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PROSTAGLANDIN TRANSPORT -AM> CATABOLISM W THE CHOROID PLEXUS DURING PERINATAL AND POSTNATAL
DEVELOPMENT IN SHEEP
Nancy Krunic
A thesis submitted in conformity with the requirements for the degree of Doetor of Philosophy, Grnduate Department of Physiology,
University of Toronto
@ Copyright by Nancy Krunic, 1999
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ABSTRACT OF THESIS
PROSTAGLANDIN TRANSPORT AND CATABOLISM IN THE CHOROiD PLEXUS DURTNG PERINATAL AND POSTNATAL DEVELOPMENT iN
SHEEP
by Nancy Kruttic
Ph.D., 1999, Department of Physiology, University of Toronto
In sheep, the marked decrease in pro staglandin (PG)E2 levels in ventricular
cerebrospinal fiuid (CSF) after birth is believed to promote continuous breathing. Factors
responsible for this decrease are unknown In adults, prostaghdins are cleared fiom
CSF to plasma by carrier- mediated, pro benecid-sensitive bansport across the choroid
plexus. Circulating prostaglandins are cleared Born plasma by a similar carrier in lungs
and kidneys. Carrier-mediated transport is rate-limiting for catabolism via 15-
hydroxyprostagiandin dehydrogenase (15-PGDH) at these sites. It is not known whether
carrier-mediated transport andor 1 5-PGDH-mediated catabolism operate in the choroid
plexus during perinatal development, or if an increase in their activities contributes to the
postnatal decrease in CSF PGE2. To address these questions, we examined in vitro 3 ~ -
PGE2 and 'H-PGF~, uptake and catabolism by the sheep choroid plexus using liquid
scintillation spectrornetry and thin-layer radiochrornatography. 15-PGDH was locahd
by immunofluorescence and immunogokl histochemistry. In the term fetus, PGE2 and
PGFzo were accumulated against a concentration-gradient in a saturable, probenecid-
sensitive, tirne- and temperature-dependent marner. Uptake was similar in fetuses (pre-
temi and terni) and kmbs (-5 and 15 d) and tended to be higher in adults. In the fenis,
both prostaglandins were extensively catabolized by 15-PGDH. PGE2 catabolism did not
change durhg the perinatal perbd, while PGF2, catabolism decreased soon &et birth.
Catabolism was probenecid-insensitive and., thus, hctioned independently of uptake. In
adults, catabolism was marginal and, accordingly, 15-PGDH was undetectable. In
fetuses, 15-PGDH was localized near the CSF-king membrane of epithelial cells. In
larnbs, 15-PGDH was localized, Uistead, in stmrnal fibroblasts. PGEz catabolism did not
change in association with the postnatal relocaluation of 15-PGDH, whereas PGF*,
catabolism decreased. This study confhns that, in the perinatal sheep as in the adult,
prostagiandins can be cleared kom CSF via carrier-mediated transport across the choroid
plexus. Contrary to the adult, in the ktus, prostagiandins are completely catabolized in
the choroid plexus. The postnatal fa11 in CSF PGE2 camot be attributed to hcreased
clearance via either transport or catabolism in the choroid plexus. However, the
concomitant activity of the prostagiandin carrier and 19-PGDH may help to maintain
PGE2 low in neonates and, by extension, to Nstaia continuous breathing.
iii
ACKNOWLEDGEMENTS
The author is indebted to Dr. F. Coceani and Dr. S.L. Adamson for their skillfiil
supervision and guidance in al1 aspects of the PkD. Prognun. The generous support of
the Canadian Foundation for the Study of Sudden Infant Deaths and the Genesis
Researc h Fo undat ion is great ly appreciated.
The author wishes to acknowledge the work of Dr. C.R. Pace-Asciak and Mr. C.
Acker le y in the analysis of sarnples by , respectively , gas chromatography/mass
spectrometry and transmission electron microsco py. Ant ibodies used in
irnrnunohistochemical studies were generously supplied by Dr. RT. Okita The
assistance of E. Seidlitz and Dr. 1. Bishai is gratefully appreciated. The author wishes to
thank Dr. P.A. Stewart, Dr. C.Y. Pang and Dr. P. Brubaker for their valuable input during
meetings of the Supervisory Cornmittee.
Material reprinted fiom Developmental Brain Research, volume 100, N. Kninic, S.L.
Adamson, 1. Bishai, F. Coceani, Prostaglandin u~take and catabolism by the choroid
plexus durine; development in sheep, 82-89, 1997, with permission fiom Elsevier
Science.
PUBLICATIONS AND WORK IN PROGRESS FROM THESIS
Journal Articles
Kninic. N.. Adamsoa S.L.. Bishai. 1.. and Coceani. F. (1997) Prostaglandin uptake and catabo h m by the choroid plexus during development in sheep. Dewlopmental Brain Research. 100: 82-89.
Knuiic. N.. Adamson. S.L.. and Coceani F. DBerentid uptake and catabolism for prostagiandin Ez versus prostaglandin FZa by the developing choroid plexus fiom sheep.
K-c. N.. Ackerlev. C.. Adamson S.L.. Okita RT. and Coceani. F. Changes at birth in the localization of immunoreactive 1 5-hydroxyprostapiandin dehydrogenase in the s heep choroid plexus.
Pu blis hed Abstracts
Kninic. N.. Adamson. S.L.. Bishai. 1.. and Coceani. F. (1996) Prostaglandin catabolism in the fetal choroid plexus: a special arrangement. Society for Neurosciences Abstracts. 22: 773.
Krunic. N.. Bishai. I., Adamson. S.L., and Coceani. F. (1995) Prostaglandin inactivation in the perinatal brain: role of the choroid plexus. Society for Neurosciences Abstracts 2 1 : 1 750.
ORAL PRESENTATIONS OF THESIS WORK
Society for Neurosciences 25' Annual Meeting (San Diego, November 1995) Frontiers in Physiology Research Day, University of Toronto (1998): Awarded Second Place Department of Physiology, University of Toronto, Mid-Program Seminar (1996) and Research Group Se& (1997) Department o f Obstetrics and Gynecology Annual Research Day, University of Toronto (1 994- 1 998) Mount Sinai Hospital, Toronto Canada, Divisional Seminars (1 995- 1998) Mount Sinai Hospital, Toronto Caaada, h u a i Retreat (1994) The Hospital for Sick Children, Toronto Canada, Divisional Seminars (1 995- 1 997)
POSTER PRESENTATIONS OF TEIESIS WORK Society for Neurosciences 26' Annual Meeting (Washington, November 1996) Ontario Quebec Perinatal Investigators Meeting (Kingston, November 1994- 1997) Frontiers in Physiology Research Day, University of Toronto (1995-1997) The Hospital for Sick Children A ~ u a l Retreat (Toronto, 1995- 1997)
TABLE OF CONTENTS
Chapter 1: Genenl Introduction
1- 1. The Eicosanoid System
1-1.1. Overview 1- 1.2. Fatty Acid Precmrs of Eicosanoids 1- 1.3. Arac hidonic Ac id Homeostasis 1- 1.4. Prostanoid Synthesis, Actions and Catabolism in the Adult
1-2. PGE2 and PGF2, in Bmin of the Adult 15
1-2.1. Overview 1-22 Presence and Actions of PGEr and PGF2, in Brain of the Aduh 1-2.3. Regulation o f PGE2 and PGFza Levels in Brain ISF of the Adult 1-2.4. Regulation of PGE2 and PGF2, Levels in Ventricular CSF of the
Adult
1-3. PGE2 and PGF2, in Brain During Perinatal Development
1-3.1. O v e ~ e w 1-3.2. Presence and Actions of PGE2 and PGF2, in Brain During
Perinatal Develo pment 1-3.3. Regdation of PGE2 and PGFta in Brain ISF and CSF During
Perinatal Develo pment
1-4. Hypothesis, Rationale and General Objectives
14.1. Hypothesis 1-4.2. Rationale 1-4.3. General Objectives
Chapter II: Study of Prostagiandin Uptake and Cataboüsm by the Choroid Plexus in the Perinatal Sheep
page
II- 1 . Background and Rat ionale 35
11-2. Materials and Methods 36
11-2.1. A~imais 11-2.2. Materiais U-2.3. General Procedure 11-2.4. Incubation Procedure 11-2.5. Determination of Uptake 11-2.6. Determination of Catabolism 11-2.7. Analysis of Data
II-4. Discussion
vii
Chapter III: Cornparison of PGEl and PGF2, Catabolism, Uptake and R e l e w by the Choroid Plexus in the Perioatal Sheep
Page
III- 1 . Background and Rationale 59
111-2. Materials and Methods 61
111-2.1. Aaimals 111-2.2. Materials 111-2.3. General Procedure 111-2.4. Incubation Procedure III-2.5. Determination of PG Catabolism 111-2.6. Determination of PG Uptake and Release 111-2.7. Analysis of Data
111-3. Results
111-3.1. PG Catabolism by the Lateral and IlIAV Ventricle Choroid PIemis
111-3.2. PG Uptake and Release by the Lateral and IIIlIV Ventricle Choroid Plexus
111-4. Discussion
viii
Cbapter IV: Locaüution of 15-PGDH Protein and Activity in the Choroid Plexus of the Perinatal Sheep
Page
IV- 1. Background and Rationale 8 1
IV-2. Materials and Methods 82
IV-2.1. animal^ IV-2.2. Materials IV-2.3. Light Microscopy Irnmunofluorescence Histochemistry IV-2.4. Transmission Electron Microscopy Immunogold Histochemistry IV-2.5. Homogenized Tissue Incubations IV-2.6. Minced Tissue incubations IV-2.7. Analysis of Cataboiism in Homogenized and Minced Tissues IV-2.8. Analysis of Data
IV-3. Results
IV-3.1. Imunofluorescent Detection of 15-PGDH IV-3.2. Immunogold Detection of 15-PGDH IV-3.3. 15-PGDH Activity in Homogenized Tissues IV-3.4. 15-PGDH Activhy in Minced Tissues
I V 4 Discussion
Chapter V: General Discussion
V-1. Mechanisms of PG Uptake and Catabolism in the Choroid Plexus
V-1.1. PGEz and PGFLa Uptake in the Choroid Plexus During Development
V-1.2. PGE2 and PGF2, Catabolism in the Choroid Plexus During Development
V- 1.3 . The Choroid Plexus and CSF hostaglandins During Development
V-2. Limitations o f the Study and Future Consideratioos
V-3. Physiological Implications of PG Uptake and Catabolism by the Choroid Plexus
V-4. Pathological Implications of PG Uptake and Catabolism By the Choroid Plexus
V-5. Concluding Rernarks
Cbapter VI: Bibliograpby
VI- 1 . Reference List
LIST OF TABLES
Table 1 . Effect of temperature on prostaglandin uptake by the sheep choroid plexus. Page 51.
Table 2. 'H-PG uptake and catabolism by the fetal choroid plexus in the presence of d a belled prostaglandin Page 71.
Table 3. 'H-PG uptake by the du i t choroid plexus in the presence of unlabelled prostaglandin. Page 73.
xii
LIST OF FIGURES
Figure 1.
Figure 2.
Figure 3.
Figure 4.
Figure 5.
Figure 6.
Figure 7.
Figure 8.
Figure 9.
Figure 10.
Figure 1 1.
T h e cowse of 'H-PGF~, and "c-sucrose uptake by the choroid plexus iiom fetal and adult sheep. Page 15.
Thin-layer radiochro matograms of sheep choro id plexus and medium fbllowing incubation with 'H-PGF~,. Page 46.
Age-related changes in uptake and catabolism of 'H-PGF~, by the chornid plexus fkom sheep. Page 48.
EKect of probenecid and oxygen on 3 ~ - ~ ~ ~ t , uptake and catabolism by the choroid plexus ftom fetal and adult sheep. Page 49.
'H-PGE~ versus 'H-PGF~, uptake by the choroid plexus from fetal and adult sheep. Page 50.
Time course of 3 ~ - ~ ~ ~ 2 and '4~-sucrose uptake by the choroid plexus fkom fetai and adult sheep. Page 52.
Effect of PGF2, concentration on 'H-PGF~, uptake and catabolism by the choroid plexus fiom sheep. Page 54-
Thin-layer radiochromatograms of the lateral ventricle choroid plexus fi0 rn sheep incubated with either 3 ~ - ~ ~ ~ t or 3 ~ - ~ ~ ~ t , .
Page 68.
'H-PGE~ verms 'H-PGF?, catabolism by the lateral and the combined third and fourtb ventricle choroid plexus fiom sheep. Page 69.
Effect of probenecid on 'H-PGE~ uptake and catabolism by the lateral venvicle choroid plexus fiom sheep. Page 72.
'H-PGE* versus 3 ~ - ~ ~ ~ 2 , uptake by the lateral and the combined third and fourth ventride choroid plexus fkom sheep. Page 75.
xiii
Figure 12. 'H-PGE* versus 3 ~ - ~ ~ ~ 2 a release by the lateral ventricle choroid plexus fiom fetal and adult sheep. Page 76.
Figure 13. Immunofluorescence detection of 15-PGDH in the choroid plexus nom the pre-term and term sheep fetus. Page 88.
Figure 14. Immunofluorescence detection of 15-PGDH in the choroid plexus fiom the 5 dand 15 d old h b . Page 89.
Figure 15. Photornicrographs of H&E stained choroid plexus sections fiom the sheep fetus and lamb. Page 90.
Figure 16. Immuwgold detection of 15-PGDH in the choroid plexus fiom the sheep fetus and lamb. Page 91.
Figure 17. Age-related changes in gofd particle density for 15-PGDH immunoreactivity in the choroid plexus epithelium and stroma. Page 92.
Figure 1 8. Thin-layer radiochromatograms of the 1 00,000 x g supernatant fiaction of the choroid plexus fiom the term fetal sheep incubated with either 'H-PGE~ or 3 ~ - ~ ~ ~ 2 a .
Page 94.
Figure 19. 'H-PGE~ and 'H-PGF~~ catabolism in homogenates and 100.000 x g supernatant fhctions of the choroid plexus fiom the sheep fetus. Page 95.
Figure 20. 'H-PGF~, cataboiisrn in homogenates of lung and choroid plexus fiom the term fetal sheep. Page 96.
Figure 21. 'H-PGE~ and 'H-PG& catabolism in miaced choroid plexus fiom the ktal sheep. Page 99.
xiv
LIST OF APPENDICES
Appendix A- 1. Thin-layer radiochromatograms comparhg mo bilities of PGE2, 15KD- PGE2 and their respect ive aikylat ion products. Page 64.
A ppendix A-2. Thin- lay er radiochromatograms CO mparing PGF2= meta bo üte peaks in intact laterai ventricle choroid plexus sarnples fiom the term fetus incubated in the absence or presence of the 100,0000 x g supernatant fraction of the contralateral ventricle choroid plexus. Page 98.
Appendix A-3. 3 ~ - ~ ~ ~ 2 uptake and catabolism by cerebral microvessels isolated fiom brain of the sheep fetus, larnb and adult. Page 121.
LIST OF DIAGRAMS AND CHARTS
Diagram 1. Enzyme pathway s in prostanoid format ion and degradat ion. Page 7.
Diagnun 2. Routes for solute exchange between blood and CSF / ISF. Page 20.
Diagram 3. Analysis of intra- versus inter-animal statistical variability. Page 42.
Diagram 4. Proposed h c t ional relationship between prostaglandin uptake and cataboiism in the choroid plexus. Page 113.
Prostanoid receptors and second messenger systems. Page 9.
Enzymes for the oxidation of the 15-hydroxyl group of PGEl and PGF2=. Page 12.
LIST OF ABBREVIATIONS IN ALPHABETICAL ORDER
9-PGR 13-PGR 15-PGDH 1SK 15KD IW AIB Al' AVP BSA CAMP cDNA CSF CNS COX D DNA DTT DP dpm ECF EDTA EP FITC FP H-FABP IP ISF ME mRNA NAD' NADP+ OVLT PG PGD2 PGE2 PGF2a PGGz PGHî PG4 PGM PLA2 PLC TLC
prostaglandin-9-reductase pros ta gland in-^' 3-reductase NAD+-dependent 1 5-hydroxyprostaglandin dehydrogenase 1 5- keto 1 3,14-dihydro- 15-keto third and fourth amino iso butyric acid area postrema arginine vasopressin bovine senun albumin cyclic AMP complementary deoxyribonucleic acid cerebrospiaal fluid central nervous system c yclooxygenase 1 3,14-dihy dro deoxyribonucleic acid dithiothreitol prostaglandin D receptors disintegrations per minute extraceiiular fluid ethy lenedinitrilo tetraacet ic acid prostaglandin E receptors fluorescein isothiocyanate prostaglandin F receptors heart-type fàtty acid binding protein prostacy clin receptors interstitial fluid median eminance messenger ribonucleic acid nicotinamide adenbe dinucleotide nico tinamide adenine dinucleotide phosphate organum vasculosum laminae tenninalis prostagiandin prostaglandin D2 prostaglandin E2 prostaglandin Ft, prostaglandin G endoperoxide prostaglandin H endoperoxide prostac yclin prostaglandin metabo iite phospholipase A2 phospholipase C thin-layer radiochromatography
t issue-to-medium ratio thromboxane receptors t hro mboxane vasoactive intestinal peptide
CHAPTER 1
GENERAL INTRODUCTION
CHAPTER 1: SUMMARY
The prMary prostaglandins (PG), PGEz and PGF2, are biologicaily active lipids derived
fiom the essentiai fatty acid, arachidonic acid. PGE2 and PGF2, genenilly exert their effects
locally in ether a paracrine or autocrine manwr. Hence, they are synthesized within cells
expressing rate-iimiting synthetic enzymes and are released into extnicellular fluids (ECF') where
they interact locally with specific plasma membrane receptors. PGs are catabolized within cells
expressing rate-limiting catabolic enzymes. If catabolic enzymes are not expressed at or near the
site of synthesis, these prostaglandins enter the general circulation fiom whic h they are rapidly
cleared via specific carriers and catabolic enzymes, primarily in the lung. Thus, in the adul,
circulat hg levels of prostag landins are very low. Ho wever, in fetal Me, prostaglandins circulate
in high concentrations and may have endocrine e ffects.
Prostaglandin levels in the central nervous system (CNS) are also higher in the term ktus
than in the adult. At both ages, the rate of PGEÎ and PGF2, appearance in brain interstitial fluids
(ISF) depends on the permeability of blood-brain barrier structures to circulating prostaghdins
and on the release of locally syntheskd prostaglandins. Due to low catabolism in adult brain,
the rate of disappearance of PGE2 and PGF2, fiom brain ISF is determined by clearance to
plasma either via specific carriers in brain microvessels or via the cerebrospinal fluid (CSF).
Clearance &om CSF, in tum, depends on CSF turnover and on carrier-mediated removal across
the chomid plexus. Similar to the adult, in the term fetus, PGE2 and PGF2, catabolism is low in
the brain parenchyma. Although CSF turnover at this stage of development is comparable to that
in the aduh, the activity of specific &ers and catabolic enzymes in brain microvesstls and the
choroid plexus remaios to be established.
Around the time of birth, changes in PGE2 and PGFh levels both in brain ISF and in CSF
have been associated with certain transitional events. One example is the proposed causal link
between the postnatal $II in PGE2 concentrations in ventricular CSF and the establishment of
continuous breatbing. However, little is known about the centrai mechanisms responsible for
decreasiag PGE2 levels in CSF. The starhg hypothesis for this thesis was that increased PGE2
removal f?om ventricular CSF by the choroid plexus contributes to the postnatal decrease in CSF
PGEl levels.
1-1. THE EICOSANOLD SYSTEM
1-1.1 Overview
Eicosanoids are a family of 20-carbon lipids derived kom essential fatty acids. The
polyunsaturated fàtty acid precursor common to most known eicosano ids is arachidonic acid.
Under basal conditions, arachidonic acid exists primarily in esterified form in plasma membrane
phospholipids. Consequently, eicosanoid synthesis depends on the availability of non-esterified
arachidonic acid. Free arachidonic acid in turn, is oxidized through one ofthree known rate-
limit ing enzyme pathway s in eicosano id fonnat ion: c yc looxy genase, lipo xy genase, and
monooxygenase. Two iso forms of cyclooxygenase have been identifed and both catalyze the
format ion of prostaglandin endo peroxide intermediates fi0 m arac hidonic acid. The
prostaglandin endoperoxide is. in tum, a substrate for prostacyclin, thromboxane, and
prostaglandin synthases; these are responsible for the format ion of bio log ically active lipids
known collectively as prostanoids. Prostacyclin and thromboxane A2 are chemically unstable
and are converted non-enzyrnat ically to stable, k t ive, byproducts. Converse1 y, hact ivat ion of
prostaglandins occurs through a series of enzyme-catalyzed oxidation / reduction reactions.
Inaftive prostaglandin metabolites are excreted fiom the body primarily through the urine and
bile.
1-1.2 Fatty Acid Precumrs of Eicosanoids
The precurson for al l eicosano ids are long-c hain, monocarbxy lic, pu lyunsaturated essent i d
fatty acids [3 7 1 1. Known precursors include arachido nic ac id, dihomo-y-ho lenic acid, adrcnic
acid, eicosapentaenoic acid, and docosahexaenoic acid, aU of which are derived nom the
desaturation and elo ngation of the essent id fatty acid, linoleic acid [3 7 1 1. The most conmion
eicosanoids in mammalian tissues are derived fiom arachidonic acid, a 20-carbon fatty acid with
four cis double bonds [26;357;371].
1- 1.3 Arachidonic Acid Homeostasis
Under normal conditions, most of the arachidonic acid in rnamrnalian tissues is esterified to
membrane phospho lipids in the sn-2 position [ 1 901. Thus, availability of k e arachidonic acid is
rate-Ming for eicosanoid synthesis [326;327]. Cleavage of arachidonic acid fiom membrane
phospholipids can be elicited by several factors including hormones, growth factors, turnour
promoters and physical stimuli [8;54;82; 124; 127; 19 1 ;247;298;3 143691. Ultimately, these
diverse fàcton activate phospho lipases, whic h mediate the deac y lat ion of esterified arachidonic
acid in membrane phospholipids [ 1 901. Accurnulating evidence suggests that an As-type
p ho spho lipase. PLA2, is the primary mediat or of arac hidonic acid release for eicosano id
synthesis [8]. Ho wever, other phospholipases, such as phospho lipase C, have also been
irnplicated in this process [2 14;3 1 21. Under basal condit ions, levels of arachidonic acid are
extremely low in the ce11 cytosol [16]. Furthermore, PLA2 activity is controiIed by several
factors endogenous to the ceil, including an inhibitory protein released in response to
glucocorticoid stimulation [12 11. Another factor regulating f?ee anichidonic acid levels is
reacylat ion to membrane phospholipids, which is a rapid process [146; 1 9O;3 771. Since &e
arachidonic acid is readily converted to eicosanoids, the activity of individual enzymes for
eicosanoid synthesis will also deierrnine its levels.
1-1.4 Prostanoid Syntbesis, Action, and Catabolism in the Adult
Eicosanoid metabo tites of arachidonic acid f o d through the action of the rate-limiting
enzyme, cyclooxygenase (COX), are generally refened to as prostaaoids. In addit ion to the
COX pathway, lipoxygenase and monooxygenase pathways for eicosanoid formation have been
identified [371]. The initial step ineach pathway is oxygenation ofanichidonic acid by a
distinctive eazyme. COX is a bifunctional enzyme with both oxygenase and peroxidase
activities [37 11. Its oxygenase act ivity catalyzes the insertion of two oxygen molecules into
arachidonic acid, thus yielding the unstable endoperoxide, PGG2. The peroxidase activity
catalyzes, instead, the reduction of PGGz to its 15-hydroxy analogue, PGH2 (Diagnun 1, pg. 7).
COX is a microsoma1 enzyme [144;228] existing in two isoforms, COXl and COX2
[90;148;186;.222;380]. COXl and COX.2 are separate gene products and have been rnapped to
different chromosomes [163]. They share 6 1% of their amiw acid sequence and have similar
kinetic pro prties for arachidouic acid oxygenation [ 1 48;273]. Ho wever, the two COX iso fonns
dSer in thek susceptibility to the inhibitory actions of non-steroidal antiinflamrnatory agents
[22O;25 3 ;27 1 3241. Furthemore, COX 1 mRNA is constitutively expressed, whereas COX2
expression is mitogen-inducible, sensitive to glucocorticoids, and abundant in pro-inflanmatory
tissues [88;88; 155;163;173;187;X5;B2;289397383]. A notable exception is the brain, where
both iso forms are constitutively expressed [252;375]. The relative importance of COX 1 and
COX2 in prostanoid formation is under active investigation.
