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Gastric opioid peptides : their biochemical forms, receptor distribution, release and actions Nishimura, Erica 1985

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GASTRIC OPIOID PEPTIDES: THEIR BIOCHEMICAL FORMS, RECEPTOR DISTRIBUTION, RELEASE AND ACTIONS BY ERICA NISHIMURA B.Sc. The University of B r i t i s h Columbia, 1981 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE FACULTY OF GRADUATE STUDIES (Department of Physiology) We accept this thesis as conforming to the required standard The University of B r i t i s h Columbia August, 1985 @ Er i c a Nishimura In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements for an advanced degree at the University of B r i t i s h Columbia, I agree that the Library s h a l l make i t f r e e l y a v a i l a b l e for reference and study. I further agree that permission for extensive copying of t h i s thesis for scholarly purposes may be granted by the head of my department or by his or her representatives. I t i s understood that copying or publication of t h i s thesis for f i n a n c i a l gain s h a l l not be allowed without my written permission. Department of / / The University of B r i t i s h Columbia 1956 Main Mall Vancouver, Canada V6T 1Y3 ABSTRACT i i G a s t r i c o p i o i d p e p t i d e s were c h a r a c t e r i z e d on the b a s i s o f t h e i r b i o c h e m i c a l forms, s i t e s o f a c t i o n , mode o f r e l e a s e and e f f e c t s on e n d o c r i n e s e c r e t i o n s of the stomach. P a r t i a l p u r i f i c a t i o n o f o p i o i d - l i k e m a t e r i a l from e x t r a c t s of the corpus/antrum r e g i o n o f the r a t stomach was c a r r i e d out by Sephadex G-50 g e l f i l t r a t i o n chromatography f o l l o w e d by a d s o r p t i o n onto A m b e r l i t e XAD-2 r e s i n . A s i n g l e peak o f o p i o i d a c t i v i t y was det e r m i n e d by both r a d i o r e c e p t o r assay (RRA) and b i o a s s a y . By h i g h performance l i q u i d chromatography (HPLC) t h i s peak was r e s o l v e d i n t o s e v e r a l d i s t i n c t components i d e n t i f i e d by t h e i r r e t e n t i o n times and measurement by RRA and/or radioimmunoassay (RIA) as c o r r e s p o n d i n g t o m e t h i o n i n e -e n k e p h a l i n (met-enk), l e u c i n e - e n k e p h a l i n ( l e u - e n k ) , met-enk-a r g - g l y - l e u , met-enk-arg-phe, as w e l l as d y n o r p h i n s 1-13, 1-17, and 1-8. T r y p s i n d i g e s t i o n o f p a r t i a l l y p u r i f i e d e x t r a c t s r e s u l t e d i n an o v e r a l l i n c r e a s e i n o p i o i d a c t i v i t y , s u g g e s t i n g the presence o f l a r g e r , i n a c t i v e forms which may f u n c t i o n as p r e c u r s o r s . The s i t e s o f o p i o i d p e p t i d e a c t i o n were i n f e r r e d from a u t o r a d i o g r a p h i c d e m o n s t r a t i o n s o f the d i s t r i b u t i o n o f t r i t i u m -l a b e l l e d o p i o i d l i g a n d b i n d i n g . I n the f u n d i c r e g i o n o f the r a t stomach mu- and d e l t a - t y p e o p i o i d r e c e p t o r s were l o c a l i z e d i n the c i r c u l a r m u s cle, m u s c u l a r i s mucosae, s u g g e s t i n g t h a t here the o p i o i d p e p t i d e s may be i n v o l v e d i n the r e g u l a t i o n o f motor a c t i v i t y . I n the corpus and antrum, the s e two o p i o i d r e c e p t o r t y p e s were found t o be a s s o c i a t e d w i t h the deep muscular p l e x u s i n some a r e a s , but p r e d o m i n a n t l y i n the submucosal p l e x u s and mucosa where the o p i o i d p e p t i d e s may a c t to d i r e c t l y a f f e c t g a s t r i c e n d o c r i n e and e x o c r i n e s e c r e t i o n s . Mu-type l i g a n d s a l s o bound t o the c i r c u l a r muscle and m y e n t e r i c p l e x u s . I n t e s t i n a l t i s s u e demonstrated mu- and d e l t a - o p i o i d b i n d i n g s i t e s t h r o u g h o u t the mucosa, e x t e n d i n g t o the t i p s of the v i l l i , i m p l i c a t i n g t h e i r i n v o l v e m e n t i n the r e g u l a t i o n o f i n t e s t i n a l f l u i d and e l e c t r o l y t e a b s o r p t i o n . U s i n g the i s o l a t e d , p e r f u s e d r a t stomach p r e p a r a t i o n , endogenous l e u - e n k was found t o be r e l e a s e d i n t o the g a s t r i c v a s c u l a t u r e i n response t o p o t a s s i u m d e p o l a r i z a t i o n and t o the n i c o t i n i c c h o l i n e r g i c a g o n i s t d i m e t h y l - p h e n y l - p i p e r a z i n i u m (DMPP). The m u s c a r i n i c c h o l i n e r g i c a g o n i s t , m e t h a c h o l i n e , had no e f f e c t . E x o g e n o u s l y i n f u s e d met-enk caused a prompt dose-dependent, naloxone s e n s i t i v e i n h i b i t i o n o f g a s t r i c i n h i b i t o r y p o l y p e p t i d e ( G l P ) - s t i m u l a t e d s o m a t o s t a t i n (SLI) s e c r e t i o n . A s i m i l a r , but reduced e f f e c t was obse r v e d w i t h d y n o r p h i n 1-13. N e i t h e r a t r o p i n e nor hexamethonium a f f e c t e d the met-enk i n h i b i t i o n o f G I P - s t i m u l a t e d S L I ; t h e r e f o r e , i t was proposed t h a t met-enk was a c t i n g d i r e c t l y on the D - c e l l s t o i n h i b i t s o m a t o s t a t i n s e c r e t i o n , p o s s i b l y by i n t e r a c t i n g w i t h mucosal o p i o i d r e c e p t o r s demonstrated by a u t o r a d i o g r a p h y . These r e s u l t s , d e m o n s t r a t i n g the p r e s e n c e o f endogenous g a s t r i c o p i o i d p e p t i d e s o f both the e n k e p h a l i n and d y n o r p h i n f a m i l i e s and the widespread d i s t r i b u t i o n o f o p i o i d r e c e p t o r s i n the g a s t r o i n t e s t i n a l t r a c t , c o u p l e d w i t h the observed r e l e a s e of endogenous g a s t r i c l e u - e n k , and p o t e n t e f f e c t s of o p i o i d p e p t i d e s on SLI s e c r e t i o n from the stomach, i n d i c a t e t h a t the i v o p i o i d p e p t i d e s a re i m p o r t a n t p h y s i o l o g i c a l r e g u l a t o r s o f g a s t r i c and i n t e s t i n a l f u n c t i o n s . SUPERVISOR.. V TABLE OF CONTENTS Abstract i i Table of Contents v L i s t of Tables xi L i s t of Figures x i i L i s t of Abbreviations xvi Acknowledgements xvi i i Dedication xix CHAPTER ONE—GENERAL INTRODUCTION I. H i s t o r i c a l Aspects 2 II. Discovery of Opioid Peptides 4 II I . Biogenesis of Opioid Peptides 5 IV. Multiple Opioid Receptors 8 V. D i s t r i b u t i o n of Opioid Peptides 14 VI. The Enteric Nervous System 17 VII. D i s t r i b u t i o n of Opioid Peptides in the Gastro-i n t e s t i n a l Tract 21 VIII. Functions of Opioid Peptides in the Gastro-i n t e s t i n a l Tract 23 IX. Rationale for the Present Studies 31 CHAPTER TWO—GENERAL METHODS AND MATERIALS I. ASSAY SYSTEMS A. Guinea Pig Ileum Bioassay 1. Rationale 35 2. Experimental Procedure 36 v i B. Radioreceptor Assay 1. Rationale 38 2. Technical Considerations 38 3. Experimental Procedure a) Brain Homogenate Preparation 39 b) Preparation of Radio-iodinated DADLE 40 c) Standards 41 d) Assay Protocol 41 e) The Use of Crude Brain Homogenate (PI) Compared to Mitochondrial-Synaptosomal Preparation (P2) 43 f) Displacement Studies 43 C. Radioimmunoassay 1. Rationale 45 2. Leu-Enk RIA a) Antiserum 45 b) Iodination of Leu-enk 47 c) Standard Curves 48 d) Assay Protocol 51 3. Dynorphin RIA a) Dynorphin antiserum 52 b) Iodination of Dynorphin 1-13 54 c) Dynorphin Standards 54 d) Assay Protocol 56 4. Somatostatin RIA a) Somatostatin Antiserum 56 b) Iodination of Somatostatin 56 c) Assay Buffer 57 d) Somatostatin Standards 58 v i i e) Assay Protocol 58 5. Gastrin RIA a) Diluent Buffer 58 b) Antiserum. 59 c) Gastrin Iodination 59 d) Gastrin Standards 60 e) Assay Protocol 60 II . CHROMATOGRAPHIC METHODS A. Gel F i l t r a t i o n 61 B. Adsorption Chromatography 61 C. Reversed Phase High Performance Liquid Chromatography 1. Rationale 62 2. HPLC Apparatus 63 3. HPLC Procedure 63 III . ISOLATED PERFUSED RAT STOMACH A. Surgical Procedure 65 B. Preparation of Perfusate 66 C. Perfusion Procedure 67 CHAPTER THREE—CHARACTERIZATION OF OPIOID PEPTIDES IN RAT STOMACH EXTRACTS INTRODUCTION 69 METHODS I. Tissue Extraction 72 II . Chromatography A. Gel F i l t r a t i o n 72 B. Adsorption Chromatography 73 v i i i C. HPLC 73 II I . Trypsin Digestion 74 IV. Assay Procedures 74 RESULTS I. Opioid A c t i v i t y in Acid Extracts of Rat Stomach 78 II . Chromatographic Separation 78 II I . E ffects of Trypsin Digestion 88 DISCUSSION 92 CHAPTER FOUR—AUTORADIOGRAPHIC LOCALIZATION OF OPIOID RECEPTORS IN THE GASTROINTESTINAL TRACT INTRODUCTION I. L o c a l i z a t i o n of Opioid Receptors 102 II . Properties of Opioid Receptors 104 II I . Opioid Receptors in the Gastrointestinal Tract 107 METHODS I. Tissue Preparation 110 II. Incubation Procedures A. Delta-Receptors 110 B. Mu-Receptors I l l C. Kappa-Receptors 112 D. GTP and Cation S e n s i t i v i t y 112 II I . Examination of Slides and Film A. Film Development 113 B. Tissue Staining 113 C. Examination of Autoradiographic Film and Slides....113 ix RESULTS I. Rat Gastrointestinal Tract A. Mu-type opioid receptor d i s t r i b u t i o n 116 B. Delta-type opioid receptor d i s t r i b u t i o n 118 C. Kappa-type opioid receptor d i s t r i b u t i o n 118 II. Guinea Pig Gastrointestinal Tract 119 III . E f f e c t of GTP and Cations on 3H-DADLE Binding 119 DISCUSSION 168 CHAPTER FIVE—RELEASE OF LEU-ENK-IR FROM ISOLATED PERFUSED RAT STOMACHS INTRODUCTION 179 METHODS 183 RESULTS I. E f f e c t of Potassium Depolarization 185 II. E f f e c t of DMPP Infusion 186 I I I . E f f e c t of Methacholine Infusion 186 DISCUSSION 190 CHAPTER SIX—REGULATION OF GASTRIC SOMATOSTATIN SECRETION BY OPIOID PEPTIDES INTRODUCTION 195 METHODS 199 RESULTS I. E f f e c t of met-enk on GIP-stimulted SLI secretion 200 II. E f f e c t of atropine and hexamethonium on met-enk induced i n h i b i t i o n of GIP-stimulated SLI secretion...203 I I I . E f f e c t s of Dynorphin 1-13 on GIP-stimulated SLI secretion 203 DISCUSSION 208 CHAPTER SEVEN—CONCLUDING REMARKS LIST OF REFERENCES x i LIST OF TABLES TABLE I. Relative potencies of some opioid peptides in displacing radio-iodinated DADLE binding to rat brain homogenates in the RRA 46 TABLE I I . Summary of autoradiographic d i s t r i b u t i o n s of opioid mu-, delta- and kappa receptors in the rat g a s t r o i n t e s t i n a l t r a c t 121 X I 1 LIST OF FIGURES FIGURE 1 Schematic r e p r e s e n t a t i o n o f human p r e p r o e n k e p h a l i n . . .. 7 2 Schematic r e p r e s e n t a t i o n o f p o r c i n e p r e p r o -d y n o r p h i n 9 3 Amino a c i d sequences o f the t h r e e o p i o i d p e p t i d e f a m i l i e s 10 4 D i a g r a m a t i c r e p r e s e n t a t i o n o f the arrangement of the e n t e r i c nervous system 18 5 Schematic diagram o f the g u i n e a p i g i l e u m b i o a s s a y a p p a r a t u s 37 6 P r o f i l e o f a DADLE i o d i n a t i o n m i x t u r e e l u t e d from Sepahdex G-10 42 7 D i s p l a c e m e n t c u r v e s comparing r a d i o i o d i n a t e d - D A D L E b i n d i n g t o crude r a t b r a i n membrane p r e p a r a t i o n s (PI) w i t h b i n d i n g t o s y n a p t o s o m a l - m i t o c h o n d r i a l p r e p a r a t i o n s (P2) 44 8 E l u t i o n p r o f i l e o f a leu-enk i o d i n a t i o n m i x t u r e from Sep-Pak c a r t r i d g e s 49 9 A sample s t a n d a r d curve f o r the l e u - e n k RIA 50 10 Comparison o f d i f f e r e n t forms o f d y n o r p h i n as s t a n d a r d s f o r RIA 53 11 P r o f i l e o f a d y n o r p h i n 1-13 i o d i n a t i o n m i x t u r e e l u t e d from Sephadex G-15 55 12 Schematic o u t l i n e o f the HPLC a p p a r a t u s 64 13 Schematic o u t l i n e o f the a p p a r a t u s f o r i n v i t r o v a s c u l a r p e r f u s i o n o f the r a t stomach 68 14 C o n c e n t r a t i o n - d e p e n d e n t i n h i b i t i o n o f e l e c t r i c a l l y -i nduced c o n t r a c t i o n s of the g u i n e a p i g i l e u m by met-enk 76 15 C o n c e n t r a t i o n - r e s p o n s e c u r v e f o r met-enk i n the g u i n e a p i g i l e u m b i o a s s a y 77 16 P a r t i a l p u r i f i c a t i o n of o p i o i d m a t e r i a l from c o r p u s / antrum e x t r a c t s by g e l f i l t r a t i o n chromatography on Sephadex G-50 79 x i i i FIGURE 17 E f f e c t o f p a r t i a l l y p u r i f i e d c orpus/antrum e x t r a c t on the e l e c t r i c a l l y - i n d u c e d c o n t r a c t i o n s o f the guin e a p i g i l e a l s t r i p 80 18 O p i o i d b i n d i n g a c t i v i t y o f Sephadex G-50 chromatographed corpus/antum e x t r a c t b e f o r e and a f t e r A m b e r l i t e XAD-2 t r e a t m e n t 82 19 O p i o i d - l i k e b i o a c t i v i t y i n Sephadex G-50 p u r i f i e d c orpus/antrum e x t r a c t s f o l l o w i n g A m b e r l i t e XAD-2 p u r i f i c a t i o n 83 20 P r o t e i n and o p i o i d b i n d i n g a c t i v i t y e l u t i o n p r o f i l e s o f corpus/antrum e x t r a c t s from Sephadex G-50 84 21 HPLC e l u t i o n p r o f i l e s o f s t a n d a r d o p i o i d p e p t i d e s . . . . 85 22 HPLC s e p a r a t i o n o f o p i o i d - l i k e components i n p a r t i a l l y p u r i f i e d corpus/antrum e x t r a c t 86 23 HPLC e l u t i o n of o p i o i d p e p t i d e s d e t e r m i n e d by RIA....89 24 HPLC e l u t i o n o f d y n o r p h i n - I R from p a r t i a l l y p u r i f i e d corpus/antrum e x t r a c t 90 25 E f f e c t o f t r y p s i n d i g e s t i o n on o p i o i d a c t i v i t y o f p a r t i a l l y p u r i f i e d corpus/antrum e x t r a c t s 91 26 Schematic diagram o f the r a t g a s t r o i n t e s t i n a l t r a c t i n d i c a t i n g the approximate r e g i o n s from which t i s s u e was ta k e n f o r a u t o r a d i o g r a p h i c l o c a l i z a t i o n o f o p i o i d r e c e p t o r s 114 27 D i a g r a m a t i c o u t l i n e o f the p r o c e d u r e s i n v o l v e d i n the a u t o r a d i o g r a p h i c l o c a l i z a t i o n o f o p i o i d r e c e p t o r s i t e s 115 28 P h o t o m i c r o g r a p h s o f r a t fundus, d e m o n s t r a t i n g mu-type o p i o i d b i n d i n g s i t e s 123 29 P h o t o m i c r o g r a p h s o f r a t fundus, d e m o n s t r a t i n g mu-type o p i o i d b i n d i n g s i t e s 125 30 P h o t o m i c r o g r a p h s of r a t c o r p u s , d e m o n s t r a t i n g mu-type o p i o i d b i n d i n g s i t e s 127 31 P h o t o m i c r o g r a p h s o f r a t c o r p u s , d e m o n s t r a t i n g mu-type o p i o i d b i n d i n g s i t e s 129 32 P h o t o m i c r o g r a p h s o f r a t antrum, d e m o n s t r a t i n g mu-type o p i o i d b i n d i n g s i t e s 131 xiv FIGURE 33 Photomicrographs of rat antrum, demonstrating mu-type opioid binding s i t e s 133 34 Photomicrographs of rat duodenum, demonstrating mu-type opioid binding s i t e s 135 35 Photomicrographs of rat ileum, demonstrating mu-type opioid binding s i t e s 137 36 Photomicrographs of rat fundus, demonstrating d e l t a -type opioid binding s i t e s 139 37 Photomicrographs of rat fundus, demonstrating delt a -type opioid binding s i t e s 141 38 Photomicrographs of rat corpus, demonstrating delt a -type opioid binding s i t e s 143 39 Photomicrographs of rat corpus, demonstrating delta-type opioid binding s i t e s 145 40 Photomicrographs of rat antrum, demonstrating delt a -type opioid binding s i t e s 147 41 Photomicrographs of rat pylorus region, demonstrating delta-type opioid binding s i t e s 149 42 Photomicrographs of rat duodenum, demonstrating delta-type opioid binding s i t e s 151 43 Photomicrographs of rat ileum, demonstrating delt a -type opioid binding s i t e s 153 44 Photomicrographs of rat ileum, demonstrating kappa-type opioid binding s i t e s 155 45 Photomicrographs of rat corpus, demonstrating kappa-type opioid binding s i t e s 157 46 Photomicrographs of guinea pig ileum, demonstrating mu-type opioid binding s i t e s 159 47 Photomicrographs of guinea pig antrum, demonstrating mu-type opioid binding s i t e s 161 48 Photomicrographs of guinea pig corpus, demonstrating mu-type opioid binding s i t e s 163 49 Photomicrographs of rat corpus, demonstrating the ef f e c t of GTP, sodium and manganese on delta-type opioid binding s i t e s 165 X V FIGURE 50 P h o t o m i c r o g r a p h s o f r a t i l e u m , d e m o n s t r a t i n g the e f f e c t o f GTP, sodium and manganese on d e l t a - t y p e o p i o i d b i n d i n g s i t e s 167 51 E f f e c t o f p o t a s s i u m d e p o l a r i z a t i o n on l e u - e n k - I R r e l e a s e from i s o l a t e d p e r f u s e d r a t stomachs 187 52 E f f e c t o f DMPP on l e u - e n k - I R r e l e a s e from i s o l a t e d p e r f u s e d r a t stomachs 188 53 E f f e c t o f m e t h a c h o l i n e o f l e u - e n k - I R r e l e a s e from i s o l a t e d p e r f u s e d r a t stomachs 189 54 E f f e c t o f met-enk on G I P - s t i m u l a t e d s e c r e t i o n o f SLI from i s o l a t e d p e r f u s e d r a t stomachs 201 55 E f f e c t o f naloxone on met-enk-induced i n h i b i t i o n o f G I P - s t i m u l a t e d SLI s e c r e t i o n from i s o l a t e d p e r f u s e d r a t stomachs 202 56 E f f e c t o f a t r o p i n e on met-enk-induced i n h i b i t i o n of G I P - s t i m u l a t e d SLI s e c r e t i o n from i s o l a t e d p e r f u s e d r a t stomachs 204 57 E f f e c t o f hexamethonium on met-enk-induced i n h i b i t i o n o f G I P - s t i m u l a t e d SLI s e c r e t i o n from i s o l a t e d p e r f u s e d r a t stomachs 205 58 E f f e c t o f 1 d y n o r p h i n 1-13 on G I P - s t i m u l a t e d SLI s e c r e t i o n from i s o l a t e d p e r f u s e d r a t stomachs 206 59 E f f e c t o f 200 nM d y n o r p h i n 1-13 on G I P - s t i m u l a t e d SLI s e c r e t i o n from i s o l a t e d p e r f u s e d r a t stomachs 207 60 P o s s i b l e pathways by which endogenous e n k e p h a l i n s i n h i b i t SLI s e c r e t i o n from g a s t r i c D - c e l l s 210 xvi LIST OF ABBREVIATIONS TERM ABBREVIATION Absorbance units f u l l scale AUFS Cholecystokinin CCK Circular muscle CM Central nervous system CNS 2 5 D-ala ,D-leu -enkephalin DADLE Dihydromorphine DHM l,l-dimethyl-4-phenyl-piperazinium DMPP Deep muscular plexus DMP Dynorphin Dyn Ethylketocyclazocine EKC Enteric nervous system ENS gamma-Aminobutyric acid GABA Gastric i n h i b i t o r y polypeptide GIP Hematoxylin and eosin H&E High performance l i q u i d chromatography HPLC Immunocytochemistry ICC Immunoreactivity IR Leucine-enkephalin Leu-enk Longitudinal muscle LM Methionine-enkephalin Met-enk Muscular is mucosae MM Myenteric plexus MP Mucosal plexus MucP Naloxone NAL xvi i TERM ABBREVIATION Pro-opiomelanocortin POMC Radioimmunoassay RIA Radioreceptor assay RRA Somatostatin-like immunoreactivity SLI Submucosal plexus SMP T r i f l u r o a c e t i c acid TFA Vasoactive i n t e s t i n a l polypeptide VIP NB: A l l other abbreviations, units, symbols and chemical names used in this thesis are in accordance with those outlined by G. E l l i s (ed) IN: Units, Symbols and Abbreviations. A Guide for  Bi o l o g i c a l and Medical Editors and Authors (1975). Royal Society of Medicine, London. x v i i i ACKNOWLEDGEMENTS F i r s t and foremost I would l i k e to express my sincere gratitude to my supervisor, Dr. Chris Mcintosh, who has guided this research project with constant enthusiasm and great insight. Also, for his painstakingly thorough re v i s i o n of this manuscript I am very much appreciative. The work presented i n this thesis has culminated from invaluable input from Dr. Mcintosh as well as many others. In particular I would l i k e to thank wholeheartedly Dr. John Brown for providing me with f a c i l i t i e s and the opportunity to work with the members of the gut hormone research group from whom I have g r a t e f u l l y acquired their expertise. To Dr. Yin Nam Kwok I am indebted for his assistance in the preparation of the bioassay apparatus and for his c r i t i c a l views on the pharmacological aspects of these experiments. Many thanks to Dr. Alison Buchan for her expert instructions i n introducing me to autoradiographic and histochemical techniques. Thanks also to Dr. Steven Vincent for many helpful suggestions regarding autoradiography and immunohistochemistry. Technical assistance from V i c k i Bakich and Angela Yeung i s greatly appreciated. F i n a l l y , I am much obliged to Mr. Kurt Henze for his excellent artwork which went into the preparation of the figures. Financial support from an H.R. MacMillan Family Fellowship i s g r a t e f u l l y acknowledged. DEDICATED TO MY PARENTS AND TO MY SISTERS, NERISSA AND LOUISA 1 CHAPTER ONE GENERAL INTRODUCTION G a s t r o i n t e s t i n a l f u n c t i o n s a re r e g u l a t e d by a complex s e r i e s o f nervous and e n d o c r i n e pathways. I n a d d i t i o n t o the p a r a s y m p a t h e t i c and s y m p a t h e t i c autonomic i n p u t s , the g a s t r o i n t e s t i n a l t r a c t c o n t a i n s i n t r i n s i c neurons which are capable o f f u n c t i o n i n g i n d e p e n d e n t l y o f the c e n t r a l nervous system. Of the e x t e n s i v e a r r a y o f the s e i n t r i n s i c e n t e r i c 7 8 neurons, which number i n the o r d e r o f 10 t o 10 (Furness and C o s t a 1980), some have been shown t o c o n t a i n o p i o i d p e p t i d e s , the endogenously o c c u r r i n g o p i a t e - l i k e s u b s t a n c e s (Elde e t a l . , 1976; P o l a k e t a l . , 1977; Alumets e t a l . , 1978; L i n n o i l a e t a l . , 1978; S c h u l t z b e r g e t a l . , 1980; Watson e t a l . , 1981; Furness e t a l . , 1983; Bu'Lock e t a l . , 1983; G i r a u d e t a l . , 1984a; V i n c e n t e t a l . , 1984; Wang e t a l . , 1984). O p i o i d p e p t i d e s have a l s o been found t o oc c u r i n g a s t r i c a n t r a l e n d o c r i n e c e l l s (Polak e t a l . , 1977; L a r s s o n and Sten g a a r d -Pedersen, 1981; Bu'Lock e t a l . , 1983; G i r a u d e t a l . , 1984a; J o n s s o n , 1985). Exogenously a d m i n i s t e r e d o p i o i d p e p t i d e s have been demonstrated t o e x e r t p o t e n t e f f e c t s on g a s t r o i n t e s t i n a l m o t i l i t y and both e n d o c r i n e and e x o c r i n e s e c r e t i o n s (reviewed by: K o n t u r e k , 1980a,b, 1981; Corder and Rees, 1981; N o r t h , 1982; N o r t h and Egan, 1983; M i l l e r , 1983). I t i s p o s s i b l e , t h e r e f o r e , t h a t t h e s e o p i o i d p e p t i d e c o n t a i n i n g e n t e r i c neurons and e n d o c r i n e c e l l s p l a y an i m p o r t a n t p h y s i o l o g i c a l r o l e i n the n e u r a l and hormonal r e g u l a t i o n o f g a s t r o i n t e s t i n a l f u n c t i o n s . 2 I . HISTORICAL ASPECTS A l t h o u g h the presence of o p i o i d p e p t i d e s i n the d i g e s t i v e system has been a r e c e n t d i s c o v e r y , the e f f e c t s o f opium on the g a s t r o i n t e s t i n a l t r a c t have been known s i n c e a n c i e n t t i m e s . Opium from the j u i c e s o f the seed c a p s u l e o f the poppy p l a n t , Papaver somniferum, had been employed i n the t r e a t m e n t of d i a r r h e a and d y s e n t e r y many c e n t u r i e s p r i o r t o i t s use as an a n a l g e s i c ( J a f f e and M a r t i n , 1980). Morphine and o t h e r p h a r m a c o l o g i c a l l y a c t i v e a l k a l o i d c o n s t i t u e n t s o f opium were p u r i f i e d i n the e a r l y n i n e t e e n t h c e n t u r y and have s i n c e been w i d e l y used c l i n i c a l l y , p r i m a r i l y f o r t h e i r a n a l g e s i c e f f e c t s . A major impetus f o r i n v e s t i g a t i o n s i n t o the a c t i o n o f these o p i a t e a l k a l o i d s has been the problem t y p i c a l of the use of n a r c o t i c d r u g s , namely t h a t o f a d d i c t i o n , t o l e r a n c e and the a s s o c i a t e d w i t h d r a w a l syndrome. Two major l i n e s o f e v i d e n c e were r e c o g n i z e d which suggested the presence i n the nervous system of some endogenous o p i a t e -l i k e s u b s t a n c e s . F i r s t l y , o p i a t e r e c e p t o r s , d e f i n e d as " t h a t e n t i t y which r e c o g n i z e s and b i n d s o p i a t e d r u g s , w i t h b i n d i n g t r a n s d u c e d i n t o v a r i o u s b i o c h e m i c a l and p h y s i o l o g i c a l s e q u e l a e " , were i d e n t i f i e d i n s e v e r a l t i s s u e s (see r e v i e w by M i l l e r and C u a t r e c a s a s 1979) . Two b i o a s s a y s , the i n h i b i t i o n of e l e c t r i c a l l y - i n d u c e d c o n t r a c t i o n s o f the g u i n e a p i g i l e u m and of the mouse vas d e f e r e n s have been commonly employed t o s t u d y o p i a t e r e c e p t o r c h a r a c t e r i s t i c s (Schaumann, 1955; P a t o n , 1957). E l e c t r i c a l l y - s t i m u l a t e d r e l e a s e o f a c e t y l c h o l i n e i n the f o r m e r , 3 and of n o r e p i n e p h r i n e i n the l a t t e r , were found to be i n h i b i t e d by the o p i a t e s , consequent ly i n h i b i t i n g muscle c o n t r a c t i o n . U s i n g these b i o a s s a y s , the a c t i o n s o f morphine and r e l a t e d drugs were found to be s t e r e o s p e c i f i c ( K o s t e r l i t z and W a t t , 1968). F u r t h e r m o r e , wi th the advent o f r a d i o l a b e l l e d l i g a n d s and t h e i r use i n measuring s t e r e o s p e c i f i c b i n d i n g i n b r a i n homogenates ( G o l d s t e i n e t a l . , 1971) s p e c i f i c , p h a r m a c o l o g i c a l l y c h a r a c t e r i z e d o p i o i d b i n d i n g s i t e s were demonstrated i n nervous t i s s u e (Pert and S n y d e r , 1973a,b; Simon et a l . , 1973; T e r e n i u s , 1973a ,b ) . S e c o n d l y , i t was shown t h a t s t i m u l a t i o n o f c e r t a i n r e g i o n s i n the b r a i n , i n p a r t i c u l a r the p e r i a q u e d u c t a l grey a r e a , caused a s t a t e o f a n a l g e s i a (Reynolds , 1969; A k i l e t a l . , 1976; Mayer and P r i c e , 1976) , an e f f e c t s i m i l a r to t h a t produced by the o p i a t e d r u g s . To e x p l a i n t h i s o b s e r v a t i o n i t was s p e c u l a t e d t h a t the e l e c t r i c a l s t i m u l a t i o n had caused the r e l e a s e o f some substance (s) p r e s e n t i n the nervous system wi th p r o p e r t i e s s i m i l a r to those o f the o p i a t e a l k a l o i d s thereby i n d u c i n g an a n a l g e s i c s t a t e . That t h i s e l e c t r i c a l l y - i n d u c e d a n a l g e s i c e f f e c t c o u l d be b l o c k e d by the o p i a t e a n t a g o n i s t , na loxone , f u r t h e r i m p l i c a t e d the p o s s i b l e r e l e a s e o f some n a t u r a l m o r p h i n e - l i k e compound ( A k i l et a l . , 1976) . T h e r e f o r e , these p h y s i o l o g i c a l f i n d i n g s of the a n a l g e s i c e f f e c t s and the b i o c h e m i c a l f i n d i n g s o f o p i a t e r e c e p t o r s , h i t h e r t o c h a r a c t e r i z e d o n l y by the b i n d i n g o f p l a n t a l k a l o i d s such as morphine , prompted a l a r g e s c a l e s e a r c h by many groups for endogenous o p i a t e - l i k e s u b s t a n c e s . 4 I I . DISCOVERY OF THE OPIOID PEPTIDES By the early 1970's brain extracts were found to contain some substances which acted as agonists at opiate receptor sit e s in the guinea pig ileum and mouse vas deferens preparations (Terenius and Whalstrom, 1974, 1975; Hughes, 1975). Soon a f t e r , the f i r s t endogenous opioid substances were i d e n t i f i e d . Two pentapeptides, methionine-enkephalin (met-enk) with the amino acid sequence H-Tyr-Gly-Gly-Phe-Met-OH and leucine-enkephalin (leu-enk) with the sequence H-Tyr-Gly-Gly-Phe-Leu-OH, were isolated and p u r i f i e d from porcine brain extracts (Hughes et a l 1975). Both peptides were observed to have potent op i a t e - l i k e a c t i v i t y , causing a dose - dependent, naloxone-reversible i n h i b i t i o n of e l e c t r i c a l l y - i n d u c e d contractions i n both the mouse vas deferens and guinea pig ileum bioassay preparations. At this time i t was noted that the met-enk amino acid sequence was i d e n t i c a l to that of residues 61-65 of the p i t u i t a r y peptide beta-lipotropin (beta-LPH) (Hughes et a l 1975). The C-terminal fragment (61-91) of beta-LPH, containing the met-enk sequence, was subsequently shown to possess potent opiate a c t i v i t y determined by both bioassay and radioreceptor assay techniques (Cox et a l . , 1976; Bradbury et a l . , 1976). This 31 amino acid peptide was l a t e r named beta-endorphin. Two shorter fragments of beta-endorphin have also been is o l a t e d , alpha-endorphin (61-76) and gamma-endorphin (61-77), both of which show opia t e - l i k e a c t i v i t y (Ling and Guillemin, 1976) . 5 In 1981 G o l d s t e i n e t a l . i s o l a t e d y e t another p i t u i t a r y p e p t i d e w i t h p o t e n t o p i a t e a c t i v i t y , d y n o r p h i n , a 17 amino a c i d p e p t i d e which was found t o c o n t a i n the l e u - e n k sequence a t i t s N-terminus ( G o l d s t e i n e t a l . , 1981; T a c h i b a n a e t a l . , 1981a,b). The s h o r t e r 1-8 d y n o r p h i n as w e l l as o t h e r p e p t i d e s w i t h C-t e r m i n a l e x t e n s i o n s of l e u - e n k , named a l p h a - and beta-neo-e n d o r p h i n have s i n c e been i s o l a t e d from p o r c i n e h y p o t h a l a m i (Minamino e t a l . , 1980; Kanagawa e t a l . , 1981; G o l d s t e i n e t a l . , 1981). I n 1982, the e x i s t e n c e o f a l e u - e n k c o n t a i n i n g t r i d e c a p e p t i d e was s i m u l t a n e o u s l y r e p o r t e d as d y n o r p h i n B ( F i s c h l i , e t a l . , 1982 ) and as r i m o r p h i n ( K i l p a t r i c k e t a l . , 1982) . A l t h o u g h much of the l i t e r a t u r e uses the terms " o p i a t e " and " o p i o i d " i n d i s c r i m i n a t e l y , the c u r r e n t consensus i s t h a t o p i a t e s h o u l d be r e s e r v e d f o r the a l k a l o i d s and o p i o i d f o r the endogenous p e p t i d e s ( K i t c h e n , 1985). T h i s c o n v e n t i o n w i l l be used t h r o u g h o u t . I l l . BIOGENESIS OF OPIOID PEPTIDES A t f i r s t i t was c o n s i d e r e d t h a t beta-LPH was the p r e c u r s o r from which both b e t a - e n d o r p h i n and met-enk were d e r i v e d and t h a t d y n o r p h i n s and neo-endorphins might be the l e u - e n k e p h a l i n p r e c u r s o r s . However, i t became a p p a r e n t t h a t t h i s was i n c o r r e c t when the a n a t o m i c a l d i s t r i b u t i o n s o f the p u t a t i v e p r e c u r s o r s were shown t o d i f f e r i n many c a s e s from those o f the e n k e p h a l i n s ( R o s s i e r , 1977; Bloom e t a l . , 1978; Watson e t a l . , 1978) . W i t h the development of recombinant DNA t e c h n i q u e s t h r e e d i s t i n c t o p i o i d p r e c u r s o r s were i d e n t i f i e d by deducing 6 the prohormone sequence from isolated messenger RNA (mRNA). Most peptide hormones are known to be formed by enzymatic cleavage of larger, often inactive, prohormones which are synthesized under the di r e c t i o n of s p e c i f i c mRNAs. Using recombinant DNA technology, small amounts of precursor mRNA can be screened and amplified by cloning the complementary DNA (cDNA). The process of cloning makes available large amounts of s p e c i f i c prohormone-encoding DNA which can then be sequenced, and from th i s information the structure of the precursor protein can be derived. Pro-opiomelanocortin (POMC), the common precursor for several neuropeptides including the endorphins, c o r t i c o t r o p i n (ACTH) and melanocyte stimulating hormones (MSHs) , was the f i r s t prohormone to be i d e n t i f i e d using t h i s recombinant DNA approach (Nakanishi et a l . , 1979). The enkephalin precursor was subsequently discovered, in brain and adrenal tissues, to be a large protein containing the sequences of six copies of met-enk and one of leu-enk (Comb et a l . , 1982; Gubler et a l . , 1982; Noda et a l . , 1982). A common feature of large precursors is that the amino acid sequence of b i o l o g i c a l l y active regions are flanked on either side by pairs of basic amino acid residues (arg or l y s ) , suggesting that they act as processing signals for trypsin l i k e enzymes. Assuming such enzymatic cleavage, examination of the proenkephalin molecule predicted the formation of four copies of met-enk, and one each of leu-enk, met-enk-arg-phe and met-enk-arg-gly-leu (see Figure 1) . The existence of these two carboxy-terminally extended sequences of met-enkephalin in brain and adrenal chromaffin 7 1 - 2 4 Signal Peptide Human Preproenkephalin 1 0 0 - 1 0 4 1 0 7 - 1 ! I 1 3 6 - 1 4 6 Met-Enkephalin Met-Enkephalin 1 8 6 - 1 9 3 • • M e t - E n k e p h a l i n - A R G 6 - G L Y 7 - L E U 8 2 1 0 - 2 1 4 LJ Met-Enkephalin 2 3 0 - 2 3 4 H Leu-Enkephalin 2 6 1 - 2 6 7 LJ M e t - E n k e p h a l i n - A R G 6 - P H E 7 FIGURE 1: SCHEMATIC REPRESENTATION OF HUMAN PREPROENKEPHALIN. The numbers t o the l e f t i n d i c a t e the p o s i t i o n o f the amino a c i d r e s i d u e s i n the sequence o f the e n k e p h a l i n p r e c u r s o r m o l e c u l e . The ex p e c t e d p r o d u c t s o f t r y p s i n - l i k e c l e a v a g e are i l l u s t r a t e d ; each o p i o i d p e p t i d e amino a c i d sequence i s f l a n k e d on e i t h e r s i d e s by p a i r s of b a s i c r e s i d u e s ( a c c o r d i n g t o Noda e t a l . , 1982; Comb e t a l . , 1982) . 8 c e l l s was l a t e r c o n f i r m e d ( R o s s i e r e t a l . , 1980; S t e r n e t a l . , 1980; K i l p a t r i c k e t a l . , 1981; Boarder e t a l . , 1982). The i d e n t i f i c a t i o n o f enzymes c a p a b l e o f g e n e r a t i n g e n k e p h a l i n s from b r a i n and a d r e n a l p r e c u r s o r s s u p p o r t s the assumption of o p i o i d p e p t i d e b i o g e n e s i s from p r o e n k e p h a l i n . A t r y p s i n - l i k e enzyme c a p a b l e o f g e n e r a t i n g f r e e met-enk from endogenous p u t a t i v e o p i o i d p r e c u r s o r s has been p a r t i a l l y p u r i f i e d from bovine a d r e n a l c h r o m a f f i n c e l l s ( L i n d b e r g e t a l . , 1982). In a d d i t i o n , a c a r b o x y p e p t i d a s e of a d r e n a l o r i g i n ( K n i g h t e t a l . , 1982; Snyder and F r i c k e r , 1984) r e f e r r e d t o as e n k e p h a l i n c o n v e r t a s e , and a b r a i n e n d o p e p t i d a s e (Hook e t a l . , 1982, 1985) have been i s o l a t e d , both o f which were found t o g e n e r a t e f r e e e n k e p h a l i n from p u t a t i v e p r e c u r s o r s f o l l o w i n g t r e a t m e n t w i t h t r y p s i n . The sequence o f the p r e c u r s o r t o the t h i r d o p i o i d group, the d y n o r p h i n s , was determined from the n u c l e o t i d e sequence of p r o d y n o r p h i n ( K a k i d a n i e t a l . , 1982) o b t a i n e d from p o r c i n e h y p o t h a l a m i c mRNA. I t was found t o encode the sequences of d y n o r p h i n A ( 1 - 1 7 ) , d y n o r p h i n A ( 1 - 8 ) , the neo-endorphins and d y n o r p h i n B ( r i m o r p h i n ) , a l l o f which c o n t a i n the leu-enk sequence a t the amino t e r m i n u s ( F i g u r e 2 ) . Thus, th e s e t h r e e p r e c u r s o r s appear t o account f o r a l l known o p i o i d p e p t i d e s d i s c o v e r e d t o d a t e and form the b a s i s f o r the p r e s e n t d e s i g n a t i o n o f t h r e e s e p a r a t e o p i o i d f a m i l i e s ( F i g u r e 3 ) . IV. MULTIPLE OPIOID RECEPTORS As p r e v i o u s l y mentioned, the e x i s t e n c e of s t e r e o s p e c i f i c and s a t u r a b l e o p i a t e r e c e p t o r s had been known l o n g b e f o r e the 9 Signal Peptide Preprodynorphin Leu Enkephalin m 0-Neo-endorphin • a-Neo-endorphin 175-179 175-183 175-184 2 0 9 - 2 1 3 m Dynorphin(l-l7) mD^g^^-Q) 2 2 8 - 2 3 2 2 0 9 - 2 2 5 | J Dynorphin B 2 2 8 - 2 4 0 FIGURE 2: SCHEMATIC REPRESENTATION OF PORCINE PREPRODYNORPHIN. The numbers s i g n i f y the position of the peptide amino acid residues in the sequence of the precursor molecule. The opioid peptides derived from the molecule which have been demonstrated to occur in the brain are indicated (according to Kakidani et a l . , 1982). 10 PRECURSOR Proopio-melanocortin OPIOID PEPTIDES alpha-endorphin gamma-endorphin beta-endorphin AMINO ACID SEQUENCE ^Tyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr j | „ jTyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-LeuJl iTyr-Gly-Gly-Phe-Met-Thr-Ser-Glu-Lys-Ser-Gln-Thr-Pro-Leu-Val-Thr-Leu-* Phe-Lys-Asn-Ala-Ile-Lys-Asn-Ala-His-Lys-Lys-Gly-Gln (END) Proenkephalin met-enkephalin ^Tyr-Gly-Gly-Phe-Met^ leu-enkephalin ^Tyr-Gly-Gly-Phe-Leu^ met-enk-arg-gly-leu ^Tyr-Gly-Gly-Phe-Met-Arg-Gly-LeuJ met-enk-arg-phe ^Tyr-Gly-Gly-Phe-Met-Arg-Phe (END) Prodynorphin dynorphin A (1-17) ^Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile-Arg-Pro-Lys-Leu-Lys-Trp-Asp-Asn-Gln^ dynorphin A (1-8) jTyr-Gly-Gly-Phe-Leu-Arg-Arg-Ile|^ alpha-neo-endorphin ^Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-Pro-Lys^ beta-neo-endorphin ^Tyr-Gly-Gly-Phe-Leu-Arg-Lys-Tyr-ProJ n dynorphin B ^Tyr-Gly-Gly-Phe-Leu-Arg-Arg-Gln-Phe-Lys-Val-Val-ThrJl FIGURE 3: AMINO ACID SEQUENCES OF THE THREE OPIOID PEPTIDE FAMILIES. Tyr i s the f i r s t amino acid of each peptide and forms the amino terminal. Bold arrows represent "normal" t r y p s i n - l i k e cleavage points at pairs of basic amino acids. Light arrows indicate "unusual" cleavage points. (END) s i g n i f i e s the f i n a l amino acid of the prohormone. (Adapted from Rossier, 1982a) 11 endogenous l i g a n d s had been i d e n t i f i e d . These b i n d i n g s i t e s are i n t e g r a l p r o t e i n s i n the membrane s i n c e t r e a t m e n t w i t h d e t e r g e n t s i s r e q u i r e d f o r t h e i r removal (Simonds e t a l . , 1980; B i d l a c k and Abood, 1980; B i d l a c k e t a l . , 1981; Simon and H i l l e r , 1984). As s t u d i e s d i r e c t e d towards the c h a r a c t e r i z a t i o n o f t h e s e o p i o i d r e c e p t o r s p r o g r e s s e d , i t became e v i d e n t t h a t the e f f e c t s of the d i f f e r e n t forms of the endogenous o p i o i d p e p t i d e s c o u l d not be mediated through i n t e r a c t i o n s w i t h o n l y one type o f o p i o i d r e c e p t o r . The concept of m u l t i p l e o p i o i d r e c e p t o r s was o r i g i n a l l y suggested by M a r t i n who noted the d i f f e r e n t i a l e f f e c t s o f v a r i o u s o p i a t e drugs i n p h a r m a c o l o g i c a l e x p e r i m e n t s performed i n c h r o n i c s p i n a l dogs ( M a r t i n e t a l . , 1976; G i l b e r t and M a r t i n , 1976). T h i s t o p i c has s i n c e been reviewed by s e v e r a l a u t h o r s ( P a t e r s o n e t a l . , 1983; Robson e t a l . , 1983; Simon and H i l l e r 1984; G o l d s t e i n and James 1984; Z u k i n and Z u k i n , 1984). K i n e t i c a n a l y s i s o f r a d i o l a b e l l e d o p i o i d l i g a n d b i n d i n g s t u d i e s and e x a m i n a t i o n o f o p i o i d p o t e n c i e s i n b i o l o g i c a l a s s a y systems were the two major approaches t a k e n t o c h a r a c t e r i z e o p i o i d b i n d i n g s i t e s . L o r d e t a l . (1977) f i r s t d emonstrated o p i o d r e c e p t o r h e t e r o g e n e i t y i n v i t r o . They found t h a t morphine and r e l a t e d o p i a t e s were s i g n i f i c a n t l y more p o t e n t t h a t e n k e p h a l i n s i n the g u i n e a p i g i l e u m ; whereas, the e n k e p h a l i n s were found t o be much more e f f e c t i v e i n the mouse vas d e f e r e n s . These s t u d i e s suggested t h a t a d i f f e r e n t c l a s s o f o p i o i d r e c e p t o r s predominated i n the two t i s s u e s . The major r e c e p t o r s p r e s e n t i n the g u i n e a p i g i l e u m were r e f e r r e d t o as mu-type because of t h e i r s i m i l a r i t y t o the mu-receptors c h a r a c t e r i z e d by M a r t i n e t 12 a l . (1976), and those i n the mouse vas d e f e r e n s were c a l l e d d e l t a - t y p e (Lord e t a l . , 1976, 1977). T h i s r e c e p t o r h e t e r o g e n e i t y was a l s o demonstrated i n b i n d i n g s t u d i e s u s i n g guinea p i g b r a i n homogenates. L o r d e t a l (1977) found t h a t e n k e p h a l i n s compete b e t t e r f o r r e c e p t o r b i n d i n g a g a i n s t a l a b e l l e d e n k e p h a l i n than a g a i n s t l a b e l l e d o p i a t e s (naloxone) w h i l e the o p i a t e s (morphine) compete more e f f e c t i v e l y a g a i n s t l a b e l l e d naloxone than a g a i n s t l a b e l l e d e n k e p h a l i n . These r e s u l t s were t h u s i n agreement w i t h t h o s e o f the b i o a s s a y s t u d i e s , s u g g e s t i n g the presence o f o p i a t e - p r e f e r r i n g (mu) and e n k e p h a l i n - p r e f e r r i n g ( d e l t a ) o p i o i d b i n d i n g s i t e s . F o l l o w i n g such i d e n t i f i c a t i o n o f d i f f e r e n t c l a s s e s o f o p i o i d r e c e p t o r s , a g o n i s t s d e f i n e d as " p r o t o t y p i c a l " have been r e g u l a r l y employed i n the c h a r a c t e r i z a t i o n o f these r e c e p t o r t y p e s ( M i l l e r 1982). 