the u r ina ry bladder is

mediated by muscarinic receptors; Jeremy e t a l . , 1986b) as well as

PDBU, thaps igarg in , A23187 and AA.

5 .2 . Hepatic portal hypertension

Hepatic po rta l hypertension (HPH) is a complication o f l i v e r

c i r rh o s is which sometimes re s u lts in severe consequences, such as

1 6 0

bleeding from esophogeal varices and asc ites formation (Bosch e t

a l . , 1992). HPH is always in t i t i a t e d by an increased vascular

resistance to po rta l blood flow (Bosch e t a l . , 1992; Murray e t a l . ,

1958; Kowalski e t a l . , 1953; Siegel e t a l . , 1974; Lee, 1989).

However, when HPH is f u l l y established there is also reduced

splanchnic a r te r io la r res istance and a marked increase in portal

blood in f lo w , which con tr ibu tes to the maintenance o f po rta l

hypertension. This hyperdynamic splanchnic c i r c u la t io n is

accompanied by increased cardiac output together w ith reduced

a r te r ia l pressure and peripheral vascular resistance (Bosch e t a l . ,

1992; Murray e t a l . , 1958; Kowalski e t a l . , 1953; Siegel e t a l . ,

1974; Lee, 1989).

S im ila r c i r c u la to ry changes are seen in experimental models

inc lud ing the hepatic porta l v e in - l ig a te d ra t (Bonzam and Blundis,

1987). Although the mechanisms underly ing these c i rc u la to ry

phenomena are s t i l l undefined, changes in vascular r e a c t iv i t y in

response to adrenergic agonists (Ramond e t a l . , 1986; Gerbes e t a l . ,

1986; Pizcueta e t a l . , 1990), 5-HT (Kaumann e t a l . , 1986; Cummings

e t a l . , 1986), angiotensin I I (Murray and P a l ie r , 1985,1986) as well

as p ros tacyc lin (PGI2 ) synthesis (Hamilton e t a l . , 1983; R i t te r e t

a l . , 1986; Sitzmann and L i , 1991; Guarner and Soriano, 1993) have

been suggested as being invo lved. I t has also been proposed th a t an

increase in the synthesis o f vascular NO, as a va so d ila to r , may play

a key ro le in the vascular adaptions to HPH (Vallance and Moncada,

1991; W h it t le and Moncada, 1991; Pizcueta e t a l . , 1992; Lee e t a l . ,

1992), although th is has recen tly been contested (Sogni e t a l . ,

1992). Some time ago, Murray and P a lie r (1985, 1986) proposed tha t

there was a post-receptor defect underly ing the vascular r e a c t iv i t y

1 6 1

changes in HPH.

With regard to PGI2 , a marked increase in output by the hepatic

portal vein in both the laboratory ra t and man has been reported

(Hamilton e t a l . , 1983; Sitzmann and L i , 1991; Guarner and Soriano,

1993). However, the mechanisms co n tro l l in g vascular PGI2 synthesis

in HPH are again not c le a r ly defined and l i t t l e is known o f PGI2

synthesis and release in systemic vasculature in HPH. In order to

elucidate the mechanisms tha t may determine a ltered vascular PGI2

synthesis in HPH, the e f fe c t o f a range o f stimulators th a t act at

d i f fe re n t s ites in the receptor activation-PG l2 synthesis pathway in

the aorta (and mesenteric vasculature) was investigated in ra ts with

HPH using an approach described above fo r the d iabetic ra t model.

5.2. RESULTS

5.2 .a. Diabetes Mellitus

Body weights (median [range]) o f the animal groups a t the time

o f s a c r if ic e were: con tro ls , 475 g (449-516; n =35); d iabe tic , 239 g

(220-280; n = 35). At s a c r i f ic e , blood glucose concentrations

(mmol/I; median [range]) were: con tro ls , 8.4 (6 .8-10.3); d iabe tic ,

30.3 (28.2-32.7).

Aortic PG%2 release (expressed as pg O-oxo-PGRj# libera ted per

mg wet tissue per min) was s ig n i f ic a n t ly reduced in d iabetic ra ts

compared to contro ls in response to adrenaline ( f i g . 5 .1 . ) , Ang I I

( f i g . 5 .2 .) , PDBU ( f i g . 5 .3 .) and A23187 ( f ig . 5 .4 .) . There were no

differences in PGI2 release in response to thapsigargin ( f i g . 5 .5 .)

or AA ( f i g . 5 .6 . ) . Since there is good evidence tha t th is reduction

o f PGI2 is due to diminished AA stores in vascular t issue of

1 6 2

diabetic ra ts , data was transformed to % maximal response to obviate

th is variab le . Transformation o f data to % maximal response revealed

a marked r ig h t s h i f t in the adrenaline-PGl2 dose-response curve in

aortae from DM ra ts (ECgQ =4.1 x 10"^M) compared to contro ls (EC5Q

= 5.4 X 10"^M; f ig 5 .1 ). S im ila r ly , there was a marked r ig h t s h i f t

in the ANG I I dose response curves (% maximal response) in aortae

from diabetic ra ts (ECgQ = 2.9 x 10"^ M) compared to contro ls (ECgQ

= 4.9 X 10*7 M; f i g . 5 .2 . ) . Following transformation o f data to %

maximal response, there was also a marked r ig h t s h i f t the PDBU-PGI2

dose-reponse curve (EC5Q = 1.4 x 10“ ^M) compared to contro ls (ECgg =

1.9 X 10“ ^M; f i g . 5 .3 . ) , whereas there were no differences in the

EC5QS (% maximal response) o f thapsigargin ( f ig . 5 .4), A23187 ( f ig .

5 .5), or AA ( f ig . 5 .6).

In contrast to the aorta there was a marked increase in PGI2

release in repense to ACh from the urinary bladder o f d iabe tic rats

compared to contro ls ( f i g . 5 .7 ). Following transformation o f data to

% maximal response there was a marked l e f t s h i f t in the Ach-PGl2

dose-response curve in urinary bladders from d iabetic ra ts (EC^q =

5.8 X 10“ ^M) compared to contro ls (EC^q = 2.2 x 10"^ M; f i g . 5 .7 ).

Although there was a generalised increase in PGI2 output in the

urinary bladders from d iabetic ra ts there were no differences in the

ECgQS of PDBU ( f ig . 5 .8 ), thapsigargin ( f ig . 5.9), A23187 ( f ig .

5.10.) or AA (data not shown) between d iabetic or control rats

fo llow ing tranformation o f data to % maximal response. In the

trachea o f the d iabe tic ra t (compared w ith contro ls) PGI2 release in

response to ACh was s ig n if ic a n t ly diminished ( f ig . 5 .11 .) .

The synthesis o f PGI2 (as G-oxo-PGFi# ) was s ig n if ic a n t ly

elevated in the mesenteric vessels and hepatic portal veins o f the

1 6 3

d ia b e t ic ra ts fed ad l ib i tu m or p a ir fed compared w ith con tro ls

( f i g . 5 .1 2 .) . In the d ia be t ic ra ts , fed ad l ib i tu m and p a ir fed,

PG%2 synthesis by aortae and ca ro t id a r te ry was s ig n i f ic a n t ly

diminished ( f i g . 5 .1 2 .) . TXB2 , PGE2 and PGP2(^ release was s im i la r ly

a lte red by blood vessels in a l l experiments ( tab le 5 .1 . ) . In su lin

adm in is tra tion prevented a l l changes o f vascular PG synthesis found

in d ia be t ic animals ( f i g . 5 .12., tab le 5 .1 . ) . 6-oxo-PGFiQ, , 1X82, PGE2

and PGF20; synthesis were a l l unaltered in the mucosal and muscular

regions o f the stomach, duodenum, jejunum and ileum, from d iabe tic

ra ts , whether fed ad l ib i tu m or p a ir fed ( tab le 5 .2 . ) . PG synthesis

by the GI t r a c t fo l lo w in g in s u l in adm in is tra tion was also not

s ig n i f ic a n t ly d i f fe r e n t from con tro ls ( ta b le 5 .2 . ) . I t is notable

th a t the re la t iv e proportions o f the various PGs studied varies from

t issue to t is s u e . Thus, in vascular t issu e the average r a t io o f 6-

oxo-PGF-joj : PGE2 : PGF2Q/ : TXB2 was 40: 15: 3: 1 whereas in GI

t issue i t was 9: 4: 2: 1. This probably re f le c ts the d i f fe r e n t ro les

o f PGs in these disparate t issue s . Analysis o f data also revealed

th a t these ra t io s were not a lte red by DM.

5.2b. Hepatic portal hypertension

12 porta l ve in -con s tr ic ted ra ts and 12 sham operated (co n tro l)

ra ts were stud ied. Portal venous pressure was s ig n i f ic a n t ly elevated

in the porta l vein con s tr ic ted ra ts compared to c o n tro ls : 13.3 ± 0.8

mm Hg vs 6.3 ± 0.8 mm Hg (p < 0 .01). Spleen weights were also

increased in po rta l vein con s tr ic ted ra ts compared to co n tro ls : 1.04

~ 0.07g vs 0.79 — 0.08 g (not s ig n i f ic a n t ) . Spontaneous release o f

PGI2 fo l low ing freeze-thawing and son ication (an index o f substrate

and synthesis ing enzymes [PLA2 , cyclooxygenase and synthase] and

1 6 4

substrate a v a i la b i l i t y ) was not s ig n i f ic a n t ly d i f fe re n t between

aortae from portal hypertensive ra ts (8.8 ± 0.86 ng 6 - o x o -P G F-|q , /

mg tissue / h) and sham-operated animals (9.2 t 0.89 6-oxo-PGF^q, /

mg tissue / h) but was s ig n i f ic a n t ly ( p < 0.001 ) elevated in

mesenteric vasculature from portal hypertensive ra ts (14.2 1 0.96 ng

6-oxo-PGF-iQ,/ mg tissue / h) and sham-operated animals (8.2 1 1.29

6-oxo-PGF-|qj / mg tissue / h).

PGI2 release by aortae from ra ts w ith portal hypertension was

enhanced in response to adrenaline compared to sham-operated animals

( f ig . 5.13). In contrast, PG%2 release by the aortae from portal

hypertensive ra ts was reduced in response to PDBU ( f ig . 5.14),

A23187 ( f ig . 5.15) and thapsigargin ( f ig . 5.16) compared to

con tro ls . There were no differences in a o rt ic PG%2 output in

response to AA between ra ts with portal hypertension and control

ra ts ( f ig 5.17). PG%2 release by the mesenteric vasculature from HPH

rats compared to contro ls was enhanced in response to adrenaline

( f ig . 5.13), PDBU ( f i g . 5.14), A23187 (5.15), thapsigargin ( f ig .

5.16) and AA ( f i g . 5.17)

5.3. DISCUSSION

5.3a Diabetes

Although i t is well established tha t PG%2 synthesis by the ra t

aorta is diminished in experimental DM (Harrison e t a l . , 1978;

Rogers e t a l . , 1981; Jeremy e t a l . , 1987a), the mechanisms th a t

determine th is reduction are not c le a r ly defined. However, i t has

been shown th a t AA stores in vascular t issue , as well c irc u la t in g

AA, are reduced in experimental DM (Holman et a l , 1983; Thomson

1980; Dang e t a l . , 1988). This probably accounts fo r the generalised

1 5 5

c 1 5 0 E

4>3V)w 100cnE

yuT0CL1OX01(D

5 0

àiP'-

- log [adrenalin^ ( M )

LUCOzoCLCOlUDC

X<

100

8 0

6 0

4 0

20

01

- log [^drenalin^ ( M )

F ig . 5 . 1 . A d r e n a l in e - s t im u la t e d PGI2 r e le a s e by a o r ta e

f ro m d i a b e t i c and c o n t r o l r a t s (O). Data a re e xp re sse d

in te rm s o f : a) a b s o lu te amounts o f PGI2 r e le a s e d (as 6 -

o x o -P G F ^ # ) and b ) as % o f maxim al re s p o n s e .

Each p o in t = mean 1 S .D . , n = 7 . * p < 0 .0 0 1 .

1 6 6

c

^ 120

(D

WOT

8 0b)E

üIlT0 Q.1OX01

(D

4 0

D)Cl

100LUWZoCL(OLUÜC

8 0

6 0

4 0

20

-log l^ngiotensin ÏÏ] ( M )

F ig . 5 *2 . A n g io te n s in I I - s t im u la te d PGI2 r e le a s e by

a o r ta e from d ia b e t i c (A) and c o n t r o l r a t s (O). Data are

exp ressed in te rm s o f : a) a b s o lu te amounts o f PGI2

re le a s e d (as 6 - o x o - P G F i# ) and b) as % o f maximal

re s p o n s e . Each p o in t = mean 1 S .D . , n = 7. * p < 0 .0 0 1 ,

1 6 7

E 2 0 0

1 50

100

5 0

0

log [phorboi este^

100

LUcozoCLmLUoc

8 0

6 0

_J<5X<

4 0

20

F ig . 5 .3 . Phorbo i e s te r - s t im u la te d PGIg re le a s e by

a o r ta e from d ia b e t i c A ) and c o n t r o l r a t s ( O ) . Data are

exp ressed in te rm s o f : a) a b s o lu te amounts o f PGIg

re le a s e d (as G-oxo-PGFj^, ) and b) as % o f maximal

re s p o n s e . Each p o in t = mean t S .D . , n = 7. * p < 0 .0 0 1 .

1 6 8

1 6 0

120

8 0

4 0

0

100

7 5

5 0

2 5

0

Fig . 5 .4 . Ca2+ ionophore A23Î87 - st imulated P6 I 2 release

by aortae from d iab et i c and control rats ( O ) . Data are

expressed in terms of : a) absolute amounts of PGI2

released (as G-oxo-PGP]# ) and b) as % of maximal

response. Each point = mean 1 S .D . , n = 7. * p < 0 .001.

1 6 9

E0) 8 03WOT

D ) 6 0E

8Ll T

4 00Q .

0X 2 00

( D

0D )Q .

- log [Thapsigargin^( M )

100

LU(0Z 8 0oÛ.wœ 60

4 0

20

- log [T h a p s ig a rg l^ ( M )

F ig . 5 .5 . T h a p s ig a rg in - s t im u la te d P6 I 2 r e le a s e by a o r ta e

from d ia b e t i c A ) and c o n t r o l r a t s (O). Data are expressed

in te rm s o f : a) a b s o lu te amounts o f P6 I 2 re le a s e d (as 6 -

oxo-PGF-jq ;) and b) as % o f maximal re sp o n se .

Each p o in t = mean 1 S .D . , n = 7. * p < 0 .0 0 1 .

1 6 0

D)E

ur0 CL1oX01

CD

D)a

120

80

40

0

100LUV)ZoCLV)LUCC

8 0

6 0

4 0

20

-log [araohldonat^ ( M )

F ig . 5 .6 . A r a c h idonate - s t im u la te d PGI2 re le a s e by a o r ta e

from d ia b e t i c (A) and c o n t r o l r a t s (O ) . Data a re expressed

in te rm s o f : a) a b s o lu te amounts o f PGI2 re le a s e d (as 6 -

0X0 -PGF1# ) and b) as % o f maximal re sp o n se .

Each p o in t = mean 1 S .D . , n = 7 . * p < 0 .0 0 1 .

1 6 1

Ê 120

80

d

J4

1I___ L0 7 6 5

100

80

0V)

d 60 V)

2gE 40

120

U 5

■log [acetylcholin^ ( M )

F ig . 5 .7 . A c e t y lc h o l in e - s t im u la te d PGI2 re le a s e by

u r in a r y b la d d e rs from d ia b e t i c (A) and c o n t r o l r a t s (O)

Data are exp ressed in term s o f ; a) a b s o lu te amounts o f

PGI2 re le a s e d (as G-oxo-PGP^# ) and b) as % o f maximal

re s p o n s e . Each p o in t = mean 1 S .D . , n = 7. * p < 0 .0 0 1 .

1 6 2

60E

d)3WOT

4 0D)E

üU TOQ . 20oXo

(D

D)a

uj 1 0 0 rV)z oû .co LUû:

5 0_j<2X< 2 5

F ig . 5 .8 . Phorbo i e s te r d ib u t y r a t e - s t im u la te d PGI2

r e le a s e by u r in a r y b la d d e rs from d ia b e t i c (A) and c o n t r o l

r a t s (O). Data are exp ressed in te rm s o f : a) a b s o lu te

amounts PGI2 re le a s e d (as 6 -oxo-PGF-| ^ ) and b) as % o f

maximal re s p o n s e . Each p o in t = mean i S .D . , n = 7. * p <

0 .0 0 1 .

1 6 3

(U3OTOT8 0

D)E

6 0

LL“0 Q.1OX01co

4 0

20

D)a

I 00 rLU(0oCLtog 60

_J< 4 0 2X< 20

log [Â23187ÿ] ( M )

F ig . 5 .9 . Ca2+ io n o p h o re A23187 - s t im u la te d P6 I 2 r e le a s e

by u r in a r y b la d d e rs from d ia b e t i c (A) and c o n t r o l r a t s

(O).. Data are exp ressed in te rm s o f : a) a b s o lu te amounts

o f PGI2 re le a s e d (as 6 -o xo -P G F i# ) and b) as % o f maximal


1 6 4

3 0E

d)3WOT

20D)E

aür-ü0 -IoX01co

10

D)a

100UJ(OzoQ.toLUOC

7 5

5 0- j<

X<2

2 5

-log [ïhapsigargi^ ( M )

F ig . 5 .1 0 . T h a p s ig a rg in - s t im u la te d PGI2 re le a s e by

u r in a r y b la d d e rs from d ia b e t i c i/S) and c o n t r o l r a t s (O)

Data are exp ressed In term s o f : a) a b s o lu te amounts o f

PGI2 re le a s e d (as G-exo-PGP^# ) and b) as % o f maximal


1 6 6

40

30

20

D)D.

