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Investigation of the protective action of the E₂ prostaglandins on the gastric mucosa 1977

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AN INVESTIGATION OF THE PROTECTIVE ACTION OF THE E 2 PROSTAGLANDINS ON THE GASTRIC MUCOSA BY JOHN PHILIP BOLTON B.Chir. Un i v e r s i t y of Cambridge 1967 M.A., M.B. Univ e r s i t y of Cambridge 1968 A THESIS SUBMITTED IN PARTIAL FULFILLMENT OF THE REQUIREMENT OF THE DEGREE OF MASTER OF SCIENCE IN THE DEPARTMENT OF SURGERY accept t h i s t h e s i s as conforming to the required standard The Un i v e r s i t y of B r i t i s h Columbia JUNE 1977 (c) John P h i l i p Bolton In presenting th i s thes is in pa r t i a l fu l f i lment of the requirements for an advanced degree at the Un ivers i ty of B r i t i s h Columbia, I agree that the L ibrary sha l l make i t 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 lar ly purposes may be granted by the Head of my Department or by his representat ives. It is understood that copying or pub l i ca t ion of th is thes is fo r f inanc ia l gain sha l l not be allowed without my writ ten permission. Department of S u r g e r y The Univers i ty of B r i t i s h Columbia 2075 Wesbrook Place Vancouver, Canada V6T 1W5 Date A P r i l 2 9 . 1977 ABSTRACT The prostaglandins are found throughout the body and are therefore thought to have some p h y s i o l o g i c a l r o l e . I t has been suggested that prostaglandin i s concerned with the maintenance of g a s t r i c mucosal i n t e g r i t y . In a previous study of the g a s t r i c e f f e c t s of the E^ prostaglandins, natural prostaglandin E^ (PGE^) , 15-methyl prostaglandin E^ (15M), and 16.16-dimethyl prostaglandin E^ (16DM), i t was observed that a l l three agents appeared to increase mucus production and that 16DM appeared to stimulate a non-acid secretion i n basal Heidenhain pouches. These properties of the prostaglandins have been studied i n d e t a i l . (Experiments 1 and 2) It has previously been shown that 15M can prevent the damaging e f f e c t of a s p i r i n and indomethaciri on the g a s t r i c mucosal b a r r i e r . Using a model i n which g a s t r i c mucosal b a r r i e r damage could be produced and sustained, the a b i l i t y of these agents to reverse established damage was studied. (Experiment 3) Experiment 1: The e f f e c t of t o p i c a l and intravenous E^ prostaglandins on g a s t r i c mucus production was studied i n ra t s by measuring both the amount of mucus shed i n t o a small volume of normal sa l i n e i n the closed stomach and the amount of mucus bound to the mucosa, over a three hour period. Mucus was measured i n d i r e c t l y by measuring the binding of A l c i a n Blue. A l l prostaglandins caused a s i g n i f i c a n t increase i n the mucus found i n so l u t i o n , but not i n that bound to the mucosa. Experiment 2: The e f f e c t of t o p i c a l and intravenous administration of the three E^ prostaglandins on non-parietal c e l l secretion was measured i n perfused canine Heidenhain pouches. The pouches were perfused with a non-acid s o l u t i o n . The increase i n volume was measured using polyethylene g l y c o l as a volume marker and the HCO^ content by the method of back t i t r a t i o n . 16DM was found to cause a s i g n i f i c a n t increase i n the volume, and the f l u i d secreted contained Na +, C l and HCO^ . In the absence of acid i t i s suggested that t h i s represents stimulation of non-parietal c e l l s . Experiment 3; The re v e r s a l of a s p i r i n . induced damage to the g a s t r i c mucosal b a r r i e r was demonstrated i n perfused canine Heidenhain pouches. The pouches were perfused f o r two hours with a s p i r i n to produce g a s t r i c mucosal b a r r i e r damage, the a s p i r i n was then withdrawn and the pouch perfused with a c i d alone. The e f f e c t s of t o p i c a l and intravenous prostaglandins and intravenous metiamide were tested during t h i s l a t t e r period when established b a r r i e r damage existed. Intravenous PGE 2 and 15M reversed the damage, but t o p i c a l prostaglandins and intravenous metiamide di d not. It i s concluded that the E^ prostaglandins have a secretory e f f e c t on .the basal g a s t r i c mucosa, causing an increase i n mucus production and i n non-parietal c e l l secretion. This previously unrecognised stimulation of active secretion has lead to the misinterpretation of permeability data f o r 16DM. These secretory e f f e c t s may have some pro t e c t i v e action. Intravenous PGE^ and 15M can reverse established g a s t r i c mucosal b a r r i e r damage i n the dog. This indicates the p o s s i b i l i t y of a therapeutic role for these agents i n the management of conditions associated with d i s r u p t i o n of the g a s t r i c mucosal b a r r i e r . i v TABLE OF CONTENTS PAGE ABSTRACT i i LIST OF TABLES v LIST OF FIGURES i x ACKNOWLEDGEMENTS x INTRODUCTION 1 REVIEW OF THE LITERATURE The prostaglandins and the stomach 4 The E^ prostaglandins and the g a s t r i c mucosal b a r r i e r 10 PURPOSE OF THE STUDY Mucus production 14 Non-parietal c e l l s e cretion 17 E f f e c t of the prostaglandins on established 19 g a s t r i c mucosal b a r r i e r damage. MUCUS PRODUCTION IN THE RAT (EXPERIMENT 1) Material and Methods 22 Results 30 NONPARIETAL CELL SECRETION IN THE DOG (EXPERIMENT 2) Mat e r i a l and Methods 36 Results 42 EFFECT ON ESTABLISHED GASTRIC MUCOSAL BARRIER DAMAGE (EXPERIMENT 3) Va l i d a t i o n of model 51 Material and Methods 55 Results 56 DISCUSSION 61 CONCLUSION 75 TABLES XVII - XLV1 76 BIBLIOGRAPHY 105 APPENDIX Antisecretory a c t i o n of Metiamide 114 E f f e c t of d i l u t i o n of A l c i a n Blue 116 LIST OF TABLES 1. A l c i a n Blue binding, volume secretion and acid output with t o p i c a l prostaglandins. 11. A l c i a n Blue binding, volume secretion and acid output with intravenous prostaglandins. 111. A l c i a n Blue binding, volume secretion and acid output with subcutaneous histamine. IV. A l c i a n Blue binding by r a t plasma. V. Volume and bicarbonate secretion from Heidenhain pouches with t o p i c a l prostaglandins. VI. Volume and bicarbonate secretion from Heidenhain pouches with intravenous prostaglandins. V l l . Net ion fluxes i n Heidenhain pouches with t o p i c a l prostaglandins. V l l l . Permeability factors i n Heidenhain pouches with t o p i c a l prostaglandins. IX. Net ion fluxes i n Heidenhain pouches with intravenous prostaglandins. X. Permeability factors i n Heidenhain pouches with intravenous.prostaglandins. XI. Net ion f l u x and permeability factors during and following t o p i c a l a p p l i c a t i o n of 20 mM a s p i r i n . X l l . Comparison of net ion flux and permeability factors i n a l l groups during exposure to 20 mM a s p i r i n . X l l l . Treatment of established g a s t r i c mucosal b a r r i e r damage with t o p i c a l prostaglandins. PAGE 31 32 34 35 44 45 46 47 49 50 54 57 58 V I XIV. Treatment of established g a s t r i c mucosal b a r r i e r damage with intravenous prostaglandins. XV. Net ion f l u x i n Heidenhain pouches with t o p i c a l 16.16-dimethyl prostaglandin E ( From the work of P.E. O'Brien and D.C. Carter ) XVI. Comparison of the volume of f l u i d produced by the Heidenhain pouches i n each group. X V l l . A l c i a n Blue binding, volume secretion and acid output with t o p i c a l prostaglandins. Controls. XV111. A l c i a n Blue binding, volume secretion and acid output with t o p i c a l 15M. XlX. A l c i a n Blue binding, volume sec r e t i o n and a c i d output with t o p i c a l 16DM. XX. A l c i a n Blue binding, volume secretion and acid output with t o p i c a l PGE^. XXI. A l c i a n Blue binding, volume secretion and acid output with intravenous prostaglandins. Controls. X X l l . A l c i a n Blue binding, volume secretion and acid output with intravenous 15M. XX111. A l c i a n Blue binding, volume secretion and acid output with intravenous 16DM. XXIV. A l c i a n Blue binding, volume secretion and acid output with intravenous PGE^. XXV. A l c i a n Blue binding, volume secretion and acid output with subcutaneous histamine. Controls. PAGE 60 68 72 76 77 78 79 80 81 82 83 84 V l l XXVI. A l c i a n Blue binding, volume secretion and acid output with subcutaneous histamine. XXVll. Bicarbonate secretion from Heidenhain pouches t o p i c a l prostaglandins. X X V l l l . Volume of secretion from Heidenhain pouches t o p i c a l prostaglandins. XXIX. Net ion fl u x and permeability f a c t o r s . Experiments with t o p i c a l prostaglandins. Controls. XXX. Net ion f l u x and permeability f a c t o r s . T o p i c a l 15M. XXXI. Net ion f l u x and permeability f a c t o r s . T o p i c a l 16DM., XXXll. Net ion fl u x and permeability f a c t o r s . X X X l l l . Topical PGE2- Bicarbonate secretion from Heidenhain pouches. Intravenous prostaglandins. XXXIV. Volume of secretion from Heidenhain pouches. Intravenous prostaglandins. XXXV. Net ion f l u x and permeability f a c t o r s . Experiments with intravenous prostaglandins. Controls. XXXVI. Net ion fl u x and permeability f a c t o r s . Intravenous 15M. XXXVll. Net ion flux and permeability f a c t o r s . Intravenous 16DM. PAGE 86 87 88 89 90 91 92 93 94 95 96 V J I L XXXVlll. Net ion f l u x and permeability f a c t o r s . Intravenous PGE^. XXXlX. Net ion f l u x , permeability factors and volumes secreted i n Damage and Recovery Periods. Controls. XL. Net ion f l u x , permeability factors and volumes secreted i n Damage and Recovery Periods. A s p i r i n Damage. XLl. Net ion f l u x , permeability factors and volumes secreted i n Damage and Recovery Periods. Topical PGE^. X L l l . Net ion f l u x , permeability factors and volumes secreted i n Damage and Recovery Periods. Topical 15M. X L l l l . Net ion f l u x , permeability factors and volumes secreted i n Damage and Recovery Periods. Intravenous PGE 2. XLlV. Net ion f l u x , permeability factors and volumes secreted i n Damage and Recovery Periods. Intravenous 15M. XLV. Net ion f l u x , permeability factors and volumes secreted i n Damage and Recovery Periods. Intravenous Metiamide. XLVl. E f f e c t of D i l u t i o n on A l c i a n Blue Binding. PAGE 97 98 99 100 101 102 103 104 117 LIST OF FIGURES The r e l a t i o n s h i p of PGE^ to other pharmacologically active substances derived from the same source. The structure of Prostaglandin E^, 15-methyl prostaglandin E^ and 16.16-dimethyl prostaglandin V Standard curve for a l c i a n Blue Measurement of free mucus. Standard curve for A l c i a n Blue Measurement of bound mucus. Perfusion system used i n the canine Heidenhain pouches. Standard curve for polyethylene g l y c o l . The experimental model used to t e s t the e f f e c t of various drugs on established g a s t r i c mucosal b a r r i e r damage. The e f f e c t of intravenous metiamide on histamine stimulated acid output. X ACKNOWLEDGEMENT I wish to thank my supervisor Dr. M.M. Cohen for h i s invaluable advice and guidance during the period of t h i s study. Expert t e c h n i c a l help has been given by Mr. D. Palmer and Mr. N. A l l a d i n a and by Mrs. C. Chan. The work would not have been po s s i b l e without t h e i r help. I wish to thank the s t a f f members of the Animal Laboratory of the Depart ment of Surgery for t h e i r help and cooperation. I am indebted to Ms. G. Henderson for her outstanding s e c r e t a r i a l help and to Mr. Frank Goudron and his s t a f f for t h e i r help with the o v e r a l l production of t h i s t h e s i s . 