Once arachidonic acid has been converted to PGHz via COX, specific enzymes catalyze the
conversion of PGH2 to prostanoids. Prostacyclin synthase and thromboxane synthase catalyze
formation of prostacyclin (PGI*) and thromboxane (TX)A2, respectively (Diagram 1, pg. 7).
Prostagiandin(PG)D synthase and PGE synthase catalyze the isomerization of PGHÎ to PGD2
Prostacyclin PGE PGF PGD Throm boxane synthase synthase synthase synthase synthase .-
V carbonyl PGF reductase synthase
nonenzymatic 15hydmxyprostag/andin dehydrogenase nonenzyrnatic h ydrolysis h yd rd ysis
.( + + prostaglandin - 413 - reductase
(13-PGR)
Diagnun 1. S ynthesis and catabolism of primary prostagiandins (PGE2, PGFza, PGD*), prostacyclin (PG12) and thromboxane (TX&). Boxed text: htty acid 1 endoperoxide precursoe. Bold tes: biologically active prostanoids. Plain text: biologicaiiy inactive prostanoid metabolites. Italicized text: synthetic / catabolic enzymes.
and PGE2, respectively (Dia- 1, pg. 7). Finally, PGF synthase catalyzes the reduction of
either PGH2 or PGDz to PGF2, (Diagnun 1, pg. 7). These enzymes have all been purified fkom
microsomal membrane fiactions [89;139;229355;354;365]. Thus, PGH2 formed in the
endopiasmic reticulum and nuclear membrane via COX is readily accessible to specitic
synthases. Almost al1 mmumlian tissues synthesize these prostanoids, their actual yield being
both organ- and species-specifc [33 71. These bio logically active lipids funct ion in a paracrine
and / or autocrine m e r in the adult, since their plasma levels are generally too low for an
endocrine effect [118].
PG12, TX& and the primary prostaglandins, PGD2, PGEr and PGF2., exert their e ffects
extracellulady via specifc receptors located in the plasma membrane. These receptors ôelong to
the family of G-protein coupled 7-transmembrane domain proteins [78]. Rostanoid membrane
recepton are classified into five groups based on their speciticities for the above mentioned
compounds (Chart 1, pg. 9). To date, recepton specinc for TX& (TP) [174], PGIz (IP) [170;246],
PGD2 (DP) [14q, and PGF2, (FP) [245;334] have k e n cloned. Four receptor nibtypes specific
for PGEz (EPi4) have also been cloned, with EP3 having several altematively spliced i s o f m
[2;4;153;287;288;335;336;345;364L The second messenger responses elicited through these
prostanoid receptors are summarized in Chart 1 (pg. 9).
To bind to their specific plasma membrane receptors, wwly synthesized prostaglaudins m u t
be translocated to the extracellular matriu. Ahhough prostaooids are lipids, they do not readily
cross plasma membranes [19;39]. Newly synthesized prostanoids are expectedly reieased into
extracellular fiuids via a specinc canier. Recently, a prostaglandin carrier bas ken cloned [161
and localized to most tissues [211;313]. This transporter has high affinity for primary
prostaglandins [ 1 571. Thus, it may play a role in tmmlocating these prostanoids fiom the
Prostanoid Endogenous Ef fecto r G Receptor Ligand protein
ca2+ channel Adenylyl Cyclase Adenylyl Cyclase Adenylyl Cyclase
Phospholipase C
Aden y lyl Cyclase
Adenylyl Cyclase Phospholipase C
Phospholipase C
Unknown Gs Gi Gs
Chart 1. List of cloned prostanoid receptors, their endogenous ligands and effector systems. For review refer to Negishi, M., Y. Sugimoto, and A Ichikawa. Molecular mec hanisms of diverse actions of pro stanoid receptors. Biochimica et Biophysca Acta, 1259: 109- 120, 1995.
intracellular to the exmicellular conpartment. At the same tirne, it may also transport
prostaglandins in the opposite direction [ 1671. Considering that this carrier has greater afEnity for
bio log ically active prostaglandins than k t ive prostaglandin metabo lites [ 1 571, it may be
responsible for removing prostaglandins fiom extniceilular fluids for catabolism by c ytosolic
enzymes. The physiological role of this carrier is still under investigation.
The actions of PG12 and TXA2 are rapidly temiinated due to spontaneous breakdown to 6-
keto-PGF,, and TXB2, respectively (Diagram 1. pg. 7) [37 11. In contrast, inactivation of PGDz,
PGE2 and PGF20, involves a series of oxidat ion / reduction reactions catalyzed by c ytosolic
enzymes. The nite-limiting step is the initial oxidation of the 15-hydroxy! group of
prostaglandins to a keto group, which markedly reduces biological activity [9;243;279]. This
reaction takes place in most adult tissues and is catalyzed by NADT-dependent 15-
hydroxyprostaglandin dehydrogenase ( 15-PGDH) [ 1 O]. 15-PGDH expression is d l y
accornpanied by expression of NADH-dependent prostaglandin-~13-reductase (1 3-PGR), which
catalyzes the subsequent reduction of the double bond between car bons 1 3 and 1 4 in the 1 5-keto
prostaglandin [9; 1 O] (Diagram 1, pg. 7). The 15-PGDH I 13-PGR system is highly active in
lungs [IO], the primary site for catabolism of circulating prostaglandins in the adult [118].
Prostaglandin catabotism in the lungs depends on carrier-mediated transport fiom extracellular to
intracellular sites [ 14;4 11. Lung 15-PGDH has been purified and has been localized to epithelial
cells luiing the bronchioles rather than the endothelhl cells of the pulmonary vasculahire
[25;259]. 1 5-PGDH has also been purified fkom the kidney [2 121. As in the lung, rend
catabolism occurs via cytosolic enzymes and depends on carrier-rnediated transport [37].
Prostaglandin metabolites undergo f ider & and m-oxidation in the kidney and liver [37 11 and
these rnetabo lites are uitimately excreted via the urine and bile [ 1 33 ;303].
A notable exception with respect to the 15-PGDH 1 13-PGR systern is the adult CNS. In the
brain and spinal corci, 15-PGDH activity is exceedingly low, h o t absent [ log 13 1 ;244], while
1 3-PGR is relat ively abundant [ 1 0 9 1 ;3 1 ;244]. Since 1 3-PGR openites in concert with 1 5-
PGDH in other tissues [IO], its role in brain is not entirely clear.
Certain enzymes which have been identified in brain, but are distinct f2om 1 SPGDH, are
able to catalyze the oxidation of the 15-hydroxyl group of prosiaglandins. However, it is
unlikely that these enzymes mediate PGE2 and PGFt, cataboüsm under normal conditions. In
particular, a PGD2-specifc, NADP+-de pendent, 1 5- hydroxyprostagiandin dehydrogenase has
been isolated fiom pig [350], but not human [256], brain (Chart 2, pg. 12). This enzyme has
very low activity towards PGEz and PGF2, [350] and is seerningly different fiom another
enzyme which cm catalyze the rate-limit ing step in PGE2 and PGF2, catabolism, namely,
NADP'-dependent carbony 1 reductase (Chart 2, pg. 1 2) [îS6;3 671. The preferred funct ion of
carbonyl reductase, however, is to catalyze the reduction of quinones [367] (Chart 2, pg. 12).
Carbonyl reductase can also reduce the 9-keto group of PGE2 to fom PGF2. [367] (Diagram 1,
pg. 7). In k t , carbonyl reductase has been identified as 9-keto-prostaglandin reductase in brain
[ 14 1 36q. This enzyme not only catalyzes the reduction of PGE2 and 15-keto, 13- 14-dihydro-
POE2 to their F-type counterparts, it also catalyzes the reverse oxidation reactions (Diagram 1,
pg. 7) [30;62; 136; 137; 14 1 ;263]. Carbonyl reductase and 1 5-PGDH are derived kom separate
gene products and share homology with other short-chah dehydrogenases [12368].
15-PGDH cDNA and genomic DNA have been cloned and the human gene has been
locaiized to chromosome 4 [ 1 l3;2 1 7;2 1 8;276]. Recently, a C-terminal tmcated isofom of
Enzyme Cofactor Ptefened Substrates
15-PGDH NAD' PGE, PGF, PGD
PGD-specific, 1 5-hydroxy- NAD?' PGD prostaglandin dehydrogenase
Carbonyl reductase NADP' quinones, alde hydes, ketones
Chart 2. Enzymes catalyzing PGE2 and PGF2. oxidation at carbon 15. For review refer to Smith, D.L., K.J. Stone, and A.L. Willis. Eicosanoid metaboliring emymes and metabolites. In: Hundbook of Ekosanoids. Volume 1. Prosiagiandins and Related lipidr. Part A. Chernical and Biochemical Aspris. AL. WilIis, ed. CRC Press, Boca Raton, 1987.
15-PGDH has been identifîed [277;278]. The physiological and I or pathophysiological role of
truncated 15-PGDH remains to be established. Studies with full-length 15-PGDH in bacterial
expression systems are underway to characterize its active site and regdation 168; 108- 1 l2;374].
15-PGDH is regulated by a variety of agents at multiple steps in the expression sequence.
Glucocorticoids are known to increase 1 5-PGDH protein levels [3 741 and catalyt ic activit y
[23 1;232;352;374]. Ghicocorticoids and other steroid hormones, mch as estrogen and
progesterone, cari aiso inhib t 1 5-PGDH activity [46;59;59;308]. Furthemore, a glucocortico id
response element and estrogen receptor bhding site have been identified in the promoter region
of the 15-PGDH gew, suggesting that steroids may also regdate gene transcription and mRNA
levels [2 1 81. Thyroid hormones can also inhibit 15-PGDH activity [ 192;230;342] as can some
fatty acids [24;60;223;242]. A wide range of exogenous substances are known to affect 15-
PGDH-mediated prostaglandin cata bolisa The non-st eroidal ant iinflammatory dmg,
indomethaci. xnay inhibit 15-PGDH activity [11;263;346], besides potently inhibiting COX
enzymes [ 1 1 7;27 1 ;324;3 581. This dual action contrasts that of the stemidal antiinflammatory
agents, which often have opposite effects on COX and 1 5-PGDH 123 1;232]. Opposing effects
on COX and 1 5-PGDH have also been noted with ant i-ulcer drugs whic h promote PGE2 release
î?om the gastnc mucosa [179;254;275]. (Mer exogcnous agents which inhibit 15-PGDH
include chernicals found in cigarette smo ke such as azobenzenes [ 1 5;28], reactive aldehydes
[208] and polyc y clic aromat ic hydrocarbons [ 1 601.
From the forego& it is clear that 1 5-PGDH activity in the aduh is present in most tissues,
with particular abundance in the lungs, and serves to maintain low basal levels of prostaglandins
in these tissues and the general circulation. The adult brain is an exception in that it has low 1 5-
PGDH expression and, consequently, M e prostaglandin catabo lism [ 10;2 1 ;244;259]. The
mechanisms by which these prostaglandkis are inactivated in adult brain are discussed in detail
in the next section.
1-2 PGEl AND PGFk IN BRAIN OF THE ADULT
1-2.1 Overview
PGEÎ and PGF2, levels in the interstitial fluid (ISF) and cerebrospinal fluid (CS0 of the
adult brain are normally low but increase in the course of certain patho logical conditions.
Accumulat ing evidence points to both neuroendoc~e and neuromodulatory roles for these
prostaglandins, w hich are synthesized in neurons, g lia, vascular endothelial and smooth muscle
cells. Blood-borne PGE2 and PGF2, nomially reach the brain sparingly due to low circulating
concentrations and low blood-brain ùarrier permeability for these compounds. COX l and COX2
have been identified in several brah regions, dong with the prostaglandin-specific carrier and
the EP and FP receptors. However, 15-PGDH activity is very low in both parenchymal and
vascular tissues of the adult b r a h It follows that the prirnary meam for terrninating the central
actions of these prostaglandins is removal fiom brain ISF either across the microvasculature or
via the CSF. Clearance fiom CSF, in t u m is mdiated both by buik CSF flow and drainage and
by der-mediated remod across the choroid plexus. Once cleared fiom brain extracellular
fluids to the general circulation, PGEl and PGF2, are removed and cataboiized p r i h l y through
the activity of the prostaglandin d e r and 1 5-PGDH located in the lungs.
1-2.2 Presence and Actions of PCEi and PGFIa in B n i n of the Adult
PGE2 and PGF2. were the 6rst eicosanoids identified in brain [77; l85;302] and have ken
implicated in many functions. Hypothaiamic PGE2 potentiates the release of several pituitary
hormones [6 1 ;95; 168; 169;257;284;293;339]. PGEt also has nociceptive effects [79;2 131,
promotes wakefilness [140] and is a central mediator of fever [320]. Both PGEz and PGF2, can
modulate the synaptic release of catecholamines [U; 142; 156;292;309;379] and, in tm
norepinephrine can stimulate PGE2 release nom the hypothalamus 12931. Acetylcholine
stimulates PGEz and PGF2, release in brah [290;291] and these prostaglaadins may regulate
centrai cholinergie receptor function [55;347]. PGEz and PGF2= can also stimulate calcium influx
into neurons [227]. In addit ion, while PGEz potentiates catecholamine-induced CAMP formation
[269], PGF2, potentiates vasoactive intest inal po lypeptide(V1P)-induced CAMP formation [3O7].
In k t , based on this action, PGF2, appears to be an obligatory intermediate in VIP-induced
dilatation of cerebral vessels [366]. PGEI and PGF2, also exert direct vasoactive effects on the
cerebral circulation [ 106;355;378]. The expression of EP and FP receptors in brain which
mediate these central effects is under investigation [96; 1 753 1 3;2 1 6;284;309;379;3 8 1 ;3 821.
Few studies have used microsamplhg techniques such as microdialysis and push 1 pu1
perfusion to measure PGE2 and PGF2, release &om distinct regions of the brain in vivo
[306;320]. Many studies have use& instead, sampling of ventricular and lumbar CSF. Brain
levels of PGE2 and PGF2, have also been inferred fiom measurements of content or release ex
vivo. However, in the latter case, both the method used for killing the animal and the condition
of the experiment may have afEected the measured values [6;66;13 11. On the 0th- hand, when
measuring CSF prostaglandios, levels may Vary with the site of sampling [74; 1351. Despite these
Limitations, measurements ex vivo and in the CSF provide a wful index for the relative rate of
PGEz and PGF*, synthesis under both normal and pathological conditions [299]. Basal levels of
PGEz and PGF2, are very low throughout the brain [1;6;80; 105; 122; 129; 13 1 ; l4S;Z6;293]. Low
prosiaglandin concentrations in the btain parenchyma result fiom low levels of fiee arachidonic
acid the tissue [66]. A sirnilar situation is found in meningeai tissues, epicortical and penetrating
blood vessels, microvessels, and the choroid plexus [l ; I32;233 ;256]. PGEz formation has been
dernonstrated in isolated neurons and glia as weîl as in cukured cerebrovascular srnooth muscle
and endothelial cells [33;234;296]. Low ùasal PGE2 and PGF*, Ievels in brain parenchyma and
the vasculature parallel levels of PGEs and PGF2, in CSF, which are generally below the 100
picograrns per ml mark [74; 104; 1 3 5;299;372].
Clinicaily, elevated CSF content of PGE2 and PGF2, has k e n associated with pyrogen
fever, semires, ischemia, tumeurs and head trauma [63; 10 1 ; 1 04; 1 1 6; 13 5; 1 84; 1 89;372]. In the
case of fever due to endotoxllis, PGEl causes hyper thed by acting on specXc sites in the
hypothalamus [74;75;94; 102;235;320]. Recent studies suggest that the source of PGE2 durhg
endotoxin fever may be the anteroventral region of the hypothalamus [306]. Prostaglandin
synthesis is also upregulated in the brain vasculature and perivascular microglia during
endo toxin induced sy stemic inflammation [ 1 07; 1 8 81. Prostaglandins may mediate increased
cerebral blood flow under mch conditions [258]. They also rnay contribute to increased
serotonin and noradrenaline release in the hippocampus in response to systernic endotoxin [207l.
Increased prostaglandin levels during ischemia, seizures and hemorrhagic injury result fkom
increased anichidonic acid release fiom membrane phospho lipids [ 1 3;22;23;286]. In the case of
ischemia and seinires, there is a h induction of CO=, and in some cases COX 1, in brain
[ 1 S2;2SO;3O 1 ;3M;3 751. inhibitkg prostaglandin synthesis during ischemia may confer
neuroprotection [250]. However, fiee oxygen radicak reieased during arachidonic acid
conversion to PGH2 via COX are most likely the mediators of cerebrovascular and neuronal
membrane damage often complicating ischernic brain injury [18O; 1 8 1 3841. Free oxygen
radicais gewrated during prostanoid formation may also mediate cerebrovascular dilat ion and
increased blood-brain M e r permeability underlying vasogenic edema associated with turuours
and head trauma [67; 1 82;3 1 81.
From the foregoing it is clear that PGE2 and PGF2, exert physiologicd effects in the aduh
brain and that their concentrations can increase during certain pathological condit ions. In either
the basal or stimulatecl me, levels of these compounds in bmin depend on local synthesis,
penetration of blood-borne PGE2 and PGF2, across the bblood-brain barria, and clearance of
these compounds from brain extracellular fluids (ECF). This is reviewed in greater detail in the
fo llowing sections.
1-22 Reguiation of PGE2 and PGFk Levels in Bnin ISF of the Adult
One factor which detemiines PGE2 and PGF*, levels in brain ISF is local synt hesis. Brain
parenchyma, cerebral microvessels, surface cerebral vessels, and the choroid plexus are able to
synthesize PGEl and PGF2, [1;29;32;34;35;130;132;337]. The rate-lirniting enzyme in their
synthesis c m be either COXl or COX2, both of which are detected in brain under basal
conditions [5 1 ;52; 107; 1 88;252;349;375]. However, COX expression in brain is generaily lower
than that in peripheml tissues such as lungs, liver, and kidneys [ 171 ;252]. COXl and COX2 are
generally found in distinct neuronal populations, and both are expressed within ce11 bodies and
dendrites [5 1 ;52]. C O U expression in brain vasculature is induced either in the presence of
serum or pro-infianimatory cytokines [52;188;296], and expression in perivascular microglia
hcreases during acute sy stemic inflammatory responses [ 1 07; 1 881. Neuronal expression of
COX2 mRNA and protein is rapidly, albeit transiently, upregulated by seizures, NMDA-linked
synaptic activity and ischemia [250;304;375]. Neuronal COX 1 expression also increases during
ischemia [ 1 521. In contrast to the relatively abundant expression in neurom, COX mRNA and
protein have not been detected in astrocytes [5 1 ;52;375]. This raises the question of whether
PGE2 and PGF2, synthesis in these celis in vitro [3 3 3 16;3 171 is artifactual. Nevertheless, it is
stiil possible tbat, in vivo, PGHz released from neurons is coaverted to PGEi and PGF2, in
astrocytes.
Release of newly synthesized PGEz and PGS, f?om neurom, rniroglia and microvessels
into ISF and CSF is presumably carrier-mediated, since these prostaghndins do not r e d i cross
the plasma membrane [39]. Indeed, a prostaglandin carrier has been identified in several brai.
regions [ 1 67;2 1 11. However, it is not known whether this carrier is localized to parenchymal or
vascular ceils and, rnoreover, whether t mediates uptake, release, or both. In vitro studies with
cultured cerebrai endothelial cells have sho wn pre ferent ial release of newly synt hesized PGE2
and PGF2. across the abluminal membrane [234]. If representative of the situation in vivo, the
latter finding would irnply that brain microvessels are a source of prostaglandins in brain ISF.
Levels of PGEl and PGF2, in brain ISF depend not only on local synthesis but also on the
extent to which blood-borne prostaglandins reach the brais Penetration of blood-borne PGE2
and PGF2, into brain is limited compared with peripheral organs such as the lungs and kidneys
[ 1 02; 1 031. In generai, there is restricted passage of hydrophiiic compounds fiom blood into
brain by diffusion due to the presence of a zonula occludens, cornmonly referred to as tight
junctions, among both capillary endothelial cells and the epithelial cells lining the choroid plexus
[53 267. Tight junctions restrict interceliular diffusion and, thus, they are the structural basis for
the blood-brain M e r 1831 (Diagram 2, pg. 20). PGE2 and PGF2, do not fieely penetrate into
regions which have a blood-brain barrier [ 1031 (Diagram 2, pg. 20). However, the
cerebrovasculat permeability coefficient for PGEz and PGF2. is almost two orders of magnitude
1 Cerebrum
SubArachnoid CSF
Choroid Plexuses
Ventricular CSF
\ Chomid plexus/ 1'7
Diagram 2. Interfaces between peripheral plasma and brain extracellular fluids (ISF and CSF). Regions containing a blood-brain bamer are denoted in bold letîers. Circumventricular organs lacking a blood-brain M e r are denoted in italic letters. ME: median eminence. OYLE organum vasculosum laminae terminalis. SFO: subfornical organ. AP: area postrema. Exchange between brain ISF and ventricular CSF across the ependymai lining of the ventricular walls is not restricted. Exchange between brain ISF and subarachnoid CSF across the pia-glia membrane is also not restricted. Ventricular CSF drains into subarachnoid CSF via the foramina of Luschke anci Magendie in the roof of the fourth ventride. Subarachnoid CSF drains into veins via the arachnoid villi and extra-eranial lymphatics.
higher than that for sucrose, an extracellular marker of comparable size [ 1 03; 1981. Even though
prostaglandins are charged molecules at physiological pH, they are more Lipophilic than sucrose
anci, consequent ly, may penetnite the plasma membrane more readily [ 103; 1 981. Special brain
regions, coIiectively known as circumventricular organs by virtue o f their proximity to the
cerebral ventricles (Diagram 2, pg. 20), lafk a blood-braiin M e r [267]. However, the
permeability of circumventricular organs to prostaglandins has not been directly measured.
Worth noting in this context is that one such organ, the! organum vasculosum laminae teminalis,
abuts the anterovenual hyporhalamus, and this rnay explain how peripherally administered PGE2
rapidly elicits fever [I02;332]. Thus, penetration of blood-borne PGE2 and PGF2, in brain ISF,
together with local synt hesis, will detemine their rate of appearance in brain. A c t a
concentrations, however, also depend on the nite of disappemce fkom brain ISF.
The primary means by which PGE2 and PGF2. are removed fiom brain ISF is through
clearance to the general circulation [42]. Unlike most other tissues, brain parenchyma and
cerebral blood vessels do not extensively catabolize PGEl and PGF2, via 15-PGDH
[ 1 ; 1 0;2 1 ;3 8;244]. Instead, these prostaglandins are rapidly cleared fiom brain IS F [IO31 either
across adjacent microvessels or via the CSF [42]. In vivo evidence suggests that removal by
brain microvessels is carrier-rnediated [42], although transport bas wt been characterized in
vitro. If the prostaglandin-specific carrier identified in brain [211] is enriched in the
microvascular component of the tissue, t may mediate prostaglandin clearance tom brain ISF.
This possibility, however, remaias to be confïrmed. PGE2 and PGF2, which are not cleared to
plasma across the microvessels are eventually cleared via the CSF.
1-2.4 Regulation of PGEl and PGFk Levels in Ventricuiar CSF in the Adult
Prostaglandins can reach the CSF ether fiorn brain ISF or from the general circulation.
Exchange of compounds between brain ISF and subarachnoid CSF across the pia-glia membrane
is unrestricted [53;285]. Similarly, there is unrestricted exchange of compounds between brain
ISF and ventricular CSF across the ependyma lining the ventricular wall [5 3 3851. Conversely,
blood-borne PGE2 and PGF2. should have limited access to subarachnoid and ventricular CSF
due to restricted diffusion across tight junctions in the arachnoid membrane and epithelial cells
Lining the choroid plexus and circumventricular organs [53;56;267] (Diagram 2, pg. 20).
