UBC Theses and Dissertations

UBC Theses Logo

UBC Theses and Dissertations

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

Your browser doesn't seem to have a PDF viewer, please download the PDF to view this item.

Item Metadata

Download

Media
831-UBC_1977_A1 D79.pdf [ 12.15MB ]
Metadata
JSON: 831-1.0094195.json
JSON-LD: 831-1.0094195-ld.json
RDF/XML (Pretty): 831-1.0094195-rdf.xml
RDF/JSON: 831-1.0094195-rdf.json
Turtle: 831-1.0094195-turtle.txt
N-Triples: 831-1.0094195-rdf-ntriples.txt
Original Record: 831-1.0094195-source.json
Full Text
831-1.0094195-fulltext.txt
Citation
831-1.0094195.ris

Full Text

IMMUNOLOGICAL TECHNIQUES IN THE INVESTIGATION OF THE PHYSIOLOGICAL FUNCTIONS OF GASTRIC INHIBITORY POLYPEPTIDE AND MOTILIN  by JILL ROBERTSON DRYBURGH B.Sc. U n i v e r s i t y of Edinburgh, 1962  A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS FOR THE DEGREE OF DOCTOR OF PHILOSOPHY IN THE DEPARTMENT OF PHYSIOLOGY  WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD  SUPERVISOR  EXTERNAL EXAMINER THE UNIVERSITY OF BRITISH COLUMBIA A p r i l , 1977 00  J i l l Robertson Dryburgh, 1977  In p r e s e n t i n g t h i s  thesis  an advanced degree at  further  fulfilment  of  the  requirements  the U n i v e r s i t y of B r i t i s h Columbia, I agree  the L i b r a r y s h a l l make it I  in p a r t i a l  freely  available  for  agree t h a t p e r m i s s i o n f o r e x t e n s i v e copying o f  of  this  representatives. thesis for  It  financial  this  thesis  The  gain s h a l l not  of  U n i v e r s i t y o f B r i t i s h Columbia  2075 Wesbrook Place Vancouver, Canada V6T 1W5  or  i s understood that copying or p u b l i c a t i o n  written permission.  Department  that  reference and study.  f o r s c h o l a r l y purposes may be granted by the Head of my Department by h i s  for  be allowed without my  ii  ABSTRACT A radioimmunoassay was motilin.  developed, s p e c i f i c for the g a s t r o i n t e s t i n a l polypeptide,  Antisera were raised i n guinea pigs and rabbits.  The  immunogen  was  porcine m o t i l i n , conjugated to bovine serum albumin by the carbodiimide condensation reaction.  The  routine antiserum behaved i d e n t i c a l l y towards endogenously-  released m o t i l i n and the pure standard preparation.  A radioactive tracer of high 125  s p e c i f i c a c t i v i t y was  obtained a f t e r incorporation  m o t i l i n molecule by the chloramine-T method. other assay variables were established Cstandard) curve.  Motilin  of  - iodine into  the  The optimum conditions for a l l  to produce the most sensitive displacement  antiserum, coupled d i r e c t l y to an agarose matrix,  retained f u l l antibody a c t i v i t y and s e n s i t i v i t y .  I t i s a feasible technique for  use i n both the radioimmunoassay and i n the extraction of m o t i l i n from both serum and  tissue  extracts.  The f a s t i n g serum l e v e l s of IR- m o t i l i n was 294  -'.44  pg/ml i n dogs (mean - SD) .  The  190  associated  after duodenal a l k a l i n i z a t i o n  with a concomitant elevation i n serum IR- m o t i l i n l e v e l s .  increase i n serum IR- m o t i l i n was exogenous administration response.  and  increase i n motor a c t i v i t y i n the  e x t r i n s i c a l l y denervated fundic pouch of the dog was  - 131 pg/ml i n men  i n the same range as that achieved by  This the  of the porcine polypeptide which produced the same motor  Duodenal a c i d i f i c a t i o n produced an apparent increase i n serum IR-  m o t i l i n with no associated  increase i n g a s t r i c motor a c t i v i t y .  m o t i l i n immunoreactivity was  detected when serum containing  Only one peak of  alkali-stimulated  motilin or a p a r t i a l l y p u r i f i e d duodenal extract were subjected to gel f i l t r a t i o n on Sephadex G-50.  The d i s t r i b u t i o n of motilin throughout the hog  gastrointestinal  tract, determined by radioimmunoassay on p a r t i a l l y p u r i f i e d extracts, agreed with  iii  the immunocytochemical findings that m o t i l i n was predominantly located i n the duodeninn and jejunum, with traces.in the upper ileum.  V i r t u a l l y the intact molecule was required f o r the expression of f u l l b i o l o g i c a l potency.  The i n d i v i d u a l amino acids were important inasmuch as they  contributed to the charge d i s t r i b u t i o n and conformation of the molecule.  The p h y s i o l o g i c a l release and function of m o t i l i n have yet to be  determined.  Elevated levels of c i r c u l a t i n g IR- m o t i l i n have not been associated with any g a s t r o - i n t e s t i n a l function, although they appear to be depressed by feeding. M o t i l i n has been implicated i n the control of the i n t e r d i g e s t i v e phase of g a s t r i c motor a c t i v i t y .  I t may be acting i n a l o c a l or paracrine manner.  M o t i l i n has not been implicated i n any .•cU'in±cal.st"at"eC&s sjffetfce i r  i  The hormonal status of g a s t r i c i n h i b i t o r y polypeptide (GIP) has been studied with the e x i s t i n g radioimmunoassay, modified to improve the l a b e l s p e c i f i c a c t i v i t y (by ion exchange chromatography).  Direct coupling of GIP antisera  to agarose beads was unsatisfactory, antibody a c t i v i t y and s e n s i t i v i t y being greatly reduced by the close proximity of the s o l i d matrix.  The postulated  role of GIP as the enterogastrone of Kosaka and Lim, suggested by studies with 1  exogenously-administered polypeptide, was  confirmed by experiments i n the dog.  Pentagastrin-stimulated g a s t r i c acid secretion was  i n h i b i t e d by intra-duodenal  infusion with glucose or f a t ; this i n h i b i t i o n being associated with a s i g n i f i cant elevation i n the c i r c u l a t i n g serum IR- GIP l e v e l s , within the range produced by ingestion of a mixed meal.  GIP does not appear to be involved i n the  i n h i b i t i o n of g a s t r i c acid secretion produced by duodenal  acidification.  iv  Endogenous GIP.stimulated by either f a t or glucose exhibited at least 3 ;  Immunoreactive components after column chromatography. i n the void volume appeared to  represent  The IR- GIP eluting  a non-specific complex between  GIP  and a serum protein and i s possibly b i o l o g i c a l l y i n a c t i v e .  GIP  component with a molecular weight of  7500-8000  the established form of GIP  (molecular weight =  to be r e l a t i v e l y unstable.  ProGIP and  extracts of hog duodenal mucosa. GIP  GIP^QQQ  (ProGIP), eluted ahead of  5105).  ProGIP has been found  have also been detected  500()  .  in  The established i n s u l i n o t r o p i c e f f e c t of  correlates best with that percentage of the t o t a l IR- GIP  ProGIP and G I P  A second IR-  The r e l a t i v e proportions  of IR- G I P  5 0 0 Q  composed of and IR- ProGIP i n  serum samples taken at d i f f e r e n t times after ingestion of either fat or glucose, suggest that ProGIP i s e i t h e r a precursor of GIP or that the ProGIP-producing c e l l s occupy a more d i s t a l region of the duodenal and j e j u n a l mucosa than the GIP- producing c e l l s .  Exogenous administration  of synthetic somatostatin i n dogs and man  w i l l inhibit  both.GIP release by either fat or glucose and the i n s u l i n o - t r o p i c action of GIP at the l e v e l of the 8/-cell. somatostatin may  Naturally-occurring i n t e s t i n a l or pancreatic  contribute to the control of GIP release and serve to modulate  the GIP- mediated response of the g a s t r i c p a r i e t a l or pancreatic S'-cell.  TABLE' OF CONTENTS  Page ABSTRACT  i i  LIST OF TABLES  x  LIST OF FIGURES ACKNOWLEDGEMENTS  •  i xiii  ^ ^ " " ^ ^ ^ ^ ^ " ^ " ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ ^ " " " " " ^ — x v i i i  INTRODUCTION  •  . 1  METHODS  19  DEVELOPMENT OF A RADIOIMMUNOASSAY FOR MOTILIN  19  A. Rationale  19  B. Iodination of m o t i l i n  20  1. Chloramine'-T method  20  2. Lactoperoxidase method  25 125  3. Estimation of s p e c i f i c a c t i v i t y of  I-motilin  25  C. Product ion- of ahtdsera-. to mot i l in  30  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 i n standards and unknowns 7. Measurement of the a f f i n i t y of the antisera  40 40  D. Conditions of the radioimmunoassay  42  1. Methods of standard curve evaluation  42  2. pH of the diluent buffer  44  3. T r a s y l o l concentration of yhe diluent buffer  44  4. Plasma concentration of the diluent buffer  46  5. Concentration of l a b e l l e d antigen added  46  6. Period of incubation  48  7. ). Protection from adsorption to glass  50  8. Routine assay conditions  52  9. Separation procedures 10. Methods of data analysis  . 54 56  vi  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  •  ;  •  A. Synthetic m o t i l i n  60 60  1. Preparation of 13-norleucine  motilin  60  2. Preparation of synthetic m o t i l i n fragments B. . Fragments of natural m o t i l i n  61 61  1. Cyanogen bromide cleavage of m o t i l i n  61  2. Tryptic and chymotryptic digestion of m o t i l i n  61  C. Modifications of natural m o t i l i n  62  1. C-terminal  residue removal  62  2. N-terminal residue removal  62  3. I d e n t i f i c a t i o n of the N-terminal residue  63  4. Acylation - acetylated derivative  65  5. Acylation - succinylated derivative  66  AFFINITY CHROMATOGRAPHY  66  —  A. A c t i v a t i o n of Sepharose 4B B. Coupling  66  of ligand to activated Sepharose 4B V  COLUMN CHROMATOGRAPHY  ;  67 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 f o r staining and destaining f o r q u a l i t a t i v e •;/v". • r determinations  73  B. Method f o r staining and destaining f o r q u a l i t a t i v e determinations ANIMAL PREPARATIONS  73 •  ' 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  vii  Page 6. Vagotomy and antrectomy  76  EXPERIMENTAL PROCEDURES .  77  A. In chronic dogs with Bickel pouches and duodenal f i s t u l a e  77  1. Bioassay for m o t i l i n  79  2. Effect of GIP on g a s t r i c acid  80  secretion  B. In chronic dogs with gastric  81  fistulae  1. Determination of the rate of g a s t r i c emptying of l i q u i d s  81  2. Determination of the rate of gastric emptying of  82  solids  83  C. In the intact dog SERUM ANALYSIS  •  —  —•*•  83  Radioimmunoassays  83  I. GIP radioimmunoassay  83  1. Iodination of GIP  84  a. Routine chloramine-T iodination and p u r i f i c a t i o n  84  b. Variation of chloramine-T iodination  87  c. Lactoperoxidase iodination  87  2. P u r i f i c a t i o n of  89  A.  125  I-GIP  3. Extension of s h e l f - l i f e  of  125  I-GIP  89  4. Production of antisera to GIP a.  93 93  Storage of antisera  b. Characterization of antisera  93  5. Assay protocol  93  6. Preparation of standards  95  7. Preparation of 8. Separation II.  95  controls  techniques  95  Insulin radioimmunoassay  1.. B$? commercially available k i t  97 97  2. By non-commercial  97  radioimmunoassay  a. Iodination of i n s u l i n b. The i n s u l i n antibody  ; / /  99 100 100  c. Assay protocol B. Serum glucose determination RESULTS —  101 102  ESTIMATION OP THE DEGREE OF CONJUGATION BETWEEN POLYPEPTIDES AND BOVINE SERUM ALBUMIN  102  a. With m o t i l i n  102  viii  Page B. With GIP  102  REPRODUCIBILITY OF IR-MOTILIN DETERMINATIONS  104  COMPARISON OF RADIOIMMUNOASSAY AND BIOASSAY FOR MOTILIN  -  104  A. Immunological comparison  104  B. B i o l o g i c a l 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 m o t i l i n and fragments  132  I I . Fragments of natural m o t i l i n  132  a. Cyanogen bromide cleavage  132  b. Tryptic and chymotryptic digestion  137  I I I . Modifications  of natural m o t i l i n .  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. B i o l o g i c a l comparison  139  I. Synthetic m o t i l i n  139  I I . Fragments of natural m o t i l i n I I I . Modification  s of natural m o t i l i n  139 142  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 A. Application  —  144  to radioimmunoassay  144  I. M o t i l i n  144  a. Antiserum d i l u t i o n and change i n a c t i v i t y  144  b. RIA standard curves and chenge i n s e n s i t i v i t y  144  I i . GIP  150  a. Antiserum d i l u t i o n and change i n a c t i v i t y  150  b. RIA: standard, curves and •change"in s e n s i t i v i t y  150  B. Application  153  to the p u r i f i c a t i o n of m o t i l i n 125  I. P u r i f i c a t i o n of I I . Extraction I I I . Extraction  I-motilin  153  of m o t i l i n added to plasma  155  of endogenous m o t i l i n from serum  155  '.'.IV. Isolation of m o t i l i n from Presekretin  159  EFFECT OF MOTILIN ON THE RATE OF GASTRIC EMPTYING  159  A. Control studies i n the g a s t r i c f i s t u l a dog  159  I. Effect of m o t i l i n on the rate of g a s t r i c emptying of l i q u id s  159  II. E f f e c t of m o t i l i n on the rate of gastric emptying of solids  162  B. Effect of m o t i l i n on the rate of emptying of l i q u i d s a f t e r truncal vagotomy and/or antrectomy  162  I. E f f e c t of m o t i l i n after truncal vagotomy  162  I I . Effect of m o t i l i n after antrectomy  162  I I I . Effect of m o t i l i n after vagotomy and antrectomy  166  MODIFICATIONS TO THE GIP RADIOIMMUNOASSAY  166  A. Antisera  166  raised to GIP  B. Iodination of GIP EFFECT OF SOMATOSTATIN ON THE CANINE RESPONSE  169 TO GIP  A. E f f e c t of somatostatin on the release of endogenous GIP  169 169  I. On the insulinotropic action of GIP released by o r a l glucose I I . On the release of endogenous GIP by oral f a t B. Effect of somatostatin on the response to exogenous GIP  169 172 172  Page RELATIONSHIP BETWEEN GIP AND GASTRIC ACID SECRETION  179  A. Effect of exogenous GIP on g a s t r i c acid secretion  179  B. Effect of endogenous GIP on g a s t r i c acid secretion  179  I. After intraduodenal infusion of f a t  179  I I . After intraduodenal infusion of glucose  187  C. Effect of an intraduodenal infusion of acid on g a s t r i c acid secretion  187  D. Effect of an intraduodenal infusion of saline on g a s t r i c acid secretion  187  STUDIES ON THE POSSIBLE HETEROGENEITY OF GIP  195  A. In serum  195  I. Immune-reactivity of GIP released II..Immunoreactivity  by f a t or glucose  of GIP a f t e r column chromatography  I I I . Immunoreactive forms of GIP released  by f a t or glucose  195 195 201  a. After o r a l f a t  201  b. After o r a l 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 c e l l u l o s e  210  c. S t a b i l i t y of ProGIP  212  I I I . Molecular weight determination DISCUSSION BIBLIOGRAPHY  212  ^  218 •  260  xi  LIST OF TABLES Table I. II. III. IV. V. VI. VII. VIII. IX. X.  Page M o t i l i n guinea pigs - immunization schedule  31  M o t i l i n rabbits - immunization schedule  33  E f f e c t of varying antibody t i t r e on assay s e n s i t i v i t y  37  E f f e c t of varying T r a s y l o l concentrations in the diluent buffer  44  Effect of varying plasma concentrations in the diluent buffer  46  E f f e c t of varying the antigen concentration on assay s e n s i t i v i t y  48  E f f e c t of varying the incubation period and type  48  Effect of s i l i c o n i z a t i o n or plasma addition on the assay system  50  Layout f o r the routine radioimmunoassay  53  E f f e c t of varying the charcoal concentration on the separation procedure in the assay f o r m o t i l i n  XI. XII.  E f f e c t of treatment of  125  I-GIP  on the NSB of that l a b e l  56 91  Results, i n pg/ml, demonstrating r e p r o d u c i b i l i t y of m o t i l i n determinations a f t e r storage f o r 3 months, with or without added Trasylol  XIII'.  Comparison  of the increase in m o t i l i t y indices a f t e r m o t i l i n  or Boots ^Pancreozymin' XIV. XV. XVI. XVII. XVIII. XIX. XX.  tract  116  Fundic motor a c t i v i t y response to duodenal infusion with a l k a l i  120  IR-motilin response to duodenal infusion with a l k a l i  121  Fundic motor a c t i v i t y response to duodenal infusion with saline  123  IR-motilin response to duodenal infusion with saline  124  Spontaneously induced fundic motor a c t i v i t y  126  IR-motilin response to spontaneously induced fundic motor 127  E f f e c t of duodenal infusion of a l k a l i , acid or saline on the incremental IR-motilin response  XXII.  133  E f f e c t of ingestion of a normal mixed meal on the serum l e v e l s of IR-motilin  XXIV.  129  E f f e c t of ingestion of o r a l glucose on the serum l e v e l s of IR-motilin  XXIII.  110  D i s t r i b u t i o n of IR-motilin throughout the hog g a s t r o i n t e s t i n a l  activity XXI.  106  Comparison motilin  134  of b i o l o g i c a l a c t i v i t i e s of natural and synthetic 140  xii  Page  Table  xxv:  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  s y n t h e t i c and n a t u r a l m o t i l i n analogues XXVI. XXVII. XXVIII.  Coupled v e r s u s uncoupled  a n t i s e r a at v a r y i n g i n c u b a t i o n volumes  E f f e c t of a s i n g l e i n j e c t i o n of s o m a t o s t a t i n on the i n c r e m e n t a l glucose  E f f e c t of a s i n g l e i n j e c t i o n of s o m a t o s t a t i n on the Incremental to o r a l f a t  to intravenous porcine  180  GIP E f f e c t of a c o n t i n u o u s  i n f u s i o n o f p e n t a g a s t r i n on the I I +  output of an e x t r i n s i c a l l y denervated XXXIII.  +  output  188  and IR-GIP r e l e a s e  +  190  output and IR-GIP r e l e a s e  E f f e c t of a duodenal i n f u s i o n o f s a l i n e on p e n t a g a s t r i n stimulated H  XXXVIII.  185  and IR-GIP r e l e a s e  E f f e c t of a duodenal i n f u s i o n of a c i d on p e n t a g a s t r i n stimulated H  XXXVII.  183  output  E f f e c t of a duodenal i n f u s i o n of g l u c o s e on p e n t a g a s t r i n s t i m u l a t e d H -output  XXXVI.  +  E f f e c t of a duodenal i n f u s i o n of f a t on p e n t a g a s t r i n stimulated H  XXXV.  182  f u n d i c pouch  E f f e c t of an i n t r a v e n o u s i n f u s i o n of p o r c i n e GIP on p e n t a gastrin-stimulated H  XXXIV.  +  192  output and IR-GIP r e l e a s e  P r o p o r t i o n s of IR-GIP m o l e c u l a r forms r e l e a s e d by f a t and  197  glucose XXXIX.  Change i n the r e l a t i v e p r o p o r t i o n s of IR-GIP m o l e c u l a r w i t h t h e time of serum sampling,  xxxx. xxxxi.  177  E f f e c t of a s i n g l e i n j e c t i o n of s o m a t o s t a t i n on the i n c r e m e n t a l I R - i n s u l i n and serum g l u c o s e response  XXXII.  173 175  to o r a l g l u c o s e  IR-GIP, I R - i n s u l i n and serum g l u c o s e response XXXI.  to o r a l  E f f e c t of an i n f u s i o n of s o m a t o s t a t i n on the i n c r e m e n t a l IR-GIP I R - i n s u l i n and serum g l u c o s e response  XXX.  146 148  C a l c u l a t i o n o f c t h e . s l o p e a t zero;dose  IR-GIP, I R - i n s u l i n and serum g l u c o s e response XXIX.  143  and fragments  forms  a f t e r o r a l f a t or glucose  202  R e l a t i v e p r o p o r t i o n s of IR-GIP m o l e c u l a r forms i n f r a c t i o n s o b t a i n e d from an e x t r a c t o f hog i n t e s t i n a l mucosa  207  A summary o f the t i s s u e e x t r a c t i o n  208  procedure  xiii  LIST OF FIGURES Figure 1.  Page Polyacrylamide g e l electrophoresis of m o t i l i n after  exposure  to chloramine-T 2.  Chloramine-T  : 22  iodination of m o t i l i n at.a peptide:iodine r a t i o  of 4 pg: 1 mCi 3.  Chloramine-T  23  iodination of m o t i l i n at a peptide:iodine r a t i o  of 2 ug: 1 mCi  24  4.  Lactoperoxidase iodination of m o t i l i n  26  5.  Standard curve for m o t i l i n i n comparison  with the d i l u t i o n  curves for labelled f r a c t i o n s . Measurement of s p e c i f i c a c t i v i t y  28  6.  M o t i l i n antiserum d i l u t i o n curves  34  7.  Effect of varying antibody t i t r e on the assay s e n s i t i v i t y  36  8.  Comparative  immunoreactivities of polypeptides with m o t i l i n  antiserum 9. 10.  Comparative  38 immunoreactivity of VIP with m o t i l i n antiserum  39  D i l u t i o n curves of serum samples containing exogenous and endogenous m o t i l i n  41  11.  M o t i l i n standard curve presented as a Scatchard .plot  43  12.  Effect of varying T r a s y l o l concentrations on m o t i l i n assay sensitivity  13  E f f e c t of varying labelled antigen concentrations on m o t i l i n assay s e n s i t i v i t y  14.  49  Effect of s i l i c o n i z a t i o n and plasma addition on m o t i l i n assay sensitivity  16.  47  Effect of v a r i a t i o n of incubation length and type on m o t i l i n assay s e n s i t i v i t y  15.  45  51  E f f e c t of varying charcoal concentrations i n separation procedure on m o t i l i n assay s e n s i t i v i t y  55  17.  Routine standard curve f o r m o t i l i n  58  18.  M o t i l i n antiserum d i l u t i o n curves before and after coupling to Sepharose 4B  19.  20.  68  GIP antiserum d i l u t i o n curves before and after coupling to Sepharose 4B  69  Diagram of the chronic dog preparation  78  xiv  Figure 21.  Page .Chloramine-T iodination of GIP.  Column p r o f i l e of separation  on Sephadex G25 22.  Lactoperoxidase  85 iodination of GIP.  Column p r o f i l e of  separation on Sephadex G25 23.  88  Chloramine-T iodination of GIP. on QAE  Column p r o f i l e of separation  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 r e s i n 25.  Effect of plasma addition on the s e n s i t i v i t y of the GIP. standard curve  26.  92 96  Effect of separation of bound antigen from free antigen by dioxane or dextran-coated charcoal, on the s e n s i t i v i t y of the GIP  27. 28. 29.  standard curve  98  Estimation of the degree of conjugation between m o t i l i n and BSA  103  Estimation of the degree of conjugation between GIP and BSA  105  •Reproducibilty of IR-motilin determinations  107  30.  Estimation of the m o t i l i n content of an extract by RIA  109  31.  Estimation of the m o t i l i n content of an extract by bioassay  111  32.  Column p r o f i l e of a motilin-containing serum on Sepahdex G50  113  33.  Column p r o f i l e 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 a c t i v i t y and c i r c u l a t i n g IR-motilin l e v e l s  36.  E f f e c t of a duodenal infusion of saline on fundic pouch motor a c t i v i t y and c i r c u l a t i n g IR-motilin l e v e l s  37.  125  Change i n c i r c u l a t i n g IR-motilin l e v e l s during a spontaneous burst of fundic pouch motor a c t i v i t y  38.  . 122  Comparison of the incremental  128  IR-motilin responses to duodenal  infusion with a l k a l i , acid or saline 39.  E f f e c t of ingestion of - glucose or a normal mixed meal on the  40.  c i r c u l a t i n g l e v e l s of IR-motilin Comparison of the immunological a c t i v i t y of natural porcine and synthetic m o t i l i n analogues and fragments  131 135 136  XV  Figure 41.  Page Comparison of the immunological a c t i v i t y of natural m o t i l i n  and  natural m o t i l i n fragments 42.  138  Comparison of the b i o l o g i c a l a c t i v i t y of natural m o t i l i n  and  . a synthetic m o t i l i n analogue 43.  •  141  Comparison of d i l u t i o n curves obtained with antiserum to m o t i l i n , under routine assay conditions or after coupling to Sepharose 4B  44.  145  Effect of coupling of m o t i l i n .antisera to Sepharose 4B on  the  s e n s i t i v i t y of the standard curve 45.  147  Graph showing the slope at zero dose for standard curves obtained with coupled and uncoupled antisera to m o t i l i n  46.  Comparison of the d i l u t i o n curves under routine  obtained with GIP  149 antisera  assay conditions or after coupling to Sepharose  4B  151  47.  Effect of coupling of GIP  48.  s e n s i t i v i t y of the assay standard curve 125 Column p r o f i l e of I-motilin obtained after a f f i n i t y chromatography 125  49.  antisera to Sepharose 4B on  Comparison of standard curves obtained with  the 152 154  I-motilin before  and a f t e r a f f i n i t y chromatography 50.  Column p r o f i l e of•IR-motilin  156  obtained after a f f i n i t y  chromatography of plasma containing exogenous m o t i l i n 51.  Column p r o f i l e of IR-motilin  157  obtained after a f f i n i t y  chomatography of a serum sample contianing endogenous m o t i l i n 52.  Column p r o f i l e of IR-motilin  obtained after a f f i n i t y  chromatography of an impure extract containing m o t i l i n 53.  Effect of an infusion of porcine m o t i l i n at two doses on  160 the  c i r c u l a t i n g l e v e l s of IR-motilin 54.  163  Fraction of a s o l i d meal emptied at d i f f e r e n t times during an infusion of 1 ^jg/kg/hour m o t i l i n  56.  161  Fraction of a l i q u i d meal emptied during, the infusion of varying doses of m o t i l i n  55.  158  164  Fraction of a l i q u i d meal emptied during infusion of varying doses of m o t i l i n , before and a f t e r truncal vagotomy  165  xvi.  Figure 57.  Page Fraction of a l i q u i d meal emptied during infusion of varying doses of m o t i l i n , before and after antrectomy  58.  167  Fraction of a l i q u i d meal emptied during infusion of varying doses of m o t i l i n , after truncal vagotomy and antrectomy  59.  168  Standard curves obtained with antisera to GIP after incubation in equilibrium and disequilibrium systems 125  60.  Standard  curves obtained with  170  I-GIP, p u r i f i e d by gel f i 1 1 -  ration, with or without subsequent p u r i f i c a t i o n on QAE : Sephadex A25 61.  .  .  171  Incremental serum glucose, IR-insulin and IR-GIP responses to oral glucose, with or without a p r i o r injection of somatostatin  62.  Incremental  174  serum glucose, IR-insulin and IR-GIP responses  to o r a l glucose, with or without a prior infusion of somatostatin 63.  176  Incremental serum glucose, IR-insulin and IR-GIP responses to o r a l f a t , with or without a p r i o r i n j e c t i o n of. somatostatin  64.  Incremental  serum glucose and IR-insulin responses to an  :  Intravenous  infusion, of porcine GIP, with or without a p r i o r  ;  i n j e c t i o n of somatostatin 65.  gastrin-stimulated H 66.  •  181  E f f e c t of an intravenous infusion of porcine GIP on penta+  output and IR-GIP l e v e l s  E f f e c t of intraduodenal f a t on pentagastrin-stimulated H  +  186  E f f e c t of intraduodenal glucose on pentagastrin-stimulated H  +  output and IR-GIP. release  189  68.  E f f e c t of intraduodenal hydrochloric acid.on pentagastrin-  69.  stimulated H output and IR-GIP release Effect of intraduodenal saline on pentagastrin-stimulated H" output and IR-GIP release +  1  70.  +  193  output and  IR-GIP release  194  Comparison of the immunoreactive forms of GIP released by ingestion of f a t or glucose  72.  191  Comparison of the e f f e c t s of intravenous GIP and intraduodenal f a t , glucose or acid on pentagastrin-stimulated H  71.  • 184  output and IR-GIP release 67.  178  196  Column p r o f i l e of serum containing glucose-released IR-GIP on Sephadadex G50  198  xv i i  Figure 73.  Page Column p r o f i l e of serum containing fat-released IR-GIP on Sephadex G50  74.  199  Relative proportions of IR-GIP molecular forms released by ingestion of f a t or glucose  75.  200  Change i n the r e l a t i v e proportions of the IR-GIP components with the time of sampling, a f t e r o r a l f a t or glucose  76.  Change i n the r e l a t i v e proportions of the IR-GIP components during an intravenous infusion of porcine GIP  77.  206  Relative proportions of the IR-GIP components in 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  79.  205  E f f e c t of 6.0M urea on the relative.proportions of the IR-GIP components in serum  78.  203  209  Column p r o f i l e of the methanol insoluble extract on Sephadex G50  211  80.  Column p r o f i l e of ProGIP I on CM c e l l u l o s e  213  81.  Column p r o f i l e of the .GIP^Q^Q-containing f r a c t i o n from Sephadex G50, on CMccellulose  82.  Repeated chromatography.of ProGIP after l y o p h i l i z a t i o n and storage at -20°C  83.  .  Relationship between V°/V  215 e  and molecular weight after  chromatography on Sephadex G50 84.  214:  217  Correlation between the b i o l o g i c a l a c t i v i t y of GIP and the r e l a t i v e proportions of the IR-GIP molecular forms  252  xviii  ACKNOWLEDGEMENTS  I should l i k e t o thank my s u p e r v i s o r , Dr John C. Brown, f o r the o p p o r t u n i t y to undertake Graduate S t u d i e s , and f o r h i s h e l p , guidance,  f o r b e a r a n c e and  f r i e n d s h i p d u r i n g t h i s time. I am a l s o g r a t e f u l f o r t h e support, p r a c t i c a l and m o r a l ,  both  g i v e n to me by members of t h e F a c u l t y and S t a f f of t h e  Department of P h y s i o l o g y .  I am a l s o indebted  to Mr Kurt Henze and Mr Ralph A s s i n a , f o r t h e p r e p a r a t i o n  of t h e 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 F o r s y t h , f o r the t y p i n g of the t e x t and t a b l e s .  F i n a n c i a l support from t h e Canadian M e d i c a l Research C o u n c i l i s g r a t e f u l l y i  acknowledged.  T h i s t h e s i s i s d e d i c a t e d to the memory of my f a t h e r , John Dryburgh, who a l l unknowingly s t a r t e d me along t h i s  path.  xix LIST OF ABBREVIATIONS APUD  - amine p r e c u r s o r , uptake and  BGP  - b r a i n g a s t r i n immunoassayable  BSA  - bovineserum albumin  CDI  -  carbodiimide  CE plasma - c h a r c o a l e x t r a c t e d CCK-PZ  —  GLI  -  IR-  -  CNBr FCA GIP  plasma  cholecystokinin-pancreozymin cyanogen  bromide  Freunds Complete Adjuvant gastric inhibitory glucagon-like  polypeptide  immunoreactivity  immunoreactive  KIU  -  Kallikrein inhibitor units  LDD  -  least detectable  IRP  M  5  ND NSB PTH PITC PLC PZn RIA Sn VIP  —  decarboxylation  insulin-releasing  pure p o r c i n e  polypeptide  dose  motilin  non-detectable non-specific binding p a r a t h y r o i d hormone phenyliso thiocyanat e proinsulin-like Pancreozymin  component  (commercial)  radioimmunoassay secretin vasoactive  intestinal  peptide  peptide  XX Sources of Reagents 125  Amersham/Searle  IMS 30  Chloramine-T  Eastman Kodak Co. Rochester, N.Y. 14650  #1022  Sodium metabisulphite  Fisher S c i e n t i f i c Co. F a i r Lawn, New Jersey  S-244  Lactoperoxidase (from milk)  Sigma Chemical Co. St. Louis, Mo. 63178  0L-2OO5  Hydrogen peroxide (30%)  Fisher S c i e n t i f i c F a i r Lawn, New Jersey  H-325  Bovine serum albumin ( f r V)  Sigma Chemical Co. St. Louis, Mo. 63178  T r a s y l o l (10,000 KlU/ml)  FBA, Boehringer Canada  Microfine s i l i c a (Quso G32)  Philadelphia Quartz Co. Valley Forge, Pa. 19482  AG 1-X10 r e s i n (200-400 mesh)  Biorad Laboratories Richmond, C a l i f o r n i a  Freunds Complete Adjuvant  Difco Laboratories D e t r o i t , Michigan  l-ethyl-3-(3-dimethyl)amino propyl carbodiimide  Calbiochem San Diego, C a l i f o r n i a  Dimethyldichlorosilane  Biorad Laboratories Richmond, C a l i f o r n i a  Charcoal (Carbon decolourT i z i n g Neutral)  Fisher S c i e n t i f i c F a i r Lawn, New Jersey  Dextran T 70  Pharmacia Uppsala, Sweden  Insulin RIA K i t  Amersham/Searle  I-sodium ( c a r r i e r free)  Ingelheim  140-1541  341006  IM 39  xxi Sephadex gels  Pharmacia Uppsala, Sweden  Sepharose r e s i n  it  CM celluloses  Whatman, England  G l a c i a l acetic acid) Hydrochlorid acid ) Ammonia solution )  A r i s t a r BDH Chemicals Poole Dorset, England  Cyanogen bromide  Eastman Organic Chemicals Rochester, N.Y.  Trypsin - TPCK  Worthington Biochemicals Freehold, New Jersey  Carboxypeptidase A  Worthington Biochemicals Freehold, New Jersey  Phenyli so thiocyanat e  Eastman Organic Chemicals Rochester, N.Y.  T r i f l u o r o a c e t i c acid  Eastman Organic Chemicals Rochester, N.Y.  Dansyl chloride  Sigma Chemical Co. St. Louis, Mo. 63178  Acetic anhydride  Eastman Organic Chemicals Rochester, N.Y.  Succinic anhydride  Eastman Organic Chemicals Rochester, N.Y.  Ethanolamine  Sigma Chemical Co. St. Louis, Mo. 63178  t  Acrylamide N N'-methylenebisacrylamide NNN'N'-tetramethylenediamine  Eastman Organic Chemicals  Fluothane -Halothane B.P.  Ayerst Laboratories, Montreal  Lipomul  Upjohn Co. of Canada , Don M i l l s , Ont. ^  Pentagastrin(Peptavlon injectionable)  Ayerst Laboratories Montreal, Canada  II  II  II  II  II  II  - 1 -  INTRODUCTION  :  In 1905 S t a r l i n g 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 o r i g i n and  carried  to. i t s target organ by the c i r c u l a t i o n .  Over  the l a s t decade the facts which have emerged about endocrine control i n general, and g a s t r o i n t e s t i n a l control i n particular,.would indicate that this c l a s s i c paradigm must undergo r e v i s i o n and re-evaluation and that the role of a chemical messenger may be more subtle than was o r i g i n a l l y envisaged.  The three generally accepted hormones with g a s t r o i n t e s t i n a l a c t i v i t y , secretin, gastrin and cholecystokiriin-pancreozymin have known chemical structures and physiological importance.  They have been joined over the l a s t ten years by  numerous other peptides of both g a s t r o i n t e s t i n a l and extra-gastrointestinal origin.  These substances have had t h e i r structures confirmed but t h e i r true  hormonal status i s uncertain, e.g., m o t i l i n , gastric i n h i b i t o r y polypeptide (GIP), vasoactive i n t e s t i n a l peptide (VIP)/, bombesin and somatostatin.  Other  workers have introduced impure extracts with b i o l o g i c a l a c t i v i t y , whose active moiety may be i d e n t i c a l with other, already i d e n t i f i e d polypeptides, e.g., i n s u l i n - r e l e a s i n g 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 r o l e s " .  The results obtained when the c l a s s i c a l physiological  methods of assessing humoral status were applied to these peptides were equi~ vocal, i n many cases.  The infusion of s u f f i c i e n t exogenous pure polypeptide  into the c i r c u l a t i o n , usually accepted as the D,_Q, i n order to mimic a physiolog i c a l event, or the t r a d i t i o n a l c r o s s - c i r c u l a t i o n experiments were no longer  -2-  enough to e s t a b l i s h true p h y s i o l o g i c a l function.  The e f f e c t i v e levels achieved  by exogenous administration of the polypeptide should be i n the range of the serum levels measured during endogenous'polypeptide release.  This requirement  i s complicated by the fact that the b i o l o g i c a l a c t i v i t y observed during the endogenous release of any g a s t r o i n t e s t i n a l polypeptide i s being modulated by the a c t i v i t y of other polypeptides,released at the same time.  Before the hormonal  status of a b i o l o g i c a l l y active p r i n c i p l e can be evaluated i t must be available in a chemically pure form, vide the confusion which has arisen around the b i o l o g i c a l a c t i v i t y 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 e s s e n t i a l .  Some of the candidate hormones which f a l l into the second category of Grossman, i.e.,  polypeptides extracted from b i o l o g i c a l tissues, which have been p u r i f i e d  and characterized, include VIP, somatostatin and m o t i l i n .  These  substances,  infused intravenously, demonstrated varied b i o l o g i c a l a c t i v i t i e s , but t h e i r endogenous release cannot be measured i n association with any of these a c t i v i t i e s by any of the methods developed for the estimation of that substance i n the circulation.  In 1953 Feyrter described h i s concept of the paracrine c e l l .  He  postulated the existence of secretory c e l l s , scattered throughout the gastroi n t e s t i n a l mucosa, adjacent to t h e i r target c e l l s .  The structures he thought  responsible, the " h e l l e Z e l l e n " or clear c e l l s were l a t e r recognized as being i d e n t i c a l with the APTJD (amine. T. precursor uptake and decarboxylation) of Pearse (1968).  cells  Under normal circumstances, he postulated, the secretions of  these c e l l s would pass to the target c e l l v i a the e x t r a c e l l u l a r f l u i d and never " s p i l l " into the c i r c u l a t i o n i n any s i g n i f i c a n t amounts.  The paracrine system  has yet to be proven to e x i s t but i t i s a plausible concept and the gut mucosa, which may be regarded.as the single largest endocrine organ i n the body, with i t s multitude of secretory and receptor c e l l s scattered over an immense area, would be i d e a l l y suited to such a mechanism. for  Some evidence  the existence of the modification of endocrine function has been obtained  with somatostatin.  This tetradecapeptide was o r i g i n a l l y i s o l a t e d from the hypothalamus of the sheep by Brazeau et a l (1973) during t h e i r search for a p i t u i t a r y growth hormone releasing factor.  Instead they were repeatedly able to demonstrate  the existence of a growth hormone release Inhibitory factor. was.extracted, p u r i f i e d , sequenced and l a t e r synthesized.  This material  I t was o r i g i n a l l y  named somatotropin-relea'se i n h i b i t o r y factor (SRIF) or growth hormone-release i n h i b i t o r y hormone (GRIH) but the findings that i t was able to i n h i b i t the release of i n s u l i n (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 s p e c i f i c name, somatostatin.  Studies with gastrin and GIP have indicated  that somatostatin did not only i n h i b i t endogenous release of these polypeptides but also blocked t h e i r action at the l e v e l of the target c e l l . levels of hypothalamically-released polypeptide were  I f effective  to occur i n the c i r c u l a t i o n  a l l these systems would be i n h i b i t e d simultaneously, and this i s an unacceptable premise.  Somatostatin-producing c e l l s have been l o c a l i z e d by immunochemical  means i n hypothalamic and pancreatic i s l e t tissue, and i n gastric and i n t e s t i n a l mucosa (Dubois, 19 75).  The location of somatostatin i n the pancreas i s d i s t i n c t  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 i n t e s t i n e .  A t e l e o l o g i c a l l y more acceptable concept would involve the modulation of hypothalamic, g a s t r i c , i n t e s t i n a l or pancreatic function by hypothalamic, g a s t r i c , i n t e s t i n a l or pancreatic somatostatin, released l o c a l l y by an appropr i a t e stimulus, where i t acted In a paracrine manner.  A second type of secretory process, postulated to exist and capable of acting i n the g a s t r o i n t e s t i n a l t r a c t ; i 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 g a s t r o i n t e s t i n a l a c t i v i t y such, as somatostatin,  substance P, VIP and gastrin, have, a l l been• (detected i n normal neural tissue. Although somatostatin has been detected i n both the hypothalamus and pancreas the p o s s i b i l i t y that some neural connection exists between these two areas seems u n l i k e l y i n view of the fact that no nerve f i b r e s to the adult pancreas have been shown to contain IR- somatostatin.  Somatostatin immunoreactivity  has also been demonstrated i n the f o e t a l pancreas by the t h i r d month of gestation. It i s more l i k e l y that this peptide i s being synthesized i n both regions and i s not merely being absorbed by pancreatic tissue after synthesis i n the brain. The evidence so f a r available favours a paracrine, rather than neurocrine r o l e for somatostatin.  On the other hand, immunoreactive  substance P has  been demonstrated i n both central and peripheral neural tissues, i n association with primary sensory neurons i n the dorsal horn, and i n non-myelinated  free  nerve endings i n 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 c e l l s i n the g a s t r o i n t e s t i n a l 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 s i g n i f i c a n t degree, i t  -5may be acting prejunctionally to modulate the response of the gut musculature to cholinergic stimulation, and be neurocrine i n i t s action.  Two polypeptides  with established g a s t r o i n t e s t i n a l properties, gastrin and VIP,  have also been located i n neural tissue.  VIP was found i n the gastrointes-  t i 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 o r i g i n i t was  l o g i c a l to look for VIP i n cultured neuroblastoma c e l l s from mice, as well as in normal canine neural t i s s u e .  In the normal extracts the highest  concentra-  tions were found i n the cerebral cortex, the hypothalamus and hippocampus. IR-VIP  was also detected  i n sympathetic ganglia, the adrenal gland and i n  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 a c t i v i t y when assayed i n v i t r o on rat stomach or guinea pig g a l l bladder s t r i p s . physiological functions have been assigned to t h i s polypeptide, t i v e l y larger concentrations  No  but the r e l a -  i n the central and peripheral nervous system, com-  pared to that i n the i n t e s t i n e , suggest that i t might function as a neurocrine transmitter.  Immunoreactivity to gastrin has also been detected  dominantly i n the c o r t i c a l grey matter.  i n the brain, pre-  Extracts from t h i s region, however,  showed a lower a f f i n i t y for the antiserum used than did heptadecapeptide g a s t r i n , and eluted behind t h i s gastrin from Sephadex G-25.  Vanderhaeghen et a l (1975)  have named t h i s material brain g a s t r i n immunoassayable p_eptide (BGP) . I t remains to be seen i f BGP corresponds to a smaller molecular form of g a s t r i n , e.g.,  the tridecapeptide found i n sera from Z o l l i n g e r - E l l i s o n patients  and S t a d i l , 1973a), and to examine the range of i t s b i o l o g i c a l a c t i v i t y .  (Rehfeld'  -6-  A model s p e c i f i c 'for the". actions of the, g a s t r o i n t e s t i n a l polypeptides,  combining  features from.both the.endocrine and paracrine systems, has been proposed by Wingate (1976).  His Eupeptide system i s based on the following f a c t s .  g a s t r o i n t e s t i n a l polypeptides  influence both motor and secretory  of the gut, and most g a s t r o i n t e s t i n a l polypeptide-producing  Most  activities  c e l l s are situated  i n close proximity to the c e l l whose secretions they influence*  However, i t  has been w e l l established that several of these factors are released into the systemic c i r c u l a t i o n i n s i g n i f i c a n t amounts.  Wingate therefore postulated a  dual action for the gut peptides and suggested that they acted at a l o c a l target c e l l as a paracrine, to promote secretion or l o c a l muscle a c t i v i t y , and at a distant target c e l l to modulate the motor control of the digestive t r a c t .  Immunological techniques such as radioimmunoassay, immunocytochemistry and a f f i n i t y 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 i n p a r t i -  cular (Berson and Yalow,1958) has proved a valuable t o o l i n monitoring polypeptide responses i n various s i t u a t i o n s , but the r e s u l t s require careful evaluation.  Some long-held b e l i e f s have had to be re-examined.  been postulated to be the polypeptide  Secretin had  responsible for the i n h i b i t i o n of g a s t r i c  acid secretion when acid passed into the duodenum. secretin did, i n fact, mimic this response.  Exogenously  administered  However, the development of a  radioimmunoassay, s p e c i f i c for s e c r e t i n , proved that the minimum e f f e c t i v e c i r c u l a t i n g IR- secretin levels achieved  a f t e r infusion of the polypeptide were  much higher than those produced by duodenal a c i d i f i c a t i o n (Ward and Bloom, 1974). When secretin was  administered  to produce serum IR- secretin levels within the  physiological range, no e f f e c t was  observed on acid secretion or g a s t r i c motor  a c t i v i t y although a s i g n i f i c a n t effect was  seen on the exocrine pancreas,  CBloom, 1975).. This f a i l u r e , to . conf irjn the role of s e c r e t i n as . the a c i d stimulated g a s t r i c i n h i b i t o r y agent, by .radioiiranunological,means, has restimulated i n t e r e s t i n 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 a c i d i f i c a t i o n of the bulbar region resulted i n a profound i n h i b i t i o n of g a s t r i c acid secretion, which was i n s t a l l e d into the lower duodenal regions.  not observed i f the acid was  However, u n t i l this material  has been p u r i f i e d and.its endogenous.release can be stimulated by physiolog i c a l secretagogues, any suggestions  that i t i s the major i n h i b i t o r y agent  released by duodenal a c i d i f i c a t i o n must be purely speculative.  The use of immunological techniques  i n the measurement of c i r c u l a t i n g poly-  peptide levels i n serum, plasma or tissue extracts has demonstrated that several polypeptides existed i n more than one molecular Tracy  form.  (1964), using c l a s s i c a l peptide extraction techniques,  Gregory and described  two  forms of heptadecapeptide gastrin, d i f f e r i n g only i n the presence of a sulphated tyrosine residue, but had the foresight to state "there may  be  present  i n 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 i n human plasma did, i n fact, exist i n more,than one' molecular  form.  This heterogeneity was  shown immunologically  when different  antisera crossreacted i n d i f f e r i n g degrees with the endogenously occurring polypeptide and the pure standard preparation.  Fractionation by g e l 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 d i s t i n c t i o n between components sharing immunoreactivity,  By 1973  Rehfeld and S t a d i l had i s o l a t e d four components of immunoreactive gastrin from the sera of Z o l l i n g e r - E l l i s o n patients by the.technique of high resolution gel f i l t r a t i o n .  1  Component 1 eluted i n 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; correlated w e l l with heptadecapeptide gastrin; was a tridecapeptide. location,,response  component I I I  component IV (minigastrin)  The d i f f e r e n t gastrins .have* been found to vary i n  to s t i m u l i , and'also perhaps i n function.  Big, b i g gastrin  i s a major component of the f a s t i n g sera.in normal subjects but occurs i n i n s i g n i f i c a n t amounts i n 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 i o n i c strength, s i g n i f i c a n t l y depressed the size of this f r a c t i o n of IR- gastrin, suggesting form may be a protein/peptide  that this largest molecular  complex (Rehfeld et a l , 1975).  The physiolo-  g i c a l function of such a complex i s not understood, although the suggestion has been made, with respect to i n s u l i n , that this type of protein-peptide binding may act as a l i m i t i n g factor i n the transport of a peptide across a c e l l u l a r membrane (Simon and Antoniades, 1975).  Component II or b i g gastrin  would appear to be the major IR- component i n the c i r c u l a t i o n a f t e r stimulation, whilst the heptadecapeptide form predominates i n a n t r a l tissue (Dockray et al,;1975).  Evaluation of the importance of.the immunoreactive forms of  gastrin under d i f f e r e n t conditions i s further complicated  by the varying  h a l f - l i v e s of the heterogeneous forms of gastrin.  P r i o r to this work, Berson and Yalow (1968) had demonstrated that human parathyroid hormone (PTH) i n tissue extracts had a molecular weight of 9000, whilst  -9-  that i n serum was mostly composed of a smaller form with a molecular weight of 7000.  In 1972  weight of 5000.  Canterbury,et  a l isolated a third PTH with a molecular  The 7000 molecular weight form was  found to represent  C-terminal portion of the larger molecule (Segre et a l , 1972)  the  and as Aurbach  et a l (1971) had postulated that the biologically, active portion of the larger molecule resided at the N- terminal, the p o s s i b i l i t y .was raised that a s i g n i f i c a n t l y large proportion of the t o t a l immunoreactive PTH i n sera was b i o l o g i c a l l y inactive.  Habener et a l (1971) have isolated a s t i l l larger  immunoreactive molecule from s l i c e s of parathyroid t i s s u e .  Biosynthesis  studies, measuring the uptake time of t r i t i a t e d amino acids into this component and the 9000 molecular weight form are suggestive of a precursorpolypeptide relationship (Cohn et a l , 1972)  and the largest molecular  form of  PTH can be enzymatically degraded, by t r y s i n to produce a polypeptide with increased b i o l o g i c a l and immunological a c t i v i t y .  The best i l l u s t r a t i o n of a precursor-hormone relationship so f a r comes from the studies on. p r o i n s u l i n and i n s u l i n .  In 1967  Steiner provided evidence that  the synthesis of i n s u l i n involved production of a precursor which was  synthe-  Isized i n the rough endoplasmic reticulum of the B c e l l s i n the pancreatic i s l e t s , and transferred to the Golgi apparatus (Steiner et a l , 1969). Approximately 95% of the p r o i n s u l i n was  converted  to i n s u l i n within the secre-  tory granules i n the cytosol, the remainder : being secreted into the c i r c u l a tion along with the i n s u l i n . (Nolan et a l , 1971)  At least two enzyme systems, one  and one similar to a carboxypeptidase  1971)appear to be necessary  trygsin^like  (Kemmlerpet a l ,  for the conversion of the p r o i n s u l i n to i n s u l i n  and a chymotryptic-like cleavage has been implicated by the work of Tageir et al,(1973) i n i n v i t r o studies i n the rat i s l e t preparation.  The p r o i n s u l i n  -10-  molecule  has  been found to  vary  only s l i g h t l y from species to species, the  average molecular weight being around  9000.  Most studies on the biosynt-  hesis of p r o i n s u l i n have been performed on the isolated i s l e t preparation (Steiner, 1967) and conversion to i n s u l i n has been found to be s t r i c t l y cellular.  -  intra-  Glucose:is an important regulator of p r o i n s u l i n synthesis, which  i s favoured under hyperglycaemic conditions over other c e l l u l a r proteins CPermitt  and Kipnis, 1972a, 1972b).  Many antisera  to  i n s u l i n also cross-  react with p r o i n s u l i n and other intermediate forms, which are together d e signated proinsulin-like-component (PLC), and therefore measurement o f IRi n s u l i n alone would b e possible only a f t e r gel f i l t r a t i o n .  P r o i n s u l i n has  been shown to' have most o f the b i o l o g i c a l properties o f i n s u l i n but only 3 5% o f i t s b i o l o g i c a l potency (Narahara, 1972) and as the IR- PLC i n f a s t i n g sera approximates 15% o f the t o t a l IR- i n s u l i n i t must b e taken into account when correlating immunological a c t i v i t y with b i o l o g i c a l a c t i v i t y .  The r a t i o o f PLC: i n s u l i n declines i n the f i r s t hour after glucose stimul a t i o n and then gradually increases.  Elevated PLC: i n s u l i n ratios have been  found i n hypokalaemias o f d i f f e r e n t aetiologies (Gorden e t a l , 1974), severe diabetes and chronic renal f a i l u r e (Mako e t a l , 1973), and are diagnostic o f 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  t o t a l material which crossreacts with antisera t o glucagon were found t o respond d i f f e r e n t l y when challenged by the same stimulus.  Samols e t a l  (1965,1966) noted that the c i r c u l a t i n g levels o f immunoreactive  glucagon  C l R - glucagon)appeared t o increase a f t e r o r a l administration o f glucose and  3  -11-  that this immunoreactivity originated from the gut rather than the pancreas. This was, confirmed by Buchanan et a l (.1967) who infused  (intrajejunally)  glucose into pancreatectomised dogs'and measured a s i g n i f i c a n t increase i n IR- glucagon l e v e l s . filtration  Valverde et a l (1968) monitored IR- glucagon.after gel  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 f r a c t i o n s i m i l a r to the second component of Valverde  was p u r i f i e d from a crude extract, of p i g ileum by Murphy et a l (1973) and was found to possess l i t t l e b i o l o g i c a l resemblance to.;pancreatic glucagon. Sasaki et a l (1975) further p u r i f i e d the small GLI component from porcine duodena/by gel f i l t r a t i o n . o n 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 i d e n t i c a l i n i t s spectrum 125 of b i o l o g i c a l a c t i v i t y .  The second f r a c t i o n eluted behind the  I-  glucagon marker, had a molecular weight of 2900, and showed varying degrees of c r o s s r e a c t i v i t y depending on the antisera used i n the assay.  Histochemi-  cal studies of the secretory c e l l s i n the g a s t r o i n t e s t i n a l mucosa i n i t i a l l y indicated a c e l l very s i m i l a r to the ej•• 7 c e l l of the pancreatic i s l e t s .  Ultra-  s t r u c t u r a l studies now suggest that, whilst these A- c e l l s in.the fundic mucosa are i d e n t i c a l i n morphology with the pancreatic a- - c e l l , those i n the i n t e s t i n a l mucosa show s l i g h t s t r u c t u r a l differences. nated A- l i k e and may  These have been desig-  correspond to the EG c e l l of Polak et a l (1971).  The  d i s t r i b u t i o n of the 3500 and 2900.molecular weight components throughout the  g a s t r o i n t e s t i n a l mucosa correspond closely to the d i s t r i b u t i o n of A  and A- l i k e c e l l s respectively.  I t i s strongly suggested that GLI, o r i g i n a l l y  defined by Unger et a l (1968) to encompass any immunoreactive material o r i g i n a -  -12-  t i n g from the gut, i s not a s i n g l e : e n t i t y , but i s made up of true glucagon of gut o r i g i n and several other peptides, more'correctly c a l l e d GLI or enteroglucagonoid, which share a degree of immunological i d e n t i t y with true glucagon.  and possible b i o l o g i c a l  An o r a l glucose.load causes a decrease i n  pancreatic glucagon plasma levels and an increase i n c i r c u l a t i n g IR-GLI levels and therefore an antiserum s p e c i f i c f o r pancreatic glucagon must be used to measure the true pancreatic response to this stimulus.  The conclusion must be drawn that any comparison between b i o l o g i c a l and immunological  a c t i v i t y of any polypeptide must take into account the presence  of immunoreactive but possibly b i o l o g i c a l l y less potent precursor forms or complexes between the peptide and a larger protein, as well as similar but functionally d i f f e r e n t molecules.  immunologically  A l l antisera should be screened  for differences i n t h e i r c r o s s r e a c t i v i t y with the b i o l o g i c a l l y occurring forms of each peptide and i n the p o s s i b i l i t y of r a i s i n g antibodies to a s p e c i f i c region of the active molecule  considered.  An obvious, though sometimes experimentally ignored, observation i s that no p h y s i o l o g i c a l l y functional peptide acts i n i s o l a t i o n , and that i t s b i o l o g i c a l effect i s modulated by the hormonal m i l i e u at that time.  A;simple example  of this fact i s shown by the combined effects of secretin and cholecystokininpancreozymin on the exocrine pancreas.  In the intact animal there i s 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  i s i n e f f e c t i v e unless i t i s washed from  the pancreatic ducts into the duodenum by the secretin-induced aqueous secretion  -13-  (Brownet a l , 1967a). i n t e s t i n a l polypeptides  I t i s also l o g i c a l to correlate the a c t i v i t y of gastroto the stage of the digestive cycle during which they  are normally released, and to measure that a c t i v i t y i n an environment of the c i r c u l a t o r y digestion products that would pertain at that stage.  Physiological  levels of IR- g a s t r i n have no e f f e c t on i n s u l i n release i n the f a s t i n g  man,,  but i n the presence of a. degree of hyperglycemia the i n i t i a l i n s u l i n response a f t e r gastrin administration i s longer and more pronounced than that seen a f t e r intravenous perfused  glucose alone (Rehfeld and S t a d i l , 1973b).  In the i s o l a t e d ,  rat pancreas Pedersonand Brown (1976) were able to demonstrate a  threshold glucose l e v e l , below which GIP had no e f f e c t on i n s u l i n release.  The major functions of the g a s t r o i n t e s t i n a l hormones so f a r discovered r e l a t e to t h e i r e f f e c t on the.secretory capacity or motor a c t i v i t y of the gastrointestinal tract.  Another property was  suggested by the discovery that gastrin  had a trophic e f f e c t on the c e l l s ofcthe g a s t r i c mucosa.  Patients treated for  duodenal ulcer by antrectomy showed atrophy of the g a s t r i c mucosa (Lees and Grandjean, 1968)  which was  (Melrose et a l , 1964). acid secretion was  not'the case i f the treatment was  vagotomy only  Disuse atrophy could not be the cause of this as the  i n i t i a l l y depressed to the same degree i n either case.  In  contrast, subjects with Z o l l i n g e r - E l l i s o n Syndrome showed hyperplasia of both -  j  the g a s t r i c and duodenal mucosa ( E l l i s o n and Wilson, 1967). was  confirmed i n r a t s , when increased RNA  and DNA  The role of gastrin  synthesis resulted from  single shots of gastrin, and chronic gastrin treatment produced a thickening of the g a s t r i c mucosa (Johnson, 1976).  Mainz et a l (1973) found that exogenous  CCK-PZ. caused an increase i n both c e l l mass and c e l l number i n the 1  pancreas, and postulated a role for CCK-PZ i n maintaining and i n t e g r i t y .  exocrine  pancreatic function  P h y s i o l o g i c a l levels of the synthetic octapeptide  of CCK-PZ  were found to have a trophic e f f e c t on duodenal mucosa, but had no e f f e c t on  -14-  g a s t r i c mucosa.  These same l e v e l s would, however, i n h i b i t the trophic e f f e c t  of gastrin on the g a s t r i c mucosa, and the same r e s u l t 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 f a t digestion products, the function of which was to i n h i b i t both g a s t r i c acid secretion and gastric motility.  This d e f i n i t i o n was l a t e r expanded to require that any candidate  polypeptide must i n h i b i t g a s t r i c acid secretion stimulated by histamine and insulin-induced hypoglycaemia,  as w e l l as gastrin and i t s analogues.  Secretin, cholecystokinin-pancreozymin, VIP and GIP h a v e . a l l been considered at some time to f i l l t h i s role,they a l l appeared to i n h i b i t the acid secretion produced by some or a l l of these s t i m u l i , and cholecystokinin-pancreozymin i s also released by the presence of f a t i n the duodenum,.but the only polypeptide which s a t i s f i e s a l l these c r i t e r i a i s GIP.  Brown.and Pederson (1970) showed that the a b i l i t y of the 10% pure preparation of cholecystokinin-pancreozymin of Jorpes and Mutt to produce g a s t r i c acid i n h i b i t i o n was s i g n i f i c a n t l y reduced by a simple p u r i f i c a t i o n step, involving gel f i l t r a t i o n on Sephadex G-50, although i t s e f f e c t on g a l l bladder contract i l i t y was unaltered.  A side f r a c t i o n , produced i n the p u r i f i c a t i o n of cholecys-  tokinin-pancreozymin was found to contain i n h i b i t o r y a c t i v i t y but had no e f f e c t on the g a l l bladder, (Brown et a l , 1969).  A polypeptide was i s o l a t e d  and p u r i f i e d , (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-IleArg-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 f o r GIP has been developed and antibodies to GIP raised in guinea pigs did not crossreact with glucagon, gastrin, m o t i l i n , cholecystokinin-pancreozymin, secretin, VIP or i n s u l i n . Studies performed on peptide fragments obtained by cyanogen bromide cleavage at the methionine residue, or on synthetic peptide fragments, suggest that the immunoreactive s i t e  lies  within the sequence 21-38. Antiserum to GIP has also been used i n the immunohi-sto.logical l o c a l i z a t i o n of the GIP-producing c e l l i n the duodenum and jejunum of man,  dog and baboon. It was tentatively i d e n t i f i e d as the Dl c e l l ,  (Polak et a l , 1973) i n the APUD series defined by Pearse (1968,1970,1974) but i s now known to be the K c e l l  (Solcia et a l , 1973).  Sera from human volunteers, taken before and a f t e r ingestion of a normal meal, were subjected to radioimmunoassay. Fasting l e v e l s 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 f o r at least 3 hours (Kuzio et a l , 1974). The major physiological secretagogues f o r GIP release were o r a l glucose (Cataland et a l , 1974) and o r a l f a t (Brown et a l , 1974).  The discovery that c i r c u l a t i n g IR-GIP l e v e l s were elevated when glucose came into contact with the duodenal and jejunal mucosa suggested that this polypeptide might be a factor i n the entero-insular axis, i . e . might be a hormone of i n t e s t i n a l o r i g i n which contributed to the regulation of the endocrine  -16-  pancreas.  Dupre et a l (1973) infused porcine GIP intravenously i n man  showed a s i g n i f i c a n t enhancement of the IR- i n s u l i n response:to  and  an i n t r a -  venous i n f u s i o n of glucose, associated with an improvement:in glucose'tolerance. The c i r c u l a t i n g levels of IR- GIP achieved during this exogenous administration of GIP were comparable to those:released endogenously by ingestion of glucose. Studies i n dogs suggested that lower levels of c i r c u l a t i n g IR- GIP were i n s u l i n o tropic only i n the presence of a degree of hyperglycaemia, although  higher,  possibly non-physiological levels stimulated i n s u l i n release i n the fasted animal.  The existence of the humoral gastric motor-activity stimulating p r i n c i p l e , l a t e r named m o t i l i n , 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 j u i c e and demonstra*i  ted  an increase i n g a s t r i c motor a c t i v i t y i n e x t r i n s i c a l l y denervated or t o t a l l y  transplanted pouches of the fundus of the stomach.  E a r l i e r , Shay and Gershon-  Cohen (1934) had described increased gastric emptying of a barium sulphate test meal a f t e r i n s t i l l a t i o n of 1% bicarbonate into the duodenum. observation coupled with the work of Thomas et a l (1934), who  This  diverted the  gastric (acidic) contents away from the duodenum and recorded an increase i n the rate of g a s t r i c emptying, suggested a pH s e n s i t i v e , duodenal r e f l e x that contributed to the control of g a s t r i c motor a c t i v i t y .  Weisbolt et a l  (1969) proposed that a r e l a t i o n s h i p existed between the rate of g a s t r i c emptying and the motor a c t i v i t y of the gastric musculature which would ensure that the contents of the stomach were delivered to the duodenum at a rate, and i n a consistency that would allow optimal duodenal and j e j u n a l digestion and absorption.  -17-  A crude duodenal extract (Pancreozymin, Boots Pure Drug Co.), administered intravenously i n dogs, produced similar changes i n the motor a c t i v i t y 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 f i v e protein peaks. Fractions 4 and 5 were i n h i b i t o r y f o r g a s t r i c motor a c t i v i t y and f r a c t i o n 5 was a potent stimulant, of pancreatic enzyme output, i . e . i t corresponded most c l o s e l y to the GIH preparation. Fraction 2 represented a 20-fold p u r i f i c a t i o n of the o r i g i n a l stimulatory material (Brown and Parkes, 1S67). M o t i l i n was eventually p u r i f i e d from a side f r a c t i o n produced in the p u r i f i c a t i o n of secretin (Brown et a l , 1972). The amino acid sequence was determined by the subtractive dansyl-Edman s technique on peptides produced 1  by cleavage of the molecule with cyanogen bromide, trypsin, chymotrypsin and thermolysin. Porcine m o t i l i n was found to be a 22 amino acid residue polypeptide with the sequence :NH -Phe-Val-Pro-Ile-Pne-Thr-Tyr-Gly-G.lu-Leu-Gln2  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 s i g n i f i c a n t increase in motor a c t i v i t y in the e x t r i n s i c a l l y denervated fundus and antrum of the canine stomach i n doses as low as. 50 ng/kg. I t had no s i g n i f i c a n t effect on g a s t r i c 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 m o t i l i n a c t i v i t y required bioassay in the chronic dog, prepared with an e x t r i n s i 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 v i t r o preparations of muscle s t r i p s from the ileum, colon and c i r c u l a r layers of the stomach of the r a t , guinea pig and rabbit were examined  but the s e n s i t i v i t y of every preparation decreased immediately after the exposure to m o t i l i n and the I n i t i a l response: could not be duplicated. ment of a radioimmunoassay was by motilin:in.the increase  first  Develop  deemed desirable to confirm the role played  i n g a s t r i c motor a c t i v i t y after duodenal a l k a l i n i -  zation and for further investigation of i t s physiological  function.  It i s the purpose of this thesis to study the hormonal.status of the  gastro-  i n t e s t i n a l p o l y p e p t i d e s G I P and m o t i l i n , and to look more closely at t h e i r possible physiological roles, bearing i n mind the following  points:-  1.  of a polypeptide  B i o l o g i c a l a c t i v i t y seen a f t e r exogenous administration  can only be considered physiological i f that same response can be  elicited  by that polypeptide when i t i s released by a physiological stimulus, and  the  c i r c u l a t i n g levels of the peptide are comparable. 2.  I n a b i l i t y to measure an increase, i n polypeptide levels i n the systemic  c i r c u l a t i o n does not necessarily preclude that polypeptide from having a physiological 3.  role.  If immunological techniques are used to measure the polypeptide levels i n  the c i r c u l a t i o n , then i t i s essential to determine what percentage of the t o t a l immunoreactivity represents the true b i o l o g i c a l a c t i v i t y . 4.  The b i o l o g i c a l a c t i v i t y of a polypeptide w i l l depend on the hormonal and  nutrient milieu pertaining at that time and results obtained i n an i s o l a t e d s i t u a t i o n , e.g.j  i n v i t r o , 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 a c t i v i t i e s need not be r e s t r i c t e d to i n f l u e n -  cing the secretory may  or motor a c t i v i t i e s i n the gastrointestinal, t r a c t .  They  also play an important metabolic role i n 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 i s based upon the s p e c i f i c relationship that exists between an antibody and i t s antigen.  Unlabelled  with labelled antigen for the binding s i t e s on the antibody.  antigen competes 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 i n the mixture.  The concen-  t r a t i o n of antigen i n an unknown sample may be determined by comparing the displacement  of l a b e l l e d antigen i t produces with that produced by a series of  standard solutions.  The mommonestl l a b e l which can be incorporated into poly125  peptides containing tyrosine or h i s t i d i n e residues i s an isotope of iodine, 131_ or I.  I  A successful polypeptide radioimmunoassay i s dependent on three absolute requirements.  F i r s t l y , the r a t i o of isotope to polypeptide i n the radio-active  tracer must be high enough so that s u f f i c i e n t tracer may be added to ensure an e f f i c i e n t counting rate without adding s i g n i f i c a n t amounts of polypeptide and obscuring the upper l i m i t s of s e n s i t i v i t y of the curve. i s an antibody of high a f f i n i t y .  The second essential  This a f f i n i t y i s expressed as a constant K.  The relationship between K and the upper l i m i t of s e n s i t i v i t y of the radioimmunoassay can be developed  as follows:-  From the 1st order Law of Mass Action B/F  =  K([A°]  -  B)  Where K i s the equilibrium ( a f f i n i t y ) constant, [A°] i s the concentration of antibody binding s i t e s and B & F are the concentrations of bound and free hormone  -20-  If b i s the f r a c t i o n of bound hormone and IH] i s the t o t a l hormone concentr a t i o n , then B  =  B/  =  bTH] _b_  =  K([Ab°]  -  b[H[)  1 1 As the hormone concentration i s increased to [H ] then B/ 4- and b[H ] t . F By d e f i n i t i o n b has a maximum of 1. 1 _ b  .". The most s e n s i t i v e assay condition p r e v a i l s when lAb°]  =  IH ] for a B/_ of 1. 1  r  Assuming a B/ of 1 with minimal tracer and no unlabelled hormone, then [Hj -* 0 i.e.,  1  =  K[Ab° ].  1  =  KIH ]  When [Ab°] approximates [H"*"] 1  or  K =  1  i . e . , the greater the value of K, the lower the concent r a t i o n of t o t a l hormone that i s detectable.  The f i n a l requirement i s that the antibody should react i d e n t i c a l l y with the unlabelled antigen, whether i t be i n the form of standard or endogenous polypeptide.  I d e a l l y , the l a b e l l e d and unlabelled peptide should also behave  i d e n t i c a l l y i n the system but this i s not an absolute necessity.  I f these  conditions are s a t i s f i e d then the optimal values for a l l other variables may be established.  B. (I)  Iodination of M o t i l i n Chloramine - T Method  A modification of the o r i g i n a l method of Hunter and Greenwood (1963) was used in routine i s o t o p i c l a b e l l i n g of m o t i l i n .  The polypeptide  contains  only  -21-  1 t y r o s y l and no h l 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 f o r each iodination a)  M o t i l i n (M ) 5  -  2 or 4 yg i n 50 y l 0.2M  sodium,phosphate buffer, pH  125  7.5  '  b)  Na  I  -  1 mCi i n 10 y l carriers-free sodium hydroxide  c)  Chloramine-T  d)  Sodium metabisulphite - 100 ;yg i n 20 y l deionized water  -  40 yg i n 10 y l deionized water  Reagents a, b & c were added i n quick succession, with bubbling to ensure rapid mixing, i n a 10 x 75 mm  s i l i c o n i z e d glass culture tube.  was added, i n l i k e manner, after a 15 sec. delay.  Reagent d  M o t i l i n contains 1  methionine residue but no tryptophan and appeared f a i r l y stable i n 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 f i n e CO.6 x 30 cms.) and eluted i n 0.2 M. acetic acid, containing 0.5% Bovine Serum Albumin T r a s y l o l per ml.  (BSA) and 100 K a l l i k r e i n Inhibitor Units (KIU)  Fractions of approximately 400 y l were collected and 10- y l  aliquots were counted for 0.1 min. i n an automatic^ counter.  The resultant  column p r o f i l e s are i l l u s t r a t e d i n F i g . 2 & F i g . 3, showing the separation of peptide-bound and free iodide.  Aliquots of the appropriate f r a c t i o n s ,  diluted to contain-«-5000 cpm/100 y l , were incubated for 24 hours at 4°C, with or without antiserum to estimate the s p e c i f i c versus non-specific binding ( N.S.B);for that f r a c t i o n .  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 i s v i s i b l e at 120 sec. The method for polyacrylamide gel electrophoresis is detailed on p. 72.  -23-  il  '  I . J  I'.  I-On  10  20 Fraction #  30  40  10 ju\ Aliquots  i  Fig. 2  Chloramine-T iodination of m o t i l i n at a r a t i o of.4"Ug:l mCi.  peptide:iodine  Separation of l a b e l l e d m o t i l i n from  free iodide on Sephadex G25 i n 0.2M per 0.1 min (••); maximum binding  acetic acid.  (x—x); NSB  Counts  (o—o).  -24-  l  I.0-,  10  20  30  Fraction #  Fig. 3  40 10/JI  50  60  Aliquots  Chloramine-T iodination o£ m o t i l i n at a peptide:iodine r a t i o of 2 ng:l mCi.  Separation of l a b e l l e d m o t i l i n from free iodide  on Sephadex G25 i n 0.2M maximum binding  (zi.-x)  acetic acid.  ; NSB  (o-o) .'  Counts per 0.1 min (•-•);  -25-  s p e c i f i c binding and lowest, non-specific binding were pooled, d i l u t e d i n 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 i n this manner was  stable for periods of up to 3 months.  Lyophilization of l a b e l proved feasible:ibut was not routinely performed.  (2)  Lactoperoxidase Method  An alternative, gentler and more e a s i l y 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). reagents were mixed i n the following order in a 10 x 75 mm.  The  siliconized,  glass, culture tube. a)  M o t i l i n (Mj) - 4 ug i n 50 u l 0.05  M -sodiumsacetate, ,p.H  5.0  125 b)  Na  1-1  mCi  in 10 u l carrier-'freeesodium  hydroxide  c)  Lactoperoxidase - 500 ng i n 10 u l .-sodiumsacetate, pH  d)  Hydrogen peroxide - 0.86  5.0  nM i n deionized water  3 x 10 u l 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 t y p i c a l column p r o f i l e i s shown i n F i g . 4. 125 (3)  Estimation of S p e c i f i c A c t i v i t y of  I - Motilin  Dose - response curves were obtained (a) by increasing the concentration unlabelled m o t i l i n and measuring the displacement of a constant amount of radioactive tracer (routine standard curve) and  (b) by adding increasing  of  -26-  I.O-i  0.8 -  Fraction #  Fig. 4  10p\  Aliquots  Lactoperoxidase iodination of m o t i l i n at a r a t i o of 2 y g : l mCi.  peptide:iodine  Separation of l a b e l l e d m o t i l i n from  free iodide on Sephadex G25 i n 0.2M acetic acid. per, 0.1 min (•—•); maximum binding  (x--x); NSB  Counts  (o-o).  -27-  amounts of l a b e l l e d motilin only and measuring the d i f f e r e n t r a t i o s of tracer bound to antibody.'  One point from the l a b e l d i l u t i o n curve was a r b i t r a r i l y placed on the standard curve and the other points f i t t e d accordingly. F i g . 5 i l l u s t r a t e s that the curves obtained when 3 d i f f e r e n t fractions of l a b e l l e d m o t i l i n (fractions.24,  25 and 26 from the column p r o f i l e shown i n F i g . 3) were  plotted on a standard curve they could be superimposed upon that standard curve.  It can be concluded that the binding k i n e t i c s of the antiserum  were v i r t u a l l y i d e n t i c a l f o r both l a b e l l e d and unlabelled antigen.  The  number of counts per minute (cpm) producing the same displacement as a standard amount of m o t i l i n can be read d i r e c t l y from t h i s curve and the value converted to mCi/mg as an index of s p e c i f i c a c t i v i t y .  Example  (from r e s u l t s shown i n F i g . 5)  .".  28 pg m o t i l i n  18,000 cpm  1 mg m o t i l i n  9 18,000 x 10 cpm 28 9 643 x 10  1 Curie .*.  3.7 x 10  1 mCi  dps (disintegrations/second) 9  1 mCi  From the e f f i c i e n c y of the y  cpm  2.2 x 10 counter  -  dpm (disintegrations /min. )  81% =  1.78 x 10  9  cpm  -28-  Motilin  Standards  Label Tube 2 4 " 2 5 26 0.8-  0.6-  0.4-  o Q_  0.2 •  Motilin  10  _i  0.0-  5 1 2 5  Fig.  5  1-Motilin  Concentration  20  40  i  -|  10  i  1  20  (pg/ml.)  80 1  160  320  1—  1—I  30 40  ( C P M x IO3)  Standard curve f o r m o t i l i n (x—x) i n comparison with l a b e l fractions 24, 25, 26. Dilutions of each f r a c t i o n from 8 x 3 3 10 cpm to 40 x 10 cpm were added and the d i l u t i o n of 3 Fraction 26 containing 25 x 10  cpm was f i t t 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. m o t i l i n  =  6.43 x 10 1.78 x 10  mCi 9  361 mCi .*. S p e c i f i c a c t i v i t y of t h i s iodination was 361 mCi/mg and the addition of 5000 cpm to each assay tube entailed the addition of 7.5 pg m o t i l i n .  The  s p e c i f i c a c t i v i t y was not measured after every iodination but was  checked at i n t e r v a l s and on every occasion when the routine  iodination was  varied i n any way.  A rough estimate of s p e c i f i c a c t i v i t y may be calculated from the percentage of the radioiodine  incorporated into the polypeptide,  Example Cfrom column p r o f i l e i n F i g . 3) %  125 , I incorporated into polypeptide 125  S p e c i f i c a c t i v i t y of  1  =  71%  = 1 4 mCi/mg  125 . .  1 mCi  I  =72  .*.•  72 ng iodine was reacted with 2 yg m o t i l i n  ng. Iodine  i . e . , 0.57 nM iodine was reacted with 0.74 nM m o t i l i n .*.  0.40 nM iodine was incorporated into 0.74 nM m o t i l i n  i . e . , 50 ng iodine was incorporated into 2 yg m o t i l i n 125 i . e . , 0.7 mCi  T was incorporated into 2 yg.motilin  125 i . e . , 350 yCi I was incorporated i n 1 yg m o t i l i n .". Specific a c t i v i t y = 350 mCi/mg. However, i t must be remembered that this c a l c u l a t i o n depends.on the assumption that iodine was incorporated into a l l the available polypeptide.  -30-  C.  Production of Antisera to M o t i l i n  Cl)  In guinea pigs  A series of guinea pigs (6) were immunized with pure porcine m o t i l i n (M,.). Conjugation of m o t i l i n to a large molecular weight protein was deemed advisable because of i t s low molecular weight.  M o t i l i n 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 m o t i l i n per animal, 80 mg BSA and 100 mg CDI were dissolved i n  100 u l , 10 mis and 1 ml deionized water, respectively.  0.5 mis each BSA  and CDI were added to the m o t i l i n , mixed gently and l e f t at room temperature for at least 1 hour.  The reaction was terminated by d i a l y s i s 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 r a t i o .  The f i n a l volume was selected  to  allow 0.5 ml emulsion per animal.  The animals were immunized subcutaneously, i n several s i t e s on the abdomen and inner thigh.  An early observation suggested that better, more s p e c i f i c  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 i n a emulsion.  (2)  FCA  The schedule followed i s shown i n Table I.  In rabbits  Ten rabbits were immunized with conjugated m o t i l i n . zation was  The route of immuni-  intradermally, i n several s i t e s , i n the supra-scapular region.  -31-  TABLE I lyiOTILIN GUINEA PIGS  ^  IMMUNIZATION SCHEDULE  DATE  IMMUNIZATION  .4.6.74  50'jif ^ / F C A  20.6.74  100 j i g M /BSA/FCA  20.7.74  200-jag l^/BSA/FCA  BLEEDING  TYPICAL TITRE  23.8.74  l:40xl0  3  5  31.2.75  50 jag M /FCA  29.9.75  l:20xl0  4  23.1.76  50 yg 1M /FCA  3.3.76  l:10xl0  5  10.8.76  50 yg  24.8.76  1:lOxlO  5  5  5  1M /FCA 5  the p u r e s t  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  i s tabulated (Table II)  (3)  Storage of Antisera  The whole blood samples were allowed to c l o t at 4°C for 20 mins., then centfifuged. assessed.  The antiserum was stored frozen at - 20°C u n t i l i t could be  Usable antiserum was aliquotted i n 200 - 500 U.1 portions, and  l y o p h i l i z e d for storage at - "20°C.  No detectable loss of s p e c i f i c i t y or  a f f i n i t y f o r at least 3 years has been observed.  As required, the l y o p h i l i z e d a l i q o t s w e r e reconstituted i n assay diluent buffer at a d i l u t i o n of 1:10 and stored at - 20°C i n 100 -ul aliquots; material was v i a b l e during the period of i t s use, usually 2 - 3  (4)  This  months.  Effect of varying antibody t i t r e  T i t r e , i n this connotation, i s defined as the f i n a l d i l u t i o n of antiserum in the incubation mixture.  This allows f o r d i r e c t comparison between  d i f f e r e n t antisera i n d i f f e r e n t assay protocols where the f i n a l incubation volumes may d i 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 s e r i a l d i l u t i o n curve for the a n t i serum.  Varying t i t r e s of the antiserum were incubated under routine assay 125  conditions with  I - m o t i l i n and the maximum binding obtained with each  d i l u t i o n plotted as % bound versus the r e c i p r o c a l of that t i t r e . d i l u t i o n curve i s shown i n F i g . 6.  A typical  From this the range of t i t r e producing  -33-  TABLE I I MOTILIN RABBITS  - • • PMONIZATION SCHEDULE  DATE  7JOTNIZAT10N  14.10.75  20 Ug M /FCA  13.11.75  50 Jig M /BSA/FCA  24.11.75  15.12.75  50 jig 1VL-/BSA/FCA  29.12.75  l:10xl0  2  23. 1.76  50 jig N /FCA  13. 2.76  l:10xl0  4  8. 4.76  1:20xl0  4  BLEEDING  TYPICAL TITRE  5  5  5  the purest preparation of natural m o t i l i n Bovine Serum Albumin FCA -  Freunds Complete Adjuvant  -34-  Fig. 6  Curve obtained with s e r i a l d i l u t i o n s of m o t i l i n antiserum GP 71.  '  Arrows indicate the t i t r e s selected for use i n the  standard curves shown i n F i g . 7.  -35-  the most sensitive assay may be roughly estimated.  Various authorities have stipulated that the most e f f e c t i v e assay i s produced at the t i t r e r e s u l t i n g i n a maximum binding of 50%, or 33% (Berson & Yalow, 1958).  However, i t i s becoming clear that no such hard and  fast r u l e can be followed and that the optimum t i t r e should be established for each RIA i n d i v i d u a l l y .  F i g 7 i s a comparison of the standard curves  obtained with varying t i t r e s of the same antiserum shown i n the d i l u t i o n curve.  The t i t r e s selected were those which resulted i n binding 50%,  33%, 27% and 13% of the l a b e l .  The results were plotted as B/„ x 100 Bo r  against the m o t i l i n 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  r e s u l t s are presented i n Table 111. The most e f f e c t i v e t i t r e was 1:5 x 10^ - - i . e . , that producing a maximum binding of 27%. The most sensitive standard curves f o r the m o t i l i n RIA under the routine conditions were obtained when the maximum binding was 25 - 30%.  {5)  Measurement of comparative  The comparative  immunoreactivity  of antiserum  immunoreactivities of natural gastric i n h i b i t o r y poly-  peptide, natural porcine secretin, natural (10% pure) cholecystokinin pancreozymin, synthetic glucagon and synthetic human gastrin with m o t i l i n antisera were investigated. On a d i f f e r e n t date the comparative  immuno-  r e a c t i v i t y of m o t i l i n antiserum with vasoactive i n t e s t i n a l peptide was examined.  No s i g n i f i c a n t c r o s s - r e a c t i v i t y between the antiserum and any  of these polypeptides was detected even when concentrations of up to 10 mg per incubation volume were employed.  The r e s u l t s are i l l u s t r a t e d i n Figs. 8 & 9.  -36-  Fig. 7  Standard curves, for m o t i l i n , demonstrating the effect of varying the antibody t i t r e on the assay s e n s i t i v i t y . least detectable dose.  LDD =  -37-  TABLE I I I E F F E C T OF VARYING  CONDITION:TITRE  ANTIBODY  MAXIMUM  "  BINDINGC%)  T I T R E ON ASSAY  SENSITIVITY  SLOPE AT ZERO (L/JMOLE)  l:1.5xl0  5  50  0.6xl0"  1:4  xlO  5  33  1:5  xlO  5  1:1  xlO  6  MIDRANGE VALUE (PG M O T I L I N )  L.D.D. (PG M O T I L I N )  82  40  -13 2.6x10  73  20  27  4.0xl0~  60  20  13  -13 2.5x10 -  105  20  13  13  LJ  -38-  o.o  J  «  \\  Fig. 8  I  5  1 10  1 100 Weight of Peptides (pg.)  1  1000  1  10000  Comparative immunoreactivities of natural m o t i l i n , synthetic human gastrin, synthetic glucagon, natural secretin, cholecystokinin-pancreozymin  (10%) and natural gastric i n h i b i t o r y poly-  peptide with antiserum to m o t i l i n . (Dryburgh and Brown; Gastroenterology 68_ : 1169-1175, 1975).  -39-  0.5.  r x  No  Motilin  added  X-^o  0.4 H  X,  I  0.3 ^ X—X O  0.2  Motilin  Stds.  VIP  oi H o.o-J  r o  - i —  10  — i  1000  100 Wt. of P e p t i d e  (pg)  Comparative immunoreactivities of natural motilin and vasoactive i n t e s t i n a l peptide with antiserum to m o t i l i n .  