1 0 0

LUCOZoû.coLUcr

75

50<iX< 25

F ig . 5 .1 1 . A c e ty lc h o l in e - s t im u la te d PGI2 r e le a s e by the

t ra c h e a from d ia b e t i c (A) and c o n t r o l r a t s ( O) . Data are

exp ressed in te rm s o f : a) a b s o lu te amounts o f PGI2

re le a s e d (as 6 -oxo-PGF^Qr ) and b) as % o f maximal


1 6 6

MESENTERIC HEPATIC PORTAL

c

03COCO

D)E

«LL0CL1OX01

CD

D)a

350

300

200

100

200

100

0

AORTA

Q

%

I

350

300

2 0 0

100

2 0 0

100

0

CAROTID

F ig . 5 .1 2 . PGI2 (as 6-oxo-P6F-|^ ) synthesis by d i f fe re n t vascular tissues o f the r a t , e igh t weeks a f te r induction o f diabetes with streptozotocin . CON = con tro l, DM = d iabe tic fed ad l ib i tu m , DM-PF = pa ir fed d iabe tic , INS-DM = in su lin - tre a te d d iabetic fed ad l ib i tu m . Each histogram represents the mean 1 SEM, n =7.# = p < 0.001 compared with con tro ls .

Table 5.1 PGE2 , PGp2#and TXB2 synthesis (pg / mg wet wt t issue / min;

mean i SEM, n = 7) by vascular t issue from d iabetic (fed ad l ib i tu m , pa ir

fed, in su lin - tre a te d ) and control ra ts e ight weeks a fte r induction o f

diabetes with s treptozotoc in . * = p < 0.001 compared with contro ls

1 6 7

Controls d iabetic

(fed ad l ib itum )

diabetic d iabetic

(pa ir fed) ( in su lin - tre a te d )

1. mesenteric vessels

PGE2 65 i 6 168 + 20* 135 i 15* 48 ± 4

PGF20! 22 1 3 78 + 15* 82 t 10* 28 ± .4

TXB2 5 1 0.5 15 + 1* 14 1 2* 8 ± 2

hepatic portal vein

PGE2 55 i 5 140 + 16* 105 t 12* 39 1 5

PGFga 16 1 3 68 + 15* 57 ± 10* 18 ± 5

TXB2 6 ± 0.5 22 + 3* 18 ± 3* 8 ± 1

aorta

PGE2 88 ± 16 58 + 12* 54 t 11* 76 t 1-

PGF2QJ 22 ± 3 13 + 2* 12 i 2* 20 1 -

TXB2 5 ± 0.5 2 + 0 .5 * 2 1 0 .3 * 6 +

caroti d

PGE2 78 ± 15 55 + 10* 50 ± 12* 66 i 21

PGF2CX 18 1 4 10 1* 11 ± 2* 16 +

TXB2 6 1 0.3 3 + 0 .3 * 3 ± 0 .3 * 7 1 0

1 6 8

Table 5.2 . PGI2 (ss G-oxo-PGF^^)* PGE2 ; PGP2( snd TXA2 (as TXB2 ) synthesis

[pg. mg"1 wet wt t issue , min” ^; mean i SEM, n = 7]) by GI tissue from

d iabetic (fed ad l ib i tu m , pa ir fed, insu lin - tre a te d ) and control ra ts eight

weeks a f te r induction o f diabetes with s treptozotocin .

Controls d iabetic (ad l ib itum )

1. Stomach muscularls6-oxo-PGF-i^TXBo PGE,PGFZa 24

2. Stomach mucosa6-oxo-PGF-j (Y 21TXBg jPGEp (PGF20Î Î

3. Duodenum (muscularls6-oxo-PG Fin/ 1 1 0 — 21

13 + 2TXB'PGEÎPGFl a

65 ± 8 54 i 10

4. Jejunal muscularlsG-oxo-PGFiof 148 ± 22TXBo 16 ± 3PGE,PGFZa

75 ± 9 24 ± 3

mucosa)120 ± 22

18 1 4 62 ± 14 64 ± 8

153 ± 24 20 i 3 84 ± 14 30 i 5

5. Jejunal mucosa6- oxo-PGF-i (y 16 — 1

8 ± 112 ± 3

6 + 1

TXBPGE:PGF:Za

6. I le a l muscularlsla6-oxo-PGFin/ 165 — 38

TXBo PGEo PGF20;

7 . I le a l mucosa6-0X0“PGFi(^TXBoPGE.PGF 20!

23 ± 3 85 1 19 23 i 3

18 + 4 5 + 0.5

10 i 3 7± 1

15 i 3 6 ± 2

10 i 4 7 ± 2

170 ± 48 21 i 2 82 i 14 29 i 5

16 i 3 6 1 1

12 ± 1 6 ± . 2

d iabetic (p a ir fed)

125 ± 18 14 i 4 51 ± 16 58 ± 13

160 i 33 17 ± 5 60 i 15 28 ± 7

14 i 2 9 i 2

10 i 5 5 1 2

170 i 18 22 i 2 92 i 25 27 i 6

20 i 4 7 + 1 9 - t 2 5 ± 2

d iabetic( insu lin - trea te d )

+ 18 146 + 28 145 + 24 130 + 19+ 2 12 T 4 16 3 15 + 2± 15 57 + 18 58 + 12 66 + 20± 3 26 i 8 28 ± 13 23 3

+ 2 23 + 3 24 + 4 23 + 2+ 0.5 4 + 1 5 + 0.5 6 1± 1 6 ± 1 7 £ 1 9 + 2+ 0.2 4 + 0.4 3 + 1 3 + 0.3

115 ± 2812 i 256 ± 11 44 ± 12

135 i 25 19 ± 6 46 1 23 27 + 4

8 i 46 i 17 ± 3 3 + 3

154 ± 33 24 ± 4 78 + 22 20 i 3

19 ^ 45 ± 1 7 i 26 1 1

cE

0)3ü)W

D)E

u_0 û.1oX01

CDU)C l

00

5 0

0

100

5 0

0

- log [jidrenalin^ ( M )

Fig* 5. 13. Adrenaline-stimulated PGI 2 release by a) aortae and b) mesenteric vasculaturefrom portal vein-constricted rats ( # ) and sham - operated rats ( ♦ ) .Each point = mean 2 S.D., n = 12. * p < 0.01.

0)CO

cÊ

o3(0coD)E

u_Oa.IoX01co

150

100

5 0

0

1 0 0

5 0

0

- log [phorboi este^ ( M )

Fig. 5. 14. Phorboi ester dibutyrate-stimulated PGI 2 release by a) aortae and b) mesenteric vasculature from portal vein-constricted rats (#) and sham-operated rats (♦). Each point - mean 1 S.D., n = 12. * p < 0.01.

cE

0DCOCO

U)E

LL0 û_1OX01co

D)a

150r

lOOr

100

50

50

Fig. 5* 15. Ca^ ionophore A23187-stimulated PGI 2 release by a) aortae and b) mesenteric

vasculature from portal vein-constricted rats (#) and sham -operated rats .Each point = mean i S.D., n = 12. * p < 0.01.

cE

m3COCO

O)E

L i.0 û -1oX01

CD

O)Q .

100

100

5050

0 06 5

FÎ&* 5* 16. Thapsigargin - stimulated PGI2 release by a) aortae and b) mesenteric

vasculature from portal vein-constricted rats (#) and sham -operated rats (^).

Each point « mean t S.D., n = 12. * p < 0.01.

ro

150cÊ

1000DCOco

g 100

gLLOû.

IOX01

CD

5 0

5 0

O)a

01 -

j4

J4 5656

-log j^rachidonat^ ( M )Fî-ê» 5. 17* Arachidonate-stimulated PGI2 release by a) aortae and b) mesenteric

vasculature from portal vein-constricted (#) and sham -operated ratsE a c h p o i n t = m ean 1 S . D . , n = 1 2 . * p < 0 . 0 1 .

NCO

174

reduction o f PGI2 re lease by aortae from d ia be t ic ra ts , ir re sp e c t ive

o f the s t im u la to r used. The exception to th is was AA-stimulated P6 I 2

synthesis, in which no d if fe rence in the output of P6 I 2 was seen

between contro l and d ia b e t ic aortae. This consolidates previous

conclusions th a t there is no a l te ra t io n o f e i th e r cyclooxygenase or

PGI2 synthase a c t i v i t y in blood vessels from d ia b e t ic ra ts (Jeremy

e t a l , 1987a). I t is also notable th a t there were no s ig n i f ic a n t

d iffe rences in PGI2 output between d ia b e t ic and con tro l aortae in

response to thapsigarg in and ove ra ll absolute output was markedly

less than th a t derived from A23187 or adrenaline. This may ind ica te

th a t in t r a c e l lu la r Ca^* e levation l ib e ra te s AA from d is t in c t l y

d i f fe re n t phospholip id stores than other s t im u la to rs . Such a

p o s s ib i l i t y warrants fu r th e r in v e s t ig a t io n .

Following transform ation o f data from absolute output o f PGI2

per u n i t weight o f t issue to % maximal response, d i f f e r e n t ia l

changes in the dose-response curves emerged fo r the d i f fe re n t

s tim u la to rs used. F i r s t l y , there were no d iffe rences in the dose-

response curves fo r thapsigarg in and A23187 between d ia b e t ic and

contro l aortae. Since thapsigarg in mobilises in t r a c e l lu la r Ca2+

(Tharstrup, 1990; Levine and Watanabe, 1991) and A23187 e l i c i t s

entry o f e x t ra c e l lu la r Ca2+ via the c rea tion o f a r t i f i c i a l Ca2+

channels (Reed and Lardy, 1972), these data ind ica te th a t Ca2+ pools

and/or PLA2 a c t i v i t y are unaltered by DM in the r a t . In con tras t,

there were marked r ig h t s h i f t s in the dose-PGl2 response curves fo r

both adrenaline, ANG I I and phorbol es te r . Since receptor-1 inked

PGI2 synthesis in the r a t aorta is mediated by PKC (Jeremy e t a l ,

1988a), these data ind ica te th a t the defect in receptor-1 inked PGI2

synthesis in the ra t aorta may be due to a l te ra t io n s o f PKC a c t iv i t y

1 7 6

ra the r than to changes o f a f f i n i t y and/or number o f receptors l inked

to PGI2 synthesis (v iz . # -adrenoceptor and ANG I I recep to rs ) . I t is

also notable th a t in the r a t aorta , the endothelium con tr ibu tes

n e g l ig ib le amounts o f the to ta l PGI2 generated by th is vessel in

response to agonists, phorbol ester and ionophores (Chapter 3 ). The

present data the re fo re re f le c ts PGI2 released by the vascular smooth

muscle component o f the aorta ra the r than th a t generated by the

endothelium.

Recent data consolidates the proposal th a t vascular PKC

a c t i v i t y is a lte red in DM. Lee e t a l . (1988) demonstrated a marked

reduction in c y to s o l ic (but an increase in membrane-associated) PKC

in aortae o f d ia b e t ic ra ts , an e f fe c t mimicked in v i t r o by high

concentrations o f glucose. Under normal cond it ions , receptor

a c t iv a t io n by agonists e l i c i t s the a c t iv a t io n o f c y to s o l ic PKC

(Iwamoto e t a l . , 1992). Activated PKC phosphorylates e f fe c to r

pro te ins and also moves in to the membrane where i t down-regulates

receptor a c t i v i t y (Iwamoto e t a l . , 1992). The data o f Lee e t a l .

(1988) the re fo re po in ts to a generalised movement o f PKC in to the

plasma membrane o f vascular c e l ls in d ia b e t ic ra ts which in tu rn

would serve to d im in ish recep to r- l inke d responses, inc lud ing PGI2

synthes is . A reduction in cy to so l ic PKC would also account fo r the

diminished response to phorbol ester reported in th is study, since

phorbol este rs e l i c i t responses by a c t iv a t in g c y to s o l ic PKC

(Nishizuka, 1988; Iwamoto e t a l . , 1992). In terms o f other recepto r-

l in ke d func tions , both a ttenuation and p o te n t ia t io n o f receptor-

l in ked con trac t ion o f aortae from d ia b e t ic ra ts has been reported

( f o r review see Tomlinson e t a l , 1992). How PKC re la te s to these

va r ia b le a l te ra t io n s in c o n t r a c t i l i t y warrants cons idera tion .

176

In d ire c t con tras t to the ao rta , there were marked increases o f

PG synthesis by mesenteric vessels and hepatic porta l veins o f

d ia be t ic ra ts whereas, in the same animals, PG synthesis by the

aorta and ca ro t id a r te r ie s was again s ig n i f ic a n t ly dim inished. Since

the increase in PG synthesis by the mesenteric and hepatic porta l

vessels was not reversed in the p a ir fed animals these data ind ica te

th a t DM per se and not the amount o f food ingested determines these

changes. This conclusion is consolidated by the prevention by

in s u l in adm in is tra tion o f changes in PG synthesis, body weights and

food intake in d ia b e t ic ra ts . The present f ind ings are also in

agreement w ith the stud ies o f F u j i i e t al (1986) who demonstrated an

increase in PG release by iso la ted perfused mesenteric vasculature

from d iabe tic ra ts .

I t has been established th a t experimental DM re s u lts in

increased GI and splanchnic blood flow (Korthuis e t a l . , 1987;

Rosenblum e t a l . , 1985) and a marked reduction in c o n t ra c t i le

responsiveness o f the mesenteric vascular bed (Longhurst e t a l . ,

1985). Given th a t splanchnic blood flow is a key mediator o f

n u tr ie n t uptake (Mailman, 1982) and th a t PGs are potent modulators

o f mesenteric and hepatic porta l c o n t r a c t i l i t y (Traverse e t a l . ,

1984; Kondo e t a l . , 1980) the marked increase in PG synthesis may

c o n s t i tu te an adapative response th a t serves to enhance n u tr ie n t

uptake. Q u a n t i ta t iv e ly , the dominant PG released by the mesenteric

and hepatic po rta l vessels is PGI2 . In tu rn , PGI2 is a vasod ila to r

o f the r a t splanchnic vascular region (Hadhazy e t a l . , 1988) and o f

the r a t hepatic po rta l vein (Traverse e t a l . , 1984). Furthermore,

although PGE2 , PGF20; and TXA2 are vascoconstictors o f mesenteric

vessels in the ra t (Hadhazy, 1988), they have also been shown to

177

i n h ib i t norepinephrine release from presynaptic term ina ls in th is

vascular region (Kuriyama and Makita, 1982). Since norepinephrine is

a vasoconstr ic tor in the mesenteric vascular bed (Kondo e t a l . ,

1980), increased loca l synthesis and release o f these vascular PGs

may the re fo re enhance the tendency towards vasod ila t ion and

increased blood flow through supression o f noradrenaline re lease.

The increased synthesis o f PGs in mesenteric and hepatic porta l

veins may be determined by a d ie ta ry component(s) or is consequent

to other adaptive changes o f these vessels. The most l i k e l y

candidate fo r a d ie ta ry fa c to r is arachidonic acid (AA), the

precursor substrate fo r the synthesis o f PGs. Fatty acid absorption

across the small gut is known to be enhanced in experimental DM

(Thomson, 1980). Thus, greater q u a n t i t ie s o f AA in "prehepatic"

blood may enhance PG synthesis in the mesenteric and hepatic porta l

ve in . The decrease in prostanoid synthesis by "post hepatic"

vessels, in tu rn , may be a t t r ib u ta b le to the documented changes in

c i r c u la t in g AA (Holman e t a l . , 1983). Thus, although greater

q u a n t it ie s o f AA may be presented to the l i v e r , reduced " f re e " AA

may a c tu a l ly enter the systemic blood stream o f the d ia be t ic r a t .

Furthermore, other d ie ta ry components, in p a r t ic u la r glucose, have

been shown to enhance vascular PGI2 synthesis (F u j i i e t a l . , 1986)

and as such may also play a ro le in these loca l changes o f vascular

PG synthesis.

Also in con tras t to the aorta , there was a generalised increase

in the release o f PGI2 by the u r in a ry bladder from d ia b e t ic ra ts ,

i r re s p e c t iv e o f the s t im u la to r used. However, fo l low ing

transformation o f data to % maximal response there was a marked l e f t

s h i f t in ACh-PGl2 dose-response curves obtained from d ia be t ic

1 7 8

bladders when compared w ith c o n tro ls . In con tras t, no s h i f t s were

seen in the dose-response curves fo r phorbol es te r , thaps iga rg in ,

A23187 or AA. Thus, in the case o f the bladder, there appears to be

a s p e c if ic increase in muscarine receptor num ber/ac t iv ity but no

changes in PKC, Ca2+ s to res, PLA^, cyclooxygenase or synthase

enzymes. These conclusions are in agreement w ith a recent study in

which marked l e f t s h i f t s o f the dose-response curves fo r Ach-

stimulated con trac t io n were found in u r in a ry bladders from d ia b e t ic

ra ts (L a t i fp o u r e t a l . , 1992). The d ia m e tr ic a l ly opposite pa tte rn o f

recep to r- l inke d PGI2 synthesis by the u r in a ry bladder and the aorta

can be ascribed to the p a r t ic u la r stresses imposed on the bladder in

experimental DM. In d ia b e t ic ra ts , the u r ina ry bladder is markedly

enlarged, a r e s u l t o f chronic d is tens ion caused by polydypsia and

po lyu r ia (L inco ln e t a l , 1984). Thus, the up-regu la tion o f muscarine

receptors (and the increase in PGI2 ou tpu t) in the u r in a ry bladder

o f d ia be t ic ra ts is probably a s p e c i f ic adaptive event r e la t in g to

increased m ic tu r i t io n ra te . Furthermore, DM does not appear to

e l i c i t a per se up -regu la t ion o f muscarine receptors l inked to PGI2

synthesis, s ince Ach-stimulated PGI2 synthesis was s ig n i f ic a n t ly

diminished in the trachea from d ia b e t ic ra ts compared w ith c o n tro ls .

