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UBC Theses and Dissertations

Fundic inhibition of acid secretion and gastrin release Soon-Shiong, Patrick 1979

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FUNDIC INHIBITION OF ACID SECRETION AND GASTRIN RELEASE by PATRICK {SOON-SHIONG M.B.,B.Ch., U n i v e r s i t y o f Witwaterstrand, 1975 A THESIS SUBMITTED IN PARTIAL FULFILMENT OF THE REQUIREMENTS OF THE DEGREE OF MASTER OF SCIENCE INi THE FACULTY OP GRADUATE STUDIES We accept t h i s t h e s i s as conforming to the required standard THE UNIVERSITY OF BRITISH COLUMBIA October, 1979 ||) Patrick Soon-Shiong, 1979 (Department of Surgery) In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Univers i ty of B r i t i s h Columbia, I agree that the L ibrary shal l make it f ree ly ava i l ab le for reference and study. I further agree that permission for extensive copying of th is thesis for scho lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is for f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of The Univers i ty of B r i t i s h Columbia 2075 Wesbrook P l a c e Vancouver, Canada V6T 1W5 - i i - ABSTRACT Despite e a r l i e r i n d i r e c t evidence that an a n t r a l chalone e x i s t s , no such i n h i b i t o r has been found i n a n t r a l e x t r a c t . Recently, i n t e r e s t i n the question of an a n t r a l i n h i b i t o r y mechanism has been revived by studies that showed that for a given r i s e i n serum g a s t r i n caused by a n t r a l d i s t e n s i o n , the response of both the innervated and denervated stomach i s g r e a t l y enhanced by vagal denervation of the antrum. While t h i s study suggested a neuro-humoral character of the a n t r a l i n h i b i t o r y mechanism, i t gave no i n d i c a t i o n as to the source of the i n h i b i t o r . Subsequent s t u d i e s , however, suggested that neither the antrum nor the CNS was the source for t h i s i n h i b i t o r . The i n i t i a l aim of t h i s study was to i n v e s t i g a t e the fundus as a p o s s i b l e source of the i n h i b i t o r by studying the e f f e c t of proximal g a s t r i c vagotomy on the a n t r a l i n h i b i t o r y mechanism i n i t i a t e d by d i s t e n s i o n . The r e s u l t s gave c l e a r i n d i c a t i o n that the i n h i b i t o r was indeed released from the fundus; indeed, the a n t r a l i n h i b i t o r y mechanism was i n r e a l i t y a fundic one. Once the fundus was shown to be the source of the i n h i b i t o r , i t was necessary to e s t a b l i s h whether t h i s i n h i b i t o r d i d i n f a c t r e s i d e i n the fundic mucosa. Four dogs were prepared with a denervated fundic pouch (or Heidenhain pouch, HP), and a f i s t u l a of the main, innervated stomach ( g a s t r i c f i s t u l a , GF). The a c i d s e c r e t o r y responses of both the HP and GF to graded doses of - i i i - p e n t a g a s t r i n and histamine was s t u d i e d . In a d d i t i o n both the s e c r e t i o n of a c i d and the response of immunoreactive g a s t r i n i n the blood i n response to a standard meal of 15% l i v e r e x t r a c t was studied. A l l these experiments were repeated a f t e r e x c i s i o n of the fundic mucosa of the main stomach. The r e s u l t s show that e x c i s i o n of the fundic mucosa reduced the GF a c i d s e c r e t i o n to the s t i m u l i by 85-100% . By c o n t r a s t , the maximal HP a c i d s e c r e t i o n increased by 247% i n response to p e n t a g a s t r i n and 200% i n response to histamine. The increase i n the response to submaximal doses of these exogenous s t i m u l i was even g r e a t e r . S i m i l a r l y , the peak 30 minutes HP output i n response to feeding increased by 418%. Fundic mucosal e x c i s i o n a l s o r e s u l t e d i n the increase i n both basal (from 36±3 to 248±37 pg/ml) and food-stimulated response (from 168±12 p r e o p e r a t i v e l y to 392±49 pg/ml p o s t o p e r a t i v e l y ) . Since the i n t r a g a s t r i c pH was held constant at 5.5 during the meal t e s t s both before and a f t e r the operation, the augmented g a s t r i n response could not be a t t r i b u t e d to reduced ac i d s e c r e t i o n caused by e x c i s i o n of the fundic mucosa. From these s t u d i e s i t can be concluded tha t : (1) a n t r a l d i s t e n s i o n r e l e a s e s an i n h i b i t o r from the fundus; (2) e x c i s i o n of the fundic mucosa r e s u l t s i n increased response of the HP to both submaximal and maximal doses of p e n t a g a s t r i n and histamine i n d i c a t i n g that both the s e n s i t i v i t y of the oxyntic c e l l and p a r i e t a l c e l l mass has increased; (3) e x c i s i o n of the fundic mucosa r e s u l t s i n increased basal and food-stimulated g a s t r i n response independent of the pH of the meal suggesting removal of an i n h i b i t o r of g a s t r i n r e l e a s e . - i v - F u r t h e r s t u d i e s a r e n e c e s s a r y t o i d e n t i f y t h e f u n d i c i n h i b i t o r ( s ) . A d d i t i o n a l s t u d i e s we have p e r f o r m e d have shown t h a t t h e i n c r e a s e d s e c r e t o r y r e s p o n s e c a u s e d by f u n d i c m u c o s a l e x c i s i o n c o u l d n o t be r e v e r s e d by t h e i n f u s i o n o f e x o g e n o u s V I P o r s o m a t o s t a t i n , p e p t i d e s known t o e x i s t i n t h e f u n d i c m u c o s a . To i n v e s t i g a t e f u r t h e r t h e r o l e o f t h e v a g u s on t h e r e l e a s e o f t h e f u n d i c i n h i b i t o r , t h e e f f e c t o f p a r e n t e r a l a t r o p i n e and o f t r u n c a l v a g o t o m y on m e a l - s t i m u l a t e d g a s t r i n and HP a c i d r e s p o n s e i n t h e p o s t f u n d u s e c t o m y s t a t e was s t u d i e d . T h e s e s t u d i e s show t h a t t r u n c a l v a g o t o m y d o e s n o t i n c r e a s e HP a c i d s e c r e t i o n f u r t h e r b u t s i g n i f i c a n t l y d e c r e a s e s g a s t r i n r e s p o n s e t o t h e m e a l . A t r o p i n e a b o l i s h e s t h e p o s t f u n d u s e c t o m y r i s e i n HP a c i d s e c r e t i o n b u t ha s no e f f e c t on t h e p o s t f u n d u s e c t o m y r i s e i n s e rum g a s t r i n . T h e s e r e s u l t s a r e c o n s i s t e n t w i t h t h e h y p o t h e s i s t h a t t h e i n h i b i t o r y m e c h a n i s m o f g a s t r i n r e l e a s e i s m e d i a t e d by a t r o p i n e - s e n s i t i v e v a g a l f i b e r s w h i c h s u p p l y t h e f u n d u s and t h a t t h e s t i m u l a t o r y a c t i o n i s m e d i a t e d by a t r o p i n e r e s i s t a n t v a g a l f i b e r s w h i c h s u p p l y t h e a n t r u m . - v - TABLE OF CONTENTS PART I; PYLORO-OXYNTIC NEUROHUMORAL INHIBITORY REFLEX 1 OF ACID SECRETION In t r o d u c t i o n 2 Purpose 5 M a t e r i a l and Methods 7 Animal P r e p a r a t i o n Experimental Design Determinations Results 12 Di s c u s s i o n 19 PART I I ; . FUNDIC INHIBITION OF ACID SECRETION AND 22 GASTRIN RELEASE In t r o d u c t i o n 23 Purpose 24 M a t e r i a l and Methods 25 Animal Pr e p a r a t i o n Experimental Design Determinations Results 36 Di s c u s s i o n 47 - v i - PART III: ANATOMY OF VAGAL INHIBITION OF GASTRIN RELEASE 51 Introduction 52 Purpose 54 Material and Methods 55 Animal Preparations Experimental Design Determinations Results 58 Discussion 66 SUMMARY AND CONCLUSIONS 71 REFERENCES 7 4 CURRICULUM VITAE - v i i - TABLE LEGEND TABLE I : Mean (±SE) Serum G a s t r i n C o n c e n t r a t i o n (pg/ml) During P e n t a g a s t r i n - I n f u s i o n Experiments - V l l l - FIGURE LEGENDS PART I: PYLORO-OXYNTIC NEUROHUMRAL INHIBITORY REFLEX OF ACID SECRETION FIGURE 1: Animal preparation showing antral pouch and gastr ic f i s t u l a before and after proximal gastr ic vagotomy. FIGURE 2: The effect of graded doses of histamine dihydrochloride on GF acid secretion prePGV (closed c i r c l e s ) and postPGV (open c i r c l e s ) . In this and each of the following figures each point represents the mean (±SE) of two experiments in each of four dogs. FIGURE 3: The effect of IV bolus inject ion of regular insu l in (0.5 U k g - 1 ) on GF acid output ( l e f t panel) and gastr in response (right panel) , prePGV (closed c i r c l e s ) and postPGV (open c i r c l e s ) . FIGURE 4: GF acid response to constant IV perfusion of pentagastrin (4.0 ug kg-"- h r - 1 ) under control condidit ions , i . e . no distension (closed c i rc l e s ) and during antral pouch distension with 0.1 M HC1 (open c i rc l e s ) in the prePGV stage. FIGURE 5: GF acid response to constant IV perfusion of pentagastrin (4.0 yg kg-"- h r - 1 ) under control conditions, i . e . no distension (closed c i r c l e ) and during antral pouch distension with 0.1 M HC1 (open c i r c l e s ) in the postPGV stage. PART I I : FUNDIC INHIBITION OF ACID SECRETION AND GASTRIN RELEASE FIGURE 1: Animal preparation demonstrating the gastr ic f i s t u l a (GF) of the innervated stomach and a vagally denervated fundic or Heidenhain pouch (HP). FIGURE 2a&b: Operative procedure demonstrating excision of the oxyntic c e l l mucosa super f i c ia l to the muscularis mucosa. FIGURE 3: PHOTO MICROGRAPHS TAKEN AT VARIOUS MAGNIFICATIONS OF A SECTION OF THE FUNDUS OF THE STOMACH OF THE DOG 3a: Normal fundic mucosa removed at operation. Note the muscularis mucosa and submucosa ( l l x magnif ication, H + E stain) 3b: High power view of surface e p i t h e l i a l c e l l s l i n i n g p i t of fundic gland (175x magnification; H + E stain) - i x - H i g h power v i e w o f f u n d i c m u c o s a s h o w i n g t h e e o s i n o p h i l i c r o u n d t o p y r a m i d a l s h a p e d o x y n t i c ( p a r i e t a l ) c e l l , a s w e l l a s t h e more b a s o p h i l i c z y m o g e n i c c e l l ( 1 7 5 x , H + E) H i g h power v i e w o f f u n d i c b a s e s h o w i n g t h e b a s o p h i l i c z y m o g e n i c c e l l s . The v a c u o l a t e d and r e t i c u l a r a p p e a r a n c e i n t h e c y t o p l a s m r e p r e s e n t e s t h e p o o r u p t a k e by t h e s e c r e t i o n g r a n u l e s o f t h e H + E s t a i n ( 1 7 5 x ) N o r m a l f u n d i c m u c o s a r emoved a t o p e r a t i o n ( l l x ) G r a n u l a t i o n t i s s u e l i n i n g f u n d u s a t a u t o p s y . T o t a l a b s e n c e o f p a r i e t a l c e l l s ( l l x ) A r e a o f r e g e n e r a t i o n o f f u n d i c m u c o s a w i t h a b s e n c e o f p a r i e t a l c e l l s . N o t e d i f f e r e n c e i n h e i g h t b e t w e e n m u c o s a i n F i g u r e s 4a & 4c ( l l x ) M e a l t e s t by t h e m e t h o d o f i n t r a g a s t r i c t i t r a t i o n . GF r e s p o n s e t o 15% l i v e r e x t r a c t m e a l by i n t r a g a s t r i c t i t r a t i o n (pH 5 . 