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The nature of the influence of various polypeptides on gastrin stimulated canine Pavlov pouch secretion Sharp, Fraser Rosslyn 1975

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THE NATURE OF THE INFLUENCE OF VARIOUS POLYPEPTIDES ON GASTRIN STIMULATED CANINE PAVLOV POUCH SECRETION BY FRASER ROSSLYN SHARP M.B., Ch.B. EDINBURGH UNIVERSITY 1967 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF SURGERY WE ACCEPT THIS THESIS AS CONFORMING TO THE REQUIRED .STANDARD THE UNIVERSITY OF BRITISH COLUMBIA 1975 In presenting th is 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 fur ther agree that permission for extensive copying of th is thes is for scho la r ly purposes may be granted by the Head of my Department or by h is representat ives . It is understood that copying or pub l ica t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my wri t ten permission. Department of 2 2 The Univers i ty of B r i t i s h Columbia Vancouver 8, Canada i i ABSTRACT The e f f e c t of simultaneous continuous i n f u s i o n of synthetic 15 leucine human g a s t r i n and 4 polypeptides on g a s t r i c H + and pepsinogen secretion i s studied. The r e s u l t s of 112 f i v e hour secretion tests on 4 Pavlov pouch dogs are reported. The doses of g a s t r i n used were 0.175, 0.35 and 0.7 ug/kg-hr. In 32 cont r o l tests the i n f u s i o n of g a s t r i n alone at the lower doses resulted i n stimulation of pepsinogen secretion. The higher dose had no apparent + e f f e c t . A l l 3 doses increased the H output. In 26 t e s t s , simultaneous i n f u s i o n of synthetic glucagon 5.0 ug/kg-hr and g a s t r i n at the three doses noted above resulted i n an i n h i b i t i o n of secretion by about 32% and pepsinogen secretion by about 40%. The c h a r a c t e r i s t i c s of the i n h i b i t i o n produced conform to a non-competitive pattern. + Porcine secretion at a dose of 1.0 ug/kg-hr. i n h i b i t e d H secretion by about 80 percent at the 3 doses of g a s t r i n used. In these 25 tests the peptic a c t i v i t y of the c o l l e c t e d secretion was reduced by 20 percent by s e c r e t i n i n f u s i o n . Closer examination of the data however suggests that peptic c e l l secretion was increased. The pepsinogen probably remained within the g a s t r i c gland lumen. The pattern of i n h i b i t i o n of H + secretion had the c h a r a c t e r i s t i c s of non-competitive i n h i b i t i o n . In 19 t e s t s , a synthetic fragment of the s e c r e t i n molecule (the N terminal amino acids 12-27) at three doses of 0.5, 1.0 and 2.5 ug/kg-hr. had no e f f e c t on g a s t r i n stimulated H + and pepsin secretion. The molecular s t r u c t u r a l s i m i l a r i t i e s between s e c r e t i n , glucagon and s e c r e t i n (12-27) and t h e i r observed e f f e c t s under the conditions of t h i s study suggests the hypothesis: the i n h i b i t o r y a c t i v i t y on acid secretion of glucagon and s e c r e t i n reside i n the C terminal 11 amino acid sequence. i i i The pepsinogen effect of secretin i s dependent on the t e r t i a r y molecular structural differences between secretin, secretin (12-27) and glucagon. In ten tests gastric i n h i b i t o r y polypeptide at a dose of 2.5 ug/kg-hr. inhibited both acid and pepsinogen secretion. The i n h i b i t o r y effect on H + secretion appeared more marked, i n this small number of tests, at a higher background stimulatory dose. This was not so for pepsinogen secretion. A l l the results reported were subjected to exhaustive s t a t i s t i c a l evaluation. i v TABLE OF CONTENTS Abstract List of Tables List of Figures Acknowledgements Introduction Methods Operative Procedures Post-Operative Treatment Complications Secretion Collection Hydrogen Ion Measurement Estimation of Peptic Activity 5 Hour Test Procedure Analysis of Results Materials Results Reproducibility of Pouch Response to Gastrin Reproducibility of Pepsin Estimation Effect of Infusion of Gastrin of H + Output Effect of Infusion of Gastrin on Pepsin Output Effect of Secretin on H + Output Effect of Glucagon on Pepsin Output Effect of Secretin (12-27) on H + Output Effect of Secretin Q-2-27) on Pepsin Output Effect of G.I.P. on Pavlov Pouch Output Page i i v l i x x i i 1 5 8 8 9 11 11 15 17 21 23 25 25 29 31 38 40 46 51 Page Discussion 52 Tables II to XXXI 72 Bibliography 102 Appendix Hormone Enzyme K i n e t i c Analogy 106 Estimation of the Parameters C.M.R. and D^^ 108 C l a s s i f i c a t i o n of I n h i b i t i o n HO Interpr e t a t i o n of Response - Response/Dose Transformation H 2 Interpr e t a t i o n of Lineweaver-Burk Transformation v i LIST OF TABLES Page I Reprod u c i b i l i t y of peptic a c t i v i t y estimation 26 II E f f e c t of i n f u s i o n of g a s t r i n on H + output 72 III E f f e c t of i n f u s i o n of g a s t r i n on pepsin output 73 IV S t a t i s t i c a l evaluation of the e f f e c t of g a s t r i n on pepsin output 74 V Inhib i t o r y e f f e c t of Secretin on H + output i n response to 0.175 ug/kg-hr g a s t r i n 7 ^ + VI Inhib i t o r y e f f e c t of s e c r e t i n on H output i n response to 0.35 ug/kg-hr g a s t r i n 76 VII Inhi b i t o r y e f f e c t of s e c r e t i n on H + output i n response to 0.7 ug/kg-hr g a s t r i n 7 7 VIII Inhi b i t o r y e f f e c t of s e c r e t i n on pepsin output i n response to 0.175 ug/kg-hr 7 8 IX Inhi b i t o r y e f f e c t of s e c r e t i n on pepsin output i n response to 0.35 ug/kg-hr 7 9 -X In h i b i t o r y e f f e c t of s e c r e t i n on pepsin output i n response to 0.7 ug/kg-hr 8 0 XI E f f e c t of Secretin on H + output Linear regression equation and Michaelis-Menten parameters 8 x XII E f f e c t of s e c r e t i n on pepsin output Linear regression equations and Michaelis-Menten parameters 82 XIII E f f e c t of glucagon on H + output i n response to 0.175 ug/kg-hr g a s t r i n 83 XIV E f f e c t of glucagon on H + output i n response to 0.35 ug/kg-hr g a s t r i n 8 ^ XV E f f e c t of glucagon on H + output i n response to 0.7 ug/kg-hr g a s t r i n 85 v i i Page XVI Effect of glucagon on pepsin output i n response to 0.175 ug/kg-hr gastrin 86 XVII Effect of glucagon on pepsin output in response to 0.35 ug/kg-hr gastrin 87 XV Effect of glucagon on pepsin output in response to 0.7 ug/kg-hr gastrin 88 + XIX The effect of glucagon on H output Linear regression equation and Michaelis-Menten parameters 89 XX The effect of glucagon on pepsin output. Linear regression equations and Michaelis-Menten parameters XXI Effect of secretin (12-27) 0.5 ug/kg-hr on H + output in ao response to gastrin 91 XXII Effect of secretin (12-27) 1.0 ug/kg-hr on H + output in response to gastrin 22 XXXIII Effect of secretin (12-27) 2.5 ug/kg-hr. on H + output in response to gastrin 93 XXIV Effect of secretin (12-27) 0.5 ug/kg-hr on pepsin output in response to gastrin 94 XXV Effect of secretin (12-27) 1.0 ug/kg-hr on pepsin output in response to gastrin 95 XXVI Effect of secretin (12-27) 2.5 ug/kg-hr on pepsin output in response to gastrin 96 XXVII Effect of secretin (12-27) on acid output Linear regression 97 equations and Michaelis-Menten parameters XXVIII Effect of secretin (12-27) on acid output Linear regression 98 equations and Michaelis-Menten parameters ;TTiii XXIX Relative values of Linear transformations in estimating Michaelis-Menten parameters . 99 XXX Effect of G.I.P. on H + output in response to 3 doses of gastrin 100 XXXI Effect of G.I.P. on pepsin output in response to 3 doses of gastrin 101 ix LIST OF FIGURES Figure Page 1 Diagram of buffer i n s t i l l a t i o n and sample collection apparatus 7 2 Standard tyrosine curve 14 3 Absorbance at 275 nm of the products of digestion of haemoglobin with standard pepsin 14 4 Example of influence of gastrin and inhibitor of Pavlov pouch secretion of acid ^ 5 Diagrammatic explanation of measured values of secretion and inhibition used in analysis 6 Linear regression of randomly paired control results demonstrating good reproducibility 4^ 7 The effect of influsion of 0.7 ug/kg-hr gastrin on H + 97 output of Pavlov pouch 8 The effect of infusion of 3 doses of gastrin on H + output 28 of Pavlov pouch 9 Example of secretin inhibition of gastrin stimulated acid 32 secretion 10 Histogram: the inhibition of acid secretion by secretin (pooled data) 11 Histogram: the inhibition of pepsin secretion by secretin (pooled data) 34 12 Linear regression: acid secretion in response to gastrin and secretin infusion. Curvilinear dose vs. response plot 35 Linear regression: a c i d secretion i n h i b i t i o n by s e c r e t i n Response vs. response/dose transformation Linear regressions: pepsin secretion i n h i b i t i o n by s e c r e t i n . Response vs. response/dose transformation Histogram: the i n h i b i t i o n of acid secretion by glucagon (pooled data) Histogram: the i n h i b i t i o n of pepsin secretion by glucagon (pooled data) Linear regression acid secretion i n h i b i t i o n by glucagon Response vs. response/dose transformation Linear regression: pepsin secretion i n h i b i t i o n by glucagon. Response vs response/dose transformation Linear regression acid secretion i n h i b i t i o n by glucagon. Lineweaver-Burk transformation : Histogram: the e f f e c t on acid secretion of s e c r e t i n (12-27) (pooled data) Histogram: the e f f e c t on pepsin secretion of s e c r e t i n (12-27) (pooled data), • Linear regression: e f f e c t of s e c r e t i n (12-27 on acid secretion (pooled data). Response vs. response/dose transformation. Linear regression: e f f e c t of s e c r e t i n (12-27) on pepsin secretion (pooled data). Response vs. response/dose transformation Diagram of the second messenger system xi Page 25 The amino acid sequence of human glucagon procine s e c r e t i n and 15 leucine human g a s t r i n I 26 T y p i c a l c u r v i l i n e a r expression of i n i t i a l v e l o c i t y vs. substrate concentration: enzyme hormone analogy 107 27 Examples of e f f e c t of i n h i b i t i o n on the response vs. response/dose l i n e a r transformation 28 Examples of e f f e c t of i n h i b i t i o n on the double r e c i p r o c a l •I 1 o l i n e a r transformation (Lineweaver-Burk) ACKNOWLEDGEMENT I wish to thank my supervisor, Dr. J.C. Brown, e s p e c i a l l y for arranging the g i f t of the polypeptides used i n t h i s study. It would not have been possible to carry out the large number of tests without the expert and meticulous technical help of Mr. Leo Yang and Mr. K.L. Leung. I would l i k e to thank Dr. I.G.H. Cleator for h i s invaluable assistance and Dr. R.C. Harrison for h i s support and encouragement. I would l i k e to thank my wife Gwen for her help i n typing the tables and the dr a f t copies of the t h e s i s . F i n a l l y I am very g r a t e f u l to the Medical Research Council of Canada who provided me with a Fellowship and also further f i n a n c i a l support of t h i s project. 1 INTRODUCTION For about 130 years, investigators of gastric secretion have noted that a multitude of physiological factors are involved in the normal function of the organ. In most cases the simple recognition of each stimulatory or inhibitory influence has, on investigation, lead to a more complex concept of the role and interplay of each factor in the normally functioning intact stomach. 4 Since the c l i n i c a l observation on Alexis St. Martin i n 1833 a series of investigative techniques have been used to identify and then characterise although even now incompletely, the factors involved in gastric secretory function. After reports of the f i r s t experimental gastric f i s t u l a (Basson 1842 36 and Blondet 1843) a wide variety of gastric pouches were developed in an attempt to isolate and c l a r i f y the separate neural and humoral mechanisms 24 involved. Heindenhain, in 1879, described a pouch from the gastric fundus which was vagally denervated (although i t retained sympathetic innervation). 37 Pavlov and Schumova-Simonovshaja in 1889 constructed a c l i n i c a l oesophag-ostomy allowing study of the effect of sham feeding. In 1910 Pavlov described a vagally innervated greater curvature pouch. Numerous experimental preparations including the entire gastric pouch denervated fundic pouch, (Bickel 1903) or transplanted fundic pouch have been used. In this century a phase of investigation by pharmacological manipulation resulted in compilation of large volumes of data sometimes contradictory. Often highly speculative deductions on the mechanisms failed to c l a r i f y the normal physiological function in man. Although tissue extracts have been known to e l i c i t gastric secretion since 1902 i t was not un t i l 1919 (Dale et a l " ^ and Popielski^) that 2 histamine was identified as partially responsible for the stimulatory actions of these extracts. Presumptive evidence for the presence of gastrin was 30 obtained by Komarov in 1938. The extraction, purification, and isolation of gastrin proved d i f f i c u l t and the synthesis of gastrin has been accomplished within the last few years. The mechanism by which small polypeptide hormones may effect c e l l function resulting in increased secretion or other c e l l function has been s i g n i f i -cantly c l a r i f i e d by Sutherland.^ The concept of specific hormone receptor 21 site interaction had been postulated for some years. Work by Robertson 43 23 et a l in 1950 and Harris and Alonso in 1965 have implicated the ubiquitous adenyl cyclase and cyclic AMP mechanism in the stimulation of parietal c e l l secretion. The possibility that reduced secretion might result from interference with this mechanism has prompted this investigation. The inhibition of gastric secretion was demonstrated by Ewaldi and 17 37 Boas in 1886 by the addition of olive o i l to a starch meal. Pavlov in 1910 showed that meat stimulated secretion from an innervated pouch could be inhibited by fat in the duodenum and a similar effect in the 18 denervated pouch was found by Feng 1929. Inhibition of vagally induced 29 gastric secretion was found to fat in the duodenum by Kosanski in 1903 1 28 and confirmed by Alley and McKenzie in 1934. Kosaka and Lim in a series of experiments from 1930 demonstrated that an extract of duocenal mucosa produced comparable inhibition to that produced by a crude preparation of CCK. Inhibition of acid secretion stimulated by exogenous histamine or gastrin by fat in the duodenum was demonstrated in the dog by Bibler et a l 5 in 1965. In man histamine and gastrin stimulated secretion was found to be inhibited by duodenal i n s t i l l a t i o n of fat only in the presence of intact 51 25 vagi. (Waddell and Wang, Johnston and Duthie ) Carbohydrate in high concentrations has also been shown to inhibit 31 gastric secretion of acid when i n s t i l l e d into the duodenum. Leconte m 1900 used a 25% glucose solution and his work was confirmed by Day and 14 Komarov in 1939 using sugar in concentrations greater than 20%. That this effect was due to the intraluminal osmotic pressure was indicated by the demonstration of the same inhibitory action induced by saline or poly-45 saccharide solutions in the duodenum. Shay et a l showed that insulin induced hypoglycaemia did not ablish this inhibitory effect of duodenal glucose suggesting that the mechanism involved intraluminal osmolarity and was not related to the absorption and subsequent systemic blood levels 46 of the osmotic solutes. Sircus in 1958 implicated a humoral agent in this response by e l i c i t i n g inhibition of histamine stimulated acid secretion by hypertonic duodenal salt and sugar solutions in both Haidenhain and Pavlov dogs. The duodenal bulb pH has been shown to influence the acid secretion of the stomach. Inhibition of stimulation in response to a meat meal 47 was el i c i t e d by Sokolov administering 0.5% HC1 intraluminally in the Pavlov pouch dog. Day and Webster"'""' were able to inhibit vagally stimulated 20 secretion (sham feeding) in a similar manner. Griffiths inhibited alcohol stimulated secretion by lowering the duodenal pH in man and Pincus et a l suggested that the pH must f a l l below 2.5 to cause inhibition of secretion. Wormsley and Grossman reported that lowering of the intraluminal pH inhibited the acid output of a Haidenhain pouch secreting in response to 2 exogenous gastrin. However Andersson et a l failed to inhibit histamine stimulated secretion by alteration of the intraluminal pH. Therefore i t appeared that the intraluminal hypertonic solutions or 4 a h i g h hydrogen i o n c o n c e n t r a t i o n i n t h e duodenum r e s u l t e d i n 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 by e i t h e r v a g a l means o r by endogenous g a s t r i n o r exogenous g a s t r i n s t i m u l a t i o n . With more p r e c i s e e x t r a c t i o n o f the duodenal hormones, the e f f e c t o f s e c r e t i n and c h o l e c y s t o k i n i n were i n v e s t i g a t e d on a c i d and p e p s i n s e c r e t i o n . The i s o l a t i o n and s y n t h e s i s o f g a s t r i n a l l o w e d more d e f i n i t i v e answers on the i n h i b i t o r y e f f e c t s o f t h e p o l y p e p t i d e s on a c i d and 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 exogenous g a s t r i n . The i d e n t i f i c a t i o n o f the amino a c i d sequence and s y n t h e s i s o f g a s t r i n a l s o a l l o w e d t h e a c t i v e segment of the g a s t r i n m o l e c u l e t o be e s t a b l i s h e d . The a v a i l a b i l i t y o f a s y n t h e s i s e d segment o f t h e s e c r e t i n m o l e c u l e has a l l o w e d i n v e s t i g a t i o n o f the a c t i v i t y o f the whole and p a r t m o l e c u l e . Glucagon has c o n s i d e r a b l e s t r u c t u r a l s i m i l a r i t y w i t h s e c r e t i n and i n t h i s s t u d y the a v a i l a b i l i t y o f s y n t h e t i c g l u c a g o n a l l o w s i n v e s t i g a t i o n o f t h e s e two p o l y p e p t i d e s under the same c o n d i t i o n s . The r e s u l t s o b t a i n e d a l l o w some s p e c u l a t i o n as t o the modes o f a c t i o n o f the p o l y p e p t i d e s and p o s s i b l y the segment of the m o l e c u l e r e s p o n s i b l e f o r the n o t e d a c t i o n s . Brown e t a l i d e n t i f i e d a n o t h e r i n h i b i t o r y p o l y p