In fa c t , the trachea more resembles the aorta in terms o f d iabetes-

induced a l te ra t io n s in receptor-1 inked PGI2 synthes is . I t is perhaps

notable th a t DM is not p a r t i c u la i r l y associated w ith d isorders o f

the airways in man (e .g . an increased incidence o f o b s tru c t ive

airways d isease). I t may, however, be o f in te re s t to study airways

function in the d ia b e t ic r a t , which in tu rn may provide in s ig h ts

in to the ro le o f PGs in basic airways physiology.

Thus, i t appears th a t changes o f PGI2 synthesis in DM are

1 7 9

o rgan -spe c if ic . For example, there are no changes in P6 I 2 synthesis

in any region o f the 61 t r a c t (a lso markedly enlarged due to

hyperphagia), whereas there is a th re e - fo ld increase in PGI2

synthesis by the splanchnic vasculature and hepatic po rta l vein o f

the d iabe tic r a t . There is also a marked output o f PGI2 from

perfused mesenteric vessels from d ia b e t ic ra ts (F u j i i e t a l , 1986).

Decreases in PGI2 have been reported in a l l regions o f the bra in and

the penis (Jeremy e t a l , 1985a, 1987b). In con tras t, Stevens e t a l .

(1992) found no s ig n i f ic a n t d if fe rences in PGI2 re lease stim ulated

by ANG I I from perfused lungs o f d ia b e t ic ra ts . Taken together,

these experimental f ind ings ind ica te th a t large a r te r ie s appear to

be more susceptib le to DM than the microvasculature. This phenomenon

may in tu rn be o f importance from a c l in i c a l po in t o f view since

vascular complications ( v iz . a therosc le ros is in large a r te r ie s ) are

a common cause o f death in DM. Thus, apart from mediating PGI2

re lease, PKC also con tro ls other vascular functions inc lud ing

c o n t r a c t i l i t y (Dantha luri and Deth; 1984; Rasmussen e t a l . , 1984)

and c e l l p r o l i f e r a t io n , the pathognomonic les ion o f a therosc leros is

(Berridge, 1984). PKC also modulates the generation o f vascular

n i t r i c oxide (NO; Lewis and Henderson, 1987; Smith and Lang, 1990),

an important va so d i la to r and in h ib i t o r o f p la te le t aggregation

(Palmer e t a l . , 1987; Radomski e t a l . , 1987). Thus, the mechanisms

and consequences o f changes in PKC in vascular t issue from d ia b e t ic

animals warrants fu r th e r in v e s t ig a t io n .

5.3b Hepatic portal hypertension.

The present study f i r s t l y demonstrates th a t there is no

1 8 0

fundamental increase in the a c t i v i t y o f PGI2 synthesising enzymes

(PLA2 , cyclooxygenase, PGI2 synthase) in the aortae o f ra ts w ith

HPH, whereas in mesenteric vessels there is a marked increase in

PGI2 synthesising capac ity . These la t t e r f ind ings concur w ith the

previous reports o f o thers: th a t PGI2 synthesis ing enzymes are

increased in the splanchnic vasculature o f ra ts w ith HPH (Hamilton

e t a l . , 1982; Sitzmann and L i , 1991; Guarner and Soriano, 1993). I t

is concluded th a t in mesenteric vessels, PGI2 may con tr ibu te to

reduced vascular r e a c t iv i t y in experimental HPH, whereas i t is

u n l ik e ly to play a s im i la r ro le in the ao rta . This fundamental

d if fe rence in the aorta and mesenteric vasculature probably re f le c ts

the d i f fe re n t stresses to which the vessels are subjected in th is

p a r t ic u la r experimental scenario. Following l ig a t io n o f the hepatic

porta l vein, there is a marked increase in blood pressure in the

splanchnic region and the re fo re d is tens ion o f blood vessels. In

tu rn , d istension has been shown to increase PGI2 synthesis ing

capac ity , not only in blood vessels, ( in c lu d in g the r a t hepatic

po rta l vein [Hamilton e t a l . , 1982] and human aorta [Tsang e t a l . ,

1988]) but also in o ther t issues (e .g . u r in a ry bladder o f the

d ia b e t ic r a t [Jeremy e t a l . , 1986d]). As was ou tl ined above, th is

phenomenon may be re la te d d i r e c t ly to hyperp lasia, an event known to

occur in blood vessels in response to hypertension in both man and

the labora tory animal (Swales e t a l . , 1991). We are unaware o f any

study th a t demonstrates hyperplasia in the splanchnic or systemic

vasculature in experimental HPH, but t h is p ropos it ion would seem to

warrant in v e s t ig a t io n .

In the aorta o f HPH ra ts , enhanced PGI2 re lease, in response to

adrenaline, but reduced output o f PGI2 in response to phorbol es te r.

181

A23187 and thapsigarg in cannot be ascribed to changes in substrate

or synthesising enzymes since the to ta l synthesis ing capacity o f

PGI2 (index o f endogenous substrate and enzymes) was not d i f fe re n t

between aortae from ra ts w ith HPH or sham-operated animals.

Therefore, there appears to be a s p e c i f ic enhancement o f

adrenoceptor-linked PGI2 synthesis in the aortae from ra ts w ith HPH

which does not seem to r e f le c t changes o f PKC a c t i v i t y or Ca^*

m o b i l isa t io n . In a p a ra l le l study on a o r t ic c o n t r a c t i l i t y in the

same animals, adrenoceptor-stimulated con trac t ion was s ig n i f ic a n t ly

reduced in HPH (Karatapanis e t a l . , 1992), in d ic a t in g th a t the

e f fe c t on adrenaline-stim ula ted PGI2 cannot be ascribed to an

increase in alpha adrenoceptors, per se. On th is p a r t ic u la r p o in t,

i t has been prev iously suggested th a t there is a d i re c t re la t io n s h ip

between con trac t ion o f blood vessels and PGI2 ( i . e th a t agonists

which s tim u la te con trac t ion also s t im u la te PG%2 v ia a shared

pathway; Jeremy e t a l . , 1985a, 1988a). However, the present data

m it iga tes against t h is . One possib le explanantion fo r t h is d is p a r i ty

is th a t adrenoceptors may be l inked d i r e c t ly to PLA2 v ia G prote ins

(DeMolle and Boeynaems, 1989); e f f e c t iv e ly by-passing PKC and Ca2+

m o b il isa t io n . Due to the ove ra ll marked increase in PGI2

synthesis ing capacity by mesenteric vessels from ra ts w ith HPH, i t

was not possib le to discern any d i f f e r e n t ia l e f fe c ts o f the various

s t im u la to rs used. However, the biochemical measurement o f PKC and

Ca2+ in the mesenteric vessels would answer whether th is vascular

t issu e resembles the aorta (v is a v is PKC and Ca2+).

The apparent reduction o f PKC a c t i v i t y and o f a c t iv a to r Ca^^, as

detected by diminished PGI2 re lease in response to phorbol es te r ,

A23187 and thaps iga rg in , may expla in (a t le a s t in pa rt) the w e l l -

182

established diminished c o n t r a c t i l i t y o f aortae in ra ts w ith HPH.

That Ca2+ is essentia l fo r e x c ita t io n -c o n tra c t io n coupling in

vascular t issue is well established (Somlyo and Somlyo, 1968). PKC

a c t iv a t io n (by phorbol esters) also e l i c i t s in v i t r o

vasoconstr ic tion o f blood vessels (an e f fe c t enhanced by Ca2+

[Danthaluri and Deth, 1984; Rasmussen e t a l . , 1984]). In tu rn ,

v i r t u a l l y every pressor agent h i th e r to investiga ted e l i c i t s

generation o f d iacyl g lycero l (DAG) and IP3 in vascular t is s u e . DAG

ac tiva tes PKC (which is a Ca2+-dependent enzyme) and IP3 l ib e ra te s

in t r a c e l lu la r stores o f Ca2+, thereby e leva ting cy to s o l ic Ca^+

le v e ls . Since both events are associated w ith vasoconstr ic t ion , i t

fo llows th a t any attenuation o f these events would re s u l t in a

tendency toward va so d ila t io n . In th is context, KCl-stimulated

con tract ion o f aortae from ra ts w ith HPH (KCl e l i c i t s

vasoconstr ic t ion v ia m o b il isa t ion o f e x t ra c e l lu la r Ca^+) was found

to be diminished in aortae o f ra ts w ith HPH (Karatapanis e t a l . ,

1992). I t is also o f in te re s t th a t phorbol ester in h ib i t s NO

synthesis in vascular t issue (Lewis and Henderson, 1987). Thus, a

reduction in PKC a c t i v i t y in vessels would be expected to increase

NO synthesis. I t would be o f in te re s t to study the converse

re la t io n s h ip ( i . e . , the e f fe c t o f NO on PKC a c t iv i t y ) in vascular

t issue from ra ts w ith HPH. However, i t is known th a t NO in h ib i t s

Ca2+ m ob il isa t ion in vascular t issue (Igna rro , 1990, Rubanyi, 1989),

which in tu rn may be expected to i n h ib i t PKC a c t i v i t y , since th is

enzyme is Ca^^-dependent (Nishizuka, 1984). The present data are

the re fo re consis tent w ith a key mediatory ro le fo r NO in HPH.

In summary, HPH e l i c i t s d i f f e r e n t ia l changes o f PGI2 synthesis

in the aorta and mesenteric vasculature o f the r a t w ith experimental

183

HPH. I t appears th a t , by v i r tu e o f the q u a n t it ie s generated, P6 I 2

may play a vasod ila to ry ro le in the splanchnic (but not systemic)

vasculature. However, a generalised reduction in PKC a c t i v i t y and

Ca2+ 'a v a i l a b i l i t y ' may account, a t le a s t in p a r t , fo r the

diminished v a s o a c t iv i ty o f the aorta in HPH. Although the mechanisms

th a t govern the adaptive changes o f the vasculature in response HPH

remain unknown, th is phenomenon is l i k e l y to be multi fa c to r ia l (NO,

PGI2 , PKC, Ca2+, G p ro te in s ) , w ith each fa c to r in te ra c t in g w ith the

other fac to rs to engender va so d ila t io n . Furthermore, by using the

approach employed in th is study, these mechanisms ( in t r in s ic

vascular and/or c i r c u la t in g tro p h ic fa c to rs ) could be dissected out.

An understanding o f the mechanisms th a t determine the vascular

changes discussed here may be o f value in understanding not only the

ae tio logy o f HPH but also o f other forms o f hyper- and hypo

tens ion. In tu rn , th is may be o f use in the design o f drugs th a t

contro l blood pressure d isorders.

184

CHAPTER 6.

GENERAL DISCUSSION AND CONCLUDING REMARKS

The ob je c t ive o f t h is thes is was to fu r th e r explore recepto r-

l inked eicosanoid synthesis in vascular t issu e and p la te le ts and to

apply these systems to the in v e s t ig a t io n o f drug action and the

mechanisms underly ing defects o f blood vessels in animal models o f

vascular d isorders ( v iz . diabetes and po rta l hypertension). Apart

from a l te ra t io n s in eicosanoid synthesis (which in themselves are o f

relevance to drug ac tion and the pathophysiology o f disease) the

methods developed also provided in s ig h ts in to a l te rn a t iv e s i te s o f

drug action and poss ib le a l te ra t io n s o f vascular func tion in

experimental pa tho log ies. Since de ta i led discussions on each top ic

have been presented a t the end o f each chapter, th is discussion

centres on some lo g is t i c aspects and possib le fu tu re d ire c t io n s fo r

the work presented in th is th e s is .

F i r s t l y , using the approach developed in th is thes is (eicosanoid

re lease in response to a ba tte ry o f s t im u la to rs ) several possible

a l te rn a t iv e s i te s o f ac tion (v iz . Ca2+ dependent processes; PKC, and

phospholipase a c t i v i t y ) were revealed fo r m ilr inone and NSAIDs. I t is

not suggested th a t t h is methodological approach supplant other

estab lished methods (e .g . p la te le t aggregation, shape change or

vascular c o n t r a c t i l i t y in organ baths) but ra the r th a t i t may prove

useful as an ad junct to these methods. Thus, by studying the e f fe c t

o f any given drug on eicosanoid re lease e l i c i t e d by a ba tte ry o f

d i f fe r e n t s t im u la to rs , one can obta in an in d ic a t io n o f the mode o f

ac tion o f the drug under in v e s t ig a t io n . This in form ation would then

186

p o in t the way to approaches using func tiona l tes ts (e .g . aggregation,

con trac t ion / re la x a t io n ) . This po in t is exemplified by a breakdown o f

the day to day lo g is t ic s o f the assessment o f TXAg release by

p la te le ts . In any one day, 10 ml o f washed p la te le ts (from 30ml o f

blood) derived from approximately an in d iv id u a l represents a t le a s t

100 separate data p o in ts . By reducing the volume o f p la te le t

suspensions fo r each te s t (drug concentra tion) th is number o f data

po in ts can e a s i ly be boosted to 200 or more. I t takes two days to

obta in f in a l r e s u l ts . Thus, fo r each in d iv id u a l , a large number o f

data po ints are qu ick ly accessib le. In con tras t, 10 ml o f p la te le t

r ic h plasma from a s in g le ind iv id ua l allows fo r 25 aggregation te s ts ,

at most. Whole blood aggregation, however, requires no preparative

stage and uses only 12 ul per t e s t . Although p la te le t function te s ts

(aggregation, shape change) are u l t im a te ly "the bottom l in e " w ith

regard to drug a c t io n , p re lim inary s tud ies w ith TXA2 release in

washed u n s t ir re d p la te le ts could help in planning approaches to

func tiona l te s ts . The same p r in c ip a l app lies to vascular t issue from

a labora tory animal. From 10 ra ts , fo r example, one can obta in as

many as 300 a o r t ic r ings (as well as la rge amounts o f t issues from

other organs). Both the large number o f samples th a t can be processed

and t h e i r homogeneity reduces in te r - and in t r a - ind iv id ua l v a r ia t io n

thereby reducing the neccessity fo r la rge numbers o f separate te s ts .

I t should be emphasised th a t although the monitoring o f

eicosanoid re lease in response to d i f fe r e n t s tim u la to rs may be

suggestive o f the s i t e o f action o f a drug, th is method is not

d e f in i t i v e . Thus, in order to r ig o ro u s ly e luc ida te s i te s o f drug

a c t io n , the present approach should be backed up w ith other

186

biochemical endpoints: v iz . PI tu rnove r, the measurement o f PKC,

phospholipase assays and Ca^^ dynamics. In th is l a t t e r con text, a

fe l lo w PhD student a t th is labo ra to ry , Ms Jasvinder G i l l , has

developed methods to inves tiga te Ca^* uptake (w ith ^^Ca^'’’ ) by human

p la te le ts and has confirmed th a t NSAIDs and m ilr inone in h ib i t

uptake (G i l l e t a l . , 1990; Jeremy e t a l . , 1993). I t should be

stressed th a t 45ca2+ uptake does not appear to r e f le c t a l te ra t io n s in

dynamics o f in t r a c e l lu la r Ca^* ( ro u t in e ly assessed using f luorescent

dyes), but ra th e r Ca^^-dependent events a t the plasma membrane (PKC

and phospholipase a c t iv a t io n , Ca%+ pumps; G i l l e t a l . , 1990; 1991).

Further work w ith ^^Ca^'*' the re fo re holds much promise as a

methodological adjunct to eicosanoid re lease and the measurement o f

other signal transduction parameters.

The va r ia t io n s in response (PGI2 re lease) to agonists by blood

vessels from d i f fe r e n t species is a lso e xp lo ita b le when studying

drugs, in p a r t ic u la r receptor an tagon is ts . For example, where one

wishes to study an adrenoceptor antagon is t, then any o f the species

inves tiga ted would be app licab le . For muscarine (a ce ty lcho line )

receptor antagonists vessels from the ra b b i t or man (but not ra t )

would be amenable. Histamine and 5-HT antagonists would have to be

stud ied in human vessels. However, recent experiments in d ica te th a t

5-HT and histamine are potent s t im u la to rs o f PGI2 re lease in the

guinea pig (Jeremy e t a l . , unpublished observations) rendering th is

species su ita b le fo r study o f antagonists o f these bioamines. On th is

p a r t ic u la r p o in t , the approach developed in th is thes is is

p a r t ic u la r ly useful in assessing drugs which are not out and out

receptor an tagon is ts . Many drugs used In the treatment o f

187

cardiovascular disease (p a r t ic u la r ly an ti hypertensive drugs) have

obvious s tru c tu ra l s im i la r i t ie s (e .g . the presence o f p y r id in e or

p irenz lp lne groups). These drugs Include phosphodiesterase

In h ib i to r s , Ca2+ channel b lockers, some ACE In h ib i to rs and ACE

antagonists. I t would be o f po ten t ia l value to form alise a study In

which drug action (using the systems described In th is th e s is )

re la te s to drug s tru c tu re . In tu rn , such a study may con tr ibu te to

drug design and the p red ic t io n o f to x ic side e f fe c ts .

