THE CARDIOVASCULAR ACTIONS OF MU AND KAPPA OPIOID AGONISTS IN

VIVO AND IN VITRO.

By

Abimbola T. Omoniyi, BSc (Hons)

A thesis submitted in accordance with the requirements of the University of Surrey for the Degree

of Doctor of Philosophy.

Department of Pharmacology, September 1998.

Cornell University Medical College,

New York, NY 10021, ProQuest Number: 27733163

All rights reserved

INFORMATION TO ALL USERS The quality of this reproduction is dependent upon the quality of the copy submitted.

In the unlikely event that the author did not send a com plete manuscript and there are missing pages, these will be noted. Also, if material had to be removed, a note will indicate the deletion. uest

ProQuest 27733163

Published by ProQuest LLC (2019). Copyright of the Dissertation is held by the Author.

All rights reserved. This work is protected against unauthorized copying under Title 17, United States C ode Microform Edition © ProQuest LLC.

ProQuest LLC. 789 East Eisenhower Parkway P.O. Box 1346 Ann Arbor, Ml 48106 - 1346 ACKNOWLEDGEMENTS

I would like to thank God through whom all things are made possible.

Many thanks to Dr. Hazel Szeto for funding this thesis.

Heartfelt gratitude to Dr. Dunli Wu for his support, encouragement and for keeping me sane.

I thoroughly enjoyed the funny stories, the relentless Viagra jokes and endless tales of the

Chinese revolution!

Thanks to Dr. Yi Soong for all her support and generous assistance and all that food!

Thanks to Dr. Ian Kitchen and Dr. Susanna Hourani for making this a successful collaborative degree.

Thanks to my family for all their support and belief in me.

To my network of support: Misan, Guy, Hesh, Yasmin, Arti, Vicky, Akin, Hicham, Emma and the rest of the wonderful people I am so fortunate to call friends; THANK YOU. You couldh \ possibly imagine how much of a contribution you made. Dedicated €o my parents, James and Do tun Omoniyi;

'pen ÿiaùtÿ me mtf Mttqe.

iii PUBLICATIONS

Publications listed below arise wholly or partly from the work described in this thesis

■ Omoniyi AT, Wu DL, Soong Y, Szeto HH. Baroreflex-mediated bradycardia is blunted by intravenous mu- but not kappa-opioid agonists. J Cardiovascular Pharmacol, 31:954-959. 1998.

■ Kett A, Omoniyi AT, Kim H, et al. Baroreflex-mediated bradycardia but not tachycardia is blunted by intravenous mu-opioid agonists in chronically-instrumented pregnant sheep. Am JObstet & Gynecol, 178:950-955, 1998.

■ Clapp JF, Kett A, Olariu N, Kim H, Omoniyi AT, Wu D and Soong Y Cardiovascular and metabolic responses to two receptor-selective opioid agonists in pregnant sheep. Am J Obstet & Gynecol, 178:397-401, 1998.

■ Omoniyi AT, Kett A, Wu D, Soong Y, Clapp JF and Szeto HH. A peripheral site of action for the attenuation of baroreflex-mediated bradycardia by intravenous mu-opioid agonists. J Cardiovascular Pharmacol. 1998. Xn »

■ Szeto HH, Soong Y, Wu DL, Omoniyi AT, Olariu N, Hyungjin K, and Clapp JF. Effect of pregnancy on cardiovascular actions of intravenously administered mu, delta and kappa- opioid agonists. AnesthAnalg, under revision, 1998.

■ Omoniyi AT, Wu DL, Soong Y, Holsey Y and Szeto HH. Development of rapid tolerance to p- opioid agonists in vivo and in vitro. Submitted to Eur. J. of Pharmacology. 1998.

■ Omoniyi AT, Wu DL, Soong Y, Holsey Y, Clapp JF and Szeto HH. The inotropic effects of selective p- opioid agonists is determined by the pre-existing condition of the heart. Submitted to J Pharmacol Exp Ther. 1998.

■ Holsey Y, Wu DL, Soong Y, Omoniyi AT and Szeto HH. Differential cardiovascular effects of a mu selective agonist in maternal and fetal sheep. Submitted to Amer J Obstet Gynecol. 1998.

■ Omoniyi AT, Wu DL, Soong Y, Pastuszak A, Holsey Y and Szeto HH. Pre-ischemic and post-reperiusion effects of two selective p- opioid agonists in the isolated guinea pig heart. Manuscript in progress.

iv ABBREVIATIONS

U50,488H trans-3,4-dichloro-N-[2-( 1 -prrrolidinyl)cyclohexyl]

benzene acetamide

DAMGO [D-Ala2,7V-Me-Phe4, Gly 5-ol] enkephalin

DPDPE [D-Pen 2,D-Pen5]-enkephalin

DALDA Tyr-D-Arg-Phe-Lys

Intravenous i.v.

Intracerebroventricular i.c.v.

Nucleus of the Tractus Solitarius NTS

Central Nervous System CNS

Sodium Nitroprusside SNP

Naloxone Methiodide NM

v ABSTRACT

The effects of two selective fi- opioid agonistsDAMGO and DALDA, and a selective k- opioid

agonist U50,488H on blood pressure and heart rate have been compared in the chronically

instrumented and awake sheèp. Differences in the heart rate response to p- and K-opioid agonists

in vivo w^edemonstrated. The mechanism behind the heart rate response to all three opioid agonists

was investigated by studying the effects of p- and K-agonists on baroreflex sensitivity and the

involvement of sympathetic activity in the heart rate response to K-opioid agonists. The site and

mechanism of action involved in the effects ofDAMGO and DALDA on the baroreflex were further

investigated in vivo. The opioid receptor agonists involved in these studies are undergoing

development as obstetrical analgesics for clinical use hence the influence of pregnancy on the effects

of p- and K-opioid agonists on blood pressure, heart rate and baroreflex sensitivity were investigated.

The mechanism responsible for the blood pressure effects of p-opioid agonists observed in vivo was

investigated in vitro using the isolated and perfused guinea pig heart. The effects of DAMGO and

DALDA on inotropy, chronotropy and coronary flow has been demonstrated in the spontaneously

beating heart. Differences in the inotropic response to DAMGO and DALDA in the absence of their

chronotropic influence was demonstrated in electrically paced hearts. Inotropic responses to

DAMGO and DALDA have been shown to be influenced by the contractile condition of the heart.

The potential therapeutic efficacy of p-opioid agonists has been investigated by studying the development of tolerance to their cardiovascular effects in vivo and in vitro. DAMGO and DALDA have been shown to induce rapid tolerance to blood pressure responses and inotropic responses in vivo and in vitro respectively. Differences in the relative potencies of DAMGO and DALDA was confirmed in all studies. SUMMARY

1. The history of the opioid receptor system is reviewed. The classification and distribution of

opioid receptors, receptor subtypes, their endogenous ligands and the developm ent o f highly

selective agonists and antagonists is discussed w ith reference to the cloning o f opioid receptors and

then transduction mechanisms. A discussion o f the regulation o f the cardiovascular system including

that o f blood pressure, heart rate and the baroreflex is presented w ith consideration o f the influence

o f pregnancy on the system. The literature on the effects o f opioids on cardiovascular regulation is

r e v i e w e d .

2. In order to assess the cardiovascular effects o f system ically adm inistered opioid peptides, changes

m blood pressure and heart rate w ere observed after intravenous adm inistration o f U 50.488H

3,4.dichloro-N-[2-(l.prrrolidinyl)cyclohexyl]benzeneacetam ide), a selective K-opioid receptor

agomst, and tw o selective p-opioid receptor agonists, D A LD A (Tyr-D-Arg-Phe-Lys) and D A M G O

(D-Ala2,7V-Me-Phe4,Gly5-ol]enkephaiin in nonpregnant and pregnant sheep. Intravenous

administration of all three agonists (U 50,488H (1.2 m g/kg), D A LD A (0.3m g/kg) and D A M G O (0.3

m g/kg)) to the awake sheep resulted in an immediate increase in blood pressure. The pressor response to U 50,488H w as accompanied by an increase in heart rate and both effects w ere naloxone- sensitive. In contrast, there was no accompanying change in heart rate to D A LD A or D A M G O . The pressor response to DAM GO and DALDA was significantly attenuated in pregnant animals. In contrast, there was no difference between the pressor response to U 50488H in nonpregnant and pregnant sheep, but the tachycardia was more pronounced in pregnant animals. It was hypothesized

that the lack of a reflex bradycardia to the pressor responses of both the p- and K-opioid receptor

agonists was due to a blunting of baroreflex-mediated bradycardia. The reflex bradycardia to

norepinephrine (0.6 pg/kg/min) was significantly reduced in the presence of DAMGO but not

U50,488H. In view of the lack of effect of U50,488H on the baroreflex, it was further hypothesized

that the tachycardia it elicited was due to an increase in sympathetic activity. Pretreatment with

propranolol (0.1 mg/kg) completely blocked the tachycardia elicited by U50,488H. These data

suggest that the lack of a reflex bradycardia to the pressor response of p-receptor agonists is due to

a blunting of baroreflex-mediated bradycardia. In contrast, the increase in heart rate caused by

U50,488H is mediated by sympathetic activation of the heart.

3. In order to assess the site and mechanism by which p-agonists cause an attenuation of baroreflex sensitivity, further investigations were carried out using the highly polar p-agonist DALDA, which has been shown to have a limited ability to cross the blood brain barrier. Furthermore, as the long term goal of the laboratory has been to develop these peptides for clinical obstetrical use, the effects of these peptides on baroreflex sensitivity in pregnancy was also investigated. The reflex bradycardia to norepinephrine (0.4 - 0.6pg / min) was significantly blunted in the presence of both DAMGO and

DALDA in pregnant and nonpregnant animals. In view of the limited ability of DALDA to get across the blood brain barrier, it was hypothesized that the blunting of baroreflex-mediated bradycardia by p-agonists occurs at a peripheral site. Pretreatment with the quaternary opioid

antagonist, naloxone methiodide (0.4 - 0.6 pg/kg/h) completely blocked the attenuation of baroreflex

sensitivity by DAMGO and DALDA in both nonpregnant and pregnant animals. In order to

investigate the baroreceptors as a possible site of action as well as address the possibility of these

drugs exacerbating hemorrhage-induced hypotension in labor and delivery, the effect of both p-

agonists on baroreflex-mediated tachycardia to a hypotensive stimulus was investigated. The reflex tachycardia to sodium nitroprusside (10 pg/kg/min) remained unchanged in the presence of DAMGO or DALDA in both nonpregnant and pregnant animals. These data suggests that the blunting of baroreflex-mediated bradycardia by DAMGO and DALDA has a peripheral site and mechanism.

Furthermore, the lack of any effect on baroreflex-mediated tachycardia indicates these p-agonists are not acting at the level of the baroreceptors.

(]3cvwe^ CiQ.pp'y 4. Further studies carried out by a collaborator^showed 44-agonists while increasing blood pressure in the sheep had no effect on peripheral resistance. However, there was a concommitant increase in cardiac output. These studies indicated the pressor responses of DAMGO and DALDA was most likely due to an increase in the contractility of the heart. The mechanism behind the pressor responses previously reported for DAMGO and DALDA was therefore investigated in vitro using the isolated and perfused guinea pig heart. The investigation was founded on the hypothesis that the increase in blood pressure observed with DAMGO and DALDA in vivo is as a result of a direct effect of these p-agonists to increase the contractility of the heart. The expectations were therefore that these agonists would produce a positive inotropic effect on the isolated heart. Pilot studies with the

isolated guinea pig heart however revealed conflicting results regarding the inotropic effects of

DAMGO and DALDA. It was further hypothesized that the pre-existing condition of the heart may

determine the inotropic effect of p-agonists on the heart. Hence the effects of DAMGO and

DALDA on the isolated and perfused guinea pig heart was further investigated under four different

conditions, two of which were "stress" paradigms: (1) Spontaneous heart rate (2) During electrical

pacing of the heart to eliminate the chronotropic effects of DAMGO and DALDA (3) Low flow

ischemia induced by rapid electrical pacing (4) and following 30-mins of no flow ischemia . DALDA

(10'"- 10'9M) caused a dose dependent decrease in the force of contraction, heart rate and coronary

flow in spontaneously beating hearts. There was a decrease in the force of contraction, coronary

flow and heart rate at 10'nM DAMGO while subsequent doses (10*9- 10* 7 M) resulted in increases

of all three parameters. Elimination of chronotropic effects with electrical pacing at spontaneous rate

unmasked dose-dependent negative inotropic effects for both DAMGO and DALDA (10"" - 1 0 ‘7 M)

that were bigger in magnitude than those observed in the spontaneously beating hearts. In hearts

stressed by ischemia or rapid pacing, administration of DAMGO or DALDA (lO9- 10’7 M) resulted

in a positive inotropic response accompanied by a decrease in heart rate and coronary flow. Both the

negative and positive inotropic effects of DAMGO and DALDA wjfere attenuated by the opioid

receptor antagonist naloxone (10* 5M). The negative inotropic effects of p-opioid receptor agonists “Hieir in the "unstressed" heart can be influenced by its chronotropic effects. In hearts stressed by ischemia

or rapid pacing, DAMGO or DALDA improve contractile performance of the heart restoring it to its pre-stressed state . The present study demonstrates that the inotropic effects of DAMGO and DALDA on the isolated heart depend on the pre-existing condition of the heart.

5. The effect of repeated exposure to DAMGO and DALDA and the possible development of

tolerance to their cardiovascular effects is investigated. In the chronically-instrumented ewe, single

exposures to DAMGO (0.3mg/kg) or DALDA (0.6mg/kg) resulted in a significant increase in blood

pressure with DAMGO eliciting a significantly larger response than DALDA. When two doses of the same agonist were administered 30 mins apart, the pressor responses to the second doses of both

DAT .DA and DAMGO was completely abolished. The possibility of cross tolerance between the two p-agonists was also investigated. Pretreatment with DAMGO completely abolished the pressor effect to DALDA. In contrast, pretreatment with DALDA resulted in a partial though significant attenuation of the pressor response to DAMGO. Hence the efficacy of DAMGO and DALDA in eliciting cardiovascular responses paralleled their ability to induce tolerance . In the isolated guinea pig heart, increasing doses of DALDA (10"n - 10* 7M) separated by a 10 min drug washout between doses resulted in a dose dependent decrease in the force of contraction. However, when the heart was continuously exposed to the same drug treatment for 30 mins without allowing for washouts between doses, the inotropic effect of DALDA was completely abolished after 10 mins. This study demonstrates acute tolerance to the cardiovascular effects of highly selective p-opioid agonists in vivo and in vitro.

xii CONTENTS

Page

ACKNOWLEDGEMENTS ü DEDICATION iii LIST OF PUBLICATIONS iv ABBREVIATIONS v ABSTRACT vi SUMMARY viii CONTENTS xiii LIST OF FIGURES xvii LIST OF TABLES xx

CHAPTER 1. A REVIEW OF THE OPIOID SYSTEM 1.1 Introduction 2 1.2 Characterization of opioid receptors 10

1 .2 .1 agonists and antagonists at ô receptors 1 0

1.2.2 agonists and antagonists at k receptors 14 1.2.3 agonists and antagonists at p receptors IB 1.3 Distribution of opioid receptors 23 1.4 Cloning of opioid receptors 25 1.5 Transduction mechanisms 28 1.6 The cardiovascular system 30 1.6.1 Cardiovascular regulation 3 0

1 .6 .2 Arterial pressure regulation 30 1.6.3 The baroreflex 32 1.6.4 Cardiovascular adaptation to pregnancy 33 1.7 Cardiovascular effects of opioids 37

xiii CHAPTER 2. THE EFFECTS OF HIGHLY SELECTIVE fi- AND K-OPIOID AGONISTS ON BLOOD PRESSURE, HEART RATE, THE BAROREFLEX, AND THEIR MODIFICATION BY PREGNANCY. 2.1 Abstract 42 2.2 Introduction 44 2.3 Materials and methods 46 2.3.1 Surgical procedure 46 2.3.2 Experimental protocol 46 2.3.3 Drugs 47 2.3.4 Drug treatments 47 2.4 Data analysis 48 2.5 Statistical analysis 49 2.6 Results 50 2.6.1 Effects of DAMGO in nonpregnant and pregnant animals 50 2.6.2 Effects of DALDA in nonpregnant and pregnant animals 51 2.6.3 Effects of U50,488H in nonpregnant and pregnant animals 51 2.6.4 Effects of U50,488H and DAMGO on baroreflex gain 52 2.6.5 Effect of propranolol on the heart rate response to U50,488H 53 2.7 Figures 54 2.8 Discussion 61

CHAPTER 3. THF, SITE AND MECHANISM OF ACTION RESPONSIBLE FOR THE BLUNTING OF BAROREFLEX-MEDIATED BRADYCARDIA BY g-OPIOH) AGONISTS.

3.1 Abstract 68 3.2 Introduction 70 3.3 Materials and methods 72 3.3.1 Drug treatments 72 3.3.2 Data analysis 73

xiv 3.3.3 Statistical analysis 74 3.4 Results 74 3.4.1 Baroreflex sensitivity in nonpregnant and pregnant animals 75 3.4.2 Effects of DAMGO and DALDA on NE challenge in 75 nonpregnant animals and pregnant animals 3.4.3 Effects of DALDA on SNP challenge in nonpregnant and 75 pregnant animals 3.4.4 Effect ofNMI on the heart rate response to NE in the 75 presence of DAMGO and DALDA (nonpregnant animals). 3.4.5 Effect ofNMI on the heart rate response to NE in the 76 presence of DAMGO and DALDA (pregnant animals) 3.5 Figures 77 3.6 Discussion 84

CHAPTER 4. THE DIRECT EFFECTS OF p-OPIOID AGONISTS ON THE ISOLATED GUINEA PIG HEART. 4.1 Abstract 91 4.2 Introduction 93 4.3 Materials and methods 95 4.31 Isolated heart perfusion 95 4.32 The Langemdorff setup 97 4.33 Drug treatments 98 4.4 Statistical Analysis 99 4.5 Results 100 4.5.1 Dose Response studies (DALDA) 100

4.5.2 Dose Response studies (DAMGO) 1 0 0 4.5.3 Arhythmic hearts 101 4.5.4 "Stressed hearts" 101

xv 4.6 Figures 102 4.7 Discussion 109

CHAPTER 5. THE DEVELOPMENT OF TOLERANCE TO THE CARDIOVASCULAR EFFECTS OF g-OPIOID AGONISTS IN VIVO AND IN VITRO. 5.1 Abstract 117 5.2 Introduction 118 5.3 Materials and methods 119 5.3.1 In vivo studies 119 5.3.2 In vitro studies 119 5.4 Data analysis 119 5.5 Statistical analysis 120 5.6 Results 121 5.6.1 In vivo experiments 121 5.6.2 In vitro experiments 122 5.7 Figures 121 5.8 Discussion 126

CHAPTER 6 , GENERAL DISCUSSION 6.1 Discussion 131 6.2 Conclusion

REFERENCES 138 LIST OF FIGURES

Page

Figure 1 Non-selective agonists and antagonists at opioid receptors. 6

Figure 2 ô-opioid receptor agonists 1 2

Figure 3 ô-opioid receptor antagonists 13

Figure 4 K-opioid receptor agonists 16

Figure 5 K-opioid receptor antagonists 17

Figure 6 p-opioid agonists 2 0

Figure 7 p-opioid antagonists 2 1

Figure 8 Effects of i.v. DAMGO (0.3 mg/kg) on mean blood pressure 54 (top panel) and heart rate (bottom panel) in nonpregnant and pregnant sheep.

Figure 9 Effects of i.v. DAMGO (0.3 mg/kg) on mean blood pressure 55 (top panel) and heart rate (bottom panel) in nonpregnant and pregnant sheep in the presence of naloxone.

Figure 10 Effects of i.v. DALDA (0.6 mg/kg) on mean blood pressure 56 (top panel) and heart rate (bottom panel) in nonpregnant and pregnant sheep.

Figure 11 Effects of i.v. U50,488H (1.2 mg/kg) on mean blood pressure 57 (top panel) and heart rate (bottom panel) in nonpregnant and pregnant sheep.

Figure 12 Effects of norepinephrine (0.6 pg/kg/min) on mean blood 58 pressure and heart rate in the absence and presence of U50,488H (0.6mg/kg) in awake sheep.

Figure 13 Effects of norepinephrine (0.6 pg/kg/min) on mean blood pressure 59 and heart rate in the absence and presence of DAMGO (0.6mg/kg) in awake sheep.

xvii Figure 14 Effects ofU50,488H (1.2 mg/kg) on heart rate in the absence and presence of propranolol ( 0 .1 mg/kg over1 0 min) in awake sheep.

Figure 15 Relationship between mean blood pressure and heart rate, in response to a hypertensive (NE, top panel) and hypotensive (SNP, bottom panel) stimulus in a nonpregnant and pregnant sheep.

Figure 16 Relationship between mean blood pressure and heart rate in response to NE in the absence or presence of DAMGO (top panel) or DALDA (bottom panel) in a nonpregnant sheep.

Figure 17 Relationship between mean blood pressure and heart rate in response to NE in the absence or presence of DAMGO (top panel) or DALDA (bottom panel) in a pregnant sheep.

Figure 18 Relationship between mean blood pressure and heart rate in response to SNP in the absence or presence of DALDA in a nonpregnant (top panel) and pregnant (bottom panel) sheep.

Figure 19 The effect of naloxone methiodide (NMI) on the relationship between mean blood pressure and heart rate in response to NE, in the absence or presence of DAMGO (top panel) or DALDA (bottom panel) in a nonpregnant sheep.

Figure 20 The effect of naloxone methiodide (NMI) on the relationship between mean blood pressure and heart rate in response to NE, in the absence or presence of DAMGO (top panel) or DALDA (bottom panel) in a pregnant sheep.

Figure 21 Effects of DALDA and DAMGO on heart rate in spontaneously beating hearts.

Figure 22 Effects of DALDA (10'n-10‘7M) on the force of contraction (top panel) and coronary flow (bottom panel) in spontaneously beating hearts and electrically paced hearts.

xviii Figure 23 Effects of DAMGO ( 1 0 "n- 1 0 '7M) on the force of contraction (top panel) and coronary flow (bottom panel) in spontaneously beating hearts and electrically paced hearts.

Figure 24 Representative traces showing the effect of DALDA (10"nM) on the force of contraction in (A) an unpaced and (B) a paced heart.

Figure 25 (A) Representative traces showing the effects of DALDA on force of contraction in pilot studies. (B) Effects of DALDA on force of contraction in hearts from pilot studies.

Figure 26 Trace showing the effects of DALDA (10'7 M) before and after rapid pacing in the same heart.

Figure 27 Effects of (A) DALDA and (B) DAMGO on force of contraction in hearts unstressed hearts and hearts subjected to rapid pacing and ischemia.

Figure 28 Effects of i.v. DAMGO (0.3 mg/kg) on mean blood pressure (top panel) and heart rate (bottom panel) in untreated animals, animals pretreated with the same agonist and animals pretreated with a different p-agonist.

Figure 29 Effects of i.v. DALDA (0.6 mg/kg) on mean blood pressure (top panel) and heart rate (bottom panel) in untreated animals, animals pretreated with the same agonist and animals pretreated with a different p-agonist.

Figure 30 Effects of DALDA (10""- 10"7M) on force of contraction (top panel), heart rate (middle panel) and coronary flow (bottom panel) with intermittent or continuous administration. LIST OF TABLES

Table 1 . Endogenous ligands of opioid receptors 7

Table 2. In vitro opioid peptide activity profile 22

Table 3. Ligands of the cloned opioid receptors 27

Table 4. Baroreflex responses to hypertensive and hypotensive 77 challenges in the presence of DAMGO and DALDA

Table 5. Methods for in vivo drug treatment 120

xx CHAPTER I

A REVIEW OF THE OPIOID SYSTEM

i 1.1 INTRODUCTION

In 1680, Sydenham wrote, “ Among the remedies which it has pleased Almighty God to give man to

relieve his sufferings, none is so efficacious as opium.” The word opium is derived from the Greek

name for juice, the drug being obtained from the juice of the poppy, Papaver somniferum. Opium

contains more than 20 distinct alkaloids. In 1806, a German chemist, Friedrich Sertumer, reported

the isolation of a pure substance in opium that he named morphine, after Morpheus, the Greek god

of dreams. This discovery was quickly followed by others such as codeine, discovered by Robiquet

in 1832 and papaverine by Merck in 1848. By the middle of the nineteenth century, the use of pure

alkaloids rather than crude opium preparations began to spread throughout the medical world.

The nature of the mood changes produced by opium has been the basis for its non-medicinal use and

abuse. The unrestricted availability of opium that prevailed until the early years of the twentieth

century coupled with the invention of the hypodermic needle led to the parenteral use of morphine which lead to a severe variety of compulsive drug abuse. The term “opioid” refers to any directly acting compound whose effects are stereospecifically antagonized by naloxone. The problem of addiction to opioids stimulated a search for potent analgesics free of addictive potential. Prior to and following World War n, synthetic compounds such as meperidine and methadone were introduced into clinical medicine, but proved to have typical morphine-like actions. Nalorphine, a derivative of morphine, was an exception. Nalorphine antagonized the effects of morphine and was used to reverse morphine poisoning in the early 1950’s. In addition, its unusual pharmacological profile ushered in the development of new drugs, such as the relatively pure antagonist naloxone and compounds with

2 mixed actions (e.g., pentazocine, butorphanol, and buprenorphine). Such agents enlarged the range

of available therapeutic entities and provide tools needed to explore the mechanisms of opioid actions.

The concept of pharmacologically relevant receptors was first elaborated by Beckett and Casy as

early as 1954. Portoghese (1965) suggested the concept of different modes of interaction of

morphine and other analgesics with opioid receptors. Goldstein and coworkers/( 1971) subsequently

proposed that radiolabelled compounds might be used to demonstrate the existence of these receptors

and to characterize them. Therefore, as soon as radioligands with high specific activities were

available, three different groups, independently, but simultaneously, showed that there are

stereospecific opioid binding sites in mammalian brain (Pert and Snyder, 1973; Simon et al, 1973;

Terenius, 1973). Opioid receptors have also been identified in various parts of the periphery such as the vagus nerve (Young et al, 1980; Zarbin et al, 1990), sympathetic nerve terminals in the heart

and blood vessels (Mantelli et al, 1987) and the sympathetic lateral columns of the spinal cord

(Goodman et al, 1980; Romagnano and Hamill, 1984).

Although it was becoming clear by the mid-1960’s that the actions of opioid agonists, antagonists and mixed agonist-antagonists could be explained best by actions on multiple opioid receptors

(Portoghese, 1965), the first convincing evidence for this concept was provided by Gilbert and Martin in 1976. Their behavioral and neurophysiological observations in the chronic spinal dog led these

3 authors to propose the existence of three types of opioid receptors. These receptors were named

after the drugs used in the studies: mu (p, for morphine), kappa ( k, for ketocyclazocine) and sigma

(o,for SKF 10,047). Various preparations of the isolated ileum of the guinea pig and of the vas

deferens from mouse; rat, rabbit and hamster have been used for more than 30 years in

pharmacological assays to assess the agonist/antagonist properties of opioids (Kosterlitz et al, 1980;

Kosterlitz et al, 1981; Wild et al, 1993). Whereas morphine is more potent than the enkephalins

in inhibiting neurotransmitter release giving rise to electrically induced contractions of the guinea pig

ileum, the reverse is true in the mouse vas deferens preparation. Moreover, the effects of the opioid

peptides on the vas deferens are relatively insensitive to naloxone. Based on these observations,

Kosterlitz and coworkers proposed that a fourth type of opioid receptor, named delta (ô for

deferens), is present in mouse vas deferens (Lord et al, 1977). The a receptor was subsequently

shown to be non-opioid in nature, thus there are three main types of pharmacologically defined opioid

receptors: p, ô and k . Their existence has recently been clearly confirmed using molecular biology techniques; three types of opioid receptors have been cloned, with binding and functional properties consistent with their identities (Reisine and Bell, 1993; Kieffer, 1995; Satoh and Minami, 1995).

Pasternak and coworkers (1986) subsequently reported the identification of two subtypes of the p- receptor. The p-receptor is thought to be a high affinity-binding site for morphine and opioids responsible for supraspinal analgesia while the p—receptor is a low affinity site responsible for other actions such as spinal analgesia and respiratory depression.

