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CENTRAL NERVOUS SYSTEM REGULATION OF INTESTINAL MOTILITY: ROLE OF ENDOGENOUS OPIOID PEPTIDES (ENDORPHINS, ENKEPHALINS)

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Galligan, James Joseph

CENTRAL NERVOUS SYSTEM REGULATION OF INTESTINAL MOTILITY: ROLE OF ENDOGENOUS OPIOID PEPTIDES

The University of Arizona PH.D. 1983

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University Microfilms International

CENTRAL NERVOUS SYSTEM REGULATION OF INTESTINAL MOTILITY: ROLE OF ENDOGENOUS OPIOID PEPTIDES

James J. Galligan

A Dissertation Submitted to the Faculty of the PROGRAM IN PHARMACOLOGY AND TOXICOLOGY

In Partial Fulfillment of the Requirements

For the degree of

DOCTOR OF PHILOSOPHY

In the Graduate College

THE UNIVERSITY OF ARIZONA

1 983 THE UNIVERSITY OF ARIZONA GRADUATE COLLEGE

As members of the Final Examir,9.tion Committee, we certify that we have read the dissertation prepared by James J. Gallisan entitled _____C_en __ tr_a_1 __ N_e_r_v_o_u_s __ S~y_s_t_e_m __ R_e~g_u_l_a_ti_o_n __ o_f_·_I_n_t_e_s_t_i_n_a_l __ M_o~t_i~l~i~ty~: _____

Role of Endogenous Opioid Peptides.

and recommend that it be accepted as fulfilling the dissertation requirement for the Degree of Doctor of Philosophy ------.----~~------

Date

Final approval and acceptance of this dissertation is contingent upon the candidate's submission of the final copy of the disser.tation to the Graduate College.

I hereby certify that I have read this dissertation prepared under my dire~~tion and recommend tha,t it be accepted as fl1lfilling the dissertation

Date J STA'rEMENT BY AUTHOR

This dissertation has been submitted in partial fulfillemnt of the requirments for an advanced degree at the University of Arizona and is deposited in the University Library to be made available to borrowers under the rules of the Library.

Brief quotations from this dissertation are allowable without special permission, provided that accurate acknowledgment of the source is made. Rpquests for permission for extended quotation from of reproduction of this manuscript in whole or in part may be granted by the head of the major department or the Dean of the Graduate College wlllm in his judgement the proposed use of the material in in the interests of scholarship. In all other instances, however, per mission must be obtai ned from th(~ author. DEDICATION

This dissertation and all the work involved in its completion is dedicated to my father.

iii ACKNOWLEDGMENTS

A special thanks to Dr. David L. Kreulen who provided expert assistance and advice on many of these experiments and also allowed the use of his laboratory during the ~n v~tro studies.

iv TABLE OF CONTENTS

Page

LIST OF ILLUSTRATIONS •••••• CI ••••••••••••••••••••••••••••••• vii LIST OF TABLES ...... x ABSTRACT ...... xi

INTRODUCTION •••••••••••••••• 0 •••••••••••••••••••••••••••••••••• 1

Hormonal Control of Intestinal Motility...... 2 Intrinsic Neural Control of Intestinal Motlity ••••••••••••• 3 Extrinsic Neural Control of Intestinal Motility...... 5 Patterns of Intestinal Motility...... 7 Opiates and Motility...... 11 Irritable Bowel Syndrome ••••••••••••••••••••••••••••••••••• 18 Statement of Problem •••••• ~...... 22 METHODS ...... 24 Surgical Preparation of Animals for Intestinal Transit Studies •••••••••••••••••••••••••••••••••••••••••••• 24 Intracerebroventricular Cannulas •••••••••••••••••••••• 26 Hypophys~ctomy •••••••••••••••••••••••••••••••••••••••• 27 Spinal Cord Section •••••••••••••••••••••••••••••••••• 27 Subdiaphramatic Vagotmy ••••••••••••••••••••••••••••••• 27 Evaluation of Intestinal Transit and Gastric Emptying •••••• 28 Gastric Emptying ••••••••••• ~...... 28 Small and Large Intestinal Transit •••••••••••••••••••• 29 Effects of Morphine on Small Intestinal Transit and Gastric Emptying ••••••••••••••••••••••••••••••••••••••• 31 Direct Measurement of Small Intestinal Motility...... 31 Effects of Opioid Peptides on Small Intestinal Transit in Castor Oil-Treated Rats ••••••••••••••••••••••••• 34 Effects of Electroconvulsive Shock on Gastrointestinal Motility and Analgesia ••••••••••••••••••••••••••••••••••••• 35 Effects of Inescapable Footshock on Gastrointestinal Motility and Analgesia ••••••••••••••••••••••••••••••••••••• 36 Kyotorphin Effects on Intestinal Transit and Analgesia ••••• 37 Determination of Opioid Receptor Selectivity of Agonists in Vitro ••••••••••••••••••••••••••••••••••••••• 38 Determination of the Opioid Receptors Mediating the Analgesic and Intestinal Motility Effects of Centrally-Administered Opioids ••••••••••••••••••••••••••••• 41

v TABLE OF CONTENTS continued

Page

RESULTS 43

Effects of Morphine on Gastric Emptying and Small Intestinal Transit and Motility...... 43 Effects of Opioid Peptides on Intestinal Transit in Castor Oil-Treated Rats ••••••••••••••••••••••••• 52 Effects of ECS on Gastrointestinal Motility and Analges 1a .•..•••...•...... ••••.•....•...•.•...••. 64 Effects of IFS on Gastrointestinal Motility and Analgesia •••••••••••••••••••••••••••••••••••••••••••••• 67 Effects of Kyotorphin of Small Intestinal Transit a nd Analgesia •••••••••••.•••.••••.••••••••••••....•.••..•.• 67 In Vitro Determination of Receptor Selectivity...... 67 Effects of Receptor Selective Agonists on Small Intestinal Transit and AnalgeSia •••••••••••••••••••••••••••••••••••••• 75 Relative Potencies In Vivo ••••••••••••••••••••••••••••••••• 84

DISCUSSION 88

REFERENCES 110

vi LIST OF ILLUSTRATIONS

Figure Page

1. Schematic drawing of the implant used in these studies to record intestinal motility from unanesthetized rats ••••••••••••••••••••••••••••••••• 33

2. Distribution of radiochromium in the small intestine of rats treated with intracerebroventricular morphine or saline ••••••••••••••••••••••• 44

3. Dose-response curves for morphine induced-inhibition intestinal transit in fasted rats •••••••••••••••• 46

4. Inhibition of gastric emptying of a radioactive marker by morphine in fasted rats ••••••••••••••••••• 48

5. Typical recording of duodenal motility on the unanesthetized rat before and after morphine treatment ..•.••..••..•.•....•.••..•••.••••..••••••..• 49

6. Dose-response curves for inhibition of intestinal motility by morphine given i.c.v. or s.c. to unanesthetized rats •••••••••••••••••••••••••••••••••• 51

7. Inhibition of intestinal transit in castor Oil-pre treated rats by a-endorphin and DALA and antagonism by naloxone •...... ••..••.••...... ••..•. 55

8. Inhibition of intestinal transit in castor oil-pre treated rats by a-endorphin and DALA and antagonism by naloxone ••••••••••••••••••••••••••••••••••• e.. 56

9. Effect of DANM-pretreatment on the antitransit actions a-endorphin, DALA and loperamide ••••••••••••••••••••• 57

10. Failure of vagotomy to alter the antitransit effects of i.e.v. a-endorphin •••••••••••.•••••••••••••••••••• 59

11. Failure of vagotomy to alter the antitransit effects of i.e.v. DALA ••••••••••••••••••••••••••••••••••••••• 60

12. Failure of spinal cord section to alter the antitransit effects of i.c.v. a-endorphin •••••••••••••••••••• 61

vii LIST OF ILLUSTRATIONS-Continued

Figure Page

13. Failure of spinal cord section to alter the antitransit effects of i.e.v. DALA •••••••••••••••••••••••••••••••• 62

14. Failure of hypophysectomy to alter the antitransit effects of i.c.v. DALA or a-endorphin ••••••••••••••••• 63

15. Increase in hot-plate response times by rats treated with electroconvulsive shock (ECS) and antagonism by naloxone •••••••••••••••••••••••••••••••• 66

16. Increase in hot-plate response times by rats treated with inescapable footshock (Shock) and antagonism by naloxone ...... •..•...... •. ~ . . • • ...... 69

17. Failure of kyotorphin to affect intestinal transit following intracerebroventricular administration •••••• 70

18. Time course of kyotorphin-induced increases in hot-plate latencies following intracerebroventricular administration of kyotorphin ••••••••••••••••• 71

19. Dose response curve for kyotorphin-induced analgesia in and antagonism by naloxone •••••••••••••••••••••••••••• 72

20. Inhibition of intestinal transit by DAGO and morphine •.•••••••••••••••••••••••••••••••••.•.•..•.•.• 76

21. Inhibition of intestinal transit by DALA and a-endorphin .•...... •.....•..•.•..•••.....•....•...•••. 77

22. Inhibition of intestinal transit by DADL and DPLCE 78

23. Failure of DPLPE and DPDPE to affect intestinal transit ...... 79 24. Failure of U-50,488H to affect intestinal transit 80

25. Time course of analgesia produced by a-endorphin, DADL, DALA and morphine ••••••••••••••••••••••••••••••• 81

viii LIST OF ILLUSTRATIONS-Continued

Figure Page

26. Time course of analgesia produced by DPLCE, DPLPE, DPDPE and DAGO ••••••••••••••••••••••••••••••••••••••• 82

27. Dose-response curves for analgesia produced by DPDPE, DPLPE, DPLCE and DAGO and antagonism by naloxone 83

28. Correlation of delta receptor selectivity with increases in analgesic EDSO and the EDSO for inhibition of small intestinal transit (S.I.T.) 87

ix LIST OF TABLES

Table Page

1. Subcutaneuous naloxone antagonism of the small intestinal antitransit effects of subcutaneous or intracerebroventricular morphine •••••••••••••••••••••••••••••••••••••••• 47

2. Frequency of contractions in two areas of the small intestine of unanesthetized rats treated with intracerebroventricu1ar of subcutaneous morphine 50

3. Effects of opioid peptides on intestinal transit in castor oil-treated rats ••••••••••••••••••••••••••••••••••••• 53

4. Effects of opioid peptides given i.c.v. to castor oil treated rats in small intestinal weight and body weight 10s8 •••••••••••••••••••••••••••••••••••••••••••• 58

5. Percent gastric emptying and geometric centers for small and large intestinal transit in sham and ECS treated rats ••••••••••••••• ~ •••••••••••••••••••••••••••• 65

6. Percent gastric emptying and geometric centers for small and large intestinal transit in IFS and sham treated rats .•...•...... •...... •....•.•.•.•...... 68

7. Inhibition of the electrically-induced contractions of the G.P.I. and M.V.D. by normorphine and several opioid peptides ••••••••••••••••••••••••••••••••••••••••••••• 74

8. ED50 values for opioid-induced inhibition of ,small intestinal transit (S.I.T.) and for producing analgesia •••••••••••••••• 86

x ABSTRACT

The complex interaction between the central nervous system, the enteric nervous system and local and endocrine hormones enables drugs affecting gastrointestinal motility to produce their effects through multiple sites and mechanisms of action. Opiates are one class of drugs which can have dramatic effects on gastrointestinal function and the mechanisms for these actions have been the subject of intense study in recent years. These changes in motility have assumed increased importance following the discovery of several endogenous opioid pep tides.

In the present studies, centrally-administered morphine was more potent than peripherally-administered morphine at inhibiting intestinal propulsion and gastric emptying in rats. Direct measurment of intesti nal motility revealed that the antipropulsive effects of morphine were due, to an inhibition of intestinal contractions.

The opioid peptide, a-endorphin, and a stabilized enkephalin analog, [D-Ala2 , Met 5]enkephalinamide, also inhibited intestinal pro pulsion only after central adminstration. These effects were not blocked by a peripherally selective opioid receptor antagonist, diallylnormorphinium.

These data indicated that there is an opioid sensitive mechanism in the brain of rats that, when activated, can inhibit intestinal moti lity. Physiological activation, by electroconvulsive shock or inesca-

xi pable footshock, or pharamcological activation by kyotorphin (Tyr-Arg)

treatment, did not affect gastrointestinal motility but did produce

naloxone-reversible analgesia. These data indicate that the opioid

mechanisms mediating analgesia and inhibition of intestinal motility

are independent and may be a function of different receptor systems.

Several opioid receptor selective agonists were used to deter

mine the specific receptors mediating the analgesic and motility

effects of centrally-administered opioids. Mu selective agonists pro

duced analgesia and inhibition of intestinal transit, while delta

receptor agonists produced'analgesia only. Kappa agonists did not pro duce analgesia or an inhibition of intestinal motility. Mu receptors mediate the ~nAlgesic and intestinal motility effects of exogenously

administered opioids, while delta receptors can mediate analgesia with out altering gut motility. It appears then, that electroconvulsive

shock, inescapable footshock and kyotorphin may produce their analgesic effects by releasing enkephalins, which are delta selective agonists.

This accounts for the failure of these treatments to alter gastroin

testinal motility while still producing the analgesic effects reported here.

xii INTRODUCTION

Control of gastrointestinal motility is a complex process and has been the subject of intense study for over 100 years. The prin cipal consequence of the many controlling factors of gastrointestinal motility is to coordinate contractions of the esophagus, stomach, small and large intestines and the intervening sphincters so that food can be digested, nutrients absorbed and waste excreted in an orderly and effi cent manner. While the fundamental basis for this process is simply an inhibition or stimulation of contractions by gut smooth muscle, the stimuli producing each of these effects can differ markedly. The pri mary concern of this discussion is the neural influences on small intestinal motility and the control of contractions in this portion of the gastrointestinal tract.

The motor functions of the mammalian small intestine are under the control of intrinsic and extrinsic neural and hormonal influences.

The intrinsic nerves are those whose cell bodies reside in the enteric nervous system, originally described by Langley (1921) as one of three divisions of the autonomic nervous system. Extrinsic innervation con sists of those nerves whose cell bodies reside in the central nervous system or in the prevertebral ganglia and form synaptic connections with the intrinsic nervous system or gut smooth muscle directly.

1 2

Hormonal Control of Intestinal Moti~

Hormonal control involves both endocrine hormones and local or paracrine hormones, both of which can influence gastrointestinal moti lity. Endocrine hormones are those substances which gain access to their site of action only after release into the systemic circulation by the hormone producing cell. In many cases, the target tissue is far removed from the source of the hormone. Paracrine hormones are released into the interstitial fluid by the hormone-producing cell and generally affect only a few surrounding cells. Paracrine hormones are locally acting substances. It is generally difficult to to make abso lute distinctions between paracrine and endocrine effects as many of these substances can serve both functions. For example, bombesin and somatostatin appear to have both endocrine and paracrine functions

(Solcia et al., 1981). To complicate this issue further, many of the endocrine/paracrine substances are also found in the intrinsic and extrinsic innervation of the small intestine. Somatostatin, chole cystokinin, substance P, serotonin and neurotensin are some of the substances that are found in the central nervous system, the enteric nervous system and in the endocrine/paracrine cells of the gut (Walsh,

1981; Solcia et al., 1981; Furness and Costa, 1982). Each of these hormone/neurotransmitter substances can have dramatic effects on intestinal motility in vivo, although it is difficult to determine if these effects are neurally mediated (either centrally or peripherally), hormonally mediated or both. Other substances such as secretin, gastrin and glucagon appear to be located exclusively in endocrine 3

cells of the gastrointestinal tract (Solcia et al., 1981; Walsh, 1981).

This has been a brief survey of the types of hormonal influences that

exist which can influence intestinal motility. The remainder of this

review will focus on the intrinsic and extrinsic neural control of

intestinal motility.

Intrinsic Neural Control of Intestinal Motility

The intrinsic innervation of the mammalian small intestine con

sists of those neurons whose cell bodies reside in one of the

ganglionated plexi located in the different layers of the gut wall.

The myenteric plexus (Aurebach's plexus) consists of very large ganglia

and an interconnecting network of nerve bundles which lies between the

longitudinal and circular muscle layers. The submucosal plexus

(Meissner's plexus) is made of smaller but more numerous ganglia and a much finer interconnecting system of nerve fibers. The submucosal

plexus resides in the connective tissue of the submucosal layer

(Gabella, 1979; Gershon, 1981; Furness and Costa, 1980). There are

other nerve bundles and ganglia found in the small intestine, however,

the prominence and fine structure of these plexi seem to vary from spe

cies to species. In all species, however, the myenteric and submucosal

plexi are the principal mediators of intestinal motility and intestinal

reflexes.