The ex v ivo studies on vascular PGIg synthesis ( e l i c i t e d with

s t im u la to rs which act a t d i f fe re n t s i te s ) In ra ts w ith e i th e r

diabetes or po rta l hypertension also I l lu s t r a te s the po ten t ia l

app lica t ions o f the approach to study drug a c t io n . Thus, animals

could receive any given drug and poss ib le e f fe c ts a t various key

s i te s (Ca^+ channels, PKC, phospholIpase, cyclooxygenase) detected.

The methods described In th is thes is are su ita b le fo r ex v ivo studies

o f drug action o f In man using p la te le t TXA2 release ( e l i c i t e d by a

ba tte ry o f s t im u la to rs ) as an endpoint. In th is l a t t e r experimental

approach. I t would perhaps be pre fe rab le to study release not only In

washed p la te le ts but also In p la te le t r ic h plasma and whole blood. By

using these d i f fe r e n t p la te le t preparations one could obta in

Inform ation on a) the amount o f drug taken up by the p la te le t , b) the

re la t iv e Impportance o f drug bound to plasma pro te ins and c) drug

e f fe c ts on In te ra c t ions o f p la te le ts w ith other blood c e l ls

(leucocytes and e ry th ro c y te s ) .

In the studies on experimental models o f diabetes and porta l

hypertension, marked a l te ra t io n s In receptors and signal transduction

mechanisms linked to PGI2 re lease became apparent. Using a ba tte ry o f

188

s tim u la to rs i t was possib le to detect not only changes in the enzymes

involved in the synthesis o f PGI2 but a lso l inked systems (receptors,

Ca2+, PKC). In the case o f po rta l hypertension, the conclusions

derived from th is study ( th a t there are a l te ra t io n s o f Ca2+

m o b il isa t ion and PKC a c t i v i t y in periphera l vasculature) were borne

out by studies ca rr ied out on c o n t r a c t i l i t y o f a o r t ic r ings in organ

baths (Karatapanis e t a l . , 1993). I t i s also apparent from these

exeperiments th a t a reduction (or increase ) in PGI2 can be a due to

a l te ra t io n s o f receptors and signal transduction mechanisms although

there are no changes in synthesis ing enzymes. The method is

app licab le in other experimental models. For example, in models o f

a therosc leros is (e .g . hyperlip idaemic Watanabe ra b b i ts ) , where c e l l

p r o l i fe ra t io n is a key event in atherogenesis, i t would be

in te re s t in g to monitor a l te ra t io n s o f PKC a c t i v i t y (v ia phorbol ester

stimulated PGI2 re lease) since PKC co n tro ls c e l l p r o l i f e r a t io n . PKC

and Ca^+ are also involved in the re lease o f mitogens and

vasoconstr ic tors from macrophages and p la te le ts . Using a ba tte ry o f

s t im u la to rs one could a lso look a t the re lease substances o r ig in a t in g

from these c e l ls taken from a th e ro s c le ro t ic animals. A s im i la r

approach could be applied to animal models o f hypertension,

homocysteinaemia, c ig a re t te smoking, ageing and gender. Furthermore,

should defects be detected in these models (as in ra ts w ith diabetes

and porta l hypertension ) i t would be fe a s ib le to use these systems

to inve s t iga te the e f f ic ia c y o f novel the rapeu tic agents. Put another

way, the potency and e f f ic a c y o f drugs could be defined using

norm alisation o f receptor (and signal t ra n sd u c t io n )- l in ke d eicosanoid

re lease by vessels and blood c e l ls a lte re d by experimental

189

patho logies. F in a l ly , the present methods are also app licab le in the

development o f improved preservation so lu t ions used in organ

tra n sp la n ta t io n . On th is general theme, these methods could also be

used to assess v i a b i l i t y o f blood vessels which have been

c ryogen ica lly frozen and then thawed fo r im p lan ta t ion . Thus,

improvements in cryogenic methods could be made using recep to r- l inked

PGI2 release as an endpoint fo r monitoring t issue in te g r i t y . This

area is o f c l in ic a l importance since vessels from donors can be

c ryogen ica lly stored and then re tr ie ve d fo r im planta tion in

re c ip ie n ts (e .g . in pa tien ts undergoing coronary a r te ry replacement)

w ith a s im i la r t issue type . I t i s hoped by the author o f t h is thes is

to address these p o s s ib i l i t ie s in the near fu tu re .

1 9 0

PUBLICATIONS DERIVED FROM WORK PRESENTED IN THIS THESIS (copies of f i r s t pages of publications have been placed In the Inside cover of th is thesis)

A) PEER REVIEWED PAPERS

1) Oeremy OY, Dandona P. E ffe c t o f endothelium removal on s t im u la to ry

and in h ib i to ry modulation o f r a t a o r t ic p ros tacyc lin synthesis.

Br 0 Pharmacol. 1989; 96: 243-250.

2) Jeremy JY, M ik h a i l id is DP, Dandona P. Muscarinic s t im u la t io n o f r a t tracheal prostanoid synthesis: calcium dependency and e f fe c t o f co r t ic o s te ro id s and c ig a re t te smoke.Eur J Pharmacol. 1989; 160: 107-115.

3) Jeremy JY, M ik h a i l id is DP, Dandona P. D i f fe re n t ia l in h ib i to r y potencies o f non-stero ida l antiin flam m atory drugs on smooth muscle prostanoid synthesis. Eur J Pharmacol, 1990, 182; 83-89.

4) Stansby G, Shukla N, Hamilton G, Jeremy JY. Comparison o f prostanoid synthesis in cu ltu red human vascular endothe lia l c e l ls derived from omentum and um bilica l ve in .Eur J Vase Surgery. 1991; 5: 501-506.

5) Jeremy JY, Stansby G, F u l le r G, Roll es K, Hamilton G. E ffe c t o f cold storage o f blood vessels in preservation so lu t ions used in organ tra n sp la n ta t io n on p ros tacyc l in synthesis .T ransp lanta tion . 1992; 53: 999-1002.

6 ) Jeremy JY, Thompson OS, M ik h a i l id is DP. D i f fe re n t ia l changes o f prostanoid synthesis by the g a s tro in te s t in a l t r a c t , mesenteric vasculature and hepatic po rta l vein o f d ia be t ic r a ts : comparison between p a ir and ad l ib i tu m feeding. In Vivo; 1992; 6 : 635-640.

191

7) Oeremy OY, G i l l 0, M ik h a i l id is DP. E f fe c t o f m ilr inone on human p la te le t cAMP phosphodiesterase, i lo p ro s t-s t im u la te d cAMP, thromboxane 2 generation and calcium uptake.Eur 0 Pharmacol; 1993. In press.

8 ) Karatapanis S, Stansby G, Roll es K, McCormick A, Oacobs M, Oeremy

OY. E ffe c t o f l i v e r and kidney preservation so lu t ions on

con trac tion and re la xa t io n and p ros tacyc lin production by the

ra b b i t aorta , in v i t r o . 0 Hepatol. 1993, In press.

9) Oeremy OY, Thompson OS, M ik h a i l id is DP. D i f fe re n t ia l changes o f adrenoceptor and muscarinic re ce p to r- l in ke d p ro s ta cy lin synthesis in the aorta and u r in a ry bladder o f d ia b e t ic ra ts .B r i t 0 Pharmacol. 1993 In press.

10) Oeremy OY, M ik h a i l id is DP, Karatapanis S, Stansby G, Roll es K, Oacobs M, MacIntyre N, Burroughs A, McCormick A. A ltered p ros tacyc lin synthesis by aortae from hepatic po rta l ve in - cons tr ic ted ra ts : evidence fo r e f fe c ts on p ro te in kinase C and calcium. 0 Hepatol, 1993 (subm itted).

11) Oeremy OY, S ifa k is G, G i l l 0, Kontoghiorghes G. Copper che lators i n h ib i t cyclooxygenase and lipoxygenase a c t i v i t y in human p la te le ts . FEBS L e tte rs . 1993 (subm itted).

12) Barradas MA, O’ Donoghue S, Oeremy OY, Mikhail ids DP. E ffe c t o f m ilr inone on human p la te le t shape change, aggregation and thromboxane ^2 synthesis: an in v i t r o study. Thromb Haem. 1993: submitted

B) BOOK CHAPTERS, REVIEWS AND EDITORIALS

1) Oeremy OY, M ik h a i l id is DP. Vascular and p la te le t eicosanoids,

smoking and a th e ro sc le ro s is . In : Tobacco Smoking and A therosc le ros is . (0. Diana, ed .) Adv Exp Med B io l . Plenum Press, New York, London. 1990; volume 273: 135-146.

19 2

2) Oeremy OY, M ikh a il id is DP. Smoking and vascular prostanoids: relevance to the pathogenesis o f atheroma and thrombosis. 0

Smoking Related D isorders. 1990; 1: 59-79.

3) Oeremy OY, M ik h a i l id is DP. NSAID e f f ic a c y and side e f fe c ts : are

they wholly prostaglandin mediated? 0 Drug Develop. 1990; 3: 3-4.

C) ABSTRACTS

1) Oeremy OY, S ifa k is G, G i l l 0, Kontoghiorghes G. Copper che la tors in h ib i t lipoxygenase and cyclooxygenase a c t i v i t y by washed human p la te le ts , in v i t r o . Br 0 Pharmacol: 1992; 105: 51P

2) Stansby G, Oeremy OY, F u l le r 8 , Roll es K, Hamilton G. Vascular p ros tacyc lin production a f te r co ld storage in organ preservation so lu t io n . Cryobiology. 1991; 28: 520.

3) Oeremy OY, G i l l 0, M ik h a i l id is DP. E ffe c t o f m ilr inone on cAMP phosphodiesterase, i lo p ro s t-s t im u la te d cAMP, thromboxane

synthesis and calcium uptake. P la te le ts . 1992; 3: 111

4) Stansby G, Oeremy OY, F u l le r B, Roll es K, Hamilton G. E ffec ts o f cold storage in organ preservation so lu t ions on in v i t r o vascular p ros tacyc lin synthesis. Br 0 Surg. 1991; 78: 1506.

5) Oeremy OY, Thompson CS, M ik h a i l id is DP. Opposite e f fe c ts on adrenoceptor and muscarinic recepto r-1 inked p ros tacyc l in synthesis in the aortae and bladders o f d ia b e t ic ra ts . Br 0 Pharmacol. 1992; 107: 89P.

1 9 3

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Effect of endothelium removal on stimulatory and inhibitory modulation of rat aortic prostacyclin synthesisU.Y. Jeremy & P. Dandona

Metabolic Unit, D epartm ent of Chemical Pathology and H um an Metabolism, Royal Free Hospital and School of Medicine, London NW3 2QG

1 Removal of the endothelium (DE) enhanced the in vitro release of prostacyclin (FGI^) by rat aortae in response to adrenaline (Ad), noradrenaline (NA), throm boxane A j analogue U46619, phorbol dibutyrate (PDBU) and sodium fluoride (NaF) when assessed at 3 h post DE. At 6h post DE, there were no differences between the dose-response curves obtained from aortic rings with or w ithout endothelium.2 At 3h post D E the antagonism of Ad- and NA-stimulated PGI^ synthesis by yohimbine and prazosin, and NA-stimulated P G I; synthesis by nifedipine was markedly reduced in aortae w ithout eudothelium when compared with controls. These eflects were reversed by protracted incubation of aortic tissue post D E (6h and 9h).3 Acetylcholine, carbachol, substance P and nitroprusside were w ithout effect on de novo or NA- stim ulated P G I; synthesis, whether or not the endothelium was present and irrespective of incubation time, post-DE.4 These results indicate that: (a) P G I; synthesis linked to excitatory receptors («-adrenoceptors, throm boxane Ai) and associated systems (G proteins, protein kinase C) in the sm ooth muscle com ponent of the rat aorta is not influenced by endothelium-derived relaxing factor (ED RF); (b) the changes of response to stim ulators and inhibitors of P G I 2 synthesis may be due to an increased reactivity of the vessels caused by the traum a of D E; and (c) vasodilators (parasympathomimetics, substance P and nitroprusside) tha t do not act directly on excitatory receptors do not influence P G I 2 synthesis.

Introduction

Removal of the endothelium (DE) of isolated blood vessels results in the abolition of relaxation elicited by vasodilators (Furchgott, 1983) and in enhanced responses to contractile agonists (e.g. Egleme et a i,1984). These observations have led to the concept that vascular endothelium produces a substance (endothelium-derived relaxing factor; EDRF), the release of which is obligatory in mediating the action of vasodilators (Furchgott, 1983).

It has been dem onstrated that vasoconstrictors («- adrenoceptor agonists, throm boxane A2 analogue U46619, fluoride, phorbol ester; G odfraind et a i, 1982; Coleman et a i, 1980; M artin et a i, 1986; D anthaluri & Deth, 1984) also stim ulate the synthesis of the endogenous prostanoid, prostacyclin (PG I2 ), by isolated vascular tissue (Stewart et a i, 1984; G olub et a i, 1985; Jeremy et a i, 1985a,b;

' Author for correspondence.

1986a; Jeremy & D andona, 1987; 1988). Since P G I 2

is both a vasodilator (at low concentrations; M oncada & Vane, 1979) and a vasoconstrictor (at high concentrations; Van Dam et a i, 1986), and there is a direct relationship between vascular contractility and P G I 2 synthesis, the pharmacological properties of blood vessels, in vitro, following removal of the endothelium may be due, in part, to changes in endogenous P G I 2 synthesis. T o explore this possibility, the effect of D E on a range of known stim ulators of ra t aortic P G I 2 synthesis (adrenaline, noradrenaline, throm boxane A 2 analogue U46619, phorbol dibutyrate [PD B U ] and sodium fluoride [N aF ]; Jeremy et a i, 1985a,b; Jeremy & D andona, 1987; 1988) and known vasodilators (yohimbine, prazosin and the calcium channel blocker, nifedipine; Jeremy et a i, 1985a; 1986a) on rat aortic P G Ij synthesis was investigated. Since acetylcholine (ACh) and substance P are established stim ulators

The Macmillan Press Ltd 1989

r.uropean Journal of Pharmacohg}', 160 (1989) 107-115 F.lscvicr

107

UJI» 50607

O'

Muscarinic sliniulalion of prostanoid synthesis by the isolated rat trachea: calcium dependency and effect of cortisol and cigarette smoke

Jamie Y. Jeremy, Dimitri P. Mikhailidis and Paresli Dandona *Meiabolic Uni(, Department of Chemical Patholv^' and Human Metabolism, Royal Free Hospital and School of Medicine,

London, N W 3 2QG, U.K.

. B

Received 29 August 1988, revised MS received 27 September 1988, accepted 18 October 1988

Tracheal prostanoid synthesis was stimulated by parasympathomimetics: a reco lin O carbachol = mcthacholine > acetylcholine » arccaidine. McNA343, dimethyl phenyl pipcrazinium (DMPP), nicotine, potassium and isoprcnalinc were without effect. Prostanoid synthesis was also stimulated by Ca^’’’ ionophore A23187 and arachidonic acid (AA), Carbachol-stimulated prostanoid synthesis was inhibited by cholinergic antagonists (atropine > ipratropium bromide

gallamine > pircnzepine); adrenaline and isoprenaline were without effect, Carbachol-stimulated prostanoid synthesis was also inhibited by the C a^Cdiannel blockers, nifedipine, diethylstilboestrol and TMB-8. Hydrocortisone and betamethasone inhibited carbachol- and A23187-stiniulated, but not AA-stimulatcd, prostanoid synthesis following an 18 h tissue culture. Cigarette smoke extracts had a biphasic effect on carbachol-, A23187- and AA-stimulatcd prostanoid synthesis (potentiation at low concentrations, inhibition at high concentrations of extracts). These data demonstrate (I) that rat tracheal prostanoid synthesis is stimulable by activation of muscarine receptor-linked Ca^* mobilisation, and (2) (hat tracheal prostanoid synthesis may be involved in secretion of mucus, the disruption of which by cigarette smoking may be related to the pathophysiology of airway disease.

Trachea; Cigarette smoke; Prostaglandin Prostaglandin p 2 „; Prostaglandin E^; Thromboxane A %; Cortisol;Muscarinic stimulation; Ca^^

& •1 ■

1. In lroducdori

In vascu lar, u rin a ry b lad d er and penile tissue it lias been d em o n s tra ted th a t those agonists w hich elic it c o n trac tio n (o r erec tion ) a lso stim u la te the syn thesis o f endogenous p ro s tan o id s th ro u g h m o b ilisa tio n of a com m on ac tiva to r ca lc ium (S tew art e t al., 1984; Jerem y et al., 1985a,b; 1986a,b,c; G o lu b e t al,, 1985). F u rth e rm o re , the ac tio n s o f these ag o n is ts w ere blocked by specific recep to r an tag o n is ts and w ere d ep en d en t on ca lc ium m ob ilisa tio n (S tew art el al., 1984; Jerem y

To whom all correspondence should be addressed: Director, Metabolic Unit, Royal Free Hospital, Pond Street, London, NW 3 2QG, U.K.

et al., 1985a,b; 1986a,b,c; G o lu b el al,, 1985), It w as therefo re p ro p o sed th a t the con co m itan t syn thesis o f p ro s tan o id s on the in itia tio n o f recep to r-linked c o n trac tio n m ay p lay a ro le in th e su b seq u en t c o n tr a c tio n /re la x a tio n phases o f sm oo th m uscle and e re c tio n /d e tu m e sc e n c e o f the pen is (Jerem y e l al,, 1985a,b; 1986a,b,c). T h e question a lso a ro se as to w hether c o n trac tile agonism s tim u la tes p ro s tan o id syn thesises in o th e r sm oo th m uscle tissues (Jerem y el al., 1985a,b ; 1986a,c).

