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Immunological techniques in the investigation of the physiological functions of gastric inhibitory polypeptide… Dryburgh, Jill Robertson 1977

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IMMUNOLOGICAL TECHNIQUES IN THE INVESTIGATION OF THE PHYSIOLOGICAL FUNCTIONS OF GASTRIC INHIBITORY POLYPEPTIDE AND MOTILIN by JILL ROBERTSON DRYBURGH B.Sc. University of Edinburgh, 1962 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD EXTERNAL EXAMINER THE UNIVERSITY OF BRITISH COLUMBIA Ap r i l , 1977 00 J i l l Robertson Dryburgh, 1977 DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF PHYSIOLOGY SUPERVISOR In present ing t h i s thes is in p a r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make it f ree ly ava i lab le for reference and study. I fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representa t ives . It is understood that copying or pub l i ca t ion of th is thes is fo r f i n a n c i a l gain sha l l not be allowed without my wri t ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 i i ABSTRACT A radioimmunoassay was developed, specific for the gastrointestinal polypeptide, motilin. Antisera were raised in guinea pigs and rabbits. The immunogen was porcine motilin, conjugated to bovine serum albumin by the carbodiimide condensa-tion reaction. The routine antiserum behaved identically towards endogenously-released motilin and the pure standard preparation. A radioactive tracer of high 125 specific activity was obtained after incorporation of - iodine into the motilin molecule by the chloramine-T method. The optimum conditions for a l l other assay variables were established to produce the most sensitive displacement Cstandard) curve. Motilin antiserum, coupled directly to an agarose matrix, retained f u l l antibody activity and sensitivity. It is a feasible technique for use in both the radioimmunoassay and in the extraction of motilin from both serum and tissue extracts. The fasting serum levels of IR- motilin was 190 - 131 pg/ml in men and 294 -'.44 pg/ml in dogs (mean - SD) . The increase in motor activity in the extrinsically denervated fundic pouch of the dog after duodenal alkalinization was associated with a concomitant elevation in serum IR- motilin levels. This increase in serum IR- motilin was in the same range as that achieved by the exogenous administration of the porcine polypeptide which produced the same motor response. Duodenal acidification produced an apparent increase in serum IR-motilin with no associated increase in gastric motor activity. Only one peak of motilin immunoreactivity was detected when serum containing alkali-stimulated motilin or a partially purified duodenal extract were subjected to gel f i l t r a t i o n on Sephadex G-50. The distribution of motilin throughout the hog gastrointestinal tract, determined by radioimmunoassay on partially purified extracts, agreed with i i i the immunocytochemical findings that motilin was predominantly located in the duodeninn and jejunum, with traces.in the upper ileum. Virtually the intact molecule was required for the expression of f u l l biolo-gical potency. The individual amino acids were important inasmuch as they contributed to the charge distribution and conformation of the molecule. The physiological release and function of motilin have yet to be determined. Elevated levels of circulating IR- motilin have not been associated with any gastro-intestinal function, although they appear to be depressed by feeding. Motilin has been implicated in the control of the interdigestive phase of gastric motor activity. It may be acting in a local or paracrine manner. Motilin has not been implicated in any .•cU'in±cal.rst"ait"eC&s sjffetfce i The hormonal status of gastric inhibitory polypeptide (GIP) has been studied with the existing radioimmunoassay, modified to improve the label specific activity (by ion exchange chromatography). Direct coupling of GIP antisera to agarose beads was unsatisfactory, antibody activity and sensitivity being greatly reduced by the close proximity of the solid matrix. The postulated role of GIP as the enterogastrone 1 of Kosaka and Lim, suggested by studies with exogenously-administered polypeptide, was confirmed by experiments in the dog. Pentagastrin-stimulated gastric acid secretion was inhibited by intra-duodenal infusion with glucose or fat; this inhibition being associated with a s i g n i f i -cant elevation in the circulating serum IR- GIP levels, within the range pro-duced by ingestion of a mixed meal. GIP does not appear to be involved in the inhibition of gastric acid secretion produced by duodenal acidification. iv Endogenous;GIP.stimulated by either fat or glucose exhibited at least 3 Immunoreactive components after column chromatography. The IR- GIP eluting in the void volume appeared to represent a non-specific complex between GIP and a serum protein and is possibly biologically inactive. A second IR-GIP component with a molecular weight of 7 5 0 0 - 8 0 0 0 (ProGIP), eluted ahead of the established form of GIP (molecular weight = 5 1 0 5 ) . ProGIP has been found to be relatively unstable. ProGIP and G I P ^ Q Q Q have also been detected in extracts of hog duodenal mucosa. The established insulinotropic effect of GIP correlates best with that percentage of the total IR- GIP composed of ProGIP and GIP 5 0 0 ( ). The relative proportions of IR- G I P 5 0 0 Q and IR- ProGIP in serum samples taken at different times after ingestion of either fat or glucose, suggest that ProGIP i s either a precursor of GIP or that the ProGIP-producing cells occupy a more distal region of the duodenal and jejunal mucosa than the GIP- producing c e l l s . Exogenous administration of synthetic somatostatin in dogs and man w i l l inhibit both.GIP release by either fat or glucose and the insulino-tropic action of GIP at the level of the 8/-cell. Naturally-occurring intestinal or pancreatic somatostatin may contribute to the control of GIP release and serve to modulate the GIP- mediated response of the gastric parietal or pancreatic S'-cell. TABLE' OF CONTENTS Page ABSTRACT i i LIST OF TABLES x i LIST OF FIGURES • x i i i ACKNOWLEDGEMENTS ^ ^ " " ^ ^ ^ ^ ^ " ^ " ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ " " " " " ^ — x v i i i INTRODUCTION • . 1 METHODS 19 DEVELOPMENT OF A RADIOIMMUNOASSAY FOR MOTILIN 19 A. Rationale 19 B. Iodination of motilin 20 1. Chloramine'-T method 20 2. Lactoperoxidase method 25 125 3. Estimation of specific activity of I-motilin 25 C. Product ion- of ahtdsera-. to mot i l in 3 0 1. In guinea pigs 30 2. In rabbits 30 3. Storage of antisera 32 4. Effect of varying antibody t i t r e 32 5. Measurement of comparative immunoreactivity of antiserum 35 6. Antibody recognition of antigen in standards and unknowns 40 7. Measurement of the a f f i n i t y of the antisera D. Conditions of the radioimmunoassay 1. Methods of standard curve evaluation 2. pH of the diluent buffer 3. Trasylol concentration of yhe diluent buffer 4. Plasma concentration of the diluent buffer 5. Concentration of labelled antigen added 6. Period of incubation 7. ). Protection from adsorption to glass 8. Routine assay conditions 9. Separation procedures 10. Methods of data analysis 40 42 42 44 44 46 46 48 50 52 . 54 56 v i Page E. Assay standards and inter-assay controls 59 1. Preparation and storage of standards 59 2. Preparation and storage of controls 59 3. Inter- and intra-assay controls 60 PREPARATION OF SYNTHETIC AND NATURAL MOTILIN FRAGMENTS AND ANALOGUES • ; • 60 A. Synthetic motilin 60 1. Preparation of 13-norleucine motilin 60 2. Preparation of synthetic motilin fragments 61 B. . Fragments of natural motilin 61 1. Cyanogen bromide cleavage of motilin 61 2. Tryptic and chymotryptic digestion of motilin 61 C. Modifications of natural motilin 62 1. C-terminal residue removal 62 2. N-terminal residue removal 62 3. Identification of the N-terminal residue 63 4. Acylation - acetylated derivative 65 5. Acylation - succinylated derivative 66 AFFINITY CHROMATOGRAPHY — 66 A. Activation of Sepharose 4B 66 B. Coupling of ligand to activated Sepharose V4B 67 COLUMN CHROMATOGRAPHY ; 67 A. Gel f i l t r a t i o n 67 B. Ion exchange chromatography 71 POLYACRYLAMIDE GEL ELECTROPHORESIS 72 A. Method for staining and destaining for qualitative •;/v". • r determinations 73 B. Method for staining and destaining for qualitative determinations 73 ANIMAL PREPARATIONS • ' 74 A. Chronic dog preparation 74 1. Bickel pouch 74 2. Mann-Bollman f i s t u l a 75 3. Gastric f i s t u l a 76 4. Truncal vagotomy 76 5. Antrectomy 76 v i i Page 6. Vagotomy and antrectomy 76 EXPERIMENTAL PROCEDURES . 77 A. In chronic dogs with Bickel pouches and duodenal f istulae 77 1. Bioassay for motil in 79 2. Effect of GIP on gastric acid secretion B. In chronic dogs with gastric fistulae 1. Determination of the rate of gastric emptying of l iquids 2. Determination of the rate of gastric emptying of solids C. In the intact dog SERUM ANALYSIS • — —•*• 83 83 83 84 84 87 87 80 81 81 82 83 A. Radioimmunoassays I. GIP radioimmunoassay 1. Iodination of GIP a. Routine chloramine-T iodination and purif ication b. Variation of chloramine-T iodination c. Lactoperoxidase iodination 2. Purif ication of 1 2 5 I - G I P 89 3. Extension of shel f - l i fe of 1 2 5 I - G I P 89 4. Production of antisera to GIP 93 a. Storage of antisera b. Characterization of antisera 5. Assay protocol 6. Preparation of standards 7. Preparation of controls 8. Separation techniques 95 II . Insulin radioimmunoassay 97 1.. B$? commercially available k i t 2. By non-commercial radioimmunoassay a. Iodination of insulin ; b. The insul in antibody / / c. Assay protocol B. Serum glucose determination 101 RESULTS — 102 ESTIMATION OP THE DEGREE OF CONJUGATION BETWEEN POLYPEPTIDES AND BOVINE SERUM ALBUMIN 102 a. With moti l in 102 93 93 93 95 95 97 97 99 100 100 v i i i Page B. With GIP 102 REPRODUCIBILITY OF IR-MOTILIN DETERMINATIONS 104 COMPARISON OF RADIOIMMUNOASSAY AND BIOASSAY FOR MOTILIN - 104 A. Immunological comparison 104 B. Biological comparison 108 MOLECULAR HOMOGENEITY OF MOTILIN 112 A. In serum 112 B. In tissue extracts 11.2 DISTRIBUTION OF MOTILIN THROUGHOUT THE HOG GASTROINTESTINAL TRACT • 115 RELATIONSHIP BETWEEN GASTRIC (FUNDIC) MOTOR ACTIVITY AND ENDOGENOUS MOTILIN RELEASE — . • 118 EFFECT OF INGESTION OF GLUCOSE OR A MIXED MEAL ON THE CIRCULATING LEVELS OF IR-MOTILIN 119 COMPARISON OF THE IMMUNOLOGICAL AND BIOLOGICAL ACTIVITY OF SYNTHETIC AND NATURAL MOTILIN FRAGMENTS AND ANALOGUES - 132 A. Immunological comparison 132 I. Synthetic motilin and fragments 132 II. Fragments of natural motilin 132 a. Cyanogen bromide cleavage 132 b. Tryptic and chymotryptic digestion 137 III. Modifications of natural motilin. 137 a. Removal of C-terminal residue(s) 137 b. After removal of N-terminal residue 137 c> Acylation - acetylation 139 d. Acylation - succinylation 139 B. Biological comparison 139 I. Synthetic motilin 139 II. Fragments of natural motilin 139 III. Modification s of natural motilin 142 a. Removal of C-terminal residue(s) 142 b. Removal of N-terminal residue 142 c. Acylation - acetylation 142 d. Agylation - succinylation 142 ix Page AFFINITY CHROMATOGRAPHY — 144 A. Application to radioimmunoassay 144 I. Motilin 144 a. Antiserum dilution and change in activity 144 b. RIA standard curves and chenge in sensitivity 144 I i . GIP 150 a. Antiserum dilution and change in activity 150 b. RIA: standard, curves and •change"in sensitivity 150 B. Application to the purification of motilin 153 125 I. Purification of I-motilin 153 II. Extraction of motilin added to plasma 155 III. Extraction of endogenous motilin from serum 155 '.'.IV. Isolation of motilin from Presekretin 159 EFFECT OF MOTILIN ON THE RATE OF GASTRIC EMPTYING 159 A. Control studies in the gastric f i s t u l a dog 159 I. Effect of motilin on the rate of gastric emptying of liqu id s 159 II. Effect of motilin on the rate of gastric emptying of solids 162 B. Effect of motilin on the rate of emptying of liquids after truncal vagotomy and/or antrectomy 162 I. Effect of motilin after truncal vagotomy 162 II. Effect of motilin after antrectomy 162 III. Effect of motilin after vagotomy and antrectomy 166 MODIFICATIONS TO THE GIP RADIOIMMUNOASSAY 166 A. Antisera raised to GIP 166 B. Iodination of GIP 169 EFFECT OF SOMATOSTATIN ON THE CANINE RESPONSE TO GIP 169 A. Effect of somatostatin on the release of endogenous GIP 169 I. On the insulinotropic action of GIP released by oral glucose 169 II. On the release of endogenous GIP by oral fat 172 B. Effect of somatostatin on the response to exogenous GIP 172 Page RELATIONSHIP BETWEEN GIP AND GASTRIC ACID SECRETION 179 A. Effect of exogenous GIP on gastric acid secretion 179 B. Effect of endogenous GIP on gastric acid secretion 179 I. After intraduodenal infusion of fat 179 II. After intraduodenal infusion of glucose 187 C. Effect of an intraduodenal infusion of acid on gastric acid secretion 187 D. Effect of an intraduodenal infusion of saline on gastric acid secretion 187 STUDIES ON THE POSSIBLE HETEROGENEITY OF GIP 195 A. In serum 195 I. Immune-reactivity of GIP released by fat or glucose 195 II..Immunoreactivity of GIP after column chromatography 195 III. Immunoreactive forms of GIP released by fat or glucose 201 a. After oral fat 201 b. After oral glucose 201 IV. Immunoreactive forms of GIP after i t s exogenous administration 201 B. In tissue extracts 204 I. I n i t i a l tissue extraction 204 II. Purification 210 a. Methanol insoluble extract on Sephadex G50 210 b. ProGIP I on CM cellulose 210 c. Stability of ProGIP 212 III. Molecular weight determination 212 DISCUSSION ^ 218 BIBLIOGRAPHY • 260 x i LIST OF TABLES Table Page I. Motilin guinea pigs - immunization schedule 31 II. Motilin rabbits - immunization schedule 33 III. Effect of varying antibody t i t r e on assay sensitivity 37 IV. Effect of varying Trasylol concentrations in the diluent buffer 44 V. Effect of varying plasma concentrations in the diluent buffer 46 VI. Effect of varying the antigen concentration on assay sensitivity 48 VII. Effect of varying the incubation period and type 48 VIII. Effect of siliconization or plasma addition on the assay system 50 IX. Layout for the routine radioimmunoassay 53 X. Effect of varying the charcoal concentration on the separation procedure in the assay for motilin 56 XI. Effect of treatment of 1 2 5I-GIP on the NSB of that label 91 XII. Results, in pg/ml, demonstrating reproducibility of motilin determinations after storage for 3 months, with or without added Trasylol 106 XIII'. Comparison of the increase in motility indices after motilin or Boots ^Pancreozymin' 110 XIV. Distribution of IR-motilin throughout the hog gastrointestinal tract 116 XV. Fundic motor activity response to duodenal infusion with a l k a l i 120 XVI. IR-motilin response to duodenal infusion with a l k a l i 121 XVII. Fundic motor activity response to duodenal infusion with saline 123 XVIII. IR-motilin response to duodenal infusion with saline 124 XIX. Spontaneously induced fundic motor activity 126 XX. IR-motilin response to spontaneously induced fundic motor activity 127 XXI. Effect of duodenal infusion of a l k a l i , acid or saline on the incremental IR-motilin response 129 XXII. Effect of ingestion of oral glucose on the serum levels of IR-motilin 133 XXIII. Effect of ingestion of a normal mixed meal on the serum levels of IR-motilin 134 XXIV. Comparison of biological a c t i v i t i e s of natural and synthetic motilin 140 x i i Table xxv: XXVI. XXVII. XXVIII. XXIX. XXX. XXXI. XXXII. XXXIII. XXXIV. XXXV. XXXVI. XXXVII. XXXVIII. XXXIX. xxxx. xxxxi. Comparison of the immunological and b i o l o g i c a l a c t i v i t i e s of synthetic and natural m o t i l i n analogues and fragments Coupled versus uncoupled antisera at varying incubation volumes Calculation ofcthe.slope at zero;dose E f f e c t of a single i n j e c t i o n of somatostatin on the incremental IR-GIP, IR - i n s u l i n and serum glucose response to o r a l glucose E f f e c t of an infusion of somatostatin on the incremental IR-GIP IR-i n s u l i n and serum glucose response to o r a l glucose E f f e c t of a single i n j e c t i o n of somatostatin on the Incremental IR-GIP, I R - i n s u l i n and serum glucose response to o r a l f a t E f f e c t of a single i n j e c t i o n of somatostatin on the incremental IR- i n s u l i n and serum glucose response to intravenous porcine GIP E f f e c t of a continuous infusion of pentagastrin on the I I + output of an e x t r i n s i c a l l y denervated fundic pouch E f f e c t of an intravenous infusion of porcine GIP on penta-gastrin-stimulated H + output E f f e c t of a duodenal infusion of f a t on pentagastrin-stimulated H + output and IR-GIP release E f f e c t of a duodenal infusion of glucose on pentagastrin-stimulated H -output and IR-GIP release E f f e c t of a duodenal i n f u s i o n of acid on pentagastrin-stimulated H + output and IR-GIP release E f f e c t of a duodenal i n f u s i o n of sa l i n e on pentagastrin-stimulated H + output and IR-GIP release Proportions of IR-GIP molecular forms released by fat and glucose Change i n the r e l a t i v e proportions of IR-GIP molecular forms with the time of serum sampling, a f t e r o r a l f a t or glucose Rela t i v e proportions of IR-GIP molecular forms i n f r a c t i o n s obtained from an extract of hog i n t e s t i n a l mucosa A summary of the tiss u e extraction procedure Page 143 146 148 173 175 177 180 182 183 185 188 190 192 197 202 207 208 x i i i LIST OF FIGURES Figure Page 1. Polyacrylamide gel electrophoresis of motilin after exposure to chloramine-T : 22 2. Chloramine-T iodination of motilin at.a peptide:iodine ratio of 4 pg: 1 mCi 23 3. Chloramine-T iodination of motilin at a peptide:iodine ratio of 2 ug: 1 mCi 24 4. Lactoperoxidase iodination of motilin 26 5. Standard curve for motilin in comparison with the dilution curves for labelled fractions. Measurement of specific activity 28 6. Motilin antiserum dilution curves 34 7. Effect of varying antibody t i t r e on the assay sensitivity 36 8. Comparative immunoreactivities of polypeptides with motilin antiserum 38 9. Comparative immunoreactivity of VIP with motilin antiserum 39 10. Dilution curves of serum samples containing exogenous and endogenous motilin 41 11. Motilin standard curve presented as a Scatchard .plot 43 12. Effect of varying Trasylol concentrations on motilin assay sensitivity 13 Effect of varying labelled antigen concentrations on motilin assay sensitivity 14. Effect of variation of incubation length and type on motilin assay sensitivity 49 15. Effect of siliconization and plasma addition on motilin assay sensitivity 51 16. Effect of varying charcoal concentrations in separation procedure on motilin assay sensitivity 55 17. Routine standard curve for motilin 58 18. Motilin antiserum dilution curves before and after coupling to Sepharose 4B 19. GIP antiserum dilution curves before and after coupling to Sepharose 4B 45 47 68 69 20. Diagram of the chronic dog preparation 78 xiv Figure Page 21. .Chloramine-T iodination of GIP. Column profile of separation on Sephadex G25 85 22. Lactoperoxidase iodination of GIP. Column profile of separation on Sephadex G25 88 23. Chloramine-T iodination of GIP. Column profile of separation on QAE Sephadex A25 90 125 24. Comparison of standard curves obtained with . I-GIP with or wothout pretreatment with s i l i c a or resin 92 25. Effect of plasma addition on the sensitivity of the GIP. standard curve 96 26. Effect of separation of bound antigen from free antigen by dioxane or dextran-coated charcoal, on the sensitivity of the GIP standard curve 98 27. Estimation of the degree of conjugation between motilin and BSA 103 28. Estimation of the degree of conjugation between GIP and BSA 105 29. •Reproducibilty of IR-motilin determinations 107 30. Estimation of the motilin content of an extract by RIA 109 31. Estimation of the motilin content of an extract by bioassay 111 32. Column profile of a motilin-containing serum on Sepahdex G50 113 33. Column profile of a motilin-containing extract on Sephadex G50 114 3.4. Desalting of a hog duodenal extract on Sephadex G25 117 35. Effect of a ./.'.duodenal infusion of a l k a l i on fundic pouch motor activity and circulating IR-motilin levels . 122 36. Effect of a duodenal infusion of saline on fundic pouch motor activity and circulating IR-motilin levels 125 37. Change in circulating IR-motilin levels during a spontaneous burst of fundic pouch motor activity 128 38. Comparison of the incremental IR-motilin responses to duodenal infusion with a l k a l i , acid or saline 131 39. Effect of ingestion of - glucose or a normal mixed meal on the circulating levels of IR-motilin 135 40. Comparison of the immunological activity of natural porcine and synthetic motilin analogues and fragments 136 X V Figure Page 41. Comparison of the immunological activity of natural motilin and natural motilin fragments 138 42. Comparison of the biological activity of natural motilin and . a synthetic motilin analogue • 141 43. Comparison of dilution curves obtained with antiserum to motilin, under routine assay conditions or after coupling to Sepharose 4B 145 44. Effect of coupling of motilin .antisera to Sepharose 4B on the sensitivity of the standard curve 147 45. Graph showing the slope at zero dose for standard curves ob-tained with coupled and uncoupled antisera to motilin 149 46. Comparison of the dilution curves obtained with GIP antisera under routine assay conditions or after coupling to Sepharose 4B 151 47. Effect of coupling of GIP antisera to Sepharose 4B on the sensitivity of the assay standard curve 152 125 48. Column profile of I-motilin obtained after a f f i n i t y chromatography 154 125 49. Comparison of standard curves obtained with I-motilin before and after a f f i n i t y chromatography 156 50. Column profile of•IR-motilin obtained after a f f i n i t y chromatography of plasma containing exogenous motilin 157 51. Column profile of IR-motilin obtained after a f f i n i t y chomatography of a serum sample contianing endogenous motilin 158 52. Column profile of IR-motilin obtained after a f f i n i t y chromatography of an impure extract containing motilin 160 53. Effect of an infusion of porcine motilin at two doses on the circulating levels of IR-motilin 161 54. Fraction of a liquid meal emptied during, the infusion of varying doses of motilin 163 55. Fraction of a solid meal emptied at different times during an infusion of 1 ^jg/kg/hour motilin 164 56. Fraction of a liquid meal emptied during infusion of varying doses of motilin, before and after truncal vagotomy 165 xvi. Figure Page 57. Fraction of a liquid meal emptied during infusion of varying doses of motilin, before and after antrectomy 167 58. Fraction of a liquid meal emptied during infusion of varying doses of motilin, after truncal vagotomy and antrectomy 168 59. Standard curves obtained with antisera to GIP after incubation in equilibrium and disequilibrium systems 170 125 60. Standard curves obtained with I-GIP, purified by gel f i 11-ration, with or without subsequent purification on QAE : Sephadex A25 . . 171 61. Incremental serum glucose, IR-insulin and IR-GIP responses to oral glucose, with or without a prior injection of somatostatin 174 62. Incremental serum glucose, IR-insulin and IR-GIP responses to oral glucose, with or without a prior infusion of somatostatin 176 63. Incremental serum glucose, IR-insulin and IR-GIP responses to oral fat, with or without a prior injection of. somatostatin 178 64. Incremental serum glucose and IR-insulin responses to an :; Intravenous infusion, of porcine GIP, with or without a prior injection of somatostatin • 181 65. Effect of an intravenous infusion of porcine GIP on penta- • gastrin-stimulated H + output and IR-GIP levels 184 66. Effect of intraduodenal fat on pentagastrin-stimulated H + output and IR-GIP release 186 67. Effect of intraduodenal glucose on pentagastrin-stimulated H + output and IR-GIP. release 189 68. Effect of intraduodenal hydrochloric acid.on pentagastrin-stimulated H + output and IR-GIP release 191 69. Effect of intraduodenal saline on pentagastrin-stimulated H1" output and IR-GIP release 193 70. Comparison of the effects of intravenous GIP and intraduodenal fat, glucose or acid on pentagastrin-stimulated H + output and IR-GIP release 71. Comparison of the immunoreactive forms of GIP released by ingestion of fat or glucose 72. Column profile of serum containing glucose-released IR-GIP on Sephadadex G50 198 194 196 xv i i Figure Page 73. Column profile of serum containing fat-released IR-GIP on Sephadex G50 199 74. Relative proportions of IR-GIP molecular forms released by ingestion of fat or glucose 200 75. Change in the relative proportions of the IR-GIP components with the time of sampling, after oral fat or glucose 203 76. Change in the relative proportions of the IR-GIP components during an intravenous infusion of porcine GIP 205 77. Effect of 6.0M urea on the relative.proportions of the IR-GIP components in serum 206 78. Relative proportions of the IR-GIP components in fractions obtained from an extract of hog intestinal mucosa 209 79. Column profile of the methanol insoluble extract on Sephadex G50 211 80. Column profile of ProGIP I on CM cellulose 213 81. Column profile of the .GIP^Q^Q-containing fraction from Sephadex G50, on CMccellulose 214: 82. Repeated chromatography.of ProGIP after lyophilization and storage at -20°C . 215 83. Relationship between V°/V e and molecular weight after chromatography on Sephadex G50 217 84. Correlation between the biological activity of GIP and the relative proportions of the IR-GIP molecular forms 252 x v i i i ACKNOWLEDGEMENTS I should l i k e to thank my supervisor, Dr John C. Brown, f o r the opportunity to undertake Graduate Studies, and f o r h i s help, guidance, forbearance and frie n d s h i p during t h i s time. I am also g r a t e f u l f o r the support, both p r a c t i c a l and moral, given to me by members of the Faculty and Staff of the Department of Physiology. I am also indebted to Mr Kurt Henze and Mr Ralph Assina, f o r the preparation of the i l l u s t r a t i o n s i n t h i s t h e s i s , and to Ms Dianne Lynch and Mrs Mary Forsyth, f o r the typing of the text and tables. F i n a n c i a l support from the Canadian Medical Research Council i s g r a t e f u l l y i acknowledged. This thesis i s dedicated to the memory of my father, John Dryburgh, who a l l unknowingly started me along t h i s path. xix LIST OF ABBREVIATIONS APUD - amine precursor, uptake and decarboxylation BGP - b r a i n g a s t r i n immunoassayable peptide BSA - bovineserum albumin CDI - carbodiimide CE plasma - charcoal extracted plasma CCK-PZ — cholecystokinin-pancreozymin CNBr - cyanogen bromide FCA - Freunds Complete Adjuvant GIP - g a s t r i c i n h i b i t o r y polypeptide GLI - glucagon-like immunoreactivity IR- - immunoreactive IRP - i n s u l i n - r e l e a s i n g polypeptide KIU - K a l l i k r e i n i n h i b i t o r units LDD - l e a s t detectable dose M 5 - pure porcine m o t i l i n ND - non-detectable NSB - non-specific binding PTH - parathyroid hormone PITC - phenyliso thiocyanat e PLC - p r o i n s u l i n - l i k e component PZn - Pancreozymin (commercial) RIA - radioimmunoassay Sn - s e c r e t i n VIP — vasoactive i n t e s t i n a l peptide XX Sources of Reagents 125 I-sodium (carrier free) Chloramine-T Sodium metabisulphite Lactoperoxidase (from milk) Hydrogen peroxide (30%) Bovine serum albumin (fr V) Trasylol (10,000 KlU/ml) Microfine s i l i c a (Quso G32) AG 1-X10 resin (200-400 mesh) Amersham/Searle Eastman Kodak Co. Rochester, N.Y. 14650 Fisher Scientific Co. Fair Lawn, New Jersey Sigma Chemical Co. St. Louis, Mo. 63178 Fisher Scientific Fair Lawn, New Jersey Sigma Chemical Co. St. Louis, Mo. 63178 FBA, Boehringer Ingelheim Canada Philadelphia Quartz Co. Valley Forge, Pa. 19482 Biorad Laboratories Richmond, California IMS 30 #1022 S-244 0L-2OO5 H-325 140-1541 Freunds Complete Adjuvant l-ethyl-3-(3-dimethyl)-amino propyl carbodiimide Difco Laboratories Detroit, Michigan Calbiochem San Diego, California 341006 Dimethyldichlorosilane Charcoal (Carbon decolourT izing Neutral) Dextran T 70 Biorad Laboratories Richmond, California Fisher Scien t i f i c Fair Lawn, New Jersey Pharmacia Uppsala, Sweden Insulin RIA Kit Amersham/Searle IM 39 xxi Sephadex gels Sepharose resin CM celluloses Glacial acetic acid) Hydrochlorid acid ) Ammonia solution ) Cyanogen bromide Trypsin - TPCK Carboxypeptidase A Phenyli so thiocyanat e Trifluoroacetic acid Dansyl chloride Acetic anhydride Succinic anhydride Ethanolamine t Acrylamide N N'-methylenebisacrylamide NNN'N'-tetramethylene-diamine Fluothane -Halothane B.P. Lipomul Pentagastrin(Peptavlon injectionable) Pharmacia Uppsala, Sweden i t Whatman, England Aristar BDH Chemicals Poole Dorset, England Eastman Organic Chemicals Rochester, N.Y. Worthington Biochemicals Freehold, New Jersey Worthington Biochemicals Freehold, New Jersey Eastman Organic Chemicals Rochester, N.Y. Eastman Organic Chemicals Rochester, N.Y. Sigma Chemical Co. St. Louis, Mo. 63178 Eastman Organic Chemicals Rochester, N.Y. Eastman Organic Chemicals Rochester, N.Y. Sigma Chemical Co. St. Louis, Mo. 63178 Eastman Organic Chemicals I I I I I I I I I I I I Ayerst Laboratories, Montreal Upjohn Co. of Canada , Don M i l l s , Ont. ^ Ayerst Laboratories Montreal, Canada - 1 -INTRODUCTION : In 1905 Starling adopted the term "hormone") f i r s t coined by W.B. Hardy, to describe a chemical substance^ .released by some physiological stimulus from i t s c e l l of origin and carried to. i t s target organ by the circulation. Over the last decade the facts which have emerged about endocrine control in general, and gastrointestinal control in particular,.would indicate that this classic paradigm must undergo revision and re-evaluation and that the role of a chemical messenger may be more subtle than was originally envisaged. The three generally accepted hormones with gastrointestinal activity, secretin, gastrin and cholecystokiriin-pancreozymin have known chemical structures and physiological importance. They have been joined over the last ten years by numerous other peptides of both gastrointestinal and extra-gastrointestinal origin. These substances have had their structures confirmed but their true hormonal status is uncertain, e.g., motilin, gastric inhibitory polypeptide (GIP), vasoactive intestinal peptide (VIP)/, bombesin and somatostatin. Other workers have introduced impure extracts with biological activity, whose active moiety may be identical with other, already identified polypeptides, e.g., insulin-releasing peptide (IRP), bulbogastrone, enterogastrone and chymodenin. These candidate hormones were described succinctly by Grossman (1974) as being either "peptide mimickers of physiological events or pure peptides seeking physiological roles". The results obtained when the classical physiological methods of assessing humoral status were applied to these peptides were equi~ vocal, in many cases. The infusion of sufficient exogenous pure polypeptide into the circulation, usually accepted as the D,_Q, in order to mimic a physiolo-gical event, or the traditional cross-circulation experiments were no longer -2-enough to establish true physiological function. The effective levels achieved by exogenous administration of the polypeptide should be in the range of the serum levels measured during endogenous'polypeptide release. This requirement is complicated by the fact that the biological activity observed during the endogenous release of any gastrointestinal polypeptide is being modulated by the activity of other polypeptides,released at the same time. Before the hormonal status of a biologically active principle can be evaluated i t must be available in a chemically pure form, vide the confusion which has arisen around the biolo-gical activity of eholecystokinin-pancreozymin, due to the studies which have been performed with a preparation of this polypeptide which was only.10-14% pure. Some method for the measurement of serum and tissue levels of the putative hormone i s also essential. Some of the candidate hormones which f a l l into the second category of Grossman, i.e., polypeptides extracted from biological tissues, which have been purified and characterized, include VIP, somatostatin and motilin. These substances, infused intravenously, demonstrated varied biological a c t i v i t i e s , but their endogenous release cannot be measured in association with any of these activities by any of the methods developed for the estimation of that substance in the circulation. In 1953 Feyrter described his concept of the paracrine c e l l . He postulated the existence of secretory cel l s , scattered throughout the gastro-intestinal mucosa, adjacent to their target cel l s . The structures he thought responsible, the "helle Zellen" or clear cells were later recognized as being identical with the APTJD (amine. T. precursor uptake and decarboxylation) cells of Pearse (1968). Under normal circumstances, he postulated, the secretions of these cells would pass to the target c e l l via the extracellular f l u i d and never " s p i l l " into the circulation in any significant amounts. The paracrine system has yet to be proven to exist but i t i s a plausible concept and the gut mucosa, which may be regarded.as the single largest endocrine organ in the body, with i t s multitude of secretory and receptor cells scattered over an immense area, would be ideally suited to such a mechanism. Some evidence for the existence of the modification of endocrine function has been obtained with somatostatin. This tetradecapeptide was originally isolated from the hypothalamus of the sheep by Brazeau et al (1973) during their search for a pituitary growth hormone releasing factor. Instead they were repeatedly able to demonstrate the existence of a growth hormone release Inhibitory factor. This material was.extracted, purified, sequenced and later synthesized. It was originally named somatotropin-relea'se inhibitory factor (SRIF) or growth hormone-release inhibitory hormone (GRIH) but the findings that i t was able to inhibit the release of insulin (Albert! et a l , 1973), glucagon (Gerich et a l , 1975), gastrin (Bloom et a l , 1974) and GIP (Pederson et al,1975) have favoured the use of a less specific name, somatostatin. Studies with gastrin and GIP have indicated that somatostatin did not only inhibit endogenous release of these polypeptides but also blocked their action at the level of the target c e l l . If effective levels of hypothalamically-released polypeptide were to occur in the circulation a l l these systems would be inhibited simultaneously, and this is an unacceptable premise. Somatostatin-producing cells have been localized by immunochemical means in hypothalamic and pancreatic i s l e t tissue, and in gastric and intestinal mucosa (Dubois, 19 75). The location of somatostatin in the pancreas i s distinct from the insulin-producing B c e l l , amd the glucagon-producing ct c e l l . It i s postulated to be the D c e l l , common to the pancreas, stomach and intestine. A teleologically more acceptable concept would involve the modulation of hypothalamic, gastric, intestinal or pancreatic function by hypothalamic, gastric, intestinal or pancreatic somatostatin, released locally by an approp-riate stimulus, where i t acted In a paracrine manner. A second type of secretory process, postulated to exist and capable of acting in the gastrointestinal tract; i s the neurocrine system, whose nerve c e l l axons extend to the target organ. Their secretions therefore have only to cross the synaptic junction,,iii a manner.analogous to that of the neurotransmitter, acetylcholine. Peptides with gastrointestinal activity such, as somatostatin, substance P, VIP and gastrin, have, a l l been• (detected in normal neural tissue. Although somatostatin has been detected in both the hypothalamus and pancreas the possibility that some neural connection exists between these two areas seems unlikely i n view of the fact that no nerve fibres to the adult pancreas have been shown to contain IR- somatostatin. Somatostatin immunoreactivity has also been demonstrated in the foetal pancreas by the third month of gestation. It i s more lik e l y that this peptide i s being synthesized in both regions and is not merely being absorbed by pancreatic tissue after synthesis in the brain. The evidence so far available favours a paracrine, rather than neurocrine role for somatostatin. On the other hand, immunoreactive substance P has been demonstrated in both central and peripheral neural tissues, in association with primary sensory neurons in the dorsal horn, and in non-myelinated free nerve endings in the skin, sweat glands and gut wall (HtJkfelt et a l , 1975,1976). It has been suggested that substance P not only has a direct stimulatory effect on smooth muscle cells in the gastrointestinal tract,:but that i t also enhances the effect of nerve stimulation.' As these same doses of substance P appear not to enhance the response to applied acetylcholine to any significant degree, i t -5-may be acting prejunctionally to modulate the response of the gut musculature to cholinergic stimulation, and be neurocrine in i t s action. Two polypeptides with established gastrointestinal properties, gastrin and VIP, have also been located in neural tissue. VIP was found in the gastrointes-ti n a l tract of several mammals (Said and Mutt,1970) and has been extracted from pancreatic tumours associated with the Werner-Morrison syndrome (Bloom et a l , 1973). As i t has also been extracted from tumours of neural origin i t was logical to look for VIP in cultured neuroblastoma cells from mice, as well as in normal canine neural tissue. In the normal extracts the highest concentra-tions were found in the cerebral cortex, the hypothalamus and hippocampus. IR-VIP was also detected in sympathetic ganglia, the adrenal gland and in extracts of the vagus nerve (Said and Rosenberg, 1976: O'Dorisio et a l , 1976). Extracts from both normal cortex and tumour tissue showed VIP-like activity when assayed in vitro on rat stomach or guinea pig gall bladder strips. No physiological functions have been assigned to this polypeptide, but the rela-tively larger concentrations in the central and peripheral nervous system, com-pared to that in the intestine, suggest that i t might function as a neurocrine transmitter. Immunoreactivity to gastrin has also been detected in the brain, pre-dominantly in the cortical grey matter. Extracts from this region, however, showed a lower a f f i n i t y for the antiserum used than did heptadecapeptide gastrin, and eluted behind this gastrin from Sephadex G-25. Vanderhaeghen et a l (1975) have named this material brain gastrin immunoassayable p_eptide (BGP) . It remains to be seen i f BGP corresponds to a smaller molecular form of gastrin, e.g., the tridecapeptide found in sera from Zollinger-Ellison patients (Rehfeld' and Stadil, 1973a), and to examine the range of i t s biological activity. -6-A model specific 'for the". actions of the, gastrointestinal polypeptides, combining features from.both the.endocrine and paracrine systems, has been proposed by Wingate (1976). His Eupeptide system is based on the following facts. Most gastrointestinal polypeptides influence both motor and secretory activities of the gut, and most gastrointestinal polypeptide-producing cells are situated in close proximity to the c e l l whose secretions they influence* However, i t has been well established that several of these factors are released into the systemic circulation in significant amounts. Wingate therefore postulated a dual action for the gut peptides and suggested that they acted at a local target c e l l as a paracrine, to promote secretion or local muscle activity, and at a distant target c e l l to modulate the motor control of the digestive tract. Immunological techniques such as radioimmunoassay, immunocytochemistry and affinity chromatography, have been, applied to the physiology of polypeptides and ^althoughs providing some of the answers about their function, they have also rained many more problems. The advent of the radioimmunoassay in par t i -cular (Berson and Yalow,1958) has proved a valuable tool in monitoring poly-peptide responses in various situations, but the results require careful evaluation. Some long-held beliefs have had to be re-examined. Secretin had been postulated to be the polypeptide responsible for the inhibition of gastric acid secretion when acid passed into the duodenum. Exogenously administered secretin did, in fact, mimic this response. However, the development of a radioimmunoassay, specific for secretin, proved that the minimum effective circulating IR- secretin levels achieved after infusion of the polypeptide were much higher than those produced by duodenal acidification (Ward and Bloom, 1974). When secretin was administered to produce serum IR- secretin levels within the physiological range, no effect was observed on acid secretion or gastric motor activity although a significant effect was seen on the exocrine pancreas, CBloom, 1975).. This failure, to . conf irjn the role of secretin as . the acid-stimulated gastric inhibitory agent, by .radioiiranunological,means, has re-stimulated interest in the work of Anderson ,et a l , (1967) on the impure extract of the bulbar region of the duodenum, bulbogastrone; They were able to show that acidification of the bulbar region resulted in a profound inhibition of gastric acid secretion, which was not observed i f the acid was installed into the lower duodenal regions. However, unt i l this material has been purified and.its endogenous.release can be stimulated by physiolo-gical secretagogues, any suggestions that i t i s the major inhibitory agent released by duodenal acidification must be purely speculative. The use of immunological techniques in the measurement of circulating poly-peptide levels in serum, plasma or tissue extracts has demonstrated that several polypeptides existed in more than one molecular form. Gregory and Tracy (1964), using classical peptide extraction techniques, described two forms of heptadecapeptide gastrin, differing only in the presence of a sulp-hated tyrosine residue, but had the foresight to state "there may be present in antral mucosa other gastrin composed of part of the peptides we have isolated, or indeed incorporating them, or the active parts, within a larger molecule". In 1971 Yalow and Berson confirmed this supposition by demonst-rating that gastrin in human plasma did, in fact, exist in more,than one' molecular form. This heterogeneity was shown immunologically when different antisera crossreacted in differing degrees with the endogenously occurring polypeptide and the pure standard preparation. Fractionation by gel f i l t r a -tion, electrophoresis or ion exchange chromatography, followed by.monitoring of the fractions obtained, by radioimmunoassay, allowed comparison of size or -8-charge distinction between components sharing immunoreactivity, By 1973 Rehfeld and Stadil had isolated four components of immunoreactive gastrin from the sera of Zollinger - Ellison patients by the.technique of high resolution gel f i l t r a t i o n . 1 Component 1 eluted in the void volume of the column and corresponded.to the "big, ,big" gastrin of Yalow and Berson, (1972); component II (big gastrin) had a molecular weight of 7000; component III correlated well with heptadecapeptide gastrin; component IV (minigastrin) was a tridecapeptide. The different gastrins .have* been found to vary in location,,response to stimuli, and'also perhaps in function. Big, big gastrin is a major component of the fasting sera.in normal subjects but occurs in insignificant amounts in normal or pathological tissue extracts, and i t s serum levels are not elevated by feeding. Treatment of the sera with 8.0M urea, or solutions of increasing ionic strength, significantly depressed the size of this fraction of IR- gastrin, suggesting that this largest molecular form may be a protein/peptide complex (Rehfeld et a l , 1975). The physiolo-gical function of such a complex is not understood, although the suggestion has been made, with respect to insulin, that this type of protein-peptide binding may act as a limiting factor in the transport of a peptide across a cellular membrane (Simon and Antoniades, 1975). Component II or big gastrin would appear to be the major IR- component in the circulation after stimulation, whilst the heptadecapeptide form predominates in antral tissue (Dockray et al,;1975). Evaluation of the importance of.the immunoreactive forms of gastrin under different conditions i s further complicated by the varying half-lives of the heterogeneous forms of gastrin. Prior to this work, Berson and Yalow (1968) had demonstrated that human para-thyroid hormone (PTH) in tissue extracts had a molecular weight of 9000, whilst -9-that in serum was mostly composed of a smaller form with a molecular weight of 7000. In 1972 Canterbury,et a l isolated a third PTH with a molecular weight of 5000. The 7000 molecular weight form was found to represent the C-terminal portion of the larger molecule (Segre et a l , 1972) and as Aurbach et al (1971) had postulated that the biologically, active portion of the larger molecule resided at the N- terminal, the possibility .was raised that a significantly large proportion of the total immunoreactive PTH in sera was biologically inactive. Habener et al (1971) have isolated a s t i l l larger immunoreactive molecule from slices of parathyroid tissue. Biosynthesis studies, measuring the uptake time of t r i t i a t e d amino acids into this com-ponent and the 9000 molecular weight form are suggestive of a precursor-polypeptide relationship (Cohn et a l , 1972) and the largest molecular form of PTH can be enzymatically degraded, by trysin to produce a polypeptide with increased biological and immunological activity. The best i l l u s t r a t i o n of a precursor-hormone relationship so far comes from the studies on. proinsulin and insulin. In 1967 Steiner provided evidence that the synthesis of insulin involved production of a precursor which was synthe-Isized in the rough endoplasmic reticulum of the B cells in the pancreatic i s l e t s , and transferred to the Golgi apparatus (Steiner et a l , 1969). Approximately 95% of the proinsulin was converted to insulin within the secre-tory granules in the cytosol, the remainder : being secreted into the circula-tion along with the insulin. At least two enzyme systems, one trygsin^like (Nolan et a l , 1971) and one similar to a carboxypeptidase (Kemmlerpet a l , 1971)appear to be necessary for the conversion of the proinsulin to insulin and a chymotryptic-like cleavage has been implicated by the work of Tageir et al,(1973) in in vitro studies in the rat i s l e t preparation. The proinsulin -10-molecule h a s been found to v a r y only slightly from species to species, the average molecular weight being around 9 0 0 0 . Most studies on the biosynt-hesis of proinsulin have been performed on the isolated i s l e t preparation (Steiner, 1967) and conversion to insulin has been found to be s t r i c t l y intra-c e l l u l a r . - Glucose:is an important regulator of proinsulin synthesis, which i s favoured under hyperglycaemic conditions over other cellular proteins C P e r m i t t and Kipnis, 1972a, 1972b). Many antisera t o insulin also cross-react with proinsulin and other intermediate forms, which are together d e -signated proinsulin-like-component (PLC), and therefore measurement o f IR-insulin alone would b e possible only after gel f i l t r a t i o n . Proinsulin has been shown to' have most o f the biological properties o f insulin but only 3 -5% o f i t s biological potency (Narahara, 1972) and as the IR- PLC i n fasting sera approximates 15% o f the total IR- insulin i t must b e taken into account when correlating immunological activity with biological activity. The ratio o f PLC: insulin declines i n the f i r s t hour after glucose stimu-lation and then gradually increases. Elevated PLC: insulin ratios have been found i n hypokalaemias o f different aetiologies (Gorden e t a l , 1974), severe diabetes and chronic renal failure (Mako e t a l , 1973), and are diagnostic o f 3 c e l l adenoma (Rubenstein e t a l , 1974). A different problem has arisen i n the studies investigating the relationship between the immunoreactive forms o f glucagon. Different components o f the total material which crossreacts with antisera t o glucagon were found t o respond differently when challenged by the same stimulus. Samols e t al (1965,1966) noted that the circulating levels o f immunoreactive glucagon C l R - glucagon)appeared t o increase after oral administration o f glucose and -11-that this immunoreactivity originated from the gut rather than the pancreas. This was, confirmed by Buchanan et al (.1967) who infused (intrajejunally) glucose into pancreatectomised dogs'and measured a significant increase in IR- glucagon levels. Valverde et a l (1968) monitored IR- glucagon.after gel f i l t r a t i o n of dog duodenum mucosal extracts and found two glucagon-like-immunoreactive (IR-GL1) components, one with a molecular weight of 3500 (small GLI) and a second, much larger molecule, with a molecular weight of 12,000 (large GLI). A fraction similar to the second component of Valverde was purified from a crude extract, of pig ileum by Murphy et al (1973) and was found to possess l i t t l e biological resemblance to.;pancreatic glucagon. Sasaki et a l (1975) further purified the small GLI component from porcine duodena/by gel filtration.on Bibgel P-10 and separated two peaks of protein, one with a molecular weight around 3500, which crossreacted completely with antisera raised to pancreatic glucagon and appeared identical in i t s spectrum 125 of biological activity. The second fraction eluted behind the I-glucagon marker, had a molecular weight of 2900, and showed varying degrees of crossreactivity depending on the antisera used in the assay. Histochemi-cal studies of the secretory cells in the gastrointestinal mucosa i n i t i a l l y indicated a c e l l very similar to the ej•• 7 c e l l of the pancreatic i s l e t s . Ultra-structural studies now suggest that, whilst these A- cells in.the fundic mucosa are identical in morphology with the pancreatic a- - c e l l , those i n the intestinal mucosa show slight structural differences. These have been desig-nated A- like and may correspond to the EG c e l l of Polak et a l (1971). The distribution of the 3500 and 2900.molecular weight components throughout the gastrointestinal mucosa correspond closely to the distribution of A and A- like cells respectively. It is strongly suggested that GLI, originally defined by Unger et a l (1968) to encompass any immunoreactive material origina--12-ting from the gut, is not a single:entity, but is made up of true glucagon of gut origin and several other peptides, more'correctly called GLI or entero-glucagonoid, which share a degree of immunological and possible biological identity with true glucagon. An oral glucose.load causes a decrease in pancreatic glucagon plasma levels and an increase in circulating IR-GLI levels and therefore an antiserum specific for pancreatic glucagon must be used to measure the true pancreatic response to this stimulus. The conclusion must be drawn that any comparison between biological and immunological activity of any polypeptide must take into account the presence of immunoreactive but possibly biologically less potent precursor forms or complexes between the peptide and a larger protein, as well as immunologically similar but functionally different molecules. A l l antisera should be screened for differences in their crossreactivity with the biologically occurring forms of each peptide and in the possibility of raising antibodies to a specific region of the active molecule considered. An obvious, though sometimes experimentally ignored, observation is that no physiologically functional peptide acts in isolation, and that i t s biological effect is modulated by the hormonal milieu at that time. A;simple example of this fact i s shown by the combined effects of secretin and cholecystokinin-pancreozymin on the exocrine pancreas. In the intact animal there is no measurable bicarbonate response to an infusion of chole^cystokinin-pancreozymin, but a combination of this peptide with secretin w i l l produce a greater secretory response than infusion of secretin alone.. The increased enzyme output which follows cholecystokinin-pancreozymin is ineffective unless i t is washed from the pancreatic ducts into the duodenum by the secretin-induced aqueous secretion -13-(Brownet a l , 1967a). It is also logical to correlate the activity of gastro-intestinal polypeptides to the stage of the digestive cycle during which they are normally released, and to measure that activity in an environment of the circulatory digestion products that would pertain at that stage. Physiological levels of IR- gastrin have no effect on insulin release in the fasting man,, but in the presence of a. degree of hyperglycemia the i n i t i a l insulin response after gastrin administration is longer and more pronounced than that seen after intravenous glucose alone (Rehfeld and Stadil, 1973b). In the isolated, perfused rat pancreas Pedersonand Brown (1976) were able to demonstrate a threshold glucose level, below which GIP had no effect on insulin release. The major functions of the gastrointestinal hormones so far discovered relate to their effect on the.secretory capacity or motor activity of the gastro-intestinal tract. Another property was suggested by the discovery that gastrin had a trophic effect on the cells ofcthe gastric mucosa. Patients treated for duodenal ulcer by antrectomy showed atrophy of the gastric mucosa (Lees and Grandjean, 1968) which was not'the case i f the treatment was vagotomy only (Melrose et a l , 1964). Disuse atrophy could not be the cause of this as the acid secretion was i n i t i a l l y depressed to the same degree in either case. In contrast, subjects with Zollinger-Ellison Syndrome-showed hyperplasia of jboth the gastric and duodenal mucosa (Ellison and Wilson, 1967). The role of gastrin was confirmed in rats, when increased RNA and DNA synthesis resulted from single shots of gastrin, and chronic gastrin treatment produced a thickening of the gastric mucosa (Johnson, 1976). Mainz et a l (1973) found that exogenous CCK-PZ.1 caused an increase in both c e l l mass and c e l l number in the exocrine pancreas, and postulated a role for CCK-PZ in maintaining pancreatic function and integrity. Physiological levels of the synthetic octapeptide of CCK-PZ were found to have a trophic effect on duodenal mucosa, but had no effect on -14-gastric mucosa. These same levels would, however, inhibit the trophic effect of gastrin on the gastric mucosa, and the same result was found with secretin (Johnson and Guthrie, 1974). In 1930, Kosaka and Lim used the term "enterogastrone" to describe a humoral agent,.released from the duodenal mucosa by fat or fat digestion products, the function of which was to inhibit both gastric acid secretion and gastric motility. This definition was later expanded to require that any candidate polypeptide must inhibit gastric acid secretion stimulated by histamine and insulin-induced hypoglycaemia, as well as gastrin and i t s analogues. Secretin, cholecystokinin-pancreozymin, VIP and GIP have.all been considered at some time to f i l l this role,they a l l appeared to inhibit the acid secretion produced by some or a l l of these stimuli, and cholecystokinin-pancreozymin i s also released by the presence of fat in the duodenum,.but the only poly-peptide which satisfies a l l these c r i t e r i a i s GIP. Brown.and Pederson (1970) showed that the ab i l i t y of the 10% pure preparation of cholecystokinin-pancreozymin of Jorpes and Mutt to produce gastric acid inhibition was significantly reduced by a simple purification step, involving gel f i l t r a t i o n on Sephadex G-50, although i t s effect on ga l l bladder contrac-t i l i t y was unaltered. A side fraction, produced in the purification of cholecys-tokinin-pancreozymin was found to contain inhibitory activity but had no effect on the gall bladder, (Brown et a l , 1969). A polypeptide was isolated and purified, (Brown et a l , 1970), sequenced (Brown, 1971: Brown and Dryburgh, 1971) and was found to be a straight chain polypeptide with 43 amino acids and a calculated molecular weight of 5105. -15-The amino acid sequence was :- | NH„-Tyr-Ala-Glu-Gly-Tnr-Phe-Ile-Ser-Asp-Tyr-Ser-Ile-Ala-Met-Asp-Lys-Ile-Arg-Gln-Gin-Asp-Phe-Val-Asn-Trp-Leu-Leu-Ala-Gln- -Lys-Gly-Lys-Lys-Ser-Asp-Tr.p-Lys-His-Asn-Ile-Thr-Gln A radioimmunoassay for GIP has been developed and antibodies to GIP raised in guinea pigs did not crossreact with glucagon, gastrin, motilin, cholecyst-okinin-pancreozymin, secretin, VIP or insulin. Studies performed on peptide fragments obtained by cyanogen bromide cleavage at the methionine residue, or on synthetic peptide fragments, suggest that the immunoreactive site l i e s within the sequence 21-38. Antiserum to GIP has also been used in the immuno-hi-sto.logical localization of the GIP-producing c e l l in the duodenum and jejunum of man, dog and baboon. It was tentatively identified as the Dl c e l l , (Polak et a l , 1973) in the APUD series defined by Pearse (1968,1970,1974) but is now known to be the K c e l l (Solcia et a l , 1973). Sera from human volunteers, taken before and after ingestion of a normal meal, were subjected to radioimmunoassay. Fasting levels of immunoreactive GIP (IR-GIP) ranged from non-detectable to 400 pg/ml and rose after eating to a mean peak of 1200 pg/ml, remaining elevated for at least 3 hours (Kuzio et a l , 1974). The major physiological secretagogues for GIP release were oral glucose (Cataland et a l , 1974) and oral fat (Brown et a l , 1974). The discovery that circulating IR-GIP levels were elevated when glucose came into contact with the duodenal and jejunal mucosa suggested that this poly-peptide might be a factor in the entero-insular axis, i.e. might be a hormone of intestinal origin which contributed to the regulation of the endocrine - 1 6 -pancreas. Dupre et al (1973) infused porcine GIP intravenously in man and showed a significant enhancement of the IR- insulin response:to an intra-venous infusion of glucose, associated with an improvement:in glucose'tolerance. The circulating levels of IR- GIP achieved during this exogenous administration of GIP were comparable to those:released endogenously by ingestion of glucose. Studies in dogs suggested that lower levels of circulating IR- GIP were insulino-tropic only in the presence of a degree of hyperglycaemia, although higher, possibly non-physiological levels stimulated insulin release in the fasted animal. The existence of the humoral gastric motor-activity stimulating principle, later named motilin, was f i r s t suspected when Brown et a l (1966) perfused the duodenum of the dog with a l k a l i : or fresh pig pancreatic juice and demonstra*-i ted an increase in gastric motor activity in extrinsically denervated or totally transplanted pouches of the fundus of the stomach. Earlier, Shay and Gershon-Cohen (1934) had described increased gastric emptying of a barium sulphate test meal after i n s t i l l a t i o n of 1% bicarbonate into the duodenum. This observation coupled with the work of Thomas et al (1934), who diverted the gastric (acidic) contents away from the duodenum and recorded an increase in the rate of gastric emptying, suggested a pH sensitive, duodenal reflex that contributed to the control of gastric motor activity. Weisbolt et a l (1969) proposed that a relationship existed between the rate of gastric emptying and the motor activity of the gastric musculature which would ensure that the contents of the stomach were delivered to the duodenum at a rate, and in a consistency that would allow optimal duodenal and jejunal digestion and absorption. -17-A crude duodenal extract (Pancreozymin, Boots Pure Drug Co.), administered intravenously in dogs, produced similar changes in the motor activity of the fundic pouches, whilst the purer GIH preparation of CCK-PZ did not (Brown, 1967). Gel f i l t r a t i o n of the crude extract on Sephadex G75 produced five protein peaks. Fractions 4 and 5 were inhibitory for gastric motor activity and fraction 5 was a potent stimulant, of pancreatic enzyme output, i.e. i t corresponded most closely to the GIH preparation. Fraction 2 represented a 20-fold purification of the original stimulatory material (Brown and Parkes, 1S67). Motilin was eventually purified from a side fraction produced in the purification of secretin (Brown et a l , 1972). The amino acid sequence was determined by the subtractive dansyl-Edman1s technique on peptides produced by cleavage of the molecule with cyanogen bromide, trypsin, chymotrypsin and thermolysin. Porcine motilin was found to be a 22 amino acid residue polypeptide with the sequence :-NH2-Phe-Val-Pro-Ile-Pne-Thr-Tyr-Gly-G.lu-Leu-Gln-Arg-Met-Gln-Glu-Lys-Glu-Arg-Asn-Lys-Gly-Gln and a molecular weight of 2700 (Brown et a l , 1973). The porcine polypeptide produced a significant increase in motor activity in the extrinsically denervated fundus and antrum of the canine stomach in doses as low as. 50 ng/kg. It had no significant effect on gastric acid secretion, but did elevate pepsin output to a higher degree than could be explained by a simple washout phenomenon (Brown et a l , 1972). The method of measuring motilin a c t i v i t y required bioassay in the chronic dog, prepared with an extrin-si c a l l y denervated pouch of the fundus and a Mann-Bollman f i s t u l a into the duodenum. In vitro preparations of muscle strips from the ileum, colon and circular layers of the stomach of the rat, guinea pig and rabbit were examined but the sensitivity of every preparation decreased immediately after the f i r s t exposure to motilin and the I n i t i a l response: could not be duplicated. Develop ment of a radioimmunoassay was deemed desirable to confirm the role played by motilin:in.the increase in gastric motor activity after duodenal a l k a l i n i -zation and for further investigation of i t s physiological function. It is the purpose of this thesis to study the hormonal.status of the gastro-intestinal polypeptidesGIP and motilin, and to look more closely at their possible physiological roles, bearing in mind the following points:-1. Biological activity seen after exogenous administration of a polypeptide can only be considered physiological i f that same response can be e l i c i t e d by that polypeptide when i t is released by a physiological stimulus, and the circulating levels of the peptide are comparable. 2. Inability to measure an increase, i n polypeptide levels in the systemic circulation does not necessarily preclude that polypeptide from having a physiological role. 3. If immunological techniques are used to measure the polypeptide levels in the circulation, then i t is essential to determine what percentage of the total immunoreactivity represents the true biological activity. 4. The biological activity of a polypeptide w i l l depend on the hormonal and nutrient milieu pertaining at that time and results obtained in an isolated situation, e.g.j in vitro, or by infusion of a polypeptide associated with digestion,in a fasting animal, may.not represent i t s true physiological activity. 5. Gastrointestinal polypeptide activities need not be restricted to influen-cing the secretory or motor activities in the gastrointestinal, tract. They may also play an important metabolic role in regulating the growth and respon-siveness of the target organ.. -19-METHODS DEVELOPMENT OF A RADIOIMMUNOASSAY FOR MOTILIN (MOTILIN RIA)  A. Rationale The concept of a radioimmunoassay is based upon the specific relationship that exists between an antibody and i t s antigen. Unlabelled antigen competes with labelled antigen for the binding sites on the antibody. The percentage of a fixed i n i t i a l amount of labelled antigen bound to the antibody gives an index of the amount of unlabelled antigen present in the mixture. The concen-tration of antigen in an unknown sample may be determined by comparing the displacement of labelled antigen i t produces with that produced by a series of standard solutions. The mommonestl label which can be incorporated into poly-125 peptides containing tyrosine or histidine residues is an isotope of iodine, I 131_ or I. A successful polypeptide radioimmunoassay i s dependent on three absolute requirements. F i r s t l y , the ratio of isotope to polypeptide in the radio-active tracer must be high enough so that sufficient tracer may be added to ensure an efficient counting rate without adding significant amounts of polypeptide and obscuring the upper limits of sensitivity of the curve. The second essential is an antibody of high a f f i n i t y . This a f f i n i t y i s expressed as a constant K. The relationship between K and the upper limit of sensitivity of the radio-immunoassay can be developed as follows:-From the 1st order Law of Mass Action B/F = K([A°] - B) Where K is the equilibrium (affinity) constant, [A°] is the concentration of antibody binding sites and B & F are the concentrations of bound and free hormone -20-If b is the fraction of bound hormone and IH] is the total hormone concent-ration, then B = bTH] B/ = _b_ = K([Ab°] - b[H[) 1 _ b 1 1 As the hormone concentration is increased to [H ] then B/ 4- and b[H ] t. F By definition b has a maximum of 1. .". The most sensitive assay condition prevails when lAb°] = IH1] for a B/_ of 1. r Assuming a B/ of 1 with minimal tracer and no unlabelled hormone, then [Hj -* 0 i.e., 1 = K[Ab° ]. When [Ab°] approximates [H"*"] 1 = KIH1] or K = 1 i.e., the greater the value of K, the lower the concen-tration of total hormone that is detectable. The f i n a l requirement is that the antibody should react identically with the unlabelled antigen, whether i t be in the form of standard or endogenous poly-peptide. Ideally, the labelled and unlabelled peptide should also behave identically in the system but this is not an absolute necessity. If these conditions are satisfied then the optimal values for a l l other variables may be established. B. Iodination of Motilin (I) Chloramine - T Method A modification of the original method of Hunter and Greenwood (1963) was used in routine isotopic labelling of motilin. The polypeptide contains only -21-1 tyrosyl and no hl s t i d y l residues. The ratios of yg polypeptide: 125 mCi Na, 1 essayed were 4:1 and 2:1. The following reagents were prepared freshly for each iodination a) Motilin (M5) - 2 or 4 yg in 50 y l 0.2M sodium,phosphate buffer, pH 7.5 125 ' b) Na I - 1 mCi in 10 y l carriers-free sodium hydroxide c) Chloramine-T - 40 yg in 10 y l deionized water d) Sodium metabisulphite - 100 ;yg in 20 y l deionized water Reagents a, b & c were added in quick succession, with bubbling to ensure rapid mixing, in a 10 x 75 mm siliconized glass culture tube. Reagent d was added, in like manner, after a 15 sec. delay. Motilin contains 1 methionine residue but no tryptophan and appeared f a i r l y stable in the presence of the oxidizing agent, withstanding exposures to chloramine-T of 2 mins. without undue fragmentation occurring, as shown by polacrylamide gel electrophoresis (Fig. 1). The reaction mixture was immediately transferred to a column of Sephadex G25 fine CO.6 x 30 cms.) and eluted in 0.2 M. acetic acid, containing 0.5% Bovine Serum Albumin (BSA) and 100 Kallikrein Inhibitor Units (KIU) Trasylol per ml. Fractions of approximately 400 y l were collected and 10- y l aliquots were counted for 0.1 min. in an automatic^ counter. The resultant column profiles are illustrated in Fig. 2 & Fig. 3, showing the separation of peptide-bound and free iodide. Aliquots of the appropriate fractions, diluted to contain-«-5000 cpm/100 y l , were incubated for 24 hours at 4°C, with or without antiserum to estimate the specific versus non-specific binding ( N.S.B);for that fraction. Those fractions showing the highest, -22-Fig. 1 Polyacrylamide gel electrophoresis of motilin after exposure to chloramine-T for 15, 30, 60 and 120 sec. No polypeptide fragmentation i s observed after 60 sec exposure but is visible at 120 sec. The method for polyacrylamide gel electrophoresis is detailed on p. 72. -23-il ' I I'. . J I -On 10 20 30 40 Fraction # 10 ju\ Aliquots i Fig. 2 Chloramine-T iodination of motilin at a peptide:iodine ratio of.4"Ug:l mCi. Separation of labelled motilin from free iodide on Sephadex G25 in 0.2M acetic acid. Counts per 0.1 min (••); maximum binding (x—x); NSB (o—o). -24-l I.0-, 10 2 0 3 0 4 0 5 0 6 0 F r a c t i o n # 10/JI A l i quo ts Fig. 3 Chloramine-T iodination o£ motilin at a peptide:iodine ratio of 2 ng:l mCi. Separation of labelled motilin from free iodide on Sephadex G25 in 0.2M acetic acid. Counts per 0.1 min (•-•); maximum binding (zi.-x) ; NSB (o-o) .' -25-specific binding and lowest, non-specific binding were pooled, diluted in the eluant buffer and aliquotted for storage at - 20°C, so that each aliquot contained ~ 2 x 10 cpm./ 2 mis. This was presumed to contain monoiodinated motilin. Label stored in this manner was stable for periods of up to 3 months. Lyophilization of label proved feasible:ibut was not routinely performed. (2) Lactoperoxidase Method An alternative, gentler and more easily controlled method of oxidizing the iodide to iodine involves the use of lactoperoxidase (Miyachi et a l , 1972). The following procedure follows the method of Holohan et a l (1973). The reagents were mixed in the following order in a 10 x 75 mm. siliconized, glass, culture tube. a) Motilin (Mj) - 4 ug in 50 ul 0.05 M -sodiumsacetate, ,p.H 5.0 125 b) Na 1 - 1 mCi in 10 ul carrier-'freeesodium hydroxide c) Lactoperoxidase - 500 ng in 10 ul .-sodiumsacetate, pH 5.0 d) Hydrogen peroxide - 0.86 nM in deionized water 3 x 10 ul at 5 min. intervals After 15 mins. the reaction mixture was transferred to a Sephadex G25 fine column and eluted, monitored, assayed and stored as previously described. on A typical column profile i s shown in Fig. 4. 125 (3) Estimation of Specific Activity of I - Motilin Dose - response curves were obtained (a) by increasing the concentration of unlabelled motilin and measuring the displacement of a constant amount of radioactive tracer (routine standard curve) and (b) by adding increasing -26-I .O-i 0.8 -Fraction # 10p\ Aliquots Fig. 4 Lactoperoxidase iodination of motilin at a peptide:iodine ratio of 2 yg:l mCi. Separation of labelled motilin from free iodide on Sephadex G25 in 0.2M acetic acid. Counts per, 0.1 min (•—•); maximum binding (x--x); NSB (o-o). -27-amounts of labelled motilin only and measuring the different ratios of tracer bound to antibody.' One point from the label dilution curve was arbitrarily placed on the standard curve and the other points fit t e d accordingly. Fig. 5 illustrates that the curves obtained when 3 different fractions of labelled motilin (fractions.24, 25 and 26 from the column profile shown in Fig. 3) were plotted on a standard curve they could be superimposed upon that standard curve. It can be concluded that the binding kinetics of the antiserum were v i r t u a l l y identical for both labelled and unlabelled antigen. The number of counts per minute (cpm) producing the same displacement as a standard amount of motilin can be read directly from this curve and the value converted to mCi/mg as an index of specific activity. Example (from results shown in Fig. 5) 28 pg motilin .". 1 mg motilin 1 Curie .*. 1 mCi From the efficiency of the y counter - 81% 1 mCi = 1.78 x 10 9 cpm 18,000 cpm 9 18,000 x 10 cpm 28 9 643 x 10 cpm 3.7 x 10 dps (disintegra-tions/second) 9 2.2 x 10 dpm (disintegra-tions /min. ) -28-0 . 8 -Mot i l in S t a n d a r d s Labe l Tube 2 4 " 2 5 2 6 0 . 6 -0 . 4 -o Q_ 0.2 • 0 . 0 -5 M o t i l i n C o n c e n t r a t i o n ( p g / m l . ) 10 2 0 4 0 8 0 160 _i i i 1 1 — 320 - | 1 1—I 10 2 0 3 0 4 0 1 2 5 1 - M o t i l i n ( C P M x I O 3 ) Fig. 5 Standard curve for motilin (x—x) in comparison with label fractions 24, 25, 26. Dilutions of each fraction from 8 x 3 3 10 cpm to 40 x 10 cpm were added and the dilution of 3 Fraction 26 containing 25 x 10 cpm was fitt e d to the standard curve, the other fractions being f i t t e d accordingly. (Dryburgh and Brown. Gastroenterology 68;1169-1175, 1975). -29-1 mg. motilin = 6.43 x 10 mCi 1.78 x 10 9 361 mCi .*. Specific activity of this iodination was 361 mCi/mg and the addition of 5000 cpm to each assay tube entailed the addition of 7.5 pg motilin. The specific activity was not measured after every iodination but was checked at intervals and on every occasion when the routine iodination was varied in any way. A rough estimate of specific activity may be calculated from the percentage of the radioiodine incorporated into the polypeptide, Example Cfrom column profile in Fig. 3) 125 % , I incorporated into polypeptide = 71% 125 Specific activity of 1 = 1 4 mCi/mg 125 . . 1 mCi I = 7 2 ng. Iodine .*.• 72 ng iodine was reacted with 2 yg motilin i.e., 0.57 nM iodine was reacted with 0.74 nM motilin .*. 0.40 nM iodine was incorporated into 0.74 nM motilin i.e., 50 ng iodine was incorporated into 2 yg motilin 125 i.e., 0.7 mCi T was incorporated into 2 yg.motilin 125 i.e., 350 yCi I was incorporated in 1 yg motilin .". Specific activity = 350 mCi/mg. However, i t must be remembered that this calculation depends.on the assumption that iodine was incorporated into a l l the available polypeptide. -30-C. Production of Antisera to Motilin  Cl ) In guinea pigs A series of guinea pigs (6) were immunized with pure porcine motilin (M,.). Conjugation of motilin to a large molecular weight protein was deemed advisable because of i t s low molecular weight. Motilin was.conjugated to bovine serum albumin by means of the carbo-diimide condensation reaction (Goodfriend et a l , 1964) using 1 - ethyl -3 - C3 - dimethyl) - amino - propyl - carbodiimide (CDI) as follows: -20 200 ug motilin per animal, 80 mg BSA and 100 mg CDI were dissolved in 100 u l , 10 mis and 1 ml deionized water, respectively. 