There is an abundance of peptide and non-peptide substances that may be neurotransmitters in the enteric nervous system (Schultzberg et

al., 1980; Furness and Costa, 1980; Furness and Costa, 1982), however, acetycholine may be the final common excitatory substance, while the 4 enteric inhibitory transmitter may be the final common inhibitory substance. Acetycholine is present in the enteric nervous system in higher concentrations than any other neurotransmitter substance

(Furness and Costa, 1982) and it is found in both intrinsic neurons which innervate the muscle layers as well as in the interneurons within the enteric ganglia. Stimulation of these enteric neurons releases acetylcholine (Paton, 1957; Szerb, 1976) which produces a contraction of intestinal smooth muscle. The direct action of acetylcholine on smooth muscle is blocked by muscarinic cholinergic antagonists, while the effects of acetylcholine released from enteric interneurons or from extrinsic parasympathetic neurons are blocked by nicotinic cholinergic antagonists (Kosterlitz and Lees, 1964; Furness and Costa, 1980).

The enteric inhibitory neurons are present in all areas of the small intestine with the cell bodies located principally in the myen teric ganglia. The inhibitory neurons appear to be involved in local intestinal relaxation as well as the in descending wave of inhibition that is part of the peristaltic reflex (Costa and Furness, 1982).

Unfortunately, the nature of this non-cholinergic, non-adrenergic inhi bitory transmitter is unknown at this time. A large volume of evidence has accumulated that indicates that this transmitter may be ATP or a related purine nucleotide (Burnstock, 1972; Burnstock, 1978). However, recent studies have provided direct evidence against a purine nucleotide being the enteric inhibitory transmitter (Westfall, et al.,

1982; Bauer and Kuriyama, 1982). Vasoactive intestinal polypeptide has also received some consideration as being this inhibitory substance 5

(Farhrenkrug et al., 1978; Furness and Costa, 1978) but more recent data indicate that this peptide is not identical to the substance pro ducing intestinal relaxation following stimulation of the non cholinergic, non-adrenergic inhibitory neuron (Mackenzie and Burnstock,

1980; Bauer and Kuriyama, 1982). Thus, the identity of the inhibitory neurotransmitter remains to be established.

Extrinsic Neural Control of Intestinal Motility

Extrinsic control of intestinal motility is mediated by the parasympathetic and sympathetic divisions of the autonomic nervous system. Parasympathetic innervation of the small intestine is derived from the vagus nerve which contains both sensory afferents and motor efferent fibers. The vagal motor efferents originate primarily in the dorsal motor nucleus of the vagus located in the brain stem (Sato et al., 1978) and synapse on intrinsic enteric neurons (Baumgarten, 1982).

Bayliss and Starling (1899) first reported that stimualtion of vagal fibers can stimulate intestinal contractions followed by an inhibition of motility. The excitatory response was blocked by atropine while the inhibitory response was not affected by cholinergic or adrenergic anta gonists. These observations, later confirmed by many others, indicate that vagal stimulation excites both the cholinergic post-ganglionic neurons and the enteric inhibitory neurons (Roman and Gonella, 1981).

Sympathetic innervation of the small intestine consists of a cholinergic preganglionic neuron which originates in the thoracic spi nal cord and a nor adrenergic neuron whose cell body is located in one 6

of the prevertebral ganglia (Furness and Costa, 1974; Baumgarten,

1982). The cholinergic preganglioinic fibe~s leave th~ spinal cord as

the splanchnic nerves and synapse with the postganglionic adrenergic

nerves located in the prevertebral ganglia (Norberg and Hamberger,

1964). Thes~ norepinephrine-containing cell bodies were identified

with flourescence-histochemical techniques and similar methods were

used to identify adrenergic nerve fibers in the intestinal wall

(Norberg, 1964; Jacobowitz,1965). These same investigators also noted

that no adrenergic cell bodies were present in the gut wall. The adre

nergic fibers innervate the myenteric and submucosal plexi and their

terminals are generally found along the edges of the ganglia (Gabella,

1979; Manber and Gershon, 1979; Llewellyn-Smith et al., 1981). A few

adrenergic fibers also terminate in the circular and longitudinal

muscle layers (Wikberg, 1977).

Stimulation of the sympathetic nerves generally inhibits

intestinal contractions, while severing these nerves results in hyper

motility. The norepinephrine released from sympathetic nerves can

affect smooth muscle directly or can alter intrinsic nervous activity

by inhibiting acetylcholine release from the intrinsic neurons. The

electrophysiological (Nishi and North, 1973; Hirst and McKirdy, 1974)

and the ultrastuctural evidence (Llewellyn-Smith et al., 1981) suggest

that the adrenergic receptors are located on cholinergic nerve ter minals. A more recent study has provided direct evidence for the pre

sence of alpha2 adrenergic receptors on cholinergic neurons of the myenteric plexus (Wikberg and Lefkowitz, 1982). Norepinephrine can

also relax smooth muscle directly by an action at both alpha and beta 7

adrenergic receptors. Alpha mediated inhibition is a result of an

increase in potasium and chloride conductance which hyperpolarizes the

cholinergic neuron (Bulbring and Tomita, 1969). The relaxation pro

duced by beta receptor stimulation is preceeded by an intracellular

accumulation of cAMP (Anderson and Mohme-Lundholm, 1970).

Patterns of Intestinal Motility

The small intestine of many species generally exhibits two pat

terns of motility (Weisbrodt, 1981). The fed pattern is difficult to

characterize and can depend on the type and quantity of food present in

the intestinal lumen. The fed pattern at a single intestinal location

consists of sequential contractions occurring at intervals of less than

one minute. These probably serve a mixing function as originally described by Cannon (1902). These phasic contractions may be superim

posed on tonic increases in intraluminal pressure as part of a pro

pulsive peristaltic contraction which travels only short distances in

the fed state. The peristaltic contractions are a result of the

peristaltic reflex or law of the intestine (Bayliss and Starling

1899) that is a fundamental principle of intestinal motility. The

peristaltic reflex consists of a descending wave of inhibition below a

point of intestinal distention that is followed by an aborally moving ring of intestinal contraction that originates above the point of distention. The peristaltic reflex appears to be mediated solely by intrinsic intestinal neurons (Costa and Furness, 1982). Myoelectric recordings obtained from the small intestine of fed animals show an almost random pattern of electrical spiking activity (Weisbodt, 1981). 8

In contrast, the fasted pattern of motility shows a regular

cyclic change in myoelectric activity. The fasted pattern of intesti

nal electric activity, originally described by Szurszewski (1969), is a

regular change in myoelectric activity that occurs in cycles along the

entire length of the intestine. This migrating myoelectric complex

(MMC) is characterized by three distinct phases in dogs and humans

(Code and Mart1ett, 1975; Vantrappen et al., 1977). Phase 1 is a period

of relative quiet that is followed by Phase 2 or a period of random

electrical activity. Phase 3 is the most striking feature of the MMC

and is easily recognized as a period of intense and regular spiking activity that may originate in the stomach or duodenum and migrates

abora11y. As electrical spiking activity is generally associated with

circular muscle contractions, Phase 3 is an abora11y moving ring of

intestinal contraction (Szurszewski, 1981). Code and Mart1ett (1975) have also described a Phase 4 as a period of declining activity but

the existence of Phase 4 is not universally agreed upon. In fact, the

intense spiking of Phase 3 generally ceases abruptly. In addition to humans and dogs, the MMC is seen in other species including sheep and

rabbits (Grive1 and Ruckebusch, 1972), pigs (Bueno et al., 1982) and

rats (Ruckebusch and Fioramonti, 1975). An understanding of the fac tors responsible for the MMC is important as much of the work con

cerning neuronal control of intestinal motility has used the MMC as a substrate for study. In addition, most of the studies dealing with drug-induced changes in motility have also been carried out in fasted animals. 9

Initiation of the MMC in the proximal small intestine appears to be under hormonal control and there is substantial evidence that moti lin may be the responsible hormone. Motilin is a 22 amino acid peptide isolated from hog intestine that was found to stimulate gastric moti lity (Brown et al., 1972). This hormone is found in highest con centrations in mucosal cells of the upper intestine (Walsh, 1981).

Intravenous administration of motilin to dogs or humans can initiate premature MMC's in fasted subjects while the same treatment does not affect the fed pattern of intestinal motility (Itoh, et al., 1978;

Ormsbee and Mir, 1978; Wingate et al., 1976). Plasma levels of motilin also show a cyclic variation with peak concentrations occurring just prior to or during Phase 3 activity (Itoh et al., 1979; Lee et al.,

1978) and stimuli which release endogenous motilin also initiate Phase

3 activity (Lee et al., 1978). Finally, treatment of fasted dogs with an antibody to motilin can inhibit the formation of MMC's (Lee et al.,

1982). Thus there is substantial evidence that intiation of the MMC is under hormonal control, however the role of intrinsic and extrinsic nerves in the orderly propagation of the MMC is less clear.

The extrinsic innervation of the small intestine mayor may not be important for the normal propagation of the MMC. Studies using iso lated loops of small intestine with .the extrinsic innervation intact have shown that the MMC will frequently pass from the intestine to the loop and back to the intestine in a normal fashion (Carlson et al.,

1972; Grivel and Ruckebusch, 1972). In addition, when the extrinsic innervation of the isolated loop is removed the MMC will not appear in the loop (Weisbrodt et al., 1975a). When a segment of intestine is 10 denervated extrinsically with the intrinsic nerves intact the MMC will pass through the segment but at a much reduced velocity (Bueno et al.,

1979). These investigators also noted that when an isolated loop of intestine was prepared and the remaining intestine reanastomosed, MMC's would move from the intestine to the loop and back to the intestine at some distance aboral to the anastomosis and after a considerable delay.

It was also noted that the number of complexes distal to the anastomo sis was greater than the number occurring on the proximal side.

Subsequent studies performed on isolate.d and denervated loops of intestine in pigs have demonstrated that MMC's could be initiated and migrate aborally in. the absence of extrinsic input (Aeberhard et al.,

1980; Itoh et al~, 1981). This effect was shown to be dependent on intrinsic cholinergic neurons (Sarna et al., 1981). These data indi cate that the mechanism for initiation and propagation of the MMC is intrinsic to the intestine but that extrinsic innervation may serve to facilitate migration of the complex in an aboral direction.

It is clear then that normal functioning of the small intestine appears to be under the direct control of the enteric nervous system and that the extrinsic innervation can modify the contractile funct tions of the gut. The extrinsic innervation of the small intestine may also serve to integrate digestive function with the other ongoing beha vioral and visceral processes of the animal. It should also beempha sized that while the enteric nervous system is relatively autonomous many of the intestinal reflexes may require intact connections to the prevertebral ganglia (Szursweski and Weems, 19776; Kreulen and

Szursweski, 1979). In additi~n, there are many excitatory and inhibi- 11 tory substances found in both the intrinsic nervous system and in the extrinsic nerves (Furness and Costa, 1980; Furness and Costa, 1982;

Dalsgaard et al., 1983) all of which serve to modify the action of the cholinergic excitatory neurons and the enteric inhibitory neurons of the intrinsic nervous system.

Opiates and Intestinal Motility

The complex interaction between the central nervous system, the intervening ganglia, the enteric nervous system and the multitude of possible neurotransmitter substances has produced many sites and mecha nisms of action for drugs to affect intestinal motility. One class of compounds which has been known for centuries to produce striking changes in gastrointestinal function is the opiates.

Opium alkaloids have been used for many centuries for the control of dysentery and other diarrheas and the mechanism of this antidiarrheal effect has been the subject of considerable investigation for 80-90 years. The reasons for such intensive study are that the opiates can produce their intestinal effects through several mecha nisms and these effects can vary from species to species and from one type of preparation to another. There is also an apparent paradox seen in many species in that the well known constipating effects of opiates are associated with increases in gastrointestinal motility.

Finally, the recent discovery of the endogenous opioid peptides has led to the possibility that some disorders in gastrointestinal moti lity may be attributed to changes in opioid peptide levels or in acti vity of opioid containing neurons. 12

The early experiments dealing with the effects of morphine on gastrointestinal motility have been reviewed by Plant and Miller

(1926). Many of the early investigators noted that subcutaneous administration of morphine would often provoke spontaneous intestinal contractions and would increase the irritability of the intestine when provoked by certain stimuli. Other investigators noted that morphine would delay the passage of food through the intestine. A principal criticism of these early experiments was the use of a general anesthe tic during the experiment which was known, even at that time, to suppress normal intestinal contractions and reflexes. Plant and Miller

(1926), using unanesthetized dogs, found that morphine would produce dose-related increases in the frequency of phasic contractions as well as tonic inc~eases in intraluminal pressure. These investigators also noted qualitatively similar effects in human subjects after morphine treatment. Following thes~ intitial observations, many other studies showed that morphine could stimulate intestinal contractions in una nesthetized animals yet still produce constipation (Vaughan Williams,

1954).

This contradiction was resolved following experiments using an isolated loop of intestine attatched at either end to a column of water. Small doses of morphine increased contractile activity of the segment yet propulsive work was reduced (Vaughan Williams and

Streeten, 1950). Morphine also increased the tone of the segment which increased resistance and reduced the flow of intraluminal contents.

This observation has been supported by many subsequent studies which indicate that morphine stimualtes a non-propulsive type of intestinal 13 motility and that the intestinal lumen becomes smaller reducing the flow of the intraluminal contents (Bass and Wiley, 1965; Bass et al.,

1973).

The work described to this point had been performed using intact animals and little information was provided concerning the site of action. A number of isolated intestinal preparations have been used to study the direct effects of opiates on intestinal motility. The

Trendelenburg preparation (Trendelenburg, 1907) and several modifica tions have been used to study the effects of opiates on the peristaltic reflex. Briefly, a small piece of guinea-pig ileum is suspended in a tissue bath with one end of the segment attatched to a tube which is connected to a buffer resevoir. The ileal segment can be distended by raising or lowering the level of the resevoir. Distention of the segment produces a contraction of the longitudinal muscle followed by progressive rings of circular muscle contraction and relaxation of the longitudinal muscle. This reflex is mediated, at least partially, by acetylcholine as hyoscine, atropine and hexamethonium will block the contractile response (Kosterlitz and Lees, 1964). Morphine and other opiates will also depress this reflex and their potency and efficacy were closely correlated with their analgesic effects (Green, 1959;

Gyang et al., 1964). It is also interesting to note that this effect was stereospecific as only levorotatory isomers of the opiates were effective (Gyang et al., 1964). This action was not due to a depress ant effect on the smooth muscle as the preparation would respond norm ally to exogenous acetylcholine. 'Instead, the effects of opiates on 14 this reflex may be attributed to inhibition of acetylcholine release from int'dnsic neurons (Schauman, 1957).

Low frequency electrical stimulation of segments of guinea-pig ileum or the of longitudinal muscle-myenteric plexus preparation also produces contractions that are inhibited stereospecifically by opiates.

This effect is also a result of an inhibition of acetylcholine release from the intrinsic nerves of the ileal tissue (Paton, 1957).

Subsequent studies have demonstrated that the depressant effects of morphine on the electrically-induced contraction are blocked by the opiate receptor antagonist, naloxone (Kosterlitz and Watt, 1968).

Much of the work concerning the effects of opiates on intesti nal contractions and reflexes in vitro has been done using strips of guinea-pig small intestine. The responses seen in these preparations are generally an inhibition of contractions or of reflex activity.

However, as pointed out previously, studies in intact animals have shown that morphine stimulates intestinal contractions. This is also true of the dog-isolated intestine as intraarterial injections of morphi~e produce both phasic and tonic contractions (Burks and Long,

1967). These investigators also reported a release of serotonin from the intestinal segment following morphine treatment and that the contractile response produced by morphine could be reduced with anta gonists of serotonin (Burks, 1973). These observations serve to illustrate the important species differences in the intestinal respon ses to exogenous opiates.

The central nervous system is also a site of action for exoge nous opiates to affect intestinal motility. An early experiment 15 showed that methadone altered intestinal motility through a vagally mediated mechanism (Scott et al., 1947) which led to the proposal that the central nervous system was the site of action for morphine-induced constipation (Vaughan Williams, 1954). Subsequently, intracerebral administration of morphine to mice was shown to produce a greater inhibition of intestinal propulsion than did subcutaneous administra tion (Margolin, 1954; Green, 1959). It was later suggested that this effect could be humorally mediated (Plekss and Margolin, 1968;

Margolin, 1963) as spinal cord section or vagotomy did not block the antipropulsive effects of intracerebally-administered morphine.

The central nervous system as a site of action for opiate induced constipation has been confirmed in subsequent studies in the rat (Parolaro et al., 1977; Stewart et al., 1978; Schulz et al.,

1979), cat (Stewart et al., 1977) and dog (Bueno and Fioramonti,

1982). Despite this considerable volume of supporting evidence the results of other studies indiciate that the central nervous system does not have a role in opiate-induced constipation and that a direct local action on the intestine is responsible (Tavani et al., ,1980).

Thus, the relative contributions of central and peripheral mechanisms to the antipropulsive effects of exogenously-administered opiates remains a point of some controversy.