5 ) b o t h p r e o p e r a t i v e l y ( c l o s e d c i r c l e s ) and p o s t f u n d u s e c t o m y ( o p e n c i r c l e s ) . I n t h i s and i n e a c h s u b s e q u e n t f i g u r e , e a c h p o i n t r e p r e s e n t s t h e mean ( ± S E ) o f two e x p e r i m e n t s i n e a c h o f f o u r d o g s . The e f f e c t o f g r a d e d d o s e s o f h i s t a m i n e ( l e f t p a n e l ) and p e n t a g a s t r i n ( r i g h t p a n e l ) on GF a c i d r e s p o n s e b e f o r e ( c l o s e d c i r c l e s ) and a f t e r e x c i s i o n o f t h e o x y n t i c c e l l m u c o s a ( o p e n c i r c l e s ) . H e i d e n h a i n p o u c h r e s p o n s e t o a 15% l i v e r e x t r a c t m e a l . The pH i s m a i n t a i n e d c o n s t a n t a t pH 5 . 5 t h r o u g h o u t t h e t e s t by i n t r a g a s t r i c t i t r a t i o n . A s i g n i f i c a n t i n c r e a s e i n b o t h b a s a l and m e a l s t i m u l a t e d r e s p o n s e i s s e e n i n t h e f u n d u s e c t o m i z e d a n i m a l s ( o p e n c i r c l e s ) as c o m p a r e d t o t h e p r e o p e r a t i v e c o n t r o l s ( c l o s e d c i r c l e s ) . H e i d e n h a i n p o u c h r e s p o n s e t o g r a d e d d o s e s o f p e n t a g a s t r i n i n f u s i o n b o t h b e f o r e ( c l o s e d c i r c l e s ) and a f t e r e x c i s i o n o f t h e o x y n t i c m u c o s a ( o p e n c i r c l e s ) HP r e s p o n s e t o g r a d e d d o s e s o f h i s t a m i n e d i h y d r o c h l o r i d e b o t h p r e o p e r a t i v e l y ( c l o s e d c i r c l e s ) and a f t e r e x c i s i o n o f t h e o x y n t i c m u c o s a ( o p e n c i r c l e s ) . Left Panel: Gastrin response to a 15% l i v e r extract meal both preoperatively (closed c i r c l e s ) and following excision of the oxyntic mucosa (open c i r c l e s ) . pH was maintained constant (5.5) throughout a l l meal tests by the method of intragastric t i t r a t i o n Right Panel: Gastrin increment over basal during the 15% l i v e r extract meal preoperatively (closed c i r c l e s ) and postoperatively (open c i r c l e s ) . Heidenhain pouch ( l e f t panel) and serum gastrin response (right panel) to a 15% l i v e r extract meal in fundusectomized animals with (open ci r c l e s ) and without (closed c i r c l e s ) infusion of 0.5 u g kg~l h r ~ l somatostatin. Heidenhain pouch ( l e f t panel) and serum gastrin response (right panel) to a 15% l i v e r extract meal in fundusectomized with (open c i r c l e s ) and without (closed c i r c l e s ) infusion of 1.0 u g kg~l h r ~ l VIP. THE ANATOMY OF VAGAL INHIBITION OF GASTRIN RELEASE Heidenhain pouch acid output ( l e f t panel) and gastrin response (right panel) after a 15% l i v e r extract meal by intragastric t i t r a t i o n . Each point represents the mean±SE of two experiments in three dogs before (closed c i r c l e s ) and after (open c i r c l e s ) fundic mucosal excision. Effect of graded doses of pentagastrin on HP acid response in control studies (closed c i r c l e s ) , in fundusectomized animals (open c i r c l e s ) and following truncal vagotomy in fundusectomized animals (open squares). Effect of graded doses of histamine dihydrochloride on HP acid response in control studies (closed c i r c l e s ) , fundusectomized animals (open ci r c l e s ) and following truncal vagotomy in fundusectomized animals (open squares). Heidenhain pouch acid output ( l e f t panel) and gastrin response (right panel) after a 15% l i v e r extract meal in control studies (closed c i r c l e s ) , following fundic mucosal excision (open ci r c l e s ) and following atropine injection in postfundusectomy dogs (open c i r c l e s ) . Heidenhain pouch (HP) ( l e f t panel) and gastrin response (right panel) to a 15% l i v e r extract meal in control studies (closed c i r c l e s ) , following fundic mucosal excision (open ci r c l e s ) and following truncal vagotomy after fundusectomy (closed squares). - xi - FIGURE 6: Vagal control of gastrin release. - x i i - ACKNOWLEDGEMENT Doctor Haile Debas who directed this study has been a constant source of inspiration and encouragement. I am indebted to him for his advice and guidance. I thank Gayle Henderson and Diane Steel for their extreme patience and s k i l l in the production of this manuscript. I wish to thank the members of the Department of General Surgery, especially Doctor W.B. Chung and Doctor A.D. Forward for the guidance, encouragement and support they have given me. Expert technical assistance has been given to me by the following people: Mr. Kwok-Lam Leung and Mr. Peter Cheung - their invaluable assistance in the animal laboratory has made this study possible; Mrs. Eileen Bonagura and Mrs. Nancy Tronsgard performed the serum gastrin assays; Miss Marie Kendall was responsible for the superb h i s t o l o g i c a l sectioning and staining; Mr. Nizar Alladina and Margaret McKinney have been of much assistance. I am grateful to the graphic and photographic sections of the Department of Biomedical Communications for their outstanding quality of work. Last but not least, a thank you to my wife, Michelle, for her understanding and encouragement. - 1 - PART I; PYLORO-OXYNTIC NEUROHUMORAL INHIBITORY REFLEX OF ACID SECRETION - 2 - INTRODUCTION D i s t e n s i o n of the p y l o r i c antrum r e s u l t s i n both s t i m u l a t i o n and i n h i b i t i o n of g a s t r i c a c i d s e c r e t i o n . S t i m u l a t i o n of g a s t r i c a c i d s e c r e t i o n a f t e r a n t r a l d i s t e n s i o n i s mediated both,by g a s t r i n release and by a gastrin-independent p y l o r o - o x y n t i c r e f l e x . The i n h i b i t o r y mechanism of acid s e c r e t i o n by a n t r a l d i s t e n s i o n however, i s l e s s c l e a r l y understood. H i s t o r i c a l l y , the concept that the antrum may play an i n h i b i t o r y r o l e was f i r s t r a i s e d by State and co-workers^ i s 1955, when they showed that r e s e c t i o n of the antrum f a c i l i t a t e d the production of histamine-induced u l c e r . This concept was given support by Har r i s o n and co-workers^ i n 1956. They transplanted h a l f of the antrum onto the colon i n dogs with Heidenhain pouches leaving the other h a l f i n i t s normal l o c a t i o n , when the p o r t i o n of the antrum i n i t s normal l o c a t i o n was excised, a c i d s e c r e t i o n from the pouch increased suggesting that a c i d bathing the antrum i n i t s normal l o c a t i o n had released an i n h i b i t o r of acid s e c r e t i o n . The hypothesis of an a n t r a l chalone was thus born. During the next decade controversy raged, some i n v e s t i g a t o r s showing a n t r a l i r r i g a t i o n released an i n h i b i t o r , ' w h i l e others re f u t e d this." 7'** i n 1974 J.C. Thompson^ reviewed the current status of the a n t r a l chalone hypothesis and concluded there was no convincing evidence for i t . - 3 - I n d i r e c t e v i d e n c e f o r an a n t r a l i n h i b i t o r y m e c h a n i s m was s u p p l i e d by Debas e t a l * when i t was u n e x p e c t e d l y n o t e d t h a t a f t e r d e n e r v a t i o n o f t h e a n t r a l p o u c h an i n c r e a s e i n r e s p o n s e o f b o t h t h e g a s t r i c f i s t u l a and H e i d e n h a i n p o u c h t o a g i v e n i n c r e m e n t o f se rum g a s t r i n o c c u r r e d d u r i n g a n t r a l d i s t e n s i o n w i t h a l k a l i . The h y p o t h e s i s was t h u s made t h a t a n t r a l d i s t e n s i o n r e l e a s e d an i n h i b i t o r t o a c i d s e c r e t i o n w h i c h r e q u i r e d v a g a l t o n e f o r i t s a c t i v i t y . S i n c e b o t h t h e H e i d e n h a i n p o u c h and t h e g a s t r i c f i s t u l a w e r e a f f e c t e d , a h u m o r a l as w e l l as a n e u r a l mechan i sm i s i m p l i c a t e d . D i r e c t e v i d e n c e c o n f i r m i n g t h e e x i s t e n c e o f t h i s a n t r a l i n h i b i t o r y m e c h a n i s m was s u p p l i e d when i t was shown t h a t a c i d d i s t e n s i o n o f t h e a n t r u m s i g n i f i c a n t l y i n h i b i t e d p e n t a g a s t r i n - s t i m u l a t e d g a s t r i c s e c r e t i o n f r o m t h e g a s t r i c f i s t u l a . • ' - O R e c e n t l y , S c h o o n e t al-'--'- d e m o n s t r a t e d t h e same phenomenon i n h e a l t h y man and i n t e r e s t i n g l y , a d e f e c t i v e i n h i b i t i o n f o r a n t r a l d i s t e n s i o n i n d u o d e n a l u l c e r p a t i e n t s . The s o u r c e o f t h e i n h i b i t o r now r e q u i r e s l o c a l i z a t i o n . W h i l e t h e a n t r u m c o u l d i t s e l f be t h e s o u r c e , i t i s e q u a l l y p o s s i b l e t h a t a n t r a l d i s t e n s i o n i n i t i a t e d a r e f l e x m e c h a n i s m w h i c h r e l e a s e s t h e i n h i b i t o r e l s e w h e r e , s u c h as t h e c e n t r a l n e r v o u s s y s t e m , t h e f u n d u s o f t h e s t o m a c h , t h e s m a l l i n t e s t i n e o r p a n c r e a s . N e i t h e r t h e a n t r u m no r t h e CNS a r e t h e s o u r c e s i n c e , when v a g a l c o m m u n i c a t i o n o n l y b e t w e e n t h e CNS and a n t r u m i s p r e s e r v e d , a l l o t h e r a b d o m i n a l o r g a n s b e i n g v a g a l l y d e n e r v a t e d , a n t r a l d i s t e n s i o n no l o n g e r c a u s e s i n h i b i t i o n o f a c i d s e c r e t i o n . 