THE ISOLATION AND PHYSIOLOGICAL ACTIONS

OF

GASTRIC INHIBITORY POLYPEPTIDE

BY

RAYMOND ARNOLD PEDERSON

B.ED.; UNIVERSITY OF CALGARY, 1964

a, I

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 JUNE, 1971 In presenting this thesis in partial fulfilment of the requirements for

an advanced degree at the University of British Columbia, I agree that

the Library shall make it freely available for reference and study.

I further agree that permission for extensive copying of this thesis

for scholarly purposes may be granted by the Head of my Department or

by his representatives. It is understood that copying or publication

of this thesis for financial gain shall not be allowed without my

written permission.

Department of ~TA.y&j ¥

The University of British Columbia Vancouver 8, Canada

Date 3^6/?/ ABSTRACT

IT IS KNOWN THAT HUMORAL MECHANISMS FOR THE INHIBITION

OF GASTRIC SECRETION AND MOTOR ACTIVITY OPERATE FROM THE

DUODENUM. THE GASTROINTESTINAL HORMONES CHOLECYSTOKININ-

PANCREOZYMIN (CCK-PZ) AND SECRETIN HAVE BEEN IMPLICATED AS

THE HORMONES RELEASED IN SOME OF THESE PROPOSED MECHANISMS.

DESPITE THE FACT THAT CCK-PZ AND SECRETIN ARE PROBABLY

INVOLVED IN GASTRIC INHIBITORY MECHANISMS, THEY FALL SHORT

OF FUL P LL L IN G THE ORIGINAL DEFINITION OF E N T E R 0 G A S T R 0 N E AS

THE INHIBITORY PRINCIPLE RELEASED FROM THE DUODENUM BY THE

PRESENCE THERE OF FAT.

IMPURE PREPARATIONS OF CCK-PZ ARE KNOWN TO STIMULATE

H+ SECRETION IN THE DOG WHEN GIVEN ALONE AND TO INHIBIT

H+ SECRETION STIMULATED BY GASTRIN. INITIAL STUDIES IN

- THIS THESIS COMPARING THE EFFECTS OF 2 PURITIES OF CCK-PZ

ON GASTRIC SECRETION PROVIDED EVIDENCE FOR THE EXISTENCE OF

A GASTRIC INHIBITOR DISTINCT FROM CCK-PZ AND SECRETIN IN

PARTIALLY PURIFIED CCK-PZ PREPARATIONS. IT WAS CONCLUDED

FROM THESE STUDIES THAT IN PARTIALLY PURIFYING CCK-PZ, AN

INHIBITORY MATERIAL COULD HAVE BEEN REMOVED. THIS CONCLU•

SION WAS SUPPORTED BY THE FINDING THAT A SIDE FRACTION FROM

THE PURIFICATION OF CCK-PZ, WITH NO SIGNIFICANT CCK OR

SECRETIN ACTIVITY, POSSESSED POTENT INHIBITORY ACTIVITY

FOR GASTRIN PENTAPEPTIDE-STIMULATED H+ AND PEPSIN SECRETION

IN THE DOG. THE SIDE FRACTION REFERRED TO IN THIS THESIS

AS EG STAGE I BECAME THE STARTING POINT FOR SEVERAL PURIFI•

CATION PROCEDURES. PURIFICATION OF THE INHIBITORY MATERIAL

I I Ml

LEO TO THE DISCOVERY THAT IT WAS A POLYPEPTIDE DISTINCT IN

ITS CHEMICAL FEATURES FROM CCK-PZ, NOTABLY BY THE ABSENCE OF

THE AMINO ACID PROLINE.

THE PURE INHIBITORY MATERIAL, GASTRIC INHIBITORY POLY•

PEPTIDE (G.I.P.), WHEN AVAILABLE, WAS SHOWN TO POSSESS NO

SECRET IN ACT 1V ITY, NEGLIGIBLE CCK ACTIVITY, AND TO HAVE NO

PYROGEN IC OR VAS0DEPRESSOR EFFECTS. G.I.P. WAS USED IN

STUDIES TO DETERMINE ITS POTENCY AS AN INHIBITOR OF GASTRIC

SECRET I ON AND MOTOR ACTIVITY IN THE DOG. STUDIES WERE

CARRIED OUT IN WHICH.G.I.P. WAS SHOWN TO BE A HIGHLY POTENT

INHIBITOR OF H+ AND PEPSIN SECRETION FROM BlCKEL POUCHES AND

MOTOR ACTIVITY FROM VAGALLY DENERVATED ANTRAL POUCHES

STIMULATED BY GASTRIN PENTAPEPTIDE AND THE WHOLE GASTRIN

MOLECULE. ANOTHER OBJECTIVE WAS TO DETERMINE IF G.I.P.

WAS EFFECTIVE AS AN INHIBITOR OF GASTRIC SECRETION AGAINST

STIMULANTS THAT WERE RESISTANT TO INHIBITION BY CCK-PZ AND

SECRETIN, E.G. HISTAMINE. RESULTS OF THESE EXPERIMENTS

PROVED G.I.P. TO BE ABOUT HALF AS EFFECTIVE AN INHIBITOR

OF H+, PEPSIN AND FUNDIC MOTOR ACTIVITY DURING HISTAMINE

INFUSION AS COMPARED WITH EXPERIMENTS IN WHICH GASTRIN

PENTAPEPTIDE WAS THE STIMULANT. THE RANGE OF EFFECTIVENESS

OF G.I.P. AS A GASTRIC INHIBITOR WAS EXTENDED TO VAGALLY—

STIMULATED H+ AND PEPSIN SECRETION WITH POTENCY OF

INHIBITION EQUALLING THAT FOUND IN HISTAMINE STUDIES.

AS A RESULT OF STRUCTURAL SIMILARITIES BETWEEN G.I.P.

AND GLUCAGON, STUDIES WERE CARRIED OUT TO DETERMINE IF

G.I.P. MIMICKED THE HYPERGLYCEMIC OR PANCREATIC INHIBITORY I V

ACTIONS KNOWN TO BE POSSESSED BY GLUCAGON. THIS WAS FOUND

NOT TO BE THE CASE. IN ADDITION TO INHIBITING STIMULATED

GASTRIC SECRETION AND MOTOR ACTIVITY, G.I.P. WAS SHOWN TO

INHIBIT FUNDIC POUCH H+ SECRETION, PEPSIN SECRETION AND

MOTOR ACTIVITY IN THE UNSTIMULATED CONDITION.

IT IS CONCLUDED THAT SINCE G.I.P. FULFILLS THE PHYSIO•

LOGICAL REQUIREMENTS AS AN EFFICIENT GASTRIC INHIBITOR AND

MIMICS THE ACTIONS OF FAT IN THE DUODENUM, IT IS AN EXCEL•

LENT CANDIDATE FOR THE HUMORAL AGENT RELEASED FROM THE

DUODENUM BY FAT AND PERHAPS BY HYDROCHLORIC ACID AND HYPER•

TONIC SOLUTIONS* HOWEVER, RESERVATIONS MUST BE HELD AS TO

ITS STATUS AS A HORMONE UNTIL IT IS DETECTED IN BLOOD AND

TISSUES. TABLE OF CONTENTS

PAGE

ABSTRACT . . . I I

LIST OF TABLES vi I I

LIST OF FIGURES xi

ACKNOWLEDGEMENTS xiv

INTRODUCTION .... 1

METHODS 18 OPERATIVE PROCEDURES 18 I CHRON ICDOGS 18 I I ACUTE CATS 21 III ACUTE GUINEA PIGS 22 IV ACUTE RABBITS 23 COLLECTION AND ANALYSES OF SAMPLES 23 I COLLECTION OF GASTRIC SECRETION .... 23 A. BICKEL POUCH COLLECTION - DOG .... 23 B. GASTRIC REMNANT COLLECTION - DOG . . 24 C. WHOLE STOMACH COLLECTION - CAT ... 24 D. PERFUSION OF ANTRAL POUCH ANO MEASUREMENT OF ANTRAL MOTOR ACTIVITY IN THE DOG 24 E. MEASUREMENT OF FUNOIC POUCH MOTOR ACTIVITY IN THE DOG 26 F. MEASUREMENT OF GALL BLADDER ACTIVITY IN THE DOG 26 I I ANALYSES OF SAMPLES 27 A. GASTRIC SECRETION 27 A) ESTIMATION OF H+ CONCENTRATION . . 27 B) ESTIMATION OF PEPSIN ...... 27 B. PANCREATIC SECRETION ...... 28 A) VOLUME 28 B) PROTEIN OUTPUT 28 C. ESTIMATION OF BLOOD GLUCOSE ..... 28 ASSAY METHODS 28 I ASSAY OF CHOLECYSTOKININ ACTIVITY IN GASTRIC INHIBITORY PREPARATIONS .... 28 II ASSAY OF ENTEROGASTRONE ACTIVITY .... 32 III ASSAY OF SECRETIN ACTIVITY • • 32 IV A. ASSAY FOR BLOOO PRESSURE CHANGES • • 34 B. ASSAY FOR PYROGENIC EFFECTS 34 SOURCE OF HORMONE PREPARATIONS 34

ANALYSIS OF DATA 36

v V I

PAGE

RESULTS 38 I MULTIPARAMETER STUDY ON THE EFFECTS OF THE INTRAVENOUS INFUSION OF TWO PREPARATIONS CONTAINING CHOLECYSTOKININ- PANCREOZYMIN (CCK-PZ) 38 A. THE EFFECT OF CCK-PZ ON INTRA-GALL BLADDER PRESSURE 39 B. THE EFFECT OF CCK-PZ ON ANTRAL MOTOR ACTI VI TY 39 C. THE EFFECT OF CCK-PZ ON PEPSIN OUTPUT FROM BlCKEL POUCHES ...... 39 D. THE EFFECT OF CCK-PZ ON H+ SECRETION FROM BlCKEL POUCHES . 44 II COMPARISON OF TWO PREPARATIONS CONTAIN• ING CCK-PZ IN TERMS OF GASTRIC SECRETORY INHIBITION 44 III PREPARATION, ASSAY AND GASTRIC INHIBITORY ACTIONS OF EG STAGE I 51 A. EFFECTS OF EGI ON GASTRIC SECRETION AND MOTOR ACITVITY . . 57 A) BICKEL POUCH H+ SECRETION . ... 57 B) BICKEL POUCH PEPSIN SECRETION . . 60 B. EFFECTS OF EGI (G25) ON GASTRIC SECRETION AND MOTOR ACTIVITY STIMU• LATED BY ENDOGENOUS GASTRIN .... 64 A) BICKEL POUCH H"*" SECRETION .... 64 B) BICKEL POUCH PEPSIN SECRETION . . 64 c) ANTRAL MOTOR ACTIVITY ...... 64 IV PREPARATION AND ASSAY OF EG STAGE II • 69 V PURIFICATION OF EG STAGE II TO EG STAGE Ml 73 VI EFFECT OF PURE GASTRIC INHIBITORY POLY• PEPTIDE ON BICKEL POUCH ACID AND PEPSIN SECRETION AND ANTRAL MOTILITY STIMU• LATED BY GASTRIN PENTAPEPTIDE 73 A) BICKEL POUCH H+ SECRETION .... 74 B) BICKEL POUCH PEPSIN SECRETION . . 74 c) ANTRAL MOTOR ACTIVITY 81

VII EFFECT OF GASTRIC INHIBITORY POLYPEP• TIDE ON GASTRIC SECRETION AND MOTOR ACTIVITY STIMULATED BY SYNTHETIC HUMAN GASTRIN (SHG-I) 81

A) BICKEL POUCH H+ SECRETION .... 87 B) BICKEL POUCH PEPSIN SECRETION • . 87 c) ANTRAL MOTOR ACTIVITY 92 VIII EFFECT OF G.I.P. ON GASTRIC SECRETION AND MOTOR ACTIVITY STIMULATED BY HISTAMINE DIHYDROCHLORIDE 92 V I I

PAGE VIII (CONT.) A) BICKEL POUCH H+ SECRETION .... 96 B) BICKEL POUCH PEPSIN SECRETION . . 100 c) BICKEL POUCH MOTOR ACTIVITY . . . 100 IX EFFECT OF G.I.P. ON INSULIN-STIMULATED GASTRIC SECRETION 104 A) GASTRIC REMNANT H* SECRETION . . 109 B) GASTRIC REMNANT PEPSIN SECRETION. 109 X EFFECT OF G.I.P. ON H+ SECRETION AND VOLUME OF PANCREATIC JUICE IN THE ANAESTHETIZED CAT DURING THE INFUSION OF GASTRIN PENTAPEPTIDE 109 A) EFFECT OF G.I.P. ON H+ SECRETION FROM THE CAT STOMACH STIMULATED BY GASTRIN PENTAPEPTIDE 115 B) EFFECT OF G.I.P. ON VOLUME OF PANCREATIC JUICE DURING THE INFUSION OF GASTRIN PENTAPEPTIDE. 115 XI EFFECT OF GASTRIC INHIBITORY POLYPEPTIDE ON FASTJNG BLOOD GLUCOSE LEVELS .... 117 A) BLOOD GLUCOSE LEVELS IN EXPERIMENTS INVOLVING THE INHIBITION OF GASTRIN PENTAPEPTIDE—STIMULATED H+ SECRE• TION . 117 B) COMPARISON OF EQUIMOLAR INJECTIONS OF GLUCAGON AND G.I.P. ON BLOOD GLUCOSE LEVELS IN THE DOG AND THE RABBIT 117 XII EFFECT OF GASTRIC INHIBITORY POLYPEPTIDE ON BICKEL POUCH PEPSIN OUTPUT STIMULATED BY SECRETIN ..... 119

XIII EFFECT OF GASTRIC INHIBITORY POLYPEPTIDE ON GASTRIC SECRETION AND MOTOR ACTIVITY IN THE UNSTIMULATED DOG 124 A) BICKEL POUCH H+ OUTPUT 124 B) BICKEL POUCH PEPSIN OUTPUT ... 124 c) ANTRAL AND BICKEL POUCH MOTOR ACTIVITY 124

DISCUSSION 127

BIBLIOGRAPHY 153 LIST OF TABLES

TABLE PAGE

I. EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ ON INTRA-GALL BLADDER PRESSURE 4-0

II. EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ ON ANTRAL MOTILITY 4-2

III. EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ ON PEPSIN SECRETION 45

IV. EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ ON H+ OUTPUT 47

V. EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ ON

H+ OUTPUT STIMULATED BY GASTRIN PENTAPEPTIDE . 52

VI. EFFECT OF INTRAVENOUS INFUSION OF EGI ON H+ OUTPUT STIMULATED BY GASTRIN PENTAPEPTIDE ... 58

VII. EFFECT OF INTRAVENOUS INFUSION OF EGI ON PEPSIN OUTPUT STIMULATED BY GASTRIN PENTAPEPT I OE 61

VIII. EFFECT OF INTRAVENOUS INFUSION OF EGI (G25) ON H+ OUTPUT STIMULATED BY PERFUSION OF ANTRAL POUCHES WITH 0.1$ ACH • 65

IX. EFFECT OF INTRAVENOUS INFUSION OF EGI (G25) ON PEPSIN OUTPUT STIMULATED BY PERFUSION OF ANTRAL POUCHES WITH 0.1$ ACH 67

X. EFFECT OF INTRAVENOUS INFUSION OF EGI (G25) ON ANTRAL MOTILITY STIMULATED BY PERFUSION OF ANTRAL POUCHES WITH 0.1$ ACH 70

XI. H+ OUTPUT IN RESPONSE TO THE INTRAVENOUS INFUSION OF GASTRIN PENTAPEPTIDE 75

XII. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. (1.0 JUG/KG/HR) ON H+ SECRETION STIMULATED BY GASTRIN PENTAPEPTIDE 76

XIII. PEPSIN SECRETION IN RESPONSE TO INTRAVENOUS INFUSION OF GASTRIN PENTAPEPTIDE 78

XIV. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. (1.0 XJG/KG/HR) ON PEPSIN OUTPUT STIMULATED BY GASTRIN PENTAPEPTIDE ... 79

VIII I X

TABLE PAGE

XV. ANTRAL MOTILITY RESPONSE TO INTRAVENOUS INFUSION OF GASTRIN PENTAPEPTIDE 82

XVI. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. (1.0 AJG/KG/HR) ON ANTRAL MOTILITY STIMULATED BY GASTRIN PENTAPEPTIDE 83

XVII. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. (2.0 AJG/KG/HR) ON H+ OUTPUT, PEPSIN OUTPUT AND ANTRAL MOTILITY STIMULATED BY GASTRIN PENTAPEPTIDE ..... 85

XVIII. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON H+ OUTPUT STIMULATED BY SYNTHETIC HUMAN GASTRIN I 88

XIX. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY SYNTHETIC HUMAN GASTRIN I 90

XX. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON ANTRAL MOTILITY STIMULATED BY SYNTHETIC HUMAN GASTR I N I 93

XXI. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON H+ OUTPUT STIMULATED BY HISTAMINE DIHYDRO• CHLORIDE (10.0 AIG/KG/HR) 97

XXII. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON hT*" OUTPUT STIMULATED BY HISTAMINE DIHYDRO• CHLORIDE (5.0/JG/KG/HR) 98

XXIII. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY HISTAMINE DIHYDROCHLORIDE (10.0 JUG/KG/HR ) 101

XXIV. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY HISTAMINE DIHYDROCHLORIDE (5.0 JUG/KG/HR) 102

XXV. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON FUNDIC MOTILITY STIMULATED BY HISTAMINE DIHYDROCHLORIDE (10.0 /JG/KG/HR) ...... 105

XXVI. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON FUND IC MOTILITY STIMULATED BY HISTAMINE DIHYDROCHLORIDE (5.0 /JG/KG/HR) 106

XXVII. COMPARISON OF THE ACTION OF G.I.P. ON H4" SECRET I ON STIMULATED BY INSULIN HYPO- GLYCAEM I A . . 110 X

TABLE PAGE

XXVIII. COMPARISON OF THE ACTION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY INSULIN HYPOGLYCAEMI A 112

XXIX. EFFECT OF G.I.P. ON H+ OUTPUT AND VOLUME OF PANCREATIC JUICE DURING THE INTRAVENOUS INFUSION OF GASTRIN PENTA• PEPTIDE IN THE CAT 116

XXX. EFFECT OF EQUIMOLAR INJECTIONS OF GLUCAGON AND G.I.P. ON FASTING BLOOD GLUCOSE LEVELS IN THE RABBIT 120

XXXI. EFFECT OF EQUIMOLAR INJECTIONS OF GLUCAGON AND G.I.P. ON FASTING BLOOD GLUCOSE LEVELS IN THE DOG . 122

XXXII. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY SECRETIN . 123

XXXIII. EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON H+ OUTPUT, PEPSIN OUTPUT, ANTRAL AND FUNDIC MOTOR ACTIVITY IN THE UNSTIMU• LATED ANIMAL 125 LIST OF FIGURES

FIGURE PAGE

1. DIAGRAM OF CHRONIC DOG SURGICAL PREPARATION NO. 3 20

2. ACRYLIC ARRANGEMENT FOR ANTRAL POUCH C ANNUL AE 25

3. (A) ABSORBANCE AT 275 NM OF THE PRODUCTS OF DIGESTION OF HAEMOGLOBIN WITH STANDARD PEPSIN

(B) STANDARD TYROSINE CURVE

(c) PLOT OF JUG TYROSINE AGAINST JUG PEPSIN . 29

4. (A) GUINEA PIG GALL BLADDER RESPONSE TO 4 DOSES OF STANOARO CCK-PZ PREPARATION

(B) LOG DOSE RESPONSE CURVE PLOTTED FROM 4(A) 31

5. EXAMPLE OF AN ASSAY FOR ENTEROGASTRONE ACTIVITY IN THE CHRONIC DOG 33

6. EXAMPLE OF AN ASSAY FOR SECRETIN ACTIVITY IN THE ACUTE CAT 35

7. GALL BLADDER PRESSURE CHANGES PRODUCED BY

10$ AND 40$ PURE CCK-PZ 41

8. CHANGES IN ANTRAL MOTOR ACTIVITY PRODUCED BY 10$ AND 40$ PURE CCK-PZ 43

9. CHANGES IN PEPSIN OUTPUT FROM BICKEL POUCHES PRODUCED BY 10$ AND 40$ PURE CCK-PZ ..... 46

10. CHANGES IN H+ SECRETION FROM BICKEL POUCHES PRODUCED BY 10$ AND 40$ PURE CCK-PZ 48

11. BICKEL POUCH H+ OUTPUT IN RESPONSE TO 4 DOSES OF GASTRIN PENTAPEPTIDE 50

12. EFFECT OF 10$ AND 40$ PURE CCK-PZ ON BICKEL POUCH H+ SECRETION STIMULATED BY GASTRIN PENTAPEPTIDE 53

13. SUMMARY OF PURIFICATION OF GASTRIC INHIBI• TORY POLYPEPTIDE . 54

XI X I I

FiGURE PAGE

14. FRACTIONATION OF 10$ PURE CCK-PZ ON SEPHADEX G-50 FINE 56

15. EFFECT OF EGI ON BICKEL POUCH H+ SECRETION STIMULATED BY GASTRIN PENTAPEPTIDE 59

16. EFFECT OF EGI ON BICKEL POUCH PEPSIN SECRE• TION STIMULATED BY GASTRIN PENTAPEPTIDE ... 62

17. CCK-PZ AND ENTEROGASTRONE ASSAYS OF FRACTIONS RESULTING FROM THE CHROMATOGRAPHY OF EGI ON SEPHADEX G25 63

18. EFFECT OF EGI (G25) ON BICKEL POUCH H+ SECRETION STIMULATED BY ENDOGENOUS GASTRIN . 66

19. EFFECT OF EGI (G25) ON BICKEL POUCH PEPSIN SECRETION STIMULATED BY ENDOGENOUS GASTRIN . 68

20. (A) EFFECT OF EGI (G25) ON ANTRAL POUCH MOTOR ACTIVITY STIMULATED BY ENDOGENOUS GASTRIN

(B) SAMPLE RECOROING OF THE EFFECT OF EGI (G25) ON ANTRAL MOTOR ACTIVITY 71

21. (A) FRACTIONATION OF EGI ON CM-CELLULOSE

(B) CCK-PZ AND ENTEROGASTRONE ASSAYS OF FRACTIONS RESULTING FROM THE CHROMA• TOGRAPHY OF EGI ON CM-CELLULOSE .... 72

22. EFFECT OF 0.25* 0,5 AND 1.0JUG/KG/HR OF G.I.P. ON BICKEL POUCH H SECRETION STIMULATED BY GASTRIN PENTAPEPTIDE 77

23. EFFECT OF 0.25, 0.5 AND 1.0JUG/KG/HR OF G.I.P. ON BICKEL POUCH PEPSIN SECRETION STIMULATED BY GASTRIN PENTAPEPTIDE 80

24. EFFECT OF 0.25» 0.5 AND 1.0JUG/KG/HR OF G.I.P. ON ANTRAL MOTOR ACTIVITY STIMULATED BY GASTRIN PENTAPEPTIDE 84

25. EFFECT OF 2.0 JUG/KG/HR OF G.I.P. ON: A. BICKEL POUCH H+ SECRETION B. BICKEL POUCH PEPSIN SECRETION C. ANTRAL POUCH MOTOR ACTIVITY STIMULATED BY GASTRIN PENTAPEPTIOE 86

26. EFFECT OF G.I.P. ON BICKEL POUCH H+ SECRETION STIMULATED BY SYNTHETIC HUMAN GASTRIN I (SHG-1 ) 89 XIII

FiCURE PAGE

27. EFFECT OF G.I.P. ON BICKEL POUCH PEPSIN SECRET I ON STIMULATED BY SHG-I 91

28. EFFECT OF G.I.P. ON ANTRAL POUCH MOTOR ACTIVITY STIMULATED BY SHG-I . 94

29. BICKEL POUCH H+ OUTPUT IN RESPONSE TO 4 DOSES OF HISTAMINE DIHYDROCHLORIDE 95

30. (A) EFFECT OF G.I.P. ON BICKEL POUCH H+ SECRETION STIMULATED BY 10.0 JUG/KG/HR OF HISTAMINE DIHYDROCHLORIDE

(B) EFFECT OF G.I.P. ON BICKEL POUCH H+ SECRETION STIMULATED BY 5.0AJG/KG/HR OF HISTAMINE DIHYDROCHLORIDE 99

31. (A) EFFECT OF G.I.P. ON BICKEL POUCH PEPSIN SECRETION DURING THE INFUSION OF 10.0 JUG/KG/HR OF HISTAMINE D I HYOROCHLORI DE

(B) EFFECT OF G.I.P. ON BICKEL POUCH PEPSIN SECRETION DURING THE INFUSION OF 5.0 /UG/KG/HR OF HISTAMINE DIHYDROCHLORIDE . 103

32. (A) EFFECT OF G.I.P. ON BICKEL POUCH MOTOR ACTIVITY DURING THE INFUSION OF 10.0 JUG/KG/HR OF HISTAMINE DIHYDROCHLORIDE

(B) EFFECT OF G.I.P. ON BICKEL POUCH MOTOR ACTIVITY DURING THE INFUSION OF 5.0 /UG/KG/HR OF HISTAMINE DIHYDROCHLORIDE . 107

33» SAMPLE RECORDING OF THE EFFECT OF G.I.P. ON BICKEL POUCH MOTOR ACTIVITY ON A BACKGROUND INFUSION OF HISTAMINE DIHYDROCHLORIDE . . . . 108

34. EFFECT OF G.I.P. ON GASTRIC REMNANT H* SECRETION STIMULATED BY INSULIN 111

35. EFFECT OF G.I.P. ON GASTRIC REMNANT PEPSIN SECRETION STIMULATED BY INSULIN 113

36. SIMILARITIES IN STRUCTURE OF SECRETIN, GLUCAGON AND G. I.P 114

37. BLOOD GLUCOSE LEVELS DURING AN EXPERIMENT IN WHICH G.I.P. INHIBITED GASTRIN PENTA- PEPTI DE-ST IMULATED H+ SECRETION IN THE DOG . 118

38. EFFECT OF G.I.P. AND GLUCAGON ON BLOOD SUGAR LEVELS IN THE RABBIT 121 ACKNOWLEDGEMENTS

I WOULD LIKE TO THANK MY SUPERVISOR DR. J.C. BROWN FOR

HIS GUIOANCE DURING MY STAY IN THE DEPARTMENT OF PHYSIOLOGY,

AND FOR THE LARGE PART OF HIS PERSONAL LIFE THAT WAS SPENT

ON MY BEHALF.

I WOULD ALSO LIKE TO THANK DR. J.A. PEARSON FOR HIS

ADVICE ANO ASSISTANCE WITH REGARO TO MY RESEARCH ANO THESIS

PREPARATION. I AM PERMANENTLY INDEBTED TO MlSS JlLL DRYBURGH

FOR HER INVALUABLE TECHNICAL ASSISTANCE AND ENCOURAGEMENT AS

A FRIEND. I AM ALSO GRATEFUL FOR THE TECHNICAL ASSISTANCE

OF MRS. HEATHER GRIFFITHS AND MR. TONY MCLINTOCK. I WOULD

LIKE TO THANK MR. KURT HENZE ANO MR. RALPH ASSINA FOR THEIR

ASSISTANCE THROUGHOUT THE COURSE OF MY RESEARCH, PARTICULARLY

IN THE PREPARATION OF THE ILLUSTRATIONS FOR THIS THESIS. I

WOULD ALSO LIKE TO THANK MlSS BEVERLY WENKSTERN FOR UNDER•

TAKING THE ARDUOUS TASK OF TYPING THE FINAL DRAFT OF THIS

THESIS.

I WOULD FINALLY LIKE TO THANK MY WIFE MARGARET FOR

TYPING THE ROUGH DRAFTS OF THIS THESIS, AND FOR HER PERSE•

VERANCE THROUGHOUT MY WORK.

FINANCIAL ASSISTANCE IN THE FORM OF A STUOENTSHIP FROM

THE MEOICAL RESEARCH COUNCIL OF CANADA IS GRATEFULLY

ACKNOWLEDGED.

X I V INTRODUCTION

MECHANISMS FOR THE INHIBITION OF GASTRIC SECRETION AND

MOTOR ACTIVITY OPERATING FROM THE DUOOENUM HAVE BEEN SHOWN

TO BE ACTIVATED DURING THE DIGESTION OF A MEAL. THOMAS,

CRIDER ANO MOGAN (1934) CONSIDERED THAT THE STOMACH PUMPED

MATERIAL INTO THE DUODENUM, WITH THE RATE OF TRANSFER FROM

STOMACH TO DUODENUM BEING LESS THAN THE MAXIMAL RATE THE

PUMP COULD ACHIEVE. THEY CONSIDERED THAT THE RESTRAINT OF

THE GASTRIC PUMP DEPENDED UPON THE ACTIVITY OF RECEPTORS IN

THE DUODENAL WALL STIMULATED BY CONSTITUENTS OF GASTRIC

CHYME. THE IMPORTANCE OF RESTRAINT OF GASTRIC EMPTYING WAS

ILLUSTRATED BY THE FACT THAT THE SMALL INTESTINE WAS THE

MAIN FOOD ABSORBING ORGAN, AND IF THE AMOUNTS WHICH LEFT THE

STOMACH EXCEEDED THE CAPACITY OF THE GUT FOR ENZYME DEGRADA•

TION AND ABSORPTION, AN OSMOTIC PURGE WOULD RESULT. MOST

INFLUENCES ON GASTRIC EMPTYING HAVE BEEN SHOWN TO BE

INHIBITORY IN NATURE, AN EXCEPTION BEING THE FINDING BY

MARBAIX (1898) THAT DISTENSION CAUSED AN INCREASE IN GASTRIC

EMPTYING. THE RATE OF EMPTYING OF THE STOMACH HAS BEEN

SHOWN TO BE DEPENDENT UPON THE VOLUME OF ITS CONTENTS ANO

TO BE HELD IN CHECK BY INHIBITORY MECHANISMS ORIGINATING IN

THE DUODENUM AND ELSEWHERE IN THE SMALL INTESTINE.

IN THE STUDY OF HUMORALLY MEDIATED GASTRIC INHIBITORY

MECHANISMS ORIGINATING IN THE DUODENUM, THE FOLLOWING STAGES

OCCURRED IN REACHING THE CURRENT LEVEL OF KNOWLEDGE OF THE

MECHANISMS: 1. ACCUMULATION OF EXPERIMENTAL EVIDENCE THAT - 2 -

HUMORALLY MEDIATED GASTRIC INHIBITORY MECHANISMS EXISTED,

2. COMPARISON OF PHYSIOLOGICAL STIMULI (FAT, HCL, HYPERTONIC

SOLUTIONS) IN TERMS OF THE SPECTRUM AND POTENCY OF GASTRIC

INHIBITORY ACTIONS, AND 3. ATTEMPTS TO ISOLATE AND PURIFY

THE ACTIVE PRINCIPLES RESPONSIBLE FOR THE GASTRIC INHIBITORY

1

ACTIONS.

EWALO AND BOAS (1886) FIRST REPORTED ON WHAT WAS

SUBSEQUENTLY PROVEN TO BE A DUODENAL MECHANISM FOR INHIBITION

OF GASTRIC SECRETION. THEY DEMONSTRATED THAT OLIVE OIL WHEN

ADDED TO A STARCH TEST MEAL WOULD DEPRESS GASTRIC SECRETION

IN HUMANS. PAVLOV (1910) FOUND THAT OLIVE OIL IN THE

DUOOENUM WOULD INHIBIT MEAT-STIMULATED GASTRIC SECRETION

FROM A PAVLOV POUCH. HE POSTULATED THAT THE OIL WAS REFLEXLY

INHIBITING THE CEPHALIC OR PSYCHIC PHASE OF GASTRIC SECRETION.

FENG, HOU AND LIM (1929) OBSERVED THAT OLIVE OIL IN THE

DUODENUM INHIBITED MEAT-STIMULATED SECRETION IN TRANSPLANTED

POUCHES OF THE WHOLE STOMACH. THE SAME GROUP INJECTED

INTRAVENOUS FATTY CHYLE FROM ONE DOG INTO ANOTHER TO DETER•

MINE WHETHER THE BLOOD BORNE AGENT WHICH OPERATED AFTER FAT

WAS INTRODUCED INTO THE DUODENUM CONSISTED OF A HORMONE

RELEASED FROM THE DUOOENUM, OR THE ABSORBED PRODUCTS OF

DIGESTION. THE ABSENCE OF SECRETORY INHIBITION PROVED THAT

THE OBSERVED EFFECT WAS NOT DUE TO ABSORPTION PRODUCTS OF

THE MEAL AND THEY CONCLUDED THAT A HORMONAL PATHWAY WAS

INVOLVED IN THE MECHANISM.

THE FIRST ATTEMPTS TO ISOLATE THE BLOOD BORNE AGENT

RESPONSIBLE FOR GASTRIC INHIBITION WERE MADE BY KOSAKA AND - 3 -

LIM (1930A). THEY FOUND THAT THE CRUDE CHOLECYSTOKININ (CCK)

OF IVY (1928) WOULD INHIBIT GASTRIC SECRETION FROM HEIDENHAIN

POUCHES STIMULATED BY A MEAT MEAL AND HISTAMINE. KOSAKA ANO

LIM SUGGESTED THAT IVY'S CCK PREPARATION CONTAINED AN IMPURITY

WHICH WAS RESPONSIBLE FOR GASTRIC SECRETORY INHIBITION AND

THEY ATTEMPTED TO ISOLATE IT. IN THE SAME YEAR (1930B),

THIS GROUP PREPARED EXTRACTS OF DUODENAL MUCOSA MADE AFTER

EXPOSURE TO OLIVE OIL AND FOUND THAT THESE EXTRACTS HAD

INHIBITORY PROPERTIES SIMILAR TO THOSE OF CCK PREPARATIONS.

THEY NAMED THE ACTIVE PRINCIPLE ENTEROGASTRONE.

THE WORK OF FENG E_T AJL (1929) HAS BEEN CONFIRMED IN

SEVERAL SPECIES USING DIFFERENT STIMULI FOR ACID SECRETION.

FAT IN THE DUODENUM WAS SHOWN BY BIBLER, HARKINS AND NYHUS

(1965) TO INHIBIT ACID SECRETION STIMULATED BY EXOGENOUS

GASTRIN IN THE DOG, BY HALVORSON, MLDDLETON, BLBLER, HARKINS

AND NYHUS (1966) TO INHIBIT HISTAMINE-STIMULATEO SECRETION

IN THE DOG, AND BY SHAY, GERSHON-COHEN AND FELS (1939) TO

INHIBIT ACID SECRETION STIMULATED BY BOTH MEANS IN MAN.

KASANSKI (1903) FOUND THAT THE PSYCHIC OR VAGALLY INDUCED

SECRETION OF GASTRIC JUICE WAS ALSO INHIBITED BY FAT IN THE

DOG. THIS WAS CONFIRMED BY ALLEY AND MACKENZIE (1934), WHO

FOUND THAT OLIVE OIL IN THE DUODENUM WOULD INHIBIT GASTRIC

SECRETION FROM THE MAIN STOMACH OF THE DOG STIMULATED BY

SHAM FEEDING. JOHNSTON ANO DU THIE (1969) FOUND THAT FAT IN

THE DUODENUM WOULD ONLY INHIBIT AC ID SECRETION IN ULCER

PATIENTS WITH INTACT VAGUS NERVES. THEY FOUND THAT INHIBI•

TION BY FAT OF BOTH GASTRIN- AND HISTAM INE-STIMULATED - 4 -

SECRET I ON WAS ABOLISHED IN PATIENTS WHO HAD UNDERGONE

VAGOTOMY. THIS WORK SUGGESTED THAT AT LEAST IN MAN, THE

VAGUS PLAYED A ROLE IN THE INHIBITION OF GASTRIC SECRETION

PRODUCED BY INTRADU0DENAL FAT. SLRCUS (1958) FOUND THAT ONE

OF THE CONDITIONS OF FAT INHIBITION OF GASTRIC SECRET I ON

WAS THAT THE FAT BE PRE INCUBATED WITH PANCREATIC JUICE.

OLIVE OIL PLACED IN ISOLATED DUODENAL LOOPS RESULTED IN NO

INHIBITION OF ACID SECRETION. SIMILARLY, MENGUY (I960)

FOUND THAT IN RATS, FAT FAILED TO INHIBIT GASTRIC SECRETION

IN THE ABSENCE OF BILE SALTS OR LIPASE.

LOBASOV (1896), A PUPIL OF PAVLOV, FIRST DEMONSTRATED

THAT MIXING FAT WITH FOODSTUFFS INHIBITED PEPTIC ACTIVITY

OF GASTRIC JUICE IN THE DOG. ALLEY AND MACKENZIE (1934)

CONFIRMED THAT OLIVE OIL IN THE DUODENUM INHIBITED THE PEPSIN

OUTPUT OF THE MAIN STOMACH STIMULATED BOTH BY HISTAMINE AND

BY SHAM FEEDING.

FROM THE TIME OF BEAUMONT (1833)» IT WAS KNOWN THAT

THE RATE OF GASTRIC EMPTYING WAS A FUNCTION OF THE TYPE

OF MEAL EATEN. IN HIS OBSERVATIONS ON HIS GASTRIC FISTULA

PATIENT, ALEXIS ST. MARTIN, HE NOTED THAT FATTY FOODS

REMAINED IN THE STOMACH THE LONGEST. ONE OF THE EARLIEST

OBSERVATIONS REGARDING THE EFFECT OF FAT ON GASTRIC EMPTYING

WAS MADE 8Y WLRSCHUBSKI (1900). HE OBSERVED THAT FAT

DELAYED THE EVACUATION OF THE STOMACH IN DOGS, A FINDING

WHICH WAS CONFIRMED BY CANNON (1911) USING A ROENTGENOLOGICAL

METHOD. UNTIL THE TURN OF THE CENTURY IT WAS THOUGHT THAT

FAT EXERTED ITS INHIBITORY ACTION ON GASTRIC MOTILITY WHILE - 5 -

IN THE STOMACH. HOWEVER LLNTWAREV (1903) OBSERVED INHIBITION

OF GASTRIC MOTILITY UPON INTRODUCING OLIVE OIL INTO THE

DUODENUM. FARRELL AND IVY (1926) ESTABLISHED THAT INHIBITION

OF GASTRIC MOTILITY BY FAT IN THE DUODENUM OPERATED AT LEAST

PARTLY VIA A HUMORAL MECHANISM WHEN THEY SHOWED THAT THE

MOTILITY OF A TRANSPLANTED FUNDIC POUCH WAS INHIBITED BY

FEEDING A FAT MEAL. LLM, LOO AND LLU (1927) CONFIRMED THAT

THE PRESENCE OF FAT IN THE DUODENUM OR JEJUNUM INHIBITED THE

MOTOR ACTIVITY OF A POUCH OF THE ENTIRE STOMACH AND OF A

STOMACH DEPRIVED OF ALL ITS EXTRINSIC NERVE SUPPLY. QUIGLEY »

ZETTLEMAN AND IVY (1934) SHOWED THAT INHIBITION OF MOTILITY

WAS NOT DUE TO ABSORBED FAT ITSELF OR TO THE PRODUCTS OF ITS

DIGESTION. THIS GROUP ALSO SHOWED CONCLUSIVELY THAT IN A

POUCH OF THE ENTIRE STOMACH, A DENERVATED POUCH, AN AUTO-

TRANSPLANTED POUCH AND IN A PAVLOV POUCH, FAT PLACED DIRECTLY

WITHIN THE STOMACH WAS WITHOUT SIGNIFICANT EFFECT ON GASTRIC

MOTILITY, WHEREAS THE INHIBITORY EFFECT ON MOTOR ACTIVITY

WAS ALWAYS MARKED WHEN FAT WAS INTRODUCED DIRECTLY INTO THE

DUODENUM. ACCORDING TO PAVLOV (1910), A NERVOUS REFLEX WAS

RESPONSIBLE FOR THE INHIBITION OF GASTRIC MOTOR ACTIVITY BY

FAT. CANNON (1911) ALSO ATTRIBUTED THE INHIBITORY EFFECT TO

THE INTERVENTION OF NERVOUS REFLEXES. IT HAD BEEN SHOWN BY

WADOELL AND WANG (1953) THAT THE EFFECT OF INTRADUODENAL FAT

ON GASTRIC MOTILITY IN MAN WAS REDUCED BY VAGOTOMY, AS WAS

THE INHIBITION OF GASTRIC SECRETION BY FAT. SIMILAR RESULTS

WERE REPORTED BY QUIGLEY AND MESCHAN (1938) WORKING WITH DOGS.

THE FIRST DEMONSTRATION THAT INTESTINAL ACIDIFICATION - 6 -

INHIBITED GASTRIC SECRETION WAS PROVIDED BY SOKOLOV (1904).

HE DEMONSTRATED THAT THE INTRODUCTION OF 0.5$ HCL INTO THE

DUODENUM MARKEDLY DIMINISHED ACID SECRETION FROM A PAVLOV

POUCH STIMULATED BY A MEAT MEAL. DAY AND WEBSTER (1935)

REINVESTIGATED THIS PROBLEM, USING DOGS WITH ESOPHAGOTOMIES

AND METAL FISTULAE IN THE STOMACH AND DUODENUM. THEY FOUND

THAT HCL OR GASTRIC JUICE WHEN INTRODUCED INTO THE DUODENUM

INHIBITED GASTRIC SECRETION STIMULATED BY SHAM FEEDING.

EXPERIMENTS WERE PERFORMED ON PAVLOV POUCH DOGS WITH GASTRIC

AND DUODENAL FISTULAE BY PlNCUS, THOMAS AND REHFUSS (1942)

TO DETERMINE WHY IN CERTAIN CASES HCL IN THE DUODENUM DID

NOT INHIBIT GASTRIC SECRETION. THEY DEMONSTRATED THAT HCL

DID NOT DEPRESS GASTRIC SECRETION UNTIL THE DUODENAL PH HAD

FALLEN TO 2.5. GRIFFITHS (1936) DEMONSTRATED IN MAN THAT

HCL IN THE DUODENUM INHIBITED GASTRIC SECRET I ON STIMULATED

BY ALCOHOL. CODE AND V/ATKINSON (1955) IMPLICATED A NERVOUS

MECHANISM IN ACID INHIBITION OF GASTRIC SECRETION WHEN THEY

FOUND THAT INFUSION OF ACID INTO THE DUODENUM INHIBITED THE

RESPONSE TO A MEAL FROM PAVLOV POUCHES BUT NOT FROM VAGALLY

DENERVATED POUCHES. IT HAS SUBSEQUENTLY BEEN ESTABLISHED

THAT A HUMORAL INHIBITORY MECHANISM DOES EXIST, SO THAT

WHETHER A VAGAL MECHANISM EXISTS IN ADDITION TO OR IN

CONJUNCTION WITH A HUMORAL MECHANISM IS STILL AN OPEN

QUESTION. IT HAS BEEN WELL ESTABLISHED PRINCIPALLY BY

ANDERSSON (1960A,B,C) THAT ACID IN THE DUODENUM CAUSES

INHIBITION OF GASTRIC SECRETION VIA A HUMORAL MECHANISM.

HE FOUND THAT ACIDIFICATION OF THE DUODENUM OF DOGS WITH EITHER VAGALLY INNERVATED OR VAGALLY OENERVATED GASTRIC

POUCHES INHIBITED SECRETORY RESPONSES FROM BOTH TYPES OF

POUCHES DURING FASTING (1960A)» AND IN RESPONSE TO A MEAL

(1960B). WORMSLEY AND GROSSMAN (1964) HAVE SHOWN THAT

ACIDIFYING THE DUODENUM INHIBITED THE HEIDENHAIN POUCH

RESPONSE TO EXOGENOUS GASTRIN. SLNCE THE SECRETORY RESPONSE

TO EXOGENOUS GASTRIN WAS INHIBITED, ACID IN THE DUODENUM

MUST HAVE RELEASED A HORMONE WHICH INHIBITED GASTRIC

SECRETION AT SOME POINT AFTER THE RELEASE OF GASTRIN.