It w as therefo re o f in te rest to investiga te the trachea o f the ra t since th is tissue syn thesis p ro s

tano id s (A lly ct al,, 1982) an d its co n trac tio n is elic ited by p a rasy m p a th o m im etic s (F ro ssa rd an d

L and ry , 1985), A n in v itro system for the stu d y o f ra t tracheal p ro s tan o id syn thesis w as therefo re

0014-2999/89/S03.50 O 1989 Elsevier Science Publishers D.V. (Biomedical Division)

Br. J. Pharmacol. (1993). 000, 0 00 -0 0 0 © Macmillan Press Lid. 1993

Differential changes of adrenoceptor- and muscarinic receptor- linked prostacyclin synthesis by the aorta and urinary bladder of the diabetic rat

'J.Y. Jeremy, C.S. Thompson & D.P. Mikhailidis

Departm ent o f Chemical Pathology and H um an M etabolism , Royal Free Hospital and School o f M edicine, University o f London, Pond Street, London N W 3 2Q G

1 The effect o f experimental diabetes m ellitus (D M ; hypcrglycaemic, non-ketototic; 2 months duration) in the rat on receptor-linked prostacyclin (P G Ij) synthesis (measured as 6-o x o -P G F i, by radioimmunoassay) was studied in the aorta and urinary bladder using adrenaline, angiotensin I I ( A l l ) and acetylcholine (A C h ). Signal transduction systems were studied via stim ulation o f P G Ij synthesis w ith phorbol ester dibutyrate (P D B U ; a protein kinase C activator [P K C ]), Ca^* ionophore A 2 3 I8 7 (A 2 3 I8 7 ) and thapsigargin (both elevate intracellular Ca^^, activating phospholipase A ; [PLA^]) and arachidonate (A A ; substrate for P G Ij synthesis).

2 In response to adrenaline. A l l and phorbol ester, aortic P G Ij release was m arkedly reduced (all > 7 5 % ) in diabetic rats compared to controls. EC%s o f the dose-response curves fo r adrenaline. A l l and P D B U were also m arkedly increased in aortae from D M rats compared to controls. A lthough there was decreased output o f P G Ij in response to A 2 3 I8 7 by aortae from diabetic rats compared to controls, there was no difference in the E C ^s (mean ± s.e.mean; diabetic, 2.7 ± 0.2 X I0 ~ ‘ M ; controls2 ± 0.18 X I0 " ‘ M ) . There were no differences in P G Ij release (or in the EC%s) in response to thapsigargin or A A between aortae from diabetic and control rats.

3 In the urinary bladder, there was a m arked increase in P G Ij output in response to A C h and a m arked decrease in EC%s for the A C h -P G Ij dose-response curves in diabetic rats (E C 50 = 5.8 ± 0.32 X 10'^ M ) compared to controls (EC% = 2.2 ± 0.15 x 10"* M ) . A lthough there was an increase in P G Ij output in the urinary bladders from diabetic rats in response to A 2 3 I8 7 , there were no differences in the EC^s (control, 1.8 ± 0.2 x 10"* M ; diabetic, 1.1 ± 0.15 x 10"® M ) . In the urinary bladders, there were no differences in P G Ij output (or the EC%s) in response to P D B U , thapsigargin or A A between diabetic or control rats.

4 These data indicate that: (i) reduced P G Ij synthesis coupled to adrenoceptors and A l l receptors in the aortae o f diabetic rats m ay be due to diminished P K C activity and no t to changes in receptor density and/or a ffn ity , Ca^*' stores, P L A j, cyclo-oxygenase or P G Ij synthase; (ii) the diam etrically opposite effect o f D M on A Ch-stim ulated P G Ij synthesis is not due to an increase in P K C activity, but possibly to an increase in muscarine receptor number and/or affinity; (iii) changes in receptor-linked P G Ij synthesis are not ubiquitous in experim ental D M and may be organ-specific.

Keywords: Prostacyclin; diabetes mellitus; rat aorta; rat urinary bladder; adrenoceptors; muscarinic receptors

Introduction

Patients w ith diabetes mellitus (D M ) are at greater risk than Jeremy el al., 1988a) an effect which in turn is m ediated by Gthe healthy population o f developing cardiovascular disease proteins, protein kinase C (P K C ) and inositol trisphosphate(C V D ; Banga & Sixm a, 1986; M ikhailid is ei al., 1988). O f the , (IP ,; Griendling el a l., 1986; H a llam el al., 1988; Jeremy etmany aetiological factors associating C V D w ith D M , a al., 1988a). The net result o f activation o f these systems ism arked attenuation o f prostacyclin (P G Ij) synthesis by blood transmembrane Ca^* influx and an increase in intracellularvessels from diabetic laboratory animals have been reported Ca^^ ([Ca^+IJ. Ca^^ then activates phospholipase A j (P L A j)(Harrison el al., 1978; Rogers el al., 1981; Jeremy el al., , which in turn liberates P G Ij substrate, arachidonic acid1987a). A p a rt from being a vasodilator and inhib itor o f i (A A ), from phospholipid stores (Irv in e , 1982). In this con-platelet aggregation (M oncada & Vane, 1979), P G Ij ' text, experimental D M in the rat is associated w ith alteredmodulates other vascular functions including cell proliféra- , receptor-linked functions in vascular tissue (Tom linson el al.,tion (H u ttn cr el al., 1977; M a ru i el al., 1990; Pom erantz & ' 1992).

Ç H a jja r, 1989) and metabolism o f cholesterol (H n m reE P s W ., Tlius, in order to elucidate further the mechanisms that In turn, disruption o f these processes have all been | determine diminished vascular P G Ij synthesis, the effects o f a

implicated in the aetiology o f C V D , in particular, range o f stimulators that act at different sites in the receptoratherogenesis and thrombosis (Jeremy & M ikhailid is , 1990). activation -P G Ij synthesis pathw ay were studied on P G Ij syn-

\ ) T lie precise mechanisms that determine reduced vascular thesis by the streptozotocin-induced diabetic rat: adrenaline P G Ij synthesis are not clearly defined, although a reduction (mediated by heterogeneous a-adrenoceptors; Jeremy ci al.,in substrate (arachidonate) levels in blood vessels has been ’ 1985a) and angiotensin I I (A l l ; Hassid & W illiam s, 1983;suggested (H o lm an el al., 1983; Jeremy ei al., 1987a; D ang el Stevens el al., 1992), phorbol ester dibuytrate (P D B U ; aal., 1988). It has also been established that vascular P G Ij P K C activator: N ish izuka, 1984; Jeremy & D andona, 1986),synthesis is stimulated by vasoactive agonists (e.g. thapsigargin (elevates intracellular C a ’ ^; Thastrup el al.,noradrenaline, histamine, acetylcholine, angiotensin 11; 1990; Levine & W atanabe, 1991), C a** ionophore A 23187

(creates artificial C a ’ channels; Reed & Lardy, 1972) and-------------------------------------------------------------------------------------------------------- arachidonic acid (A A ; substrate fo r P G Ij synthesis). In order' Author for correspondence. to examine whether other non-vascular smooth muscle tissues

Journal of Hi:paioloi>y, (1993)© 1993 Elsevier Scicnlific Publishers Ireland Ltd. All rights reserved. 0I68-8278/93/S06.Ü0

IIE P A T 01411

U N C O R R EC TtD PROOf-EC.rtliO''

p.r.o.

Effect o f hypothermic storage in liver allograft preservation solutions on vasoactivity and prostacyclin synthesis by the rabbit aorta,

' q g f a W Irfe x t'- l N 6 i- lyr.A '9 '

S. Karatapanis\ M. Jacobs’’, P.À. McCormick^ G. Stansby^ K. Relies^ N. McIntyre\A.K. Burroughs'* and J.Y. Jeremy*’

c T Â M i e"Hrpalobiliary and Liver Transplantation Unit. ^Department of Pharmacology, '^Department of Surgery. “ Department of Chemical Pathology. Royal

Free Hospital and School of Medicine. London, UK

(Received 27 April 1992)

Prim ary graft dysfunction fo llow ing o rth o p tic liver tran sp lan ta tion has been ascribed to th rom bo tic and ischemic com plications in a high p ro p o r tio n o f cases. It has been suggested th a t hypotherm ic sto rage o f livers in preservation solu

tions elicits dam age to the vascu lar endothelium . Since the endothelium co n tro ls vasoactiv ity and hem ostasis via

release o f endo thelium derived relaxing fac to r (E D R F ) and prostacyclin (P G I 2 ), s to rage in jury to the endothelium m ay predispose the a llog raft to th rom bosis, ischem ia and im paired perfusion . In rd e r to test this, the effect o f longterm hypotherm ic sto rage in m odified U niversity o f W isconsin solu tion (U W ), k idney perfusion so lu tion (KPS) and

m inim um essential m edium (M E M ) on phenylephrine (PE )-stim ulated co n trac tio n and acetylcholine (ACh)- stim ulated relaxation , as well as P G I 2 release by rab b it ao rtic rings was investigated . Follow ing cold storage for 24,

48 and 72 h, PE and A C h dose response curves were unafTected by storage in M E M , U W o r K PS. Follow ing hypother

mic storage for 24 h and 48 h, P G I 2 release (stim ulated w ith PE, A Ch, a rach id o n a te , fluoride, calcium ionophore and

p h o rbo l ester) was n o t significantly a ltered from zero time reponses. T hese results dem o n stra te th a t hypotherm ic

sto rage o f rabb it ao rtic rings in b o th U W and K PS do no t influence two key endo thelia l functions (the release o f

E D R F or P G I 2 ) w hich in tu rn ind icates th a t endothelia l dam age associated w ith reperfusion follow ing hypotherm ic

sto rage is no t camsally re lated to a lte ra tio n s in E D R F and P G I 2 release.

Key words: PL EA SE S U P P L Y 3 - 6 K E Y W O R D S FO R IN D E X IN G ^

Prim ary graft dysfunction , follow ing o rth o p tic liver

tran sp lan ta tio n , occurs in a b o u t 10% o f cases (1 -5 ) and

is a m ajor cause o f m orb id ity an d m orta lity . T he cause

o f prim ary graft dysfunction is m ultifac to ria l bu t 's to rag e in ju ry ’ may be an im p o rta n t co n tr ib u to ry factor in a subset o f patien ts (6). S tud ies using ra t livers have dem onstra ted significant m orpho log ica l dam age to endothelia l cells and K upffe r cells occurs follow ing reperfusion after hypo therm ic sto rage o f the organs in E uro-C ollins so lu tion an d to a lesser degree in U niversi

ty o f W isonsin (U W ) so lu tion (7,8). T he functional

significance o f these m orpho log ica l changes is un-

Correspondence to: J.Y. Jeremy, Department of Chemical Pathology, Royal Free Hospital and School of Medicine, London, UK.

fA lC H A t 'L

know n. H ow ever, the vascu lar endothelium controls

hem ostasis and vasoactiv ity , in p a rt, th rough the secre

tion o f p rostacyclin (P G I 2 ; inh ib its p latelet aggregation

and dilates vessels ( 9 - 11)) as well as endothelium - derived relaxing fac to r (E D R F ; is ob liga to ry for relaxation and also inh ib its p la te le t activ ity (9 -11 )). D am age to the endothelium an d d is ru p tio n o f E D R F and P G I 2

may therefore resu lt in vasoconstric tion o r throm bosis, which in tu rn m ay prejud ice g raft function . Indeed there

is a considerable body o f evidence th a t ischemic injury

is a m ajor cause o f early g raft failures (1 -5 ).

We have previously repo rted th a t cold preservation o f

ra t aortic rings h ad significant effects on vascular func

tion and th a t the effects differed w ith the preservation

solution used (1 2 -1 4 ). R a t ao rtic rings stored for up to

p . r i l b t H M c ^ o k M i C r i ) ^ S T a n s k y

X Repair îs. C.S.. nposium

DRUGS OF TODAY 1992. 28(Suppl. A): 35-43

INHIBITION OF CYCLOOXYGENASE AND LIPOXYGENASE ACTIVITY BY IRON CHELATORS: POSSIBLE USE IN THE TREATMENT OF

EICOSANOID-RELATED DISORDERS

J. Y. Jeremy*, J. Gill, T Prior, G. Sifakis, M.A. Barradas and G.J. Kontoghiorghes^

Department of Chemical Pathology and Human Metabolism and 'Dept, of Haematology. Royal Free Hospital. Pond St.. London NW3 2QG, UK

In troductionApart from the treatment of iron overload in tha

lassemia. oral iron chelators have other potential clinical uses where iron-containing enzymes regulate metabolites which contribute to the pathogenesis of disease. Two major iron-containing enzymes are cyclooxygenase (COX) and lipoxygenase (t_OX), which generate potent bioactive sutistances (viz. prostaglandins [PGs]. thromboxanes (TXs) and leukotrienes (LTsj). The activity of these enzymes has been widely implicated in the etiology of clinical disorders ranging from inflammatory to cardiovascular disease. Recent studies have also established that iron chelators inhibit the activity of COX and LOX in wfro (1. 2). The present paper examines the role of iron in mediating the activity of COX and LOX and summarizes the role of these enzymes in the pathophysiology of disease. In particular, our experimental findings on the effect of iron chelators on COX and LOX are reviewed. Iron status in relation to disorders associated with eicosanoids and the possible use of iron chelators in treating these disorders is also covered.

Eicosanoid SynthesisEicosanoid is a generic term for any metabolite

derived from the essential fatty acids (principally arachidonic acid: AA). AA is stored as an ester in the

'Correspondence

phospholipids of biomembranes and is released in response to specific extracellular signals e.g. in vascular smooth muscle, noradrenaline; in platelets, collagen; in white cells, cytokines (3, 4). AA is liberated by the hydrolytic action of phospholipase Ag (PLAg). an event mediated by activation of G proteins, protein kinase C and calcium mobilization (Fig. 1 ). Liberated AA can then be converted to PGs, TXs. LTs. hydroxyeicosatetraenoic acid (HETE) or hydroperoxyeicosatetraenoic acid (HPETE), depending on the cell type and the presence of the enzyme which generates these metabolites (Fig. 1). For example, the principal AA metabolite in vascular smooth muscle is prostacyclin (PGIg); in platelets. TXA2 and in leukocytes, LTs, HETE and HPETE (Fig. 1.).

COX is a haem containing enzyme which firstly elicits the abstraction of an hydrogen atom from AA, forming a radical structure (5, 6) followed by the incorporation of two oxygen molecules (Fig. 2). Acyclic molecule is produced, PGG, which under the peroxi- dative activity of COX is reduced to form an endoperoxide PGH, the immediate precursor for prostanoid and thromboxane synthesis, depending on the synthase/isomerase present in any given tissue (Fig. 2).

Lipoxygenase is also an iron (non-haem) containing enzyme (7-9). LOX again abstracts a hydrogen atom and incorporates an oxygen molecule into fatty acids at the C12.C5 or C15 position, producing hydroperoxyeicosatetraenoic acid (HPETE; Fig. 2). HPETE can either be converted to leukotrienes by LT synthases (in leukocytes) or spontaneously degrade

" . T: T/.'

JEiiroptan Journal of Pharmacology - Molecular Pharmacology Section,O 1993 Elsevier Science Publishers B.V. All rights reserved 0922-4106/93/S06.00

EJPMOL 90423

6 7 ^ 9 3ion, 00 (1993) E3M 904»-----------

Effect of milrinone on thromboxane synthesis, cAMP phosphodiesterase and uptake by human platelets

J a m ie Y . J e re m y , J a s v in d e r G ill a n d D im itr i M ik lia il id is

Department of Chemical Pathology and Human Metabolism, Royal Free Hospital and School of Medicine, London AOPJ 2QG, UK

Received 8 September 1992, revised MS received 11 November 1992, accepted 22 December 1992

Th e phosphodiesterase inhibitors milrinone and isobitylmethybcanthine ( IB M X ) inhibited the conversion o f p H ]c A M P to

p H lA M P by washed human platelets in concentration-dependent manners ( IC ^ : m ilrinone, 2.6 X 10 '® M ; IB M X , 4.6 X 10"^ M ). M ilrin one and IB M X increased cA M P levels when stim ulated by a single concentration (0.3 /x M ) o f iloprost. EC;,,: m ilrinone, 5.6 X 10"^ M ; IB M X , 3 X 10“ M . M ilrin one was a potent inhibitor of platelet thromboxane A ; (T X A ^ ) synthesis when stimulated by m edian stimulatory doses of collagen (ICgq: 3 X 1 0 " ’ M ), sodium fluoride (N a F ) (a non-specific G protein activator; IC ^ : 3 X 10“ M ) and phorbol ester myristate acetate (P M A ) (a protein kinase C activator; IC jg : 2.2 X 10~^ M ). In contrast, at m edian stimulatory doses o f A23187 and arachidonate there was a marked decrease in the potency of milrinone in inhibiting T X A j synthesis. M ilrinone had a weak inhibitory effect on T X A ; synthesis when elicited by freeze fracturing. In all experiments IB M X was a weaker inhibitor o f T X A ^ synthesis, although the general pattern o f effects was similar to m ilrinone. M ilrin one inhibited both collagen- and adrenaline-stim ulated “^Ca^^ uptake by human platelets in dose-dependent manners. Since p latelet T X A ; synthesis is dependent on Ca^^, and m ilrinone inhibited uptake, it is concluded that m ilrinone

- y y exerts its inhibitory effect on platelet activity, principally through an action on Ca^^ m obilisation /b in d in g to effector proteins ^ (protein kinase^jand/or phospholipase A%).