IOO00  -40-  C6)  A n t i b o d y r e c o g n i t i o n o f a n t i g e n xn atandards and unknowns  Peak IR - m o t i l i n samples from dog experiments endogenously r e l e a s e d a f t e r duodenal  i n w h i c h m o t i l i n was  a l k a l i n i z a t i o n o r exogenously  either adminis-  t e r e d by an i n t r a v e n o u s 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 o r c h a r c o a l - - e x t r a c t e d 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 t o t h e s t a n d a r d c u r v e and t h e r e m a i n i n g v a l u e s p l o t t e d accordingly ( F i g . 10).  Both serum d i l u t i o n curves c o u l d be superimposed  upon t h e s t a n d a r d  curve  i n d i c a t i n g t h a t the r e l a t i o n s h i p between t h e a n t i b o d y and t h e u n l a b e l l e d a n t i g e n i s unchanged whether t h e a n t i g e n i s t h e i s o l a t e d p o l y p e p t i d e i n t h e Standard p r e p a r a t i o n o r t h e n a t u r a l l y o c c u r r i n g form, i n t h e unknown, i . e . , i n sera.  (7)  Measurement o f a f f i n i t y o f a n t i s e r u m  From t h e 1 s t o r d e r Law o f Mass A c t i o n t h e f o l l o w i n g e q u a t i o n was by S c a t c h a r d  (1949): B/  Where B/ [A°J  developed  p  =  K([A°J  -  B°)  i s t h e r a t i o o f Bound l a b e l l e d a n t i g e n t o F r e e l a b e l l e d a n t i g e n  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 t h e f r a c t i o n o f t o t a l  a n t i g e n bound and K. i s t h e c o n s t a n t o f t h e a n t i b o d y - a n t i g e n r e a c t i o n i n the d i r e c t i o n Ab  +  Ag —^AbAg.  I n any i n d i v i d u a l assay K and {A0]  are constant  expressed l i n e a r l y i n a S c a t c h a r d p l o t .  . *. B/.^ and B°  may be  -41-  i  x  —i  5(10)  1  Motilin Standard  1  10(20) Dog Serum (/j|./ml.)  1 r 100(200)  i I  Fig. 10  Serum samples R 10 (.exogenous, motilin) and R24 (endogenous m o t i l i n ) incubated  at several dilutions, in'diluent buffer  or charcoal-extracted plasma.  The d i l u t i o n s ' o f 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 d i l u t i o n s f i t t e d  CDryburgh and Brown, Gastroenterology  accordingly.  68 :1169-1175, 1975).  -42-  I n f a c t , - any a n t i s e r u m r e p r e s e n t s a p o p u l a t i o n o f a n t i b o d i e s o f 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 t h e S c a t c h a r d p l o t i s a curve composed o f s e v e r a l straight lines.  The h i g h e s t a f f i n i t y a n t i b o d i e s a r e r e p r e s e n t e d by t h e  l i n e with the steepest s l o p e  <  The r o u t i n e RIA s t a n d a r d curve was r e p l o t t e d as a S c a t c h a r d p l o t .  The  s t a n d a r d v a l u e s were c o n v e r t e d t o a b s o l u t e a n t i g e n v a l u e s by a d d i t i o n o f t h e amount o f p o l y p e p t i d e i n c o r p o r a t i n g t h e 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 t h e s p e c i f i c a c t i v i t y o f t h a t t r a c e r .  B° i s t h e p r o d u c t  o f t h i s v a l u e , i n m o l e s , and t h e c o n c e n t r a t i o n o f l a b e l bound by i t , i . e . , B.  K i s t h e s l o p e o f t h e l i n e produced by p l o t t i n g B/  a g a i n s t B°. r  F i g s . ID shows t h e S c a t c h a r d p l o t o f a t y p i c a l a n t i s e r u m o f r e a s o n a b l e affinity.  D.: C o n d i t i o n s Of Radioimmunoassay Cl)  Methods Of s t a n d a r d 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 t h e s e n s i t i v i t y o f a s t a n d a r d curve a r e many and v a r i o u s .  Three have been s e l e c t e d and a c o m b i n a t i o n o f a t l e a s t two  of these have been used i n a l l comparisons.  The s t a n d a r d curves o b t a i n e d were n e v e r l i n e a r so t h e . " s l o p e a t z e r o dose"  was e s t i m a t e d as a S c a t c h a r d p l o t and i t s s l o p e measured as t h e  p l o t approached z e r o (Feldman & Rodbard, 1971).  The l e a s t d e t e c t a b l e dose (L.D.D.) was t a k e n as t h a t c o n c e n t r a t i o n o f u n l a b e l l e d a n t i g e n w h i c h produced a d i s p l a c e m e n t s t a n d a r d d e v i a t i o n a t maximum b i n d i n g .  of binding = 2 x the  -43-  AS  Slope  71  1:150,000  0 . 3 3 5 - 0.185  = 0.150  1.5 x 1 0 " ' 1.0 x 10 1.5  B°  Fig. 11  x 10 " '  4  L/mole  ( M o l e s ) x 10'  Standard curves with m o t i l i n antiserum  (GP 71) represented  as,a Scatchard p l o t , B/F being plotted  against the f r a c t i o n  of t o t a l antigen bound (B°) .  The slope of the l i n e 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 s e n s i t i v i t y of the standard curve at i t s upper l i m i t . The t h i r d parameter,  the midrange value, allowed  comparison  of standard curves i n the region where they were most l i k e l y to approach l i n e a r i t y and was that concentration of unlabelled m o t i l i n which displaced 50% of the maximum l a b e l bound.  (2)  pH of the diluent buffer  M o t i l i n standards i n the range 12.5 - 400 pg were incubated with antiserum 75A at a f i n a l d i l u t i o n of 1:80x10  f o r 48 hours at 4 C i n 0.04M sodium  phosphate buffer at either pH 6.5 or 7.5, and i n veronal buffer, 0.05M at pH 8.5. There was no s i g n i f i c a n t difference i n the displacement observed either at the midrange value or the L.D.D.  (3)  T r a s y l o l concentration i n the diluent buffer  T r a s y l o l i s a broad spectrum p r o t e o l y t i c enzyme i n h i b i t o r , containing 10,000 k a l l i k r e i n i n h i b i t o r u n i t s (KIU) per ml. Standard curves were incubated in 0.04M sodium phosphate buffer, pH 6.5, containing 0%, 0.25%, 0.5% or 1.0% T r a s y l o l . The resultant displacement i s shown i n Fig.12 and Table IV and 0.25% T r a s y l o l was selected as the optimum concentration.  TABLE IV  -  E f f e c t of varying the T r a s y l o l concentration i n the diluent Buffer  Condition  Midrange (pg M5)  L.D.D. (pg.M5)  Trasylol  70  25  0.25% T r a s y l o l  24  12.5  0.5%  Trasylol  47  25  1.0%  Trasylol  135  50  0.0%  -45-  Fig.  12  Effect of varying the T r a s y l o l concentrations i n the diluent buffer oh the s e n s i t i v i t y of the routine standard curve for motilin.  -46-  C4)  Plasma c o n c e n t r a t i o n jri •the d i l u e n t ' b u f f e r  Standard curves were s e t up t o compare  the e f f e c t o f v a r y i n g  the plasma  c o n c e n t r a t i o n i n the d i l u e n t b u f f e r , from 2 ^ 10%  The plasma was outdated  b l o o d bank s t o c k which had been e x t r a c t e d twice  1% (w/y) c h a r c o a l f o r 1 hour at 4°C to absorb any s m a l l p e p t i d e s present.  The c h a r c o a l was  20 mins.  The c h a r c o a l ^ e x t r a c t e d plasma was  peptide  TABLE V  ^  still  removed by c e n t r i f u g a t i o n at 5000 r.p.m. f o r  l e v e l s and s t o r e d at -20°C.  when the b u f f e r c o n t a i n e d  screened  for detectable  The g r e a t e s t s e n s i t i v i t y was  polyachieved  5% c h a r c o a l - e x t r a c t e d plasma (shown i n - T a b l e V ) .  E f f e c t o f "varying the plasma c o n c e n t r a t i o n i n the d i l u e n t b u f f e r  Condition  Midrange . (pg M5)  L.D.D. (pg M5)  2% plasma  55  25  5%  "  55  12.5  • • 10%  "  105  (5)  with  Concentration  of l a b e l l e d antigen  25  added  L a b e l l e d a n t i g e n at c o n c e n t r a t i o n s v a r y i n g from 2000 c.p.m./lOO p i to 20,000 c.p.m./100 yil  was  added to s t a n d a r d  c o n c e n t r a t i o n ofr~ 5000 c.p.m./lOO ]_tl was s e n s i t i v e curve  a t the most e f f i c i e n t  curves  i n the r o u t i n e assay.  s e l e c t e d as g i v i n g the most  counting  r a t e (Table V I , F i g . 13)  The  -47-  o—o 20.000 c p m / 1 0 0 / J l •—•  10.000  x—x  5.000  11  2.500 100L.D.D.  Midrange Value  T  12.5 pg  F i g . 13  1 25  1 50  1 100  1 200  Motilin  Effect of varying the concentration of l a b e l l e d antigen on the s e n s i t i v i t y of the routine standard curve for m o t i l i n .  -48-  TABLE VI  -  Effect of varying the antigen concentration on the assay sensitivity  Condition  Midrange  20,000 cpm/100 y l  (6)  L.D.D. (pg M5)  105  50  10,000  94  25  5,000  67  12.5  2,500  54  12.5  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. antibody alone f o r 24 hours.  Standard curve c was incubated  After l a b e l addition the incubation  with  continued  for a further 48 hours. A 48 hour incubation period was deemed to give a more s e n s i t i v e assay, from the r e s u l t s i n F i g . 14 and Table VII.  There was no advantage gained by p r i o r  incubation of cold antigen with antibody,  i . e . , under disequilibrium conditions.  Longer incubation periods of 3 - 4 days were also s a t i s f a c t o r y .  TABLE VII  -  Effect of varying the incubation period and type  Condition 24 hour equilibrium 48  "  24/48 " dis-equilibrium  Midrange (pg M5)  L.D.D. (pg M5)  145  50  53  25  53  25  -49-  0'  I  0  12.5 PG  Fig. 14  "  1  25  50  1  100  1  200  Motilin  Effect of varying the length and type of incubation on the s e n s i t i v i t y of the routine standard curve for m o t i l i n .  -50-  (7)  P r o t e c t i o n from a d s o r p t i o n to g l a s s  A d s o r p t i o n o f p e p t i d e and i o d i n a t e d m a t e r i a l on to the g l a s s tubes, used i n the assay, can be a problem.  The plasma c o n t e n t o f the d i l u e n t  b u f f e r d i d reduce the counts a d s o r b i n g to the g l a s s but s i l i c o n i z a t i o n o f the assay tubes w i t h 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 f u r t h e r improvement c o u l d be a c h i e v e d .  I t was.also  e s s e n t i a l t o determine whether i t was  n e c e s s a r y to compensate  i n the s t a n d a r d curve f o r the e x t r a p r o t e i n added i n the m o n i t o r i n g o f plasma or serum samples.  Standard curves were Incubated i n s i l i c o n i z e d  t u b e s , 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 w i t h the a d d i t i o n o f 100 T i l of c h a r c o a l - e x t r a c t e d plasma. t h a t s i l i c o n i z a t i o n o f the tubes was  The r e s u l t s o b t a i n e d suggested  not n e c e s s a r y .  The s t a n d a r d curves  o b t a i n e d a f t e r i n c u b a t i o n w i t h and w i t h o u t plasma were c o r r e c t e d f o r t h e i r i n d i v i d u a l n o n - s p e c i f i c b i n d i n g C s e e s e c t i o n s 7 and 10) and no d i f f e r e n c e was  d e t e c t e d - i . e . , the a d d i t i o n o f plasma was  significant  unnecessary.  (Table V I I I , F i g . 15)  TABLE V I I I E f f e c t o f s i l i c o n i z a t i o n o r plasma a d d i t i o n on assay  Midrange (pg  Condition Siliconized  tubes  Non-siliconized  tubes  N o n - s i l i c o n i z e d tubes & p»1asma 1  M5)  system  L.D.D. (pg  59  25  57  125  56  25  M5)  -51-  i  o-| I ,  0  !  1  1  1  1  1  12.5  25  50  100  200  j  pg Motilin  I  i  I  I  i  Fig. 15  Effect of s i l i c o n i z a t i o n of the incubation tubes or the addition of plasma on the s e n s i t i v i t y of the routine curve for m o t i l i n .  standard  -52-  C8)  Routine  assay.conditions  The diluent buffer, 0.04 % 'podium phosphate, pH 6.5; containing 5% charcoal - extracted plasma and 0.25% t r a s y l o l , was used i n a l l d i l u t i o n s and f o r correcting the f i n a l volume to 1.0 ml.  The composition  of the  incubation mixture was:125 100 y l 100  u  l  I-<motilin containing  ~5000 c.p.m.  standard m o t i l i n , range 12.5 -400 pg. or -  100 y l  interassay control or  50  200 y l  100 y l  unknown  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 i n t r i p l i c a t e and unknowns i n duplicate, i n 10 x 75 "mm  glass culture tubes at 4°C. and incubated at  4°C f o r 48 - 72 hours.  Non-specific binding (N.S.B.) was measured by s e t t i n g up tubes, minus antiserum, f o r 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  t o t a l count tubes, containing 100 y l quadruplicate.  I - m o t i l i n only, were set up i n  Table IX i l l u s t r a t e s the assay layout.  LAYOUT FOR ROUTINE RADIOIMMUNOASSAY  D.B. Total counts  LABEL  ANTIBODY  CONTROL  UNKNOWN  100 *  Standard curve NSB  900  100  Maximum binding  800  100  100  700  100  100  Interassay control NSB  800  100  Interassay control  700  100  Unknown NSB  800  100  Unknown  700  100  STDS  STD  100  100 100  100  100 100  refers to volume i n y l  _  100  -54-  (9)  Separation procedures  Both s p e c i f i c and non-specific methods exist for the separation of the antigen/ antibody complex (Bound) from the Free antigen.  The s p e c i f i c methods include  the double antibody technique and the use of a s o l i d phase antibody matrix where the antibody i s coupled to an immunologically inert material - e.g., Sephadex, Sepharose or Polyacrylamide.  The use of Sepharose-coupled  antibody  i n the m o t i l i n R.I.A. i s described i n the section on a f f i n i t y chromatography.  The non-specific methods include the addition of alcohol r e s u l t i n g i n the prec i p i t a t i o n 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 i n solution.  This l a s t method  i s that most'commonly used i n this laboratory. 1  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 p r i o r  to the addition of 200 y l to each assay tube, excluding the t o t a l count tubes. After being vortexed b r i e f l y , the tubes were centrifuged at 2800 rpm f o r 20 min.  The supernatant was  tions).  then decanted into a separate tube (for B/  Each tube was sealed with wax and counted i n an automatic' Y  Various charcoal concentrations were examined, each coated with 10% dextran.  estimac o u n  ter.  (w/w)  The results are graphed i n F i g . 16 and evaluated i n Table X.  -55-  "i  1  0  Fig. 16  1  6.25  12.5 pg  1  1  25 50 Motilin  1  100  1  200  Effect of varying charcoal concentrations i n the separation procedure on the s e n s i t i v i t y of the routine standard for m o t i l i n . with 10% (w/w)  curve  At each concentration the charcoal'was coated 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 s e n s i t i v i t y at the upper l i m i t of the curve.  TABLE X "Effect of varying the charcoal concentration i n the separation procedure  Midrange (pg  Condition  L.D.D. (pg  M5)  0.625g% charcoal  70  25  1.25  g%  "  52  6.25  2.5  g%  "  35  Recently, the dextran-coated  12.5  charcoal suspension has been prepared, 1 l i t r e at  a time, i n phosphate buffer only, and mixed for 3-4 hours. appropriate volume was  M5)  As required, an  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 i n the  r e p l i c a t i o n of t r i p l i c a t e and duplicate values.  (10)  Methods of data analysis  There are numerous methods used i n the expression of RIA r e s u l t s . used i n t h i s study include B/^, B /  T C  or % B, B /  B Q  X 100  Those  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 o f B/^, r e q u i r e s t h e c o u n t i n g o f b o t h t h e Bound and f r e e  a n t i g e n a f t e r s e p a r a t i o n and i s o b t a i n e d from t h e e x p r e s s i o n :  B/  =  p  SAMPLE  (Bound c.p.m.) -  NSB  (Bound c.p.m.)  ( f r e e c.p.m.)  ( f r e e c.p.m.)  The o t h e r methods r e q u i r e t h a t o n l y one component i s counted  after separation  but do r e q u i r e some method o f e s t i m a t i n g the T o t a l Counts (TC) i n i t i a l l y added t o each tube.  I f dextran-coated  used i t i s more convenient  t o count  c h a r c o a l i s the method o f s e p a r a t i o n  the f r e e antigen i n the c h a r c o a l p e l l e t .  T h e r e f o r e , %B i s c a l c u l a t e d from the e x p r e s s i o n :  %B  SAMPLE  (TC - FREE c.p.m.)  -  NSB  (TC - FREE c.p.m.)  TC i . e . , %B  =  TC  .(NSB) F R E E . c . p . m . ( S A M P L E )  FREE c.p.m.  TC  R e s u l t s may a l s o be expressed  as a percentage  o f t h e maximum b i n d i n g -  i . e . , B/  x 100, where B i s t h e b i n d i n g o f l a b e l a c h i e v e d when no un0 l a b e l l e d a n t i g e n i s added t o t h e system. D  Standard  U  curves a r e prepared by p l o t t i n g one o f these v a l u e s a g a i n s t t h e  c o n c e n t r a t i o n o f s t a n d a r d a n t i g e n , expressed arithmetically.  either logarithmically or  F i g . 17 i s a r o u t i n e s t a n d a r d  curve f o r m o t i l i n  obtained  a f t e r a l l t h e c o n d i t i o n s f o r a s e n s i t i v e RIA had been e s t a b l i s h e d .  A l l these c o n d i t i o n s were e s t a b l i s h e d f o r RIA w i t h a s p e c i f i c a n t i s e r u m t o  -58-  No Motilin added 0.60 •  0.50  H  0.40  0.30  0.20  0.1 0  H  0.0  l  10  1  1  1  20  40  80  Motilin  f i g . 17  1 160  1 320  l 640  (pg/ml.)  Routine standard curve f o r m o t i l i n , 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 m o t i l i n antisera and  should be re-evaluated for each  antiserum.  E.  Assay standards  and interassay controls  CI)  Preparation and storage of standards  1  Natural porcine m o t i l i n (M5) was used i n a l l standard preparations;  One - two  mg were weighed accurately on a Cahn electrobalance, dissolved i n deionized water to give a concentration of 1 yg/100 y l and aliquotted i n 100 y l amounts into s i l i c o n i z e d glass culture-tubes for l y o p h i l i z a t i o n and storage at -20°C.  Each month, or as required, a 1 -yg aliquot was reconstituted i n 0.04M sodium phosphate buffer, pH 6.5, containing 0.25% t r a s y l o l and 5% BSA,to a concentr a t i o n of 80 ng/iiil. p o l y v i n y l microtest  This s o l u t i o n was-stored at -20°C i n 1.0 ml amounts i n tubes.  At the time of assay an aliquot was d i l u t e d 1:20 - i . e . , 400 pg/100 y l and s e r i a l d i l u t i o n s 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 w e r e d i l u t e d  i n 0.04M phosphate buffer, pH 6.5,  containing 0.25% t r a s y l o l and 5% BSA, to a concentration of 1 ng/ml.  One  ml aliquots were stored at -20"C i n p o l y v i n y l microtest tubes and 100 y l samples were assayed at the beginning and end of every assay. control was discarded after being thawed.  Any remaining  -60-  (3)  Inter-and tntra-assay control  In 5 d i f f e r e n t assays-, 20 duplicate determinations of the control value were made.  The mean - S.D. was  110 ^ 23 pg-motilin/100 jal.  Results i n any  assay i n which the control lay outside these values were discarded..  Ihtra^  assay v a 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 m o t i l i n  (1)  Preparation of 13 - norleucine - m o t i l i n  The synthetic analogue, 13 - norleucine - m o t i l i n was prepared i n the laboratory by Dr. E. Wlinsch, Max-Planck I n s t i t u t e flir Eiweiss und Munich, W. Germany (Wlinsch et a l , 1973).  Lederforschung,  The RIA was used to monitor the  f i n a l p u r i f i c a t i o n 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 l a t t e r being found to  represent a f a i l e d synthesis, lacking 2 amino acid residues (-THR Further p u r i f i c a t i o n of MoB^  on SP - Sephadex C-25 resulted i n MoC^  -TYR). and M0C2,  both synthetic products being i d e n t i c a l to natural m o t i l i n with respect to amino acid composition and sequence.  -61-  (2)  Preparation of synthetic m o t i l i n fragments  During the preparation and p u r i f i c a t i o n of the synthetic analogue, the fragments containing residues 9~22 and 13--22 were also i s o l a t e d and p u r i f i e d .  B; •  Fragments of natural m o t i l i n  CT1  Cyanogen bromide cleavage of m o t i l i n  Cyanogen bromide CCNBr) treatment  of a polypeptide results i n chemical  cleavage of that polypeptide at the methionyl residue (Gross and Witkop, 1961,  1962).  The reaction was.performed i n 70% (v/v) formic acid at a  polypeptide concentration of 2.0 mg/ml and a CNBr concentration of 10 mg/ml. The reaction f l a s k , foil-covered to exclude:light, was l e f t at room temperature f o r 6 hours, then the contents were diluted 1:20 with d i s t i l l e d water p r i o r to l y o p h i l i z a t i o n .  The immunological a c t i v i t i e s of the intact m o t i l i n molecule and the unseparated mixture of cleaved and non-cleaved  CNBr-treated m o t i l i n were  compared on an equimolar basis.  C2)  Tryptic and chymotryptic digestion of m o t i l i n  Enzymatic cleavage of the polypeptide was performed i n 1% ammonium b i c a r bonate at a polypeptide concentration of 0.2% (w/v) and an enzyme:substrate r a t i o of 1:50 (w/w).  The reaction proceeded f o r 6 hours at room temperature  and was terminated by l y o p h i l i z a t i o n , redissolving i n 0.5 ml water and b o i l i n g for 6 mlns. i n a water bath.  The solution was centrifuged to remove any  p r e c i p i t a t i o n and the supernatant was l y o p h i l i z e d .  B i o l o g i c a l , and immunological  a c t i v i t i e s were e s t i m a t e * - f o r t h e unseparated  d i g e s t i o n p r o d u c t s and compared w i t h the i n t a c t m o l e c u l e on an equimolar  basis.  C.  Modifications of natural m o t i l i n  Cl)  C-terminal residue removal  The reaction involved treatment of 100 nM m o t i l i n i n 0.1M ammonium b i carbonate with 200 yg carboxpeptidase A 'DFP (Diisopropyl phospho-floridate 1  treated) i n 2.OM ammonium bicarbonate at a peptide: substrate r a t i o 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 l y o p h i l i z a t i o n 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 i n 150 JJl deionized water i n an acid-washed culture tube.  12 x 75 mm glass  Reagent (5% PITC i n pyridine) was added and the tube was  flushed with nitrogen to expel the a i r , and covered with parafilm p r i o r to incubation at 45°C for 1.5 hours.  At the end of this time the tube was un-  covered a f t e r 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 at 45°C f o r 30 mins.  t r i f l u o r o a c e t i c acid and incubation  The mixture was evaporated to dryness i n a vacuum  -63-  dessicator ionized  four  discarded  times  each  chloride the  (3) The  in  to  150  method  of  The  polypeptide,  0.6  x  and  the  tube  chloride,  of  of  N-terminal  5x5  using  approximately  45°C  residue,  organic  layer  removed  to  check  being  motilin  sulphuric  acidi.re-  for for  2-22  dansyl completeness  1%  which  20  for  u l  and h y d r o l i z e d  at  110°C  was  6M  complete  sodium hydroxide  a glass  chloride,  18  tube  and  The  contents acid,  tube  (pyrex)  then  added  water  2.5  was c e n t r i f u g e d ,  mins.  for  were  Deionized  hydrochloric  the  to  sodium bicarbonate  5 - s u l p h o n y l  tube  Bruton  of  plates.  2 ul  50  hydrolysis  the modification  was t r a n s f e r r e d  in  over  by•ex-  containing  aliquot  VI  5nM,  -  at  vacuum  upper  concentrated  and r e l y o p h i l i z e d .  and incubated and r e d i s s o l v e d  with  cms p o l y a m i d e  the  acid  10  was. removed  residue  was f o l l o w e d  to  When  and a  the  solution,  over  y± d e -  in  cycle.  added  sealed  aqueous  vacuum,  water  Cdimethylnaph,thalene  heat  acetate,  200  was r e d i s s o l v e d  phenylalanine  t h e new N - t e r m i n a l  was c e n t r i f u g e d  each were  parafilm  under  under  mms, and l y o p h i l i z e d ,  50  hilized  remaining  Gray (1967) of  mis butyl  deionized  Identification  ul  2  dryness  degradation  then  with The  ul  pellets,  phenylthiocarbamyl  determination  Hartley 0-970)  2.5  hydroxide  free  time.  evaporated  dissolved  of  The  water.  tracting  was  over,sodium  and  mg/ml  covered were  the  tube  dansyl  in  acetone)  with  again  lyop-  being  then  hours.  was c e n t r i f u g e d ,  opened  and  dried  pellets.  2 Thin  layer  systems  chromatography  was p e r f o r m e d .  on  The  5  cm  polyamide  hydrolysis  plates *  products  were  in  several  dissolved  solvent in  2.5  jal  50% aqueous pyridine and ~ 0.5 u l spotted on each side o f the p l a t e . standard solution  '<CO.5  ul) containing the dansyl derivatives  A  phenyla-  of  lanine, isoleucine, p r o l i n e , glycine, glutamic acid, serine and arginine, 1 UM each acid/ml i n acetone; one side only.  0.1M acetic acid (3:2 v/v) was spotted on  The polyamide plate was.subjectedfto  ascending chromato-  graphy i n two dimensions i n the appropriate solvent systems.  Dimension I  Solvent system 1  Water: 200  :  CWoods  XI  90% formic acid 3  and Wang, 1967)  Benzene: 9  (v/v)  :  g l a c i a l acetic acid 1  (v/v)  (Woods and Wang, 1967)  ITT  Hexane:  n-butanol:  3 : 3  g l a c i a l acetic acid  :  I  (v/v)  (Crowshaw et al,1967) IV  0.1% Ammonia: 9  :  Ethanol 1  (v/v)  After running i n solvents I and I I , the plates were viewed under shortwave u l t r a v i o l e t l i g h t and i d e n t i f i c a t i o n of the dansylated residue made. Dansyl serine/dansyl theonine, dansyl glutamic acid/ dansyl aspartic acid and  -65-  dansyl glycine/dansyl alanine may  only be d i f f e r e n t i a t e d after chromato-  graphy i n solvent III and solvent TV was  used to separate the basic residues  arginine, h i s t i d i n e and E - l y s i n e .  If lysine or tyrosine was present lysine or 0-dansyl tyrosine was  at any p o s i t i o n i n the  always seen.  polypeptide,E-dansyl-  P o s i t i v e i d e n t i f i c a t i o n of either  lysine or tyrosine as N-terminal requires the presence of bis-dansyl-lysine or bis-dansyl-tyrosine respectively.  If proline i s 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 m o t i l i n was  dissolved i n 1.0 ml 50% saturated sodium acetate. excess of acetic anhydride was reaction mixture was  added i n five portions over 1 hour and  s t i r r e d continuously  further hour the reaction mixture was Sephadex G15  A t h i r t y - f o l d molar  f i n e (0.7 x 100 cms)  After a  frozen, l y o p h i l i z e d and desalted on  i n 0.2M  6 mis/hour and 1.2 ml f r a c t i o n s i z e .  at room temperature.  acetic acid at a flow rate of  The acetylated derivative was  com-  pared with natural m o t i l i n , on an equimolar basis, for b i o l o g i c a l and immunological a c t i v i t y .  the  -66-  (5)  Acylation-succjnylated derivative  The method of Klotz (1967) was s l i g h t l y modified.  One mg motilin was  dissolved i n 2.0 mis deionized water, the pH brought to 7.0 with 0.1M sodium hydroxide and constantly monitored during the addition of a t h i r t y f o l d 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 s t i r r e d gently at room temperature for a further 1.5 hours.  The solution was  frozen, l y o p h i l i z e d and desalted on Sephadex G15 f i n e , 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 i c e , i n a fume hood.  Cyanogen bromide (CNBr) at a concentration of 100 mg/g Sepharose 4B  was added i n a volume of deionized water, equal to the t o t a l , 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 a l 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 s l u r r y at 4° f o r 1-2 weeks (Cuatrecasas, 1970).  -67-  B.  Coupling of ligand to activated Sepharose 4B  Activated Sepharose 4B was made up i n an equal^volume of 0.1M sodium b i carbonate.  Antiserum to motilin or GIF was diluted i n an equivalent volume  of bicarbonate to a f i n a l concentration of 30 jal antiserum/g Sepharose 4B. The reaction mixture was s t i r r e d 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, p r i o r 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 i l l u s t r a t e the anti-serum d i l u t i o n curves  obtained i n a t y p i c a l procedure, with v i r t u a l l y no antibody a c t i v i t y detectable i n 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 i n 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 IO  3  I x IO  4  Reciprocal Antiserum Titre  Fig. 18  M o t i l i n antiserum d i l u t i o n curves comparing t h e : a c t i v i t y of the antiserum prior to coupling to Sepharose 4B with the a c t i v i t y remaining i n the wash after completion of the coupling procedure.  I x IO  5  -69—-  2.0 i  O i  *  .0  Pre-Coupling  CM  x  DQ  Post-Coupling 0.04  I xlO*  Reciprocal of Antiserum  Fig. 19  x 10*  I x 10 Titre  GIP antiserum d i l u t i o n curves comparing the a c t i v i t y of the antiserum p r i o r to coupling to Sepharose 4B with the a c t i v i t y remaining i n 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 allowed to swell overnight before the gel was  s t i r r e d gently into excess buffer  at room temperature.  The fines were decanted  de-aerated under vacuum for 30 mins.  removed R O that the f i n a l suspension was  liquor was  and  The supernatant  a s l u r r y which would  pour e a s i l y without trapping further a i r .  The column was mounted v e r t i c a l l y , out of draughts and d i r e c t sunlight. Buffer was  injected through the outlet tubing to f i l l the space beneath  the bed support and to a l e v e l of approximately 10 cm above the support. The outlet was  closed and the s l u r r y poured gently down the column w a l l .  If necessary, a gel reservoir was gel being added at one time.  attached  to the column to ensure a l l the  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 i n subsequent  A f i l t e r paper d i s c (Whatman's 3MM)  was  operations.  inserted to s t a b i l i z e the g e l -  l i q u i d interface and the gel equilibrated i n buffer  The buffer above the l e v e l of the gel was  overnight.  removed and the sample, dissolved  i n a small volume of buffer, applied to the gel and allowed to sink to the l e v e l of the gel surface. sample volume, was  A volume of buffer, roughly equivalent  to the  similarlyy applied, washing the sample well into the body  -71-  of the gel, attached  Excess Buffer was replaced on top of the gel and the column  to the Buffer.  As the Buffer flowed through the g e l , fractions of  eluant buffer of a predetermined size were collected.  Between runs, the  column was stored i n 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 i n 0.5M hydrochloric acid f o r 30 mins. whilst cation exchangers CWhatman's CM or CE-Sephadex) were f i r s t treated i n 0.5M sodium hydroxide.  The exchangers were washed well with  d i s t i l l e d water u n t i l the intermediate pH's were 4 and 8 respectively.  The  treatments were then reversed, the anion exchanger being washed i n 0.5M sodium hydroxide and the cation exchanger i n 0.5M hydrochloric acid f o r 30 mins.  Both exchangers were well washed i n d i s t i l l e d water u n t i l the  effluent pH was near n e u t r a l i t y .  The fines were decanted and the exchangers  de-aerated under vacuum for 30 mins.  E q u i l i b r a t i o n i n the s t a r t i n g 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 l i q u i d after 10 mins. u n t i l the pH  and conductivity of the effluant were i d e n t i c a l to those of the buffer.  The columns were packed and the samples applied as described i n Section A. Development of the column was achieved by either stepwise increases i n buffer i o n i c strength or by establishing a gradient of i o n i c strength.  The most  strongly absorbed material was cleared by passage of 0.2M ammonia through the column, and the c e l l u l o s e was stored i n 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 i n the s t a r t i n g buffer to ensure replacement of the necessary counter ion.  POLYACRYLAMIDE GEL ELECTROPHORESIS The method followed i s a modification of that of Johns (1967).  The gel  solution was prepared by c a r e f u l l y mixing 10 mis of monomer (40% w/v acrylamide and 0.6% w/v NjN"*" - methylenebisacrylamide i n d i s t i l l e d water) 1 with 10 mis of catalyst I (0.5% v/v N, N , N , N in 4.6M  1 - tetramethylenediamine  acetic acid) and 6 mis of catalyst IT (0.6% w/v  in d i s t i l l e d water).  ammonium persulphate  The mixture was de-gassed under gentle vacuum for 30  mins. and 10 ml gel solution placed i n each prepared 5 x 75 mm gel column. D i s t i l l e d water was layered on top of each gel and polymerization was rated under direct l i g h t for the f i r s t hour, then allowed to continue room temperature.  acceleat  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 i n a Shandon electrophoresis apparatus, Model 12734, modified to allow cooling of the system  The buffer used was 0.01M  acid and current was passed at 320 v o l t s , for 3 hours.  acetic  The electrodes were  placed with the anode uppermost.  Samples for electrophor esis were dissolved i n 0.002M acetic acid, 1.0M respect to sucrose.  with  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  (A)  acetic acid.  Method for staining and destainjrig for q u a l i t a t i v e determinations  The samples were s e t t l e d into the gel By passing current through the gel at 320 v o l t s 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 v o l t s for a further 15 mins. rinsed and r e f i l l e d with 0.01M  The reservoirs were then carefully  acetic acid, the apparatus re-assembled, and  the gels destained by passing current at 320 v o l t s 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 s e t t l e 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 p l a s t i c and the glass cracked i n a vice.  The gels were rinsed with tap water and the marker gels were stained  in p e t r i dishes, covered with 0.5% w/v  amidoblack for 3 - 4  hours.  These  gels were destained e l e c t r i c a l l y i n an enamel or p o l y v i n y l basin, i n cotton wool saturated with 1.0M  acetic acid, by passing current at 150 v o l t s 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  appropriate sections cut from the unstained gel with 000 s i l k . section was 0.1M  the  The gel  then homogenized by passage through a 5 ml luer-lok syringe i n  a c e t i c 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 zation.  and used i n immuni--  In the l a t t e r case the polyacrylamide acted as the c a r r i e r molecule  for the hapten.  ANIMAL PREPARATIONS (A)  Chronic dog preparation  Labrador or labrador-cross breeds were selected f o r t h e i r size, nature and stamina.  The weight range used was 20 - 25 kg. A l l surgery was performed  aseptfcally.  After an 18 hour f a s t , the dog-was anaesthetized with a rapid  intravenous i n j e c t i o n of 5% sodium thiopental ("Pentothal"), given to e f f e c t , usually 9 - 1 5 mis.  An endotracheal tube was placed i n position and  anaesthesia was maintained with "Fluothane" delivered from a Foregger open c i r c u i t anaesthetic apparatus at an oxygen flowrate of 3 litres/min. and a fluothane concentration of 2.5%.  Cl)  Bickel pouch  An e x t r i n s i c a l l y denervated pouch of the body of the stomach was constructed from the greater curvature.  A stainless s t e e l and t e f l o n cannula was placed  in the pouch and brought to the exterior through a stab wound i n the dog's abdominal wall, i n the l e f t upper quadrant. removed vagal innervation and sympathetic  Sectioning of the stomach wall  denervation was achieved by stripping  the nerve plexus around the splenic artery and vein; Cthe blood supply to the pouch), f o r approximately 1 cm and removing any mesentery from the pouch.  -75-  This i s referred to i n the following study as the fundic pouch.  The fundic pouch cannula was l e f t open and draining at a l l times, except when fundic pouch motor a c t i v i t y was being monitered.  A stainless s t e e l and t e f l o n cannula (Thomas, 1941) was placed i n the most dependent portion of the stomach remnant, with a purse-string suture.  This  cannula was brought to the exterior through a flank i n c i s i o n 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 approximately 30 cm d i s t a l to the ligament of T r e i t z .  (2)  Mann-Bollman f i s t u l a  A length of terminal ileum, approximately 10 cm long was removed and intest i n a l continuity restored with an end-to-end anastomosis.  The d i s t a l 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 i n place on the right abdominal quadrant to form a stoma.  A p a r t i a l antrectomy was performed to remove gastrln-producing tissue with a resultant reduction i n water and e l e c t r o l y t e loss through the continuouslydraining fundic cannula.  -76-  (3)  Gastric f i s t u l a  A g a s t r i c f i s t u l a was constructed i n  dogs when required by i n s e r t i n g a  Thomas cannula into the most dependent portion of the whole stomach.  The  dogs were used i n the control studies.  C4)  Truncal vagotomy  Truncal vagotomy was performed by the thoracic route.  After  anaesthesia  was induced, the animals were placed on a B i r d r e s p i r a t o r and the chest opened at the 8th r i b interspace.  The oesophagus was located and the l e f t  vagus i d e n t i f i e d , divided, and 2 cm removed. was s i m i l a r i l y treated.  In a small proportion of the dogs an interconnecting  branch l i e s between the right and l e f t vagi. sectioned.  The r i g h t vagal branch  I f present, this was also  After the a i r was expelled from the thoracic cavity, nylon  sutures were used to approximate the i n t e r c o s t a l muscles.  (5)  Antrectomy  The antrum was removed i n g a s t r i c f i s t u l a dogs. was located and divided.  The pyloroduodenal junction  The junction between the antrum and body of the  stomach was i d e n t i f i e d by the subtle change i n texture on the surface of the stomach and the antrum was excised by sectioning at t h i s junction. A l l blood vessels supplying;this region were l i g a t e d and sectioned.  C6)  Vagotomy and antrectomy  In a separate operation the antrectomized dogs were vagotomized and v i c e  - 7 7 -  versa.  After a l l s u r g i c a l •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 i s shown i n Fig.  20.  EXPERIMENTAL PROCEDURES CA)  In chronic dog with B i c k e l pouches and duodenal f i s t u l a e  Dogs were fasted overnight before use.  They were harnessed  provided support whilst maintaining the dog upright. was  i n a stand which  A polyethylene b o t t l e  attached to the open Thomas cannula to c o l l e c t drainage from the stomach  and prevent passage of gastric secretions into the jejunum.  A Foley catheter ( i d . 3mms) was was  attached to a syringe b a r r e l .  Its t i p  inserted into the duodenal f i s t u l a for the approximate length of the  f i s t u l a or u n t i l saline i n the syringe b a r r e l flowed freely into the duodenum under gravity, and kept i n that position with cords around the animal's body.  Intra-duodenal infusion was performed under gravity from  an open syringe b a r r e l 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" /2" hypodermic needle attached to polyethylene tubing (PE 60) L  was  inserted into either the r a d i a l or saphenous vein f o r intravenous i n j e c t i o n or infusion.  Blood samples were taken from a permanently-indwelling  (experimental duration) intravenous cannula on a 19G Venocut Infusion Set).  /8" needle (Argyle  Patency of both cannulae was maintained with a  gravity-fed saline drip.  Blood samples were allowed to c l o t f o r 20 mins. at 4°C.  After centrifugation  for 10 mins. at 3000 rpm the serum was removed and stored at -20°C u n t i l required for RIA.  (I)  Bioassay f o r m o t i l i n  The fundic pouch cannula was  connected to a venous pressure transducer  (Statham P 23 BB) v i a a w a t e r - f i l l e d tube with a side arm, allowing c o l l e c t i o n 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  the f l u i d being changed between test procedures.  experiment,  Test procedures, either  intravenous infusion or i n j e c t i o n or duodenal infusion, were not  performed  u n t i l the fundic pouch motor a c t i v i t y had established i t s basal rhythm. Recordings of fundic pouch motor a c t i v i t y were made continuously on a polygraph (Gilson pen recorder).  M o t i l i t y indices were measured over a s p e c i f i c 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 u n t i l subjected to RIA f o r m o t i l i n , etc.  C2)  Effect of GIP on acid secretion  A 15 ml graduated test tube was attached to the fundic pouch cannula to c o l l e c t the output from the pouch over each 15 min.period.  Acid secretion  was stimulated by continuous intravenous infusion of pentagastrin or histamine dihydrochloride.  I f desired, exogenous GIP could also be administered v i a  the same, intravenous cannula.  The volume of gastric acid produced by the fundic pouch i n each period was measured, diluted 1:10 with d i s t i l l e d water and t i t r a t e d to pH 7.0 with 0.01M  sodium hydroxide i n a t i t r a t o r assembly  (Titrator I I , 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 i n t r a -  duodenally from a Harvard infusion pump, v i a 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 and 0.15N  (Lipomul-Upjohn)  hydrochloric acid were each infused at a rate of 1.91 mis./ min.  Over 30 min.  A plateau of g a s t r i c acid secretion was considered established  -81-  a f t e r continuous  intravenous  i n f u s i o n o f 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 o f H  +  s e c r e t i o n were  w i t h i n 10% o f each o t h e r .  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 s e c r e t o r y c a p a c i t y , and because o f t h e t r o p h i c e f f e c t o f g a s t r i n on t h e 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 t h e s e c r e t o r y c a p a c i t y o f one B i c k e l pouch t o v a r y t h e d u r a t i o n o f t h e study.  during  Dose-response s t 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 t h e dose o f 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 e q u a l t o 75% o f t h e maximum o u t p u t .  The v a l u e s  of the H  s e c r e t i o n were expressed  as r a t i o s o f t h e mean o f t h r e e p l a t e a u  periods.  The IR-GIP r e s p o n s e was p l o t t e d as t h e change i n IR-GIP (A IR-GIP)  f r o m t h e mean o f t h e t h r e e p e r i o d s p r i o r t o t h e s t a r t o f t h e duodenal i n f u s i o n )  CB)  I n c h r o n i c dogs w i t h g a s t r i c  fistulae  The  dogs were accustomed t o b e i n g harnessed i n t h e s t a n d and were f a s t e d  for  18 hours p r i o r t o any s t u d y .  