1 INTRODUCTION In 1933 Goldblatt"'" discovered an a c i d i c l i p i d soluble, smooth muscle stimulating compound i n seminal f l u i d . This was the f i r s t d e s c r i p t i o n of a prostaglandin and yet i t went l a r g e l y unnoticed. There followed a period of i n a c t i v i t y i n the study of t h i s compound due mainly to the lack of s u i t able methods for accurate q u a l i t a t i v e and quantitative measurement of i t s active components. The current high l e v e l of i n t e r e s t i n the prostaglandins dates back to the 1960's when these compounds were i s o l a t e d i n c r y s t a l l i n e 2 3 form and t h e i r chemical structures elucidated. In the past seventeen years a vast amount of work has been done on these substances and much information i s a v a i l a b l e regarding t h e i r pharmacological e f f e c t s . The prostaglandins are widely d i s t r i b u t e d i n the tissues of the body and are known to have a v a r i e t y of actions. Major e f f e c t s include contraction 4 5 of the uterus and the l o n g i t u d i n a l muscle of the gut, the i n h i b i t i o n of c i r c u l a r muscle i n the g u t 5 and v a s o d i l a t a t i o n , 7 the i n h i b i t i o n of g a s t r i c 8 acid secretion and the accumulation of large volumes of f l u i d i n the small . 9 i n t e s t i n e . Their wide d i s t r i b u t i o n and the v a r i e t y of t h e i r e f f e c t s has led to the assumption that they have a p h y s i o l o g i c a l r o l e . This r o l e , i f such a r o l e e x i s t s , has not yet been determined and there i s now evidence that the prostaglandins may be simply metabolites of other active agents such as the endoperoxides or one step on a metabolic pathway from arachi donic a c i d to p r o s t a c y c l i n , a highly potent agent recently i s o l a t e d from vascular t i s s u e . ^ (Figure 1) There i s no evidence that the prostaglandins or t h e i r active precursors are stored i n the t i s s u e s , biosynthesis seems to take place as required, but the stimulus for t h i s i s unknown. The agents PROSTAGLANDIN SYNTHESIS ARACHIDONIC ACID CYCLIC ENDOPEROXIDES* PGX: PROSTACYCLIN* > THROMBOXANES * PGE_* PGF. * 2a * Pharmacologically Active Substances FIGURE 1: I l l u s t r a t e s the r e l a t i o n s h i p of prostaglandin E 2 to arachidonic a c i d and other m e t a b o l i c a l l y r e l a t e d a c t i v e agents. are thought to be produced at the s i t e of t h e i r action and they are known to have an extremely short h a l f l i f e . I t i s possible that the reason why the prostaglandins have been the agents most investigated i s that they are more stable than t h e i r active precusors and have therefore been measured, i d e n t i f i e d and synthesized and have become widely a v a i l a b l e f o r laboratory experimentation. Although the debate regarding t h e i r p h y s i o l o g i c a l action continues the prostaglandins have been shown to have many pharmacological actions and they are cu r r e n t l y i n use or under i n v e s t i g a t i o n as therapeutic agents i n a number of f i e l d s . Prostaglandin i s used f o r the induction of labour"*"^ and for the 11 12 termination of pregnanacy i n the f i r s t and second trimester. ' I t also i s used to maintain a patent ductus arte r i o s u s i n neonates with major congenital cardiac abnormalities who require the maintenance of a l e f t to 13 r i g h t shunt. Controlled c l i n i c a l t r i a l s are cur r e n t l y underway to study 14 i t s value i n erosive g a s t r i t i s and the value of i t s o r a l l y active methyl analogues i n the treatment of peptic u l c e r a t i o n . ^ ' ^ 4 REVIEW OF THE LITERATURE The E^ Prostaglandins and the Stomach Prostaglandin-like material has been extracted from most portions of 17 the g a s t r o i n t e s t i n a l t r a c t , but there has been no formal c h a r a c t e r i z a t i o n of t h i s m aterial. I t i s reported that E and F prostaglandins are the main types occurring i n the gut and the material extracted from the mucosa and 18 submucosa of the stomach i s almost c e r t a i n l y PGE^. The presence of a PGE^-like material i n the g a s t r i c mucosa has stimulated considerable i n t e r e s t i n the e f f e c t of t h i s n a t u r a l l y occurring prostaglandin on g a s t r i c function. P G E 2 i-s a potent i n h i b i t o r of g a s t r i c a c i d secretion when given 8 19 20 intravenously ' but i s almost i n a c t i v e when given o r a l l y . Analogues have been prepared i n an attempt to overcome t h i s disadvantage and at the present time two such analogues have been widely investigated, 15M and 16DM. The differences between these two analogues and the parent compound are i l l u s t r a t e d i n Figure 2. The term "the E^ postaglandins" i s used to include natural PGE^ and i t s two methyl analogues and as the experimental work i n t h i s t h e s i s i s concerned with c e r t a i n actions of these agents on the g a s t r i c mucosa current knowledge of t h e i r g a s t r i c e f f e c t s i s reviewed. The E^ prostaglandins have two important actions i n the stomach: 1. I n h i b i t i o n of g a s t r i c a c i d secretion. 2. Protection of the mucosa. FIGURE 2: I l l u s t r a t e s the s i m i l a r i t y i n structure of prostaglandin E 2 and i t s methyl analogues 15-methyl prostaglandin E 2 and 16.16- dimethyl prostaglandin E 2- I n h i b i t i o n of Gastric Acid Secretion The accumulated evidence f o r i n h i b i t i o n of acid secretion by PGE^<and the c l o s e l y r e l a t e d PGE^ i s very extensive and includes experiments i n man and a v a r i e t y of animals under basal and stimulated conditions. As PGE^ was discovered before PGE 2 i t has been more extensively studied, but no important differences i n the i n h i b i t o r y actions of the two compounds have been found. Animal Studies When given p a r e n t e r a l l y PGE^ can i n h i b i t g a s t r i c a c i d secretion i n dogs stimulated by a v a r i e t y of agents i n c l u d i n g , histamine, 21 pentagastrin, food, 2-deoxy-D-glucose, reserpine and carbachol. 22 Parentral PGE^ can i n h i b i t histamine stimulated secretion. Basal 22 secretion i n rats can also be i n h i b i t e d by parenteral ~PGE^. When given o r a l l y i n high doses (0.05-5 mg) i n an isosmotic buffer (Na^HPO^, pH 7.4) PGE^ can i n h i b i t g a s t r i c acid secretion of pylorus 22 l i g a t e d r a t s , but i t i s i n e f f e c t i v e by t h i s route i n dogs. In contrast 15M and 16DM can i n h i b i t g a s t r i c a c i d secretion when given o r a l l y as well as pa r e n t e r a l l y to dogs and have a s i m i l a r 22 23 e f f e c t i n r a t s . ' These agents have been found to be much more potent than the parent compound and to exert a c t i v i t y f o r a much longer duration. The E D ^ Q (dose i n h i b i t i n g a c i d output by 50%) i n dogs for PGE^, 15M and 16DM are 10, 0.3 and 0.1 ug/Kg re s p e c t i v e l y 22 with a sing l e intravenous i n j e c t i o n . Oral 16DM i s 2.8 times as potent as 15M. 7 There i s evidence to suggest that the mode of action of the methyl analogues i s d i f f e r e n t when the d i f f e r e n t routes of administration are used. When applied t o p i c a l l y to the mucosa these agents appear to exert a l o c a l antisecretory e f f e c t rather than a systemic e f f e c t secondary to absorption. Experiments on dogs with two Heidenhain pouches demonstrated that l o c a l i n s t i l l a t i o n of 16DM to one pouch produced t o t a l i n h i b i t i o n of stimulated a c i d secretion but was without e f f e c t i n the other. At higher doses a l e s s potent systemic e f f e c t could be demonstrated on the second pouch. The systemic action was 24 les s potent and of shorter duration than the l o c a l action. Human Studies 25 19 Intravenous PGE^ w i l l i n h i b i t basal and pentagastrin stimulated 20 acid secretion but o r a l administration i s without e f f e c t . 15M on the other hand given o r a l l y to healthy subjects i n h i b i t e d pentagastrin stimulated a c i d output for several hours, the ED being approximately 50 , 2 0 1 pg/Kg. 16DM was antisecretory i n healthy volunteers when administered both o r a l l y and intravenously and both analogues were p a r t i c u l a r l y potent i n i n h i b i t i n g food induced g a s t r i c secretion both i n healthy 27 volunteers and duodenal u l c e r patients. 8 Protection of the Mucosa The evidence f o r t h i s action comes under two headings: A. Protection r e s u l t i n g from i n h i b i t i o n of acid secretion (antiulcer e f f e c t ) . B. Protection independent of a c i d secretory i n h i b i t i o n . A. Protection Resulting From I n h i b i t i o n of Acid Secretion As the E^ prostaglandins have been demonstrated to have powerful antisecretory properties i n t e r e s t has arisen i n t h e i r possible use f o r the prevention and cure of gastroduodenal u l c e r s . PGE^ or i t s methyl analogues given e i t h e r subcutaneously or o r a l l y to r a t s have been shown to i n h i b i t g a s t r i c u l c e r s produced i n a v a r i e t y of ways i n c l u d i n g : 2 2 a) Shay ulc e r s produced by pylorus l i g a t i o n . 2 2 b) Steroid induced u l c e r s . c) Ulcers produced by o r a l and i n t r a p e r i t o n e a l administration 2 8 of nonsteroidal anti-inflammatory agents, such as a s p i r i n and indomethacin. 2 9 9 PGE 2 or 16DM given e i t h e r o r a l l y or subcutaneously i n h i b i t e d i n a dose dependent manner the development of duodenal u l c e r s produced i n r a t s by: , a) A single subcutaneous i n j e c t i o n of h i s t a m i n e . ^ b) A constant subcutaneous i n f u s i o n of histamine and 22 carbachol f o r 24 hours. The doses of the prostaglandins used i n these experiments were i n the a c i d secretory i n h i b i t i n g range and s i m i l a r p r o t e c t i v e e f f e c t s have been described f o r other i n h b i t o r s of a c i d secretion, 31 32 such as the H^-receptor antagonists, ' used i n comparable i n h i b i t o r y doses. B. Protection Independent of Acid Secretory I n h i b i t i o n Recent studies suggest that the prostaglandins may also have a p r o t e c t i v e action unrelated to a c i d secretory i n h i b i t i o n . Robert has demonstrated that alcohol induced erosive l e s i o n s of the glandular p o r t i o n of the fasted r a t stomach can be completely prevented by the p r i o r administration of 16.16-dimethyl PGA2 and 15-methyl PGF which are prostaglandins that do not i n h i b i t a c i d secretion. Also 16DM administered subcutaneously i n a dose 100 times l e s s than the threshold dose i n h i b i t i n g a c i d s e c r e t i o n 33 i n the r a t w i l l a lso prevent the development of these l e s i o n s . It i s quite c l e a r therefore that the prostaglandins can exert a p r o t e c t i v e e f f e c t upon the g a s t r i c mucosa which i s independent of acid secretory i n h i b i t i o n . 