4 By the mid-1970’s, the first endogenous peptide ligands for opioid receptors (enkephalins and 0- endorphin) were isolated and sequenced (Hughes et al, 1975; Bradbury et al, 1976; Li and Chung,

1976). Another group of peptides, the first of which was named dynorphin, was then identified in the 1980’s (Goldstein et al, 1981). The endogenous opioid peptides are encoded by different genes, expressed in distinct neuronal pathways or cell types, and have differing selectivities for the various classes of opioid receptors. In the same period, it was recognized that each of the opioid peptides is made as part of a larger precursor protein. In mammals, there are three such precursors: proenkephalin A, which yields four met-enkephalins, one leu-enkephalin, one met-enkephalin-Arg 6 -

Phe7 and one met-enkephalin-Arg6-Gly7-Leu8 (Noda etal, 1982); prodynorphin (or proenkephalin

B), which gives rise to dynorphins A and B, and a- and P-neoendorphins (Kakidani et al, 1982; Noda et al, 1982); and proopiomelanocortin, which is processed into corticotropin, P-lipotropin and melanotropins along with P-endorphin (Nakanishi etal, 1979).

5 Fig. 1 Non-selective agonists and antagonists at opioid receptors

ho ,

r Morphine NCH

AGONISTS <

Heroin (diacetylmorphme) NCH.

HO.

Naloxone OH

ANTAGONISTS^ HO.

Naltrexone OH

MIXED AGONIST -ANTAGONIST HO,

Nalorphine

6 Table 1. Endogenous Ligands of Opioid receptors

Mammalian peptides

Met5-enkephalin: Tyr-Gly-Gly-Phe-Met Enkephalins Leu5-enkephalin: Tyr-Gly-Gly-Phe-Leu Met5-enkephalin-Arg 6-Phe7: Tyr-Gly-Gly-Phe-Met-Arg-Phe Met5-enkephalin-Arg 6-Gly7-Leu8: Tyr-Gly-Gly-Phe-Met-Arg-Gly-Leu

Dynorphin A:Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Trp-Asp-Asn-Gln Dynorphins Dynorphin B:Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-Thr ' : „ z - ; ^ \ Asii-Vsi

P-neoendorphin Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro

P-endorphin Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-Phe-Lys-Asn- Ala-ltjr-

Endomorphin-1 Tyr-Pro-Trp-Phe-NH2

Endomorphin-2 Tyr-Pro-Phe-Phe-NH2

Amphibian peptides Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NHz Demorphins Tyr-D-Ala-Phe-Gly-Tyr-Pro-Lys Tyr-D-Ala-Phe-Trp-Tyr-Pro-Asn

A: Tyr-D-Met-Phe-His-Leu-Met-Asp-NH2 (deltorphin, dermenkephalin,dermorphin Deltorphins gene- associated peptide) B: Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH 2 (deltorphin II) C: Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH 2 (deltorphin I)

7 CctiiaiV) Endorphins, enkephalins and dynorphins, which all have the amino-acid sequence Tyr-Gly-Gly-Phe,

bind with low to moderate specificity to the three opioid receptors. P-endorphin binds to the p and

Ô receptors with comparable affinity, but because of their selectivity, Met- and Leu-enkephalin are

considered to be the endogenous ligands for the ô receptor and dynorphins for the K-opioid receptor.

Until very recently, there was no known mammalian peptide that displayed both high affinity and

selectivity for the p receptor. A study just published describes the discovery and isolation of two

peptides from the brain named endomorphin-1 (Tyr-Pro-Trp-Phe-NFQ and endomorphin-2 (Tyr-Pro-

Phe-Phe-NLQ (Zadina etal, 1997). The high affinity (ki = 360pM) and selectivity (4000 and 15000 fold preference over the ô and k receptors) of these peptides for the p receptor make them very likely candidates for its natural ligands.

Though well defined, the nomenclature of the opioid receptors is rather confused for the following reasons: (a) although the use of Greek letters is generally accepted by pharmacologists, molecular biologists renamed the p -,ô - and K-opioid receptors MOR, DOR and KOR respectively; (b) both of these nomenclature are poorly informative regarding the nature of these receptors. For instance, the

Greek letters p and k, derived from synthetic ligands (morphine and ketocyclazocine, respectively), provide no information on the endogenous agonists acting at these receptors. Similarly, the nomenclature proposed by the molecular biologists is equally unsatisfactory because it is derived from

8 the Greek letters. Based on the guidelines defined by the International Union of Pharmacology

(IUPHAR) Committee on Receptor Nomenclature and Drug Classification, receptors should be

named after their endogenous ligands and identified by a numerical subscript corresponding to the

chronological order of the formal demonstration of their existence by cloning and sequencing

(Vanhoutte etal, 1996). Thus, the generic designation for these receptors on which all opioids act

as agonists should be OP. Because the ô receptor was the first to be cloned (Evans et al, 1992;

Kieffer etal, 1994), it should be correctly named OP l5 and the k and p receptors, which were then

successfully cloned (Reisine and Bell, 1993; Kieflfer, 1995; Satoh and Minami, 1995), should become the OP2 and OP 3 receptors, respectively. This new nomenclature unlike the other two used in the literature to date, would allow any newly discovered opioid receptor(s) to be logically named following the same informative guidelines.

9 1.2 CHARACTERIZATION OF OPIOID RECEPTORS

1.21 Agonists and antagonists at ô (OP,) receptors

With few exceptions, all ô receptor agonists are peptides, derived from enkephalins or belonging to

the class of amphibian skin opioids (table 1). D-Ala 2-D-leu5-enkephalin (DADLE) was initially found

to be a selective agonist at ô receptors using guinea pig ileum and mouse vas deferens assays

(Kosterlitz etal, 1980). However, receptor-binding studies subsequently showed that DADLE has

only a two-fold greater affinity for ô than for /A receptors (James and Goldstein, 1984). A

hexapeptide, DSLET (fig 2), was found to have at least 20-600-fold selectivity, depending on the

assay, for ô over k and p receptors. More recently, novel opioid peptide agonists have been

synthesized: DSTBULET (fig 2), and its analogues BUBU (fig 2) (Delay-Goyet et al, 1988) and

BUBUC (fig 2) (Gacel et al, 1990), which are up to 1000-fold more potent at ô than at m

receptors. The cyclic peptides DPDPE and DPLPE (fig 2) and derivatives are of comparable

selectivity for the ôreceptors (Mosbergetal, 1983; Toth et al, 1990). Certain opioid peptides such

as deltorphin (Kreil et al, 1989) from the amphibian skin also have high affinity and selectivity at ô

receptors. *0

The first non-peptidic agonist that was reported to have some selectivity for ô opioid receptors was

BW373U86 (fig 2) and although this compound is only approximately 20-fold more potent at ô than at k or p receptors (Chang etal, 1993) as well as exhibiting a degree of toxicity, it could represent

10 a lead compound for the development of non-peptidic ô receptor agonists. Indeed, the methyl ether

of one enantiomer of BW373U86, SNC 80 (fig 2) (Bilsky et al, 1995), has been recently synthesized,

and its properties are promising. Studies with SNC80 have demonstrated a 2000-fold selectivity for

Ô over ji receptors. Furthermore, in contrast to BW373U86, SNC 80 exhibits only minimal signs

of toxicity (Bilsky e/a/., 1995).

The naltrexone derivative naltrindole (NTI) was the first really selective and potent ô receptor antagonist to be synthesized (Portoghese et al, 1988). It binds to ô receptors in monkey brain with a kj value in the picomolar range and a 100-fold selectivity for these receptors as compared with p and k receptors (Emmerson et al, 1994). Substitution of tetrahydroisoquinoline (Tic) at the 2- position of a deltorphin -related tetrapeptide analogue produced a potent and highly selective ô receptor antagonist, TIPP (fig2) (Emmerson et al, 1994; Schiller et al, 1992). The Iq of TIPP was found to be approximately 1 nM making it less potent than NTI, but its selectivity for ô receptors is 80-fold higher than that of the non-peptide antagonist (Emmerson et al, 1994; Schiller et al,

1992). Reduction of the Tic 2-Phe3 peptide bond in TIPP resulted in TIPP[Y] (fig-3), which shows improved ô antagonist potency and selectivity, and p or k antagonist properties (Schiller et al,

1993).

11 Fig. 2 8-opioid receptor agonists

— PEPTIDES —

DADLE Tyr-D-Ala-Gly-Phe-D-Leu DSLET Tyr-D-Ser-Gly-Phe-Leu-Thr DTLET Tyr-D-Thr-Gly-Phe-Leu-Thr DSTBULET Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr BUBU Tyr-D-Ser(OtBu)-Gly-Phe-Leu-Thr(OtBu) BUBUC Tyr-D-Cys(StBu)-Gly-Phe-Leu-Thr-(OlBu) DPLPE Tyr-D-Pjn-Gly-Phe-L-Pen DPDPE Tyr-D-Pen-G ly-Phe-D-Pen Deltorphin Tyr-D-Met-Phe-His-Leu-Met-Asp-NHg Deltorphin 1 Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NHp Deltorphin II Tyr-D-Ala-Phe-Glu-Val-Val-Gly-NH?

— NON-PEPTIDES — O II ElgN— C Il H OH BW373U86 N^CH3

CHVXJ N v

EtgN— C X J ^ C X oCH, SNC 80 N GH,

CH3''' "N N

.CH.

SIOM OH

HO From Raynor etal., Mol Pharmacol. 45: 423-428, 1994a Mcknight, and Rees, Neurotransmissions, 7:1-7, 1991

12 Fig. 3 8-opioid receptor antagonists

OH Naltrindole (NTI)

HO O'"

,OH BNTX

HO O'"

.OH Naltrindole S'-isothiocyanate (S'-NTII) NCS

HO O'

— NON-PEPTIDES —

Naltriben

OH

HO O' N-benzy(naltrindole (BNTI* I c h 2

From Raynor et al, Mol Pharmacol. 45: 423-428, 1994a Mcknight and Rees, Neurotransmissions, 7:1-7, 1991 1.2.2 Agonists and antagonists at k (OP2) receptors

The first really selective k agonist, the arylacetamide U-50,488 (fig 4) was synthesized in 1982 by the Upjohn Company (Lahti et al, 1982; Vonvoigtlander et al, 1983). It was followed by compounds U-69,593 (Lahti et al, 1985) and U-62,066 (Vonvoigtlander and Lewis, 1988) with comparable or higher k selectivity. Several other agonists have been synthesized as derivatives of this first series of arylacetamide compounds. These include PD 117302 (Vonvoigtlander et al, 1983;

Clark etal, 1988), CI-977 (Hunter et al, 1990) ICI 197067, ICI 199441 and ICI 204448 (Costello etal, 1988; Nock et al, 1989). HMD 61753, and to a lesser extent, EMD 60400 are also selective k receptor agonists acting at the periphery (Barber et al, 1994a; Barber et al, 1994b)/ arb)7^A series of benzeneacetamido-piperazine analogues, such as GR 89696, are also potent and rather selective k receptor agonists (Hayes et al, 1990; Rogerset al, 1992).

Various structural modifications have been made in dynorphin molecules in attempts to synthesize analogues with enhanced selectivity for the k receptors. Thus, [D-Pro10]dynorphin A -(l-ll) was shown to be about 200-fold more potent than U-50,488 in stimulating k receptors (Gairin et al,

1984). Shorter (E-2078) and longer dynorphin A analogues with potent and selective k agonist properties have also been described.

The first compounds designed for blocking k receptors, such as TENA (fig 5), lacked sufficient

14 selectivity (Kosterlitz etal, 1981). However, the concept of bivalent ligands, used for the synthesis

of TENA, led to the morphine derivative nor-binaltorphimine (nor-BNI) (Portoghese et al, 1987), which has a IQ value for inhibiting [3H]U-69,593 binding to monkey brain membranes of 60pM, and a 100-fold and 200-fold preference for k over and : receptors respectively (Emmerson et al,

1994). However, in vivo, nor-BNI exhibits an unusually long duration of action as k receptor antagonist (Horan etal., 1992) and its k selectivity has been questioned (Birch et al, 1987; Levine etal, 1990). More recently, Olmsted et al. (1993) (Olmsted etal, 1993) synthesized a new series

tAtoS of NTI derivatives, among which 5'-[N -(butylamidino)-methyl] NTI revealed to be more potent than nor-BNI to block k receptors. In addition, this compound exhibits a 57-fold and 90-fold preference for k over p and 5 receptors respectively (Olmsted et al, 1993).

15 Fig. 4 K-opioid receptor agonists

NON-PEPTIDES —

— peptides — CM

m -P fo '° | Tyr-Gly-Gly-Phe-Leo-Arg-Arg-Ue-Arg-D-Pro-Lys Q- n - c - ch ,— D yn(M I) U-50.488 E-2078 (N-meThyl-TyrVN-memyl-Arg^.D-Leu8) dynorphin-AM-8l ethyiamide o °

Dynorphin la Tyr-Gly-Gly-Phe-Leu-Arg-Arg-lle-Arg-Pro-Lys-Leu- c n , Lys-Tyr-Leu-Phe-Asn-Gly-Pro

U-69,593 Q Q - n 1 - cm’- 0 — HOM-PEPTIOES — O o r-N-CH.-d

Ketocyclazoane U-62.066 iSpiradoltnei ÇQ' 1 s - " - - C ^ < & X - 2 O

Ethyketocyclazocne PD 117302 CH,CH,

N-CH,—|<] 01-877 (PD 129290, enadorma) Bremazoane CHjCH, o CH.

Tifluadom IC1 197067 o x>

From Raynor et al., Mol Pharmacol. 45: 423-428, 1994a Mcknight and Rees, Neurotransmissions, 7:1-7, 1991

16 Fig. 5 K-opioid receptor antagonists

ANTAGONISTS

— PEPTIDES —

none

— NON-PEPTIDES —

N^XI OH TENA

| HO ^ o " h ' NH-CH,-CH,.0-CH,j2

r< ll> -i

Nor-txnanorphimine (NorBNI)

5‘-(N2-(butylamidino)-methylJnaltrindole N'A. (CH2)3CH3

UPHIT OUJL.

NCS

DIPPA • n - 9 C ,

CH,

I MIXED AGONIST/ANTAGONIST j

N-CHgCH-CH,

OH

NalBzOH

N-NH-C—

17 From Raynor et al., Mol Pharmacol. 45: 423-428, 1994a Mcknight and Rees, Neurotransmissions, 7:1-7, 1991 1.2.3 Agonists and antagonists at \l (OP3) receptors

To date, the most potent and selective agonist at p receptors is the benzimidazole opioid etonitazine

(fig6 ), with a Kd value of 20pM for m receptor binding sites in monkey brain membranes, and p:K

selectivities of about 9000 and 12,000 respectively (Emmerson et al, 1994).

FK 33,824 (fig 6 , (Roemer et al, 1977) was the first peptide analogue of met-enkephalin with high

affinity for the p receptor, and p.K selectivity of approximately 30, to be synthesized. The related

compound DAMGK) (fig 6 , also referred to as DAGO or DAGOL, (Handa et al, 1981), which has A

become the most commonly used selective p receptor agonist., is almost 1 0 times more selective than

FK 33,824 and has high affinity (Kd = 0.7nM) for the p receptor. These properties led to the

development of [ 3H]DAMGO for the selective labeling of p receptors in membranes or section from

various tissues.

Naloxone, the first opioid receptor antagonist identified, has higher affinity for the p receptor than for the other opioid receptors (Magnan et al, 1982; Emmerson et al, 1994). Naltrexone is less p receptor selective (Magnan et al, 1982; Emmerson et al, 1994) but has a greater potency and longer duration of action than naloxone (Crabtree, 1984). Other long-lasting p receptor antagonists are naloxazone and naloxonazine (fig 7). The fiimarate methyl ester derivative of naltrexone, P- funaltrexamine (P-FNA) (Portoghese etal, 1980), acts as an irreversible p receptor antagonist, but

18 also as a reversible k agonist. More recently developed derivatives of naltrexone and dihydromorphinone (fig 7 ), appear to be selective irreversible antagonists at p. receptors without any agonistic action at other opioid receptors (Jiang et al, 1994).

Antagonists with the highest selectivity towards p receptors are cyclic peptides related to somatostatin. The most frequently^ compounds are CTAP and CTOP (fig 7) which inhibit

[3H]naloxone binding to rat brain membranes with an IG 50 value of about 3nM and have a 1 2 0 0 - and

4000-fold selectivity for the p receptors versus the k and 5 receptors. The more recently developed analogue D-Tic-CTOP (TCTOP) has about 10,000-fold higher affinity for p receptors than for Ô receptors (Kazmierski et al, 1988).

19 Fig. 6 |i-opioid agonists

— PEPTIDES —

CDRI 82-205 Tyr.D-Ala-Gly-MePhe-Gly-NH.C 3 H7 (i) DAMGO Tyr-D-Ala-Gly-MePhe-Gly-ol FK 33-824 Tyr-D-Ala-Gly-MePhe-Met(0)-of PL017 Tyr-Pro-r-MePhe-D-Pro-NHa Dermorphin Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NHa MorphicepUn Tyr-Pro-Phe-Pro-NHa TRIMU-5 Tyr-D-Ala-Gly-NH-(CH2)2-CH(CH3)2

— (#ON-PEPTiDES —

CHg Sufentanyi CHjCHgCO CeH5

CH,

Ohmefentanyl CHjCHgCOv O - ^ - O C.H= 0H

Etonitazene OCaH, •N

CH,

Meptazinol

OH

From Raynor et al, Mol Pharmacol. 45: 423-428, 1994a Mcknight and Rees, Neurotransmissions, 7:1-7, 1991

20 Fig. 7 |I-opioid antagonists — PEPTIDES —

CTOP D-Phe-Cys-Tyr*D-Trp-Om-Thr-Pen-Thr-NH2

CTP D-Phe-CysTyr-D-Trp-Lys-Thr-PeH-Thr-NHa

SMS-201,995 D-Phe-Cvs-Phe-D-Trp-Lys-Thr-cys-Thr-ol

CTAP D-Phe-Cys-Tyr-D-Trp-Arg-Thr-P^n-Thr-NHa

TCTOP (D-Tic-CTOP) D-Tic-Cys-Tyr-D-Trp-Om-Thr-Pen-Thr-NHa

— NON-PEPTIDES--

CHjCHaCHj CHjCH-CHj

OH HO. Naloxonazine

HO ‘N— N OH

■ r O

.OH B-Funaltrexamlne (I3-FNA) C=C. HO O' : NHCO

,n -ch 2 — <(] .NHCOCH»CH N-CPM-MET-CAMO

OH O'" CH

N—CH- NHCOCH-CH NOj MET-CAMO

OH CH

21 From Raynor et al., Mol Pharmacol. 45: 423-428, 1994a Mcknight and Rees, Neurotransmissions, 7:1-7, 1991 Table 2. In vitro opioid peptide activity profile

Bioassay Binding (nM) Peptide analog GPI MVD Kip Ki 5 K iW Tyr-Gly-Gly-Phe-Met 6.7 1.5 9.5 0.91 0.095

Tyr-D-Ala-Gly-NMePhe-Gly-ol (DAMGO) 4.5 33 1 .0 345 345

Tyr-D-Pen-Gly-Phe-D-Pen (DPDPE) 2350 2 .8 710 2.7 0.004

Tyr-D-Ala-Phe-Gly-Tyr-Pro-Ser-NH 2 1.4 2.4 0.7 62 8 8 .6

Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH 2(DELT) 845 0.147 262 1.44 0.005

Tyr-D-Arg-Phe-Lys-NH2 (DALDA) 254 781 1.69 19200 11400

Tyr-D-Nva-Phe-Om-NH2 (TNPO) 104 271 1.17 2 2 0 0 1880

GPI: Guinea pig ileum MVD: Mouse Vas Deferens

Functional and binding assays demonstrating the affinity and efficacy of opioid peptide analogs at p- and 5-opioid receptors.

/ Adapted from: Schiller et al., Proc. Natl. Acad. Sci. U.S.A. 89: 11871-11875,1992. Schiller et al., J. Med. Chem 32: 698-703, 1989.

22 1.3 DISTRIBUTION OF OPIOID RECEPTORS

Similar distribution patterns of receptors have been obtained using autoradiographical techniques with various tritiated and radioiodinated ligands (Waksman et al., 1986; Delay-Goyet et al., 1990, Renda et al, 1993). In the CNS, ô receptors have a more restricted distribution than other opioid receptors.

The highest ô receptor densities are present in olfactory bulb, neocortex, caudate putamen and nucleus accumbens. Recently, antibodies generated against selective portions of the ô receptor amino acid sequence were used to localize this receptor type in the CNS of rodents and primates. The observed immunohistochemical distribution matched perfectly that established using autoradiographical methods (Dado et al, 1993; Bausch et al, 1995; Honda and Arvidsson, 1995).

In addition, immunocytochemistry at the ultrastructural level (Cheng et al, 1995) provided the definitive proof of the existence of presynaptic opioid receptors responsible for the inhibitory influence of opioids on the release of neurotransmitters from the terminals of primary afferent fibers within the dorsal horn of the rat spinal cord (Bourgoin et al, 1994).

Species differences in the distribution of opioid receptors are particularly striking. For instance, in the guinea pig, the highest density of specific sites for the tritiated arylacetamide compounds and hence K-receptors is found in the inner layers of the cerebral cortex, the substantia nigra and the interpeduncular nucleus. By contrast, in the rat, only low levels of labeling by these radioligands

23 are found throughout the cerebral cortex, the highest densities of specific binding sites being observed in the nucleus accumbens, claustrum, dorsal endopiriform nucleus and interpeduncular nucleus (Nock etal, 1989).

The presence and localization of opioid receptors in the periphery has not been investigated to the same extent as in the CNS. The development of the guinea pig ileum and mouse vas deferens as models of opiate actions made it possible to directly demonstrate the existence of pharmacologically relevant opioid receptor binding sites in the gut (Kosterlitz and Waterfield, 1975; Henderson et al,

1972). The guinea pig ileum is often used as a bioassay system preferentially for the p-receptor while the ô-receptor is pharmacologically characterized using the mouse vas deferens, p - , ô- and k - receptors are present in a variety of peripheral tissues of most species investigated so far, including man (Nishimura et al, 1986). In the rat and guinea pig, autoradiographic studies have shown the presence of specific p- and ô-receptor binding throughout the GI-tract (Nishimura et al, 1986).

Opioid receptors have also been reported in diflferent areas of the vascular system including the heart

(Krumins et al, 1985)and vagus (Young et al, 1980; Zarbin et al, 1990), sympathetic nerve terminals in the heart and blood vessels (Mantelli et al, 1987) and the sympathetic lateral columns of the spinal cord (Goodman etal, 1980; Romagnano and Hamill, 1984)..

24 1.4 CLONING OF OPIOID RECEPTORS

Shortly after the isolation of a murine recombinant receptor from the NG108-15 cell line (Evans et "fW & rccept'C*’" al, 1992; Kieflfer etal, 1994), several groups reported identical sequences from rat and mouse brain

(Abood et al, 1994; Bzdegae/tf/., 1993; Fukudae/a/., 1993; Yasuda^a/., 1993). In addition, a

s human cDNA encoding a 372-amino-acid protein with 93% identity with the mouse and ratA receptor has been cloned (Knapp et al, 1994). These clones encoding the % receptors, when compared with binding data from studies on transfected cells reveal a fairly consistent picture with the rank order of aflSnity for the alkaloids being NTI > diprenorphine > etorphine > bremazocine » naloxone > morphine > U-50,488H. For the peptide ligands, the rank order is as follows: DSLET > DADLE >

TIPP > DPDPE > DAMGO > morphiceptin.

Several identical cDNA clones have been independently isolated and characterized as encoding the k receptor from the mouse (Yasuda et al, 1993), rat (Chen et al, 1993; Li et al, 1993) and guinea pigQ&e etal, 1994). Dynorphin and its analogues potently bind to the recombinant receptor while enkephalin and P-endorphin have low potency for this receptor. The cloned receptor binds alkaloid ligands with the following rank order of affinity: bremazocine > ethylketocyclazocine > U-50,488 > naloxone > levorphanol > naltrindole > morphine > and for the peptide ligands, the rank order of affinity is as follows: dynorphin A » P-endorphin l-31 > DPDPE > DAMGO. Based on the high affinity for U-50,488 and U-69,593, the cloned k receptor has been proposed to be identical with the

25 so called “k / 1 binding site (Raynor et al, 1994a).

The g receptor has been cloned from the rat (Zastawny et al, 1994) and from the human (Wang et al, 1994; Wang et al, 1993). Both enkephalin and p-endorphin potently bind to the recombinant p receptor, whereas this receptor has much less affinity for dynorphin.. Its rank order of affinity for alkaloid ligands is as follows: bremazocine > ethylketocyclazocine > naloxonazine > naloxone > morphine » > U-50,488 and for peptide ligands is as follows: DAMGO > DADLE > DSLET >

DPDPE. These binding data are consistent with the known pharmacological profile of p receptors.

Most of the compounds have similar affinity for the human and the rat p receptors. However, the affinities of morphine, methadone and codeine are significantly higher for the human p receptor than for the fat p receptor (Raynor et al, 1995).

26 Table 3. Ligands of the cloned opioid receptors

Receptor Selective Ligands Nonselective Ligands Endogenous

subtype Agonists AntagonistsAgonists AntagonistsLigands CM&ta - _ DAMGO CTOP Levorphanol Naloxone Enkephalin £>- Morphine Etorphine Naltrexone Endorphin EodomorpirtirtS Methadone P-fNA

Fentanyl

Dermorphin K Spiradoline Nor-BNI Levorphanol Naloxone Dynorphin A

U50,488H Etorphine Naltrexone

Dynorphin A EKC crtvte-td, z:lcu 3 Ô DPDPE Naltrindole Levorphanol Naloxone Enkephalin

Deltorphin NTB Etorphine Naltrexone

DSLET BNTX

Selective, non-selective and the putative endogenous ligands of opioid receptors.

27 1.5 TRANSDUCTION MECHANISMS

The functional coupling of the three opioid receptors with G proteins was firmly established several years ago on the bases that guanine nucleotides diminish the specific binding of agonists and that the latter compounds stimulate GTPase activity in several preparations (Childers, 1991). The predicted structures of the cloned, k and \i receptors clearly confirm that they belong to the superfamily of seven-transmembrane spanning G protein-coupled receptors (Reisine and Bell, 1993, Uhl et ah, 1994,

Kieffer, 1995; Satoh and Minami, 1995). However, it cannot be completely ruled out that opioids may also act independently of G proteins. For example, the selective p receptor agonist DAMGO, modulates a Ca2+-dependent K+ channel independently of G proteins and kinase-mediated mechanisms in cultured bovine adrenal cells (Twitchell and Rane, 1994). Studies on brain tissue indicated that stimulation of p and

Olianas and Onali, 1992).

Stimulation by opioid agonists of the cloned rat and human p receptors expressed in COS and CHO cells reduces not only forskolin-stimulated adenylyl cyclase activity but also the production of inositol triphosphate, in a naloxone sensitive manner (Wang et ah, 1994; Raynor et ah, 1995). Similarly, in

28 transfected cells, stimulation of receptors decreases the accumulation of cAMP resulting from cell exposure to forskolin (Evans et al., 1992).

A coupling of opioid receptors with Gs -proteins responsible for excitatory effects of opioid agonists on target cells has also been hypothesized (Crain and Shen, 1990). These excitatory opioid receptors would be activated by lower concentrations of opioids than the “inhibitory” receptors coupled to G0or Gq proteins. However, recent investigations demonstrated that opioid receptors can couple with various G 0 or G- proteins, and also Gz, but not with Gs.

29 1.6 THE CARDIOVASCULAR SYSTEM

1.6.1 Cardiovascular regulation

As early as 1866, the first formulated idea that the cardiovascular system may be regulated by neural

reflexes was established by Cyon and Ludwig (Cyon and Ludwig, 1866). They showed that electrical

stimulation of the cephalic end of the depressor nerve in the rabbit causes cardiac slowing and a fall

in systemic pressure. Although incorrectly concluding that this nerve arose from endings in the heart,

they recognized the significance of their observations, and a new and valid concept of circulatory

regulation was bom.

Nearly four decades passed before Koester and Tschermak (1902) proved by degeneration

experiments that the depressor reflex was not cardiac in origin. They correctly identified the

distribution of the aortic depressor nerve and introduced the concept that the arterial system

contained pressure-sensitive regions. In addition, the concept of barosensitivity was implicit in the

observations of Koester and Tschermak.