Clearance of PGE2 and PGFt, &om ventricular CSF to blood is mediated primarily by a
specifc carrier system in the choroid plexus and secondarily by bulk CSF flow and drainage
[42]. Ventricular CSF drains into subarachnoid CSF due to continuous fluid secretion by the
choroid plexus, which is driven by ion pumps and sustained through movement of electrolytes
and water through specific channels [16 1 ; 162;224;225;249;322]. Subarachnoid CSF, in tum
drains into veins via the arachnoid villi [35 11 and via extra-cranial lymphatics [49;5O;3 761. Due
to these processes, there is a continuous turnover of CSF volume [143]. However, clearance of
prostaglandins fiom ventricular CSF is more rapid and extensive than that of the reference
molecule dextran, which is cleared primarily by CSF turnover [42]. Rapid prostaglandin
clearance results &om carrier-mediated transport across the choroid plexus to the venous effluent
of the brain [42;45].
The kinetic properties of the prostaglandin-specific carrier in the choroid plexus have been
characterized through in vitro studies employing the intact tissue. These studies showed that the
choroid plexus is able to accumulate PGF2, fkom a physiological incubation medium in a
saturable, energy-, time- and sodium-dependent manner [36;91]. They also demonstrated a
component of uptake which was not saturable and likely represented diffusion [9 1 1. Non-
stemidal antiinflammatory agents such as indometbacin, ibuprofen, and aspirin may inhibit
carrier-mediated PGF2, uptake, whiie glucocorticoids are relatively ineffective [43;44]. PGF2,
uptake is competitively inhibited by PGE2, 15-keto- 13,l 4-dihydro-PGFr. and, to a lesser extent,
by organic acids such as para-amino hippuric acid [43;9 11. The conventional inhibitor of organic
acid transport. P(dipropylsuKamoyl)bef]~~ic acid (probenecid) [18], also reduces PGF2, transport
in vitro and in vivo [43;9 1 1. Coincidentally , in vivo admliistrat ion of pro benecid magnifies the
effects o f supracortically administered PGE2, supporthg the concept that carrier-mediated
removal O f prost aglandins 6om brain exirace llular fluids represent s an important homeostatic
mechanism [362;363]. It is not known Xthe prostaglandin-specific carrier of the choroid plexus
is identical to the prostaglandin carrier detected in other brain regions 12 1 11.
In the aduh, after being taken up by the choroid plexus, prostagiandins remain structuratly
intact and, hence, biologically active [38]. This daers fkom the situation in the lung and kidney,
where carrier-mediated uptake is rate-limit k g for extensive 1 5-PGDH-mediated catabolisrn
[5; l4;37;4l]. Thus, in the adulî, prostaglandins cleared kom ventricular CSF to the general
circulation are still biologically active [45] and are subsequently catabolized in peripherd
tissues, primarily the lungs [118].
In the t a m fetus, low lung perfusion [166;305325], together with relatively low 1 5-PGDH
activity at this site [265;282;353], suggests tbat the lungs do not play a major d e in the
clearance of circulating prostaglandins. Aithough other tissues, most notably the placenta,
catabolw PGE2 and PGF2, via 1 5-PGDH [65;72;73; 1 1 4; 1 59;32 11, levels of these prostaglandins
in the blood of the term fetus are high [47;172;226;241]. Likewke, PGE2 and PGF2, levels in
brain and CSF are higher during perinatal development than during adult life [165;274]. These
issues are discussed M e r in the next section,
1-3. PGEI AND PGFb IN BRALN DURING PENNATAL DEVELOPMENT
1-3.1 Overview
As in the adult, PGEl and PGF2, are synthesized in brah parenchyma and cerebrai
vasculature of the fetus and newbom and exert their effect via specific receptors. In the same
way, catabolism in brain parenchyma via 15-PGDH is very low around the tirne of birth Neither
carrier-mediat ed uptake nor catabolism have been studied in brain microvessels and the choro id
plexus at this stage of development. Thus, it is not clear how PGEz and PGF2, are inactivated in
the perinatal brain. In late-gestation, PGEz levels increase in ventricular CSF and reach peak
levels during the tram it ion &om intra- to extra-uterine life. Within 24 h of biah these levels fàll
rnarkedly and this f ' has been implicated in the establishment of continuous breathing. While a
parailel fa11 in peripheral PGE2 levels is a contributhg fktor, events w i t h the brain also govem
perinatal changes in CSF PGEt levels. However, the relative contribution of synthesis versus
removal fiom brain to this postnatal fall in CSF PGEI has not been clanfied.
1-3.2 Presence and Actions of PGEI and PGFIa in B n i n During PeRnatal Development
PGE2 and PGF2, effects in brain around the tirne of birth have been studied primarily in
sheep and pigs. A well documented effect of PGEl is the depression in breathing activity
mediated through bminstem receptors [134; 158;177; l83;239305;343;344;36 11. PGF2, can
also act as a respiratory depressant, although less effectively than PGE2 [177j. During the
perinatal period, both PGEs and PGFz, have a role in cerehl blood flow autoreguhtion
through specific receptors in cerebral microvessels [7;69;7O; 193; l97;2OO;2O 1 ;203 ;280;28 11.
Clinical data aiso suggest prostaglandin involvement in the regulation of breathing and
cerebral blood flow in the human infant [100; 1 3 8;248;260;270;283;3 1 O;356].
Studies dealing with the role of PGE2 and PGF2, in cerebral blood flow autoregdation have
been conducted primarily in the newbom pig, and have included the measurement of these
compounds in the cerebral cortex, cerebral vasculature and subanichnoid CSF. In the newborn
pig (1-3 d postnatal), levels of both POE2 and PGF2, in the cerebral cortex and vasculature are
elevated compared with the juvenile pig (4-7 weeks postnatal) [274]. Furthermore, PGEz is
released nom these sites into subanichnoid CSF [194]. No Uifomtion is available on PGFÎa
release. However, in both the newborn and juvenile pig, levels of PGF2, in the cerebral cortex
are four times higher than those of PGE2, despite their equal concentration in plasma [274]. In
cultured glial cells, cerebrovascular endothelial cells, and vascular smooth muscle cells,
synthesis is greater for PGEz than PGF2, (1 541. Likewise, cerebrovascular synthesis in vivo is
greater for PGEz compared with PGF2, 12741. The decrease in central levels of POEi and
PGFZa occurring with postnatal development in pigs has k e n associated with an increase in EP
and FP receptor density in both neurons [71302;204] and cerebral microvessels [203].
Experimental evidence also suggests that postnatal changes in EP and FP receptor demities in
cere bral microvessels under lie develo pmental changes in cerebrovascular responses to PGE2
ami PGF2= [203;274].
Studies examining the central respiratory depressant effect of PGE2 have k e n conducted
prirnarily in fetal and newbom sheep, and have dealt with rneasurements of PGE2 levels in
ventricular CSF. PGEt concentrations in third ventricular CSF are high in the fetus and low in
the newbom [165]. Smce PGE2 inhi'bits breathing through a speciflc action in the brainstem,
this change in PGE2 levels likely contniutes to the transition from intermittent breathing
movements characterstic of the term fetus to continuous breathing in the newbom
[3;84;85; 158; 165;177; 183;239;36 11. In the term fetus, PGEz concentrations in CSF are around
350 pg/rnl, while the compound is a h s t undetectable (<5 pg/ml) in the newbom (1-2 d
postnatal) and older lamb (2-2 1 d postnatal) [ 1 651. At the onset of labour, levels rise to peak
values of around 1,500 p g h i and then fall precipitously within 24 h of birth [165]. P d I e l
changes have ban O bserved in plasma PGE2, although levels are higher than those in CSF
[165]. Specifically, plasma PGE2 concentrations are around 500 pg/ml in the fetus, rise to
around 2,000 pglml at the onset of labour, and fa11 to about 50 p g / d within 24 h o f birth [165].
Elevated PGE2 concentrations in the general circulation of the term fetus result prllmarily from
the combination of high placental production and low lung catabolism
[47; 166; 1 72326;M 1 ;265;282;295;305;325;353]. However, the postnatal decrease in blood
PGE2 is proponionally srnaller than that in CSF PGE2 [165]. Consequently, the plasma-to-CSF
concentration gradient for PGEz increases fiom around 1.5 in the fetus to around 10 in the
newbom [165]. Thus, blood-borne PGE2 is not the only determinant of POEz levels in CSF of
the fetus and newbom [ 164; 1651. Although it has been established that factors within the brain
contribute to the postnatal fall in CSF PGE2, the a d identity of these factors has not been
ascertained [164]. The decrease in CSF PGE2 at birth [ 1 651 is correlated with a transient
increase in the mRNA expression of EP receptor in brainstem respiratory centres [343]. Since
PGEz is still a respiratory depressant in newborn sheep [134], central factors limiting any rise in
PGE2 concentrations in ventricular CSF may be critical for sustainhg continuous breathing.
1-33 Regdation of PGEz and PGFk in Brain ISF and CSF During Perinatal Development
As in the adult, one fkctor determining PGE2 and PGF2, levels in brain around the the of
birth is local synthesis [75; l9S;2O 1 366;274;3 191. Both COX 1 and COX2 are expressed in brain
during perinatal development [86;87;2O 1 25 1 ;274]. In the terni sheep fetus, COX 1 mRNA levels
are most abundant in the superior colliculi, hn ta l cortex and hippocampus [25 1 1.
Immunoreactive COX 1 has been localized to ceil bodies and dendrites of discrete populations of
neurons, while the wime enzyme is virtually absent in synaptic terminais and glia [25 1 1. COX 1
is also expressed in brainstem respiratory centres in the term fetus and this correlates with PGEt
release fiom brainstem slices [25 1 ;3 191. In addition, COX 1 is present in the ependyma h ing
the cerebral ventricles, stroma1 fibroblasts and vascular endothelial cells of the choroid plexus,
and the endotheliurn of both deep and surface blood vessels [25 1 1. COX2 could not be detected
in these studies [25 11. In the newborn pig, both COXl and COX2 are constitutively expressed in
neurons and brain vasculature [87;268;274]. In addition to its constitutive expression, COX2 is
selectively upregulated during ischernia in the newborn pig [87. Studies with COX2-specific
inhibitors have show that this isoform is responsible for the majority of PGEz and PGF2,
synthesis in the cerebral cortex and microvessels [20 1 ;274]. Thus, similar to the adult, brain
parenchymal and vascular cells synthesize PGE2 and PGFta. Perhaps unique to the fetal animal
is the capability of the ependyma lining the cerebral ventricles, together with stroma1 fibroblasts
of the choroid plexus, to synthesize prostaglandins. Whether PGE2 and PGF2, are a d y
formed at the latter sites is not known. Nevertheless, it is possible that PGEz released fkom these
sites contnibutes to high levels of PGE2 in ventncdar CSF of the fetal sheep.
Release of locally synthesized PGE2 and PGF2= into brain ISF and CSF is presumably
carrier-mediateci. In k t , a prostaglandin carrier is expressed in brain of the human fetu [2 1 11.
However, the precise localization of this carrier rernains to be determined. It is also not known if
this carrier mediates prostaglandin release or uptake. If it functions to remove prostaglandins
from brain ISF, then it is unlikely to be linked with 15-PGDH-mediated cataboüsm by
parenchymal tissues. Specifically, PGE2 and PGF2, catabolism in brain parenchyrna is very low
from the last third of gestation onward in sheep [266]. Altematively, the carrier may mediate
PGE2 and PGF2, clearance âom brain ISF to blood across the rnicrovessels. However, it is
possible that, like other organic acids [20], prostaglandin clearance kom brain ISF occurs only
via the CSF in the perinatal animal.
CSF content of PGEz and PGFzo depends on exchange between CSF and brain ISF and on
exchange between CSF and plasma. PGE2 synthesized in the cerebral cortex is detected in
~barachnoid CSF under normal conditions and increases during ischemia, hypoxia or
hypercapnia in the newbom pig [193;194; 1961. PGE2 syntheskd in either circumventricular
organs or the hypothalamus c m be measured in ventricular CSF in the ktal and newbom sheep,
and these levels are particularly high during fever [75;25 1 1. PGF2, has not k e n measured in any
of these studies. In add tion to locally formed prostaglandins, blood-borne prostaglandins may
also gain access to ventricular CSF around the tirne of birth [25 11. in contrast to the adult,
where l e s then 1% of blood-borne PGE2 and PGF2. appears within the brain [103], around 25%
of blood-borne PGE2 reaches ventricular CSF in the perinatal sheep [164]. These observations
are consistent with the demase in blood-brain permeabdity to miall hydrophilic compounds
fkom late-gestation through aduhhood in sheep [333]. However, only 5% of blood-borne sucrose
reaches ventricular CSF in the fetal sheep [ 1151. Sucrose is a . extracellular marker with
comparable weight (MW 4 0 0 ) and size (molecular radius -1 nm) to PGE2. Greater petration
of PGEl versus sucrose may resuh âom a reiatively greater passage through regions of the brain
concaiaing a blood-brain barrier, which has ken noted in the aduh [103; 1981.
In the sheep fetus, weil formed tight junctions between capiliary endothelial cells and choroid
plexus epithelial cells are present h m mid-gestation onward [98;23 81. Ho wever, permeabilit y
to sucrose decrease markedly during the latter half of gestation [ 1 1 51. It is possible that sucrose
penetrates the develo ping blood-brain barriers intercellular ly by crossing tight junct ional cle fis
that decrease in size with increasing age [3 3O;33 11. Altematively, sucrose may cross the blood-
brain barriers intracellularly across a network of membrane bound channels [ 1 7;238]. While
tight junct ional d e fis and intracellular charnels have been demonstrated in the develo p ing blood-
brain barrier. transfer of sucrose and compounds comparable in size across these stmctures
remains to be documented. Nevertheless, it is clear that blood-brain barrier permeability to small
molecular weight compounds decreases gradually during both prenatal and postnatal
development in sheep [115;333].
The postnatal fa11 in blood levels of PGE2 [165], possibly combined with decreased blood-
brain barrier permeability [333], contributes to the fa11 in ventricular CSF PGE2 during the
transition fiom intra- to extra-uterine life [165]. However, the fact that POE2 does not
equilibrate between blood and CSF means that PGE2 concentnitions in CSF are ody in part
determined by concentrations in plasma [ 1 64; 1 651. Furthemre, within 24 h of birth, the uphill
plasma-to-CSF concentration gradient for PGE2 increases around 5-fold [165]. Thus,
mechanisrns operathg wahin the braiu must contriiute to transitionai changes in CSF PGEt
levels [ 1651. One possibility is that there is a decrease in the rate of PGEl appearance into CSF.
Altematively, or concomitantly, there may be an increased rate of disappearance fiom CSF.
PGEl clearance fiom ventricular CSF will depend on CSF turnover and removai by the
choroid plexus. Due to the fàct that both the rate of CSF secretion a d the total CSF volume
increase after birth in sheep, CSF turnover rem& constant nom late-gestation onward [ 1 1 SI.
Thus, it is unlikely that the postnatal decrease in CSF PGE2 levels results from increased
clearance due to higher CSF turnover. However, there may be incfea~ed clearance via specific
disposal mechanisms in the choroid plexus. The activity of the prostaglandin-specific carrier in
the choroid plexus around the t h e of birth is not hown. EquaUy unknown is the activity of 15-
PGDH in the choroid plexus at thk stage of development.
From the foregoing it is clear thaî, in addition to factors goveming PGEz levels in brah ISF,
several factors which may directly regulate prostaglandin concentnitions in ventricular CSF
could contribute to the postnatal fail in CSF PGE2. One possibility is that there is a decrease in
release of either locally formed or blood- borne PGE2 into CSF fiom reg ions in proximity to the
cerebral ventricles. Altemat ively , or concomitant ly , there may be increased clearance fiom the
CSF across the choroid plexus via the prostaglandin-speci fif carrier andfor 1 5-PGDH.
1-4. HYPOTHESIS, RATIONALE AND GENERAL OBJECTIVES
1-4.1 Hypothesis
Increased PGEz removal fiom ventncular CSF by the choroid plexus via either, or bath. the
prostaglandin-specific carrier or 15-PGDH contributes to the postnatal decrease in CSF PGE2
levels in sheep.
1-4.2 Rationale
The sheep was chosen as the experimental mode1 because PGEz concentrations have been
measured in ventricu lar CSF O f this species during perinatal development [75; 1 64; 1 651.
Furthemore, extensive physiological and morphological studies on the ontogeny of the CSF-
choroid plexus system have also been carried out in this species [17;97-99; 1 l5;lB;îM-
238;333]. The rationale in implicating a postnatal increase in PGEz clearance by the
prostagiandin-specific carrier is found in studies on the related organic acid carrier [20]. In these
studies, carrier-mediated removal of para-aminohippuric acid fiom CSF appeared to be greata in
the newborn than in the older rat [20]. Thus, it is possible that there is an upregulation in carriet-
mediated PGEî clearance at bhh. It is also possible that 1 5-PGDH-rnediated catabolism is high
in the choroid plexus during a ümited period of development. The rationale behind this idea is
b d on the fiict that 15-PGDH activity is developmentally regulated in other tissues.
Specifically, in brain parenc hyma fkom sheep, 1 5-PGDH activity is high during early fetal
development but demeases to low levels characteristic of the aduh by the last third of gestation
[266]. In the same species, 15-PGDH activity in lung appears to decrease towards term and then
increase at birth [265;282;353]. Thus, one may hypothesize that 15-PGDH activity in the
choroid plexus is also developmentdy regulated.
In the present study, we used in vitro methods to examine the activity of the prostaglandin
carrier and 15-PGDH in the sheep choroid plexus. By studying these processes in preparations
of the whole tissue incubated in a physiological medium similar in composition to CSF, one rnay
extrapolate to their fùnctiod arrangement in vivo. Through immunohistochemical methods, it
was also possible to evaluate both the cellular and subcellular localization of 15-PGDH in the
choroid plexus.
1-4.3 General Objectives
The objectives of the present study were threefold:
1 . To assess the presence of the prostaglandin c h e r and 1 5-PGDH in the choroid plexus of the
tem fetus and determine ifthere are any age-related changes in transport and catabolism of
PGFZa.
2. To compare, in the same preparation, transport and catabolism of PGE2 vernis PGF2,.
3. To determine the localization of any 15-PGDH detected in the choroid plexus.
These objectives are addressed in, respectively Chapters II, III and IV.
CHAPTER II
STUDY OF PROSTAGLANDIN UfTAKE AND CATABOLISM BY THE CHOROID PLEXUS IN THE PERINATAL SHEEP
11-1 BACKGROUND AND RATIONALE
The identity of central factors contributhg to the postnatal Ml in PGE2 conceatrations in
ventricular CSF are not known. One possibility is that the activity of prostaglandin-specific
carrier in the choroid plexus increases afler birth Ahematively, 15-PGDH may be active in the
choroid plexus during the perinatal period and its activity may increase during the transit ion
fiom intra- to extra-uterine life. The purpose of the present study was to ascertain whether the
prostaglandin carrier and 1 5-PGDH are operaiionai in the choroid plexus of the near-term fetal
sheep and, if so, whether the activity of either, or both, increases after birth in association with
the decrease in CSF PGEz levels. PGF2, was used to evaluate in vitro uptake since this
prostaglandin has k e n used extensively in the adult to study the choroid plexus camer
[36;3 8;43;44;9 11. Furthemore, PGF2, is chernicaliy more stable than PGEz and, thus, better
suited for an initial evaluation of catabolisrn [48; 120;329]. In a subset of experiments, uptake
of PGE2 was compared to that of PGF2,.
11-2 MATERIALS AND METHODS
11-2.1 Anirnab
Choroid plexus specimens were collectecl from pre-term fetal sheep (man age: 100 days,
range 94- 107; terni -145 days), na-term fetal sheep (mean age: 136 days, range 133- MO),
newbom (mean age: 4 days, range 3-5 days) and older iambs (mean age: 18 days, range 15-2 1
days) of Dorset, Suffolk, or Dorset / Suffolk breed. Ewes pregnant with such fetuses were used
as a reference (age: 2 5 years). In a subset of experiments, non-pregnant ewes (age: 8 months to
5 years) were o btained fiom ou. general stock.
Pregnam ewes were anaesthetized with intravenous sodium pentobarbital (Nembutal, 30
mgkg, Abbott Laboratones, Montreal, Que.) and vedilated with a &ure of methoxyflurane,
nitrous oxide and au. Fetuses were delivered by Cesarean section in a lowsxygen environment
and then killed by exsanguination according to established methods [76]. Larnbs were killed
with an excess dose of either T-61 (2 [m-methoxyphenyl 1-2-ethylbutyl-(l)]-y-hydroxy-
butyramide, 0.3 mg/kg Lv., Hoechst, Regina, SA) or sodium pentobarbÎta1 (Euthanyl, 100 mgkg
iv., M.T.C. Pharmaceuticals, Cambridge, ON). Ewes were also killed with excess sodium
pento barbital.
11-2.2 Materiais
PGF*,, PGE2, PGA2, 1 5-keto-PGF2, (1 5K-PGF2J, 13,14-dihydro- I 5-keto-PGF2, ( 1 5KD-
PGF2=), 13,l 4-dihydro-PGF2, @-PGF2,), 1 5-keto-PGEz (1 5K-PGEz), and 13- 14dihydro- 1 5-
keto-PGE2 (15KD-PGE2) were a gifi of The Upjohn Company (Kalamazoo, MI). The organic
acid and prostaglandin traaspoa inhiiit or, p(dipropylsulfamoy1) benzoic acid (probenecid). was
a gift of Merck Frosst (Montreai, Que.). [5,6,8,9,ll, 12,14,1 S@Q~H]-PGF~~ (specific activity:
180 Cilmmol) and [5,6,8,11,12,14,150~~]-~~~2 (specific activity: 164 Cilmmol) were
purchased hom DuPont (Boston, MA), while [5,6,8,9,1l, 12,14 (N'))HI I 5KD-PGF2, (specific
activity: 179 Ci / mmol) was purchased fiom Arnersham (Arlington Heights, IL). TheÜ punty
on thin-layer radiocbrornatography (see below), was 99%, 92%, and 97%, respectively.
['4~(U)]sucrose (specific activity: 4.7 mCihnol) was purchased fiom DuPont.
Chernicals were of research grade punty and solvents were glass-distilled. Buffers were
prqared with de-ioaized, glass-distilled water on the &y of the experiment.
11-2.3 General Procedure
The choroid plexus was removed, in full or in part (see below), fiom all cerebral ventricles
withh 1 5 min of sacrifice and placed in icetold, pre-gassed (95% N2, 5% CO2) Krebs-Henseleit
buffer (1 18 mM NaCI, 4.7 rnM KCI, 2.5 mM CaC12?H20, 0.9 mM MgSOse7H20, 1 .O rnM
m P 0 4 , 1 1 mM glucose, 24.9 mM NmC03) for transportation to the laboratory. This gas
mixture was chosen to maintain low oxygen conditions until tissues were equilibrated with the
appropriate gas mixture. For equilibration, tissues were kept for an additional 60 min in the
same medium at 4OC and, depending on the protocol the oxygen concentration of the gas
mixture used was 2.5 (mean p02 16 d g , range 7-28 mmHg), 12.5 (mean p 0 2 65 mmHg,
range 50-84 d g ) , or 95% (mean p02 653 d g , range 563-739 mmHg) (plus 5% COz and,
when required, balance Ns). Gas mixtures containing 2.5% and 12.5% O2 were used to
approximate fetal and neonatal oxygenation, respectively. Gas mixtures coniaining 95% 0 2 were
used to replicate condit ions of earlier in vitro experiments in the aduh choroid plexus [36;43;9 11.
Each experiment was carried out with choroid plexuses pooled fiom a single animaL The mean
wet weight of tissw samples was 55 mg (range, 26- 10 1 ), 86 mg (range, 8- 1 !JO), 1 0 1 mg (range,
75- 1 6 1 ), 63 mg (range 39- 1 1 5), 98 mg (range, 39-272) and 1 O6 mg (range, 2 1 -287) for,
respectively, the pre-tem fetus, near-term fetus, 4 d lamb, 1 8 d lamb, non-pregnant ewe, and
pregnant ewe. Variability in weight is due to the fact that, in rnany cases, the choroid plexus
could not be retrieved intact.
11-2.4 Iacu bation Procedure
Fo llowing a 1 5-min prehcu bat ion in 2 ml Kre bs-Henseleit medium, the choroid plexus was
14 incubated in 2 ml of medium containing 3 ~ - ~ ~ ~ z a , 3 ~ - ~ ~ ~ 1 , or -'H- 1 SKPPGF~, plus C-
sucrose (0.25 pCi/ml for each compound). Both pre-incubation and incubation proper were
carried out in medium pre-equilibrated with an appropriate gas mixture (pH 7.4) at 37°C. The
incubation temperature was lower (4OC) oniy in studies whic h measured passive uptake of tracer.