Platelets; M ilrinone; Phosphodiesterase; Tliromboxane A ;

1. In tro d u c tio n

M ilrinone (2-niethyl-5-cyano-(3 ,4 '-b ipyrid in)-6(lH )- o ne) is a non-glycoside bipyrid ine derivative tha t has positive ino trop ic , lu sitrop ic and vasodilating activity (A lousi e t al., 1983; B orrow et al., 1985; Jaski e t al.,1985). M ilrinone has proved effective in the tre a tm en t o f acu te h e a rt fa ilu re (D iB ianco et al., 1989; P acker e t al., 1991). M ilrinone probably exerts its beneficial ac tion principally via inh ib ition o f cyclic ad en o sin e -3 '5 '- m o n o p h o sp h a te (cA M P ) phosphod iesterase (P D E ), the reby eleva ting in trace llu la r cA M P concen tra tions an d re d u c in g in tra c e llu la r c o n c e n tra tio n s(M ylo tte e t al., 1985; E arl et al., 1986; O lson e t al., 1987). A m rinone , a sim ilar d rug to m ilrinone, has been show n to inh ib it aggregation by rabbit and hum an p la te le ts (T an g and From jm ovic, 1980; L ipp ton e t al., 1985; P a ttison e t al., 1986; Sim pson et al., 1988; L ind- g ren e t al., 1990). C learly , the inhibition o f p la te le t

Correspondence to: Jamie Y. Jeremy, Department of Qiemlcal Pathology and Human Metatnjlism, Royal Free Hospital and School of Medicine, Pond St., London NW3 2QG, UK.

activity in pa tien ts w ith card iac failure by m ilrinone w ould constitu te a desirab le add itiona l p roperty since:(a) p la te le ts are key com p o n en ts in th rom bus fo rm ation (C hesterm an and B ernd t, 1984) and pa tien ts with h e a rt failure have an increased risk of developing th rom botic episodes if ischaem ic h ea rt d isease is involved (M ikhailidis e t al., 1990; T rip e t al., 1990); and(b) p la te le t re lease substances (viz. throm boxane A ; ) a re p o ten t vasoconstrictors (as well as being proaggregatory) and as such have been widely im plicated in the aetio logy o f card iovascu lar d isease (M ikhailidis e t al., 1987, 1990).

In light o f the above we fu rth e r explored the effects an d p roperties o f m ilrinone on various aspects of hu m an p la te le t function, in vitro. Firstly, cA M P phosphod ies te ra se activity was assessed by studying the conversion o f p H ]cA M P to p H jA M P and iloprost (a p ro s ta cyclin analogue) -stim ula ted cA M P synthesis. It has also been suggested th a t the effects o f m ilrinone-type d rugs on p la te le t activity a re not m ed ia ted by inh ib ition o f P D E (Lip7ton e t al., 1985; D origo e t al., 1991). T h e effect of m ilrinone on throm boxane A ; (T X A j) synthesis was the re fo re investigated in p la te le ts w hen synthesis o f this eicosanoid was stim ula ted with colla-

W ^ I I I J I U I . . [ MlXS.b U U J l ’U l: Wed l-ciriT l3:15:44 1993 /br2/3()5/lcnm5/ljV(p-apiil93/7371-188

Effect of Liver and Kidney Preservation Solutions on Vascular Viability: Studies on Contraction and Relaxation of Rat Aorta In Vitro

s. Knralnpanis, P.A. McCormick, M. Jacobs, G. Slatisby, K. Holies, N. Mclnlyre, A.K. Burroughs, and J.Y. Jeremy

I M M U N O LO C irA I. and technological dcvclopincnt.s

X have made liver tiansplanlalio ii an accepted therapy

for varions end stage and genetic defective liver diseases,'

1 he standard lcchni(|ne for hepatic allograft preservation

is hypotherm ic storage o f the liver after vascrtlar flush w ith

cold preservation solutions and package o f tire organ on ice

prior to rcirnplantalion o f the a l lo g r a f t .B e f o r e 1988, itsing cither Collirts or Euro-Collins (E C ) preservation

sohrtions, the "s a fe " storage time resulting in acceptable

graft function, was 8 hoirrs or less. ' I lo w cver, introduction

of the U niversity of Wiscortsin (U W ) preservation solution

has extended the safe storage time of livers for Itansplau-

talion to 15 hours and longer.'* I he U W solution differs

from Collins or E C solutions in that it contains the itnpcr-

rncant agcrrts raflirtosc and lactobionate, and the free

radical scavenger alloputinol.^’ The U W also differs con

sider al)ly from more conventional "physio log ical” buffers

in that it contains supraphysiological concentratiorrs of

potassium (!<)*■ and is calcium-free.'*

An im portant aspect of organ trarrsplantation is the

rnairrtcnance of patent vascular firnction in the grafted

orgart, so that its vessels do not tend toward vasocorrstric-

tion or throrrrbosis. W hen it occurs, hepatic a itc ry thiorn-

bosis is a potentially catastrophic early com plication in

liver trarrsplantatiorr, rcrpriring rctransplarrtation in most

cases.^ The vascular endothelium plays a key role in

prcv’cnting thronrbosis in that it sccietcs antithrom botic

factors, including prostacyclin (P G Ij) the most potent

inhibitor o f platelet aggregation known,'* and cndothcliurn-

derived ic laxing factor (E D R E ),'' which is ob ligatory iit

mediating the action of vasodilators artd also inhibits

[)latelct adhesion and aggregation."’ Dam age or rem oval of

the crrdothcliirrrr also restrlts in potcrrliation o f vasocon-

stric tiorr." I Inrs, structural arrd functiorral in tegrity o f the

vasculatirre o f transplarrtcd organs m ay be irrtpot tant con-

tribirtory factors to the success o f organ tr ansplantation. In

v itro studies w ith rat livers have demonstrated that after

prolonged periods o f cold storage the most vulnerable

tissues to reperfirsiorr injirry were sinusoidal endothelial

cells, and that U W elicits less m orphologic damage than

fo ftrtlher exam ine whether hypotherm ic storage in

flirsh .sohrtions inllucrtces vascular function, the effect o f long-term hypotherm ic storage (up to hours) in U W

solution as well as kidney perfusion solution (K P S ), K rc b ’s Ringer bicarbonate solution (IC R B ), and m inim um

essential mecliurrr (M E M ) on contraction and relaxation

patterns o f rat aortic rings was investigated. Contraction

was elicited w ith phenylephrine, and relaxation o f phenyl

ephrine-stimulated contractiorr w ith acetylcholine (E D R F -

rclease-dcpcnderrt). Th e effect o f cold storage in U W and

K PS on potassium chloride (K C I)-stim ulatcd contraction

was also investigated, since K C I elicits vasoconstriction

via a depolarisation mechanism, Independent o f receptor

activation and E D R F release.

MATERIALS AND METHODS

Phenylephrine bitartratc, acetylcholine chloride, and Dulbecco’s M EM were purchased from Sigma Chemical Co (Poole, Dorset,United Kingdom). I lie UW solution was made up try the pharmacy at the Royal Free Hospital (t.ondon. United Kingdom) and was comprised of (per mmol/L); K^, 125; sodium (N a )*, 25; magnesium (Mg)^ \ 5; lactobionate, 100; H ;P O /" , 25; S O /" , 5; allopiirinol, 1; reduced glutathione, 3; raffinosc, 30; adenosine 5; and p ll 7.4. The KPS contained (per mmol/L): K * , 80; Na^, 80;M g ^ \ 40; s o / " , 55; citrate, 55; mannltol, 185; and pH 7.2. The KRI3 was comprised of (per mmol/L): sodium chloride (NnCI),118; KCI. 4.8; MgSO,. 7 IljO , 1.2; K lljP O ,, 1,2; glucose, I t ; Na, ethylencdiamine tetraacctic acid, 0.034; N a llC O j, 24; CaCl,; and 2iljO , 2.5.

Male Sprague Dawlcy rats were killed by a blow to the head and the thoracic aortae excised. Adventitia and fatty tissue was

carefully rcrrroved arrd the aortae crrt into 2-rnrn rings. Aortic rings

were placed in cither U W , KPS, M E M , or KRB solution artd stored at 4"C for 0 hours, 24 hours, or 48 hours. Following storage, rings were washed in KRB and mounted in an organ bath. The bath medium was maintairred at 37"C, pi 1 7.4 and gassed with 95%Oj and 5% carbon dioxide. A resting tension of 1.5 g was applied and the aortic rings were equilibrated for 1.5 hours. Responses were recorded isonretrically through force displacement transducers. Concentration-resporrsc curves for phenylephrine and KCI were obtained by adding the drug directly to the bathing media in cumulative concentrations. Cumulative acetylcholine concentration-relaxation responses were obtained following stimulation of contraction with 10"’ mol/L phcnylp^mnc.

Calculation and Slalislical Analysis

The degree of contraction is expressed in absolute tensioir (g), and relaxation as % relaxation of phenylephrinc-stinrulatcrLcontrac-

___________From the I lepatobillary and Liver Transplanlallon UrriHK.K. and

R A M .); and the Departments of Ctiemlcal Patliology (fcwrTtS.S.,K.R., N.M., and A.K.B.) and Pharmacology (JJWrî^ o y a l Free - rHospltal and School of Medicine, London, tJnlted Kir^Jom " "<J

Address reprint requests to J.Y. Jeremy, Royal Free Hospital School of Medicine, Department of Chemical Pathology and Human Metabolism, Pond Street, Lbndon NW3 2QG, England.

© 1993 by Appleton & Lange004t-1345/93/$3.00/-t0

3046 Tinnsplnntitdon Piocecdings, Vol 25. No 2 (April), 1993; pp 3046-3050

In vivo 6: 635-640 ( 1992)

Differential Changes of Prostanoid Synthesis by the Gastrointestinal Tract, Mesenteric Vasculature and Hepatic Portal Vein of Diabetic Rats: Comparison between Pair and

ad Libitum Feeding

J.Y . JE R E M Y , C.S. TH O M PSO N and D .P . M IK H A IL ID IS

Department o f Chemical Pathology and Ifum an Metabolism, Royal Free Hospita l and School o f Medicine, Pond St., LondonNW3 2QG, England

A bstract. The synthesis o f the prostaglandins (PG) I 2 (measured as 6-oxo-PGF,a), E j, E2 J and thromboxane (T X ) A 2

(measured as TX B 2 ) by the mucosal and muscular portions o f the stomach, duodenum, jejunum , ileum, mesenteric vessels, hepatic po rta l vein and two arteries (carotid and aorta) was investigated in long term streptozotocin-induced diabetes mellitus (D M ; fed ad libitum and p a ir fed). In a ll regions o f the gastrointestinal tract there were no changes in PG synthesis (per unit weight o f tissue) in diabetic rats (pa ir fed o r fed ad libitum ) compared to controls. However, there were marked increases in PG synthesis (up to 3 fo ld ) by the mesenteric vascidature and hepatic p o rta l vein in diabetic animals fed ad libitum and in p a ir fed diabetic rats and decreases in the aorta and carotid artery. These data suggest that increases in PG synthesis by the splanchnic vasculature may constitute a specif ic adaptive response to D M . The sim ilarity o f the responses o f pa ir fed rats to those o f rats fe d ad libitum indicates that D M and not hyperphagia is the like ly determinant o f these adaptive

changes. Given that increa.sed .splanchnic blood f lo w enhances nutrient uptake (both known to occur in D M ), the increase in

splanchnic va.scular PG synthe.sis, in particu lar o f vasodilatory

P G I 2 , may contribute to enhanced nutrient uptake.

D iabetes mellitus (D M ) in man elicits gastrointestinal (G I) dysfunction including diarrhoea, constipation, delayed gastric em ptying and atony, disordered small bowel m ovem ent and atony and dilation of the colon (4). Increased blood flow in G I vessels is also associated with the early stages of DM (28). Aetiologically, these disorders have been largely ascribed to d iabetic autonom ic neuropathy (4). H ow ever, it is also well established that the pathophysiology of G I disorders in non-diabetics invovles endogenous prostaglandins (PG s; [2]). F urtherm ore, DM is associated with a m arked decrease in PG

Correspondence to: D t J .Y . Jeremy.

Key Words: Porstanoids, throm boxane, gastrointestinal tract, splanchnic vessels, arteries, diabetes, rat.

synthesis in several tissues, Including vascular sm ooth muscle, the penis, brain and platelets and as such has been implicated in the pathophysiology of disorders associated with DM in these tissues (22). W ith regard to the G I tract in D M , we are aw are of no systematic studies on PG synthesis in man or in experim ental animal modes. H owever, it has been dem onstrated that caloric deprivation results in differential changes in prostacyclin (PG I2 ) and throm boxane A ] (T X A ;) synthesis by the small intestine and m esenteric vasculature of the rat (12). Since these PG s influence m esenteric blood flow and gut motility (21), it was suggested that these changes in PG synthesis may play a role in the adaptation of the small gut in response to fasting through m odulation of gut motility and m esenteric blood flow (12). It is also well established tha t, in experim ental DM , the G I tract of the rat undergoes m arked adaptive changes of structure (mucosal and muscular hyperplasia; 13, 15) and function (increased nutrient uptake; 20,

23).T herefo re , in order to investigate a possible role for PGs in

the adaptation and pathology of the G I tract in DM , we assessed PG synthesis (P G I 2 , T X A 2 , P G E 2 , PG F 2 ,,) by the mucosal and muscular regions of the stom ach, duodenum , je junum , ileum as well as the m esenteric vasculature, and hepatic portal vein. PG synthesis by the aorta and carotid artery were also m easured to assess vascular PG synthesis in «post-hepatic» vessels. Since diabetic rats are markedly hyperphagic, we also examined the effect of pair feeding (am ount of food given to diabetic rats per day adjusted to the average daily intake of age-m atched controls) on PG synthesis by the above tissues. The effect of insulin adm inistration to d iabetic rats was also investigated.

M aterials and Methods

All experiments were carried out using male Sprague Dawley rats of an initial body weight of 251) g. Non-ketonuric, liyperglycaemic diabetes was induced by an intravenous injection of streptozotocin (65 nig.kg ' body wt.) which had been dissolved in citrate buffer (p it 4.5). Controls were injected with the same volume of citrate buffer alone. These rats developed glycosuria but not kctonuria or hacmaturia (for glucose levels

0258-851X/92 $2.00+.40 635

OJII I.lUV/'V.V.VIi).', (tîl!l!l<0,l(K)/()Ï U A N S n . A N I A I K U :

Cnpyriqlil l'V Willifims & WilkincVol. r.;i, !)');! lotvi, No M„y >

l ' i i i i /r ii III i ■ X .1

TIŒ EFFECT OF COLD STORAGE OF RAT THORACIC AORTIC RINGS IN ORGAN PRESERVATION SOLUTIONS— A STUDY 01'' RECEPTOR-LINKED VASCULAR PROSTACYCLIN SYNTHESIS

J. Y. Jeu iîm v ,''* G. S ta n s b y , D. F u li .e u , K. H o i.i.es , an d G. H a m i l t o n

Departm ent n f Chemical Pathology and H um an Metabolism and U nivers ity Department o f Surgery, Royal Free Hospita l and School

of Medicine, London N IFJ 2QG, England

A n im p o r ln n t a s p e c t o f o rig an p r e s e r v a t io n is t lie m a in te n a n c e o f in t r in s ic d i la t o r a n d a n t i th r o m b o t ic m e c h a n is m s o f b lo o d v e s s e ls . I31ood vesse ls s y n th e s iz e

p r o s ta c y c lin { P G I j ) , a p o te n t v a s o d ila to r a n d in h ib i t o r o f p la te le t a d h e s io n a n d a g p r c R a t io n . P G Iz s y n th e s is is c o n tr o l le d b y c o m p le x m e c h a n is m s in c lu d in g a d re n o c c p -

to r - l in k c d c a lc iu m in f lu x a n d p r o te in k in a s e C . S in c e o r g a n p r e s e r v a t io n s o lu t io n s m a y in f lu e n c e th e s e m e c h a n is m s , w e in v e s t ig a te d th e e f fe c t on in v i t r o P G G s y n th es is o f co ld s to ra g e o f r a t a o r t ic r in g s in la c to b io n a te - r a f f in o s e s o lu t io n (L U S ) a n d h y p e r to n ic c i t r a t e k id n e y p r e s e r v a t io n s o lu t io n ( K P S ) on in v i t r o P G G s y n tlie s is . A c u te in c u b a t io n o f a o r t ic t is s u e in b o th p r e s e r v a t io n s o lu t io n s a t 3 7 ® C (c o m p a r e d w i t h m in im a l e s s e n tia l m e d iu m ) c o m p le te ly in h ib i t e d P G G s y n th e s is w h e n s t im u la te d w i t h n o r a d r e n a l in e ( N A ) , p h o rb o l e s te r (a p r o te in k in a s e C a c t iv a t o r ) , N a F (a G p r o te in a c t iv a t o r ) , o r A 2 3 1 8 7 . F o l lo w in g s to r a g e o f a o r t ic r in g s a t ( fo r u p to 7 2 h r ) in L R S a n d K P S , s u b se cp ien t w a s h in g a n d in c u b a t io n in M E M , P G G s y n t lie s is w a s in i t i a l l y m a r k e d ly e n h a n c e d in re s p o n s e to N A w h e n c o m p a r e d w i t h tissues s to re d in M E M . T h e s e e n h a n c e d re s p o n s e s d is a p p e a re d , a n d P G G s y n th e s is r e tu r n e d to n o r m a l f o l lo w in g 1 h r in c u b a t io n o f tis s u e s in M E M a t 3 7 ° C . T h e s e d a ta d e m o n s tra te th a t c o ld s to r a g e in p r e s e r v a t io n f lu id s

e x e r ts m in im a l d e le te r io u s e f fe c ts , n o t o n ly on PCÀG s y n th e s is , b u t p o s s ib ly on o th e r k e y p ro c ess es (c a lc iu m h o m e o s ta s is , p r o te in k in a s e C a c t iv i t y ) in b lo o d vesse ls .

