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The isolation, structure and physiological actions of motilin Cook, Michael Arnold 1972

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THE ISOLATION, STRUCTURE AND PHYSIOLOGICAL ACTIONS OF MOT IL IN by MICHAEL ARNOLD COOK B . S c . U n i v e r s i t y o f L o n d o n , 1967 A THESIS SUBMITTED IN THE REQUIREMENTS DOCTOR OF PARTIAL FULFILMENT OF J FOR THE DEGREE OF PHILOSOPHY IN THE DEPARTMENT OF PHYSIOLOGY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED STANDARD SUPERVISOR EXTERNAL EXAMINER THE UNIVERSITY OF BRIT ISH COLUMBIA M a y , 1972 In presenting t h i s thesis i n p a r t i a l f u l f i l m e n t of the requirements f o r an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L i b r a r y s h a l l make i t f r e e l y a v a i l a b l e f o r reference and study. I f u r t h e r agree that permission f o r extensive copying of t h i s thesis f o r s c h o l a r l y purposes may be granted by the Head of my Department or by h i s representat ives . It i s understood that copying or p u b l i c a t i o n of t h i s thes is f o r f i n a n c i a l gain s h a l l not be allowed without my wri t ten permission. Department of PtfyS't O £0 fcy The U n i v e r s i t y of B r i t i s h Columbia Vancouver 8 , Canada Date MfiUr 7 % ABSTRACT The c o n t r o l of g a s t r i c motor a c t i v i t y has been shown to be exer ted by both neural and humoral mechanisms. Of the humoral mechanisms, the best known i s the e f f e c t of a c i d in the duodenum in i n h i b i t i n g g a s t r i c motor a c t i v i t y v i a the hormone e n t e r o g a s t r o n e . A l k a l i n e s o l u t i o n s in the duodenum have been shown t o i n c r e a s e the r a t e of g a s t r i c emptying in man and t o s t i m u l a t e the motor a c t i v i t y of a t r a n s p l a n t e d f u n d i c pouch in the dog. These f i n d i n g s have suggested t h a t a g a s t r i c m o t o r - s t i m u l a t i n g agent might be r e l e a s e d from the duodenal mucosa on a IkaI i n i z a t i o n . In support of t h i s h y p o t h e s i s , g a s t r i c m o t o r - s t i m u l a t i n g f r a c t i o n s have been separated from crude e x t r a c t s of the duodenal mucosa. T h i s t h e s i s d e a l s w i th the i s o l a t i o n , amino a c i d sequence d e t e r m i n a t i o n and some p h y s i o l o g i c a l a c t i o n s of m o t i l i n , a g a s t r i c m o t o r - s t i m u l a t i n g p o l y p e p t i d e prepared from an e x t r a c t of hog upper -i n t e s t i n a l mucosa. The i s o l a t i o n of m o t i l i n from hog i n t e s t i n a l mucosa e x t r a c t s was achieved by a column chromatographic system i n v o l v i n g both ion exchange stages and gel f i l t r a t i o n . The p u r i t y of the i s o l a t e d p o l y p e p t i d e was e s t a b l i s h e d by t h i n - l a y e r chromatography on s i l i c a gel and by poIyacryI amide gel e l e c t r o p h o r e s i s as wel l as by the demonst rat ion of a homogeneous amino a c i d c o m p o s i t i o n a c r o s s the e l u t i o n peak of the f i n a l p r e p a r a t i v e s t a g e . End-group a n a l y s e s revea led the N- and C - t e r m i n a l amino a c i d r e s i d u e s of m o t i l i n to be p h e n y l a l a n i n e and g lu tamine r e s p e c t i v e l y . Cyanogen bromide and t r y p s i n were used to c l e a v e the molecu le and the fragments r e s u l t i n g from these procedures were sub jec ted to amino a c i d sequence a n a l y s i s by the Edman d e g r a d a t i v e procedure . P e p t i d e s w i th o v e r l a p p i n g sequences were produced by d i g e s t i o n of m o t i l i n w i th chymotryps in and t h e r m o l y s i n . Sequence i i a n a l y s i s of these pept ides led to the e l u c i d a t i o n of the complete amino a c i d sequence of m o t i l i n . The sequence of m o t i l i n , a 22 amino a c i d r e s i d u e p o l y p e p t i d e , showed no s i m i l a r i t i e s to any of the known g a s t r o i n t e s t i n a l hormones. Chemical m o d i f i c a t i o n s of the molecu le were made in o rder t o determine which f e a t u r e s were e s s e n t i a l f o r i t s b i o l o g i c a l a c t i v i t y . Removal of t he C - t e r m i n a l r e s i d u e s d i d not a l t e r the a c t i v i t y w h i l e o x i d a t i o n of the meth ion ine r e s i d u e r e s u l t e d in a s i g n i f i c a n t l o s s of b i o l o g i c a l a c t i v i t y . A c y l a t i o n of the l y s i n e r e s i d u e s by a c e t y l a t i o n and succ iny I a t ion a l s o r e s u l t e d in s i g n i f i c a n t l o s s of b i o l o g i c a l a c t i v i t y . The most s p e c i f i c a c t i o n of m o t i l i n , determined by in t ravenous a d m i n i -s t r a t i o n in c h r o n i c dog pouch p r e p a r a t i o n s , was the s t i m u l a t i o n of f u n d i c and a n t r a l motor a c t i v i t y . Peps in output from the pouches was increased d u r i n g i n f u s i o n s of m o t i l i n w h i l e H + ion output was not s i g n i f i c a n t l y a l t e r e d . A decrement in the motor a c t i v i t y response t o m o t i l i n was n o t i c e d d u r i n g i n f u s i o n s , and i n t e r r u p t e d i n f u s i o n s conf i rmed t h i s t a c h p h y l a x i s . In fus ions of m o t i l i n a g a i n s t background H + ion s e c r e t i o n s t i m u l a t e d by s y n t h e t i c human g a s t r i n r e s u l t e d in no s i g n i f i c a n t a l t e r a t i o n of H + ion ou tput of the pouches thus e s t a b l i s h i n g t h a t m o t i l i n does not possess any i n h i b i t o r y o r s t i m u l a t o r y p r o p e r t i e s w i t h regard t o a c i d s e c r e t i o n . A t r o p i n e was shown to b lock the motor a c t i v i t y response to m o t i l i n w h i l e g a n g l i o n i c b lockade us ing p e n t o l i n i u m d i d not r e s u l t in any s i g n i f i c a n t decrement in t h i s response . The p o s s i b i l i t y t h a t m o t i l i n i s the agent r e l e a s e d when the duodenum becomes a l k a l i n e and i s thus p a r t of a dual humoral , pH dependent, duodenal c o n t r o l mechanism f o r g a s t r i c motor a c t i v i t y must a w a i t i t s i d e n t i f i c a t i o n in the blood and t i s s u e s . i i i TABLE OF CONTENTS Page ABSTRACT i i L I S T OF TABLES v i i i L I S T OF FIGURES x i ACKNOWLEDGEMENTS x v INTRODUCTION I METHODS 10 COLUMN CHROMATOGRAPHY 10 i Ge l F i l t r a t i o n 10 i i Ion E x c h a n g e C h r o m a t o g r a p h y I I HIGH VOLTAGE ELECTROPHORESIS • 12 i E q u i p m e n t and R e a g e n t s 12 i i S t a n d a r d s and M a r k e r s 13 i i i P r o c e d u r e f o r Q u a l i t a t i v e E l e c t r o p h o r e s i s 15 i v P r o c e d u r e f o r P r e p a r a t i v e E l e c t r o p h o r e s i s 15 v D e t e c t i o n R e a g e n t s 16 PAPER CHROMATOGRAPHY 17 i E q u i p m e n t 17 i i P r o c e d u r e f o r Q u a l i t a t i v e C h r o m a t o g r a p h y 17 i i i P r o c e d u r e f o r P r e p a r a t i v e C h r o m a t o g r a p h y 17 i v S o l v e n t S y s t e m 18 THIN LAYER CHROMATOGRAPHY 18 QUALITATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS 19 ELUTION FROM PAPER 20 AMINO ACID ANALYSIS 20 i E q u i p m e n t and R e a g e n t s 20 i i P r e p a r a t i o n o f S a m p l e s 22 i i i C a l c u l a t i o n o f Amino A c i d C o m p o s i t i o n s 23 AMIDE DETERMINATIONS 24 POLYPEPTIDE DEGRADATION PROCEDURES 24 A N o n - E n z y m a t i c C l e a v a g e 24 B E n z y m a t i c C l e a v a g e 27 i P r e p a r a t i o n o f R e a g e n t s • 27 i i D i g e s t i o n P r o c e d u r e s 27 C-TERMINAL RESIDUE DETERMINATION 27 N-TERMINAL RESIDUE DETERMINATION 28 AMINO ACID SEQUENCE DETERMINATION 30 i P r e p a r a t i o n o f R e a g e n t s 30 i i D e g r a d a t i o n P r o c e d u r e 33 NOMENCLATURE OF PEPTIDES 34 S P E C I F I C COLOUR REACTIONS 34 i v Pa je CHEMICAL MODIFICATION REACTIONS 36 i O x i d a t i o n w i th Perform i c A c i d 36 i i O x i d a t i o n w i t h Hydrogen P e r o x i d e 36 i i i Reduct ion w i th C y s t e i n e 36 i v A c e t y l a t i o n 37 v S u c c i n y l a t i o n 37 ANIMAL PREPARATIONS 38 i Chron ic Dog P r e p a r a t i o n s 38 A B i c k e l Pouch 38 B A n t r a l Pouch 38 i i Acute Guinea P i g P r e p a r a t i o n s 39 i i i Acute Cat P r e p a r a t i o n s 41 i v Acute R a b b i t P r e p a r a t i o n s 41 EXPERIMENTAL PROCEDURES 42 i C h r o n i c Dog Experiments 42 i i A n a l y s i s of G a s t r i c Samples 44 A E s t i m a t i o n of H+ ion c o n c e n t r a t i o n 44 B E s t i m a t i o n of Peps in 44 i i i C a l c u l a t i o n of M o t i l i t y Ind ices 44 i v Assay of M o t i l i n A c t i v i t y u s i n g C h r o n i c Dog P r e p a r a t i o n s 46 v Assay of C h o l e c y s t o k i n i n a c t i v i t y of M o t i l i n us ing Acute Guinea P i g P r e p a r a t i o n s 46 v i Assay of S e c r e t i n a c t i v i t y of M o t i l i n us ing Acute Cat P r e p a r a t i o n s 47 v i i Assay f o r Blood P r e s s u r e e f f e c t s of M o t i l i n u s i n g Acute R a b b i t P r e p a r a t i o n s 47 v i i i Assay f o r Py rogen ic e f f e c t s of M o t i l i n us ing C h r o n i c Dog P r e p a r a t i o n s 49 ANALYSIS OF DATA 49 RESULTS 50 PURIFICATION OF MOTILIN 50 i S t a r t i n g M a t e r i a l 50 i i F r a c t i o n a t i o n o f P r e s e c r e t i n on CM Cel l u l o s e • 50 i i i F r a c t i o n a t i o n of Ml on Sephadex G-25 52 i v F r a c t i o n a t i o n of M2 on TEAE C e l l u l o s e 52 v F r a c t i o n a t i o n of M3 on Sephadex G -25 55 v i D e s a l t i n g of M4 on Sephadex G -25 55 v i i Chromatography of M3 on SE Sephadex C -25 55 DETERMINATION OF THE AMINO ACID COMPOSITION OF MOTILIN 58 THIN LAYER CHROMATOGRAPHY AND QUALITATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS OF MOTILIN 58 DETERMINATION OF N-TERMINAL RESIDUE 61 DETERMINATION OF C-TERMINAL RESIDUE 61 OXIDATION OF MOTILIN WITH PERFORMIC ACID 61 CYANOGEN BROMIDE CLEAVAGE OF MOTILIN - 64 CHYMOTRYPTIC CLEAVAGE OF MOTILIN 64 v Page TRYPTIC CLEAVAGE OF MOTILIN 68 THERMOLYSIN CLEAVAGE OF MOTILIN 74 AMIDE DETERMINATIONS 82 CHEMICAL MODIFICATIONS OF MOTILIN 82 i O x i d a t i o n o f M o t i l i n w i t h P e r f o r m i c A c i d 82 i i O x i d a t i o n of M o t i l i n w i th Hydrogen P e r o x i d e — 82 i i i Reduct ion of Hydrogen P e r o x i d e o x i d i s e d M o t i l i n w i th C y s t e i n e 82 iv C leavage of M o t i l i n w i th T r y p s i n and Cyanogen Bromide 84 v Removal of C - t e r m i n a l Res idues w i th Carboxypept idase A 84 v i Removal of N - te rmina l r e s i d u e 84 v i i A c e t y l a t i o n of M o t i l i n 86 v i i i Succ i ny I a t ion of M o t i l i n 90 EFFECTS OF INTRAVENOUS INFUSION OF MOTILIN AT TWO DOSE LEVELS ON BICKEL POUCH MOTOR ACTIVITY, H + ION SECRETION, PEPSIN SECRETION AND ANTRAL MOTOR ACTIVITY 90 i B i c k e l - P o u c h Motor A c t i v i t y 90 i i B i c k e l Pouch H + ion S e c r e t i o n 94 i i i B i c k e l Pouch Peps in S e c r e t i o n 94 i v B i c k e l Pouch motor a c t i v i t y 94 EFFECTS OF INTERRUPTED INTRAVENOUS INFUSION OF MOTILIN ON BICKEL POUCH MOTOR ACTIVITY, H ION SECRETION, PEPSIN SECRETION AND ANTRAL MOTOR ACTIVITY 109 i B i c k e l pouch Motor A c t i v i t y 109 i i B i c k e l pouch H + ion s e c r e t i o n 109 i i i B i c k e l pouch Peps in s e c r e t i o n •• 114 iv A n t r a l pouch Motor A c t i v i t y 114 EFFECTS OF INTRAVENOUS INFUSION OF MOTILIN ON BICKEL POUCH MOTOR ACTIVITY, H+ ION SECRETION, PEPSIN SECRETION AND ANTRAL POUCH MOTOR ACTIVITY AGAINST A BACKGROUND OF H + ION SECRETION STIMULATED BY SYNTHETIC HUMAN GASTRIN I 14 i B i c k e l pouch Motor A c t i v i t y I 19 i i B i c k e l pouch H* ion s e c r e t i o n 119 i i i B i c k e l pouch Peps in s e c r e t i o n 119 iv A n t r a l pouch motor a c t i v i t y 119 EFFECTS OF INTRAVENOUS INFUSION OF MOTILIN ON BICKEL POUCH MOTOR ACTIVITY, H+ ION SECRETION, PEPSIN SECRETION AND ANTRAL POUCH MOTOR ACTIVITY AGAINST A BACKGROUND PEPSIN SECRETION STIMULATED BY SECRETIN 119 — i B i c k e l pouch motor a c t i v i t y 128 i i B i c k e l pouch H + ion s e c r e t i o n 128 i i i B i c k e l pouch peps in s e c r e t i o n 128 iv A n t r a l pouch motor a c t i v i t y 128 vi Page EFFECTS OF CHOLINERGIC AND GANGLIONIC BLOCKADE ON THE BICKEL POUCH MOTOR ACTIVITY, H + ION SECRETION AND PEPSIN SECRETION IN RESPONSE TO INTRAVENOUS INJECTIONS OF MOTILIN 137 i B i c k e l pouch motor a c t i v i t y 137 i i B i c k e l pouch H + ion s e c r e t i o n 141 i i i B i c k e l pouch Peps in s e c r e t i o n 141 ASSAY OF CHOLECYSTOKININ EFFECTS OF MOTILIN 141 ASSAY OF SECRETIN EFFECTS OF MOTILIN 141 ASSAY OF BLOOD PRESSURE AND PYROGENIC EFFECTS OF MOTILIN 150 DISCUSSION 155 BIBLIOGRAPHY 170. APPENDIX 179 vii LIST OF TABLES Tab I e Page I Summary of s tages of p u r i f i c a t i o n o f mot i I i n 59 II Amino a c i d a n a l y s e s and c o m p o s i t i o n s 60 III Amino a c i d c o m p o s i t i o n s of CNBr f ragments 66 IV Amino a c i d c o m p o s i t i o n s of c h y m o t r y p t i c p e p t i d e s of moti I in 70 V Amino a c i d c o m p o s i t i o n s of t r y p t i c p e p t i d e s of moti I in 75 VI Amino a c i d c o m p o s i t i o n s of t r y p t i c p e p t i d e s of CN2 77 VII Amino a c i d c o m p o s i t i o n s of t h e r m o l y s i n p e p t i d e s of m o t i l i n 80 VI I I Amides of the t r y p t i c p e p t i d e s of m o t i l i n • 83 IX Amino a c i d c o m p o s i t i o n of ACTR2 89 X E f f e c t of m o t i l i n i n f u s i o n o f I u g / k g - h . on f u n d i c pouch motor a c t i v i t y • 92 XI E f f e c t of m o t i l i n i n f u s i o n a t 2 u g / k g - h . on f u n d i c pouch motor a c t i v i t y 95 XII E f f e c t of m o t i l i n i n f u s i o n a t I u g / k g - h . on f u n d i c pouch H"1" ion output 97 XI I I E f f e c t of m o t i I i n i n f u s i o n a t 2 u g / k g - h . on f u n d i c pouch H + ion output 99 XIV E f f e c t of m o t i l i n i n f u s i o n a t I u g / k g - h . on f u n d i c pouch peps in output 101 XV E f f e c t of m o t i l i n i n f u s i o n a t 2 u g / k g - h . on f u n d i c pouch peps in output 103 XVI E f f e c t of m o t i l i n i n f u s i o n a t I u g / k g - h . on a n t r a l pouch motor a c t i v i t y 7 105 XVII E f f e c t of m o t i l i n i n f u s i o n a t 2 u g / k g - h . on a n t r a l pouch motor a c t i v i t y 107 v i i I Tab I e Page XVIII E f f e c t of i n t e r r u p t e d i n f u s i o n of m o t i l i n on f u n d i c pouch motor a c t i v i t y 110 XIX E f f e c t of i n t e r r u p t e d i n f u s i o n of m o t i l i n on f u n d i c pouch H + ion output 112 XX E f f e c t of i n t e r r u p t e d i n f u s i o n of m o t i l i n on f u n d i c pouch peps in output 115 XXI E f f e c t o f i n t e r r u p t e d i n f u s i o n of m o t i l i n on a n t r a l pouch motor a c t i v i t y 117 XXII E f f e c t of m o t i I i n i n f u s i o n a g a i n s t i n f u s i o n of s y n t h e t i c human g a s t r i n on f u n d i c pouch motor a c t i v i t y 120 XXII I E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s y n t h e t i c human g a s t r i n on f u n d i c pouch H + ion output 122 XXIV E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s y n t h e t i c human g a s t r i n on f u n d i c pouch peps in output 124 XXV E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s y n t h e t i c human g a s t r i n on a n t r a l pouch motor a c t i v i t y 126 XXVI E f f e c t o f m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s e c r e t i n on f u n d i c pouch motor a c t i v i t y 129 XXVII E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s e c r e t i n on f u n d i c pouch l-T1" ion output 131 XXVIII E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s e c r e t i n on f u n d i c pouch peps in output 133 XXIX E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s e c r e t i n on a n t r a l pouch motor a c t i v i t y 135 XXX E f f e c t of a t r o p i n e and a n s o l y s e n on f u n d i c pouch motor a c t i v i t y responses t o i n j e c t i o n s of mot i I i n 138 XXXI E f f e c t of a t r o p i n e and a n s o l y s e n on f u n d i c pouch H + ion output reponses t o i n j e c t i o n s o f moti I in 142 XXXII E f f e c t of a t r o p i n e and a n s o l y s e n on f u n d i c pouch peps in output responses t o i n j e c t i o n s of moti I i n 145 ix Tab le Page XXXIII E f f e c t of in t ravenous i n j e c t i o n s of m o t i l i n on f u n d i c pouch motor a c t i v i t y , rf1" ion s e c r e t i o n and peps in s e c r e t i o n 148 XXXIV C o n t r o l data - Fundic motor a c t i v i t y 151 XXXV C o n t r o l data - H + ion output 152 XXXVI C o n t r o l data - Peps in output 153 XXXVII C o n t r o l data - A n t r a l motor a c t i v i t y 154 x LIST OF FIGURES F i g u r e Page 1 . Standards and Markers used on High V o l t a g e E l e c t r o p h o r e s i s 14 2 . E l e c t r o p h o r e t i c M o b i l i t i e s a t pH 6 . 5 25 3 . E l e c t r o p h o r e t i c M o b i l i t i e s a t pH 1.9 26 4 . P o s i t i o n s of Dansyl Amino A c i d s a f t e r Chromatography 31 5 . P o s i t i o n s of Dansyl Amino A c i d s a f t e r Chromatography 32 6 . R e a c t i o n s of Edman Degradat i ve Procedure 35 7 . C h r o n i c Dog P r e p a r a t i o n 40 8 . A n t r a l Pouch Cannula Arrangement 43 9 . Standard Curves f o r Peps in E s t i m a t i o n 45 10. Records and Standard c u r v e f o r C h o i e c y s t o k ? n i n assay 48 11. E l u t i o n prof i I e o f P r e s e c r e t i n on CM ee l IuIose 51 12. E l u t i o n p r o f i l e of Ml on Sephadex G-25 53 13. E l u t i o n p r o f i l e o f M2 on TEAE Cel l u l o s e 54 14. E l u t i o n p r o f i l e of M3 on Sephadex G-25 56 15. E l u t i o n p r o f i l e of M4 on Sephadex G-25 57 16. S ta ined pol yacry I am i de gel of M o t i l i n 62 17. P l o t showing r a t e of r e l e a s e o f C - t e r m i n a l amino a c i d r e s i d u e s o f m o t i l i n 63 18. High v o l t a g e e lec t rophore tog ram of m o t i l i n c l e a v e d w i t h CNBr 65 19. Amino a c i d sequences o f CNBr f ragments of m o t i l i n 67 2 0 . High v o l t a g e e lec t rophore tog ram of c h y m o t r y p t i c d i ges+ of mot i I i n 69 2 1 . Descending paper chromatogram of CHT2 • — 71 2 2 . Amino a c i d sequences of c h y m o t r y p t i c p e p t i d e s of moti I in 72 xi F i g u r e Page 23 . High v o l t a g e e lec t rophore togram of t r y p t i c d i ges t of mot i I i n 73 24. Amino a c i d sequences of t r y p t i c p e p t i d e s of m o t i l i n 76 2 5 . Amino a c i d sequences of t r y p t i c p e p t i d e s of CN2 78 26. High v o l t a g e e lec t rophore togram of t h e r m o l y s i n d i g e s t of moti I i n 79 27. Amino a c i d sequences of t h e r m o l y s i n p e p t i d e s of moti I in 81 28. P l o t s showing r a t e of r e l e a s e of C - t e r m i n a l amino a c i d r e s i d u e s o f m o t i l i n w i t h m o t i l i t y i n d i c e s 85 29 . E l u t i o n p r o f i l e of a c e t y l a t e d m o t i l i n on Sephadex G -15 — 87 30 . High v o l t a g e e lec t rophore tog ram of t r y p t i c d i g e s t s of i n t a c t , a c e t y l a t e d and s u c c i n y l a t e d m o t i l i n 88 3 1 . E l u t i o n p r o f i l e of s u c c i n y l a t e d m o t i l i n on Sephadex G-15 91 32 . E f f e c t o f m o t i l i n i n f u s i o n a t I y g / k g - h . on f u n d i c pouch motor a c t i v i t y 93 3 3 . E f f e c t of m o t i l i n i n f u s i o n a t 2 u g / k g - h . on f u n d i c pouch motor a c t i v i t y • 96 34 . E f f e c t of m o t i l i n i n f u s i o n a t I u g / k g - h . on f u n d i c pouch H + ion output 98 3 5 . E f f e c t o f m o t i l i n i n f u s i o n a t 2 u g / k g - h . on f u n d i c pouch H + ion ou tput 100 36. E f f e c t o f m o t i l i n i n f u s i o n a t I y g / k g - h . on f u n d i c pouch peps in ou tput 102 37. E f f e c t of m o t i l i n i n f u s i o n a t 2 y g / k g - h . on f u n d i c pouch peps in ou tput 104 3 8 . E f f e c t of m o t i l i n i n f u s i o n a t I y g / k g - h . on a n t r a l pouch motor a c t i v i t y 106 39 . E f f e c t of m o t i l i n i n f u s i o n a t 2 y g / k g - h . on a n t r a l pouch - motor a c t i v i t y 108 4 0 . E f f e c t of i n t e r r u p t e d i n f u s i o n of m o t i l i n on f u n d i c pouch motor a c t i v i t y III 4 1 . E f f e c t of i n t e r r u p t e d i n f u s i o n o f m o t i l i n on f u n d i c pouch K4" ion o u t p u t I 13 xii F i g u r e Page 4 2 . E f f e c t of i n t e r r u p t e d i n f u s i o n of m o t i l i n on f u n d i c pouch peps in ou tput 116 4 3 . E f f e c t of i n t e r r u p t e d i n f u s i o n of m o t i l i n on a n t r a l pouch motor a c t i v i t y 118 4 4 . E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s y n t h e t i c human g a s t r i n on f u n d i c pouch motor a c t i v i t y 121 4 5 . E f f e c t o f m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s y n t h e t i c human g a s t r i n on f u n d i c pouch H + ion output 123 4 6 . E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s y n t h e t i c human g a s t r i n on f u n d i c pouch peps in output 125 4 7 . E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s y n t h e t i c human g a s t r i n on a n t r a l pouch motor a c t i v i t y 127 4 8 . E f f e c t o f m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s e c r e t i n on f u n d i c pouch motor a c t i v i t y 130 4 9 . E f f e c t o f m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n o f s e c r e t i n on f u n d i c pouch H + ion ou tput 132 50 . E f f e c t o f m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s e c r e t i n on f u n d i c pouch peps in output 134 5 1 . E f f e c t of m o t i l i n i n f u s i o n a g a i n s t i n f u s i o n of s e c r e t i n on a n t r a l pouch motor a c t i v i t y 136 52. E f f e c t of a t r o p i n e on f u n d i c pouch motor a c t i v i t y responses t o i n j e c t i o n s o f m o t i l i n 139 53 . E f f e c t of anso lysen on f u n d i c pouch motor a c t i v i t y responses to i n j e c t i o n s o f m o t i l i n 140 54. E f f e c t o f a t r o p i n e on f u n d i c pouch H + ion ou tput responses t o i n j e c t i o n s of m o t i l i n 143 5 5 . E f f e c t of a n s o l y s e n on f u n d i c pouch H + i o n output responses t o i n j e c t i o n s o f m o t i l i n 144 56. E f f e c t of a t r o p i n e on f u n d i c pouch peps in output responses to i n j e c t i o n s of m o t i l i n 146 57. E f f e c t of a n s o l y s e n on f u n d i c pouch peps in ou tput responses t o i n j e c t i o n s of m o t i l i n 147 x i i i F i g u r e Page 58. E f f e c t of s e c r e t i n and m o t i l i n on p a n c r e a t i c volume output of c a t 149 59. A l ignment o f t r y p t i c p e p t i d e s of m o t i l i n 158 60 . P e p t i d e sequences of m o t i l i n arranged t o d i s p l a y o v e r l a p p i n g sequences 160 6 1 . Complete amino a c i d sequence of m o t i l i n 161 x i v ACKNOWLEDGEMENTS I wish to thank my s u p e r v i s o r , Dr . J . C . Brown, f o r the a d v i c e , a s s i s t a n c e and i n s t r u c t i o n which he a f f o r d e d me d u r i n g the course of these s t u d i e s . I am g r a t e f u l to Dr . C O . Parkes f o r h i s i n t e r e s t and encouragement and to the o t h e r members of the F a c u l t y of the Department of P h y s i o l o g y f o r t h e i r i n s t r u c t i o n d u r i n g my graduate s t u d i e s . S p e c i a l thanks a r e due t o M iss J i l l Dryburgh f o r her a s s i s t a n c e and encouragement and to Mr . A . M . McL in tock f o r h i s e x p e r t t e c h n i c a l a s s i s t a n e e . I am indebted to Mr. K. Henze f o r h i s a s s i s t a n c e in p r e p a r i n g the f i g u r e s in t h i s t h e s i s and f o r o t h e r c o n t r i b u t i o n s too numerous to ment ion . I am a l s o g r a t e f u l t o Mr . R. Ass inna f o r h i s a s s i s t a n c e w i t h photography. I would l i k e t o thank M r s . Jean Marchant f o r u n d e r t a k i n g the arduous t a s k of t y p i n g the f i n a l d r a f t of t h i s t h e s i s and t o thank my w i f e f o r her encouragement, understanding and p a t i e n c e d u r i n g t h e performance of these i n v e s t i g a t i o n s . F i n a n c i a l a s s i s t a n c e , i n the form of a Medica l Research C o u n c i l S t u d e n t s h i p , i s g r a t e f u l l y acknowledged. xv FOR MEGAN, WHO CAME WITH, STEVEN, WHO ARRIVED, AND THE MONKEY, WHO WAS OFF. - I -INTRODUCTION One of the major f u n c t i o n s of the stomach i s t h a t of moderat ing the f low of m a t e r i a l through the g a s t r o i n t e s t i n a l t r a c t . The l a r g e volumes which the stomach r e c e i v e s over r e l a t i v e l y s h o r t p e r i o d s of t ime a r e d e l i v e r e d t o the duodenum over very much longer p e r i o d s of t i m e . T h i s f u n c t i o n i s e s s e n t i a l in o r d e r to match the r a t e of d e l i v e r y of n u t r i e n t s t o the i n t e s t i n e t o the a b s o r p t i v e c a p a c i t y of the i n t e s t i n a l mucosa. I t can t h e r e f o r e be s a i d t h a t the stomach i s an organ of s t o r a g e . Food i s n o t , however, s t o r e d p a s s i v e l y in the stomach, but i s kneaded and mixed w i t h g a s t r i c j u i c e by c o n t r a c t i o n s of the g a s t r i c m u s c u l a t u r e . A l though such c o n t r a c t i o n s have been known f o r c e n t u r i e s , Cannon (1898) repor ted the f i r s t d e s c r i p t i v e r e c o r d , made by H a l l e r in 1764, of a l t e r n a t e c o n t r a c t i o n and r e -l a x a t i o n of the stomachs of v a r i o u s a n i m a l s . Beaumont (1833) p ioneered the s c i e n t i f i c study of g a s t r i c motor a c t i v i t y when he noted the movements of m a t e r i a l p laced in the stomach of a s u b j e c t w i th an a c c i d e n t a l l y c r e a t e d g a s t r i c f i s t u l a . Cannon (1898) d e s c r i b e d the e f f e c t s of g a s t r i c motor a c t i -v i t y on rad io -opaque c o n t e n t s u s i n g a r o e n t g e n o l o g i c a l t e c h n i q u e and repor ted the p e r i s t a l t i c c o n t r a c t i o n s which moved m a t e r i a l toward and through the p y l o r u s as we l l as back i n t o t h e body of the stomach. K l e i n (1926) observed the stomachs of a n a e s t h e t i z e d dogs and repor ted g r e a t v a r i a t i o n in the types of c o n t r a c t i o n s seen , some waves pass ing p e r i s t a 1 1 i c a I Iy t o the p y l o r u s w h i l e o t h e r s ended in a v i g o r o u s segmental c o n t r a c t i o n . The v a r i o u s forms of g a s t r i c motor a c t i v i t y were g i v e n a c l a s s i f i c a -t i o n by Hightower and Code (1950) . In t h e i r sys tem, t h r e e t ypes of c o n t r a c t i o n s can be recorded manometricaI Iy from the stomach. Types I and II a r e c o n s i d e r e d t o be p e r i s t a l t i c c o n t r a c t i o n s w h i l e t ype III i s d e s c r i b e d as a tone c o n t r a c -t i o n . These c l a s s i f i c a t i o n s do n o t , however, r e p r e s e n t an exac t f u n c t i o n a l - 2 -d e s c r i p t i o n . P r e c i s e c o r r e l a t i o n be tween m i x i n g m o v e m e n t s , p r o p u l s i v e movements and t h e t h r e e t y p e s o f c o n t r a c t i o n has n o t , t h u s f a r , been made . Thomas ( 1 9 5 7 ) c o n c l u d e d t h a t g a s t r i c e m p t y i n g i s r e l a t e d t o p e r i s t a l s i s w h i l e S m i t h , Code and S c h l e g e l ( 1 9 5 7 ) , u s i n g s i m u l t a n e o u s k y m o g r a p h i c and c i n e r a d i o g r a p h i c t e c h n i q u e s , s t a t e d t h a t p e r i s t a l t i c c o n t r a c t i o n s c o n t r i -b u t e t o b o t h m i x i n g and p r o p u l s i o n . More r e c e n t l y , W e i s b o d t e t a l ( 1 9 6 9 ) have p r o p o s e d a r e l a t i o n s h i p between c o n t r a c t i o n s o f t h e g a s t r i c m u s c u l a t u r e and g a s t r i c e m p t y i n g b u t t h e e x a c t c o r r e l a t i o n i s s t i l l l a c k i n g . I t i s e v i d e n t , h o w e v e r , t h a t w h a t e v e r t h e p r e c i s e n a t u r e o f g a s t r i c d y n a m i c s m i g h t b e , t h e c o n t r a c t i o n s o f t h e s t o m a c h o c c u r u n d e r t h e d i r e c t i o n o f a w e l l r e g u l a t e d c o n t r o l s y s t e m . T h i s s y s t e m s e r v e s t o s u b j e c t g a s t r i c c o n t e n t s t o t h e c o r r e c t d e g r e e o f m i x i n g a c c o r d i n g t o t h e i r n a t u r e and t o p r o p o r t i o n t h e i r r a t e o f d e l i v e r y t o t h e duodenum i n s u c h a way a s t o a l l o w a d e q u a t e t i m e and o p t i m u m c o n d i t i o n s f o r d i g e s t i o n and a b s o r p t i o n . If t h i s c o n t r o l o v e r g a s t r i c m o t o r a c t i v i t y were n o t e x e r t e d , c o n d i t i o n s s u c h a s a n o s m o t i c p u r g e c o u l d r e s u l t . M e c h a n i s m s w h i c h r e g u l a t e t h e m o t o r a c t i v i t y o f t h e s t o m a c h have been shown t o i n v o l v e n e u r a l and humora l p a t h w a y s a s w e l l a s i n v o l v i n g t h e i n t r i n s i c p l e x u s w i t h i n t h e g a s t r i c smooth m u s c l e . They have been e x t e n s i v e l y r e v i e w e d by Thomas and B a l d w i n ( 1 9 6 8 ) . The n e u r a l r e g u l a t i o n o f g a s t r i c m o t o r a c t i v i t y e m b r a c e s b o t h i n t r i n s i c and e x t r i n s i c r e f l e x p a t h w a y s . The i n t r i n s i c r e f l e x e s a r e m e d i a t e d by two p l e x u s e s w h i c h a r e l o c a t e d be tween t h e m u s c l e l a y e r s o f t h e s t o m a c h and w h i c h c o n s i s t o f an e x t e n s i v e n e t w o r k o f n o n - m y e l i n a t e d n e r v e f i b r e s . The m o r p h o l o g y o f t h e s e p l e x u s e s has been r e v i e w e d by B u r n s t o c k ( 1 9 7 0 ) . The e l e c t r i c a l a c t i v i t y i n t h e p l e x u s e s s e r v e s t o c o n t r o l t h e c o n t r a c t i o n s of g a s t r i c smooth m u s c l e s i n c e i t i s o n l y t h e s e f i b r e s w h i c h d i r e c t l y i n n e r v a t e - 3 -t h e m u s c l e . P l e x u s a c t i v i t y i s , l i k e any o t h e r r e f l e x a c t i v i t y , s u b j e c t t o i n h i b i t i o n and f a c i l i t a t i o n , so t h a t a p r e c i s e d e s c r i p t i o n o f c o n t r o l a t t h i s l e v e l c a n n o t be p r e s e n t e d . I t i s known, h o w e v e r , t h a t t h e v i s c e r a l e f f e r e n t f i b r e s whose a c t i v i t y c a n m o d i f y t h e m o t o r a c t i v i t y o f t h e s t o m a c h , t e r m i n a t e i n t h e p l e x u s e s . Thus t h e p l e x u s e s a c t a s a f i n a l common pathway f o r t h e n e u r a l c o n t r o l o f g a s t r i c m o t o r a c t i v i t y . I n t r i n s i c r e f l e x e s a r e c a p a b l e o f e x e r t i n g a c o o r d i n a t e d c o n t r o l o v e r g a s t r i c m o t o r a c t i v i t y a s e v i d e n c e d by t h e r e t u r n , w i t h i n a few w e e k s , o f a p p a r e n t l y normal g a s t r i c p e r i s t a l s i s f o l l o w i n g vagotomy (Thomas and B a l d w i n ( 1 9 6 8 ) ) . I t i s e v i d e n t t h a t a h i g h d e g r e e o f i n t e g r a t i o n o c c u r s w i t h i n t h e p l e x u s e s so t h a t t h e r e g u l a t i o n o f g a s t r i c m o t o r a c t i v i t y i n v o l v e s b o t h c e n t r a l i n t e g r a t i o n and i n t e g r a t i o n a t t h e l e v e l o f t h e e f f e c t o r o r g a n . The d e t a i l e d f u n c t i o n i n g o f t h e two p l e x u s e s i s n o t w e l l u n d e r s t o o d . K o t t e g o d a ( 1 9 6 9 ) p r e s e n t e d a model i n v o l v i n g r e c i p r o c a l a c t i v a t i o n o f t h e two m u s c l e c o a t s , b u t t h e p r e c i s e m e c h a n i s m o f t h i s c o m p l e x s y s t e m i s n o t known. I t i s c l e a r , h o w e v e r , t h a t m o d i f i c a t i o n o f t h e a c t i v i t y o f t h e p l e x u s e s , and t h e r e b y o f t h e e x t r i n s i c a f f e r e n t f i b r e s , must o c c u r i f t h e c o n t r o l o f g a s t r i c m o t o r a c t i v i t y i s t o be r e s p o n s i v e t o t h e s t i m u l i p r e s e n t e d by t h e p h y s i c a l and c h e m i c a l c h a r a c t e r i s t i c s o f t h e g a s t r i c c o n t e n t s . T h a t t h i s o c c u r s i s e v i d e n c e d by t h e w e a l t h o f o b s e r v a t i o n s c o r r e l a t i n g v a r i o u s s t i m u l i w i t h c h a n g e s i n g a s t r i c m o t o r a c t i v i t y o r e m p t y i n g t i m e . T h e s e o b s e r v a t i o n s s u g g e s t e d t h e p r e s e n c e o f r e c e p t o r s i n t h e g a s t r i c and i n t e s t i n a l mucosa w h i c h w o u l d m e d i a t e t h e s p e c i f i c s t i m u l i p r e s e n t e d by t h e g a s t r i c c o n t e n t s . C a r n o t and C h a s s e v a n t ( 1 9 0 5 ) o b s e r v e d t h a t h y p e r t o n i c o r h y p o t o n i c s o l u t i o n s i n c o n t a c t w i t h i n t e s t i n a l mucosa d e l a y e d g a s t r i c e m p t y i n g . The e x i s t e n c e o f o s m o r e c e p t o r s was s u g g e s t e d and t h e work o f Hunt and Knox ( 1 9 6 8 ) has l e d t o t h e i r r e c o g n i t i o n a s e n t i t i e s . R e c e p t o r s s e n s i t i v e t o h y d r o g e n i o n s have a l s o been r e c o g n i s e d . - 4 -S e r d i u k o v ( 1 8 9 8 ) d e m o n s t r a t e d t h a t a c i d i n t h e i n t e s t i n e d e l a y s g a s t r i c e m p t y i n g and S c h a p i r o and Woodward ( 1 9 5 9 ) showed t h a t pH s e n s i t i v e r e c e p t o r s a r e l o c a t e d i n t h e duodenum and j e j u n u m b u t n o t i n t h e i l e u m . Iggo ( 1 9 5 7 ) r e c o r d e d f r o m t h e v a g u s n e r v e o f c a t s and n o t e d t h a t t h e e l e c t r i c a l a c t i v i t y i n c r e a s e d when s o l u t i o n s h a v i n g a pH b e l o w 3 . 0 o r a b o v e 8 . 