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Opioid-induced ocular hypotension: actions at pre-and postjunctional sitesDuanran WangClark Atlanta University
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Recommended CitationWang, Duanran, "Opioid-induced ocular hypotension: actions at pre- and postjunctional sites" (1994). ETD Collection for AUC RobertW. Woodruff Library. Paper 1002.
OPIOID-INDUCED OCULAR HYPOTENSION: ACTIONS AT
PRE- AND POSTJUNCTIONAL SITES
A THESIS
SUBMITTED TO THE FACULTY OF CLARK ATLANTA UNIVERSITY
IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR
THE DEGREE OF MASTER OF SCIENCE
BY
DUANRAN WANG
DEPARTMENT OF BIOLOGICAL SCIENCES
ATLANTA, GEORGIA
DECEMBER 1994
i
%
© 1994
DUANRANWANG
All Rights Reserved
ABSTRACT
BIOLOGICAL SCIENCES
WANG, DUANRAN M.D., QINGDAO SCHOOL OF MEDICINE
P.R. CHINA, 1984
OPIOID-INDUCED OCULAR HYPOTENSION; ACTIONS AT
PRE- AND POSTJUNCTIONAL SITES
Advisor: Dr. David E. Potter
Thesis dated December 1994
This study examined the ocular actions of an opioid
agonist. Experiments were performed to evaluate the effects
of DPDPE ([D-pen2, D-pen5] enkephalin), a delta opioid
agonist on: 1) intraocular pressure (IOP) in rabbits; 2)
cAMP accumulation in rabbit iris ciliary bodies (ICBs); 3)
3H-norepinephrine (NE) overflow from electrically stimulated
sympathetic nerves in ICBs. DPDPE Lowerd IOP in normal
rabbits but not in sympathectomized (SX) eyes. Naloxone did
not inhibit the effect of DPDPE on IOP in normal rabbits.
DPDPE inhibited 3H-NE overflow and suppressed cAMP
accumulation in ICBs. The presence of naltrindole, a delta
receptor antagonist, did not prevent the suppression of cAMP
levels by DPDPE. Pertussis toxin (PTX) did not prevent the
inhibition of cAMP levels by DPDPE. The data suggest that
the lowering of IOP by DPDPE is mediated at both pre-
(neuronal) and postjunctional (ciliary body) sites and may
involve an atypical opioid receptor. In addition, the
actions of DPDPE in the anterior segment may involve a PTX-
insensitive G protein.
ACKNOWLEDGEMENTS
Firstly, I must sincerely express my gratitude to my
major professor, Dr. David E. Potter and my other committee
members, Drs. Isabella Finkelstein and Juarine Stewart for
their advice and encouragement during my work on this
project, matriculation at Clark Atlanta University and the
preparation of this thesis. Special and deepest appreciation
go to Dr. David E. Potter for giving me a chance to enter
this project, for supplying research assistantship to me and
for his guidance and patience.
Secondly, I owe thanks to Dr. Teh-Ching Chu and Miller
J. Ogidigben for teaching me all the methods used in this
research. I also thank other members in the Department of
Pharmacology and Toxicology at Morehouse School of Medicine,
including Dr. Robin R. Socci and Zenovia Pearson. I am
grateful to all my teachers at Clark Atlanta University for
their competent teaching and my graduate classmates and
friends for help, friendship, and wonderful times.
Finally, I thank my husband for giving me the strength
and courage to perform this work; I thank my son for his
understanding during the entire work.
This work was supported, in part, by MBRS grant
GM08248.
ii
TABLE OF CONTENTS
PAGE
ACKNOWLEDGEMENTS ii
LIST OF FIGURES V
LIST OF ABBREVIATIONS vi
CHAPTER
I. INTRODUCTION 1
II. REVIEW OF LITERATURE 4
Glaucoma and Aqueous Humor Dynamics 4
Opioids and Their Receptors 6
Peripheral Actions of Opioids 7
Ocular Actions of Opioids 9
Prejunctional Actions of Opioids 10
Postjunctional Actions of Opioids 11
III. EXPERIMENTAL DESIGN AND METHODS 13
Experimental Plan 13
Methods 15
Animals 15
Drug Preparation 16
Drug Administration 16
IOP Measurement 17
3H-NE Overflow in Rabbit ICBs 18
cAMP Assay in Isolated Rabbit ICBs 20
Statistical Analysis 21
IV. RESULTS 22
IOP Experiments 22
iii
PAGE
The Prejunctional Action of DPDPE:
3H-NE Overflow Measurement in ICBs 28
The Postjunctional Action of DPDPE:
Measurement of cAMP Accumulation in ICBs . . 31
V. DISCUSSION 36
VI. SUMMARY AND CONCLUSIONS 47
CITATION OF REFERENCES 48
iv
LIST OF FIGURES
FIGURE PAGE
1. Aqueous Humor Dynamics 5
2. IOP Response to Topical Administration of
DPDPE (160ng) in Normal Rabbit 23
3. IOP Response to Intravitreal Injection of
DPDPE (10|xg) in Normal Rabbit 24
4. IOP Response to Topical Naloxone Plus DPDPE ... 26
5. IOP Response to Topical Administration of
DPDPE (16Ojig) in SX Rabbit 27
6. A Typical Experiment of DPDPE Inhibition of
3H-NE Overflow From Electrically Stimulated
Rabbit ICBs 29
7. A Summary Result of Four Experiments of
DPDPE Inhibition of 3H-NE Overflow FromElectrically Stimulated Rabbit ICBs 30
8. Effect of DPDPE on Basal cAMP Accumulation
in Rabbit ICBs 32
9. Dose-dependent Effects of DPDPE on
ISO-stimulated cAMP Accumulation
in Rabbit ICBs 33
10. Effect of Antagonist on the Inhibition
of cAMP Level by DPDPE 34
11. Effect of PTX on the Inhibition of cAMP
Level by DPDPE 35
LIST OF ABBREVIATIONS
AVP Arginine vasopressin
°C Degree Celsius
cAMP Cyclic adenosine monophosphate
CNS Central nervous system
DPDPE [D-pen2, D-pen5] enkephalin
g Gram(s)
Gi Guanine nucleotide regulatory protein
Gs Guanine nucleotide regulatory protein
h Hour(s)
Hz Hertz
125I Radioactive isotope of iodine
ICB Iris-ciliary body
IBMX 3-isobutyl-l-methylxanthine
i.m. Intramuscular injection
IOP Intraocular pressure
ISO Isoproterenol
kg Kilogram(s)
M Molar
mg Microgram(s)
min Minute(s)
ml Milliliter(s)
vi
mm Millimeters
mM Millimolar
mmHg Millimeters of mercury
NE Norepinephrine
ng Nanogram(s)
NG108-15 Mouse neuroblastoma x rat glioma hybrid cell
nM Nanomolar
NTI Naltrindole
NZW New Zealand White
pmol Picomolar
PGE Prostaglandin E
PTX Pertussis toxin
SX Sympathectomized
|xg Microgram(s)
Hi Microliter(s)
|xM Micromolar
VII
CHAPTER I
INTRODUCTION
Glaucoma is one of the common causes for blindness,
particularly among African-Americans (Quigley, 1993).