1.6.2 Arterial Pressure regulation

Blood pressure is the force exerted by the blood against any unit area of the vessel wall and is almost

always measured in millimeters of mercury (mmHg). The mean arterial pressure (MAP) is the average pressure throughout each cycle of the heartbeat. Arterial pressure is not regulated by a single

30 pressure controlling system but instead by several interrelated systems that perform specific functions.

Any condition that alters either the cardiac output or total peripheral resistance (i.e. the impediment

to blood flow in the blood vessels) will result in a change in MAP, since MAP is a product of the two

factors. Thus, both these factors are often manipulated in the control of arterial pressure.

Cardiac output is the quantity of blood pumped into the aorta each minute by the heart and is equal

to venous return (the quantity of blood flowing from the veins into the right atrium each minute)

except for a few heartbeats at a time when blood is temporarily being stored in or removed from the

heart and lungs. Cardiac output = stroke volume x heart rate. The heart plays quite a permissive role

in the regulation of cardiac output, permitting the output to be regulated at any level up to the limit

of its pumping capability. Under normal circumstances the major factor determining the cardiac

output is the rate of venous return.

Heart rate is normally determined by the interrelationship between the intrinsic automaticity of the

sinoatrial node and the activity of the autonomic nervous system. Sympathetic stimulation increases

= and parasympathetic stimulation decreases the heart rate.

Another important component in the regulation of blood pressure is the renin-angiotensin system. Renin released from the kidney acts on circulating Angiotensinogen to produce Angiotensin I. Angiotensin His in turn formed from Angiotensin I in a reaction catalyzed by Angiotensin Converting Enzyme (ACE). Angiotensin II has a generalised vasoconstrictor action which acts directly to produce a rise in systolic and diastolic blood pressure. The production of renin hence Angiotensin I is increased when blood pressure falls therefore helping to maintain blood pressure.

31 1.6.3 The Baroreflex

The best known of the mechanisms for arterial pressure control is the baroreceptor reflex. Reflex circulatory regulation is carried out by the two baroreceptor systems namely the aortic and carotid reflex systems (see-Bvans Downing, 1979). The reflex systems consist of stretch receptors, known as baroreceptors, located in the wall of the carotid arteries (carotid sinus) and aortic arch. A rise in pressure stretches the baroreceptors and causes them to transmit signals into the central nervous system, and “feedback” signals are then sent back through the autonomic nervous system to the circulation to reduce arterial pressure. Signals from the carotid sinus are transmitted via the Hering’s nerve to the glossopharyngeal nerve (Hering, 1923), while those from the aortic arch are transmitted through the vagus nerves (Koester and Tschermak, 1902). Both sets of signals end up in the nucleus of the tractus solitarius (NTS). Once the baroreceptor signals reach the NTS, secondary signals inhibit the vasoconstrictor center of the medulla and excite the vagal center causing peripheral vasodilation and a decrease in heart rate and strength of contraction. Conversely, low pressure has opposite effects, reflexly causing the pressure to rise back towards normal.

32 1.6.4 CARDIOVASCULAR ADAPTATION TO PREGNANCY

The cardiovascular system undergoes profound changes during pregnancy. The first report on

maternal cardiovascular function in pregnancy was published in 1915 (Lindhard, 1915) in which he

observed an increase in cardiac output during pregnancy by as much as 50 percent. However, it is

only in the last decade that it has become feasible, through the use of modern echocardiographic

methods combined with Doppler, to fully determine the timing and magnitude of the pregnancy-

related increase in cardiac output. Thus, several investigators have confirmed Lindhard’s observation

of an increase in cardiac output to be the case in various stages of pregnancy starting from as early

as the first few weeks to the late stages of pregnancy (Atkins et al, 1981; Capeless and Clapp, 1989;

Duvekotefa/., 1993; Robson etal, 1989; Walters et al, 1966). Because cardiac output is elevated

in pregnancy, peripheral resistance is also decreased (Easterling et al, 1990; Mashim et al, 1987;

Robson et al, 1989). It should however be pointed out that the magnitude of changes observed varies enormously between studies. These differences are thought to be due to a number of reasons

such as differences in methods and number of subjects used, as well as variations in the reference point for baseline data and measurement points during pregnancy.

In addition to changes in cardiac output, an initial rise in heart rate followed by an increase in stroke volume has also been reported to occur in pregnancy (Capeless and Clapp, 1989; Duvekot et al,

1993; Robson et al, 1989). Furthermore, mean arterial pressure is decreased relative to the

33 preconceptional reference (Clapp et al., 1988). The increases in both stroke volume and heart rate

lead to an additional rise in cardiac output. These profound changes may be due, in part, to structural

changes in the heart during pregnancy. There is an increase in left ventricular mass during pregnancy

(Capeless and Clapp, 1989; Katz et al, 1978; Robson et al, 1989), suggesting a presence of

myocardial hypertrophy. Left atrial diameter, which has been demonstrated to correlate well as an

indirect estimate of vascular filling state (Duvekot et al, 1994), has been shown to increase gradually

through the progressive stages of pregnancy (Duvekot et al, 1993; Katz et al, 1978; Robson et al,

1989; Vered etal, 1991). This pattern of increase in the left atrial diameter, suggests a rise in both

preload and circulating blood volume, and in fact closely follows the rise in blood volume during

pregnancy (Duvekot et al, 1994). It would seem, that the hemodynamic demands in pregnancy are

consistent with the effects of chronic strain put upon the heart as the structural cardiac changes

observed in pregnancy, though brought about by different mechanisms, are comparable to those seen in athletes undergoing physical training (Robson et al, 1989).

The role of the pregnancy hormones, such as estrogen, progesterone, prolactin, human chorionic gonadotropin (hCG) and human placental lactogen (hPL) in the altered cardiovascular state of pregnancy cannot be ruled out as yet and remains to be fully elucidated. In man, the guinea pig and ewe, estradiol administration has been shown to produce physiologic evidence of ventricular and vascular remodeling (Katz et al, 1978; Robson et al, 1989).

34 Pregnancy-induced change in baroreflex homeostasis is an area that has received a lot of attention

in recent years. Not surprising, since the state of pregnancy poses challenges to arterial pressure

regulation such as in pregnancy-induced hypertension and hemorrhage, a normal consequence of

every delivery. The methods used to investigate the baroreflex include generation of curves to

examine the relationship between arterial pressure and heart rate in response to a pressor or depressor

stimulus. The numerous reports on the status of baroreflex sensitivity in pregnancy are however,

conflicting. Several investigators report a decrease in baroreflex sensitivity (Tabsh et al, 1986;

Olsson etal, 1987; Brooks and Keil, 1994a) while others report an enhanced baroreflex sensitivity

(Seligman, 1971; Magness and Rosenfeld, 1988; Conrad and Russ, 1992). Some of the conflicts in these reports may be due to the fact that only the bradycardic responses to increases in arterial pressure was examined in some studies, while the entire baroreflex was examined in others. Other sources of conflict in baroreflex investigations include the use of anesthesia in some studies, which can itself, compromise cardiovascular function. In one study, changes to the bradycardic response to a pressor stimulus was compared to overall baroreflex sensitivity in conscious rabbits, and it was found that while the former appeared to be enhanced in pregnancy, the latter was significantly blunted

(Brooks et al, 1995), emphasizing the importance of examining the entire baroreflex curve when evaluating differences in pregnancy.

Hormones have also been implicated in the altered status of baroreflex sensitivity seen in pregnancy.

35 Studies indicate that reflex increases in vasopressin, adrenocorticotropic hormone (ACTH) and

glucocorticoids are also blunted in pregnant dogs and sheep (Brooks and Keil, 1994a, b,

Keller-Wood, 1994a, b), suggesting a general blunting of arterial baroreflex activity in pregnancy.

It is also possible that any of a number of hormones increased in pregnancy, including estrogen,

progesterone, angiotensin n, atrial natriuretic factor (ANF), testosterone, oxytocin or prolactin, may

be mediators (see Brooks and Keil, 1994b). There is no information on the role of hormones in baroreflex or cardiovascular regulation in general to make a valid conclusion at present, thus further

studies are required in this area of the physiology of pregnancy.

Despite the numerous reports that are now available on the hemodynamic changes in pregnancy and the recent increase in methodological possibilities, the physiological meaning of these changes, as well as the mechanisms involved, remain to be understood. Further understanding of the mechanisms involved in the cardiovascular changes observed in pregnancy is of utmost importance, especially in the field of obstetrical drug development, as vascular changes and altered hormonal milieu in the physiology of the pregnant animal may account for differences in its response to vasoactive drugs.

Furthermore, there is relatively little information regarding the response to opioids in pregnancy. An altered response to opioids in pregnancy will play a significant role in the development of opioid drugs for obstetrical use.

36 1.7 CARDIOVASCULAR EFFECTS OF OPIOIDS

The discovery of endogenous opiate systems precipitated a resurgence of interest in opiate-

cardiovascular interactions . One of the earlier observations on the biological activity of opiates

besides analgesia, was the demonstration of potent cardiorespiratory effects of morphine and other

opiate alkaloids. In the second half of the 19th century full reports on the cardiac, respiratory and

blood pressure effects of morphine were available (Jackson, 1914). Subsequently, opioid peptides

and their receptors were found to be present in brain areas important for cardiovascular control, in

heart, in autonomic ganglia, and in adrenal medulla (Goodman et ah, 1980; Lewis et ah, 1985;

Schultzberg et al, 1979; Hanbauer et al, 1982). Opioid receptors were shown to be densely

distributed in the brainstem and hypothalamus in close proximity to cardiovascular centers.

Administration of opioid peptides, intravenously, intracerebrally or applied directly upon brainstem areas produce potent cardiovascular responses. The cardiovascular effects of pharmacologically administered opioid peptides are complex and dependent upon species, doses, route and site of administration, presence or absence of anesthetics and other interactions (Feuerstein, 1985). Hence, the presence or absence of anesthetics can change a bradycardic, hypotensive opioid effect into a tachycardia and hypertension (Pfeiffer et al, 1983b; Pfeiffer et al, 1983a; Faden and Feuerstein,

1983). Systemic versus central administration results in differences that are often interpreted to indicate a lack of penetration in sufficient quantities to the cardiovascular centers in the brain.

37 However, this controversy can also be due to opposite effects of the peptide in different brain sites.

For example, pressor and depressor sites were found for morphine and Dala 2,D-Leu5-enkephalm IX

(DADL) in neighboring hypothalamic nuclei, which were less than 1mm apart from each other

(Feuerstein and Faden, 1982). The relationship between opioid peptides and the autonomic system suggests involvement of both the sympathetic and parasympathetic nervous systems in the cardiovascular actions of these peptides. Activation of the sympathoadrenomedullary axis has been implicated in the pressor and tachycardie responses to the mu agonist DAMGO or the delta agonist

DADL in the conscious rat (Pfeiffer et al, 1983b; Feuerstein eta/., 1983). On the other hand, the bradycardic effects of various enkephalin analogs have been attributed to an activation of the vagus

(Pfeiffer etal., 1982,1983b) although this may also be due to the potent respiratory depressant effect

(Pfeiffer e t al, 1983a, b; Faden and Feuerstein, 1983). Morphine and enkephalins were found to inhibit the bradycardia elicited in response to vagal stimulation (Musha et al, 1989; Weitzell et al,

1984), as well as the release of acetylcholine from rabbit atria (Weitzell et al, 1984).

«

The heart is known to contain both opioid peptides and opioid receptors (Krumins et a/., 1985; Kiang

and Wei, 1984). The direct effects of p,ô and K-opioid agonists have been investigated in a variety

of cardiac systems, though there is disagreement regarding the direction of change. Ô- and k- but not

H- agonists were found to decrease contractility in rat (Ventura et al, 1992) and hamster (Kasper

etal, 1992) ventricular myocytes. I n h u m a n atria strips, p, ô and k-agonists also produced (Llobe

38 andLaorden, 1995) negative inotropic effects. The K-opioid agonist U50,488H produced negative

inotropic effects in the isolated rat heart (Xia et ah, 1994). In contrast the naturally occurring ô -

opioid agonists leucine- and methionine-enkephalin produced positive inotropic effects in chick

embryo ventricular cells (Laurent et al., 1985; Laurent et al., 1986). The negative inotropic effects

of U50,488H were correlated with a decrease in Ca* transient (Ventura et al, 1992). Ventura and

co-workers (Ventura et al, 1991) demonstrated that a short exposure of myocytes to U50,488H

resulted in both an increase in Ca* transient and enhancement of the myofilament responsiveness that

resulted in an increase in contractility while longer exposures caused a depletion of SR calcium

resulting in a decrease in C% transient and negative inotropic effects. This indicates the level of

agonist exposure may determine the direction of change and may explain some of the discrepancies

in the literature. In the isolated rat atria, p, ô and k- agonists were shown to reduce the spontaneous

rate in a naloxone-sensitive manner (Micol and Laorden, 1994). Another study demonstrated the

p-agonist fentanyl produced negative chronotropic effects in the rabbit sinoatrial node that was

completely antagonized by the p-opioid antagonist P-FNA (Saeki et al, 1995). A study on the effects of leu-enkephalin on the Ca2+ current (7Ca) of L-type Ca2+ channels concluded the negative inotropic effects of the opioid peptide are due in part to a reduction in 7Ca. In addition, opioid peptides have been shown to inhibit Na+ - K+ and Ca2+ - dependent ATPase activities in bovine cardiac sarcolemma (Ventura etal, 1987). As Ca2+ entry into the myocardial cell is thought to be regulated by Ca2+ - dependent ATPase (Dhalla et al, 1982), this may explain, the cardiodepressant effects of

39 opioids. Although there is some conflict between studies, they clearly show that opioid peptides have a primary action on the heart.. However, while there are numerous studies on the effects of opioid receptor agonists on cardiac cells, relatively few studies have been done on the direct effects of selective opioid agonists in the whole heart isolated from systemic influences.

AIMS OF THESIS PROJECT The aim of this study is to evaluate the cardiovascular effects of highly selective opioid agonists. This study aims to resolve the disagreements from previous studies which arise from the use of anesthesia, poorly selective ligands and/or route of administration. The cardiovascular effects of highly selective ligands for all three classes of opioid receptors will be examined in vivo. In contrast to the majority of studies on opioids, these ligands will be administered systemically and not centrally. All experiments will be done without anesthesia in order to avoid its compounding effects on cardiovascular function. If any significant cardiovascular effects are observed in vivo, further studies will be carried out in vitro using the isolated guinea pig heart in order to investigate the mechanisms responsible for the effects observed in vivo. Furthermore, as the long term goal of the laboratory has been to develop an obstetrical analgesic that produces minimal side effects to the mother and the fetus, the cardiovascular effects of these candidate opioid ligands will be evaluated in pregnancy to determine if the physiology of the pregnant animal alters the cardiovascular effects of opioids and if these effects could be deleterious to the mother or the fetus. In summary the scope of the study will be as follows: • Highly selective opioid ligands: DAMGO (p), DALDA (p), U50,488H (k) and DPDPE (5) • Awake and unanesthetized animals', chronically instrumented and conscious sheep • Systemic administration of ligands: intravenous administration of drugs e Three indices of cardiovascular function to be Bloodmeasured: pressure, heart rate and baroreflex sensitivity • Comparative studies with pregnant animals • Further investigation of the results observed in vivo with the isolated guinea pig heart.

0

40 CHAPTER 2

THE EFFECTS OF HIGHLY SELECTIVE |> AND K-OPIOID RECEPTOR AGONISTS ON BLOOD PRESSURE, HEART RATE, THE BAROREFLEX, AND THEIR MODIFICATION BY PREGNANCY.

41 2.1 ABSTRACT

In order to assess the cardiovascular effects of systemically administered opioid peptides, changes in blood pressure and heart rate were observed after intravenous administration of U50,488H (trans-

3,4-dichloro-N-[2-(l-prrTolidinyl)cyclohexyl]benzeneacetamide), a selective K-opioid receptor agonist, and two selective |i-opioid receptor agonists, DALDA (Tyr-D-Arg-Phe-Lys) and DAMGO

(D - Ala2, A-Me-Phe4, Gly5-ol] enkephalin in nonpregnant and pregnant sheep. Intravenous administration of all three agonists (U50,488H (1.2 mg/kg), DALDA (0.3mg/kg) and DAMGO (0.3 mg/kg)) to the awake sheep resulted in an immediate increase in blood pressure. The pressor response to U50,488H was accompanied by an increase in heart rate and both effects were naloxone- sensitive. In contrast, there was no accompanying change in heart rate to DALDA or DAMGO. The pressor response to DAMGO and DALDA was significantly attenuated in pregnant animals. In contrast, there was no difference between the pressor response to U50488H in nonpregnant and pregnant sheep, but the tachycardia was more pronounced in pregnant animals. It was hypothesized that the lack of a reflex bradycardia to the pressor responses of both the p- and K-opioid receptor agonists was due to a blunting of baroreflex-mediated bradycardia. The reflex bradycardia to norepinephrine (0.6 pg/kg/min) was significantly reduced in the presence of DAMGO but not

U50,488H. In view of the lack of effect of U50,488H on the baroreflex, it was further hypothesized that the tachycardia it elicited was due to an increase in sympathetic activity. Pretreatment with propranolol (0.1 mg/kg) completely blocked the tachycardia elicited by U50,488H. These data

42 suggest that the lack of a reflex bradycardia to the pressor response of p-receptor agonists is due to a blunting of baroreflex-mediated bradycardia. In contrast, the increase in heart rate caused by

U50,488H is mediated by sympathetic activation of the heart.

43 2.2 INTRODUCTION

A number of studies have demonstrated that \i- and K-opioid receptor agonists elicit significant

cardiovascular responses (Hassen etal., 1983; Bell et al., 1990; Morita et al, 1987; Ogutman et al,

1995), while ô-opioid receptor agonists do not appear to modulate cardiovascular function

(Kiritsy-Roy et al, 1989; Bachelard and Pitre, 1995). Opioid peptide analogs are generally thought to have difficulty crossing the blood brain barrier after i.v. administration. In most studies, these agonists have been administered directly into the CNS in anesthetized animals and the resulting effects interpreted to be centrally mediated. Microinjection of the p-selective agonist DAMGO ([D-Ala2,

JV-Me-Phe4,Glycol]enkephalin) into the nucleus tractus solitarius (NTS) was found to increase both blood pressure and heart rate in anesthetized rats (Hassen et al, 1982) while another study reported hypotension and tachycardia (Faden and Feuerstein, 1983). May and colleagues^found intracerebroventricular (i.c.v.) administration of DAMGO to the conscious rabbit caused hypertension accompanied by an initial bradycardia followed by a tachycardia while U69,593, a K-selective agonist, had no effect on blood pressure or heart rate. In a more recent study, bilateral injection of DAMGO into the hypothalamic paraventricular nuclei of conscious rats resulted in an increase in blood pressure and tachycardia while U50,488H had no significant effect on either blood pressure or heart rate

(Bachelard and Pitre, 1995).

However, little is known about the effects of systemically administered p - and k- opioid agonists on

44 blood pressure and heart rate regulation. In the present study, the effects of intravenously

administered \i- and K-receptor agonists on blood pressure and heart rate in awake sheep were

compared. All three agonists utilized significantly increased blood pressure but were accompanied by different heart rate changes. DAMGO and DALDA increased blood pressure without affecting the heart rate while U50,488H increased both blood pressure and heart rate. It was hypothesized that the lack of a pressor-induced bradycardia to both p- and K-receptor agonists support an inhibition of the baroreflex. In this study, there is evidence that p-agonists significantly depressed baroreflex- mediated bradycardia while K-agonists had no effect on baroreflex function. In view of the lack of effect by U50,488H on the baroreflex, it was further hypothesized that the tachycardia it elicited is due to direct sympathetic activation of the heart.

45 2.3 MATERIALS AND METHOD

2.3.1 Surgical procedure

Chronic indwelling catheters were implanted in ewes of mixed Western breed in accordance with guidelines approved by the Institution for the Care and Use of Animals at Cornell University Medical

College. Polyvinyl catheters were placed in the distal aorta and inferior vena cava in all animals. All catheters were tunneled and exteriorized to the maternal flank and stored in a pouch. Surgery was performed with the ewe under intrathecal lidocaine anaesthesia and supplemented with intravenous sodium pentobarbital. Intraoperatively, Ig of ampicillin was placed in the peritoneal cavity of the ewe. This preparation permitted continuous recording of all above parameters in an unrestrained unanesthetized animal.

2.3.2 Experimental Protocol

Experiments were performed a minimum of 5 days after surgery to ensure complete recovery from surgical stress. The health status of all animals was regularly monitored by analysis of their blood respiratory gases, daily activities, weight and feeding habits. On the day of the study, the ewe was placed in a cart with free access to food and water throughout the duration of the study and care taken to provide an environment free of external stressful stimuli. The ewe was allowed a period of

2h to acclimate to the study conditions prior to drug administration. Continuous recording of blood pressure and EKG were obtained with a Gould 2800S analog recorder and appropriate amplifiers.

46 /

Experimental Protocol (continued) j

The sheep were allowed a minimum of five days to adjust to the animal facilities before surgery j commenced. Feeding of the animals was suspended for 24 hours prior to surgery. Water supply was maintained throughout this period. Surgery on pregnant animals was carried out around the gestational ageof 115-117 days. The gestational age o fthe pregnant sheep at the tune o fexperiments ranged from 120-135 days. Experiments with nonpregnant sheep consisted of a mix of originally

nonpregnant animals and previously pregnant animals at 5 days post-partum. Control studies showed

there were no differences in the basal blood pressure and heart rate of both groups of nonpregnant

sheep (data not shown) like that observed between pregnant and nonpregnant animals.

Blood pressure was measured with a Baxter micro-pressure transducer coupled to a Gould

preamplifier. The transducer was calibrated against a mercury column prior to each experiment.

Heart rate was obtained from the arterial pressure wave and transposed using a biotachometer. To

avoid postural artifact, the pressure transducer was mounted at the level of the maternal heart on the

flank of the animal. All amplifier outputs were recorded on a Gould 2800, 8 channel recorder at a

speed of 2 mm/sec. The precision and accuracy of the entire system was ± 2mmHg for blood pressure

and ± 4 b.p.m. for heart rate. and stored on FM tape (TEAC XR-310). Heart rate was obtained from blood pressure or EKG by

use of a Gould Biotach coupler.

2.3.3 Drugs

U50,488H,DAMGO and DALDA (supplied by the National Institute on Drug Abuse, Rockville,

MD.) were administered as an i.v. bolus at a dose of 1.2 mg/kg , 0.3 mg/kg and 0.6mg/kg respectively. These doses were chosen based on rodent studies with DAMGO (Gordon, 1986;

Randich etal, 1993), as well as preliminary dose ranging studies (0.3 - 0.9 mg/kg for DAMGO and

DALDA and 0.3 -1.2 mg/kg for U50,488H) carried out in our laboratory. Each animal was allowed a minimum of 3 days between drug studies and only received each drug treatment once. In some studies, naloxone (gift from the Dupont-Merck Pharmaceutical Co., Wilmington, DE) was infiised i.v. (0 .3 mg/kg/h) starting Ih prior to the administration of the opioid receptor agonist and maintained for a total of3h.

2.3.4 Drug treatments

To explore the hypothesis that DAMGO and U50,488H cause a blunting of the baroreflex, a challenge dose of norepinephrine ( 0 .6 pg/kg given over1 min) was administered to the ewe before and after the i.v. dose of U50,488H or DAMGO at the time of their peak pressor effect. The dose of norepinephrine was determined from preliminary dose range studies ( 0 .2 - 1 .0 pg/kg) in order to

47 produce a pressor response comparable in magnitude to that of U50,488H or DAMGO.

Norepinephrine was chosen for its short duration of action compared to phenylephrine. In addition, the dose of norepinephrine used in this study has been shown to produce similar blood pressure and heart rate responses to phenylephrine in the awake sheep (Magness and Rosenfeld, 1988), and to have no direct stimulatory actions on the heart (Magness and Rosenfeld, 1988; Assali et ah, 1981; Woods et al, 1977). Furthermore, the effects of comparable doses of norepinephrine were found to be completely abolished by the alpha antagonist, phenoxybenzamine (Oakes et al, 1980), eliminating the possibility of residual beta effects by norepinephrine at these doses.

To test whether the effects of U50,488H on heart rate involved an increase in sympathetic activity, propranolol was administered to the ewe by continuous i.v. infusion ( 0 .1 mg/kg for1 0 min starting

5 min before the U50,488H bolus).

2.4 Data analysis.

Blood pressure and heart rate were analyzed using 1 min intervals and control values were averaged over 5 min prior to the administration of DAMGO or U50,488H. Mean arterial pressure was calculated as the diastolic pressure plus 1/3 of the pulse pressure. For the baroreflex studies, measurements of baroreflex gain were carried out according to the ‘steady state’ method described by Komer and colleagues (Komer et al, 1974). Blood pressure and heart rate were determined every

48 5s and averaged over 20 seconds for a total of 6 consecutive minutes (2 min before and 4 min after norepinephrine administration). Maximum heart rate and blood pressure changes were observed

60-100s after onset of norepinephrine infusion and the baroreflex gain was calculated as the ratio of the decrease in heart rate to the rise in mean blood pressure (bpm/mmHg).

2.5 Statistical analysis

All data are reported as mean ± S.E.M. One-way analysis of variance (ANOVA) with repeated measure (factor = time) was used to examine the effects of U50,488H and DAMGO on blood pressure and heart rate. In cases where test of normality failed, ANOVA by ranks was used.

Dunnett's test was used for post-hoc comparison of each time point to the pre-drug control. The ability of propranolol to modify the heart rate response to U50,488H was evaluated using Friedman's two-way ANOVA with repeated measure (factors = treatment, time). The effects of norepinephrine on blood pressure and heart rate before and after the i.v. bolus of U50,488H or DAMGO was compared using paired t-tests. Differences were considered significant when P <0.05.

49 2.6 RESULTS

2.6.1 Effects of DAMGO in nonpregnant and pregnant animals.

Mean blood pressure and heart rate in nonpregnant animals prior to DAMGO administration were

81.2 ± 3.2 mmHg and 87.6 ± 1.3 bpm, respectively (n = 4). Intravenous administration of DAMGO

(0 .3 mg/kg) to the nonpregnant ewe resulted in an immediate increase in blood pressure with no change in heart rate (Fig. 8 ). The effect of DAMGO on mean blood pressure was highly significant

(P < 0.001), and post-hoc comparison revealed that mean blood pressure was significantly elevated compared to pre-drug values for up to 15 min. Pretreatment with naloxone completely blocked this pressor response to DAMGO (Fig. 9).

Basal mean blood pressure was lower (74.4 ± 1.8 mmHg) and heart rate was higher (99.8 + 4.0 bpm) in the pregnant animals (n = 4). The same dose of DAMGO (0.3mg/kg) significantly increased blood pressure (P < 0.01), and the effect lasted 11 min after injection (Fig. 8 ). However, the response in t pregnant animals was significantly less compared to nonpregnant animals (P = 0.01). This pressor response was completely blocked by naloxone pretreatment (Fig. 9). In the pregnant animals, there was a tendency for heart rate to decrease after DAMGO administration, although the changes did not reach statistical significance (fig. 8 ).

50 2.6.2 Effects of DALDA in nonpregnant and pregnant animals

In nonpregnant animals, control mean blood pressure and heart rate were 76.1 ± 1.3 mmHg and 78.8

±3.1 bpm, respectively (n= 5). The administration of DALDA resulted in a significant increase in

' mean pressure (P< 0.001), with peak effect occurring slightly later, and being mor-e-significantly

smaller, than after the same dose of DAMGO (Fig. 10). The peak increase in blood pressure was

only 32.2 ± 4% over control values. Heart rate did not change appreciably after DALDA

administration. The pressor effect to DALDA was also completely blocked by naloxone

pretreatment, fbata *ot

In pregnant animals, mean blood pressure increased from 70.3 ± 2.9 mmHg to 8 6 .1 ± 3.5 mmHg at

2 min, representing an increase of 18.1 ±3.1% which is significantly less than in nonpregnant animals

(Fig. 1 0 ). This increase in pressure was completely abolished by naloxone (results not shown).