ANDERSSON (1960C), ANDERSSON AND GROSSMAN (1965) AND JOHNSON

AND GROSSMAN (1968) CONFIRMED THAT DUOOENAL ACIDIFICATION

DID NOT INHIBIT HISTAM INE-ST I MULATED SECRETION IN THE DOG.

ACIDIFICATION OF THE DUODENUM HAS BEEN SHOWN TO STIMU•

LATE PEPSIN SECRETION BY STENING E_T AJ. (1969A) IN CONTRAST TO

THE INHIBITORY EFFECT OF FAT IN THE DUODENUM ON THIS

PARAMETER.

PAVLOV (1910) ESTABLISHED THAT THE PASSAGE OF CHYME FROM

THE PYLORIC ANTRUM OF THE STOMACH INTO THE DUODENUM WAS

INTERMITTENT. IT APPEARED THAT THE PASSAGE OF FOOD FROM THE

STOMACH TO THE DUODENUM WAS REGULATED QUANTITATIVELY BY A

REFLEX FROM THE LATTER. TH I S WAS FIRST DEMONSTRATED BY

SERDIUKOV (1903), A COLLEAGUE OF PAVLOV, WHO SHOWED THAT

INHIBITION OF GASTRIC EMPTYING WAS CAUSED BY A CHEMICAL

EFFECT PRODUCED BY CONTACT OF ACID CHYME WITH THE DUODENUM.

HE FOUND THAT A SOLUTION OF SODIUM BICARBONATE PREVIOUSLY

INTRODUCED INTO THE STOMACH REMAINED THERE INDEFINITELY IF

SMALL QUANTITIES OF ACID SOLUTION OR GASTRIC JUICE WERE - 8 -

CONTINUOUSLY INTRODUCED INTO THE DUODENUM. IF NO ACID WAS

PRESENT, THE ALKALINE SOLUTION WAS EMPTIED FROM THE STOMACH

VERY QUICKLY. HENCE EACH TIME THAT THE INTESTINE RECEIVED

ACID CONTENTS FROM THE STOMACH, A MECHANISM WAS TRIGGERED

WHICH TEMPORARILY BLOCKED GASTRIC EMPTYING. PAVLOV CON•

SIDERED THIS TO BE A VERY LOGICAL PROCESS AND OF GREAT

ASSISTANCE IN THE FUNCTIONS OF THE DIGESTIVE TRACT. THE

ACIDIFIED FOOD ALLOWED TO PASS THROUGH THE PYLORUS WOULD

CAUSE AN INCREASED FLOW OF PANCREATIC JUICE ANO BILE WHICH

WOULD RAPIDLY LEAD TO NEUTRALIZATION OF DUOOENAL CONTENTS.

ONLY WHEN THIS HAD BEEN ACCOMPLISHED WOULD THE ESCAPE OF A

FURTHER PORTION OF CONTENTS FROM THE STOMACH BE PERMITTED.

SHAY AND GERSHON-COHEN (1934) REPORTED THAT THE MECHANISM OF

INHIBITION OF GASTRIC MOTILITY BY ACID WAS PRESENT IN HUMANS.

THEY NOTED A DELAY IN THE EVACUATION OF THE HUMAN STOMACH

WHEN HCL WAS ADDED TO A TEST MEAL. THOMAS (1947) FOUND THAT

THE STOMACH WOULD CONTINUE TO EMPTY, BUT AT A DIMINISHING

RATE, AS THE PH OF THE DUODENAL CONTENTS DECREASED FROM

PH 3.0 TO PH 2.0, WITH THE STOMACH CEASING TO EMPTY AT PH 2.0.

INVOLVEMENT OF THE VAGUS IN THE GASTRIC INHIBITORY MECHANISM

TRIGGERED BY ACID WAS INDICATED BY THOMAS (1947) AND QUIGLEY

AND MESCHAN (1938) WHEN THEY FOUND THAT THE REFLEX MOTOR

INHIBITORY RESPONSE TO ACID IN DOGS IS PRACTICALLY ABOLISHED

BY VAGOTOMY.

LECONTE (1900) DEMONSTRATED THAT THE INTRODUCTION OF

A 25$ SOLUTION OF GLUCOSE INTO THE DUODENUM INHIBITED BOTH

THE SECRET I ON AND MOTILITY OF THE STOMACH. OKADA ET AL (1927) - 9 -

BELIEVED THAT THE INHIBITORY MECHANISM WAS MEDIATED BY

HYPERGLYCAEMI A. THIS WAS DISPROVED AS A RESULT OF THE

DEMONSTRATION BY ROHOLM (1930) THAT GASTRIC SECRETION DIO

NOT CHANGE DURING ADRENAL INE—INDUCED HYPERGLYCAEM I A OR THE

INTRAVENOUS INFUSION OF GLUCOSE (KALK AND MEYER, 1939).

IT WAS SHOWN BY DAY AND KOMAROV (1939) THAT INSTILLATION

OF 20$ OR HIGHER CONCENTRATIONS OF GLUCOSE INTO THE DUODENUM

OF DOGS INHIBITED THE RESPONSE TO HISTAMINE. THIS INHIBITION

WAS INDICATIVE OF A COMMON OSMORECEPTOR MECHANISM SENSITIVE

TO CERTAIN LEVELS OF DUODENAL OSMOLARITY , BECAUSE OF THE

FACT THAT OTHER SUGARS, POLYSACCHARIDES AND SALINE HAD

SIMILAR ACTIONS. IN A FURTHER ATTEMPT TO ELUCIDATE THE

MECHANISM OF ACTION OF GLUCOSE, SHAY, GERSHON-COHEN, FELS

AND SLPLET (1942) SHOWED THAT INSULIN HYPOGLYCAEMI A DID NOT

COMPLETELY ABOLISH THE EFFECT OF DUODENAL GLUCOSE INSTILL•

ATION ON ACID SECRETION, ALTHOUGH IT WAS REDUCED. TH IS

GROUP CONCLUDED THAT THE MAIN EFFECT WAS DUE TO DUODENAL

OSMORECEPTORS WITH A SMALL PART DUE TO HYPERGLYCAEMI A.

SLRCUS (1958) PERFORMED EXPERIMENTS IN WHICH 5$ SALINE,

FRUCTOSE ANO GLUCOSE IN THE DUODENUM INHI8ITED HIS T A M I NE•

ST IMULATED SECRET I ON FROM BOTH INNERVATED AND DENERVATEO

POUCHES. SINCE INHIBITION WAS PRODUCED IN TRANSPLANTED

POUCHES, IT WAS CONCLUDED THAT HYPERTONIC SOLUTIONS IN THE

DUODENUM RELEASED AN INHIBITORY HORMONE.

SOME OF THE EARLIEST OBSERVATIONS OF OSMOTIC EFFECTS

ON GASTRIC MOTILITY WERE MADE BY SHAY ANO GERSHON-COHEN

(1934) EMPLOYING RADIOGRAPHIC TECHNIQUES. THEY N0TE0 THAT - 10 -

HYPERTONIC SOLUTIONS OF SALTS OR GLUCOSE SLOWEO EMPTYING OF

THE STOMACH. QUIGLEY ANO HALLARAN (1932) FIRST DEMONSTRATED

THAT THE INHIBITION OF GASTRIC MOTILITY WAS NOT DUE TO

HYPERGLYCAEMIC EFFECTS. THEY FOUND THAT THE INTRAVENOUS

ADMINISTRATION OF GLUCOSE FAILED TO ALTER SPONTANEOUS

GASTROINTESTINAL MOTILITY IN DOGS. QUIGLEY AND PHELPS (1934)

FOUND THAT SUGARS IN THE INTESTINE INHIBITED THE MOTILITY OF

TRANSPLANTED AND THEREFORE DENERVATED GASTRIC POUCHES,

INDICATING THAT A HUMORAL MECHANISM WAS INVOLVED IN THE

INHIBITION OF GASTRIC MOTILITY BY CARBOHYDRATES IN THE

DUODENUM. QUIGLEY AND MESCHAN (1938) FOUND THAT INHIBITION

OF GASTRIC MOTILITY AND SECRETION IN DOGS, PRODUCED BY

PLACING HYPERTONIC SOLUTIONS IN THE DUODENUM, WAS ABOLISHED

BY VAGOTOMY.

AS STATED EARLIER, PAVLOVJS GROUP ASSUMED THAT MECHAN•

ISMS OF INHIBITION OF GASTRIC SECRETION AND MOTILITY WERE

MEDIATED BY NERVOUS REFLEXES. EVEN THOUGH HUMORAL FACTORS

HAD LATER BEEN IMPLICATED IN THE ACTION OF FAT, ACID AND

HYPERTONIC SOLUTIONS ON GASTRIC SECRETION AND MOTILITY,

EVIDENCE EXISTED FOR THE IMPLICATION OF NERVOUS MECHANISMS

AS WELL. THOMAS AND MOGAN (1931) PROPOSED AN "ENTEROGASTRIC

REFLEX" THROUGH WHICH INHIBITION OF GASTRIC PERISTALSIS

COULD BE BROUGHT ABOUT BY APPROPRIATE CHEMICAL OR MECHANICAL

STIMULATION OF THE MUCOSA OF THE UPPER INTESTINE. THIS

REFLEX WAS IMPLICATED IN SITUATIONS MENTIONED EARLIER WHERE

INHIBITION OF ACID SECRETION AND MOTILITY FROM THE DUODENUM

WAS MARKEDLY REDUCED BY SECTIONING THE VAGUS NERVES. A - 11 -

NERVOUS MECHANISM HAS THUS BEEN IMPLICATED IN THE INHIBITION

OF GASTRIC SECRET I ON BY FAT AND ACID IN THE DUODENUM, AND

IN THE INHIBITION OF GASTRIC MOTILITY BY FAT, ACID AND

HYPERTONIC SOLUTIONS IN THE DUODENUM. NO EXPERIMENTAL

DESIGN, HOWEVER, HAS BEEN PROPOSED TO ISOLATE A NERVOUS

MECHANISM FROM THE HUMORAL INHIBITORY MECHANISMS THAT ARE

KNOWN TO EXIST.

AFTER THE INITIAL DISCOVERY, ATTEMPTS BY WORKERS TO

ISOLATE THE ENTEROGASTRONE OF KOSAKA AND LlM (1930B) MET

WITH LITTLE SUCCESS. WALAWSKI (1928) PRODUCED A "BIODIALY-

SATE1' BY SOAKING SMALL ANO LARGE INTESTINES OF DOGS IN

RINGER-LOCKE'S SOLUTION AT 37°C FOR ONE HOUR. THE BIO-

DIALYSATE PRODUCED INHIBITION OF HISTAMINE-STIMULATED SECRE•

TION IN GASTRIC FISTULA DOGS. KOSAKA, LLM, LLNG ANO LLU

(1932) REPEATED WALAWSKL'S EXPERIMENT ANO FOUND THAT THE

MATERIAL EXTRACTED WOULD INHIBIT THE RESPONSE OF A HEIDENHAIN

POUCH SECRETING IN RESPONSE TO A MEAL. FURTHER ATTEMPTS WERE

MADE BY GRAY, BRADLEY AND IVY (1937) AND GREENGARD, ATKINSON,

GROSSMAN AND IVY (1946) TO ISOLATE ENTEROGASTRONE, BUT EFFORTS

TO CONCENTRATE THE ACTIVE PRINCIPLE MET WITH LITTLE SUCCESS.

AT THE END OF THESE PURIFICATION PROCEDURES, SECRETIN AND

CCK ACTIVITY WERE STILL RETAINED, MAKING IT IMPOSSIBLE TO

DETERMINE WHETHER OR NOT THE INHIBITORY EFFECT OF FAT IN

THE DUODENUM WAS DUE TO SECRETIN, CCK, A COMBINATION OF THESE

TWO, OR DUE TO SOME OTHER HUMORAL AGENT. AS A RESULT OF

WORK DONE SUBSEQUENTLY ON GASTRIC INHIBITION, THE CLASSICAL

DEFINITION OF ENTEROGASTRONE AS NAN INHIBITORY HORMONE LIBERATED FROM THE DUODENUM WHEN FAT IS INTRODUCEO THERE11

WAS EXPANDED BY GREGORY (1967) TO "A HORMONE OF THE UPPER

INTESTINAL MUCOSA WHICH IS LI BERATED BY FAT OR ITS DIGESTION

PRODUCTS, HYPERTONIC SOLUTIONS, OR ACID, AND WHICH INHIBITS

GASTRIC SECRETION ANO MOTILITY."

WORK ON ISOLATION OF A DUODENAL INHIBITORY HORMONE

WANED UNTIL THE 1950'S WHEN GREENLEE, LONGHI, GUERRERO,

NELSON, EL-BEDRI AND DRAGSTEDT (1957), USING A CRUDE COMMER•

CIAL SECRETIN PREPARATION, OBSERVED MARKED INHIBITION OF ACID

SECRETION FROM A HEIDENHAIN POUCH STIMULATED TO SECRETE BY

IRRIGATION OF A SEPARATE ANTRAL POUCH WITH LIVER EXTRACT

TO RELEASE ENDOGENOUS GASTRIN. THE WORK OF GREENLEE WAS

CONFIRMED BY KENNEDY AND HALLENBECK (1963) WHO ALSO DEMON•

STRATED THAT THE GASTRIC SECRETORY INHIBITION BY SECRETIN

PERSISTED AFTER PANCREATECTOMY. THIS ELIMINATED THE

POSSIBILITY THAT THE INHIBITION WAS SECONDARY TO THE

PASSAGE OF ALKALINE PANCREATIC SECRETION INTO THE DUODENUM.

FURTHER WORK ON GASTRIC SECRETORY INHIBITION WAS CARRIED

OUT BY WORMSLEY AND GROSSMAN (1964). THEY FOUND THAT AN

ESSENTIALLY PURE SECRETIN PREPARATION (JORPES ANO MUTT,

1962) INHIBITED ACID SECRETION STIMULATED BY EXOGENOUS

GASTRIN. THEY CONCLUDED FROM THESE STUDIES THAT SECRETIN

WAS BLOCKING THE ACTION OF GASTRIN AT THE LEVEL OF THE

PARIETAL CELL. THESE FINDINGS WERE CONFIRMED BY GILLESPIE

AND GROSSMAN (1964) WHO ALSO FOUND THAT THE CCK PREPARATION

OF JORPES AND MUTT (1964) WOULD INHIBIT SECRETION OF A

HEIDENHAIN POUCH STIMULATED BY EXOGENOUS GASTRIN BUT WOULD - 13 -

INHIBIT ONLY LOW DOSES OF HISTAMINE. AT THAT TIME, THE

INHIBITORY ACTIONS OF CCK AND SECRETIN WERE INCOMPLETELY

DEFINED, MAINLY BECAUSE PURE PREPARATIONS WERE NOT THEN

AVAILABLE. ALTHOUGH INHIBITION OF GASTRIC SECRETION AND

MOTILITY BY THE RELEASE OF ENDOGENOUS HORMONES WAS WELL

DOCUMENTED, IT WAS STILL UNDECIDED WHETHER THE INHIBITION

PRODUCED BY THE PRESENCE OF ACID, FAT AND HYPERTONIC

SOLUTIONS IN THE DUODENUM WERE MEDIATED BY THE SAME

DUODENAL HORMONE, OR THROUGH SEPARATE MECHANISMS.

THE FIRST OF THE DUODENAL INHIBITORY HORMONES TO HAVE

ITS STRUCTURE ELUCIDATED WAS SECRETIN, THE AMINO ACID

SEQUENCE BEING PUBLISHED BY MUTT AND JORPES (19^6). VAGNE,

STENING, BROOKS AND GROSSMAN (1968) COMPARED THE PHYSIO•

LOGICAL ACTIONS OF NATURAL SECRETIN AND SYNTHETIC MATERIAL

PRODUCED BY BODANSKY, ONDETTI AND LEVINE (1966). THEY

FOUND THE INHIBITORY ACTIONS OF THE SYNTHETIC MATERIAL TO

BE THE SAME AS NATURAL SECRETIN, ESTABLISHING THAT THE

INHIBITION OF THE HEIDENHALN POUCH RESPONSE TO GASTRIN IN

FACT WAS DUE TO SECRETIN AND NOT AN IMPURITY. THIS OBSER•

VATION HAS BEEN CONFIRMED BY JOHNSON AND GROSSMAN (1969)

USING PURE SECRETIN PREPARATIONS. WAY (1970) ADMINISTERED

SECRETIN TO PAVLOV POUCH DOGS IN WHICH ACID SECRETION WAS

STIMULATED BY INSULIN AND A SUBMAXIMAL DOSE OF HISTAMINE,

THE RESULTS INDICATED THAT SECRETIN DID NOT INHIBIT VAGALLY

STIMULATED ACID SECRETION IN DOGS THAT HAD BEEN ANTRECTOM I ZED.

THE EVIDENCE REGARDING THE EFFECT OF SECRETIN ON

GASTRIC MOTILITY IS NOT AS DECISIVE AS THAT PERTAINING TO ITS ACTION ON GASTRIC SECRETION. JOHNSON ANO MAGEE (1965)

FOUND THAT SECRETIN PRODUCED SOME INHIBITION OF GASTRIC

MOTILITY, BUT NOT AS CONSISTENTLY AS DID CCK-PZ. VAGNE

£1 AL (1968) COMPARED THE EFFECTS OF NATURAL AND SYNTHETIC

SECRETIN ON GASTRIC MOTOR ACTIVITY AND FOUND THAT THEY BOTH

INHIBITED ANTRAL AND FUNDlC POUCH MOTOR ACTIVITY IN THE DOG

TO AN EQUAL EXTENT.

IN THE ABSENCE OF A BLOOD ASSAY CAPABLE OF DETECTING

ALTERED BLOOD LEVELS OF A PARTICULAR HORMONE OR HORMONES

DURING DUODENAL ACIDIFICATION, THE ONLY MEANS OF ELUCIDATING

THE PRINCIPLE INVOLVED WOULD BE TO COMPARE THE EFFECTS OF

KNOWN GASTROINTESTINAL HORMONES WITH THOSE OF DUODENAL ACID•

IFICATION. THE RESULTS OF A LARGE NUMBER OF INDEPENDENT

STUDIES REPORTED EARLIER INDICATED THAT SECRETIN AND

DUODENAL ACIDIFICATION BOTH I NHIBIT GASTRIC SECRETION AND

MOTILITY UNDER SIMILAR CIRCUMSTANCES. PRESHAW, COOK AND

GROSSMAN (1966) OBSERVED THAT WHEN THE DUODENUM WAS PERFUSED

WITH ACID, PANCREATIC ENZYME OUTPUT INCREASED. THEY

CONCLUDED THAT CCK-PZ MAY HAVE BEEN RELEASED UNDER THESE

CIRCUMSTANCES. NAKAJIMA AND MAGEE (1970) FOUND THAT ACID

AT PH 1.0 IN DUODENAL POUCHES INHIBITED PEPSIN SECRETION

AS WELL AS ACID SECRETION FROM HEIDENHAIN POUCHES, SUGGESTING

THAT CCK PLAYS A MORE IMPORTANT ROLE IN THIS MECHANISM.

THE FACT THAT SECRETIN STIMULATED PEPSIN SECRETION AND CCK

INHIBITED IT (NAKAJIMA AND MAGEE, 1970), PLUS THE OBSERVATION

BY HONG, MAGEE AND CREWDSON (1956) THAT ACID IN THE DUODENUM

CAUSED GALL BLADDER CONTRACTION, IMPLICATED THE RELEASE OF - 15 -

CCK-PZ AS WELL AS SECRETIN. JOHNSON AND GROSSMAN (1968)

COMPARED THE INHIBITORY EFFECTS OF PURE SECRETIN AT DOSES

SUBMAXIMAL FOR PANCREATIC SECRETION AND HYDROCHLORIC ACID

IN THE DUODENUM, ON GASTRIN- AND HISTAM INE-STIMULATED ACID

SECRETION. THE CLOSE SIMILARITY BETWEEN THE INHIBITORY

EFFECTS OF THESE TWO PROCEDURES LED THEM TO CONCLUDE THAT

SECRETIN WAS PROBABLY THE ONLY ENTEROGASTRONE RELEASED BY

ACID IN THE DUODENUM. IN SUPPORT OF THIS CONCLUSION WERE

THE FINDINGS OF STENING, JOHNSON AND GROSSMAN (1969A) THAT

BOTH SECRETIN AND DUODENAL ACIDIFICATION STIMULATED PEPSIN

SECRETION IN THE DOG AND CAT TO A SIMILAR DEGREE.

AS MENTIONED EARLIER, IT WAS ESTABLISHED BY GILLESPIE

AND GROSSMAN (1964) THAT AN IMPURE CCK-PZ PREPARATION WOULD

INHIBIT GASTRIN-STIMULATED ACID SECRETION. THE WORK OF

JORPES AND MUTT (1966) SUGGESTED THAT CHOLECYSTOKININ AND

PANCREOZYMIN, THE POSTULATED PANCREATIC ENZYME-STIMULATING

HORMONE OF HARPER AND RAPER (1943)» MAY BE ONE ANO THE SAME

HORMONE. THE ORIGINAL OBSERVATIONS ON THE GASTRIC EFFECTS

OF CCK-PZ WERE EXTENDED BY BROWN AND MAGEE (1967) WHO

DEMONSTRATED THAT AN IMPURE CCK-PZ PREPARATION WOULD INHIBIT

ACID SECRETION FROM HEIDENHAIN POUCHES WHICH HAD BEEN

STIMULATED BY ANTRAL PERFUSION WITH ACETYLCHOLINE OR

PEPTONE SOLUTIONS. THE CCK-PZ PREPARATIONS EMPLOYED IN

THESE STUDIES CONTAINED APPROXIMATELY 250 IVY DOG UNITS

(l.D.U.) PER MG., AND WERE ONLY 10$ PURE. To QUALIFY AS

ENTEROGASTRONE, CCK-PZ MUST BE SHOWN TO INHIBIT GASTRIC

MOTOR ACTIVITY AS WELL AS GASTRIC SECRETION. JOHNSON AND - 16 -

MAGEE (1965) DEMONSTRATED THAT A PREPARATION OF CCK-PZ

INHIBITED GASTRIC MOTILITY OF DENERVATED GASTRIC POUCHES

AS DID FAT AND HCL. THIS WAS SUPPORTED BY JOHNSON, BROWN

AND MAGEE (1966) WHO FOUND THAT CCK-PZ WAS A POTENT INHIBITOR

OF MOTILITY IN MAN.

WHILE STUDYING PANCREATIC ENZYME SECRETION, PRESHAW

AND GROSSMAN (1965) FOUND THAT SMALL DOSES OF CCK-PZ WHEN

GIVEN ALONE STIMULATED ACID SECRETION. THIS OBSERVATION

WAS CONFIRMED BY TWO GROUPS, MURAT AND WHITE (1966) AND MAGEE

AND NAKAMURA (1966) WHO SUGGESTED THAT CCK-PZ BEHAVED LIKE

GASTRIN, ACTING IN SMALL DOSES AS A STIMULANT FOR ACID

SECRETION AND IN LARGE DOSES AS AN INHIBITOR. NAKAMURA,

NAKADLMA AND MAGEE (1968) AGAIN STUDIED CCK-PZ AND ATTRIBUTED

THE STIMULATORY ACTION OF THIS MATERIAL TO THE FACT THAT BOTH

MOLECULES POSSESSED IOENTICAL AMINO ACID SEQUENCES OF THE

C-TERMINAL PENTAPEPTIDE. SINCE A SYNTHETIC PREPARATION OF

CCK-PZ WAS NOT AVAILABLE, IT COULD NOT BE STATED WITH

ASSURANCE THAT ALL ACTIONS OF CCK-PZ PREPARATIONS WERE

PROPERTIES OF THE HORMONE ITSELF.

BY METHODS ANALAGOUS TO THOSE EMPLOYED IN ATTEMPTING

TO SHOW THAT SECRETIN WAS RELEASEO DURING DUODENAL ACIDIFI•

CATION, EXPERIMENTAL EVIDENCE HAS BEEN REPORTED IN SUPPORT

OF THE HYPOTHESIS THAT CCK-PZ WAS RELEASED WHEN FAT WAS

PLACED IN THE DUODENUM. THE PRESENCE OF FAT IN THE DUODENUM

HAS BEEN SHOWN BY IVY (1934) TO BE A POTENT STIMULUS FOR GALL

BLADDER CONTRACTION AND BY WANG AND GROSSMAN (L951) TO

RELEASE PANCREATIC ENZYMES. THE FACT THAT THE GALL BLADDER - 17 -

AND PANCREATIC EFFECTS OF I NTRADUODENAL FAT WERE MIMICKED

BY CCK-PZ AND THAT GASTRIC INHIBITION BY THIS HORMONE HAD

BEEN WELL DOCUMENTED LED TO THE SUGGESTION THAT CCK-PZ

MIGHT BE IN PART, IF NOT ENTIRELY, RESPONSIBLE FOR THE

INHIBITION OF GASTRIC SECRET I ON AND MOTILITY CAUSED BY FAT

IN THE SMALL INTESTINE.

AS MENTIONED ABOVE, IN THE ABSENCE OF DEFINITIVE ASSAYS

FOR THESE POLYPEPTIDES IT IS IMPOSSIBLE TO MAKE A STATEMENT

AS TO WHICH INHIBITORY HORMONE IS RELEASED FROM THE DUOOENUM

UPON ADMINISTERING SECRETOGOGUES SUCH AS FAT OR ACID. THUS

THE CIRCUMSTANTIAL EVIDENCE CONCERNING THE ROLE OF SECRETIN

AND CCK-PZ IN GASTRIC SECRETORY AND MOTOR INHIBITION DOES

NOT RULE OUT A SEPARATE AND DISTINCT ENTEROGASTRONE OR

ENTEROGASTRONES WHOSE ROLE MAY BE SEPARATE FROM OR SUPPLE•

MENTARY TO THE ESTABLISHED DUODENAL HORMONES.

THIS INTRODUCTION HAS POINTED OUT THAT THE DUODENAL

HORMONES CCK-PZ AND SECRETIN DO NOT COMPLETELY SATISFY THE

REQUIREMENTS PROPOSED FOR THE DEFINITION OF ENTEROGASTRONE.

NEITHER HORMONE EFFECTIVELY INHIBITS HISTAMINE- OR VAGALLY-

STIMULATED ACID SECRETION, A MECHANISM WHICH OPERATES WHEN

FAT IS PLACED IN THE DUODENUM. THIS, TOGETHER WITH THE

INABILITY OF MANY WORKERS TO ISOLATE AND CHARACTERIZE AN

ENTEROGASTRONE DISTINCT FROM OTHER DUODENAL HORMONES, HAS

PROVIDED THE JUSTIFICATION FOR THE PRESENT WORK. METHODS

OPERATIVE PROCEDURES

I CHRONIC DOGS

DOGS WEIGHING 20 - 30 KG WERE USED. THE ANIMALS WERE

FASTED 18 HOURS PRIOR TO SURGERY. ANAESTHESIA WAS INDUCED

WITH 5$ SODIUM PENTOTHAL TO EFFECT (7 - 10 ML), AND MAIN•

TAINED WITH 0.5$ FLUOTHANE UTILIZING AN 02 FLOW RATE OF

2.5 - 3.0 L/MIN.

A. PREPARATION NO. 1: USED IN THE MULTIPARAMETER STUDY

OF THE ACTIONS OF 2 PREPARATIONS

CONTAINING CCK-PZ

A) BICKEL POUCH

BICKEL POUCHES WERE PREPARED WHICH WERE VAGALLY AND

SYMPATHETICALLY DENERVATED. A POUCH OF THE FUNDIC GLAND

AREA WAS CONSTRUCTED FROM THE GREATER CURVATURE AND ALLOWED

TO DRAIN TO THE EXTERIOR BY MEANS OF A METAL CANNULA (FLG. 1),

IN CONSTRUCTING THE POUCH THE VAGAL FIBRES WERE SECTIONED AND

THE SYMPATHETIC FIBRES WERE REMOVED BY STRIPPING THE NERVE

PLEXUS FROM THE SPLENIC ARTERY AND VEIN AND SEVERING ALL

MESENTERIC CONNECTIONS TO THE POUCH. THE SPLEEN WAS REMOVED.

B) ANTRAL POUCH

ALL DOGS PREPARED WITH BICKEL POUCHES WERE ALSO

PREPARED WITH VAGALLY DENERVATED POUCHES OF THE ANTRUM OF

THE STOMACH (FLG. 1). THE LATTER OPERATION WAS CARRIED OUT

APPROXIMATELY 3 - 4 WEEKS AFTER THE BLCKEL POUCH WAS PREPARED,

THE STOMACH WAS FIRST EXAMINED FOR THE SUBTLE CHANGE IN

- 18 - - 19 -

TEXTURE WHICH MAY BE USED AS AN INDICATION OF THE JUNCTION

BETWEEN THE ANTRUM AND THE FUNDUS. THE BLOOD VESSELS TO

THE LESSER CURVATURE, FROM THE PYLORUS TO THE ANGULARIS,

WERE SECTIONED. THIS ALSO REMOVED THE VAGAL FIBRES WHICH

RUN TO THE ANTRUM ALONGSIDE THE RIGHT GASTRIC ARTERY. THE

ANTRUM WAS ISOLATED BY SECTIONING BELOW THE PYLORUS AND AT

THE OBSERVED JUNCTION WITH THE FUNDUS. BEFORE CONSTRUCTING

THE POUCH, THE MUCOSA WAS EVERTED AND TESTED WITH UNIVERSAL

PH PAPER TO ENSURE THAT NO ACID-SECRETING TISSUE WAS BEING

INCORPORATED INTO THE ANTRAL POUCH. As ADDED INSURANCE

AGAINST THIS, A CUFF OF 2 — 3 CM OF ANTRAL TISSUE WAS

RESECTED. THE CUT EDGES OF THE ANTRUM WERE STITCHED TOGETHER

AND THE POUCH BROUGHT TO THE EXTERIOR THROUGH A BUTTON

WOUND IN THE ANIMAL'S FLANK. BY INCORPORATING THE PYLORUS

INTO THE POUCH, THE SPHINCTER ACTED AS A NATURAL VALVE TO

PREVENT THE LEAKAGE OF FLUID DURING PERFUSION. THE POUCH

RECEIVED AN AOEQUATE BLOOD SUPPLY VIA THE RIGHT GASTRO•

EPIPLOIC BRANCH OF THE GASTRODUODENAL ARTERY WHICH SUPPLIED

THE GREATER CURVATURE. ALTHOUGH THE POUCH WAS VAGALLY

DENERVATED, NO ATTEMPT WAS MADE TO REMOVE THE SYMPATHETIC

INNERVATION. GASTROINTESTINAL CONTINUITY WAS RESTORED BY

AN END-TO-SIDE GASTRO-JEJUNOSTOMY 30 CM DISTAL TO THE

LIGAMENT OF TREITZ (FlG. 1).

c) GALL BLADDER CANNULA

DOGS USED IN EXPERIMENTS IN WHICH GALL BLADDER

PRESSURE WAS MONITORED WERE PREPARED WITH CANNULAE OF PE 260

POLYETHYLENE TUBING INSERTED INTO THE FUNDUS OF THE GALL - 20 -

FI GURE 1 . DIAGRAM OF SURGICAL PREPARATION NO. 3 (CHRONIC OOG) USEO IN ALL STUOIES INVOLVING GASTRIC INHIBITORY POLYPEPTI0E . BLADDER. THE CANNULA WAS SECURED BY MEANS OF A PURSE STRING

SUTURE WHICH DID NOT PENETRATE THE LUMEN, AND WAS EXTERIOR•

ISED LOW ON THE ANIMAL'S FLANK.

B. PREPARATION NO. 2: USED IN THE INVESTIGATION OF THE

COMPARISON OF TWO PREPARATIONS

CONTAINING CCK-PZ IN TERMS OF

GASTRIC SECRETORY INHIBITION AND

GASTRIC SECRETORY STUDIES INVOLVING

EG STAGE I

DOGS USED IN THIS STUDY WERE PREPARED WITH BICKEL POUCHES

AND ANTRAL POUCHES ONLY.

C. PREPARATION NO. 3S USED IN ALL STUDIES INVOLVING THE

PURIFIED GASTRIC INHIBITORY POLY•

PEPTIDE DESIGNATED EG III

DOGS USED IN THESE STUDIES WERE PREPARED WITH BICKEL AND

ANTRAL POUCHES. DURING THE SURGICAL PROCEDURE INVOLVING

CONSTRUCTION OF AN ANTRAL POUCH, A THOMAS CANNULA FITTED

WITH A NYLON CAP WAS INSERTED INTO THE GASTRIC REMNANT BEFORE

THE GASTRO—JEJUNAL ANASTOMOSIS WAS COMPLETED. THIS PROVIDED

A MEANS OF DRAINING THE GASTRIC REMNANT TO THE EXTERIOR.

I I ACUTE CATS

ANAESTHESIA WAS INDUCED WITH FLUOTHANE AND MAINTAINED BY

INTRAVENOUS INJECTION OF 2$ CHLORALOSE (80 MG/KG) INJECTED

VIA A CANNULA IN THE SAPHENOUS VEIN OF THE ANIMAL. SATIS•

FACTORY ANAESTHESIA COULD BE MAINTAINED FOR 9 - 10 HOURS BY

THIS METHOD. THE TRACHEA WAS INTUBATED. THE STANDARD

PROCEDURE IN ACUTE CAT PREPARATIONS INVOLVED INTUBATION OF - 22 -

THE STOMACH AND CANNULATI ON OF THE PANCREATIC DUCT.

THE GREATER AND LESSER SPLACHNIC NERVES WERE CUT EXTRA-

PERI TONEALLY THROUGH INCISIONS IN THE FLANKS. THE ESOPHAGUS

WAS EXPOSED IN THE NECK AND INTUBATED WITH A FLEXIBLE RUBBER

TUBE, WHICH WAS PASSED INTO THE STOMACH. A MIDLINE INCISION

WAS MADE IN THE ABDOMEN AND A TAPE LIGATURE TIED TIGHTLY

AROUND THE PYLORUS, CARE BEING TAKEN NOT TO OCCLUDE THE

GASTRODUODENAL BLOOD VESSELS. THIS PROCEDURE ENSURED THAT

NONE OF THE STOMACH CONTENTS COULD BE EXPELLED INTO THE

SMALL INTESTINE. THE POSITION OF THE STOMACH TUBE WAS THEN

ADJUSTED SO THAT THE TIP WAS IN THE MOST DEPENDENT PART OF

THE STOMACH. THE TUBE WAS TIED IN POSITION WITH A THREAD

LIGATURE AT ITS ENTRANCE INTO THE ESOPHAGUS. THE PANCREATIC

DUCT WAS CANNULATED AT A POINT BEFORE IT PIERCED THE DUODENAL

WALL. A BRISK FLOW OF PANCREATIC JUICE WAS STIMULATED BY AN

INTRAVENOUS INJECTION OF SECRETIN TO AID CANNULATI ON OF THE

DUCT. IN ALL EXPERIMENTS THE VAGI WERE SECTIONED IN THE

NECK. IF TWO ROUTES OF INTRAVENOUS INJECTION WERE REQUIRED

A RADIAL VEIN WAS ALSO CANNULATED. THROUGHOUT THE COURSE OF

EXPERIMENTS BODY TEMPERATURE WAS MAINTAINED AT 36.5°C USING

A HEATING PAD CONTROLLED THROUGH A TELE-THERMOMETER (YELLOW

SPRINGS INSTRUMENT CO., MODEL 73) WHICH MONITORED BODY

TEMPERATURE VIA A RECTAL PROBE.

I I I ACUTE GUINEA PIGS

GUINEA PIGS WERE ANAESTHETIZED WITH 30$ URETHANE

(300 MG/100 G) ADMINISTERED SUBCUTANEOUSLY. WHEN SURGICAL

ANAESTHESIA WAS ACHIEVED (|N APPROXIMATELY 45 MINUTES) THE - 23 -

TRACHEA WAS INTUBATED WITH PE 260 TUBING. THE EXTERNAL

JUGULAR VEIN WAS CANNULATED TO FACILITATE INJECTIONS DURING

THE COURSE OF THE EXPERIMENT. A MIDLINE INCISION 3 - 4 CM

LONG, WHICH JUST REVEALED THE TIP OF THE X IPHISTERNUM ,

EXPOSED THE GALL BLADDER. A LIGATURE WAS TIED AROUND THE

APEX OF THE FUNDUS OF THE GALL BLADDER FOR SUBSEQUENT

ATTACHMENT TO A STRAIN GAUGE TO RECORD FORCE OF GALL BLADDER

CONTRACTION. BODY TEMPERATURE WAS MAINTAINED BY THE SAME

METHOD AS THAT USED FOR CATS.

IV ACUTE RABBITS

ANAESTHESIA WAS INDUCED WITH NEMBUTAL (35 MG/KG BODY

WEIGHT), ADMINISTERED INTRAVENOUSLY VIA THE MARGINAL EAR

VEIN AND MAINTAINED WITH 30$ URETHANE GIVEN TO EFFECT AS

REQUIRED. A MIDLINE INCISION IN THE NECK WAS MADE AND A

GLASS CANNULA INSERTED IN A TRACHEAL INCISION. AN EXTERNAL

JUGULAR VEIN WAS CANNULATED WITH POLYETHYLENE TUBING PE 60

FOR TAKING BLOOD SAMPLES AND GIVING INJECTIONS. A PE 240

POLYETHYLENE CANNULA WAS PLACEO IN THE CAROTID ARTERY AND

CONNECTED TO A SALINE-FILLED STATHAM HIGH PRESSURE TRANS•

DUCER (P23 AA) WITH BLOOD PRESSURE RECORDED ON A GlLSON PEN

RECORDER WHEN THE EXPERIMENTS INVOLVED ARTERIAL BLOOD

PRESSURE MONITORING.

COLLECTION AND ANALYSIS OF SAMPLES

I COLLECTION OF GASTRIC SECRETION

A. BICKEL POUCH COLLECTION - DOG

COLLECTIONS WERE MADE AT EITHER 10 OR 15 MINUTE INTERVALS. - 24 -

TWENTY-FIVE ML OF WATER WERE INTRODUCED INTO THE FUNDIC

POUCH AND ALLOWED TO REMAIN THERE FOR THE DURATION OF THE

PERIOD. AT THE END OF THE PERIOD THE FLUID FROM THE POUCH

WAS DRAINED AND THE POUCH WASHED OUT WITH A FURTHER 25 ML

OF WATER WHICH WAS POOLED WITH THE ORIGINAL SAMPLE. ALIQUOTS

OF THIS COMBINED SAMPLE WERE USED FOR H+ AND PEPSIN ESTIMATION.

B. GASTRIC REMNANT COLLECTION - DOG

IN THE STUDIES INVOLVING SECRETION OF THE VAGALLY INNER•

VATED GASTRIC REMNANT, GASTRIC SECRETION WAS COLLECTED

DIRECTLY BY GRAVITY THROUGH THE THOMAS CANNULA INTO A 250 ML

POLYETHYLENE BOTTLE. GASTRIC SAMPLES WERE DILUTED 1 : 10 AND

SAMPLES WERE TAKEN FOR H+ AND PEPSIN ESTIMATION.

C. WHOLE STOMACH COLLECTIONS - CAT

GASTRIC COLLECTIONS WERE MADE FROM THE WHOLE STOMACH OF

THE ANAESTHETIZED CAT IN THE SAME MANNER AS DESCRIBED FOR

FUNDIC POUCH DOGS.

D. PERFUSION OF ANTRAL POUCH AND MEASUREMENT OF ANTRAL

MOTILITY IN THE DOG

IN ORDER TO PERFUSE THE ANTRAL POUCH WITH TEST SOLUTIONS

AND TO MEASURE THE ANTRAL POUCH MOTILITY, A SERIES OF POLY•

ETHYLENE CANNULAE WERE INTRODUCED INTO THE POUCH BY WAY OF

THE PYLORIC SPHINCTER. AN ACRYLIC SUPPORT WAS MOULDED AROUND

THE TUBING (FLG. 2) AND SHAPED TO CONFORM TO THE ANIMAL'S

BODY TO HOLD THE CANNULAE SECURELY IN PLACE. THE SUPPORT

WAS HELD IN POSITION BY BINDINGS TIED AROUND THE ABDOMEN.

ONE TUBE INSERTED WELL INTO THE LUMEN OF THE POUCH AND

ATTACHED EXTERNALLY TO A SYRINGE WAS USED TO PERFUSE THE - 25 -

FIGURE 2. ACRYLIC ARRANGEMENT FOR CANNULAE FOR SIMULTANEOUS PERFUSION, DRAINAGE AND MOTOR ACTIVITY RECORDING OF ANTRAL POUCHES. A-D PERFUSION CANNULA B-C DRAINAGE CANNULA E MOTOR ACTIVITY RECORDING CANNULA F TIES G ACRYLIC SUPPORT - 26 -

ANTRAL POUCH WITH FLUID. A SECOND SALINE-FILLED TUBE

POSITIONED SLIGHTLY BEHIND THE FIRST WAS USED TO TRANSMIT

PRESSURE CHANGES IN THE POUCH TO A STATHAM HIGH PRESSURE

TRANSDUCER (P23 AA) ANO GlLSON PEN RECORDER. PERFUSION

FLUID WAS ALLOWED TO DRAIN CONTINUOUSLY FROM AROUND THE

ACRYLIC SUPPORT. WHEN ANTRAL MOTILITY WAS BEING RECORDED

THE RECORDING CANNULA WAS PERFUSED WITH SALINE AT A RATE OF

0.275 ML/MIN WITH 0.9$ SALINE TO PREVENT ITS BLOCKAGE WITH

MUCIN.

AN INDEX OF MOTOR ACTIVITY HAS BEEN EMPLOYED TO ANALYZE

THE MOTOR ACTIVITY OF ANTRAL AND FUNDIC POUCHES. THE INDEX

OF MOTOR ACTIVITY WAS CALCULATED AS FOLLOWS:

SUM OF (AMPLITUDE IN MM HG X DURATION IN SECS) OF EACH WAVE TIME OF PERIOD IN MINS X 10

E. MEASUREMENT OF FUNDIC POUCH MOTOR ACTIVITY IN THE DOG

A WATER-FILLED POLYETHYLENE TUBE IN CONTINUITY WITH THE

FUNDIC POUCH CONTENTS WAS POSITIONED BETWEEN THE METAL FUNDIC

POUCH CANNULA AND A STATHAM LOW PRESSURE TRANSDUCER (P23 BB)

CONNECTED TO A GlLSON PEN RECORDER. FUNDIC POUCH MOTOR

ACTIVITY WAS RECORDED AS PRESSURE WAVES FROM WHICH AN INDEX

OF MOTOR ACTIVITY WAS DERIVED USING THE FORMULA DESCRIBED

FOR ANTRAL MOTILITY.

F. MEASUREMENT OF GALL BLADDER ACTIVITY IN THE DOG

CHANGES IN GALL BLADDER PRESSURE IN THE DOG WERE MONITORED

USING A PERMANENT INDWELLING POLYETHYLENE CANNULA. AT THE

BEGINNING OF EXPERIMENTS IN WHICH GALL BLADDER PRESSURE WAS

MONITORED, BILE WAS ALLOWED TO FLOW OUT OF THE CANNULA AND

THE PRESSURE IN THE GALL BLADDER ADJUSTED TO APPROXIMATELY - 27 -

5 MM HG. THE CANNULA WAS THEN ATTACHED TO A STATHAM LOW

PRESSURE TRANSDUCER (P23 BB) AND PRESSURE CHANGES MONITORED

ON THE GlLSON PEN RECORDER.