Increased intraocular pressure (IOP), optic nerve head
damage and visual field loss are considered hallmarks of
this disease. Current therapeutic measures attempt to lower
IOP in an effort to protect against optic nerve head damage.
Although opioids, such as morphine, have been reported
to have significant effects on iris function (Murray et al.,
1983) and IOP in man (Drago et al., 1985), much less is
known about their effects on other structures in the
anterior segment of the eye. One of the prominent ocular
effects of morphine-like drugs is pupillary constriction
(miosis), but this is attributed to an action in the brain
(nucleus of the oculomotor nerve). Experimental evidence
from rabbits has suggested the presence of opioid receptors
in the iris (Drago et al., 1980), but there is limited
information on the effect of opioids on aqueous humor
dynamics. Opioids have been demonstrated to lower IOP in
humans and conjunctival instillation of naloxone, a
nonselective opioid receptor antagonist, reversed the ocular
hypotension produced by morphine (Drago, 1985). Clearly, the
1
2
ocular hypotensive action of selective opioid agonists
requires further investigation. Moreover, it is possible
that the endogenous peptides, such as enkephalins, may play
a role in regulating aqueous humor dynamics.
Opioid peptides are endogenous or synthetic compounds
that have a spectrum of pharmacological activity similar to
that of morphine. Their predominant use in the clinic is for
analgesia. Currently, it is known that synthetic and
endogenous opioids interact with at least three types of
receptors: mu, delta and kappa. Opioid peptides and their
receptors function not only in nerves within the brain but
also in peripheral parasympathetic and sympathetic nerve
terminals. Opioid receptors are present at terminals of
prejunctional sympathetic neurons in a number of smooth
organs (Starke, 1977). Illes and Bettermann (1986) suggested
that the rabbit ear artery contains both prejunctional
delta- and kappa-receptors. Opioid peptides decrease
noradrenaline release and blood pressure in the rabbit at
peripheral receptors as has been demonstrated by Szabo et
al. (1986). Opioid receptors have been suggested to be
negatively coupled to adenylyl cyclase through a pertussis
toxin (PTX)-sensitive G protein in a variety of tissues
(Childers, 1991).
Because opioids have been demonstrated to lower IOP in
clinical situations by undefined mechanisms, the primary
goal of this research was to investigate the ocular actions
3
of opioids which may have a role in the modulation of
aqueous humor dynamics. Moreover, opioids, which are
relatively selective for a particular receptor subtype, may
have potential as therapeutic agents for glaucoma.
The hypotheses to be tested by this research were: 1)
opioid receptors play a role in modulation of aqueous humor
dynamics; 2) opioid peptides, by interacting with their
receptors, lower IOP by actions mediated at prejunctional
(neuronal) and/or postjunctional sites in the iris ciliary
bodies (ICBs). To test these hypotheses, specific opioid
agonist and antagonists were examined alone and in
combination for effects on IOP, sympathetic neuronal
function and ICB function. The following specific aims were
proposed for this study:
1. evaluation of a relatively selective opioid receptor
agonist and non-selective and relatively selective
antagonists on IOP;
2. assessment of opioid receptor agonist and
antagonist action at prejunctional (neuronal) sites
in the isolated ICBs;
3. elucidation of effect on a specific signal
transduction pathway (cAMP) involved in the
postjunctional action of opioid peptides in the
ICBs.
CHAPTER II
REVIEW OF LITERATURE
Glaucoma and Aqueous Humor Dynamics
Glaucoma is a group of diseases which is characterized
by progressive optic nerve damage, usually associated with
increased IOP and loss of visual field(s). In normal
subjects, aqueous humor is formed in the posterior chamber
by the bi-layered epithelial layer of the ciliary processes.
The bulk of formation of aqueous humor is a product of
active cellular secretion by the outer, nonpigmented ciliary
epithelium. Once formed, the aqueous humor flows from the
posterior chamber through the pupil into the anterior
chamber and exits at the anterior chamber angle of the
cornea and iris via the trabecular (conventional) and
uveoscleral (unconventional) routes (see Figure 1). In
primary open angle glaucoma, elevated pressure usually
results from increased resistance to outflow through the
trabecular meshwork and canal of Schlemm. IOP is the only
measurable risk factor that is easily identified, quantified
and treated. However, the modulation of the ocular humor
dynamics by neuroendocrine mechanisms requires further
investigation so that more effective therapeutic agents for
glaucoma can be discovered.
4
AQUEOUS HUMOR DYNAMICS
5stf^~^"'* Cornea
Anterior
chamber
Ciliary
process
U)
6
Opioids and Their Receptors
Morphine was the first natural opioid to be identified
and characterized. This alkaloid was isolated as one of the
analgesic components of opium; it can bind
stereospecifically to receptors in the central and
peripheral nervous systems where it produces dramatic
effects.
From clinical observations, it was shown that patients
addicted to morphine had lower IOP than nonaddicted subjects
(Drago, 1985). Morphine has been known for sometime to
produce ocular effects such as miosis (Green, 1975). These
events led to investigate other ocular actions of opioids.
Within the past decade, investigation of mechanisms of
narcotic and analgesic action has revealed specific opiate
receptors. Subsequent neurophysiologic studies and other
receptor binding studies suggested the existence of
endogenous substances that could bind to such receptors.
Typical substances first described in the central nervous
system (CNS) were met-enkephalin and leu-enkephalin (Hughes,
1975b). Following the discovery of the enkephalins, multiple
opioid receptors, originally demonstrated by Martin et al.
(1976), using the alkaloid opiates, were established as a
focus in opioid biochemistry. Recently, there has been
considerable progress in providing a firm basis for the
characterization of the various opioids and their possible
physiologic and pathophysiologic roles.