2.6.3 Effects of U50,488H in nonpregnant and pregnant animals

Intravenous administration of U50,488H (1.2 mg/kg) to the ewe resulted in an immediate increase

in blood pressure in both nonpregnant and pregnant animals which was longer-lasting than that after

DAMGO or DALDA (Fig. 11). Mean pressure increased from 76.5 ±2.1 mmHg to 97.3 ± 4.7

mmHg (n = 4) in nonpregnant animals (n = 4) which lasted for 15-30min (P< 0.05) . Heart rate also

significantly increased after U50,488H (P < 0.001), This increase in pressor response was highly

51 significant and post-hoc comparison revealed that mean blood pressure was significantly elevated

compared to pre-drug values for up to 30 min. Both the blood pressure and heart rate responses were CbOfarttTl completely blocked by naloxone. In pregnant animals (n = 4), mean pressure increased from 72.6 ±

2.0 mm Hg to 98.0 ± 4.2 mmHg. Heart rate was significantly elevated after U50,488H, increasing

from 102.5 ±5.3 bpm to 135.7 ± 8.2 bpm, which is significantly greater than that seen in the nonpregnant animals. In contrast to DAMGO, DALDA AND U50,488H, the 5-agonist DPDPE

(0 .6 mg/kg) had no effect on blood pressure or heart rate (data not shown).

2.6.4 Effects of U50,488H and DAMGO on baroreflex gain.

U50,488H. Norepinephrine alone increased mean blood pressure by 17.0 ± 0.8 mmHg and

decreased heart rate by 19.4 ± 1.1 bpm. Following the administration of U50,488H, the same dose

of norepinephrine increased the mean blood pressure by 22.1 ± 0.6 mmHg and lowered the heart rate

by 27.7 ±2.6 bpm (Fig. 12). The calculated baroreceptor gain with norepinephrine was -1.2 ±0.1

bpm/mmHg before and -1.3 ±0.1 bpm/mmHg after the administration of U50,488H. Comparison

by the paired t-test showed there was no significant difference in the baroreceptor gain before and

after U50,488H.

DAMGO. Norepinephrine alone increased mean blood pressure by 15.0 ± 2.7 mmHg and decreased

heart rate by 20.3 ± 1.5 bpm. In the presence of DAMGO, norepinephrine increased mean blood pressure by 17.1 ± 2.2 mmHg and heart rate fell by 10.5 ± 1.6 bpm (Fig. 13). The calculated baroreceptor gain was -1.5 ± 0.2 bpm/mmHg before DAMGO and -0.6 ± 0.0 bpm/mmHg afterwards.

X 52 Baroreceptor gain was significantly attenuated in the presence of DAMGO (P < 0.05).

2.6.5 Effect of propranolol on the heart rate response to U50,488H.

Administration of propranolol alone had no effect on basal mean blood pressure and heart rate.

However, pretreatment with propranolol completely blocked the increase in heart rate following

U50,488H administration (Fig. 14). Two-way ANOVA confirmed that propranolol pretreatment significantly altered the heart rate response to U50,488H (P < 0.05 ).

53 Fig. 8 Effects of i.v. DAMGO (0.3mg/kg) on mean blood pressure (top panel) and heart rate (bottom panel) in nonpregnant and pregnant sheep (n = 4). Data are presented as means'S.E. A

O Nonpregnant # Pregnant

10 12 14 16 10 -,

0 ------

£ -10 -

c - 3 0 - O) ” - 4 0 -

- 5 0 -

-6 0

Time (min) À

3 0 -,

E 2 5 Q_ O Nonpregnant -Q 2 0 -J # Pregnant 3 0

S E E Q) 20 -j

O Nonpregnant

I # Pregnant 1 10 X sc E 0 O)< D C x:(C O Fig. 11 effects of i.v. U50,488H (1.2mg/kg) on mean blood pressure (top panel) and heart rate (bottom panel) in nonpregnant and pregnant sheep (n = 4). Data are presented as means"S.E.

O Nonpregnant o> # Pregnant

4 0 -

3 0 -

20 -

10 -

o>

0 2 4 6 8

4 0 - E CL

3 0 -

20 -

O) 10 -

-10

0 2 4 6 8 Time (min) Fig. 12 Effects of NE (0.6ggZkg/mm) on mean blood pressure and heart rate in the absence and presence of U50,488H (0.6mg/kg) in nonpregnant sheep (n = 5). Data are presented as means S.E.

140 -I BP, HR/-Wf0,4393 BP, HR/ +U5fei4®H

Q. 120 -

o 100 -

80 -

60

0 60 120 180 240 300 360 Time (s) Blood pressure (mmHg) or heart rate (bpm) 100 120 4 -| 140 0 - 60 aaaepeetda meansS.E. are presentedasData 0 - 80 in the absence and presence ofDAMGO (0.6mg/kg, n = 6).(0.6mg/kg, n= inthe and absence presenceofDAMGO Fig. 13 Effects of of (0.6gg/kg/min)on NE blood mean Effects and rate heart pressure 13Fig. - - 0 60 120 Time(s) 180 240 300 360 BP,HR/-:-D-ÂJM50; O A P R M.MCO BP,HR/ + # A

Change in heart rate (bpm) sheep (n = 4). Data are presented as f meansTS.E. presentedare as Data f sheep(n 4). = and presence of andof presencepropanoiol (0.1 mg/kg10over mins) innonpregnant Fig. 14 Effects of of U50,488H(1.2mg/kg) Effects on heart rateabsence inthe 14Fig. -10 20 0 2 4 Time (min) Time 6 8 0 2 4 16 14 12 10 + propanoiol+ - propanoiol-

2.6 DISCUSSION

The results of these studies demonstrate a clear difference not only in the cardiovascular response but also in the mechanisms involved in the heart rate response to systemically administered p- and k- opioid agonists. DAMGO, DALDA and U50,488H produced significant increases in blood pressure but had different effects on heart rate. The p-agonists had no effect on heart rate while the k- agonist, U50,488H caused a significant tachycardia. The pressor effects of all three agonists and tachycardia elicited by U50,488H were sensitive to naloxone. The results of the norepinephrine challenge suggest that DAMGO significantly attenuated baroreflex sensitivity, thus explaining the lack of a reflex bradycardia despite a significant increase in blood pressure. In contrast, U50,488H did not alter baroreflex sensitivity, and the accompanying tachycardia is mediated by sympathetic activation of the heart as demonstrated by its sensitivity to propranolol.

The results with DAMGO are different from those previously reported. Gordon (1986) reported that i.v. DAMGO produces a profound reduction in arterial pressure and heart rate in urethane- anesthetized rats which is naloxone-sensitive. Randich et al. (1993) also found a dose-dependent decrease in arterial pressure and heart rate in pentobarbital-anesthetized rats. In contrast to these studies, the present study found a significant increase in arterial pressure after i.v. DAMGO administration. Since the doses of DAMGO used in the rodent studies were in the same range as the present study, this discrepancy is most likely due to the use of anesthesia in the rat studies. In

61 conscious sheep, i.v. administration of FK33-824, another p-selective agonist, had no effect on

arterial pressure (Wang et al, 1989), thus suggesting that the hypotensive action of p-opioid agonists

may be due to the presence of anesthesia.

A pressor response has been reported after administration of DAMGO into the CNS, however, the

extent of distribution of these peptides into the CNS after i.v. administration is thought to be very

limited. A recent study showed that only 0.08% of an i.v. dose of [D-Pen 2,D-Pen5]-enkephalin

(DPDPE), another pentapeptide, was found in the brain at 60 min (Weber et al, 1992). In addition,

in the present study, i.v. administration of DALDA (Tyr-D-Arg-Phe-Lys), which is extremely polar

in nature (Schiller et al, 1989), also results in a rapid elevation of arterial pressure. There is evidence

that the distribution of DALDA across the blood-brain barrier is very slow (Samii et al, 1994) and

only very weak activity could be detected in the mouse hot plate test l- 2 h after subcutaneous

administration (Schiller et al, 1990). It is therefore likely that these p-selective peptide analogs may

also produce a pressor effect via peripheral mechanisms. The idea of a peripheral mechanism is

supported by a recent report which demonstrated that DAMGO exerts direct vasoconstrictive action

on vascular smooth muscle as well as potentiating the vasoconstrictive action of norepinephrine

(Parra ef a/., 1995).

The pressor response to systemic administration of vasoconstrictive agents is known to be

62 accompanied by a baroreflex-mediated bradycardia (Oakes et al, 1980; Magness and Rosenfeld,

1988; Assali et al, 1981; Woods et al, 1977) and this was confirmed in the present study with

norepinephrine. In contrast, there was no change in heart rate in the presence of a comparable

pressor response to DAMGO. This suggests that DAMGO may be inhibiting the baroreflex and this

was confirmed by the inhibition of the baroreflex response to norepinephrine. Considerable evidence

shows that baroreflex activity can be influenced by central administration of opioid peptides (see

review, (Gordon, 1994). These studies have generally reported a decrease in reflex-mediated

bradycardia provoked by increases in arterial pressure (Numao et al, 1985; Petty and Reid, 1982;

Schaz et al, 1980; Yukimura et al, 1981) with the exception of Matsumura et al. (1992)

(Matsumura et al, 1992) who found enhanced rather than reduced baroreflex sensitivity in rabbits

after intracistemal administration of DAMGO. Others have also reported a decrease in sympathetic

nerve activity in response to opioid peptides (Gordon, 1986; Numao et al, 1985). These studies

suggest that opioids depress baroreflex activity through a central mechanism most likely at the level

of the NTS. Microinjection of DAMGO into the NTS, was found to reduce the increase in blood

pressure and heart rate that occurred with bilateral carotid occlusion (Hassen and Feuerstein, 1987).

Other studies found that aortic baroreflexes were significantly attenuated when morphine (Wang and

Li, 1988) or DAMGO (Gordon, 1990) was microinjected into the NTS of rabbits or rats, respectively. Furthermore, opioid-induced baroreflex inhibition was completely reversed by NTS injections of the opioid receptor antagonist naloxone.

63 The precise location and mechanism by which systemic administration of p-receptor peptide analogs attenuate the baroreflex are not known, but it is likely to be mediated by a peripheral mechanism, p opioid receptors located on vagal afférents (Zarbin et al, 1990) may be responsible for the mediation of DAMGO effects on the baroreflex. It is also possible that DAMGO causes an attenuation of baroreflex sensitivity by modifying the transmission of impulses in efferent pathways, resulting in modulation of vagal or cardiac sympathetic activity such as inhibition of acetylcholine release by the vagus which would in turn inhibit baroreflex-mediated bradycardia. Opioid receptors have been identified in various parts of the periphery such as the vagus nerve (Young et al, 1980;

Zarbin et al, 1990), sympathetic nerve terminals in the heart and blood vessels (Mantelli et al, 1987) and the sympathetic lateral columns of the spinal cord (Goodman et al, 1980; Romagnano and

Hamill, 1984). The precise location and mechanism by which i.v. DAMGO inhibits the baroreflex remain to be investigated.

Relatively few studies have examined the effects ofU50,488H on blood pressure and heart rate and the mechanisms involved. Some studies suggest that K-opioid agonists have no effect on blood pressure (May et al, 1989; Bachelard and Pitre, 1995) while others reported hypotension (Hall et al,

1988; Pugsley et al, 1992). Anesthesia was employed in the majority of these studies with the exception of Carter and Lightman (1985) who administered U50,488H into the NTS of conscious adult rats and found it to produce a significant pressor effect. In the present study, there was clearly

64 a significant increase in mean pressure in response to the administration of U50,488H. The tachycardia elicited, instead of the expected reflex bradycardia in response to the pressor effect of

U50,488H, led to the investigation of its effect on baroreflex sensitivity. In previous studies, injections of K-opioid agonists into the cistema magna of anesthetized rabbits have been reported to increase the sensitivity of baroreflex control of heart rate in response to a rise in arterial pressure, while reducing reflex sensitivity when arterial pressure was lowered (Petty and Reid, 1982). In the anesthetized adult rat, the pressor effect of U50,488H was accompanied by a decrease in heart rate, consistent with a baroreflex response (Carter and Lightman, 1985). In the present study, it was found that U50,488H had no effect on the heart rate response to a challenge dose of norepinephrine indicating that K-opioid agonists do not inhibit the baroreflex in unanesthetized animals. The observed tachycardia further suggests that there is an increase in sympathetic activity, and this was confirmed by the complete reversal of the tachycardia by propanoiol. Since U50,488H, unlike

DAMGO, is highly lipid soluble, the increase in blood pressure and heart rate is most likely due to a centrally-mediated increase in sympathoadrenal outflow leading to an increase in vascular resistance and cardiac output which in turn causes an increase in blood pressure.

In summary, these data show that systemic administration of p - and k - opioid agonists in the awake animal results in cardiovascular effects that are different from the majority of those reported with centrally administered agonists and those carried out in the presence of anesthesia. Furthermore, the

65 effects of these agonists on heart rate appear to be mediated via different mechanisms. Effects of k- opioid agonists on heart rate clearly involve sympathetic activation of the heart while p-opioid agonists significantly attenuate baroreflex-mediated bradycardia via a peripheral mechanism which warrants further investigation.

Basal blood pressure was reducedm pregnancy and heart rate significantly elevated confirming previous reports on cardiovascular changes in pregnancy (Brooks and Keil,1994c; Clapp et al., 1988; Keller Wood, 1994b). The effects of DAMGO, DALDA and U50,488H were found to be different when compared to nonpregnant animals. The pressor response to DAMGO and DALDA was significantly attenuated in pregnancy while pregnant animals had a higher tachycardia in response to U50,488H.

66 CHAPTER 3

THE SITE AND MECHANISM OF ACTION RESPONSIBLE FOR

THE ATTENUATION OF BAROREFLEX - MEDIATED

BRADYCARDIA BY INTRAVENOUS ^-OPIOID AGONISTS

67 3.1 ABSTRACT

As previously reported, DAMGO (Tyr-D-Ala-Gly-NMePhe-Gly-ol) and DALDA (Tyr-D-Arg-Phe-

Lys) caused an increase in blood pressure with no accompanying reflex bradycardia and it was

consequently demonstrated that DAMGO attenuates baroreflex-mediated bradycardia in chronically instrumented and unanesthetized sheep. In order to assess the site and mechanism by which p- agonists cause an attenuation of baroreflex sensitivity, further investigations were carried out using the highly polar p-agonist DALDA, which has been shown to have a limited ability to cross the blood brain barrier. Furthermore, as the long term goal of the laboratory has been to develop these peptides for clinical obstetrical use, the effects of these peptides on baroreflex sensitivity in pregnancy were also investigated. The reflex bradycardia to norepinephrine (0.4 - 0.6pg / min) was significantly blunted in the presence of both DAMGO and DALDA in pregnant and nonpregnant animals. In view of the limited ability of DALDA to get across the blood brain barrier, it was hypothesized that the blunting of baroreflex-mediated bradycardia by p-agonists occurs at a peripheral site. Pretreatment with the quaternary opioid antagonist, naloxone methiodide (0.4 - 0.6 pg/kg/h) completely blocked the attenuation of baroreflex sensitivity by DAMGO and DALDA in both nonpregnant and pregnant animals. In order to investigate the baroreceptors as a possible site of action as well as address the possibility of these drugs exacerbating hemorrhage-induced hypotension in labor and delivery, the effect of both p-agonists on baroreflex-mediated tachycardia to a hypotensive stimulus was investigated. The reflex tachycardia to sodium nitroprusside (10 pg/kg/min) remained unchanged in the presence of DAMGO or DALDA in both nonpregnant and pregnant animals. These data

68 suggests that the blunting of baroreflex-mediated bradycardia by DAMGO and DALDA has a peripheral site and mechanism. Furthermore, the lack of any effect on baroreflex-mediated tachycardia indicates these p-agonists are not acting at the level of the baroreceptors.

69 3.2 INTRODUCTION

The laboratory has been investigating the potential use of p and ô-selective opioid peptide analogs

for pain control during labor and delivery, therefore an understanding of the peripheral effects of

opioids on baroreflex control is important for the use of these p-selective opioid peptide analogs as

clinical analgesic agents. This, coupled with the importance of baroreflex control during labor and

delivery, and the relative lack of information on the effect of p-opioid peptide agonists during

pregnancy, led to an extension of the investigations into the cardiovascular role of DAMGO and (cUupT

Ala-Gly-NMePhe-Gly-ol) resulted in a rapid increase in blood pressure with no significant change in

heart rate in awake sheep (Omoniyi et al., 1998b). The lack of a baroreflex-mediated bradycardia in the presence of a significant pressor response suggested that these p-opioid agonists must inhibit the baroreflex, although the site and mechanism were not understood. Previous work by numerous investigators have shown that opioids can inhibit the baroreflex, and it is generally thought to be mediated by opioid receptors in the central nervous system (CNS), especially those in the nucleus tractus solitaiius (NTS) (Gordon, 1986). However, a similar pressor effect without bradycardia was observed with the i.v. administration of DALDA (Tyr-D-Arg-Phe-Lys-NH2), a p-selective opioid agonist that is highly polar because of its 3+ charge at physiologic pH. This led to the suspicion that these synthetic opioid peptides may also inhibit the baroreflex via a peripheral mechanism.

To test the hypothesis that DAMGO and DALDA cause a blunting of the baroreflex, heart rate

70 responses to changes in blood pressure using norepinephrine (NE) and sodium nitroprusside (SNP) were determined before and after i.v. administration of DAMGO or DALDA in both nonpregnant and pregnant sheep. In addition, the quartenary naloxone analog, naloxone methiodide, was used to demonstrate that these peptide analogs suppress the baroreflex via peripheral mechanisms.

71 3.3 MATERIALS AND METHOD

Surgical procedure. See 2.3.1

Experimental Protocol. See 2.3.2

3.3.1 Drug treatments

DAMGO and DALDA were administered as an i.v. bolus at a dose of 0.6 mg/kg, and 0.3mg/kg

respectively. These doses were chosen based on dose- ranging studies (0.3 - 0.9 mg/kg) with both

agonists carried out in our laboratory (Szeto et al., submitted for publication). Each animal was

allowed a minimum of 2 days between drug studies and only received each drug treatment once. In

some studies, naloxone methiodide (provided by NDDA and supplied by Research Triangle Institute,

Research Triangle Park, NC) was infused i.v. (0.6mg/kg/h) starting Ih prior to the administration of

DAMGO or DALDA and maintained for a total of 1.5h.

To test the hypothesis that DAMGO and DALDA cause a blunting of baroreflex function, blood pressure challenges using ME (0.6 pg/kg for DAMGO and 0.4 pg/kg for DALDA studies) or SNP

(10 pg/kg) were administered to the ewe to produce hypertensive or hypotensive pressor stimuli respectively. Thirty minutes later, an i.v. dose of DAMGO (0.6mg/kg) or DALDA (0.3mg/kg) was administered, and the pressor challenges repeated to coincide with the peak pressor effect of each p-opioid agonist. The doses of NE used were determined from preliminary dose range studies (0.2 -

72 1.Ong/kg) in order to produce a blood pressure response comparable in magnitude to that observed

with each opioid agonist. The dose of SNP used was based on previous studies in the adult sheep

(Keller-Wood, 1994a). Both NE and SNP were administered over 1 min by the use of an infusion

pump. Norepinephrine was chosen over phenylephrine for the pressor challenge due to its shorter

duration of action compared to phenylephrine. In addition, the doses of NE used in this study have been shown to produce similar blood pressure and heart rate responses to phenylephrine in the adult

sheep (Magness and Rosenfeld, 1988), and to have no direct stimulatory actions on the heart

(Magness and Rosenfeld, 1988; Assali et al, 1981; Woods et al, 1977). Furthermore, the effects of comparable doses of NE were found to be completely abolished by the alpha antagonist, phenoxybenzamine (Pfeiffer et al, 1982), eliminating the possibility of residual beta effects by NE at these doses.

3.3.2 Data analysis

Arterial blood pressure and heart rate were monitored continuously throughout the study. Mean arterial pressure was calculated as the diastolic pressure plus 1/3 of the pulse pressure. Blood pressure and heart rate responses to the NE and SNP challenges were determined using 10 sec intervals for a total of 220 consecutive seconds (120 secs before and 100 secs after NE or SNP administration). Maximum heart rate and blood pressure changes were observed 60-100s after onset of drug infusion. The slope of the regression line representing the change in heart rate (bpm) versus change in blood pressure (mmHg) was used as an index of baroreflex sensitivity. Because of the delay

73 in heart rate responses to changes in blood pressure, heart rate was plotted against the preceding

blood pressure change according to the ‘ramp’ method of baroreflex sensitivity assessment (Smythe

etal, 1969).

3.3.3 Statistical analysis

Regression lines representing the responses to NE and SNP, as well as responses to NE and SNP in the presence of DAMGO or DALDA, were plotted for each animal. The slopes of the regression lines for each animal before and after opioid agonist treatment were compared using paired t-tests.

Differences were considered significant when P < 0.05.

74 3.4 RESULTS

3.4.1 Baroreflex sensitivity in nonpregnant and pregnant animals

Fig 15 illustrates the initial differences in baroreflex sensitivity in response to a hypertensive (NE, top panel) and hypotensive (SNP, bottom panel) stimulus in a pregnant and nonpregnant animal.

Baroreflex sensitivity in response to the NE stimulus was significantly enhanced in pregnant animals compared to nonpregnant animals (see Table 3). In contrast, reflex sensitivity was significantly attenuated in pregnant animals in response to the hypotensive (SNP) challenged t^o-o5)

3.4.2 Effects of DAMGO and DALDA on NE challenge in nonpregnant animals and pregnant animals

Fig. 16 summarizes the mean blood pressure and heart rate data before and after DAMGO (top panel) or DALDA (bottom panel) in a representative nonpregnant animal while Fig. ITsummarizes the blood pressure and heart rate data before and after DAMGO (top panel) or DALDA (bottom panel) in a representative pregnant animal. The baroreflex response to the NE challenge was significantly attenuated in the presence of DAMGO or DALDA in both nonpregnant and pregnant animals (Table 3).

3.4.3 Effects of DALDA on SNP challenge in nonpregnant and pregnant animals .

In contrast to the NE challenge, baroreflex sensitivity to the hypotensive challenge of SNP remained

75 unchanged in the presence of DALDA as illustrated by a representative nonpregnant (Fig. 18, top

panel) and pregnant animal (bottom panel). DALDA had no effect on the heart rate response to SNP in either nonpregnant or pregnant animals (see table 3). Baroreflex sensitivity also remained unchanged in the presence of DAMGOC^cd4 ncf* -skouon) •

3.4.4 Effect of naloxone methiodide on the heart rate response to NE in the presence of

DAMGO and DALDA (nonpregnant animals).

The attenuation of the heart rate response to the NE stimulus observed with DAMGO and DALDA was completely blocked by naloxone methiodide (NMI) in nonpregnant animals (Table 3). Figure 19 illustrates the blood pressure and heart rate data for a representative animal after the administration of DAMGO (top panel) and DALDA (bottom panel) in the absence and presence of NMI.

3.4.5 Effect of naloxone methiodide on the heart rate response to NE in the presence of

DAMGO and DALDA (pregnant animals).

The attenuation of the heart rate response to the NE stimulus observed with DAMGO and DALDA was also completely blocked by naloxone methiodide (NMI) in pregnant animals (Table 3). Figure

20 illustrates the blood pressure and heart rate data for a representative animal after the administration of DAMGO (top panel) and DALDA (bottom panel) in the absence and presence of NMI.

76 TABLE 3 Baroreflex responses to hypertensive (NE) and

hypotensive (SNP) challenges

NONPREGNANT CONTROL + DRUG STUDY NP/ PREGNANT P beats/min/mmHg beats/min/mmHg

NE (hypertensive) NP -0.81 ±0.22 -

P -2.08 ±0.40** -

SNP (hypotensive) NP -2.77 ±0.48 -

P -0.53 ±0.04** - DAMGO + NE NP -1.10±0.35 -0.44 ±0.08* P -2.18 ±0.48 -0.65 ±0.17* DALDA + NE NP -1.03 ±0.30 -0.38 ±0.02* P -2.26 ±0.39 -0.94 ±0.27* DALDA + SNP NP -2.10 ±0.45 -1.73 ±0.36 P -0.86 ±0.18 -0.96 ±0.20 DAMGO + NE+NM NP -1.24 ±0.32 -0.98 ±0.21 P -2.60 ±0.42 -2.95 ±0.48 DALDA + NE + NM NP -1.77 ±0.40 -1.73 ±0.36 P -2.50 ±0.40 -2.00 ±0.36 NE:Norepinephrine NM: Naloxone Methiodide SNP: Sodium Nitroprusside *P < 0.02 when compared to control values. **P < 0.05 when compared to nonpregnant animals. N = 4 to 8 animals per study group.

77 ina nonpregnant andpregnant sheep toa hypertensive (NE,top panel) andhypotensive bottom(SNP, panel) stimulus i.1 Relationshipbetween meanblood 15 pressureFig.andresponserate in heart A.

Tachycardie Response (bpm/mmHg) Bradycardic Response (bpm/mmHg) ■ 4 - ■ 3 - -2 -1 0-- 2 3 1 -j 0 - - - - - • •

Nonpregnant ] Pregnant a

Fig. 16 Relationship between mean blood pressure and heart rate in response to NE in the absence or presence of DAMGO (A) or DALDA (B) injionpregnant sheep.

i 1 -D A M G O — +D A M G O

i 1 -D A LD A ■ ■ + DALDA

9 1 Fig. 17 Relationship between mean blood pressure and heart rate in response to NE in the absence or presence of DAMGO (A) or DALDA (B) in pregnant sheep.

E5553 -DAMGO ■ ■ +D A M G O

F5553 -DALDA ■ ■ +DALDA

SO Fig. 18 Relationship between mean blood pressure and heart rate in response to SNP in the absence or presence of DALDA in a nonpregnant (A) or pregnant (B) sheep.

-DALDA +DALDA

-D A LD A ■ ■ + DALDA

8 1 presence of presenceof (A) or DAMGO(B) DALDAanonpregnant in sheep. pressureand heart rate response in toNEor intheabsence Fig. 19 The effect of of on effect NMI Therelationship the betweenblood19mean Fig. A.

Bradycardic Response (bpm/mmHg) Bradycardic Response (bpm/mmHg) -2 3 2 1 0 1 o - 2 8 +MI + DA LD A +D I, +NM 3 Z Z C ——1 D DA A LD -DA 1 — r— DAMGO O G M A -D N , DAMGO G M A +D I, +NM

Fig. 20 The effect of NMI on the relationship between mean blood pressure and heart rate in response to NE in the absence or presence of (A) DAMGO or (B) DALDA in a pregnant sheep.

A.

CL 1

CL 2 V)

•3

E3533 -DAMGO 4 n m +NMI, +DAMGO

B.

0 I I CL 1 .O Ï 2

1 .2 E ■3 cessa -DALDA p-771 +NMI, +DALDA Ico 4

S 3 3.6 DISCUSSION

There is considerable evidence to suggest that baroreflex activity can be influenced by opioid agonists

(Gordon, 1994). Numerous investigators have studied the effect of centrally administered opioids on baroreflex function and generally reported a decrease in baroreflex-mediated bradycardia in response to increases in arterial pressure (Numao et ah, 1985; Petty and Reid, 1982; Schaz et al., 1980;

Yukimura et al, 1981). These inhibitory effects on baroreflex function have most often been attributed to activation of central p-opioid receptors because agonists relatively selective for ô - or k- opioid receptors either had little or no effect on baroreflex function (Gordon, 1986; Matsumura et al,

1992; Numao et al, 1985; Petty and Reid, 1982). In the previous study, it was also found that the

K-opioid agonist, U50,488H, had no effect on the heart rate response to NE in nonpregnant sheep

(Omoniyi et al, 1998b).

The results of the present study demonstrate for the first time that systemically-administered p-opioid peptide analogs can significantly alter baroreflex sensitivity in both nonpregnant and pregnant sheep.

Interestingly, both DAMGO and DALDA blunted the baroreflex-mediated bradycardia to pressor stimuli, but had no effect on the baroreflex-mediated tachycardia to a hypotensive stimulus. These results also suggest that, in addition to opioid receptors in the CNS, peripheral opioid receptors may also play an important role in inhibiting the baroreflex.