Incubations lasted 60 min except in experiments dealhg with the t h e course of uptake where
dserent intervals (1 to 90 min) were used. At the end of the incubation, tissues were pulled out
fiom the medium, dragged over alumtoum foi1 to remove the adhering fluid, and weighed.
Merwards, they were dried ovemight at 60°C, weighed again d e r drying, and homogenîzed
(Polytron) for 30 seconds in phosphate buffer (50 mM KH2P04 1 NaOH, pH 7.4). Tritium and
'"c activities in media and tissues were measured simultaneously in a liquid scintillation
spectrometer (Beckman mod. LS-3800, Fullerton, CA) and counts were corrected for isotope
spillover (counting efficiency about 25% and 70% for 'H and I4c, respectively). Mean
recoveries of radioactivity were 95% and 9 1 % for 3~ and 14c, respectively. Aliquots of the
samples were stored ai -20°C for aiialysis of PG catabolism (see below).
ïI-2.5 Determination of Uptake
PG uptake was assessed according to established methods [36]. Specifically, tissue-to-
medium ratios (TM; T = dpm in 100 mg tissue, M = dpm in 100 pl medium) were calculated for
the 3~-labelled PG and for the extracellular marker, '4~-sucrose. A TIM for 3~ greater thau
unity measured concornitantly with a T/M for 14c l e s than unity signifies PG accumulation
against a concentration gradient in the absence of tissue sweiiing [36]. Tissues with T M values
for %-sucrose above 2 standard deviations of the mean for the group were not inc luded in the
fuial tabulation. This criterion was chosen since T/M values for '4~-sucrose are an indicator of
the extracellular space and, thus, the viabilit y of the preparation [Ml. Mean T/M values for
sucrose ranged between 0.2 and 0.5 for aii incubations. Only 3 of 394 tissues were excluded due
to a b n o d l y high T M values for 14~-sucrose. In the majority of these incubations, 'H-PGF~,
was the tracer. In a subset of experiments, the uptake of 'H-PGF~, was compared with that of
'H-1 SKD-PGF~, and 'H-PGE~. To determine specificity of uptake, some experiments with 'H-
PGF2, were carried out in the presence of probenecid ( 1 mM). This vansport inhibitor and
concentration was chosen based on earlier studies demonstrating effective cornpetit ive and non-
cornpetitive inhibitionofprostaglandin uptake by the choroid plexus [43;44;91]. In addition, the
capacity of the carrier was ascertained by adding inmeashg concentrations (1 - 60 IrM) of
unlabelled PGF2, to incubation medium containing 'H-PGF*,. Separate studies using 10 nM
unlabelled PGF2, were done to approximate the PG concentration of the fetai CSF in vivo [Ml.
The water content of specimens was evduated as the ciifference between wet and dry weight,
divided by the wet weight. Water content was around 9 1 % for aii ages with the highest Ievels
king in the older Lamb (97%) and the lowest in the pregnant adult (88%).
II-2.6 Determination of Catabolism
Catabolism was examined only in experiments where 'H-PGF~, was used as tracer because
'H-PGE~ yielded a chemically unstable product that did not aiiow, with the methods given
below, accurate quantification A detailed discussion of this issue is given in Chapter III of the
thesis, where rnethodologies were revised to quantfi 'H-PGE~ catabolism. For analysis of 'H-
PGF2. catabolism, fiozen aliquots of the medium and tissue homogenate were thawed and
partitioned with 2 volumes of petroleurn ether (boiling point 35-60°C). The resulting aqueous
phase was acidifted to pH 3-4 with 1 M citric acid and then extracted three times with 2 volumes
of ethyl acetate. ûrganic phases were washed with distilled water until neutrai, were combined,
and then evaporated to dryness under a Stream of nitrogen. The residue was dissolved in ethyl
acetate for thin-layer radiochromatography (TLC). Mean recovery of tritium was 82%. Extracts
were spotted on silica ge CG60 plates alongside authentic PGF2, and prostaglandin metabo iites
(1 5K-PGFz, 1 SKD-PGF2, D-PGF2,, 1 SKD-PGEi) and plates were developed once at room
temperature in a chlorofom: methanol: acetic acid: water (909: 1 :0.65 by volume) system
Reference standards were visualized by spraying with a 10% solution of phosphomolybdic acid
in ethanol and gentle heating. The distribution of radioactivity was ascertained by d g the
plates on a Berthold scanner (mode1 LB 2722-2, Wildbad, Germany) and eluting appropriate
zones with 1 ml methanol-water (1 : 1 by volume) for determination of counts by liquid
scintillation spectrometry. Medium incubaîed with 3 ~ - ~ ~ ~ 2 , in the absence of tissue was used
as a negative control Catabolism is expressed as the % total radioactivity rnigrating with
inactive PGF2. metabolites (PGM). in any instance with catabolism, peaks coinciding with
metabolites were included in the &ai tabulation only if they were two standard deviations above
background levels of radioactivity (Le. greater than 8% total radioactivity on the plate). In cases
where no metabolite peaks were above this cutoff, only the peak correspondhg with the primary
metabolite was included in caiculations of PGM.
For mass spectrometnc identification of rnetabo lites, 'H-PGF*, was incubated for 60 min at
3 7OC with the c horoid plexus of the near-term fetus in the presence of saturat h g concentrations
(60 pM) of unlabelled PGF2,. Tissues were processed as describecl above, and then subjected to
TLC analysis. Appropriate sections of plates were eluted with 1.5 ml methanoCwater (9:1, by
vol) and the eluate was cenaifuged (10,000 g) br 20 mix~ at 4°C to remove any silica The fluid
was recovered, dried and then resuspended in methanol-water for a second centrifbgatioa The
fluid was dried again and the residue was dissolved in water for extraction with ethyl acetate
under acidic conditions (see above). The extract was applied to a silicic acid column (200400
mesh, Sigma, St. Louis, MO) and eluted wîth ethyl acetate pnor to preparation of a methyl ester
denvative (2941. An appropriate reference standard was processed dong with the unknown and
both samples were analyzed wit h a Hewlett-Packard (mode1 5988) gas chromatograph-mass
spectrometer-computer assernbly in the EI mode.
11-2.7 Analysis of Data
Al1 data are expressed as the mean * S.E.M. where n is the nurnber of anixnals. Tests used
for statistical cornparisons are indicated in figure legends. DifEerences are considered signifiant
when P < 0.05,
Preliminary results suggested large variability in T/M values. To determine whether this was
due to inter- or intra-animal variability, the intact choroid plexus was dissected into six parts
according to Diagram 3 @g. 42). Parts of the original plexuses were interchanged (see Diagram
3, pg. 42) and the resulting composite specimens were incubated with 3 ~ - ~ ~ ~ 2 , for either 5, 15,
Third and Fourth Ventricle Choroid Plexuses
i Lateral Ventricle Choroid Plexuses
D i a m 3. Diagrammatic representation of the dissection procedures used in determining the source of variability in measurements of in vitro prostaglandin uptake and catabolism. Lines indicate location where specimens were cut. The resulting parts were pooled according to the common letter (A vs. B) and used as described in Chapter II, Section II-2.7.
30 or 60 min at 37OC. A repeaîed measufes ANOVA was carried out at each tirne point to
compare TM values between choroid plexus specirnens Li group A with specimens in group B
(see Diagram 3, pg. 42). There was no sigdicant difference between the two groups at any of
the time points. Furthermore, omega squared tests were carried out to detennuie the proportion
of variance in T M values which resulted from intra- vernis inter-animal variability. The
proportion of total variance accounted for by intra-animal differences ( i.e. expdental mor)
was modest (IO?!) compared to that accounted for by inter-animal differences (36%) and was
considered unimportant. This was also the case with PGM values fiom 60-min incubations,
where intra-animal differences accounted for 1 6% of to ta1 variance and inter-animal differences
accounted for 4 1 % of total variance. Equally non-signifiant was the daerence in either T/M or
PGM between choroid plexus samples in the upper and lower 25' percentile of wet weights (see
above). Consequently, samples were not excluded on the basis of wet weight.
-'H-PGS, was accumulated against a concentration-gradient in a the-dependent manner by
the isolateci choroid plexus of the n a - t e m sheep fetus (Fig. 1, pg. 45). Steady-state levels of
uptake were attained &er 15 min in the fetus compared with 60 min in the pregnant adult (Fig.
1, pg. 45). At both ages, T/M values for 14~-sucrose were less than unity (4 .4) and stable
throughout the period of observation (Fig. 1, pg. 45). Based on the tirnecourse of 'H-PGF~.
uptake in the fetus and adult, 60-min incubations were chosen to represent steady-state uptake in
al1 subsequent experiments. Steady-state levels of accumulated 'H-PGF~. were greater in the
adult compared with the fetus (Fig. 1, pg. 45).
TLC analysis of the term fetal choroid plexus and medium after 60-min incubations with 'H-
PGF*, proved the presence of less polar products which rnigrated with authentic l SK-PGF2,
ISKD-PGF2, , and D-PGF2, (Fig. 2, pg. 46). 1 SKD-PGF2, was the major rnetabolic product
(8 1% of total metabolites fomed) and was noted in al1 cases. When present, peaks migrating
witb either I SK-PGF2, D-PGF2,, and in some cases 1 SKD-PGE?, represented around 18 % of
the total rnetabolic product. 'H-PGF~, incubated m medium aione or with the choroid plexus
f ~ o m the adult was recovered intaci upon TLC analysis (Fig. 2, pg. 46). Mass spectrometnc
analysis of fetal choroid plexus samples f?om a separate experiment (see Methods) showed that
the mass spectnim and relative intensity of ions of the major PGF2, byproduct matched those of
authentic I SKD-PGF2, The coincidence between unknown and I 5KD-PGF2, standard included
the presence of a peak at 330 d z which is characteristic of this metabolite.
Uptake and catabolism of 3 ~ - ~ ~ ~ z , changed differently with age. Uptake was unchanged
during the perinatai period but was higher in the pregnant adult when compared with the fetus
Near-Terrn Fetus + 'H-PGF,,
+ "c-sucrose
f O
5
A L A A A I I 1 I I I
Li.
O 15 30 45 60 75
Time (min)
Pregnant Adult
Time (min)
Figure 1 . Time course of 3 ~ - ~ ~ ~ 2 , and '"c-sucrose uptake (expressed as the tissue-to-medium ratio for radioactivity, TM) by the choroid plexus fiom the fetal (lefi panel) and adult (right panel) sheep. Incubations were carried out at 37°C and pûL of the incubation medium was 563-739 d g . In either group of animals, the effect of time on T/M values, assessed with a one way analysis of variance (ANOVA), was significant. Comparisons among time points were niade using the Student-Newman-Keuls test with different letters denoting a significant difference. Comparisons between the fetus and adult were made using Bonferroni corrected t-tests. A significant difference between fetal and adult values was attained only at 60 min. Values are the mean f S.E.M. for the nurnber of animals given below each time point for 'H- PGF2n. "'c-sucrose uptake was measured in the same specirnens.
i%
Tissue I
I Medium
a a . . a v iv iii ii i +
Figure 2. Thin-layer radiochromatograms of sheep choroid plexus and Krebs medium following a 60-min incubation at 37 O C with 'H-PGE~. pO? of the incubation medium was 563 - 739 mmHg. a: choroid plexus h m the near-term fmis. b: medium afkr incubation with choroid plexus hom the neaFterm fenis. c: choroid plexus of the pregnant aduit. d: medium afier incubation without tissue. Markers are (i) PGF2=, (ii) D-PGF2,, (iii) 1 SK-PGFt., (iv) 15KD-PGh, and (v) 1 SKD-PGEL Ongin is at arrow.
and 4 d lamb (Fig. 3, pg. 48). In contrast, catabolism decreased with postnatal development to
aimost undetectable levels in the adult (Fig. 3, pg. 48). Robenecid (1 mM) reduced uptake of
'H-PGF~, to a comparable degree in the near-terrn fetus and pregnant adult (Fig. 4, pg. 49). This
transport inhibitor hsd no effect on fetal catabolism and adult catabolism remained negligible
(Fig. 4, pg. 49).
Lowe- p02 levels of the incubation medium fiom hyperoxia to levels better approximating
normoxia did not change uptake or catabolism in either the fetus or the adult (Fig. 4, pg. 49).
Uptske and catabolism by the choroid plexus of the near-term fehis measured at f12 leveis
ranging fiom 1 4-20 rnrnHg (Fig. 4, pg. 49) were sirnilar to those measured at oxygen levels
approximating the newborn conditions @O2 57-67 mmHg, TM: 4.4H.5; PGM: 57f4%, n=lO).
Under both nonnoxic and hyperoxic conditions, 'H-PGE~ uptake was lower than that of 'H-
PGF2, at 37°C (Fig. 5 , pg. 50). The dBerence between 'H-PGF~~ and 'H-PGE~ uptake was not
seen when incubations were carried out at 4OC (Table 1, pg. 5 1). 3 ~ - ~ ~ ~ 2 uptake by the aduh
choroid plexus at 4OC was lower compared with the fetus (Table 1, pg. 51 ). At the same low
temperature, 3 ~ - ~ ~ ~ z a uptake tended to be lower in the adult than in the fetus but dinerences
between the two ages did w t attain statistical significance (Table 1, pg. 5 1). For both
prostaghdins, uptake at 4OC was significantly lower than that at 37°C (Table 1, pg. 5 1).
Despite lower levels of steady-state uptake compared with 3 ~ - ~ ~ ~ 2 a , the the-course of 3 ~ -
PGE2 uptake (Fig. 6, pg. 52) resembled that of 3 ~ - ~ ~ ~ 2 , at 37OC (Fig. 1, pg. 45) . At the same
temperatw, in the near-term fetus, T M values at 60 min for 3 ~ - 1 SKD-PGF~~ (TM: 3.9 f 0.3,
n= 6, p@ 14- 1 8 d g , 37OC) were similar to those for 'H-PGF~, (Table 1, pg. 5 1). Thus, it is
unlikely that the format ion of this metabolite afTected T/M values for the parent oompound.
14 'H-PGF, Uptake. 95% 0, 13 b
pre-lem near-term 4 d 18 d non-pregnant pregnant fetus fetus iamb iamb adult adult
'H-PGF, Catabolism. 05% 0,
pre-term near-ten 4 d 18 d non-pregnant pregnant fetus fetus bmb bmb adult adult
Figure 3. Age-rehted changes in uptake (measured as the tissue-to-medium raîio for radioactivity, TM) and catabolism (rneasured as % total radioactivity migrating with PG metabolites, PGM) of 'H-PGF~, by the choroid plexus nom sheep. Measurements were made after a 60-min incubation at 37°C. f i of the incubation medium was 563-739 mmHg. Values are means f S.E.M. for number of animals given above each column. For both uptake (upper panel) and catabolism (lower panel), a one way analysis of variance (ANOVA), followed by the Student-Newman-Keuls test for individual comparisons, was carried out to test the effect of age on T M and PGM values, respectively. WhiIe uptake increased sigaincantly with age, catabolism decreased signincantly. In either panel dif5erent letters denote significant ciifferences among ages.
neer-terni pmgflaflt fetus adut
near-term pregnant fetus aduit
- near-terni pregnant near-terni pregnant
fetus adult fetus adult
Figure 4. Effect of probenecid (lefi panels) and oxygen concentrations (right panels) on 3 H-PGF2, uptake (measured as tissue-to-medium ratio for radioactivity, TM) and catabolism (rneamed as % total radioactivity migrating with PG metabolites, PGM) by the choroid plexus fkom fetal and adult sheep. Measurements were made &er a 6emi.n incubation at 37OC. Control open columns. Treatment, closed columns. In left panels, treatment was the addition of 1 mM probenecid to the incubation medium. In right panels, treatment was lowering the p02 of the incubation medium Eom hyperoxic levels @û2: 563-739 rnmHg) to levels approximating normoxia in the fetus (14-20 mmHg) and adult (5 1-71 mmHg). Values are meam f S.E.M. for nurnber of animals given above each column. For each panel the effects of treatment and age were tested with a two way analy sis of variance (ANOVA). SiBnificant ciifferences between control and treatment groups are denoted with an asterisk. Significant Werences between the fetus and dui t for a given group are denoted with a spade.
Near-Term Fetus 1 'H-PGE~
'H-PGF,,
norm oxia hyperoxia
Pregnant AduIt
normoxia hyperoxia
Figure 5. 'H-PGE~ versus 'H-PGF~, uptake (measured as tissue-to-medium ratios for radioactivity, T/M) by the choroid plexus fiom the fetal (lefi panel) and adult (right panel) sheep. Measurements were made after a 60-min incubation at 37OC. Nonnoxic p02 values were 14- 20 mmHg and 5 1-71 mmHg for fetal and adult tissues, respectively. Hyperoxic p02 values were 563-739 mmHg for both ages. Values are means f S.E.M. for number of animals given above each column. At either age, the effect of labelled PG and oxygen levels on Th4 values was assessed with a two way analysis of variance (ANOVA). Fetal values were log transformed to normalize variance. Significant differences between 3 ~ - ~ ~ ~ 2 and 'H-PGF~, are denoted with an asterisk and these differences were independent of oxygen levels at both ages. With either prostaglandin, ihere was no significant difference between uptake under normoxic and hyperoxic conditions.
Table 1 Effect of temperature on prostaglandin uptake by the choroid plexus fiom fetal and adult sheep
Prostaglandin 4°C 37°C
Near-Term Fet us Pregnant Adult Significance Near-TermFetus PregnantAduh Significance
'H-PGF~, 0.90 f 0.08 (5) 0.68 f 0.19 (4) N.S. 4.6 f 0.4 (1 1) 13.7 + 1.9 (15) P < 0.05
'H-PGE~ 0.64 I 0.02 (6) 0.30 f O. 11 (4) P < 0.05 2.1&0.2(15)* 4.6&0.6(8)' P < 0.05
Choroid plexus was incubated with either prostaglandin for 60 min at the stated temperature. pOz of the incubation medium was 14-28 and 64-84 mmHg for fetal and aduh groups, respeciively. Uptake is expressed as the tissue-to-medium ratio for radioactivity (TM) and values are means f S.E.M. for no. of animals given in parentheses. At either temperature, the effects of labelled PG and age on T/M values were assessed with a two way analysis o f variance (ANOVA). Values at 37OC were log transformed IO noomiaze variance. Differences between the two PGs are indicated with an asterisk and did not depend on age. While ï /M values for either PG in the adult were significantly higher than those in the fetus at 37OC, they were significantly lower for 3H-PGE2 at 4°C.
Near-Term Fetus
I I 1 I 1 I V
O 15 30 45 60 75
Tirne (min)
Pregnant AduIt
l I I I 1 I
O 20 40 60 80 100
Time (min)
Figure 6. Time course of 'H-PGE~ and "c-sucrose uptake (measured as the tissue-to-medium ratio for radioactivity, T/M) by the choroid plexus From the fetal (left panel) and adult (right panel) sheep. Incubations were carried out at 37°C and p02 of the incubation medium was 13-23 and 563-739 mmHg for fetal and adult groups, respectively. In either group of anirnals, the effect of tirne on T/M values, assessed with a one way analysis of variance (ANOVA), was significant. Fetal values were log transformed and adult values were reciprocal transformed to nomlize variance. Comparisons among tirne points were made using the Student-Newman-Keuls test with different letters denoting a significant difference. Comparisons beiween the fetus and adult were made using Bonferroni correcteci t-tests. A significant difference between fetal and adult values was attained at 30 and 60 min. Values are the mean i S.E.M for the number of animals given below each time point for 3 ~ - ~ ~ ~ 2 . '4~-sucrose uptake was measured in the same specimens.
Vi w
S teady-state uptake of 3 ~ - ~ ~ ~ 2 , d e c d prognssivel y with increasing concentrations of
unlabelled PGF2, in the incubation medium (Fig. 7, pg. 54). Cornparison between the fetus ancl
adult (see Fig. 7, pg. 54) revealed a significant ciifference in uptake only in the absence of
unlabelled PGF2,. At both ages, in the presence of excess (i.e. 60 CiM) unlabelled PGFIa, 3 ~ -
PGF2, was d l accumulated against a concentration gradient (Fig. 7, pg. 54). Also at both ages,
TIM ratios were sUnilar in the absence of unlabelled PGF2, and in the presence of 1 pM PGF2,
(Fig. 7, pg. 54), despite a decrease in specific activity fiom 4 x 1 014 dprn/mmol to 6 x 10' ' dpm/mmol. T'us, dilution of label is unlikely to account for changes in T/M ratios. In the fetus,
the total quantity of PGF*, being accumulated by the choroid plexus at steady-state (60 min)
increased signZcantly with increasing concentrations of uniabelled PGF2, (P = 4 x 10~7 fiom
0.006 pmVg (no unlabelled PGF2, added), to 5 pmovg (1 pM uniabelled PGF2, added), to 36
pmoVg (10 FM), to 72 pmVg (20 pM), and to a plateau of around 150 pmoVg (40 and 60 IiM).
A similar increase was seen in the adult (P = 1 x 1 c3'). Thus, decreasing steady-state T/M
values for kbelled PGFIa (Fig. 7, pg. 54) likely resulted fkom cornpetit ion by unlabelled PGF2,
for a saturable mechanism. Concomitant with demashg T/M values in the fetus, levels of 3 ~ -
1 5KD-PGF2, decreased with increasing concentnttions of uniabelled PGFza in incubation
medium (Fig. 7, pg. 54). However, at equal PGF2, concentrations. 3 ~ - ~ ~ ~ 2 , catabolism was
reiatively more affected than uptake (Fig. 7, pg. 54). In the adult, negiigible 3 ~ - ~ ~ ~ 2 ,
catabolism in the choroid plexus under control conditions (see Fig. 3, pg. 48) precluded analysis
of catabolism in the preseme increasing concentrations of unlabeiled PGFza. In separate
experiments in the temi-fetus, addition of physiological concentrations (1 0 nM) of PGFz, to the
incubation medium did not alter contml values of 'H-PGF~, uptake and catabolisxn.
Near-Tm Fetus: 'H-PGF, Uptake Pregnant Adult: 'H-PGF, Uptake 16 16
a a
12 . l2 ' 15
1
Figure 7. Effect of PGFÎa concentration (O, 1, 1 O, 24 40 and 60 pMJ on 3 ~ - ~ ~ ~ t , uptake (measured as the tissue-to-medium ratio for radioact ivit y, T/M) and catabolism (measured as % total radioactivity rnigrating with PG metabolites, PGM) by the choroid plexus from fetal and adult sheep. Measurements were made idter a 60-min incubation at 37'C with either 'H-PGF~, alone or together wiîh unlabelled PGF2.. pOz of the incubation medium was 14-21 and 51-77 mmHg for fetal and aduh tissues, respectively. Note that changes in 3 ~ - ~ ~ ~ t , catabolism could only be studied in the fetus where metabolite levels were above the limit of detection In the fetu, the effect of unlabelled PG concentration on either T/M or PGM values was assessed with a one way analysis of variance (ANOVA) foiiowed by the Student-Newman-Keuls test for individual comparisons. In the aduh, variance could not be normalized by transforming data and, hence, the effect of unlabelled PG concentrations on T M values was assessed with a Kniskal Wallis ANOVA on Ranks foiiowed by Dunn's test for individual comparisons. In all cases, there was a significant treatment effect and different letters denote signiscant differences among concentrations. Cornparison of T M values between the two ages by Bonfenoni corrected t-tests revealed a signifiant merence only in the absence of unlabelled PGF*,. Values are the mean t S.E.M. for the number of animais given below each point.
11-4 DISCUSSION
This study demonsvates that the choroid plexus acquires the capability to accumulate
prostaglandh to a level exceeding that due to fkee diffusion by at least 1 00 d gestation in sheep
(tenn -145 days). This accumulation was temperature-dependent, saturable, and amenable to
inhibition by probenecid, suggesting that it was mediated by the PG-specific carrier. Contrary to
the original hypothesis of the thesis, in vitro PGFt, uptake was similar in the fetus and h b .
Somewhat unexpectedly , stead y-state levels of accumulated PGE2 were Io wer than those for
PGF2,, despite a similar t h e course of uptake. No nich dnerence was observed when
incubations were c h e d out at a low temperature to reduce energy-dependent uptake [9 11.
