O sistema grelina-GHSR no globo ocular: regulação local e ... · work that they do to keep the department ... o gato e o cavalo, ... acrescentam novos argumentos a favor de um papel

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Paulo Jorge Pereira da Silva

O sistema grelina-GHSR no globo ocular: regulação local e implicações fisiopatológicas.

Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications.

Porto 2012

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Paulo Jorge Pereira da Silva

Master Degree Course in Cardiovascular Pathophysiology Orientador: Prof. Doutor Amândio António Rocha Dias de Sousa

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To my parents,

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To Sara,

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and in memory of my uncle…

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Acknowledgements

The elaboration of this master thesis would not have been possible

without the support, guidance and sympathy of innumerous persons

that either directly or indirectly contributed to this work and to whom I

extend my deepest and sincere gratitude. Among them I would like to

express some particular acknowledgements.

To Prof. Doutor Adelino Leite Moreira, head of the department of

Physiology and Cardiothoracic Surgery of the Faculty of Medicine of the

University of Porto, for providing me the opportunity to work at the

department and grow as a scientist and as an individual. This

acknowledgment is too modest to express my entire gratitude.

To Prof. Doutor Amândio Rocha Sousa, my supervisor, for trusting in me

at first impression, for the guidance provided, for all the support during

the investigation, for the patience, tolerance and for entrusting me with

responsibility that lead to my current self as a scientist. For all the

friendship I have received, I cannot find enough words for my gratitude.

To Professor Sónia Pinho, for the help she had given me in my

experimental protocols and for the patience to discuss them, I also am

very grateful.

One of the main acknowledgments goes to my work team. To Marta

Silva, for all the support, for the long hours and the good moments

shared in the lab, for being always one of the first critics of my work, for

all the sympathy and friendship dispensed, here goes a special thank

you. To Joana Rodrigues Araújo and to my recent team colleagues Ana

Rita and Rita for all the laughs and support. I am sincerely grateful. Also,

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I would like to express special thanks to Glória Almeida, for being one of

the most happiest “bambinas”, for all the “singing”, amazing support and

incredible patience.

To all my teachers in the Master’s Course, most of them also researchers

at the department, for all the knowledge and experience transmitted.

To all the staff in the department with a special thanks to the “technical”

and “TDT” staff namely Drs. Maria José Mendes, Marta Oliveira, Dulce

Fontoura and Sara Leite for all the patience and the amazing “invisible”

work that they do to keep the department running smoothly, for all the

laughs, discussions and “bate-papo”, and also to Dr. Francisco Nóvoa for

all the support in animal perfusion.

To the rest of the staff of the department, Dr. Armando Jorge, Mr. André

Alves, Mrs. Rosa Gonçalves, Mrs. Margarida, Mr. Alberto Sampaio, and a

special thanks to Mrs. Francelina for being such an amazing person and

for all the strength that she has given me since the first day.

To Professor António Avelino and Professor Carlos Reguenga from the

Experimental Biology department, for all the support regarding

cryosections and immunofluorescence microscopy.

To Dr. Zé Pedro from the department of Anatomical Pathology of

Hospital de São João for precious advice regarding cryosections and to

Prof. Russel Foster and Dr. Steven Hughes from the Nuffield Laboratory

of Ophthalmology for all the advice and discussion regarding

immunofluorescence protocols.

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To Dra. Luisa Guardão, our veterinary. For all the support with animal

manipulation as well for maintenance of animal well fare.

A different but not less important and special thank you goes to three of

the most amazing persons in my life, Ms. Gisela Madureira, Ms. Cláudia

Amorim and Ms. Ana Magalhães. For being always there for me, for all

the amazing and joyful moments that I cannot count, for being part of

my life, for the friendship, support and also for tolerating my absence in

a more or less comprehensive fashion. To them I express my true

gratitude.

My most sincere and profound acknowledgment goes to my parents and

to my girlfriend Sara. To my parents for all the efforts they have made to

enable my further education, for tolerating my temper, for making me

what I am today, for all the understanding and support despite all of my

absence. I cannot thank them enough for all they have always given me

and still do…

To my girlfriend Sara, for being the most outstanding woman, for all the

love, strength, patience, comprehension, support, for being a flawless

pillar, for appearing in my life, for the role model she is and for tirelessly

helping in my work. For being everything…

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“Nature hides her secret because of her essential loftiness, but not by

means of ruse.”

Albert Einstein

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Sumário

Revisão da literatura: A grelina é um peptideo acilado com 28

aminoácidos isolado pela primeira vez a partir da mucosa gástrica do

rato, sendo o seu principal local de produção as células X/A deste tecido.

Sistemicamente, a grelina afeta vários sistemas como o endócrino,

gastrointestinal, o cardiovascular, o pulmonar, o reprodutor, o sistema

nervoso central entre outros1, 2.

A grelina é o ligando endógeno do recetor dos secretagogos da hormona

do crescimento (GHSR-1a) e promove a libertação desta hormona por

parte da hipófise de forma independente do tradicional recetor da

hormona libertadora de hormona do crescimento1. Para além da forma

acilada da grelina, existe também uma forma desacilada denominada

des-acil grelina. Esta variante apresenta uma constituição aminoacídica

igual à grelina, mas não tem o grupo acil acoplado à serina 3, estando

por isso impedida de se ligar ao GHSR-1a3. Face à comprovada existência

de efeito fisiológicos atribuíveis à des-acil grelina, que em várias

situações são partilhados com a grelina, foi sugerida a existência de

outro recetor para além do tradicional GHSR-1a4.

Recentemente, foram atribuídas acções importantes à grelina no globo

ocular, quer no segmento anterior como no segmento posterior. No

segmento anterior o seu ARNm foi identificado, encontrando-se este

principalmente na face posterior da íris e no epitélio ciliar não

pigmentado. Foi demonstrado que a grelina relaxa os músculos

constritor e dilatador da pupila5. A presença de grelina foi ainda

detetada no humor aquoso, estando os seus níveis diminuídos em

doentes com diferentes tipos de glaucoma6, 7

. No segmento posterior, a

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grelina foi identificada na retina e implicada na fisiopatologia da

retinopatia da prematuridade8.

Objetivo: Considerando assim que este peptídeo parece desempenhar

um papel tanto na fisiologia como na fisiopatologia ocular, o objetivo

deste trabalho é investigar a presença de grelina e do seu recetor neste

órgão e avaliar o efeito deste sistema na modulação da pressão

intraocular.

Métodos: A deteção de grelina e do seu recetor no globo ocular de rato

foi efetuada através de immunofluorescência. Adicionalmente avaliou-se

a sua distribuição relativamente aos componentes musculares

intraoculares e às células endoteliais vasculares. Com vista ao estudo da

modulação da pressão intraocular, a sua medição foi realizada com um

tonómetro de impacto comercial usado em veterinária. Visto este

dispositivo estar calibrado apenas para o cão, o gato e o cavalo,

procedeu-se à sua calibração para os modelos animais utilizados neste

protocolo, com vista à obtenção de medições mais fidedignas. Esta

calibração foi feita comparando as medidas do tonómetro com

manometria intraocular in vivo. Em ambas as espécies o tonómetro

apresentou uma boa correlação com a manometria. A indução de

hipertensão ocular foi realizada em dois modelos animais (coelho e rato)

através da adaptação de um modelo previamente descrito no coelho9.

Com as alterações introduzidas verificou-se um aumento da pressão

intraocular semelhante à previamente descrita, mas neste caso com uma

maior estabilidade e durante um período de tempo mais longo.

Posteriormente procedeu-se à implementação deste modelo no rato,

tendo sido demostrada a aplicabilidade da técnica, com visível

desenvolvimento de hipertensão ocular. Uma vez validado o modelo,

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procedeu-se ao estudo dos efeitos da grelina e da des-acil grelina na

modulação da pressão intraocular através da administração

subconjuntival de um dos peptídeos. Com vista à avaliação das vias sub-

celulares envolvidas neste processo, foi realizado o bloqueio seletivo da

via da ciclooxigenase ou da via da sintase do óxido nítrico.

Resultados/Discussão: Os resultados destes estudos revelam que a

grelina é produzida pelo globo ocular, nomeadamente pelos processos

ciliares e pela retina. O recetor da grelina também está presente no

globo ocular, tendo sido a sua expressão detetada na rede trabecular,

estroma dos processos ciliares, epitélio corneano, íris e em toda a

coróide. Nos modelos animais de hipertensão ocular, tanto a grelina

como a des-acil grelina demonstraram ter um efeito hipotensor, sendo

que a des-acil grelina apenas o conseguiu no rato. Estes estudos

acrescentam novos argumentos a favor de um papel do sistema grelina-

GHSR na fisiologia ocular. A produção de grelina pelos processos ciliares

aliada à presença do seu recetor no tecido ocular, nomeadamente em

componentes responsáveis pela dinâmica do humor aquoso, fortalece a

hipótese do envolvimento deste peptídeo na fisiopatologia do glaucoma.

Fazendo a ponte para uma potencial aplicação clínica, podemos

considerar que o efeito hipotensor demonstrado por este peptídeo

poderá ser utilizado como futuro alvo terapêutico nas situações de

glaucoma, uma das principais causas de cegueira no mundo ocidental.

Palavras-chave: Grelina, des-acil grelina, GHSR, glaucoma, pressão

intraocular, imunofluorescência.

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Abstract

Background: Ghrelin is a 28 amino acid acylated peptide first isolated

from the rat’s stomach and is mainly produced by the X/A cells of the

gastric mucosa. Ghrelin exerts its action on several organ systems,

namely the endocrine, the gastrointestinal, the cardiovascular, the

pulmonary, the reproductive and the central nervous system, among

others1, 2.

Ghrelin is the endogenous ligand for the growth hormone secretagogues

receptor (GHSR-1a), promoting growth hormone release of growth

hormone from the pituitary independently from the growth hormone

release hormone receptor1. Another variant of ghrelin is its unacylated

form des-acyl ghrelin. This variant is identical to ghrelin, except for the

acyl group in the serine 3, thus being unable to bind GHSR-1a3. Since

some effects have been attributed to both ghrelin and des-acyl ghrelin,

the existence of a different receptor responsible for ghrelin’s actions

besides GHSR-1a has been proposed4.

In recent studies, ghrelin has been proposed to play important roles in

the ocular tissue, both in the anterior and posterior segments.

Regarding the anterior segment, ghrelin’s mRNA was identified in the

posterior surface of the iris and in the non-pigmented ciliary epithelium.

This peptide was also shown to induce the relaxation of the iris sphincter

and dilator muscles5. Ghrelin has also been implicated in glaucoma,

being its levels decreased in the aqueous humour of patients suffering

from different types of this pathology6, 7. Regarding the posterior

segment, ghrelin has been identified in the retina and implicated in the

pathophysiology of retinopathy of prematurity8.

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Aims: Bringing together ghrelin’s involvement in the ocular physiology

and in the pathophysiology of glaucoma, the aim of this work is to

investigate the presence of ghrelin and its receptor in the ocular tissue

and to evaluate the effect of the ghrelin-GHSR-1a system in the IOP

modulation.

Methods: The detection of ghrelin and GHSR-1 expression in the ocular

tissue was performed through the immunofluorescence technique. The

position of these distribution patterns relatively to the localization of the

ocular contractile elements and vascular endothelial cells was also

assessed. In order to study the modulation of intra-ocular pressure, its

measurement was performed using commercial available rebound

tonometer. The tonometer was calibrated for both experimental models

as it does not possess calibration for the animals used in our protocols.

The tonometer correlated very well with in vivo manometry and in the

experimental protocols it proved to be very efficient, allowing an easy,

fast and user-independent measurement of the IOP. Ocular hypertension

was induced in two different animal models (rabbit and rat) through the

adaptation of an experimental model previously described in the rabbit.

The alterations introduced to the protocol proved to induce a similar

increase in the IOP, but in a more sustained and stable fashion and

during a longer time period. After the model validation in the rabbit,

intraocular hypertension was also successfully induced in the rat through

the same experimental protocol, highlighting its technical applicability.

Once the model was optimized, ghrelin and des-acyl ghrelin’s effect in

the modulation of the intraocular tension was evaluated through

subconjunctival administration of either of the peptides. In order to

assess the sub-cellular pathways involved in this process, either the

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cyclooxygenase or the nitric oxide synthase pathway was selectively

blocked.

Results/Discussion: The results of the above presented studies reveal

that ghrelin is produced by the ocular tissue, namely by the ciliary

processes and the retina. Ghrelin receptor is also expressed in the eye,

more precisely in the trabecular meshwork, ciliary proceses stroma,

corneal epithelium, iris and throughout the choroid. In the ocular

hypertension models both ghrelin and des-acyl ghrelin were able to

significantly decrease the intraocular pressure, being that des-acyl

ghrelin was only able to do so in the rat. These data increase the already

existing evidence for a role for the ghrelin-GHSR system in the ocular

physiology. The endogenous production of ghrelin by the ciliary body

together with the presence of the ghrelin receptor in the ocular tissue,

namely in the components responsible for aqueous humour dynamics,

strengthens previous data that associated ghrelin with the

pathophysiology of glaucoma. Translating these findings into a potential

clinical application, the IOP-lowering effect of ghrelin points to a possible

therapeutic role in glaucoma, one of the main causes of blindness in

western world.

Keywords: Ghrelin, des-acyl ghrelin, GHSR, glaucoma, intraocular

pressure, immunofluorescence.

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List of abbreviations

AKT – protein kinase B

AMP – adenosine–

monophosphate

ANOVA – analysis of variance

AqH – aqueous humour

BSA – bovine serum albumin

cAMP – cyclic adenosine

monophosphate

CB – ciliary body

cGMP – cyclic guanosine

monophosphate

CGRP – calcitonin gene related

peptide

CM – ciliary muscle

CNS – central nervous system

COX – cyclooxygenase

CP – conventional pathway

CRH – corticotropin-releasing

hormone

DAG – diacylglycerol

DAPI – 4’,6-diamino-2-

pheylindole

eNOS – endothelial nitric oxide

synthase

ERK – extracellular signal

regulated protein kinase

ET-1 – endothelin-1

ETA – endothelin receptor type

A

ETB – endothelin receptor type

B

GC – guanylate cyclase

GH – growth hormone

GHRHR – growth hormone

releasing hormone receptor

GHSR – growth hormone

secretagogue receptor

Gln – glutamine

GnRH – gonadotropin-releasing

hormone

GOAT – ghrelin octanoyl-

-acyltransferase

GPCR – G protein coupled

receptor

GTP – guanosine triphosphate

HCSMC – human ciliary smooth

muscle cells

HDL – high density lipoprotein

IDM – iris’ dilator muscle

IF – immunofluorescence

IL-1β – interleukin 1 beta

IL-6 – interleukin 6

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IM – intramuscular

iNOS – inducible nitric oxide

synthase

IOP – intraocular pressure

IP – intraperitoneal

IP3 – inositol trisphosphate

ISH – in situ hybridization

ISM – iris’ sphincter muscle

IV – intravitreous/

intravitreously

LDL – low density lipoproteins

L-NAME – L-NG-Nitroarginine

methyl ester

MAPK – mitogen activated

protein kinase

MBOAT – membrane bound

octanoyl-acyltransferases

Min – Minute

mRNA – messenger ribonucleic

acid

nNOS – neuronal nitric oxide

synthase

NO – nitric oxide

NOS – nitric oxide synthase

NPCE – non-pigmented ciliary

epithelium

NTG – normal tension glaucoma

OCT – optimum cutting

temperature medium

OHT – ocular hypertension

PACAP – pituitary adenylate

cyclase-activating peptide

PBS – phosphate buffer saline

PCR – polymerase chain

reaction

PFA – paraformaldehyde

PG – prostaglandin

PGI2 – prostacyclin

PI3K – phosphatidylinositol 3-

kinase

PIP2 – phosphatidylinositol 4,5-

bisphosphate

PKC – protein kinase C

PLC – phospholipase C

PNS – parasympathetic nervous

system

POAG – primary open angle

glaucoma

PPARγ2 – peroxisome-

proliferator activated receptor

gamma 2

RGC – retinal ganglion cell

ROP – retinopathy of

prematurity

RT – room temperature

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SC – subconjunctival

SEM – standard error of the mean

Ser – serine

SNS – sympathetic nervous system

TM – trabecular meshwork

TNF-α – tumor necrosis factor alpha

TRL – triglyceride rich proteins

US – uveoscleral pathway

VHDL – very high density lipoproteins

VIP – vasoactive intestinal peptide

PCE – pigmented ciliary epithelium

XXVII

TABLE OF CONTENTS

CHAPTER I - INTRODUCTION AND AIMS ........................................... XXXI

1. GHRELIN AND RELATED PEPTIDES ............................................................. 3

2. GROWTH HORMONE SECRETAGOGUE RECEPTOR (GHSR) ............................. 6

3. GHRELIN’S ACTIONS ............................................................................ 10

3.1 Acylated ghrelin...................................................................... 10

3.2. Des-acyl ghrelin’s actions ...................................................... 13

4. GENERAL ANATOMY OF THE EYE ............................................................ 14

4.1 Cornea and sclera ................................................................... 15

4.2 Uveal tract .............................................................................. 15 4.2.1 Iris .................................................................................................... 16

4.2.2 Ciliary body .......................................................................... 19 4.2.3 Choroid ............................................................................................ 24

4.3 Retina ..................................................................................... 26

5. GHRELIN IN THE EYE ............................................................................ 28

5.1 Iris muscles ............................................................................. 28

5.2 Ghrelin production in the eye ................................................. 30

5.3 Ghrelin-GHSR system role in ocular pathophysiology ............. 30 5.3.1 Glaucoma ........................................................................................ 30 5.3.2 Retinopathy of prematurity (Ocular Angiogenesis) ..................... 32

AIMS ..................................................................................................... 33

CHAPTER II - MATERIALS AND METHODS ............................................. 36

Animals ........................................................................................ 37

Reagents....................................................................................... 37

Statistical Analysis ........................................................................ 37

1. IMMUNOFLUORESCENCE DETECTION OF GHRELIN AND GHSR IN THE RAT’S

OCULAR TISSUE...................................................................................... 38

1.1 Animal perfusion and tissue fixation ...................................... 39

1.2 Double immunofluorescence protocols .................................. 40

1.3 Controls .................................................................................. 41

1.4 Epifluorescence microscopy .................................................... 42

2. TONOMETER CALIBRATION ................................................................... 43

2.1 TonoVet® calibration in New Zealand White rabbits .............. 43

2.2 TonoVet®’s calibration in Wistar rats ..................................... 44

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3. ACUTE GLAUCOMA MODEL .................................................................. 45

3.1 Acute glaucoma model in the rabbit ...................................... 45

3.2 Acute glaucoma model in the rat ........................................... 46

CHAPTER III - RESULTS .......................................................................... 50

1. IMMUNOFLUORESCENCE ..................................................................... 51

1.1 Immunofluorescence for ghrelin ............................................ 51

1.2 Immunofluorescence for GHSR ............................................... 54

2. TONOMETER CALIBRATION .................................................................. 58

3. ACUTE GLAUCOMA MODEL .................................................................. 60

4. EFFECTS OF GHRELIN AND DES-ACYL GHRELIN IN IOP ................................. 61

4.1 Rabbit model of OHT .............................................................. 61 4.1.1 Evaluation of the systemic influence in ghrelin’s hypotensive

effect .......................................................................................................... 63 4.1.2 Effects of L-NAME and Ketorolac in ghrelin’s action .................... 63

CHAPTER IV - DISCUSSION .................................................................... 67

1. LOCALIZATION OF GHRELIN AND GHSR IN THE RATS’ OCULAR TISSUE ........... 69

2. VALIDATION OF THE TONOVET® REBOUND TONOMETER ............................ 74

3. ANIMAL MODEL OF OHT .................................................................... 76

4. Effects of ghrelin and des-acyl ghrelin in animal models of OHT

..................................................................................................... 78 4.1 Role of nitric oxide and prostaglandins in the effect of ghrelin ..... 81

CONCLUSION ........................................................................................ 87

BIBLIOGRAPHY ..................................................................................... 91

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List of tables

Table 1. Aqueous humour levels of ghrelin and des-acyl ghrelin in

patients with glaucoma.....................................................…32

Table 2. Primary and secondary antibodies, as well as vascular

endothelial cell markers used in immunofluorescence

protocols………………………………………………………………………….…38

Table 3 Comparison of both animal models of OHT……………………………..61

Table 4 Mean arterial pressure in rabbits injected with ghrelin or des-acyl

ghrelin…………………………………………………………………………………….63

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List of images

Fig.1 Overview of the ghrelin gene and derived peptides………………………………… 5

Fig. 2 Overview of the GHSR gene and derived receptor isoforms…………………… 7

Fig.3 Intracellular pathways involved in ghrelin’s effects…………………………………… 9

Fig. 4 Summary of ghrelin’s action……………………………………………………………….. 13

Fig. 5 Neuro-humoral pathways regulating iris’ sphincter muscle contraction…. 18

Fig. 6 Neuro-humoral pathways regulating iris’ sphincter muscle relaxation….. 18

Fig. 7 Neuro-humoral pathways regulating iris’ dilator muscle contraction and

relaxation …………………………………………………………………………………………………………20

Fig.8 Retinal functional morphology………………………………………………………………… 28

Fig.9 Illustration of the setup used in the tonometer calibration…………………..... 44

Fig. 10 Localization of ghrelin in the gastric mucosa……………………………………… 51

Fig.11 Localization of ghrelin in the ciliary processes………………………………………. 52

Fig.12 Localization of ghrelin in the ciliary processes stroma…………………………… 53

Fig.13 Localization of ghrelin in the retina………………………………………………………. 53

Fig.14 Localization of GHSR in transverse sections of the brain……………………….. 54

Fig.15 Localization of GHSR in the ciliary body………………………………………………… 55

Fig.16 I Localization of GHSR in the cornea……………………………………………………… 55

Fig.17 Double Immunofluorescence for GHSR and α-SMA or lectin in the ciliary

body……………………………………………………………………………………………………………… 56

Fig.18 Localization of GHSR in the iris………………………………………………………………. 57

Fig.19 Localization of GHSR in the retina…………………………………………………………. 57

Fig. 20 Linear correlation established for the rabbit between the manometric

and tonometric IOP…………………………………………………………………………………………. 59

Fig. 21 Linear correlation established for the rat between the manometric and

tonometric IOP………………………………………………………………………………………………… 59

Fig. 22 Effect of ghrelin in rabbits’ hypertensive eyes……………………………………… 62

Fig. 23 Effect of des-acyl ghrelin in rabbits’ hypertensive eyes……………………… 62

Fig. 24 Effect of L-NAME in ghrelin’s action in rabbits’ hypertensive eyes……... 64

Fig. 25 Effect of Ketorolac in ghrelin’s action in rabbits’ hypertensive eyes…….64

Fig. 26 Effect ghrelin in rats’ hypertensive eyes.………...................................65

Fig. 27 Effect of des-acyl ghrelin in rats’ hypertensive eyes………………………….. 66

Figures 1-9 were produced using Servier Medical Art

(http://www.servier.com/Powerpoint-image-bank)

Chapter I - Introduction and aims

Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications

Chapter I - Introduction

3

1. Ghrelin and related peptides

Ghrelin is a 28 amino acid peptide that presents a fatty acid at serine 3

and was first isolated from the rat’s gastric mucosa. It is the endogenous

ligand of the somatosecretagogue’s receptor type 1a (GHSR-1a),

promoting growth hormone’s release from the pituitary1.