0 were a p p l i e d t o t h e g a s t r i c m u c o s a . Thus t h e s e r e c e p t o r s a p p e a r t o have w e l l d e f i n e d t h r e s -h o l d s . O t h e r c h e m o r e c e p t o r s have been i m p l i c a t e d i n n e u r a l c o n t r o l m e c h a n i s m s . Thomas and C r i d e r ( 1 9 3 9 ) have s u g g e s t e d t h a t t h e p r o d u c t s o f p r o t e i n d i g e s t i o n i n h i b i t g a s t r i c m o t o r a c t i v i t y v i a a n e u r a l m e c h a n i s m w h i l e t h e p r o d u c t s o f f a t d i g e s t i o n have been s i m i l a r l y i m p l i c a t e d , ( Q u i g l e y e t a l ( 1 9 3 4 ) ) . M e c h a n o -r e c e p t o r s a r e c e r t a i n l y p r e s e n t i n t h e s t o m a c h and i n t e s t i n e ( H u k u h a r a e t a l ( 1 9 5 9 ) , P a i n t a l ( 1 9 5 4 ) ) b u t t h e i r r e l a t i o n t o t h e c o n t r o l o f g a s t r i c m o t o r a c t i v i t y i s n o t c l e a r . H e r r i n e t a I ( 1 9 3 3 ) d e m o n s t r a t e d d e l a y e d g a s t r i c e m p t y i n g i n r e s p o n s e t o t h e d i s t e n t i o n o f an i n t e s t i n a l segment and Iggo ( 1 9 5 7 a ) has r e c o r d e d a f f e r e n t i m p u l s e s i n t h e v a g u s i n r e s p o n s e t o s t r e t c h i n g t h e g a s t r i c m u s c u l a t u r e . O t h e r r e c e p t o r s w h i c h may i n f l u e n c e g a s t r i c c o n t r a c -t i o n s i n c l u d e t h o s e s e n s i t i v e t o p a i n ( P i c k e r i n g ( 1 9 5 1 ) ) and t e m p e r a t u r e ( S l e e t h and Van L i e r e ( 1 9 3 7 ) ) . A f f e r e n t i m p u l s e s f r o m t h e s e v a r i o u s r e c e p t o r s a r e c a r r i e d by v a g a l a f f e r e n t n e r v e s and by a f f e r e n t n e r v e s w h i c h accompany s y m p a t h e t i c e f f e r e n t s a s do o t h e r v i s c e r a l a f f e r e n t s . T h e s e n e r v e s c o n d u c t i m p u l s e s t o t h e c e n t r a l n e r v o u s s y s t e m where i n t e g r a t i o n and t h e i n i t i a t i o n o f e f f e r e n t c o n t r o l a c t i v i t y o c c u r s . The c e n t r a l a r e a s w h i c h have been i m p l i c a t e d i n g a s t r i c mote-p c o n t r o l s y s t e m s a r e w i d e s p r e a d and i n c l u d e e v e r y l e v e l o f t h e c e n t r a l n e r v o u s s y s t e m . Thomas and B a l d w i n ( 1 9 6 8 ) have p r e s e n t e d a model s h o w i n g p o s s i b l e l e v e l s o f i n t e g r a t i o n a l o n g w i t h f e e d b a c k c o n t r o l . E f f e r e n t p a t h w a y s o f t h e n e u r a l c o n t r o l o f g a s t r i c m o t o r a c t i v i t y i n c l u d e v a g a l f i b r e s and t h e s p l a n c h n i c n e r v e s . M c S w i n e y ( 1 9 3 1 ) r e p o r t e d t h e r e s u l t s o f c l a s s i c a l s e c t i o n and s t i m u l a t i o n e x p e r i m e n t s o n t h e v a g u s and M a r t i n s o n e t a l ( 1 9 6 3 , 1 9 6 5 , 1965a) have added q u a n t i t a t i v e r e f i n e m e n t s t o t h e e a r l i e r w o r k . They d e m o n s t r a t e d t h e p r e s e n c e o f b o t h e x c i t a t o r y and i n h i b i t o r y f i b r e s i n t h e v a g u s . Semba and H i r a o k a ( 1 9 5 7 ) s t u d i e d s y m p a t h e t i c n e r v e s t i m u l a t i o n and r e p o r t e d t h a t s t r o n g s t i m u l a t i o n o f t h e s p l a n c h n i c n e r v e s i n d o g s r e s u l t e d i n a n i n h i b i t i o n o f g a s t r i c m o t o r a c t i v i t y w h i l e w e a k e r s t i m u l a t i o n l e d t o c o n t r a c t i o n s . The t e r m i n a t i o n o f t h e s e e f f e r e n t f i b r e s i n t h e m y e n t e r i c p l e x u s c o m p l e t e s t h e e x t r i n s i c r e f l e x p a t h w a y , t h e e l e c t r i c a l a c t i v i t y o f t h e s e e f f e r e n t s m o d i f y i n g t h e a c t i v i t y o f t h e p l e x u s i n some manner a s y e t n o t c l e a r l y d e f i n e d . The r e g u l a t i o n o f g a s t r i c m o t o r a c t i v i t y by humoral m e c h a n i s m s i s somewhat l e s s e v i d e n t t h a n n e u r a l r e g u l a t i o n . T h i s i s due t o t h e d i f f i c u l t y i n s e p a r a t i n g t h e two i n f l u e n c e s i n t h e i n t a c t a n i m a l . N e v e r t h e l e s s , humora l r e g u l a t i n g s y s t e m s have been r e c o g n i s e d , t h e most o u t s t a n d i n g e x a m p l e b e i n g t h e e f f e c t o f f a t i n t h e duodenum; t h e e n t e r o g a s t r o n e humoral s y s t e m . The e v i d e n c e f o r t h e e x i s t e n c e o f t h e i n h i b i t o r y hormone e n t e r o g a s t r o n e has been r e v i e w e d by G r o s s m a n ( 1 9 5 0 ) , G r e g o r y ( 1 9 6 7 ) and Thomas and B a l d w i n ( 1 9 6 8 ) . F a t i n t h e duodenum has been shown t o d e l a y g a s t r i c e m p t y i n g (Cannon ( 1 9 2 0 ) ) w h i l e f a t c o n f i n e d t o t h e s t o m a c h has no e f f e c t o n g a s t r i c m o t o r a c t i v i t y ( Q u i g l e y e t a l ( 1 9 3 4 ) ) . A l t h o u g h s e v e r a n c e o f t h e v a g u s g r e a t l y d i m i n i s h e s t h e i n h i b i t i o n o f m o t o r a c t i v i t y o f t h e s t o m a c h ( Q u i g l e y and M e s c h a n ( 1 9 3 8 ) ) , and S i r e u s ( 1 9 5 8 ) r e p o r t e d t h a t t r e a t m e n t o f t h e i n t e s t i n a l mucosa w i t h p r o c a i n e wou ld a b o l i s h s u c h i n h i b i t i o n , t h e d e m o n s t r a t i o n by F a r r e l and Ivy ( 1 9 2 6 ) o f t h e i n h i b i t i o n by f a t i n t h e I n t e s t i n e o f an a u t o t r a n s p l a n t e d pouch f i r m l y e s t a b l i s h e d t h e e x i s t e n c e o f a humora l m e c h a n i s m . K o s a k a and L i m ( 1 9 3 0 ) named t h e a g e n t r e s p o n s i b l e e n t e r o g a s t r o n e . T h i s hormone c l e a r l y has a p r o f o u n d e f f e c t o n g a s t r i c m o t o r a c t i v i t y and m u s t be - 6 -c o n s i d e r e d o n e o f t h e p r i m a r y m e c h a n i s m s r e g u l a t i n g g a s t r i c c o n t r a c t i o n s . A l t h o u g h i s o l a t i o n o f a hormone i d e n t i f i a b l e a s e n t e r o g a s t r o n e has n o t y e t o c c u r r e d , a g a s t r i c i n h i b i t o r y p o l y p e p t i d e has been p u r i f i e d and i t s a m i n o a c i d s e q u e n c e e l u c i d a t e d (Brown and D r y b u r g h ( 1 9 7 1 a ) ) . T h i s m a t e r i a l i s an e x c e l l e n t c a n d i d a t e f o r t h e humora l a g e n t r e l e a s e d by f a t i n t h e duodenum. Of t h e o t h e r g a s t r o i n t e s t i n a l h o r m o n e s , o n l y g a s t r i n has been i m p l i c a t e d a s h a v i n g any s i g n i f i c a n t c o n t r o l o v e r g a s t r i c m o t o r a c t i v i t y . G r e g o r y and T r a c e y ( 1 9 6 4 ) showed t h a t g a s t r i n c a u s e d r a p i d and p o w e r f u l c o n t r a c t i o n s o f a d e n e r v a t e d pouch i n t h e dog and t h e s e f i n d i n g s were c o n f i r m e d by I s e n b e r g and G r o s s m a n ( 1 9 6 9 ) . M i s i e w i c z e t a l ( 1 9 6 7 ) d e m o n s t r a t e d s i m i l a r m o t o r s t i m u l a t i n g p r o p e r t i e s i n man u s i n g t h e p e n t a p e p t i d e a n a l o g u e o f g a s t r i n . B e n n e t t ( 1 9 6 5 ) and M i k o s and Vane ( 1 9 6 7 ) have shown t h a t g a s t r i n s t i m u l a t e d c o n t r a c t i o n i n i s o l a t e d smooth m u s c l e p r e p a r a t i o n s . Cameron e t a l ( 1 9 7 0 ) have c o n f i r m e d t h i s f i n d i n g u s i n g human g a s t r i c smooth m u s c l e p r e p a r a t i o n s . I t i s c l e a r t h a t g a s t r i n c o n t r i b u t e s , t o some e x t e n t , t o t h e o v e r a l l c o n t r o l o f g a s t r i c m o t o r a c t i v i t y . The i n f l u e n c e o f t h e r e m a i n i n g known g a s t r o i n t e s t i n a l hormones o n g a s t r i c m o t o r a c t i v i t y i s l e s s c l e a r l y d e f i n e d . C h o i e c y s t o k i n i n -P a n c r e o z y m i n ( C C K - P Z ) was r e p o r t e d t o i n h i b i t g a s t r i c m o t o r a c t i v i t y i n dogs ( J o h n s o n and Magee ( 1 9 6 5 ) ) and i n man ( J o h n s o n , Brown and Magee ( 1 9 6 6 ) ) and t o s l o w g a s t r i c e m p t y i n g i n man (Chey e t a l ( 1 9 6 8 ) ) . In c o n t r a s t , Nakayama and Fukuda ( 1 9 6 6 ) h a v e r e p o r t e d s t i m u l a t i o n o f g a s t r i c m o t o r a c t i v i t y by t h i s ho rmone . Brown and P a r k e s ( 1 9 6 7 ) r e p o r t e d t h e s e p a r a t i o n o f a g a s t r i c m o t o r -s t i m u l a t i n g f r a c t i o n f r o m c r u d e C C K - P Z and showed t h a t t h i s a c t i v i t y was d i s t i n c t f r o m t h e C C K - P Z a c t i v i t y . They a l s o s u g g e s t e d t h a t t h e i n h i b i t o r y a c t i o n s o f o t h e r somewhat p u r e r p r e p a r a t i o n s u s e d were due t o a n o t h e r s e p a r a t e a c t i v i t y . T h i s l a t t e r s u p p o s i t i o n has g a i n e d c r e d e n c e i n t h e l i g h t o f t h e r e p o r t by Brown and P e d e r s o n ( 1 9 7 0 ) t h a t d u r i n g t h e p u r i f i c a t i o n o f C C K - P Z an i n h i b i t o r s u b s t a n c e was r e m o v e d . T h i s s u b s t a n c e was f u r t h e r p u r i f i e d ( B r o w n , M u t t and P e d e r s o n ( 1 9 7 0 ) ) and was shown t o p o s s e s s e n t e r o -g a s t r o n e a c t i v i t y . A l t h o u g h s t i m u l a t i o n o f human g a s t r i c smooth m u s c l e i n v i t r o by C C K - P Z has been r e p o r t e d (Cameron e t a I ( 1 9 7 0 ) ) , t h e p r e c i s e e f f e c t o f t h i s hormone must a w a i t i t s u s e i n a p u r e f o r m . The e l u c i d a t i o n o f i t s a m i n o a c i d s e q u e n c e ( M u t t and J o r p e s ( 1 9 7 2 ) ) s u g g e s t s t h a t s y n t h e t i c p r o d u c t w i l l be f o r t h c o m i n g . S e c r e t i n was i n i t i a l l y r e p o r t e d t o have l i t t l e e f f e c t on g a s t r i c m o t o r a c t i v i t y ( J o h n s o n and Magee ( 1 9 6 5 ) , J o h n s o n e t a l ( 1 9 6 6 ) ) . I t was s u b s e q u e n t l y f o u n d t o c a u s e i n h i b i t i o n o f d i s t e n t i o n - i n d u c e d m o t o r a c t i v i t y i n a n t r a l p o u c h e s o f d o g s (Chey e t a I ( 1 9 6 9 ) ) . Vagne e t a I ( 1 9 6 8 ) , c o m p a r i n g t h e p h y s i o l o g i c a l a c t i o n s o f n a t u r a l and s y n t h e t i c s e c r e t i n , r e p o r t e d i n h i b i t i o n o f b o t h a n t r a l and f u n d i c m o t o r a c t i v i t y i n d o g s . An i n h i b i t i o n o f g a s t r i c m o t o r a c t i v i t y i n man, a s m e a s u r e d by a d e l a y i n g a s t r i c e m p t y i n g , was n o t e d by D i n o s o e t a l ( 1 9 6 6 ) and Chey e t a l ( 1 9 7 0 ) f o l l o w i n g t h e i n t r a v e n o u s a d m i n i s t r a t i o n o f s e c r e t i n . I n h i b i t o r y e f f e c t s o f s e c r e t i n o n human g a s t r i c smooth m u s c l e i n v i t r o w e r e r e p o r t e d by Cameron e t a I ( 1 9 7 0 ) . T h e s e v a r i o u s hormonal e f f e c t s o n g a s t r i c m o t o r a c t i v i t y have n o t , a s y e t , been c o r r e l a t e d i n t o a c o o r d i n a t e d r e g u l a t o r y m e c h a n i s m . Some c i r c u m s t a n t i a l e v i d e n c e , t h e w e l l known e f f e c t o f a c i d i n t h e duodenum i n r e l e a s i n g s e c r e t i n f o r e x a m p l e , s u g g e s t s t h a t some o f t h e s e hormones m i g h t be p r e s e n t a t t h e most o p p o r t u n e s t a g e i n g a s t r i c d i g e s t i o n t o e x e r t some c o o r d i n a t e d c o n t r o l . The e x t r e m e d i f f i c u l t y , h o w e v e r , o f s e p a r a t i n g n e u r a l f r o m humora l e f f e c t s has t h u s f a r c o m p l i c a t e d s u c h a n a p p r o a c h . N e v e r t h e l e s s , - 8 -some p o s s i b i l i t i e s have been i n v e s t i g a t e d and t h e e x i s t e n c e o f a d u a l h u m o r a l , pH d e p e n d e n t , d u o d e n a l c o n t r o l o f g a s t r i c m o t o r a c t i v i t y has been s u g g e s t e d (Brown and P a r k e s ( 1 9 6 7 ) ) . The i n h i b i t i o n o f g a s t r i c m o t o r a c t i v i t y by a c i d i n t h e duodenum has been w e l l documented ( B o l d y r e f f ( 1 9 0 4 ) , B r u n e m e i e r and C a r l s o n ( 1 9 1 5 ) ) and c a n , t o some e x t e n t , be e x p l a i n e d i n t h e l i g h t o f r e c e n t work w i t h p u r e hormone p r e p a r a t i o n s , f o r e x a m p l e , s e c r e t i n . Shay and G e r s h o n - C o h e n ( 1 9 3 4 , 1935) n o t e d t h a t i n s t i l l a t i o n o f a \% s o d i u m b i c a r b o n a t e s o l u t i o n i n t o t h e duodenum o f human s u b j e c t s r e s u l t e d i n r a p i d g a s t r i c e m p t y i n g o f a b a r i u m s u l p h a t e s u s p e n s i o n . Thomas e t a I ( 1 9 3 4 ) showed t h a t d i v e r s i o n o f g a s t r i c c o n t e n t s f r o m t h e duodenum r e s u l t s i n a n i n c r e a s e i n g a s t r i c m o t o r a c t i v i t y and a c o n c o m m i t a n t d e c r e a s e i n g a s t r i c e m p t y i n g t i m e . T h e s e o b s e r v a t i o n s c o u l d be i n t e r p r e t e d a s e v i d e n c e f o r t h e n e u r a l c o n t r o l , v i a t h e p r e v i o u s l y m e n t i o n e d d u o d e n a l pH r e c e p t o r s , o f g a s t r i c m o t o r a c t i v i t y . The e x i s t e n c e o f h u m o r a l l y m e d i a t e d s t i m u l a t i o n o f g a s t r i c m o t o r a c t i v i t y i n r e s p o n s e t o an e l e v a t e d d u o d e n a l pH was d e m o n s t r a t e d by Brown e t a I ( 1 9 6 6 ) . I t was shown t h a t t h e p r e s e n c e o f a n a l k a l i n e b u f f e r i n t h e duodenum r e s u l t e d i n a n i n c r e a s e i n t h e m o t o r a c t i v i t y o f a t r a n s p l a n t e d f u n d i c p o u c h . The n a t u r e o f t h e p r e p a r a t i o n l e f t no d o u b t t h a t a humora l m e c h a n i s m was i n v o l v e d b u t t h e d i s t i n c t i o n be tween i n h i b i t i o n o f t h e r e l e a s e o f an i n h i b i t o r y a g e n t o r t h e r e l e a s e o f a s t i m u l a t o r y a g e n t c o u l d n o t be made . In o r d e r t o t e s t t h e l a t t e r p o s s i b i l i t y , s e v e r a l c o m m e r c i a l d u o d e n a l e x t r a c t s were a s s a y e d f o r g a s t r i c m o t o r - s t i m u l a t i n g p r o p e r t i e s (Brown ( 1 9 6 7 ) ) . The C C K - P Z p r e p a r a t i o n o f B o o t s P u r e Drug C o . ( B o o t s P a n c r e o z y m i n ) was shown t o p o s s e s s a p o w e r f u l g a s t r i c m o t o r - s t i m u l a t i n g a c t i v i t y and i t was s u g g e s t e d t h a t t h i s a c t i v i t y was due t o a s e p a r a t e m o t o r - s t i m u l a t i n g p r o p e r t y . The s u b s e q u e n t d e m o n s t r a t i o n by Brown and P a r k e s ( 1 9 6 7 ) t h a t t h e m o t o r - s t i m u l a t i n g a c t i v i t y c o u l d be s e p a r a t e d f r o m t h e C C K - P Z a c t i v i t y s u p p o r t e d t h i s h y p o t h e s i s . The - 9 -p o s s i b i l i t y t h a t t h i s a c t i v i t y was i d e n t i c a l w i t h t h e i n t e s t i n a l m o t o r -s t i m u l a t i n g s u b s t a n c e p r o p o s e d by v o n - E u l e r and Gaddum ( 1 9 3 1 ) and r e f e r r e d t o a s s u b s t a n c e P was r e j e c t e d i n t h e l i g h t o f t h e p u r i f i c a t i o n (Chang and Leeman ( 1 9 7 0 ) and e l u c i d a t i o n o f t h e a m i n o a c i d s e q u e n c e ( C h a n g , Leeman and NIa 11 ( 1 9 7 1 ) ) o f t h i s p o l y p e p t i d e . P o s s i b l e i d e n t i t y w i t h t h e v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e ( V I P ) o f S a i d and M u t t ( 1 9 7 0 ) was e l i m i n a t e d b e c a u s e o f c h e m i c a l d i s s i m i l a r i t i e s w h i c h became e v i d e n t d u r i n g t h i s w o r k . The n e c e s s i t y f o r i s o l a t i n g any b i o l o g i c a l l y a c t i v e g a s t r o i n t e s t i n a l p o l y p e p t i d e and d e t e r m i n i n g i t s a m i n o a c i d s e q u e n c e i s e v i d e n t i n v i e w o f t h e m u l t i p l i c i t y o f a c t i o n s w h i c h have been d e s c r i b e d f o r t h e known g a s t r o -i n t e s t i n a l h o r m o n e s . O v e r l a p p i n g a c t i o n s be tween h o r m o n e s , f o r e x a m p l e t h e a c i d s t i m u l a t i n g a b i l i t y o f b o t h g a s t r i n a n d C C K - P Z , have been e x p l a i n e d o n p u r e l y s t r u c t u r a l g r o u n d s . The need f o r t h e i s o l a t i o n and c h e m i c a l c h a r a c t e r -i s a t i o n o f t h e m o t o r - s t i m u l a t i n g a c t i v i t y r e p o r t e d by Brown was t h u s e v i d e n t . The u s e o f B o o t s P a n c r e o z y m i n a s a s t a r t i n g m a t e r i a l f o r t h e i s o l a t i o n o f t h i s m o t o r s t i m u l a t i n g a c t i v i t y p r o v e d u n s a t i s f a c t o r y . Brown e t a l ( 1 9 7 1 ) r e p o r t e d p a r t i a l p u r i f i c a t i o n o f a g a s t r i c m o t o r s t i m u l a t i n g p o l y p e p t i d e f r o m a s i d e - f r a c t i o n p r o d u c e d d u r i n g t h e c o m m e r c i a l p r e p a r a t i o n o f s e c r e t i n f r o m e x t r a c t s o f hog d u o d e n a l m u c o s a . The p o l y p e p t i d e was named m o t i l i n b e c a u s e o f t h e i n i t i a l o b s e r v a t i o n t h a t i t s t i m u l a t e d f u n d i c pouch m o t o r a c t i v i t y . T h e s e i n v e s t i g a t i o n s d e a l w i t h t h e i s o l a t i o n o f m o t i l i n i n a p u r e f o r m s u i t a b l e f o r c h e m i c a l a n a l y s i s , t h e e l u c i d a t i o n o f i t s c o m p l e t e a m i n o a c i d s e q u e n c e , t h e e f f e c t s o f some m o l e c u l a r m o d i f i c a t i o n s t o t h e b i o l o g i c a l a c t i v i t y o f t h e p o l y p e p t i d e and some p r e l i m i n a r y s t u d i e s o n i t s p h y s i o l o g i c a l a c t i o n s . - 10 -METHODS COLUMN CHROMATOGRAPHY ( i ) Ge l F i l t r a t i o n P u r i f i c a t i o n s t a g e s i n v o l v i n g m o l e c u l a r s i e v i n g were p e r f o r m e d by c o l u m n c h r o m a t o g r a p h y u s i n g c r o s s l i n k e d d e x t r a n g e l s ( S e p h a d e x , P h a r m a c i a , Sweden) a s t h e s o l i d p h a s e . T h i s t e c h n i q u e , w h i c h i s known a s g e l f i l t r a t i o n , was d e s c r i b e d by P o r a t h ( 1 9 5 9 , I 9 6 0 ) and i s o n e o f t h e m o s t common ways o f s e p a r a t i n g m o l e c u l e s a c c o r d i n g t o s i z e . In a t y p i c a l p r o c e d u r e , Sephadex beads were s t i r r e d i n t o a c o n v e n i e n t vo lume o f b u f f e r s o l u t i o n and a l l o w e d t o s w e l l o v e r n i g h t . A f t e r r e m o v a l o f f i n e s e x c e s s l i q u i d was d e c a n t e d , t h e g e l s t i r r e d i n t o a s l u r r y and p o u r e d i n t o a c h r o m a t o g r a p h i c c o l u m n c o n t a i n i n g 5 - 1 0 c m . o f b u f f e r s o l u t i o n a t t h e b o t t o m o f t h e c o l u m n . The s l u r r y was a l l o w e d t o s e t t l e u n d e r g r a v i t y u n t i l t h e g e l was ' p a c k e d ' t o a p p r o x i m a t e l y h a l f t h e l e n g t h o f t h e c o l u m n and t h e t o p o f t h e c o l u m n was c o n n e c t e d t o a r e s e r v o i r o f b u f f e r s o l u t i o n w h i c h was a l l o w e d t o f l o w t h r o u g h t h e c o l u m n u n d e r g r a v i t y . T h i s f l o w o f b u f f e r s o l u t i o n c o m p l e t e d t h e p a c k i n g o f t h e c o l u m n . F u r t h e r a d d i t i o n s o f s l u r r y were c a r r i e d o u t u n t i l t h e p a c k e d c o l u m n o f g e l r e a c h e d t h e h e i g h t d e s i r e d . F i l t e r p a p e r (3MM) was c u t t o s i z e and p l a c e d o n t h e t o p o f t h e g e l i n o r d e r t o s t a b i l i z e t h e g e l - l i q u i d i n t e r f a c e . B u f f e r s o l u t i o n was a l l o w e d t o r u n t h r o u g h t h e c o l u m n f o r s e v e r a l h o u r s , u s u a l l y o v e r n i g h t , p r i o r t o u s e . B u f f e r s o l u t i o n was removed f r o m t h e t o p o f t h e c o l u m n t o t h e l e v e l o f t h e g e l . A s o l u t i o n o f t h e m i x t u r e f o be s e p a r a t e d d i s s o l v e d i n a s s m a l l a v o l u m e o f b u f f e r s o l u t i o n a s p o s s i b l e was a p p l i e d t o t h e t o p o f t h e c o l u m n and a l l o w e d t o s i n k i n t o t h e g e l . B u f f e r was t h e n r e p l a c e d on t o p o f t h e g e l and t h e t o p o f t h e c o l u m n r e c o n n e c t e d t o t h e b u f f e r r e s e r v o i r . B u f f e r was a l l o w e d t o f l o w t h r o u g h t h e c o l u m n , s u i t a b l e f r a c t i o n s o f t h e - 11 -e f f l u e n t v o l u m e b e i n g c o l l e c t e d on a f r a c t i o n c o l l e c t o r , (LKB U l t r o r a c , LKB p r o d u c t u r , Sweden) . C o l u m n s were s t o r e d a f t e r u s e i n b u f f e r s o l u t i o n s c o n t a i n i n g 0.1% o c t a n o i c a c i d . T h i s p r e v e n t e d b a c t e r i a l g r o w t h . ( i i ) Ion Exchange C h r o m a t o g r a p h y Ion e x c h a n g e r s b a s e d on Sephadex and c e l l u l o s e (Whatman, E n g l a n d ) were u s e d . A n i o n e x c h a n g e r s were s w o l l e n i n 0 . 5 M HCl and c a t i o n e x c h a n g e r s i n 0 . 5 M ammonia s o l u t i o n . A f t e r s w e l l i n g , f i n e s were r e m o v e d , t h e e x c h a n g e r s washed w i t h w a t e r , and t h e n w i t h 0 . 5 M ammonia s o l u t i o n and 0 . 5 M HCl r e s p e c t i v e l y . A f i n a l wash was t h e n g i v e n t o e a c h e x c h a n g e r s o t h a t t h e n e c e s s a r y c o u n t e r i o n was s u p p l i e d . A n i o n e x c h a n g e r s were washed w i t h ammonium b i c a r b o n a t e s o l u t i o n a t t h e i o n i c s t r e n g t h w h i c h was t o be used a s t h e s t a r t i n g c o n d i t i o n f o r t h e c h r o m a t o g r a p h y , and c a t i o n e x c h a n g e r s s i m i l a r l y were washed w i t h s o l u t i o n s o f ammonium b i c a r b o n a t e o r ammonium a c e t a t e . C o l u m n s were p o u r e d i n t h e u s u a l manner and s a m p l e s , d i s s o l v e d i n s t a r t i n g b u f f e r s o l u t i o n , were a p p l i e d . D e v e l o p m e n t o f t h e c o l u m n s was p e r f o r m e d e i t h e r by s t e p w i s e i n c r e a s e s i n i o n i c s t r e n g t h o r by g r a d i e n t s o f i n c r e a s i n g i o n i c s t r e n g t h . F i n a l e l u t i o n o f a d s o r b e d m a t e r i a l was e n s u r e d by p a s s a g e o f 0 . 2 M ammonia s o l u t i o n t h r o u g h t h e c o l u m n s and t h i s s o l u t i o n was used t o s t o r e c o l u m n s . The p r e c y c l i n g o f i o n e x c h a n g e r s d e s c r i b e d a b o v e was a l w a y s p e r f o r m e d when a new c o l u m n was p r e p a r e d . F o r s u b s e q u e n t u s e o f a l r e a d y p r e p a r e d c o l u m n s i t was o n l y n e c e s s a r y t o wash s t a r t i n g b u f f e r t h r o u g h t h e c o l u m n u n t i l t h e e f f l u e n t pH was t h e same a s t h e b u f f e r . T h i s r e p l a c e d t h e n e c e s s a r y c o u n t e r i o n s on t h e i o n e x c h a n g e r . - 12 -HIGH VOLTAGE ELECTROPHORESIS ( i ) E q u i p m e n t and R e a g e n t s H i g h v o l t a g e e l e c t r o p h o r e s i s ( L e g g e t t - B a i I e y ( 1 9 6 7 ) ) was p e r f o r m e d u s i n g t h e v e r t i c a l s t r i p a p p a r a t u s o f M i c h l ( 1 9 5 1 , 1959) a s m o d i f i e d by R y l e e t a I ( 1 9 5 5 ) . The a p p a r a t u s c o n s i s t e d o f g l a s s c h r o m a t o g r a p h i c t a n k s 2 2 . 5 i n . x 2 2 . 5 i n . deep ( C h r o m o t a n k s , S h a n d o n , E n g l a n d ) . T h e s e were f i t t e d w i t h g l a s s b u f f e r t r o u g h s a t t h e t o p , s u p p o r t e d on g l a s s r a c k s , a g l a s s r o d t o p r e v e n t t h e p a p e r t o u c h i n g t h e w a l l s and p e r s p e x l i d s s u p p o r t i n g g l a s s c o o l i n g c o i l s . P l a t i n u m e l e c t r o d e s mounted i n g l a s s h o l d e r s were p l a c e d i n t h e u p p e r t r o u g h ( c a t h o d e ) and a t t h e b o t t o m o f t h e t a n k s w h i c h were f i l l e d w i t h b u f f e r t o a d e p t h o f 4 c m . The e l e c t r o d e s were c o n n e c t e d , v i a c o n n e c t i n g b l o c k s mounted e x t e r n a l l y on t h e t a n k s , t o a C a n a d i a n R e s e a r c h I n s t i t u t e (Don M i l l s , O n t a r i o ) model E P S K - 2 0 0 D . C . power s u p p l y . The t a n k s were l o c a t e d i n a fume hood w h i c h was e q u i p p e d w i t h p e r s p e x d o o r s a l l o w i n g o b s e r -v a t i o n o f t h e p a p e r d u r i n g a r u n . The two v o l a t i l e b u f f e r s y s t e m s w h i c h were used were d e s c r i b e d by A m b l e r ( 1 9 6 3 ) and a r e a s f o l l o w s : pH I .9 B u f f e r : 2% F o r m i c a c i d (v/v ) 8% A c e t i c a c i d (v/v ) i n d e i o n i s e d w a t e r C o o l a n t : V a r s o l pH 6 . 5 B u f f e r 8 7 9 m l . D e i o n i s e d w a t e r 100 m l . R e d i s t i l l e d p y r i d i n e 3 m l . G l a c i a l a c e t i c a c i d C o o l a n t : 92% T o l u e n e , 8% r e d i s t i l l e d p y r i d i n e (by v o l u m e ) A l l r e a g e n t s were F i s h e r c e r t i f i e d g r a d e . V a r s o l was o b t a i n e d f r o m I m p e r i a l O i l C o . ( i i ) S t a n d a r d s and M a r k e r s Two s t a n d a r d m i x t u r e s o f a m i n o a c i d s were p r e p a r e d c o n t a i n i n g 2 nM/uI o f e a c h . The s t a n d a r d s were a s f o l l o w s : S t a n d a r d ' R 1 A r g i n i n e C y s t e i c a c i d Leuc i ne L y s i n e M e t h i o n i n e P h e n y l a l a n i n e P r o I i ne T y r o s i ne V a I i ne S t a n d a r d ' T ' A l a n i n e A s p a r t i c a c i d G l u t a m i c a c i d G l y c i ne H i s t i d i n e I s o l e u c i n e S e r i ne T a u r i ne T h r e o n i n e S t a n d a r d s were a p p l i e d a s a 5 y1 • s p o t o n e a c h s i d e o f t h e s a m p l e a r e a on t h e o r i g i n l i n e . The p o s i t i o n s o f t h e s t a n d a r d a m i n o a c i d s a f t e r e l e c t r o p h o r e s i s a t pH 6 . 5 and I . 9 a r e shown i n f i g . I . The s t a n d a r d s were used b o t h a s a c h e c k o f t h e d e t e c t i o n r e a g e n t s and t o c a l c u l a t e r e l a t i v e m o b i l i t i e s . C o l o u r e d m a r k e r s were u s e d on a l l r u n s s o t h a t u n i f o r m r u n n i n g c o n d i t i o n s c o u I d be a c h i e v e d r e g a r d l e s s o f v o l t a g e g r a d i e n t . The m a r k e r s used were M e t h y l G r e e n ( C . l . n o . 4 2 5 9 0 F i s h e r C e r t i f i e d B i o l o g i c a l S t a i n ) and e - D i n i t r o p h e n y l L y s i n e (Ca l B i o c h e m , L o s A n g e l e s ) . A t pH 6 . 5 r u n s were t e r m i n a t e d when t h e m e t h y l g r e e n had moved 20 c m . f r o m t h e o r i g i n . The e - D i n i t r o p h e n y l l y s i n e i s n e u t r a l a t t h i s pH and any movement o f t h i s m a r k e r away f r o m t h e o r i g i n i s due t o e l e c t r o -- 1 4 -© OLYS O ARG OHIS V/ (1 1 1 I DNP LYS O- ° O l i 1 2 • 3 OASP Q G L U o CYSO,H p H 6 .5 0 OLYS O ARG OHIS QMG 11 OGLY OALA 11 11 OVAL OLEU pMET HPRO ° PHE °TYR QSER OILE °THR °ASP OGUU o £ DNP LYS TAU — O — — 1 1 1 2 i 3 o CYSO,H © p H 1.9 F i g u r e I. S t a n d a r d s and M a r k e r s used o n h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 and pH 6 . 5 . I. - M a r k e r s . M . G . , M e t h y l G r e e n . e -DNP L Y S , e - D i n i t r o p h e n y I L y s i n e . 2 . - S t a n d a r d R. 3 - S t a n d a r d T . - 15 -e n d o s m o s i s . The p o s i t i o n o f t h e e - D i n i t r o p h e n y l l y s i n e was t h e r e f o r e t a k e n t o be t h e n e u t r a l l i n e . A t pH 1 . 9 r u n s were t e r m i n a t e d when t h e m e t h y l g r e e n had moved 30 c m . f r o m t h e o r i g i n . The n e u t r a l l i n e was i n d i c a t e d by t h e p o s i t i o n o f t a u r i n e i n s t a n d a r d ' T ' w h i c h i s n e u t r a l a t t h i s p H . ( i i i ) P r o c e d u r e f o r Q u a l i t a t i v e E l e c t r o p h o r e s i s S h e e t s o f Whatman 3MM p a p e r (57 x 46 c m . ) were c u t t o an a p p r o p r i a t e w i d t h and t h e o r i g i n l i n e marked on t h e p a p e r w i t h p e n c i l . F o r r u n s a t pH 1 . 9 t h e o r i g i n was p l a c e d 10 c m . up f r o m t h e a n o d e ( b o t t o m ) e n d . A t pH 6 . 5 t h e o r i g i n was p o s i t i o n e d 32 c m . down f r o m t h e c a t h o d e e n d . The s a m p l e , s t a n d a r d s and m a r k e r s were s p o t t e d o n t h e p a p e r u s i n g c o o l a i r f r o m a h a i r d r y e r t o d r y t h e s p o t s . The p a p e r was t h e n w e t t e d w i t h t h e r e q u i r e d b u f f e r , t a k i n g c a r e t h a t e a c h s i d e o f t h e o r i g i n l i n e was w e t t e d i n t u r n t o a v o i d movement o f t h e s p o t s by f l u i d d r a g . A f t e r p o s i t i o n i n g i n t h e t a n k and t h e p a s s a g e o f c u r r e n t f o r t h e r e q u i r e d t i m e , t h e p a p e r was hung t o d r y i n a g e n t l e a i r c u r r e n t i n a fume h o o d . When d r y t h e p a p e r c o u l d t h e n be t r e a t e d w i t h d e t e c t i o n r e a g e n t s t o l o c a l i s e p e p t i d e s . ( i v ) P r o c e d u r e f o r P r e p a r a t i v e E l e c t r o p h o r e s i s . The p r o c e d u r e f o r p r e p a r a t i v e e l e c t r o p h o r e s i s d i f f e r e d f r o m t h a t f o r q u a l i t a t i v e e l e c t r o p h o r e s i s o n l y i n t h e s a m p l e a p p l i c a t i o n . The s a m p l e was a p p l i e d a s a band a l o n g t h e o r i g i n l i n e and 1 . 0 c m . i n l e n g t h f o r e a c h 100 nM. o f a p p l i e d p e p t i d e . A f t e r e l e c t r o p h o r e s i s and d r y i n g o f t h e p a p e r a g u i d e s t r i p was c u t , up t o and i n c l u d i n g t h e edge o f t h e s a m p l e z o n e , a l o n g t h e l e n g t h o f t h e p a p e r . The g u i d e s t r i p , a f t e r d e v e l o p m e n t w i t h d e t e c t i o n r e a g e n t s , was used t o l o c a t e t h e p o s i t i o n o f t h e p e p t i d e z o n e s o n t h e r e m a i n i n g p o r t i o n o f t h e p a p e r . T h e s e p e p t i d e z o n e s w e r e c u t o u t and e l u t e d . - 16 -( v ) D e t e c t i o n R e a g e n t s The u s u a l r e a g e n t f o r t h e l o c a t i o n o f p e p t i d e s was t h e C a d m i u m -N i n h y d r i n r e a g e n t o f H e i l m a n e t a l ( 1 9 5 7 ) . T h i s r e a g e n t was s e n s i t i v e 2 t o between 1 . 0 - 5 . 0 nM/cm and y i e l d e d u s e f u l i n f o r m a t i o n a b o u t N - t e r m i n a l a m i n o a c i d r e s i d u e s . The r e a g e n t c o n s i s t e d o f a s t o c k s o l u t i o n o f 5 . 0 g . cadmium a c e t a t e ( F i s h e r C e r t i f i e d g r a d e ) i n 250 m l . o f g l a c i a l a c e t i c a c i d and 500 m l . d i s t i l l e d w a t e r . S t o c k s o l u t i o n (15 m l . ) was added t o 100 m l . o f 0 . 5 ? (w/v) N i n h y d r i n ( T r i k e t o h y d r i n d a n t i n , P i e r c e C h e m i c a l C o . ) i n a c e t o n e ( F i s h e r R e a g e n t g r a d e ) . P a p e r s were d i p p e d i n t h i s r e a g e n t and a l l o w e d t o d r y i n a c u r r e n t o f a i r i n a fume h o o d . The c o l o u r s w h i c h d e v e l o p e d depended on t h e N - t e r m i n a l amino a c i d r e s i d u e o f t h e p e p t i d e . In g e n e r a l t h e f o l l o w i n g scheme a p p l i e d : Y e l l o w .> Red ( S t a b l e one w e e k ) . C y s S O ^ H , C y s , G l y , P r o , S e r , T h r . O r a n g e H i s , S e r . S low Red ( o v e r n i g h t ) l i e , V a l . F a s t Red A l a , A r g , A s p , G l u , L e u , L y s , M e t , M e t S 0 2 , Phe T r p , T y r . On o c c a s i o n , t h e PAQ r e a g e n t o f Yamada and I t a n o ( 1 9 6 6 ) was used t o l o c a l i s e a r g i n i n e c o n t a i n i n g p e p t i d e s . The r e a g e n t was p r e p a r e d by m i x i n g e q u a l v o l u m e s o f a s o l u t i o n c o n t a i n i n g 0 . 0 2 ? (w/v) p h e n a n t h r e n e q u i n o n e ( F i s h e r ) i n a n h y d r o u s e t h a n o l and 10? (w/v) s o d i u m h y d r o x i d e s o l u t i o n i n 6 0 ? ( v/v ) e t h a n o l . P a p e r s were d i p p e d i n t h i s m i x t u r e and a l l o w e d t o a i r d r y . A r g i n i n e c o n t a i n i n g p e p t i d e s were l o c a l i s e d by t h e i r b l u e f l u o r e s c e n c e u n d e r u l t r a - v i o l e t l i g h t . The PAQ r e a g e n t c o u l d be f o l l o w e d by a s p e c i a l n i n h y d r i n r e a g e n t p r e p a r e d a s f o l l o w s : 20 m l . o f a s o l u t i o n c o n t a i n i n g 1 . 0 g . cadmium a c e t a t e i n 150 m l . o f g l a c i a l a c e t i c a c i d and 50 m l . d i s t i l l e d w a t e r were m i x e d w i t h 80 m l . o f 0 . 5 $ (w/v) n i n h y d r i n i n a c e t o n e . A f t e r d r y i n g t h e d i p p e d p a p e r s , a l l p e p t i d e s p r o d u c e d a v i o l e t - r e d c o l o u r w i t h t h i s r e a g e n t . PAPER CHROMATOGRAPHY ( i ) E q u i p m e n t D e s c e n d i n g p a p e r c h r o m a t o g r a p h y was p e r f o r m e d i n a g l a s s c h r o m a -t o g r a p h i c t a n k ( A m e r i c a n O p t i c a l C o . R i c h m o n d , C a l i f o r n i a ) 12 i n . x 12 i n . x 24 i n . d e e p . The t a n k was e q u i p p e d w i t h a s t a i n l e s s s t e e l r a c k w h i c h s u p p o r t e d t h e s o l v e n t t r o u g h and a g l a s s r o d a t t h e t o p o f t h e t a n k . The w a l l s o f t h e t a n k were l i n e d w i t h f i l t e r p a p e r w h i c h was s o a k e d i n s o l v e n t p r i o r t o a r u n . T h i s k e p t t h e a t m o s p h e r e i n t h e t a n k s a t u r a t e d . The t a n k was e q u i p p e d w i t h a g l a s s p l a t e a s a l i d ; t h i s p l a t e f i t t e d a g a i n s t g r o u n d g l a s s e d g e s o f t h e t a n k w a l l s t o make a s e a l . ( i i ) P r o c e d u r e f o r Q u a l i t a t i v e C h r o m a t o g r a p h y . S h e e t s o f Whatman 3MM p a p e r (57 x 46 c m . ) were c u t t o an a p p r o -p r i a t e w i d t h a l o n g t h e s h o r t e r a x i s o f t h e p a p e r so t h a t s o l v e n t f l o w c o u l d o c c u r ' a c r o s s t h e g r a i n ' o f t h e p a p e r . An o r i g i n s p o t was marked a t t h e c e n t e r o f t h e s t r i p , 7 c m . down f r o m t h e t o p . A f t e r s p o t t i n g t h e s a m p l e , t h e s t r i p was hung i n t h e t a n k f r o m t h e s o l v e n t t r o u g h , s o l v e n t p l a c e d i n t h e t r o u g h and c h r o m a t o g r a p h y a l l o w e d t o p r o c e e d f o r 14 h o u r s . A t t h e end o f t h i s t i m e p a p e r s were r e m o v e d , d r i e d i n a g e n t l e a i r c u r r e n t i n a fume hood and d e v e l o p e d w i t h t h e u s u a l d e t e c t i o n r e a g e n t s . ( i i i ) P r o c e d u r e f o r P r e p a r a t i v e C h r o m a t o g r a p h y P r e p a r a t i v e d e s c e n d i n g c h r o m a t o g r a p h y was p e r f o r m e d i n a s i m i l a r manner t o q u a l i t a t i v e c h r o m a t o g r a p h y e x c e p t t h a t t h e s a m p l e was a p p l i e d a s a l i n e a t t h e o r i g i n 1 . 