II ANALYSIS OF SAMPLES

A. GASTRIC SECRETION

A) ESTIMATION OF H+ CONCENTRATION

H+ CONCENTRATION OF GASTRIC SAMPLES WAS DETERMINED BY

TITRATION TO PH 7.0 WITH 0.01 M NAOH USING A RADIOMETER

TITRATOR ASSEMBLY (TlTRATOR 11) AND A MAGNETIC VALVE. H+

WAS EXPRESSED AS/JEQ/10 OR 15 MIN PERIOD.

B) ESTIMATION OF PEPSIN

PEPSIN OUTPUT WAS DETERMINED BY A MODIFICATION OF THE

METHOD OF ANSON AND MLRSKY (1932). IN THIS TECHNIQUE THE

GASTRIC SAMPLE WAS ALLOWED TO DIGEST A HAEMOGLOBIN SUBSTRATE

AND THE AMOUNT OF TYROSINE LIBERATED WAS MEASURED SPECTRO•

PHOTOMETRY AL L Y AT 275 NM. VALUES FOR TYROSINE WERE READ

FROM A STANDARD CURVE FOR TYROSINE CONCENTRATION. IN THE

METHOD OF ANSON AND MlRSKY (1932) THE PEPSIN DIGESTION OF

THE HAEMOGLOBIN SUBSTRATE WAS STOPPED BY THE ADDITION OF

TRICHLORACETIC ACID AND THE RESULTANT MIXTURE FILTERED.

FOLIN-CIOCALTEAU REAGENT WAS THEN ADDED TO THE FILTRATE AND

THE LIGHT ABSORBANCE OF THE PRODUCT OF THE COLOR REACTION

WAS RE A 0 AT 640 NM. THE MODIFICATION OF THIS METHOD INVOLVED

MEASURING THE LIGHT ABSORBANCE OF THE FILTRATE OF THE

HAEMOGLOBIN DIGESTION MIXTURE DIRECTLY, ELIMINATING THE

COLOR REACTION. WHEN PEPSIN ESTIMATIONS USING THE MODIFIED

METHOD WERE COMPARED WITH THE ORIGINAL TECHNIQUE, NO - 28 -

DIFFERENCE IN RESULTS WAS OBTAINED. THE HAEMOGLOBIN SUB•

STRATE HAD BEEN DIGESTED WITH DIFFERENT CONCENTRATIONS OF

A STANDARD PEPSIN PREPARATION (WORTHINGTON B I 0 C HE M I C A L S ) ,

(FIG. 3A). EMPLOYING THIS CURVE AND THE STANDARD TYROSINE

CURVE (FIG. 3B), PEPSIN VALUES WERE PLOTTED AGAINST JJQ OF

TYROSINE (FIG. 3C) SO THAT PEPSIN OUTPUT COULD BE EXPRESSED

EITHER AS AMOUNT OF TYROSINE LIBERATED OR AS pQ PEPSIN.

B. PANCREATIC SECRETION

A) VOLUME WAS MEASURED DIRECTLY BY COLLECTION IN

GRADUATED TUBES.

B) PROTEIN OUTPUT WAS MEASURED AS FOLLOWS: THE

ASSUMPTION WAS MADE THAT AT A WAVELENGTH OF 280 NM A SOLUTION

OF MIXED PANCREATIC PROTEIN CONTAINING 1 MG/ML HAD AN OPTICAL

DENSITY OF 1.8. THIS PROVIDED AN INDEX FOR DETERMINING

PROTEIN CONCENTRATION OF SAMPLES OF PANCREATIC JUICE.

OD OF PJ / -—£ X OILUTION FACTOR = MG/ML PROTEIN IN PANCREATIC JUICE

C. ESTIMATION OF BLOOD GLUCOSE

BLOOD GLUCOSE LEVELS WERE DETERMINED BY THE 0 - TOLUIDINE

METHOD OF DUBOWSKI (1962). THIS WAS A COLORIMETRIC METHOD

BASED ON THE FACT THAT 0 - TOLUIDINE, A PRIMARY AROMATIC AMINE,

REACTED RELATIVELY SELECTIVELY WITH GLUCOSE, YIELDING A STABLE

GREEN COLOUR WHICH ADHERED TO THE BEER-LAMBERT LAW OVER A WIDE

RANGE OF CONCENTRATIONS.

ASSAY METHODS

I ASSAY OF CHOLECYSTOKININ ACTIVITY OF GASTRIC INHIBITORY

PREPARAT I ONS - 29 -

/Jfl. Pepsin

FIGURE 3. (A) THE ABSORBANCE AT 275 NM OF THE PRODUCTS OF DIGESTION OF HAEMOGLOBIN SUBSTRATE WITH DIFFERENT CONCENTRATIONS OF A STANDARD PEPSIN PREPARATION.

(B) STANDARD TYROSINE CURVE, ADHERING TO BEER'S LAW OVER THE RANGE OF CONCENTRATIONS USED.

(c) PLOT OF UG TYROSINE AGAINST JUG PEPSIN FROM THE DATA OF FlGURE 3 (A) AND (B). - 30 -

CHOLECYSTOKININ ASSAYS WERE CARRIED OUT ON THE GUINEA PIG

GALL BLADDER PREPARATION OESCRIBEO ON PAGES 21 AND 22. THE

ASSAY USED WAS A MODIFICATION OF THE METHOD OF LJUNGBERG (1964).

THE SILK LIGATURE TIED TO THE APEX OF THE GALL BLADDER WAS

ATTACHED TO A STATHAM 3 02 STRAIN GAUGE (G 10B - 3 - 350±).

THE STRAIN GAUGE WAS CONNECTED TO A GILSON PEN RECORDER AND

CALIBRATED SO THAT A WEIGHT OF 2.0 GRAMS PRODUCED A PEN

DEFLECTION OF 3 CM.

THE CCK-PZ PREPARATION USED AS A STANDARD IN ALL CCK-PZ

ASSAYS WAS OBTAINED FROM THE G.I.H. RESEARCH LABORATORY,

STOCKHOLM (BATCH NO. 26841). THE MATERIAL CONTAINEO APPROXI•

MATELY 250 I.D.U. CCK-PZ/MG AND is REFERRED TO THROUGHOUT

THIS THESIS AS 10$ PURE CCK-PZ.

IN CARRYING OUT AN ASSAY, A LOG DOSE-RESPONSE CURVE WAS

FIRST OBTAINED USING THE STANDAR0 CCK-PZ PREPARATION. FLG. 4A

SHOWS RECORDINGS OF THE GALL BLAODER RESPONSE TO 4 DOSES OF

STANDARD CCK-PZ PREPARATION. FORCE OF GALL BLADDER CONTRACTION

IN GRAMS WAS PLOTTED AGAINST LOG DOSE OF CCK-PZ IN I.D.U.

(FIG. 4B). THIS RELATIONSHIP WAS SHOWN TO BE LINEAR IN A

RANGE OF ABOUT 0.05 I.D.U. - 1.0 I.D.U. OF CCK-PZ IN THE

GUINEA PIG. WEIGHED SAMPLES OF UNKNOWN WERE THEN INJECTED

AND THE CCK-PZ ACTIVITY/MG DETERMINED BY MEASURING THE GALL

BLADDER RESPONSE IN GRAMS AND CONVERTING THE RESPONSE TO I.D.U.

OF CCK-PZ USING THE DOSE-RESPONSE CURVE. AFTER FORCE OF

CONTRACTION HAD RETURNED TO PREINJECTION LEVELS (APPROXIMATELY

5 MINUTES), AN INJECTION OF STANDARD WAS GIVEN TO ENSURE THAT

THE DOSE RESPONSE RELATIONSHIP TO THE STANDARD CCK-PZ PREPAR•

ATION STILL CONFORMED TO THE STANDARD CURVE. WHEN THE RESPONSE - 31 -

FIGURE 4. (A) RESPONSE OF THE GUINEA-PIG GALLBLADDER IN GRAMS FORCE TO INTRAVENOUS INJECTIONS OF FOUR DOSES OF THE STANDARD CCK PREPARATION ('10$', 250 I.D.U./MG BATCH NO* 26841).

(B) LOG DOSE RESPONSE CURVE PREPARED FROM THE DATA REPRESENTED IN FlGURE 4 (A). - 32 -

OF THE ANIMAL PREPARATION BEGAN TO DECREASE (3 - 4 HOURS),

THE EXPERIMENT WAS TERMINATED.

II ASSAY OF ENTEROGASTRONE ACTIVITY

ASSAYS FOR ENTEROGASTRONE ACTIVITY WERE CARRIED OUT ON

DOGS PREPARED AS DESCRIBED IN PREPARATIONS 2 AND 3, PAGE 20.

INHIBITORY MATERIAL FROM.EG STAGE II TO PURE GASTRIC INHIBI•

TORY POLYPEPTIDE (EG III) WAS ASSAYED FOR ENTEROGASTRONE

ACTIVITY IN DOGS WITH A THOMAS CANNULA IN THE GASTRIC REMNANT

(PREPARATION 3)•

INHIBITORY MATERIAL WAS GIVEN AS A ONE HOUR INTRAVENOUS

INFUSION AGAINST A PLATEAU OF BLCKEL POUCH H+ SECRETION

STIMULATED BY 1.5 JUG/KG/HR OF GASTRIN PENTAPEPTIDE. ENTERO•

GASTRONE ACTIVITY WAS EXPRESSED AS "$ INHIBITION11, WHICH WAS

CALCULATED BY COMPARING H+ OUTPUT DURING THE PERIODS OF

GREATEST ACID INHIBITION WITH THE MEAN OUTPUT OF THE 3

PLATEAU PERIOOS OF H+ SECRETION PRIOR TO THE START OF THE

INFUSION OF THE INHIBITOR. AN EXAMPLE OF AN ASSAY FOR

ENTEROGASTRONE ACTIVITY OF A FRACTION RESULTING FROM THE

FRACTIONATION OF EG STAGE I ON CM-CELLULOSE IS SHOWN IN

FIG. 5. THIS FRACTION PRODUCED ONLY 39$ INHIBITION OF ACID

SECRETION BY THE ABOVE CALCULATION.

III ASSAY OF SECRETIN ACTIVITY

SECRETIN ACTIVITY WAS ASSAYED FOR IN THE ACUTE CAT,

PREPARED AS DESCRIBED ON PAGES 20 AND 21. A CONTINUOUS

INTRAVENOUS INFUSION OF SECRETIN WAS GIVEN AT A DOSE WHICH

WOULD YIELD AT LEAST 1.0 ML OF PANCREATIC JUICE PER 15 MINUTE - 33 -

Gastrin Pentapeptide

I.O/jg./kg./hr.

I i I i i i i 1 1 1 1 1 1 1 0 2 4 6 8 10 12 14 15 min. periods

FIGURE 5. EXAMPLE OF AN ASSAY FOR ENTEROGASTRONE ACTIVITY IN THE CHRONIC DOG. AN INFUSION OF 1.0/)G/KG/HR OF THE FRACTION TO BE ASSAYED WAS INFUSED FOR ONE HOUR ON A PLATEAU OF BlCKEL POUCH H+ SECRETION STIMULATED BY 1.5 /JG/KG/HR OF GASTRIN PENTAPEPTIDE. - 34 -

PERIOD. AFTER AT LEAST THREE PERIODS DURING WHICH THE VOLUME

WAS CONSTANT (±10$) AND THE PROTEIN OUTPUT LOW (<2.0 MG

PROTEIN/15 MIN), RAPID INTRAVENOUS INJECTIONS OF SAMPLES OF

MATERIAL TO BE ASSAYED WERE GIVEN. THESE INJECTIONS WERE

FOLLOWED BY INJECTIONS OF SECRETIN. ALL PREPARATIONS USED

WERE RESPONSIVE TO AN INJECTION OF 0.5 UNITS OF SECRETIN OR

LESS. THIS REPRESENTED THE SENSITIVITY IN DETERMINING

SECRETIN ACTIVITY OF A GIVEN FRACTION.

FIG. 6 SHOWS THE RESULTS OF ONE SUCH ASSAY EXPERIMENT.

SINCE SECRETIN ACTIVITY WAS REMOVED AT AN EARLY STAGE IN THE

PURIFICATION OF G.I.P. (FIG. 13), THE SECRETIN ASSAY WAS

PRINCIPALLY DESIGNED TO QUALITATIVELY CONFIRM THE ABSENCE OF

SECRETIN ACTIVITY IN THE PRODUCTS OF THE LATER PURIFICATION

STAGES.

IV ASSAY FOR BLOOD PRESSURE CHANGES AND PYROGENIC EFFECTS

A. BLOOD PRESSURE

ARTERIAL BLOOD PRESSURE WAS RECORDED ON A GILSON PEN

RECORDER DURING THE INJECTION OF WEIGHED QUANTITIES OF

MATERIALS TO BE ASSAYED. THE PREPARATION USED WAS THE ACUTE

RABBIT PREPAREO AS DESCRIBED ON PAGE 22.

B. PYROGENIC EFFECTS

PYROGENIC EFFECTS OF SAMPLES WERE ASSAYED FOR IN DOGS BY

MONITORING RECTAL TEMPERATURE BEFORE, DURING AND AFTER

INFUSION OF THE MATERIAL BEING TESTED.

SOURCE OF HORMONE PREPARATIONS

GASTRIN PENTAPEPTIDE: AYERST LABORATORIES (MONTREAL, CANADA) - 35 -

6.0 units /hr Secretin

3.0r 0.5 U 10.0 jag 0.5 U 25.0/jg 0.5 U Secretin G.I.P Secretin G.I.R Secretin

ID O 1 I I I 2.0

oc CL *o- 1.0

0.0 _L 8 10 12 14 16 18 15 min. periods

FIGURE 6. EXAMPLE OF AN ASSAY FOR SECRETIN ACTIVITY IN THE

ACUTE CAT. SINGLE I'.V. INJECTIONS OF 0.5 u SECRETIN AND 10 AND 25 JUG OF G.I.P. WERE GIVEN ON A BACKGROUND OF PANCREATIC JUICE STIMULATED

BY A CONSTANT INFUSION OF 6 u/HR OF SECRETIN. - 36 -

SYNTHETIC HUMAN GASTRIN It IMPERIAL CHEMICAL INDUSTRIES

(MACCLESFIELD, ENGLAND)

INSULIN: CONNAUGHT LABORATORIES (TORONTO, CANADA)

GLUCAGONt ELI LILLY AND COMPANY (TORONTO, CANADA)

CCK-PZ (250 I.D.U./MG) BATCH NO. 26841 : G.I.H. RESEARCH

LABORATORIES (STOCKHOLM, SWEDEN)

CCK-PZ (1500 I.D.U./MG) BATCH No. 26751: G.I.H. RESEARCH

LABORATORIES (STOCKHOLM, SWEDEN)

SECRETIN BATCH No. 17042: G.I.H. RESEARCH LABORATORIES

(STOCKHOLM, SWEDEN)

ANALYSIS OF DATA

IN ALL STUDIES INVOLVING INFUSION OF A GASTRIC INHIBITOR

AGAINST A CONSTANT INFUSION OF A STIMULANT, THE VALUES OF H+

SECRETION AND MOTOR ACTIVITY INDEX WERE EXPRESSED AS RATIOS

OF THE MEAN OF THREE PLATEAU PERIODS. IN ANY ONE EXPERIMENT

THE RATIOS CONSISTED OF EACH OBSERVATION/MEAN OF 3 PLATEAU

PERIODS. THE CRITERION FOR A PLATEAU WAS 3 CONSECUTIVE

PERIODS WITH LEVELS OF H+ SECRETION ± 10$ OF ONE ANOTHER.

THIS METHOD WAS EMPLOYED BECAUSE OF VARYING SECRETORY CAPA•

CITIES OF 0 I FFERENT BlCKEL POUCHES TO THE SAME DOSE OF

STIMULANT. THIS POINT IS ILLUSTRATED BY REFERENCE TO THE

RAW DATA WHICH IS INCLUDED IN TABULAR FORM. PEPSIN LEVELS

WERE NOT EXPRESSED AS RATIOS BECAUSE IN MANY CASES, INFUSION

OF GASTRIC INHIBITORY POLYPEPTIDE REDUCED PEPSIN LEVELS TO

ZERO.

THE RATIOS OF VALUES .OF H+ SECRETION AND MOTOR ACTIVITY

WERE TRANSFORMED INTO LOGARITHMS IN OROER TO APPLY TESTS OF - 37 -

SIGNIFICANCE. LOGARITHMS OF RATIOS OF H+ AND MOTOR ACTIVITY

VALUES FOR THE THIRD PLATEAU PERIOD WERE COMPARED WITH THE

VALUES OF THE PERIOD OF MAXIMUM INHIBITION USING THE STUDENT

T TEST FOR PAIRED DATA. WHERE TESTS OF SIGNIFICANCE WERE

CARRIED OUT ON PEPSIN DATA, COMPARISONS WERE MADE OF PEPSIN

VALUES (MG TYROSINE/10 OR 15 MIN) OF THE PERIOD OF MAXIMUM

INHIBITION WITH THE CORRESPONDING VALUES FOR THAT PERIOD

IN CONTROL STUDIES, USING THE STUDENT T TEST FOR UNPAIRED

DATA . RESULTS

I MULTIPARAMETER STUDY ON THE EFFECTS OF THE INTRAVENOUS

INFUSION OF 2 PREPARATIONS CONTAINING CHOLECYSTOKININ-

PANCREOZYMIN (CCK-PZ)

ANIMALS USED IN THIS STUDY WERE PREPARED AS DESCRIBED

ON P. 18 OF THE METHODS SECTION, PREPARATION NO. 1. IN

THESE ANIMALS IT WAS POSSIBLE TO RECORD SIMULTANEOUSLY

H+ SECRETION, PEPSIN SECRETION ANO MOTOR ACTIVITY IN BlCKEL

POUCHES, GALL BLADDER PRESSURE AND MOTOR ACTIVITY IN ANTRAL

POUCHES. THE PARAMETERS WERE MEASURED AND THE DATA

ANALYZED AS DESCRIBED IN THE METHODS SECTION, P. 23 • AT

THE BEGINNING OF THE EXPERIMENTS, CONTROL DATA WERE

OBTAINED FOR AT LEAST THREE 1O-MIN PERIODS. CONDITIONS WERE

CONSIDERED TO BE BASAL IF THE LEVELS OF H+ SECRETION FROM

THE BICKEL POUCHES WERE < 100 /JEQ/10 MIN AND INTRA-GALL

BLADDER PRESSURE WAS <6.0 MM HG. AFTER THE ESTABLISHMENT

OF BASAL CONDITIONS, THE ANIMALS RECEIVED AN INTRAVENOUS

INFUSION OF EITHER 10$ CCK-PZ (250 I.D.U./MG) OR 40$ CCK-PZ

(1,500 I.D.U./MG) FOR A 10-MIN PERIOD. THE CCK-PZ PREPARA•

TIONS WERE ASSAYED PRIOR TO THEIR USE IN THIS STUDY,

USING THE GUINEA PIG GALL BLADDER PREPARATION, DESCRIBED ON

P. 22 OF THE METHODS SECTION AND THE ASSAY TECHNIQUE

DESCRIBED ON P. 28 . IN THIS ASSAY, THE 10$ PURE CCK-PZ

WAS CONSIDERED TO POSSESS THE POTENCY ASCRIBED TO IT ANO

THE 40$ PURE MATERIAL WAS ASSAYED AGAINST THIS STANDARD.

THIS INITIAL ASSAY OF GALL BLADDER ACTIVITY WAS ESSENTIAL

- 38 - - 39 -

IN CALCULATING COMPARABLE DOSES OF BOTH PREPARATIONS.

WHERE LEVELS OF SIGNIFICANCE ARE REPORTED, COMPARISONS WERE

MADE BETWEEN DATA OBTAINED FROM THE PERIODS IMMEDIATELY

PRIOR TO THE INFUSION OF THE CCK-PZ AND THOSE IMMEDIATELY

AFTER THE CESSATION OF THE INFUSION.

A) THE EFFECT OF CCK-PZ ON INTRA-GALL BLADDER PRESSURE

COMPARABLE DOSES OF BOTH PREPARATIONS (0.2 I.D.U./KG/

MIN) WERE INFUSED AND THE RESULTS PRESENTED IN FIG. 7 (TABLE

1). EACH POINT IN THE FIGURE REPRESENTS THE MEAN i S.E. OF

13 OBSERVATIONS IN 3 ANIMALS. THERE WAS NO SIGNIFICANT

DIFFERENCE (P y 0.50) IN THE GALL BLADDER RESPONSES BETWEEN

THE 2 PREPARATIONS. THIS INDICATED THAT BOTH PREPARATIONS

WERE EQUALLY POTENT AS STIMULANTS OF GALL BLADDER CONTRACTION.

THESE RESPONSES REPRESENTED APPROXIMATELY 50 - 60$ OF

MAXIMUM GALL BLADDER PRESSURE IN THESE ANIMALS. A GALL

BLADDER PRESSURE LESS THAN CONTROL LEVELS WAS FOUND TO

OCCUR FOLLOWING THE CESSATION OF THE CCK-PZ INFUSION.

B) THE EFFECT OF CCK-PZ ON ANTRAL MOTOR ACTIVITY

BOTH CCK-PZ PREPARATIONS WERE FOUND TO HAVE EFFECTS

ON THE INDEX OF ANTRAL MOTOR ACTIVITY WHICH WERE NOT

SIGNIFICANTLY DIFFERENT (P> 0.30). THE RESULTS ARE SHOWN

IN FIG. 8 AND SUMMARIZED IN TABLE II. EACH POINT IN THE

FIGURE REPRESENTS THE MEAN jt S.E. OF 13 OBSERVATIONS IN 3

AN IMALS.

c) THE EFFECT OF CCK-PZ ON PEPSIN OUTPUT FROM

BlCKEL POUCHES

THE PEPSIN LEVELS OF THE PERIOD PRIOR TO CCK-PZ TABLE I

EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ1 ON INTRA-GALL BLADDER PRESSURE (MM HG)

CONTROL PERIODS CCK-PZ POST- i NFUSION PERIODS' DOG EXP NO. 1

K 61 3.0 3.0 5.0 9.0 2.0 1.0 2.0 4.0 K 109 6.0 6.0 6.0 14.0 8.0 2.0 0.0 0.0 K 33 0.0 1.4 0.4 3.7 0.0 o K 30 4.8 0.0 0.0 10.4 2.0 0.0 0.0 0.0 C 38 6.0 4.4 4.8 17.2 4.8 0.0 o 0.0 o J 37 1.8 2.0 2.0 10.0 2.0 0.0 7^ 0.0 0.0 I J 36 2.4 3.6 4.4 10.4 4.0 D 0.0 J 35 1.8 1.8 2.0 14.0 7.0 0.0 INI 0.0 J 34 4.8 5.2 6.4 10.8 3.0 0.0 3.4 3.0 3.4 11.1 3.6 0.3 0.7 i S.E. 0.7 0.6 0.8 1.3 0.9 0.2 0.5

CONTROL PERIOOS CCK-PZ POST-INFUSION PERIOOS 0.0 0.0 K 29 4.0 5.2 5.2 10.4 2.0 4.0 9.3 1.8 0.0 0.0 K 32 3.6 4.7 CD 0.0 0.0 K 39 8.8 8.0 6.4 12.8 3.6 0.0 0.0 K 46 7.2 7.6 9.2 11.6 1.2 3.6 0.0 0.0 K 53 6.0 4.8 4.8 13.6 o 6.4 0.0 0.0 K 63 2.0 2.4 2.4 6.8 0.0 0.0 K 61 5.0 4.0 5.0 8.0 0.0 o 6.0 5.0 K 108 6.0 6.0 6.0 10.0 6.0 o 0.0 0.0 7K J 44 2.4 3.6 3.6 12.8 1.6 I 0.0 0.0 T3 J 48 1.6 3.2 3.6 10.0 0.0 0.0 0.0 rsi J 51 5.2 4.8 5.6 15.6 4.4 0.0 0.0 0.0 J 52 4.8 3.2 4.0 12.0 4.8 0.0 0.0 C 43 4.8 3.6 4.8 13.6 4.7 4.7 5.0 11.0 2.7 t S.E. 0.6 0.5 0.5 0.8 0.6

1 0.2 I .D.U./KG/MIN 2 10 MIN DURATiON - 41 -

0.2 u/kg / min. CCK-PZ I I

10 min. periods

FIGURE 7. GALLBLADDER PRESSURE CHANGES PRODUCED BY THE INTRAVENOUS INFUSION OF M0$' AND '40$' PURE CCK-PZ (0.2 I.D.U./KG/MIN FOR 10 MIN). EACH POINT REPRESENTS THE MEAN OF 13 OBSERVATIONS ON 3 DOGS. IN THIS AND SUBSEQUENT FIGURES THE VERTICAL BARS ARE THE STANDARD ERRORS OF THE MEAN. DATA REPRESENTED IN THIS GRAPH IS TAKEN FROM TABLE I.

THESE PERCENTAGE PURITIES WERE GIVEN BY THE SUPPLIER AND ARE ARBITRARY. ACTUAL IVY DOG UNIT NUMBERS USED IN THE THESIS ARE CORRECT. TABLE I I

1 2 EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ ON ANTRAL MOTILITY

CONTROL PERIODS' CCK-PZ POST—INFUSION PERIOOS' DOG EXP NO. 1

J 8 7.3 7.3 7.3 91.7 11.7 11.2 10.1 J 12 7.4 18.6 26.6 125.2 97.7 18.1 6.4 J 34 63.2 68.8 61.9 115.3 70.5 42.2 24.6 J 35 39.6 22.1 31.8 131.0 72.4 6.4 0.0 J 36 70.3 7.7 32.7 146.5 63.3 15.3 2.0 o J 37 30.4 31.8 17.3 61.6 29.9 4.8 0.0 •A K 30 137.3 0.0 64.0 45.1 8.6 20.1 o 0.0 o K 33 26.8 153.9 2.8 112.8 104.0 12.2 3.7 K 61 0.0 0.0 0.0 25.8 16.0 4.0 0.0 -v H 105 26.5 38.5 56.8 155.8 100.0 53.6 H 106 56.3 47.6 34.0 151 .2 200.0 64.8 25.0 H 108 12.9 15.8 23-4 45.0 26.6 25.5 40.7 C 38 19.0 18.1 142.2 97.9 45.4 68.6 25.JL 4» 38.8 31.5 22.6 102.6 67.0 23.2 22.2 10.6 12.6 5.2 6.6 I S.E. 8.9 9-9 4.9

CONTROL PERIOPS' CCK-PZ POST-iNFUSiON PERIOOS'

J 39 60.0 59-0 28.9 64.0 12.5 25.2 43.6 CD J 44 74.8 118.8 42.3 116.0 59.7 41.7 72.0 J 48 135.0 103.1 81.6 102.3 38.8 103.0 171.1 J 51 79-2 109-3 143.9 183.1 53.8 40.1 87.2 o J 52 145.1 121.0 98.1 39.4 8.4 44.6 73.8 51.5 70.4 20.5 5.5 K 29 8.3 0.0 12.3 o K 32 0.0 11.4 18.8 53.4 25.9 14.2 12.9 o 174.0 124.2 38.1 10.8 7Z K 46 12.3 41.6 26.0 I 10.0 53 18.5 23-2 8.3 177.1 95.7 8.7 T3 K M K 63 14.0 14.4 17.7 18.5 14.6 0.0 18.5 K 61 7.3 0.0 5.2 35.9 11.5 0.0 2.8 34.6 C 108 20.4 16.3 9.6 52.5 47.3 8.9 47.9 51.0 43.2 90.9 53.7 36.3 49.9 t S.E. 14.7 12.9 11.8 16.1 11.8 9.0 15.1

1 0.2 I .O.U./KG/MIN 2 MOTOR ACTIVITY INOEX 3 10 MIN 0URAT I ON - 43 -

02 u/ kg /min CCK - PZ

40% CCK-PZ

10 % CCK-PZ

0 I 2 3 4 5 6 7 10 min. periods

FIGURE 8. CHANGES IN ANTRAL MOTOR ACTIVITY AFTER THE INTRA VENOUS INFUSION OF '10$' AND '40$' PURE CCK-PZ (0.2 I.D.U./KG/MIN FOR 10 MIN). THIRTEEN OBSER• VATIONS ON 3 DOGS WITH EACH HORMONE PREPARATION. DATA IS TAKEN FROM TABLE II. - 44 -

INFUSION WERE SIGNIFICANTLY DIFFERENT (P< 0.05) IN THE

TWO SETS OF EXPERIMENTS INVOLVING THE 2 PURITIES OF CCK-PZ

(FIG. 9; TABLE III). THIS DIFFICULTY AROSE IN PART BECAUSE

IT WAS NOT POSSIBLE TO DETERMINE PEPSIN OUTPUTS UNTIL THE

END OF THE EXPERIMENTS AND THUS FLUCTUATING PEPSIN LEVELS

COULD NOT BE DETECTED BEFORE CCK-PZ INFUSION. ALTHOUGH THE

ABOVE PRECLUDED MAKING A DEFINITIVE STATEMENT ABOUT PEPSIN

RESPONSE, IT APPEARED THAT THE 40$ PURE MATERIAL MAY HAVE

STIMULATED PEPSIN OUTPUT TO A GREATER DEGREE THAN THE 10$

MATERIAL. EACH POINT IN THE FIGURE REPRESENTS THE MEAN

jt S.E. OF 13 OBSERVATIONS IN 3 DOGS.

o) THE EFFECT OF CCK-PZ ON H+ SECRETION FROM THE

BICKEL POUCHES

ADMINISTRATION OF BOTH THE 10$ AND THE 40$ PURE CCK-

PZ INCREASED H+ OUTPUT FROM THE BlCKEL POUCHES. THE PURER

MATERIAL WAS MUCH MORE POTENT THAN THE 10$ PURE MATERIAL

IN STIMULATING H+ OUTPUT, THE PEAK OUTPUTS BEING SIGNIFI•

CANTLY DIFFERENT (P< 0.005) (F|G. 10; TABLE IV). THE 40$

CCK-PZ PREPARATION PRODUCED A MEAN PEAK ACID OUTPUT OF

155.5 /JEQ rT*"/1 0 MIN AS COMPARED TO 111.9 JUEQ H+/"l 0 MIN

AFTER INFUSION OF 10$ CCK-PZ. EACH POINT IN THE FIGURE

REPRESENTS THE MEAN - S.E. OF 13 OBSERVATIONS IN 3 DOGS.

THE H+ RESPONSE TO THE PURER MATERIAL WAS ALSO OF MUCH

LONGER DURATION.

II COMPARISON OF TWO PREPARATIONS CONTAINING CCK-PZ IN

TERMS OF GASTRIC SECRETORY INHIBITION

THIS STUDY WAS UNDERTAKEN AS A RESULT OF THE FINDING THAT TABLE I I I 1 2 EFFECT OF- NTRAVENOUS INFUSION OF CCK-PZ ON PEPSIN OUTPUT

CONTROL PERIODS' CCK-PZ POST-iNFUS i ON PERIOOS' Doc EXP NO. 1 J 3 0.825 0.350 0.475 1.026 0.600 0.325 0.225 J 12 0.356 0.300 0.375 0.503 0.250 0.100 0.075 J 35 1.122 0.850 0.624 0.618 0.400 0.200 0.250 T J 36 0.257 0.202 0.153 0.212 0.102 0.0 0.102 J 37 0.780 0. 300 0.100 0.714 0.306 0.1 56 0.300 - K 30 1.716 0.936 0.416 0.468 0.312 0.153 0.153 £ K 33 0.312 0.350 0.450 0.756 0.408 0.300 0.364 0 K 61 0.202 0.202 0.300 0.364 0.536 0.312 0.306 o K 109 0.400 0.312 0.324 0.432 0.309 0.104 0.052 ? K 107 2.236 1 .224 0.936 1 .288 0.927 0.600 1.377 £ H 105 0.663 0.480 0.832 0.833 0.404 0.400 H 106 0.0 0.053 0.106 0.159 0.052 0.104 0.108 H 108 0.500 0.300 0.530 0.0 0.0 0.0 0.0 J 34 0.867 0.850 0.714 0.459 0.306 0.306 0.208 0.770 0.520 0.500 0.610 0.380 0.260 0.280 t S.E. 0.160 0.090 0.080 0.100 0.060 0.050 0.090

CONTROL PERIOOS' CCK-PZ POST-INFUSION PERIODS K 29 0.500 0.357 0.300 0.572 0.702 0.312 0.156 • K 32 1.050 0.663 0.350 0.770 0.702 0.306 0.102 K 46 0.0 0.0 0.0 0.153 0.094 0.0 0.153 CD K 53 0.104 0.106 0.051 0.378 0.265 0.104 0.0 1.122 0.900 0.714 1.484 1 .007 0.200 K 63 0.594 o K 61 0.210 0.103 0.0 0.156 0.327 0.104 0.156 0.416 0.780 K 108 0.416 0.306 0.361 0.578 0.550 o J 39 0.159 0.208 0.468 0.204 0.106 0.102 o 0.105 1.070 1 .240 J 44 0.0 0.561 0.196 0.900 0.800 I 0.330 0.150 0.350 0.250 0.102 ~v J 48 0.100 0.102 Kl 0.200 1.530 2.080 0.561 0.364 J 51 0.312 0.520 0.300 J 52 0.0 0.052 0.350 0.0 1 .224 0.0 1.352 0.720 C 43 0.500 0.350 0.364 0.364 0.340 0.310 0.330 0.750 0.550 0.290 0.330 0.160 0.090 0.070 0.100 I S.E. 0.110 0.080 0.110

1 0.2 I.D.U./KG/MIN 2 MG TYROS INE/10 M IN 3 10 MIN DURATION - 46 -

FIGURE 9. CHANGES IN PEPSIN OUTPUT FROM BICKEL POUCHES AFTER THE INTRAVENOUS INFUSION OF '10$' AND '40$' CCK-PZ (0.2 I.D.U./KG/MIN FOR 10 MIN). THIRTEEN OBSER• VATIONS ON 3 DOGS WITH EACH HORMONE PREPARATION. DATA IS TAKEN FROM TABLE III. TABLE IV

EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ^ ON H+ OUTPUT

3 CONTROL PERIODS^ CCK-PZ POST-iNFUSiON PERIODS DOG EXP NO. 1 2 3 4 5 6 7 J 8 33 33 25 67 58 35 25 J 12 100 106 75 121 70 60 52 J - 34 43 45 45 152 117 71 57 > J 35 117 112 109 146 135 70 63 ' J 36 110 106 102 217 132 63 63 J 37 71 75 65 163 135 119 120 K 30 104 83 46 67 78 56 46 K 33 33 37 35 102 102 62 52 o K 61 95 95 90 119 164 114 89 ? K 109 45 42 41 60 72 62 57 .1 K 107 114 64 36 46 54 35 41 ^ C 38 107 112 110 183 130 100 105 H 106 37- 48 58 70 36 52 60 H 105 43 43 52 108 35 49 H 108 15 15 14 57 • 16 26 40 71.1 67.3 60.2 111.9 88.9 64.9 61.4 + S.E. 9.4 8.3 7.9 13.4 11.6 7.0 7.0

3 OST- NFUS ON CONTROL PERIOOS' CCK-PZ P i i "ERIODS K 29 50 43 45 78 162 96 42 K 32 45 38 35 107 129 92 61 CD K- 46 119 110 115 198 263 224 183 * K 53 68 64 51 119 180 177 161 K 63 43 35 33 90 148 105 70 o K 61 89 84 76 135 237 187 163 >P> K 108 57 54 46 68 114 74 45 o J 39 103 99 187 202 175 100 102 9. J 44 180 82 44 122 78 80 80 -L, J 48 39 25 30 100 70 41 46 M J 52 52 56 47 109 105 78 173 H 43 100 105 338 280 275 145 x 74.2 54.0 65.5 136.2 155.5 122.6 104.1 + S.E. 12.2 7.8 12.6 20.2 21.4 19.5 14.7

1 0.2 I.O.U./KG/MIN 2 pEo H+/10 MIN 3 10 MIN CURAT I ON FIGURE 10. CHANGES IN H SECRETION FROM THE BICKEL POUCHES AFTER THE INTRAVENOUS INFUSION OF '10$' AND '40$' CCK-PZ (0.2 t.D.U./KG/MiN FOR 10 MIN). THIRTEEN OBSERVATIONS ON 3 DOGS WITH EACH HORMONE PREPAR• ATION. DATA IS TAKEN FROM TABLE IV. - 49 -

THE 40$ PURE CCK-PZ PREPARATION HAD A GREATER ACID STIMU•

LATORY EFFECT ON BLCKEL POUCHES THAN THE 10$ PURE CCK-PZ

PREPARATION. A POSSIBLE EXPLANATION FOR THIS DIFFERENCE

WAS THAT AN INHIBITORY MATERIAL WAS PROPORTIONATELY REMOVED

DURING THE FURTHER PURIFICATION. A COMPARISON OF THE

INHIBITORY ACTIVITY OF THE TWO CCK-PZ PREPARATIONS AGA INST

GASTRIN PENTAPEPTIDE-STIMULATED BLCKEL POUCH H+ SECRETION

WAS UNDERTAKEN TO TEST THIS HYPOTHESIS. THIS DOSE WAS

CALCULATED FROM THE DATA PRESENTED IN F|G. 11, OBTAINED

FROM THE INTRAVENOUS INFUSION OF GASTRIN PENTAPEPTIDE IN

DOSES OF 0.75» 1.5, 3.0 AND 6.0 JUG/KG/HR ON 2 OCCASIONS

TO EACH OF 2 DOGS. DOSES OF 3.0 JUG/KG/HR AND ABOVE INDUCED

VOMITING IN OVER HALF OF THE EXPERIMENTS AT THOSE LEVELS.

A DOSE OF 1.5 AJG/KG/HR WAS SELECTED AS YIELDING 65 - 70$

OF MAXIMUM H+ SECRETION.

AFTER AT LEAST 3 PERIODS OF BASAL H+ SECRETION,

(<100/JEQ H+/10 MIN) A CONTINUOUS INTRAVENOUS INFUSION OF

1.5 UG/KG/HR OF GASTRIN PENTAPEPTIDE WAS STARTED AND MAIN•

TAINED THROUGHOUT THE COURSE OF THE EXPERIMENT. UPON

ACHIEVING A PLATEAU OF H+ SECRETION (l.E., H+ VALUES OF

3 PERIODS t 10$ OF ONE ANOTHER), A 10 MINUTE INFUSION OF

CCK-PZ (EITHER 10$ OR 40$) AT A DOSE LEVEL OF 0.2 I.D.U./

KG/MIN WAS GIVEN. FOUR TO SIX FIFTEEN MINUTE PERIODS OF

INFUSION OF GASTRIN PENTAPEPTIDE WERE REQUIRED TO ESTABLISH

A PLATEAU OF H+ SECRETION. THE CCK-PZ PREPARATIONS WERE

ASSAYED IN THE GUINEA PIG GALL BLAODER PREPARATION TO CHECK

THE UNITAGE PRIOR TO EXPERIMENTATION. BOTH PREPARATIONS - 50 -

FIGURE 11. THE H OUTPUT FROM BICKEL POUCHES OF 2 DOGS, L AND P, IN RESPONSE TO THE I.V. INFUSION OF GASTRIN PENTAPEPTIDE IN DOSES OF 0.75» 1»5» 3.0 AND 6.0 /JG/KG/HR. EACH POINT REPRESENTS THE MEAN OF 6 OBSERVATIONS IN EACH ANIMAL MADE AFTER A PLATEAU OF H+ SECRETION HAD BEEN ESTABLISHED. - 51 -

SIGNIFICANTLY INHIBITED CONTROL LEVELS OF H+ SECRETION

STIMULATED BY GASTRIN PENTAPEPTIDE (10$ CCK-PZ - P< 0.0005,

40$ CCK-PZ - P < 0.0005). THE PURER MATERIAL WAS LESS

POTENT IN INHIBITING GASTRIN STIMULATED H+ SECRETION

(48$) THAN THE 10$ PURE PREPARATION (64$) (F|G. 12;

TABLE V). THE LEVELS OF H+ SECRETION AT THE PERIOD OF PEAK

INHIBITION WERE SIGNIFICANTLY DIFFERENT (P < 0.025).

LEVELS OF H+ WERE REDUCED FROM A MEAN OF 613.1 JUEQ H+/10 MIN

TO 240.0 JUEQ H+/10 MIN BY 10$ CCK-PZ, WHEREAS 40$ CCK-PZ

ONLY REDUCED H+ SECRETION FROM 600.4 AIEQ H+/1 0 MIN TO

372.8 JUEQ H+/10 MIN. EACH POINT IN THE FIGURE REPRESENTS

THE MEAN ±S.E. OF 9 OBSERVATIONS IN 3 DOGS. IN THIS AND

SUBSEQUENT STUDIES, PERCENTAGE INHIBITION WAS CALCULATED

BY COMPARING THE VALUE OF THE PARAMETER UNDER STUDY IN THE

PERIOD OF GREATEST INHIBITION WITH THE CORRESPONDING MEAN

VALUE IN CONTROL STUDIES. PERCENTAGE INHIBITION WAS CAL•

CULATED FROM RATIOS WHERE RESULTS WERE EXPRESSED AS RATIOS.