The effects of exogenous opiates on gastrointestinal motility have assumed increased interest and importance following the iden tification of stereospecific opiate binding sites or receptors in the gastrointestinal tract and central nervous system (Pert and Snyder,

1973; Simon et al., 1973) and the isolation and characterization of 16

several endogenous peptides with potent opiate-like activity (Hug~es et

al., 1975; Li and Chung, 1976; Cox et al., 1976; Goldstein et al.,

1979). It has recently become apparent that there are three distinct

classes of opioid peptides; the endorphins, the enkephalins and the dynorphin-related peptides (Cox, 1982). The endorphins, including e

endorphin, are found largely in the pituitary and hypothalamus (Bloom

et al., 1978) and are derived from a larger precursor, proopiomelano

cortin (Mains et al., 1977). The enkephalins are the second class of

opioid peptide and are widely distributed in the brain, spinal cord

and peripheral tissues, including the gastrointestinal tract (Hughes et

al., 1977; Schultzberg et al., 1978; Miller and Pickel, 1980). Although methionine enkephalin is the N-terminal pentapeptide of e

endorphin, the distribution (Stengaard-Pedersen and Larsson, 1981) and

biosynthetic pathways of these peptides differ markedly. Methionine

enkephalin, and in smaller quantities leucine enkephalin, are derived

from proenkephalin A (Kakidani et al., 1982). The dynorphin-related

peptides, including a-neoendorphin and to a lesser extent leucine

enkephalin, are derived from proenkephalin B (Kakidani et al., 1982).

These peptides are also widely'distributed in the brain, pituitary and

peripheral tissues (Goldstein et al., 1979; Tachibana et al., 1982).

Each class of opioid peptides has been shown to inhibit the

electrically-induced contractions of the guinea-pig ileum (Cox et al.,

1976; Hughes et al., 1975; Goldstein et al., 1979) and the presence of

the enkephalins and dynorphin in intestinal nerves suggests that these

peptides participate in the regulation of intestinal motility. There

is also some indirect functional evidence supporting a role for these 17

peptides in control of intestinal contractions. Dynorphin levels have

been shown to increase in the bathing medium during fatigue of the

peristaltic reflex in vitro, while incubating the preparation with

naloxone increases the frequency of peristatltic waves (Kromer and

pretzlaff, 1979; Kromer, 1980).

The effects of opioid peptides on intestinal contractions that have been described here have been found in isolated tissue prepara

tions only and very little is known about the actions of these peptides on intestinal motility in the intact animal. Gillan and Pollock (1981) have found that, in the rat, morphine, methionine and leucine enkepha lins can inhibit colonic contractions produced by stimulating the motor efferents of the spinal cord but would also produce contractions of the unstimulated colon. In these studies the opioids were given systemically and no conclusions could be made as to the site of action. A previous study (Cowan et al., 1976) had demonstrated an inhibition of intestinal transit following intracerebroventricular administration to mice of methionine and leucine enkephalin. However, an antipropulsive effect was seen only with very high doses and again it would be difficult to make firm conclusions concerning the site of action. These peptides are also unstable in vivo and the high doses may have been required to overcome rapid degradation of the peptides.

Since that time a number of stabilized enkephalin analogs have been synthesized which possess a longer biological half-life in vivo. One such analog has been shown to inhibit intestinal transit in the rat following central administration (Schulz et al., 1979) but there is still no information concerning the effects of the other classes of 18

opioid peptides or their analogs on intestinal transit. Another point

to consider when discussing the opioid peptides is the existence of

several subclasses of opioid receptor. At least three distinct types

of opioid receptor have been identified using pharmacological (Martin

et al., 1976; Gilbert et al., 1976; Lord et al., 1977) and biochemical

(Chang et al., 1979; Chang and Cuatrecasas, 1979) techniques. The

responses mediated at each type of receptor are unknown at this time

and the biological effects produced by the three classes of opioid

peptides may differ based on their relative affinity for each receptor

subclass.

The Irritable Bowel Syndrome

Opioid peptide control of intestinal motility at both local and

central sites may be an important topic for both basic and clinical

research. There are a number of motility disorders, including the

irritable bowel syndrome, that appear to be related to the emotional

state of the individual. In addition, the symptoms of this disorder

are exacerbated by emotional or psychological stress, a clear

illustration of the central nervous system producing changes in

gastrointestinal motility. The irritable bowel syndrome is the most

common disorder of bowel motility seen in medical clinics in the

United States and Great Britain (Almy, 1957; Ruoff, 1973). The irri

table bowel syndrome (IBS) is a collection of symptoms which include abdominal cramps, diarrhea, constipation or diarrhea alternating with

constipation. The diagnosis of IBS is generally made by exclusion of all other possible diseases and a careful patient history (Kirsner, 19

1981). In addition to the gastrointestinal problems, IBS patients are generally found to be very anxious individuals who score high on a variety of psychological tests for emotional disorders (Young et al.,

1976; Whitehead et al., 1980; Latimer et al., 1981).

The relationship of emotional state to colonic motility had been established in early studies of IBS patients. These patients demonstrated a marked increase in motility and spastic contractions of the sigmoid colon during a discussion of emotionally charged topics

(Almy et al., 1949). Similar changes could be produced in healthy individuals by discussion of emotion provoking topics or by producing experimental stress (Almy and Tubin, 1947; Almy et al., 1949). In addition to this experimental evidence for emotional influences on colonic motility, many IBS patients report an onset or worsening of their symptoms during stressful periods (Chaurdonay and Truelove, 1962;

Young et al., 1976). More recent studies of colonic motility in IBS patients have shown a marked alteration in contractile and myoelectric activity. Snape and coworkers (1977) have reported an increase in the frequency of 3 cycle/minute contractions and colonic slow wave activity in IBS subjects when compared to controls. Subsequent studies have confirmed this observation that a slower contractile frequency predomi nates in IBS patients (Whitehead et al., 1980; Latimer et al., 1981).

Motility of the small intestine has not been as extensivley studied in relationship to IBS due to the difficulty of using endoscopic pro ceedures for examining the small intestine without first sedating the patient. The use of a sedative in this situation poses a problem due 20 to the relationship of intestinal motility to the emotional state of the subject.

Although blood levels of several gut hormones known to influence gastrointestinal motility, including gastrin, neurotensin and motilin, are unchanged in IBS patients, the role of other neuropeptides in this disorder has not been investigated. There are several conflicting reports concerning the efficacy of naloxone treatment for the symptoms of irritable bowel (Ambinder et al., 1980; Fielding and

O'Malley, 1979), however, there is a considerable amount of circumstan tial evidence suggesting that the opioid peptides may at least par ticipate in this symptom complex.

a-Endorphin has been proposed to function as a neuroendocrine peptide released into the circulation along with ACTH during stressful situations (Guillemin et al., 1977) and a-endorphin release is under the same neurochemical control as ACTH (Vale et al., 1981). In addi tion, methionine and leucine enkephalin and several C-terminally extended enkephalins with opioid activity have been identified in the adrenal medulla (Lewis et al., 1980) and these peptides are released concomittantly with catecholamines following cholinergic stimulation

(Viveros et al., 1979). No target tissue has been established for these circulating endorphins or enkephalins but it is possible that gastrointestinal function may be affected by these circulating peptides or biologically active fragments whose levels can rise during stress.

Enkephalinergic or endorphinergic containing neurons within the brain may also participate in the regulation of autonomic outflow from the CNS to the gut. Endorphinergic neurons originating within the 21

hypothalamus project to several brainstem regions including the reticu

lar formation, periaqueductal gray and locus ceruleus (Childers, 1980).

The periaqueductal grey has shown to be a possible site for morphine's

intestinal antipropulsive effects (Sala et al., 1983). Enkephalin

containing neurons have also been located· in the anterior hypothalamus which also contains a high density of opiate binding sites. In addi

tion, the amygdyla sends enkephalin-containing processes to the stria

terminalis and its nulcei (Uhl et al., 1978) and contains the highest density of opiate receptors in the brain (Simantov et al., 1976).

These are important observations as early work on CNS control of gastrointestinal motility has shown that electrical stimulation of the anterior hypothalamus enhances gastric (Fennegan and Puiggari, 1965), small intestinal and colonic motility (Wang et al., 1940) while electrical stimualtion of the amygdyla inhibits gastric motility

(Fennegan and Puiggari, 1965). Autoradiographic studies of opiate receptor distribution in the medulla have revealed high densities of opiate binding sites in the solitary nuclei, nucleus ambiguus, dorsal motor nucleus of the vagus and on the vagus nerve itself (Atweh and

Kuhar, 1977) suggesting that the endogenous opioid peptides can modu late afferent input from the viscera as well as efferent outflow to a number of visceral structures including the gut.

The data described above indicate that the endorphins and enkephalins may be important neuromodulators of autonomic outflow to the gastrointestinal tract and of the emotional state of the individual as indicated by the high density of opiate receptors on limbic struc- 22

tures such as the amygdyla. It is in both of these areas that the

symptoms of the irritable bowel syndrome arise.

Statement of the Problem

Previous studies have shown that morphine could alter gastroin-

testinal motility by an action within the central nervous system. The

present study was designed to provide further support for the

centrally-mediated effect by comparing the relative potencies for inhibition of intestinal transit by morphine given by several routes of administration. While much of the work concerni.ng the site of morphine's action on gut motility has been done in the rat, very little is known about the contractile state of the intestine following morphine treatment. A system was developed for direct measurement of intestinal contractions in the unanesthetized rat before and after morphine treatment.

The effect of endogenous opioid peptides on intestinal transit in the rat was also unknown and intestinal transit was evaluated in rats treated intracerebroventricularly and peripherally with several opioid peptides as a means of determining a site of action. The cerebrally-mediated effects of opioids on intestinal• motility• suggests the existence of an opioid sensitive mechanism in the brain that when stimulated can alter gut motility. Several physiological and pharmacological stimuli were used in an attempt to activate this system with the intent of developing an animal model for the irritable bowel syndrome. Finally, the possibility that a single class of opioid 23 receptor is mediating the intestinal effects of centrally-administered opioids was investigated using several opioid agonists which were highly selective for a single class of receptor. METHODS

Surgical Preparation of Animals for Intestinal Transit Studies

In all experiments male or female Sprague-Dawley rats were used

and the preparation was similar for each type of experiment. These

techniques were a modification of the procedures developed by Poulakos

and Kent (1973) for intraluminal instillation of non-absorbable

radioactive markers. In some experiments, only small intestinal can

nulas were implanted while in others intragastric, small intestinal and

large intestinal cannuals were implanted. In each case, silastic can

nulas were used. The small intestinal cannula consisted of a 20 cm

long piece of silastic tubing (Dow Corning, Midland, MI; 0.02 in. I.D. x 0.037 in. O.D.) with a small bulb of silicone rubber (General

Electric RTV-112, Waterford, N.Y.) fixed 2 em from the intestinal end.

The intestinal end of the cannula was sealed with a small plug of

petroleum jelly to prevent efflux of the intestinal contents.

Each rat was anesthetized with ketamine HCI (Ketalar, Parke

Davis, Detroit MI) 100 mg/kg given intraperitoneally and the proximal small intestine was exposed through a midline abdominal incision. The intestinal end of the cannula was pulled through a cutaneous puncture in the midlumbar region of the animal's back and was brought sub cutaneously to the abdominal incision. A small stab wound was made in the abdominal wall and the cannula was pulled into the abdominal cavity. The tip of the cannula was introduced into the intestinal lumen through a small incision (approximately 2 cm from the pyloric

24 25 region) and was fastened in place by tying a suture (4-0 silk) around the silicone bulb. The abdominal incision was closed with a single set of 4-0 silk sutures that passed through the abdominal musculature and the skin. A second suture was used to close the cutaneous puncture and to secure the exposed end of the intestinal cannula. The cannula was then coiled under a gauze sponge and was kept in place by a masking tape harness. Implantation of the intragastric and colonic cannulas was essentially identical to this procedure with only a few modifica tions. The intragastric cannula was of the same inside and outside diameter except the length was greater than 30 cm. This longer cannula distinguished it from the small intestinal cannula in animals that had been implanted with both intestinal and intragastric cannulas. The intragastric cannula was implanted in the fundic region of the stomach and was secured by passing a suture through the stomach wall and tying it around the silicone bulb.

The colonic cannula was also made of silastic tubing but of a larger diameter (0.025 in I.D. x 0.04 in O.D.) and was approxiamtely 20 cm in length. The larger diameter cannula distinguished it from the small intestinal and intragastric cannulas in animals that had been implanted with each type of cannula. The larger diameter also per mitted instillation of a more viscous marker used for measurement of colonic transit as will be described in more detail. The colonic can nula was also fitted with a small silicone bulb approximately 0.5 cm from the intestinal end which had been sealed with petroleum jelly.

The cannula was inserted into the colonic lumen through an incision 26 approximately 1 em from the colonic-cecal junction and was secured in a manner similar to that used for the small intestinal cannula.

Intracerebroventricular Cannulas

Direct administration of drugs into the cerebral ventricles required prior implantation of a ventricular cannula. The method used in these studies was a modification of the procedure developed by

Robison et al. (1969). Polyethylene tubing (PE-10, Clay Adams,

Parsipanny, N.Y.) was passed through a small metal coil of a device designed to pass electrical current through the coil to generate heat.

When heated, a small expansion of the tubing was produced inside the coil. The tubing was removed and was cut on one end 4 mm from the base of the raised portion and 5 em from this area on the other end. The cannula was inserted (under ketamine anesthesia) into the right lateral cerebral ventricle (4.0 mm below the skull surface) through a small hole drilled in the skull surface. The hole was drilled with a hand held pin vise 2.0 mm lateral and 2.0 mm posterior to bregma. A second hole was drilled 2.0 mm anterior and lateral to bregma and a small stainless steel screw (J. I. Morris Co. Framingham, MA) was inserted.

The cannula was secured to the skull with a small mound of dental acry lic (Codesco Supply, Tucson, AZ) and the head wound was closed with wound clips. The cannula was filled with 5.0 ~l of saline to flush out any blood or cerebrospinal fluid and the tip was sealed closed with a heated forceps. 27

Hypophysectomy

Hypophysectomized rats and aged matched, sham-operated controls were purchased from Taconic Farms Animal Breeders (Taconic, N. Y.).

Upon arrival at the local animal facility the operated rats were pro vided with drinking water containing 5.0% glucose and 1.0% NaCI. All animals were allowed to stay in their cages for 3-4 days prior to implantation of small intestinal and intracerebroventricular cannulas.

Spinal Cord Section

Some of the motor efferents from the central nervous system leave the spinal cord as the splanchnic nerves and in the rat they emerge from the cord below thoracic vertabrae number 4. This input to the gut was eliminated by severing the spinal cord between thoracic vertabrae 2 and 3. This was accomplished under an operating microscope using a number 11 scapel blade. Sham-operated animals had their spinal cord exposed but not severed. Following spinal cord section small intestinal and i.c.v. cannulas were implanted as described previously and each rat was placed in a cage which rested on a heating pad. This procedure helped to maintain body temperature during the two day reco very period between surgery and the experiment. Also during the these two days each rat was fed twice daily with 5.0 m1 of a 5% glucose solu tion via a feeding needle.

Subdiaphramatic Vagotomy

Elimination of the vagal input to the intestine was performed in rats that had been prepared two days previously with small intestinal 28

and i.c.v. cannulas. On the morning of the experiment each rat was

anesthetized lightly with ether and the esophagus was ,exposed by

removing the sutures that had closed the midline abdominal incision.

Two sutures were placed around the esophagus, one close to the diaphram

and the second just proximal to the esophageal-gastric junction. The

sutures were pulled tight and the esophagus was severed. In addition,

all surrounding connective tissue was also cut. Sham-operated animals

had the sutures placed only loosely around the esophagus. These ani

mals were allowed to recover from this procedure for two hours prior to

initiation of the experiment.

Evaluation of Intestinal Transit and Gastric Emptying

Gastric Emptying

Changes in gastric in response to drug or other treatment were

evaluated by instilling approximately 0.6 ~Ci of [3H]-polyethylene gly

col 900 (New England Nuclear, Boston, MA) 0.5m1 saline into the gastric

lumen via the implanted cannula. Thirty five minutes after instilla

tion of the non-absorbable marker the rat was killed by cervical dislo

cation and the stomach was removed. The stomach was placed into a large centrifuge tube and brought to a final volume of 20 m1 with nor mal saline. A standard sample was prepared by adding 0.5 m1 of the

tritiated marker to 19.5 m1 of saline. The stomach samples and the standard were homogenized (Tekmar) and centrifuged (15 minutes, 6800 x g). A 200 ~l aliquot of the supernatant of each tube was added to 5 ml of scintillation cocktail (Aquamix, Westchem, Tucson, Az). and each sample was counted for 5 miuntes (Beckman L8-250, 1.0% error). The 29 number of disintegrations per minute in each stomach sample was divided by the number of disintegrations per minute in the standard sample to determine the percentage of administered marker that remained in each stomach. Percent gastric emptying was calculated by subtracting from

100 the percentage of administered marker remaining in each sample.