0 c m . l o n g f o r e a c h 100 nM. o f p e p t i d e . A f t e r - 18 -c h r o m a t o g r a p h y and d r y i n g , a g u i d e s t r i p was c u t a d j a c e n t t o t h e s a m p l e z o n e a l o n g t h e l e n g t h o f t h e p a p e r and was d e v e l o p e d w i t h a d e t e c t i o n r e a g e n t . The g u i d e s t r i p was used t o l o c a t e p e p t i d e c o n t a i n i n g z o n e s on t h e r e m a i n i n g p o r t i o n o f t h e p a p e r so t h a t t h e s e c o u l d be c u t o u t and e l u t e d . ( i v ) S o l v e n t Sys tem The s o l v e n t s y s t e m used i n t h i s s t u d y was t h a t d e s c r i b e d by Whaley and Watson ( 1 9 5 4 ) and c o n s i s t e d o f B u t a n o l , A c e t i c a c i d , P y r i d i n e and Water ( 3 0 : 6 : 2 0 : 2 4 ) . A l l r e a g e n t s were F i s h e r C e r t i f i e d g r a d e and t h e p y r i d i n e was r e d i s t i l l e d b e f o r e u s e . THIN LAYER CHROMATOGRAPHY T h i n l a y e r c h r o m a t o g r a p h y was p e r f o r m e d i n an a s c e n d i n g s o l v e n t s y s t e m u s i n g Eastman p r e c o a t e d 20 x 20 c m . 6061 S i l i c a G e l p l a t e s . S a m p l e s were s p o t t e d o n t h e p l a t e s 3 . 0 c m . f r o m t h e b o t t o m and t h e p l a t e p l a c e d between two g l a s s p l a t e s w h i c h had 3 o f t h e i r s i d e s p r o u d o f t h e p l a n e o f t h e g l a s s s o t h a t a s e a l e d s a n d w i c h was f o r m e d . T h i s s a n d w i c h was p l a c e d v e r t i c a l l y , o p e n end down , i n a s o l v e n t t r o u g h c o n t a i n i n g s o l v e n t and a s c e n d i n g c h r o m a t o g r a p h y a l l o w e d t o o c c u r u n t i l t h e s o l v e n t f r o n t r e a c h e d 1 . 0 c m . f r o m t h e t o p o f t h e p l a t e . The s a n d w i c h was t h e n o p e n e d , t h e p l a t e removed a n d , a f t e r d r y i n g i n a i r , t h e u s u a l d e t e c t i o n r e a g e n t s were a p p l i e d by s p r a y i n g . The s o l v e n t s y s t e m s used f o r t h i n l a y e r c h r o m a t o g r a p h y were e i t h e r t h a t o f B r e w e r e t a l ( 1 9 6 8 ) c o n s i s t i n g o f B u t a n o l , P y r i d i n e , W a t e r , A c e t i c a c i d (I 5 :1 0 : 1 2 : 3 ) o r t h e s y s t e m o f Whaley and Watson ( 1 9 5 4 ) p r e v i o u s l y d e s c r i b e d . - 19 -QUALITATIVE POLYACRYLAMIDE GEL ELECTROPHORESIS P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s was p e r f o r m e d a s d e s c r i b e d by J o h n s ( 1 9 6 7 ) . The g e l s were p r e p a r e d i n 5 x 7 5 mm. g l a s s c o l u m n s u s i n g I . 0 m l . o f a s o l u t i o n c o n t a i n i n g 10 m l . o f s t o c k monomer s o l u t i o n , ( 4 0 ? (w/v) a c r y l a m i d e , 0 . 6 ? (w/v) N, N ' - m e t h y I e n e b i s a c r y I a m i d e ( E a s t m a n , R o c h e s t e r , New Y o r k ) i n d i s t i l l e d w a t e r ) 10 m l . o f c a t a l y s t I ( 0 . 5 ? ( v/v ) r e d i s t i l l e d N , N , N ' , N ' - t e t r a m e t h y l e n e d i a m i n e i n 4 . 6 M a c e t i c a c i d ) and 6 m l . o f c a t a l y s t 2 ( 0 . 6 ? (w/v) ammonium p e r s u l p h a t e , ( F i s h e r r e a g e n t g r a d e ) i n e a c h t u b e . The g e l s o l u t i o n was p l a c e d i n a d e s s i c a t o r and d e -g a s s e d u n d e r m o d e r a t e vacuum f o r 30 m i n u t e s b e f o r e p l a c i n g i n t h e t u b e s . Water was c a r e f u l l y l a y e r e d o n t o p o f t h e g e l s and t h e y were a l l o w e d t o p o l y m e r i s e a t room t e m p e r a t u r e , b u t u n d e r a 60 w a t t lamp f o r t h e f i r s t h o u r . G e l s were s t o r e d a minimum o f 3 d a y s b e f o r e u s e and were n e v e r used beyond 2 weeks a f t e r p r e p a r a t i o n . E l e c t r o p h o r e s i s was c a r r i e d o u t i n a Shandon model 12734 e l e c t r o p h o -r e s i s a p p a r a t u s w h i c h p r o v i d e d f o r c o o l i n g t h e g e l s d u r i n g a r u n . The g e l s were p l a c e d i n t h e a p p a r a t u s , t h e r e s e r v o i r s f i l l e d w i t h 0 . 0 1 M a c e t i c a c i d and t h e e l e c t r o d e s c o n n e c t e d so t h a t t h e a n o d e was u p p e r m o s t . E q u i l i b r a t i o n o f t h e g e l s was a l l o w e d t o t a k e p l a c e by p a s s i n g c u r r e n t a t 320 v o l t s f o r t h r e e h o u r s . The b u f f e r s were r e m o v e d , s a m p l e s ( 5 - 1 0 0 u g . ) d i s s o l v e d i n a s o l u t i o n o f I .0M s u c r o s e i n 0 . 0 0 2 M a c e t i c a c i d a p p l i e d t o t h e t o p o f t h e g e l s , t h e b u f f e r s r e p l a c e d and c u r r e n t p a s s e d a t 320 v o l t s f o r 25 m i n u t e s . Amido b l a c k ( M e r c k ) ( 2 . 0 m l . o f a 0 . 5 ? (w/v) s o l u t i o n i n I .0M a c e t i c a c i d ) was p l a c e d i n t h e b u f f e r c o n t a i n e d i n t h e l o w e r r e s e r v o i r and c u r r e n t p a s s e d f o r a f u r t h e r 15 m i n u t e s . The d y e s o l u t i o n was t h e n removed f r o m t h e l o w e r r e s e r v o i r and r e p l a c e d w i t h f r e s h 0 . 0 I M a c e t i c a c i d . C u r r e n t was p a s s e d a t 320 v o l t s u n t i l s u c h t i m e a s t h e band o f d y e had moved up t h e g e l and d i s p e r s e d i n t h e u p p e r r e s e r v o i r l e a v i n g s t a i n e d p r o t e i n bands v i s i b l e i n t h e g e l . - 20 -ELUTION FROM PAPER E l u t i o n o f p e p t i d e s f r o m p a p e r a f t e r h i g h v o l t a g e e l e c t r o p h o r e s i s o r c h r o m a t o g r a p h y was p e r f o r m e d i n a c o v e r e d b u f f e r t r o u g h s y s t e m . The end o f t h e s t r i p o f p a p e r f u r t h e s t f r o m t h e s a m p l e zone was t a p e r e d , t h e o p p o s i t e end p l a c e d between two c l e a n g l a s s s l i d e s and t h e s l i d e s p l a c e d i n t h e b u f f e r t r o u g h so t h a t t h e p a p e r hung down o v e r t h e s i d e o f t h e t r o u g h . A t e s t t u b e was p l a c e d i n a r a c k s o t h a t t h e t i p o f t h e p a p e r r e s t e d a g a i n s t t h e w a l l o f t h e t u b e and t h e b u f f e r a l l o w e d t o f l o w by c a p i l l a r i t y and syphon a c t i o n . The a p p a r a t u s was c o v e r e d w i t h a p e r s p e x l i d d u r i n g e l u t i o n and t h e a t m o s p h e r e i n s i d e k e p t s a t u r a t e d by w e t t i n g f i l t e r p a p e r a t t h e b o t t o m w i t h b u f f e r . The b u f f e r used f o r e l u t i o n i n t h i s s t u d y was 1 . 0 $ (v/v) a c e t i c a c i d C A r i s t a r 1 , B . D . H . , P o o l e , E n g l a n d ) . A f t e r a s u i t a b l e v o l u m e had been c o l l e c t e d , t h e t u b e s were removed and l y o p h i l i z e d . A c h e c k was made f o r c o m p l e t e e l u t i o n by c u t t i n g a s m a l l edge s t r i p o f f o f t h e p a p e r and d i p p i n g i t i n n i n h y d r i n . AMINO ACID ANALYSIS ( i ) E q u i p m e n t and R e a g e n t s A u t o m a t e d a m i n o a c i d a n a l y s e s were p e r f o r m e d u s i n g t h e method o f Spackman e t a l ( 1 9 5 8 , 1 9 6 7 ) . The e q u i p m e n t used was a B i o c h r o m BC200 a u t o m a t i c a m i n o a c i d a n a l y s e r ( B i o C a l I n s t r u m e n t C o . , R i c h m o n d , C a l i f o r n i a ) w h i c h , when p u r c h a s e d , was c a p a b l e o f q u a n t i t a t i n g 5 0 - 2 5 0 nM. o f a m i n o a c i d . In o r d e r t o a l l o w f o r q u a n t i t a t i o n ~ T n t h e 5 - 1 0 nM. r a n g e s e v e r a l m o d i f i c a t i o n s t o t h i s e q u i p m e n t were made. The p h o t o m u l t i p l i e r t u b e s i n t h e c o l o u r i m e t e r , w h i c h were i n h e r e n t l y n o i s y , were r e p l a c e d by p h o t o t r a n s i s t o r s (FPT 1 0 0 , F a i r c h i l d S e m i c o n d u c t o r , M o u n t a i n V i e w , C a l i f o r n i a ) w h i c h were b o t h c h e a p and e l e c t r o n i c a l l y q u i e t . Cook e t a I ( 1 9 6 9 ) d e s c r i b e d t h e u s e o f t h e s e d e v i c e s i n a w h e a t s t o n e b r i d g e t y p e o f d e t e c t i o n c i r c u i t . A s i n g l e c o l u m n s y s t e m , s i m i l a r t o t h a t o f D e v e n y i ( 1 9 6 8 ) was used and s e v e r a l o f t h e p r e c a u t i o n s m e n t i o n e d by H a m i l t o n ( 1 9 6 7 ) w i t h r e g a r d t o h i g h s e n s i t i v i t y a n a l y s i s were a d o p t e d . In o r d e r t o m i n i m i s e b a s e l i n e f l u c t u a t i o n s an a m p l i f i e r w i t h a l o g a r i t h m i c o u t p u t ( T e c h n i c a l E l e c t r o n i c S e r v i c e s , V a n c o u v e r , B . C . ) was i n s t a l l e d . T h i s improved t h e s i g n a l t o n o i s e r a t i o and b r o u g h t t h e e f f e c t i v e q u a n t i t a t a b l e r a n g e o f t h e a n a l y s e r down t o 2nM. A f u l l a c c o u n t o f t h e v a r i o u s s t a g e s i n m o d i f i c a t i o n o f t h e a n a l y s e r was r e p o r t e d by O ' D o r ( 1 9 7 1 ) . The two b u f f e r s y s t e m s w h i c h were used t o e l u t e a m i n o a c i d s were f o r m u l a t e d a s f o l l o w s : pH 5 . 2 5 pH 4 . 4 Sod ium C i t r a t e d i h y d r a t e 7 8 . 4 g . 7 8 . 4 g . (Amino a c i d a n a l y s i s g r a d e , B i o - R a d L a b o r a t o r i e s , R i c h m o n d , C a l i f o r n i a ) C o n e . H y d r o c h l o r i c A c i d 4 9 . 4 m l . 3 0 . 0 m l . ( B a k e r - A d a m s o n R e a g e n t , A l l i e d C h e m i c a l s , C a n a d a , L t d . ) Sod ium C h l o r i d e - 1 4 0 . 4 g . ( F i s h e r , R e a g e n t g r a d e ) O c t a n o i c A c i d 0 . 5 m l . 0 . 5 m l . ( A . A . A . g r a d e , B i o - R a d ) F i n a l Vo lume 4 . 0 L . 4 . 0 L. - 22 -The n i n h y d r i n r e a g e n t was p r e p a r e d a s f o l l o w s : M e t h y l C e l l o s o l v e 3 . 0 L. ( S e q u a n a l G r a d e , P i e r c e C h e m i c a l C o . R o c k f o r d , I l l i n o i s . ) 4 . 0 M . Sod ium A c e t a t e 1 . 0 L. ( F i s h e r , R e a g e n t G r a d e ) Ni n h y d r i n 80 g . ( R e a g e n t G r a d e , P i e r c e ) S t a n n o u s c h l o r i d e d i h y d r a t e I . 6 g . ( A . A . A . g r a d e , B i o - R a d ) ' C o m p l e t e s o l u t i o n o f t h e n i n h y d r i n was e n s u r e d by s t i r r i n g b e f o r e r e d u c t i o n w i t h s t a n n o u s c h l o r i d e . Oxygen was removed by b u b b l i n g w i t h n i t r o g e n (L g r a d e , C a n a d i a n L i q u i d A i r L t d . ) f o r 30 m i n u t e s . The i o n e x c h a n g e r e s i n s used d u r i n g t h e c o u r s e o f t h i s s t u d y were e i t h e r A m i n e x A - 5 ( B i o - R a d ) o r C u s t o m R e s e a r c h R e s i n A A I 5 ( B e c k m a n . ) . ( i i ) P r e p a r a t i o n o f S a m p l e s d e s c r i b e d by Moore and S t e i n ( 1 9 6 3 ) . P e p t i d e s ( 5 - 1 0 n M . ) were l y o p h i l i z e d i n t o t h e b o t t o m o f c u l t u r e t u b e s ( P y r e x , 12 x 75 mm.) and 2 5 0 - 3 0 0 u I . o f 5 . 7 M h y d r o c h l o r i c a c i d C A r i s t a r " , B . D . H . ) a d d e d . The t u b e s were h e a t e d a t t h e neck w i t h a h o t o x y - g a s f l a m e and drawn o u t t o y i e l d a n a r r o w h e a v y -w a l l e d n e c k . The c o n t e n t s were t h e n f r o z e n i n a d r y i c e - m e t h y l c e l l o s o l v e - 2 m i x t u r e t o p r e v e n t b u b b l i n g , e v a c u a t e d t o 10 t o r r and s e a l e d w i t h a m o d e r a t e o x y - g a s f l a m e . H y d r o l y s i s a t I 10°C was p e r f o r m e d u s i n g a Temp-B l o k ( L a b - L i n e i n s t r u m e n t s , M e l r o s e P a r k , I l l i n o i s ) f o r 1 8 - 2 2 h o u r s . A f t e r h y d r o l y s i s t h e t u b e s were o p e n e d and r o t a r y e v a p o r a t e d u n d e r v a c u u m . S a m p l e s f o r a m i n o a c i d a n a l y s i s were p r e p a r e d e s s e n t i a l l y a s - 23 -The p y r e x t u b e s used f o r h y d r o l y s i s were washed i n s t r o n g d e t e r g e n t , r i n s e d w i t h t a p and d i s t i l l e d w a t e r , and a l l o w e d t o s t a n d o v e r n i g h t i n I.OM h y d r o c h l o r i c a c i d b e f o r e b e i n g d r i e d i n a 60°C o v e n d i r e c t l y f r o m t h e a c i d r i n s e . ( i i i ) C a l c u l a t i o n o f Amino A c i d C o m p o s i t i o n s Amino a c i d c o m p o s i t i o n s were c a l c u l a t e d u s i n g t h e a r e a m e t h o d . The h e i g h t o f t h e o p t i c a l d e n s i t y peak f r o m b a s e l i n e t o a p e x was measured i n c e n t i m e t r e s and a h o r i z o n t a l l i n e drawn a c r o s s t h e peak a t t h e h a l f h e i g h t . The number o f d o t s and f r a c t i o n s o f a d o t o c c u r i n g a b o v e t h i s l i n e was m e a s u r e d and t h i s c o n s t i t u t e d t h e w i d t h o f t h e p e a k . The h e i g h t was t h e n m u l t i p l i e d by t h e w i d t h and t h e p r o d u c t d i v i d e d by a c o n s t a n t C t o y i e l d t h e number o f n a n o m o l e s o f a m i n o a c i d . The c o n s t a n t C was d e r i v e d f r o m t h e mean h e i g h t t i m e s w i d t h p e r nanomole o b t a i n e d f r o m s t a n d a r d r u n s u s i n g known amounts o f e a c h amino a c i d . The n a n o m o l a r c o n c e n t r a t i o n s o f e a c h amino a c i d were t h e n d i v i d e d by t h e number o f n a n o m o l e s o f v a l i n e o c c u r i n g i n t h e p e p t i d e . T h i s f i g u r e , t h e m o l e r a t i o , y i e l d e d t h e number o f r e s i d u e s o f e a c h amino a c i d and was rounded o f f t o t h e n e a r e s t i n t e g e r number , t h e m o l e i n t e g e r . In t h e e v e n t o f t h e p e p t i d e b e i n g d e v o i d o f v a l i n e , l y s i n e was used a s t h e d e n o m i n a t o r . T h e s e amino a c i d s were c h o s e n b e c a u s e o f t h e i r known r e s i s t a n c e t o d e s t r u c t i o n by a c i d h y d r o l y s i s . In t h e e v e n t t h a t t h e c o n c e n t r a t i o n o f one o r more amino a c i d s was s u f f i c i e n t t o y i e l d f u l l s c a l e o p t i c a l d e n s i t y r e a d i n g s , t h e s u p p r e s s e d c h a n n e l was r e a d u s i n g c o n s t a n t s c a l c u l a t e d f r o m t h e s u p p r e s s e d c h a n n e l r e a d i n g s o f s t a n d a r d r u n s . - 24 -AMIDE DETERMINATIONS The p r e s e n c e o f a s p a r a g i n e o r g l u t a m i n e r e s i d u e s i n a p e p t i d e was d e t e r m i n e d by t h e h i g h v o l t a g e e l e c t r o p h o r e t i c method o f Of f o r d ( 1 9 6 6 ) . The r e l a t i v e e l e c t r o p h o r e t i c m o b i l i t i e s o f p e p t i d e s a f t e r h i g h v o l t a g e e l e c t r o p h o r e s i s , when p l o t t e d a g a i n s t t h e i r m o l e c u l a r w e i g h t s , f a l l i n t o d i s t i n c t p o p u l a t i o n s a c c o r d i n g t o t h e c h a r g e on t h e p e p t i d e . T h i s method was t h e r e f o r e used t o d e t e r m i n e t h e c h a r g e on p e p t i d e s o f known c o m p o s i t i o n . F i g u r e s 2 and 3 show t h e r e l a t i v e m o b i l i t i e s a t pH 6 . 5 ( r e l a t i v e t o a s p a r t i c a c i d ) and a t pH 1 . 9 ( r e l a t i v e t o s e r i n e ) r e s p e c t i v e l y . P e p t i d e s w h i c h c o n t a i n e d o n l y o n e g l u t a m i c a c i d o r a s p a r t i c a c i d • r e s i d u e were d e a l t w i t h a s o u t l i n e d a b o v e ; h o w e v e r , when more t h a n o n e o f t h e s e r e s i d u e s was p r e s e n t , t h e e l e c t r o p h o r e t i c m o b i l i t i e s were m e a s u r e d b e f o r e and a f t e r t h a t s t a g e o f Edman d e g r a d a t i o n w h i c h removed o n e o f t h e r e s i d u e s . C a r e was t a k e n t o e n s u r e t h a t i n c a l c u l a t i n g t h e m o l e c u l a r w e i g h t o f t h o s e p e p t i d e s w h i c h had u n d e r g o n e Edman d e g r a d a t i o n , t h e i n c r e -ment due t o t h e p r e s e n c e o f t h e e - p h e n y l t h i o c a r b a m y I g r o u p o f l y s i n e was i n c l u d e d . POLYPEPTIDE DEGRADATION PROCEDURES (A) N o n - E n z y m a t i c (Cyanogen B r o m i d e ) C l e a v a g e C y a n o g e n b r o m i d e (CNBr ) c l e a v a g e o f p e p t i d e s a t m e t h i o n y l r e s i d u e s ( G r o s s and W i t k o p ( 1 9 6 1 , 1 9 6 2 ) ) was p e r f o r m e d i n 10% ( v/v ) f o r m i c a c i d a t a p o l y p e p t i d e c o n c e n t r a t i o n o f 2 . 0 m g / m l . and a c y a n o g e n b r o m i d e c o n c e n t r a t i o n o f 1 0 . 0 m g / m l . The r e a c t i o n was c a r r i e d o u t i n a s m a l l , s e a l e d l y o p h i l i z i n g f l a s k w h i c h was c o v e r e d w i t h a l u m i n i u m f o i l t o e x c l u d e l i g h t . A f t e r 6 h o u r s a t room t e m p e r a t u r e t h e r e a c t i o n m i x t u r e was d i l u t e d 20 t i m e s w i t h d i s t i l l e d w a t e r and l y o p h i l i z e d . - 25 -F i g u r e 2 . L o g a r i t h m i c p l o t o f e l e c t r o p h o r e t i c m o b i l i t y a t pH 6 . 5 r e l a t i v e t o a s p a r t i c a c i d v e r s u s p e p t i d e m o l e c u l a r w e i g h t . A l i g n m e n t s c o r r e s p o n d t o i n d i c a t e d u n i t c h a r g e s . ( A f t e r O f f o r d ( 1 9 6 6 ) ) . - 26 -M O L E C U L A R W E I G H T X 10 F i g u r e 3. L o g a r i t h m i c p l o t o f e l e c t r o p h o r e t i c m o b i l i t y a t pH I.9 r e l a t i v e t o s e r i n e v e r s u s p e p t i d e m o l e c u l a r w e i g h t . A l i g n m e n t s c o r r e s p o n d t o i n d i c a t e d u n i t c h a r g e s . ( A f t e r O f f o r d (I 966)). - 27 -C y a n o g e n b r o m i d e ( E a s t m a n ) was s t o r e d a t - 2 0 ° C and f r e s h c y a n o g e n b r o m i d e r e a g e n t was a l w a y s u s e d f o r e a c h c l e a v a g e . (B) E n z y m a t i c C l e a v a g e ( i ) P r e p a r a t i o n o f R e a g e n t s . T r y p s i n ' T P C K ' ( t o IuenesuI phony I am i d o p h e n y I e t h y I c h I o r o m e t h y I k e t o n e t r e a t e d ) , ( W o r t h i n g t o n B i o c h e m i c a I s ) was p r e p a r e d a s a 2 . 0 m g / m l . s o l u t i o n i n \% ammonium b i c a r b o n a t e . T h e r m o l y s i n ( S i g m a ) was p r e p a r e d a s a 2 . 0 mg/ml . s o l u t i o n i n \% ammonium b i c a r b o n a t e . A l l enzyme s o l u t i o n s were s t o r e d a t - 2 0 ° C u n t i l i m m e d i a t e l y p r i o r t o u s e . ( i i ) D i g e s t i o n P r o c e d u r e s T r y p t i c and c h y m o t r y p t i c d i g e s t i o n s were p e r f o m e d i n \% ammonium b i c a r b o n a t e s o l u t i o n w i t h a p o l y p e p t i d e c o n c e n t r a t i o n o f 0 . 2 ? ( w / v ) . The e n z y m e / s u b s t r a t e r a t i o was 1/50 (w/w). D i g e s t i o n was a l l o w e d t o p r o c e e d f o r a t o t a l o f 6 h o u r s . A t t h e end o f t h i s t i m e , t h e r e a c t i o n m i x t u r e was l y o p h i l i z e d , t h e d i g e s t i o n p r o d u c t s r e d i s s o l v e d i n 0 . 5 m l . o f w a t e r and h e a t e d f o r 6 m i n u t e s i n a b o i l i n g w a t e r b a t h . The s o l u t i o n was t h e n c e n t r i f u g e d , a n y p r e c i p i t a t e was removed and t h e s u p e r n a t a n t r e l y o p h i I i z e d . T h i s t r e a t m e n t e f f e c t i v e l y d e n a t u r e d t h e e n z y m e s . D i g e s t i o n w i t h t h e r m o l y s i n was p e r f o r m e d a s o u t l i n e d a b o v e e x c e p t t h a t t h e e n z y m e / s u b s t r a t e r a t i o was l/20(w/w) and t h e r e a c t i o n a l l o w e d t o p r o c e e d f o r 2 h o u r s a t 22°C . C -TERMINAL RESIDUE DETERMINATION C - t e r m i n a l r e s i d u e s were d e t e r m i n e d by d i g e s t i o n o f t h e p o l y p e p t i d e w i t h c a r b o x y p e p t i d a s e A and k i n e t i c a n a l y s i s o f t h e a p p e a r a n c e o f f r e e a m i n o a c i d s . - 28 -C a r b o x y p e p t i d a s e A ' D F P ' ( D i i s o p r o p y l p h o s p h o f I u o r i d a t e t r e a t e d , W o r t h i n g t o n ) was p r e p a r e d a s a 1 0 . 0 m g / m l . s o l u t i o n i n 2 .0M.ammonium b i c a r b o n a t e s o l u t i o n . M o t i l i n (100 nM) was d i s s o l v e d i n 360 y l . o f w a t e r and 20 y l . o f 2 . 0 M . ammonium b i c a r b o n a t e a d d e d . Enzyme s o l u t i o n (20 y l . ) was added and t h e r e a c t i o n a l l o w e d t o p r o c e e d a t 22°C. A l i q u o t s o f 40 y l . ( c o n t a i n i n g a p p r o x i m a t e l y 10 nM o f p o l y p e p t i d e ) were removed a t 2 , 4 , 6 and 12 h o u r s r e s p e c t i v e l y , l y o p h i l i z e d and s u b j e c t e d t o amino a c i d a n a l y s e s w i t h no f u r t h e r t r e a t m e n t . N-TERMINAL RESIDUE DETERMINATION N - t e r m i n a l r e s i d u e s w e r e d e t e r m i n e d by t h e d a n s y l (DNS) method o f G r a y ( 1 9 6 7 ) and G r a y and H a r t l e y ( 1 9 6 3 ) . The method used i n c l u d e d t h e 2 m o d i f i c a t i o n s o f B r u t o n and H a r t Iey ( I 970) i n w h i c h 5 cm p o l y a m i d e p l a t e s a r e u s e d . A p p r o x i m a t e l y 5 nM o f p o l y p e p t i d e were l y o p h i l i z e d i n t o t h e b o t t o m o f a s m a l l c u l t u r e t u b e ( P y r e x , 6 x 50 m m . ) , 2 . 0 y l . o f \% a q u e o u s s o d i u m b i c a r b o n a t e s o l u t i o n a d d e d , t h e t u b e c e n t r i f u g e d and r e l y o p h i I i z e d . D e i o n i s e d d i s t i l l e d w a t e r ( 2 . 5 y l . ) and 2 . 5 y l . o f d a n s y l c h l o r i d e ( D i m e t h y l -n a p h t h a I e n e - 5 - s u I phony I c h l o r i d e , S i g m a , 2 . 5 m g / m l . i n a c e t o n e ) were a d d e d , t h e t u b e c e n t r i f u g e d , s e a l e d w i t h p a r a f i l m and i n c u b a t e d 20 m i n u t e s a t 4 5 ° C . A f t e r t h i s t i m e t h e t u b e was o p e n e d , c e n t r i f u g e d and l y o p h i l i z e d . 50 y l . o f 5 . 7 M . h y d r o c h l o r i c a c i d were added t o t h e t u b e w h i c h was c e n t r i f u g e d , s e a l e d i n a f l a m e and h e a t e d i n a T e m p - B l o k a t I00°C f o r 18 h o u r s . A f t e r h y d r o l y s i s t h e t u b e was c e n t r i f u g e d , opened and l y o p h i l i z e d o v e r s o d i u m h y d r o x i d e p e l l e t s . D a n s y l amino a c i d s were i d e n t i f i e d a f t e r t h i n l a y e r c h r o m a t o g r a p h y o n p o l y a m i d e l a y e r p l a t e s (Chen C h i n T r a d i n g C o . T a i p e i , T a i w a n ) a s d e s c r i b e d by Woods and Wang ( 1 9 6 7 ) . A q u e o u s 50% p y r i d i n e ( 2 . 5 y l . ) was added t o t h e t u b e and 0 . 2 5 y l . s p o t t e d on e a c h s i d e - 29 -o f a 5 x 5 c m . p o l y a m i d e p l a t e . A s t a n d a r d s o l u t i o n c o n t a i n i n g t h e d a n s y l d e r i v a t i v e s o f a r g i n i n e , g l y c i n e , g l u t a m i c a c i d , i s o l e u c i n e , p h e n y l a l a n i n e , p r o l i n e and s e r i n e was s p o t t e d o n one s i d e o f t h e p l a t e s u c h t h a t a b o u t 0 . 5 nM o f e a c h were p r e s e n t . A s c e n d i n g c h r o m a t o g r a p h y was p e r f o r m e d i n two d i m e n s i o n s u s i n g t h e f o l l o w i n g s o l v e n t s y s t e m s : D i m e n s i o n So l v e n t 1 ( I ) W a t e r / 9 0 ? F o r m i c A c i d ( 2 0 0 / 3 v / v ) 2 (2 ) B e n z e n e / G l a c i a l A c e t i c A c i d (9/1 v/v ) 2 • (3) H e x a n e / B u t a n o l / G l a c i a I A c e t i c A c i d (3/3/1 v/v/v ) 2 (4 ) O . IM A m m o n i a / E t h a n o l (9/1 v/v ) S o l v e n t s ( I ) and (2) a r e f r o m Woods and Wang ( 1 9 6 7 ) , and s o l v e n t (3 ) f r o m Crowshaw e t a l ( 1 9 6 7 ) . The p l a t e s were r o u t i n e l y r u n i n s o l v e n t s ( I ) and (2) and were t h e n v i e w e d u n d e r a s h o r t wave u l t r a v i o l e t s o u r c e , i d e n t i f i c a t i o n b e i n g made i f p o s s i b l e . O v e r l a p p i n g p a i r s s u c h a s d a n s y l s e r i n e / d a n s y I - t h r e o n i n e , d a n s y l -a s p a r t i c a c i d / d a n s y I - g I u t a m i c a c i d and d a n s y I - g I y c i n e / d a n s y I - a I a n i n e w e r e s e p a r a t e d by r u n n i n g i n s o l v e n t ( 3 ) . S o l v e n t (4) was used t o d i s t i n g u i s h d a n s y I - a r g i n i n e , e - d a n s y l - l y s i n e and a - d a n s y l - h i s t i d i n e . P o s i t i v e i d e n t i f i c a t i o n was a i d e d by t h e p r e s e n c e o f t h e s t a n d a r d s on o n e s i d e o f t h e p l a t e . C o i n c i d e n c e o f a p a r t i c u l a r s p o t o n b o t h s i d e s o f t h e p l a t e o r t h e p r e s e n c e o f an e x t r a s p o t on t h e s t a n d a r d s i d e e n a b l e d p r e c i s e i d e n t i t y t o be a t t a c h e d t o t h e unknown d a n s y l a m i n o a c i d . I f l y s i n e was p r e s e n t i n t h e p e p t i d e , e - d a n s y I - 1 y s i n e was a l w a y s s e e n a s was 0 - d a n s y l - t y r o s i n e when t h i s amino a c i d was p r e s e n t . B i s - d a n s y I - I y s i n e was - 30 -o n l y s e e n when t h i s a m i n o a c i d was N - t e r m i n a l f o r t h e p e p t i d e . B i s - d a n s y l -h i s t i d i n e was n e v e r s e e n s i n c e t h e i m i d a z o l e g r o u p o f t h i s a m i n o a c i d i s d e s t r o y e d by h y d r o l y s i s . When p r o l i n e was s u s p e c t e d a s N - t e r m i n a l f o r a p e p t i d e , t h e h y d r o l y s i s t i m e was s h o r t e n e d t o 4 h o u r s s i n c e d a n s y I - p r o l i n e i s l a b i l e u n d e r p r o l o n g e d a c i d h y d r o l y s i s c o n d i t i o n s . F i g u r e s 4 and 5 show t h e p o s i t i o n s o f t h e d a n s y l a m i n o a c i d s a f t e r t h i n l a y e r c h r o m a t o g r a p h y . AMINO ACID SEQUENCE DETERMINATION The a m i n o a c i d s e q u e n c e s o f p e p t i d e s were d e t e r m i n e d by t h e d a n s y l -Edman m o d i f i c a t i o n o f t h e Edman d e g r a d a t i v e p r o c e d u r e (Edman ( 1 9 5 6 ) ) . T h i s m e t h o d , w h i c h was d e s c r i b e d by G r a y ( 1 9 6 7 ) and G r a y and H a r t l y ( 1 9 6 3 ) and w h i c h was r e v i e w e d by H a r t l e y ( 1 9 7 0 ) , i n v o l v e d s e q u e n t i a l r e m o v a l o f t h e N -t e r m i n a l r e s i d u e o f a p e p t i d e f o l l o w e d by i d e n t i f i c a t i o n o f t h e N - t e r m i n a l r e s i d u e o f t h e new p e p t i d e , ( i ) P r e p a r a t i o n o f R e a g e n t s P h e n y i s o t h i o c y a n a t e ( E a s t m a n ) was d i s t i l l e d u n d e r m o d e r a t e vacuum u s i n g a Q i i i c k f i t t h r e e way a d a p t o r (RA 5 / 2 1 , Q u i c k f i t , E n g l a n d ) w i t h B I 9 neck f i t t i n g . The d i s t i l l a t e was c o l l e c t e d i n t h e 72° -78°C r a n g e , s e a l e d i n v i a l s (2 m l . ) and s t o r e d a t - 2 0 ° C . P y r i d i n e ( F i s h e r ) was r e f I u x e d w i t h p o t a s s i u m h y d r o x i d e ( I . 5 L . w i t h 5 0 0 g ) f o r 3 h o u r s and d i s t i l l e d a t M 4 0 - I I 6 ° C . S t o r a g e was a t room t e m p e r a -t u r e i n a brown b o t t l e . P h e n y l i s o t h i o c y a n a t e r e a g e n t f o r t h e Edman r e a c t i o n was p r e p a r e d a s a 5% s o l u t i o n i n p y r i d i n e . T h i s r e a g e n t was s t o r e d a t - 2 0 ° C and was made up m o n t h l y . T r i f I u o r o a c e t i c a c i d ( E a s t m a n ) was d i s t i l l e d i n 10 m l . l o t s u s i n g an o i l h e a t i n g b a t h t o e n s u r e e v e n h e a t i n g . The d i s t i l l a t e was c o l l e c t e d - 31 -• ILE BIS-TYR •LEU • , » P H E B1S-HIS • PRO • VAL @ D N S - N H 2 • ALA • 8 , S - L Y S . W E T •SLY • TRP N-TYR O-TYR *GLU .THR E -LYS - f c v ^ c V & O 2 ? ^ * A S P . S E R , « - H I S CYS CYSO3H DNS-OH A R e I , • 2 l L E .PRO #, FH VAL BIS-TYR 'LEU • • 'PH* © D N S - N H 2 BIS-HIS « M E T " GLY* » T H R y _ L v o • BIS-LYS • L L T b r M 1.SER C C- H' S •TRP 0 - T Y R * G L U # A R 6 • ASP N-TYR # CYS 0» ^ ^ ^ D N S - O H CYS - > l , t 2 , * 3 F i g u r e 4 . P o s i t i o n o f D a n s y l amino a c i d s a f t e r p o l y a m i d e l a y e r c h r o m a t o g r a p h y . S o l v e n t s ( s e e t e x t ) and d i m e n s i o n s a s i n d i c a t e d . D N S - N h ^ , D a n s y l a m i n e . D N S - O H , D a n s y l - - h y d r o x i d e . - 32 -© DNS -NH 2 • CC- HIS • I -LYS • A R G -» I, • 2, 4 3 , 1 4 F i g u r e 5 . P o s i t i o n o f D a n s y l a m i n o a c i d s a f t e r p o l y a m i d e l a y e r c h r o m a t o g r a p h y . S o l v e n t s ( s e e t e x t ) and d i m e n s i o n s a s i n d i c a t e d . - 33 -i n t h e 7 0 ° - 7 l ° C r a n g e and was r e d i s t i l l e d u s i n g t h e same p r o c e d u r e . The f i n a l d i s t i l l a t e was s t o r e d i n a d e s s i c a t o r o v e r c o n c e n t r a t e d s u l p h u r i c a c i d f o r 24 h o u r s b e f o r e d i v i s i o n i n t o 1 . 0 m l . a l i q u o t s i n s e a l e d v i a l s and s t o r a g e a t - 2 0 ° C . B u t y l A c e t a t e ( F i s h e r ) was washed i n a s e p a r a t o r y f u n n e l w i t h 2 1 . 5 ? s o d i u m c a r b o n a t e s o l u t i o n ( I . 5 L . w i t h 500 m l . ) , t h e a q u e o u s p h a s e removed and t h e o r g a n i c p h a s e rewashed w i t h 500 m l . o f 2 1 . 5 ? c a l c i u m c h l o r i d e s o l u t i o n . The b u t y l a c e t a t e was s t o r e d o v e r 250 g . o f p o t a s s i u m c a r b o n a t e o v e r n i g h t and t h e n d i s t i l l e d w i t h t h e p o t a s s i u m c a r b o n a t e s t i l l p r e s e n t , t h e d i s t i l l a t e between I 2 3 ° - I 2 6 ° C b e i n g c o l l e c t e d . S t o r a g e was a t room t e m p e r a t u r e i n a brown b o t t l e , ( i i ) D e g r a d a t i o n P r o c e d u r e . A p p r o x i m a t e l y 100 nM o f p e p t i d e were l y o p h i l i z e d a f t e r c e n t r i f u g a -t i o n i n t o t h e b o t t o m o f an a c i d - w a s h e d P y r e x 12 x 75 mm. t u b e and d i s s o l v e d i n 150 y l . o f d e i o n i s e d d i s t i l l e d w a t e r . An a l i q u o t ( I 0 P I . ) was removed f o r N - t e r m i n a l d e t e r m i n a t i o n by t h e d a n s y l method d e s c r i b e d p r e v i o u s l y . P h e n y l i s o t h i o c y a n a t e r e a g e n t (150 y l . ) was t h e n a d d e d , t h e t u b e f l u s h e d w i t h n i t r o g e n , s e a l e d w i t h p a r a f i l m and i n c u b a t e d f o r 1.5 h o u r s a t 4 5 ° C . A f t e r i n c u b a t i o n t h e t u b e was c e n t r i f u g e d , u n s e a l e d and p l a c e d i n a d e s s i c a t o r c o n t a i n i n g c o n c e n t r a t e d s u l p h u r i c a c i d , s o d i u m h y d r o x i d e and f r e s h p h o s p h o r u s p e n t o x i d e . The d e s s i c a t o r was e v a c u a t e d t o a p p r o x i m a t e l y I mm.Hg p r e s s u r e and p l a c e d i n a 60°C o v e n f o r 3 0 - 4 5 m i n u t e s . The t u b e was t h e n removed f r o m t h e d e s s i c a t o r , I 5 0 y I. o f t r i f I u o r o a c e t i c a c i d a d d e d , t h e t u b e f l u s h e d w i t h n i t r o g e n , s e a l e d w i t h p a r a f i l m and i n c u b a t e d a t 45°C f o r 30 m i n u t e s b e f o r e d r y i n g i n a vacuum d e s s i c a t o r o v e r s o d i u m h y d r o x i d e f o r 10 m i n u t e s . D e i o n i s e d d i s t i l l e d w a t e r (200 y l . ) was added t o t h e t u b e a n d , a f t e r e n s u r i n g d i s s o l u t i o n o f t h e c o n t e n t s , e x t r a c t e d f o u r t i m e s w i t h 2 m l . o f b u t y l a c e t a t e e a c h t i m e . A l a r g e s u r f a c e a r e a f o r e x t r a c t i o n was - 34 -e n s u r e d by v i b r a t i n g t h e t u b e i n a v o r t e x m i x e r d u r i n g e a c h w a s h . F o l l o w i n g remova l o f a s much o f t h e f i n a l b u t y l a c e t a t e wash a s p o s s i b l e , t h e t u b e was p l a c e d i n a vacuum d e s s i c a t o r o v e r c o n c e n t r a t e d s u l p h u r i c a c i d and t h e c o n t e n t s e v a p o r a t e d . The r e a c t i o n p r o d u c t s were t h e n d i s -s o l v e d i n 150 y l . - o f w a t e r , 10 y l . removed f o r N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n and t h e r e m a i n d e r t r e a t e d a s d e s c r i b e d f o r f r e s h ' p e p t i d e . A summary o f t h e r e a c t i o n s i n v o l v e d i s shown i n f i g . 6 . C o u p l i n g , c l e a v a g e and e x t r a c t i o n r e a c t i o n s were p e r f o r m e d d u r i n g t h e e a r l y p a r t o f t h e a f t e r n o o n . The a l i q u o t f o r i d e n t i f i c a t i o n was d a n s y l a t e d and h y d r o l y s e d o v e r n i g h t . P o l y a m i d e l a y e r c h r o m a t o g r a p h y and i d e n t i f i c a t i o n t o o k p l a c e t h e n e x t m o r n i n g so t h a t a p o s i t i v e i d e n t i f i c a -t i o n was made b e f o r e t h e p e p t i d e was s u b j e c t e d t o t h e n e x t c y c l e o f d e g r a d a t i o n . NOMENCLATURE OF PEPTIDES P e p t i d e s s e p a r a t e d by h i g h v o l t a g e e l e c t r o p h o r e s i s were numbered a r b i t r a r i l y a c c o r d i n g t o t h e i r p o s i t i o n a f t e r e l e c t r o p h o r e s i s s t a r t i n g a t t h e a n o d e . P e p t i d e s s e p a r a t e d c h r o m a t o g r a p h i c a I Iy were numbered f r o m t h e o r i g i n and were p r e f i x e d ' W W . The p r o c e d u r e used t o p r e p a r e p e p t i d e s i s i n d i c a t e d by t h e u s e o f t h e f o l l o w i n g p r e f i x e s : CN - p e p t i d e p r o d u c e d by c y a n o g e n b r o m i d e c l e a v a g e CHT - p e p t i d e p r o d u c e d by c h y m o t r y p t i c d i g e s t i o n TR - p e p t i d e p r o d u c e d by t r y p t i c d i g e s t i o n Th - p e p t i d e p r o d u c e d by t h e r m o l y s i n d i g e s t i o n S P E C I F I C COLOUR REACTIONS S p e c i f i c c o l o u r r e a c t i o n s were u s e d t o d e t e c t t h e p r e s e n c e o f v a r i o u s a m i n o a c i d r e s i d u e s i n p e p t i d e s . T h e s e i n c l u d e d t h e E h r l i c h ' s r e a c t i o n f o r t r y p t o p h a n , t h e P a u l y r e a c t i o n f o r h i s t i d i n e and t h e S a k a g u c h i - 35 -u r e 6 . Summary o f r e a c t i o n s i n v o l v e d i n Edman d e g r a d a t i v e p r o c e d u r e . - 36 -r e a c t i o n f o r a r g i n i n e . The methods used were a s q u o t e d by A m b l e r ( 1 9 6 3 ) . The s p e c t r o p h o t o m e t r i c method f o r t h e d e t e r m i n a t i o n o f t r y p t o p h a n d e s c r i b e d by G a i t o n d e and Dovey ( 1 9 7 0 ) was a l s o u s e d . CHEMICAL MODIFICATION REACTIONS ( i ) O x i d a t i o n w i t h P e r f o r m i c A c i d O x i d a t i o n o f m e t h i o n i n e r e s i d u e s was p e r f o r m e d u s i n g t h e p e r f o r m i c a c i d r e a g e n t o f Moore ( 1 9 6 3 ) . 500 u g . o f p o l y p e p t i d e were d i s s o l v e d i n 1 . 