Ghrelin’s precursor is encoded by a gene composed by 5 exons and 4

introns located on chromosome 3 (3p25-26)10. There have been

described several transcriptional variants associated with the ghrelin

gene: the first variant encodes a 117 amino acid preprohormone with

82% homology between species named preproghrelin1; the second

variant is 5’ truncated at exon 2 and encodes prepro des-Gln14-ghrelin11,

12; the last variant has exon 3 deleted and encodes exon 3 deleted-

preproghrelin 13, 14.

After preproghrelin has been synthetized, the 23 amino acid signal

peptide is cleaved resulting in a 94 amino acid peptide named

proghrelin. This prohormone undergoes a post-translational modification

in which an acyl group is added to the Ser 31, 15. This modification is

carried out by ghrelin octanoyl-acyltransferase (GOAT), an enzyme

belonging to a family of membrane bound O-acyltransferases (MBOAT)

16, 17. The localization of GOAT overlaps the localization of ghrelin17, 18

and among all the enzymes of the family only GOAT is able to add the

acyl group to ghrelin. This modification, along with the first five amino

acids, is necessary for ghrelin’s linkage to GHSR-1a19.

4 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction

After acylation, proghrelin is cleaved into the 28 amino acid acylated

ghrelin and a 66 amino acid propeptide named C-ghrelin12. This process

is mediated by prohormone convertase 1/3 (PI3K)15.

Posteriorly C-ghrelin can either be processed into smaller peptides,

namely a 23 amino acid peptide designated obestatin20, or circulate as a

full peptide21. Although ghrelin and C-ghrelin derive from the same

prohormone, there does not appear to be a direct relation between

ghrelin and C-ghrelin levels, neither in human serum21 nor rat plasma

and tissues22, suggesting that C-ghrelin may be an independently

regulated gene-derived hormone with distinct functions. Obestatin also

derives from proghrelin and was initially discovered through

bioinformatics techniques. It has posteriorly been isolated from the rat’s

stomach and was first described as a hormone possessing contrary

effects to ghrelin, namely in reducing food intake, bodyweight gain and

gastric emptying in rats20. Furthermore, it has attributed a role in the

activation of GPR39, a GPCR related to the ghrelin receptor family. This

activation was subsequently questioned by other studies and there is still

some controversy regarding obestatin’s actions and its receptor23, 24.

As said above, the second splice variant originates preprodes-Gln14-

ghrelin. This preprohormone is processed similarly to preproghrelin, but

originates a variant of ghrelin devoid of glutamine at position 14 named

des-Gln14-ghrelin11. Interestingly, although the levels of this peptide are

negligible in humans, des-Gln14-ghrelin maintains the ability to stimulate

GHSR-1a with the same potency of ghrelin11, 12.

The last splice variant encodes exon 3-deleted preproghrelin. This

preprohormone is also able to originate functional acylated ghrelin but



5

completely lacks the sequence coding for obestatin, generating a

truncated C-ghrelin peptide with a novel 16 amino acid C-terminal

peptide (∆3D)13.

Fig.1 Overview of the ghrelin gene and derived peptides. Exons are illustrated as colored boxes with

corresponding numbers. Blue box) ghrelin’s gene can originate different mRNA transcripts through

alternative splicing. Left part of red box) Preproghrelin is encoded by exons 1-4. Exon 1 and part of exon

2 encode ghrelin whereas the remainder of exon 2 and exons 3 and 4 encode C-ghrelin. Left part of

purple and light blue boxes) Proghrelin is acylated and then cleaved by PI3K originating ghrelin and C-

ghrelin. White box) Ghrelin can be posteriorly deacylated and originate des-acyl ghrelin. Right part of

white box) Exon 3 deleted transcript is unable to originate obestatin but is capable of originating ghrelin

or des-acyl ghrelin.

Once acylated ghrelin is produced, it can be further processed and

originate des-acyl ghrelin, a peptide structurally equal to ghrelin except

for the octanoil group at serine 3. 3, 19, 25. Although some studies have

tried to identify the enzymes responsible for the deacylation of ghrelin26-

28, no conclusion has been drawn yet (Figure.1). Once released into the

bloodstream, acylated ghrelin circulates associated with triglyceride rich

lipoproteins (TRL), high density lipoproteins (HDL), very high density

lipoproteins (VHDL) and, to some extent, with low density lipoproteins


(LDL), while des-acyl-ghrelin circulates as a free peptide in plasma.25,27, 29

Des-acyl ghrelin levels represent about 90% of the total circulating

ghrelin11, 19, 25, being the plasma concentration of acylated ghrelin 10-20

fmol/mL and the total concentration 100-150 fmol/mL (including

acylated and non-acylated forms) 30,31.

Regarding ghrelin production, two thirds of the total ghrelin are

produced by the X/A cells of the gastric oxyntic mucosa32. The remaining

one third is produced by the intestine, pancreas, kidney, placenta,

endometrium, lymphatics, gonads, adrenal glands, thyroid gland, heart,

lung, pituitary gland, hypothalamus, B and T cells, neutrophils and the

eye1,33,34,5, 32.

2. Growth hormone secretagogue receptor (GHSR)

GHSR-1a was first discovered in the pituitary and in the hypothalamus

previously to the identification of its endogenous ligand ghrelin35. The

stimulation of this receptor by ghrelin promotes the release of GH

independently from the stimulation of GHRHR1, 30, 35, 36. GHSR-1a is a

typical GPCR, presenting seven transmembrane domains, and belongs to

a small sub-family of GPCRs that also includes the motilin receptor, the

neurotensin receptors, the neuromedin U receptors and GPR3930, 35, 37

.

The human gene encoding GHSR is located on chromosome 3q26.2.

There are currently two identified splice variants of the GHSR gene. One

variant encodes the full length isoform of the receptor, GHSR-1a, which

contains 366 amino acids and the seven transmembrane domains. The

second variant encodes a C-terminal truncated isoform composed of



7

only 289 amino acids and five transmembrane domains35, 38 (Figure.2).

Although the two isoforms have been described, GHSR-1b is unable to

bind ghrelin or other known growth hormone secretagogues35. However,

this truncated isoform has been identified in numerous organs such as

the heart, thyroid, pancreas, spleen and adrenal glands33,2 and has been

proposed to interact with GHSR-1a and to modulate its activity39,40.

Regarding GHSR-1a, it is also widely expressed in tissues such as the

stomach, intestine, pancreas, spleen, thyroid, gonads, adrenal glands,

kidney, heart, lung, liver, adipose tissue and bone4, 30, 33, 41-43.

Fig. 2 Overview of the GHSR gene and derived receptor isoforms. Exons are illustrated as colored boxes

with corresponding numbers. Red rectangle represents part of the intron between exons 1 and 2. Blue

box) GHSR gene can originate two splice variants. One variant contains the two exons and originates the

full-length receptor. The second contains only exon 1 and part of the intron and originates a truncated

form of the receptor. Left part of red box) This variant encodes the full-length GHSR-1a receptor. Exon 1

encodes domains I-V and exon 2 encodes domains VI-VII. Right part of red box) This variant has a stop

codon in the intron and generates a truncated version of the receptor with only 5 domains encoded by

exon 1.

GHSR-1a is constitutively active44 and presents two binding sites: one

that is located in the transmembrane domain number 3 and binds

ghrelin, as well as other peptide and non-peptide synthetic agonists19, 45;

and a second one that was proposed to bind adenosine46

. In the


somatotropes, the activation of the GHSR-1a stimulates the enzyme

phospholipase C (PLC), which promotes the hydrolysis of

phosphatidylinositol 4,5-bisphosphate (PIP2) into diacylglycerol (DAG)

and IP3. IP3 subsequently binds its receptor on the sarcoplasmic

reticulum, promoting a transient increase in the intracellular calcium

concentration. DAG activates the PKC and promotes the inhibition of

potassium channels, leading to the membrane depolarization and

posterior opening of voltage dependent calcium channels. This process

generates a persistent elevation of intracellular calcium levels47.

Nevertheless, this is not the only subcellular pathway activated by

ghrelin. This hormone is able to promote cellular proliferation through

GHSR-1a activation of subcellular signaling pathways such as the 5’

adenosine-monophosphate activated protein kinase pathway48, the

MAPKs pathway, in particular MAPK p44/p42, (also known as the

extracellular signal regulated protein kinase ERK 1/2)49-51, the

transcriptional factor Elk 150, the Akt/PI3K pathway49 and tyrosine

kinases pathways51

. Moreover, it appears to inhibit several inflammatory

and pro-apoptotic pathways52. Ghrelin also stimulates the activation of

the NO/cGMP pathway53, 54, as well as the increase of the PPARγ2 in

differentiated adipocytes, promoting adipogenesis through a GHSR-1a

dependent pathway55 (Figure 3).



9

Fig. 3 Intracellular pathways involved in ghrelin’s effects. Top left). Ghrelin binds to the GHSR-1a, a Gq

protein coupled receptor, leading to the activation of PLC. This enzyme converts PIP2 into IP3 and DAG.

IP3 promotes Ca2+ release from the sarcoplasmatic reticulum; DAG inhibits K

+ channels, inducing the

opening of voltage-dependent L-type Ca2+ channels in the cellular membrane. The increase of

intracellular Ca2+ results in membrane depolarization. Top right). Ghrelin activates a tyrosine kinase

receptor leading to the activation of the Ras protein. The double phosphorylation of the Ras protein

results in the MAPK, which needs another phosphorylation to enter the nucleus and regulate cell

proliferation. Ghrelin also binds to an unknown receptor which activates ERK1 and ERK2 and Akt/PI3K,

resulting in an anti-apoptotic effect. Bottom left). In endothelial cells ghrelin stimulates a G-protein-

coupled system which activates GC. This enzyme transforms GTP into cGMP, which leads to the

activation of NOS, increasing NO levels. NO then promotes relaxation of the smooth muscle cell by

entering it. Bottom right). In adipocytes ghrelin binds GHSR-1a and stimulates the PPARγ, a transcription

factor that regulates genetic transcription and promotes adipogenesis. From Ghrelin: production, action

mechanisms & physiological effects56

.


3. Ghrelin’s actions

3.1 Acylated ghrelin

Ghrelin is responsible for multiple actions in numerous organs and

tissues. As the endogenous ligand for GHSR-1a, ghrelin promotes GH

release from the pituitary through the stimulation of this receptor1, 47, 57,

58. In addition, ghrelin is able to stimulate the release of GH dependently

on GHRHR59, 60. It is also able to enhance the secretion of corticotropin-

releasing hormone (CRH), adrenocorticotropic hormone (ACTH) and

prolactin and to inhibit the release of GnRH and gonadotropins2, 61-66.

Another well documented effect of ghrelin is the regulation of

hunger/satiety cycle. Ghrelin has an orexigenic effect and its levels

increase before meals and decrease post-prandially67-70. Furthermore,

ghrelin seems to play an important role in body weight regulation, as its

levels increase in response to weight loss and low-calorie diets67 and

decrease in response to weight gain, forced overfeeding71 or high-fat

diets72

.

Ghrelin presents significant effects in the metabolism of glucose and

lipids. It increases plasma glucose levels due to direct effect on

hepatocytes, where it modulates glycogen synthesis and

gluconeogenesis, but also through inhibition of insulin secretion73-75

. This

effect is regulated by of a negative feedback loop, since insulin and

glucose decrease ghrelin levels76, 77. Regarding the adipose tissue, ghrelin

is responsible for preadipocytes proliferation and for an increase in

insulin-induced glucose uptake in developed adypocites, which is



11

associated with a promotion of lipogenesis78-80. It has also been shown to

inhibit lipolysis in vitro81.

In the central nervous system (CNS) ghrelin interferes in the

enhancement of memory and learning82-85, modulation of sleep86-91 and

control of the response to stress82, 92.

In the gastrointestinal system, it is able to stimulate gastric emptying and

promote jejunal motility. Ghrelin also exerts a protective effect in the

gastric mucosa and is involved in the regulation of acid secretion93-97.

The human lung has been indicated as one of the organs that produce

ghrelin42, 98. In an experimental model of pulmonary hypertension,

administration of exogenous ghrelin attenuates pulmonary vasculature

remodeling and right ventricle hypertrophy99.

Concerning the muscular tissue, all three, skeletal, smooth and cardiac

muscles were described to be affected by ghrelin. In the skeletal muscle,

ghrelin increases the permeability to chloride, which leads to a decrease

in membrane’s resting potential100. In endothelin-1 (ET-1) precontracted

internal mammary arteries101 ghrelin induces vascular smooth muscle

relaxation, being this hypotensive effect dependent on calcium activated

potassium channels and associated with a decrease in the nitric oxide’s

bioavailability102.

In the heart, this hormone has negative inotropic and lusitropic

effects103,104. The negative inotropic effect occurs as a response to

ghrelin, des-Gln14-ghrelin and des-acyl ghrelin103. Ghrelin’s effect is

dependent on calcium activated potassium channels and independent

from GHSR-1a103, 104. These potassium channels also participate in the


negative lusitropic effect 104. This hormone also decreases the afterload

and increases cardiac output without altering the heart rate105; increases

coronary perfusion pressure and the blood flow on its

microvasculature106 and inhibits the cytokines produced after an

increase in NO bioavailability, ameliorating endothelial function107.

Recently it was reported by Zhang et al. that ghrelin might protect the

heart from disease induced by oxidative stress, since it prevented H9c2

cardiac myocytes apoptosis52.

In male reproductive system, ghrelin is present in Leydig cells since birth

and it plays a role in their survival108, 109. However, the main effect of

ghrelin in this system is the suppression of the reproductive axis during

periods of hunger and negative energetic metabolism110

. In the female

reproductive system, ghrelin is expressed at the ovary in all phases of the

reproductive cycle and seems to play a role in embryogenesis33, 111.

The immune system is also affected by ghrelin’s actions. Chronic

administration of exogenous ghrelin preserves thymus architecture and

influences T lymphocytes production112. In addition, ghrelin is expressed

in T lymphocytes and upon their activation both ghrelin and des-acyl

ghrelin are released by these cells34. Regarding pro-inflammatory

cytokines, ghrelin has been shown to inhibit the synthesis of IL-1β, IL-6

and TNF-α suggesting an anti-inflammatory role for this peptide34, 107, 113

.

Finally, ghrelin interferes in bone physiology, promoting both the

formation and differentiation of osteoblast and osteoblast cell lines as

well as an increase in bone mineral density114-117 (Figure 4).



13

Fig.4 Summary of ghrelin’s action. Adapted from: Rocha-Sousa, A., et al. 2010

118.

3.2. Des-acyl ghrelin’s actions

As previously mentioned, des-acyl ghrelin is a non-acylated form of

ghrelin that is unable to bind GHSR-1a. Although it was initially

considered to be biologically inactive, it has currently been attributed

diverse effects in numerous organ systems. Depending on the

experimental conditions, some of these effects are similar, contrary or

independent when compared to ghrelin’s effect.

In the cardiovascular system, des-acyl ghrelin has a negative inotropic

effect that is even more pronounced than that exhibited by ghrelin. This

effect is modulated by cyclooxygenase (COX), due to the production of

prostacyclin (PGI2), and dependent on the endocardial endothelium103. In

H9c2 cardiomyocytes, which do not express GHSR-1a, both ghrelin and


des-acyl ghrelin bind with the same affinity to a common site and inhibit

apoptosis through the same pathway (Figure.3 top right)119. In HIT-T15

pancreatic β-cells120 and human osteoblast cells, both peptides are able

to promote cellular proliferation121. Contrary to ghrelin, des-acyl ghrelin

is able to inhibit gastric emptying as well as food intake122, 123. In primary

hepatocytes, des-acyl ghrelin inhibits glucose output and opposes

ghrelin’s effect on glucose release124.

All the previous data suggest the existence of alternative receptors

responsible for such actions. It is possible that there is a common

receptor responsible for ghrelin and des-acyl ghrelin’s analogous actions,

as well as an independent receptor for des-acyl ghrelin. However, to

date this question remains to be clarified.

4. General anatomy of the eye

The eye is divided in 3 compartments: the anterior chamber, the

posterior chamber and the vitreous cavity. Both anterior and posterior

chambers are filled with aqueous humour (AqH) while the vitreous cavity

is filled with vitreous humour. Regarding its constitution, the ocular

globe is composed by 3 concentric layers. The outermost is the structural

layer and is composed by the cornea anteriorly and sclera posteriorly.

The middle layer is the vascular layer of the eye, also known as uvea.

Finally the innermost layer is the neurosensory layer and is composed by

the retina.



15

4.1 Cornea and sclera

The cornea is a completely transparent structure that occupies the

center of the anterior pole of the eye and contributes to the focusing

power of the eye. In the most anterior part, it is composed by a stratified

squamous epithelium with a columnar basal layer that is attached to the

basal lamina. The other components include the Bowman’s layer,

stroma, Descemet’s membrane and the corneal endothelium. At the

periphery, the cornea is separated from the sclera by a gray and

translucent border called the limbus.

The sclera covers the remaining ocular tissue and presents a posterior

opening for the optic nerve. It is composed by bundles of collagen,

fibroblasts and a moderate amount of ground substance. Contrarily to

the cornea, the sclera is opaque and white due to the different degree of

interweave between fibrils and the presence of emissaria that are

responsible for the passage of arteries veins and nerves125.

4.2 Uveal tract

The uvea is the middle layer of the eye, standing beneath the sclera. It is

composed by the iris, ciliary body (CB), and choroid. As the vascular layer

of the ocular globe, it is responsible for the nourishment of the posterior

segment, namely the retina. Moreover, its structures control many eye

functions, such as the adjustment to different levels of light,

accommodation, decrease of optical aberrations, regulation of ocular

tension, production and drainage of aqueous humour (AqH). The

muscular components of the uvea are present in the iris, and in the CB.

http://www.ncbi.nlm.nih.gov/pubmedhealth/n/pmh_adam/A002295/





4.2.1 Iris

The iris is the most anterior extension of the uveal tract. It is perforated

at the pupil and separates the anterior from the posterior chamber. The

iris stroma contains blood vessels and connective tissue as well as

melanocytes and non-pigmented cells. The functions of the iris include

the control of retinal illumination, reduction of optical aberrations and

improvement in the depth of focus. The iris regulates the amount of light

reaching the retina by altering pupil diameter, and thus maximizes visual

perception. Also, by reducing the pupil diameter it limits the light rays

entering the optical system to the central cornea and lens avoiding more

peripheral portions of these structures where aberrations are greater.

When focusing close objects, a decrease in pupil diameter produces a

pinhole effect that decreases refractive errors and improves the depth of

focus. The modulation of pupil diameter is accomplished by the two iris

muscles, the iris sphincter muscle and the iris dilator muscle125.

4.2.1.1 Iris sphincter muscle

The iris’ sphincter muscle (ISM) is located in the most anterior part (0.75-

1 mm from the pupilar margin) of the iris and is organized in circular

muscle bundles126. This muscle is mainly innervated by the

parasympathetic nervous system (PNS), being the type 3 muscarinic

receptors127 (M3) the most abundant, followed by the M1 and M5

types128. Despite this parasympathetic predominance, it also presents

some innervation by the sympathetic nervous system SNS129. Other

mediators have also been attributed a role in the kinetics of the ISM,



17

being these usually defined as the non-adrenergic non-cholinergic

system.