Ill PREPARATION, ASSAY, AND GASTRIC INHIBITORY ACTIONS

OF EG STAGE 1

RESULTS OF THE ABOVE STUDIES LED TO ATTEMPTS TO

SEPARATE A GASTRIC INHIBITOR DISTINCT FROM CCK-PZ USING THE

CRUDER 10$ CCK-PZ PREPARATIONS. THE STARTING MATERIAL FOR

THIS PURIFICATION WAS THE CCK-PZ PREPAREO BY THE METHOD OF

JORPES AND MUTT (1961). A FLOW CHART OF THE PURIFICATION

OF GASTRIC INHIBITORY POLYPEPTIDE IS SHOWN IN FIG. 13. IN

A TYPICAL EXPERIMENT, 1.0 G OF STARTING MATERIAL WAS SUBJECTED

TO GEL FILTRATION USING SEPHADEX G-50 FINE. THE COLUMN

SIZE WAS 5 X 90 CM AND THE MATERIAL WAS ELUTED WITH TABLE V

EFFECT OF INTRAVENOUS INFUSION OF CCK-PZ ON \V OUTPUT1- STIMULATED BY GASTRIN PENTAPEPTIDE"'

PLATEAU PERIODS CCK-PZ POST-INFUSION PERIODS DOG EXP NO. 1 2 3 4 5 6 7 M 134 481 462 416 342 182 259 • 323 M 150 532 599 615 572 389 275 275 M 110 583 666 660 432 224 270 370 o K 145 335 297 354 254 196 238 331 K 148 406 370 380 270 167 198 292 o 302 342 286 127 181 231 o K 149 313 7^ H 151 661 644 633 443 194 308 504 I ~0 H 147 843 869 900 534 470 291 456 M H 146 1165 1336 1218 646 211 329 608 261 .0 376.6 X 589.7 617.3 613.1 419.8 240.0 + S.E. 91.1 109.5 97.5 47.2 37.6 16.2 40.8

PLATEAU PERIODS CCK-PZ POST-INFUSION PERIOOS

M 138 . 558 577 543 462 364 413 431 00 M 137 666 585 700 528 330 487 609 M 136 1035 1038 986 787 564 560 600 o K 139 434 374 410 364 280 290 330 K 140 403 405 353 267 195 202 200 364 o K 141 445 468 450 329 208 3H o 682 616 551 496 609 696 H 142 614 I 378 448 494 H 143 748 767 671 475 540 502 600 H 144 604 700 675 535 611.8 621.7 600.4 477.5 372.8 425.0 480.4 53.8 + S.E. 65.2 69.6 63.7 50.9 45.4 44.5

1 0.2 I.D.U;/KG/MIN 2 JJEQ H+/10 MIN 3 1.5 JUG/KG/HR - 53 -

l.5/jg./kg./hr. Gastrin Pentapeptide •o o 1096 or 40% CCK-Pz 0.2 I.D.U./kg./min. 0. T

c 1 o 1.2 O tO o c o 0> 0.8 4> .C

o o .4 - o 0 © © Control data

CO A--A 10% CCK-Pz(250 I.D.U./mg.) o 40% « ( 1500 . .. )

CL O.O ~i 1 1 r~ 4— -i r 6 8 O + 2 10 min. periods X

FIGURE 12. EFFECT OF 10 MINUTE I.V. INFUSIONS OF 0.2 I.D.U./ MIN OF '10$' CCK-PZ (250 I.D.U./MG) AND '40$' CCK-PZ (1500 I.D.U./MG) ON BICKEL POUCH H+ SECRE• TION STIMULATED BY 1.5JUG/KG/HR OF GASTRIN PENTA• PEPTIDE. DUE TO VARIABILITY IN ABSOLUTE AMOUNTS OF H+ PRODUCED BY DIFFERENT DOGS TO THE SAME DOSE OF STIMULI, H+ IS EXPRESSED AS A RATIO OF THE MEAN OF 3 PLATEAU PERIODS. CONTROLS RECEIVED ONLY GASTRIN PENTAPEPTIDE THROUGHOUT THE EXPERIMENT. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 9 OBSERVATIONS IN 3 DOGS. EXPERIMENTAL DATA FROM WHICH RATIOS WERE CALCULATED IS PRESENTED IN TABLE V. - 54 -

FIGURE 13 SUMMARY OF PURIFICATION OF GASTRIC INHIBITORY POLYPEPTIDE

CCK-PZ H INHI•I tI 0* PROCEDURE ACTIVITY OF OUtPU*' I .D.U./MO

HEAT COACULATEO HOC OUOOENQ-JEJUNAL MUCOSA

ACETIC ACID

ACETIC ACID EXTRACT

J^^ALGINIC ACID ADSORPTION

NACL PRECIPITATE CONTAINING SN, CCK-PZ AND EG ACTIVITY

METHANOL

METHANOL SQ L U B LE FRACTION (SN)

METHANOL INSOLUBLE FRACTION

CM-CELLULOSE PH 6.5 i CRUOE "CCK-PZ" 22-25

TEAE-CELLULOSE PH 9.1 — •

10# PURE CCK-PZ 250-275 SEPHADEX G50 > (PHOSPHATE) PH 8.0 404 PURE CCK-PZ 1 ,500

EG STAGE I 10

CM-CELLULOSE FRACTION A 6 30 AMMONIUM BICARBONATE (0.02M>M)) Ip H 7.8 FRACTION C 120 0 (& o.,2M; ) EG STAGE I I (FRACTI ON B) 7.5 70

SEPHADEX G25 FINE (ACETIC ACID)

EG STAGE I I I 2.0 75

GASTRIC INHIBITORY POLYPEPTIDE

1 INHIBITORY ACTIVITY ASSAYED AS DESCRIBED ON P. 32 0.25 M PHOSPHATE BUFFER, PH 8.0. FRACTIONS OF 50 ML WERE

COLLECTED. ABSORBANCE WAS MEASURED AT 280 NM THROUGH A

1.0 CM LIGHT PATH. FlG. 14 SHOWS THE CHROMATOGRAM OBTAINED.

EACH FRACTION WAS ASSAYED FOR CCK ACTIVITY USING THE GUINEA

PIG GALL BLADDER TECHNIQUE AND EMPLOYING THE ASSAY PROCEDURE

DESCRIBED ON P. 28 OF THE METHODS SECTION. ALL FRACTIONS

NOT DEMONSTRATING CCK ACTIVITY WERE P00LE0,DESALTED AND

CONCENTRATED USING ALGINIC ACID ANO DEAE - SEPHADEX.

THE FRACTIONS FROM THE SEPHADEX G-50 COLUMN WHICH POSSESSED

LITTLE CCK ACTIVITY WERE POOLED AND DILUTED 10X WITH

DISTILLED WATER. THE PH OF THE SOLUTION WAS LOWERED TO

2.5 WITH 2.0 M HCL AND THE ABSORBANCE AT 215 NM, THROUGH A

1.0 CM LIGHT PATH WAS MEASURED. IN A TYPICAL PROCEDURE THIS

WAS FOUND TO BE ABOUT 2.0. ALGINIC ACID (7.0 - 10.0 G

WET WEIGHT) WHICH HAD BEEN PREVIOUSLY WASHED WITH ACID-

ETHANOL, FOLLOWED BY DILUTE AQUEOUS HCL AND WATER, WAS ADDED

TO THE OILUTED SOLUTION AND STIRRED FOR 15 MIN. THE

ABSORBANCE AT 215 NM WAS READ AND ADSORPTION CONSIDERED

COMPLETE WHEN THIS WAS <. 0.10. THE ALGINIC ACID WITH

ADSORBED POLYPEPTIDES WAS COLLECTED BY VACUUM FILTRATION ON

2 LAYERS OF WHATMAN 541 FILTER PAPER AND WASHED SEVERAL TIMES

WITH ICE COLD 0.005 M HCL. THE ADSORBED POLYPEPTIDES WERE

ELUTED FROM THE ALGINIC ACID WITH APPROXIMATELY 20 - 30 ML

ICE COLD 0.2 M HCL, APPLIED IN SMALL ALIQUOTS. THE ELUATE

WAS PASSED THROUGH A 1.5 X 10 CM COLUMN OF DEAE - SEPHADEX

WHICH HAD BEEN EQUILIBRATED WITH 0.2 M ACETIC ACID. THE

EFFLUENT, ABOUT 40 ML ANO CL- FREE, WAS LYOPHILIZED FOR 24 - 56 -

0.25 M Phosphate Buffer pH 8.0

t

10 12 14 16 18 20 22 24 26 26 30 Sample Number

FIGURE 14. ' FRACTIONATION OF STARTING MATERIAL ON SEPHADEX G-50 FINE. 1.0 G OF STARTING MATERIAL APPLIED TO A COLUMN 5 X 90 CM AND ELUTED WITH 0.25 M PHOSPHATE BUFFER, PH 8.0. FRACTION SIZE 50 ML. ABSORBANCE MEASURED AT 280 NM THROUGH A 1.0 CM LIGHT PATH. SHADED AREA DEMONSTRATED ENTERO- GASTRONE ACTIVITY. - 57 -

HOURS. YIELDS AT THIS STAGE RANGED FROM 150 - 250 MG OF

MATERIAL. THIS PREPARATION WAS REFERRED TO AS EG STAGE I

(EG I). EG I POSSESSED ^10 I.D.U. CCK-PZ/MG AND WAS FOUND

TO HAVE NO SECRETIN ACTIVITY, EMPLOYING THE SECRETIN ASSAY

PROCEDURE OUTLINED ON P. 32 OF THE METHODS SECTION.

A. EFFECTS OF EG I ON GASTRIC SECRETION AND MOTOR ACTIVITY

DOGS USED IN THIS STUDY WERE PREPARED AS DESCRIBED ON

P. 21 OF THE METHODS SECTION. GASTRIC JUICE WAS COLLECTED

AND ANALYZED FOR H AND PEPSIN AS OUTLINED IN THE METHODS

SECTION. THE EXPERIMENTAL DESIGN WAS THE SAME AS IN THE

PREVIOUS STUDY, WITH EG I BEING GIVEN AS A 10 MINUTE INTRA•

VENOUS INFUSION (1.0 JUG/KG/MIN) AGAINST A PLATEAU OF H+

SECRET I ON STIMULATED BY 1.5>IG/KG/HR OF GASTRIN PENTAPEPTIDE.

A; BICKEL POUCH H SECRETION

H+ OUTPUT IS EXPRESSED AS A RATIO OF THE MEAN OF 3

CONTROL PERIODS AS IN THE„PREVIOUS STUDY. INFUSI ON OF 1.0

JUG/KG/MIN OF EG I PRODUCED 70$ (P < 0.05) INHIBITION OF

CONTROL LEVELS OF H+ SECRETION (F|G. 15, TABLE V I ). As

WITH THE 10$ CCK-PZ, MAXIMUM INHIBITION DID NOT OCCUR UNTIL

THE PERIOD AFTER THE INFUSION OF THE INHIBITOR. EG I

REDUCED LEVELS OF H+ SECRETION FROM A MEAN OF 618.2 /JEQ H+/

10 MIN IN CONTROL STUDIES TO A LOW OF 168.1 /jEQ H /10 MIN

IN THE FIRST P0ST-INFUSI 0N PERIOD. EACH CONTROL POINT IN

THE FIGURE REPRESENTS THE MEAN 1 S.E. OF 9 OBSERVATIONS IN

3 DOGS. EACH EG I POINT REPRESENTS THE MEAN i S.E. OF 7

OBSERVATIONS IN 3 DOGS. TABLE VI

EFFECT OF INTRAVENOUS INFUSION OF EG I ON H+ OUTPUT1 STIMULATED BY GASTRIN PENTAPEPTIDE

PLATEAU PERIOOS' DOG EXP No. 1 2 3 4 5 6 7 8 9

K 156 286 270 281 292 281 297 313 297 260 K 155 249 254 262 275 276 275 273 275 300 K 154 208 167 214 232 278 257 210 212 212 588 M 153 672 636 627 695 610 577 545 554 M 152 481 493 488 486 470 487 452 475 487 M 151 605 553 594 554 555 550 554 575 497 738 785 H 1 59 748 803 817 823 872 839 773 1168 1200 H 160 1043 1386 1183 1407 1334 1266 1269 870 H 1 50 788 900 1050 1035 888 888 851 900 564.0 606.8 612.8 644.3 618.2 604.0 575.5 580.8 573.8 X 107.1 108.2 + S.E. 94.1 127.9 115.8 130.8 119.1 112.7 104.9

PLATEAU PERIODS' EG I POST-iNFUSiON PERIODS 120 K 173 187 200 205 200 100 114 153 179 286 352 H 185 545 635 651 730 354 150 180 348 506 H 170 1363 1081 1192 826 166 260 404 178 248 375 M 187 465 519 521 302 157 297 228 398 437 M 188 594 591 610 432 190 323 283 402 350 H 722 260 300 302 265 77 182 161 183 172 191 K 172 297 260 228 212 133 423.8 168.1 181.8 240.7 301.7 341.4 X 530.1 512.3 529.8 .4 18.5 31.5 40.5 45.4 ± S.E. 150.2 114.5 129.8 96 34.3

1 >>EQ H+/10 MIN 2 1.5 UG/KG/HR 3 GASTRIN PENTAPEPTI DE ONLY 4 10 MIN INFUSION (1 .0 JJG/K G/M I N ) - 59 -

CO l.5/jg./kg./hr. Gostrin Pentapeptide "CT kO_

^2 1.2 c— o o rO o 0.8 o s: 1 n o 0.4 -i' r o Control data o or A—A 1.0 /jg./kg./min. ro o 0.0 T r— -1 T 1 1 1 r 8 10 3 2 4 Q. 15 min. periods O + I

FIGURE 15. EFFECT OF A 10 MINUTE INTRAVENOUS INFUSION OF 1.0 JUG/KG/MIN OF EG STAGE I ON BICKEL POUCH H+ SECRETION STIMULATED BY 1.5 JUG/KG/HR OF GASTRIN PENTAPEPTIDE. EACH POINT ON THE CONTROL CURVE REPRESENTS 9 EXPERIMENTS IN 3 DOGS. EACH POINT ON THE EGI CURVE REPRESENTS 7 EXPERIMENTS IN 3 DOGS. THE RATIOS ARE CALCULATED FROM DATA IN TABLE VI. - 60 -

B) BICKEL POUCH PEPSIN SECRETION

TEN MINUTE INFUSION OF EG 1 PRODUCED 70$ (P < 0.005)

INHIBITION OF CONTROL LEVELS OF PEPSIN SECRETION (F|G. 16;

TABLE VII). PEPSIN LEVELS DROPPED FROM A CONTROL VALUE OF

0.501 MG TYROSINE/10 MIN TO 0.091 MG TYROSINE/10 MIN IN

THE FI RST POST-INFUSI ON PERIOD. EACH CONTROL POINT IN THE

FIGURE REPRESENTS THE MEAN — S.E. OF 7 OBSERVATIONS IN 3

DOGS. EACH EG 1 POINT REPRESENTS THE MEAN iS.E. OF 5

OBSERVATIONS IN 3 DOGS. As MENTIONED EARLIER, MEAN PEPSIN

VALUES WERE PLOTTED AS MG TYROSINE/15 MIN RATHER THAN RATIOS.

EG STAGE 1 BECAME THE STARTING POINT FOR SEVERAL

PURIFICATION PROCEDURES IN AN ATTEMPT TO ACHIEVE A HIGHLY

PURIFIED AND POTENT GASTRIC INHIBITOR DISTINCT FROM THE

GASTROINTESTINAL HORMONES SECRETIN AND CCK-PZ. ONE SUCH

PROCEDURE INVOLVED THE RECHROMAT0GRAPHY OF EG 1 ON SEPHADEX

G25 FINE, ELUTING WITH 0.2 M ACETIC ACID (FLG. 13). THREE

FRACTIONS WERE OBTAINED AND LYOPHILIZED. THESE FRACTIONS

WERE ASSAYED FOR CCK-PZ AND ENTEROGASTRONE ACT I V I TY (F|G. 17).

INFUSION OF FRACTION 2 YIELDED 73$ INHIBITION OF GASTRIN

PENTAPEPTIDE STIMULATED SECRETION AND POSSESSED 11 I.D.U.

CCK-PZ/MG. THIS WAS THE PUREST PREPARATION OF THE INHIBITOR.

THIS FRACTION IS REFERRED TO AS EG1(G25). THE FACT THAT

EG 1 PRODUCED 70$ INHIBITION OF GASTRIN PENTAPEPTIDE

STIMULATED H+ SECRETION (FlG. 15) INDICATED THAT NO SIGNIFI•

CANT INCREASE IN POTENCY OF THE INHIBITORY MATERIAL WAS

ACHIEVED AS A RESULT OF FRACTIONATION OF EG 1 ON SEPHADEX

G25. FOR THIS REASON THE TWO PREPARATIONS WERE LOOKED UPON TABLE VI I

EFFECT OF INTRAVENOUS INFUSION OF EGI ON PEPSIN OUTPUT STIMULATED BY GASTRIN PENTAPEPTIDE

,3 PLATEAU PERIODS DOG EXP NO. 1 2 3 4 5 6 7 8 9 K 1 56 0.572 0.424 0.429 0.385 0.530 0.371 0.318 0.371 0.432 K 155 0.212 0.212 0.159 0.216 0.212 0.108 0.159 0.162 0.162 H 150 2.451 1.458 0.840 0.901 1 .092 0.840 0.448 0.336 M 112 1.450 0.689 0.741 0.605 0.616 0.535 0.606 0.707 0.650 K 79 0.336 0.265 0.318 0.300 0.840 0.212 0.300 0.226 0.350 K 78 0.110 0.108 1.020 0.054 0.108 0.106 0.106 0.159 0.378 M 110 0.159 0.106 0.053 0.161 0.108 0.110 0.162 0.399 0.230 X 0.755 0.466 0.466 0.374 0.501 0.326 0.299 0.335 0.367 S.E. 0.332 0.182 0.182 0.110 0.144 0.104 0.060 0.070 0.063

3 4 3 PLATEAU PERIODS EGI POST-iNFUSiON PERIODS K 173 0.364 0.364 0.312 0.364 0.300 0.212 0.208 0.312 0.208 H 185 0.550 0.580 0.360 0.351 0.156 0.116 0.107 0.162 0.110 M 187 0.165 0.171 0.056 0.054 0.0 0.0 0.0 0.0 0.0 M 188 0.162 0.108 0.108 0.0 0.0 0.050 0.0 0.212 0.195 H 172 1.762 1.166 1.040 0.0 0.0 0.0 0.0 0.0 0.0 0.600 0.477 0.477 0.154 0.091 0.080 0.063 0.137 0.103 + S.E. 0.298 0.190 0.190 0.080 0.060 0.031 0.041 0.060 0.045

1 MG TYROSINE/10 MIN 2 1.5 PG/KG/HR 3 GASTRIN PENTAPEPTIDE ONLY 4 10 MIN INFUSION (1.0 JUG/KG/MIN) - 62 -

FIGURE 16. EFFECT OF A 10 MINUTE I.V. INFUSION OF 1.0 JJG/KG/ MIN OF EG I ON PEPSIN OUTPUT FROM BLCKEL POUCHES STIMULATED BY 1.5 ^UG/KG/HR OF GASTRIN PENTAPEPTIDE. EACH PLOT ON THE CONTROL CURVE REPRESENTS THE MEAN OF 7 EXPERIMENTS IN 3 DOGS. EACH PLOT ON THE CURVE WITH EG I REPRESENTS 5 EXPERIMENTS IN 3 DOGS. PEPSIN VALUES TAKEN FROM TABLE VII. - 63 -

% Inhibition I.D.U. CCK/mg.

8O-1 r 160

60 M20 •D • -•C—L o> o E •J- N X 40 h 80 O O

o ci

•£ 20 h 40 c

^ 0 E.G.I. E.G.I. E.G.I. (G-25) (G-25) 3 (G-25) Fraction I Fraction 2 Fraction 3

FIGURE 17. RESULTS OF CCK-PZ AND ENTEROGASTRONE ASSAYS OF FRACTIONS RESULTING FROM THE CHROMATOGRAPHY OF EG I ON SEPHADEX G25. - 64 -

AS BEING EQUAL IN INHIBITORY STUOIES.

B. EFFECTS OF EG I (G25) ON GASTRIC SECRETION AND MOTOR

ACTIVITY STIMULATED BY ENDOGENOUS GASTRIN

ANIMALS USED IN THESE EXPERIMENTS WERE AS OESCRIBEO IN

PREPARATION NO. 2, P. 21 . ENDOGENOUS GASTRIN WAS RELEASED

BY PERFUSING ISOLATED ANTRAL POUCHES WITH 0.1$ ACETYLCHOLINE

IN 0.9$ NACL SOLUTION AT A RATE OF 0.5 ML/MIN. AFTER 3

CONSECUTIVE 10 MINUTE PERIODS OF PLATEAU LEVELS OF H+ /

SECRET I ON FROM THE BlCKEL POUCHES, EG I (G25) WAS GIVEN

AS A 10 MINUTE INTRAVENOUS INFUSION (1.0 JUG/KG/MIN).

A) BlCKEL POUCH H+ SECRET I ON

EG I (G25) PRODUCED 66$ (P < 0.0025) INHIBITION OF CONTROL LEVELS OF H SECRETION (FlG. 18; TABLE VIII).

LEVELS OF H SECRETION FELL FROM A MEAN OF 433.9 JJEQ H /

10 MIN IN THE CONTROL STUDIES TO 121.6 JUEQ H+/1 0 MIN IN

THE SECOND POST INFUSION PERIOD. EACH POINT IN THE FIGURE

REPRESENTS THE MEAN i S.E. OF 8 OBSERVATIONS IN 3 DOGS.

B) BlCKEL POUCH PEPSIN SECRETION

EG I (G25) PRODUCED 65$ (P <0.01) INHIBITION OF

CONTROL LEVELS OF PEPSIN SECRETION STIMULATED BY ENDOGENOUS

GASTRIN (FIG. 19; TABLE IX). PEPSIN LEVELS DROPPED FROM

A CONTROL VALUE OF 0.511 MG TYROSINE/10 MIN TO 0.179 MG

TYROSINE/10 MIN IN THE SECOND POST INFUSION PERIOD.

c) ANTRAL MOTOR ACTIVITY

EG I (G25) PRODUCED 70$ (P < 0.05) INHIBITION OF

CONTROL VALUES OF THE INDEX OF ANTRAL MOTOR ACTIVITY TABLE VI I I

1 EFFECT OF INTRAVENOUS INFUSION OF EGI (G25) ON H+ OUTPUT STIMULATED BY PERFUSION OF ANTRAL POUCHES WITH 0.1$ ACH

PLATEAU PERIODS DOG EXP NO. ,1 2 3 4 5 6 7 8 9

J 106 440 473 386 417 386 340 545 465 525 H ' 120 642 562 778 773 703 738 710 719 720 H 127 290 310 365 446 477 604 691 455 486 H 126 519 470 704 629 676 559 551 718 637 J 97 444 435 336 294 309 319 244 244 363 M 75 350 341 329 315 316 371 416 351 337 K 74 237 194 270 212 1 56 146 151 148 165 K 73 286 355 290 308 354 396 379 424 465 X 400.6 392.5 432.2 424.2 422.1 433.9 460.9 440.5 462.3 + S.E. 48.2 40.8 68.9 67.1 66.4 66.5 71.0 71.7 62.1

3 EGI PLATEAU PERIODS POST-iNFUSiON PERI OPS (G25) M 104 549 620 562 453 113 125 188 196 265 K 96 265 300 319 230 120 97 186 265 340 221 M 95 224 265 280 197 135 82 163 245 250 K 94 215 235 216 190 164 225 259 267 158 260 313 K 93 571 577 605 405 233 235 213 M 91 270 291 340 260 110 120 175 209 130 196 227 317 K 86 201 239 260 193 135 78 36 98 181 222 K 72 620 567 541 306 327.8 386.8 390.3 276.8 136.0 121.6 187.5 228.3 270.6 19.6 11.6 16.6 + S.E. 60.7 59.7 54.3 34.1 16.3 17.1

1 JJEQ H+/10 MIN 2 ANTRAL PERFUSION WITH ACH ONLY 3 10 MIN INFUSION (1.0 /JG/KG/MIN) - 66 -

0.1% Ach (Antral Perfusion)

FIGURE 18. EFFECT OF A 10 MINUTE I.V. INFUSION OF 1.0 JUG/KG/ MIN OF EGI (G25) ON BLCKEL POUCH H+ SECRETION STIMULATED BY ENDOGENOUS GASTRIN RELEASED BY PERFUSION OF ANTRAL POUCHES WITH 0.1$ ACETYL• CHOLINE. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 8 OBSERVATIONS IN 3 DOGS. RATIOS CAL• CULATED FROM EXPERIMENTAL DATA IN TABLE VIII. TABLE IX

EFFECT OF INTRAVENOUS INFUSION OF EGI (G25) ON PEPSIN OUTPUT STIMULATED BY PERFUSION OF ANTRAL POUCHES WITH 0.1$ ACH

2 PLATEAU PERIODS 8 DOG EXP NO. 1 2 3 4 5 6 7 9 2.360 J 106 0.594 0.206 0.309 0.206 0.567 0.864 0.803 2.279 0.530 J 97 3.216 0.689 0.392 0.368 0.416 0.364 0.208 0.416 0.378 M 75 0.540 0.416 0.336 0.371 0.399 0.324 0.318 0.378 K 74 0.368 0.212 0.106 0.156 0.208 0.106 0.153 0.155 0.156 0.817 0.825 1.375 K 73 0.643 0.636 0.520 0.810 0.648 0.856 0.638 K 41 0.884 0.663 0.530 0.695 1.696 0.754 0.540 0.638 0.108 M 76 0.696 0.810 0.412 0.208 0.159 0.315 0.212 0.204 0.402 0.435 0.685 0.805 X 1.277 0.518 0.372 0.584 0.511 0.300 + S.E. 0.379 0.089 0.044 0.094 0.194 0.114 0.104 0.280

3 2 EGI 2 PLATEAU PERIODS (G25) POST-1NFUS1 ON PERIODS 0.0 M 104 0.106 0.105 0.055 0.103 0.0 0.0 0.0 0.0 0.648 K 96 0.742 0.459 0.550 0.306 0.408 0.408 0.530 0.510 M 95 0.832 0.424 0.216 0.141 0.0 0.102 0.050 0.0 0.0 0.200 K 94 0.104 0.108 0.100 0.051 0.050 0.053' 0.105 0.214 K 93 1.904 1.320 1 .026 0.594 0.318 0.153 0.810 0.771 0.810 0.306 M 91 0.663 0.408 0.350 0.100 0.150 0.0 0.102 0.052 0.648 K 86 0.990 0.780 1.144 0.795 0.520 0.300 0.530 1.664 0.416 0.318 K 72 1.166 1.026 1.140 0.459 - 0.520 0.416 0.275 0.578 0.572 0.318 0.245 0.179 0.343 0.315 0.461 X 0.813 0.054 0.094 0.100 0.200 + S.E. 0.204 0.151 0.164 0.094 0.077

1 MG TYROS INE/10 MIN 2 ANTRAL PERFUSION WITH ACH ONLY 3 10 MIN INFUSION (1.0 JUG/KG/HIN) - 68 -

0.1% Ach (Antral Perfusion)

(G25-Fr2)

1.6

1.4- E g 1.2 - \ CD c l.O- 'coo 0.8- E

0.6 CL o 0.4

to CL CD 0_ 0.2 H o—o control data 1.0 jug./kg./min

0.0 —r- I 4 n 1 10 min. periods 8 9

FIGURE 19. EFFECT OF A 10 MINUTE I.V. INFUSION OF 1.0 JUG/KG/ HR OF EG I (G25) ON BICKEL POUCH PEPSIN SECRETION STIMULATED BY ENDOGENOUS GASTRIN. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 8 OBSERVATIONS IN 3 DOGS. PEPSIN VALUES TAKEN FROM TABLE IX. - 69 -

(FIG. 20AJ TABLE X). THE DATA REPRESENT THE MEAN t S.E.

OF 7 OBSERVATIONS IN 3 DOGS. FIG. 20B SHOWS A SAMPLE

RECORDING OF ANTRAL MOTOR ACTIVITY FROM AN EXPERIMENT IN

WHICH A 10 MINUTE INFUSION OF 1.0 JUG/KG/MIN OF EG I (G25)

WAS GIVEN ALONG WITH ANTRAL PERFUSION OF 0.1$ ACH.

IV PREPARATION AND ASSAY OF EG STAGE II

IN A TYPICAL EXPERIMENT 60 MG EG STAGE I WAS DISSOLVED

IN 5.0 ML 0.01 M AMMONIUM BICARBONATE AND THE PH ADJUSTED

TO 7.8 WITH 0.01 M NH3 IN WATER. A SLIGHT PRECIPITATE

FORMED, WHICH WAS REMOVEO BY CENTRIFUGATI ON ANO DISCARDED.

THE CLEAR SOLUTION WAS ALLOWED TO SINK INTO A COLUMN

(1.5 X 19 CM) OF CM-CELLULOSE (WHATMAN CM 11), EQUILIBRATED

AND DEVELOPED WITH 0.01 M NH4HCO3 BUFFER, PH 7.8, THEN WITH

0.2 M NH4HCO3 BUFFER PH 8.0. THE ELUATE WAS COLLECTED IN

FRACTIONS OF 5.0 ML AT A FLOW RATE OF 130 ML/HR.

THE ABSORBANCE OF THE FRACTIONS WAS MEASURED AT 280

NM THROUGH A 1.0 CM LIGHT PATH. THE RESULTS ARE SHOWN IN

FIG. 21A. THE SAMPLES MAKING UP EACH OF THE 3 FRACTIONS

WERE POOLED, LYOPHILIZED AND ASSAYED FOR CCK ANO ENTERO•

GASTRONE ACTIVITY. THE RESULTS OF THE ASSAYS (FIG. 21B)

I NO I CATEO THAT FRACTION B (SAMPLES' 10 - 48) POSSESSED THE

MOST SIGNIFICANT ENTEROGASTRONE ACTIVITY. THE YIELDS OF

FRACTIONS A, B AND C WERE 6.05* 20.6 AND 24.3 MG RESPECT•

IVELY. FRACTION B WAS REFERRED TO AS EG STAGE II (EG I I). TABLE X

1 EFFECT OF INTRAVENOUS INFUSION OF EGI (G25) ON ANTRAL MOTILITY STIMULATED BY PERFUSION OF ANTRAL POUCHES WITH 0.1$ ACH

PLATEAU PERIODS DOG EXP NO. 1 8

J 106 214.9 330.1 266.0 289.0 316.0 243.4 195.8 190.1 172.0 H 120 22.1 52.6 57.6 38.2 48.8 60.8 42.9 40.3 50.4 H 126 69.1 81.7 85.6 115.6 98.9 90.4 50.4 79.6 96.4 J 97 97.5 188.3 176.7 153.3 187.2 138.2 176.0 177.5 120.6 M 75 48.5 81.2 57.1 52.3 55.2 50.0 71.5 50.4 47.6 K 74 13.1 182.1 199.7 204.4 146.4 103.7 87.4 103.7 95.6 K 73 16.3 135.7 142.0 120.4 129.3 138.4 106.8 98.0 100.4 o 97.6 68.7 150.2 140.6 139.0 140.3 117.8 104.4 105.7 16.1 ± S.E. 27.0 35.9 29.8 32.9 34.7 24.6 22.7 22.0

3 EGI PLATEAU PERIODS POST-iNFUSiON PERIODS (G25) M 104 197.3 106.5 102.4 47.5 4.3 10.0 22.7 32.7 29.5 30.6 K 96 171.4 90.0 87.1 112.3 46.8 71.6 36.0 50.4 27.9 80.6 M 95 57.6 59.4 78.5 23.7 26.3 15.8 13.7 82.4 83.1 K 94 151.6 132.8 142.2 158.4 118.1 82.8 82.4 41.4 54.4 51.1 K 93 320.7 394.9 206.6 196.2 170.3 85.3 9.4 60.8 45.2 M 91 57.2 45.0 57.6 50.1 44.6 15.5 42.8 48.2 26.6 K 72 305.2 158.1 130.4 220.7 69.2 44.3 52.2 x 180.1 140.9 114.9 115.5 68.5 44.2 35.6 48.9 8.4 + S.E. 40.1 44.9 18.8 29.6 21.7 13.7 7.1

1 MOTOR ACTIVITY INDEX/10 MIN 2 ANTRAL PERFUSION WITH ACH ONLY 3 10 MI,', INFUSION (1.0 /JG/KG/MIN) - 71 -

B

f 0 E E.

: 200 r

i L_J k_XJ

200 r

nl —>-

Time in Minutes

FIGURE 20. (A) EFFECT OF A 10 MINUTE I.V. INFUSION OF 1.0 JJG/KG/HR OF EGI (G25) ON ANTRAL POUCH MOTOR ACTIVITY STIMULATED BY ENDOGENOUS GASTRIN. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 7 OBSERVATIONS IN 3 DOGS. RATIOS CALCULATED FROM THE EXPERIMENTAL DATA IN TABLE X.

(B) SAMPLE RECORDING OF ANTRAL MOTOR ACTIVITY BEFORE, DURING AND AFTER THE INFUSION OF EGI (G25) ON A BACKGROUND OF MOTOR ACTIVITY STIMULATED BY ENDOGENOUS GASTRIN. B | I.D.U CCK/mg

•$ 60 - o

80 o

E.G.I. E.GJ. E.O.I (CM- II) (CM-II) (CM-II) Froctien A IFroction C

FIGURE 21. (A) FRACTIONATION OF EG STAGE I ON CM-CELLULOSE. FIFTY MG EGI WAS APPLIED TO A COLUMN 1.5 x 19 CM AND ELUTED WITH 0.01 M NH4HCO3 BUFFER PH 7.8 AND 0.2 M NH4HCO3 BUFFER PH 8.0. FRACTION SIZE WAS 5.0 ML. ABSORBANCE MEASURED AT 280 NM THROUGH A 1.0 CM LIGHT PATH. SHADED AREA DEMONSTRATED THE MOST POTENT ENTEROGASTRONE ACTIVITY.

(B) RESULTS OF CCK AND ENTEROGASTRONE ASSAYS OF 3 FRACTIONS RESULTING FROM FRACTIONATION OF EG STAGE I ON CM-CELLULOSE (FlGURE 21 (A)). - 73 -

V PURIFICATION OF EG STAGE II TO EG STAGE III

THE FINAL STAGE OF PURIFICATION INVOLVED THE USE OF

SEPHADEX G25 FINE. IN A TYPICAL EXPERIMENT 12.0 MG OF

EG STAGE II WERE DISSOLVED IN 1.0 ML OF 0.2 M ACETIC ACID,

APPLIED TO A COLUMN OF SEPHADEX G25 FINE (0.6 X 120 CM) AND

ELUTED WITH 0.2 M ACETIC ACIO. THE ELUATE WAS COLLECTED IN

2.0 ML FRACTIONS AT A FLOW RATE OF 8.0 ML/HR. THE ABSOR•

BANCE OF THE FRACTIONS WAS MEASURED AT 280 NM ANO FRACTIONS

FROM THE SINGLE RESULTANT PEAK WERE POOLED AND LYOPHILIZED.

THIS FRACTIONATION YIELDED 4.1 MG OF LYOPHILIZED MATERIAL

WHICH WAS REFERRED TO AS EG STAGE III. THIS WAS PURE POLY•

PEPTIDE AND WAS SUBSEQUENTLY REFERRED TO AS "GASTRIC INHIBI•

TORY POLYPEPTIDE" (G.I.P.). ASSAY FOR CCK-PZ ACTIVITY

REVEALED THERE TO BE 2.0 I.D.U./MG.

TWO EXPERIMENTS WERE CARRIED OUT IN THE ACUTE RABBIT TO

DETERMINE IF G.I.P. HAD ANY VASODEPRESSOR ACTIVITY, USING

THE PREPARATION DESCRIBED ON P. 23 OF THE METHODS SECTION.

INJECTIONS OF UP TO 20 JUG/KG PRODUCED NO CHANGE IN ARTERIAL

BLOOD PRESSURE.

VI EFFECT OF PURE GASTRIC INHIBITORY POLYPEPTIDE ON BICKEL

POUCH ACID AND PEPSIN SECRETION AND ANTRAL MOTILITY

STIMULATED BY GASTRIN PENTAPEPTIDE

ANIMALS USED IN THE STUDY WERE PREPARED AS DESCRIBED ON

P. 21 OF THE METHODS SECTION (PREPARATION NO. 3). EXPERI•

MENTS WERE CONDUCTED WITH THE THOMAS CANNULA OPEN. DURING

THESE EXPERIMENTS, RECTAL TEMPERATURES WERE MONITORED IN

3 DOGS BEFORE, DURING AND AFTER THE INTRAVENOUS INFUSION OF - 74 -

G.I.P. THE ABSENCE OF ANY TEMPERATURE INCREASE WAS TAKEN

TO INDICATE AN ABSENCE OF PYROGENIC EFFECTS.

A) BICKEL POUCH H SECRETION

THE EFFECT OF THREE DOSES OF G.I.P. (0.25* 0.5, AND

1.0 JJG/KG/HR) ON A PLATEAU OF H+ SECRETION STIMULATED BY 1.5

JUG/KG/HR OF GASTRIN PENTAPEPTIDE IS SHOWN IN FlG. 22;

TABLES XI AND XI I. THE DOSES OF 0.25» 0.5» AND 1.0 JUG/KG/HR

PRODUCED 25$ (P < 0.01), 55$ (P < 0.0005), AND 75$ (P < 0.0005)

INHIBITION OF CONTROL LEVELS OF H+ OUTPUT. THE CONTROL

VALUE OF 888 ^JEQ H /15 MIN WAS REDUCED TOt 856.0 IN THE

FOURTH PERIOD OF G.I.P. INFUSION AT 0.25 JUG/KG/HRJ 453.8 pEQ H /15 MIN AT 0.5 JUG/KG/HR, AND 270.7 /UEQ H /15 MIN AT

1.0 JUG/KG/HR. INCREASING THE DOSE OF G.I.P. TO 2.0 /JG/KG/HR

PRODUCEO 83$ (P < 0.0005) INHIBITION OF H SECRETION

STIMULATED BY 1.5 UG/KG/HR OF GASTRIN PENTAPEPTIDE (F|G. 25A;

TABLE XVIII). G.I.P. AT A DOSE OF 2.0JUG/KG/HR REDUCED

LEVELS OF H+ FROM A CONTROL VALUE OF 888 JUEQ H+/1 5 MIN TO

159.8 pEo H /15 MIN. EACH POINT IN THE FIGURE REPRESENTS

THE MEAN OF 6 EXPERIMENTS IN 3 DOGS. CONTROL VALUES FOR

FIGS. 22 AND 25A WERE CALCULATED FROM DATA IN TABLE XI.

B) BICKEL POUCH PEPSIN SECRETION

THE EFFECTS OF THREE DOSES OF G.I.P. ON PEPSIN OUTPUT

STIMULATED BY GASTRIN PENTAPEPTIDE ARE SHOWN IN F|G. 23?

TABLE XIV. INFUSION OF 0.5 AND 1.0 JUG/KG/HR OF G.I.P.

PRODUCED 50$ (P < 0.05) AND 78$ (P < 0.0125) INHIBITION OF

CONTROL LEVELS OF PEPSIN. AT THE 0.25 JUG/KG/HR LEVEL, PEPSIN

OUTPUTS WERE NOT REDUCED FROM CONTROL LEVELS. INCREASING TABLE XI 1 2 H OUTPUT IN RESPONSE TO THE INTRAVENOUS INFUSION OF GASTRIN PENTAPEPTIDE

PLATEAU PERIODS

8 DOG EXP NO 1 7 446 K 1 56 429 405 423 428 437 446 470 390 K 155 374 381 393 413 4U 413 410 413 450 K 1 54 31 2 251 321 350 417 366 31 5 31 8 31 8 M 155 1008 954 1111 1043 91 5 866 818 882 831 71 3 M 152 722 740 732 739 705 731 678 731 863 746 M 151 908 830 891 831 833 825 831 1107 11 76 H 159 1122 1205 1226 1235 1 308 1259 11 60 1752 1800 H 1 60 1565 2079 1775. 2110 2001 1899 1 904 1350 1332 1 332 1277 1350 I 305 H 1 50 1182 1575 1553 v_n 725 797 797 804 P 406 717 814 806 667 719 590 412 41 2 P 407 396 419 450 490 581 386 680 675 675 660 P 404 630 680 690 700 670 1450 1276 1276 L 390 1505 1493 1 211 1525 1534 1375 1220 1200 1 200 1220 L 391 1274 1260 1044 1157 1168 1050 1100 1100 II L 392 960 903 1109 1109 1 31 6 50

888 887 889 874 918 917 956 957 924

1 30 113 112 106 11 2 +S.E. 105 128 112 121

1 H+ OUTPUT MEASURED IN JJEQ/15 MIN 2 INTRAVENOUS INFUSION OF 1.5JUG/KG/HR 3 PERIODS OF 15 MIN DURATION TABLE XI I

T OF INTRAVENOUS INFUSION OF G.I.P. ON H* SECRETION STIMUL.ATEO BY GASTRIN PENTAPEPTIOE

PLATEAU PERIODS G. I .P. (1 .0 ;UG/KG POST-INFUSION' DOG EXP NO. 1 __2 .—it. - 5 6 7 9 H 191 904 871 823 941 609 505 420 465 838 H 190 866 990 1050 764 373 305 312 388 410 M 194 466 468 483 388 207 173 172 275 355 M 193 624 575 671 554 341 281 247 366 448 M 204 514 504 462 . 418 250 188 194 246 335 M 183 342 407 375 329 241 175 143 135 135 P 507 515 435 542 533 232 235 181 228 375 L_ _ _ _54J - 1643 _ 1659 J287__ _5J1 572 497 794 1015 X 73~3.8 71 5*. 6 758.1 651 ."7 361 .¥ 304.2 270.7 362.1 488.8 + S.E.. . 1 32JLP__J.5P_.J _ IJ 5__8 61.1 ... 5-4-2. 45.5 71.7 102.3

PLATEAU PERIODS^ J3.J.• P. (0.5 JUG/KG /HR) POST-INFUS10N^

H 192 575 622 605 550 386 307 289 286 350 M 196 739 725 683 577 392 316 292 423 500 M 195 470 407 420 442 260 206 195 275 335 L 800 1490 1525 1543 1501 1172 1144 1139 1506 1 506 P 801 480 . 466 585 506 316 246 332 471 501 P 802 528 698 723 632 424 416 489 518 536 P 803 643 907 941 858 518 413 406 432 484 P 804 528 699 724 633 424 416 489 518 536 X 681.6 756.0 778.0 712.4 468.0 433.0 453.8 553.6 593.5 + S.E. 119.6 122.8 121.6 120.7 101.6 105.4 104.3 139.9 133.2

3 POST-INFUSION PLATEAU PERIODS ____G.J_ .P. (0.25 JJG/KG/HR) M 197 393 400 394 ~371 286 286 297 410 459 M 198 638 710 714 608 476 437 406 432 498 P 011 611 621 594 580 406 382 380 486 529 L 009 1142 1281 1687 1765 1151 1288 1350 1425 1500 L 010 1469 1646 1386 1409 1309 1288 1134 1327 1497 L__ 012 1575 1596 1752 1653 1652 1698 1 566 1663 1440 X 971.3 1042.3 1082.8 1064.3 880.0 896.0 856.0 957.0 987.1 + S.E. 201.4 218.2 239.3 250.3 230.4 245.2 229.0 221.6 220.3

1 pEa H+/15 MIN 2 1.5 JUC/KG/HR 3 15 MIN DURATI ON - 77 -

FIGURE 22. EFFECT OF ONE HOUR INFUSION OF 0.25, 0.5 AND 1.0 JUG/KG/HR OF GASTRIC INHIBITORY POLYPEPTIDE (G.I.P.) ON BICKEL POUCH H+ SECRETION STIM• ULATED BY 1.5 JUG/KG/HR OF GASTRIN PENTAPEPTIDE. CONTROLS RECEIVED ONLY GASTRIN PENTAPEPTIDE THROUGHOUT THE EXPERIMENT. RATIOS CALCULATED FROM DATA IN TABLES XI AND XII. TABLE XI

1 < PEPSIN SECRETION IN RESPONSE TO INTRAVENOUS INFUSION OF GASTRIN PENTAPEPTIDE'

PLATEAU PERIODS DOG EXP NO, 1 A. 8 K 1 56 0.858 0.636 0.636 0.578 0.795 0.557 0.477 0.557 0.648 K 155 0.318 0.318 0.239 0.324 0.318 0.162 0.239 0.243 0.243 H 150 3.677 2.187 1.260 1.350 1.638 1.260 0.672 0.501 M 112 2.175 1.034 1.112 0.908 0.924 0.803 0.909 1 .050 0.975 K 79 0.504 0.398 0.477 0.450 0.126 0.318 0.450 0.330 0.525 K 78 0.165 0.162 1.530 0.081 0.162 0.159 0.159 0.239 0.567 M 110 2.385 0.159 0.080 0.242 0.162 0.165 0.243 0.599 0.345 P 527 0.310 0.250 0.200 0.290 0.200 0.320 0.280 0.300 0.260 L 590 Oo290 0.170 0.640 0.290 0.170 0.170 0.170 0.170 0.170 L 392 3.520 2.800 0.120 0.600 0.600 0.120 0.290 0.410 0.410 L 393 1.240 1.480 0.890 0.560 0.770 1 .280 0.580 1.160 0.510 1.301 0.872 0.653 0.515 0.533 0.483 0.406 0.505 0.465 + S.E. 0.356 0.273 0.144 0.104 0.141 0.130 0.070 0.094 0.070

1 M G TYROS INE/15 MIN 2 1.5 /JG/KG/HR 3 15 MIN DURATI ON TABLE XIV

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY GASTRIN PENTAPEPTIDE

3 3 PLATEAU PERIODS G. I.P. (1 .0 >UG/KG/ HR ) POST-INFUSION . DOG EXP No. 1 2 3 4 5 6 7 8 9 H 191 . 0.570 0.650 0.750 0.0 0.0 0.0 0.0 0.0 0.0 H 190 0.165 0.055 0.600 0.0 0.0 0.0 0.0 0.0 0.0 M 194 0.722 , 0.0 0.0 0.0 0.0 0.0 0.0 0.0 0.0 M 193 0.944 0.214 0.055 0.055 0.0 0.0 0.0 0.0 0.0 M 204 0.110 0.056 0.055 0.055 0.0 0.0 0.0 0.0 0.0 M 183 0.385 0.405 0.500 0.0 0.0 0.0 0.0 0.0 0.0 L 547 0.930 0.708 1.525 0.580 0.055 0.432 0.378 0.378 0.896 L 542 0.600 1.440 0.236 0.0 0.0 0.0 0.440 1.640 0.684 P 507 0.860 0.795 0.800 0.700 0.435 0.360 0.306 0.500 0.700 X 0.635 0.360 0.536 0.167 0.070 0.099 0.085 0.279 0.250 + S. E. 0.100 0.114 0.182 0.104 0.060 0.065 0.056 0.181 0.122

3 PLATEAU PERIOOS' G. •P. (0. 5 JUG/KG/KR) POST-INFUSION H 192 0.330 0.224 0.165 0.165 0.162 0.106 0.161 0.159 0.053 M 196 0.392 0.168 0.112 0.056 0.0 0.055 0.0 0.0 0.0 M 195 1.188 0.560 0.165 0.109 0.112 0.0 0.0 0.0 0.0 L 800 0.950 0.640 0.810 0.560 0.320 0.280 0.390 1.460 1.790 P 801 0.090 0.090 0.090 0.090 0.0 0.0 0.080 0.097 0.093 P 803 0.590 1.400 0.740 1.760 0.760 0.800 0.780 0.810 1.100 P 804 0.275 0.495 0.864 0.825 0.636 0.520 0.605 O..864 0.935 X 0.545 0.511 0.564 0.509 0.284 0.252 0.288 0.609 0.567 + S. E. 0.149 0.168 0.233 0.235 0.115 0.115 0.117 0.213 0.269

3 3 PLATEAU PERIOOS ' G. .P. (0.25 JUG/KG/HR) POST-INFUSION M 197 0.660 0.336 0.324 0.165 0.162 0.0 0.054 0.110 0.112 M 198 0.550 0.110 0.0 0.0 0.0 0.0 0.0 0.0 0.0 P 011 1.600 0.270 0.270 0.0 0.100 0.270 0.420 0.810 0.920 L 009 0.392 1.450 2.066 2.660 1.653 2.166 3.591 2.280 1.653 L 010 1.416 1.180 2.280 2.900 1.711 1.566 1.482 1.298 2.464 L 012 2.006 1.860 1.740 1.403 1.770 1.800 1.382 1.770 1.980 x 1.104 0.867 1.106 1.188 0.899 0.967 1.154 1.044 1.188 + S. E. 0.268 0.294 0.414 0.547 0.363 0.401 0.553 0.371 0.412

1 MG TYROS INE/15 MIN 2 1.5 JJG/KG/HR 3 15 MIN CURAT I ON - 80 -

1.5/jg./kg./hr. Gastrin Pentapeptide

FIGURE 23. EFFECT OF A 60 MINUTE INFUSION OF 0.25, 0.5 AND 1.0,UG/KG/HR OF GASTRIC INHIBITORY POLYPEPTIDE ON BlCKEL POUCH PEPSIN SECRET I ON STIMULATED BY 1.5 JUG/KG/HR OF GASTRIN PENTAPEPTIDE. PEPSIN VALUES TAKEN FROM TABLES XIII AND XIV. - 81 -

THE DOSE OF G.I.P. TO 2.0/JG/K'G/HR PRODUCED 90$ (P < 0.0005)

INHIBITION OF PEPSIN SECRETION STIMULATED BY 1.5 JUG/KG/HR

OF GASTRIN PENTAPEPTIDE (FlG. 25B} TABLE XVIl). THE CONTROL

VALUE OF 0.483 MG TYROSINE/15 MIN FELL TO 0.252 MG TYROSINE/

15 MIN IN THE THIRD PERIOD OF G.I.P. INFUSION AT A DOSE OF

0.5 JUG/KG/HR AND 0.099 MG TYROSINE/15 MIN WITH A DOSE OF

1 .0 JUG/KG/HR. G.I.P. AT A DOSE OF 2.0 JUG/KG/HR REDUCED

LEVELS OF PEPSIN FROM A CONTROL VALUE OF 0.483 MG TYROSINE/

15 MIN TO 0.053 MG. TYROS INE/15 MIN. CONTROL VALUES FOR

FIGS. 23 AND 253 WERE CALCULATED FROM DATA IN TABLE XIII.

c) ANTRAL MOTOR ACTIVITY

THE EFFECTS OF THREE DOSES OF G.I.P. ON ANTRAL

MOTOR ACTIVITY ARE SHOWN IN FIG. 24; TABLE XVI. No

INHIBITION COULD BE DEMONSTRATED BETWEEN CONTROL LEVELS OF

MOTOR ACTIVITY AND LEVELS AFTER INFUSION OF 0.25 AND 0.5

JUG/KG/HR OF G.I.P. A DOSE OF 1.0 JUG/KG/HR OF G.I.P.