1 0 - 4 - This study sought to investigate the effect of vagal denervation of the oxyntic mucosa or proximal gastric vagotomy (PGV) on the inh i b i t i o n of pentagastrin-stimulated acid secretion caused by antral distension. The results show that PGV completely abolishes the inhibitory effect of antral distension and suggests that the fundus is the source of the inhibitory substance(s) released by antral distension. - 5 - PURPOSE To i n v e s t i g a t e t h e e f f e c t o f v a g a l d e n e r v a t i o n o f t h e o x y n t i c m u c o s a i e . , p r o x i m a l g a s t r i c v a g o t o m y (PGV) on t h e i n h i b i t i o n o f p e n t a g a s t r i n - s t i m u l a t e d a c i d s e c r e t i o n c a u s e d by a n t r a l d i s t e n s i o n . FIGURE 1: Animal p r e p a r a t i o n showing a n t r a l pouch and g a s t r i c f i s t u l a before and a f t e r proximal g a s t r i c vagotomy. - 7 - MATERIAL AND METHODS ANIMAL P R E P A R A T I O N : F o u r f e m a l e m o n g r e l d o g s ( 2 0 - 2 6 kg) w e r e s u r g i c a l l y p r e p a r e d w i t h an i n n e r v a t e d p o u c h o f t h e p y l o r i c a n t r u m ( A P ) . The AP was p r e p a r e d by d i v i d i n g t h e a n t r u m c o m p l e t e l y f r o m t h e b o d y o f t h e s t o m a c h e x c e p t f o r a b r i d g e o f 2 cm a t t h e l e s s e r c u r v e w h e r e o n l y t h e m u c o s a was d i v i d e d and a d o u b l e - m u c o s a l s e p t u m c r e a t e d . T h i s p r o c e d u r e e n s u r e d c o m p l e t e p r e s e r v a t i o n o f v a g a l i n n e r v a t i o n t o t h e a n t r u m . The p y l o r u s was t r a n s e c t e d and a G r e g o r y c a n n u l a i n s e r t e d i n t o t h e body o f t h e A P . 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 a n a s t o m o s i n g t h e body o f t h e s t o m a c h t o t h e duodenum. A Thomas c a n n u l a was i n s e r t e d i n t o t h e body o f t h e s t o m a c h p r o x i m a l t o t h i s a n a s t o m o s i s t o s e r v e as a g a s t r i c f i s t u l a ( G F ) . A s m a l l amount o f a c i d s e c r e t i n g m u c o s a was d e l i b e r a t e l y l e f t w i t h t h e a n t r a l p o u c h t o p r e v e n t e x c e s s i v e b a s a l g a s t r i n s e c r e t i o n . ( F i g u r e 1, S t a g e I ) F o l l o w i n g r e c o v e r y , p r e - P G V c o n t r o l s t u d i e s w e r e p e r f o r m e d b e f o r e t h e a n i m a l s w e r e s u b j e c t e d t o a s e c o n d o p e r a t i o n . I n t h e s e c o n d o p e r a t i o n , PGV was p e r f o r m e d by d i v i d i n g a l l t h e n e u r o v a s c u l a r c o n n e c t i o n s a l o n g t h e l e s s e r c u r v a t u r e f r o m t h e a n t r u m - b o d y j u n c t i o n t o t h e g a s t r o - e s o p h a g e a l j u n c t i o n l e a v i n g i n t a c t b o t h t h e a n t e r i o r and p o s t e r i o r n e r v e s o f L a t e r j e t t o t h e a n t r u m . The l a s t 2 i n c h e s o f t h e e s o p h a g u s was a l s o d e n u d e d o f a l l n e r v e f i b e r s t o e n s u r e c o m p l e t e n e s s o f P G V . ( F i g u r e 1 , S t a g e I I ) - 8 - EXPERIMENTAL DESIGN; No experiments were performed for at l e a s t 3 weeks a f t e r each oper a t i o n . The animals were fasted of food but not of water for 18 hours before each t e s t . No t e s t s were done w i t h i n 2 days of each other i n a given animal. The f o l l o w i n g experiments were performed before and a f t e r Proximal G a s t r i c Vagotomy: (1) Histamine Dose-response to c h a r a c t e r i z e GF Response: This was performed by constant intravenous i n f u s i o n of hist a m i n e - d i h y d r o c h l o r i d e i n doses doubling from 5 to 160 u g kg-1 h r - 1 . (2) I n s u l i n Test For Completeness of Vagotomy: Both GF aci d s e c r e t i o n and serum g a s t r i n concentration i n response to an IV bolus i n j e c t i o n of regular i n s u l i n (0.5 U kg-"-) was stud i e d . (3) C o n t r o l P e n t a g a s t r i n Plateau of S e c r e t i o n : A c i d s e c r e t i o n i n response to a continuous i n f u s i o n of p e n t a g a s t r i n (4 yg k g - l hr -"-) was determined over a 3 hour p e r i o d . During these c o n t r o l s t u d i e s , the AP was f i l l e d with 0.1 M HC1 but d i s t e n s i o n was prevented by keeping the l e v e l of the barostat from which the AP was f i l l e d at the l e v e l of the pouch. - 9 - (4) Effect of Antral Distension on Pentagastrin Plateau: These experiments were similar to the control pentagastrin plateau experiments except the AP was distended by l i f t i n g the barostat to 40 cm during the second hour of the pentagastrin infusion. In these test, the AP was kept f i l l e d with 0.1 M HC1 in the f i r s t and third hours of infusion but AP distension was prevented by keeping the barostat at the level of AP. - 10 - DETERMINATIONS ACID OUTPUT; GF s e c r e t i o n was c o l l e c t e d continuously by g r a v i t y and d i v i d e d i n t o 15 minute samples. Two basal samples were obtained before a d m i n i s t r a t i o n of a stimulus. The volume of each sample t- was measured to the nearest 0.1 ml and a c i d concentration determined by t i t r a t i n g 0.5 ml of each sample to pH 7.0 with 0.1 M NaOH on an automatic t i t r a t o r (Radiometer, Copenhagen). A c i d output was c a l c u l a t e d by m u l t i p l y i n g the volume by the c o n c e n t r a t i o n . SERUM GASTRIN CONCENTRATION: Venous blood was obtained b a s a l l y and every 30 minutes a f t e r the s t a r t of s t i m u l a t i o n i n a l l p e n t a g a s t r i n and i n s u l i n t e s t s . > Serum was separated by c o l d c e n t r i f u g a t i o n and stored at -40°C u n t i l assayed. Serum g a s t r i n concentration was measured by r a d i o - immunoassay using antibody 1296 (the kind g i f t of Dr. John H. Walsh from the Center for Ulcer Research and Education, Los Angeles). The assay was s e n s i t i v e to 5 pg/ml and measured the t o t a l c o n centration of immunoreactive g a s t r i n . STATISTICAL ANALYSIS; The s i g n i f i c a n c e of the d i f f e r e n c e of the mean aci d s e c r e t i o n during the c o n t r o l and d i s t e n s i o n experiments was analysed using - 11 - t - t e s t for p a i r e d values. A p-value of l e s s than 0.05 was considered s i g n i f i c a n t . - 12 - RESULTS EFFECT OF PGV ON GF RESPONSES (1) Histamine Dose-Response; Figure 2 shows that the e f f e c t of PGV on the dose-response to histamine i s s m a l l . Submaximal but not maximal responses are a f f e c t e d . A s i g n i f i c a n t decrease was only seen at the 20 yg k g - 1 h r ~ l dose. (2) I n s u l i n Test: PGV completely abolished the GF a c i d response to i n s u l i n hypoglycemia (Figure 3, l e f t p a n e l ) . An increase i n g a s t r i n response to i n s u l i n occurred f o l l o w i n g PGV, but t h i s change was not s t a t i s t i c a l l y s i g n i f i c a n t (Figure 3, r i g h t p a n e l ) . EFFECT OF ANTRAL DISTENSION ON PENTAGASTRIN BEFORE PGV A n t r a l d i s t e n s i o n with 0.1 M HCI caused s i g n i f i c a n t i n h i b i t i o n of a c i d response during the time the d i s t e n s i o n was applied (Figure 4). The two h a l f hourly responses during the second hour of p e n t a g a s t r i n i n f u s i o n f e l l s i g n i f i c a n t l y from the c o n t r o l values of 15.2±1.1 and 14.8 mEq to 9.9±1.2 and 8.5±1.4 mEq r e s p e c t i v e l y with d i s t e n s i o n . Once a n t r a l d i s t e n s i o n was discontinued, there was recovery i n a c i d response. - 13 - PGV lowered the plateau response to p e n t a g a s t r i n (4 ug kg~l h r ~ l ) only s l i g h t l y , the change being s t a t i s t i c a l l y n o n - s i g n i f i c a n t . However, f o l l o w i n g PGV, i n h i b i t i o n of GF response to p e n t a g a s t r i n , by a n t r a l d i s t e n s i o n , was abolished. (Figure 5) SERUM GASTRIN RESPONSES; The e f f e c t of a n t r a l d i s t e n s i o n with a c i d on g a s t r i n responses both before and a f t e r PGV are given i n Table I . As might be expected, a n t r a l d i s t e n s i o n with a c i d had no s t i m u l a t o r y e f f e c t on g a s t r i n r e l e a s e , and i n f a c t , serum g a s t r i n l e v e l s were s i g n i f i c a n t l y lower when a c i d d i s t e n s i o n was a p p l i e d . Changes in serum g a s t r i n concentration were of no consequence in the r e s u l t s shown above. - 14 - E 8000 C • mmm E jo LU D Q. D O < 6000- 4000 - o 2000 -l/r .... T 1̂—* Control I / T 7 f PGV 0 L L * p < 0.05 B 5 10 20 40 80 160 Histamine (jug kg"̂  hH) FIGURE 2: The e f f e c t of graded doses of histamine d i h y d r o c h l o r i d e on GF a c i d s e c r e t i o n prePGV (closed c i r c l e s ) and postPGV (open c i r c l e s ) . In t h i s and each of the f o l l o w i n g f i g u r e s each p o i n t represents the mean (±SE) of two experiments i n each of four dogs. - 15 - ACID GASTRIN O" LI 1 4000 r 1 r 1 "5 Q_ 3000 - D o 2000 - < LL. o 1000- • — • Pre PGV U | n s u l i n o o Post PGV ^ 200 • T * p < 0.05 3: g 150 o u •E ioo o 4-1 % * * Q'...a..n...rS B 1 3 5 15 min Periods </> O O E D 0) CO 50 0 Insulin * I i y,* 'I B 1 2 3 30 min Periods FIGURE 3: The e f f e c t of IV bolus i n j e c t i o n of regular i n s u l i n (0.5 U kg" 1) on GF a c i d output ( l e f t panel) and g a s t r i n response ( r i g h t panel)j prePGV (closed c i r c l e s ) and postPGV (open c i r c l e s ) . - 16 - 20 { Pentagastrin 4.0 ;ug kg-1 hrl f Distension ? Pre PGVl c E o co cr LU J . "5 a "5 O < 16 - 12 8 0 L •"ft -T T X Control o»»o Distension * p < 0.05 J B 1 2 3 4 5 30 min Intervals FIGURE 4: GF acid response to constant IV perfusion of pentagastrin (4.0 yg k g - 1 h r " 1 ) under c o n t r o l c o n d i d i t i o n s , i . e . no d i s t e n s i o n (closed c i r c l e s ) and during a n t r a l pouch d i s t e n s i o n with 0.1 M HC1 (open c i r c l e s ) i n the prePGV stage. - 17 - J Pentagastrin 4.0//g kg"l hr'l J d 12 CO cr 3 9 a 8 6 < J 0' Post PGV distension I — I I X V J • - • Control 0"«o Distension B 1 30 min Intervals FIGURE 5: GF a c i d response to constant IV p e r f u s i o n of p e n t a g a s t r i n (4.0 yg k g - 1 h r " 1 ) under c o n t r o l c o n d i t i o n s , i . e . no d i s t e n s i o n ( c l o s e d c i r c l e ) and during a n t r a l pouch d i s t e n s i o n with 0.1 M HCI (open c i r c l e s ) i n the postPGV stage. - 18 - TABLE I: Mean (±SE) Serum G a s t r i n Concentration (pg/ml) During P e n t a g a s t r i n - I n f u s i o n Experiments EFFECT OF DISTENSION (PRE PGV): BASAL PRE-DIST CONTROL (0 DISTENSION) 71±17 46±9 40 cm DISTENSION 47±4 51±8 DISTENSION 44±8 38±3 POST-DIST 54±12 32±3* EFFECT OF VAGOTOMY: PRE-PGV (DISTENSION) POST-PGV (DISTENSION) BASAL PRE-DIST 47±4 52±8 69±13 58±12 DISTENSION 38±3 46±4 POST-DIST 32±3 41±5 * p < 0.05 - 19 - DISCUSSION The impetus for t h i s study came from the unexpected observation that the responsiveness of both the innervated and denervated stomach to a given increment i n serum g a s t r i n concentration f o l l o w i n g a n t r a l d i s t e n s i o n was g r e a t l y increased a f t e r vagal denervation of the antrum. 