2 5 Dihydromorphine i s used as a (mu)-agonist; D - a l a ,D-leu -enk (DADLE) i s c o n s i d e r e d a ( d e l t a ) - a g o n i s t and the k e t a z o c i n e - l i k e compounds e t h y l k e t o c y c l a z o c i n e (EKC) and bremazocine are c o n s i d e r e d k a p p a - a g o n i s t s . A l t h o u g h s e v e r a l r e c e p t o r t y p e s have been p o s t u l a t e d — m u , d e l t a , kappa, sigma and e p s i l o n , o n l y the mu-, d e l t a - and k a p p a - s i t e s have been c h a r a c t e r i z e d t o any degree and some i n f o r m a t i o n r e g a r d i n g the s p e c i f i c i t i e s and p o t e n c i e s of o p i o i d p e p t i d e s a t these t h r e e r e c e p t o r s i t e s has been o b t a i n e d . The POMC-derived e n d o r p h i n s have been shown t o e q u i p o t e n t l y d i s p l a c e r a d i o l a b e l l e d d e l t a and mu l i g a n d s ( K o s t e r l i t z e t a l . , 1982), but t o have l i t t l e o r no e f f e c t on the k a p p a - r e c e p t o r (McKnight e t a l . , 1982). B o t h met- and l e u -enk have h i g h e r a f f i n i t i e s f o r the d e l t a - r e c e p t o r than f o r the 13 mu-receptor (Lord e t a l . , 1977) and a g a i n have l i t t l e e f f e c t a t k a p p a - s i t e s . The l o n g e r , C - t e r m i n a l l y extended forms o f met-enk appear t o b i n d e q u a l l y w e l l t o bo t h the mu- and d e l t a -r e c e p t o r s , but m e t - e n k - a r g - g l y - l e u i s a p p r o x i m a t e l y f i v e times more p o t e n t than met-enk-arg-phe (Magnan e t a l . , 1982). N e i t h e r i s a c t i v e a t the k a p p a - s i t e s (Chavkin e t a l . , 1982). The b i o l o g i c a l l y a c t i v e p r o d u c t s o f p r o d y n o r p h i n a re the o p i o i d p e p t i d e s which appear t o be the endogenous k a p p a - l i g a n d s ( C o r b e t t e t a l . , 1982; C h a v k i n e t a l . , 1982; Oka e t a l . , 1982; James e t a l . , 1984), a l t h o u g h t h e i r a f f i n i t i e s f o r the r e c e p t o r are v a r i a b l e . D e s p i t e t h e i r common le u - e n k amino t e r m i n a l , any e x t e n s i o n o f the le u - e n k sequence appears t o d e c r e a s e i t s c h a r a c t e r i s t i c p o t e n c y a t the d e l t a - r e c e p t o r s . A l t h o u g h the premise t h a t the spectrum o f o p i o i d e f f e c t s are mediated by s e v e r a l t y p e s o f o p i o i d s p e c i f i c r e c e p t o r s appears t o be f i r m l y e s t a b l i s h e d , s e v e r a l key a s p e c t s c o n c e r n i n g the n a t u r e o f the endogenous l i g a n d - r e c e p t o r r e l a t i o n s h i p s t i l l need c l a r i f i c a t i o n . Whether t h e s e m u l t i p l e o p i o i d r e c e p t o r s a re indeed s e p a r a t e e n t i t i e s o r whether they r e p r e s e n t d i f f e r e n t c o n f o r m a t i o n a l s t a t e s o f a s i n g l e r e c e p t o r ( Q u i r i o n e t a l . , 1981) r e q u i r e s much f u r t h e r i n v e s t i g a t i o n . I n a d d i t i o n , the q u e s t i o n o f the " t r u e " endogenous l i g a n d f o r each o p i o i d r e c e p t o r type i s s t i l l u n s e t t l e d . The a t t e m p t s which have been made t o p a i r r e c e p t o r t y p e s w i t h o p i o i d p e p t i d e s may prove u n n e c e s s a r y , however, once the a n a t o m i c a l d i s t r i b u t i o n s of the o p i o i d p e p t i d e s and t h e i r r e c e p t o r s have been c l e a r l y d e t e r m i n e d . 14 V. DISTRIBUTION OF OPIOID PEPTIDES F o l l o w i n g the d i s c o v e r y o f endogenous o p i o i d p e p t i d e s i n b r a i n e x t r a c t s and t h e i r i d e n t i f i c a t i o n as n a t u r a l l i g a n d s o f the o p i a t e r e c e p t o r s , s t u d i e s were d i r e c t e d towards d e t e r m i n i n g t h e i r d i s t r i b u t i o n and l o c a l i z a t i o n t h r o u g h o u t the body. Much of t h i s work has been c a r r i e d o u t by means o f i m m u n o l o g i c a l l y based t e c h n i q u e s such as radioimmunoassay (RIA) and immunocytochemistry ( I C C ) , u s i n g a n t i s e r a which r e c o g n i z e s p e c i f i c o p i o i d p e p t i d e s . The o p i o i d s o f a l l t h r e e f a m i l i e s have been found t o o c c u r p r i m a r i l y i n neurons: both i n the c e n t r a l and p e r i p h e r a l nervous systems, i n c l u d i n g the e n t e r i c nervous system. As i n the case w i t h o t h e r n e u r o p e p t i d e s , the o p i o i d s are s y n t h e s i z e d i n c e l l b o d i e s o f neurons and s t o r e d i n v e s i c u l a r compartments which are t r a n s p o r t e d down the axon t o the nerve t e r m i n a l where they are presumed t o be r e l e a s e d as n e u r o t r a n s m i t t e r s or neurohormones ( H o k f e l t e t a l . , 1980). The d i s t i n c t d i s t r i b u t i o n s o f the e n k e p h a l i n s , e n d o r p h i n s and dynophins i n the c e n t r a l nervous system have been e x t e n s i v e l y r e v i e w e d (Snyder, 1979; M i l l e r and P i c k e l , 1980; M i l l e r , 1981; F r e d e r i c k s o n and Geary, 1982; C u e l l o , 1983; Watson e t a l . , 1984). B r i e f l y , the major s i t e o f e n d o r p h i n b i o s y n t h e s i s o c c u r s i n the p i t u i t a r y g l a n d , and i t i s p r e s e n t i n the c o r t i c o t r o p h s and melanotrophs a l o n g w i t h the o t h e r p r o d u c t s o f POMC (Bloom e t a l . , 1977). I n the b r a i n , the major e n d o r p h i n - c o n t a i n i n g n e u r o n a l systems o c c u r i n the b a s a l hypothalamus w i t h p r o j e c t i o n s r u n n i n g t h r o u g h o u t the b r a i n ( A k i l and Watson, 1983). D y n o r p h i n c o n c e n t r a t i o n s are h i g h e s t 15 i n the p i t u i t a r y , p a r t i c u l a r l y i n the p o s t e r i o r l o b e s but are a l s o d i s t r i b u t e d w i d e l y i n the hypothalamus, m e d u l l a - p o n s , mid-b r a i n and s p i n a l c o r d ( G o l d s t e i n and G h a z a r o s s i a n , 1980). I n g e n e r a l , the d i s t r i b u t i o n o f the neo-endorphins appear t o be s i m i l a r t o t h a t o f both d y n o r p h i n s A and B which might be expected on the b a s i s o f t h e i r common p r e c u r s o r o r i g i n (Watson e t a l . , 1983). Of the c e n t r a l o p i o i d p e p t i d e s , the e n k e p h a l i n s have been shown t o have the w i d e s t d i s t r i b u t i o n , o c c u r r i n g t h roughout the p i t u i t a r y , b r a i n and s p i n a l c o r d ( M i l l e r and P i c k e l , 1980) . Some i n s i g h t c o n c e r n i n g f u n c t i o n can be ga i n e d from knowledge o f the d i s t i n c t n e u r a l pathways f o r each o p i o i d p e p t i d e f a m i l y d e t e r m i n e d by RIA and ICC. However, i t i s not p o s s i b l e t o a s s i g n a s p e c i f i c p h y s i o l o g i c a l r o l e t o any p a r t i c u l a r o p i o i d p e p t i d e s i n c e each c e n t r a l f u n c t i o n i s r e g u l a t e d by s e v e r a l n e u r o n a l systems i n v o l v i n g d i f f e r e n t n e u r o t r a n s m i t t e r s , neuromodulators and p o s s i b l y neurohormones. Hughes (1984) has suggested t h a t the e n k e p h a l i n s and s m a l l e r o p i o i d p e p t i d e s may f u n c t i o n d i r e c t l y as n e u r o t r a n s m i t t e r s , t h e i r i n d i v i d u a l a c t i o n s b e i n g dependent on r e c e p t o r type s e l e c t i v i t y . I n c o n t r a s t , the e n d o r p h i n s , d y n o r p h i n s and the ot h e r l o n g e r o p i o i d p e p t i d e s , which a re l e s s s u s c e p t i b l e t o enzymatic d e g r a d a t i o n , a re c o n s i d e r e d t o be s t r a t e g i c a l l y l o c a t e d t o f u n c t i o n as neurohormones. As they a re p r e s e n t i n neurons p r o j e c t i n g from the hypothalamus t o the v e n t r i c u l a r system, they may s e r v e t o l i n k the b r a i n , p i t u i t a r y and the p e r i p h e r a l e n d o c r i n e system. As f o r s p e c i f i c a c t i o n s , s u f f i c e i t t o mention here t h a t the c e n t r a l l y o c c u r r i n g o p i o i d s have 16 been a s s o c i a t e d w i t h numerous c e n t r a l f u n c t i o n s ( r e c e n t r e v e i w s : O l s o n e t a l . , 1983; G o l d s t e i n 1984; Watson e t a l . , 1984) of which a n t i n o c i c e p t i o n has been most i n t e n s e l y s t u d i e d . A s i d e from t h e i r c e n t r a l d i s t r i b u t i o n s and f u n c t i o n s , the o p i o i d s have been found t o occur i n the p e r i p h e r y , p r i m a r i l y a s s o c i a t e d w i t h the p e r i p h e r a l nervous system. High c o n c e n t r a t i o n s o f o p i o i d s have been demonstrated i n s e v e r a l s y m p a t h e t i c and p a r a s y m p a t h e t i c g a n g l i a , and i n e n t e r i c neurons and g a n g l i a ( D i G u i l i o , 1978; S c h u l t z b e r g e t a l . , 1980; Hervonen et a l . , 1981; D a l s g a a r d e t a l . , 1982, 1983; N o r t h and Egan, 1983; V i n c e n t e t a l . , 1984). I n a d d i t i o n , o p i o i d p e p t i d e s have been found t o o c c u r i n the a d r e n a l m e d u l l a ( S c h u l t z b e r g e t a l . , 1978; L i n n o i l a e t a l . , 1980; Hexum e t a l . , 1980) a p p a r e n t l y s t o r e d t o g e t h e r w i t h c a t e c h o l a m i n e s . I t has been demonstrated t h a t e n k e p h a l i n - l i k e i m m u n o r e a c t i v i t y i s r e l e a s e d upon s p l a n c h n i c nerve s t i m u l a t i o n , an e f f e c t which can be i n h i b i t e d by the g a n g l i o n i c b l o c k e r hexamethonium (Govoni e t a l . , 1981). The a d r e n a l m e d u l l a can t h e r e f o r e be c o n s i d e r e d as an e x t e n s i o n o f the s y m p a t h e t i c nervous system, comparable t o a s y m p a t h e t i c g a n g l i o n w i t h the c h r o m a f f i n c e l l s a c t i n g as " s p e c i a l i z e d neurons." F u r t h e r m o r e , e n k e p h a l i n - i m m u n o r e a c t i v i t y has a l s o been found t o o c c u r i n f i b r e s o f the s p l a n c h n i c nerve and i s b e l i e v e d t o be c o - r e l e a s e d w i t h a c e t y l c h o l i n e (Kumakura et a l . , 1980a,b; C o s t a e t a l . , 1981). Thus the p e r i p h e r a l l o c a l i z a t i o n o f o p i o i d p e p t i d e s i s i n agreement w i t h t h e i r proposed r o l e s as n e u r o t r a n s m i t t e r s and neurohormones. 17 V I . THE ENTERIC NERVOUS SYSTEM (ENS) B e f o r e c o n s i d e r i n g the d i s t r i b u t i o n s and f u n c t i o n s o f the o p i o i d p e p t i d e s i n the g a s t r o i n t e s t i n a l t r a c t , the o r g a n i z a t i o n of the e n t e r i c nervous system (ENS) s h o u l d f i r s t be b r i e f l y d e s c r i b e d . The ENS i n n e r v a t e s the g a s t r o i n t e s t i n a l smooth muscle l a y e r s , b l o o d v e s s e l s and g l a n d s . The uni q u e n e s s o f the g a s t r o i n t e s t i n a l i n n e r v a t i o n had been noted i n 1899 when B a y l i s s and S t a r l i n g r e c o g n i z e d t h a t i n t e s t i n a l r e f l e x e s such as m o t i l i t y induced by m e c h a n i c a l s t i m u l a t i o n o f the gut c o u l d be m a i n t a i n e d i n the absence o f c e n t r a l i n n e r v a t i o n ( B a y l i s s and S t a r l i n g 1899, reviewed by Gershon and Erde 1981). The e n t e r i c nervous system has s i n c e been shown t o c o n t a i n a l l components r e q u i r e d o f a r e f l e x , i n c l u d i n g s e n s o r y neurons, i n t e g r a t i v e i n t e r n e u r o n s , and e f f e c t o r motor neurons. The complex arrangement o f the ner v e s which form the ENS, a s u b s e c t i o n o f the autonomic nervous system, has been d e f i n e d (Furness and C o s t a 1980). I n F i g u r e 4, a s c h e m a t i c r e p r e s e n t a t i o n o f the the ENS i s p r e s e n t e d . I t c o n s i s t s of i n t e r c o n n e c t e d nerve f i b r e s and g a n g l i a which a re found i n a l l l a y e r s o f the d i g e s t i v e t r a c t , e x t e n d i n g from the esophagus t o the i n t e r n a l a n a l s p h i n c t e r . D e s p i t e some minor r e g i o n a l and s p e c i e s d i f f e r e n c e s i n the degree o f i n n e r v a t i o n and i n the s i z e of g a n g l i a , the o r g a n i z a t i o n o f the ENS i s e s s e n t i a l l y the same throughout the g a s t r o i n t e s t i n a l t r a c t . The neurons which form the ENS a r i s e from two major i n t e r c o n n e c t e d g a n g l i o n a t e d p l e x i , the m y e n t e r i c or Auerbach's and the submucosal or M e i s s n e r ' s . The m y e n t e r i c p l e x u s i s 18 SMP FIGURE 4: DIAGRAMATIC REPRESENTATION OF THE ARRANGEMENT OF THE ENTERIC NERVOUS SYSTEM. The serosal surface i s to the l e f t and the mucosa i s shown to the r i g h t . LM=longitudinal muscle, MP=myenteric plexus (ganglionated), CM=circular muscle, DMP=deep muscular plexus, SMP=submucosal plexus (ganglionated), MM=muscularis mucosae and MucP=mucosal plexus. (Adapted from Furness and Costa, 1980). 19 s i t u a t e d between the l o n g i t u d i n a l and c i r c u l a r muscle l a y e r s w i t h f i b r e s e x t e n d i n g a l o n g the p l e x u s and o n l y a few f i b r e s d i r e c t e d towards the l o n g i t u d i n a l muscle. The m a j o r i t y o f f i b r e s p r o j e c t through the c i r c u l a r muscle t o the u n d e r l y i n g submucosal p l e x u s . I n a d d i t i o n t o the t r a n s v e r s i n g nerve f i b r e s , the c i r c u l a r muscle i s d e n s e l y i n n e r v a t e d w i t h m u l t i -a x o n a l nerve bundles r u n n i n g p a r a l l e l t o the muscle b u n d l e s . I n c e r t a i n r e g i o n s o f the g a s t r o i n t e s t i n a l t r a c t , i n p a r t i c u l a r the s m a l l i n t e s t i n e , axons from t h e s e nerve b u n d l e s form the n o n - g a n g l i o n a t e d deep muscular p l e x u s a l o n g the i n n e r (mucosal) boundary of the c i r c u l a r muscle l a y e r . The submucosal p l e x u s l i e s w i t h i n the c o n n e c t i v e t i s s u e u n d e r l y i n g the mucosa. Pathways from the submucosal p l e x u s p r o j e c t t o the mucosa, t o form the mucosal p l e x u s , as w e l l as t o the m y e n t e r i c p l e x u s . The b l o o d v e s s e l s s u p p l y i n g the g a s t r o i n t e s t i n a l t r a c t are i n n e r v a t e d by a p e r i v a s c u l a r p l e x u s which s u r r o u n d s the v e s s e l s and by p a r a v a s c u l a r n e r v e s which run a l o n g s i d e the a r t e r i e s and a r t e r i o l e s . The nervous r e g u l a t i o n o f g a s t r o i n t e s t i n a l f u n c t i o n i s thus performed by t h e s e i n t r i n s i c e n t e r i c n e u r o n s , which have t h e i r c e l l b o d i e s l o c a t e d i n e n t e r i c g a n g l i a , and e x t r i n s i c neurons which have t h e i r c e l l b o d i e s o u t s i d e the g a s t r o i n t e s t i n a l t r a c t . The c l a s s i c a l p i c t u r e o f the autonomic i n n e r v a t i o n o f the g a s t r o i n t e s t i n a l t r a c t c o n s i s t e d o f p r e g a n g l i o n i c c h o l i n e r g i c p a r a s y m p a t h e t i c i n p u t s and p o s t g a n g l i o n i c s y m p a t h e t i c a d r e n e r g i c i n p u t s . However, the d i v e r s i t y o f neuron t y p e s i n the e n t e r i c nervous system has s i n c e been d i s t i n g u i s h e d on e l e c t r i c a l , p h a r m a c o l o g i c a l , 20 h i s t o c h e m i c a l , b i o c h e m i c a l and u l t r a s t r u c t u r a l grounds (Furness and C o s t a 1980). A s i d e from the e x t r i n s i c c h o l i n e r g i c and a d r e n e r g i c neurons which e n t e r the ENS, c o n t r i b u t i n g t o the network of f i b r e s i n the p l e x i ( K o s t e r l i t z and L e e s , 1964), s e v e r a l types o f e n t e r i c neurons have been proposed t o e x i s t . These i n c l u d e neurons c o n t a i n i n g p u r i n e s such as adenosine t r i p h o s p h a t e (ATP) ( B u r n s t o c k , 1975, 1977), s e r o t o n i n (5-h y d r o x y t r y p t a m i n e , 5-HT) (Dreyfuss e t a l . , 1977a,b; C o s t a and F u r n e s s , 1979), gamma-aminobutyric a c i d (GABA) and o t h e r amino a c i d s (Jessen e t a l . , 1979), and v a r i o u s n e u r o p e p t i d e s (Furness and C o s t a , 1980; P o l a k and Bloom, 1981; Gershon e t a l . , 1981). The o c c u r r e n c e o f p e p t i d e - c o n t a i n i n g axons i n the ENS was f i r s t proposed on the b a s i s o f m o r p h o l o g i c a l s i m i l a r i t i e s between some m y e n t e r i c neuron t e r m i n a l s and p o s t e r i o r p i t u i t a r y p e p t i d e r g i c n e u r o s e c r e t o r y neurons, both o f which c o n t a i n l a r g e dense-cored s y n a p t i c v e s i c l e s (Baumgarten e t a l . , 1979). With the e v e n t u a l i d e n t i f i c a t i o n o f b i o l o g i c a l l y a c t i v e p e p t i d e s i n neurons o f the g a s t r o i n t e s t i n a l t r a c t came the n o t i o n o f p e p t i d e n e u r o t r a n s m i t t e r s as r e g u l a t o r s o f g a s t r o i n t e s t i n a l f u n c t i o n s . The d i s c o v e r y o f o p i o i d p e p t i d e s i n neurons o f the g a s t r o i n t e s t i n a l t r a c t was not s u r p r i s i n g i n l i g h t o f the d u a l l o c a l i z a t i o n i n gut and b r a i n t i s s u e o f s e v e r a l o t h e r n e u r o p e p t i d e s such as substa n c e P, v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e ( V I P ) , s o m a t o s t a t i n , c h o l e c y s t o k i n i n (CCK), g a s t r i n , bombesin and a n g i o t e n s i n , some o f which were f i r s t i d e n t i f i e d i n the g a s t r o i n t e s t i n a l t r a c t as p e p t i d e hormones (Pearse, 1978; P o l a k and Bloom, 1981; B i s h o p e t a l . , 1982; 21 C o s t a and F u r n e s s , 1982; Fur n e s s e t a l . , 1982). The term b r a i n - g u t a x i s has been c o i n e d t o c l a s s i f y the common a n a t o m i c a l d i s t r i b u t i o n o f t h i s ever i n c r e a s i n g group o f n e u r o p e p t i d e s which p u t a t i v e l y f u n c t i o n as n e u r o t r a n s m i t t e r s , neurohormones or hormones. A l t h o u g h o p i o i d p e p t i d e s have been l o c a l i z e d i n e n t e r i c neurons and have been shown t o a f f e c t o t h e r e n t e r i c n e u r o n s , i t i s not known whether t h e y f u n c t i o n as n e u r o t r a n s m i t t e r s , n e u r o m o d u l a t o r s , or hormones i n the g u t . The main approaches t a k e n t o c h a r a c t e r i z e g a s t r o i n t e s t i n a l o p i o i d p e p t i d e s have been t o d e t e c t t h e i r p r e s e nce by b i o c h e m i c a l or h i s t o c h e m i c a l methods, and t o examine t h e i r g a s t r o i n t e s t i n a l e f f e c t s when a p p l i e d e x o g e n o u s l y . V I I DISTRIBUTION OF OPIOID PEPTIDES IN THE GASTROINTESTINAL  TRACT Immunocytochemical t e c h n i q u e s u s i n g s p e c i f i c a n t i s e r a t o met- and l e u - e n k e p h a l i n have been a p p l i e d t o i d e n t i f y numerous e n k e p h a l i n e r g i c neurons i n the ENS of s e v e r a l s p e c i e s (Elde e t a l . , 1976; P o l a k e t a l . , 1977; Alumets e t a l . , 1978; L i n n o i l a e t a l . , 1978; S c h u l t z b e r g e t a l . , 1978; S c h u l t z b e r g e t a l . , 1980; L a r s s o n and St e n g a a r d - P e d e r s e n , 1982; F u r n e s s e t a l . , 1982, 1983; Bu'Lock e t a l . , 1983, Wang e t a l . , 1984; G i r a u d e t a l . , 1984). The e n k e p h a l i n - c o n t a i n i n g e n t e r i c neurons o c c u r throughout the g a s t r o i n t e s t i n a l t r a c t b ut are most d e n s e l y l o c a l i z e d i n the stomach and upper i n t e s t i n e (Smith e t a l . , 1976; Hughes e t a l . , 1977; P o l a k e t a l . , 1977; P o l a k and Bloom 1981). Met- and leu - e n k immunoreactive neurons are most abundant i n the m y e n t e r i c p l e x u s where they have been e s t i m a t e d 22 t o comprise 25 p e r c e n t o f the f i b r e s (Furness e t a l . , 1980, 1983). C e l l b o d i e s of almost a l l e n k e p h a l i n e r g i c e n t e r i c neurons have been found i n the m y e n t e r i c g a n g l i a ( J e s s e n e t a l . , 1980 F u r n e s s e t a l . , 1983) w i t h p r o c e s s e s i n the m y e n t e r i c p l e x u s , c i r c u l a r muscle, deep muscular p l e x u s and submucosal p l e x u s (Alumets e t a l . , 1977; P o l a k e t a l . , 1976; L i n n o i l a e t a l . , 1978; F u r n e s s e t a l . , 1983). Only a few e n k e p h a l i n -c o n t a i n i n g nerve c e l l b o d i e s have been i d e n t i f i e d i n the submucosal p l e x u s ( L o l o v a e t a l . , 1984; Wang e t a l . , 1984). T h e r e f o r e , most e n k e p h a l i n e r g i c f i b r e s i n the submucosal g a n g l i a a r i s e from the o v e r l y i n g m y e n t e r i c p l e x u s . S m a l l a r t e r i e s i n the g a s t r o i n t e s t i n a l t r a c t have a l s o been found t o be i n n e r v a t e d by m e t - e n k e p h a l i n immunoreactive t e r m i n a l s ( L a r s s o n and Stengaard-Pedersen 1982). E n k e p h a l i n e r g i c f i b r e s e i t h e r c o u l d not be i d e n t i f i e d i n the mucosa (Furness e t a l . , 1983; Bu'Lock e t a l . , 1983; V i n c e n t e t a l , 1984) or were found to occur v e r y o c c a s i o n a l l y i n the r a t g a s t r o i n t e s t i n a l mucosa (Wang e t a l . , 1984). There have, however been r e p o r t s o f e n k e p h a l i n s i n e n d o c r i n e c e l l s , p o s s i b l y c o e x i s t i n g w i t h g a s t r i n i n G - c e l l s i n the g a s t r i c a n t r a l mucosa (P o l a k e t a l . , 1977; L a r s s o n and S t e n g a a r d - P e d e r s e n , 1981; Bu'Lock e t a l . , 1983; G i r a u d e t a l . , 1984a; J o n s s o n , 1985). E n k e p h a l i n i m m u n o r e a c t i v i t y has a l s o been demonstrated i n e n t e r o c h r o m a f f i n c e l l s o f the gut mucosa (Alumets e t a l . , 1978). The e n k e p h a l i n e r g i c p r o c e s s e s i n the g a s t r o i n t e s t i n a l t r a c t appear t o be of i n t r i n s i c e n t e r i c o r i g i n s i n c e e n k e p h a l i n - c o n t a i n i n g f i b r e s can be demonstrated i n i n v i t r o c u l t u r e systems 23 ( S c h u l t z b e r g e t a l . , 1978; J e s s e n e t a l . , 1979, 1980b; J e s s e n , 1982). High c o n c e n t r a t i o n s o f d y n o r p h i n - i m m u n o r e a c t i v i t y (dyn-IR) have been measured by RIA i n the stomach and upper i n t e s t i n e (Spampinato and G o l d s t e i n 1983). Immunocytochemical s t u d i e s (Watson e t a l . , 1981) have demonstrated a heavy c o n c e n t r a t i o n o f d y n o r p h i n p o s i t i v e f i b r e s i n the submucosal p l e x u s o f the g u i n e a p i g i l e u m , w i t h some f i b r e s p e n e t r a t i n g the c i r c u l a r muscle, p r o j e c t i n g towards the m y e n t e r i c p l e x u s . V i n c e n t e t a l . (1984) have demonstrated d y n o r p h i n - I R i n the e n t e r i c p l e x i , c i r c u l a r muscle l a y e r and the l a m i n a m u s c u l a r i s o f the mucosa. While the p a t t e r n s of e n k e p h a l i n and d y n o r p h i n d i s t r i b u t i o n s have been found t o be s i m i l a r i n some a r e a s of the g a s t r o i n t e s t i n a l t r a c t , they are d i s t i n c t i n o t h e r s , s u g g e s t i n g t h a t a t l e a s t two o p i o i d n e u r o n a l systems are p r e s e n t . Endorphins and r e l a t e d p e p t i d e s have not been e x t e n s i v e l y examined i n the g a s t r o i n t e s t i n a l t r a c t . A l t h o u g h no e n d o r p h i n - c o n t a i n i n g f i b r e s c o u l d be i d e n t i f i e d by Alumets e t a l (1978), o t h e r s have demonstrated the p r e s e n c e o f e n d o r p h i n -l i k e p e p t i d e s by RIA i n the g a s t r i c antrum and i n the pancreas (Smyth, 1983) . V I I I . FUNCTIONS OF OPIOID PEPTIDES IN THE GASTROINTESTINAL  TRACT The f u n c t i o n s o f the ENS i n c l u d e c o o r d i n a t i n g r e f l e x c o n t r a c t i o n s o f the g a s t r o i n t e s t i n a l m u s c u l a t u r e t o ensure proper m i x i n g and p r o p u l s i o n o f gut c o n t e n t s , r e g u l a t i n g e n d o c r i n e and e x o c r i n e s e c r e t i o n s r e q u i r e d f o r d i g e s t i o n , and 24 r e g u l a t i n g g a s t r o i n t e s t i n a l b l o o d f l o w t o p e r m i t e f f i c i e n t n u t r i e n t a b s o r p t i o n . The p e p t i d e r g i c e n t e r i c n e u r o n a l systems have been i m p l i c a t e d i n a l l a s p e c t s o f the autonomic and r e f l e x c o n t r o l o f g a s t r o i n t e s t i n a l f u n c t i o n s , i n c l u d i n g m o d u l a t i o n o f e x t r i n s i c i n p u t s and p a r t i c i p a t i o n i n l o c a l r e f l e x mechanisms as s e n s o r y neurons, i n t e r n e u r o n s or e f f e c t o r neurons. The d i s t r i b u t i o n o f e n k e p h a l i n s and d y n o r p h i n s i n i n t r i n s i c e n t e r i c neurons s u g g e s t s they are w e l l - p o s i t i o n e d t o i n f l u e n c e the a c t i v i t y of the smooth muscle l a y e r s as w e l l as t o a f f e c t the a c t i v i t y of neurons i n the m y e n t e r i c p l e x u s . The p r e c i s e r o l e of the endogenous g a s t r o i n t e s t i n a l o p i o i d n e u r o n a l systems i s u n c l e a r , but based on the e f f e c t s o f e x o g e n o u s l y a d m i n i s t e r e d o p i a t e s and o p i o i d s and the e f f e c t s o f the o p i a t e a n t a g o n i s t , n a l o x o n e , some i n f o r m a t i o n as to the a c t i o n s o f the endogenous o p i o i d p e p t i d e s has been o b t a i n e d . The e f f e c t s o f morphine on g a s t r o i n t e s t i n a l m o t i l i t y , t o n e , p e r i s t a l s i s and emptying have been shown t o be mimicked by m e t - e n k e p h a l i n (Konturek et a l . , 1980; K o n t u r e k , 1980b, 1981). Depending on the s p e c i e s , r e g i o n o f the g a s t r o i n t e s t i n a l t r a c t and r o u t e of a d m i n s t r a t i o n the o p i o i d s have been d e s c r i b e d as e i t h e r i n h i b i t o r y or s t i m u l a t o r y . The c e n t r a l e f f e c t s on m o t i l i t y appear t o be i n h i b i t o r y . I n t r a c e r e b r o v e n t r i c u l a r a d m i n i s t r a t i o n o f o p i o i d p e p t i d e s was found t o d e c r e a s e the i n t e s t i n a l t r a n s i t o f r a d i o i s o t o p e markers ( G a l l i g a n and B u r k s , 1983; B u r k s e t a l . , 1984) and the f r equency o f i n t e s t i n a l c o n t r a c t i o n s (Ruckebusch e t a l . , 1984b). The i n c r e a s e i n t r a n s i t time o b s e r v e d i n r a t s f o l l o w i n g 25 i n t r a v e n o u s l y a d m i n i s t e r e d morphine has been suggested as a "gut-mediated" event ( B i a n c h i e t a l . , 1983). I n v i t r o , o p i o i d p e p t i d e s e x h i b i t an i n h i b i t o r y a c t i o n , as o r i g i n a l l y demonstrated by the i n h i b i t i o n o f e l e c t r i c a l l y induced c o n t r a c t i o n s o f the g u i n e a p i g i l e u m ( P a t o n , 1957). S i m i l a r l y , met-enk in d u c e s a dose-dependent t r a n s i e n t r e l a x a t i o n i n r a t duodenal s t r i p s (Furukawa e t a l . , 1980). I n t r a c e l l u l a r r e c o r d i n g s from neurons i n i s o l a t e d g u i n e a p i g i l e u m m y e n t e r i c p l e x u s have g i v e n some i n s i g h t as t o the mechanism and s i t e o f o p i o i d a c t i o n (North e t a l . , 1982). I t appears t h a t the e n k e p h a l i n s can a c t p r e s y n a p t i c a l l y t o cause a r e d u c t i o n i n t r a n s m i t t e r r e l e a s e and p o s t - s y n a p t i c a l l y t o i n h i b i t the f i r i n g o f m y e n t e r i c neurons ( D i n g l e d i n e and G o l d s t e i n , 1975; E h r e n p r e i s e t a l . , 1976; N o r t h and W i l l i a m s , 1976). In a d d i t i o n , i t has r e c e n t l y been demonstrated t h a t exogenouly a d m i n i s t e r e d o p i o i d p e p t i d e s can s t r o n g l y i n h i b i t the r e l e a s e o f s u b s t a n c e P from the m y e n t e r i c p l e x u s ( G i n t z l e r and S c a l i s i , 1982). Membrane h y p e r p o l a r i z a t i o n r e s u l t i n g from the a c t i v a t i o n o f p o t a s s i u m c h a n n e l s n o r m a l l y gated by i n t r a c e l l u l a r c a l c i u m c o n c e n t r a t i o n s (North e t a l . , 1979; Tokimasa e t a l . , 1981; M o r i t a and N o r t h , 1982) has been shown to be the u n d e r l y i n g mechanism r e s p o n s i b l e f o r t h i s i n h i b i t i o n . A l s o , i t has been demonstrated by f o c a l i o n t o p h o r e s i s of e n k e p h a l i n t h a t t h i s h y p e r p o l a r i z i n g a c t i o n o f the o p i o i d s does not occur a t the c e l l soma but r a t h e r a t the c e l l p r o c e s s e s where a c t i o n p o t e n t i a l p r o p a g a t i o n can be p r e v e n t e d (North e t a l 1979). T h i s mechanism o f a c t i o n i s c o n s i s t e n t w i t h e n k e p h a l i n - i n d u c e d i n h i b i t i o n o f the e l e c t r i c a l l y evoked 26 release of acetylcholine and the resultant i n h i b i t i o n of contractile a c t i v i t y in the guinea pig ileum (Waterfield et a l . , 1977), and the i n h i b i t i o n of acetylcholine release from the submucosal plexus of rat colon (Gaginella and Wu, 1983). The most predominant g a s t r o i n t e s t i n a l motor response to the opioids i n intact animals, man included, i s stimulatory. Generally, the constipating effects of opiate narcotics and opioid peptides are considered to be produced by the increase in non-propulsive muscle a c t i v i t y in the g a s t r o i n t e s t i n a l t r a c t . This leads to the slowing of i n t e s t i n a l t r a n s i t time, allowing for more e f f i c i e n t f l u i d and e l e c t r o l y t e absorption through the mucosa (Mil l e r , 1983). In the dog, morphine and opioid peptides were found to produce dose-dependent contractions of the isolated vascularly perfused small intestine (Burks et a l . , 1982). S i m i l a r l y , i t was found that intravenous (i.v.) administrations of fentanyl, a mu-receptor agonist, induced a dose-related increase i n both tone and frequency of contractions in the d i s t a l portion of the colon; whereas, a period of decreased m o t i l i t y was e l i c i t e d in the proximal colon (Bardon and Ruckebusch, 1985). The i . v . administration of the kappa-agonist, EKC, i n h i b i t e d colonic m o t i l i t y while DADLE, a delta-agonist, stimulated colonic contractions. Therefore, not only do there appear to be regional differences in g a s t r o i n t e s t i n a l motor responses to opioid substances, but activation of s p e c i f i c opioid receptor types appears to e l i c i t d i s t i n c t motor e f f e c t s . In sheep, kappa-, mu- and delta-type opioid agonists caused a transient increase in duodenal motor a c t i v i t y (Ruckebusch et a l . , 1984a). 27 By p l a c i n g t h i n - w a l l e d f l a c c i d p o l y e t h y l e n e bags i n the stomach of dogs t o r e c o r d g a s t r i c c o n t r a c t i o n s and by s i m u l t a n e o u s l y r e c o r d i n g the m y o e l e c t r i c a l a c t i v i t y u s i n g i m p l a n t e d e l e c t r o d e s , met-enk was found t o d o s e - d e p e n d e n t l y i n c r e a s e the amp l i t u d e and s t r e n g t h , and t o d e c r e a s e the f r e q u e n c y of g a s t r i c c o n t r a c t i o n s (Konturek 1981). The r e c o r d e d e l e c t r i c a l a c t i v i t y was a l s o i n c r e a s e d . Thus, i t has been proposed t h a t the d e l a y e d g a s t r i c emptying induced by the o p i o i d s might be due t o both p y l o r o s p a s m and i n c r e a s e d duodenal c o n t r a c t i o n s . In man, d i r e c t d e m o n s t r a t i o n s o f the i n h i b i t i o n o f g a s t r i c emptying i n response t o a s y n t h e t i c e n k e p h a l i n analogue have been made ( S u l l i v a n e t a l . , 1981). F o l l o w i n g i n g e s t i o n of a meal c o n t a i n i n g a r a d i o a c t i v e t r a c e r , a subcutaneous i n j e c t i o n of 0.5 mg of D - A l a 2 - M e P h e 4 - M e t ( 0 ) - 0 1 - e n k e p h a l i n (DAMME) was found t o cause a s i g n i f i c a n t s l o w i n g o f g a s t r i c emptying as rec o r d e d by a s c i n t i l l a t i o n camera. A g a i n i n man, o p i a t e s and o p i o i d s have been shown t o r e s u l t i n an i n c r e a s e i n stomach and i n t e s t i n a l muscle tone as w e l l as an i n c r e a s e i n segmental but not p r o p u l s i v e c o n t r a c t i o n s (Corder and Rees 1981). The profound a c t i o n s o f morphine on the s m a l l i n t e s t i n e have a l s o been demonstrated by c i n e r a d i o g r a p h i c s t u d i e s . Upon morphine a d m i n i s t r a t i o n t h e r e was observed an i n c r e a s e i n c o n t r a c t i l e a c t i v i t y and tone f o l l o w e d by d i s t e n t i o n , r e s u l t i n g i n a de l a y e d i n t e s t i n a l t r a n s i t time and an accompanying i n c r e a s e d i n t e s t i n a l a b s o r p t i o n ( S i l b i g e r and Donner 1968). T h e r e f o r e , the d e l a y i n g a s t r i c emptying and i n t e s t i n a l t r a n s i t , i n c o n j u n c t i o n w i t h the a s s o c i a t e d i n c r e a s e i n i n t e s t i n a l 28 a b s o r p t i o n , may c o n t r i b u t e t o the w e l l known c o n s t i p a t i n g e f f e c t s o f the o p i a t e n a r c o t i c s and o p i o i d p e p t i d e s . The e x t r e m e l y complex, i n t e r a c t i n g c o n t r o l l i n g mechanisms i n v o l v e d i n the r e g u l a t i o n o f g a s t r i c a c i d s e c r e t i o n have c o m p l i c a t e d the s t u d y o f the e f f e c t s of o p i o i d s on g a s t r i c a c i d s e c r e t i o n . Depending on the e x p e r i m e n t a l model and doses a d m i n i s t e r e d , v a r y i n g e f f e c t s o f the o p i o i d s have been r e p o r t e d . I n human s u b j e c t s , the s y n t h e t i c met-enk analogue FK 33-824 caused an i n c r e a s e i n b a s a l and p e n t a g a s t r i n - s t i m u l a t e d a c i d s e c r e t i o n (Olsen e t a l . , 1981). Serum g a s t r i n l e v e l s were 2 unchanged. I n a s i m i l a r s t u d y , D - a l a - e n k e p h a l i n i n h i b i t e d b a s a l and v a g a l l y - or p e n t a g a s t r i n - induced g a s t r i c s e c r e t i o n (Konturek e t a l . , 1983). The e n k e p h a l i n analogue a l s o i n c r e a s e d serum g a s t r i n l e v e l s d u r i n g m o d i f i e d sham f e e d i n g . U s i n g c o n s c i o u s dogs w i t h v a g a l l y d e n e r v a t e d H e i d e n h a i n pouches and g a s t r i c f i s t u l a s , morphine was found t o i n c r e a s e b oth b a s a l and h i s t a m i n e - s t i m u l a t e d g a s t r i c a c i d s e c r e t i o n but caused a decrease i n p e n t a g a s t r i n - s t i m u l a t e d s e c r e t i o n (Magee 1975). T h i s i n h i b i t o r y e f f e c t has been proposed t o be a r e s u l t of o p i a t e - i n d u c e d s u p p r e s s i o n o f a c e t y l c h o l i n e r e l e a s e from c h o l i n e r g i c n e r v e s i n the stomach. Sham f e e d i n g i n d u c e d g a s t r i c s e c r e t i o n , a v a g a l l y - m e d i a t e d e v e n t , was found t o be suppressed by m e t - e n k e p a h l i n (Konturek e t a l . , 1982). S i n c e t h e r e have been r e p o r t s o f e n k e p h a l i n - I R i n the vagus nerve (Elde e t a l . , 1976; Lundberg e t a l . , 1979) i t has been suggested t h a t o p i o i d s may be i n v o l v e d i n the c e p h a l i c phase, perhaps by l i m i t i n g the degree of v a g a l l y - s t i m u l a t e d g a s t r i c s e c r e t i o n . I n a more r e c e n t s t u d y , employing a s i m i l a r dog 29 p r e p a r a t i o n , i t was found t h a t morphine and e n k e p h a l i n g i v e n i n a s m a l l e r dose than by Magee (1975) produced a dose-dependent i n c r e a s e i n b a s a l a c i d s e c r e t i o n (Konturek 1981). An i n c r e a s e i n serum g a s t r i n l e v e l s was not d e t e c t e d ; t h e r e f o r e , i t was suggested t h a t the o p i a t e s and o p i o i d s may d i r e c t l y s t i m u l a t e the o x y n t i c g l a n d s . On the o t h e r hand, i n v e s t i g a t i o n s u s i n g c o n s c i o u s r a t s w i t h g a s t r i c f i s t u l a e showed t h a t met-enk c o u l d i n h i b i t h i s t a m i n e - , but not p e n t a g a s t r i n - , s t i m u l a t e d or b a s a l s e c r e t i o n s ( E l Munshid e t a l . , 1980). S i n c e i n these e xperiments the o p i o i d s were a d m i n i s t e r e d i n t r a v e n o u s l y or i n t r a - a r t e r i a l l y the r e s u l t s may have been c o m p l i c a t e d by c e n t r a l e f f e c t s caused by some o p i a t e s which can c r o s s the b l o o d - b r a i n - b a r r i e r . Naloxone a l o n e i n h i b i t e d submaximal h i s t a m i n e or p e n t a g a s t r i n s t i m u l a t e d a c i d s e c r e t i o n , s u g g e s t i n g t h a t endogenous o p i o i d s may have a " t o n i c " a c t i o n on the a c i d s e c r e t i n g p a r i e t a l c e l l (Konturek 1980a, b) . However, the e f f e c t s of the o p i a t e s and o p i o i d p e p t i d e s can be r e a d i l y i n h i b i t e d not o n l y by naloxone but a l s o by a n t i c h o l i n e r g i c s or H 2 - r e c e p t o r a n t a g o n i s t s (Konturek 1980b), s u g g e s t i n g an i n d i r e c t e f f e c t . I t has been p r o p o s e d , t h e r e f o r e , t h a t enhancement o f g a s t r i c s e c r e t i o n s and mucosal b l o o d f l o w a re due t o o p i a t e - and o p i o i d - s t i m u l a t e d r e l e a s e o f endogenous h i s t a m i n e (Walus e t a l . , 1981). S i n c e the f i n a l outcome o f a s i n g l e i n h i b i t o r y or e x c i t a t o r y agent i s a r e s u l t o f the combined e f f e c t s o f s e v e r a l i n t e r a c t i n g endogenous mechanisms c o n v e r g i n g on the p a r i e t a l c e l l , more i n v e s t i g a t i o n s i n t o the complex r e g u l a t o r y pathways are r e q u i r e d i n o r d e r t o e l u c i d a t e the r o l e o f e n k e p h a l i n s i n the r e g u l a t i o n o f a c i d s e c r e t i o n . 