0.5 mis each BSA and CDI were added to the motilin, mixed gently and l e f t at room temperature for at least 1 hour. The reaction was terminated by dialysis of the reaction mixture against d i s t i l l e d water overnight at 4°C. The volume was corrected by addition of deionized water and then emulsified with Freund's Complete Adjuvant (FCA) at a 1:1 ratio. The f i n a l volume was selected to allow 0.5 ml emulsion per animal. The animals were immunized subcutaneously, in several sites on the abdomen and inner thigh. An early observation suggested that better, more specific antisera were produced i f the immunization with conjugated material was preceded by an i n i t i a l "priming" dose of polypeptide alone in a FCA emulsion. The schedule followed is shown in Table I. (2) In rabbits Ten rabbits were immunized with conjugated motilin. The route of immuni-zation was intradermally, in several sites, in the supra-scapular region. -31-TABLE I lyiOTILIN GUINEA PIGS ^ IMMUNIZATION SCHEDULE DATE IMMUNIZATION BLEEDING TYPICAL TITRE .4.6.74 50'jif ^ / F C A 20.6.74 100 jig M5/BSA/FCA 20.7.74 200-jag l^/BSA/FCA 23.8.74 l : 4 0 x l 0 3 31.2.75 50 jag M5/FCA 29.9.75 l : 2 0 x l 0 4 23.1.76 50 yg 1M5/FCA 3.3.76 l : 1 0 x l 0 5 10.8.76 50 yg 1M5/FCA 24.8.76 1:lOxlO 5 the purest preparation of natural m o t i l i n Bovine Serum Albumin Treunds Complete Adjuvant -32-Blood was obtained by marginal ear vein venepuncture. The schedule followed is tabulated (Table II) (3) Storage of Antisera The whole blood samples were allowed to clot at 4°C for 20 mins., then centfifuged. The antiserum was stored frozen at - 20°C unt i l i t could be assessed. Usable antiserum was aliquotted in 200 - 500 U.1 portions, and lyophilized for storage at - "20°C. No detectable loss of specificity or af f i n i t y for at least 3 years has been observed. As required, the lyophilized aliqotswere reconstituted in assay diluent buffer at a dilution of 1:10 and stored at - 20°C in 100 -ul aliquots; This material was viable during the period of i t s use, usually 2 - 3 months. (4) Effect of varying antibody t i t r e Titre, in this connotation, is defined as the f i n a l dilution of antiserum in the incubation mixture. This allows for direct comparison between different antisera in different assay protocols where the f i n a l incubation volumes may di f f e r . It must be established for each antiserum and checked after each immunization and i t s subsequent bleeding. The i n i t i a l procedure was to establish a serial dilution curve for the anti-serum. Varying titr e s of the antiserum were incubated under routine assay 125 conditions with I - motilin and the maximum binding obtained with each dilution plotted as % bound versus the reciprocal of that t i t r e . A typical dilution curve is shown in Fig. 6. From this the range of t i t r e producing -33-TABLE II MOTILIN RABBITS - • • PMONIZATION SCHEDULE DATE 7JOTNIZAT10N BLEEDING TYPICAL TITRE 14.10.75 20 Ug M5/FCA 13.11.75 50 Jig M5/BSA/FCA 24.11.75 15.12.75 50 jig 1VL-/BSA/FCA 29.12.75 l:10xl0 2 23. 1.76 50 jig N5/FCA 13. 2.76 l:10xl0 4 8. 4.76 1:20xl04 FCA -the purest preparation of natural motilin Bovine Serum Albumin Freunds Complete Adjuvant -34-Fig. 6 Curve obtained with ser i a l dilutions of motilin antiserum GP 71. Arrows indicate the titres selected for use in the ' standard curves shown in Fig. 7. -35-the most sensitive assay may be roughly estimated. Various authorities have stipulated that the most effective assay is produced at the t i t r e resulting in a maximum binding of 50%, or 33% (Berson & Yalow, 1958). However, i t i s becoming clear that no such hard and fast rule can be followed and that the optimum t i t r e should be established for each RIA individually. Fig 7 is a comparison of the standard curves obtained with varying titr e s of the same antiserum shown in the dilution curve. The titres selected were those which resulted in binding 50%, 33%, 27% and 13% of the label. The results were plotted as B/„ x 100 r Bo against the motilin standards and the curves evaluated by the c r i t e r i a , slope at zero dose,midrange value and least detectable dose. The results are presented in Table 111. The most effective t i t r e was 1:5 x 10^ - - i . e . , that producing a maximum binding of 27%. The most sensitive standard curves for the motilin RIA under the routine conditions were obtained when the maximum binding was 25 - 30%. {5) Measurement of comparative immunoreactivity of antiserum The comparative immunoreactivities of natural gastric inhibitory poly-peptide, natural porcine secretin, natural (10% pure) cholecystokinin -pancreozymin, synthetic glucagon and synthetic human gastrin with motilin antisera were investigated. On a different date the comparative immuno-reactivity of motilin antiserum with vasoactive intestinal peptide was examined. No significant cross-reactivity between the antiserum and any of these polypeptides was detected even when concentrations of up to 10 mg per incubation volume were employed. The results are illustrated in Figs. 8 & 9. -36-F i g . 7 Standard curves, for motilin, demonstrating the effect of varying the antibody t i t r e on the assay sensitivity. LDD = least detectable dose. -37-TABLE I I I EFFECT OF VARYING ANTIBODY TITRE ON ASSAY SENSITIVITY CONDITION:-TITRE MAXIMUM " BINDINGC%) SLOPE AT ZERO (L/JMOLE) MIDRANGE VALUE (PG MOTILIN) L.D.D. (PG MOTILIN) l:1.5xl0 5 50 0.6xl0" 1 3 82 40 1:4 xlO 5 33 -13 2.6x10 73 20 1:5 xlO 5 27 4.0xl0~ 1 3 60 20 1:1 xlO 6 13 -13 2.5x10 - L J 105 20 -38-o.o J « I 1 1 1 1 \\ 5 10 100 1000 10000 Weight of Peptides (pg.) Fig. 8 Comparative immunoreactivities of natural motilin, synthetic human gastrin, synthetic glucagon, natural secretin, cholecysto-kinin-pancreozymin (10%) and natural gastric inhibitory poly-peptide with antiserum to motilin. (Dryburgh and Brown; Gastroenterology 68_ : 1169-1175, 1975). -39-r N o M o t i l i n a d d e d 0 . 5 . x 0.4 H I 0 . 3 ^ 0 .2 o i H o.o-J X - ^ o X , X — X M o t i l i n S t d s . O V I P r o - i — 10 100 W t . o f P e p t i d e ( p g ) — i 1 0 0 0 I O O 0 0 Comparative immunoreactivities of natural motilin and vasoactive intestinal peptide with antiserum to motilin. -40-C6) Antibody r e c o g n i t i o n of antigen xn atandards and unknowns Peak IR - m o t i l i n samples from dog experiments i n which m o t i l i n was e i t h e r endogenously r e l e a s e d a f t e r duodenal a l k a l i n i z a t i o n or exogenously adminis-tered by an intravenous i n f u s i o n were s e r i a l l y d i l u t e d i n assay d i l u e n t b u f f e r or charcoal--extracted plasma r e s p e c t i v e l y . A f t e r RIA, one v a l u e from each s e r i e s was f i t t e d to the standard curve and the remaining values p l o t t e d a c c o r d i n g l y ( F i g . 10). Both serum d i l u t i o n curves could be superimposed upon the standard curve i n d i c a t i n g that the r e l a t i o n s h i p between the antibody and the u n l a b e l l e d antigen i s unchanged whether the antigen i s the i s o l a t e d p o lypeptide i n the Standard p r e p a r a t i o n or the n a t u r a l l y o c c u r r i n g form, i n the unknown, i . e . , i n s era. ( 7 ) Measurement of a f f i n i t y of antiserum From the 1st order Law of Mass A c t i o n the f o l l o w i n g equation was developed by Scatchard (1949): B/ p = K([A°J - B°) Where B/ i s the r a t i o of Bound l a b e l l e d antigen to Free l a b e l l e d antigen [A°J i s the c o n c e n t r a t i o n of t o t a l antibody; B° i s the f r a c t i o n of t o t a l antigen bound and K. i s the constant of the antibody - antigen r e a c t i o n i n the d i r e c t i o n Ab + Ag —^AbAg. In any i n d i v i d u a l assay K and {A0] are constant . *. B/.^  and B° may be expressed l i n e a r l y i n a Scatchard p l o t . -41-i x Motilin Standard —i 1 1 1 r 5(10) 10(20) 100(200) Dog Serum (/j|./ml.) i I Fig. 10 Serum samples R 10 (.exogenous, motilin) and R24 (endogenous motilin) incubated at several dilutions, in'diluent buffer or charcoal-extracted plasma. The dilutions'of R 10 and R 24 respectively, at 50 (100) 'D.l/ml were, f i t t e d to the standard curve and the other dilutions f i t t e d accordingly. CDryburgh and Brown, Gastroenterology 68 :1169-1175, 1975). -42-In fact,- any antiserum represents a po p u l a t i o n of antibodies of v a r y i n g a f f i n i t i e s and t h e r e f o r e the Scatchard p l o t i s a curve composed of s e v e r a l s t r a i g h t l i n e s . The highest a f f i n i t y a n t i b o d i e s are represented by the l i n e w i t h the steepest s l o p e < The r o u t i n e RIA standard curve was r e p l o t t e d as a Scatchard p l o t . The standard values were converted to absolute antigen values by a d d i t i o n of the amount of polypeptide i n c o r p o r a t i n g the added r a d i o a c t i v e t r a c e r , c a l c u l a t e d from the s p e c i f i c a c t i v i t y of that t r a c e r . B° i s the product of t h i s v a l u e , i n moles, and the concentration of l a b e l bound by i t , i . e . , B. K i s the slope of the l i n e produced by p l o t t i n g B/ against B°. r F i g s . ID shows the Scatchard p l o t of a t y p i c a l antiserum of reasonable a f f i n i t y . D.: Conditions Of Radioimmunoassay Cl ) Methods Of standard curve e v a l u a t i o n The c r i t e r i a f o r e v a l u a t i n g the s e n s i t i v i t y of a standard curve are many and v a r i o u s . Three have been s e l e c t e d and a combination of at l e a s t two of these have been used i n a l l comparisons. The standard curves obtained were never l i n e a r so the."slope at zero dose" was estimated as a Scatchard p l o t and i t s slope measured as the p l o t approached zero (Feldman & Rodbard, 1971). The l e a s t d e t e c t a b l e dose (L.D.D.) was taken as that concentration of u n l a b e l l e d antigen which produced a displacement of b i n d i n g = 2 x the standard d e v i a t i o n at maximum b i n d i n g . -43-A S 71 1:150,000 S l o p e 0 . 3 3 5 - 0 .185 = 0.150 1.5 x 10"' 1.0 x 10 1.5 x 10 " ' 4 L / m o l e B ° ( M o l e s ) x 10' Fig. 11 Standard curves with motilin antiserum (GP 71) represented as,a Scatchard plot, B/F being plotted against the fraction of total antigen bound (B°) . The slope of the line gives the a f f i n i t y constant of this antiserum (K). -44-Both these c r i t e r i a evaluate the sensitivity of the standard curve at i t s upper limit. The third parameter, the midrange value, allowed comparison of standard curves in the region where they were most l i k e l y to approach linearity and was that concentration of unlabelled motilin which displaced 50% of the maximum label bound. (2) pH of the diluent buffer Motilin standards in the range 12.5 - 400 pg were incubated with antiserum 75A at a f i n a l dilution of 1:80x10 for 48 hours at 4 C in 0.04M sodium phosphate buffer at either pH 6.5 or 7.5, and in veronal buffer, 0.05M at pH 8.5. There was no significant difference in the displacement observed either at the midrange value or the L.D.D. (3) Trasylol concentration in the diluent buffer Trasylol i s a broad spectrum proteolytic enzyme inhibitor, containing 10,000 kallikrein inhibitor units (KIU) per ml. Standard curves were incubated in 0.04M sodium phosphate buffer, pH 6.5, containing 0%, 0.25%, 0.5% or 1.0% Trasylol. The resultant displacement i s shown in Fig.12 and Table IV and 0.25% Trasylol was selected as the optimum concentration. TABLE IV - Effect of varying the Trasylol concentration in the diluent Buffer Condition Midrange (pg M5) L.D.D. (pg.M5) 0.0% Trasylol 70 25 0.25% Trasylol 24 12.5 0.5% Trasylol 47 25 1.0% Trasylol 135 50 -45-Fig . 12 Effect of varying the Trasylol concentrations in the diluent buffer oh the sensitivity of the routine standard curve for motilin. -46-C4) Plasma concentration jri •the d i l u e n t ' b u f f e r Standard curves were set up to compare the e f f e c t of varying the plasma concentration i n the diluent b u f f e r , from 2 ^ 10% The plasma was outdated blood bank stock which had been extracted twice with 1% (w/y) charcoal f o r 1 hour at 4°C to absorb any small peptides s t i l l present. The charcoal was removed by c e n t r i f u g a t i o n at 5000 r.p.m. for 20 mins. The charcoal^extracted plasma was screened for detectable poly-peptide l e v e l s and stored at -20°C. The greatest s e n s i t i v i t y was achieved when the bu f f e r contained 5% charcoal-extracted plasma (shown i n - Table V). TABLE V ^ E f f e c t of "varying the plasma concentration i n the diluent buffer Condition Midrange . (pg M5) L.D.D. (pg M5) 2% plasma 55 25 5% " 55 12.5 • • 10% " 105 25 (5) Concentration of l a b e l l e d antigen added Labelled antigen at concentrations varying from 2000 c.p.m./lOO p i to 20,000 c.p.m./100 yil was added to standard curves i n the routine assay. The concentration ofr~ 5000 c.p.m./lOO ]_tl was selected as giving the most s e n s i t i v e curve at the most e f f i c i e n t counting rate (Table VI, F i g . 13) -47-o—o 20.000 cpm /100/Jl • — • 10.000 x—x 5.000 11 2.500 100-L.D.D. Midrange Value T 1 1 1 1 12.5 25 50 100 200 pg Motilin Fig. 13 Effect of varying the concentration of labelled antigen on the sensitivity of the routine standard curve for motilin. -48-TABLE VI - Effect of varying the antigen concentration on the assay sensitivity Condition Midrange L.D.D. (pg M5) 20,000 cpm/100 y l 105 50 10,000 94 25 5,000 67 12.5 2,500 54 12.5 (6) Period of incubation Standard curves, a, b and c were set up and iodinated antigen, containing ^ 5000 cpm/100 y l was added immediately to a and b which were then incubated for 24 hours and 48 hours respectively. Standard curve c was incubated with antibody alone for 24 hours. After label addition the incubation continued for a further 48 hours. A 48 hour incubation period was deemed to give a more sensitive assay, from the results in Fig. 14 and Table VII. There was no advantage gained by prior incubation of cold antigen with antibody, i.e., under disequilibrium conditions. Longer incubation periods of 3 - 4 days were also satisfactory. TABLE VII - Effect of varying the incubation period and type Condition Midrange (pg M5) L.D.D. (pg M5) 24 hour equilibrium 145 50 48 " 53 25 24/48 " dis-equilibrium 53 25 -49-I " 1 1 1 12.5 25 50 100 200 P G Motilin Fig. 14 Effect of varying the length and type of incubation on the sensitivity of the routine standard curve for motilin. 0' 0 -50-(7) Protection from adsorption to glass Adsorption of peptide and iodinated material on to the glass tubes, used i n the assay, can be a problem. The plasma content of the diluent b u f f e r did reduce the counts adsorbing to the glass but s i l i c o n i z a t i o n of the assay tubes with 1% (y/v) d j c h l o r o s i l a n e i n benzene was also performed to see i f any further improvement could be achieved. It was.also e s s e n t i a l to determine whether i t was necessary to compensate i n the standard curve for the extra protein added i n the monitoring of plasma or serum samples. Standard curves were Incubated i n s i l i c o n i z e d tubes, n o n - s i l i c o n i z e d tubes and i n n o n - s i l i c o n i z e d tubes with the addition of 100 Til of charcoal - extracted plasma. The r e s u l t s obtained suggested that s i l i c o n i z a t i o n of the tubes was not necessary. The standard curves obtained a f t e r incubation with and without plasma were corrected f or t h e i r i n d i v i d u a l non-specific binding C s e e sections 7 and 10) and no s i g n i f i c a n t difference was detected - i . e . , the addition of plasma was unnecessary. (Table VIII, F i g . 15) TABLE VIII E f f e c t of s i l i c o n i z a t i o n or plasma addition on assay system Condition Midrange (pg M5) L.D.D. (pg M5) S i l i c o n i z e d tubes Non-siliconized tubes Non-siliconized tubes & p »1 59 57 asma 56 25 125 25 1 - 5 1 -i o-| 1 1 1 1 1 I , 0 12.5 25 50 100 200 j ! pg Motilin I i I I i Fig. 15 Effect of siliconization of the incubation tubes or the addition of plasma on the sensitivity of the routine standard curve for motilin. -52-C8) Routine assay.conditions The diluent buffer, 0.04 % 'podium phosphate, pH 6.5; containing 5% charcoal - extracted plasma and 0.25% trasylol, was used in a l l dilutions and for correcting the f i n a l volume to 1.0 ml. The composition of the incubation mixture was:-125 100 y l I-<motilin containing ~5000 c.p.m. 100 u l standard motilin, range 12.5 -400 pg. or -100 y l interassay control or 50 200 y l unknown 100 y l antiserum at the appropriate i n i t i a l dilution Diluent buffer to a volume of 1.0 ml A l l assays were set up, with standards in tr i p l i c a t e and unknowns in duplicate, in 10 x 75 "mm glass culture tubes at 4°C. and incubated at 4°C for 48 - 72 hours. Non-specific binding (N.S.B.) was measured by setting up tubes, minus antiserum, for the standard curve, the interassay controls, each group of sera from one subject and a l l other unknowns. In assays where only one separated component was to be counted (see section 9) 125 total count tubes, containing 100 y l I - motilin only, were set up in quadruplicate. Table IX illustrates the assay layout. LAYOUT FOR ROUTINE RADIOIMMUNOASSAY D.B. LABEL ANTIBODY STD CONTROL UNKNOWN Total counts 100 * Standard curve NSB 900 100 Maximum binding 800 100 100 STDS 700 100 100 100 Interassay control NSB 800 100 100 Interassay control 700 100 100 100 Unknown NSB 800 100 100 Unknown 700 100 100 _ 100 refers to volume in y l -54-(9) Separation procedures Both specific and non-specific methods exist for the separation of the antigen/ antibody complex (Bound) from the Free antigen. The specific methods include the double antibody technique and the use of a solid phase antibody matrix where the antibody is coupled to an immunologically inert material - e.g., Sephadex, Sepharose or Polyacrylamide. The use of Sepharose-coupled antibody in the motilin R.I.A. i s described in the section on af f i n i t y chromatography. The non-specific methods include the addition of alcohol resulting in the pre-cipitation of large molecular weight proteins, including the antigen/antibody complex and the use of dextran-coated charcoal which w i l l adsorb the free antigen, leaving ,the antigen/antibody complex in solution. This last method is 1 that most'commonly used in this laboratory. Phosphate buffer, 0.04M, pH 6.5, containing 2% plasma, was cooled to 4°C. The dextran was mixed well to ensure a complete suspension before the addition of charcoal. The suspension was mixed gently at 4°C for at least 1 hour prior to the addition of 200 y l to each assay tube, excluding the total count tubes. After being vortexed br i e f l y , the tubes were centrifuged at 2800 rpm for 20 min. The supernatant was then decanted into a separate tube (for B/ estima-tions). Each tube was sealed with wax and counted in an automatic' Y c o u n t e r . Various charcoal concentrations were examined, each coated with 10% (w/w) dextran. The results are graphed in Fig. 16 and evaluated in Table X. -55-"i 1 1 1 1 1 1 0 6.25 12.5 25 50 100 200 pg Motilin Fig. 16 Effect of varying charcoal concentrations in the separation procedure on the sensitivity of the routine standard curve for motilin. At each concentration the charcoal'was coated with 10% (w/w) dextran T-70 -56-1.25g% (w/v) charcoal was f i n a l l y selected because i t was the concentration producing the greatest sensitivity at the upper limit of the curve. TABLE X "Effect of varying the charcoal concentration in the separation procedure Condition Midrange (pg M5) L.D.D. (pg M5) 0.625g% charcoal 70 25 1.25 g% " 52 6.25 2.5 g% " 35 12.5 Recently, the dextran-coated charcoal suspension has been prepared, 1 l i t r e at a time, in phosphate buffer only, and mixed for 3-4 hours. As required, an appropriate volume was removed, the plasma added, and the suspension mixed for -v 15 mins. before use. This suspension keeps well at 4°C for 1-2 weeks and provides a more homogenous suspension, as demonstrated by an improvement in the replication of t r i p l i c a t e and duplicate values. (10) Methods of data analysis There are numerous methods used in the expression of RIA results. Those used in this study include B/^, B / T C or % B, B / B Q X 100 and a l l include a correction to account for the non-specific binding of labelled antigen to glass or plasma protein. -57-The c a l c u l a t i o n of B/^ , requires the counting of both the Bound and f r e e antigen a f t e r separation and i s obtained from the expression: B/ p = SAMPLE (Bound c.p.m.) - NSB (Bound c.p.m.) (free c.p.m.) (free c.p.m.) The other methods require that only one component i s counted a f t e r separation but do require some method of estimating the Total Counts (TC) i n i t i a l l y added to each tube. I f dextran-coated charcoal i s the method of separation used i t i s more convenient to count the f r e e antigen i n the charcoal p e l l e t . Therefore, %B i s calculated from the expression: %B SAMPLE (TC - FREE c.p.m.) - NSB (TC - FREE c.p.m.) TC TC i . e . , %B = .(NSB) F R E E . c . p . m . ( S A M P L E ) FREE c.p.m. TC Results may also be expressed as a percentage of the maximum binding -i . e . , B/ x 100, where B i s the binding of l a b e l achieved when no un-D0 U l a b e l l e d antigen i s added to the system. Standard curves are prepared by p l o t t i n g one of these values against the concentration of standard antigen, expressed e i t h e r l o g a r i t h m i c a l l y or ar i t h m e t i c a l l y . F i g . 17 i s a routine standard curve f o r m o t i l i n obtained a f t e r a l l the conditions f o r a s e n s i t i v e RIA had been established. A l l these conditions were established f o r RIA with a s p e c i f i c antiserum to -58-0.60 • 0.50 H 0.40 0.30 0.20 0.1 0 H 0.0 No Motilin added l 1 1 1 1 1 l 10 20 40 80 160 320 640 Motilin (pg/ml . ) f i g . 17 Routine standard curve for motilin, obtained .after the optimum conditions had been established. Each point represents the mean(- SD)•for 7'observations $ (Dryburgh and Brown, Gastroenterology 68: 1169-1175, 1975) -59-motilin; they do not necessarily hold true for a l l motilin antisera and should be re-evaluated for each antiserum. E. Assay standards and interassay controls 1 CI) Preparation and storage of standards Natural porcine motilin (M5) was used in a l l standard preparations; One - two mg were weighed accurately on a Cahn electrobalance, dissolved in deionized water to give a concentration of 1 yg/100 y l and aliquotted in 100 yl amounts into siliconized glass culture-tubes for lyophilization and storage at -20°C. Each month, or as required, a 1 -yg aliquot was reconstituted in 0.04M sodium phosphate buffer, pH 6.5, containing 0.25% trasylol and 5% BSA,to a concent-ration of 80 ng/iiil. This solution was-stored at -20°C in 1.0 ml amounts in polyvinyl microtest tubes. At the time of assay an aliquot was diluted 1:20 - i.e., 400 pg/100 y l and serial dilutions prepared over the range''6.25 - 400 pg/100 yl. Any remaining standard was. discarded after thawing. C2) Preparation and storage of controls One yg aliquots of M5 werediluted in 0.04M phosphate buffer, pH 6.5, containing 0.25% trasylol and 5% BSA, to a concentration of 1 ng/ml. One ml aliquots were stored at -20"C in polyvinyl microtest tubes and 100 y l samples were assayed at the beginning and end of every assay. Any remaining control was discarded after being thawed. -60-(3) Inter-and tntra-assay control In 5 different assays-, 20 duplicate determinations of the control value were made. The mean - S.D. was 110 ^ 23 pg-motilin/100 jal. Results in any assay in which the control lay outside these values were discarded.. Ihtra^ assay va r i a b i l i t y was checked by having duplicate measurements of the control at the beginning and end of each.individual assay and applying the same conditions' to their evaluation., PREPARATION OF SYNTHETIC AND NATURAL ~M0TILIN FRAGMENTS AND ANALOGUES  A. Synthetic motilin (1) Preparation of 13 - norleucine - motilin The synthetic analogue, 13 - norleucine - motilin was prepared in the labora-tory by Dr. E. Wlinsch, Max-Planck Institute flir Eiweiss und Lederforschung, Munich, W. Germany (Wlinsch et a l , 1973). The RIA was used to monitor the f i n a l purification stages. The i n i t i a l crude synthesis product,/MoA was separated by column chromato^-graphy on QAE Sephadex A-25 into MoB^ and MoB^, the latter being found to represent a failed synthesis, lacking 2 amino acid residues (-THR -TYR). Further purification of MoB^  on SP - Sephadex C-25 resulted in MoC^ and M0C2, both synthetic products being identical to natural motilin with respect to amino acid composition and sequence. -61-(2) Preparation of synthetic motilin fragments During the preparation and purification of the synthetic analogue, the fragments containing residues 9~22 and 13--22 were also isolated and purified. B; • Fragments of natural motilin CT1 Cyanogen bromide cleavage of motilin Cyanogen bromide CCNBr) treatment of a polypeptide results in chemical cleavage of that polypeptide at the methionyl residue (Gross and Witkop, 1961, 1962). The reaction was.performed in 70% (v/v) formic acid at a polypeptide concentration of 2.0 mg/ml and a CNBr concentration of 10 mg/ml. The reaction flask, foil-covered to exclude:light, was l e f t at room tempera-ture for 6 hours, then the contents were diluted 1:20 with d i s t i l l e d water prior to lyophilization. The immunological activities of the intact motilin molecule and the un-separated mixture of cleaved and non-cleaved CNBr-treated motilin were compared on an equimolar basis. C2) Tryptic and chymotryptic digestion of motilin Enzymatic cleavage of the polypeptide was performed in 1% ammonium bicar-bonate at a polypeptide concentration of 0.2% (w/v) and an enzyme:substrate ratio of 1:50 (w/w). The reaction proceeded for 6 hours at room temperature and was terminated by lyophilization, redissolving in 0.5 ml water and boiling for 6 mlns. in a water bath. The solution was centrifuged to remove any precipitation and the supernatant was lyophilized. B i o l o g i c a l , and immunological a c t i v i t i e s were estimate*-for the unseparated digestion products and compared with the i n t a c t molecule on an equimolar basis. C. Modifications of natural motilin Cl) C-terminal residue removal The reaction involved treatment of 100 nM motilin in 0.1M ammonium b i -carbonate with 200 yg carboxpeptidase A 'DFP1 (Diisopropyl phospho-floridate treated) i n 2.OM ammonium bicarbonate at a peptide: substrate ratio of 38:2 (y/v) for 6 hours. Kinetic studies had shown that after"6 hours at 22°C 80% of the C-terminal glutamine and 20% of the penultimate C-terminal residue, glycine had been removed. The reaction was terminated by lyophilization of the mixture. (2)- Nonterminal residue removal Removal of the N-terminal phenylalanine was achieved by one cycle of: the Edman degradative procedure (Edman, 1956; Gray, 1967). Coupling of the phenylisothiocyanate (PITC) was accomplished by dissolving 100 nM of the polypeptide in 150 JJl deionized water in an acid-washed 12 x 75 mm glass culture tube. Reagent (5% PITC in pyridine) was added and the tube was flushed with nitrogen to expel the air , and covered with parafilm prior to incubation at 45°C for 1.5 hours. At the end of this time the tube was un-covered after centrifugation, and dried over fresh phosphorus pentoxide,-under vacuum at 60°C. The coupled phenylthiocarbamyl residue was cleaved from the peptide by treatment with 150 yil trifluoroacetic acid and incubation at 45°C for 30 mins. The mixture was evaporated to dryness in a vacuum -63-d e s s i c a t o r o v e r , s o d i u m h y d r o x i d e p e l l e t s , t h e n w a s r e d i s s o l v e d i n 200 y± d e -i o n i z e d w a t e r . The f r e e p h e n y l t h i o c a r b a m y l p h e n y l a l a n i n e w a s . r e m o v e d by•ex-tracting f o u r t i m e s w i t h 2 m i s b u t y l a c e t a t e , t h e u p p e r o r g a n i c l a y e r b e i n g d i s c a r d e d e a c h t i m e . The r e m a i n i n g a q u e o u s s o l u t i o n , c o n t a i n i n g m o t i l i n 2-22 w a s e v a p o r a t e d t o d r y n e s s u n d e r v a c u u m , o v e r c o n c e n t r a t e d s u l p h u r i c a c i d i . r e -d i s s o l v e d i n 150 u l d e i o n i z e d w a t e r a n d a 10 VI a l i q u o t r e m o v e d f o r d a n s y l c h l o r i d e d e t e r m i n a t i o n o f t h e n e w N - t e r m i n a l r e s i d u e , t o c h e c k f o r c o m p l e t e n e s s o f t h e d e g r a d a t i o n c y c l e . (3) Identification o f N - t e r m i n a l r e s i d u e The m e t h o d o f Gray (1967) w a s f o l l o w e d w i t h t h e m o d i f i c a t i o n o f Bruton a n d Hartley 0-970) o f u s i n g 5 x 5 cms p o l y a m i d e p l a t e s . The p o l y p e p t i d e , a p p r o x i m a t e l y 5nM, w a s t r a n s f e r r e d t o a g l a s s t u b e ( p y r e x ) 0.6 x 50 m m s , a n d l y o p h i l i z e d , 2 u l 1% s o d i u m b i c a r b o n a t e w e r e t h e n a d d e d a n d t h e t u b e w a s c e n t r i f u g e d a n d r e l y o p h i l i z e d . Deionized w a t e r a n d d a n s y l c h l o r i d e , C d i m e t h y l n a p h , t h a l e n e - 5 - s u l p h o n y l c h l o r i d e , 2.5 m g / m l i n a c e t o n e ) 2.5 u l e a c h w e r e a d d e d t o t h e t u b e w h i c h w a s c e n t r i f u g e d , c o v e r e d w i t h p a r a f i l m a n d i n c u b a t e d a t 45°C f o r 20 m i n s . The c o n t e n t s w e r e a g a i n l y o p -h i l i z e d a n d r e d i s s o l v e d i n 50 u l 6M h y d r o c h l o r i c a c i d , t h e t u b e b e i n g t h e n h e a t s e a l e d a n d h y d r o l i z e d a t 110°C f o r 18 h o u r s . When a c i d h y d r o l y s i s w a s c o m p l e t e t h e t u b e w a s c e n t r i f u g e d , o p e n e d a n d d r i e d u n d e r v a c u u m o v e r s o d i u m h y d r o x i d e p e l l e t s . 2 Thin l a y e r c h r o m a t o g r a p h y o n 5 cm p o l y a m i d e p l a t e s * i n s e v e r a l s o l v e n t s y s t e m s w a s p e r f o r m e d . The h y d r o l y s i s p r o d u c t s w e r e d i s s o l v e d i n 2.5 j a l 50% aqueous pyridine and ~ 0.5 ul spotted on each side o f the plate. A standard solution '<CO.5 ul) containing the dansyl derivatives o f phenyla-lanine, isoleucine, proline, glycine, glutamic acid, serine and arginine, 1 UM each acid/ml in acetone; 0.1M acetic acid (3:2 v/v) was spotted on one side only. The polyamide plate was.subjectedfto ascending chromato-graphy in two dimensions in the appropriate solvent systems. Dimension Solvent system I 1 Water: 90% formic acid 200 : 3 (v/v) CWoods and Wang, 1967) XI Benzene: glacial acetic acid 9 : 1 (v/v) (Woods and Wang, 1967) ITT Hexane: n-butanol: glacial acetic acid 3 : 3 : I (v/v) (Crowshaw et al,1967) IV 0.1% Ammonia: Ethanol 9 : 1 (v/v) After running in solvents I and II, the plates were viewed under short-wave ultra violet light and identification of the dansylated residue made. Dansyl serine/dansyl theonine, dansyl glutamic acid/ dansyl aspartic acid and -65-dansyl glycine/dansyl alanine may only be differentiated after chromato-graphy in solvent III and solvent TV was used to separate the basic residues arginine, histidine and E -lysine. If lysine or tyrosine was present at any position in the polypeptide,E-dansyl-lysine or 0-dansyl tyrosine was always seen. Positive identification of either lysine or tyrosine as N-terminal requires the presence of bis-dansyl-lysine or bis-dansyl-tyrosine respectively. If proline is the N-terminal amino acid, the hydrolysis must be limited to a 4 hour period. (4) Acylatlon^acetylated derivative Acetylation of the polypeptide with acetic anhydride was performed by a modification of the method of Riordan and Valee (1967). One mg motilin was dissolved in 1.0 ml 50% saturated sodium acetate. A thirty-fold molar excess of acetic anhydride was added in five portions over 1 hour and the reaction mixture was stirred continuously at room temperature. After a further hour the reaction mixture was frozen, lyophilized and desalted on Sephadex G15 fine (0.7 x 100 cms) in 0.2M acetic acid at a flow rate of 6 mis/hour and 1.2 ml fraction size. The acetylated derivative was com-pared with natural motilin, on an equimolar basis, for biological and immunological activity. -66-(5) Acylation-succjnylated derivative The method of Klotz (1967) was slightly modified. One mg motilin was dissolved in 2.0 mis deionized water, the pH brought to 7.0 with 0.1M sodium hydroxide and constantly monitored during the addition of a thirty-fold molar excess of succinic anhydride over 15 mins. The pH was maintained at 7.5 by addition of 0.1M sodium hydroxide and the mixture was stirred gently at room temperature for a further 1.5 hours. The solution was frozen, lyophilized and desalted on Sephadex G15 fine, as described for the acetylated derivative. AFFINITY CHROMATOGRAPHY A. Activation of Sepharose 4B Equal volumes ( ~ 20 mis) of Sepharose 4B (Pharmacia, Uppsala, Sweden) slurry and deionized d i s t i l l e d water were mixed together gently over ice, in a fume hood. Cyanogen bromide (CNBr) at a concentration of 100 mg/g Sepharose 4B was added in a volume of deionized water, equal to the total, and the pH was immediately raised to pH 9 - 11 and maintained i n this range by the addition of 4.0M sodium hydroxide. When the pH had remained stable for 10 mins. with no further addition of al k a l i , the reaction was considered terminated. Ice was added to the mixture and the gel was washed on a Buchner f i l t e r under gentle suction with at least 10 volumes of cold 0.1M sodium bicarbonate. The CN Br-activated Sepharose 4B could be stored as a moist slurry at 4° for 1-2 weeks (Cuatrecasas, 1970). -67-B. Coupling of ligand to activated Sepharose 4B Activated Sepharose 4B was made up in an equal^volume of 0.1M sodium b i -carbonate. Antiserum to motilin or GIF was diluted in an equivalent volume of bicarbonate to a fi n a l concentration of 30 jal antiserum/g Sepharose 4B. The reaction mixture was stirred gently for 24 hours at 4°C. The coupled gel was washed well with 20 volumes cold deionized water on a Buchner f i l t e r . Aliquots of the diluted antiserum, prior to coupling, and the i n i t i a l wash, after coupling, were put aside for RIA, to determine the efficacy of the coupling reaction. Figs. 18 and 19 illustrate the anti-serum dilution curves obtained in a typical procedure, with virtually no antibody activity de-tectable in the wash. Unreacted active groups on the gel matrix were blocked by treatment with excess ethanolamine as follows: ethanolamine (MW 61.1, 16.4M, pH 12.7) was brought to pH 9.0 by addition of 5.0 hydrochloric acid. Sufficient ethanolamine was added to a known volume of coupled Sepharose 4B such that the f i n a l molarity, with respect to ethanolamine, was 1.0M. The reaction was complete after 4 hours at 4°C and the excess ethanolamine removed by washing the gel with 10 alternating cycles of 0.1M sodium acetate, pH 4.0, and 0.1M sodium phosphate, pH 8.0. The f i n a l product was stored in an equal volume of 0.1M sodium bicarbonate at 4°C. At this stage 0.01% sodium azide was added as a preservative. COLUMN CHROMATOGRAPHY  A. Gel f i l t r a t i o n The technique of using a cross-linked dextran gel as a molecular sieve was -68-I x IO3 I x IO4 I x IO5 Reciprocal Antiserum Titre Fig. 18 Motilin antiserum dilution curves comparing the:activity of the antiserum prior to coupling to Sepharose 4B with the activity remaining in the wash after completion of the coupling procedure. -69—-2.0 i O i CM DQ .0 0.04 * Pre-Coupling x Post-Coupling I xlO* I x 10 Reciprocal of Antiserum Titre x 10* Fig. 19 GIP antiserum dilution curves comparing the activity of the antiserum prior to coupling to Sepharose 4B with the activity remaining in the wash after completion of the coupling procedure. -70-f i r s t described by Porath and Flodin (1959) and has become one of the most commonly used methods of separating the components of a mixture by molecular size. The appropriate weight of the gel was stirred gently into excess buffer and allowed to swell overnight at room temperature. The fines were decanted before the gel was de-aerated under vacuum for 30 mins. The supernatant liquor was removed R O that the f i n a l suspension was a slurry which would pour easily without trapping further air. The column was mounted vertically, out of draughts and direct sunlight. Buffer was injected through the outlet tubing to f i l l the space beneath the bed support and to a level of approximately 10 cm above the support. The outlet was closed and the slurry poured gently down the column wall. If necessary, a gel reservoir was attached to the column to ensure a l l the gel being added at one time. The i n i t i a l packing of the gel occurred under gravity u n t i l the gel reservoir could be removed, then the buffer reservoir was attached and the column packing was completed with the outlet open, at the hydrostatic pressure which would be used in subsequent operations. A f i l t e r paper disc (Whatman's 3MM) was inserted to stabilize the gel-liquid interface and the gel equilibrated in buffer overnight. The buffer above the level of the gel was removed and the sample, dissolved in a small volume of buffer, applied to the gel and allowed to sink to the level of the gel surface. A volume of buffer, roughly equivalent to the sample volume, was similarlyy applied, washing the sample well into the body -71-of the gel, Excess Buffer was replaced on top of the gel and the column attached to the Buffer. As the Buffer flowed through the gel, fractions of eluant buffer of a predetermined size were collected. Between runs, the column was stored in buffer containing 0.01% sodium azide as a preservative B. Ion exchange chromatography Ion exchangers require precycling through acid and a l k a l i to provide the necessary counter ion. An ion exchanger, e.g., Whatman's DE celluloses or Sephadex AE resins, were treated f i r s t in 0.5M hydrochloric acid for 30 mins. whilst cation exchangers CWhatman's CM or CE-Sephadex) were f i r s t treated in 0.5M sodium hydroxide. The exchangers were washed well with d i s t i l l e d water until the intermediate pH's were 4 and 8 respectively. The treatments were then reversed, the anion exchanger being washed in 0.5M sodium hydroxide and the cation exchanger in 0.5M hydrochloric acid for 30 mins. Both exchangers were well washed in d i s t i l l e d water unt i l the effluent pH was near neutrality. The fines were decanted and the exchangers de-aerated under vacuum for 30 mins. Equilibration in the starting buffer