10 A possible mechanism f o r t h i s p r o t e c t i v e e f f e c t i s tightening of the g a s t r i c mucosal b a r r i e r . T h e _ l 0 Prostaglandins and the Gastric Mucosal B a r r i e r In 1964 Davenport""** demonstrated that the apparent i n h i b i t i o n of acid secretion caused by the t o p i c a l a p p l i c a t i o n of eugenol to the g a s t r i c mucosa of a Heidenhain pouch was i n f a c t due to back d i f f u s i o n of hydrogen ion from the g a s t r i c lumen to the i n t e r s t i t i a l f l u i d of the mucosa. He subsequently postulated that the e f f e c t of b a r r i e r damaging agents, such as a s p i r i n , on the g a s t r i c mucosa was the r e s u l t of the back d i f f u s i o n of hydrogen ion. The high concentration of hydrogen ion i n the i n t e r s t i t i a l f l u i d damaged the mucosal c e l l s and the c a p i l l a r i e s and caused a release of histamine which l e d to v a s o d i l a t a t i o n and the secretion of more hydrogen ion. This sequence of events led to the shedding of mucosal c e l l s , bleeding 35 from the mucosal c a p i l l a r i e s and the t y p i c a l changes of erosive g a s t r i t i s . This work l e d to the r e v i v a l of the concept of the g a s t r i c mucosal 36 b a r r i e r . Under normal circumstances the concentration of hydrogen ion and sodium ion are unevenly d i s t r i b u t e d on e i t h e r side of the g a s t r i c mucosa. There i s a high concentration of hydrogen ion i n the lumen and a high concentration of sodium i n the i n t e r s t i t i a l f l u i d . Despite the concentration gradients that therefore e x i s t f o r these ions there i s l i t t l e movement of hydrogen ion out of the lumen or sodium ion i n t o the lumen. The mucosa i s almost impermeable to the d i f f u s i o n of these ions down t h e i r negative concentration gradients. This r e l a t i v e impermeability of the mucosa i s r e f e r r e d to as the g a s t r i c mucosal b a r r i e r . The small movement 11 of ions that does occur under normal circumstances can be measured and represents the basal l e v e l of the g a s t r i c mucosal permeability. Agents, such as a s p i r i n , increase the permeability of the mucosa and the measured movement of ions. A s i g n i f i c a n t increase i n g a s t r i c mucosal permeability above the basal l e v e l indicates d i s r u p t i o n of the g a s t r i c mucosal b a r r i e r . A v a r i e t y of agents are known to break the g a s t r i c mucosal b a r r i e r , 37 38 38 39 the important ones being a s p i r i n , ' indomethacin, b i l e s a l t s 37 40 and a l c o h o l , ' and a l l these agents cause erosive and hemorrhagic l e s i o n of the g a s t r i c mucosa. Hemorrhagic l e s i o n s associated with stress have also been shown to be associated with d i s r u p t i o n of the g a s t r i c mucosal 41 b a r r i e r . Disruption of the g a s t r i c mucosal b a r r i e r and the back d i f f u s i o n of hydrogen ion are thought to be major factors i n the pathogenesis of 42 acute hemorrhagic l e s i o n of the g a s t r i c mucosa. The g a s t r i c mucosal b a r r i e r i s a p h y s i o l o g i c a l concept rather than a true anatomical e n t i t y , and i t i s not known how the various b a r r i e r breakers produce t h e i r e f f e c t on the b a r r i e r . I t has been suggested that the b a r r i e r i s formed by the t i g h t junctions that e x i s t between the l a t e r a l borders of the mucosal c e l l s and that b a r r i e r damage i s associated with separation of the t i g h t junctions which allows the passage of ions between the c e l l s . There i s ele c t r o n microscopic evidence that when d i t h i o t h r e i t o l , a b a r r i e r breaker that also causes plasma shedding, i s applied to the mucosa p r o t e i n i s l o s t by passing between adjacent mucosal c e l l s and t h e i r 43 disrupted t i g h t junctions. I t i s probable that i o n i c d i f f u s i o n occurs by the same route. 12 The possible r o l e of prostaglandins i n maintaining the g a s t r i c mucosal b a r r i e r was r a i s e d a f t e r Vane et a l demonstrated that c e r t a i n nonsteroidal 44 45 anti-inflammatory agents, i n p a r t i c u l a r a s p i r i n and indomethacin, could i n h i b i t the synthesis of prostaglandins. This i n v i t r o e f f e c t has 46 since been demonstrated i n vivo, and i t has been suggested that these two agents produce damage to the g a s t r i c mucosal b a r r i e r by the i n h i b i t i o n of endogenous prostaglandin synthesis, and therefore that the prostaglandins are responsible f o r the maintenance of g a s t r i c mucosal i n t e g r i t y . 47 Support f o r t h i s concept was provided by the experiments of Cohen. He studied g a s t r i c mucosal permeability i n canine Heidenhain pouches and demonstrated a highly s i g n i f i c a n t increase when a s p i r i n i n a concentration of 20 mM and indomethacin i n a concentration 2 mM were added to the acid perfusate. In subsequent experiments he added 15M i n a dose of 5 ug/Kg to the acid and a s p i r i n and acid and indomethacin perfusates and was able to demonstrate that i n the presence of t h i s prostaglandin a s p i r i n and indomethacin did not cause damage to the g a s t r i c mucosal b a r r i e r . The dose of 15M was the E D , _ Q f o r ac i d secretory i n h i b i t i o n , but t h i s same e f f e c t was achieved with a dose as low as 1.25 yg/Kg. Cohen also recorded i n t h i s experiment that when 15M was applied to the mucosa i n the absence of the b a r r i e r breakers i t decreased g a s t r i c mucosal permeability below the cont r o l l e v e l , suggesting that exogenous prostaglandin could not only prevent damage to the g a s t r i c mucosal b a r r i e r but could also tighten the g a s t r i c mucosal b a r r i e r . Further studies of the permeability e f f e c t s of the E ^ prostaglandins have been reported and have produced c o n f l i c t i n g data. O'Brien and Carter 13 studied the e f f e c t of 16DM both t o p i c a l l y and intravenously on the canine Heidenhain pouch. They found that 16DM applied t o p i c a l l y to the mucosa i n a concentration of 15 ug/ml broke the g a s t r i c mucosal b a r r i e r , but d i d not 49 produce t h i s damaging e f f e c t when given systemically. Bolton and Cohen subsequently studied the permeability e f f e c t s of a l l three prostaglandins administered t o p i c a l l y and systemically. They were unable to confirm Cohen's previous f i n d i n g that 15M tightened the g a s t r i c mucosal b a r r i e r when applied t o p i c a l l y . Using the same dose, and larger doses up to ten times the o r i g i n a l dose, they found no s i g n i f i c a n t a l t e r a t i o n i n g a s t r i c mucosal permeability. However they d i d f i n d that 15M tightened the b a r r i e r when given intravenously, an e f f e c t also produced by intravenous PGE 2. They also confirmed O'Brien and Carter's data demonstrating d i s r u p t i o n of the g a s t r i c mucosal b a r r i e r by t o p i c a l 16DM with a concentration as low as 1 ug/ml and also found that i t had the same e f f e c t systemically i f given by constant intravenous i n f u s i o n as opposed to the single bolus i n j e c t i o n used by O'Brien and Carter. The apparent detrimental e f f e c t of 16DM i s hard to reconcile with the fact that i t i s a potent protector of the g a s t r i c mucosa. 14 PURPOSE OF THE STUDY ' Evidence has been presented that the prostaglandins have a p r o t e c t i v e action on the g a s t r i c mucosa, an e f f e c t which i s unrelated to acid secretory i n h i b i t i o n and whose mechanism i s uncertain. As a r e s u l t of previous observations c e r t a i n aspects of the g a s t r i c e f f e c t s of the E^ prostaglandins have been investigated i n greater depth. 1. Mucus production. 2. Non-parietal c e l l secretion. 3. The e f f e c t of E^ prostaglandins on established g a s t r i c mucosal b a r r i e r damage. 1. Mucus Production When studying the permeability e f f e c t s of the prostaglandins 49 i n canine Heidenhain pouches i t was noticed that i n the experiments i n which prostaglandins were used the amount of stringy mucus appearing i n the perfusate was greater than i n the control experiments. I t seemed probable that the prostaglandins were stimulating mucus production and an attempt has been made to quantify t h i s e f f e c t . There have been two previous references to the e f f e c t of prostaglandins on g a s t r i c mucus production. Robert et a l ~ ^ when measuring the e f f e c t of prosta glandin E^ on g a s t r i c secretion i n the r a t noted a reduction i n mucus output as measured by fucose, hexosamine and s i a l i a c a c i d concentrations. The dose at which these observation were made was 0.5 ug/Kg/minute. 15 At larger doses the marked i n h i b i t i o n or o v e r a l l g a s t r i c secretion produced volumes of j u i c e too small for biochemical measurement of the components of mucus. These findings by no means exclude the p o s s i b i l i t y that at a higher dose stimulation of mucus was produced but was masked by the a c i d i n h i b i t o r y e f f e c t s of the prostaglandin. I t has also been reported that 15M increased g a s t r i c mucus formation i n patients with e i t h e r g a s t r i c u l c e r s or g a s t r i t i s . This was assessed v i s u a l l y and h i s t o l o g i c a l l y , but no measurements were made.^ Mucus i s a d i f f i c u l t component of g a s t r i c secretion to study because of the problem of recovery. Mucus i s found i n the stomach i n two forms, that adherent to the mucosa c a l l e d b a r r i e r mucus and that i n the g a s t r i c j u i c e , r e f e r r e d to as soluble or free mucus. Not a l l the mucus i n the g a s t r i c j u i c e i s i n s o l u t i o n , and therefore the term free mucus i s more accurate. Most studies of the action of various secretory agents such as histamine, h i s t a l o g and pentagastrin have measured only the free f r a c t i o n , and because of the problems of recovery and measurement have l a r g e l y ignored the b a r r i e r f r a c t i o n . A study of mucus production should i d e a l l y include measurement of both f r a c t i o n s . Because of the problems of recovery of b a r r i e r mucus any form of chronic pouch preparation has severe l i m i t a t i o n s , and i t was because of t h i s that the r a t was chosen for t h i s study despite the f a c t that the i n i t i a l observations were made i n canine Heidenhain pouches. Two methods have been described for the measurement of b a r r i e r 16 mucus i n the r a t . One involves assessment by weight and the other 53 uses a technique of binding to a h i s t o l o g i c dye, A l c i a n Blue. The l a t t e r method was chosen because A l c i a n Blue can also be used to measure the free f r a c t i o n of mucus. I t not only binds to glycoprotein, the main constituent of b a r r i e r mucus, but also binds to soluble 54 mucopolysaccharides, the main constituent of the soluble f r a c t i o n , to form insoluble dye-mucus complexes. This method allows for the determination of the e f f e c t of the prostaglandins on both f r a c t i o n s of mucus as well as on the t o t a l amount of mucus produced. In order to assess the magnitude of the secretory action of the prostaglandins, studies were undertaken using another agent known to stimulate mucus production, namely histamine. Unfortunately the other agents that stimulate mucus production a l l stimulate g a s t r i c a c i d secretion and so the conditions i n the two sets of experiments could not be i d e n t i c a l . Mucus secretion during a c i d stimulation was measured as a convenient parameter for comparison. 17 2. Non-Parietal C e l l Secretion Topical and intravenous 16DM has been demonstrated to damage the gastric mucosal barrier in perfused canine Heidenhain pouches, an action which would indicate a disruptive rather than a protective effect on the gastric mucosa as damage to the gastric mucosal barrier is associated with damage to mucosal ce l l s and bleeding. In direct contrast to this finding i t has been demonstrated that 16DM can protect the gastric mucosa from damage f i r s t l y by i t s antiulcer effect when used in doses which i n h i b i t acid secretion and secondly by i t s protective effect in doses 100 times less than the acid inhibitory dose. This discrepancy has been studied by re-examining the effect of 16DM in the canine Heidenhain pouch. 