Small and Large Intestinal Transit

Approximately 0.5 ~Ci of radiochromium as Na5lCr04 (New England

Nuclear, Boston, ~~) in 0.2 ml of saline was instilled into the duode num via the previously implanted cannula. The marker for large bowel transit consisted of Na5lCr04 saline/5.0% xanthum gum which producad a marker that was similar in consistency to normal colonic contents.

This marker was instilled into the large bowel via the cannula (0.5

~Ci, 0.2 ml volume). Twenty five or thirty five minutes after marker instillation the rats were killed by cervical dislocation and the small and large intestines were excised. The small and large intestines were each divided into ten equal segments on a ruled template. The intesti nal segments were placed consecutively into culture tubes and the amount of radioactivity in each segment was determined by gamma counting (Tracor Analytic. Elk Grove IL). The amount of radioactivity in each small or large intestinal segment was then expressed as a frac tion of the total radiaoctivity that was found in the small intestine or large intestine. Intestinal transit was then quantitated by calcu lating the geometric center of the distribution of radioactive marker in the small or large intestine using the following formula:

Geometric Center = ~(fraction of counts in segment X segment no.) 30

The geometric center is the center of gravity of the distribution of marker in the intestine and can range from a value of 1.0 where all the marker is in the first intestinal segment to 10.0 with all the marker in the last intestinal segment. Treatments which inhibit intestinal transit decrease the value of the geometric center while treatments which stimulate intestinal transit increase the value of the geometric center. This technique has proven to be a reliable measure of drug induced changes in intestinal transit and it is sensitive to changes in both the distribution and leading edge of the marker (Miller et al.,

1981). As there is a maximum inhibition of transit as indicated by a geometric center of 1.0, calculation of the EDSO value for drugs affecting intestinal transit was greatly simplified. The EDSO is that dose of drug which produces a half-maximal inhibition of intestinal transit. This value is calculated from a linear regression on the dose-response curve for percent maximum inhibition of intestinal tran sit produced by each dose of drug. Percent maximum inhibition is calculated as follows:

% Maximum inhibition of Transit = (drug-control/1.0-control) X 100 where drug is the geometric center of each drug treated animal and control is the mean geometric center of the control for each experiment and 1.0 is the maximum inhibitIon of intestinal transit. This calcula tion allows a direct comparison of potencies for a drug given by dif ferent routes of administration or between different drugs given by the same route. 31

Effects of Morphine on Small Intest~aal Transit and Gastric Emptying.

Female rats were prepared with small intestinal and intragastric cannulas as previously described. Intracerbroventricular cannulas were also implanted in some rats. All animals were placed in individual cages and allowed to recover for 72 hours. These experiments were done in rats that had been fasted 18 hours prior to the experiment.

Morphine sulfate dissolved in saline was administered subcutaneously

(1.0 ml/kg volume) and i.c.v. (5.0 ~l) 20 minutes prior to marker while intragastric morphine (2.0 ml/kg volume) was given 30 minutes prior to marker instillation. Thirty five minutes after the intragastric and intestinal markers had been instilled the rats were killed and gastric emptying and intestinal transit were determined. Naloxone (2 mg/kg s.Cw) antagonism of the intestinal effects of morphine was investigated in animals that had been implanted with intestinal cannulas only.

Differences between treated and control groups were assessed using Dunnett's t-test for comparing several groups to a single control and Student's t-test for grouped data.

Direct Measurement of Small Intestinal Motility

A simple and inexpensive technique for measurement of small intestinal motility in the unanestheitzed rat was developed. An implant conSisting of silastic tubing, 23-gauge hypodermic needles, a 6 cc syringe and dental acrylic was used for these studies. A small length (12 cm) of silas tic tubing was fixed to a 23 gauge hypodermic needle cut to 0.5 cm in length. The connection was sealed with silicone rubber. Two of these tubing-needle combinations were used for 32 each implant and were fixed in a small rubber mold. The plunger was removed from a 6 cc plastic syringe and the top 2 cm of the barrel was used as a support matrix inserted into the rubber mold. Dental acrylic was placed into the mold, allowed to set and the implant was removed from the mold. A small bulb of silicone rubber was fixed approximately

1 cm from the intestinal end of the cannulas (see figure 1). The implant was soaked in 70% ethanol before implantation. Rats were anesthetized with ketamine and a midline abdominal incision as well as a small incision between the shoulders were made. The cannulas were brought subcutaneously from the shoulders into the abdominal cavity through a puncture in the abdominal wall. The dental acrylic plug was secured between the shoulders using a purse string suture. The intestinal ends of each cannula were inserted through small incisions in the proximal duodenum and proximal jejunum and were secured by fastening a silk suture around the silicone bulb. Most of the radioac tive marker in the intestinal transit studies was in the proximal 50 % of the intestine (see figure 2). In order to correlate changes in transit with alterations in intestinal contractions, motility was recorded from the proximal portion of the small intestine. Animals fitted with recording cannulas were housed and fasted on the same sche dule as that used in the intestinal transit experiments. On the day of the experiment, rats were placed in a plastic restrainer for 30 minutes after which the cannulas were connected, via the 23 gauge needles, to an infusion pump (Harvard Apparatus) and a pressure transducer (Statham

P23Db) using a three-way stopcock. The cannulas were perfused at a 33

23 gao Needles I

6 cc Syringe Silastic Tubing

RTV 112 Silicone Rubber

Figure 1. Schematic drawing of the implant used to record intestinal motility from unanesthetized rats. 34 rate of 0.04 ml/min with distilled water and motility was recorded as pressure increases resulting from changes in outflow resistance as the intestine contracted. Pressure tracings were recorded on a Beckman 511

Dynograph (9853A coupler, 30 Hz high frequency cut-off). The rate of pressure increase in this system when the lumen of a cannula was abruptly occluded was 2.9 cm of H20 per second. The fall in pressure was more rapid with a rate of 12.6 cm of H20 per second. A control recording was obtained for 30 minutes, efter which morphine was administered either s.c. or i.c.v. and intestinal contractions were recorded for an additional 60 minutes. The records were analyzed by visual inspection and the number of contractions occurring in both areas of the small intestine was recorded. Changes in the frequency of contractions were expressed as a percentage of that occurring in the thirty minute control period for each rat. Data were analyzed by

Student's t-test for paired data.

Effects of Opioid Peptides on Intestinal Transit in Castor Oil Treated Rats

Si1astic small intestinal cannulas were implanted into the duodenum of female rats and some animals were also prepared with i.c.v. cannulas. All animals were housed individually for 48 hours and were fasted for 18 hours prior to the start of the experiment. These experiments were initiated by instilling 0.5 ml of castor oil (Fisher

Scientific Products, Tustin, CA) into the duodenum. Thirty minutes later the opioid peptides, a-endorphin and [D-a1a2-methionine5]enkeph- a1inamide (Beckman Bioproducts, Palo Alto, CA) were given either intra- cerebroventricu1a1ry (i.c.v.) or intraperitonea1ly (i.p.), while the 35

peptides dynorphin (1-13) and [D-ala2-leucineS]enkephalinamide

(Peninsula Laboratories, San Carlos, CA) were given i .. c.v. Thirty

minutes after the peptides were administered radiochromium was

instilled into the intestine and after an additional 25 minutes the

rats were killed·and small intestinal transit in each rat was determined. A separate group of animals was used to determine the anti-

diarrheal effects of the opioid peptides. Small intestinal weight and

percent body weight loss were determined following castor oil and pep-

tide treatment. The animals were treated using the same protocol

(without radiochromium instillation) used to evaluate intestinal tran-

sit. Differences in body weight loss and intestinal transit between groups were assessed using analysis of variance and Student's t-test

for grouped data.

Effects of Electroconvulsive Shock on Gastrointestinal Motility and Analgesia

Intragastric, small and large intestinal cannulas were implanted in male Sprague-Dawley rats (280-340 g, Division of Animal Resources,

University of Arizona). The animals were housed individually and allowed to recover for 72 hours and were fasted 18 hours prior to the experiment. The rats were pretreated with saline or naloxone (1.0 mg/kg s.c.) followed after 10 minutes by transocular electroconvulsive shock (ECS, 150 rnA, 0.5 sec duration) or sham-ECS. Five minutes after

ECS the radioactive markers were instilled into the gastrointestinal tract. The animals were killed thirty-five minutes later and gastric emptying, small intestinal and large intestinal transit were evaluated.

A separate group of animals was used to determine whether ECS treatment could produce naloxone-reversible analgesia as had been reported

previously (Lewis et al., 1981; Holaday and Belenky, 1980). Thermal

analgesia was determined using a 52°C hot-plate test. Groups of 6-9

rats were pretreated with saline or naloxone (1.0 or 5.0 mg/kg s.c.)

followed after 10 minutes by ECS or sham-ECS. Analgesia was tested at

5 and 35 minutes after ECS. The time to rear-paw lick or an escape

attempt from the plexiglass box that surrounded the hot-plate surface

was timed and a 60 second maximum cut-off time was used. Response

latencies were converted to percent maximum possible effect (% M.P.E.)

using the following formula:

% M.P.E.·= (Test Latency-Control Latency/60-Control Latency) X 100

where test latency is the time to rear-paw lick or an escape attemtpt

following ECS treatment, control is the pretreatment latency obtained

for each rat and 60 is the maximum time each rat could remain on the hot-plate. Data were analyzed by analysis of variance and Student's t-

test for grouped data.

Effects of Inescapable Footshock on Gastrointestinal Motility and Analgesia

Intragastric, small intest~nal and large intestinal cannulas were implanted in the gastrointestinal tract of male Sprague-Dawley

rats (280-340 g, Division of Animal Resources, University of Arizona).

Each animal was housed individually and allowed to recover for 72 hours and fasted 18 hours prior to the experiment. Each rat was then placed

in a plexiglass box with a grid floor and a shock scrambler was used to apply electrical current (3.75 mA, 1 shock/5 sec) to the grid floor for

20 minutes. 37

Sham treated animals were placed in the box for 20 minutes with no

current applied to the floor. Immediately following the cessation of

shock or sham treatment the radioactive markers were instilled into

the gastrointestinal tract. After an additional 35 minutes, the rats

were killed and small intestinal and large intestinal transit and

gastric emptying were evaluated. Previous studies (Akil et al.,1976;

Watkins et sl., 1980) have shown that inescapable footshock (IFS) could

produce a naloxone-reversible analgesia. The analgesic effects of IFS were determined in animals that had been pretreated with saline or

naloxone (10 mg/kg s.c.). Twenty minutes after pretreatment, rats were placed in the plexiglass box for either shock or sham treatment for twenty minutes. Immediately following and at 10 minutes after removal from the shock box, the rats were placed on the 52 0C hot-plate and the latency to rear-paw lick or an escape attempt was timed. Percent

M.P.E. was calculated as described previously. Data were analyzed by analysis of variance and Student's t-test for grouped data.

Kyotorphin Effects on Intestinal Transit and Analgesia

Kyotorphin is a dipeptide first isolated from bovine brain and produces opioid effects by promoting enkephalin release from enkephalinergic neurons (Takagi et al., 1979). Silastic small intestinal cannulas and polyethylene i.c.v. cannulas were implanted into female

Sprague-Dawley rats (200-240 g, Division of Animal Resources,

University of Arizona). The rats were housed individually for 72 hours and were fasted 18 hours prior to the experiment. Kyotorphin 38

(Peninsula Laboratories, San Carlos, CA) was given i.c.v. and ten minutes later radiochromium was instilled into the duodenum. After an additional 35 minutes the rats were killed and small intestinal transit was evaluated.

The analgesic effects of kyotorphin were determined in a separate group of animals in which only i.c.v. cannulas had been implanted. Rats were pretreated with naloxone (2.0 or 5.0 mg/kg s.c.) or saline followed after 10 minutes by i.c.v. kyotorphin (15, 30, 60 or

120 ~g). The analgesic effects of kyotorphin were determined at 10,

20, and 40 minutes post-peptide treatment on the 52 0C hot-plate.

Percent maximum possible effect was calculated as described previously and data were analyzed by Dunnett's t-test and Student's t-test for grouped data.

Determination of Opioid Receptor Selectivity of Agonists In Vitro

The opioid agonists examined in these studies included normorphine, a-endorphin, [D-Ala2 , Met 5]enkephalinamide (DALA), [D-ala2 ,

MePhe4 , Gly-oI5]enkephalin (DAGO), cyclic [D-pen2 , L-Cys5]enkephalin

(DPLCE), cyclic [D-Pen2 , L-pen5]enk~phalin (DPLPE), [D-Pen2 ,

D-Pen5]enkephalin (DPDPE) and [D-Ala2 , D-Leu5]enkephalin (DADL). All drugs except the cyclic enkephalins were obtained commercially. The cyclic enkephalins were synthesized by solid-phase methods that have been described elesewhere (Mosberg et al., 1983a,b). Receptor selectivity of each of these agonists was estimated by comparing the potencies of each compound for inhibition of the electrically-induced contractions of the guinea-pig ileum longitudinal muscle, myenteric 39

plexus (G.P.I.) to that of the mouse vas deferens (M.V.D.). The M.V.D.

is believed to contain predominately delta type opioid receptors while

the G.P.I. contains predominately the mu type of receptor (Lord et al.,

1977). The IC50 is the concentration of agonist required to produce a half-maximal inhibition of the contraction height and is an indicator

of affinity of a drug for its receptor. The ratio of IC50 values for each of the compounds in the G.P.I. and M.V.D. can be used as an index of the preference of each agonist for the receptors found in each of

the preparations. A large G.P.I./M.V.D. ratio indicates a greater

selectivity for the receptor found in the M.V.D., the delta receptor.

The vasa deferentia of male CD-I and ICR (25-35 g) mice were removed and mounted in a tis'sue bath after the procedure developed by

~enderson et al. (1972). Briefly, the vasa were removed and stripped of any connective tissue and blood vessels and a pair of vasa were used for each preparation. The two tissues were tied together and were fastened at each end to a 14 K gold chain using 5-0 silk thread. The tissue was then connected to the bottom of the tissue bath and to a

Grass isometric force transducer (Model FT030). The tissue was bathed in Mg++ free Krebs' bicarbonate buffer warmed to 37 °C and bubbled with

95% 02 5% C02. The preparation was stimulated transmurally (100 V,

1100 ~A, 0.1 Hz, 2.0 msec duration) using platinum electrodes and a

Grass S44D stimulator. Contractile responses were recorded on a Grass oscillographic recorder (Model 2200S). The preparations were stimu lated for 30 minutes during which time the buffer in the bath was

changed several times. Agonists were added in volumes of 10-300 ~l and 40

remained in contact with the tissue for 3 minutes after which the

buffer was changed until the pre-drug twitch height was restored.

Subsequent doses were added at 15 minute intervals. The ICsO was

calculated from the regression line of the dose response curve for each preparation. Naloxone Ke values (the dissociation constant of the antagonist) was calculated using the single dose method of Kosterlitz and Watt (1968). The Ke was calculated using the following formula:

Ke = a/DR-1 where a is the agonist concentration in riM and DR is the dose ratio of

ICsO values obtained in the presence and absence of naloxone.

The G.P.I. was prepared after the methods used by Kosterlitz et ale (1970). A glass rod was inserted into the lumen of a 3 em segment of guinea-pig (Hartley, either sex) ileum. A scapel blade was used to make a small cut through the longitudinal muscle with attatched myen teric plexus along the mesenteric attatchment. The longitudinal muscle, myenteric plexus was then separated from the rest of the ileal segment hy brushing lightly with a cotton swab around the circum ference of the segment. The tissue was then suspended in the bath as described for the M.V.D. in normal Krebs' buffer consisting of the following in roM concentrations: NaCI 115, KCl 2.0, CaCl2 2.5; MgCl2,

2.5; KHP04,1.2; NaHC03, 23.8 and glucose 5.0.

The G.P.I. was stimulated (80 V, 900 ~A, 0.5 msec duration) and stretched until the contraction was maximized (Lo). Drugs were added on the same cycle as described above and ICsO and Ke values were calcu lated as described for the vas deferens. 41

Determination of the Opioid Receptors Mediating the Analgesic and Intestinal Motility Effects of Centrally Administered Opioids

Several opioid agonists which possess a high degree of opioid receptor selectivity were given i.c.v. to female Sprague-Dawley rats

(200-220 g, Division of Animals Resources, University of Arizona) in order to determine the relative contribution of each of the opioid receptor subtypes to the centrally-mediated intestinal motility effects and the analgesic effects of exogenously-administered opioids. These experiments were done in rats that had been prepared with polyethylene i.c.v. cannulas and silas tic small intestinal cannulas for the intestinal transit studies and i.c.v. cannulas alone for the analgesic experiments. All animals were housed individually for 72 hours and only those rats used for intestinal transit experiments were fasted for 18 hours. The intestinal transit experiments were initiated by giving all the agonists, except a-endorphin and morphine, ten minutes prior to marker administration. Morphine and a-endorphin were administered 20 minutes prior to the instillation of radiochromium into the intestine.