e p t i d e p r e s e n t i n duodenal mucosa e x t r a c t s . I t was hoped to e s t a b l i s h the mode o f i t s a c t i o n i n t h i s s t u d y . U n f o r t u n a t e l y the s m a l l s u p p l y o f 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 was exhausted b e f o r e a s t a t i s t i c a l l y s i g n i f i c a n t number of t e s t s c o u l d be c a r r i e d o ut. 5 METHOD Operative procedures Healthy mongrel dogs weighing between 13 and 25 kg were used. In a l l instances the dogs were fasted for at l e a s t 16 hours p r i o r to surgery. Sodium pentothal 0.5 gm/kg was given by rapid intravenous i n j e c t i o n to induce anaesthesia. Fluothane at a v a r i a b l e concentration (approximately 0.75% i n 1 - 1 1/2 l i t r e s oxygen per minute was given v i a an oro-tracheal tube to maintain the appropriate depth of anaethesia. Pre-medication of morphine 15 mg IM and atrophine 10 mg IM was given 3/4 hour before induction. Pavlov Pouch preparation Vagal and sympathetic innervation of the greater curvature pouch of the body of the stomach was retained as described by Cleator and Harrison. Procedure The abdomen and lower chest were clipped of h a i r and s k i n preparation was with t i n c t u r e of iodine. S t e r i l e drapes were then applied i n the usual manner. A midline i n c i s i o n was made extending from the ziphoid i n f e r i o r l y for some 15 - 20 cm. Bleeding vessels were clipped with artery forceps and l i g a t e d with 0000 s i l k sutures. The peritoneum was then grasped with artery forceps and a f t e r ensuring that bowel was not adherent, i n c i s e d . The abdominal f a t pad was dissected from the p e r i t o n e a l edges and amputated, the upper edge being f i r m l y l i g a t e d a f t e r d i v i s i o n . The spleen was then delivered at the wound, wrapped i n a s a l i n e soaked sponge and placed on the dogs l e f t , without tension on the vessels. The stomach presented at the wound and was spread so that l i g h t occlusion clamps could be applied along the l e s s e r curvature and also across the short g a s t r i c and g a s t r o - e p i p l o i c vessels. The superior end of the pouch was marked by i n s e r t i n g a 2.0 black s i l k suture through a l l layers of the greater curvature of the stomach at the l e v e l 6 of the second most cranial short gastric artery. The lower end of the pouch was decided upon considering the overall size of the pouch and the normal proximal margin of the antrum. The arteries supplying the greater curvature at this point were divided and ligated; the main gastroepiploic arch was not disturbed. Using large Mayo scissors a straight incision was made at right angles to the greater curvature dividing both anterior and posterior walls of the stomach as far as the midpoint between greater and lesser curvature. The greater curvature from this point up to the suture marking the upper end of the presumptive pouch was then everted through the gastrotomy incision. Using a scalpel, an incision through the mucosa only was made by joining the ends of the right angle incision through the anterior and posterior stomach walls to the suture marking the upper end of the pouch. The mucosa was then stripped from the submucosa 2 cm on either side of this incision. Starting at the upper end marker the mucosa was sutured to mucosa forming a double layer mucosal bridge separating the stomach from the pouch. The titanium cannula were then inserted in the stomach and in the pouch and secured with chromic catgut 0000 sutures. A portion of greater omentum was then insinuated between the double mucosal bridge layers and held in place by one loosely tied suture as shown. The cannulae were then led out through a stab wound in the anterior abdominal wall so that no tension was applied to the pouch or stomach and as normal an anatomic position as possible was achieved. The titanium cannulae were then held in place by polystyrene collars, the stomach and pouch cannulae being discriminated by different coloured collars, (see f i g . l ) 7 Pavlov pouch abdominal wall //////////, teflon collar — titanium cannula-"' plastic Y connector spring clip small air outlet -—* to automatic biuret -collection test tube lightweight transparent test tube holder FIGURE 1 DIAGRAM OP BUFFER INSTILLATION AND SAMPLE COLLECTION APPARATUS USED IN ALL SECRETION TESTS 8 The abdominal incision was then closed by 0 mono filament polypropylene suture to fascial and muscle layers and 00 chromic catgut suture for sub-cuticular closure of the skin. The operative time was normally 1 1/2 to 2 hours although in the earlier operations 2 to 2 1/2 hours were necessary. Only one death occurred in the early postoperative period and was due to acute respiratory obstruction. Post-Operative Treatment Maintenance fluids were provided by intravenous infusion for a variable period but usually five days. A total of 750 cc with an appropriate propor-tion of isotonic dextrose saline and Ringer's lactate were given daily. No oral f l u i d was allowed in the f i r s t 24 hours. Following this, small amounts of water for 24 hours, and milk for a further 24 hours were introduced. In most cases by the fourth post-operative day the dog was tolerating a semi-solid milk shake preparation well and was tolerating a normal diet by the sixth or seventh post-operative day. During the test series the diet used was Purina dog meal. Complications Two significant early post-operative complications only arose. One dog died within an hour of the operation from anoxia secondary to pharyngeal obstruction. A further dog had an abdominal wound dehiscence on the four-teenth post-operative day. Late As would be expected the continuing presence of cannulae in each dog led to a number of complications. Fortunately in a l l cases except one, the problem was related to the cannula draining the stomach not the Pavlov pouch. It was therefore possible to operate and reposition the cannula without disturbing the size, position, vasculature or nervous upply of the pouch. 9 The immunity of the pouch cannula from trouble was fortunate, and surprising. A collection bag was continually attached to this cannula and although supported by a further linen bag and circumferential ties, considerable movement and stress must have occurred at times. The signs related to malfunction of cannula were of decreased drainage during testing or of a very small amount of blood in the sample collected from the stomach. In every case, the repositioning of the cannula and repair of original skin incision and gastrostomy wound was entirely successful although technically d i f f i c u l t . This was evidenced by return of good drainage and loss of blood staining of the gastric secretion. Following a l l repair procedures methylene blue testing of the mucosal bridge between the pouch and stomach was used to verify i t s integrity. An occasional subcutaneous abscess occurred in relation to the cannulae. These were not associated with marked systemic upset and were relieved by surgical drainage Secretion Collection A modified washout technique was used to collect accurately the secretion of the Pavlov pouch. The apparatus used for delivery and i n s t i l l a t i o n of 20 ml glycine buffer into the Pavlov pouch i s shown in f i g . 1. It can be seen that an automatically r e f i l l a b l e biuret of either 25 ml or 50 ml capacity was used in conjunction with a reservoir. This allowed accurate and reproducible measurement of the i n s t i l l e d buffer to within 0.1 ml. The 20 ml mark of the biuret was placed for each experiment, by direct measurement, at the level of the cannula skin junction. This ensured that there was no marked distension of the pouch, that delivery every 15 minutes took place at the same pressure and that 10 this pressure within the pouch was rela t e d to i n t r i n s i c muscle tone and was not influenced by v a r i a t i o n s i n f i l l i n g pressure. P r i o r to i n s t i l l a t i o n of the glycine buffer, the drainage limb of the Y connector was clamped immediately below the titanium cannula. Release of the clamped tubing between biuret allowed f i l l i n g of the pouch from the biu r e t . This was regulated by the biuret tap and flow stopped when the glycine buffer miniscus l e v e l reached 20 ml. The de l i v e r y tubing was then clamped f o r further s e c u r i t y . At the end of each 15 minute period the clamp on the tubing connecting the drainage limb of the Y connector to the test tube holder was released and f l u i d allowed to drain by gravity into the test tube. Small a i r holes i n the upper sides of the test tube holder allowed e x i t of a i r and ensured rapid and complete emptying of the pouch into the c o l l e c t i o n t e s t tube. It i s obvious however that during each c o l l e c t i o n period a f r a c t i o n of the i n s t i l l e d buffer was retained i n the cannula and tubing and not i n the pouch. This volume was measured i n animals subjected to post mortem with an i d e n t i c a l pouch configuration, and found to be about 3 ml. The e f f e c t i v e buffer within the pouch therefore was 17 ml. It was f e l t that i f adequate mixing of the buffer retained i n the cannula and that obtained from the pouch i t s e l f was ensured, no s i g n i f i c a n t error i n r e s u l t s would be introduced by t h i s constant small l o c u l a t i o n of bu f f e r . Measurement of the volume of buffer and secretion c o l l e c t e d was ca r r i e d out i n a 25 ml measuring cylinder measuring to within 0.2 ml. Repr o d u c i b i l i t y of de l i v e r y and c o l l e c t i o n was checked under experimental conditions with a f a s t i n g unstimulated dog. The maximum v a r i a b i l i t y was 0.5 ml although usually l e s s than t h i s . This error due to the apparatus alone of a maximum 4% and usually 2% was considered to be very s a t i s f a c t o r y . 11 Hydrogen Ion Measurement Following adequate mixing of the buffer and g a s t r i c secretion by i n v e r s i o n of the test tube, an a l i q u o t of 2 ml was pipetted into the t i t r a t i o n cup of Radiometer automatic t i t r i m e t e r . P r i o r to t i t r a t i o n of the sample from each 5 hour t e s t , the pH electrode was c a l i b r a t e d using two b u f f e r s . The appropriate temperature compensation was made to the pH meter and a number of t e s t t i t r a t i o n s c a r r i e d out. Each day a number of aliquots of 2 ml of the buffer alone were t i t r a t e d against 0.1 N sodium hydroxide. Any discrepancy with the expected r e s u l t might suggest errors i n the compositions of the b u f f e r , the d i l u t i o n or contamination of the t i t r a n t or the function of the pH electrode or t i t r i m e t e r . If these were suspected t i t r a t i o n of a standard s o l u t i o n of 0.1 N HC1 was c a r r i e d out to pin point the source of e r r o r . 1 A f t e r ensuring proper function of the t i t r i m e t e r each day an a l i q u o t of each 15 minute c o l l e c t i o n had i t s pH measured. It was then t i t r a t e d automatically against a standard s o l u t i o n of 0.1 N sodium hydroxide. T i t r a t i o n was c a r r i e d to two end points 7.0 and 7.4 and both r e s u l t s recorded. Ratio m u l t i p l i c a t i o n of these r e s u l t s allowed c a l c u l a t i o n of t o t a l hydrogen ion present. From previous t i t r a t i o n of the buffer used for each 5 hour test the contribution of that buffer to the t o t a l hydrogen ion present could be c a l c u l a t e d , subtraction of t h i s value allowed c a l c u -l a t i o n of the hydrogen ion due to g a s t r i c secretion i n the washout f l u i d . Estimation of Peptic A c t i v i t y 3 A modification of the method described by Anson and Mirsky was used to measure peptic a c t i v i t y of the pouch secretion. At the end of each 15 minute c o l l e c t i o n period, the b u f f e r / s e c r e t i o n mixture was allowed to drain into the c o l l e c t i o n tube. The volume was measured and the tube was cooled i n i c e water and maintained at t h i s temperature u n t i l i t s peptic a c t i v i t y was measured. Peptic a c t i v i t y was measured by the a b i l i t y of 1 ml aliqu o t of the washout f l u i d to degrade a standard a c i d i f i e d s o l u t i o n of bovine haemoglobin. A large portion of the degradation products formed have a carbon r i n g s t r u c -ture (e.g. tyrosine) and s e l e c t i v e l y absorb u l t r a v i o l e t l i g h t at 275 nm. The standard haemoglobin s o l u t i o n i t s e l f and the reagents used also have an absorbance at t h i s frequency. Therefore each specimen was read against a c o n t r o l sample containing the same reagents but i n which the pepsin had been i n a c t i v a t e d and prevented from degrading the haemoglobin. This required the measurement of the U.V. absorbance i n a double beam spectro-photometer reading each sample against i t s own co n t r o l . The haemoglobin substrate s o l u t i o n was made up the night p r i o r to each experiment by adding 12.5 gm of bovine haemoglobin to 500 cc d i s t i l l e d water ( i . e . 2.5%). This s o l u t i o n was then f i l t e r e d through glass wool and 12.5 cc of 0.3 N HC1 was added. The other reagents used were 10% t r i c h l o r o - a c e t i c a c i d and 0.04 N hydrochloric a c i d . A l l the above were spectrophotometric grade reagents. The 1 ml aliquots of each c o l l e c t i o n specimen were added to two test tubes designated c o n t r o l and sample. The sample tube contained exactly 5 ml 0.04 N hy d r o c l o r i c acid; the control tube contained i n addi t i o n 5 ml of t r i c h l o r a c e t i c a c i d . (The measurement of these solutions was by a multiple d e l i v e r y pipette the accuracy of which was frequently checked). To both tubes, which were standing i n a water bath at 25° C was added 5 cc of 2.5% haemoglobin s o l u t i o n . After exactly 10 minutes of incubation at t h i s temperature the degradation of haemoglobin i n the sample tube was stopped by the addition of 5 ml 10% t r i c h l o r o a c e t i c acid and the e f f e c t i v e mixing by repeated inversion. Both sample and control solutions were f i l t e r e d 13 through Whatman 50 f i l t e r paper. The U.V. absorbance of each sample f i l t r a n t was then read against i t s own control f i l t r a n t in the double beam spectrophotometer. The difference in absorbance at 275 nm was recorded for each specimen. The relationship between the ultraviolet absorbance at 275 nm and the concentration of tyrosine in solution was investigated over the range of absorbance found in this experiment. Standard solutions of tyrosine made up in the reagents used were read against blank solution containing only the reagents. The results when plotted showed a linear relationship as shown in f i g . 2. It was also f e l t necessary to investigate the relationship between the amount of pepsin present in a sample and the concentration of degradation products present after incubation with the substrate. This was carried out by the preparation of a number of solutions of pepsin of varying concentration and using 1 ml aliquots for estimation using the technique described above. The concentration of products of degradation as represented by U.V. absorbance was directly proportional to the pepsin concentration or peptic activity of the standard solution used. The linear plot of values is shown in f i g . 3. Therefore the concentration of degradation products or the U.V. absorbance at 275 nm using the method described i s a direct function of the concentration of pepsin in the sample under the conditions used. As the total peptic activity of the whole sample, and not just of an aliquot, was required multiplication of the concentration of degradation products (as read from graph) by the volume of each sample was a precise measurement of the peptic activity of the sample. A l l values of peptic activity used were calculated in this manner. The units used are jigm of tyrosine or tyrosine equivalent. Although the method of estimation of pepsin used is generally accepted .70 .60 -i .50 -io o 10 20 30 40 50 60 70 80 90 100 fjgm Tyrosine / 10 ml. FIGURE 3 ABSORBANCE AT 275 n.m» °P THE PRODUCTS OF DIGESTION OF HAEMOGLOBIN WITH STANDARD PEPSIN as reliable and accurate multiple estimations on a single sample was carried out to confirm the reproducibility. Five Hour Test Procedure Prior to any testing each animal was trained to stand in a specially constructed stand. This was raised about two and one-half feet so that the level of the dogs1 legs and the collection apparatus were at a convenient height for continuous close observation. Similarly, the biuret used for measuring the i n s t i l l e d buffer and the Harvard pumps for infusion of hormones were placed conveniently beside the dog stands. The two dog stands, f a c i l i t i e s for pepsin estimation, and the automatic titrimeter allowed two 5 hour tests to be done each day. Each dog was therefore tested either twice of three times per week. There was always at least 48 hours between each test and usually more." Before each test the dog was fasted for twelve to fourteen hours. For one hour before each test the dog was allowed to exercise freely with the gastric f i s t u l a open. This ensured that the dog's stomach was entirely empty and that the f i s t u l a was draining properly. The dog was then allowed to stand in the dog stand in a comfortable position. It was not necessary to restrain any dog although a support was tied loosely under the dog's abdomen only to prevent the dog from sittin g down and thereby potentially damaging i t s e l f on the collecting apparatus. During testing there was no evidence of fatigue in any dog other than during insulin testing when reducing blood sugar resulted in some unsteadiness in a l l dogs. At the start of each test, an intravenous infusion line was introduced usually into the large vein on the anterior aspect of the fore limb. Disposable 18 gauge needles were tightly fitted to thin polythene tubing. At the other end the tubing was f i t t e d to a stub end Luer connector which was i t s e l f f i t t e d snugly to a 50 ml disposable syringe. This was driven by a double Harvard pump. The speed of the Harvard pump was adjustable and was set to achieve an i n f u s i o n rate of 43 ml an hour. This was rate measured to ensure accuracy of d e l i v e r y on a number of occasions. This rate on i n f u s i o n was chosen for a number of reasons. At t h i s rate, any inadvertent extravascular i n f u s i o n would be recognized very r a p i d l y and the test appropriately terminated. At t h i s high rate the change of syringes which i s an opportunity for minimal i n t e r r u p t i o n of i n f u s i o n , i s much less c r i t i c a l than at a slow rate. Any leak i n the system would also be picked up quickly. As each test required i n f u s i o n of g a s t r i n i n about 200 ml s a l i n e , i t was possible to keep most of the s o l u t i o n at approximately 0° C u n t i l i n f u s i o n . As each animal had a small d a i l y loss of g a s t r i c s ecretion from the Pavlov pouch and had been fasted for twelve to fourteen hours p r i o r to each t e s t , i t was f e l t to be of some benefit to replace f l u i d and e l e c t r o l y t e s by i n f u s i o n of 200 -250 ml s a l i n e during each t e s t . (43 ml/hr) The i n s e r t i o n and maintenance of the intravenous i n f u s i o n l i n e s were accomplished without d i f f i c u l t y . This was probably because of the expertise developed by such frequent performance of the t e s t s . As the needles used were disposable, i t was possible to use extremely sharp needles and thereby inconvenience the dogs as l i t t l e as p o s s i b l e . No i n d i c a t i o n of d i s l i k e for the procedures was observed. As the influence of emotion on g a s t r i c s ecretion may be profound t h i s f actor was f e l t to be of considerable importance. Most tests involved the i n f u s i o n of g a s t r i n continuously for four hours including i n f u s i o n of a polypeptide for one hour. Separate s o l u t i o n syringe and intravenous routes were used for each s o l u t i o n . Each te s t therefore 17 required the use of two separate veins. Ipsilateral fore limb and hind limb veins were used for each test and the side used was alternated. This allowed completion of the series of tests without great d i f f i c u l t y . A small number of tests were abandoned for some reason. Only one or two of these were because of d i f f i c u l t y of establishing or maintaining satisfactory infusion lines. Analysis of Results Continuous infusion of gastrin results in a plateau of secretion from the pouch which i s maintained for at least three hours. When this plateau had been established continuous infusion of a second polypeptide "inhibitor" for a period of one hour modified the level of secretion as shown in f i g . 