49 In previous studies of gastric mucosal permeability, which were performed by perfusing a nonsecretory canine Heidenhain pouch with a solution containing H + , Na + , CI and PEG, the volume of any f l u i d produced by the pouch could be assessed at the end of the three hour period of perfusion by subtracting the expected f i n a l volume ( i n i t i a l volume - sampling volume) from the measured f i n a l volume, calculated from the PEG concentration. The pouches produced some f l u i d even during the control perfusions, but i t was noted that in the presence of 16DM the volume of f l u i d produced was greatly increased. Similar increases were not observed with the other two prostaglandins. 18 There are two possible explanations f o r the cause of t h i s f l u i d production. Agents which damage the g a s t r i c mucosal b a r r i e r also cause a production of f l u i d by the pouch, so i t i s possible that the increase i n f l u i d production occurred by the same mechanism of an increase i n g a s t r i c mucosal permeability and the subsequent damaging e f f e c t of the hydrogen ion on the mucosa. Doubt i s cast upon t h i s explanation by the f a c t that the degree of damage produced by 16DM, assessed by the increase i n g a s t r i c mucosal permeability, was small compared with known b a r r i e r breakers, yet the volume of f l u i d produced was greater. A second explanation i s that 16DM stimulated active secretion by nonparietal c e l l s i n the mucosa. Such an e f f e c t could account f o r the volume of f l u i d produced, and active secretion of t h i s sort i n a system i n which c a l c u l a t i o n of the permeability factor i s based on the assumption that the pouch i s e n t i r e l y nonsecretory could lead to discrepancies i n the permeability data. If the f i r s t explanation i s correct increased production of f l u i d would only occur i n the presence of exogenous a c i d which could d i f f u s e i n t o the mucosa as a r e s u l t of the increase i n permeability. In the absence of acid there should be no increased production of f l u i d . On the other hand i f the f l u i d were due to a c t i v e secretion then i t would occur i n the presence or absence of a c i d . Perfusion experiments were therefore performed i n canine Heidenhain pouches i n which the e f f e c t of t o p i c a l and intravenous E^ prostaglandins was studied when the perfusate contained no acid. The sol u t i o n used was isosmotic, and contained L i + , Na +, C l and PEG. Lithium behaves i n + 55 the same way as H i n respect of permeability and can be used as an 19 i n d i c a t o r of permeability i n the absence of H*". Sodium would not be an accurate i n d i c a t o r of permeability i f nonparietal c e l l secretion were occurring as the secreted f l u i d would contain sodium ions. PEG i s a volume marker, and the absence of a c i d allowed the HCO^ content of the perfusate to be measured. The production of f l u i d containing Na +, CI and HCO^ i n response to 16DM i n the absence of exogenous acid would indicate d i r e c t stimula t i o n of nonparietal c e l l s . 3. The E f f e c t of the E^ Prostaglandins on Established G a s t r i c Mucosal B a r r i e r Damage The i n h i b i t o r y e f f e c t of the E ^ prostaglandins on a c i d secretion and the proven a n t i u l c e r e f f e c t i n experimental animals has l e d to t h e i r study i n man as therapeutic agents i n peptic u c l e r a t i o n . PGE^ i s i n e f f e c t i v e when given o r a l l y , but the two methyl analogues, 15M and 16DM, are highly potent i n h i b i t o r s by the o r a l route. Controlled t r i a l s have demonstrated that 15M w i l l reduce both the s e v e r i t y of the 16 15 pain and the s i z e of the u l c e r i n patients with g a s t r i c and duodenal u l c e r s , s i g n i f i c a n t l y better then a placebo. However the agent i s not c u r r e n t l y i n general use, and i t seems l i k e l y that the recent introduction of the H^-receptor antagonists on to the European market w i l l g r e atly reduce i n t e r e s t i n the prostaglandins unless serious side e f f e c t of the H^-receptor antagonists emerge. However, the prostaglandins may f i n d a r o l e i n the treatment of acute g a s t r i c mucosal l e s i o n s as t h e i r known g a s t r i c e f f e c t s are p a r t i c u l a r l y 20 appropriate to t h i s condition. The pathogenesis of acute g a s t r i c mucosal lesio n s i s complex but two fa c t o r s are of p a r t i c u l a r importance, the 4 2 presence of a c i d and di s r u p t i o n of the g a s t r i c mucosal b a r r i e r . The medical management of bleeding from acute g a s t r i c mucosal lesi o n s has changed l i t t l e over the past 30 years and i s aimed p r i m a r i l y at reducing the amount of g a s t r i c a c i d present by n e u t r a l i z a t i o n and more recently by i n h i b i t i o n . An agent which combined acid i n h i b i t i o n with the a b i l i t y to tighten the g a s t r i c mucosal.barrier would t h e o r e t i c a l l y be of great v a l u e . ^ I t has previously been demonstrated that 15M can prevent damage to the g a s t r i c mucosal b a r r i e r by a s p i r i n and indomethacin, and t h i s could i n d i c a t e a pos s i b l e prophylactic r o l e . Before suggesting a therapeutic r o l e i t would be necessary to demonstrate that the prostaglandins could also reverse g a s t r i c mucosal b a r r i e r damage once i t was established. An attempt has been made to do t h i s using the perfused canine Heidenhain pouch. I n i t i a l l y i t was necessary to design a model i n which g a s t r i c mucosal b a r r i e r damage could be produced and i n which the damage would p e r s i s t a f t e r withdrawal of the damaging agent, a s i t u a t i o n analagous to that encountered c l i n i c a l l y . This was done by perfusing the pouch fo r two hours with an a c i d and a s p i r i n s o l u t i o n and then perfusing with acid alone f o r the t h i r d hour. The persistence of damage during the t h i r d hour would provide the model on which the e f f e c t of the prostaglandins could be tested by adding them to the perfusate and giv i n g them intravenously during the t h i r d hour. As evidence has been 21 presented that the p r o t e c t i v e e f f e c t of the prostaglandins i s not r e l a t e d to a c i d secretory i n h b i t i o n i t was decided to investigate simultaneously the e f f e c t of another type of antisecretory agent, an H^-receptor antagonist, on established g a s t r i c mucosal b a r r i e r damage. The drug metiamide was used. This agent i n s o l u t i o n acts as a b u f f e r and i s not suitable for addition to a c i d perfusates, and therefore only i t s intravenous e f f e c t s were studied. 22 STUDY OF THE EFFECTS OF THE PROSTAGLANDINS ON GASTRIC MUCUS PRODUCTION IN THE RAT (EXPERIMENT 1) Material and Methods (i) Preparation of the Animals: The study was c a r r i e d out i n male Wistar rats weighing approximately 300 gm. A f t e r an 18 hour f a s t a laparotomy was performed under ether anaesthesia, and the esophagus l i g a t e d . This was done to prevent s a l i v a contaminating the g a s t r i c contents. Vagotomy was accomplished by i n c l u d i n g the vagi i n the esophageal l i g a t u r e . A needle was passed from the duodenum through the pylorus, and 4 ml. of the t e s t s o l u t i o n were i n j e c t e d i n t o the stomach, the needle withdrawn and the pylorus l i g a t e d . A f t e r laparotomy, the rats were allowed to recover and then s a c r i f i c e d at three hours. Group I: The e f f e c t of T o p i c a l A p p l i c a t i o n of the Prostaglandins: The t e s t s o l u t i o n consisted of 150 mM sodium chloride i n the controls and 150 mM sodium chloride plus the prostaglandin i n the experimental groups. 15M and 16DM were added i n a concentration of 10 pg/ml and PGE^ i n a concentration of 100 ug/ml. These concentrations are smaller than the concentrations used i n experiments to demonstrate the a n t i u l c e r properties of these agents and represent doses of 0.13 mg/Kg for 15M and 16DM and 1.3 mg/Kg for PGE 2 which are le s s than the E D g g f o r i n h i b i t i o n of a c i d secretion i n pylorus l i g a t e d r a t s . There were i n i t i a l l y 10 r a t s i n the c o n t r o l group and i n each of the three experimental groups, one r a t i n the PGE^ group died during the course of the experiment. 23 Group I I ; The E f f e c t of Intravenous Administration of Prostaglandins: The t e s t s o l u t i o n was 150 mM sodium chl o r i d e i n both c o n t r o l and experimental groups. A fine polyethylene cannula was placed cephalad i n the i n f e r i o r vena cava and brought to the surface through the po s t e r i o r abdominal wall for the repeated administration of the prostaglandins. The prostaglandins were given by bolus intravenous i n j e c t i o n through the cannula. Three i n j e c t i o n s were given: the f i r s t when the t e s t s o l u t i o n was placed i n the stomach, and then at one and two hours afterwards. Each bolus dose of 15M and 16DM was 0.6 ug/Kg and each dose of PGE 2 0.6 mg/Kg. These doses are below the ED^^ for i n h i b i t i o n of a c i d secretion i n pylorus l i g a t e d r a t s when these agents are given subcutaneously. There were i n i t i a l l y f i v e r a t s i n the con t r o l and each of the three experimental groups, one r a t i n the con t r o l group died, and i n one r a t i n the 15M group the cannula became dislodged. Group I I I : The E f f e c t of Subcutaneous Administration of Histamine di-HCl: The preparation of the animals was the same as for the other groups. The histamine was given by intermittent subcutaneous i n j e c t i o n i n 1 ml of normal s a l i n e . Three subcutaneous i n j e c t i o n s were given: the f i r s t when the t e s t s o l u t i o n was placed i n the stomach, and then at one and two hours afterwards. Each bolus dose was 6 mg/Kg. The con t r o l animals received normal s a l i n e . There were f i v e r a t s i n the con t r o l and experimental group. 24 ( i i ) C o l l e c t i o n of Samples: At the completion of the experiment the rats were s a c r i f i c e d . The stomachs were removed, opened down the le s s e r curvature and the contents c o l l e c t e d . The g a s t r i c contents were homogenised i n a Dounce homogeniser and the volume recorded. Analysis 1. Hydrogen Ion: The hydrogen ion concentration was measured by t i t r a t i o n against 0.1 N sodium hydroxide i n an automatic t i t r i m e t e r (Radiometer Copenhagen). 