1 The i m p l i c a t i o n was that a n t r a l d i s t e n s i o n r e s u l t s i n the release not only of g a s t r i n but a l s o of a substance that i s an i n h i b i t o r to the a c t i o n of g a s t r i n and that vagal denervation of the antrum abolished the release of the i n h i b i t o r but not of g a s t r i n . While i t was c e r t a i n the antrum was the source of the g a s t r i n , that study would not determine whether the i n h i b i t o r was released from the antrum or e x t r a - a n t r a l s i t e s . These other s i t e s could be the CNS, the oxyntic mucosa, or other v a g a l l y - s u p p l i e d abdominal organs. A recent study has provided evidence that neither the antrum nor the CNS are the source of the i n h i b i t o r , s u g g e s t i n g that the i n h i b i t o r was released r e f l e x l y from the other v a g a l l y - i n n e r v a t e d s i t e s . The present study explored the p o s s i b i l i t y that the oxyntic c e l l mucosa might be the source of the i n h i b i t o r and that the vagal branches to the oxyntic mucosa may be the e f f e c t o r f i b e r s of the neuro-hormonal pathway. Indeed, t h i s study has shown that PGV completely abolished the i n h i b i t o r y a c t i o n of a n t r a l d i s t e n s i o n . The choice to distend the antrum with a c i d rather than a l k a l i was based on e a r l i e r demonstration that a c i d d i s t e n s i o n was more - 20 - e f f e c t i v e than a l k a l i d i s t e n s i o n . A p o s s i b l e explanation for these d i f f e r e n c e s might be that a l k a l i n e d i s t e n s i o n releases g a s t r i n thus adding to the stimulus of s e c r e t i o n against which the i n h i b i t o r has to act. In the present study a dose of pen t a g a s t r i n was se l e c t e d that gave near maximal a c i d response i n order to minimize the e f f e c t of any decrease i n serum g a s t r i n concentration that might r e s u l t from ac i d d i s t e n s i o n of the antrum. Denervated pouches were not used i n the present study for the sake of s i m p l i c i t y and the statement that a humoral agent i s released i s based on previous studies of t h i s mechanism. 1' 1 0 We now know that v a r i o u s i n h i b i t o r y peptides are found i n the vagal f i b e r s and i n endocrine c e l l s w i t h i n the oxyntic mucosa. Substance P, VIP, and somatostatin have been shown to e x i s t i n the vagal nerve endings. 12,13,14,15,19 v i P , somatostatin and glucagon are also found i n the endocrine c e l l s of the oxyntic mucosa. 1^ The i n h i b i t i o n caused by a n t r a l d i s t e n s i o n could be mediated by release of these neurocrine and/or endocrine substances. Of course, an e n t i r e l y d i f f e r e n t substance than those mentioned above might be involved. The f a c t that a Heidenhain pouch i s i n h i b i t e d suggests that the i n h i b i t o r can be transmitted v i a the c i r c u l a t i o n . Since substance P and somatostatin do not appear to have p e r s i s t e n c e i n the c i r c u l a t i o n , they are weak candidates. Further studies are required to define the nature of the i n h i b i t o r ( s ) involved i n t h i s a n t r a l neuro-humoral i n h i b i t o r y mechanism. The question of whether an a n t r a l chalone e x i s t s has been the subject of much heated debate since Harrison et a l ^ suggested i t s - 21 - e x i s t e n c e . Some i n v e s t i g a t o r s have been able to show that a n t r a l a c i d i f i c a t i o n i n h i b i t s a c i d secretion,4,5,6 others have not.7'8 I t i s l i k e l y that these d i s c r e p a n t f i n d i n g s were due to f a i l u r e to c o n t r o l the element of a n t r a l d i s t e n s i o n , so that only those who i n a d v e r t e n t l y distended the antrum had p o s i t i v e r e s u l t s . The present study along with s e v e r a l others p r e v i o u s l y published 1»!0 i n d i c a t e that neither a n t r a l a c i d i f i c a t i o n nor a n t r a l d i s t e n s i o n release an a n t r a l chalone, and that the a n t r a l i n h i b i t o r y mechanism i s i n r e a l i t y a fundic one. - 22 - PART I I : FUNDIC INHIBITION OF ACID SECRETION AND GASTRIN RELEASE - 23 - INTRODUCTION: This proof of a pyloro-oxyntic neurohumoral inhibitory reflex strengthens the hypothesis that inhibitory mechanism(s) of acid secretion reside in the fundus. In addition three types of evidence point to the fundus as a possible source of an inhibitor of acid secretion and/or gastrin release. F i r s t , proximal gastrectomy, fundic mucosal s t r i p p i n g ^ and proximal gastric vagotomy-^! a l l result in a marked increase in 24 hour Heidenhain pouch (HP) acid secretion in dogs. This increase had been attributed to the rise in pH following loss of parietal c e l l a c t i v i t y . Second, vagal denervation of the oxyntic mucosa results in increases in basal,22,23,24,25 food-24,25 a n ( j insulin-stimulated23 gastrin release in man. This postvagotomy hypergastrinaemia occurs independent of change in pH in both man25 and dog.26 Third, endocrine c e l l s containing peptides inhibitory to acid secretion and gastrin release have been ide n t i f i e d in the mucosa of the proximal stomach.1^,17,18 This study aimed to define the inhibitory role of the fundus by investigating the effect of fundic (oxyntic c e l l ) mucosal excision on acid secretion and gastrin release in response to endogenous and exogenous stimuli. The results indicate that removal of the fundic mucosa may result in the withdrawal of inhibitory substance(s). - 24 - PURPOSE To define the i n h i b i t o r y r o l e of the fundus by i n v e s t i g a t i n g the e f f e c t of fundic (oxyntic c e l l ) mucosa e x c i s i o n on a c i d s e c r e t i o n and g a s t r i n release i n response to endogenous and exogenous s t i m u l i . - 25 - HP GF FIGURE 1: Animal p r e p a r a t i o n demonstrating the g a s t r i c f i s t u l a (GF) of the innervated stomach and a v a g a l l y denervated f u n d i c or Heidenhain pouch (HP). - 26 - METHODS AND MATERIAL ANIMAL PREPARATIONS Four female dogs (15-20 kg) were provided with a g a s t r i c f i s t u l a (GF) and a v a g a l l y denervated Heidenhain pouch (HP). (Figure 1) Following completion of c o n t r o l experiments the dogs underwent a second operation i n which, through an a n t e r i o r gastrostomy, the oxyntic c e l l mucosa was excised at a plane s u p e r f i c i a l to the muscularis mucosa. The e n t i r e oxyntic c e l l mucosa was removed (Figure 2) except for a small rim near the esophageal j u n c t i o n and at the margins of the GF. Figure 3 i s a photomicrograph of normal fundic mucosa. Figure 4 shows the specimens of mucosa before and a f t e r fundusectomy, demonstrating the extent of mucosal e x c i s i o n . The dogs t o l e r a t e d the procedure remarkably w e l l and resumed normal d i e t w i t h i n 5-6 days. EXPERIMENTAL DESIGN Three weeks were allowed for recovery from the 1st operation, and two weeks from the 2nd. The dogs were fasted for 18 hours before each t e s t . No t e s t s were done w i t h i n 48 hours of each other. The f o l l o w i n g t e s t s were done before and a f t e r e x c i s i o n of the fundic mucosa: (1) Meal Test by I n t r a g a s t r i c T i t r a t i o n : A f t e r removal of the cork from the GF, the i n s i d e of the stomach was r i n s e d with - 27 - t a p w a t e r . Two 15 m i n u t e c o l l e c t i o n s o f b a s a l s e c r e t i o n w e r e o b t a i n e d f r o m t h e GF and H P . A l l c o l l e c t i o n s o f HP s e c r e t i o n w e r e made u s i n g t h e t e c h n i q u e o f i r r i g a t i o n w i t h 50 m l s a l i n e e a c h 15 m i n u t e s . A m e a l o f 300 m l . o f 15% s o l u t i o n o f l i v e r e x t r a c t ( w / v ) a d j u s t e d t o pH 5 . 5 was i n t r o d u c e d i n t o t h e GF w h i c h was t h e n c o n n e c t e d t o an a u t o m a t i c i n t r a g a s t r i c t i t r a t i o n s y t e m . ( F i g u r e 5) The l i v e r e x t r a c t u s e d was d r i e d w a t e r e x t r a c t o f mammal i an l i v e r ( R e h e c k C h e m i c a l Company , P h o e n i x , A r i z o n a ) . The t e c h n i q u e o f i n t r a g a s t r i c t i t r a t i o n was a m o d i f i c a t i o n o f t h e me thod o f F o r d t r a n and W a l s h 2 7 and e m p l o y e d a pH s t a t a s s e m b l y ( e l e c t r o d e G K 3 2 2 1 C , T i t r a t o r T T T l l , A u t o b u r e t t ABV13 w i t h 25 m l . b u r e t t e , R e c o r d e r S B R 2 C , R a d i o m e t e r , C o p e n h a g e n , D e n m a r k ) , a p i s t o n pump ( B r e w e r , M o d e l 6 0 4 5 3 , D i c k i n s o n , M a r y l a n d ) w i t h t h e s p e e d a d j u s t e d a t 10 s t r o k e s p e r m i n u t e , and 0 . 5 M NaHCC»3 as t i t r a n t . I n t r a g a s t r i c t i t r a t i o n was p e r f o r m e d f o r two h o u r s w i t h t h e e n d - p o i n t a d j u s t e d t o 5 . 5 . A c i d o u t p u t f o r e a c h 30 m i n u t e s was c a l c u l a t e d f r o m t h e t i t r i g r a p h w h i c h r e c o r d e d t h e amount o f 0 . 5 M NaHCC>3 u s e d t o m a i n t a i n i n t r a g a s t r i c pH a t 5 . 5 . D u r i n g e a c h i n t r a g a s t r i c t i t r a t i o n t e s t v e n o u s b l o o d was o b t a i n e d a t - 1 5 , 0 , 3 0 , 6 0 , 90 and 120 m i n u t e s . A f t e r c l o t t i n g s e r u m was s e p a r a t e d by c o l d c e n t r i f u g a t i o n and s t o r e d a t - 4 0 ° C u n t i l a s s a y e d f o r g a s t r i n . P e n t a g a s t r i n D o s e R e s p o n s e S t u d i e s ; A c o n t i n u o u s i n t r a v e n o u s i n f u s i o n o f 0 . 1 5 M N a C l i s g i v e n u s i n g a H a r v a r d p e r i s t a l t i c pump a t a r a t e o f 28 m l p e r h o u r . A f t e r two 15 m i n u t e b a s a l p e r i o d s o f c o l l e c t i o n o f GF and HP s e c r e t i o n , p e n t a g a s t r i n i s a d d e d t o t h e i n f u s a t e s t a r t i n g a t 0 . 