The inhibition of baroreflex function by p-opioid agonists has generally been thought to be mediated

84 by opioid receptors in the CNS, particularly at the level of the NTS (Gordon, 1990; Hassen and

Feuerstein, 1987; Wang and Li, 1988). This is consistent with the presence of endogenous opioid

peptides and opioid receptors in this area of the CNS (Goodman et ah, 1980; Kalia et al, 1984).

Hence, several investigators have studied effects of centrally administered opioids on baroreflexes and

have generally, reported a decrease in baroreflex-mediated bradycardia (Numao et al, 1985; Petty

and Reid, 1982; Schaz et al, 1980; Yukimura et al, 1981) in response to increases in arterial pressure. In contrast, relatively few studies have addressed the effects of systemically-administered opioid agonists or the possibility of peripheral opioid-receptor mediated effects on baroreflex function.

This is generally because opioid peptide analogs are thought to have difficulty crossing the blood brain barrier to any appreciable extent. In our studies however, the rapid onset of the response to DAMGO led us to believe that its actions were probably peripherally-mediated. This is further supported by the lack of a bradycardia to the pressor response induced by DALDA. DALDA is a potent and highly selective p-agonist that is very polar because of its 3+ charge at physiologic pH (Schilleret al, 1989), and the distribution of DALDA across the blood brain barrier has been shown to be highly restrictive

(Samii et al, 1994; Schiller et al, 1990). This study demonstrates that DALDA like DAMGO, blunted the heart rate response to pressor stimuli. In addition, the effect of DAMGO and DALDA on baroreflex function can be completely antagonized by i.v. administration of naloxone methiodide, providing support for the hypothesis that peripheral mechanisms are also involved in the modulation of baroreflex function by opioids.

85 The heart rate response to pressor agents is determined by the underlying autonomic tone, so that the predominance of the sympathetic or parasympathetic tone over one another will determine the direction of the heart rate response to pressor stimuli. Previous studies on the autonomic control of heart rate in the adult sheep demonstrated that the conscious ewe is under parasympathetic dominance hence, the heart rate response to the pressor stimulus of agents such as NE is accompanied by a baroreflex-mediated bradycardia (Oakes et ah, 1980; Magness and Rosenfeld, 1988; Assali et ah,

1981; Woods et ah, 1977). This can be contrasted with the sheep fetus which is under sympathetic dominance and therefore responds to the pressor stimulus of NE with a tachycardia (Woods et ah,

1977; Assali N.S. et ah, 1977).

The specific site and mechanism by which systemic administration of p-opioid agonists attenuate the heart rate response to pressor stimuli remain to be determined. Baroreceptors located in the carotid sinus and aortic arch are responsible for the detection of changes in arterial pressure, resulting in afferent impulses to the NTS. The lack of any effect on the heart rate response to a hypotensive stimulus suggests DAMGO and DALDA are not suppressing baroreceptor fimction at the level of the baroreceptors. It is possible that DAMGO and DALDA are acting to modulate the efferent signal from the NTS to the heart, resulting in an attenuated bradycardic response to a pressor stimulus. A direct inhibition of vagal activity would explain the lack of effect on heart rate response to a hypotensive stimulus which is instead dependent on cardiac sympathetic nerve activity. This hypothesis is supported by previous reports that morphine and enkephalins inhibit the bradycardia

86 elicited in response to vagal stimulation (Musha et ah, 1989; Weitzell et al., 1984), as well as the

release of acetylcholine from rabbit atria (Weitzell et al, 1984). In addition, p-opioid receptors have

also been demonstrated on vagal nerves (Young et ah, 1980; Zarbin et ah, 1990). Although the lack

of a bradycardia may also be due to enhanced sympathetic activity, and p-opioid receptors have been identified on sympathetic nerve terminals in the heart (Mantelli et al., 1987) and sympathetic lateral columns of the spinal cord (Goodman et ah, 1980; Romagnano and Hamill, 1984), this is unlikely since the tachycardie response to SNP was not enhanced by DAMGO or DALDA

This is the first report on the effects of opioid agonists on baroreflex sensitivity in pregnancy.

Pregnancy is known to be associated with marked changes in the cardiovascular system at rest.

Cardiac output, heart rate and blood volume increases, whereas systemic vascular resistance and arterial pressure decreases (Weinstock and Rosin, 1984; Oakes et ah, 1980). In addition to these changes in the cardiovascular system of the pregnant animal, other studies have also reported a state of altered baroreflex sensitivity. However, there is some conflict among investigators regarding the direction of change probably arising from differences in the methods employed, animal species studied, and the indices used for assessment of baroreflex sensitivity. Hence, attenuated baroreflex sensitivities were reported for smaller increases in maternal pressure and peripheral resistance produced by decreases in pressure in the isolated carotid sinus of anesthetized pregnant rabbits

(Omoniyi et ah, 1998b), as well as smaller reflex increases in lumbar sympathetic activity in anesthetized pregnant rats (Cox and Bagshaw, 1979). In contrast, bradycardic responses to increases

87 in arterial pressure, were found to be enhanced in pregnancy (Conrad and Russ, 1992; Magness and

Rosenfeld, 1988). Other indices used for measurement of baroreflex sensitivity include baroreflex-

mediated changes in renal sympathetic activity as well as reflex-mediated changes in the level of plasma hormones such as vasopressin, adrenocorticotropic hormone, cortisol and angiotensin II. The use of anesthesia in some of these studies is also a potential source of conflict as demonstrated by reports on anesthetic effects on baroreflexes (Cox and Bagshaw, 1979; Murthy et al., 1982). The present study revealed pregnant animals had an enhanced heart rate response to the norepinephrine stimulus when compared to nonpregnant animals. In contrast, the heart rate response to SNP was significantly attenuated in pregnancy. This is consistent with a previous report by Brooks et al.,

(1995) (Brooks et al, 1995) which demonstrated the same phenomenon in pregnant, conscious dogs.

Hemorrhage-induced hypotension is a normal consequence of every delivery, therefore any potential use of these peptides in clinical obstetrics would be greatly compromised by deleterious effects on baroreflex control. The present study showed that DAMGO and DALDA also inhibited the heart rate response to NE in pregnant sheep, and the data suggests that the magnitude of inhibition was greater X in pregnant animals. The enhanced baroreflex sensitivity to the pressor stimulus in pregnancy observed in this study, may explain why DAMGO and DALDA caused a higher degree of blunting in pregnant animals, as there was a greater potential for suppression of reflex sensitivity when compared to the nonpregnant animals. Fortunately, neither DAMGO nor DALDA inhibited the heart

88 rate response to a hypotensive challenge, thus ruling out the risk of these peptides compromising baroreflex control in delivery.

In summary, these data demonstrate for the first time that systemically-administered p-opioid agonists suppress baroreflex-mediated bradycardia while having no effect on reflex-tachycardia in nonpregnant and pregnant sheep. In addition, the suppression of baroreflex-mediated bradycardia by p-agonists occurs via a peripheral mechanism. The specific site and mechanism by which p-opioid peptide analogs attenuate baroreflex sensitivity remains to be elucidated.

89 CHAPTER 4.

THE DIRECT EFFECTS OF ji-OPIOID AGONISTS ON THE

ISOLATED AND PERFUSED GUINEA PIG HEART.

90 4.1 ABSTRACT

The mechanism behind the pressor responses previously reported for DAMGO and DALDA was investigated in vitro using the isolated guinea pig heart. The investigation was based on the hypothesis that the increase in blood pressure observed with DAMGO and DALDA in vivo is as a result of a direct effect of these p-agonists on the contractility of the heart. Pilot studies revealed conflicting results regarding the inotropic effects of DAMGO and DALDA. It was further hypothesized that the pre-existing condition of the heart may determine the inotropic effect of mu- agonists on the heart. Hence the effects of DAMGO and DALDA on the isolated and perfused guinea pig heart were further investigated under four different conditions, two of which were "stress" paradigms: (1) Spontaneous heart rate (2) Elimination of chronotropic Punctuations by electrical pacing of the heart (3) Low flow ischemia induced by rapid electrical pacing (4) and following 30- mins of no flow Ischemia . DALDA (10"11 - 10"9M) caused a dose dependent decrease in the force of contraction, heart rate and coronary flow in spontaneously beating hearts. There was a decrease in the force of contraction, coronary flow and heart rate at 10*nM DAMGO while subsequent doses

(10*9- 10'7M) resulted in increases of all three parameters. Elimination of chronotropic effects with electrical pacing at spontaneous rate unmasked dose-dependent negative inotropic effects for both

DAMGO and DALDA (10 " - 10"7M) that were bigger in magnitude than those observed in the spontaneously beating hearts. In hearts stressed by ischemia or rapid pacing, administration of

DAMGO or DALDA (109 - 10'7M) resulted in a positive inotropic response accompanied by a decrease in heart rate and coronary flow. Both the negative and positive inotropic effects of DAMGO

91 and DALDA where attenuated by the opioid receptor antagonist naloxone (10'5 M). The negative -tketlr inotropic effects of p-opioid receptor agonists in the "unstressed" heart can be influenced by'its-

chronotropic effects. In hearts stressed by ischemia or rapid pacing, DAMGO or DALDA improve contractile performance of the heart restoring it to its pre-stressed state . The present study demonstrates that the inotropic effects of DAMGO and DALDA on the isolated heart depend on the pre-existing condition of the heart.

92 4.2 INTRODUCTION

The inotropic effects of p, ô and K-opioid agonists have been investigated in a variety of cardiac

systems, however there is disagreement regarding the direction of change, ô- and k- but not p - agonists were found to decrease the contractility of rat (Ventura et al., 1992) and hamster (Kasper et al, 1992) ventricular myocytes. In human atria strips, p, ô and K-opioid agonists produced (Llobell and Laorden, 1995) negative inotropic effects and while the K-opioid agonist U50,488H produced negative inotropic effects in the isolated rat heart (Xia et al., 1994), it was found to increase contractility in ventricular myocytes (Ventura et al., 1992; Tai et al., 1992). The naturally occurring

ô-opioid agonists leucine- and methionine-enkephalin were also found to produce positive inotropic effects in chick embryo ventricular cells (Laurent et ah, 1985; Laurent et ah, 1986). The majority of these studies involved the use of high agonist doses of up to lO^M and this in addition to a relative lack of receptor selectivity of most of the agonists studied can make it difficult to determine the receptor-specific effects of opioid agonists on inotropy.

There are relatively few studies demonstrating the direct inotropic effects of opioid agonists on the heart itself. While the use of isolated myocytes is more useful than multicellular cardiac preparations for the purpose of mechanistic studies, the varying conditions under which these studies are carried out creates inconsistencies that may affect the inotropic response of the cardiac cell making it difficult to scale the findings up to the level of the heart. Furthermore, the contractile state and hence inotropic response of the myocardium can be altered by a variety of conditions. In addition,

93 myocardial changes such as chronotropic effects can have confounding effects on the direction of inotropic changes. In the present study, the effects of the highly potent and selective p-opioid agonists DAMGO and DALDA are compared under different inotropic and chronotropic conditions of the heart.

94 4.3 MATERIALS AND METHODS

4.3.1 Isolated heart perfusion

Female Hartley guinea pigs weighing 350 to 400 g were sacrificed by cervical dislocation under light

anesthesia with C02 vapor. The rib cage was rapidly dissected away, exposing the heart. A cannula

was inserted into the aorta and the heart was perfused at constant pressure (40 cm of H20) with

Krebs-Henseleit solution equilibrated with 95% 0 2 and 5% C02 and maintained at 32°C (pH 7.4).

The composition of the solution was as follows (in millimolar amounts) : NaCl, 118.1; KC1, 4.8; CaCl2

2.5;MgS04 2.4; KHfO,, 1.0; NaHC03, 25.0; and glucose, 11.1. The heart was then transferred to

a Langendorff apparatus, where it was suspended by the aortic cannula and allowed to beat

spontaneously. The sinus rate was determined from surface electrograms recorded from the right

atrium and left ventricle and recorded at a paper speed of 25mm/sec on a polygraph (Gould model

2400). A resting tension of Ig was applied and isometric left ventricular contractile force was

measured with a force-displacement transducer (Grass FT03C) and continuously recorded on a Grass

polygraph (model 78). A period of thirty minutes was allowed for stabilization of the heart rate and

contractile amplitude before drug treatments were applied. For the paced hearts, once mounted, the

hearts were continuous paced using bipolar platinum electrodes and stimulated with a Grass stimulator

(model S44) at 2.5Hz with square wave pulses, 0.4 msec in duration and of supramaximal intensity

(1IV). The frequency of pacing was increased to 5Hz for hearts subjected to rapid pacing. For the

ischemia-reperfusion studies, following a 30-min pre-ischemic stabilization period, global ischemia was induced by 30-min of complete interruption of coronary perfusion. This was followed by a

95 reperfusion period of 1 hour.

Coronary flow waa measured by collecting timed samples of the coronary effluent from the heart.

96 4.3.2 THE LANGERDORFF SETUP

OXYGENATED RESERVOIRS

EL.

TO OVERFLOW

BUFFER IN

l-WAY SWITCH

HEAT EXCHANGER-

TO RESERVOIRS

OXYGENATED *■ BUFFERS ISOLATED TO PUMP HEART

TRANSDUCER-

TIMED CORONARY EFFLUENT COLECTIONS-

PUMP WATER BATH (37°C)

97 4.3.3 Drug treatments

"Non-stressed hearts". Following the stabilization period, hearts were perfused with DAMGO or

DALDA in increasing doses of 10*n , 10'9 and 10‘7M. Each dose was administered for 10 min followed by a 10 min recovery period during which the heart was perfused with Krebs-Henseleit buffer alone followed by the next dose. In order to eliminate confounding chronotropic effects, dose response studies were conducted in hearts paced at spontaneous rate using the same protocol. Only one dose response study was carried out per heart.

Arrhythmic hearts. In early pilot studies, several hearts developed arrhythmias due to poor coronary perfusion caused by insufficient hydrostatic pressure. Dose response studies were carried out in these hearts with DALDA at increasing doses from 10*7 to 10"5Musing the same protocol as described above.

"Stressed hearts". Following the stabilization period of 30 min, hearts were subjected to either one of two stress paradigms: Rapid pacing at twice the spontaneous rate or complete cessation of coronary perfusion, with each lasting for a period of 30 min. The hearts were allowed a further 10 min to recover spontaneous rate and contractile amplitude after the completion of the stress paradigm before drug administration. Due to the time-related changes that occur in the myocardium following ischemia, only one drug dose was administered per stressed heart to allow for interpretation of data.

The point of administration (10 min post-reperfusion) was chosen to avoid the early disturbances brought about by ischemia and late changes from post-reperfusion damage.

98 4.4 Statistical Analysis

All data are reported as mean ± S.E.M. One-way analysis of variance (ANOVA) with repeated

measure was used to examine the effects of DAMGO and DALDA on force of contraction, heart rate

and coronary flow. In cases where test of normality failed, ANOVA by ranks was used. Dunnett s test was used for post-hoc comparison of each concentration to the pre-drug control. The ability of electrical pacing of the heart to modify the inotropic and coronary flow response to DAMGO and

DALDA was evaluated using Friedman's two-way ANOVA. The difference between inotropic effects in "stressed" versus "non-stressed" hearts was analyzed using paired t-tests. Differences were considered significant whenP < 0.05.

99 4.5 RESULTS

4.5.1 Dose Response studies (DALDA) f Perfusion with DALDA (10"n- 10"7M) resulted in a significant dose dependent decrease in heart rate

(p < 0.001, Fig. 21). The force of contraction and coronary flow were also significantly reduced (P

< 0.02, Fig. 22). Since the decrease in heart rate may modify any direct action of DALDA on contractility, additional dose response studies were carried out under constant pacing of the heart at spontaneous rate. The negative inotropic effect of DALDA was significantly greater in paced hearts when compared to unpaced hearts (P < 0.03), resulting in a 50% reduction in the force of contraction at at 10‘7M (Fig. 22). - ;

4.5.2 Dose Response studies (DAMGO)

In hearts perfused with DAMGO, the first drug administration (10 nM) resulted in a 34% drop in heart rate compared with 15% at the same dose of DALDA (fig. 21). This profound chronotropic effect of DAMGO resulted in a rise in the baseline tension of the heart which in turn masked the inotropic effects of subsequent doses of DAMGO. Hence the higher doses of 10 *M and 10 7M of

DAMGO appeared to have smaller effects on the force of contraction, coronary flow and heart rate

(Fig. 23). When the confounding chronotropic effects of DAMGO was eliminated by pacing, a significantly larger, dose-dependent decrease in inotropy (P < 0.001) was unmasked (Fig. 23).

100 Similarly to DALDA, the negative inotropic effect of DAMGO in paced hearts (n = 4) was

significantly greater than in unpaced hearts (P < 0.001), resulting in a 55% reduction in the force of

contraction at 10'7M.

4.5.3 Arrhythmic hearts

In pilot studies, low flow ischemic hearts resulting from poor coronary perfusion responded with a

dose-dependent increase in the force of contraction (Fig. 25a, b) following the administration of

DALDA (10"7- 10'5M). The contractile performance of the heart was significantly increased (P <

0.002) resulting in a maximum increase of 30% at 10*5M. Furthermore, following the first dose of

DALDA administered, the arrhythmias resulting from poor coronary perfusion ceased (Fig. 25a)

4.5.4 "Stressed hearts"

Following ischemia or rapid pacing, the force of contraction of the heart was significantly reduced (P

< 0.05) prior to any drug administration. Perfusion of these compromised hearts with DALDA or

DAMGO resulted in increases in the force of contraction (Figx27a, b). In ischemic hearts perfused

with DALDA or DAMGO, the force of contraction was increased by 32% ( P < 0.02) and 53% ( P

/< 0.03) respectively compared to pre-drug values. In rapidly paced hearts, the force of contraction

was increased by 52% with DAMGO (P < 0.05) and 64% with DALDA (P< 0.05). The positive

inotropic effects of both DAMGO and DALDA were also accompanied by decreases in the coronary

flow and heart rate.

The effect of naloxone on the inotropic response of DAMGO and DALDA in the normal and "stressed" heart was inconclusive in the present study. Naloxone at 10‘5M initially appeared to attenuate the inotropic effects of DALDA. However, this relatively high dose of naloxone by itself produced an inotropic response in the heart.

101 % change from control (heart rate) J 0 7 - - 0 6 - - 0 5 - - 0 4 - - 0 3 - -20 10 -1 s meansTS.E. as arepresented Data spontaneously beatinghearts (n = 4). Fig. 21 Effects of Effectsof DALDA and hearton rateDAMGO in Fig.21 Log [Drug] (M)[Drug] Log f02. DALDA DAMGO

Fig. 22 Effects of DALDA (10“11-10"7M) on force of contraction (top panel) and cvow y -fiou (bottom panel) in spontaneously beating (unpaced) + and electrically paced hearts (n = 4). Data are presented as means S.E.

Log [Drug] (M)

-11 - 9 - 7

• Paced O Unpaced

-30 -

- 5 0 - O)

-7 0

10 ->

-10 -

- 4 0 -

c - 5 0 -

-70 -

103 Fig. 23 Effects ofDAMGO (10'n-10’7M) on force of contraction (top panel) and jCoroo*gfw (bottom panel) in spontaneously beating (unpacedj and electrically paced hearts (n = 4). Data are presented as means"S.E.

Log [Drug] (M)

-11 -9 -7

c o 0 1 8 # Paced o O Unpaced g £ 30 -

t -50- O) ■5 -60-

-70 -I

104- Fig. 24 Representative traces showing the effect of DALDA (10'MM) on the force of contraction in (A) an unpaced heart and (B) an electrically paced heart.

A . 1 ^ O U T 10 MINS

B.

OUT

'3

\0i> Fig. 25 (A) Representative traces showing the effect of DALDA on the force of contraction of the heart in pilot studies. (B) Effect of DALDA on % change in force of contraction in pilot studies (n = 3).

Control 10*7M lO^M 10 M

^ 60-, oT O) ro 50 - S £ 40- oC 30 -

8 20 - o

10 - o LL

-8 7 6 5 ■4 Log [DALDA] (M) Fig. 26 Trace showing the effect of DALDA (10"7M) before and after rapid pacing in the same heart.

Spontaneous rate ~150bpm

j DALDA 10-7M

After pacing at 300 bpm DALDA 10'7M Fig. 27 Effects of (A) DALDA and (B) DAMGO on force of contraction in "unstressed” hearts and hearts subjected to "stress" by rapid pacing or ischemia. *P < 0.05 for hearts vs control **P < 0.005 for each control vs untreated hearts.

Untreated hearts

] Unstressed heart + 10 M drug E#:;# Rapidly paced heart + 10'7M drug Eaaa Ischemic heart + 10"9M drug A.

B.

Normal Hearts Rapidly paced ischemic hearts hearts

iOS 4.7 DISCUSSION

The present study demonstrates highly selective p-opioid agonists can significantly alter the force of

contraction of the heart. The inotropic effects of the p-agonists employed in this study can occur in

either direction depending on the pre-existing condition of the heart. DAMGO and DALDA caused

a negative inotropic response in the "unstressed" heart accompanied by significant negative

chronotropic effects and a reduction in coronary flow. When the chronotropic effects were eliminated by electrical pacing of the heart at spontaneous rate, a much larger negative inotropic effect was unmasked. In contrast, hearts stressed by ischemia or rapid pacing responded to DAMGO and

DALDA with positive inotropic effects that were also accompanied by a reduction in heart rate and coronary flow. The p-opioid agonists used in the present study were found to be highly potent, working at doses far below those previously reported for other opioid-agonists. -

The normal contraction of the heart is caused by a large Ca release from the main intracellular Ca store, the sarcoplasmic reticulum (Bers, 1985). This results in a rise in the cytoplasmic Ca concentration known as the "C% transient". The rise in the Ca, transient activates the myofilaments to slide resulting in an increase in the force of contraction of each cardiac muscle cell. When this is scaled up to the level of the whole heart, the increased contraction of each individual cell is translated

109 into a greater strength of contraction of the whole ventricle. Ca release from the sarcoplasmic reticulum (SR) is triggered by a small Ca entry into the cell that occurs during the normal cardiac action potential. This Ca entry in turn triggers a much larger SR Ca release via the mechanism of "Ca- induced-Ca release"(Fabiato, 1983; 1985a,b,c) summing up the excitation-contraction process. There are a number of sites in the excitation-contraction coupling process at which drugs may exert inotropic action. Modulation of the excitation-contraction process can occur in one of four ways: modulation of the trigger stimulus which cause Ca release from the SR, alteration of sensitivity of the

SR release mechanism for a given trigger stimulus, alteration of the releasable Ca content of the SR and finally myofilament sensitivity may be modulated to cause an increased or decreased responsiveness to Ca.

Several studies in cardiac cells have documented the point at which opioid agonists modulate the excitation-contraction coupling process to produce inotropic effects. The negative inotropic effects of U50,488H as well as Leu- and met-enkephalin were correlated with a decrease in Ca* transient

(Ventura et al, 1992). Morphine was also shown to increase both the calcium influx and C% transient in rat myocytes (Ela etal, 1993). In another study, dynorphin and U50,488H were found to increase

Caj transient by mobilizing calcium from an intracellular pool most likely the SR (Tai et al, 1992).

Ventura and co-workers (Ventura etal, 1991) demonstrated that a short exposure of myocytes to

U50,488H resulted in both an increase in Ca, transient and enhancement of the myofilament responsiveness that resulted in an increase in contractility while, longer exposures caused a depletion

110 of SR calcium resulting in a decrease in Cai transient and negative inotropic effects. A study on the

effects of leu-enkephalin on the Ca2+ current (/Ca) of L-type Ca2+ channels concluded the negative inotropic effects of the opioid peptide are due in part to a reduction in /Ca. In addition, opioid peptides have been shown to inhibit Na+ - K+ and Ca2+ - dependent ATPase activities in bovine cardiac sarcolemma (Ventura etal, 1987). As Ca2+ entry into the myocardial cell is thought to be regulated by Ca2+ - dependent ATPase (Dhalla et al, 1982), this may explain, the Jno4ropic .a'.t effects of opioids. It is noteworthy that previous studies on the effect of opioids on contractile amplitude and mechanisms have employed much higher drug doses than those used in the present study. In addition, while there are numerous studies on the contractile effects of opioid receptor agonists on cardiac cells, relatively few studies have been done on the inotropic effects of selective opioid agonists in the whole heart isolated from systemic influences.

In ischemia and experimental heart failure induced by rapid pacing the homeostasis of the heart is altered (For review, see (Zucchi and Ronca-Testoni, 1997). Contractility of cardiac muscle cells is impaired as confirmed in the present study. Other alterations in the heart include a change in the Cai transient, SR Ca2+ channel receptor and Ca2+ uptake (Zucchi and Ronca-Testoni, 1997). Therefore a drug with inotropic effects administered under the different contractile states of the "stressed" versus "unstressed" heart may have altered effects as observed in the present study. However, the mechanism of action responsible for the difference in the inotropic effects ofDAMGO and DALDA under these conditions of the heart is unclear at the present time. From the present study, it appears

111 the controversy surrounding the different direction of inotropic effects observed with opioids in

previous studies is probably due to a lack of consistency in the investigative methods employed leading to different contractile conditions of cardiac systems. A study comparing inotropic actions of U50,488H between normal and cardiomyopathic ventricular cells showed no difference in the contractile response to U50,488H in the two groups (Kasper et al, 1992) indicating this phenomenon might be receptor-specific.

Glycosides and other traditional positive inotropic drugs such as P-adrenoceptor agonists and phosphodiesterase inhibitors used for the treatment of congestive heart failure (CHF), improve cardiac contractility by enhancing intracellular calcium (Packer, 1988; Levi et al., 1994). Their mechanisms of action include receptor-mediated stimulation of the adenylate cyclase-cAMP second messenger system, inhibition of K+, Na+-ATPase and inhibition of cAMP breakdown. Inhibition of the K+, Na+-ATPase will elevate intracellular Na+ leading to an increase in calcium influx through the

Na7 Ca2+ exchanger, a phenomenon which has been reported to also occur in response to opioid peptides in isolated sarcolemma from ovine hearts (Ventura et al, 1987). The effects ofDAMGO and DALDA in the pathologic heart may also be indicative of a change in the underlying Ca^ transient which will in turn produce a secondary effect on Na and Ca fluxes via the Na+/ Ca2+ exhanger so that the altered response is a reflection of the different starting conditions of a lower Na^ and smaller contraction. It is interesting to note that our pilot studies showed that in addition to the positive inotropic effects observed with DAMGO and DALDA, they also eliminated the arrhythmias resulting

112 from impaired coronary perfusion. This is similar to the action of Digitalis glycosides which are both positively inotropic in the failing heart as well as anti-arrhythmic via a depression of atrioventricular node conduction (for review, see Levi et al, 1994).

Contractility of the heart is pH-sensitive therefore the mechanisms that regulate intracellular pH are important in controlling cardiac contractility. A number of studies have shown that intracellular acidosis during ischemia increases sarcolemmal Na+/ H+ exchange during reperfiision (Deitmer and

Ellis, 1980; Ellis and MacLeod, 1985; Kaila and Vaughan-Jones, 1987). The sarcolemmal Na-H exchange regulates pH by extruding H ions and taking up Na ions. This decreases the myofilament responsiveness to calcium in addition to a possible depletion of the SR Ca2+ pool (Atar et al, 1995;

G&o et al, 1995; Ela etal, 1993) . Hence acidosis results in negative inotropy (Fabiato and Fabiato,

1978), whereas alkalosis produces a positive effect. Opioids have been shown to produce both intracellular acidosis (Ela etal, 1993) and alkalosis (Kiang and Colden-Stanfield, 1990; Isom et al,

1987). However, a study on the activation of NaV H+ exchange revealed acidosis-induced activation of the Na+/ H+ exchanger can result in different contractile effects depending on extracellular pH

(Vaughan-Jones et al, 1989). Hence, if intracellular acidosis is accompanied by a reduction in extracellular pH, a negative inotropic response will occur while the same fall in intracellular pH at normal extracellular pH results in an increase in contractility. This difference in contractile response was found to be due to an acidosis-induced elevation of intracellular Na at normal extracellular pH resulting in aninerease in Ca influx via the Na-Ca exchanger. Hence, it is feasible that the difference

113 in the contractile response ofDAMGO and DALDA in the "stressed" versus "unstressed" hearts is

a reflection of a difference in extracellular pH under the two conditions.