0 m l . o f p e r f o r m i c a c i d r e a g e n t and t h e r e a c t i o n a l l o w e d t o p r o c e e d f o r 4 h o u r s a t 0°C . The r e a c t i o n m i x t u r e was t h e n l y o p h i l i z e d . M e t h i o n i n e , i n an a l i q u o t o f t h e r e a c t i o n m i x t u r e , was i d e n t i f i e d a s m e t h i o n i n e s u l p h o n e a f t e r h y d r o l y s i s i n 5 . 7 M h y d r o c h l o r i c a c i d i n v a c u o f o r 18 h o u r s and s u b s e q u e n t a m i n o a c i d a n a l y s i s o f t h e o x i d i s e d s a m p l e . ( i i ) O x i d a t i o n w i t h H y d r o g e n P e r o x i d e M i l d o x i d a t i o n o f m e t h i o n i n e r e s i d u e s w i t h h y d r o g e n p e r o x i d e was p e r f o r m e d a c c o r d i n g t o t h e method o f M u t t ( 1 9 6 4 ) . P o l y p e p t i d e s were d i s -s o l v e d i n h y d r o g e n p e r o x i d e r e a g e n t ( 1 . 0 m l . o f 30? h y d r o g e n p e r o x i d e , F i s h e r , made up t o 100 m l . w i t h 0 . 0 5 M a c e t i c a c i d ) t o a c o n c e n t r a t i o n o f 1 . 0 m g / m l . The r e a c t i o n was a l l o w e d t o p r o c e e d f o r 4 5 m i n u t e s a t 22°C a f t e r w h i c h t h e s o l u t i o n was l y o p h i l i z e d . No a t t e m p t was made t o q u a n t i t a t e t h e o x i d a t i o n c h e m i c a My. ( i i i ) R e d u c t i o n w i t h C y s t e i n e P e p t i d e s i n w h i c h t h e m e t h i o n i n e r e s i d u e had been o x i d i s e d w i t h h y d r o g e n p e r o x i d e were r e d u c e d w i t h c y s t e i n e h y d r o c h l o r i d e ( C a l B i o c h e m ) a c c o r d i n g t o t h e method o f M u t t ( 1 9 6 4 ) . O x i d i s e d p e p t i d e was d i s s o l v e d i n 5 0 ? a q u e o u s c y s t e i n e h y d r o c h l o r i d e (w/v) t o a c o n c e n t r a t i o n o f 2 . 0 m g / m l . and - 37 -t h e s o l u t i o n was s t o r e d a t 22°C i n an a t m o s p h e r e o f n i t r o g e n f o r 72 h o u r s . The s o l u t i o n was t h e n l y o p h i l i z e d . ( i v ) A c e t y l a t i o n A c e t y l a t i o n o f a m i n o g r o u p s was p e r f o r m e d e s s e n t i a l l y a s d e s c r i b e d by R i o r d a n and V a l l e e ( 1 9 6 7 ) w i t h m o d i f i c a t i o n s . P o l y p e p t i d e ( 1 . 0 m g . ) was d i s s o l v e d i n 1 .0 m l . o f h a l f s a t u r a t e d a q u e o u s s o d i u m a c e t a t e s o l u t i o n . The s o l u t i o n was s t i r r e d w i t h a m a g n e t i c s t i r r e r and a t h i r t y f o l d m o l a r e x c e s s o f a c e t i c a n h y d r i d e ( E a s t m a n ) was a d d e d , a t 2 2 ° C , i n 5 e q u a l p o r t i o n s o v e r I h o u r . The s o l u t i o n was s t i r r e d f o r a f u r t h e r h o u r and t h e r e a c t i o n was s t o p p e d by f r e e z i n g . The a c y i a t e d p e p t i d e was s e p a r a t e d f r o m t h e r e a c t i o n m i x t u r e by d e s a l t i n g on Sephadex G - 1 5 . ( v ) S u c c i n y l a t i o n S u c c i n y l a t i o n o f a m i n o g r o u p s was p e r f o r m e d a c c o r d i n g t o m o d i -f i c a t i o n s o f t h e method d e s c r i b e d by K l o t z ( 1 9 6 7 ) u t i l i z i n g t h e s p e c i f i -c a t i o n s f o r pH a d j u s t m e n t m e n t i o n e d f o r m a l e y l a t i o n by B u t l e r e t a l ( 1 9 6 9 ) . P o l y p e p t i d e ( 1 . 0 m g . ) was d i s s o l v e d i n 2 . 0 m l . o f w a t e r and t h e pH a d j u s t e d t o 7 . 0 w i t h 0 . IM s o d i u m h y d r o x i d e . The s o l u t i o n was s t i r r e d w i t h a m a g n e t i c s t i r r e r and t h e pH m o n i t o r e d by means o f a g l a s s e l e c t r o d e p o s i t i o n e d i n t h e r e a c t i o n v e s s e l . A 30 f o l d m o l a r e x c e s s o f s u c c i n i c a n h y d r i d e ( E a s t m a n ) was added i n s m a l l p o r t i o n s o v e r 15 m i n u t e s and t h e pH m a i n t a i n e d a t 7 . 5 by a d d i t i o n s o f 0 . IM s o d i u m h y d r o x i d e f r o m a H a m i l t o n l O u l . s y r i n g e e q u i p p e d w i t h a t i p o f f i n e b o r e p o l y e t h y l e n e t u b i n g . The s o l u t i o n was s t i r r e d a t 22°C f o r a t o t a l o f 1.5 h o u r s b e f o r e t h e r e a c t i o n was s t o p p e d by f r e e z i n g . The a c y i a t e d p e p t i d e was s e p a r a t e d f r o m t h e r e a c t i o n m i x t u r e on Sephadex G - 1 5 . - 38 -ANIMAL PREPARATIONS ( i ) C h r o n i c Dog P r e p a r a t i o n s Dogs o f t h e l a b r a d o r v a r i e t y w e i g h i n g 2 0 - 3 0 k g . were used i n t h i s s t u d y . S u r g e r y was p e r f o r m e d u n d e r a s e p t i c c o n d i t i o n s and t h e a n i m a l s were f a s t e d 18 h o u r s p r i o r t o s u r g e r y . A n a e s t h e s i a was i n d u c e d by r a p i d i n t r a -v e n o u s i n j e c t i o n o f 5% s o d i u m t h i o p e n t a l ( ' P e n t o t h a I ' ) t o e f f e c t ( 7 - 1 2 m l . ) . A 10 mm. e n d o t r a c h e a l t u b e was p a s s e d and t h e c u f f i n f l a t e d . T h i s a l l o w e d m a i n t e n a n c e o f a n a e s t h e s i a w i t h F l u o t h a n e ( A y e r s t , M o n t r e a l ) a t an o x y g e n , f l o w r a t e o f 3 . 0 L / m i n u t e . (A) B i c k e l pouch B i c k e l p o u c h e s o f t h e f u n d u s o f t h e s t o m a c h were c o n s t r u c t e d f r o m t h e g r e a t e r c u r v a t u r e , a s t a i n l e s s s t e e l c a n n u l a b e i n g i n c o r p o r a t e d d u r i n g t h i s c o n s t r u c t i o n . The c a n n u l a was e x t e r i o r i s e d t h r o u g h a s t a b wound 3 c m . f r o m t h e m i d - l i n e i n t h e u p p e r l e f t a b d o m i n a l q u a d r a n t . D u r i n g c o n s t r u c t i o n o f t h e p o u c h , v a g a l f i b r e s s u p p l y i n g t h e pouch t i s s u e were s e c t i o n e d and s y m p a t h e t i c f i b r e s were removed by s t r i p p i n g t h e n e r v e p l e x u s f r o m t h e s p l e n i c a r t e r y and v e i n and s e v e r i n g a l l m e s e n t e r i c c o n n e c t i o n s t o t h e p o u c h . (B) A n t r a l P o u c h A s e c o n d o p e r a t i o n was p e r f o r m e d o n dogs w i t h B i c k e l p o u c h e s t o c o n s t r u c t a v a g a l l y d e n e r v a t e d pouch o f t h e a n t r a l r e g i o n o f t h e s t o m a c h . T h i s p r o c e d u r e was c a r r i e d o u t 3 - 4 weeks a f t e r t h e p r e p a r a t i o n o f t h e B i c k e l p o u c h . A f t e r d e t e r m i n i n g t h e p o s i t i o n o f t h e j u n c t i o n between a n t r a l and f u n d i c r e g i o n s by e x a m i n i n g f o r t h e s u b t l e c h a n g e i n t e x t u r e on t h e s u r f a c e o f t h e s t o m a c h , t h e a n t r u m was i s o l a t e d by s e c t i o n i n g a l o n g t h i s j u n c t i o n and j u s t b e l o w t h e p y l o r u s . The b l o o d v e s s e l s a l o n g t h e l e s s e r - 39 -c u r v a t u r e f r o m t h e p y l o r u s t o t h e a n g u l a r i s were s e c t i o n e d t h u s r e m o v i n g t h e v a g a l c o n n e c t i o n s w h i c h r u n w i t h t h e r i g h t g a s t r i c a r t e r y . In o r d e r t o e n s u r e t h a t no a c i d s e c r e t i n g t i s s u e was i n c o r p o r a t e d i n t o t h e p o u c h , t h e mucosa was e v e r t e d and t e s t e d w i t h u n i v e r s a l pH p a p e r . As a p r e c a u t i o n , 2 - 3 c m . o f a n t r a l t i s s u e were r e s e c t e d b e f o r e t h e c u t e d g e s were s u t u r e d . The pouch was e x t e r i o r i s e d by s t i t c h i n g t h e p y l o r u s i n t o a b u t t o n wound 3 c m . f r o m t h e m i d - l i n e i n t h e r i g h t u p p e r a b d o m i n a l q u a d r a n t . B l o o d s u p p l y t o t h e pouch was e n s u r e d v i a t h e r i g h t g a s t r o e p i p l o i c b r a n c h o f t h e g a s t r o -d u o d e n a l a r t e r y w h i c h s u p p l i e d t h e g r e a t e r c u r v a t u r e . A s t a i n l e s s s t e e l Thomas ( 1 9 4 1 ) c a n n u l a w i t h t e f l o n p l u g was i n s e r t e d i n t o t h e g a s t r i c r e m n a n t and s e c u r e d w i t h a d o u b l e p u r s e - s t r i n g s u t u r e . The c a n n u l a was e x t e r i o r i s e d v i a a b u t t o n wound o n t h e l e f t u p p e r a b d o m i n a l q u a d r a n t 3 c m . f r o m t h e m i d - l i n e and 5 c m . a n t e r i o r t o t h e f u n d i c c a n n u l a . G a s t r o i n t e s t i n a l c o n t i n u i t y was r e s t o r e d by p e r f o r m i n g a n e n d - t o -s i d e g a s t r o j e j u n o s t o m y 1 0 - 2 0 c m . d i s t a l t o t h e l i g a m e n t o f T r e i t z . F i g u r e 7 i l l u s t r a t e s t h e c h r o n i c dog p r e p a r a t i o n , ( i i ) A c u t e G u i n e a P i g P r e p a r a t i o n s G u i n e a p i g s w e i g h i n g 4 0 0 - 6 0 0 g . were used i n t h i s s t u d y and were a n a e s t h e t i s e d w i t h 3 0 ? u r e t h a n e (300 mg/100 g . ) a d m i n i s t e r e d s u b c u t a n e o u s l y . Body t e m p e r a t u r e was m a i n t a i n e d a t 37°C by means o f a h e a t i n g pad c o n t r o l l e d by a t e l e - t h e r m o m e t e r ( Y e l l o w S p r i n g s I n s t r u m e n t C o . , Model 7 3 ) w h i c h m o n i t o r e d body t e m p e r a t u r e u s i n g a r e c t a l p r o b e . The t r a c h e a was i n t u b a t e d w i t h p o l y e t h y l e n e t u b i n g (PE 2 6 0 ) and t h e e x t e r n a l j u g u l a r v e i n c a n n u l a t e d t o f a c i l i t a t e i n j e c t i o n d u r i n g t h e e x p e r i -m e n t . A m i d - l i n e i n c i s i o n 3 - 4 c m . l o n g was made s u c h t h a t t h e t i p o f t h e x i p h i s t e r n u m was r e v e a l e d . T h i s e x p o s e d t h e g a l l b l a d d e r . A l i g a t u r e was - 40 -F i g u r e 7 . D i a g r a m s h o w i n g c h r o n i c dog p r e p a r a t i o n used i n t h e s e s t u d i e s . - 41 -t i e d a r o u n d t h e apex o f t h e f u n d u s o f t h e g a l l b l a d d e r t o a l l o w a t t a c h m e n t t o a s t r a i n guage s o t h a t g a l l b l a d d e r s h o r t e n i n g c o u l d be m e a s u r e d . ( i i i ) A c u t e C a t P r e p a r a t i o n s . A n a e s t h e s i a was i n d u c e d w i t h F l u o t h a n e and m a i n t a i n e d by i n t r a -v e n o u s a d m i n i s t r a t i o n o f 2% c t - c h l o r a l o s e s o l u t i o n (80 mg/kg) v i a a c a n n u l a t e d s a p h e n o u s v e i n . The t r a c h e a was i n t u b a t e d and t h e g r e a t e r and l e s s e r s p l a n c h n i c n e r v e s s e c t i o n e d e x t r a p e r i t o n e a I l y v i a f l a n k i n c i s i o n s . A m i d - l i n e a b d o m i n a l i n c i s i o n was made and a t a p e l i g a t u r e t i e d round t h e p y l o r u s t o p r e v e n t s t o m a c h c o n t e n t s f r o m d r a i n i n g i n t o t h e duodenum. C a r e was t a k e n t o e x c l u d e t h e g a s t r o d u o d e n a I b l o o d v e s s e l s f r o m t h e l i g a t u r e . S e c r e t i n was i n j e c t e d i n t r a v e n o u s l y i n o r d e r t o s t i m u l a t e t h e f l o w o f p a n c r e a t i c j u i c e and e n a b l e t h e p a n c r e a t i c d u c t t o be i d e n t i f i e d . The d u c t was t h e n c a n n u l a t e d a t a p o i n t p r o x i m a l t o i t s i n s e r t i o n i n t h e d u o d e n a l w a l l u s i n g a s t a i n l e s s s t e e l c a n n u l a o f s u i t a b l e d i a m e t e r ( 2 - 3 m m . ) . A f t e r t y i n g i n t h e c a n n u l a , t h e v a g i were c u t i n t h e neck and t h e a n i m a l was p o s i t i o n e d o n i t s l e f t s i d e so t h a t t h e p a n c r e a t i c j u i c e d r a i n i n g f r o m t h e c a n n u l a c o u l d be c o l l e c t e d . ( i v ) A c u t e R a b b i t P r e p a r a t i o n s R a b b i t s w e i g h i n g a p p r o x i m a t e l y 1 . 5 k g . were u s e d . A n a e s t h e s i a was p r o d u c e d w i t h Nembuta l (35 m g / k g . ) a d m i n i s t e r e d i n t r a v e n o u s l y v i a t h e m a r g i n a l e a r v e i n . A g l a s s c a n n u l a was i n s e r t e d i n t o t h e t r a c h e a and t h e e x t e r n a l j u g u l a r v e i n c a n n u l a t e d w i t h p o l y e t h y l e n e (PE 6 0 ) t u b i n g . E i t h e r a c a r o t i d o r a f e m o r a l a r t e r y was c a n n u l a t e d w i t h p o l y e t h y l e n e (PE 90) t u b i n g and c o n n e c t e d t o a s a l i n e - f i l l e d h i g h p r e s s u r e t r a n s d u c e r ( S t a t h a m , P 2 3 AA) t o a l l o w r e c o r d i n g s o f h e a r t r a t e and b l o o d p r e s s u r e t o be made. - 42 -EXPERIMENTAL PROCEDURES ( i ) C h r o n i c Dog E x p e r i m e n t s Dogs were f a s t e d 18 h o u r s p r i o r t o e x p e r i m e n t s and were b r o u g h t t o t h e l a b o r a t o r y u n d e r c o n d i t i o n s w h i c h were d e s i g n e d t o p r e v e n t undue e x c i t e m e n t . The dog was h a r n e s s e d i n t o a s t a n d s u c h t h a t i t was s u p p o r t e d b u t n e v e r t h e l e s s s t a n d i n g e r e c t , and t h e Thomas c a n n u l a o p e n e d . A p o l y -e t h y l e n e b o t t l e was a t t a c h e d t o t h i s c a n n u l a t o p r e v e n t s p i l l a g e o f r e m n a n t s e c r e t i o n s and t h e f u n d i c pouch c a n n u l a was a t t a c h e d t o a low p r e s s u r e t r a n s d u c e r ( S t a t h a m , P23 BB) v i a a w a t e r - f i l l e d t u b e . T h i s t u b i n g was e q u i p p e d w i t h a s i d e - a r m t o a l l o w c o l l e c t i o n s o f pouch w a s h o u t f l u i d t o be made. The a n t r a l pouch was i n t u b a t e d w i t h p e r f u s i o n , d r a i n a g e and p r e s s u r e m o n i t o r i n g c a n n u l a e and t h e s e were h e l d i n p l a c e by an a c r y l i c m o u l d i n g s e c u r e d t o t h e a n i m a l w i t h t i e s . F i g u r e 8 shows t h e a r r a n g e m e n t u s e d . The a n t r a l pouch was p e r f u s e d w i t h 0 . 9 ? s a l i n e a t a r a t e o f 0 . 2 4 7 m l / m i n . t o p r e v e n t b l o c k a g e o f t h e p r e s s u r e - m o n i t o r i n g c a n n u l a w i t h m u c i n . P r e s s u r e i n t h e a n t r a l pouch was r e c o r d e d v i a a h i g h p r e s s u r e t r a n s d u c e r ( S t a t h a m P23 A A ) . I n f u s i o n s and i n j e c t i o n s w e r e p e r f o r m e d v i a a 21 G 1 . 5 i n . n e e d l e a t t a -ched t o p o l y e t h y l e n e (PE 6 0 ) t u b i n g i n s e r t e d i n t o a s u i t a b l e v e i n on t h e a n i m a l ' s l e g . The n e e d l e was c o n n e c t e d t o a s a l i n e d r i p b o t t l e be tween i n f u s i o n s t o p r e v e n t i t s b l o c k a g e w i t h b l o o d . The f u n d i c pouch was f i l l e d w i t h 25 m l . o f w a t e r a t t h e b e g i n n i n g o f an e x p e r i m e n t a l p e r i o d , e m p t i e d a t t h e e n d , and r e f i l l e d and washed o u t w i t h a f u r t h e r 25 m l . w h i c h was p o o l e d w i t h t h e f i r s t w a s h o u t . The t o t a l w a s h o u t f l u i d f o r e a c h p e r i o d was s u b j e c t e d t o a n a l y s i s f o r p H , H + i o n and p e p s i n c o n c e n t r a t i o n . P e r i o d s were o f 15 m i n u t e d u r a t i o n and m o t o r a c t i -v i t y r e c o r d i n g s f r o m t h e f u n d i c and a n t r a l p o u c h e s were made c o n t i n u o u s l y t h r o u g h o u t t h e e x p e r i m e n t e x c e p t a t t h e c h a n g e o v e r o f e a c h p e r i o d . - 43 -A n t r a l pouch c a n n u l a a r r a n g e m e n t . A D , P e r f u s i o n c a n n u l E , P r e s s u r e r e c o r d i n g c a n n u l a . E C , D r a i n a g e c a n n u l a . G , A c r y l i c m o u l d i n g . F , T i e s . - 44 -( i i ) A n a l y s i s o f G a s t r i c S a m p l e s (A) E s t i m a t i o n o f H + i o n c o n c e n t r a t i o n A l i q u o t s o f g a s t r i c w a s h o u t s a m p l e s were t i t r a t e d t o pH 7 . 0 w i t h 0 . 0 I M s o d i u m h y d r o x i d e s o l u t i o n . A t i t r a t o r a s s e m b l y w i t h m a g n e t i c v a l v e ( T i t r a t o r I I , R a d i o m e t e r ) was used and r e s u l t s were e x p r e s s e d a s u E q . o f H + i o n p e r 15 m i n u t e p e r i o d . (B) E s t i m a t i o n o f P e p s i n The p e p s i n c o n c e n t r a t i o n o f g a s t r i c w a s h o u t s a m p l e s was d e t e r m i n e d by a m o d i f i c a t i o n o f t h e method o f A n s o n and M i r s k y ( 1 9 3 2 ) . An a l i q u o t o f t h e s a m p l e was a l l o w e d t o d i g e s t a h a e m o g l o b i n s u b s t r a t e ( W o r t h i n g t o n ) and t h e r e l e a s e d t y r o s i n e e s t i m a t e d s p e c t r o p h o t o m e t r i c a I Iy a t 275 nm. by r e f e r e n c e t o a s t a n d a r d a b s o r b a n c e c u r v e f o r t y r o s i n e ( F i g . 9 B ) . In t h e o r i g i n a l method t h e t y r o s i n e p r o d u c e d was e s t i m a t e d c o l o u r i m e t r i c a I Iy u s i n g t h e F o l i n - C i o c a I t e a u r e a g e n t . The m o d i f i c a t i o n i n v o l v e d d i r e c t r e a d i n g a t 275 nm. and t h e t y r o s i n e l i b e r a t e d by d i g e s t i o n was r e l a t e d t o p e p s i n by r e f e r e n c e t o a s t a n d a r d a b s o r b a n c e c u r v e f o r d i g e s t i o n p r o d u c t s u s i n g known c o n c e n t r a t i o n s o f a s t a n d a r d p e p s i n p r e p a r a t i o n ( W o r t h i n g t o n ) , ( F i g . 9 A ) . The r e l a t i o n s h i p between t y r o s i n e p r o d u c e d by d i g e s t i o n and t h e amount o f p e p s i n w h i c h r e l e a s e d t h e same amount o f t y r o s i n e i s shown i n F i g . 9 C . R e s u l t s were e x p r e s s e d a s m g . t y r o s i n e p e r 15 m i n u t e p e r i o d . ( i i i ) C a l c u l a t i o n o f M o t i l i t y I n d i c e s R e c o r d i n g s o f p r e s s u r e c h a n g e s i n a n t r a l and f u n d i c p o u c h e s were made o n a p o l y g r a p h r e c o r d e r ( G i l s o n ) . A n t r a l p r e s s u r e was r e c o r d e d d i r e c t l y whi-l-e t h e s i g n a l f r o m t h e f u n d i c p r e s s u r e t r a n s d u c e r was f i l t e r e d t h r o u g h a n 0 . 5 y F c a p a c i t o r so t h a t a r t i f a c t s due t o r e s p i r a t i o n and movements o f t h e a n i m a l were e l i m i n a t e d . - 45 -0.4 0.0 10 20 30 /jg. Pepsin 0.0 jug. Tyrosine 10 ZO 30 yua Pepsin e F i g u r e 9 . (A) P l o t o f a b s o r b a n c e a t 275 nm. o f p r o d u c t s o f d i g e s t i o n o f h a e m o g l o b i n s u b s t r a t e v e r s u s p e p s i n c o n c e n t r a t i o n . (B) P l o t o f a b s o r b a n c e a t 275 nm. v e r s u s t y r o s i n e c o n c e n t r a t i o n . (C) P l o t o f t y r o s i n e c o n c e n t r a t i o n v e r s u s p e p s i n c o n c e n t r a t i o n f r o m d a t a o f A and B. - 46 -M o t i l i t y i n d i c e s f o r e a c h p e r i o d were c a l c u l a t e d u s i n g t h e f o l l o w i n g e x p r e s s i o n : ( A m p l i t u d e i n mm.Hg x D u r a t i o n i n s e e s . M o t i l i t y i n d e x = o f e a c h wave) D u r a t i o n o f P e r i o d i n m i n s . x 10 ( i v ) A s s a y o f M o t i l i n A c t i v i t y u s i n g C h r o n i c Dog P r e p a r a t i o n s M o t i l i n a c t i v i t y was a s s a y e d i n t h e c h r o n i c dog p r e p a r a t i o n by d e t e r m i n i n g t h e f u n d i c m o t o r a c t i v i t y r e s p o n s e t o t h e m a t e r i a l t o be a s s a y e d a s compared w i t h t h e r e s p o n s e t o a s t a n d a r d p a n c r e o z y m i n ( B o o t s , E n g l a n d ) p r e p a r a t i o n . The p r o c e d u r e was a s o u t l i n e d a b o v e e x c e p t t h a t p e r i o d s were o f 10 m i n u t e s d u r a t i o n . D o s e s o f t e s t m a t e r i a l were i n j e c t e d i n t r a v e n o u s l y a t t h e b e g i n n i n g o f a p e r i o d and two o r t h r e e f u r t h e r p e r i o d s a l l o w e d t o p a s s b e f o r e i n j e c t i o n o f o t h e r t e s t m a t e r i a l o r t h e s t a n d a r d p r e p a r a t i o n . O n l y t h e f u n d i c m o t o r a c t i v i t y r e c o r d e d d u r i n g t h e p e r i o d o f i n j e c t i o n was used i n c a l c u l a t i n g t h e m o t i l i t y i n d e x . T h i s i n d e x was t a k e n a s a m e a s u r e o f m o t i I i n a c t i v i t y . The s t a n d a r d p r e p a r a t i o n o f p a n c r e o z y m i n was a s s i g n e d a s t r e n g t h o f 1 .0 m o t i l i n u n i t p e r mg. Doses f o r i n j e c t i o n were a d j u s t e d s u c h t h a t t h e y c o n t a i n e d a p p r o x i m a t e l y 1 .0 u n i t p e r k g . o f dog w e i g h t . C o m p a r i s o n s c o u l d t h e n be made between t h e p o t e n c y o f v a r i o u s p r e p a r a t i o n s . R e s u l t s were e x p r e s s e d a s u n i t s p e r mg. o f m a t e r i a l . ( v ) A s s a y o f C h o I e c y s t o k i n i n a c t i v i t y o f M o t i l i n u s i n g A c u t e G u i n e a - p i g P r e p a r a t i o n s A s s a y s o f c h o I e c y s t o k i n i n (CCK) a c t i v i t y were p e r f o r m e d u s i n g t h e a c u t e g u i n e a - p i g p r e p a r a t i o n e s s e n t i a l l y a s d e s c r i b e d by L j u n g b e r g ( 1 9 6 4 ) . The I i g a t u r e t i e d round t h e a p e x o f t h e g a l l b l a d d e r v/as a t t a c h e d t o a 3 o z . s t r a i n gauge ( S t a t h a m , G IOB-3-350+_) and t h e f o r c e d i s p l a y e d on a p o l y g r a p h w h i c h had been c a l i b r a t e d s u c h t h a t a f o r c e o f 2 g . p r o d u c e d a 3 c m . d e f l e c -t i o n . - 47 -A I o g - d o s e / r e s p o n s e c u r v e was o b t a i n e d t o i n j e c t i o n s o f a s t a n d a r d CCK p r e p a r a t i o n ( G . I . H . R e s e a r c h L a b o r a t o r i e s , Sweden , B a t c h n o . 2 6 8 4 1 ) c o n t a i n i n g 250 Ivy dog u n i t s ( I . D . U . ) p e r mg. F i g u r e 10 shows a t y p i c a l s e r i e s o f r e s p o n s e s t o s t a n d a r d d o s e s and a c u r v e c o n s t r u c t e d f r o m t h i s d a t a . M a t e r i a l f o r a s s a y was d i l u t e d so t h a t t h e CCK a c t i v i t y f e l l i n t o t h e r a n g e c o v e r e d by t h e l o g - d o s e / r e s p o n s e c u r v e and t h e f o r c e o f g a l l b l a d d e r s h o r t e n i n g t o a p a r t i c u l a r d o s e o f t e s t m a t e r i a l was m e a s u r e d . I n j e c t i o n s were o n l y g i v e n a f t e r t h e r e s p o n s e t o a p r e v i o u s i n j e c t i o n had r e t u r n e d t o c o n t r o l l e v e l s . C h o I e c y s t o k i n i n a c t i v i t i e s o f t e s t s a m p l e s were c a l c u l a t e d f r o m t h e l o g - d o s e / r e s p o n s e c u r v e f o r t h a t p a r t i c u l a r a n i m a l u s e d , and were e x p r e s s e d a s I . D . U . p e r mg. ( v i ) A s s a y o f S e c r e t i n a c t i v i t y o f M o t i l i n u s i n g A c u t e C a t p r e p a r a t i o n s A s s a y s o f s e c r e t i n a c t i v i t y were p e r f o r m e d u s i n g t h e a c u t e c a t p r e p a r a t i o n by m e a s u r i n g t h e v o l u m e r e s p o n s e o f p a n c r e a t i c j u i c e o u t p u t t o i n t r a v e n o u s i n j e c t i o n s o f t e s t m a t e r i a l s . I n j e c t i o n s o f m a t e r i a l s t o be a s s a y e d were b r a c k e t e d b e t w e e n i n j e c t i o n s o f 5 c l i n i c a l u n i t s o f s e c r e t i n ( G . I . H . R e s e a r c h L a b o r a t o r i e s , B a t c h N o . 1 7 1 5 2 ) . E x p e r i m e n t a l p e r i o d s were o f 10 m i n u t e d u r a t i o n and i n j e c t i o n s were o n l y g i v e n when t h e r e s p o n s e t o a p r e v i o u s s t i m u l u s had r e t u r n e d t o r e s t i n g v a l u e s . ( v i i ) A s s a y f o r B l o o d P r e s s u r e e f f e c t s o f M o t i l i n u s i n g A c u t e R a b b i t p r e p a r a t i o n s The b l o o d p r e s s u r e o f a c u t e r a b b i t p r e p a r a t i o n s was m o n i t o r e d u s i n g a h i g h p r e s s u r e t r a n s d u c e r and a p o l y g r a p h . I n t r a v e n o u s i n j e c t i o n s o f m a t e r i a l s t o be a s s a y e d were made v i a t h e j u g u l a r c a n n u l a and b l o o d p r e s s u r e c h a n g e s were r e c o r d e d . The t r a n s d u c e r was c a l i b r a t e d t o 100 mm.Hg. u s i n g a p r e s s u r e b o t t l e . - 48 -F i g u r e 1 0 . R e c o r d s s h o w i n g f o r c e o f s h o r t e n i n g o f g u i n e a - p i g g a l l b l a d d e r i n r e s p o n s e t o i n t r a v e n o u s i n j e c t i o n s o f c h o I e c y s t o k i n i n ( C C K ) . R i g h t : P l o t o f peak s h o r t e n i n g f o r c e v e r s u s Ivy Dog U n i t s ( IDU) o f CCK p r e p a r e d f r o m t h e s e d a t a . - 4 9 -( v i i i ) A s s a y f o r P y r o g e n i c e f f e c t s o f M o t i l i n u s i n g C h r o n i c Dog p r e p a r a t i o n s The r e c t a l t e m p e r a t u r e o f c h r o n i c dog p r e p a r a t i o n s was m o n i t o r e d u s i n g a t e l e - t h e r m o m e t e r and r e c t a l p r o b e s y s t e m d e s c r i b e d f o r t h e a c u t e g u i n e a - p i g p r e p a r a t i o n . R e c t a l t e m p e r a t u r e was m e a s u r e d b e f o r e , d u r i n g and a f t e r i n f u s i o n s o f m o t i I i n . ANALYSIS OF DATA i n s t u d i e s i n v o l v i n g i n f u s i o n o f m o t i l i n , v a l u e s f o r f u n d i c and a n t r a l m o t o r i n d i c e s , H + i o n o u t p u t and p e p s i n o u t p u t were e x p r e s s e d a s r a t i o s o f t h e mean o f t h e v a l u e s f o r t h r e e c o n s e c u t i v e c o n t r o l p e r i o d s o b t a i n e d j u s t p r i o r t o t h e i n f u s i o n . T h i s method was e m p l o y e d i n o r d e r t o e l i m i n a t e d i f f e r e n c e s i n a b s o l u t e maxima o b t a i n a b l e f r o m d i f f e r e n t a n i m a l s . R a t i o s were t r a n s f o r m e d i n t o l o g a r i t h m s i n o r d e r t o a p p l y t e s t s o f s t a t i s t i c a l s i g n i f i c a n c e . L o g a r i t h m s o f t h e r a t i o s f o r t h e t h i r d c o n t r o l p e r i o d were compared w i t h t h o s e f o r t h e p e r i o d o f maximum r e s p o n s e o c c u r i n g d u r i n g t h e i n f u s i o n u s i n g t h e STUDENT t - t e s t f o r p a i r e d d a t a . R a t i o s o f v a r i a n c e s ( F - t e s t ) were o b t a i n e d b e f o r e a p p l i c a t i o n o f t h e t - t e s t , a n d , i n t h o s e i n s t a n c e s where t h e F v a l u e s were g r e a t e r t h a n a l l o w a b l e , a m o d i f i e d t - t e s t w i t h r e d u c e d d e g r e e s o f f r e e d o m ( B a i l e y ( 1 9 5 9 ) ) was a p p l i e d . - 50 -RESULTS PURIF ICATION OF MOTILIN ( i ) S t a r t i n g M a t e r i a l The s t a r t i n g m a t e r i a l f o r t h i s s t u d y was a s i d e f r a c t i o n p r o d u c e d d u r i n g t h e p r e p a r a t i o n o f s e c r e t i n f r o m hog d u o d e n a l mucosa d e s c r i b e d by M u t t ( 1 9 5 9 ) . T h i s p r o c e d u r e was s l i g h t l y m o d i f i e d a s d e s c r i b e d by Brown e t a I ( 1 9 7 1 ) . T h i s s i d e f r a c t i o n was r e f e r r e d t o a s p r e s e c r e t i n and c o n t a i n e d 3 5 - 5 0 m o t i I i n u n i t s p e r mg. The f o l l o w i n g p r e p a r a t i o n p r o c e d u r e s a r e n o t n e c e s s a r i l y c o n s e c u t i v e t r e a t m e n t s o f a s i n g l e b a t c h o f m a t e r i a l . ( i i ) F r a c t i o n a t i o n o f P r e s e c r e t i n o n CM c e l l u l o s e In a t y p i c a l e x p e r i m e n t 507 mg. o f p r e s e c r e t i n were d i s s o l v e d i n 15 m l . o f 0 . 0 2 M ammonium b i c a r b o n a t e w h i c h was g a s s e d w i t h CO2 t o a pH o f 6 . 5 . The s t a r t i n g b u f f e r was m a i n t a i n e d a t t h i s pH by h o l d i n g i n an a t m o s p h e r e o f CO2. The s o l u t i o n was a p p l i e d t o a 2 . 5 x 10 c m . c o l u m n o f CM 22 (Whatman) w h i c h had been p r e v i o u s l y e q u i l i b r a t e d w i t h t h e s t a r t i n g b u f f e r , and was d e v e l o p e d w i t h t h e same b u f f e r . S t r o n g l y a d s o r b e d m a t e r i a l was e l u t e d by d e v e l o p i n g t h e c o l u m n w i t h 0 . 2 M ammonium b i c a r b o n a t e a t pH 8 . 0 . The e l u a t e was c o l l e c t e d i n 6 m l . f r a c t i o n s a t a f l o w r a t e o f 360 m l / h o u r . The a b s o r b a n c e o f t h e f r a c t i o n s a t 280 nm. was m e a s u r e d t h r o u g h a 1 . 0 c m . l i g h t p a t h and t h e r e s u l t s a r e shown i n f i g . l l . F r a c t i o n s 1 5 - 2 5 i n c l u s i v e were p o o l e d and l y o p h i l i z e d . T h i s p r o c e d u r e y i e l d e d 37 mg. o f l y o p h i l i z e d m a t e r i a l w h i c h was r e f e r r e d t o a s M l . On a s s a y , t h i s m a t e r i a l was f o u n d t o c o n t a i n a p p r o x i m a t e l y 2 , 0 0 0 m o t i l i n u n i t s p e r mg. - 51 -| N H 4 H C O 3 0 . 0 2 M p H 6 . 5 | 0 . 2 M pH8.0| 1 1 v 0 20 <W 60 Fraction Number F i g u r e I I . E l u t i o n p r o f i I e o f P r e s e c r e t i n on CM e e l I u l o s e . Co lumn s i z e , 2 . 5 x 10 c m . F l o w r a t e , 360 m l / h . F r a c t i o n s i z e , 6 m l . C o l u m n t e m p e r a t u r e , 22°C. F r a c t i o n s i n d i c a t e d by s h a d e d a r e a were p o o l e d and l y o p h i I i z e d . - 52 -( i i i ) F r a c t i o n a t i o n o f Ml on Sephadex G - 2 5 In a t y p i c a l e x p e r i m e n t 40 mg. o f Ml were d i s s o l v e d i n 3 . 0 m l . o f 0 . 2 M a c e t i c a c i d and a p p l i e d t o a 0 . 9 x 115 c m . c o l u m n o f Sephadex G - 2 5 f i n e . The c o l u m n was d e v e l o p e d w i t h t h e same b u f f e r and t h e e l u a t e c o l l e c t e d i n 2 . 0 m l . f r a c t i o n s a t a f l o w r a t e o f 18 m l / h o u r . The a b s o r b a n c e o f t h e f r a c t i o n s a t 280 nm. was m e a s u r e d t h r o u g h a 1 . 0 c m . l i g h t p a t h and t h e r e s u l t s a r e shown i n f i g . 1 2 . F r a c t i o n s 2 0 - 2 3 i n c l u s i v e were p o o l e d and l y o p h i l i z e d . T h i s p r o c e d u r e y i e l d e d 12 mg. o f l y o p h i l i z e d m a t e r i a l w h i c h was r e f e r r e d t o a s M 2 . On a s s a y , t h i s m a t e r i a l was f o u n d t o c o n t a i n a p p r o x i m a t e l y 4 , 0 0 0 m o t i l i n u n i t s p e r mg. ( i v ) F r a c t i o n a t i o n o f M2 on TEAE c e l l u l o s e In a t y p i c a l e x p e r i m e n t 13 mg. o f M2 were d i s s o l v e d i n 1 . 0 m l . o f 0 . 0 I M ammonium b i c a r b o n a t e and t h e pH a d j u s t e d t o 8 . 1 w i t h t h e a d d i t i o n o f 0 . 0 I M ammonium s o l u t i o n . The s o l u t i o n was a p p l i e d t o a 0 . 9 x 5 . 0 c m . c o l u m n o f TEAE c e l l u l o s e ( S e r v a , H e i d e l b e r g ) w h i c h had been p r e v i o u s l y e q u i l i b r a t e d w i t h 0 . 0 I M ammonium b i c a r b o n a t e pH 7 . 8 and was d e v e l o p e d w i t h t h e same b u f f e r . S t r o n g l y a d s o r b e d m a t e r i a l was e l u t e d by d e v e l o p i n g t h e c o l u m n w i t h 0 . 2 M ammonium b i c a r b o n a t e pH 8 . 0 . The e l u a t e was c o l l e c t e d i n 2 . 0 m l . f r a c t i o n s a t a f l o w r a t e o f 60 m l / h o u r . The a b s o r b a n c e o f t h e f r a c t i o n s a t 280 nm. was m e a s u r e d t h r o u g h a 1 . 0 c m . l i g h t p a t h and t h e r e s u l t s a r e shown i n f i g . 1 3 . F r a c t i o n s 3 - 7 i n c l u s i v e were p o o l e d and l y o p h i l i z e d . T h i s p r o c e d u r e y i e l d e d 9 . 2 mg. o f l y o p h i l i z e d m a t e r i a l w h i c h was r e f e r r e d t o a s M 3 . On a s s a y t h i s m a t e r i a l was f o u n d t o c o n t a i n a p p r o x i m a t e l y 8 , 0 0 0 m o t i l i n u n i t s p e r mg. - 53 -1.5 i Fraction Number F i g u r e 1 2 . E l u t i o n p r o f i l e o f Ml on Sephadex G - 2 5 . Co lumn s i z e , 0 . 9 x 115 c m . B u f f e r , 0 . 2 M a c e t i c a c i d . F l o w r a t e , 18 m l / h . F r a c t i o n s i z e , 2 . 0 m l . Co lumn t e m p e r a t u r e , 22°C. F r a c t i o n s i n d i c a t e d by shaded a r e a were p o o l e d and l y o p h i l i z e d . - 54 -F i g u r e 1 3 . E l u t i o n p r o f i l e o f M2 on TEAE c e l l u l o s e . Co lumn s i z e , 0 . 9 x 5 . 0 c m . F l o w r a t e , 60 m l / h . F r a c t i o n s i z e , 2 . 0 m l . C o l u m n t e m p e r a t u r e , 22°C . F r a c t i o n s i n d i c a t e d by shaded a r e a were p o o l e d and I y o p h i I i z e d . - 55 -( v ) F r a c t i o n a t i o n o f M3 on Sephadex G - 2 5 In a t y p i c a l e x p e r i m e n t 6 . 8 mg. o f M3 were d i s s o l v e d i n 2 . 0 m l . o f 0 . 2 M ammonium a c e t a t e and a p p l i e d t o a 0 . 9 x 118 c m . Sephadex G - 2 5 f i n e c o l u m n . The c o l u m n was d e v e l o p e d w i t h 0 . 2 M a c e t i c a c i d and t h e e l u a t e c o l l e c t e d i n 1 . 5 m l . f r a c t i o n s a t a f l o w r a t e o f 18 m l / h o u r . The a b s o r b a n c e o f t h e f r a c t i o n s a t 280 nm. was measured t h r o u g h a 1 . 0 c m . l i g h t p a t h and t h e r e s u l t s a r e shown i n f i g . 1 4 . F r a c t i o n s . 2 1 - 2 3 i n c l u s i v e were p o o l e d and l y o p h i l i z e d . T h i s p r o c e d u r e y i e l d e d a m a t e r i a l r e f e r r e d t o a s M4. W e i g h i n g s and a s s a y s ( o t h e r t h a n f o r l o c a t i o n o f a c t i v i t y ) were n o t p e r f o r m e d a t t h i s s t a g e . ( v i ) D e s a l t i n g o f M4 on Sephadex G - 2 5 In a t y p i c a l e x p e r i m e n t , t h e p o o l e d l y o p h i l i z e d p r o d u c t f r o m t h e g e l f i l t r a t i o n o f 6 . 8 mg. o f M3 on Sephadex G - 2 5 was d i s s o l v e d i n 2 . 0 m l . o f 0 . 2 M a c e t i c a c i d and a p p l i e d t o a 0 . 9 x 118 c m . c o l u m n o f Sephadex G - 2 5 f i n e . The c o l u m n was d e v e l o p e d w i t h 0 . 2 M a c e t i c a c i d and t h e e l u a t e c o l l e c t e d i n 2 . 0 m l . f r a c t i o n s a t a f l o w r a t e o f 18 m l / h o u r . The a b s o r b a n c e o f t h e f r a c t i o n s a t 230 nm. was measured t h r o u g h a 1 . 0 c m . l i g h t p a t h and t h e r e s u l t s a r e shown i n f i g . I 5 . F r a c t i o n s 2 2 - 2 5 i n c l u s i v e were l y o p h i l i z e d i n d i v i d u a l l y . T h i s p r o c e d u r e y i e l d e d 2 . 2 mg. o f l y o p h i l i z e d m a t e r i a l w h i c h was r e f e r r e d t o a s M5. On a s s a y t h i s m a t e r i a l was f o u n d t o c o n t a i n g r e a t e r t h a n 1 0 , 0 0 0 m o t i l i n u n i t s p e r mg. ( v i i ) C h r o m a t o g r a p h y o f M3 on SE Sephadex C - 2 5 In t h i s e x p e r i m e n t 8 . 0 mg. o f M3 were d i s s o l v e d i n 2 . 0 m l . o f 0 . 0 I M ammonium a c e t a t e pH 6 . 5 and a p p l i e d t o a 0 . 9 x 8 . 0 c m . c o l u m n o f SE Sephadex C - 2 5 w h i c h had been p r e v i o u s l y e q u i l i b r a t e d w i t h t h e same b u f f e r . The - 56 -0.3-. Fraction Number F i g u r e 14. E l u t i o n p r o f i l e o f M3 d i s s o l v e d i n 2 . 