30 In a d d i t i o n t o g a s t r i c a c i d s e c r e t i o n , the o p i o i d s may be i n v o l v e d i n the r e g u l a t i o n o f p a n c r e a t i c e x o c r i n e and e n d o c r i n e s e c r e t i o n s . O p i o i d p e p t i d e s have been found t o be p r e s e n t i n e x t r a c t s o f g u i n e a p i g pancreas ( S t e r n e t a l . , 1982), and e n k e p h a l i n - c o n t a i n i n g neurons have been found t o i n n e r v a t e the c a t pancreas ( L a r s s o n , 1979) . Met-enk and morphine have been shown t o suppress s e c r e t i n - and CCK- s t i m u l a t e d b i c a r b o n a t e and enzyme r e l e a s e (Konturek e t a l . , 1978; Chey e t a l . , 1980). F u r t h e r m o r e , p a n c r e a t i c s e c r e t i o n s t i m u l a t e d by duodenal a c i d i f i c a t i o n or f e e d i n g was i n h i b i t e d by e n k e p h a l i n or morphine. T h i s was accompanied by a d r o p i n plasma s e c r e t i n l e v e l s , s u g g e s t i n g t h a t e n k e p h a l i n s i n h i b i t both the r e l e a s e and a c t i o n o f s e c r e t i n . I s l e t c e l l f u n c t i o n s may a l s o be r e g u l a t e d by the o p i o i d s s i n c e the r e l e a s e o f i n s u l i n , g l u c a g o n and s o m a t o s t a t i n have been shown t o be a f f e c t e d by morphine, b e t a - e n d o r p h i n and e n k e p h a l i n (Ipp e t a l . , 1978; P i e r l u i s s i e t a l . , 1981). I n the dog p a n c r e a s , e n k e p h a l i n s have been found t o d o s e - d e p e n d e n t l y s t i m u l a t e i n s u l i n and i n h i b i t s o m a t o s t a t i n i n a n a l o x o n e - r e v e r s i b l e manner (Hermansen, 1983). R e c e n t l y , i t has been demonstrated u s i n g the i s o l a t e d p e r f u s e d r a t pancreas p r e p a r a t i o n t h a t low c o n c e n t r a t i o n s o f m e t - e n k e p h a l i n ( l e s s than 10 ^ M) i n h i b i t e d , w h i l e h i g h c o n c e n t r a t i o n s s t i m u l a t e d , s o m a t o s t a t i n r e l e a s e ( D i S c a l a - G u e n o t and M c i n t o s h , 1985). I n s u l i n s e c r e t i o n was s t i m u l a t e d a t a l l c o n c e n t r a t i o n s , and naloxone a n t a g o n i z e d the e f f e c t s o f m e t - e n k e p h a l i n on s o m a t o s t a t i n s e c r e t i o n , but i n s u l i n s e c r e t i o n was o n l y p a r t i a l l y b l o c k e d . 31 O p i o i d p e p t i d e s are known t o a f f e c t g a s t r o i n t e s t i n a l b l o o d f l o w ; e n k e p h a l i n i n f u s i o n i n t o the g a s t r i c a r t e r y r e s u l t s i n an i n c r e a s e d g a s t r i c mucosal b l o o d f l o w (Konturek e t a l . , 1978). I t has a l s o been r e p o r t e d t h a t met-enk i n c r e a s e s oxygen consumption and b l o o d f l o w t o muscle l a y e r s (Walus e t a l . , 1981). F i n a l l y , i t has been demonstrated t h a t o p i a t e a l k a l o i d s and o p i o i d p e p t i d e s i n f l u e n c e i o n t r a n s p o r t a c r o s s r a b b i t i l e a l mucosa (McKay e t a l . , 1981; B i n d e r e t a l . , 1984) and i n h i b i t f l u i d a c c u m u l a t i o n induced by p r o s t a g l a n d i n s (Coupar 1978) or VIP (Beubler and Lembeck, 1979). F u r t h e r m o r e , e n k e p h a l i n s have been found t o reduce the t r a n s e p i t h e l i a l p o t e n t i a l d i f f e r e n c e and the s h o r t c i r c u i t c u r r e n t a c r o s s i s o l a t e d mucosal segments of the g u i n e a p i g i l e u m , r e s u l t i n g i n the i n h i b i t i o n o f i l e a l f l u i d s e c r e t i o n (Kachur and M i l l e r , 1982; V i n a y e k e t a l . , 1983). Thus, i t has been suggested t h a t the a n t i d i a r r h e a l a c t i o n s of the o p i o i d s may be b r o u g h t about by changes i n mucosal i o n t r a n s p o r t i n a d d i t i o n t o changes i n g a s t r o i n t e s t i n a l motor a c t i v i t y . From t h e s e known e f f e c t s of o p i o i d s on g a s t r o i n t e s t i n a l f u n c t i o n i t can be i n f e r r e d t h a t the endogenous o p i o i d p e p t i d e s must p l a y a s i g n i f i c a n t p h y s i o l o g i c a l r o l e i n g a s t r o i n t e s t i n a l r e g u l a t i o n . IX. RATIONALE FOR THE PRESENT STUDIES The m a j o r i t y of the e x t e n s i v e volume of knowledge c o n c e r n i n g the d i s t r i b u t i o n , b i o g e n e s i s , r e c e p t o r t y p e s , and a c t i o n s o f o p i o i d p e p t i d e s which has accumulated i n the p a s t 32 decade has f o c u s e d on c e n t r a l o p i o i d p e p t i d e s . W i t h r e s p e c t t o the g a t r o i n t e s t i n a l o p i o i d p e p t i d e s t h e r e are many unanswered q u e s t i o n s . The b i o g e n e s i s of the t h r e e f a m i l i e s of o p i o i d p e p t i d e s i n the b r a i n and a d r e n a l g l a n d has been w e l l c h a r a c t e r i z e d based on m o l e c u l a r b i o l o g i c a l t e c h n i q u e s , but v e r y l i t t l e i s known c o n c e r n i n g the forms o f g a s t r o i n t e s t i n a l o p i o i d s . W h i l e i t might be p r e d i c t e d t h a t the o p i o i d s i n the g a s t r o i n t e s t i n a l t r a c t are s i m i l a r t o t h o s e i n the b r a i n and a d r e n a l g l a n d , the i d e n t i f i c a t i o n of the e x a c t t y p e s o f endogenous g a t r o i n t e s t i n a l o p i o i d s i s r e q u i r e d i n o r d e r t o determine the s i g n i f i c a n c e o f s t u d i e s r e l a t e d t o t h e i r a c t i o n s . T h e r e f o r e the endogenous o p i o i d s i n the r a t stomach have been p a r t i a l l y c h a r a c t e r i z e d (Chapter 3) . T h i s work c o n c e n t r a t e d on the r a t stomach s i n c e t h i s i s one o f the r e g i o n s i n the g a s t r o i n t e s t i n a l t r a c t where e n k e p h a l i n s are p r e s e n t i n h i g h amounts (Polak e t a l . , 1976; Hughes e t a l . , 1977). P a r t i a l p u r i f i c a t i o n o f o p i o i d - l i k e s u b s t a n c e s from crude g a s t r i c e x t r a c t s was a c h i e v e d by chromatography, and s e v e r a l d i f f e r e n t a s s a y systems were u t i l i z e d i n o r d e r t o d e t e c t o p i o i d p e p t i d e s and o p i o i d a c t i v i t y . Chapter f o u r a d d r e s s e s the problem o f m u l t i p l e o p i o i d r e c e p t o r s and t h e i r d i s t r i b u t i o n i n the g a s t r o i n t e s t i n a l t r a c t . P r e s e n t u n d e r s t a n d i n g o f g a s t r o i n t e s t i n a l o p i o i d r e c e p t o r s i s based almost e x c l u s i v e l y on p h a r m a c o l o g i c a l b i n d i n g s t u d i e s conducted on the i s o l a t e d g u i n e a p i g i l e u m p r e p a r a t i o n . The wide d i s t r i b u t i o n o f e n k e p h a l i n e r g i c and d y n o r p h i n e r g i c n e u r o n a l systems i n the ENS c o u p l e d w i t h the o b s e r v e d p o t e n t o p i o i d e f f e c t s t h r o u g h o u t the g a s t r o i n t e s t i n a l t r a c t suggest 33 the presence o f c o r r e s p o n d i n g o p i o i d r e c e p t o r s . A u t o r a d i o g r a p h i c methods i n c o n j u n c t i o n w i t h l i g h t m i c r o s c o p y have proven u s e f u l i n d e m o n s t r a t i n g the d i s t r i b u t i o n o f o p i o i d r e c e p t o r s i n the b r a i n (Herkenham and P e r t , 1982). T h i s t e c h n i q u e was t h e r e f o r e adapted t o l o c a l i z e the d i s t r i b u t i o n of o p i o i d b i n d i n g s i t e s i n the v a r i o u s l a y e r s o f the g a s t r o i n t e s t i n a l t r a c t . Having d e t e r m i n e d the predominant t y p e s o f g a s t r i c o p i o i d p e p t i d e s and the s p e c i f i c r e g i o n a l d i s t r i b u t i o n o f t h e i r r e c e p t o r s , the r e l e a s e o f endogenous o p i o i d p e p t i d e s from the i s o l a t e d p e r f u s e d r a t stomach p r e p a r a t i o n was s t u d i e d (Chapter 5 ) . D e m o n s t r a t i o n of the r e l e a s e o f endogenously o c c u r r i n g o p i o i d s i s c r u c i a l e v i d e n c e t o s u b s t a n t i a t e a r o l e f o r these n e u r o p e p t i d e s i n the r e g u l a t i o n o f p h y s i o l o g i c a l f u n c t i o n s . P r e v i o u s l y , o n l y i n d i r e c t e v i d e n c e has been a v a i l a b l e s u g g e s t i n g the r e l e a s e o f endogenous g a s t r o i n t e s t i n a l o p i o i d s based on the e f f e c t o f naloxone (Puig e t a l . , 1977). A l s o , i t has been shown t h a t t h e r e i s a s i g n i f i c a n t r e d u c t i o n i n the e n k e p h a l i n c o n t e n t i n the m y e n t e r i c p l e x u s f o l l o w i n g e l e c t r i c a l s t i m u l a t i o n ( C o r b e t t e t a l 1981). There has been no d i r e c t d e m o n s t r a t i o n o f g a s t r i c o p i o i d r e l e a s e made t o d a t e . The i s o l a t e d p e r f u s e d stomach system p r o v i d e s an e x e l l e n t model f o r d e t e c t i n g the r e l e a s e o f endogenous s u b s t a n c e s i n t o the g a s t r i c v a s c u l a t u r e . I t has been s u c e s s f u l l y employed i n t h i s l a b o r a t o r y t o measure the s e c r e t i o n o f p e p t i d e hormones such as s o m a t o s t a t i n ( M c i n t o s h e t a l . , 1981) and g a s t r i n (Pederson e t a l . , 1984) from g a s t r i c e n d o c r i n e c e l l s and the s e c r e t i o n o f VI P , known t o be p r e s e n t i n e n t e r i c neurons (Pederson e t a l . , 34 1981.) Due t o the r a p i d e n z y m a t i c d e g r a d a t i o n o f o p i o i d p e p t i d e s and t h e i r r e l a t i v e l y s m a l l q u a n t i t i e s i n the stomach, i n comparison w i t h the o t h e r g a s t r i c p e p t i d e s , i t was n e c e s s a r y t o t a k e s e v e r a l p r e c a u t i o n a r y s t e p s t o f a c i l i t a t e t h e i r d e t e c t i o n . The mechanisms c o n t r o l l i n g g a s t r i c o p i o i d p e p t i d e r e l e a s e were a l s o s t u d i e d by examining t h e i r response t o p h a r m a c o l o g i c a l a g e n t s . In the s i x t h c h a p t e r , s t u d i e s on the e f f e c t s o f exogenously a d m i n i s t e r e d o p i o i d p e p t i d e s on the s e c r e t i o n o f the p e p t i d e s o m a t o s t a t i n are d e s c r i b e d . O r i g i n a l l y e x t r a c t e d from the hypothalamus as an i n h i b i t o r o f growth hormone r e l e a s e , s o m a t o s t a t i n has a l s o been found t o be l o c a t e d throughout the g a s t r o i n t e s t i n a l t r a c t and t o i n h i b i t the r e l e a s e o f s e v e r a l g a s t r o i n t e s t i n a l and p a n c r e a t i c hormones as w e l l as g a s t r i c a c i d s e c r e t i o n . C h i b a e t a l . (1980) demonstrated t h a t e n k e p h a l i n i n h i b i t s b a s a l s o m a t o s t a t i n s e c r e t i o n , but e x t e n s i v e s t u d i e s were not performed. T h e r e f o r e , the e f f e c t s and mechanism o f a c t i o n o f the o p i o i d p e p t i d e s , e n k e p h a l i n and d y n o r p h i n , on s o m a t o s t a t i n r e l e a s e from the i s o l a t e d p e r f u s e d r a t stomach were examined. The work p r e s e n t e d i n t h i s t h e s i s t h u s a d d r e s s e s s e v e r a l key a s p e c t s c o n c e r n i n g the c h a r a c t e r i z a t i o n o f g a s t r o i n t e s t i n a l , i n p a r t i c u l a r g a s t r i c , o p i o i d p e p t i d e s , f o c u s i n g on t h e i r b i o c h e m i c a l forms, r e c e p t o r d i s t r i b u t i o n s , r e l e a s e , a c t i o n s and mode of a c t i o n . 35 CHAPTER TWO  GENERAL METHODOLOGY AND MATERIALS I . ASSAY SYSTEMS A. GUINEA PIG ILEUM BIOASSAY 1. R a t i o n a l e The g u i n e a p i g i l e u m b i o a s s a y system i s based on e a r l y o b s e r v a t i o n s r e p o r t i n g t h a t morphine d e c r e a s e s the spontaneous and e l e c t r i c a l l y - s t i m u l a t e d r e l e a s e o f a c e t y l c h o l i n e from e n t e r i c neurons i n the g u i n e a p i g i l e u m (Paton, 1957). The re s p o n s e , d e t e r m i n e d as a r e d u c t i o n i n muscular c o n t r a c t i o n , i s dose-dependent and naloxone s e n s i t i v e . Both the whole g u i n e a p i g i l e u m and the m y e n t e r i c p l e x u s -l o n g i t u d i n a l muscle p r e p a r a t i o n s have been r o u t i n e l y employed i n the d e t e c t i o n and q u a n t i f i c a t i o n o f o p i o i d s u b s t a n c e s . A l t h o u g h the b i o a s s a y i s not as s e n s i t i v e as radioimmunoassays or r e c e p t o r b i n d i n g a s s a y s , i t does have the advantage of s p e c i f i c i t y i n t h a t o n l y b i o l o g i c a l l y a c t i v e m a t e r i a l i s d e t e c t e d . Moreover, t o ensure s p e c i f i c i t y , each sample i s u s u a l l y checked f o r naloxone antagonism. One drawback, however, i s t h a t the mu- and kappa-type o p i o i d r e c e p t o r s a re the dominant ones i n t h i s t i s s u e (Lord e t a l . , 1977); t h e r e f o r e , d i f f e r e n t i a l r esponses a re o b t a i n e d depending on the a g o n i s t r e c e p t o r s p e c i f i c i t y and a f f i n i t y . 36 2. E x p e r i m e n t a l P r o c e d u r e Male a d u l t g u i n e a p i g s w e i g h i n g 300-400 g were f a s t e d o v e r n i g h t ( a p p r o x i m a t e l y 16 hours) b e f o r e b e i n g k i l l e d by a blow t o the neck. The abdomen was opened by an i n c i s i o n a t the m i d l i n e and the t e r m i n a l i l e u m d i s s e c t e d o ut and imm e d i a t e l y p l a c e d i n a m o d i f i e d K r e b s 1 b i c a r b o n a t e b u f f e r gassed w i t h 95% 0 2 and 5% C 0 2 t o m a i n t a i n a pH o f 7.4. The c o m p o s i t i o n o f the b u f f e r was as f o l l o w s (mM): NaCl 120.0 KC1 4.4 C a C l 2 2.5 MgS0 4 1.2 K H 2 P 0 4 1.5 NaHC0 3 25.0 D e x t r o s e 8.8 A f t e r d i s c a r d i n g a 10 cm l e n g t h p r o x i m a l t o the i l e o c e c a l j u n c t i o n , 1.5-2.0 cm segments o f the r e m a i n i n g whole i l e u m , trimmed o f f a t and mesentery, were mounted i n t o 10 ml t i s s u e baths f i l l e d w i t h Krebs' b u f f e r . The s o l u t i o n i n the bath was m a i n t a i n e d a t 37 °C and c o n t i n u o u s l y gassed w i t h the 95% 0 2 and 5% C 0 2 gas m i x t u r e . I s o m e t r i c muscular c o n t r a c t i o n s were d e t e c t e d by means o f a f o r c e d i s p l a c e m e n t t r a n s d u c e r (Statham G10B) and were r e c o r d e d on a G i l s o n P o l y g r a p h . A f t e r a b a s a l t e n s i o n of 1 g was s e t , the i l e a l segments were e q u i l i b r a t e d f o r a minimum of 1 hour w i t h r e g u l a r washes e v e r y 15 m i n u t e s . Two p a r a l l e l p l a t i n u m e l e c t r o d e s h e l d i n a l u c i t e chamber were i n t r o d u c e d i n t o the organ bath through which e l e c t r i c a l f i e l d s t i m u l a t i o n (100 V, 0.3 Hz, 0.5 msec) was a p p l i e d . F i g u r e 5 37 Heated Water Bath 37°C 95 % 0 2 5 % C 0 2 Transducer Gilson Pen Recorder Stimulator 100 V, 03 Hz 0.5 msec FIGURE 5: SCHEMATIC DIAGRAM OF THE GUINEA PIG ILEUM BIOASSAY APPARATUS. The i l e a l s t r i p i s maintained i n oxygenated Krebs' bicarbonate buffer and i s attached to the strain-gauge transducer by means of a s i l k thread. The temperature of the organ bath i s maintained at 37 C by means of heated water which i s pumped through the outer sleeve of the organ chamber. F i e l d stimulation (100 v o l t s , 0.3 hertz for 0.5 msec) i s applied through p a r a l l e l platinum electrodes placed on either side of the tis s u e . " 38 schematically outlines the bioassay apparatus. Known concentrations of met-enk (Peninsula Laboratories Inc., San Carlos, C a l i f o r n i a ) , dissolved in Krebs' buffer containing 1% BSA (bovine serum albumin, Sigma, Fraction V) , were used as standard references for q u a n t i f i c a t i o n . B. RADIORECEPTOR ASSSAY (RRA) 1. Rationale Receptor binding assays are in v i t r o methods of peptide quantification, u t i l i z i n g the p r i n c i p l e s of competitive binding of radio-labelled and nonlabelled peptides for s p e c i f i c receptor s i t e s . The l a b e l l e d "tracer" peptide binding in the presence of unknown samples can be compared to standard curves prepared by measuring the tracer binding i n the presence of varying known opioid peptide concentrations. 2. Technical considerations Several technical requirements must be met in order to apply receptor binding to the detection of opioid peptides. F i r s t l y , a suitable receptor preparation i s required. For the opioid RRA, homogenates of whole brains less the cerebellum, which demonstrates no appreciable opioid binding (Pert and Snyder, 1973b), are most commonly used as a source of opioid receptors. Although i n t e s t i n a l preparations may be employed, Pert and Snyder (1973b) have shown that the greatest amount of opiate receptor binding occurs in the brain while the guinea pig small intestine displays only one-half as much binding. In place of crude brain membranes, more p u r i f i e d mitochondrial-synaptosomal fractions (P2) have been used by 39 some investigators (Somoza et a l . , 1981; Hazum et a l . , 1981). Secondly, to determine the amount of tracer which i s bound to the membrane receptors, a rapid means of separating the unbound or "free" tracer from those bound to the membrane binding s i t e s must be available. F i l t r a t i o n through glass f i b r e f i l t e r s and centrifugation are most frequently used. In the RRA, the amount of s p e c i f i c binding, described as that portion of the to t a l measured binding which relates d i r e c t l y to the b i o l o g i c a l a c t i v i t y , cannot be assessed. Therefore, for the purpose of the RRA, s p e c i f i c binding i s assumed to be that which can be displaced by a high concentration of non-radioactive ligand. The remaining non-displaceable binding i s assumed to be bound nons p e c i f i c a l l y to glass, connective tissue and non-receptor membrane structures, etc. A f i n a l consideration when performing receptor binding studies with membrane homogenates i s the p o s s i b i l i t y of ligand breakdown as a re s u l t of degradation by membrane bound proteases. Stable enkephalin analogues resistant to enzymatic degradation or protease i n h i b i t o r s are often employed i n the assay i n order to avoid this problem. 3. Experimental Procedure a) Brain Homogenate Preparation Fresh, whole rat brains minus the cerebellum were homogenized with a Sor v a l l Omni-mixer in 50 mM Tris-HCl buffer pH 7.4 (25 ml/brain). The homogenate was centrifuged at 4 °C for 50 minutes at 27,500 g (Sorvall, RC-5B) . The supernatant was discarded and the remaining p e l l e t (PI) resuspended by hand with 6 strokes of a teflon Thomas 40 homogenizer using the same buffer (15 ml/brain). This mixture was continuously s t i r r e d at 4 °C u n t i l pipetted into the assay tubes. In cases in which a mitochondrial-synaptosomal preparation (P2) was used, the whole brain minus the cerebellum was homogenized i n 10 volumes of isosmotic sucrose (0.32 M) using a teflon-glass homogenizer (Thomas)—10 gentle strokes per brain. This homogenate was centrifuged at 1000 g (Sorvall) for 10 minutes at 4 °C. The supernatant was decanted and recentrifuged, at 17,000 g for 30 minutes. The supernatant was discarded and the p e l l e t (P2) was reconstituted in 50 mM T r i s -HCl pH 7.4 (7.5 ml/original brain). b) Preparation of Radio-iodinated DADLE  1 2 5 I - l a b e l l e d D-ala 2,D-leu 5-enkephalin (DADLE) was chosen as the radio-labelled tracer since DADLE i s an enkephalin analogue which i s not readily degraded by proteo l y t i c enzymes which may be present in the rat brain homogenate preparations. The iodination procedure used was a modification of that described by M i l l e r et a l . (1978). Four to ten micrograms of DADLE (Peninsula laboratories Inc) were dissolved in lOjil of d i s t i l l e d water to which was added 1 mCi 12 5 Na I (Amersham) and 10 jul of chloramine-T (0.5 mg/ml) . The oxidative reaction was allowed to take place for 20 seconds before being terminated by the addition of lOjil of sodium metabisulphite (1 mg/ml). 500 jul of 50 mM Tris-HCl pH 7.4 containing 0.8% BSA (Sigma, Fraction V) eluting buffer was added to the reaction mixture before application to a Sephadex G-10 (Pharmacia) column (1 x 17 cm) i n order to remove the free, unreacted iodine. The eluting buffer was applied to the 41 column to achieve a flow rate of 0.5 ml/min. Two minute fractions were collected and 10 pi aliquots of each were counted for 0.1 min using a gamma-spectrometer (Searle 1285). The elution p r o f i l e of a t y p i c a l iodination mixture i s given in Figure 6. The f i r s t peak of r a d i o a c t i v i t y (A) consists of 125 I-labelled DADLE while the second peak (B) i s composed of •I O C i o r free, unreacted I. The peak I-DADLE f r a c t i o n and the one following on the descending shoulder were pooled, and aliquots were stored at -20°C. A fresh aliquot was used for each set of assays. The s p e c i f i c a c t i v i t y of th i s l a b e l was calculated by means of tracer d i l u t i o n curves to be 1.83 x 10 mCi/mg. c) Standards Frozen 100 pi aliquots of DADLE (2 pg/ml) were dissolved i n 900 pi of 50 mM Tris-HCl pH 7.4 assay buffer from which s e r i a l d i l u t i o n s were made to obtain the following standard concentrations (ng/ml): 100, 50, 25, 12.5, 6.25, 3.1, 1.56, 0.78, and 0.39. d) Assay Protocol The radioreceptor assays were conducted i n 12x75 mm test tubes. 300 pi of brain homogenate were incubated with 100 jj l of standard DADLE or 100 pi of sample in the presence of 100 pi of I-DADLE (30,000 cpm) . Incubation of the assay mixture was carried out at room temperature and was terminated after 90 min with the addition of 2 ml of ice cold T r i s buffer. The assay was immediately centrifuged at 1800g (Damon/IEC DPR 6000) for 30 minutes at 4 °C. The supernatant was decanted and 125 the p e l l e t containing the membrane-bound I-DADLE was counted for 2 minutes. The binding was calculated as a percentage 42 Fraction Number FIGURE 6: PROFILE OF DADLE IODINATION MIXTURE ELUTED FROM SEPHADEX G-10. Gel f i l t r a t i o n chromatographic separation of iodinated DADLE on a Sephadex G-10 column (1 x 17 cm). Peak A corresponds to 1 2 5 I - l a b e l l e d DADLE and Peak B represents the 125 elution of unreacted I. of the t o t a l counts placed i n each tube and binding i n the presence of 20 ng DADLE was considered as non-specific. e) The Use of Crude Brain Homoqenates (PI) Compared  to Mitochondrial-Synaptosomal Preparations (P2) Two standard curves, one using the crude brain membrane preparation and the second with the "purer" mitochondrial-synaptosomal preparation, were prepared under id e n t i c a l conditions. As shown in Figure 7, the curves were p a r a l l e l , and both preparations displayed a similar s p e c i f i c binding (maximum binding minus the nonspecific binding). Although the absolute binding to the synaptosomal preparation was higher, assay s e n s i t i v i t y was lower than that obtained using the crude membrane f r a c t i o n . The I(-"50 (DADLE 125 concentration required to i n h i b i t the binding of I-DADLE to brain membranes by 50%) was 5 ng/ml for the crude membrane preparation and 10 ng/ml for the synaptosomal preparation. The reason for t h i s difference i s unknown, but the increased 125 binding and the decreased displacement suggest that the I-DADLE may have a greater a f f i n i t y for the opioid binding s i t e s in the synaptosomal preparation than those in the crude homogenate. In view of these findings and the greater ease i n preparation, the crude membrane homogenate was used i n a l l assays. f) Displacement Studies The a b i l i t y of several opioid peptides to 125 displace the binding of I-DADLE to crude rat brain homogenates was examined. Increasing concentrations of met-enk, leu-enk, met-enk-arg-gly-leu, met-enk-arg-phe, dynorphin 44 ng Dadle 0.037 0.15 0.62 2.5 10 50 . I 1—I 1 1 1 1 1 1 1 1 1 FIGURE 7: DISPLACEMENT CURVES COMPARING x z : >I-DADLE BINDING TO CRUDE RAT BRAIN MEMBRANE PREPARATIONS (PI) WITH BINDING TO SYNAPTOSOMAL-MITOCHONDRIAL PREPARATIONS (P2). The "purer" P2 f r a c t i o n demonstrates a greater percent binding, but the s p e c i f i c binding for both are si m i l a r . Each point i s a mean of t r i p l i c a t e values. 45 " I O C 1-8, and dynorphin 1-13 a l l inhibited binding of I-labelled DADLE. However, each peptide displayed d i f f e r e n t a f f i n i t i e s as was demonstrated by their respective I C 5 Q values (Table 1) . -9 Compared to the 1.3 x 10 M concentration required to i n h i b i t 125 the binding of I-DADLE to half maximum, the other opioids required a greater concentration. Nevertheless, since these d i f f e r e n t opioid peptides were able to compete with the binding 125 of I-DADLE to varying degrees, t h i s assay system could be used to give an estimate of opioid peptide a c t i v i t y . The values obtained for opioid a c t i v i t y using t h i s RRA were thus expressed as DADLE equivalents by reference to a standard DADLE curve prepared with each assay. C. RADIOIMMUNOASSAY (RIA) 1. Rationale Radioimmunoassay, a competitive binding assay f i r s t devised by Berson and Yalow (1960), i s based on the binding of a ligand to s p e c i f i c antibodies. The RIA can be extremely sensitive, capable of measuring minute l e v e l s of peptide hormones. However, the antiserum, l a b e l l e d peptide and assay conditions must be c a r e f u l l y selected in order to maximize the s e n s i t i v i t y and s p e c i f i c i t y of the assay. 2. Leu-Enkephalin RIA a) Antiserum The leu-enkephalin rabbit antiserum (RAS 8601) used in this RIA was purchased from Peninsula Laboratores Inc., C a l i f o r n i a (lot no. 006145). In agreement with data reported by Peninsula, this antiserum cross reacted s l i g h t l y 46 I C 5 0 (M) DADLE 1.3 X I O " 9 Met-enk 1.0 X I O - 8 D y n o r p h i n 1-13 2.2 X 10~ 8 M e t - e n k - a r g - p h e 3.0 X I O " 8 Leu-enk 4.2 X I O - 8 M e t - e n k - a r g - g l y - l e u 5.0 X I O " 8 D y n o r p h i n 1-8 3.3 X I O " 7 TABLE I : RELATIVE POTENCIES OF SOME OPIOID PEPTIDES IN DISPLACING 1 2 5 I - D A D L E BINDING TO RAT BRAIN HOMOGENATES IN THE RRA. The I C ^ g j v a l u e s r e p r e s e n t the c o n c e n t r a t i o n o f u n l a b e l l e d p e p t i d e r e q u i r e d to i n h i b i t the s p e c i f i c b i n d i n g of 125I-DADLE by 50 p e r c e n t . 47 with met-enk and n e g l i g i b l y with peptides of the dynorphin family: % C r o s s - r e a c t i v i t y Leu-enkephalin 100.0 Met-enkephalin 2.5 Dynorphin A(1-17) 0.001 Dynorphin A(1-8) 0.01 Alpha-neo-endorphin 0.001 Dynorphin A(1-6) 0.02 Dynorphin A(l-7) 0.04 Dynorphin A(1-13) 0.01 The l y o p h i l i z e d antiserum mixture was reconstituted with 50 ml of 0.1% Triton X-100 in d i s t i l l e d water and was divided into 10 ml aliquots which were stored at -20°C u n t i l used. b) Iodination of leu-enk 125 I-leu-enk was prepared according to the method of M i l l e r et a l (1978) for DADLE. Four to eight micrograms of leu-enk (Peninsula), dissolved in 10 p i of 0.1 M acetic acid, were added to 25 yul of 0.25 M phosphate buffer pH 7.5. 1 mCi 125 of Na I (Amersham) and 10 p i of chloramine-T (2 mg/ml) were added and allowed to stand for 20 sec before 10 pi of sodium metabisulphite (5 mg/ml) were added to terminate the reaction. The gel f i l t r a t i o n chromatographic procedure for p u r i f i c a t i o n of 1 2 5I-DADLE described by M i l l e r et a l (1978) was found to 125 give poor results when applied to the separation of the I-leu-enk iodination mixture. Therefore, a separation method was developed which involved the use of disposable o c t a d e c y l - s i l y l -s i l i c a reverse phase cartridges (Sep-Pak C-18, Waters Associates, Inc.) These Sep-Pak cartridges which contain approximately 0.8 cm3 Q f C-18 reverse phase packing have been 48 designed such that the mobile phase can be applied with a syringe. They have been described as being useful for the rapid separation of enkephalin and endorphin (Gay and Ahti 1981). The cartridges were prepared by f i r s t flushing with 10 ml of a c e t o n i t r i l e containing 0.1% t r i f l u r o a c e t i c acid (TFA) followed by a 10 ml wash with deionized water (dE^O) , containing 0.1% TFA. The cartridges were then dried with 10 ml of a i r . The iodination mixture was taken up i n 500 ul of dB^O plus 0.1% TFA, applied to the cartridge and flushed with f i v e separate 1 ml washes with d H 2 ° t o elute free iodine. Subsequently, f i v e 1 ml washes with each of 10%, 20%, 30%, and 40% a c e t o n i t r i l e were coll e c t e d from which 10 pi samples were 12 5 counted for r a d i o a c t i v i t y . The I-leu-enk, which eluted with the second 30% wash (Figure 8) , was pipetted into 20 ul amounts and frozen u n t i l used. The s p e c i f i c a c t i v i t y of the 125 I-leu-enk was calculated by means of l a b e l d i l u t i o n curves to be 618 mCi/mg. c) Standard Curves Frozen 100 jul aliquots of 96 nM leu-enk were diluted to give 1 ml of 9.6 nM which was s e r i a l l y d i luted to give concentrations (nM) of: 4.8, 2.4, 1.2, 0.6, 0.3, 0.15, 0.075 and 0.0375. A sample standard curve i s shown i n Figure 9. The I C 5 Q f calculated as the concentration of unlabelled leu-enk required to i n h i b i t the binding to 50 percent, was calculated to be 200 pM (34 pg/tube); the lowest detectable l e v e l was 4.3 pg/tube. The inter-assay v a r i a b i l i t y was determined from a series of 10 assays as an average deviation. 49 O 3.6 r E 2.8 -Q . O to O 2.0 ^ 1-2 > o o o TD O or 0.8 0.0 Acetonitrile % 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 1 2 3 4 5 0 10 20 30 Fraction Number n — 2 3 4 5 40 FIGURE 8: ELUTION PROFILE OF A LEU-ENK IODINATION MIXTURE FROM SEP-PAK CARTRIDGES. Five, sequential, 1 ml washes were conducted for each a c e t o n i t r i l e concentration. The elution of radioactive material with 0% a c e t o n i t r i l e represents unreacted 1 2 5 I . The r a d i o a c t i v i t y which eluted with 30% 125 a c e t o n i t r i l e corresponds to I-labelled leu-enk. 50 Leu Enk Standards pM 37.5 150 600 2400 9600 0 J FIGURE 9: A SAMPLE STANDARD CURVE FOR THE LEU-ENK RIA. Each point represents the mean of t r i p l i c a t e s . 51 For concentrations of 150 pM and 2.4 nM, there occurred an 8% and 17% var i a t i o n , respectively. d) Assay Protocol The RIA procedure used was a modified version of that recommended by Peninsula Laboratories for use with their antiserum. The assay buffer consisted of 0.1 M sodium phosphate pH 7.4, 0.1% BSA (Sigma, RIA grade) and 0.1% Tri t o n X-100. The BSA and Tri t o n were added to the phosphate buffer just prior to each assay. One hundred m i c r o l i t r e s of standard 125 or unknown, 100 yul of rehydrated antiserum and 100 jul of I-leu-enk (approximately 10,000 cpm) were added to 12 x 75 mm culture tubes which were incubated for 48 hours at 4 °C. The 125 bound and free I-leu-enk were separated by means of adsorption of free peptide onto dextran coated charcoal rather than by the double antibody procedure suggested by Peninsula. The dextran (molecular weight 60,000 to 90,000) i s presumed to block the large pores i n the charcoal thereby preventing the adsorption of the antibody complex. Charcoal extracted plasma (1 ml) was added to a continuously s t i r r e d mixture of 3.5 g charcoal (Fisher, carbon decolourizing neutral Norit) and 0.2 g dextran T-70 (Pharmacia) in 100 ml of 0.1 M phosphate buffer. The plasma proteins also serve to coat the charcoal, and in addition, upon centrifugation, the increased protein concentration f a c i l i t a t e s the separation of the charcoal p e l l e t from the supernatant. The charcoal suspension was prepared fresh each time and was allowed to s t i r for at least 90 min at 4 °C before 1 ml aliquots were dispensed into each test tube. The tubes were vortexed and centrifuged at 1800 g for 30 52 minutes (Damon/IEC). The supernatants were decanted and the pe l l e t s allowed to dry with the tubes inverted before the ra d i o a c t i v i t y was counted for two min on a Searle 1285 gamma-spectrometer. The binding was calculated as a percentage of the t o t a l number of counts placed into each tube. The non-125 s p e c i f i c binding (NSB), determined as the amount of leu-enk bound in the absence of antiserum, was obtained for the assay buffer and for assay samples. 3. Dynorphin RIA a) Dynorphin antiserum The dynorphin antiserum (20004 Dynorphin 1-13 rabbit antiserum, Lot No 002448) was purchased from Peninsula Laboratories and was reconstituted and stored as for the leu-enk antiserum (section C 2 [a]). As reported by Peninsula, this antiserum, although raised against dynorphin A (l - 1 3 ) , cross reacted completely with dynorphin A(1-17): % Cross-Reactivity Dynorphin 1-13 100 Dynorphin (porcine) 100 Leu-enkephalin 0 Beta-human-endorphin 0 The binding of the shorter fragment, dynorphin 1-8, to thi s antiserum was not tested by Peninsula. Therefore, a standard curve using dynorphin 1-8 was performed (Figure 10). The 100 percent c r o s s - r e a c t i v i t y with dynorphin 1-17 was reproduced, and the dynorphin 1-8 was found to exhibit a much poorer a f f i n i t y for the antiserum. The i n h i b i t i o n of binding obtained with 10, 20 and 40 nM dynorphin 1-8 was comparable to that 53 FIGURE 10: COMPARISON OF DIFFERENT FORMS OF DYNORPHIN AS STANDARDS FOR RIA. To the l e f t i s given a t y p i c a l standard curve using dynorphin 1-13 as the displacing ligand. In the right panel, dynorphin 1-17 i s shown to cross-react completely with the antiserum; whereas, dynorphin 1-8 exhibits a much poorer a f f i n i t y . The percentage of dynorphin 1-8 cross-r e a c t i v i t y (6.25%) was determined from the equivalent dynorphin 1-13 values which correspond to the i n h i b i t i o n of binding obtained with 40, 20, and 10 nM dynrophin 1-8. A l l points are means of t r i p l i c a t e s . 5'4 obtained with 0.625, 1.25 and 2.5 nM dynorphin 1-13 respectively, indicating a 6.25% c r o s s - r e a c t i v i t y with this dynorphin antiserum. b) Iodination of Dynorphin 1-13 A modification of the procedure outlined by Ghazarossian et a l . , (1980) was used. Five micrograms of dynorphin 1-13 was dissolved in 25 jul 0.1 M phosphate buffer pH 7.5 to which 1.5 mCi Na I (Amersham) and 20 ul chloramine-T (0.5 mg/ml) were introduced. After 20 seconds had elapsed, 100 p.1 of sodium metabisulphite (1 mg/ml) and 200 p.1 of 0.25 M acetic acid containing 0.1% BSA (Sigma, Fraction V) were added to terminate the reaction. The reaction mixture was applied to a Sephadex G-15 (Pharmacia) column (1 x 17 cm) which had been equilibrated with 0.25 M acetic acid plus 0.1% BSA. The flow rate was 0.5 ml/min and 2 minute fractions were co l l e c t e d . When 10 j i l aliquots were counted for r a d i o a c t i v i t y , the p r o f i l e 125 given i n Figure 11 was obtained, I-dynorphm 1-13 eluting in 125 peak A and the free I in peak B. The peak fr a c t i o n plus the one following on the descending limb were pooled, and aliquots of 15 ul were stored at -20 °C. c) Dynorphin Standards One hundred m i c r o l i t r e s of frozen 400 nM aliquots of dynorphin 1-13 (Peninsula) were diluted to 40 nM with assay buffer. S e r i a l d i l u t i o n s were then made to obtain concentrations of 20, 10, 5, 2.5, 1.25, 0.62, 0.31 and 0.15 nM. A sample standard curve i s given in Figure 10. The I C 5 n was calculated as 667 pM (320 pg/tube); the lower detection l i m i t of this assay was 24 pg/tube. Inter-assay v a r i a b i l i t y was .55 Fraction Number FIGURE 11: PROFILE OF A DYNORPHIN 1-13 IODINATION MIXTURE ELUTED FROM SEPHADEX G-15. Gel f i l t r a t i o n chromatographic separation of iodinated dynorphin 1-13. Peak A corresponds to 1 2 5 I - l a b e l l e d dynrophin 1-13 and Peak B to unreacted 1 2 5 I . determined from a set of 10 assays as being 7% and 16% for concentrations of 0.625 and 10 nM, respectively, d) Assay Protocol The procedure recommended by Peninsula for use with this antiserum was followed. The assay buffer, conditions, separation procedure and calculations were as described for the leu-enk RIA (section C 2 [d]). 5. Somatostatin RIA a) Somatostatin Antiserum The antiserum Go 26 3.2 used in this RIA was raised in rabbits against c y c l i c somatostatin conjugated to bovine serum albumin (Mcintosh et a l . , 1978). This antiserum did not cross-react with LH-RH (l e u t i n i z i n g hormone releasing hormone), TSH (thyrotropin), i n s u l i n , p r o i n s u l i n , secretin, glucagon, CCK (cholecystokinin), m o t i l i n or gastrin. Aliquots of 250 p i l y o p h i l i z e d antiserum were diluted with 25 ml of d i s t i l l e d water and aliquoted into 1 ml portions which were stored at -20 °C. For use in the assay, each aliquot was further diluted to 30 ml with the assay buffer in order to give a f i n a l antibody t i t r e of 1:30,000. b) Iodination of Somatostatin Five micrograms of synthetic tyr-1 somatostatin (Serono) were dissolved in 10 p i of d i s t i l l e d water. To this was added 10 p i of 0.5 M sodium phosphate buffer pH 7.5, 1 mCi N a x " i (Amersham) and 10 p i of chloramine-T (2 mg/ml) in phosphate buffer. The mixture was vortexed and after 30 seconds had elapsed, 10 p i of sodium metabisulphite in phosphate buffer was added to terminate the reaction. To 57 125 separate the free I from the la b e l l e d peptide, 1 ml charcoal extracted human plasma and 20 mg of microfine S i l i c a (QUSO G32; Philadelphia Quartz Co.) were added to the reaction mixture which was then centrifuged. The supernatant was discarded and the resulting p e l l e t was washed twice with 1 ml 12 5 d i s t i l l e d water to remove unreacted I. The la b e l l e d somatostatin was eluted with 1 ml acetic acid/ acetone/ water (0.1: 3.9: 4.0 v:v:v) and diluted to 5 x 10 5 cpm/10 pi with 0.1 M acetic acid containing 0.5% BSA (Sigma, RIA grade). Aliquots of 100 jul were l y o p h i l i z e d and stored at -20 °C. On 125 the day of assay the l y o p h i l i z e d I-somatostatin was further p u r i f i e d by ion-exchange chromatography on a CM-cellulose 125 (Whatman CM 52) column (0.7 x 8 cm). The I-somatostatin was dissolved in 0.002 M ammonium acetate buffer pH 4.6 and applied to the column which had been equilibrated in the same buffer. After the column was washed at a rate of 1 ml/min with 30 ml of 12 5 this buffer, the I-somatostatin was eluted with 0.2 M ammonium acetate pH 4.6. Two minute fractions were collected and the r a d i o a c t i v i t y in 100 pi aliquots were counted. The peak fr a c t i o n and those following on the descending limb were pooled, neutralized with 2 M sodium hydroxide and diluted with assay buffer to obtain 3000-3500 cpm/100 jul. c) Assay Buffer The diluent buffer consisted of 50 mM sodium bar b i t a l pH 7.4 with the addition of 0.01% merthiolate, 500 KlU/ml aprotinin (Trasylol) and 1% charcoal-extracted human plasma. t 58 d) Somatostatin Standards Ten microgram aliquots of l y o p h i l i z e d somatostatin (synthetic c y c l i c , Kabi) were s e r i a l l y diluted to obtain concentrations of 250, 125, 62.5, 31.3, 15.6, 7.8 and 3.9 pg/ml. e) Assay Protocol Qne hundred m i c r o l i t r e s of standard or l o t : -i sample, 100 p i antiserum, 100 p i I-tyr somatostatin (3000-3500 cpm) and 100 ^pl diluent buffer were allowed to incubate for 72 hours at 4 °C. Bound and free I-somatostatin were separated by means of dextran-coated charcoal. One ml aliquots of a mixture of 1.25% activated charcoal (Fisher), 0.25% dextran T-70 (Pharmacia) and 1% charcoal extracted human plasma in 0.05 M phosphate buffer pH 7.5 were added to each tube. Following vortexing, the tubes were l e f t at 4*C for 15 - 20 min before being centrifuged at 1800g for 30 min. The supernatant was decanted and the p e l l e t counted for 3 min. Non-specific binding for the buffer and for randomly pooled samples was determined for each assay. The minimum detection l e v e l was 0.4 pg/tube, and the inter-assay v a r i a t i o n was calculated from 10 assays as 16% and 12% for concentrations of 15 and 125 pg/ml, respectively (Mcintosh et a l . , 1978). 