4. In this example, the inhibitor was infused from the beginning of period 7 u n t i l the end of period 10. Each test was used to provide two sets of data; the secretory response of the pouch to continuous infusion of a specific dose of gastrin and the response when a continuous infusion of another polypeptide inhibitor was added. A representation of the measurements obtained from each test and used for analysis i f shown in f i g . 5. The level of secretory response A to gastrin alone was measured by the value of either peptic activity or hydrogen ion present in the two 15 minute samples immediately prior to starting infusion of the inhibitor. The last two 15 minute samples obtained during the one hour infusion of the "inhibitor" were taken as the response to gastrin + inhibitor and are shown as B in f i g . 5. The percentage of inhibition C was caluculated from the values A + B. In this figure the data used to establish the effect of gastrin alone would be the values of acid or pepsin measured in period 7 and 8. The measured levels during period 11 and 12 would represent the effect of simultaneous infusion of gastrin and inhibitor. Clearly, the GASTRIN FIGURE 4 EXAMPLE OF EFFECT OF CONTINUOUS INFUSION OF A POLYPEPTIDE INHIBITOR ON THE PAVLOV POUCH SECRETION OF ACID UNDER CONTINUOUS STIMULATION BY 15 LEUCINE HUMAN GASTRIN GASTRIN FIGURE 5 A DIAGRAMMATIC EXPLANATION OF THE MEASUREMENTS USED IN DETERMINING THE EFFECT OF AN INHIBITOR A = Pre-inhibition plateau secretion level B = Secretion level under influence of gastrin and inhibitor C = Calculated inhibition 20 r e s u l t s o f bo t h hydrogen i o n s e c r e t i o n and p e p t i c a c t i v i t y a r e a n a l y z e d e n t i r e l y s e p a r a t e l y and the r e s u l t s w i l l be d i s c u s s e d s e p a r a t e l y . The d a t a f o r each i n h i b i t o r a t the t h r e e doses o f g a s t r i n used were c a l c u l a t e d as d e s c r i b e d . However l i n e a r t r a n s f o r m a t i o n s o f the d a t a from a l l dogs a t each dose o f g a s t r i n were a l s o c a r r i e d out i n a s s o c i a t i o n w i t h the Department o f Surgery programmer and u s i n g the U.B.C. computer s e r v i c e . The d a t a was key punched onto c a r d s and s t o r e d i n t h i s manner. U s i n g a s t a n d a r d l i n e a r r e g r e s s i o n programme, S I M R E G (unweighted l e a s t square method) t h r e e s e p a r a t e l i n e a r t r a n s f o r m a t i o n s o f the a c i d s e c r e t i o n d a t a and o f the p e p s i n s e c r e t i o n d a t a were o b t a i n e d . A l i n e a r r e g r e s s i o n o f the d a t a o b t a i n e d d u r i n g i n f u s i o n o f the t h r e e d i f f e r e n t doses of g a s t r i n was c a l c u l a t e d . A second l i n e a r r e g r e s s i o n o f the d a t a o b t a i n e d d u r i n g the l a s t two p e r i o d s o f i n f u s i o n o f bo t h g a s t r i n and i n h i b i t o r was computed f o r each i n h i b i t o r . Three l i n e a r t r a n s f o r m a t i o n s r e s p o n s e v s . dose, r e s p o n s e v s . re s p o n s e d i v i d e d by dose, and r e c i p r o c a l r e s p o n s e v s . r e c i p r o c a l dose were each computed and e q u a t i o n s o b t a i n e d . The same t h r e e l i n e a r t r a n s f o r m a t i o n s o f the d a t a o b t a i n e d d u r i n g i n f u s i o n o f g a s t r i n and i n h i b i t o r were computed f o r t h e p o l y p e p t i d e s i n v e s t i g a t e d i n t h i s s t u d y , except G.I.P. The e q u a t i o n s o f each s e t o f l i n e a r t r a n s f o r m a t i o n s r e p r e s e n t i n g the response o f the pouch t o g a s t r i n a l o n e , and to g a s t r i n and i n h i b i t o r were then compared f o r common s l o p e and common e q u a t i o n a g a i n by t h e s t a n d a r d computer programme S I M R E G. C o n s i d e r a t i o n o f the an a l o g y between hormone c e l l i n t e r a c t i o n and enzyme s u b s t r a t e k i n e t i c s and an e x p l a n a t i o n of t e r m i n o l o g y and i n t e r p r e t a t i o n o f the l i n e a r e q u a t i o n s i s found i n the appendix. MATERIALS 15 leucine human Gastrin This hormone was kindly gifted by Dr. E.S. Wiinsch. It had been synthesised at the Max Plank Institue, Munich and was supplied as a dry white powder. The original sample supplied was divided into small aliquots of approximately 100 ugm and stored in small sealed test tubes kept refrigerated. The weight of each aliquot was carefully obtained using an electronic microbalance. Immediately prior to each five hour secretion test, the gastrin was dissolved in alkalinised d i s t i l l e d water. Alkalinisation was achieved by addition of a small accurately measured volume of dilute ammonium hydrochloride. Accurate concentration was obtained by the use of micro pipettes to deliver the solvent. The stock gastrin solution was then added to sterile normal saline to obtain the correct dosage for each dog. Secretin This was obtained from the Gastro-intestinal Hormone Laboratory, Karolinska as pure natural porcine secretin. (Batchl7221) The contents of each sterile sealed ampoule were dissolved in sterile physiological saline immediately prior to each experiment. Each ampoule contained 75 c l i n i c a l units or 18.75 pgm of pure peptide. The stock solution was then dissolved in further sterile saline to obtain appropriate dosage for each dog. If the stock solution was not used immediately the remainder was frozen and kept refrigerated, but for no longer than 48 hours. Glucagon This was kindly gifted by Dr. E.S. WUnsch and had been synthesised at the Max Plank Institute, Munich. Small aliquots were accurately weighted in a micro balance and stored i n a sealed glass test tube. Prior to each experiment, the glucagon was dissolved i n s t e r i l e water and made up with s t e r i l e s a l i n e to a su i t a b l e concentration for in f u s i o n of each dog. The stock s o l u t i o n was kept frozen. Secretin 12 - 27 This was synthesised at the Max Plank I n s t i t u t e , Munich and g i f t e d by Dr. E.S. Wunsch. Small aliquots were accurately weighted and stored i n a manner s i m i l a r to other polypeptides. Stock solutions and d i l u t e solutions were made up i n an i d e n t i c a l manner. Stock solutions were used as soon as possible and kept frozen. G.I.P. This was kindly g i f t e d by Dr. J.C. Brown, Department of Physiology, University of B.C. I t had been prepared i n hi s laboratory by the standard published method. As with the other hormones used, the f a c i l i t i e s f o r accurate weighing and storage were generously provided by Dr. Brown. The preparation of stock solutions and in f u s i o n solutions was however ca r r i e d out i n the Department of Surgery, Vancouver General H o s p i t a l . 23 RESULTS Reproduc i b i l i t y of measured pouch response to Gastrin M u l t i p l e factors both recognized and not, influence the measured response of the pouch to i n f u s i o n of a stimulant. C l e a r l y , the experimental methods used must t r y to eliminate errors i n c a l c u l a t i o n or administration of stimulant dosage, and c o l l e c t i o n and measurement of sample volumes and concentrations. The presence of such errors could render i n t e r p r e t a t i o n of the r e s u l t s u n r e l i a b l e or valueless. In an attempt to evaluate possible errors the control r e s u l t s for each dog at each dose l e v e l were paired. P l o t t i n g of each 15 minute r e s u l t of the calculated a c i d response i n that period was plotted against the r e s u l t i n the same period i n the paired t e s t . Thirteen paired tests resulted i n a t o t a l of 208 pairs of r e s u l t s . Linear regression of these paired r e s u l t s was c a r r i e d out by computer and the p l o t obtained shown i n f i g . 6. Examination of the s i g n i f i c a n c e of the c o r r e l a t i o n c o e f f i c i e n t 0.85 indicated a very high s i g n i f i c a n c e to the obvious c o r r e l a t i o n between each of the paired 15 minute r e s u l t s . This r e s u l t indicates that the method used does not have inherent experimental errors which s i g n i f i c a n t l y influence the measured acid secretion response of the pouch. By implication therefore there was a reproducible and adequate accuracy i n d e l i v e r y of stimulant dosage, and c o l l e c t i o n and measurement of sample volume and hydrogen.ion concentration. As described multiple estimations of pepsin concentration i n the same sample indicated good r e p r o d u c i b i l i t y for the method of measurement used for the measurement of peptic a c t i v i t y . Therefore i t would appear that the r e s u l t s obtained probably have not been influenced by any marked experimental error. 24 1000 T REPRODUCIBILITY TEST A vs TEST B GASTRIN ALL CONTROL RESULTS FIGURE 6 LINEAR REGRESSION OF 208 MATCHED 15 MINUTE ACID SECRETION VALUES FROM 26 RANDOMLY PAIRED CONTROL SECRETION TESTS 25 R e p r o d u c i b i l i t y of P e p s i n E s t i m a t i o n The r e s u l t s of m u l t i p l e e s t i m a t i o n s o f p e p t i c a c t i v i t y on two s e p a r a t e samples a r e shown on T a b l e I . As can be seen t h e r e i s l i t t l e v a r i a t i o n i n t h e r e s u l t s o b t a i n e d . The c o n c l u s i o n t h a t the method used w i t h the sample o b t a i n e d gave r e p r o d u c i b l e r e s u l t s was c o n f i r m e d many times d u r i n g the s e r i e s o f t e s t s as f r e q u e n t m u l t i p l e t e s t i n g o f the same sample y i e l d e d s i m i l a r r e s u l t s t o the examples shown. E f f e c t o f I n f u s i o n o f G a s t r i n on a c i d o utput A l l dogs used i n the st u d y were t e s t e d by c o n t i n u o u s i n f u s i o n of g a s t r i n a l o n e f o r a p e r i o d o f f i v e h o u r s . The same t h r e e dose l e v e l s were used i n each dog (0.7, 0.35, 0.175 ug/kg-hr) and the c o n t r o l t e s t s were c a r r i e d out b o t h a t the b e g i n n i n g of the s e r i e s o f t e s t s and a t the end. The a c i d and p e p s i n p r e s e n t i n the s e c r e t i o n were measured a s d e s c r i b e d . T h i r t y - t w o c o n t r o l t e s t s were completed e n t i r e l y s a t i s f a c t o r i l y . W i t h each dog i n s p i t e o f an o v e r n i g h t f a s t , o c c a s i o n a l l y the b a s a l c o l l e c t i o n s o b t a i n e d i n d i c a t e d t h a t t h e r e was s i g n i f i c a n t ongoing s e c r e t i o n a t the time of t e s t i n g . I t i s not c l e a r what the s t i m u l u s was i n t h e s e c a s e s b u t t h e s e t e s t s a l t h o u g h c o n c l u d e d w i t h o u t t e c h n i c a l d i f f i c u l t y , were not i n c l u d e d i n t h e 32 t e s t s used f o r a n a l y s i s o f the a c i d r e s ponse t o g a s t r i n a l o n e . The a c i d o utput f o r each f i f t e e n month p e r i o d f o r each dog were averaged and the d a t a o b t a i n e d shown i n T a b l e I I . The r e s u l t s o b t a i n e d f o r BC1 a t the dose o f 0.7 ug/kg-hr a r e graphed and shown i n f i g . 7 F i g . 8 shows the means of v a l u e s o b t a i n e d a t the t h r e e dose l e v e l s f o r BC3 i n graph form. I n g e n e r a l , the r e s u l t s o b t a i n e d show t h a t a l l t h r e e doses used produce a s u s t a i n e d p l a t e a u o f a c i d s e c r e t i o n i n a l l dogs. The l e v e l o f p l a t e a u s e c r e t i o n produced a t each dose l e v e l d i d v a r y from dog to dog, and i n each REPRODUCIBILITY OF PEPTIC ACTIVITY ESTIMATION SAMPLE A SAMPLE B U.V. Absorbance at 275 n.m. .375 , .3^5 . 3 ^ .353 .333 .339 .330 . .333 .343 Peptic Activity 460 443 44-2 453 434 435 443 424 428 439 U.V. absorbance at 275 n.m. .133 .131 .132 . .119 .121 .118 .115 .115 .118 .116 Peptic Activity 171 168 164 1.53. 155 1.52' 148 148 152 149 mean S.D. 440.1 10.8 mean S.D. 156 8.5 Table I 27 Gastrin 0 .7 / ig /kg-hr Dog BC 1 15 Minute Periods FIGURE 7 THE EFFECT OF INFUSION OF 0.7 jag/kg-hr. 15 LEUCINE HUMAN GASTRIN ON H + OUTPUT OF PAVLOV POUCH Mean of 5 tests and S.E.M. indicated 28 ACID SECRETION IN RESPONSE TO 15 LEUCINE HUMAN GASTRIN A . B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 15 minute periods FIGURE 8 THE EFFECT OF CONTINUOUS INFUSION OF THREE DIFFERENT DOSES OF 15 LEUCINE HUMAN GASTRIN ON H + OUTPUT OF PAVLOV POUCH 0.175 mean of two tests 0.35 mean of four tests 0.7 mean of two tests 29 dog with each experiment. However for each individual experiment, the plateaus were sustained with minimal or no f a l l - o f f after four hours. During periods 5 to 13, a particularly f l a t plot was obtained. Combining the values for plateau periods 8 to 11 in a l l tests, the level of acid secretion produced by 0.35 ug/kg-hr was 72 percent of the level in response to 0.7 ug/kg-hr. A dose of 0.175 ug/kg-hr produced a response of 58 percent. Effect of Infusion of Gastrin on Pepsin output The same thirty-two five hour tests used to determine the effect on acid output were usedr.to assess the effect of the three doses of gastrin used on pepsin output of the Pavlov pouch. The results of the measurement of peptic activity of each 15 minute sample have been analysed and are reported in a number of ways. The means of a l l control tests at each dose level of gastrin have been calculated for each dog and the values obtained for each 15 minute period are shown in Table I I I . In these experiments to determine the effect of continuous infusion of gastrin, but with many undetermined variables, i t was f e l t that using each test as i t s own control would yield most meaningful results. Therefore the peptic activity of the basal periods were compared to the results of measurement of peptic activity of gastric secretion after a sustained period of continuous infusion of gastrin. The values in periods 10 and 11 were therefore compared to the basal periods C and D. In each case, 10 was compared to C and 11 to D, yielding two match pairs of results for analysis. In view of the apparently inconsistent effect a number of s t a t i s t i c a l tests were used to analyse the pairs of results. These are detailed in Table IV. 30 The conclusion of the e f f e c t of i n f u s i o n of g a s t r i n on the measured peptic a c t i v i t y of the secretion of the Pavlov pouch i s deduced for the s t a t i s t i c a l evaluations shown i n t h i s table. The r e s u l t s of comparing the peptic a c t i v i t y of the basal periods C and D to that of periods 10 and 11 i s that the secretion of pepsin appears to be markedly dose r e l a t e d . Infusion of g a s t r i n at dose l e v e l s of 0.35 and 0.175 ug/kg-hr would appear to r e s u l t i n increased peptic a c t i v i t y of the secretion. However, the l a r g e r dose of 0.7 has e i t h e r no e f f e c t or an i n h i b i t o r y e f f e c t as measured by the method used i n t h i s s e r i e s of experiments. Results of i n f u s i o n of I n h i b i t o r s The measured Pavlov pouch se c r e t i o n of a c i d and pepsin i n response to g a s t r i n and an i n h i b i t o r y polypeptide are d e t a i l e d i n the Tables section, (see l i s t of tables) The periods during which both g a s t r i n and the i n h i b i t o r were continuously infused are indicated i n these t a b l e s . The values recorded i n these tables are used for a l l graphs and s t a t i s t i c a l a n a l y s i s . Conclusions therefore are based e n t i r e l y on evaluation of these r e s u l t s . For each i n h i b i t o r investigated, the r e s u l t s obtained for a l l dogs were pooled at each g a s t r i n dose l e v e l . The values obtained during i n f u s i o n of g a s t r i n alone and during i n f u s i o n and i n h i b i t o r were analysed separately. Linear transformations were computed and the equations obtained were compared as indicated i n the appendix. The e f f e c t of continuous i n f u s i o n of each i n h i b i t o r on the pouch output of a c i d and pepsin under the influence of g a s t r i n w i l l be considered separately. The l i n e a r ^transformations and Michaelis-Menten constants are d e t a i l e d and I the i n t e r p r e t a t i o n of these values given. Further deductions from these in t e r p r e t a t i o n s are expanded i n the discussion. 