In clin ica l organ tra n s p la n ta tio n , h ypo therm ic storage of

organs, such as the liver and k id ney, is ro u tin e ly carried ou t by

a vascular flush w ith cold so lutions follow ed by storage in ice. U n til recently , the safe cold-storage tim e for livers used in

tran sp la n ta tio n was considered to be about 10 hr, using a

citrate-based so lution (J ), b u t the in tro d u ctio n o f new flush

solutions has increased the m ax im u m "safe" storage tim e to 15

hr and longer ( 2 ). These p reservatio n flu ids d iffe r considerab ly

from more co nventiona l “physio log ic” buffers in th a t they

contain additives sucli as lac tob ion ate , ra ffinosc, and supra- physiologic co ncentration s o f K \

One im p o rta n t aspect o f organ tra n s p la n ta tio n is th e m a in tenance o f p a ten t vascular fu nction in the g rafted organ, which

dictates th a t vessels should n o t tend tow ard vasoconstriction

or throm bosis. F u rth erm o re , since a rte r ia l throm bosis is an

in lre (|u cn t hut. p o te n tia lly catast rophic co m p lica tion o f tra n s

plant proccduies (.7), it is essential th a t endogenous vascular

an tith ro m b o tic m echanism s rem ain in tac t fo llow ing organ s to r

age. B lood vessels synthesize and release a num ber o f a n t i

th rom botic factors, in c lud ing prostacyclin (P G I j (/f |) . P G l, is a

key vascular eicosanoid th a t in h ib its ])la te le t adhesion and

aggregation and is a p o te n t vasodilator (^). P G I j synthesis in

blood vessels is co ntro lled by specific endogenous receptors

(e.g., a lpha-adrenoceptors) as well as receptor-linked in tra c e l

lu lar m echanism s in vo lv in g ca lcium m obilizatio n , ac tiva tio n o f G proteins and prote in k in ase C (P K C )* (5 -9 ) . Because o f the

im p o rta n t role o f P G B in m a in ta in in g norm al vascular fu nctio n , we investigated the e ffec t o f cold storage of blood vessels

in two com m only used organ-p reservation solutions: lactob ion- ate-raffinose solution (L R S ) and citrate-based k idney perfusion

solution (K P S ; JO) on P G G synthesis by the ra t aorta . T h re e aspects were investigated: ( 1) acute effects o f L R S and K P S on

P G lj synthesis w hen s tim u la te d w ith a range o f agents (9 ) includ ing arachidonic acid (A A , substrate for P G L synthesis), calcium ionophore A 23187 (creates a rtific ia l calcium channels ), N a F (G prote in a c tiv a to r), noradrena line ( [N A ] co ntractile

agonist), and phorbol ester d ib u ty ra te (P K C ac tiva to r); (2 )

revers ib ility o f acute effects o f L R S and K P S on ra t aortic

PG Ij synthesis (s tim u la ted by no radrena line ) fo llow ing cold

storage for 12 hr; and (3 ) effect o f longer term cold storage (21

hr, 48 hr, and 72 hr) in L R S , K P S , and M E M on no radrena line -

stim u lated PG Ij synthesis.

MATERIALS AND METHODS

Reagents. Noradreimline bitartratc, sodium fluoride, phorbol ester dibutyrate, calcium ionojriiorc A 23187, arachidonic acid (calcium salt; 99.9% pure), and Duibccco’s minimum essential medium were purchased from Sigma Chemical Co. (Poole, Dorset, U K ). LR S was made up by the pharmacy at the Royal Free Hospital and consists of the following (in mmol/L): K *, 125; Na'', 25; Mg’ ', 5; lactobionate, 100; H jP O /" , 25; S0«’", 5; nllopurinol, 1; reduced glutathione, 3; raffinosc, 30; adenosine 5; pH 7.L KPS contained (in m mol/L): K ', 80; N a ', 80; Mg’ ', 40; S O / ', 55; citrate, 55; mannitol, 185; ])H 7.2.

Tissue preparation. Male S|rraguc Dawlcy rats (300 g) were decapitated, and their thoracic aortae excised. Having removed the adventitia and fatty tissue, the aortae were cut into 2-mm rings with a scalpel blade on a Teflon block. The rings pooled and randomised in Dulbecco’s minimum essential medium (pregossed with 95% 0 /5 % CO,). The rings were then incubated for 6 hr in M E M in stoppered llasks at 37'C in a shaking water bath, with changes of medium every 30 min, to allow innslanoid release elicited by preparative handling to subside (5-8). The following cx|)crimcnls were then carried out.

' Address correspondence to: Dr. -J. Y. .Jeremy, Department, of Chemical Pathology and Human Metabolism, Royal Free Hospital and School nf Medicine, Pond Street, London N W 3 2()C, England.

’ Department of Chemical Pathology and Human Metabolism.

' Abbreviations: AA, arachidonic acid; EDRF, endotheliumderived relaxing factor; KPS, citrate-based kidney preservation solution; LRS, laclobionate-raffinosc solution; NA, noradrenaline; NaF, (! protein activator; PKC, protein kinase C.

999

J. Drug De ! 1990:3U);3-A

GUEgT EDITORIAL NSAID Efficacy and side-effects; Are they wholly prostaglandin-mediated?J.Y. Jeremy, D.P. MikhailidisDept Chemical Pathology and Human Metabolism, Royal Free Hospital and School o f Medicine, Pond St., London, UK

Non-steroidal anti-inflammatory drugs (NSAIDs) are the principal agents used for the alleviation of inflammation and pain associated w ith rheumatoid arthritis and osteoarthritis, ankylosing spondylitis and gout. However, NSAIDs also elicit serious side-effects including 'NSAID gastropathy,' nephrotoxicity, hypertension and disturbances of the central nervous system (1-4).

Since NSAIDs inhibit the activity o f cyclooxygenase [5] (an enzyme involved in prostaglandin synthesis) it is widely held that their therapeutic action is prostaglandin (PG)-mediated. The ability of NSAIDs to inhibit PG synthesis does not often correlate w ith their anti-inflammatory efficacy or the incidence and severity of side-effects (6). Thus, it is of interest that there is an increasing body of evidence that NSAIDs exert effects on non-PG- related bfosystems. For example, several recent studies have shown that NSAIDs disrupt calcium mobilisation in biological membranes (7). It is well established that calcium is a universal second messenger which links receptor activation to endpoint function in virtually every mammalian cell type. The possibility that NSAID effects are mediated, at least in part, through disruption of calcium dynamics is potentially of extreme importance in rationalising NSAID action and future drug design.

NSAIDs readily partition into biological membranes [8] and have been shown to inhibit a variety o f membrane-associated processes (7). NSAIDs were found to inhibit calcium binding to plasma membranes nearly 20 years ago (9). Although

the implications o f this observation were overlooked until recently, Abramson et al. [101 demonstrated that aspirin, indomethacin and piroxicam are potent inhibitors of the early steps of receptor- activated neutrophil function, including the uptake and mobilisation of calcium at the plasma membrane of these cells. It was proposed by these authors that NSAIDs may act by disrupting a "trigger" pool of membrane-bound calcium linked to signal transduction mechanisms. These signal transduction mechanisms include the sequential activation of G proteins, phospholipase C and protein kinase C, all of which are modulated by calcium and ultimately determine the response of the neutrophil. In this context, NSAIDs have also been shown to inhibit cell proliferation at the G, phase through a non PG mediated mechanism [11). In turn, the G, phase of cell division also involves Ca^^ mobilisation as well as phospholipases, protein kinase C and G proteins [12]. Further evidence for effects on activator calcium have also been reported in the control of receptor-linked PG synthesis (also mediated by calcium, G proteins, and protein kinase C) [13] in smooth muscle tissues. In these latter studies it was found that indomethacin, ibuprofen and tiaprofenic acid inhibited receptor-PG synthesis coupling at concentrations lower than those required to inhibit cyclooxygenase activity [14,15]. The disruption of membrane- associated calcium was thought to be responsible for this phenomenon. In this context, it is of interest that indomethacin and ibuprofen are also potent inhibitors of calcium uptake by isolated human platelets at lower concentrations than

Eur J Vase Surg 5, 501-506 (1991)

Comparison of Prostanoid Synthesis in Cultured Human Vascular Endothelial Cells Derived from Omentum and Umbilical Vein

G. Stansby\ N. Shukla', G. Hamilton' and J. Jeremy'

'Academic Deparlmettl of Surgery and 'DepartmenI of Clinical Chemistry and Human Melabolism, Royal Free Hospital andSchool of Medicine, London, U.K.

Endothelial seeding of vascular grajls may reduce thrombogenicity and significantly itnprove graft patency. For clinical studies endothelial cells derived from human omentum may be the ideal source of cells as they are obtainable in large numbers. This study UKis designed to assess their ability to produce the antithrombogenic substance prostacyclin (PGL).

Human omeittal microvascular endothelial cells (HOTMECs) were grown and their ability to produce prostacyclin (PGL), PGE2 , PGp2 a und thromboxane A 2 (TXA 2 ) in response to a range of agonists was measured by radioimmunoassay. Control exjKriments were performed using human umbilical vein endothelial cells (HUVECs).

Both HUVECs and HOTMECs synthesised similar (juantities o f PGL basally and both increased production after adding A23187 (calcium ionophore), arachidonic acid, sodium fluoride and phorbol ester. In contrast, both thrombin and histamine were potait stimulators o f PGL release in HUVECs but were without effect on HOTMECs. In both cell types serotonin, carbachol and noradremline were without effect. The pattern of release ofPGE2 , PGp2 a and TXA 2 were identical to those of PGL in both cell types but the quantities released were lower.

These results show that HOTMECs can produce the antithrombogenic agent PGL in significant quantities and that they do so by mechanisms similar to those of large vessel endothelium. This sufrports the proposal that they would be a suitable cell source fo r vasadar graft seeding.

Key VVort/s; Endolhclium; Microvascular; Prostacyclin; Human.

Introduction

Occlusive arterial disease affecting the lower limb is an increasing problem in our aged population and bypass of occluded arteries in the leg saves many patients from amputation or severe rest pain. Autologous vein is the graft material of choice but unfortunately this is unavailable in a significant number of patients’ and for bypass below the knee joint longterm patency results are disappointing when prosthetic material is used.^ This problem has been ascribed, at least in part, to the absence of the vascular endothelium on the luminal surface of prosthetic grafts and this concept has prompted work with the seeding of

Please address all correspondence lo: M r G. Ham ilton, Consultant Vascular Surgeon, Academic Department of Surgery, The Royal Free Hospital, London N W 3 2QG, U.K.

0950-821X/91/050501+06 $03.00 /0© 1991 Grune & Stratton Ltd.

endothelial cells onto the luminal surface of artificial graft materials in an attempt to produce an antithrombogenic surface similar to that possessed by normal vessels. Such endothelial seeding has experimentally been shown to reduce thrombogenicity and significantly improve graft patency even in low flow conditions.^

Cells must be derived from the recipient of the graft or immunosuppression is needed and it has been suggested that autologous microvascular endothelial cells derived from human adipose tissue could provide the ideal source of cells for clinical seeding trials in humans.'*"^ Currently, however, little is known about endothelial cells derived from this source, and whether they retain their ability to produce antithrombotic agents such as prostacyclin (PGI2).

PGI2 is the predominant prostaglandin released

:;* iAljfllDB$|fib

European Journal of Pharmacology, 182 (1990) 83-89 Elsevier

EJP 51340

83

Differential inhibitory potencies of non-steroidal antiinflammatory drugs on smooth muscle prostanoid synthesis

Jamie Y. Jeremy, Dimitri P. Mikhailidis, Paresh DandonaMeiabolic Unit, Department of Chemical Pathology and Human Metabolism, The Royal Free Hospital and School of Medicine,

Pond Street, London N IV 3 2QG, V.K,

Received 11 December 1989, revised MS received 7 March 1990, accepted 20 March 1990

In isolated rat aorta and urinary bladder, indom ethacin inhibited the synthesis of the prostaglandins (PG ) P G Ij. P G E j, P G F j, and T X A j equipotently when PG synthesis was stim ulated with excitatory receptor agonists (noradrenaline and carbachol), fluoride (a G protein activator), phorbol ester (a protein kinase C (PKC) activator) and calcium ionophore A23187 (a creator of artificial calcium channels). However, there was a marked right shift (30 fold) in the indom ethacin concentration-inhibition curves when PG synthesis was stim ulated by arachidonate (PG substrate) and traum a (freeze fracturing and sonication). Although less potent than indom ethacin, the N SA ID s tiaprofenic acid and ibuprofen showed a similar disparity between the ICjqS with the same PG stim ulators. Since PG synthesis stim ulated by receptor agonists, fluoride, phorbol ester and A23187 is dependent on calcium channel activation whereas traum a and arachidonate-stim ulated PG synthesis bypass calcium channel activation, these data indicate that N SAID s inhibit not only cyclooxygenase but also (and more potently) the m obilisation of Ca** linked to PG synthesis in these tissues.

Non-steroidal antiinflam m atory drugs; C a“ ^; Prostaglandins; Smooth muscle

1. Introduction

Non-steroidal antiinflammatory drugs (N SA IDs) are the principal drugs used in the treatment of osteoarthritis, rheumatoid arthritis, ankylosing spondylitis, gout and pain. However, NSAIDs also elicit deleterious side effects, including gastric erosion (Atwater et al., 1965; Roth and Bennett, 1987), nephrotoxicity (Lipsett and Goldman, 1954; SwainSon. 1984) and hypertensive effects (Durao et al., 1977; Brown el al., 1986). Since NSAIDs reduce prostanoid synthesis through inhibition of cyclooxygenase (Vane, 1971), their actions and

Correspondence lo: J.Y. Jeremy, Metabolic Unit, Department of Chemical Pathology and Human Metabolism. The Royal Free Hospital and School of Medicine. Pond Street, London

N W 3 2QG, U .K .

side effects are largely interpreted as being prostanoid-mediated. However, there are many reports that NSAIDs exert effects on other biosystems, including phosphodiesterases (Weinryb ..ct al., 1972), binding at the plasmalcmma (North- over, 1971) and phospholipase C (Bomalasaki,1986). NSAIDs have also been shown to inhibit tumour growth (Hial et al., 1976; 1977) and cell proliferation (at the G I phase) through a nonprostanoid mediated mechanism (De Mello et al„ 1980). In turn the G I phase of cell division involves mobilisation, protein kinase C (PKC), G proteins, and phospholipid diesterases (Rosen- gurt, 1984; Wickremasinghe, 1988). In this context, it has recently been established that excitatory receptor prostanoid synthesis coupling in smooth muscle involves the sequential activation of G protein, PKC, Ca^^ mobilisation, phos-

0014-2999 /90 /503 .50 ® 1990 Elsevier Science Publishers B.V. (Biomedical Division)

J. Smoking-Related Dis. 1990: 1(1), 5 9 -6 9

SMOKING AND VASCULAR PROSTANOIDS: relevance to the pathogenesis of atheroma and thrombosisJ.Y. Jeremy, D.P. MikhailidisDepartment of Chemical Pathology and Human Metabolism, Royal Free Hospital and School of Medicine, London, UK

A b s t r a c t'

This review covers the current state of knowledge on the role of the .vascular prostanoids in the pathogenesis of smoking-related atheroma and thrombosis. The relationship between prostanoids, srnoking apd bther risk factors for atherdclerosis, is discussedîThe conclusion;: is that -cigareüe^ smokë ' œntains'iÿne pr; more

,componen^jwhicK..are^càpaWëîqf (nhibitlng ^ ^ ^ !s)^ht!:jesis^,whichr.in

' 'H

In t ro d u c tio n

The present review considers the role of vascular prostanoids in the pathogenesis of smoking-related atheroma and thrombosis. As many readers may not be familiar with this field there follows a brief introduction to three topics

(a) Smoking and the various types of vascular disease;

(b) Mechanisms responsible for atherogenesis and thrombosis;

(c) Synthesis and actions of vascular prostanoids.

(a) Smoking and the various types o f vascular disease

Epidemiological evidence has convincingly linked smoking to CHD, but the mechanisms responsible for this association remain undefined [1 -7 ]. This

topic is reviewed in the current issue of the Journal (see Doll, page 3). It would appear that several consequences of smoking accelerate atherogenesis and increase the risk of thrombosis. These factors are briefly considered later in this text.

The epidemiological evidence does present some interesting insights into the role of smoking in the pathogenesis of vascular disease. For example, smoking is more strongly associated with PVD than with CHD [8,9]. In contrast, smoking does not appear to be a major risk factor for stroke [10] . Even more surprising is the evidence that smoking is not a risk factor for venous thrombosis [7 ,1 1 -1 3j. Smokers may even carry a lower risk of postoperative thromboembolism [11] and myocardial infarction [12,13]. These

The following abbreviations have been used throughoutA A , arachidonic acid; ADP, adenosine diphosphate; CHD, coronary heart disease; PDGF, platelet derived growth factor; PG, prostaglandin; PGIz, prostacyclin; PKC, protein kinase C; PLAz, phospholipase A^; PVD, peripheral vascular disease; NEFA, non-esterified fatty acids; SMC, smooth muscle cell; TXAz, thromboxane A^.