6. Gastrin RIA. a) Diluent buffer Buffer, composed of 0.02 M sodium b a r b i t a l pH 8.4 and 0.5% BSA (Sigma, RIA grade), was used in the d i l u t i o n of antiserum, standards and radiolabelled g a s t r i n . 59 b) Antiserum The gastrin antiserum TR5 was obtained from Dr. Norman Track (Department of Surgery and Pathology, McMaster University Medical Centre, Hamilton, Ontario) and was used at a f i n a l d i l u t i o n of 1:80,000. This antiserum cross reacts equally with gastrin-17 and gastrin-34. c) Gastrin Iodination The iodination procedure was a modification, of that described by S t a d i l and Rehfeld (1972). Five micrograms of synthetic human gastrin I (Research Plus) were dissolved i n 10 j i l of 0.4 M phosphate buffer pH 7.4. To this •jot: were added 0.2 mCi Na I (Amersham) and 10 pi of chloramine-T in 0.04 M phosphate buffer pH 7.4 (0.5 mg/ml). The mixture was allowed to incubate for 60 sec at room temperature before 10 pi of sodium metabisulphite in 0.04 M phosphate buffer (0.5 mg/ml) and a few drops of 0.05 M imidazole buffer pH 7.5 were added to I O C stop the reaction. The p u r i f i c a t i o n of I-gastrin was carried out by ion-exchange chromatography using DEAE-Sephadex A25 (Pharmacia) (Brown et a l 1976). The mixture was applied to the column (1 x 12 cm) which had been equilibrated with 0.05 M 125 imidazole buffer pH 7.5. El u t i o n of the I-gastrin was accomplished using a lin e a r gradient from 0 to 1 M NaCl in the imidazole buffer. The flow rate was maintained at 2 ml/min by means of a p e r i s t a l t i c pump (Pharmacia, P-3) and 1 min fractions were c o l l e c t e d . The second and/or t h i r d f r a c t i o n following the peak of r a d i o a c t i v i t y were retained for use in the assay. 60 d) Gastrin Standards Synthetic human gastrin (SHG-I) (Research Plus) was used to prepare standards. 1 ml aliquots of 400, 200, 100, 50, 25, 12 and 6 pg/ml were prepared in the assay buffer and stored at -20 °C. e) Assay Protocol One hundred m i c r o l i t r e s of standard or sample, I O C 100 p i antiserum, 100 p i I-gastrin (2000 cpm/100 pi) and 700 pi diluent buffer were incubated at 4 °C for 48 hours. Separation was performed by the addition of 200 p i of a charcoal s l u r r y containing 12.5% activated charcoal (Fisher) 2.5% dextran T-70 (Pharmacia) and 5% charcoal extracted human plasma in 0.04 M sodium phosphate buffer pH 6.5. The assays were centrifuged at 1800 g for 30 minutes, decanted, and the pe l l e t s counted for 3 minutes. NSB and calculations were performed in the same manner as for the previously described RIAs. The lowest l i m i t of d e t e c t a b i l i t y was 0.6 pg/tube. Inter-assay variations based on a series of 10 assay standard curves were found to be 4% and 11% for concentrations of 125 and 200 pg/ml, respectively. I I . CHROMATOGRAPHIC METHODS The separation of s p e c i f i c peptides from a complex mixture of b i o l o g i c a l material can be achieved by employing chromatographic methods which s e l e c t i v e l y separate on the basis of differences in molecular properties. Many commercially available matrices have been developed to separate proteins and peptides on the basis of si z e , charge, p o l a r i t y ( s o l u b i l i t y ) or i 61 bi o l o g i c a l a c t i v i t y . Of the many d i f f e r e n t chromatographic techniques available, including paper, gas and column chromatography, l i q u i d chromatographic methods using columns containing chromatographic material have been most extensively applied in peptide research. A. GEL FILTRATION CHROMATOGRAPHY Gel f i l t r a t i o n (permeation) separates proteins according to molecular s i z e . The column i s f i l l e d with a matrix which consists of a material with small pores. As the solute i s passed down thi s gel matrix, movement of the molecules i s dependent on their a b i l i t y to pass through the pores which contain a "stationary phase." Large molecules which cannot enter into the gel pores move rapidly down the column with the l i q u i d phase, therefore eluting f i r s t . The passage of the smaller molecules which can enter the pores in the gel i s impeded by the stationary phase and these elute according to their s i z e . Since the opioid peptides are small molecules, gels with a low molecular size fractionation range were used: Sephadex G-10 to 700 daltons Sephadex G-15 to 1,500 daltons Sephadex G-50 1,500-30,000 daltons B. ADSORPTION CHROMATOGRAPHY The a b i l i t y of the enkephalins to adsorb to polystyrene and their hydrophobic nature were taken into consideration when the neutral polystyrene r e s i n , Amberlite XAD-2 (BDH Chemicals), was selected by Smith et a l . , (1976) as a simple method of i s o l a t i n g the enkephalins from tissue homogenates. Before use, 62 the r e s i n was p u r i f i e d by washing t h r e e t i m e s w i t h methanol (10 ml/g) f o l l o w e d by s e v e r a l r i n s e s w i t h d i s t i l l e d water t o remove a l l t r a c e s o f the a l c o h o l . The r e s i n was poured i n t o columns (0.7 x 9 cm). The samples were adsorbed onto the column and washed w i t h 15 ml 0.1 M HC1 and 30 ml d i s t i l l e d water t o remove h y d r o p h i l i c compounds. The o p i o i d p e p t i d e s were e l u t e d w i t h 20 ml o f 90% m ethanol. C. REVERSED PHASE HIGH PERFORMANCE LIQUID CHROMATOGRAPHY 1) R a t i o n a l e Reversed phase h i g h performance l i q u i d chromatography s e p a r a t e s p e p t i d e s a c c o r d i n g t o t h e i r h y d r o p h o b i c i t y . I n r e v e r s e d phase chromatography, i n c o n t r a s t t o c o n v e n t i o n a l c h r o m a t o g r a p h i c methods, the s t a t i o n a r y m a t r i x c o n s i s t i n g o f s i l i c a p a r t i c l e s c o a t e d w i t h c h e m i c a l l y bonded hydrocarbon c h a i n s r e p r e s e n t s the h y d r o p h o b i c phase w h i l e the aqueous, o r g a n i c m i x t u r e r e p r e s e n t s the h y d r o p h i l i c phase. As the s o l u t e passes down the column, depending on the h y d r o p h o b i c i t y of the e l u e n t , the sample w i l l be r e t a i n e d on the s t a t i o n a r y phase. The h y d r o p h i l i c compounds w i l l pass through the column f i r s t s i n c e the s t a t i o n a r y phase i s i n i t i a l l y more hy d r o p h o b i c than the s o l v e n t . The unique f e a t u r e o f HPLC i s t h a t the sample can be passed through an e x t r e m e l y t i g h t l y packed m a t r i x by means of v e r y h i g h p r e s s u r e s , r e s u l t i n g i n h i g h r e s o l u t i o n s e p a r a t i o n s which cannot be a c h i e v e d u s i n g o t h e r methods. P e p t i d e s d i f f e r i n g by one amino a c i d such as met and l e u -e n k e p h a l i n can be r e a d i l y r e s o l v e d . 63 2) HPLC A p p a r a t u s A Waters A s s o c i a t e s HPLC system was employed. The column m a t r i x (jj-Bondapak C-18) c o n s i s t e d o f hydrocarbons 18 carbons i n l e n g t h a t t a c h e d t o a s i l i c a base. B e f o r e use, the s o l v e n t s , HPLC grade water and a c e t o n i t r i l e , were f u r t h e r p u r i f i e d through a Waters m i l l i p o r e f i l t r a t i o n system; 0.1% TFA was added t o each. The s o l v e n t s were housed i n s e a l e d b o t t l e s and were degassed and m a i n t a i n e d under a r e g u l a t e d f l o w o f h e l i u m gas. A Waters 660 model s o l v e n t programmer c o n t r o l l e d the speeds o f two s e p a r a t e pumps so as t o m a i n t a i n a c o n s t a n t f l o w r a t e w h i l e the s o l v e n t c o n c e n t r a t i o n s were v a r i e d . I n t h i s manner, s e l e c t e d g r a d i e n t s o f a c e t o n i t r i l e c o n c e n t r a t i o n s c o u l d be o b t a i n e d over d i f f e r i n g time spans. The column e f f l u e n t was passed through a model 450 v a r i a b l e w a v e l e n t h d e t e c t o r s e t a t 225 nm t o m o n i t o r p r o t e i n o u t f l o w and the absorbances were r e c o r d e d on a H e w l e t t - P a c k a r d 3380A i n t e g r a t o r - c h a r t r e c o r d e r (see F i g u r e 1 2 ) . 3. HPLC P r o c e d u r e S t a n d a r d o p i o i d p e p t i d e s ( P e n i n s u l a L a b o r a t o r i e s , Inc) were d i s s o l v e d i n HPLC water c o n t a i n i n g 0.1% TFA t o g i v e a c o n c e n t r a t i o n o f 5 pg/lOpl. S i n g l e p e p t i d e i n j e c t i o n s o f 5 pi, or a l a r g e r volume o f a m i x t u r e o f s e v e r a l p e p t i d e s were i n j e c t e d i n t o the m i x i n g chamber such t h a t 2.5 pg of each p e p t i d e was a p p l i e d o n t o the column. I n o r d e r t o determine s u i t a b l e c o n d i t i o n s whereby the o p i o i d p e p t i d e s c o u l d be s e p a r a t e d , s e v e r a l runs were made, m a n i p u l a t i n g the i n i t i a l or f i n a l a c e t o n i t r i l e c o n c e n t r a t i o n s or the time span o f the g r a d i e n t . An a p p r o x i m a t i o n o f the a c e t o n i t r i l e c o n c e n t r a t i o n 64 Solvent Programmer Chart Recorder Wavelength Detector Solvent Stabilization System Pump A Pump B —TZZ Solvent Solvent A B A H e I i u m FIGURE 12: SCHEMATIC OUTLINE OF THE HPLC APPARATUS. Solvent A consisted of HPLC grade water, containing 0.1% TFA, and solvent B consisted of HPLC grade a c e t o n i t r i l e , also containing 0.1% TFA. A reversed phase C-18 column was employed. The wavelength detector was set for 205 nm and the chart recorder was set to 0.2 absorbance units f u l l scale (AUFS). r e q u i r e d t o e l u t e the o p i o i d s t a n d a r d s was determined by f i r s t r u n n i n g a l i n e a r g r a d i e n t from 0% - 70% a c e t o n i t r i l e over 30 min. From t h i s r e s u l t , a r e l a t i v e l y narrow a c e t o n i t r i l e c o n c e n t r a t i o n range c o u l d be e s t a b l i s h e d . I n subsequent runs the i n i t i a l or f i n a l c o n c e n t r a t i o n s were a l t e r e d i n o r d e r t o a c h i e v e complete r e s o l u t i o n o f a l l o p i o i d p e p t i d e s t a n d a r d s . The e x a c t parameters used a re d e s c r i b e d i n the " R e s u l t s " s e c t i o n o f Chapter 3. A s e t o f s t a n d a r d s was run p r i o r t o each s e t o f stomach e x t r a c t samples as r e t e n t i o n times were found t o v a r y s l i g h t l y (+ 0.2 minutes) between r u n s . I l l . ISOLATED PERFUSED RAT STOMACH I s o l a t e d organ systems a re o f t e n employed i n the s t u d y of p e p t i d e a c t i o n s . That the e f f e c t o f the p e p t i d e can be examined i n r e l a t i v e i s o l a t i o n i s a p a r t i c u l a r advantage when s t u d y i n g the o p i o i d s s i n c e the r e s u l t s cannot be c o m p l i c a t e d by c e n t r a l e f f e c t s . I n the i s o l a t e d p e r f u s e d r a t stomach p r e p a r a t i o n , the g a s t r i c c i r c u l a t i o n i s c o m p l e t e l y i s o l a t e d , a l l o w i n g c o n t r o l o f the c o m p o s i t i o n o f the c i r c u l a t i n g p e r f u s a t e and c o l l e c t i o n o f the e f f l u e n t f o r a n a l y s i s . A. S u r g i c a l P r o c e d u r e Male W i s t a r r a t s (250-350 g) were housed i n a l i g h t c o n t r o l l e d room and g i v e n f r e e a c c e s s t o l a b o r a t o r y r a t chow and w a t e r . The r a t s were f a s t e d o v e r n i g h t (16-18 h) and were a n a e s t h e t i z e d w i t h an i n t r a p e r i t o n e a l i n j e c t i o n o f sodium p e n t o b a r b i t a l (60 mg/kg). A m i d l i n e i n c i s i o n was made t o expose the v i s c e r a and the abdominal a o r t a was l o c a t e d . The l e f t r e n a l a r t e r y and v e i n , and the s u p e r i o r m e s e n t e r i c a r t e r y 66 were sectioned before preparing the aorta below the area of the coeliac branch for insertion of an a r t e r i a l cannula (PE 160) . The spleen and the head of the pancreas were cut away from the greater curvature of the stomach and the branches of the coeliac artery supplying the l i v e r and esophagus were then sectioned. The duodenum was sectioned to permit the placement of a drainage cannula into the lumen of the stomach through the pylorus. The colon was sectioned at the rectum which allowed the removal of the small and large i n t e s t i n e s , the pancreas and spleen. After the right renal artery and vein were sectioned, the a r t e r i a l cannula was inserted adjacent to the coeliac artery through which a single dose of heparinized saline (Sigma, 800 U) was administered. A second cannula was placed into the portal vein in order to c o l l e c t the venous e f f l u e n t . It has been demonstrated (Mcintosh et a l . , 1981a) that following removal of the pancreas, the small remaining portion attached to the portal vein and b i l e duct gives only 1% of the in s u l i n response of an i n t a c t , isolated pancreas to a stimulus of 300 mg% glucose. Therefore, this remaining pancreas i s assumed to contribute neglegibly to the peptide secretion into the portal e f f l u e n t . B. PREPARATION OF PERFUSATE The perfusing solution consisted of the following: NaCl 120.0 mM KC1 4 .4 mM CaCl 9 2.5 mM MgSO^ 1.2 mM KH PO. 1.5 mM NaHCO^ 25.0 mM Dextrose 4.4 mM Dextran 3.0 percent BSA 0.2 percent 67 , The d e x t r a n (Sigma C h e m i c a l Co) , BSA (Sigma, RIA grade) and d e x t r o s e ( F i s h e r S c i e n t i f i c ) were d i s s o l v e d o v e r n i g h t . i n s a l i n e s o l u t i o n . On the morning o f the e x p e r i m e n t s , a p p r o p r i a t e volumes o f Kre b s ' c o n c e n t r a t e and sodium b i c a r b o n a t e were added t o the s a l i n e m i x t u r e t o g i v e the above f i n a l c o n c e n t r a t i o n s . C. PERFUSION PROCEDURE The p e r f u s a t e , which was c o n t i n u o u s l y gassed w i t h a water vapour s a t u r a t e d m i x t u r e o f 95% oxygen and 5% car b o n d i o x i d e , was pumped through a h e a t i n g chamber t o a bubble t r a p i n which a temperature probe was i n s e r t e d . The temperature o f the p e r f u s a t e e n t e r i n g the stomach was r e g u l a t e d t o 40 °C such t h a t the stomach temperature was m a i n t a i n e d a t 37 *C. The p e r f u s a t e f l o w e d i n t o the g a s t r i c c i r c u l a t i o n t h r ough the a o r t i c c a n n u l a and the venous e f f l u e n t was c o l l e c t e d . The p e r f u s i o n p r e s s u r e was m o n i t o r e d by means o f a 10 v o l t Statham t r a n s d u c e r c o n v e r t e r and was m a i n t a i n e d between 50 and 70 mm Hg. P e p t i d e s and o t h e r c h e m i c a l s were i n t r o d u c e d i n t o the g a s t r i c c i r c u l a t i o n t h r ough a s i d e arm i n f u s i o n e n t e r i n g the a o r t i c c a n n u l a . T h i s was a c c o m p l i s h e d u s i n g a H a r v a r d i n f u s i o n pump s e t t o d e l i v e r a f l o w r a t e o f 0.2 ml/min. F i g u r e 13 i s a sc h e m a t i c r e p r e s e n t a t i o n o f t h e p e r f u s i o n a p p a r a t u s . 68 Apparatus for In Vitro Vascular Perfusion of the Rot Stomach 9 5 % O2 596 Ct>2 Perfusion / Coeliac Axis k FIGURE 13: SCHEMATIC OUTLINE OF THE APPARATUS FOR IN VITRO VASCULAR PERFUSION OF THE RAT STOMACH. M o d i f i e d K r e b s ' b i c a r b o n a t e b u f f e r was p e r f u s e d through the c o e l i a c a r t e r y i n t o the g a s t r i c v a s c u l a t u r e and was c o l l e c t e d from the p o r t a l v e i n . T e s t compounds were i n t r o d u c e d i n t o the g a s t r i c c i r c u l a t i o n v i a a s i d e arm i n f u s i o n e n t e r i n g the a o r t i c c a n n u l a . 69 CHAPTER THREE CHARACTERIZATION OF OPIOID PEPTIDES IN RAT STOMACH EXTRACTS INTRODUCTION Immediately f o l l o w i n g the i d e n t i f i c a t i o n o f e n k e p h a l i n s as endogenous o p i o i d l i g a n d s i n the b r a i n (Hughes e t a l . , 1975) o p i o i d p e p t i d e s were demonstrated i n g a s t r o i n t e s t i n a l t i s s u e s (Smith e t a l . , 1975; E l d e e t a l . , 1976; Hughes e t a l . , 1977; P o l a k e t a l . , 1977). The pronounced e f f e c t s o f o p i a t e drugs on g a s t r o i n t e s t i n a l f u n c t i o n s and the presence o f o p i a t e s p e c i f i c r e c e p t o r s i n the gut had been w e l l documented; t h e r e f o r e , i t was not unexpected t h a t s u b s t a n t i a l amounts o f e n k e p h a l i n s were found i n g a s t r o i n t e s t i n a l t i s s u e s . E a r l y s t u d i e s d e t e c t e d met-and l e u - e n k e p h a l i n i n e x t r a c t s o f the s m a l l i n t e s t i n e o f s e v e r a l s p e c i e s by means o f b i o a s s a y (Smith e t a l . , 1976; Hughes e t a l . , 1977). Met-enk a c t i v i t y was d i s t i n g u i s h e d from t h a t o f le u - e n k by t h i n l a y e r chromatography or by measuring the o p i o i d b i o a c t i v i t y b e f o r e and a f t e r cyanogen bromide t r e a t m e n t , which causes a 92% t o 95% l o s s i n met-enk a c t i v i t y due t o the f o r m a t i o n o f the homoserine analogue. E n k e p h a l i n a c t i v i t y was found t h r o u g h o u t the g a s t r o i n t e s t i n a l t r a c t w i t h the h i g h e s t c o n c e n t r a t i o n s o c c u r r i n g i n the duodenum and g a s t r i c antrum. The o p i o i d s were found t o be a s s o c i a t e d w i t h n e u r o n a l elements as the a c t i v i t y was c o n c e n t r a t e d i n the m y e n t e r i c p l e x u s l a y e r . The met-enk t o leu - e n k r a t i o s were found t o v a r y from r e g i o n t o r e g i o n , but i n a l l cases the met-70 enk c o n c e n t r a t i o n s were a t l e a s t two times g r e a t e r than those o f l e u - e n k . W i t h the a v a i l a b i l i t y o f a n t i s e r a r e c o g n i z i n g the o p i o i d p e p t i d e s e n k e p h a l i n - l i k e i m m u n o r e a c t i v i t y was l o c a l i z e d by ICC i n nerve f i b r e s t h r o u g h o u t the g a s t r o i n t e s t i n a l t r a c t w i t h p a r t i c u l a r l y h i g h c o n c e n t r a t i o n s i n the antrum and upper s m a l l i n t e s t i n e (Elde e t a l . , 1976; P o l a k e t a l . , 1977; Bu'Lock e t a l . , 1983; F u r n e s s e t a l . , 1983). S i m i l a r d i s t r i b u t i o n s were measured by RIA (Smith e t a l . , 1976; Hughes e t a l . , 1977). The d i s t r i b u t i o n o f e n k e p h a l i n - and d y n o r p h i n - c o n t a i n i n g n e u r o n a l systems i n the g a s t r o i n t e s t i n a l t r a c t have been d e s c r i b e d i n the f i r s t c h a p t e r ( s e c t i o n V I I ) . I d e n t i f i c a t i o n o f the v a r i o u s o p i o i d p e p t i d e s by imm u n o l o g i c a l means has l e d t o some c o n f u s i o n as t o the forms i n the g a s t r o i n t e s t i n a l t r a c t s i n c e a l l p o s s e s s a common amino t e r m i n a l e n k e p h a l i n sequence. The p o s s i b i l i t y o f c r o s s -r e a c t i o n betweeen a n t i b o d i e s d i r e c t e d towards the met- or l e u -enk sequence and l o n g e r forms o f o p i o i d p e p t i d e s , hampers the p r e c i s e d e t e r m i n a t i o n o f the o p i o i d forms which e x i s t i n g a s t r o i n t e s t i n a l t i s s u e . F u r t h e r m o r e , s i n c e the p r e c u r s o r s f o r the g a s t r o i n t e s t i n a l o p i o i d p e p t i d e s have not been i d e n t i f i e d , i t i s not known f o r c e r t a i n whether the same o p i o i d f a m i l i e s found i n the b r a i n and a d r e n a l g l a n d e x i s t i n the g u t . The a p p l i c a t i o n o f r e c e n t developments i n HPLC methods f o r the s e p a r a t i o n o f p e p t i d e s , c o u p l e d w i t h b i o l o g i c a l and im m u n o l o g i c a l assay methods may prove u s e f u l i n the i d e n t i f i c a t i o n o f v a r i o u s o p i o i d p e p t i d e s . T h e r e f o r e , i n the f o l l o w i n g s t u d y , r a t stomach e x t r a c t s were examined by s e v e r a l •, 71 c h r o m a t o g r a p h i c methods, i n c l u d i n g HPLC, i n c o n j u n c t i o n w i t h s e v e r a l o p i o i d d e t e c t i o n m e t h o d s — b i o a s s a y , r e c e p t o r b i n d i n g assay and RIA. I n t h i s manner, the t y p e s o f o p i o i d p e p t i d e s p r e s e n t i n the r a t stomach c o u l d be c h a r a c t e r i z e d . METHODS I . TISSUE EXTRACTION Male W i s t a r r a t s , f a s t e d o v e r n i g h t (15 t o 18 h) , were a n a e s t h e t i z e d w i t h sodium p e n t o b a r b i t a l (60 mg/kg). The stomachs were r a p i d l y removed and r i n s e d w i t h s a l i n e . The fundus was s e p a r a t e d from the r e m a i n i n g corpus-antrum t o a l l o w the two r e g i o n s o f the stomach t o be e x t r a c t e d s e p a r a t e l y . The t i s s u e from 8 t o 10 r a t s was p l a c e d i n t o b o i l i n g 2 M a c e t i c a c i d (5 ml/g t i s s u e ) f o r 30 min. The t i s s u e was then c o o l e d and homogenized w i t h a S o r v a l l Omni-Mixer. A f t e r the homogenate was c e n t r i f u g e d under r e f r i g e r a t i o n a t 27,500 g f o r 50 min ( S o r v a l l , RC-5B) the s u p e r n a t a n t was r e t a i n e d and f i l t e r e d under vacuum (Whatman, Q u a l i t a t i v e 1) . The f i l t r a t e was l y o p h i l i z e d and s t o r e d a t -20°C. I I . CHROMATOGRAPHY A. G e l F i l t r a t i o n S i x t y m i l l i g r a m samples o f the l y o p h i l i z e d t i s s u e e x t r a c t s were d i s s o l v e d i n 2 ml o f 0.2 M a c e t i c a c i d w i t h the a d d i t i o n o f one or two drops o f 2 M sodium h y d r o x i d e . T h i s m i x t u r e was a p p l i e d t o a Sephadex G-50 column (0.8 x 53 cm) and e l u t e d w i t h 0.2 M a c e t i c a c i d . The f l o w r a t e was 0.8 ml/min and 2 min f r a c t i o n s were c o l l e c t e d . An i n d i c a t i o n o f the p r o t e i n c o n t e n t was o b t a i n e d by measuring the absorbance o f the column e f f l u e n t s a t 280 nm (Pye Unicam SP8-100 S p e c t r o p h o t o m e t e r ) . One m i l l i g r a m o f s y n t h e t i c met-enk (Serva) was used as the s t a n d a r d . • < 73 B. Adsorption Chromatography Effl u e n t fractions of Sephadex G-50 p u r i f i e d g a s t r i c extract, pooled on the basis of their opioid binding a c t i v i t y , were further p u r i f i e d by adsorption onto Amberlite XAD-2, according to the procedure described in Chapter 2 (section II B) . The methanol eluted f r a c t i o n containing the opioid peptides was placed under a stream of forced a i r in order to f a c i l i t a t e evaporation of the alcohol. The remaining aqueous phase was l y o p h i l i z e d and stored at -20 °C. The l y o p h i l i z e d samples were reconstituted with 1 ml of water when assayed for opioid a c t i v i t y . Complete recovery of 2 jug of standard DADLE (Peninsula Laboratories) was measured by means of RRA. Using s p e c i f i c RIA, recovery of 6 ng of leu-enk (Peninsula) was found to be 88 - 7 % (n=5). C. HPLC Amberlite XAD-2 treated, l y o p h i l i z e d samples which were reconstituted with 1 ml HPLC grade water containing 0.1% TFA were examined by HPLC. Before application onto the column, the samples were centrifuged for 10 minutes (Eppendorf). A 50 - 80 pi sample of the supernatant was used for each run. The outflow was collected for 1.5 min u n t i l approximately 5 min prior to the estimated elution of the opioids at which time fractions at smaller intervals of 0.5 min were co l l e c t e d . A l l traces of a c e t o n i t r i l e were removed from HPLC effluent fractions by placing them under a stream of nitrogen gas. The remaining aqueous phase was l y o p h i l i z e d and reconstituted to 0.5 ml with d i s t i l l e d water pr i o r to assay. 74 I I I . TRYPSIN DIGESTION One m i l l i l i t r e samples of p o o l e d Sephadex G-50 f r a c t i o n s or .Amberlite XAD-2 t r e a t e d Sephadex f r a c t i o n s were n e u t r a l i z e d i f n e c e s s a r y w i t h 2 M sodium h y d r o x i d e . The d i g e s t i o n p r o c e s s was a l l o w e d t o t a k e p l a c e o v e r n i g h t f o r 16 hours a t 37°C i n the presence o f 1 jug T P C K - t r y p s i n (Worthington B i o c h e m i c a l Corp.) which was d i s s o l v e d i n 1 ml o f 50 mM T r i s - H C l b u f f e r pH 8.5 a c c o r d i n g t o the method of Lewis e t a l . , (1980). The t r y p t i c d i g e s t s were l y o p h i l i z e d and r e c o n s t i t u t e d w i t h 1 ml d i s t i l l e d water p r i o r t o a s s a y . As c o n t r o l s , e q u a l volumes of Sephadex G-50 column b u f f e r (0.2 M a c e t i c a c i d ) were t r e a t e d i n an i d e n t i c a l manner and a s s a y e d . IV. ASSAY PROCEDURES The o p i o i d RRA, the l e u - e n k and d y n o r p h i n RIAs and the b i o a s s a y were performed as d e s c r i b e d i n Chapter 2. For the b i o a s s a y employing the g u i n e a p i g i l e u m s t r i p p r e p a r a t i o n , the s t i m u l a t i o n parameters used were 0.3 Hz, 0.5 msec a t supramaximal v o l t a g e . A f t e r a s t e a d y c o n t r a c t i l e response was r e c o r d e d , s t a n d a r d or sample was a d m i n i s t e r e d t o the s t r i p . When the maximum i n h i b i t o r y response was r e a c h e d , the s t r i p s were washed t w i c e w i t h f r e s h , Krebs' s o l u t i o n (37°C) and were a l l o w e d t o r e c o v e r f o r a minimum o f 15 min between s t i m u l a t i o n s . To q u a n t i t a t e o p i o i d b i o a c t i v i t y , a dose-response c u r v e u s i n g s y n t h e t i c met-enk was o b t a i n e d b e f o r e t e s t i n g o f any e x t r a c t samples. F i g u r e 14 i s an example of the 75 s t a n d a r d i n h i b i t o r y r e sponses o b t a i n e d , and F i g u r e 15 i s the c o r r e s p o n d i n g l o g - c o n c e n t r a t i o n response c u r v e . 76 Ix l0- 8 M Ix l0 - 7 M FIGURE 14: CONCENTRATION-DEPENDENT INHIBITION OF ELECTRICALLY-INDUCED CONTRACTIONS OF THE GUINEA PIG ILEUM BY MET-ENK. S t i m u l a t i o n parameters a p p l i e d were 0.3 Hz, 0.5 msec and supramaximal v o l t a g e (100 V) . The response was det e r m i n e d as the d i f f e r e n c e between the peak t e n s i o n s o b s e r v e d p r i o r and d u r i n g maximal met-enk i n h i b i t i o n . The t i s s u e was washed t w i c e w i t h f r e s h Krebs' b u f f e r when the maximum response t o each dose of met-enk was o b t a i n e d . 77 3 . 0 MET-ENK ( 1 0 I 2 . 5 5 .0 J I I -8 M) 10 _L_ 2 0 _l FIGURE 15: CONCENTRATION-RESPONSE CURVE FOR MET-ENK IN THE GUINEA PIG ILEUM BIOASSAY. Each point i s a mean of three values. The response i s given as an absolute decrease in tension. 78 RESULTS I . OPIOID ACTIVITY IN ACID EXTRACTS OF RAT STOMACH RRA was used t o measure o p i o i d c o n t e n t i n crude r a t f u n d i c and c o r p u s - a n t r a l a c e t i c a c i d e x t r a c t s . The c o r p u s -antrum r e g i o n e x t r a c t e x h i b i t e d a much h i g h e r c o n c e n t r a t i o n o f o p i o i d m a t e r i a l (377 pmole/g wet weight) than d i d the f u n d i c r e g i o n which c o n t a i n e d o n l y m i n i m a l amounts (1.7 pmole/g wet w e i g h t ) . A l l subsequent s t u d i e s were t h e r e f o r e undertaken u s i n g e x t r a c t s o f the corp u s - a n t r u m . I I . CHROMATOGRAPHIC SEPARATION In F i g u r e 16A i s shown the p r o t e i n e l u t i o n p r o f i l e o b t a i n e d from Sephadex G-50 g e l f i l t r a t i o n chromatography o f a corpus-antrum e x t r a c t , d e m o n s t r a t i n g one f a i r l y b r o a d , major peak. Peak o p i o i d - l i k e b i n d i n g a c t i v i t y , d e t e c t e d by RRA ( F i g u r e 16C), o c c u r r e d i n f r a c t i o n s 20-23 c o r r e s p o n d i n g w i t h the e l u t i o n volume o f s y n t h e t i c met-enk ( F i g u r e 16B). The d e t e r m i n a t i o n of o p i o i d b i o a c t i v i t y i n the s e Sephadex f r a c t i o n s was c o m p l i c a t e d by the presence o f a s t r o n g c o n t r a c t i l e component which masked the a c t i o n s o f any i n h i b i t o r y s u b s t a n c e s which may have been p r e s e n t i n the samples. F i g u r e 17 i s an example o f such an e x c i t a t o r y r e s p o n s e . Maximal c o n t r a c t i o n s c o u l d not be d e t e c t e d as the s e n s i t i v i t y o f the c h a r t r e c o r d e r was s e t so as t o r e c o r d i n h i b i t o r y r e s p o n s e s . I n an attempt t o remove t h i s c o n t r a c t i l e component, p o o l e d groups o f Sephadex G-50 f r a c t i o n s were f u r t h e r p u r i f i e d by a d s o p t i o n chromatography 79 Protein Elution A -, Standard Met-enk Elu. Fraction Number FIGURE 16: PARTIAL PURIFICATION OF OPIOID PEPTIDES FROM CORPUS/ANTRUM EXTRACTS BY SEPHADEX G-50 GEL FILTRATION CHROMATOGRAPHY. The top panel i l l u s t r a t e s protein elution , determined as the absorbance at 280 nm. The second panel demonstrates the elution of 1 mg synthetic met-enk which corresponds with the elution of opioid binding a c t i v i t y determined by RRA shown in the bottom panel. 80 200p\ Fraction 22 Sephadex G-50 Wash FIGURE 17: EFFECT OF PARITIALLY PURIFIED CORPUS/ANTRUM EXTRACT ON THE ELECTRICALLY-INDUCED CONTRACTIONS OF THE GUINEA PIG ILEAL STRIP. F i e l d stimulation (100V, 0.3 Hz, 0.5 msec) was applied to the i l e a l tissue to induced muscular contractions. Fraction 22 was obtained by Sephadex G-50 gel f i l t r a t i o n of the ga s t r i c extract and had been demonstrated previously by RRA to possess opioid binding a c t i v i t y . 81 on Amberlite XAD-2 columns. It was found that amberlite XAD-2 treatment completely removed substance(s) which had induced i l e a l contractions. Following Amberlite XAD-2 adsorption chromatography, opioid binding a c t i v i t y was found to be retained (Figure 18) , and corresponding opioid b i o a c t i v i t y could also be detected (Figure 19) as demonstrated by an in h i b i t i o n of the e l e c t r i c a l l y induced contractions of the guinea pig ileum s t r i p . Responses were blocked by the — fi administration of 10 M naloxone. In order to obtain s u f f i c i e n t material to allow opioid detection following separation by HPLC, i t was necessary to pool opioid containing fractions from 4 separate Sephadex G-50 runs (Figure 20) . The opioid content of the Sephadex G-50 column effluents was measured by RRA and those with the greatest a c t i v i t y were selected and pooled accordingly as A-D. These Sephadex pools were further p u r i f i e d by adsorption onto Amberlite XAD-2 resin before examination by reversed phase HPLC. A set of standard opioid peptides was always run prior to application of each sample in order to assess proper resolution. In Figure 21 the standard elution p r o f i l e s which correspond to the d i f f e r e n t separation conditions are presented. When a sample of Amberlite XAD-2 treated .pool "C" was applied to the HPLC column using a gradient of 17 to 28% a c e t o n i t r i l e over 25 min, four d i s t i n c t major peaks of opioid binding a c t i v i t y (measured by RRA) were revealed (Figure 22) . These peaks in opioid a c t i v i t y were found to elute with 82 Protein Elution <D o c o i_ O co < E c O CO CM .6-0 . 8 -0.0 Opiate-Like Binding Activity > < cn c c CO ^ c UJ CD I I Q I o < I Q £ O g O o a. 4 . 0 2.0 0.0 Post-Amberlite X A D - 2 6.0-t Opiate-Like Binding Activity 4 . 0 -2 . 0 -0.0 0 12 20 28 36 4 4 Fraction Number FIGURE 18: OPIOID BINDING ACTIVITY IN SEPHADEX G-50 PURIFIED CORPUS/ANTRUM EXTRACTS FOLLOWING AMBERLITE XAD-2 TREATMENT. The o p i o i d - l i k e b i n d i n g a c t i v i t y determined by RRA (B) was r e t a i n e d f o l l o w i n g e x t r a c t i o n on A m b e r l i t e XAD-2 columns ( C ) . 83 B. Opiate-Like Binding Activ. c r U J UJ _1 Q < Q c n c Synthetic Met-Enk • Sephadex G-50 c a> % E C ' 8-1 4-Post Amberlite XAD-2 Opiate-Like Bioactivity ~l 8 j'6 24 Fraction Number FIGURE 19: OPIOID-LIKE BIOACTIVITY IN SEPHADEX G-50 PURIFIED CORPUS/ANTRUM EXTRACTS FOLLOWING AMBERLITE XAD-2 TREATMENT. The e l u t i o n o f o p i o i d b i n d i n g a c t i v i t y i n Sephadex G-50 chromatography o f e x t r a c t d e t e r m i n e d by RRA (B) i s compared t o the o p i o i d b i o a c t i v i t y measured by the g u i n e a p i g i l e u m b i o a s s a y f o l l o w i n g A m b e r l i t e XAD-2 t r e a t m e n t ( C ) . 84 > Fraction Number FIGURE 20: PROTEIN AND OPIOID BINDING ACTIVITY ELUTION PROFILES OF CORPUS/ANTRUM EXTRACT FROM SEPHADEX G-50. Four s e p a r a t e a p p l i c a t i o n s o f 60 mg o f l y o p h i l i z e d e x t r a c t are d e p i c t e d as runs #l-#4. The shaded a r e a s r e f e r t o o p i o i d a c t i v i t y d e t ermined by RRA. The unshaded a r e a s r e p r e s e n t the p r o t e i n e l u t i o n d e t e r m i n e d by the absorbance a t 280 nm. The r e s p e c t i v e f r a c t i o n s from each run were p o o l e d a c c o r d i n g t o the breaks i l l u s t r a t e d on the p r o t e i n e l u t i o n p r o f i l e s and were l a b e l l e d A through D w i t h the p o o l e d peak i n o p i o i d a c t i v i t y b e i n g *C'. 85 0.05 AUFS FIGURE 21: HPLC ELUTION PROFILES OF STANDARD OPIOID PEPTIDES. Protein elution i s given on the ordinate (0.2 absorbance units f u l l scale [AUFS]) and the abcissa corresponds to time in minutes. The i n i t i a l peak in a l l panels (A-C) i s the injec t i o n a r t i f a c t s . (A) Gradient=17-28% a c e t o n i t r i l e , time=25 minutes, flow rate=2 ml/min and chart speed=l cm/min. l=met-enk, 2=leu-enk, 3=dyn 1-13, 4=met-enk-arg-gly-leu, 5=met-enk-arg-phe. (B) The conditions were as described for (A) except that the chart speed was 0.5 cm/min. Standards 1 - 5 are as for (A). In (C), the gradient=10-40% a c e t o n i t r i l e , time=30 min, flow rate=l ml/min and the chart speed=l cm/min. l=dyn 1-13, 2=dyn 1-8 and 3=dyn 1-17. ,86 A. CO LL Z> < CM o 1 met - enk 2 leu-enk 3 Dynorphin 1-13 4 met-enk-org-gly-leu 5 met-enk-arg-phe B. 0.6 LJ o.4 a 0.2 I 2 3 4 5 I I '1 1 1 24 32 40 Fraction Number FIGURE 22: HPLC SEPARATION OF VARIOUS OPIOID-LIKE COMPONENTS IN PARTIALLY PURIFED CORPUS/ANTRUM EXTRACT, DETERMINED BY OPIOID BINDING ACTIVITY. The gra d i e n t = 1 7 - 2 8 % a c e t o n i t r i l e , time=25 min, f l o w rate=2 ml/min and c h a r t speed=l cm/min. (A) P r o t e i n e l u t i o n p r o f i l e o f 80 u l o f A m b e r l i t e XAD-2 p u r i f i e d f r a c t i o n 'C . (B) O p i o i d a c t i v i t y measured by RRA i n 1 ml f r a c t i o n s . Time 0 c o r r e s p o n d s t o the time o f sample i n j e c t i o n and the r e t e n t i o n t i m e s o f the s y n t h e t i c s t a n d a r d s are denoted by the numbered a r r o w s . 87 r e t e n t i o n t i m e s c o r r e s p o n d i n g t o those o f s t a n d a r d met-enk, l e u - e n k , m e t - e n k - a r g - g l y - l e u and met-enk-arg-phe. No o p i o i d b i n d i n g a c t i v i t y was observed i n those f r a c t i o n s c o r r e s p o n d i n g to the e l u t i o n time o f s y n t h e t i c d y n o r p h i n 1-13. However, when the HPLC e f f l u e n t f r a c t i o n s were assayed f o r the presence o f d y n o r p h i n by s p e c i f i c RIA, a d i s t i n c t peak i n d y n o r p h i n -i m m u n o r e a c t i v i t y (dynorphin-IR) was found t o o c c u r , e l u t i n g w i t h the same r e t e n t i o n time as s y n t h e t i c d y n o r p h i n 1-13 ( F i g u r e 23C). The presence o f leu - e n k was c o n f i r m e d by RIA ( F i g u r e 23D). As suggested by t h e i r r e t e n t i o n t i m e s , the f i r s t s m a l l peak i n l e u - e n k - i m m u n o r e a c t i v i t y ( l e u - e n k - I R ) appeared t o be due t o the presence o f met-enk which c r o s s r e a c t s s l i g h t l y (2.5%) w i t h the leu - e n k a n t i s e r u m , w h i l e the second, l a r g e r peak corresponded t o the e l u t i o n of g a s t r i c l e u - e n k . Some d y n o r p h i n - I R was p r e s e n t i n HPLC f r a c t i o n s which preceded or f o l l o w e d the major peak ( F i g u r e 23C), s u g g e s t i n g the p o s s i b l e presence o f o t h e r forms o f g a s t r i c d y n o r p h i n . F u r t h e r m o r e , s i n c e the a n t i s e r u m used i n the d y n o r p h i n RIA c r o s s r e a c t e d e q u a l l y w e l l w i t h both d y n o r p h i n 1-13 and 1-17, i t was not p o s s i b l e t o d i s c r i m i n a t e between th e s e two p e p t i d e s under these s e p a r a t i o n c o n d i t i o n s . A c l o s e r e x a m i n a t i o n o f the corpus-antrum e x t r a c t was t h e r e f o r e made a f t e r h a v i n g a d j u s t e d the g r a d i e n t c o n d i t i o n s such t h a t d y n o r p h i n s 1-17, 1-13, and 1-8 c o u l d be r e s o l v e d . Changing the g r a d i e n t from 17-38% a c e t o n i t r i l e over 25 min t o 10-40% over 30 min and r e d u c i n g the f l o w r a t e from 2 t o 1 ml/min p e r m i t t e d the s e p a r a t i o n o f these t h r e e d y n o r p h i n s by a p p r o x i m a t e l y one min i n r e t e n t i o n time ( F i g u r e 21C) . U s i n g t h i s g r a d i e n t , the same p o o l "C" was 88 examined by HPLC and found t o c o n t a i n p e p t i d e s c o - e l u t i n g w i t h the t h r e e d y n o r p h i n s t a n d a r d s . As i l l u s t r a t e d i n F i g u r e 24, both d y n o r p h i n 1-13 and d y n o r p h i n 1-17 appeared t o be p r e s e n t i n s i m i l a r q u a n t i t i e s . A s m a l l , but d e f i n i t e peak o f d y n o r p h i n - I R , c o r r e s p o n d i n g t o the e l u t i o n o f d y n o r p h i n 1-8 was a l s o o b s e r v e d . I l l . EFFECTS OF TRYPSIN DIGESTION Upon d i g e s t i o n w i t h the p r o t e o l y t i c enzyme, t r y p s i n , an i n c r e a s e i n o p i o i d b i n d i n g a c t i v i t y d e t e r m i n e d by RRA was observed i n a l l p o o l e d samples o f Sephadex G-50 chromatographed corpus/antrum e x t r a c t ( F i g u r e 25A). These same Sephadex G-50 p o o l s were f u r t h e r p u r i f i e d by a d s o r p t i o n chromatography and were s u b j e c t e d t o t r y p s i n d i g e s t i o n as w e l l . There was a pronounced i n c r e a s e i n o p i o i d a c t i v i t y i n p o o l "C" o f the A m b e r l i t e XAD-2 p u r i f i e d Sephadex G-50 f r a c t i o n s f o l l o w i n g t r y p s i n d i g e s t i o n , w h i l e o n l y s m a l l i n c r e a s e s were observed i n p o o l s "A" and "B" ( F i g u r e 25B). ?89 B. I 2 345 1 met-enk 2 leu -enk 3 Dynorphin 1-13 4 met-enk-arg-gly- leu 5 met-enk-arg-phe Fraction Number FIGURE 23: HPLC ELUTION OF GASTRIC OPIOID PEPTIDES DETERMINED BY RIA. Gradient=17-28% a c e t o n i t r i l e , time=25 min, f l o w rate=2 ml/min, c h a r t speed=0.5 cm/min. (A) P r o t e i n e l u t i o n o f 80 jil o f A m b e r l i t e XAD-2 p u r i f i e d f r a c t i o n "C". (B) O p i o i d a c t i v i t y measured by RRA i n 1 ml f r a c t i o n . (C) Dynorphin-IR e l u t i o n d e t e c t e d by RIA and (D) E l u t i o n o f Leu-enk-IR determined by RIA. The arrows r e f e r t o the r e t e n t i o n times o f the s t a n d a r d s and time 0 denotes the p o i n t o f sample i n j e c t i o n . 90 FIGURE 24: HPLC ELUTION OF DYNORPHIN-IR FROM PARTIALLY PURIFIED CORPUS/ANTRUM EXTRACT. Gradient=10-40%, time=30 min, flow rate=l ml/min and chart speed=l cm/min. (A) Protein elution of 80 p i of Amberlite XAD-2 fr a c t i o n ' C. (B) Elution of dynorphin-IR measured by RIA. Time 0 indicates the point of inject i o n and the numbered arrows represent the retention times of synthetic dynorphin standards. 