was ensured by repeatedly s t i r r i n g the exchanger into 15 volumes of that buffer and decanting the supernatant liquid after 10 mins. until the pH and conductivity of the effluant were identical to those of the buffer. The columns were packed and the samples applied as described in Section A. Development of the column was achieved by either stepwise increases in buffer ionic strength or by establishing a gradient of ionic strength. The most strongly absorbed material was cleared by passage of 0.2M ammonia through the column, and the cellulose was stored in this buffer between runs. Further pre-cycling was not necessary before re-use of the column, but i t was essential that the column be well-equilibrated in the starting buffer to ensure re-placement of the necessary counter ion. POLYACRYLAMIDE GEL ELECTROPHORESIS The method followed is a modification of that of Johns (1967). The gel solution was prepared by carefully mixing 10 mis of monomer (40% w/v acrylamide and 0.6% w/v NjN"*" - methylenebisacrylamide in d i s t i l l e d water) 1 1 with 10 mis of catalyst I (0.5% v/v N, N, N , N - tetramethylenediamine in 4.6M acetic acid) and 6 mis of catalyst IT (0.6% w/v ammonium persulphate in d i s t i l l e d water). The mixture was de-gassed under gentle vacuum for 30 mins. and 10 ml gel solution placed in each prepared 5 x 75 mm gel column. Di s t i l l e d water was layered on top of each gel and polymerization was accele-rated under direct light for the f i r s t hour, then allowed to continue at room temperature. Gels were stored at least 3 days before use and could be kept for up to a month i f dehydration was prevented. Gels were equilibrated in a Shandon electrophoresis apparatus, Model 12734, modified to allow cooling of the system The buffer used was 0.01M acetic acid and current was passed at 320 volts, for 3 hours. The electrodes were placed with the anode uppermost. Samples for electrophor esis were dissolved in 0.002M acetic acid, 1.0M with respect to sucrose. The apparatus reservoirs were emptied, the sample layered onto the gel surface from a Lang-Levy micropipette, and the reservoirs -73-r e f i l l e d with fresh 0.01M acetic acid. (A) Method for staining and destainjrig for qualitative determinations The samples were settled into the gel By passing current through the gel at 320 volts for 15 mins. The dye, 1.0 ml amidoBlack (0.5% w/v in 1.0M acetic acid) was mixed throughout the lower reservoir buffer, and the current run at 320 volts for a further 15 mins. The reservoirs were then carefully rinsed and r e f i l l e d with 0.01M acetic acid, the apparatus re-assembled, and the gels destained by passing current at 320 volts u n t i l the gel was cleared of dye, except for the stained protein bands. CB) Method for staining and destairiirig for quantitative determinations The paired samples were allowed to settle into the gel as previously described, except that the time was extended to 25 - 50} mins. The gel tubes were removed from the apparatus, protected with plastic and the glass cracked in a vice. The gels were rinsed with tap water and the marker gels were stained in petri dishes, covered with 0.5% w/v amidoblack for 3 - 4 hours. These gels were destained electrically in an enamel or polyvinyl basin, in cotton wool saturated with 1.0M acetic acid, by passing current at 150 volts across the gels. The remaining gels were stored moist at 4°C u n t i l the destaining process was complete. The marker gels were aligned along the unstained gels and the appropriate sections cut from the unstained gel with 000 s i l k . The gel section was then homogenized by passage through a 5 ml luer-lok syringe i n 0.1M acetic acid or d i s t i l l e d water. The peptide was then either extracted -74-into the acetic acid overnight at 4°C for RIA determination or the water/gel mixture was emulsified with Freund's Complete Adjuvant and used in immuni--zation. In the latter case the polyacrylamide acted as the carrier molecule for the hapten. ANIMAL PREPARATIONS (A) Chronic dog preparation Labrador or labrador-cross breeds were selected for their size, nature and stamina. The weight range used was 20 - 25 kg. A l l surgery was performed aseptfcally. After an 18 hour fast, the dog-was anaesthetized with a rapid intravenous injection of 5% sodium thiopental ("Pentothal"), given to effect, usually 9 - 1 5 mis. An endotracheal tube was placed in position and anaesthesia was maintained with "Fluothane" delivered from a Foregger open circuit anaesthetic apparatus at an oxygen flowrate of 3 litres/min. and a fluothane concentration of 2.5%. C l ) Bickel pouch An extrinsically denervated pouch of the body of the stomach was constructed from the greater curvature. A stainless steel and teflon cannula was placed in the pouch and brought to the exterior through a stab wound in the dog's abdominal wall, in the l e f t upper quadrant. Sectioning of the stomach wall removed vagal innervation and sympathetic denervation was achieved by stripping the nerve plexus around the splenic artery and vein; Cthe blood supply to the pouch), for approximately 1 cm and removing any mesentery from the pouch. -75-This i s referred to in the following study as the fundic pouch. The fundic pouch cannula was le f t open and draining at a l l times, except when fundic pouch motor activity was being monitered. A stainless steel and teflon cannula (Thomas, 1941) was placed in the most dependent portion of the stomach remnant, with a purse-string suture. This cannula was brought to the exterior through a flank incision on the same side and ~ 5-6 cms posterior to the fundic pouch cannula. Except during experimental procedures, this cannula was kept closed with a teflon plug. Gastrointestinal continuity was restored with a gastro-jejunostomy approxi-mately 30 cm distal to the ligament of Treitz. (2) Mann-Bollman f i s t u l a A length of terminal ileum, approximately 10 cm long was removed and intes-ti n a l continuity restored with an end-to-end anastomosis. The distal end of the terminal ileum segment was attached to the duodenuum approximately 3 cm below the pylorus with an end-to-side anastomosis, and the proximal end brought to the exterior through a stab wound and stitched in place on the right ab-dominal quadrant to form a stoma. A partial antrectomy was performed to remove gastrln-producing tissue with a resultant reduction in water and electrolyte loss through the continuously-draining fundic cannula. -76-(3) Gastric f i s t u l a A gastric f i s t u l a was constructed in dogs when required by inserting a Thomas cannula into the most dependent portion of the whole stomach. The dogs were used in the control studies. C4) Truncal vagotomy Truncal vagotomy was performed by the thoracic route. After anaesthesia was induced, the animals were placed on a Bird respirator and the chest opened at the 8th rib interspace. The oesophagus was located and the l e f t vagus identified, divided, and 2 cm removed. The right vagal branch was similarily treated. In a small proportion of the dogs an interconnecting branch li e s between the right and l e f t vagi. If present, this was also sectioned. After the air was expelled from the thoracic cavity, nylon sutures were used to approximate the intercostal muscles. (5) Antrectomy The antrum was removed in gastric f i s t u l a dogs. The pyloroduodenal junction was located and divided. The junction between the antrum and body of the stomach was identified by the subtle change in texture on the surface of the stomach and the antrum was excised by sectioning at this junction. A l l blood vessels supplying;this region were ligated and sectioned. C6) Vagotomy and antrectomy In a separate operation the antrectomized dogs were vagotomized and vice - 7 7 -versa. After a l l surgical •procedures,, dogs were maintained by- intravenous therapy for 3 days post-operatively and allowed a 2 week recovery period before experimentation began. A diagrammatic representation of the chronic dog preparation is shown in Fig. 20. EXPERIMENTAL PROCEDURES CA) In chronic dog with Bickel pouches and duodenal fistulae Dogs were fasted overnight before use. They were harnessed in a stand which provided support whilst maintaining the dog upright. A polyethylene bottle was attached to the open Thomas cannula to collect drainage from the stomach and prevent passage of gastric secretions into the jejunum. A Foley catheter (id. 3mms) was attached to a syringe barrel. Its tip was inserted into the duodenal f i s t u l a for the approximate length of the fi s t u l a or un t i l saline in the syringe barrel flowed freely into the duo-denum under gravity, and kept in that position with cords around the animal's body. Intra-duodenal infusion was performed under gravity from an open syringe barrel or at a pre-determined rate with the syringe driven by an infusion pump (Dual Infusion/Withdrawal Pump, Harvard Apparatus Co. Inc. Diver, T^ass, U.S.A.). -78-Duodenal Blind Loop ic Pouch Diagram of Chronic Dog Preparation. f i g . 20 Diagrammatic representation of the chronic dog preparation. A 21 G l" L/2" hypodermic needle attached to polyethylene tubing (PE 60) was inserted into either the radial or saphenous vein for intravenous injection or infusion. Blood samples were taken from a permanently-indwelling (experimental duration) intravenous cannula on a 19G /8" needle (Argyle Venocut Infusion Set). Patency of both cannulae was maintained with a gravity-fed saline drip. Blood samples were allowed to clot for 20 mins. at 4°C. After centrifugation for 10 mins. at 3000 rpm the serum was removed and stored at -20°C until required for RIA. (I) Bioassay for motilin The fundic pouch cannula was connected to a venous pressure transducer (Statham P 23 BB) via a water-filled tube with a side arm, allowing collection of f l u i d from the pouch. The pouch was f i l l e d with ~25 mis tap water at the start of an experiment, the f l u i d being changed between test procedures. Test procedures, either intravenous infusion or injection or duodenal infusion, were not performed until the fundic pouch motor activity had established i t s basal rhythm. Recordings of fundic pouch motor activity were made continuously on a poly-graph (Gilson pen recorder). Motility indices were measured over a specific time period from the formula: M.I. = Amplitude (mm Hg) x Duration (sees.) of each contraction Duration of period (mins.) x 10 -80-Unless otherwise stated, no two tests were performed less than 40 mins. apart and the response to any test was measured over the 10 min. period immediately following that test. If required, serum samples were obtained and stored at -20°C until subjected to RIA for motilin, etc. C2) Effect of GIP on acid secretion A 15 ml graduated test tube was attached to the fundic pouch cannula to collect the output from the pouch over each 15 min.period. Acid secretion was stimulated by continuous intravenous infusion of pentagastrin or histamine dihydrochloride. If desired, exogenous GIP could also be administered via the same, intravenous cannula. The volume of gastric acid produced by the fundic pouch in each period was measured, diluted 1:10 with d i s t i l l e d water and titrated to pH 7.0 with 0.01M sodium hydroxide in a titrator assembly (Titrator II, Radiometer). The H~*~ ion concentration was expressed as uEq. of H~*~ ion per 15 mins. In the appropriate experiments , glucose, fat or acid were infused intra-duodenally from a Harvard infusion pump, via a catheter, passed through the Mann-Bollman f i s t u l a . Glucose was administered as a 20% dextrose solution in d i s t i l l e d water at a rate of lg/Kg over 30 min ."Whilst fat (Lipomul-Upjohn) and 0.15N hydrochloric acid were each infused at a rate of 1.91 mis./ min. Over 30 min. A plateau of gastric acid secretion was considered established -81-a f t e r continuous intravenous i n f u s i o n of p e n t a g a s t r i n had r e s u l t e d i n three consecutive periods during which the l e v e l s of H + s e c r e t i o n were w i t h i n 10% of each other. The B i c k e l pouches i n d i f f e r e n t dogs v a r i e d i n s i z e and se c r e t o r y c a p a c i t y , and because of the t r o p h i c e f f e c t of g a s t r i n on the g a s t r i c mucosa i t was a l s o p o s s i b l e f o r the se c r e t o r y c a p a c i t y of one B i c k e l pouch t o vary during the d u r a t i o n of the study. Dose-response st u d i e s w i t h p e n t a g a s t r i n were performed i n each dog, and the dose of p e n t a g a s t r i n s e l e c t e d which r e s u l t e d i n a g a s t r i c a c i d output equal to 75% of the maximum output. The values of the H s e c r e t i o n were expressed as r a t i o s of the mean of three p l a t e a u p e r i o d s . The IR-GIP response was p l o t t e d as the change i n IR-GIP (A IR-GIP) from the mean of the three periods p r i o r to the s t a r t of the duodenal i n f u s i o n ) CB) In chronic dogs w i t h g a s t r i c f i s t u l a e The dogs were accustomed to being harnessed i n the stand and were f a s t e d f o r 18 hours p r i o r to any study. Blood samples f o r RIA were obtained and intravenous i n f u s i o n s administered as described i n S e c t i o n A. (I) Determination of the r a t e of g a s t r i c emptying of l i q u i d s Sodium c h l o r i d e , 0.15M, con t a i n i n g 60 m g / l i t r e phenol red as an i n d i c a t o r , was i n s t i l l e d i n the stomach v i a the g a s t r i c f i s t u l a and then drained at the end of a 10 min. p e r i o d . Various cencentrations of m o t i l i n were administered as intravenous i n f u s i o n s . The phenol red con c e n t r a t i o n i n the f l u i d meal was determined i n both the -82-i n i t i a l meal p r i o r to i t s i n s t i l l a t i o n i n t o the stomach and i n the f l u i d drained from the stomach. Phenol red determinations were performed as fo l l o w s :-" 1 ml of the l i q u i d meal p l u s 2 mis of sodium phosphate (27.5 g Na^PO^/litre) Were made up to 10 mis w i t h d i s t i l l e d water and mixed w e l l . The O.D. of the s o l u t i o n was read at 550 nm i n a 1 cm l i g h t path and used to c a l c u l a t e the r a t e of g a s t r i c emptying from the formula. Rate of emptying (mis/10 mms) = (V-P,) - ( V P ) 1 1 r r ( P i + P r) 12 Where V. and V are the volumes of f l u i d i n s t i l l e d and recovered and P. and i r x P r are the concentrations of phenol red i n the i n s t i l l e d and recovered f l u i d s r e s p e c t i v e l y . This c a l c u l a t i o n i s based on the assumption that the concent-r a t i o n of phenol red l e a v i n g the stomach i s the mean of the i n i t i a l and f i n a l c o ncentrations. C2) . Determination of the r o l e of g a s t r i c emptying of s o l i d s Dogs were fed a p r o p r i e t a r y canned dog food equivalent to a 3 g dry weight/kg. The amount of s o l i d m a t e r i a l that remained i n the stomach at various time i n t e r v a l s (weighed a f t e r d e s s i c a t i o n ) was expressed as a f r a c t i o n of the i n i t i a l weight. M o t i l i n was administered as an intravenous i n f u s i o n of 1,0 yg/kg/hour -83-(C) In the i n t a c t dog Dogs, i n the weight range 3CH35 kg^with no s u r g i c a l i n t e r f e r e n c e , were t r a i n e d to remain harnessed i n the experimental stand. A l l animals were fa s t e d f o r 18 hours. Blood samples f o r RIA and glucose determinations were c o l l e c t e d and t e s t i n f u s i o n s were administered as described i n Section A. Test substances were given o r a l l y from a glass syringe f i t t e d w i t h a f l e x i b l e catheter. When the catheter t i p was h e l d i n s i d e the dog's cheek by the p o s t e r i o r molars, any l i q u i d deposited there induced swallowing \ without undue trauma to the dog. Unless otherwise s t a t e d , glucose was administered o r a l l y as a 20% dextrose s o l u t i o n i n d i s t i l l e d water at a dose of 1 g/kg. The o r a l f a t used was Lipomul, a p a l a t a b l e emulsion c o n t a i n i n g 66g t r i g l y c e r i d e s per 100 mis. Results were expressed as change from c o n t r o l t(A.),the c o n t r o l value being defined as the mean of three f a s t i n g serum values of that parameter, measured at 15 min. i n t e r v a l s at the s t a r t of the experiment. A c o n t r o l was only acceptable i f the v a r i a t i o n i n serum glucose was l e s s than 2%. S t a t i s t i c a l s i g n i f i c a n c e was measured using the Student t t e s t . SERUM ANALYSIS (A) Radioimmunoassays (I) GIP radioimmunoassay A radioimmunoassay f o r GIP was developed by Kuzio et a l (1974) but has undergone repeated m o d i f i c a t i o n s i n c e then i n an attempt to improve the -84-specific activity and s t a b i l i t y of the labelled tracer, the af f i n i t y of the antisera and the efficiency and reproducibility of the separation technique. Ct) Iodination of GIP Ca) Routine chloramine-T iodiriatibri and purification 125 Originally, when 6 yg GIP was reacted with 2 mCi T-Na, the best tracer did not share complete identity with the peak of radioactivity but was as-sociated with the descending limb of the radiochromatogram and therefore with reduced counts. If the ratio of polypeptide : iodine was increased to 6:1, the peak immunoreactivity coincided more closely with the radioactive peak, with no loss in specific activity. The reagents were added in the following order in a siliconized 10 x 75 mm glass tube, mixing being ensured by bubbling air through the reaction mixture:-6 j(g GIP in 100 y l 0.4M sodium phosphate, pH 7.5 125 1 -mCi 1-Na in 10 "yl carrier-free NaOH 40 yg chloramine-T in 10 y l 0.4M sodium phosphate, pH 7.5 15 sec. exposure 252 ^lg sodium metabisulphite in 20 y l 0.4M sodium phosphate, pH 7.5 125 Separation of the free 1 from the labelled peptide was routinely achieved by transferring the reaction mixture to a column of Sephadex G25 fine, CO.9 x 28 cm) and eluting the radioactive material with 0.2M acetic acid, containing 2000 Kill Trasylol/100 ml and 0.5% B.S.A. Approximately 40 x 400 y l fractions were collected and 10 y l aliquots were counted for 0.1 min. to produce he radiochromatogram in Fig. 21. A charcoal-bi ding assay was . 21 Chloramine-T iodination of GIP at a peptide:iodine ratio of 6 ug: 2 mCi. Separation of labelled GIP from free iodide on Sephadex G25 in 0.2M acetic acid. Counts per 0.1 min (•—•) : NSB (6—o). LEAF 86 OMITTED IN PAGE NUMBERING. -87-performed by adding 200 yl ..of the routine dextran-coated charcoal suspension 3 to tubes containing 5 x 10 cpm/100 yl in a total volume of 1 ml. The fractions displaying the lowest maximum binding to charcoal were pooled, diluted with column eluant buffer to a concentration of ~ 1.6 x 10 cpm/2 ml and stored at -20°C in siliconized glass tubes. The average specific activity of labelled GIP purified in this manner was 50-90 mCi/mg and i t s s h e l f - l i f e was 3-4 weeks. (b) Variations of chloramine-T iodination It had been shown that decreased concentrations of chloramine-T for the same time exposure only served to decrease the degree of incorporation of iodine into the peptide (Kuzio, 1973). The effect of decreased concentrations of chloramine-T on the degree of GIP iodination, when the exposure time was 125 increased, was measured by reacting 2 yg porcine GIP with 1 mCi I-Na in the presence of 4 yg chloramine-T. After 30, 60, and 120 sec, 20 y l aliquots were removed from the reaction mixture and added to 25 yg sodium metabisulphite in 20 y l phosphate buffer, along with 100 y l of the column eluant buffer. Each aliquot was purified in the routine manner on Sephadex G25 fine and the specific activity of each calculated approximately from the % incorporation of the iodine into the peptide, estimated from the approp-riate radiochromatogram. After 30 sec exposure the specific activity was 45 mCi/mg, after 60 sec i t was 112 mCi/mg and after 120 sec i t was 350 mCi/mg. Cc) Lactoperdxidase method of iodination As lactoperoxidase provided a gentler method of oxidation of iodide to -88-10 2 0 3 0 4 0 Fraction # - IO/JI Fig. 2'2 Lactoperoxidase iodination of GIF at a peptide:iodine ratio of 6 3ig;l mCi. Separation of labelled GIF from free iodide on Sephadex G25 in 0.2M acetic acid. Counts per 0.1 min (x-x); NSB Co-o) -89-iodine, i t was expected to be preferable as the oxidizing agent in the iodin-ation of GIP. The procedure was performed exactly as was outlined for motilin. The radiochromatogram, in Fig. 22, was used to calculate the specific activity from the % incorporation of iodine into the polypeptide. The specific activity, calculated this way. was 62 mCi/mg and therefore this method showed no improve-ment over the chloramine-T mediated oxidation. (2) Purification of GIP In an attempt to separate labelled from unlabelled GIP the fractions selected after gel f i l t r a t i o n were pooled, lyophilized and subjected to ion exchange chromatography. The label was reconstituted in 2 Ml 0.06M Tris buffer, pH corrected to 8.5 with 6.0M HCI, and applied to a column of QAE Sephadex A25, (0.6 x 15 cm), well-equilibrated with the Tris/HCl buffer, containing 1% Trasylol and 0.5% B;S.A. The column was developed with the same buffer and 1 ml fractions were collected at a flowrate of 20 ml/hour. The radiochroma-togram obtained by counting these fractions in an automatic <y counter i s 125 shown in Fig. 23. The I-GIP peak was located by determining the region of lowest charcoal-binding, as previously described. The appropriate fractions were pooled and diluted 1:4 in acid-ethanol, (15 ml ethanol : 5 ml d i s t i l l e d water : 0.3 ml concentrated HCI). The specific activity was determined by assaying ser i a l dilutions of the labelled GIP. (3) Extension of the s h e l f - l i f e of 1-GIP The charcoal binding (NSB)ofthe routine label preparation was 5 - 9 % B. Internal decay during the storage of this label raised this value to 20 % B -90-Fraction # Fig. 23 Chloramine-T iodination of GIF.at a peptide:iodine ratio of 5 jig: ImCi. Separation of labelled GIF from unlabelled GIF:On QAE Sephadex A25 in 0.06M Tris, pH8.5. Counts per 0.1 min (• • ) . Column calibrated with porcine GIP). -91-a f t e r 3 weeks at -20° and t h i s was unacceptable. ^ p u r i f i c a t i o n of the 125 l a b e l was attempted by two d i f f e r e n t methods. A 2 ml a l i q u o t of 1-GIP con t a i n i n g 2,1 x lO** cpm, one month o l d and w i t h a NSB of 24.2% B, was tre a t e d 125 w i t h 10 mg r e s i n CAG 1-XIO) to adsorb any f r e e 1 present. The mixture was vortexed w e l l , c e n t r i f u g e d at 3000 rpm f o r 10 min. and the supernatant kept. A second l a b e l a l i q u o t was t r e a t e d w i t h 10 mg m i c r o f i n e s i l i c a (QUSO) which absorbed the l a b e l l e d antigen. A f t e r mixing and c e n t r i f u g a t i o n , the supernatant was discarded and the p e l l e t washed w i t h d i s t i l l e d water. The l a b e l l e d peptide was eluted from the s i l i c a i n t o 2 mis 40% acetone: 1% a c e t i c a c i d : 60% d i s t i l l e d water (v/v) and the supernatant was kept. Both supernatants and an untreated v i a l of the same l a b e l were d i l u t e d w i t h d i l u e n t b u f f e r to the same concentration of cpm/100 u l and standard curves prepared w i t h each l a b e l . There was no s i g n i f i c a n t d i f f e r e n c e i n the standard curves obtained at e i t h e r the LDD or midrange values but treatment w i t h AG 1-XIO s u b s t a n t i a l l y reduced the NSB of the l a b e l and could be used to prolong the s h e l f l i f e (Table X I , T i g . 24) TABLE XI 125 E f f e c t of treatment of I-GIP on NSB of that l a b e l Label treatment LDD Midrange NSB Pg GIP pg GIP . % B Untreated 25 160 24.2 AG I-XIO 25 200 14.1 quso 25 140 26.0 -92-Comparison of GIP standard curves obtained w i t h untreated l a b e l , l a b e l t r e a t e d w i t h Quso and l a b e l t r e a t e d w i t h ItG l--xlO r e s i n . -93-(4^ Production of a n t i s e r a to GIP x A n t i s e r a to GIP were r a i s e d i n r a b b i t s and guinea pigs by the methods p r e v i o u s l y d e t a i l e d f o r m o t i l i n . (a) Storage of a n t i s e r a A n t i s e r a a t t a i n i n g a usable t i t r e was s t o r e d , l y o p h i l i z e d i n 200 y l a l i q u o t s at -20°C. As r e q u i r e d , an a l i q u o t was r e c o n s t i t u t e d at a 1:10 d i l u t i o n i n d i l u e n t b u f f e r and kept frozen i n 50 - 200 y l amounts i n s i l i c o n i z e d 10 x 75 mm glass tubes at -20°C. Cb) C h a r a c t e r i z a t i o n of a n t i s e r a A l l a n t i s e r a to GIP were checked f o r cross r e a c t i v i t y w i t h other p o l y p e p t i d e s , i e s p e c i a l l y those of the s e c r e t i n f a m i l y , i . e . , v a s o a c t i v e i n t e s t i n a l peptide, glucagon and s e c r e t i n i t s e l f , w i t h which GIP s h a r e s . s t r u c t u r a l s i m i l a r i t i e s . The r o u t i n e assay antiserum, Van 8, d i d not cross react w i t h any of these, or w i t h c h o l e c y s t o k i n i n - pancreozymin, g a s t r i n , m o t i l i n or i n s u l i n at concen-t r a t i o n s up to 10 ng/100 y l . 05.) As say p r o t o c o l A l l d i l u t i o n s and volume c o r r e c t i o n s were made w i t h 0.04 M sodium phosphate, pH 6.5 c o n t a i n i n g 7500 KIU T r a s y l o l per 100 y l and 5% (w/v) ch a r c o a l - e x t r a c t e d plasma. The composition of the in c u b a t i o n volume was:--94-125 100 yL I rvSJP containing- 5000 cpm 100 pi GIP standard over the range 12.5-400 pg or 100 VI assay control or 50-200 -pi unknown 100 pi antiserum at the appropriate i n i t i a l dilution Diluent buffer to a f i n a l volume of 1.0 ml. Assays were set up in a cold tray at 4°C, in t r i p l i c a t e , in siliconized 10 x 75 mm glass tubes. Incubation was of the equilibrium type, at 4°C, for 48-72 hours. The incubation volume has recently been altered to 300 p i , with the resultant decrease in the antiserum volume required. After the normal incubation period, the volume was corrected to 1.0 ml with diluent buffer prior to the separation of bound and free antigen. In both cases, NSB tubes and total count tubes were included and the analysis of data was performed as described for motilin. The standardization of the incubation milieu was examined by preparing standard curves with and without the addition of 100 yl charcoal-extracted plasma, to compensate for the protein added in the remainder, of the assay when serum samples were being-monitored. There was no significant difference in the -95-curves obtained (Fig.25) and the addition of plasma was deemed unnecessary. (6) Preparation of standards Working stock standards were prepared by dissolving 1 ug GIP in 0.04M sodium phosphate buffer, pH 6.5, containing 7500 KIU Trasylol per 100 ml and 0.5% BSA (w/v) to a f i n a l concentration of 8 ng/ml. These standards were stored in 1.5 ml polypropylene mictrotest tubes at -20°C and were used in the assay after being diluted 1:1 in diluent buffer, i.e to 400 pg/100 u l . The other standards were prepared by serial dilution. Working stock standards were discarded after being thawed. (7) Preparation of controls A r t i f i c i a l control sera were prepared by dissolving porcine GIP in 0.04M sodium phosphate buffer, pH 6.5, containing 2000 KIU Trasylol per 100 ml and 5% (v/v) charcoal-extracted plasma, to a concentration of 200 pg/100 ul. The controls were stored in 1.5 ml polypropylene microtest tubes at -20°C and assayed at the beginning and end of every assay, to provide an estimate of intra- and inter-assay v a r i a b i l i t y . In 50 arbit r a r i l y chosen assays, the i n i t i a l control value was 254 * 43 pg/100 u l (mean ^  SD) and the f i n a l control was 258 i 50 pg/100 u l . Any assay in which the controls varied more than 1 SD from these values was suspect and was discarded. (8) Separation techniques 125 Separation of the free I-GIP from the labelled peptide bound to the anti-serum was routinely performed using dextran-coated charcoal, exactly as - 9 6 -CD 100-® 8 0 8 6 0 H 2 0 H 0 -X o Normal Standard Curve Standard Curve after Addi t ion of 100 M C - E Plasma 12.5 2 5 pg G I P 5 0 T 1 100 2 0 0 4 0 0 Fig. 25 Effect of the addition of charcoaL--extracted plasma on the sensitivity of the.routine.standard curve for GIP. -97-described for motilin. An alternative method involyes the precipitation of the antibody/antigen complex with the alcohol, dioxane. When standard curves were incubated under routine conditions and then separated by the addition of either 200 ul dextran-coated charcoal suspension or 1 ml dioxane, there was no significant difference in curves obtained (Fig. 26), but at this time the dioxane method has proved less reliable. IT. Insulin radioimmunoassay Cl) By commercially available k i t Measurement of immunoreactive insulin (TRI) was performed with the Amersham Searle Insulin Immunoassay Kit (IM-39) - developed from the method of Hales 125 and Randle (1973). Insulin tracer, labelled with - I at a minimum specific activity of 50 mCi/mg reacted with insulin antibody, provided as a dessicate already bound to a second antibody. The standards were human insulin, over the range 2.5 - 160 UTJ/ml. A l l dilutions were made in isotonic sodium phosphate buffer at pH 7.4, containing 0.5% BSA and trace thiomersal as a preservative. The assay was of the disequilibrium type with 6 hour incubation of antibody and unlabelled antigen, followed by label addition and incubation for a further 18 hours. After centrifugation,. the supernatant was discarded and the pellet consisting of the Bound antibody/antigen complex was counted in an automatic gamma counter. Results were expressed as a percentage of the i n i t i a l l y added counts (%B). (2) By non-commercial radioimmunoassay A laboratory radioimmunoassay was developed from the method of Dr. K.D. Buchanan (personal communication). -98-Fig. 26 Effect of separation of.bound from free antigen by dextran-coated charcoal or dioxane on the sensitivity of the routine standard curve for GIP. -99-(a) Iodiriatibri of insulin-The following reagents were prepared just prior to use and mixed together in a siliconized glass 12 x 75 mm tube in the order given:-1 2 5 -I 1 mCi in 10 y l InsulinCporcine) 5 -jag in 20 jil 0.2M sodium phosphate pH 7.4 Chloramine-T 100 yg in 20 yil 0.2M sodium phosphate pH 7.4 10 sec exposure Sodium metabisulphite 240 yg in 100 -yl 0.2M sodium phosphate pH 7.4 45 sec exposure Sodium iodide 1% in 50 ^yl 0.2M sodium phosphate pH 7.4 125 The Iv- insulin was purified by adsorption onto 10 mg microfine s i l i c a (QUSO) in a total volume of 2.0 mis sodium phosphate buffer. After vortexing and centrifugation, the free iodide was discarded with the supernatant. The s i l i c a complex was washed with 3.0 mis d i s t i l l e d water and the labelled insulin was eluted with 5.0 mis acid-ethanol (15 ml ethanol; 5.0 ml d i s t i l l e d water; 0.3 ml concentrated hydrochloric acid) and stored at -20°C. The specific activity of the label was calculated from the percentage iodine incorporated into the insulin, by counting aliquots of the i n i t i a l supernatant, the water wash, the f i n a l labelled product and the s i l i c a pellet, and correcting 125 for the volume, i.e., a typical incorporation of 75% -I into the polypeptide represented an approximate specific activity of 150 mCi/mg. -100-(b) The insulin antibody The antiserum, obtained from a guinea pig, was kindly donated by Dr. K.D. Buchanan. C c ) The assay protocol Sodium phosphate buffer (0.04M, pH 7.5, 5% charcoal-extracted plasma) was used in a l l dilutions and for correcting the f i n a l volume to 1.0 ml. The composition of the incubation volume was:-100 y l standard human or porcine insulin, range 1.25-80 yU/ml or 50-200 y l unknown 100 y l antiserum at the appropriate i n i t i a l dilution Diluent buffer to a fi n a l volume of 0.9 ml | 24 hour incubation at 4°C 125 100 yi -I insulin, containing ~12,000 cpm 24 hour incubation at 4°C Non-specific binding and total count tubes were included (see motilin RIA). Separation of counts bound to the antibody/antigen complex was achieved by adsorption of the free and damaged antigen onto dextran-coated charcoal (5% w/v charcoal, 0.5% w/v dextran in 0.04M sodium phosphate buffer pH 7.5 -200 yl/tube) and centrifugation at 2800 rpm for 20 min. After the supernatant was discarded, the free cpm (the charcoal pellet) was counted in an automatic counter and the results expressed as % Bound. -101-B. Serum glucose Serum glucose determinations were performed in a Beckman glucose analyzer. A precise volume of sample 0.0 pi) was pipetted into a standard amount of well-aerated enzyme reagent solution, containing glucose oxidase, ethanol, molybdate and iodide. The D-glucose in the sample reacted with the oxygen in solution to produce gluconic acid and hydrogen peroxide. Glucose Oxidase Glucose + 0 Gluconic Acxd + H20 The presence of ethanol, iodide and molybdate in the reagent solution pre-vented the destruction of the peroxide by a pathway resulting in further oxygen production. The rate of oxygen consumption was directly proportional to the glucose concentration in the sample and the change of oxygen concentration in the solution was measured by an oxygen electrode and converted to produce a direct d i g i t a l read out in mg% glucose. - 1 0 2 -RESULTS ESTIMATION OF THE DEGREE OF CONJUGATION BETWEEN A POLYPEPTIDE AND BOVINE  SERUM ALBUMIN A. With motilin A motilin/BSA conjugate was prepared as previously described with one difference 125 i.e., the addition of an aliquot of 1-motilin, containing 9000 cpm, to the reaction mixture. The reaction was terminated by freezing the mixture. 125 A column of Sephadex G25 fine CO.9 x 100 cm) was calibrated with I-motilin in 0.2M acetic acid, 1.5 ml fractions being collected. The conjugate reaction products were eeparated by chromatography on this same column, under identical conditions. The protein content of each fraction was determined spectrop- ^ hotometrically at 280 nm in a 1 cm light path, and the radioactive content of each fraction was estimated by counting for 1 min in an automatic y counter. The column profiles obtained are illustrated in Fig. 27. The assumption was made that the labelled and unlabelled polypeptide would behave identically under the conditions of the conjugation procedure. Therefore, the percentage of the total reactivity eluting in the void volume of the column, with the major protein peak would give a measure of the percentage of poly-peptide conjugated to the BSA in this reaction. From Fig.27 i t was estimated that at least 60% of the motilin was conjugated. B, With GIP 125 The method was as described for motilin except that I-GIP 0-2,000 cpm) was added to the reaction mixture during the conjugation of GIP and BSA -103-Flg. 27 Column profile obtained after elution of Irmotilin: motilin/BSA conjugate from Sephadex C25 in 0.2M acetic acid. 125 The column had been previously calibrated with 1- motilin -104-by the carbodiimide method. The column profile obtained indicates that 100% of the GIP was conjugated to the BSA as illustrated in f i g . 28. REPRODUCIBILITY OF IR-'MOTILIN DETERMINATIONS Serum samples, containing motilin endogenously released by duodenal a l k a l i n i -zation, were obtained from 2 different studies in dogs. Sera from one experiment was immediately treated with the protease inhibitor, Trasylol, 100 Jul per ml of serum. Both groups of sera were assayed for IR- motilin content, stored at -20°C and re-assayed 3 months later. Table X l l shows the results, i n pg/ml IR'- motilin, obtained in the two assays, after correction for the dilution by Trasylol in the appropriate experiment. In Fig. 29, the assay results have been plotted against each other. Protection against enzymatic degradation did not appear necessary for motilin. On re-assay only 1 sample deviated more than 25% from the line of identity. COMPARISON OF RIA AND BIOASSAY OF "MOULIN The motilin content of the commercially prepared duodenal extract "Pancreo--zymin" (PZN) estimated by RIA under routine conditions and by bioassay in the chronic dog preparation. A. •Immunological comparison serial dilutions of Boots "Pancreozymin" (PZN), ranging from 0.25 - 2.0 yg/ -105-Fraction # Tig. 28 Column profile obtained after elution of I-GIP:CIP/BSA conjugate from Sephadex C25 in 0.2M acetic acid.. The column 125 had been previously calibrated with I-GIP. TABLE XII pg/ml IR-motilin, demonstrating reproducibility of motilin determinations after storage for three months, with or without added Trasylol Sample Time Sample : With Trasylol Without Trasylol 24/6/74 9/10/74 24/6/74 9/10/74 -30 min 1 530 580 680 670 -15 min 2 560 520 660 670 0 min 3 410 490 700 690 2 min 4 870 760 850 940 5 min 5 700 640 1100 1200 7 min 6 960 940 1100 1120 10 min 7 900 890 960 830 15 min 8 760 690 830 840 20 min 9 870 820. 