7
There appear to be at least three different types of
opioid receptors upon which morphine and opioid peptides act
(Lord et al., 1977; Martin, 1983). The mu receptor has a
greater affinity for morphine and is exemplified by effects
of morphine in the guinea pig ileum. The mu receptor is
believed to mediate the analgesic effects of opioid
agonists. The delta receptor has a greater affinity for
enkephalin and is the predominant type of receptor
characterized in the mouse vas deferens assay; the delta
receptor also mediates other behavioral effects of opioids.
Another receptor, termed the kappa receptor, has been
demonstrated by preferential binding to ketocyclazocine and
related benzomorphans; such receptors also appear to be
present in guinea pig ileum. Kappa receptors in the CNS are
involved in mediating hallucinatory and sedative states of
opioids. Therefore, opioid receptors function both in the
central nervous system and in peripheral systems.
Peripheral Actions of Opioids
There are many peripheral structures containing opioid
receptors, including the heart and blood vessels, the
gastrointestinal tract, the reproductive tract, and the
immune system. Opioids can reduce gastrointestinal
secretions and/or increase non-propulsive contractions,
which contribute to the antidiarrheal actions of morphine-
like drugs. These actions have generally been assumed to be
8
exerted, in part, locally in the gastrointestinal tract on
both nerves and smooth muscle. Peripheral actions of opioids
on the function of the sympathetic nervous system within
various tissues have also been studied. For example,
morphine can inhibit contractions of the cat nictitating
membrane elicited by stimulation of the cervical sympathetic
nerves (Trendelenburg, 1957). Because sympathetic nerves
from the superior cervical ganglion innervate both
extraocular and intraocular structures, one could
hypothesize opioid receptors on nerve ending at multiple
sites. Some of the peripheral effects of opioids appear to
be related, in part, to an action on the postganglionic
sympathetic nerve terminals. Moreover, the presence of
enkephalin-like material in sympathetic ganglia has been
demonstrated (Schultzberg et al., 1979). Enkephalins depress
vasoconstrictor responses to sympathetic nerve stimulation
in the rabbit isolated ear artery as was first noted by
Knoll (1976). Reflex regulation of heart rate by opioids has
also been observed. Moreover, it has been suggested, that
ethylketocyclazocine decreases blood pressure in the rabbit
through an inhibition of norepinephrine (NE) release from
peripheral sympathetic nerves (Ensinger et al., 1984). Thus,
a potential role for opioids, physiologically released from
sympathetic ganglia, appears probable.
9
Ocular Actions of Qpioids
Opioids have significant effects on iris function
(Murray et al.f 1983) and IOP in man (Drago et al., 1985).
With regard to iris function, there are species specific and
characteristic effects on pupillary size. In humans, rabbits
and dogs, opioid agonists cause miosis, whereas mydriasis
follows their administration to rodents, monkeys and cats
(Martin, 1983). Some investigators have attributed the iris
response to morphine to a central action on the oculomotor
nuclear complex. However, evidence from rabbits has
suggested the presence of opioid receptors in the iris
(Drago et al., 1980). Thus, pupillary effects of
systemically administered opioids are species-dependent and
primarily mediated by centrally evoked effects although a
peripheral component may also contribute.
Intravenous diacetyl morphine (heroin) produced a
decrease on IOP in rabbits, and this effect was accompanied
by a 50% increase in aqueous outflow facility; a dose-
related miosis was also observed (Green, 1975). Intravenous
morphine hydrobromide produced similar results (Uusitalo,
1972). Opioids can also lower IOP in humans. For example,
intramuscularly administered morphine sulfate, decreased IOP
both in normal patients and in glaucomatous eyes of various
etiologies (Leopold, 1948). In another study, it was
observed that: 1) the longer the duration of opium
addiction, the lower the mean IOP (Aminlari and Hashem,
10
1985), and 2) conjunctival instillation of naloxone, a
nonselective opioid receptor antagonist, would reverse the
ocular hypotension (Drago, 1985). It was also noted that
topical naloxone decreased in aqueous outflow facility and
induced mydriasis (Fanciullacci et al., 1980), suggesting
the existence of an opiate receptor mechanism in the human
eye. Clearly, the mechanism for the ocular hypotensive
action of receptor-selective opioid agonists requires
further investigation. Because endogenous opioid peptides
are believed to serve as neuromodulators in the peripheral
and CNS, it is possible that these endogenous peptides may
play a role in regulating aqueous humor dynamics.
Preiunctional Actions of Ooioids
Previous studies have demonstrated the sympathetic
terminals in the rabbit ICB contain regulatory prejunctional
receptors of various type. These receptors have been
postulated to mediate the lOP-lowering effects of several
drugs, such as alpha-2 adrenergic and dopamine (DA2)
receptor agonists (Potter and Burke, 1984; Jumblatt et al.,
1993). Earlier studies had noted that electrically evoked NE
release was depressed by opioid receptor agonists in vas
deferens of mouse, rat, rabbit, and hamster (Henderson et
al., 1972; Hughes et al., 1975a; Henderson and North, 1976),
and cat nictating membrane (Dubocovich and Langer, 1980).
However, demonstration of this effect is often dependent on
11
the antagonism of alpha-2 prejunctional receptors. It has
been shown that naloxone can increase, dose-dependently,
electrically-evoked NE release in guinea-pig isolated distal
colon (Marino et al., 1993). However, there is, at present,
no evidence whether this effect occurs in the field-
stimulated ICB of rabbits.
Post1unctional Actions of Qpioids
Opioid receptors have been demonstrated to be
negatively coupled to adenylyl cyclase in a variety of
tissues. Acutely, opiate agonists inhibit the activity of
adenylyl cyclase in plasma membranes from brain tissue
(Hamprecht, 1978; Childers, 1991). Moreover, there are
alterations in cAMP metabolism in brain tissues from animals
treated with opioid drugs chronically. The acute and chronic
actions of opiate drugs in NG108-15 cells are mediated by
the delta class of receptors (Law et al., 1982, 1983). There
is also some evidence from biochemical, pharmacological and
neurophysiological studies that some of the biological
responses to opioid receptor activation are dependent on a
PTX-sensitive G protein (Kurose et al., 1982). However, no
such experiments have been done on ocular tissues, such as
the rabbit ICB.
Delta opioid receptors are one of the major opioid
receptor subtypes (mu, delta, kappa). Agonists, such as
DPDPE, have relatively high affinity for and efficacy on
12
delta receptor. Moreover, delta opioid agonists are more
receptor-selective drugs than morphine, and this specificity
of action promoted examination of their effect on ICB
function and IOP lowering activity in rabbits. Therefore,
experiments were performed to determine: 1) if DPDPE can
induce ocular hypotension and 2) what mechanisms might be
involved at pre- and postjunctional sites in the ciliary
body. One potential application of this knowledge could be
that opioid analogs may eventually be developed as drugs for
treating one of the most common cause of blindness:
glaucoma. Moreover, endogenous opioids may play a role in
modulation aqueous flow. In glaucoma, there may be
dysfunction of the opioidergic system which contributes to
abnormalities in aqueous humor dynamics.