The PNS and the trigeminal system are the best characterized systems in

the contraction of the ISM. The stimulation of M3 receptors activates

PLC which promotes the hydrolysis of PIP2 into DAG and IP3. DAG

inhibits K+ channels, inducing the opening of voltage -dependent L-type

Ca2+ channels in the cellular membrane. IP3 promotes intracellular Ca2+

increase and free Ca2+ ions will bind calmodulin forming the calcium-

calmodulin complex. This complex activates the myosin light-chain

kinase (MLCK) promoting myosin phosphorylation and muscle

contraction. Other mediators of the ISM contraction include the

substance P130

, PACAP130, 131

, Calcitonin gene related peptide (CGRP)130

,

neurokinines132, bradykinin133, 134, PGF2α, PGE2 and PGD2135 thromboxane

A2135

, ET-1136, angiotensin II (ATII)135 and luminous stimulation137 (Figure

5).

The relaxation of the ISM is modulated by the inhibition of acetylcholine

(Ach) release in the synaptic cleft or by the increase of intracellular cAMP

or cGMP levels138-140. Mediators that inhibit Ach release include

adenosine141, galanin and somatostatin142. VIP143, 144, adrenomedullin145,

146 and sympathetic stimulation147, 148 lead to an increase in cAMP

intracellular levels. Adrenomodullin145, 146

is able to increase cGMP

intracellular levels, as do nitric oxide149 and natriuretic peptides A and

C140. Finally, the specific stimulation of type B2 endothelin receptor (ETB2)

also relaxes the ISM through prostaglandins (PGs) synthesis and NO

release150 (Figure.6).


Fig. 5 Neuro-humoral pathways regulating iris’ sphincter muscle contraction. ET-1 – endothelin 1; ETB –

endothelin receptor type B; Pg – prostaglandin; Tx – thromboxane; CGRP –calcitonin gene related

peptide; PACAP –pituitary adenilate cyclase activator peptide; CCK-cholecystokinin; ATII – angiotensin II;

NK2,3 – Neurokinins’ receptor type 2 or 3; AT? – angiotensin receptor unknown; PLC – phospholipase C;

PIP2 – phosphatidylinositol 4,5-bisphosphate; IP3 –inositol trisphosphate; DAG – diacylglycerol; AA –

arachidonic acid. From Ghrelin: production, action mechanisms & physiological effects56

.

Fig. 6 Neuro-humoral pathways regulating iris’ sphincter muscle relaxation. VIP – vasointestinal peptide;

P Sub –substance P; ANP – auricular natriuretic peptide; CNP – cerebral natriuretic peptide; NPA –

natriuretic peptides receptor type A; ET – endothelin; ETB – endothelin receptor type B; Pg –

prostaglandin; Tx – thromboxane; NO – nitric oxide; NOS – nitric oxide synthase; Gc – guanylate cyclase;

cGMP – cyclic GMP; Ac – adenylate cyclase; cAMP – cyclic AMP. From Ghrelin: production, action

mechanisms & physiological effects56

.



19

4.2.1.2 Iris dilator muscle

The iris’ dilator muscle (IDM) is organized in radial muscle bundles and is

located in the basal part of the iris126. Contrary to the ISM, the dilator

muscle is primarily innervated by the SNS and its contraction occurs

mainly by the stimulation of α1 receptors. The stimulation of α1

receptors activates Gq protein with a consequent mobilization of both

intracellular and extracellular Ca2+ leading to muscle contraction. This

contraction is potentiated by neuropeptide Y151. Type A endothelin

receptor (ETA) stimulation by ET-1 also lead to the contraction of this

muscle152.

The PNS seems to influence both the contraction and the dilation of the

IDM. If the concentration of Ach is high, the stimulation of M3 type

receptors leads to muscle contraction. However, if the concentration of

Ach is low or the stimulation of the muscle is made with pilocarpine, the

muscle relaxes153. PACAP154 and CGRP155 also promote dilator muscle

relaxation (Figure.7).

4.2.2 Ciliary body

The ciliary body (CB) is a structure that bridges the anterior and posterior

segments. It presents a triangular shape in transverse cross-section and

is composed by the ciliary processes and the ciliary muscle (CM). The

base of the CB gives rise to the iris and attaches to the scleral spur via

longitudinal muscle fibers.

The main functions of the CB are: the production of AqH, lens

accommodation and maintenance of the zonules. It also plays a role in


both the trabecular and uveoscleral drainage pathways. The CB has been

described as a “neuroendocrine gland”, since it participates not only in

the secretion of AqH, but also in the synthesis of biological peptides,

metabolism of steroid hormones156, 157.

Fig.7 Neuro-humoral pathways regulating iris’ dilator muscle contraction and relaxation. NpY –

neuropeptide Y; ET-1 – endothelin 1; ETB – endothelin receptor type B; Pg – prostaglandin; Tx –

thromboxane; CGRP – calcitonin gene related peptide; PACAP –pituitary adenylate cyclase activator

peptide; CCK-cholecystokinin; ATII – angiotensin II; NK2,3 – Neurokinins’ receptor type 2 or 3; AT? –

angiotensin receptor unknown; PLC – phospholipase C; PIP2 – phosphatidylinositol 4,5-bisphosphate;

IP3 –inositol trisphosphate; DAG – diacylglycerol; AA – arachidonic acid56

.

4.2.2.1 Aqueous humour production and drainage

The secretion of aqueous humor (AqH) and regulation of its outflow are

physiologically important processes for the normal function of the eye.

The structure responsible for AqH production is the CB, more precisely

the ciliary processes. The ciliary processes are localized in the innermost

part of the ciliary body and are composed by a double layered



21

epithelium, the non-pigmented layer and the pigmented layer. The apical

surfaces of both layers lie in apposition to each other and are joined

together by gap junctions, leading to the formation of a syncytium158.

The non-pigmented layer stays in contact with the AqH from the

posterior chamber and the pigmented layer adjoins the ciliary processes

stroma and blood vessels159-161.

Aqueous humour is a clear fluid that provides nutrition, removes

excretory products from metabolism, transports neurotransmitters,

stabilizes the ocular structure and contributes to the regulation of

homeostasis of the ocular tissues. It also plays an important role in

pathological conditions as it allows the distribution of inflammatory

mediators as well as drugs by the different ocular structures. There are

three mechanisms responsible for AqH formation: diffusion,

ultrafiltration and active secretion, being the latter the only active

process and the major contributor for aqueous humour formation162.

The non-pigmented ciliary epithelium is the main site responsible for

active transport, providing passage of anions, cations and other

molecules such as amino acids. There are two enzymes involved in this

process: sodium-potassium-activated adenosine triphosphatase (Na+-K+

ATPase) and carbonic anhydrase (CA). Na+-K+ ATPase provides the energy

for the metabolic pump which transports sodium into the posterior

chamber. CA catalyzes the formation of H2CO3 from CO2 and H2O. H2CO3

is will originate HCO3- that is necessary for active secretion of aqueous

humour. Active transport produces an osmotic gradient across the ciliary

epithelium favoring the movement of other plasma constituents by

ultrafiltration and diffusion163

. After its secretion, AqH flows from the


posterior chamber through the pupil into the anterior chamber and then

exits the eye by one of two ways: the conventional pathway (CP) or the

unconventional pathway (US).

In the CP, the AqH flows through the trabecular meshwork (TM) into the

Schlemm’s canal and from there it is drained to the episcleral veins.

There is a pressure gradient responsible for the flow from the TM to the

Schlemm’s canal. In normal conditions, the production of AqH is enough

to generate the tension needed in the TM to promote this flow in a

passive way generating an average intraocular pressure (IOP) of about 15

mmHg for the general population. The TM is composed mainly of

collagen and elastic fibers organized in lamellae164 and is responsible for

about 75% of AqH outflow resistance165

. This resistance can be

influenced by the muscular tone of the CM, as it is inserted in the scleral

spur and maintains a close relation with the TM. Thus, when this muscle

contracts, it moves inward and anteriorly promoting a spread between

the fibers of the TM. This leads to increased space between the fibers

and a consequent decrease in TM resistance, promoting AqH outflow.

On the contrary, when this muscle relaxes the resistance to AqH outflow

increases166. The TM also possesses contractile elements that can

regulate its tonus. The modulation of these elements may be involved in

a regulation of the TM, and thus in the modulation of AqH outflow,

independent from the CM164, 167.

The other pathway involved in the drainage of aqueous humour is the

unconventional pathway, also called the uveoscleral pathway. In this

pathway, the AqH produced by the ciliary processes circulates normally

to the anterior chamber but then exits through the CB and iris root to



23

the CM and suprachoroidal space being drained to the choroid or

episcleral tissue. Contrary to the CP, the IOP is not determinant in the US

pathway because the sclera offers little resistance to flow and the

choroidal vessels absorb the amount of AqH delivered168. The CM, as an

integral part of this drainage pathway, plays a pivotal role in its

regulation. It is considered as the main site of resistance for AqH outflow

in this route and its contraction impairs US outflow169.

4.2.2.2 Ciliary muscle

The CM has three components: an external and longitudinal portion

(Brucke muscle), an intermediate and obliquous portion, and an internal

and circular portion (Muller muscle). The longitudinal portion originates

from the scleral spur and maintains a close relation with the TM.

The CB is well supplied with nerves, both myelinated and unmyelinated

of parasympathetic, sympathetic, and sensory types. The

parasympathetic nerve fibers originate in the Edinger-Westphal nucleus

and travel in the oculomotor nerve. They synapse in the ciliary ganglion

and in a few local ganglia within the ciliary nerves, before forming

extensive plexuses around the CM fibers170, 171. The M1, M2 and M3 type

muscarinic receptors are all present in the CM but the M2 type is only

present in the longitudinal portion of the muscle, indicating that

accommodation may not be regulated by this receptor subtype172, 173.

Besides this neuronal regulation, the CM also appears to be affected by

other peptides. One such example is ET-1. Stimulation of HCSMCs with

ET-1 or carbachol resulted in an increase of intracellular Ca2+

levels.


However, in the presence of norepinephrine or isoproterenol (a β-

agonist), ET-1-induced Ca2+ elevation was reduced. This effect seems to

be related to an elevation of cAMP promoted by the adrenergic

system174.

The CM is responsible for the accommodation mechanism. During this

process, CM tone influences the tension on the zonule fibers that attach

to the lens and hence influence the lens thickness. When focusing closer

objects the CM contracts, leading to a decrease in the tension of the

zonules and a consequent increase in the thickness of the lens. When

focusing distant objects, the muscle relaxes and the thickness of the lens

decreases.

4.2.3 Choroid

The choroid is the posterior portion of the uveal tract and lies between

the sclera and the retina. Its main function is to nourish the outer retina

and to remove waste products. Blood supply comes from both the long

and short posterior ciliary arteries and venous blood drains through the

vortex veins. The choroidal circulation constitutes 85% of the blood

circulating through the eye. Choroidal blood flow is higher than that in

most body tissues, including the retina and brain. The choroid extends

from the optic nerve to the ora serrata and is composed of four layers:

the Bruch’s membrane, the choriocapillaris, a middle layer of small

vessels (Sattler’s layer) and an outer layer of large vessels (Haller’s layer).

The Bruch’s membrane is the innermost layer and results from the fusion

of the basal lamina of the retinal pigmented epithelium and the



25

choriocapillaris. The choriocapillaris lies in a single plane beneath the

Bruch’s membrane and contains fenestrated vessels facing the retinal

pigmented epithelium (RPE). On the contrary, Sattler and Haller’s layers

are constituted by larger and non-fenestrated vessels. Abundant

melanocytes, some macrophages, lymphocytes, mast cells and plasma

cells appear in the choroid stroma as well as connective tissue that

support its structure125.

The smooth muscle cells of the choroidal vessels walls of the choroid are

innervated by both the PNS and SNS. The fibers from the PNS

innervation are rich in vasodilators like VIP and NO and are responsible

by an increase in blood flow. The SNS nervous system innervates both

vascular an non-vascular smooth muscle cells and is responsible for

vasoconstriction.175. Autoregulation of blood flow is the ability of the

tissue to maintain a constant blood flow despite changes in the perfusion

pressure. The blood flow in the ocular tissue depends on the local

arterial blood pressure, the local venous pressure and the resistance to

flow. A rise in IOP increases the resistance of this flow. Autoregulation of

the choroidal circulation has been proposed but is still under debate.

One argument against the autoregulation of the choroid lies on the fact

that choroidal blood flow greatly exceeds the metabolic needs of the

retina and so even if the perfusion pressure drops significantly, retinal

metabolism would not be compromised. Other studies however show

that the choroid presents an autoregulation mechanism dependent on

the IOP and that occurs only at IOP values lower than 5 mmHg, which is

in agreement with the myogenic mechanism of regulation176.


4.3 Retina

The retina is the innermost of the three layers that compose the eye and

is the structure responsible for phototransduction. The neural retina is

composed of six neuron types: photoreceptor cells, horizontal cells,

bipolar cells, amacrine cells, interplexiform cells, and ganglion cells. The

neurons are in close contact and supported by radial glial cells, the

Müller cells.

The retina is structurally organized in ten layers that, from the outer to

inner retina are: RPE; rods and cones inner and outer segments; external

limiting membrane; outer nuclear layer (nuclei of photoreceptors); outer

plexiform layer; inner nuclear layer; inner plexiform layer; ganglion cell

layer; nerve fiber layer (axons of ganglion cells) and finally the internal

limiting membrane. (Figure 8)

The RPE separates the photoreceptor outer segments from the choroid.

This epithelium is highly pigmented and can absorb excessive light from

reaching the photoreceptors. Furthermore, it supports and maintains the

functions of the photoreceptor outer segments and creates a selective

barrier between the choroidal circulation and the retina.

The nuclei of photoreceptor cells are localized in the outer nuclear layer.

The external limiting membrane limits these nuclei from the inner

segments of the photoreceptors that are in the photoreceptor layer.

Photoreceptors are responsible for phototransduction and consist of two

different cell types, the rods that are responsible for scotopic vision and

the cones that are responsible for photopic vision.



27

The inner nuclear layer consists of four cell types: horizontal cells,

bipolar cells, amacrine cells and Müller cells. The horizontal and bipolar

cells from this region form synapses with photoreceptor cells in the

outer plexiform layer.

The inner plexiform layer is where bipolar, amacrine and ganglion cells

synapse. Ganglion cells and some displaced amacrine cells constitute the

ganglion cell layer. Ganglion cells are responsible for collecting visual

information processed in the retina and send it to the brain. Axons of the

ganglion cells form the nerve fiber layer as they converge from all parts

of the retina toward the optic disc. Finally, the inner limiting

membrane is the boundary between the retina and the vitreous body

and is constituted by astrocytes and the end feet of Müller cells.

Glial cells are an important component of the retina. There are four

types of glial cells present in the retina: Müller cells; astrocytes present

mainly in the inner limiting membrane; microglial cells with phagocytic

properties; and oligodendrocytes that surround ganglion cell axons when

they are myelinated. Müller cells are the most abundant glial cells in the

retina. These cells transverse the retina from the inner limiting

membrane to the outer nuclear layer. Throughout this traject, Müller

cells branch in order to relate with the retinal neurons, other glia or

blood vessels. They are responsible for structural support of the neural

retina and are also capable of insulating the neurons both chemically and

electrically.125

http://en.wikipedia.org/wiki/Retina

http://en.wikipedia.org/wiki/Vitreous_body

http://en.wikipedia.org/wiki/Astrocytes

http://en.wikipedia.org/wiki/Muller_glia


Fig.8 Retinal functional morphology. RPE – retinal pigmented epithelium; PhL – photoreceptor layer;

ELM – external limiting membrane; ONL – outer nuclear layer; OPL – outer plexiform layer; INL – inner

nuclear layer; IPL – inner plexiform layer; GCL – ganglion cell layer; NFL - nerve fiber layer; ILM –

internal limiting membrane. Glial elements are also illustrated. Astrocytes – star-shaped cells; Müller cell

– tall cell passing from the ILM to ONL..

5. Ghrelin in the eye

5.1 Iris muscles

Recently ghrelin has been attributed a role in the ocular muscle kinetics.

More precisely, ghrelin is able to relax rabbit’s ISM and IDM5.

In ISM precontracted with carbachol, ghrelin promotes a decrease in

active tension in the first 1.5-3 minutes, with return to the initial active

tension in the following 10 minutes5. Moreover, in a contraction

mediated by electrical field stimulation, it is able to decrease the active



29

tension when compared to control177. Finally, ghrelin is also able to

decrease ISM basal tension5, 177.

The relaxation promoted by ghrelin in carbachol precontracted ISM does

not seem to be dependent on GHSR-1a because des-acyl ghrelin is also

able to promote ISM relaxation. Moreover, when antagonizing GHSR-1a

with Lys3-GHRP-6, ghrelin’s-induced decrease in the active tension is

more than three-fold when compared to non-antagonized receptor.

Since GHSR-1a blockade potentiated this hormone’s effect in this

muscle, it is possible that GHSR-1a acts as a modulator of ghrelin’s

effect5. Relaxation of this muscle is dependent on PGs release and

independent from NO production. Pretreatment of this muscle with

indomethacin, a COX inhibitor, blunts ghrelin-induced relaxation, while

pretreatment with L-nitro-L-arginine, a NOS inhibitor, has no effect on

ghrelin’s effect5. Finally, this effect is not species dependent since the

results have been replicated in isolated rat’s sphincter muscles both with

ghrelin and des-acyl ghrelin5.

Regarding the dilator muscle, ghrelin is able to decrease the active

tension developed by rabbit’s IDM once it has been contracted with

epinephrine. Contrary to the mechanism underlying rabbit’s ISM

relaxation, the effect on the IDM muscle is associated with GHSR-1a,

since pretreatment with Lys3-GHRP-6 abolishes ghrelin’s relaxing effect.

Moreover, des-acyl ghrelin is unable to decrease active tension in this

muscle5. Concerning dilator muscle basal tension, ghrelin was shown to

have no effect on it177.


These data show that ghrelin is able to inhibit both cholinergic and

adrenergic mediated contractions, being these effects possibly mediated

by different types of ghrelin receptors5, 177.

5.2 Ghrelin production in the eye

In addition to the data mentioned above, ghrelin production in the

ocular tissue has also been demonstrated. In 2006, a study

demonstrated ghrelin’s production in the rat’s ocular tissue for the first

time. Based on in situ hybridization (ISH) studies directed to ghrelin’s

mRNA, the presence of ghrelin transcripts in the iris’ posterior

epithelium and in the CBNPE was detected5. Later, the presence of

ghrelin in the AqH of patients with glaucoma has also been verified6, 7.

Regarding the posterior segment, Zaniolo et al. identified ghrelin and

GHSR-1a expression in the retina. Ghrelin production was assessed by

real time PCR and immunofluorescence (IF), showing that ghrelin is

produced in the retina, namely in the Müller cells end-feet. As for GHSR-

1a, this group has shown through IF that it is expressed in the retina,

mainly in Müller cells, and in the endothelial cells of the choroid8.

5.3 Ghrelin-GHSR system role in ocular pathophysiology

5.3.1 Glaucoma

Glaucoma is a progressive neurodegenerative disease characterized by

visual impairment as a result of optic nerve degeneration and retinal

ganglion cell loss (RGC) loss. It is the second leading cause of blindness



31

worldwide and it is estimated that it will affect 79.6 million individuals by

the year 2020178. Impairment in AqH production and/or its drainage is

the main cause for OHT. Elevated IOP is the most common cause

responsible for glaucoma179. As there appears to be a relation between

ghrelin and some of the ocular components responsible for AqH

dynamics, ghrelin’s presence in the AqH of patients with glaucoma was

assessed by two independent research groups (Table 1). The

experimental conditions included control patients, patients with chronic

open-angle glaucoma6, 7 and patients with pseudo-exfoliation glaucoma7.

The results show that ghrelin’s levels are decreased in patients with both

types of glaucoma compared to control. However, the plasmatic levels of

ghrelin did not differ between normal and glaucomatous patients,

pointing to a local mechanism of action6, 7. Nevertheless, there seems to

be no relation between ghrelin AqH levels and the severity of the

disease7. Regarding des-acyl ghrelin, its AqH and plasma levels in normal

and pathological conditions were also measured6. Interestingly, neither

AqH nor plasmatic levels of des-acyl ghrelin showed any differences

between normal and pathologic conditions6.

The current clinical therapies for glaucoma focus on the reduction of IOP

by either surgical or pharmacological methods. Even though not all

clinical scenarios of glaucoma exhibit elevated IOP, lowering IOP remains

the only option to prevent vision loss.


Table 1. Comparison between ghrelin and des-octanoyl ghrelin levels in plasma and aqueous humour

described by two different research teams6, 7

.

5.3.2 Retinopathy of prematurity (Ocular Angiogenesis)

Recent studies have attributed a role for the ghrelin-GHSR-1a system in

the pathogenesis of ROP. Zaniolo et al. described that ghrelin’s mRNA

levels are decreased during the vaso-obiterative phase of the disease,

while being two-fold increased in the vaso-proliferative phase. This

group also demonstrated that ghrelin has a protective vascular effect

during the vaso-obliterative phase. However, during the vaso-

proliferative phase, ghrelin’s exerts deleterious effects. Moreover, this

effect was described as being dependent of GHSR-1a stimulation, since

the blockage of this receptor with specific antagonists blunted ghrelin’s

effect.8

Ischemic proliferative retinopathies include the diabetic retinopathy (DR)

and the retinopathy of prematurity (ROP) and are the main causes of

blindness in the working age and pediatric populations of industrialized

countries respectively180, 181.