920 910 25 min 10 940 1100 840 730 30 min 11 830 940 840 720 45 min 12 720* 980* 600 590 60 min 13 520 600 550 580 Deviates more than 25% from the line of indentity -107-i I • Serum Samples, 10% Trasylol j o Serum Samples,No Trasylol 4 0 0 800 1200 Motilin Concentration (pg./ml.)in Assay 2 Fig. 29 Reproducibility of motilin determinations on serum samples stored between assays,at -20°C, with (•) or without (o) Trasylol. Only 1 sample deviated more than '25% from the line of identity on reassay. (Dryburgh and Brown. Gastro-enterology 68: 1169-1175, 1975). -108-100 y l were incubated with motilin antiserum in a routine RIA. The resul-125 tant displacement of I - motilin obtained with 1 yg/100 y l PZN was fitted to the standard curve, the other dilutions being plotted accordingly. The immunoreactive material in the duodenal extract shared 100% immunological identity with the pure polypeptide and the motilin content in 4 separate estimates was 140 - 40 pg/ yg Boots PZN (Fig. 30). B. Biological comparison In 2 experiments in each of 3 dogs, matched responses of increased fundic pouch motor activity were produced by bolus, intravenous injections of natural porcine motilin or Boots PZN given at least 40 mins. apart. The order of the injections was randomized. Fig. 31 and Table XIII il l u s t r a t e the increase in motor activity, expressed as motility indices, in the 10 min. period immediately following the injection compared to the 10 min. period immediately prior to i t , for 2 different doses of pure polypeptide and impure extract. Equivalent responses were observed after 1 and 2 yg pure motilin or 10 and 20 mg PZN (Table XIII, Fig. 311). Therefore, by both immunological or biological estimations, the pure polypeptide represents a 10,000 fold purification over the impure extract. \ -109-o.o 25 Motilin (pg) 50 100 500 250 500 Boots 1000 2000 PzN (ng) Fig. 30 Comparative immungireactiyities of porcine motilin and a crude duodenal extract (BootsPZN), the 1 ug dilution of the PZN being fi t t e d to the standard curve and the other points fit t e d accordingly. (Dryburgh and Brown. Gastroenterology 68: 1169-1175, 1975). -110-TABLE XIII Comparison in the increase in Motility Indices after single intravenous - j -injections of motilin or Boots PZN A. 1 pg motilin / 10 mg Boots PZN Dog Experiment •k Motility Index Control Post motilin Post PZN P 2/5/74 15.6 71.4 76.1 S 9/5/74 24.7 90.8 69.3 A 14/5/74 19.8 75.7 85.2 Mean - SE i 20.0 - 2.6 79.3 t 5.8 76.8 - 4.6 B. 2 jag motilin / 20 mg Boots PZN Dog Experiment •k Motility Index Control Post motilin Post PZ.N P 9/5/74 11.0 169.0 180.8 S 14/5/74 18.3 195.1. 184.7 A 2/5/74 16.2 173.7 175.4 Mean - SE 15.2 - 2.2 179.3 - 8.0 176.9 - 5.9 * represents a 10 min period + PZN - Boots 'Pancreozymin' -111-2 0 0 n I I Control ^ 3 Natural Porcine Motilin E Z 2 Boots P z n I60H S '20H c "o 80-^ 4 0 J 0-1 rf i i B. I I Comparison of biological activity (expregised as motility indices) of pure motilin and a crude duodenal.extract '. (Boots PZN) in the chronic dog bioassay, at 2 doses, (a) 1 ug/kg porcine motilin and 10 mg/kg PZN and (b) 2 ug/kg pure motilin and 20 mg/kg PZN. + Each group of results represents the mean - SE of 4 experiments in 2 dogs. -112-MOLECULAR HOMOGENEITY OF .MOTILIN A. • In ' serum To determine whether motilin existed in more than one detectable form sera containing endogenous-motilin was subjected to gel f i l t r a t i o n as follows:-2 mis serum containing 900 pg/ml IR - motilin was applied to a colum of Sephadex G50 fine CO.9 100 cms), the column was developed with 0.2M acetic acid, and the eluant was collected in 1.0 ml fractions at a flow-rate of 10 mis/hour. Samples were stored at -20°C u n t i l 100 yil aliquots could be assayed. The column profile C'Fig. 32) indicates that only one detectable form of motilin existed in this serum which was taken at the peak of a canine response to duodenal alkalinization. B. In tissue extract Presekretin, the starting material in the purification of motilin, was estima-ted, by RIA, to contain 13.5 ng/ jig IR - motilin. Three yg of this material in 2.0 mis 0.2M acetic acid was eluted from Sephadex G50 fine under the same conditions as in CA). The column profile in Fig. 33 was obtained after monitoring each fraction at 1:1000 dilution for IR - motilin content. 125 The chromatography system was calibrated routinely with 1- albumin and 125 1- motilin, 50,000 cpm of each in 0.2M acetic acid. -113-Fig. ;3_2' • . Column profile obtained after chromatography of 2 ml serum containing 900 pg/ml IR- motilin on Sephadex G-50 (0.9 x 100 cm) 125 in 0.2M acetic acid. The column was calibrated with I-t < 1 . . 125 _ ' . •motilin and Iodine -114-125 I-Albumin I25T . . . . 125 T I-Motilin I 8 T 6H E \ O) c •- 4-i CH 2H - | — 10 I 20 I 30 I 40 I 50 - r ~ 60 I 80 I 90 -1 100 70 ml Column eluant Fig..33 Column profile obtained after chromatography of 3 ug Presekretin in 2 ml elutlng buffer on Sephadex G-50 in 0.2M acetic acid. The X25 125 125 column was calibrated with I- albumin, I-m o t i l i n and Iodine. -115-DISTRIBUTION Of IR* W H I N THROUGHOUT THE HOG GASTROINTESTINAL TRACT The tissue was collected fresh and divided into appropriate sections. It was boiled Briefly, cleared of connective tissue, frozen, coarsely minced and extracted into acid-ethanol. Composition of acid^ethanol:-3.75 l i t r e s 95% ethanol 75 ml glacial acetic acid 1.25 l i t r e s d i s t i l l e d water The f i l t r a t e was cleared By successive passage through several layers of cheese cloth and nylon gauze, and the pH lowered to 2.5 by addition of 2.0M hydrochloric acid. Adsorption onto alginic acid was allowed to pro-ceed for 1 hour at 4°C and the alginic sediment was then removed by gentle f i l t r a t i o n under vacuum and washed well with 0.2M acetic acid. The com-bined washings were cooled to 4°C and the protein was precipitated by saturation with sodium chloride (35 g / l i t r e ) , redissolved in d i s t i l l e d water and lyophilized. After lyophilization, each extract was desalted and partially purified by chromatography on Sephadex G25 coarse in 0.2M acetic acid. Fig. 34 shows a typical column profile, obtained in the purification of a duodenal extract. The appropriate fractions were pooled as indicated, lyophilized and monitored for IR-motilin content at several dilutions. The results are presented in Table XIV. -116-TABLE XIV Distribution of IR-motilin throughout the hog gastrointestinal tract Region IR-motilin in pg/ug Ratio dry weight extract Duodenum (Fr 1) 94 7 Jejunum (Fr 1) 1300 100 Ileum (Fr 1) 0.6 0.04 Fundus *ND 0 Antrum *ND 0 Oesophagus *ND 0 * ND - non-detectable -117-i Fig. 34 10 20 30 40 50 60 Fraction # Desalting of a crude extract of hog duodenal mucosa on Sephadex G-25 coarse (2,5 x 100 cm) in 0.2M acetic acid. Absorbance at 280 nm in a 1 cm light path-and-conductivity - - - . . . . in millimho was determined for each fraction. Fractions 28-38 were pooled and lyophilized and subsequently referred to as Fr I. i -118-The jejunal extract content was. assigned an arbitrary value of 100 and in descending order anatomically, the following ratios were obtained:-Oesophagus 0 'Fundus 0 Antrum 0 Duodenum 7 Jej unum 100 Ileum 0.04 RELATIONSHIP BETWEEN GASTRIC CTTJNDIC) EMPTOR ACTIVITY AND ENDOGENOUS MOTILIN  RELEASE Six dogs prepared for the chronic bioassay study were used in this study. Blood samples were obtained whilst the dogs exhibited low spontaneous activity in the fundic pouch, during infusion of 50 mis 0.3M Tris buffer, pH 9.5, and whilst the motor activity was returning to Cor below) basal levels. In 3 experiments, 50 mis 0.1M hydrochloric acid was substituted for the alk a l i and control experiments were performed in 4 dogs, during duodenal infusion with 50 mis 0.9% saline.Blood sampling and gastric motor activity recording were performed as in the al k a l i experiments. On 2 different occasions dogs exhibited a spontaneous and significant i n -crease in fundic motor activity. The experiment was continued and blood samples taken to monitor any changes in IR- motilin levels. J . The fasting serum level of IR- motilin in the dogs was 412 - 44 pg per ml + Cmean - SE) and the duodenal pH was 7.5. Passage of the infusion into the duodenum was complete in 3 mins. After a l k a l i , the circulating IR--119-motilin had increased to 498 <r 100 pg per ml within 2 -mins, the duodenal pH having reached 8.2. At the end of 5 mins the IR- motilin levels were at their peak value of 916 i 96 pg/ml. The gastric motor activity also reached i t s maximum within the f i r s t 5 min post-infusion and the duodenal pH had returned to i t s pre-infusion level (Fig 35 and Tables XV, XVI). There was no increase in either IR- motilin or fundic pouch motor activity when the duodenum was perfusedn with saline, CFig 36 and Tables XVII, XVIII). In the 2 instances, when the fundic pouch motor activity increased spon-taneously, a concomitant and spontaneous elevation in IR- motilin was also noted, CFig 37 and Tables XIX, XX). The results obtained after duodenal perfusion with 0.1M hydrochloric acid were equivocal. The increase in circulating IR- motilin was not as great, was slower in reaching i t s peak value and was also slower in returning to the pre-infusion levels. There was no associated increase in fundic motor activity. The comparison between the incremental IR-motilin responses to acid, a l k a l i and saline is summarized in Fig 38 and Table XXI. EFFECT OF INGESTION OF GLUCOSE OR A MIXED MEAL ON THE CIRCULATING LEVELS  OF IR- MOTILIN These studies were performed in 6 fasted, human volunteers (aged 20 -36 years) with no history of gastrointestinal disorder. On the f i r s t occasion the subjects were given, orally, lg/kg glucose in a 20% solution. Blood samples were taken during the control period and at 5, 10, 15, 30, er, 60, 75 and 90 min after the ingestion of the glucose. On a second occasion, these same subjects were given a normal meal, consisting of TABLE XV Fundic motor activity response to duodenal infusion with a l k a l i . DOG CONTROL Minutes after onset of duodenal infusion I.D. -20 -15 -15 -10 -10 -5 -5 -0 0 5 5 10 10 15 15 20 20 25 25 30 30 35 35 40 40 45 45 50 50 55 55 60 P l 31.4 0 10.7 4.8 94.9 41.2 0 13.2 0 8.1 P2 69.5 73.0 42.2 44.0 97.2 23.3 34.8 23.2 45.8 35.0 16.0 17.2 26.8 R l 19.0 55.3 6.4 7.6 124.8 154.0 66.2 56.3 24.6 30.4 7.8 11.0 5.0 9.8 4.8 0 S l 14.0 35.7 36.1 42.7 127.8 65.0 40.6 27.1 67.6 46.6 41.1 38.4 35.0 0 16.0 14.6 S2 17.1 51.4 3.3 0 226.6 107.5 65.5 55.1 33.2 72.6 53.7 54.0 28.4 33.2 R2 64.7 62.5 46.3 45.4 179.8 84.2 48.6 30.6 42.8 33.4 18.8 35.3 8.4 0 0 5.1 X 35.9 46.3 24.1 24.0 141.8 79.2 42.6 34.2 35.6 37.6 27.4 31.2 20.7 10.7 6.9 6.5 SE 10.1 10.5 7.9 8.9 21.0 19.3 9.9 7.1 9.2 8.6 8.5 7.7 5.9 7.8 4.7 4.2 TABLE XVI IR-motilin (pg/ml) response to duodenal infusion with a l k a l i . DOG I.D. CONTROL Minutes after start of duodenal infusion -20 -10 0 2 4 6 8 10 15 30 45 60 P l 450 550 200 200 800 600 500 400 300 P2 450 450 600 1800 1300 800 550 400 350 R l 300 280 280 280 450 960 540 560 620 380 300 280 S l 310 270 460 500 650 1100 600 550 520 700 S2 377 311 277 622 544 477 577 440 300 280 R2 890 570 560 970 1100 940 980 940 740 560 430 400 X 412 405 396 728 807 855 690 655 430 426 445 315 SE 44 55 67 241 135 106 145 71 101 39 89 28 -122-50 ml 0.3 M TRIS r900 Time in Minutes i I i i ! I Fig . 35 Fundic pouch motor act iv i ty , expressed as an index pf m o t i l i t y and circulating levels of IR- moti l in in pg/ml after intraduodenal infusion :of 0.3M t r i s buffer. Each mot i l i ty index represents the mean (- SE) for a.5 min period, whilst the serum levels of motil in (mean. - . SE) are measured at.a specific time. (Dryburgh and Brown (1975), Gastroenterology _68: 1169-1176) n= 6 TABLE XVII Fundic motor activity response to duodenal infusion with saline. DOG CONTROL Minutes duodenal after start infusion of of saline I.D. 20 -15 -10 -5 0 5 10 15 20 25 15 10 5 0 5 10 15 20 25 30 R3 54.0 28.4 33.2 58. 3 56.7 47.0 27.5 26.0 14.8 12.8 P3 23.6 28.4 14.6 11. 2 4.7 4.7 0 0 11.6 7.8 R4 62.1 54.7 30.6 0 0 0 0 11.4 6.5 11.7 P4 20.4 20.9 11.3 12. 6 7.8 13.4 14.8 9.6 18.4 20.6 X 40.2 33.1 22.4 20. 5 17.3 16.3 10.5 11.7 12.8 13.2 SE 10.5 7.4 5.5 12. 9 13.2 10.6 6.6 5.3 2.5 2.6 TABLE XVIII IR-motilin (pg/ml) response to duodenal infusion with saline. DOG I.D. CONTROL Minutes after start of duodenal infusion -20 -10 0 2 4 6 8 10 15 30 45 60 R3 320 470 510 410 390 240 350 360 400 280 280 280 P3 450 390 380 125 310 350 125 300 280 280 350 280 R4 380 280 240 280 320 310 260 200 125 200 280 300 P4 410 440 450 380 350 380 270 310 300 400 350 300 X 390 395 395 298 342 320 251 292 276 290 315 290 SE 27 41 58 64 17 30 46 33 56 41 20 5 -125-36 Fundic pouch motor activity, expressed as an index of; motility, and circulating levels of IR-motilin in pg/ml after intraduodenal infusion of 0.15M' saline; Each.motility index represents the mean (- SE) for a 5 min period, whilst the serum levels of motilin are measured at a specific time, as mean - SE (n = 4) TABLE XIX Spontaneously induced fundic motor activity. DOG CONTROL Spontaneously induced fundic motor activity I.D. . -20 -15 -10 -5 0 5 10 15 20 25 -15 -10 5 0 5 10 15 20 25 30 R5 82.2 63.1 143.8 127.7 25.7 11.6 0 0 P7 38.4 14.2 67.8 65.8 16.0 14.6 39.2 40.6 X 60.3 38.6 105.8 96.7 20.8 8.1 19.6 20.3 SE 21.9 24.4 38.0 30.9 4.8 6.5 19.6 20.3 TABLE XX IR-motilin (pg/ml) response to spontaneously increased motor activity. DOG I.D. CONTROL Time after onset of spontaneous activity -20 -10 0 2 4 6 8 10 15 30 45 R5 460 520 800 720 1000 430 400 400 270 350 300 P7 390 520 700 1100 2600 640 520 680 500 300 300 X 425 520 750 910 1800 535 460 540 385 325 300 SE 35 0 50 190 800 105 60 140 115 25 0 -128-120 100 x 80 CD TD rl800 1600 hl400 1200 E \ O) a. > 60+ 600 1000 800 40+ 400 20+ 200 0-L 0 I 1 1 1 1 1 1 T 1 1 r 0 10 20 30 40 50 60 Time in Minutes •37 Fundic pouch motor ac.tlv.ityi-expressed as an index of motility, and circulating levels of IRr- motilin in pg/ml, during a spontaneous burst of fundic motor activity. Each motility index-represents the mean - SE for a 5 min period, whilst the serum levels of motilin (mean - SE) are measured at a specific time, (n = 2) TABLE XXI Effect of duodenal infusion of a l k a l i , acid or saline on the incremental IR-motilin (AlR-motilin) response. Time in min. after duodenal infusion of a l k a l i • CONTROL 2 5 7 10 15 30 45 60 400 -200 400 - 200 100 0 - -100 500 1300 800 300 - -100 - -150 50 280 0 170 680 280 340 100 20 0 350 150 300 750 250 200 170 350 -50 320 302 224 157 250 120 -20 20 -40 570 400 530 33- 370 170 -10 -130 -170 Mean 325 404 451 270 138 48 22 -77 ±SE 214 95 115 28 59 37 89 31 TABLE XXI (Cont.) Time in min. after duodenal infusion of acid. CONTROL 2 5 7 10 15 30 45 60 165 155 355 395 365 285 185 35 165 160 100 200 270 180 200 5 130 70 330 40 -80 220 0 80 160 30 20 266 34 144 164 234 159 69 -86 -26 175 105 25 165 115 105 105 25 115 Mean 86 129 243 179 166 105 28 69 ±SE 22 74 43 60 36 32 34 34 Time in min. after duodenal infusion of saline • CONTROL 2 5 7 10 15 30 45 60 430 -20 -40 -190 -70 -30 -150 -150 -150 400 -275 -90 -40 -100 -120 -120 -50 -120 300 -20 20 10 -100 -175 -100 -20 0 430 -50 -80 -50 -120 -130 -30 -80 -130 Mean -91 -48 -67 -97 -113 -100 -75 -100 ±SE 61 24 42 10 30 25 27 33 -131-• S a l me 5 0 0 - i 4 0 0 -3 0 0 -1 2 0 0 -a + 1 0 0 -0 • I 0 0 - | 2 0 0 rnrT Alkali ) Duodenal Infusion Acid 1 1 1 1 1 1 1 r~ 2 5 7 10 15 3 0 4 5 6 0 Time in Minutes from Start of Duodenal Infusion Fig . 38 A comparison of the incremental IR- motilin responses C IR- motil in in pg/ml to Intraduodenal infusion of a l k a l i , acid or saline, each point representing the mean - SE for 5 experiments of each type. -132-orange juice, bacon and eggs with fried potatoes, toast with conserves and coffee. Blood samples were taken before breaking the fast and at 15 min. intervals thereafter for 2 hours. No increase in systemic serum IR- motilin levels was observed.' A significant (P >0.025) decrease in IR- motilin (from the control values) was noted at 30 and 45 min. after ingestion of either glucose or the meal. The IR- motilin levels then tended to return to the control values (Tables XXII and XXIII; f i g . 39). COMPARISON OF THE IMMUNOLOGICAL AND BIOLOGICAL ACTIVITIES OF SYNTHETIC AND  NATURAL MOTILIN FRAGMENTS AND ANALOGUES A. Immunological comparison. (I) Synthetic motilin and fragments. The i n i t i a l purification products, 1foB^ and M0B2, were found to contain 50% and 10% of the immunological activity of natural motilin respectively. The polypeptide MoC^ shared complete immunological identity with the natural polypeptide whereas M0C2, although chemically identical, was immunologically inactive, and may represent an isomer of the active form. Synthetic fragments, motilin 9-22 and 13-22 passed no immunological activity. The results are summarized in f i g . 40. (II) Fragments of natural motilin  (a) Cyanogen bromide cleavage The immunological cross-reactivity was -measured on the mixture of products TABLE XXII Effect of ingestion of oral glucose on serum levels of IR-motilin in pg/ml CONTROL Post glucose ingestion -30 -15 0 5 10 15 30 45 60 75 90 RAP 280 280 260 260 230 240 235 190 160 200 160 JRD 200 210 180 200 190 170 180 180 200 210 200 JCB 300 310 260 240 255 260 200 170 125 100 125 KM 190 200 160 140 125 100 100 100 100 140 160 HS 160 100 140 125 125 160 125 100 140 135 140 TM 200 200 230 215 190 160 170 135 140 200 210 Mean 221 216 205 196 185 181 168 145 144 164 165 ±SE 23 30 21 17 22 24 20 16 14 18 13 TABLE XXIII Effect of ingestion of a normal mixed meal on serum levels of IR-motilin in pg/ml. CONTROL Post meal ingestion -30 -15 0 15 30 45 60 75 90 105 120 HS 230 280 200 300 190 160 200 220 200 250 225 KM 160 280 210 210 200 100 200 210 240 190 200 JRD 140 260 180 260 160 140 230 205 235 255 260 RAP 100 160 200 240 140 125 215 190 210 235 190 JCB 330 230 310 230 225 210 200 180 190 160 210 TM 280 235 240 180 100 125 170 160 140 200 205 Mean 206 240 223 236 169 143 202 194 202 215 215 ±SE 36 18 19 1 1 7 18 15 8 9 15 15 10 -135-Fig. 39 The serum IR- motilin levels (mean - SE) in pg/ml measured after ingestion df either 1 g/kg 20% dextrose or a normal', .mixed meal in 6 normal subjects. Points marked * indicate: a significance of >0.05 by the Mann-Whitney U test. J -136-Fig. 40 Standard curve to motilin showing comparative immunoreactivities with synthetic 13-norleu-motilins and and synthetic fragments 9-22 and 13-22. Synthetic motilin showed 100% crossreactivity with natural porcine -motilin. (Dryburgh and Brown (1975) Gastroenterology 68: 1169-1176) -137-produced bycyanogen bromide cleavage of motilin. The degree of cross-reactivity observed was approximately 30% of that seen with the intact molecule (Jig. 41). This can be explained by the presence in the mixture of uncleaved material, the cleavage being incomplete due to the presence of a small percentage of methionine sulphoxide residues which are resistant to the action of cyanogen bromide (Gross, 1967). (b) Tryptjc and chymotryptic digestion After enzymatic degradation of the molecule by trypsin and chymotrypsin, acting specifically at the carboxyl terminals of the basic and aromatic amino acide respectively, tryptic peptides showed no cross-reactivity with motilin antiserum (Fig* 41). ( I l l ) Modifications of natural motilin  (a) Removal of the C-terminal residue Cs) A 6 hour digestion of motilin with carboxypeptidase A resulted in release + of 80% of the C-terminal glutamine and 30% of the penultimate C-terminal glycine. The degree of immunological activity remaining, approximately 18% that of the intact molecule, may be accounted for by the remaining un-digested polypeptide. Cb) After removal of the N- terminal residue After one cycle of the Edman's degradation reaction and the removal of the N- terminal phenylalanine only 18% of the original immunological activity remained. This decrease in immunoreactivity may be due to the loss of the aromatic N- terminal. -138-I l I 12.5 25 50 100 200 400 1000 pg Motilin 'i Fig. 41 Standard curve to motilin showing comparative immunoreactivities with the products of tryptic:and chymotryptic digestion of motilin, or cleavage with cyanogen bromide. -139-(c) Acylation - acetylation Treatment of the molecule with acetic anhydride resulted in acetylation of the lysine residues, the acyl derivatives having no charge, and loss of 50% of the immunoreactivity. Cd) Acylation - succinylation Acyl derivatives obtained after treatment of motilin with succinic an-hydride bear unit negative charge and have only 50% of the immunoreactivity of the original molecule. B. Biological comparison Cl) Synthetic motilin Comparison of the biological activity of the synthetic and natural poly-peptides was achieved by matching the increase in fundic pouch motor activity in the chronic dog preparation produced by single intravenous injections of 1 fig or 2 ug of natural motilin with Injections of the test material. No significant difference in the biological activity of MoC^ 0-3- norlencine -motilin) or natural motilin could be detected CFig. 42 and Table XXIV). No biological activity was observed after injections of the motilin fragments 9-22 or 13-22, even in doses equivalent to 10 times that of the natural peptide, on an equimolar basis. Cll) Fragments of natural -motilin The mixture of peptides produced by tryptic digestion were biologically inactive In doses equimolar with 10 -jig natural motilin. -140-TABLE XXIV Comparison of biological a c t i v i t i e s of natural and synthetic motilin Dog ID. Motility Index 2pg dose dose Natural Synthetic Natural Synthetic motilin motilin motilin motilin Polly 161.4 147.3 50.8 47.3 Abraham 178.2 168.7 59.1 76.5 X 169.8 158.0 59.1 61.9 ;'+SE 8.4 10.7 8.3 14.6 * represents a 10 min period -141-Dog A Dog B S 1601 o Q-o § 120 u. c > i o o 40 x c f in / jg | | Natural Motilin in / jg Synthetic Motilin f i g . 42.'. Comparison of biological activities of synthetic 12-norleu-motilin and natural porcine motilin, assayed by measuring the increase in fundic pouch motor a c t i v i t y a f t e r bolus injections of 1 or 2 yg of each peptide. ' + Each pair of results represents.the mean - SE of 4 experiments in 2 dogs. (Bryburgh and Brown (1975). Gastroenterology 68: 1169-1176). -142-CIII) Modifications of natural-motilin Ca) Removal of the C- terminal residue(s) There was no.loss in Biological activity- observed after the release of 80% of the glutamine residues and 30% of the glycine residues. Cb) Removal of the N- terminal residue Comparable increases in fundic pouclv motor activity were produced by l.ug intact motilin and 10 Jig motilin 2 - 22, i.e., removal of phenylalanine or the net loss of positive charges on the lysine residues reduced the activity by 90%. Cc) Acetylation The biological activity was reduced by 90% after the net loss of positive charge in the acyl derivative. Cd) Succinylation The biological activity of motilin was almost completely destroyed, i.e., to less than 5%, in the acyl derivative bearing negatively charged succinyl groups. The comparative immunological and biological a c t i v i t i e s o f the natural and synthetic peptides are summarized in Table XXV. -143-TABLE XXV Comparison of the immunological and biological a c t i v i t i e s of synthetic and natural motilin fragments and analogues Material % Immunological activity MoBj^  MoB? MoC1 MoC2 Mo t i l . in 9-22 Motilin 13 - 22 50 10 100 0 0 0 % Biological activity 90 - 100 Not tested 100 Not tested 0 0 CNBr. motilin Tryptic digest Chymotryptic digest 30 0 0 Not tested 0 0 After C-terminal removal After N-terminal removal Acetylated derivative Succinylated derivative 18 18 50 50 100 10 10 5 -144-ATTFINITT CHROMATOGRAPHY  A. Application to RIA  CI) Motilin Ca) Antiserum dilution and change in activity 125 Serial dilutions of the coupled ligand were incubated with 1- motilin in a total volume of 1.0 ml, for 48 hours at 4°C. A l l other variables were identical to those of the routine RIA. The tubes were centrifuged at 2800 rpm for 30 min., the supernatants discarded and the pellets counted. The antiserum dilution curve Ccoupled ligand) i s shown in Fig. 43 in comparison with an antiserum dilution curve (uncoupled ligand) obtained under identical conditions in a routine RIA. There was no significant loss in activity produced by coupling the antibody to a solid matrix. Cb) RIA standard curves and change in sensitivity 125 Motilin standards over the range 12.5 - 400 pg were incubated with 1-motilin and equivalent f i n a l dilutions of coupled or uncoupled antisera as follows:-CD routine RIA conditions, including uncoupled antiserum and usual charcoal separation; ( i i ) at 1.0 ml incubation volume, with coupled antiserum; Cii i ) at 0.3 ml incubation volume, with coupled antiserum; Civ) at 0.3 ml incubation volume, with coupled antiserum, in siliconized tubes. The standard curves obtained (Fig.44) were judged by the slope at zero dose Ccalculated as shown in Fig. 45 and Table XXVII) and L.D.D. The greatest sensitivity was established when coupled antiserum was incubated with radio-active tracer in a total volume of 0.3 ml, in non-siliconized tubes CTable XXVI). -145-70 n 60 4 50 A o CD 40H 30A 20 A Uncoupled Antiserum Sepha rose Ant iserum I O H o 100 Reciprocal of Ant iserum Titre IxlO 3 2 . 5 x l 0 3 5 x l 0 3 IOXIO3 2 0 x l 0 3 50 25 10 m g / m l coupled ligand 5 2 1.6 Fig. 43 Comparison of dilution curves obtained with antiserum to motilin, performed on aliquots taken before and after coupling of the anti-serum to CNBr-activated Sepharose 4B. -146-TABLE XXVI Coupled versus uncoupled antisera to motilin at varying incubation volumes Standard curve "Criteria for Evaluation Slope at Zero Dose Midrange L.D.D. a. 4.5xl0" 1 3l/mole 88 pg 25 pg b. 2.5xl0 - 1 3l/mole 320 pg 25 pg c. 7.5x10"13l/mole 120 pg 12.5 pg d. 5.5xl0~ 1 3l/niole 150 pg 25 pg -147-Flg. 44 Comparison of the standard curves obtained with antiserum to motilin under routine assay conditions or after .coupling to Sepharose 4B, at various incubation volumes,:in siliconized or.non-siliconized tubes. -148-TABLE XXVII Calculation of the slope at zero dose. CURVE STD CONC in moles BOUND B/F B x CONC SLOPE AT in pg (x 10" 1 5) (%) (x 10" 1 5) (L/mole) 0 1.3 0.315 0.46 0.42 a 12.5 5.9 0.309 0.40 1.8 25.0 10.5 0.267 0.36 2.6 50.0 19.8 0.223 0.28 4.4 4.5 x 10' -13 0 1.3 0.309 0.44 0.40 b 12.5 5.9 0.305 0.43 1.8 25.0 10.5 0.284 0.39 2.9 50.0 19.8 0.251 0.33 4.9 2.5 X 10 0 1.3 0.448 0.81 0.58 c 12.5 5.9 0.398 0.66 1.3 25.0 10.5 0.359 0.56 3.7 50.0 19.8 0.298 0.42 5.9 7.5 X 10' 0 1.3 0.428 0.74 0.55 d 12.5 5.9 0.402 0.67 2.3 25.0 10.5 0.367 0.57 3.8 50.0 19.8 0.323 0.44 6.4 5.5 X 10' -13 -13 -13 -149-A- Routine RIA .siliconized tubes B x Cone. ( Moles) x IO" 1 5 f i g . '45 Standard curves, from f i g . 44, presented as Scatchard.plots, demonstrating the.slope at zero dose.'. -150-II Gastric inhibitory polypeptide a. Antiserum dilution and change in activity Comparison of the antiserum dilution . 1 curves obtained with coupled and 125 uncoupled antisera to GIP after incubation with I- GIP under routine RIA conditions i s shown in Fig. 46. Coupling of GIP antiserum to a solid matrix resulted in a recovery of only 10% (approximately) of the anti-body activity. b. RIA standard curves and change in sensitivity Standard GIP in the range 12.5 - 400 pg was incubated with different f i n a l dilutions of coupled and uncoupled antisera, to give approximately the same maximum binding. Not only did the coupled antiserum have to be used at a much lesser dilution but i t also produced a less sensitive assay curve, with a midrange value of 340 pg and LDD af 50 pg' compared to values of 170 pg and 25 pg respectively obtained with the same antiserum when used uncoupled, (Fig. 47). -151-mg/ml coupled ligand 100 50 20 10 5 2 J ' ^ ' ' ^ 10 10 Reciprocal of Antiserum Titre Fig. 46 Comparison of dilution curves obtained with antiserum to GIP, performed on.aliquots taken before and after coupling of the antiserum to CNBr-activated Sepharose 4B. -152-Fig. 47 Comparison of the.standard curves obtained with antiserum to GIP, under routine assay conditions or after coupling to Sepharose 4B, demonstrating the loss of both:antiserum activity:and .sensitivity potential after the coupling procedure. -153-B. Application to purification of motilin 125 CI) Purification of I- Motilin A column of activated Sepharose 4B, coupled to-motilin antiserum, was pre-pared in a Pasteur pipette, the Bed volume being 1.0 ml. The gel was well equilibrated in 0.04M sodium phosphate ..Buffer, pH 6.5. A lyophilized sample of laBelled -motilin, containing 2 t x 10 cpm (estimated to contain 4 ng IR- motilin from the laBel specific activity) was applied in 0.5 ml of the equiliBrating Buffer and the column developed in this Buffer u n t i l no further 125 counts were eluted. That portion of the T- motilin which had been bound to the gel was eluted with 0.2M acetic acid. One ml fractions were collected throughout and the column profile i n 'Fig. 48 obtained by plotting cpm/fraction against ml of eluant. Eighty percent of the i n i t i a l l y applied cpm remained bound to the gel until the pH of the eluting buffer was lowered to 2.4, when 78% of that bound material was eluted. The remaining 22% was distributed between the gel and the glass wool at the bottom of the column. An aliquot of this same radioactive tracer was treated with coupled Sepharose 4B in a batchwise manner, as follows:- 2x10^ cpm I- motilin was diluted in 5.0 mis 0.04M phosphate buffer, pH 6.5 and mixed with 1.0 ml Sepharose slurry, equilibrated in the phosphate buffer, for 18 hours at 4°C. The super-nate was discarded and the gel washed with 5x5 ml phosphate buffer, i.e., u n t i l the counts in the wash were negligible. The gel was resuspended in 5 mis 0.2M acetic acid and mixed, by rotation, for 1 hour. The supernate was diluted in assay diluent buffer to give ~ 5000 cpm/100 y l . An aliquot of -154-125 Fig. 48 Column profile obtained after elution of I- motilin, adsorbed to Sepharose 4B, with 0.04M phosphate, pH 6.5 and 0.2M acetic acid', pH 2.4. One ml samples were collected and counted for 1 min in an automaticrty Counter. -155-untreated A"^J 1- motilin was similarity diluted to produce the same concen-tration and standard curves were prepared with both labels. The non-specific binding of the untreated label was 18.9%B, that of the treated portion was 10.6%. This improvement was due to the removal of free iodine and damaged, iodinated but non-immunoreactive fragments. There was no significant difference in the curves produced (see Fig. 49), the iodinated motilin appeared unaffected by i t s passage through the gel. (II) Extraction of motilin added to plasma Natural porcine motilin was diluted in charcoal-extracted plasma to a concentration of 4 ng/100 y l . When 4 ng was applied to a column of Sepharose 4B and the column developed as previously described, RIA was used to monitor the recovery of motilin. The column profile, in Fig. 50, is a plot of pg/ml IR- motilin against ml eluant. I n i t i a l l y 83% of the motilin was bound to the gel and 100% of that amount was recovered. (III) Extraction of endogenous motilin from serum Two mis serum (subject R.K.), which contained 680 pg IR- motiliri/ml was subjected to affinity chromatography as in II. Virtually 100% of the applied motilin was bound to the gel and then recovered by elution with a lower pH buffer (Fig. 51). - 1 5 6 --157-ml eluant Fig. 50 Column profile obtained after elution .of a motilin-containing plasma, adsorbed onto Sepharose 4B, with 0.04M phosphate, pH 6.5 and 0.2M acetic acid, pH 2.4. One ml samples were collected and monitored for TR-.-.motilin by radioimmunoassay. -158-Fig. 51 Column profile obtained after elution of a motilin-containing serum, adsorbed onto Sepharose 4B, with 0.04M phosphate, pH 6.5 and 0.2M acetic acid, pH 2.4. One ml samples were collected and monitored for IR- motilin by radioimmunoassay. -159-(IV) Isolation of motilin from presekretin Presekretin, a side fraction produced during the purification of secretin, i s the starting material i n the isolation of motilin (Brown et a l , 1971). The original purification procedure involved 5 stages of column chromato-graphy, was monitored i n the chronic dog bi'oassay and resulted i n produc-tion of.an active moiety representing 0.5-1.0% of the starting material by weight. Two yg Presekretin, estimated to contain 4 ng IR- motilin, was dissolved in 0.04M phosphate buffer, pH 6.5 and treated as previously described. The IR- motilin content was 90% recovered and the yield'represented a 5% recovery of the starting material by weight, (Fig. 52). EFFECTS OF MOTILIN ON THE RATE OF GASTRIC EMPTYING It was desirable to perform the studies on the rate of gastric emptying during a relatively constant background of circulating motilin. Pilot studies indicated that IR- motilin levels reached a plateau within 20 mins. of the start of the infusion and therefore measurements of the rate of gastric emptying were performed i n the last 10 mins, of a 30 mins. infusion (Fig. 53). A. Control studies in the gastric f i s t u l a dog (I) Effect of motilin on the rate of gastric emptying of liquids Motilin infusions in the range 0.125 - 2.0 yj;/kg/hour accelerated the rate D f emptying of the test liquid in a dose-related manner. The basal rate (C) -160-700 - j | 600-o t 500-| 1 400--*— o z 1 300-cc Q- 2  H 100 H 0 0.04 M P0 4 0.2 M HAc pH 6.5 T 5 10 ml eluant Fig. 52 Column profile obtained after.elution of a motilin-containing duodenal extract (Presekretin), adsorbed onto Sepharose 4B, with 0.04M phosphate pH 6.5 and'0.2M acetic acid, pH 2.4. One ml samples were collected and .-monitored for IR- -motilin by radioi-mmunoassay. -161-Fig. 53 Mean - SE serum concentrations of IR- motilin (pg/ml) following infusion of pure natural motilin 0.5 and 1.0 ug/kg/hour. 'Each point is the mean,of two experiments on each of six dogs (Debas et a l , Gastroenterology in press, 1977). -162-was.measured during the infusion of 0.15M saline, (Fig. 54). The lowest effective dose was 0.25 ug/kg/hour motilin (p< 0.001) and the maximum effect was achieved at 0.5 ug/kg/hour,motilin. (II) Effect of motilin on the rate of gastric emptying of solids The fraction of the solid meal remaining in the stomach :I 30^ ' 60, 90 and 120 mins after itss ingestion was virtually the same whether the infusion was 1.0 ug/kg/hour motilin or 0.T5M saline (Fig. 55). B. Effects of motilin on the rate of gastric emptying of liquids after truncal vagotomy and/or antrectomy (I) Effect of motilin after truncal vagotomy ^ The dose of motilin producing the maximum effect in the control dogs, 0.5 -ug/kg/hour, was ineffective after vagotomy. The doses.of 1.0 and 2.0 ug/kg/hour, however, did produce a significant increase in the fraction of the liquid meal emptied from the stomach. The effect of vagotomy, therefore, was to decrease the sensitivity of the response to motilin. The basal rate of gastric emptying, measured during saline infusion,, was significantly lower in the vagotomized animal - as shown in Fig. 56. (II) Effect of motilin after antrectomy• There was no difference in the response to 0.25, 1.0 and 2.0 ug/kg/hour motilin in the control and post-antrectomy•dogs. There was an, as yet, i n --163-"D OJ E LU c o o D 100 90-1 8 0 7 0 6 0 -5 0 -4 0 A 0 A I 1 * * p < 0.001 "1 1 1.0 2.0 Motilin (jug/kg/hr) C 1 1 ' 0.125 0 . 2 5 0 .5 f i g . 54 Mean - SE fraction ;of liquid meal emptied while different'doses .of motilin were infused:intravenously. Each point i s the mean of two experiments on each of six dogs; (Debas et a l , Gastroenterology, in press, 1977). -164-100 - | 0 30 60 90 120 Minutes after Meal Fig. 55 Mean - SE fraction of solid meal emptied at different'time periods.following ingestion.• Each point is the; mean of two experiments on each of six dogs (Debas et a l , Gastroenterology, in press, 1977) . -165-100-i 8 0 H 60 H • CL E L U c .2 40 H o o L L 20 -0 J Prevagotomy I 1 i •I Post Vagotomy 0.125 0.25 0.5 1.0 Motilin (jug/kg/hr) 2.0 Fig. 56 Mean - SE fraction of liquid meal emptied while different doses of motilin were:infused I.V before and after truncal vagotomy. Each.point i s the mean of two experiments on each of three dogs (Debas et a l , Gastroenterology, in press, 1977). -166-explicable, but significant.decrease in the rate of gastric emptying.at 0.5 ug/kg/hour motilin in the:antrectomized animal, as shown in Fig. 57. The basal rates were hot altered by removal of the antrum. (I l l ) Effect of motilin after truncal vagotomy arid antrectomy The basal rate of gastric emptying was significantly increased in the vago-tomized, antrectomized animal, compared to that in the control animal. There was a significant increase in the rate of gastric emptying of the liquid meal after 0.5 and 1.0 jig/kg/hour motilin (P<0.01). This was less significant, however, than the increase in the control animals (P <0.001) -(Fig. 58) MODIFICATIONS TO GIP RADIOIMMUNOASSAY A^  Antisera to GIP A l l animals received at least 3 immunizations with 50 yg porcine gtP, conjugated to BSA and emulsified with FCA. After 6 months, 10% of the animals' 3 were producing antisera usable at titres of 1:20x10 . The aff i n i t y constants (K) of the best guinea pig (Van 8) and best rabbit (Go 5) were 7. 5xl0 1 Z t and 1.0x10"'"^  X./mole respectively. Rabbit antiserum Ro7 demonstrated a maximum binding of >30% at a t i t r e of 3 1:30x10 but the displacement of label by standard antigen was unsatisfactory after an incubation of the equilibrium type. When this antiserum was incubated -167-100 9 0 TD Q> 'jZ 8 0 CL E L U c 7 0 o o o 6 0 5 0 4 0 0 J Control r 1— 0.125 0.25 Antrectomy - i 1 1 0.5 1.0 2.0 Motilin (jug/kg/hr) Fig. 57 Mean,- SE fraction of liquid meal emptied while different doses j of motilin were infused I.V. before and after antrectomy. Each point is the mean of two experiments on each of three.dogsi (Debas et a l , Gastroenterology, in press, 1977). -168-0> Q. E L U lOO - i 9 0 H 8 0 H 70H c eo-o o 2 50-LL 4 0 4 J ** I * • * p <0.0I * * * p < 0.001 o J I I 1 1 1 C 0.125 0.25 0.5 1.0 2.0 Motilin (jug/kg /hr) Fig. 58 Mean - SE fraction of a liquid meal emptied while different doses of motilin were infused'I.V'before and after truncal vagotomy1and ": antrectomy. Each, point i s the -mean of two experiments ion each of 6 dogs, (Debas et a l , Gastroenterology, in press, 1977). -169-125 with cold antigen for 24 hours prior to the addition of I- GIP, and for another 48 hours after the label addition a satisfactory,standard curve was obtained, the antiserum affinity.constant (K).being 2.5xl0 1^ 1/mole.