31 The Effect of Secretin on Pavlov pouch secretion The continuous infusion of Secretin 1.0 ug/kg-hr resulted in the inhibition of acid and apparently pepsin secretion of the Pavlov pouch already secreting in response to a continuous infusion of gastrin. The detailed results for these tests are shown in the Tables V - X. A typical example of the effect on acid secretion i s shown in f i g . 9 where the results of both tests on BC1 at 1.35 ug/kg-hr gastrin infusion rate are shown together with standard deviations. Fig. 10 shows in histogram form the relative inhibitions of acid secretion by the standard dose of secretin at each of the three doses of gastrin used. A l l results for a l l dogs are pooled and the standard errors of the mean are indicated. The effect on pepsin secretion is shown in the histogram f i g . 11. The linear transformation dose/response is shown in f i g . 12. The curvilinear nature of this linear regression is clearly indicated (for emphasis of this, two straight lines are included on the graph). However, conclusion on this inhibitory action are based on the interpretation of the linear transformations shown using the method described in the appendix. These regression equations are summarised in Tables XI and XII and the characteristic Michaelis-Menten parameters are also shown. Graphical representations of the equations are shown in f i g . 13 and f i g . 14. Acid Secretion Referring to the equation of the r - r/d plot in Table XI, a comparison of the slopes of the two linear regressions (in the presence of gastrin alone and gastrin with secretin) by computer gave a p value of .00. As this is less than .05, i t is considered that the linear regressions have different slopes. Therefore one of the two characteristics Michaelis-Menten parameters had been significantly altered by the presence of the inhibitor. 32 SECRETIN INHIBITION OF GASTRIN STIMULATED ACID SECRETION Gastrin 0.35 jug/kg-hr Dog BC 1 n = 2 T Secretin 1.0 ;ug/kg-hr t t X UJ 15 Minute Periods FIGURE 9 AN EXAMPLE OF THE EFFECT OF CONTINUOUS INFUSION OF 1.0 ug/kg-hr. PORCINE SECRETIN ON H + OUTPUT OF PAVLOV POUCH STIMULATED BY 0.35 ug/kg-hr. 15 LEUCINE HUMAN GASTRIN Mean of two tests and S.E.M. are shown 33 THE INHIBITION OF ACID SECRETION BY SECRETIN 10 PERCENTAGE INHIBITION OF H + OUTPUT OF PAVLOV POUCH BY 1.0 ug/kg-hr. PORCINE SECRETIN Three per hour doses of 15 leucine human gastrin are indicated Histogram represents mean of results of 8 tests at doses 0.175 and 0.35 and 10 tests at 0.7 ug/kg-hr. Standard errors of mean are shown. 34 THE INHIBITION OF PEPSIN SECRETION BY SECRETIN GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN 0.175>ig/kg + 0.35yg/kg + 07i;g/k g + SECRETIN SECRETIN SECRETIN FIGURE 11 PERCENTAGE INHIBITION OF MEASURED PEPTIC ACTIVITY OF PAVLOV POUCH SECRETION BY 1.0 ug/kg-hr. PORCINE SECRETIN Three per hour doses of 15 leucine human gastrin background stimulation are indicated Histogram represents mean of results of 8 tests at doses of 0.175 and 0.35 and 10 tests at 0.7 ug/kg-hr. Standard errors of mean are shown. 35 Z to + x cr LU 3. lOOOn 800H 600-\ 400H 200H GASTRIN y- 510.8 x+ 369.1 r= .45 1 GASTRIN + SECRETIN y- 70.58 x + 120 .6 r= .1 0.175 0.35 DOSE GASTRIN (jug/kg-hr. 0.7 FIGURE 12 CURVILINEAR DOSE RESPONSE PLOTS OF H + OUTPUT OF PAVLOV POUCH IN RESPONSE TO THREE DOSES OF 15 LEUCINE HUMAN GASTRIN ALONE AND TOGETHER WITH INFUSION OF 1.0 ug/kg-hr. PORCINE SECRETIN The curvilinear nature of the transformation is emphasized by the presence of two straight lines present for illustration 36 ACID SECRETION INHIBITION BY SECRETIN RESPONSE vs R E S P O N S E / D O S E R E S P O N S E / D O S E FIGURE 13 RESPONSE - RESPONSE/DOSE LINEAR TRANSFORMATION OF REGRESSION EQUATIONS OBTAINED BY ANALYSIS OF RESULTS OF INFUSION OF PORCINE SECRETIN ON H + OUTPUT OF PAVLOV POUCH STIMULATED BY 15 LEUCINE HUMAN GASTRIN (26 tests) 37 PEPSIN SECRETION INHIBITION BY SECRETIN RESPONSE vs RESPONSE/DOSE 2000-1 FIGURE 14 RESPONSE - RESPONSE/DOSE LINEAR TRANSFORMATION OF REGRESSION EQUATIONS OBTAINED BI ANALYSIS OF RESULTS OF INFUSION OF PORCINE SECRETIN ON MEASURED PEPTIC ACTIVITY OF PAVLOV POUCH SECRETION STIMULATED BY 15 LEUCINE HUMAN GASTRIN (26 tests) 38 The other parameter,Jthe C.M.R. was obtained, with i t s standard error, from the computer linear regression program print out. A Students' t test comparison of the C.M.R. in the presence of gastrin alone (489 standard error of 85.2) and gastrin and secretin (19.1 standard error of 20.7) demonstrated a highly significant difference p<002. It has therefore been demonstrated that the presence of secretin alters both of the Michaelis-Menten parameters as calculated from the response vs. response/dose linear transformation. This indicated that the inhibition of acid secretion by secretin in the Pavlov pouch secreting in response to gastrin has the characteristics of mixed inhibition. Pepsin secretion Referring to the equations of the r - r/d plot in Table XII, computer comparison of the slopes of the two linear regression lines obtained in the presence of gastrin and gastin and secretin yielding a probability value of 0.04. This indicated significantly different slopes for the two lines and therefore a change in the D,-Q induced by the presence of secretin. The C.M.R. values in the presence of gastrin (1049 f 138.4 S.E.) and gastrin and secretin (510 + 141.6 S.E.) were compared by Students' t test and found to be significantly different p<0.002. As both of the parameters D,.Q and C.M.R. have been significantly changed by the presence of secretin, the inhibition of pepsin secretion in response to gastrin in the Pavlov pouch had the characteristics of mixed inhibition. Further examination of this result in the discussion indicates this conclusion may be too simplistic. The Effect of Glucagon on _P_aylov pouch secretion The continuous infusion of glucagon 5.0 ug/kg-hr resulted in the inhibition of both acid and pepsin secretion of the Pavlov pouch stimulated 39 by continuous infusion of gastrin. The effect on acid and pepsin secretion are considered separately below. The results on acid and pepsin secretion are shown in Tables XIII to XVIII. Acid Secretion The results of the percentage inhibition of acid at each infusion dose of gastrin are shown by the histograms in f i g . 15. The mean and standard error of the mean of tests on a l l dogs was calculated. The equations of the linear regression computed of the results obtained during infusion of gastrin and gastrin and glucagon are shown in Table XIX. Graphical representation of the response vs. response/dose transformations are shown in f i g . 17. Referring to the response vs. response/dose plot, i t can be seen that the D^Q in absence and presence of glucagon are very similar (0.1267 and 0.1348). A computer comparison of the equation indicated that the slopes are not significantly different (common slope p = 0.83). For il l u s t r a t i o n only, the Limeweaver-Burke transformation i s shown in f i g . 19. The values for C.M.R. in the presence of gastrin alone (427.9 ± 87.28 S.E.) and gastrin and glucagon (286.6 ± 55.57 S.E.) were compared by Students' t test and found to be significantly different (p<0.05) The characteristic parameter C.M.R. in the Michaelis-Menten equation as calculated from the response vs. response/dose linear transformation has been changed by the presence of glucagon. However, there was no alteration in the D^Q as calculated from the same linear transformation. It must be concluded therefore that the inhibition of gastrin stimulated acid production in the Pavlov pouch by glucagon is of the non-competitive type. Pepsin secretion The percentage inhibition of pepsin at each infusion dose of gastrin in shown in f i g . 16. The standard errors of the mean are indicated. 40 The equations of the results obtained during infusion of gastrin and gastrin and glucagon are shown in Table XX. As for a l l other series of tests in this study, conclusions are based on the response vs. response/dose linear transformation. Referring to the Table XX, i t can be seen that there appears to be l i t t l e change in the calculated D ^ Q by the infusion of glucagon during stimulations by gastrin. Computer analysis of the slope of the two lines confirmed this and indicated parallelism (common slope .21). The calculated C.M.R. values in the presence of gastrin alone (1626 ± 191.6 S.E.) and gastrin and glucagon (805.1 ± 133.2 S.E.) were compared by Students' t test and found to be significantly different p<0.002. Therefore as with acid secretion, i t must be concluded that the inhibitory pattern of glucagon on gastrin stimulated pepsin secretion of the Pavlov pouch is non-competitive. The Effect of Secretin (12-27) on Pavlov pouch secretion Three different doses of secretin (12-27) were infused. The results of acid and pepsin measurement during test and periods during which secretin (12-27) was infused are shown in Tables XXI to XXVI. Acid Secretion The effect of infusion of the three different doses of secretin (12-27) on the Pavlov pouch output of acid is shown in the histogram f i g . 20. As can be seen the mean change is small and in a l l cases the standard deviation is sufficiently large to easily include the secretin in response to gastrin infusion only (i.e. 100%). This finding is born out in the computer analysis of the computed linear regression shown in Table XXVII. Comparison of the response vs. response/dose transformations indicated a common slope (p=.85) and also 41 THE INHIBITION O F ACID SECRETION BY G L U C A G O N GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN O.I75j,g/kg + 0.35pg/kg + 07ijg/kg + GLUCAGON GLUCAGON GLUCAGON FIGURE 15 PERCENTAGE INHIBITION OF H + OUTPUT OF PAVLOV POUCH BI 5.0 ug/kg-hr. SYNTHETIC GLUCAGON Three per hour doses of 15 leucine human gastrin background stimulation are indicated Histogram represents mean of results of 9 tests at 0.175t 8 tests at 0.35 and 8 tests at 0 .7 ugm/kg-hr. Standard errors of mean are shown. 42 THE INHIBITION O F PEPSIN SECRETION BY G L U C A G O N GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN 0.175yg/kg + 0.35ijg/kg + 07ijg/kg + GLUCAGON GLUCAGON GLUCAGON FIGURE 16 PERCENTAGE INHIBITION OF MEASURED PEPTIC ACTIVITY OF PAVLOV POUCH SECRETION BY 5.0 ug/kg-hr. SYNTHETIC GLUCAGON Three per hour doses of 15 leucine human gastrin background stimulation are indicated. Histogram represents mean of results of 9 tests at 0.175» 8 tests at 0.35 and 8 tests at 0.7 ugm/kg-hr. Standard errors of mean are shown. 43 A C I D S E C R E T I O N I N H I B I T I O N BY G L U C A G O N R E S P O N S E vs R E S P O N S E / D O S E 1000 2000 3000 R E S P O N S E / D O S E FIGURE 17 RESPONSE - RESPONSE/DOSE LINEAR TRANSFORMATION OF REGRESSION EQUATION OBTAINED BY ANALYSIS OF RESULTS OF INFUSION OF GLUCAGON ON H + OUTPUT OF PAVLOV POUCH STIMULATED BY 15 LEUCINE HUMAN GASTRIN (26 tests) 44 PEPSIN SECRETION INHIBITION BY G L U C A G O N RESPONSE vs R E S P O N S E / D O S E 500 1000 1500 Response/dose FIGURE 18 RESPONSE - RESPONSE/DOSE LINEAR TRANSFORMATION OF REGRESSION EQUATIONS OBTAINED BY ANALYSIS OF RESULTS OF INFUSION OF GLUCAGON ON MEASURED PEPTIC ACTIVITY OF PAVLOV POUCH SECRETION STIMULATED BY 15 LEUCINE HUMAN GASTRIN (25 tests) 45 LINEWEAVER-BURK PLOT GLUCAGON 1 D FIGURE 19 DOUBLE RECIPROCAL OR LINEWEAVER-BURK LINEAR TRANSFORMATION OF REGRESSION EQUATIONS OBTAINED BX ANALYSIS OF RESULTS OF INFUSION OF GLUCAGON ON H + OUTPUT OF PAVLOV POUCH STIMULATED BY 15 LEUCINE HUMAN GASTRIN 46 common equation (p=.62). This i s shown c l e a r l y i n f i g . 22. It i s cl e a r that under the conditions used i n t h i s study and at the doses of g a s t r i n and s e c r e t i n (12-27) used there i s no demonstrable e f f e c t on the g a s t r i n stimulated acid output of the Pavlov pouch exerted by the se c r e t i n (12-27). Pepsin secretion The percentage change i n pepsin output induced by s e c r e t i n (12-27) on the continuous stimulated Pavlov pouch i n shown i n histogram f i g . 21. From t h i s histogram l i t t l e consistent change i s indicated. The l i n e a r transformations are shown i n Table XXVIII. Reference to the response vs. response/dose transformation shows very s i m i l a r equations and c h a r a c t e r i s t i c Michaelis-Menten parameters C.M.R. and D^Q- Computer comparison of the two l i n e a r transformations with g a s t r i n i n f u s i o n only and g a s t r i n and s e c r e t i n (12-27) y i e l d s both a common slope (p=.69) and equation (p=.69) and equation (p=.64). The graphical representation f i g . 23 c l e a r l y indicates t h i s . This confirms that under the conditions of t h i s study s e c r e t i n (12-27) i n the doses used had no s i g n i f i c a n t e f f e c t on pepsin output of the Pavlov pouch. 47 THE INHIBITION OF ACID SECRETION BY SECRETIN (12-27) T 100 - i 9 0 -8 0 -7 0 -z O 6 0 -i— Oi (J 5 0 -co Q 4 0 -U < 3 0 -2 0 -1 0 -I GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN 0.35yg/l<g 0.35^g/kg 0.35jjg/kg 0.35ug/kg 0.35|jg/kg 0.35pg/kg SECRETIN (12-27; O.tyg/kg SECRETIN (12-27) 'Ong/kg SECRETIN (12.27) 2>g /kg FIGURE 20 PERCENTAGE INHIBITION OF H + OUTPUT OF PAVLOV POUCH BI THREE DOSES OF SECRETIN (12 - 27) Background stimulation 0*35 ug/kg-hr. 15 leucine human gastrin Histograms represent mean of results of 6 tests at 0.5, 7 tests at 1.0 and 6 tests at 2.5 ug/kg-hr Secretin (12- 27) infusion rate. 48 THE INHIBITION O F PEPSIN S E C R E T I O N BY S E C R E T I N (12-27) 6? z O t— U J OL V LU to Z CO 2 0 0 - i 1 8 0 -1 6 0 -1 4 0 -1 2 0 -1 0 0 -8 0 -6 0 -4 0 -2 0 -GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN GASTRIN 0.35jug/kg 0.35>ig/kg 0.35pg/kg O ^ g / k g 0.35yg/kg 0.35«g/kg + + + SECRETIN (12-27) SECRETIN (12-27) SECRETIN (12-27) 0.5>jg/kg l.t>g/kg 2.5><g/kg FIGURE 21 PERCENTAGE INHIBITION OF MEASURED PEPTIC ACTIVITY OF PAVLOV POUCH SECRETION BY THREE DOSES OF SECRETIN ( 12 - 27) Background stimulation 0.35 ug/kg-hr 15 leucine human gastrin Histograms represent mean of results of 6 tests at 0.5» 7 tests at 1.0, and 6 tests at 2.5 ug/kg-hr secretin ( 12 - 27) infusion rate. Standard errors of mean are shown. 49 ACID SECRETION INHIBITION BY SECRETIN (12-27) 1000 2000 Response/dose FIGURE 22 RESPONSE - RESPONSE/LOSE LINEAR TRANSFORMATION OF REGRESSION EQUATION OBTAINED BY ANALYSIS OF RESULTS OF INFUSION OF THREE DOSES OF SECRETIN (12 - 27) ON THE H + OUTPUT OF A PAVLOV POUCH STIMULATED BY 0.35 ug/kg-hr. 15 LEUCINE HUMAN GASTRIN (19 tests) 50 PEPSIN SECRETION INHIBITION BY SECRETIN (12-27) RESPONSE vs RESPONSE/DOSE 1000 2000 3000 4000 5000 Response/dose FIGURE 23 RESPONSE - RESPONSE/DOSE LINEAR TRANSFORMATION OF REGRESSION EQUATION OBTAINED BY ANALYSIS OF RESULTS OF INFUSION OF THREE DOSES OF SECRETIN ( 12-27) ON THE MEASURED PEPTIC ACTIVITY OF THE SECRETION OF A PAVLOV POUCH STIMULATED BY 0.35 ug/kg-hr. 15 LEUCINE HUMAN GASTRIN (19 tests) 51 The Effect of Gastric Inhibitory Polypeptide A limited supply of GIP was available. Unfortunately i t was only possible to carry out ten technically satisfactory tests using this material. The results of four tests were discarded as the gastric f i s t u l a drainage was much less than would normally be expected from previous tests at the same gastrin dose. The effect of endogenous inhibition due to passage of acid in the duodenum would not be predictable in these tests. (See Table XXX and XXXI) Consistent inhibition of acid secretion by 2.5 ug/kg-hr G.I.P. was found. The percentage inhibition was 49 percent at the higher dose of gastrin 0.7 ug/kg-hr, 35 percent at 0.25 and 29 percent at the low dose 0.175 ug/kg-hr. The inhibition of pepsin was also quite marked; 69 percent at 0.7 ug/kg-hr gastrin infusion, 34 percent at 0.35 and 40 percent at 0.175 ug/kg-hr. The small number of tests at dose of gastrin infusion (either 2 or 3) did not allow s t a t i s t i c a l evaluation. It i s therefore not possible to speculate on the mode of action of this polypeptide. However i t is possible to confirm the potency of this dose of 2.5 ug/kg-hr in inhibiting both acid and pepsin stimulated by gastrin infusion in the doses used. It was also surprising, although only a few test results were available, that the inhibition of secretion was more marked when the stimulatory dose of gastrin was highest. 52 DISCUSSION Clearly, any surgical interference with the pouch throughout the series of tests would have influenced i t s size, drainage and possibly innervation in a completely unpredictable manner. It was therefore of marked importance that although there were minimal problems associated with the pouch cannulae (i.e. gastric erosions or subcutaneous abscesses) in no case was i t necessary to even consider revising the pouch drainage cannula in any dog. The unknown influence on the results after any revision would probably have prevented valid interpretation. It was necessary to revise the gastric fistulae cannulae and following any such procedure the dog was allowed to convalesce for three weeks prior to further testing. In a l l tests used in the analysis of results the influence of endogenous and therefore uncertain inhibition was minimized by ensuring good drainage from the gastric cannula. Measurement of the gastric f i s t u l a output during the five hour test was carried out by collection in a totally supported football bladder. If this collection contained food or was not consistent with previous collections at the same dose of gastrin, indicating poor drainage of the stomach, then the results were discarded. During the i n i t i a l tests, i t was found that the dog was not basal at the start of the test and a number of tests were discarded for this reason As was mentioned, insulin testing ensured that the pouches were innervated. Employment of the procedure described by Cleator and Harrison"''''' to strengthen the mucosal bridge probably contributed to the lack of problem with leaks from the stomach to pouch both on methylene blue testing and as noted by close observation of test results. 