intravenous  (I)  i n f u s i o n s administered  Determination  Blood samples f o r RIA were o b t a i n e d and as d e s c r i b e d i n S e c t i o n A.  o f t h e r a t e o f g a s t r i c emptying o f l i q u i d s  Sodium c h l o r i d e , 0.15M, c o n t a i n i n g 60 m g / l i t r e p h e n o l r e d as an i n d i c a t o r , was  instilled  i n t h e stomach v i a t h e g a s t r i c f i s t u l a and t h e n d r a i n e d a t t h e  end o f a 10 min. p e r i o d .  V a r i o u s c e n c e n t r a t i o n s o f m o t i l i n were  administered  as i n t r a v e n o u s i n f u s i o n s .  The p h e n o l r e d c o n c e n t r a t i o n i n t h e f l u i d m e a l was d e t e r m i n e d i n b o t h t h e  -82-  initial  meal p r i o r t o i t s i n s t i l l a t i o n  d r a i n e d from t h e stomach.  i n t o t h e stomach and i n t h e f l u i d  P h e n o l r e d d e t e r m i n a t i o n s were performed as  f o l l o w s :-" 1 m l o f t h e l i q u i d meal p l u s 2 mis o f sodium phosphate (27.5 g N a ^ P O ^ / l i t r e ) Were made up t o 10 m i s w i t h d i s t i l l e d w a t e r and mixed w e l l .  The O.D. o f t h e  s o l u t i o n was r e a d a t 550 nm i n a 1 cm l i g h t p a t h and used t o c a l c u l a t e t h e r a t e o f g a s t r i c emptying from t h e f o r m u l a .  Rate o f emptying (mis/10 mms)  =  (V-P,) 11  -  (Pi + P ) r  (VP) r r 12  Where V. and V a r e t h e volumes o f f l u i d i n s t i l l e d and r e c o v e r e d and P. and i r x P  r  a r e t h e c o n c e n t r a t i o n s o f p h e n o l r e d i n t h e i n s t i l l e d and r e c o v e r e d f l u i d s  respectively.  T h i s c a l c u l a t i o n i s based on t h e assumption t h a t t h e concent-  r a t i o n o f p h e n o l r e d l e a v i n g t h e stomach i s t h e mean o f t h e i n i t i a l  and f i n a l  concentrations.  C2) . D e t e r m i n a t i o n o f t h e r o l e o f g a s t r i c emptying o f s o l i d s Dogs were f e d a p r o p r i e t a r y canned dog f o o d e q u i v a l e n t t o a 3 g d r y w e i g h t / k g . The amount o f s o l i d m a t e r i a l t h a t remained i n t h e stomach a t v a r i o u s t i m e intervals  (weighed a f t e r d e s s i c a t i o n ) was e x p r e s s e d as a f r a c t i o n o f t h e  i n i t i a l weight.  M o t i l i n was a d m i n i s t e r e d as an i n t r a v e n o u s i n f u s i o n o f 1,0 yg/kg/hour  -83-  (C)  I n t h e i n t a c t dog  Dogs, i n t h e w e i g h t range 3CH35 k g ^ w i t h 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 t o remain h a r n e s s e d i n t h e e x p e r i m e n t a l s t a n d . f a s t e d f o r 18 h o u r s .  A l l animals were  B l o o d samples f o r RIA and g l u c o s e d e t e r m i n a t i o n s  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 a d m i n i s t e r e d as d e s c r i b e d i n S e c t i o n A.  Test substances were g i v e n o r a l l y from a g l a s s s y r i n g e f i t t e d w i t h  a f l e x i b l e catheter.  When t h e c a t h e t e r t i p was h e l d i n s i d e t h e dog's cheek  by t h e p o s t e r i o r m o l a r s , any l i q u i d d e p o s i t e d t h e r e induced s w a l l o w i n g  \  w i t h o u t undue trauma t o t h e dog.  U n l e s s o t h e r w i s e s t a t e d , g l u c o s e was a d m i n i s t e r e d o r a l l y as a 20% d e x t r o s e s o l u t i o n i n d i s t i l l e d water a t a dose o f 1 g/kg.  The o r a l f a t used was  L i p o m u l , a p a l a t a b l e e m u l s i o n c o n t a i n i n g 66g t r i g l y c e r i d e s p e r 100 m i s .  R e s u l t s were expressed as change from c o n t r o l (A.),the c o n t r o l v a l u e b e i n g t  d e f i n e d as t h e mean o f t h r e e f a s t i n g serum v a l u e s o f t h a t parameter, a t 15 min. i n t e r v a l s a t t h e s t a r t o f t h e experiment.  measured  A c o n t r o l was o n l y  a c c e p t a b l e i f t h e v a r i a t i o n i n serum g l u c o s e 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 u s i n g t h e Student t t e s t .  SERUM ANALYSIS (A)  Radioimmunoassays  (I)  GIP radioimmunoassay  A radioimmunoassay f o r GIP was developed by K u z i o e t a l (1974) b u t has undergone r e p e a t e d m o d i f i c a t i o n s i n c e then i n an attempt t o improve t h e  -84-  s p e c i f i c a c t i v i t y and s t a b i l i t y of the labelled tracer, the a f f i n i t y of the antisera and the e f f i c i e n c y and r e p r o d u c i b i l i t y of the separation technique.  Ct)  Iodination of GIP  Ca)  Routine chloramine-T iodiriatibri and p u r i f i c a t i o n 125  O r i g i n a l l y , when 6 yg GIP was reacted with 2 mCi  T-Na,  the best tracer  did not share complete i d e n t i t y with the peak of r a d i o a c t i v i t y but was associated with the descending limb of the radiochromatogram and therefore with reduced counts.  I f the r a t i o of polypeptide : iodine was increased to  6:1, the peak immunoreactivity coincided more closely with the radioactive peak, with no loss i n s p e c i f i c a c t i v i t y . The reagents were added i n the following order i n a s i l i c o n i z e d 10 x 75 mm glass tube, mixing being ensured by bubbling a i r through the reaction mixture:-  6 j(g GIP i n 100 y l 0.4M  sodium phosphate, pH  7.5  125 1 -mCi  1-Na i n 10 "yl c a r r i e r - f r e e NaOH  40 yg chloramine-T i n 10 y l 0.4M  sodium phosphate, pH  7.5  15 sec. exposure  252 ^lg sodium metabisulphite i n 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 containing 2000 Kill Trasylol/100 ml and 0.5% B.S.A.  acetic acid,  Approximately 40 x 400  y l fractions were collected and 10i gy.l21. aliquots were counted for 0.1 was min. to produce the radiochromatogram in F A charcoal-binding assay  . 21  Chloramine-T iodination of GIP at a peptide:iodine  ratio  of 6 ug: 2 mCi. Separation of l a b e l l e d GIP from free iodide on Sephadex G25 i n 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  i n a t o t a l 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 i n s i l i c o n i z e d glass tubes. The average s p e c i f i c a c t i v i t y of l a b e l l e d GIP p u r i f i e d i n 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 e f f e c t 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 n the presence of 4 yg chloramine-T.  I-Na  After 30, 60, and 120 sec, 20 y l  aliquots were removed from the reaction mixture and added to 25 yg sodium metabisulphite eluant buffer.  i n 20 y l phosphate buffer, along with 100 y l of the column Each aliquot was p u r i f i e d i n the routine manner on Sephadex  G25 f i n e and the s p e c i f i c a c t i v i t y of each calculated approximately from the % incorporation of the iodine into the peptide, estimated from the appropr i a t e radiochromatogram.  After 30 sec exposure the s p e c i f i c a c t i v i t y was  45 mCi/mg, a f t e r 60 sec i t was 112 mCi/mg and a f t e r 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  20 Fraction #  Fig. 2'2  30 -  40  IO/JI  Lactoperoxidase iodination of GIF at a peptide:iodine r a t i o of 6 3ig;l mCi.  Separation of l a b e l l e d GIF from free iodide  on Sephadex G25  i n 0.2M  (x-x); NSB  Co-o)  acetic acid.  Counts per 0.1  min  -89-  iodine, i t was expected to be preferable as the oxidizing agent i n the i o d i n ation of GIP. The procedure was performed exactly as was outlined for m o t i l i n . The radiochromatogram, i n F i g . 22, was used to calculate the s p e c i f i c a c t i v i t y from the % incorporation of iodine into the polypeptide.  The s p e c i f i c a c t i v i t y ,  calculated this way. was 62 mCi/mg and therefore this method showed no improvement over the chloramine-T mediated oxidation.  (2)  P u r i f i c a t i o n of GIP  In an attempt to separate l a b e l l e d from unlabelled GIP the fractions selected a f t e r gel f i l t r a t i o n were pooled, l y o p h i l i z e d and subjected to ion exchange chromatography.  The l a b e l was reconstituted i n 2 Ml 0.06M T r i s 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 b u f f e r , containing 1% T r a s y l o l 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 i n an automatic <y counter i s 125 shown i n F i g . 23. The  I-GIP peak was located by determining  of lowest charcoal-binding, as previously described.  The appropriate fractions  were pooled and diluted 1:4 i n acid-ethanol, (15 ml ethanol water : 0.3 ml concentrated HCI).  the region  : 5 ml d i s t i l l e d  The s p e c i f i c a c t i v i t y was determined by  assaying s e r i a l d i l u t i o n s 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 l a b e l preparation was 5 - 9 % B. Internal decay during the storage of this l a b e l raised this value to 20 % B  -90-  Fraction #  Fig. 23  Chloramine-T iodination of GIF.at a peptide:iodine r a t i o of 5 jig: ImCi.  Separation of labelled GIF from unlabelled GIF:On QAE  Sephadex A25 i n 0.06M T r i s , pH8.5. Column calibrated with porcine GIP).  Counts per 0.1 min (• • ) .  -91-  a f t e r 3 weeks a t -20° and t h i s was u n a c c e p t a b l e .  ^ 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 o f  1-GIP  c o n t a i n i n g 2,1 x lO** cpm, one month o l d and w i t h a NSB o f 24.2% B, was t r e a t e d 125 w i t h 10 mg r e s i n CAG 1-XIO) t o adsorb  any f r e e  1 present.  The m i x t u r e  was v o r t e x e d w e l l , c e n t r i f u g e d a t 3000 rpm f o r 10 m i n . and t h e s u p e r n a t a n t 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)  w h i c h absorbed  the l a b e l l e d antigen.  A f t e r m i x i n g and c e n t r i f u g a t i o n , t h e  s u p e r n a t a n t was d i s c a r d e d and t h e p e l l e t washed w i t h d i s t i l l e d w a t e r .  The  l a b e l l e d p e p t i d e was e l u t e d from t h e s i l i c a i n t o 2 mis 40% acetone: 1% acetic acid: supernatants  60% d i s t i l l e d w a t e r (v/v) and t h e s u p e r n a t a n t was k e p t .  Both  and an u n t r e a t e d v i a l o f t h e 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 t o t h e same c o n c e n t r a t i o n o f cpm/100 u l and s t a n d a r d curves w i t h each l a b e l .  prepared  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 t h e s t a n d a r d  curves  o b t a i n e d a t e i t h e r t h e LDD o r midrange v a l u e s b u t treatment w i t h AG 1-XIO s u b s t a n t i a l l y reduced shelf l i f e  t h e NSB o f t h e l a b e l and c o u l d be used t o p r o l o n g t h e  (Table X I , T i g . 24)  TABLE X I 125 E f f e c t o f treatment  Label  treatment  of  I-GIP on NSB o f t h a t l a b e l  LDD Pg GIP  Midrange pg GIP .  NSB % B  Untreated  25  160  24.2  AG I-XIO  25  200  14.1  quso  25  140  26.0  -92-  Comparison of GIP s t a n d a r d curves o b t a i n e d w i t h u n t r e a t e d 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  o f a n t i s e r a t o GIP  x  A n t i s e r a t o GIP were r a i s e d i n r a b b i t s and g u i n e a p i g s by t h e methods previously detailed f o r motilin.  (a)  Storage o f 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 u s a b l e 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 a t 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 f r o z e n i n 50 - 200 y l amounts i n s i l i c o n i z e d 10 x 75 mm g l a s s tubes a t -20°C.  Cb)  Characterization of antisera  A l l a n t i s e r a t o GIP were checked f o r c r o s s r e a c t i v i t y w i t h o t h e r i e s p e c i a l l y those o f t h e 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  polypeptides,  intestinal  peptide,  glucagon and s e c r e t i n i t s e l f , w i t h w h i c h 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 a n t i s e r u m , Van 8, d i d n o t c r o s s r e a c t w i t h any o f t h e s e , o r w i t h c h o l e c y s t o k i n i n - p a n c r e o z y m i n , g a s t r i n , m o t i l i n o r i n s u l i n a t concent r a t i o n s up t o 10 ng/100 y l .  05.)  As say  protocol  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 p e r 100 y l and 5% (w/v) c h a r c o a l - e x t r a c t e d The c o m p o s i t i o n o f t h e i n c u b a t i o n volume was:-  plasma.  -94-  100 yL 100  125  IrvSJP  containing- 5000 cpm  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 d i l u t i o n Diluent buffer to a f i n a l volume of 1.0  ml.  Assays were set up i n a cold tray at 4°C, i n t r i p l i c a t e , i n s i l i c o n i z e d 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 i n the antiserum volume required. the volume was  After the normal incubation period,  corrected to 1.0 ml with diluent buffer prior to the separation  of bound and free antigen.  In both cases, NSB  tubes and t o t a l count tubes were included and the analysis  of data was performed as described for m o t i l i n .  The standardization of the incubation m i l i e u was curves with and without the addition of 100 yl  examined by preparing  standard  charcoal-extracted plasma,  to compensate for the protein added i n the remainder, of the assay when serum samples were being-monitored.  There was  no s i g n i f i c a n t difference i n 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 i n 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 i n 1.5 ml polypropylene mictrotest tubes at -20°C and were used i n the assay after being diluted 1:1 i n diluent buffer, i . e to 400 pg/100 u l . The other standards were prepared by s e r i a l d i l u t i o n . 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 i n 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 u l . The controls were stored i n 1.5 ml polypropylene microtest tubes at -20°C and assayed at the beginning and end of every assay, to provide an estimate of i n t r a - and inter-assay v a r i a b i l i t y . In 50 a r b i t 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 i n 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 l a b e l l e d peptide bound to the a n t i -  serum was routinely performed using dextran-coated charcoal, exactly as  -96-  100-®  80  8  6  0H X  o  CD  Normal Standard C u r v e Standard C u r v e after A d d i t i o n of 100 M C - E Plasma  20H 0-  12.5  Fig. 25  25 pg  GIP  50  T  100  200  Effect of the addition of charcoaL--extracted plasma on the s e n s i t i v i t y of the.routine.standard curve f o r GIP.  1  400  -97-  described for m o t i l i n .  An alternative method involyes the p r e c i p i t a t i o n  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 u l dextran-coated charcoal suspension or 1 ml dioxane, there was no s i g n i f i c a n t difference i n curves obtained (Fig. 2 6 ) , but at this time the dioxane method has proved less r e l i a b l e .  IT.  Insulin radioimmunoassay  Cl)  By commercially available k i t  Measurement of immunoreactive  i n s u l i n (TRI) was performed with the Amersham  Searle Insulin Immunoassay K i t (IM-39) - developed from the method of Hales 125 and Randle (1973).  Insulin tracer, labelled with  - I at a minimum s p e c i f i c  a c t i v i t y of 50 mCi/mg reacted with i n s u l i n antibody, provided as a dessicate already bound to a second antibody. range 2.5 - 160 UTJ/ml.  The standards were human i n s u l i n , over the  A l l d i l u t i o n s were made i n 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 l a b e l addition and incubation for a further 18 hours.  After centrifugation,. the supernatant was discarded and the p e l l e t  consisting of the Bound antibody/antigen complex was gamma counter. counts (2)  counted i n an automatic  Results were expressed as a percentage of the i n i t i a l l y added  (%B). By non-commercial radioimmunoassay  A laboratory radioimmunoassay was developed from the method of Dr. K.D. (personal  communication).  Buchanan  -98-  Fig.  26  Effect of separation of.bound from free antigen by dextrancoated charcoal or dioxane on the s e n s i t i v i t y of the routine standard curve for GIP.  -99-  (a)  Iodiriatibri of insulin-  The following reagents were prepared just p r i o r to use and mixed together i n a s i l i c o n i z e d glass 12 x 75 mm 1 2 5  -I  1 mCi  InsulinCporcine) Chloramine-T  5 -jag 100 yg  tube i n the order given:-  in  10 y l  in  20 jil 0.2M  sodium phosphate pH  7.4  in  20 yil  sodium phosphate pH  7.4  0.2M  10 sec exposure Sodium metabisulphite 240 yg i n 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  I v - i n s u l i n was p u r i f i e d by adsorption onto 10 mg microfine s i l i c a  (QUSO) i n a t o t a l 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 i n s u l i n was water;  eluted with 5.0 mis acid-ethanol (15 ml ethanol; 5.0 ml d i s t i l l e d  0.3 ml concentrated hydrochloric acid) and stored at -20°C.  The s p e c i f i c a c t i v i t y of the l a b e l was  calculated from the percentage iodine  incorporated into the i n s u l i n , by counting aliquots of the i n i t i a l  supernatant,  the water wash, the f i n a l l a b e l l e d product and the s i l i c a p e l l e t , and correcting 125 for the volume, i . e . , a t y p i c a l incorporation of 75%  -I into the polypeptide  represented an approximate s p e c i f i c a c t i v i t y of 150 mCi/mg.  -100-  (b)  The i n s u l i n  antibody  The antiserum, obtained from a guinea p i g , was kindly donated by Dr.  K.D.  Buchanan.  Cc)  The assay protocol  Sodium phosphate buffer (0.04M, pH 7.5,  5% charcoal-extracted plasma) was  used i n a l l d i l u t i o n s and for correcting the f i n a l volume to 1.0 ml. composition  of the incubation volume  The  was:-  100 y l standard human or porcine i n s u l i n , 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 f i n a l volume of 0.9 ml  |  24 hour incubation at 4°C 125 100 y i  -I i n s u l i n , containing ~12,000 cpm 24 hour incubation at 4°C  Non-specific binding and t o t a l count tubes were included (see m o t i l i n 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 i n 0.04M sodium phosphate buffer pH 7.5  200 yl/tube) and centrifugation at 2800 rpm for 20 min. was  discarded, the free cpm  (the charcoal p e l l e t ) was  counter and the results expressed as % Bound.  -  After the supernatant  counted i n an automatic  -101-  B.  Serum glucose  Serum glucose determinations were performed i n a Beckman glucose analyzer. A precise volume of sample 0.0 p i ) was pipetted into a standard amount of well-aerated enzyme reagent solution, containing glucose oxidase, ethanol, molybdate and iodide.  The D-glucose i n the sample reacted with the oxygen  in solution to produce gluconic acid and hydrogen peroxide.  Glucose + 0  Glucose Oxidase  Gluconic Acxd +  H0 2  The presence of ethanol, iodide and molybdate i n the reagent solution prevented the destruction of the peroxide by a pathway r e s u l t i n g i n further oxygen production.  The rate of oxygen consumption was d i r e c t l y proportional to the glucose concentration i n the sample and the change of oxygen concentration i n the solution was measured by an oxygen electrode and converted to produce a direct d i g i t a l read out i n mg% glucose.  -102-  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 reaction mixture.  1-motilin, containing 9000 cpm, to the  The reaction was terminated by freezing the mixture. 125  A column of Sephadex G25 fine CO.9 x 100 cm) was calibrated with i n 0.2M acetic acid, 1.5 ml fractions being collected.  I-motilin  The conjugate reaction  products were eeparated by chromatography on this same column, under i d e n t i c a l conditions.  The protein content of each fraction was determined spectrop-  ^  hotometrically at 280 nm i n a 1 cm l i g h t path, and the radioactive content of each f r a c t i o n was estimated by counting for 1 min i n an automatic y counter. The column p r o f i l e s obtained are i l l u s t r a t e d i n F i g . 27. The assumption was made that the labelled and unlabelled polypeptide would behave i d e n t i c a l l y under the conditions of the conjugation procedure.  Therefore,  the percentage of the t o t a l r e a c t i v i t y eluting i n the void volume of the column, with the major protein peak would give a measure of the percentage of polypeptide conjugated to the BSA i n 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 f o r 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 p r o f i l e obtained after elution of  Irmotilin:  motilin/BSA conjugate from Sephadex C25 i n 0.2M acetic acid. 125 The column had been previously calibrated with 1- m o t i l i n  -104-  by the carbodiimide method.  The column p r o f i l e obtained indicates that 100% of the GIP was conjugated to the BSA as i l l u s t r a t e d i n f i g . 28.  REPRODUCIBILITY OF IR-'MOTILIN DETERMINATIONS Serum samples, containing m o t i l i n endogenously released by duodenal a l k a l i n i zation, were obtained from 2 d i f f e r e n t studies i n dogs.  Sera from one  experiment was immediately treated with the protease i n h i b i t o r , T r a s y l o l , 100 Jul per ml of serum.  Both groups of sera were assayed f o r IR- m o t i l i n  content, stored at -20°C and re-assayed 3 months l a t e r .  Table X l l shows the r e s u l t s , i n pg/ml IR'- m o t i l i n , obtained i n the two assays, after correction for the d i l u t i o n by Trasylol i n the appropriate experiment. In F i g . 29, the assay results have been plotted against each other.  Protection  against enzymatic degradation did not appear necessary f o r m o t i l i n .  On re-  assay only 1 sample deviated more than 25% from the l i n e of i d e n t i t y .  COMPARISON OF RIA AND BIOASSAY OF "MOULIN The m o t i l i n content of the commercially prepared duodenal extract "Pancreo-zymin" (PZN) estimated by RIA under routine conditions and by bioassay i n the chronic dog preparation.  A.  •Immunological comparison  s e r i a l d i l u t i o n s of Boots "Pancreozymin" (PZN), ranging from 0.25 - 2.0 yg/  -105-  Fraction  T i g . 28  #  Column p r o f i l e obtained after elution of  I-GIP:CIP/BSA  conjugate from Sephadex C25 i n 0.2M acetic acid.. The column 125 had been previously calibrated with I-GIP.  TABLE XII pg/ml IR-motilin, demonstrating  r e p r o d u c i b i l i t y of m o t i l i n determinations after storage f o r  three months, with or without added T r a s y l o l  Sample Time  Sample  :  With T r a s y l o l 24/6/74  Without T r a s y l o l  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 l i n e of indentity  -107-  i  I  •  Serum Samples, 10% Trasylol  j  o  Serum S a m p l e s , N o Trasylol  400  800  1200  Motilin Concentration (pg./ml.)in Assay 2  Fig. 29  Reproducibility of m o t i l i n determinations on serum samples stored between assays,at -20°C, with (•) or without (o) Trasylol.  Only 1 sample deviated more than '25% from the  l i n e of i d e n t i t y on reassay. (Dryburgh and Brown. enterology 68: 1169-1175, 1975).  Gastro-  -108-  100 y l were incubated with m o t i l i n antiserum i n a routine RIA.  The r e s u l -  125 tant displacement  of  I - m o t i l i n obtained with 1 yg/100 y l PZN was  fitted  to the standard curve, the other d i l u t i o n s being plotted accordingly. immunoreactive material i n the duodenal extract shared 100%  The  immunological  i d e n t i t y with the pure polypeptide and the m o t i l i n content i n 4 separate estimates was B.  140 - 40 pg/ yg Boots PZN  (Fig. 30).  B i o l o g i c a l comparison  In 2 experiments i n each of 3 dogs, matched responses of increased fundic pouch motor a c t i v i t y were produced by bolus, intravenous injections of natural porcine m o t i l i n or Boots PZN given at least 40 mins. apart. order of the injections was  randomized.  The  F i g . 31 and Table XIII i l l u s t r a t e  the increase i n motor a c t i v i t y , expressed as m o t i l i t y indices, i n the 10 min. period immediately period immediately  following the i n j e c t i o n compared to the 10  min.  p r i o r to i t , for 2 different doses of pure polypeptide  and impure extract.  Equivalent responses were observed after 1 and 2 yg  pure m o t i l i n or 10 and 20 mg PZN  (Table XIII, F i g . 311).  Therefore, by both immunological or b i o l o g i c a l estimations, the pure polypeptide represents a 10,000 f o l d p u r i f i c a t i o n over the impure extract.  \  -109-  Motilin (pg) 50  25  o.o 250  500  1000  Boots PzN  Fig. 30  500  100 2000  (ng)  Comparative immungireactiyities of porcine motilin and a crude duodenal extract (BootsPZN), the 1 ug d i l u t i o n of the PZN being f i t t e d to the standard curve and the other points f i t t e d accordingly. (Dryburgh and Brown. Gastroenterology 68: 1169-1175, 1975).  -110-  TABLE XIII Comparison i n the increase i n M o t i l i t y Indices after single intravenous -j-  injections of m o t i l i n or Boots PZN  A.  1 pg m o t i l i n / 10 mg Boots PZN •k  Dog Experiment Control  Post m o t i l i n  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  B.  M o t i l i t y Index  i  20.0 - 2.6  79.3  t  5.8  76.8 - 4.6  2 jag m o t i l i n / 20 mg Boots PZN •k  Dog Experiment  M o t i l i t y Index Control  Post m o t i l i n  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-  200n  I  I  Control  ^3  Natural Porcine Motilin  EZ2  Boots P z n  B.  I60H  S '20H c  "o  80-^  40J  rfi  i  I  I  0-1  Comparison of b i o l o g i c a l a c t i v i t y (expregised as m o t i l i t y indices) of pure m o t i l i n and a crude duodenal.extract'.(Boots PZN) i n the chronic dog bioassay, at 2 doses, (a) 1 ug/kg porcine m o t i l i n and 10 mg/kg PZN and (b) 2 ug/kg pure m o t i l i n and 20 mg/kg PZN.  + Each group of r e s u l t s represents the mean - SE of 4 experiments i n 2 dogs.  -112-  MOLECULAR HOMOGENEITY OF .MOTILIN A.  • In ' serum  To determine whether m o t i l i n existed i n 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 - m o t i l i n was applied of Sephadex G50 fine CO.9  100 cms),  to a colum  the column was developed with 0.2M  acetic acid, and the eluant was collected i n 1.0 ml fractions at a flowrate of 10 mis/hour. could be assayed.  Samples were stored at -20°C u n t i l 100 yil aliquots  The column p r o f i l e C'Fig. 32) indicates that only one  detectable form of m o t i l i n existed i n this serum which was taken at the peak of a canine response to duodenal a l k a l i n i z a t i o n .  B.  In tissue  Presekretin,  extract  the s t a r t i n g material  i n the p u r i f i c a t i o n of m o t i l i n , was estima-  ted, by RIA, to contain 13.5 ng/ jig IR - m o t i l i n .  Three yg of t h i s  material  in 2.0 mis 0.2M acetic acid was eluted from Sephadex G50 fine under the same conditions as i n CA). The  column p r o f i l e i n Fig. 33 was obtained after monitoring each f r a c t i o n at  1:1000 d i l u t i o n for IR - m o t i l i n content. 125 The chromatography system was calibrated routinely with 125 1- m o t i l i n , 50,000 cpm of each i n 0.2M acetic acid.  1- albumin and  -113-  F i g . ;3_2' • . Column p r o f i l e obtained after chromatography of 2 ml serum containing 900 pg/ml IR- m o t i l i n on Sephadex G-50  (0.9 x 100 cm) 125 in 0.2M acetic acid. The column was calibrated with I. . 125 _ '. •motilin and Iodine t < 1  -114-  125  I25  I-Albumin  ....  T  I-Motilin  125  T  I  8T  6H E \  O)  c •- 4-  i CH  2H  -|—  10  I  20  I  30  I  40  I  50  ml Column  Fig..33  -r~  60  eluant  I  70  80  I  90  -1  100  Column p r o f i l e obtained after chromatography of 3 ug Presekretin i n 2 ml elutlng buffer on Sephadex G-50 i n 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 f r e s h and divided into appropriate  sections. I t  was boiled B r i e f l y , cleared of connective tissue, frozen, coarsely minced and extracted into  acid-ethanol.  Composition of acid^ethanol:-  3.75  litres  95% ethanol  75 ml  g l a c i a l acetic acid  1.25  d i s t i l l e d water  litres  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 a l g i n i c acid was allowed to pro-  ceed for 1 hour at 4°C and the a l g i n i c 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 i n d i s t i l l e d water and l y o p h i l i z e d .  After l y o p h i l i z a t i o n , each extract was desalted and p a r t i a l l y p u r i f i e d by chromatography on Sephadex G25 coarse i n 0.2M acetic acid.  F i g . 34 shows a  t y p i c a l column p r o f i l e , obtained i n the p u r i f i c a t i o n of a duodenal extract. The appropriate  fractions were pooled as indicated, l y o p h i l i z e d and monitored  for I R - m o t i l i n  content at several d i l u t i o n s .  Table XIV.  The results are presented i n  -116-  TABLE XIV D i s t r i b u t i o n of IR-motilin throughout the hog g a s t r o i n t e s t i n a l tract  Region  IR-motilin i n 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-  10  Fig. 34  20  30  40  Fraction  i  50  60  #  Desalting of a crude extract of hog duodenal mucosa on Sephadex G-25 coarse (2,5 x 100 cm) i n 0.2M acetic acid. Absorbance at 280 nm i n a 1 cm l i g h t path-and-conductivity -  -  -  .  .  .  .  in millimho was determined f o r each f r a c t i o n . Fractions 28-38 were pooled and l y o p h i l i z e d and subsequently referred to as Fr I.  i  -118-  The j e j u n a l extract content was. assigned an a r b i t r a r y value of 100 and i n descending order anatomically,  the following r a t i o s were obtained:-  Oesophagus  0  'Fundus  0  Antrum  0  Duodenum  7  Jej unum Ileum  100 0.04  RELATIONSHIP BETWEEN GASTRIC CTTJNDIC) EMPTOR ACTIVITY AND ENDOGENOUS MOTILIN RELEASE Six dogs prepared for the chronic bioassay study were used i n this study. Blood samples were obtained whilst the dogs exhibited low spontaneous a c t i v i t y i n the fundic pouch, during infusion of 50 mis 0.3M T r i s buffer, pH 9.5, and whilst the motor a c t i v i t y was returning to Cor below) basal l e v e l s . In 3 experiments, 50 mis 0.1M hydrochloric acid was substituted for the a l k a l i and control experiments were performed i n 4 dogs, during duodenal infusion with 50 mis 0.9% saline.Blood sampling and g a s t r i c motor a c t i v i t y  recording  were performed as i n the a l k a l i experiments.  On 2 d i f f e r e n t occasions dogs exhibited a spontaneous and s i g n i f i c a n t i n crease i n fundic motor a c t i v i t y .  The experiment was continued and blood  samples taken to monitor any changes i n IR- m o t i l i n l e v e l s .  J.  The fasting serum l e v e l of IR- m o t i l i n i n 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 i n 3 mins.  After a l k a l i , the c i r c u l a t i n g IR-  -119-  m o t i l i n 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- m o t i l i n levels were  at t h e i r peak value of 916 i 96 pg/ml.  The g a s t r i c motor a c t i v i t y 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 l e v e l (Fig 35 and Tables XV,  XVI).  There was no increase i n either IR- m o t i l i n or fundic pouch motor a c t i v i t y when the duodenum was perfusedn with s a l i n e , CFig 36 and Tables XVII, XVIII). In the 2 instances, when the fundic pouch motor a c t i v i t y increased spontaneously, a concomitant and spontaneous elevation i n IR- m o t i l i n was noted, CFig 37 and Tables XIX,  XX).  The results obtained after duodenal perfusion with 0.1M were equivocal. great, was  hydrochloric acid  The increase i n c i r c u l a t i n g IR- m o t i l i n was  slower i n reaching i t s peak value and was  returning to the pre-infusion l e v e l s . i n fundic motor a c t i v i t y .  also  not as  also slower i n  There was no associated increase  The comparison between the incremental  IR-  m o t i l i n responses to acid, a l k a l i and s a l i n e i s summarized i n F i g 38 and Table  XXI.  EFFECT OF INGESTION OF GLUCOSE OR A MIXED MEAL ON THE  CIRCULATING LEVELS  OF IR- MOTILIN These studies were performed i n 6 fasted, human volunteers 36 years) with no h i s t o r y of g a s t r o i n t e s t i n a l disorder.  (aged 20 -  On the f i r s t  occasion the subjects were given, o r a l l y , lg/kg glucose i n a 20% solution. Blood samples were taken during the control period and at 5, 10, 15, er,  60, 75 and 90 min after the ingestion of the glucose.  30,  On a second  occasion, these same subjects were given a normal meal, consisting of  TABLE XV Fundic motor a c t i v i t y response to duodenal infusion with a l k a l i . DOG I.D.  Minutes a f t e r onset of duodenal infusion  CONTROL -20 -15  -15 -10  -10 -5  31.4  0  69.5  -5  0  5  10  20  15  25 30  16.0  17.2  26.8  24.6  30.4  7.8  11.0  5.0  27.1  67.6  46.6  41.1  38.4  35.0  0  65.5  55.1  33.2  72.6  53.7  54.0  28.4  33.2  84.2  48.6  30.6  42.8  33.4  18.8  35.3  8.4  0  141.8  79.2  42.6  34.2  35.6  37.6  27.4  31.2  20.7  21.0  19.3  9.9  7.1  9.2  8.6  8.5  7.7  5.9  4.8  94.9  41.2  0  13.2  0  8.1  73.0  42.2  44.0  97.2  23.3  34.8  23.2  45.8  19.0  55.3  6.4  7.6  124.8 154.0  66.2  56.3  14.0  35.7  36.1  42.7  127.8  65.0  40.6  17.1  51.4  3.3  0  226.6 107.5  64.7  62.5  46.3  45.4  179.8  X  35.9  46.3  24.1  24.0  SE  10.1  10.5  7.9  8.9  2  R  l  S  l  S  2  R  2  50  35.0  10.7  P  45 45  10  l  40  35 40  5  P  30 35  -0  15  20  25  55 60  50  55  9.8  4.8  0  16.0  14.6  0  5.1  10.7  6.9  6.5  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. P  l  P  2  R  l  S  l  S  2  R  2  X SE  CONTROL  Minutes after s t a r t of duodenal  -20  -10  0  2  4  450  550  200  200  800  450  450  600  1800  1300  800  550  300  280  280  280  450  960  540  310  270  460  500  650  377  311  277  622  890  570  560  412  405  44  55  6  8  infusion  10  15  30  600  500  400  45  60 300  400  350  560  620  380  300  1100  600  550  520  700  544  477  577  440  300  970  1100  940  980  940  740  560  430  400  396  728  807  855  690  655  430  426  445  315  67  241  135  106  145  71  101  39  89  28  280  280  -122-  50 ml 0.3 M TRIS  r900  Time in Minutes  i I i i  ! I  F i g . 35  Fundic pouch motor a c t i v i t y , expressed as an index pf m o t i l i t y and c i r c u l a t i n g levels of IR- m o t i l i n i n pg/ml after intraduodenal :of 0.3M t r i s buffer.  infusion  Each m o t i l i t y index represents the mean (- SE) for  a.5 min period, whilst the serum levels of m o t i l i n (mean. - . SE) are measured a t . a s p e c i f i c _68: 1169-1176)  n= 6  time.  (Dryburgh and Brown (1975),  Gastroenterology  TABLE XVII Fundic motor a c t i v i t y response to duodenal infusion with s a l i n e . DOG I.D.  R  3  P  3  R  4  P  4  Minutes after start of duodenal infusion of saline  CONTROL 20  -15 10  -10  15  5  0  0 5  10  15  20  25  30  54.0  28.4  33.2  58. 3  56.7  47.0  27.5  26.0  14.8  12.8  23.6  28.4  14.6  11. 2  4.7  4.7  0  0  11.6  7.8  62.1  54.7  30.6  0  0  0  11.4  6.5  11.7  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  -5  0  5  10  20  15  25  TABLE XVIII IR-motilin (pg/ml) response to duodenal infusion with saline. DOG I.D.  R  3  P  3  R  4  P  4  X SE  CONTROL  Minutes after s t a r t of duodenal  infusion  -20  -10  0  2  4  6  8  10  15  30  45  60  320  470  510  410  390  240  350  360  400  280  280  280  450  390  380  125  310  350  125  300  280  280  350  280  380  280  240  280  320  310  260  200  125  200  280  300  410  440  450  380  350  380  270  310  300  400  350  300  390  395  395  298  342  320  251  292  276  290  315  290  27  41  58  64  17  30  46  33  56  41  20  5  -125-  36  Fundic pouch motor a c t i v i t y , expressed as an index of; m o t i l i t y , and c i r c u l a t i n g levels of I R - m o t i l i n i n pg/ml after intraduodenal infusion of 0.15M' saline;  Each.motility index represents the mean (- SE) f o r  a 5 min period, whilst  the serum levels of m o t i l i n are measured at a  s p e c i f i c time, as mean - SE  (n = 4)  TABLE XIX Spontaneously induced fundic motor a c t i v i t y . DOG I.D. .  -20  -15 -15  R  5  P  7  Spontaneously induced fundic motor a c t i v i t y  CONTROL -10 -10  0  -5  10  5  20  15  25  5  0  5  10  15  20  82.2  63.1  143.8  127.7  25.7  11.6  0  0  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  25  30  TABLE XX IR-motilin (pg/ml) response to spontaneously increased motor a c t i v i t y . DOG I.D.  R  5  P  7  X SE  CONTROL  Time after onset of spontaneous a c t i v i t y  -20  -10  0  2  460  520  800  720  390  520  700  425  520  35  0  6  8  10  15  30  45  1000  430  400  400  270  350  300  1100  2600  640  520  680  500  300  300  750  910  1800  535  460  540  385  325  300  50  190  800  105  60  140  115  25  0  4  -128-  r  l800 1600  hl400 120  1200 E \  100  1000  80  800  O)  x  CD TD  > 60+  a.  600  40+ 400 20+ 200 0-L  0  I  0  •37  T1  10  1 1  r  1 1  1  20 30 40 T i m e in M i n u t e s  Fundic pouch motor ac.tlv.ityi-expressed  1  50  1  60  as an index of m o t i l i t y , and  c i r c u l a t i n g levels of IRr- motilin i n pg/ml, during a spontaneous burst of fundic motor a c t i v i t y .  Each m o t i l i t y index-represents the mean  - SE f o r a 5 min period, whilst  the serum levels of m o t i l i n  are measured at a s p e c i f i c time, (n = 2)  (mean - SE)  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 i n min. a f t e r duodenal infusion of CONTROL  2  5  7  10  15  400  -200  400  -  200  500  1300  800  300  -  alkali •  30  45  100  0  -  -100  -  -150  60 -100 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 i n min. a f t e r duodenal infusion of acid. CONTROL  2  5  7  10  15  30  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  45  60  Time i n min. a f t e r duodenal infusion of saline • CONTROL  2  5  430  -20  -40  400  -275  300  7  10  15  -190  -70  -90  -40  -20  20  430  -50  Mean ±SE  30  45  60  -30  -150  -150  -150  -100  -120  -120  -50  -120  10  -100  -175  -100  -20  -80  -50  -120  -130  -30  -80  -130  -91  -48  -67  -97  -113  -100  -75  -100  61  24  42  10  30  25  27  33  0  -131-  •  Sal  me  Alkali 500-i  )  Duodenal Infusion  Acid  400-  300-  1 a  200-  +100-  rnrT  0  • I 00-|  200  1  2  1  5  1  1  1  7  10  15  Time in Minutes from Start of  F i g . 38  1  1  30  45  Duodenal  r~ 60  Infusion  A comparison of the incremental IR- m o t i l i n responses C IR- m o t i l i n in pg/ml to Intraduodenal infusion of a l k a l i , acid or s a l i n e ,  each  point representing the mean - SE for 5 experiments of each type.  -132-  orange j u i c e , bacon and eggs with f r i e d potatoes, toast with conserves and coffee.  Blood samples were taken before breaking the f a s t and at  15 min. intervals thereafter for 2 hours.  No increase i n systemic serum IR- m o t i l i n levels was observed.' A s i g n i f i c a n t (P >0.025) decrease i n IR- m o t i l i n (from the control values) was noted at 30 and 45 min. a f t e r ingestion of either glucose or the meal. The IR- m o t i l i n 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 m o t i l i n and fragments.  The i n i t i a l p u r i f i c a t i o n products,  1foB^  and  M0B2,  were found to contain 50%  and 10% of the immunological a c t i v i t y of natural m o t i l i n respectively.  The  polypeptide MoC^ shared complete immunological i d e n t i t y with the natural polypeptide whereas  M0C2,  although chemically i d e n t i c a l , was immunologically  inactive, and may represent an isomer of the active form.  Synthetic fragments, m o t i l i n 9-22 and 13-22 passed no immunological a c t i v i t y . The results are summarized i n f i g . 40.  (II)  Fragments of natural m o t i l i n  (a)  Cyanogen bromide cleavage  The immunological c r o s s - r e a c t i v i t y was -measured on the mixture of products  TABLE XXII E f f e c t of ingestion  of o r a l glucose  on serum l e v e l s of IR-motilin i n pg/ml CONTROL  Post glucose ingestion  -30  -15  0  5  RAP  280  280  260  JRD  200  210  JCB  300  KM  10  15  30  45  60  75  90  260  230  240  235  190  160  200  160  180  200  190  170  180  180  200  210  200  310  260  240  255  260  200  170  125  100  125  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  23  30  21  17  22  24  20  16  14  18  13  ±SE  TABLE XXIII E f f e c t of ingestion of a normal mixed meal on serum levels of IR-motilin i n 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  36  18  19  18  15  8  9  15  15  10  ±SE  1  1 7  -135-  Fig. 39  The serum IR- m o t i l i n levels (mean - SE) i n 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.  -136J  Fig. 40  Standard curve to m o t i l i n showing comparative immunoreactivities with synthetic 13-norleu-motilins 9-22 and 13-22.  Synthetic m o t i l i n  and  and synthetic fragments  showed 100% c r o s s r e a c t i v i t y  with natural porcine -motilin. (Dryburgh and Brown (1975) Gastroenterology 68: 1169-1176)  -137-  produced bycyanogen bromide cleavage of m o t i l i n . r e a c t i v i t y observed was molecule ( J i g . 41).  The degree of cross-  approximately 30% of that seen with the intact  This can be explained by the presence i n the mixture  of uncleaved m a t e r i a l , 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 s p e c i f i c a l l y at the carboxyl terminals of the basic and aromatic amino acide respectively, t r y p t i c peptides showed no c r o s s - r e a c t i v i t y with m o t i l i n antiserum (Fig* 41).  (Ill)  Modifications of natural m o t i l i n  (a)  Removal of the C-terminal residue Cs)  A 6 hour digestion of m o t i l i n with carboxypeptidase A resulted i n release + of 80% of the C-terminal glutamine and 30% of the penultimate C-terminal glycine.  The degree of immunological a c t i v i t y remaining, approximately  18% that of the intact molecule, may be accounted for by the remaining undigested 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 o r i g i n a l immunological remained.  This decrease i n immunoreactivity may  aromatic N- terminal.  activity  be due to the loss of the  -138-  I l  I  12.5  25 50 pg Motilin  100  200  400  1000  'i  Fig. 41  Standard curve to m o t i l i n showing comparative immunoreactivities with the products of tryptic:and chymotryptic digestion of m o t i l i n , 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 m o t i l i n with succinic anhydride bear unit negative charge and have only 50% of the immunoreactivity of the o r i g i n a l molecule.  