-Fig.- 59_. .illustrates the Improvement obtained when Ro7 was incubated in a disequilibrium assay system, i n contrast, assays containing Van 8 showed no significant difference when incubated under either equilibrium or dis-equilibrium conditions;-Bj Iodination of GIP 125 The specific activity of the I- GIP, purified by gel f i l t r a t i o n was only 70 mCi/mg. After further purification by ion exchange chromatography on QAE Sephadex A-25 the specific activity was greatly improved, the value being 250 mCi/mg. A comparison of the standard curves obtained with the label at each stage i s shown in Fig. 60. EFFECT OF SOMATOSTATIN ON THE CANINE RESPONSE TO GIP A. Effect of somatostatin on the release of endogenous GIP (I) On the insulinotropic action of GIP released by oral glucose These studies were performed i n the intact, conscious dog. Somatostatin was administered intravenously either as a single rapid injection (3 ug/kg) immediately prior to the oral administration of glucose, or as an infusion of 6 ug/kg over 1 hour, the glucose load being given after 30 mins. The glucose load in either case was lg/kg of 20% dextrose in d i s t i l l e d water and was administered alone in control studies. -170-i n g . ' 59 The,effect of incubations of the equilibrium and disequilibrium type on the sensitivity-; of the'standard curves obtained with GIP antisera Van 8 and Ro7, -171-Fig . 60 Comparison of the standard curves for GIP obtained with radioactive tracer isolated by gel f i l t r a t i o n on Sephadex G25 only and after subsequent elution from Sephadex QAE A25. -172-A f t e r the s i n g l e bolus i n j e c t i o n of somatostatin, the f a s t i n g l e v e l s of serum glucose, IR- GIP and IR- i n s u l i n were i n h i b i t e d and the response to the o r a l glucose was delayed. There was a s i g n i f i c a n t reduction from the control values of a l l parameters at 15 mins. (IR-GIP, P\ <0.0025; IR-i n s u l i n P. <0.0005, glucose, p <0.0005), (Fig. 61, Table XXVIII). -When somatostatin was administered as an in f u s i o n , the IR-GIP and IR-i n s u l i n responses to o r a l glucose were suppressed u n t i l the in f u s i o n had ended at 60 mins. when the IR i n s u l i n response rebounded to values s i g n i f i -cantly above the c o n t r o l . The delayed elevation of the serum glucose l e v e l s was s i m i l a r to that seen a f t e r the'bolus i n j e c t i o n of of somatostatin and by 60 mins. there was no s i g n i f i c a n t difference from the control values (Fig. 62, Table XXIX). (II) On the release of endogenous GIP by f a t The,effect of a s i n g l e , rapid intravenous i n j e c t i o n of 3 ug/kg somatostatin i n the IR-GIP response to ingestion of 100 mis Lipomul was compared with control studies when fat alone was administered. The IR-GIP response a f t e r somatostatin was found to be delayed and reduced (P . <0.05) a f t e r somato-s t a t i n and then rebounded above the control values (as may be observed i n Fi g . 63 and Table XXXT). B. E f f e c t of somatostatin on the response to exogenous GIP A comparison was drawn between the responses of IR i n s u l i n and serum glucose to an intravenous i n f u s i o n of 1.5 ug/kg porcine GIP over 5 mins, given with and without a p r i o r s i n g l e , rapid, intravenous i n j e c t i o n of 3 yg/kg somato-s t a t i n . There was an 80% reduction i n the peak IR i n s u l i n response to GIP TABLE XXVIII The e f f e c t of a single rapid i . v . i n j e c t i o n of somatostatin on the incremental IR-GIP, IRI and serum glucose response to oral glucose. n = 7 Incremental Response (A) Time (mins) 5 10 15 20 25 30 45 60 75 90 105 120 135 150 Serum Glucose Control X ±SE 10.2 4.5 19.6 5.4 32.0 4.8 33.7 4.2 46.6 8.7 45.7 5.8 43.9 4.6 31.1 4.3 24.6 4.0 14.1 3.9 11.3 3.3 10.2 2.8 8.7 3.7 5.7 3.3 mg% Test X ±SE •7.6 2.9 3.8 4.9 5.9 6.4 11.9 5.6 24.4 5.8 24.8 6.2 36 11 39 8.2 36.1 5.7 25 5.2 22 7.1 14 5.3 10.3 3 9.1 3 , IR-GIP Control X ±SE 205 95 520 149 551 156 980 202 1063 132 909 239 1348 134 1188 182 910 117 673 97 .518 123 336 124 221 85 o i 60 75 Pg/ml Test X ±SE -192 120 -128 101 87 148 136 134 316 210 357 172 842 367 1314 295 1007 298 750 241 668 240 614 261 578 332 378 117 IRI Control X ±SE 22.8 11.3 54.6 14.2 68.7 9.1 63.0 5.9 72.8 9.4 62.6 8.0 42.5 7.0 36.4 7.6 20.4 4.7 8.2 7.7 1.3 4.1 -2.9 1.7 -2.7 4.8 -5.4 4.2 yu/mi Test X ±SE -6.5 2.4 -4.4 2.8 -0.5 6.8 7.5 5.2 39 12 ' 58 9.2 59 13 49 16 37.4 12 25.2 10.5 5.5 4.9 2.3 5.3 1.4 2.4 -1.8 1.8 -174-Fig. 61 The effect of an Intravenous injection of 3 jig/kg somatostatin on the incremental IR-GIP, IR- insulin and serum glucose responses to the ingestion of glucose. (1 g/kg) (Pederson at a l , Can J. Physiol. Phamacolv. 53j: :1200-l205, 1975) TABLE XXIX of a 1 hr infusion of somatostatin on the incremental IR-GIP, IRI and serum glucose response to oral gli n = 6 Incremental response (A) Time (mins) 5 10 15 20 25 30 35 40 45 50 55 60 75 90 105 120 135 150 165 180 Serum glucose Control X ±SE 10.2 A.5 19.6 5.4 32.0 4.8 33.7 4.2 46.0 8.7 45.7 5.8 43.9 4.6 31.1 4.3 24.7 4.0 14.1 3.9 11.3 3.3 10.2 2.8 8.7 3.7 5.7 3.3 6.4 3.5 6.3 4.1 mg% Test X ±SE 2.4 0.7 4.7 3.1 2.2 2.1 0.5 2.6 4.6 5.0 3.0 3.4 5.0 3.9 0.2 1.7 6.0 3.7 10 4.2 18.2 5.6 25 6 43 6.8 27 5.2 29 5.5 35.4 5.4 32.3 4.7 21.5 7.9 20 6.3 11.4 2.1 IR-GIP Control X + SE 208 95 520 149 551 156 980 202 1063 132 909 238 1348 134 1188 182 910' 117 673 97 518. 123 336 124 221 85 60 75 143 121 115 74 pg/ml Test X + SE 72 89 115 339 150 339 843 128 176 58 802 200 752 183 684 156 763 210 886 212 750 143 500 200 324 136 IRI Control X +SE 22.8 11.3 54.6 14.2 68.7 9.1 63.0 5.9 72.8 9.4 62.6 8.0 42.5 7.0 36.4 7.6 20.4 4.7 8.2 7.7 1.3 4.1 -2.9 1.7 -2.7 4.8 -5.4 4.2 -5.0 4.8 2.7 5.1 yU/ml Test X ±5E 0 2.4 -3 1.8 -1.6 2.9 -9.6 3.7 -10 3.3 -6.5 3.6 -6.8 3.6 -6.6 3.1 -5.6 2.6 -7.6 2.2 . i -1.8 4.7 119.8 13 43.2 8.4 3.5 9.2 34.6 7.6 17 5.6 10.2 4.7 7.1 5 8.3 6 -176-Fig. 62 The effect of an infusion of.somatostatin (6 ug/kg/hour) on the incremental IR-GIP j IR-^ , insulin: and serum glucose .responses to the ingestion of 1 g/kg glncose; (Pederson.et a l , Can.: J.Physiol. Pharmacol. 53: 1200-1205, 1975) < T A B J ' - E X X X Effect of a single rapid i.v. injection of somatostatin on the incremental IR-GIP, IRI and serum glucose response to oral fat. Incremental Response (A) Time (mins) - 5 10 15 20 25 30 35 45 60 75 90 105 120 135 150 165 180 Serum glucose Control X ±SE 3.8 1.3 3.6 3.1 5.8 3.3 3.7 2.0 3.7 2.6 7.5 3.6 8.3 2.5 6.5 4.0 7.5 4.4 4.3 4.6 5.5 3.6 7.5 3.4 mg% Test X ±SE 0 1.2 0.4 1.9 5.0 1.9. 3.3 1.3 4.2 1.4 3.9 4.2 l .0 1.3 3.0 1.8 1.9 1.5 1.8 0.7 -0.3 1.4 2.0 2.0 0 1-5 0.2 1.1 3.4 1.2 3.0 ' 2.2 4.8 3.7 Control X ±SE 674 174 1210 274 1712 441 2641 497 2696 560 3083 614 3035 612 3325 605 3270 635 . 3548 782 3526 707 2704 565 IR-GIP pg/ml Test X ±SE 29 43 12 30 30 35 -5 29 -10 46 242 154 210 140 394 164 946 257 1235 323 1585 122 1492 103 1642 215 1435 221 1903 414 1721 416 1964 322 IRI Control X ±SE -1.0 0.45 0.83 1.6 . -0.3 1.6 4.1 2.9 5.6 1.8 6.5 3.1 9.5 3.8 3.1 0.9 3.9 1.9 3.6 1.2 5.0 2.2 4.5 0.8 yU/ml Test X +SE -6 2.8 -9 1.8 -5 2.0 -3 3.0 -3 3.0 -2 2.9 -1.3 2.8 4.2 4.3 9.3 4.6 17.6 3.4 12.8 5.4 5.8 2.7 8.3 2.6 7.8 2.3 5.6 2.5 1.6 1.9 8.8 7.0 -178-F3 u g / k g Somatostatin 100 ml Lipomul 4000n Time in Minutes I i I •Fig. 63 The effect of an intravenous injection of 3 Jig/kg somatostatin on the incremental IR-GIP, IR-; Insulin :• and .serum gliicos e responses to the ingestion of 100 mis Lipomul.(Pederson et a l , Can. J. Physiol. Pharmacol. 53: 1200-1205, 1975). -179-after somatostatin, with a concomitant and significant (P\ <0.01) reduction in the depression of the serum glucose values (Fig. 64, Table XXXI). RELATIONSHIP BETWEEN GIP AND GASTRIC ACID SECRETION A. Effect of exogenous GIP on gastric acid secretion The effect of a 60 min. intravenous infusion of 1.0 ug/kg/hour porcine GIP on a gastric acid plateau stimulated by pentagastrin (2.0 - 4.0 ug/kg/hour) in 9 experiments in 3 dogs i s illustrated in Fig. 65 and Table XXXIII. A 60% inhibition of gastric acid secretion was observed in the second half of the GIP infusion, associated with IR-GIP levels in the range 1200-1400 pg/ml above the control value. During the post-infusion period,.the IR-GIP gradually declined back toward the pre-infusion values and the H + output retarned toward the control plateau levels. Control experiments were per-formed in 3 dogs which received pentagastrin only (Table XXXII). B. Effect of endogenous GIP on gastric acid secretion  (I) After an intraduodenal infusion of fat A triglyceride emulsion (Lipomul) was infused intraduodenally at 1.91 ml/min. over 30 mins. after a plateau of gastric acid secretion had been achieved by intravenous infusion of pentagastrin in 3 experiments in each of 3 dogs. The results (Fig. 66 and Table XXXIV) showed that a marked increase in IR-GIP occurred to levels of 800 - 1000 pg/ml above the pre-infusion levels, co-incident with a 68% inhibition of gastric acid secretion. During the post-infusion period both IR-GIP and H + values returned toward the control levels. i i TABLE XXXI Effect of a single rapid i.v. injection of somatostatin on the incremental IRI and serum glucose response, to i.v. porcine GIP. n = 4 Incremental Response (A) Time (mins) 3 4 5 7 10 15 20 25 30 45 P r m t - T * n 1 X 0.6 -3.6 -1.6 -5.0 -8.4 -9.8 -6.8 -4.2 -0. 2 2.6 L - U I 1 L L U 1 ±SE 1.5 3.3 1.4 2.4 3.1 7.3 7.7 2.5 3. 7 1.3 Serum glucose mg% Test X 3.7 4.7 2.2 1.5 0.25 -1.7 -1.25 -1.25 3. 0 4.7 ±SE 1.8 1.3 1.7 1.8 1.7 1.4 2.4 5.3 1. 5 . 3.9 X 25.4 25.4 30.0 16.4 9.4 -1.4 -3.8 -2.6 -2. 2 -1.4 ^ U U L L U l ±SE 11.9 6.4 6.9 3.3 2.1 1.3 1.4 1.1 1. 2 2.1 IRI uU/ml Test X -1.25 -1.75 5 15 5.8 •_1.7 -2.0 1.0 1. 25 1.25 ±SE 1.3 1.0 3.7 2.2 1.7 4.4 0.8 3.1 1. 0 1.0 -181-Fig. 64 The effect of an intravenous injection of 3 J-ig/kg somatostatin on the /incremental IR* insulin:and serum glucose responses to an intravenous infusionof 1 ^ g/kg porcine GIP oyer 5 min. - CPederson et a l , Can. J. Physiol. Pharmacol. 53: 1200-1205, 1975) TABLE XXXII Effect of a continuous infusion of pentagastrin on H+1 output of an extrinsically denervated fundic pouch. Expt. # INTRAVENOUS PENTAGASTRIN INFUSION °1 °2 °3 15 30 45 60 75 90 105 120 Ma 161 149 140 220 198 208 261 202 213 198 201 Di 330 326 310 264 238 235 280 274 264 251 283 Be 1720 1705 1669 1647 1324 1307 1120 1205 1409 1460 1103 1. uEq H /15 min. TABLE XXXIII of an intravenous infusion of porcine GIP on pentagastrin-stimulated H + output 2. Experiment CONTROL Intravenou 3 Porcine GIP Infusion Post Infusion Period ii °1 °2 °3 : 5 10 15 20 25 30 45 60 75 90 105 120 Ma 1 H+ IR-GIP3 329 340 374 400 314 320 800 137 1475 1500 1650 106' 1675 97 1750 120 1325 103 1225 145 1425 193 1050 143 920 Ma 2 H+ IR-GIP 378 380 389 320 297 420 210 620 217 780 950 1300 89 1050 124 1750 216 450 235 700 233 230 228 220 Ma 3 H+ IR-GIP 269 125 288 125 245 125 370 440 195 310 1500 1450 75 1300 60 1500 48 1050 40 1600 84 140 128 130 171 130 Be 1 H+ IR-GIP 292 280 351 230 313 430 320 500 248 540 1200 1150 102 1200 158 1700 79 1150 162 430 149 310 283 400 Be 2 H+ IR-GIP 235 125 205 125 205 125 440 1350 124 1500 1850 1800 85 1750 123 2600 90 2400 61 1550 117 570 159 550 165 580 Be 3 H+ IR-GIP 275 125 237 125 259 125 125 460 202 840 1450 1900 130 1400 151 1900 99 2400 102 700 156 300 186 350 197 480 Di 1 H+ IR-GIP 252 200 224 150 225 125 140 700 197 830 1200 1700 153 1450 146 1900 109 1700 84 1800 155 1050 166 620 199 320 _ Di 2 H+ IR-GIP 300 125 329 125 292 125 125 470 236 720 650 1000 184 ' 1620 134 2000 124 1450 163 1750 171 1200 234 650 200 270 1. 1 ug/kg/hr GIP over 60 min. 2. uEq . H+/15 min. 3. IR-GIP in pg/ml. -184-I | 1 1 1 — i 1 — i 1 1 L-zoo<l 0 30 60 90 120 Time in Min. after Start of Infusion Fig. 65 The incremental IR-GIP response and inhibition ;of pentagastrin-induced gastric acid'secretion caused by an intravenous > infusion of 1 pg/kg/hour porcine GIP. The results represent the mean -SE of 8 experiments in 3 dogs. TABLE XXXIV Effect of duodenal infusion of fat on pentagastrin-stimulated H output and IR-GIP release. Experiment « CONTROL Duodenal Fai : Infusion Post Infusion Period °1 °2 °3 5 10 : 15 '20 25 30 45 60 75 90 105 120 Ma 1 H+ IR-GIP 499 125 528 600 533 300 358 820 342 720 167 1650 162 1175 420 1050 486 440 416 660 440 385 Ma 2 H+ IR-GIP 270 520 234 420 218 500 520 920 50-730 690 1025 82 25 116 125 168 245 282 Ma 3 H+ IR-GIP 555 130 508 220 518 330 200 460 78 470 610 690 157 840 40 420 25 1125 80 1875 155 1425 178 1200 185 900 Be 1 H+ IR-GIP 17 6 150 162 190 128 120 220 500 150 800 98 1550 68 1550 . 145 1650 86 1550 230 700 209 500 Be 2 H + IR-GIP 220 910 230 1000 250 800 275 360 162 840 70 1700 117 1450 146 1250 160 1150 192 1000 335 860 Be 3 H+ IR-GIP 298 650 273 710 297 600 620 860 185 1250 1450 1500 186 1600 166 1750 216 1900 249 1350 201 930 278 700 286 360 Di 1 H+ IR-GIP 127 180 110 ' 125 156 500 700 590 168 1600 1500 1750 1800 71 1400 21 1350 70 1250 172 1350 177 1350 210 1330 Di 2 H+ IR-GIP 262 125 250 125 299 125 125 125 340 550 970 1550 65 1350 46 680 74 580 68 400 210 340 245 170 198 125 1. 100 mis Lipomul over 30 min. 2. yEq H+/15 min. 3. IR-GIP in pg/ml. -186-i I D Fat I I • • Time in Min. after Start of Infusion Fig. 66 The:incremental IR-GIP:response and inhibition of pentagastrin-induced gastric acid.secretion caused.by.an:intraduodenal infusion of 1.9 ml/min Lipomul oyer 30 min. The results represent the mean + - SE of 8 experiments in 3 dogs. -187-(II) After an intraduodenal infusion of glucose A 30 min. duodenal infusion of l.Og/kg 20% dextrose was performed when a plateau of gastric acid secretion had been achieved by pentagastrin ad-ministration. The H + output was reduced to 52% of the pre-infusion plateau levels with a concomitant increment in IR-GIP of 400 - 600 pg/ml. Each point represents the mean of 9 experiments in 3 dogs (Fig. 67, Table XXXV). C. Effect of an intraduodenal infusion of acid on gastric acid secretion A duodenal infusion of 1.91 ml/min 0.15M hydrochloric acidfover 30 min. inhibited the H + output stimulated by pentagastrin to 48% of the pre-infusion levels. This reduction in the acid secretion was not accompanied by any significant change in IR-GIP, in 4 experiments in 2 dogs (Fig. 68, Table XXXVI). D. Effect of ah intraduodenal infusion of saline bri gastric acid secretion In 7 experiments in 3 dogs, a duodenal infusion of 0.9% saline at 1.91 ml/ min. over 30 min. resulted in a small (27%) non-significant, inhibition of H + output, preceded by a slight, transient increase in IR-GIP (Fig. 69, Table XXXVII). These results are summarized in Fig. 70, which compares the maximum in-hibition of gastric acid achieved with the concomitant circulating level of IR-GIP, TABLE XXXV Effect of duodenal infusion of glucose on pentagastrin-stimulated H + output 2 and IR-GIP release 3. Experiment CONTROL Duodenal Glucose Infusion Post Infusion Period # Ol o 2 °3 5 10 15 20 25 30 45 60 75 90 105 120 Ma 1 203 202 182 82 42 33 55 197 107 163 IR-GIP 125 125 125 125 125 1020 460 500 300 680 235 145 150 430 Ma 2 H + 329 374 314 137 • 106 97 120 249 148 193 243 IR-GIP 340 400 320 - 800 1475 1500 1650 1675 1750 1325 1225 1425 1020 920 Be 1 H + 298 273 297 185 186 166 216 163 201 278 286 IR-GIP 480 460 440 370 320 690 940 930 740 740 1150 1350 125 370 275 Be 2 ' H + 145 156 124 119 167 90 130 217 172 IR-GIP 125 125 125 125 125 625 380 415 250 260 125 • 145 125 Be 3 H + 394 314 355 310 150 190 193 196 236 234 IR-GIP 125 125 125 125 125 725 490 330 420 240 125 125 125 - 125 Di 1 H + 277 270 260 161 199 . 243 208 186 235 229 287 IR-GIP 350 340 220 140 340 480 480 650 1400 750 340 280 240 125 125 . Di 2 H + 326 272 298 232 232 202 112 103 206 143 135 IR-GIP 125 125 125 125 170 200 680 820 600 540 190 125 125 160 125 1. 20% dextrose - 1 g/kg over 30 min. 2. uEq H+/15 min. 3. IR-GIP in pg/ml. -189-The incremental IR-GIP response and inhibition of penta-gastrin-induced gastric acid secretion caused by an intra-duodenal infusion of 1 g/kg glucose over 30 min. The results represent the mean "t SE of 7 experiments in 3 dogs. TABLE XXXVI Effect of a duodenal infusion of acid 1 on pentaga 3trin-stimulated H + output 2 and IR-GIP release 3. Experiment it CONTROL Be 1 Be. 2 Ro 1 Ro 2 H IR-GIP H IR-GIP H IR-GIP H IR-GIP Ol 0 2 0 3 2626 2608 2522 500 315 310 2714 2831 2760 390 310 250 1534 1800 1746 760 840 835 1780 1750 1830 350 410 300 1. 0.15M HCl at 1.91 ml/min for 30 min. 2. In UEq H+/15 min. 3. In pg/ml IR-GIP. Duodenal Acid Infusion 10 15 .'.0 25 30 2060 315 2173 200 1100 700 1210 300 1823 290 1714 250 918 850 942 260 Post Infusion Period 45 60 75 90 105 120 1944 1735 2013 2112 2257 2070 400 335 150 150 490 1802 1691 1990 2431 2461 200 360 225 150 200 255 2406 200 1120 1299 1415 1508 1368 1573 720 730 590 1100 1160 920 1100 1198 1460 1490 1600 1570 280 240 200 300 265 280 O Fig. 68 The incremental IR-GIP response and inhibition ,of pentagastrin-induced gastric'. acid secretion caused by an intraduodenal infusion of 1.91 ml/min O.lM.Hcl. The results represent the mean - SE of 4:experiments in 3.dogs. TABLE XXXVII Effect of a duodenal infusion of saline on pentagastrin-stimulated H output and IR-GIP release . Expt. CONTROL Duodenal Infusion of Saline Post Infusion Period °1 o 2 °3 5 10 15 20 25 30 45 60 75 90 105 120 Ma 1 H+. IR-GIP 326 270 294 260 250 260 180 140 220 370 164 330 129 250 135 240 128 190 184 170 220 300 Ma 2 H+ IR-GIP 216 530 257 240 182 330 136 280 129 440 105 1150 142 670 170 630 260 360 264 340 251 410 Ma 3 H+ IR-GIP 275 350 256 330 249 370 195 380 134 300 159 340 232 320 216 320 280 168 141 Be 1 H+ IR-GIP 143 125 178 125 223 125 297 125 200 125 118 125 184 125 233 125 189 125 215 125 198 125 Be 2 + H IR-GIP 256 125 242 125 264 125 222 125 192 125 198 125 133 125 169 125 121 125 159 125 168 125 Di 1 H+ IR-GIP 294 315 292 130 239 380 250 125 266 1100 206 125 199 125 142 125 205 125 108 125 125 Di 2 H+ IR-GIP 275 250 256 280 249 220 195 135 134 125 15.9 125 232 150 216 210 280 210 . 168 125 141 125 1. 0.15M saline, 100 mis over 30 min. 2. in uEq H+/15 min. 3. IR-GIP in pg/ml. -193-to O <D D_ <= O o ro c o 0) £1 o o o or OT o CL o Soline Pentagastrin Infusion 1 Control After ID Saline T 1 1 1 r 0 3 0 6 0 9 0 120 Time in Min. after Start of Infusion E \ 6 0 0 2 o_ + 2 0 0 o - 2 0 0 < Fig. 69 The incremental IR-GIP response 'and inhibition of pentagastrin-induced gastric acid secretion caused by an -intraduodenal infusion of 1.91 ml/min 0.15M saline. The results represent the mean — SE of 7 experiments in 3 dogs. -194-Fig. 70 The fundic pouch H output and incremental serum IR-GIP response achieved a f t e r i . v . infusion of pentagastrin (pg) only - control - compared with these same parameters dur-ing a concomitant i . v . infusion of porcine GIP or i n t r a -duodenal infusions of f a t , glucose or ac i d . -195-af ter intraduodenal infusion with f at * glucose andacid or intravenous i n -fusion of porcine GIP. STUDIES ON THE POSSIBLE HETEROGENEITY OF GIP  A. In serum (I) Immunoreactivity of GIP released by glucose or fat Serum samples were obtained from the same human subject (JRD) either 45 min. after ingestion of 100 ml 20% dextrose or 90 min. after ingestion of 100 ml of the. triglyceride emulsion, Lipomul, representing the i n i t i a l peak response of IR- GIP to either stimulus. They were serially diluted in diluent buffer and monitored by RIA. Neither of the serum dilution curves showed any significant difference from the standard curve obtained by diluting pure porcine GIP. The anti-serum used in the routine RIA did not, therefore, differentiate between the GIP released by glucose or fat given orally, as shown in Fig. 71. • (II) Immunoreactivity of GIP after column chromatography The apparent immunoreactive homogeneity-of•the GIP released by glucose or fat was further-examined by.chromatography of 2 ml aliquots of these same serum samples on a 1x200 cm column of Sephadex G50 fine in 0.2M acetic acid. The column\was calibrated prior to each run with dextran-blue and 125 I- GIP ( ~ 60,000 cpm) in 2 mis charcoal-extracted plasma and the con-ductivity monitored to determine-the position of the salt peak. One ml -196-Fig. 71 A comparison of the immunoreactivities of porcine GIP and the IR-GIP in human sera after the ingestion of glucose or Lipomul. (Brown et a l , Rec Prog. Horm.Res. 31 : 487-532, 1975). -197-fractions were collected and the IR-GIP content of each fraction measured by RIA. At least three immunoreactive regions were detectable after this treatment, one eluting in the void volume of the column GIP^0 : and a significant immunoreactive component which eluted ahead of the GIP , - Q Q Q arbitrarily named PROGIP. These..three immunoreactive peaks were present in serum samples after either glucose or fat stimulation (as shown in Fig. 72 and Fig. 73). The relative proportions of each IR-GIP component were given by expressing the area under each peak (approximately ^~/2 height x width) as a percentage of the total (Fig. 74, Table XXXVIII). TABLE XXXVIII Proportions of IR-GIP components released by fat and glucose After glucose stimulation After fat stimulation Total IR-GIP content 1400 pg/ml 2700 pg/ml % Total as GIP 0 v 14.4 31.4 % Total as ProGIP 35.3 40.8 % Total as GIT^ 50.3 27.8 These .results indicated that .there might be a difference in the GIP response to glucose and fat, the major component of IR-GIP in the i n i t i a l peak res-ponse to glucose being GIP,-nnn whilst that after fat was ProGIP. -198-. 72 Regions of IR-GIP obseryed after chromatography of 2 ml serum, obtained 45 min after:ingestion of glucose, pn Sephadex G50 Cl x 200 cm) in O.ZM acetic acid. The column was previously 125 calibrated with dextran blue and I-GIP. The enclosed numbers refer to the percentage of the total IR-GIP represented by that region. -199-Fig. 73 Regions 1of IR-G1P observed after chromatography,of 2 ml serum . obtained 90 min after ingestion of Lipomul, on Sephadex G50 in 0.2M acetic acid. The column was previously calibrated with dextran blue and v I-GIP. The enclosed numbers refer to the percentage of total IR-GIP represented by that region. > -200-Fig. 74 The ehfomatograms from Fig. : 71- and Fig- 72 expressed in histogram form, the heterologies of GIP being represented as percentages of the total IR-GIP'response. - 2 0 1 -(III) Immunoreactive forms of GIP released by oral fat or glucose  (a.) After oral fat Serum samples were obtained from both normal .human rsubjects and., dogs at various time intervals after ingestion of fat in.the form of 100 ml Lipomul. After routine RIA of these samples a 2 ml aliquot of each was chromatographed on Sephadex G50 fine in 0..2Kacetic acid and 1 ml fractions collected. The 125 125 125 column was routinely calibrated with I*- albumin, I- GIP and I in 2 ml extracted plasma. The results obtained after RIA of the column fractions are typified in Fig. 7/5; and Table XXXIX. In both species the proportion of the total IR- GIP represented by the larger molecular form (ProGIP.) increased with increase in time after the ingestion of fat. (3bL) After oral glucose Sera from human subjects 45 min and 100 min. after oral administration of 100 ml 20% dextrose were eluted from Sephadex G50 fine as previously des-cribed. The percentage of the total IR- GIP existing in the ProGIP form increased with increase in time after the ingestion of glucose (Fig. 75 Table XXXIX). These results indicate that the important factor in determining the relative proportions of the different forms of IR- GIP is the time of sampling the serum after the stimulus and not the nature of the stimulus i t s e l f . i (IV) Immunoreactive forms of GIP after i t s exogenous administration Normal, fasted, dogs were given intravenous infusions of 1.5 pg/kg/hour -202-TABLE XXXIX Change in relative proportions of IR-GIP components with the time of serum sampling after oral fat and glucose. Type of stimulus Oral.Glucose Time (min) of sampling after stimulus 45 100 Oral fat in man *Total IR-GIP content % Total as GIP 0 v % Total as ProGIP % Total as GIP 5 0 0o Oral fat in dog *Total IR-GIP content % Total as GIP 0 v % Total as ProGIP % Total as G I P 5 0 0 Q Oral glucose in man *Total IR-GIP content % Total as GIP v D % Total as ProGIP % Total as G I P 5 0 Q 0 1400 14. 4 35.3 50.3 680 16.1 42.5 41.4 * Expressed in pg/ml IR-GIP. -203-IOO-i 80-60-40-20-0-80-i 3 60 i £ 40H o >° 20H o-J Min. 90 150 Oral Fat in Dog Min. 20 35 60 Oral Fat in Man • G I P V o ^ Pro-GIP 5000 60-i 40 20 0-> n i Min. 45 100 Oral Glucose in Man Fig . 75 The relative proportions of IR-GIP y 0 , I R - G I F 5 Q 0 Q and IR-ProGIP, 'expressed as percentages ;of the total IR-GIP response,',observed Ca) 20,35 and 60 ^ninJafter fat : ingest ion: in man, .(b)-90 and 150 min after fat ingestion in dogs and (c) 45and 100'min after glucose ingestion In man.-.. -204-natural porcine GIP over one hour. Blood samples were taken after 15, 30, 50 min. and 15 min. after the infusion had ended. . The total IR- GIP of each serum sample was determined by RIA and 2 ml aliquots were subjected to gel f i l t r a t i o n on Sephadex G50 fine prior to further RIA. Two major regions of immunoreactivity were observed, one corresponding to GIP 5000 , as might be expected by reason of the procedures involve in the purification of GIP. The other immunoreactive peak eluted in the void volume of the column, i.e.jGIP^o, representing a form or complex with a molecular weight^7-50,000, and was too large to be explained in terms of the circulating GIP present in the serum of the fasted dog, (Fig.76_). Pretreatment of serum samples from the series with 6.0M urea prior to chroma-tography resulted in the conversion of a significant proportion of the GIP^o to GIP^ QQQ suggesting that the IR- GIP eluting in the void volume represents an immunoreactive complex formed by the binding of GIP,-QQQ to a large molecular weight, serum protein, e.g., albumin or globulin (Fig.776). B. In tissue extracts (I) I n i t i a l tissue extraction Extracts of the duodenal and jejunal mucosa of dogs were partially purified in the laboratory of Dr. V. Mutt (Karolinska Institutet, Stockholm, Sweden). The tissue was boiled briefly and extracted into acetic acid. The protein was adsorbed onto alginic acid, eluted with 0.2M hydrochloric acid and pre-cipitated from solution with saturated sodium chloride. This precipitate contained secretin and cholecystokinin - pancreozymin, as well as GIP. After -205-100 Cu 3 8 0 cr " 6 0 4— o 4 0 -I 2 0 0 • • GIP Vo P ro -G IP GIP 5 0 0 0 3 0 5 0 Min. Infusion 5 post Fig. 76 The relative proportions of IR<-GIPy0, IR-GIP 5 0 Q 0 and IR-ProGiP, expressed as percentages of the.total IR-GIP response, in serum taken 15, 30 and 50 min after the start of an infusion of porcine GIP, 1 ug/kg/hour, and-15 min after the termination of the infusion. -206-100 T 80H CL i—i © I w 6 0 H • G I P V o \WX Pro-GIP GIP 5 0 0 0 o 4 0 J 2.0 A Without Urea With Urea Fig. 77 The relative proportions of IR-GIP y 0,'lR-GlP 5 0 0 Q and IR-ProCIP, ex-pressed as percentages of the total IR-GIP response,;in a serum sample containing exogenously administered GIP,'with or without pre-treatment with 6.OK urea. -207-desalting on Sephadex G25, a fraction preceding IR-GIP <_QQQ (Fr 1-8) was selected for f urther extraction. Ten g of this material (SPC I G25 Fr 1—8) was dissolved in 200 ml ammonium acetate, pH 6.5, and the pE corrected to 7.0 by addition of 2.0M ammonia. The mixture was centrifuged at 7000 rpm for 30 min at 4°C and the supernatant was decanted. The insoluble precipitate was redissolved im 0.1M acetic acid and lyophilized - neutral insoluble material.HHethanoi'-;(w9 volumes .) ..' was added to the supernatant and the insoluble precipitate removed by centrifugation, redissolved in 0.1M acetic acid and lyophilized - methanol insolublemmatefial.The protein remaining in solution was precipitated by the addition of 4 volumes of acetone at 4°C, and the precipitate was f i l t e r e d out on Whatman's 3MM paper, redissolved in 0.1M acetic acid and lyophilized;-d methanol soluble material. ,These.procedures are summarized i n Table XXXXI. The. fractions designated neutral insoluble, methanol insoluble and methanol soluble were chromatographed on Sephadex G50 fine (1x100 cm) in 0.2M acetic acid and the IR-GIP content of each 1.0 ml fraction was measured by RIA. The relative proportions of each component as a percentage of the total IR-GIP cont-ent is shown in Table XXXX and Fig. 78. Table XXXX Relative proportions of the IR-GIP components in fractions obtained from an extract of hog intestinal mucosa Fraction % Total IR-GIP content GIP„ Vo ProGIP G I P5000 Neutral insoluble 0 33.3 66.7 Methanol insoluble 0 72.2 27.8 Methanol soluble 0 0.0 100.0 Further purification was performed on the methanol insoluble fraction. -208-TABLE XXXXI Summary of Tissue Extraction Procedure Heat coagulated hog duodeno-jejunal mucosa Acetic acid Acetic acid extract |^  Algihic acid adsorption NaCl precipitate containing Sn, CCK-PZ and GIP activity ^ Sephadex G25 Fraction I containing IR-GIP 0.04M Ammonium acetate .pH 7.0 Neutral Insoluble Neutral Soluble fraction fraction Methanol Methanol Soluble fraction Methanol Insoluble fraction -209-100 -t CL 8 0 H © I 60 H o o 4 0 <4— O. 2 0 A 0-1 • GIP Vo Pro-GIP G I P 5 0 0 0 1 Neutral Methanol t Insoluble Methanol Soluble Fig. 78 The'relative proportions of IR-GIP y 9, IR-GTP^^ and TR-ProGIP, . expressed as percentages of the total IR-GIP, in partially puri-fied extracts of the hog duodenal and jejunal mucosa. -210-(II) Purification (a;) ' Methanol insoluble on Sephadex G50 In a typical experiment 300 mg methanol insoluble was dissolved in 5 ml 125 125 0.2M acetic acid containing I- albumin and I- GIP. The column of Sephadex G50 fine (2.5x90 cm) was developed with 0.2M acetic acid and 5.0 ml fractions were collected at a flowrate of 80 ml/hour. The void volume of the column and the elution volume of GIP^QQQ were determined by counting 0.5 ml aliquots of each fraction for 1 min. in an automatic gamma counter. The region between these peaks was pooled, lyophilized and designated ProGIP I. The column profile of such a column, obtained by plotting absorbance at 280 nm in a 1 cm light path against ml eluant, i s shown.in Fig. 79; with the regions of GIP immunoreactivity determined by RIA, superimposed. This procedure yielded approximately 100 mg lyophilized material with an IR- GIP content of 30 ng/mg. (Ib) ProGIP I on CM cellulose Thirty mg ProGIP I was dissolved in 5 ml 0.01M ammonium bicarbonate and the pH adjusted to 7.05 with carbon dioxide. The solution was applied to a column of cellulose CM II (1.5x13 cm) which was developed with 0.01M ammonium bicarbonate, pH 7.8. The more strongly absorbed material was eluted with 0.2M ammonium bicarbonate. The eluate was collected in 5 ml fractions at a flow rate of 120 ml/hour. The column was calibrated by chromatographirig porcine GIP under identical conditions. The absorbance of each fraction was measured at 280 nm in a 1 cm light path and the IR- GIP content estimated by radioimmunoassay with two different anti-sera. There was no significant IR- GIP in this sample, the major immuno-5000 -211-Flg. 79 Column profile obtained after elution of the methanol insoluble fraction from Sephadex G50:and /measurement of absorbance at 280 nm. The regions\o% GIP immunoreactivity were determined by RIA. Fr II was designated ProGIP I. The column was'calib-rated with 1- albumin, I-GIP and ^Iodine. -212-reactive peak.being less basic than GIP^^QQ. The alternative antiserum Ro 7, appeared to cross react to a different'degree with this molecular form of IR- G I P , compared with the antiserum, Van 8, routinely used in the assay (Fig. 80 ). Both antisera seemed to cross-react with the standard G I P prepara-tion to the same degree. The interassay control value was 254- 43 pg/ml IR- G I P (mean - SE in 50 determinations) according to Van 8, and 242- 14 pg/ml IR- G I P (mean - SE in 18 determinations) according to Ro7.. When Fraction III, from chromatography on Sephadex G50, i.e., that fraction corresponding to G I P 5 Q 0 q , was eluted from cellulose CM II under identical conditions, the major portion of the immunoreactivity eluted in the same position as natural porcine G I P ^ 0 Q Q (Fig. 81,). (g) Stability of ProGIP The material containing ProGIP, and selected to contain no G I P ^ Q Q Q was routinely lyophilized and stored at -20°C. After the yield from several columns had been pooled, 2 mg of the material was re-run on Sephadex G50 fine (1x100 cm) in 0.2M acetic acid as previously described. Radioimmunoassay on the fractions obtained revealed that a third of this material now existed in the GIP,-QQQ form, as shown in Fig. 82. ( I l l ) Molecular weight determination A series of chromatpgrams were run on Sephadex G50 fine (1x100 cm) in 0.2M 125 acetic acid. The samples were ~ 50,000 cpm I- albumin and .« ,50,000 cpm Df one of the following iodinated markers, I- motilin, I-GIP, I-125 'Insulin or I- parathyroid hormone, in 2 mis 0.2M acetic acid. The 1 ml -213-G I P 5 0 0 0 0.01 M N H 4 H C 0 3 P o o l e d a L y p h o l i z e d l . 5 0 0 n E c O 00 OJ CD o c 1.000 H 0 . 5 0 0 4 0.000 J I i — I R - G I P ( R o * 7 ) • O D a t 2 8 0 n m I R - G I P ( V a n ff8) n— 10 2 0 F r a c t i o n # _^ 0,2M NH4HCO3 4 0 r 9 . 0 8 . 0 I- 7.0 I h 6.0 o c I 5 0 - 5 . 0 - 4 . 0 3 . 0 h 2 . 0 I . 0 0 .0 Fig. 80 Column profile obtained after elution of ProGIP I from CM cellulose. The IR-GIP:content was determined by RIA with antisera Van 8 and Ro7. The column was calibrated with porcine GIP. -214-0 10 20 30 Fraction # Fig. 81 Column profile obtained after elution of the GIP,-x„„ - < <r 5000 . containing fraction from Sephadex G50, on CM cellulose.. The column' was ' calibrated with', porcine GIF -215-Fig. 82 Column profile obtained after rechromatography of ProGIP I on Sephadex G50 in 0.2M acetic acid, 'demonstrating the .reappearance of IR-GIP, 5000' -216-fractions collected were counted for 1 min. in an automatic gamma counter and plotted against ml eluant. The void volume (V°) and elution volume 125 (V„) were taken as the volumes corresponding to the peak tube of the I-125 albumin and I marker respectively, and V E/V° versus molecular weight was plotted for each marker (Fig. 83). V"E/V° was determined for the IR-GIP in ProGIP I and this was found to correspond to a molecular weight of 7500-8000 iii four separate determinations. -217-2.0 - i .8 I .6 1.4 H 1 2 H 1.0 4 x^Motilin x Glucagon \ x GIP \ x insulin Parathyroid s- Hormone T— i—n—i—i 3 4 5 6 8 10 M . W . X 10 3Fig. 83 Curve showing the relationship between V./V and molecular weight for various'polypeptides, obtained by elution of the iodinated polypeptides from Sephadex G50 (1 x 100 cm) in 0.2M acetic acid'and counting of these fractions for 1 min in an automatic y counter. -218-DISCUSSION Studies on the composition and structure of motilin revealed that i t was quite distinct from the previously isolated and characterized gastrointestinal polypeptides of duodenal mucosal origin. The major property of motilin appeared to be i t s stimulatory effect on the motor activity of the stomach. Unlike gastrin and cholecystokinin-pancreozymin, motilin stimulated the motor activity of the fundus as well as that of the antrum. Another action of motilin, suggested by studies with exogenously administered polypeptide, was stimulation of pepsin secretion in dogs. It did not, however, appear to have any effect on the exocrine pancreas or on gastric acid secretion (Brown et al,1972). Recognition of the physiological role played by motilin in regula-ting gastric motor and secretory act i v i t i e s , and support for the hypothesis that motilin was the humoral agent released upon alkalinization of the duodenal mucosa both required the development of some method for the identification and measurement of changes in the levels of motilin in the systemic circulation. A radioimmunoassay has been developed for the measurement of IR- motilin in sera and tissue extracts (Dryburgh and Brown, 1975). Antisera were raised in albino guinea pigs and New Zealand white rabbits. As a general rule, any substance with a molecular weight ofV <1000 may be regarded as non-immunogenic, whilst those with molecular weights in the range 1000-6000 are poor immunogens. Motilin, with a molecular weight of 2700, f a l l s into the latter group and no useful antisera to this polypeptide have been obtained by immunization with the polypeptide alone. This was overcome by covalently coupling the molecule (hapten) to a larger protein. The most commonly used method of achie-ving this is the carbodiimide condensation reaction. Carbodiimide w i l l react with a -219-number of weak acids but the predominant reaction at room temperature is with carboxylic acid, usually provided by the hapten. The activated carboxyl group then reacts with the free amino groups on the protein to form a peptide bond as schematically shown by the formula:-R-COOH + CH3-CH2-N=C=N(CH2)3 -^-(CH^ (HAPTEN) (CDI) + H20 CH3-CH2-NH-C=N-(CH2)3 -fi-(CH 3) 2 O-C-R II 0 + R'NH2 (PROTEIN) R-C-NH-R' + CH3-CH2-NH-C-NH-(CH2)3 4i-(CH3)2 0 (CONJUGATE) Some degree of condensation w i l l occur between the protein molecules, via their activated carboxyl groups. This may be reduced somewhat by activating the haptenic carboxylic acids before the addition of the protein. Bovine and human serum albumin are the most'commonly used proteins. Immunization of both rabbits and guinea pigs, the schedule of injections involving both motilin, conjugated to BSA, and non-conjugated motilin, resulted in the production of acceptable antisera in the majority of animals. 