9 1 LU _ l Q < Q c A. 10 r- Sephadex G - 5 0 8 -6 -E > 0 CT UJ 2 -B. 24 20 16 12 8 4 0 L Before • After Trypsinization A B C D 13-21 2 2 - 3 3 3 4 - 4 0 41-50 Amberlite X A D - 2 JZXS. A B C D FIGURE 25: EFFECT OF TRYPSIN DIGESTION ON OPIOID ACTIVITY OF PARTIALLY PURIFIED CORPUS/ANTRUM EXTRACT. (A) O p i o i d a c t i v i t y d e termined by RRA i n p o o l s of Sephadex G-50 chromatographed e x t r a c t s . The c o r r e s p o n d i n g column f r a c t i o n numbers are i n d i c a t e d under each p o o l e d group. (B) O p i o i d a c t i v i t y i n the same samples f o l l o w i n g A m b e r l i t e XAD-2 t r e a t m e n t . 92 DISCUSSION U s i n g the RRA the o p i o i d c o n t e n t i n e x t r a c t s o f the a c i d s e c r e t i n g and e n d o c r i n e corpus-antrum r e g i o n o f the r a t stomach was found t o be g r e a t e r than i n the n o n - g l a n d u l a r fundus, a d i s t r i b u t i o n which has been s u p p o r t e d by numerous immunocytochemical and radioimmunoassay s t u d i e s d e m o n s t r a t i n g the g r e a t e s t c o n c e n t r a t i o n o f e n k e p h a l i n and d y n o r p h i n i m m u n o r e a c t i v i t i e s i n the antrum and upper i n t e s t i n e (Polak e t a l . , 1977; Alumets e t a l . , 1978; L a r s s o n and Stengaard-Pedersen 1981; Watson e t a l . , 1981; Bu'Lock e t a l . , 1983; Wang e t a l . , 1984; J o n s s o n , 1985). G e l f i l t r a t i o n o f the crude corpus-antrum a c i d e x t r a c t s on a Sephadex G-50 column made p o s s i b l e the p a r t i a l p u r i f i c a t i o n o f s u b s t a n c e s p o s s e s s i n g o p i o i d b i n d i n g a c t i v i t y by removal o f p r i m a r i l y l a r g e , i n a c t i v e components. A l t h o u g h the o p i o i d a c t i v i t y appeared t o have e l u t e d w i t h a p p r o x i m a t e l y the same volume as t h a t o f s y n t h e t i c met-enk, o t h e r o p i o i d p e p t i d e s p r e s e n t i n the e x t r a c t are presumed t o have had a s i m i l a r e l u t i o n volume s i n c e the r e s o l v i n g power o f t h i s g e l m a t r i x was not g r e a t enough t o s e p a r a t e s m a l l m o l e c u l e s i n the s i z e range o f the o p i o i d p e p t i d e s . Sephadex G-50 g e l r e s o l v e s p e p t i d e s and p r o t e i n i n the 1,500 t o 30,000 m o l e c u l a r weight range; t h e r e f o r e , the o p i o i d s o f the e n k e p h a l i n f a m i l y , a l l o f which are s m a l l e r than the lower r e s o l u t i o n l i m i t would have e l u t e d w i t h an e l u t i o n volume c l o s e t o the bed volume. Only the l a r g e r d y n o r p h i n s 1-17 (2,148 d a l t o n s ) and 1-13 (1,608 93 d a l t o n s ) might have been r e s o l v e d , e l u t i n g towards the end o f the f r a c t i o n a t i o n range. However, s i n c e the peak i n o p i o i d a c t i v i t y d e termined by RRA was f a i r l y b r o a d , s p r e a d i n g over 4 or 5 f r a c t i o n s i n most c a s e s , i t was most l i k e l y t h a t d y n o r p h i n s were i n c l u d e d w i t h the p o o l e d o p i o i d peak. F u r t h e r p u r i f i c a t i o n o f the p o o l e d Sephadex G-50 o p i o i d peak was a c h i e v e d by a d s o r p t i o n onto A m b e r l i t e XAD-2 r e s i n as demonstrated by the removal o f the i n t e r f e r i n g c o n t r a c t i l e component ( s ) . P a s s i n g the Sephadex G-50 o p i o i d peak through the A m b e r l i t e beads removed h y d r o p h i l i c s u b s t a n c e s , a l l o w i n g the h y d r o p h o b i c compounds such as the o p i o i d s t o be r e t a i n e d and e l u t e d w i t h methanol. That t h i s p a r t i a l l y p u r i f i e d e x t r a c t p o ssessed not o n l y o p i o i d r e c e p t o r b i n d i n g a c t i v i t y but a l s o b i o l o g i c a l a c t i v i t y , d e t e r m i n e d by i t s n a l o x o n e - s e n s i t i v e i n h i b i t i o n o f the e l e c t r i c a l l y i n d u ced c o n t r a c t i o n s of the g u i n e a p i g i l e u m s t r i p , added f u r t h e r s u p p o r t t o the c o n c l u s i o n t h a t the m a t e r i a l b e i n g p u r i f i e d was indeed o p i o i d - l i k e . By HPLC, p e p t i d e s w i t h e l u t i o n c h a r a c t e r i s t i c s matching those o f met-enk, l e u - e n k , m e t - e n k - a r g - l e u , met-enk-arg-phe were demonstrated t o be p r e s e n t i n the p a r t i a l l y p u r i f i e d r a t corpus-antrum e x t r a c t . The i d e n t i f i c a t i o n o f t h e s e p e p t i d e s was based on t h e i r unique r e t e n t i o n t i m e s on the r e v e r s e d phase 12 5 column and on t h e i r a b i l i t i e s t o d i s p l a c e the b i n d i n g o f ^^-"L-l a b e l l e d DADLE i n the r e c e p t o r b i n d i n g a s s a y . RIA c o n f i r m e d the presence o f l e u - e n k and met-enk p r e v i o u s l y i d e n t i f i e d by t h e i r r e t e n t i o n times i n c o n j u n c t i o n w i t h RRA. A l t h o u g h complete c h a r a c t e r i z a t i o n would r e q u i r e sequence d e t e r m i n a t i o n s , from these r e s u l t s i t appears t h a t the o p i o i d - 94 p e p t i d e s b e l o n g i n g t o the e n k e p h a l i n f a m i l y a re p r e s e n t i n the r a t stomach. However, due t o t h e i r v a r i a b l e a f f i n i t i e s f o r the o p i o i d b i n d i n g s i t e r e c o g n i z e d by the DADLE t r a c e r i n t h i s RRA (Table 1) , i t was not p o s s i b l e t o q u a n t i f y e i t h e r s p e c i f i c a l l y or r e l a t i v e l y the c o n t e n t o f each o p i o i d p e p t i d e p r e s e n t i n t h i s p a r t i a l l y p u r i f i e d e x t r a c t p r e p a r a t i o n . T h e r e f o r e , i n t h i s s t u d y , i t was e s t a b l i s h e d t h a t these f o u r o p i o i d p e p t i d e s , p o s s i b l y d e r i v e d from a s i n g l e p r e c u r s o r m o l e c u l e such as the p r o e n k e p h a l i n demonstrated i n the b r a i n and a d r e n a l s , e x i s t i n the r a t stomach. I n a s i m i l a r s t u d y i n which a c i d e x t r a c t s o f human g a s t r i c c o r p us and a n t r a l mucosa and muscular i s were examined, met-enk and leu - e n k i m m u n o r e a c t i v i t i e s were found t o be p r e s e n t i n a l l t i s s u e s w i t h the h i g h e s t c o n c e n t r a t i o n o c c u r r i n g i n the mucosa o f the body o f the stomach ( F e u r l e e t a l . , 1982). A n t i b o d i e s s p e c i f i c f o r each form o f the o p i o i d p e p t i d e s a re r e q u i r e d i n o r d e r t o q u a n t i t a t e the r e l a t i v e amounts o f each form o f e n k e p h a l i n by RIA as has been r e p o r t e d by Sakamoto e t a l . (1983a,b). T h i s group had a v a i l a b l e f o u r d i f f e r e n t a n t i s e r a which s p e c i f i c a l l y r e c o g n i z e d o n l y one o f the p e p t i d e s met-enk, l e u - e n k , met-enk-arg-phe or m e t - e n k - a r g - g l y - l e u , a l l o w i n g the d e t e r m i n a t i o n o f the r a t i o o f molar c o n c e n t r a t i o n s of these o p i o i d forms i n e x t r a c t s o f human g a s t r i c a n t r a o b t a i n e d a f t e r a u t o p s y . I t was found t h a t the c o n c e n t r a t i o n s of l e u - e n k , m e t - e n k - a r g - g l y - l e u and met-enk-arg-phe were a p p r o x i m a t e l y e q u a l and were 4 t o 5 t i m e s lower than t h a t o f met-enk. T h i s 1:1:1:4 r a t i o agrees w i t h the r e s u l t s o f sequencing s t u d i e s o f human b r a i n p r o e n k e p h a l i n (Comb e t a l . , i 95 1982). T h i s s u g gests t h a t the g a s t r i c , c e n t r a l and a d r e n a l o p i o i d p e p t i d e s a re g e n e r a t e d from the same or s i m i l a r e n k e p h a l i n p r e c u r s o r . S i m i l a r l y , i t has been r e p o r t e d t h a t p r o - e n k e p h a l i n gene d e r i v e d p e p t i d e s o c c u r i n the p o r c i n e stomach muscle and a n t r a l mucosa ( G i r a u d e t a l . , 1984a). The i n a b i l i t y t o d e t e c t any d y n o r p h i n - l i k e substance by RRA i n the p a r t i a l l y p u r i f i e d c orpus-antrum e x t r a c t f o l l o w i n g HPLC was somewhat s u r p r i s i n g i n view o f p r e v i o u s r e p o r t s i n the l i t e r a t u r e o f g a s t r o i n t e s t i n a l d y n o r p h i n - I R measured by e i t h e r RIA or ICC (Tachibana e t a l . , 1981; Watson e t a l . , 1981; Spampinato and G o l d s t e i n 1983; V i n c e n t e t a l . , 1984). I t was o n l y by s p e c i f i c RIA t h a t the presence o f d y n o r p h i n i n the e x t r a c t c o u l d be d e t e c t e d . Under the HPLC c o n d i t i o n s o p t i m i z e d f o r s e p a r a t i o n o f the e n k e p h a l i n s , d y n o r p h i n - I R was found t o e l u t e w i t h a r e t e n t i o n time c o r r e s p o n d i n g t o t h a t o f s y n t h e t i c d y n o r p h i n 1-13. An e x p l a n a t i o n f o r t h i s i n a b i l i t y t o d e t e c t d y n o r p h i n by RRA i s not o b v i o u s but may have been a r e s u l t o f a co m b i n a t i o n o f s m a l l q u a n t i t i e s and r e l a t i v e l y low p o t e n c i e s of 125 the d y n o r p h i n s i n d i s p l a c i n g I-DADLE b i n d i n g t o o p i o i d b i n d i n g s i t e s i n t h i s p a r t i c u l a r RRA system. I n o r d e r t o examine the t y p e s o f d y n o r p h i n p r e s e n t i n the p a r t i a l l y p u r i f i e d e x t r a c t , i t was n e c e s s a r y t o m a n i p u l a t e the g r a d i e n t c o n d i t i o n s t o a l l o w f o r complete s e p a r a t i o n o f d y n o r p h i n 1-13, 1-8 and 1-17. T h i s was a c h i e v e d by a l t e r i n g the i n i t i a l and f i n a l a c t e t o n i t r i l e c o n c e n t r a t i o n s t o 10-40%. U s i n g the RIA the one major peak i n d y n o r p h i n - I R d e t e c t e d u s i n g the p r e v i o u s g r a d i e n t c o n d i t i o n s o f 17-38% was r e s o l v e d i n t o t h r e e peaks w i t h r e t e n t i o n t i m e s e q u a l t o those o f d y n o r p h i n 1-13, d y n o r p h i n 1-8 and d y n o r p h i n 1-17. Because i t had been found t h a t d y n o r p h i n s 1-13 and 1-17 d i s p l a y e q u a l p o t e n c i e s i n 125 d i s p l a c i n g the I - d y n o r p h m 1-13 t r a c e r from the a n t i s e r u m ( F i g u r e 10, Chapter 2 ) , the q u a n t i t i e s o f the s e two d y n o r p h i n s were assumed t o be e q u i v a l e n t . Moreover, s i n c e d y n o r p h i n 1-8 demonstrates o n l y a s l i g h t c r o s s - r e a c t i v i t y w i t h t h i s d y n o r p h i n 1-13 a n t i s e r u m ( F i g u r e 10, Chapter 2) i t i s p o s s i b l e t h a t t h i s s m a l l peak i n d y n o r p h i n - I R o c c u r r i n g w i t h a r e t e n t i o n time s i m i l a r t o s y n t h e t i c d y n o r p h i n 1-8 r e p r e s e n t s a much g r e a t e r v a l u e than measured by t h i s RIA system. A l t h o u g h i t was not p o s s i b l e t o q u a n t i t a t e the e x a c t d y n o r p h i n 1-8 c o n t e n t , by t a k i n g i n t o c o n s i d e r a t i o n t h a t t h e r e i s o n l y a 6.25% c r o s s r e a c t i o n between d y n o r p h i n 1-8 and the d y n o r p h i n 1-13 a n t i s e r u m , the measured v a l u e o f 1 nM would a c t u a l l y r e p r e s e n t 16 nM. T h e r e f o r e , the 1-8 form would appear t o be p r e s e n t i n amounts s i m i l a r t o d y n o r p h i n s 1-13 and 1-17 i n the r a t c o r p u s -antrum. I n v a r i o u s r e g i o n s o f the r a t b r a i n , Weber e t a l . (1982) found, by u s i n g h i g h l y s p e c i f i c radioimmunoassays, t h a t on a molar b a s i s the c o n c e n t r a t i o n s o f d y n o r p h i n 1-8 were s e v e r a l times (up t o 10 t i m e s ) g r e a t e r than those o f d y n o r p h i n 1-17. The f i n d i n g i n the p r e s e n t s t u d y o f s i m i l a r c o n c e n t r a t i o n s o f d y n o r p h i n s 1-8, 1-13, and 1-17 i n the r a t stomach s u g g e s t s t h e r e may be some organ d i f f e r e n c e s i n the p r o c e s s i n g o f the d y n o r p h i n p r e c u r s o r . There has been a r e c e n t a c c u m u l a t i o n o f e v i d e n c e t o i n d i c a t e the o c c u r r e n c e o f r e g i o n a l d i f f e r e n c e s i n the p a t t e r n s o f o p i o i d p r e c u r s o r p o s t - t r a n s l a t i o n a l p r o c e s s i n g . Zamir e t a l . (1984) have demonstrated d i f f e r e n t i a l p r o c e s s i n g o f 97 p r o d y n o r p h i n and p r o e n k e p h a l i n i n s p e c i f i c r e g i o n s o f the r a t b r a i n . S i m i l a r l y , G i r a u d e t a l . (1984b) have p r e s e n t e d r a d i o i m m u n o l o g i c a l e v i d e n c e f o r d i f f e r e n t p a t t e r n s o f p o s t -t r a n s l a t i o n a l p r o c e s s i n g o f p r o - e n k e p h a l i n i n the b o v i n e a d r e n a l g l a n d , c o l o n and s t r i a t u m by u s i n g r e g i o n - s p e c i f i c a n t i s e r a t o met-enk-arg-phe and m e t - e n k - a r g - g l y - l e u . F u r t h e r m o r e , the p a t t e r n s o f the m o l e c u l a r forms of e n k e p h a l i n s i n e x t r a c t s o f hog a n t r a l mucosa have been shown t o d i f f e r from those i n e x t r a c t s o f a n t r a l and f u n d i c muscle l a y e r s (Giraud e t a l . , 1984a). T h e r e f o r e , i t was suggested t h a t the e n k e p h a l i n p r e c u r s o r i n the stomach i s p r o c e s s e d by d i s t i n c t pathways i n d i f f e r e n t p o p u l a t i o n s o f e n d o c r i n e c e l l s and neurons. I n view of these r e p o r t s o f the d i f f e r e n t i a l r e g i o n a l d i s t r i b u t i o n o f the p r o e n k e p h a l i n and p r o d y n o r p h i n gene d e r i v e d p e p t i d e s , i t i s not s u r p r i s i n g t h a t , i n the p r e s e n t s t u d y , the r e l a t i v e l e v e l s of d y n o r p h i n s 1-8, 1-13 and 1-17 i n r a t g a s t r i c e x t r a c t s were found t o d i f f e r from t h o s e demonstrated i n r a t b r a i n r e g i o n s by o t h e r s (Weber e t a l . , 1982). I t i s p o s s i b l e t h a t the g a s t r o i n t e s t i n a l t r a c t , o r perhaps s p e c i f i c r e g i o n s o f the g a s t r o i n t e s t i n a l t r a c t , p o s s e s s e s unique pathways f o r the p r o c e s s i n g o f the d y n o r p h i n p r e c u r s o r , d i f f e r i n g from those e s t a b l i s h e d as o c c u r r i n g i n the b r a i n . Moreover, i t i s a l s o p o s s i b l e t h a t the g a s t r o i n t e s t i n a l o p i o i d p r e c u r s o r s themselves may be d i f f e r e n t from those p r e s e n t i n the b r a i n and a d r e n a l g l a n d s . Both d y n o r p h i n s 1-17 and 1-8 have been d e t e c t e d i n p i t u i t a r y and b r a i n (Suda e t a l . , 1982,1983; I v e r s e n , 1982; Weber e t a l . , 1982; Spampinato and G o l d s t e i n , 1983; Cone e t 98 a l . , 1983). S m a l l amounts o f d y n o r p h i n 1-13 have been found i n r a t and human p i t u i t a r y e x t r a c t s (Suda e t a l . , 1982, 1983). Dynorphin 1-13 was o r i g i n a l l y employed by G o l d s t e i n ' s group (Watson e t a l . , 1981) f o r the p r e p a r a t i o n o f d y n o r p h i n a n t i s e r a because o n l y the f i r s t 13 amino t e r m i n a l r e s i d u e s o f d y n o r p h i n were i d e n t i f i e d . The remainder o f the m o l e c u l e was unknown a t t h a t t i m e . A c c o r d i n g t o the p r e s e n t dogma o f o p i o i d p e p t i d e b i o g e n e s i s , i n o r d e r f o r d y n o r p h i n 1-13 t o be formed, t h e r e must be an e n z y m a t i c c l e a v a g e between r e s i d u e s 13 and 14 o f the d y n o r p h i n 1-17 sequence, a l y s - t r p bond (see F i g u r e 3, Chapter 1 ) . A l t h o u g h t h i s i s not a c o n v e n t i o n a l c l e a v a g e p o i n t f o r a t r y p s i n - l i k e enzyme, o t h e r o p i o i d p e p t i d e s such as a l p h a - and gamma-endorphins, d y n o r p h i n 1-8 and d y n o r p h i n B are known t o be c l e a v e d from t h e i r r e s p e c t i v e p r e c u r s o r s a t p e p t i d e bonds i n v o l v i n g one or no b a s i c amino a c i d r e s i d u e . T h e r e f o r e , i t i s p o s s i b l e t h a t d y n o r p h i n 1-13 i s a t r u e b i o l o g i c a l p r o d u c t d e r i v e d from the s p e c i f i c p r o c e s s i n g o f d y n o r p h i n 1-17. A l t e r n a t i v e l y , however u n l i k e l y , the d y n o r p h i n - I R measured c o r r e s p o n d i n g t o d y n o r p h i n 1-13 may have been due t o the presence o f some o t h e r o p i o i d p e p t i d e o f the d y n o r p h i n f a m i l y . D y n o rphin B and the neo-endorphins are d e r i v e d from the same p r e c u r s o r as d y n o r p h i n 1-17, but o n l y the l e u - e n k sequence i s common t o a l l . S i n c e l e u - e n k does not c r o s s - r e a c t w i t h t h i s d y n o r p h i n a n t i s e r u m and s i n c e d y n o r p h i n B and the neo-endorphins a l l p o s s e s s c a r b o x y t e r m i n a l e x t e n s i o n s o f leu-enk which are c o m p l e t e l y d i f f e r e n t from d y n o r p h i n 1-17, i t i s improbable t h a t the observed d y n o r p h i n - I R w i t h a r e t e n t i o n time c o r r e s p o n d i n g t o d y n o r p h i n 1-13 r e p r e s e n t s these o t h e r ' 99 d y n o r p h i n - r e l a t e d p e p t i d e s . However, i t i s p o s s i b l e t h a t the observed d y n o r p h i n 1-13 might have been an a r t i f a c t o f the e x t r a c t i o n and/or p u r i f i c a t i o n p r o c e d u r e s , r e s u l t i n g from the breakdown o f d y n o r p h i n 1-17. N e v e r t h e l e s s , the e x i s t e n c e o f endogenous g a s t r i c d y n o r p h i n 1-13 i s a s t r o n g p o s s i b i l i t y s i n c e t h e r e have been ample r e p o r t s d e m o n s t r a t i n g t h a t d y n o r p h i n 1-13 po s s e s s e s p o t e n t b i o l o g i c a l a c t i v i t y i n the g u i n e a p i g i l e u m ( G o l d s t e i n e t a l . , 1979; C h a v k i n and G o l d s t e i n 1981; Vaught 1981; C h a v k i n e t a l . , 1982; Huid o b r o - T o r o and Way 1982; Garzon e t a l . , 1982; Yoshimura e t a l . , 1982a,b; Yuang-Xiang Zhu e t a l . , 1982; I v e r s e n , 1982; Kamikawa and Shimo, 1983). Dynorphin 1-17 ( I v e r s e n 1982; Yoshimura e t a l . , 1982; Cox and C h a v k i n , 1983) and d y n o r p h i n 1-8 (Minamino e t a l . , 1980; C o r b e t t e t a l . , 1982) a l s o p o s s e s s p o t e n t o p i o i d b i o a c t i v i t y and a l l t h r e e appear t o a c t on the kappa o p i o i d r e c e p t o r subtype (Chavkin e t a l . , 1982; C o r b e t t e t a l . , 1982). Which o f these a c t s as the t r u e p h y s i o l o g i c a l l y f u n c t i o n a l d y n o r p h i n ( s ) has, however, s t i l l t o be d e t e r m i n e d . The i n c r e a s e i n o p i o i d a c t i v i t y d e t e c t e d f o l l o w i n g t r y p s i n d i g e s t i o n o f Sephadex G-50 and A m b e r l i t e XAD-2 p o o l s suggests the presence o f some l a r g e r i n a c t i v e form from which s m a l l e r , a c t i v e p e p t i d e s were r e l e a s e d by enzy m a t i c d e g r a d a t i o n . P o o l s "A" and "B" ( F i g u r e 25A) were expected t o c o n t a i n the l a r g e r m o l e c u l a r weight s u b s t a n c e s s i n c e these samples c o n s i s t e d o f the e a r l y f r a c t i o n e l u t i n g from Sephadex G-50 g e l permeation chromatography. B o t h were found t o c o n t a i n a much h i g h e r c o n c e n t r a t i o n o f o p i o i d b i n d i n g a c t i v i t y a f t e r t r y p s i n d i g e s t i o n , i n d i c a t i n g t h a t some l a r g e , p o s s i b l y 100 p r e c u r s o r m o l e c u l e s were p r e s e n t i n thes e p o o l e d Sephadex samples. The i n c r e a s e i n a c t i v i t y o b s e r v e d i n Sephadex p o o l "C" might have been due t o the breakdown o f l a r g e r , l e s s a c t i v e or i n a c t i v e m o l e c u l e s t o a c t i v e , s h o r t e r forms. The i n c r e a s e i n p o o l "D" was unexpected, as t h i s sample t h e o r e t i c a l l y s h o u l d c o n s i s t o f v e r y s m a l l m o l e c u l e s which e l u t e f o l l o w i n g the e n k e p h a l i n s . Some degree o f a d s o r p t i o n o f l a r g e r m o l e c u l e s onto the Sephadex g e l , c a u s i n g a d e l a y e d e l u t i o n may account f o r t h i s o b s e r v a t i o n . T r y p s i n d i g e s t i o n o f the A m b e r l i t e XAD-2 p u r i f i e d Sephadex G-50 p o o l s a l s o r e s u l t e d i n an i n c r e a s e i n o p i o i d a c t i v i t y , However, the most marked i n c r e a s e i n a c t i v i t y was observed i n p o o l "C" ( F i g u r e 25B) w i t h much s m a l l e r i n c r e a s e s o c c u r r i n g i n p o o l s "A" and "B" which a g a i n presumably c o n t a i n l a r g e r m o l e c u l e s . I t t h e r e f o r e appears as though the l a r g e r , i n a c t i v e p r o t e i n s which were p r e s e n t i n the Sephadex G-50 p o o l s "A" and "B" were not adsorbed by the a m b e r l i t e XAD-2 r e s i n and were e l u t e d w i t h the a c i d or water washes r a t h e r than the a l c o h o l e l u t i o n which was r e t a i n e d and assay e d . From th e s e t r y p s i n d i g e s t i o n s t u d i e s , the p o s s i b l e e x i s t e n c e o f g a s t r i c o p i o i d p e p t i d e p r e c u r s o r s was e s t a b l i s h e d , but the p r e c i s e n a t u r e o f thes e prohormones c o u l d not be det e r m i n e d . The f i n d i n g i n r a t g a s t r i c e x t r a c t s o f a l l forms of e n k e p h a l i n s d e r i v e d from p r o e n k e p h a l i n , the e n k e p h a l i n p r e c u r s o r i n the b r a i n and a d r e n a l g l a n d , s u g g e s t s t h a t the same or s i m i l a r p r e c u r s o r s might be p r e s e n t i n the r a t stomach. The o c c u r r e n c e o f d y n o r p h i n s 1-17, 1-13 and 1-8 a l s o suggests the p o s s i b i l i t y o f p r o d y n o r p h i n i n the stomach. F u r t h e r 101 i n v e s t i g a t i o n s i n t o the presence i n the stomach of the neo-endorphins and d y n o r p h i n B are s t i l l r e q u i r e d t o demonstrate the complete s e t o f p r o d y n o r p h i n d e r i v e d o p i o i d s . 102 CHAPTER FOUR AUTORADIOGRAPHIC LOCALIZATION OF OPIOID RECEPTORS  IN THE GASTROINTESTINAL TRACT INTRODUCTION The concept t h a t c h e m i c a l messengers produce t h e i r b i o l o g i c a l e f f e c t s by i n t e r a c t i n g w i t h s p e c i f i c r e c e p t o r s l o c a t e d e x c l u s i v e l y on r e s p o n s i v e c e l l s has been w e l l e s t a b l i s h e d on both b i o c h e m i c a l and p h a r m a c o l o g i c a l grounds. The e f f e c t s o f the o p i a t e n a r c o t i c s and endogenous o p i o i d p e p t i d e s are l i k e w i s e mediated by t h e i r b i n d i n g t o s p e c i f i c r e c e p t o r s which e x i s t i n a m u l t i p l i c i t y o f forms (Chapter 1, s e c t i o n I V ) . I . LOCALIZATION OF OPIOID RECEPTORS E a r l y i n v e s t i g a t i o n s i n t o o p i a t e a c t i o n s , u s i n g e i t h e r whole a n i m a l s t o m o n i t o r a n t i n o c i c e p t i v e e f f e c t s or i s o l a t e d t i s s u e s t o measure i n h i b i t i o n o f n e u r o t r a n s m i s s i o n , demonstrated the presence o f s p e c i f i c o p i a t e r e c e p t o r s i n b r a i n , g u i n e a p i g i l e u m and mouse vas d e f e r e n s ( B e c k e t t and Casy, 1954; Schaumann, 1955; P a t o n , 1957). S u b s e q u e n t l y , s t e r e o s p e c i f i c b i n d i n g o f r a d i o l a b e l l e d o p i o i d s was demonstrated i n b r a i n homogenates ( P e r t and Snyder, 1973a; T e r e n i u s , 1973a; Simon e t a l . , 1973) and i n g u i n e a p i g i l e a l homogenates (Creese and Snyder, 1975). By means o f c e l l f r a c t i o n a t i o n , o p i o i d b i n d i n g s i t e s were l o c a l i z e d t o s y n a p t i c r e g i o n s o f the b r a i n membrane homogenates ( P e r t e t a l . , 1974). 103 The r e g i o n a l d i s t r i b u t i o n s o f o p i o i d r e c e p t o r s i n the b r a i n have been determined by measuring t r i t i u m l a b e l l e d o p i a t e b i n d i n g i n g r o s s l y d i s s e c t e d a r e a s o f the b r a i n (Kuhar e t a l . , 1973; H i l l e r e t a l . , 1973; Lee e t a l . , 1975). A u t o r a d i o g r a p h i c d e t e r m i n a t i o n o f o p i o i d b i n d i n g s i t e s has proven t o be a more u s e f u l approach i n t h a t i t a l l o w s the m i c r o s c o p i c e x a m i n a t i o n of r e c e p t o r b i n d i n g t o t i s s u e s e c t i o n s . I n v i v o e x p e r i m e n t s were performed w h e r e i n r a d i o l a b e l l e d o p i o i d l i g a n d s were a d m i n i s t e r e d i n t r a v e n o u s l y and b i n d i n g o f the t r a c e r was determined by e x p o s i n g t i s s u e s e c t i o n s t o a u t o r a d i o g r a p h i c f i l m . H i s t o c h e m i c a l l o c a l i z a t i o n o f o p i o i d r e c e p t o r s r e p r e s e n t e d by dark s i l v e r g r a i n s on the f i l m r e v e a l e d i n f a r g r e a t e r d e t a i l the s p e c i f i c d i s t r i b u t i o n s than d i d i n v i t r o b i n d i n g s t u d i e s u s i n g t i s s u e homogenates ( f o r r e v i e w s , see Robson e t a l . , 1983; Atweh and Kuhar, 1983; Simon and H i l l e r , 1984). Q u a l i t a t i v e l y , the r e s u l t s o f a u t o r a d i o g r a p h i c s t u d i e s of o p i o i d r e c e p t o r d i s t r i b u t i o n s i n the c e n t r a l nervous system ( P e r t e t a l . , 1976; Atweh and Kuhar, 1977a,b,c; Pearso n e t a l . , 1980) were s i m i l a r t o those o b t a i n e d by measuring b i n d i n g t o b r a i n homogenates. However, the use o f i n v i v o a u t o r a d i o g r a p h i c t e c h n i q u e s was l i m i t e d s i n c e o n l y compounds which were not r e a d i l y m e t a b o l i z e d , which p o s s e s s e d slow d i s s o c i a t i o n r a t e s and which c r o s s e d the b l o o d - b r a i n b a r r i e r ( f o r c e n t r a l l o c a l i z a t i o n s ) c o u l d be employed. T h e r e f o r e , a u t o r a d i o g r a p h i c t e c h n i q u e s i n which r a d i o l a b e l l e d o p i o i d l i g a n d i s a l l o w e d t o b i n d t o t i s s u e s e c t i o n s i n v i t r o have been de v e l o p e d . U s i n g t h i s i n v i t r o method, e x t e n s i v e l i g h t m i c r o s c o p i c h i s t o c h e m i c a l mappings o f o p i o i d r e c e p t o r 104 d i s t r i b u t i o n s i n the b r a i n have been made (Young and Kuhar, 1979; Herkenham and P e r t , 1980, 1981, 1982; Meibach and Maayani, 1980; Goodman e t a l . , 1980, Le w i s e t a l . , 1982; Goodman and Snyder, 1982; Wamsley e t a l . , 1982; Wamsley, 1983). A d d i t i o n a l l y , a u t o r a d i o g r a p h i c l o c a l i z a t i o n o f o p i o i d r e c e p t o r s i n the mammalian r e t i n a (Wamsley e t a l . , 1981) and r a t s p i n a l c o r d ( S l a t e r and P a t e l , 1983) have been r e p o r t e d . Advantages of i n v i t r o l a b e l l i n g o f r e c e p t o r s a re t h a t i t i s p o s s i b l e t o reduce n o n - s p e c i f i c b i n d i n g t o low l e v e l s by washing, s e r i a l s e c t i o n s can be i n c u b a t e d w i t h d i f f e r e n t o p i o i d l i g a n d s t o p r o v i d e i n f o r m a t i o n on m u l t i p l e o p i o i d r e c e p t o r d i s t r i b u t i o n s (Goodman e t a l . , 1980), and the c o n d i t i o n s o f t i s s u e l a b e l l i n g can be r e a d i l y a l t e r e d t o s t u d y b i n d i n g r e g u l a t i o n (Bowen e t a l . , 1981; O l g i a t i e t a l . , 1982). I I . PROPERTIES OF OPIOID RECEPTORS L i g a n d b i n d i n g t o o p i o i d r e c e p t o r s i s a f f e c t e d by temperature and pH and i s r e g u l a t e d by d i v a l e n t and monovalent c a t i o n s and guanine n u c l e o t i d e s , i n p a r t i c u l a r , guanosine 5'-t r i p h o s p h a t e (GTP) (Pa s t e r n a k e t a l . , 1975a; Simantov e t a l . , 1976). Sodium has been found t o be the monovalent c a t i o n which most p o t e n t l y a f f e c t s r e c e p t o r - l i g a n d i n t e r a t i o n s . Sodium i o n s (100 mM) have been shown t o cause a d e c r e a s e i n the s p e c i f i c 3 b i n d i n g o f such a g o n i s t s as [ H]-dihydromorphme t o 40% o f 3 c o n t r o l v a l u e s , w h i l e the a n t a g o n i s t [ H]-naloxone b i n d i n g was u n a f f e c t e d or s l i g h t l y i n c r e a s e d (Simon e t a l . , 1973, 1975; P e r t and Snyder, 1973b, 1974; P e r t e t a l . , 1973; P a s t e r n a k e t 3 a l . , 1975a). S i m i l a r l y , the b i n d i n g o f the e n k e p h a l i n s , [ H]-105 3 met-enk and [ H ] - l e u - e n k , (Morin e t a l . , 1976; Simantov and Snyder, 1976; Meunier and Moisand, 1977, Simantov e t a l . , 1978) and [ 1 2 5 I ] - D - a l a 2 , D - l e u 5 - e n k e p h a l i n ( M i l l e r e t a l . , 1978) has been shown t o be reduced i n the presence of 100 mM N a C l . C h i l d e r s e t a l . (1979) have demonstrated t h a t sodium causes s i m i l a r e f f e c t s on o p i o i d b i n d i n g i n the g u i n e a p i g i l e u m . I n c o n t r a s t , d i v a l e n t c a t i o n s such as magnesium, manganese, and c a l c i u m have been found t o s i g n i f i c a n t l y enhance a g o n i s t , but not a n t a g o n i s t , b i n d i n g ( P a s t e r n a k e t a l . , 1975a; Simantov and Snyder, 1976; P f e i f f e r and H e r z , 1982). F u r t h e r m o r e , i n the presence o f 100 mM N a C l , these e f f e c t s o f d i v a l e n t c a t i o n s were even more pronounced; the d e c r e a s e observed i n the presence of sodium a l o n e was not o b s e r v e d ( P a s t e r n a k e t a l . , 1975a; Simantov e t a l . , 1976). There does not appear t o be agreement i n the l i t e r a t u r e as t o the mechanism by which sodium d i f f e r e n t i a l l y e f f e c t s a g o n i s t and a n t a g o n i s t b i n d i n g . Some i n v e s t i g a t o r s have r e p o r t e d e v i d e n c e s u g g e s t i n g t h a t sodium a l t e r s the number o f b i n d i n g s i t e s ( P e r t and Snyder, 1974; P a s t e r n a k and Snyder 1975; M o r i n e t a l . , 1976), w h i l e o t h e r s c l a i m t h a t r e c e p t o r a f f i n i t y i s a l t e r e d (Simon e t a l . , 1975). Guanine n u c l e o t i d e s p l a y a d u a l r o l e i n r e c e p t o r - l i g a n d i n t e r a c t i o n s : f i r s t l y by s e n s i t i z i n g o p i o i d r e c e p t o r s by d e c r e a s i n g the a f f i n i t y f o r r e c e p t o r b i n d i n g and by i n c r e a s i n g a g o n i s t d i s s o c i a t i o n r a t e s and s e c o n d l y by r e g u l a t i n g the c o u p l i n g o f the r e c e p t o r t o a d e n y l a t e c y c l a s e (Blume 1978a,b; Blume e t a l . , 1979; R o d b e l l , 1980; C h i l d e r s , 1983). I t has been r e p o r t e d t h a t GTP i n h i b i t s the b i n d i n g o f e n k e p h a l i n s most c p r o f o u n d l y i n the p r e s ence o f 100 mM NaCl ( C h i l d e r s and Snyder, 106 1978, 1980; Snyder e t a l . , 1979), the e f f e c t s o f sodium-and GTP b e i n g a d d i t i v e . T h i s GTP and N a + e f f e c t has been demonstrated 2+ 2+ to be a n t a g o n i z e d by the d i v a l e n t c a t i o n s Mn and Mg (Barnard and Demouliou-Mason, 1983; Lambert and C h i l d e r s , 1984). I t has been suggested t h a t c o n t r a r y t o e a r l y p r o p o s a l s , the e f f e c t s o f GTP and c a t i o n s may not be t o d i f f e r e n t i a t e a g o n i s t - a n t a g o n i s t b i n d i n g but r a t h e r t o d i f f e r e n t i a t e r e c e p t o r s u b t y p e s . P e r t ' s group have r e p o r t e d e v i d e n c e s u g g e s t i n g t h a t d i s t i n c t mu and d e l t a s i t e s i n r a t s t r i a t u m c o u l d be d i s t i n g u i s h e d by t h e i r s e n s i t i v i t i e s t o GTP i n h i b i t i o n ( P e r t e t a l . , 1980; Bowen e t a l . , 1981; Q u i r i o n e t a l . , 1981). O p i o i d r e c e p t o r s i t e s d e m o n s t r a t i n g m u - l i g a n d s e l e c t i v i t y were found t o be s e n s i t i v e t o GTP w h i l e those d e m o n s t r a t i n g d e l t a - l i g a n d s e l e c t i v i t y were i n s e n s i t i v e t o GTP; the two s i t e s have been d e s i g n a t e d as Type I and Type I I . S i m i l a r l y , Chang e t a l . (1981) have demonstrated t h a t GTP has a r e l a t i v e l y g r e a t e r e f f e c t i n i n h i b i t i n g mu ( m o r p h i n e ) - b i n d i n g s i t e s than d e l t a ( e n k e p h a l i n ) - b i n d i n g s i t e s . F u r t h e r m o r e , the s e n s i t i v i t y o f o p i o i d r e c e p t o r t y p e s t o r e g u l a t i o n by sodium and GTP was found to v a r y , w i t h mu b i n d i n g s i t e s b e i n g more s e n s i t i v e than d e l t a s i t e s w h i l e kappa s i t e s were the l e a s t s e n s i t i v e t o both r e g u l a t o r s ( W e r l i n g e t a l . , 1984). These r e s u l t s suggest t h a t t h e r e i s v a r i a t i o n i n the a b i l i t y o f o p i o i d r e c e p t o r s t o i n t e r a c t w i t h guanine n u c l e o t i d e s . As mentioned p r e v i o u s l y , the c o u p l i n g o f the o p i o i d l i g a n d - r e c e p t o r b i n d i n g t o a d e n y l a t e c y c l a s e i s r e g u l a t e d by GTP. Other hormone r e c e p t o r - c o u p l i n g s w i t h a d e n y l a t e c y c l a s e have been demonstrated t o be mediated by a c o u p l i n g p r o t e i n ( N - p r o t e i n ) which b i n d s GTP, and i t i s t h i s 107 N - p r o t e i n which a c t u a l l y i n h i b i t s or a c t i v a t e s the enzyme, depending on the n a t u r e o f the system. A l t h o u g h the c o u p l i n g p r o t e i n a s s o c i a t e d w i t h the o p i a t e - i n h i b i t e d a d e n y l a t e c y c l a s e has not been c h a r a c t e r i z e d as y e t , the c o u p l i n g r e a c t i o n i s known t o be GTP dependent and so i s presumed t o i n v o l v e such a r e g u l a t o r y p r o t e i n . Whether the a c t i o n o f GTP on r e c e p t o r b i n d i n g i s a l s o mediated v i a t h i s N - p r o t e i n or i s a d i r e c t e f f e c t on the r e c e p t o r s i t e i t s e l f i s u n c e r t a i n but s t u d i e s by C h i l d e r s and L a R i v i e r e (1984) i n d i c a t e t h a t t h e r e are d i f f e r e n c e s between the membrane components which c o u p l e r e c e p t o r s t o a d e n y l a t e c y c l a s e and those t h a t r e g u l a t e o p i o i d b i n d i n g . R e c e n t l y , a GTPase has been found t o be a s s o c i a t e d w i t h the r e g u l a t o r y p r o t e i n which may f u n c t i o n t o t e r m i n a t e the c o u p l i n g between the r e c e p t o r s and a d e n y l a t e c y c l a s e (Enomoto and Asakawa, 1984). T h e r e f o r e , i t i s p o s s i b l e t h a t i n h i b i t i o n o f a d e n y l a t e c y c l a s e a c t i v i t y i s mediated through s t i m u l a t i o n o f GTPase t o c o n v e r t the r e g u l a t o r y p r o t e i n from the a c t i v e GTP- t o the i n a c t i v e GDP-complexed s t a t e . Hoss and F r a n k l i n (1984) have demonstrated the s t e r o s p e c i f i c s t i m u l a t i o n of b r a i n GTPase a c t i v i t y by o p i o i d s , s u p p o r t i n g t h i s h y p o t h e s i s t h a t o p i o i d - i n d u c e d i n h i b i t i o n o f a d e n y l a t e c y c l a s e a c t i v i t y might be due t o s t i m u l a t i o n o f GTP h y d r o l y s i s . I l l . OPIOID RECEPTORS IN THE GASTROINTESTINAL TRACT I t has been w e l l - e s t a b l i s h e d t h a t the g u i n e a p i g i l e u m c o n t a i n s o p i o i d r e c e p t o r s , and the i n v i t r o b i o a s s a y employing t h i s t i s s u e has been i n s t r u m e n t a l i n the d i s c o v e r y o f endogenous o p i o i d p e p t i d e s (Hughes e t a l . , 1975). The o p i o i d ' 108 r e c e p t o r s p r e s e n t i n the g u i n e a p i g i l e u m have p r i m a r i l y been p h a r m a c o l o g i c a l l y c h a r a c t e r i z e d as mu- and kappa-types i n the m y e n t e r i c p l e x u s ( K o s t e r l i t z and W a t e r f i e l d , 1975; L o r d e t a l . , 1977; C h a v k i n and G o l d s t e i n , 1981; Yoshimura e t a l . , 1982a,b; C h a v k i n e t a l . , 1982), a l t h o u g h t h e r e have been some i n d i c a t i o n s as t o the presence o f d e l t a o p i o i d r e c e p t o r s i n t h i s t i s s u e ( G i n t z l e r and S c a l i s i , 1982). The r a b b i t i l e u m (Dobbins e t a l . , 1980; McKay e t a l . , 1981; B i n d e r e t a l . , 1984) and the g u i n e a p i g i l e a l mucosa (Kachur e t a l . , 1980; Kachur and M i l l e r , 1 9 8 2 ; V i n a y e k e t a l . , 1983) appear t o p o s s e s s o p i o i d r e c e p t o r s i n v o l v e d i n the r e g u l a t i o n o f i n t e s t i n a l e l e c t r o l y t e t r a n s p o r t . These i l e a l r e c e p t o r s have been suggested t o be of the d e l t a t y p e . O p i o i d r e c e p t o r s have a l s o been demonstrated to o c c u r on g a s t r o i n t e s t i n a l smooth muscle and t o d i r e c t l y a f f e c t motor a c t i v i t y . I s o l a t e d c i r c u l a r muscle c e l l s from the guinea p i g stomach ( B i t a r and M a k h l o u f , 1982) have been shown 2 to respond t o d y n o r p h i n , met-enk, l e u - e n k , D - a l a -met-enkephalinamide and morphine. F u r t h e r m o r e , s i n c e the e x c i t a t o r y e f f e c t o f d y n o r p h i n on the spontaneous a c t i o n p o t e n t i a l s r e c o r d e d from duodenal smooth muscle c o u l d not be b l o c k e d by a t r o p i n e (Ohkawa, 1985), i t was suggested t h a t d y n o r p h i n d i r e c t l y a f f e c t s muscle c e l l s by a c t i n g on kappa o p i o i d r e c e p t o r s o c c u r r i n g on the muscle membrane. I n c o n t r a s t , H i r n i n g e t a l . , (1985) have r e p o r t e d t h a t mu, but not kappa, o p i o i d a g o n i s t s cause c o n t r a c t i o n s o f the c a n i n e s m a l l i n t e s t i n e , s u g g e s t i n g t h a t the m i n i m a l e f f e c t s o bserved w i t h the k a p p a - a g o n i s t s EKC were mediated through mu o p i o i d r e c e p t o r s . 109 The types of opioid receptors present An the gastr o i n t e s t i n a l tract and their regional d i s t r i b u t i o n s are far from c l e a r . Therefore, in the following studies in v i t r o autoradiographic techniques which previously have been employed to v i s u a l l y demonstrate opioid receptors in the central nervous system have been applied to l o c a l i z e the d i s t r i b u t i o n s of opioid receptors in the rat and guinea pig gas t r o i n t e s t i n a l 2 5 t r a c t s . The binding of t r i t i u m l a b e l l e d D-ala ,D-Leu -enkephalin (DADLE), naloxone or dihydromorphine, and ethylketocyclazocine were used to locate delta, mu and kappa opioid binding s i t e s respectively. In addition, the effects of GTP, and sodium and manganese ions on DADLE binding were examined in order to compare the regulation of ga s t r o i n t e s t i n a l opioid receptors to that which has been described for central opioid receptors. 110 METHODS I . TISSUE PREPARATION Male W i s t a r r a t s (250-300 g) , f a s t e d o v e r n i g h t , were a n e s t h e t i z e d w i t h p e n t o b a r b i t a l (60 mg/kg) and p e r f u s e d through the h e a r t w i t h 50 ml of 0.1 M phosphate b u f f e r e d s a l i n e (PBS) pH 7.4, c o n t a i n i n g 1000 u n i t s o f h e p a r i n (Sigma). Male guinea p i g s (350-400 g) were p e r f u s e d w i t h 150 ml PBS. The stomach, duodenum and i l e u m were r a p i d l y d i s s e c t e d , mounted onto c o r k b l o c k s w i t h O.C.T. compound ( T i s s u e Tek I I , embedding media f o r f r o z e n t i s s u e specimens) and f r o z e n i n F r e o n 22 ( i s o p e n t a n e ) which was c o o l e d i n l i q u i d n i t r o g e n . The f r o z e n t i s s u e was p l a c e d i n t o the c r y o s t a t chamber f o r a t l e a s t one hour p r i o r t o c u t t i n g i n o r d e r t o a l l o w the temperature o f the t i s s u e t o e q u i l i b r a t e w i t h t h a t o f the c r y o s t a t (-16°C). S e c t i o n s 18 pm t h i c k were thaw-mounted onto s l i d e s which had been co a t e d w i t h chrome-alum (0.05%) and g e l a t i n ( 0 . 