B.  B i o l o g i c a l comparison  Cl)  Synthetic m o t i l i n  Comparison of the b i o l o g i c a l a c t i v i t y of the synthetic and natural polypeptides was  achieved by matching the increase i n fundic pouch motor a c t i v i t y  i n the chronic dog preparation produced by single intravenous injections of 1 fig or 2 ug of natural m o t i l i n with Injections of the test material. No s i g n i f i c a n t difference i n the b i o l o g i c a l a c t i v i t y of MoC^ motilin) or natural m o t i l i n could be detected b i o l o g i c a l a c t i v i t y was 9-22  0-3-  norlencine  CFig. 42 and Table XXIV).  observed after injections of the m o t i l i n fragments  or 13-22, even i n doses equivalent to 10 times that of the natural  peptide, on an equimolar basis.  Cll)  No  Fragments of natural -motilin  The mixture of peptides produced by t r y p t i c digestion were b i o l o g i c a l l y inactive In doses equimolar with 10 -jig natural m o t i l i n .  -  -140-  TABLE XXIV Comparison of b i o l o g i c a l a c t i v i t i e s of natural and synthetic m o t i l i n Dog ID.  M o t i l i t y 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  169.8  158.0  59.1  61.9  8.4  10.7  8.3  14.6  X ;'+SE  * represents a 10 min period  -141-  Dog  S  Dog  A  B  1601  o Qo § 120 u.  c >  i  f  o o x  40  c  in |  f i g . 42.'.  in  /jg  /jg  Synthetic Motilin  | Natural Motilin  Comparison of b i o l o g i c a l a c t i v i t i e s of synthetic 12-norleu-motilin and natural porcine m o t i l i n , assayed by measuring the increase i n 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 i n 2 dogs. (Bryburgh and Brown (1975).  Gastroenterology 68: 1169-1176).  -142-  CIII)  Modifications  Ca)  Removal of the C- terminal  There was  of natural-motilin residue(s)  no.loss i n B i o l o g i c a l activity- observed a f t e r the release of  80% of the glutamine residues and 30% of the glycine  Cb)  Removal of the N- terminal  Comparable increases  residues.  residue  i n fundic pouclv motor a c t i v i t y were produced by  l.ug  intact m o t i l i n and 10 Jig m o t i l i n 2 - 22, i . e . , removal of phenylalanine or the net loss of positive charges on the l y s i n e residues reduced the a c t i v i t y by  Cc)  90%.  Acetylation  The b i o l o g i c a l a c t i v i t y was  reduced by 90% a f t e r the net loss of p o s i t i v e  charge i n the acyl derivative.  Cd)  Succinylation  The b i o l o g i c a l a c t i v i t y of m o t i l i n was to less than 5%,  almost completely destroyed, i . e . ,  i n the acyl derivative bearing negatively  charged succinyl  groups.  The  comparative immunological and b i o l o g i c a l a c t i v i t i e s o f the natural  synthetic peptides are summarized i n Table  XXV.  and  -143-  TABLE XXV 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 fragments and analogues  Material  % Immunological a c t i v i t y  % Biological activity  MoBj^  50  90 - 100  MoB  ?  10  Not tested  MoC  1  100  100  MoC  0  Not tested  Mo t i l . in 9 - 2 2  0  0  M o t i l i n 13 - 22  0  0  CNBr. motilin  30  Not tested  Tryptic digest  0  0  Chymotryptic digest  0  0  After C-terminal removal  18  100  After N-terminal removal  18  10  Acetylated derivative  50  10  Succinylated derivative  50  5  2  -144-  ATTFINITT CHROMATOGRAPHY A.  Application to RIA  CI)  Motilin  Ca)  Antiserum d i l u t i o n and change i n a c t i v i t y 125  S e r i a l dilutions of the coupled ligand were incubated with in a t o t a l volume of 1.0 ml, f o r 48 hours at 4°C. i d e n t i c a l to those of the routine RIA.  1- m o t i l i n  A l l other variables were  The tubes were centrifuged at 2800  rpm f o r 30 min., the supernatants discarded and the p e l l e t s counted.  The  antiserum d i l u t i o n curve Ccoupled ligand) i s shown i n F i g . 43 i n comparison with an antiserum d i l u t i o n curve (uncoupled ligand) obtained under i d e n t i c a l conditions i n a routine RIA.  There was no s i g n i f i c a n t  loss i n a c t i v i t y  produced by coupling the antibody to a s o l i d matrix. Cb)  RIA standard curves and change i n s e n s i t i v i t y 125  M o t i l i n standards over the range 12.5 - 400 pg were incubated with  1-  m o t i l i n and equivalent f i n a l d i l u t i o n s of coupled or uncoupled antisera as follows:CD  routine RIA conditions, including uncoupled antiserum and usual charcoal separation;  (ii)  at 1.0 ml incubation volume, with coupled antiserum;  Ciii)  at 0.3 ml incubation volume, with coupled antiserum;  Civ)  at 0.3 ml incubation volume, with coupled antiserum, i n s i l i c o n i z e d tubes.  The standard curves obtained (Fig.44) were judged by the slope at zero dose Ccalculated as shown i n F i g . 45 and Table XXVII) and L.D.D.  The greatest  s e n s i t i v i t y was established when coupled antiserum was incubated with radioactive tracer i n a t o t a l volume of 0.3 ml, i n non-siliconized tubes XXVI).  CTable  -145-  70 n 60 4 50  o  CD  A  Uncoupled Antiserum  40H Sepharose  30A  20  A  IOH  Reciprocal of A n t i s e r u m Titre IxlO  o  Fig. 43  Antiserum  100  50  3  2.5xl0  3  25  10  mg/ml  coupled  5xl0  3  5  IOXIO  3  20xl0 2  3  1.6  ligand  Comparison of d i l u t i o n curves obtained with antiserum to m o t i l i n , performed on aliquots  taken before and after coupling of the a n t i -  serum to CNBr-activated Sepharose 4B.  -146-  TABLE XXVI Coupled versus uncoupled antisera to m o t i l i n at varying incubation volumes Standard curve  " C r i t e r i a for Evaluation Slope at Zero Dose  Midrange  L.D.D.  a.  4.5xl0" l/mole  88 pg  25 pg  b.  2.5xl0 l/mole  320 pg  25 pg  c.  7.5x10" l/mole  120 pg  12.5 pg  d.  5.5xl0~ l/niole  150 pg  25 pg  13  -13  13  13  -147-  F l g . 44  Comparison of the standard curves obtained with antiserum to m o t i l i n under routine assay conditions or a f t e r .coupling to Sepharose 4B, at various incubation volumes,:in s i l i c o n i z e d or.non-siliconized tubes.  -148-  TABLE XXVII Calculation of the slope at zero dose. CURVE  a  STD  CONC i n moles  BOUND  in pg  (x 10" )  (%)  0  1.3  0.315  0.46  0.42  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  1.3  0.309  0.44  0.40  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  1.3  0.448  0.81  0.58  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  1.3  0.428  0.74  0.55  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  0 b  0 c  0 d  15  B/F  B x CONC  SLOPE AT  (x 10" )  (L/mole)  15  -13 4.5 x 10'  -13 2.5 X 10  -13 7.5 X 10'  -13 5.5 X 10'  -149-  A- Routine  RIA  .siliconized tubes  B x Cone. ( Moles) x  f i g . '45  IO"  15  Standard curves, from f i g . 44, presented as Scatchard.plots, demonstrating the.slope at zero dose.'.  -150-  II  Gastric i n h i b i t o r y polypeptide  a.  Antiserum d i l u t i o n and change i n a c t i v i t y  Comparison of the antiserum d i l u t i o n . 1 curves obtained with coupled and 125 uncoupled antisera to GIP a f t e r incubation with RIA conditions i s shown i n F i g . 46.  I- GIP under routine  Coupling of GIP antiserum to a s o l i d  matrix resulted i n a recovery of only 10% (approximately) of the a n t i body a c t i v i t y .  b.  RIA standard curves and change i n s e n s i t i v i t y  Standard GIP i n the range 12.5 - 400 pg was incubated with d i f f e r e n t  final  d i l u t i o n s of coupled and uncoupled antisera, to give approximately the same maximum binding.  Not only d i d the coupled antiserum have to be used  at a much lesser d i l u t i o n 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-  100  J  50  mg/ml coupled ligand 20 10 5  '  ^  10 Reciprocal  Fig. 46  ' '  10 of Antiserum Titre  2  ^  Comparison of d i l u t i o n 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 . s e n s i t i v i t y potential after the coupling procedure.  -153-  B.  Application  to p u r i f i c a t i o n of m o t i l i n 125  CI)  P u r i f i c a t i o n of  I- Motilin  A column of activated Sepharose 4B,  coupled to-motilin antiserum, was  pared i n a Pasteur pipette, the Bed volume being 1.0 ml. equilibrated i n 0.04M sodium phosphate ..Buffer, pH 6.5. of laBelled -motilin, containing 2 t x 10  cpm  pre-  The gel was  well  A l y o p h i l i z e d sample  (estimated to contain 4 ng  IR- m o t i l i n from the laBel s p e c i f i c a c t i v i t y ) was  applied i n 0.5 ml of  the  equiliBrating Buffer and the column developed i n this Buffer u n t i l no 125 counts were eluted. to the gel was  T- m o t i l i n which had been bound  That portion of the  eluted with 0.2M  further  acetic acid.  One ml fractions were collected  throughout and the column p r o f i l e i n 'Fig. 48 obtained by p l o t t i n g cpm/fraction against ml of eluant. Eighty percent of the i n i t i a l l y applied cpm the pH of the e l u t i n g buffer was was  eluted.  The  remained bound to the gel u n t i l  lowered to 2.4,  remaining 22% was  when 78% of that bound material  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 i n a batchwise manner, as follows:-  cpm  i n 5.0 mis 0.04M phosphate buffer, pH 6.5  2x10^  I- motilin was  and mixed with 1.0 ml Sepharose  s l u r r y , equilibrated i n the phosphate buffer, for 18 hours at 4°C. nate was  discarded and  The  super-  the gel washed with 5x5 ml phosphate buffer, i . e . ,  u n t i l the counts i n the wash were n e g l i g i b l e . 0.2M  diluted  The  gel was  acetic acid and mixed, by rotation, for 1 hour.  The  resuspended i n 5 mis supernate  diluted i n assay diluent buffer to give ~ 5000 cpm/100 y l .  was  An aliquot of  -154-  125 F i g . 48  Column p r o f i l e 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 f o r 1  min i n an automaticrty Counter.  -155-  untreated "^ A  J  1- m o t i l i n was  s i m i l a r i t y diluted to produce the same concen-  t r a t i o n and standard curves were prepared with both l a b e l s .  The non-specific binding of the untreated treated portion was  10.6%.  l a b e l was  This improvement was  18.9%B, that of the  due to the removal of  free iodine and damaged, iodinated but non-immunoreactive fragments. was  no s i g n i f i c a n t difference i n the curves produced (see F i g . 49),  There the  iodinated motilin appeared unaffected by i t s passage through the gel.  (II)  Extraction of m o t i l i n added to plasma  Natural porcine m o t i l i n was concentration  d i l u t e d i n charcoal-extracted  of 4 ng/100 y l .  When 4 ng was  plasma to a  applied to a column of Sepharose  4B and the column developed as previously described, RIA was the recovery of m o t i l i n .  The column p r o f i l e , i n F i g . 50, i s a plot of pg/ml  IR- m o t i l i n against ml eluant. the gel and 100%  (III) Two  used to monitor  I n i t i a l l y 83% of the m o t i l i n was bound to  of that amount was  recovered.  Extraction of endogenous m o t i l i n from serum  mis serum (subject R.K.), which contained  subjected  680 pg IR- motiliri/ml was  to a f f i n i t y chromatography as i n I I .  V i r t u a l l y 100%  of the applied m o t i l i n was  bound to the gel and then recovered  by elution with a lower pH buffer (Fig. 51).  -156-  -157-  ml  Fig. 50  eluant  Column p r o f i l e obtained a f t e r 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-  F i g . 51  Column p r o f i l e 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 f o r IR- motilin by radioimmunoassay.  -159-  (IV)  I s o l a t i o n of m o t i l i n from presekretin  Presekretin, a side f r a c t i o n produced during the p u r i f i c a t i o n of secretin, i s the s t a r t i n g material i n the i s o l a t i o n of m o t i l i n (Brown et a l , 1971). The o r i g i n a l p u r i f i c a t i o n procedure involved 5 stages of column chromatography, was monitored i n the chronic dog bi'oassay and resulted i n product i o n of.an active moiety representing 0.5-1.0% of the s t a r t i n g material by weight.  Two yg Presekretin, estimated to contain 4 ng IR- m o t i l i n , was dissolved i n 0.04M phosphate buffer, pH 6.5 and treated as previously The IR- m o t i l i n content was 90% recovered  described.  and the yield'represented a 5%  recovery of the s t a r t i n g material by weight, ( F i g . 52).  EFFECTS OF MOTILIN ON THE RATE OF GASTRIC EMPTYING It was desirable to perform the studies on the rate of g a s t r i c emptying during a r e l a t i v e l y constant background of c i r c u l a t i n g m o t i l i n .  Pilot  studies indicated that IR- m o t i l i n 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 l a s t 10 mins, of a 30 mins. infusion (Fig. 53).  A.  Control studies i n the g a s t r i c f i s t u l a dog  (I)  E f f e c t of m o t i l i n on the rate of g a s t r i c emptying of l i q u i d s  M o t i l i n infusions i n the range 0.125  - 2.0 yj;/kg/hour accelerated the rate  D f emptying of the test l i q u i d i n a dose-related manner.  The basal rate (C)  -160-  0.04 M P 0 pH 6.5  0.2 M HAc  4  700-j  | 600o t 500-|  1  400-  -*— o  z  1  Q-  cc  00 H 320 100 H  0  T  5  10 ml  Fig. 52  eluant  Column p r o f i l e obtained a f t e r . e l u t i o n of a duodenal extract  (Presekretin),  motilin-containing  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 f o r IR- -motilin by radioimmunoassay.  -161-  Fig. 53  Mean - SE serum concentrations of IR- motilin (pg/ml) following infusion of pure natural m o t i l i n 0.5 and 1.0 ug/kg/hour. 'Each point i s the mean,of two experiments on each of s i x dogs (Debas et a l , Gastroenterology i n press, 1977).  -162-  was.measured during the infusion of 0.15M s a l i n e , ( F i g . 54).  The lowest e f f e c t i v e dose was 0.25 ug/kg/hour m o t i l i n (p< 0.001) and the maximum effect was achieved at 0.5 ug/kg/hour,motilin.  (II)  Effect of motilin on the rate of g a s t r i c emptying of solids  The f r a c t i o n of the s o l i d meal remaining i n the stomach  :I 30^' 60, 90 and  120 mins a f t e r i t s s ingestion was v i r t u a l l y the same whether the infusion was 1.0 ug/kg/hour m o t i l i n or 0.T5M saline (Fig. 55).  B.  Effects of m o t i l i n on the rate of gastric emptying of l i q u i d s after truncal vagotomy and/or antrectomy  (I)  Effect of motilin after truncal vagotomy  ^  The dose of m o t i l i n producing the maximum effect i n the control dogs, 0.5 -ug/kg/hour, was i n e f f e c t i v e a f t e r vagotomy.  The doses.of 1.0 and 2.0  ug/kg/hour, however, did produce a s i g n i f i c a n t increase i n the f r a c t i o n of the l i q u i d meal emptied from the stomach.  The effect of vagotomy, therefore,  was to decrease the s e n s i t i v i t y of the response to m o t i l i n .  The basal rate of g a s t r i c emptying, measured during s a l i n e infusion,, was s i g n i f i c a n t l y lower i n the vagotomized animal - as shown i n F i g . 56.  (II)  Effect of m o t i l i n a f t e r antrectomy•  There was no difference i n the response to 0.25, 1.0 and 2.0 ug/kg/hour motilin i n the control and post-antrectomy•dogs.  There was an, as yet, i n -  -163-  100 "D OJ  90-1  E  80  c  70  LU o o  D  60 -  I1  * *  p < 0.001  50-  40  0  A  A  C  1  1  0.125 0 . 2 5  '  0.5  "1 1.0  1 2.0  Motilin (jug/kg/hr)  f i g . 54  Mean - SE f r a c t i o n ;of l i q u i d meal emptied while different'doses .of m o t i l i n were infused:intravenously.  Each point i s the mean of  two experiments on each of s i x dogs; (Debas et a l , Gastroenterology, i n press, 1977).  -164-  100 - |  0  30  60  90  120  Minutes after Meal  Fig. 55  Mean - SE f r a c t i o n of s o l i d meal emptied at different'time periods.following ingestion.• Each point i s the; mean of two experiments on each of s i x dogs (Debas et a l , Gastroenterology, i n press, 1977) .  -165-  Prevagotomy 100-i  I  1  •I  i  80H •  CL  E 60 H LU  Post Vagotomy  c  .2 o o LL  40  H  20 -  0  J  0.125 0.25 0.5  1.0  2.0  Motilin (jug/kg/hr)  Fig. 56  Mean - SE f r a c t i o n of l i q u i d meal emptied while different doses of m o t i l i n 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, i n press, 1977).  -166-  explicable, but significant.decrease i n the rate of gastric  emptying.at  0.5 ug/kg/hour m o t i l i n i n the:antrectomized animal, as shown i n F i g . 57.  The basal rates were hot altered by removal of the antrum.  (Ill)  Effect of m o t i l i n a f t e r truncal vagotomy arid antrectomy  The basal rate of gastric emptying was s i g n i f i c a n t l y increased i n the vagotomized, antrectomized animal, compared to that i n the control animal.  There was a s i g n i f i c a n t increase i n the rate of g a s t r i c emptying of the l i q u i d meal a f t e r 0.5 and 1.0 jig/kg/hour m o t i l i n (P<0.01).  This was less  s i g n i f i c a n t , however, than the increase i n 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 t i t r e s of 1:20x10 . The a f f i n i t y constants (K) of the best guinea p i g (Van 8) and best rabbit (Go 5) were 7. 5 x l 0  1Zt  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 l a b e l by standard antigen was unsatisfactory  a f t e r an incubation of the equilibrium type.  When this antiserum was incubated  -167-  100  90 TD Q> 'jZ CL  Antrectomy  80  E  LU  c o o o  Control  70 60 50  J  40  0  r  1—  0.125 0 . 2 5  - i  0.5  1  1  1.0  2.0  Motilin (jug/kg/hr)  Fig. 57  j  Mean,- SE f r a c t i o n of l i q u i d meal emptied while d i f f e r e n t doses of motilin were infused I.V. before and after antrectomy.  Each  point i s the mean of two experiments on each of three.dogsi (Debas et a l , Gastroenterology, i n press, 1977).  -168-  lOO-i  ** 0>  Q. E  90H  8 0 H  LU  70H  o c  eo-  * • * p <0.0I  2  * * * p < 0.001  I  o  LL  J  50-  404  o  J  C  I 0.125  I  1  1  0.25  0.5  1.0  1 2.0  Motilin (jug/kg / h r )  F i g . 58  Mean - SE f r a c t i o n of a l i q u i d meal emptied while different doses of motilin were infused'I.V'before and after truncal vagotomy and 1  ": antrectomy.  Each, point i s the -mean of two experiments ion each of  6 dogs, (Debas et a l , Gastroenterology, i n press, 1977).  -169-  with cold antigen for 24 hours p r i o r to the addition of 125 I - GIP, and for another 48 hours after the l a b e l addition a satisfactory,standard curve was obtained, the antiserum affinity.constant (K).being 2.5xl0 ^ 1/mole.1  Fig.- 59_. . i l l u s t r a t e s the Improvement obtained when Ro7 was incubated i n a disequilibrium assay system,  i n contrast, assays containing Van 8 showed  no s i g n i f i c a n t difference when incubated under either equilibrium or d i s equilibrium conditions;-  Bj  Iodination of GIP 125  The s p e c i f i c a c t i v i t y of the only 70 mCi/mg.  I- GIP, p u r i f i e d by gel f i l t r a t i o n was  After further p u r i f i c a t i o n by ion exchange chromatography  on QAE Sephadex A-25 the s p e c i f i c a c t i v i t y was greatly improved, the value being  250 mCi/mg.  A comparison of the standard curves obtained with the  l a b e l at each stage i s shown i n F i g . 60.  EFFECT OF SOMATOSTATIN ON THE CANINE RESPONSE TO GIP A.  Effect of somatostatin on the release of endogenous GIP  (I)  On the i n s u l i n o t r o p i c action of GIP released by o r a l glucose  These studies were performed i n the i n t a c t , conscious dog.  Somatostatin  was administered intravenously either as a single rapid i n j e c t i o n (3 ug/kg) immediately p r i o r to the o r a l administration of glucose, or as an infusion of 6 ug/kg over 1 hour, the glucose load being given a f t e r 30 mins.  The  glucose load i n either case was lg/kg of 20% dextrose i n d i s t i l l e d water and was administered alone i n control studies.  -170-  i  ng.'  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-  F i g . 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 QAE A25.  subsequent elution from Sephadex  -172-  After  the s i n g l e b o l u s i n j e c t i o n o f s o m a t o s t a t i n , t h e f a s t i n g l e v e l s o f  serum g l u c o s e , IR- GIP and IR- i n s u l i n were i n h i b i t e d and t h e response to the o r a l g l u c o s e was d e l a y e d .  There was a s i g n i f i c a n t r e d u c t i o n from the  c o n t r o l v a l u e s o f a l l parameters a t 15 mins. (IR-GIP, P\ <0.0025; i n s u l i n P. <0.0005, g l u c o s e , p <0.0005),  ( F i g . 61, T a b l e XXVIII). -  When s o m a t o s t a t i n was a d m i n i s t e r e d as an i n f u s i o n , i n s u l i n responses  t o o r a l g l u c o s e were suppressed  ended a t 60 mins. when t h e IR i n s u l i n response cantly  above t h e c o n t r o l .  was s i m i l a r t o t h a t  IR-  t h e IR-GIP and IRu n t i l t h e i n f u s i o n had  rebounded t o v a l u e s  The delayed e l e v a t i o n  signifi-  o f t h e serum g l u c o s e  levels  seen a f t e r t h e ' b o l u s i n j e c t i o n o f o f s o m a t o s t a t i n and  by 60 mins. t h e r e was no s i g n i f i c a n t d i f f e r e n c e  from t h e c o n t r o l  values  ( F i g . 62, T a b l e XXIX).  (II)  On t h e r e l e a s e o f endogenous GIP by f a t  The,effect of a single, i n t h e IR-GIP response control  rapid  i n t r a v e n o u s i n j e c t i o n o f 3 ug/kg s o m a t o s t a t i n  to ingestion  o f 100 mis Lipomul was compared w i t h  s t u d i e s when f a t alone was a d m i n i s t e r e d .  s o m a t o s t a t i n was found t o be delayed and reduced s t a t i n and then rebounded above the c o n t r o l  The IR-GIP response  after  (P . <0.05) a f t e r somato-  values  (as may be observed i n  F i g . 63 and T a b l e XXXT).  B.  E f f e c t o f s o m a t o s t a t i n on t h e response  A comparison was drawn between t h e responses t o an i n t r a v e n o u s i n f u s i o n and w i t h o u t statin.  a prior single,  t o exogenous GIP o f IR i n s u l i n and serum g l u c o s e  o f 1.5 ug/kg p o r c i n e GIP over 5 mins, g i v e n w i t h rapid,  i n t r a v e n o u s i n j e c t i o n o f 3 yg/kg somato-  There was an 80% r e d u c t i o n i n t h e peak IR i n s u l i n response  t o GIP  TABLE XXVIII The e f f e c t of a s i n g l e  rapid  i . v . i n j e c t i o n o f s o m a t o s t a t i n on t h e i n c r e m e n t a l  IR-GIP, IRI and serum glucose response to o r a l glucose. n = 7  Incremental Response (A)  Time (mins)  Serum Glucose mg%  IR-GIP Pg/ml  10  15  20  25  30  45  60  75  90  105  120  135  150 5.7 3.3  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  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  X Control ±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  60 75  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  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  Test  X ±SE  -6.5 2.4  -4.4 2.8  -0.5 6.8  7.5 5.2  39 12  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  IRI  yu/mi  5  ' 58 9.2  9.1 3, o i  -174-  F i g . 61  The effect of an Intravenous i n j e c t i o n of 3 jig/kg somatostatin on the incremental IR-GIP, IR- i n s u l i n and serum glucose responses to the ingestion of glucose. (1 g/kg) Physiol. Phamacolv. 53j: :1200-l205,  (Pederson at a l , Can J .  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  Time (mins)  Serum glucose mg%  IR-GIP pg/ml  IRI yU/ml  5  10  15  20  25  30  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  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  Control  X + SE  208 95  520 149  551 156  980 202  1063 132  909 238  Test  X + SE  72 89  115 339  150 339  Control  X +SE  22.8 11.3  54.6 14.2  68.7 9.1  63.0 5.9  72.8 9.4  Test  X ±5E  0 2.4  -3 1.8  -1.6 2.9  -9.6 3.7  -10 3.3  35  40  45  50  response (A) 55  43.9 4.6 5.0 3.9  0.2 1.7  6.0 3.7  10 4.2  18.2 5.6  1348 134  -6.5 -6.8 3.6 3.6  42.5 7.0 -6.6 3.1  -5.6 2.6  75  90  31.1 4.3  24.7 4.0  25 6  105  120  135  150  165  180  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  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  910' 117  673 97  518. 123  336 124  221 85  60 75  143 121  115 74  176 58  802 200  752 183  684 156  763 210  886 212  750 143  500 200  324 136  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  -1.8 119.8 4.7 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  1188 182 843 128  62.6 8.0  60  -7.6 2.2  . i  -176-  F i g . 62  The e f f e c t of an infusion of.somatostatin (6 ug/kg/hour) on the incremental IR-GIP j IR-^, i n s u l i n : and serum glucose .responses to the ingestion of 1 g/kg glncose; (Pederson.et a l , Can.: J.Physiol. Pharmacol. 53: 1200-1205, 1975) <  TABJ'-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 f a t . Incremental Response (A) Time (mins) -  5 Control  Serum glucose mg%  IR-GIP pg/ml  IRI yU/ml  ±SE X  Control  X  Test  X  Test  ±SE  0 1.2  0.4 1.9  29 43  12 30  ±SE +SE  25  5.0 1.9.  30 35  -6 2.8  -9 1.8  -5 2.0  30  35  3.6 3.1 3.3 1.3  4.2 1.4  3.9 4.2  .0 1.3 l  1210 274 -5 29  -10 46  -1.0 0.45  X  X  20  674 174  ±SE ±SE  15 3.8 1.3  X  Test  Control  10  242 154  210 140  0.83 1.6 -3 3.0  -3 3.0  -2 2.9  -1.3 2.8  45  60  75  90  105  120  135  150  165  180  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  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  1712 441  2641 497  2696 560  3083 614  3035 612  3325 605  3270 3548 635 . 782  3526 707  2704 565  394 164  946 257  1235 323  1585 122  1492 103  1642 215  1435 221  1903 414  1721 416  1964 322  . -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  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-  F  4000  3ug/kg  Somatostatin  100 ml Lipomul  n  Time in Minutes  I i  I  •Fig. 63  The effect of an intravenous i n j e c t i o n 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 Pharmacol. 53: 1200-1205, 1975).  et a l , Can. J . Physiol.  -179-  a f t e r somatostatin, with a concomitant  and s i g n i f i c a n t (P\ <0.01) reduction  i n 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) i n 9 experiments i n 3 dogs i s i l l u s t r a t e d i n F i g . 65 and Table XXXIII.  A 60%  i n h i b i t i o n of gastric acid secretion was observed i n the second half of the GIP infusion, associated with IR-GIP levels i n 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 retarned toward the control plateau l e v e l s .  +  output  Control experiments were per-  formed i n 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 t r i g l y c e r i d e emulsion  (Lipomul) was infused intraduodenally at 1.91 ml/min.  over 30 mins. a f t e r a plateau of gastric acid secretion had been achieved by intravenous infusion of pentagastrin i n 3 experiments i n each of 3 dogs.  The  results (Fig. 66 and Table XXXIV) showed that a marked increase i n IR-GIP occurred to levels of 800 - 1000 pg/ml above the pre-infusion l e v e l s , co-incident with a 68% i n h i b i t i o n of gastric acid secretion. During the post-infusion period both IR-GIP and H  i i  +  values returned toward the control l e v e l s .  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)  Serum glucose mg%  IRI uU/ml  3  4  5  7  10  15  20  25  30  45  -8.4 3.1  -9.8 7.3  -6.8 7.7  -4.2 2.5  -0. 2 3. 7  2.6 1.3  Prmt-T*n1 L-UI1LLU1  X ±SE  0.6 1.5  -3.6 3.3  -1.6 1.4  -5.0 2.4  Test  X ±SE  3.7 1.8  4.7 1.3  2.2 1.7  1.5 1.8  0.25 1.7  -1.7 1.4  -1.25 2.4  -1.25 5.3  ^ U U L L U l  X ±SE  25.4 11.9  25.4 6.4  30.0 6.9  16.4 3.3  9.4 2.1  -1.4 1.3  -3.8 1.4  -2.6 1.1  Test  X ±SE  -1.25 1.3  -1.75 1.0  5 3.7  15 2.2  5.8 1.7  •_1.7 4.4  -2.0 0.8  1.0 3.1  3. 0 1. 5 .  -2. 2 1. 2 1. 25 1. 0  4.7 3.9  -1.4 2.1 1.25 1.0  -181-  Fig. 64  The effect of an intravenous i n j e c t i o n of 3 J-ig/kg somatostatin on the /incremental IR* insulin:and serum glucose responses to an intravenous i n f u s i o n o f 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 e x t r i n s i c a l l y denervated fundic pouch.  INTRAVENOUS PENTAGASTRIN INFUSION  Expt. #  30  45  60  75  90  105  120  140  220  198  208  261  202  213  198  201  310  264  238  235  280  274  264  251  283  1324  1307  1460  1103  °2  °3  Ma  161  149  Di  330  326  Be  1.  15  °1  1720  1705  uEq H /15 min.  1669  1647  1120  1205  1409  TABLE XXXIII of an intravenous infusion of porcine GIP CONTROL  Experiment ii  Intravenou 3 Porcine GIP Infusion :  °1  °2  °3  329 340  374 400  314 320  5  +  output . 2  Post Infusion Period  10  15  20  25  30  45  60  75  90  105  120  800  137 1475  1500  1650  106' 1675  97 1750  120 1325  103 1225  145 1425  193 1050  143 920  Ma 1  H IR-GIP  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  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 ' 134 1620 2000  124 1450  163 1750  171 1200  234 650  200 270  +  +  +  +  +  +  +  +  1. 2. 3.  on pentagastrin-stimulated H  3  1 ug/kg/hr GIP over 60 min. uEq . H+/15 min. IR-GIP i n pg/ml.  171 130  -184-  Fig. 65  1  1  1—i  1—i  1  I  |  0  30 60 90 120 Time in Min. after Start of Infusion  1  L  -zoo  <l  The incremental IR-GIP response and i n h i b i t i o n ;of pentagastrininduced gastric acid'secretion of 1 pg/kg/hour porcine GIP. SE of 8 experiments i n 3 dogs.  caused by an intravenous > infusion The results represent the mean -  TABLE XXXIV Effect of duodenal infusion of fat on pentagastrin-stimulated  «  and IR-GIP  °1  °2  °3  5  10  : 15  '20  25  release.  Post Infusion Period 30  45  60  75  90  342 720  167 1650  162 1175  420 1050  486 440  416 660  440 385  82  25  116  125  168  245  282  157 840  40 420  25 1125  80 1875  155 1425  178 1200  185 900  105  120  Ma 1  H IR-GIP  499 125  528 600  533 300  Ma 2  H IR-GIP  270 520  234 420  218 500  520  920  50730  690  1025  Ma 3  H IR-GIP  555 130  508 220  518 330  200  460  78 470  610  690  Be 1  H IR-GIP  17 6 150  162 190  128 120  220 500  150 800  98 1550  68 145 1550 . 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  Di 1  H IR-GIP  127 180  110 ' 156 125 500  Di 2  H IR-GIP  262 125  250 125  +  +  +  +  +  +  +  +  1. 2. 3.  output  Duodenal Fai: Infusion  CONTROL  Experiment  H  100 mis Lipomul over 30 min. yEq H /15 min. IR-GIP in pg/ml. +  299 125  358 820  860  185 1250  1450  1500  186 1600  166 1750  216 1900  249 1350  201 930  278 700  286 360  700  590  168 1600  1500  1750  1800  71 1400  21 1350  70 1250  172 1350  177 1350  210 1330  125  125  340 550  970  1550  65 1350  46 680  74 580  68 400  210 340  245 170  198 125  620  -186-  i  I D Fat  •  I  I •  Time in Min. after Start of Infusion  Fig. 66  The:incremental IR-GIP:response induced gastric acid.secretion  and i n h i b i t i o n of pentagastrincaused.by.an:intraduodenal  of 1.9 ml/min Lipomul oyer 30 min. + - SE of 8 experiments i n 3 dogs.  infusion  The results represent the mean  -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 g a s t r i c acid secretion had been achieved by pentagastrin administration.  The H  +  output was reduced to 52% of the pre-infusion  levels with a concomitant increment i n IR-GIP of 400 - 600 pg/ml.  plateau Each  point represents the mean of 9 experiments i n 3 dogs (Fig. 67, Table XXXV).  C.  Effect of an intraduodenal infusion of acid on gastric acid  A duodenal infusion of 1.91 ml/min 0.15M hydrochloric i n h i b i t e d the H  +  infusion l e v e l s .  acidfover  secretion 30 min.  output stimulated by pentagastrin to 48% of the preThis reduction i n the acid secretion was not accompanied  by any s i g n i f i c a n t change i n IR-GIP, i n 4 experiments i n 2 dogs (Fig. 68, Table XXXVI).  D.  Effect of ah intraduodenal infusion of saline bri gastric acid  secretion  In 7 experiments i n 3 dogs, a duodenal infusion of 0.9% saline at 1.91 ml/ min. H  +  over 30 min.  resulted i n a small (27%) non-significant,  i n h i b i t i o n of  output, preceded by a s l i g h t , transient increase i n IR-GIP (Fig. 69,  Table XXXVII).  These results are summarized i n F i g . 70, which compares the maximum i n h i b i t i o n of g a s t r i c acid achieved with the concomitant c i r c u l a t i n g l e v e l of IR-GIP,  TABLE XXXV Effect of duodenal infusion of glucose CONTROL  Experiment Ol  o  IR-GIP  203 125  Ma 2  H IR-GIP  Be 1  #  5  202 125  182 125  125  329 340  374 400  314 320  H IR-GIP  298 480  273 460  Be 2 '  H IR-GIP  145 125  Be 3  H IR-GIP  Di 1 Di 2 1. 2. 3.  +  output and IR-GIP release . 2  10  3  Post Infusion Period  15  20  25  30  45  60  75  90  125  82 1020  460  500  42 300  33 680  55 235  197 145  107 150  163 430  -  800  137 • 1475 1500  1650  106 1675  97 1750  120 1325  249 1225  148 1425  193 1020  243 920  297 440  370  320  185 690  940  930  186 740  166 740  216 1150  163 1350  201 125  278 370  286 275  156 125  124 125  125  125  119 625  380  415  167 250  90 260  130 125  217 • 145  172 125  394 125  314 125  355 125  125  125  310 725  490  330  150 420  190 240  193 125  196 125  236 125  -  234 125  H IR-GIP  277 350  270 340  260 220  140  340  161 480  480  650  199 1400  . 243 750  208 340  186 280  235 240  229 125  287 125  H IR-GIP  326 125  272 125  298 125  125  170  232 200  680  820  232 600  202 540  112 190  103 125  206 125  143 160  135 125  +  +  +  +  +  +  .  H  Duodenal Glucose Infusion °3  Ma 1  on pentagastrin-stimulated  2  20% dextrose - 1 g/kg over 30 min. uEq H+/15 min. IR-GIP i n pg/ml.  105  120  -189-  The incremental IR-GIP response and i n h i b i t i o n of pentagastrin-induced g a s t r i c acid secretion caused by an i n t r a duodenal infusion of 1 g/kg glucose over 30 min. The r e s u l t s represent the mean "t SE of 7 experiments 3 dogs.  in  TABLE XXXVI Effect of a duodenal infusion of a c i d on pentaga trin-stimulated 1  3  CONTROL  Experiment it  Ol  0  2  H  +  output and IR-GIP release . 2  Duodenal Acid Infusion 0  3  10  15  .'.0  25  3  Post Infusion Period 30  45  60  75  90  105  120  Be 1  H IR-GIP  2626 500  2608 315  2522 310  2060 315  1823 290  1944 400  1735 335  2013 150  2112 150  2257 490  2070 255  Be. 2  H IR-GIP  2714 390  2831 310  2760 250  2173 200  1714 250  1802 200  1691 360  1990 225  2431 150  2461 200  2406 200  Ro 1  H IR-GIP  1534 760  1800 840  1746 835  1100 700  918 850  1120 720  1299 730  1415 590  1508 1100  1368 1160  1573 920  Ro 2  H IR-GIP  1780 350  1750 410  1830 300  1210 300  942 260  1100 280  1198 240  1460 200  1490 300  1600 265  1570 280  1.  0.15M HCl at 1.91 ml/min for 30 min.  2.  In UEq H+/15 min.  3.  In pg/ml IR-GIP.  O  F i g . 68  The incremental IR-GIP response and i n h i b i t i o n ,of pentagastrininduced gastric'. acid secretion caused by an intraduodenal infusion of 1.91 ml/min O.lM.Hcl. 4:experiments i n 3.dogs.  The results represent the mean - SE of  TABLE XXXVII Effect of a duodenal infusion of saline  °1  o  2  H  output  °3  5  10  15  20  25  and IR-GIP release . Post Infusion Period  Duodenal Infusion of Saline  CONTROL Expt.  on pentagastrin-stimulated  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  256 125  242 125  264 125  222 125  192 125  198 125  133 125  169 125  121 125  159 125  168 125  +  +  +  +  Be 2  +  H IR-GIP  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. 2. 3.  0.15M saline, 100 mis over 30 min. i n uEq H+/15 min. IR-GIP in pg/ml.  -193-  Soline  Pentagastrin  1  Infusion  to O  <D D_ <= O  o  ro  Control  oc  After ID Saline  0) £1  o o  o  or  E  OT  \  600  o  CL  o_  o + 200 T  0  Fig. 69  2  1  1  1  o  r  30 60 90 120 Time in Min. after Start of Infusion  -200<  The incremental IR-GIP response 'and i n h i b i t i o n of pentagastrininduced gastric acid secretion caused by an -intraduodenal infusion of 1.91 ml/min 0.15M s a l i n e .  The results represent the mean —  SE of 7 experiments i n 3 dogs.  -194-  Fig.  70  The f u n d i c pouch H response  output and i n c r e m e n t a l serum  IR-GIP  a c h i e v e d a f t e r i . v . i n f u s i o n of p e n t a g a s t r i n (pg)  o n l y - c o n t r o l - compared w i t h t h e s e same parameters during a concomitant  i . v . i n f u s i o n of p o r c i n e GIP or i n t r a -  duodenal i n f u s i o n s of f a t , g l u c o s e or a c i d .  -195-  af ter intraduodenal infusion with f at * glucose a n d a c i d 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 f a t  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. t r i g l y c e r i d e emulsion, Lipomul, representing the i n i t i a l peak response of IR- GIP to either stimulus. diluent buffer and monitored by  They were s e r i a l l y diluted i n  RIA.  Neither of the serum d i l u t i o n curves showed any s i g n i f i c a n t difference from the standard curve obtained by d i l u t i n g pure porcine GIP.  The a n t i -  serum used i n the routine RIA did not, therefore, d i f f e r e n t i a t e between the GIP released by glucose or f a t given o r a l l y , as shown i n F i g . 71. •  (II)  Immunoreactivity  of GIP after column chromatography  The apparent immunoreactive homogeneity-of•the fat was  GIP released by glucose or  further-examined by.chromatography of 2 ml aliquots of these same  serum samples on a 1x200 cm column of Sephadex G50 f i n e i n 0.2M acid.  acetic  The column\was calibrated p r i o r to each run with dextran-blue and  125 I- GIP  ( ~ 60,000 cpm)  i n 2 mis charcoal-extracted plasma and the con-  d u c t i v i t y monitored to determine-the position of the s a l t peak.  One ml  -196-  F i g . 71  A comparison of the immunoreactivities of porcine GIP and the IR-GIP i n human sera after the ingestion of glucose or Lipomul. (Brown et a l , R e c Prog. Horm.Res. 31 : 487-532, 1975).  -197-  fractions were collected and the IR-GIP content of each f r a c t i o n measured by RIA.  At least three immunoreactive regions were detectable a f t e r this treatment, one eluting i n the void volume of the column GIP^  0  immunoreactive component which eluted ahead of the PROGIP.  : and a s i g n i f i c a n t GIP,-QQQ  a r b i t r a r i l y named  These..three immunoreactive peaks were present i n serum samples  a f t e r either glucose or f a t stimulation (as shown i n F i g . 72 and F i g . 7 3 ) .  The r e l a t i v e 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 t o t a l (Fig. 74, Table  XXXVIII).  TABLE XXXVIII Proportions of IR-GIP components released by f a t and glucose  After glucose stimulation  Total IR-GIP content  1400 pg/ml  After f a t stimulation  2700  pg/ml  % Total as GIP v  14.4  31.4  % Total as ProGIP  35.3  40.8  % Total as  50.3  27.8  0  GIT^  These .results indicated that .there might be a difference i n the GIP response to glucose and f a t , the major component of IR-GIP i n the i n i t i a l peak response to glucose being GIP,-  nnn  whilst that after f a t was ProGIP.  -198-  . 72  Regions of IR-GIP obseryed a f t e r chromatography of 2 ml serum, obtained 45 min after:ingestion of glucose, pn Sephadex G50 C l x 200 cm) i n O.ZM acetic acid. calibrated with dextran blue and  The column was previously 125 I-GIP.  The enclosed numbers  refer to the percentage of the t o t a l IR-GIP represented by that region.  -199-  Fig. 73  Regions of IR-G1P observed after chromatography,of 2 ml serum 1  . obtained 90 min after ingestion 0.2M acetic acid. dextran blue and v  of Lipomul, on Sephadex G50 i n  The column was previously I-GIP.  calibrated with  The enclosed numbers refer to the  percentage of t o t a l IR-GIP represented by that region. >  -200-  Fig. 74  The ehfomatograms from Fig. 71- and Fig- 72 expressed i n histogram :  form, the heterologies of GIP being represented as percentages of the t o t a l  IR-GIP'response.  -201-  (III)  Immunoreactive forms of GIP released by o r a l fat or glucose  (a.)  After o r a l 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 f i n e i n 0..2Kacetic column was routinely calibrated with  acid and 1 ml fractions collected. 125  I*- albumin,  125  I- GIP and  The  125  I in  2 ml extracted plasma.  The  results obtained after RIA of the column fractions are t y p i f i e d i n Fig.  7/5; and Table XXXIX.  In both species the proportion of the t o t a l IR- GIP  represented by the larger molecular form (ProGIP.) increased with increase i n time after the ingestion  (3bL)  of f a t .  After oral glucose  Sera from human subjects 45 min and 100 min. after o r a l administration of 100 ml 20% dextrose were eluted from Sephadex G50 f i n e as previously described.  The percentage of the t o t a l IR- GIP existing i n the ProGIP form  increased with increase i n time after the ingestion  of glucose (Fig. 75  Table XXXIX).  These results indicate that the important factor i n determining the r e l a t i v e proportions of the d i f f e r e n t forms of IR- GIP i s the time of sampling the serum a f t e r 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  -202TABLE XXXIX Change i n r e l a t i v e proportions of IR-GIP components with the time of serum sampling after o r a l f a t and glucose. Type of stimulus Time (min) of sampling after stimulus  Oral.Glucose 45  100  Oral f a t i n man *Total IR-GIP content % Total as GIP v  0  % Total as ProGIP % Total as  GIP o 500  Oral f a t i n dog *Total IR-GIP content % Total as GIP v  0  % Total as ProGIP % Total as G I P  5 0 0 Q  Oral glucose i n man *Total IR-GIP content  1400  680  14. 4  16.1  % Total as ProGIP  35.3  42.5  % Total as G I P  50.3  41.4  % Total as G I P  vD  5 0 Q 0  * Expressed i n pg/ml IR-GIP.  -203-  IOO-i  806040200Min. 20 35 Oral Fat in Man 80-i  3  60  i £ 40H o >° 20H  60  •  GIP  ^  Pro-GIP  V o  5000  o-J  Min. 9 0 150 Oral Fat in Dog  60-i 40 20  ni  0-> Min. 4 5 100 Oral Glucose in Man  Fig.  75  The r e l a t i v e proportions of IR-GIP y 0 , I R - G I F Q 0 Q and IR-ProGIP, 5  'expressed as percentages ;of the t o t a l IR-GIP response,',observed Ca) 20,35 and 60 ^ninJafter f a t : i n g e s t i o n : i n man, .(b)-90 and 150 min after fat ingestion i n dogs and (c) 45and 100'min after glucose ingestion In man.-..  -204-  natural porcine GIP over one hour. 50 min.  and 15 min.  Blood samples were taken a f t e r 15,  a f t e r the infusion had ended. . The t o t a l IR- GIP  each serum sample was  30,  of  determined by RIA and 2 ml aliquots were subjected  to gel f i l t r a t i o n on Sephadex G50  f i n e p r i o r 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 i n the  p u r i f i c a t i o n of GIP.  