Rocha-Sousa et al., 2009 Katsanos et al., 2011

Control (pg/ml)

Open angle glaucoma (pg/ml)

Control (pg/ml)

Open angle glaucoma

(pg/ml)

Pseudo-exfoliation glaucoma

(pg/ml)

Ghrelin AqH levels

14.36±8.63 7.69±3.51* 123.4 ±25,5 84.2±14.8* 88.6 ± 17.6*

Grelin plasma levels

32.12±14.87 24.88±9.81 482.2±125.4 459.8±140.3 577.5±178.4

Ghrelin plasma/AqH levels ratio

10.21±7.25 12.34±7.25 4.00±1.04 5.60±2.05 6.19±1.55

Des-acyl-ghrelin AqH levels

61.10±15.96 62.27±22.13 - - -

Des-acyl-ghrelin plasma levels

254,10±128,81 229.634±100,81 - - -


Chapter I - Aims

33

Aims

Regarding all previously said, the aim of this work is to assess the

presence of the ghrelin-GHSR system in the ocular tissue and its role in

the modulation of IOP. Specifically the objectives are:

1. The identification of a local production of ghrelin and the

distribution of its receptor.

Ghrelin’s presence in AqH and its decreased levels in patients with

glaucoma, along with its physiological role in the eye led to the

hypothesis of the existence of a local regulatory system by ghrelin.

Through immunofluorescence studies we characterized the

distribution of ghrelin and its receptor in the ocular tissue.

2. Characterization of the ghrelin-GHSR system role in AqH drainage.

2.1 The establishment of a consistent and reproducible model of

OHT.

Due to the lack of reproducibility of acute models of OHT we

adapted a previously described model and applied it in two

different animal models. This allowed us to study the

hypotensive effect of ghrelin.

2.2 The characterization of the effect of ghrelin in hypertensive

eyes.

Considering previous studies that implicate ghrelin in the

physiology of the eye and in the pathophysiology of glaucoma,

we investigated ghrelin’s hypotensive effect and its association

with GHSR-1a. These studies were performed in two animal

models of acute glaucoma.

Chapter II - Materials and methods


Chapter II – Materials and methods

37

Animals

All animal procedures were conducted in accordance with the

Association for Research in Vision and Ophthalmology’s (ARVO)

statement for the use of animals in Ophthalmic and Visual Research.

Wistar rats were purchased from Charles River Laboratories. Wistar rats

were housed 2 per cage, in a 12 hour light-dark cycle with lights on at 8

a.m., controlled temperature and humidity. New Zealand white rabbits

were housed 1 per cage, in a 12 hour light-dark cycle with lights on at 8

a.m., controlled temperature and humidity. Further description of

animals will be provided in each protocol description.

Reagents

Imalgene 1000® was obtained from Merial (Lyon, France). Rompun® was

obtained from Bayer (Kiel, Germany). Anestocil® was obtained from Edol

(Lisbon, Portugal). Ghrelin and des-acyl ghrelin were obtained from

Peptides International (Louisville, Kentucky, USA). The peptides were

prepared in aliquots and stored at -20°C. Further description of

particular reagents used in each protocol will be provided in protocol

description.

Statistical Analysis

Statistical analysis was performed using SYSTAT’s Sigmaplot 11.0. Linear

regression was used to establish a relation between the manometric IOP

and tonometric IOP measurements. One-way ANOVA was used to test

38 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter II - Materials and methods

differences between test groups and Student’s T-test or the

correspondent non-parametric test was used to compare between

control and ghrelin or des-acyl ghrelin groups in acute glaucoma studies.

A p-value < 0.05 was considered statistically significant.

1. Immunofluorescence detection of ghrelin and GHSR in

the rat’s ocular tissue

Reagents

The antibodies and vascular endothelial cells marker used in the

immunofluorescence protocols are summarized in table 2.

Correspondent secondary antibodies used for each primary antibody are

shown in the same column.

Table 2. List of primary, and correspondent secondary, antibodies and vascular endothelial cell marker

used in the immunofluorescence protocols. N.A – not applicable.

Target Ghrelin GHSR 1 α-SMA

Vascular

endothelial

cells

Company H-40 Santa Cruz

Biotechnology

D-16 Santa Cruz

Biotechnology Ab5694 Abcam

L-5264

Sigma-Aldrich

Clonality Rabbit polyclonal Goat polyclonal Rabbit

polyclonal N.A

Dilution 1:150 1:150 1:250 1:50

Secondary

antibody Anti-rabbit Anti-goat Anti-rabbit N.A

Fluorochrome

conjugated Alexa Fluor® 488 Alexa Fluor® 488

Alexa Fluor®

568 TRITC

Host Goat Donkey Goat N.A

Company A11008 Invitrogen A11055 Invitrogen A11036

Invitrogen N.A

Dilution 1:1000 1:1000 1:1000 N.A



39

1.1 Animal perfusion and tissue fixation

Male Wistar rats weighing 200-350 g were anaesthetized with an

intraperitoneal injection of a mixture containing ketamine chlorhydrate

(Imalgene 1000®, 75mg/Kg) and xylazine chlorhydrate (Rompun®, 5

mg/Kg). After deep anaesthesia had been confirmed, a thoracotomy was

performed to expose the whole chest cavity. A catheter was inserted

through the apex of the heart across the left ventricle and placed into

the ascending aorta. Once the catheter had been hold in place, a cut was

made in the right atrium and the animals were perfused with ± 150 mL

of phosphate buffered saline (PBS, pH 7.4), at a rate between 5 and 10

mL/min to clean the vasculature. After that, the animals were perfused

with 400 mL of cold 4% paraformaldehyde (PFA) in PBS (pH 7.4) at a rate

of 5 mL/min to avoid vascular dilation. The efficiency of the perfusion

was controlled by monitoring liver color change and spontaneous

“formalin dance”. Once perfusion had been completed, the eyes and the

stomach were collected and placed in the same 4% PFA solution for an

additional 1 hour period at 40C. After this period, the samples were

transferred to sucrose solutions of 10 and 20% during 30 minutes and 2

hours at room temperature (RT) respectively and finally to a 30% sucrose

solution containing 0.01% sodium azide during an overnight period at

4oC. The sucrose solutions were made in PBS (pH 7.4).

Following cryopreservation in sucrose solutions, samples were rinsed

with PBS to remove external sucrose residues, placed in custom made

silicon molds filled with OCT (Optimum Cutting Temperature medium,

Neg50® Thermo Scientific Richard-Allan Scientific) at room temperature,

left to equilibrate with OCT for 10 min and then transferred to the


cryostat for cryosectioning or stored at -80ºC until use. Transversal serial

cryosections 10 µm thick were obtained and placed in Superfrost plus®

(Thermo Scientific) slides. The slides were then stored at -80ºC or

subjected to immunofluorescence.

Slides were left to air-dry at room temperature for 1 hour and then

washed in 0.1 M PBS pH 7.4 for 20 min. After that, tissue was

permeabilized with PBS Triton X- 100 0.2% for 20 min before blocking

non-specific binding with 3% bovine serum albumin (BSA) also in PBS

Triton X-100 0.2%. Slides were encircled with a pap pen (Vector

Laboratories, Burlingame, CA) and incubated overnight with primary

antibodies at 4oC. Primary antibodies were diluted in 1% BSA in PBS

Triton X-100 0.2%.

Following incubation with primary antibodies, slides were washed 3

times, 10 min each, with PBS Triton X-100 0.05% and incubated with

secondary antibodies during 1 hour at room temperature and in the

dark. Secondary antibodies were diluted in the same manner as primary

antibodies. Finally, slides were subjected to 2 washes with PBS Triton X-

100 0.05%, 10 min each and one final wash with PBS for 10 min before

mounting with Prolong Gold® antifade reagent contaning 4’,6-diamino-2-

pheylindole (DAPI) to label the nuclei.

1.2 Double immunofluorescence protocols

Double labeling experiments were performed to assess the possible co-

localization of ghrelin or GHSR with vascular endothelial cells.



41

Additionally, double labeling was also used to evaluate the possible co

localization of GHSR with smooth muscle cells.

Double labeling experiments regarding ghrelin or GHSR and vascular

endothelial cells were performed according to the protocol previously

described up to 2 initial washes following the incubation with the

secondary antibody. After this step, slides were incubated with Lectin

from Bandeiraea simplicifolia during one hour at room temperature and

in the dark. Slides were then washed and mounted as already described.

For the double labeling experiments regarding GHSR and α-SMA, a

sequential protocol was followed. Slides were subjected to the

immunofluorescence protocol described above, first directed to smooth

muscle localization, through utilization anti-α-SMA and the

corresponding secondary antibody. Afterwards, slides were subjected to

the same protocol, including the blocking step, and incubated with

primary anti-GHSR and corresponding secondary antibodies. In the end

slides were washed and mounted.

1.3 Controls

Both positive and negative controls were performed. Positive controls

for ghrelin included extracts from the rat’s stomach and positive control

for GHSR included extracts from rat’s brains.

Negative controls for single labeling were obtained by omitting the

primary antibody in the incubation serum. For double labeling

experiments, besides the omission of the primary antibodies used,

additional negative controls were performed.


To control cross-reaction between primary and non-correspondent

secondary, primary antibodies were incubated separately with the non-

-corresponding secondary used in the double labeling experiment (i.e.

rabbit anti-α-SMA primary with donkey anti-goat secondary and goat

anti-GHSR primary with goat anti-rabbit secondary).

To control cross-reaction between secondary antibodies, double labeling

was performed normally by omitting one of the primary antibodies but

maintaining both secondary antibodies.

Negative control for lectin was performed by pre-incubating Lectin with

500mM of galactose (G0625; Sigma-Aldrich) during one hour.

Controls for autofluorescence were also performed by omitting both

primary and secondary antibodies.

1.4 Epifluorescence microscopy

Epifluorescence microscopy was performed on an inverted microscope

(Axio imager Z1; Carl Zeiss), using 10x, 20x, or 40x air objectives. Photos

were taken with a CCD camera (Axiocam MRM), using the AxioVision®

software version 4.8.2.



43

2. Tonometer calibration

2.1 TonoVet® calibration in New Zealand White rabbits

Validation of the TonoVet® rebound tonometer (Icare, Helsinki, Finland)

was achieved by comparing tonometric measurements with those

obtained through in vivo manometry of the anterior chamber.

Male New Zealand White rabbits (Oryctolagus cuniculus 2.0-3.0 Kg) were

anaesthetized with a mixture containing ketamine chlorhydrate

(Imalgene 1000®; 0.4 g/Kg) and xylazine chlorhydrate (Rompun®; 8

mg/Kg). Experimental protocol started once deep anaesthesia had been

confirmed through paw pinching and absence of corneal reflex. A

scheme of the setup used in this protocol is represented on Figure 9.

The setup consisted in a 3-way stopcock linked to a pressure transducer

connected to different parts; one way was connected to a monitor

(Datascope 2000A); another was connected to a saline solution bag; and

the last way was connected to a 25 gauge needle used to cannulate the

anterior chamber of the eye. IOP was manipulated by altering the height

of the saline reservoir relatively to the eye. The alteration of the IOP was

monitored in real time through the Datascope 2000A monitor. The IOP

range used to calibrate the TonoVet® was between 5 and 60 mmHg

using intervals of 5 mmHg.

With the setup in the closed position, the 25 Gauge needle was inserted

through the peripheral cornea with care to avoid iris injury and as

horizontal as possible. There was no contact between the needle and the

cornea except for the entry point as this could alter tonometer’s

readings in the impact point. Once the anterior chamber had been


cannulated, the needle position was kept fixed until the end of the

protocol. The reservoir height was set to induce a 60 mmHg IOP. Five

measurements were made with the tonometer in each pressure level. As

the TonoVet® gives the mean of six measures, in each interval there was

a total of 30 valid measurements. Only values with low or no standard

deviation were considered. TonoVet® possesses internal calibration

tables for some species. The “d” calibration, which is used for cat’s and

dog’s eyes, was used in this calibration. The manometric pressure and

the TonoVet® measurements were then compared.

Fig. 9 Illustration of the setup used in the tonometer calibration.

2.2 TonoVet®’s calibration in Wistar rats

Validation of the TonoVet® rebound tonometer (Icare, Helsinki, Finland)

was also made in Wistar rats using a similar protocol similar to that

described above for New Zealand White rabbits.

3-way stopcock and

transducer

Datascope 2000A

NaCl 0.9%

25 G needle



45

Male Wistar rats (Rattus norvegicus 200-350g) were anaesthetized

through an intraperitoneal injection of mixture containing ketamine

chlorhydrate (Imalgene 1000®; 75 mg/Kg) and xylazine chlorhydrate

(Rompun®; 5 mg/Kg). Experimental protocol started once deep

anaesthesia had been confirmed through paw pinching and absence of

corneal reflex. A scheme of the setup used in this protocol is represented

on Figure 9. The IOP range used to calibrate the TonoVet® was between

5 and 80 mmHg using intervals of 5 mmHg.

Five TonoVet® measurements were made in each pressure level tested.

Only values with low or no standard deviation were considered. The “d”

calibration was used in this calibration. The manometric and tonometric

pressure measurements were then compared.

3. Acute glaucoma model

3.1 Acute glaucoma model in the rabbit

To test the effect of ghrelin and des-acyl ghrelin in the IOP, acute

glaucoma was induced by adapting a method firstly described by

Orihashi et al. 9.

New Zealand albino rabbits weighing 2.0-3.0 Kg were anaesthetized with

an intramuscular mixture containing ketamine chlorhydrate (Imalgene

1000® 0.4 g/Kg) and xylazine chlorhydrate (Rompun® 8 mg/Kg). A drop of

4% oxybuprocaine (Anestocil®) was instilled in each eye for corneal

anaesthesia and the basal IOP was measured with TonoVet®. After deep

anaesthesia was confirmed, the basal IOP was measured with the


Tonometer and subsequently increased through the injection of a

hypertonic saline solution (20% NaCl, 50μL) in the vitreous body of both

eyes, with care to avoid lens injury. Either ghrelin (10-4M, n=6) or des-

acyl ghrelin (10-4M, n=6) were subconjunctivally injected in one eye

randomly selected immediately after the intravitreous injection. The

control group (n=7) received a subconjunctival injection containing only

vehicle solution. Five readings were obtained every 15 minutes during 5

hours and the mean IOP value for each time point was determined. To

evaluate a possible systemic effect in the administration of ghrelin, the

arterial pressure was also monitored through the insertion of a catheter

into the central artery of the ear. The catheter was connected to a

pressure transducer and the transducer was connected to a monitor

(Datascope 2000A) in order to record the mean blood pressure.

To study the influence of NO and PGs in ghrelin’s action, a

subconjunctival injection of either L-NAME (500 µL; 150 mg/Kg; n=9) or

ketorolac (500 µL; 30 mg/mL; n=7) was delivered 30 min before the

intravitreous injection of hypertonic saline solution. Control groups for

these protocols (L-NAME n=6; Ketorolac n=6) also received a

subconjunctival injection of L-NAME or ketorolac 30 min before the

elevation of IOP.

3.2 Acute glaucoma model in the rat

To test the effect of ghrelin and des-acyl ghrelin in the IOP, acute

glaucoma was induced according to the previously described protocol.



47

Briefly, male Wistar rats weighing 200-350g were anaesthetized through

an intraperitoneal injection of a mixture containing ketamine

chlorhydrate (Imalgene 1000® 75mg/Kg) and xylazine chlorhydrate

(Rompun® 5 mg/Kg). A drop of 4% oxybuprocaine (Anestocil®) was

instilled in each eye for corneal anaesthesia. After deep anaesthesia has

been confirmed, basal IOP was measured with TonoVet® and

subsequently increased through the injection of a hypertonic saline

solution (20% NaCl, 16μL) in the vitreous body of both eyes with care to

avoid lens injury. Either ghrelin (10-4M, n=13) or des-acyl ghrelin (10-4M,

n=18) was subconjunctivally injected in one eye randomly selected

immediately after the intravitreous injection. The control group (n=12)

received a subconjunctival injection containing vehicle solution. Five

readings were obtained at 5, 10 15, 20 and 30 minutes and then every 15

minutes for a total 2 hours. In the end, the mean IOP value for each time

point was determined.

Chapter III - Results



51

1. Immunofluorescence

1.1 Immunofluorescence for ghrelin

The expression of ghrelin in the ocular tissue was determined through

immunofluorescence studies. Positive signal for this peptide was

detected in the ocular tissue, namely in the inside of the ciliary processes

facing the ciliary body stroma (Figures 11 and 12) and in the retina

(Figure 13). Figure 10 presents the tissue used as positive control.

Fig. 10 Immunofluorescence for ghrelin in the gastric mucosa (positive control). A, B) Ghrelin

immunoreactive cells (green). C,D) Negative control. Counterstain with DAPI (blue)

A

B D

C

52 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter III - Results

C F

A

B E

D

Fig.11 Immunofluorescence for ghrelin in the ciliary processes. A, B, C) Ghrelin immunoreactive cells.

(green). D, E, F) Negative control. Counterstain with DAPI (blue) and lectin (red).



53

Fig.12 Immunofluorescence for ghrelin in the ciliary processes epithelium facing the stroma. A) Ghrelin

immunoreactive cells (green). B) Negative control. Counterstain with DAPI (blue) and lectin (red).

Fig.13 Immunofluorescence for ghrelin in the retina. A, B) Ghrelin immunoreactive cells (green) appear

to be localized to Müller cells (Arrow). B, D) Negative controls without primary antibodies against ghrelin

but maintaining lectin. Counterstain with DAPI (blue) and lectin (red). GCL – ganglion cell layer; INL –

inner nuclear layer; ONL – outer nuclear layer; PhL – photoreceptor layer.

A B

A

B D

C

ONL ONL

ONL ONL

PhL PhL

PhL PhL

INL

INL INL

INL GCL

GCL

GCL

GCL


1.2 Immunofluorescence for GHSR

The expression of GHSR in the rat’s ocular tissue was assessed by

immunofluorescence. GHSR is expressed in the TM (Figure 17), ciliary

processes (Figures 15 and 17), corneal epithelium (Figure 16), iris, (Figure

15 and 18), retina and choroid (Figure 19). When assessing GHSR

localization relative to the ocular contractile elements, although these

structures did not co-localize, positive signal for receptor expression was

present in the base of the ciliary muscle, a region comprehending the TM

and the transition between the ciliary body and the sclera. Positive

controls are shown in Figure 14.

Fig. 14 Immunofluorescence for GHSR in transverse sections of the brain (positive control). A, B) Positive

signal for GHSR in the arcuate nucleus (green). C, D) Negative control. Counterstain with DAPI (blue).

A

B D

C



55

Fig.15 Immunofluorescence for GHSR in the ciliary body. A, B) Positive signal for GHSR in the ciliary

processes stroma, iris and in the ciliary muscle area (green). C, D) Negative control. Counterstain with

DAPI (blue).

Fig.16 Immunofluorescence for GHSR in the cornea. A) Positive signal for GHSR in the corneal epithelium

(green). B) Negative control. Counterstain with DAPI (blue).

A

B D

C

A B


Fig.17 Double Immunofluorescence for GHSR and α-SMA or lectin. Images on the right side are a

magnified view (20X) of the correspondent image on the left side. A, D) Double labeling for GHSR (green)

and lectin (red). A strong expression of GHSR is found on the TM. There is co-localization with vascular

endothelial cells in the ciliary processes. B,E) Double labeling for GHSR and α-SMA. There is no co-

localization of GHSR with the ciliary muscle. However, there is a strong expression in the TM and in the

border of the CM with the sclera. C,F) Negative controls. Counterstain with DAPI (blue).

C F

A

B E

D



57

.

Fig.18 Immunofluorescence for GHSR in the iris. A, B) Positive signal for GHSR is distributed through the

entire iris but appears to be more intense near the posterior epithelium (green). C, D) Negative control.

Counterstain with DAPI (blue).

Fig.19 Immunofluorescence for GHSR in the retina. A) Double labeling for GHSR (green) and vascular

endothelial cells (red). GHSR is expressed in the inner plexiform layer but does not colocalize with

vascular endothelial cells in this area. B, C) Double immunofluorescence for GHSR (green) and α-SMA

(red). GHSR is abundantly expressed in the retina. C) Most of the expression is localized in the inner

plexiform layer. B) This area is closer to the fovea and shows a strong expression in the photoreceptor

area. There is also expression for GHSR in the choroid (arrow). D) Negative control. Counterstain with

DAPI (blue).

A

B D

C

A

B D

C

ONL

PhL

INL

GCL

ONL PhL

INL

GCL

PhL

IPL IPL


2. Tonometer calibration

Both in the rabbit and in the rat, tonometer measurements

underestimated the manometric pressure measurements as shown

(Figures 20 and 21). A linear relation was established between the

manometric IOP and the IOP given by the tonometer. In the rabbit

(n=14), the determination coefficient (R2) for this relation was 0.87 and

the slope was 0.750. In the rat (n=11), these values were 0.98 and 0.459

respectively.

To establish a more accurate relation with real IOP, all tonometric

measurements obtained during the intraocular hypertension protocols

were adjusted according to the following equations:

Rabbit:

( )

Rat:

( )



59

Fig. 20 Linear correlation established for the rabbit between the manometric IOP and the IOP measured

with the TonoVet®.

Fig. 21 Linear correlation established for the rat between the manometric IOP and the IOP measured

with the TonoVet®.