0 m l . ammonium a c e t a t e on Sephadex G - 2 5 . Co lumn s i z e , 0 . 9 x 118 cm. B u f f e r , 0 . 2 M. a c e t i c a c i d . F l o w r a t e , 18 m l / h . F r a c t i o n s i z e , 1 .5 m l . Co lumn t e m p e r a t u r e , 22°C. F r a c t i o n s i n d i c a t e d by shaded a r e a were p o o l e d and I y o p h i I i z e d . • - 57 -T 16 24 Fraction Number ti e < x CO c *= — c o r-> 10,000 >- 0 F i g u r e 15 . E l u t i o n p r o f i l e o f M4 on Sephadex G - 2 5 . Co lumn s i z e , 0 . 9 x 118 c m . B u f f e r , 0 . 2 M. a c e t i c a c i d . F l o w r a t e , 18 m l / h . F r a c t i o n s i z e , 2 . 0 m l . Co lumn t e m p e r a t u r e 22°C. F r a c t i o n s i n d i c a t e d by a r r o w s were l y o p h i l i z e d i n d i v i d u a l l y and a s s a y e d f o r b i o l o g i c a l a c t i v i t y . - 58 -c o l u m n was d e v e l o p e d w i t h t h e s t a r t i n g b u f f e r and a s t e p w i s e g r a d i e n t o f t h i s b u f f e r i n i n c r e m e n t s o f 0 . 0 5 M . The e l u a t e was m o n i t o r e d f o r a b s o r b a n c e a t 2 8 0 nm. t h r o u g h a 1 . 0 c m . l i g h t p a t h and i n c r e m e n t s were made i n i o n i c s t r e n g t h when f o u r c o l u m n v o l u m e s had been c o l l e c t e d . The f i n a l i n c r e m e n t , t o 0 . 2 M (pH 6 . 5 ) , f a i l e d t o e l u t e any UV (280 nm. ) a b s o r b i n g e l u a t e and 0 . 2 M ammonium a c e t a t e pH 8 . 0 was a l l o w e d t o f l o w t h r o u g h t h e c o l u m n . T h i s r e s u l t e d i n t h e e l u t i o n o f a s i n g l e p e a k . The peak f r a c t i o n s were l y o p h i l i z e d , d i s s o l v e d i n 2 . 0 m l . o f 0 . 2 M a c e t i c a c i d and a p p l i e d t o a 0 . 9 x 118 c m . c o l u m n o f Sephadex G - 2 5 f i n e w h i c h was d e v e l o p e d w i t h t h e same b u f f e r . The e l u t i o n p r o f i l e r e s u l t i n g f r o m t h i s p r o c e d u r e was e x a c t l y s i m i l a r t o t h a t shown i n f i g . 1 4 . A summary o f t h e p u r i f i c a t i o n o f m o t i l i n i s shown i n t a b l e I. DETERMINATION OF THE AMINO ACID COMPOSITION OF MOTILIN Amino a c i d a n a l y s e s were p e r f o r m e d o n t h e f r a c t i o n s i n d i c a t e d by t h e a r r o w s i n f i g . 1 5 . The r e s u l t s , e x p r e s s e d a s m o l e r a t i o s o f e a c h a m i n o a c i d t o g e t h e r w i t h t h e assumed m o l e i n t e g e r , a r e shown i n t a b l e I I . The a b s e n c e o f t r y t o p h a n was d e m o n s t r a t e d u s i n g t h e method o f G a i t o n d e and Dovey ( 1 9 7 0 ) a s w e l l a s by a n e g a t i v e r e s u l t w i t h t h e E r l i c h ' s r e a g e n t . The a b s e n c e o f h i s t i d i n e was c o n f i r m e d u s i n g t h e P a u l y r e a g e n t . THIN LAYER CHROMATOGRAPHY AND QUALITATIVE POLYACRYLAMIDE GEL  ELECTROPHORESIS OF MOTILIN T h i n l a y e r c h r o m a t o g r a p h y o f M o t i l i n (M5) o n s i l i c a g e l u s i n g t h e s o l v e n t s y s t e m B u t a n o l : A c e t i c a c i d : P y r i d i n e : W a t e r , 3 0 : 6 : 2 0 : 2 4 (v/v/v/v) y i e l d e d a s i n g l e n i n h y d r i n p o s i t i v e , p h e n a n t h r e n e q u i n o n e p o s i t i v e s p o t . P o I y a c r y I a m i d e g e l e l e c t r o p h o r e s i s o f m o t i l i n u s i n g 0 . 0 I M a c e t i c a c i d and s u b s e q u e n t s t a i n i n g w i t h a m i d o s c h w a r t z r e s u l t e d i n t h e p r o d u c t i o n - 59 -TABLE I SUMMARY OF STAGES OF PURIFICATION OF MOTILIN $ y i e l d a t e a c h s t a g e $ y i e l d f rom P r e s e c r e t i n M o t i I i n u n i t s / m g . * PRESECRETIN vie Ml v M2 M3 M4 M5 Ion e x c h a n g e c h r o m a t o g r a p h y on CM e e l I u I o s e pH 6 . 5 Ge l f i I t r a t i o n on Sephadex G - 2 5 Ion e x c h a n g e c h r o m o t a g r a p h y on TEAE e e l I u l o s e pH 7 . 8 Ge l f i I t r a t i o n on Sephadex G - 2 5 . added ( S a l t t o s a m p l e ) D e s a l t i n g on Sephadex G - 2 5 100$ 3 5 - 5 0 7 . 3 $ 3 0 . 2 $ 71$ 3 2 . 4 $ 7 . 3 $ 2000 2 . 2 $ 4 0 0 0 I . 7 $ 8 0 0 0 0 . 5 4 $ >10000 *These u n i t a g e s a r e a p p r o x i m a t e . - 60 -TABLE I I AMINO ACID ANALYSES AND COMPOSITIONS M o l e R a t i o s ( F i g u r e s i n b r a c k e t s a r e assumed m o l e s i n t e g e r s ) AMINO FRACTION* PROBABLE ACID 22 23 24 25 COMPOSITION A l a n i n e 0 0 0 0 0 A r g i n i n e 2 . 2 ( 2 ) 2 . 3 ( 2 ) 2 . 1 ( 2 ) 2 . 2 ( 2 ) 2 A s p a r t i c a c i d 1 . 0 ( 1 ) 1 . 1 ( 1 ) 1 .0 (1 ) 1 . 1 ( 1 ) 1 1/2 c y s t i n e 0 0 0 0 0 G l u t a m i c a c i d 5 . 8 ( 6 ) 6 . 3 ( 6 ) 5 . 9 ( 6 ) 5 . 7 ( 6 ) 6 G l y c i ne 1 . 9 ( 2 ) 2 . 1 ( 2 ) 2 . 2 ( 2 ) 2 . 3 ( 2 ) 2 H i s t i d i n e 0 0 0 0 0 1 s o l e u c i ne 0 . 9 ( 1 ) 1 .0 (1 ) 1 . 1 ( 1 ) 1 . 0 ( 1 ) 1 Leuc i ne 1 . 0 ( 1 ) 1 . 1 ( 1 ) 1 . 2 ( 1 ) 1 . 3 ( 1 ) 1 L y s i n e 2 . 0 ( 2 ) 2 . 1 ( 2 ) 2 . 2 ( 2 ) 2 . 2 ( 2 ) 2 Meth i o n i ne** 0 . 4 ( 1 ) 0 . 8 ( 1 ) 0 . 5 ( 1 ) 0 . 8 ( 1 ) 1 P h e n y l a l a n i n e 1 . 8 ( 2 ) 1 . 9 ( 2 ) 2 . 0 ( 2 ) 2 . 1 ( 2 ) 2 P r o 1i ne 1 . 1 ( 1 ) 1 . 3 ( 1 ) 1 . 3 ( 1 ) 1 .2 (1 ) 1 S e r i ne 0 0 0 0 0 T h r e o n i n e | 0 . 8 ( 1 ) 1 . 0 ( 1 ) 0 . 9 ( 1 ) 1 . 0 ( 1 ) 1 T r y p t o p h a n 0 0 0 0 0 T y r o s i n e 0 . 9 ( 1 ) 1 . 1 ( 1 ) 1 . 0 ( 1 ) 1 . 1 ( 1 ) 1 Va1 i ne § 1 . 0 ( 1 ) 1 . 0 ( 1 ) 1 . 0 ( 1 ) 1 . 0 ( 1 ) 1 T o t a l number o f r e s i d u e s : 22 C a l c u l a t e d m o l e c u l a r w e i g h t : 2700 * I n d i c a t e d by a r r o w s i n F i g . 1 5 + Mean o f 4 a n a l y s e s ** M e t h i o n i n e s u l p h o x i d e s and s u l p h o n e p r e s e n t ++ D e t e r m i n e d by t h e method o f G a i t o n d e and Dovey ( 1 9 7 0 ) § A s s i g n e d m o l e i n t e g e r o f 1 . 0 - 61 -o f a s i n g l e b a n d . The s t a i n e d g e l i s shown i n f i g . 1 6 . DETERMINATION OF N-TERMINAL RESIDUE D a n s y l a t i o n o f m o t i l i n and s u b s e q u e n t h y d r o l y s i s and p o l y a m i d e l a y e r c h r o m a t o g r a p h y r e v e a l e d t h a t t h e N - t e r m i n a l r e s i d u e was p h e n y l a l a n i n e . T h i s p r o c e d u r e a l s o r e v e a l e d t h e p r e s e n c e o f l y s i n e and t y r o s i n e i n t h e m o l e c u l e . DETERMINATION OF C -TERMINAL RESIDUE The C - t e r m i n a l r e s i d u e o f m o t i l i n was d e t e r m i n e d u s i n g t h e c a r b o x y p e p t i d a s e t e c h n i q u e . M o t i l i n (100 n M . ) was d i s s o l v e d i n 360 p l . o f w a t e r and 20 p | . o f 2 . 0 M ammonium b i c a r b o n a t e a d d e d . To t h i s s o l u t i o n 20 p | . o f c a r b o x y p e p t i d a s e A (10 m g / m l . i n 2 . 0 M ammonium b i c a r b o n a t e ) were added and t h e r e a c t i o n a l l o w e d t o p r o c e e d a t 22°C. A l i q u o t s o f 4 0 p | . w e r e removed a t 2 , 4 , 6 and 12 h o u r i n t e r v a l s and were s u b j e c t e d t o a m i n o a c i d a n a l y s i s w i t h no f u r t h e r t r e a t m e n t . A f t e r c o r r e c t i n g f o r t h e d e c r e m e n t i n v o l u m e a f t e r e a c h s a m p l i n g , t h e r e s u l t s were e x p r e s s e d a s m o l e s o f a m i n o a c i d r e l e a s e d p e r m o l e o f p o l y p e p t i d e and a r e shown i n f i g . 1 7 . G l u t a m i n e , t h e a m i n o a c i d w i t h t h e g r e a t e s t r a t e o f r e l e a s e , was r e a d a s e l u t e d ( p r i o r t o g l u t a m i c a c i d ) o n t h e a m i n o a c i d a n a l y s e r u s i n g a c o n s t a n t o b t a i n e d by s t a n d a r d r u n s w i t h known a m o u n t s o f g l u t a m i n e . OXIDATION OF MOTILIN WITH PERFORMIC ACID M o t i l i n was o x i d i s e d w i t h p e r f o r m i c a c i d i n o r d e r t o d e t e r m i n e t h e m e t h i o n i n e r e s i d u e s . M o t i l i n ( 0 . 5 m g . ) was d i s s o l v e d i n 1 . 0 m l . o f p e r -f o r m i c a c i d r e a g e n t and t h e r e a c t i o n a l l o w e d t o p r o c e e d f o r 6 h o u r s a t 0 ° C . The r e a c t i o n m i x t u r e was l y o p h i l i z e d and a m i n o a c i d a n a l y s e s p e r f o r m e d o n - 62 -F i g u r e 16 . S t a i n e d p o l y a c r y l a m i d e g e l s h o w i n g m o t i I i n r u n n i n g a s s i n g l e b a n d . Anode u p p e r m o s t . - 6 3 -0 2 4 6 8 10 12 Digestion Time in Hours F i g u r e 17 . P l o t s h o w i n g r a t e o f r e l e a s e o f C - t e r m i n a l a m i n o a c i d r e s i d u e s o f m o t i l i n w i t h c a r b o x y p e p t i d a s e A d i g e s t i o n . - 64 -h y d r o l y s e d a l i q u o t s . M e t h i o n i n e s u l p h o n e e l u t e d f r o m t h e a m i n o a c i d a n a l y s e r p r i o r t o t h r e o n i n e and was d e t e r m i n e d u s i n g a c o n s t a n t o b t a i n e d by s t a n d a r d r u n s w i t h known a m o u n t s o f m e t h i o n i n e s u l p h o n e . T h i s p r o c e d u r e r e s u l t e d i n t h e d e t e r m i n a t i o n o f a m o l e r a t i o o f 0 . 7 5 and a m o l e i n t e g e r o f I f o r m e t h i o n i n e s u l p h o n e . CYANOGEN BROMIDE CLEAVAGE OF MOTILIN MOTILIN ( 1 . 0 m g . ) was c l e a v e d w i t h c y a n o g e n b r o m i d e and an a l i q u o t o f t h e l y o p h i l i z e d r e a c t i o n m i x t u r e c o n t a i n i n g a p p r o x i m a t e l y 50 nM. was s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 . S u b s e q u e n t s t a i n i n g o f t h e c h r o m a t o g r a m w i t h n i n h y d r i n r e v e a l e d two m a j o r p e p t i d e s and a m i n o r amount o f s t a i n e d m a t e r i a l be tween t h e m . The s t a i n e d c h r o m a t o g r a m i s shown i n f i g . 1 8 . The r e m a i n d e r o f t h e r e a c t i o n m i x t u r e was s u b j e c t e d t o p r e p a r a t i v e h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 and t h e t h r e e p e p t i d e z o n e s e l u t e d and l y o p h i l i z e d . Amino a c i d a n a l y s e s and N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n s w e r e p e r f o r m e d o n t h e p e p t i d e s and t h e r e s u l t s a r e shown i n t a b l e I I I . The m i n o r s t a i n i n g p e p t i d e had a n i d e n t i c a l a m i n o a c i d c o m p o s i t i o n t o t h a t o f t h e w h o l e m o l e c u l e and was assumed t o be u n c l e a v e d m a t e r i a l . The two m a j o r p e p t i d e s w e r e r e f e r r e d t o a s CNI and CN2 and were s u b j e c t e d t o Edman d e g r a d a t i o n . The s e q u e n c e s o f t h e s e p e p t i d e s a r e shown i n f i g . 1 9 . N . B . M o b i l i t i e s o f a l l e l e c t r o p h o r e t i c a I Iy p r e p a r e d p e p t i d e s a r e shown i n A p p e n d i x (A). CHYMOTRYPTIC CLEAVAGE OF MOTILIN M o t i l i n ( 1 . 0 m g . ) was c l e a v e d w i t h c h y m o t r y p s i n and a n a l i q u o t o f t h e r e a c t i o n m i x t u r e c o n t a i n i n g a p p r o x i m a t e l y 50 nM. was s u b j e c t e d t o h i g h 65 -F i g u r e 18 . H i g h v o l t a g e e l e c t r o p h o r e t o g r a m o f c y a n o g e n b r o m i d e c I e a v e d m o t i I i n a t pH 1 . 9 . I. M a r k e r s . 2. S t a n d a r d R. 3. CNBr p e p t i d e s . 4 . S t a n d a r d T . - 66 -TABLE I I I AMINO ACID COMPOSITIONS OF THE TWO CNBr FRAGMENTS MOLE RATIOS PEPTIDE AMINO ACID ARGININE ASPARTIC ACID GLUTAMIC ACID GLYCINE ISOLEUCINE LEUCINE LYSINE PHENYLALANINE PROLINE THREONINE TYROSINE VALINE CNI CN2 I . 0 ( 1 ) 2 . 5 ( 2 ) * I . 3 ( 1 ) I . 3 ( 1 ) I . 2 ( 1 ) I . 7 ( 2 ) + I . 0 ( 1 ) I . 0 ( 1 ) 0 . 9 ( 1 ) I . 0 ( 1 ) I . 0 ( 1 ) 0 . 9 ( 1 ) 3 . 8 ( 4 ) + I . 0 ( 1 ) 2 . 0 ( 2 ) F i g u r e s i n b r a c k e t s a r e assumed m o l e i n t e g e r s . * H i g h g l u t a m i c a c i d v a l u e s due t o h o m o s e r i n e e l u t i n g w i t h g l u t a m i c a c i d on a m i n o a c i d a n a l y s e r . + N - t e r m i n a l r e s i d u e f o r t h e p e p t i d e . - 67 -C N I P h e - V a l - P r o - l i e - P h e - T h r - T y r - G ly - G l x - L e u - Glx - A r g - Met C N 2 G l x - G l x - L y s - G l x - A r g - A s x - L y s - G l y - G i n g u r e 19. Amino a c i d s e q u e n c e s o f t h e two c y a n o g e n b r o m i d e f r a g m e n t s o f m o t i I i n . - 68 -v o l t a g e e l e c t r o p h o r e s i s a t pH 6 . 5 . S u b s e q u e n t s t a i n i n g o f t h e c h r o m a t o -gram w i t h n i n h y d r i n r e v e a l e d 4 p e p t i d e s , 2 o f w h i c h were j u d g e d t o be m a j o r and 2 m i n o r . The r e m a i n d e r o f t h e r e a c t i o n m i x t u r e was s u b j e c t e d t o p r e p a r a t i v e h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 6 . 5 and t h e p e p t i d e z o n e s were e l u t e d and l y o p h i l i z e d . The s t a i n e d c h r o m a t o g r a m i s shown i n f i g . 2 0 . Amino a c i d a n a l y s e s and N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n s were p e r f o r m e d o n t h e p e p t i d e s and t h e r e s u l t s a r e shown i n t a b l e IV. D a n s y l a t i o n d e m o n s t r a t e d t h a t CHT2 was a m i x t u r e o f p e p t i d e s and a p p r o x i m a t e l y 50 nM. o f t h i s p e p t i d e were s u b j e c t e d t o d e s c e n d i n g p a p e r c h r o m a t o g r a p h y i n t h e s y s t e m o f WhaIey and Watson ( 1 9 5 4 ) . S u b s e q u e n t s t a i n i n g o f t h e c h r o m a t o g r a m w i t h n i n h y d r i n r e v e a l e d 3 p e p t i d e s , 2 o f w h i c h were j u d g e d t o be m a j o r c o m p o n e n t s and I m i n o r . The s t a i n e d c h r o m a t o -gram i s shown i n f i g . 2 1 . Amino a c i d a n a l y s e s and N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n s were p e r f o r m e d o n t h e two m a j o r p e p t i d e s CHT2WW2 and CHT2WW3. The r e s u l t s a r e i n c I u d e d i n t a b Ie I V . Edman d e g r a d a t i o n was p e r f o r m e d o n p e p t i d e s C H T I , CHT2WW2 and CHT2WW3. The s e q u e n c e s o f t h e s e p e p t i d e s a r e shown i n f i g . 2 2 . TRYPTIC CLEAVAGE OF MOTILIN M o t i I i n ( 2 . 0 m g . ) was c l e a v e d w i t h t r y p s i n and a n a I i q u o t o f t h e r e a c t i o n m i x t u r e c o n t a i n i n g a p p r o x i m a t e l y 50 nM. was s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 . S u b s e q u e n t s t a i n i n g o f t h e c h r o m a t o g r a m w i t h ninhydr '^n_ r e v e a I ed 6 p e p t i d e s . The s t a i n e d c h r o m a t o g r a m i s shown i n f i g . 2 3 . The r e m a i n d e r o f t h e r e a c t i o n m i x t u r e was s u b j e c t e d t o p r e p a r a t i v e h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 and t h e p e p t i d e z o n e s were e l u t e d and l y o p h i I i z e d . - 69 -F i g u r e 20. H i g h v o l t a g e e l e c t r o p h o r e t o g r a m o f c h y m o t r y p t i c d i g e s t o f m o t i l i n a t pH 6.5. 1. M a r k e r s . 2. S t a n d a r d R. 3. C h y m o t r y p t i c p e p t i d e s . 4. S t a n d a r d T. - 70 -TABLE IV AMINO ACID COMPOSITIONS AND N-TERMINAL RESIDUES OF SOME CHYMOTRYPTIC PEPTIDES OF MOTILIN PEPTIDE COMPOSITION N-TERMINAL RESIDUE CHTI G L U - 2 . 3 ( 2 ) G L Y - I . 0 ( 1 ) L E U - I . 0 ( 1 ) GLY CHT2WW2 T H R - I . 1 ( 1 ) T Y R - I . 0 ( 1 ) THR CHT2WW3 I L E - 0 . 9 C I ) P H E - I . 7 ( 2 ) P R O - I . I ( I ) V A L - I . 0 ( 1 ) PHE F i g u r e s i n b r a c k e t s a r e assumed m o l e i n t e g e r s . - 71 -F i g u r e 2 1 . D e s c e n d i n g c h r o m a t o g r a m o f c h y m o t r y p t i c p e p t i d e CHT2 on p a p e r . F o r s o l v e n t and c o n d i t i o n s s e e t e x t . OR, O r i g i n . Oil - 72 -CHTI CHT2WW2 CHT3WW3 Gly - Glx -Leu -Glx — ^ * -» * Thr -Tyr — T * Phe - Vol - Pro - he - Phe ^ ^ ^ ^ F i g u r e 2 2 . Amino a c i d s e q u e n c e s o f some c h y m o t r y p t i c p e p t i d e s o f m o t i I i n . - 73 -t : : t F i g u r e 2 3 . H i g h v o l t a g e e l e c t r o p h o r e t o g r a m o f t r y p t i c d i g e s t o f m o t i I i n a t pH 1 . 9 . 1 . M a r k e r s . 2 . S t a n d a r d R. 3 . T r y p t i c p e p t i d e s . 4 . S t a n d a r d T . 0 o 1 2 3 4 1 Amino a c i d a n a l y s e s and N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n s were p e r f o r m e d on t h e p e p t i d e s and t h e r e s u l t s a r e shown i n t a b l e V . Edman d e g r a d a t i o n s were p e r f o r m e d o n p e p t i d e s T R I , T R 2 , T R 3 , T R 4 , TR5 and T R 6 . The s e q u e n c e s o f t h e s e p e p t i d e s a r e shown i n f i g . 2 4 . M o t i l i n c y a n o g e n b r o m i d e f r a g m e n t CN2 (200 n M . ) was s u b j e c t e d t o t r y p t i c d i g e s t i o n , and q u a l i t a t i v e h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 o f an a l i q u o t r e v e a l e d 3 p e p t i d e s . The p e p t i d e s were p r e p a r e d e l e c t r o -p h o r e t i c a l l y and were s u b j e c t e d t o amino a c i d a n a l y s e s and N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n s . The r e s u l t s a r e shown i n t a b l e V I . Edman d e g r a d a t i o n s were p e r f o r m e d o n p e p t i d e s C N 2 T R I , CN2TR2 and CN2TR3. The s e q u e n c e s o f t h e s e p e p t i d e s a r e shown i n f i g . 2 5 . THERMOLYSIN CLEAVAGE OF MOTILIN M o t i l i n ( 2 . 5 m g . ) was c l e a v e d w i t h t h e r m o l y s i n and a n a l i q u o t o f t h e r e a c t i o n m i x t u r e c o n t a i n i n g a q p r o x i m a t e l y 50 nM. was s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 . S u b s e q u e n t s t a i n i n g o f t h e c h r o m a t o g r a m w i t h n i n h y d r i n r e v e a l e d 7 p e p t i d e s , 3 o f w h i c h were j u d g e d t o be m a j o r c o m p o n e n t s and 4 m i n o r . The s t a i n e d c h r o m a t o g r a m i s shown i n f i g . 2 6 . The r e m a i n d e r o f t h e r e a c t i o n m i x t u r e was s u b j e c t e d t o p r e p a r a t i v e h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 and t h e p e p t i d e z o n e s were e l u t e d and Iyoph i I i z e d . Amino a c i d a n a l y s e s and N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n s were p e r f o r m e d on t h e 3 m a j o r p e p t i d e s and t h e r e s u l t s a r e shown i n t a b l e V I I . Edma-R-degradations were p e r f o r m e d on p e p t i d e s T h 2 , Th4 and T h 7 . The s e q u e n c e s o f t h e s e p e p t i d e s a r e shown i n f i g . 2 7 . - 75 -TABLE V AMINO ACID COMPOSITIONS AND N-TERMINAL RESIDUES OF THE TRYPTIC PEPTIDES OF MOTILIN PEPTIDE TRI COMPOSITION A R G - I . 2 ( 1 ) G L U - 2 . I ( 2 ) G L Y - I . 0 ( 1 ) I L E - I . 2 ( 1 ) L E U - 1 . 1 ( 1 ) P H E - I . 6 ( 2 ) P R 0 - 0 . 7 U ) T H R - 0 . 9 U ) T Y R - 0 . 8 U ) V A L - I . 0 ( 1 ) N-TERMINAL RESIDUE PHE TR2 G L U - f . 0 ( 1 ) G L Y - I . O ( I ) GLY TR3 G L U - I . 9 ( 2 ) M E T - 0 . 8 ( I ) L Y S - I . 0 ( 1 ) MET TR4 A S P - I . O ( I ) G L Y - 0 . 9 U ) GLU- I.2 (1 ) L Y S - I . 0 ( 1 ) ASX TR5 G L U - I . 0 ( 1 ) A R G - I . O ( I ) GLU TR6 A S P - I . O ( I ) L Y S - I . 0 ( 1 ) ASX F i g u r e s i n b r a c k e t s a r e assumed m o l e i n t e g e r s . - 76 -T R I Phe - Val - Pro - lie - Phe - Thr - Tyr - Gly - Glx - L e u - G l x - Arg , — . y »- T ^ 3^  ^ ^ 7 TR 2 Gly - G l x — TR 3 Met - G l x - G l x - Lys TR 4 Asx - L y s -G l y - G l x TR 5 Glx - A r g T R 6 Asx - L y s F i g u r e 2 4 . Amino a c i d s e q u e n c e s o f t h e t r y p t i c p e p t i d e s o f m o t i l i n . - 77 -TABLE VI AMINO ACID COMPOSITIONS AND N-TERMINAL RESIDUES OF TRYPTIC PEPTIDES OF PEPTIDE CN2 PEPTIDE COMPOSITION N-TERMINAL RESIDUE CN2TRI A S P - 0 . 9 U ) ASX GLY -1 . 1 ( 1 ) G L U - I . 0 ( 1 ) L Y S - 0 . 8 U ) CN2TR2 G L U - 2 . 0 ( 2 ) GLX L Y S - I . 0 ( 1 ) CN2TR3 A R G - I . O ( I ) GLU G L U - I . 0 ( 1 ) F i g u r e s i n b r a c k e t s a r e assumed m o l e i n t e g e r s i - 78 -CN2TRI C N 2 T R 2 C N 2 T R 3 Asx - Lys - Gly - Gin T T S - J » Glx - Glx - Lys —T —^ —•—^ Glx - Arg — ^ — ^ F i g u r e 2 5 . Amino a c i d s e q u e n c e s o f t h e t r y p t i c p e p t i d e s o f m o t i l i n c y a n o g e n b r o m i d e p e p t i d e C N 2 . - 79 -F i g u r e 2 6 . H i g h v o l t a g e e l e c t r o p h o r e t o g r a m o f t h e r m o l y s i n d i g e s t o f m o t i I i n a t pH 1 . 9 . 1 , M a r k e r s . 2 , S t a n d a r d R, 3 , T h e r m o l y s i n p e p t i d e s . 4 , S t a n d a r d T . 4. • * * r -* w 1 t 3 4 1 - 80 -TABLE V I I AMINO ACID COMPOSITIONS AND N-TERMINAL RESIDUES OF SOME THERMOLYSIN PEPTIDES OF MOTILIN PEPTIDE COMPOSITION N-TERMINAL RESIDUES Th2 I L E - I . 0 ( 1 > PHE P H E - 2 . K 2 ) P R O - I . O ( I ) T H R - I . 1 ( 1 ) V A L - I . 0 ( 1 ) Th4 G L U - I . 0 ( 1 ) TYR G L Y - I . 0 ( 1 ) T Y R - 0 . 9 U ) Th7 A R G - 2 . 0 ( 2 ) LEU A S P - I . 3 ( 1 ) G L Y - 1 . 2 ( 1 ) G L U - 4 . 7 ( 5 ) L E U - 0 . 8 ( I ) L Y S - 2 . 0 ( 2 ) M E T - 0 . 6 ( I ) F i g u r e s i n b r a c k e t s a r e assumed m o l e i n t e g e r s - 81 -T H 2 P h e - V a l - Pro - lie - Phe - Thr T H 4 T y r - G l y - G l x T H 7 Leu - Glx - A r g - Met - Glx - G l x - L y s - Glx - Arg - Asx - L y s - G l y - Gin F i g u r e 2 7 . Amino a c i d s e q u e n c e s o f some t h e r m o l y s i n p e p t i d e s o f m o t i I i n . - 82 -AMIDE DETERMINATIONS A m i d a t e d g l u t a m i c o r a s p a r t i c a c i d r e s i d u e s were d e t e r m i n e d by t h e e l e c t r o p h o r e t i c m o b i l i t i e s o f t h e 6 t r y p t i c p e p t i d e s a t pH 6 . 5 . P e p t i d e s T R 2 , T R 3 , T R 4 , TR5 and TR6 were s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 6 . 5 i n t a c t . P e p t i d e TRI was s u b j e c t e d t o Edman d e g r a d a t i o n u n t i l a g l u t a m i c a c i d o r g l u t a m i n e r e s i d u e became N - t e r m i n a l , i . e . a f t e r 8 and 10 c y c l e s o f d e g r a d a t i o n , and t h e n s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 6 . 5 . The e l e c t r o p h o r e t i c m o b i l i t i e s a l l o w e d t h e c h a r g e s on t h e p e p t i d e s t o be c a l c u l a t e d and t h i s d a t a , t o g e t h e r w i t h a s s i g n m e n t o f f r e e a c i d o r a m i d e s t a t u s i s shown i n t a b l e V I I I . CHEMICAL MODIFICATIONS OF MOTILIN ( i ) O x i d a t i o n o f M o t i l i n w i t h P e r f o r m i c A c i d M o t i l i n (200 n M . ) was o x i d i s e d w i t h p e r f o r m i c a c i d and a n a l i q u o t o f t h e l y o p h i l i z e d p r o d u c t s u b j e c t e d t o a m i n o a c i d a n a l y s e s . The a n a l y s e s showed t h a t t h e m e t h i o n i n e r e s i d u e had been o x i d i s e d t o m e t h i o n i n e s u l p h o n e . The o x i d i s e d m a t e r i a l was a s s a y e d i n t h e dog f o r m o t i l i n a c t i v i t y and was f o u n d t o p o s s e s s a p p r o x i m a t e l y 500 m o t i l i n u n i t s p e r mg. ( i i ) O x i d a t i o n o f M o t i l i n w i t h Hydrogen P e r o x i d e M o t i l i n (200 n M . ) was o x i d i s e d w i t h h y d r o g e n p e r o x i d e and an a l i q u o t o f t h e l y o p h i l i z e d p r o d u c t a s s a y e d i n t h e dog f o r m o t i l i n a c t i v i t y . The o x i d i s e d m a t e r ia I was f o u n d t o p o s s e s s a p p r o x i m a t e l y 500 m o t i l i n u n i t s p e r mg. ( i i i ) R e d u c t i o n o f H y d r o g e n P e r o x i d e o x i d i s e d M o t i l i n w i t h C y s t e i n e H y d r o g e n p e r o x i d e o x i d i s e d m o t i l i n (150 nM) was r e d u c e d w i t h c y s t e i n e h y d r o c h l o r i d e and an a l i q u o t o f t h e l y o p h i l i z e d p r o d u c t a s s a y e d i n t h e dog - 8 3 -TABLE V I I I AMIDES OF THE TRYPTIC PEPTIDES OF MOTILIN PEPTIDE NET CHARGE* ASSIGNED SEQUENCE WITH AMIDES TR2 NEUTRAL G L Y - G L N TR3 NEGAT IVE ( I ) M E T - G L U - G L U - L Y S TR4 P O S I T I V E ( I ) A S N - L Y S - G L Y - G L N TR5 NEUTRAL GLU-ARG TR6 P O S I T I V E ( I ) A S N - L Y S TRI (Edman s t a g e 8 ) NEUTRAL 10 I TRI (Edman s t a g e 10) P O S I T I V E ( I ) G L U - L E U - G L N - A R G * D e t e r m i n e d by method o f O f f o r d ( 1 9 6 6 ) - 84 -f o r m o t i l i n a c t i v i t y . The r e d u c e d m a t e r i a l was f o u n d t o p o s s e s s g r e a t e r t h a n 10,000 m o t i l i n u n i t s p e r mg. C y s t e i n e h y d r o c h l o r i d e , when a s s a y e d i n t h e d o g , p o s s e s s e d no m o t o r s t i m u l a t i n g a c t i v i t y . ( i v ) C l e a v a g e o f M o t i l i n w i t h T r y p s i n and C y a n o g e n B r o m i d e A l i q u o t s o f t r y p t i c and c y a n o g e n b r o m i d e c l e a v e d m o t i l i n were a s s a y e d i n t h e dog f o r m o t i l i n a c t i v i t y b e f o r e s e p a r a t i o n p r o c e d u r e s were u n d e r t a k e n . No m o t o r s t i m u l a t i n g a c t i v i t y was d e t e c t e d when d o s e s o f 2,000 t i m e s t h o s e r e q u i r e d f o r ' f u i I a c t i v i t y w i t h i n t a c t m o t i l i n were g i v e n . ( v ) Removal o f C - t e r m i n a l R e s i d u e s w i t h C a r b o x y p e p t i d a s e A M o t i l i n (1.0 m g . ) was d i g e s t e d w i t h c a r b o x y p e p t i d a s e A and a I i q u o t s removed f r o m t h e r e a c t i o n m i x t u r e a t 2, 4, 6 and 12 h o u r i n t e r v a l s f o r a m i n o a c i d a n a l y s i s and a s s a y i n t h e d o g . The r a t e o f r e l e a s e o f t h e C - t e r m i n a l amino a c i d r e s i d u e s e x p r e s s e d a s p e r c e n t removed i s shown i n f i g . 2 8 . The m o t i l i t y i n d e x o f e a c h a l i q u o t a f t e r a s s a y i n t h e dog i s shown i n t h e same f i g u r e . T h e s e i n d i c e s r e p r e s e n t a c t i v i t i e s o f g r e a t e r t h a n 10,000 m o t i l i n u n i t s p e r mg. C a r b o x y p e p t i d a s e A , when a s s a y e d i n t h e dog i n d o s e s e q u i v a l e n t t o t h o s e c a l c u l a t e d t o be p r e s e n t i n t h e t e s t a I i q u o t s , p o s s e s s e d no m o t o r s t i m u l a t i n g a c t i v i t y . ( v i ) Removal o f N - t e r m i n a l r e s i d u e M o t i l i n (50 nM) was s u b j e c t e d t o o n e c y c l e o f Edman d e g r a d a t i o n and an a l i q u o t o f t h e p r o d u c t d a n s y l a t e d and s u b j e c t e d t o N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n . The r e a c t i o n p r o d u c t p o s s e s s e d an N - t e r m i n a l v a l i n e r e s i d u e - 8 5 -— 6 0 0 -O 2 0 0 -l 0 0 - i Time in Hours F i g u r e 2 8 . P l o t s s h o w i n g r a t e o f r e l e a s e o f C - t e r m i n a l a m i n o a c i d r e s i d u e s o f m o t i l i n w i t h c a r b o x y p e p t i d a s e A and f u n d i c pouch m o t i l i t y i n d i c e s measured f o r a l i q u o t s o f t h e d i g e s t i o n m i x t u r e i n t h e c h r o n i c dog p r e p a r a t i o n . - 8 6 -and showed no c o n t a m i n a t i o n w i t h p h e n y l a l a n i n e , t h e N - t e r m i n a l r e s i d u e o f i n t a c t mot i I i n . When a s s a y e d i n t h e dog t h e p r o d u c t was f o u n d t o p o s s e s s l e s s t h a n 1 , 0 0 0 m o t i l i n u n i t s p e r mg. ( v i i ) A c e t y l a t i o n o f M o t i l i n M o t i l i n ( 1 . 0 m g . ) was a c e t y l a t e d w i t h a c e t i c a n h y d r i d e and t h e r e a c t i o n m i x t u r e l y o p h i l i z e d . The p r o d u c t s were d i s s o l v e d i n I . 0 m l . o f 0 . 2 M a c e t i c a c i d and a p p l i e d t o a 0 . 7 x 106 c m . S e p h a d e x G - 1 5 f i n e c o l u m n w h i c h was d e v e l o p e d w i t h t h e same b u f f e r . F r a c t i o n s o f 1 . 2 m l . were c o l l e c t e d a t a f l o w r a t e o f 6 . 0 m l / h o u r and t h e a b s o r b a n c e o f t h e f r a c t i o n s a t 230 nm. and 280 nm. was m e a s u r e d t h r o u g h a 1 . 0 c m . l i g h t p a t h . F r a c t i o n s 1 1 - 1 5 i n c l u s i v e were p o o l e d and l y o p h i l i z e d . The r e s u l t s a r e shown i n f i g . 2 9 . The l y o p h i l i z e d p r o d u c t , when a s s a y e d i n t h e dog f o r m o t i l i n a c t i v i t y , was f o u n d t o p o s s e s s a p p r o x i m a t e l y 1 , 0 0 0 m o t i l i n u n i t s p e r mg. V e r i f i c a t i o n o f t h e a c y l a t i o n o f f r e e amino g r o u p s was d e m o n s t r a t e d by t r y p t i c d i g e s t i o n o f a n a l i q u o t o f t h e l y o p h i l i z e d p r o d u c t . An a l i q u o t o f t h e d i g e s t i o n m i x t u r e was s u b j e c t e d t o h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH 1 . 9 . S u b s e q u e n t s t a i n i n g o f t h e c h r o m a t o g r a m w i t h n i n h y d r i n r e v e a l e d 2 p e p t i d e s w h i c h w e r e r e f e r r e d t o a s ACTRI and ACTR2. T r e a t m e n t o f t h e c h r o m a t o g r a m w i t h Sakaguch j r e a g e n t r e v e a l e d a n i n h y d r i n n e g a t i v e , S a k a g u c h i p o s i t i v e p e p t i d e r u n n i n g b e h i n d A C T R I . The c h r o m a t o g r a m , w h i c h a l s o d e m o n s t r a t e s t h e t r y p t i c p e p t i d e s o f i n t a c t m o t i l i n , i s shown i n f i g . 3 0 . A f t e r p r e p a r a t i v e h i g h v o l t a g e e l e c t r o p h o r e s i s a t pH I . 9 o f a t r y p t i c d i g e s t o f a c e t y l a t e d m o t i I i n , p e p t i d e ACTR2 was e l u t e d , l y o p h i l i z e d and s u b j e c t e d t o a m i n o a c i d a n a l y s i s and N - t e r m i n a l r e s i d u e d e t e r m i n a t i o n . The r e s u l t s a r e shown i n t a b l e IX . - 87 -F i g u r e 2 9 . E l u t i o n p r o f i l e o f a c e t y l a t e d m o t i l i n on Sephadex G - 1 5 . Co lumn s i z e , 0 . 7 x 106 c m . B u f f e r , 0 . 2 M a c e t i c a c i d , F l o w r a t e , 6 . 0 m l / h . F r a c t i o n s i z e , 1 . 2 m l . C o l u m n t e m p e r a t u r e , 22°C. F r a c t i o n s b r a c k e t e d by a r r o w s were p o o l e d and l y o p h i l i z e d . - 88 -s w $ i s F i g u r e 3 0 . H i g h v o l t a g e e l e c t r o p h o r e t o g r a m o f t r y p t i c d i g e s t o f i n t a c t , a c e t y l a t e d and s u c c i n y l a t e d m o t i l i n a t pH I . 9 . I, M a r k e r s . 2 , S t a n d a r d R. 3 , T r y p t i c p e p t i d e s o f i n t a c t m o t i l i n . 4 , T r y p t i c p e p t i d e s o f a c e t y l a t e d m o t i l i n . 5 , T r y p t i c p e p t i d e s o f s u c c i n y l a t e d m o t i l i n . 6 , S t a n d a r d T . f 1 2 3 4 S 6 1 - 89 -TABLE IX AMINO ACID COMPOSITION AND N-TERMINAL RESIDUE PEPTIDE ACTR2 COMPOSITION N-TERMINAL RESIDUE A R G - I . O ( I ) MET G L U - 2 . 7 ( 3 ) L Y S - 0 . 9 ( I ) M E T - I . K I ) F i g u r e s i n b r a c k e t s a r e assumed m o l e i n t e g e r s . - 90 -( v i i i ) S u c c i n y l a t i o n o f M o t i I i n M o t i l i n ( 1 . 0 m g . ) was s u c c i n y l a t e d w i t h s u c c i n i c a n h y d r i d e and t h e r e a c t i o n m i x t u r e l y o p h i l i z e d . The p r o d u c t s were d i s s o l v e d i n I . 0 m l . o f 0 . 2 M a c e t i c a c i d and a p p l i e d t o a 0 . 7 x 106 c m . Sephadex G - 1 5 f i n e c o l u m n w h i c h was d e v e l o p e d w i t h t h e same b u f f e r . F r a c t i o n s o f 1 . 2 m l . were c o l l e c t e d a t a f l o w r a t e o f 6 . 0 m l / h o u r and t h e a b s o r b a n c e o f t h e f r a c t i o n s a t 230 nm. and 280 nm. was measured t h r o u g h a 1 . 0 c m . l i g h t p a t h . F r a c t i o n s 1 1 - 1 5 i n c l u s i v e were p o o l e d and l y o p h i l i z e d . The r e s u l t s a r e shown i n f i g . 3 1 . The l y o p h i l i z e d p r o d u c t , when a s s a y e d i n t h e dog f o r m o t i l i n a c t i -v i t y , was f o u n d t o p o s s e s s l e s s t h a n 500 m o t i l i n u n i t s p e r mg. V e r i f i c a t i o n o f t h e a c y l a t i o n o f f r e e a m i n o g r o u p s was d e m o n s t r a t e d i n a n e x a c t l y s i m i l a r manner t o t h a t f o r a c e t y l a t i o n . F i g u r e 30 i n c l u d e s t h e p e p t i d e s p r o d u c e d by t r y p t i c d i g e s t i o n o f s u c c i n y l a t e d m o t i l i n . EFFECTS OF INTRAVENOUS INFUSION OF MOTILIN AT TWO DOSE LEVELS ON  B ICKEL POUCH MOTOR A C T I V I T Y , H + ION SECRETION, P E P S I N SECRETION  AND ANTRAL MOTOR ACTIV ITY T h e s e s t u d i e s were c a r r i e d o u t u s i n g c h r o n i c dog p r e p a r a t i o n s a s d e s c r i b e d i n M e t h o d s , pages 38 and 4 2 . A minimum o f 3 c o n t r o l p e r i o d s was r u n a t t h e b e g i n n i n g o f e a c h e x p e r i m e n t . I n f u s i o n s o f e i t h e r 1 . 0 u g . / K g -h r . o r 2 . 0 u g . / K g - h r . o f m o t i l i n were g i v e n o v e r o n e h o u r f o l l o w i n g t h e c o n t r o l p e r i o d s . A l l e x p e r i m e n t s were p e r f o r m e d w i t h t h e Thomas c a n n u l a o p e n . ( i ) B i c k e l pouch M o t o r A c t i v i t y The e f f e c t o f i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 1 . 0 u g . / K g - h r . i s shown i n t a b l e X and f i g u r e 3 2 . T h i s d o s e o f m o t i I i n s i g n i f i c a n t l y - 91 -Fraction Number F i g u r e 3 1 . E l u t i o n p r o f i l e o f s u c c i n y l a t e d m o t i l i n on Sephadex G - 1 5 . Co lumn s i z e , 0 . 7 x 106 c m . B u f f e r , 0 . 2 M a c e t i c a c i d . F low r a t e , 6 . 0 m l / h . F r a c t i o n s i z e , 1 . 2 m l . C o l u m n t e m p e r a t u r e , 22°C. F r a c t i o n s b r a c k e t e d by a r r o w s were p o o l e d and l y o p h i l i z e d . TABLE X EFECT OF SINGLE INTRAVNOUS INFUSION OF MOTILIN AT 1.0 ig/Kg./hr. ON FUNDIC MOTILITY MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS") DOG EXPT. I + 2 3 4 5 6 7 8 9 10 CONTROL PERIODS INFUSION POST INFUSION P 27/7/71 36.0(1.92) 9.3(0.49) 10.8(0.57) 244.0(13.05) 199.0(10.64) 150.0(8.02) 118.0(6.31 ) 20.2(1.06) 0.0( - ) 0.0( - ) P 3/8/71 11.5(0.81 ) 17.4(1.23) 13.5(0.95) 252.0(17.83) 198.0(14.0) 128.0(9.05) 114.0(8.06) 35.5(2.51 ) 0.0( - ) 0.0( - ) P 9/7/71 107.1(0.68) 214.2(1.37) 146.2(0.94) 248.1( 1.59) 231.4( 1.48) 128.9(0.83) 187.2(1.20) 315.7(2.02) 218.3(1.40) 101.9(0.65) P 29/7/71 141.5(0.69) 320.0(1.56) 154.5(0.75) 323.4( 1.57) 175.4C 0.85) 153.0(0.74) 101.3(0.49) 38.1(0.18) 81.1(0.39) 136.4(0.66) L 12/7/71 79.3(1.02) 131.6(1.69) 22.9(0.29) 2.4( 0.03) 0.0( - ) 0.0( - ) 0.0( - ) 0.0( - ) 0.0( - ) 0.0( - ) L 16/7/71 2.7(0.25) 12.7(1.18) 16.9(1.57) 30.0( 2.78) 62.4( 5.79) 22.8(2.12) 4.3(0.39) 4.6(0.43) 0.0( - ) 0.0( - ) L 19/7/71 1.8(0.58) 2.6(0.84) 4.9(1.58) 48.1(15.51) 35.5(11.45) 3.2(1.03) 0.0( - ) 0.0( - ) 0.0( - ) 0.0( - ) A 2/11/71 49.2(1.31 ) 47.7(1.26) 16.2(0.43) 166.1 ( 4.41) 124.7( 3.31) 117.6(3.12) 103.3(2.74) 42.3(1.12) 12.9(0.34) 11.0(0.30) A 4/11/71 0.0( - ) 2.9(0.22) 36.2(2.77) 154.1(11.82) 75.7( 5.81) 56.8(4.36) 69.8(5.35) 41.2(3.16) 22.0(1.69) 8.1(0.62) X 0.81 1.16 1.0 7.62 5.93 3.25 2.73 1.17 0.42 0.23 + S.E. 0.19 0.18 0.26 2.30 1.69 1.0 1.03 0.38 0.22 0.10 • RATIOS OF MEAN OF THE 3 CONTROL PERIODS + AL  PERIODS ARE OF 15 MIN. DURATION - 93 -Motilin Ijug/kg/hr 0 2 4 6 8 10 15 Min. Periods F i g u r e 3 2 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g - h . o n f u n d i c pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m 9 e x p e r i m e n t s i n 3 dogs ( T a b l e X) - 94 -i n c r e a s e d t h e m o t o r a c t i v i t y o f t h e B i c k e l pouch (p = < . 