5 y g k g - 1 h r - 1 and - 28 - doubling the dose stepwise every 30 minutes to 16 u g k g - 1 h r - 1 . The lowest dose was infused for 45 minutes. GF c o l l e c t i o n was done by g r a v i t y and HP s e c r e t i o n was c o l l e c t e d by an i r r i g a t i o n method as described above. The volume of each c o l l e c t i o n was measured to the nearest 0.1 ml, and a c i d concentration was determined i n 0.5 ml and 10 ml samples of GF and HP c o l l e c t i o n s r e s p e c t i v e l y using an automatic t i t r a t o r (Autoburette, Radiometer, Copenhagen). A c i d output was c a l c u l a t e d by m u l t i p l y i n g the volume by the c o n c e n t r a t i o n . The output i n the l a s t 15 minutes c o l l e c t i o n at each dose was taken as the response for that dose of p e n t a g a s t r i n . To minimize d i f f e r e n c e s i n HP s i z e , HP output was expressed as % of the maximal response to histamine. HP response to meal were handled s i m i l a r l y . (3) Histamine Dose-Response Studies; These t e s t s were performed i n a manner s i m i l a r to the p e n t g a s t r i n t e s t s . Histamine d i h y d r o c h l o r i d e (Sigma Chemical Co., S t . Louis, MO) was employed at doses s t a r t i n g at 5 pg k g ~ l h r - 1 and doubling stepwise every 30 minutes to the highest dose used which was 160 yg k g - 1 h r - 1 . Again the lowest dose was infused for 45 minutes. DETERMINATION OF SERUM GASTRIN CONCENTRATION: Serum g a s t r i n concentrations were determined by radioimmunoassay using antibody 1296 which was the kind g i f t of Dr. J . Walsh from the Center for Ulcer Research and Education (CURE), Los Angeles, C a l i f o r n i a . T h i s antibody c r o s s - r e a c t s with - 29 - both the G-17 ( l i t t l e ) and the G-34 (large molecular forms of g a s t r i n . The assay was s e n s i t i v e to 5 pg/ml. SOMATOSTATIN AND VASOACTIVE INTESTINE POLYPEPTIDE (VIP) INFUSION STUDIES ON MEAL TEST In a d d i t i o n f o l l o w i n g e x c i s i o n of fundic mucosa, the e f f e c t s of i n f u s i o n of e i t h e r somatostatin (Peninsula Lab, San Carlos) or VIP (Peninsula Lab, San Carlos) on a c i d s e c r e t i o n and g a s t r i n release were s t u d i e d . The meal t e s t by i n t r a g a s t r i c t i t r a t i o n i s performed as described above but i n a d d i t i o n intravenous i n f u s i o n of somatostatin (0.5 yg k g - 1 h r - 1 ) or VIP (1.0 yg kg~l h r - 1 ) i s commenced 10 minutes p r i o r to the i n s t i l l a t i o n of the l i v e r e x t r a c t meal and continued throughout the t e s t . I n f u s i o n of each peptide i s performed on separate t e s t days. - 30 - FIGURE 2a&b: Operative procedure demonstrating excision of the oxyntic c e l l mucosa su p e r f i c i a l to the mucularis mucosa. FIGURE 2a FIGURE 2b - 32 - FIGURE 3: PHOTO MICROGRAPHS TAKEN AT VARIOUS MAGNIFICATIONS OF A SECTION OF THE FUNDUS OF THE STOMACH OF THE DOG 3a: Normal fundic mucosa removed at operation. Note the muscularis mucosa and submucosa ( l l x m a g n i f i c a t i o n , H + E stain) 3b: High power view of surface e p i t h e l i a l c e l l s l i n i n g p i t of fundic gland (175x m a g n i f i c a t i o n ; H + E sta i n ) 3c: High power view of fundic mucosa showing the e o s i n o p h i l i c round to pyramidal shaped oxyntic ( p a r i e t a l ) c e l l , as w e l l as the more b a s o p h i l i c zymogenic c e l l (175x, H + E) 3d: High power view of fundic base showing the b a s o p h i l i c zymogenic c e l l s . The vacuolated and r e t i c u l a r appearance i n the cytoplasm representes the poor uptake by the s e c r e t i o n granules of the H + E s t a i n (175x)  1 -34- FIGURE 4a: Normal f u n d i c mucosa removed at o p e r a t i o n ( l l x ) FIGURE 4b: G r a n u l a t i o n t i s s u e l i n i n g fundus at autopsy. T o t a l absence of p a r i e t a l c e l l s ( l l x ) FIGURE 4c: Area of r e g e n e r a t i o n of f u n d i c mucosa w i t h absence of p a r i e t a l c e l l s . Note d i f f e r e n c e i n h e i g h t between mucosa i n F i g u r e s 4a & 4c (11c) - 35 - FIGURE 5: Meal test by the method of intragastric t i t r a t i o n . - 36 - RESULTS EFFECT OF FUNDIC MUCOSAL EXCISION ON GF RESPONSES: E x c i s i o n of the fundic mucosa from the main stomach drained by the GF abolished a c i d response to meal completely (Figure 6). The maximal response to pe n t a g a s t r i n was reduced by 83% and to histamine by 8 4% i n d i c a t i n g that some a c i d s e c r e t i n g mucosa was l e f t behind. (Figure 7) EFFECT OF FUNDIC MUCOSAL EXCISION ON HP RESPONSES Basal HP S e c r e t i o n : E x c i s i o n of the fundic mucosa r e s u l t e d i n s i g n i f i c a n t increases i n basal a c i d s e c r e t i o n from the HP. Taking the mean of a l l basal c o l l e c t i o n taken before each experiment the mean basal 15 minute output increased from 5.5±1.6% of the maximal histamine response before e x c i s i o n to 50.1±4.6% a f t e r e x c i s i o n , an increase of 909%. Meal Test (Figure 8): Following e x c i s i o n of the fundic mucosa from the main stomach, a s i g n i f i c a n t increase i n HP response to the meal occurred, (p < 0.05) The peak 30 minute output increased from 38% of maximal histamine output before e x c i s i o n to 159% a f t e r . T h i s represents a r i s e of 418%. Pent a g a s t r i n Dose Response (Figure 9): S i m i l a r l y , HP response to a l l doses of pe n t a g a s t r i n s i g n i f i c a n t l y increased, (p < 0.05) The - 37 - increases were more marked with lower doses. The response to the 0.5 yg dose increased by 558% while that to the 16 yg dose increased by only 247%. Histamine Dose-Response (Figure 10): The exaggeration of the response to histamine was s i m i l a r to that seen with p e n t a g a s t r i n . The response to a l l doses was increased, but the response to the lower doses was more markedly elevated compared to the response to the higher doses. Thus, fundic mucosal e x c i s i o n increased the response to the 0.5 yg dose by over 10 times while the response to the 16.0 yg dose was only doubled. EFFECT OF FUNDIC MUCOSAL EXCISION ON GASTRIN RELEASE: Basal G a s t r i n Concentration (Figure 11, l e f t p a n e l ) : The basal g a s t r i n c oncentration increased from a mean of 36±3 before fundusectomy to a mean of 248±47 pg/ml a f t e r , an increase of 688%. The increase i n basal g a s t r i n was evident even with the f i r s t experiments done 2 weeks a f t e r the o p e r a t i o n . Basal g a s t r i n concentration d i d not appear to r i s e with time a f t e r e x c i s i o n of the fundic mucosa. G a s t r i n Response to Meal (Figure 11): The g a s t r i n response to meal was s i g n i f i c a n t l y elevated a f t e r fundic mucosal e x c i s i o n . The peak response was 168±12 pg/ml before and 39 2±49 pg/ml a f t e r . Since i n t r a g a s t r i c pH was i d e n t i c a l (5.5) i n the preoperative and postoperative experiments, t h i s r i s e could not be a t t r i b u t e d to decreased a c i d response during the meal i n the experiments a f t e r - 38 - fundic mucosal e x c i s i o n . Tests were commenced 14 days a f t e r operation and a l l meal t e s t s completed w i t h i n 23 days a f t e r o peration. Although the absolute serum g a s t r i n response to the meal was higher a f t e r fundusectomy, (Figure 11, l e f t panel) the increment of serum g a s t r i n over basal was not changed because of the high basal g a s t r i n concentration a f t e r the operation. (Figure 11, r i g h t p a n e l ) . EFFECT OF SOMATOSTATIN AND VIP INFUSION ON MEAL TEST RESPONSE; Ac i d S e c r e t i o n ; Both somatostatin (Figure 12) and VIP (Figure 13) i n f u s i o n d i d not r e v e r t the meal stimulated HP response to prefundusectomy l e v e l s . With both peptides no s i g n i f i c a n t d i f f e r e n c e was noted from the c o n t r o l (postfundusectomy) l e v e l s . G a s t r i n Release: S i m i l a r l y VIP i n f u s i o n had noH e f f e c t on g a s t r i n release a f t e r a meal t e s t when compared to c o n t r o l (postfundusectomy) l e v e l s (Figure 13, r i g h t p a n e l ) . Somatostatin however r e s u l t e d i n s i g n i f i c a n t lowering of the g a s t r i n l e v e l s (Figure 12, r i g h t p a n e l ) . Despite t h i s , the g a s t r i n response was s t i l l s i g n i f i c a n t l y higher than that of the prefundusectomy meal response. - 39 - 15% LE IGT (pH 5.5) f * LU 51 a o -g < 8000 6000 4000 - 2000 - r GF Response Pre-op * p < 0.05 * * * * * * po c f Q I i F v O i i i i M i n , . . . . . i r > . . . . . . . n .n. i.n 'O rOM-op B 1 2 3 4 5 6 20 min Intervals FIGURE 6: GF response to 15% l i v e r e x t r a c t meal by i n t r a g a s t r i c t i t r a t i o n (pH 5.5) both p r e o p e r a t i v e l y (closed c i r c l e s ) and postfundusectomy (open c i r c l e s ) . In t h i s and i n each subsequent f i g u r e , each p o i n t represents the mean (±SE) of two experiments i n each of four dogs. - 40 - GF "? 6000 E jo 3 a 4000 6 2000 -a < 0 L Pre-op J 1 I L B 5 10 20 40 80 160 Histamine (fxg kg"̂  hH) I GF i / / Pre-op . > s . . . " t " " * P o s , o p * * » ? : • B 0.5 1.0 2.0 4.0 8.0 16.0 Pentagastrin (/ugkg^hr"1) FIGURE 7: The e f f e c t of graded doses of histamine ( l e f t panel) and p e n t a g a s t r i n ( r i g h t panel) on GF a c i d response before ( c l o s e d c i r c l e s ) and a f t e r e x c i s i o n of the oxyntic c e l l mucosa (open c i r c l e s ) . - 41 - 15%LE IGT (pH 5.5) | C E o i X o 3 Q. "5 O *G < HP RESPONSE 200 r J Post Op *p<0.05 I Pre Op B 1 2 3 4 30 min Intervals FIGURE 8: Heidenhain pouch response to a 15% l i v e r e x t r a c t meal. The pH i s maintained constant at pH 5.5 throughout the t e s t by i n t r a g a s t r i c t i t r a t i o n . A s i g n i f i c a n t increase i n both basal and meal stimulated response i s seen i n the fundusectomized animals (open c i r c l e s ) as compared to the preoperative c o n t r o l s (closed c i r c l e s ) . - 42 - 2- * O i < 3: x o 150 r s 1 2 0 o a fl> HP 90 60 - 30 - J* 5- *o Post Op *0' p<0.05 i Pre Op B0.5 1.0 2.0 4.0 8.0 16.0 Pentagastrin (jug kg~̂  hr"̂ ) FIGURE 9 : Heidenhain pouch response to graded doses of pen t a g a s t r i n i n f u s i o n both before (c l o s e d c i r c l e s ) and a f t e r e x c i s i o n of the oxyntic mucosa (open c i r c l e s ) * - 43 - 3 Q. D o < to O a C £ c 'e o *± x' o T .ô Post Op ,§ Pre Op * p < 0 . 