Manometric IOP (mmHg)

0 5 10 15 20 25 30 35 40 45 50 55 60

To

no

metr

ic I

OP

(m

mH

g)

0

5

10

15

20

25

30

35

40

45

50

55

60

Manometric IOP (mmHg)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80

To

no

metr

ic I

OP

(m

mH

g)

0

5

10

15

20

25

30

35

40

45

50

Y= 0.750X – 0.331

R2

= 0.87

Y= 0.459X + 2.947

R2

= 0.98


3. Acute glaucoma model

The method used in glaucoma induction was an adaptation from the

model previously described by Orihashi et al9. Data regarding the rabbit

experimental protocols are summarized in table 3 as mean ± SEM. In this

animal model, the injection of hypertonic saline promoted a consistent

increase of the IOP that was observed at 15 min post-injection. The

increase in intraocular pressure did not present any statistically

significant difference between the different groups (p = 0.962). Thus, an

increase of 559.4 ± 125.25% was observed in the control group, of

479.90 ± 27.0% in the ghrelin group and of 463.37 ± 56.01% in the des-

acyl ghrelin group. In the control group, elevated IOP was maintained

during 150 min when IOP value was 41.47 ± 3.56 mmHg. After this

period, the IOP slowly began to decrease returning to baseline values

300 min post injection.

In the rat, the injection of hypertonic saline also promoted a consistent

increase in the IOP without any statistical difference between groups

(p=0.192). This elevation was noticed at 5 min post-injection and

corresponds to an increase of 663.02 ± 90.48% in the control group,

508.80 ± 73.44% in the ghrelin group and 559.56 ± 78.07% in the des-

acyl ghrelin group. In this model, the control group maintained elevated

IOP during a period of 30 min, returning to baseline values 120 min after

the intravitreous injection.



61

Table 3 Comparison of both animal models of OHT. Basal IOP was measured in all animals after deep

anaesthesia had been confirmed and immediately before the induction of OHT.

4. Effects of ghrelin and des-acyl ghrelin in IOP

4.1 Rabbit model of OHT

In the rabbit, subconjunctival injection of ghrelin promoted a decrease of

the IOP when compared to control. This decrease reached statistical

significance between 90 and 165 min. The maximal IOP decrease

occured at min 135 and corresponded to a negative difference of 18.98 ±

5.22 mmHg (43.8 ± 12.05%) when compared to control. Des-acyl ghrelin

was not able to significantly decrease the intraocular pressure. The

maximal decrease promoted by des-acyl ghrelin of -7.29 ± 3.70 mmHg

also at min 135 and accounts for a decrease of 16% ± 8.52% when

compared to control. Moreover, in the des-acyl ghrelin group IOP values

Rabbit Rat

Basal IOP (mmHg)

IOP increase at 15 min (mmHg)

Peak IOP (mmHg)

Basal IOP (mmHg)

IOP increase at 5 min (mmHg)

Peak IOP (mmHg)

Control 9.965±1.855 34.82±4.12 45.62±2.52 12.34±1.14 59.28±4.26 71.69±4.22

Ghrelin 9.33±0.74 34.98±2.7 44.84±3.32 14.41±1.28 49.63±4.31 64.04±4.61

Des-acyl ghrelin

10.71±1.13 36.09±3.11 46,80±3.06 14.021±1.29 49.70±3.61 63.72±2.83

L-Name 14.71±0.63 28.98±1.94 44.66±5.58 - - -

L-NAME + ghrelin

12.26±1.04 30.87±3.19 50.01±2.90 - - -

Ketorolac 15.46±1.20 25.2±3.47 47.33±2.31 - - -

Ketorolac + ghrelin

11.83±0.9 36.95±5.18 51.60±3.49 - - -


did not return to baseline values contrary to what happened both in

ghrelin and control groups.

Fig. 22 Effect of ghrelin in rabbits’ hypertensive eyes. *p ≤ 0.05

Fig. 23 Effect of des-acyl ghrelin in rabbits’ hypertensive eyes. *p ≤ 0.05

Time (min)

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300

IOP

(m

mH

g)

0

10

20

30

40

50

60

Control

Ghrelin* * * *

*

*

Time (min)

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300

IOP

(m

mH

g)

0

10

20

30

40

50

60

Control

Des-acyl ghrelin

* * * * *



63

4.1.1 Evaluation of the systemic influence in ghrelin’s

hypotensive effect

To evaluate the influence of a possible systemic response to ghrelin’s

administration on its intraocular hypotensive effect, the arterial pressure

was monitored through the catheterization of the central artery of the

ear. Data concerning the mean arterial pressure are summarized in table

4. The results suggest that the influence of ghrelin in the IOP is not

influenced systemically but is due to a local mechanism of action.

Mean arterial pressure

Initial 15 min P value

Ghrelin 58.3±5.7 58.2±6.0 0.985

Des-acyl ghrelin 72.7±4.4 69.4±5.4 0.486

Table 4 Data regarding mean arterial pressure in rabbits injected with ghrelin or des-acyl ghrelin.

4.1.2 Effects of L-NAME and Ketorolac in ghrelin’s action

To study the influence of NO and PGs on ghrelin’s effect, L-NAME or

ketorolac were subconjunctivally injected 30 min before intraocular

hypertension induction.

Ghrelin’s action was completely blunted in the presence of either L-

NAME or ketorolac. In the presence of L-NAME, IOP values in the

presence of ghrelin were overall higher when compared to control. At

min 165, the IOP in the ghrelin group was in fact 10.40 ± 4.54 higher than

in the control group (Figure 24). When ketorolac was administered, the

IOP pattern in the ghrelin group resembled that of the control group


(maximal decrease of 4.58 ± 3.40 at 225 min), demonstrating that

ghrelin’s effect was blunted (Figure 25).

Fig. 24 Effect of L-NAME in ghrelin’s action in rabbits’ hypertensive eyes.

Fig. 25 Effect of Ketorolac in ghrelin’s action in rabbits’ hypertensive eyes.

Time (min)

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300

IOP

(m

mH

g)

0

10

20

30

40

50

60

Control

Ghrelin + L-NAME

Time (min)

0 15 30 45 60 75 90 105 120 135 150 165 180 195 210 225 240 255 270 285 300

IOP

(m

mH

g)

0

10

20

30

40

50

60

Control

Ketorolac + Ghrelin



65

4.2 Rat model of OHT

In the rat, subconjunctival ghrelin injection also promoted a decrease on

the IOP when compared to control, reaching statistical significance

between 10 and 25 min (Figure 26). This decrease was maximum at 20

min and corresponded to a negative difference of 21.24 ± 4.13 mmHg

(33.28 ± 6,47%) when compared to control.

Contrary to the observed in the rabbit, in the rat des-acyl ghrelin

promoted a decrease in the IOP. This decrease was similar to that of

ghrelin but occurred in a broader time range (between 5 and 45 min)

(Figure 27). The maximal decrease promoted by des-acyl ghrelin was

registered at 30 min and accounted for –21.09 ± 3.94 mmHg (39.09

±7.30%) in IOP when compared to control.

Fig. 26 Effect ghrelin in rats’ hypertensive eyes. *p ≤ 0.05.

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120

IOP

(m

mH

g)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

Control

Ghrelin

* *

*

*


Fig. 27 Effect of des-acyl ghrelin in rats’ hypertensive eyes. *p ≤ 0.05

Time (min)

0 5 10 15 20 25 30 35 40 45 50 55 60 65 70 75 80 85 90 95 100 105 110 115 120

IOP

(m

mH

g)

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

Control

Des-acyl ghrelin

*

*

*

*

*

*

Chapter IV - Discussion



69

1. Localization of Ghrelin and GHSR in the rats’ ocular tissue

Recent studies have implicated ghrelin in the ocular physiology5, 177 as

well as in the pathophysiology of glaucoma6, 7 and ROP8. Ghrelin’s mRNA

has been identified in the anterior segment and this hormone is also

present in the AqH. Interestingly, ghrelin’s levels in the AqH of glaucoma

patients are decreased and this decrease is not accompanied by a similar

alteration in plasmatic levels6, 7

. Taken together, these data suggest a

local role for ghrelin in the anterior segment. Regarding GHSR presence

in the anterior segment, no data had previously been described.

Both ghrelin and GHSR-1a localization in the posterior segment had

already been characterized. Ghrelin is expressed in the retina,

particularly in Müller cells end-feet. GHSR-1a is expressed in the retina as

well, namely in Müller cells like ghrelin, but also in retinal endothelial

cells. Moreover, the ghrelin GHSR-1a system has been implicated in the

pathophysiology of oxygen-induced ROP. In this condition, this system

has been proved beneficial in the vaso-obliterative phase and

deleterious in the vaso-proliferative-phase8.

We investigated the presence of ghrelin and GHSR in the rat’s ocular

tissue by immunofluorescence. Our results demonstrate the expression

of ghrelin in the anterior and posterior segments of the rat’s eye.

In the anterior segment ghrelin’s expression was confined to the ciliary

processes, particularly in the epithelium and in the stroma. In the stroma

ghrelin did not co-localized with vascular endothelial cells marker. These

results indicate a local production of ghrelin by the ciliary processes of

normal Wistar rats, which is in agreement with previous studies where

70 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter IV - Discussion

ghrelin’s mRNA was identified in the non-pigmented ciliary epithelium5.

Based on the present study results, it is not possible to ascertain

ghrelin’s localization to the non-pigmented ciliary epithelium. However,

because ghrelin’s mRNA was identified in this type of epithelium, and

also because the non-pigmented epithelium is the main site of AqH

production by active secretion, it is logical to infer that ghrelin’s

expression is confined to this epithelium.

Rocha-Sousa et. al also found ghrelin’s mRNA in the iris’ stroma5. In the

currently presented studies we were not able to find ghrelin’s expression

in the iris. A similar situation has been described in the case of some

prostatic carcinomas, where ghrelin mRNA was detected but the protein

presence was not confirmed by immunohistochemistry182

. It is possible

for cellular regulation at the protein level to be involved in this

discrepancy.

In the posterior segment ghrelin’s expression was detected in the retina.

Positive signal for ghrelin appeared throughout the retina in a pattern

most likely corresponding to the localization of Müller cells. These results

are in agreement with a recent study where ghrelin was identified in the

retina, particularly in Müller cells end-feet8. However, in our study we

did not perform double-labeling with Müller cells specific marker Cellular

Retinaldehyde-Binding Protein (CRALBP) and therefore cannot state that

ghrelin’s expression is indeed located in these cells. Further evaluation is

needed to confirm these data. Nevertheless, these results point to a role

for this peptide in the posterior segment. Regarding the choroid, there

was no positive signal for ghrelin’s expression.



71

Focusing now on GHSR, its expression was detected throughout the

ocular tissue. The major expression sites for GHSR are the ciliary

processes, TM, corneal epithelium, iris and choroid.

In the anterior segment a marked expression is found in the ciliary

processes stroma and in the TM. The presence of GHSR in these

locations may indicate a role in the regulation of the production of

aqueous humour or in TM functionality.

Active secretion is the main process responsible for the production of

AqH. There are two enzymes abundantly present in the NPE that are

responsible for this process: Na+-K+-ATPase and CA183. NO is a well-

known mediator that has been proved to reduce IOP184. Moreover,

cGMP-NO pathway is responsible for a reduction in aqueous humour

production by inhibiting Na+-K+ ATPase activity in the NPE185, 186. The

stimulation of GHSR-1a can activate the Gc pathway, with formation of

cGMP and consequent NO release53, 54. In our study we co-localized

GHSR with VECs in the ciliary processes. These data suggest that this

receptor may play an important role in the physiology of this tissue by an

autocrine or paracrine regulation.

The TM is one of the major structures responsible for AqH drainage. Its

relation with the CM is responsible for the modulation of AqH drainage.

However, there have been described contractile elements in the scleral

spur, which present some characteristics of both vascular smooth muscle

cells and myofibroblasts. These contractile elements probably function

independently from those of the CM and are involved in the regulation

of trabecular outflow167, 187. Once relaxation of the TM contractile

elements occurs, there is a decrease in trabecular resistance, with a


consequent increase in AqH outflow164, 188. NO and the PKC pathway

have been involved in the modulation of isolated TM strips tonus. NO led

to the relaxation of TM strips 189, while PKC promoted their

contraction190. As mentioned above, GHSR-1a stimulation can promote

NO release through cGMP formation or activate the PKC pathway. Since

the inhibition of Na+-K+ ATPase by NO reduces AqH formation, one can

speculate that this reduction together with the relaxing effect of NO in

TM cells may lead to a reduction in IOP by a synergic mechanism. On the

other hand, if PKC signaling is simultaneously activated, there might

occur a decrease in the trabecular outflow. These data support the

hypotheses of GHSR-1a involvement in the regulation of AqH dynamics

and may present an interesting therapeutic target for glaucoma.

Still regarding the anterior segment, a strong expression for GHSR was

found in the corneal epithelium. The cornea is the first refractive

structure of the eye and is also a barrier between the outer environment

and the interior of the ocular globe. The corneal epithelium is composed

of three cell types: basal cells, in which mitosis occurs; wing cells that

result from the differentiation and migration of daughter cells originated

by basal cells mitosis; and a more external layer of completely

differentiated cells that degenerate and are sloughed from the

epithelium. The corneal epithelium is continuously renewed and

interacts with the tear film in order to maintain a smooth optical surface.

This renewal is supported by the migration of basal cells from the limbus

to the center of the cornea.

The regulation of the corneal epithelium plays a central role in cornea

healing, responding promptly to injury. Cell migration and proliferation



73

are the two main processes responsible normal regulation of the

epithelium and its response to injury. These mechanisms are under

control of a great variety of growth factors and signaling pathways191.

Signaling pathways involved in this regulation include the MAPK signaling

cascade192, and intracellular mediators like PKC193, 194 and cAMP195. Upon

stimulation, GHSR-1a presents the capacity to trigger these signaling

pathways. The expression of GHSR in corneal epithelium may therefore

indicate a possible role for this receptor in the maintenance of the

corneal epithelium functional structure.

In the iris, GHSR-1a presents a moderate expression particularly near the

posterior epithelium. The role of GHSR-1a in the contraction of the iris’

sphincter and dilator muscle has previously been studied5. In these

studies, GHSR-1a was shown to be involved in the relaxation of the

dilator muscle, but not the sphincter. Moreover, ghrelin’s mRNA was

identified in the iris’ stroma, particularly near the posterior epithelium.

Regarding the already proved effects of ghrelin-GHSR-1a system in the

muscular kinetics, it is possible that a local regulatory loop between

ghrelin and GHSR-1a occurs in this tissue. However, since ghrelin’s

expression in the iris was not detected in the present study, further

investigation is needed in order to clarify this situation.

Finally, GHSR is also expressed throughout the choroid, being these data

consistent with previous studies that had already characterized GHSR

expression in the vascular layer8. However, contrary to our data, in

previous studies this receptor co-localized with endothelial cells. The

presence of this receptor in the choroid and the presence of ghrelin in

Müller cells of the retina may suggest a a local regulation loop regarding


this tissue. However, further studies are needed to evaluate this

hypothesis and to determine the precise localization of GHSR.

2. Validation of the TonoVet® rebound tonometer

Two animal species were used to develop an acute glaucoma model, the

rabbit and the rat. To measure the intraocular pressure in these animal

models we used a commercial rebound tonometer used in veterinary,

the TonoVet®. This tonometer was chosen because it does not require

special training and the IOP is measured easily, rapidly and in a non-

invasive way, and thus causing no discomfort to animals. In fact, the

impact of the light-weight probe of the TonoVet® in the cornea is so

despicable that it does not trigger corneal reflex.

TonoVet® has been designed for veterinary use and calibrated for dogs,

cats and horses. Therefore, the validation of the tonometer by

establishing a linear relation with manometric values allows a more

accurate appreciation of the IOP, which is particularly important when

using the tonometer in different species such as the rabbit and the rat.

Both in the rabbit and in the rat, a relation between TonoVet®’s

measurements and IOP measured by in vivo manometry was established.

The TonoVet® clearly underestimates the IOP in both animals models. In

the rabbit, this result is in agreement with other tonometers196-199,

namely the Tono-pen XL®. Moreover, the underestimation exhibited by

the TonoVet® becomes more evident with IOP increase, which is also in

accordance with other reports199. However, TonoVet®’s accuracy seems



75

to vary according to species. Thus, in monkeys and cats, TonoVet®

proved to have a good correlation with manometric values200, 201.

In our study we also validated TonoVet®’s measurements in rat. In this

animal, when compared to our validation in the rabbit, the

underestimation of the IOP by the TonoVet® was much more

pronounced and more evident in high pressure values. However, for the

manometric values of 5 and 10 mmHg exceptionally, the rat’s tonometric

measurements are overestimated when compared with the rabbit’s.

To our knowledge this was the first evidence of successful utilization of

TonoVet® in the rat. Rebound tonometry has an error associated with

central corneal thickness. It has been reported that when measuring IOP

with rebound tonometry, thicker corneas result in overestimated

values202, 203. Because the rabbit presents a thicker cornea than the rat204,

it is it possible that the difference encountered between the values is

associated to this difference. Moreover, our correlation with in vivo

manometry was performed using the internal calibration for cats and

dogs. Thus, because cats and dogs have similar central corneal

thickness,205, 206 and this thickness is greater than the rabbits, it

strengthens our hypothesis.

The same company who fabricates TonoVet® also offers another

tonometer specifically designed for the use in rodents, the TonoLab®.

There have been discrepancies regarding TonoLab®’s accuracy in rats.

Two independent studies in Wistar rats reported a very good correlation

between TonoLab®’s measurements and manometry207, 208. However, a

study by Morrison et al. in Brown Norway rats described an

underestimation of IOP by the TonoLab® when compared to manometry.


Moreover, this study also classified the TonoLab® as non-sensitive for

measures below 20 mmHg209. Our findings with the TonoVet® in the rat

resemble most the latter study. In fact, although we did not find a

plateau below 20 mmHg, we noticed that most of TonoVet® readings

below 10 mmHg failed, requiring many attempts in order to obtain a

valid measurement. It is possible that due to the small size of the rats

eye when compared to the rabbits, at low pressures the probe is not

sensitive enough to perform accurate measurements. Moreover, this can

be an explanation for the discrepancies found at 5 and 10 mmHg in the

rat, which are the only overestimated values when compared with the

rabbit. Fortunately this was not a limitation to our study because rat’s

normal IOP is around 17-18 mmHg207, 210.

3. Animal model of OHT

To study the effects of ghrelin and des-acyl ghrelin in the acute elevation

of IOP, we adapted a previously described model9. Although several

other models have been used to transiently increase the IOP211-213, we

were not able to reproduce them in a consistent way. On the contrary,

when testing the model described by Orihashi et al9 we obtained similar

results (data not shown).

The injection of 0.1 mL of 5% NaCl in the vitreous body of rabbit eyes

produces an increase in the IOP that is evident in the first 15 min post-

injection. Ocular hypertension is maintained for a period of 90 min and

then starts to decrease slowly, maintaining IOP above normal values



77

until min 180 and returning to baseline values between 240 min and 300

min9, 214, 215. In the rabbit, the modification of the saline solution’s

concentration from 5% to 20% and the reduction of the volume injected

IV from 100µL to 50 µL conducted a more sustained increase in the IOP.

This modification did not alter per se the peak IOP at 15 min (44.31 ±

3.15 mmHg vs 49.7 ±2.5 mmHg216; 45.0 ±2.2 mmHg214), promoting an

increase of 34.98 ± 2.7 mmHg from baseline levels, similar to the values

reported by Orihashi9. However, this modification produced an evident

sustain in ocular hypertension that prolonged until min 150. After this

time, the pressure slowly started to decrease and remained above

normal values until min 240, reaching baseline values by min 300.

The same model was used in the rat, with a reduction of the volume

injected from 50 µL to 16 µL. The amount of volume injected was based

in the volume of the rat’s eye when compared with the rabbit’s. The

intravitreous injection of hypertonic saline in this animal also promoted a

sustained increase in the IOP. When compared to the rabbit, this

increase is much more pronounced and evident as soon as 5 min post-

injection (59.28 ± 4.26 mmHg). Indeed, at 15 min this model still exhibits

a marked difference (44.31 ± 3.15 mmHg in the rabbit vs 59.35 ± 4.72

mmHg in the rat). However, ocular hypertension in this animal model

remained constant only until 30 min post injection, then starting to

decrease, reaching baseline values after 120 min. Nevertheless,

hypertensive values were observed until min 60. The consistency of

these results indicates the stability and high reproducibility of this model

for the induction of intraocular hypertension.


The mechanism that explains the elevation of IOP in this model is the

osmolarity gradient created dependent on salt concentration9. When

injected, the salt deposits on the vitreous body and a gradient is

established allowing water entrance to the posterior segment. This

volume increase in the posterior segment pushes the lens anteriorly and

increases the IOP. Therefore, by increasing the salt concentration from

5% to 20% we were able to increase the gradient and maintain ocular

hypertension for a longer period of time. Nevertheless, it is possible that

the volume increase in the posterior segment can lead to an anterior

displacement of the iris and as well as a rotation in the CB, with

subsequent shallowing of the anterior chamber. This phenomenon is

known as malignant glaucoma217.

4. Effects of ghrelin and des-acyl ghrelin in animal models

of OHT

Ocular hypertension is the main controlled etiologic factor for glaucoma

and the reduction of IOP remains the best and only proved treatment.