CHAPTER III
EXPERIMENTAL DESIGN AND METHODS
Experimental Plan
AIM 1. Evaluation of Relatively Selective Qpioid Receptor
Agonist and Antagonist on IOP
Delta opioid receptor agonist and antagonist, given
alone and in combination, were used in .in vivo experiments
testing effects on IOP in normal and sympathectomized (SX)
rabbit eyes. Drugs were given by topical administration or
intravitreal injection. This design sought to characterize
the actions of a relatively selective opioid receptor
agonist DPDPE in lowering IOP.
AIM 2. Assessment of Opioid Receptor Agonist and Antagonist
Action at Preiunctional fNeuronal) Site in the Anterior
Segment of the Eve
After confirming the ocular hypotensive activity of a
delta receptor agonist, subsequent experiments assessed the
effects of the drugs on ciliary body function including NE
release from sympathetic nerves and signal transduction in
rabbit ICBs. Isolated ICBs from rabbits were used for in
vitro study of prejunctional (neuronal) activities by
electrical field stimulation as determined by NE overflow.
13
14
This technique has been utilized to study a variety of
receptors, whose ocular actions are mediated, in part, by
effects at prejunctional (neuronal) sites. Because
peripheral functions of opioids are believed to be modulated
also by actions at postganglionic sympathetic neuron
terminals in other organs, it was considered that the ocular
actions of an opioid receptor agonist might be mediated by a
similar mechanism.
AIM 3. Elucidation of Signal Transduction Pathways Involved
in the Action of Qpioid Peptides at Postiunctional Sites
riCBs)
Because effects of opioids on prejunctional neurons in
the ICBs may be only partially responsible for the ocular
hypotension induced by opioid peptides, it seemed possible
that postjunctional (ciliary epithelial) effects could also
be involved. Therefore, the signal transduction pathways at
postjunctional sites (ICBs) were elucidated by evaluating
cAMP accumulation under basal and isoproterenol (ISO)-
stimulated conditions. To test delta opioid receptor
coupling to adenylate cyclase and G protein system, ICBs
were pretreated with PTX, a Gi protein inhibitor, in in
vitro studies.
15
Methods
Animals
New Zealand White (NZW) rabbits (2-4kg) of either sex
were maintained on a schedule in which rabbits were on a 12
h dark/12 h light cycle at a constant room temperature. One
group of rabbits underwent unilateral, surgical
sympathectomy (SX). Rabbits were anesthetized with a
combined dose of 44mg/kg ketamine and 8mg/kg xylazine (i.m.)
and the preganglionic sympathetic nerve and superior
cervical ganglion on the right side were exposed and
sectioned. To be certain that the separated nerve trunk
contained the sympathetic nerves, a stimulating electrode
was placed on the nerve bundle for electric stimulation with
a square-wave pulse of 4 Hz (0.7 ms duration, 5 s train) at
4 volts using a bipolar electrode connected to a Grass SD-9
stimulator; a widening of the rabbit's pupil confirmed
successful nerve isolation. After surgical sympathectomy,
the rabbits were allowed to recover for 10 days. SX eyes
were examined for unequivocal miosis and ptosis before
rabbits were used in experiments. This SX model served as a
test for initial evidence for involvement of sympathetic
nerves in the action of the delta opioid receptor agonist,
DPDPE ([D-pen2, D-pen5] enkaphelin). Animal care and
treatment were conducted according to the NIH and ARVO
resolutions on the care and use of animals in research, and
protocols were approved by the Institutional Animal Care and
16
Use Committee of Morehouse School of Medicine.
Drug Preparation
DPDPE (Peninsula Laboratories, INC.)/ a relatively
selective opioid delta receptor agonist, was dissolved in
0.05M NaH2PO4. Naloxone, a nonselective opioid antagonist,
was dissolved in distilled water; and naltrindole (NTI,
Research Biochemicals Incorporated), a delta opioid
antagonist, was dissolved in 45% 2-hydroxypropyl-fi-
cyclodextrin. All agents were prepared in their respective
vehicles on the day of experiments.
(1) Topical administration:
In the IOP experiments, DPDPE (160^g) was applied
topically and unilaterally to the unanesthetized rabbits.
Naloxone was applied the same way 1/2 h before DPDPE
administration to determine if opioid receptors were
involved. Furthermore, DPDPE (160ng) was administered
topically to SX eyes of rabbits to determine whether the
lowering IOP response was dependent upon intact sympathetic
nerves. The contralateral eyes received equal volumes of
vehicle. The drug administration in the IOP experiments was
performed in a masked design, i.e., the person performing
the IOP measurements had no prior knowledge of the
solutions' contents.
17
(2) Intravitreal injection:
Rabbits were restrained in rabbit holders and
anesthetized with intramuscular injections of ketamine
(25mg/kg). After topical application of 0.05% tetracaine
hydrochloride, eyelids were refracted with a speculum. In a
masked fashion, lOug DPDPE in lOul of sterile NaH2PO4
(0.05M) was injected in one eye 3mm posterior to the limbus,
on the upper pole of the eye, and an equal volume of NaH2PO4
was injected in the other eye. The injection was directed
toward the center of the vitreous by means of a 30-gauge
needle on a 50-^1 syringe, with care being taken to avoid
hitting the lens (see Berthe et al., 1994; MacCumber et al.,
1991).
IOP Measurement
IOP (mm Hg) was measured in normal and unilaterally SX
rabbits using a manometrically calibrated BIO-RAD Digilab
pneumatonometer (Cambridge, MA). Tetracaine hydrochloride
(0.05%, Alcon, Fort Worth, TX) was applied to each cornea
before the IOP measurements to minimize discomfort. Baseline
(control) readings were taken before drug administration;
post drug IOP determinations were made at 1/2, 1, 2, 3, 4,
and 5 h after topical administration. In experiments
involving intravitreal injection, IOP was measured by a
pneumatonometer after topical tetracaine hydrochloride
(0.05%) anesthesia: 1 h before, immediately before and 10
18
min after the injection of drug or vehicle. Additional IOP
measurements were made following the injection at 1/2, 1, 2,
3, 4, 5 h and at 1, 2, 3, 4, 5, 8 days post drug.