0 5 ) . The e f f e c t o f i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 2 . 0 p g . / K g - h r . i s shown i n t a b l e XI and f i g u r e 3 3 . T h i s d o s e o f m o t i l i n s i g n i f i c a n t l y i n c r e a s e d t h e m o t o r a c t i v i t y o f t h e B i c k e l pouch (p = < . 0 0 1 ) . ( i i ) B i c k e l pouch H + i o n S e c r e t i o n . The e f f e c t o f i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 1 .0 p g . / K g - h r . i s shown i n t a b l e X I I and f i g u r e 3 4 . T h i s d o s e o f m o t i l i n d i d n o t s i g -n i f i c a n t l y a l t e r t h e H + i o n o u t p u t o f t h e pouch (p = > . 2 ) . The e f f e c t o f i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 2 . 0 p g . / K g - h r . i s shown i n t a b l e X I I I and f i g u r e 3 5 . T h i s d o s e o f m o t i l i n s i g n i f i c a n t l y i n c r e a s e d t h e H + i o n o u t p u t o f t h e pouch (p = < . 0 0 5 ) . ( i i i ) B i c k e l pouch P e p s i n S e c r e t i o n The e f f e c t o f i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 1 . 0 p g . / K g - h r . i s shown i n t a b l e X IV and f i g u r e 3 6 . T h i s d o s e o f m o t i l i n r e s u l t e d i n a n i n c r e a s e i n p e p s i n o u t p u t o f t h e pouch w h i c h was p r o b a b l y n o t s i g n i f i c a n t (p = < . 1 ) . The e f f e c t o f i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 2 . O p g . / K g - h r . i s shown i n t a b l e XV and f i g u r e 3 7 . T h i s d o s e o f m o t i l i n s i g n i f i c a n t l y i n c r e a s e d t h e p e p s i n o u t p u t o f t h e pouch (p = < . 0 1 ) . ( i v ) A n t r a l pouch m o t o r a c t i v i t y The e f f e c t o f i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 1 . 0 p g . / K g - h r . i s shown i n t a b l e XVI and f i g u r e 3 8 . T h i s d o s e o f m o t i l i n a l t e r e d t h e p a t t e r n o f a n t r a l m o t i l i t y b u t t h e v a r i a n c e i n t h e d a t a was s u c h t h a t no t e s t s o f s t a t i s t i c a l s i g n i f i c a n c e were a p p l i e d . The e f f e c t o f i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 2 . 0 p g . / K g - h r . i s shown i n t a b l e XV I I and f i g u r e 3 9 . T h i s d o s e o f m o t i l i n s i g n i f i c a n t l y i n c r e a s e d t h e m o t o r a c t i v i t y o f t h e a n t r a l TABLE XI EFECT OF SINGLE INTRAVENOUS INFUSION OF MOTILIN AT 2.0 yg/kg/hr. ON FUND IC MOTILITY MOTILITY INDEX ( FIGURES IN BRACKETS ARE RATIOS") DOG EXPT. 2 3 4 5 6 7 8 9 10 CONTROL PERIODS INFUSION POST INFUSION P 8/6/71 58.2(0.71) 84.2(1.57) 38.1(0.71 ) 127.1( 2.27) 73.8( 1.38) 60.3( 1.13) 37.0(0.69) 3.4(0.06) 8.8(0.16) 6.9(0.13) P 24/6/71 86.0(1.63) 49.0(0.93) 23.0(0.44) 177.0( 3.36) 219.0( 4.16) 114.0( 2.16) 96.0(1.82) 36.0(0.68) 3.0(0.06) 5.3(0.10) P 22/6/71 161.0(1.92) 31.2(0.37) 58.7(0.70) 125.2( 1.50) 54.1( 0.65) 44.4( 0.53) 56.8(0.68) 4.0(0.05) 0.0( - ) 9.1(0.1 1 ) L 25/5/71 15.0(1.00) 15.0(1.00) 15.0(1.00) 223.8(14.92) 150.4(10.02) 77.5( 5.16) 32.3(2.15) 7.4(0.49) 0.0( - ) 0.0( - ) L 22/6/71 8.1(1.12) 5.6(0.77) 8.0(1.11) 211.0(29.17) 114.0(15.76) 58.0( 8.02) 44.3(6.12) 8.6(1.19) 0.0( - ) 0.0( - ) L 17/6/71 8.7(0.97) 7.8(0.87) 10.3(1.15) 241.8(27.07) 174.2(19.50) 102.7(1 1 .50) 50.9(5.70) 24.1(2.70 10.7(1.20) 0.0( - ) L 17/5/71 13.9(1.15) 8.2(0.91 ) 4.8(0.53) 113.5(12.66) 79.8( 8.90) 45.8( 5.1 1 ) 29.9(3.33) 2.9(0.32) 0.0( - ) 0.0( - ) A 8/11/71 124.2(1.30) 100.7(1.06) 60.7(0.64) 127.2( 1.34) 80.9( 0.85) 53.5( 0.56) 74.0(0.78) 33.0(0.35) 6.4(0.07) 7.8(0.08) A 15/11/71 131.4(1.98) 38.9(0.58) 28.9(0.43) 190.9( 2.87) 132.3( 1.99) 73.7( 1.1 1 ) 76.9(1.16) 32.1(0.48) 9.1(0.14) 12.4(0.19) X 1.31 0.90 0.75 10.57 7.02 3.92 2.49 0.70 0. 18 0.06 -+ S.E. 0.15 0. II 0.09 3.71 2.32 1.28 0.71 0.27 0. 13 0.02 " RATIOS OF MEAN OF THE 3 CONTROL PERIODS + AL  PERIODS ARE OF I 5 MINS. DURATION - 96 -Motilin 2 jug/kg/hr o s c 1 2 . 0 -oO F i g u r e 3 3 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 2 y g / k g - h . o n f u n d i c pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 9 e x p e r i m e n t s i n 3 dogs ( T a b l e X I ) TABLE XI I EFECT OF SINGLE INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/kg./hr. on H+ ION SECRETION p Eq.H+/PERIOD (FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. I + 2 3 4 5 6 7 8 9 10 CONTROL PERIODS INFUSION POST INFUSION P 27/7/71 40.8(1.30) 31.8(1.01) 21.6(0.69) 21.2(0.67) 21.2(0.67) 20.8(0.66) 25.5(0.81) 20.8(0.66) 20.8(0.66) 21 .2(0.67) P J/8/71 21.2(1.22) 15.3(0.88) 15.6(0.90) 26.5(1.52) 32.4(1.86) 20.8(1.20) 20.8(1.20) 15.3(0.88) 15.6(0.90) 20.8(1.20) P 9/7/71 37.1(0.62) 68.9(1.16) 72.8(1.22) 113.4(1.90) 129.6(2.17) 64.2(1 .24) 86.4(1.45) 113.4(1.90) 48.6(0.81) 36.4(0.61) P 29/7/71 53.0(0.55) 148.4(1.54) 88.4(0.91) 74.2(0.77) 37.1(0.38) 20.8(0.21) 31 .8(0.33) 15.3(0.16) 21.2(0.22) 21.2(0.22) L 12/7/71 27.0(0.80) 30.6(0.91) 43.2(1.28) 21.6(0.64) 15.9(0.47) 52.0(1.54) 31.2(0.93) 15.6(0.46) 20.4(0.61) 20.8(0.62) L 16/7/71 25.5(0.59) 31.2(0.72) 72.8(1.69) 75.6(1.75) 66.3(1.53) 27.0(0.62) 15.6(0.36) 25.5(0.59) 15.6(0.36) 15.9(0.37) L 19/7/71 10.2(0.66) 25.5(1.65) 10.6(0.69) 20.8(1.35) 15.6(1.01) 15.9(1.03) 15.6(1.01 ) 15.9(1.03) 10.4(0.67) 10.4(0.67) A 2/11/71 20.8(1.08) 15.9(0.83) 20.8(1.08) 20.8(1.08) 20.8(1.08) 21.2(1.11) 15.9(0.83) 10.6(0.55) 10.4(0.54) 10.4(0.54) A 4/11/71 37.8(0.76) 58.3(1.18) 52.0(1.05) 70.2(1.42) 25.5(0.52) 20.4(0.41) 15.6(0.32) 21.6(0.44) 16.2(0.33) 21.2(0.43) X 0.84 1.10 1.06 1.23 1.07 0.89 0.80 0.74 0.57 0.59 -+ S.E. 0.09 0.16 0.10 0.15 0.22 0. 14 0.13 0. 17 0.08 0.09 * RATIOS OF MEAN OF THE 3 CONTROL PERIODS + AL  PERIODS ARE OF 15 MINS. DURATION - 98 -Motilin I jjg /kg /hr c o ° - ° T 1 P 1 1 1 1 1 1 1 1 ° O 2 4 6 8 1 0 "x 15 Min. Periods F i g u r e 3 4 . E f f e c t o f a I h . i n t r a v e n o u s + i n f u s i o n o f m o t i l i n a t I u g / k g - h . o n f u n d i c pouch H i o n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m 9 e x p e r i m e n t s i n 3 dogs ( T a b l e X I I ) TABLE XIII EFFECT OF SINGLE INTRAVENOUS INFUSION OF MOTILIN AT 2.0 yg/Kg./hr. ON H+ ION SECRETION pEq.HVPERIOD (FIGURES IN BRACKETS ARE RATIOS*) COG EXPT. I* 2 3 4 5 6 7 8 9 10 CONTROL PERIODS INFUSION POST INFUSION L 17/6/71 20.8(1.20) 15.6(0.90) 15.6(0.90) 31.2(1.80) 25.5(1.47) 25.5(1.47) 15.6(0.90) 20.8(1.20) 20.4(1.17) 20.0(1.15) L 22/6/71 25.5(1.05) 26.0(1.08) 20.8(0.86) - ( - ) 52.0(2.15) 35.0(1 .45) 26.0( 1.08) 30.0(1.24) 20.0(0.83) 25.5(1.05) L 25/5/71 20.0(1.06) 20.4(1.08) 16.2(0.85) 20.8(1.10) 25.5(1.35) 20.4(1.08) 15.3(0.81 ) 15.3(0.01) 10.6(0.56) 16.2(0.85) L 17/5/71 26.0(1.16) 20.4(0.91) 20.4(0.91 > 26.0(1.16) 26.0(1.16) 24.5(1.10) 20.8(0.93) 20.4(0.91 ) 20.8(0.93) 21 .2(0.68) P 22/6/71 189.0(2.06) 59.4(0.64) 26.5(0.29) 47.7(0.52) 53.0(0.58) 37.8(0.41) 26.0(0.28) 10.8(0.12) 15.6(0.17) 16.2(0.18) P 24/6/71 40.8(1.41) 25.5(0.88) 20.4(0.70) 27.0(0.93) 52.0(1.80) 30.6(1.05) 25.5(0.88) 21.2(0.73) 20.8(0.72) 15.3(0.53) P 8/6/71 46.8(0.88) 68.9(1.30) 42.4(0.80) 52.0(0.98) 46.8(0.88) 41.6(0.79) 39.6(0.75) 25.0(0.47) 20.4(0.38) 15.9(0.30) A 8/11/71 46.8(1.68) 31.2(1.12) 5.3(0.19) 42.4(1.52) 30.6(1.10) 32.4(1.16) 31 .8( 1.14) 32.4(1.16) 20.8(0.74) 21 .2(0.76) A 15/11/71 10.4(0.73) 16.2(1.15) 15.6(1.10) 20.0(1.42) 27.5(1.95) 15.6(1 .10) 16.2(1.15) 21.2(1.50) 15.9(1.13) 15.6(1.10) X 1.25 1.12 0.73 1.18 1.38 1.07 0.88 0.90 0.74 0.73 i S.E. 0. 14 0.12 0.10 0.14 0.17 0. 1 1 0.09 0. 14 0. II 0.12 • RATIOS OF MEAN OF THE 3 CONTROL PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION - 100 -Mofilin 2jug/kg/hr 15 Min. Periods F i g u r e 3 5 . E f f e c t o f a I h . i n t r a v e n o u s + i n f u s i o n o f m o t i l i n a t 2 y g / k g - h . o n f u n d i c pouch H i o n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 9 e x p e r i m e n t s i n 3 dogs ( T a b l e X I I I ) TABLE XIV EFFECT OF SINGLE INTRAVENOUS INFUSION OF MOTILIN 1.0 wg/Kg./hr. ON PEPSIN SECRETION MG. TYROS INE/PERIOD (FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. 1 + i 3 4 5 6 7 8 9 16 CONTROL PERIODS INFUSION POST INFUSION P 27/7/71 0.612(0.83) 0.742(1.00) 0.864(1.17) 1.271(1.72) 3.286(4.45) 3.640(4.92) 3.417(4.62) 2.288(3.09) 1.820(2.46) 1.537(2.07) P 3/8/71 1.219(0.90) 1.275(0.94) 1.560(1.15) 3.074(2.28) 2.862(2.12) 1.820(1.35) 1 .716(1.27) 1.275(0.94) 1.508(1.12) 1.300(0.96) P 9/7/71 0.901(0.77) 1.431(1.22) 1.196(1.02) 1.890(1.61) 1.566(1.33) ( - ) 1.350(1.15) 2.430(2.07) 1.24211.06) 1.092(0.93) P 29/7/71 2.544(1.16) 2.491(1.13) 1.560(0.71) 2.915(1.33) 2.279(1.04) 2.444(1.11 ) 1.855(0.84) 1 .071(0.49) 1.537(0.70) 1.855(0.84) L 12/7/71 2.646(1.09) 2.703(1.11) 1.944(0.80) 1.782(0.73) 1.272(0.52) 1.924(0.79) 0.884(0.36) 0.676(0.28) 0.408(0.17) 0.884(0.36) L 16/7/71 1.377(1.16) 1.508(1.27) 0.676(0.57) 0.648(0.55) 1.275(1.07) 1.026(0.86) 1.I96(1.01) 1 .530(1.29) 2.08011.75) 1.961(1.65) L 19/7/71 2.397(0.99) 2.703(1.13) 2.120(0.88) 2.444(1.02) 1.456(0.61) 1.696(0.71) 1.508(0.63) 1 .431(0.60) 1.040(0.43) 1.092(0.45) A 2/11/71 0.832(1.06) 0.795(1.01) 0.728(0.93) 0.364(0.46) 1.092(1.39) 1.696(2.16) 1.885(2.36) 0.901(1.15) 0.416(0.53) 0.260(0.33) A 4/11/71 0.162(1.02) 0.159(1.00) 0.156(0.98) 1.196(7.52) 0.459(2.89) 0.561(3.53) 0.885(5.57) 1.566(9.85) 1.242(7.81) 0.742(4.67) X 1.00 1.09 0.91 1.91 1.71 1.93 1.98 2.19 1 .78 1.36 i S.E. 0.05 0.04 0.06 0.73 0.42 0.54 0.62 1.00 0.79 0.46 « RATIOS OF MEAN OF THE 3 CONTROL PERIODS + AL  PERIODS ARE OF 15 MIN. DURATION - 102 -Motilin I jag/kg/tir 2 . 4 -1 0 o c o . 6 -1 . 2 -°1 O w o o a. _ 0 . 8 « s l o 0.4-1 0 . 0 -1 0 4 6 15 Min. Periods F i g u r e 3 6 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I y g / k g - h . o n f u n d i c pouch p e p s i n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m 9 e x p e r i m e n t s i n 3 dogs ( T a b l e X I V ) TABLE XV EFECT OF SINGLE INTRAVENOUS INFUSION OF MOTILIN AT 2.0 yg/Kg./hr. ON PEPSIN SECRETION MG. TYROSINE/PERIOD (FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. I+ 2 3 4 5 6 7 8 9 10 CONTROL PERIODS INFUSION POST INFUSION P 8/6/71 0.988(1.06) 1.272(1.37) 0.53 (0.57) 1.664(1.79) 1.716(1.84) 1.04 (1.12) 0.462(0.50) 0.45 (0.48) 1.377(1.48) 1.961(2.11 ) P 24/6/71 1.27 (1.17) 1.12 (1.03) 0.86 (0.79) 1.56 (1.44) 2.44 (2.25) 1.47 (1.36) 1.47 (1.36) 1.16 (1.07) 1.30 (1.20) 1.63 (1.50) P 22/6/71 2.916(1.81) 0.864(1.53) 1.06 (0.66) 4 . 346(2.69)-2.12 (1.31) 0.918(0.57) 0.884(0.55) 1.08 (0.67) 1 .82 (1.13) 2.53 (1.57) 25/5/71 1.55 (0.55) 3.06 (1.09) 3.78 (1.35) 4.16 (1.49) 2.91 (1.04) 2.55 (0.91) 2.49 (0.89) 2.39 (0.85) 2.43 (0.87) 2.86 (1.00) 22/6/71 1.887(1.05) 1.82 (1.02) 1.664(0.93) ( - ) 1.092(0.61) 0.75 (0.42) 0.78 (0.43) 1.35 (0.75) 1.35 (0.75) 1.58 (0.88) 17/6/71 2.2J (1.20) 1.92 (1.04) 1.40 (0.76) 2.18 (1.18) 1.22 (0.66) 0.96 (0.52) 0.93 (0.50) 1 . 14 (0.62) 1.2 (0.60.) 1.15 (0.62) 17/5/71 1.248(0.83) 1.53 (1.02) 1.734(1.15) 3.172(2.11 ) 2.08 (1.38) 1.52 (1.01) 1.51 (1.00) 1.581(1.05) 2.02 (1.34) 2.01 (1.34) 8/11/71 0.624(0.92) 0.832(1.22) 0.583(0.86) 1.484(2.18) 1.071(1.58) 1.08 (1.59) 0.848(1.25) 0.648(0.95) 0.572(0.84) 0.742(1.09) 15/11/71 1.456(1.17) 1.242(1.00) 1.04 (0.84) 0.80 (0.64) 0.935(0.75) 0.884(0.71 ) 0.648(0.52) 0.583(0.47) 0.424(0.34) 0.572(0.46) X 1.09 1.5 0.88 1.69 1.27 0.91 0.78 0.77 0.95 1.17 i S.E. 0.1 1 0.06 0.08 0.23 0.19 0.13 0.12 0.08 0.12 0.17 » RATIOS OF MEAN OF THE 3 CONTROL PERIODS + AL  PERIODS ARE OF 15 MINS. DURATION - 104 -Motilin 2 / jg /kg/hr F i g u r e 3 7 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 2 u g / k g - h . o n f u n d i c pouch p e p s i n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 9 e x p e r i m e n t s i n 3 dogs ( T a b l e XV) TABLE XV I EFECT OF SINGLE INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/Kg/hr. ON ANTRAL MOTILITY MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS") DOG EXPT. I+ 2 3 4 5 6 7 8 9 10 CONTROL PERIODS INFUSION POST INFUSION P 27/7/71 28.0(0.43) 77.4(1.19) 89.6(1.38) 196.0(3.01) 206.5( 3.18) 151.8( 2.33) 78. 1 ( 1.20) 15.9(0.24) 78.4tr.2l ) 107.4(1.65) P 3/8/71 26.0(0.73) 26.4(0.75) 53.7(1.52) 200.9(5.68) 171.It 4.84) 113.2( 3.20) 45.8( 1 .29) 22.4(0.63) 0.0( - ) 0.0( - ) P 9/7/71 5.6(0.81 ) 3.2(0.46) 12.0(1.73) 10.3(1.48) 1.6( 0.23) l.6( 0.23) 5.5( 0.79) 6.3(0.91) 4.1(0.59) 0.0( - ) P 29/7/71 7.5(0.23) 36.1(1.10) 54.4(1.66) 138.3(4.23) 46.3( 1 .42) 69. Of 2.1 1 ) 69.1 ( 2. 1 1 ) 38.6(1.18) 50.0(1.53) 41.7(1.28) L 12/7/71 240.6(1.71 ) 98.5(0.70) 82.9(0.59) 292.5(2.08) 31 3.4( 2.23) 260.1 ( 1.85) 241.2( 1.71 ) 294.1(2.09) 155.5(1.10) 288.3(2.05) L 16/7/71 238.7(1.16) 173.1(0.84) 203.1(0.99) 272.7(1.33) 113.7( 0.55) 93.3( 0.45) 14.9( 0.07) 47.7(0.23) 0.0( - ) 10.3(0.05) L 19/7/71 150.6(0.84) 385.9(2.16) 0.0( - ) 5.7(0.03) 18.K 0.10) 94. K 0.53) 270.9( 1.51 ) 131.2(0.73) 102.0(0.57) 65.3(0.36) A 2/11/71 4.0(0.97) 5.3(1.28) 3.1(0.75) 38.0(9.19) 177.3(42.90) 248.1(60.17) 112.3(27.17) 5.3(1.28) 0.0( - ) 7.7(1.86) A 4/11/71 47.9(0.39) 182.3(1.50) 133.3(1.10) 86.9(0.72) 121.9( 1.01) 38.6( 0.32) 27.1 ( 0.22) 5.9(0.05) 63.1(0.52) 7.7(0.06) X 0.81 I.I 1 1.08 3.08 6.27 7.91 4.01 0.82 0.61 0.81 i S.E. 0.15 0.17 0.19 0.96 4.61 6.54 2.90 0.21 0.19 0.29 * RATIOS OF MEAN OF THE 3 CONTROL PERIODS • AL  PERIODS ARE OF 15 MINS. DURATION - 106 -Motilin I jug/kg/hr ws^^vvvvwvws<vs.v« 1.2 -i to o F i g u r e 3 8 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g - h . on a n t r a l pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m 9 e x p e r i m e n t s i n 3 dogs ( T a b l e X V I ) TABLE XVII EFFECT OF SINGLE INTRAVENOUS INFUSION OF MOTILIN AT 2.0 wg/Kg./hr. ON ANTRAL MOTILITY MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS") DOG EXPT. 1 2 3 4 5 6 7 8 9 10 CONTROL PCRIODS INFUSION POST INFUSION P 8/6/71 120.1(0.93) 171.8(1.33) 94.1(0.73) 204.5(1.59) 190.5(1.48) 166.8(1.30) 222.5(1.73) 195.6(1.52) 104.3(0.81) 103.2(0.80) P 24/6/71 19.9(0.70) 10.7(0.37) 55.1(1.93) 63.1(2.21) 74.4(2.60) 10.0(0.35) 48.2(1.69) 61.7(2 .16) 2 .4(0 .08) 5 .4(0 .19) P 22/6/71 42 .1(1 .85) 11.2(0.49) 15.0(0.66) 36.1(1.58) 29.3(1.29) 19.5(0.86) 59.3(2.60) 10.7(0.47) 0.0( - ) .10.1 (0.44) L 25/5/71 206.6(1.56) 97.1(0.73) 92.3(0.70) 187.7(1.42) 5.4(0.04) 4 .3(0 .03) 7.1(0.05) 8 .2 (0 .06) 0.0( - ) 0.0( - ) L 22/6/71 21.7(1 .47) 4 .0(0.27) 18.5(1.25) 32.7(2.22) 27.2(1.85) 19.9(1.35) 7.3(0.49) 2 .7(0 .18) 1.9(0.13) 2 .0 (0 .14) L 17/6/71 12.1(0.52) 22.1 (0.94) 36.1(1.54) 164.7(7.03) 166.7(7.11 ) 188.6(8.05) 176.0(7.51) 170.8(7.25) 68 .7(2 .93) 76 .8(3 .28) L 17/5/71 21.5(0.81 ) 24.5(0.92) 33.8(1.27) 73.0(2.74) 90.2(3.39) 55.5(2.09) 78.6(2.95) 25.5(0.96) 54.8(2 .06) 176.8 (6 .65) A 8/11/71 50.8(0 .58) 102.9(1.17) 109.9(1.25) 185.7(2.11) 189.5(2.16) 1 15.7(1.32) 46.8(0.53) 63.4(0 .72) 37.5(0 .43) 1.2(0.01) A 15/11/72 69 .9 (0 .86) 82.3(1.01) 91.3(1.12) 132.1(1.63) 185.1(2.28) 154.8(1.91 ) 174.3(2.15) 102.7(1.26) 0.0( - ) 9 .6 (0 .12) X 1.03 0.81 1.16 2.50 2.47 1.92 2.19 1.62 0.71 1.29 i S . E . 0. 16 0.12 0.14 0.58 0.66 0.80 0.74 0.74 0.35 0.75 « RATIOS OF MEAN OF THE 3 CONTROL PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION - 108 -Motilin 2>jg/kg/hr F i g u r e 3 9 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t 2 y g / k g - h . o n a n t r a l pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e m e a n . . D a t a f r o m 9 e x p e r i m e n t s i n 3 dogs ( T a b l e X V I I ) - 109 -pouch (p = < . 0 0 1 ) . C o n t r o l d a t a , o b t a i n e d f r o m e x p e r i m e n t s i n w h i c h no i n f u s i o n was g i v e n , i s shown i n t a b l e s XXXIV - X X X V I I . EFFECTS OF INTERRUPTED INTRAVENOUS INFUSION OF MOTILIN ON BICKEL POUCH  MOTOR A C T I V I T Y , H* ION SECRETION, P E P S I N SECRETION AND ANTRAL MOTOR  ACT IV ITY T h i s s t u d y was p e r f o r m e d u s i n g c h r o n i c dog p r e p a r a t i o n s and by i n t e r r u p t i n g a 1 . 5 h o u r i n f u s i o n o f m o t i l i n w i t h a 30 m i n u t e c o n t r o l p e r i o d . M o t i l i n was i n f u s e d a t 1 . 0 u g / k g - h . f o r I h o u r f o l l o w i n g t h e 3 c o n t r o l p e r i o d s . A f t e r 2 more e x p e r i m e n t a l p e r i o d s m o t i l i n was a g a i n i n f u s e d a t t h e same d o s e f o r 30 m i n u t e s . A l I e x p e r i m e n t s were p e r f o r m e d w i t h t h e Thomas c a n n u l a o p e n . ( i ) B i c k e l pouch M o t o r A c t i v i t y The e f f e c t o f i n t e r r u p t e d i n f u s i o n o f m o t i l i n i s shown i n t a b l e X V I I I and f i g u r e 4 0 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e f u n d i c m o t o r a c t i v i t y s i g n i f i c a n t l y (p = < . 0 1 2 5 ) d u r i n g t h e f i r s t i n f u s i o n . The c h a n g e i n m o t o r a c t i v i t y d u r i n g t h e s e c o n d i n f u s i o n was n o t s t a t i s t i c a l l y s i g n i f i c a n t (p = < 0 . 2 ) . ( i i ) B i c k e l pouch H + i o n s e c r e t i o n The e f f e c t o f i n t e r r u p t e d i n f u s i o n o f m o t i l i n i s shown i n t a b l e X I X and f i g u r e 4 1 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e H + i o n o u t p u t o f t h e pouch s i g n i f i c a n t l y (p = < . 0 2 5 ) d u r i n g t h e f i r s t i n f u s i o n . The o u t p u t o f H + i o n was n o t s i g n i f i c a n t l y c h a n g e d d u r i n g t h e s e c o n d i n f u s i o n (p = < . 2 ) . TABLE XVI11 EFFECT OF INTERRUPTED INTRAVENOUS INFUSION OF MOTILIN AT 1.0 pg/Kg./hr. ON FUNDIC MOTILITY MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. I+ 2 3 4 5 6 7 8 9 10 11 12 CONTROL PERIODS INFUSION INFUSION A 13/10/71 129.8(0.85) 151.1 (1.04) 154.6(1.06) 181.9( 1.25) 118.4( 0.81 ) 116.4(0.80) 1 11.6(0.77) 56.7(0.39) 8 .3 (0 .06) 113.7(0.78) 150.4(1.04) 60.8<0.4?) P 14/10/71 96.9(1 .14) 120.3(1.42) 36.5(0.43) 116.B( 1.38) 62.8( 0.74) 52.4(0.62) 34.0(0.40) 6 .2(0 .07) 6 .7(0 .08) 85 .2(1 .01) 75.6(0 .89) 29.9(0 .35) L 15/10/71 0.0( - ) 13.9(1.72) 10.4(1.28) 73.8( 9.1 1 ) 38.0( 4.69) 9.0(1.11) 4 .2(0 .52) 0.0( - ) 0.0( - ) 3.1 (0.38) 15.5(1.91) 1.3(0.16) A 18/10/71 2 .5(0 .06) 4 .7 (0 .12) 1 13.3(2.82) 146.8( 3.65) 76.4( 1.90) 70.5(1.75) 78.2(1.95) 29.0(0.72) 6 .3(0.16) 76.1(1 .89) 51.8(1 .29) 5.7(0.14 ) P 8/12/71 50.3(2 .12) 8 .3 (0 .35) 12.5(0.53) 135.1( 5.70) 63.7( 2.69) 73.6(3.10) 62.2(2.62) 21.4(0.90) 1.5(0.06) 107.0(4.51 ) 71.3(3 .01) 14.7(0.62) L 9/12/71 3.3(0.91 ) 2 .1(0 .58) 5.5(1.51 ) 78.6(21.63) 50.7(13.95) 25.5(7.02) 9.7(2.67) 0.0( - ) 0.0( - ) 5 .1(1 .40) 2 5 . 3 ( 6 . % ) 5 .4(1 .47) X 0.85 0.87 1.27 7.07 4.13 2.40 1.49 0.35 0.06 1.66 2.52 0.53 i S . E . 0.32 0.26 0.35 3.15 2.05 0.99 0.43 0.16 0.02 0.61 0.94 C.20 * RATIOS OF MEAN OF THE 3 CONTROL PERIODS + ALL PERIODS ARE OF 15 MIN. DURATION - I l l -Motilin ljug/kg/hr Motilin l/jg/kg/hr F i g u r e 4 0 . E f f e c t o f an i n t e r r u p t e d 1 . 5 h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g - h . o n f u n d i c pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 6 e x p e r i m e n t s i n 3 dogs ( T a b l e X V I I I ) TABLE X I X EFFECT OF INTERRUPTED INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/Kg./hr. ON H* ION SECRET ION U Eg. HVPERIOO(FIGURES IN BRACKETS ARE RATIOS") O X EXPT. l + 2 3 4 5 6 7 8 9 10 11 ( 2 CONTROL PERIODS INFUSION INFUSION A 13/10/71 1 3 7 . 5 ( 1 . 0 2 ) 1 1 1 . 3 ( 0 . 8 3 ) 153.7(1 .14) 1 9 2 . 4 ( 1 . 4 3 ) 1 8 8 . 7 ( 1 . 4 1 ) 1 4 0 . 4 ( 1 . 0 5 ) 1 6 1 . 2 ( 1 . 2 0 ) 1 3 0 . 0 ( 0 . 9 7 ) 7 9 . 5 ( 0 . 5 9 ) 4 1 . 6 ( 0 . 3 1 ) 7 4 . 2 ( 0 . 5 5 ) 7 9 . 5 ( 0 . 5 9 ) P 14/10/71 6 6 . 0 ( 0 : 7 2 ) 1 6 7 . 4 ( 1 . 8 3 ) 4 0 . 8 ( 0 . 4 5 ) 4 1 . 6 ( 0 . 4 5 ) 4 2 . 8 ( 0 . 4 6 ) 3 1 . 2 ( 0 . 3 4 ) 3 7 . 1 ( 0 . 4 0 ) 3 0 . 6 ( 0 . 3 3 ) 2 6 . 0 ( 0 . 2 8 ) 5 2 . 0 ( 0 . 5 7 ) 9 5 . 4 ( 1 . 0 4 ) 6 1 . 2 ( 0 . 6 7 ) L 15/10/71 3 4 . 3 ( 0 . 6 3 ) 3 5 . 0 ( 0 . 6 4 ) 9 3 . 6 ( 1 . 7 2 ) 1 2 4 . 8 ( 2 . 3 0 ) 5 1 . 0 ( 0 . 9 4 ) 2 9 . 4 ( 0 . 5 4 ) 2 0 . 8 ( 0 . 3 8 ) 1 5 . 0 ( 0 . 2 8 ) 2 1 . 2 ( 0 . 3 9 ) 2 0 . 0 ( 0 . 3 7 ) 2 0 . 8 ( 0 . 3 8 ) 1 5 . 3 ( 0 . 2 8 ) A 18/10/71 1 2 4 . 2 ( 0 . 9 6 ) 1 4 0 . 4 ( 1 . 0 9 ) 1 2 1 . 9 ( 0 . 9 5 ) 1 1 0 . 0 ( 0 . 8 5 ) 6 2 . 4 ( 0 . 4 8 ) 2 5 . 5 ( 0 . 2 0 ) 2 0 . 4 ( 0 . 1 6 ) 1 6 . 5 ( 0 . 1 3 ) 1 5 . 6 ( 0 . 1 2 ) 1 4 . 7 ( 0 . 1 1 ) 1 6 . 2 ( 0 . 1 2 ) 1 5 . 6 ( 0 . 1 2 ) L 12/10/71 3 7 . 8 ( 1 . 3 5 ) 2 5 . 5 ( 0 . 9 1 > 2 0 . 8 ( 0 . 7 4 ) 4 4 . 0 ( 1 . 5 7 ) 3 1 . 2 ( 1 . 1 1 ) 2 0 . 0 ( 0 . 7 1 ) 2 5 . 5 ( 0 . 9 1 ) 1 5 . 6 ( 0 . 5 6 ) 1 5 . 6 ( 0 . 5 6 ) 1 5 . 3 ( 0 . 5 4 ) 2 0 . 8 ( 0 . 7 4 ) 2 1 . 2 ( 0 . 7 5 ) P 8/12/71 6 6 . 0 ( 1 . 5 5 ) 3 0 . 6 ( 0 . 7 2 ) 3 1 . 2 ( 0 . 7 3 ) 3 9 . 2 ( 1 . 1 8 ) 3 0 . 0 ( 0 . 7 0 ) 3 7 . 1 ( 0 . 8 7 ) 2 5 . 5 ( 0 . 6 0 ) 1 5 . 9 ( 0 . 3 7 ) 1 6 . 2 ( 0 . 3 8 ) 6 9 . 6 ( 1 . 6 3 ) 6 7 . 6 ( 1 . 5 9 ) 3 0 . 6 ( 0 . 7 2 ) L 9/11/71 4 2 . 4 ( 1 . 4 3 ) 2 6 . 0 ( 0 . 8 7 ) 2 0 . 8 ( 0 . 7 0 ) 5 0 . 4 ( 1 . 6 9 ) 3 7 . 1 ( 1 . 2 5 ) 2 6 . 0 ( 0 . 8 7 ) 2 1 . 6 ( 0 . 7 3 ) 2 0 . 8 ( 0 . 7 0 ) 15.3(0.51 ) 1 5 . 6 ( 0 . 5 2 ) 1 5 . 3 ( 0 . 5 1 ) 1 5 . 3 ( 0 . 5 1 ) X 1.09 0.98 0.92 1.35 0.91 0.65 0.62 0.48 0.40 0.58 0 .71 0.52 + 0.13 0.15 0.16 0.23 0.14 0.12 0.13 0. II 0.06 0.18 0.18 0.09 • RATIOS OF MEAN OF THE 3 CONTROL PERIODS + A L L PERIODS ARE OF 15 MINS. DURATION Motilin I jjg /kg /hr Motilin I/jg/kg/hr E f f e c t o f an i n t e r r u p t e d 1 . 5 h . i n t r a v e n o u s i n f u s i o n m o t i l i n a t I y g / k g - h . o n f u n d i c pouch H i o n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 7 e x p e r i m e n t s i n 3 dogs ( T a b l e X I X ) - 1 1 4 -( i i i ) B i c k e l pouch P e p s i n s e c r e t i o n The e f f e c t o f i n t e r r u p t e d i n f u s i o n o f m o t i l i n i s shown i n t a b l e XX and f i g u r e 4 2 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e p e p s i n o u t p u t o f t h e pouch s i g n i f i c a n t l y (p = < . 0 5 ) d u r i n g t h e f i r s t i n f u s i o n . The v a r i a n c e o f p e p s i n o u t p u t d u r i n g t h e s e c o n d i n f u s i o n was s u c h t h a t t e s t s o f s i g n i f i -c a n c e were n o t a p p l i e d . ( i v ) A n t r a l pouch M o t o r A c t i v i t y The e f f e c t o f i n t e r r u p t e d i n f u s i o n o f m o t i l i n i s shown i n t a b l e XXI and f i g u r e 4 3 . The v a r i a n c e o f a n t r a l m o t o r a c t i v i t y d u r i n g t h e f i r s t and s e c o n d i n f u s i o n s was s u c h t h a t t e s t s o f s i g n i f i c a n c e were n o t a p p l i e d . EFFECTS OF INTRAVENOUS INFUSION OF MOTILIN ON BICKEL POUCH MOTOR A C T I V I T Y . H + ION SECRETION. PEPSIN SECRETION AND ANTRAL POUCH MOTOR ACTIV ITY AGAINST  A BACKGROUND H + ION SECRETION STIMULATED BY SYNTHETIC HUMAN GASTRIN T h i s s t u d y was p e r f o r m e d u s i n g c h r o n i c dog p r e p a r a t i o n s and i n f u s i n g i n t r a v e n o u s l y s y n t h e t i c human g a s t r i n I (SHGI) ( I . C . I . M a c c l e s f i e l d , E n g l a n d ) a t 0 . 4 u g . / k g - h . i n o r d e r t o e s t a b l i s h a p l a t e a u o f H + i o n s e c r e t i o n . T h i s d o s e o f SHGI was c h o s e n s i n c e i t s t i m u l a t e d H + i o n o u t p u t t o a p p r o x i -m a t e l y 75$ o f t h e max imal a c i d s e c r e t o r y c a p a c i t y o f t h e p o u c h e s . SHGI was i n f u s e d c o n t i n u o u s l y f r o m t h e s t a r t o f t h e e x p e r i m e n t u n t i l 3 p e r i o d s had p a s s e d i n w h i c h H + i o n o u t p u t was c o n s t a n t * 10$ T h e s e 3 p l a t e a u p e r i o d s were t r e a t e d i n a s i m i l a r manner t o t h e 3 c o n t r o l p e r i o d s i n t h e p r e v i o u s s t u d i e s . M o t i l i n was i n f u s e d i n t r a v e n o u s l y a t 1 . 0 u g . / k g - h . f o r I h o u r t o g e t h e r w i t h t h e o n g o i n g g a s t r i n i n f u s i o n w h i c h was c o n t i n u e d f o r 2 e x p e r i -m e n t a l p e r i o d s a f t e r t h e end o f t h e m o t i l i n i n f u s i o n . A l l e x p e r i m e n t s were p e r f o r m e d w i t h t h e Thomas c a n n u l a o p e n . TABLE XX EFFECT OF INTERRUPTED INTRAVENOUS INFUSION OF MOTILIN AT 1.0 wg/Kg./hr ON PEPSIN SECRETION MG. TYROSINE/PERIOD (FIGURES IN BRACKETS ARE RATIOS") DOG EXPT. 2 3 4 5 6 7 8 9 10 11 12 CONTROL PERIODS INFUSION INFUSION A 13/10/71 2.475(1.01) 2.544(1.04) 2.332(0.95) 2.028(0.83) 1.122(0.46) 0.756(0.31) 0.624(0.25) 0.208(0.08) 0.053(0.02) 0 .052(0.02) 1.325(0.54) 1.007(0.41) P 14/10/71 0.66 (0.65) 2.214(2.19) 0.153(0.15) 2.236(2.22) 1.484(1.47) 1.30 (1.29) 0.424(0.42) 0.204(0.20) 0.156(0.15) 0 .572(0.57) 0 .371(0.37) 0 .408(0.40) L 15/10/71 0.539(0.49) 0.65 (0.60) 2.08 (1.91) 2.08 (1.91 ) 0.867(0.80) 1.421(1.30) 1.664(1.53) 1.60 (1.47) 2.332(2.14) 2.00 (1.84) 1.508(1.38) 1.02 (0.94) L 12/10/71 0.648(0.93) 0.714(1.02) 0.728(1.04) 1.595(2.29) 1.04 (1.49) 1.20 (1.72) 1 .836(2.64) 2.236(3.21) 1.664(2.39) 1.989(2.86) 1.924(2.76) 1.272(1.83) P 8/12/71 0.66 (0.88) 0.714(0.95) 0.884(1.17) 2.184(2.90) 3.25 (4.32) 2.915(3.88) 2.55 (3.39) 1.537(2.04) 1.134(1.51 ) 2.03 (2.70) 1.196(1.59) 1.02 (1.36) L 9/12/71 0.583(0.95) 0.468(0.77) 0.78 (1.28) 1.792(2.94) 1.431(2.34) 1.664(2.73) 2.592(4.25) 1.872(3.07) 1.683(2.76) 2.236(3.66) 1.632(2.67) 1.224(2.01) X 0.82 1.09 1.08 2.18 1.81 1.87 2.08 1.68 1.49 1.94 1.55 1.16 i S . E . 0.08 0.23 0.23 0.32 0.57 0.51 0.66 0.55 0.47 0.57 0.42 0.28 " RATIOS OF MEAN OF THE 3 CONTROL PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION - 1 1 6 -Mofility l / jg/kg/hr Motility ljug/kg/hr ^ W V V A W A W v W V J K V W Y W W 3 2.4 -° - 0.0-1 1 1 1 1 1 1 1 1 1 1 1 1 0 2 4 6 8 1 0 1 2 15 Min. Periods F i g u r e 4 2 . E f f e c t o f an i n t e r r u p t e d 1 . 5 h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g - h . o n f u n d i c pouch p e p s i n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 6 e x p e r i m e n t s i n 3 dogs ( T a b l e XX) TABLE XXI EFFECT OF INTERRUPTED INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/Kg/hr. ON ANTRAL MOTILITY MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS") CONTROL PERIODS INFUSION 10 INFUSION 12 0.0< - ) 0.0( - ) 22.5(3.00) 70.7(2 .12) 5 .3(0.16) 23.9(0.72) 37.1(0.43) 106.2(1.23) 116.3(1.34) 100.2(0.87) 115.3(1.00) 130.3(1.13) 16.9(2.43) 4 .0 (0 .57) 0.0( - ) 52.5(0 .82) 47.6(0 .74) 92.1(1.44) 33.6( 4.48) 24.2( 3.23) 27.5( 3.67) 8.0( 1.07) 129.6( 3.89) I 11.0< 3.33) 61.51 1.86) 125.8( 3.78) 136 .K 1.57) 267.7( 3.09) 188.K 2.17) 151.5C 1.75) 2 2 1 . K 1.92) 202.4( 1.75) 148.41 1.29) 200.I< 1.73) 198.5(28.49) 179.3(25.74) 184.3(26.46) 172.3(24.73) 134.6( 2.10) 85.2( 1.33) 146.6( 2.29) 17.3( 0.27) I .1 I 0.39 0.62 0.19 1.27 0.41 7.08 4.31 6.41 3.88 6.29 4.05 5.55 3.86 2.1(0.28) 30.0(0.90) 22.4(0.26) 91 .1(0.79) 21.4(3.07) 0.0( - ) 9 .2(0.28) 2 .8(0 .03) 17. I (0.15) 0.0( - ) 22.3(0 .35) 54.4(0.85) 0.94 0.44 0.22 0.13 0.0( - ) 7.7( 1.03) 142.6( 4.28) 103.Of 3.09) 49.8( 0.57) 60.9( 0.07) 46.0( 0.40) 5 6 . K 0.49) 128.4(18.43) 149.3(21.43) 81.7( 1.27) I39.3( 2.17) 4.16 2.93 4.82 3.35 4 .3 (0 .57) 29 .2(0 .88) 0.0< - ) 38.5(0 .33) 35.6(5.1 I ) 56 .7(0 .88) 1.29 0.7B » RATIOS OF MEAN OF THE 3 CONTROL PERIOOS + ALL PERIODS ARE OF 15 MINS. DURATION - 1 1 8 -Motilin Ijug/kg/hr Motilin l/jg/kg/hr ° 12 . 0 -| o o £ 1 0 . 0 -< 0 . 0 I i i i i 1 1 1 1 i I I I 0 2 4 6 8 1 0 1 2 15 Min. Periods F i g u r e 4 3 . E f f e c t o f an i n t e r r u p t e d 1 . 5 h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g - h . o n a n t r a l pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 6 e x p e r i m e n t s i n 3 dogs ( T a b l e X X I ) - I 19 -( i ) B i c k e l pouch M o t o r A c t i v i t y The e f f e c t o f i n f u s i o n o f m o t i l i n a g a i n s t a b a c k g r o u n d H + i o n s e c r e t i o n i s shown i n t a b l e XX I I and f i g u r e 4 4 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e m o t o r a c t i v i t y o f t h e pouch s i g n i f i c a n t l y (p = < . 0 2 5 ) . ( i i ) B i c k e l pouch H + i o n s e c r e t i o n The e f f e c t o f i n f u s i o n o f m o t i l i n a g a i n s t a b a c k g r o u n d H + i o n s e c r e -t i o n i s shown i n t a b l e X X I I I and f i g u r e 4 5 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e H + i o n o u t p u t o f t h e pouch s i g n i f i c a n t l y (p = < . 0 5 ) . ( i i i ) B i c k e l pouch P e p s i n s e c r e t i o n 4. The e f f e c t o f i n f u s i o n o f m o t i l i n a g a i n s t a b a c k g r o u n d H i o n s e c r e t i o n i s shown i n t a b l e XXIV and f i g u r e 4 6 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e p e p s i n o u t p u t o f t h e pouch s i g n i f i c a n t l y (p = < . 0 1 ) . ( i v ) A n t r a l pouch m o t o r a c t i v i t y The e f f e c t o f i n f u s i o n o f m o t i l i n a g a i n s t a b a c k g r o u n d H + i o n s e c r e t i o n i s shown i n t a b l e XXV and f i g u r e 4 7 . The v a r i a n c e o f a n t r a l m o t o r a c t i v i t y d a t a was s u c h t h a t t e s t s o f s i g n i f i c a n c e were n o t a p p l i e d . EFFECTS OF INTRAVENOUS INFUSION OF MOTILIN ON BICKEL POUCH MOTOR A C T I V I T Y .  H + ION SECRETION, PEPSIN SECRETION AND ANTRAL POUCH MOTOR ACTIV ITY AGAINST  A BACKGROUND PEPSIN SECRETION STIMULATED BY SECRETION T h i s s t u d y was p e r f o r m e d u s i n g c h r o n i c dog p r e p a r a t i o n s and a s i m i l a r p r o t o c o l t o t h e p r e v i o u s s t u d y . P o r c i n e s e c r e t i n ( G . I . H . r e s e a r c h l a b o r a t o r i e s , Sweden , B a t c h n o . 1 7 1 5 2 ) was i n f u s e d i n t r a v e n o u s l y a t 2 . 0 u / k g - h . t h r o u g h o u t t h e e x p e r i m e n t . S i n c e s i m u l t a n e o u s p e p s i n m e a s u r e m e n t s were n o t a v a i l a b l e d u r i n g e a c h e x p e r i m e n t ( p e p s i n e s t i m a t i o n s were p e r f o r m e d TABLE XXI I EFFECT OF INTRAVENOUS INFUSION OF MOTILIN AT 1.0 wg/Kg./hr. ON FUNDIC MOTILITY AGAINST BACKGROUND INFUSION OF SYNTHETIC HUMAN GASTRIN AT 0.4 pg/Kg/hr. MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. I + 2 3 4 5 6 7 8 9 PLATEAU PERIODS 1 INFUSION S POST INFUSION 1 A 21/10/71 5 .5(0 .13) 107.0(2.54) 13.9(0.33) 127.6(3.03) 156.7(3.72) 129.6(3.07) 127.7(3.03) 54.0(1.28) 8 .1 (0 .19) P 20/10/71 38.1(1 .00) 29.3(0.77) 47.0(1.23) 156.3(4.10) 72.7(1.91) 44.6(1.16) 30.7(0.80) 3 .0(0.08) 0.0( - ) L 19/10/71 0 .0( - ) 3 .1(0.17) 52.3(2.83) 80.8(4.37) 22.0(1.19) 7 .3(0.39) 2 .6(0 .14) 0.0( - > 0.0( - ) A 27/10/71 13.1(0.36) 83.0(2.29) 12.8(0.35) 166.1(4.57) 175.0(4.82) 111.3(3.07) 129.6(3.57) 48.5(1 .34) 2 .0(0 .06) L 28/10/71 29.8(2 .12) 11.1(0.79) 1.2(0.08) 61.4(4.37) 58.3(4.15) 17.2(1.22) 14.6(1.04) 0.0( - ) 0 .0( - ) X 0.72 1.31 0.97 4.09 3.16 1.79 1.72 0.54 0.05 t S . E . 0.39 0.46 0.50 0.28 0.69 0.54 0.67 0.31 0.04 * RATIOS OF MEAN OF THE 3 PLATEAU PERIODS + ALL PERIODS ARE OF I 5 MINS-DURATION II GASTRIN ONLY S GASTRIN + MOTILIN - 121 -Motilin I ,/ug/kg/hr 15 Min. Periods F i g u r e 4 4 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g - h . a g a i n s t a n i n t r a v e n o u s i n f u s i o n o f s y n t h e t i c human g a s t r i n I (SHGI) a t 0 . 4 u g / k g - h . on f u n d i c pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . — D a t a f r o m 5 e x p e r i m e n t s i n 3 dogs ( T a b l e X X I I ) TABLE XXII I EFFECT OF INTRAVENOUS INFUSION OF MOTILIN AT 1.0 wg/Kg/hr. ON H + ION SECRETION AGAINST BACKGROUND INFUSION OF SYNTHETIC HUMAN GASTRIN AT 0.4 ug/Kg./hr. M Eq. HV PER I OP (FIGURES IN BRACKETS ARE RATIOS') DOG EXPT. I + 2 3 4 5 6 7 8 9 PLATEAU PERIODS INFUSION 5 POST INFUSION' A 21/10/71 402.0(0.90) 502.2(1.12) 434.5(0.97) 437.4(0.98) 845.6(1.89) 761.6(1.70) 803.0(1 .80) 440.8(0 .99) 379.5(0.85) P 20/10/71 286.2(0.92) 302.1(0.97) 348.4(1.11) 498.2(1.59) 486.0(1.56) 365.7(1.17) 249.1(0.80) 147.9(0.47) 125.0(0.40) L 19/10/71 1129.6(0.93) 1185.9(0.90) 1542.0(1.17) 1701.9(1.29) 1450.0(1.10) 1357.2(1.03) 1145.7(0.87) 884.8(0.67) 1019.2(0.77) A 27/10/71 912.0(1 .06) 880.0(1.02) 784.0(0.91 ) 851.2(0.99) 1032.4(1.20) 877.8(1.02) 985.6(1.15) 599.4(0.70) 378.0(0.44) L 28/10/71 1043.1(1.07) 1024.8(1.05) 856.8(0.88) 940.5(0.96) 1015.0(1.04) 912.0(0.93) 935.2(0.96) 759.0(0.78) 577.5(0.59) X 0.98 1.01 1.01 1.16 1.36 1.17 I .U 0.72 0.61 i S . E . 0.04 0.04 0.06 0.12 0.16 0.14 0.18 0.08 0 .09 « RATIOS OF MEAN OF THE 3 PLATEAU PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION 1 GASTRIN ONLY S GASTRIN + MOTILIN - 123 -Motilin Ijug/kg/hr > v ' SHG 1 0.4/jg/kg/hr Ploteau Period ro c o 15 Min. Periods F i g u r e 4 5 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g - h . a g a i n s t an i n t r a v e n o u s i n f u s i o n o f s y n t h e t i c human g a s t r i n I (SHGI) a t 0 . 4 y g / k g / h . on f u n d i c pouch H i o n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m 5 e x p e r i m e n t s i n 3 dogs ( T a b l e X X I I I ) TABLE XXIV EFECT OF INTRAVENOUS INFUSION OF MOTILN AT 1.0 ug/Kg./hr. ON PEPSIN SECRET ION AGAINST BACKGROUND INFUSION OF SYNTHETIC HUMAN GASTRIN AT 0.4 ug/Kg/hr. MG.TYROS INE/PER 10  (FIGURES IN BRACKETS ARE RATIOS*) COG EXPT. 1 + 2 J 4 5 6 7 8 9 PLATEAU PERIODS 11 INFUSION 1 POST INFUSION * A 21/10/71 0.0 ( - ) 0.0 ( - ) 0.0 ( - ) 0.0 ( - ) 0.392(0.39) 0.672(0.67) 0.605(0.60) 0.058(0.06) 0.0 ( - ) P 20/10/71 0.795(0.94) 0.742(0.88) 0.988(1.17) 1.855(2.20) 1.728(0.47) 1.219(1.45) 0.636(0.76) 0.357(0.42) 0.25 (0.30) L 19/10/71 1.276(0.72) 1.8 (0.67) 2.82 (1.60) 4.514(2.57) 3.364(1.91 ) 2.842(1.62) 2.109(1.20) 0.952(0.54) 0.672(0.38) A 27/10/71 0.0 ( - ) 0.0 ( - ) 0.0 ( - ) 0.616(0.67) 1.102(1.10) 0.855(0.85) 1.176(1.18) 0.81 (0.81) • 0.0 ( - ) L 28/10/71 2.451(1.41 ) 1.624(0.94) 1.2 (0.65) 3.13 (1.81 ) 3.712(2.14) 2.451(1.41) 2.632(1.52) 1.32 (0.76) 0.88 (0.51 ) X 0.62 0.50 0.68 1.44 1.20 1.20 1.05 0.52 0.24 -+ S.E. 0.27 0.21 0.32 0.49 0.36 0.18 0.16 0.13 0.10 * RATIOS OF MEAN OF THE 3 PLATEAU PERIODS + AL  PERIODS ARE OF 15 MINS. DURATION 1 GASTRIN ONLY S GASTRIN + MOTILN - 125 -F i g u r e 4 6 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g / h . a g a i n s t a n i n t r a v e n o u s i n f u s i o n o f s y n t h e t i c human g a s t r i n I (SHGI) a t 0 . 4 u g / k g / h . on f u n d i c pouch p e p s i n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m 5 e x p e r i m e n t s i n 3 dogs ( T a b l e XX IV ) TABLE XXV EFFECT OF INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/Kg./hr ON ANTRAL MOTILITY AGAINST BACKGROUND INFUSION OF SYNTHETIC HUMAN GASTRIN AT 0.4 ug/Kg./hr MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. I+ 2 3 4 5 6 7 8 9 PLATEAU PERIODS 1 INFUSION § POST INFOS lO* * A 21/10/71 5.3(1.53) 5.1(1.47) 0.0( - ) 0.0( - ) 0.0( - ) 77.9(22.47) 98.3(28.36) 9 .0 (2 .60) 0.0( - ) P 20/10/71 60.5(0 .92) 75.2(1.15) 60.5(0.92) 189.9(2.90) 240.1(3.67) 125.4( 1.92) 56.5( 0.86) 21.5(0.33) 12.7(0.19) L 19/10/71 193.1 (1.1 1 ) 211.1(1.22) 116.5(0.67) 125.5(0.72) 0.0( - ) 0.0( - ) 8.5( 0.05) 0.0( - ) 6 .9 (0 .04) A 27/10/71 15.5(1.16) 24.6(1.84) 0.0( - ) 19.4(1.45) 56.2(4.20) 59.4( 4.44) 41.2( 3.08) 2 .0(0 .15) 0.0( - ) L 28/10/71 189.8(1.32) 55.9(0.39) 185.1(1.29) 206.8(1.44) 69.5(0.48) 72.8( 0.51) 222.7( 1.55) 47 .5 (0 .33) 58.3(0.40) X 1.21 1.21 0.58 1.30 1.67 5.87 6.78 0.68 0.13 i S . E . 