A newer experimental approach in the treatment of CHF is the development of calcium-sensitizing drugs such as sulmazole and levosimendan which directly improve myocardial contractile force by increasing the affinity of the regulatory site of contractile proteins for calcium rather than enhancing intracellular calcium concentrations (for review, see Vegh et al, 1995). Calcium sensitizers improve myocardial contractility by stabilizing the calcium-induced conformational changes in the contractile component of the myofilament troponin-C (Vegh et al, 1995). It is possible that the difference in the inotropic actions ofDAMGO and DALDA in the normal versus stressed heart is due to different conformational changes in troponin-C in the normal versus stressed heart resulting in a decreased sensitization to calcium in the normal heart and increased sensitization in the stressed heart. This however, remains to be investigated.

In conclusion, this study confirms the differences in the contractile state of the normal heart and hearts subjected to stress. These differences result in an altered response of the inotropic effects of p- opioid peptides on the heart. Biochemical studies on the effect of opioid peptides on phosphodiesterase activity, intracellular cyclic A M P level, C a2+ binding to sarcolemma and C a2+ uptake by the SR and of possible effects on the contractile apparatus to mention a few are required to clarify the mechanisms by which p-opioid receptor peptides exert inotropic effects on the heart.

114 CHAPTER 5

THE DEVELOPMENT OF RAPID TOLERANCE TO THE

CARDIOVASCULAR EFFECTS OF p-OPIOID AGONISTS IN

VIVO AND IN VITRO.

116 5.1 ABSTRACT

As previously reported, the highly selective p-opioid receptor agonists DAMGO ([D-Ala2, 7V-Me-

Phe4,Gly5-ol]enkephalin) and DALDA (Tyr-D-Arg-Phe-Lys) have profound cardiovascular effects in the chronically instrumented ewe. In the present study, the effect of repeated exposure to these agonists and the possible development of tolerance to their cardiovascular effects is investigated. In the chronically-instrumented ewe, single exposures to DAMGO (0.3mg/kg) or DALDA (0.6mg/kg) resulted in a significant increase in blood pressure with DAMGO eliciting a significantly larger response than D AT D A When two doses of the same agonist were administered 30 min apart, the pressor responses to the second doses of both DALDA and DAMGO was completely abolished. The possibility of cross tolerance between the two p-agonists was also investigated. Pretreatment with

DAMGO completely abolished the pressor effect to DALDA. In contrast, pretreatment with DALDA resulted in a partial though significant attenuation of the pressor response to DAMGO. Hence the efficacy of DAMGO and DALDA in eliciting cardiovascular responses paralleled their ability to induce tolerance . In the isolated guinea pig heart, increasing doses of DALDA (10'n - 10'7M) separated by a 10 min drug washout between doses resulted in a dose dependent decrease in the force of contraction. However, when the heart was continuously exposed to the same drug treatment for

30 min without allowing for washouts between doses, the inotropic effect of DALDA was completely abolished after 10 min. This study demonstrates acute tolerance to the cardiovascular effects of highly selective p-opioid agonists in vivo and in vitro.

117 5.2 INTRODUCTION

While opioid agonists are the drugs of choice in the management of severe and chronic pain, a prominent characteristic of their action is their ability to induce tolerance and dependence thus limiting their therapeutic efficacy. The p-opioid receptor is of critical importance in the development of tolerance and addiction (Loh et al., 1988; Di Chiara and North, 1992) and previous studies have reported the development of tolerance to the effects of morphine on heart rate and respiratory rate following a single dose exposure in rabbits (Hovav and Weinstock, 1987). More recently, it has been appreciated that tolerance can develop to opioid drugs even after brief exposure. Tolerance to the analgesic effect of alfentanil and remifentanil develops during constant rate infusion of these drugs in rats and humans (Kissing a/., 1996; Vinik and Kissin, 1998) and this rapid development of tolerance has been termed acute or short-term tolerance. The experimental conditions known to influence the development of acute tolerance to opioid drugs include dose, magnitude of effect, duration of drug action, and the time interval between doses (Hovav and Weinstock, 1987).

In the studies reported previously, the profound cardiovascular effects of the highly selective p-opioid receptor agonists DAMGO and DALDA have been demonstrated in vivo (Omoniyi et aL, 1998b,

Clapp et al, 1996; Clapp et al, 1998). In the present study, the possibility of tolerance to the cardiovascular effects ofDAMGO and DALDA are investigated in vivo and in vitro.

118 5.3 MATERIALS AND METHOD

5.3.1 In vivo studies (chronically instrumented sheep)

Following a 2 hour control period, each animal received one of the four drug treatments given in

Table 4. The second dose was administered 30 min after the first dose. Each animal was allowed a on -tucd

of drug treatment was randomized.

5.3.2 In vitro studies (isolated guinea pig hearts)

Following the stabilization period, hearts were perfused with DALDA in increasing doses of 10 ,

10"9 and 10"7M. Each dose was administered for 10 min followed by a 10 min recovery period during

which the heart was perfused with drug free Krebs-Henseleit buffer. To investigate the possibility of

tolerance to DALDA, all three doses were administered consecutively without the 10 min washout between doses. Only one dose response study was carried out per heart.

5.4 Data analysis

Blood pressure was analyzed using 1 min intervals and control values were averaged over 5 min prior to the administration ofDAMGO or DALDA. Mean arterial pressure was calculated as the diastolic pressure plus 1/3 of the pulse pressure. Force of contraction of the isolated heart was analyzed using two min intervals. Control values were averaged over 5 min prior to the administration of DALDA.

119 5.5 Statistical analysis

All data are reported as mean ± S.E.M. One-way analysis of variance (ANOVA) with repeated measure (factor = time) was used to examine the effects ofDAMGO or DALDA on blood pressure.

In cases where test of normality failed, ANOVA by ranks was used. Dunnett's test was used for post- hoc comparison of each time point to the pre-drug control. The differences between first and second drug administrations were evaluated using Friedman's two-way ANOVA with repeated measure

(factors = treatment, time). Differences were considered significant whenP <0.05.

TABLE 4:Methods for in vivo drug treatment

Drug treatment First drug Second drug n I DAMGO DAMGO 3 n DAMGO DALDA 3 m DALDA DALDA 3 IV DALDA DAMGO 3

120 5.6 RESULTS

5.6.1 In vivo experiments

The first dose ofDAMGO (0.3 mg/kg) resulted in a significant increase in blood pressure (P < 0.001),

and post-hoc comparison showed blood pressure was significantly elevated up to 8 min after drug

administration (Fig. 28, top panel). When a second dose ofDAMGO was administered 30 min after

the dose of DAMGO, there was no change in blood pressure (Fig. 28, top panel). However, a

significant pressor response was observed when DAMGO was administered 30 min after DALDA,

although the response was significantly attenuated when compared to a single dose ofDAMGO alone

(P< 0.002).

The first dose of DALDA (0.3 mg/kg) also significantly increased mean blood pressure (P < 0.001),

but the increase was smaller in magnitude and was only significant for 4 min after administration (Fig.

29, top panel). Administration of DALDA 30 min after either DALDA or DAMGO produced no

effect on blood pressure (Fig. 29, top panel).

DAMGO had no effect on heart rate when administered singly or following pretreatment with itself

or DALDA (Fig. 28, bottom panel). In contrast, while a single exposure to DALDA had no effect

on heart rate, a significant bradycardia (P < 0.05) was observed when DALDA was administered 30

min after DAMGO (Fig. 29, bottom panel).

In contrast to DAMGO and DALDA, a repeat dose of U50,488H (0.6mg/kg) produced the same pressor effect and increase in heart rate as that observed with the first dose (data not shown).

121 ! 5.6.2 In vitro experiments

Perfusion of the heart with DALDA (10"n - 10*7M) separated by a 10 min washout resulted in a dose-

dependent decrease in force of contraction (Fig. 30, top panel), heart rate (middle panel) and coronary flow (bottom panel). The decrease in force of contraction was highly significant (P < 0.02) and post- hoc comparison revealed that force of contraction was significantly decreased with all three drug concentrations. In contrast, the same doses of DALDA administered consecutively without allowing for drug washout between doses resulted in a loss of the negative inotropic response following the first dose. There was a significant difference between the two inotropic dose-response curves (P <

0.001). There was no significant difference in the heart rate and coronary flow responses(fig. 30).

122 % % change in heart rate % change in blood pressure 0 4 - -20 40 -4 -20 20 - 0 4 -I 0 6 20 - 0 4 -, 0 6 - - - - DALDA (n = 3).DALDA Data asmeansare presented S.E.+ animalspretreated andwith animals DAMGO pretreatedwith (toppanel)and heart rate(bottom panel) in untreatedanimals, of (0.3mg/kg)i.v. Effects DAMGO on meanbloodpressure 28Fig. 0 0 2 2 4 4 6 6 Time (mins) Time 8 8 12-3 10 12 1210 14 14 16 6 1 18 8 1 untreated o DALDA pretreated DALDA DAMGO pretreated DAMGO

Fig. 29 Effects of i.v. DALDA (0.6mg/kg) on mean blood pressure (top panel) and heart rate (bottom panel) in untreated animals, animals pretreated with DALDA and animals pretreated with DAMGO (n = 3). Data are presented as means + S.E.

untreated DALDA pretreated DAMGO pretreated

CL Dû c 0) O) c CO ■5 o -----

-20 -

-4 0

0 2 4 6 8 10 12 14 1 6 1 8

60 -

40 - ÛC =c c 20 - 0) O) c (0 _c 0 - o £

-20 -

-40 10 12 14 16 18 Time (mins) Fig. 30 Effects of DALDA (10"n-10"7M) on force of contraction (top panel), heart rate (middle panel) and coronary flow (bottom panel) when administered intermittently and continuously in the guinea pig heart (n = 6). Data are presented as : meansTS.E. o intermittent # Continuous 2 0 -I

c -10 - 8 -20 - C -30 - -40 - -50 - -60 -11 9 7 20 i

10 - 0 ---

O) -30 -

■E -40 - -50 - -60 -11 ■9 ■7 20 -j

0 10 - 5=

1 °- O -10 - q ° -20 - c CD -30 - O) c ca -40 - o s o - 5 0 - -60 - -11 9 7 Log [Drug] (M) 5.8 DISCUSSION

The present study demonstrates rapid tolerance to the cardiovascular effects of selective p-opioid

agonists in vivo and in vitro. As reported previously, both DAMGO and DALDA caused a significant increase in blood pressure with no effect on heart rate when administered to the unanesthetized ewe

(Clapp et al, 1996; Clapp et al, 1998). DAMGO was found to be more potent than DALDA when administered intravenously, and this is in accordance with the relative potency of these two p-opioid agonists in the guinea pig ileum (Schiller et al, 1989). The lack of a bradycardia in the presence of a pressor response was shown to be due to blunting of the baroreflex (Omoniyi et al, 1998a; Omoniyi etal, 1998b). Tolerance to the pressor effect ofDAMGO and DALDA was observed after exposure to a single dose of the drug, with the second dose producing no measurable effect on blood pressure.

The dose interval used in this study (30 min) was much less than the shortest interval that was thought to be necessary for the development of acute tolerance to morphine (4 h) (Hovav and Weinstock,

1987). Furthermore, administration of one p-agonist resulted in cross-tolerance to the other, with the more potent DAMGO producing complete tolerance to DALDA but DALDA resulting only in partial tolerance to the pressor effect ofDAMGO. In the isolated perfused heart studies, the rapid development of tolerance made it impossible to obtain a dose-response curve for DALDA using an escalating dose design. However, when a 10 min washout period was allowed between doses, a dose- dependent decrease in force of contraction became apparent.

The attenuation of responses to an agonist can occur at the level of the agonist, the receptor, the G-

126 proteins, and at numerous steps downstream of the signaling pathway. Mechanisms for the attenuated

response to repeated agonist response include receptor desensitization, receptor endocytosis

(internalization), receptor phosphorylation and receptor downregulation (for review, see Grady et aL,

1997). All four mechanisms have been implicated in the reduced responsiveness to repeated or

continuous exposure to opioid agonists. Thus, internalization of the p-receptor following agonist

exposure has been reported in human embryonic kidney (HEK) cells (Burford NT et ah, 1998) and

neurons in vivo (Stemini et aL, 1996). The p-receptor was also shown to be due to internalization

via clathrin-coated vesicles (Kato et aL, 1998). The desensitization observed with DAMGO in one

study was correlated with a down regulation of p-receptors (Pak et aL, 1996), while numerous other

studies have shown that repeated exposure to opioid agonists results in receptor phosphorylation.

The mechanism involved in the attenuation of agonist response is dependent on the length of exposure to the stimulus. Receptor desensitization occurs during short term exposure to agonists and is mediated by uncoupling of activated receptors from G proteins. With continuous exposure, the receptor becomes sequestered depleting the plasma membrane of high affinity receptors. When the stimulation is chronically persistent, this results in downregulation of receptors and hence a reduction in receptor density. Desensitization appears to develop very rapidly for both p and ô opioid receptor, within 3 min of exposure to opioid agonists (Zhang et aL, 1996; Pei et aL, 1995). It is therefore not surprising that tolerance to the negative inotropic action of DALDA was apparent within 10 min of perfusion in the isolated perfused heart system, and complete tolerance to the pressor effect of

127 DAMGO and DALDA was observed 30 min after drug exposure. It is also interesting that it was

possible to overcome the development of tolerance in the isolated heart by a 10 min washout period, just as it was reported that ô opioid receptors in NG108 cells can be re-sensitized with a time constant

of 6.7 min (Morikawa et al, 1998).

G-protein cascades activated by p receptors can also reduce adenylyl cyclase activity, alter inositol triphosphate turnover, activate G-protein linked, inwardly rectifying potassium channels and mitogen-

activated protein kinase, and close calcium channels (Childers, 1991; Loh and Smith, 1990).

Attenuated responses to several of these effectors have also been demonstrated for repeated opioid agonist exposure in cells containing stably transfected opioid receptors. In agreement with the present study, desensitization was observed with DAMGO-induced potassium channel responses in Xenopus oocyte (Zhang etal, 1996), G protein activation in NG108-15 cells (Fan GH et al, 1998) and HEK cells (Burford NT et al, 1998), and forskolin-stimulated cAMP accumulation in CHO cells (Pak et al, 1996).

Another mechanism suggested for desensitization of the p-opioid receptor is receptor phosphorylation. Increased phosphorylation of the p-opioid receptor protein was demonstrated after as little as 5 min of exposure to DAMGO \n Xenopus (Zhang et al, 1996). The rank order for various opioid agonists in desensitization generally paralleled the rank order for phosphorylation of the p- opioid receptor in CHO cells (Yu et al, 1997). Receptor phosphorylation has also been shown to

128 be coupled to desensitization of the ô-opioid receptor (Pei et al, 1995; Hasbi et al, 1998). Several studies have suggested the involvement of protein kinases in opioid receptor desensitization.

Activation ofPKC was found to reduce the responsiveness of p-opioid receptors in Xenopus oocytes

(Chen and Yu, 1994) and another study demonstrated the involvement ofPKC in the functional uncoupling of ô-opioid receptors from G-proteins (Fukushima et al, 1994). The overexpression and inhibition of PARK1 has also been shown to enhance and inhibit opioid receptor desensitization respectively (Raynor et al, 1994b; Pei et al, 1995).

The present data suggest that DAMGO is also more potent in producing tolerance than DALDA since pretreatment with DALDA was only able to partially attenuate the pressor response to DAMGO.

Few cellular experiments have examined the phenomenon of desensitization with different opioid agonists. There appears to be a correlation between the efficacies of these opioid agonists in increasing blood pressure and their efficacies in inducing desensitization, and is in agreement with previous studies that show a correlation between agonist efficacy and level of desensitization

(Yabaluri and Medzihradsky, 1997; Yu et al, 1997).

In conclusion, this study demonstrates a rapid tolerance to highly selective p-opioid agonists in vivo and in vitro. The specific mechanism by which these p-opioid agonists cause receptor desensitization in these systems remains to be investigated.

129 CHAPTER 6

A GENERAL DISCUSSION

130 6.1 Discussion

In the intact and unanesthetized animal, the p-opioid agonists DAMGO and DALDA, as well as the

K-agonist, U50,488H had significant effects on blood pressure. These results contradicted that of

several other studies involving the use of anesthesia confirming earlier reports on the ability of anesthetics to reverse the cardiovascular effects of opioids. The pressor effects of all three agonists was sensitive to naloxone. Furthermore, the pressor effects of DAMGO and DALDA was also sensitive to the quaternary opioid antagonist naloxone methiodide, in contrast to the generally held belief that these agonists affect blood pressure via a central site.

The investigation into the cardiovascular effects of these p-agonists was then focused on an in vitro of setup with the aim^elucidating the mechanism involved in their profound pressor effects. The initial expectation had been that DAMGO and DALDA would increase myocardial contractility thus explaining the pressor responses observed in vivo. This hypothesis was based on preliminary data from James F. Clapp'^laboratory which indicated that these agonists increased cardiac output in the ewe while having no effect on peripheral resistance. Considering the lack of any effect on peripheral resistance, it was thought that the increase in cardiac output hence blood pressure was due to a direct action of p-agonists on the heart to increase contractility and/or venous return. In light of the isolated heart results, the increase in blood pressure is most likely due to an increase in venous return. In fact, preliminary data now confirms that DALDA significantly increases venous return in the unanesthetized ewe (unpublished data from collaborator). This is in agreement with a study in the

131 conscious rat which demonstrated a concomitant increase in blood pressure and cardiac output with

no accompanying change in total peripheral resistance (Siren and Feuerstein, 1991).

The isolated heart study however still demonstrated highly selective p-opioid agonists can significantly

alter the force of contraction of the heart. Whatimore, these p-opioid agonists were found to be highly potent, working at doses far below those previously reported for other opioid-agonists. It is also interesting that the effect of DAMGO and DALDA on myocardial contractility can occur in either direction depending on the pre-existing condition of the heart and that its chronotropic effects can significantly masks/its inotropic response. Both the negative and positive inotropic actions of

DAMGO and DALDA were attenuated by the non-selective opioid antagonist naloxone indicating the guinea pig heart may contain p-opioid receptors in contrast to that reported for the rat (Krumins et al, 1985). Alternatively, it ispossible that the effects of p-opioid agonists is not as a result of a direct effect on the myocardium. Instead the cardiodepressant effects of DAMGO and DALDA on the heart may be due to actions on sympathetic and/or parasympathetic nerve terminals. This would be consistent with reports which demonstrate the presence of opioid receptors in the vagus nerve

(Young et al, 1980; Zarbin et al, 1990) and sympathetic nerve terminals in the heart and blood vessels (Mantelli et al, 1987). The mechanism behind the unusual difference in the inotropic response of the "stressed" versus "unstressed" heart is not understood at the present time and warrants fiirther investigation. It is almost certain that it could be explained by the effects of these p-agonists on calcium fluxes in the heart. The use of cardiac myocytes would be ideal in the mechanistic

132 evaluation of the effects of DAMGO and DALDA on the force of contraction of the heart.

The negative chronotropic effects of p-opioid agonists observed in vitro were in sharp contrast to the

lack of a bradycardia seen in vivo. It is posiible that in vivo, the lack of a bradycardia may be partially // z

due to an underlying increase in sympathetic activity in response to DAMGO and DALDA.

Furthermore the differences in the cardiovascular state of in vivo and in vitro setups i.e. the absence

of autonomic tone, baroreflexes etc may account for the differences observed between the effects of

p-opioid agonists in vivo and in vitro.

These studies also demonstrated a significant attenuation of the cardiovascular effects of opioid

agonists in pregnancy. As previously explained, significant vascular changes occur in pregnancy.

These profound changes in the physiology of the pregnant animal may account for differences in its response to vasoactive drugs. Many of these changes are thought to be mediated by estrogen receptors located throughout the vascular tree. However, progesterone probably also plays a role as progesterone receptors are also present throughout the vascular tree and have been shown to modify responses to estrogen. Thus, it is logical to assume that many of the altered responses to vasoactive drugs during pregnancy are the result of an altered hormonal milieu, however this has not been confirmed in either an animal or human model.

133 ^-agonists had no effect on heart rate while the k - agonist, U50,488H caused a significant tachycardia. The results of the baroreflex study demonstrate for the first time that systemically- administered g-opioid peptide analogs can significantly alter baroreflex sensitivity in both nonpregnant and pregnant animals. Interestingly, both DAMGO and DALDA blunted the baroreflex-mediated bradycardia to pressor stimuli, but had no effect on the baroreflex-mediated tachycardia to a hypotensive stimulus. These results also suggest that, in addition to opioid receptors in the CNS, peripheral opioid receptors may also play an important role in inhibiting the baroreflex. In contrast

to p-agonists, the k - agonist, U50,488H had no effect on the baroreflex and its tachycardie effect was found to be mediated by sympathetic activation of the heart. This indicated that unlike p- agonists, systemically administered K-agonists appear to possess a central component to their cardiovascular effects.

The attenuation of baroreflex-mediated bradycardia observed with p-agonists could be as a result of inhibition of vagal activity by DAMGO and DALDA. Other opioid receptor agonists such as morphine and the enkephalins have been shown to inhibit the bradycardia elicited in response to vagal stimulation (Musha et al, 1989; Weitzell et al, 1984). In addition, enkephalins were also found to inhibit the release of acetylcholine from rabbit atria (Weitzell et al., 1985; Wong-Dusting et al., 1985).

To further support this hypothesis are previous studies documenting the presence of opioid receptors on the vagus (Young et al, 1980; Zarbin et al, 1990). Blunting of the bradycardic response to pressor stimuli may also be due to a direct inhibition of the baroreceptors, or modulation of the

134 afferent or efferent pathways to and from the NTS. However, data from these study showed that the

baroreflex-mediated tachycardia to hypotensive stimuli was not affected, suggesting that p-agonists

must be inhibiting vagal efferents to the sino-atrial node, p-agonists may inhibit vagal activity either

prejuktionally by reducing acetylcholine release or postjuntionally by interfering with the action of

acetylcholine at its receptors. It would be expected that an inhibition of the bradycardic response to vagal nerve stimulation by these p-opioid agonists would be significantly blunted in pregnant animals. Finally, if DAMGO and DALDA shift the dose-response to exogenously administered acetylcholine, pregnant and hormonally-treated hearts will show less of a rightward shift. Likewise, since_p-opioid agonists exert a direct effect on the myocardial contractility in the nonpregnant guinea pig heart, it is likely that the dose-response relationship in hearts from pregnant animals will be shifted to the right. These differences may be associated with a reduction of p-receptors in the heart or vagal terminals of the pregnant animal, or a change in binding affinity. These findings could then be confirmed by opioid receptor binding assays in the vagus and cardiac tissues.

The rapid development of tolerance to the cardiovascular effects of DAMGO and DALDA found in vivo and in vitro may also be limited to p-agonists as preliminary data with U50,488H and another

K-opioid agonist orphanin indicates that k- unlike p-opioid agonists do not induce tolerance following repeated systemic administration. The specific mechanism by which DAMGO and DALDA induce tolerance also remains to be investigated. While desensitization at the level of the receptor seems the most likely cause of the attenuation to the cardiovascular effects of DAMGO and DALDA, other

135 causes of tolerance cannot be ruled out. These include receptor endocytosis (internalization),

receptor phosphorylation and receptor downregulation. All four mechanisms have been implicated

in the reduced responsiveness to repeated or continuous exposure to opioid agonists (Burford NT et al, 1998; Stemini et al., 1996; Kato et al, 1998; Pak et al, 1996).

6.2 Conclusion

In conclusion, these studies together demonstrate p- and K-opioid agonists have significant cardiovascular actions in the intact animal and directly at the level of the heart. While both p- and

K-opioid agonists have similar effects on blood pressure, the heart rate response to K-opioid agonists differ significantly from those of p-agonists in site and mechanism of action. These studies also demonstrate for the first time that the highly selective p-opioid agonistsDAMGO and DALDA blunt baroreflex-mediated bradycardia but not tachycardia following systemic administration. The effects o f p-agonists on the baroreflex like its pressor effects also occur at a peripheral site. The cardiovascular effects of both p- and K-opioid agonists were significantly attenuated in pregnancy.

For the first time, the inotropic and chronotropic effects of p-agonists on the heart are shown to be subject to the state of the myocardium. Furthermore, repeated or continuous exposure to these agonists results in the rapid development of tolerance to the cardiovascular effects observedin vivo and in vitro. Finally, the two p- agonists DAMGO and DALDA though producing similar effects in all studies carried out, displayed different levels of potency. DAMGO blunted the baroreflex to a higher degree, produced bigger chronotropic and inotropic effects in the isolated heart and induced

136 a higher level of tolerance than DALDA in accordance with the relative potency of these two p-opioid

agonists in the guinea pig ileum (Schiller et al, 1989).

These studies demonstrate that highly selective p and K-opioid agonists in the absence of the confounding effects of anesthesia, produce profound cardiovascular effects. In contrast, 5-opioid agonists do not appear to have any effects on either blood pressure or heart rate. These studies clearly resolve the confusion regarding the direction of change of blood pressure, heart rate and baroreflex sensitivity in response to opioid agonists. The direct actions of DALDA and DAMGO on the force of contraction of the heart, isolated from systemic influences is also demonstrated for the first time. It is particularly interesting that these opioid agonists have a different and possibly protective effect in the stressed heart indicating a possible therapeutic role for opioid agonists in the compromised heart.

y The pressor, heart rate and baroreflex effects of these opioid agonists are also demonstrated in pregnancy and shown to be altered when compared to nonpregnant animals. More importantly, the present study demonstrates there are no serious cardiovascular side effects produced in the mother when exposed to these opioid agonists increasing their potential for clinical use. However, like its counterparts, there is a development of rapid tolerance following exposure to the (i-opioid agonists DAMGO and DALDA.

137 REFERENCES

138 ABOOD, M. E., NOEL, M. A., FARNSWORTH, J. S. AND TAO, Q. Molecular cloning and

expression of a delta-opioid receptor from rat brain. J. Neurosci. Res. 37: 714-719, 1994.

ASSALI N.S., BRINKMAN III C R , WOODS, J. J. R., DANDAVTNO A. AND NUWAYHID B.

Development of neurohumoral control of fetal, neonatal, and adult cardiovascular functions. Am. J.

Obstet. Gynecol. 129: 748-759, 1977.

ASSALI, N. S., NUWAYHID, B., BRINKMAN, 3. ,C.R., TABSH, K., ERKKOLA, R. AND

USHIODA, E. Autonomic control of the pelvic circulation: In vivo and in vitro studies in pregnant

and nonpregnant sheep. Am. J. Obstet. Gynecol. 141: 873-882, 1981.

ATAR, D., GAO, W. D. AND MARBAN, E. Alterations of excitation-contraction coupling in stunned myocardium and in failing myocardium. J. Mol. Cell Cardiol. 27: 783-791, 1995.

ATKINS, A. F., WATT, J. M., MILAN, P., DAVIES, P. AND CRAWFORD, J. S. A longitudinal study of cardiovascular dynamic changes throughout pregnancy. Eur. J. Obstet. Gynecol. Reprod.

Biol. 12: 215-224, 1981.

BACHELARD, H. AND PITRE, M. Regional haemodynamic effects of mu-, delta-, and kappa-opioid

139 agonists microinjected into the hypothalamic paraventricular nuclei of conscious, unrestrained rats.

Br. J. Pharmacol. 115: 613-621, 1995.

BARBER, A., BARTOSZYK, G. D., BENDER, H. M., GOTTSCHLICH, R., GREINER, H. E.,

HARTING, J., MAULER, F., MINCK, K. O , MURRAY, R. D , SIMON, M. AND ET AL: A pharmacological profile of the novel, peripherally-selective kappa-opioid receptor agonist, emd

61753. Br. J. Pharmacol. 113: 1317-1327, 1994a.

BARBER, A., BARTOSZYK, G. D , GREINER, H. E., MAULER, F., MURRAY, R. D.,

SEYFRIED, C. A , SIMON, M., GOTTSCHLICH, R , HARTING, J. AND LUES, I. Central and peripheral actions of the novel kappa-opioid receptor agonist, emd 60400. Br. J. Pharmacol. I ll:

843-851, 1994b.

BAUSCH, S. B., PATTERSON, T. A., APPLEYARD, S. M. AND CHAVKIN, C.

Immunocytochemical localization of delta opioid receptors in mouse brain. J. Chem. Neuroanat. 8.

175-189, 1995.