In control experiments, both eyes of rabbits received
10nl NaH2PO4 by intravitreous injection. The time of IOP
measurement was similar to that of DPDPE-treated animals.
3H-NE Overflow in Rabbit ICBs
In these .in vitro experiments, rabbit ciliary body
(ICB) preparations were perfused with physiological salt
solution containing increasing concentration of DPDPE
(0.01(iM, 0.1|iM and l\iM) to investigate 3H-NE overflow in
response to electrical field stimulation.
Rabbits of either sex were sedated (ketaraine, lOmg/kg
i.m.) and then euthanized by an overdose of sodium
pentobarbital injected into the marginal vein of the ear.
The eyes were rapidly enucleated, dissected and placed in
Ringer's solution. During dissection, the eye was opened
with a pair of scissors and the anterior portion (cornea,
lens and ciliary body) removed from the rest of the globe.
The ICB was removed from the rest of the anterior segment
tissue using fine forceps and then cut into three to four
pieces of approximately equal size. Pieces of ICBs were
incubated for 1 h at 37°C in a oxygenated (95% 02: 5% C02
mixture) bicarbonate-rich, HEPES-buffered solution
(composition in mM: NaCl, 115; KC1, 5; CaCl2, 1.8; MgSO4,
19
0.8; NaH2PO4, 0.9; NaHCO3, 26; glucose, 10; HEPES, 10;
indomethacin 2.2 mg%, pH 7.4) containing 2.5 uCi/ml 3H-NE.
After 3H-NE loading, ICBs were subsequently rinsed three
times with non-radioactive, normal Ringer's solution to
remove excess 3H-NE. ICBs were transferred to temperature-
controlled (37°C) plexiglass superfusion chambers containing
built-in electrodes and superfused at a rate of 2 ml/min
with the normal Ringer's solution containing ljxM desipramine
to block re-uptake of NE. During superfusion, the tissues
were subjected to a series of electrical field stimulation
(12 V/cm, 5 Hz, 1 min). Fractions (2 ml) of the superfusate
were collected at 1 min intervals and analyzed for
radioactivity. Samples (0.5 ml) were counted on a Beckman LS
5801 liquid scintillation counter and results reported as
DPM per fraction.
Results have demonstrated, that under controlled
conditions, the 3H-NE release during S1 through S4 were of
similar magnitude. In an ICB preparation run in parallel,
doses of DPDPE were given in a cumulative manner in a series
of three doses (O.OlfxM, O.luM, lfxM). Prior to and subsequent
to each dose of DPDPE, tissues were stimulated electrically
to release 3H-NE from sympathetic nerve endings. After a
control stimulation (SJ, concentrations of DPDPE (O.OlfiM,
O.lfAM and l\M) were added sequentially to the superfusion
medium 10 min before other stimulations (S2, S3, S4). Ratios
of 3H-NE overflow were calculated as: S2/Si, S3/Slf S^S^
20
CAMP Assay in Isolated Rabbit ICBs
In addition to an action on prejunctional (neuronal)
sites, a possible postjunctional site of action by DPDPE was
evaluated. Fresh ICBs were dissected from rabbits as
previously described to determine the effects of DPDPE on
basal and ISO-induced cAMP accumulation. Pieces of ICB were
incubated with modified Earle's/Ringer solution (mM): NaCl,
115; KC1, 5; CaCl2, 1.8; MgSO4/ 0.8; NaH2PO4, 0.9; NaHCO3, 26;
glucose, 10; HEPES, 10; indomethacin 2.2 mg%. Incubation of
ICBs were carried out for 30 min in a humidified incubator
mixed with 5% CO2 and air. The tissues were treated with
IBMX (1 mM, a phosphodiesterase inhibitor) for additional 10
min under conditions stated above. DPDPE concentrations
(O.OljiM, O.luM and ljiM), with or without the beta agonist,
ISO (Sigma Co., St. Louis), were added to the tissues at 10
min intervals in the presence or absence of delta opioid
antagonist, naltrindole (NTI), which was used to confirm the
delta receptor selectivity of the agonist. To examine the
possibility of Gi protein involvement in ocular action of a
delta opioid receptor agonist, ICBs were pretreated with PTX
(150ng/ml) for 4 h. All experiments were conducted in
duplicate or triplicate.
After completion of the entire incubation series,
tissues were withdrawn quickly from the reaction vessel,
placed in vials and frozen by immersion in liquid N2.
Frozen samples were stored at -80°C and tissue extraction
21
for cAMP assay were performed within 2 weeks. Samples were
homogenized in 300 |xl of 10% TCA at 4°C. After
centrifugation of homogenates for 10 min, 175 |xl of
supernatant were removed for determination of cAMP levels by
an Amersham cAMP [125I]radio immunoassay system. NaOH (IN,
300 pi) was added to the remaining pellets for determination
of protein concentration by a Bio-Rad assay.
After separation of bound and unbound cAMP by double
antibody precipitation and counting of the respective
samples on a gamma counter, the amounts of cAMP accumulation
were calculated in terms of pmol/mg protein.
Statistical Analysis
The statistical analysis of experimental data used was
the Student's t-test for non-paired or paired data. Values
were reported as the mean ± SE. The level of significance
was chosen as P < 0.05.
CHAPTER IV
RESULTS
IOP Experiments
Figure 2 illustrates the IOP response to topical,
unilateral administration of DPDPE (160ng) in normal
rabbits. The control IOP was variable with time because
rabbit IOP has circadian rhythm. The ocular hypotensive
response in ipsilateral eyes to DPDPE (160fig) was
significant but of short duration. The maximum IOP decrease
was about 3.5 + 1 mmHg (compared to control) at 1 and 2 h,
then, the IOP began to recover. In contralateral eyes, the
onset of hypotensive response was observed a little later
and the only point significantly different from control was
at 2 h. The maximal decrease in IOP in the contralateral and
ipsilateral eyes was about the same.
Figure 3 illustrates the IOP response to intravitreal
injections of DPDPE (10\ig) in 10[xl of NaH2PO4 (0.05M).
Injection of DPDPE caused a greater IOP decrease than did
intravitreal injection of IOjaI NaH2PO4 (0.05M). The IOP
began to decrease 0.5 h after the injection of DPDPE as
compared to (vehicle-treated) control eyes, in which the IOP
increased immediately after the injection of NaH2PO4. The
maximum IOP decrease (4+1 mmHg) was at 1 to 3 days. The
reduction in IOP lasted approximately one week.