The other immunoreactive peak eluted i n the void  volume of the column, i.e.jGIP^o, representing molecular weight^7-50,000, and was  a form or complex with a  too large to be explained  c i r c u l a t i n g GIP present i n the serum of the fasted dog,  Pretreatment of serum samples from the series with 6.0M tography resulted i n the conversion  i n terms of the  (Fig.76_).  urea p r i o r to chroma-  of a s i g n i f i c a n t proportion of the GIP^o  to GIP^QQQ suggesting that the IR- GIP e l u t i n g i n 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  B.  In tissue extracts  (I)  I n i t i a l tissue extraction  (Fig.776).  Extracts of the duodenal and j e j u n a l mucosa of dogs were p a r t i a l l y p u r i f i e d i n the laboratory of Dr. V. Mutt (Karolinska I n s t i t u t e t , Stockholm, Sweden). The  tissue was  boiled b r i e f l y and extracted into a c e t i c acid.  was  adsorbed onto a l g i n i c acid, eluted with 0.2M  hydrochloric acid and pre-  c i p i t a t e d from solution with saturated sodium chloride. contained  The protein  This p r e c i p i t a t e  secretin and cholecystokinin - pancreozymin, as well as GIP.  After  -205-  •  GIP  Vo  Pro-GIP 100  GIP  Cu  3  5000  80  cr  "  60  4—  40  o  -I  20 0• 30  Fig.  76  50 Min. Infusion  5  The r e l a t i v e proportions of IR<-GIPy0, I R - G I P 5 0 Q 0 and IR-ProGiP, expressed as percentages of t h e . t o t a l IR-GIP response, i n 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.  post  -206-  100 T  • GIP \WX Pro-GIP V o  CL i—i  ©  80H  GIP 5 0 0 0  I w  o  60 H 40 J 2.0 A  Without Urea  Fig. 77  With Urea  The r e l a t i v e proportions of I R - G I P , ' l R - G l P y0  500Q  and IR-ProCIP, ex-  pressed as percentages of the t o t a l IR-GIP response,;in a serum sample containing exogenously treatment with 6.OK  urea.  administered GIP,'with or without pre-  -207-  desalting on Sephadex G25,  a f r a c t i o n preceding IR-GIP <_QQQ (Fr 1-8)  selected f o r f urther extraction. Ten g of t h i s material dissolved i n 200 ml ammonium acetate, pH 6.5, addition of 2.0M  ammonia. The mixture was  4°C and the supernatant was im 0.1M  a c e t i c acid and  volumes .) ..' was  and  (SPC I G25  was by  for 30 min  insoluble p r e c i p i t a t e was  at  redissolved  l y o p h i l i z e d - neutral insoluble material.HHethanoi'-;(w9  added to the supernatant and  by centrifugation, redissolved i n 0.1M  the insoluble p r e c i p i t a t e removed  acetic acid and  l y o p h i l i z e d - methanol  insolublemmatefial.The protein remaining i n solution was addition of 4 volumes of acetone at 4°C, on Whatman's 3MM  Fr 1—8)  the pE corrected to 7.0  centrifuged at 7000 rpm  decanted. The  was  and  paper, redissolved i n 0.1M  precipitated by  the p r e c i p i t a t e was  the  filtered  out  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 acid and  f i n e (1x100 cm)  the IR-GIP content of each 1.0 ml f r a c t i o n was  r e l a t i v e proportions  i n 0.2M  acetic  measured by RIA.  The  of each component as a percentage of the t o t a l IR-GIP cont-  ent i s shown i n Table XXXX and F i g . 78.  Table XXXX Relative proportions extract of hog  of the IR-GIP components i n fractions obtained from an  i n t e s t i n a l mucosa  Fraction  % Total IR-GIP content GIP„ Vo  Neutral  insoluble  ProGIP  GIP  5000  0  33.3  66.7  Methanol insoluble  0  72.2  27.8  Methanol soluble  0  0.0  100.0  Further  p u r i f i c a t i o n was  performed on the methanol insoluble f r a c t i o n .  -208-  TABLE XXXXI Summary of Tissue Extraction Procedure  Heat coagulated hog duodeno-jejunal mucosa Acetic acid Acetic acid extract |^  A l g i h i c acid adsorption  NaCl p r e c i p i t a t e containing Sn, CCK-PZ and GIP a c t i v i t y ^  Sephadex G25  Fraction I containing IR-GIP 0.04M Ammonium acetate .pH 7.0  Neutral Soluble  Neutral Insoluble  fraction  fraction  Methanol Methanol Soluble fraction  Methanol Insoluble fraction  -209-  • 100  GIP Vo Pro-GIP  -t  G I P  CL  80 H  5000  © I  o o  60 H  40  <4—  O.  20 A 0-1  1  Neutral  Methanol  Methanol  t  Insoluble  Fig. 78  Soluble  The'relative proportions of IR-GIP , I R - G T P ^ ^ and TR-ProGIP, y9  . expressed as percentages of the t o t a l IR-GIP, i n p a r t i a l l y p u r i f i e d extracts of the hog duodenal and j e j u n a l mucosa.  -210-  (II)  Purification  (a;) ' Methanol insoluble on Sephadex G50 In a t y p i c a l experiment  300 mg methanol insoluble was dissolved i n 5 ml  0.2M a c e t i c acid containing  125  I - albumin and  125  I- GIP. The column of  Sephadex G50 f i n e (2.5x90 cm) was developed with 0.2M acetic acid and 5.0 ml fractions were collected at a flowrate of 80 ml/hour. column and the elution volume of  GIP^QQQ  The void volume of the  were determined by counting 0.5 ml  aliquots of each f r a c t i o n f o r 1 min. i n an automatic gamma counter.  The  region between these peaks was pooled, l y o p h i l i z e d and designated ProGIP I. The column p r o f i l e of such a column, obtained by p l o t t i n g absorbance at 280 nm i n a 1 cm l i g h t path against ml eluant, i s shown.in F i g . 79; with the regions of GIP immunoreactivity determined by RIA, superimposed.  This procedure  yielded approximately 100 mg l y o p h i l i z e d material with an IR- GIP content of 30 ng/mg.  (Ib)  ProGIP I on CM c e l l u l o s e  Thirty mg ProGIP I was dissolved i n 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. rate of 120 ml/hour.  The eluate was collected i n 5 ml fractions at a flow The column was calibrated by chromatographirig porcine  GIP under i d e n t i c a l conditions.  The absorbance of each f r a c t i o n was measured at 280 nm i n a 1 cm l i g h t path and the IR- GIP content estimated by radioimmunoassay with two different a n t i sera.  There was no s i g n i f i c a n t IR- GIP i n this sample, the major immuno5000  -211-  Flg. 79  Column p r o f i l e obtained after elution of the methanol insoluble f r a c t i o n 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.  rated with  1- albumin,  I-GIP and  The column was'calib^Iodine.  -212-  reactive peak.being less basic than G I P ^ ^ Q Q .  The a l t e r n a t i v e antiserum  Ro 7, appeared to cross react to a different'degree with t h i s molecular form of IR- G I P , compared with the antiserum, Van 8, routinely used i n 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  IR- G I P (mean - SE i n 50 determinations) according to Van IR- G I P (mean - SE i n 18 determinations) according to  When Fraction I I I , from chromatography on Sephadex G50, corresponding to G I P Q 5  0 q  , was  254-  43 pg/ml  8, and 242- 14 pg/ml  Ro7..  i . e . , that f r a c t i o n  eluted from c e l l u l o s e CM II under i d e n t i c a l  conditions, the major portion of the immunoreactivity eluted i n the same p o s i t i o n as natural porcine G I P ^ Q 0 Q  (g)  (Fig. 81,).  S t a b i l i t y of ProGIP  The material containing ProGIP, and selected to contain no G I P ^ Q Q was Q  routinely l y o p h i l i z e d and stored at -20°C.  After the y i e l d from several  columns had been pooled, 2 mg of the material was f i n e (1x100 cm) i n 0.2M  re-run on Sephadex G50  acetic acid as previously described.  Radioimmunoassay  on the fractions obtained revealed that a t h i r d of this material now  existed i n  the GIP,-QQQ form, as shown i n F i g . 82.  (Ill)  Molecular weight  determination  A series of chromatpgrams were run on Sephadex G50 f i n e (1x100 cm) i n  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- m o t i l i n , I-GIP, I125 'Insulin or I- parathyroid hormone, i n 2 mis 0.2M a c e t i c acid. The 1 ml  -213-  GIP 5000 _^ Pooled a  0.01 M Lypholized  N H  4  HC0  0,2M  NH4HCO3  3  r  9.0  I 8.0 l.500n  i —  IR-GIP • OD  E c O 00 OJ  1.000  (Ro*7) I-  at 2 8 0 n m  7.0  I h  6.0  -  5.0  -  4.0  c  H  CD  IR-GIP  o c  (Van  8)  ff  3.0 0.500 4  h  2.0  I .0  0.000 J  n— 10  20 Fraction  F i g . 80  40  I 50  0.0  #  Column p r o f i l e obtained after elution of ProGIP I from CM cellulose.  o  The IR-GIP:content was determined by RIA with  antisera Van 8 and Ro7. porcine GIP.  The column was calibrated with  -214-  0  10  20 Fraction  Fig. 81  30 #  Column p r o f i l e obtained after elution of the GIP,-x„„ - < <r 5000 . containing f r a c t i o n from Sephadex G50, on CM c e l l u l o s e . . The column' was ' calibrated with', porcine GIF  -215-  F i g . 82  Column p r o f i l e obtained after rechromatography of ProGIP I on Sephadex G50 i n 0.2M of IR-GIP, 5000'  acetic acid, 'demonstrating the .reappearance  -216-  fractions collected were counted f o r 1 min. i n 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 /V° versus molecular weight  was plotted f o r each marker ( F i g . 83).  E  V" /V° was determined f o r the IR-GIP E  i n ProGIP I and t h i s was found to correspond to a molecular weight of 7500-8000 iii four separate determinations.  -217-  2.0  - i  x^Motilin x Glucagon  \  .8  x GIP  \  I .6  x insulin  1.4 H  12  H  1.0  4  Parathyroid sHormone  T—i—n—i—i 3  M.W.  F i g . 83  4  5  X  10  6  8  10  3  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) i n 0.2M acetic acid'and counting of these fractions f o r 1 min i n an automatic y counter.  -218-  DISCUSSION Studies on the composition and structure of m o t i l i n revealed that i t was quite d i s t i n c t from the previously isolated and characterized g a s t r o i n t e s t i n a l polypeptides of duodenal mucosal o r i g i n . appeared  The major property of m o t i l i n  to be i t s stimulatory e f f e c t on the motor a c t i v i t y of the stomach.  Unlike gastrin and cholecystokinin-pancreozymin, m o t i l i n stimulated the motor a c t i v i t y of the fundus as well as that of the antrum. m o t i l i n , suggested by studies with exogenously was stimulation of pepsin secretion i n dogs.  Another action of  administered polypeptide, I t did not, however, appear to  have any effect on the exocrine pancreas or on g a s t r i c acid secretion (Brown et al,1972).  Recognition of the physiological r o l e played by m o t i l i n i n regula-  ting gastric motor and secretory a c t i v i t i e s , and support f o r the hypothesis that m o t i l i n was the humoral agent released upon a l k a l i n i z a t i o n of the duodenal mucosa both required the development of some method f o r the i d e n t i f i c a t i o n and measurement of changes i n the levels of m o t i l i n i n the systemic c i r c u l a t i o n .  A radioimmunoassay has been developed for the measurement of IR- m o t i l i n i n sera and tissue extracts (Dryburgh and Brown, 1975).  Antisera were raised  i n albino guinea pigs and New Zealand white rabbits.  As a general r u l e , any  substance with a molecular weight ofV <1000 may be regarded as non-immunogenic, whilst those with molecular weights i n the range 1000-6000 are poor immunogens. M o t i l i n , with a molecular weight of 2700, f a l l s into the l a t t e r group and no useful antisera to this polypeptide have been obtained by immunization with the polypeptide alone. molecule  This was overcome by covalently coupling the  (hapten) to a larger protein.  The most commonly used method of achie-  ving t h i s i s the carbodiimide condensation reaction.  Carbodiimide w i l l react with a  -219-  number of weak acids but the predominant reaction at room temperature i s 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  +  CH -CH -N=C=N(CH ) 3  2  2  3  (CDI)  (HAPTEN) +  H0 2  CH -CH -NH-C=N-(CH ) - f i - ( C H ) 3  -^-(CH^  2  2  3  3  2  O-C-R  II 0 + R-C-NH-R'  +  R'NH  (PROTEIN)  2  CH -CH -NH-C-NH-(CH ) 3  2  2  3  4i-(CH ) 3  2  0 (CONJUGATE) Some degree of condensation w i l l occur between the protein molecules, v i a t h e i r activated carboxyl groups.  This may be reduced somewhat by activating  the haptenic carboxylic acids before the addition of the protein. human serum albumin are the most'commonly used proteins.  Bovine and  Immunization of  both rabbits and guinea pigs, the schedule of injections involving both m o t i l i n , conjugated to BSA, and non-conjugated m o t i l i n , resulted i n the production of acceptable antisera i n the majority of animals. 12 The range of a f f i n i t y constants (K) of m o t i l i n antisera was 1 x 10 L  14 to 1 x 10  l/mole, calculated as shown i n F i g . 10. When a radioimmunoassay i s being estab-  l i s h e d i t must be accepted that there i s an inherent  l i m i t to the s e n s i t i v i t y  -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 i n t e r v a l since the immunization (Eisen and Siskind, 1964). present  It i s assumed that the heterogeneity  of antibodies  i n an antiserum i s the r e s u l t of t h e i r production by-a heterogeneous  population of antigen-sensitive small lymphocytes.  Low  to moderate doses  of immunogen w i l l p r e f e r e n t i a l l y stimulate c e l l s with high a f f i n i t y  receptors  and their progeny w i l l , i n turn, produce high a f f i n i t y antibodies.  With the  passage of time the antibodies produced w i l l tend to n e u t r a l i z e some of the antigen and as the l e v e l f a l l s , i t w i l l be the higher a f f i n i t y c e l l s which w i l l continue to be stimulated.  Antisera to m o t i l i n have been raised by  immunization with 20-50 pg polypeptide, a reasonable t i t r e was immunogen, at 3 - 6  achieved.  given at monthly i n t e r v a l s u n t i l  Booster immunizations with low doses of  month intervals,maintained or increased the t i t r e .  Animals were bled 10 - 12 days a f t e r each immunization and at monthly i n t e r v a l s ' thereafter.  M o t i l i n antiserum Mo7  4 had a t i t r e of 1: 10 x 10 , 10 days a f t e r 4  i t s fourth immunization, which had increased to 1: 20 x 10  two months l a t e r ,  with no intervening booster (Table I I ) .  The immunization schedules and r e s u l t s  are summarized i n Table I and Table I I .  No c r o s s r e a c t i v i t y has been demonst-  rated between any m o t i l i n antiserum and g a s t r i n , GIP, cholecystokininpancreozymin,  s e c r e t i n , glucagon,  VIP or i n s u l i n , as shown i n Figs. 8 and  9.  T h e o r e t i c a l l y the iodination of. any.polypeptide containing tyrosine requires 125 only 3 basic ingredients:-- the isotope, usually and some method -for o x i d i z i n g the iodide to iodine.  I-Na,  the pure polypeptide,  Variables, such as the  -221-  r e l a t i v e r a t i o s 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 l a b e l l e d polypeptide,/have to be established f o r each i n d i v i d u a l polypeptide.. The majority of iodination procedures, however, vary only s l i g h t l y from the o r i g i n a l method proposed by Hunter and Greenwood i n 1963.  Motilin.was iodinated by a s l i g h t modification of their  chloramine-T method and radioactive tracer with: a s p e c i f i c a c t i v i t y of 400 mCi/mg was routinely produced.  A t h e o r e t i c a l iodination of this polypeptide,  r e s u l t i n g i n the incorporation of 1 atom of iodine into each mole of peptide, 125 would produce  I- m o t i l i n with a s p e c i f i c a c t i v i t y of 626 mCi/mg, assuming an  isotope abundance of 96%.  The actual r e s u l t s obtained would suggest that the  tyrosine i n p o s i t i o n 7 i n m o t i l i n i s r e l a t i v e l y accessible to incorporation of iodine.  Storage of  I- m o t i l i n , at a dilutioniof 1 x 10  acetic acid, containing 0.5%  cpm/ml, i n 0.2M  B.S.A. at -20°C, resulted i n a l a b e l , stable  without r e p u r i f i c a t i o n , for periods of up to 3 months.  With the antiserum so  far available, the most sensitive assay has resulted from an incubation which i s 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 i s adsorption of the antigen.onto dextran-coated charcoal, 2.5 and 0.5 mg dextran being added per tube, as i l l u s t r a t e d i n F i g . 16.  mg  A  s o l i d phase antibody has-',h.een developed by coupling m o t i l i n antiserum to a beaded form.of agarose, a cross-linked dextran with high porosity (Sepharose 4B). This matrix i s activated by treatment with cyanogen bromide at high pH.  It i s  then reacted with the unprotonated amino groups on the ligand, i n this case the IgG i n the.antiserum, to form a stable complex by formation of hydrogen bonds.  -222-  R -NIL,  NRH  Ligand coupled  CNBr-Sepharose' 4B  to;Sepharose  No detectable antibody a c t i v i t y was measured i n the washings of the s o l i d after coupling to the antiserum was complete ( F i g . 18). The coupled antibody could be used i n the radioimmunoassay with no apparent loss i n antibody a c t i v i t y or.sensitivity potential.  No s i g n i f i c a n t difference was observed i n the  antiserum d i l u t i o n 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 i n the further development of the m o t i l i n radioimmunoassay.  P i l o t studies were performed  on the  extraction of m o t i l i n from sera and tissue extracts by chromatography of the motilin-containing material on columns of.Sepharose 4B, coupled to m o t i l i n antiserum.  The results were favourable f o r the development of this technique  on a larger scale, both for the concentration of m o t i l i n from sera and the i s o l a t i o n of the peptide from.its s t a r t i n g material, Presekretin, i n a more economical fashion than can be achieved by the s e r i a l stages of chromatography, at present i n use (Fig. 51 and F i g . 52). M o t i l i n , adsorbed to this matrix can be eluted by a lowering of the pH with no apparent damage to the molecule, as i l l u s t r a t e d by the s i m i l a r i t y of the standard curves obtained 125 with  I - m o t i l i n , before.and a f t e r 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 m o t i l i n . < • The fasting serum m o t i l i n concentrations  i n man,  measured i n 45 normal subjects.in the age range 20-35 years, was 190 - 131 pg/  +  ml IR m o t i l i n (mean - S.D.). ration  +  The mean - S.D. f a s t i n g serum /motilin concent-  i n 8:dogs was. 294 - 44 pg/ml IRT/motilin.. When! serum samples, containing  exogenous or endogenous m o t i l i n , were.assayed at several d i l u t i o n s , the r e s u l t s obtained  could be f i t t e d to the standard curve, as shown'in T i g . 10.  This  antibody would not therefore appear to d i f f e r e n t i a t e between unlabelled i n the form of the standard or antigen i n the form.of the. endogenous  antigen  polypeptide,  s a t i s f y i n g one of the basic requirements for.the development of a s e n s i t i v e radiomunoassay.  Addition of 5000 cpm per tube of a radioactive tracer with a  s p e c i f i c a c t i v i t y of 400 mCi/mg e n t a i l s the addition of only 7 pg m o t i l i n and s t i l l allows an e f f i c i e n t 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 s a t i s f i e d , as l a i d down i n the r a t i o n a l e , the other variables have been established at t h e i r optimal values.  The apparent homogeneity of IR-motilin, suggested by the serum d i l u t i o n curves, was further examined by chromatography of either a l k a l i - s t i m u l a t e d IR- m o t i l i n i n serum or an impure duodenal extract on Sephadex G-50.  As i l l u s t r a t e d i n  F i g . 31 and F i g . 32, only one region of IR- m o t i l i n was detectable, e l u t i n g i n 125 the same p o s i t i o n as  I- motilin.  The m o t i l i n content of 2 d i f f e r e n t preparations, 13-norleucine-motilin,  one the synthetic analogue,  the other an impure duodenal extract, was measured by  bioassay.and immunoassay, i n comparison with natural porcine m o t i l i n .  The  natural and synthetic motilins were found to be i d e n t i c a l i n both.biological  -224and immunological a c t i v i t y - ( F i g . 39 and F i g . 41).  The natural polypeptide  represented a 10,000 fold p u r i f i c a t i o n of the crude extract,  measured by  either bioassay or immunoassay, as i l l u s t r a t e d i n . F i g . 29 and F i g . 30. These results and the apparent homogeneity of IR- m o t i l i n , suggest that the b i o l o g i c a l a c t i v i t y of m o t i l i n may be f a i r l y closely correlated with  its  immunological a c t i v i t y , as measured by radioimmunoassay.  The IR- m o t i l i n of various regions of the hog g a s t r o i n t e s t i n a l measured i n p a r t i a l l y p u r i f i e d acid-ethanol extracts of the mucosa.  tract was  gastrointestinal  The region with the highest m o t i l i n content (per g dry weight of  the extract) was the jejunum, followed by the duodenum and upper ileum, as summarized i n Table XIV. stomach or lower ileum. (1974). first  No detectable m o t i l i n was found i n the oesophagus,  These findings agree with the results of Pearse et a l  Using the i n d i r e c t sandwich technique, with m o t i l i n antiserum as the  layer,and fluorescein-labelled goat antirabbit IgG as the second l a y e r ,  they were able to detect fluorescent motilin-cbntaining c e l l s i n the duodenum, jejunum and upper ileum of the dog, p i g , baboon and man.  The c e l l of o r i g i n  was i d e n t i f i e d as the enterochromaffin (EC) c e l l , 85% of the motilin-containing c e l l s showing p o s i t i v e argentaffinity. argyrophyl.  The remaining 15% of the c e l l s were  No EC c e l l i n the stomach or lower intestine could be demonstrated  as containing m o t i l i n .  The EC c e l l s of the upper gastrointestinal  tract have  also been shown to contain serotonin, substance P and melatonin, a l l of which are also found i n neural tissue. classified  Although the EC c e l l i s unlike the other c e l l s  as APUD i n that i t does not derive from the neural c r e s t , i t does  appear to originate from neuroectodermally derived t i s s u e . should be investigated that m o t i l i n has neural connections.  The p o s s i b i l i t y The r e l a t i o n s h i p ,  i f any, between m o t i l i n and the other substances of EC c e l l o r i g i n i s not c l e a r .  -225-  D i f f e r e n t i a l staining techniques, applied to mammalian duodenal tissue, have demonstrated that m o t i l i n and substance P are present i n -different EC c e l l s (Polak et al,..1976). _ Forssman et a l (1976) confirmed  that not a l l serotonin-  containing c e l l s ih'thms region contained m o t i l i n but were.: ruhable to 'rule out t  the p o s s i b i l i t y that m o t i l i n - containing c e l l s do.contain.:serotonin. must postulate that EC c e l l s f a l l into subpopulations,  One  c l a s s i f i e d by the poly-  peptide they produce.  The production of a synthetic analogue (Wunsch et a l , 1973)  permitted some  insight into the r e l a t i o n s h i p betweent. 'the structure of m o t i l i n and i t s b i o l o g i c a l potency.  The methionine residue at p o s i t i o n 13 was  to be e s s e n t i a l for the expression of b i o l o g i c a l a c t i v i t y .  o r i g i n a l l y thought When the methionine  was  oxidized by treatment with-hydrogen peroxide, 95% of the b i o l o g i c a l .activity  was  lost.  F u l l potency was  with cysteine (Cook,. 1972).  restored, however-, after reduction of the residue 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, i n either b i o l o g i c a l or immunological a c t i v i t y was  observed (Fig. 39 and F i g . 41)  suggesting that i t was not the methionine residue per se which was conferring f u l l b i o l o g i c a l potency.  important  in  It was more l i k e l y that some conformational  change, which resulted from the oxidation of the sulphur-containing residue, was  responsible for the loss of a c t i v i t y .  In the course of the synthesis and  p u r i f i c a t i o n of 13-norleueine-motilin, the intermediate compounds Mol^ and were found to be Inactive.  M0C2  Mol^ resulted from a f a i l e d synthesis, i n 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  p o s i t i o n 5 being i n the D-configuration,, rather than the  L-configuratidn found:in the active molecule.  Differences were observed i n the 'electrophoretic mobilities.',p.ftheitryptic ;  digestion products of the natural and synthetic motilins. peptide 3 (Tr 3), reported by Brown et a l (1973) was material.  When the t r y p t i c digestion was  motilin, Tr 3 was  The a c i d i c t r y p t i c  absent i n the synthetic  repeated on freshly prepared porcine  i s o l a t e d and subjected' to electrophoresis at pH 6.5.  ran as a neutral peptide.  It  now  K i n e t i c studies with leucine aminopeptidase and  3-cycle Edman degradation, indicated that the peptide contained position 14 and not glutamic acid as o r i g i n a l l y stated. that deamidation of the residue had occurred m o t i l i n (Schubert and Brown;, 1974)-  a  glutamine at  It must be supposed  during the early preparation  of  This deamidation had no e f f e c t on b i o l o -  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 i s o l a t i o n of a fragment containing any s i g n i f i c a n t b i o l o g i c a l or munological a c t i v i t y .  im-  The immunological a c t i v i t y observed after cyanogen  bromide cleavage of m o t i l i n , seen i n F i g . 40, can be accounted for by  the  presence of uncleaved material remaining i n the reaction mixture.  Modification of the naturally-occurring molecule by acylation d r a s t i c a l l y reduced the potency of m o t i l i n .  Acetylation neutralized the p o s i t i v e charges  of the £ -amino groups and the N-terminal phenylalanine, produced a net negative charge on the molecule.  One  whilst succinylation  cycle of the Edman's de-  gradation procedure also resulted i n a loss of b i o l o g i c a l potency.  It i s  -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 a c t i v i t y associated with the change i n the net charge on the molecule would suggest that m o t i l i n binds to i t s receptor s i t e by formation of ion pair bonds.  The b i o l o g i c a l a c t i v i t y of -motilin was seemingly unimpaired by the  removal of the C-terminal and penultimate amino acids, after treatment with carboxypeptidase A.  I t should be remembered, however, that this i s not a com-  plete degradation procedure and u n t i l synthetic peptides 1-20 and 1-21 can be prepared, no firm conclusions may.be drawn, regarding the importance of these amino acids i n the b i o l o g i c a l a c t i v i t y of m o t i l i n .  From these r e s u l t s ,  summarized i n Table XV,, i t must, be concluded that v i r t u a l l y the intact molecule i s required f o r the expression of f u l l b i o l o g i c a l potency.  The i n d i v i d u a l  amino acids are important inasmuch as they contribute to the charge d i s t r i b u t i o n and probable confirmation of the molecule.  Brown et a l (1966) reported an increase i n the motor a c t i v i t y of an e x t r i n s i c a l l y denervated or transplanted pouch of the fundus of the stomach after  duodenal  infusion of i s o t o n i c a l k a l i or fresh pig pancreatic j u i c e , s u f f i c i e n t to raise the pH from the basal l e v e l of 7.5 to 8.2.  Intravenous infusion of a pure poly-  peptide, isolated from hog duodenal mucosa, mimicked this response.  No other  g a s t r o i n t e s t i n a l polypeptide has been discovered which w i l l produce the reported increase i n fundic motor a c t i v i t y ; gastrin and cholecystokinin-pancreozymin having their motor effect only on the antrum of the stomach. of a/radioimmunoassyi  The development  s p e c i f i c f o r m o t i l i n , confirmed the supposition that the  increased fundic motor a c t i v i t y -observed after duodenal a l k a l i n i z a t i o n was accompanied by a concomitant increase i n the c i r c u l a t i n g levels-of IR- m o t i l i n ,  -228-  comparable to those achieved during exogenous administration of the polypeptide :CFig. 34). An increase i n serum IR- m o t i l i n levels was also reported by Hellemans et a l C1976) a f t e r 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 a l 0-976) were unable to detect any increase  i n c i r c u l a t i n g IR- m o t i l i n l e v e l s . a f t e r intraduodenal T r i s buffer, pH 10.2, in human volunteers.  They claimed a f a l l i n plasma m o t i l i n levels occurred  after duodenal alkalinia'atlon but examination of their, results f a i l e d to reveal that the depression of TR-motilin. levels was s i g n i f i c a n t i n view of the v a r i a t i o n i n IR- m o t i l i n levels measured during the pre-infusion control period.  Control studies i n dogs', with an intraduodenal i n f u s i o n of 0.15M  saline showed no increase i n fundic pouch motor a c t i v i t y and no change i n IR- m o t i l i n from the basal l e v e l s , throughout the duration of the experiment, as shown i n F i g . 35.  Similar findings i n mart-have been reported by Mitznegg  et a l (1976).  In two experiments, the dogs exhibited spontaneous increases i n fundic pouch motor a c t i v i t y , very s i m i l a r to those obtained after a l k a l i . IR- m o t i l i n accompanied this Increased motor a c t i v i t y . m o t i l i n release i s unclear.  An increase i n  The cause of this  The a c t i v i t y occurred after the dogs had exhibited  a period of normal basal motor a c t i v i t y 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 i n s e r t i o n of the Foley catheter into the MannBollman f i s t u l a .  -229-  A somewhat anomalous observation was that duodenal a c i d i f i c a t i o n appeared to produce an increase i n serum IR- m o t i l i n i n dogs.  This elevation was  less than that seen after duodenal a l k a l i n i z a t i b n , but was more prolonged, as i l l u s t r a t e d i n F i g . 37. in these animals.  No increase i n fundic pouch a c t i v i t y was observed  This r e s u l t was unexpected i n view of the finding by  Brown et a l (1967) that intraduodenal acid suppressed alkali-induced fundic pouch motor a c t i v i t y . observation.  Recent studies by Itoh et a l (1976) supported  this  They demonstrated that the i n t e r - d i g e s t i v e pattern of gastric  motor a c t i v i t y i n the fundus, antrum and lower oesophageal sphincter i n dogs was exactly mimicked by i n f u s i o n of 0.1 - 2.7 ug/kg/hour synthetic m o t i l i n (13-methionine-motilin) tude and frequency  when compared with respect to duration, ampli-  of the contractions, and t h e i r v e l o c i t y at different distan-  ces along the g a s t r o i n t e s t i n a l t r a c t .  This i n t e r d i g e s t i v e pattern, whether  natural or motilin-indueed, was interrupted by feeding, duodenal a c i d i f i c a t i o n or an infusion of pentagastrin.  One possible explanation of these contra-  dictory findings i s that the substance released by duodenal a c i d i f i c a t i o n i s not m o t i l i n , but some other substance which shares immunological but not b i o l o g i c a l i d e n t i t y with m o t i l i n . acylation almost completely approximately  I t has already been noted that  although  abolished the b i o l o g i c a l a c t i v i t y of the molecule,  50% of the immunological a c t i v i t y s t i l l remained (Table XXV).  Investigation of this acid-released m o t i l i n - l i k e immunoreactivity  by electrop-  horesis or column chromatography would provide more information r e l a t i v e to any size or charge difference between i t and the alkali-induced, motor stimulatory m o t i l i n .  I t i s i n t e r e s t i n g to speculate whether the a n t i - m o t i l i n  e f f e c t observed after duodenal a c i d i f i c a t i o n i s due, i n part, to competitive antagonism by this m o t i l i n - l i k e material.  r  -230-  The p h y s i o l o g i c a l r o l e ( s ) . o f m o t i l i n w i l l remain a subject of  controversy  u n t i l a p h y s i o l o g i c a l secretagogue or other mechanism f o r i t s release can be demonstrated.  The lack of any s i g n i f i c a n t increase i n the systemic  levels  of IR- m o t i l i n a f t e r ingestion of either glucose or a mixed meal Csee F i g . 38) would suggest that m o t i l i n . p l a y e d ' l i t t l e r o l e i n the normal digestive processes. The apparent increase i n IR- m o t i l i n reported by Mitzrtegg et a l (1976) after ingestion of f a t , with the accompanying i n h i b i t i o n of fundic motor a c t i v i t y , may  be due to the. same m o t i l i n - l i k e m a t e r i a l released by duodenal a c i d i f i c a t i o n .  In 1966,  when Brown et a l f i r s t described the alkali-induced increase i n fundic  pouch motor a c t i v i t y , they postulated that, under these conditions, some humoral agent was  being released which would counteract  other g a s t r o i n t e s t i n a l polypeptides, gastrin,  the i n h i b i t o r y e f f e c t of the cholecystokinin-pancreozymin  (and, of course, GIP) which were released by the ingestion of various nutrients. Studies by Hoelzel (1925) and Reinke et a l (1969) i n dogs, indicated that the duodenal contents  tended to an a l k a l i n e pH during the f a s t i n g periods.  As  previously described by Ttoh et a l (1976) exogenous m o t i l i n exactly reproduced the normal pattern of mechanical i n t e r d i g e s t i v e a c t i v i t y *  .This consisted of  bands of contractions, a r i s i n g simultaneously  i n the fundus and duodenum and  t r a v e l l i n g aborally the length of the ileum.  Each cycle took approximately  20 mins. to pass one point and consecutive This pattern was  interrupted by feeding.  cycles were about 90 mins apart. Preliminary studies have suggested  that m o t i l i n levels were depressed i n i t i a l l y  after feeding, as shown i n F i g . 38.  The purpose of this i n t e r d i g e s t i v e c y c l i c a c t i v i t y i s postulated to be cleaning from the digestive  the  and absorbent,t regions of the upper gastrointes-  t i n a l tract of the extra mucus'and desquamated from the processing of the previous meal.  epithelial cells resulting  This "cleaning up" operation i s  -231-  probably mediated i n part by Immoral means; in part by neural mechanisms. :  'r  The  appearance of a single cycle of a c t i v i t y at a time i n the upper 70%  the g a s t r o i n t e s t i n a l tract may upper and  be due  lower regions of the t r a c t .  of  to some i n h i b i t o r y r e f l e x between the The  tachyphylaxis described by Coot-  (1972) as occurring when a second i n j e c t i o n of m o t i l i n was  given less than  40 mins after the f i r s t , might be explained by an i n h i b i t o r y . r e f l e x of t h i s type.  The  fact that g a s t r i c a l k a l i n i z a t i o n w i t h or without p r i o r g a s t r i c a c i d i f i c a -  tion, has been reported as causing an increase i n the pressure of the lower oesophageal sphincter that motilin was pressure was  (LESP) (Castell and Levine, 1971)  involved  raised the p o s s i b i l i t y  i n the control of this region.  The  increased  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 a l (1974) or Kline et a l (1975) who gastrin a f t e r antral a l k a l i n i z a t i o n .  could show no increase i n serum IRJennewein et a l (1975) measured changes  i n LESP i n dogs after bolus injections or intravenous infusions of motilin over 30 mins.  The most e f f e c t i v e doses were 30 ng/kg or 100 ng/kg/hour  respectively.  The m o t i l i n resulted i n increased a c t i v i t y i n both the antrum  and  the fundus,,and low frequency phasic a c t i v i t y i n the LES,  a c t i v i t y i n the fundus.  When m o t i l i n was  phasic a c t i v i t y i n the LES  ceased within  related to  administered as an infusion, 10 mins of the end of the  the  infusion.  Duodenal a l k a l i n i z a t i o n resulted i n an increase (non-significant)  i n the  LESP i n 4 dogs.  the  Similar results were obtained i n studies  i n man,  the  LES  responding to graded doses of the synthetic nor-leucine analogue i n a doserelated 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 m o t i l i n was  performed i n normal subjects and patients who  had undergone truncal vagotomy  with B i l l r o t h I or B i l l r o t h II antrectomies (McCallum et a l , 1977). subjects ingested either 0.4M preparation.  Increased LESP was  witTt"Billroth I anastomoses. r  serum IR- gastrin was The  sodium bicarbonate or a commercial  The  antacid  observed i n the normal subjects and  those  No increase i n either serum IR- m o t i l i n or -  detected i n association with this pressure  lack of any response i n the B i l l r o t h II patients  of the duodenum i n e l i c i t i n g t h i s response.  increase.  indicated the importance  Hellemans et a l (1976) were  able to demonstrate a slight.increase i n IR- m o t i l i n a f t e r either gastric a l k a l i n i z a t i o n or a c i d i f i c a t i o n . The motilin response to a l k a l i was i n onset and s l i g h t l y preceded the peak response i n the LES. was  rapid  This finding  i n agreement with the postulate of, Jennewein that m o t i l i n exerted i t s  effect on the LES  i n d i r e c t l y , v i a i t s e f f e c t on fundic motor a c t i v i t y and  g a s t r i c intraluminal pressure. fundus was  The  increase i n intraluminal pressure i n the  then the d i r e c t cause of the increased LESP.  motilin response to antral a c i d i f i c a t i o n was  In contrast,  the  much slower i n reaching i t s  peak (approximately 45 mins.) and correlated d i r e c t l y with the peak LESP. These differences were not remarked upon by the authors. rates of motilin release were due  I f the d i f f e r e n t  to different rates of passage of the acid  or a l k a l i into the duodenum, the authors' postulate that m o t i l i n , l i k e acid, has  a r o l e i n the i n h i b i t i o n of g a s t r i c emptying loses support.  Increased LESP a f t e r exogenous administration synthetic motilin has been conclusively The  of low doses of natural  demonstrated i n both dogs and  results obtained after endogenous released m o t i l i n are f a r less  or man. 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 i n an increased LESP, which correlated with the release of IR-motilin. However, the increase i n LESP associated with o r a l ingestion of a l k a l i could ;  not be related td; any detectable increase i n IR- m o t i l i n .  This f i n d i n g does  not necessarily preclude m o t i l i n from playing a role i n regulating LESP. may  It  exert i t s e f f e c t at levels not detectable by radioimmunoassay.  In 1967  Brown and Parkes postulated the existence of a duodenal, pH dependent,  dual hormonal mechanism f o r the control of g a s t r i c motor a c t i v i t y .  In such a  mechanism, low duodenal pH would i n h i b i t g a s t r i c motor a c t i v i t y and  delay  gastric emptying, by the release of some humoral agent, whilst a high duodenal pH would increase g a s t r i c motor a c t i v i t y and possibly increase rate of g a s t r i c emptying. was  The e f f e c t of m o t i l i n on the rate of g a s t r i c emptying  investigated i n dogs equipped with g a s t r i c f i s t u l a e .  of porcine m o t i l i n was meal ( F i g . 53).  the  Intravenous infusion  found to accelerate the emptying of a neutral test  I t had no e f f e c t on the rate of emptying of s o l i d s (Fig. 54).  The s i t e of action of the m o t i l i n during the emptying of/ the l i q u i d was  deter-  mined to be the fundus, antrectomy having l i t t l e e f f e c t on the response, as shown i n F i g . 56.  The decrease i n s e n s i t i v i t y of this response to m o t i l i n  after truncal vagotomy (shown i n F i g . 55) suggested that the response depended... on some i n t e r a c t i o n between m o t i l i n and a cholinergically-mediated' neural r e f l e x , at the l e v e l of the fundic musculature (Debas et a l , 1977).  These  r e s u l t s were i n d i r e c t contradiciton to those obtained by Ruppirt et a l (1975) i n man.  . They described a decrease i n the rate of g a s t r i c emptying of an acid  test l i q u i d , a f t e r an infusion of 13-norleu-motilin. - on the rate of emptying of the s o l i d meal may  The lack of any e f f e c t  be due to an over-riding or an  i n h i b i t i o n of the motilin-induced response by the humoral f a c t o r s released 1  from the duodenum by the constituents of that meal.  As yet, an increased  -234-  release of endogenous m o t i l i n has not been demonstrated to be part of the pattern associated with increased rates of gastric emptying, and the p h y s i o l o g i c a l importance of this action i s uncertain.  Investigation of the mode of action at the c e l l u l a r l e v e l has involved the development of an i n v i t r o preparation for the assay of m o t i l i n a c t i v i t y . Domschke et a l (1976) have reported the development of such a preparation u t i l i z i n g either rabbit duodenal muscle or s t r i p s of human fundus.  They were  able to demonstrate a dose-related c o n t r a c t i l e response to natural or synthetic motilin.  This response was  unaffected by ganglionic blockade, axonal blockade,  atropine or antihistamine, suggesting  that m o t i l i n acted at a receptor on or  i n the muscle c e l l .  M o t i l i n may exert i t s e f f e c t on muscle by influencing the I | transport of calcium (Ca ) within the cytosol. The action of m o t i l i n was I | . . blocked by the Ca antagonist, verapamil. Increased c y c l i c guanosine 3'5' I | monophosphate (cGMP) l e v e l s , associated with the rapid release of Ca  from  microsomal f r a c t i o n s , were observed during the response to m o t i l i n . Strunz et a l (1976) found that the response of i s o l a t e d rabbit p y l o r i c muscle to acetylcholine was motilin.  enhanced by pretreatment with subthreshold  This augmentation was  levels of  not associated with either increased acetyl-  choline synthesis or decreased acetylcholine degradation.  