Ghrelin has been implicated in the pathophysiology of glaucoma6, 7. Its

AqH levels are decreased in patients with different types of glaucoma,

while ghrelin’s plasmatic levels remain normal. Plasma and AqH levels of

des-acyl ghrelin are also normal in these patients6. Moreover, ghrelin has

been shown to relax both the iris’ sphincter and dilator muscle and

ghrelin’s mRNA has been identified in the eye5. Cuncurrent with all

previously said, this work enhances the hypothesis of a role for ghrelin in



79

the pathophysiology of glaucoma, since it demonstrates the lowering

effect of ghrelin in the IOP of hypertensive eyes.

Both in the rabbit and in the rat, ghrelin is responsible for a significant

decrease in the IOP of hypertensive eyes. In the rabbit, this effect was

maximum at 135 min post-elevation of IOP and corresponded to a

decrease of 18.98 ± 5.22 mmHg (43.81 ± 10.81%) when compared to

control. In the rat, the maximum decrease in the IOP obtained with

ghrelin was at 20 min and corresponded to a difference of 21.24 ± 4.13

mmHg (33.28 ± 6.47%).

The subconjunctival route was used to deliver both ghrelin and des-acyl

ghrelin to the eye. This route relies on sclera permeability for the

diffusion of drugs to the eye. Nevertheless, this route poses a risk for

systemic effects. We evaluated a possible systemic influence in the

hypotensive effects of these peptides. Both ghrelin and des-acyl ghrelin

are known potent vasodilators105, 218. Indeed, when injected systemically,

ghrelin decreases the mean arterial pressure by 12 mmHg and the

hypotensive effect is evident five minutes after injection. In our studies,

neither ghrelin nor des-acyl ghrelin were capable of decreasing the mean

arterial pressure. This indicates that the hypotensive effect of ghrelin in

the IOP is not systemically mediated.

The current therapeutic options to decrease IOP in primary open-angle

glaucoma include α2-adrenoreceptor agonists, β-blockers, carbonic

anhydrase inhibitors (CAIs) and hypotensive lipids (prostaglandin

analogs). All these drugs show a less effective decrease in the IOP than

ghrelin. In growing order of efficacy, Brinzolamide and Dorzolamide

(CAIs) decrease IOP 17 and 22 % respectively, Brimonidine (α-2 agonist)


decreases IOP 25%, Betaxolol and Timolol (β-adrenergic blockers)

decrease 23 and 27% respectively and finally Travoprost, Latanoprost

and Bimatoprost (prostaglandin analogs) that decrease 31, 31 and 33%

respectively219.

Glaucoma is not accompanied by a decrease in des-acyl ghrelin’s levels.

However, some studies have attributed biological effects to des-acyl

ghrelin similar to ghrelin in some organs. Similarly to ghrelin, we tested

des-acyl ghrelin effect in both rabbit and rat’s hypertensive eyes. In the

rabbit, contrary to ghrelin, this peptide failed to significantly decrease

the IOP. The maximum decrease achieved by this peptide was 7.29 ±

3.70 mmHg (16.80 ± 8.52%) at 135 min post-elevation of the IOP.

Moreover, des-acyl ghrelin’s eyes were not able to return to baseline

levels of IOP after 300 min and had a significant increase in IOP when

compared to control. Interestingly, in the rat des-acyl ghrelin was able to

significantly decrease the IOP in hypertensive eyes similarly to ghrelin.

The maximum decrease achieved by this peptide was at 30 min and

corresponded to a decrease of 21.09 ± 3.94 mmHg (39.09 ± 7.3%) when

compared to control.

Ghrelin and des-acyl ghrelin are similar peptides with the exception that

des-acyl ghrelin lacks the acyl group at Ser 3 and therefore it is unable to

bind GHSR-1a. Ghrelin’s hypotensive effect in the rabbit seems to be

mediated by GHSR-1a because des-acyl ghrelin failed to decrease the

IOP. However, in the rat this effect was not concurrent as des-acyl

ghrelin was also able to decrease the IOP with the same potency as

ghrelin. There is a great deal of uncertainty regarding ghrelin and des-

acyl ghrelin’s mechanism of action. On the one hand, both peptides have



81

been shown to have opposing effects in numerous systems122-124. On the

other hand, there also have been described similar effects78, 81, 119, or no

effects for des-acyl ghrelin when compared to ghrelin3, 107. In the eye,

des-acyl ghrelin has also been shown to have different effects depending

on the muscle. Thus, in the iris’ sphincter it is able to promote relaxation,

whereas in the dilator it has no effect5. Moreover, ghrelin was shown to

exert effects in tissues were GHSR-1a was not present220, 221. All this data

lead to the hypothesis of other receptors responsible for ghrelin and des-

acyl ghrelin’s actions. CD36, a multifunctional scavenger receptor with

widespread distribution has been pointed as one of the receptors that

may be indirectly involved in ghrelin’s actions due to its affinity to bind

growth hormone secretagogues222, 223. However, this hypothesis still

requires further studies. Another possible explanation is that possibly

the different hypotensive effect of des-acyl ghrelin, as well as the cellular

pathways activated, in these animal models is species dependent. In the

rabbit the effect seems to be mediated by GHSR-1a while in the rat it is

possible that the effect is associated to another receptor that remains

unknown.

4.1 Role of nitric oxide and prostaglandins in the effect of ghrelin

Nitric oxide and prostaglandins are two of the mediators involved in

some of ghrelin’s actions53, 224.In addition, in the eye ghrelin is

responsible for a relaxation of the iris’ sphincter muscle and this effect is

dependent on prostaglandins’ synthesis but independent from NO

release5. Because ghrelin has shown a hypotensive effect, we decided to

investigate the influence of NO and prostaglandins. In the rabbit, the


inhibition of NOS or COX with L-NAME or ketorolac completely blunted

ghrelin’s effect. In the L-NAME group, the maximum decrease in IOP

achieved was 0.55 ± 2.90 mmHg (1.25 ± 6.65%) at 20 min, while in the

ketorolac group it was 4.58 ± 3.40 mmHg (15.48 ± 11.50%) at 225 min.

These results suggest the involvement of these two mediators in

ghrelin’s effect in the rabbit animal model.

Nitric oxide is an important mediator in ocular physiology and pathology.

It can be synthetized by three isoforms of NOS: neuronal NOS (nNOS),

endothelial NOS (eNOS) and inducible NOS (iNOS). The first two are

constitutive enzymes and the latter is mainly expressed following

exposure to pro-inflammatory stimuli. Both nNOS and eNOS are active in

the anterior segment of the eye, particularly eNOS. Moreover, eNOS

activity is enriched in sites responsible for AqH outflow regulation such

as the CM, the TM and the Schlemm’s canal225. In addition, NO has been

implicated in the reduction of IOP184, 226. Regarding the rabbit animal

model, ghrelin’s hypotensive effect was shown to be related to NO

release. Ghrelin has been shown to phosphorylate eNOS and lead to the

synthesis of nitric oxide53. Nitric oxide in its turn is responsible for an

intracellular increase in cGMP that is responsible for an IOP lowering

effect184, 227. This increase in cGMP can induce the relaxation of the CM

and increase the US pathway promoting a decrease in IOP. Another

explanation relies on the fact that the TM has intrinsic contractile

elements164 that can be relaxed by NO. This would promote a decrease in

the trabecular tension and promote the drainage of AqH. These results

are in concordance with other studies where some compounds

promoted an IOP decrease dependent on NO synthesis214-216

.



83

Prostaglandins are hormones that play an important role in the ocular

tissue. They are produced by cells related with the AqH outflow pathway

and have been suggested as having a role in the regulation of AqH

outflow228, 229. Various prostaglandins’ receptors have also been located

in the ocular tissue230, 231. Moreover, PGF2α analogs such as Latanoprost,

Bimatoprost and Travoprost are currently the most effective drugs in the

treatment of glaucoma219. These drugs increase US outflow first by

relaxing the CM and then by activating matrix-metalloproteinases

(MMPs) which in long-term are responsible for the degradation of

extracellular matrix within the CM and a consequent increase in the US

pathway232-235. However, some studies indicate that MMPs are also

expressed by TM and that their activation may be involved in an increase

in the outflow by this route236-239. Cyclooxygenases are involved in the

pathway of prostaglandin formation. They are expressed in the ocular

tissue240, 241 and in our studies the blockage of these mediators with

ketorolac abolished ghrelin’s relaxing effect. The release of

prostaglandins promoted by ghrelin is known and has already been

verified in the ocular tissue5. As said above, PGF2α analogs are

responsible for an increase US outflow. These act primarily through FP

receptors and Gq signaling increasing intracellular Ca2+ 242. Another type

of prostaglandin, PGE2, can bind to 4 subtypes of receptor (EP1, EP2, EP3

and EP4). EP1 and EP3 receptors promote an increase in intracellular

Ca2+ and lead to contraction. EP3 is also able to inhibit cAMP

formation242. However, the stimulation of the EP2 in the CM activates

the Gs and triggers a signaling cascade that leads to an increase in

intracellular levels of cAMP. This increase is responsible for a relaxation

of the CM and a consequent increase in the US outflow243. Regarding the


trabecular outflow, a recent study showed that the stimulation of the

EP4 receptor present in the Schlemm’s canal improved this route244. The

mechanism responsible for prostaglandins’ reduction in IOP is still

unclear. The fact that PGF2α analogs act through FP receptors and Gq

signaling is not concordant with the theory of CM relaxation, as Ca2+

would lead to a contraction of this muscle. However, the increase in US

outflow is still considered the main mechanism of action for PGs analogs.

Some studies proposed that PGF2α can promote a PG-induced PG release

and that this may be an explanation for the mechanism of action of PGs

analogs235. Moreover, although PGs have their respective receptor,

natural PGs can bind with less affinity to the other PG receptors245.

Therefore, the hypotensive effect of ghrelin related to prostaglandins

release may be explained by an activation of receptors involved in the

relaxation of the CM or the TM, decreasing resistance and increasing

outflow. Although the effect related to prostaglandins’ analogs is highly

related to a remodeling of the extracellular matrix by MMPs, it is unlikely

that this effect could contribute to ghrelin’s decrease in IOP because

ghrelin is responsible for an acute decrease in IOP. Nevertheless,

because nothing is known about the effect of chronic administration of

ghrelin in the IOP, the possibility of MMP formation cannot be discarded.

Conclusion|


Conclusion

87

Conclusion

The equilibrium between AqH production and drainage is responsible for

a physiological IOP. When this equilibrium is disturbed either by an

excessive production or an insufficient drainage the IOP rises. Ocular

hypertension is the main cause for glaucoma which is one of the leading

causes of blindness in the western world. The current therapy for

glaucoma consists in surgical or pharmacological methods that focus in

the reduction of IOP. The CM plays a pivotal role in the drainage

mechanisms of the eye and most drugs that reduce the IOP have effects

in this muscle.

In our studies we have demonstrated ghrelin as potent hypotensive

peptide. Indeed, this hypotensive effect was superior than that of

current drugs used in the management of glaucoma. We have confirmed

that ghrelin is locally produced in the ocular tissue. This production,

along with the fact that this peptide‘s levels are reduced in glaucoma

patients strongly suggests that this peptide may be involved in the

pathophysiology of glaucoma.

In addition to ghrelin, also GHSR is present in the ocular tissue, namely in

the major components responsible for the regulation of aqueous

humour. Considering ghrelin’s effect in IOP and the presence of its

receptor in the ocular tissue, further studies should be performed to

elucidate the mechanism responsible for ghrelin’s action.

Taken together, these data suggest that the ghrelin and its receptor may

play an important role in the ocular physiology. Also, they should be

considered as potential therapeutic targets for glaucoma.

Bibliography|


Bibliography

91

Bibliography

1. Kojima M, Hosoda H, Date Y, Nakazato M, Matsuo H, Kangawa K. Ghrelin is a growth-hormone-releasing acylated peptide from stomach. Nature 1999;402:656-660. 2. Leite-Moreira AF, Soares JB. Physiological, pathological and potential therapeutic roles of ghrelin. Drug Discov Today 2007;12:276-288. 3. Hosoda H, Kojima M, Matsuo H, Kangawa K. Ghrelin and des-acyl ghrelin: two major forms of rat ghrelin peptide in gastrointestinal tissue. Biochem Biophys Res Commun 2000;279:909-913. 4. Soares JB, Leite-Moreira AF. Ghrelin, des-acyl ghrelin and obestatin: three pieces of the same puzzle. Peptides 2008;29:1255-1270. 5. Rocha-Sousa A, Saraiva J, Henriques-Coelho T, Falcao-Reis F, Correia-Pinto J, Leite-Moreira AF. Ghrelin as a novel locally produced relaxing peptide of the iris sphincter and dilator muscles. Exp Eye Res 2006;83:1179-1187. 6. Rocha-Sousa A, Alves-Faria P, Falcao-Pires I, Falcao-Reis F, Leite-Moreira AF. Analyses of aqueous humour ghrelin levels of eyes with and without glaucoma. Br J Ophthalmol 2009;93:131-132. 7. Katsanos A, Dastiridou A, Georgoulias P, Cholevas P, Kotoula M, Tsironi EE. Plasma and aqueous humour levels of ghrelin in open-angle glaucoma patients. Clin Experiment Ophthalmol 2011;39:324-329. 8. Zaniolo K, Sapieha P, Shao Z, et al. Ghrelin modulates physiologic and pathologic retinal angiogenesis through GHSR-1a. Invest Ophthalmol Vis Sci 2011;52:5376-5386. 9. Orihashi M, Shima Y, Tsuneki H, Kimura I. Potent reduction of intraocular pressure by nipradilol plus latanoprost in ocular hypertensive rabbits. Biol Pharm Bull 2005;28:65-68. 10. Wajnrajch MP, Ten IS, Gertner JM, Leibel RL. Genomic Organization of the Human GHRELIN Gene. International Journal on Disability and Human Development 2000;1:231-234.

92 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Bibliography

11. Hosoda H, Kojima M, Matsuo H, Kangawa K. Purification and characterization of rat des-Gln(14)-ghrelin, a second endogenous ligand for the growth hormone secretagogue receptor. Journal of Biological Chemistry 2000;275:21995-22000. 12. Hosoda H, Kojima M, Mizushima T, Shimizu S, Kangawa K. Structural divergence of human ghrelin. Identification of multiple ghrelin-derived molecules produced by post-translational processing. J Biol Chem 2003;278:64-70. 13. Yeh AH, Jeffery PL, Duncan RP, Herington AC, Chopin LK. Ghrelin and a novel preproghrelin isoform are highly expressed in prostate cancer and ghrelin activates mitogen-activated protein kinase in prostate cancer. Clin Cancer Res 2005;11:8295-8303. 14. Jeffery PL, Murray RE, Yeh AH, et al. Expression and function of the ghrelin axis, including a novel preproghrelin isoform, in human breast cancer tissues and cell lines. Endocrine-related cancer 2005;12:839-850. 15. Zhu X, Cao Y, Voogd K, Steiner DF. On the processing of proghrelin to ghrelin. J Biol Chem 2006;281:38867-38870. 16. Gutierrez JA, Solenberg PJ, Perkins DR, et al. Ghrelin octanoylation mediated by an orphan lipid transferase. Proc Natl Acad Sci U S A 2008;105:6320-6325. 17. Yang J, Brown MS, Liang G, Grishin NV, Goldstein JL. Identification of the acyltransferase that octanoylates ghrelin, an appetite-stimulating peptide hormone. Cell 2008;132:387-396. 18. Sakata I, Yang J, Lee CE, et al. Colocalization of ghrelin O-acyltransferase and ghrelin in gastric mucosal cells. Am J Physiol Endocrinol Metab 2009;297:E134-141. 19. Bednarek MA, Feighner SD, Pong SS, et al. Structure-function studies on the new growth hormone-releasing peptide, ghrelin: minimal sequence of ghrelin necessary for activation of growth hormone secretagogue receptor 1a. J Med Chem 2000;43:4370-4376. 20. Zhang JV, Ren PG, Avsian-Kretchmer O, et al. Obestatin, a peptide encoded by the ghrelin gene, opposes ghrelin's effects on food intake. Science 2005;310:996-999.


Bibliography

93

21. Pemberton C, Wimalasena P, Yandle T, Soule S, Richards M. C-terminal pro-ghrelin peptides are present in the human circulation. Biochem Biophys Res Commun 2003;310:567-573. 22. Bang AS, Soule SG, Yandle TG, Richards AM, Pemberton CJ. Characterisation of proghrelin peptides in mammalian tissue and plasma. J Endocrinol 2007;192:313-323. 23. Lauwers E, Landuyt B, Arckens L, Schoofs L, Luyten W. Obestatin does not activate orphan G protein-coupled receptor GPR39. Biochem Biophys Res Commun 2006;351:21-25. 24. Holst B, Egerod KL, Schild E, et al. GPR39 signaling is stimulated by zinc ions but not by obestatin. Endocrinology 2007;148:13-20. 25. Patterson M, Murphy KG, le Roux CW, Ghatei MA, Bloom SR. Characterization of ghrelin-like immunoreactivity in human plasma. J Clin Endocr Metab 2005;90:2205-2211. 26. De Vriese C, Gregoire F, Lema-Kisoka R, Waelbroeck M, Robberecht P, Delporte C. Ghrelin degradation by serum and tissue homogenates: identification of the cleavage sites. Endocrinology 2004;145:4997-5005. 27. De Vriese C, Hacquebard M, Gregoire F, Carpentier Y, Delporte C. Ghrelin interacts with human plasma lipoproteins. Endocrinology 2007;148:2355-2362. 28. Satou M, Nishi Y, Yoh J, Hattori Y, Sugimoto H. Identification and characterization of acyl-protein thioesterase 1/lysophospholipase I as a ghrelin deacylation/lysophospholipid hydrolyzing enzyme in fetal bovine serum and conditioned medium. Endocrinology 2010;151:4765-4775. 29. Beaumont NJ, Skinner VO, Tan TM, et al. Ghrelin can bind to a species of high density lipoprotein associated with paraoxonase. J Biol Chem 2003;278:8877-8880. 30. Kojima M, Kangawa K. Ghrelin: Structure and function. Physiol Rev 2005;85:495-522. 31. Ariyasu H, Takaya K, Tagami T, et al. Stomach Is a Major Source of Circulating Ghrelin, and Feeding State Determines Plasma Ghrelin-Like Immunoreactivity Levels in Humans. J Clin Endocrinol Metab 2001;86:4753-4758.


32. Date Y, Kojima M, Hosoda H, et al. Ghrelin, a novel growth hormone-releasing acylated peptide, is synthesized in a distinct endocrine cell type in the gastrointestinal tracts of rats and humans. Endocrinology 2000;141:4255-4261. 33. Gnanapavan S, Kola B BS, Morris DG, et al. The tissue distribution of the mRNA of ghrelin and subtypes of its receptor, GHS-R, in humans. J Clin Endocrinol Metab 2002;86:2988- 2991. 34. Dixit VD, Schaffer EM, Pyle RS, et al. Ghrelin inhibits leptin- and activation-induced proinflammatory cytokine expression by human monocytes and T cells. J Clin Invest 2004;114:57-66. 35. Howard AD, Feighner SD, Cully DF, et al. A receptor in pituitary and hypothalamus that functions in growth hormone release. Science 1996;273:974-977. 36. Pong SS, Chaung LYP, Dean DC, Nargund RP, Patchett AA, Smith RG. Identification of a new G-protein-linked receptor for growth hormone secretagogues. Mol Endocrinol 1996;10:57-61. 37. Holst B, Holliday ND, Bach A, Elling CE, Cox HM, Schwartz TW. Common structural basis for constitutive activity of the ghrelin receptor family. J Biol Chem 2004;279:53806-53817. 38. McKee KK, Palyha OC, Feighner SD, et al. Molecular analysis of rat pituitary and hypothalamic growth hormone secretagogue receptors. Mol Endocrinol 1997;11:415-423. 39. Chan CB, Cheng CH. Identification and functional characterization of two alternatively spliced growth hormone secretagogue receptor transcripts from the pituitary of black seabream Acanthopagrus schlegeli. Mol Cell Endocrinol 2004;214:81-95. 40. Leung PK, Chow KBS, Lau PN, et al. The truncated ghrelin receptor polypeptide (GHS-R1b) acts as a dominant-negative mutant of the ghrelin receptor. Cell Signal 2007;19:1011-1022. 41. Camina JP. Cell biology of the ghrelin receptor. Journal of Neuroendocrinology 2006;18:65-76. 42. Katugampola SD, Pallikaros Z, Davenport AP. I-125-His(9) -Ghrelin, a novel radioligand for localizing GHS orphan receptors in human and rat tissue; up-regulation of receptors with atherosclerosis. Br J Pharmacol 2001;134:143-149.