2. Mucus: The mucus content was estimated by measuring the amount of A l c i a n Blue bound to the whole stomach and to a sample of the g a s t r i c contents. The A l c i a n Blue used was A l c i a n Blue GX8. A) Free Mucus: The dye binding i n the g a s t r i c contents was 57 measured by the method of Piper et a l . They studied a v a r i e t y of conditions which could influence the A l c i a n Blue binding properties of g a s t r i c j u i c e mucoproteins and reached the following conclusions: a) Concentration of A l c i a n Blue: The concentration was v a r i e d between 2 and 100 mg/100 ml and maximum p r e c i p i t a t i o n was obtained at 40 mg/100 ml. b) pH of the Reaction Mixture: Maximum p r e c i p i t a t i o n of the dye was obtained at pH 6. c) Temperature of the Reaction Mixture: No difference was found between binding at 20°C and 37° C. 25 d) Time of Incubation: I t was found that dye binding increased with time, the amount of dye p r e c i p i t a t e d at the end of the 24 hours was almost double that at one hour - e) Ionic Strength of the Reaction Mixture: Varying the molarity of sodium chl o r i d e was found to have no e f f e c t on dye binding. The optimal conditions ou t l i n e d here were used i n these experiments. One m i l l i l i t r e of the homogenised g a s t r i c contents was mixed with 3.3 ml. of Mcllvaines c i t r a t e phosphate buffer and 0.2 ml. of A l c i a n Blue of concentration 10 mg/ml and made to 5 ml. with d i s t i l l e d water. The A l c i a n Blue s o l u t i o n was f r e s h l y prepared each day.. The concentration of A l c i a n Blue i n the reac t i o n mixture was 40 mg/100 ml and the pH 5.8. The reaction mixture was incubated at 20°C fo r 24 hours and then centrifuged at 2500 revolutions per minute for 10 minutes. The concentration of A l c i a n Blue i n the supernatent f r a c t i o n was estimated spectrophotometrically at 615 nm and compared with that i n a tube containing the i d e n t i c a l reagents except that the g a s t r i c j u i c e was replaced by buffer. Dye binding was expressed as the amount of Al c i a n Blue i n milligrams p r e c i p i t a t e d from the reaction mixture during incubation. The standard curve for A l c i a n Blue i n t h i s reaction mixture i s shown i n Figure 3. The curve was constructed SO a> u c o JO o JQ < A l c i a n B lue (mg/5 ml) FIGURE 3: The standard curve f o r A l c i a n Blue prepared i n the re a c t i o n mixture used f o r the measurement of free mucus. 27 from t r i p l i c a t e assays of each concentration tested and can be seen to be biphasic. One m i l l i l i t r e of of the supernatant of each sample was d i l u t e d with one m i l l i l i t r e of buffer p r i o r to measuring the absorbance so that a l l the absorbance readings f e l l on the f i r s t part of the curve. An error could occur i f A l c i a n Blue bound to plasma proteins. There i s always a small leak of plasma proteins into the g a s t r i c lumen and t h i s may be increased by an increase i n mucosal blood flow. Studies were performed i n which plasma from a r a t was used to replaced the g a s t r i c contents i n the r e a c t i o n mixture and the binding measured. B) B a r r i e r Mucus; This was estimated by a modification 53 of the method of Corne et a l . The everted stomach was incubated i n 25 ml. of Mcllvaines c i t r a t e phosphate buffer containing A l c i a n Blue i n a concentration of 40 mg/100 ml, for two hours. The stomach was then removed and the s o l u t i o n centrifuged at 2500 revolutions per minute f o r 10 minutes and the concentration of A l c i a n Blue estimated spectrophotometrically. Dye binding was expressed as the amount of A l c i a n Blue i n milligrams p r e c i p i t a t e d from the s o l u t i o n during incubation. 28 The standard curve for A l c i a n Blue i n Mcllvaines c i t r a t e phosphate buffer i s shown i n Figure 4. I t was constructed i n the same way as the other standard curve and i s s i m i l a r l y biphasic. The supernatant samples were also d i l u t e d with buffer to b r i n g the absorbance readings i n t o the f i r s t part of the curve. Corne demonstrated by h i s t o l o g i c a l examination of the r a t stomach stained for two hours with A l c i a n Blue that there was no penetration of the dye in t o the mucosal t i s s u e . (iv) S t a t i s t i c a l A nalysis: The r e s u l t s were assessed using Student's t - t e s t f o r paired values and applying the H o t e l l i n g c o r r e c t i o n . FIGURE 4: The standard curve f o r A l c i a n Blue used i n the measurement of bound mucus. 30 Results The r e s u l t s of the t o p i c a l a p p l i c a t i o n of the prostaglandins are tabulated i n Table I and those f o r intravenous administration are tabulated i n Table I I . Prostaglandin E^ had the same e f f e c t on mucus production when given by eith e r route. As can be seen from Table I and II i t had no e f f e c t on b a r r i e r mucus as measured by A l c i a n Blue bound to the g a s t r i c mucosa but caused a highly s i g n i f i c a n t increase i n mucus i n the g a s t r i c contents (p < 0.01) and the t o t a l amount of mucus produced (p < 0.01). 16DM had a s i m i l a r e f f e c t when applied t o p i c a l l y (Table I ) , having no e f f e c t on b a r r i e r mucus but causing a highly s i g n i f i c a n t increase i n free (p < 0.01) and t o t a l (p < 0.01) mucus. Given intravenously (Table II) the e f f e c t was les s marked. There was a s i g n i f i c a n t increase i n free mucus (p < 0.05) but the o v e r a l l increase was not s i g n i f i c a n t . 15M given intravenously caused the l a r g e s t increase i n free mucus (p < 0.01) and consequently i n the t o t a l mucus produced (p < 0.01) but was without e f f e c t on b a r r i e r mucus. Given t o p i c a l l y (Table I) i t caused a reduction i n b a r r i e r mucus (p < 0.01) and a corresponding increase i n free mucus (p < 0.01) but without a s i g n i f i c a n t increase i n the t o t a l amount. The a l t e r a t i o n i n the volume and acid output produced by the three agents i s shown i n the Tables. Four m i l l i l i t r e s was the volume of t e s t s o l u t i o n i n s t i l l e d i n t o the stomach at the onset of the experiments. Topical a p p l i c a t i o n of the prostaglandins caused an increase i n volume of between 1 and 3 ml., being greatest with PGE 0 and l e a s t with 16DM. EXPERIMENT CONTROL 15M 10 yg/ml 16DM 10 yg/ml PGE 100 yg/ml NO. 10 10 10 VOLUME i n ml. 4.05 ±0.03 5.45** ±0.27 5.07** ±0.08 6.82** ±0.41 ACID OUTPUT yeq H /3 Hr 70.4 ±10.2 23.1** ±5.9 20.4** ±3.2 12.7** ±5.82 ALCIAN BLUE BOUND (in mg.) MUCOSA CONTENTS TOTAL 0.80 ±0.06 0.45** ±0.08 0.67 ±0.13 0.71 ±0.19 0.57 ±0.09 1.44** ±0.19 1.66** ±0.30 2.16** ±0.505 1.37 ±0.11 1.89 ±0.25 2.33* ±0.35 2.88* ±0.55 ** = p < 0.01 TABLE I: E f f e c t of t o p i c a l E 2 prostaglandins on A l c i a n Blue binding, volume secreted and acid output i n the closed r a t stomach over 3 hours (Mean ± S.E.). EXPERIMENT CONTROL 15M 0.3 yg/Kg/Hr 16DM 0.3 yg/Kg/Hr PGE 2 0.3 mg/Kg/Hr NO. 4 VOLUME i n ml. 3.88 ±0.12 4.33 ±0.02 4.04 ±0.02 4.30 ±0.05 ACID OUTPUT yeq H /3 Hr 40.75 ±7.78 82.4* ±13.2 45.1 ±7.7 36.1 ±2.07 ALCIAN BLUE BOUND (in mg.) MUCOSA CONTENTS TOTAL 0.71 ±0.04 0.98 ±0.03 0.61 ±0.05 0.97 ±0.12 1.60 ±0.18 4.72** ±0.11 2.82* ±0.46 3.74** ±0.47 2.31 ±0.22 5.82* ±0.04 3.42 ±0.49 4.70* ±0.54 * = p < 0.05 p < 0.01 TABLE I I : E f f e c t of intravenous E prostaglandins on A l c i a n Blue binding, volume secreted and acid output i n the closed r a t stomach over 3 hours (Mean ± S.E.). 33 A l l these increases were highly s i g n i f i c a n t (p < 0.01). Intravenous administration however di d not cause a s i g n i f i c a n t increase i n the volume of f l u i d i n the stomach. Topical a p p l i c a t i o n of each prostaglandin caused a highly s i g n i f i c a n t (p < 0.01) reduction i n a c i d output. When given i n t r a venously 16DM and PGE 2 had not e f f e c t on acid output, but 15M caused an increase i n acid output that j u s t reached s i g n i f i c a n c e (p < 0.05). Table III shows the r e s u l t s of repeated subcutaneous i n j e c t i o n s of histamine. Histamine produced a s i g n i f i c a n t increase (p < 0.05) i n volume secreted, but the acid output was v a r i a b l e between the i n d i v i d u a l animals at t h i s dose and the increase i n acid output was not s i g n i f i c a n t . There was a s i g n i f i c a n t (p < 0.05) reduction i n A l c i a n Blue bound to the mucosa, but no a l t e r a t i o n i n that bound to the g a s t r i c contents, or the t o t a l amount bound. Ulcers d i d not develop i n any of the stomachs. Table IV shows the degree of binding of A l c i a n Blue that occurred i n r a t plasma. In two specimens of pooled plasma 0.14 mg. A l c i a n Blue was bound per ml. of plasma. EXPERIMENT CONTROL HISTAMINE S.C. 6 mg/Kg/Hr ALCIAN BLUE BOUND (in mg.) VOLUME ACID OUTPUT NO. i n ml. yeq H /3 Hr MUCOSA CONTENTS TOTAL 5 4.14 147.34 0.51 0.45 0.96 ±0.01 ±23.38 ±0.11 ±0.06 ±0.16 5 4.80* 246.84 0.26* 0.49 . 0.75 ±0.34 ±95.03 ±0.04 ±0.06 ±0.09 p < 0.05 TABLE I I I : E f f e c t of subcutaneous histamine on A l c i a n Blue binding, volume secreted and acid output i n the closed r a t stomach over 3 hours (Mean ± S.E.). Absorbance mg AB/5 ml. Correction f o r D i l u t i o n mg. AB ppt. PLASMA A 1.0 0.75 1.50 PLASMA B 1.0 0.75 1.50 BLANK 1.1 0.82 1.64 0.14 0.14 TABLE IV: A l c i a n Blue binding by r a t plasma. 36 A STUDY OF THE EFFECT OF THE E PROSTAGLANDINS ON NONPARIETAL CELL SECRETION IN CANINE HEIDENHAIN POUCHES (EXPERIMENT 2) Material and Methods (i) Preparation of the Animals; The study was c a r r i e d out i n a t o t a l of six female mongrel dogs weighing approximately 20 Kg., four dogs being used i n each set of experiments. Each dog underwent a laparotomy at which an antrectomy and gastroduodenal anastomosis were performed and a Heidenhain pouch prepared. The pouch opened to the surface through a wide bore metal cannula. The dogs were allowed four weeks to recover from surgery before any t e s t s were performed. ( i i ) C h a r a c t e r i s t i c s of the Model: In t h i s model the Heidenhain pouch should not secrete acid except i n response to an exogenous stimulus. Each dog was tested to demonstrate f i r s t l y that the pouch could respond to an intramuscular dose of pentagastrin by a c i d secretion, and secondly that following three hours perfusion with normal s a l i n e no acid was secreted into the perfusate. ( i i i ) The Perfusion System: Perfusion studies were performed using a system i l l u s t r a t e d schematically i n Figure 5. The pouches f i l l e d by g r a v i t y i n f u s i o n from a r e s e r v o i r set at the height of the pouch, to avoid di s t e n s i o n . The perfusate entered at the bottom and l e f t from the top and thereby attained maximum contact with the mucosa. The p e r i s t a l t i c pump was placed on the withdrawal l i n e to return the 37 FIGURE 5: I l l u s t r a t e s the perfusion system used i n the canine Heidenhain pouches. 38 f l u i d to the r e s e r v o i r and a sampling tap allowed regular samples to be taken. P r i o r to each experiment the dogs were fasted for 18 hours and during the experiment stood comfortably supported on a Pavlov t a b l e . The pouches were perfused for three hours with 60 ml. of a so l u t i o n that contained 5 mEq/L lithium, 5 gm/L poly ethylene g l y c o l (PEG) as a volume marker and was made isosmotic at 300 mOsm/L with NaCl. Samples (3.5 ml.) were taken at 30 minute + + + - -i n t e r v a l s and were analysed f o r Na , L i , K , CI , HCO^ and PEG. (iv) Analysis 1) Sodium, Lithium and. Potassium: These ions were analysed by flame photometry using the Corning Flame Photometer. 2) Chloride: This ion was measured on a Corning c h l o r i d e meter. 3) Bicarbonate: This was measured by the method of back t i t r a t i o n . One m i l l i l i t e r of the sample was added to 0.5 ml. of 0.1 N HCl, heated to drive o f f the CO^, d i l u t e d with 10 ml. d i s t i l l e d water and t i t r a t e d against 0.1 N sodium hydroxide using an automatic t i t r i m e t e r (Radiometer Copenhagen). 