22
23
11
—o-- Control (n=8)
—£— DPDPE160ng(n=4)
.....Q-... Contralateral (n=4)
2 3
Time (h)
24
5-
EE,
o.
O
o—• NaH2PO4 (control) lOjai (n=4)
-9
2 3 4 5
Time (days)
8
25
Topical administration of a non-selective antagonist,
naloxone (910^g), did not block the effect of DPDPE on IOP
(Figure 4).
DPDPE (160fig), applied topically, had no hypotensive
effect on the SX eyes; instead, there was a slight increase
in IOP (Figure 5). In contrast, DPDPE (160|xg) decreased IOP
in the contralateral (normal, vehicle-treated) eyes as
observed previously (see Figure 2).
26
X
E
Q.
O
0
-1-
-2-
-3"
-4-
-5-
-6-
-7-
-8
-O-- Control (n=8)
DPDPE 160ng (n=4)
DPDPE 160^ig + naloxone 910}ig (n=4)
1 2 3
Time (h)
27
O)
X
E
a.
O
1-
-1-
-2-
■3-
-4-
-5"
-6-
-7-
\ ?%\ ^ '
•\ —I^i i
■
* «
■
r
f^7 ■' Contro.... dpdpe
DPDPE
I (n=8)
:i60^ig(SX)(n=4)
: 160{ig (n=4)
■ i
Time (h)
28
The Preiunctional Action of DPDPE; 3H-NE Overflow
Measurement in ICBs
The prejunctional (neuronal) action of DPDPE was
studied in ICB perfusion experiments. Electrical field-
stimulation of the isolated, superfused rabbit ICB,
preincubated with 3H-NE, increased the rate of tritium
overflow from sympathetic nerve endings into the superfusion
medium. In a typical experiment, by calculating fractional
tritium overflow, cumulative concentrations of DPDPE
(0.01|xM, O.luM, or ljiM) were shown to cause inhibition of
3H-NE overflow (18.2%, 28.3%, or 57.1%) in response to
electrical field stimulation (Figure 6). A summary of
results from four sets of experiments shows the percent
inhibition of 3H-NE release from ICBs. DPDPE in the lowest
concentration (O.OluM) had no significant inhibition
(17.4%), but in the intermediate (0.1(iM) and high
concentrations (ljxM), DPDPE inhibited the 3H-NE overflow
from ICBs significantly (38.1% and 52.8%, respectively)
(Figure 7). These results are consistent with the events
observed in SX rabbits in that a site of action, implicated
by these findings, is on sympathetic nerves.
29
(a)
2000n
1500
1000-
500-
CONTROL OF 3 H-NE RELEASE FROM ICB
S-1 S-2 S-3 S-4
10 20 30
Fraction Numbers
40 50
Q.Q
(b) DPDPE ON 3H-NE RELEASE FROM ICB
auu-
800-
700-
600-
500-
400-
300-
A
S-1
A hA/v
S-2
S-1
S-2
S-3
S-4
I h
S-3I i
= Control
= DPDPE
= DPDPE
= DPDPE
a hVS-4
l
0.01 |iM
0.1 (iM
1^M
VKM10 20 30
Fraction Numbers
40 50
30
DPDPE INHIBITED 3 H-NE RELEASE FROM ICB
31
The Post-functional Actions of DPDPE: Measurement of cAMP
Accumulation in ICBs
The postjunctional action of DPDPE was examined by
radio-immunoassay of cAMP accumulation in rabbit ICBs after
pretreatment with DPDPE alone and following stimulation of
cAMP formation by ISO. DPDPE (O.l^M) caused a 35.7%
inhibition of basal (without ISO stimulation) cAMP level
(Figure 8). ISO (l\M) stimulated cAMP accumulation by three
fold above basal levels. DPDPE (O.OluM, O.lfxM, or l^iM)
caused a dose-dependent inhibition (30%, 45%, or 55%) of
ISO-stimulated cAMP levels in ICBs (Figure 9). In addition,
the presence of naltrindole (NTI, lOfiM) did not block the
suppression of cAMP levels by DPDPE (O.ljiM) in ICBs (Figure
10). In subsequent experiments, pretreatment with PTX
(150ng/ml) for 4 h in ICBs did not prevent DPDPE inhibition
of ISO-stimulated cAMP accumulation (Figure 11).
pmol
cAMP/mg
protein
oI
o o 3
to
33
2001
o 150
Q.
E
<o
o
Ea
100-
50-
Control
DPDPE ^|
pmol
cAMP/mg
protein
S8
O1
O o
O o 3
35
0>
Control < iso 0.1
DPDPE 0.
PTX
CHAPTER V
DISCUSSION
It has been known for some time that opioid receptors
exist in various major organs of the body, including the
brain, where the receptors mediate morphine-induced
analgesia (mu receptor), behavioral changes (delta
receptor), hallucinatory and sedative states (kappa
receptor) (Pasternak, 1988a). Moreover, opioid receptors
also exist in many peripheral tissues where some of the
opioid receptors have been demonstrated to be negatively
coupled to adenylate cyclase as occurs in some regions of
the brain. In some peripheral organs, the effects of opioids
appear to be mediated, in part, by an action on the
postganglionic sympathetic nerve terminals (Trendelenburg,
1957), which usually results in inhibition of
neurotransmitter release. In this research, the approaches
were: 1) to evaluate the effect of the relatively selective
opioid receptor agonist (DPDPE) on IOP and 2) to test its
actions at both pre- and postjunctional sites in the ICBs.
There is limited information regarding the effects of
opiates on aqueous humor dynamics. Drago (1985) suggested
morphine- or heroin-addicted patients had increased aqueous
outflow facility and lowered intraocular pressure. A similar
36
37
unreferenced statement can be found in pharmacology
textbooks such as the eighth edition of the Pharmacological
Basis of Therapeutics (Jaffe and Martin, 1990a). After
conjunctival instillation of naloxone, the aqueous outflow
facility and IOP in the addicted patients returned to normal
levels. However, in the current study, naloxone applied
topically, did not antagonize the IOP lowering effect of
DPDPE in rabbits. Therefore, this result with an opioid
peptide in rabbits, did not agree with that of Drago's
(1985) using opioids of an alkaloid nature.
DPDPE, applied topically, to normal rabbits lowered IOP
both in treated and contralateral (vehicle-treated) eyes.
Because DPDPE is a peptide, which has a relatively high
molecular weight (645.57), one might question if ocular
absorption of this peptide can occur from the corneal
surface. Although absorption through the cornea would be
limited, it is probable that DPDPE is absorbed via blood
vessels in the conjunctiva and nasopharynx, thereby reaching
the systemic circulation. Thus, the drug could be
transferred by the blood to the contralateral eye or to
sites in the CNS, causing hypotension in both eyes.