The doses of m o t i l i n  required to stimulate the i n v i t r o preparations were r e l a t i v e l y larger than those which promoted g a s t r o i n t e s t i n a l m o t i l i t y i n the intact animal. possible that c i r c u l a t i n g , subthreshold  It i s  l e v e l s of m o t i l i n i n the intact animal  may be contributing to the cholinergically-maintained tone i n the g a s t r o i n t e s t i n a l nusculature  and that functionally s i g n i f i c a n t changes i n serum IR- m o t i l i n levels  -235-  are.not detectable in,the radioimmunoassay system.. A.second p o s s i b i l i t y i s that motilin i s not transported to i t s target c e l l v i a the c i r c u l a t i o n but i s merely passed v i a the e x t r a c e l l u l a r f l u i d to the adjacent c e l l where i t acts, i n a paracrine fashion.. The i n a b i l i t y of other groups, to establish an in v i t r o assay using tissue from rats, guinea pigs and dogs can only be explained'at this time- by species differences..  It i s now  suggested  that there i s no evidence that m o t i l i n normally plays  any p h y s i o l o g i c a l role at the levels of IR- m o t i l i n 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 i s that the role of motilin. i s to maintain the interdigestive pattern of g a s t r i c motor activity-, the e f f e c t i v e - l e v e l s being much lower than those achieved during these experiments. during- a continuous  A study of IR- motilin levels  recording'of the interdigestive- and digestive patterns  of motor a c t i v i t y would help confirm or deny this supposition.. A second question which should be answered i s whether m o t i l i n i s acting independently  i n controlling  c o n t r a c t i l e a c t i v i t y or whether i t i s acting i n concert with some other humoral or neural reflex, probably cholinergically-mediated. I f this i s the case, the effective- increases*in m o t i l i n levels-may only occur at the c e l l u l a r l e v e l and may neyer be reflected by changes i n the systemic levels of the polypeptide. The p h y s i o l o g i c a l 'stimulant for m o t i l i n release:would be the r i s e i n duodenal and jejunal pH occurring i n the fasted state.  The absence of increased systemic  levels of IR- m o t i l i n associated with any s p e c i f i c part of the digestive cycle and the lack of evidence regarding the existence of any hypersecretory syndrome set m o t i l i n apart from the other known gastrointestinal hormones and would suggest that m o t i l i n may  be acting as a l o c a l hormone or i n a paracrine manner,  rather than as a c l a s s i c a l endocrine hormone.  -236-  The polypeptide i s o l a t e d from the i n t e s t i n a l mucosa of hogs by Brown et a l (1969) was named gastric i n h i b i t o r y polypeptide (GIP) because of the early observation that the pure porcine material, infused intravenously i n dogs, i n h i b i t e d stimulated gastric acid secretion,.in a dose related manner (Pederson and Brown, 1972).  A second, at least equally important, b i o l o g i c a l a c t i v i t y  of this polypeptide was demonstrated by Dupre et a l (1973).  They infused  porcine GIP i n men and were able to measure an enhanced i n s u l i n response to an intravenous glucose infusion. finding i n dogs.  Their results would suggest'that GIP might also be i n t e r -  preted as glucose-dependent 1977).  Pederson et a l (1975A, 1975B) confirmed this  i n s u l i n o t r o p i c polypeptide (Brown and  Dryburgh,  A radioimmunoassay, s p e c i f i c for GIP, was developed by Kuzio et a l  (1974) to determine the physiological secretagogues for GIP and the r e l a t i v e importance of the endogenously-released polypeptide i n gastric acid secretion or i n s u l i n release under normal or pathological conditions.  Antisera to GIP have been obtained i n guinea pigs and rabbits after immunization with GIP conjugated to BSA.  GIP would not appear to be a good immunogen,  no s a t i s f a c t o r y antisera having been produced i n animals immunized with nonconjugated GIP, and even the conjugate has produced good antisera i n 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 p a r t i c u l a r antiserum.  When an  alternative antiserum, Ro7,.was introduced, the assay system had to be changed to the disequilibrium type to achieve a s a t i s f a c t o r y degree of label displacement, as shown i n F i g . 59.  -237-  When i t was revealed that IR-GIP d i d not exist i n a homogeneous'• form, the crossreactivity of the available antisera was examined with the. various heterologues of IR-GIP. immunoreactive  The o r i g i n a l antiserum, Van 8, crossreacted with 3 d i f f e r e n t  forms of GIP:  GIP^, G I P  5 0 0 Q  and ProGIP CFig. 72 and F i g . 73)  and antiserum GO 5 behaved i n a s i m i l a r fashion.  Antiserum Ro7 and antiserum  Van 8 crossreacted with GIP^QQQ i n the interassay control preparations to the same degree, but did not agree i n the measurement of ProGIP, as i l l u s t r a t e d i n F i g . 80. Antiserum Ro7 appeared to have.a greater a f f i n i t y f o r this form of IR-GIP than did antiserum Van 8.  The t o t a l IR-GIP response to any stimulus  consists of varying proportions of at least 3 components, as determined by these antisera.  I t i s obvious'that comparisons between IR-GIP responses to  s t i m u l i may only be made i f the studies are performed with antisera whose behaviour with the various IR-GIP heterologues i s also comparable, or better s t i l l , with the same antiserum throughout.  The usual technique used i n 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, p i l o t studies were performed, varying the concentration of chloramine-T and the length of the exposure time.  The s p e c i f i c  a c t i v i t y of the resulting l a b e l , 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 f o r 120 sec.  This compares well with the routine l a b e l ,  whose s p e c i f i c a c t i v i t y , calculated i n the same way, was 120 mCi/mg, (from the radiochromatogram  i n F i g . 21). An improved l a b e l i s l i k e l y to r e s u l t from  further studies with reduced concentrations of chloramine-T and varied periods of exposure.  I t should be noted that this method of calculating the s p e c i f i c  a c t i v i t y i s much less r e l i a b l e f o r GIP than i t was f o r m o t i l i n .  With m o t i l i n  there was l i t t l e difference i n the values obtained, whether they were calculated  -238-  from the radiochromatogram or from a separate assay, requiring the addition of l a b e l at several d i l u t i o n s .  This i s not the case with GIP.  The l a b e l  obtained i n the procedure i l l u s t r a t e d i n F i g . 21 had an apparent s p e c i f i c a c t i v i t y of 120 mCi/mg.  The same l a b e l , after a l a b e l d i l u t i o n assay, had  estimated s p e c i f i c a c t i v i t y of 68 mCi/mg. portion of the l a b e l l e d polypeptide  an  This indicates that a smaller pro-  eluting in the f i r s t peak represented  125 mono-iodinated  I-GIP.  The molecule has two t y r o s y l residues,  N-terminal, the other at position 10.  At present, there i s no way  one of knowing  which tyrosine i s the more available to the incorporation of iodine, or whether the t e r t i a r y structure of the molecule i s such that the N-terminal portion of the peptide i s indeed r e a d i l y accessible to the incorporation of iodine at a l l .  The lack of improvement i n the s p e c i f i c a c t i v i t y of the l a b e l  after the gentler lactoperoxidase  method of iodination would suggest that  neither tyrosine i s r e a d i l y accessible to iodine incorporation. P u r i f i c a t i o n of the l a b e l by gel f i l t r a t i o n on Sephadex G-25 iodinated polypeptide  w i l l separate the  from the unincorporated isotope but f a i l s to separate the  labelled and unlabelled peptides from each other, and i s barely adequate for separation of mono-iodinated GIP from the d i - and t r i - i o d i n a t e d forms. of uniodinated  GIP  i n the l a b e l l e d preparation  The presence  i s a contributing factor to the  low  s p e c i f i c a c t i v i t y , blunting the s e n s i t i v i t y of the upper end of the standard curve and l i m i t i n g the concentration  of l a b e l l e d 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 s p e c i f i c a c t i v i t y in the range 200-250 mCi/mg (estimated  by the l a b e l d i l u t i o n method).  This improve-  ment i s probably due to the removal of the unlabelled polypeptide, which elutes  -239-  ahead of the labelled material i n the system employed, (Fig. 22). The routine i n c l u s i o n of interassay controls:in the•assay have made i t easier to monitor the" performance  of the assay system and.acts as a r e l i a b l e Index for the estima-  tion of the .effect of variations: i n procedure on the s e n s i t i v i t y of the assay;  Antisera to porcine GIP, raised i n rabbits, were covalently coupled to Sepharose 4B and tested as an alternative to dextran-eoated charcoal i n the separation of bound antigen.from free antigen i n the incubation mixture.  The binding of the  antibodies to the agarose was complete, as indicated by.the lack of antibody a c t i v i t y i n the wash, (Fig. 19). The coupled antiserum, tested i n the radioimmunoassay, appeared to possess only a small percentage of the o r i g i n a l a n t i body a c t i v i t y .  This was indicated by.the reduction i n the t i t r e of the coupled  antiserum, required to produce a maximum binding of 30% of the labelled tracer, shown i n F i g . 46. The coupled antiserum at this lower t i t r e also showed a diminished s e n s i t i v i t y 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 a c t i v i t y , i n the radioimmunoassay system, was unaltered by the presence of the s o l i d matrix or the coupling process (Bolton and Hunter, 1973). The Sepharose-coupled  antiserum i n the radioimmunoassay f o r m o t i l i n , another  small polypeptide, also demonstrated no loss i n s e n s i t i v i t y p o t e n t i a l .  a high recovery of antibody a c t i v i t y and  The same was not true f o r i n s u l i n , human growth  hormone, human thyroid stimulating hormone or GIP. A l l these radioimmunoassay systems showed a loss i n antibody a c t i v i t y and s e n s i t i v i t y when the antibody was coupled d i r e c t l y 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 s t e r i c hindrance prevented the molecule  -240from having complete access to the binding s i t e s on the antibody. c r i t i c a l s i z e must l i e between 2700 (motilin) and 5105 coupling of GIP antisera to.Sepharose was  (GIP).  an uneconomical way  This  Direct of u t i l i s i n g  the antisera, and this technique has not been used i n studies on the extraction and p u r i f i c a t i o n of  GIP.  This problem may well be overcome by interposing a hydrocarbon chain between the ligand and the s o l i d matrix.  The use of such a hydrocarbon spacer has  dramatically improved the effectiveness of several Sepharose systems i n the p u r i f i c a t i o n of enzymes (Cuatrecasas,  1970), e s p e c i a l l y i n low a f f i n i t y 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 coupling of the ligand to the spacer by the carbodiimide  reaction.  1  The i n c -  rease i n distance between the s o l i d and the antibody reduces the s t e r i c 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 p o s s i b i l i t y of a coupled ligand for GIP for the following reasons:  The separation procedure i n the routine assay be-  comes more rapid, e n t a i l i n g no extra addition step, as i n the  dextran-charcoal  separation, and no further incubation, as i n the double antibody system. fugation for 5 min.  Centri-  at 2000 rpm i s adequate for packing the s o l i d , allowing the  supernatant liquor to be decanted.  The system i s unaffected by the plasma  concentration in,the normal radioimmunoassay, and i s • l e s s disruptive to the primary antigen/antibody.reaction  than charcoal addition.  Reduction of the  incubation volume to a minimum obviated the need f o r r o t a t i o n of the incubation tubes and therefore they do not require  stoppering.  -241With 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 i n h i b i t o r of g a s t r i c 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 respons i b l e f o r the i n h i b i t i o n of g a s t r i c acid secretion and the delay i n g a s t r i c emptying.  Pederson and Brown (1972) demonstrated that porcine GIP was e f f e c t i v e  i n dogs i n i n h i b i t i n g gastric acid secretion, whether that secretion was stimulated by infusion of pentagastrin or histamine, insulin-mediated hypoglycaemia).  or by vagal stimulation (induced by  In studies where the e x t r i n s i c a l l y denervated  fundic pouch was stimulated to produce ~75% of i t s maximum secretory capacity, a s i g n i f i c a n t degree of i n h i b i t i o n was observed at doses of 1 ug/kg/hour.  When serum samples, obtained from human volunteers breakfast, were subjected  a f t e r ingestion of a normal  to radioimmunoassay, they were found to r i s e from a  mean f a s t i n g l e v e l of 237 - 14 pg/ml IR-GIP (mean - SE) to a mean l e v e l of 1200 pg/ml IR-GIP, and they remained elevated above basal l e v e l s for periods i n excess of 3 hours, (Kuzio et a l , 1974).  When the various components of the meal  were tested i n d i v i d u a l l y , o r a l ingestion of both glucose (Cataland et a l , 1974) and f a t , i n the form o f a t r i g l y c e r i d e emulsion (Brown et a l , 1974) were found to produce a s i g n i f i c a n t elevation i n the c i r c u l a t i n g levels of serum IR-GIP. Ingestion of protein, i n the form of either a meat, extract or a fat-trimmed f i l e t steak, produced no such increase i n the c i r c u l a t i n g l e v e l s of IR-GIP (Brown et a l , . 1975).  When the o r i g i n a l studies were duplicated i n dogs, the c i r c u l a t i n g l e v e l s  of IR-GIP, achieved  during the exogenous infusion of GIP, s u f f i c i e n t to produce  a s i g n i f i c a n t i n h i b i t i o n of the g a s t r i c acid output, were determined to l i e within the range of serum IR-GIP l e v e l s released by ingestion of f a t .  Similar IR-GIP  responses were obtained i n dogs when the stimulus, "either fat or glucose, was  -242-  administered  as an intraduodenal infusion.  Confirmation of the i n h i b i t o r y action of endogenous GIP was in dogs.  obtained i n studies  Acid, stimulated by a continuous infusion of pentagastrin, was i n -  h i b i t e d by intraduodenal i n f u s i o n of f a t (Fig. 66), glucose c h l o r i c acid (Fig. 68) but not s a l i n e (Fig. 69). pouch acid secretion by fat or glucose was of serum IR-GIP.  (Fig- 67) or hydro-  The i n h i b i t i o n of fundic  accompanied by a concomitant elevation  There was no s i g n i f i c a n t change i n IR-GIP levels related to  the acid-induced i n h i b i t i o n , and intraduodenal perfusion with s a l i n e produced neither i n h i b i t i o n of g a s t r i c . a c i d secretion nor change i n the serum IR-GIP levels.  It would seem that GIP i s , i n strong l i k e l i h o o d , the enterogastrone  tulated by KOsaka and Um, evidence i n man,  according to the evidence obtained i n dogs.  pos-  The  regarding the i n h i b i t o r y r o l e played by GIP released by f a t  digestion, i s less strong. CTeatbr and-Gourlay(1975) found that exogenous GIP T  would i n h i b i t g a s t r i c acid secretion i n man  at a dose of 2 ug/kg/ 30 min. which  resulted i n c i r c u l a t i n g IR-GIP levels w e l l above those achieved by ingestion of fat i n the same subjects;  GIP does not appear to play any part i n the autoregulation of g a s t r i c 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 pentagastrinstimulated acid into the ^duodenum (Cleator and Gourlay, 1975).  The evidence supporting the claims of the other g a s t r o i n t e s t i n a l polypeptides be enterogastrone  has gradually been diminished.  to  Secretin i s not released i n any  s i g n i f i c a n t amounts by ingestion of f a t , and has'.been shown to have l i t t l e inhibi t o r y effect on the secretory or motor a c t i v i t y of the stomach when infused i n  -243-  doses which mimicked the c i r c u l a t i n g levels of IR- secretin achieved by duodenal a c i d i f i c a t i o n .  Cholecystokinin-pancreozymin  i s released by f a t , but  much of the i n h i b i t o r y a c t i v i t y reported as occurring after infusion of the polypeptide can be accounted for by the GIP contamination i n the impure preparation of cholecystokinin-pancreozymin used i n these studies. A t h i r d possible enterogastrone, VIP, i s a potent i n h i b i t o r of g a s t r i n - or histaminestimulated acid secretion, when i t i s 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 i n the humoral r e f l e x so designated.  The actual mecha-  nism of GIP release after ingestion of f a t remains to be elucidated.  The  reduced IR-GIP response to a test meal i n patients with coeliac disease would suggest that the rate of absorption of the nutrients i s an important factor. As yet, no s i g n i f i c a n t reduction i n the absolute number of GIP-producing  cells  has been detected i n biopsy samples from the jejunal mucosa of these subjects (Creutzfeldt et a l , 1976).  The search for the duodenal factor involved i n a second humoral r e f l e x dates back to the work of Moore et a l (1906).  They were able to r e l i e v e the glyco-  suria of patients with diabetes mellitus by o r a l l y administering an extract of the duodenal mucosa. secretin preparation. and was not i n s u l i n .  A hypoglycaemic  f r a c t i o n was  separated from a crude  It had no s e c r e t i n - l i k e effect on the exocrine pancreas This f r a c t i o n was named " i n c r e t i n " by La Barre (1932)  when he postulated a possible r o l e for t h i s duodenal factor i n the treatment of diabetes mellitus.  With the advent of the radioimmunoassay for plasma i n s u l i n (Yalow and 1958)  Berson,  i t became possible to compare the IR- i n s u l i n response to glucose adminis-  -244tered o r a l l y or intravenously ( E l r i c k et a l , 1964).  The greater i n s u l i n  response: and: improved glucose tolerance which '.accompanied: glucose  administra-  tion by the o r a l route was ..postulated to be due to the release of some i n s u l o 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".  I f a g a s t r o i n t e s t i n a l polypeptide i s to be seriously•considered as a  candidate  for the r o l e of i n c r e t i n i n this enteroinsular r e f l e x i t must s a t i s f y the following c r i t e r i a . i t s release.  I t must be demonstrated that glucose i s a stimulant f o r  It should be shown that exogenous administration of this poly-  peptide, i n doses achieving c i r c u l a t i n g immunoreactive levels within the physiological range, administered  i n p a r a l l e l with an  intravenous'glucose  load, w i l l mimic the pattern of serum IR- i n s u l i n release and glucose observed a f t e r oral or duodenal administration of glucose alone.  tolerance  In l i g h t  of these requirements mast of the established g a s t r o i n t e s t i n a l peptides have been ruled out as possible candidates.  Secretin and cholecystokinin-  pancreozymin are not released by ingestion of glucose i n p h y s i o l o g i c a l l y e f f e c t i v e l e v e l s , as demonstrated by the lack of effect on the exocrine pancreas (Mahler and Weisberg, 1968) whilst only a s l i g h t elevation i n serum IR- gastrin levels were observed (Rehfeld and S t a d i l , 1973). secretin (Lerner and Porte, 1972)  When g a s t r i n ,  or the synthetic octapeptide of cholecystokinin-  pancreozymin (Frame et a l , 1975) were infused intravenously, i n conjunction with intravenous'glucose,  a l l three peptides produced a t r a n s i t o r y , enhanced  i n s u l i n response, c h a r a c t e r i s t i c of the i n i t i a l phase of i n s u l i n relaase. response was continued.  over within 10-15  min. even when the polypeptide infusion was  The  -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 i n an;enhanced IR- i n s u l i n response to the glucose infusion,during both the i n i t i a l phase of i n s u l i n release and the l a t e r sustained phase.  This same dose of GIP, without the glucose, had no i n s u l i n o -  tropic action.  The levels of c i r c u l a t i n g IR-GIP reached during this infusion  were comparable to those achieved i n the same subjects a f t e r the ingestion of 50 g glucose.  The effect of endogenous GIP on i n s u l i n release i n man  reported by Brown et a l , CL975).  was  The IR- i n s u l i n response to intravenous  glucose was potentiated by GIP released after the ingestion of f a t , i n the form of a t r i g l y c e r i d e emulsion.  Although GIP- mediated i n s u l i n release has been demonstrated i n the fasted dog (Pederson et a l , 1975b) i t i s probable that the e f f e c t i v e levels of IR-GIP achieved were pharmacological rather than p h y s i o l o g i c a l . some degree of hyperglycaemia was were to be i n s u l i n o t r o p i c .  In  man,  e s s e n t i a l i f physiological levels of GIP  Studies i n the i s o l a t e d rat pancreas  prepa-  ration have confirmed that GIP i s capable of augmenting the sustained i n s u l i n response to glucose, i n a dose-related manner, (Pederson and Brown, 1976). Their findings suggest that GIP i s only e f f e c t i v e as an i n s u l i n o t r o p i c agent i n the presence of a glucose concentration which i s i t s e l f capable of stimulat i n g i n s u l i n release from the pancreas. GIP was  In the presence of 8.9 mMglucose,  e f f e c t i v e i n doses as low as 1 ng/ml perfusate and i n the presence  of a fixed GIP concentration, increasing glucose concentrations stimulated i n s u l i n release i n an exponential manner.  -246-  GIP has also been shown to potentiate the i n s u l i n response to glucose i n the i s o l a t e d rat pancreatic i s l e t preparation (.Schauderet a l , 1975) at glucose concentrations above a. threshold l e v e l , which lay between 6-8 mM. The e f f e c t i v e dose of GIP, however, was.lOyg/ml incubate at the lower glucose concentration, and 1 jag/ml at the higher concentrations.  The  reason for t h i s much greater GIP requirement.in t h i s preparation, compared to that of the isolated pancreas or the i n t a c t animal, i s not degradation of GIP during the incubation period.  I t i s possible that exposure of the  i s l e t s to collagenase and pancreatic p r o t e o l y t i c enzymes, during their i s o l a t i o n , causes some a l t e r a t i o n . t o t h e i r membrane structure and reduces their s e n s i t i v i t y to the action of GIP. An alternative explanation i s that GIP requires the presence of some intermediate f o r f u l l expression of i t s b i o l o g i c a l potency and this substance has been destroyed or l o s t i n the i s o l a t i o n procedure.  In both i n Vivo and i i i v i t r o systems GIP has s a t i s f i e d the  c r i t e r i a for i t s establishment as a major factor i n the entero-insular axis. T e l e o l o g i c a l l y , i t i s desirable that GIP should have no i n s u l i n o t r o p i c action i n the fasted animal.  I t would be inappropriate f o r i n s u l i n to be released  when serum glucose l e v e l s were not elevated.  In 1973, Raptis et a l reported that an intraduodenal infusion of a mixture of amino acids was a stimulant for i n s u l i n release, whilst intravenous i n fusion of these same acids was f a r less e f f e c t i v e .  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 l e v e l s achieved were much higher, (Thomas et a l , 1976). In t h i s s i t u a t i o n GIP was shown.to be i n s u l i n o t r o p i c i n the absence of measurable hyperglycemia  and the authors suggested  that GIF w i l l also act to enhance the  -247pancreatic i n s u l i n response i n the presence of hyperaminoacidaemia.  However,  they did not measure serum IR- gastrin:levels i n this study and i t i s possible that the gastrin response.to an amino acid i n f u s i o n would contribute to the i n s u l i n o t r o p i c response.observed.after protein  ingestion.  Evidence supporting the claims of GXP to be the major humoral factor i n both the enterogastrone and entero-ins^lar GIP released a f t e r ingestion  reflexes i s gradually accumulating.  IR-  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 r e l a t i n g the i n i t i a l response to  the glucose content of the.meal, correlating i t with the increase i n serum glucose and the period j u s t p r i o r to the peak response of IR- i n s u l i n .  The  second peak compares well with the IR-GIP response to o r a l l y administered f a t . The GIP released by either f a t or glucose appeared to be e f f e c t i v e as an enterogastrone or an i n s u l l n o t r o p i c agent.  However, i f the serum glucose and  IR-GIP levels achieved after o r a l glucose were duplicated  by an intravenous  glucose infusion with either intraduodenal f a t infusion or intravenous porcine GIP infusion, the IR- i n s u l i n response after f a t was s i g n i f i c a n t l y lower than that produced by either o r a l glucose or exogenous GIP, et a l , 1975B).  From the  feeleological  angle i t i s desirable  (Pederson  to have an immediate  i n s u l i n response to a carbohydrate-containing meal but i t would be most inappropriate f o r the gastric acid secretion  to be i n h i b i t e d this early i n the  digestion  of the meal.  In l i g h t of these findings i t seemed advisable to i n -  vestigate  the nature of the IR-GIP released after these different s t i m u l i .  The antiserum routinely used i n the GIP assay, Van 8,was unable to d i f f e r e n t i a t e  -248between  t h e serum IR-GIP  r e l e a s e d by f a t o r g l u c o s e  ( F i g . 71) b u t i f t h e s e  same serum samples were s u b j e c t e d . t o chromatography on Sephadex  G-50, t h r e e  r e g i o n s .of i m m u n o r e a c t i y i t y were d e t e c t e d i n t h e f r a c t i o n s o b t a i n e d , and F i g . 73)(Dryburgh and Brown, 1976).  The f i r s t  region  (IR-GIP  CFig-  72  ) eluted  i n t h e v o i d volume o f the.column and was s i g n i f i c a n t l y d i m i n i s h e d i f t h e serum was p r e t r e a t e d by b o i l i n g o r w i t h 6.OM u r e a . that G I P  v o  r e p r e s e n t e d a complex  to a serum p r o t e i n .  CFig'  77)  T h i s would suggest  formed by t h e n o n - s p e c i f i c b i n d i n g o f GIP  A second r e g i o n corresponded t o t h e e l u t i o n p a t t e r n o f  n a t u r a l p o r c i n e GIP C G I P ^ Q Q Q )  and a t h i r d  immunoreactive r e g i o n e l u t e d  ahead  o f t h e normal GIF and was determined t o have a m o l e c u l a r weight o f 7500-8000, as i l l u s t r a t e d  i n F i g . 83.  T h i s form o f IR-GIP was d e s i g n a t e d ProGIP.  attempt has y e t been made t o examine and G I P ^ Q Q Q i n f a s t i n g serum.  the r e l a t i v e r a t i o s o f G I P  r  GIP _ _ 5000 r  the p e r c e n t a g e o f G l P increased. reversed.  Still An a l l  v o  rt(  ProGIP  v q 5  The e a r l y GIP response t o e i t h e r g l u c o s e o r  f a t was c h a r a c t e r i z e d by t h e p r o p o r t i o n s o f t h e IR-GIP r e l a t i o n s h i p GIP > vo  No  >  ProGIP.  components  b e a r i n g the  As t h e s t i m u l a t i o n was c o n t i n u e d ,  remained r e l a t i v e l y  constant, w h i l s t  later,the r e l a t i v e proportions of 1 5 Q O O g  P  that o f G I P , - Q Q Q ProGIP were  s t u d i e s , i n man o r dog, a f t e r g l u c o s e o r f a t , t h e p e r c e n t a g e  o f t h e t o t a l IR-GIP  r e p r e s e n t e d by ProGIP i n c r e a s e d w i t h i n c r e a s e i n time a f t e r  the s t i m u l u s , as t y p i f i e d  i n F i g . 75.  Chromatography o f p a r t i a l l y p u r i f i e d e x t r a c t s from t h e duodenal mucosa o f hogs demonstrated t h a t they a l s o c o n t a i n e d IR-GIP i n t h e ProGIP and forms.  The h i g h e s t r a t i o o f ProGIP  methanol i n s o l u b l e e x t r a c t  ; GIP Q 5  0 0  GIP  was found i n t h e n e u t r a l  5 0 0 Q  soluble,  ( F i g . 7 8 ) . Attempts which have been made t o p u r i f y  ProGIP from t h i s e x t r a c t have n o t been s u c c e s s f u l .  Rechromatography o f  m a t e r i a l , supposedly. c o n t a i n i n g no G I P ' Q Q Q , r e s u l t e d i n a p p r o x i m a t e l y 3 0 % o f 5  t h e t o t a l IR-GIP.recovered b e i n g i n t h e G I P , - Q  0 0  form, as shown i n F i g . 8 2 ,  -249-  i n d i c a t i n g that ProGIP had y e t : t o b e . i s o l a t e d i n a stable form.  From i t s  behaviour on CM-cellulose, ProGIP was determined to be less basic than GIP^QQQ,  CFig. 80).  One might expect a functionally different/molecule  to be more stable than ProGIP has so: far proved to be. this molecule represents  a precursor form of  GIP^^QQ.  It i s possible  that  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  GIP^QQQ.  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^QQQ,  .diminished and increased precursor was released  the synthesis of GIP accelerated.  as  Biosynthesis studies, with t r i t i a t e d  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  GIP^QQQ  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 c e l l s , s p a t i a l l y separated. require that  GIP^QQQ  This hypothesis would  be synthesized and secreted by c e l l s predominantly  located i n the upper region of the duodenum, whilst the ProGIP c e l l s would be situated more d i s t a l l y .  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-QQQ-  containing c e l l s .  Later this mixture of nutrients would come into contact with the lower, ProGIPproducing c e l l s .  The b i o l o g i c a l potency of ProGIP, r e l a t i v e to that of G I P 5 0 0 Q  i s d i f f i c u l t to estimate i n view of the i n s t a b i l i t y of the material.  A second unknown factor i s the b i o l o g i c a l potency of that proportion of the t o t a l IR-GIP complexed to serum .protein. with other polypeptide hormones,, e . g . , i n s u l i n (Sramakova et a l , 1975).  This phenomenon has been demonstrated  gastrin Of alow and Berson,,1972) and  The function of this type of complex was  -250studied by Simon and Antonlades (1975).  They measured the transport of  i n s u l i n across the i s o l a t e d rat mesentery, i n the presence.of human serumbound i n s u l i n .  They foundsthat the i n s u l i n transport'was i n h i b i t e d i n a  s p e c i f i c manner.  One might extend this, finding to postulate that n a t u r a l l y -  occurring serum protein/polypeptide the polypeptide  complexes would l i m i t the transport of  across*the membrane of the target c e l l or reduce i t s a c t i v i t y  at that membrane by competitively binding with the receptor s i t e s thereon. The complex might also act by sequestering, peptide i n the serum.  temporarily, some of the poly-  "Big, b i g " gastrin was found to be a major component  of the t o t a l IR- gastrin in.the f a s t i n g serum of men, dogs and pigs. I t s 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 feeding.  acid response to  The existence of a s i m i l a r "big, b i g " i n s u l i n i n normal subjects i s  less well documented.  I t has, however, been demonstrated to comprise a large  percentage of the t o t a l IR- i n s u l i n i n the f a s t i n g and stimulated serum i n certain pathological conditions, (not insulinomas).  These patients have ex-  tremely high basal and stimulated IR- i n s u l i n l e v e l s but rarely experience hypoglycemic attacks a f t e r prolonged f a s t i n g or l i m i t e d food intake.  The  most common time for hypoglycaemia to occur i n these subjects was a few hours a f t e r a substantial meal.  Sramkova et a l (1975) postulated that these findings  could be accounted for i f the IR- i n s u l i n was predominantly i n an inactive form, the hypoglycaemic attacks being due to disruption of this complex and the l i b e r a t i o n of the b i o l o g i c a l l y active i n s u l i n a f t e r the stress of the large meal.  In the l i g h t o f these observations,  i t was postulated that  i s either b i o l o g i c a l l y inactive or .has reduced potency.  G  lP  v o  The b i o l o g i c a l a c t i v i t y  of GIP would not then correlate d i r e c t l y to the t o t a l IR-GIP response  measured.  Bearing this i n mind, the experiments performed by Pederson et a l , comparing  -251-  the IR- i n s u l i n response to matched serum glucose and IR-GIP l e v e l s , obtained by various means, were reconsidered. The peak .levels of IR-GIP and the Integrated i n s u l i n response achieved after intravenous glucose and porcine GIP were a r b i t r a r i l y considered.to be 100% of the possible response.  The  various IR-GIP components of the t o t a l IR-GIP response to o r a l fat were determined by column chromatography and were expressed as percentages of the t o t a l IR-GIP response;  The integrated IR- i n s u l i n response achieved after  intravenous glucose and o r a l f a t was expressed as a percentage of the integrated IR- i n s u l i n response obtained with intravenous glucose and GIP. these values were plotted in', histogram form ( F i g . 84), the closest to the b i o l o g i c a l a c t i v i t y ( i . e . , the i n s u l i n response) was bining the I R - G I P (Fig.  500Q  When correlation  obtained by com-  and IR-ProGIP, and ignoring the IR-GIP^ component.  84)  The existence of several molecular forms of IR-GIP cannot yet account for the apparently d i f f e r e a t  functions of GIP, i . e . , i t s i n i t i a l i n c r e t i n - l i k e  and the l a t e r enterogastrone e f f e c t .  effect  Another, as yet unexplained, phenomenon  i s the reduced IR-GIP response stimulated by o r a l f a t i n the presence of an i n t r a venous glucose infusion, et a l , 1976).  compared to that produced by o r a l f a t alone, (Crockett  The answer to these problems may  l i e i n a study of the other  humoral mechanisms acting at the same time, or i n the i d e n t i f i c a t i o n of some factor which i n h i b i t s the action of GIP at the l e v e l of either the pancreatic 3 c e l l s .  the p a r i e t a l or  The w e l l documented effect of intravenously, adminis-  tered .somatostatin on the pancreatic secretion of both i n s u l i n and glucagon i n Vivo, (Alberti et a l , 1973  : Mortimer et a l , 1974  i n v i t r o , (Gerich et a l , 1975)  stimulated interest  : Koerker et a l , 1974)  and  i n the possible effects of  somatostatin on the i n s u l i n response to GIP and on GIP release after physiolo-  -252-  c o  CL CO Q_ CD  ® 06  § s O CO Q- c  r  o C  CD  IR-Insulin  0  IR  -  GIP  5000  Fig. 84  H" > IR  GIP  vo  60H  40H  / / /  20H  L_  CD  IR-Pro-GIP  80H  O  CL  [[]]]  100 n  CO  or  [3  0-»  7 A  A.  .  B.  The i n s u l i n response and serum IR-GIP l e v e l s associated with that response, showing the r e l a t i v e proportions of the d i f f e r e n t heterologues of IR-GIP. The serum glucose and IR-GIP l e v e l s 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 o r a l f a t administration.  -253-  gical.stimulation.  Somatostatin was administered as a bolus i n j e c t i o n ,  immediately p r i o r to the exogenous administration of an intravenous GIP i n fusion, normally i n s u l i n o t r o p i c i n the fasted dog (Pederson et a l , 1975). The i n s u l i n response was delayed and the serum glucose values measured r e f l e c t e d t h i s i n s u l i n i n h i b i t i o n , as shown.in F i g . .63.  The e f f e c t of a bolus  i n j e c t i o n of the synthetic somatostatin was to delay.the release of IR-GIP, stimulated by either o r a l glucose or f a t .  In the case of the studies with  o r a l glucose the i n s u l i n response was also delayed ( F i g . 60 and F i g . 62). If the somatostatin was administered as an infusion, the IR-GIP and IRi n s u l i n 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 o r a l glucose alone.  The i n s u l i n response, however, r e -  bounded to values s i g n i f i c a n t l y above the control values, a phenomenon not explicable,in terms of the p r e v a i l i n g serum glucose levels ( F i g . 61). A s i m i l a r rebound response of i n s u l i n 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 i n h i b i t the action o f . c i r c u l a t i n g GIP.at the l e v e l of the c e l l .  The effect of somatostatin, on GIP- mediated gastric a c i d . i n h i b i t i o n has not been examined but:the presence of somatostatin-containlng c e l l s i n the gastric mucosa has been demonstrated by Dubois (1975).  I t would not be unexpected i f  intravenous infusion of somatostatin, was also found to have a. modulating luence on the response of the p a r i e t a l c e l l to endogenous GIP.  inf-  The/possibility  should be considered that the disparate actions of GIP on the gastric p a r i e t a l  -254and pancreatic 8 c e l l are being influenced separately by somatostatin of g a s t r i c and pancreatic o r i g i n respectively..  The stimulus.for, and the time  of release of the peptide from these d i f f e r e n t regions need not be i d e n t i c a l . No information i s available about the endogenous release of somatostatin at the present time.  . . . .  i.  The p o s s i b i l i t y that some i n h i b i t o r y feedback mechanism existed between the endocrine pancreas and GIP releasewjas^,. suggested by. the observation that subjects with maturity onset diabetes exhibited an abnormally elevated IRGIP response to o r a l glucose or f a t .  The i n h i b i t o r y factors implicated were  i n s u l i n , glucagon or the degree of hyperglycaemia  achieved.  When an i n s u l i n  i n j e c t i o n was administered to normal human volunteers, the serum glucose levels being clamped i n the fasting range, the IR-GIP response to f a t ingestion was s i g n i f i c a n t l y less than that observed i n the control s i t u a t i o n .  The time  course of the peak serum IR- i n s u l i n response did not, however, correlate well with the IR-GIP depression (Brown et a l , 1975).  I t would have been pre-  ferable i f the i n s u l i n had been given as an infusion rather than a bolus i n jection.  Ebert et a l (1976) infused glucagon intravenously for 2 hours during the i n gestion of a provocative test meal and recorded a s i g n i f i c a n t depression of the IR-GIP response.  This e f f e c t could not have been due to the i n s u l i n  released, there being no s i g n i f i c a n t difference in; the c i r c u l a t i n g IR- i n s u l i n 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 s i g n i f i c a n t l y higher levels of serum  -255-  glucose demonstrate an exaggerated GIP response to the same challenge.  In  normal subjects, the evidence so f a r accumulated, regarding endocrine pancrea t i c control of GIP release i s most persuasive with respect to glucagon. Creutzfeldt and Ebert (1976) have also confirmed  the i n h i b i t o r y effect of  somatostatin on GIP release and GIP-medlated response i n man.  Further information about the mechanism of the action of GIP and i t s control was  obtained from studies performed i n subjects with abnormal digestive  metabolism, or who IR-GIP response was  had undergone g a s t r o i n t e s t i n a l surgery.  The stimulated  s i g n i f i c a n t l y reduced i n patients with^coeliac disease,  with a concomitant reduction i n the IR- i n s u l i n response (Creutzfeldt et a l , 1976).  The most l i k e l y cause of the diminished GIP output i s 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 t h e i r IR-GIP response to glucose, administered with or without p h l o r i z i n .  The addition of the p h l o r i z i n v i r t u a l l y abolished the  release of IR-GIP.  An a l t e r n a t i v e explanation for the low GIP levels measured  i s the p o s s i b i l i t y that the absolute number of GIP-producing c e l l s has been reduced because of the v i l l o u s atrophy,  c h a r a c t e r i s t i c of coeliac disease.  The majority of the GIP c e l l s are found, however, i n the crypts of the i n t e s t i n a l mucosa and v i l l o u s atrophy would probably result i n only an i n s i g n i f i c a n t reduction i n the GIP c e l l population..  Exaggerated GIP responses to stimulation by a mixed meal were observed after any s u r g i c a l procedure which resulted i n 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 i n duodenal  -256-  ulcer patients (Creutzfeldt and Ebert, 1976). stimulated IR-GIP levels was  A s i m i l a r elevation i n  the general- rule i n chronic pancreatitics,  possibly due to the loss of some feedback control by glucagon or i n s u l i n (Botha et a l , 1976  : Ebert et a l , 1976).  The most marked IR-GIP response, however,  was observed i n patients with moderate hypoinsulinaemia, whereas those with severe i n s u l i n depression had an IR-GIP response which approached values.  the normal  This apparent anomaly i s probably due to a combination of factors,  a f f e c t i n g the release of GIP In different ways.  The lack of i n s u l i n would  lead to an overproduction of GIP but the associated gross exocrine pancreatic deficiencey would result i n an abnormal f a t metabolism,  leading to malabsor-  ption of f a t (with associated-steatorrhea) and a reduction i n the release of IR-GIP. The r e l a t i v e hyperglucagonaemia,  reportedly occurring i n severe cases  of chronic pancreatitis (Kalk et a l , 1974) could also contribute to the reduction i n 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 i n the levels of IR-GIP released a f t e r a p a r t i a l duodenopancreatectomy  would r e s u l t from the accelerated rate of g a s t r i c  emptying,  following s u r g i c a l interference.  