4) PEG: The PEG concentration was measured by the method of 58 Malawer and Powell which i s a modification of the turbimetric method of analysis. The turbimetric method i s based on the creation of an oil - i n - w a t e r emulsion of the water soluble PEG when t r i c h l o r o a c e t i c a c i d (TCA) i s added. In t h i s method - 59 described by Hyden the emulsion lacks s t a b i l i t y and the plateau of maximum t u r b i d i t y i s short l i v e d so that precise timing i s required from the addition of the TCA reagent u n t i l 39 the o p t i c a l density i s read. The i n s t a b i l i t y i n Hyden's method r e s u l t s from the f a c t that i n the concentration used the droplets coalesce. Coalescence can be prevented by the addition of a emulsifying agent which can enter the oil-water i n t e r f a c e and produce a f i l m around the droplets. Gum arabic i s such an agent. The addition of gum arabic i s the basis of Malawer and Powell's modification and gives a stable and prolonged peak of maximum t u r b i d i t y which makes precise timing unnecessary and provides a l i n e a r PEG concentration - o p t i c a l density r e l a t i o n s h i p over the range of PEG concentration used i n t h i s experiment which was 500-300 mg/100 ml (Figure 6). Group I: The E f f e c t of To p i c a l A p p l i c a t i o n of the Prostaglandins: In the contr o l studies the pouches were perfused with the lithium/saline/PEG s o l u t i o n alone. In the experimental studies the prostaglandins were added to the perfusate i n the following concentrations: 15M and 16DM, 10 yg/ml; PGE 2, 100 yg/ml. Group I I : The. E f f e c t of Intravenous Administration of Prostaglandins: In these studies the pouches were perfused e x c l u s i v e l y with the lithium/saline/PEG s o l u t i o n . In the co n t r o l studies 25 ml. normal s a l i n e was given intravenously each hour. In the experimental studies the prostaglandins were administered i n 75 ml. normal s a l i n e at a rate of 0.6 yg/Kg/hour for 15M and 16DM and 0.6 mg/Kg/hour for PGE 2. These concentrations and i n f u s i o n rates correspond to those used i n previous studies of the permeability e f f e c t s . The con t r o l experiments and each of the prostaglandin experiments were performed twice i n each dog. 0.5 r 1 I _ L _ 1 I I I 1 0 100 200 300 400 500 600 700 800 PEG mg/100 ml FIGURE 6: A representative standard curve for polyethylene g l y c o l . 41 C a l c u l a t i o n of Data: Net ion fluxes (NIF) and permeability factors (K) were calculated f o r each ion from the following equations: Vo = V i (PEGi)/(PEGo) VI = (Vo - SV)(PEGo/PEGi) Vj = ( V j - i - SV) (PEGj/PEGj-1) NIF = VjCj - (Vj-1 - SV) Cj-1 where V = volume of perfusate C = concentration of ion PEG = concentration of PEG SV = sample volume i = i n s t i l l e d s o l u t i o n o = zero time s o l u t i o n . For each ion s i x 30-minute f l u x determinations were made, and the NIF for the experiment was taken as the mean of the l a s t f i v e values. Permeability factors f o r each ion were calculated from the formula: K (ml/30 minute) = n e t i o n f l u X ^ e & 3 0 "»i™tes) 1/2(Cj - Cj-1) (yeq/ml) Six permeability factors were c a l c u l a t e d for each ion corresponding to the six 30-minute periods. The permeability f a c t o r for the experiment was taken 55 as the mean of the l a s t f i v e values. For each concentration or dose of each prostaglandins eight sets of NIF and K values were a v a i l a b l e f o r each i o n . The r e s u l t s are expressed as the mean and SE of these eight values. 42 S t a t i s t i c a l Analysis: The s t a t i s t i c a l s i g n i f i c a n c e of any diffe r e n c e between the co n t r o l and experimental values was assessed using Student's t - t e s t f or paired values. 43 Results The volume secreted and the bicarbonate output for the t o p i c a l a p p l i cation of the prostaglandins i s shown i n Table V and for intravenous administration i n Table VI. When given by e i t h e r route 16DM caused a highly s i g n i f i c a n t (p < 0.005) increase i n the volume secreted over the three hour period, i n each case being almost double the amount produced i n the control experiments. 15M and PGE^ given by e i t h e r route did not cause a s i g n i f i c a n t a l t e r a t i o n i n the volume of f l u i d produced. In both groups the increase i n f l u i d produced by 16DM was associated with a highly s i g n i f i c a n t (p < 0.005) increase i n bicarbonate secretion over the three hour period. When applied t o p i c a l l y 15M caused a highly s i g n i f i c a n t (p < 0.01) increase i n bicarbonate output but had no e f f e c t when given intravenously. PGE^ had no e f f e c t on bicarbonate secretion when given by ei t h e r route. The e f f e c t of t o p i c a l a p p l i c a t i o n of the three prostaglandins on the four ions measured i s shown i n Tables VII and VIII, Table VII showing the net ion fl u x (NIF) for each ion and Table VIII the permeability factor (K). 16DM caused a highly s i g n i f i c a n t increase (p < 0.0005) i n the NIF of Na + and CI but did not a l t e r the L i + f l u x . These a l t e r a t i o n s i n NIF are also r e f l e c t e d i n the changes i n the permeability factors of these ions and the permeability factor for K + was also s i g n i f i c a n t l y increased (p < 0.05). 15M and PGE^ were without e f f e c t on NIF and permeability factors of a l l the ions. NO. VOLUME PRODUCED ml/3 hours HCO yEq/3 hours CONTROL 18.16 ±3.31 121.99 ±31.96 15M 10 yg/ml 18.42 ±1.39 231.15* ±23.93 16DM 10 yg/ml 37.68** ±3.42 483.37** ±110.99 PGE 100 yg/ml 22.30 ±2.65 55.95 ±22.79 = p < 0.01 p < 0.005 TABLE V: E f f e c t of t o p i c a l E prostaglandins on volume and bicarbonate secretion from Heidenhain pouch (Mean ± S.E.). NO. VOLUME PRODUCED ml/3 hours HCO uEq/3 hours CONTROL 13.43 ±2.01 65.91 ±18.52 15M 6.6 yg/Kg/Hr 11.91 ±1.01 30.13 ±10.11 16DM 0.6 yg/Kg/Hr 24.47**' ±1.60 317.78** ±66.27 PGE2 0.6 mg/Kg/Hr 12.10 ±1.22 62.37 ±31.88 ** = p < 0.005 TABLE VI: E f f e c t of intravenous E prostaglandins on volume and bicarbonate secretion from Heidenhain pouch (Mean ± S.E.) 46 NO. CONTROL 8 15M 8 10 yg/ml 16DM 8 10 yg/ml PGE 2 8 100 yg/ml • + C l L i 119.60 -3.22 ±56.30 ±0.80 125.50 -1.46 ±29.84 ±1.08 553.28** -0.90 ±87.38 ±1.56 236.48 -3.14 ±82.85 ±1.15 + + Na K 161.74 26.94 ±58.05 ±2.47 229.79 21.09 ±28.77 ±2.79 666.42** 33.48 ±87.57 ±3.27 267.76 30.72 ±54.22 ±6.37 ** = p < 0.0005 TABLE VII: E f f e c t of t o p i c a l E 2 prostaglandins on net ion (yeq/30 minutes) from Heidenhain pouches (Mean fluxes ± S.E.) NO. C l .+ L l + Na + K CONTROL 8 0.80 -0.82 1.08 14.88 ±0.38 ±0.17 ±0.38 ±1.73 15M 8 0.85 -0.40 1.65 15.86 10 yg/ml ±0.17 ±0.28 ±0.20 ±0.80 16DM 8 4.02** -0.49 4.76** 19.06* 10 yg/ml ±0.64 ±0.53 ±0.61 ±1.36 PGE 8 1.50 -0.93 1.86 18.53 100 yg/ml ±0.50 ±0.34 ±0.37 ±1.84 * = p < 0.05 ** = p < 0.0005 TABLE VIII: E f f e c t of t o p i c a l E 2 prostaglandin on permeability factors K (ml/minute) (Mean ± S.E.). 48 The e f f e c t of intravenous administration of the prostaglandins i s shown i n Tables IX and X, Table IX showing the NIF for each ion and Table X the K f a c t o r s . The e f f e c t of 16DM on Na + and C l i s s i m i l a r intravenously as when applied t o p i c a l l y causing a h i g l y s i g n i f i c a n t (p < 0.0005) increase i n NIF, and i n addition a s i g n i f i c a n t (p < 0.05) increase i n NIF of K +. However the NIF of L i + was s i g n i f i c a n t l y (p < 0.05) reduced. 15M caused a reduction i n the NIF of Na + and C l , there being a net loss of these ions from the pouch during the perfusion period. The reduction i n Na + f l u x was s i g n i f i c a n t (p < 0.05). 15M d i d not a f f e c t the L i + or K + fluxes. Prostaglandin was without e f f e c t on the NIF of a l l the ions. S i m i l a r a l t e r a t i o n s were produced i n the permeability f a c t o r s . 16DM caused a highly s i g n i f i c a n t (p < 0.0005) increase i n K + and K - and ^ 1 ^ * Na C l a s i g n i f i c a n t (p < 0.05) reduction i n K .+. The increase i n K + was not La K s i g n i f i c a n t . With 15M the reductions i n K + and K - were s i g n i f i c a n t 3 Na C l (p < 0.05), but 15M was without e f f e c t on K .+ and K+. PGE was without L i JC 2 e f f e c t on the permeability f a c t o r s . NO. CONTROL 8 15M 8 0.6 yg/Kg/Hr 16DM 8 0.6 yg/Kg/Hr PGE 8 0.6 mg/Kg/Hr C l L i + 31.74 -5.25 ±34.55 ±1.02 -31.78 -4.85 ±19.12 ±0.86 363.21** -2.48* ±41.28 ±0.74 39.66 -4.66 ±28.46 ±0.88 + + Na K 49.91 17.16 ±37.20 ±1.61 -40.35* 17.34 ±18.03 ±3.76 421.85** 24.98* ±66.79 ±1.85 30.73 20.34 ±27.80 ±2.09 * = p < 0.05 ** = p < 0.0005 TABLE IX: E f f e c t of intravenous E^ prostaglandins on net (yeq/30 minutes) from Heidenhain pouches (Mean ion fluxes ± S.E.) NO. C l L i + Na + K + CONTROL 8 0.23 -1.39 . 0.32 14.76 ±0.24 ±0.28 ±0.26 ±1.21 15M 8 -0.22* -1.30 -0.29* 13.11 0.6 yg/Kg/Hr ±0.10 ±0.22 ±0.10 ±2.38 16DM 8 2.55** -0.73* 3.02** 17.27 0.6 yg/Kg/Hr ±0.28 ±0.17 ±0.43 ±1.70 PGE 2 8 0.28 -1.19 0.22 16.29 0.6 yg/Kg/Hr ±0.20 ±0.22 ±0.17 ±1.03 * = p < 0.05 ** = p < 0.0005 TABLE X: E f f e c t of intravenous E prostaglandins on permeability factors K (ml/minute) (Mean ± S.E.) 51 THE EFFECT OF E 2 PROSTAGLANDINS ON THE REVERSAL OF ESTABLISHED GASTRIC MUCOSAL BARRIER DAMAGE (EXPERIMENT 3) 1. Preparation and V a l i d a t i o n of a Suitable Model Material and Methods: The experiments were c a r r i e d out on f i v e antrec tomized dogs with Heidenhain pouches. The perfusion system used was as described and i l l u s t r a t e d i n the previous chapter but various modifications i n the method were introduced. The three hour perfusion period was divided i n t o three separate one hour periods, samples were taken at 10 minute i n t e r v a l s and the perfusing s o l u t i o n was changed at the end of each hour. In t h i s way i t was possible to c a l c u l a t e the permeability f a c t o r f o r each hour. The r e s u l t s f o r the f i r s t two hours were averaged. Control experiments were performed i n which the pouches were perfused f or each of the three one hour periods with an a c i d / s a l i n e s o l u t i o n containing 120 mEq/L hydrogen ion, 5 gm/L PEG as a volume marker and made isosmotic at 300 mOsm/L with sodium c h l o r i d e . In subsequent experiments damage of the g a s t r i c mucosal b a r r i e r was produced by perfusing the pouches during the f i r s t two hours (Damage Period) with acid s a l i n e solutions containing 20 mM a s p i r i n . During the t h i r d hour (Recovery Period) only an a c i d / s a l i n e perfusate was used. (Figure 7) THE EXPERIMENTAL MODEL FIRST TWO HOURS POUCH PERFUSED WITH (DAMAGE PERIOD) HCl AND ASA THIRD HOUR POUCH PERFUSED WITH (RECOVERY PERIOD) HCl ALONE FIGURE 7: I l l u s t r a t i o n of the experimental model used to tes t the e f f e c t of various drugs on established g a s t r i c mucosal b a r r i e r damage. 53 Analysis: The following analyses were c a r r i e d out on each 10-minute sample, by the methods previously described. 