Address correspondence to Dr D.P. Mikhailidis, Dept o f Chemical Pathology and Human Metabolism, Royal Free Hospital and School o f Medicine, Pond St, London NW32QC, UK. (Accepted for publication 9.7.90.)

59

VASCULAR AND PLATELET EICOSANOIDS, SMOKING AND ATHEROSCLEROSIS

J.Y. Jeremy and D.P, Mikhailidis

Department of Chemical Pathology and Human Metabolism Royal Free Hospital and School of Medicine University of London Pond Street, London NW3 2QG, UK

INTRODUCTION

Vascular tissue synthesizes prostanoids (PGs) that modulate contractility, myocyte and fibroblast proliferation, platelet aggregation and leucocyte function, all of which are key components in the pathophysiology of atherogenesis. Furthermore, platelet and leucocyte release substances have been shown to stimulate the synthesis and release of PGs from vascular cells. It is possible, therefore, that vascular PGs are part of a protective response against atherogenic events. Consequently, any disruption of PG synthesis (viz. as a result of smoking) may accelerate the atherogenic state. Although smoking is a major risk factor in the development of atherosclerosis, the combination of smoking with other risk factor (e.g., diabetes, hypertension, hyper- cholesterolaemia) markedly increases the likelihood of death from atherosclerotic disease. The present paper therefore discusses the following:

1) How PGs relate to pathophysiology of atherosclerosis2) Experimental findings on the effects of cigarette smoking

on vascular and platelet prostanoid synthesis.3) How other risk factors may interact with smoking to

Influence vascular permeability4) Future directions and emphases on research into eicosanoids

and smoking.

Before discussing the effects of cigarette smoking on vascular PG synthesis, it is relevant to briefly review current concepts on the pathogenesis of atherosclerosis and how they may relate to PGs.

The key event in the initiation and perpetuation of the fibrous plaque, the pathognomonic lesion of atherosclerosis, is the proliferation of arterial smooth muscle cells, followed by deposition of lipid and the accumulation of collagen, elastic fibres and proteoglycans (1). Although the trigger for these events is unknown, it has been proposed that atherogenesis is initiated by endothelial cell injury leading to adherence of platelets resulting in local release of platelet constituents including TXAg, platelet derived growth factor (PDGF), platelet activating factor (PAF), serotonin (5HT) and histamine (2).

Tobacco Sm oking and Atherosclerosis Edited by J. N . Diana Plenum Pres*, New York, 1990

135

1 A/L, UUI- lUJ .)5 1 P COPPER C H ELA TO R S IN H IB IT C Y C LO O X Y G E N A S E A N D LIP O X Y G E N A S E A C T IV IT Y BY W A S H E D H U M A N

PLATELETS, IN VITRO

J.Y. Jeremy, G Sifakis, J. G ill & 'G . K ontoghiorghes, D epartm ent of C hem ical Pathology and H um an M etabolism and

1 Departm ent of Haem atology, Royal Free Hospital and School of M edicine, Pond Street, London N W 3 2QG

Copper im balance is associated w ith cardiovascular disease (C V D ). D ivalent copper has also been shown to m odulate eicosanoid synthesis (Elliot el al, 1987; M itchell et al, 1988). Platelet thromboxane synthesis and lipoxygenase activity has been Im plicated in the pathophysiology of C V D . In order to explore the role of copper in m odulating eicosanoid synthesis by platelets, the effect o f the Cu^+ chelators, d im ethyld ithiocarbam ic acid (D M tC ), d iethyld ith iocarbam ic acid (D E tC ) and tetraethylthiuram disulphide (TETD ) on T X A 2 synthesis and hydroxy-eicosatelraenoic acid (H E TE ) synthesis by isolated human platelets was investigated. Platelets were obtained from healthy volunteers. Platelets were washed w ith HEPES containing apyrase and resuspended in HEPES buffer. Aliquots of platelets were incubated w ith D M tC , D EtC and T E T D for 15 m in at 37 ®C prior to the stim ulation of T X A ; synthesis w ith the fo iiow ing stimulators: arachidonic acid (A A ; 10 p M ), calcium ionophore A23187 (10 pM ), N aF (10 p M ), phorbol ester d ibutyrate (1 pM ); and spontaneous release. Platelets were further incubated for 20 m in at 37°C. The reaction was stopped by addition of ethanol, and aliquots of supernatant taken for measurement of TXB ] by radioimmunoassay. Conversion of A A to H E TE was assessed by the addition of P “*CJ-AA to platelets suspended in HEPES

containing various concentrations of Cu^+ chelators. Following incubation for 15 m in at 37 "C, the reaction was stopped and H ETE and unchanged A A extracted w ith ch lo ro form /m ethano l (2:1). H ETE was separated from A A by thin layer chrom atography, counted for radioactivity and percentage inhibition of A A conversion fo H E TE calculated.

D M tC inhibited TXA% synthesis in a concentration-dependent manner. IC 5 0 S (concentration at which D M tC inhibited T X A 2

synthesis by 50%; derived from 10 experim ents) were sim ilar for all stim ulators (A A , 33 pM ; A23187, 35 pM ; N aF , 34 pM ; phorbol ester, 30 pM ; spontaneous, 28 pM ). The sim ilarity in the IC 50 is indicative of an effect of chelator on cyclooxygenase, rather than on other enzymes or systems (phospholipases, protein kinase C, calcium m obilisation). D M tC also inhibited the conversion of [•'’C j-A A to J'^CJ-HETE (IC 5 0 , 38.5 pM ). DEtC and TE TD inhibited T X A 2 synthesis and lipoxygenase activity in a sim ilar fashion to D M tC and at sim ilar concentrations. The inhibitory effect of all Cu^^ chelators was reversed by the addition

of approxim ately equim olar concentrations of Cu^+ but not by Fe^+, Fe^+, Zn^+, A P + or Sn^+. These data indicate that Cu^+ (as is the case w ith Fe^+) m ay play an obligatory role in cyclooxygenase and lipoxygenase activity. The present data also support the concept that copper imbalance m ay be invo lved in the pathophysiology of C V D and that eicosanoids may play a role in m ediating this relationship.

Elliot, G R , van Batenburg, M.J. & Bonta, I.L. Prostaglandins, 1987; 34: 657-668.Mitchell, L.L., A llen, K.G.D. k Mathias, M .M . Prostaglandins, 1988,35: 977-986.

5 2 P M O D U L A T IO N OF T H E A C T IV A T IO N O F PLATELETS BY G E N IS T E IN , A N IN H IB IT O R OF TY R O S IN E K INASE(S)

C .T . M urphy & J. Westwick, Department of riiarm acology, University of Bath, Claverton Down, Bath, Avon, BA2 7 A Y

W e have explored the role o f tyrosine kinases in (he signal transduction of PAF-activated rabbit platelets. Rabbit platelets were prepared, signal molecules quantitated and dense granule release dctennined as described previously (M urphy et al, 1991). Tyrosine phosphorylation of rabbit platelet proteins was determined by Western blotting o f platelet lysates with P Y 20, a monoclonal a n ti- phosphotyrosine antibody (Glcnney et al, 1988). Resting platelets exhibited at least four tyrosine phosphorylatcd (TP)-pro teins of 5 2 -6 2 kDa. Stimulation with 300 nM PA F induced a rapid increase in TP-protcins visible w ithin 5 secs of both low (3 5 -4 5 kDa) and high (6 6 -9 0 kD a) molecular weight. Pretrcatmcnt of platelets with gcnistcin (1 0 -3 0 0 /rM ) for 20 min produced a dose related inhibition of tyrosine phosphorylation of a number o f the 6 6 -1 5 0 kDa and 3 5 -4 5 kDa proteins, although was much less effective against the proteins of 5 2 -6 2 kDa. Genistcin (A kiyam a ct al, 1987) was an effective inhibitor o f PA F-induccd calcium elevation (|C a+ + Ji), inositol 1,4,5-trisphosphate ( IP 3 ) formation, thromboxane (T x ) B2 generation and the release o f dense granules ( 1 4 c - 5 H T ) see table.

Treatment [Ca++Jj(nM) lP3(pmol/109pI) TxB2(pmol/108pl) 14C-5HT (%)

Unstirnulatcd 107 ± 12 7.5 ± 1 .5 <0.1 <0.1Vehicle + PAF 490 ± 22 34 ± 5.5 44 ± 3.2 58 ± 1 .510 / ,M Gcnistcin + P A F 434 ± 1 7 .5 - 25.5 ± 0.31 46 ± 0.730 /,M Genistein + PA F 421 ± 15 22 ± 2 3.4 ± 1.6 39 ± 2.8100 /rM G en is te in + PA F 341 ± 24 15 ± 0 .8 <0.1 13.8 ± 2.33 0 0 /rM Genistein + PA F 135 ± 13 10.2 ± 2.7 <0.1 3 + 0.5

We have demonstrated that PAF induces a rapid tyrosine phosphorylation of approximately 17 proteins in three molecular weight ranges. Ib is probably involves a number of tyrosine kinases as genistein is an effective inhibitor of only a limited number of proteins. However, these TP proteins appear to be crucial in the very early signal transduction events of PA F activated platelets as genistein is an effective inhibitor o f these events.

W e are grateful to the British Heart Foundation for support.

Akiyam a, T ., Ishida, 1., Nakagawa, S., Ogawara, I I . , Watanabe, S., Itoh, N ., Shibuya, M . & Fukami, Y . (1 98 7 )7 . Biol. Chem. 262, 5 59 2 -5 59 5 .Glcnney, J.R. Jr., Tokas, L. & Kamps, M .P . (1 98 8 )7 . Immunol. Me(h. 109, 2 7 7 -2 8 5 .M urphy, C .T ., Elmore, M ., Kellie, S. & W estwick, J. (1991) Biochem. 7. 278, 2 5 5 -2 6 1 .

*' . - .. .j - , . .' : .

89P OPPOSITE CHANGES OF ADRENOCEPTOR- AND MUSCARINE-RECEPTOR-LINKED PROSTACYCLIN SYNTTHESIS BY THE AORTA AND BLADDER OF THE DIABETIC RAT

J.Y. Jeremy. D.P, Mikhailidis & C.S. Thompson. Department of Chemical Pathology and Human Metabolism, Royal Free Hospital and School of Medicine. Pond Street, London NW3 2QG, UK

Although diabetes mellitus (DM) Is associated with diminished vascular prostacyclin (PGIg) synthesis, enhanced synthesis of PGI2 by other tissues (e.g. urinary bladder jUBl) has been reported (Jeremy et aL, 1986a). In turn, PGÎ2 synthesis by the aorta and UB of the rat Is stimulated by activation of a-adrenoceptors and muscarine receptors, respectively (Jeremy et a i , 1985. 1986b). In order to Investigate this area further, the effect of experimental DM In the rat on adrencoceptor- and muscarine receptor-linked PGI2 synthesis was studied. Intracellular second messenger systems were studied following stimulation of PGI2 synthesis with phorbol ester (PE; a protein kinase C (PKCj) activator; Ca^+ Ionophore A23187 (A23187) and thapsigargin (both elevate intracellular Ca^+, activating phospholipase A2 IPLA2 I); and arachidonate (AA; substrate for PGI2 synthesis),

DM was induced In male Sprague Dawley rats with streptozotocin (65 mg/kg. l.v ). After 8 weeks, rats were killed and aortae and UBs excised. Aortae were cut Into I mm rings and UBs Into approximately 2x2 mm segments and processed for assessment of agonist-stimulated PGI2 synthesis as previously described (Jeremy et aL, 1985.1986b). PGI2 synthesis was stimulated In the aortae with adrenaline and in the UBs by acetylcholine (Ach), PGI2 release was measured by radlommunoassay of 6-oxo-PGFi^. and dose-response curves compiled.

Transformation of data to % maximal response revealed a marked right shift In the adrcnallne-PGIi dose-response curve In aortae from DM rats (EC50 = 4.1 x 10 ® M) compared to controls (EC50 = 5.4 x 10'^ M). In contrast, Uiere was a marked left shift In the Ach-PGl2 dose-response curve In UBs from DM rats (EC50 = 5.8 x 10^ M) compared to controls (EC50 = 2.2 x 10 ® M), In the aortae from DM rats there was also a marked right shift In the PE-PGI2

dose-response curve (EC50 = 1 4 x 10 ® M) compared to controls (EC50 = 1,9 x 10'^ M), whereas there were no differences In the ECbqs of thapsigargin. A23187 or AA. In the UBs there were no differences In the EC50S of PE, thapsigargin, A23187 or AA between diabetic and control rats.

These data Indicate that: (1) reduced PGIg synthesis coupled to adrenoceptors In the aorta of the diabetic rat may be due to diminished PKC activity and not to Ca^+ mobilising systems. PLA2 . cyclooxygenase or PGIg synthase; and (11) the diametrically opposite effect of DM on muscarine receptor-linked PGI2 synthesis consolidates that changes In PGI2 are organ-specific. What determines these marked differential changes In the activity of different receptor types in DM warrants further investigation. >

Jeremy. J.Y,. Mikhailidis. D.P. & Dandona. P. (1985) Eur. J. Pharmacol. 114: 33-40, 'Jeremy. J.Y,. Tliompson, C.S,. Barradas, M.A., Mikhailidis, D.P. & Dandona. P. (1986a) Prog. Llpld Res, 25: 505-507, Jeremy. J.Y,. Mikhailidis. D.P. & Dandona, P. Naunyn Schmied, Arch, Pharmacol, (1966b) 332: 70-73,

90P A COMPARISON OF THE INHIBITORY EFFECTS OF PROSTANOID EP2 RECEPTOR AGONISTS AND p2- ADRENOCEPTOR AGONISTS ON HUMAN MYOMETRIUM FROM PREGNANT DONORS

IL L . Ycard lcy. R .A . C o lem an ^ J.K . C layton. K . Marshall and J. Senior. Postgraduate Studies in Pharmacology, School o f Pharmacy, University o f Bradford. Bradford BD7 ID P . U .K . ^Cardiovascular and Respiratory Pharmacology. Glaxo Group Research Ltd., Ware, Herts. S G 1 2 0 D P .U .K . ,

(i2 *adrcnoccptor agonists arc used clinically to inhibit premature labour, but they arc not very effective (Calixto & Cimas, 1984), and have high sidc-cffccl liab ility (Bcsingcr ei a l., 1991). E-scrics prostanoids can both contract and relax human isolated myometrium, contraction being mediated by E P p and EPj-rcceptors. and relaxation by BP2 -reccptors. (Senior et a l., 1991). In the present study, the tocolytic activities o f two selective prostanoid EP2 -receptor agonists, butaprost (Gardiner, 1986) and A H 13205 (Niais el a l., 1990) have been compared with PGE2 andtlie P-adrcnoccptor agonists, isoprenaline (IS O ), salbutainol (SA L), tcrbutaline (TE R B ) and ritodrinc (R IT ), on human isolated myometrium.

. ! ■ ■

Human myometrium from consenting pregnant donors (elective Caesarean sections at term, non-labour), was supcrfuscd with Krebs solution (2m l m in"*) containing 2 .8 p M indomethacin. and oxygenated with 95% 02/5% CO2 a l3 7 * C (Massclc and Senior, 1981). Each agonist was infused at a rate o f 0.02m l.m in"* over a range o f concentrations (O .ln M -lO u M ) for 15 min. Inhibition was seen as a decrease in frequency and/or amplitude o f spontaneous contractions. Effectiveness was expressed in terms o f potency (m inim um concentration significantly Inlnbiting myogenic activity), and recovery time (period o f inhibition following cessation o f agonist infusion until return of control myogenic activity). 1 ,

A ll agonists except R IT . inhibited spontaneous contractions in a conccntraiion-dcpendcnl manner and none o f tlic agonists caused spasmogenic effects. PGE2 . butaprost. IS O and TERB significantly inhibited myogenic contractions at 0.1 p M . whereas A H 13205 and S A L had no effect at concentrations < l< iM . The mean recovery times (m in 4 S E M ) following a lO nM infusion o f agonist were: butaprost (> 120. n=7), A H 13205 (8 9 .0 * 11.8. n = l 1), S A L (7 3 .6 * 15.2. n=9). T E R B (7 3 .3 * 16.6, n=9), ISO (7 1 .5 * 15.9, n=8) and PGE2 (34.1 * 16.4, n=5), R IT was inactive (n=9).

Both E p 2 *rcccptor and p2 -»<*rcnoccptor agonists, with the exception o f R IT , inhibit spontaneous uterine contractions in human myometrium from pregnant donors. The selective EP2 -receptor agonist, butaprost is the most effective o f the agonists tested. The fact that EP-rcccpior agonists cause inhibition o f myométrial activity without excitatory effects could have clinical implications for the prevention and/or treatment o f premature labour, ,

Besingcr. R E.. N iebyl. J R.. Keyes. W .G . and Johnson. T.R .B . (1991). Am. J. Obstet. Gynecol. 981-988.Calixto. J.B. and Cimas. C M . (1984). Biology and Reproduction 30; 1117-1123.Gardiner. P.J, (1986), Br. J. Pharmacol., 45-56.Massele. A Y . and Senior, J. (1981), Br. J. Clin. Pharmac. 1 2 .285P.Niais. A .T .. Coleman. R .A .. Hartley. D. and Shcldrick. R.L.G. (1991). Br. J. Pharmacol., 102. 24P.Senior, J., Marshall, K ., Sangha, R., Baxter, G.S. and Clayton, J.K. (1991), Br. J, Pharmacol., 102.747-753.

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MtDiCAL LiliK^'^> by - UCL Discovery€¦ · MtDiCAL LiliK^'^> ROYAL FREE nUSMlAL HAMPSTEAD. JAMIE YANCEY JEREMY Department of Chemical Pathology and Human Metabolism, The Royal Free