53 As noted previously, the polypeptides were infused in sterile saline at a rate of 43 ml/hr. This rate was chosen to ensure even at high gastric secretion rates that the dogs did not establish a reduced intra-vascular volume with resultant change in gastric mucosa perfusion. Also, this high rate of infusion allowed any inadvertent extravascular infusion to be noted immediately. This latter problem was almost non existent. Further the supply of sodium and chloride ions prevented any serious deficit occurring during testing. The dose of 15 leucine human gastrin was chosen such that the secretory response would be sufficient at the low dose (0.175 ug/kg-hr) to eliminate serious inaccuracies due to collection but such that the high dose did not produce a maximal response. In fact the dose 0.7 ug/kg-hr of human gastrin I produced about 50 to 60 percent secretory response for acid and pepsin 12 when used in gastric f i s t u l a dogs by A.R. Cooke (1967). It was f e l t that this level of response to the stimulant would enable any augmentation rather than the expected inhibition of secretin to be readily recognized. The dose of secretin used was double that found by Brooks et a l ^ (1970) to inhibit acid secretion 65 percent in Pavlove pouch dogs. In a separate study Johnson and Grossman found that the dose used in this study 1.0 ug/kg-hr completely inhibited acid secretion evoked by gastrin when the response was 70 percent maximal. The inhibitory dose of glucagon (5 ug/kg-hr) used in this study was 1/200 that given by Clarke et a l " ^ and found to inhibit meat extract stimu-lated secretion from a canine innervated pouch when given a single intravenous dose. It was also considerably less than that which Lin and Warrick used (50 jig/kg-hr) and demonstrated a 40 percent reduction of pentagastrin stimulated acid output of a vagally innervated gastric f i s t u l a . 54 As the effect of secretin 0-2-27) was entirely unknown, the dosages used were roughly 100, 200, and 500 percent on a molar basis of the dose of secretin known to inhibit markedly the acid output of the Pavlov pouch. The dose of gastric inhibitory polypeptide used 2.5 ug/kg-hr was known from a thesis: The Isolation and Physiological Actions of Gastric Inhibitory Polypeptide by R.A. Pederson (University of B.C.) and from many publications from Dr. J.C. Brown's laboratory to markedly inhibit acid secretion from the canine gastric pouch. The polypeptides used in this study were a l l administered by continuous infusion. As they are also either believed or known to have short half lives, the steady states or plateaus of secretions noted probably occur because of the rapid establishment of a dynamic equilibrium of intravenous delivery and breakdown or excretion of the hormone. Without this situation, the analogy with enzyme substrate kinetics would not be soundly based. The apparatus used for collection of the gastric secretion and buffer was very simple and lightweight. As mentioned earlier, the reproducibility of delivery of buffer to the pouch and collection from the pouch was found to be excellent and an error of less than 2 percent was normally found. The reproducibility of delivery of stimulant by intravenous infusion of an accurate dosage was obviously not amenable to direct testing. However, the random pairing of results, and plotting as in Fig. 6 demonstrates that there was a very significant positive correlation. This attest to the overall accuracy of delivery of stimulant, reproducibility of secretory response of the pouch and efficient collection of the secretion. 55 The use of the same test stands, a constant attendant during a l l test and quiet surroundings must have tended to reduce psychic factors possibly influencing the response of the pouch. The buffer delivery apparatus was arranged to ensure that a constant and small pressure only was used to i n s t i l l the buffer. The post mortem studies confirm that the volume of buffer used did not cause significant distension of the pouch. The possibility of inadvertant mechanical stimulation of secretion i t is f e l t was very small. 48 The use of glycine/hydrochloric acid buffer pH 3.0 probably contri-bute markedly to the validity of the results obtained. First completeness of collection especially at small rates of secretion i s much improved by this method. Secondly, the measured acid output is the difference between the secreted hydrogen ions and those which have back diffused through the mucosa. The use of a buffer and maintenance of an almost constant pH at a l l levels of secretion of acid ensures very l i t t l e variation in hydrogen ion gradient across the gastric mucosa. Possible effect of back diffusion on the results are thereby minimised. Thirdly, the measured peptic activity of the pouch secretion i s dependent on the concentration of active pepsin in the sample. Pepsinogen is secreted by the mucosa and a number of different pepsins are produced by auto-catalytic breakdown of the various pepsinogens secreted. This breakdown is highly pH dependent occuring maximally in the range of 2.0 to 3.5. The pesinogen i s irreversibly inactivated at or close to neutrality, Thus, at basal acid secretion, inspite of significant pepsinogen secretion, the measurement of peptic activity of the sample would indicate negligible activity and encourage the deduction that there was no pepsinogen being secreted. 56 It i s clear that the maintenance of an optimal pH for conversion of pepsinogen to pepsin and the constant conditions of storage prior to estimation of peptic activity are essential for the accurate determination of peptic activity. These constant conditions also enable the deductions about secretion of pepsinogen from the measure peptic activity to be made with more confidence. A disadvantage of the use of the buffer i s that i t s effect on acid and pepsin secretion has not been carefully studied. However, basal acid levels are low and well within the normal range for this type of preparation. It would therefore appear that any stimulatory action must be negligible although, the unlikely possibility of inhibition or augmentation of the secretory response to the polypeptides used does not exist. Again as this factor was constant throughout, even i f present, i t probably would not invalidate the results. The influence of the buffer on the measured peptic activity of the pouch secretion i s of some interest. The reported effect of most stimulants of acid secretion i s a concomitant increase in measured peptic activity. It i s commonly assumed that this i s due either to a release of pepsinogen in response to the stimulus or a passive washout of pepsin because of the increased volume of secretion. Clearly i f the effect of the acid stimulant i s to lower the pH of the secretion from 5.5 to 2.5 for example, the activation of pepsinogen would very markedly increase. This might be erroneously interpreted as an increase in pepsinogen secretion. For this reason, with the constant low pH in the pouch lumen, i t i s f e l t that the measured peptic activity accurately mirrors the secretion of pepsinogen. 57 In the present study, the collected secretion buffer mixture had a pH in the range 2.2 to 3.0. The extremes noted, only on a very few occasions, were 0.3 pH units above or below this. Grossman and Marks investigating the effect of pH on the measured peptic activity of pyloric juice and solutions of crystalline pH note that this range 2.2 to 3.0 appeared optimal for measurement of total peptic activity. They also noted that acidified secretion containing pepsinogen lost almost a l l peptic activity i f exposed to an alkaline pH. Measured activity varied by up to 300 percent i f the pH. was changed from 3.0 to 4.0 but in the range 2.2 to 3.0, changes of only 10 to 15 percent in measured activity occurred. It would therefore not seem reasonable to draw conclusions on the effect of acid stimulants on the secretion of pepsinogen unless the effect of concomitant pH decrease is known or as in this study, kept to a minimum. It would seem that any report on peptic activity of gastric secretion without pH control, or compen-sation for i t s lack, would not be an accurate representation of the secretion of pepsinogen. Discrepancies between the results obtained in this study and others without control would therefore be expected. Previous work on gastric secretion and inhibition have been handicapped by the availability of hormones of uncertain purity. It was most fortunate for this study that synthetic glucagon, 15 leucine human gastrin and secretin (12-27) were kindly gifted by Dr. E.S. WUnsch. The other hormones used were highly purified extracts; the porcine secretin was purchased from G.I.H. Laboratory Karolinska (Batch 17222) and the Gastric Inhibitory Polypeptide was gifted by Dr. J.C. Brown. 58 In the gastrin used the methionine of the active tetrapeptide was 34 replaced by leucine. However, Morley and Smith have established that no loss of activity occurs with: the simple replacement. It i s presumed that interaction of the hormone at the specific c e l l receptor site i s identical to the normal methionyl gastrin and that interference with this interaction would be very similar i f not identical for both of these gastrins. The method by which, a hormone e l i c i t s a response from a target c e l l has recently been c l a r i f i e d . Using c e l l homogenates, Sutherland"^ in a series of classic experiments, showed that interaction of small polypeptide hormones with a c e l l membrane bound enzyme, adenyl cyclase, results in an increased intracellular level of a high energy compound, cyclic AMP. This in turn acts intracellularly to alter the rate of one or more cellular processes. Glucagon and epinephrine i n i t i a l l y and then ACTH, TSH, secretin, gastrin and parathormone were found to activate adenyl cyclase i n cells from their normal target organ. This second messenger system i s diagramatically illustrated in Fig. 24. In the intact animal, i t i s well recognized that a complex interplay of neural and hormonal factors may influence the physiological response of any target organ. A physiological stimulus such as eating a meal may imitate simultaneous release of a number of gastrointestinal hormones. It i s now accepted that these polypeptides, although having a primary target organ, may have actions on other organs and may interact with other 21 hormones influencing these organs. This interaction of pairs of hormones may result in an augmented or inhibited response by the target organ. 59 THE SECOND MESSENGER SYSTEM INVOLVING ADENYL CYCLASE ENDOCRINE GLAND + STIM. 5 •) EFFECTOR CELL j ATP 5'-AMP H O R M O N E — (ISt MESSENGER) INACTIVATED H O R M O N E j + Mg / Mg M E T H Y L vV P H O S P H O D I E S T E R A S E [ X A N T H I N E S / \ ADENINE ( O R Y) C Y C L I C 3',5-AMP (2nd MESSENGER) E N Z Y M E S . ^ P E R M E A B I L I T Y E T C • PHYSIOLOGICAL" R E S P O N S E I N C L U D I N G S T E R O I D S , T 4 , E T C FIGURE 24 ADAPTED FROM: "Some aspects of the Biological Role of Adenosine 3 > 5 ? monophosphate Cyclic AMP" by E.W. Sutherland et al . Circulation XXXVII, 279 (1968) 60 23 Harris and Alonso have reported a series of experiments using frog gastric mucosa which indicate that gastrin stimulated HCL secretion is mediated by intracellular cyclic AMP increases. The percise method by which the secretion i s stimulated i s not clear however further indirect evidence using prostaglandin E, which i s known to inhibit accumulation of cyclic AMP, suggests that gastrin acts by alteration of the level of intracellular AMP. Prostaglandin E inhibits gastric secretion in 41 42 1 mammals (Ramwell and Shaw 1968, Robert et a l 1970) and man (Carter et a l 1971) as well as in frog gastric mucosa preparations. In the latter, secretion stimulated by gastrin i s abolished and secretion stimulated by histamine reduced. It i s of particular interest that although glucagon and secretin have similar molecular structures and both activate cyclic AMP, i t would appear that they operate by way of separate receptor sites. Trypsin treatment of adipose tissue, which prevented the action of glucagon, has been shown not to influence response of that tissue to secretin. Also, in liver preparations, secretin did not stimulate adenyl cyclase and did not 44 interfere with the action of glucagon. Ross 1970 provided experimental evidence that the hormones interacted with separate receptor sites in at least some vascular beds. It would not be improbable therefore i f the inhibitory effects of the two hormones, on an adenyl cyclase stimulated mechanism, had different characteristics. Wlodek and Leach found hydrogen ion fluxes were similar in innervated 35 and denervated canine fundic pouches. However Overholt et a l found that significant back diffusion occurred on i n s t i l l i n g 100 mM hydrochloric acid into the stomachs of vagotomised rats. Sham vagotomy only abolished the observed back diffusion. The concentration and volume of secretion in response to large doses of gastrin tetrapeptide was found to mask the back diffusion although the measured acid output was obviously only a fraction of the acid secretory response to the gastrin stimulus. In this study, the pouch was innervated. The presence of the buffer and relatively constant pH in the lumen w i l l tend to minimise changes in back diffusion. There i s no doubt that unrecognized and unexpected factors may have influenced back diffusion. This possibility i t i s hoped i s remote elimination of a l l possible influences on back diffusion would clearly be impossible. Because of the many factors influencing secretion in the intact Pavlov pouch, i t was expected that relatively large variations in measured response would be found both within each test, and from dog to dog. The acid secretion by dog BC3 in response to three different doses of gastrin, shown in Fig 8, illustrates this variation. Only two tests at 0.175 and 0.7 ug/kg-hr were carried out and the graph shows a plateau with some variation. However the plateau maintained at the dose 0.35 represents the mean of four tests and clearly i s subject to much smaller changes. Fortunately large number of tests were carried out at a l l doses of gastrin and with each inhibitor polypeptide. This enable pooling of data and use of standard s t a t i s t i c a l analysis in the evaluation of the results. 62 The results on pepsin output of the pouch in response to gastrin were surprising and prompted very thorough s t a t i s t i c a l examination (see Table IV). The fact that large doses of gastrin greater than that required to e l i c i t a maximal response may cause a dimunition of acid and pepsin 19 output, has been demonstrated by many authors (e.g. Gillespie J.E., 20 33 12 Grossman M.I., Master et a l , Cooke ). In this study although infusion of 0.175 and 0.35 ug/kg-hr gastrin resulted in increased measured peptic activity the dose 0.7 inhibited peptic activity to a level well below that occurring while the animal was basal. As discussed earlier i t i s the author's belief that these measured values accurately represent the secretion of pepsinogen by the pouch. This phenomenon i s not consistent with most reports on the subject. However, other workers, not controlling the conditions under which pepsinogen is converted to pepsin, might easily f a i l to observe this effect. It has to be concluded that continuous infusion of synthetic 15 leucine human gastrin at doses of 0.175 and 0.35 ug/kg-hr results in an increase of pepsinogen secretion of 65 percent and 90 percent respectively over basal levels. A dose of 0.7 ug/kg-hr resulted in a secretion of pepsinogen 25 percent less than the basal secretion. A possible explanation for this observation i s the application of Paton's theory of drug action. In this theory, the response of a target c e l l i s dependent on the rate of association of a drug and i t s receptor site, rather than the absolute number of receptor sites occupied at one time. Under conditions where rapid dissociation of the drug, or hormone, occurs the receptor sites are quickly available for reoccupation. If slow dissociation occurs for example in presence of high concentrations of drug (or hormone) the response of the target c e l l i s relatively less. 63 If this conjectural theory i s true, i t i s surprising that i t should occur at a dose of gastrin 0.7 ug/kg-hr which would be expected to produce only a 50 percent maximal acid response. Nethertheless the results clearly indicate inhibition of pepsinogen secretion at this dose under the conditions used in this experiment. Infusion of both porcine secretin and synthetic glucagon resulted in significant inhibition of acid (Fig 10 and Fig 15) and pepsin (Fig 11 and Fig 16) secretion. Infusion of secretin 1.0 ug/kg-hr resulted in a 74 percent inhibiton of acid output (mean for three doses of gastrin). Glucagon however at a dose of five times that (5.0 ug/kg-hr) reduced the acid output by 32 percent (mean of three doses of gastrin. It can therefore be stated that secretin, on a molar basis i s a considerably more potent inhibitor of acid secretion stimulated by synthetic human leucine gastrin. There was more variation in the percentage inhibition of pepsin secretion resulting from infusion of glucagon or secretin on the three different background doses of gastrin (Fig 11 and Fig 16). The mean percentage inhibition of pepsin secretion (for the three levels of background stimulation) was 40 percent for glucagon and 26 percent for secretin. The interpretation of these results in combination with the results for acid inhibiton encourage interesting speculation. The theories of the modes of stimulation of pepsin secretion allow for at least two possible explanations of the finding that secretin inhibits acid by a mean of 74 percent (at the three doses of gastrin used) but pepsin by only 26 percent. 