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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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