5 % ) . The c u t s e c t i o n s were p l a c e d under vacuum at 4°C o v e r n i g h t t o d r y . The g e n e r a l a r e a s of the r a t g a s t r o i n t e s t i n a l t r a c t which were d i s s e c t e d and examined by a u t o r a d i o g r a p h i c t e c h n i q u e s are demonstrated i n F i g u r e 26. I I . INCUBATION PROCEDURES A. DELTA RECEPTORS D i s t r i b u t i o n o f g a s t r o i n t e s t i n a l o p i o i d d e l t a -3 2 r e c e p t o r s i t e s was d etermined by the b i n d i n g o f [ H]-D-Ala ,D-L e u 5 - e n k e p h a l i n (DADLE) (46.9 Ci/mmol, New England N u c l e a r , I l l B o s t o n , Mass). E s s e n t i a l l y , the i n c u b a t i o n c o n d i t i o n s d e s c r i b e d by Herkenham and P e r t (1982) were f o l l o w e d w i t h the e x c e p t i o n o f some m o d i f i c a t i o n s . The t i s s u e s e c t i o n s were p r e i n c u b a t e d f o r 15 minutes i n 50 mM T r i s - H C l b u f f e r pH 7.4 t o a l l o w removal o f endogenous l i g a n d s i n the t i s s u e which may compete w i t h the b i n d i n g o f l a b e l l e d o p i o i d s . The s e c t i o n s 3 were then c o m p l e t e l y c o v e r e d w i t h 2 - 3 drops o f 24 nM H-DADLE prepared i n T r i s - H C l b u f f e r c o n t a i n i n g 1% BSA. N o n - s p e c i f i c b i n d i n g was determined by i n c u b a t i n g s e q u e n t i a l s e c t i o n s i n the 3 same H-DADLE media but w i t h the a d d i t i o n o f 20 }M u n l a b e l l e d DADLE. A f t e r 30 minutes a t room t e m p e r a t u r e , the i n c u b a t i o n was t e r m i n a t e d by 3 s u c c e s s i v e washes i n 400 ml o f i c e - c o l d T r i s - H C l b u f f e r , each l a s t i n g 20 t o 25 seconds. The s e c t i o n s were i m m e d i a t e l y d r i e d under a f o r c e d stream o f c o l d , d r y a i r and p l a c e d i n t o X-ray c a s s e t t e s i n a p p o s i t i o n t o t r i t i u m s e n s i t i v e f i l m (LKB 2208-190, U l t r o f i l m ) f o r 28 t o 30 days. The c a s s e t t e s were s t o r e d i n d a r k n e s s a t 4 °C. C o n t a c t between f i l m and t i s s u e was m a i n t a i n e d by a p p l y i n g even p r e s s u r e t o the c a s s e t t e s . B. MU-RECEPTORS 3 [N-methyl- H ] - d i h y d r o m o r p h i n e (79 Ci/mmol) or [ N - A l l y l -3 2,3- H]-naloxone (40 Ci/mmol) was used t o i d e n t i f y mu- type r e c e p t o r s i n g a s t r o i n t e s t i n a l t i s s u e . The i n c u b a t i o n procedure was i d e n t i c a l t o t h a t d e s c r i b e d f o r the d e l t a - r e c e p t o r s e x cept 0.1 M phosphate b u f f e r e d s a l i n e (PBS) pH 7.4 was used f o r 3 p r e i n c u b a t i o n s , i n c u b a t i o n s and washes. 5nM H-dihydromorphme 3 . . . and 25 nM H-naloxone were p r e p a r e d i n PBS c o n t a i n i n g 1% BSA. The n o n - s p e c i f i c b i n d i n g was de t e r m i n e d as t h a t b i n d i n g which 1 1 2 o c c u r r e d i n the presence o f 1 0 pM morphine s u l p h a t e or 1 5 0 jM na loxone . C . KAPPA-RECEPTORS 1 9 nM [ 9 - 3 H ] - e t h y l k e t o c y c l a z o c i n e (EKC) ( 1 9 Ci /mmol) prepared i n 5 0 mM T r i s b u f f e r c o n t a i n i n g 1% BSA was employed to determine o p i o i d kappa s i t e s . A l t h o u g h EKC i s a p r o t o t y p i c a l kappa a g o n i s t , i t a l s o demonstrates a f f i n i t y f o r both mu and 3 d e l t a r e c e p t o r s . T h e r e f o r e , i t was n e c e s s a r y to add to the H -EKC s o l u t i o n 3 0 nM morphine and lOOnM DADLE to s e l e c t i v e l y b lock mu r e c e p t o r s and d e l t a r e c e p t o r s r e s p e c t i v e l y . C o n t r o l t i s s u e s e c t i o n s were i n c u b a t e d wi th the same s o l u t i o n to which was added excess ( 2 5 JJM) u n l a b e l l e d EKC methanesu l fonate ( S t e r l i n g - W i n t h r o p Research I n s t i t u t e , R e n s s e l a e r , New Y o r k ) . I n c u b a t i o n , r i n s i n g and d r y i n g p r o c e d u r e s were as d e s c r i b e d f o r the d e l t a r e c e p t o r s . The t i s s u e s e c t i o n s were p l a c e d i n 3 a p p o s i t i o n to the LKB H - u l t r o f i l m for a p e r i o d o f 6 weeks p r i o r to be ing d e v e l o p e d . D . GTP AND CATION SENSITIVITY The c o n d i t i o n s d e s c r i b e d by Bowen et a l . ( 1 9 8 1 ) as be ing optimum f o r v i s u a l i z a t i o n o f Type I G T P - s e n s i t i v e o p i o i d r e c e p t o r s were employed. The t i s s u e was p r e i n c u b a t e d i n 5 0 mM T r i s - H C l pH 7 . 4 w i th the a d d i t i o n o f 1 0 0 mM NaCl and 2 pM GTP (Sigma). The i n c u b a t i o n medium c o n s i s t e d o f T r i s - H C l b u f f e r , 3 mM manganous a c e t a t e , 1 0 0 mM N a C l , 2 yuM GTP, 1% BSA and 2 0 nM 3 H-DADLE. A l l subsequent s teps were c a r r i e d out i n the same manner as f o r d e l t a r e c e p t o r i d e n t i f i c a t i o n . I l l . EXAMINATION OF SLIDES AND FILM 113 A. FILM DEVELOPMENT The LKB- H - U l t r o f i l m was developed u s i n g Kodak D-19 d e v e l o p e r f o r 2 t o 4 minutes depending on the g r a i n d e n s i t y and was f i x e d u s i n g Kodak R a p i d f i x . B. TISSUE STAINING A l l t i s s u e s were examined by h e m a t o x y l i n - e o s i n s t a i n i n g . N u c l e i and o t h e r b a s i c m a t e r i a l were s t a i n e d dark bl u e w h i l e the c y t o p l a s m was s t a i n e d p i n k w i t h e o s i n . S l i d e s were immersed i n h e m a t o x y l i n f o r 4 min and washed under r u n n i n g water f o r 5 min. One s h o r t d i p i n a c i d a l c o h o l was s u f f i c i e n t t o c l e a r the s t a i n from the g l a s s s l i d e . The s e c t i o n s were then dipped 10 times w i t h a g i t a t i o n i n l i t h i u m c a r b o n a t e i n o r d e r t o f i x the h e m a t o x y l i n s t a i n i n g . A f t e r a 10 min r i n s e under r u n n i n g w a t e r , the t i s s u e was s t a i n e d 5 min w i t h e o s i n f o l l o w e d by a r i n s e c o n s i s t i n g o f 3 s h o r t d i p s i n t o r u n n i n g w ater. The s l i d e s were then p l a c e d t h rough a s e r i e s o f graded a l c o h o l s (70%, 90%, 100%) and were c l e a r e d i n x y l e n e b e f o r e e x a m i n a t i o n . C. EXAMINATION OF AUTORADIOGRAPHIC FILM AND SLIDES The s l i d e s and c o r r e s p o n d i n g a u t o r a d i o g r a m s were examined under a l i g h t m i c r o s c o p e and photographed f o r p r e s e n t a t i o n o f c o l o u r p r i n t s . 100 ASA K o d a c o l o u r f i l m was used, and p h o t o m i c r o g r a p h s were t a k e n under b r i g h t , b l u e f i l t e r e d l i g h t . A summary of the a u t o r a d i o g r a p h i c p r o c e d u r e s i s o u t l i n e d s c h e m a t i c a l l y i n F i g u r e 27. 114 FIGURE 26: DIAGRAMATTC REPRESENTATION OF THE RAT GASTROINTESTINAL TRACT, INDICATING THE APPROXIMATE REGIONS FROM WHICH TISSUE WAS TAKEN FOR AUTORADIOGRAPHIC LOCALIZATION OF OPIOID RECEPTORS. The numbers r e f e r t o f i g u r e s of the c o r r e s p o n d i n g a u t o r a d i o g r a m s p r e s e n t e d i n the " R e s u l t s " s e c t i o n o f t h i s c h a p t e r . 115 Examine { Photograph FIGURE 27: DIAGRAMATIC OUTLINE OF THE PROCEDURES INVOLVED IN THE AUTORADIOGRAPHIC LOCALIZATION OF OPIOID RECEPTOR SITES. 116 RESULTS A summary of the autoradiographically determined d i s t r i b u t i o n s of mu, delta and kappa type opioid receptors in the rat gas t r o i n t e s t i n a l t r a c t i s presented in Table 2. Figures 28 to 50 are photographs of autoradiograms in which 3 binding of H-opioid ligands to rat and guinea pig gast r o i n t e s t i n a l tissue can be vis u a l i z e d by dark s i l v e r grains. Unless otherwise stated, the results have been presented in sets of two photographs with the f i r s t being the autoradiographic f i l m which has been superimposed over the hematoxylin-eosin stained tissue section and the second being the corresponding "control" f i l m of an adjacent section which was incubated with excess unlabelled ligand. In some figures, a t h i r d photomicrograph of the H&E stained section alone has been presented in order to provide clearer d e f i n i t i o n of the tissue components. I. RAT GASTROINTESTINAL TRACT A. MU-TYPE OPIOID RECEPTOR DISTRIBUTIONS The binding d i s t r i b u t i o n s of the prototypic mu 3 3 3 receptor ligands, H-naloxone ( H-Nal) and H-dihydromorphine (3H-DHM), in ga s t r o i n t e s t i n a l tissue were found to be i d e n t i c a l . Therefore, photographs of either naloxone or dihydromorphine have been presented with the pa r t i c u l a r ligand employed being denoted in each figure legend. In the rat 3 gastric fundus dark s i l v e r grains, corresponding to [ H]-mu-ligand binding, were found to occur over the c i r c u l a r muscle layer (Figure 28) . This d i s t r i b u t i o n was rather sparce and 117 patchy with some s i l v e r grains appearing to coincide with the areas between muscle bundles as can be seen in the corresponding control section. Binding s i t e s were present in the myenteric plexus between the longitudinal and c i r c u l a r muscle layers (Figure 2 8 ) . S i l v e r grains occurring just beneath the mucosa may represent binding to the submucosal plexus or to the muscularis mucosae (Figures 28 and 29) . A l l binding was determined as s p e c i f i c in that high concentrations of unlabelled ligand completely abolished the dark s i l v e r staining, leaving only the granular appearing background. In the rat corpus, dense mu-ligand binding s i t e s were present throughout the submucosal plexus with the s i l v e r grains c l e a r l y outlining the region of this plexus and scattered throughout the mucosa (Figure 3 0 ) . In c e r t a i n areas of the corpus, binding occurred in the deep muscular plexus; an example of such an area i s presented in Figure 30. In Figure 31, mu-type opioid binding s i t e s are demonstrated to occur in the myenteric plexus and the c i r c u l a r muscle layer of the rat corpus. The d i s t r i b u t i o n of mu-type binding s i t e s in the antrum was found to be very similar to that observed in the corpus. The region demonstrating the highest density of s i l v e r grain accummulation was the submucosal plexus (Figure 32 and 33) with patchy receptor binding occurring in the mucosa. Some regions of the antrum demonstrated binding s i t e s in the deep muscular plexus (Figure 3 2 ) , while in others, none were observed (Figure 3 3 ) . Binding s i t e s were found in the antral myenteric plexus and c i r c u l a r muscle layer, but they occurred less frequently than 3 in the corpus of the stomach. In the duodenum, extensive [ H]-118 3 naloxone and [ H]-dihydromorphine b i n d i n g o c c u r r e d throughout the mucosa, p r i m a r i l y w i t h i n the v i l l i and a t t h e i r b a s e s , p o s s i b l y a s s o c i a t e d w i t h the submucosal p l e x u s ( F i g u r e 34). No b i n d i n g s i t e s were apparent i n the muscle l a y e r s , m y e n t e r i c p l e x u s or Brunner's g l a n d s ( F i g u r e 3 4 ) . S i m i l a r l y , i n the i l e u m , a dense d i s t r i b u t i o n o f s p e c i f i c mu-agonist b i n d i n g s i t e s was l o c a l i z e d i n the mucosa ( F i g u r e 3 5 ) . B. DELTA-TYPE OPIOID RECEPTOR DISTRIBUTIONS D e l t a type o p i o i d r e c e p t o r s i d e n t i f i e d by the 3 b i n d i n g o f [ H]-DADLE were found t o be d i s t r i b u t e d s p a r s e l y i n the c i r c u l a r muscle l a y e r o f the fundus ( F i g u r e 36) , and b i n d i n g s i t e s were a l s o p r e s e n t i n the submucosal p l e x u s ( F i g u r e 37) . I n the corpus and antrum r e g i o n s the submucosal 3 p l e x u s was found t o be the predominant s i t e o f H-DADLE b i n d i n g ( F i g u r e s 38, 39 and 40). S i l v e r g r a i n s were found s c a t t e r e d throughout the mucosa but i n the deep muscular p l e x u s d e l t a -type b i n d i n g s i t e s were not apparent i n a l l a r e a s ( F i g u r e 40). There was no l i g a n d b i n d i n g i n the muscle l a y e r s nor i n the m y e n t e r i c p l e x u s . The muscle o f the p y l o r i c s p h i n c t e r ( F i g u r e 41) was c o m p l e t e l y d e v o i d of d e l t a type b i n d i n g s i t e s nor d i d i t demonstrate any o t h e r o p i o i d a g o n i s t b i n d i n g . I n the 3 . duodenum and i l e u m , i n t e n s e H-DADLE b i n d i n g was r e s t r i c t e d t o the mucosa and the submucosal p l e x u s ( F i g u r e s 42 and 43). C. KAPPA-TYPE OPIOID RECEPTOR DISTIBUTIONS Kappa b i n d i n g s i t e s were v i s u a l i z e d by l a b e l l i n g 3 w i t h H-EKC w h i l e the mu and d e l t a s i t e s were b l o c k e d w i t h morphine and DADLE r e s p e c t i v e l y . S p e c i f i c kappa s i t e s c o u l d not be demonstrated i n the muscle l a y e r s nor i n any p l e x i o f 119 the r a t g a s t r o i n t e s t i n a l t i s s u e s sampled. However, i n a l l r e g i o n s except f o r the f u n d u s , d i f f u s e b i n d i n g o c c u r r e d throughout the mucosa ( F i g u r e s 44 and 45) which c o u l d o n l y be p a r t i a l l y b l o c k e d by the a d d i t i o n of excess u n l a b e l l e d E K C . I I . GUINEA PIG GASTROINTESTINAL TRACT 3 U s i n g H - n a l o x o n e , mu type o p i o i d r e c e p t o r s i t e s were v i s u a l i z e d i n the submucosal p l e x u s o f the gu inea p i g i l e u m , antrum and corpus as dark s i l v e r g r a i n s o u t l i n i n g the p l e x u s (F igures 46 -48) . T h i s dark s t a i n i n g was c o m p l e t e l y a b o l i s h e d by i n c u b a t i o n wi th u n l a b e l l e d n a l o x o n e . In c o n t r a s t to the g r a n u l a r a p p e a r i n g d i s t r i b u t i o n o f d i s c r e t e s i l v e r g r a i n s observed i n the submucosal p l e x u s , a d i f f u s e , but u n i f o r m l y dense s i l v e r g r a i n d i s t r i b u t i o n was p r e s e n t over both l o n g i t u d i n a l and c i r c u l a r muscle l a y e r s , i n c l u d i n g the m y e n t e r i c p l e x u s ( F i g u r e s 46 -48 ) , which was reduced c o n s i d e r a b l y i n the c o n t r o l s e c t i o n s . 3 . . . . . No H-DADLE b i n d i n g to gu inea p i g g a s t r o i n t e s t i n a l t i s s u e c o u l d be v i s u a l i z e d under the c o n d i t i o n s used i n the p a r a l l e l 3 s t u d i e s wi th r a t t i s s u e . Only n o n - d i s p l a c e a b l e H-EKC b i n d i n g to gu inea p i g t i s s u e was o b s e r v e d . I I I . EFFECT OF GTP, N a + , AND M n 2 + ON 3 H-DADLE BINDING In the presence o f 2 uM GTP, 100 mM NaCl and 3 mM M n ( C H 3 c 0 0 ) 2 , s p e c i f i c b i n d i n g o f 3 H-DADLE was c o m p l e t e l y a b o l i s h e d . F i g u r e 49A demonstrates the t y p i c a l d e l t a o p i o i d 3 r e c e p t o r d i s t r i b u t i o n i n the r a t corpus o b t a i n e d wi th H-DADLE, s i l v e r g r a i n s o c c u r r i n g i n the submucosal p l e x u s , deep muscular 120 p l e x u s and mucosa. As i l l u s t r a t e d i n F i g u r e 49B and C, the + 2+ b i n d i n g i n the presence o f GTP, Na and Mn was reduced t o c o n t r o l l e v e l s ( F i g u r e 49C). A s i m i l a r e f f e c t was found i n the r a t i l e u m ( F i g u r e 50) as w e l l as i n antrum, fundus, and duodenum (not shown). 121 REGION FUNDUS D E L T A - T Y P E (DADLE) - C i r c u l a r M u s c l e - S u b m u c o s a l P l e x u s - M y e n t e r i c P l e x u s " M U - T Y P E (NALOXONE/DIHYDROMORPHINE) - C i r c u l a r M u s c l e , possibly between m u s c l e b u n d l e s .'' - M u s c u l a r i s Mucosae -- S u b m u c o s a l P l e x u s ' K A P P A - T Y P E CEKC) - N o n - s p e c i f i c b i n d i n g * o n l y PYLORIC SPHINCTER - S u b m u c o s a l P l e x u s - M u c o s a - D e e p M u s c u l a r P l e x u s - S u b m u c o s a l P l e x u s - M u c o s a - D e e p M u s c u l a r P l e x u s . - V e r y dense i n Mucosa t h r o u g h o u t V i l l i - S u b m u c o s a l P l e x u s - A b s e n t i n D r u n n e r ' s G l a n d - S u b m u c o s a l P l e x u s - C i r c u l a r M u s c l e - M u c o s a - D e e p M u s c u l a r P l e x u s - M y e n t e r i c P l e x u s - S u b m u c o s a l P l e x u s - C i r c u l a r M u s c l e - M u c o s a - D e e p M u s c u l a r P l e x u s i n some a r e a s - M y e n t e r i c P l e x u s - V e r y d e n s e i n Mucosa t h r o u g h o u t v i l l i - S u b m u c o s a l P l e x u s - A b s e n t i n B r u n n e r ' s G l a n d s - M u c o s a , b u t v e r y h i g h n o n -s p e c i f i c b i n d i n g - M u c o s a , b u t v e r y h i g h n o n -s p e c i f i c b i nd i ng - M u c o s a , but v e r y h i g h n o n -s p e c i f i c b i n d i n g - D e n s e in Mucosa . -Dense i n Mucosa - M u c o s a , b u t v e r y h i g h n o n -s p e c i f i c b i nd i ng TABLE I I : SUMMARY OF AUTORADIOGRAPHIC DISTRIBUTIONS OF OPIOID MU-, DELTA-, AND KAPPA-RECEPTORS IN THE RAT GASTRONTESTINAL TRACT. 122 FIGURE 28: PHOTOMICROGRAPHS OF RAT FUNDUS, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. The 3 a u t o r a d i o g r a m d e m o n s t r a t i n g the b i n d i n g o f [ H]-NAL t o a l o n g i t u d i n a l s e c t i o n o f r a t fundus i s superimposed over the c o r r e s p o n d i n g H&E s t a i n e d t i s s u e s e c t i o n ( A ) . Mu-type b i n d i n g s i t e s are p r e s e n t i n the c i r c u l a r muscle (CM), p o s s i b l y between muscle b u n d l e s , and a s s o c i a t e d w i t h the m y e n t e r i c p l e x u s which runs between the o u t e r l o n g i t u d i n a l muscle l a y e r and the i n n e r c i r c u l a r muscle. S i l v e r g r a i n s are p r e s e n t i n the r e g i o n o f the submucosal p l e x u s (SMP), but as th e y a re o r i e n t e d towards the mucosal s u r f a c e , these b i n d i n g s i t e s may oc c u r on the m u s c u l a r i s mucosae (MM) which runs j u s t beneath the mucosal l a y e r . I n (B) , an a d j a c e n t s e c t i o n i s p r e s e n t e d o v e r w h i c h the c o n t r o l a u t o r a d i o g r a m o b t a i n e d i n the presence o f exce s s u n l a b e l l e d NAL i s superimposed. The dense s i l v e r g r a i n s are absent from t h i s c o n t r o l a u t o r a d i o g r a m (B) ; background g r a i n s remain. The H&E s t a i n e d s e c t i o n i s p r e s e n t e d on i t s own (C) i n ord e r t h a t the s t r u c t u r a l arrangement o f the t i s s u e can be more r e a d i l y o b s e r v e d . (x 40) 123 124 FIGURE 29: PHOTOMICROGRAPHS OF RAT FUNDUS, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. (A) I n t h i s 3 c r o s s - s e c t i o n , s i l v e r g r a i n s r e p r e s e n t i n g the b i n d i n g o f [ H ] -DHM are l o c a t e d t h r o u g h o u t the submucosal p l e x u s (SMP) w i t h some i n the m u s c u l a r i s mucosae. B i n d i n g s i t e s are a l s o s c a t t e r e d through the c i r c u l a r muscle l a y e r (CM). (B) The dense s i l v e r g r a i n s are not p r e s e n t i n the a u t o r a d i o g r a m o f the a d j a c e n t t i s s u e i n c u b a t e d i n the presence o f exce s s u n l a b e l l e d DHM. (x40) 126 FIGURE 30: PHOTOMICROGRAPHS OF RAT CORPUS, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. (A) B i n d i n g 3 o f [ H]-NAL t o a t a n g e n t i a l l y c u t s e c t i o n o f r a t corpus t i s s u e o c c u r s as a dense network o f s i l v e r g r a i n s c l e a r l y o u t l i n i n g the submucosal p l e x u s (SMP) and the deep muscular p l e x u s (DMP) which u n d e r l i e s the c i r c u l a r muscle. S c a t t e r e d b i n d i n g s i t e s are l o c a t e d i n the mucosa. I n the c o n s e c u t i v e s e c t i o n employed as c o n t r o l , the dense s i l v e r g r a i n s a re c o m p l e t e l y absent (B) . (x40) 128 FIGURE 31: PHOTOMICROGRAPHS OF RAT CORPUS, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. Dense s i l v e r 3 g r a i n s which r e p r e s e n t the b i n d i n g o f [ H]-NAL t o a l o n g i t u d i n a l s e c t i o n through r a t co r p u s t i s s u e are p r e s e n t i n the m y e n t e r i c p l e x u s (MP). The l o n g i t u d i n a l muscle may a l s o p ossess mu-type b i n d i n g s i t e s , but i t i s not p o s s i b l e t o c l e a r l y r e s o l v e t h e i r d i s t r i b u t i o n w i t h l i g h t m i c r o s c o p i c e x a m i n a t i o n . Some b i n d i n g s i t e s are p r e s e n t i n the c i r c u l a r muscle l a y e r (CM). A g a i n , dense s i l v e r g r a i n s a re l o c a t e d i n the submucosal p l e x u s and s c a t t e r e d i n the mucosal r e g i o n (Muc). The c o n t r o l s e c t i o n (B) demonstrates no s i l v e r g r a i n s above background l e v e l s . (x40) 129 130 FIGURE 32: PHOTOMICROGRAPHS OF RAT ANTRUM, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. Binding of 3 [ H]-NAL to a tangentially cut cross-section of rat antrum (A) can be observed i n the submucosal plexus, mucosa (Muc) and the deep muscular plexus (DMP) which i s displaced by excess unlabelled naloxone (B). (x 40) 131 132 FIGURE 33: PHOTOMICROGRAPHS OF RAT ANTRUM, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. S i l v e r grains 3 which demonstrate the binding of [ H]-NAL to a tangentially cut cross-section of rat antrum (A) are most predominant in the submucosal plexus (SMP). Scattered binding s i t e s are present in the mucosa, c i r c u l a r muscle, and the myenteric plexus or possibly longitudinal muscle (LM). An adjacent section, employed as the control (B), demonstrates no dark s i l v e r grains. The orientation of the tissue can be determined from the micrograph of the corresponding H&E stained section (C) . (x 40) 134 FIGURE 34: PHOTOMICROGRAPHS OF RAT DUODENUM, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. 3 B i n d i n g o f [ H]-DHM t o a c r o s s - s e c t i o n through r a t duodenal t i s s u e o c c u r s t h r o u g h o u t the mucosa (A) . S i l v e r g r a i n s are l o c a t e d a t the base and extend through the c o r e t o the apex o f the v i l l i . The Brun n e r ' s g l a n d (BG) i s surrounded by mu-type b i n d i n g s i t e s , but none are obser v e d w i t h i n the g l a n d . The c o n t r o l a u t o r a d i o g r a m d i s p l a y s no dark s i l v e r g r a i n s (B) . The o r i e n t a t i o n o f the t i s s u e i s demonstrated by the 'S' which denotes the s e r o s a l s u r f a c e . (x 40) 136 FIGURE 35: PHOTOMICROGRAPHS OF RAT ILEUM, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. Dark s i l v e r 3 g r a i n s i n d i c a t i n g the b i n d i n g o f [ H]-NAL t o c r o s s - s e c t i o n a l l y c u t r a t i l e a l t i s s u e o c c u r t h r o u g h o u t the mucosal v i l l i , c o n c e n t r a t e d a t the apex o f each v i l l i ( A ) . Some b i n d i n g s i t e s are s c a t t e r e d i n the submucosal p l e x u s . No dark s i l v e r g r a i n s are p r e s e n t i n the muscle l a y e r s . I n c u b a t i o n af an a d j a c e n t s e c t i o n w i t h e x c e s s u n l a b e l l e d NAL c o m p l e t e l y a b o l i s h e d the oc c u r r e n c e o f dark s i l v e r g r a i n s (B) . The s e r o s a l s u r f a c e i s marked by an 'S*. (x 40) 138 FIGURE 36: PHOTOMICROGRAPHS OF RAT FUNDUS, DEMONSTRATING DELTA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. (A) A spar s e d i s t r i b u t i o n o f s i l v e r g r a i n s r e p r e s e n t i n g the b i n d i n g 3 of [ H]-DADLE t o a t a n g e n t i a l l y c u t s e c t i o n i s observed i n the c i r c u l a r muscle l a y e r (cm), and p o s s i b l y i n the l o n g i t u d i n a l muscle. A few b i n d i n g s i t e s can be seen i n the submucosal p l e x u s (smp) . The dark g r a i n s a re absent from the au t o r a d i o g r a m of the c o n t r o l t i s s u e ( B ) . (x 40) CM 140 FIGURE 37: PHOTOMICROGRAPHS OF RAT FUNDUS, DEMONSTRATING DELTA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. I n ( A ) , 3 the a u t o r a d i o g r a m demonstrates [ H]-DADLE b i n d i n g t o the submucosal p l e x u s (smp) and the m u s c u l a r i s mucosae (mm) o f c r o s s - s e c t i o n a l l y c u t r a t fundus. I n the c o n t r o l s e c t i o n (B) , no dark s i l v e r g r a i n s a re e v i d e n t . (x 40) 141 A 142 FIGURE 38: PHOTOMICROGRAPHS OF RAT CORPUS, DEMONSTRATING DELTA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. [ 3H]-DADLE b i n d i n g s i t e s c l e a r l y o u t l i n e the submucosal p l e x u s (smp) o f the r a t corpus (A) and are s c a t t e r e d t h rough the mucosa (muc). No dark s i l v e r g r a i n s are p r e s e n t i n the muscle l a y e r s . I n the c o n t r o l a u t o r a d i o g r a m (B) , the s i l v e r g r a i n s a re reduced t o background i n t e n s i t y . The t i s s u e i s c u t t a n g e n t i a l l y . (x 40) 143 144 FIGURE 39: PHOTOMICROGRAPHS OF RAT CORPUS, DEMONSTRATING DELTA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. Dark s i l v e r g r a i n s which i n d i c a t e d e l t a - t y p e b i n d i n g s i t e s a r e l o c a l i z e d i n the submucosal p l e x u s (smp) and s p a r s e l y i n the mucosal r e g i o n (muc). A u t o r a d i o g r a p h i c g r a i n s a re i n t e n s e i n the submucosal p l e x u s g a n g l i a denoted by the l i g h t arrow heads (g) . Some may be a s s o c i a t e d w i t h the o v e r l y i n g deep muscular p l e x u s . No s i l v e r g r a i n s o c c u r i n the c i r c u l a r muscle (cm) . Dark s i l v e r g r a i n s a re absent i n the c o n t r o l a u t o r a d i o g r a m (B) of an a d j a c e n t s e c t i o n . The s t r u c t u r a l o r g a n i z a t i o n o f the r a t corpus i s i n d i c a t e d i n ( C ) , the H&E s t a i n e d l o n g i t u d i n a l / t a n g e n t i a l s e c t i o n c o r r e s p o n d i n g t o the aut o r a d i o g r a m p r e s e n t e d i n (A). (x 40) FIGURE 40: PHOTOMICROGRAPHS OF RAT ANTRUM, DEMONSTRATING DELTA OPIOID BINDING SITES BY AUTORADIOGRAPHY. (A) [ 3H]-DADLE b i n d i n g s i t e s are p r e s e n t i n the mucosa (muc), submucosal p l e x u s (smp) and deep muscular p l e x u s (dmp). (B) Dark s i l v e r g r a i n s are absent from a u t o r a d i o g r a m s o f c o n t r o l s e c t i o n s . (C) The o r g a n i z a t i o n o f the t i s s u e can be seen i n the H&E s t a i n e d s e c t i o n o f the s e l o n g i t u d i n a l s e c t i o n s through the r a t antrum (C) . (x 40) 148 FIGURE 41: PHOTOMICROGRAPHS OF RAT PYLORUS REGION, DEMONSTRATING DELTA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. These are l o n g i t u d i n a l s e c t i o n s through the p y l o r i c r e g i o n such t h a t the a n t r a l mucosa i s p r e s e n t i n the lower r i g h t and the duodenal mucosa o c c u r s i n the upper l e f t q u adrant. The p y l o r i c s p h i n c t e r (PS) i s c o m p l e t e l y d e v o i d o f 3 [ H]-DADLE b i n d i n g s i t e s but are p r e s e n t i n the duodenal mucosa (A). The s e r o s a l s u r f a c e i s i n d i c a t e d by the 'S' i n the c o r r e s p o n d i n g c o n t r o l s e c t i o n ( B ) . (x 40) 150 FIGURE 42: PHOTOMICROGRAPHS OF RAT DUODENUM, DEMONSTRATING DELTA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. The mucosa o f the r a t duodenum i s marked by a 3 dense d i s t r i b u t i o n o f [ H]-DADLE b i n d i n g s i t e s ( A). S i l v e r g r a i n s are l o c a t e d w i t h i n the v i l l i , but none are p r e s e n t i n the muscle l a y e r s on the s e r o s a l s i d e ( S ) . I n a s e r i a l c r o s s -s e c t i o n used as a c o n t r o l , no dark s i l v e r g r a i n s a re p r e s e n t (B). (x 40) 151 152 FIGURE 43: PHOTOMICROGRAPHS OF RAT ILEUM, DEMONSTRATING DELTA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. Dark s i l v e r g r a i n s i n d i c a t i n g the l o c a t i o n o f d e l t a o p i o i d b i n d i n g s i t e s are p r e s e n t t h r o u g h o u t the i l e a l mucosa (A) w i t h g r a i n s c o n c e n t r a t e d a t the apex o f v i l l i and a t t h e i r b a s e s . I n f r e q u e n t b i n d i n g s i t e s can be observed i n the muscle l a y e r which o c c u r s towards the s e r o s a l s i d e (S) . The d e n s i t y o f s i l v e r g r a i n s i s reduced t o background l e v e l s i n the c o r r e s p o n d i n g c o n t r o l l o n g i t u d i n a l s e c t i o n ( B ) . (x 40) 153 154 FIGURE 44: PHOTOMICROGRAPHS OF RAT ILEUM, DEMONSTRATING KAPPA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. The dense, d i f f u s e b i n d i n g o f [3H]-EKC t o r a t i l e a l mucosa (A) i s o n l y s l i g h t l y d i m i n i s h e d i n the a d j a c e n t c o n t r o l s e c t i o n (B) . The o r g a n i z a t i o n o f the i l e a l t i s s u e i s p r e s e n t e d i n (C) by the H&E s t a i n e d l o n g i t u d i n a l s e c t i o n . (x 40) 155 156 FIGURE 45: PHOTOMICROGRAPHS OF RAT CORPUS, DEMONSTRATING KAPPA-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. Dense, 3 n o n - s p e c i f i c b i n d i n g o f [ H]-EKC t o mucosa i s demonstrated i n t h i s c r o s s - s e c t i o n through r a t stomach c o r p u s (A) . The i n t e n s i t y o f b i n d i n g i s u n a f f e c t e d by e x c e s s u n l a b e l l e d EKC (B) . The s e r o s a l s u r f a c e i s i n d i c a t e d by the 'S' . (x 40) 158 FIGURE 46: . PHOTOMICROGRAPHS OF GUINEA PIG ILEUM, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. The b i n d i n g o f [3H]-NAL i s obser v e d i n a "patchy" manner i n the submucosal p l e x u s (SMP), d e m o n s t r a t i n g a d i s t i n c t g r a n u l a r appearance ( A ) . The b i n d i n g i n the muscle l a y e r s (ML) i s a l s o dense, but demonstrates a much more " d i f f u s e " d i s t r i b u t i o n , o c c u r r i n g over both the c i r c u l a r and l o n g i t u d i n a l muscle l a y e r s . The s i l v e r g r a i n s a r e absent i n the submucosal p l e x u s and t h e i r d e n s i t y g r e a t l y reduced i n the muscle l a y e r o f the c o n t r o l s e c t i o n (B) . Both (A) and (B) are t a n g e n t i a l l y c u t s e c t i o n s . (x 40) A 160 FIGURE 47: PHOTOMICROGRAPHS OF GUINEA PIG ANTRUM, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. •5 Dark s i l v e r g r a i n s r e p r e s e n t i n g the b i n d i n g o f [ H]-NAL are p r e s e n t i n the submucosal p l e x u s (smp) o f the g u i n e a p i g antrum (A) . A d i f f u s e d i s t r i b u t i o n o f dense s i l v e r g r a i n s can be observed over the c i r c u l a r muscle (cm) and, o f g r e a t e r i n t e n s i t y , over the l o n g i t u d i n a l muscle. The s i l v e r g r a i n s are reduced t o background l e v e l s i n the c o r r e s p o n d i n g c o n t r o l s e c t i o n ( B ) . The t i s s u e i s c u t t a n g e n t i a l l y . (x 40) S M P 162 FIGURE 48: PHOTOMICROGRAPHS OF GUINEA PIG CORPUS, DEMONSTRATING MU-TYPE OPIOID BINDING SITES BY AUTORADIOGRAPHY. 3 Dense s i l v e r g r a i n s r e p r e s e n t i n g the b i n d i n g o f [ H]-NAL i s l o c a l i z e d t o the submucosal p l e x u s (smp) i n a "patchy" manner, 'while the b i n d i n g i n the muscle l a y e r s (ml) i s " d i f f u s e " (A) . The dark g r a i n s i n the submucosal p l e x u s and the muscle l a y e r s i s g r e a t l y reduced i n the a d j a c e n t s e c t i o n (B) which s e r v e s as a c o n t r o l . The t i s s u e i s t a n g e n t i a l l y c u t . (x 40) 164 FIGURE 49: PHOTOMICROGRAPHS OF RAT CORPUS, DEMONSTRATING THE EFFECT OF GTP, SODIUM AND MANGANESE ON DELTA-TYPE OPIOID BINDING SITES. The c h a r a c t e r i s t i c d i s t r i b u t i o n o f [ 3H]-DADLE b i n d i n g i s g i v e n i n ( A ) , w i t h s i l v e r g r a i n s o c c u r r i n g i n the submucosal p l e x u s (smp), deep muscular p l e x u s (dmp) and the mucosa (A). I n the presence o f 2 uM GTP, 100 mM N a C l , and 3 mM manganese a c e t a t e (B) , the i n t e n s i t y o f s i l v e r g r a i n s i s not d i f f e r e n t from t h a t o f the c o n t r o l s e c t i o n s (C) . These are t a n g e n t i a l l y c u t c r o s s - s e c t i o n s . (x 40) 165 166 FIGURE 50: PHOTOMICROGRAPHS OF RAT ILEUM, DEMONSTRATING THE EFFECT OF GTP, SODIUM, AND MANGANESE ON DELTA-TYPE BINDING SITES. The d e l t a - t y p e o p i o i d b i n d i n g s i t e s i n the mucosa o f the r a t i l e u m (A) i s c o m p l e t e l y a b o l i s h e d i n the presence o f 2 uM GTP, 100 mM NaCl and 3 mM manganese a c e t a t e (B) , a p p e a r i n g no d i f f e r e n t from the c o n t r o l a u t o r a d i o g r a m (C) . These are t a n g e n t i a l l y c u t l o n g i t u d i n a l s e c t i o n s o f r a t i l e u m . (x 40) 168 DISCUSSION The d e m o n s t r a t i o n o f the presence o f s p e c i f i c o p i o i d b i n d i n g s i t e s t h r o u g h o u t the r a t and g u i n e a p i g g a s t r o i n t e s t i n a l t r a c t s i s i n d i c a t i v e o f a widesp r e a d r o l e f o r o p i o i d p e p t i d e s i n the r e g u l a t i o n o f g a s t r o i n t e s t i n a l f u n c t i o n . However, the s i g n i f i c a n c e o f these f i n d i n g s must ta k e i n t o c o n s i d e r a t i o n the d i f f e r e n c e between a u t o r a d i o g r a p h i c b i n d i n g s i t e s and p h y s i o l o g i c a l r e c e p t o r s . R e c e p t o r s f u n c t i o n not o n l y as r e c o g n i t i o n s i t e s f o r t h e i r s p e c i f i c l i g a n d but must a l s o u l t i m a t e l y t r a n s l a t e the b i n d i n g event t o a b i o l o g i c a l r e s p o nse. A u t o r a d i o g r a p h i c b i n d i n g s t u d i e s p r o v i d e e s s e n t i a l i n f o r m a t i o n c o n c e r n i n g the d i s t r i b u t i o n and l o c a l i z a t i o n o f l i g a n d r e c o g n i t i o n s i t e s , b u t the s e do not n e c e s s a r i l y r e p r e s e n t f u n c t i o n a l r e c e p t o r s . O p i o i d r e c e p t o r t y p e s are f u r t h e r d e f i n e d by t h e i r l i g a n d s p e c i f i c i t i e s . A l t h o u g h p r o t o t y p i c delta-(DADLE) , mu-(Nal o r DHM) and kappa-(EKC) a g o n i s t s were employed, some degree o f c r o s s r e a c t i o n amongst r e c e p t o r t y p e s i s i n e v i t a b l e . T h e r e f o r e , i n t h i s c h a p t e r , two b a s i c assumptions have been made when d e s c r i b i n g the a u t o r a d i o g r a p h i c g r a i n s as r e p r e s e n t i n g d e l t a , mu or kappa r e c e p t o r s : f i r s t l y t h a t the b i n d i n g s i t e s c o r r e s p o n d t o r e c e p t o r s i t e s , and s e c o n d l y t h a t mu, d e l t a and kappa o p i o i d l i g a n d s p r e d o m i n a n t l y r e c o g n i z e t h e i r r e s p e c t i v e r e c e p t o r t y p e s . I n t h i s s t u d y , the l o c a l i z a t i o n s o f o p i o i d r e c e p t o r s t o s p e c i f i c s t r u c t u r e s i n the g a s t r o i n t e s t i n a l w a l l have been assumed as s i t e s o f o p i o i d p e p t i d e a c t i o n from which s p e c u l a t i o n s as t o t h e i r f u n c t i o n s have been made. I n the r a t g a s t r o i n t e s t i n a l t r a c t , the fundus o f the stomach was the p r i m a r y r e g i o n i n which o p i o i d r e c e p t o r s were d e t e c t e d i n smooth muscle. B o t h mu- and d e l t a - b i n d i n g s i t e s o c c u r r e d i n the c i r c u l a r muscle l a y e r , p o s s i b l y l o c a t e d between the muscle b u n d l e s . Some a u t o r a d i o g r a p h i c g r a i n s were found t o be a s s o c i a t e d w i t h the muscular i s mucosae, a t h i n band of smooth muscle which o c c u r s a t the base o f the mucosa. I n the corpus and antrum r e g i o n s , mu-, but not d e l t a - t y p e o p i o i d r e c e p t o r s were found t o be p r e s e n t i n c i r c u l a r smooth muscle. These b i n d i n g s i t e s i n the c o r p u s and a n t r a l muscle l a y e r were s p a r s e l y d i s t r i b u t e d , an o b s e r v a t i o n which may have r e s u l t e d from the p l a n e o f t i s s u e s e c t i o n . O p i o i d r e c e p t o r s i n the c i r c u l a r muscle are presumed t o be the t a r g e t s i t e s o f o p i o i d p e p t i d e s r e l e a s e d from e n k e p h a l i n e r g i c nerve t e r m i n a l s , a r i s i n g from the m y e n t e r i c p l e x u s and t r a n s v e r s i n g t h rough the muscle l a y e r . T h e r e f o r e , depending on the p l a n e o f s e c t i o n , t e r m i n a t i o n s o f these neurons may or may not be apparent i n a p a r t i c u l a r t i s s u e s e c t i o n . These o p i o i d p e p t i d e r e c o g n i t i o n s i t e s on smooth muscle o f the fundus would be e x p e c t e d t o be i n v o l v e d i n the r e g u l a t i o n o f m o t i l i t y . Moreover, B i t a r and Makhlouf (1982) have demonstrated f u n c t i o n a l o p i o i d r e c e p t o r s on i s o l a t e d g u i n e a p i g g a s t r i c smooth muscle c e l l s . C o n t r a c t i o n s i n response t o a c t i v a t i o n o f the o p i o i d r e c e p t o r s were measured by i m a g e - s p l i t t i n g m i c r o m e t r y and a rank o r d e r o f potency was determined f o r d i f f e r e n t o p i o i d l i g a n d s . D y n o r p h i n e x h i b i t e d the h i g h e s t p o t e n c y , f o l l o w e d by m e t - e n k e p h a l i n , morphine and l e u - e n k e p h a l i n . A l t h o u g h the e x a c t r e g i o n o f the stomach from which these c e l l s were i s o l a t e d was not r e p o r t e d , 170 the a u t o r a d i o g r a p h i c l o c a l i z a t i o n o f mu and d e l t a o p i o i d b i n d i n g s i t e s i n the r a t fundus i s c o n s i s t e n t w i t h t h e i r f i n d i n g s . A l t h o u g h kappa o p i o i d b i n d i n g s i t e s i n the r a t or guin e a p i g stomach c o u l d not be demonstrated 3 a u t o r a d i o g r a p h i c a l l y u s i n g H-EKC, i t does not n e c e s s a r i l y s i g n i f y the absence o f kappa r e c e p t o r s i n t h i s t i s s u e . S i n c e the c o n c e n t r a t i o n s o f mu- and d e l t a - l i g a n d s used t o b l o c k t h e i r r e s p e c t i v e r e c e p t o r s were based on p r e v i o u s s t u d i e s , examining the d i s t r i b u t i o n o f kappa r e c e p t o r s i n b r a i n t i s s u e , i t i s p o s s i b l e t h a t these c o n c e n t r a t i o n s o f u n l a b e l l e d o p i o i d l i g a n d s were s u f f i c i e n t l y h i g h enough t o b l o c k the kappa-type r e c e p t o r s as w e l l . A l t e r n a t i v e l y , the use o f a d i f f e r e n t l i g a n d such as 3 H-dynorphin may prove more s u c c e s s f u l i n the a u t o r a d i o g r a p h i c l o c a l i z a t i o n o f kappa-type o p i o i d r e c e p t o r s i n the g a s t r o i n t e s t i n a l t r a c t . The presence o f o p i o i d b i n d i n g s i t e s i n the r a t f u n d i c submucous a r e a , e i t h e r a s s o c i a t e d w i t h the submucosal p l e x u s or the m u s c u l a r i s mucosae s u g g e s t s t h a t i n a d d i t i o n t o d i r e c t e f f e c t s on smooth m u s c l e , the motor responses t o o p i o i d s i n the f u n d i c r e g i o n may a l s o be mediated by i n d i r e c t e f f e c t s t h r ough submucosal neurons. The absence o f o p i o i d r e c e p t o r s i n smooth muscle of the remainder o f the r a t g a s t r o i n t e s t i n a l t r a c t was somewhat s u r p r i s i n g i n view o f immunocytochemical s t u d i e s which have demonstrated e x t e n s i v e systems o f e n k e p h a l i n and d y n o r p h i n immunoreactive nerve f i b r e s i n the c i r c u l a r muscle o f the r a t g a s t r o i n t e s t i n a l t r a c t (Bu'Lock e t a l . , 1983; Wang