Thirty five minutes later the animals were killed and small intestinal transit was evaluated.

The analgesic effects of the receptor selective agonists were determined by placing each rat on a SS °C hot-plate and timing the latency to rear-paw lick or to an escape attempt from the plexiglass box surrounding the hot-plate surface. Saline or naloxone (2.0 mg/kg s.c.) were administered 10 minutes prior to peptide treatment and the rats were tested at 10, 20 and 40 minutes post-agonist administration. 42

The analgesic response for each animal was expressed as the % M.P.E. as described previously. All drugs except DPDPE and DPLPE were dissolved in saline. These cyclic enkphalins were first dissolved in a small drop of dimethyl sulfoxide (10-20 ~l) and then brought to the final concentration with saline. The final concentration of dimethyl sulfoxide never exceeded 1.0%. All centrally-administered drugs given i.c.v. in these experiments were given in a volume of 10 ~l. Data were analyzed by Dunnett's t-test and Student's t-test for grouped data. RESULTS

Effects of Morphine on Gastric.~mptying, Small Intestinal Transit and Motilit~

Morphine effectively inhibited small intestinal transit regardless of the route of administration. Morphine retarded the movement of marker down the small intestine and affected both the distance travelled and the pattern of distribution (Figure 2). Saline-treated animals showed a bimodal pattern of distribution with approximately

50-60% of the marker in the proximal two intestinal segments. Smaller percentages were found in the next two to three segments, with the sixth and seventh segments containing higher percentages of radioactivity. Intracerebroventricular morphine changed this pattern to one containing a single peak of radioactvity in the first two segments with the amount of marker in succeeding segments diminshing gradually. The highest dose of morphine prevented any marker from moving past the third intestinal segment. Both s.c. and intragastric morphine produced similar effects. When the distributions of marker obtained for each route of administration were converted to the geometric center, the dose-response curve for i.c.v. morphine was far to the left of that for both s.c. and intragastric morphine (Figure 3). The ED50 values in milligrams per kilogram with 95% confidence limits for i.cav., s.c. and intragastric morphine respectively were 0.06 (0.04-0.09), 3.7

(2.7-4.9) and 22.3 (12.4-40.2). Intragastric morphine was approxima-

43 44

5 }JI Saline 5}Jg Morphine 70 70 60 60 50 50 40

30 1/1 c 20 -:::J 0 10 10 0

111 2 5 8 7 8 -0 I- c 20 }J9 Morphine 80 }J9 Morphine -Q) ...tJ Q) a.

40 40 30 30

20 20 10 10

5 6 7 8 3 4 5 6 7 8 Segment Number

Figure 2. Distribution of radiochromium in the small intestine of rats treated intracerebroventricularly with morphine or saline. The amount of radioactivity in each intestinal segment is expressed as a percentage of the total amount present in the small intestine. Distributions represent the mean ± s.e.m of 4-8 animals for each treatment. 45 tely 375-fold less potent than i.c.v. morphine whereas s.c. was 60-fold less potent than the the central route. The antitransit effects of i.c.v. and s.c. morphine were blocked by naloxone pretreatment (Table

1). Naloxone treatment alone did not affect small intestinal transit.

Saline-treated animals emptied approximately 70 (intragastric saline) to 90 (i.c.v. saline) percent of the administered [3H]-polyethylene glycol in the thirty five minute test period. Morphine given i.c.v. and s.c. produced a dose-related decrease in gastric emptying, with the highest doses reducing gastric emptying to 30 percent (Figure 4).

Intragastric morphine did not significantly reduce gastric emptying at doses up to 500 mg/kg.

A mean frequency of intestinal contractions in both areas of the small intestine was calculated by averaging the contractile frequency occurring in the thirty minute predrug control period for all rats tested. The duodenum contracted at a frequency of 7.3 ± 0.4 contractions/minute, whereas the jejunal frequency was 6.3 ± 0.6 contractions/minute. Figure 5 shows a typical recording of duodenal motility in the rat before and after morphine treatment. Both s.c. and i.c.v. morphine produced dose-related decreases in the frequency of contractions in both areas of the intestine. The dose response curve for inhibition of intestinal motility after i.c.v. morphine was far to the left of the curve for s.c. morphine. The jejunum may also be more sensitive to the inhibitory effects of morphine on intestinal motility as this portion of the intestine showed a greater reduction in contrac tile fr.equency 30-60 minutes after morphine treatment (Figure 6). This was true for both i.c.v. and s.c. administered morphine. It is also 46

2.6 ...... CD I: CD Intra gastric (J .!:!..... 2.2 CD E 0 CD .....CJ ::: III 1.8 I: ...as !- iii .5.. * III ..CD 1.4 .5 .1 •

1.0~~...... ~ ...... ~ ...... ~~ ...... •01 . 0.1 1.0 10.0 100.0

Morphine Sulfate (mg/kg)

Figure 3. Dose-response curves for morphine-induced inhibition of intestinal transit in fasted rats.

Data points represent the mean ± s.e.m. for each dose of morphine. *Significantly different from saline-treated controls (p < .05, Dunnett's t-test). 47

Table 1. Subcutaneous naloxone anatagonism of the small intestinal antitransit effects of subcutaneous of intracerebroventricu lar morphine.

Intestinal transit is expressed as the geometric center ± s.e.m. for each group of rats. *Indicates significantly different from morphine alone (p < .05) by Student's t-test for grouped data. Naloxone was given at a dose of 2.0 mg/kg.

Intestinal Transit Pretreatment Dose of Morphine (Geometric Center) S.c.

Saline 0.0 3.34 ± 0.4 Naloxone 0.0 2.46 ± 0.26

Saline 1.5 mg/kg 2.41 ± 0.33 Naloxone 1.5 mg/kg 2.57 ± 0.12

Saline 3.0 mg/kg 1.61 ± 0.09 Naloxone 3.0 mg/kg 2.49 ± 0.06*

Saline 6.0 mg/kg 1.49 ± 0.17 Naloxone 6.0 mg/kg 2.78 ± 0.13*

Saline 12.0 mg/kg 1.43 ± 0.13 Naloxone 12.0 mg/kg 3.96 ± 0.22*

I.C.V.

Saline 0.0 2.57 ± 0.23 Naloxone 0.0 2.83 ± 0.29

Saline 1.25 Jlg 2.64 ± 0.38 Naloxone 1.25 Jlg 3.13 ± 0.28

Saline 5.0 Jlg 2.08 ± 0.18 Naloxone 5.0 Jlg 3.21 ± 0.47*

Saline 20.0 Jlg 1.90 ± 0.25 Naloxone 20.0 Jlg 3.09 ± 0.50*

Saline 80.0 Jlg 1.62 ± 0.21 Naloxone 80.0 Jlg 2.15 ± 0.13* 48

100 5 5 90 5 6 Ct c 80 6 >- 70 7 _5,- Q. 5 - 7 E 60 w 5 u 50 * 5 ~ ...... II) 40 - 4 aJ * ~ 30 * ~ 20 10

o 0.020.080.32 o 3.0 6.0 12.0 o 20 100 500 I.C.V. S.C. Intragastrlc Morphine Sulfate (mg/kg)

Figure 4. Inhibition of gastric emptying of a radioactive marker by morphine in fasted rats.

Gastric emptying is expressed as the percentage of admi nistered marker that had left the stomach in the thirty five minute test period. *Significantly different from saline-treated controls (p < .05, Dunnett's t-test). 49

Control

30 sec.

Morphine 80 JAg i.e.v. (30 min.)

Figure 5. Typical recording of duodenal motility in the unanesthe tized rat before and after morphine treatment. 50

Table 2. Frequency of contractions in two areas of the small intestine of unanesthetized rats treated with intracerebroventricular or subcutaneous morphine.

Data are expressed as the mean ± s.e.m. frequency of intestinal contractions. The data shown are for 4 time periods; the 30 minute pre-drug control, 0-10 minutes after treatment, 11-30 minutes after treatment and 31-60 minutes after treatment. *Indicates significantly different from the control period (p < .05, Student's t-test for paired data).

Control 0-10 min 11-30 min 31-60 min I.C.V. Saline (5.0 J.ll)

Duodenum 6.4 ± 1.4 6.4 ± 1.5 5.9 ± 1.3 6.1 ± 1.3 Jejunum 5.6 ± 1.1 5.6 ± 0.9 5.1 ± 0.7 5.8 ± 1.2 Morphine (5.0 J.lg) Duodenum 8.4 ± 1.9 7.4 ± 1.4 7.0 ± 1.5 7.9 ± 1.8 Jejunum 4.9 ± 0.5 4.2 ± 4.2 3.4 ± 2.3 3.7 ± 2.3 Morphine (20 J.lg) Duodenum 6.0 ± 1.2 6.1 ± 1.6 5.0 ± 1.8 2.8 ± 0.7* Jejunum 7.4 ± 1.1 3.7 ± 1.3 4.1 ± 1.8 2.4 ± 0.8* Morphine (80 J.lg) Duodenum 8.9 ± 2.3 11.1 ± 3.1 9.6 ± 2.4 6.3 ± 1.9* Jejunum 12.0 ± 3.4 7.7 ± 3.5 4.9 ± 1.9* 3.9 ± 2.2* S.C. Saline (1.0 ml/kg)

Duodneum 5.7 ± 0.9 4.8 ± 0.9 4.5 ± 0.7 5.4 ± 0.6 Jejunum 5.2 ± 1.4 5.3 ± 1.2 5.3 ± 1.2 4.6 ± 0.8 Morphine (3.0 mg/kg) Duodenum 6.2 ± 0.9 4.6 ± 1.5 5.5 ± 1.3 4.6 ± 0.8 Jejunum 4.9 ± 0.7 3.5 ± 1.1 3.3 ± 1.1 2.7 ± 0.7* Morphine (6.0 mg/kg) Duodenum 7.9 ± 0.9 5.2 ± 0.9 4.5 ± 0.8* 5.3 ± 1.4 Jejunum 5.4 ± 1.2 5.1 ± 2.3 3.4 ± 1.3* 2.8 ± 1.1*

Duodenum 8.2 ± 1.1 5.4 ± 1.4 4.4 ± 1.4* 4.1 ± 1.2* Jejunum 5.6 ± 1.9 3.4 ± 2.6 2.0 ± 1.6* 1.4 ± 1.0* 51

120

I.C.V. S.C. 100 -...0 duodenum duodenum c 80 -0 0 * -0 60 ~ * .... * >- u c 40 jejunum CD jejunum ::J tT • ...CD 20 LL

0.01 0.1 1.0 10.0

Morphine Sulfate (mg/kg)

Figure 6. Dose-response curves for inhibition of intestinal motility by morphine given i.c.v. or s.c. to unanesthetized rats.

lntestinal motility is expressed as the frequency of contractions in both areas of the small intestine during the 31-60 minute period following morphine treatment. *Indicates significantly different from the control recording (p < .05, Student's t-test for paired data). 52 and s.c. morphine. It is also important to note that the inhibition of intestinal motility did not become siginificant until thirty minutes after morphine administration (Tables 2). This is the same time frame used to assess the antitransit actions of morphine in the other set of experiments (20-55 minutes post-morphine). Also, the time required for inhibition of motility to develop was the same for both i.c.v. and s.c. morphine.

Effects of Opioid Peptides on Intestinal Transit in Castor Oil-Treated Rats

Both a-endorphin and [D-Ala2 , Met5] enkephalinamide (DALA) produced dose-related decreases in intestinal transit when administered into the cerebral ventricles (Table 3). The relative potency of the two peptides was determined on a molar basis. The ED50 for a-endorphin and DALA with 95% confidence limits was 1.33 nmol (0.79-2.2) and

19.1 nmol (11.2-32.5) respectively. a-Endorphin was approximately 14 times more potent in producing a delay in transit through the intestine when compared to DALA following i.c.v. administration.

Neither peptide produced any significant inhibition of intestinal transit when given i.p. (Table 3), although the doses used were many times greater than those given directly into the cerebral ventricles.

Dynorphin (1-13) (50 ~g) and [D-ala2 , Leu5]enkephalinamide (125 ~g) administered i.c.v. did not affect intestinal transit (Table 3). At these doses however, some animals showed mild seizures and barrel- rolling behavior that precluded the use of higher doses of either peptide. The antitransit actions of both a-endorphin and DALA were sen- 53 Table 3. Effects of opioid peptides on intestinal transit in castor oil-treated rats.

Values represent the mean ± s.e.m. of the geometric center for each group of animals and *represents significantly different from saline- treated controls (p < .05, Student's t-test for grouped data). Peptides were administered either intracerebroventricularly (I.C.V.) or intraperitoneally (I.P.).

Intestinal Transit Peptide Dose (Geometric Center) I.C.V.

Saline 5.0 III 1.7 ± 0.1

a-endorphin 0.2 Ilg 1.9 ± 0.2 1.0 Ilg 1.7 ± 0.08 5.0 llg 1.6 ± 0.05* 50.0 llg 1.3 ± 0.09*

·DALA 0.2 llg 1.9 ± 0.02 5.0 llg 1.6 ± 0.08* 25.0 llg 1.6 ± 0.07* 125.0 llg 1.2 ± 0.07*

Dynorphin (1-13) 50.0 llg 2.0 ± 0.1

[D-Ala2, Leu5] enkephalinamide 125.0 llg 1.8 ± 0.3

I.P.

Saline 1.0 ml/kg 1.7 ± 0.1

a-endorphin 50 llg/kg 1.9 ± 0.5 250 llg/kg 1.8 ± 0.1 1000 llg/kg 1.7 ± 0.1

DALA 250 llg/kg 1.7 ± 0.2 1000 llg/kg 1.5 ± 0.1 5000 llg/kg 1.5 ± 0.09 54

sitive to blockade by the opioid receptor antagonist, na1trexone (5-25

~g) when both drugs were given i.c.v. A much higher dose of

naltrexone was required to reduce the inhibitory effects of centrally

administered DALA than was required to block the effects of a-endorph

in at equiactive doses of the peptides (Figure 7). Naloxone (2.0 mg/kg i.p.), an opioid receptor antagonist which readily crosses the

blood-brain barrier when administered peripherally, significantly reduced the antitransit actions of both peptides (Figure 8). Naloxone or naltrexone treatment alone did not affect small intestinal transit.

As both antagonists used here are capable of blocking both peripheral and centrally located opioid receptors the site of action of the anta gonist in blocking the effects of the two opioid p~ptides was not readily apparent. N,N-Dia1lynormorphinium (DANM) is an opioid recep tor antagonist that contains a quarternary-amine function. This com pound is poorly permeable across the blood-brain barrier due to the cationic charge and was used for selective blockade of peripheral opioid receptors. DANM (10 mg/kg) given i.p. did not significantly antagonize the antitransit action of either centrally-administered peptide. Loperamide is an opiate agonist with established anti diarrheal actions. Loperamide acts via stimulation of opiate recep tors located on myenteric neurons and is without central nervous system effects at conventional doses (Niemegeers et a1., 1976; Wuster and Herz, 1978). Loperamide (5.0 mg/kg i.p.) produced a significant but submaxima1 reduction in transit. DANM was effective in atte nuating this inhibitory effect indicating that the dose of antagonist used was sufficient to block peripheral opioid receptors (Figure 9). 55

o Saline icy. Geometric Center ~ Naltrex~ne 5 1-19 icy. 2.2 1::-:::::1 Naltrexone 25 1-19 icy.

2.0

1.8

1.6

1.4

1.2

1.0 51-1 1 1 1-19 5 1-19 50 1-19 25 1-19 125 1-19 Saline iCY. J3-endorphin icy. DALA icy.

Figure 7. Inhibition of intestinal transit in castor oil-pretreated rats by a-endorphin and DALA and antogonism by naltrexone.

All drugs were given i.c.v. with naltrexone given in the same injection as the peptide. alndicates Significantly different from saline-treated controls and bindicates significantly different from rats treated with peptides alone (p < .05, Student's t-test for grouped data). 56

Geometric Center

3. o Saline i.p. ~ Naloxone 2 mg/kg i.p. 2.5

.2.

1.5

1.0 5 1-11 5 1-19 50 I-Ig 25 J,lg I-Ig Saline B-endorphin DALA icv. icv. icv.

Figure 8. Inhibition of intestinal transit in castor oil-pretreated rats by B-endorphin and DALA and antagonism by naloxone.

Rats were given naloxone 10 minutes prior to peptide treatment. Data are the mean ± s.e.m. for each group. aRepresents significantly different from saline-pretreated animals while breptesents significantly different from rats treated with peptide alone (p < .05, Student's t-test for grouped data). 57

Geometric Center

2.0 o Vehicle i.p. ~ DANM 10 mg/kg i.p. 1.8

1.6

1.4

1.2

1.0 5 ~I 5 mg/kg 50 ~g 125 ~g Saline Loperamide p-end. DALA icY. i.p. icy. icy.

Figure 9. Effects of DANM-pretreatment on the antitransit actions of a-endorphin, DALA and loperamide.