Bibliography

95

43. Iglesias MJ, Pineiro R, Blanco M, et al. Growth hormone releasing peptide (ghrelin) is synthesized and secreted by cardiomyocytes. Cardiovascular Research 2004;62:481-488. 44. Holst B, Cygankiewicz A, Jensen TH, Ankersen M, Schwartz TW. High constitutive signaling of the ghrelin receptor--identification of a potent inverse agonist. Mol Endocrinol 2003;17:2201-2210. 45. Feighner SD, Howard AD, Prendergast K, et al. Structural requirements for the activation of the human growth hormone secretagogue receptor by peptide and nonpeptide secretagogues. Mol Endocrinol 1998;12:137-145. 46. Carreira MC, Camina JP, Smith RG, Casanueva FF. Agonist-specific coupling of growth hormone secretagogue receptor type 1a to different intracellular signaling systems. Role of adenosine. Neuroendocrinology 2004;79:13-25. 47. Malagon MM, Luque RM, Ruiz-Guerrero E, et al. Intracellular signaling mechanisms mediating ghrelin-stimulated growth hormone release in somatotropes. Endocrinology 2003;144:5372-5380. 48. Andersson U, Filipsson K, Abbott CR, et al. AMP-activated protein kinase plays a role in the control of food intake. J Biol Chem 2004;279:12005-12008. 49. Rossi F, Castelli A, Bianco MJ, Bertone C, Brarna M, Santiemma V. Ghrelin induces proliferation in human aortic endothelial cells via ERK1/2 and PI3K/Akt activation. Peptides 2008;29:2046-2051. 50. Mousseaux D, Le Gallic L, Ryan J, et al. Regulation of ERK1/2 activity by ghrelin-activated growth hormone secretagogue receptor 1A involves a PLC/PKCvarepsilon pathway. Br J Pharmacol 2006;148:350-365. 51. Nanzer AM, Khalaf S, Mozid AM, et al. Ghrelin exerts a proliferative effect on a rat pituitary somatotroph cell line via the mitogen-activated protein kinase pathway. Eur J Endocrinol 2004;151:233-240. 52. Zhang Q, Huang WD, Lv XY, Yang YM. Ghrelin protects H9c2 cells from hydrogen peroxide-induced apoptosis through NF-


kappa B and mitochondria-mediated signaling. European Journal of Pharmacology 2011;654:142-149. 53. Xu X, Jhun BS, Ha CH, Jin ZG. Molecular mechanisms of ghrelin-mediated endothelial nitric oxide synthase activation. Endocrinology 2008;149:4183-4192. 54. Rodriguez-Pacheco F, Luque RM, Tena-Sempere M, Malagon MM, Castano JP. Ghrelin induces growth hormone secretion via a nitric oxide/cGMP signalling pathway. Journal of Neuroendocrinology 2008;20:406-412. 55. Choi KC, Roh SG, Hong YH, et al. The role of ghrelin and growth hormone secretagroogues receptor on rat adipogenesis. Endocrinology 2003;144:754-759. 56. Yamada H, Takahashi K. Ghrelin : production, action mechanisms & physiological effects. New York: Nova Science; 2012:xi, 153 p. 57. Arvat E, Di Vito L, Broglio F, et al. Preliminary evidence that Ghrelin, the natural GH secretagogue (GHS)-receptor ligand, strongly stimulates GH secretion in humans. J Endocrinol Invest 2000;23:493-495. 58. Takaya K, Ariyasu H, Kanamoto N, et al. Ghrelin strongly stimulates growth hormone (GH) release in humans. J Clin Endocr Metab 2000;85:4908-4911. 59. Hataya Y, Akamizu T, Takaya K, et al. A low dose of ghrelin stimulates growth hormone (GH) release synergistically with GH-releasing hormone in humans. The Journal of clinical endocrinology and metabolism 2001;86:4552. 60. Popovic V, Miljic D, Micic D, et al. Ghrelin main action on the regulation of growth hormone release is exerted at hypothalamic level. The Journal of clinical endocrinology and metabolism 2003;88:3450-3453. 61. Broglio F, Benso A, Castiglioni C, et al. The endocrine response to ghrelin as a function of gender in humans in young and elderly subjects. J Clin Endocrinol Metab 2003;88:1537-1542. 62. Mozid AM, Tringali G, Forsling ML, et al. Ghrelin is released from rat hypothalamic explants and stimulates


Bibliography

97

corticotrophin-releasing hormone and arginine-vasopressin. Horm Metab Res 2003;35:455-459. 63. van der Lely AJ, Tschop M, Heiman ML, Ghigo E. Biological, physiological, pathophysiological, and pharmacological aspects of ghrelin. Endocr Rev 2004;25:426-457. 64. Fernandez-Fernandez R, Tena-Sempere M, Navarro VM, et al. Effects of ghrelin upon gonadotropin-releasing hormone and gonadotropin secretion in adult female rats: in vivo and in vitro studies. Neuroendocrinology 2005;82:245-255. 65. Schmid DA, Held K, Ising M, Uhr M, Weikel JC, Steiger A. Ghrelin stimulates appetite, imagination of food, GH, ACTH, and cortisol, but does not affect leptin in normal controls. Neuropsychopharmacology : official publication of the American College of Neuropsychopharmacology 2005;30:1187-1192. 66. Kluge M, Schussler P, Schmidt D, Uhr M, Steiger A. Ghrelin suppresses secretion of luteinizing hormone (LH) and follicle-stimulating hormone (FSH) in women. J Clin Endocrinol Metab 2012;97:E448-451. 67. Cummings DE, Weigle DS, Frayo RS, et al. Plasma ghrelin levels after diet-induced weight loss or gastric bypass surgery. New England Journal of Medicine 2002;346:1623-1630. 68. Tschop M, Wawarta R, Riepl RL, et al. Post-prandial decrease of circulating human ghrelin levels. J Endocrinol Invest 2001;24:RC19-RC21. 69. Drazen DL, Woods SC. Peripheral signals in the control of satiety and hunger. Current Opinion in Clinical Nutrition and Metabolic Care 2003;6:621-628. 70. Cowley MA, Smith RG, Diano S, et al. The distribution and mechanism of action of ghrelin in the CNS demonstrates a novel hypothalamic circuit regulating energy homeostasis. Neuron 2003;37:649-661. 71. Williams DL, Grill HJ, Cummings DE, Kaplan JM. Overfeeding-induced weight gain suppresses plasma ghrelin levels in rats. J Endocrinol Invest 2006;29:863-868. 72. Otukonyong EE, Dube MG, Torto R, Kalra PS, Kalra SP. High-fat diet-induced ultradian leptin and insulin hypersecretion


are absent in obesity-resistant rats. Obesity research 2005;13:991-999. 73. Egido EM, Rodriguez-Gallardo J, Silvestre RA, Marco J. Inhibitory effect of ghrelin on insulin and pancreatic somatostatin secretion. Eur J Endocrinol 2002;146:241-244. 74. Broglio F, Gottero C, Benso A, et al. Effects of ghrelin on the insulin and glycemic responses to glucose, arginine, or free fatty acids load in humans. J Clin Endocr Metab 2003;88:4268-4272. 75. Broglio F, Arvat E, Benso A, et al. Ghrelin, a natural GH secretagogue produced by the stomach, induces hyperglycemia and reduces insulin secretion in humans. J Clin Endocrinol Metab 2001;86:5083-5086. 76. Flanagan DE, Evans ML, Monsod TP, et al. The influence of insulin on circulating ghrelin. Am J Physiol Endocrinol Metab 2003;284:E313-316. 77. Shiiya T, Nakazato M, Mizuta M, et al. Plasma ghrelin levels in lean and obese humans and the effect of glucose on ghrelin secretion. J Clin Endocrinol Metab 2002;87:240-244. 78. Thompson NM, Gill DA, Davies R, et al. Ghrelin and des-octanoyl ghrelin promote adipogenesis directly in vivo by a mechanism independent of the type 1a growth hormone secretagogue receptor. Endocrinology 2004;145:234-242. 79. Patel AD, Stanley SA, Murphy KG, et al. Ghrelin stimulates insulin-induced glucose uptake in adipocytes. Regul Pept 2006;134:17-22. 80. Tschop M, Smiley DL, Heiman ML. Ghrelin induces adiposity in rodents. Nature 2000;407:908-913. 81. Muccioli G, Pons N, Ghe C, Catapano F, Granata R, Ghigo E. Ghrelin and des-acyl ghrelin both inhibit isoproterenol-induced lipolysis in rat adipocytes via a non-type 1a growth hormone secretagogue receptor. Eur J Pharmacol 2004;498:27-35. 82. Carlini VP, Monzon ME, Varas MM, et al. Ghrelin increases anxiety-like behavior and memory retention in rats. Biochem Biophys Res Commun 2002;299:739-743.


Bibliography

99

83. Carlini VP, Varas MM, Cragnolini AB, Schioth HB, Scimonelli TN, de Barioglio SR. Differential role of the hippocampus, amygdala, and dorsal raphe nucleus in regulating feeding, memory, and anxiety-like behavioral responses to ghrelin. Biochem Biophys Res Commun 2004;313:635-641. 84. Carlini VP, Gaydou RC, Schioth HB, de Barioglio SR. Selective serotonin reuptake inhibitor (fluoxetine) decreases the effects of ghrelin on memory retention and food intake. Regul Pept 2007;140:65-73. 85. Diano S, Farr SA, Benoit SC, et al. Ghrelin controls hippocampal spine synapse density and memory performance. Nature neuroscience 2006;9:381-388. 86. Obal F, Jr., Alt J, Taishi P, Gardi J, Krueger JM. Sleep in mice with nonfunctional growth hormone-releasing hormone receptors. Am J Physiol Regul Integr Comp Physiol 2003;284:R131-139. 87. Weikel JC, Wichniak A, Ising M, et al. Ghrelin promotes slow-wave sleep in humans. Am J Physiol Endocrinol Metab 2003;284:E407-415. 88. Schussler P, Uhr M, Ising M, et al. Nocturnal ghrelin, ACTH, GH and cortisol secretion after sleep deprivation in humans. Psychoneuroendocrinology 2006;31:915-923. 89. Szentirmai E, Hajdu I, Obal F, Jr., Krueger JM. Ghrelin-induced sleep responses in ad libitum fed and food-restricted rats. Brain Res 2006;1088:131-140. 90. Szentirmai E, Kapas L, Krueger JM. Ghrelin microinjection into forebrain sites induces wakefulness and feeding in rats. Am J Physiol Regul Integr Comp Physiol 2007;292:R575-585. 91. Steiger A. Ghrelin and sleep-wake regulation. Am J Physiol Regul Integr Comp Physiol 2007;292:R573-574. 92. Asakawa A, Inui A, Kaga T, et al. A role of ghrelin in neuroendocrine and behavioral responses to stress in mice. Neuroendocrinology 2001;74:143-147. 93. Dornonville de la Cour C, Lindstrom E, Norlen P, Hakanson R. Ghrelin stimulates gastric emptying but is without effect on acid secretion and gastric endocrine cells. Regul Pept 2004;120:23-32.


94. Date Y, Nakazato M, Murakami N, Kojima M, Kangawa K, Matsukura S. Ghrelin acts in the central nervous system to stimulate gastric acid secretion. Biochem Biophys Res Commun 2001;280:904-907. 95. Sibilia V, Muccioli G, Deghenghi R, et al. Evidence for a role of the GHS-R1a receptors in ghrelin inhibition of gastric acid secretion in the rat. J Neuroendocrinol 2006;18:122-128. 96. Yakabi K, Ro S, Onouhi T, et al. Histamine mediates the stimulatory action of ghrelin on acid secretion in rat stomach. Digestive diseases and sciences 2006;51:1313-1321. 97. Brzozowski T, Konturek PC, Drozdowicz D, et al. Role of central and peripheral ghrelin in the mechanism of gastric mucosal defence. Inflammopharmacology 2005;13:45-62. 98. Volante M, Fulcheri E, Allia E, Cerrato M, Pucci A, Papotti M. Ghrelin expression in fetal, infant, and adult human lung. J Histochem Cytochem 2002;50:1013-1021. 99. Henriques-Coelho T, Correia-Pinto J, Roncon-Albuquerque R, Jr., et al. Endogenous production of ghrelin and beneficial effects of its exogenous administration in monocrotaline-induced pulmonary hypertension. Am J Physiol Heart Circ Physiol 2004;287:H2885-2890. 100. Pierno S, De Luca A, Desaphy JF, et al. Growth hormone secretagogues modulate the electrical and contractile properties of rat skeletal muscle through a ghrelin-specific receptor. Br J Pharmacol 2003;139:575-584. 101. Wiley KE, Davenport AP. Comparison of vasodilators in human internal mammary artery: ghrelin is a potent physiological antagonist of endothelin-1. Br J Pharmacol 2002;136:1146-1152. 102. Shinde UA, Desai KM, Yu C, Gopalakrishnan V. Nitric oxide synthase inhibition exaggerates the hypotensive response to ghrelin: role of calcium-activated potassium channels. Journal of hypertension 2005;23:779-784. 103. Bedendi I, Alloatti G, Marcantoni A, et al. Cardiac effects of ghrelin and its endogenous derivatives des-octanoyl ghrelin and des-Gln (14)-ghrelin. European Journal of Pharmacology 2003;476:87-95.


Bibliography

101

104. Soares JB, Rocha-Sousa A, Castro-Chaves P, Henriques-Coelho T, Leite-Moreira AF. Inotropic and lusitropic effects of ghrelin and their modulation by the endocardial endothelium, NO, prostaglandins, GHS-R1a and K-Ca channels. Peptides 2006;27:1616-1623. 105. Nagaya N, Kojima M, Uematsu M, et al. Hemodynamic and hormonal effects of human ghrelin in healthy volunteers. American Journal of Physiology-Regulatory Integrative and Comparative Physiology 2001;280:R1483-R1487. 106. Pemberton CJ, Tokola H, Bagi Z, et al. Ghrelin induces vasoconstriction in the rat coronary vasculature without altering cardiac peptide secretion. American Journal of Physiology-Heart and Circulatory Physiology 2004;287:H1522-H1529. 107. Li WG, Gavrila D, Liu XB, et al. Ghrelin inhibits proinflammatory responses and nuclear factor-kappa B activation in human endothelial cells. Circulation 2004;109:2221-2226. 108. Tena-Sempere M, Barreiro ML, Gonzalez LC, et al. Novel expression and functional role of ghrelin in rat testis. Endocrinology 2002;143:717-725. 109. Barreiro ML, Gaytan F, Castellano JM, et al. Ghrelin inhibits the proliferative activity of immature Leydig cells in vivo and regulates stem cell factor messenger ribonucleic acid expression in rat testis. Endocrinology 2004;145:4825-4834. 110. Garcia MC, Lopez M, Alvarez CV, Casanueva F, Tena-Sempere M, Dieguez C. Role of ghrelin in reproduction. Reproduction 2007;133:531-540. 111. Caminos JE, Tena-Sempere M, Gaytan F, et al. Expression of ghrelin in the cyclic and pregnant rat ovary. Endocrinology 2003;144:1594-1602. 112. Koo GC, Huang C, Camacho R, et al. Immune enhancing effect of a growth hormone secretagogue. J Immunol 2001;166:4195-4201. 113. Chang L, Zhao J, Yang J, Zhang Z, Du J, Tang C. Therapeutic effects of ghrelin on endotoxic shock in rats. European Journal of Pharmacology 2003;473:171-176.


114. Delhanty PJ, van der Eerden BC, van der Velde M, et al. Ghrelin and unacylated ghrelin stimulate human osteoblast growth via mitogen-activated protein kinase (MAPK)/phosphoinositide 3-kinase (PI3K) pathways in the absence of GHS-R1a. J Endocrinol 2006;188:37-47. 115. Lago R, Gomez R, Dieguez C, Gomez-Reino JJ, Lago F, Gualillo O. Unlike ghrelin, obestatin does not exert any relevant activity in chondrocytes. Ann Rheum Dis 2007;66:1399-1400. 116. Fukushima N, Hanada R, Teranishi H, et al. Ghrelin directly regulates bone formation. J Bone Miner Res 2005;20:790-798. 117. Maccarinelli G, Sibilia V, Torsello A, et al. Ghrelin regulates proliferation and differentiation of osteoblastic cells. J Endocrinol 2005;184:249-256. 118. Rocha-Sousa A, Tavares-Silva M, Fonseca S, Falcão M, Falcão-Reis F, Leite-Moreira AF. Diabetes, Ghrelin And Related Peptides: From Pathophysiology To Vasculopathy. The Open Circulation and Vascular Journal 2010;3:17-29. 119. Baldanzi G, Filigheddu N, Cutrupi S, et al. Ghrelin and des-acyl ghrelin inhibit cell death in cardiomyocytes and endothelial cells through ERK1/2 and PI 3-kinase/AKT. Journal of Cell Biology 2002;159:1029-1037. 120. Granata R, Settanni F, Biancone L, et al. Acylated and unacylated ghrelin promote proliferation and inhibit apoptosis of pancreatic beta-cells and human islets: involvement of 3',5'-cyclic adenosine monophosphate/protein kinase A, extracellular signal-regulated kinase 1/2, and phosphatidyl inositol 3-Kinase/Akt signaling. Endocrinology 2007;148:512-529. 121. Delhanty PJ, van Koetsveld PM, Gauna C, et al. Ghrelin and its unacylated isoform stimulate the growth of adrenocortical tumor cells via an anti-apoptotic pathway. Am J Physiol Endocrinol Metab 2007;293:E302-309. 122. Chen CY, Chao Y, Chang FY, Chien EJ, Lee SD, Doong ML. Intracisternal des-acyl ghrelin inhibits food intake and non-nutrient gastric emptying in conscious rats. Int J Mol Med 2005;16:695-699.


Bibliography

103

123. Asakawa A, Inui A, Fujimiya M, et al. Stomach regulates energy balance via acylated ghrelin and desacyl ghrelin. Gut 2005;54:18-24. 124. Gauna C, Delhanty PJ, Hofland LJ, et al. Ghrelin stimulates, whereas des-octanoyl ghrelin inhibits, glucose output by primary hepatocytes. J Clin Endocrinol Metab 2005;90:1055-1060. 125. Kaufman PL, Alm A, Adler FH. Adler's physiology of the eye : clinical application. 10th ed. St. Louis: Mosby; 2003:xvii, 876 p. 126. Yamaji K, Yoshitomi T, Usui S, Ohnishi Y. Mechanical properties of the rabbit iris smooth muscles. Vision research 2003;43:479-487. 127. Choppin A, Eglen RM, Hegde SS. Pharmacological characterization of muscarinic receptors in rabbit isolated iris sphincter muscle and urinary bladder smooth muscle. Br J Pharmacol 1998;124:883-888. 128. Ishizaka N, Noda M, Yokoyama S, Kawasaki K, Yamamoto M, Higashida H. Muscarinic acetylcholine receptor subtypes in the human iris. Brain Res 1998;787:344-347. 129. Ehinger B. Ocular and orbital vegetative nerves. Acta Physiol Scand Suppl 1966;268:1-35. 130. Tajti J, Uddman R, Moller S, Sundler F, Edvinsson L. Messenger molecules and receptor mRNA in the human trigeminal ganglion. Journal of the Autonomic Nervous System 1999;76:176-183. 131. Wang ZY, Alm P, Hakanson R. Distribution and effects of pituitary adenylate cyclase-activating peptide in the rabbit eye. Neuroscience 1995;69:297-308. 132. Beding-Barnekow B, Brodin E, Hakanson R. Substance-p, neurokinin-a and neurokinin-b in the ocular response to injury in the rabbit. Br J Pharmacol 1988;95:259-267. 133. Bynke G, Hakanson R, Horig J, Leander S. Bradykinin contracts the pupillary sphincter and evokes ocular inflammation through release of neuronal substance-p. European Journal of Pharmacology 1983;91:469-475.


134. El Sayah M, Calixto JB. New evidence on the mechanisms underlying bradykinin-mediated contraction of the pig iris sphincter in vitro. Peptides 2003;24:1045-1051. 135. Lu W, Okamura T, Bian K, Inatomi A, Toda N. Prostaglandins involved in contractions by angiotensin-ii and bradykinin of isolated dog sphincter pupillae. Br J Pharmacol 1988;95:544-550. 136. Osborne NN, Barnett NL. Endothelin-1 stimulates phosphatidylinositol hydrolysis in the iris ciliary complex and is a potent constrictor of the sphincter muscle. Exp Eye Res 1992;54:285-290. 137. Barr L, Gu FJ. A quantitative model of myosin phosphorylation and the photomechanical response of the isolated sphincter pupillae of the frog iris. Biophysical Journal 1987;51:895-904. 138. Tachado SD, Akhtar RA, Abdellatif AA. Activation of beta-adrenergic receptors causes stimulation of cyclic-amp, inhibition of inositol trisphosphate, and relaxation of bovine iris sphincter smooth-muscle - biochemical and functional interactions between the cyclic-amp and calcium signaling systems. Invest Ophthalmol Vis Sci 1989;30:2232-2239. 139. Tachado SD, Akhtar RA, Zhou CJ, Abdellatif AA. Effects of isoproterenol and forskolin on carbachol-induced and fluoroaluminate-induced polyphosphoinositide hydrolysis, inositol trisphosphate production, and contraction in bovine iris sphincter smooth-muscle - interaction between camp and ip3 2nd messenger systems. Cell Signal 1992;4:61-75. 140. Ding KH, AbdelLatif AA. Actions of C-type natriuretic peptide and sodium nitroprusside on carbachol-stimulated inositol phosphate formation and contraction in ciliary and iris sphincter smooth muscles. Invest Ophthalmol Vis Sci 1997;38:2629-2638. 141. Gustafsson LE, Wiklund NP. Adenosine-modulation of cholinergic and nonadrenergic noncholinergic neurotransmission in the rabbit iris sphincter. Br J Pharmacol 1986;88:197-204.