64 Again, the efficacy of the secretin and gastrin molecules on the peptic cells may be markedly different from the effect of parietal c e l l s , because of dissimilar receptor site interactions or different intracellular 49 responses. Stening et a l found a profound reduction in secretion volume occurring during infusion of secretin in Heidenhain pouch dogs stimulated by infusion of gastrin. This reduction was so large to prevent, in their experimental model, reliable values for pepsin output from being obtained. The use of glycine buffer in this study and the resultant good collection of samples, enables the author to conclude that the measured peptic activity of the secretion during infusion of secretin i s reduced, by only by 26 percent. j Secretin has been shown in two studies, during which the gastric pH was not controlled, to increase measured pepsin output in dogs with ongoing 52 secretion in response to bethanechol (Way L.W. ) and histamine (Stening et a l 4 9 ) . The relatively small inhibition of pepsin secretion in this study could therefore be explained by the interaction of two components, an increased cellular response, but a markedly reduced volume of secretion. The pepsinogen secreted may not be delivered to the pouch lumen, remaining withing the gastrin gland lumen. If this i s so, when the secretin inhibition is removed and the volume of secretion returns to the pre-inhibition level a large increase in the level of peptic activity measured in the secretion would be expected. This theory was tested by examination of the measured peptic activity of secretion in the half hour immediately before infusion of secretin and in the period from 15 to 45 minutes after stopping infusion c f . Tables VIII - X. The values at a l l three levels of gastrin background stimulus were pooled, giving fifty-two 15 minute values before and 50 after infusion of secretin. The mean and standard deviation of the 15 minute values of measured peptic activity immediately prior to secretin infusion was 1980 ± 114.1. However, the secretion in the fifteen to forty-five minute period after the infusion of secretin had been stopped was 55 percent highter at 3076 ± 1701.8. This i s a highly significant difference (p < 0.002). Clearly i f the secretion of pepsin has remained constant during infusion of secretin, presuming no influence of peptic c e l l secretion, then the reduction in noted peptic activity would have been identical to the reduction in secretion volume and probably more than the 26 percent observed. Also, the rebound in level of pepsin secretion on resumption of the pre-inhibition volume of secretion would have been approximately equal to the noted 26 percent reduction during infusion of secretin. The observed rebound i s more than twice the measured inhibition of pepsin secretion. An attractive explanation for these results is that secretin does in fact increase the peptic c e l l output of pepsinogen but that the very pronounced inhibition of volume of secretion allows this pepsin to remain within the gastric gland lumen and avoid collection. Following removal of the inhibitory influences of the accumulated pepsin is "washed out" and, as noted in this study, i t results in an apparent rebound in secretion. Consideration of the linear transformations and Michaelis-Menten parameters in Table XII suggest a mixed inhibition. Pure mathematical analysis of the results yield this interpretation of the recorded data. However i t i s the author's view that this analysis is too simplistic and that in fact secretin has had a stimulatory effect on peptic c e l l s , already under the influence of gastrin. 66 The stimulatory effect of pepsinogen secretion was entirely masked by the more profound influence of the volume of gastric secretion and reduced washout of the pepsin. Considering the effect of glucagon on the acid and pepsin secretion of the Pavlov pouch i t is of interest that the percentage inhibition of acid i s 23 and of measured peptic activity 40. The inhibition of acid secretion usually involves dimunition of both concentration and volume. The reduction in volume of secretion could therefore result in diminished peptic activity in the collected secretion because of accumulation of secreted pepsin in the gastric gland lumen. If this were the only factor responsilbe however, the noted reduction in peptic activity would presumably be less than that noted for total acid output. (Reduction in hydrogen ion concentration also contributing to the measured reduction in acid output) Also the return of pre-inhibition volume of secretion would l i k e l y result in a transient increase in peptic activity because of washout of the accumulated pepsin. The mean 15 minute peptic activity in the immediate pre-inhibition periods i s 2952.6 ± 1392.8 S.D. In the 30 minute period 15 to 45 minutes after stopping infusion of glucagon the mean 15 minute output i s 2311 ± 1160. Rather than a transient rebound, the measured peptic activity i s significantly less (p < 0.01), than the pre-inhibition periods. This leads to a conclusion that the noted inhibition i s not related to poor washout of pepsinogen secreted into the gastric gland lumen but represents a definite reduction of pepsinogen secretion during infusion of the synthetic glucagon. 67 Classification of type of inhibition by consideration of the Michaelis-Menten parameters Table XX shows that marked change in the calculated maximal response (C.M.R.) occurs by introduciton of glucagon (1626 - 805). However the change in D^ ^ i s numerically small and test for common slope yields .21. As this i s larger than 0.05, the slopes of the two lines, which in the response-response/dose transformation are equal to the D^ Q» are parallel. These are the characteristics of non-competitive inhibition. The implication i s therefore that the presence of glucagon does not interfere with the specific peptic c e l l receptor site a f f i n i t y for gastrin. The inhibiton may occur because of interference with intracellular activity. If so, on a prior grounds, a different intracellular mechanism for the actions of the two hormones might be suspected. Examination of the Michaelis-Menten characteristics related to inhibition of acid secretion by glucagon reveals (Table XIX plot response-response/dose) that the presence of glucagon reduces the C.M.R. from 427.9 to 286.6. The linear transformations however have a common slope and therefore D^ Q- This indicated that the af f i n i t y of the parietal c e l l for gastrin remains unaffected by the presence of glucagon. These are the characteristics of non-competitive inhibition. 26 Johnson and Grossman reported that acid secretion in Heidenhain pouch dogs was inhibited in response to infusion of impure gastrin by porcine secretin (G.I.H.). They noted that the presence of secretin reduced the C.M.R. and did not change the d^Q- They therefore classified the inhibition as noncompetitive. 68 The data i n t h i s study i s not as easy to i n t e r p r e t . From consideration of the Michaelis-Menten charact e r i s t i c s Table XI, i t can be seen that the presence of s e c r e t i n markedly reduces the C.M.R. (489.1 to 19.14. The D,_Q does also appear to be changed (.61 to .30). Unfortunately there was marked v a r i a t i o n i n the plateaus of secretion p r i o r to i n f u s i o n of s e c r e t i n . This has resulted i n a low c o r r e l a t i o n c o e f f i c i e n t (.18) for the l i n e a r regression equation. Perhaps the best i n t e r p r e t a t i o n i s that the i n h i b i t i o n of a c i d secretion by s e c r e t i n markedly reduces the maximum rate of secretion as i n non-competitive i n h i b i t i o n . The p o s s i b i l i t y of a l t e r a t i o n i n the a f f i n i t y of the s p e c i f i c p a r i e t a l c e l l receptor s i t e for g a s t r i n c e r t a i n l y appears to be al t e r e d as i n mixed i n h i b i t i o n but t h i s must remain open to doubt on the data a v a i l a b l e . It was disappointing to f i n d that three d i f f e r e n t doses of the second h a l f (12-27) of the s e c r e t i n molecule had no demonstrable influence on ga s t r i n stimulated pepsin or acid secretion. The doses used are approxi-mately equimolar, twice and f i v e times the molar dose of s e c r e t i n causing profound acid secretion i n h i b i t i o n . Although F i g 20 and F i g 21 suggest that s e c r e t i n (12-27) at the low dose of 0.5 /ug/kg-hr may have increased the acid and pepsin output the f a i r l y large standard error of the mean do not allow t h i s deduction. The l i n e a r transformations of the data on acid and pepsin secretion are shown i n Table XXVII and XXVIII. I t i s seen that the test for common equation i s p o s i t i v e on comparison of the equations i n the presence of g a s t r i n and g a s t r i n and s e c r e t i n for both acid (0.62) and pepsin (0.64) secretion. This confirms the impression that there was no s i g n i f i c a n t e f f e c t on the ga s t r i n stimulated secretion by s e c r e t i n (12-27). 69 This allows a number of conclusions regarding the observed actions of both porcine secretin and human glucagon. The amino acid sequences are shown in Fig 25. As noted in this study under identical conditions both procine secretin and human glucagon had a significant influence on secretion of acid and pepsin. The two molecules have considerable similarity in the C terminal end of the molecule having 8 of the f i r s t 11 amino acids in the same position. As they also have a marked inhibitiory action on acid secretion i t i s an attractive hypothesis that the C terminal end of the molecules i s responsible for this activity. The small differences in structure could account for the observed differences in efficacy. The actions of these two polypeptides on pepsinogen secretion as shown by this study are markedly different; secretin enhancing the gastrin stimulated secretion and glucagon inhibiting i t . The N terminal end of the two molecules are relatively dissimilar. When numbered conventionally from the C terminal end they have six animo acids in the same position. If numbered from the N terminal end, only one amino acid i s in the same positions. It would therefore appear to f i t the observed facts that i t is a either the amino acid sequence of the N terminal end of the two molecules or their tertiary structure which i s responsible for the activity noted when infused with gastrin. But a number of doses of Secretin (12-27) were infused with no significant activity, even at a molar dose five time the dose of secretin which apparently produced a marked increase in pepsinogen secretion. It would seem then that the amino acid structure (12-27) of the N terminal alone was not responsible for the pepsigogue effect. This would tend to suggest that the tertiary structure of the molecule i s essential for this action. 70 The inab i l i t y of Secretin (12-27) to influence acid secretion stimulated by gastrin i n this study would tend to support the hypothesis, above, that i t i s the structure of the C terminal end of the secretin and glucagon molecule which i s responsible under the conditions of this experiment for the action on acid output. 71 HUMAN GLUCAGON His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Tyr-Ser-Lys-Tyr-Leu-Asp-Ser-Arg-Arg-Ala-Gln-Asp- Phe-Val-Gln-Trp-Leu-Met-Asn-Thr 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 His-Ser-Asp-Gly-Thr-Phe-Thr-Ser-Glu-Leu-Ser-Arg-Leu-Arg-Asp-Ser-Ala-Arg-Leu-Gln-Arg-Leu-Leu-Gln-Gly- Leu-Val-NH2 PORCINE SECRETIN Glu-Gly-Pro-Trp-Leu-Glu-Glu-Glu-G!u-Glu-Ala-Tyr-Gly-Trp-Leu-Asp-Phe-NH2 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 1 5 Leucine Human Gastrin I FIGURE 25 THE AMINO ACID SEQUENCE OF HUMAN GLUCAGON PORCINE SECRETIN AND 15 LEUCINE HUMAN GASTRIN I CONTROL RESULTS (Acid output to Gastrin) Dose Dog number tests of h g C i. 2 3 4 / 0 7 8 Q 10 11 12 13 14 1= 16 1? 0.7 EC1 . 5 27 11 13 33 114 338 529 703 729 740 769 785 771 7S3 777 809 731 703 645 '691 699 BC3 ' 2 50 5? 76 81 178 427 478 542 512 571 459 514 424 4-70 451 4-24 334 446 473 369 464 BC4 36- 16 28 35 69 213 359 4-40 519 512 568 599 481 537 517 515 537 540 564 607 587 BC8 1 100 34 8 19 649 947 893 1025 1210 IO67 1071 968 684 903 589 803 844 704 720 583 0.35 BC1 3 46 7 3 3 53 255 333 447 543 526 490 544 535 679 588 510 471 505 477 472 448 BC3 4 27 9 12 16 83 205 295 334- 356 369 332 300 311 317 348 351 343 360 355 335 336 BC4 2 12 14 25 ?~ 48 183 368 338 420 472 586 496 512 508 528 533 521 462 467 515 492 BC8 1 50 0 22 25 134 363 517 527 593 601 675 686 535* 495 478 572 400 510 457 429 363 0.175 BC1 4 31 16 15 7 24 106 225 342 391 412 433 376 358 348 319 398 44-5 431 424 354 367 BC3 2 14 19 1 7 30 106 129 178 170 173 188 198 209 217 255 245 199 203 203 234 241 BC4 3 4 6 5 1 8 . 53 120 207 319 315 447 458 426 433 423 378 363 355 373 431 450 H in u Eq Table II I-I c; o c t -P w O ft co a, CQ[ 8 O o i-H o ON H 1227 Cv-•rr u\ i-i i-i N.O o CM O N C O C N CM U N cn i-i ~v 3r CV! cn ON v-i co i-i CO i-H CN-CM 1-1 ON •ir 1-4 UN O cn cn VO H o N O 1-1 CM CO CO r>--c}' CO V O i-4 i - l o-U N CM o -cr 1-1 r-i N.O O j CM co o o cn UN O O Cvl O CO CN-CM r>-00CO 1-1 UN CM cn cn U N r H O CM C O 1-4 ON & UN o VD i - l i-H •CT i - l CVI UN 00 cn i-H O cn vO i-i 03 U N i-i cn cn o CM UN UN CVI cn CM cn cn 1-4 CM NO CV) on r H O-O 1—1 CM CN-o U N r H CO 1-1 -y T-4 O co o-CNi o ev-en i-t -rr ON VO 1-1 O N CV! C O CM o i-i O J o CM CM cn UN C O ON T- l o-00 ON CM cn r H O CM CO TH it CO 1-4 1-1 UN i-4 C N i-4 CO CM ON CM CM i-H 1-1 -cj-O J TH ON ON I-H cn i—4 00 o CV! co VO i-H cn CO UN O 1-1 ON O cn cn C\J r - l cn -cf o CM U N r-l O r H U N i-< CM i-l O i-H i - l i - l >n i-i cn r>-U N i - l 00 O N CO CM U N o CO CM CO i-4 o cn 1-1 1-1 CM i-4 i-i ON -cr cn r-l r H CM SI i - l O CM ON O N O-CNI I-I o-CV1 CM CM O-C O cn i-i VO ON cn i-H ON o O N cn UN ON CO 1-1 o i-i CM CM O CM 1-4 o o-1-4 O r H VO C N O -CM VO CO 00 VO U N O r-1 cn O N cn i-i co o o CM cnCM O •t-l i-i vo t>-CM UN C^-O N I-4 UN CM CM UN O r H t-l -cr ~X-CVI -c}-ON O-T - l CM i - l ON r>-C N O • H U N co m CM U N cn vO i-i CN-m CO U N rl VO CM UN UN O CM f^-Cv-vo O J vO ON T-4 u-'x ev-en CO CM vO O J CM cn cn i—i i-i N-O r-o i-i - V ON CM ^t i-( D -i—t CM t>-C N C O ON i - l ON i-l CN-CM o cn cn CM U N CO CO Cv-o en cn CN- O 1~l o CM - V CM t>-CM o iH CM co U N M f t O T - l C O •>-4 ON o U N U N CM O O N U N CM 1-4 CO UN CM 3N CO CO i-i Cv-CM N O VO i-i iH U N U N C O O-O N C N O-cn CM VO 1-4 o-T~t T-H UN O-UN. -3-C O i-t O CN-1-1 CO ev-en CM CO CO vo o-T-4 U N ON o O N i - l 0-C N [N-C N UN i~4 i - l O i-i C O i-H V Q vn VO i - l cn o CO U N vo cn T - l CM •3" IN-iH CO VO CN-1-4 O N O O- cn CO T - l -~> i~4 00 co T-4 o C O o TH Cv-Cn o 1-1 CM T-4 O CM o CM O J i-H UN UN vO •H O-O H cn VO 1-4 Cv! i-1 CO •t-4 CM CO co cn i-i cn 1—1 cn C O i-H o i-H T-4 U N 01 i-i i-i O-U N CM i - l CO O N C N ON VO ON CN 1-4 UN cn VO CM i - l CM NO 1-1 o NO o 1-4 CO CO CM T-4 CM o CN-C N T - l CM NO co TH CM ON CO i-4 VO CO VO cn CO o i-H CM i - l U N H D -vo O-i-l CN-VO VO CM CO N-O UN i-H i-4 i-H UN T-4 VO ON T-4 TH ^± V O CO CM o UN. VO 1-1 C"— O N C O cn ON VO cn CM vO r H r H CM i - l —V TH CO vO C-1-1 VO C O c-0J O UN O J 0J OC O -cf i-4 -ct-U'N UN T-4 O C O CO O N Cv! •3-cn T-i i-i o O N vo iH i-H cn C O 1-1 T-4 cn U N ON vn o-CM CM r H ON O r-l Cvl 0J cn CM 1-4 O O-t>-VO UN i—l u C O T-4 ON 1-1 ON o VO i-H UNev-en i H 1-1 -cr o CM T l O U N VO vo -~r U N ON CM CM CM cn -3-1-4 0J iH CO vo O o cn r H VO U N CO 1-1 O J i - l CM i-H CM o o H CM CM C N CM u> V O iH CM -cr O UN O CM CM U N CO VOUN i-4 O J CO o-CM 1-4 UN r-l r H r - l number of tests A -cf o o CM U N cn VO i-i VO 03 CN-H o-UN vO O-CO ON TH vo vo O N O U N i-4 CV! T-4 UN vn cn cn UN UN i-l i-H i-l o CM UN ON r H r-l number of tests A U N CM UN. r-l cn CM r H CVI -3- en to o o i - l o C? cq -cf u 8 CQ cq £? cq cq 8 cq d cq cn o pq 3 cq Dose « o UN cn • o UN o-r H o o 73 5 w o - p to =L m oi *g U a CD U - p •ri • p O o •rl a © H H © I 74 The Effect of 15 Leucine Human Gastrin on Peptic Activity  of Pavlov Pouch Secretion Sign Test Dose number of increase in decrease in statistical ug/kg-hr paired results peptic activity peptic activity evaluation probability 0 .7 26 14 12 no change > .1 0 .35 20 17 3 increase ^0 .01 0 .175 18 16" 2 increase <0.01 Wilcoxon's Signed Rank Test A. Analysis by pooling of data at a l l three doses for each individual dog gave the following results: BC1 Secretion during gastrin infusion periods not significantly different from basal p >0.1 BC3 As above p >0.05<" 0.1 BC4 As above p >0.05<0.1 BCS Insufficient data (4 pairs only available) B. Analysis by pooling of data from a l l dogs at each dose: Dose n Results JJ 0.7 26 no change > 0.1 0 .35 20 increase <0.02 0 .175 18 increase <0.02 Student's T Test Results for a l l dogs were pooled for each dose: Dose Basal periods Gastrin periods increase or ug/kg-hr n mean S.D. mean S.D. decrease j> 0.7 26 2108 1498 1591 946 0 >0.1 0 .35 20 997 924 1855 955 + < 0 .002 0 .175 18 1580 1432 2614 1767 + <0.05 peptic activity reported as ug tyrosine Table IV Measured Pavlov pouch secretion of acid in./j Eg, Stimulus; Continuous infusion of Gastrin 0.175 jug/kg-hr.  Inhibitor: Continuous infusion of Secretin 1.0 jug/kg-hr. during periods indicated 5 hr. Dog Gastric Date Number A JJ> C D 1 2 3 4 c 6 7 - 8 9 10 11 12 1? 14 l e 16 17 Fistu: Jan 18 BC1 53 88 12? 111 107 102 173 182 201 307 280 270 242 178 67 64 . 92 133 283 240 258 225 Jan 5 BC1 84 50 69 87 202 3^9 429 506 501 471 511 ^64 203 84 102 135 18.5 246 257 227 276 220 Apr 12 3C3 151 156 158 14S 177 321 388 372 431 436 470 384 407 230 22n 218 257 387 337 406 275 Apr 5 BC4 109 63 ' 39 53 69 52 187 337 184 342 433 419 539 428 119 OA 1 V 80 131 479 619 610 200 Jun 12 BC4 0 0 23 26 15 39 111 207 292 304 334 449 330 97 46 33 69 380 536 457 501 Apr 26 BCS 0 0 0 0 64 343 450 480 445 435 606 551 386 126 69 2 '69 156 200 191• 210 Mar 28 BCS 141 155 96 61 34 81 110 153 I65 262 247 296 266 163 39 4 3 19 6 53 75 Jun 6 BC3 207 135 216 166 157 208 418 327 310 299 353 263 329 218 162 190 186 281 290 280 312 312 Table V Measured Pavlov pouch secretion of acid in ;g Sq. timulus:' Continuous infusion of Gastrin 0.3^ Ag/kg-hr. Inhibitor: Contim IOUS infusion of Secret! •n L O /ig/kg-hr ...d luring oeriods indicated 5 hr. Dog Gastric Ds it e Number A B C D 1 2 -3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Fistula Jan 20 BC1 118 103 42 17 85 202 342 436 534 526 497 557 496 •340 143 14S 182 175 4 J 3 576 552 275 Jan 10 BC1 73 70 40 53 177 377 554 594 601 655 679 599 535 388 182 195 212 218 501 524 543 200 Apr Jan c BC3 199 267 410 420 563 549 569 660 630 605 571 689 615 527 461 506 543 667 691 710 150 18 BC3 27 3 6 23 65 241 531 655 546 635 544 567 496 403 397 426 447'448 495 491 443 400 Apr 7 BC4 76 148 I 6 5 53 4 2 117 167 296 451 432 791 750 334 398 458 423 138 43 58 1^3 550 250 Jun 14 BC4 3 9 6 0 k 64 306 480 539 675 805 790 930 626 ISO 64 41 4 4 251 611 619 702 300 Apr BCS 22 14 18 21 84 159 298 465 585 638 74-9 616 527 90 12 0 0 133 377 500 365 . 