0.10 0.24 0.25 0.48 0.93 4.22 5.42 0.48 0.08 * RATIOS OF MEAN OF THE 3 PLATEAU PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION II GASTRIN ONLY 5 GASTRIN + MOTILIN - 127 -F i g u r e 4 7 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I y g / k g / h . a g a i n s t a n i n t r a v e n o u s i n f u s i o n o f s y n t h e t i c human g a s t r i n I (SHGI) a t 0 . 4 y g / k g / h . on a n t r a l pouch — m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 5 e x p e r i m e n t s i n 3 dogs ( T a b l e XXV) - 128 -a t t h e end o f e a c h e x p e r i m e n t ) t h e c r i t e r i a f o r a p l a t e a u o f p e p s i n s e c r e t i o n were e s t a b l i s h e d i n p i l o t s t u d i e s . T h e s e showed t h a t t h e i n f u s i o n o f s e c r e t i n f o r 4 5 m i n s . p r i o r t o t h e 3 p e r i o d s w h i c h were used a s p l a t e a u p e r i o d s wou ld be s u f f i c i e n t t o e s t a b l i s h a p l a t e a u o f p e p s i n s e c r e t i o n . M o t i l i n was i n f u s e d i n t r a v e n o u s l y a t 1 . 0 u g . / k g - h . f o r I h o u r a f t e r t h e p l a t e a u p e r i o d s t o g e t h e r w i t h t h e o n g o i n g s e c r e t i n i n f u s i o n w h i c h was c o n t i n u e d f o r 3 e x p e r i m e n t a l p e r i o d s a f t e r t h e end o f t h e m o t i l i n i n f u s i o n . A l l e x p e r i m e n t s were p e r f o r m e d w i t h t h e Thomas c a n n u l a o p e n . ( i ) B i c k e l pouch m o t o r a c t i v i t y The e f f e c t o f i n f u s i o n o f m o t i l i n a g a i n s t a b a c k g r o u n d p e p s i n s e c r e t i o n i s shown i n t a b l e XXVI and f i g u r e 4 8 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e m o t o r a c t i v i t y o f t h e pouch s i g n i f i c a n t l y (p = < . 0 5 ) . ( i i ) B i c k e l pouch H + i o n s e c r e t i o n The e f f e c t o f i n f u s i o n o f m o t i l i n a g a i n s t a b a c k g r o u n d p e p s i n s e c r e t i o n i s shown i n t a b l e XXVI I and f i g u r e 4 9 . T h i s d o s e o f m o t i l i n d i d n o t s i g n i f i c a n t l y a l t e r t h e H + i o n o u t p u t o f t h e pouch (p = > . 2 ) . ( i i i ) B i c k e l pouch p e p s i n s e c r e t i o n The e f f e c t o f i n f u s i o n o f m o t i l i n a g a i n s t a b a c k g r o u n d p e p s i n s e c r e t i o n i s shown i n t a b l e X X V I I I and f i g u r e 5 0 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e p e p s i n o u t p u t o f t h e pouch s i g n i f i c a n t l y (p = < . 0 1 ) . ( i v ) A n t r a l pouch m o t o r a c t i v i t y The e f f e c t o f i n f u s i o n o f m o t i l i n a g a i n s t a b a c k g r o u n d p e p s i n s e c r e t i o n i s shown i n t a b l e X X I X and f i g u r e 5 1 . T h i s d o s e o f m o t i l i n i n c r e a s e d t h e a n t r a l m o t o r a c t i v i t y s i g n i f i c a n t l y (p = < . 0 2 5 ) . TABLE XXVI EFFECT OF INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/Kg./hr. ON FUNDIC MOTILITY AGAINST BACKGROUND INFUSION OF SECRETIN AT 2 .0 u/Kg./hr. MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS") DOG EXPT. l + 2 3 4 5 6 7 8 9 PLATEAU PERIODS 11 INFUSION 5 POST INFUSION t P 1/10/71 23.6(0.85) 42.5(1.54) 16.8(0.61) 54.9( 1.99) 21-OC 0.76) 3.7( 0.13) l .0 ( 0.04) 0.0< - ) 0.0( - ) L 29/9/71 2 .0(0 .71) 2.8(1.00) 3.4(1.22) 28.9(10.20) 26.6( 0.39) 5.7( 2.01) l .8 ( 0.63) O.Ot - ) 0.0( - ) A 4/10/71 0.0( - ) 0.0( - ) 3.0(1.28) 175.9(75.40) 105.7(45.31) 100.0(42.86) 101.9(43.68) 38.8(16.63) 4 .5 (1 .93) P 6/10/71 22.1(0.46) 63.1(1.31) 59.2(1.23) 86.7( 1.80) 27.3( 0.57) 9.8( 0.20) 7 . K 0.15) 0.0( - ) O.Ot - ) L 7/10/71 3.0(0.79) 3 .8(1.00) 2.6(0.69) 59.5(15.52) 39.0(10.17) 16.7( 4.36) 3.5( 0.91 ) 0.0( - ) O.Ot - ) X 0.26 0.57 0.62 20.98 13.33 9.91 9.08 3.33 0.36 + 0.18 0.35 0.28 13.85 8.26 8.27 8 .65 3.33 0.38 » RATIOS OF MEAN OF THE 3 PLATEAU PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION H SECRETIN ONLY I SECRETIN + MOTILIN - 130 -F i g u r e 4 8 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I y g / k g - h . a g a i n s t an i n t r a v e n o u s i n f u s i o n o f s e c r e t i n a t 2 u / k g - h . o n f u n d i c pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e m e a n . D a t a f r o m 5 e x p e r i m e n t s i n 3 dogs ( T a b l e X X V I ) TABLE X X V I I EFFECT OF INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/Kg./hr. ON H + ION SECRETION AGAINST BACKGROUND INFUSION OF SECRETIN AT 2.0 u/Kg./hr. M Eq HVPERIOD (FIGURES IN BRACKETS ARE RATIOS") DOG EXPT. I + 2 3 4 5 6 7 8 9 PLATEAU PERIODS 11 INFUSIONS POST INFUSION* P 1/10/71 4 6 . 8 ( 1 .18) 4 0 . 0 ( 1 . 0 1 ) 3 2 . 4 ( 0 . 8 1 ) 3 5 . 0 ( 0 . 8 3 ) 2 5 . 0 ( 0 . 6 3 ) 1 5 . 0 ( 0 . 3 8 ) 1 6 . 2 ( 0 . 4 1 ) 1 4 . 7 ( 0 . 3 7 ) 1 5 . 0 ( 0 . 3 8 ) L 29/ 9/71 1 5 . 0 ( 0 . 9 7 ) 1 5 . 9 ( 1 . 0 5 ) 1 5 . 3 ( 0 . 9 9 ) 2 0 . 8 ( 1 . 3 5 ) 2 5 . 5 ( 1 . 6 5 ) 2 0 . 4 ( 1 . 3 2 ) 1 5 . 3 ( 0 . 9 9 ) 1 9 . 2 ( 1 . 2 5 ) 1 6 . 8 ( 1 . 0 9 ) A 4/10/71 2 1 . 2 ( 1 . 5 0 ) 1 0 . 2 ( 0 . 7 2 ) 1 1 . 0 ( 0 . 7 8 ) 1 6 . 2 ( 1 . 1 5 ) 2 0 . 4 ( 1 . 4 4 ) 2 0 . 8 ( 1 . 4 7 ) 2 0 . 4 ( 1 . 4 4 ) 1 5 . 6 ( 1 . 1 0 ) 1 0 . 8 ( 0 . 7 6 ) P 6/10/71 4 2 . 4 ( 0 . 9 5 ) 5 7 . 1 ( 0 . 8 5 ) 5 4 . 0 ( 1 . 2 1 ) 4 4 . 0 ( 0 . 9 9 ) 3 7 . 1 ( 0 . 8 3 ) 3 0 . 6 ( 0 . 6 9 ) 3 0 . 0 ( 0 . 6 7 ) 3 3 . 0 ( 0 . 7 4 ) 2 6 . 0 ( 0 . 5 8 ) L 7/10/71 2 0 . 4 ( 1 . 2 0 ) 1 5 . 6 ( 0 . 9 2 ) 1 5 . 0 ( 0 . 8 8 ) 2 0 . 8 ( 1 . 2 2 ) 2 0 . 4 ( 1 . 2 0 ) 1 5 . 3 ( 0 . 9 0 ) 2 0 . 8 ( 1 . 2 2 ) 1 5 . 9 ( 0 . 9 3 ) 1 5 . 3 ( 0 . 9 0 ) X 1.16 0.90 0.94 I.I 1 1.15 0.95 0.95 0.88 0.74 i S.E. 0.10 0.06 0.08 0.09 0.19 0.20 0.18 0.15 0.12 » RATIOS OF MEAN OF THE 3 PLATEAU PERIODS + A L L PERIODS ARE OF 15 MINS DURATION 1 SECRETIN ONLY S SECRETIN + MOTILIN - 132 -Motilin l/jg/kg/hr p i o ^ P e T i o d S e c r e f i n 2 u / k 9 / h r K> ° 0 . 0 - ) 1 1 , , 1 1 1 1 1 , + O 2 4 6 8 10 15 Min. Periods F i g u r e 4 9 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I u g / k g - h . a g a i n s t an i n t r a v e n o u s i n f u s i o n o f s e c r e t i n a t 2 u / k g - h : on f u n d i c pouch H + i o n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m 5 e x p e r i m e n t s i n 3 dogs ( T a b l e X X V I I ) TABLE XXVI 11 EFFECT OF INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/Kg./hr. ON PEPSIN SECRETION AGAINST BACKGROUND INFUSION OF SECRETIN AT 2.0 u/Kg./hr. MG. TYROSINE/PERIOD (FIGURES IN BRACKETS ARE RATIOS") DOG EXPT. I + 2 3 4 5 6 7 8 9 PLATEAU PERIODS 1 INFUSION § POST INFUSION " P 1/10/71 2.808(0.63) 4.95 ( I . I I ) 5.562( 1.25) 14.63 (3.29) 10.35 (2.33) 5.65 (1.27) 2.916(0.66) 1.96 (0.44) 1.80 (0.40) L 29/ 9/71 1.20 (0.90) 1.431(1.07) 1.3771 1.03) 3.796(2.84) 4.18 (3.13) 3.672(2.75) 3.264(2.44) 5.472(4.09) 3.36 (2.51) A 4/10/71 0.795(0.63) 1.02 (0.81 ) 1.98 ( 1.56) 7.02 (5.55) 9.384(7.42) 7.592(6.00) 6.528(5.16) 4 .42 (3.49) 3.078(2.43) P 6/10/71 4.929(0.80) 5.51 (0.89) 8.10 ( 1.31 ) 14.30 (2.31) 9.964(1.61) 4.08 (0.66) 2.85 (0.46) 2.64 (0.43) 1.92 (0.31) L 7/10/71 1.785(0.77) 2.288(0.99) 2.85 ( 1.23) 7.384(3.20) 8.823(3.82) 6.579(2.85) 9.984(4.33) 7.261(3.15) 5.763(2.50) X 0.74 0.87 1.28 3.44 3.66 2.71 2.61 2.32 1.63 i S . E . 0.05 0.06 0.09 0.55 l .o l 0.92 0.95 0.78 0.52 • RATIOS OF MEAN OF THE 3 PLATEAU PERIODS + ALL PERIODS ARE OF 15 MIN. DURATION * SECRETIN ONLY i SECRETIN + MOTILIN - 134 -F i g u r e 5 0 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I y g / k g - h . a g a i n s t an i n t r a v e n o u s i n f u s i o n o f s e c r e t i n — a t 2 u / k g - h . on f u n d i c pouch p e p s i n o u t p u t . B a r s a r e s t a n d a r d e r r o r s o f t h e m e a n . D a t a f r o m 5 e x p e r i m e n t s i n 3 dogs ( T a b l e X X V I I I ) TABLE XXIX EFFECT OF INTRAVENOUS INFUSION OF MOTILIN AT 1.0 ug/Kg./hr. ON ANTRAL MOTILITY AGAINST BACKGROUND INFUSION OF SECRETIN AT 2.0 u/Kg./hr. MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS") UOG EXPT. I + 2 3 4 5 6 7 8 9 PLATEAU PERIODS fl INFUSION § POST INFUSION 1 P I/IO/7I 8 .0 (1 .06) 5 .6(0.74) 9.1(1.20) 70.1(9.26) 138.9(18.36) 86.5(11.43) 47 .6(6 .29) 2 .7(0.36) O.Ot -L 29/9/71 23.7)0.84) 35.2(1.25) 25.7(0.91) 4 .0(0.14) 108.6( 3.85) 74.8( 2.65) 42.5(1.51) 8 .8 (0 .31) 2 .7 (0 .09) A 4/10/71 15.3(1.21 ) 13.9(1.10) 8 .7(0 .69) 41.3(3.27) 118.8( 9.40) 89.0( 7.04) 45.6(3.61) 15.1(1.19) 0.0( - ) P 6/10/71 0.0( - ) 4 .0(0 .76) 10.7(2.04) 14.9(2.85) 17.9( 3.42) 13.6( 2.60) 1.6(0.30) 1.6(0.30) 0.01 - ) L 7/10/71 187.7(0.79) 260.8(1.09) 266.7(1.12) 468.9(1.97) 423.2( 1.77) 394.7( 1.65) 285.5(1.20) 4 .3(0 .02) 44 .9(0 .19) X 0.82 0.99 1.19 3.50 7.36 5.08 2.58 0.44 0.06 - + S . E . 0.17 0.10 0.23 1.54 3.03 1 .84 1.07 0.20 0.04 » RATIOS OF MEAN OF THE 3 PLATEAU PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION H SECRETIN ONLY s SECRETIN + MQTILIN - 136 -Motil in I jag / k g / h r » w w w w w w w v W I 15 Min. Periods F i g u r e 5 1 . E f f e c t o f a I h . i n t r a v e n o u s i n f u s i o n o f m o t i l i n a t I y g / k g - h . a g a i n s t an i n t r a v e n o u s i n f u s i o n o f s e c r e t i n a t 2 u / k g - h . o n a n t r a l pouch m o t o r a c t i v i t y . B a r s a r e s t a n d a r d e r r o r s o f t h e m e a n . D a t a f r o m 5 e x p e r i m e n t s i n 3 dogs ( T a b l e X X I X ) - 137 -EFFECTS OF CHOLINERGIC AND GANGLIONIC BLOCKADE ON THE BICKEL POUCH MOTOR  A C T I V I T Y , H + ION SECRETION AND P E P S I N SECRETION IN RESPONSE TO , INTRAVENOUS INJECTIONS OF MOTILIN T h i s s t u d y was p e r f o r m e d u s i n g c h r o n i c dog p r e p a r a t i o n s w i t h o p e n Thomas c a n n u l a e . E x p e r i m e n t a l p e r i o d s o f 15 m i n u t e s d u r a t i o n were r u n and a t t i m e z e r o a n i n t r a v e n o u s i n j e c t i o n o f 2 u g . o f m o t i l i n was g i v e n . I n j e c -t i o n s o f b l o c k i n g d r u g s were g i v e n a t t h e b e g i n n i n g o f t h e s e c o n d p e r i o d , i . e . a t 15 m i n u t e s , and f u r t h e r 2 y g . i n j e c t i o n s o f m o t i l i n were g i v e n a t 3 0 , 60 and 90 m i n u t e s . C o n t r o l e x p e r i m e n t s were p e r f o r m e d i n t h e same manner e x c e p t t h a t b l o c k i n g d r u g s were n o t g i v e n . The r e s p o n s e s o f t h e p a r a m e t e r s u n d e r i n v e s t i g a t i o n were measured f o r t h e p e r i o d s o f m o t i l i n i n j e c t i o n . The r e s p o n s e t o t h e f i r s t m o t i l i n i n j e c t i o n was d e f i n e d a t 100? and t h e r e s p o n s e s d u r i n g t h e s u b s e q u e n t p e r i o d s o f m o t i l i n i n j e c t i o n w e r e e x p r e s s e d a s p e r c e n t a g e s o f t h e c o n t r o l r e s p o n s e . The raw d a t a i s i n c l u d e d i n t h e t a b l e s i n o r d e r t o c l a r i f y t h e s e m a n i p u l a t i o n s . ( i ) B i c k e l pouch m o t o r a c t i v i t y The e f f e c t o f a d m i n i s t r a t i o n o f a t r o p i n e a t 0 . 1 and 0 . 2 m g . / k g i s shown i n t a b l e XXX and f i g u r e 5 2 . B o t h t h e s e d o s e s p r o d u c e d s i g n i f i c a n t r e d u c t i o n s i n t h e m o t o r a c t i v i t y r e s p o n s e t o m o t i l i n (p = < . 0 2 5 and p = < . 0 0 2 5 r e s p e c t i v e l y ) . The e f f e c t o f a d m i n i s t r a t i o n o f a n s o l y s e n ( P e n t o l i n i u m t a r t r a t e , P o u l e n c , M o n t r e a l ) a t 0 . 5 m g / k g . i s shown i n t a b l e XXX and f i g u r e 5 3 . T h i s d o s e d i d n o t r e s u l t i n any s i g n i f i c a n t c h a n g e i n t h e m o t o r a c t i v i t y r e s p o n s e t o m o t i l i n (p = < . 2 ) . The d a t a f r o m c o n t r o l e x p e r i m e n t s i s shown i n t a b l e X X X I I I . - 138 -TABLE XXX EFFECT OF ATROPINE AND ANSOLYSEN* ON FUND IC POUCH MOTOR ACTIVITY STIMULATED BY INTRAVENOUS INJECTIONS* OF MOTILIN DRUG TIME(mins.) EXPT. 0 CONTROL RESPONSE(C) MOT 1L1TY INDEX(M.I.) 30 M. 1 % of C 60 M.I. J of C 90 M.I. i of C 0.1 mg/kg ATROPINE A15/9/71 A30/9/7I P29/9/7I P2I/12/71 197.5 129.8 121.0 107.1 43.5 22.03 21.4 16.49 8.0 6.61 1.4 1.31 165.3 83.7 40.6 31.28 17.4 14.38 11.7 10.92 264 . 9 1 34.1 67.0 51.62 22.2 18.35 9.6 8.96 X i S.E. 1 38.85 20.1 18.57 11.61 9.29 4.6 58.75 35.07 36.0 16.8 90.92 53.25 59.2 28.4 0.2 mg/kg ATROPINE A22/9/7I P29/12/71 A3/I/72 A5/I/72 168.6 80.1 101.7 90.2 I5..7 9.31 2.3 2.87 1.0 0.98 2.9 3.22 23.3 13.82 3.6 4.49 4.6 4.52 1.0 1 .1 1 43.7 25.92 6.0 7.49 10.0 9.83 1.9 2.11 X i S.E. 110.15 19.9 5.47 4.09 3.43 1.8 8.12 5.98 5.11 2.73 15.4 11.33 9.57 5.1 0.5 mg/kg ANSOLYSEN P27/9/7I A24/9/7I A28/9/7I PI9/I/72 L20/I/72 76.3 149.0 196.7 90.5 46.7 112.7 147.7 132.7 89.06 176.6 89.78 54.7 60.44 25.3 54.18 103.1 135.1 178.5 119.8 185.5 94.31 85.5 94.48 39.0 83.51 117.1 153.4 143.4 96.24 148.1 75.29 115.1 127.18 37.4 80.09 X i S.E. 1 1 1.84 26.9 100.4 88.23 27.2 16.5 118.3 105.43 28.0 9.5 112.2 106.44 19.8 14.3 * DRUGS GIVEN AT 15 mins. + INJECTIONS OF 2 ug NOT IL IN GIVEN AT THE STATED TIMES - 1 3 9 -1 0 0 -£ <° CO c o o ca 9-c o 6 0 -°S -5 § i= o O o •3 c 8 0 -4 0 -2 0 -Drugs given | | Control (no drugs) ^ Atropine 0.1 mg/kg Atropine 0.2 mg/kg ! 1 3 0 6 0 Time in Minutes 1 9 0 F i g u r e 5 2 . E f f e c t o f i n t r a v e n o u s i n j e c t i o n s o f a t r o p i n e a t 0 . 1 and 0 . 2 m g . / k g . o n f u n d i c pouch m o t o r a c t i v i t y r e s p o n s e s t o i n t r a v e n o u s i n j e c t i o n s o f m o t i l i n (2 y g . ) a t t h e s t a t e d t i m e s . R e s p o n s e s a t t i m e 0 d e f i n e d a s 1 0 0 $ . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m T a b l e XXX - 140 -| [ Control (No Drug) Ansolysen (0.5 mg/kg) Drugs given 3 0 ! 6 0 Time in Minutes lp 9 0 E f f e c t o f i n t r a v e n o u s i n j e c t i o n o f a n s o l y s e n a t 0 . 5 m g . / k g . on f u n d i c pouch m o t o r a c t i v i t y r e s p o n s e s t o i n t r a v e n o u s i n j e c t i o n s o f m o t i l i n (2 y g . ) a t t h e s t a t e d t i m e s . R e s p o n s e s a t t i m e 0 d e f i n e d a s 1 0 0 ? . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m T a b l e XXX - 141 -( i i ) B i c k e l pouch H + i o n s e c r e t i o n The e f f e c t o f a d m i n i s t r a t i o n o f a t r o p i n e a t 0 . 1 and 0 . 2 mg/kg i s shown i n T a b l e XXXI and f i g u r e 5 4 . The e f f e c t o f a d m i n i s t r a t i o n o f a n s o l y s e n a t 0 . 5 mg/kg i s shown i n t a b l e XXXI and f i g u r e 5 5 . The d a t a f r o m c o n t r o l e x p e r i m e n t s i s shown i n t a b l e X X X I I I ( i i i ) B i c k e l pouch P e p s i n s e c r e t i o n The e f f e c t o f a d m i n i s t r a t i o n o f a t r o p i n e a t 0 . 1 and 0 . 2 mg/kg i s shown i n t a b l e XXXI I and f i g u r e 5 6 . The e f f e c t o f a d m i n i s t r a t i o n o f a n s o l y s e n a t 0 . 5 mg/kg i s shown i n t a b l e XXXI I and f i g u r e 5 7 . The d a t a f r o m c o n t r o l e x p e r i m e n t s i s shown i n t a b l e X X X I I I . ASSAY OF CHOLECYSTOKININ EFFECTS OF MOTILIN C h o I e c y s t o k i n i n a c t i v i t y o f m o t i l i n was a s s a y e d u s i n g t h e a c u t e g u i n e a p i g p r e p a r a t i o n . The e x p e r i m e n t a l p r o c e d u r e i s d e s c r i b e d i n M e t h o d s , P . 4 6 . A l o g d o s e / r e s p o n s e c u r v e was o b t a i n e d t o c h o I e c y s t o k i n i n , and i n j e c t i o n s o f 10 and 20 y g . o f m o t i l i n were g i v e n . The c h o I e c y s t o k i n i n a c t i v i t y o f m o t i l i n , a s r e v e a l e d by 4 e x p e r i m e n t s , i s i n t h e r a n g e 3 - 8 I . D . U . p e r mg. ASSAY OF SECRETIN EFFECTS OF MOTILIN S e c r e t i n a c t i v i t y o f m o t i l i n was a s s a y e d u s i n g t h e a c u t e c a t p r e p a r a t i o n . The e x p e r i m e n t a l p r o c e d u r e i s d e s c r i b e d i n M e t h o d s , P . 4 7 . I n j e c t i o n s o f 10 and 20 y g . o f m o t i l i n were g i v e n , b r a c k e t e d be tween s t a n d a r d d o s e s o f s e c r e t i n . No i n c r e a s e i n t h e v o l u m e o f p a n c r e a t i c j u i c e was d e t e c t e d i n r e s p o n s e t o t h e s e d o s e s o f m o t i l i n . The p a n c r e a t i c j u i c e v o l u m e c h a n g e s d u r i n g a t y p i c a l e x p e r i m e n t a r e shown i n f i g . 5 8 . - 142 -TABLE XXXI EFFECT OF ATROPINE ANO ANSOLYSEN* ON H + ION SECRETION IN THE PRESENCE OF INTRAVENOUSLY INJECTED MOTILIN DRUG T I M E i m i n s . ) EXPT. 0 CONTROL RESPONSE(C) Vi Eq H + ion 30 H + % of C 60 H + % o f C 90 H + % o f C O.I mg/kg ATROPINE L20/12/71 L22/I2/71 P2I/I2/7I P29/9/7I A30/9/7I A15/9/71 106.4 6 1 . 2 4 9 . 5 . 7 9 . 8 15.9 10.2 3 0 . 0 2 8 . 1 9 2 5 . 5 4 1 . 6 6 2 0 . 5 41.41 2 1 . 6 2 7 . 0 6 11.2 70 .44 15.6 152.94 2 7 . 0 2 5 . 3 7 2 0 . 8 33 .98 2 7 . 0 54 .54 17.1 2 1 . 4 2 10.8 6 7 . 9 2 10.8 105.88 3 3 . 0 31.01 2 0 . 8 3 3 . 9 8 2 6 . 5 5 3 . 5 3 10.8 13 .53 5 . 4 3 3 . 9 6 10.6 103.92 X i S . E . 5 3 . 8 3 15.1 2 0 . 7 3 6 0 . 2 8 2 .74 19.6 18.91 51.51 2 .997 13.08 17 .85 4 4 . 9 8 4 . 3 6 12.87 0 . 2 mg/kg ATROPINE A22/9/7I P23/9/7I P29/12/71 L28/I2/71 A3/I/72 A5/I/72 16.5 148.5 4 1 . 6 4 1 . 6 2 2 . 4 1 1.0 10.8 6 5 . 4 5 3 1 . 2 21.01 2 7 . 5 6 6 . 1 0 3 1 . 8 7 6 . 4 4 16.2 7 2 . 3 2 5 .6 5 0 . 9 16.2 9 8 . 1 8 2 2 . 4 15.08 4 7 . 7 114.66 2 6 . 0 6 2 . 5 2 2 . 0 98.21 16 .5 150.0 16 .5 100.0 2 2 . 0 14.81 4 1 . 6 100.0 2 1 . 2 5 1 . 9 2 15 .9 7 0 . 9 8 10.8 9 8 . 1 8 X i S . E . 4 6 . 9 3 2 0 . 9 7 20.51 5 8 . 7 0 4 . 5 6 8 . 3 3 2 5 . 1 3 8 9 . 6 7 4 . 7 6 18.87 2 1 . 3 3 3 7 2 . 6 4 4 . 3 7 8 14 .05 0 . 5 mg/kg ANSOLYSEN P27/9/7I A24/9/7I A28/9/7I PI 9/1/72 LI8/I/72 L20/I/72 108.0 16 .5 15 .9 145.6 6 3 . 6 3 6 7 . 2 2 1 . 2 19.62 16.5 100.0 10.6 6 6 . 6 6 32 .4 2 2 . 2 5 3 3 . 0 51 .88 4 2 . 4 11.54 10.8 10.0 2 1 . 6 130.9 16.5 103.77 2 1 . 2 14.56 2 1 . 6 3 3 . 9 6 2 0 . 8 5 . 6 6 15 .9 14.72 10.4 6 3 . 0 3 5 . 3 3 3 . 3 3 2 1 . 6 14 .83 2 2 . 0 3 4 . 5 9 2 6 . 0 7 .08 X i S . E . 119.46 53 .74 26.01 4 5 . 3 2 4 . 8 6 13.93 18.75 4 9 . 8 0 1.77 2 2 . 0 16.866 2 7 . 9 3 3 . 2 0 8 . 3 3 * DRUGS GIVEN AT 15 MINS. INJECTIONS OF 2 ug MOTILIN GIVEN AT THE STATED TIMES - 143 -1 8 0 - i < n 1 6 0 -c o o 1 4 0 • 1 2 0 • 1 0 0 • er U i 5. o + X 6 0 -4 0 -2 0 - 1 1 Drugs given | | Control (No Drugs) ^ Atropine (0.1 mg/kg) Atropine (0.2mg/kg) 3 0 6 0 Time in Minutes F i g u r e 5 4 . E f f e c t o f i n t r a v e n o u s i n j e c t i o n s o f a t r o p i n e a t 0 . 1 and 0 . 2 m g / k g . o n f u n d i c pouch H + i o n o u t p u t r e s p o n s e s t o i n t r a v e n o u s i n j e c t i o n s o f m o t i l i n (2 y g . ) a t t h e s t a t e d t i m e s . R e s p o n s e s a t t i m e 0 d e f i n e d a s 1 0 0 ? . B a r s a r e s t a n d a r d e r r o r s o f t h e m e a n . D a t a f r o m T a b l e XXXI - 144 -| | Control (No Drug) Ansolysen (0.5 mg/kg) o 9 O C <D U W V „« 2 2 2 0 - 1 £ . £ 1 8 0 -s | §1 •2 o © w o o o CO c o 1 4 0 -1 0 0 -6 0 -2 0 -Drugs given rh 3 0 6 0 Time in Minutes F i g u r e 5 5 . E f f e c t o f i n t r a v e n o u s i n j e c t i o n o f a n s o l y s e n a t 0 . 5 m g / k g . o n f u n d i c p o u c h H + i o n o u t p u t r e s p o n s e s t o i n t r a v e n o u s i n j e c t i o n s o f m o t i l i n (2 y g . ) a t t h e s t a t e d t i m e s . R e s p o n s e s a t t i m e 0 d e f i n e d a s 1 0 0 ? . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m T a b l e XXXI - 145 -TABLE XXXI I EFFECT OF ATROPINE AND ANSOLYSEN* ON PEPSIN SECRETION IN THE PRESENCE OF INTRAVENOUSLY INJECTED MOTILIN+ DRUG T IME(mins . ) EXPT. 0 CONTROL RESPONSE(C) PEPSIN (mg.TYR) 30 mg.TYR % of C 60 mg.TYR % of C 90 mg.TYR % of C 0.1 mg/kg ATROP1NE L20/12/71 L22/I2/7I P2I/I2/7I P29/9/7I A30/9/7I A15/9/71 2 . 6 8 8 1.632 2 . 2 0 2 . 7 3 6 0 . 6 8 9 0 . 9 6 9 1.5 5 5 . 8 1.53 9 3 . 7 5 2.491 113.22 1.89 6 9 . 0 7 0 . 8 4 0 121.91 0 . 9 8 8 101.96 2 . 9 7 110.49 2 .132 130.63 1.89 8 5 . 9 0 . 7 9 8 2 9 . 1 6 0 . 7 0 2 101.88 0 . 4 8 6 5 0 . 1 5 2 . 3 6 3 8 7 . 9 1.872 114.7 1.166 5 3 . 0 1.026 3 7 . 5 0 . 2 7 3 9 . 1 8 0 . 0 0 X i S . E . 1 .819 0 .354 1.539 9 2 . 6 2 0 . 2 4 7 10 .46 1.494 8 4 . 7 0 0 . 4 0 15.65 I.I 16 5 5 . 3 8 0 . 3 6 9 1 6 . 5 0 . 2 mg/kg ATROPINE A22/9/7I P23/9/7I P29/12/71 L28/I2/71 A3/I/72 A5/I/72 3 . 5 7 5 1.87 1.664 1 . 0 9 2 0 . 6 1 6 0 . 3 8 5 0 . 6 4 8 18.12 1.716 9 1 . 7 6 1.815 109.07 2 . 1 2 194.13 0 .054 8 . 7 6 0 0 0 0 2 .072 110.8 0 0 1.872 171.42 0 . 2 2 35.71 0 0 0 0 2 . 1 4 5 114.7 0 0 1.272 116.48 0 . 1 5 9 25.81 0 0 X i S . E . 1 . 5 3 3 0.471 1.058 7 0 . 3 0 0 . 384 3 0 . 9 0 .694 5 2 . 9 8 0 . 4 0 2 9 . 4 0 . 5 9 6 4 2 . 8 3 0 . 3 6 9 2 3 . 3 7 0 . 5 mg/kg ANSOLYSEN P27/9/7I A24/9/7I A28/9/7I PI 9/1/72 LI 8/1/72 L20/1/72 0 . 4 1 6 1 . 0 0 7 1 .196 0.371 1.728 1.325 3 1 8 . 5 0 . 7 7 0 . 8 4 8 84.21 1.08 9 0 . 3 0 . 3 8 5 103.77 0 . 2 1 2 11 .89 1.728 4 2 8 . 3 6 0 . 4 3 2 0 . 9 3 5 9 2 . 8 5 1.272 106.35 0 .864 2 3 2 . 8 8 0 . 7 8 4 3 . 7 7 1.007 2 4 2 . 0 6 0 . 5 7 2 0 . 4 7 7 4 7 . 3 6 1.62 135.45 1.10 2 9 6 . 4 9 2 . 0 8 116.72 X i S . E . 0 . 9 4 3 0 .254 0 . 7 7 121.73 0 . 1 7 5 1 . 7 2 1.001 180.84 0 . 1 8 2 6 9 . 3 1.142 167 .6 0.251 4 4 . 8 5 * DRUGS GIVEN AT 15 MINS. + INJECTIONS OF 2 u q . MOTILIN GIVEN AT THE STATED STIMES - 146 -160 -140 -120 -o 100 -80 -60 20-Drugs given I I 30 Time in i 60 | [ Control (No Drug) [^ ) Atropine (0.1 mg/kg) Atropine (0.2 mg/kg) ! 90 Minutes F i g u r e 5 6 . E f f e c t o f i n t r a v e n o u s i n j e c t i o n s o f a t r o p i n e a t 0 . 1 and 0 . 2 m g . / k g . o n f u n d i c pouch p e p s i n o u t p u t r e s p o n s e s t o i n t r a v e n o u s i n j e c t i o n s o f m o t i l i n (2 y g . ) a t t h e s t a t e d t i m e s . R e s p o n s e s a t t i m e 0 d e f i n e d a s 1 0 0 ? . B a r s a r e s t a n d a r d e r r o r s o f t h e mean . D a t a f r o m T a b l e XXXI I - 147 -[ | Control (No Drug) Y/A Ansolysen (0.5 mg/kg) 240 i „ 200 .5 c in o o o . w at >s <o »- ac e» — E 2 .9 6 a a. 120 -80 40 • 0 J Drugs given 1 1 30 60 Time in Minutes 1 90 F i g u r e 5 7 . E f f e c t o f i n t r a v e n o u s i n j e c t i o n o f a n s o l y s e n a t 0 . 5 m g / k g . on f u n d i c pouch p e p s i n o u t p u t r e s p o n s e s t o i n t r a v e n o u s i n j e c t i o n s o f m o t i l i n (2 y g . ) a t t h e s t a t e d t i m e s . R e s p o n s e s — a t t i m e 0 d e f i n e d a s 1 0 0 ^ . B a r s a r e s t a n d a r d e r r o r s o f t h e mean. D a t a f r o m T a b l e XXXI I - 148 -TABLE XXXIII EFFECT OF INTRAVENOUS INJECTIONS OF MOTILIN* ON FUNDIC POUCH MOTOR ACTIVITY, H + ION SECRETION AND PEPSIN SECRETION TIME(mins.) 0 30 60 90 CONTROL RESPONSE(C) MOTILITY -EXPT. INDEXCM.I.) M. 1. % of C M.I. % of C M. 1 • i of C J16/9/71 152.0 101.5 66.77 126.6 83. 28 87. 9 57.82 A9/9/7I 221.9 166.2 74.89 265.5 119. 64 196. 5 88.55 J10/9/71 143143.6 100.8 70.19 1 17.8 82. 03 94. 8 66.01 A2/9/7I 134.7 128.5 95.39 133.9 99. 4 134. 0 99.48 J8/9/7I 153.0 137.5 89.86 106.0 69. 28 104. 7 68.43 Al 3/9/71 175.3 139.1 79.34 1 18.1 67. 37 135. 4 77.23 X 163.41 128.93 79.4 144.65 86. 83 125. 55 76.25 i S.E. 12.93 10.18 4.58 24.47 8. 07 16. 33 6.32 W Eq.H+ ION 4 (C) H + { o f C H + % of C H { of C J16/9/71 81.6 40.8 50.0 26.0 31. 86 21 . 6 25.98 A9/9/7I 15.9 16.2 101.88 17.1 107. 54 10. 4 65.4 J10/9/71 16.2 21 .6 133.33 21 .6 133. 33 20. 8 128.39 A2/9/7I 26.5 1 5.9 60.0 15.6 60. 0 1 1 . 0 41.5 J8/9/7I 10.4 22.0 21 1.53 15.9 152. 88 28. 5 274.03 A13/9/71 10.2 15.9 155.88 10.4 101. 96 53. 0 510.6 X 26.8 22.06 118.77 17.76 97. 92 24. 15 175.81 i S.E. 11.25 3.9 24.94 2.202 18. 49 6. 41 78.06 PEPSIN mg. TYR (C) mg.TYR % of C mg.TYR % of C mg.TYR J of C J16/9/71 1.173 4.437 378.26 3.588 305. 88 3. 657 31 1.76 A9/9/7I 0.583 0.162 27.7 0.285 48. 88 0 832 142.71 J10/9/71 2.376 2.646 1 1 1 .36 2.376 100. 0 2. 028 87.62 A2/9/7I 1.007 0.477 47.36 0.624 63. 75 0. 66 65.54 J8/9/7I 3.392 3.52 103.77 2.597 76. 56 2. 508 73.93 A13/9/71 0.867 0.265 29.52 0.26 29. 98 0. 742 85.58 X 1.566 1.917 116.32 1 .621 104. 17 1 . 737 127.85 i S.E. 0.443 0.76 54.45 0.578 41 . 5 0. 492 38.40 INJECTIONS OF 2 ug. MOTILIN GIVEN AT THE STATED TIMES - 149 -Secretin Motilin Secretin Motilin 5u lOjug 5u 20 >jg 0 2. 4 6 8 10 10 Min. Periods F i g u r e 5 8 . E f f e c t o f i n t r a v e n o u s i n j e c t i o n s o f s e c r e t i n and m o t i l i n o n p a n c r e a t i c j u i c e v o l u m e o u t p u t o f an a c u t e c a t . - 150 -ASSAY OF BLOOD PRESSURE AND PYROGEN IC EFFECTS OF MOTILIN M o t i l i n was a s s a y e d u s i n g a c u t e r a b b i t p r e p a r a t i o n s f o r b l o o d p r e s s u r e e f f e c t s . No c h a n g e s c o u l d be d e t e c t e d i n h e a r t r a t e o r c a r o t i d a r t e r y b l o o d p r e s s u r e w i t h d o s e s o f 10 and 25 u g . o f m o t i l i n . P y r o g e n i c e f f e c t s were m o n i t o r e d by m e a s u r i n g t h e r e c t a l t e m p e r a t u o f c h r o n i c dog p r e p a r a t i o n s b e f o r e , d u r i n g and a f t e r i n f u s i o n s o f m o t i l i n a t 1 . 0 p g . / k g - h . No t e m p e r a t u r e c h a n g e s were d e t e c t e d i n 4 e x p e r i m e n t s . In 2 e x p e r i m e n t s i n t h e same d o g , r e c t a l t e m p e r a t u r e i n c r e a s e d by 0 . 4 ° C d u r i n g t h e i n f u s i o n o f m o t i l i n . TABLE XXXIV CONTROL DATA (NO INFUSION) FUNDIC MOTILITY INDEX ( FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. 1 + 2 3 4 5 6 7 8 9 10 P 7/1/72 74.6(0 .93) 78.4(0.98) 87.8(1.09) 81.9(1.02) 89.9(1.09) 86.6(1.08) 89.7(1.12) 79.2(0.99) 88 .3(1 .10) 78.5(0.98) A 8/2/72 1 17.3(0.93) 125.4(0.99) 134.9(1 .07) ' 124.1(0.99) 112.4(0.89) 108.7(0.86) 113.8(0.90) 139.5(1.11) 101.1(0.80) 104.1(0.83) P 13/1/72 118.0(1.12) 86.4(0.82) 112.7(1.07) 121.4(1.15) 31.9(0.30) 73.7(0.70) 55.5(0.52) 33.8(0.32) 43 .8(0 .41) 84 .6(0 .80) P 7/1/72 74.6(0 .93) 78.4(0.98) 87.8(1.09) 81.9(1.02) 89.9(1.12) 86.6(1.08) 89.7(1.13) 79.2(0.99) 88 .9(1 .10) 78.5(0 .98) P 29/2/72 91.8(1 .10) 75.7(0.91) 83.4(1.00) 92.5(1.11) 100.9(1.21) 88.5(1.06) 114.0(1.36) 84.3(1.01) 94.6(1 .13) 85.9(1 .03) A 25/2/72 160.8(1.10) 122.5(0.84) 154.5(1.06) 139.1(0.95) 162.1(I.I 1) 123.5(0.85) 131.2(0.90) 146.8(1.00) 140.1(0.96) 142.0(0.97) X 1.02 0.92 1.06 1.04 J . 9 5 0.94 0.99 0.90 0.92 0 .93 i S . E . 0.04 0.03 0.02 0.03 0.14 0.06 0.11 0.12 0.11 0.04 « RATIOS OF MEAN OF 3 CONTROL PERIODS + ALL PERIODS ARE OF 15 MINS. OURATION TABLE XXXV CONTROL DATA (NO INFUSION) uEq. H* ION ( FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. I+ 2 3 4 5 6 7 ' 8 9 10 P 7/1/72 313.5(1.08) 324.5(1.12) 233.2(0.80) 226.8(0.78) 216.0(0.74) 194.4(0.67) 164.3(0.56) 145.8(0.50) 135.0(0.46) 111.3(0.38) A 17/1/72 30.6(1.48) 15.6(0.76) 15.6(0.76) 21.6(1.05) 16.2(0.79) 15.3(0.74) 15.6(0.76) 15.9(0.77) 15.9(0.77) 1 6 . 8 ( 0 . 8 1 ) P 13/1/72 180.2(1.18) 159.5(1.04) 118.8(0.78) 37.1(0.24) .27.0(0.18) 21.6(0.14) 20.8(0.14) 43 .2(0 .28) 53.0(0.35) 41. .MO.27) L 11/1/72 318.0(1.58) 159.0(0.79) 127.2(0.63) 218.4(1.08) 330.4(1.64) 379.5(1.88) 426.4(2 .12) 621.3(3.08) 498.4(2 .47) 205.2(1 .02) A 10/1/72 21.6(1.02) 20.8(0.98) 21.2(1.00) 16.5(0.78) 11.0(0.52) 15.6(0.73) 15.6(0.73) 16.2(0.76) 15.9(0.75) 15.3(0 .7?) L 6/1/72 67.6(1 .49) 37.1(0.82) 31.2(0.69) 42.4(0.93) 132.5(2.92) 102.0(2.25) 43.2(0 .95) 26.5(0.58) 21.6(0 .48) 22 .0(0 .46) L 11/2/72 57.2(1.57) 30.6(0.84) 21.6(0.59) 21.6(0.59) 21.2(0.58) 58.3(1.59) 88 .4(2 .42) 161.2(4.42) 165.0(4.52) 93 .6 (2 .57) P 10/2/72 42.4(1 .28) 31.2(0.94) 26.0(0.78) 20.8(0.63) 63.6(1.91) 128.8(3.88) 66 .3(1 .99) 37.1(1 .12) 26 .0(0 .78) 21 .6 (0 .65) A 8/2/72 15.6(1.01) 15.9(1.13) 10.8(0.76) 15.9(1.13) 10.6(0.75) 15.9(1.13) 15.6(1.11) 31.2(2 .21) 38.5(2.73) 56 .0(3 .97) * RATIOS OF MEAN OF 3 CONTROL PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION TABLE XXXVI CONTROL DATA (NO INFUSION) PEPSIN MO. TYROSINE ( FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. |+ 2 5 4 5 6 7 8 9 10 P 7/1/72 1.485(1.36) 1.155(1.06) 0.636(0.58) 0.756(0.69) 0.756(0.69) 0 . 4 8 M 0 . 4 4 ) 0 . J 7 K 0 . 3 4 ) 0.378(0.35 ) 0.270<0.25> 0.583(0.53) A 17/1/72 0.204(0.99 ) 0.208(1.01) 0.208(1.01) 0.216(1.05 ) 0.486(2.36) 1.071(5.20) 1.300(6.31 ) 1.74')(8.40) 1.590(7.72) 1.512(7.34) P 13/1/72 1.431(0.86) 1.815(1.09) 1.728(1.04) 1.272(0.77) 1.512(0.91) 1.350(0.81) I . I9M0.72) 1.134(0.68) 0.136(0.38) 0.728(0.44) L 11/1/72 1.325(2.34) 0.159(0.28) 0.212(0.37) 0.952(1.68) 0.952(1.68) 0.440(0.7%) 0.624(1.10) 2.950(5.04) 2 .240(3.9t ) 0.918(1.62) A 10/1/72 0.216(1.11) 0.156(0.80 ) 0.212(1.09 ) 0.330(1.70) 1.540(7.94 ) 2.496(12,86 > 1.508(7.77 ) 0.864(4.45 ) 0.477(2.46 ) 0.408(2.10) L 6/1/72 1.300(1.70) 0.371(0.48) 0.624(0.81) 0.795(1.04) 1.590(2.08) 0.663(0.87) 0.00 < - 1 0.159(0.21) 0.648(0.85) 1.650(2.16) L 11/2/72 0.782(1.02) 0.714(0.93) 0.810(1.05) 0.864(1.12) 0.848(1.10) 1.007(1.31) 0 .884(1.15) 0 . 7 8 ( 1 . 0 1 ) 1.265(1.65) 0.988(1.29) P 10/2/72 1.060(1.21) 0.780(0.89) 0.780(0.89) 0.832(0.95) 1.696(1.94) 2.856(3.27) 2.040(2.34) 1.431(1.64) 1.092(1.25) 0 .810(0.93) A 8/2/72 1.092(1.51) 0.477(0.66) 0.594(0.82) 0.583(0.81) 0.371(0.51) 0.424(0.59) 0.208(0.29) 0.208(0.29) 0.035(0.07) 0.056(0.08) 1.35 0.80 0.85 1.09 2.13 2.90 2.24 2.46 2.06 1.83 » RATIOS OF MEAN OF 3 CONTROL PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION TABLE XXXV11 CONTROL DATA (NO INFUSION) ANTRAL MOTILITY INDEX (FIGURES IN BRACKETS ARE RATIOS*) DOG EXPT. 2 3 4 5 6 7 8 9 10 P 7/1/72 82 .0 (0 .91) 95.6(1.06) 92.3(1.(J2) 71.3(0.79) 74.6(0.83) 96.5(1.07) 140.9(1.57) 124.8(1.39) 130.7(1.45) 68 .1(0 .76) A 17/1/72 8 .5 (1 .35) 6.9(1.09) 3 .5(0.55) 6 .7(1.06) 8.0(1 .27) 6 .5(1 .03) 5 .9(0.94) 5 .1(0 .81) 6 .4 (1 .01) 7 .6(1 .21) P 13/1/72 132.1(0.67) 180.1(0.91) 278.0(1.41) 93.5(0.47) 124.5(0.63) 45.6(0.23) 100.1(0.50) 84 .1(0 .43) 98 .9(0 .50) 37.9(0 .19) L 11/1/72 173.7(2.47) 9 .3(0.13) 27.5(0.39) 69.7(0.99) 131.9(1.88) 210.3(2.99) 63.4(0.90) 138.6(1.97) 124.6(1.77) 129.7(1.85) A IO/I/72 31.6(0.84) 49.9(1.33) 30.6(0.82) 73.8(1.97) 100.2(2.68) 47.2(1.26) 37.7(1.01) 51.5(1.38) 53.6(1 .43) 47 .6(1 .27) L 6/1/72 7 .7(2 .04) 2.6(0.69) 0.0( - ) 2.8(0.74) 8.4(2.23) 1.9(0.50) O.Ot - ) 10.9(2.89) 40.0(10.62) 15.2(4.04) L 11/2/72 68.7(0 .98) 46.4(0.67) 94.1(1 .35) 130.9(1.88) 148.8(2.14) 228.0(3.28) 153.1(2.20) 113.5(1.63) 37.9(0.54) 19.9(0.29) P 10/2/72 29.2(0.91) 13.9(0.43) 52.7(1.65) 38.3(1.20) 120.4(3.77) 215.6(6.75) 226.8(7.10) 123.6(3.87) 51.9(1.62) 53.3(1 .67) A 8/2/72 105.1(1.00) 96.4(0.92) 114.1(1.08) 126.9(1.20) 111.0(1.05) 154.9(1.47) 107.5(1.02) 113.9(1.08) 121.3(1.15) 167.7(1.59) X 1.24 0.80 0.95 1.15 1.83 2.07 1.72 1.72 2.23 1.43 + 0.21 0.12 0.16 0.17 0.33 0.69 0.70 0.36 1.05 0. S? " RATIOS OF MEAN OF 3 CONTROL PERIODS + ALL PERIODS ARE OF 15 MINS. DURATION - 155 -DISCUSSION The b i o l o g i c a l l y a c t i v e g a s t r o i n t e s t i n a l p o l y p e p t i d e s r e p r e s e n t a g r o u p o f m o i e t i e s w i t h i n w h i c h some a r e e s t a b l i s h e d hormones w h i l e o t h e r s have a l e s s w e l l d e f i n e d s t a t u s . S e c r e t i n , c h o l e c y s t o k i n i n - p a n c r e o z y m i n ( C C K - P Z ) and g a s t r i n a r e a c c e p t e d hormones whose p h y s i o l o g i c a l a c t i v i t i e s have been e x t e n s i v e l y s t u d i e d and whose c h e m i c a l s t r u c t u r e s a r e known. The a m i n o a c i d s e q u e n c e s o f g a s t r i n ( G r e g o r y e t a I. ( 1 9 6 4 ) ) and C C K - P Z ( M u t t and J o r p e s ( 1 9 7 2 ) ) i n p a r t i c u l a r , showed c o m p l e t e i d e n t i t y i n t h e i r C - t e r m i n a l s e q u e n c e s and g a v e a b i o c h e m i c a l b a s i s f o r t h e p r e v i o u s l y d e s c r i b e d o v e r -l a p p i n g o f some o f t h e a c t i o n s o f t h e s e h o r m o n e s . Of t h e l e s s w e l l d e f i n e d p o l y p e p t i d e s o f g a s t r o i n t e s t i n a l o r i g i n , g a s t r i c i n h i b i t o r y p o l y -p e p t i d e ( G . I . P . ) (Brown and D r y b u r g h ( 1 9 7 1 ) ) and s u b s t a n c e P (Chang e t a l . ( 1 9 7 1 ) ) have been s e q u e n c e d w h i l e a v a s o a c t i v e i n t e s t i n a l p o l y p e p t i d e has been p u r i f i e d and i t s amino a c i d c o m p o s i t i o n e l u c i d a t e d ( S a i d and M u t t ( 1 9 7 0 ) . S i m i l a r i t y i n amino a c i d s e q u e n c e , f o r e x a m p l e between G . I . P . and g l u c a g o n , i s i n f o r m a t i o n w h i c h may l e a d t o t h e b e t t e r u n d e r s t a n d i n g o f t h e p h y s i o -l o g i c a l a c t i o n s o f t h e s e p o l y p e p t i d e s p a r t i c u l a r l y i n a r e a s where t h e s e a c t i v i t i e s o v e r l a p . I t i s c l e a r t h a t k n o w l e d g e o f t h e a m i n o a c i d s e q u e n c e o f a new g a s t r o i n t e s t i n a l p o l y p e p t i d e i s n o t o n l y o f b i o c h e m i c a l i n t e r e s t b u t may s e r v e t o d i r e c t i n v e s t i g a t i o n s i n t o p h y s i o l o g i c a l a c t i o n s w h i c h m i g h t o t h e r w i s e n o t have been o b v i o u s . M o t i l i n i s a new g a s t r o i n t e s t i n a l p o l y p e p t i d e and t h i s s t u d y commenced w i t h i t s i s o l a t i o n i n a p u r e f o r m s u i t a b l e f o r c h e m i c a l a n a l y s i s . The f r a c t i o n a t i o n o f p r e s e c r e t i n on CM c e l l u l o s e y i e l d e d a p r o d u c t whose a c t i v i t y was 40 t i m e s t h a t o f t h e p r e s e c r e t i n and t h e r e f o r e r e p r e s e n t e d a s i g n i f i c a n t d e g r e e o f p u r i f i c a t i o n . The 2 s u b s e q u e n t p u r i f i c a t i o n s t a g e s y i e l d e d a p r o d u c t (M3) whose a c t i v i t y was 8 , 0 0 0 u n i t s p e r mg. and w h i c h was — I 56 — s u i t a b l e f o r p r e l i m i n a r y p h y s i o l o g i c a l s t u d i e s (Brown e t a l . ( 1 9 7 1 ) ) . H o w e v e r , amino a c i d a n a l y s e s d e m o n s t r a t e d t h a t t h i s m a t e r i a l was n o t p u r e and t h e r e f o r e n o t i n a s u i t a b l e s t a t e f o r c h e m i c a l a n a l y s i s . F u r t h e r p u r i f i c a t i o n o f t h e p o l y p e p t i d e was t h u s u n d e r t a k e n . An a t t e m p t t o f r a c t i o n a t e M3 on SE Sephadex f a i l e d , but i n d e s a l t i n g t h e p r o d u c t on Sephadex G - 2 5 an a p p r e c i a b l e r e s o l u t i o n i n t o two o p t i c a l d e n s i t y p e a k s was n o t e d . A p r o t o c o l was t h e r e f o r e e s t a b l i s h e d i n w h i c h M3 was f r a c t i o n a t e d o n S e p h a d e x G - 2 5 w i t h ammonium a c e t a t e i n t h e s a m p l e z o n e . T h i s r e s u l t e d i n e q u i v a l e n t r e s o l u t i o n , ( F i g . 