0 5 B 5 10 2 0 4 0 8 0 160 Histamine ( fig kg"̂  hr"̂ ) FIGURE 10: HP response to graded doses of histamine d i h y d r o c h l o r i d e both p r e o p e r a t i v e l y (closed c i r c l e s ) and a f t e r e x c i s i o n of the oxynt i c mucosa (open c i r c l e s ) . - 44 - O) c o U </) D o 400 375 - 300 225 150 75 0 15% LE (IGT: pH 5.5) f 1 ' ' f t "<i 5 Post op J. Pre-op ft *p<0.05 I I I I B 1 2 3 4 30 min Intervals 200 175 - 150 E u> 125 100 o O < 75 50 15% LE (IGT pH 5.5 ) * 6 Post-op B 1 2 3 4 30 min Intervals FIGURE 11: L e f t Panel: G a s t r i n response to a 15% l i v e r e x t r a c t meal both p r e o p e r a t i v e l y (closed c i r c l e s ) and f o l l o w i n g e x c i s i o n of the oxyntic mucosa (open c i r c l e s ) . pH was maintained constant (5.5) throughout a l l meal t e s t s by the method of i n t r a g a s t r i c t i t r a t i o n Right Panel: G a s t r i n increment over basal during the 15% l i v e r e x t r a c t meal p r e o p e r a t i v e l y (closed c i r c l e s ) and p o s t o p e r a t i v e l y (open c i r c l e s ) . - 45 - % M A O 15% LE IGT (PH5.5) , , 15% LE IGT (PH5.5) , py / mL r I 200rmpi I 4 0 Qrr^ASTRiN] 0 160 120 3 a O '9 80 K^i""* 00 350 300 h 5 ° C O 4 0 h j ° •—•Control 200 - o o Somatostatin QL I i j i B l 0 L L B l > 1 I I * P < 0.05 l l 30 min Periods FIGURE 12: Heidenhain pouch ( l e f t panel) and serum g a s t r i n response ( r i g h t panel) to a 15% l i v e r e x t r a c t meal i n fundusectomized animals with (open c i r c l e s ) and without (closed c i r c l e s ) i n f u s i o n of 0.5 yg kg~l h r ~ l somatostatin. - 46 - 15% LE IGT (pH5.5) . , 15% LE IGT (pH5.5) r x pg/mL r — — 200 r 160 I 120 h O 3 80 6* $ 300 E S 250 CO 40 h J 0 L L BI 2 3 4 30 min Intervals 200 0 L L HP 400 " GASTRIN I > 350 c I f - e-« Control o-o VIP BI 2 3 4 30 min Intervals FIGURE 13: Heidenhain pouch ( l e f t panel) and serum g a s t r i n response ( r i g h t panel) to a 15% l i v e r e x t r a c t meal i n fundusectomized with (open c i r c l e s ) and without (closed c i r c l e s ) i n f u s i o n of 1.0 yg k g - 1 h r - 1 VIP. - 47 - DISCUSSION; This study has shown that e x c i s i o n of the oxyntic c e l l mucosa of the main stomach causes marked e l e v a t i o n s both in basal serum g a s t r i n and basal HP a c i d s e c r e t i o n . In a d d i t i o n , there i s marked enhancement i n the response to meal, and to p e n t a g a s t r i n and histamine. These r e s u l t s are c o n s i s t e n t with the presence of t o n i c and phasic fundic i n h i b i t i o n of a c i d s e c r e t i o n and/or g a s t r i n r e l e a s e . Loss of phasic fundic i n h i b i t i o n f o l l o w i n g e x c i s i o n of the oxyntic c e l l mucosa i s a p o s s i b l e explanation for the increase i n HP a c i d output f o l l o w i n g a meal. This elevated response cannot be due to e l e v a t i o n s i n pH i n that the i n t r a d i g e s t i v e pH changes were elim i n a t e d by the method of i n t r a g a s t r i c t i t r a t i o n . Is t h i s p o s t u l a t e d i n h i b i t o r a c t i n g d i r e c t l y on the p a r i e t a l c e l l , ( p a r i e t a l c e l l i n h i b i t o r ) or i n d i r e c t l y , by i n h i b i t i n g g a s t r i n release ( G - c e l l i n h i b i t o r ) ? The design of our study does not allow us to d e f i n i t i v e l y answer t h i s question. However, despite the s i g n i f i c a n t increase i n g a s t r i n concentration f o l l o w i n g the meal i n the postfundusectomy dogs, we f i n d no s i g n i f i c a n t change i n the increment i n g a s t r i n over b a s a l . T h i s suggests that the e l e v a t i o n i n the HP i s as a r e s u l t of l o s s of i n h i b i t i o n of the p a r i e t a l c e l l d i r e c t l y . Evidence for removal of a t o n i c fundic i n h i b i t o r y substance(s) of a c i d s e c r e t i o n and g a s t r i n release i s supported by the s i g n i f i c a n t e l e v a t i o n i n basal a c i d output and basal g a s t r i n - 48 - l e v e l s noted f o l l o w i n g e x c i s i o n of the oxyntic c e l l mucosa. A second explanation for t h i s f i n d i n g could be that the hypochlorhydria that f o l l o w s these operations may remove the negative feedback c o n t r o l of g a s t r i n release r e s u l t i n g i n basal hypergastrinaemia and subsequently increased basal HP output. Both mechanisms may be involved. However a l l basal g a s t r i n l e v e l s were measured w i t h i n 23 days of the operation - whether G - c e l l h y p e r p l a s i a could occur w i t h i n t h i s period of time to account fo r the 688% increase i n basal g a s t r i n and 909% increase i n basal HP output i s unknown. To date there are no s t u d i e s reported on G - c e l l turnover i n dogs, but Lehy and Williams^** reported that i n mice the G - c e l l turnover time was 2 to 4 months. The increased s e n s i t i v i t y of the HP to exogenous p e n t a g a s t r i n and histamine a l s o suggests that fundic i n h i b i t i o n r equires t o n i c a c t i v i t y . The observation that the HP response to submaximal doses of p e n t a g a s t r i n and histamine i s increased, suggests that the s e n s i t i v i t y of the oxyntic c e l l to these s t i m u l i i s increased. While the increased s e n s i t i v i t y to histamine can be explained by the presence of basal hypergastrinaemia f o l l o w i n g e x c i s i o n of the fundic mucosa, the increased s e n s i t i v i t y to p e n t a g a s t r i n cannot be explained on the same b a s i s . The r e s u l t s suggest that an i n h i b i t o r ( s ) of a c i d s e c r e t i o n has been removed. Glucagon, somatostatin and VIP are candidate i n h i b i t o r s that may have been removed by e x c i s i o n of the oxyntic c e l l mucosa. I t appears u n l i k e l y that the removal of the l a t t e r two peptides can e x p l a i n - 49 - the l o s s of i n h i b i t i o n since exogenous i n f u s i o n of these peptides i n high doses d i d not r e v e r t the HP a c i d output nor the serum g a s t r i n l e v e l s to that of the prefundusectomy stat e i n response to a meal. While we have not excluded glucagon, the p o s s i b i l i t y remains that a completely new hormone, yet to be i d e n t i f i e d , i s responsible f o r these changes. The observation that the maximal histamine response i s increased i n d i c a t e s that the p a r i e t a l c e l l mass i n the HP has increased and i s compatible with the hypothesis that e i t h e r a fundic f a c t o r important i n the t r o p h i c r e g u l a t i o n of the p a r i e t a l c e l l has been removed or the r e s u l t a n t hypergastrinaemia has produced a marked t r o p h i c e f f e c t on the HP. There i s growing evidence that g a s t r i n has such a t r o p h i c a c t i o n - chronic a d m i n i s t r a t i o n of p e n t a g a s t r i n to r a t s r e s u l t s i n increased p a r i e t a l c e l l population,29,30 a n a - in m a n # p a t i e n t s with the Z o l l i n g e r - E l l i s o n syndrome have a higher p a r i e t a l c e l l mass.^l The design of our study does not allow us to choose between these f a c t o r s . Indeed, both f a c t o r s ma;y be important. An important question as yet unanswered by t h i s study i s the r o l e of the vagus i n the release of t h i s fundic i n h i b i t o r y substance. VIP, somatostatin and substance P have been i d e n t i f i e d i n vagal nerve endings. The question thus a r i s e s : Is t h i s i n h i b i t o r y substance released by the fundic mucosa (endo or p a r a c r i n e ) , and i f so, i s vagal a c t i v i t y required for i t s release or i s t h i s i n h i b i t o r y substance(s) released d i r e c t l y from the vagal ending supplying the fundus (neurocrine). With regard to - 50 - fundic i n h i b i t i o n of a c i d s e c r e t i o n , ( p a r i e t a l c e l l i n h i b i t o r ) the f a c t that the HP i s a f f e c t e d suggests that t h i s i n h i b i t o r i s endocrine (humoral) rather than para or neurocrine. Our study however does not enable us to conclude the same for the fundic i n h i b i t o r of g a s t r i n release from the antrum ( G - c e l l i n h i b i t o r ) . In summary, we have shown that w i t h i n the fundic mucosa may r e s i d e substance(s) which both t o n i c a l l y and p h a s i c a l l y i n h i b i t a c i d s e c r e t i o n and g a s t r i n r e l e a s e . Glucagon, VIP and somatostatin are candidates for these substances. Our study suggests that n e i t h e r VIP nor somatostatin subserve t h i s i n h i b i t o r y mechanism, but does not exclude the p o s s i b i l i t y that glucagon or another as y e t u n i d e n t i f i e d substance(s) may be involved. In a d d i t i o n to i t s r o l e as i n h i b i t o r of s e c r e t i o n , t h i s fundic substance(s) may a l s o be t r o p h i c to the p a r i e t a l c e l l mucosa. - 51 - PART I I I ; THE ANATOMY OF VAGAL INHIBITION OF GASTRIN RELEASE INTRODUCTION We have provided evidence that i n h i b i t o r y substance(s) of a c i d s e c r e t i o n and g a s t r i n release r e s i d e i n the fundus. The r o l e of the vagus i n the release of these substances r e q u i r e s c l e a r e r d e f i n i t i o n . The vagus both stimulates and i n h i b i t s g a s t r i n r e l e a s e . Evidence f o r vagal i n h i b i t o r y f i b r e s was provided by Farooq and Walsh32 who showed that atropine enhanced the g a s t r i n response to hypoglycaemia i n man. Since the pH i n the stomach was held constant, t h i s enhancement could not have been promoted by a l k a l i n i t y . S i m i l a r l y , atropine enhances g a s t r i n release stimulated by food.33,34 Further evidence for these i n h i b i t o r y f i b e r s i s provided by the f i n d i n g s that s e l e c t i v e c o o l i n g of the c e r v i c a l vagi r e s u l t s i n increased g a s t r i n response to i n s u l i n hypoglycemia,35 that sham feeding i n h i b i t s p e n t a g a s t r i n - stimulated a c i d s e c r e t i o n i n dogs,36 a n c ; that t r u n c a l vagotomy increases basal,22,23,24,25 f 0 0 d , 2 4 ' 2 5 and i n s u l i n - s t i m u l a t e d 2 3 g a s t r i n r e l e a s e . T h i s postvagotomy hypergastrinemia occurs independent of change i n pH i n both man^S and dog. The release of g a s t r i n by vagal s t i m u l a t i o n represents t h e r e f o r e a balance between the e f f e c t s of st i m u l a t o r y and i n h i b i t o r y f i b e r s . A c l e a r e r d e f i n i t i o n of both the anatomic d i s t r i b u t i o n and the p h y s i o l o g i c r o l e of the vagus with respect to the c o n t r o l of g a s t r i n release i s req u i r e d . T h i s study aims to do t h i s by i n v e s t i g a t i n g the e f f e c t of atropine and t r u n c a l vagotomy - 53 - on the s e c r e t o r y patterns i n fundusectomized animals. The r e s u l t s of our study suggest that the i n h i b i t o r y mechanism of g a s t r i n r e l e a s e i s mediated by vagal f i b e r s supplying the fundus and that the s t i m u l a t i o n of g a s t r i n release i s mediated by vagal f i b e r s supplying the antrum. In a d d i t i o n evidence i s supplied for the presence of a nongastrin stimulant of a c i d s e c r e t i o n i n dogs. - 54 - PURPOSE To define the role of the vagus in the fundic inhibitory mechanism by investigating the effect of atropine and truncal