Sirnilar T/M ratios for the two compounds at low temperatures suggests similar bhding to
extracellular matrix components. Thus, it would appear that greater PGF2, uptake at
physiological temperatures is due to greater uptake by the prostaglandin carrier. However,
earlier kinetic studies in the adult choroid plexus have shown that the 6 i t y of this carrier is
similar for PGEz and PGF2, [91]. In the present study? rather than attaining equal concentrations
in tissue and medium, 3 ~ - ~ ~ ~ 2 a was still accumulated against a twofold concentration gradient
in the presence of excess unlabelled PGF2,. These results imply a component of PGF2, uptake
which is not sustained by either the saturable PG d e r or diaision It remains to be
ascertained whether there is a similar component of PGE2 uptake. An add it ional question is
whether energy-dependent PGE2 uptake is mediated by the same carrier responsible for PGF2,
uptake in the choroid plexus kom the perinatal sheep.
In the fetus, both prostaglandins attained an essentially even distnîbution be tween the tissue
and medium at a low temperature, suggesting that PGs diffuse across the apical surface of the
choroid plexus. In k t , a diffusional component of PG accumulation has k e n reported
previously in the adult choroid plexus [91]. Results of the present study suggest that PO
diffusion is more restricted in the adult than in the fetus. Namely, at low temperature, PGE2
uptake in the adult was signiticantly less than that in the feîus. A similar tendency was seen with
PGFZa, altho ugh dserences were no t statist icall y different. Whether diffusion was intercellular
(Le. through tight junct ions) or intraceliular (i.e. through epithelial ceils) remains to be
established. However, the tendency for PG diffision to be more restricted in the adult is
consistent with the fact that, in vivo, peripherally adrninistered PGs peneûate the brain more
resdily in the fetus [164] than in the adult [103].
Besides king actively accumulated by the fetal choroid plexus, PGF2, was extensively
catabolized, primarily to 15KD-PGF2, by this tissue. The structure of this metabolite implies
cataboiism through the 15-PGDH / 13-PGR enzyme pathway. In some cases, metabohes with
the chromotagraphic mobility of DPGF*, and 1 SKD-PGEt were also detected They likely
onginate fiom, respect ively, 1 SKD-PGF2, reduction at car bon 1 5 and oxidation at carbon 9.
Carbonyl reductase, which is expressed in brain, likely catalyzes these reactions
[141;256;263;367]. However, in all cases, ISKD-PGF2, was the major metabolite formed by the
choroid plexus of the perinatal sheep. 1 5KPPGF2, uptake was similar to that of PGF2. in the
choroid plexus fiom the fetus. Thus, formation of this metabolite is unlikely to alter the overall
level of uptake. Uptake of 15KD-PGE2 could not be quantined due to the chernical instability of
the compound. This instability also posed dittculties in assessing PGE2 catabolism. The latter
issue was resolved in the following study (see Chapter III).
Contrary to the originai hypothesis, PGF2, catabolism was sigdicantly lower in the lamb
than in the fetus, imply h g a loss of catabo tic enzymes soon d e r birth. On the other hand, the
very low catabolic activity in the adult is consistent with data in the literature [1;38].
In the near-terni fetus, PGF2, cataboiisrn appears to operate independently of carrier-
mediated uptake. Specüically, catabolism was not aEected by the traosport inhibitor,
probenecid. This is contrary to the situation in lungs and kidneys, where carrier-mediated,
probenecid-sensitive PGE2 and PGF2, uptake is rate-iimiting for catabolisrn [5; 1437;41].
Furthermore, in saturation studies, 3 ~ - ~ ~ ~ 2 , catabolism was significantly reduced in the
presence of 10 pM unlabelled PGF2,, while a significant reduction in uptake was not seen until
concentrations of unlabel led PGF*, were increased to 20 pM. Taken together, these
observations support the conclusion that catabolisrn operates independently of carrier-mediated
uptake in the choroid plexus of the fetus. Although one would expect a sirnilar functiouai
arrangement for uptake and catabolism of PGEz, no conclusions can be made based on results
presented in this chapter. Thus, unlike the adult, where carrier-mediated uptake is the primary
means by which the choroid plexus regulates prostaglandin levels in ventricuiar CSF [42], the
present study dernonstrates the coexistence of two independent mechanisms in the fetus,
namely, der-mediated uptake and 1 5-PGDH-mediated catabolism. Whether PGE2 uptake
and catabo lism are mediated independently by, respectively, the prostaglandin &er and 1 5-
PGDH is addressed in Chapter m.
CHAPTER III
COMPARISON OF PGE2 AND PGFh CATABOLISM, UPTAKE AND RELEASE BY THE CHOROID PLEXUS IN THE PERINATAL
SHEEP
III-1 BACKGROUND AND RATIONALE
Results in C hapter II demonstrated that, in the fetal sheep, PGF2, removal by the choroid
plexus fiom the surroundhg incubation medium is mediated by two independent mec hanisms:
uptake and catabolism. W e uptake in the lamb was sirnila, to that in the fetus, catabolism was
much lower in the older animal. Results suggested that PGF2, uptake and catabolism are
mediated by the prostaglandin carrier and the 1 5-PGDH / 1 3-PGR complex, respective1 y.
Spontaneous breakdown of the PGEl metabolite formed through the above enzyme complex
complicated quantification of PGEz catabo lism. Considering the marked postnatal decrease in
PGF2. catabolism, and the possible implications of this decrease, it was important to assess the
extent and developmental profile of PGE2 catabolism. Since 15-PGDH catalyzes oxidation of
the two compounds with high affinity [243], one would expect PGE2 and PGF2, catabolism in
the choroid plexus to be sllnilar. However, differences were O bserved in uptake of the two
compounds (see Chapter II), despite the high a f i i t y of the prostaglandin carrier for both PGE2
and PGF2, [9 11. Thus, it is plausible that diffient mechanisms contribute to uptake and
catabolism of these prostagiandins.
The primary objective of the present study was to examine PGE2 catabolism by the perinatal
sheep choroid plexus. In brief, technical difficulties were overcome be chemically treating
tissues [48; 1201 prior to d y s i s of catabolism by thin-layer radiochromatography. An
additional objective was to determine whether PGE2 and PGFÎa compete with each other for the
sarne carrier and enzyme systems. To address these questions, lateral ventricle choroid plexuses
fiom the same animal were incubated separately with, respectively, 3 ~ - ~ ~ ~ 2 and 3 ~ - ~ ~ ~ z , In
experiments where specincity of uptake and catabolism was asse& both choroid plexws
were incubated with the same tracer. Separate experiments were h e d out with the combined
third and fourth (IIVIV) venbncle c horoid plexusa to ascertain whether PG disposal mechanisms
are similarly organizRd at al1 sites.
III-2 MATERIALS AND METHODS
111-2.1 Aaimals
Choroid plexuses were collected &O in near-terni fetal sheep (rnean age 1 3 8 days, range 1 34-
142 days; term -145 d); iambs (mean age 17 days, range 13-2 1 days) and pregnant adult sheep
(> 1 year of age) according to the methods reported in Chapter 11. A subgroup of fetuses were
delivered b y Cesarean section immediately d e r g iving excess sodium pento barbital to the ewes.
Cornparison of results fiom these fetuses with results nom fetuses delivered under normal
anesthesia (see C hapter II Section 11-2.1 ) did no t reveal any sîgnificant difXerence (F==û.867 and
0.443 for uptake and catabolism, respectively). In the present study al1 lambs were killed with
excess sodium pentobiirbital.
111-2.2 Materials
AU materials used in the present study have been described in Chapter II, Section II-2.2.
Purity of 'H-PGE~ and 'H-PGF~, on TLC was 95% and 96%, respectively.
III-2.3 General Procedure
Choroid plexuses were removed and transported to the laboratory as previously demibed
(see Methods, Chapter II). Tissues were pre-equilibrated in Krebs-Henseleit medium on ice with
either 2.5% 9 (fetus: mean p02 17 mmH& range 13-25) or 12.5% 0 2 (lamb and adult: mean
p9: 67 rnmHg, range 54-83) plus 5% Ca and balance N2. Each lateral ventricle choroid plexus
sample was incubated separately. Being srnali in size, specimens fiom the third and fourth
(m/rV) ventncle were pooled In some cases, ody part of the choroid plexus could be retrieved.
The mean wet weight for the lateral ventricle choroid plexus was 37 mg (range, 9 - 74), 42 mg
(rage, 14 - 85) and 66 mg (range: 22 - 1 94), for, respectively, the fetus, lamb, and adult. The
mean wet weight for the IIIAV ventricle choroid plexus fiom these age groups was 30 mg (range,
20 - 41)- 45 mg (range, 32 - 54), and 44 mg (range, 1 O - 1 18), respectively. Mean T/M d u e s
for 14c-sucrose (see Chapter II Section 11-2.5) were around 0.4 regardless of age and tissue
source. Four of 107 animals were excluded on the basis of abnomially high T/M values for
sucrose (see Chapter II, Section II-2.5).
III-2.4 h c u bation Procedu re
Each lateral ventricle choroid plexus and the IIYIV ventricle choroid plexuses was incubated
separately with 3 ~ - ~ ~ ~ z or 3 ~ - ~ ~ ~ 2 , plus '4~-sucrose as reported in Chapter II, Section 11-2.4.
In some tissues where 3 ~ - ~ ~ ~ 2 was used as tracer, probenecid was also added to the incubation
medium ( fml concentration, I mM). In separate experiments, unlabelled PGF2. was added
instead (final concentration, 60 W . Altematively, the same concentrations of unlabelled PGE2
was added to the incubation medium of tissues when 'H-PGF~, was used as tracer. The
concentration of uniabelled prostaglandin was chosen to ensure satunition of both uptake and
catabolism based on the saturation studies reported in Chapter II. However. control incubations
with PGE2 (60 CiM) were also done to connmi 3 ~ - ~ ~ ~ 2 seifkaturation. All incubations lasted
60 m . to m m e steady-state uptake (see Chapter II). For the release study (see below), tissues
were removed fkom medium a f k 60-& incubations, rinsed briefly, and then placed in fiesh
medium over difFêrent t h e intervals (5, 30,60,90 and 120 min). Incubations were terminated
by retrieving tissue specimens fiom the medium and dragging them over aiuminum foil to
remve the adhering fluid. Media and tissues were processed for iiquid scintillation
spectrornetry according to methods reported in Chapter II, Section il-2.5. The mean recovery of
radioactivity aAer combining counts in tissue and medium was 87% and 70% for 'H and I4c,
respectively. In the release study, radioactivity was measured in both the original incubation
medium and the series of washout media.
III-2.5 Determination of PG Catabolism
In preliminary experiments with choroid plexus specimens that had been incubated with 'H-
PGE2 and then subjected to TLC analysis, a major peak was obsemed having greater mobility
than PGA2 (Le. the non-enzymatic breakdown product of PGE2). This suggested the presence of
the highly lipophilic, non-enzymatic breakdown product of 1 SKD-PGE2, namely, 1 1 -deoxy-
13,14-dihydro- 1 5-keto-1 1 p, 1 6c-cyclo-PGE2 (bicycle-PGE*) [48; 1 201. This compound
originates fiom spontaneous re-anaagement of 1 5KD-PGA2, the dehydration product of 1 5KD-
PGE2 [120]. To verify that breakdown did not occw during tissue incubation, storage, or
extraction, the 100,000 x g supernatant hction of fetal lung was incubated with 'H-PGE* to
generate 3 ~ - 1 SKD-PGF via the 15-PGDH / 13-PGR system [265]. There was no breakdown of
1 SKD-PGE2 in lung samples (see Appendix A-1, pg. 64). Thus. one may surmise that
breakdown occurs during the drying of tissues. To confirm that the breakdown product in
choroid plexus samples was bicyclo-PGE2, lung sarnples were treated chemicaily to promote
conversion of 1 SKD-PGE2 to bicyclo-PGE2. Namly, samples were alkylated to pH 1 1 - 12 for 1
h at room temperanire [ 1201. TLC analysis of these samples revealed a major peak with mo bility
equal to bicyclo-PGE2, which was generated by alkylating authentic lSKPPGE2 (Appendix A-
l, pg. 64). Medium incubated with 'H-PGE~ alone and processeci in the same way showed two
major peaks rnigrating with PGEz and its dehydration product, PGA2 (Appedk A- 1, pg. 64).
ControI C
-1 J d C
O a m. * t * . a . v IV Ill II I V IV III 11 1 t
Appendix A- 1. Thin-layer radiochromatograms of Krebs medium (a&b) and homogenates ( 1 :20 wlv) of pre-term fetal lung (c & d) incubated with N-PGE2. a & c: wunples extracted under acidic conditions. b &d : samples extracted under acidic conditions following alkylation (pH 1 1-12) for 1 h at rwrn temperature. Markers are ( 1 ) PGE*, (II) ISK-PGE,, ( III) 15KD-PGE2, (IV) PGA2, and (V) alkylated ISKD-PGE2. Ongin is at arrow.
+1 N NaOH
d 3
v IV III II v IV III 11
To standardize quantification of 3 ~ - ~ ~ ~ 2 cataboiism in the choroid plexus, al1 tissues were
treated chemically in the manwr described above. Tissues were then e m t e d three times with
ethy l acetate, under acidic condit ions, and subject to TLC analysis accord& to reported methods
(Chapter II, Section 11-2.6). With the exception of the alkylation step, 'H-PGF~, samples were
processed in the same mannet. M m recovery of tritium following ethyl acetate extraction was
69%. For TLC anaiysis of 'H-PGE~ incu bates, plates were developed in a diethy 1 ether-acetic
acid system (99: 1, by vol.). This system gave the best mobility for authentic PGE2 (Rf- 0.05)
and the best resolution between PGEl and its enzymatic and non-enzymatic breakdown products
(IV= 0.4,0.6,0.75 and 0.9 for, respectively, 15K-PGE2, lSKPPGE2, PGA2, and bicyclo-PGE*).
Often, an addit ionai, more polar, peak was seen just ahead of bicyclo-PGEs and likely represents
a second epirner of this cornpound [120]. TLC analysis of 'H-PGF~, samples and preparation of
TL.C plates for iiquid scintillation spectronietry has been described (Chapter II, Section 11-2.6).
Mean recovery of tritium h m TLC plates was 87%. Caiabolism is expressed as the percent
total radioactivity migrating with inactive PG metabohes (PGM). In any instance with
catabolism, metabolite peaks were included in the final tabulation oniy ifradioactivity was two
standard deviations above background leveis of radioactivity (Le. greater than 8% total
radioactivity on the pke). In cases where no metabolite peaks were above this cutoff.
tabulatioas of PGM included oniy the radioactivity migrating with the primary metabolite.
III-2.6 Determination of PG Uptake and Release
'H-PG and '4~-sucrose uptake was quantified as the tissue-to-medium ratio (TM) for
radioactivity (see Chapter II, Methods). Release was quantifieci as the total dpm in washout
media per mg wet choroid plexus weight (dpm x mg") at each time interval.
iII-2.7 Anaiysis of Data
Al1 data are expressed as the mean & S E M . where n is the number of animals. Tests used
for statistical cornparisons are described in figure legends. DXerences were considered
significant when P c 0.05.
II-3.1 PG Catabolism by the Lateral and III 1 IV Ventriele Choroid Plexus
To compare PGE2 catabo lism with that of PGF2, lateral ventricle choroid plexus samples
fiom the same animals were incubated separately with, respectively, 3 ~ - ~ ~ ~ 2 and 'H-PGF~,. In
the fetw and larnb, -'H-PGEI was converted ptimarily to a peak coinciding with the less polar
epirner of the non-enzymatic breakdown product of 1 SKD-PGE, (i.e. bicyclo-PGE2) (Fig. 8, pg.
68). A second peak migrating with the more polar epimer of bicyclo-PGEz was also observed in
the majonty of fetal samples (Fig. 8, pg. 68). In addition, bicyclo-PGE2 was detected in the
lateral ventric le c horoid plexus of the adult, though in that case the rnajority of radioact ivity
migrated with PGE2 and its non-enzymatic breakdown product, PGA2 (Fig. 8, pg. 68). TLC
scans of tissues incubated with 'H-PGF~, (Fig. 8, pg. 68) confirmed earlier results (see Chapter
II). Whether in the lateral or LII/IV ventricle choroid plexus, PGE2 catabolism in the ktus was
similar to that in the lamb (Fig. 9, pg. 69). This sharply con- the large decrement in PGF2,
cataboiism between these two age groups (Fig. 9, pg. 69). In general catabolism was lower in
the adult than in the perinatai animal (Fig. 9, pg. 69). The only exception was PGF2, catabolism
in the m/N ventricle choroid plexus, where equally low levels were observed in the lamb and
adult (Fig. 9, pg. 69). The only case where catabolism in the aduh exceeded background leveis
was with PGE2 in the lateral ventncle choroid plexus (Fig. 9, pg. 69).
To ver@ that PGEz and PGFz, compete with each other for the same catabolic eraymes in
the choroid plexus of the fetus, catabolism of each compound was assessed in the presence of
hi& concentrations of the other compound. In both the lateral and LII/IV ventricle choroid
plexus, 'H-PGE~ catabolism was reduced in the presence of excess PGF2, and 3 ~ - ~ ~ ~ 2 ,
. . VI v IV III II Y 1
VI v IV III Il I
Fetus d R
. ..a a v iv iii ii i
Lamb
a.. a v iv iii ii i
Adult 1
Figure 8. Thin-layer radiochromatograms of lateral ventricle sheep c horoid plexus incubated with 3H-PGEz (lefi panels) and 3 ~ - ~ ~ ~ 2 a (right panels). a & d: near-term fetus. b & e: 17 d lamb. c & fi pregnant adult Tissues were incubated with the appropriate tracer at 37 O C for 60-min. pOz of the incubation medium was 14- 17 and 56-76 de; with prenabl and postnatal groups, respectively. Markers on the lefl are (I) PGEt, (II) ISK-PGE2, (III) I SKD-PGE2, (IV) PGA2 , (V and VI) epimers of alkylated 1 SKD-PGE 2 . Markers on the right are (i) PGFIa, (ii) D-PGF& (iii)lSK-PGF*,, (iv) ISKPPGF2a, and (v) 15-PGE2. On'gin is at arrow.
Lateral Ventricle Cho raid Plexus 111 4 IV Ventrlcle Choroid Plexus
* - 7 8
1
I T b
I 17 d lem b
p i e g n a n l ad ult
near - t srm f s i u i
17 d p r e ~ n a n t lem b adult
Figure 9. .'H-PGE~ versus 'H-PGF~, catabolism (measured as the % total radioactivity migrating with PG metabolites, PGM) by the lateral (left panel) and IlInV (right panel) ventricle choroid plexus from sheep. Measurements were made afler a 60-min incubation at 37°C. p02 of the incubation medium was 14- 17 and 56-76 mmHg with prenatal and postnatal groups, respectively. For the lateral ventricle choroid plexus, transforming data could not normalize the variance and, hence, a two way repeated measures analysis of variance (ANOVA) could not be perforrned. Individual one way ANOVAs (PGE*, Kruskal Wallis ANOVA on Ranks with Dunnts test; PGF2,, ANOVA with Student-Newman-Keuls test) showed a significant effect of age on PGM values. Different letters (PGE2, bold; PGF2,, italics) denote significant differences among ages. Differences between the two PGs were assessed with Bonferroni corrected t-tests and are denoted with an asterisk. For the IlIlIV ventricle choroid plexus, the effects of age and labelled PG on PGM values were assessed with a two way ANOVA. Both variables haà a significant effect and the efTect of age depended on the labelled PG. lndividual cornparisons were made using the Student-Newman-Keuls test, with different letters (PGE2, bold, PGF*,, italics) denoting significant differences among ages and an asterisk denoting a significant difference between the two PGs. Values are the mean f S.E.M. for the number of animals given above each column.
catabolism was reduced in the presence of excess PGEt (Table 2, pg. 71). With either
prostaglandin, the reduction in catabo h m was similar to that O bserved under se fisaturahg
conditions (Table 2, pg. 71). However, regardless of the unlabelled prostaglandin, inhibition of
'H-PGE~ catabolism was only partid, whereas that of 3 ~ - ~ ~ ~ 2 , catabolism was nearly complete
(Table 2, pg. 71).
To evaluate whether PGEz catabolism depends on carrier-mediated uptake, the effect of the
transport inhibitor probenecid was assessed. Consistent with earlier findings with 'H-PGF~, (see
Chapter II), 'H-PGE~ catabolism in the fetus was not affected by probenecid (Fig. 10, pg. 72).
This was aiso the case in the iamb (Fig. 10, pg. 72). In contrast to the perinatal animal PGE2
catabolism in the adult was reduced in the presence of probenecid (Fig. 10, pg. 72).
III-3.2 PG Uptake and Release by the Lateral and III f IV Ventricle Choroid Plexus
The specificit y of 'H-PGE* uptake was assessed by measuring T/M nitios in the presence of
either probenecid or excess prostaglandin. The cross-saturability of 'H-PGF~, uptake was also
evaluated. In ôoth the perinatal animal and the adult, 'H-PGE? uptake by the choroid plexus
was reduced in the presence of probenecid (Fig. 10, pg. 72). In the fetus, 'H-PGE~ uptake was
also reduced by saturaîing concentrations of either PGE2 or PGF2, and 'H-PGF~, uptake was
reduced by saturating concentrations of PGE2 (Table 2, pg. 71). In generaI, results nom cross-
saturation studies in the adult were similar to those in the fehis (Table 3, pg. 73). The ody
exception was 'H-PGF~, uptake in the WiV venîricle choroid plexus, where the addition of
excess PGE2 did not cause a statistically significant reduction in uptake (Table 3, pg. 73).
However, T/M values in these tissues were less than 50% of control values (Table 3, pg. 73).
in both the adult and fenis, 3 ~ - ~ ~ ~ 2 , uptake under saturathg conditions tended to be higher
Table 2 'H-PG uptake and catabolism by the choroid plexus fiom fetal sheep in the absence and presence of unlabelled prostaglandins - -- - - --
NEAR TERM FETUS LATERAL VENTRICLE 111 / 1V VENTRICLE
Control 8.0 f 1 .O (6) 7.4 I 1.1 (5)t 60 PM PGE2 1.3 f O. 1 (6)* 1.9 f 0.3 (5)*
Control 3.5 I 0.5 ( 5 ) [4.6 t 0.4 (1 l)] 60 pM PGF2, 1.1 f 0.1 (5)* [1.8 f O. 1 ( 1 3)]*
'H-PGE~ 'H-PGF*. Catabolism (PCM) 'H-PGE~ 'H-PGF~,
Control 69 + 3% (6) 73 i 3% (5) 60 FM PGE2 48 k 6% (6)* 1 1 f 4% (5)* t
ControI 73 f 1 % ( 5 ) [58 f 3% (IO)] 60 pM PGF*, 41 f 5% (5)* [8 t 2%(13)]*
61 f 2% ( 5 ) [58 f 3% (IO)] 21 f 4%(4)* [8*2%(13)]*
Choroid plexus was incubated with labelled prostaglandin (0.25 pCVml) alone or together with 60 pM unlabelled prostaglandin for 60 min at 37°C. Uptake is expressed as the tissue-to-medium ratio for radioactivity (TM). Catabolism is expressed as the % total radioactivity migrating with PG metabolites (PGM). pOz of the incubation medium was 14-2 1 mmHg. Values are means f S.E.M. for no. of animals given in parentheses. Values in square brackets are fiom saturation studies in Chapter II (specimens pooled fkom al1 cerebral ventricles). For PGE2 sel f-inhibit ion studies (lateral ventricle), cornparisons bet ween control ( 3 ~ - ~ ~ ~ 2 alone) and treatment (60 pM PGE2) were made with a paired t-test. For cross-inhibition studies in either the lateral or W i V ventricle choroid plexus, a two way (repeated measures) analysis of variance (ANOVA) was used to assess the effects of treatment (control vs. 60 pM unlabelled PG) and labelled PG (PGE2 vs. PGF2,) on either T/M or PGM. In al1 cases, both effects were significant and the treatment effect depended on the labelled PG. Differences between control end treatment groups are indicated with an nsterisk. In either group, differences between the two PGs are denoted with a spade. r!