1) Hydrogen Ion 2) Sodium and Potassium 3) Chloride 4) Polyethylene G l y c o l C a l c u l a t i o n of Data: The r e s u l t s are expressed as permeability factors (K) f o r each ion which were calculated from each experiment from the regression l i n e of the log of the concentration of the ion against time. The slope of the regression l i n e i s m u l t i p l i e d by the average volume of the perfusate to give the K values. Results: The addition of 20 mM a s p i r i n to the perfusate caused a s i g n i f i c a n t (p < 0.005) increase i n K + and K + during the Damage H Na Period, i n d i c a t i n g damage to the g a s t r i c mucosal b a r r i e r (Table XI). In the Recovery Period when only an acid s a l i n e perfusate was used the pouches that had been previously perfused with the a c i d / s a l i n e and a s p i r i n exhibited evidence of continuing g a s t r i c mucosal b a r r i e r damage as both K + and K + remained s i g n i f i c a n t l y (p < 0.05) elevated above H Na the control l e v e l s . (Table 11) In the pouches exposed to a s p i r i n there was no s i g n i f i c a n t d i f f e r e n c e between the K + and K + values i n the H Na Damage and Recovery Periods. Conclusions: This model provides a period of one hour i n which g a s t r i c mucosal b a r r i e r damage p e r s i s t s despite withdrawal of the damaging agent. In subsequent experiments t h i s model was used. DAMAGE PERIOD NIF- ueq/minute K EXPERIMENT CONTROL DAMAGE PERFUSING SOLUTION NO ACID SALINE 20 ACID SALINE 20 + 20 mM ASA Na 14.39 ±1.47 H -2.49 ±1.44 22.50** -16.64** ±2.50 ±2.54 Na 0.30 ±0.03 H -0.13 ±0.01 0.42** -0.29** ±0.30 ±0.04 RECOVERY PERIOD CONTROL DAMAGE ACID SALINE 10 ACID SALINE 10 14.27 ±2.08 20.30 ±3.28 -0.56 ±2.53 -9.20* ±3.51 0.27 ±0.03 0.41* ±0.05 -0.12 ±0.02 -0.20* ±0.04 = p < 0.05 p < 0.005 TABLE XI: E f f e c t of 20 mM a s p i r i n during the Damage Period on net ion f l u x _£NIF peq/minute) and permeability factor (K) f o r Na and H (Mean ± S.E.). 55 2. Study of the E f f e c t s of PGE 2, 15M and Metiamide on Established Gastric Mucosal B a r r i e r Damage Material and Methods: Using the model j u s t described and val i d a t e d experiments were performed to study the e f f e c t of t o p i c a l PGE 2 and 15M and intravenous pGE 2' 15M and metiamide. Group I: The E f f e c t of Topical Applications of the Prostaglandins: PGE 2 and 15M were added to the acid s a l i n e perfusate during the Recovery Period, PGE^ i n a concentration of 100 yg/ml and 15M i n a concentration of 10 yg/ml. Group I I : The E f f e c t of Intravenous Administration of the Prostaglandins: PGE^ and 15M were given by intravenous i n f u s i o n i n 25 ml. normal s a l i n e during the Recovery Period - PGE^ at a rate of 0.3 mg/Kg/hour, 15M at a rate of 0.6 yg/Kg/hour. Group I I I : The E f f e c t of Intravenous Administration of Metiamide: Metiamide was given at a rate of 3 mg/Kg/hour. This dose of metiamide i s above the ED for the i n h i b i t i o n of stimulated acid secretion i n a canine 50 Heidenhain pouch^^ and i s comparable i n i t s antisecretory e f f e c t with the doses of the prostaglandins used. 56 Results In Table XII the amount of the damage produced during the Damage Period i n a l l the experiments i s shown i n terms of permeability factors K and K and the net ion flux of H + and Na +. H Na The experiments i n which the e f f e c t of t o p i c a l 15M was tested K +, K +, and H + flux i n the Damage Period were less than those i n the H Na v a l i d a t i o n experiments, i n d i c a t i n g that the degree of damage was of a less e r degree. In the experiments i n which the agents were given intravenously the H + f l u x was s i g n i f i c a n t l y (p < 0.005) reduced i n the 15M experiment and the Na + flux (p < 0.05) i n the metiamide experiment, but there was no dif f e r e n c e i n the permeability f a c t o r s , and i t i s therefore considered that the amount of damage c a r r i e d over i n t o the Recovery Period was comparable i n a l l the experimental groups, except that f o r t o p i c a l 15M. Table XII shows the e f f e c t of t o p i c a l PGE and 15M on K + and 2. H K N a + values and NIF during the Recovery Period. There was no s i g n i f i cant d i f f e r e n c e between the K values or the NIF when no treatment was given and when PGE,, was applied t o p i c a l l y . T opical 15M d i d not cause a s i g n i f i c a n t reduction i n K + or the Na + f l u x , but K + was reduced H ' Na (p < 0.005) as was the H + f l u x (p < 0.05). However as there was no differen c e between the K + or H + f l u x values i n the Damage and Na Recovery Period f o r the t o p i c a l 15M experiments, the low values i n the Recovery Period can not be taken to ind i c a t e any re v e r s a l of damage. DAMAGE PERIOD NIF ueq/minute K EXPERIMENT NO. Na + H + Na + H + No Treatment 20 22.50 -16.64 0.42 -0.29 ±2.50 ±2.54 ±0.03 ±0.04 Top. PGE 2 18 19.28 -11.35 0.35 -0.25 ±1.85 ±1.83 ±0.04 ±0.01 Top. 15M 16 17.08 -7.17**. 0.24** -0.21* ±2.49 ±1.86 ±0.03 ±0.02 I.V. PGE 2 24 21.20 -13.44 0.43 -0.27 ±1.62 ±2.72 ±0.01 ±0.03 I.V. 15M 18 20.44 -4.63** 0.35 -0.23 ±2.42 ±3.53 ±0.05 ±0.03 I.V. Metiamide 12 17.09* -11.53 0.36 -0.22 ±1.60 ±1.80 ±0.03 ±0.02 p < 0.05 ** = p < 0.005 TABLE XII: Comparison of the a l t e r a t i o n i n NIF (yeq/minute) and K occurring i n the Damage Period i n a l l the groups i n which a s p i r i n damage was produced (Mean ± S.E.). RECOVERY PERIOD PERFUSING SOLUTION DAMAGE RECOVERY Acid Saline Acid Saline + ASA 20 mM Acid Saline Acid Saline + ASA 20 mM Acid Saline Acid Saline + ASA 20 mM TREATMENT DURING R.P. N i l Top. PGE„ 100 yg/ml Top. 15M 10 yg/ml NO. 10 10 NIF yeq/minute Na 20.30 ±3.28 23.80 ±3.69 17.68 ±2.95 H -9.20 ±3.51 -3. ±1. 41 86 -0.39* ±3.48 Na 0.41 ±0.05 0.34 ±0.06 0.22** ±0.03 K H -0.21 ±0.04 -0.22 ±0.03 -0.17 ±0.03 * = p < 0.05 p < 0.005 TABLE XIII E f f e c t of treatment with t o p i c a l PGE and 15M on est a b l i s h e d GMB damage NIF and K (Mean ± S.E.). 59 Table XIV shows the e f f e c t of the three intravenously administered agents on K + and K + i n the Recovery Period. PGE caused a highly ri Ncl 2 s i g n i f i c a n t (p < 0.005) reduction i n K + and K + during t h i s period and H Na 15M a s i g n i f i c a n t reduction (p < 0.025). Metiamide on the other hand did not a l t e r the permeability fa c t o r s , there being no s i g n i f i c a n t d i f f e r e n c e between K + and K + when treatment with metiamide was compared with no H Na treatment. The data f o r the NIF of Na + supports the permeability f a c t o r data, there being a s i g n i f i c a n t (p < 0.025) reduction with PGE^ and 15M but no change with metiamide. The NIF of H + was reduced by PGE 2 and 15M but these values d i d not quite reach s i g n i f i c a n c e . Metiamide d i d not a l t e r the NIF of H +. PERFUSING SOLUTION DAMAGE RECOVERY TREATMENT DURING R.P. NO. RECOVERY PERIOD NIF yeq/minute Na H + Na K H Acid Saline Acid Saline + ASA 20 mM N i l 10 20.30 ±3.28 -9.20 ±3.51 0.41 ±0.05 -0.21 ±0.04 Acid Saline Acid Saline + ASA 20mM I.V. PGE 0.3 mg/Kg/Hr 12 10.52* ±1.58 -2.14 ±1.88 0.23** ±0.04 -0.10** ±0.01 Acid Saline Acid Saline + ASA 20 mM I.V. 15M 0.6 yg/Kg/Hr 12.13* ±1.98 -1.23 ±3.90 0.23* ±0.05 -0.10* ±0.03 Acid Saline Acid Saline IV Metiamide 6 + ASA 20 mM 3 mg/Kg/Hr 16.95 ±1.75 -5.97 ±1.39 0.37 ±0.03 -0.17 ±0.02 * = p < 0.025 ** = p < 0.005 TABLE XIV: E f f e c t of treatment with intravenous PGE 2, 15M and Metiamide on esta b l i s h e d GMB damage. NIF and K (Mean ± S.E. o 61 DISCUSSION The prostaglandins have been shown capable of preventing damage to the g a s t r i c mucosa by a mechanism which i s unknown but which i s not r e l a t e d to t h e i r action as i n h i b i t o r s of a c i d secretion. Because of the potency of the E^ prostaglandins as i n h i b i t o r s of acid secretion, i t was not u n t i l these agents were studied i n basal pouch prepara tions that i t was appreciated that they also had a secretory action on other c e l l s i n the g a s t r i c mucosa. These e f f e c t s on mucus and nonparietal c e l l secretion have been studied as both e f f e c t s could be regarded as p r o t e c t i v e i n nature and may play a p a r t i n the p r o t e c t i v e e f f e c t of the prostaglandins. This secretory action may also be responsible for the mis i n t e r p r e t a t i o n of some permeability data. Most previous studies have measured only one component of g a s t r i c mucus. The majority have measured free mucus, t h i s being the e a s i e s t to c o l l e c t and measure, but at l e a s t two studies have measured b a r r i e r mucus. In t h i s study an attempt was made to measure the e f f e c t of the prostaglandins i n both f r a c t i o n s . The r e s u l t s i n d i c a t e that when given t o p i c a l l y and intravenously the prostaglandins cause an increase i n the free f r a c t i o n of mucus but are without e f f e c t on the b a r r i e r f r a c t i o n , but there was some v a r i a t i o n with the i n d i v i d u a l agents. Prostaglandin E^ produced the same e f f e c t s both t o p i c a l l y and intravenously being without e f f e c t on the b a r r i e r mucus but causing an increase i n the free f r a c t i o n and an o v e r a l l increase i n mucus 62 production. This same pattern was shown by t o p i c a l 16DM and intravenous 15M. Intravenous 16DM caused a s i m i l a r trend but the t o t a l amount of mucus produced was not s i g i n f i c a n t l y greater than the controls. The only r e s u l t s which d i d not f i t t h i s pattern were those with t o p i c a l 15M i n which there was no increase i n the t o t a l amount of mucus produced, but there was a s i g n i f i c a n t f a l l i n b a r r i e r mucus and a corresponding increase i n free mucus suggesting that t h i s agent stripped mucus from the mucosa so inc r e a s i n g the free f r a c t i o n without stimulating mucus production. An attempt was made to compare the stimulant e f f e c t of the prostaglandins with another stimulant of g a s t r i c mucus (histamine), but t h i s was not s a t i s f a c t o r y because of the d i f f e r e n t e f f e c t s of the stimulants on acid s e c r e t i o n . A combination of p y l o r i c l i g a t i o n and parenteral histamine i s ulcergenic i n the r a t , and i t was f e l t that i n the presence of muosal u l c e r a t i o n A l c i a n Blue would bind to the areas of mucosal destruction, so g i v i n g f a l s e l y high values for b a r r i e r mucus. Unfortunately the dose of histamine chosen d i d not produce a s i g n i f i c a n t increase i n acid output, but i t d i d not cause u l c e r s . Histamine caused a reduction i n b a r r i e r mucus but no a l t e r a t i o n i n free mucus or the t o t a l amount produced. The increase i n free mucus produced by the prostaglandins was greater than the e f f e c t produced by histamine but as the dose of histamine was not maximal - i t was not pos s i b l e to draw any conclusion regarding the magnitude of the response evoked by the prostaglandins. As dose-response studies with the prostaglandins were not performed the maximal mucus response to prostaglandin i s not known. Because the r o l e of g a s t r i c mucus i s unclear the i n t e r p r e t a t i o n of t h i s data i s d i f f i c u l t . G astric mucus i s f r e e l y permeable to hydrogen i on and has only minimal b u f f e r i n g capacity, and i t plays no part i n the 63 g a s t r i c mucosal b a r r i e r or i n the n e u t r a l i z a t i o n of i n t r a l u m i n a l a c i d . I t probably acts as a l u b r i c a n t on the g a s t r i c mucosa and prevents minor degrees of mechanical damage to the underlying c e l l s . I t i s b a r r i e r mucus therefore which i s probably the important f r a c t i o n while free mucus probably represents denatured and shed b a r r i e r muucs. If f r e s h l y secreted mucus f i r s t appears as b a r r i e r mucus adherent to the mucosa, and then as a r e s u l t of the action of aci d , pepsin and l o c a l enzymes i s broken down and shed as free mucus, one might expect an increase i n the b a r r i e r f r a c t i o n i n response to a stimulant of mucus secretion. If however the b a r r i e r f r a c t i o n stays more or less constant then an increase i n the free mucus f r a c t i o n would indic a t e that e x i s t i n g b a r r i e r mucus had been shed and replaced by f r e s h l y secreted mucus. The prostaglandins d i d not increase b a r r i e r mucus and a l l except 15M caused no change i n t h i s f r a c t i o n but an o v e r a l l increase i n mucus produc t i o n . The replacement of e x i s t i n g b a r r i e r mucus by f r e s h l y secreted mucus which would perform i t s function more e f f i c i e n t l y could be regarded as prot e c t i v e , at l e a s t as f a r as minor mechanical trauma i s concerned. The reason for the reduction i n b a r r i e r mucus caused by t o p i c a l 15M i s not clear and may represent an inaccuracy i n the method. The method of measurement used i n t h i s study i s probably le s s accurate than many of the biochemical methods of ana l y s i s , but has the advantage that both f r a c t i o n s can be measured. I t was chosen for t h i s reason and i t s s i m p l i c i t y , on the understanding that i f the prostaglandins were confirmed as stimulants of mucus production then other more so p h i s t i c a t e d techniques could be applied to the problem i n the future. 