PRODUCED 23$ (P < 0.05) INHIBITION OF CONTROL LEVELS OF

ANTRAL MOTOR ACTIVITY. DOUBLING THE DOSE OF G.I.P.

(FIG. 25C; TABLE XVII) YIELDED 21$ INHIBITION (P <0.05).

CONTROL VALUES OF FlGS. 24 AND 25B WERE CALCULATED FROM

TABLE XV.

VII EFFECT OF GASTRIC INHIBITORY POLYPEPTIDE ON GASTRIC

SECRETION AND MOTOR ACTIVITY STIMULATED BY SYNTHETIC

HUMAN GASTRIN (SHG-l)

THE AVAILABILITY OF A SMALL QUANTITY OF SYNTHETIC

HUMAN GASTRIN I MADE POSSIBLE A COMPARISON OF THE INHIBITORY TABLE XV

1 = ANTRAL MOTILITY RESPONSE TO INTRAVENOUS INFUSION OF GASTRIN PENTAPEPTIDE

3 PLATEAU PERIODS DOG EXP NO. 1 4 5 6 7 8 9

K 1 56 189 194 162 108 153 186 134 113 134 K 155 147 107 110 117 86 104 87 95 74- K 1 54 243 209 122 120 81 47 45 50 65 M 153 105 104 111 84 102 75 93 84 110 68 M 152 59 74 66 89 63 66 68 69 38 M 151 41 39 30 30 42 47 36 59 H 159 90 57 84 80 66 86 75 71 65 128 H 160 126 146 '120 113 132 152 125 119 oo 144 ro H 1 50 254 251 231 197 131 143 210 135 416 392 P 400 535 564 413 408 710 354 548 178 157 P 407 156 145 102 148 168 112 138 261 210 221 P 404 254 198 211 301 221 250 1 32.2 134.1 183.3 174.1 146.8 155.9 162.9 135.2 151.9 x 51 .8 26.3 40.7 29.7 27.3 S.E. 38.3 40.3 29.2 32.9

1 MOTOR ACTIVITY INDEX/15 MIN 2 1.5 JUG/KG/HR 3 15 MIN DURATION TABLE XVI

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON ANTRAL MOTILITY STIMULATED BY GASTRIN PENTAPEPTIOE 3 3 G. I .P. (1.0 >UG/KG/HR) PLATEAU PERIODS POST-INFUS1 ON DOG EXP NO. l_ 1 2 3 4 5 6 7 8 9 H 191 53.8 60.5 77.6 54.9 33.6 55.0 59.5 70.6 95.4 H 190 ' 92.9 107.5 106.0 70.8 55.7 53.3 48.7 87.1 78.7 M 194 44.1 29.1 23.5 25.4 18.7 15.7 17.0 13.3 19.6 M 193 120.7 78.2 46.8 41.0 47.1 56.7 43.2 59.5 45.3 M 204 5.4 4.1 3.3 2.6 9.6 6.7 7.9 7.6 6.6 P 507 200.7 281.9 347.2 186.5 110.2 88.5 263.8 501.2 305.6 L 541 113.8 177.5 160.7 116.0 97.2 63.0 70.2 110.2 175.0 K 189 87.7 112.0 62.1 45.4 30.7 38.5 38.2 45.2 52.1 K 188 126.9 156.8 139.7 72.8 75.6 72.0 62.7 77.1 89.4 K 179 29.2 16.0 13.3 13.7 13.7 15.2 16.2 12.9 7 87.5 102.3 98.0 63.2 49.2 46.4 98.4 98.4 96.4 + S.E. 18.0 27.0 32.2 11.0 11.0 23.3. _ , 46.0 _ 46.0 30.8

3 3 G. I.P. (0.5 JUG/KG/HR) PLATEAU PERIODS POST-INFUSION H 192 27.9 34.3 30.4 12.4 15.1 56.3 23.9 35.2 31.8 M 196 58.8 70.0 54.8 56.1 63.4 54.0 43.6 40.1 51.1 M 195 83.8 78.2 70.4 80.6 70.6 42.2 41.6 51.6 70.4 P 801 25.4 18.4 36.3 46.4 48.0 40.3 30.5 49.6 41.9 P 803 77.1 101.2 146.9 205.9 214.9 180.6 234.6 177.0 ' 149.4 P 804 223.7 186.6 192.1 159.1 120.1 141.7 298.9 227.3 201.9 X 82.8 8f.4 88.5 93.4 88.6 87.2 112.2 96.8 68.8 + S.E. 29.8 „24._3_ 26^9_ 30.2 28.8 25.5 49.6 34.0 28.1

3 G. I .P. (0.25 JUG/KG/HR) 3 PLATEAU PERIODS POST-INFUSION M 197 49.7 43.7 31.8 50.3 61.0 35.3 43.9 50.1 M 198 132.0 110.4 111.6 74.7 70.8 73.4 68.2 49.4 P 011 49.6 34.4 48.0 42.1 42.6 30.9 49.7 54.4 L 009 273.0 129.9 154.5 148.4 132.9 156.5 198.3 164.6 L 010 236.3 220.7 328.9 303.1 167.9 247.8 126.1 119.1 58.2 L 012 124.8 132.9 160.4 174.4 152.6 133.7 120.2 173.1 191.3 7 127.6 112.0 139.2 133". 3 104 ,T~ ~Ti'2.9 ~ TOTTO 101.7 + S.E. 42.5 27.8 43.6 40.5 21.5 34.0 24.1 23.8

1 MOTOR ACTIVITY INDEX/15 MIN 2 1.5 JJG/KG/HR 3 15 MIN OURATION - 84 -

FIGURE 24. EFFECT OF 60 MINUTE INFUSION OF 0.25» 0.5 AND 1.0 /JG/KG/HR OF GASTRIC INHIBITORY POLYPEPTIDE ON ANTRAL MOTOR ACTIVITY STIMULATED BY 1.5 JUG/ KG/HR OF GASTRIN PENTAPEPTIDE. RATIOS CALCULATED FROM DATA IN TABLES XV AND XVI. TABLE XVI I

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON H+ OUTPUT A STIMULATED BY GASTRIN PENTAPEPTIOE

3 PLATEAU PERIODS G. .P. (2.0 UG/KG/HR ) POST-INFUS 1 0N^ DOG EXP NO. 1 2 3 4 5 6 7 8 ? M 200 589 546 566 559 185 119 80 112 254 M 201 718 616 616 389 183 135 127 234 342 M 354 673 633 658 546 223 154 127 227 378 L 457 13H 1375 1470 795 307 228 216 364 706 L 458 1508 1525 1600 869 382 329 249 627 986 X 960.4 939.0 982.0 631.6 256.0 193.0 159.8 312.8 533.2 + S.E. 187.6 210.4 227.1 87.8 38.6 38.7 31.3 88.0 136.6

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY GASTRIN PENTAPEPTIOE

3 3 PLATEAU PERIOOS G. I.P. (2.0 JUG/KG/HR) UOG EXP NO. 1 2 3 4 5 6 7 POST-8 iNFUS I O9 N

M 200 0.456 0.224 0.224 0.0 0.0 0.0 0.0 0.053 0.154 M 201 0.918 0.224 0.224 0.162 0.052 0.054 0.053 0.208 0.216 M 354 0.627 0.560 0.448 0.392 0.212 0.162 0.100 0.214 0.216 L . 457 1.500 1.711 1.440 0.168 0.0 0.053 0.0 0.168 0.560 L 458 1.276 1.159 1.260 0.440 0.106 0.0 0.0 0.0 0.560 X 0.944 0.775 0.719 0.232 0.074 0.053 0.031 0.129 0.341 + S.E. 0.204 0.288 0.260 0.077 0.031 0.001 0.020 0.043 0.089

1 c EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON ANTRAL MOTILITY STIMULATED BY GASTRIN PENTAPEPTIOE

3 PLATEAU PERIODS' G.I.P. (2.0 JUG/KG/HR) POST-INFUSION DOG EXP NO. 1 2 3 4 5 6 7 8 9

M 200 46.5 30.5 34.7 27-9 37.4 38.2 48.0 57.8 31.5 M 201 38.1 36.6 47.4 46.1 52.3 27.7 17.7 20.3 28.1 M 354 30.8 40.0 29.2 20.3 15.3 10.5 11.3 25.7 35.6 L 457 25.7 21.6 31.9 34.9 33.1 24.1 18.5 24.4 20.6 L 458 18.6 20.9 21.4 16.1 20.2 11.2 11.2 16.2 22.4 28.8 27.6 X 31.9 29.9 32.9 29.0 31.6 22.3 21.3 + S.E. 4.8 3.8 4.2 5.4 6.6 5.2 6.8 7.4 2.8

1 A. ,UEQ H+/15 MIN B. MG TYROSINE/15 MIN C. MOTOR ACTIVITY INDEX/15 MIN 2 1.5 JJG/KG/HR 3 15 MIN DURATION - 86 -

FIGURE 25. EFFECT OF A 60 MINUTE I.V. INFUSION OF 2.0 JLIG/KG/ HR GASTRIC INHIBITORY POLYPEPTIDE ON:' A. BLCKEL POUCH H+ SECRETION B. BLCKEL POUCH PEPSIN SECRETION C. ANTRAL POUCH MOTOR ACTIVITY STIMULATED BY 1.5 JUG/KG/HR OF GASTRIN PENTAPEPTIDE. RATIOS OF THE G.I.P. STUDIES CALCULATED FROM DATA IN TABLE XVII. CONTROL DATA OBTAINED FROM TABLES XI, AND XV. H+ OUTPUT ANO ANTRAL MOTILITY EXPRESSEO AS RATIOS CALCULATED FROM DATA IN TABLES XI, XV AND XVII. PEPSIN VALUES TAKEN DIRECTLY FROM TABLES XIII AND XVII. - 87 -

POTENCY OF G.I.P. AGAINST GASTRIC SECRETION ANO MOTOR

ACTIVITY STIMULATED BY GASTRIN PENTAPEPTIDE AND THE

WHOLE GASTRIN MOLECULE. DOGS USED IN THESE EXPERIMENTS

WERE AS DESCRIBED ON P. 21 OF THE METHODS SECTION (PREPARA•

TION No. 3). THE DOSE OF 0.5 JJG/KG/HR OF SHG-I WAS CHOSEN

AS BEING THAT WHICH WOULD GIVE THE SAME LEVEL OF H+

SECRETION FROM BlCKEL POUCHES AS 1.5 JJG/KG/HR OF GASTRIN

PENTAPEPTIDE. LIMITED QUANTITIES OF SHG-I DICTATED THE

USE OF ONLY ONE DOSE OF G.I.P., 1.0 JJG/KG/HR GIVEN AS A

ONE HOUR INFUSION. EACH POINT IN THE FIGURES OF THIS

STUDY REPRESENTS 2 EXPERIMENTS IN EACH OF 2 DOGS.

A) BICKEL POUCH H+ SECRETION

THE EFFECT OF 1.0 JUG/KG/HR G.I.P. ON A PLATEAU OF

H+ SECRETION STIMULATED BY 0.5 /JG/KG/HR SHG-1 is SHOWN

IN FIG. 26; TABLE XVIII. THIS DOSE OF G.I.P. PRODUCED

81$ (P < 0.0005) INHIBITION OF H+ SECRETION AS COMPARED TO

75$ AGAINST A C0MPARA8LE DOSE OF GASTRIN PENTAPEPTIDE.

LEVELS OF H+ WERE REDUCED FROM A CONTROL VALUE OF 959.7

PEQ H+/1 5 MIN TO A MEAN VALUE OF 189.0 JJEQ H+/1 5 MIN IN

THE THIRD PERIOD OF G.I.P. INFUSION.

B) BICKEL POUCH PEPSIN SECRETION

THE EFFECT OF 1.0 >UG/KG/HR G.I.P. ON A PLATEAU OF

PEPSIN OUTPUT STIMULATED BY 0.5/JG/KG/HR SHG-I IS SHOWN

IN FIG. 27; TABLE XIX. A DOSE OF SHG-I, WHICH GAVE COMPARABLE

LEVELS OF H+ OUTPUT TO 1.5 JUG/KG/HR GASTRIN PENTAPEPTIDE,

STIMULATED PEPSIN OUTPUT TO A GREATER EXTENT THAN THE

PENTAPEPTIDE (TABLE XIX). G.I.P. PRODUCED 85$ (P< 0.0125) TABLE XVI I I

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON H+ OUTPUT STIMULATED BY SYNTHETIC HUMAN GASTRIN I2

3 PLATEAU PERIODS DOG EXP NO. -1 2 3 4 5 6 7 8 9 P 570 470 500 550 496 500 505 520 530 500 P 571 480 500 490 520 570 580 560 580 575 L 572 1009 1109 1227 1183 1350 1482 1475 1452 1770 L 573 1134 1183 1350 1134 1180 1272 1026 1042 1066 X 773.3 823.0 904.2 833.2 900.0 959.7 895.2 901.0 977.7 + S.E. 174.1 187.0 223.6 188.1 214.0 245.1 224.8 216.7 292.3

3 3 G. I .P. (1.0 JUG/KG/HR) PLATEAU PERIODS POST-iNFUSiON P 575 589 610 554 218 146 100 143 151 228 P 576 380 400 390 208 94 52 47 120 230 L 577 1066 1086 1062 790 513 297 329 513 672 L 578 1566 1566 1500 1288 545 307 383 902 1114 7. 900.2 915.5 876.5 626.0 324.5 189.0 225.5 421.5 561.0 ± S.E. 264.3 260.0 252.5 259.1 118.6 66.0 78.5 183.2 211.8

1 /JEQ H+/15 MIN 2 0.5 /JG/KG/HR 3 15 MIN DURATiON - 89 -

FIGURE 26. EFFECT OF A ONE HOUR I.V. INFUSION OF. 1.0 /JG/KG/ HR OF GASTRIC INHIBITORY POLYPEPTIDE ON BLCKEL POUCH H+ SECRETION STIMULATED BY 0.5JUG/KG/HR OF SYNTHETIC HUMAN GASTRIN I, (SHG-l). CONTROLS RECEIVED SHG-l ONLY. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 4 EXPERIMENTS IN 2 DOGS. RATIOS WERE CALCULATED FROM DATA IN TABLE XVIII. TABLE XIX

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY SYNTHETIC HUMAN GASTRIN I

3 PLATEAU PERIODS DOG EXP NO. 1-2 3 4 5 6 7 8 9

P 570 1.375 1.344 1.595 1.296 1.350 1.560 1.700 1.600 1.560 P 571 1.836 1.456 1.570 1.650 1.760 1.800 1.760 1.800 1.670 L 572 1.856 1.475 2.832 3.074 2.940 4.712 3.953 3.780 3.339 L 573 1.325 1.550 2.040 1.627 1.684 1.440 1.276 1.232 1.824 X 1.593 1.456 2.009 1.911 1.933 2.378 2.172 2.103 2.098 + S.E. 0.137 0.031 0.293 0.396 0.346 0.781 0.603 0.570 0.415

3 PLATEAU PERIODS G. I .P. (1.0 JUG/KG/HR ) POST-1NFUSION P 575 1.836 1.140 1.432 0.312 0.0 0.0 0.265 0.416 0.795 P 576 1.605 1.275 1.275 0.364 0.052 0.104 0.260 0.624 0.800 L 577 1.539 1.344 1.375 1.060 0.810 0.616 0.324 0.810 1.232 L 578 1.566 2.160 2.400 2.204 1.045 0.530 O.648 1.512 1.508 X 1.636 1.479 1.620 0.985 0.476 0.312 0.374 0.840 1.083 + S.E. 0.063 0.230 0.260 0.439 0.264 0.151 O.O89 0.236 0.176

1 MG TYROSINE/15 MIN 2 0.5 /JG/KG/HR 3 15 MIN DURATION 0.5/jg./kg./hr. Synthetic Human Gastrin I

c 'E m a> c '5o5

E

3 Q.

o 0.8 c 'in a. CL

0.0

15 min. periods

FIGURE 27. EFFECT OF A 60 MINUTE I.V. INFUSION OF 1.0 JUG/ KG/HR OF GASTRIC INHIBITORY POLYPEPTIDE ON BICKEL POUCH PEPSIN SECRETION STIMULATED BY 0.5 JUG/KG/ HR OF SHG-l. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 4 EXPERIMENTS IN 2 DOGS. PEPSIN VALUES TAKEN FROM TABLE XIX.

- 91 - - 92 -

INHIBITION OF THE HIGHER PLATEAU OF PEPSIN OUTPUT STIMULATED BY SHG-l. PEPSIN LEVELS WERE REDUCED FROM A CONTROL VALUE

OF 2.172 MG TYROSINE/15 MIN TO 0.374 MG TYROSINE/15 MIN

IN THE FOURTH PERIOD OF G.I.P. INFUSION. c) ANTRAL MOTOR ACTIVITY

G.I.P. IN A DOSE OF 1.0 JUG/KG/HR PRODUCED 51$ (P < 0.05) INHIBITION OF ANTRAL MOTILITY STIMULATED BY SHG-l (FIG. 28; TABLE XX). ANTRAL MOTOR ACTIVITY INDEX WAS REDUCED FROM A MEAN OF 40.5 IN THE CONTROL STUDIES TO

A MEAN OF 13.6 DURING G.I.P. INFUSION.

VIII EFFECT OF G.I.P. ON GASTRIC SECRETION AND MOTOR ACTIVITY STIMULATED BY HISTAMINE DIHYDROCHLORIDE THE ANIMAL PREPARATION AND EXPERIMENTAL DESIGN WERE AS USED IN THE GASTRIN PENTAPEPTIDE STUDY INVOLVING THE

USE OF G.I.P. THE CRITERION FOR CHOOSING THE DOSE OF HISTAMINE USED WAS THE SAME AS THAT INVOLVED IN SELECTING THE DOSE OF GASTRIN PENTAPEPTIDE, NAMELY THAT WHICH WOULD YIELD 60 - 70$ OF MAXIMUM POUCH SECRETION TO HISTAMINE. FIG. 29 SHOWS THE H OUTPUT FROM BICKEL POUCHES OF 4 DOGS IN RESPONSE TO THE INTRAVENOUS INFUSION OF HISTAMINE DIHYDROCHLORIDE IN DOSES OF 5.0, 10.0, 20.0, AND 40.0 JUG/KG/HR. EACH POINT IN THE FIGURE REPRESENTS THE MEAN S.E. OF 3 OBSERVATIONS. ALL OF THE DOGS VOMITED WHEN

THE INFUSION OF HISTAMINE WAS INCREASED TO 20.0 JUG/KG/HR

AND ABOVE. A DOSE OF 10.0 JUG/KG/HR WAS SELECTED AS BEING

THAT DOSE WHICH GAVE APPROXIMATELY 60$ OF MAXIMUM H+ TABLE XX

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON ANTRAL MOTILITY

STIMULATED BY SYNTHETIC HUMAN GASTRIN I2

PLATEAU PERIODS^ DOG EXP NO. 12 3 4 5 67 8 9 P 570 55.0 60.0 59.0 57.0 61.5 58.6 60.3 57.1 61.5 P 571 59.3 53.4 59.6 63.0 54.7 48.9 58.6 60.1 58.7 L 572 25.5 39.9 35.8 32.8 23.6 23.8 40.8 43.4 44.9 L 573 37.6 48.1 75.9 45.9 32.7 30.7 27.9 37.9 24.8

X 44.4 50.3 57.5 49.6 43.1 40.5 46.9 49.6 47.4 + S.E. 7.8 4.3 8.3 6.7 8.9 8.1 7.6 5.3 8.4

3 3 I .P. /JG/KG/HR) PLATEAU PERIODS G. (1.0 POST- I N FUS ION P 575 61.0 48.9 53.0 22.7 16.6 26.9 18.6 41.8 35.1 P 576 19.7 23.7 33.7 10.3 10.0 tO.O 10.0 12.9 21.5 L 577 14.2 11.5 10.2 13.1 21.8 7.8 16.0 24.3 22.0 L 578 40.4 49.3 68.6 52.9 37.1 19.7 26.4 36.6 57.8 7. 33.8 33.3 41.3 24.7 18.8 13.6 15.2 28.9 34.1 t S.E. 10.6 9.4 12.6 9.7 7.6 6.0 5.5 6.4 8.4

1 >JEQ H+/15 MIN 2 0.5 JUG/KG/HR 3 15 M I N DURATI ON - 94 -

FIGURE 28. EFFECT OF A 60 MINUTE I.V. INFUSION OF 1.0 JUG/ KG/HR OF GASTRIC INHIBITORY POLYPEPTIDE ON ANTRAL MOTOR ACTIVITY STIMULATED BY 0.5 JUG/KG/HR OF SHG-l. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 4 EXPERIMENTS IN 2 DOGS. RATIOS WERE CALCULATED FROM DATA IN TABLE XX. - 95 -

O 5 10 20 Histamine Dihydrochloride /jg/kg/hr (I.V.)

FIGURE 29. THE H+ OUTPUT FROM BICKEL POUCHES OF 4 DOGS, L, P, S AND HU, IN RESPONSE TO THE INFUSION OF HISTAMINE DIHYDROCHLORIDE IN DOSES OF 5.0, 10.0, 20.0 AND 40.0 /JG/KG/HR. EACH POINT REPRESENTS THE MEAN OF 6 OBSERVATIONS IN EACH ANIMAL MADE AFTER A PLATEAU OF H+ SECRETION HAD BEEN ESTABL I SHED. - 96 -

OUTPUT FROM THE POUCHES TO HISTAMINE STIMULATION. THESE

H+ OUTPUTS WERE SIMILAR TO THOSE OBTAINED WITH THE INFUSION

OF 1.5/UG/KG/HR GASTRIN PENTAPEPTIDE (FIG. 11). THE ACTION

OF G.I.P. ON AN INFUSION OF 5.0JUG/KG/HR OF HISTAMINE WAS

ALSO OBSERVED. TWO DOSES OF G.I.P. (2.0 AND 4.0 JUG/KG/HR)

WERE USED IN THE LATTER STUDY. THE RELATIVELY LOW LEVELS

OF INHIBITION OF H+ SE C RE T I 0 N P ROD UCE D BY 2.0/JG/KG/HR OF

G.I.P. AGAINST HISTAMINE (45$) WAS THE BASIS FOR USING 4.0

JUG/KG/HR AGAINST HISTAMINE AT THE 10.0 JUG/KG/HR LEVEL. AT

THE 5.0 UG LEVEL» EACH POINT IN THE FIGURES REPRESENTS 6

OBSERVATIONS IN 3 DOGS. AT THE 10.0 JUG LEVEL, EACH POINT

IN THE FIGURES REPRESENTS 8 OBSERVATIONS IN 4 DOGS.

A) BICKEL POUCH H+ SECRETION

G.I.P. IN A DOSE OF 4.0 >UG/KG/HR PRODUCED 40$

(P <0.05) INHIBITION OF H+ SECRETION STIMULATED BY 10.0

/JG/KG/HR HISTAMINE (FIG. 30A; TABLE XXI). LEVELS OF H+

STIMULATED BY 10.0 /JG/KG/HR OF HISTAMINE WERE REDUCED FROM

787 J-EQ H+/15 MIN TO 528 BY 4.0 /JG/KG/HR OF G.I.P. IN

DOSES OF 2.0 AND 4.0 JUG/KG/HR, G.I.P. PRODUCED 45$ (P<0.005)

AND 55$ (P<0.0025) INHIBITION OF H* SECRETION STIMULATED

BY 5.0 /JG/KG/HR OF HISTAMINE (FIG. 30B; TABLE XXI I).

LEVELS OF H+ STIMULATED BY 5.0 JUG/KG/HR OF HISTAMINE WERE

REDUCED FROM 556 JUEQ H+/15 MIN TO 330 JUEQ H+/15 MIN BY

2.0 JUG/KG/HR OF G.I.P., AND TO 287 BY 4.0 AJG/KG/HR OF G.I.P.

AT THE PERIOD OF MAXIMUM SECRETORY INHIBITION, SIGNIFICANT

DIFFERENCES EXISTED BETWEEN THE VALUES AT THE 2.0 AND 4.0 TABLE XXI

1 EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON H* OUTPUT STIMULATED BY HISTAMINE DIHYDROCHLORIDE--

PLATEAU PERIODS' 6 8 DOG EXP NO. _i______3 4_ 7 530 589 ~ 500 530 528 535 664 518 598 530 580 675 Hu 501 371 504 599 585 680 424 809 418 418 Hu 502 434 454 312 350 416 421 430 432 408 281 350 S 503 334 440 473 477 402 562 519 s 504 462 462 518 549 539 616 627 1357 1516 1317 1334 1479 1398 L 505 1333 1379 1357 1151 1354 1351 1516 1548 1539 L 506 1066 1014 1151 906 906 890 920 860 820 870 P 507 1009 847 810 780 790 800 850 820 P 508 907 800 810 732 789 760 787 811 797 X 719 714 720 122.9 131.5 147.5 139.3 ± S.E. 121.4 108.1 110.4 117.4 131.7

POST-INFUSION' PLATEAU PERIODS' G.I.P. 491 550 Hu 510 753 745 713 778 600 524 385 660 530 495 562 756 Hu 511 887 936 908 675 887 699 890 870 905 L 512 1542 1344 1314 678 969 958 792 754 889 950 L 513 1133 1009 1044 695 543 493 508 650 767 P 514 1009 809 812 904 709 572 540 684 853 P 515 1103 1034 889 474 440 383 318 400 443 S 516 473 523 512 535 429 386 334 380 390 S 517 497 481 513 547 528 616 702 925 860 838 738 629 63.2 60.2 50.1 71.4 68.8 75.6 ± S.E. 125.4 100.5 95.0

1 JUEQ H+/15 MIN 2 10.0 /JG/KG/HR 3 HISTAMINE ONLY . 4 ONE HOUR INFUSION (4.0 JUG/KG/HR; TABLE XXI I EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON H+ OUTPUT STIMULATED BY HISTAMINE OIHYOROCHLORIDE^

PLATEAU PERIODS DOG EXP NO. 1 2 5 4 5 6 7 8 9 Hu 700 405 447 528 484 491 550 525 681 499 Hu 701 546 600 652 614 678 764 626 626 626 L 702 - 472 424 413 380 410 523 405 387 387 L 705 632 631 582 556 545 594 622 638 578 P 704 541 529 529 540 570 485 535 535 509 p 705 838 742 562 610 683 707 622 540 700 7. 522.1 562.3 541.0 530.6 562.8 600.5 555.8 567.6 549.8 + S. E. 61.7 49.1 30.1 35.9 43.4 45.6 35.5 43.1 44.7

3 3 G.I.P. (2.0 JUG/KG/ HR ) PLATEAU PERIODS POST-iNFUSiON Hu 711 355 360 369 326 290 205 220 281 347 Hu 710 375 421 408 404 305 349 374 374 427 Hu 712 354 374 387 385 367 257 260 275 372 L 715 1080 1264 1216 935 589 398 372 474 616 P 750 575 536 557 575 463 451 494 610 610 P 714 482 514 469 429 366 218 188 311 456 P 713 626 622 572 578 504 448 402 549 539 x 549.5 584.4 568.2 518.5 412.0 329.4 350.0 410.5 481.0 + S.E . 97.5 118.7 112.1 78.1 41.6 40.9 41.6 51.0 41 .3

3 3 G.I.P. (4.0 JUG/KG/HR) PLATEAU PERIOOS POST-iNFUSiON Hu 717 355 307 311 253 227 160 150 133 200 Hu 716 413 403 416 378 245 220 194 254 260 M 777 600 596 550 525 442 366 551 435 520 L 721 1116 1155 1392 1534 810 352 276 306 437 L 731 579 477 518 523 535 451 512 316 375 P 718 525 543 517 385 290 259 244 386 424 P 720 540 559 591 538 403 519 324 405 468 p 719 975 946 884 772 594 564 465 664 765 637.8 620.7 647.3 613.5 445.2 331.3 287.0 362.3 431.1 + s. E. 94.5 100.9 121.2 142.1 70.5 46.3 34.0 54.8 60.7

1 JJEQ H+/15 MIN 2 5.0 JJG/KG/HR 3 HISTAMINE ONLY - 99 -

10 O Aig/kg/hr. Histomine Dihydrochloride

4.0/ug/kg/hr G.I.P

°—• Control dota G.I.P.

01 23«5«7e» 15 min. periods

B

o—o Control doto a-.-a 20 jug/kg/hr •—• 4.0 « -T- -i 1 1 r- I i 2 3 4 9 e 9 15 mm periods

FIGURE 30. (A) EFFECT OF A ONE HOUR INFUSION OF 4.0 /JG/KG/ HR OF GASTRIC INHIBITORY POLYPEPTIDE ON BLCKEL POUCH H+ SECRETION STIMULATED BY 1 0.0 JU G/K G/HR OF HISTAMINE DIHYDROCHLORIDE. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 8 EXPERIMENTS IN 4 DOGS. RATIOS WERE CALCULATED FROM DATA IN TABLE XX I. 5 (B) EFFECT OF INFUSIONS OF 2.0 AND 4.0/JG/KG/ HR OF G.I.P. ON BLCKEL POUCH H+ SECRET I ON STIMU• LATED BY THE INFUSION OF 5.0 JU G/K G/HR. OF HISTAMINE DIHYDROCHLORIDE. EACH PLOT ON THE GRAPH REPRE• SENTS THE MEAN OF 6 EXPERIMENTS IN 3 DOGS. RATIOS WERE CALCULATED FROM DATA IN TABLE XXII. - 100 -

JUG/KG/HR LEVELS (P<0.05).

B) BICKEL POUCH PEPSIN SECRETION

G. LP. IN A DOSE OF 4.0 JUG/KG/HR PRODUCEO 55$

(P<0.05) INHIBITION OF PEPSIN OUTPUT DURING 10.0 JUG/KG/HR

HISTAMINE INFUSIONS (F|G. 31A; TABLE XX I I I ). PEPSIN LEVELS

DURING THE INFUSION OF 10.0 JUG/KG/HR OF HISTAMINE WERE

REDUCED FROM 0.838 MG TYROSINE/15 MIN TO 0.332 MG TYROSINE/

15 MIN BY 4.0 JUG/KG/HR OF G. I.P. DOSES OF 2.0 AND 4.0 JUG/

KG/HR OF G.I.P. PRODUCED 75$ (P<0.0005) AND 83$ (P< 0.0125)

INHIBITION OF PEPSIN SECRETION STIMULATED BY 5.0JUG/KG/HR

OF HISTAMINE (F|G. 31BJ TABLE XXIV) BUT THE DIFFERENCE

BETWEEN THE PEPSIN VALUES AT THE TWO LEVELS OF G.I.P

INFUSION WERE NOT SIGNIFICANT (P>0.10). PEPSIN LEVELS

DURING THE INFUSION OF 5.0 JUG/KG/HR OF HISTAMINE WERE

REDUCED FROM 1.255 MG TYROSINE/15 MIN TO 0.304 MG TYROSINE/

15 MIN BY 2.0 JUG/KG/HR OF G.I.P. AND TO 0.216 MG TYROSINE/

15 MIN BY 4.0 JUG/KG/HR OF G.I.P.

C) BlCKEL POUCH MOTOR ACTIVITY

G.I.P. IN A DOSE OF 4.0 JUG/KG/HR PRODUCED 60$

(P<0.05) INHIBITION OF BlCKEL POUCH MOTOR ACTIVITY DURING

INFUSION OF 10.0 JUG/KG/HR OF HISTAMINE (FlG. 32A; TABLE XXV).

VALUES FOR MOTOR ACTIVITY INDEX DURING THE INFUSION OF

10.0 JUG/KG/HR OF HISTAMINE WERE REDUCED FROM 61.4 TO 28.3

BY 4.0 JUG/KG/HR OF G.I.P. IN DOSES OF BOTH 2.0 AND 4.0 JUG/

KG/HR G.I.P. PRODUCED INHIBITION OF BlCKEL POUCH MOTOR

ACTIVITY DURING INFUSION OF 5.0jUG/KG/HR OF HISTAMINE

(FIG. 32BJ TABLE XXVI). A SAMPLE RECORDING OF THE EFFECT TABLE XXI I I

1 EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY HISTAMINE DIHYDROCHLORIDE

PLATEAU PERIODS' DOG EXP NO. 1 8

Hu 500 0.324 0.330 0.162 0.324 0.432 0.312 0.171 0.540 0.208 Hu 501 0.0 0.0 0.280 0.280 0.100 0.0 0.0 0.050 0.216 Hu 502 0.117 0.119 0.052 0.051 0.058 0.084 0.070 0.055 0.570 0.795 S 503 1.026 0.440 0.385 0.424 0.432 0.605 0.530 1.026 0.660 S 504 0.969 0.550 0.216 0.392 0.550 1.210 0.728 0.106 1.392 L 505 1.121 0.855 0.600 0.812 0.885 0.690 1.218 1.450 1.120 L 506 0.342 0.560 1.083 1.254 1.197 1.488 1.487 2.520 1 .000 P 507 2.850 1.682 0.969 1.040 0.916 0.978 2.296 1.711 0.700 0.680 P 508 0.650 0.500 0.610 0.480 0.620 0.700 1.050 -o 7 0.822 0.559 0.484 0.562 0.576 0.674 0.838 0.906 0.742 0.126 ± S.E. 0.286 0.161 0.118 0.130 0.122 0.164 0.250 0.284

PLATEAU PERIODS' G.I.P. POST-INFUSION'

Hu 571 0.385 0.572 0.440 0.350 0.150 0.104 0.051 0.0 0.224 1.100 L 512 1.121 0.984 1.218 O.696 0.684 0.550 0.371 1.197 0.448 1.276 L 513 3.840 1.260 1.200 0.580 0.371 0.530 0.986 0.616 0.378 1 .711 P 514 3.068 2.850 1.682 0.969 0.448 2.296 0.162 1.682 P 515 1.003 0.855 0.741 0.986 0.285 0.053 0.342 0.107 0.108 0.432 S 516 1.080 0.605 0.440 0.436 0.212 0.108 0.530 0.810 0.908 1.219 S 517 0.864 1.080 1.458 1.113 0.550 1.092 1.623 1.172 1.025 0.732 0.382 0.406 0.332 0.833 0.298 0.214 + S.E. 0.487 0.293 0.184 0.109 0.070 0.094 0.094

1 MG TYROS INE/15 MIN 2 10.0 >JG/KG/HR 3 HISTAMINE ONLY 4 ONE HOUR INFUSION (4.0 /JG/KG/HR) TABLE XXIV EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT 1 STIMULATED BY HISTAMINE 0 IHYOROCHLOR I OE^

.« u rtnn Doc EXP NO. 1 2 ? „ 4 5 6 7 8 9 Hu 700 0.791 1.220 0.989 0.887 0.776 1.130 0.887 0.887 0.887 Hu 701 1.766 0.623 0.611 0.611 0.600 0.588 0.611 0.611 0.611 L 7035 0.997 1.162 0.840 0.810 0.825 0.795 0.840 0.905 0.489 L 702 0.884 0.959 1.158 1.826 2.558 2.511 2.698 2.093 1.116 P 705 1.847 0.659 0.494 0.513 1.499 0.923 0.485 0.923 0.923 P 704 1.360 0.820 0.820 0.791 1.385 1.583 2.770 1.979 1.612 X 1.274 . 0.907 0.818 0.906 1.273 1.255 1.381 1.233 0.939 + S.E. 0.184 0.100 0.094 0.192 0.294 0.284 0.431 0.256 0.161

PLATEAU PERIOOS' G.I.P. (2.0 /JG/KG/HR ) POST-iNFUSiON' Hu 711 0.686 1.272 1.040 0.663 0.350 0.400 0.550 0.530 0.880 Hu 710 0.150 0.104 0.265 0.157 0.057 0.057 0.157 0.180 0.210 L 715 1.782 2.376 2.695 0.880 0.385 0.102 0.102 0.357 2.430 P 730 1.564 1.485 1.786 1.380 1.300 1.060 1.300 1.590 1 .590 P 714 4.187 4.187 2.915 1.595 0.371 0.156 0.114 1.326 2.568 P 713 0.756 0.935 0.884 0.715 0.168 0.054 0.051 0.056 0.220 7 1.520 1.727 1.597 0.898 0.438 0.304 0.379 0.673 1.316 ± S.E. 0.580 0.577 0.430 0.212 0.178 0.158 0.197 0.256 0.427

PLATEAU PERIODS' G. I .P. (4.0 AJG/KG/HR) POST-INFUSION' Hu 717 0.530 0.208 0.153 0.385 0.112 0.0 0.0 0.051 0.371 Hu 716 0.742 0.848 0.810 0.324 0.051 0.100 0.102 0.106 0.364 M 777 0.594 0.375 0.935 0.832 0.260 0.0 0.105 0.053 0.622 L 721 1.110 1.540 2.880 3.000 0.570 0.255 0.636 0.714 0.848 P 718 3.074 2.016 0.935 0.520 0.100 0.052 0.530 1 .680 1 .272 P 720 2.226 2.310 1.925 1.568 1.590 0.825 1.070 1.242 2.080 1.566 1.472 P 719 2.337 1.904 1.425 0.583 0.285 0.750 2.072 1.516 1.3H 1.294 1.170 0.466 0.216 0.456 0.845 1.004 t I.E. 0.380 0.316 0.334 0.361 0.202 0.109 0.148 0.314 0.238

1 MG TYROSINE/15 MIN 2 5.0 /JG/KG/HR 3 HISTAMINE ONLY - 103 -

FIGURE 31. (A) EFFECT OF A 60 MINUTE INFUSION OF 4.0 JJG/KG/ HR OF GASTRIC INHIBITORY POLYPEPTIDE ON BLCKEL POUCH PEPSIN SECRETION DURING THE INFUSION OF 10.0 JUG/KG/HR OF HISTAMINE DIHYDROCHLORIDE. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 7 EXPERI• MENTS IN 4 DOGS. PEPSIN VALUES TAKEN FROM TABLE XXI I I.

(B) EFFECT OF INFUSIONS OF 2.0 AND 4.0JUG/KG/HR OF G.I.P. ON BLCKEL POUCH PEPSIN SECRETION DURING THE INFUSION OF 5.0JUG/KG/HR OF HISTAMINE DIHYDRO• CHLORIDE. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 6 EXPERIMENTS IN 3 DOGS. PEPSIN VALUES TAKEN FROM TABLE XXIV. - 104 -

OF G.I.P. ON FUNDIC MOTOR ACTIVITY IS SHOWN IN FlG. 33.

AS WITH PEPSIN, NO SIGNIFICANT DIFFERENCE WAS PRODUCED BY

DOUBLING THE DOSE OF G.I.P. FROM 2.0 TO 4.0 /JG/KG/HR.

VALUES FOR MOTOR ACTIVITY INDEX DURING THE INFUSION OF

5.0/JG/KG/HR OF HISTAMINE WERE REDUCED FROM 56.2 TO 39.2

BY 2.0 /JG/KG/HR OF G.I.P. AND TO 38.0 BY 4.0/JG/KG/HR OF

G.I.P.