Lateral Ventricle C horoid Plexus
'H-PG E,, Control -- -
'H-PG E,, Probenecld * . . Uptake 4 - a
- near-term I f d pregnant
fetus lamb adult
'O0 Catabolism
near-terni 17 d pregnant fetus lamb adult
Figure 10. Effect of probenecid (1 mM) on 'H-PGE* uptake (measwd as the tissue-to- medium ratio for radioactivity, T/M) and catabolisn (rneasured as the % total radioactivity migrating with PG metabolites, PGM) in the lateral ventricle choroid plexus fiom sheep. Measurements were made d e r a 60-min incubation at 37OC. & of the incubation medium was 13-20 and 56-75 mmHg for prenatal and postnatal groups, respectively. Control, open bars. Treatment, closed bars. For both uptake and catabolism, a two way repeated maures analysis of variance (ANOVA), foiiowed by the Student-Newman- Keuls test for individuai comparisons, was c d e d out to test the effects of treatment and age on, respectively, T M and PGM values. T M values were log transformed to normalize variance. The significant effèct of probenecid on both uptake and cataboiism depended on age. For each group, different letters (control, bold; treatment, italics) denote significant differences among ages. DifTerences between control and treatment groups are denoted with an asterisk Values are the mean f S.E.M. for number of anirnals given above each column
than 'H-PGE~ uptake under equivalent conditions (Table 2, pg. 7 1 and Table 3, pg. 73).
Despite the fact that the two prostagiandins competed with each 0 t h for the same saturable
uptake mechanism, steady-state levels of accumulated PGEz were lower dian those for PGF2, in
the perinatal sheep (Fig. 1 1, pg. 75). A similar trend was O bserved in the adult, although
dflerences were not statistically significant (Fig. I 1, pg. 75). In contrast to dserences in
uptake, steady-state release was similar for the two prostaglandins in the fetus and the adult
(Fig. 12, pg. 76).
Lateral Ventricle C horold Plexus
near-terrn tetus
1 1 B
1
1 3 8
I 1
,
,
I
\
pregnant adull
III 1 IV Ventricle Choroid Plexus
'H-PGE,
near-terrn 17 d lelus lam b
.a
I pregnant
adult
Figure 1 1 . 'H-PGE~ versus 3 ~ - ~ ~ ~ 2 , uptake (measured as the tissue-to-medium ratio for radioactivity, TM) by the lateral (left panel) and IIVIV (right panel) ventricle choroid plexus fiom sheep. Measurements were made afier a 60-min incubation at 37OC. p02 of the incubation medium was 14-17 and 56-76 mmHg with prenatal and postnatal groups, respectively. For the lateral ventricle choroid plexus, transforming data could not normalize the variance and, hence, a two way repeated measures analysis of variance (ANOVA) could not be performed. For either PG, a one way ANOVA did not reveal a significant change with age (PGE2, bold letters; PGF2,, italic letters). Differences between the two PGs were assessed with Bonferroni corrected t- tests and are denoted with an asterisk. For the Il InV ventricle choroid plexus, the effects of age and labelled PG on TM values were assessed with a two way ANOVA. Values were log transformed to normalize variance. Both variables had a significant effect and the effect of age depended on the labelled PG. lndividual cornparisons were made using the Student-Newman-Keuls test, with different letters (l'GE2, bold, PGF2,, italics) denoting significant differences among ages and an asterisk denoting a significant difference between the two PGs. Values are the mean f S.E.M. for the number of animals given above each column.
Near-Term Fetus Pregnant Adult Lateral Ventricle Choroid Plexus Lateral Ventricle C horoid Plexus
a 3 ~ - ~ ~ ~ , 'H-PGE, r 'H-PGF,, A 'H-PGF,~
. "c-sucrose . ''~-sucrose 15 - "c-sucrose 15 = ' ' ~ - ~ ~ ~ r o ~ e
Time (min) Time (min)
Figure 12. 3 ~ - ~ ~ ~ 2 versus 'H-PGF~, release (measured as dpm in washout medium per mg wet choroid plexus weight) by the lateral ventricle choroid plexus fiom sheep following a 60-min incubation at 37OC with labelled compound (see Methods). ' ~ ~ u c r o s e (open symbols) release was measured in the same tissues. pOI of the original hcuôation and subsequent washout media was 16-25 and 61 -83 mmHg for fetal and adult groups, respectively. For either group, variance could not be normalized by transforming data and, hence, a two way repeated measures (RM) ANOVA could not be perforrned. For PGE2 release in the fetus, a one way RM ANOVA, followed by the Student-Newman-Keuls test for individual comparisons, showed release changed significantly with tirne. For al1 other curves, a Friedman Rh4 ANOVA on Ranks, followed by Dunn's test for individual cornparisons, also showed that release changed significantly with tirne. Ditierent letten (bold, PGE2; italics, PGFZa) denote significant diflerences arnong time points. At either age, comparisons between the two PGs at each time point were made with Bonferroni corrected paired t-tests and did not reveal a statistical significance. Values are the mean f S.E.M. of 4 animais for each group.
LII-4 DISCUSSION
The present study confÏrms that POE2 is converted to l e s polar metabolites in boîb the lateral
and IWIV ventricle choroid plexus of the near-terni sheep fetus. Although the identity of these
metabo lit es was not assessed by mass-spectrometry, several pieces of evidence suggest that this
conversion was catdyzed by 15-PGDH followed by 13-PGR First, PGF2, was catabolized to
ISKD-PGF*. by tissues fkom the same animals, and the identity of this metabolite was confirmed
by mass spectrometry (see Chapter II). Secondly, it was shown that PGE2 and PGF2, cornpete
with each 0 t h for the same catabolic system. Thirdly, the product detected in alkylated fetal
choroid plexus samples following incubations with 'H-PGE~ had the sanie chromatographie
mobility of the metabolite recovered fiom similady treated lung samples (see Appendix A-1, pg.
64). It is hown that 15-PGDH and 13-PGR are both functional in lung of the fetal sheep
[265;353]. Lastly, lung samples which were not alkylated had a major peak migrating with
authentic 1 SKD-PGEt (Appendix A-1, pg. 64). Taken together, these findings form a strong
case for the existence of the 15-PGDH / 13-PGR compkx in the choroid plexus of the fetd
sheep.
Whether in the lateral or the m/lV ventncle choroid plexus, PGE2 caîabolism did not dSer
between the term fetus and the kmb. In con- PGF2. catabolism was markedly reduced in the
lamb compared with the fetus. The postnatal decrease in PGF2, catabolism is consistent with
resuhs h m Chapter II. However, if this change for PGF2, had ken the result of decreased 15-
PGDH activdy, one would have expected similar age-reiated changes for PGE2. A possible
explanation for this apparent discrepancy is that PGE2 and PGF2. are king catabolized by
dSerent enyme pools in the choroid plexus and tbat the individual pools undergo differentiai
development. However, this hypothesis is inconsistent with the fïnding that the two
prostaglanduis compete for the same cataboiic emymes. Altemtively, PGEr and PGFza could
be catabolized by a single enzyme pool which may be e q d y accessible to the two compounds
in the fetus but not in the lamb. Any such dserence in accessibility could, in turn, be related to
dserences in the mechanisms of PGEI and PGF2, uptake.
As with studies in choroid plexus samples pooled fiom ail cerebral ventricles (see Chapter
U), steady-date levels of accumulated PGE2 were lower than those for PGF2, in both the lateral
and iII/IV ventricle choroid plexus fkom the perinatal animal. In con- to steady-state uptake,
stead y-state release of the two pro staglandins was equal and, thus, cannot account for differences
in levels of the accumulated compounds. Furthermore, PGE2 and PGF2, competed for the same
saturable carrier. Based on these k t s , the two compounds should have be taken up to an equal
degree [9 1 1. Yet, uptake was generally pater for PGFt, than PGE2. One possible explanat ion
for this apparent discrepanc y is that there is an additional component of PGF2, uptake which is
not shed by PGE2. Worth noting in this context is that, in the presence of excess udabelled
prostaglandin, only 'H-PGF~, was accumulated against a concentration gradient. Taken together
with earlier fïndings (see Chapter II), these results suggest that PGE2 uptake in the perinatal
sheep chomid plexus is mediated by the prostaglandin c h e r and by diffusion, and that PGFt.
uptake is mediated by the same mechanisms plus an accessory carrier. Validation of this
hypothesis would require M e r experimentation in vitro. Furthermore, one wouid need to
assess the fiinctional relat ionship between uptake by this hypothetical carrier and catabolism by
15-PGDH,
Consistent with kdings for PGF2, (see Chapter II), PGEz catabolism did not depend on
carrier-mediateci, probenecid-sensitive uptake in the choroid plexus nom the perinatal h e p . In
contrast, carrier-mediated uptake was necessary for catabolism in the adult. Thus, foilowing the
perinatal period, there is both a decrease in catabo lism and a change in its funct ional relat ionship
with uptake. This functional re-arrangement may be linked to the apparent decrease in PG
diffusion tollowing the perinatal perïod (see Chapter II). Specifcally, in the absence of Carnec-
mediated uptake in the younger animal PGEz rnay still gain access to 1 5-PGDH by difision.
However, in the aduh, where diffusion may be more restricted, catabolism will depend on
carrier-mediated uptake.
If PGFID is less accessible to 15-PGDH in the lamb compared to the fetu because of its
interaction with an accessory uptake mechanism then one of two things rnay take place: either
the site of the accessory c h e r or the site of 15-PGDH changes &er birth The latter possibility
is addressed in Chapter IV.
CHAPTER IV
LOCALIZATION OF 15-PGDH PROTEIN AND ACTMTY IN THE CHOROID PLEXUS OF THE PERINATAL SHEEP
IV-1 BACKGROUND AND RATIONALE
The k t that PGEz catabolism in the choroid plexus does not change during the p h t a 1
period suggests thst the decrease in PGF2, catabolism o c c e at this t h e is not due to a loss
in 15-PGDH activity (see Chapter DI). It is possible that the different age-related profiles in
catabolism of the two compounds result fiom a change in the site of 15-PGDH expression
Namely, in the fetus, both prostaglandins are extensively catabolized, suggesting that 15-PGDH
is localized at a site readily accessible to these compounds. In contrasi, in the lamb, PGE2
catabolism is extensive whereas PGF2, catabolism is moderate to low. It is possible that, &er
birth, 15-PGDH is localized to a site which is readiiy accessible to PGE2, but not PGF2, To test
this possibility, the distribution of 15-PGDH protem and catalytic activity was examined ushg
immun0 histochemical met hods and bioc hemical assays on tissue h t ions.
IV-2 MATERIALS AND METHODS
IV-2.1 Aaimab
Experiments were c b e d out in sheep choroid plexuses fiom the pre-texm fetus (mean age:
102 d gestation, range 10 1 - 103 d; term -1 45 d), near-term fetus (man age: 1 36 d gestation,
range 134-1 38 d), newborn (mean age: 5 ci, range 5-6 d) and older lamb (mean age: 15 d, range
1 5- 1 8 d), and the pregnant ewe (> 1 year). Lung samples were collected from the near-term fetus
as a positive control. Fetuses were delivered by Cesarean section, and both fetuses and ewes
were killed as described previously (Chapter II). Lambs were killed with excess sodium
pentobarbital (see Chapter II).
IV-2.2 Materials
Polyclonal antibodies raised in guinea pig against NAD'-dependent 15-PGDH piained fiom
pregnant rabbit lung were generously supplied by Dr. Richard T. Okita (College of Pharmacy,
Washington Staîe University). Ant ibodies were prepared according to a published method [259].
Antibody stock was verified with immunofluorescence histochemimy in lung samples ikom
pregnant rabbits (25 d gestation, term -28 d) taken fkom out general stock. Fluorescein
isothiocyanate (FITC)-labelled anti-guinea pig IgG, nicotinamide adenine diaucleotide (NAD'),
nicotinamde adenine dinucleotide phosphate (NADP'), bovine serum albumin (BSA, Cohn
hction V), and indomethacin were all purchased fiom Sigma (St. Louis, MO). Colloidal gold-
labelled protein A (particle size, 10 nm) was purchased fiom Amershm (Arlington Heights, Ill.).
Leupeptin, 1,4dithiothreitol @TT), and chymostatin were purchased fiom Boehringer
Mannheim (Mannheim, G e m y ) . Chernicals and solvents used for immunohîstochemical
-dies were of analytical grade purity and buffers were prepared with ultrapute water. For ail
other materials refer to Chapter II, Section U-2.2.
IV-2.3 Light Microscopy Immunofluorescence Histoehemistcy
Choroid plexuses were removed h m al1 cerebral ventricles and p k e d in ice-cold, pre-
equilibrated Krebs-Henseleit buffer for transportation to the laboratory (see Chapter 11). Samples
were cleaned of meningeal tissue, flash fiozen in OCT, and stored at -70°C for cryosectioning.
Cryosections (5 PM) were fixed in 2% paraformaldehyde for 30 min, washed thoroughly with
BSA / glycine (0.5% / 0.15% in phosphate buffered saline, PBS) and blocked with BSA (0.5% in
PBS). The sections were then incubated with a 1:75 dilution (in 5% milk stock) of guinea pig
anti-senun against rabbit huig NAD+-dependent 15-PGDH for 60 min. Following a thorough
wash in BSA (0.5% in PBS), sections were incubated with a 1 :75 dilution (in PBS) of FITC-
conjugated anti-guinea pig IgG for 60 min in the dark. Sections were thoroughly washed with
PBS and mounted. Negative controls were sections incubated with either 0.5% BSA or
preimrnune guinea pig semm instead of the primary antibody. Positive controls were lung
simples collected fiom the near-term fetus. A positive reaction in fetal sheep lung and pregnant
rabbit lung (see above) confïxmed previous hdings [259]. AU immunohistochemical staining
was carried out in a humid chamber. Images were acquired on a confocal microscope (Leica,
Mode1 DM 1 RB). Images of sections where primary antibody was omitted were overexposed in
order to improve visibility of morpho logical elements.
IV-2.4 Transmission Electron Microscopy Immunogold Bistochem istry
Choroid plexus samples were retrieved nom all cerebral ventricles and irnmediatefy placed in
4% paraformaidehyde. This fixative was chosen to minimize cross-linking and Unprove access
to epitopes. Fixed tissues were transferred to a Petri dish, stretched out, and cut into pieces of
about 0.5 mm length. They were then placed in &esh fixative and lefi for 4 h at room
temperature. Fixative was rinsed off and replaced with 0.1 M PO4 1 0.02 M sodium azide for
storage at 4OC. Ultrathin cryosections (90 nrn) were prepared us& a Reichert Jung Ultracut E
microtome (Vienna, Austria) equipped with a FC4 cryochamber. Sections were then transferred
to glow-discharged carbon-Fonnvar nickel grîds ushg a loo p of rno lten sucrose.
Immunolabeiling was carried out according to rnethods outlined earlier (Section IV-2.3, this
chapter), except that the secondary antibody was colloid gold-conjugated protein A (1 :20 in
PBS). M e r incubation with the secondary antibody, sections were washed thoroughly with PBS
followed with double-distilled water. They were then incubated in neutrai m y l acetate for 10
min, washed with double-distilted water and stabilized in 0.2% uranyl acetate in methyl cellulose
for 10 min. Specirnens were viewed and photographed in a JEOL mode1 1200 ExlI transmission
electron microscope. Quantification of gold particle density was carried out in 3 samples for
each age group. After counting the nurnber of particles per field @m2) at a rnagnification of
25,000 ushg NIH Image, an average of 25 randody chosen fields was tabulated.
N-2.5 Homogenized Tissue Incubations
Fetal choroid plexus samples were collected accordhg to methods descnbed previously
(Chapter II, Section II-2.3). Lung samples nom the near-term fetus were used as a positive
control. Tissues were transferred to icecold phosphate buffer (50 mM -PO4 I NaOH, pH 7.4,
1 :20 wth l ) and homogenized (Polytron) on ice. In some studies, the homogeaization buffer
contained one of the fo llowing compouods: indometbacin (2.8 or 28 pM), NAD+ (5 mM), D?T
(1 mM), EDTA (1 mM), glycerol(20% by vol), leupeptin (1 or 10 ~LM), or chymostatin (10 or
100 @Pl). For studies in the 100,000 x g supernatant fmctions, 1 : 10 (wt/vol) homogenates were
prepared according to the above methods and centrifuged at 1 0,000 x g for 1 5 min at 4'C. The
10,000 x g supernatant fiaction was then centrifiiged at 100,000 x g for 1 h at 4OC. Protein
concentraiion was measured in total hornogenates and 100,000 x g supematant fktions
according to the method of Lowry [2 1 O]. Mean protein concentrations were 1.34 @ml and
1 .O6 mglm1 for choro id plexus and lung preparations, respect ively.
Total homogenates and 100,000 x g supernatant fiactions were incubated with either 'H-
PGEz or 'H-PGF*, (0.25 pcilrnl) and NAD+ or NADP+ (5 rnM) for 60 min at 37OC in ambient
air. In experiments in whic h the homogenizît ion buffer was supplemented with NAD+ (see
above), no addit ional CO factor was added. Reactions were terminated either by acidifcation wit h
1 M citric acid followed by immediate extraction with ethyl acetate or by rapid iieezing in a dry
ice-acetone bath. Frozen sarnples were stored at -20°C, thawed and acidified on ice for ethyl
acetate extraction.
IV-2.6 Minced Tissue Incubations
Fetal choroid plexus samples were retrieved kom al1 cerebral ventricles and pre-equilibrated
in Krebs-Henseleit medium according to the reported methods (Chapter II, Section II-2.3). For
preparation of tissue minces, choroid plexuses were chopped into 4 1 pieces on a glas plate on
ice, transferred to 250 pl pre-equilibrated Krebs-Henseleit bufTer and centrifbged at 2,000 x g for
5 min in sealed tubes flushed with the appropriate gas d u r e (pH 7.4). Tissue and fluid
fiactions were then separated and each was incubated with the tracer (0.25 pcilrnl) for 60 min at
37T and the appropriate oxygen concentration @û2, 13-2 1 mmHg). Reactions were termioated
either by ticidifkat ion foiiowed by immediate ethyl acetate extraction or by rapid b z i n g for
storage at -20°C. Frozen samples were thawed and acidifed on ice for ethyl acetate extraction
IV-2.7. Analysis of Catabolism in Homogeiiized and Minced Tissues
The procedures for ethyl acetate extraction and TLC analysis have k e n d ~ m b e d (Chapter
II, Section II-2.6). S ince spontaneous breakdo wn of PGE2 was negligible in these preparations,
tissues were not dkylated to ensure uniform breakdown (see Chapter III, Section 11-2.9.
Solvent systerns used for developing TLC plates were diethy 1 ether-acet ic acid (99: 1 by vol) and
chloro form-methano 1-acet ic ac id-water (90:9: 1 :0.65 by vo 1) for PGE2 and PGF2, incubations,
respectively. Catabolism was quantified as the % total radioactivity migrating with PG
metabolites (PGM). In cases where catabolism was observed, only peaks which were 2 standard
deviations above background radioactivity (Le. greater than 8% of total radioactinty on plate)
were included in final tabulations. In cases where no metabolite peaks were above this cutoff,
tabulat ions of PGM included only the primary metabo lite f o m d .
IV-2.8 Analysis of Data
Data are expressed as the mean f S.E.M., where n is the number of animals. Tests used for
statistical cornparisons are described in figure legends. Differences were considered significant
when P < 0.05.
IV93 RESULTS
IV-3.1 Imrnunofiuorescen t Detection of 15-PGDH
Immunoreactive 15-PGDH was detectable in the choroid plexus of both the fetus (Fig. 13,
pg. 88) and lamb (Fig. 14, pg. 89). However, its localization differed greatly between the two
ages. In both the pre-terni and near-term fetus, there was abundant staining in the choroid plexus
epithelium with predominaace near the apical srnface ofepithelial cells (Fig. 13, pg. 88). In
addition, there was sparse mrnal staining in the near-terni fetus (Fig. 1 3, pg. 88). Conversely,
in the lamb at both 5 and 15 d, stalliuig was essentially absent in the epithelium but abundant in
the stroma (Fig. 14, pg. 89). Staullng in the adult choroid plexus did not exceed background
kvels, while immunoreactivity in the fetal lung, used as a positive control was locabd in
epithelial cells lining the bronchioles (data not shown). Histological staining of the choroid
plexus showed a well forrned epitheiium and stroma at ali ages (Fig. 15, pg. 90).
IV-3.2 Immunogold Detection of 15-PGDH
When viewed under the transmission electron microscope, immunoreactive I 5-PGDH was
localized near the bnish-border, CSF-facing membrane of epithelial celis of the fetal choroid
plexus (Fig. 16, pg. 91). Epithelial staining was cytosolic and was not associated with any
organelle. Stroma1 staining in the near-term fetus was also cytosolic and was localized to
fibro blasts (Fig. 16, pg. 9 1). Immunoreactivity in stromal fibro blasts was sparse in the near-term
fetus. In contrast, stroma1 staining in the lamb was abundant (Fig. 16, pg. 9 1). The gold particle
density increased with age in stromal fibroblasts, while an opposite trend occurred in epithelid
cek (Fig. 17, pg. 92).
Pre-Terr n Fetus
Near-Ter &rn Fetus
Figure 13. Immunofluorescence detection O
the a) pre-tem and b) near-term sheep fetuz antisera (l:75 dilution) raised in guinea pig Negative controls are c) pre-tem and d) ne: prirnary antibody was omitted. Arrows indi Scale bar is 25pm in a) and b) and 15 pm in
f 15-PGDH in the choroid plexus h m ;. Tissue sections ( 5 ~ ) were incubated with against purified rabbit lung I 5-PGDH. ir terni fetal choroid plexus sections where icate epithelium. Asterisks indicate stroma. C) and d).
5 Day Lamb
15 Day Lamb
Figure 14. Immunofluorescence detection from the a) 5 d and b) 15 d lamb. Tissue s ( 1 :75 dilution) raised in guinea pig againsi controls are c) 5 d and d) 1 5 d lamb chom was omitîed. Arrows indicate epithelium. epithelium in a) and b) is not visible due t( Scale bar is 25 pm in a) and b) and 15 pn
1 of 15-PGDH in the choroid plexus fiom ections (5 pm) were incubated with antisera : purified rabbit lung 15-PGDH. Negative id plexus sections where prirnary antibody Asterisks indicate stroma- Note that the low immunoreactivity at this site. in c) and d).
LAMB
Figure 15. Photomicrograpbs of H&E fetus (a: pre-terni; c: near-term) and la Asterisks iodicate stroma Scaie bars i
stained chon mb (b: 5 d; d are 25 p.
aid plexus sections from the sheep : 15 d). h w s indicate epithelium.
Figure 1 6. Electron micrograp h of the near-term sheep fetus choroid plexus (A) epithelial ce11 and (B) stromal fibroblast. (C) stromal fibroblast of the 15 d lamb choroid plexus. ZA = zonule occludens. N = nucleus. Note the pmence of gold particles in the epithelial cytosol near the bnish-border membrane of the fetal choroid pIexus (A, arrowheads). Cytosolic staining in stromal fibroblasts was sparse in the fetus (B. mwheads) and abundant in the lamb (C, asterisks). Epithelial staining was not detectable in the larnb. Scale bars are 0 2 5 pm in (A); 0.40 pm in (B); and 0.50 pm in (0.
Epithelial Cells Stroma1 Fibroblasts
1 I
I pre-term
fetus
b
I near-term
fetus
C
O 5 d 15 d
lamb lamb
Figure 17. Age-related changes in yold particle density for 15-PGDH
pre-term near-term 5 d fetus fetus lam b
C
$5 d lam b
immunoreactivity in choroid plexus epithelial cells (left panel) and stromal fibroblasts (right panel). Values are the means i S.E.M. for three samples in each age group. Gold pariicle densiiy was determined in 25 randomly chosen fields fiom each sample. In each ce11 type, the effect of age on gold particle density was evaluated with a one way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test for individual comparisons. In epithelial cells, density decreased significantly with agr whereas, in stromal fibroblasts, it increased significantly with age. Different letters denote significant differences among ages.
TV-33 15-PGDH Activity in Bomogeaized Tissues
To assay 15-PGDH activity in the cytosolic fiaction of the fetal choroid plexus, 'H-PGE~ and
'H-PGF~, catabolism was measured in the 100,000 x g supernatant fiaction. Unexpectedly, the
abundant catabolism seen in the intact tissue (see Chaptea II and III) was nearly absent in the
100,000 x g supernatant fraction (Fig. 18, pg. 94). M y PGE2 was cataboîized to a relatively
high degree in the total homogenate and the 100,000 x g supernatant fiom the pre-terrn fetus
(Fig. 19, pg. 95). Catabolism was marginal for PGF2, both before term and at te- and for
PGEz at term (Fig. 19, pg. 95). No dBerence was noted when NAD' was replaced with NADP'
as cofactor (Fig. 19, pg. 95). The lack of catabolism in choroid plexus homogenates was not
methodological since lung homogenates prepared in the same marner were able to catabolize
PGF2, extensively (Fig. 20, pg. 96). Furthemore, PGF2. catabolism in the choroid plexus did
not appear to be inhibited by excess formation of endogenous prostaglaiadins during
homogenization. Supporthg the latter point is the fact th addition of indomethacin to buffer
prior to homogenuation did not restore catabolism to values observed in the intact tissue (Fig.