Data are the mean ± s.e.m. for each group. alndicates significantly different from saline treatment alone while bindicates significantly different from opioid treatment alone (p < .05, Student's t-test for grouped data). 58

Table 4. Effects of opioid peptides on small intestinal weight and body weight loss in castor oil-pretreated rats.

The opioid peptides were given i.c.v. Small intestinal weight is expressed as the percentage of total body weight and body weight loss is expressed as the percentage of pretreatment body weight. Values are the mean ± s.e.m. for each group and *represents significnat1y different from saline-treated controls (p < .05, Student's t-test for grouped data)

Treatment (dose) Small intestinal weight %Body weight loss

Saline (5.0 Jl1) 3.5 ± 0.3 5.3 ± 1.1 a-endorphin (50 Jlg) 3.4 ± 0.4 4.1 ± 1.2

DALA (125 Jlg) 3.6 ± 0.4 1.6 ± 0.8* 59

...

'- .- -0) . en -c: c: -QJ CO '- () t- o CO c: '- -QJ en E -QJ 0 c: QJ - (!) - o Saline Saline P-E P-E Sham Vagotomy Sham Vagotomy

Figure 10. Failure of vagotomy to alter the antitransit effects of i.c.v. a-endorphin. 60

2.2 -- -I-

~ ... - :: -Q) 1.8 rn c:: c:: as -Q) ... (,) I- 1.6 - ~ 0 as 0-... c:: 0- Q) rn -E 1.4 -Q) 0 Q) t: - (!) - 102

Saline Saline DALA DALA Sham Vagotomy Sham Vagotomy

Figure 11. Failure of vagotomy to alter the antitransit effects of i.c.v. DALA. 61

... ~en -Q) c c ... -Q) I-'" 0 0 ~ .~ - c '" Q) 1.6 +=en -E Q) 0 c Q) --~ 1.4

Saline Saline I3-E I3-E Sham Cord Sham Cord Section Section

Figure 12. Failure of spinal cord transection to alter the antitran sit effects of i.c.v. a-endorphin. 62

:: "- en -Q) c: c: as -Q) "- I- 0 1.8 as 0.: c: . Q) 1.6 en -E -Q) 0 c: Q) --C!) 1.4

Saline Saline DALA DALA Sham Cord Sham Cord Section Section

Figure 13. Failure of spinal cord section to alter the antitransit effects of i.c.v. DALA. 63

.... "en- -2 c c .=u~ Q) - u c~ "!::: -en Q)E Q) 0 E(!)- -Q) 0 Saline Saline DALA DALA ~-E ~-E Sham Hypox Sham Hypox Sham Hypox

Figure 14. Failure of hypophysectomy to alter the antitransit effects of i.c.v. DALA or a-endorphin. 64

a-Endorphin 'and DALA did not alter the fluid accumulation in the

small intestine produced by castor 'oil treatment. DALA did inhibit the

castor oil-induced diarrhea, as evidenced by a significant decrease in body weight loss (Table 6).

Subdiaphramatic vagotomy did not block the antitransit effects of a-endorphin (Figure 10) or DALA (Figure 11). Spinal cord section at a high thoracic level also did not abolish the intestinal effects of either peptide (Figures 12 and 13). Similar results were obtained in hypophysectomized rats (Figure 14).

Effects of ECS on Gastrointestinal Motility and Analagesia

Transocular electroconvulsive shock (ECS) produced both tonic and clonic seizures followed by a period of catalepsy. The tonic phase was characterized by full hind limb extension (in most animals) and m!Je~I"lar rigidity which lasted 18-30 seconds. The clonic phase was characterized by a loss of the righting reflex which lasted from 0.5-2 minutes. ECS-treated animals also showed catalepsy following the clonic phase as indicated by maintenance for 3 seconds or longer of a hindlimb placed on a bar elevated 3 cm above the table top. Naloxone pretreated animals receiving ECS also showed some tonic-clonic seizures. In addition to these behavioral effects, ECS also produced a significant increase in hot-plate latencies (Figure 15, 5 min.).

Naloxone blocked this ECS-induced increase in hot-plate latency at the two doses of antagonist studied. This decreased responsiveness to the thermal stimulus had dissipated by 35 minutes. Gastric emptying, small 65

Table 5. Percent gastric emptying and geometric centers for small and large intestinal transit in sham and ECS-treated rats.

Values are the mean ± s.e.m. and the number of animals in each group is given in parenthesis. Data were analyzed by a two way analysis of variance with no main effects or interactions revealed.

Intestinal Transit Treatment Gastric emptying Small Large

Saline-Sham (5) 93.8 ± 1.7 3.1 ± 0.2 3.5 ± 0.4

Naloxone-Sham (5) 91.4 ± 2.6 3.5 ± 0.3 4.2 ± 0.5

Saline-ECS (6) 74.0 ± 7.8 3.1 ± 0.2 3.2 ± 0.2

Naloxone-ECS (5) 75.0 ± 9.9 3.1 ± 0.2 4.1 ± 0.5 66

100 5 min 35 min -E a 80 Q) til -+I 60 W 40 a.: b ~ 20 cP. 0 c:::::. c:::::::, 0 Dc:::=,DDoc:J SAL NX NXs SAL NX 1 1 NXs SAL NX1 NXs SAL NX1 NXs Sham Sham Sham ECS ECS ECS Sham Sham Sham ECS ECS ECS

Figure 15. Increase in hot-plate response times by rats treated with electroconvulsive shock (ECS) and antagonism by naloxone.

Naloxone (1.0 or 5.0 mg/kg) was given s.c. ten minutes prior to ECS or sham treatment. Rats were tested on the hot plate at 5 minutes and again at 35 minutes after ECS. aIndicates significantly different from saline-sham and bIndicates significantly different from saline-ECS (p < .05). Data were analyzed by analysis of variance and Student's t-test for grouped data. 67 intestinal and large intestinal transit were not altered by ECS when the markers were administered after 5 minutes (Table 5).

Effects of IFS on Gastrointestinal Motility and Analgesia

Inescapable footshock (IFS) did not produce any notable behavioral changes following twenty minutes of shock treatment. IFS also did not affect gastric emptying, small intestinal or large intestinal transit when the markers were administered at 0 minutes following the cessation of shock treatment (Table 6). IFS did produce a naloxone- reversible analgesia at 0 minutes but this effect was not present at 10 minutes following the cessation of shock treatment (Figure 16).

Effects of Kyotorphin on Small Intestinal Transit and Analgesia

Kyotorphin given i.c.v. 10 minutes prior to radiochromium instillation did not affect small intestinal transit at doses up to 120

~g (Figure 17). Kyotorphin did, however, produce a long-lasting (up to

40 minutes), dose-related analgesic effect with the peak response occurring at 10 minutes post-injection (Figure 18). Naloxone blocked the analgesic effects of kyotorphin at an antagonist dose of 5.0 mg/kg s.c. but not 2.0 mg/kg (Figure 19). The antagonism by naloxone was evaluated at the time of peak response for kyotorphin.

In Vitro Determination of Receptor Selectivity

Each of the opioid agonists were effective at inhibiting the electrically-induced contractions of the G.P.I. and M.V.D. and the inhibitory effects were antagonized by prior incubation of the preparation 68

Table 6. Percent gastric emptying and geometric centers for small and large intestinal transit in IFS and sham-treated rats.

Values are the mean ± s.e.m. and the number of animals in each group is given in parenthesis. Data were analyzed by Student's t-test for grouped data and no significant differences were found.

Intestinal Transit Treatment Gastric emptying Small Large

Sham (7) 85.2 ± 4.1 3.3 ± 0.3 3.7 ± 0.2

IFS (6) 73.6 ± 13.3 3.6 ± 0.6 4.4 ± 0.5 69

Saline+ Naloxone Saline + Naloxone Sham + Sham Shock + Shock 100

'"• E 80 • CI) en• +1 60 '"'"• W 40 a.:. ~ 20 ?ft 0 t::::J 0 c:::. 0 0 0 0' 10' 0' 10' 0' 10' 0' 10' Time Post-Shock (min)

Figure 16. Increase in hot-plate response times by rats treated with ineecapable footshock (Shock) and antagonism by naloxone.

Rats were tested at 0 minutes and at 10 minutes following the cessation of IFS. Naloxone (10 mg/kg s.c.) was given 10 minutes prior to the start of IFS. 70

4.2 'C" :!:O) cCen""" 3.4 ~(3 F _0 c.....,ns-t: 2.6 --wE0) 0)0 1.8 """0) ..5~ "-"' 1.0~--~~~1------~1------~1------~1~ o 15 30 60 120 IJg Kyotorphin (i.e.v.)

Figure 17. Failure of kyotorphin to affect small intestinal transit following intracerebroventricular administration. 71

Kyotorphin (i.e. v.) o saline 100 • 30 1019 • 60 1019 ~ 120 1019 80

~ ~ Q) 60 W. en Q. . +1 ~ c: 40 as #. Q) E - 20

0 5 10 15

Time Post injection (min)

Figure 18. Time course for kyotorphin-induced increases in hot-plate latencies following intracerebroventricular administration of kyotorphin. 72

100

~ 80 E. Q). w· CIJ a.· 60 ~· +1 t: cP- as Q) 40 E -- 20

o 30 60 120

J,l9 Kyotorphin (Le. v.)

Figure 19. Dose-response curves for kyotorphin-induced analgesia and antagonism by naloxone.

Kyotorphin-induced analgesia was measured as increases in hot-plate response times ten minutes post-kyotorphin admi nistration. Closed circles are saline-pretreated rats. Squares are naloxone (2.0 mg/kg) and circles are naloxone 5.0 mg/kg)-pretreated rats. Naloxone was given i.p. ten minutes prior to kyotorphin. *Indicates significantly different from kyotorphin alone (p < .05, Student's t-test for grouped data). 73 with naloxone. There were major differences, however, in the potencies of many of the agonists in the two preparations (Table 7). DAGO, DALA and DADL were equipotent in the G.P.I. while DADL was the most potent of these analogs in the M.V.D. DAGO was much less potent than either

DADL or DALA in this tissue. DADL was 90-fold more selective for the delta receptor while DAGO and normorphine were several times more selective for the receptor in the G.P.I., as indicated by the

G.P.I./M.V.D. ratios. DALA was modestly selective for the delta receptor although its potency in the M.V.D. was ten-fold less than that seen with DADL. DPLCE was more selective for the delta receptor (666x) and was equipotent with DADL in the M.V.D. Although DPLPE and DPDPE were somewhat less potent than DADL or DPLCE in the M.V.D., their rela tive inactivity in the G.P.I. (ICSO values greater than 2000 oM) indi cated that these cyclic enkephalins are highly selective for the delta opioid receptor. a-Endorphin was essentially equipotent in the two tissues and is a non-selective opioid agonist.

The dissociation constant of naloxone in competition with each opioid agonist was calculated as the naloxone Ke. The cyclic eokepha lins, as well as DALA and DADL, exhibited uniformly high Ke values

(33-49 oM) in the M.V.D. while the mu agonists, DAGO and normorphine, both exhibited much lower Ke values (2 oM). In the M.V.D., a-endorphin exhibited a naloxone dissociation constant that was intermediate bet ween that seen for the mu or delta selective agonists. The naloxone

Ke values for all the agonists in the G.P.I. were not different (Table 7). 74

Table 7. Inhibition of the electrically-induced contractions of the G.P.I. and M.V.D. by norpmorphine and several opioid peptides.

Data are the mean IC50 values ± s.e.m. of 3-4 preparations for each agonist. Naloxone Ke is the mean ± s.e.m. dissociation constant obtained in the same preparation as the IC50 and was calculated as described in the text.

IC50 (nM) G.P.I. Naloxone Ke (nM) Agonist G.P.I. M.V.D. M.V.D. G.P.I. M.V.D.

DAGO 23.4 ± 3.5 44.6 ± 6.7 0.5 2.0 ± 0.4 3.4 ± 0.7 a-endorphin 47.8 ± 5.9 28.2 ± 5.7 1.6 0.7 ± .08 13 .8 ± 4.0

Normorphine 91.0 ± 19 540 ± 113 0.2 2.5 ± 0.4 4.3 ± 0.5

DALA 23.4 ± 5.3 3.7 ± .04 6.2 2.7 ± 0.6 33.5 ± 6.3

DADL 24.3 ± 5.3 0.3 ± .06 90 2.1 ± 0.6 36.2 ± 11

DPLCE 213 ± 63 0.3 ± .03 666 2.5 ± 1.3 33.8 ± 2.7

DPLPE 2717 ± 50 2.5 ± .03 1078 1.1 ± 0.2 49.4 ± 1.9

DPDPE 6929 ± 123 2.2 ± 0.3 3164 2.7 ± 0.7 45.7 ± 9.1 75

Effects of Receptor Selective Agonists on Small Intestinal Transit and Analgesia

The mu selective agonists DAGO (0.02-2.0 ~g), and morphine

(1.25-80 ~g) as well as the nonselective agonists, a-endorphin (5.0-80

~g) and DALA (5.0-125 ~g) produced dose-related decreases in small intestinal transit following central administration (Figures 20 and

21). DADL also inhibited intestinal transit in a dose-related manner while DPLCE delayed intestinal transit only at the highest dose administered (Figure 22). The highly selective delta agonists, DPLPE and

DPDPE did not affect significantly affect intestinal transit at doses up to 50 ~g (DPLPE) and 125 ~g for DPDPE (Figure 23). Higher doses of these analogs were not tested as some rats showed mild seizures and barrel-rolling behavior. Animals that showed these toxic effects were excluded from the results on intestinal transit and analgesia. The kappa receptor agonist, U-50,4884 (VonVoightlander et aI, 1983) did not affect intestinal transit at doses up to 125 ~g i.c.v. (Figure

24). a-Endorphin (0.2-5.0 ~g), DALA (1.0-10 ~g), DADL (0.25-5.0 ~g) and morphine (0.25-5.0 ~g) produced dose-related increases response times for rats placed on the 55 0C hot plate following i.c.v. administration of the agonist. The analgesic effect lasted between 20 and

40 minutes with peak effects occurring at 10 minutes following treatment (Figure 25). DPLCE (1.0-10 ~g), DPLPE (5.0-50 ~g), DPDPE (5.0-125

~g) and DAGO produced similar effects (Figure 26). The analgesic effect of these peptides was antagonized by pretreatment with naloxone

(2.0 mg/kg i.p.) when evaluated at peak effect (10 minutes post-peptide treatment, Figure 27). 76

DAGO MORPHINE ... 4.0 :!:: -Q) en c: c: -Q) to ... (,) 3.0 I- (J to ... • c: Q) • en -E 2.0 -Q) 0 • Q) • -c: -(!) 1.0 00 Do 0 0 0.08 0.4 2.0 1.25 5.0 20 80

Dose (1-I9 i.c.v.)

Figure 20. Inhibition of intestinal transit by DAGO and morphine.

Agonists were given ten minutes prior to radiochromium instillation. *Indicates significantly different from saline-treated controls (p < .05, Dunnett's t-test). 77

DALA f3-ENDORPHIN r... .:: -Q) en c:: c:: Cd -0) r... 0 I- 0 Cd r... c:: .- 0) * en - E -0) 0 * * c:: 0) * --~ * 1.0 0 0 0 0 0 5.0 25 125 5.0 20 80

Dose (1J9 i.e.v.)

Figure 21. Inhibition of intestinal transit by DALA and a-endorphin.

*Indicates significantly different from saline-treated controls (p < .05 Dunnett's t-test). 78

DADL DPLCE 4.0 ... :t::en -Q) c: c: ...tU -Q) 3.0 t- 0 o tU .~ c: Q) en -E * • -Q) 0 Q) -c: C!) • - 0 0 0 0 1.25 5.0 20 80 10 50 100

Dose <1-19 i.e.v.)

Figure 22. Inhibition of intestinal transit by DADL and DPLCE.

*Indicates significantly different from saline-treated controls (p < .05, Dunnett's t-test). 79

DPLPE DPDPE 4.0

~ -Q) :t:::: en c: c: as -Q) 3.0 ~ (,) I- 0 as ~ .~ -Q) 2.0 en E -Q) 0 Q) c: - ~ - 1.0 o 5.0 25 50 5.0 25 125

Dose (~g i.e.v.)

Figure 23. Failure of DPLPE and DPDPE to affect intestinal transit. 80

U-50488H 4.0

:!:: "'"'~ en .....(]) c: c: as (]) 3.0 ~ I- 0 -as .-0 c: .....~ ..... (]) 2.0 en E .....(]) 0 c: (]) CJ "'" 1.0 0 5 25 125

Dose (loig i.e. v.)

Figure 24. Failure of U-50,488H to affect intestinal transit 81

OSaline o Saline p-Endorphin • 25.0 ,..g .5.0,..g A5.0 ,..g DADL A 1.25,..g .1.0,..g .0.25,..g 100 * 100 * 80 60 -E 40 a) 20 en 0 +t I I i i 0 10 20 40 - o Saline oSaline W .10.0,..g .5.0,..g a.: DALA A3.3,..g Morphine A 1.25,..g ~ .1.0,..g .0.25,..g 100 * 100 rF. * * * 80 80 * 60 40 20 20 * 0 0 I i i , i i i 0 10 20 40• 0 10 20 40 Time Postinjection (min)

Figure 25. Time course of analgesia produced by a-endorphin, DADL, DALA and morphine.