250 Apr- 28 BCS 24 71 0 0 3 6 3 2 2 566 649 593 751 885 933 694 115 225 8 6 185 467 679 832 275 Table VI ON Measured Pavlov pouch secretion of acid in /u Eq. Stimulus: Continuous infusion of Gastrin 0.7 ug/kg-hr. Inhibitor: Continuous infusion of Secretin 1.0 jug/kg-hr during periods indicated Dog 15 Minute Perioc3 Date Number A D c D 1 2 3 4 5 6 7 8 Q 10 11 12 1? 14 15 16 17 f i s t u l a Jan 7 BC1 7 29 102 60 135 322 528 725 796 711 842 607 160 214 269 586 725 873 786 784 280 Jan 13 BC1 106 201 94 64 201 414 760 579 512 701 759 647 533 436 202 168 195 224 454 840 695 350 Apr 7 3C3 200 248 202 162 361 774 829 370 928 863 903 814 768 909 835 919 684 626 705 719 758 200 Jan 13 BC3 32 9 33 36 171 526 636 580 519 509 522 472 434 410 366 300 342 377 375 386 469 450 Jun 9 BC4 0 5 3 2 53 109 143 90 146 302 295 361 138 35 29 67 367 529 477 507 150 Jun 30 3C4 25 2 0 65 ISO 422 633 692 561 500 569 4C? 137 55 118 230 543 657 629 628 325 Mar 24 BC4 432 514 261 54 29 148 290 649 651 725 710 669 699 574 245 82 45 103 451 621 639 225 Apr 19 • BCS 15 13 3 4 15S 713 1471 1377 801 1288 1478 1373 775 101 20 17 14. 18 417 1002 1092 325 Apr 13 BCS 25 10 l l 162 5Q2 738 860 923 918 871 843 462 90 20 Q S 8 200 641 802 841 250 Apr 17 BC8 0 62 52 38 77 277 401 689 852 844 1085 818 613 842 1052 145 19 17 13 13 403 200 Table VII Measured Pavlov Pouch secretion of Pepsin , Stimulus; Continuous infusion of Gastrin 0.17g ug/kg-hr.  In h i b i t o r : Continuous infusion .'of Secretin 1,0 pg/kg-hr. during periods indicated 15 Minute Periods Number A B C B . . 1 . 2 3 4 5 6 7 8 0 / 10 11 12 1 H 14 1 c , , •? , - 1 1 ,~, ,',„ * /• iO 17 Jan 18 BC1 1952 1891 2719 3252 3430 1138 1004 1149 961 1606 1330 1903 371 938 801 1696 2438 3346 4596 2470 2176 Jan 5 • BC1 22?1 1262 1378 1173 1929 1829 2380 3494 2845 2675 2726 1979 532 3 4 9 1006 1963 3581 3716 2604 1204 1^48 Apr 12 BC3 1 0 0 2 745 919 812 922 .713 635 636 800 1071 1614 1220 1397 1008 1258 2222 2243 3186 3738 2809 2023 Apr 5 BC4 2339 1353 I 6 7 I 3503 4789 3 4 3 ? 3800 2548 1003 1698 2597 2784 3300 2807 850 l'j-03 2835 4135 6304 58 00 4932 Jun 12 BC4 2576 1036 3523 24-4 I830 1467 1640 3031 4008 3743 4476 5 < « 4643 2651 1475 1864 3311 6118 6443 5 5 6 I 6034 Apr 26 BG3 2841 1374 1175 1424 2874 4.368 3655 3479 2814 2011 2219 2 l 6 l 2251 588 314 389 697 1868 2782 2269 1556 Mar 28 BCS 383 3 1 6 393 421 372 1058 660 466 338 432 719 454 300 127 46 23 5 46 81 377 332 Jun 6 BC3 1477 2391 4332 3^24 2796 1764 2598 1806 1794 2169 2897 2512 2906 3228 3272 4665 4934 6030 4970 351 6 3204 peptic activity reported as jug tyrosine Table VIII Measured Pavlov pouch secretion of pepsin Stimulus: Continuous infusion of Gastrin 0.35 ug/kg-hr.  Inhibitor: Continuous infusion of Secretin 1.0 ug/kg-hr during periods indicated Date Dog Number A B C D 1 2 3 15 Minute periods 5 6 7 8 9 10 11 12 13 14 15 16 17 Jan 20 BC1 4490 3819 23/45 1899 1603 884 ' 883 1693 1710 2007 2793 3810 1898 1227 1082 1439 23-56 2316 3843 3503 2824 Jan 10 EC1 1925 2031 1.555 I.58O 1176 729 1254 1393 1509 2856 2941 2745 1966 1890 1050 1803 3114 3390 5467 3527 3673 Apr 5 BC3 709 1489 1322 1511 841 957 1277 1194' 926 1300 1249 1485 1700 2734 2611 3584 3174 2888 3640 2626 Jan 13 3C3 2488 2202 1367 1126 2186 2650 2142 1717 1048 1534 1403 2207 2287 1240 2687 3577 3650 3365 2504 1171 2543 Apr 7 BC4 3109 2182 I859 1769 1795 1750 1145 938 973 886 2344 1560 525 853 975 940' 333 217 435' 1163 3221 Jun 14 BC4 2719 1629 1110 1118 2486 3412 1967 1741 3007 3133 3219 5056 3255 1590 752 706 1055 4490 5704 4447 4196 Apr 6 . BCS 3148 1407 1099 1387 1873 1386 1385 1470 ,1617 922 1704 1239 1279 143 62 66 82 883 1022 1613 921 Apr 28 BCS 2494 1595 994 948 1693 2341 1638 14?5 1031 1387 2056 1742 1715 350 143 120 205 1925 1825 2015 1893 peptic ac t i v i t y reported as p.g tyrosine Table IX ' 0 Measured Pavlov pouch secretion c f pepsin . Stimulus: Continuous i n f u s i o n of Gastrin 0 . 7 pg/kg-hr. I n h i b i t o r : Continuous i n f u s i o n of Secretin 1 . 0 ug/kg-hr during periods indicated Date Dog NumDer A B C D •1 J . 2 3 h. 15 Minute periods 6 ? 8 Q 10 11 12 11 14 IS 16 17 Jan 7 BC1 1717 1502 2522 2653 2788 1395 1880 2655 2247 2126 3816 2617 1135 883 2036 2585 4912 3426 3905 3155 3216 Jan 13 BC1 3472 4641 2962 2656 3275 977 2127 864 641 1149 2180 1426 911 1250 817 857 1116 1040 1784 2958 2236 Apr 17 BC3 S34 1317 1149 1188 1840 990 644 1016 1144 1219 1908 1470 1504 2502 2611 2926 2730 3477 3162 2639 Jan 13 BC3 3245 2404 3515 3846 5220 4177 2023 1070 703 733 1423 Il6l 1046 1085 1643 2080 3038 2651 2172 1354 1544 Jun 30 BC4 1065 606 863 907 2182 1920 1294 1333 847 1239 1899 m?3 1243 576 369 1014 1970 3424 2521 2215 2089 Kar 29 BC4 2801 3154 3705 1817 120 6 24-97 1766 1974 1438 1361 1708 1408 1772 1493 810 633 615 1616 4960 3809 2575 Apr 19 BC3 2313 1325 13-48 1816 3834 3891 2069 2335 876 1694 2334 1545 1422 224 131 110 191 2687 2538- 2295 2316 Apr, 13 BCS 812 956 650 564 1398 727 487 509 479 467 469 514 257 31 23 23 41 690 918 825 860 Apr 17 BCS 1714 1576 1394 1453 2406 2932 1811 1476 1147 1243 1544 777 496 1060 1612 197 88 143 85 510 917 Jun 9 BC4 2071 2533 1924 1370 866 12.58 1505 2463 1859 2102 2055 I838 1540 725 315 302 697 2343 2745 1373 1939 peptic a c t i v i t y reported as fig tyrosine CO o Table X X^e_jaffgot of Secretin on Aeid output of the Pavlov pouch Lijieag^^eg|i^^ion__equations_and^j|icha®1 is-Menten parameters GASTRIN GASTRIN + SECRETIN 1.0 ug/kg-hr. fa/ s common slope equation PLOT EQUATION r C.M.R. EQUATICII r C.M.R. D ^ p p Response - r = 369.! + 510o8(d) A5 curvilinear r = 120o6 + 70«,58(d) .1 Curvilinear .01 — Dose l/Response - i/r= 0o0012 + 0.0003(l/d) .57 33«33 -0.25 l/r= 0.028 - 0 . 0 0 0 5 ( l / d ) o 02 35.71 0.018 .81 .00 i/Dose Response - r = 489.1 + 0.6l(r/d) .18 489.1 0.61 r = 19»14 + 0.30(r/d) .78 19.14 0.30 .00 — • Response/Dose Table XI The Effect of Secretin on the pepsin output of the Pavlov pouch  Linear regression equations and Michaelis-Menten parameters GASTRIN GASTRIN + SECRETIN common slope equation PLOT EQUATION r C.M.R. EQUATION r C.M.R. p p Response - r = 2552 - 1326(d) .2? Curvilinear r = 2148 -1565(d) .27 Curvilinear .80 .03 Dose 1/Response - l/r= 0.0008 - 0.00001(l/d) .15 1250 0.0125 l/r= 0.0067 - 0.0004(l/d) .07 149.3 0.060 .62 .00 1/Dose Response- r = 1049 + 0.1376(r/d) .79. 1049 0.1376 r = 510.4+ 0.l856(r/d) .82 510 .I856 .04 — Response/Dose Table XII 00 fo Measured Pavlov pouch secretion of acid in p Eg.  Stimulus: Continuous infusion of Gastrin 0.175 ug / kg-hr.  Inhibitor: Continuous infusion of Glucagon 5.0 jag/kg-hr during periods indicated Date : Dog Number A B C D 1 2 3 4 5 6 15 Minute:;Periods 7 8 9 10 11 12 13 14 15 16 17 5 : Gast: Fisti May 18 BCl 422 195 179 224 371 541 758 858 760 979 912 764 790 917 767 481 275 353 4-50 418 495 175 M*y 25 BCl 716 247 230 231 343 815 961 961 985 1023 932 1091 1053 1041 1039 658 932 864 982 1036 1004 225 Flay 24 BC3 290 253 152 153 344 409 445 544 506 S74 569 524 467 343 265 235 320 346 339 478 478 300 Jun 2 BC3 18 151 71 82 196 340 425 348 294 302 282 289 293 222 206 144 144 155 197 205 246 200 Jun 5 BC4 135 198 83 16 0 49 150 157 188 161 237 311 256 368 324 366 273 228 287 130 90 200 Jun 7 BC4 0 0 0 4 139 163 54 38I 413 533 511 439 464 426 469 463 409 320 242 228 351 225 May 31 BC3 556 93 34- 75 139 408 644 655 640 536 639 490 340 408 259 193 163 172 149 I63 265 260 May 24 BC8 73 122 132 68 72 337 546 471 931 893 1090 1034 995 405 657 545 269 319 269 445 473 225 Jun 2 BCS 196 133 286 167 145" 290 2^0 369 374 587 396 398 404 374 329 276 I87 246 146 210 350 150 hr. ric Table XIII Measured Pavlov pouch secretion of acid in g Eg  Stimulus: Continuous infusion of Gastrin 0.35 jug/kg-hr Inhibitor; Continuous Infusion of Gluoagon 5.0 Mg/kg-hr during periods indicated Date Dog Number A B C D 1 2 3 4 s 6 15 Minute Periods 7 8 9 10 11 12 13 14 1«J- 16 17 5 hr. Gastrio Fistula May 15 BC1 255 173 135 224 422 543 564 477 490 546 676 912 558 511 459 346 343 390 399 388 363 125 May 15 BC3 626 390 222 191 355 705 791 761 647 680 729 587 543 443 333 259 219 277 368 567 504 600 May 29 BC5 299 296 156 79 164 234 352 479 582 571 673 553 563 392 338 335 285 345 380 402 522 225 May 30 BC4 18 100 28 14- 54 204 333 434 409 501 696 616 566 562 663 874 739 553 518 481 655 300 May 23 BC4 0 0 16 38 69 125 384 397 340 212 382 477 514 490 545 676 596 532 358 330 456 350 May 29 BCS 187 68 47 79 230 594 896 1240 1217 1068 905 710 764 590 401 330 317 292 224 243 243 350 May 11 BC8 0 61 138 75 160 667 1259 1405 1640 1480 1476 1409 1380 1258 960 847 611 440 578 752 896 250 Jun 30 BC1 7 83 64 33 10 132 400 421 679 599 769 735 673 715 614 675 488 186 287 391 449 225 Table XIV Measured Pavlov pouch seoretion of acid in n Eg, Stimulus; Continuous infusion of Gastrin 0.7 ug/kg-hr. Inhibitor; Continuous infusion of Glucagon 5.0 ug/kg-hr during periods indicated Date Dog Number A B "C D 1 2 3, 4 5 15 Minute Periods 6 7 8 9 10 11 12 14 1? 16 17 j • Gastric Fistula May 30 BC1 24 72 27 40 96 3 5 1 574 700 8 8 7 990 642 500 565 752 8 7 7 722 400 5 3 2 464 380 3 8 6 180 Jun 6 BC1 0 0 9 31 5 9 228 525 665 512 510 510 439 351 568 422 300 282 3 0 2 311 313 534 160 May 31 BC3 2 0 2 283 255 177 296 476 6 8 9 5 8 2 530 589 558 540 412 383 357 322 251 3 9 2 285 253 219 550 May 16 BC3 3 6 0 3 96 188 189 427 907 847 986 1011 922 843 722 995 920 918 576 597 600 732 986 1031 200 May 25 BC4 172 108 91 52 71 231 386 609 735 583 694 803 902 763 653 431 422 463 504 677 799 475 Jun 1 BC4 0 21 7 6 73 191 3 6 2 342 348 375 535 536 607 611 523 459 345 361 437 517 508 300 May 17 BC8 3 3 6 106 37$ 81 458 1001 2002 1137 1600 954 990 1599 2272 2265 1901 1531 1125 1727 952 952 509 350 Jun 5 BC8 53 50 26 3 7 172 700 8 5 8 603 927 1008 1114 976 842 496 367 371 367 329 3 4 3 561 718 150 Table XV CO 86 VO I w a u • p a S O o c o •H (0 3 40 8 g ca 3 H SI 1 co o P u n •H T3 I til o W rt o O c o •H W 3 •H 8} 3 O s •ri •s o o 3 ^3-R cn VO cn co co 5> ON ON UN cn cn vo cn -=>• o O-cn ON -3-CM COl UN to I Q -~J~ CM ON O r1 S3 U N H US cm CM VO CO o cn UN UN CM CM ON CO cn & s fc cn cn t-4 3^" vo CM Cv ev-en cn CM i H « . ON 1-4 VO cn cn CM ON CO S3 3 UN cn UN cn 1-4 O-UN vo T-l o UN CM cn co o <»-o cn UN cn UN ON ON cn CM ON m CM VO CM VO VO CM CM 3" O-i - l vS UN 1-1 o cn ON CM UN CO T-4 3^" 2862 o UN vo 1-4 cn i-» cn CM . . * i-« o-CVl ON CM i - i CM vO UN UN cn tv UN UN 1-4 ^ VO o o UN o VO & CO t>-i - i CM CO cn VO CM CO cn CM •»-» 1-4 O-CM 1-4 UN CO CO cn ON ON CM i - l 1-H CM cn co CO UN CM 3-o o i H cn UN O--3-o VO cn o CO 1-1 cn 1-H 1-4 VO » CM o o cn 1-4 co m UN UN cn UN UN i - l cn 1-4 UN CM 1-4 VO UN cn o CM O-1-4 UN CO i - * © cn cn o-CO UN cn o-o 1-1 1-1 00 o cn cs-1-4 cn •»-« -er 1-4 VO -d-s> £ UN -3-UN ON i H i H UN UN i H cn* CM UN Cvl UN tv 00 cn i -4 1-4 vo UN O fc i - l ev-en O N UN CN- CO vo <r CM 1-4 I & & § S? <? t>- .ch .3-1-4 CM i-« -d" CO O . 3 - 1 - 4 1 - 4 vo cn UN 1-4 CM O N ON -d-UN cn cn UN CM CM VO i-« UN O Cn *H -rt CM O UN VO cn ^ - ON cn i-< cn o cn CM cn r i cn CM CO ON 1-4 CM CM CM VO ON i-4 cn CM VO -3" CM O ON 3; cn cn vo .3- CM *H co co -3-o n N co cn ON cn CM CM O- O vO v o OO u> CM i - l CM cn O- t> UN O CO c> cv-cn *-* o UN £ ON CM ON CM cn cn CO cn 1-4 CM CM UN UN O VO o VO UN 1-4 ON UN O Sv vo i -4 UN -3" UN 1-4 -4-VO ON CM T-4 CM; O N CO CM cn UN -3" o o CM cn CM Sv co cn CM cn CM cn o CO UN CM O cn VO o CO VO O - ON cn CM ON vo CM cn VO cn o o -3" CM cn VO VO CM UN ON UN CM UN «N ON cn UN ON ev-en CM cn vo 1-1 s o cn CM CM fc ON ON o-CM VO vo 3 cn o K? cn fc i - i UN 1-4 vS cn co o 1-4 CM O CO CO vo cn CM CM cn 8N cn CO cn i - i --3-cn BCl BCl 8 $ i § § PA s. BC8 CO r-l UN CM CM CM UN i H cn CN! CM May May May Jun Jun Jun May May Jun 10 O 4-> faO a. to ed "8 +> u a * 4 >» -P •4-> O tt O •H u CO Dog Date Number .Kay 25 BC1 Jun 1 BC1 May 15 BC3 May 29 BC3 May 30 BC4 May 23 BC4 May 29 BC8 May 11 BC8 Jun 30 BC1 Stimulus: Continuous infusion of Gastrin 0a35 ug/kg-hr.  Inhibitor: Continuous Infusion of Glucagon 5«0 xtg/kg-hr during periods indicated 15 Minute Periods B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 BC4 3812 4352 2373 1785 2216 2167 2201 2076 1739 2603 4186 3624 3693 3?8l 3841 4539 3299 2812 2670 2650 4610 Jun 30 BC1 3692 4498 4216 3755 2607 2119 2687 2221 3303 2382 5207 3872 4264 3878 1901 3965 1988 1931 4020 4044 4405 peptic activity reported as ug tyrosine Table XVII co vj 88 "8 •P rt o •rt "8 •rt 10 *D O •rt f. CO P 8 I «H bi *< 3 T> c w •H a U W si P o i o « p o c bi o •rt a u s o o « • •rt rt +> O <D G *, <T-t O O O hi © ct) (0 c o o 3 J3 .rt H e> 3 3 Q t! P C. | o > •n c O eo o 1-4 3 •rt > O W rt 3 3 Cv, C •bl •H 2 -P •rt © C 3 cS US w O rt 3 © C to •rl 3 •P H C nw Co CO 8 -p .rt CM T H | co PQl CN-ON NO » * • cn CM cn 2212 NO T H NO CM CM T-l CM o cn o CM cn CM O NO cn CM cn o CN-T-l St NO T-l O-T-t cn T H T H ON 0. T H o T-l CM o T-l CM NO T-l co T H T H NO CM T-l 8N NO T-l T-l CM ON rn T H T H CM CO T-l T-l o CM O O-CO cn CO CM cn cv NO T-l ON cn o « ON cn NO tN-CM NO NO »n CO o T-l CM vn NO CM CM cn \0 NO vn vn cn CO T-l « T H cn CM & CM CM CM cn T-l cn CM O-T-l i CM • * CM vn cn 8 -=)• cn cn o cn T-l CM CO cn o CM T-l cn cn T-l "vn vn o o ON t>-CM NO cn CM NO CM vn o\ ON CM CO T-l «>-T H * NO T-l CM T-l 2029 NO CM ON ON NO cn T-l * CM ON ON o T-l ON CN-CM cn vn o NO T H co T-l ON NO CO cn T-l ON ON CM CM o T-l CM cn T-l CM T-l vn CM CM CO UN CM T H CM *D NO CO vn T-l O T-l CM T-l JO ON o T H cn vn co T-l NO vn co T-l T-l JO ON CM CM • * vn oo T-l T-l O t>- Tn "« vn NO T-l T-l O cn NO on vn T-l NO CM vn T-l NS co ct NO T-l NO cn CO T-» NO NO vn CM ON cn cn vn T-l cn T-l o cn I T-l E T-l CM & T-l T H R CM ON CM NO T H o , co :« cn o T-l CM cn SN cn • cn o < o cn vn 3 T-l -3-T-l ON CM CM NO O-T-l NO cn T-l vn CO ft CM co CO CM tn CM vn ON T-l CM T-l CM ON T-I cn ' » - * T-l CM cn NO CM CM cn vo T-l T-l*0 NO CM T-l CM CM T-l on T-l vn 1 NO CSN T H o-vn o T-T T-l ON CM cn CM ON T-l NO CM a cn cn ON cn CM vn NO o T-l cn CM CN? cnNO vn CM -* T-l ON CM T-l cn gvn SN r>-CM ON c^ -T-l O CP m PQ « cq •« PQ 8 PQ •8 PQ June 6 T-l cn >» it NO iH >» vn CM >» T-l o c 4 o-H >> •iS vn CD a 4 © •S (0 O fc +> bO a . rt •8 -P a © I H )S •p -p o rt u CD P, M M M ti E-i The effect of Glucagon on the acid output of the Pavlov pouoh Linear regression equations and Michaelis-Menten parameters GASTRIN GASTRIN + GLUCAGON common slope equation EQUATION r C.M.R. EQUATION r C.M.R. p p 530.3 + 464.5(d) .26 curvilinear r = 393.5 + 281.8(d) .21 curvilinear .56 .00 =0.00125 + 0.00015(l/d) .32 800 -.1200 l/r*0.0019 + 0.00002l(l/d) .27 526.3 - O i l .63 .00 427.9 + 0.1267(r/d) .49 427.9 .1267 r = 286.6 + 0.1348(r/d) .57 286.6 .1348 .83 .03 Table XEC CO vo The effect of Glucagon on the pepsin output of the Pavlov pouch  Linear regression equations and Michaelis-Menten parameters GASTRIN PLOT EQUATION r C.M.R. Response - r = 3974 - 255*Kd) ,39 curvilinear Dose l/Response - l / r =0.00059 -0.000046(l/d) .35 I695 .0078 l/Dose Response - r = I626 + 0.1227(r/d) .79 I626 .1227 Response/Dose Table XX GASTRIN + GLUCAGON common slope equation EQUATION r C.M.R. p p r = 2797 - 2311(d) .41 curvilinear .81 .00 l/r= 0.0016 -0.00019(l/d) .41 625 .1188 .02 — r = 805.1 + 0.l476(r/d) .84 805.1 .1476 .21 .00 Measured Pavlov pouoh secretion of aold in u Eg. Stimulus: Continuous infusion of Gastrin 0f1^ ug/kg-hrf r: Continuous infusion of Secretin {12-27) 0.5 ug/kg-hr. during periods indicated Date Dog Number A B C D 1 2 „ 3 4 15 Minute Periods 5 6 7 8 0 10 11 12 13 14 15 16 17 Feb 14 BCl 0 0 0 16 37 113 158 290 400 289 266 320 403 394 472 431 402 276 333 294 307 Feb 10 BCl 0 14 0 2 130 439 418 327 393 290 333 208 550 531 617 863 826 524 470 308 302 Jan 26 BC3 0 0 4 28 120 414 499 492 571 492 506 550 527 579 498 487 431 499 541 572 590 Apr 18 BC4 0 0 0 0 114 i l l 165 261 365 436 503 566 615 620 647 615 561 429 416 517 687 Apr 20 BC4 719 22 3 0 46 245 361 441 396 451 551 641 ,643 576 624 618 640 700 589 657 644 Feb 10 BCS 0 0 0 0 9 157 260 322 401 443 532 571 656 616 567 586 579 537 523 579 572 Table XXI Measured Pavlov pouch secretion of acid in u Eg.  Stimulus: Continuous infusion of Gastrin 0.35 »g/kg-hr.  Inhibitor: Continuous infusion of Secretin (12 -27) 1.0 Mg/kg°hr. during, periods Indicated Date Dog Number A B C D 1 ? 4 15 Minute Periods •5 6 7 8 9 10 11 12 11 14 *1 16 17 Apr 17 BCl 101 238 90 60 83 258 279 353 475 656 712 793 764 763 673 699 855 705 580 477 570 Jan 26 BCl 102 51 37 191 401 959 1208 972 1011 1003 881 705 760 894 1023 950 1049 930 684 620 677 Apr 14 BC3 286 143 104 90 215 523 684 853 766 881 870 971 953 948 993 908 725 691 656 644 55k Apr 20 BC3 0 0 5 0 113 256 399 560 580 638 546 504 527 491 525 550 555 555 599 520 471 Apr 20 BC4 291 228 176 280 473 464 552 589 557 640 636 832 757 636 597 763 884 920 643 657 769 Apr 14 BC4 369 120 18 48 96 133 390 438 500 569 615 606 771 694 642 664 617 641 630 724 684 Feb 14 BC8 504 572 17 3 32 233 360 404 477 466 538 528 513 537 601 624 569 450 424 458 7^5 Table XXII Measured Pavlov pouch secretion of acid In m Eg. Stimulus; Continuous infusion of Gastrin 0.35 ug/kg-hr.  Inhibitor: Continuous infusion of Secretin (12-27) 2*5 ug/kg-hr. during periods indicated Dog 15 Minute Periods Date Number A B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Apr 19 BCl 0 0 0 0 83 212 361 259 150 276 399 640 500 46l 586 653 490 422 394 393 456 Apr 13 BCl 0 0 22 7 62 194 273 475 559 508 473 500 464 433 373 572 393 345 381 349 43Q May 1 BQ3 347 375 316 256 395 484 620 591 498 533 491 435 349 396 288 341 275 341 299 310 266 Apr 18 BC3 279 200 232 409 435 494 448 414 410 454 459 444 409 377 357 378 374 301 367 347 316 Jan 27 BC4 66 74 22 11 117 305 392 371 371 444 415 444 465 546 504 612 618 599 438 383 493 May 1 BC4 126 28 53 194 403 621 635 649 595 572 58I 679 748 738 677 820 720 903 782 830 852 Table XXIII Measured Pavlov pouoh secretion of pepsin  Stimulus: Continuous infusion of Gastrin 0«35 ug/kg-hr.  