IX EFFECT OF G.I.P. ON INSULIN-STIMULATED GASTRIC SECRETION

DOGS USED IN SECRETORY STUDIES INVOLVING THE PURE

INHIBITORY POLYPEPTIDE (G.I.P.) WERE PREPARED WITH A

THOMAS CANNULA IN THE VAGALLY INNERVATED GASTRIC REMNANT

(PREPARATION NO. 3). INSULIN HYPOGLYCAEMIA WAS USED AS A

MEANS OF VAGAL STIMULATION. INSULIN WAS GIVEN AS A RAPID

INTRAVENOUS INJECTION OF 0.5 UNITS/KG IN ALL EXPERIMENTS.

G.I.P. WAS GIVEN AS AN INTRAVENOUS INFUSION OVER ONE HOUR

AT A DOSE OF 4.0 JUG/KG/HR. COLLECTION FROM THE GASTRIC

REMNANT AND ANALYSIS OF H AND PEPSIN ARE DESCRIBED IN

THE METHODS SECTION, P. 23 . A LARGE DEGREE OF VARIABILITY

OCCURRED IN THE MAXIMUM H+ AND PEPSIN OUTPUTS IN DIFFERENT

DOGS TO THE SAME DOSE PER KG OF INSULIN. THIS COULD BE

EXPLAINED BY VARIATION IN THE. SIZE OF THE GASTRIC REMNANT.

BECAUSE OF THE LARGE DEGREE OF VARIATION IN RESULTS ANO

THE RELATIVELY SMALL NUMBERS OF OBSERVATIONS COMPARED TO

OTHER SECRETORY STUDIES, THE RESULTS OF ALL EXPERIMENTS

ARE PRESENTED ONLY IN TABULAR FORM, WITH FIGURES REPRESENTING

THE RESULTS OF ONE EXPERIMENT. A TOTAL OF 6 EXPERIMENTS TABLE XXV 1 EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON FUNDIC MOTILITY STIMULATED BY HISTAMINE DIHYDROCHLORIDE2

PLATEAU PERIODS' DOG EXP NO. 1 8 88.8 Hu 500 38.9 48.3 75.3 96.5 88.6 93.7 88.8 93.7 Hu 501 79-4' 117.4 137.8 111.1 84.0 83.1 102.2 120.7 110.0 Hu 502 75.0 82.0 95.6 85.0 87.0 79.0 81 .2 79.0 83.0 L 505 12.3 6.8 7.9 7.9 16.2 7.5 17.2 13.7 18.5 L 506 13.3 18.1 23.1 29.8 39.1 26.7 18.6 67.7 64.1 86.0 P 507 85.0 79.0 67.6 87.0 72.0 70.0 83.0 75.0 66.0 P 508 65.0 67.0 58.0 69.0 53.0 70.0 67.0 65.0 77.8 52.7 59.8 70.7 69.4 62.8 61.4 65.4 73.7 12.6 + S.E. 11.7 14.6 17.0 14.1 10.5 12.0 12.8 10.9

PLATEAU PERIODS' G.I.P. POST-INFUSION' 47.8 Hu 510 48.0 63.0 73.9 83.2 52.6 48.8 67.5 85.5 26.0 69.0 Hu 511 65.0 63.0 69.0 46.0 37.0 24.0 85.0 12.5 14.8 10.0 L 512 77.5 22.5 34.0 10.0 10.0 10.0 14.3 38.0 L 513 20.7 20.2 18.0 22.7 18.5 15.3 47.7 66.3 44.6 132.6 90.0 P 514 107.7 117.6 87.9 51.9 57.2 61.8 28.8 66.0 110.5 P 515 100.0 50.8 80.6 91.4 24.5 68.2 69.8 56.2 60.6 49.2 39.4 28.3 29.0 66.3 x 16.7 14.5 11.4 14.2 10.6 8.6 6.0 15.7 + S.E. 13.3

1 MOTOR ACTIVITY INDEX/15 MIN 2 10.0 JUG/KG/HR 3 HISTAMINE ONLY 4 ONE HOUR INFUSION (4.0 ^UG/KG/HR) TABLE XXVI

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON FUNOIC MOTILITY STIMULATED BY HISTAMINE DIHYDROCHLORIDE

3 PLATEAU PERIOOS DOG EXP NO. 1 2 3 4 5 6 7 8 9 Hu 700 40 59 45 48 44 59 12 53 60 Hu 701 41 70 34 62 59 86 69 66 66 P 702 15 9 7 11 7 8 7 11 8 P 703 9 7 7 9 7 7 15 15 12 L 704 37 27 80 94 73 41 38 45 32 L 705 63 29 40 40 54 136 77 34 69 X 34.2 33.5 35.5 44.0 40.6 56.2 46.3 37.3 41.2 + S. E. 8.0 10.6 11.1 13.1 11.3 20.2 12.5 8.8 11.2

3 I 3 PLATEAU PERIOOS G. I .P (2.0 UG/KG/HR) POST- i NFUSiON Hu 710 54 102 106 47 51 55 34 69 73 Hu 711 91 76 67 76 68 71 81 82 127 Hu 712 48 40 68 54 48 39 36 57 49 P 713 102 91 92 66 71 37 84 78 96 P 714 117 102 96 58 52 28 43 115 124 L 715 52 61 30 2 4 5 10 18 47 X 77.3 78.6 76.5 50.5 49.0 39.2 46.3 69.8 56.0 + S. E. 12.1 10.1 11.7 10.5 9.8 9.2 12.9 13.1 14.5

3 3 PLATEAU PERIODS G.1.P. (4.0 UG/KG /HR) POST-INFUSION H 716 . 71 84 84 44 26 26 34 48 62 P 718 92 71 71 38 31 66 89 176 60 H 717 60 56 56 88 66 49 39 60 78 P 719 72 49 49 56 16 36 61 148 62 P 720 71 72 72 95 52 45 74 99 99 L 721 65 40 40 41 3 6 2 14 55 y 72.6 52.0 52.0 60.3 32.3 38.0 49.8 90.8 69.3 A + S. E. 4.5 6.7 6.7 10.2 9.5 8.4 12.7 25.3 6.7

1 MOTOR ACTIVITY INDEX/15 MIN 2 5.0 JUG/KG/HR 3 HISTAMINE ONLY - 107 -

I

—© Control doto 2.0 jUQ./koVhr. -•• 4,0 • • • —i 1 1 1— IS min. periods 0 2 104 min . periods

FIGURE 32. (A) EFFECT OF A ONE HOUR INFUSION OF 4.0 /JG/KG/ HR OF GASTRIC INHIBITORY POLYPEPTIDE ON BLCKEL POUCH MOTOR ACTIVITY DURING THE INFUSION OF 10.0 /JG/KG/HR OF HISTAMINE DIHYDROCHLORIDE. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 6 EXPERIMENTS IN 3 DOGS. RATIOS WERE CALCULATED FROM DATA IN TABLE XXV.

(B) EFFECT OF A 60 MINUTE INFUSION OF 2.0 AND 4.0 JJG/KG/HR OF G.I.P. ON BICKEL POUCH MOTOR ACTIVITY DURING THE INFUSION OF 5.0 JUG/KG/HR OF HISTAMINE DIHYDROCHLORIDE. EACH PLOT ON THE GRAPH REPRESENTS THE MEAN OF 6 EXPERIMENTS IN 3 DOGS. RATIOS WERE CALCULATED FROM DATA IN TABLE XXVI. - 108 -

60

0

I 20 JUQ Aq/hr Gostric Inhibitory Polypeptide

1 60 j

0

30 ."5 Time in Minutes

FIGURE 33. SAMPLE RECORDING OF BICKEL POUCH MOTOR ACTIVITY BEFORE, DURING AND AFTER THE 60 MINUTE INFUSION OF 2.0 /JG/KG/HR OF GASTRIC INHIBITORY POLY• PEPTIDE ON A BACKGROUND INFUSION OF 5.0 IMG/KG/HR OF HISTAMINE DIHYDROCHLORIDE. - 109 -

WERE CARRIED OUT IN 3 DOGS.

A) GASTRIC REMNANT H SECRETION

G.I.P. IN A DOSE OF 4.0 JJG/KG/HR PRODUCED 40 - 70$ +

INHIBITION OF GASTRIC REMNANT H SECRETION STIMULATED BY

0.5 UNITS/KG OF INSULIN (TABLE XXVI I). FIG. 34 SHOWS THE

RESULTS OF A PAIR OF EXPERIMENTS. IN THIS FIGURE A PEAK

CONTROL H VALUE OF 1,868 JJEQ H /15 MIN WAS PRODUCED

COMPARED TO 708 JUEQ H /1 5 MIN DURING G.I.P. INFUSION.

B) GASTRIC REMNANT PEPSIN SECRETION

G.I.P. PRODUCED 30 - 45$ INHIBITION OF PEPSIN

STIMULATED BY INSULIN HYPOGLYCAEMI A (TABLE XXVI I l). FlG. 35

SHOWS THE RESULTS OF A PAIR OF EXPERIMENTS. IN THIS FIGURE

A PEAK CONTROL PEPSIN LEVEL OF 34.9 MG TYROSINE/15 MIN WAS

PRODUCED COMPARED TO 17.1 MG TYROSINE/15 MIN DURING G.I.P.

I NFUS I ON.

X EFFECT OF G.I.P. ON H+ SECRETION AND VOLUME OF PANCREATIC

JUICE IN THE ANAESTHETIZED CAT DURING THE INFUSION OF

GASTRIN PENTAPEPTIDE

CATS WERE PREPARED ACCORDING TO THE PREPARATION DESCRIBED

ON P. 21 OF THE METHODS SECTION. GASTRIC COLLECTIONS

WERE MADE ACCORDING TO THE PROCEDURE DESCRIBED ON P. 24

AND ANALYSIS OF PANCREATIC SECRETION DESCRIBED ON P. 28 .

A BACKGROUND INFUSION OF 11.6 U/HR OF SECRETIN WAS GIVEN

TO STIMULATE FLOW OF PANCREATIC JUICE IN THE FASTED CATS.

AFTER THREE PERIODS IN WHICH VOLUME OF PANCREATIC JUICE

WAS APPROXIMATELY 1.0 ML/15 MINUTES, AND H+ LEVELS WERE

<100 /JEQ H+/15 MIN, INFUSION OF 1.5 PG/KG/HR GASTRIN

PENTAPEPTIDE WAS BEGUN. ONCE A PLATEAU OF H+ SECRETION - 110 -

TABLE XXVI I

COMPARISON OF THE ACTION OF G.I.P.1 ON H+ SECRETION 2 STIMULATED BY INSULIN HYPOGLYCAEMI A

1 NSUL1N POST -INSULIN PERIODS^

DOG 0 1 2 3 4 5 6

P I NSUL 1 N - 1705 1868 1566 1466 1003 328

1 NSUL1N + - 538 708 340 558 359 G. I.P.

io INHIBITION - 68 62 78 62 64

H 1NSUL1N - 3385 8630 8062 7304 6397 4329

1 NSUL1N + - 1923 5916 5324 4029 4127 G.I.P.

io INHIBITION - 43 31 46 45 ' 35

L . 1 NSUL1N - 3781 8854 8809 7962 7567 5470

1 NSUL1N + - 1001 5350 5323 4768 4754 G. I.P.

io INHIBITION - 74 40 40 40 37

1 G.I.P. INFUSED AT A DOSE OF 4.0/JG/KG/HR 2 0.5 UNITS/KG INSULIN INTRAVENOUSLY 3 EACH VALUE REPRESENTS THE MEAN H+ OUTPUT IN 2 EXPERIMENTS, IN JJEQ/15 MIN. - 111 -

-Insulin 0.5 units/kg.

G.I.P. 4.0/j.g/kg/hr

2000n

15 min. periods

FIGURE 34. EFFECT OF A 60 MINUTE INFUSION OF 4.0 JUG/KG/HR OF GASTRIC INHIBITORY POLYPEPTIDE ON SECRETION OF H+ FROM THE GASTRIC REMNANT STIMULATED BY RAPID INTRAVENOUS INJECTION OF 0.5 UNITS/KG OF INSULIN. DATA FROM A SINGLE EXPERIMENT (DOG P OF TABLE XXVI I). CONTROL RECEIVED ONLY INSULIN. - 112 -

TABLE XXVI I I

COMPARISON OF THE ACTION OF G.I.P.^ ON PEPSIN OUTPUT

STIMULATED BY INSULIN HYP0GLYCAEMIA^

3 1 1 P - 1 NSUL N OST NSULIN PER 1 ODS DOG 0 1 2 3 4 5 6

P INSUL1N - 25.1 34.9 23.8 19.6 14.9 6.6

1NSUL1N + - 15.3 17.1 12.4 14.5 9.5 G.I.P.

io INHIBITION - 39 51 48 26 36

H 1 NSUL 1 N - 120.8 154.0 67.5 48.2 30.3 21.9

1NSUL1N + - 52.2 60.4 38.3 30 78.9 G.I.P.

io INHIBITION - 57 61 43 38 38

L 1NSUL 1 N - 61 .7 77.8 62.5 57.5 48.3 38.2

1NSUL1N + - 29.3 51.9 50.1 41.9 37.3 G.I.P.

io INHIBITION - 53 33 20 27 23

1 G.I.P. INFUSED AT A DOSE OF 4.0 /JG/KG/HR 2 0.5 UNITS/KG INSULIN INTRAVENOUSLY 3 EACH VALUE REPRESENTS THE MEAN PEPSIN OUTPUT IN 2 EXPERIMENTS, IN MG TYROSINE/15 MIN. - 113 -

- Insulin 0.5 units/kg.

O.I.P. 4.0/jg/kg/hr.

40.0-,

00 -f 1 1 1 1 1 1 1 0 I 2 3 4 5 6 7 15 min. periods

FIGURE 35. EFFECT OF A 60 MINUTE INFUSION OF 4.0 JUG/KG/HR OF GASTRIC INHIBITORY POLYPEPTIDE ON PEPSIN SECRETION FROM THE GASTRIC REMNANT STIMULATED BY 0.5 UNITS/KG OF INSULIN. DATA FROM A SINGLE EXPERIMENT (DOG P OF TABLE XXVI I L). - 114 -

Glucag<

G.I.P.

Secre

G.I.P. - Gastric Inhibitory Polypeptide

FIGURE 36. SIMILARITIES IN STRUCTURE OF SECRETIN, GLUCAGON AND G.I.P. - 115 -

ANO PANCREATIC JUICE FLOW RATE HAD BEEN ESTABLISHED, ONE

HOUR INFUSIONS OF G.I.P. WERE GIVEN. THE DOSE OF PENTA•

PEPTIDE WAS CHOSEN AS THAT WHICH WOULD YIELD -^2-60$ OF

MAXIMUM H SECRETION IN RESPONSE TO GASTRIN PENTAPEPTIDE.

DOSE RESPONSE CURVES TO GRADED DOSES OF PENTAGASTRIN

REPORTED BY KONTUREK ET AL (1969) WERE USED TO DETERMINE

THE APPROXIMATE VALUES FOR MAXIMUM H+ OUTPUT FROM THE CAT

STOMACH. THE RESULTS OF EXPERIMENTS INVOLVING 4 CATS ARE

PRESENTED IN TABLE XXIX.

A) EFFECT OF G.I.P. ON H+ SECRETION FROM THE CAT

STOMACH STIMULATED BY GASTRIN PENTAPEPTIDE

THE DATA FROM EXPERIMENTS IN WHICH 4.0 JJG/KG/HR

OF G.I.P. WAS INFUSED AGAINST A PLATEAU OF H+ SECRETION

STIMULATED BY GASTRIN PENTAPEPTIDE IS PRESENTED IN TABLE

XXIX. NO SIGNIFICANT INHIBITION OF PREINFUSION LEVELS OF

H+ SECRETION RESULTED FROM THE INFUSION OF G.I.P. (P> 0.10).

H LEVELS OF 687 - 748 JJEQ H /15 MIN STIMULATED BY GASTRIN

PENTAPEPTIDE FELL TO A LOW OF ONLY 650 /JEQ H+/1 5 MIN DURING

G.I.P. INFUSI ON.

B) EFFECT OF G.I.P. ON VOLUME OF PANCREATIC JUICE

DURING THE INFUSION OF GASTRIN PENTAPEPTIDE

THE DATA PRESENTED IN TABLE XXIX SHOWS THAT INFUSION

OF G.I.P. DID NOT INHIBIT THE VOLUME OF PANCREATIC JUICE

BEING STIMULATED BY SECRETIN. MEAN PLATEAU VALUES OF

2.4 - 2.5 ML OF PANCREATIC JUICE/15 MIN WERE MAINTAINED

DURING AND AFTER G.I.P. INFUSION. TABLE XXIX

EFFECT OF G.I.P. ON H+ OUTPUT AND VOLUME OF PANCREATIC JUICE DURING THE INTRAVENOUS INFUSION OF GASTRIN PENTAPEPTIDE^ IN THE CAT

PLATEAU PERIODS G. I .P. (4.0 >JG/KG/HR) POST-1NFUS1 ON PERIOOS EXP.No. 1 2 3 4 5 6 7 8 9 10 11

A 110 930 896 1050 823 848 828 820 756 870 910 875 A 111 655 622 735 777 837 733 670 710 725 680 700 A 112 718 715 700 680 680 616 715 700 675 680 690 A 113 495 515 506 515 396 481 392 385 396 360 x 699.5 687.0 747.7 698.7 690.2 664.7 649.2 637.7 666.5 657.5 755.0 + S.E. 90.0 80.7 112.6 68.2 105.3 75.2 91.3 85.2 99.1 113.0 60.0

PLATEAU PERIODS G. .P. (4.0 JUG/KG/ HR ) POST -INFUSION PERIODS EXP NO. 1 2 3 4 5 6 7 8 9 10 11

A 110 2.6 2.6 2.5 2.2 2.6 3.0 2.8 2.6 2.4 2.3 A 111 2.4 2.3 •2.3 2.1 2.0 2.4 2.3 2.5 2.6 2.3 A 112 2.3 2.5 2.4 2.3 2.2 2.5 2.5 2.5 2.1 2.6 A 113 2.6 2.7 2.8 3.0 2.7 2.8 2.9 2.8 2.6 2.7 X 2.4 2.5 2.5 2.4 2.4 2.7 2.6 2.6 2.4 2.5 ± S.E. 0.3 0.1 0.1 0.2 0.2 0.1 0.1 0.1 0.1 0.1

1 JJEQ H+/15 MIN 2 ML/1 5 MIN 3 1.5 UG/KG/HR - 117 -

XI EFFECT OF GASTRIC INHIBITORY POLYPEPTIDE ON FASTING

BLOOD GLUCOSE LEVELS

BLOOD GLUCOSE LEVELS WERE MONITORED IN CONSCIOUS DOGS

(PREPARATION NO. 3»P. 21 ) AND IN ACUTE RABBITS, PREPARED

AS DESCRIBED ON P. 23 OF THE METHODS SECTION. BLOOD

GLUCOSE LEVELS WERE DETERMINED ACCORDING TO THE METHOD

DESCRIBED ON P. 28 .

A) BLOOD GLUCOSE LEVELS IN EXPERIMENTS INVOLVING

THE INHIBITION OF GASTRIN PENTAPEPTIDE STIMULATED

H SECRETION

IN 2 SEPARATE OBSERVATIONS IN ONE DOG, BLOOD GLUCOSE

LEVELS WERE MONITORED THROUGHOUT EXPERIMENTS IN WHICH A +

PLATEAU OF H SECRETION WAS ESTABLISHED BY A CONSTANT

INFUSION OF 1.5 JUG/KG/HR OF GASTRIN PENTAPEPTIDE FOLLOWED

BY A ONE HOUR INFUSION OF 1.0 JJG/KG/HR GASTRIC INHIBITORY

POLYPEPTIDE. FlG. 37 SHOWS THAT A DOSE OF G.I.P. WHICH

YIELDED 70$ INHIBITION OF H SECRETION PRODUCED NO HYPER-

GLYCAEMIC RESPONSE IN THE DOG. WHEREAS LEVELS OF H+

SECRETION FELL FROM 1,700 JJEQ H+/1 5 MIN TO 650 JJEQ H+/1 5 MIN,

BLOOD GLUCOSE LEVELS REMAINED BETWEEN 80 AND 90 MG/100 ML.

8) COMPARISON OF EQUIMOLAR INJECTIONS OF GLUCAGON

AND G.I.P. ON BLOOD GLUCOSE LEVELS IN THE DOG

AND THE RABBIT

SINGLE INTRAVENOUS INJECTIONS OF 10.0/JG/KG

GLUCAGON WERE GIVEN BEFORE AND AFTER SINGLE INJECTIONS OF

EQUIMOLAR AMOUNTS OF G.I.P. (H.O /JG/KG). THE DOSE OF

GLUCAGON USED PRODUCED A HYPERGLYCAEMIC RESPONSE (70 - 100$ - 118 -

1.5 /jg./kg./hr. Gastrin Pentopeptide

O.I.P. 1.0 /jg/kg/hr

I800n

o-\ 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 1 O 2 4 6 8 10 12 14 16

15 min. periods

FIGURE 37. LEVELS OF BICKEL POUCH H+ OUTPUT IN UEQ/15 MIN, AND BLOOD GLUCOSE LEVELS IN MG/100 ML DURING THE INFUSION OF 1.0/JG/KG/HR OF G.I.P. ON A PLATEAU OF H+ OUTPUT STIMULATED BY 1.5 /JG/KG/HR OF GASTRIN PENTAPEPTIDE. - 119 -

INCREASE IN FASTING BLOOD GLUCOSE LEVELS) IN THE DOG AND

RABBIT WHICH LASTED UP TO ONE HOUR. TABLE XXX GIVES THE

BLOOD GLUCOSE LEVELS FOLLOWING GLUCAGON AND G.I.P. INJECTIONS

IN 6 EXPERIMENTS CARRIEO OUT IN ACUTE RABBITS. FLG. 38

SHOWS A PLOT OF THE DATA OF ONE OF THESE SIX EXPERIMENTS.

IN THIS EXPERIMENT BLOOD GLUCOSE LEVELS ROSE FROM A PRE-

INJECTION LEVEL OF 115 MG/100 ML TO A PEAK OF 175 MG/100 ML-

30 MINUTES AFTER GLUCAGON INJECTION. IN THE SAME EXPERIMENT,

G.I.P. INJECTION PRODUCEO NO INCREASE IN THE PRE—INJECTION

BLOOD GLUCOSE LEVEL OF 135 MG/100 ML. ONE EXPERIMENT OF

THE SAME DESIGN AS ABOVE WAS CARRIED OUT IN EACH OF TWO

CONSCIOUS DOGS (TABLE XXXI ). THESE RESULTS INDICATE THAT,

BOTH IN THE CONSCIOUS DOG AND ACUTE RABBIT, G.I.P. IN

DOSES UP TO 14 TIMES THAT REQUIRED TO PRODUCE HIGHLY

SIGNIFICANT INHIBITION OF ACID SECRETION, HAD NO HYPERGLY-

CAEMIC EFFECTS.

XII EFFECT OF GASTRIC INHIBITORY POLYPEPTIDE ON BICKEL

POUCH PEPSIN OUTPUT STIMULATED BY SECRETIN

DOGS USED IN THIS STUOY WERE PREPARED AS IN PREPARATION

NO. 3> P. 21 OF THE METHODS SECTION. G.I.P. WAS GIVEN AS

A 1 HOUR INFUSION AT A DOSE OF 2.0 JUG/KG/HR AGAINST A

PLATEAU OF PEPSIN OUTPUT STIMULATED BY THE INTRAVENOUS

INFUSION OF 2 UNITS/KG/HR OF SECRETIN. TABLE XXXII SHOWS

THAT G.I.P. PRODUCED NO SIGNIFICANT INHIBITION OF CONTROL

LEVELS OF PEPSIN OUTPUT STIMULATED BY SECRETIN (P> 0.15).

THE LOWEST MEAN VALUE FOR PEPSIN OUTPUT DURING G.I.P. TABLE XXX

EFFECT OF EQUIMOLAR INJECTIONS OF GLUCAGON AND G.I.P. 3 ON FASTING BLOOD GLUCOSE LEVELS IN THE RABBIT

TIME IN MIN 0 20 30 45 60 90 120 0 20 30 45 60 90 120 0 20 30 45 60 EXP NO. GLUCAGON G.I.P. GLUCAGON

200 1 127 208 219 238 232 182 130 150 160 158 140 146 135 168 160 240 260 230 168 290 220 1 1 105 110 200 212 130 105 130 132 140 168 148 168 155 160 296 285 11 1 89 100 HO 110 95 95 80 89 80 90 95 112 107 104 107 145 132 147 125 IV 110 135 175 175 135 135 135 135 120 127 135 131 135 130 135 220 230 205 180 180 250 210 160 V 115 250 275 225 190 160 115 105 95 85 80 80 85 80 90 VI 100 185 170 175 155 120 95 95 100 105 95 93 95 98 95 210 260 195 150 •

1 GLUCAGON ADMINISTERED AS A SINGLE INTRAVENOUS INJECTION 2 G.I.P. ADMINISTERED AS A SINGLE INTRAVENOUS INJECTION 3 BLOOD GLUCOSE AS MG/100 ML - 121 -

lO.O/jg./kg. Glucagon r- l4.0/jg./kg. G.I.P. r IO.O/jg./kg. Glucagon

c 280i E O O \ di £ 2 00- w v > CD

CD CO o o I 20- A^ O 'A ^A-A-A---A- --A T3 O O o—© Glucagon CO. 40 A-- A G.I.P.

i i i i r —r T—i—r -1 —I 1 1— -1 —i—r r -i 1 30 60 90 120 0 30 60 90 120 30 60 Time in Minutes

FIGURE 38. EFFECT OF SINGLE RAPID INTRAVENOUS INJECTIONS OF 10.0 JUG/KG OF GASTRIC INHIBITORY POLYPEPTIDE AND H.O JUG/KG OF GLUCAGON ON BLOOD SUGAR LEVELS IN THE ANAESTHETIZED RABBIT. DATA FROM EXPERI• MENT No. V OF TABLE XXX. TABLE XXXI

EFFECT OF EQUIMOLAR INJECTIONS OF GLUCAGON AND G.I.P. 3 ON FASTING BLOOD GLUCOSE LEVELS IN THE DOG

TIME IN M I N 0 20 30 43 60 90 120 0 20 30 43 60 90 120 0 20 30 45 60 EXP NO. GLUCAGON G.I.P. GLUCAGON

P. 400 88 120 155 95 88 80 87 97 83 86 89 85 85 83 82 130 165 120 100

S.401 81 131 190 140 100 82 81 73 67 67 70 80 72 73 75 125 185 130 95

1 GLUCAGON ADMINISTERED AS A SINGLE INTRAVENOUS INJECTION 2 G.I.P. ADMINISTERED AS A SINGLE INTRAVENOUS INJECTION I 3 BLOOD GLUCOSE AS MG/100 ML __ ro ro i TABLE XXXI I

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON PEPSIN OUTPUT STIMULATED BY SECRETIN

3 PLATEAU PERIODS DOG EXP NO. 1 2 3 45 6 7 8 9 S 001 2.145' 2.646 2.080 1.890 1.890 2.438 2.340 2.915 3.360 H 002 2.160 4.240 4.505 4.600 2.912 4.620 2.600 2.236 4.900 P 003 2.500 2.230 2.340 2.650 2.475 2.850 2.560 2.490 2.600 L 004 1.961 2.916 1.500 2.080 2.226 2.397 2.544 2.040 2.464 K 005 2.170 2.560 3.180 2.900 2.800 2.300 2.356 2.400 2.490 7 2.187 2.918 2.721 2.824 2.460 2.921 2.480 2.416 3.162 ± S.E. 0.083 0.347 0.520 0.479 0.187 0.433 0.044 0.144 0.464

3 4 3 PLATEAU PERIODS G.I.P. POST-INFUSION S 006 1.092 1.166 1.300 1.537 1.325 0.676 0.883 0.930 1.306 H 007 1.612 1.820 1.700 2.756 2.365 1.326 1.508 1.092 0.884 L 008 0.475 0.930 0.890 0.880 1.005 0.620 0.825 0.690 0.825 P 009 1.683 3.100 5.050 6.477 6.188 4.576 4.056 3.672 5.044 K 010 2.173 2.597 3.180 2.916 3.339 3.021 2.968 2.544 2.385 1.407 1.922 2.424 2.913 2.844 2.043 2.048 1.785 2.088 + S.E. 0.288 0.412 0.761 0.967 0.931 0.767 0.632 0.571 0.789

1 MG TYROSINE/15 MIN 2 2.0 UNITS/KG/HR 3 SECRETIN ONLY 4 ONE HOUR INFUSION - 124 -

INFUSION WAS 2.043 MG TYROSINE/15 MIN COMPARED TO A

CORRESPONDING MEAN VALUE OF 2.921 IN THE CONTROL STUDIES.

ONE EXPERIMENT WAS CARRIED OUT IN EACH OF FIVE DOGS.

XIII EFFECT OF GASTRIC INHIBITORY POLYPEPTIDE ON GASTRIC

SECRETION AND MOTOR ACTIVITY IN THE UNSTIMULATED DOG

DOGS USED WERE PREPARED ACCORDING TO PREPARATION

NO. 3» P. 21 OF THE METHODS SECTION. THE DOSE OF 2.0

JUG/KG/HR OF G.I.P. WAS THAT WHICH GAVE 83$ INHIBITION OF

GASTRIN PENTAPEPTIDE STIMULATED H+ SECRET ION IN THE SAME

AN I MALS.

A) BICKEL POUCH H+ OUTPUT

A ONE HOUR INFUSION OF 2.0 /JG/KG/HR OF G.I.P.

PRODUCED 57$ (P < 0.05) INHIBITION OF UNSTIMULATED BlCKEL

+ POUCH H"*" : SECRET I ON (TABLE XXX I I I ) . MEAN LEVELS OF H

SECRETION VARYING FROM 53 TO 59 UEQ H+/15 MIN WERE REDUCED + TO A LOW VALUE OF 24 *JEQ H /15 MIN DURING G.I.P. INFUSION.

B) BICKEL POUCH PEPSIN OUTPUT

G.I.P. IN A DOSE OF 2.0 JJG/KG/HR PRODUCED 66$

(P < 0.05) INHIBITION OF UNSTIMULATED PEPSIN SECRETION FROM

BlCKEL POUCHES (TABLE XXXI I I ). MEAN LEVELS OF PEPSIN

OUTPUT VARYING FROM 0.415 TO 0.697 MG. TYROSINE/15 MIN WERE

REDUCED TO A LOW VALUE OF 0.155 MG TYROSINE/15 MIN DURING

G.I.P.INFUSION.

c) ANTRAL AND BICKEL POUCH MOTOR ACTIVITY

G.I.P. IN A DOSE OF 2.0 JJG/KG/HR PRODUCED NO INHIBITION TABLE XXXI I I

EFFECT OF INTRAVENOUS INFUSION OF G.I.P. ON H+ OUTPUT, PEPSIN OUTPUT, ANTRAL AND FUNOIC MOTOR ACTIVITY* IN THE UNSTIMULATED ANIMAL

BASAL G.I.P. POST—IN FUSION Doc EXP NO. 1 2 3 4 5 6 7 8 9 10 11 12 13 M 540 42 28 22 25 24 26 29 27 26 25 23 18 21 P 541 24 38 36 38 36 28 11 16 20 60 38 30 31 P 542 77 88 62 27 27 27 28 28 26 47 H 543 110 106 106 99 76 48 37 32 59 70 90 90 L 544 42 36 42 38 41 42 42 26 25 31 35 36 43 X 53.0 56.6 59.0 52.4 39.8 31.4 24.0 26.2 40.6 38.4 44.2 46.2 + S.E. 19.0 15.2 15.9 13.1 9.4 6.3 2.6 1.8 7.7 8.3 12.3 15.2

BASAL G.I.P.4 POST—INFUSION M 540 0.906 0.807 0.370 0.297 0.390 0.160 0.120 0.100 0.095 0.110 0.435 0.630 0.850 P 541 1.045 1.312 0.850 0.620 0.401 0.301 0.250 0.250 0.360 0.720 0.850 P 542 0.450 0.350 0.210 0.200 0.195 0.100 0.080 0.090 0.070 0.100 0.150 0.190 0.260 H 543 0.650 0.200 0.310 0.320 0.330 0.295 0.200 0.195 0.200 0.260 0.290 0.300 0.340 L 544 0.785 0.650 0.330 0.370 0.310 0.265 0.270 0.150 0.160 0.200 0.190 0.265 0.310 0.697 0.501 0.453 0.499 0.415 0.288 0.214 0.167 0.155 0.184 0.285 0.421 0.522 + I.E. 0.097 0.137 0.148 0.202 0.109 0.089 0.054 0.031 0.033 0.034 0.053 0.106 0.134 4 BASAL POST-INFUSION G.I.P. M 540 80.0 50.0 61.2 62.4 45.6 79.2 62.0 56.4 65.2 64.4 70.3 65.5 70.1 P 541 36.9 19.5 32.1 54.4 37.7 15.0 20.2 19.7 10.8 92.5 85.5 55.4 67.2 P 542 33.8 22.8 48.2 53.4 14.6 39.6 17.9 34.7 39.4 51.7 42.0 H 543 20.0 40.7 80.4 63.7 48.1 64.7 96.0 80.7 35.6 19.4 59.6 50.1 X 29.8 41.9 55.0 48.8 48.9 40.3 52.9 43.6 56.8 53.6 58.0 57.3 + S.E. 10.1 6.7 12.0 5.3 13.1 13.3 16.1 17.2 13.7 14.9 3.2 6.8

BASAL G.I.P.4 POST-INFUSION M 540 47.2 53.0 86.0 47.6 52.0 35.6 21.0 18.6 17.2 25.6 46.0 57.6 48.9 P 541 30.7 31.6 25.5 35.6 25.0 15.0 9.6 10.0 18.7 22.6 35.6 22.0 P 542 85.7 62.3 25.6 37.5 42.0 30.6 15.7 18.6 14.5 20.1 32.6 37.5 42.0 H 543 15.6 9.7 14.7 13.6 10.5 8.4 5.6 8.7 15.6 27.2 13-4 15.8 66.4 40.4 38.2 31.3 35.8 25.4 15.0 13.1 12.6 20.0 32.1 36.0 32.1 + S.E. 27.2 10.5 16.5 7.1 8.0 5.4 2.5 3.2 1.9 2.0 5.0 9.0 7.9

1 JJEQ H+/15 MIN 2 MC TYROS INE/15 MIN 3 MOTOR ACTIVITY INOEX/15 MIN 4 2.0 /JG/KC/HR - 126 -

OF SPONTANEOUS LEVELS OF ANTRAL MOTOR ACTIVITY, HOWEVER

THE INFUSION PRODUCED 63$ (P < 0.025) INHIBITION OF BlCKEL

POUCH MOTOR ACTIVITY. MEAN VALUES OF MOTOR ACTIVITY INDEX

VARYING FROM 31.3 TO 66.4 WERE REDUCED TO A LOW VALUE OF

12.6 DURING G.I.P. INFUSION. DISCUSSION

PREPARATIONS CONTAINING CCK-PZ, IN ADOITION TO CAUSING

GALL BLAOOER CONTRACTION ANO ENZYME SECRETION BY THE PANCREAS,

HAVE BEEN SHOWN TO EXERT VARIOUS EFFECTS ON THE GASTRO•

INTESTINAL TRACT. THEY HAVE BEEN DESCRIBED AS POSSESSING

INHIBITORY ACTIVITY FOR EXOGENOUS GASTRIN-STIMULATE0 H+

SECRETION (GILLESPIE AND GROSSMAN, 1964) AND ENDOGENOUS

GASTRIN-STIMULATED H+ SECRETION (BROWN AND MAGEE, 1967).

SIMILAR CCK-PZ PREPARATIONS, HOWEVER, HAVE BEEN SHOWN BY

MAGEE AND NAKAMURA (1966) ANO MURAT AND WHITE (1966) TO

STIMULATE ACID SECRETION WHEN GIVEN ALONE, AS WELL AS TO

INHIBIT BOTH BASAL AND STIMULATED MOTOR ACTIVITY OF THE

FUNDUS (JOHNSON ANO MAGEE, 1965; BROWN, JOHNSON AND MAGEE,

1967). IN AN ATTEMPT TO EXPLAIN THE APPARENTLY AMBIGUOUS

EFFECTS OF CCK-PZ ON ACID SECRET I ON, STENING, JOHNSON AND

GROSSMAN (1969B) PUT FORWARD AN HYPOTHESIS OF COMPETITIVE

INHIBITION BETWEEN THE CCK-PZ AND GASTRIN MOLECULES FOR A

COMMON RECEPTOR SITE INVOLVED IN THE STIMULATION OF H+.

THIS THEORY WAS SUPPORTED BY IDENTIFICATION OF THE SIMILAR•

ITIES BETWEEN THE C— TERM INAL PENTAPEPTIDES OF BOTH MOLECULES.

SINCE ALL OF THE ABOVE-CITED EXPERIMENTAL FINDINGS

INVOLVED THE USE OF IMPURE CCK-PZ PREPARATIONS, THE GASTRIC

EFFECTS MAY HAVE BEEN PRODUCED BY AGENTS OTHER THAN CCK-PZ.

THE INITIAL STUDY REPORTED IN THIS THESIS, WHICH PROVIDED

THE BASIS FOR THE' SUBSEQUENT ISOLATION OF GASTRIC INHIBITORY

POLYPEPTIDE, WAS UNDERTAKEN TO DETERMINE WHETHER IN FACT ALL

- 127 - - 128 -

OF THE DESCRIBED EFFECTS OF CCK-PZ PREPARATIONS ON THE

UNSTIMULATED ANIMAL WERE DUE TO THE HORMONE, OR WHETHER

SOME OF THESE EFFECTS WERE DUE TO OTHER MATERIALS PRESENT IN

THE EXTRACTS.

THE APPROACH TO THIS PROBLEM INVOLVED A COMPARISON OF

THE EFFECTS OF TWO PURITIES OF CCK-PZ DESIGNATED AS 10$

PURE (250 I.D.U./MG) AND 40$ PURE (1,500 I.D.U./MG) ON THE

GASTRIC PARAMETERS OF H+ SECRETION, PEPSIN SECRETION AND

ANTRAL MOTOR ACTIVITY (BROWN AND PEDERSON, 1970). RESULTS

INDICATED THAT THE 40$ PURE CCK-PZ HAD A GREATER ACID-

STIMULATORY EFFECT ON SYMPATHETICALLY AND VAGALLY DENERVATED

FUNDIC POUCHES THAN THE 10$ PURE MATERIAL (FLG. 10), THE

GALL BLADDER EFFECT BEING IDENTICAL. AT THE TIME OF THIS

STUDY POSSIBLE EXPLANATIONS FOR THE RESULTS WERE THAT,

EITHER A GASTRIC SECRETORY STIMULANT WAS SELECTIVELY CONCEN•

TRATED DURING THE PURIFICATION PROCEDURES, OR THAT AN

INHIBITORY MATERIAL WAS PROPORTIONATELY REMOVEO. DETRACTING

FROM THE FORMER POSSIBILITY WAS THE EXISTING EVIDENCE

CONCERNING THE SIMILARITIES BETWEEN THE GASTRIN AND CCK-PZ

MOLECULES, WHICH SUGGESTED THAT ACID-SECRETORY STIMULATION

WAS AN INHERENT ACTION OF CCK-PZ. THE FACT THAT BOTH

PREPARATIONS OF CCK-PZ EXERTED A SIMILAR DEGREE OF STIMULA•

TION OF ANTRAL POUCH MOTILITY SUGGESTED THAT THIS STIMULA•

TORY EFFECT WAS A FEATURE OF THE CCK-PZ MOLECULE. THE PEPSIN

RESPONSE (FIG. 7? TABLE I) UNDERWENT CHANGES SIMILAR IN KIND

TO THOSE OF H+ SECRETION UPON INCREASING THE PURITY OF

CCK-PZ PREPARATIONS INJECTED. THE OBSERVATIONS IN THIS - 129 -

STUDY MADE TENABLE THE HYPOTHESIS THAT IN PURIFYING CCK-PZ

FROM 10$ TO 40$, AN INHIBITORY MATERIAL FOR GASTRIC ACID

SECRETION WAS REMOVED.

ONCE IT HAD BEEN ESTABLISHED THAT TWO PURITIES OF CCK-PZ

PRODUCED DIFFERENT DEGREES OF ACID STIMULATION, A COMPARISON

OF THE INHIBITORY POTENCY OF THESE TWO PREPARATIONS ON

GASTRIN PENTAPEPTIDE-STIMULATED H+ SECRETION WAS UNDERTAKEN.

ALL OF THE ACTIONS OF THE WHOLE GASTRIN MOLECULE HAVE BEEN

DESCRIBED AS BEING POSSESSED BY THE C-TERMINAL TETRAPEPTIDE

AMIDE: TRP - MET - ASP - PHE - NH2 (TRACY AND GREGORY, 1964),

ALTHOUGH IT HAS BEEN SHOWN TO BE ONLY ONE TWELFTH AS POTENT

ON A MOLAR BASIS AS THE WHOLE MOLECULE (GROSSMAN, 1970).

SOME ACYL DERIVATIVES HAVE ACTIVITY SEVERAL TIMES THAT OF

THE TETRAPEPTIDE. ONE OF THESE IS THE B-ALANYL DERIVATIVE

OF THE C-TERMINAL TE TRAPEPTIDE AMIDE, N-T-BUTYLOXYCARBONYL-

B - ALA - TRP - MET - ASP - PHE - NH2» OTHERWISE KNOWN AS

GASTRIN PENTAPEPTIDE OR PENTAGASTRIN. THIS MATERIAL WAS

USED AS A STIMULANT OF GASTRIC SECRETION BECAUSE OF ITS

COMMERCIAL AVAILABILITY AND THE FACT THAT IT HAD BEEN WIDELY

USEO IN BOTH HUMAN AND ANIMAL STUDIES. THE REASONING INVOLVED

WAS THAT IF AN INHIBITORY MATERIAL WAS REMOVED DURING PURIFI•

CATION OF CCK-PZ, THEN THIS WOULD BE INDICATED BY A DECREASE

IN INHIBITION OF GASTRIN PENTAPEPTI 0E-STIMULATED H+ SECRETION.

THE RESULTS PRESENTED IN FIG. 12; TABLE V INDICATED THAT THIS

WAS IN FACT THE CASE, WITH THE 40$ PURE MATERIAL YIELDING A

SIGNIFICANTLY LESSER OEGREE OF INHIBITION OF H+ SECRETION

THAN THE 10$ PURE MATERIAL (P<0.005). - 130 -

THE OBSERVATION THAT PARTIAL PURIFICATION OF CCK-PZ

RESULTED IN INCREASED POTENCY FOR H+ STIMULATION AND

DIMINISHED POTENCY FOR THE INHIBITION OF GASTRIN-STIMULATED

H+ SECRETION LED TO ATTEMPTS TO SEPARATE A GASTRIC INHIBITOR

DISTINCT FROM CCK-PZ USING THE 10$ PURE PREPARATION AS

STARTING MATERIAL. THE INITIAL STAGES OF THIS PURIFICATION

PROCEDURE, THE PRODUCT OF WHICH WAS CALLED EGI, WERE

DESCRIBED IN THE RESULTS SECTION, PAGE 51 AND SUMMARIZED IN

FIG. 13. SINCE THE CHEMICAL NATURE OF EGI WAS NOT COMPLETELY

ELUCIDATED, IT WAS IMPORTANT TO DETERMINE WHETHER OR NOT THE

INHIBITORY POTENCY OF THE MATERIAL COULD BE EXPLAINED BY

CCK-PZ AND/OR SECRETIN ACTIVITY. ASSAYS IN THE GUINEA PIG

AND CAT INDICATED THAT LESS THAN 10 I.D.U./MG OF CCK-PZ WERE

PRESENT AND NO SECRETIN COULD BE DETECTED BY THE ASSAY

PROCEDURE EMPLOYED. THE ASSAYS (F|GS. 15 AND 16) INDICATED

THAT THE FIRST STAGE IN THE PURIFICATION OF GASTRIC INHIBI•

TORY POLYPEPTIDE PRODUCED A FRACTION (EG I ) WHICH WOULD

SIGNIFICANTLY INHIBIT H+ AND PEPSIN OUTPUT STIMULATED BY

GASTRIN PENTAPEPTIDE. H+ OUTPUTS DECREASED FROM A MEAN OF

618 JUEQ/10 MIN TO 168/JEQ/10 MIN, AND PEPSIN OUTPUTS FELL

FROM 0.326 MG TYROSINE/10 MIN TO 0.080 MG TYROSINE/10 MIN.