12 14 The range of af f i n i t y constants (K) of motilin antisera was 1 x 10 to 1 x 10 Ll/mole, calculated as shown in Fig. 10. When a radioimmunoassay is being estab-lished i t must be accepted that there is an inherent limit to the sensitivity -220-of the assay that i s dependent on the a f f i n i t y constant characterizing the predominant antibodies in. the antiserum. . The a f f i n i t y ;of the antiserum has been determined to be a function of the dose of the immunogen.employed (Parker'et". a l , 1967), and the time interval since the immunization (Eisen and Siskind, 1964). It is assumed that the heterogeneity of antibodies present in an antiserum is the result of their production by-a heterogeneous population of antigen-sensitive small lymphocytes. Low to moderate doses of immunogen w i l l preferentially stimulate cells with high a f f i n i t y receptors and their progeny w i l l , in turn, produce high affinity antibodies. With the passage of time the antibodies produced w i l l tend to neutralize some of the antigen and as the level f a l l s , i t w i l l be the higher a f f i n i t y cells which w i l l continue to be stimulated. Antisera to motilin have been raised by immunization with 20-50 pg polypeptide, given at monthly intervals un t i l a reasonable t i t r e was achieved. Booster immunizations with low doses of immunogen, at 3 - 6 month intervals,maintained or increased the t i t r e . Animals were bled 10 - 12 days after each immunization and at monthly intervals' 4 thereafter. Motilin antiserum Mo7 had a t i t r e of 1: 10 x 10 , 10 days after 4 i t s fourth immunization, which had increased to 1: 20 x 10 two months later, with no intervening booster (Table II). The immunization schedules and results are summarized in Table I and Table II. No crossreactivity has been demonst-rated between any motilin antiserum and gastrin, GIP, secretin, glucagon, cholecystokininpancreozymin, VIP or insulin, as shown in Figs. 8 and 9. Theoretically the iodination of. any.polypeptide containing tyrosine requires 125 only 3 basic ingredients:-- the isotope, usually I-Na, the pure polypeptide, and some method -for oxidizing the iodide to iodine. Variables, such as the -221-relative ratios of the various reagents, the constituents' of the diluent buffer, the f i n a l volume of the reaction -mixture, and the method for separating the unincorporated isotope from the labelled polypeptide,/have to be estab-lished for each individual polypeptide.. The majority of iodination procedures, however, vary only slightly from the original method proposed by Hunter and Greenwood in 1963. Motilin.was iodinated by a slight modification of their chloramine-T method and radioactive tracer with: a specific activity of 400 mCi/mg was routinely produced. A theoretical iodination of this polypeptide, resulting in the incorporation of 1 atom of iodine into each mole of peptide, 125 would produce I- motilin with a specific activity of 626 mCi/mg, assuming an isotope abundance of 96%. The actual results obtained would suggest that the tyrosine in position 7 in motilin is relatively accessible to incorporation of iodine. Storage of I- motilin, at a dilutioniof 1 x 10 cpm/ml, in 0.2M acetic acid, containing 0.5% B.S.A. at -20°C, resulted in a label, stable without repurification, for periods of up to 3 months. With the antiserum so far available, the most sensitive assay has resulted from an incubation which is allowed to reach equilibrium over 48-72 hours at 4°C. (Fig. 14). The routine method of separating the free antigen from the antibody/antigen complex is adsorption of the antigen.onto dextran-coated charcoal, 2.5 mg and 0.5 mg dextran being added per tube, as illustrated in Fig. 16. A solid phase antibody has-',h.een developed by coupling motilin antiserum to a beaded form.of agarose, a cross-linked dextran with high porosity (Sepharose 4B). This matrix is activated by treatment with cyanogen bromide at high pH. It is then reacted with the unprotonated amino groups on the ligand, in this case the IgG in the.antiserum, to form a stable complex by formation of hydrogen bonds. -222-R -NIL, CNBr-Sepharose' 4B NRH Ligand coupled to;Sepharose No detectable antibody activity was measured in the washings of the solid after coupling to the antiserum was complete (Fig. 18). The coupled antibody could be used in the radioimmunoassay with no apparent loss in antibody activity or.sensitivity potential. No significant difference was observed in the antiserum dilution curves or assay standard curves, obtained with coupled or uncoupled antiserum, a l l other assay conditions being the same (Fig. 42 and Fig. 43). Solid phase antibodies are a feasible prospect in the further develo-pment of the motilin radioimmunoassay. Pilot studies were performed on the extraction of motilin from sera and tissue extracts by chromatography of the motilin-containing material on columns of.Sepharose 4B, coupled to motilin antiserum. The results were favourable for the development of this technique on a larger scale, both for the concentration of motilin from sera and the isolation of the peptide from.its starting material, Presekretin, in a more economical fashion than can be achieved by the serial stages of chromato-graphy, at present in use (Fig. 51 and Fig. 52). Motilin, adsorbed to this matrix can be eluted by a lowering of the pH with no apparent damage to the molecule, as illustrated by the similarity of the standard curves obtained 125 with I- motilin, before.and after treatment by a f f i n i t y chromatography (Fig. 49). -223-Antisera to porcine motilin;appear to crossreact completely with porcine, canine and human motilin. <• The fasting serum motilin concentrations in man, measured in 45 normal subjects.in the age range 20-35 years, was 190 - 131 pg/ + + ml IR motilin (mean - S.D.). The mean - S.D. fasting serum /motilin concent-ration in 8:dogs was. 294 - 44 pg/ml IRT/motilin.. When! serum samples, containing exogenous or endogenous motilin, were.assayed at several dilutions, the results obtained could be fitted to the standard curve, as shown'in Tig. 10. This antibody would not therefore appear to differentiate between unlabelled antigen in the form of the standard or antigen in the form.of the. endogenous polypeptide, satisfying one of the basic requirements for.the development of a sensitive radiomunoassay. Addition of 5000 cpm per tube of a radioactive tracer with a specific activity of 400 mCi/mg entails the addition of only 7 pg motilin and s t i l l allows an efficient rate of counting. The f i n a l absolute essential, a high a f f i n i t y antiserum, has also been approached, and with these conditions satisfied, as laid down in the rationale, the other variables have been estab-lished at their optimal values. The apparent homogeneity of IR-motilin, suggested by the serum dilution curves, was further examined by chromatography of either alkali-stimulated IR- motilin in serum or an impure duodenal extract on Sephadex G-50. As illustrated in Fig. 31 and Fig. 32, only one region of IR- motilin was detectable, eluting in 125 the same position as I- motilin. The motilin content of 2 different preparations, one the synthetic analogue, 13-norleucine-motilin, the other an impure duodenal extract, was measured by bioassay.and immunoassay, in comparison with natural porcine motilin. The natural and synthetic motilins were found to be identical in both.biological -224-and immunological ac t iv i ty- (F ig . 39 and Fig . 41). The natural polypeptide represented a 10,000 fold purif icat ion of the crude extract, measured by either bioassay or immunoassay, as i l lustrated i n . F i g . 29 and Fig . 30. These results and the apparent homogeneity of IR- moti l in, suggest that the biologica l act iv i ty of motilin may be f a i r ly closely correlated with i t s immunological ac t iv i ty , as measured by radioimmunoassay. The IR- motil in of various regions of the hog gastrointestinal tract was measured in part ia l ly purified acid-ethanol extracts of the gastrointestinal mucosa. The region with the highest motil in content (per g dry weight of the extract) was the jejunum, followed by the duodenum and upper ileum, as summarized in Table XIV. No detectable motilin was found in the oesophagus, stomach or lower ileum. These findings agree with the results of Pearse et a l (1974). Using the indirect sandwich technique, with motil in antiserum as the f i r s t layer,and fluorescein-labelled goat antirabbit IgG as the second layer, they were able to detect fluorescent motilin-cbntaining cel l s in the duodenum, jejunum and upper ileum of the dog, pig , baboon and man. The c e l l of origin was identif ied as the enterochromaffin (EC) c e l l , 85% of the motilin-containing cel ls showing positive argentaffinity. The remaining 15% of the cel ls were argyrophyl. No EC c e l l in the stomach or lower intestine could be demonstrated as containing moti l in . The EC cells of the upper gastrointestinal tract have also been shown to contain serotonin, substance P and melatonin, a l l of which are also found in neural tissue. Although the EC c e l l is unlike the other cel ls c lass i f ied as APUD in that i t does not derive from the neural crest, i t does appear to originate from neuroectodermally derived tissue. The poss ib i l i ty should be investigated that motil in has neural connections. The relationship, i f any, between motilin and the other substances of EC c e l l origin is not clear. -225-Differential staining techniques, applied to mammalian duodenal tissue, have demonstrated that motilin and substance P are present in -different EC cells (Polak et al,..1976). _ Forssman et al (1976) confirmed that not a l l serotonin-containing cells ih'thms region contained motilin but were.:truhable to 'rule out the possibility that motilin - containing cells do.contain.:serotonin. One must postulate that EC cells f a l l into subpopulations, classified by the poly-peptide they produce. The production of a synthetic analogue (Wunsch et a l , 1973) permitted some insight into the relationship betweent. 'the structure of motilin and i t s biolo-gical potency. The methionine residue at position 13 was originally thought to be essential for the expression of biological activity. When the methionine was oxidized by treatment with-hydrogen peroxide, 95% of the biological .activity was lost. Full potency was restored, however-, after reduction of the residue with cysteine (Cook,. 1972). During the synthesis.leucine or norleucine were substituted for the methionine, because of the d i f f i c u l t i e s involved i n a synthesis containing a central arginyl-me.thionyl bond.. . No. loss, in either biological or immunological activity was observed (Fig. 39 and Fig. 41) suggesting that i t was not the methionine residue per se which was important in conferring f u l l biological potency. It was more lik e l y that some conformational change, which resulted from the oxidation of the sulphur-containing residue, was responsible for the loss of activity. In the course of the synthesis and purification of 13-norleueine-motilin, the intermediate compounds Mol^ and M0C2 were found to be Inactive. Mol^ resulted from a failed synthesis, in which the amino-acids, threonine and tyrosine; at-positions 6 and 7, were not incorporated. MoCii was determined to be a diastereomeric form of the active polypeptide, the -226-phenylalanineat position 5 being in the D-configuration,, rather than the L-configuratidn found:in the active molecule. Differences were observed in the 'electrophoretic ; mobilities.',p.ftheitryptic digestion products of the natural and synthetic motilins. The acidic tryptic peptide 3 (Tr 3), reported by Brown et al (1973) was absent in the synthetic material. When the tryptic digestion was repeated on freshly prepared porcine motilin, Tr 3 was isolated and subjected' to electrophoresis at pH 6.5. It now ran as a neutral peptide. Kinetic studies with leucine aminopeptidase and a 3-cycle Edman degradation, indicated that the peptide contained glutamine at position 14 and not glutamic acid as originally stated. It must be supposed that deamidation of the residue had occurred during the early preparation of motilin (Schubert and Brown;, 1974)- This deamidation had no effect on biolo-gical activity. Studies on the synthetic fragments 9-22 and 13-22 or on fragments of natural motilin produced by either chemical or enzymatic digestion'have not resulted in the isolation of a fragment containing any significant biological or im-munological activity. The immunological activity observed after cyanogen bromide cleavage of motilin, seen in Fig. 40, can be accounted for by the presence of uncleaved material remaining in the reaction mixture. Modification of the naturally-occurring molecule by acylation drastically reduced the potency of motilin. Acetylation neutralized the positive charges of the £ -amino groups and the N-terminal phenylalanine, whilst succinylation produced a net negative charge on the molecule. One cycle of the Edman's de-gradation procedure also resulted in a loss of biological potency. It is -227-unclear whether this was due.tg:the loss.of the N-terminal aromatic residue, phenylalanine, or to the acylation of ,the E-amino groups on the lysine residues, caused by•exposure of the-molecule to phenylisothlbcyanate. The loss of activity associated with the change in the net charge on the molecule would suggest that motilin binds to i t s receptor site by formation of ion pair bonds. The biological activity of -motilin was seemingly unimpaired by the removal of the C-terminal and penultimate amino acids, after treatment with carboxypeptidase A. It should be remembered, however, that this is not a com-plete degradation procedure and unti l synthetic peptides 1-20 and 1-21 can be prepared, no firm conclusions may.be drawn, regarding the importance of these amino acids in the biological activity of motilin. From these results, summarized in Table XV,, i t must, be concluded that v i r t u a l l y the intact molecule is required for the expression of f u l l biological potency. The individual amino acids are important inasmuch as they contribute to the charge distribution and probable confirmation of the molecule. Brown et al (1966) reported an increase in the motor activity of an extrinsically denervated or transplanted pouch of the fundus of the stomach after duodenal infusion of isotonic a l k a l i or fresh pig pancreatic juice, sufficient to raise the pH from the basal level of 7.5 to 8.2. Intravenous infusion of a pure poly-peptide, isolated from hog duodenal mucosa, mimicked this response. No other gastrointestinal polypeptide has been discovered which w i l l produce the reported increase in fundic motor activity; gastrin and cholecystokinin-pancreozymin having their motor effect only on the antrum of the stomach. The development of a/radioimmunoassyi specific for motilin, confirmed the supposition that the increased fundic motor activity -observed after duodenal alkalinization was accompanied by a concomitant increase in the circulating levels-of IR- motilin, -228-comparable to those achieved during exogenous administration of the poly-peptide :CFig. 34). An increase in serum IR- motilin levels was also reported by Hellemans et a l C1976) after i n s t i l l a t i o n of bicarbonate into the antrum of the stomach In man, but no such response was detected i f the perfusate was sodium hydroxide. Mitznegg et al 0-976) were unable to detect any increase in circulating IR- motilin levels.after intraduodenal Tris buffer, pH 10.2, in human volunteers. They claimed a f a l l in plasma motilin levels occurred after duodenal alkalinia'atlon but examination of their, results failed to reveal that the depression of TR-motilin. levels was significant in view of the variation in IR- motilin levels measured during the pre-infusion control period. Control studies in dogs', with an intraduodenal infusion of 0.15M saline showed no increase in fundic pouch motor activity and no change in IR- motilin from the basal levels, throughout the duration of the experiment, as shown in Fig. 35. Similar findings in mart-have been reported by Mitznegg et al (1976). In two experiments, the dogs exhibited spontaneous increases in fundic pouch motor activity, very similar to those obtained after a l k a l i . An increase in IR- motilin accompanied this Increased motor activity. The cause of this motilin release i s unclear. The activity occurred after the dogs had exhibited a period of normal basal motor activity for at least 20 mins* and was therefore unlikely to be caused by either distension of the fundic pouch whilst i t was being f i l l e d with water or by insertion of the Foley catheter into the Mann-Bollman f i s t u l a . -229-A somewhat anomalous observation was that duodenal acidification appeared to produce an increase in serum IR- motilin in dogs. This elevation was less than that seen after duodenal alkalinizatibn, but was more prolonged, as illustrated in Fig. 37. No increase in fundic pouch activity was observed in these animals. This result was unexpected in view of the finding by Brown et al (1967) that intraduodenal acid suppressed alkali-induced fundic pouch motor activity. Recent studies by Itoh et al (1976) supported this observation. They demonstrated that the inter-digestive pattern of gastric motor activity in the fundus, antrum and lower oesophageal sphincter in dogs was exactly mimicked by infusion of 0.1 - 2.7 ug/kg/hour synthetic motilin (13-methionine-motilin) when compared with respect to duration, ampli-tude and frequency of the contractions, and their velocity at different distan-ces along the gastrointestinal tract. This interdigestive pattern, whether natural or motilin-indueed, was interrupted by feeding, duodenal acidification or an infusion of pentagastrin. One possible explanation of these contra-dictory findings is that the substance released by duodenal acidification is not motilin, but some other substance which shares immunological but not biological identity with motilin. It has already been noted that although acylation almost completely abolished the biological activity of the molecule, approximately 50% of the immunological activity s t i l l remained (Table XXV). Investigation of this acid-released motilin-like immunoreactivity by electrop-horesis or column chromatography would provide more information relative to any size or charge difference between i t and the alkali-induced, motor stimulatory motilin. It i s interesting to speculate whether the anti-motilin effect observed after duodenal acidification is due, in part, to competitive antagonism by this motilin-like material. r -230-The physiological role(s).of motilin w i l l remain a subject of controversy u n t i l a physiological secretagogue or other mechanism for i t s release can be demonstrated. The lack of any significant increase in the systemic levels of IR- motilin after ingestion of either glucose or a mixed meal Csee Fig. 38) would suggest that motilin .played'little role in the normal digestive processes. The apparent increase in IR- motilin reported by Mitzrtegg et a l (1976) after ingestion of fat, with the accompanying inhibition of fundic motor activity, may be due to the. same motilin-like material released by duodenal acidification. In 1966, when Brown et al f i r s t described the alkali-induced increase in fundic pouch motor activity, they postulated that, under these conditions, some humoral agent was being released which would counteract the inhibitory effect of the other gastrointestinal polypeptides, gastrin, cholecystokinin-pancreozymin (and, of course, GIP) which were released by the ingestion of various nutrients. Studies by Hoelzel (1925) and Reinke et al (1969) in dogs, indicated that the duodenal contents tended to an alkaline pH during the fasting periods. As previously described by Ttoh et a l (1976) exogenous motilin exactly reproduced the normal pattern of mechanical interdigestive activity* .This consisted of bands of contractions, arising simultaneously in the fundus and duodenum and travelling aborally the length of the ileum. Each cycle took approximately 20 mins. to pass one point and consecutive cycles were about 90 mins apart. This pattern was interrupted by feeding. Preliminary studies have suggested that motilin levels were depressed i n i t i a l l y after feeding, as shown in Fig. 38. The purpose of this interdigestive cyclic activity i s postulated to be the cleaning from the digestive and absorbent,t regions of the upper gastrointes-ti n a l tract of the extra mucus'and desquamated epithelial cells resulting from the processing of the previous meal. This "cleaning up" operation is -231-probably mediated in part by Immoral means;:in part by neural mechanisms. 'r The appearance of a single cycle of activity at a time in the upper 70% of the gastrointestinal tract may be due to some inhibitory reflex between the upper and lower regions of the tract. The tachyphylaxis described by Coot-(1972) as occurring when a second injection of motilin was given less than 40 mins after the f i r s t , might be explained by an inhibitory.reflex of this type. The fact that gastric a l k a l i n i z a t i o n w i t h or without prior gastric a c i d i f i c a -tion, has been reported as causing an increase in the pressure of the lower oesophageal sphincter (LESP) (Castell and Levine, 1971) raised the possibility that motilin was involved in the control of this region. The increased pressure was i n i t i a l l y postulated to be the result of an increased gastrin output from the antrum. This was not however supported by the work of Debas et al (1974) or Kline et al (1975) who could show no increase in serum IR-gastrin after antral alkalinization. Jennewein et a l (1975) measured changes in LESP in dogs after bolus injections or intravenous infusions of motilin over 30 mins. The most effective doses were 30 ng/kg or 100 ng/kg/hour respectively. The motilin resulted in increased activity in both the antrum and the fundus,,and low frequency phasic activity in the LES, related to the activity in the fundus. When motilin was administered as an infusion, the phasic activity in the LES ceased within 10 mins of the end of the infusion. Duodenal alkalinization resulted in an increase (non-significant) in the LESP in 4 dogs. Similar results were obtained in studies in man, the LES responding to graded doses of the synthetic nor-leucine analogue in a dose-related manner. This response was depressed by infusion of atropine sulphate, suggesting a cholinergic involvements (Rtfsch et a l , 1976). -232-An attempt to correlate increased LESP with endogenous release of motilin was performed in normal subjects and patients who had undergone truncal vagotomy with Bi l l r o t h I or B i l l r o t h II antrectomies (McCallum et a l , 1977). The subjects ingested either 0.4M sodium bicarbonate or a commercial antacid preparation. Increased LESP was observed in the normal subjects and those witTt"Billroth rI anastomoses. No increase in either serum IR- motilin or -serum IR- gastrin was detected in association with this pressure increase. The lack of any response in the B i l l r o t h II patients indicated the importance of the duodenum in e l i c i t i n g this response. Hellemans et a l (1976) were able to demonstrate a slight.increase in IR- motilin after either gastric alkalinization or acidification. The motilin response to a l k a l i was rapid in onset and slightly preceded the peak response in the LES. This finding was in agreement with the postulate of, Jennewein that motilin exerted i t s effect on the LES indirectly, via i t s effect on fundic motor activity and gastric intraluminal pressure. The increase in intraluminal pressure in the fundus was then the direct cause of the increased LESP. In contrast, the motilin response to antral acidification was much slower in reaching i t s peak (approximately 45 mins.) and correlated directly with the peak LESP. These differences were not remarked upon by the authors. If the different rates of motilin release were due to different rates of passage of the acid or a l k a l i into the duodenum, the authors' postulate that motilin, li k e acid, has a role in the inhibition of gastric emptying loses support. Increased LESP after exogenous administration of low doses of natural or synthetic motilin has been conclusively demonstrated in both dogs and man. The results obtained after endogenous released motilin are far less conclusive. Direct i n s t i l l a t i o n of a l k a l i into the antrum or duodenum has been shown to -233-result in an increased LESP, which correlated with the release of IR-motilin. However, ;the increase in LESP associated with oral ingestion of a l k a l i could not be related td; any detectable increase in IR- motilin. This finding does not necessarily preclude motilin from playing a role in regulating LESP. It may exert i t s effect at levels not detectable by radioimmunoassay. In 1967 Brown and Parkes postulated the existence of a duodenal, pH dependent, dual hormonal mechanism for the control of gastric motor activity. In such a mechanism, low duodenal pH would inhibit gastric motor activity and delay gastric emptying, by the release of some humoral agent, whilst a high duodenal pH would increase gastric motor activity and possibly increase the rate of gastric emptying. The effect of motilin on the rate of gastric emptying was investigated in dogs equipped with gastric fistulae. Intravenous infusion of porcine motilin was found to accelerate the emptying of a neutral test meal (Fig. 53). It had no effect on the rate of emptying of solids (Fig. 54). The site of action of the motilin during the emptying of/ the liquid was deter-mined to be the fundus, antrectomy having l i t t l e effect on the response, as shown in Fig. 56. The decrease in sensitivity of this response to motilin after truncal vagotomy (shown in Fig. 55) suggested that the response depended... on some interaction between motilin and a cholinergically-mediated' neural reflex, at the level of the fundic musculature (Debas et a l , 1977). These results were in direct contradiciton to those obtained by Ruppirt et al (1975) in man. . They described a decrease in the rate of gastric emptying of an acid test liquid, after an infusion of 13-norleu-motilin. The lack of any effect - on the rate of emptying of the solid meal may be due to an over-riding or an inhibition of the motilin-induced response by the humoral factors 1 released from the duodenum by the constituents of that meal. As yet, an increased -234-release of endogenous motilin has not been demonstrated to be part of the pattern associated with increased rates of gastric emptying, and the physiological im-portance of this action is uncertain. Investigation of the mode of action at the cellular level has involved the development of an in vitro preparation for the assay of motilin activity. Domschke et al (1976) have reported the development of such a preparation u t i l i z i n g either rabbit duodenal muscle or strips of human fundus. They were able to demonstrate a dose-related contractile response to natural or synthetic motilin. This response was unaffected by ganglionic blockade, axonal blockade, atropine or antihistamine, suggesting that motilin acted at a receptor on or in the muscle c e l l . Motilin may exert i t s effect on muscle by influencing the I | transport of calcium (Ca ) within the cytosol. The action of motilin was I | . . blocked by the Ca antagonist, verapamil. Increased cyclic guanosine 3'5' I | monophosphate (cGMP) levels, associated with the rapid release of Ca from microsomal fractions, were observed during the response to motilin. Strunz et a l (1976) found that the response of isolated rabbit pyloric muscle to acetylcholine was enhanced by pretreatment with subthreshold levels of motilin. This augmentation was not associated with either increased acetyl-choline synthesis or decreased acetylcholine degradation. The doses of motilin required to stimulate the in vitro preparations were relatively larger than those which promoted gastrointestinal motility in the intact animal. It is possible that circulating, subthreshold levels of motilin in the intact animal may be contributing to the cholinergically-maintained tone in the gastrointestinal nusculature and that functionally significant changes in serum IR- motilin levels - 2 3 5 -are.not detectable in,the radioimmunoassay system.. A.second possibility is that motilin is not transported to i t s target c e l l via the circulation but is merely passed via the extracellular f l u i d to the adjacent c e l l where i t acts, in a paracrine fashion.. The i n a b i l i t y of other groups, to establish an in vitro assay using tissue from rats, guinea pigs and dogs can only be explained'at this time- by species differences.. It is now suggested that there is no evidence that motilin normally plays any physiological role at the levels of IR- motilin achieved by infusion of 1 ug/kg/hour of polypeptide or after duodenal infusion with a l k a l i at pH 10.2. The hypothesis is that the role of motilin. is to maintain the interdigestive pattern of gastric motor activity-, the effective-levels being much lower than those achieved during these experiments. A study of IR- motilin levels during- a continuous recording'of the interdigestive- and digestive patterns of motor activity would help confirm or deny this supposition.. A second question which should be answered is whether motilin is acting independently in controlling contractile activity or whether i t is acting in concert with some other humoral or neural reflex, probably cholinergically-mediated. If this is the case, the effective- increases*in motilin levels-may only occur at the cellular level and may neyer be reflected by changes in the systemic levels of the polypeptide. The physiological 'stimulant for motilin release:would be the rise in duodenal and jejunal pH occurring in the fasted state. The absence of increased systemic levels of IR- motilin associated with any specific part of the digestive cycle and the lack of evidence regarding the existence of any hypersecretory syndrome set motilin apart from the other known gastrointestinal hormones and would suggest that motilin may be acting as a local hormone or in a paracrine manner, rather than as a classical endocrine hormone. -236-The polypeptide isolated from the intestinal mucosa of hogs by Brown et a l (1969) was named gastric inhibitory polypeptide (GIP) because of the early observation that the pure porcine material, infused intravenously in dogs, inhibited stimulated gastric acid secretion,.in a dose related manner (Pederson and Brown, 1972). A second, at least equally important, biological activity of this polypeptide was demonstrated by Dupre et al (1973). They infused porcine GIP in men and were able to measure an enhanced insulin response to an intravenous glucose infusion. Pederson et a l (1975A, 1975B) confirmed this finding in dogs. Their results would suggest'that GIP might also be inter-preted as glucose-dependent insulinotropic polypeptide (Brown and Dryburgh, 1977). A radioimmunoassay, specific for GIP, was developed by Kuzio et a l (1974) to determine the physiological secretagogues for GIP and the relative importance of the endogenously-released polypeptide in gastric acid secretion or insulin release under normal or pathological conditions. Antisera to GIP have been obtained in guinea pigs and rabbits after immuni-zation with GIP conjugated to BSA. GIP would not appear to be a good immunogen, no satisfactory antisera having been produced in animals immunized with non-conjugated GIP, and even the conjugate has produced good antisera in only a small percentage of the animals injected. A l l the studies reported from this laboratory, up to the present time, have been based on a radioimmunoassay u t i l i z i n g one antiserum, Van 8, and the assay conditions have been established to produce the most sensitive assay for this particular antiserum. When an alternative antiserum, Ro7,.was introduced, the assay system had to be changed to the disequilibrium type to achieve a satisfactory degree of label displace-ment, as shown in Fig. 59. -237-When i t was revealed that IR-GIP did not exist in a homogeneous'• form, the crossreactivity of the available antisera was examined with the. various hetero-logues of IR-GIP. The original antiserum, Van 8, crossreacted with 3 different immunoreactive forms of GIP: GIP^, G I P 5 0 0 Q and ProGIP CFig. 72 and Fig. 73) and antiserum GO 5 behaved i n a similar fashion. Antiserum Ro7 and antiserum Van 8 crossreacted with GIP^QQQ in the interassay control preparations to the same degree, but did not agree in the measurement of ProGIP, as illustrated in Fig. 80. Antiserum Ro7 appeared to have.a greater af f i n i t y for this form of IR-GIP than did antiserum Van 8. The total IR-GIP response to any stimulus consists of varying proportions of at least 3 components, as determined by these antisera. It is obvious'that comparisons between IR-GIP responses to stimuli may only be made i f the studies are performed with antisera whose behaviour with the various IR-GIP heterologues is also comparable, or better s t i l l , with the same antiserum throughout. The usual technique used in the iodination of GIP has been a modified version of the chloramine-T method. In an attempt to reduce the damage done to the GIP molecule by the oxidising agent, pilot studies were performed, varying the concentration of chloramine-T and the length of the exposure time. The specific activity of the resulting label, calculated from the percentage incorporation 125 of I into the molecule was 350 mCi/mg when GIP was exposed to a chloramine-T concentration of 4 Ug for 120 sec. This compares well with the routine label, whose specific activity, calculated in the same way, was 120 mCi/mg, (from the radiochromatogram in Fig. 21). An improved label i s lik e l y to result from further studies with reduced concentrations of chloramine-T and varied periods of exposure. It should be noted that this method of calculating the specific activity is much less reliable for GIP than i t was for motilin. With motilin there was l i t t l e difference in the values obtained, whether they were calculated -238-from the radiochromatogram or from a separate assay, requiring the addition of label at several dilutions. This i s not the case with GIP. The label obtained in the procedure illustrated in Fig. 21 had an apparent specific activity of 120 mCi/mg. The same label, after a label dilution assay, had an estimated specific activity of 68 mCi/mg. This indicates that a smaller pro-portion of the labelled polypeptide eluting in the f i r s t peak represented 125 mono-iodinated I-GIP. The molecule has two tyrosyl residues, one N-terminal, the other at position 10. At present, there is no way of knowing which tyrosine is the more available to the incorporation of iodine, or whether the tertiary structure of the molecule is such that the N-terminal portion of the peptide is indeed readily accessible to the incorporation of iodine at a l l . The lack of improvement in the specific activity of the label after the gentler lactoperoxidase method of iodination would suggest that neither tyrosine is readily accessible to iodine incorporation. Purification of the label by gel f i l t r a t i o n on Sephadex G-25 w i l l separate the iodinated polypeptide from the unincorporated isotope but f a i l s to separate the labelled and unlabelled peptides from each other, and is barely adequate for separation of mono-iodinated GIP from the d i - and tri-iodinated forms. The presence of uniodinated GIP in the labelled preparation is a contributing factor to the low specific activity, blunting the sensitivity of the upper end of the standard curve and limiting the concentration of labelled antigen which may be added to the assay. 