Bibliography

105

142. Yamaji K, Yoshitomi T, Usui S. Effect of somatostatin and galanin on isolated rabbit iris sphincter and dilator muscles. Exp Eye Res 2003;77:609-614. 143. Hayashi K, Masuda K. Effects of vasoactive intestinal polypeptide (vip) and cyclic-amp on the isolated sphincter pupillae muscles of the albino rabbit. Japanese Journal of Ophthalmology 1982;26:437-442. 144. Hayashi K, Mochizuki M, Masuda K. Effects of vasoactive intestinal polypeptide (vip) and cyclic-amp on isolated dilator pupillae muscle of albino rabbit eye. Japanese Journal of Ophthalmology 1983;27:647-654. 145. Yousufzai SYK, Ali N, Abdel-Latif AA. Effects of Adrenomedullin on Cyclic AMP Formation and on Relaxation in Iris Sphincter Smooth Muscle. Invest Ophthalmol Vis Sci 1999;40:3245-3253. 146. Uchikawa Y, Okano M, Sawada A, et al. Relaxant effect of adrenomedullin on bovine isolated iris sphincter muscle under resting conditions. Clinical and Experimental Pharmacology and Physiology 2005;32:675-680. 147. Geyer O, Bar-Ilan A, Nachman R, Lazar M, Oron Y. beta(3)-Adrenergic relaxation of bovine iris sphincter. Febs Letters 1998;429:356-358. 148. Toda M, Okamura T, Fujimiya M, Azuma I, Toda N. Mechanisms underlying the neurogenic relaxation of isolated porcine sphincter pupillae. Exp Eye Res 1999;68:505-512. 149. Pianka P, Oron Y, Lazar M, Geyer O. Nonadrenergic, noncholinergic relaxation of bovine iris sphincter: Role of endogenous nitric oxide. Invest Ophthalmol Vis Sci 2000;41:880-886. 150. Rocha-Sousa A, Saraiva J, Amaral M, Alves-Faria P, Falcao-Reis F, Leite-Moreira AF. ETB2 receptor subtype stimulation relaxes the iris sphincter muscle. Physiological research / Academia Scientiarum Bohemoslovaca 2009;58:835-842. 151. Piccone M, Littzi J, Krupin T, Stone RA, Davis M, Wax MB. Effects of neuropeptide Y on the isolated rabbit iris dilator muscle. Invest Ophthalmol Vis Sci 1988;29:330-332.


152. Satoh M, Yamamoto Y, Takayanagi I. Characterization of endothelin receptor subtypes mediating Ca2+ mobilization and contractile response in rabbit iris dilator muscle. Br J Pharmacol 1996;117:1277-1285. 153. Masuda Y, Yamahara NS, Tanaka M, et al. Characterization of muscarinic receptors mediating relaxation and contraction in the rat iris dilator muscle. Br J Pharmacol 1995;114:769-776. 154. Yoshitomi T, Yamaji K, Ishikawa H, Ohnishi Y. Effect of pituitary adenylate cyclase-activating peptide on isolated rabbit iris sphincter and dilator muscles. Invest Ophthalmol Vis Sci 2002;43:780-783. 155. Yousufzai SY, Abdel-Latif AA. Calcitonin gene-related peptide relaxes rabbit iris dilator smooth muscle via cyclic AMP-dependent mechanisms: cross-talk between the sensory and sympathetic nervous systems. Curr Eye Res 1998;17:197-204. 156. Escribano J, Coca-Prados M. Bioinformatics and reanalysis of subtracted expressed sequence tags from the human ciliary body: Identification of novel biological functions. Molecular Vision 2002;8:315-332. 157. Coca-Prados M, Escribano J. New perspectives in aqueous humor secretion and in glaucoma: The ciliary body as a multifunctional neuroendocrine gland. Progress in Retinal and Eye Research 2007;26:239-262. 158. Raviola G, Raviola E. Intercellular junctions in the ciliary epithelium. Invest Ophthalmol Vis Sci 1978;17:958-981. 159. Smelser GK. Electron microscopy of a typical epithelial cell and of the normal human ciliary process. Transactions - American Academy of Ophthalmology and Otolaryngology American Academy of Ophthalmology and Otolaryngology 1966;70:738-754. 160. Tormey JM. The ciliary epithelium: an attempt to correlate structure and function. Transactions - American Academy of Ophthalmology and Otolaryngology American Academy of Ophthalmology and Otolaryngology 1966;70:755-766. 161. Do CW, Civan MM. Species variation in biology and physiology of the ciliary epithelium: Similarities and differences. Exp Eye Res 2009;88:631-640.


Bibliography

107

162. Mark HH. Aqueous humor dynamics in historical perspective. Survey of ophthalmology 2010;55:89-100. 163. Tornquist P, Alm A, Bill A. Permeability of ocular vessels and transport across the blood-retinal-barrier. Eye (Lond) 1990;4 ( Pt 2):303-309. 164. Tamm ER. The trabecular meshwork outflow pathways: structural and functional aspects. Exp Eye Res 2009;88:648-655. 165. Grant WM. Further studies on facility of flow through the trabecular meshwork. AMA archives of ophthalmology 1958;60:523-533. 166. Barany EH. The mode of action of miotics on outflow resistance. A study of pilocarpine in the vervet monkey Cercopithecus ethiops. Transactions of the ophthalmological societies of the United Kingdom 1966;86:539-578. 167. Tamm E, Flugel C, Stefani FH, Rohen JW. Contractile cells in the human scleral spur. Exp Eye Res 1992;54:531-543. 168. Alm A, Nilsson SF. Uveoscleral outflow--a review. Exp Eye Res 2009;88:760-768. 169. Bill A, Phillips CI. Uveoscleral drainage of aqueous humour in human eyes. Exp Eye Res 1971;12:275-281. 170. Warwick R. The ocular parasympathetic nerve supply and its mesencephalic sources. Journal of anatomy 1954;88:71-93. 171. Ruskell GL, Griffiths T. Peripheral nerve pathway to the ciliary muscle. Exp Eye Res 1979;28:277-284. 172. Gupta N, Drance SM, McAllister R, Prasad S, Rootman J, Cynader MS. Localization of M3 muscarinic receptor subtype and mRNA in the human eye. Ophthalmic Res 1994;26:207-213. 173. Gupta N, McAllister R, Drance SM, Rootman J, Cynader MS. Muscarinic receptor M1 and M2 subtypes in the human eye: QNB, pirenzipine, oxotremorine, and AFDX-116 in vitro autoradiography. Br J Ophthalmol 1994;78:555-559. 174. Prasanna G, Dibas AI, Yorio T. Cholinergic and adrenergic modulation of the Ca2+ response to endothelin-1 in human ciliary muscle cells. Invest Ophthalmol Vis Sci 2000;41:1142-1148. 175. Nickla DL, Wallman J. The multifunctional choroid. Prog Retin Eye Res 2010;29:144-168.


176. Kiel JW, Shepherd AP. Autoregulation of choroidal blood flow in the rabbit. Invest Ophthalmol Vis Sci 1992;33:2399-2410. 177. Rocha-Sousa A, Falcao-Reis F, Leite-Moreira AF. The obestatin/ghrelin system as a novel regulatory mechanism of iris muscle contraction. Curr Eye Res 2008;33:73-79. 178. Quigley HA, Broman AT. The number of people with glaucoma worldwide in 2010 and 2020. Br J Ophthalmol 2006;90:262-267. 179. Boland MV, Quigley HA. Risk factors and open-angle glaucoma: classification and application. J Glaucoma 2007;16:406-418. 180. Kempen JH, O'Colmain BJ, Leske MC, et al. The prevalence of diabetic retinopathy among adults in the United States. Arch Ophthalmol 2004;122:552-563. 181. Gilbert C, Rahi J, Eckstein M, O'Sullivan J, Foster A. Retinopathy of prematurity in middle-income countries. Lancet 1997;350:12-14. 182. Cassoni P, Ghe C, Marrocco T, et al. Expression of ghrelin and biological activity of specific receptors for ghrelin and des-acyl ghrelin in human prostate neoplasms and related cell lines. Eur J Endocrinol 2004;150:173-184. 183. Riley MV, Kishida K. ATPases of ciliary epithelium: cellular and subcellular distribution and probable role in secretion of aqueous humor. Exp Eye Res 1986;42:559-568. 184. Kotikoski H, Vapaatalo H, Oksala O. Nitric oxide and cyclic GMP enhance aqueous humor outflow facility in rabbits. Curr Eye Res 2003;26:119-123. 185. Shahidullah M, Yap M, To CH. Cyclic GMP, sodium nitroprusside and sodium azide reduce aqueous humour formation in the isolated arterially perfused pig eye. Br J Pharmacol 2005;145:84-92. 186. Shahidullah M, Delamere NA. NO donors inhibit Na,K-ATPase activity by a protein kinase G-dependent mechanism in the nonpigmented ciliary epithelium of the porcine eye. Br J Pharmacol 2006;148:871-880.


Bibliography

109

187. Tamm ER, Koch TA, Mayer B, Stefani FH, Lutjen-Drecoll E. Innervation of myofibroblast-like scleral spur cells in human monkey eyes. Invest Ophthalmol Vis Sci 1995;36:1633-1644. 188. Wiederholt M, Thieme H, Stumpff F. The regulation of trabecular meshwork and ciliary muscle contractility. Prog Retin Eye Res 2000;19:271-295. 189. Wiederholt M, Sturm A, Lepple-Wienhues A. Relaxation of trabecular meshwork and ciliary muscle by release of nitric oxide. Invest Ophthalmol Vis Sci 1994;35:2515-2520. 190. Thieme H, Nass JU, Nuskovski M, et al. The effects of protein kinase C on trabecular meshwork and ciliary muscle contractility. Invest Ophthalmol Vis Sci 1999;40:3254-3261. 191. Imanishi J, Kamiyama K, Iguchi I, Kita M, Sotozono C, Kinoshita S. Growth factors: importance in wound healing and maintenance of transparency of the cornea. Prog Retin Eye Res 2000;19:113-129. 192. Imayasu M, Shimada S. Phosphorylation of MAP kinase in corneal epithelial cells during wound healing. Curr Eye Res 2003;27:133-141. 193. Chandrasekher G, Bazan NG, Bazan HE. Selective changes in protein kinase C (PKC) isoform expression in rabbit corneal epithelium during wound healing. Inhibition of corneal epithelial repair by PKCalpha antisense. Exp Eye Res 1998;67:603-610. 194. Hirakata A, Gupta AG, Proia AD. Effect of protein kinase C inhibitors and activators on corneal re-epithelialization in the rat. Invest Ophthalmol Vis Sci 1993;34:216-221. 195. Jumblatt MM, Neufeld AH. Characterization of cyclic AMP-mediated wound closure of the rabbit corneal epithelium. Curr Eye Res 1981;1:189-195. 196. Lim KS, Wickremasinghe SS, Cordeiro MF, Bunce C, Khaw PT. Accuracy of intraocular pressure measurements in new zealand white rabbits. Invest Ophthalmol Vis Sci 2005;46:2419-2423. 197. Acosta AC, Espana EM, Nose I, et al. Estimation of intraocular pressure in rabbits with commonly used tonometers.


Ophthalmic surgery, lasers & imaging : the official journal of the International Society for Imaging in the Eye 2007;38:43-49. 198. Abrams LS, Vitale S, Jampel HD. Comparison of three tonometers for measuring intraocular pressure in rabbits. Invest Ophthalmol Vis Sci 1996;37:940-944. 199. Kalesnykas G, Uusitalo H. Comparison of simultaneous readings of intraocular pressure in rabbits using Perkins handheld, Tono-Pen XL, and TonoVet tonometers. Graefes Arch Clin Exp Ophthalmol 2007;245:761-762. 200. Yu W, Cao G, Qiu J, et al. Evaluation of monkey intraocular pressure by rebound tonometer. Molecular Vision 2009;15:2196-2201. 201. McLellan GJ, Kemmerling JP, Kiland JA. Validation of the TonoVet((R)) rebound tonometer in normal and glaucomatous cats. Vet Ophthalmol 2012. 202. Iliev ME, Goldblum D, Katsoulis K, Amstutz C, Frueh B. Comparison of rebound tonometry with Goldmann applanation tonometry and correlation with central corneal thickness. Br J Ophthalmol 2006;90:833-835. 203. Sahin A, Basmak H, Yildirim N. The influence of central corneal thickness and corneal curvature on intraocular pressure measured by tono-pen and rebound tonometer in children. J Glaucoma 2008;17:57-61. 204. Schulz D, Iliev ME, Frueh BE, Goldblum D. In vivo pachymetry in normal eyes of rats, mice and rabbits with the optical low coherence reflectometer. Vision research 2003;43:723-728. 205. Gilger BC, Whitley RD, McLaughlin SA, Wright JC, Drane JW. Canine corneal thickness measured by ultrasonic pachymetry. American journal of veterinary research 1991;52:1570-1572. 206. Gilger BC, Wright JC, Whitley RD, McLaughlin SA. Corneal thickness measured by ultrasonic pachymetry in cats. American journal of veterinary research 1993;54:228-230. 207. Wang WH, Millar JC, Pang IH, Wax MB, Clark AF. Noninvasive measurement of rodent intraocular pressure with a


Bibliography

111

rebound tonometer. Invest Ophthalmol Vis Sci 2005;46:4617-4621. 208. Pease ME, Hammond JC, Quigley HA. Manometric calibration and comparison of TonoLab and TonoPen tonometers in rats with experimental glaucoma and in normal mice. J Glaucoma 2006;15:512-519. 209. Morrison JC, Jia L, Cepurna W, Guo Y, Johnson E. Reliability and sensitivity of the TonoLab rebound tonometer in awake Brown Norway rats. Invest Ophthalmol Vis Sci 2009;50:2802-2808. 210. Kurata K, Nishida E, Tsukuda R, Suzuki T, Sato S. Evaluation of Perkin's applanation tonometer and the normal range of intraocular pressure in anesthetized rats. The Journal of toxicological sciences 1996;21:249-252. 211. Shah GB, Sharma S, Mehta AA, Goyal RK. Oculohypotensive effect of angiotensin-converting enzyme inhibitors in acute and chronic models of glaucoma. J Cardiovasc Pharmacol 2000;36:169-175. 212. Bonomi L, Tomazzoli L, Jaria D. An improved model of experimentally induced ocular hypertension in the rabbit. Investigative ophthalmology 1976;15:781-784. 213. Torngren L, Lundgren B, Madsen K. Intraocular pressure development in the rabbit eye after aqueous exchange with ophthalmic viscosurgical devices. J Cataract Refract Surg 2000;26:1247-1252. 214. Krauss AH, Impagnatiello F, Toris CB, et al. Ocular hypotensive activity of BOL-303259-X, a nitric oxide donating prostaglandin F2alpha agonist, in preclinical models. Exp Eye Res 2011;93:250-255. 215. Impagnatiello F, Borghi V, Gale DC, et al. A dual acting compound with latanoprost amide and nitric oxide releasing properties, shows ocular hypotensive effects in rabbits and dogs. Exp Eye Res 2011;93:243-249. 216. Borghi V, Bastia E, Guzzetta M, et al. A novel nitric oxide releasing prostaglandin analog, NCX 125, reduces intraocular


pressure in rabbit, dog, and primate models of glaucoma. J Ocul Pharmacol Ther 2010;26:125-132. 217. Ruben S, Tsai J, Hitchings RA. Malignant glaucoma and its management. Br J Ophthalmol 1997;81:163-167. 218. Moazed B, Quest D, Gopalakrishnan V. Des-acyl ghrelin fragments evoke endothelium-dependent vasodilatation of rat mesenteric vascular bed via activation of potassium channels. Eur J Pharmacol 2009;604:79-86. 219. Webers CA, Beckers HJ, Nuijts RM, Schouten JS. Pharmacological management of primary open-angle glaucoma: second-line options and beyond. Drugs & aging 2008;25:729-759. 220. De Vriese C, Gregoire F, De Neef P, Robberecht P, Delporte C. Ghrelin is produced by the human erythroleukemic HEL cell line and involved in an autocrine pathway leading to cell proliferation. Endocrinology 2005;146:1514-1522. 221. Caminos JE, Gualillo O, Lago F, et al. The endogenous growth hormone secretagogue (ghrelin) is synthesized and secreted by chondrocytes. Endocrinology 2005;146:1285-1292. 222. Bodart V, Febbraio M, Demers A, et al. CD36 mediates the cardiovascular action of growth hormone-releasing peptides in the heart. Circ Res 2002;90:844-849. 223. Avallone R, Demers A, Rodrigue-Way A, et al. A growth hormone-releasing peptide that binds scavenger receptor CD36 and ghrelin receptor up-regulates sterol transporters and cholesterol efflux in macrophages through a peroxisome proliferator-activated receptor gamma-dependent pathway. Mol Endocrinol 2006;20:3165-3178. 224. Sibilia V, Pagani F, Rindi G, et al. Central ghrelin gastroprotection involves nitric oxide/prostaglandin cross-talk. Br J Pharmacol 2008;154:688-697. 225. Nathanson JA, McKee M. Identification of an extensive system of nitric oxide-producing cells in the ciliary muscle and outflow pathway of the human eye. Invest Ophthalmol Vis Sci 1995;36:1765-1773.


Bibliography

113

226. Kotikoski H, Alajuuma P, Moilanen E, et al. Comparison of nitric oxide donors in lowering intraocular pressure in rabbits: role of cyclic GMP. J Ocul Pharmacol Ther 2002;18:11-23. 227. Carreiro S, Anderson S, Gukasyan HJ, Krauss A, Prasanna G. Correlation of in vitro and in vivo kinetics of nitric oxide donors in ocular tissues. J Ocul Pharmacol Ther 2009;25:105-112. 228. Weinreb RN, Mitchell MD, Polansky JR. Prostaglandin production by human trabecular cells: in vitro inhibition by dexamethasone. Invest Ophthalmol Vis Sci 1983;24:1541-1545. 229. Weinreb RN, Mitchell MD. Prostaglandin production by cultured cynomolgus monkey trabecular meshwork cells. Prostaglandins, leukotrienes, and essential fatty acids 1989;36:97-100. 230. Schlotzer-Schrehardt U, Zenkel M, Nusing RM. Expression and localization of FP and EP prostanoid receptor subtypes in human ocular tissues. Invest Ophthalmol Vis Sci 2002;43:1475-1487. 231. Mukhopadhyay P, Geoghegan TE, Patil RV, Bhattacherjee P, Paterson CA. Detection of EP2, EP4, and FP receptors in human ciliary epithelial and ciliary muscle cells. Biochem Pharmacol 1997;53:1249-1255. 232. Schachtschabel U, Lindsey JD, Weinreb RN. The mechanism of action of prostaglandins on uveoscleral outflow. Current opinion in ophthalmology 2000;11:112-115. 233. Lindsey JD, Kashiwagi K, Kashiwagi F, Weinreb RN. Prostaglandins alter extracellular matrix adjacent to human ciliary muscle cells in vitro. Invest Ophthalmol Vis Sci 1997;38:2214-2223. 234. Weinreb RN, Kashiwagi K, Kashiwagi F, Tsukahara S, Lindsey JD. Prostaglandins increase matrix metalloproteinase release from human ciliary smooth muscle cells. Invest Ophthalmol Vis Sci 1997;38:2772-2780. 235. Yousufzai SY, Ye Z, Abdel-Latif AA. Prostaglandin F2 alpha and its analogs induce release of endogenous prostaglandins in iris and ciliary muscles isolated from cat and other mammalian species. Exp Eye Res 1996;63:305-310.


236. Wan Z, Woodward DF, Cornell CL, et al. Bimatoprost, prostamide activity, and conventional drainage. Invest Ophthalmol Vis Sci 2007;48:4107-4115. 237. Stamer WD, Piwnica D, Jolas T, et al. Cellular basis for bimatoprost effects on human conventional outflow. Invest Ophthalmol Vis Sci 2010;51:5176-5181. 238. Alexander JP, Samples JR, Van Buskirk EM, Acott TS. Expression of matrix metalloproteinases and inhibitor by human trabecular meshwork. Invest Ophthalmol Vis Sci 1991;32:172-180. 239. Bradley JM, Vranka J, Colvis CM, et al. Effect of matrix metalloproteinases activity on outflow in perfused human organ culture. Invest Ophthalmol Vis Sci 1998;39:2649-2658. 240. Damm J, Rau T, Maihofner C, Pahl A, Brune K. Constitutive expression and localization of COX-1 and COX-2 in rabbit iris and ciliary body. Exp Eye Res 2001;72:611-621. 241. Maihofner C, Schlotzer-Schrehardt U, Guhring H, et al. Expression of cyclooxygenase-1 and -2 in normal and glaucomatous human eyes. Invest Ophthalmol Vis Sci 2001;42:2616-2624. 242. Bos CL, Richel DJ, Ritsema T, Peppelenbosch MP, Versteeg HH. Prostanoids and prostanoid receptors in signal transduction. Int J Biochem Cell Biol 2004;36:1187-1205. 243. Nilsson SF, Drecoll E, Lutjen-Drecoll E, et al. The prostanoid EP2 receptor agonist butaprost increases uveoscleral outflow in the cynomolgus monkey. Invest Ophthalmol Vis Sci 2006;47:4042-4049. 244. Millard LH, Woodward DF, Stamer WD. The role of the prostaglandin EP4 receptor in the regulation of human outflow facility. Invest Ophthalmol Vis Sci 2011;52:3506-3513. 245. Sharif NA, Kelly CR, Crider JY, Williams GW, Xu SX. Ocular hypotensive FP prostaglandin (PG) analogs: PG receptor subtype binding affinities and selectivities, and agonist potencies at FP and other PG receptors in cultured cells. J Ocul Pharmacol Ther 2003;19:501-515.

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