vagotomy in fundusectomized animals, on acid secretion and gastrin release in response to endogenous and exogenous stimuli. - 55 - MATERIAL AND METHODS ANIMAL PREPARATION; Three dogs each prepared with a g a s t r i c f i s t u l a (GF) and Heidenhain pouch (HP) underwent c o n t r o l s t u d i e s before e x c i s i o n of the o x y n t i c c e l l mucosa was performed as d e s c r i b e d p r e v i o u s l y . The s t u d i e s were repeated f o l l o w i n g recovery from the o p e r a t i o n . In a d d i t i o n , the e f f e c t of a t r o p i n e on the postfundusectomy response was s t u d i e d . F o l l o w i n g complet ion of these s t u d i e s b i l a t e r a l t r a n s t h o r a c i c t r u n c a l vagotomy was performed. EXPERIMENTAL DESIGN: The dogs were fas ted of food but not of water for 18 hours before each t e s t . No t e s t s were done w i t h i n 48 hours of each o t h e r . A . CONTROL STUDIES; (1) Meal Tes t by I n t r a g a s t r i c T i t r a t i o n ; The GF was opened, and the i n s i d e of the stomach washed wi th tap water . Two 15-minute b a s a l c o l l e c t i o n s were obta ined from the GF and the HP. The techniques of a c i d c o l l e c t i o n and c a l c u l a t i o n of a c i d output were the same as p r e v i o u s l y d e s c r i b e d . During each i n t r a g a s t r i c t i t r a t i o n t e s t venous b lood was obta ined at - 1 5 , 0, 30, 60, 90 and 120 minutes . A f t e r c l o t t i n g the serum was separated by c o l d - 56 - centrifugation and stored at -40°C u n t i l assayed for gastin. (2) Pentagastrin Dose Response Study; The acid response from the GF and HP to graded doses of pentagastrin was studied as described before. (3) Histamine Dose Response Study; Similarly the effects of graded doses of histamine dihydrochloride was assessed as described previously. B. THE EFFECT OF FUNDUSECTOMY: Following excision of oxyntic c e l l mucosa, the control studies above were repeated. In addition the effect of atropine on the meal test was assessed. Effect of Atropine on Meal Test: Two 15-minute basal collections were obtained from the GF and HP. Atropine sulphate (0.2 mg/kg) (Galaxo, Toronto) was then injected subcutaneously. Thirty minutes after atropine injection the meal test was commenced. Acid and blood collections were performed as in the control study. C. THE EFFECT OF TRUNCAL VATOTOMY: Following completion of the above studies, truncal vagotomy was performed. Three weeks were allowed for recovery from surgery. The following tests were then performed: - 57 - (1) meal test by intragastric t i t r a t i o n (2) pentagastrin dose response studies (3) histamine dose response studies. DETERMINATION OF SERUM GASTRIN CONCENTRATION; This has been described in d e t a i l previously. STATISTICAL SIGNIFICANCE The significance of the difference of the mean acid output and gastrin responses during the control and postoperative experiments was analysed using a t-test for unpaired values. A p-values of less than 0.05 was considered s i g n i f i c a n t . - 58 - RESULTS A. EFFECT OF FUNDUSECTOMYt (1) Meal Response: HP Acid Output: Fundic mucosal excision resulted in a s ign i f i cant increase in both basal and meal stimulated acid secretion from the HP (Figure 1, l e f t panel) , as seen in Part I I . Gastr in Response: S imi lar ly a s ign i f i cant increase in basal and meal stimulated gastr in release occurred following the operation (Figure 1, r ight panel) . (2) Pentagastrin and Histamine Dose Response Studies: The HP acid output in response to exogenous pentagastrin and histamine increased s i g n i f i c a n t l y after excision of the oxyntic mucosa (Figures 2 and 3). B. EFFECT OF ATROPINE SULPHATE ON MEAL RESPONSE AFTER FUNDUSECTOMY: HP Acid Output: The HP response in the fundusectomized animals in response to a meal was s i g n i f i c a n t l y decreased (74%) after subcutaneous atropine in jec t ion . The peak secretion - 59 - d e c r e a s e d f r o m an o u t p u t o f 1 9 8 ± 5 1 % m a x i m a l h i s t a m i n e a c i d o u t p u t (MAO) b e f o r e t o 5 2 ± 2 1 % a f t e r a t r o p i n e i n j e c t i o n ( F i g u r e 4 , l e f t p a n e l ) . The e f f e c t o f a t r o p i n e was t o r e t u r n t h e m e a l r e s p o n s e o f t h e HP t o t h e p r e f u n d u s e c t o m y l e v e l (peak a c i d o u t p u t 46±10% M A O ) . G a s t r i n R e l e a s e : The e l e v a t e d g a s t r i n r e s p o n s e t o t h e m e a l s e e n i n t h e f u n d u s e c t o m i z e d a n i m a l s p e r s i s t e d a f t e r a t r o p i n e s u l p h a t e i n j e c t i o n . No f u r t h e r e l e v a t i o n i n g a s t r i n r e s p o n s e was n o t e d , t h e r e s p o n s e i n t h e f u n d u s e c t o m i z e d d o g s w i t h a t r o p i n e s h o w i n g no s i g n i f i c a n t d i f f e r e n c e f r o m t h o s e w i t h o u t a t r o p i n e ( F i g u r e 4 f r i g h t p a n e l ) . C . E F F E C T OF TRUNCAL VAGOTOMY A F T E R FUNDUSECTOMY (1) M e a l T e s t by I n t r a g a s t r i c T i t r a t i o n : HP A c i d O u t p u t : No s i g n i f i c a n t d i f f e r e n c e was n o t e d b e t w e e n t h e m e a l r e s p o n s e s o f t h e f u n d u s e c t o m i z e d d o g s b e f o r e and a f t e r t r u n c a l v a g o t o m y ( F i g u r e 5 , l e f t p a n e l ) , t h e peak r e s p o n s e b e i n g v i r t u a l l y i d e n t i c a l ( 1 9 8 ± 5 1 % maximum h i s t a m i n e a c i d o u t p u t c o m p a r e d t o 1 9 8 ± 4 8 % r e s p e c t i v e l y ) . I n t h e f u n d u s e c t o m i z e d a n i m a l s , t h e e f f e c t o f a t r o p i n e c o m p a r e d t o t h e e f f e c t o f t r u n c a l v a g o t o m y - 60 - d i f f e r e d s i g n f i c a n t l y , the peak response being 42±17% and 198±49% maximal h i s tamine a c i d output r e s p e c t i v e l y . G a s t r i n Re lease : A f t e r t r u n c a l vagotomy i n the fundusectomized dogs the g a s t r i n response was s t i l l s i g n i f i c a n t l y e l evated when compared to the prefundusectomy study (F igure 5, r i g h t p a n e l ) . However t h i s e l e v a t i o n in g a s t r i n r e l e a s e was s i g n i f i c a n t l y lower than i n the fundusectomized animals before t r u n c a l vagotomy. T h i s drop i n serum g a s t r i n l e v e l s a f t e r t r u n c a l vagotomy (F igure 5, r i g h t panel) was not accompanied by a corresponding drop in HP output (F igure 5, l e f t p a n e l ) , suggest ing the presence of a n o n g a s t r i n s t i m u l a n t of a c i d s e c r e t i o n . When compared to the e f f e c t of a t r o p i n e , both HP a c i d output and g a s t r i n re l ease to a meal s i g n i f i c a n t l y d i f f e r e d . P e n t a g a s t r i n and Histamine Dose Response S t u d i e s Both the p e n t a g a s t r i n and his tamine dose response s t u d i e s in the fundusectomized animals d i d not not change a f t e r t r u n c a l vagotomy ( F i g u r e 2 and F i g u r e 3 ) . - 61 - 15% LE IGT (PH5.5) E H f 200 c E o 160 ± X o 120 80 Q. D o 40 < 0 -! B 1 1 o—o Post Op £ •-•Control i i 15% LE IGT (PH5.5) f } 500 ^ 400h 3 300 c O = 200 O 1001- I GASTRIN 0 L B 1 I" -5. -1 * p<0.05 30 min Intervals FIGURE 1: Heidenhain pouch a c i d output ( l e f t panel) and g a s t r i n response ( r i g h t panel) a f t e r a 15% l i v e r e x t r a c t meal by i n t r a g a s t r i c t i t r a t i o n . Each p o i n t represents the mean±SE of two experiments i n three dogs before (cl o s e d c i r c l e s ) and a f t e r (open c i r c l e s ) fundic mucosal e x c i s i o n . - 62 - 0) </> c o a 0) £ - a> O £ . — CO u • -< ̂ o 65 150 r 120 90 60 30 0 H P o *6 Post Op A Post Op + TV A* T . 5 T - i - ^ I-- * Pre Op »- . 5 ' *p<0.05 L» . B0.5 1.0 2.0 4.0 8.0 16.0 Pentagastrin (jug kg"^ hr"̂ ) FIGURE 2: E f f e c t of graded doses of pe n t a g a s t r i n on HP a c i d response i n c o n t r o l s t u d i e s ( c l o s e d c i r c l e s ) , i n fundusectomized animals (open c i r c l e s ) and f o l l o w i n g t r u n c a l vagotomy i n fundusectomized animals (open squares). - 63 - 3 a. O < O *n 0) C £ o o T * D Post Op + TV / . a P o s t Op .« Pre Op *p<0.05 B5 10 20 40 80 160 Histamine ( //gkg^hr"1) FIGURE 3: E f f e c t o f g r a d e d d o s e s o f h i s t a m i n e d i h y d r o c h l o r i d e on HP a c i d r e s p o n s e i n c o n t r o l s t u d i e s ( c l o s e d c i r c l e s ) , f u n d u s e c t o m i z e d a n i m a l s ( open c i r c l e s ) and f o l l o w i n g t r u n c a l v a g o t o m y i n f u n d u s e c t o m i z e d a n i m a l s ( open s q u a r e s ) . - 64 - 15% LE IGT (PH5.5) m 200 c E 5 160 x i 120 r so z> CL O 401- 72 < 0 L * 4 5 B 1 1 o—o Post Op o -o Post op + Atropine L .oO 1 ...••2 •-•Control 15% LE IGT (PH5.5) £ 500 E 400 c O o 300 200 S ioo OL- IO ASTRINlj T I* o* B 1 I" * P<0,05 30 min Intervals FIGURE 4: H e i d e n h a i n p o u c h a c i d o u t p u t ( l e f t p a n e l ) and g a s t r i n r e s p o n s e ( r i g h t p a n e l ) a f t e r a 15% l i v e r e x t r a c t m e a l i n c o n t r o l s t u d i e s ( c l o s e d c i r c l e s ) , f o l l o w i n g f u n d i c m u c o s a l e x c i s i o n ( o p e n c i r c l e s ) and f o l l o w i n g a t r o p i n e i n j e c t i o n i n p o s t f u n d u s e c t o m y d o g s ( o p e n c i r c l e s ) . - 65 - on 15% LE IGT (PH5.5) f } . E I 160 x § 120 ~ 80 a O 40 ~D \) < 0 1 9 B 1 * j * ,iii'*'B)* • u. o * i o - o Post O p a-a Post op + TV •-•Control J L_ 1 15% LE IGT (pH5.5) 500 I ~ IGASTRIN1 1 400 CO a. <J c o u 300- * 200 o 100 0 I'* * f T B 1 I * 5 ^ * P<0.05 30 min Intervals FIGURE 5: Heidenhain pouch (HP) ( l e f t panel) and g a s t r i n response ( r i g h t panel) to a 1.5% l i v e r e x t r a c t meal i n co n t r o l studies (closed c i r c l e s ) , f o l l o w i n g fundic mucosal e x c i s i o n (open c i r c l e s ) and fol l o w i n g t r u n c a l vagotomy a f t e r fundusectomy (closed squares). - 66 - DISCUSSION Excision of the oxyntic mucosa resulted in increased Heidenhain pouch response to meal and exogenous pentagastrin and histamine. These results are the same as those seen in Part II. Similarly, an increased gastrin response to the meal occurred. These findings confirm the presence of a fundic inhibitory mechanism of acid secretion and gastrin release. The enhanced gastrin response to a meal seen after excision of the oxyntic c e l l mucosa was neither decreased nor increased further by systemic atropine. This suggests that the enhancing effect of atropine on food stimulated gastrin release noted by previous investigators-^ 134 m a y j-,e