e t a l . , 1984; Watson e t a l . , 1981; V i n c e n t e t a l . , 1984). The muscle i n the p y l o r i c r e g i o n o f the f e l i n e ( E d i n e t a l . , 1980; L o l o v a ,171 et a l . , 1984), rat and guinea pig (Vincent et a l . , 1984) stomachs have been found to contain a dense network of enkephalin immunoreative f i b r e s , but no s i g n i f i c a n t opioid ligand binding was observed in the muscle of the p y l o r i c sphincter. The l o c a l i z a t i o n of high densities of autoradiographic grains corresponding to the binding of mu and delta opioid ligands in the submucosal plexus of the rat antrum and corpus was also unexpected since very few nerve bodies and fibres immunoreactive for enkephalins have been observed to occur in the submucosal plexus of the rat antrum and corpus (Wang et a l . , 1984). In contrast, most enkephalin-IR c e l l bodies and f i b r e s have been found in the myenteric plexus (Schultzberg et a l . , 1980; Furness et a l . , 1983; Bu'Lock et a l . , 1983; Wang et a l . , 1984). In the present study some mu-, but not delta-, binding s i t e s were i d e n t i f i e d in the myenteric plexus. There i s , therefore, an apparent incongruity between opioid receptor d i s t r i b u t i o n s and previously described opioid peptide d i s t r i b u t i o n s . Enkephalinergic f i b r e s have been found to occur primarily i n the myenteric plexus and c i r c u l a r muscle layer, and infrequently in the submucosal plexus and mucosa; whereas, opioid receptors were found to be most prominant in the submucosal plexus and mucosa, occurring only occasionally in the c i r c u l a r muscle and myenteric plexus. Although extensive enkephalin-immunoreactivity containing neuronal networks have been described in the c i r c u l a r muscle, the exact terminations of these f i b r e s are not c e r t a i n . It i s therefore possible that the majority of enkephalinergic f i b r e s in the muscle layer do not terminate on smooth muscle c e l l s but rather ,0.72 r e p r e s e n t p r o c e s s e s from the m y e n t e r i c p l e x u s which t r a v e r s e the muscle t o make s y n a p t i c c o n t a c t w i t h the neurons i n the submucosal p l e x u s . A l t e r n a t i v e l y , r e c e p t o r s which mediate the e f f e c t s o f o p i o i d p e p t i d e s may be d i f f u s e l y d i s t r i b u t e d i n comparison w i t h those i n the p l e x i . The e l e c t r i c a l l y c o u p l e d smooth muscle c e l l s may r e q u i r e the a c t i v a t i o n o f few r e c e p t o r s t o i n i t i a t e a c o n t r a c t i l e r e s p o n s e . However, d i s t i n c t " d i f f u s e " b i n d i n g o f [ H]-naloxone was o b s e r v e d i n smooth muscle o f the g u i n e a p i g g a s t r o i n t e s t i n a l t i s s u e , which suggests t h e r e may be a s p e c i e s r e l a t e d d i f f e r e n c e i n o p i o i d r e c e p t o r d i s t r i b u t i o n s . M u - o p i o i d r e c e p t o r s may t h e r e f o r e p l a y a more i m p o r t a n t r o l e i n the r e g u l a t i o n o f g a s t r o i n t e s t i n a l motor a c t i v i t y i n the g u i n e a p i g than i n the r a t . S i n c e o p i o i d p e p t i d e s have been shown t o i n h i b i t the f i r i n g o f m y e n t e r i c neurons i n the g u i n e a p i g i l e u m (North and W i l l i a m s , 1976; N o r t h e t a l . , 1982; N o r t h and Egan, 1983), mu-r e c e p t o r s were expected i n the m y e n t e r i c p l e x u s o f t h i s t i s s u e . Due t o the d i f f u s e d i s t r i b u t i o n o f s i l v e r g r a i n s over the l o n g i t u d i n a l and c i r c u l a r muscle l a y e r s i t was not p o s s i b l e t o d i s t i n g u i s h d i s t i n c t r e c e p t o r s l o c a t e d s p e c i f i c a l l y i n the m y e n t e r i c p l e x u s . However, i t appeared t h a t d i f f u s e b i n d i n g s i t e s o c c u r r e d e v e n l y over the p l e x u s t h r o u g h o u t the g u i n e a p i g g a s t r o i n t e s t i n a l t r a c t . I n the r a t , the corpus and antrum were the o n l y r e g i o n s i n which any o p i o i d b i n d i n g s i t e s c o u l d be l o c a l i z e d i n the m y e n t e r i c p l e x u s , and these were r e s t r i c t e d t o m u - r e c e p t o r s . No b i n d i n g s i t e s o c c u r r e d i n the m y e n t e r i c p l e x u s o f the r a t i l e u m , s u g g e s t i n g t h a t o p i o i d i n h i b i t i o n o f m y e n t e r i c neurons may not n e c e s s a r i l y a p p l y t o a l l s p e c i e s and 173 t o a l l r e g i o n s o f the g a s t r o i n t e s t i n a l t r a c t . Moreover, s i n c e mu-receptors have been found t o be the most prominant type i n the g u i n e a p i g ( K o s t e r l i t z and W a t e r f i e l d , 1975; L o r d e t a l . , 1977) and s i n c e o n l y mu-binding s i t e s were a s s o c i a t e d w i t h the m y e n t e r i c p l e x u s o f the r a t co r p u s and antrum, t h i s i n h i b i t o y e f f e c t o f o p i o i d p e p t i d e s on m y e n t e r i c neurons may be e x c l u s i v e l y mediated by m u - r e c e p t o r s . The reason f o r the apparent d i s c r e p e n c i e s between o p i o i d p e p t i d e and r e c e p t o r d i s t r i b u t i o n s might be c l a r i f i e d by c o n d u c t i n g immunocytochemical and a u t o r a d i o g r a p h i c s t u d i e s on s e r i a l t i s s u e s e c t i o n . I n t h i s manner, a c o r r e l a t i o n between the a n a t o m i c a l d i s t r i b u t i o n s o f o p i o i d r e c e p t o r s and p e p t i d e s c o u l d be made. However, an a p p r o p r i a t e means o f f i x a t i o n must be developed such t h a t the t i s s u e i s f i x e d f o r ICC w i t h o u t damaging or changing the p r o p e r t i e s o f the b i n d i n g s i t e s . The e x t e n s i v e d i s t r i b u t i o n o f " p a t c h e s " o f mu and d e l t a o p i o i d r e c e p t o r s i n the mucosa o f the r a t g a s t r o i n t e s t i n a l t r a c t i s s u g g e s t i v e o f a r o l e f o r o p i o i d p e p t i d e s i n the r e g u l a t i o n o f mucosal f u n c t i o n . I n the stomach, o p i o i d p e p t i d e s have been found t o a f f e c t g a s t r i c a c i d s e c r e t i o n (see Chapter 1, s e c t i o n V I I I ) . S i n c e an i n c r e a s e i n serum g a s t r i n l e v e l s does not n e c e s s a r i l y accompany e n k e p h a l i n - i n d u c e d s t i m u l a t i o n o f a c i d s e c r e t i o n , a d i r e c t a f f e c t on o x y n t i c c e l l s has been proposed (Konturek, 1981b). The r e s o l u t i o n o f t h i s l i g h t m i c r o s c o p i c a u t o r a d i o g r a p h i c t e c h n i q u e i s not s u f f i c i e n t t o d i s t i n g u i s h the c e l l t y p e s a s s o c i a t e d w i t h the s i l v e r g r a i n s . F i n e s t r u c t u r a l l o c a l i z a t i o n o f the b i n d i n g s i t e s r e q u i r e s v i s u a l i z a t i o n by e l e c t r o n m i c r o s c o p i c a u t o r a d i o g r a p h y . 174 N e v e r t h e l e s s , the p a t c h y d i s t r i b u t i o n o f a u t o r a d i o g r a p h i c g r a i n s i n the mucosa of the c o r pus s u g g e s t s these s i t e s may mediate the observed e f f e c t s o f e n k e p h a l i n s on a c i d s e c r e t i o n . O p i o i d b i n d i n g s i t e s i n the mucosa of the a n t r a l r e g i o n may be a s s o c i a t e d w i t h e n d o c r i n e c e l l s , p o s s i b l y s o m a t o s t a t i n s e c r e t i n g D - c e l l s as w i l l be suggested i n Chapter 6. The presence of e n d o c r i n e c e l l s c o n t a i n i n g e n k e p h a l i n - I R ( L a r s s o n and S t e n g a a r d - P e d e r s e n , 1981; Bu'Lock e t a l . , 1983; G i r a u d e t a l . , 1984a; J o n s s o n , 1985) i n r a t and p o r c i n e antrum s u g g e s t s t h a t e n k e p h a l i n s may a c t i n e i t h e r a p a r a c r i n e or e n d o c r i n e manner t o d i r e c t l y a c t i v a t e o p i o i d r e c e p t o r s i n the a n t r a l mucosa. Immunocytochemical e x a m i n a t i o n s of the mucosal d i s t r i b u t i o n s o f e n k e p h a l i n e r g i c neurons i n the r a t stomach have e i t h e r f a i l e d t o l o c a t e f i b r e s (Alumets e t a l . , 1978; V i n c e n t e t a l . , 1984) or have observed o n l y s c a n t immunoreactive f i b r e s (Wang e t a l . , 1984). T h e r e f o r e , a n e u r a l l y mediated a c t i v a t i o n o f g a s t r i c mucosal o p i o i d r e c e p t o r s i s l e s s p r o b a b l e . I n the r a t i l e u m and duodenum, the s t r i k i n g l y dense d i s t r i b u t i o n o f mu- and d e l t a - b i n d i n g s i t e s i n the mucosa, throughout the v i l l i , i n d i c a t e s t h a t o p i o i d s are p r o b a b l y i m p o r t a n t r e g u l a t o r s o f i n t e s t i n a l f u n c t i o n s . T h i s f i n d i n g i s i n agreement w i t h the r e p o r t by Dashwood e t a l . (1985) who 3 3 have d e s c r i b e d the b i n d i n g o f [ H]-naloxone and [ H ] -d i hydromorphine t o v i l l i and c r y p t s i n the r a t s m a l l i n t e s t i n e . Thus the o c c u r r e n c e o f o p i o i d mu- and d e l t a - r e c e p t o r s l o c a l i z e d t o the mucosa of r a t duodenum and i l e u m tend t o support r e c e n t i n d i c a t i o n s t h a t o p i o i d p e p t i d e s are i n v o l v e d i n 175 the regulation of water and e l e c t r o l y t e transport across the guinea pig and rabbit i l e a l mucosa (Dobbins et a l . , 1980; Kachur et a l . , 1980; McKay et a l . , 1981; Kachur and M i l l e r , 1982; Vinayek et a l . , 1983; Binder et a l . , 1984). In the present study, s i l v e r grains occurred within the core of the v i l l i and were concentrated at the t i p s , suggesting that these binding s i t e s may be associated with mucosal neurons to i n d i r e c t l y a f f e c t i n t e s t i n a l absorption or with i n t e s t i n a l e p i t h e l i a l c e l l s to mediate d i r e c t e f f e c t s . The source of the endogenous opioid peptides which would act at these i n t e s t i n a l receptors i s unknown. Although extensive VIP-containing neuronal networks have been demonstrated to occur within i n t e s t i n a l v i l l i (Buchan et a l . , 1985), enkephalinergic nerve fibres have not been i d e n t i f i e d in the mucosa of the rat in t e s t i n e . Enkephalin-IR f i b r e s have been found to occur in the i n t e s t i n a l submucosal plexus of the cat (Lolova et a l . , 1984), suggesting that these may a f f e c t mucosal opioid receptors. Also, c i r c u l a t i n g opioid peptides from endocrine c e l l s may function through these i n t e s t i n a l receptors. Although some evidence has been presented which suggests that the antisecretory effects of the opioids in the guinea pig i l e a l mucosa are mediated by a delta receptor (Kuchar and M i l l e r , 1982; Vinayek et a l . , 1983), no delta binding s i t e s were found to occur in the mucosa of the guinea pig ileum. The presence of mu-binding s i t e s in the submucosal regions suggests, however, that opioids may have an i n d i r e c t action on guinea pig i l e a l mucosal function. Furthermore, since binding of radiolabelled opioid peptides could not be demonstrated to 176 b i n d t o r a b b i t i l e a l e n t e r o c y t e s ( B i n d e r e t a l . , 1984), o p i o i d p e p t i d e s t i m u l a t i o n o f a c t i v e sodium and c h l o r i d e a b s o r p t i o n was proposed t o r e q u i r e an u n i d e n t i f i e d i n t e r m e d i a t e a g o n i s t . I n c o n t r a s t t o the g u i n e a p i g , d e f i n i t e mu- and d e l t a - b i n d i n g s i t e s o c c u r r e d i n the r a t i l e u m , a g a i n i n d i c a t i n g t h a t t h e r e are d i s t i n c t s p e c i e s d i f f e r e n c e s . The a c t i o n s o f o p i o i d p e p t i d e s on these i n t e s t i n a l r e c e p t o r s are presumed t o c o n t r i b u t e t o the well-known c o n s t i p a t i n g e f f e c t s o f o p i o i d s . O p i o i d r e c e p t o r s i n the duodenal mucosa may be i n v o l v e d i n the r e g u l a t i o n o f water and e l e c t r o l y t e a b s o r p t i o n as i n the i l e u m but may a l s o p l a y a r o l e i n the r e g u l a t i o n o f i n t e s t i n a l e n d o c r i n e and e x o c r i n e s e c r e t i o n s as i n the stomach. An e x t e n s i v e network of e n k e p h a l i n e r g i c (Wang e t a l . , 1984) and d y n o r p h i n e r g i c ( V i n c e n t e t a l . , 1984) neurons has been demonstrated i n the r a t duodenal m y e n t e r i c p l e x u s , y e t no r e c e p t o r s were found t o o c c u r i n t h i s p l e x u s or i n the muscle l a y e r . A g a i n , i t i s p o s s i b l e t h a t the m y e n t e r i c f i b r e s send p r o j e c t i o n s through the c i r c u l a r muscle t o a f f e c t o p i o i d r e c e p t o r s i n the mucosa. A l s o , by immunocytochemistry, numerous beaded f i b e r s were obse r v e d t o form a band around the duodenal B r u n n e r ' s g l a n d s (Bu'Lock e t a l . , 1983; F e r r i e t a l . , 1984). A l t h o u g h no b i n d i n g s i t e s were p r e s e n t i n the g l a n d s , b i n d i n g s i t e s i n the mucosa s u r r o u n d i n g the g l a n d may be a s s o c i a t e d w i t h the e n k e p h a l i n e r g i c r e g u l a t i o n o f i t s f u n c t i o n as suggested by the d i s t r i b u t i o n o f e n k e p h a l i n - c o n t a i n i n g f i b r e s . The d e t e c t i o n o f the r e l e a s e o f o p i o i d - l i k e compounds (Smith e t a l . , 1983) and d y n o r p h i n (Donnerer e t a l . , 1984) i n t o the p o r t a l c i r c u l a t i o n o f i s o l a t e d p e r f u s e d s m a l l i n t e s t i n e 177 p r e p a r a t i o n s i n d i c a t e t h a t these p e p t i d e s may f u n c t i o n i n an e n d o c r i n e f a s h i o n to a c t i v a t e the mucosal r e c e p t o r s . S i m i l a r l y , the appearance o f o p i o i d p e p t i d e s i n the v a s c u l a t u r e may a l s o r e f l e c t r e l e a s e i n t o the e x t r a c e l l u l a r space v i a p a r a c r i n e s e c r e t i o n . Whereas few e n k e p h a l i n e r g i c neurons occur i n the i n t e s t i n a l submucosal p l e x u s and mucosa, d y n o r p h i n -immunoreactive f i b r e s appear to be h e a v i l y c o n c e n t r a t e d i n the submucosal p l e x u s (Watson et a l . , 1981) . T h e r e f o r e , d y n o r p h i n e r g i c f i b r e s may a c t at the mucosal b i n d i n g s i t e s . However, s p e c i f i c a u t o r a d i o g r a p h i c k a p p a - b i n d i n g c o u l d not be 3 demonstrated u s i n g [ H ] - E K C . The absence o f d e t e c t a b l e d e l t a - t y p e b i n d i n g i n the gu inea p i g i l eum i s c o n s i s t e n t w i th p r e v i o u s b i n d i n g s t u d i e s u s i n g guinea p i g i l e a l homogenates (Creese and S n y d e r , 1976) t h a t mu-type r e c e p t o r s predominate i n t h i s t i s s u e . F u n c t i o n a l s t u d i e s have a l s o demonstrated the predominance o f mu- over d e l t a -r e c e p t o r s i n t h i s t i s s u e ( K o s t e r l i t z and W a t e r f i e d , 1975; L o r d et a l . , 1977) . However, n e g a t i v e r e s u l t s i n a u t o r a d i o g r a p h i c s t u d i e s such as these need not be i n t e r p r e t e d as an i n d i c a t i o n of the absence o f r e c e p t o r s i n the t i s s u e . As demonstrated by the e f f e c t s o f GTP, M n + 2 and N a + on the b i n d i n g o f [ 3 H ] - D A D L E , the k i n e t i c s o f b i n d i n g can be r e a d i l y a l t e r e d by the i n c u b a t i o n c o n d i t i o n s . T h e r e f o r e , i t i s important tha t the e x p e r i m e n t a l c o n d i t i o n s are comparable w i th those observed by the r e c e p t o r s i n v i v o . The f i n d i n g t h a t i n c u b a t i o n c o n d i t i o n s d e s c r i b e d as optimum f o r Type 1 DADLE r e c e p t o r c h a r a c t e r i z a t i o n i n r a t s t r i a t u m (Bowen e t a l . , 1981; O l g i a t i et a l . , 1982) c o m p l e t e l y a b o l i s h e d a l l s p e c i f i c DADLE b i n d i n g 178 i n g a s t r o i n t e s t i n a l t i s s u e s u g g e s t s t h a t mechanisms i n v o l v e d i n the r e g u l a t i o n o f b i n d i n g t o g a s t r o i n t e s t i n a l o p i o i d r e c e p t o r s d i f f e r c o n s i d e r a b l y from those i n the b r a i n . I t would be o f i n t e r e s t t o examine s y s t e m a t i c a l l y the e f f e c t s o f N a + , GTP and d i v a l e n t c a t i o n s on the b i n d i n g o f o p i o i d l i g a n d s t o g a s t r o i n t e s t i n a l b i n d i n g s i t e s . I n a d d i t i o n t o p r o v i d i n g i n f o r m a t i o n c o n c e r n i n g the mechanisms u n d e r l y i n g the c o n t r o l o f g a s t r o i n t e s t i n a l o p i o i d r e c e p t o r s , the r e s u l t s o f such s t u d i e s may uncover the presence o f r e c e p t o r s i n r e g i o n s h i t h e r t o u n d e t e c t e d w i t h the p r e s e n t c o n d i t i o n s . 179 CHAPTER FIVE RELEASE OF LEU-ENK IMMUNOREACTIVITY FROM  THE ISOLATED PERFUSED RAT STOMACH INTRODUCTION The presence o f numerous e n t e r i c neurons c o n t a i n i n g o p i o i d p e p t i d e s (Elde e t a l . , 1976; P o l a k e t a l . , 1977, Alumets e t a l . , 1978, S c h u l t z b e r g e t a l . , 1978; Watson e t a l . , 1980; L a r s s o n and S t e n g a a r d - P e d e r s o n , 1982; F u r n e s s e t a l . , 1983; Bu'Lock e t a l . , 1983; V i n c e n t e t a l . , 1984; Wang e t a l . , 1984) suggests t h a t t hey may f u n c t i o n as n e u r o t r a n s m i t t e r s , neuromodulators or neurohormones i n the r e g u l a t i o n o f g a s t r o i n t e s t i n a l f u n c t i o n s . F u r t h e r m o r e , the i d e n t i f i c a t i o n o f e n k e p h a l i n - I R i n a n t r a l e n d o c r i n e c e l l s , p o s s i b l y c o - e x i s t i n g w i t h g a s t r i n ( L a r s s o n and S t e n g a a r d - P e d e r s e n , 1981; Bu'Lock e t a l . , 1983; G i r a u d e t a l . , 1984a; J o n s s o n , 1985), s u g g e s t s t h a t endogenous o p i o i d p e p t i d e s may a l s o f u n c t i o n as g a s t r i c hormones. However, i n o r d e r t o e s t a b l i s h the p h y s i o l o g i c a l r e l e v a n c e o f o p i o i d p e p t i d e s as r e g u l a t o r s i n the e n t e r i c nervous system, i t i s n e c e s s a r y t o demonstrate t h e i r r e l e a s e . P o t a s s i u m d e p o l a r i z a t i o n - i n d u c e d r e l e a s e o f o p i o i d s has been d i r e c t l y demonstrated i n p e r f u s e d b r a i n s l i c e s ( I v e r s e n e t a l . , 1978; L i n d b e r g and D a h l , 1981; P a t e y e t a l . , 1983; L i n d b e r g e t a l . , 1985), and synaptosomal p r e p a r a t i o n s (Henderson e t a l . , 1978). S i m i l a r l y , e n k e p h a l i n r e l e a s e from the i s o l a t e d p e r f u s e d a d r e n a l g l a n d s o f s e v e r a l s p e c i e s i n response t o 180 e i t h e r c h o l i n e r g i c a g o n i s t s or s p l a n c h n i c nerve s t i m u l a t i o n has been r e p o r t e d ( V i v e r o s e t a l . , 1979; K i l p a t r i c k e t a l . , 1980; Corder e t a l . , 1982; B a r r o n and Hexum, 1984), s u p p o r t i n g a r o l e f o r endogenous o p i o i d p e p t i d e s as p u t a t i v e n e u r o t r a n s m i t t e r s i n these t i s s u e s . I n d i r e c t d e m o n s t r a t i o n s o f o p i o i d p e p t i d e r e l e a s e from the e n t e r i c nervous system have been made based on the e f f e c t s o f the o p i a t e r e c e p t o r a n t a g o n i s t , n a l o x o n e . Van Neuten e t a l . , (1976) showed t h a t d u r i n g the f a t i g u e o f p e r i s t a l t i c a c t i v i t y i n the i l e u m , a subst a n c e was r e l e a s e d whose a c t i o n s c o u l d be b l o c k e d by n a l o x o n e . F u r t h e r m o r e , t h i s p e r i s t a l t i c f a t i g u e i t s e l f c o u l d be r e v e r s e d by n a l o x o n e . I n a s i m i l a r s t u d y , P u i g e t a l . , (1977) showed t h a t b r i e f b u r s t s o f h i g h f r e q u e n c y s t i m u l a t i o n o f the g u i n e a p i g i l e u m i n h i b i t e d the c h o l i n e r g i c t w i t c h i n a naloxone r e v e r s i b i l e manner, s u g g e s t i n g the r e l e a s e o f endogenous o p i o i d r e c e p t o r l i g a n d s . In r e c e n t s t u d i e s employing the i s o l a t e d , p e r f u s e d r a t stomach p r e p a r a t i o n , i t was found t h a t the v a g a l i n h i b i t i o n o f g a s t r i c i n h i b i t o r y p o l y p e p t i d e ( G I P ) - s t i m u l a t e d s o m a t o s t a t i n s e c r e t i o n c o u l d be p a r t i a l l y r e v e r s e d by naloxone ( M c i n t o s h e t a l . , 1983), f u r t h e r s u g g e s t i n g the r e l e a s e o f endogenous o p i o i d s u b s t a n c e s . A de c r e a s e i n t i s s u e o p i o i d c o n t e n t f o l l o w i n g e l e c t r i c a l f i e l d s t i m u l a t i o n has been employed as an i n d i c a t i o n o f e n k e p h a l i n r e l e a s e from the m y e n t e r i c p l e x u s - l o n g i t u d i n a l muscle p r e p a r a t i o n o f the g u i n e a p i g s m a l l i n t e s t i n e (McKnight e t a l . , 1978; C o r b e t t e t a l . , 1980). When the i n t r a m u r a l n erves were s t i m u l a t e d i n the presence o f c y c l o h e x i m i d e , t o pr e v e n t de novo p e p t i d e s y n t h e s i s , a p p r o x i m a t e l y h a l f o f the measured e n k e p h a l i n s t o r e s were found t o be r e l e a s a b l e , and 181 t h i s r e l e a s e c o u l d be p r e v e n t e d by t e t r o d o t o x i n . The amount of e n k e p h a l i n l o s s was dependent on the f r e q u e n c y o f s t i m u l a t i o n , w i t h m e t - e n k e p h a l i n more r e a d i l y r e l e a s e d than l e u - e n k e p h a l i n . D i r e c t measurements o f o p i o i d p e p t i d e r e l e a s e from v a r i o u s i n v i t r o i n t e s t i n a l p r e p a r a t i o n s have been r e p o r t e d . U s i n g the m y e n t e r i c p l e x u s - l o n g i t u d i n a l muscle o f the g u i n e a p i g i l e u m , McKnight e t a l . , (1981) were a b l e t o measure the r e l e a s e o f endogenous e n k e p h a l i n s i n t o the p e r i f u s i n g b a t h f l u i d i n response t o e l e c t r i c a l f i e l d s t i m u l a t i o n . The r e l e a s e o f immunoreactive d y n o r p h i n from the i s o l a t e d g u i n e a p i g i l e u m i n t o the b a t h i n g s o l u t i o n was found t o be maximal under r e s t i n g c o n d i t i o n s and was s i g n i f i c a n t l y reduced f o l l o w i n g i n d u c t i o n o f i l e a l p e r i s t a l s i s d u r i n g s u s t a i n e d e l e v a t i o n o f i n t r a l u m i n a l p r e s s u r e (Kromer e t a l . , 1981) which suggested a r o l e f o r d y n o r p h i n i n the c o n t r o l o f p e r i s t a l s i s . B a s a l and s t i m u l a t e d r e l e a s e o f endogenous i n t e s t i n a l o p i o i d s have been d e t e c t e d i n the p o r t a l c i r c u l a t i o n o f the i s o l a t e d , p e r f u s e d r a t i l e u m -jejunum (Smith e t a l . , 1983). R e l e a s e o f o p i o i d a c t i v i t y , d etermined by r a d i o r e c e p t o r a s s a y u s i n g crude r a t b r a i n homogenates, was measured i n response t o i n c r e a s e d i n t r a l u m i n a l p r e s s u r e , e l e v a t e d c a l c i u m l e v e l s , and t o l b u t a m i d e . S i m i l a r l y , d y n o r p h i n was found t o be r e l e a s e d i n t o the venous e f f l u e n t o f the i s o l a t e d v a s c u l a r l y p e r f u s e d g u i n e a p i g s m a l l i n t e s t i n e f o l l o w i n g an i n c r e a s e i n i n t r a l u m i n a l p r e s s u r e (Donnerer e t a l . , 1984). T h i s r e l e a s e was c o m p l e t e l y p r e v e n t e d by the a d d i t i o n o f t e t r o d o t o x i n , i n d i c a t i n g the n e u r a l o r i g i n o f the d y n o r p h i n . 5 182 D e s p i t e the many r e p o r t s o f o p i o i d p e p t i d e presence i n g a s t r i c neurons and e n d o c r i n e c e l l s , the r e l e a s e o f o p i o i d s u b s t a n c e s from the stomach has never been d i r e c t l y d emonstrated. T h e r e f o r e , the o b j e c t i v e o f the f o l l o w i n g s t u d y was t o determine whether endogenous o p i o i d p e p t i d e s c o u l d be r e l e a s e d from the v a s c u l a r l y p e r f u s e d r a t stomach p r e p a r a t i o n , which o f f e r s an i d e a l system w h e r e i n the r e l e a s e o f s u b s t a n c e s from g a s t r i c neurons and e n d o c r i n e c e l l s i n t o the c i r c u l a t i o n can be measured. One major o b s t a c l e encountered when a t t e m p t i n g t o measure the r e l e a s e o f p e p t i d e s u b s t a n c e s i s the p o s s i b i l i t y o f r a p i d e n z y m a t i c d e g r a d a t i o n . S e v e r a l enzymes are known t o be i n v o l v e d i n the i n a c t i v a t i o n o f e n k e p h a l i n s (Hughes, 1983). The p r i m a r y pathways i n v o l v e the c l e a v a g e o f the T y r - G l y bond by an a m i n o p e p t i d a s e and the Gly-Phe bond by a s p e c i f i c e n d o peptidase r e f e r r e d t o as e n k e p h a l i n a s e . T h i s G l y -Phe l i n k a g e i s a l s o s u s c e p t i b l e t o c l e a v a g e by the enzyme a n g i o t e n s i n - c o n v e r t i n g enzyme. A c o c k t a i l o f enzyme i n h i b i t o r s c o n s i s t i n g o f b e s t a t i n , a g e n e r a l a m i n o p e p t i d a s e i n h i b i t o r , t h i o r p h a n , a s p e c i f i c i n h i b i t o r o f e n k e p h a l i n a s e (Schwartz, 1983) and c a p t r o p r i l which i n h i b i t s a n g i o t e n s i n - c o n v e r t i n g enzyme was t h e r e f o r e employed t o p r e v e n t the breakdown of endogenously r e l e a s e d e n k e p h a l i n s . A d d i t i o n a l l y , i n o r d e r t o i n v e s t i g a t e the mechanism(s) r e g u l a t i n g the r e l e a s e o f endogenous o p i o i d p e p t i d e s from the r a t stomach, the e f f e c t s o f a c e t y l c h o l i n e r e c e p t o r a g o n i s t s were examined. 183 METHODS The s u r g i c a l i s o l a t i o n and p e r f u s i o n o f the r a t g a s t r i c v a s c u l a t u r e was performed as d e s c r i b e d i n Chapter 2, s e c t i o n I I I A. The p e r f u s a t e was p r e p a r e d a c c o r d i n g l y w i t h the a d d i t i o n o f the f o l l o w i n g enzyme i n h i b i t o r s : 0.1 juM t h i o r p h a n ( P e n i n s u l a L a b o r a t o r i e s ) , 1 JJM c a p t o p r i l and 2 juM b e s t a t i n (Sigma) . The f l o w r a t e was s e t a t 2 ml/min and the p r e s s u r e was m a i n t a i n e d a t 50 - 70 mm Hg. F o l l o w i n g a 30 min s t a b i l i z a t i o n p e r i o d , the p e r f u s a t e from the p o r t a l v e i n c a n n u l a was c o l l e c t e d f o r 5 min i n t e r v a l s i n c h i l l e d s c i n t i l l a t i o n v i a l s each c o n t a i n i n g 1 ml T r a s y l o l (1000 K a l l i k r e i n i n h i b i t o r u n i t s KlU/ml a p r o t i n i n , M i l e s , Canada). S t i m u l a t i o n w i t h h i g h p o t a s s i u m was a c h i e v e d by exchanging a t the l e v e l o f the bubble t r a p , r e g u l a r p e r f u s a t e w i t h p e r f u s a t e i n which the KC1 c o n c e n t r a t i o n was i n c r e a s e d t o 50 mM and the NaCl reduced a p p r o p r i a t e l y t o m a i n t a i n a c o n s t a n t o s m o l a l i t y . Both the h i g h p o t a s s i u m and r e g u l a r p e r f u s a t e s were c o n t i n u o u s l y gassed w i t h 95% oxygen/ 5% carbon d i o x i d e and heated t o 40°C. A l l o t h e r t e s t s u b s t a n c e s ( 1 , l - d i m e t h y l - 4 -p h e n y l - p i p e r a z i n i u m [DMPP], and m e t h a c h o l i n e ) were i n t r o d u c e d i n t o the g a s t r i c c i r c u l a t i o n by means o f a s i d e - a r m i n f u s i o n a t a r a t e c a l c u l a t e d t o g i v e the f i n a l c o n c e n t r a t i o n s i n d i c a t e d i n the t e x t . A 500 j i l a l i q u o t o f each sample was r e s e r v e d f o r d e t e r m i n a t i o n o f I R - g a s t r i n by RIA as d e s c r i b e d i n Chapter 2, s e c t i o n I . C ( 6 ) . For the d e t e r m i n a t i o n o f l e u - e n k i m m u n o r e a c t i v i t y , each 5 minute f r a c t i o n was e x t r a c t e d f o r o p i o i d s u s i n g A m b e r l i t e XAD-2 -184 columns (Chapter 2, s e c t i o n I I . B) . A r e c o v e r y o f 88 - 7 % (n=5) was a c h i e v e d when 5 ng o f s y n t h e t i c l e u-enk was a p p l i e d t o these columns. The l y o p h i l i z e d A m b e r l i t e XAD-2 t r e a t e d samples were r e c o n s t i t u t e d w i t h 1 ml o f d i s t i l l e d water and assayed f o r l e u - e n k - I R by RIA a c c o r d i n g t o the p r o t o c o l o u t l i n e d i n s e c t i o n I . C (2) o f Chapter 2. The mean and s t a n d a r d e r r o r o f the mean f o r each t e s t p e r i o d were c a l c u l a t e d f o r the d i f f e r e n t s t i m u l i and s i g n i f i c a n t changes i n leu-enk r e l e a s e were determined i n comparison t o p r e - t e s t p e r i o d s by the p a i r e d S t u d e n t ' s t - t e s t . P o i n t s which were found t o d i f f e r s i g n i f i c a n t l y a r e l a b e l l e d i n the f i g u r e s w i t h a s t e r i s k s (* f o r p<0.05 and ** f o r p<0.01). 185 RESULTS D i r e c t measurements of l e u - e n k - I R i n the p o r t a l o u t f l o w o f the p e r f u s e d r a t stomach were p o s s i b l e o n l y i n the presence of the c o c k t a i l o f enzyme i n h i b i t o r s . B a s a l l e v e l s o f l e u - e n k - I R v a r i e d from 18 - 75 f m o l e / m l . However, the d e t e c t e d l e v e l s p r o b a b l y accounted f o r o n l y a p o r t i o n o f the a c t u a l amounts r e l e a s e d from the stomach, s i n c e i t was observed t h a t when 6.2 ng/min s y n t h e t i c l eu-enk was i n f u s e d through the s i d e arm i n t o the a r t e r i a l p e r f u s i o n , o n l y 40% was r e c o v e r e d i n the venous o u t f l o w . I . EFFECT OF POTASSIUM DEPOLARIZATION R a i s i n g the p o t a s s i u m c o n c e n t r a t i o n o f the p e r f u s a t e from 4.4 t o 50 mM r e s u l t e d i n a s i g n i f i c a n t (p < 0.01, n=7) i n c r e a s e i n l e u - e n k - I R which was s u s t a i n e d over the f u l l 10 minute s t i m u l a t i o n p e r i o d ( F i g u r e 51A). There was a t w o - f o l d i n c r e a s e observed i n the f i r s t K + - s t i m u l a t e d c o l l e c t i o n p e r i o d ( f r a c t i o n 3) f o l l o w e d by a f o u r - f o l d i n c r e a s e i n f r a c t i o n 4. Leu-enk-IR l e v e l s r e t u r n e d i m m e d i a t e l y t o b a s a l upon removal o f the K + s t i m u l a t i o n . P o t a s s i u m d e p o l a r i z a t i o n caused a concommitant f o u r - f o l d i n c r e a s e i n I R - g a s t r i n s e c r e t i o n from e n d o c r i n e c e l l s ( F i g u r e 51B). The g a s t r i n l e v e l s , however, remained s i g n i f i c a n t l y h i g h e r than b a s a l i n f r a c t i o n s 5 and 6 d e s p i t e the r e t u r n t o r e g u l a r p o t a s s i u m c o n c e n t r a t i o n s d u r i n g these p e r i o d s . 186 I I . EFFECT OF DMPP INFUSION -5 The n i c o t i n i c , c h o l i n e r g i c a g o n i s t DMPP (10 M) caused a pronounced t h r e e - f o l d i n c r e a s e (p < 0.01, n=9) i n l e u - e n k - I R r e l e a s e over b a s a l l e v e l s which was accompanied by a s i m i l a r i n c r e a s e i n I R - g a s t r i n ( F i g u r e 5 2). The l e v e l s o f both p e p t i d e s i m m e d i a t e l y r e t u r n e d t o p r e - s t i m u l a t i o n v a l u e s when the DMPP i n f u s i o n was t e r m i n a t e d . I I I . EFFECT OF METHACHOLINE INFUSION — 5 A l t h o u g h 10 M m e t h a c h o l i n e , a m u s c a r i n i c c h o l i n e r g i c a g o n i s t , caused a s i g n i f i c a n t i n c r e a s e (p < 0.01, n=13) i n IR-g a s t r i n s e c r e t i o n from the stomach, no s i g n i f i c a n t change i n le u - e n k - I R was observed ( F i g u r e 5 3 ) . A. = a; o E or i—i i UJ i Z) LU 100 8 0 6 0 4 0 2 0 0 ** - JU ra K +  m 50 mM X X B. 100 8 0 E \ cn Q_ — 6 0 S 4 0 O i or 2 0 0 .••AY l I 2 3 4 5 6 FIGURE 51: EFFECT OF POTASSIUM DEPOLARIZATION ON LEU-ENK-IR SECRETION FROM ISOLATED PERFUSED RAT STOMACHS. P e r f u s a t e c o n t a i n i n g 50 mM p o t a s s i u m was i n t r o d u c e d i n f r a c t i o n s 3 and 4. (A) Leu-enk-IR l e v e l s measured by RIA i n A m b e r l i t e XAD-2 t r e a t e d p e r f u s i o n e f f l u e n t s r i s e s i g n i f i c a n t l y (p<0.01) d u r i n g these p e r i o d s as does the s e c r e t i o n o f IR-g a s t r i n (B) . (n=7) 188 A . £ 250 1 2 0 0 E ^ I 50 1—1 1 LU 1 Z) LU _1 0 0 50 -0 L ** D M P P I 0 " 5 M B . 75 60 45 30 £ . 15 E \ CL If) a O 1 1 I 0 L 1 2 3 4 5 FIGURE 52: EFFECT OF DMPP ON LEU-ENK-IR 'SECRETION FROM ISOLATED PERFUSED RAT STOMACHS. DMPP was introduced during the thir d f i v e minute c o l l e c t i o n period such that a f i n a l concentration of 1 0 - 5 M was obtained. (A) Leu-enk-IR leve l s were measured in portal effluents following Amberlite XAD-2 treatment. (B) Corresponding IR-gastrin l e v e l s in portal effluents were measured d i r e c t l y . (n=9) 189 A. £ 75 \ d) 0 60 E >-*— — 45 cr i—i 1 30 2Z LU i LU B. 15 0 50 h >E 40 Q. ~ 30 g 20 CD i 1 0 0 Methacholine I 0 " 5 M KK I - J L 1 2 3 4 5 FIGURE 53: EFFECT OF METHACHOLINE ON LEU-ENK-IR SECRETION FROM ISOLATED PERFUSED RAT STOMACHS. M e t h a c h o l i n e was i n f u s e d d u r i n g f r a c t i o n 3. (A) L e u - e n k - I R l e v e l s were measured i n p o r t a l e f f l u e n t s f o l l o w i n g e x t r a c t i o n o f o p i o i d s by A m b e r l i t e XAD-s t r e a t m e n t . C o r r e s p o n d i n g I R - g a s t r i n l e v e l s are p r e s e n t e d i n (B) . (n=13) 190 DISCUSSION From these r e s u l t s i t i s e v i d e n t t h a t under r e s t i n g c o n d i t i o n s t h e r e i s r e l e a s e o f endogenous l e u - e n k e p h a l i n from g a s t r i c neurons and/or e n d o c r i n e c e l l s i n t o the stomach v a s c u l a t u r e . That t h i s r e l e a s e c o u l d o n l y be measured i n the presence o f a c o c k t a i l o f p e p t i d a s e i n h i b i t o r s demonstrates the presence o f e f f i c e n t o p i o i d p e p t i d e i n a c t i v a t i n g mechanisms i n the r a t stomach which most l i k e l y resemble those r e s p o n s i b l e f o r the breakdown o f e n k e p h a l i n s i n the b r a i n and a d r e n a l g l a n d (Schwartz, 1983). The a d d i t i o n o f enzyme i n h i b i t o r s , however, was o n l y p a r t i a l l y e f f e c t i v e i n p r e v e n t i n g e n k e p h a l i n d e g r a d a t i o n s i n c e the r e c o v e r y o f exogenously a d m i n i s t e r e d l e u -enk i n t o the g a s t r i c v a s c u l a t u r e was o n l y 40%. The need t o e x t r a c t and c o n c e n t r a t e the o p i o i d c o n t e n t from r e a s o n a b l y l a r g e samples (10 ml) o f p o r t a l e f f l u e n t r e f l e c t s the v e r y minute l e v e l s o f p e p t i d e r e l e a s e d i n t o the g a s t r i c v a s c u l a t u r e . E n k e p h a l i n s r e l e a s e d from e n d o c r i n e c e l l s p l u s a s m a l l f r a c t i o n o f the e n k e p h a l i n s r e l e a s e d from nerve t e r m i n a l s would be expected t o e n t e r the c i r c u l a t i o n . The b a s a l r e l e a s e o f l e u -enk-IR demonstrated c o n s i d e r a b l e day t o day v a r i a b i l i t y w i t h mean v a l u e s r a n g i n g from 18 t o 75 fmole/ml. The t h r e e - f o l d i n c r e a s e i n l e u - e n k - I R o b s e r v e d f o l l o w i n g g e n e r a l membrane d e p o l a r i z a t i o n w i t h K C l might have a r i s e n from the r e l e a s e o f p e p t i d e s s t o r e d i n any c e l l t y p e ; i t cannot be determined whether the l e u - e n k - I R measured i n the p o r t a l o u t f l o w was o f n e u r a l or e n d o c r i n e o r i g i n . The concommitant 191 r e l e a s e o f I R - g a s t r i n c o n f i r m s the d e p o l a r i z i n g e f f e c t o f the hi g h p o t a s s i u m . However, i n c o n t r a s t t o the leu-enk response which p r o m p t l y r e t u r n e d t o b a s a l l e v e l s upon removal o f the po t a s s i u m s t i m u l u s , I R - g a s t r i n remained s i g n i f i c a n t l y h i g h e r than the b a s a l l e v e l s o b t a i n e d p r i o r t o the p o t a s s i u m p u l s e . A l t h o u g h the reason f o r t h i s phenomenon i s not a p p a r e n t , i t may be t h a t e n d o c r i n e c e l l s such as the g a s t r i n s e c r e t i n g a n t r a l G-c e l l s r e q u i r e a l o n g e r p e r i o d o f time f o r t h e i r membrane p o t e n t i a l s t o reach r e s t i n g l e v e l s f o l l o w i n g p e r t u r b a t i o n by h i g h p o t a s s i u m . From the d a t a p r e s e n t e d , i t appears t h a t the r e l e a s e o f leu - e n k - I R from the r a t stomach i s mediated by a c h o l i n e r g i c n i c o t i n i c r e c e p t o r . DMPP, a g a n g l i o n i c n i c o t i n i c r e c e p t o r a g o n i s t was found t o cause a t h r e e - f o l d i n c r e a s e d r e l e a s e o f endogenous l e u - e n k , w h i l e m e t h a c h o l i n e , a m u s c a r i n i c r e c e p t o r a g o n i s t , caused no change. B o t h DMPP and m e t h a c h o l i n e caused a s i g n i f i c a n t i n c r e a s e i n I R - g a s t r i n r e l e a s e which i s i n aggreement w i t h p r e v i o u s r e p o r t s t o t h i s a f f e c t (Schubert and Makhlou f , 1982; Pederson e t a l . , 1984). The i d e n t i f i c a t i o n o f numerous e n k e p h a l i n c o n t a i n i n g nerve c e l l b o d i e s i n the m y e n t e r i c g a n g l i a i s c o n s i s t e n t w i t h the proposed c h o l i n e r g i c g a n g l i o n i c s t i m u l a t i o n o f the s e neurons. I n a d d i t i o n , the r e l e a s e o f e n k e p h a l i n from the i s o l a t e d p e r f u s e d a d r e n a l g l a n d has a l s o been shown t o be s t i m u l a t e d by DMPP (Govoni e t a l . , 1981; B a r r o n and Hexum, 1984) . The g a n g l i o n i c s t i m u l a t e d r e l e a s e o f g a s t r i c e n k e p h a l i n s may be mediated by l o c a l c h o l i n e r g i c i n t r a m u r a l neurons or by e x t r i n s i c autonomic i n n e r v a t i o n . P r e g a n g l i o n i c p a r a s y m p a t h e t i c i n p u t s i n t o the 192 stomach are c a r r i e d by the vagus nerve w h i l e p r e g a n g l i o n i c s y m p a t h e t i c imputs are r a r e i n the g a s t r o i n t e s t i n a l t r a c t . V a g a l r e l e a s e o f a c e t y l c h o l i n e may d i r e c t l y s t i m u l a t e e n k e p h a l i n e r g i c f i b r e s p r e s e n t i n the g a n g l i a or i n d i r e c t l y by means o f an i n t e r n e u r o n . The r e p o r t s o f e n k e p h a l i n - l i k e i m m u n o r e a c t i v i t y i n G - c e l l s o f the a n t r a l mucosa ( L a r s s o n and S t e n g a a r d - P e d e r s e n , 1981; Bu'Lock e t a l . , 1983; G i r a u d e t a l . , 1984a; J o n s s o n , 1985) suggest t h a t e n k e p h a l i n s may be c o - r e l e a s e d w i t h g a s t r i n . However, i n the p r e s e n t s t u d y , the r e l e a s e o f I R - g a s t r i n and leu - e n k - I R c o u l d be uncoupled i n t h a t m e t h a c h o l i n e was found t o s t i m u l a t e g a s t r i n r e l e a s e w i t h o u t c a u s i n g any i n c r e a s e i n e n k e p h a l i n r e l e a s e as might be e x p e c t e d i f the two p e p t i d e s were c o - r e l e a s e d from the same c e l l . S i n c e Jonsson (1985) has demonstrated t h a t a s u b - p o p u l a t i o n (6-10%) o f hog a n t r a l G-c e l l s c o n t a i n e n k e p h a l i n - I R and t h a t 30% o f e n k e p h a l i n -c o n t a i n i n g e n d o c r i n e c e l l s are G - c e l l s , d i f f e r e n t i a l r e l e a s e o f g a s t r i n and e n k e p h a l i n might be p o s s i b l e . I t i s more l i k e l y , however, t h a t l e u - e n k - I R d e t e c t e d f o l l o w i n g n i c o t i n i c r e c e p t o r s t i m u l a t i o n o r i g i n a t e d p r e d o m i n a n t l y from e n k e p h a l i n e r g i c nerves i n the stomach w a l l or from e n d o c r i n e c e l l s o t h e r than the G - c e l l s . S t u d i e s u s i n g the i d e n t i c a l v a s c u l a r l y p e r f u s e d r a t stomach model have a l s o p r o v i d e d i n d i r e c t e v i d e n c e f o r v a g a l l y mediated e n k e p h a l i n r e l e a s e . M c i n t o s h e t a l . , (1983) demonstrated t h a t naloxone c o u l d p a r t i a l l y r e v e r s e v a g a l l y -induced i n h i b i t i o n o f G I P - s t i m u l a t e d s o m a t o s t a t i n s e c r e t i o n . F u r t h e r m o r e , i t has been found t h a t post-vagotomy h y p e r g a s t r i n e m i a c o u l d be i n h i b i t e d by m e t - e n k e p h a l i n (Pederson 193 e t a l . , 1984) s u g g e s t i n g t h a t removal of v a g a l tone from the stomach a t the same time removes an e n k e p h a l i n e r g i c tone which may n o r m a l l y f u n c t i o n t o lower g a s t r i n l e v e l s . Now t h a t t h i s system f o r d e t e c t i n g l eu-enk r e l e a s e from the r a t stomach has been d e v i s e d the r e l e a s e o f endogenous g a s t r i c o p i o i d s i n response t o v a g a l s t i m u l a t i o n can be examined. However, the immunofluorescent s t u d i e s d e m o n s t r a t i n g the presence o f e n k e p h a l i n - l i k e immunoreactive m a t e r i a l i n the human vagus nerve (Lundberg e t a l . , 1979) must be taken i n t o c o n s i d e r a t i o n when i n t e r p r e t i n g such r e s u l t s . Other experiments can a l s o be proposed which might c l a r i f y the mechanism(s) i n v o l v e d i n the r e g u l a t i o n o f endogenous g a s t r i c o p i o i d p e p t i d e r e l e a s e . To examine the p u t a t i v e r o l e o f e n k e p h a l i n as a n e u r o t r a n s m i t t e r , the c a l c i u m dependency of the s t i m u l a t e d l eu-enk r e l e a s e s h o u l d be d e t e r m i n e d . I n a d d i t i o n , the e f f e c t o f the n e u r o t o x i n t e t r o d o t o x i n would i n d i c a t e whether a n e u r a l pathway i s i n v o l v e d i n the r e l e a s e o f endogenous o p i o i d s from the stomach. I t would a l s o be p o s s i b l e to examine the c o n t e n t o f the p o r t a l v e i n e f f l u e n t f o r o t h e r p r o e n k e p h a l i n d e r i v e d o p i o i d p e p t i d e s u s i n g the HPLC methods d e s c r i b e d i n Chaper 3 f o r the c h a r a c t e r i z a t i o n o f o p i o i d p e p t i d e s i n r a t stomach e x t r a c t s . I t i s p r o b a b l e t h a t some o f the r e l e a s e d l e u - e n k - I R d e t e c t e d by RIA was due t o the presence of met-enk which demonstrates a 2.5% c r o s s - r e a c t i v i t y w i t h the leu - e n k a n t i s e r u m . However, by HPLC, i t would be p o s s i b l e t o s e p a r a t e the two p e p t i d e s , as w e l l as o t h e r o p i o i d p e p t i d e s which might a l s o be r e l e a s e d . A l t h o u g h e x t e n s i v e e x a m i n a t i o n of the f a c t o r s c o n t r o l l i n g the r e l e a s e o f o p i o i d p e p t i d e s from 194 the r a t stomach are s t i l l r e q u i r e d , these p r e l i m i n a r y s t u d i e s d e m o n s t r a t i n g the r e l e a s e o f endogenous g a s t r i c l e u - e n k from the p e r f u s e d r a t stomach i n response t o p o t a s s i u m - i n d u c e d membrane d e p o l a r i z a t i o n and t o g a n g l i o n i c s t i m u l a t i o n l e n d s u p p o r t t o e n k e p h a l i n ' s p u t a t i v e r o l e as a r e g u l a t o r y n e u r o t r a n s m i t t e r i n the stomach. 