20, pg. 96). Protecting the cofactor binding domain of 15-PGDH by adding either NAD' or DïT
to the hornogenization buffer, or by stabilizing the enzyme with glycerol did not restore
catabolic activity (Fig. 20, pg. 96). Similarly, the addit ion of the protease inhibitors, EDTA,
leupeptin and chymostatin, did not result in greater catabolism (Fig. 20, pg. 96). Lung
catabolism was w t af5ected by these treatments, a single exception king the inhibitory effect of
chymostatin at hîgh concentrations (Fig. 20, pg. 96). The reason for the latter fhding is not
known, however, chymostatin at lower concentrations and indomethacin at high concentrations
a h àad a moderate inhibitory eE'ect in the lung (Fig. 20, pg. 96). The hding with
indomethacin accords with earlier studies in the lung [ I l ;263 3461.
Near-Tenn Fetus
b
Pre-lem Fetus
Figure 18 . Thin-layer radiochromatograms of 100,000 x g supernatant fractions of the fetal sheep choroid plexus incubated with 0.25 p Ci/ml of either 3H-PGE 2
(a & b) 0 r ' k I - p ~ ~ ~ . (c & d) plus the cofactor, NAD+(S mM). Incubations lasted 60 min at 37°C and under ambient air. Markers on the left are (I) ?GE 2, (II) ISK-PGE2, (III) ISKD-PGE2, (IV) PGA*, and (V) akylated 1 SKD-PGE2. Markers on the right are (i) PGF2=, (ii) D-PGFZa, (5) 1 5K-PGFza, (iv) 1 SKD-PGL and (v) 1 SKD-PGE2. Origin is at arrow.
a c
Pre-Tenn Fetus Near-Tenn Fetus
'H-PGE~ + NAD' . 'H-PGE~ + NAD' 1 O0 %-PGF, + NAD* IO0 'H-PGF~ + NAD*
1 :20 100,000 x g homogenate supematant
t :20 100,000 x Q homogenate supematant
Near-Term Fetus
1 2 0 100,000 x g homogenate supematant
Figure 19. 'H-PGE~ and 'H-PGF~, catabolism (measured as the % radioactivity migra@ with PG metabolites, PGM) by the total homogenate and 100,000 x g supematant M i o n of the choroid plexus using either NAD' (upper panels) or NADP' (lower panel) as cofhctor. Measurements were made &er a 60-min incubation at 37'6 in ambient air with the appropriate tracer (0.25 pCi / ml) and cofactor (5 mM). Values are the means k S.E.M. for the number of animals given above each column. For incubations where NAD+ was cofkctor (upper panels), a two way d y s i s of variance (ANOVA), foiIowed by the Student-Newman-Keuls test, was used to assess the effects of tissue M i o n (homogenate vs. supernatant) and labeiled PG (PGE2 vs. PGF2,) on PGM values. In incubations where NADP' was cofactor (lower panel) variance could not be normalized by traosforrning data and, hence, a two way ANOVA could wt be performed. Instead, the effects of tissue hction and labellecl PG were assessed with Bonferroni corrected t-tests. Significant differences between PGE2 and PGF2, are indicated with an asterisk. For either prostaglandin, dserences between the total homogenate and 100,000 x g supernatant fiaction are indicated with a spade.
Near-Term Fetus: 1:20 Hornogenate
i , luno: 'H-PGF,,, + NAD* choroid plexus: 'H-PGF,,, + NAD'
- control indo lndo NAD' OTT (2.0pM) (28pM) (5mM) ( ImM)
Figure 20. 3 ~ - ~ ~ ~ 2 , catabolism by the total homogenate of the lune (open bars) and choroid plexus (closed bars) fiom the sheep fetus. Measurements were made afier a 60-min incubation with 'H-PGF~, (0.25 pCi / ml) and NAD' ( 5 mM). One of the following compounds was added to the buffer prior to tissue homogenization: indomethacin (indo), NAD', DTT, EDTA, glycerol, leupeptin (leup) or chymostatin (chyrn). In studies where NAD' was added to the homogenization buffer, there was no subsequent addition prior to incubation. Each bar represents the mean i S.E.M. for 3 animals. A two way analysis of variance ( ANOVA), followed by the Student-Newman-Keuls test for individual comparisons, was carried out to test the effect of the above treatments and o f tissue source (lung vs. choroid plexus) on PGM. Both effects were significant and the treatment effect depended on the tissue source. For either tissue, different letters ( M d , h g ; italics, choroid plexus) denote significant differences among treatments. Significant differences between lung and choroid plexus homogenates are indicated with an asterisk. No significant difference was noted among choroid plexus homogenates regardless of treatment.
Additional controk (Appendix A-2, pg. 98) were done to d e t e d e whether soluble inhibitors of
1 5-PGDH were being released during homogenizat ion [24;60;223 ;242]. In these controls, the
intact latemi ventricle choroid plexus was incubated with 'H-PGF*. in the presence of the
100,000 x g supernatant fiaction of the contralateral choroid plexus fiom the same animal.
However, catabolism in this preparation was similar to that in the intact choroid plexus incubated
with 'H-PGF*, alone ( Appendk A-2, pg. 98).
IV-3.4 15-PGDH Activity in Minced Tissues
To assess whether low catabolic activity in tissue hornogenates is due to the loss of structural
htegrity, catabolism was measured in rninced specimens. Mincing does not dismpt the tissue as
extensively as homogenization. Furthemore, the minced tissue was incu bated in a small volume
of physiological buffer to better approximate conditions in the intact tissue. Parallel incubations
with the supernatant fiom minced tissues served as a negative control. In contrast to the low
catabolism in homogenates, minced spechens of the choroid plexus fiom the near-term fetus
showed high catabolic activity (Fig. 21, pg. 99). PGEz and PGF2, were not extensively
catabolized in the supernatant k t i o n of rninced tissues (Fig. 2 1, pg. 99). Thus, high catabolism
in minced tissue specimens is unlikely to be artifactual. The dinerence between 3 ~ - ~ ~ ~ 2 and
3 H-PGF2, catabolism seen in the homogenized choroid plexus fkom the pre-term fetus (Fig. 19,
pg. 95) was not observed in the minced tissue (Fig. 21, pg. 99). Furthermore, there was no
dinerence between the pre-term and near-tem fetus with ether prostaglandin (Fig. 21, pg. 99).
a: lntact Choroid Plexus
a a e . . v iv iii ii i
Appendix A-2. Thin-layer radiochromatograms of the intact lateral ventricle choroid plexus fiom the near-terni sheep fetus incubated with %I-PGF~~. a: conwl. b: after addition of the 100,000 x g supernatant fraction ( 10% by vol.) h m the contralateral ventricle choroid plexus. Markers are (i) PGh,, ( i i ) D-PGfia, (iii) 1 5K-PGF2a, (iv) 1 SKD-PGF?,, (v) 1 SHI-PGEt . Origin is at arrow.
b: Intact Choroid Plexus + 100,000 x g supernatant
C
LII
1 . - a a œ v iv
3 ~ - ~ ~ ~ ,
i , mince, whole tissue I mince, supernatant I
pre-terni fe tus
l
1 3
near-terrn fe tus
. mince, whole tissues mince, supematant
pre-term near-term fe tus fe tus
Figure 21. 'H-PGE~ (lefi panel) and 'H-PGF*, (right panel) catabolism by the minced choroid plexus and supernatant hction fiom the sheep fetus. Tissues and fluids were separated by low speed-centrifugation and each hction was incubated separately (see Methods). Measurements were made after a 60-min incubation. p02 of the incubation medium was 14 -21 mmHg. Values are the means f S.E.M. for the number of anirnals given above each column. For each PG, a two way analysis of variance (ANOVA), followed by the Student- Newman-Keuls test for individual comparisons, was carried out to detennine the effect of age (pre-term vs. near-term fetus) and tissue fraction (mince vs. supernatant) on PGM values. Significant differences between minced tissues and supernatants are indicated with an asterisk. No significant difference was noted between the two age groups. Bonferroni corrected t-tests comparing PGM values for 3 ~ - ~ ~ ~ 2
and "-PGF~, did not reveal any significant differences.
IV-4 DISCUSSION
The present study establishes that 1 5-PGDH irnmunoreactivity is present in the perinatal
sheep c horoid plexus. However, its location dMered markedly between the fetus and the lamb.
In the fetus, 15-PGDH was predominantly localized in the choroid plexus epithelium. and only
weak immunoreactivity was seen in the stroma Ln contrast, in the h b , 15-PGDH staining was
abundant in stroma1 fibroblasts and essentially absent in the epithelium. in both ceu types, 15-
PGDH irnmunoreactivity was cytosolic. However, while irnmunoreactivity was distributed
diffisely throughout the cytosol of stroma1 fibroblasts, in the epithelium it was rnainly evident in
proximity of the apical, CSF-king, s d c e . Enzyme localization in the fetus implies that, both
in the CSF in vivo and in the incubation medium in vitro, prostaglandins can easily gain access
to epithelial 15-PGDH.
In the fetus, the close association of 15-PGDH with the brush border membrane of epithelial
cells may explain the scarcity of emyme activity in the homogenized tissue. Specincally,
localization of immunoreagtive 15-PGDH at this age suggests that this enzyme forms a
functional unit with the brush border membrane of the epithelium. Thus, it is possible that high
enzyme activity in minced tissues is due to the fact that membranes were only partially disnipted,
whereas Iow activity in homogenized tissues results fiom the nearly complete disniption of
membranes. Furthemore, the present study does not support the idea that 15-PGDH is
deactivated, or rendered otherwise inoperative, during tissue homogenization. Thus, low
catabolic activity in the homogenized tissue fiom the term fenis does not appear to be artifktual.
In contrast to the term fetus, catabolism was measunible in the homogenized choroid plexus of
the pre-term fetus. The reason for this discrepancy is not immediately apparent. Higher
catabolism in the younger animal may be associated with the greater abundance of 15-PGDH in
the choroid plexus epithelium at this age. in the intact choroid plexus (see Chapter II),
catabolism tended to be higher in the pre-term versus near-term fetus, although differences were
not statistically significant. Coiacidentally, catabolism was greater for PGE2 than for PGFzo in
homogenized tissues. The latter findiag is likely due to the greater afnnity of 15-PGDH for
PGE2 [Z; 1 59; 1 921.
In con- to the close association of 15-PGDH with the CSF-facing membrane in the fetal
choroid plexus, this enzyme was confined to stroma1 fibroblasts in the iamb. Thus,
prostaglandins present either in the CSF in vivo or in the incubation medium in vitro, must cross
the choroid plexus epithelium to gain access to 15-PGDH in the underlying stroma If PGF2, is
king accumulated within the choroid plexus epithelium by an accessory uptake mechanism (see
Chapters 11 and ID), then this hietion of PGF2, may not reach stroma1 fibroblasts in its fkee
form. According to the scheme put forth in Chapter III, PGEI would not be accumulated by this
mechanism and, thus, would be entirely accessible to stroma1 15-PGDH.
Results presented in the current chapter demonstrate that the site of 15-PGDH expression in
the cho roid plexus changes during perinatal develo pment . Whether there is m e r relocalizat ion
after the perinatal period couid not be determined since immunoreactive staining in the aduh did
not exceed background levels. However, the redistribution of 15-PGDH fiom the epitheliurn to
the stroma soon afler birth may explain the dBerent age-related profiles in PGEl and PGF2,
catabolism. This issue is fûrther discussed in Chapter V.
CHAPTER V
GENERAL DISCUSSION
CHAPTER V: SUMMARY
This chapter includes a general discussion of resuks presented in Chapters U-IV. Studies
forming this thesis establish that two mechanisms for prostaglandin disposal are operational in
the cho ro id plexus during perinatal develo pment, nameiy, carrier-mediated uptake and 1 5-
PGDH-mediated catabolism. This is the first demonstration of the prostaglandin carrier in the
choroid plexus of the developing animal. Results also suggest that prostaglandins can diffuse
across the CSF-facing membrane of the choroid plexus epithelium and that diffusion decreases
with postnatal development. In addition to uptake via the prostaglandin carrier and diffusion,
PGFIa, but not PGE2, may be taken up by an accessory carrier. Uptake by the prostaglandin
carrier rnay operate in parallel with catabolism by 15-PGDH to mediate prostaglandin clearance
fkom ventricular CSF. Abundant 15-PGDH activity has not been previously documented in
studies with the adult choroid plexus. In k t , the present study c o n h m earlier hdings by
showing that the prostaglandin carrier is the primary disposal mec hanism in the adult c horoid
plexus, since 1 5-PGDH expression and activity feu to marginal levels in the adult. During the
perinatal period, the site of 15-PGDH expression chaxged fiom the epitheliurn in the fetus to the
stroma in the lamb. PGE2 catabolism was unaffected by this relocalization, king extensive at
both ages. PGF2, catabolism, on the other han& decreased fkom high levels in the fetus to
moderate, even low, levels in the lamb.
Remhs of the thesis do not support the original hypothesis, which stated that increased PGE2
removal fkom CSF by the choroid plexus via either, or both, the prostaglandin-specific carrier
and 15-PGDH contributes to the postnatal decrease in CSF PGEz levels. Nevertheless, the
presence of two independent disposal mechanisms durhg a limited period of postnatal
develo pment may have bo th p hy sio log ical and patho logical implications. The present discussio n
d l integrate hdings kom each chapter, will fùrther examine mechanisms of prostaglandin
uptake and catabolism in the choroid plexus, and will consider the implication of findings to
prostaglandin homeostasis in the brain during development. Particular emphasis will be placed
on the possible association between homeostatic mechanisms Ui the choroid plexus and the
respiratory depressant effect of PGE2 typical of the perinatai period.
V-1 MECHANISMS OF PG UPTAKE AND CATABOLISM IN THE
CHOROID PLEXUS
V-1.1 PGEl and PGF- Uptake in the Choroid Plexus Durhg Development
The present study is the first demonstration of carrier-mediated prostaglandin uptake in the
choroid plexus during the perinatal period. Proof that in vitro PGE2 and PGF2, uptake is carrier-
mediated was provided by the demonstnition that the accumulation of both mmpounds takes
place against a concentration gradient in a self- saturable, pro benecid-sensit ive, t ime- and
temperature-dependent rnanner. In both the perinatai and adult sheep, each prostaglaadin
inhibited uptake of the other. Thus, the two compounds are taken up by the same saturable
carrier. Consistent with previous in vitro studies m the adult [44;91], PGE2 and PGF2, uptake
was inhibited by probenecid in the perinatal animal. In hct, recent in vivo studies have
demonstrat ed that PGE2 clearance fiom ventricular CSF is mediated b y a pro benecid-sensit ive
mechanism in the term fetd sheep [360]. PGEz is also c leared by this mechanism during the
early postnatal period and in the adult sheep (S.L. Adamson, D. Engelberts, N. M c , and F.
C o c M unpublished observations). These studies are consistent with an earlier dernonstration
of PGF2, clearance fiom CSF of the adult f?om another species [43]. Thus, the carrier shown to
be operational in the isolated choroid plexus during perinatal development appears to conm%ute
to prostaglandin clearance fiom CSF in vivo.
This thesis demonstrates that the prostaglandin c d e r is fùnctional in the fetal sheep fkom at
least 0.7 gestation. However, contrary to the onginal hypothesis, steady-state in vitro uptake of
PGEl and PGF2, did not increase during the early postnatal period. This could not be explained
by in vitro oxygenation conditions since uptake by the fetal choroid plexus was similar under
conditions of oxygenation rnimicking the fetal and neunatal condition. In addition, in vitro
uptake was not sahirated by prostaglandin concentrations approximat ing elevated levels in CSF
around the tirne of birth. Taken together, these in vitro results predict that, in vivo, postnatal
changes in oxygenation or CSF prostaglandin levels do not alter carrier activity.
In addition to demnstrating PGEr and PGF2, uptake by the prostaglandin carrier in the
choroid plexus, the present study suggests that boa compounds may diaise across the apical
surface of the c horoid plexus epithelium at this stage of development. Specificdly, at low
temperatures, the two compounds were not excluded fkom the tissue like the extracellular marker
sucrose. Whether dinusion is predorninantly intracellular (i.e. across the brush-border
membrane) or intercellular (Le. across tight junctions) was not established. Findings do suggest,
however, that diffusion may become more restricted with age. Specifcally, at low temperatures,
TM values for PGE2 were significantly lower, and those for PGF2, tended to be lower, in the
adult compared with the fetus. These in vitro findings suggest that, in vivo, exchange of
prostaglandins between ventncular CSF and plasma via difision across the choroid plexus
epithelium is less restricted around the tirne of birth than in adulthood. This speculation is
consistent with the k t that blood-borne prostaglandlis reach brain extracellular fluids in highes
amounts in the perinatal animal than in the adult [103;I 641.
In the choroid plexus of the perinatal sheep, POEz and PGF2, competed for the same carrier
and appeared to difise to a similar extent; yet, steady-state accumulation of PGEz was less than
that of PGF2.. This dBerence was seen in choroid plexuses combined fkom all cerebral
ventricles and in choroid plexuses isolated fiom either the lateral or IIIW ventride. In the adult.
daerences were statisticdiy signincant ody in choroid plexuses combined fiom a l l cerebral
ventricles. Lower steady-staîe uptake of PGEz was not due to either Iowa passive uptake or
greater steady-state release of this compound. Taken together, these findings imply that the
dflerence between PGE2 and PGF2, accumulation may decrease with age and may result from a
difference in active uptake. Grester steady state-uptake of PGF2, has also been obsemed in the
adult of other species and it does not result fiom a greater afTimity O f the prostaglandin carrier for
PGFZa [38;9 11. Thus, the reason for the observed difference in uptake remains unclear.
One possible eicplanation for differences between PGF2. and PGE2 uptake is that an
accessory carrier contributes to the uptake of the former, but not the latter, compound. This
speculat ion is supported by the fact that there was residual accwnulat ion of 3 ~ - ~ ~ ~ t , in the
presence of 60 pM unlabelled prostaglandin, a concentration which should saturate the
prostaglandin carrier [9 1 1. Under the same conditions, 3 ~ - ~ ~ ~ 2 was not accumulated aga- a
concentration-gradient. Thus, it would appea. that when the prostaglandin carrier is saturated
with unlabelled compound 3 ~ - ~ ~ ~ 2 only diffises into the choroid plexus, whereas 'H-PGF~. is
still behg accumulated by a carrier. The identity of this hypothetiai accessory uptake
mechanism remains to be established. Theoretically, an organic acid carrier may accumulate
PGFza with low afnnity, which has k e n observed in the kidney [40]. Carrier-mediated transport
of several orgaaic acids has been demonstrated in the choroid plexus, and the group includes
monoamine metabolites [8 11, dmgs such as peniciilin and AZT [3 3 8;3 731, and non-prostanoaie
eicosano ids includhg the leukotrienes 132 81. Furthermore, an organic acid transport protein has
been localized to the cytosol of choroid plexus epithelial cells in the newbom rat and to the
h h - border membrane in the adult [ 1 50; 1 5 11. Alternat ively, the accessory uptake mechanism
rnay be a cytosolic carrier protein specific for long-chain, polyunsaturated fàîty acids. One of
them, the heart-type fàtty acid binding protein (Il-FABP) which binds oleic acid wah high
atfinity [272], is abundantiy expressed in the epithelium of the developing muse choroid plexus
[3 151. Furthermore, cornpetition studies have shown that PGF2,, but not PGE2, can displace
oleic acid fkom RFABP with low affinity [272]. To prove that PGFZa hteracts with either an
organic acid or a fatty acid carrier would require M e r work. In pursuhg this h e of
investigation, it would also be possible to determine whether PGF*, uptake by this hypothetical
accessory carrier accounts for the greater accumulation PGF2, compared to PGE2.
In the lateral venîricle choroid plexus, PGF2, and PGEz uptake tended to increase with
postnatal development. In contrast, in the IWIV ventricle choroid plexus, PGF2. uptake
decreased, and POEl uptake remained constant, after the perinatal perïod. The reason for this
discrepancy between the two sites is not immediately apparent. One possible explmation is that
PGF2, uptake by the putative accessory mechanism in the IIYIV ventricle choroid plexus is
lower in the dui t than in the lamb. Assuming that this carrier is responsible for the difference
between PGE2 and PGF2, uptake, this speculation is consistent with the fact that PGFt, uptake
was only slightly greater than PGE2 uptake in the IWIV ventricle choroid plexus of the adult.
Regardless of whether measurements were made in the lateral or 11I/IV ventricle choroid
plexus, overall levels of both PGE2 and PGF2, uptake did not change during the perinatal period.
Thus, contrary to the original hypothesis of this thesis, it is unlikely that the postnatal decrease in
CSF PGEz results fkom an increase in carrier-mediated removal at either site. However, the
c horoid plexus of the perinatai animal was capable no t only of carrier-mediat ed prostaglandin
uptake, but also of substant id 1 5-PGDH-mediated prostaglandin catabo lism.
V-1.2 PGE2 and PGFk Catabolism in the Cboroid Plexus During Development
Biochemical and imm~~1ohistochemical results of this thesis establish the presence of an
active 15-PGDH I 13-PGR cornplex in the choroid plexus of the perinatal sheep. This is in sharp
contrast to 15-PGDH activity in brain parenchyma, which decreases to low levels by the Last
third of gestation in sheep [266]. Likewise, in the choroid plexus fiom the adult, POE2 and
PGF2, catabo üsm via 1 5-PGDH is very low [ 1 ;3 81. In bt, the present study confirmed low
cataboiisrn in the adult while, in the fetus, there was essentially cornplete catabolism of both
PGEz and PGF2,. In the lamb, extensive catabolism was observecl with PGE2 whereas moderate
to low catabolism was obsewed with PGF2=. Different age-related changes in PGEl and PGF2,
catabolism during the perinatal period was an unexpected bding.
One explanation for the dBerent developmental profiles for PGEz and PGF2. catabolism is
that distinct rate-limiting enzymes catalyze the initial oxidation of these two prostaglandins. For
example, carbony 1 reductase (see Chart 2, pg. 1 2) could be active in the developing sheep
choroid plexus. This enzyme catalyzes the oxiciation of the hydroxyl group at carbon 15 in both
PGE2 and PGF2, [192]. One could argue that carbonyl reductase and 15-PGDH, respectively,
catalyze the rate-limiting step in PGF2. and PGEt catabolism, because the former enzyme has a
much greater affinity for PGF2, and the latter enzyme has a slightly greater ailhity for PGE2
[159; 192;308]. As an extension, the postnatal decrease in PGF2, catabolisrn would result fiom a
decrease in car bony l reductase expression. Altho ugh attractive, this hypothesis is inconsistent
with the fact that 'H-PGF~, catabolism was inhibited by unlabelled PGE2 as effectively as it was
inhibited by unlabelled PGF2,. An equal lack of selectivity was noted with 'H-PGE~ catabolism.
Thus, it is more plausible that both PGE2 and PGFr, are cataboüzed by 15-PGDH.
Coasequently, one may speculate that, in the fetus, 15-PGDH is localized to a site equally
accessible to PGE2 and PGF2. in the incubation medium whereas, in the lamb, it is localized to a
site more accessible to PGE2. This speculation is consistent with the k t that 15-PGDH was
expressed at different sites in the f e u and lamb.
in the choroid plexus, cytosolic 15-PGDH protein was abundantly expressed in both the fetus
and lamb, albeit at markedly different locations. In the fetus, 1 5-PGDH expression was
predominant ly epitheliai and was closely associateci with the apical, CSF- facing membrane. In
contrast, in the lamb, this enzyme was confined to fibroblasts in the underlying stroma
Alt hou& the density of stroma1 fibro blast s increases graduall y during postnatal deve lopment
[83], this is unlikely to acwunt for the rapid hcrease in 15-PGDH expression in these cells
within 5 days of bah. in the epithelium, the close association of 15-PGDH with the brush-
border membrane may explain how the abundant catalytic activity observed in the intact fetal
choroid plexus was lost upon homogenization. Specifically, 15-PGDH may form a fùnctional
unit with the brush-border membrane and, thus, may require structural kitegrity for its operation
This idea is ~pported by the fact that PGE2 and PGF2, were catabolized in minced tissues.