t n e r e s u i t of loss of fundic inhibitory mechanisms requiring vagal, atropine-sensitive innervation. Following fundusectomy atropine reverted the HP acid response to a meal to control levels but f a i l e d to abolish acid secretion completely. This finding is at variance with previous work in which atropine was found to abolish acid responses of gastric pouches to feeding.34,40 & possible explanation for this discrepancy is the presence of a nongastrin stimulator of acid secretion, now unmasked by fundusectomy, which is atropine resistant. We cannot exclude the p o s s i b i l i t y that this meal stimulated HP response after atropine reflects an increased parietal c e l l mass. - 67 - Our s t u d i e s showed that t r u n c a l vagotomy does not change HP response to meal, to p e n t a g a s t r i n or to histamine i n fundusectomized dogs. In nonfundusectomized dogs, t r u n c a l vagotomy (TV) has been shown to enhance HP a c i d response. Bearing i n mind that fundusectomy has already enhanced HP response to these s t i m u l i , the lack of f u r t h e r e f f e c t of TV may i n d i c a t e that denervation of the fundus may be r e s p o n s i b l e for the e f f e c t of TV i n nonfundusectomized dogs. A study of the e f f e c t of p a r i e t a l c e l l vagotomy on HP response to these s t i m u l i i s required to confirm t h i s hypothesis. We have shown that the i n h i b i t i o n of a c i d s e c r e t i o n from the main stomach caused by a n t r a l d i s t e n s i o n with a c i d i s abolished by p a r i e t a l c e l l vagotomy. I t i s p o s s i b l e , but unproven, that the i n h i b i t o r y e f f e c t of a n t r a l d i s t e n s i o n may be mediated by a r e f l e x release of t h i s i n h i b i t o r from the fundic mucosa. The g a s t r i n response to a meal i n the fundusectomized dogs dropped s i g n i f i c a n t l y f o l l o w i n g t r u n c a l vagotomy. This i m p l i e s that c u t t i n g the vagus nerves removes not only the i n h i b i t o r y f i b e r s to the fundus but a l s o s t i m u l a t o r y f i b e r s to the antrum. This supports the f i n d i n g s that i n dogs the HP response to 2-deoxy-glucose, which presumably acts the vagal release of g a s t r i n , was g r e a t l y decreased a f t e r vagal denervation of the antrum. 29 That atropine f a i l e d to i n h i b i t the s t i m u l a t o r y f i b e r s i n response to food, suggest that the mechanism for g a s t r i n r e l e a s e i s n o n c h o l i n e r g i c . This concurs with f i n d i n g s of Csendes who showed that g a s t r i n release by feeding i s r e s i s t a n t to atropine.34 - 68 - V A G A L C O N T R O L O F G A S T R I N R E L E A S E a g u s "*«»••«•»** P y l o r o - P y l o r i c R e f l e x , = = A t r o p i n e R e s i s t a n t A t r o p i n e S e n s i t i v e • ' • A t r o p i n e S e n s i t i v i t y U n k n o w n ) I n h i b i t i o n f + j S t i m u l a t i o n Vagal c o n t r o l of g a s t r i n r e l e a s e . - 69 - The amount of gastrin released by any mode of vagal stimulation is substantially smaller than can be released by food or topical acetylcholine in the antrum. This suggests that gastrin release represents a balance between the effect of stimulatory and inhibitory vagal fibers. Gastrin is released by direct vagal stimulation during the cephalic phase^S and by both long and short neural r e f l e x e s , 4 1 r 4 2 a s w e n a s by direct chemical action of food on the G-cell durng the gastric phase.^7 Both atropine^3 » 3 4 a n ( j truncal vagotomy increases food stimulated gastrin release giving support to the presence of vagal inhibitory f i b e r s . The results of this study suggest that inhibitory mechanism of gastrin release is mediated by vagal fibers which supply the fundus and that the stimulatory action is mediated by vagal fibers which supply the antrum. The former are atropine sensitive, the latt e r atropine resistant. This hypothesis, in combination with the known mechanisms of gastrin release, allows the description of a model for the mechanism of gastrin release (Figure 6 ) . This model offers an explanation for several findings. Parietal c e l l vagotomy results in hypergastrinemia in both man 25 and dog^G which cannot be explained by changes in pH. A possible explanation is that the PCV removes inhibitory fibers which subserve a fundic inhibitory mechanism. Fundic mucosal excision might remove the same inhibitory agent. In man, increase in gastrin release in response to feeding is greater in proximal vagotomy than in selective vagotomy.24 These findings can be explained by the hypothesis that inhibitory mechanisms of gastrin release mediated - 70 - by vagal fiber which supply the fundus and that the stimulatory fibers of gastrin release supply the antrum. Truncal vagotomy in the fundusectomized animal causes sig n i f i c a n t decrease in meal stimulated gastrin release. A corresponding f a l l in HP secretion does not occur. Indeed the HP acid output is v i r t u a l l y identical before and after truncal vagotomy. These findings are in keeping with the hypothesis that removal of the fundic mucosa unmasks a nongastrin mechanism of stimulation of acid secretion. Alternatively, this observation might be explained that TV has interrupted extra-gastric vagal fibers which subserve release of an i n t e s t i n a l inhibitor (vagogastrone) In summary, these physiological observations have served to define the anatomy of the distribution of vagal fibers to the stomach. The inhibitory action of the vagus is mediated by atropine-sensitive fibers that go to the fundus, while the stimulatory action is mediated by atropine resistant fibers that go to the antrum. - 71 - SUMMARY AND CONCLUSIONS SUMMARY AND CONCLUSIONS This study provides evidence for a servo c o n t r o l mechanism between the fundus and the antrum. I t supports the hypothesis that the fundus acts as an i n h i b i t o r y organ. The r e s u l t s of t h i s study have shown that a c i d a n t r a l d i s t e n t i o n i n h i b i t s a c i d s e c r e t i o n from the main stomach. T h i s i n h i b i t i o n i s abolished by p a r i e t a l c e l l vagotomy, suggesting that a p y l o r o - o x y n t i c vagal r e f l e x mechanism i s involved. Previous s t u d i e s have shown that a n t r a l d i s t e n s i o n i n h i b i t s HP responses suggesting that a neurohumoral mechanism i s i n v o l v e d . Thus, the a n t r a l i n h i b i t o r y mechanism i s i n r e a l i t y a fundic one. Excison of the fundic (oxyntic c e l l ) mucosa of the main stomach causes marked e l e v a t i o n both i n basal and stimulated g a s t r i n and HP a c i d responses. These r e s u l t s are c o n s i s t e n t with the presence of substance(s) i n the oxyntic c e l l mucosa which both t o n i c a l l y and p h a s i c a l l y i n h i b i t a c i d s e c r e t i o n and/or g a s t r i n r e l e a s e . The f a c t that HP a c i d s e c r e t i o n i s a f f e c t e d implies that the p a r i e t a l c e l l i n h i b i t o r i s humorally mediated. This study i s unable to define whether the G - c e l l i n h i b i t o r i s endocrine, paracrine or neurocrine i n nature. An increased HP response to maximal histamine s t i m u l a t i o n implies an increased p a r i e t a l c e l l mass. Whether t h i s increase i s due to hypergastrinemia or to l o s s of a t r o p h i c substance with removal of the fundic mucusa remains to be answered. - 73 - The r e s u l t s of the e f f e c t of atropine and t r u n c a l vagotomy on fundusectomized dogs support the hypothesis that the i n h i b i t o r y mechanism of g a s t r i n release i s mediated by vagal, atropine s e n s i t i v e f i b e r s which supply the fundus and that the st i m u l a t o r y a c t i o n i s mediated by vagal, atropine r e s i s t a n t f i b e r s which supply the antrum. 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Polak JM, Pearse AGE, Grimelius , et a l : Growth hormone release - i n h i b i t i n g hormone i n g a s t r o i n t e s t i n a l and p a n c r e a t i c D c e l l s . Lancet 1:1220, 1975. 18. Sasaki H, Rubaclava B, Bacteus D, et a l : I d e n t i f i c a t i o n of glucagon i n the g a s t r o i n t e s t i n a l t r a c t . J C l i n Invest 56:135-145, 1975. 19. Said S, Rosenberg R: Vasoactive I n t e s t i n a l Polypeptide: Abundant immunoreactivity i n neural c e l l l i n e s and normal nervous t i s s u e . Science 192:907-908, 1976. 20. Landor JH, Ross J L , Gay GR: The importance of a c i d i n h i b i t i o n i n the r e g u l a t i o n of g a s t r i c s e c r e t i o n . Arch Surg 85:695-700, 1972. 21. Amdrup BM, G r i f f i t h CA: S e l e c t i v e vagotomy of the p a r i e t a l c e l l mass: Part I: With p r e s e r v a t i o n of the innervated antrum and p y l o r u s . Ann Surg 170(2) :207-214, 1969. 22. 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Yamagishi T, Debas HT, Walsh JH: Fundic i n h i b i t i o n of g a s t r i n r e l e a s e . Gastroenterology 72:1152, 1977. 27. Fordtran JS, Walsh JH: G a s t r i c a c i d s e c r e t i o n rate and bu f f e r content of the stomach a f t e r e a t i n g . J C l i n Invest 52:645-659, 1973. 28. Lehy T, Willems G: Population k i n e t i c s of a n t r a l g a s t r i n c e l l s i n the mouse. Gastroenterology 71:614-619, 1976. 29. Cream GP, Marshall MW, Rumsey RDE: P a r i e t a l c e l l h y p e r p l a s i a induced by the a d m i n i s t r a t i o n of p e n t a g a s t r i n (1C150,123) to r a t s . Gastroenterology 57:147-155,1969. - 78 - 30. Stanley MD, Coalson RE, Grossman MI, et a l : Influence of s e c r e t i o n and p e n t a g a s t r i n on a c i d s e c r e t i n and p a r i e t a l c e l l number i n r a t s . Gastroenterology 63(2):264-268, 1972. 31. 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Debas HT, Walsh JH, Grossman MI: In: Symposium on G a s t r o i n t e s t i n a l Hormones, e d i t e d by JC Thompson, A u s t i n , U n i v e r s i t y of Texas Press, pp. 425-436, 1975. 42. Debas HT, Walsh JH, Grossman MI: Evidence for o x y n t o p y l o r i c r e f l e x for release of a n t r a l g a s t r i n . Gastroenterology 687-690, 1975. 43. Debas HT, Walsh JH, Grossman MI: A f t e r vagotomy atropine suppresses g a s t r i n release by food. Gastroenterology 70:1082-1084, 1976. - 80 - 44. Emmas S, Grossman MI: E f f e c t pepsin responses to histamine P h y s i o l 212:1007-1012, 1967. of t r u n c a l vagotomy on aci d and and g a s t r i n i n dogs. Amer J

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