125 Subsequent ion exchange chromatography on QAE Sephadex A-25 of I-GIP, i n i t i a l l y isolated by gel f i l t r a t i o n , .has produced an iodinated GIP with a specific activity in the range 200-250 mCi/mg (estimated by the label dilution method). This improve-ment i s probably due to the removal of the unlabelled polypeptide, which elutes -239-ahead of the labelled material in the system employed, (Fig. 22). The routine inclusion of interassay controls:in the•assay have made i t easier to monitor the" performance of the assay system and.acts as a reliable Index for the estima-tion of the .effect of variations: in procedure on the sensitivity of the assay; Antisera to porcine GIP, raised in rabbits, were covalently coupled to Sepharose 4B and tested as an alternative to dextran-eoated charcoal in the separation of bound antigen.from free antigen in the incubation mixture. The binding of the antibodies to the agarose was complete, as indicated by.the lack of antibody activity in the wash, (Fig. 19). The coupled antiserum, tested in the radio-immunoassay, appeared to possess only a small percentage of the original anti-body activity. This was indicated by.the reduction in the t i t r e of the coupled antiserum, required to produce a maximum binding of 30% of the labelled tracer, shown in Fig. 46. The coupled antiserum at this lower t i t r e also showed a diminis-hed sensitivity to the addition of unlabelled antigen. (Fig. 47) The results obtained with Sepharose-coupled antisera to the steroid hormones and the low molecular weight polypeptide hormones, e.g., gastrin, would suggest that the antibody activity, in the radioimmunoassay system, was unaltered by the presence of the solid matrix or the coupling process (Bolton and Hunter, 1973). The Sepharose-coupled antiserum in the radioimmunoassay for motilin, another small polypeptide, also demonstrated a high recovery of antibody activity and no loss in sensitivity potential. The same was not true for insulin, human growth hormone, human thyroid stimulating hormone or GIP. A l l these radioimmunoassay systems showed a loss in antibody activity and sensitivity when the antibody was coupled directly to the Sepharose matrix. Bolton and Hunter suggested that there was a c r i t i c a l size of antigen, above which steric hindrance prevented the molecule -240-from having complete access to the binding sites on the antibody. This c r i t i c a l size must l i e between 2700 (motilin) and 5105 (GIP). Direct coupling of GIP antisera to.Sepharose was an uneconomical way of u t i l i s i n g the antisera, and this technique has not been used in studies on the extra-ction and purification of GIP. This problem may well be overcome by interposing a hydrocarbon chain between the ligand and the solid matrix. The use of such a hydrocarbon spacer has dramatically improved the effectiveness of several Sepharose systems in the purification of enzymes (Cuatrecasas, 1970), especially in low affinity systems. The reaction involves the coupling of the spacer, such as ethylene diamine or the tripeptide GLY-GLY-TYR to the activated Sepharose, followed by the 1 coupling of the ligand to the spacer by the carbodiimide reaction. The inc-rease in distance between the solid and the antibody reduces the steric hindrance imposed by the presence of the matrix and increases the f l e x i b i l i t y and mobility of the ligand. It would be advantageous to pursue the possibility of a coupled ligand for GIP for the following reasons: The separation procedure in the routine assay be-comes more rapid, entailing no extra addition step, as in the dextran-charcoal separation, and no further incubation, as in the double antibody system. Centri-fugation for 5 min. at 2000 rpm is adequate for packing the solid, allowing the supernatant liquor to be decanted. The system is unaffected by the plasma concentration in,the normal radioimmunoassay, and is•less disruptive to the primary antigen/antibody.reaction than charcoal addition. Reduction of the incubation volume to a minimum obviated the need for rotation of the incubation tubes and therefore they do not require stoppering. -241-With the advent of a radioimmunoassay i t became possible to investigate the physiological function of GIP, f i r s t l y as an inhibitor of gastric acid secretion and strong candidate for the role of enterogastrone. The term "enterogastrone" was defined by Kosaka and Lim to describe the humoral agent postulated to be released from the duodenal mucosa by fat or fat digestion products and respon-sible for the inhibition of gastric acid secretion and the delay in gastric emptying. Pederson and Brown (1972) demonstrated that porcine GIP was effective in dogs in inhibiting gastric acid secretion, whether that secretion was stimu-lated by infusion of pentagastrin or histamine, or by vagal stimulation (induced by insulin-mediated hypoglycaemia). In studies where the extrinsically denervated fundic pouch was stimulated to produce ~75% of i t s maximum secretory capacity, a significant degree of inhibition was observed at doses of 1 ug/kg/hour. When serum samples, obtained from human volunteers after ingestion of a normal breakfast, were subjected to radioimmunoassay, they were found to rise from a mean fasting level of 237 - 14 pg/ml IR-GIP (mean - SE) to a mean level of 1200 pg/ml IR-GIP, and they remained elevated above basal levels for periods in excess of 3 hours, (Kuzio et a l , 1974). When the various components of the meal were tested individually, oral ingestion of both glucose (Cataland et a l , 1974) and fat, in the form of a triglyceride emulsion (Brown et a l , 1974) were found to produce a significant elevation in the circulating levels of serum IR-GIP. Ingestion of protein, in the form of either a meat, extract or a fat-trimmed f i l e t steak, produced no such increase in the circulating levels of IR-GIP (Brown et a l , . 1975). When the original studies were duplicated in dogs, the circulating levels of IR-GIP, achieved during the exogenous infusion of GIP, sufficient to produce a significant inhibition of the gastric acid output, were determined to l i e within the range of serum IR-GIP levels released by ingestion of fat. Similar IR-GIP responses were obtained in dogs when the stimulus, "either fat or glucose, was -242-administered as an intraduodenal infusion. Confirmation of the inhibitory action of endogenous GIP was obtained in studies in dogs. Acid, stimulated by a continuous infusion of pentagastrin, was i n -hibited by intraduodenal infusion of fat (Fig. 66), glucose (Fig- 67) or hydro-chloric acid (Fig. 68) but not saline (Fig. 69). The inhibition of fundic pouch acid secretion by fat or glucose was accompanied by a concomitant elevation of serum IR-GIP. There was no significant change in IR-GIP levels related to the acid-induced inhibition, and intraduodenal perfusion with saline produced neither inhibition of gastric.acid secretion nor change in the serum IR-GIP levels. It would seem that GIP i s , in strong likelihood, the enterogastrone pos-tulated by KOsaka and Um, according to the evidence obtained in dogs. The evidence in man, regarding the inhibitory role played by GIP released by fat digestion, i s less strong. CTeatbrTand-Gourlay(1975) found that exogenous GIP would inhibit gastric acid secretion in man at a dose of 2 ug/kg/ 30 min. which resulted in circulating IR-GIP levels well above those achieved by ingestion of fat in the same subjects; GIP does not appear to play any part in the autoregulation of gastric acid secretion by duodenal acid. This finding was supported by the lack of any IR-GIP response to ingestion of protein or alcohol, or the passage of pentagastrin-stimulated acid into the ^duodenum (Cleator and Gourlay, 1975). The evidence supporting the claims of the other gastrointestinal polypeptides to be enterogastrone has gradually been diminished. Secretin is not released in any significant amounts by ingestion of fat, and has'.been shown to have l i t t l e inhib-itory effect on the secretory or motor activity of the stomach when infused in -243-doses which mimicked the circulating levels of IR- secretin achieved by duo-denal acidification. Cholecystokinin-pancreozymin is released by fat, but much of the inhibitory activity reported as occurring after infusion of the polypeptide can be accounted for by the GIP contamination in the impure pre-paration of cholecystokinin-pancreozymin used in these studies. A third possible enterogastrone, VIP, is a potent inhibitor of gastrin- or histamine-stimulated acid secretion, when i t is administered exogenously (Barbezat and Grossman, 1971) but no mechanism for the physiological release of VIP has yet been described. GIP may not be the only enterogastrone but i t would appear to be a major factor in the humoral reflex so designated. The actual mecha-nism of GIP release after ingestion of fat remains to be elucidated. The reduced IR-GIP response to a test meal in patients with coeliac disease would suggest that the rate of absorption of the nutrients is an important factor. As yet, no significant reduction in the absolute number of GIP-producing cells has been detected in biopsy samples from the jejunal mucosa of these subjects (Creutzfeldt et a l , 1976). The search for the duodenal factor involved in a second humoral reflex dates back to the work of Moore et a l (1906). They were able to relieve the glyco-suria of patients with diabetes mellitus by orally administering an extract of the duodenal mucosa. A hypoglycaemic fraction was separated from a crude secretin preparation. It had no secretin-like effect on the exocrine pancreas and was not insulin. This fraction was named "incretin" by La Barre (1932) when he postulated a possible role for this duodenal factor in the treatment of diabetes mellitus. With the advent of the radioimmunoassay for plasma insulin (Yalow and Berson, 1958) i t became possible to compare the IR- insulin response to glucose adminis--244-tered orally or intravenously (Elrick et a l , 1964). The greater insulin response: and: improved glucose tolerance which '.accompanied: glucose administra-tion by the oral route was ..postulated to be due to the release of some insulo-tropic factor from the duodenal-jejunal mucosa (Mclntyre et a l , 1965). This postulated intestinally-mediated regulation of endocrine pancreatic function has been termed "the enterbinsular axis". If a gastrointestinal polypeptide is to be seriously•considered as a candidate for the role of incretin in this enteroinsular reflex i t must satisfy the following c r i t e r i a . It must be demonstrated that glucose is a stimulant for i t s release. It should be shown that exogenous administration of this poly-peptide, in doses achieving circulating immunoreactive levels within the physiological range, administered in parallel with an intravenous'glucose load, w i l l mimic the pattern of serum IR- insulin release and glucose tolerance observed after oral or duodenal administration of glucose alone. In light of these requirements mast of the established gastrointestinal peptides have been ruled out as possible candidates. Secretin and cholecystokinin-pancreozymin are not released by ingestion of glucose in physiologically effective levels, as demonstrated by the lack of effect on the exocrine pancreas (Mahler and Weisberg, 1968) whilst only a slight elevation in serum IR- gastrin levels were observed (Rehfeld and Stadil, 1973). When gastrin, secretin (Lerner and Porte, 1972) or the synthetic octapeptide of cholecystokinin-pancreozymin (Frame et a l , 1975) were infused intravenously, in conjunction with intravenous'glucose, a l l three peptides produced a transitory, enhanced insulin response, characteristic of the i n i t i a l phase of insulin relaase. The response was over within 10-15 min. even when the polypeptide infusion was continued. -245-In 1973, Dupre et a l measured: the IRT-;insulin response:in normal, volunteers to intravenous infusion of 0.5 g/min glucose alonej glucose infusion with the addition of 1 ug/min pure porcine GIP and GIP infusion alone. The addition of GIP resulted in an;enhanced IR- insulin response to the glucose infusion,during both the i n i t i a l phase of insulin release and the later sus-tained phase. This same dose of GIP, without the glucose, had no insulino-tropic action. The levels of circulating IR-GIP reached during this infusion were comparable to those achieved i n the same subjects after the ingestion of 50 g glucose. The effect of endogenous GIP on insulin release in man was reported by Brown et a l , CL975). The IR- insulin response to intravenous glucose was potentiated by GIP released after the ingestion of fat, in the form of a triglyceride emulsion. Although GIP- mediated insulin release has been demonstrated in the fasted dog (Pederson et a l , 1975b) i t is probable that the effective levels of IR-GIP achieved were pharmacological rather than physiological. In man, some degree of hyperglycaemia was essential i f physiological levels of GIP were to be insulinotropic. Studies in the isolated rat pancreas prepa-ration have confirmed that GIP is capable of augmenting the sustained insulin response to glucose, in a dose-related manner, (Pederson and Brown, 1976). Their findings suggest that GIP is only effective as an insulinotropic agent in the presence of a glucose concentration which i s i t s e l f capable of stimula-ting insulin release from the pancreas. In the presence of 8.9 mMglucose, GIP was effective in doses as low as 1 ng/ml perfusate and in the presence of a fixed GIP concentration, increasing glucose concentrations stimulated insulin release in an exponential manner. -246-GIP has also been shown to potentiate the insulin response to glucose in the isolated rat pancreatic i s l e t preparation (.Schauderet a l , 1975) at glucose concentrations above a. threshold level, which lay between 6-8 mM. The effective dose of GIP, however, was.lOyg/ml incubate at the lower glucose concentration, and 1 jag/ml at the higher concentrations. The reason for this much greater GIP requirement.in this preparation, compared to that of the isolated pancreas or the intact animal, is not degradation of GIP during the incubation period. It i s possible that exposure of the islets to collagenase and pancreatic proteolytic enzymes, during their iso-lation, causes some alteration.to their membrane structure and reduces their sensitivity to the action of GIP. An alternative explanation is that GIP requires the presence of some intermediate for f u l l expression of i t s biolo-gical potency and this substance has been destroyed or lost in the isolation procedure. In both i n Vivo and i i i vitro systems GIP has satisfied the cr i t e r i a for i t s establishment as a major factor in the entero-insular axis. Teleologically, i t i s desirable that GIP should have no insulinotropic action in the fasted animal. It would be inappropriate for insulin to be released when serum glucose levels were not elevated. In 1973, Raptis et a l reported that an intraduodenal infusion of a mixture of amino acids was a stimulant for insulin release, whilst intravenous i n -fusion of these same acids was far less effective. This same intraduodenal amino acid perfusate was found to be a stimulant for GIP release, whereas an intravenous infusion resulted i n no detectable IR-GIP production, although the serum a-amino nitrogen levels achieved were much higher, (Thomas et a l , 1976). In this situation GIP was shown.to be insulinotropic i n the absence of measurable hyperglycemia and the authors suggested that GIF w i l l also act to enhance the -247-pancreatic insulin response in the presence of hyperaminoacidaemia. However, they did not measure serum IR- gastrin:levels in this study and i t is possible that the gastrin response.to an amino acid infusion would contribute to the insulinotropic response.observed.after protein ingestion. Evidence supporting the claims of GXP to be the major humoral factor in both the enterogastrone and entero-ins^lar reflexes i s gradually accumulating. IR-GIP released after ingestion of a mixed meal demonstrated a biphasic pattern, with the early peak occurring approximately 45 min. after ingestion of the meal,,and a second, more prolonged response being seen between 120-180 min. (Brown et a l , 1975). Ample evidence exists relating the i n i t i a l response to the glucose content of the.meal, correlating i t with the increase in serum glucose and the period just prior to the peak response of IR- insulin. The second peak compares well with the IR-GIP response to orally administered fat. The GIP released by either fat or glucose appeared to be effective as an enterogastrone or an insullnotropic agent. However, i f the serum glucose and IR-GIP levels achieved after oral glucose were duplicated by an intravenous glucose infusion with either intraduodenal fat infusion or intravenous porcine GIP infusion, the IR- insulin response after fat was significantly lower than that produced by either oral glucose or exogenous GIP, (Pederson et a l , 1975B). From the feeleological angle i t is desirable to have an immediate insulin response to a carbohydrate-containing meal but i t would be most inap-propriate for the gastric acid secretion to be inhibited this early in the digestion of the meal. In light of these findings i t seemed advisable to i n -vestigate the nature of the IR-GIP released after these different stimuli. The antiserum routinely used in the GIP assay, Van 8,was unable to differentiate -248-between the serum IR-GIP released by f a t or glucose ( F i g . 71) but i f these same serum samples were subjected.to chromatography on Sephadex G-50, three regions .of immunoreactiyity were detected i n the fr a c t i o n s obtained, CFig- 72 and F i g . 73)(Dryburgh and Brown, 1976). The f i r s t region (IR-GIP ) eluted i n the void volume of the.column and was s i g n i f i c a n t l y diminished i f the serum was pretreated by b o i l i n g or with 6.OM urea. CFig' 77) This would suggest that GIP v o represented a complex formed by the non-specific binding of GIP to a serum prot e i n . A second region corresponded to the e l u t i o n pattern of natu r a l porcine GIP C G I P ^ Q Q Q ) and a t h i r d immunoreactive region eluted ahead of the normal GIF and was determined to have a molecular weight of 7500-8000, as i l l u s t r a t e d i n F i g . 83. This form of IR-GIP was designated ProGIP. No attempt has yet been made to examine the r e l a t i v e r a t i o s of G I P v q 5 ProGIP and G I P ^ Q Q Q i n f a s t i n g serum. The early GIP response to e i t h e r glucose or f a t was characterized by the proportions of the IR-GIP components bearing the re l a t i o n s h i p GIP > GIPr_rt(_ > ProGIP. As the stimulation was continued, r vo 5000 the percentage of G l P v o remained r e l a t i v e l y constant, whilst that of G I P , - Q Q Q increased. S t i l l l a t e r , t h e r e l a t i v e proportions of g 1 P 5 Q O O ProGIP were reversed. An a l l studies, i n man or dog, a f t e r glucose or f a t , the percentage of the t o t a l IR-GIP represented by ProGIP increased with increase i n time a f t e r the stimulus, as t y p i f i e d i n F i g . 75. Chromatography of p a r t i a l l y p u r i f i e d extracts from the duodenal mucosa of hogs demonstrated that they also contained IR-GIP i n the ProGIP and G I P 5 0 0 Q forms. The highest r a t i o of ProGIP ; G I P 5 Q 0 0 was found i n the neutral soluble, methanol insoluble extract ( F i g . 7 8 ) . Attempts which have been made to p u r i f y ProGIP from t h i s extract have not been successful. Rechromatography of mater i a l , supposedly. containing no G I P ' 5 Q Q Q , r esulted i n approximately 3 0 % of the t o t a l IR-GIP.recovered being i n the G I P , - Q 0 0 form, as shown i n F i g . 8 2 , -249-indicating that ProGIP had yet:to be.isolated in a stable form. From i t s behaviour on CM-cellulose, ProGIP was determined to be less basic than G I P ^ Q Q Q , CFig. 80). One might expect a functionally different/molecule to be more stable than ProGIP has so: far proved to be. It i s possible that this molecule represents a precursor form of G I P ^ ^ Q Q . In this case, the i n i t i a l IR-GIP response to any stimulus might then consist, predominantly, of already pre-formed G I P ^ Q Q Q . As the stimulus persisted the IR-GIP response would gradually change to contain increasing amounts of the precursor form, as the preformed pool of GIP ^ Q Q Q , .diminished and increased precursor was released as the synthesis of GIP accelerated. Biosynthesis studies, with t r i t i a ted amino acids, would help to answer some of these questions about the actual rate of GIP synthesis; under various conditions, and might also elucidate the relationship between G I P ^ Q Q Q and ProGIP. An alternative explanation for this phenomenon might be that the different forms of IR-GIP are being produced by different populations of APUD ce l l s , spatial ly separated. This hypothesis would require that G I P ^ Q Q Q be synthesized and secreted by cel l s predominantly located in the upper region of the duodenum, whilst the ProGIP cells would be situated more d i s ta l ly . As the stomach contents pass into the upper intestine they w i l l i n i t i a l l y stimulate primarily GIPJ - Q Q Q - containing ce l l s . Later this mixture of nutrients would come into contact with the lower, ProGIP-producing ce l l s . The biologica l potency of ProGIP, relative to that of G I P 5 0 0 Q is d i f f i cu l t to estimate in view of the ins tab i l i ty of the material. A second unknown factor i s the biological potency of that proportion of the tota l IR-GIP complexed to serum .protein. This phenomenon has been demonstrated with other polypeptide hormones,, e .g . , gastrin Of alow and Berson,,1972) and insul in (Sramakova et a l , 1975). The function of this type of complex was -250-studied by Simon and Antonlades (1975). They measured the transport of insulin across the isolated rat mesentery, in the presence.of human serum-bound insulin. They foundsthat the insulin transport'was inhibited in a specific manner. One might extend this, finding to postulate that naturally-occurring serum protein/polypeptide complexes would limit the transport of the polypeptide across*the membrane of the target c e l l or reduce i t s activity at that membrane by competitively binding with the receptor sites thereon. The complex might also act by sequestering, temporarily, some of the poly-peptide in the serum. "Big, big" gastrin was found to be a major component of the total IR- gastrin in.the fasting serum of men, dogs and pigs. Its release was not stiniulated by feeding (Yalow, 1974), leading one to suspect that i t did not play an active role'in the gastrin-mediated acid response to feeding. The existence of a similar "big, big" insulin in normal subjects is less well documented. It has, however, been demonstrated to comprise a large percentage of the total IR- insulin in the fasting and stimulated serum in certain pathological conditions, (not insulinomas). These patients have ex-tremely high basal and stimulated IR- insulin levels but rarely experience hypoglycemic attacks after prolonged fasting or limited food intake. The most common time for hypoglycaemia to occur in these subjects was a few hours after a substantial meal. Sramkova et a l (1975) postulated that these findings could be accounted for i f the IR- insulin was predominantly in an inactive form, the hypoglycaemic attacks being due to disruption of this complex and the liberation of the biologically active insulin after the stress of the large meal. In the light of these observations, i t was postulated that G l P v o is either biologically inactive or .has reduced potency. The biological activity of GIP would not then correlate directly to the total IR-GIP response measured. Bearing this in mind, the experiments performed by Pederson et a l , comparing -251-the IR- insulin response to matched serum glucose and IR-GIP levels, obtained by various means, were reconsidered. The peak .levels of IR-GIP and the Integrated insulin response achieved after intravenous glucose and porcine GIP were arbitrarily considered.to be 100% of the possible response. The various IR-GIP components of the total IR-GIP response to oral fat were determined by column chromatography and were expressed as percentages of the total IR-GIP response; The integrated IR- insulin response achieved after intravenous glucose and oral fat was expressed as a percentage of the integra-ted IR- insulin response obtained with intravenous glucose and GIP. When these values were plotted in', histogram form (Fig. 84), the closest correlation to the biological activity (i.e., the insulin response) was obtained by com-bining the IR-GIP 5 0 0 Q and IR-ProGIP, and ignoring the IR-GIP^ component. (Fig. 84) The existence of several molecular forms of IR-GIP cannot yet account for the apparently differeat functions of GIP, i.e., i t s i n i t i a l incretin-like effect and the later enterogastrone effect. Another, as yet unexplained, phenomenon is the reduced IR-GIP response stimulated by oral fat in the presence of an intra-venous glucose infusion, compared to that produced by oral fat alone, (Crockett et a l , 1976). The answer to these problems may l i e in a study of the other humoral mechanisms acting at the same time, or in the identification of some factor which inhibits the action of GIP at the level of either the parietal or the pancreatic 3 c e l l s . The well documented effect of intravenously, adminis-tered .somatostatin on the pancreatic secretion of both insulin and glucagon in Vivo, (Alberti et a l , 1973 : Mortimer et a l , 1974 : Koerker et a l , 1974) and in vitro, (Gerich et a l , 1975) stimulated interest in the possible effects of somatostatin on the insulin response to GIP and on GIP release after physiolo--252-[3 IR-Insulin [[]]] IR-Pro-GIP 0 I R- G I P5000 H IR"GIP> vo CO c o CL CO Q_ or CD ® 06 § s O CO Q- c o C CD O L_ CD CL 100 n 80H 6 0 H r 40H 20H 0-» / / / 7 A . A. B. Fig. 84 The insulin response and serum IR-GIP levels associated with that response, showing the relative proportions of the different heterologues of IR-GIP. The serum glucose and IR-GIP levels were comparable after (A) i.v. glucose and i.v GIP infusions (considered as producing 100% of the possible response) and (B) i.v. glucose and oral fat administration. -253-gical.stimulation. Somatostatin was administered as a bolus injection, immediately prior to the exogenous administration of an intravenous GIP i n -fusion, normally insulinotropic in the fasted dog (Pederson et a l , 1975). The insulin response was delayed and the serum glucose values measured re-flected this insulin inhibition, as shown.in Fig. .63. The effect of a bolus injection of the synthetic somatostatin was to delay.the release of IR-GIP, stimulated by either oral glucose or fat. In the case of the studies with oral glucose the insulin response was also delayed (Fig. 60 and Fig. 62). If the somatostatin was administered as an infusion, the IR-GIP and IR-insulin responses were delayed u n t i l the end of that infusion. The IR-GIP response was also diminished, when compared to the control values achieved after oral glucose alone. The insulin response, however, re-bounded to values significantly above the control values, a phenomenon not explicable,in terms of the prevailing serum glucose levels (Fig. 61). A similar rebound response of insulin was observed by Mortimer et a l , (1974) and the same phenomenon was observed with gastrin (Bloom et a l , 1974), when an infusion of somatostatin, administered during the ingestion of a provacative meal, was terminated. Somatostatin, therefore, appears to block the endogenous release of GIP and also to inhibit the action of.circulating GIP.at the level of the c e l l . The effect of somatostatin, on GIP- mediated gastric acid.inhibition has not been examined but:the presence of somatostatin-containlng cells in the gastric mucosa has been demonstrated by Dubois (1975). It would not be unexpected i f intravenous infusion of somatostatin, was also found to have a. modulating inf-luence on the response of the parietal c e l l to endogenous GIP. The/possibility should be considered that the disparate actions of GIP on the gastric parietal -254-and pancreatic 8 c e l l are being influenced separately by somatostatin of gastric and pancreatic origin respectively.. The stimulus.for, and the time of release of the peptide from these different regions need not be identical. No information i s available about the endogenous release of somatostatin at the present time. . . . . i . The possibility that some inhibitory feedback mechanism existed between the endocrine pancreas and GIP releasewjas^,. suggested by. the observation that subjects with maturity onset diabetes exhibited an abnormally elevated IR-GIP response to oral glucose or fat. The inhibitory factors implicated were insulin, glucagon or the degree of hyperglycaemia achieved. When an insulin injection was administered to normal human volunteers, the serum glucose levels being clamped in the fasting range, the IR-GIP response to fat ingestion was significantly less than that observed in the control situation. The time course of the peak serum IR- insulin response did not, however, correlate well with the IR-GIP depression (Brown et a l , 1975). It would have been pre-ferable i f the insulin had been given as an infusion rather than a bolus i n -jection. Ebert et al (1976) infused glucagon intravenously for 2 hours during the i n -gestion of a provocative test meal and recorded a significant depression of the IR-GIP response. This effect could not have been due to the insulin released, there being no significant difference in; the circulating IR- insulin response to the test meal, whether the glucagon was being infused or not. The hyperglycaemia stimulated by the glucagon was also unlikely to be the modulating influence. Diabetics.with significantly higher levels of serum -255-glucose demonstrate an exaggerated GIP response to the same challenge. In normal subjects, the evidence so far accumulated, regarding endocrine pancre-atic control of GIP release i s most persuasive with respect to glucagon. Creutzfeldt and Ebert (1976) have also confirmed the inhibitory effect of somatostatin on GIP release and GIP-medlated response in man. Further information about the mechanism of the action of GIP and i t s control was obtained from studies performed in subjects with abnormal digestive metabolism, or who had undergone gastrointestinal surgery. The stimulated IR-GIP response was significantly reduced in patients with^coeliac disease, with a concomitant reduction in the IR- insulin response (Creutzfeldt et a l , 1976). The most l i k e l y cause of the diminished GIP output is the defective absorption of nutrients, symptomatic of this disorder. Creutzfeldt and Ebert (1976) studied the importance of adequate nutrient absorption i n rats, by comparing their IR-GIP response to glucose, administered with or without phlorizin. The addition of the phlorizin v i r t u a l l y abolished the release of IR-GIP. An alternative explanation for the low GIP levels measured i s the possibility that the absolute number of GIP-producing cells has been reduced because of the villous atrophy, characteristic of coeliac disease. The majority of the GIP cells are found, however, in the crypts of the intes-t i n a l mucosa and villous atrophy would probably result in only an insignificant reduction in the GIP c e l l population.. Exaggerated GIP responses to stimulation by a mixed meal were observed after any surgical procedure which resulted in accelerated gastric emptying (dumping) e.g., gastrojejunostomy or vagotomy and pyloroplasty. This mechanism has also been put forward to explain the elevated IR-GIP levels measured in duodenal -256-ulcer patients (Creutzfeldt and Ebert, 1976). A similar elevation in stimulated IR-GIP levels was the general- rule in chronic pancreatitics, possibly due to the loss of some feedback control by glucagon or insulin (Botha et a l , 1976 : Ebert et a l , 1976). The most marked IR-GIP response, however, was observed in patients with moderate hypoinsulinaemia, whereas those with severe insulin depression had an IR-GIP response which approached the normal values. This apparent anomaly i s probably due to a combination of factors, affecting the release of GIP In different ways. The lack of insulin would lead to an overproduction of GIP but the associated gross exocrine pancreatic deficiencey would result in an abnormal fat metabolism, leading to malabsor-ption of fat (with associated-steatorrhea) and a reduction in the release of IR-GIP. The relative hyperglucagonaemia, reportedly occurring in severe cases of chronic pancreatitis (Kalk et a l , 1974) could also contribute to the re-duction in the GIP response. In the f i n a l outcome these factors would balance each other, and the IR-GIP response would appear to be f a i r l y normal. The further exaggeration in the levels of IR-GIP released after a p a r t i a l duodeno-pancreatectomy would result from the accelerated rate of gastric emptying, following surgical interference. The situation, in the cases of maturity onset diabetes (Brown et a l , 1975) and obesity (Ebert et a l , 1977) is slightly different. In these situations the GIP response to a test meal i s abnormally high, in the presence of relatively high insulin levels. The insulin deficiency may be regarded as functional rather than absolute, the GI.P-producing c e l l being unresponsive to the inhi-bitory action of that insulin. The possibility exists, and should be investi-gated, that this lack of sensitivity.to the insulin may be due to high percen-tage of the total IR-insulin measured being in the form of the relatively -257-inactive proinsulin. This .insensitivity would appear to be.reversible, by sulphonylureas in the diabetics or by diet-mediated weight reduction in the obese subjects. In a l l the previously mentioned abnormal conditions; only the stimulated IR-GIP levels were abnormal. In juvenile onset diabetes the fasting IR-GIP >.• levels were in excess of 1 ng/ml. The fasting IR-GIP levels in.the obese subjects, after prolonged starvation, approach this value. Both these situa-tions are characterized by insulin deficiency and a high level of circula-ting ketone bodies. Treatment with insulin or food, respectively; reverses both these symptoms and reduces the IR-GIP output. The role of hyperketonaemia in the control of GIF release requires further investigation. In summary, several factors have been implicated in the regulation of GIP production. The response of the GIP c e l l may be depressed by glucagon, insulin, somatostatin or a reduction in the absorption of nutrients. The IR-GIP output would appear to be increased by surgical disruption of gastrointestinal continuity, absolute or functional deficiency in insulin or glucagon and possibly by the presence of elevated levels of ketone bodies in the circulation. If the GIP release is being inhibited by some endocrine pancreatic factor, either insulin or glucagon, this could explain the reduced IR-GIP response to oral fat in the presence of an intravenous glucose infusion, relative to that response observed when fat was administered alone and there was no pancre-atic stimulation. It could also account for the fact that the IR-GIP response to an intraduodenal infusion of glucose.is not only delayed but is also diminis-hed when i t i s preceded by a somatostatin infusion. As shown in Fig. 62, the IR-insulin response rebounded to levels significantly above the control -258-values at the termination of the somatostatin infusion and this could be responsible for the reduction in the' (SIP response. It is:interesting to speculate whether hyperinsulinaemia or hyperglucagonaemia might not only effect an:inhibition of IR-GIP release but also inhibit the activity of circulating'GIP at the level of the 8 c e l l (cf somatostatin). Another facet of the role played by.GIP in carbohydrate metabolism is i l l u s -trated in obesity. The observation that complete starvation or a low caloric diet resulted in an eventual decrease in both the IR-GIP and IR- insulin responses in obese individuals suggested that carbohydrate intake played a role in regulation of the sensitivity of the GIF- producing c e l l . It i s also possible that the number of GIP cells i s controlled by the nature of the diet. If this is so, one might question i f any pathological condition, characterized by malabsorption of nutrients (e.g., coeliac disease) might not also result in a reduction of the absolute number of GIP- producing cell s , which would contribute to the reduction in IR-GIP output observed in this circumstance . (see page 243). GIP alone was found to have a weak l i p o l y t i c effect on isolated rat adipocytes, but GIP, administered in conjunction with glucagon, was found to be strongly an t i l i p o l y t i c . It was possible to block the l i p o l y t i c action of glucagon and to displace glucagon from i t s binding sites on the adipocyte plasma membrane by the addition of GIP to the incubate (Dupre et a l , 1976 : Ebert and Brown, 1977). GIP was, however, much less effective in antagonizing the l i p o l y t i c action of secretin, and was ineffective against adrenocorticotropic hormone (ACTR), adrenaline, noradrenaline and theophylline. Obesity is a pathological condition of many aetiologies, but an impaired carbohydrate metabolism, -259-resulting:in or from an increased.carbohydrate intake, is li k e l y to produce obesity directly v i a the elevated GIP.'response and indirectly via the eleva-ted insulin response induced by GIP. The status of GIP as a hormone is established. Its predominant role would appear to be that of the major gastrointestinal regulator of carbohydrate metabolism. This is supported by the alteredTR-r-GIP response measured in c l i n i c a l conditions'related to impaired carbohydrate metabolism. GIP also plays a part in controlling the secretory and motor activity of the stomach in dogs and probably in man, but the c l i n i c a l evidence for GIP involvement in hypersecretory conditions, e.g. duodenal ulcer and Zollinger-Ellison syndrome,ofrthe'?hy.posecretory states, e.g., Werner-Morrison syndrome and . achlorhydria, is virtually non-existent. In a l l the pathological conditions so far investigated, abnormal GIP responses are symptomatic, rather than causative, of the disorder. -260-BIMQGRAPHY. 1. Alberti, K.G.M., Christensen, S.E., Tversen, J., Sever-Hansen, K., Christensen, N.J., Prange Hansen, A., Lundbaek, K., Ovsrov, H. (1973). Inhibition of insulin secretion by somatostatin. Lancet 11 : 1299-1301 2. Andersson, S., Nilsson, G., Uvn'as, B. (1967). Effect of acid on gastric secretory responses to gastrin and histamine. Acta Physiol. Scand. 7_1_ : 368-378 3. Aurbach, G.D., Keutmann, H.T., N i a l l , H.D., Tregear, G.W., O'Riordan, J.L.H., Marcus, R., Marx, S.J., Potts, J.T. (1971). Structure, synthesis and mechanism of action of parathyroid hormone. Rec.Prog.Horm. Res. 2&. : 353-398 4. Barbezat, G.O., Grossman, M.I. (1971). Intestinal secretion : stimulation by peptides. Science 174 : 422-424 j5. La Barre, J. (1932). Sur les possibilities d'un traitment du diabete par l'incretine. 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