Interestingly, this ocular (conjunctival) route has been
exploited as an alternative route for delivery and
absorption of a variety of peptides (Lee, 1985). Enkephalin
with molecular weight of 600 has been examined by Chiou et
al. (1988). The data showed that enkephalin can be
38
effectively absorbed into the systemic circulation following
topical application to eyes without using a surfactant as
absorption enhancer. Likewise, Ahmed and Patton (1985)
demonstrated that certain peptides, especially the high
molecular weight compounds like inulin (molecular weight:
5500), reached the ICB when applied to the
conjunctival/scleral surfaces. It is noteworthy that, the
conjunctival and the nasal mucosa have extensive vascular
networks which provide a good site for extensive absorption
of both lipophilic drugs and hydrophilic drugs.
Comparing the IOP lowering effect of DPDPE by different
routes of administration, the IOP decreased more following
intravitreal injection than following topical
administration. IOP in the contralateral (vehicle-treated)
eye changed minimally following intravitreal injection.
These results suggest that administration and absorption of
DPDPE by different routes alters ocular function
quantitatively more than qualitatively. DPDPE, injected into
the vitreous, produced an IOP lowering effect that was of
much longer duration than that of topical administration of
DPDPE. This result may be related, in part, to the gradual
diffusion of DPDPE from the gel-like vitreous body to the
ciliary body in the anterior segment of the eye.
In other experiments, an attenuated ocular hypotensive
response of DPDPE was noted in SX eyes, suggesting that
peripheral sympathetic nerves have to be intact for the
39
decrease of IOP in response to DPDPE. It is known that drugs
acting prejunctionally and/or centrally to suppress
sympathetic neuron function can lower IOP (Potter, 1981).
Therefore, to determine whether a prejunctional (neuronal)
site is involved in the IOP lowering effect of DPDPE,
electrically stimulated 3H-NE overflow from ICBs was
measured. Other investigators have demonstrated that opioid
receptors are present on the terminals of postganglionic
sympathetic neurons in a number of smooth muscle organs
(Starke, 1977) including the cat nictitating membrane
(Dubocovich and Langer, 1980). The activation of
prejunctional receptors by opioids leads to depression of
action potential-induced transmitter release from
sympathetic nerves. Illes et al. (1985) suggested that
rabbit ear artery contains only prejunctional delta and
kappa but not mu receptors. Ensinger et al. (1984) showed
ethylketocyclazocine could decrease noradrenaline release
and blood pressure in the rabbit by acting at a peripheral
opioid receptor. DPDPE, in the experiments described here,
inhibited 3H-NE release from ICBs. This effect is probably
caused by activating a prejunctional opioid receptor.
There is evidence of interaction between opioid and
other prejunctional receptors in other tissues. For example,
in pithed rats, naloxone-induced depression of neurogenic
tachycardia was completely blocked by phentolamine,
suggesting that the interaction of naloxone with the
40
sympathetic nervous system involves prejunctional inhibitory
alpha-adrenoceptors located on cardiac sympathetic nerves
(Slavik and Szilagyi, 1992). The cardiovascular hypotensive
effect of methionine-enkephalin was partially blocked by
phentolamine, a non-specific adrenoceptor antagonist (Eulie
and Rhee, 1984). These data suggest that met-enkephalin's
action could be mediated by opioid receptors but that alpha-
adrenoceptors may be involved in modulating met-enkephalin's
hypotensive action. Because alpha-2 adrenoceptors act as
autoreceptors on sympathetic nerves, their inhibition might
cause a functional antagonism of agonists (e.g., opioids)
that act on prejunctional (neuronal) heteroreceptors.
There is additional evidence suggesting an interaction
between alpha-2 adrenoceptors and opioid receptors. For
example, clonidine is used clinically to ameliorate symptoms
associated with opioid withdrawal (Jaffe and Martin, 1990b).
Clonidine, an alpha-2 adrenergic receptor agonist, produced
additive effects to the met-enkephalin-induced
cardiovascular actions: reduction in blood pressure, heart
rate and multi-unit renal nerve activity (Rhee and Tyler,
1986). Therefore, it is possible that DPDPE inhibits 3H-NE
release by either interacting indirectly with alpha-2
receptors and/or directly with opioid receptors. In some
cases, it may be necessary to unmask the activity of
prejunctional opioid receptors by antagonizing the
physiologically active (autoreceptors) alpha-2 adrenoceptors
41
on sympathetic neurons.
There are many extracellular messengers that rely on G
proteins to direct the signal from the membrane receptor to
the rest of the cell. G proteins have been isolated in about
20 distinct forms. In the resting state, the alpha, beta and
gamma subunits form a complex, and GDP is bound to the alpha
subunit. After hormones or other extracellular "first
messengers" bind to their receptors at the cell membrane,
activation or inhibition of the G protein complex (attached
to the inner surface of cell membrane) occurs. The alpha
subunit releases GDP, then GTP binds to the alpha subunit,
altering its shape and activating it. Once activated, the
GTP-bound alpha subunit dissociates from the beta and gamma
subunits and diffuses along the inner surface of the plasma
membrane until it links up with an effector, such as
adenylyl cyclase. Afterward, the alpha subunit hydrolyzes
GTP to form GDP, thereby shutting the process off.
Activation of the receptor and its associated G protein, in
turn, acts on membrane-bound intermediaries (effectors, such
as adenylyl cyclase). The second messenger generated by
activation of adenylyl cyclase triggers a cascade of
molecular reactions (e.g., protein kinase) leading to a
change in cellular function (Linder and Gilman, 1992).
To test the postjunctional action of DPDPE, cAMP
accumulation in ICBs was measured. With second messenger
systems, there is presumed to be no direct interaction
42
between the opioid peptide and the signal transducing
enzymes. In contrast, the membrane transduction mechanisms
(e.g., adenylyl cyclase, 6 protein) are linked to opioid
receptors. In general, neurotransmitter and hormone
receptors that are coupled to adenylyl cyclase can be
divided into two categories: 1) beta-adrenergic, glucagon,
adenosine (A2), and dopamine (Dl) agonists, which stimulate
the enzyme when the appropriate receptor is activated,
causing increased levels of intracellular cAMP; 2) alpha-2
adrenergic, adenosine (Al), dopamine (D2), and opioid
agonists which causes inhibition of adenylyl cyclase
activity. Guanine nucleotides (GTP) mediate receptor-
adenylyl cyclase coupling through specific GTP-binding
proteins, Gs-(stimulatory) and Gi-(inhibitory) proteins,
respectively. G proteins consist of three subunits: alpha,
beta and gamma. PTX can bind to alpha-i subunit, catalyzing
ADP-ribosylation of the proteins, thereby, inactivating the
inhibitory function of the subunit. In some systems, PTX can
cause stimulation of adenylyl cyclase by removing the
inhibitory component of the cycle (Katada and Ui, 1982).