1 4 ) . E x p l a n a t i o n s o f t h i s phenomenon a r e o f n e c e s s i t y s p e c u l a t i v e b u t t h e a f f i n i t y o f d e x t r a n g e l s f o r ir - e l e c t r o n r i c h compounds i s w e l l known ( J a n s o n ( 1 9 6 7 ) ) . The a r o m a t i c c h a r a c t e r o f t h e N-t e r m i n a l r e g i o n o f m o t i l i n wou ld have c o n t r i b u t e d t o i t s r e t a r d a t i o n o n t h e g e l and t h e p r e s e n c e o f ammonium a c e t a t e m i g h t have i n t e r f e r e d w i t h t h i s a d s o r p t i o n t h u s a c c e l e r a t i n g i t s r e s o l u t i o n f r o m t h e o t h e r f r a c t i o n . The m a t e r i a l i s o l a t e d by t h i s p r o c e d u r e (M4) was s u b j e c t e d t o a n o t h e r p a s s a g e t h r o u g h S e p h a d e x G - 2 5 i n o r d e r t o e n s u r e c o m p l e t e r e m o v a l o f s a l t and t h e p r o d u c t (M5) e l u t e d a s a s i n g l e peak ( F i g . 1 5 ) p o s s e s s i n g b i o l o g i c a l a c t i v i t y i n e x c e s s o f 1 0 , 0 0 0 u n i t s p e r mg. The y i e l d o f M5 (32$ o f t h e M3) a l o n g w i t h t h e h i g h u n i t a g e o f t h i s m a t e r i a l l e d t o t h e p r e m i s e t h a t a s i g n i f i c a n t d e g r e e o f p u r i f i c a t i o n had been a c h i e v e d o v e r t h e M3 s t a g e . A c c o r d i n g l y , t e s t s o f p u r i t y were a p p l i e d . T h i n l a y e r c h r o m a t o g r a p h y o n s i l i c a g e l , whose p r i n c i p l e i s d i f f e r e n t f r o m t h e s i z e and c h a r g e s e p a r a t i o n s w h i c h had been p e r f o r m e d up t o t h e M5 s t a g e and w h i c h t h e r e f o r e m i g h t have been e x p e c t e d t o d e m o n s t r a t e any r e m a i n i n g i m p u r i t y , r e s u l t e d i n a s i n g l e n i n h y d r i n - p o s i t i v e s p o t . P o l y a c r y l a m i d e g e l e l e c t r o p h o r e s i s y i e l d e d a s i n g l e band ( F i g . 1 6 ) and d a n s y l a t i o n had r e v e a l e d a c l e a r p h e n y l a l a n i n e N - t e r m i n a l r e s i d u e . Amino a c i d a n a l y s e s were p e r f o r m e d a c r o s s t h e M5 e l u t i o n p e a k and t h e r e s u l t s ( t a b l e I) i n d i c a t e d t h a t t h i s p r o d u c t was - 157 -homogeneous . These c r i t e r i a l e d t o t h e t e n e t t h a t M5 ( h e r e i n a f t e r r e f e r r e d t o a s m o t i l i n ) was a p u r e p o l y p e p t i d e . The amino a c i d c o m p o s i t i o n i n d i c a t e d a 22 amino a c i d r e s i d u e p o l y -p e p t i d e and d i g e s t i o n w i t h c a r b o x y p e p t i d a s e A ( F i g . 1 7 ) r e v e a l e d a g l u t a m i n e C - t e r m i n a l r e s i d u e . O x i d a t i o n o f m o t i l i n w i t h p e r f o r m i c a c i d and s u b s e q u e n t amino a c i d a n a l y s e s o f t h e o x i d i s e d p r o d u c t c o n f i r m e d t h e a m i n o a c i d c o m p o s i -t i o n w i t h r e s p e c t t o t h e p r e s e n c e o f a s i n g l e m e t h i o n i n e r e s i d u e . T h i s i n f o r m a t i o n , t o g e t h e r w i t h t h e k n o w l e d g e t h a t m e t h i o n i n e was n e i t h e r N - no r C - t e r m i n a l l e d t o t h e use o f c y a n o g e n b r o m i d e t o c l e a v e t h e m o l e c u l e a t t h e m e t h i o n i n e r e s i d u e . The e x p e c t e d two p e p t i d e s were p r o d u c e d and were a l i g n e d a s a N - t e r m i n a l p e p t i d e (CNI ) and a C - t e r m i n a l p e p t i d e ( C N 2 ) . The p r e s e n c e o f 4 b a s i c r e s i d u e s s u g g e s t e d t h a t d i g e s t i o n o f m o t i l i n w i t h t r y p s i n w o u l d y i e l d 5 p e p t i d e s . 6 p e p t i d e s were p r o d u c e d by t h i s method i n d i c a t i n g t h a t s l o w e r r a t e s o f c l e a v a g e were o c c u r i n g a t o n e s u s c e p t i b l e p e p t i d e b o n d . The k n o w l e d g e o f t h e a m i n o a c i d c o m p o s i t i o n s and N - t e r m i n a l r e s i d u e s o f t h e s e 8 p e p t i d e s ( T a b l e s II and IV) e n a b l e d p r e c i s e a l i g n m e n t o f t h e t r y p t i c p e p t i d e s i n t h e m o l e c u l e t o be made , ( F i g . 5 9 ) . The a m i n o a c i d c o m p o s i t i o n s o f CNI and TRI were i d e n t i c a l e x c e p t f o r t h e m e t h i o n i n e r e s i d u e o f CNI w h i c h was c l e a r l y t h e C - t e r m i n a l r e s i d u e f o r t h a t p e p t i d e . T r y p t i c p e p t i d e TRI was t h e r e f o r e p l a c e d a t t h e N - t e r m i n a l o f t h e m o l e c u l e and i t s C - t e r m i n a l a r g i n i n e r e s i d u e p o s i t i o n e d b e f o r e t h e m e t h i o n i n e . The o n l y p e p t i d e w i t h a m e t h i o n i n e N - t e r m i n a l r e s i d u e was TR3 and t h i s p e p t i d e was p o s i t i o n e d a f t e r TRI i n t h e a l i g n m e n t . Of t h e r e m a i n i n g p e p t i d e s o n l y TR5 had an N - t e r m i n a l g l u t a m i c a c i d t h u s p r e c l u d i n g i t s p o s i t i o n i n g a t t h e C - t e r m i n a l end o f t h e m o l e c u l e . I t was t h e r e f o r e p o s i t i o n e d a f t e r T R 5 , and TR4 was c l e a r l y t h e C - t e r m i n a l t r y p t i c p e p t i d e . P e p t i d e s TR6 and TR2 were i d e n t i f i e d a s t h e N - and C - t e r m i n a l h a l v e s o f TR4 and were p o s i t i o n e d a c c o r d i n g l y . - 158 -PHE • < PHE •CN1-" T R I • MET -ARG - T R 3 -MET - •LYS < - T R 5 - » G L U - A R 6 T R 4 -< - T R 6 - » A S X - L Y S - G L N GLY — GLN F i g u r e 5 9 . The a l i g n m e n t o f t h e t r y p t i c p e p t i d e s o f m o t i l i n . - 159 -C h y m o t r y p t i c and t h e r m o l y s i n d i g e s t i o n s were p e r f o r m e d and CN2 was c l e a v e d w i t h t r y p s i n . The p e p t i d e s p r o d u c e d by t h e s e p r o c e d u r e s a l o n g w i t h t h e t r y p t i c and c y a n o g e n b r o m i d e p e p t i d e s were s e q u e n c e d and t h e r e s u l t s , a r r a n g e d i n o r d e r t o d i s p l a y o v e r l a p p i n g s e q u e n c e s , a r e s u m m a r i s e d i n F i g . 6 0 . A f t e r t h e a s s i g n m e n t o f a m i d e s t a t u s t o t h e g l u t a m i c and a s p a r t i c a c i d r e s i d u e s t h e u n i q u e s e q u e n c e o f m o t i l i n was r e v e a l e d and i s shown i n F i g . 6 1 . The amino a c i d s e q u e n c e o f m o t i l i n i s r e m a r k a b l e i n t h a t i t b e a r s no r e s e m b l a n c e t o any o f t h e s t r u c t u r a l l y u n d e r s t o o d g a s t r o i n t e s t i n a l p o l y p e p t i d e s . The N - t e r m i n a l r e g i o n o f t h e m o l e c u l e i s h y d r o p h o b i c w h i l e t h e C - t e r m i n a l h a l f i s b a s i c . The c e n t r a l p o s i t i o n o f t h e s i n g l e m e t h i o n i n e r e s i d u e l e d t o t h e p r e m i s e t h a t i t m i g h t be i m p o r t a n t f o r t h e b i o l o g i c a l a c t i v i t y o f t h e m o l e c u l e . M u t t ( 1 9 6 4 ) had shown t h a t o x i d a t i o n o f t h e m e t h i o n i n e r e s i d u e s o f C C K - P Z w i t h h y d r o g e n p e r o x i d e l e d t o t h e l o s s o f a t l e a s t 98% o f t h e a c t i v i t y o f t h i s hormone . O t h e r s h a v e shown t h a t s i m i l a r b e h a v i o u r c a n be e l i c i t e d f r o m o t h e r h o r m o n e s , M . S . H . ( L o , ( 1 9 6 2 ) ) , A . C . T . H . (Dedman e t a l . ( 1 9 5 7 ) ) and t h e p a r a t h y r o i d hormone (Rasmussen and C r a i g ( 1 9 6 2 ) ) . M u t t f u r t h e r showed t h a t r e d u c t i o n o f h y d r o g e n p e r o x i d e o x i d i s e d C C K - P Z l e d t o r e s t o r a t i o n o f f u l l a c t i v i t y . I n a c t i v a t i o n o f t h e s e hormones r e s u l t e d f r o m t h e o x i d a t i o n o f m e t h i o n i n e r e s i d u e s t o t h e c o r r e s p o n d i n g s u l p h o x i d e . R e d u c t i o n r e s t o r e s t h e m e t h i o n i n e r e s i d u e and t h e b i o l o g i c a l a c t i v i t y . M o t i l i n , when o x i d i s e d w i t h h y d r o g e n p e r o x i d e , s u f f e r e d a 9556 l o s s o f a c t i v i t y w h i c h was r e s t o r e d o n r e d u c t i o n w i t h c y s t e i n e . S t r o n g e r o x i d a t i o n o f j h e m e t h i o n i n e r e s i d u e t o t h e s u l p h o n e w i t h p e r f o r m i c a c i d r e s u l t e d i n an e q u i v a l e n t l o s s o f a c t i v i t y . The m e t h i o n i n e r e s i d u e o f m o t i l i n i s e v i d e n t l y e s s e n t i a l f o r f u l l b i o l o g i c a l a c t i v i t y . - 160 -CN1 PHE - V A L - P R O - I L E - PHE-THR - T Y R - 6 L Y - G U I - L E U - G L N - ARG - MET* T R I PHE - V A L - P R O - I L E -PHE—THR -TYR - G L Y - G L U - LEU - GLN — ARG CHT2WW3 - PHE - V A L - P R O - I L E - P H E Th 2 PHE - V A L - P R O - I L E - P H E - T H R CHT2WW2 THR-TYR Th 4 TYR - GLY - GLU CHT1 G L Y - G L U - L E U - G L N Th 7 LEU - G L N - A R G —MET-GLU —GLU — LYS —GLU - A R G - A S N - L Y S — GLY — GLN TR 3 MET-GLU - G L U —LYS CN 2 GLU - GLU -LYS - G L U - A R G - A S N - L Y S -GLY —GLN C N 2 T R 2 GLU - GLU —LYS C N 2 T R 3 GLU - A R G TR 5 GLU - A R G T R 4 A S N - L Y S - G L Y - G L N C N 2 T R 1 A S N - L Y S - G L Y - G L N TR 6 A S N - L Y S TR 2 GLY - G L N F i g u r e 6 0 . Amino a c i d s e q u e n c e s o f m o t i l i n p e p t i d e s a r r a n g e d i n o r d e r t o d i s p l a y o v e r l a p p i n g s e q u e n c e s . * i d e n t i f i e d a s h o m o s e r i n e . - 1 6 1 -5 10 IS 22 I I I > HjN - P H E - V A L - P R O - I LE — PHE—THR -TYR —GLY —GLU —LEU -GLN — ARG —MET —GLU -GLU -LYS - G L U - A R G - A S N - L Y S -GLY - G L N - C O O H F i g u r e 6 1 . The c o m p l e t e a m i n o a c i d s e q u e n c e o f m o t i l i n . - 162 -O t h e r c h e m i c a l m o d i f i c a t i o n s were made t o m o t i l i n i n o r d e r t o d e t e r m i n e w h e t h e r o t h e r f e a t u r e s o f t h e m o l e c u l e were i m p o r t a n t f o r i t s b i o l o g i c a l a c t i v i t y . The two C - t e r m i n a l amino a c i d r e s i d u e s were removed by d i g e s t i o n w i t h c a r b o x y p e p t i d a s e A and t h i s r e s u l t e d i n no d e t e c t a b l e l o s s o f b i o l o g i c a l a c t i v i t y ( F i g . 2 8 ) . I t was d e c i d e d t o i n v e s t i g a t e t h e n e c e s s i t y f o r t h e i n t a c t d i s t r i b u t i o n o f p o i n t c h a r g e s w i t h i n t h e m o l e c u l e . The C - t e r m i n a l end o f m o t i l i n p o s s e s s e s two l y s i n e r e s i d u e s and a c c o r d i n g l y t h e s e were m o d i f i e d by a c y l a t i o n . A c e t y l a t i o n o f t h e m o l e c u l e r e s u l t e d i n a p r o d u c t w h i c h had l o s t 90% o f i t s b i o l o g i c a l a c t i v i t y w h i l e s u c c i n y l a t i o n r e s u l t e d i n a l o s s o f a t l e a s t 95% o f t h e a c t i v i t y . I t i s known t h a t a c e t y l a t i o n o f amino g r o u p s r e s u l t s i n an a c y l d e r i v a t i v e b e a r i n g no c h a r g e w h i l e s u c c i n l y l a t e d d e r i v a t i v e s b e a r a u n i t n e g a t i v e c h a r g e . The c l e a r p r o g r e s s i o n p r e s e n t e d h e r e , r a n g i n g f r o m f u l l b i o l o g i c a l a c t i v i t y w i t h i n t a c t p o s i t i v e l y c h a r g e d N - t e r m i n a l and e - a m i n o g r o u p s t o 10$ a c t i v i t y w i t h n e u t r a l a c e t y l a t e d amino g r o u p s and t o l e s s t h a n 5% a c t i v i t y w i t h n e g a t i v e l y c h a r g e d s u c c i n y l g r o u p s , o f f e r s some i n s i g h t i n t o t h e n a t u r e o f t h e r e c e p t o r s y s t e m f o r m o t i l i n . Two i n t e r p r e t a t i o n s o f t h i s e f f e c t a r e p o s s i b l e . I t i s known t h a t s m a l l p o l y p e p t i d e s c a n have c o n s i d e r a b l e t h r e e d i m e n s i o n a l s t r u c t u r e ( V e n k a t a c h o c l a m and Ramachandran ( 1 9 6 9 ) ) . The h y p o t h e s i s c a n be a d v a n c e d t h a t i n t r a m o l e c u l a r i o n i c b o n d i n g w i t h i n t h e m o t i l i n m o l e c u l e s t a b i l i z e s i t s c o n f o r m a t i o n i n s u c h a way a s t o p r e s e n t e s s e n t i a l s t r u c t u r e s t o t h e r e c e p t o r s i t e and s o a c h i e v e f u l l b i o l o g i c a l a c t i v i t y . The r e v e r s a l o f s i d e c h a i n c h a r g e s w o u l d c r e a t e i n t r a m o l e c u l a r r e p u l s i o n t h u s d i s t o r t i n g t h e c o n f o r m a t i o n and c o n t r i b u t i n g t o w a r d t h e o b s e r v e d l o s s o f a c t i v i t y . If i t i s s u b s e q u e n t l y shown t h a t m o t i l i n d o e s n o t p o s s e s s an i n t e r n a l l y f o l d e d s t r u c t u r e b u t e x i s t s s u b s t a n t i a l l y i n an o p e n c h a i n a - h e l i x c o n f o r m a t i o n , a n o t h e r e x p l a n a t i o n must be a d v a n c e d . - 163 -The o t h e r p o s s i b l e c o n s t r u c t i o n w h i c h c a n be p u t upon t h e s e r e s u l t s i s t h a t t h e r e c e p t o r s i t e i t s e l f b e a r s n e g a t i v e c h a r g e s w h i c h i n t e r a c t w i t h t h e i n t a c t p o s i t i v e p o i n t c h a r g e s o f m o t i l i n . Removal o r r e v e r s a l o f t h e s e c h a r g e s w o u l d o f n e c e s s i t y i n t e r f e r e w i t h s u c h i n t e r a c t i o n s and l e a d t o r e d u c t i o n o r l o s s o f b i o l o g i c a l a c t i v i t y . The n e c e s s i t y f o r t h e i n t a c t d i s t r i b u t i o n o f p o i n t c h a r g e s i n m o t i l i n h a v i n g been t h u s d e m o n s t r a t e d , i t was d e c i d e d t o i n v e s t i g a t e t h e i m p o r t a n c e o f t h e a r o m a t i c N - t e r m i n a l end o f t h e m o l e c u l e . To t h i s e n d , t h e p h e n y l a l a n i n e N - t e r m i n a l r e s i d u e was removed by o n e c y c l e o f Edman d e g r a d a t i o n . The p r o d u c t was a t l e a s t 90$ i n a c t i v a t e d b u t i n t e r p r e t a t i o n o f t h i s r e s u l t must be made c a r e f u l l y . I t i s p o s s i b l e t h a t t h e a r o m a t i c n a t u r e o f t h e N - t e r m i n a l r e g i o n o f m o t i l i n i s i m p o r t a n t and t h a t r e m o v a l o f t h e p h e n y l a l a n i n e r e s i d u e d o e s c o n t r i b u t e t o t h e r e d u c t i o n i n a c t i v i t y . H o w e v e r , d u r i n g t h e Edman r e a c t i o n s i t i s known t h a t t h e e - a m i n o g r o u p s o f l y s i n e a r e a c y i a t e d t o t h e c o r r e s p o n d i n g e - p h e n y I t h i o c a r b a m y I d e r i v a t i v e s . I t i s e q u a l l y p o s s i b l e t h e r e f o r e t h a t a c y l a t i o n o f t h e l y s i n e r e s i d u e s o f m o t i l i n d u r i n g t h e Edman p r o c e d u r e c o n t r i b u t e d t o t h e o b s e r v e d l o s s o f a c t i v i t y . I n v e s t i g a t i o n s i n t o t h e p h y s i o l o g i c a l a c t i o n s o f m o t i l i n began w i t h a s e r i e s o f e x p e r i m e n t s i n w h i c h m o t i l i n was i n f u s e d i n t o c h r o n i c dog p r e p a r a t i o n s a t two d o s e l e v e l s . The d o s e s used w e r e 1 . 0 and 2 . 0 u g / k g - h r . B o t h d o s e l e v e l s p r o d u c e d s i g n i f i c a n t i n c r e a s e s i n f u n d i c pouch m o t o r a c t i v i t y w i t h i n o n e p e r i o d o f t h e b e g i n n i n g o f t h e i n f u s i o n ( F i g s . 3 2 and 3 3 ) . The s e c r e t i.cux o f H + i o n was n o t s t i m u l a t e d by e i t h e r d o s e o f m o t i l i n ( F i g . 34 and 3 5 ) , t h e s t a t i s t i c a l s i g n i f i c a n c e o f t h e v a l u e s f o r t h e h i g h e r d o s e b e i n g a c o n s e q u e n c e o f t h e d a t a f o r m a t u s e d . R e f e r e n c e t o t h e d a t a ( t a b l e s XI and X I I ) c l a r i f i e s t h i s p o i n t . P e p s i n s e c r e t i o n was s i g n i f i c a n t l y - 164 -I n c r e a s e d a t t h e h i g h e r d o s e ( F i g . 3 7 ) , w h i l e t h e v a r i a b i l i t y o f t h e d a t a a t t h e l o w e r d o s e l e v e l ( F i g . 3 6 ) p r e c l u d e d t h e a t t a c h m e n t o f any s t a t i s t i c a l s i g n i f i c a n c e t o t h e r e s p o n s e . I t i s e v i d e n t t h o u g h t h a t an i n c r e a s e o f p e p s i n o u t p u t i s o c c u r r i n g i n r e s p o n s e t o m o t i l i n . A n t r a l pouch m o t i l i t y s u f f e r e d f r o m v a r i a b i l i t y i n t h e d a t a ( F i g s . 3 8 and 39) c a u s e d , i n p a r t , by t h e r e c o r d i n g t e c h n i q u e u s e d . The r e s u l t s i n d i c a t e , h o w e v e r , t h a t t h e m o t i l i t y i s c e r t a i n l y p e r t u r b e d d u r i n g t h e i n f u s i o n o f m o t i l i n , and t h e h i g h e r d o s e r e s u l t e d i n a s i g n i f i c a n t i n c r e a s e i n t h i s p a r a m e t e r . T h i s s e r i e s o f e x p e r i m e n t s d e m o n s t r a t e d t h a t p r o b a b l y t h e most s p e c i f i c a c t i o n o f m o t i l i n i s t o s t i m u l a t e f u n d i c m o t o r a c t i v i t y . H + i o n i s c l e a r l y n o t s t i m u l a t e d a l t h o u g h m o t i l i n c a u s e d a n i n c r e a s e i n i t s o u t p u t . T h i s i n c r e a s e was p r o b a b l y due t o t h e i n c r e a s e d m o t o r a c t i v i t y o f t h e pouch w h i c h c o u l d e x p r e s s more H + i o n t h a n wou ld a p p e a r d u r i n g p e r i o d s o f q u i e s c e n c e . The same a r g u m e n t c a n be a d v a n c e d f o r t h e i n c r e a s e i n p e p s i n o u t p u t , h o w e v e r , t h e r e s u l t s d i s c u s s e d h e r e do n o t a l l o w any c o n c l u s i o n t o be made. The most s t r i k i n g f e a t u r e o f t h i s s e r i e s o f e x p e r i m e n t s was t h e o b v i o u s t a c h y p h y l a x i s w h i c h o c c u r e d t o t h e f u n d i c m o t o r r e s p o n s e t o m o t i l i n . Hav i ng eI i m i n a t e d t h e p o s s i b i I i t y t h a t mot i I i n , i n d i I u t e so I u t i on i n s a I i ne a t room t e m p e r a t u r e , w o u l d decompose d u r i n g o n e h o u r , a s e r i e s o f e x p e r i -ments was d e s i g n e d i n o r d e r t o i n v e s t i g a t e t h i s d e c r e m e n t i n t h e r e s p o n s e . To t h i s e n d , m o t i l i n was a d m i n i s t e r e d t o c h r o n i c dogs i n t h e f o r m o f an i n t e r r u p t e d i n f u s i o n . The r e s u l t s o f t h e i n t e r r u p t e d i n f u s i o n s t u d y d e m o n s t r a t e d t h a t t h e f u n d i c and a n t r a l m o t o r r e s p o n s e s ( F i g s . 4 0 and 4 3 ) were r e d u c e d d u r i n g t h e s e c o n d i n f u s i o n so t h a t a t r u e t a c h y p h y l a x i s was o c c u r i n g . F u r t h e r , t h e H + i o n o u t p u t ( F i g . 4 1 ) s u f f e r e d a p a r a l l e l r e d u c t i o n i n mean v a l u e d u r i n g t h e s e c o n d i n f u s i o n l e n d i n g w e i g h t t o t h e p r e m i s e t h a t i t i s b e i n g e x p r e s s e d a s a r e s u l t o f t h e m o t o r a c t i v i t y r a t h e r t h a n a c t i v e l y s t i m u l a t e d . The p e p s i n - 165 -r e s u l t s ( F i g . 4 2 ) d i d n o t p r e s e n t any e v i d e n c e f o r o r a g a i n s t t h i s h y p o t h e s i s . The s e r i e s o f e x p e r i m e n t s i n w h i c h m o t i l i n was i n f u s e d a g a i n s t a b a c k -g r o u n d o f H + i o n s e c r e t i o n s t i m u l a t e d by SHGI d e m o n s t r a t e d t h a t m o t i l i n d i d n o t i n h i b i t H + i o n s e c r e t i o n by t h e p o u c h , ( F i g . 4 5 ) . On t h e c o n t r a r y , t h e now f a m i l i a r s l i g h t i n c r e m e n t i n H + i o n o u t p u t was s e e n . The o t h e r p a r a m e t e r s , f u n d i c m o t i l i t y ( F i g . 4 4 ) , a n t r a l m o t i l i t y ( F i g . 4 7 ) and p e p s i n o u t p u t ( F i g . 4 6 ) , showed no a l t e r a t i o n i n t h e i r p a t t e r n s o f r e s p o n s e f r o m t h a t o b s e r v e d i n t h e f i r s t s e r i e s o f e x p e r i m e n t s . E v i d e n t l y a h i g h l e v e l o f H + i o n s e c r e t o r y a c t i v i t y i n no way i n t e r f e r e s w i t h t h e a c t i o n s o f m o t i l i n . In o r d e r t o t e s t t h e r e c u r r i n g q u e s t i o n o f w h e t h e r p e p s i n o u t p u t i s s t i m u l a t e d by m o t i l i n o r m e r e l y e x p r e s s e d a s a r e s u l t o f t h e i n c r e a s e d m o t o r a c t i v i t y , m o t i l i n was i n f u s e d a g a i n s t a b a c k g r o u n d p e p s i n s e c r e t i o n s t i m u l a t e d by s e c r e t i n . The H + i o n ( F i g . 4 9 ) and a n t r a l m o t i l i t y ( F i g . 5 1 ) r e s p o n s e s were t y p i c a l , b u t p e p s i n o u t p u t ( F i g . 5 0 ) was i n c r e a s e d more t h a n was s e e n w i t h t h e same d o s e o f m o t i l i n i n t h e a b s e n c e o f s e c r e t i n ( F i g . 3 6 ) . T h e s e r e s u l t s m i g h t l e n d w e i g h t t o t h e p r e m i s e t h a t p e p s i n i s b e i n g e x p r e s s e d by t h e m o t o r a c t i v i t y , h o w e v e r , i t c a n a l s o be a r g u e d t h a t m o t i l i n a c t s s y n e r g i s t i c a I Iy w i t h s e c r e t i n . R e g a r d i n g t h e a p p a r e n t l y l a r g e i n c r e a s e i n f u n d i c m o t o r a c t i v i t y , i t must be remembered t h a t s e c r e t i n i n h i b i t s t h e normal s p o n t a n e o u s m o t o r a c t i v i t y o f t h e f u n d i c pouch s o t h a t t h e ' n o i s e ' l e v e l o f t h e c o n t r o l p e r i o d s i s c o n s i d e r a b l y l o w e r t h a n i t wou ld be w i t h o u t s e c r e t i n . The f o r m a t o f t h i s d a t a t h e r e f o r e a m p l i f i e s t h e c o n t r a s t between s t i m u j a t e d and u n s t i m u l a t e d m o t i l i t y . The s u s c e p t i b i l i t y o f t h e m o t i l i n r e s p o n s e t o c h o l i n e r g i c and g a n g l i o n i c b l o c k a d e was t e s t e d w i t h a s e r i e s o f e x p e r i m e n t s i n w h i c h i n j e c t i o n s o f m o t i l i n were g i v e n b e f o r e and a f t e r t h e a d m i n i s t r a t i o n o f - 166 -b l o c k i n g d r u g s . The c r i t e r i a a p p l i e d t o o b t a i n a s i g n i f i c a n t l e v e l o f b l o c k a d e were t h a t t h e n i c t i t a t i n g membrane s h o u l d be e l e v a t e d , and t h a t s p o n t a n e o u s m o t o r a c t i v i t y . o f t h e pouch s h o u l d be a b o l i s h e d . The d o s e s o f b l o c k i n g d r u g s were c h o s e n a c c o r d i n g l y . The r e s u l t s d e m o n s t r a t e d t h a t c h o l i n e r g i c b l o c k a d e w i t h a t r o p i n e c a u s e d a s e v e r e d e c r e m e n t i n t h e f u n d i c m o t o r a c t i v i t y r e s p o n s e t o m o t i l i n ( F i g . 5 2 ) . G a n g l i o n i c b l o c k a d e w i t h a n s o l y s e n ( F i g . 5 3 ) d i d n o t r e s u l t i n any s i g n i f i c a n t a l t e r a t i o n t o t h e m o t o r a c t i v i t y r e s p o n s e t o m o t i l i n . V e r y l i t t l e i n f o r m a t i o n c a n be g a t h e r e d f r o m t h e H + i o n and p e p s i n o u t p u t r e s u l t s f r o m t h i s s e r i e s o f e x p e r i m e n t s , b u t t h e r e s u l t s a r e d i s p l a y e d ( F i g s . 5 4 - 5 7 ) f o r c o m p l e t e n e s s . I t i s p r o b a b l e t h a t m o t i l i n d o e s n o t e x e r t i t s a c t i o n a t t h e p r e g a n g l i o n i c l e v e l . The e x a c t mechan ism o f i t s a c t i o n r e m a i n s u n c l e a r , e x c e p t t o s a y t h a t i t i s a p p a r e n t l y c h o l i n e r g i c a I Iy m e d i a t e d . S e v e r a l h y p o t h e s e s on t h e mechan ism c a n be p o s t u l a t e d , t h e s i m p l e s t b e i n g t h a t m o t i l i n s t i m u l a t e s t h e m u s c a r i n i c r e c e p t o r on t h e smooth m u s c l e d i r e c t l y . O b j e c t i o n s t o t h i s p o s t u l a t e a r i s e i n l i g h t o f t h e a p p a r e n t s p e c i f i c i t y o f a c t i o n o f m o t i l i n . I t m i g h t a l s o be a r g u e d t h a t , a t a m o l e c u l a r l e v e l , t h e l i k e l i h o o d o f a p o l y p e p t i d e s t i m u l a t i n g a r e c e p t o r w h i c h a l s o r e s p o n d s t o t h e c h o l i n e b a s e s i s r e m o t e . T h e r e r e m a i n s t h e n , t h e e q u a l l y u n l i k e l y p o s s i b i l i t y t h a t g a s t r i c smooth m u s c l e p o s s e s s e s a s p e c i f i c m o t i l i n r e c e p t o r w h i c h c a n be b l o c k e d w i t h a t r o p i n e . W i t h o u t b e i n g t o o p e r f u n c t o r y i n t h e d i s m i s s a l o f t h e s e a r g u m e n t s i t wou ld a p p e a r t h a t more p l a u s i b l e m e c h a n i s m s f o r t h e a c t i o n o f m o t i l i n c a n be a d v a n c e d i f i t were n o t r e q u i r e d t o . a c t d i r e c t l y b u t by e i t h e r r e l e a s i n g an i n t e r m e d i a t e o r by some a c t i o n on n e r v o u s t i s s u e . One o f t h e most s t r i k i n g r e s u l t s o b t a i n e d i n t h i s s t u d y i s t h e t a c h y p h y l a x i s w h i c h o c c u r s i n t h e f u n d i c m o t o r a c t i v i t y r e s p o n s e t o m o t i l i n . A f t e r t h i r t y m i n u t e s w i t h no i n f u s i o n , t h e r e s p o n s e t o m o t i l i n i s c l e a r l y r e d u c e d ( F i g . 4 0 ) . T h a t t h i s m i g h t s u g g e s t d e p l e t i o n o f a - 167 -s e c r e t e d m o i e t y i s e v i d e n t . The i n t e r m e d i a t e wou ld have t o be s t o r e d a t some s i t e , r e l e a s e d by an a c t i o n o f m o t i l i n a t a r e c e p t o r , and have a s t i m u l a t i n g a c t i o n on smooth muse I e w h i c h wou ld be b l o c k e d by a t r o p i n e . T h e s e c r i t e r i a c l e a r l y i n d i c a t e a c e t y l c h o l i n e a s t h e i n t e r m e d i a t e . A n o t h e r mechan ism w h i c h c a n be p o s t u l a t e d i s t h a t t h e i n t e r m e d i a t e i t s e l f , w h i l e b e i n g r e l e a s e d c o n t i n u o u s l y i n t h e p r e s e n c e o f m o t i l i n , has a s t i m u l a t i n g a c t i o n o n smooth m u s c l e w h i c h u n d e r g o e s t a c h y p h y l a x i s . T h e s e c r i t e r i a f o r t h e i d e n t i t y o f an i n t e r m e d i a t e a r e n o t a s e a s y t o f u l f i l a s i n t h e p r e v i o u s c a s e , h o w e v e r , i t i s i n t e r e s t i n g a t t h i s j u n c t u r e t o c o n s i d e r some o f t h e p h y s i o l o g i c a l e f f e c t s o f 5 - h y d r o x y t r y p t a m i n e ( 5 - H T ) . I t i s known t h a t 5 -HT c a u s e s c o n t r a c t i o n i n a w i d e v a r i e t y o f smooth m u s c l e p r e p a r a t i o n s ( B o r n ( 1 9 7 0 ) ) . I t i s f u r t h e r known t h a t a p r o g r e s s i v e d e c l i n e i n r e s p o n s i v e n e s s o f smooth m u s c l e t o 5 -HT i s e x h i b i t e d when t h e same d o s e o f 5 -HT i s g i v e n r e p e a t e d l y ( B u l b r i n g and B u r n s t o c k , ( I 9 6 0 ) ) . W h i l e t h i s e v i d e n c e i s somewhat c i r c u m s t a n t i a l , c o n s i d e r a t i o n o f t h e r o l e o f 5 -HT i n t h e s t i m u l a t i o n o f smooth m u s c l e and e s p e c i a l l y i n i t s b e i n g i m p l i c a t e d a s a n i n t e r m e d i a t e o f m o t i l i n a c t i o n , i s m e r i t e d . A f u r t h e r p r o p e r t y o f 5 -HT b r i n g s a b o u t c o n s i d e r a t i o n o f t h e p o s s i b i l i t y t h a t m o t i l i n m i g h t a c t o n n e r v o u s t i s s u e . Gaddum and P i c a r e l I i ( 1 9 5 7 ) p r o p o s e d t h a t 5 -HT a c t s a t two r e c e p t o r s i n t h e g u i n e a - p i g i l e u m , o n e o f w h i c h i s b l o c k e d by l y s e r g i c a c i d d i e t h y a m i d e (LSD) and i t s a n a l o g u e s , and t h e o t h e r , w h i c h i s b l o c k e d by a t r o p i n e and m o r p h i n e , w h i c h was r e f e r r e d t o a s t h e M r e c e p t o r . T h e s e a u t h o r s c o n c l u d e d t h a t t h e M r e c e p t o r s a r e i n n e r v o u s t i s s u e . The p o s s i b i l i t y e x i s t s t h e n , t h a t m o t i l i n m i g h t a c t a t a n e r v o u s r e c e p t o r s i t e and t h a t t h e ' i n t e r m e d i a t e ' i n i t s m e c h a n i s m o f a c t i o n w o u l d be a t r a n s m i t t e r r e l e a s e d by a c t i o n p o t e n t i a l s i n n e u r o n e s . A c e t y l c h o l i n e w o u l d a g a i n be t h e p r i m e c a n d i d a t e . The s u s c e p t i b i l i t y o f m o t i l i n e f f e c t s t o a t r o p i n e b l o c k a d e c o u l d t h e n be - 168 -l o c a l i s e d t o t h e m u s c a r i n i c s i t e o r t o t h e n e r v o u s r e c e p t o r o r t o b o t h . Much t e s t i n g o f t h e s e h y p o t h e s e s a r e n e c e s s a r y b e f o r e any i n s i g h t i n t o t h e m e c h a n i s m o f a c t i o n o f m o t i l i n c a n be g a i n e d . I t w i l l be n e c e s s a r y t o i n v e s t i g a t e t h e e f f e c t o f m o t i l i n on a c o m p l e t e l y d e n e r v a t e d smooth m u s c l e p r e p a r a t i o n i n o r d e r t o a s s e s s t h e n e c e s s i t y f o r i n t a c t i n t r i n s i c i n n e r v a t i o n i n o r d e r t o s t i m u l a t e c o n t r a c t i o n . E x p e r i m e n t s u s i n g 5 -HT i n h i b i t o r s wou ld i m p l i c a t e o r d i s m i s s 5 -HT a s a p o s s i b l e i n t e r m e d i a t e i n t h e a c t i o n o f m o t i l i n . I t i s i m p o s s i b l e a t t h e p r e s e n t s t a t e o f k n o w l e d g e t o s a y more a b o u t t h e p o s s i b l e m e c h a n i s m s o f a c t i o n o f m o t i l i n , s u f f i c e i t t o add t h a t m o t i l i n i s c l e a r l y an e x t r e m e l y p o t e n t b i o l o g i c a l e n t i t y . L e s s t h a n 0 . 3 x I 0 ~ ^ M . o f t h e p o l y p e p t i d e d i s t r i b u t e d i n t h e e n t i r e p l a s m a v o l u m e o f a dog a r e n e c e s s a r y f o r an e f f e c t . M o t i l i n a p p e a r e d t o have no s e c r e t i n o r c h o I e c y s t o k i n i n e f f e c t s and no a p p a r e n t c a r d i o v a s c u l a r e f f e c t s . The s l i g h t p y r o g e n i c e f f e c t s n o t e d i n some e x p e r i m e n t s may be e x p l a i n e d i n l i g h t o f t h e d e m o n s t r a t i o n (Brown and C o o k , u n p u b l i s h e d o b s e r v a t i o n s ) t h a t m o t i l i n s t i m u l a t e s c o n t r a c t i o n o f i s o l a t e d c i r c u l a r m u s c l e s t r i p s f r o m human c o l o n . A c t i v e l y c o n t r a c t i n g m u s c l e l a y e r s i n t h e v i c i n i t y o f a r e c t a l t e m p e r a t u r e p r o b e w o u l d r e s u l t i n t h e o b s e r v e d i n c r e m e n t i n t e m p e r a t u r e . I t r e m a i n s t o c o n s i d e r t h e p o s s i b l e hormonal s t a t u s o f m o t i l i n and t h e p a r t w h i c h i t m i g h t p l a y i n a d u o d e n a l c o n t r o l o f g a s t r i c m o t o r a c t i v i t y . The e l e v a t i o n o f m o t i l i n t o hormonal s t a t u s must a w a i t i t s i d e n t i f i c a t i o n i n t h e b l o o d and i n i t s presumed t i s s u e o f o r i g i n , t h e d u o d e n a l m u c o s a . I t i s o f i n t e r e s t t o add t h a t s e v e r a l d i f f e r e n t t y p e s o f e n d o c r i n e p o l y p e p t i d e c e l l s have r e c e n t l y been d e s c r i b e d i n t h e human u p p e r i n t e s t i n a l mucosa ( P e a r s e e t a l . ( 1 9 7 0 ) ) . E v i d e n t l y t h e duodenum i s a p o t e n t i a l l y r i c h s o u r c e o f g a s t r o i n t e s t i n a l p o l y p e p t i d e s . I t i s p o s s i b l e - 169 -t h a t m o t i l i n i s t h e hummoral a g e n t r e l e a s e d f r o m t h e d u o d e n a l mucosa when t h e l a t t e r becomes a l k a l i n e (Brown e t a I . ( 1 9 6 6 ) ) . T h i s w o u l d i m p l i c a t e m o t i l i n i n a d u a l humoral c o n t r o l mechan ism f o r g a s t r i c m o t o r a c t i v i t y i n w h i c h low d u o d e n a l pH i n h i b i t s g a s t r i c m o t i l i t y and h i g h d u o d e n a l pH s t i m u l a t e s i t . The low pH h a l f o f t h i s m e c h a n i s m wou ld f u n c t i o n v i a t h e r e l e a s e f r o m t h e d u o d e n a l mucosa o f e n t e r o g a s t r o n e i n i t s s i n g l e o r s e v e r a l f o r m s w h i l e a h i g h d u o d e n a l pH w o u l d r e l e a s e m o t i l i n and so s t i m u l a t e m o t i l i t y . If t h i s s p e c u l a t i o n i s l o g i c a l l y c o n t i n u e d , i t i s p o s s i b l e t o a r g u e t h a t t h e g a s t r i c m o t o r e f f e c t s o f b o t h t h e s e e n t i t i e s wou ld have d i r e c t b e a r i n g o n t h e r a t e o f g a s t r i c e m p t y i n g . In t h i s way a d u a l n e g a t i v e f e e d b a c k s y s t e m wou ld o p e r a t e t o p r o p o r t i o n t h e f l o w o f g a s t r i c c o n t e n t s i n t o t h e duodenum and m a i n t a i n i t a t a n o p t i m a l l e v e l . I t i s l i k e l y t h a t s u c h a mechan ism e x i s t s and d o e s i n f l u e n c e g a s t r i c e m p t y i n g , h o w e v e r , o n a more i n t e g r a t e d l e v e l , i t must be remembered t h a t t h e s e a r g u m e n t s i g n o r e t h e i n f l u e n c e o f n e u r a l c o n t r o l s y s t e m s . The humora l c o n t r o l s y s t e m s a r e p h y l o g e n e t i c a l l y more p r i m i t i v e t h a n t h e i r n e u r a l c o u n t e r p a r t s and t h e r e f o r e p r e s u m a b l y o f l e s s e r o v e r a l l i m p o r t a n c e . In a n y c a s e t h e p r e m i s e r e s t s o n t h e i d e n t i f i c a t i o n i n t h e b l o o d o f t h e a g e n t r e l e a s e d upon a l k a l i n i z a t i o n o f t h e duodenum. If c r o s s i m m u n o r e a c t i v i t y i s d e t e c t e d between s u c h a n a g e n t and a n t i b o d i e s r a i s e d a g a i n s t m o t i l i n , t h e a r g u m e n t w i l l have g a i n e d c o n s i d e r a b l e c r e d e n c e . O t h e r s u p p o r t i n g e v i d e n c e c o u l d be o b t a i n e d i f a p o s s i b l e t a c h y p h y I a c t i c m o t o r a c t i v i t y r e s p o n s e were i n v e s t i g a t e d when d u o -d e n a l pH i s m a i n t a i n e d a t a n e l e v a t e d l e v e l f o r a l o n g e r p e r i o d t h a n was p r e v i o u s l y a t t e m p t e d . In t h e i n t e r i m i t i s o n l y p o s s i b l e t o s t a t e t h a t q u a l i t a t i v e l y , i n t e r m s o f s p e c i f i c e f f e c t and l a t e n t c y o f a c t i o n t h e e f f e c t s o f t h e a l k a l i n e r e l e a s e d humoral a g e n t and m o t i l i n a p p e a r t o be t h e same. - 170 -BIBLIOGRAPHY 1 . A m b l e r , R . P . ; The Amino a c i d s e q u e n c e o f Pseudomonas c y t o c h r o m e C - 5 5 1 . B i o c h e m . J . 8 9 , 349 ( 1 9 6 3 ) 2 . A n s o n , M . L . and M i r s k y , A . E . ; The e s t i m a t i o n o f p e p s i n f r o m h a e m o g l o b i n . J . G e n . P h y s i o l . J_6, 59 ( 1 9 3 2 ) 3 . B a i l e y , N . T . J . ; i n S t a t i s t i c a l M e t h o d s i n B i o l o g y , 1 7 3 , ( E n g l i s h U n i v e r s i t i e s P r e s s , L o n d o n , 1 9 5 9 ) . 4 . 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