195 CHAPTER SIX REGULATION OF GASTRIC SOMATOSTATIN SECRETION BY OPIOID PEPTIDES INTRODUCTION From the r e s u l t s p r e s e n t e d i n the p r e v i o u s c h a p t e r s , d e m o n s t r a t i n g the presence o f endogenous o p i o i d p e p t i d e s and s p e c i f i c o p i o i d r e c e p t o r s i n the r a t stomach and the r e l e a s e o f e n k e p h a l i n s from the p e r f u s e d r a t stomach, i t i s e v i d e n t t h a t g a s t r i c o p i o i d s s h o u l d f u n c t i o n as p h y s i o l o g i c a l r e g u l a t o r y p e p t i d e s i n t h i s o rgan. As mentioned i n the i n t r o d u c t o r y c h a p t e r , o p i o i d s appear t o be i n v o l v e d i n the r e g u l a t i o n o f g a s t r i c a c i d s e c r e t i o n and g a s t r i c motor a c t i v i t y . F u r t h e r m o r e , e n k e p h a l i n s have been found t o a f f e c t g a s t r i c e n d o c r i n e s e c r e t i o n , i n h i b i t i n g the b a s a l r e l e a s e o f s o m a t o s t a t i n - l i k e i m m u n o r e a c t i v i t y (SLI) (Chiba e t a l . , 1980). S o m a t o s t a t i n has been demonstrated i n d i s t i n c t e n d o c r i n e D - c e l l s i n the v i c i n i t y o f a c i d - s e c r e t i n g o x y n t i c c e l l s and a n t r a l g a s t r i n - p r o d u c i n g c e l l s ( P o lak e t a l . , 1975; L a r s s o n e t a l . , 1979; Alumets e t a l . , 1979). From the base o f D - c e l l s a r i s e l o n g s l e n d e r p r o c e s s e s o f t e n a b u t t i n g on G - c e l l s i n the antrum and o x y n t i c c e l l s , c h i e f c e l l s or e n t e r o c h r o m a f f i n c e l l s i n the c o r p u s . I n the stomach, as i n i t s v a r i o u s o t h e r l o c a t i o n s , s o m a t o s t a t i n appears t o have a g e n e r a l i z e d i n h i b i t o r y e f f e c t , i n c l u d i n g i n h i b i t i o n o f g a s t r i c e n d o c r i n e and e x o c r i n e s e c r e t i o n s (Konturek, 1980a), a c t i n g through the 196 c i r c u l a t i o n or l o c a l l y i n a p a r a c r i n e f a s h i o n . The r e l e a s e o f SLI i s i t s e l f under complex c o n t r o l , s t r o n g l y i n f l u e n c e d by n u t r i e n t s i n the stomach and by e n d o c r i n e and nervous e f f e c t o r s . I n v i t r o s t u d i e s on the r a t stomach have demonstrated t h a t p h y s i o l o g i c a l l e v e l s o f GIP i n c r e a s e SLI s e c r e t i o n ( M c i n t o s h e t a l . , 1981a). However, i n v i v o s t u d i e s i n dogs showed t h a t CCK-8 was the most p o t e n t SLI secretagogue w h i l e GIP induced a s m a l l , but i n s i g n i f i c a n t , r i s e i n f u n d i c v e i n l e v e l s ( R o u i l l e r e t a l . , 1980). The reason f o r t h i s d i f f e r e n c e between the i n v i v o and i n v i t r o s i t u a t i o n s i s u n c e r t a i n but i t has been p o s t u l a t e d t h a t i t may be due t o the i n h i b i t o r y e f f e c t s o f the vagus on SLI s e c r e t i o n ( M c i n t o s h e t a l . , 1981b; M c i n t o s h e t a l . , 1983). A r o l e f o r the s y m p a t h e t i c nervous system i n the c o n t r o l o f D - c e l l a c t i v i t y has been proposed based on the o b s e r v a t i o n s t h a t c a t e c h o l a m i n e s s t i m u l a t e SLI r e l e a s e from the p e r f u s e d r a t stomach (Koop e t a l . , 1981; Goto e t a l . , 1981; Koop e t a l . , 1983). F u r t h e r m o r e , p r e g a n g l i o n i c s p l a n c h n i c nerve s t i m u l a t i o n i n the presence o f a t r o p i n e , but not i n i t s absence, has been found t o produce a marked i n c r e a s e i n SLI s e c r e t i o n ( M c i n t o s h e t a l . , 1981b; M c i n t o s h , 1985). I n v i t r o e x p e r i m e n t s have demonstrated i n h i b i t i o n o f b a s a l and G I P - s t i m u l a t e d r a t g a s t r i c SLI i n response t o e l e c t r i c a l s t i m u l a t i o n o f the vagus ne r v e s ( M c i n t o s h e t a l . , 1981a,b; M c i n t o s h e t a l . , 1983) or by i n f u s i o n o f parasympathometics (M c i n t o s h e t a l . , 1981a,b; S a f f o u r i e t a l . , 1980; M a r t i n d a l e e t a l . , 1982; S c h u s d z i a r r a e t a l . , 1984; Sue e t a l . , 1985). The i n h i b i t o r y e f f e c t o f c h o l i n e r g i c a g o n i s t s c o u l d be r e v e r s e d by 197 a t r o p i n e , hexamethonium and I ^ - r e c e p t o r b l o c k a d e s u g g e s t i n g the involv e m e n t o f m u s c a r i n i c , n i c o t i n i c and h i s t a m i n e r g i c r e c e p t o r a c t i v a t i o n ( S a f f o u r i e t a l . , 1980; M a r t i n d a l e e t a l . , 1982; S c h u s d z i a r r a e t a l . , 1984). A t r o p i n e , a p p l i e d d u r i n g p e r i o d s o f v a g a l s t i m u l a t i o n , d i d not a b l a t e the i n h i b i t i o n o f S L I ; r a t h e r , a s t i m u l a t o r y component was observed ( M c i n t o s h e t a l . , 1985). S i n c e hexamethonium r e v e r s e d v a g a l i n h i b i t i o n w i t h o u t c a u s i n g any f u r t h e r s t i m u l a t i o n , and a t r o p i n e a l o n e d i d not a f f e c t SLI s e c r e t i o n ( M c i n t o s h e t a l . , 1981b), i t was suggested t h a t a p o s t - g a n g l i o n i c i n h i b i t o r y pathway and a n o n - m u s c a r i n i c s t i m u l a t o r y pathway are a c t i v a t e d by v a g a l s t i m u l a t i o n . From s t u d i e s based on the e f f e c t s o f the n i c o t i n i c a g o n i s t DMPP (Schubert and Mak h l o u f , 1982), i t has a l s o been proposed t h a t c h o l i n e r g i c and n o n - c h o l i n e r g i c pathways a c t on g a s t r i c D-c e l l s . A b o m b e s i n - l i k e p e p t i d e has been suggested as the non-c h o l i n e r g i c t r a n s m i t t e r . The v a g a l i n h i b i t i o n o f G I P - s t i m u l a t e d SLI r e l e a s e were o n l y p a r t i a l l y b l o c k e d by a t r o p i n e ( M c i n t o s h e t a l . , 1979). S i n c e naloxone was a l s o found t o p a r t i a l l y b l o c k the v a g a l i n h i b i t i o n , o p i o i d p e p t i d e s may be i n v o l v e d ( M c i n t o s h e t a l . , 1983). L i t t l e i s known c o n c e r n i n g the e f f e c t s o f o t h e r o p i o i d p e p t i d e s which have been demonstrated t o be p r e s e n t i n the g a s t r i c n e r v e s and e n d o c r i n e c e l l s . The aim o f the s t u d i e s p r e s e n t e d i n t h i s c h a p t e r was t w o - f o l d : f i r s t l y t o examine more c l o s e l y the e f f e c t s o f e n k e p h a l i n on s t i m u l a t e d SLI r e l e a s e and t o examine the p o s s i b l e mechanism f o r i t s i n h i b i t o r y a c t i o n s , and s e c o n d l y t o determine the e f f e c t s o f exogenously a d m i n i s t e r e d d y n o r p h i n 1-13. S i n c e the presence o f d y n o r p h i n 198 i n r a t stomach e x t r a c t s was demonstrated (Chapter t h r e e ) i t was of i n t e r e s t t o i n v e s t i g a t e i t s a c t i o n on SLI s e c r e t i o n , p a r t i c u l a r l y i n comparison w i t h those o f the e n k e p h a l i n s . 199 METHODS The s u r g i c a l and p e r f u s i o n t e c h n i q u e s were as d e s c r i b e d i n Chapter 2, s e c t i o n I I I . A. The p e r f u s a t e was p r e p a r e d as o u t l i n e d p r e v i o u s l y (Chapter 2, s e c t i o n I I I . B ) . P e p t i d e s and drugs were a d m i n i s t e r e d i n t o the a r t e r i a l p e r f u s a t e by means o f a s i d e arm i n f u s i o n . P e r f u s i o n was m a i n t a i n e d a t 3 ml/min. F o l l o w i n g a 30 min s t a b i l i z a t i o n p e r i o d , the p o r t a l v e i n e f f l u e n t was c o l l e c t e d a t one minute i n t e r v a l s i n t o c h i l l e d tubes c o n t a i n i n g 1000 k a l l i k r e i n i n h i b i t o r u n i t s (KIU)/ml a p r o t i n i n ( T r a s y l o l , M i l e s , Canada). Samples were s t o r e d a t -20 °C u n t i l a s sayed. SLI was measured by radioimmunoassay (Chapter 2, s e c t i o n I . C [ 5 ] ) . The r e s u l t s were e x p r e s s e d as r a t e o f SLI s e c r e t i o n (pg/min) and have been p r e s e n t e d as means and s t a n d a r d e r r o r s o f the means (SEMs). For the d e t e r m i n a t i o n of s i g n i f i c a n t p e p t i d e or drug e f f e c t s , the v a l u e s o b t a i n e d under t e s t s i t u a t i o n s were compared t o p r e - i n f u s i o n l e v e l s by the p a i r e d S t u d e n t ' s t - t e s t . 200 RESULTS I . EFFECT OF ENKEPHALIN ON GIP-STIMULATED SLI SECRETION C o n s i s t e n t w i t h p r e v i o u s r e p o r t s , ( M c i n t o s h e t a l . , 1981b) , the a d m i n s t r a t i o n o f 1 nM GIP r e s u l t e d i n a prompt and s u s t a i n e d i n c r e a s e i n SLI s e c r e t i o n . I n f u s i o n o f met-e n k e p h a l i n caused an immediate i n h i b i t i o n o f the G I P - s t i m u l a t e d SLI s e c r e t i o n which was shown t o be dose-dependent ( F i g u r e 54). A t a f i n a l c o n c e n t r a t i o n o f 1 pR, met-enk c o m p l e t e l y b l o c k e d the G I P - s t i m u l a t e d SLI s e c r e t i o n , w h i l e the lower dose o f 100 pM caused a much s m a l l e r i n h i b i t i o n o f S L I . Upon removal o f the e n k e p h a l i n , SLI s e c r e t i o n i n c r e a s e d , r e t u r n i n g t o l e v e l s which were s l i g h t l y l e s s than those o b t a i n e d p r e v i o u s t o the e n k e p h a l i n i n f u s i o n . T h i s i n h i b i t o r y e f f e c t o f met-enk was found t o be naloxone s e n s i t i v e i n t h a t the i n f u s i o n o f 100 nM naloxone p r i o r t o the i n t r o d u c t i o n o f met-enk c o m p l e t e l y b l o c k e d the i n h i b i t o r y e f f e c t o f 1 nM met-enk ( F i g u r e 55A) . Naloxone on i t s own ( p e r i o d s 13-17) d i d not a f f e c t the SLI r e l e a s e s t i m u l a t e d by GIP. F o l l o w i n g removal o f naloxone ( p e r i o d s 28-38) the i n h i b i t o r y e f f e c t o f met-enk was a g a i n o b s e r v e d . A t h i g h e r met-enk doses ( F i g u r e 55B) , t h e r e was a s l i g h t r e d u c t i o n i n the r a t e o f SLI s e c r e t i o n even i n the presence o f 100 nM n a l o x o n e ; however, when the naloxone i n f u s i o n was d i s c o n t i n u e d , SLI r e l e a s e dropped down t o b a s a l l e v e l s and r e t u r n e d t o G I P - s t i m u l a t e d l e v e l s f o l l o w i n g t e r m i n a t i o n o f met-enk i n f u s i o n . 201 A. GIP ( 1 n M ) Met-Enk (IjuM) • t V 1600' ~ 1200' E cn Q - 800-400-B. 2400 | 1800-1 cn CL 1200-600-GIP (InM) Met-Enk 1 (100 PM) 10 -r— 20 —I 1— 30 40 Time in Minutes FIGURE 54: EFFECT OF MET-ENK ON GIP-STIMULATED SECRETION OF SLI FROM ISOLATED PERFUSED RAT STOMACHS. SLI was s t i m u l a t e d d u r i n g the i n f u s i o n o f 1 nM GIP (12-35). Met-enk was i n t r o d u c e d d u r i n g p e r i o d s 20-28 t o g i v e a f i n a l c o n c e n t r a t i o n o f 1 juM (A) or 100 pM (B) . The SLI s e c r e t i o n r a t e was s i g n i f i c a n t l y reduced d u r i n g p e r i o d s 21-29 p<0.01 i n (A) and 22-28 p<0.05 i n ( B ) . (n=6) 202 1600 1200 c E 800 CO 400 Gastric Inhibitory Poly peptide (I nM) Noloxone(IOOnM)|  | M e t - E n | l l n E r r ~ | B. 1600 -1200 E \ cn 9- 800 CO 400 Gastric Inhibitory Polypeptide (I nM) tory NaloxoneOOOnM fiSBafflaflHOI 10 I 20 —r— 30 I 40 \ 50 Time in Minutes FIGURE 55: EFFECT OF NALOXONE ON MET-ENK-INDUCED INHIBITION OF GIP-STIMULATED SLI SECRETION FROM ISOLATED PERFUSED RAT STOMACHS. SLI was s t i m u l a t e d d u r i n g p e r i o d s 9-46. Naloxone (100 nM) was i n t r o d u c e d d u r i n g p e r i o d s 13-19 and met-enk was i n f u s e d (18-38) at a dose o f 1 nM (A) and 1 u^M (B) . The r a t e o f s e c r e t i o n was s i g n i f i c a n t l y reduced (p<0.01) d u r i n g p e r i o d s 33-41 (A) and 30-39 (B) . (n=5) 203 I I . EFFECT OF ATROPINE AND HEXAMETHONIUM ON ENKEPHALIN INDUCED INHIBITION OF GIP-STIMULATED SLI RELEASE The a d m i n i s t r a t i o n o f 1 pM a t r o p i n e , a m u s c a r i n i c c h o l i n e r g i c a n t a g o n i s t , d i d not a f f e c t e n k e p h a l i n - i n d u c e d i n h i b i t i o n o f G I P - s t i m u l a t e d SLI r e l e a s e ( F i g u r e 56) . A l t h o u g h complete i n h i b i t i o n o f SLI s e c r e t i o n t o b a s a l l e v e l s was not a c h i e v e d w i t h the a d m i n s t r a t i o n o f 1 pM met-enk ( F i g u r e 57) as was observed p r e v i o u s l y , the i n t r o d u c t i o n o f the g a n g l i o n i c b l o c k e r , hexamethonium (400 pM) , d u r i n g p e r i o d s 31 - 40 caused no s i g n i f i c a n t change i n the i n h i b i t i o n o f SLI s e c r e t i o n by met-enk. I I I . EFFECT OF DYNORPHIN 1-13 ON GIP-STIMULATED SLI SECRETION Dynorphin 1-13 was found t o i n h i b i t the G I P - s t i m u l a t e d r e l e a s e o f S L I . W i t h a h i g h dose o f 1^pM ( F i g u r e 5 8 ) , the r a t e of SLI s e c r e t i o n was reduced by 53 - 6% w h i l e a lower dose o f 200 nM ( F i g u r e 59) d y n o r p h i n 1-13 caused a s i m i l a r 58 — 6% drop i n SLI s e c r e t i o n . 204 1500-1 .£ 1000-E CL if) 500 Gastric Inhibitory Polypeptide (InM) Met-Enkephal in ( I / J M ) Atropine (IyuM) o 0 8 16 24 32 Time in Minutes 40 48 FIGURE 56: EFFECT OF ATROPINE ON MET-ENK-INDUCED INHIBITION OF SLI SECRETION FROM ISOLATED PERFUSED RAT STOMACHS. SLI was stimulated during periods 10-45 with 1 nM GIP. Met-enk (1 pM) was introduced from 20-40, and atropine (1 jmM) was administered during periods 31-40. There was no s i g n i f i c a n t difference between the secretion rates between periods 20-30 and 31-40. (n=5) 205 Gastric Inhibitory Polypeptide (I nM) Met-Enkephalin (\pM) Hexamethonium ( 4 0 0 / J M ) 2 0 0 0 - 1 "i 1 n — 1 1 • 1 1 1 • r 0 8 16 2 4 3 2 4 0 Time in Minutes FIGURE 57: EFFECT OF HEXAMETHONIUM ON MET-ENK-INDUCED INHIBITION OF GIP-STIMULATED SLI SECRETION FROM ISOLATED PERFUSED RAT STOMACHS. SLI was stimulated during periods 10-45 with 1 nM GIP. Met-enk (1 juM) was introduced from 20-40 and hexamethonium (400 juM) during periods 31-40. No s i g n i f i c a n t difference was observed between periods 31-40 and 20-30. (n=4) 206 FIGURE 58: EFFECT OF 1 pM DYNORPHIN 1-13 ON GIP-STIMULATED SLI SECRETION FROM ISOLATED PERFUSED RAT STOMACHS. SLI s e c r e t i o n was s t i m u l a t e d d u r i n g p e r i o d s 10-35 w i t h 1 nM GIP. The a d m i n i s t r a t i o n o f 1 uM dyn 1-13 (20-28) was found t o cause a s i g n i f i c a n t (*= p<0.05, **=p<0.01) r e d u c t i o n i n GIP-s t i m u l a t e d SLI s e c r e t i o n d u r i n g p e r i o d s 22-29. (n=4) 207 FIGURE 59: EFFECT OF 200 nM DYNORPHIN 1-13 ON G I P -STIMULATED SLI SECRETION FROM ISOLATED PERFUSED RAT STOMACHS. SLI s e c r e t i o n was s t i m u l a t e d d u r i n g p e r i o d s 10-35 w i th 1 nM G I P . The a d m i n i s t r a t i o n of 200 nM dyn 1-13 (per iods 20-28) caused a s i g n i f i c a n t r e d u c t i o n (*=p<0.05) i n SLI s e c r e t i o n d u r i n g p e r i o d s 21-28. A s i g n i f i c a n t i n c r e a s e (p<0.05) over p r e - d y n o r p h i n GIP s t i m u l a t e d SLI s e c r e t i o n o c c u r r e d at p e r i o d 32. (n=5) 208 DISCUSSION The p o t e n t i n h i b i t o r y a c t i o n s o f exogenously a d m i n i s t e r e d e n k e p h a l i n and d y n o r p h i n suggest t h a t endogenous o p i o i d p e p t i d e s may f u n c t i o n as r e g u l a t o r s o f g a s t r i c s o m a t o s t a t i n s e c r e t i o n . I n h i b i t i o n o f G I P - s t i m u l a t e d SLI s e c r e t i o n by both met-enk and d y n o r p h i n 1-13 i n d i c a t e s t h a t o p i o i d p e p t i d e s are a b l e t o a f f e c t s t i m u l a t e d D - c e l l a c t i v i t y as w e l l as b a s a l a c t i v i t y (Chiba e t a l . , 1980). The s p e c i f i c i t y o f a c t i o n o f e n k e p h a l i n was demonstrated by i t s c o n c e n t r a t i o n dependence and s e n s i t i v i t y t o the o p i o i d a n t a g o n i s t , n a l o x o n e . D y n o rphin 1-13 was l e s s p o t e n t than met-enk. W h i l e 1 jiM met-enk c o m p l e t e l y i n h i b i t e d G I P - s t i m u l a t e d SLI r e l e a s e , the same dose o f d y n o r p h i n 1-13 r e s u l t e d i n o n l y a 53% r e d u c t i o n . Dynorphins have been demonstrated t o be p o t e n t kappa a g o n i s t s i n b r a i n and the g u i n e a p i g i l e u m (Chavkin and G o l d s t e i n , 1981; C h a v k i n e t a l . , 1982; Garzon e t a l . , 1982; Yoshimura e t a l . , 1982a,b; Cox and C h a v k i n , 1983) . I n the r e c e p t o r l o c a l i z a t i o n s t u d i e s i t was not p o s s i b l e t o d e t e c t s p e c i f i c kappa r e c e p t o r s i n the g a s t r i c c o r p us or antrum, but i n l i g h t o f the d e m o n s t r a t i o n o f d y n o r p h i n - l i k e m o l e c u l e s i n g a s t r i c e x t r a c t s and smooth muscle e f f e c t s o f d y n o r p h i n s they are l i k e l y t o be p r e s e n t . T h e r e i s , however, no a v a i l a b l e e v i d e n c e t h a t g a s t r i c D - c e l l s have kappa r e c e p t o r s . A t h i g h c o n c e n t r a t i o n s o p i o i d a g o n i s t s are c a p a b l e o f i n i t i a t i n g a response by i n t e r a c t i n g w i t h o t h e r r e c e p t o r t y p e s . T h e r e f o r e , a p o s s i b l e reason f o r the s i m i l a r i n h i b i t o r y e f f e c t s o f 200 nM and 1 pM d y n o r p h i n 1-13 i s t h a t a t h i g h c o n c e n t r a t i o n s a f i f t y p e r c e n t i n h i b i t i o n i s the 209 maximum e f f e c t a t t a i n a b l e by a kappa a g o n i s t a c t i n g through mu or d e l t a r e c e p t o r s . A c h o l i n e r g i c pathway was e a r l i e r proposed t o be p o t e n t i a l l y i n v o l v e d i n the i n h i b i t i o n o f SLI r e l e a s e by e n k e p h a l i n s ( M c i n t o s h e t a l . , 1 9 8 3 ) . The major e v i d e n c e f o r t h i s was t h a t v a g a l i n h i b i t i o n o f G I P - s t i m u l a t e d SLI r e l e a s e c o u l d be p a r t i a l l y b l o c k e d by a t r o p i n e , as w e l l as by n a l o x o n e , and t h a t a c e t y l c h o l i n e mimicked the i n h i b i t o r y e f f e c t o f v a g a l s t i m u l a t i o n ( M c i n t o s h e t a l . , 1 9 8 1 a ) . A t t h a t time the m a j o r i t y of d a t a i n d i c a t e d t h a t the p r i m e , i f not the o n l y , s i t e o f a c t i o n o f e n k e p h a l i n s was i n the m y e n t e r i c p l e x u s . I n l i g h t o f the c u r r e n t r e c e p t o r d i s t r i b u t i o n s t u d i e s i t now seems p o s s i b l e t h a t the e n k e p h a l i n s c o u l d a c t d i r e c t l y on the D - c e l l or v i a a c t i o n s on neurons i n the m y e n t e r i c or submucosal p l e x i . The pathways o r i g i n a l l y s uggested ( M c i n t o s h e t al.,1983) w i l l be used f o r f u r t h e r d i s c u s s i o n ( F i g u r e 6 0 ) . The most d i r e c t pathway by which e n k e p h a l i n e r g i c neurones c o u l d a c t on the c h o l i n e r g i c system would be by s t i m u l a t i n g c h o l i n e r g i c i n t e r n e u r o n s ( F i g u r e 60A). However, i n the e n t e r i c nervous system of the g u i n e a p i g i l e u m , o p i o i d s have been shown to p r e s y n a p t i c a l l y i n h i b i t the f i r i n g o f c h o l i n e r g i c neurons (North and Egan, 1983). T h e r e f o r e , a second, more p l a u s i b l e pathway would be a d i s i n h i b i t i o n system whereby e n k e p h a l i n e r g i c neurons i n h i b i t an i n t e r n e u r o n o f unknown i d e n t i t y , which t o n i c a l l y i n h i b i t s the i n f l u e n c e o f c h o l i n e r g i c neurons on D-c e l l s ( F i g u r e 60B). A s i m i l a r d i s i n h i b i t o r y s i t u a t i o n i n v o l v i n g e n k e p h a l i n e r g i c neurons has been d e s c r i b e d i n the hippocampus ( Z i e g e l g a n s b e r g e r e t a l . , 1979; N i c o l l e t a l . , 1 9 8 0 ) . 210 A. B. C. © 0 Enkephalinergic Neurone (3 Cholinergic Neurone Unknown Transmitter (^ j Enkephalin Endocrine Ce Somatostatin Cell Parasymathetic Nerves Myenteric Plexus Mucosa FIGURE 60: POSSIBLE PATHWAYS BY WHICH ENDOGENOUS ENKEPHALINS INHIBIT SLI SECRETION FROM GASTRIC D-CELLS. 4211 The f i n d i n g t h a t the i n h i b i t o r y e f f e c t o f e n k e p h a l i n on SLI s e c r e t i o n was u n a f f e c t e d by c h o l i n e r g i c m u s c a r i n i c or n i c o t i n i c r e c e p t o r a n t a g o n i s t s p r o v i d e d no e v i d e n c e f o r the i n v o l v e m e n t o f c h o l i n e r g i c pathways. Such a pathway can n o t , however, be d e f i n i t e l y e x c l u d e d s i n c e i t i s p o s s i b l e t h a t , w i t h the r e l a t i v e l y h i g h c o n c e n t r a t i o n s o f e n k e p h a l i n used, the predominant a c t i o n was a d i r e c t i n h i b i t i o n a t the l e v e l o f the D - c e l l . T h i s p o s s i b i l i t y i s s u p p o r t e d by the a u t o r a d i o g r a p h i c e v i d e n c e p r e s e n t e d i n Chapter 4 d e m o n s t r a t i n g the o c c u r r e n c e o f s p e c i f i c o p i o i d b i n d i n g s i t e s i n the mucosa o f the r a t antrum and c o r p u s . A l t h o u g h w i t h l i g h t m i c r o s c o p i c t e c h n i q u e s the e x a c t c e l l u l a r l o c a l i z a t i o n o f the b i n d i n g s i t e s c o u l d not be r e s o l v e d , i t i s p o s s i b l e t h a t some of the mucosal s i t e s were a s s o c i a t e d w i t h s o m a t o s t a t i n s e c r e t i n g D - c e l l s , and t h a t t h e s e s i t e s r e p r e s e n t the f u n c t i o n a l r e c e p t o r s m e d i a t i n g the i n h i b i t i o n o f D - c e l l a c t i v i t y by exogenous e n k e p h a l i n s . The p h y s i o l o g i c a l e v e n t s l e a d i n g t o i n h i b i t i o n o f D - c e l l f u n c t i o n by endogenous o p i o i d p e p t i d e s i s more d i f f i c u l t t o d i s c e r n and the problem of the o r i g i n o f e n k e p h a l i n d e t e c t e d i n the g a s t r i c v a s c u l a t u r e a g a i n a r i s e s . I t i s p o s s i b l e f o r endogenous o p i o i d p e p t i d e s , o r i g i n a t i n g from e n t e r i c n eurons, t o f u n c t i o n i n a n e u r o c r i n e or n e u r o e n d o c r i n e f a s h i o n such t h a t f o l l o w i n g r e l e a s e from nerve t e r m i n a l s they a c t v i a the e x t r a c e l l u l a r space or v a s c u l a t u r e r e s p e c t i v e l y . The a l t e r n a t i v e pathway ( F i g u r e 63C) , i n which a n t r a l e n d o c r i n e c e l l s may r e l e a s e o p i o i d p e p t i d e s t o a c t d i r e c t l y on s o m a t o s t a t i n c e l l s i n a p a r a c r i n e or e n d o c r i n e manner, w i l l be d i s c u s s e d f i r s t . 212 The well-documented m e t a b o l i c i n s t a b i l i t y o f e n k e p h a l i n i s d i f f i c u l t t o r e c o n c i l e w i t h a n e u r o e n d o c r i n e or hormonal r o l e . Indeed, as d e t a i l e d i n Chapter 5, the r e l e a s e o f endogenous l e u - e n k - I R from the i s o l a t e d v a s c u l a r l y p e r f u s e d r a t stomach c o u l d o n l y be d e t e c t e d i n the pre s e n c e o f p e p t i d a s e i n h i b i t o r s . I t i s , however, p o s s i b l e t h a t the l a r g e r more s t a b l e p r o d u c t s of p r o e n k e p h a l i n , demonstrated t o be p r e s e n t i n r a t stomach e x t r a c t s (Chapter 3) are f u n c t i o n a l o p i o i d p e p t i d e s a c t i n g v i a the v a s c u l a t u r e . There have been some i n c o n s i s t e n c i e s i n the immunocytochemical s t u d i e s d e m o n s t r a t i n g e n k e p h a l i n - I R i n a n t r a l G - c e l l s . E a r l y s t u d i e s (Polak e t al.,1977) r e p o r t e d the presence o f met-enk i n human a n t r a l G - c e l l s . However, these s t u d i e s were performed b e f o r e the d i s c o v e r y o f l a r g e r forms o f met-enk; t h e r e f o r e , t h e r e may have been some c r o s s - r e a c t i v i t y w i t h the met-enk a n t i s e r u m employed and the C - t e r m i n a l l y extended forms o f e n k e p h a l i n . I n s e v e r a l r e c e n t s t u d i e s employing a n t i s e r a s p e c i f i c f o r the unique c a r b o x y - t e r m i n a l l y extended o p i o i d p e p t i d e s ( L a r s s o n and S t e n g a a r d - P e d e r s e n , 1981; Bu'Lock e t a l . , 1983), i t has been demonstrated t h a t o n l y met-enk-arg-phe or o t h e r extended forms o f met-enk oc c u r i n r a t a n t r a l G - c e l l s ; met-enk and l e u - e n k c o u l d not be d e t e c t e d . O t h e r s , ( G i r a u d e t a l . , 1984a; J o n s s o n , 1 9 8 5 ) , a l s o u s i n g r e g i o n s p e c i f i c a n t i s e r a , have found met-enk, met-enk-arg-phe and met-e n k - a r g - g l y - l e u t o oc c u r i n G - c e l l s . D e s p i t e these i n c o n s i s t e n c i e s , most l i k e l y a r i s i n g from d i f f e r e n c e s i n a n t i b o d y c h a r a c t e r i s i t i c s and s p e c i e s d i f f e r e n c e s , i t i s e v i d e n t t h a t l a r g e r p r o e n k e p h a l i n - d e r i v e d p e p t i d e s a re p r e s e n t i n a n t r a l e n d o c r i n e c e l l s and the s e may be the p h y s i o l o g i c a l l y 213 r e l e v a n t endogenous o p i o i d s u b s t a n c e s which a c t h u m o r a l l y , or i n a p a r a c r i n e manner, t o i n h i b i t SLI s e c r e t i o n from g a s t r i c D-c e l l s d i r e c t l y . A l t h o u g h o p i o i d p e p t i d e s occur i n e n d o c r i n e c e l l s , the major so u r c e o f endogenous g a s t r i c o p i o i d p e p t i d e s would be from i n t r i n s i c n e r v e s s i n c e most e n k e p h a l i n - I R , determined by immunocytochemistry, i s c o n t a i n e d i n e n t e r i c neurons ( L a r s s o n and S t e n g a a r d - P e d e r s e n , 1981; Bu'Lock e t a l . , 1983; Wang e t a l . , 1984). From d i r e c t e v i d e n c e d e m o n s t r a t i n g t h a t endogenous l e u - e n k - I R i s r e l e a s e d from the r a t stomach i n response t o the n i c o t i n i c g a n g l i o n i c a g o n i s t , DMPP (Chapter 5 ) , and from i n f e r e n c e s made from s t u d i e s d e m o n s t r a t i n g a p a r t i a l r e v e r s a l of v a g a l l y i n h i b i t e d SLI r e l e a s e by naloxone ( M c i n t o s h e t a l . , 1983), i t can be i m p l i e d t h a t autonomic s t i m u l a t i o n would cause r e l e a s e o f o p i o i d p e p t i d e s from e n t e r i c neurons. Because i t i s u n l i k e l y t h a t much o f the e n k e p h a l i n s r e l e a s e d from nerve t e r m i n a l s would n o r m a l l y escape e n z y m a t i c d e g r a d a t i o n and e n t e r the g a s t r i c c i r c u l a t i o n i t i s more p r o b a b l e t h a t they a c t as n e u r o t r a n s m i t t e r s or n e u r o m o d u l a t o r s . Most c e l l b o d i e s o f e n k e p h a l i n e r g i c e n t e r i c neurons a re l o c a t e d i n the m y e n t e r i c p l e x u s w i t h f i b r e s p r o j e c t i n g w i t h i n the p l e x u s and i n the d i r e c t i o n o f the submucosal p l e x u s ( S c h u l t z b e r g e t a l . , 1 9 8 0 ; F u r n e s s e t a l . , 1 9 8 2 ) . A few e n k e p h a l i n - c o n t a i n i n g c e l l b o d i e s and f i b r e s have been l o c a t e d i n the submucosal p l e x u s (Wang e t a l . , 1984). There have been no r e p o r t s o f e n k e p h a l i n e r g i c f i b r e s i n the mucosa o f the stomach. As s t a t e d p r e v i o u s l y , d e s p i t e the l a c k o f e f f e c t o f c h o l i n e r g i c r e c e p t o r a n t a g o n i s t s on e n k e p h a l i n - i n d u c e d i n h i b i t i o n o f SLI r e l e a s e , the 214 i n v o l v e m e n t o f c h o l i n e r g i c pathways, as o u t l i n e d i n F i g u r e 60A and B, cannot be e x c l u d e d . N e v e r t h e l e s s , the r e c e p t o r l o c a l i z a t i o n s t u d i e s suggest a number of a l t e r n a t i v e s . The dense d i s t r i b u t i o n o f b i n d i n g s i t e s i n the mucosa su g g e s t s t h a t o p i o i d p e p t i d e s c o u l d a c t d i r e c t l y i f some o f these s i t e s are l o c a t e d on D - c e l l s . S i n c e t h e r e i s no a v a i l a b l e d a t a f o r an e x t e n s i v e e n k e p h a l i n e r g i c i n n e r v a t i o n o f the mucosa the most l i k e l y s ource f o r o p i o i d s a c t i n g i n t h i s r e g i o n would be e n d o c r i n e c e l l s . Such a c o n c l u s i o n c o u l d be v a l i d f o r the antrum but t h e r e has been no d e m o n s t r a t i o n o f e n k e p h a l i n -c o n t a i n i n g e n d o c r i n e c e l l s i n the c o r p u s . I t i s t h e r e f o r e p o s s i b l e t h a t neurons do e x i s t but s i m p l y have not as y e t been d e t e c t e d . An a l t e r n a t i v e p o s s i b i l i t y i s t h a t a n t r a l l y d e r i v e d o p i o i d p e p t i d e s i n f l u e n c e the c o r p o r e a l D - c e l l s v i a a g a s t r i c p o r t a l system (Torrence and T a y l o r , 1975). A second a l t e r n a t i v e i s t h a t e n k e p h a l i n e r g i c neurons a c t i n d i r e c t l y a t the l e v e l o f the m y e n t e r i c and/or submucosal p l e x u s . S i n c e the major s i g h t o f both mu- and d e l t a - o p i o i d l i g a n d b i n d i n g i n the stomach was the submucosal p l e x u s t h i s l o c u s would now be f a v o u r e d . I f the f i n a l i n h i b i t o r y neuron i s indeed n o n - c h o l i n e r g i c then i t s i d e n t i t y i s u n c l e a r . Substance P a l s o i n h i b i t s g a s t r i c SLI r e l e a s e but i t s e f f e c t i s u n a f f e c t e d by naloxone (Kwok e t a l . , 1 9 8 5 ) . I t i s , t h e r e f o r e , u n l i k e l y t o be the t r a n s m i t t e r concerned and f u r t h e r s t u d i e s are needed. 215 CHAPTER SEVEN  CONCLUDING REMARKS The combined e v i d e n c e f o r the e x i s t e n c e o f endogenous g a s t r i c o p i o i d p e p t i d e s (Chapter 3) , t h e i r r e l e a s e from the stomach (Chapter 5) and e f f e c t s on g a s t r i c s o m a t o s t a t i n s e c r e t i o n (Chapter 6) , c o u p l e d w i t h the presence o f s p e c i f i c o p i o i d r e c e p t o r s i n the g a s t r o i n t e s t i n a l t r a c t (Chapter 4) , p r o v i d e s a s t r o n g case f o r o p i o i d p e p t i d e s as b e i n g i m p o r t a n t r e g u l a t o r s o f g a s t r i c f u n c t i o n s . That the p r o d u c t s o f two s e p a r a t e o p i o i d p e p t i d e f a m i l i e s were found t o oc c u r i n the r a t stomach e x t r a c t s i n t r o d u c e s a fundamental q u e s t i o n i n o p i o i d r e s e a r c h , namely t h a t o f why t h e r e e x i s t s such a m u l t i t u d e o f o p i o i d p e p t i d e s w i t h apparent o v e r l a p p i n g d i s t r i b u t i o n s and a c t i o n s . Are a l l o f the s e o p i o i d p e p t i d e s p h y s i o l o g i c a l l y r e l e v a n t or are some b i o s y n t h e t i c i n t e r m e d i a t e s or b y - p r o d u c t s ? I n the g a s t r o i n t e s t i n a l t r a c t i t i s c o n c e i v a b l e t h a t each o p i o i d p e p t i d e p l a y s a unique r o l e i n c o n t r o l l i n g a s p e c i f i c f u n c t i o n . S m a l l o p i o i d p e p t i d e s such as met- and leu-enk may a c t as n e u r o t r a n s m i t t e r s or neuromodulators i n the e n t e r i c nervous system. The l a r g e r o p i o i d p e p t i d e s may a l s o f u n c t i o n as n e u r o t r a n s m i t t e r s , but by v i r t u e o f t h e i r m e t a b o l i c s t a b i l i t y , t h ey may a c t as neurohormones or hormones s e c r e t e d by g a s t r i c e n d o c r i n e c e l l s . However, s i n c e o p i o i d p e p t i d e s are d e r i v e d from common p r e c u r s o r s , i t must be dete r m i n e d whether a l l p r o d u c t s c o - e x i s t 216 in the same c e l l or whether d i f f e r e n t cell-types demonstrate d i f f e r e n t i a l processing. The demonstration of ganglionic cholinergic stimulation of gastric leu-enk i s v i t a l information for the understanding of the regulation of g a s t r i c opioid release, yet this knowledge i s only the basis from which to begin examining the complex nervous and hormonal pathways involved. How i s the autonomic nervous system involved? Which enteric interneurons are involved? F i n a l l y , the widespread d i s t r i b u t i o n of opioid receptors in the g a s t r o i n t e s t i n a l t r a c t implies that the actions of endogenous opioid peptides must likewise be extensive. Due to the i d e n t i f i c a t i o n of the s t r i k i n g number of opioid binding s i t e s in the submucosal plexus and mucosa of the rat stomach and intestine, the functions of g a s t r o i n t e s t i n a l opioids must be re-examined. Emphasis has been placed on their effects in the myenteric plexus, but in view of the present findings, the scope of opioid peptide actions in the g a s t r o i n t e s t i n a l tract should be broadened. In the case of enkephalinergic control of somatostatin secretion, inferences were made (Chapter 6) as to the dual role for opioid peptides as neurotransmitters acting through submucosal receptors to i n d i r e c t l y a f f e c t g a s t r i c D-c e l l s and as hormones d i r e c t l y a f f e c t i n g somatostatin secretion by means of mucosal receptors. Therefore, i f the precise anatomical d i s t r i b u t i o n s of the multiple opioid receptors and the d i s t r i b u t i o n s of the multitude of g a s t r o i n t e s t i n a l opioid peptides can be determined, their physiological function might be more c l e a r l y defined. 217 Thus, i n c o n c l u s i o n , the e l u c i d a t i o n o f the m o l e c u l a r forms of g a s t r i c o p i o i d p e p t i d e s , the v i s u a l i z a t i o n o f t h e i r r e c e p t o r d i s t r i b u t i o n s and t h e i r p h y s i o l o g i c a l a c t i o n s demonstrated by the p r e s e n t s t u d i e s c o n t r i b u t e d t o the i n c r e a s i n g w e a l t h o f knowledge i n the f i e l d o f o p i o i d p e p t i d e r e s e a r c h . However, w i t h each new p i e c e o f i n f o r m a t i o n s e v e r a l more q u e s t i o n s a r i s e c o n c e r n i n g the r e g u l a t i o n o f the r e l e a s e o f g a s t r i c o p i o i d p e p t i d e s and the n e u r a l and hormonal pathways i n which these p e p t i d e s are t hemselves i n v o l v e d . The work i n t h i s t h e s i s i s t h e r e f o r e p r e s e n t e d i n the hope t h a t these q u e s t i o n s may promote the s e a r c h f o r more answers and the g e n e r a t i o n o f f u r t h e r q u e s t i o n s towards the u n d e r s t a n d i n g o f endogenous o p i o i d p e p t i d e s as r e g u l a t o r s o f g a s t r o i n t e s t i n a l f u n c t i o n , f o r i n the words of John L o c k e , "the improvement o f the u n d e r s t a n d i n g i s f o r two ends: f i r s t , f o r our own i n c r e a s e o f knowledge; s e c o n d l y t o e n able us t o d e l i v e r and make out t h a t knowledge t o o t h e r s " . 218 LIST OF REFERENCES ,AKIL, H., D.J. MAYER, and J.C. LIEBESKIND (1976). Antagonism o f s t i m u l a t i o n - p r o d u c e d a n a l g e s i a by na l o x o n e , a n a r c o t i c a n t a g o n i s t . S c i e n c e 191:961-962. AKIL, H. and S.J. WATSON (1983). 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Characteriza-tion of Gastric Opioid Peptides. Canadian Journal of Physiology and Pharmacology 61:AXViii. E. Nishimura, A.M.J. Buchan and C.H.S. Mcintosh (1984). Autoradiographic localization of gastric opioid peptides and the release of opioid peptides from the perfused rat stomach. Canadian Journal of Physiology and Pharmacology 62:AR21-AR22. E. Nishimura, V. Bakich and C.H.S. Mcintosh (1985). The regulation of somato-statin and gastrin secretion from the isolated perfused rat stomach by opioid peptides. Canadian Journal of Physiology and Pharmacology 63. 4198A/54A 

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