Inhibitor: Continuous infusion of Secretin (12-27) 0»5 ug/kg-hr, during periods Indicated Dog 15 Minute Periods Date Number A B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Feb 14 BCl 3504 2604 2680 3429 2843 3464 3472 2755 2631 3384 2169 2178 2644 2121 2869 3329 3137 2155 2926 2418 3156 Feb 10 BCl 358O 4143 2721 2729 3591 2818 2207 2420 2581 1966 1766 1746 3609 2179 4387 3881 4753 2135 3166 1819 2207 Jan 26 BC3 1725 2206 1216 14£9 3757 3370 1309 715 535 694 810 1246 815 1553 1394 702 790 627 983 1260 1529 Apr 18 BC4 2046 1258 863 1005 3718 1979 1312 1464 1965 2368 3060 3327 3417 4113 4655 4593 4691 3606 3034 3422 4856 Apr 20 BC4 3043 2956 1565 1289 1694 2340 1862 2099 2333 2504 3815 4633 5201 4230 4941 5167 5516 5530 4535 5321 5172 Feb 10 BCS 706 408 347 467 872 994 627 452 495 418 554 565 802 669 777 937 1038 907 924 975 948 peptic activity reported as ug tyrosine Table XXIV Measured Pavlov pouch secretion of Pepsin  Stimulus: Continuous Infusion of Gastrin Oe35 jug/kg-hr*  Inhibitor: Continuous infusion of Secretin (12-27) 1«0 ug/kg-hr during periods indicated Dog 15 Minute Periods Date Number A B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 IS 16 17 Apr 17 BCl 2687 8076 4166 4576 4932 4619 2661 2471 3065 4080 4937 5204 5357 5171 *ff!5 **051 5581 5021 4367 2841 4307 Jan 26 BCl 3013 3114 1244 3984 3400 3891 5280 4190 2729 4053 *068 2448 2768 3817 4256 3840 4551 4409 2388 2570 3794 Apr 14 BC3 716 814 952 1056 2122 1870 1525 1986 1776 3015 367^ 4420 4678 4417 4444 3372 2637 2516 2375 2188 3625 Apr 20 BC3 2249 2101 2179 3699 2982 1200 1131 1512 1452 1703 1799 1683 1745 1781 1461 1522 1980 3116 3093 2530 1792 Apr 20 BC4 4919 2564 1412 1134 4109 3759 4077 5101 4672 4918 4367 5187 5124 4425 4189 5029 5884 6593 4913 4700 4 4 3 6 Apr 14 BC4 253^ 925 724 3198 4515 2992 2157 1598 1911 1675 1824 2411 1479 2452 4143 3133 2524 2983 3785 *O02 5226 Feb 14 BC8 563 852 727 478 791 937 408 319 346 568 843 576 711 726 727 1002 1118 670 647 667 676 peptic activity reported as jag tyrosine Table XXV Measured Pavlov pouch secretion of pepsin  Stimulus; Continuous infusion of Gastrin 0o35 ug/kg-hr.  Inhibitor; Continuous infusion of Secretin (12-27) 2.5 ug/kg-hr during periods indicated Dog 15 Minute Periods ^Date Number A B C D 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 Apr 19 BCl 2125 4355 9194 4376 4596 5126 5367 4891 4879 5260 5133 6633 5215 5085 5919 6279 5354 5353 4769 3879 5000 Apr 13 BCl 3661 3114 3452 4078 3888 3374 2557 3954 3560 2958 2339 3568 3128 2578 1611 4210 2278 2217 2182 1660 2653 May 1 BC3 2426 4245 2575 1976 2299 IO87 1050 824 812 776 756 963 866 1024 534 846 639 7 6 9 980 1363 915 Apr 18 BC3 2333 1979 1823 2740 2232 1271 791 563 502 682 883 1020 1021 881 1068 1011 1102 964 1209 1218 1204 Jan 27 BC4 4768 3735 1471 1502 2545 2 9 6 9 2284 1859 2164 2888 2114 2755 3151 3895 3377 3957 3996 4342 4749 2859 3851 May 1 BC4 4869 1495 1682 2380 2820 3083 2433 3451 3351 3152 3013 3549 4560 4413 4110 4566 4725 5854 4660 4905 4351 peptic activity reported as pg tyrosine Table XXVI CTi The effect of Secretin (12 - 27) on the aold output of the Pavlov pouch  Linear regression equations and Mlohaelis - Menten parameters PLOT Response Dose GASTRIN EQUATION r = 631.6 - 37.26(d) r C.M.R. D Q^ .20 curvilinear GASTRIN + SECRETIN (12 - '27) common slope equation r = EQUATION 681.9 - 55.97(d) r C.M.R. D, '50 p p .27 curvilinear .68 .50 1/Response - l/r= 0.0017 + 0.00015(i/d) .15 588.2 -.088 l/r= 0.0020 - 0.000l6(i/d) .11 500 .080 *i6 .60 1/Dose Response - r = 467.8 + 0.1769(r/d) .42 467.8 .1769 - r = 476.2 + 0.190(r/d) .48 476.2 .190 .85 .62 Response/Dose Table XXVII VO The effect of Secretin (12 - 27) on the pepsin output of the Pavlov pouch  Linear regression equations and Michaelis-Menten parameters GASTRIN GASTRIN + SECRETIN (12 - 27) common slope equation PLOT EQUATION r C.M.R. EQUATION r C.M.R. p p Response - r = 2593 + 167.7(d) . 0 9 curvilinear r = 3131 + 29.37(d) . 0 1 curvilinear .76 .37 Dose 1/Response - l/r= 0.00049 + 0.00007(l/d) .10 2040 -.014 l/r= 0 . 0 0 0 5 3 -0.000007(l/d) .01 1887 .013 .63 . 61 1/Dbse Response- r = 1589 + 0.406o(r/d) . 5 9 1589 .^>6 r = 1899 + 0.356*9(r/d) .61 1899 .35^9.69 .64 Response/Dose vO OO Table XXVHI 99 THE RELATIVE VALUES OF THREE LINEAR TRANSFORMATIONS IN ESTIMATING THE MICHAELIS-MENTEN CONSTANTS C.M.R. AND D ^ Plot variable large or constant Error of r Error of r small large increasing 1/r - 1/d (1) worthless poor poor r - r/d (2) definitely superior reasonably definitely superior to (3) accurate to (3) d/r - d (3) reasonably accurate slightly superior satisfactory to (2) adapted from: "A comparison of the estimates of Michaelis-Menten kinetic constants from various linear transformations" J.E. Ebwd and D.S. Riggs J. Biol. Chem 240. 2. 863 (I965) Table XXIX Measured Pavlov pouch secretion of acid in n Eq  Stimulus: Continuous infusion of Gastrin at dose indicated in »g/kg-hr. Inhibitort Continuous infusion of G.I.P. 2.5 ug/kg-hr during periods indicated Dog Date Number A B C D 1 2 3 4 15 Minute periods 5 6 7 8 9 10 11 12 13 14 15 16 17 Dose 0.175 Dec 6 BC4 45 22 13 5 16 18 24 70 115 105 114 116 67 100 120 162 198 282 343 343 370 Dec 13 BC3 43 82 26 30 136 235 339 376 363 332 336 354 351 273 240 346 399 391 333 369 Dec 15 BCl 333 159 145 230 523 669 501 620 527 631 502 487 465 327 249 249 345 472 524 Dose 0.35 Dec 29 BCl 8 1 3 4 60 252 604 512 466 489 493 580 613 580 595 401 594 709 601 534 473 496 Nov 29 BC4 50 59 72 48 25 24 110 90 152 223 298 257 187 175 212 245 294 427 537 559 597 Nov 26 BC5 0 41 71 50 6? 161 264 257 316 407 444 301 209 220 284 238 366 775 672 466 340 Nov 25 BCl 59 11 0 2 144 377 384 450 501 525 610 485 232 229 216 234 342 530 582 681 565 Dose 0,7 Dee 13 BCl 49 27 12 24 133 543 1181 1180 848 1426 1566 1502 1081 580 523 756 779 843 988 1019 911 Dec 16 BC5 — 4 3 9 254 811 1424 1820 1949 2256 2145 1889 1651 1274 1217 1239 1345 1919 1539 1779 1729 1552 Dec 9 BC3 0 0 10 36 179 453 401 614 602 547 425 342 212 220 169 155 190 307 285 268 218 Table XXX H O o Measured Pavlov pouch secretion of pepsin  Stimulus: Continuous infusion of Gastrin at dose indicated in jag/kg-hr.  Inhibitor: Continuous infusion of G.I,P. 2 . 5 ng/kg-hr during periods indicated Dog Date Number A B C D 1 2 3 4 15 Minute periods "5 6 7 8 9 10 11 12 14 15 16 Dose 0„175 Dec 6 BC4 586 472 531 806 897 758 1108 687 973 779 644 612 521 544 576 1006 1036 1279 1856 1956 2824 Dec 13 BC3 688 1918 1468 575 594 740 751 553 581 545 3^3 546 509 290 133 183 369 582 605 635 5^ 5 Dec 15 BCl 3902 1885 2791 3232 2939 2708 1865 2553 1592 2391 2112 1995 2088 1280 1104 1274 2311 3019 Dose p f35 Dec 29 BCl 1083 1021 1363 1892 4781 4210 3105 2618 2457 2525 3767 3715 2740 3421 2025 3266 4389 2887 1492 2119 2488 Nov 29 BC4 1099 1230 1618 1607 2153 1160 1792 621 712 769 1160 834 579 598 1015 1089 1602 2374 3113 3357 3423 Nov 26 BC5 1302 1769 1763 1429 1613 1949 1864 1228 1145 1201 1436 77k 324 347 596 817 638 1944 1395 864 560 Nov 25 BCl 2489 1029 669 679 2077 1254 828 930 715 1013 1342 1032 312 413 3^ 9 352 535 1219 938 153^ 976 Dose 0 . 7 Dec 13 BCl 621 790 777 1185 1759 886 897 912 7^ 9 1870 1950 2377 1058 323 500 1153 953 933 1492 1372 1105 Dec 16 BC5 1428 874 1341 2500 2355 1753 2051 2637 2679 3637 3364 2277 836 784 1233 1409 4788 3953 5580 4760 3232 Deo 9 BC3 1507 1200 685 1098 1947 1279 274 206 158 217 276 194 56 24 20 76 97 301 279 242 193 peptic activity reported as ug tyrosine Table XXXI 102 BIBLIOGRAPHY 1. 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Gastroenterology 47, 72 (1964) APPENDIX Hormone Enzyme Kinetic Analogy If i t i s considered that the response of a biological target to a hormone i s , under certain conditions, analogous to enzyme substrate interactions then the parameters of the biological response may conform to Michaelis-Menten kinetics The Michaelis-Menten equation expresses the relationship of the i n i t i a l velocity of the reaction, to the concentration of the substrate. Two parameters characterise the equation the V max of maximum i n i t i a l velocity and the K m or Michaelis constant. The equation is of the form; v = V C / ( K + C ) max s m s where v = i n i t i a l velocity V = maximum i n i t i a l velocity where C i s i n f i n i t e l y large max s C = concentration of substrate s K = Michaelis constant (equals concentration of substrate when m v = h V ) max The plot of v describes a rectangular hyperbola and f i g . 26 is a representative plot. The terminology used in hormone target c e l l interactions i s different from that used in enzyme kinetics. The Michaelis-Menten equation would be expressed as below for such hormone kinetics, r = C.M.R. x d / (P 5 Q + d> where r = response of target CMR = calculated maximum response where d i s optimally large d = dose of hormone D^Q = dose of hormone where r ?= ^  C.M.R. The analogous terms are shown on f i g . 26 beside the terms used in enzyme kinetics. HORMONE/ENZYME KINETICS ANALOGY FIGURE 26 TYPICAL CURVILINEAR EXPRESSION OF RELATIONSHIP BETWEEN INITIAL VELOCITY AND SUBSTRATE CONCENTRATION IN ENZYME KINETICS The analogous terms used in this study of hormone kinetics are also shown. 108 Estimation of the Parameters C.M.R. and The Michaelis-Menten equation i s characterised by the two parameters K and V which i n hormone terminology would be C.M.R. and D..,.. As shown m max b J 50 i n f i g . 26, a plot of response against dose ( v against C ) i s c u r v i l i n e a r s and the estimation of C.M.R. and D^Q from the p l o t i s inaccurate. However three l i n e a r transformations of the equation f a c i l i t a t e estimation of the c h a r a c t e r i s t i c parameters C.M.R. and D^ Q> These are: A. 1/r against 1/d = Double r e c i p r o c a l or Lineweaver-Burk B. r against r/d C. d/r against d 16 Dowd and Riggs i n 1965 published a study i n which they compared the accuracy with which the above transformations would estimate the c h a r a c t e r i s t i c parameters i n the Michaelis-Menten equation. Although t h e i r data was e n t i r e l y biochemical and t h e i r terminology r e l a t e d to enzyme k i n e t i c s , the conclusions are v a l i d f o r a l l data which conforms to the Michaelis-Menten equation. In t h e i r study, i t was assumed that errors i n measurement of the i n i t i a l v e l o c i t y might occur, (e.g. because of experimental error) The influence of small and large errors of t h i s measured value on the estimated constants K and V was investigated, m max ° The conditions i n the present study are e n t i r e l y analogous where, although possible, i t i s u n l i k e l y that s i g n i f i c a n t errors i n hormone dosage used have occurred. The m u l t i f a c t o r i a l influences on the response, to t h i s dosage, w i l l produce a more marked v a r i a b i l i t y i n t h i s measured value. Whether these errors are large and v a r i a b l e , or small and constant, i t i s not possible to asc e r t a i n . 109 Table XXIX i s a summary of the findings of the study of Dowd and Riggs. Replacement of the enzyme terminology by the response-dose notation applicable to th i s study has been c a r r i e d out i n t h i s table. With large error (constant or increasing) of r (for 95% confidence of better) estimates of the constants D<-Q and C.M.R. derived by p l o t t i n g r - r/d always gave smaller confidence l i m i t s than p l o t t i n g d/r -d. I f the large underestimates or overestimates of r are feared then r - r/d transformation i s preferred. Cert a i n l y i f the in v e s t i g a t o r i s unsure about the magnitude of such errors t h i s transformation should be used because of small errors i n r eith e r r - r/d or d/r - d w i l l give accurate r e s u l t s . I t i s of i n t e r e s t that the double r e c i p r o c a l or Lineweaver-Burk plo t which has been used for many years i s so i n f e r i o r f o r estimation of the c h a r a c t e r i s t i c parameters C.M.R. and D,-Q. I f however the experimental values are plotted and the "best l i n e " assessed v i s u a l l y there i s no doubt that the Lineweaver-Burk plot appears to obtain the best f i t . I t i s possible that t h i s f a c i l i t y for providing a good f i t t i n g l i n e with even extremely v a r i a b l e experimental data has ensured i t s popularity and continued use. In t h i s experiment although the data has been subjected to two l i n e a r transformations, and constants have been calculated f o r both of these, the conclusions are based on the r e s u l t s of the r - r/d transformation and not on the Lineweaver-Burk transformation. 110 The C l a s s i f i c a t i o n of I n h i b i t i o n The b i o l o g i c a l response to a hormone may be analogous to biochemical enzyme substrate i n t e r a c t i o n . The l a t t e r may be i n t e r f e r e d with by another component r e s u l t i n g i n either augmentation or i n h i b i t i o n . The phenomenon of i n h i b i t i o n i n t h i s biochemical context has been widely studied and the c h a r a c t e r i s t i c s of the i n h i b i t i o n produced have been c l a s s i f i e d . The Michaelis-Menten equation which expresses the r e l a t i o n s h i p of i n i t i a l v e l o c i t y of reaction to the concentration of substrate contains two c h a r a c t e r i s t i c parameters the V (maximum i n i t i a l v e l o c i t y ) and K max m (the substrate concentration when the i n i t i a l v e l o c i t y i s exactly % V ). J max The introduction of an i n h i b i t o r into the system w i l l r e s u l t i n a d i f f e r e n t Michaelis-Menten equation with v a r i o u s l y d i f f e r e n t V max and K parameters. Examination of the changes i n these two parameters produced by an i n h i b i t o r allow c l a s s i f i c a t i o n of the type of i n h i b i t i o n produced. In t h i s experiment the analogy with the biochemical system has been used to attempt to c l a s s i f y the type of i n h i b i t i o n produced by a s p e c i f i c polypeptide i n h i b i t o r on the stimulant action of g a s t r i n . As mentioned previously the graphical representation of the Michaelis-Menton equation i s c u r v i l i n e a r , d i f f i c u l t to i n t e r p r e t v i s u a l l y and inaccurate f o r c a l c u l a t i o n of the c h a r a c t e r i s t i c parameters C.M.R. (or V ) and D c „ (or K ). max 50 m The l i n e a r transformations r - r/d and 1/r - 1/d are therefore used i n t h i s study. F i g . 27 and 28 show the c h a r a c t e r i s t i c changes occurring i n the plots with the addi t i o n of s p e c i f i c types of i n h i b i t o r to the system. The v i s u a l i n t e r p r e t a t i o n of such plo t s has been shown to be les s accurate than s t a t i s t i c a l examination of the c h a r a c t e r i s t i c parameters. Although for demonstration purposes the plots of various l i n e a r I l l t r a n s f o r m a t i o n s a r e shown i n graph form a l l c o n c l u s i o n s a r e based on the m a t h e m a t i c a l comparisons o f the M i c h a e l i s - M e n t e n e q u a t i o n s and the changes i n d u c e d i n t h e i r c h a r a c t e r i s t i c parameters C.M.R. and D^^ by t h e a d d i t i o n of an i n h i b i t o r to the system. I n t e r p r e t a t i o n of the r - r/d Transformation Representative graphs of the main type of i n h i b i t i o n are given i n f i g . 27. In h i b i t i o n r e s u l t s i n a l t e r a t i o n of the c h a r a c t e r i s t i c s of the l i n e obtained by p l o t t i n g the values obtained i n response to the presence of a stimulus only. The presence of both i n h i b i t o r and stimulus r e s u l t s i n a l i n e a r p l o t with d i f f e r e n t slopes and or intercepts with the two axes. In t h i s transformation non-competitive i n h i b i t i o n r e s u l t s i n a l t e r e d intercepts with the r and r/d axes but i n an unchanged slope ( - D ^ Q ) . Competitive i n h i b i t i o n a l t e r s the slope but the intercept with the r axis (C.M.R.) remains unchanged. Mixed i n h i b i t i o n r e s u l t s i n an a l t e r a t i o n i n both intercepts and slope. The e f f e c t s of i n h i b i t i o n on the l i n e a r transformation and the c h a r a c t e r i s t i c Michaelis-Menten parameters are summarised below. 112 Type of I n h i b i t i o n non competitive competitive Mixed Slope of l i n e " D50 unchanged increased increased Intercept with r axis C.M.R. decreased unchanged increased Intercept with r/d axis decreased decreased decreased This transformation provides accurate estimates of the Michaelis-Menten parameters D,-Q and C.M.R. under a l l conditions of uncontrolled experimental measurement errors and i s used for conclusions on the influence of i n h i b i t o r s i n t h i s study. 113 r - r/d PLOT C.M.R. Gastrin + Inhibitor Gastrin C.M.R. Ns^slope = D 50 0 r/d Non Competitive C.M.R. vs slope = D 5 Q 0 r/d 0 Competitive r/d Mixed F I G U R E 2 7 DIAGRAMMATIC EXAMPLES OF THE E F F E C T OF I N H I B I T I O N ON THE RESPONSE - RESPONSE/DOSE L I N E A R TRANSFORMATION DOUBLE RECIPROCAL PLOT (1/r - 1/d) Gastrin + Inhibitor Gastrin 1/r 1/C.M.R. •^l/C.M.R. >5» 1/C.M.R 1/r / / , 1/C.M.R. / y f y > y ^1/C.M.R. J 50 1/d Non Competitive - D 5 r j 0 1/d Competitive D50 0 1/d Mixed F I G U R E 28 DIAGRAMMATIC EXAMPLES OF THE E F F E C T O F I N H I B I T I O N ON THE DOUBLE R E C I P R O C A L L I N E A R TRANSFORMATION 114 Interpretaion of the Double Reciprocal (Lineweaver-Burk) Transformation Representative graphs for each of the main types of i n h i b i t i o n are given i n f i g . 28. In t h i s transformation the e f f e c t of i n h i b i t i o n i s to increase the slope of the l i n e and with non-competitive i n h i b i t i o n to change i t s intercept with the y (or 1/r) axis. Competitive i n h i b i t i o n r e s u l t s i n a change i n the intercept with the x (or 1/d) a x i s . A mixed i n h i b i t o r w i l l produce a change i n the intercepts with both these axes. In t h i s transformation the values at the two intercepts represent the c h a r a c t e r i s t i c Michaelis-Menten parameters indicated i n f i g . 28. Type of I n h i b i t i o n Slope of l i n e Intercept with 1/d axis Intercept with -D,-Q 1/r axis non competitive increased unchanged alt e r e d competitive increased a l t e r e d unchanged mixed increased al t e r e d a l t e r e d Although t h i s transformation has been used for many years i t does not provide an accurate estimate of C.M.R. or D,.Q. It does produce v i s u a l l y s a t i s f a c t o r y r e s u l t s by minimising small deviations and producing a good " f i t t i n g " l i n e from u n r e l i a b l e experimental values. 

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