THE FINDING THAT EGI INHIBITED PEPSIN SECRETION SUPPORTED

THE RESULTS OF THE FIRST STUDY (FlG. 9) WHICH SUGGESTED THAT

DURING THE PURIFICATION OF CCK-PZ AN INHIBITOR OF PEPSIN

OUTPUT MAY HAVE BEEN REMOVED.

IN AN ATTEMPT TO PURIFY THE GASTRIC INHIBITOR PRESENT

IN EGI, SEVERAL PURIFICATION PROCEDURES WERE ATTEMPTED. - 131 -

CHROMATOGRAPHY OF EGI ON SEPHADEX G25 YIELDED 3 FRACTIONS,

(BROWN, PEDERSON, JORPES AND MUTT, 1969). THE RESULTS OF

THE CCK-PZ AND ENTEROGASTRONE ASSAYS ON THESE FRACTIONS

(FIG. 17) INDICATED THAT FRACTION 2, REFERRED TO AS EGI

G25-FR 2, WAS THE PUREST PREPARATION OF THE INHIBITORY

MATERIAL. ASSAYS FOR ENTEROGASTRONE ACTIVITY, HOWEVER,

REVEALED NO SIGNIFICANT INCREASE IN INHIBITORY ACTIVITY

COMPARED TO EGI. THE EFFECTS OF EGI G25~FR 2 ON ENDOGENOUS

GASTRIN RELEASED BY PERFUSING AN ISOLATED ANTRAL POUCH

WITH 0.1$ ACH WERE INVESTIGATED. THE RESULTS OF THIS STUDY

INDICATED THAT THIS FRACTION PRODUCED 70$ INHIBITION OF

H+ AND PEPSIN OUTPUTS AND ANTRAL MOTOR ACTIVITY (FLGS. 18,

19 AND 20). THIS STUDY GAVE THE FIRST INDICATION THAT THE

INHIBITORY MATERIAL UNDER INVESTIGATION WOULD INHIBIT

GASTRIC MOTOR ACTIVITY. IT IS KNOWN THAT GASTRIN TETRA-

PEPTIDE AND GASTRIN STIMULATE ANTRAL MOTOR ACTIVITY (VJACOBY

AND MARSHALL, 1969). THESE OBSERVATIONS, PLUS THE DATA IN

THE FIRST STUDY (BROWN AND PEDERSON, 1970) SHOWING THAT

CCK-PZ PREPARATIONS STIMULATED ANTRAL MOTOR ACTIVITY,

SUGGESTED THAT THE SHARED C-TERMINAL PENTAPEPTIDE OF CCK-PZ

AND GASTRIN WAS RESPONSIBLE FOR THE STIMULATION. THE RESULTS

SUGGESTED THAT THE INHIBITOR BEING PURIFIED ACTS ON ALL

GASTRIC PARAMETERS AFFECTED BY GASTRIN WHICH HAD BEEN

INVESTIGATED, NAMELY H+ OUTPUT, PEPSIN OUTPUT AND ANTRAL

MOTOR ACTIVITY. THE FINDING THAT EGI G25"FR 2 INHIBITED

ANTRAL MOTOR ACTIVITY DID NOT FIT WITH THE OBSERVATION

(FIG. 8) THAT BOTH PURITIES OF CCK-PZ STIMULATED ANTRAL - 132 -

MOTOR ACTIVITY TO A SIMILAR DEGREE. POSSIBLE EXPLANATIONS

COULD BE THAT THE AMOUNT OF INHIBITOR REMOVED BY PURIFI•

CATION FROM THE 10$ TO THE 40$ PURE STAGE WAS NOT SUFFICIENT

TO PRODUCE A DIFFERENCE IN DEGREE OF STIMULATION OF ANTRAL

MOTOR ACTIVITY, BUT SUFFICIENT TO EFFECT A SIGNIFICANT

INCREASE IN STIMULATION OF H+ OUTPUT, OR THAT THE DOSE OF

CCK-PZ USED WAS AT OR ABOVE MAXIMUM FOR STIMULATION OF

ANTRAL MOTOR ACTIVITY. IN THE LATTER CASE REMOVAL OF THE

AMOUNT OF G.I.P. INVOLVED IN THE PURIFICATION STAGE WOULD t NOT INCREASE THE DEGREE OF STIMULATION OF ANTRAL MOTILITY.

IN STUDIES WHICH INVOLVED GASTRIN PENTAPEPTIDE OR SYNTHETIC

HUMAN GASTRIN I (SHG-l), THE EFFECT OF G.I.P. ON BlCKEL

POUCH MOTOR ACTIVITY COULD NOT BE ASCERTAINED. THE REASON

FOR THIS WAS THAT AT THE COMMENCEMENT OF GASTRIN INFUSION,

ALL SPONTANEOUS MOTOR ACTIVITY PRESENT IN THE POUCHES

BECAME COMPLETELY INHIBITED.

THE STAGES IN PURIFICATION AND ASSAYS OF EGI TO EGII I

(PURE GASTRIC INHIBITORY POLYPEPTIDE) WERE OUTLINED IN THE

RESULTS SECTION, PAGE 69, AND SUMMARIZED IN FLG. 13. THE

FINAL PURIFICATION PROCEDURES ARE AS REPORTED BY BROWN, MUTT

AND PEDERSON (1970). THIS WORK REVEALED THAT THE MATERIAL

WAS A POLYPEPTIDE AND ALSO DESCRIBED SOME OF THE STRUCTURAL

FEATURES WHICH ARE DISCUSSED LATER. WORK ON THE CHEMISTRY

OF THE POLYPEPTIDE WAS CONTINUED BY BROWN (1971) WITH THE

ELUCIDATION OF THE AMINO ACID COMPOSITION AND THE SEQUENCE

OF THE TRYPTIC PEPTIDES. THE INHIBITORY POLYPEPTIDE WAS

HENCEFORTH REFERRED TO AS GASTRIC INHIBITORY POLYPEPTIDE. - 133 -

THE SEQUENCE OF PORCINE GASTRIC INHIBITORY POLYPEPTIDE

(G.I.P.) HAS NOW BEEN ELUCIDATED. IT IS A 43 AMINO ACID

RESIDUE POLYPEPTIDE WITH THE AMINO ACID SEQUENCE TYR-ALA-

GLU-GLY-THR-PHE-ILE-SER-ASP-TYR-SER-ILE-ALA-MET-ASP-LYS-ILE-

ARG-GLN-GLN-ASP-PHE-VAL-ASN-TRP-LEU-LEU-ALA-GLN-GLN-LYS-GLY-

LYS-LYS-SER-ASP-TRP-LYS-HIS-ASN-ILE-THR-GLN (BROWN AND

DRYBURGH, 1971). THE CALCULATED MOLECULAR WEIGHT OF THE

POLYPEPTIDE IS 5»105• DURING ALL STAGES OF THE ELUCIDATION

OF THE CHEMICAL NATURE OF G.I.P., COMPARISONS WERE MADE

BOTH STRUCTURALLY AND PHYSIOLOGICALLY TO THE CHEMICALLY

IDENTIFIED GASTRO-INTESTINAL HORMONES SECRETIN AND CHOLE-

CYSTOKININ-PANCREOZYM I N. PRELIMINARY AMINO ACID ANALYSES

REPORTED BY BROWN EJT AJ- (1970) FIRST DEMONSTRATED DIFFERENCES

BETWEEN THE INHIBITORY POLYPEPTIDE AND CCK-PZ. THE 33 AMINO

ACID CCK-PZ MOLECULE HAD BEEN SHOWN TO HAVE 2 PROLINE

RESIDUES (MUTT AND JORPES, 1968). IN ADDITION, THE ANALYSES

OF G.I.P. SHOWED A HIGH GLUTAMIC ACID/GLUTAMINE CONTENT AND

A PREPONDERANCE OF LYSINE OVER ARGININE. THIS WAS IN

CONTRAST TO CCK-PZ WHICH POSSESSED A SINGLE GLUTAMIC AC I D/

GLUTAMINE RESIDUE, 3 ARGININES AND A SINGLE LYSINE. THIS

DISTINCTION FROM CCK-PZ WAS IMPORTANT IN THAT IMPURE PREPAR•

ATIONS OF CCK-PZ HAD SERVED AS THE STARTING MATERIAL FOR

THE PURIFICATION OF G.I.P. (FLG. 13).

ONCE THE AMINO ACID SEQUENCE WAS COMPLETED, COMPARISONS

WERE MADE TO THE STRUCTURE OF OTHER KNOWN GL HORMONES AND

RELATED POLYPEPTIDE HORMONES. IT WAS SHOWN BY BROWN AND

DRYBURGH (1971) THAT 9 OF THE FIRST 26 AMINO ACIOS OF G.I.P. - 134 -

OCCURRED IN THE SAME POSITION AS IN SECRETIN (F|G. 36). AS

A RESULT OF WELL—DOCUMENTED STRUCTURAL SIMILARITIES BETWEEN

GLUCAGON AND SECRETIN (JORPES, 1968), A COMPARISON OF THE

STURCTURES OF GLUCAGON, SECRETIN AND GASTRIC INHIBITORY

POLYPEPTIDE WAS MADE. THIS COMPARISON (BROWN AND DRYBURGH,

1971) REVEALED THAT 15 OF THE FIRST 26 AMINO ACIDS OF G.I.P.

OCCURRED IN SIMILAR POSITIONS AS IN PORCINE GLUCAGON.

ONCE IT HAD BEEN ASCERTAINED THAT THE POLYPEPTIDE WAS

PURE, IT WAS OF INTEREST TO LOOK AT THE POTENCY OF G.I.P.

AS AN INHIBITOR OF GASTRIC PARAMETERS AGAINST WHICH THE

GASTROINTESTINAL HORMONES SECRETIN AND-CCK-PZ HAD BEEN USED

IN THE PAST. ANOTHER OBJECTIVE OF STUDIES USING THE PURE

MATERIAL WAS TO DETERMINE IF G.I.P. WAS EFFECTIVE AS AN

INHIBITOR OF GASTRIC SECRETION AGAINST STIMULANTS THAT

WERE RESISTANT TO INHIBITION BY SECRETIN AND CCK-PZ, E.G.

HISTAMINE. IT WAS HOPED THAT EVIDENCE AS TO THE MODE OF

ACTION OF G.I.P. WOULD BE REVEALED BY LOOKING AT THE RANGE

OF ITS EFFECTIVENESS AGAINST GASTRIC SECRETION STIMULATED BY

BOTH NERVOUS AND HORMONAL PATHWAYS. AS A RESULT OF THE

STRUCTURAL SIMILARITIES OF G.I.P. TO SECRETIN AND GLUCAGON,

ATTEMPTS WERE MADE TO DETERMINE IF G.I.P. MIMICKED ANY OF

THE PHYSIOLOGICAL ACTIONS OF THESE HORMONES. THIS POSSIBIL•

ITY WAS HEIGHTENED BY THE FACT THAT BOTH SECRETIN AND GLUCA•

GON SHARED WITH G.I.P. THE ABILITY TO INHIBIT GASTRIN-

STIMULATED H+ SECRETION (LLN AND SPRAY, 1968), AS WELL AS

SHARING STRUCTURAL FEATURES.

THE PURE POLYPEPTIDE WAS GIVEN IN DOSES UP TO 20.0 JUG/KG - 135 -

IN THE ACUTE RABBIT WHILE MONITORING ARTERIAL BLOOD PRESSURE.

EVEN THE LARGEST DOSES YIELDED NO DETECTABLE CHANGE IN BLOOD

PRESSURE. DOSES OF G.I.P. USED IN SECRETORY STUDIES IN

DOGS PRODUCED NO CHANGES IN RECTAL TEMPERATURES, INDICATING

AN ABSENCE OF PYROGENIC EFFECTS.

GLUCAGON HAS BEEN SHOWN BY DYCK, RUDICK, HOEXTER AND

JANOWITZ (1969) TO INHIBIT SECRETIN-STIMULATED PANCREATIC

EXOCRINE SECRETION IN THE DOG, AN OBSERVATION THAT WAS IN

SHARP CONTRAST TO THE OTHER PHYSIOLOGICAL PROPERTIES WHICH

IT POSSESSED IN COMMON WITH SECRETIN, NAMELY THE CHOLERETIC

(MORRIS, SARDI AND BRADLEY, 1967)» 1NSUL 1 NOTROP1c (SAMOLS,

MARRI AND MARKS, 1965) AND INHIBITORY EFFECTS ON GASTRIC ACID

SECRETION (LIN AND SPRAY, 1968). FOR THIS REASON THE EFFECT

OF INFUSION OF G.I.P. ON VOLUME OF PANCREATIC JUICE WAS

OBSERVED. BECAUSE OF THE STRUCTURAL SIMILARITIES BETWEEN

GLUCAGON AND G.I.P., BLOOD GLUCOSE LEVELS WERE MONITORED

DURING EXPERIMENTS IN WHICH G.I.P. WAS INFUSED.

THE AVAILABILITY OF A SMALL QUANTITY OF SHG-I MADE

POSSIBLE A COMPARISON OF THE POTENCY OF G.I.P. WITH GASTRIC

PARAMETERS STIMULATED BY BOTH GASTRIN PENTAPEPTIDE AND THE

WHOLE GASTRIN MOLECULE. ONE OF THE REASONS THAT SHG-I WAS

EMPLOYED WAS THAT IT WOULO MORE CLOSELY MIMIC THE NORMAL

PHYSIOLOGICAL SITUATION.

ONE STUDY INVOLVED THE INTRAVENOUS INFUSION OF G.I.P.

IN DOGS THAT HAD BEEN FASTED. IN THESE EXPERIMENTS, ANTRAL

AND FUNDIC MOTOR ACTIVITY WERE MONITORED ALONG WITH H+ AND

PEPSIN LEVELS. THE RATIONALE FOR THIS STUOY WAS THE FACT - 136 -

THAT AT LEAST ONE OTHER GASTROINTESTINAL HORMONE, CCK-PZ,

WHEN GIVEN INTRAVENOUSLY, HAD BEEN SHOWN TO EXERT OPPOSITE

EFFECTS ON GASTRIC SECRETION WHEN GIVEN ALONE (PRESHAW AND

GROSSMAN, 1965) FROM THOSE WHEN GIVEN IN CONJUNCTION WITH

GASTRIN (JOHNSON AND GROSSMAN, 1970).

PURE GASTRIC INHIBITORY POLYPEPTIDE WAS ADMINISTERED IN

DOSES OF 0.25* 0.5, 1.0 AND 2.0 JUG/KG/HR AGAINST A PLATEAU

OF H+ SECRETION STIMULATED BY GASTRIN PENTAPEPTIDE. THE

PLATEAU OF H+ SECRETION ACHIEVED WAS 60 - 70$ OF MAXIMUM

POUCH OUTPUT TO THE STIMULANT, THE AIM BEING TO REMAIN

WITHIN A PHYSIOLOGICAL RANGE OF ACID SECRETION. THE AC I 0

RESPONSES OF 2 DOGS TO DIFFERENT DOSES OF GASTRIN PENTA•

PEPTIDE (FLG. 11) ILLUSTRATED THAT THE DOSE USED ACHIEVED

60 - 70$ OF MAXIMUM H+ OUTPUT.' SINCE GASTRIN PENTAPEPTIDE

HAD BEEN SHOWN ALSO TO STIMULATE PEPSIN OUTPUT AND ANTRAL

MOTOR ACTIVITY (HIRSCHOWITZ, 1967; JACOBY AND MARSHALL, 19^9),

THESE PARAMETERS WERE MONITORED. THE DATA INDICATED THAT AT

A DOSE OF 2.0JUG/KG/HR G.I.P. PRODUCED 85$ INHIBITION OF

PENTAPEPT I DE-STIMULATED H AND 90$ INHIBITION OF PEPSIN

OUTPUT (FIG. 25). THE RESULTS SUGGESTED THAT THE THRESHOLD

J. DOSE LEVEL FOR SIGNIFICANT INHIBITION OF H SECRETION WAS

LESS THAN 0.25" JJG/KG/HR , WHEREAS FOR SIGNIFICANT PEPSIN

INHIBITION THE THRESHOLD LEVEL WAS BETWEEN 0.25 AND 0.5

JUG/KG/HR. THE LATTER FINDING MAY BE A FUNCTION OF THE

VARIABILITY OF PEPSIN LEVELS RATHER THAN AN INDICATION THAT

THE THRESHOLD FOR SIGNIFICANT INHIBITION OF PEPSIN DIFFERS

FROM THAT OF H+. IT APPEARED THAT G.I.P. WAS LESS POTENT - 137 -

AS AN INHIBITOR OF ANTRAL MOTOR ACTIVITY THAN FOR THE PARA•

METERS OF H+ AND PEPSIN SECRETION. THIS WAS INDICATED BY THE

FACT THAT A DOSE OF 1.0/JG/KG/HR OF G.I.P. WAS REQUIRED TO

PRODUCE SIGNIFICANT INHIBITION OF ANTRAL MOTOR ACTIVITY. AS

SUCH, THE DEGREE OF INHIBITION (23$) WAS VERY SMALL COMPARED

WITH H+ AND PEPSIN (75 - 80$), IN ADDITION, DOUBLING THE

DOSE OF G.I.P. TO 2.0 /JG/KG/HR PRODUCED NO FURTHER INHIBITION

OF ANTRAL ACTIVITY, INDICATING THAT MAXIMUM LEVELS OF

INHIBITION HAD BEEN ACHIEVED. ONE POSSIBLE EXPLANATION FOR

THE SMALL AMOUNT OF INHIBITION COULD BE THAT GASTRIN PENTA•

PEPTIDE WAS A MORE POTENT STIMULANT FOR ANTRAL MOTOR ACTIVITY

THAN OF THE OTHER PARAMETERS STUDIED, AND THAT THE DOSE

EMPLOYED WAS PRODUCING MAXIMAL MOTOR ACTIVITY RESPONSES. AN

ALTERNATE POSSIBILITY COULD BE THAT THE ACTION OF THE PENTA•

PEPTIDE ON MOTOR ACTIVITY WAS MORE RESISTANT TO INHIBITION

BY G.I.P. THAN H+ OR PEPSIN SECRETION. THE FORMER POSSIBIL•

ITY WAS SUPPORTED BY THE WORK OF JACOBY ANO MARSHALL (1969)

WHO FOUND THAT A PEAK RESPONSE OF ANTRAL MOTOR ACTIVITY IN

THE DOG WAS PRESENT AT DOSES OF GASTRIN PENTAPEPTIDE AS LOW

AS 1.0 JUG/KG/HR.

GREGORY AND TRACY (1964) WERE THE FIRST TO REPORT THAT

THERE WERE 2 GASTRINS, GASTRIN I AND GASTRIN II. BOTH

GASTRINS ARE SIMILAR IN AMINO ACIO SEQUENCE EXCEPT THAT

GASTRIN II HAS A SULPHATE ESTERIFIED TO THE TYROSYL RESIDUE

AT POSITION 12. SHG-L WAS GIVEN IN SUCH A DOSE AS TO YIELD

EQUIVALENT LEVELS OF H+ SECRETION FROM BLCKEL POUCHES AS

ACHIEVED WITH 1.5 JJG/KG/HR OF GASTRIN PENTAPEPTIDE. A DOSE - 138 -

OF 0.5 /JG/KG/HR OF SHG-I WAS FOUND TO BE SUITABLE. THIS

AGREED WITH THE FINDINGS OF HlRSCHOWITZ (1968) WHO SHOWED

THAT GASTRIN I WAS ABOUT 3 TIMES AS POTENT AS GASTRIN

PENTAPEPTIOE ON A WEIGHT BASIS IN STIMULATING H+ SECRETION,

ANO THE STATEMENT OF GROSSMAN (1970) THAT THE TETRAPEPTIDE

AMIDE OF GASTRIN POSSESSED ONE-TWELFTH THE ACTIVITY OF THE

WHOLE GASTRIN MOLECULE ON A MOLAR BASIS.

THE DATA PRESENTED IN FlGS. 22 AND 26 INDICATED THAT

G.I.P. WAS MORE EFFECTIVE AS AN INHIBITOR OF THE WHOLE

MOLECULE THAN GASTRIN PENTAPEPTIDE WITH RESPECT TO H

SECRETION. A DOSE OF G.I.P. THAT PRODUCED 75$ INHIBITION OF

GASTRIN PENTAPEPTIDE-STIMULATED H SECRETION YIELDED 85$

INHIBITION OF ACID LEVELS STIMULATED BY THE WHOLE GASTRIN

MOLECULE. OF INTEREST WAS THE OBSERVATION THAT A DOSE OF

SHG-I THAT PRODUCED COMPARABLE LEVELS OF H+ OUTPUT TO GASTRIN

PENTAPEPTIDE YIELOED PEPSIN LEVELS THAT WERE APPROXIMATELY

3i_ TIMES HIGHER (TABLES XIII AND XIX). TH I S WAS IN CONTRAST

TO THE FINDINGS OF HlRSCHOWITZ (1968) WHO REPORTED THAT

GASTRIN PENTAPEPTIDE STIMULATED PEPSIN SECRETION 3 TIMES AS

STRONGLY AS GASTRIN I. G.I.P. PRODUCED SLIGHTLY GREATER

INHIBITION OF THE HIGHER LEVELS OF PEPSIN THAN IN THE PENTA•

PEPTIDE STUDIES (85$ AS COMPARED WITH 78$). G.I.P. REDUCED

PEPSIN LEVELS FROM A MEAN OF 2.378 MG TYROSINE/15 MIN TO A

MEAN OF 0.312 MG TYROSINE/15 MIN IN THE SHG-I STUDIES AND

FROM A MEAN OF 0.500 MG TYROSINE/15 MIN TO A MEAN OF 0.050

MG TYROSINE/15 MIN IN THE GASTRIN PENTAPEPTIDE STUOIES.

IF THE ASSUMPTION IS MADE THAT PARIETAL CELLS HAVE - 139 -

RECEPTORS FOR THE GASTRIN MOLECULE, ONE GASTRIN MOLECULE

PRODUCES THE SAME DEGREE OF STIMULATION OF THE RECEPTOR AS

12 GASTRIN PENTAPEPTIDE MOLECULES AT SUBMAXIMAL DOSES. THE

GREATER INHIBITORY POTENCY OF G.I.P. AGA INST SHG-I-STIMULATED

H+ SECRETION MAY BE DUE TO THE FACT THAT IN THE DOSES USED

IN THE STUDY, G.I.P. WITH A MOLECULAR WEIGHT OF 5,105 CAME

MUCH CLOSER TO EQUALLING THE NUMBER OF MOLECULES OF SHG-l

(MW 2,114) INFUSED, THAN IT DID GASTRIN PENTAPEPTIDE (MW

APPROXIMATELY 750). IF G.I.P. AND GASTRIN WERE COMPETING

FOR THE SAME RECEPTOR SITE, THEN THE ABOVE NUMERICAL CONSID•

ERATION COULD BE OF IMPORTANCE. ALTERNATIVELY, G.I.P. MAY

INTERACT WITH GASTRIN EITHER IN THE BLOOD OR AT THE LEVEL

OF THE PARIETAL CELL. THE PART OF THE GASTRIN MOLECULE

ABSENT FROM THE PENTAPEPTIDE MAY CONTRIBUTE TO THE INTER•

ACTION BETWEEN THE WHOLE GASTRIN MOLECULE AND G.I.P.,

INCREASING ITS EFFICIENCY AS AN INHIBITOR.

ALTHOUGH ANTRAL MOTILITY WAS INHIBITED TO A MUCH LESSER

DEGREE (51$) THAN WERE THE PARAMETERS OF H+ OR PEPSIN (FLGS.

26, 27 ANO 28), THIS LEVEL OF INHIBITION WAS TWICE THAT

PRODUCED AGAINST PENTAPEPT I DE-STIMULATED ANTRAL MOTILITY.

THE SAME ARGUMENT USED IN THE DISCUSSION OF THE GREATER

POTENCY OF G.I.P. AGAINST H+ AND PEPSIN OUTPUT STIMULATED

BY SHG-L AS COMPARED WITH GASTRIN PENTAPEPTIDE COULD APPLY

TO ANTRAL MOTOR ACT I V I TY. ANOTHER POSSIBLE REASON COULD

BE THE FACT THAT PLATEAU LEVELS OF ANTRAL MOTOR ACTIVITY

STIMULATED BY SHG-L WERE ONE THIRD OF THOSE PRODUCED BY

PENTAPEPTIDE STIMULATION. THE LATTER FINDING WAS THE DIRECT - HO -

OPPOSITE TO THE OBSERVATION THAT SHG-I WAS A MORE POTENT

STIMULATOR OF PEPSIN SECRETION, ANO NO EXPLANATION CAN BE

OFFERED AT PRESENT FOR EITHER PHENOMENON.

HISTAMINE IS ONE OF THE MOST POTENT STIMULANTS OF

GASTRIC H+ SECRETION, BUT IT IS NOT KNOWN FOR CERTAIN IF

HISTAMINE PARTICIPATES IN A PHYSIOLOGICAL MECHANISM FOR THE

STIMULATION OF H+ SECRETION. THE USE OF HISTAMINE AS A

GASTRIC SECRETORY STIMULANT IN THE CONTEXT OF THIS THESIS

WAS NECESSARY AS A RESULT OF PREVIOUS EXPERIMENTAL WORK

INVOLVING THE SEARCH FOR ENTEROGASTRONE. FENG, HOU AND

LLM (1929) WERE THE FIRST TO OBSERVE THAT FAT IN THE

DUODENUM WOULD INHIBIT HISTAM INE-STIMULATED H+ SECRETION FROM

DENERVATED STOMACH POUCHES, THEREFORE BY A HUMORAL MECHANISM.

KOSAKA AND LlM (1930A) FOUND THAT THE CRUOE CHOLECYST0KININ

OF IVY AND OLDBERG ((1928) WOULD INHIBIT HI STAM I NE-ST I MULATED

H+ OUTPUT. SINCE FAT IN THE DUODENUM WAS SHOWN BY IVY (1934)

TO BE A POTENT STIMULUS FOR GALL BLADOER CONTRACTION,

PRESUMABLY 8Y THE RELEASE OF ENDOGENOUS CCK-PZ, IT COULD BE

HYPOTHESIZED THAT CCK-PZ WAS THE ACTIVE AGENT RESULTING IN

INHIBITION OF HISTAMINE-STIMULATED H+ SECRETION WHEN FAT

WAS PLACED IN THE DUODENUM. THIS WAS NOT, HOWEVER, THE

CONCLUSION OF KOSAKA AND LlM (1930B), WHO THOUGHT THAT IVY'S

CHOLECYSTOKININ PREPARATION CONTAINED AN IMPURITY WHICH WAS

RESPONSIBLE FOR THE INHIBITION OF HI STAM I NE-STIMULATED H

SECRETION. THEY ATTEMPTED TO ISOLATE THE MATERIAL WHICH

THEY NAMED ENTEROGASTRONE. THE HYPOTHESIS OF KOSAKA AND

LlM HAD BEEN GIVEN SUPPORT BY THE FINDING THAT A HIGHLY - HI -

PURIFIED CCK-PZ PREPARATION DID NOT INHIBIT HISTAMI NE•

ST IMULATED H+ SECRETION IN THE DOG (JOHNSON AND GROSSMAN,

1969). THE SAME GROUP SHOWED THAT H I STAM I NE-STIMULATED H+

SECRETION WAS NOT INHIBITED BY SECRETIN, AND CONCLUDED THAT

A HORMONE OTHER THAN CCK-PZ OR SECRETIN WAS RELEASED BY THE

PRESENCE OF FAT IN THE DUODENUM. FROM THE ABOVE, IT CAN BE

STATED THAT ANY PRINCIPLE SEEKING TO FULFILL THE CLASSICAL

DEFINITION OF ENTEROGASTRONE (KOSAKA AND LlM, 1930B) MUST BE

EFFECTIVE IN INHIBITING HISTAMINE-STIMULATED H+ SECRETION IN

EXTRINSICALLY DENERVATED STOMACH POUCHES IN THE DOG. IN

STUDIES INVOLVING THE USE OF HISTAMINE, THE DOSE USED WAS

ONLY ONE QUARTER THAT WHICH WAS OFTEN QUOTED IN THE LITER•

ATURE AS BEING USED IN SECRETORY STUDIES IN DOGS. THE POUCH

RESPONSES TO DIFFERENT DOSES OF HISTAMINE INDICATE THAT A

OOSE OF 20.0 JJG/KG/HR OF HISTAMINE YIELDED NEAR-MAXIMAL

POUCH RESPONSE FOR THIS STIMULUS (F|G. 29). ANOTHER FACT

IN SUPPORT OF THE USE OF LOWER DOSES OF HISTAMINE WAS THAT

THE PLATEAU LEVELS OF H+ OUTPUT (TABLE XX I ) (APPROXIMATELY

0.8 MEQ H+/15 MIN) COMPARED FAVOURABLY TO PLATEAU LEVELS OF

H+ OUTPUT CITED IN THE LITERATURE (JOHNSON AND GROSSMAN,

1969) EVEN THOUGH THE HISTAMINE DOSE IN THE CITED STUDY

WAS 40.0 JUG/KG/HR. THESE H+ OUTPUTS ALSO COMPARED FAVOUR•

ABLY WITH THOSE ACHIEVED FOR GASTRIN PENTAPEPTIDE. AN

ADDITIONAL FACTOR RULING AGAINST THE HIGHER DOSES OF

HISTAMINE WAS THAT ALL DOGS VOMITED WHEN THE INFUSION OF

HISTAMINE WAS I NCREASED TO AND ABOVE 20. 0 JU G/K G/H R .

ALTHOUGH G.I.P. WAS FOUND TO INHIBIT' HISTAMINE- - 142 -

STIMULATED H+ SECRET I ON FROM DENERVATED FUNDIC POUCHES, IT

WAS FOUND TO BE MUCH LESS EFFECTIVE THAN AGAINST GASTRIN

PENTAPEPTIDE- OR SHG-I-ST IMULATED H+ SECRETION. USING A

DOSE OF HISTAMINE (10.0 JUG/KG/HR ) THAT YIELDED COMPARABLE

LEVELS OF H+ OUTPUT TO 1 • 5 JU G/K G/H R OF GASTRIN PENTAPEPTIDE,

G.I.P. WAS LESS THAN 50$ AS EFFECTIVE AS AN INHIBITOR.

TWICE THE DOSE OF G.I.P. REQUIRED TO YIELD 85$ INHIBITION

OF GASTRIN PENTAPEPTIDE-STIMULATED H+ SECRETION RESULTED IN

ONLY 55$ INHIBITION OF HISTAMINE-STIMULATED H+ SECRETION

(FIGS. 25A AND 30A). DOUBLING THE DOSE OF G.I.P. DID NOT

GREATLY INCREASE THE DEGREE OF INHIBITION OF H+ STIMULATED

BY HISTAMINE INFUSION (FlG. 30s). THIS INDICATED THAT

MAXIMUM INHIBITION BY G.I.P. WAS BEING PRODUCED. IT WAS

POSSIBLE THAT THE LEVELS OF HISTAMINE USED WERE UNPHYSIO-

LOGICAL AND THAT NO INHIBITOR COULD PRODUCE A GREATER

DEGREE OF INHIBITION. COMPARISONS OF THIS NATURE CANNOT

BE MADE SINCE THE CHEMICALLY IDENTIFIED HORMONES SECRETIN

AND CCK-PZ BOTH FAIL TO INHIBIT HISTAMINE-STIMULATED H+

SECRETION. ONE PIECE OF CIRCUMSTANTIAL EVIDENCE IMPLICATING

G.I.P. IN A KNOWN INHIBITORY MECHANISM WAS THE FACT THAT

G.I.P. PRODUCED THE SAME DEGREE OF INHIBITION OF HISTAMINE—

STIMULATED H+ SECRETION AS DID FAT IN THE DUODENUM (JOHNSON

AND GROSSMAN, 1969). IN THEIR STUDIES 50$ INHIBITION OF H+

SECRETION STIMULATED BY HISTAMINE WAS THE MAXIMUM OBTAINED.

HlRSCHOWITZ (1968) REPORTED THAT GASTRIN PENTAPEPTIDE

WAS A MUCH BETTER STIMULANT FOR PEPSIN SECRETION THAN

HISTAMINE. THE DATA PRESENTED IN TABLES XIII AND XXIII - 143 -

INDICATED THAT IN STUDIES USING HISTAMINE AT A DOSE LEVEL OF

10.0 pG/KG/HR AND GASTRIN PENTAPEPTIDE, OF 1.5 JUG/KG/HR,

PLATEAU LEVELS WERE APPROXIMATELY EQUAL. G.I.P. PRODUCED

55 - 80$ INHIBITION OF PEPSIN SECRETION IN THE HISTAMINE

STUDIES. DOUBLING THE DOSE OF G.I.P. FROM 2.0 TO 4.0 JUG/KG/

HR PRODUCED NO SIGNIFICANT INCREASE IN THE INHIBITION OF

PLATEAU LEVELS OF PEPSIN SECRETION (FlG. 31B), INDICATING

THAT A NEAR-MAXIMUM DEGREE OF INHIBITION WAS BEING EXERTED

AT THE 2.0/JG/KG/HR LEVEL. SlNCE THE BLOOD-BORNE AGENT

RELEASED WHEN FAT WAS PLACED IN THE DUODENUM HAD BEEN SHOWN

TO INHIBIT PEPSIN SECRETION STIMULATED BY HISTAMINE (ALLEY

E_T A_L, 1934), CANDIDATES FOR THIS ROLE MUST BE CAPABLE OF

THIS ACTION. HISTAMINE HAD BEEN SHOWN BY JACOBY AND MARSHALL

(1969) TO BE A POOR STIMULANT OF GASTRIC ANTRAL MOTILITY.

THIS WAS FOUND TO BE THE CASE IN THESE STUDIES, AND THEREFORE

THIS PARAMETER HAS NOT BEEN QUANT ITATED IN INHIBITORY STUDIES

INVOLVING HISTAMINE. INFUSION OF GASTRIN PENTAPEPTIDE OR

SHG-L INHIBITED UNSTIMULATED FUNDIC POUCH MOTOR ACTIVITY.

FOR THIS REASON THERE WAS NO MEANS OF DETERMINING IF G.I.P.

INHIBITED FUNDIC MOTOR ACTIVITY DURING GASTRIN STIMULATION

OF OTHER PARAMETERS. BECAUSE OF THE NORMALLY HIGH DEGREE OF

VARIATION IN BLCKEL POUCH MOTOR ACTIVITY, IT WAS DIFFICULT

TO DETERMINE IF HISTAMINE HAD ANY EFFECT ON THIS PARAMETER.

SUBJECTIVELY, IT APPEARED AS IF SPONTANEOUSLY OCCURRING

BURSTS OF MOTOR ACTIVITY BECAME MORE REGULAR AFTER THE

COMMENCEMENT OF HISTAMINE INFUSION. TH I S POINT IS ILLUS•

TRATED BY THE SMALL DIFFERENCES IN THE MEAN VALUES FOR - U4 -

UNSTIMULATED BlCKEL POUCH MOTOR ACTIVITY (TABLE XXXI I I ) AND

THE LEVELS OF BlCKEL POUCH MOTOR ACTIVITY DURING HISTAMINE

INFUSION (TABLE XXV). INFUSION OF G.I.P. PRODUCED 55 - 60$

INHIBITION OF MOTOR ACTIVITY, AND AS WITH PEPSIN, DOUBLING

THE DOSE OF G.I.P. DID NOT PRODUCE INCREASED INHIBITION.

G.I.P., UNLIKE THE GASTROINTESTINAL HORMONES CCK-PZ

AND SECRETIN, WILL INHIBIT H+ SECRETION, PEPSIN SECRETION

AND FUNDIC MOTOR ACTIVITY STIMULATED BY HISTAMINE. LUCIEN,

ITOH, SUN, MEYER, CARLTON AND SCHALLY (1969) EMPLOYING

TECHNIQUES SIMILAR TO THOSE USED IN THE PURIFICATION OF THE

ESTABLISHED GASTROINTESTINAL POLYPEPTIDES, ISOLATED AN

INHIBITORY SUBSTANCE OR ENTEROGASTRONE FROM THE DUODENAL

MUCOSA. THE AUTHORS HAD NOT PROCEEDED WITH PURIFICATION TO

THE STAGE OF IDENTIFYING THE CHEMICAL NATURE OF THE INHIBITORY

MATERIAL. LUCIEN, ITOH AND SCHALLY (1970) DEMONSTRATED THAT

A SINGLE INJECTION OF 0.5 MG OF THEIR INHIBITOR REDUCED ACID

SECRETION STIMULATED BY HISTAMINE DIHYDROCHLORIDE BY 18$. A

DOSE OF 2.0 MG WAS REQUIRED TO PRODUCE A MAXIMUM INHIBITION

OF 35$. EXTRANEOUS FACTORS MAY HAVE BEEN OPERATING TO

MODIFY SECRETION DURING HISTAMINE INFUSION IN THE STUDY OF

LUCIEN EJ_ M. (1970). THE DOG PREPARATION USED IN THIS STUDY

WAS SUCH THAT THE ANIMALS STILL POSSESSED ANTRA WITH INTACT

INNERVATION. IT HAD BEEN SHOWN BY ANDERSSON AND GROSSMAN

(1965) THAT BACKGROUND VAGAL RELEASE OF ENDOGENOUS GASTRIN

FROM AN INNERVATED ANTRUM POTENTIATED STIMULATION OF H+

SECRETION BY HISTAMINE. SINCE THE DEGREE OF INHIBITION

PRODUCED BY THE INHIBITOR OF LUCIEN ___T M._ (1970) IS SO - 145 -

SLIGHT, IT COULD HAVE BEEN PRODUCED BY INHIBITING THE ACIO

RESPONSE PRODUCED BY ENDOGENOUSLY RELEASED GASTRIN. THE

NECESSITY FOR USING SUCH HIGH DOSES OF MATERIAL (0.5 AND

2.0 MG) INDICATED EITHER THAT THE ENTEROGASTRONE WAS NOT

PURE, OR ELSE DIFFERENT FROM GASTRIC INHIBITORY POLYPEPTIDE.

THE LATTER HYPOTHESIS COULD WELL BE SO, BUT BIOCHEMICAL

DATA ON THE ENTEROGASTRONE OF LUCIEN JE_T AJL (1970) MUST BE

MADE AVAILABLE BEFORE THIS CAN BE ASCERTAINED.

SECRETIN HAS BEEN SHOWN BY WAY (1970) TO EXERT NO

INHIBITION ON PAVLOV POUCH H+ SECRETION STIMULATED BY

INSULIN OR 2-DEOXYGLUCOSE. NO DATA WERE AVAILABLE REGARDING

THE EFFECTS OF CCK-PZ ON VAGALLY INDUCED ACID SECRETION.

OLIVE OIL IN THE DUODENUM WAS SHOWN BY ALLEY JET AJ. (1934)

TO INHIBIT GASTRIC SECRETION FROM THE MAIN STOMACH STIMU•

LATED BY SHAM FEEDING, AND THEREFORE BY A VAGAL MECHANISM.

DOGS USED IN STUDIES INVOLVING G.I.P. WERE PREPARED WITH

THOMAS CANNULAE IN THE GASTRIC REMNANT, THUS PROVIDING A

MEANS OF COLLECTING VAGALLY INDUCED GASTRIC SECRETION.

WAY (1970) DEMONSTRATED THAT SECRETIN WOULD INHIBIT THE

ACID SECRETORY RESPONSE OF PAVLOV POUCHES TO 2-DEOXY-D-

GLUCOSE AND INSULIN HYPOGLYCAEMI A IF THE VAGALLY INNER•

VATED ANTRUM WAS LEFT INTACT. ANTRECTOMY ELIMINATED INHI•

BITION OF ACID SECRETION BY SECRETIN IN THE SAME DOGS,

THE AUTHOR INTERPRETED THE ABOVE EVIDENCE AS INDICATING

THAT IN THE FORMER CASE, SECRETIN HAD BEEN INHIBITING

VAGALLY RELEASED ANTRAL GASTRIN. IN THE STUDY IN WHICH

SECRETION FROM THE GASTRIC REMNANT WAS MEASURED, THE DOGS -146-

USED WERE PREPARED WITH ISOLATED VAGALLY DENERVATED ANTRAL

POUCHES, A PREPARATION WHICH PRECLUDED THE VAGAL RELEASE OF

GASTRIN. AS AN ADDED PRECAUTION, IN CONTROL EXPERIMENTS IN

WHICH INSULIN ALONE WAS GIVEN, THE ANTRAL POUCH WAS PER•

FUSED WITH 0.1M HCL, A PROCEDURE WHICH HAD BEEN SHOWN TO

BLOCK ALL MECHANISMS OF GASTRIN RELEASE (WOODWARD, L.YON,

LANDOR AND DRAGSTEDT, 1954). THE ABSENCE OF ANY DECREASE IN

PEAK ACID RESPONSE TO INSULIN INDICATED THAT THERE HAD BEEN

NO BACKGROUND VAGAL RELEASE OF GASTRIN. G.I.P. PRODUCED

APPROXIMATELY 50$ INHIBITION OF GASTRIC REMNANT H+ AND PEPSIN

SECRETION STIMULATED BY INSULIN H YPOGL YCAEM I A (FLGS. 34 AND 35).

G.I.P. WAS LESS EFFECTIVE AGAINST INSULIN-STIMULATED H+

SECRETION THAN AGAINST GASTRIN PENTAPEPTIDE, AN EFFECT

SIMILAR TO THAT SEEN WITH HISTAMINE.

G.I.P. HAS BEEN SHOWN TO BE A POTENT INHIBITOR OF ACID

SECRETION FROM DENERVATED POUCHES IN THE DOG STIMULATED BY

ENDOGENOUS GASTRIN, EXOGENOUS GASTRIN PENTAPEPTIDE, SHG-L,

HISTAMINE, AND INSULIN HYP0GLYCAEMI A. THE FACT THAT G.I.P.

WAS EFFECTIVE AGAINST DIFFERENT STIMULANTS SUGGESTED THAT

THE INHIBITOR MAY HAVE BEEN ACTING AT THE LEVEL OF THE

PARIETAL CELL DIRECTLY OR AT A FINAL PATHWAY COMMON TO ALL

OF THE STIMULANTS, AS OPPOSED TO BEING A SPECIFIC ANTAGONIST

OF GASTRIN AS HAS BEEN POSTULATED FOR SECRETIN (GROSSMAN,

1970). IN OPPOSITION TO THE CONCEPT OF A GENERALIZED

INHIBITORY ACTION AT THE PARIETAL CELL ARE THE RESULTS

DEMONSTRATING THE GREATER POTENCY OF G.I.P. AS AN INHIBITOR

OF GASTRIC SECRETION STIMULATED BY THE WHOLE GASTRIN MOLECULE - 147 -

OR GASTRIN PENTAPEPTIDE AS COMPARED WITH HISTAMINE OR

VAGALLY INDUCED SECRET I ON.

ONE HOUR INTRAVENOUS INFUSIONS OR SINGLE INJECTIONS

OF G.I.P. PRODUCED NO INHIBITION OF H+ SECRETION STIMULATED

BY GASTRIN PENTAPEPTIDE IN THE ANAESTHETIZED CAT (TABLE XXIX).