In the eye, inhibition of cAMP formation by alpha-2
adrenoceptor agonists has been demonstrated to be a
contributing factor to the decrease in IOP (Potter, 1990).
Inhibition of cAMP formation in ICBs, in many cases,
involves the Gi alpha subunit. More effectors than adenylyl
cyclase have also been investigated and may be implicated in
43
the action of opioids. For example, the cellular effects of
opioids on neurons, except inhibition of cAMP formation
(Childers, 1991), may involve inhibition of calcium entry
through voltage-sensitive calcium channels (Porzig, 1990),
and stimulation of an outward potassium conductance (North
et al., 1987). Thus, the coordinated changes in
intracellular cAMP, calcium and hyperpolarization may
modulate neurotransmission at both central and peripheral
sites (Leslie, 1987). In the case of opioids, it is possible
that ocular actions of DPDPE may be regulated by a PTX-
insensitive G protein (Cripps and Bennett, 1991) or by the
actions on ion channels (Pasternak, 1988b).
In the experiments described herein, DPDPE inhibited
basal and ISO-stimulated cAMP level in ICBs. Pretreatment
with NTI did not inhibit the suppressive effect of DPDPE on
adenylyl cyclase. Moreover, PTX pretreatment did not alter
the DPDPE-induced inhibition of cAMP accumulation. It has
been shown that opioid inhibition of adenylyl cyclase in
NG108-15 cells occurs not only for PGE, adenosine, and
cholera toxin-stimulated activity, but also for basal
adenylyl cyclase activity (Propst and Hamprecht, 1981).
Fantozzi et al. (1981), using the irreversible antagonist
beta-chlornaltrexamine, blocked 95% of opiate receptor
binding sites but this maneuver did not alter the inhibition
of adenylate cyclase by opiate agonists, suggesting there
may be a large population of spare receptors in the NG108-15
44
cells. The same possibility might exist in ICBs, but there
are also other possibilities to be considered. One
possibility is that delta opioid receptors, as with other
opioid receptors, exist as subtypes; this possibility has
been demonstrated by Mattia (1991a) and Horan et al. (1993).
In vivo pharmacological characterization of interaction
between [D-Ala2] deltorphin II and DPDPE by using the
development of tolerance as the index of biological
activity, suggested that these compounds produced
antinociception via interaction with subtypes of delta
receptors because of the failure to develop cross-tolerance.
Mattia et al. (1991b) termed the receptor subtypes delta-1
(DPDPE- and DALCE-sensitive) and delta-2 ([D-Ala2, Glu4]
deltorphin- and 5'-NTII-sensitive). In an analogous
situation, it is possible, therefore, that DPDPE acts at a
receptor in the ICB which is NTI-insensitive because NTI did
not block the inhibitory effect of DPDPE on cAMP
accumulation.
Minneman and Iversen (1976) showed that enkephalin
increased intracellular cyclic GMP levels in slice
preparations of rat striatum. In NG 108-15 cells,
stimulation of cGMP levels appears to be mediated by a delta
receptor response (Gwynn and Costa, 1983). Thus, in ICBs,
occupation of delta or other possible receptor(s) may also
increase cGMP levels. Ross and Cardenas (1977) and End et
al. (1981) showed that opiate agonists reduce calcium
45
content in brain. Therefore, it is of interest to
investigate what other second messenger systems are also
coupled to opioid receptor(s) in ICBs.
Calcagnetti et al. (1990) suggested that DPDPE produced
analgesia at site other than delta receptors. Therefore, it
is possible that the ocular actions of DPDPE may not act on
delta receptors. There are other endogenous substances upon
which opioids may have effects: substance P and AVP (an
antidiuretic hormone). Bioassay and radio-immunoassay
studies have revealed substance P-like immunoreactivity in
mammalian retinas (Eskay et al., 1980). Stimulation of the
trigeminal ganglion in rabbits causes miosis and release of
a substance P-like material into the aqueous humor;
injection of synthetic substance P into the anterior chamber
of rabbits causes a marked increase in IOP (Stell et al.,
1980). Substance P-induced scratching of the hindlimbs in
the mouse is potently antagonized by delta agonist (Hylden
and Wilcox, 1983). One interesting question relates to
whether DPDPE lowered IOP by antagonizing the release of
substance P. Another substance, AVP, can also increase IOP
in rabbits when administered by intravenous infusion (Cole
and Nagashbramanian, 1973). Desmopressin, a synthetic analog
of vasopressin, was noted to increase IOP and aqueous humor
production (Wallace et al. 1988). Recently, it has been
demonstrated that endogenous opioid peptides exert a tonic
inhibitory control on AVP secretion in the rat.
46
Interestingly, naloxone significantly enhanced AVP secretion
(Yamada et al., 1989). Therefore, opioids may be involved in
regulating AVP release which could, in turn, affect IOP.
Whether DPDPE lowered IOP, in part, by decreasing AVP or
substance P secretion needs to be investigated in greater
detail.
CHAPTER VI
SUMMARY AND CONCLUSIONS
This investigation involved evaluation of the ocular
actions of a delta opioid receptor agonist, DPDPE, at both
pre- (neuronal) and postjunctional (ICB) sites in the
anterior segment of the eye. DPDPE was shown to:
1) lower IOP in normal rabbits but not in SX rabbits;
naloxone, a nonselective opioid receptor antagonist,
did not block the IOP lowering effect;
2) inhibit 3H-NE overflow from sympathetic nerves in ICBs
in response to electrical field stimulation;
3) suppress cAMP accumulation in ICBs, but this effect was
not antagonized by NTI, a delta receptor antagonist;
PTX did not prevent the inhibition of cAMP levels by
DPDPE.
These preliminary data suggest that the mechanism of
lowering of IOP by the delta opioid receptor agonist, DPDPE,
is complex and may be mediated at both pre- and
postjunctional sites in the rabbit eye. In addition, the
cellular actions of DPDPE may involve a PTX-insensitive G
protein or be due to a direct action on ion channels. Other
possible mechanisms need to be investigated in order to
better understand the ocular actions of opioids.
47
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