BECKETT, A. H. AND CASY, A. F. Synthetic analgesics:stereochemical considerations. J. Pharm.

Pharmacol. 6: 986-1001, 1954.

140 BELL, L. B., DORWARD, P. K. AND RUDD, C. D. Influence of cardiac afférents on time-dependent changes in the renal sympathetic baroreflex of conscious rabbits. Chn. Exp.

Pharmacol. Physiol. 17: 545-555, 1990.

BERS, D. M. Ca influx and sarcoplasmic reticulumCa release in cardiac muscle activation during postrest recovery. Am. J. Physiol. 248: H3 66-81, 1985.

BDLSKY, E. J., CALDERON, S. N., WANG, T., BERNSTEIN, R. N., DAVIS, P., HRUBY, V. J.,

MCNUTT, R. W., ROTHMAN, R. B., RICE, K. C. AND PORRECA, F. Snc 80, a selective, nonpeptidic and systemically active opioid delta agonist. J. Pharmacol. Exp. Ther. 273. 359-366,

1995.

BIRCH, P. J., HAYES, A. G , SHEEHAN, M. J. AND TYERS, M. B. Norbinaltorphimine: antagonist profile at kappa opioid receptors. Eur. J. Pharmacol. 144. 405-408, 1987.

BOURGOIN, S., BENOLIEL, J. J., COLLIN, E., MAUBORGNE, A., POHL, M., HAMON, M.

AND CESSELIN, F. Opioidergic control of the spinal release of neuropeptides, possible significance for the analgesic effects of opioids. Fundam. Clin. Pharmacol. 8: 307-321, 1994.

141 BRADBURY, A. F., SMYTH, D. G , SNELL, C. R , BIRDSALL, N. J. M. AND HULME, E. C.

C-Fragment of lipoprotein has a high affinity for brain opiate receptors. Nature 260: 793-795, 1976.

BROOKS, V. L., QUESNELL, R. R., CUMBEE, S. R. AND BISHOP, V. S. Pregnancy attenuates

activity of the baroreceptor reflex. Clin. Exp. Pharmacol. Physiol. 22: 152-156, 1995.

BROOKS, V. L. AND KEEL, L. C. Hemorrhage decreases arterial pressure sooner in pregnant

compared with nonpregnant dogs: role of baroreflex. Am. J. Physiol. 266: HI 610-9, 1994a.

BROOKS, V. L. AND KEIL, L. C. Changes in the baroreflex during pregnancy in conscious dogs: heart rate and hormonal responses. Endocrinology 135: 1894-1901, 1994b.

BURFORD NT, TOLBERT LM AND SADEE W: Specific G protein activation and mu-opioid receptor internalization caused by morphine, DAMGO and endomorphin I. Eur. J. Pharmacol. 342:

123-126, 1998.

BZDEGA, T., CHIN, H , KIM, H , JUNG, H. H , KOZAK, C. A. AND KLEE, W. A. Regional expression and chromosomal localization of the delta opiate receptor gene. Proc. Natl. Acad. Sci. U.

S. A. 90: 9305-9309, 1993.

142 CAPELESS, E. L. AND CLAPP, J. F. Cardiovascular changes in early phase of pregnancy. Am. J.

Obstet. Gynecol. 161: 1449-1453, 1989.

CARTER, D. A. AND LIGHTMAN, S. L. Selective cardiovascular and neuroendocrine effects of

a kappa-opioid agonist in the nucleus tractus solitarii of rats. J. Physiol. (Lond) 367: 363-375, 1985.

CHANG, K. J., RIGDON, G. C., HOWARD, J. L. AND MCNUTT, R. W. A novel, potent and

selective nonpeptidic delta opioid receptor agonist BW373u86. J. Pharmacol. Exp. Ther. 267.

852-857, 1993.

CHEN, Y., MESTEK, A., LIU, J. AND YU, L. Molecular cloning of a rat kappa opioid receptor reveals sequence similarities to the mu and delta opioid receptors. Biochem. J. 295: 625-628, 1993.

CHEN, Y. AND YU, L. Differential regulation by camp-dependent protein kinase and protein kinase

C of the mu opioid receptor coupling to a g protein-activated k+ channel. J. Biol. Chem. 269:

7839-7842, 1994,

CHENG, P. Y., SVINGOS, A. L., WANG, H., CLARKE, C. L., JENAB, S., BECZKOWSKA, I.

W., INTURRISI, C. E. AND PICKEL, V. M. Ultrastructural immunolabeling shows prominent

143 presynaptic vesicular localization of delta-opioid receptor within both enkephalin- and nonenkephalin-containing axon terminals in the superficial layers of the rat cervical spinal cord. J.

Neurosci. 15: 5976-5988, 1995.

CHILDERS, S. R. Opioid receptor-coupled second messenger systems. Life Sci. 48: 1991-2003,

1991.

CLAPP, J. P., SOONG, Y , WU, D. L., OLARIU, N , YEE, J. AND SZETO, H. H. Maternal cardiovascular and metabolic responses to intravenous Tyr-D-Ala-Gly-Phe(N-Me)-Gly-ol (DAMGO) in chronically -instrumented pregnant sheep. Analgesia 2: 137-143, 1996.

CLAPP, J. F., KETT, A., OLARIU, N., OMONIYI, A. T., WU, D., KIM, H. AND SZETO, H. H.

Cardiovascular and metabolic responses to two receptor-selective opioid agonists in pregnant sheep.

Am. J. Obstet. Gynecol. 178: 397-401, 1998.

CLAPP, J. F.,3D, SEAWARD, B. L., SLEAMAKER, R. H. AND RISER, J. Maternal physiologic adaptations to early human pregnancy. Am. J. Obstet. Gynecol. 159: 1456-1460, 1988.

CLARK, C. R , BIRCHMORE, B., SHARIF, N. A., HUNTER, J. C., HILL, R. G. AND HUGHES,

144 J. Pdl 17302: a selective agonist for the kappa-opioid receptor. Br. J. Pharmacol. 93: 618-626, 1988.

CONRAD, K. P. AND RUSS, R. D. Augmentation of baroreflex-mediated bradycardia in conscious pregnant rats. Am. J. Physiol. 262: R472-7, 1992.

COSTELLO, G. P., MAIN, B. G., BARLOW, J. J., CARROLL, J. A. AND SHAW, J. S. A novel series of potent and selective agonists at the opioid kappa-receptor. Eur. J. Pharmacol. 151: 475-478,

1988.

COX, R. H. AND BAGSHAW, R J. Influence of anesthesia on the response to carotid hypotension in dogs. Am. J. Physiol. 237: 571-577, 1979.

CRABTREE, B. L. Review of naltrexone, a long-acting opiate antagonist. Clin. Pharm. 3: 273-280,

1984.

CRAIN, S. M. AND SHEN, K. F. Opioids can evoke direct receptor-mediated excitatory effects on sensory neurons. Trends. Pharmacol. Sci. 11: 77-81, 1990.

CYON, E. AND LUDWIG, C. Die reflexe eines der sensiblen Nerven des Herzens auf die

145 Motorischen der Blutgefusse. Ber. Saeschs. Ges. Akad. Wissenschaften 18: 307-329, 1866.

DADO, R. J., LAW, P. Y., LOH, H. H. AND ELDE, R. Immunofluorescent identification of a delta

(delta)-opioid receptor on primary afferent nerve terminals. Neuroreport. 5: 341-344, 1993.

DEITMER, J. W. AND ELLIS, D. Interactions between the regulation of the intracellular ph and

sodium activity of sheep cardiac purkinje fibres. J. Physiol. (Lond) 304: 471-488, 1980.

DELAY-GOYET, P., SEGUIN, C , GACEL, G. AND ROQUES, B. P.

[3h][d-ser2(o-tert -butyl),Ieu5]enkephalyl-thr6 and

[d-ser2(o-tert-butyl),leu5]enkephalyl-thr6(o-tert-butyl). two new enkephalin analogs with both a good

selectivity and a high affinity toward delta-opioid binding sites. J. Biol. Chem. 263: 4124-4130, 1988.

DELAY-GOYET, P., ZAJAC, J. M. AND ROQUES, B. P. Improved quantitative radioautography

of rat brain delta opioid binding sites using [3H]DSTBULET, a new and highly potent and selective

linear enkephalin analogue. Neurochem. Int. 16: 341-368, 1990.

DHALLA, N. S., PIERCE, G. N , PANAGIA, V., SINGAL, P. K. AND BEAMISH, R. E. Calcium movements in relation to heart function. Basic. Res. Cardiol. 77: 117-139, 1982.

146 DI CHIARA, G. AND NORTH, R. A. Neurobiology of opiate abuse. Trends. Pharmacol. Sci. 13:

185-193, 1992.

DUVEKOT, J. J., CHERIEX, E. C , PIETERS, F. A., MENHEERE, P. P. AND PEETERS, L. H.

Early pregnancy changes in hemodynamics and volume homeostasis are consecutive adjustments triggered by a primary fall in systemic vascular tone. Am. J. Obstet. Gynecol. 169: 1382-1392, 1993.

DUVEKOT, J. J., CHERIEX, E. C , TAN, W. D., HEIDENDAL, G. A. AND PEETERS, L. L.

Volume-dependent echocardiographic parameters are not useful for estimating baseline blood volume but are useful for detecting acute changes in vascular filling state. Basic. Res. Cardiol. 89: 270-277,

1994.

EASTERLING, T. R , BENEDETTI, T. J., SCHMUCKER, B. C. AND MILLARD, S. P. Maternal hemodynamics in normal and preeclamptic pregnancies: a longitudinal study [see comments]. Obstet.

Gynecol. 76: 1061-1069, 1990.

ELA, C , HASIN, Y. AND EDLAM, Y. Opioid effects on contractility, Ca(2+)-transients and intracellular PH in cultured cardiac myocytes. J. Mol. Cell Cardiol. 25: 599-613, 1993.

147 ELLIS, D. AND MACLEOD, K. T. Sodium-dependent control of intracellular PH in purkinje fibres

of sheep heart. J. Physiol. (Lond) 359: 81-105, 1985.

EMMERSON, P. I , LIU, M. R., WOODS, J. H. AND MEDZIHRADSKY, F. Binding affinity and

selectivity of opioids at mu, delta and kappa receptors in monkey brain membranes. J. Pharmacol.

Exp. Ther. 271: 1630-1637, 1994.

EVANS, C. J., KEITH, D. E.,JR., MORRISON, H., MAGENDZO, K. AND EDWARDS, R. H.

Cloning of a delta opioid receptor by functional expression [see comments]. Science 258: 1952-1955,

1992.

FABIATO, A. Calcium-induced release of calcium from the cardiac sarcoplasmic reticulum. Am. J.

Physiol. 245: Cl-14, 1983.

FABIATO, A. Rapid ionic modifications during the aequorin-detected calcium transient in a skinned canine cardiac purkinje cell. J. Gen. Physiol. 85: 189-246, 1985a.

FABIATO, A. Time and calcium dependence of activation and inactivation of calcium-induced release of calcium from the sarcoplasmic reticulum of a skinned canine cardiac purkinje cell. J. Gen. Physiol.

148 85: 247-289, 1985b.

FABIATO, A. Simulated calcium current can both cause calcium loading in and trigger calcium

release from the sarcoplasmic reticulum of a skinned canine cardiac purkinje cell. J. Gen. Physiol. 85.

291-320, 1985c.

FABIATO, A AND FABIATO, F. Effects o f # on the myofilaments and the sarcoplasmic reticulum

of skinned cells from cardiace and skeletal muscles. J. Physiol. (Lond) 276: 233-255, 1978.

FADEN, A. I. AND FEUERSTEIN, G. Hypothalamic regulation of the cardiovascular and respiratory

systems: role of specific opiate receptors. Br. J. Pharmacol. 79: 997-1002, 1983.

FAN GH, ZHAO J, WU YL, LOU LG, ZHANG Z, JING Q, MA L AND PEI G:

N-Methyl-D-Aspartate attenuates opioid receptor mediated G protein activation and this process

involves protein kinase C. Mol. Pharmacol. 53: 684-690, 1998.

FEUERSTEIN, G, ZERBE, R L. AND FADEN, A. I. Opiate receptors and cardiovascular control in conscious shr and wky rats. Hypertension 5: 663-671, 1983.

149 FEUERSTEIN, G. The opioid system and central cardiovascular control: analysis of controversies.

Peptides 6 Suppl 2: 51-56, 1985.

FEUERSTEIN, G. AND FADEN, A. I. Differential cardiovascular effects of mu, delta and kappa

opiate agonists at discrete hypothalamic sites in the anesthetized rat. Life Sci. 31: 2197-2200, 1982.

FUKUDA, K., KATO, S., MORI, K , NISEI, M. AND TAKESHIMA, H. Primary structures and

expression from ci>MA of rat opioid receptor delta- and mu-subtypes. FEES Lett. 327: 311-314, 1993.

FUKUSHMA, N , UEDA, H., HAYASHI, C , KATAYAMA, T., MTYAMAE, T. AND MISU, Y.

Species and age-dependent differences of functional coupling between opioid delta-receptor and

Ç --proteins and possible involvement of protein kinase Ç in striatal membranes. Neurosci. Lett. 176:

55-58, 1994.

GACEL, G. A , FELT ION, E., BAAMONDE, A , DAUGE, V. AND ROQUES, B. P. Synthesis, biochemical and pharmacological properties of bubuc, a highly selective and systemically active

agonist for in vivo studies of delta-opioid receptors. Peptides 11: 983-988, 1990.

GAIRIN, J. E., GOUARDERES, C , MAZARGUIL, H., ALVINERIE, P. AND CROS, J.

150 [d-prolO]dynorphin-(l-l 1) is a highly potent and selective ligand for kappa opioid receptors. Eur. J.

Pharmacol. 106: 457-458, 1984.

GAO, W. D., ATAR, D., BACKX, P. H. AND MARBAN, E. Relationship between intracellular calcium and contractile force in stunned myocardium, direct evidence for decreased myofilament Ca2+ responsiveness and altered diastolic function in intact ventricular muscle. Circ. Res. 76. 1036-1048,

1995.

GILBERT, P. E. AND MARTIN, W. R. The effects of morphine and nalorphine-like drugs in the nondependent, morphine-dependent and cyclazocine-dependent chronic spinal dog. J. Pharmacol.

Exp. Ther. 198: 66-82, 1976.

GOLDSTEIN, A , LOWNEY, L. I. AND PAL, B. K. Stereospecific and nonspecific interactions of the morphine congener levorphanol in subcellular fractions of mouse brain. Proc. Natl. Acad. Sci. U.

S. A. 68: 1742-1747, 1971.

GOLDSTEIN, A, FISCHLI, W., LOWNEY, L. I., HÜNKAPILLER, M. AND HOOD, L. Porcine pituitary dynorphin: complete amino acid sequence of the biologically active heptadecapeptide. Proc.

Natl. Acad. Sci. U. S. A. 78: 7219-7223, 1981.

151 GOODMAN, R. R , SNYDER, S. H., KUHAR, M. J. AND YOUNG, W. S.,3D: Differentiation of

delta and mu opiate receptor localizations by light microscopic autoradiography. Proc. Natl. Acad.

Sci. U. S. A. 77: 6239-6243, 1980.

GORDON, F. J. Central opioid receptors and baroreflex control of sympathetic and cardiovascular

function. J. Pharmacol. Exp. Ther. 237: 428-436, 1986.

GORDON, F. J. Opioids and central baroreflex control: a site of action in the nucleus tractus

solitarius. Peptides 11: 305-309, 1990.

GORDON, F. J. Opioids and the nucleus of the tractus solitarius: effects on cardiovascular and

baroreflex function. In Nucleus of the solitary tract, ed. by A. I. Robin, pp. 283-287, CRC press Inc,

1994.

GRADY, E. F., BOHM, S. K. AND BUNNETT, N. W. Turning off the signal: mechanisms that

attenuate signaling by 6 protein-coupled receptors. Am. J. Physiol. 273: G5 86-601, 1997.

HALL, E. D., WOLF, D. L. AND MCCALL, R. B. Cardiovascular depressant effects of the kappa

opioid receptor agonistsU50488h and spiradoline mesylate. Circ. Shock 26: 409-417, 1988.

152 HANBAUER, I, KELLY, G. D., SAIANI, L. AND YANG, H. Y. [met5]-enkephalin-like peptides of the adrenal medulla: release by nerve stimulation and functional implications. Peptides 3: 469-473,

1982.

HANDA, B. K., LAND, A. C , LORD, J. A., MORGAN, B. A , RANCE, M. J. AND SMITH, C.

F. Analogues of beta-lph61-64 possessing selective agonist activity at mu-opiate receptors. Eur. J.

Pharmacol. 70: 531-540, 1981.

HASBI, A, POLASTRON, J., ALLOUCHE, S., STANASILA, L., MAS SOTTE, D. AND JAUZAC,

P. Desensitization of the delta-opioid receptor correlates with its phosphorylation in sk-n-be cells: involvement of a § protein-coupled receptor kinase. J. Neurochem. 70: 2129-2138, 1998.

HASSEN, A. H., FEUERSTEIN, G. AND FADEN, A. I. Mu receptors and opioid cardiovascular effects in the nts of rat. Peptides 3: 1031-1037, 1982.

HASSEN, A H., FEUERSTEIN, G. AND FADEN, A I. Differential cardiovascular effects mediated by mu and kappa opiate receptors in hindbrain nuclei. Peptides 4: 621-625, 1983.

HASSEN, A. H. AND FEUERSTEIN, G. Mu-opioid receptors in nts elicit pressor responses via

153 sympathetic pathways. Am. J. Physiol. 252: HI 56-62, 1987.

HAYES, A. G., BIRCH, P. J., HAYWARD, N. J., SHEEHAN, M. J , ROGERS, H , TYERS, M.

B., JUDD, D. B., SCOPES, D. I. AND NAYLOR, A. A series of novel, highly potent and selective agonists for the kappa-opioid receptor. Br. J. Pharmacol. 101: 944-948, 1990.

HENDERSON, G., HUGHES, J. AND KOSTERLITZ, H. W. A new example of a morphine-sensitive neuro-effector junction: adrenergic transmission in the mouse vas deferens. Br.

J. Pharmacol. 46: 764-766, 1972.

HERING, H. E. Der Karotisdruckversuch. Muench. Med. Wochschr 70: 1287-1290, 1923.

HONDA, C. N. AND ARVIDSSON, U. Immunohistochemical localization of delta- and mu-opioid receptors in primate spinal cord. Neuroreport. 6: 1025-1028, 1995.

HORAN, P., TAYLOR, J., YAMAMURA, H. I. AND PORRECA, F. Extremely long-lasting antagonistic actions of nor-binaltorphimine (nor-bni) in the mouse tail-flick test. J. Pharmacol. Exp.

Ther. 260: 1237-1243, 1992.

154 HOVAV, E. AND WEINSTOCK, M. Temporal factors influencing the development of acute tolerance to opiates. J. Pharmacol. Exp. Ther. 242: 251-256, 1987.

HUGHES, J., SMITH, T. W., KOSTERLITZ, H. W., FOTHERGELL, L. A, MORGAN, B. A. AND

MORRIS, H. R. Identification of two related pentapeptides from the brain with potent opiate agonist activity. Nature 258: 577-580, 1975.

HUNTER, J. C , LEIGHTON, G. E., MEECHAM, K. G, BOYLE, S. J., HORWELL, D. C , REES,

D. C. AND HUGHES, J. Ci-977, a novel and selective agonist for the kappa-opioid receptor. Br. J.

Pharmacol. 101: 183-189, 1990.

ISOM, L. L., CRAGOE, E. I,JR. AND LIMBIRD, L. E. Multiple receptors linked to inhibition of adenylate cyclase accelerate8ja+/H+ exchange in neuroblastoma x glioma cells via a mechanism other c bftf than decreased accumulation [published erratum appears in j biol chem 1988 nov 5;

263(31):16513]. J. Biol. Chem. 262: 17504-17509, 1987.

JACKSON, D. E. A note on the pharmacological action of opium alkaloids. J. Pharmacol. Exp. Ther

6: 57-72, 1914.

155 JAMES, I. F. AND GOLDSTEIN, A. Site-directed alkylation of multiple opioid receptors, i. binding selectivity. Mol. Pharmacol. 25: 337-342, 1984.

JIANG, Q , SEBASTIAN, A., ARCHER, S. AND BIDLACK, J. M. 5 beta-methyl-14 beta-(p-nitrocinnamoylamino)-7,8-dihydromorphinone and its corresponding n-cyclopropylmethyl analog, n-cyclopropylmethylnor-5 beta-methyl-14 beta-(p-nitrocinnamoylamino)-

7,8-dihydromorphinone: mu-selective irreversible opioid antagonists. J. Pharmacol. Exp. Ther. 268:

1107-1113, 1994.

KAILA, K. AND VAUGHAN-JONES, R. D. Influence of sodium-hydrogen exchange on intracellular

Si, sodium and tension in sheep cardiac purkinje fibres. J. Physiol. (Lond) 390: 93-118, 1987.

KAKIDANI, H , FURUTANI, Y, TAKAHASHI, H., NOD A, M., MORIMOTO, Y., HIROSE, T.,

ASAI, M., INAYAMA, S., NAKANISHI, S. AND NUMA, S. Cloning and sequence analysis of-eWA for porcine beta-neo-endorphin/dynorphin precursor. Nature 298: 245-249, 1982.

KALIA, M., FUXE, K., HOKFELT, T., JOHANSSON, O., LANG, R., GANTEN, D., CUELLO,

C. AND TERENIUS, L. Distribution of neuropeptide immunoreactive nerve terminals within the subnuclei of the nucleus of the tractus solitarius of the rat. J. Comp. Neurol. 222: 409-444, 1984.

156 KASPER, E., VENTURA, C., ZIMAN, B. D., LAKATTA, E. G , WEISMAN, H. AND

CAPOGROSSI, M. C Effect ofU-50,488h on the contractile response of cardiomyopathic hamster ventricular myocytes. Life Sci. 50: 2029-2035, 1992.

KATO, S., FUKUDA, K., MORIKAWA, H., SKODA, T., MIMA, H. AND MORI, K. Adaptations to chronic agonist exposure of mu-opioid receptor-expressing Chinese hamster ovary cells. Eur. J.

Pharmacol. 345: 221-228, 1998.

KATZ, R., KARLDNER, J. S. AND RESNIK, R. Effects of a natural volume overload state

(pregnancy) on left ventricular performance in normal human subjects. Circulation 58: 434-441, 1978.

KAZMIERSKI, W., WIRE, W. S., LUI, G. K., KNAPP, R. J., SHOOK, J. E., BURKS, T. R,

YAMAMURA, H. I. AND HRUBY, V. J. Design and synthesis of somatostatin analogues with topographical properties that lead to highly potent and specific mu opioid receptor antagonists with greatly reduced binding at somatostatin receptors. J. Med. Chem. 31: 2170-2177, 1988.

KELLER-WOOD, M. Corticotropin responses to hypoglycemia and hypotension during ovine pregnancy. Am. J. Physiol. 266: R180-7, 1994a.

157 KELLER-WOOD, M. Vasopressin response to hyperosmolality and hypotension during ovine

pregnancy. Am. J. Physiol. 266: R188-93, 1994b.

KIANG, j . G. AND COLDEN-STANFIELD, M. Morphine induces an intracellular alkalinization in

bovine aortic endothelial cells (baecs). Prog. Clin. Biol. Res. 328: 137-140, 1990.

KIANG, J. G. AND WEI, E. T. Peripheral opioid receptors influencing heart rate in rats: evidence

for endogenous tolerance. Regul. Pept. 8: 297-303, 1984.

KIEFFER, B. L., BEFORT, K., GAVERIAUX-RUFF, C. AND HIRTH, C. G. The delta-opioid

receptor: isolation of a cQH&by expression cloning and pharmacological characterization. Proc. Natl.

Acad. Sci. U. S. A. 91: 11931994.

KIEFFER, B. L. Recent advances in molecular recognition and signal transduction of active peptides:

receptors for opioid peptides. Cell Mol. Neurobiol. 15: 615-635, 1995.

KIRITSY-ROY, J. A., MARSON, L. AND VAN LOON, G. R. Sympathoadrenal, cardiovascular and blood gas responses to highly selective mu and delta opioid peptides. J. Pharmacol. Exp. Ther. 251:

1096-1103, 1989.

158 KISSIN, L, LEE, S. S., ARTHUR, G. R AND BRADLEY, E. L. Time course characteristics of acute tolerance development to continuously infused alfentanil in rats. Anesth. Analg. 83. 600-605,

1996.

KNAPP, R J., MALATYNSKA, E , FANG, L., LI, X., BABIN, E , NGUYEN, M., SANTORO, G ,

VARGA, E. V., HRUBY, V. J., ROESKE, W. R. AND ET AL: Identification of a human delta opioid receptor: cloning and expression. Life Sci. 54: PL463-9, 1994.

KOESTER, G. AND TSCHERMAK, A. Uber Ursprung und Endigung des Nervus depressor und

Nervus laryneus superior beim Kaninchien. Arch. Anat. Entwickelungeschicht 25. 255-294, 1902.

KORNER, P. I., WEST, M. J., SHAW, J. AND UTHER, J. B. 'steady-state' properties of the baroreceptor-heart rate reflex in essential hypertension in man. Clin. Exp. Pharmacol. Physiol. 1.

65-76, 1974.

KOSTERLITZ, H. W., LORD, J. A., PATERSON, S. J. AND WATERFIELD, A. A. Effects of changes in the structure of enkephalins and of narcotic analgesic drugs on their interactions with mu- and delta-receptors. Br. J. Pharmacol. 68: 333-342, 1980.

159 KOSTERLITZ, H. W., PATERSON, S. J. AND ROBSON, L. E. Characterization of the kappa-subtype of the opiate receptor in the guinea-pig brain. Br. J. Pharmacol. 73. 939-949, 1981.

KOSTERLITZ, H. W. AND WATERFIELD, A. A. In vitro models in the study of structure-activity relationships of narcotic analgesics. Annu. Rev. Pharmacol. 15: 29-47, 1975.

KREIL, G , BARRA, D., SIMMACO, M., ERSPAMER, V., ERSPAMER, G. F., NEGRI, L.,

SEVEREST, C , CORSI, R AND MELCFUORRI, P. Deltorphin, a novel amphibian skin peptide with high selectivity and affinity for delta opioid receptors. Eur. J. Pharmacol. 162: 123-128, 1989.

KRUMNS, S. A, FADEN, A. I. AND FEUERSTEIN, G. Opiate binding in rat hearts: modulation of binding after hemorrhagic shock. Biochem. Biophys. Res. Commun. 127: 120-128, 1985.

LAHTI, R. A., VON VOIGTLANDER, P. F. AND BARSUHN, C. Properties of a selective kappa agonist, U-50,488h. Life Sci. 31: 2257-2260, 1982.

LAHTI, R. A., MICKELSON, M. M., MCCALL, J. M. AND VON VOIGTLANDER, P. F.

[3h]u-69593 a highly selective ligand for the opioid kappa receptor. Eur. J. Pharmacol. 109: 281-284,

1985.

160 LAURENT, S., MARSH, J. D. AND SMITH, T. W. Enkephalins have a direct positive inotropic effect on cultured cardiac myocytes. Proc. Natl. Acad. Sci. U. S. A. 82: 5930-5934, 1985.

LAURENT, S., MARSH, J. D. AND SMITH, T. W. Enkephalins increase cyclic adenosine monophosphate content, calcium uptake, and contractile state in cultured chick embryo heart cells.

J. Clin. Invest. 77: 1436-1440, 1986.

LEVI, A. J., BOYETT, M. R. AND LEE, C. O. The cellular actions of digitalis glycosides on the heart. Prog. Biophys. Mol. Biol. 62: 1-54, 1994.

LEVINE, A. S, GRACE, M„ BÏÏJL1NGTON, C. J. AND PORTOGHESE, P S. Nor-binaltorphimine decreases deprivation and opioid-induced feeding. Brain Res. 534: 60-64, 1990.

LEWIS, M. E., KHACHATURIAN, H. AND WATSON, S. J. Combined autoradiographic-immunocytochemical analysis of opioid receptors and opioid peptide neuronal systems in brain. Peptides 6 Suppl 1: 37-47, 1985.

LI, C. H. AND CHUNG, D. Isolation and structure of an untriakontapeptide with opiate activity from camel pituitary glands. Proc. Natl. Acad. Sci. U. S. A. 73: 1145-1148, 1976.