THE DOSE OF G.I.P. EMPLOYED WAS 4 TIMES THAT REQUIRED TO

PRODUCE 75$ INHIBITION OF DENERVATED POUCH H+ SECRETION

STIMULATED BY GASTRIN PENTAPEPTIDE IN THE DOG. THIS LACK OF

INHIBITION MAY NOT BE CONSIDERED UNUSUAL IN THE LIGHT OF THE

FACT THAT IN THE CAT, GASTRIN-STIMULATED GASTRIC SECRETION

WAS FOUND BY STENING E_T M_ (1969A) TO BE MUCH LESS SENSITIVE

TO INHIBITION BY SECRETIN THAN IN THE DOG. IN ADDITION,

CCK-PZ PREPARATIONS WHICH WERE WELL-D0CUMENTED INHIBITORS

OF GASTRIN-STIMULATED H+ SECRETION IN THE DOG DID NOT INHIBIT

SECRETION STIMULATED BY GASTRIN PENTAPEPTIDE IN THE CAT (WAY

AND GROSSMAN* UNPUBLISHEO, 1971).

DYCK J__T J__L (19"9) HAD SHOWN THAT GLUCAGON IN DOSES AS

LOW AS 1.0 UG/KG/HR WOULD INHIBIT VOLUME AND PROTEIN OUTPUT

OF PANCREATIC JUICE STIMULATED BY SECRETIN AND CCK-PZ IN THE

DOG. BECAUSE OF THE SIMILARITIES OF G.I.P. TO GLUCAGON

(FIG. 36) G.I.P. INFUSIONS WERE GIVEN INTRAVENOUSLY TO THE

ANAESTHETIZED CAT ON A BACKGROUND OF SECRETIN-STIMULATED

FLOW OF PANCREATIC JUICE. GASTRIC COLLECTIONS WERE MADE

CONCURRENTLY WITH THE PANCREATIC STUDIES IN THESE EXPER•

IMENTS, WITH ACID SECRETION STIMULATED BY A CONSTANT INFUSION

OF GASTRIN PENTAPEPTIDE. GASTRIN PENTAPEPTIDE IN THE DOSE

USED DID NOT PRODUCE ANY OBSERVABLE INCREASES IN VOLUME - 148 -

EVEN THOUGH THIS HAS BEEN DEMONSTRATED TO BE A PHYSIOLOGICAL

ACTION OF GASTRIN (PRESHAW, COOKE AND GROSSMAN, 1965).

LACK OF INHIBITION OF SECRETIN-STIMULATED VOLUME OF

PANCREATIC JUICE INDICATED THAT G.I.P. AT THE DOSES USED IN

THIS STUDY DID NOT HAVE A GLUCAG0N-LIK£ ACTION ON THIS

PARAMETER IN THE CAT.

IN EXPERIMENTS INVOLVING BOTH'CONSCIOUS DOGS AND

ANAESTHETIZED RABBITS, BLOOD GLUCOSE LEVELS WERE MONITORED

DURING INTRAVENOUS ADMINISTRATION OF BOTH GLUCAGON AND G.I.P.

G.I.P. WAS GIVEN BOTH AS A SINGLE INJECTION AND AS A ONE

HOUR INFUSION. DURING A TYPICAL EXPERIMENT IN THE DOG IN

WHICH G.I.P. WAS GIVEN AS A ONE HOUR INFUSION AGAINST GASTRIN

PENTAPEPTIDE-STIMULATED H+ SECRETION, NO INCREASE IN BLOOD

SUGAR LEVELS WAS NOTED (F|G. 57). IN THE RABBIT AND THE

DOG, SINGLE INJECTIONS OF G.I.P. WERE GIVEN AT A DOSE OF

14.0 JUG/KG. THIS DOSE WAS CALCULATED AS BEING EQUIMOLAR TO

A DOSE OF 10.0 JUG/KG OF GLUCAGON, G.I.P. HAVING A MOLECULAR

WEIGHT OF 5,105 AND GLUCAGON, 3,485. THE RESULTS PRESENTED

INDICATED THAT 10.0/JG/KG OF GLUCAGON YIELDED 70 - 100$

INCREASES IN BLOOD SUGAR LEVELS (FlG. 38, TABLES XXX AND

XXXI). RESULTS THEREFORE INDICATE THAT IN DOSES UP TO 14

TIMES THOSE AT WHICH G.I.P. IS AN EFFECTIVE INHIBITOR OF

ACID SECRETION, IT YIELDS NO HYPERGLYCAEM I C NOR PANCREATIC

INHIBITORY EFFECTS DESPITE STRUCTURAL SIMILARITIES TO

GLUCAGON.

EXPERIMENTS IN WHICH THE EFFECTS OF G.I.P. ON PEPSIN

OUTPUT WERE MONITORED HAD INVOLVED THE STIMULATION OF H+ - 149 -

SECRET I ON AND MOTOR ACTIVITY. THE ACTION OF SECRETIN AS A

STIMULANT OF PEPSIN SECRETION IS DIFFERENT TO ITS OTHER

GASTRIC EFFECTS, I.E. INHIBITION OF GASTRIC H+ SECRETION

AND MOTOR ACTIVITY. SECRETIN THUS PROVIDED A MEANS OF

STIMULATING PEPSIN WITHOUT STIMULATING H+ SECRETION OR

GASTRIC MOTOR ACTIVITY. THE DOSE OF SECRETIN USED WAS

SHOWN BY JOHNSON AND GROSSMAN (1968) TO BE SUBMAXIMAL FOR

PANCREATIC STIMULATION IN THE DOG, BUT STILL PRODUCED

POTENT INHIBITION OF GASTRIN—ST IMULATED H+ SECRETION. G.I.P.

IN A DOSE SUFFICIENT TO PRODUCE 85$ INHIBITION OF PEPSIN

OUTPUT STIMULATED BY GASTRIN PENTAPEPTIDE PRODUCED NO SIGNIF•

ICANT INHIBITION OF SECRETIN-STIMULATED PEPSIN LEVELS (TABLE

XXXII). NO EXPLANATION FOR THIS OBSERVATION WAS READILY

AVAILABLE, BUT SEVERAL POSSIBILITIES EXISTEO. IT WAS

POSSIBLE THAT SECRETIN STIMULATED PEPSIN OUTPUT BY A

DIFFERENT MECHANISM THAN GASTRIN OR HISTAMINE, WHICH WAS

RESISTANT TO THE ACTION OF G.I.P., OR SOME OTHER ACTION(S)

OF SECRETIN MAY HAVE BEEN INTERFERING WITH THE INHIBITORY

ACTION OF G.I.P. ON PEPSIN SECRETION.

THE ADVANTAGES AND MORE SPECIFICALLY THE NECESSITY OF

HAVING PHYSIOLOGICAL GASTRIC INHIBITORY MECHANISMS HAVE

BEEN OUTLINED. TE LEOLOGI CALLY, ONE COULD SPECULATE AS TO

WHAT AN EFFECTIVE GASTRIC INHIBITORY MECHANISM SHOULD

ENTAIL. CLINICAL AND EXPERIMENTAL EVIDENCE SUPPORTED THE

STATEMENT THAT THE MUCOSA OF THE SMALL INTESTINE IS MUCH

LESS RESISTANT TO CHEMICAL AND MECHANICAL STRESS THAN THAT

OF THE STOMACH. EVEN SO, GASTRIC JUICE OF LOW PH AND HIGH - 150 -

CONCENTRATION OF PEPSIN, IF IN CONTACT WITH THE GASTRIC

MUCOSA FOR PROLONGED PERIOOS OF TIME, CAN SE FATALLY ULCER•

OGENIC, E.G. THE SHAY RAT PREPARATION (SHAY, SUN AND

GRUENSTEIN, 1954). IN ADDITION, THERE IS THE FACT THAT A

NEUTRAL MEDIUM WILL PROVIDE OPTIMUM CONDITIONS FOR DIGESTIVE

ACTIVITY IN THE DUODENUM. THUS AS A SAFETY FACTOR AND TO

CREATE OPTIMUM DIGESTIVE CONOITIONS, AN INHIBITORY MECHANISM

IDEALLY SHOULD INHIBIT H+ AND PEPSIN SECRETION, DECREASE THE

RATE OF GASTRIC EMPTYING AND NEUTRALIZE H+. G.I.P. MAY

CONTRIBUTE TO THE NEUTRALIZATION OF H+ IN THE SMALL BOWEL

BY THE STIMULATION OF JEJUNAL SECRETION. ALTHOUGH G.I.P.

HAS SO FAR BEEN SHOWN TO POSSESS NO GLUCAG0N-L I KE HYPE R —

GLYCAEMIC OR PANCREATIC EFFECTS, IT HAS BEEN SHOWN BY

GROSSMAN (PERSONAL COMMUNICATION) TO STIMULATE INTESTINAL

SECRETION IN DOGS PREPARED WITH JEJUNAL LOOPS AT A DOSE OF

150 JUG/15 MIN. IN A PREVIOUS SERIES OF EXPERIMENTS GROSSMAN

(PERSONAL COMMUNICATION) REPORTED THAT GLUCAGON WAS SHOWN

TO EXERT THE SAME EFFECT AT A SIMILAR DOSE. THE DOSE OF

G.I.P. USED WAS ABOUT 20 TIMES HIGHER THAN THAT REQUIRED TO

PROOUCE 75$ INHIBITION OF GASTRIN PENTAPEPTIDE-STIMULATED

H+ SECRETION FROM FUNDIC POUCHES IN DOGS, AND THE DOSE OF

GLUCAGON FAR ABOVE LEVELS THAT WILL PRODUCE PHYSIOLOGICAL

CHANGES IN BLOOD SUGAR LEVELS. THE SIGNIFICANCE OF THIS

MUST AWAIT FURTHER STUDY, PARTICULARLY TO DETERMINE IF G.I.P.

IS EFFECTIVE AS A STIMULANT OF INTESTINAL SECRETION IN

DOSES COMPARABLE TO THOSE WHICH PRODUCE INHIBITION OF

GASTRIC SECRETION. - 151 -

THE GASTROINTESTINAL HORMONES SECRETIN AND CCK-PZ HAVE

BEEN CLASSIFIED AS ENTEROGASTRONES BECAUSE OF THEIR ABILITY

TO INHIBIT GASTRIN-STIMULATED H+ SECRETION. SECRETIN, THE

ENTEROGASTRONE WHICH IS PROBABLY RELEASED BY THE PRESENCE OF

HCL IN THE DUODENUM (JOHNSON AND GROSSMAN, 1968), INHIBITED

GASTRIC ACID SECRETION AND MOTOR ACTIVITY BUT STIMULATED

PEPSIN OUTPUT (NAKAJIMA AND MAGEE, 1970). SlNCE THE

STRUCTURE OF CCK-PZ HAS NOT BEEN COMPLETELY ELUCIDATED,

THE ACTIONS OF THE PURE MATERIAL ON THE STOMACH CANNOT BE

STATED WITH COMPLETE ASSURANCE. THIS IS PARTICULARLY TRUE

WITH RESPECT TO STUDIES INVOLVING THE USE OF THE 10$ PURE

CCK-PZ, IN THAT THIS MATERIAL ACTED AS THE STARTING MATERIAL

FOR THE PURIFICATION OF G.I.P. THE LAST POINT IS GIVEN

CREDENCE BY THE WORK OF SPINGOLA AND GROSSMAN (1971) WHO

FOUND THAT ENDOGENOUSLY RELEASED CCK-PZ IN THE DOG WOULD NOT

INHIBIT HEIDENHAIN POUCH SECRETION STIMULATED BY GASTRIN PEN•

TAPEPTIDE, ALTHOUGH THE PANCREATIC RESPONSE CHARACTERISTIC

OF CCK-PZ WAS ELICITED. THE FACT THAT AN EXOGENOUS PORCINE

CCK-PZ PREPARATION (250 I.D.U./MG) WOULD INHIBIT GASTRIN

PENTAPEPTIDE-STIMULATED H+ SECRETION WAS ATTRIBUTED BY

THEM TO BE DUE TO EITHER THE PRESENCE OF GASTRIC INHIBITORY

POLYPEPTIDE OR A DIFFERENCE IN CANINE AS OPPOSED TO PORCINE

CCK-PZ IN TERMS OF ITS ABILITY TO INHIBIT GASTRIN PENTA•

PEPT I D E- S T I M UL A T E D H+ SECRETION IN THE DOG. IN ADDITION TO

NOT INHIBITING ALL OF THE GASTRIC PARAMETERS MENTIONED,

SECRETIN AND CCK-PZ FALL SHORT OF COMPLETELY FULFILLING

THE ROLE OF ENTEROGASTRONE BY NOT INHIBITING HISTAMINE- - 152 -

STIMULATED GASTRIC SECRET I ON (JOHNSON AND GROSSMAN, 19^9)»

A RESPONSE ELICITED BY PLACING FAT IN THE DUODENUM.

G.I.P. HAS BEEN SHOWN IN THE STUDIES OF THIS THESIS TO

INHIBIT GASTRIC H , PEPSIN AND MOTOR ACTIVITY STIMULATED

BY GASTRIN PENTAPEPTIDE, GASTRIN, INSULIN HYP0GLYCAEMI A

AND HISTAMINE. THUS IT FULFILLS THE PHYSIOLOGICAL REQUIRE•

MENTS DESCRIBED ABOVE FOR AN EFFICIENT GASTRIC INHIBITORY

MECHANISM AND MIMICS THE ACTIONS PRODUCED BY THE PRESENCE

OF FAT IN THE DUODENUM. FOR THESE REASONS IT IS FELT THAT

G.I.P. IS AN EXCELLENT CANDIDATE FOR THE HUMORAL AGENT

RELEASED FROM THE DUODENUM BY FAT AND PERHAPS BY HYORO—

CHLORIC ACID AND HYPERTONIC SOLUTIONS WHICH INHIBITSGASTRIC

SECRETION AND MOTOR ACTIVITY.

GASTRIN IS THE ONLY ONE OF THE CHEMICALLY IDENTIFIED

GASTROINTESTINAL HORMONES FOR WHICH THERE IS A SPECIFIC

ASSAY ALLOWING ITS DETECTION IN BLOOD ANO TISSUES. IF THE

DETECTION OF SECRETION AND CHANGES OF BLOOD LEVELS OF A

HUMORAL AGENT ARE REQUIRED AS FINAL PROOF OF HORMONAL STATUS,

THEN SECRETIN AND CCK-PZ FALL SHORT OF ACHIEVING THIS STATUS.

IN THAT THE SAME CONDITIONS HAVE BEEN MET FOR G.I.P. AS HAVE

BEEN FULFILLED FOR SECRETIN AND CCK-PZ, NAMELY THE MIMICKING

OF THE EFFECTS OF AN ENDOGENOUSLY RELEASED HORMONE BY AN

EXOGENOUS MATERIAL, G.I.P. HAS MET THE SAME REQUIREMENTS

FOR HORMONAL STATUS AS SECRETIN AND CCK-PZ. HOWEVER, THE

ESTABLISHMENT OF G.I.P. AS A HORMONE PARTICIPATING IN A

PHYSIOLOGICAL GASTRIC INHIBITORY MECHANISM MUST AWAIT ITS

DETECTION IN BLOOD AND TISSUES. BIBLIOGRAPHY

1. ALLEY, A. AND MACKENZIE, D.W. DISSOCIATION OF THE FUNC• TIONAL PROPERTIES OF GASTRIC GLANDS UNDER THE INFLUENCE OF FAT. AM. J. DIG. DIS. ANO NUT. 1_, 333 (1934-35).

2. ANDERSSON, S. INHIBITORY EFFECTS OF ACID IN ANTRUM- DUODENUM ON FASTING GASTRIC SECRETION IN PAVLOV AND HEIDENHAIN POUCH DOGS. ACTA. PHYSIOL. SCAND. 49, 42 (1960A). —

3. ANDERSSON, S. INHIBITORY EFFECTS OF HYDROCHLORIC ACID IN ANTRUM AND DUODENUM ON GASTRIC SECRETORY RESPONSES TO TEST MEAL IN PAVLOV AND HEIDENHAIN POUCH DOGS. ACTA. PHYSIOL. SCAND. 49, 231 (1960B).

4. ANDERSSON, S. INHIBITORY EFFECTS OF HYDROCHLORIC ACID IN ANTRUM AND DUOOENUM ON HISTAMINE STIMULATED GASTRIC SECRETION IN PAVLOV AND HEIDENHAIN POUCH DOGS. ACTA. PHYSIOL. SCAND. 50, 186 (1960c).

5. ANDERSSON, S. AND GROSSMAN, M.I. EFFECT OF DENERVATION AND SUBSEQUENT RESECTION OF ANTRAL POUCHES ON SECRETION FROM HEIDENHAIN POUCHES IN RESPONSE TO GASTRIN AND HISTAMINE. GASTROENTEROLOGY 5__, 4 (1965).

6. ANDERSSON, S. , NILSSON, G. AND UVNHS, B. INHIBITION OF GASTRIC SECRETION BY ACID IN PROXIMAL AND DISTAL DUODENAL POUCHES. ACTA. PHYS I OL. SCAND. 6j>, 191 (1965).

7. ANSON, M.L. AND MIRSKY, A.E. THE ESTIMATION OF PEPSIN FROM HEMOGLOBIN. J. GEN. PHYSIOL. 1j5, 59 (1932).

8. BAYLISS, W.M. AND STARLING, E.N. THE MECHANISM OF PANCREATIC SECRETION. J. PHYSIOL. 28, 325 (1902).

9. BEAUMONT, W. EXPERIMENTS AND OBSERVATIONS ON THE GASTRIC JUICE AND THE PHYSIOLOGY OF DIGESTION. (PLATTSBURG, NEW YORK, 1833).

10. BIBLER, D.D., HARKINS, H.N. AND NYHUS, L.M. INHIBITION OF EXOGENOUS GASTRIN STIMULATED SECRETION BY FAT IN THE DUOD,ENUM. REV. SURG. 22, 455 (1965).

11. BLAIR, E.L., HARPER, A.A., PEARSON, J.A. AND REED, J.D. STIMULATION OF PEPSIN SECRETION BY EXTRACTS OF INTESTINAL MUCOSA. J. PHYSIOL. 175, 60 (1964).

12. BODANSKY, M., ONDETTI, M.A. AND LEVINE, S.D. SYNTHESIS OF A HEPTACOSAPEPTIDE AMIDE WITH THE HORMONAL ACTIVITY OF SECRETIN. CHEM. INDUSTR. 42, 1757 (1966).

- 153 - - 154 -

13. BROWN, J.C. A GASTRIC INHIBITORY POLYPEPTIDE. I. THE AMINO ACID COMPOSITION AND THE TRYPTIC PEPTIDES. CAN. J. BIOCHEM. 49, 255 (1971).

14. BROWN, J.C. AND DRYBURGH, J.R. A GASTRIC INHIBITORY POLYPEPTIDE. II. THE COMPLETE AMINO ACID SEQUENCE. CAN. J. BIOCHEM. IN PRESS.

15. BROWN, J.C. AND MAGEE, D.F. INHIBITORY ACTION OF CHOLECYSTOK I N I N ON ACID SECRETION FROM HEIDENHAIN POUCHES INDUCED BY ENDOGENOUS GASTRIN. GUT 8, 29 (1967).

16. BROWN, J.C, MUTT, V. AND PEDERSON, R.A. FURTHER PURIFI• CATION OF A POLYPEPTIDE DEMONSTRATING ENTEROGASTRONE ACTIVITY. J. PHYSIOL. 209, 57 (1970).

17. BROWN, J.C. AND PEDERSON, R.A. A MULTIPARAMETER STUDY ON THE ACTION OF PREPARATIONS CONTAINING CHOLECYSTOKININ- PANCREOZYM I N. SCAND. J. GASTROENT. j), 537 (1970).

18. BROWN, J.C, PEDERSON, R.A., JORPES, E. AND MUTT, V. PREPARATION OF HIGHLY ACTIVE ENTEROGASTRONE. CAN. J. PHYSIOL. PHARMAC. 47, 113 (1969).

19. CANNON, W.B. THE MECHANICAL FACTORS OF DIGESTION. (LONGMANS, GREEN & Co., NEW YORK, 1911). "~

20. CODE, C.F. AND WATKINSON, G. IMPORTANCE OF VAGAL INNER• VATION IN THE REGULATORY EFFECT OF ACID IN THE DUODENUM ON GASTRIC SECRETION OF ACID. J. PHYSIOL. 130 » 233 (1955).

21. DAY, J.J. AND KOMAROV, S.A. GLUCOSE AND GASTRIC SECRE• TION. AMER. J. DIG. DIS. 6, 169 (1939).

22. DAY, J.J. ANO WEBSTER, D.R. THE AUTO REGULATION OF THE GASTRIC SECRETION. AMER. J. DIGEST. DlS. NUTR. 2, 527 (1935).

23. DUBOWSKI, K.M. AN 0 - TOLUIDINE METHOD FOR BODY-FLUID GLUCOSE DETERMINATION. J. CLIN. CHEM. 8, 215 (1962).

24. DYCK, W.P., RUDICK, J., HOEXTER, B. AND JANOWITZ, H.D. INFLUENCE OF GLUCAGON ON PANCREATIC EXOCRINE SECRETION. GASTROENTEROLOGY 5.6, 531 (1969).

25. EWALD, CA. AND BOAS, J. VIRCHOWS ARCH. 104, 271 (1886). CITED BY GREGORY, R.A. SECRETORY MECHANISMS OF THE

GASTRO-INTESTINAL TRACTF 118 (EDWARD ARNOLD LTD., NEW YORK, 1962).

26. FARRELL, J.E. AND IVY, A.C STUDIES ON THE MOTILITY OF THE TRANSPLANTED GASTRIC POUCH. AMER. J. PHYSIOL. 76, 227 (1926). - 155 -

27. FENG, T.P., Hou, H.C. AND LIM, R.K.S. ON THE MECHANISM OF THE INHIBITION OF GASTRIC SECRETION BY FAT. CHINESE J. PHYSIOL. 3, 371 (1929).

28. GILLESPIE, I.E. AND GROSSMAN, M.I. INHIBITORY EFFECT OF SECRETIN AND CHOLECYST0KININ ON HEIDENHAIN POUCH RESPONSES TO GASTRIN EXTRACT AND HISTAMINE. GUTJ), 342 (19-4).

29. GRAY, J.S., BRAOLEY, W.B. ANO IVY, A.C. ON THE PREPAR• ATION AND BIOLOGICAL ASSAY OF ENTEROGASTRONE. AM. J. PHYSIOL. JJ_S, 463 (1937).

30. GREENGARD, H.A., ATKINSON, A.J., GROSSMAN, M.I. AND IVY, A.D. THE EFFECTIVENESS OF PARENTERALLY ADMINISTERED ENTEROGASTRONE IN THE PROPHYLAXIS OF RECURRENCES OF EXPERIMENTAL AND CLINICAL PEPTIC ULCER. GASTROENTEROLOGY 7, 625 (1946).

31. GREENLEE, H.B., LONGHI, E.H., GUERRERO, J.D., NELSON, T.S., EL-BEDRI, A.L., AND DRAGSTEDT, L.R. INHIBITORY EFFECT OF PANCREATIC SECRETIN ON GASTRIC SECRETION. AM. J. PHYSIOL. J_90, 396 (1957).

32. GREGORY, R.A. ENTEROGASTRONE - A REAPPRAISAL OF THE PROBLEM. IN GASTRIC SECRETION, 477, SHNITKA, T.K., GILBERT, J.A. AND HARRISON, R.C, ED. (PERGAMON PRESS, NEW YORK, 1967).

33. GREGORY, R.A. AND TRACY, H.J. CONSTITUTION AND PROPER• TIES OF TWO GASTRINS EXTRACTED FROM HOG ANTRAL MUCOSA. GUT 5, 103 (1964).

34. GRIFFITHS, W.J. THE DUODENUM AND THE AUTOMATIC CONTROL OF GASTRIC ACIDITY. J. PHYSIOL. 87, 34 (1936).

35. GROSSMAN, M.I. GASTRIN AND ITS ACTIVITIES. NATURE 228, 1147 (1970).

36. HALVORSON, H.C, MIDDLETON, M.D., BIBLER, D.D., HARKINS, H.N. AND NYHUS, L.M. INFLUENCE OF THE VAGUS NERVE ON THE INHIBITORY EFFECT OF FAT IN THE OUODENUM. AM. J. DIG. DIS. 1_1_, 911 (1966).

37. HARPER, A.A. AND RAPER, H.S. PANCREOZYMIN, A STIMULANT OF THE SECRETION OF PANCREATIC ENZYMES IN EXTRACTS OF THE SMALL INTESTINE. J. PHYSIOL. 102, 115 (1943).

38. HIRSCHOWITZ, B.l. THE SECRETION OF PEPSINOGEN. HANDBOOK OF PHYSIOLOGY, ALIMENTARY CANAL, VOL. II, CHAPTER 50 (AMERICAN PHYSIOLOGICAL SOCIETY HANDBOOK SERIES, WASHINGTON D.C, 1967).

39. HIRSCHOWITZ, B.l. GASTRIN I, PENTAGASTRIN AND HISTAMINE IN THE FISTULA DOG. FED. PROC 27, 1318 (1968). - 156 -

40. HlRSCHOWITZ, B.I. AND SACHS, G. ATROPINE INHIBITION OF INSULIN-, HISTAMINE— AND PENTAGASTRIN-STIMULATED GASTRIC ELECTROLYTE AND PEPSIN SECRETION IN THE DOG. GASTRO• ENTEROLOGY 56, 693 (1969).

41. HONG, S.S., MAGEE, D.F. AND CREWDSON, J.F. THE PHYSIO• LOGICAL REGULATION OF GALL BLADDER EVACUATION. GASTRO• ENTEROLOGY 30, 625 (1956).

42. IVY, A.C. PHYSIOLOGY OF THE GALL BLADDER. PHYSIOL. REV. U, 1 (1934).

43. IVY, A.C. AND OLDBERG, E. A HORMONE MECHANISM FOR GALL BLADDER CONTRACTION AND EVACUATION. AMER. J. PHYSIOL. 86, 599 (1928).

44. JACOBY, H.I. AND MARSHALL, CH. GASTRIC MOTOR-STIMULA• TING ACTIVITY OF GASTRIN TE TRAPEPT I DE IN OOGS. GASTRO• ENTEROLOGY .56, 80 (1969).

45. JOHNSTON, D. AND DUTHIE, H.L. EFFECT OF FAT IN THE DUODENUM ON GASTRIC ACID SECRETION BEFORE AND AFTER VAGOTOMY IN MAN. SCAND. J. GASTROENT. 4, 561 (1969).

46. JOHNSON, L.P., BROWN, J.C. AND MAGEE, D.F. EFFECT OF SECRETIN AND CHOLECYST0KININ-PANCRE0ZYMIN EXTRACTS ON GASTRIC MOTILITY IN MAN. GUT 7, 52 (1966).

47. JOHNSON, L.P. AND MAGEE, D.R. CHOLECYSTOKININ-PANCREO- ZYMIN EXTRACTS AND GASTRIC MOTOR INHIBITION. SURG. GYNEC. OBSTET. V21_, 557 (1965).

48. JOHNSON, L.R. AND GROSSMAN, M.I. SECRETIN: THE ENTERO• GASTRONE RELEASED BY ACID IN THE DUODENUM. AMER. J. PHYSIOL. 21_5, 885 (1968).

49. JOHNSON, L.R. AND GROSSMAN, M.I. EFFECTS OF FAT, SECRETIN AND CHOLECYSTOKININ ON HI STAM INE-STIMULATED GASTRIC SECRETION. AMER. J. PHYSIOL. 216, 1176 (I969).

50. JOHNSON, L.R. AND GROSSMAN, M.I. ANALYSIS OF INHIBITION OF ACID SECRETION BY CHOLECYST0KININ IN DOGS. AMER. J. PHYSIOL. 21J3, 550 (1970).

51. JORPES, J.E. THE ISOLATION AND CHEMISTRY OF SECRETIN AND CHOLECYSTOK I N I N. GA S T R 0 E N TE R 0 L 0 G Y _55 , 157 (1968).

52. JORPES, J.E. AND MUTT, V. GASTROINTESTINAL HORMONES, SECRETIN AND CHOLECYST0KININ-PANCRE0ZYM I N. ANN. INTERN. MED. 55, 395 (1961 ).

53. JORPES, J.E. AND MUTT, V. CHOLECYSTOKININ AND PANCREO• ZYMIN, ONE SINGLE HORMONE? ACTA. PHYSIOL. SCAND. 66, 196 (1966). - 157 -

JORPES, J.E., MUTT, V., MAGNUSSON, S. ANO STEELE, B.B. AMINO ACID COMPOSITION AND TERMINAL AMINO ACID SEQUENCE OF PORCINE SECRETIN. BLOCHEM. BLOPHYS. RES. COMM. 9, 275 (1962).

JORPES, J.E., MUTT, V. AND TOCZKO, K. FURTHER PURIFI• CATION OF CHOLECYSTOKlNIN AND PANCREOZYMIN. ACTA. CHEM.

SCAND. J_8, 2408 (1964).

KALK, H. AND MEYER, P.F. BLUTZUCKERSPIEGEL UND MAGEN- SEKRET 1 ON. Z. KLIN. MED. J_20, 693 (1932).

KASANSKI, P. JAHRESBER. FORTSCHR. TIERCHEM. J53, 552 (1903). CITEO IN IVY, A.D. AND GRAY, J.S. ENTEROGASTRONE, COLO SPRING HARBOR SYMP. QUANT. BIOL. 5, 405 (1937).

KENNEDY, J.A. AND HALLENBECK, G.A. THE PANCREAS ANO GASTRIC SECRETION. FAILURE OF PANCREATECTOMY TO PREVENT INHIBITION OF GASTRIC SECRETION BY SECRETIN. GUT 4, 58 (1963).

KONTUREK, S.J., GA8RYS, B. ANO DUBIEL, J. EFFECT OF EXOGENOUS AND ENDOGENOUS SECRETIN ON GASTRIC ANO PANCREATIC SECRETION IN CATS. AMER. J. PHYSIOL. 217, 1110 (1969).

KOSAKA, T. AND LlM, R.K.S. ON THE MECHANISM OF THE INHIBITION OF GASTRIC SECRETION BY FAT. THE ROLE OF BILE AND CYSTOK|NIN. CHINESE J. PHYSIOL. 4, 213 0930A).

KOSAKA, T. AND LlM, R.K.S. DEMONSTRATION OF THE HUMORAL AGENT IN FAT INHIBITION OF GASTRIC SECRETION. PROC. Soc. EXP. BIOL. MED. 27, 890 (1930s).

KOSAKA, T., LIM, R.K.S., LING, S.M. AND LIU, A.C. ON THE MECHANISM OF THE INHIBITION OF GASTRIC SECRETION BY FAT. A GASTRIC INHIBITORY AGENT OBTAINED FROM THE INTESTINAL MUCOSA. CHINESE J. PHYSIOL. 6, 107 (1932).

LECONTE, P. FONCTIONS GASTR0-INTESTINALES. LA CELLULE 17, 307 (1900). CITED BY BABKIN, B.P. IN THE SECRETORY MECHANISMS OF THE DIGESTIVE GLANDS, 2ND ED., 651 (PAUL B. HOEBER INC., NEW YORK, 1950).

LIM, R.K.S. , Loo, CT. AND LIU, A.C OBSERVATIONS ON THE SECRETION OF THE TRANSPLANTED STOMACH. CHINESE J. PHYSIOL. 1_, 51 (1927).

LIN, T.M. AND SPRAY, G.F. EFFECT OF GLUCAGON ON GASTRIC HCL SECRETION (ABSTR.). GASTROENTEROLOGY^, 1254 (1968).

LINTWAREV, I. BIOCHEM. CENTR. 1_, 96 (1903). CITED IN IVY, A.D. AND GRAY, J.S. ENTEROGASTRONE, COLD SPRING HARBOR SYMP. QUANT. BIOL. 5, 405 (1937). - 158 -

66A. LJUNGBERG, S. BlOLOGISK STYRKEBESTAMNING AV CHOLECYSTO- KININ. SVENSK. FARM. TIDSKR. 68, 351 (1964).

67. LOBASOV, 1.0. THESIS. ST. PETERSBURG (1896). CITED BY BABKIN, B.P. IN THE SECRETORY MECHANISMS OF THE OIGESTIVE GLANDS, 2ND ED., 536 (PAUL B. HOEBER INC., NEW YORK, 1950).

68. LUCIEN, H.W., ITOH, Z., SUN, D.C.H., MEYER, J., CARLTON, N. AND SCHALLY, A.V. THE PURIFICATION OF ENTEROGASTRONE FROM PORCINE GUT. ARCH. BIOCHEM. BIOPHYS. 134, 180 (1969).

69. LUCIEN, H.W., ITOH, Z. AND SCHALLY, A.V. INHIBITORY EFFECTS OF A PURIFIED ENTEROGASTRONE, SECRETIN, AND CHOLECYSTOKININ ON HISTAMINE—STIMULATED GASTRIC ACID SECRETION. GASTROENTEROLOGY 59, 707 (1970).

70. MAGEE, O.F. ANO NAKAMURA, M. ACTION OF PANCREOZYMIN PRE• PARATIONS ON GASTRIC SECRETION. NATURE 212, 1487 (1966).

71. MARBAIX, 0. LE PASSAGE PYLORIQUE. LA CELLULE 14, 249 (1898). —

72. MENGUY, R. STUDIES ON THE ROLE OF PANCREATIC AND BILIARY SECRETIONS IN THE MECHANISM OF GASTRIC INHIBITION BY FAT. SURGERY 48, 195 (1960).

73. MORRIS, T.Q., SAROI, G. AND BRADLEY, S.E. CHARACTER OF GLUCAGON-INDUCED CHOLERESIS. FED. PROC. 26, 774 (1967).

74. MURAT, J.E. AND WHITE, T.T. STIMULATION OF GASTRIC SECRETION BY COMMERCIAL CHOLECYSTOKININ EXTRACTS. PROC. SOC. EXPTL. BIOL. MED. 1_23, 593 (1966).

75. MUTT, V. AND JORPES, J.E. SECRETIN ISOLATION AND DETER• MINATION OF STRUCTURE (ABSTR.). PROCEEDINGS OF THE FOURTH INTERNATIONAL SYMPOSIUM ON THE CHEMISTRY OF NATURAL PROOUCTS. STOCKHOLM, SWEOEN (1966).

76. MUTT, V. AND JORPES, J.E. STRUCTURE OF PORCINE CHOLE- CYSTOKININ—PANCREOZYMIN. EUROPEAN J. BlOCHEM. 6, 156 (1968).

77. NAKAJIMA, S. AND MAGEE, D.R. INFLUENCES OF DUODENAL ACIDIFICATION ON ACID ANO PEPSIN SECRETION OF THE STOMACH

IN DOGS. AMER. J. PHYSIOL. __J_8, 545 (1970).

78. NAKAMURA, M., NAKAJIMA, S. AND MAGEE, D.F. ACTION OF PANCREOZYMIN PREPARATIONS ON GASTRIC ACID SECRETION. GUT 9, 405 (1968).

79* OKAOA, S., KURAMOCHI, I., TSUKAHARA, T. AND OOINOUE, T. PANCREATIC FUNCTION. IV. THE HUMONEURAL REGULATION OF THE GASTRIC, PANCREATIC AND BILIARY SECRETIONS. ARCH. INTERN. MED. 43» 446 (1929). - 159 -

80. PAVLOV, I.P. THE WORK OF THE DIGESTIVE GLANDS, 2ND ED, TRANSLATED BY W.H. THOMPSON (CHARLES GRIFFIN, LONDON, 1910).

81 . PINCUS, I.J., THOMAS, J.E. AND REHFUSS, M.E. A STUDY OF GASTRIC SECRETION AS INFLUENCED BY CHANGES IN DUODENAL ACIDITY. PROC. Soc. Exp. BlOL. N.Y. 51, 367 (1942).

82. PRESHAW, R.M., COOK, A.R. AND GROSSMAN, M.I. QUANTITA• TIVE ASPECTS OF RESPONSE OF CANINE PANCREAS TO DUODENAL ACIDIFICATION. AMER. J. PHYSIOL. 210, 629 (1966).

83. PRESHAW, R.M. AND GROSSMAN, M.I. COMPARISON OF SUBCU• TANEOUS AND INTRAVENOUS ADMINISTRATION OF PANCREATIC

STIMULANTS. AMER. J. PHYSIOL. 209, 803 (1965).

84. QUIGLEY, J.P. AND HALLARAN, W.R. THE INDEPENDENCE OF SPONTANEOUS GASTRO-INTESTINAL MOTILITY AND BLOOD SUGAR

LEVELS. AMER. J. PHYSIOL. 100, 102 (1932).

85. QUIGLEY, J.P. AND MESCHAN, I. THE ROLE OF THE VAGUS IN THE REGULATION OF THE PYLORIC SPHINCTER AND ADJACENT PORTIONS OF THE GUT, WITH SPECIAL REFERENCE TO THE PROCESS OF GASTRIC EVACUATION. AMER. J. PHYSIOL. 123, 166 (1938).

86. QUIGLEY, J.P. AND PHELPS, K.R. THE MECHANISM OF GASTRIC MOTOR INHIBITION FROM INGESTED CARBOHYDRATE. AMER. J. PHYSIOL. 109, 133 (1934).

87. QUIGLEY, J.P., ZETTLEMAN, H.J. AND IVY, A.D. ANALYSIS OF THE FACTORS INVOLVED IN GASTRIC MOTOR INHIBITION BY

FATS. AMER. J. PHYSIOL. J_08, 643 (1934). 88. SAMOLS, E., MARRI, G. AND MARKS, V. PROMOTION OF

INSULIN SECRETION BY GLUCAGON. LANCET 2, 415 0965). 89. SERDIUKOV, A.S., CITED IN PAVLOV, ..I.P. . THE WORK OF THE DIGESTIVE GLANDS, 2ND ED. TRANSLATETRANS D BY W.H. THOMPSON (CHARLES GRIFFIN, LONDON, 1910).

SHAY, H. AND GERSHON-COHEN, J. EXPERIMENTAL STUDIES IN 90. GASTRIC PHYSIOLOGY IN MAN. II. A STUDY OF PYLORIC CONTROL. THE ROLES OF ACID ANO ALKALI. SURG. GYNEC. OBSTET. 58, 935 (1934).

SHAY, H., GERSHON-COHEN, J. AND PELS, S.S. THE ROLE OF 91. THE UPPER SMALL INTESTINE IN THE CONTROL OF GASTRIC SECRETION, THE EFFECT OF NEUTRAL FAT, FATTY ACID AND SOAPS: THE PHASE OF GASTRIC SECRETION INFLUENCED ANO THE RELATIVE IMPORTANCE OF THE PSYCHIC AND CHEMICAL PHASES. ANN. INTERN. MED. 13, 294 (1939). - 160 -

92. SHAY, H., GERSHON-COHEN, J., FELS, S.S. AND SIPLET, H. CONCERNING THE INFLUENCE OF GLUCOSE ON THE RESPONSE OF THE HUMAN STOMACH TO TEST MEALS. AMER. J. DLG. DLS. 9, 363 (1942).

93. SHAY, H. , SUN, D.C.H. AND GRUENSTEIN, M.A. QUANTITA• TIVE METHOD FOR MEASURING SPONTANEOUS GASTRIC SECRETION IN THE RAT. GASTROENTEROLOGY 26, 906 (1954).

94. SIRCUS, W. STUDIES ON THE MECHANISMS IN THE DUODENUM INHIBITING GASTRIC SECRETION. QUART. J. EXP. PHYSIOL. 43, 114 (1958).

95. SOKOLOV, A.P. THESIS. ST. PETERSBURG (1904). CITED BY BABKIN, B.P. IN THE SECRETORY MECHANISMS OF THE DIGESTIVE GLANDS, 2ND ED., 642 (PAUL B. HOEBER INC., NEW YORK, 1950).

96. SPINGOLA, L.J. AND GROSSMAN, M.I. FAILURE OF ENDOGENOUS CHOLECYSTOKININ TO INHIBIT PENTAGASTRIN-STIMULATED ACID SECRETION (ABSTR.). GASTROENTEROLOGY, IN PRESS.

97. STENING, G.F., JOHNSON, L.R. AND GROSSMAN, M.I. EFFECT OF SECRETIN ON ACID AND PEPSIN SECRETION IN CAT ANO DOG. GASTROENTEROLOGY _5_6, 468 (1969A).

98. STENING, G.F., JOHNSON, L.R. AND GROSSMAN, M.I. EFFECT OF CHOLECYSTOKININ AND CAERULEIN ON GASTRIN- AND HISTAMINE-EVOKED GASTRIC SECRETION. GASTROENTEROLOGY 57, 44 (1969B).

99. THOMAS, J.E. THE INTESTINAL PH THRESHOLD FOR REGULATION OF GASTRIC EMPTYING. FED. PROC. j5, 214 (1947).

100. THOMAS, J.E., CRIDER, J.O. AND MOGAN, J.C. A STUDY OF REFLEXES INVOLVING THE PYLORIC SPHINCTER AND ANTRUM AND THEIR ROLE IN GASTRIC EVACUATION. AMER. J. PHYSIOL. 108, 683 (1934).

101. THOMAS, J.E. AND MOGAN, C.J. THE ENTEROGASTRIC REFLEX. PROC. SOC. EXP. BIOL. N.Y. 28, 968 (1931 ).

102. TRACY, H.J. AND GREGORY, R.A. PHYSIOLOGICAL PROPERTIES OF A SERIES OF SYNTHETIC PEPTIDES STRUCTURALLY RELATED TO GASTRIN I. NATURE 204, 935 (1964).

103. VAGNE, M., STENING, G.F., BROOKS, F.P. AND GROSSMAN, M.I. SYNTHETIC SECRETIN: COMPARISON WITH NATURAL SECRETIN FOR POTENCY AND SPECTRUM OF PHYSIOLOGICAL ACTIONS. GASTROENTEROLOGY jj5, 260 (1968).

104. WADDELL, W.R. AND WANG, CC. THE EFFECT OF VAGOTOMY ON GASTRIC EVACUATION OF HIGH FAT MEALS. J. APP. PHYSIOL. 5, 705 (1953). - 161 -

105. WALAWSKI, J. LES BIOOIALYSATES INTESTINAUX AGENTS INHIBITEURS DE LA SECRETION GASTRIQUE. CR. SOC. BlOL. PARIS 99, 1169 (1928). CITED BY BABKIN, B.P. IN THE SECRETORY MECHANISMS OF THE DIGESTIVE GLANDS, 2ND ED., 635 (PAUL B. HOEBER INC., NEW YORK, 1950).

106. WANG, CC AND GROSSMAN, M.I. PHYSIOLOGICAL DETER• MINATION OF THE RELEASE OF SECRETIN AND PANCREOZYMIN FROM THE INTESTINE OF DOGS WITH TRANSPLANTED PANCREAS.

AMER. J. PHYSIOL. __54, 527 (1951 ).

107. WAY, L.W. EFFECT OF SECRETIN ON GASTRIC ACID SECRETION IN RESPONSE TO CHOLINERGIC STIMULI. GASTROENTEROLOGY 59, 510 (1970).

108. WIRSCHUBSKI, I. JAHRESBER. FORTSCHR. TIERCHEM. 30, 374 (1900). CITED IN IVY, A.D. AND GRAY, J.S. ENTERO- GASTRONE, COLD SPRING HARBOR SYMP. QUANT. BIOL. 5, 405 (1937).

109. WOODWARD, E.R., LYON, E.S., LANDOR, J. AND DRAGSTEDT, L.R. THE PHYSIOLOGY OF THE GASTRIC ANTRUM. GASTRO• ENTEROLOGY 27, 766 (1954).

110. WORMSLEY, K.C AND GROSSMAN, M.I. INHIBITION OF GASTRIC ACID SECRETION BY SECRETIN AND BY EXOGENOUS ACID IN THE DUODENUM. GASTROENTEROLOGY 47, 72 (1964).