Analgesia is expressed as the maximum possible increase in hot-plate latency. *Indicates significantly different from saline-treated controls at each time point (p < .05, Dunnett's t-test). 82

DPLCE DAGO o Saline 100 100 o Saline '" • 10.0/-lg .0.4/-lg 80 to 3.3/-lg 80 to 0.08 /-lg 60 • 1.0/-lg 60 .0.016/-lg ,.... 40 20 E· 0 0 I , Q)· I I en 0 10 20 40 0 10 20 40 +I DPLPE DPDPE o Saline o Saline • 125 J-lg -W· 25.0/-lg 100 • 50.0/-lg 100 * 5.0/-lg a; to 25.0/-lg 80 80 • 5.0/-lg ::E 60 60 ?P. 40 40 20 20 0 0 0 10 20 40 0 10 20 40

Time Postinjection (Min)

Figure 26. Time course of aolagesia produced by DPLCE, DPLPE, DPDPE and DAGO.

Analgesia is expressed as the maximum possible increase in hot-plate latency. *Indicates significantly different from saline-treated controls at each time point (p < .05, Dunnett's t-test). 83

DPDPE DPLCE 100 100 80 80 60 60

...... 40 40 ~ IU 20 20 uj ~ II ii 0 , , II 0 I , z I cs:: 5.0 25.0 125.0 1.0 3.3 w 10,0 ...... :a DPLPE DAGO 100 u.i 0: ::E 80 ~ 60 40 20 20 • 0 0 I I j ..----- 5.0 25.0 125.0 DOSE 119 ICV

Figure 27. Dose-response curves for analgesia produced by DPLCE, DPLPE, DPDPE and DAGO and antagonism by naloxone.

Analgesia was measured at ten minutes post-agonist admi nistration and is expressed as the maximum possible increase in hot-plate latency. Circles represent saline pretreated animals and squares represent naloxone (2.0 mg/kg i.p.) pretreated animals. *Indicates significantly different saline pretreated animals (p <.05, Student's t-test for grouped data). 84 Relative Potencies In Vivo

The relative potency of the opioid agonists were compared in the two in vivo assays (inhibition of small intestinal transit and the hot plate test). Morphine and DAGO produced a delay in intestinal transit and increased the repsonse times in the hot-plate test. a-Endorphin was slightly less potent than DAGO in these assays followed by DADL in the hot plate and morphine in the intestinal transit assay. The cyclic eokephalin analogs were the least potent agonists in producing analge sia while DPLCE was only slightly effective in delaying intestinal transit. The EDSO values for DPLPE and DPDPE could only be estimated due to their lack of effect on small intestinal transit. U-SO,488H produced analgesia only at the highest dose and the EDSO for this drug in the hot plate test may also be an estimate only.

It should also be noted that there was approximately a 2-4 fold difference in potency for inhibition of intestinal transit and for pro duction of analgesia by DAGO, morphine DALA and a-endorphin, However, there was a marked difference in potency (16-46 fold) for inhibition of transit and for producing analgesia for those agonsits showing selec tivity for the delta rec~ptor (DADL, DPLCE, DPLPE and DPDPE). Based on this observation, delta receptor selectivity was correlated with an increase in the EDSO calculated for inhibition of intestinal transit and for producing analgesia using the ratio of ICSO values in the

G.P.I. and M.V.D. as a measure of delta receptor selectivity. There was a significant positive correlation between delta selectivity and 8S and an increase in the analgesic EDSO (decrease in potency) with delta selectivity as well as with an increase in the antitransit EDSO and delta selectivity (Figure 28). 86

Table 8. ED50 values for opioid-induced inhibition of small intestinal transit (S.I.T.) and for producing analgesia.

ED50 for each end point was calculated as described in the text. Numbers in parenthesis are 95% confidence limits.

ED50 nanomoles/rat Agonist Inhibition of S.LT. Analgesia

DAGO 0.6 (0.25, 1.3) 0.14 (0.07, 0.3) e-endorphin 1.1 (0.28, 4.14) 0.53 (0.3, 0.9)

Morphine 5.7 (2.7, 12.2) 2.02 (0.95, 4.3)

DALA 17.4 (8.4, 36.1) 5.1 (2.8, 9.0)

DADL 26.9 (17.3, 41.8) 0.58 (0.42, 0.81)

DPLCE 114.9 (79.4, 166) 6.2 (2.8, 13.8)

DPLPE IV 300 18.2 (10.3, 32.0)

DPDPE IV 1000 35.5 (18.2, 69.0)

U-50,488H IV 1000 120.8 (93.3, 154.8) 87

2.0 r = 0.747 .! P<0.05 fIj o Q) .., 01 • eli 1.0 w c: < oClI- as - E ~ 0.0 Q) s= l- -1.0 •

3.0 r = 0.919 ~ P< 0.01 en c: 2.0 0 ;: :c :c .E 1.0 o on C W 01 0.0 .2

-1.0 -1.0 0.0 1.0 2.0 3.0 4.0

log IC so GPllICso MVD

Figure 28. Correlation of delta receptor selectivity with increases in analgesic EDSO and the EDSO for inhibition of small intestinal transit (S.1.T.). DISCUSSION

Although exogenously administered opiates consistently delay passage of intraluminal contents through the gastrointestinal tract in humans and experimental animals, multiple sites and mechanisms of action are involved in opiate-induced constipation in different spe cies. In the dog, cat, monkey and human, morphine produces an increase in segmenting contractions of the small and large bowel (Plant and

Miller; Weinstock, 1971; Pruitt et al., 1974; Bueno and Fioramonti,

1982). These contractions occlude the lumen and increase resistance to flow of intraluminal contents (Vaughan Williams, 1954; Bass and Wiley,

1965; Bass et al., 1973). Alternatively, contractions of the guinea pig intestine are inhibited by exogenously administered morphine

(Pruitt et al., 1974). The contractile response of the rat intestine has been inferred largely from myoelectric recordings in unanesthetized rats in which systemic morphine inhibits electrical spiking (Weisbrodt et al., 1980). This is in contrast to results obtained in dogs and cats in which morphine is known to stimulate both contractions and electrical spiking of the intestine in these species (Stewart et al,

1977; Konturek et al., 1980). As this type of electrical activity has been correlated with circular muscle contractions (Weisbrodt, 1981), morphine may inhibit small intestinal motility in the rat. Multiple sites of action must also be considered when discussing opiate effects

88 89 on intestinal motility and propulsion as both central nervous system and local intestinal effects have been described. Morphine stimulates contractions of some isolated small intestinal (Burks and Long, 1967) and colonic preparations (Gillan and Pollock, 1980; Huidobro-Toro and

Way, 1981) and systemic treatment o.f the anesthetized rat can produce a brief contractile response; an effect that persists after decapita tion, suggesting that a local intestinal action is responsible (Burks,

1976). A peripheral mechanism has also been supported by studies in which low doses of morphine given intraperitoneally inhibited movement of an orally administered charcoal meal along the intestine. The same doses given i.v. had no effect on gastrointestinal transit (Tavani et a1., 1980).

The intestinal mucosa may be a peripheral site at which opiates can produce their antidiarrheal effect, which is unrelated to changes in intestinal motility (Powell, 1981). In vitro studies have shown that opiate agonists can stereospecifically enhance electrolyte absorb tion by the intestinal mucosa and that this effect is naloxone sen sitive (McKay, et al., 1981; Dobbins et al., 1980). Electrolyte absorbtion would also promote water reabsorption by the mucosa, decreasing the volume of intraluminal content and fecal output.

Morphine-induced changes in gastrointestinal motility resulting from an effect within the central nervous system have been reported in a number of species including the cat (Stewart, et al., 1977), dog

(Bueno and Fioramonti, 1982), and rat (Margolin, 1963; Parolaro et al,

1977; Stewart et a1., 1978; Schulz et al., 1979). Evidence for a central site of action in the rat is based on the ability of low doses 90 morphine given intracerebrally to inhibit intestinal transit, while much larger systemic doses are required to produce a comparable antitransit effect. A recent study used a quarternary amine con taining opiate receptor antagonist, diallynormorphinium, given i.c.v. to selectively block central nervous system opiate receptors. The effects on intestinal motility produced by s.c. administration of morphine were inhibited by this pretreatment, whereas diallylnor morphinium pretreatment did not alter the anti transit effects of peripherally administered morphine (Burleigh et al., 1981).

The results of the present investigation suggest that a central nervous system mechanism was responsible for morphine-induced inhibi tion of intestinal motility in the rat. Intracerebroventricular morphine was more potent in its inhibition of intestinal transit than either s.c. or intragastrically administered morphine. These data support the idea that morphine can act within the central nervous system to alter autonomic or humoral outflow to the gastrointestinal tract and delay intestinal transit. The markedly reduced potency of intragastrically administered mor.phine also argues in favor of a central site of action. If the anti transit effects of opiates were exerted exclusively on myenteric neurons or on the intestinal mucosa, it might be expected that intragastric morphine would be considerably more potent than the other routes of administration. Rapid con jugation of morphine to glucuronic acid in the intestinal epithelium and liver certainly accounts for the reduction in systemic availabilty of orally-administered opiates (Jaffe and Martin, 1980). However, if 91 the principal action was directly on the mucosa, metabolism would not be a factor. These results suggest that a mucosal mechanism is not sufficient to account for the antipropulsive effects of opioids and opiates in the absence of a secretory diarrhea. A secretory diarrhea is an increase in fecal output resulting from a disruption of the nor mal absorption/secretion processes of the intestinal epithelium. The result being a dramatic increase in the amount of intraluminal con tents reaching the large bowel as well as in the fluid content of this material. Although it is likely that the antisecretory effects of opiates are mediated, at least partially, at the mucosa and that this contributes to their antidiarrheal actions, a mucosal effect is pro bably not the primary action under physiological conditions as altera tions in intestinal motility alone appear be sufficient to reduce intestinal transit.

Alterations in motility alone may also account for the antipro pulsive effects of opioids in some pathological conditions. Castor oil is a commonly used laxative that promotes fluid accumulation in the intestinal lumen by damaging the mucosal epithelium (Gaginella and

Bass, 1979). DALA and a-endorphin both inhibited intestinal transit in castor oil treated rats, yet neither peptide altered the amount of fluid that had accumulated in the intestine. Although the peptides were given centrally, opioids have been shown to alter fluid transport by a central mechanism (Brown and Miller, 1983). If the antisecretory effects were responsible for the centrally-mediated delay in transit, it would be expected that the amount of fluid would be reduced in 92

opioid-treated rats when compared to saline-injected controls. While

the antisecretory effects of centrally administered opioids may not be

active against castor oil-induced secretion as the absorption/secretion

mechanisms may destroyed by this laxative, the motility effects alone

are sufficent to delay intestinal transit even in the presence of a

secretory diarrhea.

Gastric emptying was not affected by intragastrically admini

stered morphine whereas s.c. and i.c.v. morphine effectively reduced

gastric emptying. This observation suggests that a local mechanism for

opiate-induced changes in gastric motility may not be important in the

rat. These data also suggest that the small intestine may be more sen

sitive than the stomach to the inhibitory effects of intragastric

morphine. Due to first pass metabolism, intragastric morphine would

not achieve the circulating level of free morphine that may be required

to alter gastric motility whereas sufficient morphine may be available

to alter small intestinal motility. Parenterally administered morphine

is not subject to this large first-pass effect and sufficient amounts

of unconjugated morphine can reach the site of action responsible for morphine-induced changes in gastric motility. The finding that paren

terally administered morphine delayed gastric emptying is also signifi

cant to technical aspects of studies involving gastrointestinal tran

sit. When the marker is placed directly into the stomach, inhibition

of gastric emptying can be responsible for delays in propulsion that may be incorrectly attributed to changes in small intestinal motility.

The apparent difference in sensitivity of gastric and intestinal moti lity to the effects of morphine makes this distinction highly relevant. 93

It should also be noted that liquid markers were used to study intesti···

nal transit and gastric emptying. It is generally accepted that tran

sit and emptying of liquids and solids may occur at different rates

(Kelly, 1981). The results presented here may only apply to intesti

nal transit and gastric emptying of liquid markers.

Previously, alterations in small bowel motility after morphine

treatment of the rat have been inferred from changes in small intesti

nal transit and myoelectric activity. The technique developed here

has allowed direct measurement of small intestinal motility in unan

esthetized rats. The experimental protocols measuring intestinal

motility and transit were carried out in parallel to allow correlation

of changes in motility with changes in transit. Morphine treatment

resulted in a dose-related decrease in the frequency of contractions in

both the duodenum and jejunum. With a decrease in contractile fre

quency, it can be assumed that there would be also be a reduction in

the propulsive forces applied to the intraluminal contents which may

account for the decreased intestinal transit after morphine treatment.

The distal portion of the small intestine was more sensitive to the

inhibitory effects of morphine as the jejunum showed a greater reduc

tion in contractile frequency than the more proximal duodenum. It

should also be noted that the doses of morphine that did not signifi

cantly alter transit also did not affect intestinal motility. This was true for both i.c.v and s.c. administered morphine.

The reduction in contractile frequency was also closely asso ciated in time with inhibition of intestinal transit. Previous studies 94

have shown that the time to peak effect for inhibition of intestinal

transit by morphine in the rat was approximately 30 minutes (Stewart et al., 1978). In the present set of experiments, a significant reduc

tion in contractile frequency was not seen consistently until 30 minu tes after morphine administration. These observations also support the hypothesis that an inhibition of motility is responsible for the antitransit effects of morphine in the rat. In addition, the greater potency of i.c.v. morphine at inhibiting both intestinal transit and motility indicates th~t morphine is acting within the brain to alter humoral or autonomic influences on myenteric neurons.

It is also interesting to note that the time course of inhibi tion of intestinal contractions was the same for both s.c. and i.c.v. administered morphine. This observation suggests that although morphine is acting in the central nervous system, it may be at sites that are removed from the periventricular space. Subcutaneous morphine would be carried to these areas by the systemic circulation while i.c.v. morphine would gain access to these areas only by the much slower process of simple diffusion. These pharmacokinetic con siderations would account for the similarities in time course.

In addition to morphine, the opioid peptides, a-endorphin and

DALA, can also delay the propulsion of a radioactive marker through the small intestine of unanesthetized rats. Although a-endorphin was several times more potent than DALA, both peptides produced their effects by an action within the central nervous system. This is evi denced by the failure of relatively high doses of the opioid peptides 95 to inhibit intestinal transit while much smaller quantities given int~acerebrally were effective. The opioid peptides [D-Ala2 , Leu5] enkephalinamide and dynorphin (1-13) did not alter intestinal transit when given i.c.v. at the doses studied. This result is consistent with the belief that the different classes of opioid peptides differ in the~r receptor selectivity and in the responses produced by each type of peptide. Dynorphin (1-13) is believed to be an agonist at the kappa opioid receptor (Huidobro-Toro et al., 1981; Wuster et al.,

1981; Chavkin et al., 1982). These data coupled with the data demonstrating a failure of U-50488H, a non-peptide kappa agonist

(VonVoightlander et al., 1983) to affect small intestinal transit in the rat indicate that the kappa receptor does not participate in the brain-mediated opioid effects on intestinal motility. The specific receptors mediating these effects will be discussed in more detail.

The intestinal responses produced by DALA and a-endorphin were antagonized by naltrexone ~~d nslaxone indicating that the response to i.c.v. administered opioid peptide is mediated specifically at opiate receptors. Naloxone was given i.p. in these experiments to establish that centrally-located opioid receptors are accessible to peripherally administered drugs. The opioid peptides also show a limited per meability to the blood-brain barrier (Rapoport et al., 1980) and hence the central opioid receptors should be modestly accessible to peptides given i.p., however, a systemic antipropulsive effect for DALA and a-endorphin could not be demonstrated. Dairman et ale (1979) reported similar results with methionine enkephalin but obtained an anti diarrheal effect with intravenously administered a-endorphin. The 96 dose of peptide used in those experiments was several fold higher than

the highest dose used in the present studies and it is probable that _ at much higher blood levels a significant fraction of the opioid pep tide would penetrate into the brain (Kastin et al., 1980).

Although a-endorphin was many times more potent in producing a delay in intestinal transit, five times as much naltrexone was required to antagonize the antitransit effects of DALA. This may be related to the extended half-life of the synthetic analog or to its smaller size which may permit diffusion to areas not accessible to a-endorphin. In addition, DALA but not a-endorphin reduced the body weight loss pro duced by castor oil-induced diarrhea. This antidiarrheal effect was mediated at a site distal to the small intestine as the fluid accumula tion in the small intestine was not altered by DALA or a-endorphin.