161 LI, S., ZHU, J., CHEN, C , CHEN, Y. W., DERIEL, J. K., ASHBY, B. AND LIU-CHEN, L. Y.

Molecular cloning and expression of a rat kappa opioid receptor. Biochem. J. 295: 629-633, 1993.

LINDHARD, J. Uber das Minutenvolume des Herzens bei Rube und bei Muskelarbeit. Pflugers Arch

161: 223-227, 1915.

LLOBELL, F. AND LAORDEN, M. L. Characterization of the opioid receptor subtypes mediating the negative inotropic effects of Bamgo, Qpdpe and Ü-50, 48814 in isolated human right atria strips.

Neuropeptides 29: 115-119, 1995.

LOH, H. H., TAO, P. L. AND SMITH, A. P. Role of receptor regulation in opioid tolerance mechanisms. Synapse 2: 457-462, 1988.

LOH, H. H. AND SMITH, A. P. Molecular characterization of opioid receptors. Annu. Rev.

Pharmacol. Toxicol. 30: 123-147, 1990.

LORD, J. A, WATERFIELD, A. A., HUGHES, J. AND KOSTERLITZ, H. W. Endogenous opioid peptides: multiple agonists and receptors. Nature 267: 495-499, 1977.

162 MAGNAN, J., PATERSON, S. J., TAVANI, A. AND KOSTERLITZ, H. W. The binding spectrum of narcotic analgesic drugs with different agonist and antagonist properties. Naunyn Schmiedebergs

Arch. Pharmacol. 319: 197-205, 1982.

MAGNES S, R. R. AND ROSENFELD, C. R. Mechanisms for attenuated pressor responses to alpha-agonists in ovine pregnancy. Am. J. Obstet. Gynecol. 159: 252-261, 1988.

MANTELLI, L., CORTI, V. AND LEDDA, F. On the presence of opioid receptors in guinea-pig ventricular tissue. Gen. Pharmacol. 18: 309-313, 1987.

MASH1NI, I. S., ALBAZZAZ, S. J., FADEL, H. E., ABDULLA, A. M., HADI, H. A., HARP, R.

AND DEVOE, L. D. Serial noninvasive evaluation of cardiovascular hemodynamics during pregnancy. Am. J. Obstet. Gynecol. 156: 1208-1213, 1987.

MATSUMURA, K., ABE, L, TOMTNAGA, M., TSUCHMASHI, T., KOBAYASHI, K. AND

FUJISHIMA, M. Differential modulation by mu- and delta-opioids on baroreceptor reflex in conscious rabbits. Hypertension 19: 648-652, 1992.

MAY, C. N., DASHWOOD, M. R., WHITEHEAD, C. J. AND MATHIAS, C. J. Differential

163 cardiovascular and respiratory responses to central administration of selective opioid agonists in conscious rabbits: correlation with receptor distribution. Br. J. Pharmacol. 98: 903-913, 1989.

MICOL, J. A. AND LAORDEN, M. L. Effects of mu-, delta- and kappa-agonists on isolated right atria of the rat. Neuropeptides 26: 365-370, 1994.

MORIKAWA, H., FUKUDA, K., MIMA, H., SHODA, T., KATO, S. AND MORI, K.

Desensitization and resensitization of delta-opioid receptor-mediated Ga2+ channel inhibition in ngl08-15 cells. Br. J. Pharmacol. 123: 1111-1118, 1998.

MORITA, H., NISHIDA, Y., UEMURA, N. AND HOSOMI, H. Effect of pentobarbital anesthesia on renal sympathetic nerve activity in the rabbit. J. Auton. Nerv. Syst. 20: 57-64, 1987.

MOSBERG, H. I., HURST, R , HRUBY, V. 1, GEE, K., YAMAMURA, H. L, GALLIGAN, J. J.

AND BURKS, T. F. Bis-penicillamine enkephalins possess highly improved specificity toward delta opioid receptors. Proc. Natl. Acad. Sci. U. S. A. 80: 5871-5874, 1983.

MURTHY, V. S., ZAGGER, M. E., VOLLMER, R. R. AND SCHMIDT, D. H.

Pentobarbital-induced changes in vagal tone and reflex vagal activity in rabbits. Eur. J. Pharmacol.

164 84:41-51, 1982.

MUSHA, T., SATOH, E , KOYANAGAWA, H., KMURA, T. AND SATOH, S. Effects of opioid agonists on sympathetic and parasympathetic transmission to the dog heart. J. Pharmacol. Exp. Ther.

250: 1087-1091, 1989.

NAKANISHI, S., INDUE, A, KITA, T., NAKAMURA, M., CHANG, A. C., COHEN, S. N. AND

NUMA, S. Nucleotide sequence of cloned dEMAfor bovine corticotropin-beta-lipotropin precursor.

Nature 278: 423-427, 1979.

NISHMURA, E., BUCHAN, A. M. AND MCINTOSH, C H. Autoradiographic localization of mu- and delta-type opioid receptors in the gastrointestinal tract of the rat and guinea pig. Gastroenterology

91: 1084-1094, 1986.

NOCK, B., GIORDANO, A. L. AND CICERO, T. J. Ici 197067 is a selective ligand for the u-69593-sensitive kappa opiate binding site. Eur. J. Pharmacol. 162: 385-386, 1989.

NODA, M , FURUTANI, Y., TAKAHASHI, H., TOYOSATO, M., HIROSE, T., INAYAMA, S.,

NAKANISHH, S. AND NUMA, S. Cloning and sequence analysis of cDNA for bovine adrenal

165 preproenkephalin. Nature 295: 202-206, 1982.

NUMAO, Y., SIATO, M., TERUI, N. AND KUMADA, M. The aortic nerve-sympathetic reflex in the rat. J. Auton. Nerv. Syst. 13: 65-79, 1985.

OAKES, G. K., EHRENKRANZ, R. A., WALKER, A. M., MCLAUGHLIN, M. K., BRENNAN,

S. C. AND CHEZ, R A. Effect of alpha-adrenergic agonist and antagonist infusion on the umbilical and uterine circulations of pregnant sheep. Biol. Neonate 38: 229-237, 1980.

OGUTMAN, C, KAPUTLU, I. AND SAD AN, G. Cardiovascular effects of intrathecally injected mu, delta and kappa opioid receptor agonists in rabbits. J. Auton. Pharmacol. 443-446, 1995.

OLIANAS, M. C. AND ONALI, P. Characterization of opioid receptors mediating stimulation of adenylate cyclase activity in rat olfactory bulb. Mol. Pharmacol. 42: 109-115, 1992.

OLIANAS, M. C. AND ONALI, P. Activation of opioid and muscarinic receptors stimulates basal adenylyl cyclase but inhibits Ch2+/calmodulin- and forskolin-stimulated enzyme activities in rat olfactory bulb. J. Neurochem. 63: 161-168, 1994.

166 OLMSTED, S. L, TAKEMORI, A. E. AND PORTOGHESE, P. S. A remarkable change of opioid

receptor selectivity on the attachment of a peptidomimetic kappa address element to the delta

antagonist, natrindole: 5,-[n2-alkylamidino)methyl]naltrindole derivatives as a novel class of kappa opioid receptor antagonists. J. Med. Chem. 36: 179-180, 1993.

OLSSON, K., ANDEN, N. E., JOHANSSON, K. AND THORNSTROM, U. Effects of acute haemorrhagic hypotension during pregnancy and lactation in conscious goats. Acta Physiol. Scand.

129: 479-487, 1987.

OMONIYI, A. T., KETT, A , WU, D., SOONG, Y. AND SZETO, H. H. A peripheral site and mechanism of of action for the blunting of baroreflex-mediated bradycardia by intravenous mu agonists. J. Cardiovasc. Pharmacol. 1998a.

OMONIYI, A. T., WU, D. L., SOONG, Y. AND SZETO, H. H. Baroreflex-mediated bradycardia is blunted by intravenous mu- but not kappa-opioid agonists. J. Cardiovasc. Pharmacol. 1998b.

PACKER, M. Vasodilator and inotropic drugs for the treatment of chronic heart failure: distinguishing hype from hope. J. Am. Coll. Cardiol. 12: 1299-1317, 1988.

167 PAK, Y., KOUVELAS, A., SCHEIDELER, M. A., RASMUSSEN, J., O'DOWD, B. F. AND

GEORGE, S. R. Agonist-induced functional desensitization of the mu-opioid receptor is mediated

by loss of membrane receptors rather than uncoupling from Ci protein. Mol. Pharmacol. 50:

1214-1222, 1996.

PARRA, L., PEREZ-VIZCAINO, F., ALSASUA, A., MARTIN, M. I. AND TAMARGO, J. Mu- and delta-opioid receptor-mediated contractile effects on rat aortic vascular smooth muscle. Eur. J.

Pharmacol. 277: 99-105, 1995.

PEI, G , KIEFFER, B. L., LEFKOWITZ, R. J. AND FREEDMAN, N. J. Agonist-dependent phosphorylation of the mouse delta-opioid receptor: involvement of Ci protein-coupled receptor kinases but not protein kinase 0. Mol. Pharmacol. 48: 173-177, 1995.

PERT, C. B. AND SNYDER, S. H. Opiate receptor: demonstration in nervous tissue. Science 179:

1011-1014, 1973.

PETTY, M. A. AND REID, J. L. The effect of opiates on arterial baroreceptor reflex function in the rabbit. Naunyn Schmiedebergs Arch. Pharmacol. 319: 206-211, 1982.

168 PFEIFFER, A, FEUERSTEIN, G , FADEN, A. AND KOPIN, I. J. Evidence for an involvement of

mu-, but not delta- or kappa-opiate receptors in sympathetically and parasympathetically mediated

cardiovascular responses to opiates upon anterior hypothalamic injection. Life Sci. 31: 1279-1282,

1982.

PFEIFFER, A, FEUERSTEIN, G, KOPIN, I. J. AND FADEN, A. I. Cardiovascular and respiratory

effects of mu-, delta- and kappa-opiate agonists microinjected into the anterior hypothalamic brain area of awake rats. J. Pharmacol. Exp. Ther. 225: 735-741, 1983a.

PFEIFFER, A, FEUERSTEIN, G, ZERBE, R. L., FADEN, A. I. AND KOPIN, I. J. Mu-receptors mediate opioid cardiovascular effects at anterior hypothalamic sites through sympatho-adrenomedullary and parasympathetic pathways. Endocrinology 113: 929-938, 1983b.

POLASTRON, J., BOYER, M. J., QUERTERMONT, Y., THOUVENOT, J. P., MEUNIER, J. C.

AND JAUZAC, P. Mu-opioid receptors and not kappa-opioid receptors are coupled to the adenylate cyclase in the cerebellum. J. Neurochem. 54: 562-570, 1990.

PORTOGHESE, P. S. A new concept on the mode of interaction of narcotic analgesics with receptors. J. Med. Chem. 8: 605-616, 1965.

169 PORTOGHESE, P. S., LARSON, D. L., SAYRE, L. M., FRIES, D. S. AND TAKEMORI, A. E.

A novel opioid receptor site directed alkylating agent with irreversible narcotic antagonistic and

reversible agonistic activities. J. Med. Chem. 23: 233-234, 1980.

PORTOGHESE, P. S., LIPKOWSKI, A. W. AND TAKEMORI, A. E. Bimorphinans as highly

selective, potent kappa opioid receptor antagonists. J. Med. Chem. 30: 238-239, 1987.

PORTOGHESE, P. S., SULTANA, M. AND TAKEMORI, A. E. Naltrindole, a highly selective and potent non-peptide delta opioid receptor antagonist. Eur. J. Pharmacol. 146: 185-186, 1988.

PUGSLEY, M. K , PENZ, W. P., WALKER, M. J. AND WONG, T. M. Cardiovascular actions of the kappa-agonist, U-50,488h, in the absence and presence of opioid receptor blockade. Br. J.

Pharmacol. 105: 521-526, 1992.

RANDICH, A., ROBERTSON, J. D. AND WILLINGHAM, T. The use of specific opioid agonists

and antagonists to delineate the vagally mediated antinociceptive and cardiovascular effects of

intravenous morphine. Brain Res. 603: 186-200, 1993.

RAYNOR, K., KONG, H , CHEN, Y., YASUDA, K., YU, L., BELL, G. I. AND REISINE, T.

170 Pharmacological characterization of the cloned kappa-, delta-, and mu-opioid receptors. Mol.

Pharmacol. 45: 330-334, 1994a.

RAYNOR, K„ KONG, H., HINES, J., KONG, G, BENOVIC, J., YASUDA, K., BELL, G. I. AND

REISINE, T. Molecular mechanisms of agonist-induced desensitization of the cloned mouse kappa opioid receptor. J. Pharmacol. Exp. Ther. 270: 1381-1386, 1994b.

RAYNOR, K , KONG, H , MESTEK, A., BYE, L. S., TIAN, M., LIU, I , YU, L. AND REISINE,

T. Characterization of the cloned human mu opioid receptor. J. Pharmacol. Exp. Ther. 272: 423-428,

1995.

REISINE, T. AND BELL, G. I. Molecular biology of opioid receptors [see comments]. Trends.

Neurosci. 16: 506-510, 1993.

REND A, T., NEGRI, L , TOOYAMA, L, CASU, C. AND MELCHIORRI, P. Autoradiographic study on [3h]-[d-ala2]-deltorphin-i binding sites in the rat brain. Neuroreport. 4: 1143-1146, 1993.

ROBSON, S. C., HUNTER, S., BOYS, R. J. AND DUNLOP, W. Serial study of factors influencing changes in cardiac output during human pregnancy. Am. J. Physiol. 256: H I060-5, 1989.

171 ROEMER, D., BUES CHER, H. H., HILL, R. C, PLESS, J., BAUER, W., CARDINAUX, F.,

CLOSSE, A., HAUSER, D. AND HUGUENIN, R A synthetic enkephalin analogue with prolonged parenteral and oral analgesic activity. Nature 268: 547-549, 1977.

ROGERS, H., BIRCH, P. J., HARRISON, S. ML, PALMER, E., MANCHEE, G. R , JUDD, D. B.,

NAYLOR, A., SCOPES, D. I. AND HAYES, A. G. Gr94839, a kappa-opioid agonist with limited access to the central nervous system, has antinociceptive activity. Br. J. Pharmacol. 106: 783-789,

1992.

ROMAGNANO, M. A. AND HAMILL, R. W. Spinal sympathetic pathway: an enkephalin ladder.

Science 225: 737-739, 1984.

SAEKI, T., NISHMURA, M., SATO, N., FUJINAMI, T. AND WATANABE, Y.

Electrophysiological demonstration and activation of mu-opioid receptors in the rabbit sinoatrial node.

J. Cardiovasc. Pharmacol. 26: 160-168, 1995.

SAME, A, BICKEL, U., STROTH, U. AND PARDRIDGE, W. M. Blood-brain barrier transport of neuropeptides: analysis with a metabolically stable dermorphin analogue. Am. J. Physiol. 267:

E124-31, 1994.

172 SATOH, M. AND MINAM, M. Molecular pharmacology of the opioid receptors. Pharmacol. Ther.

68: 343-364, 1995.

SCHAZ, K., STOCK, G., SIMON, W., SCHLOR, K. H., UNGER, T., ROCKHOLD, R. AND

GANTEN, D. Enkephalin effects on blood pressure, heart rate, and baroreceptor reflex. Hypertension

2: 395-407, 1980.

SCHILLER, P. W., NGUYEN, T. M., CHUNG, N. N. AND LEMEUX, C. Dermorphin analogues carrying an increased positive net charge in their "message" domain display extremely high mu opioid receptor selectivity. J. Med. Chem. 32: 698-703, 1989.

SCHILLER, P. W., NGUYEN, T. M., CHUNG, N. N., DIONNE, G. AND MARTEL, R. Peripheral antinociceptive effect of an extremely mu-selective polar dermorphin analog (dalda). Prog. Clin. Biol.

Res. 328: 53-56, 1990.

SCELLER, P. W., NGUYEN, T. M., WELTROWSKA, G, WILKES, B. C , MARSDEN, B. I ,

LEMEUX, C AND CHUNG, N. N. Differential stereochemical requirements of mu vs. delta opioid receptors for ligand binding and signal transduction: development of a class of potent and highly delta-selective peptide antagonists. Proc. Natl. Acad. Sci. U. S. A. 89: 11871-11875, 1992.

173 SCHILLER, P. W., WELTROWSKA, G., NGUYEN, T. M., WILKES, B. C , CHUNG, N. N. AND

LEMEUX, C. Tippfpsi]: a highly potent and stable pseudopeptide delta opioid receptor antagonist with extraordinary delta selectivity. J. Med. Chem. 36: 3182-3187, 1993.

SCHULTZBERG, M., HOKFELT, T., TERENIUS, L., ELFVIN, L. G., LUNDBERG, J. M.,

BRANDT, J., ELDE, R. P. AND GOLDSTEIN, M. Enkephalin immunoreactive nerve fibres and cell bodies in sympathetic ganglia of the guinea-pig and rat. Neuroscience 4: 249-270, 1979.

SELIGMAN, S. A. Baro-receptor reflex function in pre-eclampsia. J. Reprod. Fertil. 26: 2811971.

SIMON, E. J., HILLER, J. M. AND EDELMAN, I. Stereospecific binding of the potent narcotic analgesic (3h) etorphine to rat-brain homogenate. Proc. Natl. Acad. Sci. U. S. A. 70: 1947-1949,

1973.

SIREN, A. L. AND FEUERSTEIN, G. Hypothalamic opioid mu-receptors regulate discrete hemodynamic functions in the conscious rat. Neuropharmacology 30: 143-152, 1991.

SMYTHE, H. S., SLEIGHT, P. AND PICKERING, G. W. Reflex regulation of arterial pressure during sleep in man: a quantitative method of assessing barorefiex sensitivity. Circ. Res. 24: 109-121,

174 1969.

STERMNI, C , SPANN, M., ANTON, B., KEITH, D. E.,JK, BUNNETT, N. W., VON

ZASTROW, M., EVANS, C. AND BRECHA, N. C Agonist-selective endocytosis of mu opioid

receptor by neurons in vivo. Proc. Natl. Acad. Sci. U. S. A. 93: 9241-9246, 1996.

TABSH, K , RUDELSTORFER, R , NUWAYHID, B. AND ASSALI, N. S. Circulatory responses

to hypovolemia in the pregnant and nonpregnant sheep after pharmacologic sympathectomy. Am. J.

Obstet. Gynecol. 154: 411-419, 1986.

TAI, K. K., BIAN, C. F. AND WONG, T. M. Kappa-opioid receptor stimulation increases intracellular free calcium in isolated rat ventricular myocytes. Life Sci. 51: 909-913, 1992.

TERENIUS, L. Stereospecific interaction between narcotic analgesics and a synaptic plasm a membrane fraction of rat cerebral cortex. Acta Pharmacol. Toxicol. (Copenh) 32: 317-320, 1973.

TOTH, G , KRAMER, T. H , KNAPP, R., LUI, G , DAVIS, P., BURKS, T. F., YAMAMURA, H.

I. AND HRUBY, V. J. [d-pen2,d-pen5]enkephalin analogues with increased affinity and selectivity for delta opioid receptors. J. Med. Chem. 33: 249-253, 1990.

175 TWITCHELL, W. A. AND RANE, S. G. Nucleotide-independent modulation of ca(2+)-dependent k+ channel current by a mu-type opioid receptor. Mol. Pharmacol. 46: 793-798,1994.

UHL, G. K , CHILDERS, S. AND PASTERNAK, G. An opiate-receptor gene family reunion.

Trends. Neurosci. 17: 89-93, 1994.

VANHOUTTE, P. M., HUMPHREY, P. P. AND SPEDDING, M. X. international union of pharmacology recommendations for nomenclature of new receptor subtypes. Pharmacol. Rev. 48: 1-2,

1996.

VAUGHAN-JONES, R. D , WU, M. L. AND BOUNTRA C. Sodium-hydrogen exchange and its role in rnntrnlling contractility during acidosis in cardiac muscle. Mol. Cell Biochem. 89: 157-162,

1989.

VEGH, A , PAPP, J. G., UDVARY, E. AND KASZALA K. Hemodynamic effects of calcium-sensitizing agents. J. Cardiovasc. Pharmacol. 26 Suppl 1: S20-31, 1995.

VENTURA C., MUSCARI, C., SPAMPINATO, S., BERNARDI, P. AND CALDARERA, C. M.

Inhibitory action of opioid peptides on ouabain-sensitive tia+-K+ and €a2+-dependent atpase activities

176 in bovine cardiac sarcolemma. Peptides 8: 709-713, 1987.

VENTURA, C, CAPOGROSSI, M. C, SPURGEON, H. A. AND LAKATTA, E. G. Kappa-opioid peptide receptor stimulation increases cytosolic Bti and myofilament responsiveness to

VENTURA, C., SPURGEON, H., LAKATTA, E. G., GUARNIERI, C. AND CAPOGROSSI, M.

C. Kappa and delta opioid receptor stimulation affects cardiac myocyte function and €a2+ release from an intracellular pool in myocytes and neurons. Circ. Res. 70: 66-81, 1992.

VERED, Z , POLER, S. M., GIBSON, P., WLODY, D. AND PEREZ, J. E. Noninvasive detection of the morphologic and hemodynamic changes during normal pregnancy. Clin. Cardiol. 14: 327-334,

1991.

VINIK, H. R. AND KISSIN, I. Rapid development of tolerance to analgesia during remifentanil infusion in humans. Anesth. Analg. 86: 1307-1311, 1998.

VONVOIGTLANDER, P. P., LAHTI, R. A. AND LUDENS, J. H. U-50,488: a selective and structurally novel non-mu (kappa) opioid agonist. J. Pharmacol. Exp. Ther. 224: 7-12, 1983.

177 VONVOIGTLANDER, P. F. AND LEWIS, R. A. Analgesic and mechanistic evaluation of spiradoline, a potent kappa opioid. J. Pharmacol. Exp. Ther. 246: 259-262, 1988.

WAKSMAN, G., HAMEL, E., FOURNIE-Z ALU SKI, M. C. AND ROQUES, B. P.

Autoradiographic comparison of the distribution of the neutral endopeptidase "enkephalinase" and of mu and delta opioid receptors in rat brain. Proc. Natl. Acad. Sci. U. S. A. 83: 1523-1527, 1986.

WALTERS, W. A., MACGREGOR, W. G. AND HILLS, M. Cardiac output at rest during pregnancy and the puerperium. Clin. Sci. 30: 1-11, 1966.

WANG, J. B., MAI, Y., EPPLER, C. M., GREGOR, P., SPIVAK, C. E. AND UHL, G. R. Mu opiate receptor: cdna cloning and expression. Proc. Natl. Acad. Sci. U. S. A. 90: 10230-10234, 1993.

WANG, J. B., JOHNSON, P. S., PERSICO, A. M., HAWKINS, A. L., GRIFFIN, C. A. AND UHL,

G. R. Human mu opiate receptor. cBMA and genomic clones, pharmacologic characterization and chromosomal assignment. FEBS Lett. 338: 217-222, 1994.

WANG, Q. AND LI, P. Inhibition of barorefiex following microinjection of GABA or morphine into the nucleus of the tractus solitarii of rabbits. J. Auton. Nerv. Sys. 165-171, 1988.

178 WANG, X , TRESHAM, J. J., SCOGGINS, B. A. AND COGHLAN, J. P. Effects of enkephalins and the analogue FK-33824 on mean arterial pressure and heart rate in conscious sheep. Clin. Exp.

Hypertens. 427-431, 1989.

WEBER, S. J., GREENE, D. L., HRUBY, V. J., YAMAMURA, H. L, PORRECA, F. AND DAVIS,

T. P. Whole body and brain distribution of [3h]cyclic [d-pen2,d-pen5] enkephalin after intraperitoneal, intravenous, oral and subcutaneous administration. J. Pharmacol. Exp. Ther. 263: 1308-1316, 1992.

WEINSTOCK, M. AND ROSIN, A. J. Relative contributions of vagal and cardiac sympathetic nerves to the reflex bradycardia induced by a pressor stimulus in the conscious rabbit: Comparison of'steady state' and 'ramp' methods. Clin. Exp. Pharmacol. Physiol. 11: 133-141, 1984.

WEITZELL, R., ILLES, P. AND STARKE, K. Inhibition via opioid mu- and delta-receptors of vagal transmission in rabbit isolated heart. Naunyn Schmiedebergs Arch. Pharmacol. 328: 186-190, 1984.

WILD, K. D., CARLISI, V. J., MOSBERG, H. I., BOWEN, W. D„ PORTOGHESE, P. S.,

SULTANA, M., TAKEMORI, A. E., HRUBY, V. J. AND PORRECA, F. Evidence for a single functional opioid delta receptor subtype in the mouse isolated vas deferens. J. Pharmacol. Exp. Ther.

264: 831-838, 1993.

179 WOODS, J. R, DANDAVINO, A., MURAYAMA, K., BRINKMAN, C. R.,3D AND ASSALI, N.

S. Autonomie control of cardiovascular functions during neonatal development and in adult sheep.

Circ. Res. 40: 401-407, 1977.

XIA, Q., SHENG, J. Z., TAI, K. K. AND WONG, T. M. Effects of chronic U50,488h treatment on binding and mechanical responses of the rat hearts [published erratum appears in j pharmacol exp ther

1994 aug; 270(2):839]. J. Pharmacol. Exp. Ther. 268: 930-934, 1994.

XŒs, G. X., MENG, F., MANSOUR, A, THOMPSON, R. C , HOVERSTEN, M. T., GOLDSTEIN,

A., WATSON, S. J. AND AKIL, H. Primary structure and functional expression of a guinea pig kappa opioid (dynorphin) receptor. Proc. Natl. Acad. Sci. U. S. A. 91: 3779-3783, 1994.

YABALURI, N. AND MEDZIHRADSKY, F. Down-regulation of mu-opioid receptor by full but not partial agonists is independent of^protein coupling. Mol. Pharmacol. 52: 896-902, 1997.

YASUDA, K., RAYNOR, K., KONG, H., BREDER, C. D., TAKEDA, J., REISINE, T. AND

BELL, G. I. Cloning and functional comparison of kappa and delta opioid receptors from mouse brain. Proc. Natl. Acad. Sci. U. S. A. 90: 6736-6740, 1993.

180 YOUNG, W. S., WAMSLEY, J. K., ZARBIN, M. A. AND KUHAR, M. J. Opioid receptors undergo axonal flow. Science 210: 76-78, 1980.

YU, Y., ZHANG, L , YIN, X , SUN, H., UHL, G. R. AND WANG, J. B. Mu opioid receptor phosphorylation, desensitization, and ligand efficacy. J. Biol. Chem. 272: 28869-28874, 1997.

YUKIMURA, T., STOCK, G , STUMPF, H , UNGER, T. AND GANTEN, D. Effects of

[d-ala2]-methionine-enkephalin on blood pressure, heart rate, and baroreceptor reflex sensitivity in conscious cats. Hypertension 3: 528-533, 1981.

ZADINA, J. E., HACKLER, L., GE, L. J. AND KASTIN, A. J. A potent and selective endogenous agonist for the mu opiate receptor. Nature 386: 499-502, 1997.

ZARBIN, M. A., WAMSLEY, J. K. AND KUHAR, M. J. Anterograde transport of opioid receptors in rat vagus nerves and dorsal roots of spinal nerves: pharmacology and sensitivity to sodium and guanine nucleotides. Exp. Brain Res. 81: 267-278, 1990.

ZASTAWNY, R. L., GEORGE, S. R., NGUYEN, T., CHENG, R., TSATSOS, J.,

BRIONES-URBINA, R. AND ODOWD, B. F. Cloning, characterization, and distribution of a

181 mu-opioid receptor in rat brain. J. Neurochem. 62: 2099-2105, 1994.

ZHANG, L., YU, Y , MACKIN, S., WEIGHT, F. F., UHL, G. R. AND WANG, J. B. Differential

mu opiate receptor phosphorylation and desensitization induced by agonists and phorbol esters. J.

Biol. Chem. 271: 11449-11454, 1996.

ZUCCFH, R. AND RONCA-TESTONI, S. The sarcoplasmic reticulum Ca2+ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states. Pharmacol. Rev. 49: 1-51,

1997.

UNIVERSITY OF SURREY LIBRARY