I
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
III
Paulo Jorge Pereira da Silva
Master Degree Course in Cardiovascular Pathophysiology Orientador: Prof. Doutor Amândio António Rocha Dias de Sousa
V
To my parents,
VII
To Sara,
IX
and in memory of my uncle…
XI
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,
XII
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.
XIII
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
XVII
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,
XIX
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.
XXI
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.
XXIII
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
XXIV
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
XXV
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
XXIX
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
XXX
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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
6 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
(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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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
8 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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).
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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
.
10 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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
12 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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).
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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
14 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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.
16 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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,
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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).
18 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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
.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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
20 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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
22 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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.
24 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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.
26 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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
28 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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.
30 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter I - Introduction
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.
32 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter I - Introduction
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 - - -
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter II – Materials and methods
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
40 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter II - Materials and methods
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.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter II – Materials and methods
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.
42 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter II - Materials and methods
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.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter II – Materials and methods
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
44 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter II - Materials and methods
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter II – Materials and methods
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
46 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter II - Materials and methods
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.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter II – Materials and methods
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
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).
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter III - Results
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
54 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter III - Results
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter III - Results
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
56 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter III - Results
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter III - Results
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
58 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter III - Results
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:
( )
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter III - Results
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
60 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter III - Results
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.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter III - Results
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 - - -
62 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter III - Results
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
* * * * *
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter III - Results
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
64 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter III - Results
(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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter III - Results
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
* *
*
*
66 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter III - Results
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
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.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter IV - Discussion
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
72 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter IV - Discussion
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter IV - Discussion
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
74 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter IV - Discussion
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter IV - Discussion
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.
76 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter IV - Discussion
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter IV - Discussion
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.
78 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter IV - Discussion
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter IV - Discussion
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)
80 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter IV - Discussion
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
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter IV - Discussion
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
82 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter IV - Discussion
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
.
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
Chapter IV - Discussion
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
84 Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications Chapter IV - Discussion
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|
Ghrelin-GHSR system in the eye: local regulation and pathophysiological implications
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|
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