The s i t u a t i o n , i n the cases of maturity onset diabetes (Brown et a l , 1975) obesity (Ebert et a l , 1977) i s s l i g h t l y d i f f e r e n t .  and  In these situations the  GIP response to a test meal i s abnormally high, i n the presence of r e l a t i v e l y high i n s u l i n l e v e l s .  The i n s u l i n deficiency may be regarded as functional  rather than absolute, the GI.P-producing c e l l being unresponsive to the i n h i b i t o r y action of that i n s u l i n .  The p o s s i b i l i t y exists, and should be i n v e s t i -  gated, that this lack of s e n s i t i v i t y . t o the i n s u l i n may be due to high percentage of the t o t a l IR-insulin measured being i n the form of the r e l a t i v e l y  -257-  inactive p r o i n s u l i n .  This .insensitivity would appear to be.reversible,  by sulphonylureas i n the diabetics or by diet-mediated weight  reduction  i n the obese subjects.  In a l l the previously mentioned abnormal conditions; only the IR-GIP levels were abnormal.  stimulated  In juvenile onset diabetes the f a s t i n g IR-GIP >.•  levels were i n excess of 1 ng/ml.  The  f a s t i n g IR-GIP l e v e l s in.the obese  subjects, after prolonged starvation, approach this value.  Both these s i t u a -  tions are characterized by i n s u l i n deficiency and a high l e v e l of c i r c u l a ting ketone bodies.  Treatment with i n s u l i n or food, respectively; reverses  both these symptoms and reduces the IR-GIP output.  The role of hyperketonaemia  i n the control of GIF release requires further investigation.  In summary, several factors have been implicated i n the regulation of production.  The response of the GIP  c e l l may  somatostatin or a reduction i n the absorption  GIP  be depressed by glucagon, i n s u l i n , of nutrients.  The IR-GIP  output would appear to be increased by s u r g i c a l disruption of g a s t r o i n t e s t i n a l continuity, absolute or functional deficiency i n i n s u l i n or glucagon and possibly by the presence of elevated l e v e l s of ketone bodies i n the c i r c u l a t i o n .  I f the GIP release i s being i n h i b i t e d by some endocrine pancreatic  factor,  either i n s u l i n or glucagon, t h i s could explain the reduced IR-GIP response to o r a l f a t i n the presence of an intravenous glucose infusion, r e l a t i v e to that response observed when fat was a t i c stimulation.  administered alone and there was  no pancre-  I t could also account for the fact that the IR-GIP response  to an intraduodenal infusion of glucose.is not only delayed but i s also diminished when i t i s preceded by a somatostatin infusion.  As shown i n F i g . 62,  the IR-insulin response rebounded to l e v e l s s i g n i f i c a n t l y above the control  -258-  values at the termination of the somatostatin infusion and this could be responsible for the reduction i n the' (SIP response.  It i s : i n t e r e s t i n g to  speculate whether hyperinsulinaemia or hyperglucagonaemia might not only effect an:inhibition of IR-GIP release but also i n h i b i t the a c t i v i t y of circulating'GIP at the l e v e l of the 8 c e l l (cf somatostatin).  Another facet of the role played by.GIP i n carbohydrate metabolism i s i l l u s trated i n obesity.  The observation that complete starvation or a low c a l o r i c  diet resulted i n an eventual decrease i n both the IR-GIP and IR- i n s u l i n responses i n obese individuals suggested that carbohydrate intake played a r o l e i n regulation of the s e n s i t i v i t y of the GIF- producing c e l l .  It  is  also possible that the number of GIP c e l l s i s controlled by the nature of the diet.  I f this i s so, one might question i f any pathological condition,  characterized by malabsorption of nutrients (e.g., coeliac disease) might not also result i n a reduction of the absolute number of GIP-  producing  c e l l s , which would contribute to the reduction i n IR-GIP output observed i n this circumstance . (see page 243).  GIP alone was  found to have a weak l i p o l y t i c effect on i s o l a t e d rat adipocytes,  but GIP, administered i n conjunction with glucagon, was antilipolytic.  found to be strongly  I t 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 s i t e s on the adipocyte plasma membrane by the addition of GIP to the incubate (Dupre et a l , 1976 1977).  GIP was,  : Ebert and Brown,  however, much less e f f e c t i v e i n antagonizing the l i p o l y t i c  action of secretin, and was  i n e f f e c t i v e against a d r e n o c o r t i c o t r o p i c hormone  (ACTR), adrenaline, noradrenaline and theophylline.  Obesity i s a pathological  condition of many aetiologies, but an impaired carbohydrate metabolism,  -259-  r e s u l t i n g : i n or from an increased.carbohydrate intake, i s l i k e l y to produce obesity d i r e c t l y v i a the elevated GIP.'response and i n d i r e c t l y v i a the elevated i n s u l i n response induced by GIP.  The status of GIP as a hormone i s established.  Its predominant  role would  appear to be that of the major g a s t r o i n t e s t i n a l regulator of carbohydrate metabolism.  This i s supported by the alteredTR-r-GIP response measured i n  c l i n i c a l conditions'related to impaired carbohydrate metabolism.  GIP also  plays a part i n c o n t r o l l i n g the secretory and motor a c t i v i t y of the stomach i n dogs and probably i n man, i n hypersecretory  but the c l i n i c a l evidence f o r GIP involvement  conditions, e.g. duodenal ulcer and Z o l l i n g e r - E l l i s o n  syndrome,ofrthe'?hy.posecretory states, e.g., Werner-Morrison achlorhydria, i s v i r t u a l l y non-existent.  In a l l the pathological conditions  so f a r investigated, abnormal GIP responses are symptomatic, causative, of the disorder.  syndrome and .  rather than  -260BIMQGRAPHY.  1.  A l b e r t i , K.G.M., Christensen, S.E., Tversen, J . , Sever-Hansen, K., Christensen, N.J., Prange Hansen, A., Lundbaek, K., Ovsrov, H. i n s u l i n secretion by somatostatin.  2.  (1973).  Inhibition of  Lancet 11 : 1299-1301  Andersson, S., Nilsson, G., Uvn'as, B.  (1967).  secretory responses to gastrin and histamine.  Effect of acid on gastric 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).  mechanism of action of parathyroid hormone.  Structure, synthesis and Rec.Prog.Horm.  Res. 2&.  :  353-398 4.  j5.  Barbezat, G.O., Grossman, M.I.  (1971).  I n t e s t i n a l secretion : stimulation  by peptides.  Science 174 : 422-424  La Barre, J .  (1932).  l'incretine.  B u l l e t i n de l'Academie Royale de Medicine de Belgique (Bruxelles)  Sur les p o s s i b i l i t i e s d'un traitment du diabete par  12 : 620-634 6.  Berson, S.A., Yalow, R.S.  (1958).  Isotropic tracers i n the study of diabetes.  Adv. B i o l . Med.Phys. 6 : 349-430 i  7.  Berson, S.A., Yalow, R.S. (1968). hormone i n plasma.  8.  Immunochemical heterogeneity of parathyroid  J.Clin.Endocrinol. Metab. 28 : 1037-1047  Berson, S.A., Yalow, R.S.  (1971).  Nature of immunoreactive  from tissues of the g a s t r o i n t e s t i n a l t r a c t . 9.  Bloom, S.R., Polak, J.M., Pearse, A.G.E. . and watery- diarrhoea syndrome.  10.  gastrin extracted  Gastroenterology j>0 : 215-222  (1973).  Vasoactive i n t e s t i n a l peptide  Lancet 2_ : 14-16  Bloom, S.R. Mortimer, C H . Thorner, M.O., Besser, G.M., Coy, CM., H a l l , R., Gomez-Pan,.A., Roy, V.TC., Russell, R.C.G., Coy, D.H., Kastin, A.J., Schally, A.V.  (1974).  I n h i b i t i o n of gastrin and g a s t r i c acid secretion by growth-  hormone release i n h i b i t o r y hormone.  Lancet j i i : 1106-1109  -261Bloom, S.R.  (1975).  Radioimmunoassay f o r s e c r e t i n .  In " G a s t r o i n t e s t i n a l  Hormones" (ed. J.C. Thomson. U n i v e r s i t y o f Texas P r e s s . ) , Bodansky, M.  (1974).  G a s t r o i n t e s t i n a l hormones : F a m i l i e s o f o l i g o e l e c t r o -  l y t e s . . . I n " E n d o c r i n o l o g y o f the Gut", Slack Inc.)  pps.  B o l t o n , A.E.,  pps. 257-268  (eds. W.Y.  Chey and F.P. B r o o k s ,  3-13  H u n t e r , W.M.  (1973).  The use o f a n t i s e r a c o v a l e n t l y c o u p l e d  t o agarose, c e l l u l o s e and Sephadex i n RIA systems f o r p r o t e i n s and Biochem. B i o p h y s . A c t a 329  i n chronic pancreatitis.  G u i l l e m i n , R.  o f immunoreactive  zation  p i t u i t a r y growth hormone.  Johnson, L.P., Magee, D.F.  (1967).  duodenal e x t r a c t s . Brown, J . C ,  L i n g , N., B u t c h e r , M.,  Rivier, J.,  Hypothalamic p o l y p e p t i d e t h a t i n h i b i t s the s e c r e t i o n  on g a s t r i c m o t i l i t y .  Brown, J.C.  (1976) The e f f e c t o f m o t i l i n on the  Ann.Roy.Coll.Surg.Canada 9^ : 39  Burgus, R.,  (1973).  Gastric i n h i b i t o r y polypeptide  J . C l i n . E n d o c r i n o l . Metab. hl_ : 791-797  sphincter.  B r a z e a u , P., V a l e , W.,  Brown, J . C ,  (1976).  M e i s s n e r , A . J . , Zwick, R.  l o w e r oesophageal  haptens.  : 318-330  B o t h a , J.L. V i n i k , A . I . , Brown, J.C.  Bowes, K.C.,  CB.  S c i e n c e 179  (1966).  : 77-79  E f f e c t o f duodenal  G a s t r o e n t e r o l o g y 50  alkalini-  : 333-339  P r e s e n c e o f a g a s t r i c motor s t i m u l a t i n g p r o p e r t y i n G a s t r o e n t e r o l o g y 5_2_ : 225-229  Parkes, CO.  (1967).  E f f e c t on f u n d i c pouch motor a c t i v i t y o f  s t i m u l a t o r y and i n h i b i t o r y f r a c t i o n s s e p a r a t e d from pancreozymin.  Gastro-  e n t e r o l o g y _53 : 731-736 Brown, J . C ,  Harper, A.A.,  S c r a t c h a r d , T.  s t i m u l a t i o n o f the p a n c r e a s .  (1967a).  P o t e n t a t i o n of s e c r e t i n  J . P h y s i o l . . 1 9 0 : 519-530  Brown, J.C., 'J^hn_son,.-.L.?P., Magee,HD,. F. £ (1967b-),;. :  The"'^inhibition o f induced  motor I - a c t i y i t y ^ f i n 'transplanted f u n d i c pouches,,.' J.PhysioL., 188 ,: 45-52  -262Brown, J . C , Pederson, R.A., Jorpes, J.E., Mutt, V. (1969).  The preparation  of a highly active enterogastrone.  Can.J.Physiol. Pharmacol.  Brown, J.C. Pederson, R.A. (1970).  A multiparameter  47 : 113-114  study on the actions of  preparations containing cholecystokinin-pancreozymin.  Scand.J.Gastroenterology  5 : 537-541  Brown, J . C , Mutt, V., Pederson, R.A. polypeptide demonstrating Brown, J.C.  (1971).  (1970).  enterogastrone  Further p u r i f i c a t i o n of a  activity.  J.Physiol. 209  : 57-64  A gastric inhibitory polypeptide. I. The amino acid  composition and t r y p t i c peptides Brown, J . C , Dryburgh, J.R.  Can. J . Biochem. 49_ : 255-261  (1971).  The further p u r i f i c a t i o n of m o t i l i n ,  a gastric motor a c t i v i t y stimulating polypeptide from the mucosa of the small intestine of hogs.  Can. J.Physiol Pharmacol. 4£ : 399-405  Brown, J . C , Mutt, V., Dryburgh, J.R. II.  (1971).  The complete amino acid sequence.  Brown, J . C , Cook, M.A., Dryburgh, J.R.  A g a s t r i c i n h i b i t o r y polypeptide,  Can.J.Biochem. 49_ : 867-872  (1972).  M o t i l i n , a gastric motor  a c t i v i t y polypeptide : f i n a l p u r i f i c a t i o n , amino acid composition and Cterminal residues.  Gastroenterology  62 : 401-404  Brown, J . C , Cook, M.A., Dryburgh, J.R.  (1973).  M o t i l i n , a gastric motor  a c t i v i t y stimulating polypeptide : the complete amino acid sequence.  Can.J.  Biochem. 51 : 533-537 Brown, J . C , Dryburgh, J.R., Pederson, R.A. peptide (GIP).  (1974).  In "Eridocririolggy of the gut"  Gastric inhibitory poly-  (eds. W.Y. Chey and F.P. Brooks,  C.B..Slack, Inc.) pps. 76-82  Brown, J . C , Dryburgh, J . R . Ross, S'.A., Dupre, J . (1975)'. actions of gastric i n h i b i t o r y polypeptide.  Bruton, C J . Hartley, B.S. from E.Coli. J.Molec.Biol..52  (1970).  I d e n t i f i c a t i o n and  Rec.Prog.Horm.Res. 31•••: 487-532  Chemical studies on methionyl-RNA synthetase  : 165-178  -263Buchanan, K.D., Vance, J.E., Aoki, T., Williams, R.H.  (1967).  Rise i n  serum immunoreactive glucagon a f t e r i n t r a j e j u n a l glucose i n pancreatectomized dogs.  Proc.Soc.Biol. Med. 126 : 813-815  Canterbury,  J.M., Reis, E.  (1972).  Multiple immunoreactive molecular forms  of parathyroid hormone i n human serum.  C a s t e l l , D.O., Levine, S.M.  (1971).  Proc.Soc.Exptl.Biol.Med.  140 : 1393-1398  LES response to gastric a l k a l i n i z a t i o n .  A new method for the treatment of heartburn with antacids.  Ann.Int.Med. _74 :  223-227  Cataland, S., Crockett, S.E., Brown, J . C , Mazzaferri, L. i n h i b i t o r y polypeptide.  (1974).  Gastric  Stimulation by o r a l glucose i n man. J.Clin.Endocrinol.  Metab. 39 : 223-228  Chisholm, D.J., Young, J.D., Lazarus, L. stimulus to i n s u l i n release.  (1969).  The gastrointestinal  J.Clin.Invest. 4j5 : 1453-1460  Cleator, I.CM., Gourlay, R.H.  (1975).  Release of immunoreactive gastric i n -  h i b i t o r y polypeptide (IR-GIP) by o r a l ingestion of substances.  Am.J.Surg. 130 :  128-135.  Cohn, D.V., MacGregor, R.R., Chu, L.L.H., Kimmel, J.R., Hamilton, J.W., Calcaemic  (1972).  f r a c t i o n - A : Biosynthetic peptide precursor of parathyroid hormone.  Proc.Nat.Acad.Sci. Cook, M.A. (1972).  (USA) 69 : 1521-1525 Ph.D. Thesis, University of B.C.  Creutzfeldt, W., Ebert, R. (1976).  Release of gastric inhibitory polypeptide  to a test meal under normal and pathological conditions i n man. Internatl.Diabeti Fed.Congress, New Delhi, India. (Excerpta Medica).  In press  Creutzfeldt, W., Ebert, R., Arnold, R., Frerichs, H., Brown, J.C.  (1976).  Gastric i n h i b i t o r y polypeptide CGIP), gastrin. and i n s u l i n : Response to ..test meal i n coeliac disease and a f t e r duodenopancreatectomy.  Djabetologia 12 :  279-286  Crockett, S.E., Cataland, S., Falko, J.M., Mazzaferri, E.L. J . Clin.Endocrinol.Metab. 42 : 1098-  (1976)  -264Crowshaw, K. , Jessup, S., Ramwell, P.W.  (1967).  Thin layer  chromatography  of l-dimethyl-aminonaphthalene-5-sulphonyl derivatives of amino acids present i n superfusates of cat cerebral cortex.  Biochem.J. 103 : 79-85  Cuatrecasas, P., Wilchek, M.j Anfinsen, C B . p u r i f i c a t i o n by a f f i n i t y chromatography. Cuatrecasas, P. (1970).  (1968).  Selective enzyme  Biochem. 61 : 636-643  P u r i f i c a t i o n by a f f i n i t y chromatography.  tives of agarose and polyacrylamide beads.  J.Biol.Chem. 245 : 3059-3065  Debas, H.T., Csendes, A., Walsh, J.H., Grossman, M.I. (1974). antral gastrin.  In "Endocrinology of the gut"  Brooks, C B . Slack, Inc.)  Deriva-  Release of  (eds. W.Y. Chey and F.P.  pps. 222-232  Debas, H.T., Yamagishi, T., Dryburgh, J.R. gastric emptying of l i q u i d s i n dogs.  (1977).  M o t i l i n enhances  Gastroenterology (in press)  Dockray, G.J., Debas, H.T., Walsh, J.H., Grossman, M.I. (1975). forms of gastrin i n antral mucosa and serum of dogs.  Molecular  Proc.Soc.Exptl.Biol.  Med. 149 : 550-553 Domschke, W., Strunz, U., Mitznegg, P., Domschke, S., Wunsch, E., Demling, L. (1976). 11  M o t i l i n and m o t i l i n analogues : mode of action. Scand. J.Gastroenterology  (Supp.39) : 25-28  Dryburgh, J.R., Brown, J . C  (1975).  Radioimmunoassay for m o t i l i n .  Gastroen-  terology 68 : 1169-1176 Dryburgh, J.R., Brown, J.C. (1976).  Immunoreactive forms of gastric i n h i b i t o r y  polypeptide i n serum and tissue. Can.Physiol. 1_ : 28 Dubois, M.P.,  (1975).  Immunoreactive somatostatin i s present i n discrete  c e l l s of the endocrine pancreas.  Proc.Nat.Acad.Sci. (USA) 72-': 1340-1343  Dubois, P.M., Paulin, C., Assan, R., Dubois,"M.P.  (1975).  Evidence f o r  immunoreactive somatostatin i n the endocrine c e l l s of the human foetal pancreas. Nature 256 : 731-732  -265Dupre, J .  (1964).  An i n t e s t i n a l hormone a f f e c t i n g glucose disposal i n man.  Lancet 2_ : 672-673 Dupre, J . , Rojas,.L., White, J . J . , Unger, R.H., Beck, J . C ,  (1966).  Effects of secretin on i n s u l i n and glucagon i n p o r t a l and peripheral blood i n man.  Lancet 2^ :  26-27  Dupre, J . , Beck, J . C  (1966).  extract of i n t e s t i n a l mucosa.  Stimulation of release of i n s u l i n by an Diabetes 15_ : 555-559  Dupre, J . , C u r t i s , D.J., Unger, R.H., Waddell, R.W.,  Beck, J.C. (1969).  Effects of secretin, pancreozymin or gastrin on the response of the endocrine pancreas to administration of glucose or arginine i n man.  J.Clin.Invest. 4J3 ;  745-757 Dupre, J . , Ross, S.A., Watson, D., Brown, J.C.  (1973).  Stimulation of  i n s u l i n secretion by g a s t r i c i n h i b i t o r y polypeptide i n man.  J.Clin.Endocrinol.  Metab.J7 : 826-828 Dupre, J . , Greenidge, T.J., McDonald, T.J., Ross, S.A., Rubinstein, D. (1976). I n h i b i t i o n of actions of glucagon i n adipocytes by g a s t r i c i n h i b i t o r y  polypeptide.  Diabetologia 12: Ebert, R., Willms. B., Brown, J . C , Creutzfeldt, W. (1976). i n h i b i t o r y polypeptide  (GIP) l e v e l s i n obese subj.ects and after weight reduction  Edman, P.  (1956).  peptides.  Acta.Chem.Scand.  Eisen, H.N.,  Serum g a s t r i c  On the mechanism of phenylisothiocyanate  Siskind, G.W.  degradation of  10 : 761-768 (1964).  during the immune response.  E l l i s o n , E.H., Wilson, S.D.  Variations i n a f f i n i t i e s of antibodies  Biochem. _3 : 996-1008  (1967).  Further observations on factors influencing  the symptomatology manifest by patients with Z o l l i n g e r - E l l i s o n syndrome. "Gastric Secretion" (eds. T.K. Shnitka, J.A.L. G i l b e r t , and R.C York, Pergamon)  pps 363-369  In  Harrison, New  -26666.  Elrick,,U.,Stimmler, L . , Hiad, C . J . , A r a i , Y. (1964). to o r a l and intravenous  glucose administration.  Plasma i n s u l i n responses  J . C l i n . E n d o c r i n o l . 24 :  1076-  1082 67.  Farrell, J.I.,  Ivy, A . C .  gastric pouch. 68.  (1926).  Am.J.Physiol.  Feldman, H . , Rodbard, D.  Studies on the m o t i l i t y of the transplanted  76 : 227-228  (1971).  Mathematical theory of radioimmunoassay.  In " P r i n c i p l e s of Competitive Protein-Binding Assays" W.H. Daughaday, 69.  Feyrter, F. Menschen.  70.  J.B.  L i p p i n c o t t , Co.)  (1953).  (eds.  pps 158-173  Uber die peripheren endokrinen (parakrinin)  Drusen des  W.Maundrich,.Wien-Dusseldorf  Forssman, W.G., Yanahaira, N . , Helmstaedter,  V . , Grube, D. (1976).  demonstration of the m o t i l i n c e l l and the enterochromaffin Gastroenterology 71.  W.D. Odell and  11 (Supp.39)  cell.  Differential Scand.J.  43-45.  Frame, C M . , Davidson, M . B . , Sturdevant, R . A . L .  (1975).  Effects of the  octapeptide of cholecystokinin on i n s u l i n and glucagon secretion i n the dog. Endocrinology 97 : 549-553 72.  Gerich, J . E . ,  Lovinger, R., Grodsky, M. (1975).  I n h i b i t i o n by somatostatin of  glucagon and i n s u l i n release from the perfused rat pancreas i n response to arginine,  isoproterenol  on glucagon secretion. 73.  effect  Endocrinology 96 : 749-754  Goodfriend, T . L . Levine, L . , Fasman, G.D. (1964). and angiotension  74.  and theophylline : evidence for a preferential  : a use of carbodiimides  Gorden, P.,Sherman, B . M . , Roth, J .  Antibodies to bradykinin  in immunology.  (1971).  Science 144  : 1344-1346  P r o i n s u l i n - l i k e components of  c i r c u l a t i n g i n s u l i n i n the basal state and i n patients with i s l e t c e l l tumours. J.Clin.Invest.50 75.  : 2113-2122: :  Gorden, P . , Sherman, B . M . , Sinopoulos, with hypokalaemia component.  A.P.  (1974).  Glucose  intolerance  : an increased proportion of c i r c u l a t i n g p r o i n s u l i n - l i k e  J.Clin.Endocrinol.Metab. 34  : 235-240  -26776.  Gray, W.R. (1967).  Sequential degradation plus dansylation.  In "Methods  i n Enzymolbgy" XI (ed. C.H.W. Hirs. Academic Press, New York) 77.  Gregory, R.A., Tracy, H.J.  (1964).  The constitution and properties of  two gastrins extracted from hog antral mucosa.  Gross, E., Witkop, B. (1961).  I. The i s o l a t i o n of two  Gut j> : 103-104  gastrins from hog antral mucosa.  78.  Selective cleavage of the methionyl peptide  bonds i n ribonuclease with cyanogen bromide. J.Am.Chem.Soc.  79.  Gross, E., Witkop, B. (1962).  83 : 1510-1511  Non-enzymatic cleavage of peptide bonds :  the methionine residues of bovine pancreatic ribonuclease. 237  p.469  J.Biol.Chem.  1856-1860  80.  Grossman, M.I.  (1974).  Candidate hormones of the gut.  !  .67 : 730-755  81.  Habener, J.F., Powell, D., Murray, T.M., Mayer, G.P., Potts, J.T. Parathyroid hormone : Secretion and metabolism i n vivo. (USA)  82.  83.  Gastroenterology  (1971).  Proc.Soc.Acad. S c i .  68 : 2986-2991  Hales,- C.N., Randle, P.J. (1963).  Immunoassay of i n s u l i n with i n s u l i n -  antibody p r e c i p i t a t e .  j58_ : 137-146  Biochem. J .  H a l l , R.j Besser, G.M., Schally, A.V., Coy, D.H., Evered, D., Goldie, D.J., Kastin, A.J., McNeilly, A.S., Mortimer, C.H., Phenekos, C , Tunbtoidge, W.M.G., Weightman, D. (1973). Lancet  ' 84.  Sept.15  Action of GHRIH i n healthy men and i n acromegaly.  : 581-584  Hedqvist,„P., von Euler, U£S (1975).  Influence of substance P on the response  of guinea pig ileum to transmural nerve stimulation.  Acta Physiol. Scand.  9_5 :  341-343  85.  Hellemans, J . , Vantrappen, G., Bloom, S .R. (1976).  Endogenous m o t i l i n and the  lower oesophageal sphincter pressure. .'Scahd.J.Gastroenterology 11 (supp 39) : 69-73  -26886.  Hoelzel, F. (1925).  The r e l a t i o n between the secretory and motor a c t i v i t y i n  the f a s t i n g stomach (man). 87.  AmJ.Physiol.7_3:  463-469  HUkfelt, T., K e l l e r t h , J.O., Nilsson, G., Pernow, B. (1975).  Substance P :  Localization i n the central nervous system and i n some primary sensory neurons. Science 190 : 889-890 88.  HBkfelt, T., Elde, R., Johansson, 0., Luft, R., Nilsson, G., Arimura, A. (1976).  Immunohistochemical  evidence for separate populations of somato-  statin-containing and substance P-containing primary afferent neurons i n the rat. 89. '  Neuroscience 1 : 131-136.  Holohan, K.N.-, Murphy, R.F., Flanagan, R.W.J., Buchanan, K.D., (1973).  Elmore,  D.T.  Enzymic iodination of the h i s t i d y l residue of secretin : a radio-  immunoassay of the hormone.  Biochem.Biophys.Acta  322  : 178-180 131  90.  Hunter, W.M.,  Greenwood, F.C. (1963).  Preparation of  growth hormone of high s p e c i f i c a c t i v i t y . ^ Biochem.J.89_ 91.  : 114-123  • Itoh, Z., Honda, R., Hiwatashi, K., Takenchi, S., Aizawa, I., Takayanagi, Couch,.E.F. (1976). tary t r a c t .  92.  I - l a b e l l e d human  R.,  Motilin-induced mechanical a c t i v i t y i n the caniite alimen-  Scand. J.Gastroenterology 1_1 (supp 39) : 93-110  Jennewein, H.M.,  Hummett, H., Siewert, R., Waldeck, F. (1975).  The motor-  stimulating effect of natural m o t i l i n on the lower oesophageal sphincter, fundus, antrum and duodenum i n dogs. 93.  Johns, E.W.  (1967).  Digestion _L3 : 246-250  The electrophoresis of histones i n polyacrylamide gel  and their quantitative determination. Biochem. J . 104 94.  Johnson, L.R.,  Guthrie, P.  (1974).  of the trophic action o f . g a s t r i n .  : 78-82  Mucosal DNA synthesis : a short.term index GAstroenterology 6T  : 453-459  ~\  95.  Johnson, L.R.  (1976). _The.._trophic action of g a s t r o i n t e s t i n a l hormones.  Gastroenterology 70 : 278-288  j  -269Johnson, L.R.,  Guthrie, P.  (1976).  The effect of CCK and 16, 16-dimethyl  prostoglandin E2 on RNA and DNA of gastric and duodenal mucosa. Gastroenterology 70 : 59-65  Kalk, W.J.,  Vinik, A.I., Bank, S., Hayes, J.R., A r d i l l , J . , Buchanan,  K e l l e r , P.,  Jackson, W.P.U. (1974).  chronic p a n c r e a t i t i s .  Kemmler, W.,  Kline, M.M.,  Plasma gastrin responses to arginine i n  Diabetes 23 : 264-267  Peterson, J.D., Steiner, D.F.  of proinsulin to i n s u l i n . peptidase.  K.D.,  1.  (1971).  Studies on the conversion  Conversion i n v i t r o  with trypsin and carboxy-  J.Biol.Chem.246:: 6786-6791  McCallum, R.W.,  Curry, N., Sturdevant, R.A.L. (1975).  Effect of  gastric a l k a l i n i z a t i o n on lower oesophageal sphincter pressure and serum gastrin. Gastroenterology J>8 : 1137-1139  Klotz, I.M.  (1967).  Succinylation.  In "Methods i n Enzymology" XI (ed.  C.H.W. Hirs. Academic Press, New York) p. 576  Koerker, D.J. Ruch,.W., Chideckol, E., Palmer, J . , Goodman, C.J. J . , Gale, C.C. pancreas.  (1974).  Somatostatin : hypothalamic i n h i b i t o r of the endocrine  Science 184 : 482-484  Kosaka, T., Lim, R.K.S. (1930).  On the mechanism of the i n h i b i t i o n of gastric  secretion by f a t . The r o l e of b i l e and cystokinin. Kuzio, M.D.  (1973).  M.Sc.  Ch.J.Physiol.IV : 213-220.  Thesis, University of B.C.  Kuzio, M., Dryburgh, J.R., Malloy, K.M.  Brown, J.C. (1974).  for gastric inhibitory polypeptide. Gastroenterolgoy 66 :  Lees, F., Grandjean, L.C. (1968). patients with p a r t i a l gastrectomy.  Lerner, R.L., Porte D. glucose i n  Ensinck.  Radioimmunoassay 357-364  The gastric and jejunal mucosae i n healthy Arch.Int.Med. 101;  (1972)Acute:and steady  nonobese diabetic subjects.  ;  9436-9451  state i n s u l i n responses to  J.Clin.Invest. 51 : 1624-1631  -270107.  Luft, R. Effendic, S., HBkfelt, T., Johansson, 0., Arimura, A Immunohistochemical  (.1974).  evidence f o r the l o c a l i z a t i o n of somatostatin-like  immunoreactivity i n a c e l l population of the pancreatic i s l e t s .  Med.Biol. 52  : 428-430 108.  McCallum, R.W.,  Kline, M.M.,  Sturdevant, R.A.L., Dryburgh, J.R.  (1977).  Studies on the mechanism of lower oesophageal sphincter pressure response to a l k a l i ingestion. 109.  Submitted.  Mclntyre, N., Holdsworth, CD.,  Turner, D.S.,  (1965).  I n t e s t i n a l factors  i n the control of i n s u l i n secretion. J.Clin.Endocrinol.Metab. 25 : 1217-1224  110.  Mahler, R.J. Weisberg, H. (1968).  Failure of endogenous stimulation of secretin  and pancreozymin release to influence serum i n s u l i n . 111.  Mainz, D.L.,  Black, 0., Webster, P.D.,  a t i c growth. 112.  Hormonal influences on pancre-  Gastroenterology 64 : Abs. 83  Mako, M.E. Block, M., (1973).  (1973).  Lancet 1 : 448-451  Starr, J. , Nielson, D., Friedman, E., Rubenstein,  A.H.,  P r o i n s u l i n i n chronic renal f a i l u r e and hepatic f a i l u r e : a r e f l e c t i o n  of the r e l a t i v e contribution of the l i v e r and the kidney to i t s metabolism. Clin.Res. 21 : 631  113.  Melani, F., Rubenstein, A.H., J.Clin.Invest. 49  114.  Melrose, A.G.,  (1970).  Russell, R.I., Dick, A.  Mitznegg, P., Bloom, S.R.,  (1964).  Christofides, N., Besterman, H., Domschke,  Scand. J . Gastroenterology 11  Miyachi,  W.,  Release of motilin i n man.  (supp 39) : 53-56  T a i t u i k a l t i s , J.L., Nieschlag, E., L i p s e t t , M.B.  Enzymatic radioiodination of gonadotropins. 23-28  Gastric mucosal structure  Gut _5 : 546-549  Domschke, S., Wlinsch, E., Demling, L. (1976).  116.  Human serum p r o i n s u l i n .  : 497-507  and function after vagotomy. 115.  Steiner, D.F.  (1972).  J.Clin.Endocrinol .'Metab.34  :  -271-  117.  Moore, B., Edie, E.S., Abram, J.H. C1906).  On the treatment of diabetes  mellitus by acid extract of duodenal mucous membrane.  Biochem. J . 1 '• 28-  38.  118.  Mortimer, C.H.,  Tunbridge, W.M.G., Carr, D., Yeomans, L., Lind, T.,  D.H.,  Bloom, S.R.,  Kastin, A., Mallinson, C.N.,  A.V.,  H a l l , R. (1974).  Besser, G.M.,  Coy,  Schally,  Effects of growth-hormone release i n h i b i t i n g -  hormone on c i r c u l a t i n g glucagon i n s u l i n and growth hormone i n normal, diabetic, acromegalic and hypo-pituitary patients, Lancet 1_ : 697-701  119.  Murphy, R.F., Buchanan, K.D.,  Elmore, D.T.  (1973).  Isolation of gluca-  gon l i k e immunoreactivity of. gut by a f f i n i t y chromatography antibodies coupled to Sepharsoe 4B. 120.  Narahara, H.T.  (1972).  Biochim, Biophys Acta 303  B i o l o g i c a l a c t i v i t y of p r o i n s u l i n .  Action" (ed. I. F r i t z . Academic Press, New York)  121.  122.  O'Dorisio, T.M.,  123.  In "Insul  (1971).  in  The structure  J.Biol.Chem.246 : 2780-2795.  Sirinek, K.R.,  Vasoactive i n t e s t i n a l peptide: nervous tissues.  : 118-127  pps 63.  Nolan, C. Margoliash, E., Peterson, J.D., Steiner, D.F. of bovine p r o i n s u l i n .  on antiglucagon  Mazzaferri, E.L., Cataland, S. (1976). i t s presence i n the vagus nerve and other  1st Int. Symposium of GI Hormones, ( i n press).  Palkovits, M., Brownstein, M.J., Arimura, A., Sato, E., Schally, Kizer, J.S. (1976).  A.V.,  Somatostatin content dif the hypothalamic ventromedial  and arcuate n u c l e i and the circumventricular organs i n the r a t . Brain Res. 109 : 430-434.  124.  Pearse, A.G.E. (1968).  Common cytochemical and u l t r a s t r u c t u r a l c h a r a c t e r i s t i c s  of c e l l s producing polypeptide hormones (The APUD Series) and t h e i r relevance to thyroid and ultimobranchial C c e l l s and c a l c i t o n i n .  Proc.Roy.Soc.B. 170 :  71-80.  125.  Pearse, A.G.E., Coulling, I., Weavers, B., Friesen, S. (1970). polypeptide c e l l s of the human stomach, duodenum and jejunum.  The endocrine Gut 11 : 649-658  -272-  126.  Pearse, A.G.E.  CL974)  .  Cytochemical and ultrastructural* characteristics of  c e l l s producing polypeptide hormones and t h e i r relevance to gut hormones. In "Endocrinology of the Gut" (ed. W.Y.  Chey and E.P. Brooks.  C.B. Slack, Inc.)  pps. 24-34 127.  Pearse, A.G.E., Polak, J.M., Bloom, S.R., Adams, C., Dryburgh, J.R., Brown, J.C. (1974).  Enterochromaffin c e l l s of the mammalian small intestine as the source  of motilin. 128.  Virchows Arch.B.  Pederson, R.A., Brown, J.C. (1972). insulin-stimulated peptide.  129.  Celi.Path.  16  : 111-120  Inhibition of histamine-pentagastrin-, and  canine gastric secretion by pure g a s t r i c i n h i b i t o r y poly-  Gastroenterology 62_  : 393-400  Pederson, R.A., Dryburgh, J.R., Brown, J.C. (1975a).  The effect of somato-  s t a t i n on release and i n s u l i n o t r o p i c action of g a s t r i c i n h i b i t o r y polypeptide. Can.J.Physiol.Pharmacol. 53 : 1200-1205  130.  131.  Pederson, R.A., Schubert, H.E., Brown, J.C.  (1975b).  action of GIP, Can.J.Physiol.Pharmacol. _53  : 217-223  Pederson,,R.A., Brown; J.C. (1976) The i n s u l i n o t r o p i c action of GIP i n the perfused isolated rat pancreas.  132.  Permutt, M.A., Kipnis, D.M.  Can.J.Physiol.Pharmacol. _5_3 : 217-223  (1972a) Insulin biosynthesis.  mechanism of glucose stimulation. 133.  The i n s u l i n o t r o p i c  Permutt, M.A., Kipnis, D.M.  1.  J.Biol.Chem. 247 : 1194-1199  (1972b).  Insulin biosynthesis.  glucose on ribonucleic acid synthesis i n i s o l a t e d rat i s l e t s . ,247 134.  On the  2.  Effect of  J.Biol.Chem.  : 1200-1207  Polak, J.M., Bloom, S.R., Coulling, I., Pearse, A.G.E. (1971).  Immunofluores-  cent l o c a l i z a t i o n ..of entefoglucagon c e l l s i n , the gastrointestinal tract of the dog.  135.  Gut 12  :  311-318  Polak, J.M., Bloom, S.R., Kuzio, M., Brown, J . C , Pearse," A.G.E. ,0973). C e l l u l a r l o c a l i z a t i o n of GIP i n the duodenum and jejunum.'  136  Porath, J . , Flodin, P.  (1959)  Gek f i l t r a t i o n :  Gut 14  : 284-288.  a method f o r desalting  -273group separation. 137.  Nature 183 : 1657-1659  Raptis, S., Dollinger, "M.,  Schleyer, 6. (1973).  Differences i n i n s u l i n ,  growth hormone and pancreatic enzyme secretion a f t e r intravenous and i n t r a duodenal administration of mixed amino acids i n man.  New  Eng. J.Med. 288 :  119-120 138.  Rehfeld, J.F. (1972).  Three components of gastrin inihuman sera.  Gel  f i l t r a t i o n studies on the molecular size of immunoreactive serum gastrin. Biochim. Biophys. Acta 285 : 365-372 139.  Rehfeld, J.F., S t a d i l , F. (1972). Lancet 2 :  140.  "Big" gastrin i n the ZE syndrome.  1200  Rehfeld, J.F., S t a d i l , F. (1973a). gastrin i n serum from Z-E patients.  141.  Rehfeld, J.F., S t a d i l , F. (1973b). stimulated i n s u l i n secretion i n man.  142.  Gel f i l t r a t i o n studies on immunoreactive Gut  14_ :  The effect of gastrin on basal and J.Clin.Invest. 5J2 : 1415-1426  Rehfeld, J.F., S t a d i l , F., Malmstr^m J . (1975). serum and tissue :  a progress report.  Reinke, D.A.  Rosenbaum, A.H.,  Bennett, D.R.  144.  Am.J.Dig.Dis.12  Riprdan, J.F., Vallee, B.L.  (1967).  pps 43-58  (1969).  g a s t r o i n t e s t i n a l c o n t r a c t i l e a c t i v i t y monitored force transducers.  Gastrin heterogeneity i n  In "Gastrointestinal Hormones"  (ed. J.C. Thompson, University of Texas Press.) 143.  369-373  Patterns of dog  i n vivo  with extraluminal  : 113-141 Acetylation. In "Methods i n Enzymology"  XI (ed. C.H.W. Hirs. Academic Press, New York,)pps 565 145  R8sch, W.,Lux, G. Domschke, S.,Domschke, W., Demling, L. (1976).  Wuhsch, E., Jaeger, E.,  Effect of 13-norleu-motilin on lower oesophageal  sphincter pressure (LESP) iii mam  Gastroenterology 70  : 931  -274146.  Rubenstein, A.H., Mako, M.E.,  Starr, J . J . , Juhn, D.J., Horwitz, D.L.  C i r c u l a t i n g p r o i n s u l i n i n patients with i s l e t c e l l tumours. Eighth Congress of Internatl. Diabetes Fed." Ecerpta Medica.) 147.  In '^Pfoc. of  (ed. W.J. Malaisse. Amsterdam,  pps 736  Ruppin, H., Domschke, S., Domschke, W., (1975).  (1974).  Wlinsch, E., Jaeger, E., Demling, L.  Effects of 13-norleu-motilin i n man.  t i o n and stimulation of pepsin secretion.  I n h i b i t i o n of gastric evacua-  Scand. J . Gastroenterology 10  : 199-  202 148.  Said, S., Mutt, V. (1970).  Polypeptide with broad b i o l o g i c a l a c t i v i t y ;  i s o l a t i o n from small i n t e s t i n e . 149.  Said, S., Rosenberg, R.N.,  Science 169 : 1217-1218  (1976).  Vasoactive i n t e s t i n a l peptide:  immunoreactivity i n neural c e l l lines and normal nervous tissue.  abundant  Science  192 : 907 - 908 150.  Samols, E., Tyler, J . , Marri, G., Marks, V. (1965). glucagon secretion by o r a l glucose.  151.  Immunochemical glucagon  Lancet i i : 727  Sasaki, H., Ito-h, M., Hebitani, I., Sako, K., Unger, R.H. pancreatic glucagon. J.JapDiabetes Soc.  153.  : 1257  Samols, E., Tyler, J . , Megyesi, C., Marks, V. (1966). i n human pancreas, gut and plasma.  152.  Lancet i i  Stimulation of  Scatchard G. (1949)  JL8  (1975).  Extra-  (supp 94)  The a t t r a c t i o n of proteins f o r small molecules and ions.  Ann. N.Y. Acad. S c i . 51 : 660-672 154.  Schauder, P., Brown, J . C , Frerichs, H., Creutzfeldt, W.  (1975).  Gastric  i n h i b i t o r y polypeptide : e f f e c t on glucose-induced i n s u l i n release from isolated r a t pancreatic i s l e t s i n v i t r o .  155.  Schubert, H.E., porcine m o t i l i n .  Brown, J.C; (1974)..  Diabetologia 11  : 483-484  Correction to the,amino acid sequence of  Can. J. B jo chem •• 5 2 r 7-8' •  -275156.  S c h u b e r t , E., M i t z n e g g , P.,  S t r u n z , ' U . , Domschke, W.,  J a e g e r , E., Demling, L., Helm, F. C1975).  Domschke, S., Wttnsch,  I n f l u e n c e o f t h e hormone analogue,  1 3 - n o r l e u - m o t i l i n and o f l - m e t h y l - 3 - I s o b u t y i x a n t h i n e on t h e tone and 3'5*-AMP c o n t e n t o f a n t r a l and duodenal muscle i n t h e r a b b i t . 16 157.  :  Segre, G.V.,  Habener, J . F . , P o w e l l , D.,  biological implications. Shay, H., i n man.  Life Sci.  T r e g e a r , W.,  J.Clin.Invest.  Simon, J.D.,  :  (1972). and  51 : 3163-3172  Experimental studies i n g a s t r i c physiology  I I . A study of p y l o r i c c o n t r o l . 58  P o t t s , J.T.  Immunochemical c h a r a c t e r i z a t i o n  Gershpn-Cohen, J . (1934).  (Synec. O b s t e t . 159.  cyclic  263-272  P a r a t h y r o i d hormone i n human plasma.  158.  E.,  The r o l e s o f a c i d and a l k a l i .  Surg.  935-955  A n t o n i a d e s , H.N.  i s o l a t e d r a t mesentery.  (1975).  T r a n s p o r t o f p e p t i d e hormones a c r o s s  E f f e c t o f human serum-bound i n s u l i n .  D i a b e t e s __4  :  997-1004 160.  S o l c i a , E., P e a r s e , A.G.E., Grube, D., C r e u t z f e l d t , W.,  Gepts, W.  (1973).  K u b a y a s h i , S., B u s s o l a t i ,  C e l l u l a r l o c a l i z a t i o n of g a s t r i c  p o l y p e p t i d e i n t h e duodenum and jejunum. 161.  Sramkova, J . , Pav. J . , E n g e l b e r t h , 0. serum immunoreactive  G.,  Gut 14  (1975).  i n s u l i n i n monoclonal  inhibitory  284-288  Inordinately high l e v e l s of  immunoglobulinaemia.  D i a b e t e s 2A_  :  214-224 162.  S t a r l i n g , E.H., body.  163.  On t h e c h e m i c a l c o r r e l a t i o n ' o f the f u n c t i o n s o f t h e  Lancet _2 : 339-341  S t e i n e r , D.F., insulin.  164.  (1905).  (1967).  Trans N.Y.  Evidence f o r a p r e c u r s o r i n the b i o s y n t h e s i s of  Acad. S c i . Ser I I 30:  60-68  S t e i n e r , D . F . , ' C l a r k , ' J . L . , N o l a n , C , R u b e n s t e i n , A.R., A t e n , B.  Oyer, P.E.  (1969). P r o i n s u l i n ^ a n d t h e b i o s y n t h e s i s o f i n s u l i n .  "Recent P r o g r e s s i n Hormone;]Research: '. (ed. E.B. 25  pps  207  M a r g o l i a s h , E.,  Atwood, .Acadmic P r e s s , New  In York)  -276165.  Steiner, D.F.,  Clark, J.L., Nolan, C., Rubenstein, A.H., .Marggliash, E.,  Melani, F., Oyer, P.E.  (1970).  The biosynthesis of i n s u l i n and some specula-  tions regarding the pathogenesis of human diabetes.  Iri"The Pathogenesis of  Diabetes M e l l i t u s " - Proc. XIII Nobel Symposium, (eds. Almqvist and Wiksell, Stockholm.) 166.  pps  123.  Strunz, U., Domschke, W.,  Domschke, S., Mitznegg, P.,  E., Demling, L.  Potentiation between 13-norleu-motilin and acetylcho-  (1976).  l i n e on rabbit p y l o r i c muscle i n v i t r o .  Wlinsch, E., Jaeger,  Scand J . Gastroenterology 11 (supp.39)  : 29-33 167.  Tager, H.S.,  Endin, S.O.,  Clark, J.L., Steiner, D.F.,  conversion of p r o i n s u l i n to i n s u l i n .  II.  (1973).  Studies on the  Evidence f o r a chymotrypsin-like  cleavage i n the connecting peptide region of i n s u l i n precursors i n the r a t . J.Biol.Chem. 248: 3476-3482 168.  Thomas, J.E., Crider, J.O.,  Morgan, C.J., (1934).  A study of reflexes  involving the p y l o r i c sphincter and antrum and t h e i r r o l e i n gastric evacuation. Am.J.Physiol. 169*  108  : 683-700  Thomas, J.E., (1941).  An improved cannula for g a s t r i c and i n t e s t i n a l f i s t u l a s .  Proc.Soc.Exptl.Biol.Med. 46: 260-261 170.  Thomas, F.B. Mazzaferri, E.L. Crockett, S.E., Mekhjian, H.S., Cataland, S. (1976).  171.  H.D.,  Stimulation of secretion of g a s t r i c i n h i b i t o r y poly-  peptide and i n s u l i n by intraduodenal amino acid perfusion. 70  Gruemer,  Gastroenterology  : 523-527  Unger, R.H.,  Ohneda, A., Valverde, I., Eisentraut, A.M.,  Exton, J.E.  (1968).  Characterization of the responses of c i r c u l a t i n g glucagon-like immunoreactiv i t y to intraduodenal and Intravenous administration:of glucose. J.Clin.Invest, 47 172.  : 48-65  Valverde, I., Rigopoulou, D., Exton, J . , Ohneda, A., Eisentraut, Unger, R.H.  &968).  Demonstration and characterization of a second factor of_  glucagon-like immunoreactivity  173.  A.M.,  i n jejunal extracts.  Vanderhaeghen, J . J . , Signeau, J . C ,  Gepts, W.  (1975).  brate CNS reacting with antigastrin antibodies.  Am.J.Med.Sci. 255  New  Nature 257  : 415-420  peptide i n verte: 604-605  -277-  4.  Ward, A.S., Bloom, S.R.  (1974).  The r o l e of secretin  g a s t r i c secretion by intraduodenal acid. 5.  Weisbodt, N.W.,  Wiley, T.H.,  i n the i n h i b i t i o n of  Gut 15 : 889-897  Overholt, B.F., Bass, P.  (1969).  A relation  between gastroduodenal muscle contractions and g a s t r i c emptying.  Gut 10  :  543-548 176.  Wingate, D. (1976). t i n a l hormones.  177.  Woods, K.R.,  The eupeptide system : a general theory of gastrointes-  Lancet 1 : 529-532  Wang, K.T.,  (1967).  Separation of dansyl-amino acids by poly-  ;rylamide layer chromatography.  Biochim. Biophys. Acta 133:  aci  178.  Wttnsch, E., Brown, J.C. Diemer, K-H.,  Drees, F., Jaeger, E., Musiol, J . ,  SJcharf, R. Stocker, H., Thamm, P., Wendelberger, of Norleucine-13-motilin. Z. Naturforsch. 28. 179.  Yalow, R.S.,  Berson, S.A.  (1970).  369-371  :  G. (1973).  The t o t a l synthesis  235-240  Size and charge d i s t i n c t i o n s between  endogenous human plasma gastrin i n peripheral blfiod and heptadecapeptide gastrins. 180.  Gastroenterology \58  Yalow, R.S.,  Berson, S.A.  (1971).  c t i v e gastrin i n human plasma. 181.  Yalow, R.S., Berson, S.A. Res. Commun. 48  182.  Yalow, R.S.  : 609-615 Further studies on the nature of immunorea-  Gastroenterology 60 : 2?03-214  (1972).  And now "big, b i g " gastrin.  Biochim. Biophys  : 391-395  (1974).  the Gut" (eds. W.Y.  Gastrins : small, b i g and big-big. Chey and F.P. Brooks, C B .  In "Endocrinology of  Slack, Inc.)  pps 261-276  

Cite

Citation Scheme:

        

Citations by CSL (citeproc-js)

Usage Statistics

Share

Embed

Customize your widget with the following options, then copy and paste the code below into the HTML of your page to embed this item in your website.
                        
                            <div id="ubcOpenCollectionsWidgetDisplay">
                            <script id="ubcOpenCollectionsWidget"
                            src="{[{embed.src}]}"
                            data-item="{[{embed.item}]}"
                            data-collection="{[{embed.collection}]}"
                            data-metadata="{[{embed.showMetadata}]}"
                            data-width="{[{embed.width}]}"
                            async >
                            </script>
                            </div>
                        
                    
IIIF logo Our image viewer uses the IIIF 2.0 standard. To load this item in other compatible viewers, use this url:
http://iiif.library.ubc.ca/presentation/dsp.831.1-0094195/manifest

Comment

Related Items