Colonic as well as small intestinal motility can be affected by sti mulation of cerebellar nuclei (Martner, 1975). These areas are removed from the ventricular space and may be the site the additional effect produced by DALA.

The delay in transit is probably not the result of opioid pep tides from the adrenal medulla (Viveros et al.,_ 1979) or pituitary

(Guillemin et al., 1977) in response to stimulation of CNS opioid receptors by i.c.v. peptide with a subsequent effect on myenteric neurons. The antitransit effects of DALA or a-endorphin were not affected by hypophysectomy. In addition, the quarternary amine con taining antagonist, DANM, was used to selectively block peripherally located opioid receptors (Schulz et al., 1979). The cationic charge 97 restricted the distribution of DANM to the periphery. DANM did not affect the antitransit effects of either a-endorphin or DALA. The dose of DANM used was sufficient to block peripheral opioid receptors as the antitransit effects of peripherally-administered loperamide was attenuated. Similar results with other quarternary amine-containing opioid receptor antagonists have been obtained by other investigators

(Russell et al., 1982). These investigators used N-methyl-naltrexone to block the antidiarrheal effects of peripherally-administered morphine but only at those doses of antagonist that would also block the analgesic effects of morphine in the rat. These same investigators obtained somewhat different results in the dog, however. The quarter nary antagonist would block the intestinal effects of morphine but would not produce signs of abstinence in dependent dogs. The results presented here, along with those of others, demonstrate that the intestinal inhibitory actions produced by opiates and opioid peptides are not the result of peripheral release of opioid peptides into the systemic circulation with a subsequent effect on myenteric neurons and also point out the important species differences in the gastroin testinal responses to opiates. The failure of hypohysectomy to abo lish these intestinal inhibitory effects also shows that the central effect of the opioids are not due exclusively to release of a non opioid substance from the pituitary with a subsequent direct effect on the small intestine.

Spinal cord section at a high thoracic level or subdiaphramatic vagotomy also did not abolish the antitransit effects of a-endorphin or 98

DALA. Similar results have been obtained by others in mice following intracerebral administration of morphine (Margolin, 1963). However, subdiaphramatic vagotomy has also been shown to abolish the anti pro pulsive effects of i.c.v. morphine in the rat (Stewart et al., 1978).

These investigators used a similar method for vagotomy to that used in the present study. In addition, the vagus nerve adheres closely to to the serosal surface of the esophagus at this level (Legros and

Griffth, 1968) and severing the esophagus would also severe the vagus nerve. The failure of this procedure to block the intestinal effects of the opioid peptides should be attributed to factors other than incomplete vagotomy.

The sympathetic innervation of the small intestine leaves the spinal cord as the splanchnic nerves below thoracic level four in the rat (Furness and Costa, 1974). Presumably cutting the spinal cord at the second thoracic vertabra would remove this input. However, another study has shown that the intestinal effects of centrally administered morphine could be blocked by cutting the spinal cord at the fourth cervical vertabra (Margolin, 1963). It is possible that some sympathetic fibers emerge from the spinal cord above this level and pass down the sympathetic chain before joining the splanchnic ner ves. ~his would bypass the cut at thoracic level two and the inhibi tory effects of centrally~administered opioids would persist.

Alternatively, the antipropulsive effects of the opioids could be mediated simultaneously by hormonal, sympathetic and parasympathetic mechanisms and elimination of only one system may be insufficient to reduce significantly the antipropulsive effects. 99

The results described to this point clearly indicate the pre sence of an opiate-sensitive mechanism within the brain of rats that, when stimulated by exogenously administered opiates or opioids, can alter small intestinal motility. The distribution of opioid peptides and their receptors in brain areas associated with autonomic control suggests that this may be an important system for regulation of intestinal motility. Naloxone and naltrexone given centrally or peripherally had no effects of their own on intestinal transit, indi cating that this opioid peptide system may not be tonically active and may require certain stimuli or conditions for activation. Several treatments were tested in an attempt to provide direct evidence for a physiological role of the opioid peptides in control of intestinal motility.

Electroconvulsive shock (ECS) given to rats produces analgesia, catalepsy, hyperthermia and respiratory depression, all of which are characteristic responses of opiate treatment (Holaday et al.,1981;

Holaday and Belenky, 1980). These effects were naloxone sensitive, again suggesting an effect mediated specifically at opioid receptors.

ECS produced a naloxone-reversible analgesia in the present study but did not affect gastrointestinal motility as was indicated by no changes in gastric emptying, small intestinal transit or colonic transit. An important point to note is the relatively short duration of the analgesic effects, lasting between 5 and 35 minutes. A similar duration has been reported by others (Lewis et al., 1981). These investigators also reported that the analgesic effects of ECS also are

.;.. 100 not blocked by hypophysectomy, indicating that this response is mediated by brain endorphin or enkephalinergic systems and not by cir culating opioids. These data indicate that the brain systems involved with opioid-induced analgesia and constipation may be independent and may require different stimuli for activation. It is possible that a more protracted activation of central opioid systems, that is longer than five minutes, is required to produce a significant alteration in gastrointestinal propulsion. The timing of marker administration may also be an important factor as the duration of the analgesic response was relatively short and it is possible that an effect on gastroin testinal motility was missed by giving the markers at 5 minutes after

ECS. However, the analgesic response is maximal at this point and it would be expected that inhibition of GI motility would also be maximal.

This would produce a delay in the propulsion of marker in ECS-treated rats that would not be present in 'sham-treated animals; the result being a decrease in gastric emptying and intestinal transit.

Other studies have shown that stressors such as inescapable footshock can produce naloxone-sensitive analgesia (Akil et al., 1976) and it is possible that the CNS mechanism for modulation of intestinal motility may also require an extended period of stress for full activa tion. The results obtained here using footshock also indicate that the analgesic and intestinal mechanisms in the brain which respond to opiates are independent. The naloxone-sensitive analgesia resulting from IFS is produced within the brain as hypophysectomy does not abo lish footshock-induced analgesia (Watkins et al., 1982). The duration 101 of the analgesic effect produced by IFS is relatively brief (less than ten minutes) and this may not be of sufficient duration to produce a significant inhibition of gastric emptying or small and large intesti nal transit.

Kyotorphin also produced a naloxone-reversible analgesia of relatively long duration (greater than IS minutes at the highest dose).

Kyotorphin did not inhibit small intestinal transit, however, at any dose. It is possible that this disparity between the gastrointestinal and analgesic effects of endogenous opioids could be based on a dif ference in opioid receptor SUbtypes mediating each type of response.

The receptors mediating the analgesic and intestinal effects of exoge nously administered opioids were investigated using several highly selective agonists for the opioid receptors.

The opioid selectivity of the cyclic analogs of the enkepha lins, DPLCE, DPLPE and DPDPE and the other opioid agonists discussed here, was established by comparison of the ICSO values of each agonist in the M.V.D. and G.P.I. The G.P.I. contains predominately mu recep tors with lower numbers of kappa receptors and no demonstrated func tional delta receptors, while the M.V.D. contains predominately delta receptors with lower numbers of mu and kappa receptors (Lord et ale,

1977; Leslie et al., 1980). The ratio of ICSO values was used, there fore as a biological indicator of mu versus delta selectivity. In the present study, DAGO and normorphine showed the greatest selectivity for the receptor found in the G.P.I., in agreement with the results of others showing a high mu receptor selectivity for DAGO (Handa et al., 102

1981). Others have also demonstrated with binding experiments that

DAGO acts exclusively at mu receptors with little or no activity at the kappa or delta receptors (Kosterlitz and Paterson, 1981). The mu receptor selectivity of 2-5 fold obtained in these experiments for

DAGO and normorphine is relatively small in comparison with delta receptor selectivity obtained with DPLPE and DPDPE (1000-3000 fold), based on potency differences in the G.P.I. and M.V.D. This method may underestimate mu receptor selectivity as there is a significant number of mu receptors in the M.V.D. (Lord et ru .• , 1977) and a mu agonist can therefore be active in this tissue. The ratio of activity in the

G.P.I and M.V.D. may provide a more accurate assessment of delta selectivity than of mu selectivity. This is evidenced by the low naloxone Ke values (2 oM) for DAGO and normorphine in the M.V.D., indicating an action at mu receptors, which are more sensitive to naloxone blockade than delta (Lord et al., 1977) or kappa receptors

(Chavkin and Goldstein, 1981). The high Ke values (33-49 oM) in the

M.V.D. for the eokephalin analogs, other than DAGO, indicate an action at the delta receptor. There is no difference among the Ke values for the agonists tested in the G.P.I. suggesting an action at a single receptor. The complete lack of kappa activity for the cyclic eokepha lins (S.J. Paterson, personal communication) and the uniformly low Ke values (0.7-2.7 oM) for all the agonists in the G.P.I., indicate that this is a mu receptor interaction. It should be emphasized that although the cyclic eokephalins show some mu activity in the G.P.I., they are very weak agonists at this receptor with IC50 values as high 103

as 2700-6000 nM for the bis-penicillamine analogs. They are, however,

potent delta receptor agonists with ICSO values of less than 3 nM in

the M.V.D. These observations establish the cyclic enkephalin analogs

as being highly selective for the delta opioid receptor and also pro

vide further evidence for the absence of functional delta receptors

in the G.P.I. preparation.

S-endorphin was equipotent in the M.V.D. and the G.P.I., indica

tive of equivalent actions on mu and delta receptors. The naloxone

Ke for S-endorphin in the G.P.I. was very low (0.7 nM) suggesting a mu

receptor interaction only, while in the M.V.D. the Ke (13 nM) was

intermediate between that obtained for the mu and delta agonists suggesting an effect at both mu and delta receptors in this tissue.

DALA was about 6-fold more potent in the M.V.D. than in the G.P.I. but

this preference for the delta receptor is weak in comparison with other delta receptor agonists we have examined (90-3000 fold). a-endorphin and DALA are therefore non-selective opioid agonists.

DADL, with a G.P.I./M.V.D. ratio of 90, was relatively more selective for the delta receptor.

Although the kappa receptor selectivity of U-SO,488H was not tested directly in the present study, its inability to inhibit S.I.T. after i.c.v. administration supports the recent report that this com pound acts exclusively at kappa receptors (VonVoightlander et al.,

1983).

Central administration of the mu selective opioid agonists, DAGO 104 and morphine, and the non-selective agonists, a-endorphin and DALA pro duced significant decreases in small intestinal transit. Normorphine was used to characterize mu selectivity in vitro because of its lower lipid solubility and ready reversiblity. However, morphine and nor morphine are similar in their in vivo effects (Lockett and Davis, .1958;

Miller and Anderson, 1954) and are equipotent in the G.P.I.

~Waterfield et al., 1977). The highly selective delta receptor ago nists, DPLPE and DPDPE, did not significantly affect intestinal tran sit at any dose tested. The failure of these cyclic enkephalins to alter transit is not due to rapid degradation in vivo as their analge sic effects were of the same duration as those of the enkephalin ana logs which did produce a delay in intestinal transit. The kappa agon nist U-50,488H, did not alter small intestinal transit at any dose used in this study. Other kappa agonists, such as the ketazocines, also do not affect gastrointestinal motility following i.c.v. administration (Porecca et al., 1983a). These data indicate that delta and kappa receptors do not participate in the brain-mediated opioid effects on small intestinal motility. A previous study has suggested that opioid-induced inhibition of gastrointestinal transit can be mediated by mu receptors located in the central nervous system (Ward and Takemori, 1983a). The data presented here indicate that the intestinal motility effects of i.c.v. administered opioids are mediated exclusively by mu receptors.

DADL, an enkephalin analog previously regarded as a selective delta agonist, was a potent inhibitor of intestinal transit. While the 105

G.P.I/M.V.D. ratio indicates that DADL possesses delta receptor agon

ist properties, it is also a potent mu agonist as indicated by its low

IC50 in the G.P.I. DPLCE is 6-fold more selective than DADL as a delta

agonist yet DPLCE produced a moderate inhibition of intestinal transit

at the highest dose (100 ~g). Although the ICSO for inhibition of

contractions in the G.P.I. by DPLCE was much higher than that seen for

the other enkephalin analogs (DADL, DAGO and DALA), it was still only

double that obtained for normorphine (213 vs. 90 nM). It is probable

therefore, that the antitransit effects of DPLCE, like DADL, are mu

mediated.

The receptor or receptors mediating the analgesic effects of

these peptides is not as clear. The analgesic effects of the cyclic

enkephalins were blocked by pretreatment of the animal with naloxone.

Naloxone antagonism indicates that the analgesic effects of the confor

mationally constrained enkephalins are produced by binding to opioid

receptors regardless of the type or anatomical location. The inact

ivity of the cyclic enkephalins as kappa agonists and the relative

absence of kappa receptors from the rat brain (Hiller and Simon, 1980)

seems to exclude this population as mediators of analgesia; at least

at the supraspinal level. U-50,488H is believed to produce its

analgesic effects at the spinal level (VonVoightlander et al., 1983).

In the present study U-50,488H produced analgesia only at the highest

dose (125 ~g, data not shown) while lower doses (5 and 25 ~g) were

ineffective. It is conceivable that at the highest dose a significant amount of the compound could reach the spinal cord via the cerebrospi

nal fluid, accounting for the ana~gesic response. 106

Our finding that highly selective delta agonists induce analge

sia, indicating a role of brain delta receptors mediating analgesia, is

consistent with previous suggestions to this effect (Fredrickson et al,

1981; Ward and Takemori, 1983b). The cyclic enkephalins, DPLCE, DPLPE

and DPDPE were all effective analgesics, although considerably less

potent than the mu selective or non-selective compounds. There was in

fact a significant inverse correlation between delta selectivity and analgesic potency. The lower absolute potency of the delta agonists as analgesics may be attributed to the lower numbers of delta receptors

in brain regions normally associated with opiate-induced analgesia

(Chang et al., 1979; Ninkovic et al., 1981). This would require more agonist to produce analgesic responses comparable to those mediated by the more abundant mu receptors. Studies in vitro, have shown that receptor number can be an important factor in enhancing potency of opioid agonists (Cox and Chavkin, 1983) and it is believed that this is also an important determinant of potency in vivo. Alternatively, the delta mediated analgesia may also be a spinal cord event (Ling and

Pasternak, 1983) and significant fractions of i.c.v. administered pep tide can reach the spinal cord level (Ohlsson et al., 1982).

Distribution of the peptide to the cord may require relatively high doses of the cyclic enkephalins in order to produce an analgesic effect. ,Indeed, intrathecal adminstration to mice of cyclic enkepha-

1ins can produce a potent and long-lasting analgesia (Porreca et al.,

1983b). However, these data are also consistent with an action of the cyclic enkephalins on brain delta receptors mediating analgesia but not 107

inhibition of small intestinal transit. In fact, the large dose ratios

between analgesia and inhibition of intestinal transit indicate a great

selectivity of the delta receptors agonists for analgesic receptors in

the brain.

The considerably lower EDsO values for the mu selective agonists

(DAGO and morphine) and non-selective agonists (a-endorphin and DALA)

suggests that these compounds produce analgesia by acting at brain mu

receptors. The correlation of delta receptor selectivity with

increases in analgesic EDsO, while significant (r= 0.747), is not as dramatic as that obtained with delta selectivity and increases in EDsO for inhibition of intestinal transit (r= 0.919). An important point to consider is the .value of r2 (0.5 for analgesia and 0.82 for inhibition of intestinal transit). The r2 value allows an estimation of the frac tion of change in the dependent variable, the EDsO for each assay, that can be explained by changes in the independent variable, delta selec tivity (Steel and Torrie, 1960). Approximately 50% of the increase in

EDsO for producing analgesia is explicable by preference for the delta receptor. The remainder may be attributed to other variables, such as distribution to the spinal cord, lower receptor number or experimental error. However, less than 20% of the increase in EDsO for the antitransit effects of these compounds can be attributed to factors other than delta receptor selectivity. It should also be noted that the EDsO values for inhibition of intestinal transit by the most selec tive compounds (DPLPE and DPDPE) were calculated from small but insignificant decreased in the geometric center when compared to 108 control. This yielded only an estimation of the EDSO that may in fact be much larger. A larger value would thereby strengthen the rela tionship between preference for the delta receptor and lack of effect on intestinal motility.

These studies may provide evidence for a physiological role for the delta opioid receptor and at least a pharmacological role for the mu opioid receptor. However, as the nature of the endogenous mu ligand is unknown at this time, characterization of this substance may be required before a physiological function for the mu receptor can be established. The enkephalins are believed to be the endogenous ligands for the delta receptor (Lord et al., 1977), while the dynorphin-related pep tides may be the agonists for the kappa receptor

(Chavkin and Goldstein, 1981). These observations are consistent with the data presented here. The physiological and pharmacological sti muli used to activate the endogenous opioid systems produced their analgesic effects by releasing eokephalins and possibly dynorphins with subsequent actions on delta and kappa receptors. This is also consistent with the failure of these stimuli to alter gastrointestinal motility, a mu receptor event.

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