Further support cornes fiom the fact that loss of catabolic activity during tissue homogenization
did not result fiom enzyme deactivation or saturation. Whether catabolic activity in the lamb is
lost upon tissue homogenization was not detemüwd. Furthermore, the intrinsic mechanisms
regulating the change in 15-PGDH localization diniag the transition fiom intra- to extra-ut-
life remain unknown
One possible mechanism underlying perinatal changes in choroidal 1 bPGDH expression is
increasing plasma cortisol levels durhg the hst third of gestation [205]. Specificaily, this
increase in cortisol may upregulate 15-PGDH in the choroid plexus epithelium. As a corollary,
the decrease in plasma cortisol after birth [57;348] may downregulate 15-PGDH expression in
the epitheliurn. Glucocorticoids are known to regulate 1 5-PGDH protein levels and catalytic
activity in other tissues [46;23 1 s2;352;374]. Furthermore, a glucocorticoid response element
Iias been identified in the promoter region of the 1 5-PGDH gene [2 181. In addition,
glucocorticoid receptors have been localized in the choroid plexus epithelium, but not the
stroma, during perinatal development [ 1 761. Thus, it is plausible that, in the chomid plexus,
blood-borne cortisol regulates 1 5-PGDH expression only in the epithelium
Decreasing concentrations of thyroid hormones in blood after the immediate postnatal period
[57;5 81 may, theoreticall y, result in hcreased stroma1 expression of 1 5-PGDH. Thyro id
hormones can inhibit 1 5-PGDH activity [ 192;230;342] and may reduce protein expression 12301.
Although it remains to be established that thyroid hormone receptors are expressed in the choroid
plexus stroma, it is not hconceivable that these hormones may selectively affect stromal, and not
epitheliai, 15-PGDH. This speculation is based on the hct that the chomid plexus epithelium
actively secretes thyro id hormones into ventricular CSF via the abundant ly expressed carrier,
transthyretin [92;93;3 111. Thus, one could surmise that thyroid hormones would have W e d
effects in the choroid plexus epithelium In con- blood-borne thyroid hormones would
readily interact with the choroid plexus stroma.
In contrast to the perinatai animal, 15-PGDH protein expression did not exceed background
leveh in the adult choroid plexus. The intrinsic mechanisms underlying the decrease in 15-
PGDH expression d e r the perinatal period rernain unknowu. According to the scheme
discussed above, the sharp decrease in plasma thyroid hormones during the neonatal period [57]
would upregulate stromal 15-PGDH during the first two postnatal weeks. It follows, then, that
the gradual increase in plasma thyroid hormones during the tira postnatd month in sheep 1571
might result in a downregulation of stromal 15-PGDH sometime der the perinatal period.
Verifkation that 15-PGDH expression in the choroid plexus is developmentaily regulateà by
glucocorticoids and/or thyroid hormones would require fùrther experimentation both in vivo and
in vitro.
Despite the relocalization of 15-PGDH during the perinatal period, PGE2 catabolism was
unchanged during t h tirne. In the fetus, the close proxirnity of 1 5-PGDH to the apical
membrane of the epithelium suggests that PGE2 in the incubation medium is almost
instantaneously accessible to epithelial 1 5-PGDH once it crosses the apical surface of the
choroid plexus. Since PGE2 appears to cross the apical surface both by facilitated transport via
the prostaglandin carrier and by di fision, it will be catabolized as long as one of these routes is
available. In the lamb, 15-PGDH is confined to fibroblasts in the underlying stroma and,
consequently, the available substrate pool would be PGE2 within the choroid plexus. Thus,
similar to epithelial cataboiism, PGE2 catabolism in the stroma will be observed as long as PGE2
can reach the stroma1 cornpartment either via the prostaglandin carrier or diffusion. This scheme
is outlined in Diagram 4 (pg. 1 13) and is supported by results kom probenecid studies.
Specifically, in the fetus and lamb, PGE2 catabolism was unaffected when carrier-mediated
uptake was reduced by probenecid. In contrast, catabolism in the adult depended on probenecid-
sensitive uptake and this may be related to the k t that diffusion appeared to be more restncted
in the adult than in the perinatal animal. The fact that PGEz catabolism was equal in the fetus
and lamb suggests that 15-PGDH activity is the same whether it is expressed in the epithelium or
in the stroma.
Aithough 15-PGDH relocalization during the peruiatal period does not appear to be
associated with a change in enzyme activity, PGF2, catabolism decreased fiom levels equal to or
greater than PGE2 catabolism in the fetus to levels substantially lower in the larnb. Hi& PGF2,
catabolisrn in the fetus suggests that, like PGE2, the available substrate pool for epithelial 15-
PGDH is PGF2, present in the incubation medium. However, it appears that PGF2, available
for stroma1 catabolism is limited relative to PGE2. Specifically, like PGE2, PGF2, taken up
FETUS
PG Carrier
O Diffusion
LAMB ADULT
CSF
Tight Junction
Choroid Plexus
Plasma
8 Accessory Carrier Fenestrated Capillary for PGFz,
Diagram 4. Proposed ~c t iona i relationship between PG uptake and catabolism in the choroid plexus of the sheep fetus, lamb, and adult. The present study establishes that PGE2 and PGF2. are taken up by the PG carrier in these tissues. Results also suggest that PGs cross the CSF-king membrane of the epithelium by diaision. Diffusion may be more restricted in the adult compared with the perinatai animal. PGF*, may also be taken up by an accessory carrier. In the fetus, PG catabolism via 15-PGDH takes place in the epithelium near the CSF- k ing membrane. Conversely, in the lamb, catabo lism takes place in stromal fibro blasts. 1 5-PGDH is not detectable in the adult c horoid plexus. We speculate that PGEz and PGFî, taken up either by the PG carrier or by diffusion are readily accessible to 15-PGDH regardless o f site. In contrast, PGF2. taken up by the hypotheticai accessory d e r may not be accessible to stromal 15-PGDH.
either by the prostaglandin d e r or difision should be readily accessible to strornai 1 5-PGDH.
In contrast to PGE2, however, the possible accumulation of PGF2, within the epithelium or
underlying stroma by an accessory carrier rnay render this hction of PGF2, inaccessible to 15-
PGDH in stroma1 fibroblasts (Diagram 4, pg. 1 13). Thus, in the h b , where enzyme expression
is still high, this reduced accessibility would resuh in reduced catabolism. In fkt, PGF2,
catabolism in the lateral ventncle choroid plexus of the larnb was moderate, while PGE2
catabolism was high Interestingly, PGF2, catabolism in the III/IV ventricle choroid plexus was
negligible in the h b , despite high levels of PGEz catabolism. The wiusually low PGF2,
catabolism rnay be related to unusually high uptake at this site, presuming that a large k t i o n of
thk uptake is mediated by an accessory carrier (Diagram 4, pg. 1 13). Before testing the
hypothesis that PGF2, taken up by an accessory carrier is not available for 1 WGDH catabo lism,
it would be necessary to demonstrate that PGF2, a c t d y interacts with such a carrier.
V-1.3 The Cboroid Plexus and CSF Prostaglandins During Development
The k t that neither uptake nor catabolism of PGEl c h g e d during the perinatal period does
not support the original hypothesis that the postnatal decrease in CSF PGEt levels [165] results
kom increased clearance through either mechanism. It is also unWrely that there is increased
clearance resuhing fiom greater CSF tunover. Specifically, although CSF secret ion by the
choroid plexus increases with postnatal developrnent [115;323], the concomitant Uicrease in CSF
volume results in a constant turnover rate [ 1 151. By exclusion, one must assume that there is
reduced PGE2 release into CSF. A possible site fiom which CSF PGE2 may originate is the
choroid plexus itself. in the present study, we demonstrated that PGE2 accumulated by the
choroid plexus can be subsequently released into the incubation medium Translated to the
2
physiological situation, either blood-borne or locdy synthesized PGE2 m y enter ventricular
CSF from this site. Worth n o t h in this context is that upregulation of 15-PGDH in stroma1
fibroblasts after birth may iudirectly contribute to the postnatal Ml in CSF PGE, by causing a
reduction in PGEz released fiom these cells.
Stroma1 fibroblasts of the choroid plexus express COXl protein in the near-term sheep fetus
[25 11 and, thus, may synthesh and release PGE2. Whether these ceUs are a source of PGEi in
the fetus and lamb is not known Presuming that PGEî is synthesized in stroma1 fibroblasts
during perinatal develo pment, the upregulat ion of 1 5-PGDH expression m these cells after birth
rnay result in decreased release of aewly formed PGE2. 15-PGDH is known to directly regulate
PGEi release fiom other tissues [ 1 79;22 1 ;240;254;275;32 1 1. If the increase in stromal 1 5-PGDH
expression were to coincide with a decrease in COX expression and activity, then the reduction in
PGEl release into CSF would be even greater. Whether COX expression in strornai fibroblasts is
Iower in the newbom compared with the fetus is not known However, COX is not expressed at
this site in the adult [5 1;52; 1881. Thus. it is conceivable that the transient postnatal hcrease in
I 5-PGDH expression in stromal fibro blasts is coupled with a decrease in COX expression-
Decreased PGEz release across the apical surnice of the choroid plexus hto CSF would, in tum,
contribute to the postnatal fa11 in PGE2 concentrations in ventricular CSF. Validation of this
hypothesis would require demomtration that there is Iower release of PGEz f?om the choroid
plexus when 15-PGDH is localized in strornal fibroblssts rather than the epithelium.
V-2 LIMITATIONS OF THE STUDY AND FUTURE
CONSIDERATIONS
The advantage of the present in vitro model in which the intact choroid plexus is incubated
in a physiological medium, is that it provides a simplifed system for studying prostaglandin
uptake and catabo lism without loss of structural integrity. The main difference fiom in vivo
conditions is that the choroidal vasculature is not king pefised. Thus, possible humoral
influences are missing. Furthermore, prostaglandins taken up fiom the incubation medium at
steady-state are codned to the intracellular and extracellular compartments of the choroid
plexus. While this is beneficial for assessing catabolism, it Ieaves open the question of whether
prostaghdins taken up by specific carriers would, in the nonnal situation, be cleared to the
venous emuent. Nevertheless, in vitro uptake of pro staglandins fiom a phy sio log ical incubation
medium provides evidence of carrier-mediated clearance h m CSF [36].
Recently, an in vivo study in the near-term sheep fetus has shown tbat PGE2 is rapidly
cleared fiom ventricular CSF by a pro benecid-sensit ive mec hanism [360]. Similar remlts were
obtained in the newborn sheep through studies in our laboratmies (S.L. Adamson, D. Engelberts.
N. Kninic, and F. Coceani, unpublished observations). Ho wever, one limitation of these in vivo
studies is that they could not separate clearance across brain microvessels koom clearance across
the choroid plexus. Furthermore, they did w t d y z e catabohm of the POEz fiaction reaching
the venous effluent of the brain via the choroid plexus [360].
Proving that 15-PGDH in the choroid plexus of the perinatal sheep regdates CSF
concentrations of prostaglandins independently O f the pro staglandin carrier would require in vivo
studies in which 15-PGDH is selectively inhibited. One possible approach would be to
administer monoclonal mti-15-PGDH antihdies, which can inhib it 1 5-PGDH activity [34 11,
during a ventriculocistenial perfusion study designed to measure prostaglandin clearance fkom
ventricular CSF [42].
The postnatal change in 15-PGDH localization in the choroid plexus did not alter the
magnitude of PGEz catabolism and, thus, it is unlikely to lead to increased clearance of PGE2
fiom ventricular CSF. However, it may contribute to the postnatal fàll in CSF PGE2 levels by
reducing the amount of PGE2 king released 6orn the choroid plexus into ventricular CSF. To
ver@ this possibility, it would be necessary to measure first the release of endogenous PGEr
fiom the choroid plexus in vitro and determine if'this release decreases &er birth. if this is
indeed the case, one couid immunologically inhibit 15-PGDH and then compare PGE2 release in
the fetus versus the Iamb.
The precise interaction between 1 5-PGDH-mediated catabo lism and a hypo thet ical accessory
carrier which may contribute to PGF2, uptake remains to be elucidated. The first step in
answering this question would be to analyze the kinetics of PGE2 and PGFta uptake in order to
distinguish between different components of transport. If these studies were to CO& that
transport of PGE2 is mediated only by the prostaglandin carrier and by diffusion, while that of
PGFza is mediated by these mechanisms plus an accessory carrier, then PGFZa catabolism could
be assessed under conditions where uptake by the accessory carrier is minimized. According to
the scheme outlined in Diagram 4 @g. 113), reducing PGF2, uptake by this accessory carrier
should not affect catabolism by epithelial 15-PGDH but should enhance catabolism by stroma1
15-PGDH.
Rapid relocalization of choroidal 15-PGDH f?om the epithelium to the underlying stroma at
birth implies the operation of specinc regdatory mechanism. To evaluate the importance of
hormones on this differentiai expression of 1 5-PGDH, a suitable approach would be to use ceil
cultures. Recently, culture systerns for choroid plexus epithelial cells have been developed
[126;128;359]. In one of these systems [126], stromal fibroblasts are a h present and ractively
secrete extracellular matrix proteins. This system would be a good mode1 for studying effects of
glucocorticoids and thyroid hormones on 15-PGDH expression in epitbelial cells versus
fibro blasts. If these studies were to demonsûate hormonal regdation, then M e r in vivo studies
would be warnuited to examine the impact of manipulating plasma concentrations of these
hormones on 15-PGDH expression Ui the choroid plexus.
V-3: PHYSIOLWICAL IMPLICATIONS OF PG UPTAKE: AND
CATABOLISM BY THE CHOROID PLEXUS
Results obtained in the present study suggest that central effects of CSF PGs specifically
PGE2 and PGF2., around the time of birth are terminated in vivo by both carrier-mediated
removal and 1 5-PGDH-mediated catabolism O perating in the c horoid plexus. In vivo studies
from another laboratory have confumed that probenecid-inhibition of carrier-mediated PGE2
clearance fiom ventricular CSF reduces the incidence and arnp litude of spontaneous breathing
movements in the near-terni sheep fetus [360]. Whether the sarne effect occurs in the lamb is not
known. Furthermore, it rernains to be seen if inhibit ion of choroidal 1 5-PGDH would affect the
drive to breathe in the perinatal animal. Nevertheless, I 5-PGDH may be particularly important
in the lamb during systemic hypotension, since, unlike the fetus, blood flow to the choroid plexus
would be select ively attenuated [340]. Thus, under such conditions, prostaglandin clearance
across the choroid plexus to the venous effluent of the brain would be impaired. The presence of
15-PGDH in the choroid plexus, however, should prevent biologically active prostaglamlios
£iom returning to ventricular CSF and, consequent ly , fi0 rn reac hing brainstern respiratory
centres.
Choroid plexus disposal mechanisms, together with clearance via buik CSF flow, rnay be the
only means of regulating levels of PGEz and PGF2, in the perinatal brain. Brain parenchyma
has, in k t , extremely low 15-PGDH activity during this stage of development [266].
Furthermore, it rem- to be established with certainty that PGEî and PGF2, are cleared fiom
brain ISF to plasma via a carrier system located in the microvessels. 15-PGDH activity has w t
been studied in microvessels fiom the developing animal but its activity is low in the aduh [3 51.
Prelhimry results obtained in the course of this study could not demonstrate temperature-
dependent PGE2 uptake in isulated cerebral microvessels fiom the sheep fetus, larnb, or adult
(Appendk A-3, pg. 1 2 1). Furthemore, PGEz incubated with the same microvessels was
recovered intact ( Appendix A-3, pg. 1 2 1 ). In the adult nom other species, there is evidence
suggesting that, in vivo, prostaglandins are cleared fiom brain ISF into plasma via carrier-
mediated transport across microvessels [42]. It is possible that the prostaglandin c h e r becomes
operational in brain microvessels later in development compared with the choroid plexus. This
appears to be the case with an organic acid carrier whose activity in the choroid plexus of the rat
is evident soon after birth, but becomes operational only d e r the first postnatal month in brain
microvessels [20]. I f PGE2 and PGF2, are not cleared tiom brain ISF across microvessels during
perinatal development, then the choroid plexus-CSF system would be the only means for
regulating levels of these compounds in brain. That is to say, prostaglandins in brain ISF,
whether locally formed or blood-borne, would be cleared to blood via the subarachno id and
ventricular CSF.
In the perinatal animal, the k t i o n of PGE2 and PGF2, cleared kom ventricuiar CSF
through bulk CSF flow and drainage enters the general circulation in a biologically active form
[3 601. In contrast, the fiaction cleared across the chomid plexus should have reduced activity
due to the presence of 15-PGDH. Any biologically active prostaglandins reaching the general
circulation fiom the brain are catabolized at different sites in the fehis and newborn In the
fetus, the lung does not appear to be a major site for pmstagkndin inactivation due to low blood
flow [166;305;325] and, possibly, reduced 15-PGDH activity in this tissue [265;282;353].
Instead, other peripheml tissues such as the kidneys [264] and the placenta
[65;72;73; 1 14; 1 S9;E 11 catabolize circulating PGE2 and PGF2,. In contrast to the fetus,
a 'O 2-0 Adult
Markers on TLC Scan of cerebral microvessefs isolated from brain of the sheep fetus:
(i) PGE2 (ii) 15K-PGE2 (iii) 1 SKD-PGE2 (iv) PGA2 (v) bicycl O-PG €2
1 Orig in is at arrow.
rime (min)
Appendix A-3. 3 ~ - ~ ~ ~ 2 uptake and catabolism by cerebral microvessels isolated fkom brain of the sheep. Uptake (quantified as the dpm per mg protein) was measured in the fetus (pre-term and term), lamb (5 and 15 d) and adult (non-pregnant ewe), following incubations in Ringer-Hepes medium at either 37°C or 4°C. Tissues were sepmted fkom fluids by rapid filtration. Each point is the mean k S.E.M. of 3 anirnals. There was no significant effect of the or temperature on 'H-PGE~ uptake at any age. There was also no catabolism in microvessels isolated 6om the fetus. For methods of microvessel isolation refer to Bishai, L, Dinarello, C.A. and Coceani, F. Prostaglandin formation in feline cerebral microvessels: effect of endotoxin and interleukin- 1. Canadian Journal of PhysioZogy and Phannacology, 65: 2225-2230, 1987.
the lung is likely the prirnary site for inactivating circulating prostaglandins in the newbom, as
it is in the adult [IO; 1 181. However, unlike the adult, the newbom brain may be protected
agauist the central actions of blood-borne prostaglandins not only by the presence of an
inactivation mec hanism in lungs but also by the operation of an enzymat ic barrier within the
choroid plexus. Considering that the choroid plexus receives a large portion of total brain
blood flow [340], catabolism of any blood-borne prostagiandins at this site may be important
for homeostasis of these compowids in brain.
Results kom this thesis, together with earlier studies measuring CSF turnover [115], suggest
that there is not a postnatal increase in PGE2 clearance kom CSF which would contribute to the
postnatal decrease in CSF PGE2 levels [ 1 651. Aitematively, there may be accelerated removal
from brain ISF across cerebral microvessels. However, the preliminary experiments mentioned
above (see Appendix A-3, pg. 12 l), together with the fact that brain plasma volume does not
increase between late gestation and 3 d postnatal in sheep [3 331, do not support this possibilit y.
Most plausibly, a decrease in PGEz synthesis within brain is the main factor contributing to the
postnatal fa11 in concentrations of this compound in ventricular CSF.
Although the perinatal sheep brain has the enzymes for PGEz synthesis [75;25 1 ;266;3 191,
little is known about the actual formation of this compound within specific brain regions. Of
particular interest in the current context is the expression of COX 1 protein in reg ions which
border the cerebral ventricles, namely, circurnventncular organs, the hypothalamus, bminstern,
ventricular ependyma, and choroid plexus stroma [Z 11. If PGE2 is fomed at these sites, then
decreased synthesis after birth, together with decreased influx of blood-borne PGE2, codd be
responsible for the postnatal fàll in PGE2 concentrations in ventricular CSF [165]. In fàct, there
is evidence that, in the perinatal sheep, PGE2 is released f?om the brainstem, hypothalamus,
d o r circumventncular organs under basal conditions [75;3 1 91.
V-4: PATHOLOGICAL IMPLICATIONS OF PG UPTAKE AND
CATABOLISM BY THE CHOROID PLEXUS
There is strong evidence that PGEl synthesized either in the circumventricular organs or the
hypothalamus, in response to bacterial pyrogens, acts as a neural mediator of fever
[34;94; 102;300;320;332;370]. This increased synthesis is associated with an increase in PGEz
levels in third ventricular CSF [74; 1 89;23 51. uitravenous administration of bacterial pyrogens
causes a larger increase in CSF PGEz in the newborn sheep than it does in the aduh [75]. In the
newbom, PGEz inhibits the central dnve to breathe via specific receptors located in respiratory
control region ofthe brainstem [134;343;344]. Thus, ifthe choroid plexus does not adequately
clear PGEt released into third ventricular CSF during fever, this compound may reach the
brainstem through bulk CSF flow and diffusion and may exert its respiratory depressant effect.
Meaningful in this context is the fact that fever is one of the many risk factors linked to sudden
infant death syndrome (SIDS) [ 1491. Furthemore, awther risk factor in SIDS is a viral
infection [ 1491 which is associated with exaggerated apnea, particularly in infants l e s then two
months of age [178;206].
A composite risk mode1 put forth to explain the occurrence of SIDS states that three events
need to coincide in precipitating the respiratory arrest [ 1 191. They are: (1) an underly ing
abnormality linked to cardioventilatory control in the infànt; (2) the occurrence of an exogenous
stressor and (3) an age-Linked susceptibility to tbat particular stressor. D e . wodd occur only if
these tbree &etors intenect and the infant happens to be vulnerable to the stressor [119]. Thus,
the identity of the stressor may d s e r in each case of SIDS [119]. The speculation here is that
reduced PGE2 clearance by the choroid plexus makes some infants vulnerable to SIDS. For
example, these infants may have reduced choro idal 1 5-PGDH activity in association with
matemal smoking during pregnancy, a well established risk factor for SIDS [149]. Specifcally,
chemicals in c igaretie smo ke such as azo benzenes, reactive aldehydes and po lycyclic aromatic
hydrocarbons are kwwn to inhibit 1 5-PGDH activity [ 1 5;28; 160;208]. Translated to the above
triple risk mode1 for SIDS, ihis hypothesis would ident% reduced 15-PGDH activity in the
choroid plexus as the underlying abnonnality. The stressor wodd be high levels of CSF PGE2,
suc h as those seen with fever in the newbom [75]. The cntical stage of development during
which this stressor can result in death would be the postnatal period when PGE2 is able cause
apnea [64;134;209;2 1 91. Supporthg this set of events is the observation that therapeutic
administration of PGE2 has k e n noted to cause both fever and apnea in newborn infants
[125;199;248;260-262;310]. Conversely, inhibition of prostaglandin synthesis with
indomethach may reverse apnea in newboms [ 1 381.
Contrary to the hypothesis presented at the start of the thesis, there is no change in either the
magnitude or the fictional arrangement of PGE2 uptake and catabolism in the iso ked choroid
plexus duhg the perinatal period. However, the relocaiization of 15-PGDH at the t h e of birth
may indirectly contribute to the postnatal f d in CSF PGE2 by reducing release of this compound
fiom the choroid plexus into ventncular CSF. Thus, 15-PGDH in the newbom choroid plexus
may have a dual role in replat h g prostaglandin concentrations. On the one han& it may operate
in para1 el with the PG-carrier to facilitate prostaglandin clearance f?om ventricular CSF. On the
other k d , it may be coupled to prostagiandin synthesis within the choroid plexus itself and
may , thus, control the arnount of endogenous prostaglandins released into ventricular CSF.
Furthemore, 15-PGDH rnay control levels of blood-borne prostagkndins reaching ventricular
CSF via the choroid plexus around the t h e of birth These difTerent functions may bave
physiological and pathological implications associated specifically with the centrai control of
breathing by PGE*. Although the original hypothesis of this thesis was not confirmecl, findings
reveal novel rnechanisms by which the choroid plexus can regulate central levels of
prostaglandins. They also point to a potential hormonal control of choroid plexus function which
may be developmentally regulated. Thus, not only does this study add to the knowledge of how
the choroid plexus contributes to prostagiandin homeostasis in the developing bmh, it a h
provides a basis for M e r investigation of homeostatic mecbanisms within the choroid plexus
itself,
CHAPTER VI
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