64 The other important f i n d i n g i n t h i s study was that when applied t o p i c a l l y to the mucosa a l l three prostaglandins caused an increase i n volume despite a f a l l i n a c i d output. In these experiments i t was impos s i b l e to measure bicarbonate secretion, but there was i n d i r e c t evidence that the f l u i d secreted was a l k a l i n e . P G E 2 applied t o p i c a l l y i s a poor i n h i b i t o r of a c i d secretion and yet i n a dose well below the ED i t caused 50 a greater reduction i n a c i d output than i t s more potent methyl analogues. It also caused the l a r g e s t volume increase and therefore the apparent reduction i n a c i d output and the unexpected potency could be due to neutra l i z a t i o n of the small amount of acid secreted i n these vagotomised r a t s by the a d d i t i o n a l secretion of an a l k a l i n e g a s t r i c j u i c e . There are at l e a s t three possible explanations f o r these observed e f f e c t s : damage to the g a s t r i c mucosal b a r r i e r , an increase i n mucosal blood flow or stimulation of nonparietal and mucus secr e t i n g c e l l s . Damage to the g a s t r i c mucosal b a r r i e r could cause an increase i n the f l u i d transudate i n t o the g a s t r i c lumen and an apparent reduction i n a c i d ouput due to back d i f f u s i o n . However of the prostaglandins studied only 16DM has been suspected of breaking the g a s t r i c mucosal b a r r i e r and other experiments i n t h i s t h e s i s refute t h i s suggestion. The e f f e c t of a l t e r a t i o n i n blood flow may be more important. The o r i g i n of nonparietal c e l l secretion remains uncertain. Is i t a plasma transudate containing Na +, HCO^ and p r o t e i n , 6 1 , 6 2 or do nonparietal c e l l s ~ 63 a c t i v e l y secrete HCO^ and mucus i n response to s p e c i f i c s t i m u l i ? I f the former were true an increase i n mucosal blood flow accompanied by an increase 65 i n hydrostatic pressure could lead to an increased transudation of f l u i d and protein. A l c i a n Blue has been shown not to bind s i g n i f i c a n t l y to plasma protein and therefore the measured increase i n A l c i a n Blue binding i n the g a s t r i c contents i s not s o l e l y due to an increase i n the plasma p r o t e i n i n the g a s t r i c lumen. Also i t i s to be expected that the vasoactive properites of those agents would be more apparent with intravenous rather than t o p i c a l administration. The increases i n mucus production following intravenous administration occured i n the absence of any volume changes and so must be independent of the secretion of a l k a l i n e j u i c e . This evidence strongly suggests that i n the r a t these agents stimulate mucus and nonparietal c e l l secretion. Further evidence that the increase i n f l u i d i n the r a t stomachs was due to nonparietal c e l l secretion was gained from the second s e r i e s of experiments, but these experiments, performed i n dogs, i n d i c a t e that there may be some species v a r i a t i o n i n response to the i n d i v i d u a l agents. In the dogs only 16DM stimulated nonparietal c e l l secretion but did so when given by both the t o p i c a l and intravenous routes. In the dog experiments the e f f e c t of the three prostaglandins on permeability and nonparietal c e l l secretion was studied i n the presence of a pouch perfusate that was a c i d free. In t h i s system only 16DM caused an increase i n the f l u i d produced by the pouches, but t h i s amounted to almost double the c o n t r o l volume. Associated with t h i s was a s i g n i f i c a n t increase i n the amount of bicarbonate secreted and i n the i n f l u x of Na + and C l . Using t o p i c a l 16DM the t o t a l i n f l u x of cation i n yEq/30 minutes (Na + and K +) was approximately 700 yEq. The t o t a l i n f l u x of anion i n yEq/30 minutes was 66 553 yEq Cl plus one sixth of the bicarbonate output over the three hours, i . e . 80 yEq/30 minutes, a total of 633 yEq/30 minutes. Using intravenous 16DM the corresponding figures are for cation 445 yEq/30 minutes and for anion 416 yEq/30 minutes. Minor degrees of pouch distension could lead to the secretion of H + and the resulting neutralization of bicarbonate could account for the anion d e f i c i t . The evidence suggests that 16DM stimulates nonparietal c e l l secretion in the dog, the f l u i d produced being r ich i n Na + and C l and containing some bicarbonate. 15M and PGE^ both topical ly and intravenously had no effect on f l u i d production and PGE^ and intravenous 15M were without effect on bicarbonate secretion. Topical 15M however caused a s ignificant increase i n bicarbonate secretion. This result i s not readily explained but the other values may be underestimates as a result of distension and p a r t i a l neutralization. The effect of the prostaglandins on gastric mucosal permeability was + + measured by studying the movement of L i . The net efflux of L i from the 55 Heidenhain pouch of antrectomized dogs has been studied by Chung et a l in relation to the net efflux of hydrogen. In the presence of various barrier breakers i t was found that the ratio K .+/K + remained unchanged L l H and i t was concluded that K .+ is a useful indirect measure of mucosal L i permeability. The accuracy of the relationship K .+/K+ has been questioned L i H in human studies where a variety of uncontrolled factors may interfere , but in the closed perfusion system of a Heidenhain pouch the relationship i s val id and has been confirmed by extensive studies in this laboratory. 67 2 A l l three prostaglandins applied t o p i c a l l y and 15M and prostaglandin E given intravenously were without e f f e c t on net ion f l u x of L i + and K +. L i whereas the intravenous administration of 16DM caused a s i g n i f i c a n t reduction i n NIF of L i + and K +. This indicates that 16DM tightens the g a s t r i c mucosal b a r r i e r . This f i n d i n g i s contrary to that previously reported but i s more i n keeping with the other recorded e f f e c t s of 16DM. 48 O'Brien and Carter reported an increase i n permeability i n canine Heidenhain pouches exposed to 16DM by d i r e c t l y measuring the loss of H + from the pouch. They used a system of repeated i n s t i l l a t i o n s rather than the continuous perfusion system used i n these experiments. In t h e i r experi mental system each experiment consisted of s i x 30-minute periods. Periods 1 and 2 were controls and Period 3 the t e s t period; Periods 4, 5 and 6 were further c o n t r o l periods. The net ion f l u x of H + i n Period 3 i s compared with those i n Periods 1 and 2, and from t h e i r data (Table XV) i t can be seen that the NIF of H + i n Periods 1 and 2 p r i o r to the a p p l i c a t i o n of 16DM was abnormally low. Had the value been i n the same range as i n t h e i r c o n t r o l experiments there would have been no s i g n i f i c a n t d i f f e r e n c e . No signficance can be attached to the Na + f l u x data i n view of the stimulation of nonparietal c e l l s e c r e t i o n . Their claim that 16DM increases g a s t r i c mucosal permeability i s based on questionable data. However t h i s f i n d i n g did receive support from the work of Bolton and 49 Cohen, i n which they claimed that not only did t o p i c a l 16DM break the g a s t r i c mucosal b a r r i e r but the same e f f e c t was produced by continuous infusion of the agent. The method by which t h e i r data was c a l c u l a t e d d i d not allow for the p o s s i b i l i t y of active nonparietal c e l l s e c r e t i o n , and t h i s H BACK DIFFUSION (MEAN AND S.E.) NET Na OUTPUT (MEAN AND S.E.] EXPERIMENTS PERIODS 1 AND 2 PERIOD 3 P VALUE PERIODS 1 AND 2 PERIOD 2 P VALUE n = 12 n = 6 n = 1 2 n = 6 CONTROL (basal solution) -81 ± 46 -85 ± 57 0.47 190 ± 57 134 ± 41 0.40 16 DM 300 yg/20 ml i n pouch (basal solution) -13 ± 19 187 ± 76 >0.001 132 ± 61 781 ± 64 >0.001 TABLE XV: Comparision of i o n i c fluxes between c o n t r o l and t e s t periods (from O'Brien PE, Carter DC, Gut 16: 437-442, 1975. 69 has probably led to erroneous conclusions. Mucosal permeability (K) was determined by measuring the slope of the regression l i n e for the log of the concentration of each ion against time, and m u l t i p l y i n g t h i s by the average volume of the perfusate during the experiment. This method of c a l c u l a t i o n i s v a l i d only i f the average volumes i n the groups under comparison are the same. I f i n one group the volume increase i s much la r g e r then the concen t r a t i o n of the various ions w i l l change not only as a r e s u l t of movement across the mucosa but also due to a d i l u t i o n a l f a c t o r . This e f f e c t probably accounts for the f i n d i n g of Bolton and Cohen and t h e i r data i s c u r r e n t l y under re-evaluation by the method used i n t h i s study, which has already been described. In t h i s method NIF and K are c a l c u l a t e d for each 30 minute i n t e r v a l taking i n t o account the volume changes over that period. Two conclusions can be drawn from t h i s study. F i r s t l y that 16DM stimulates a nonparietal c e l l secretion containing Na +, C l " and HC0^~ and secondly that 16DM does not break the g a s t r i c mucosal b a r r i e r . The production of an increased amount of f l u i d by the pouches i n the absence of H + indicates that t h i s cannot be the r e s u l t of back d i f f u s i o n and damage, and therefore must came from some other source. The f a c t that the f l u i d contains bicarbonate suggests that i t i s the r e s u l t of nonparietal c e l l secretion and confirms what was suspected from the r a t studies, although there appears to be some species v a r i a t i o n . The L i + data confirms that 16DM does not break the b a r r i e r and i n f a c t suggests that i t may tighten i t . I t has been demonstrated that despite t h e i r a c i d i n h i b i t o r y action the E prostaglandins can stimulate c e r t a i n g a s t r i c mucosal c e l l s leading to 70 the production of mucus and nonparietal c e l l s ecretion. I t i s p o s s i b l e that these e f f e c t s contribute s i g n i f i c a n t l y to the p r o t e c t i v e action of the prostaglandins. Although the amount of HCO^ secretion i s small and not enough to cause s i g n i f i c a n t n e u t r a l i z a t i o n of acid, the exact r o l e of the mucus remains uncertain. The protective properties of the prostaglandins demonstrated by Robert were for stronger l o c a l i r r i t a n t s , a s i t u a t i o n where increased mucus production would be b e n e f i c i a l . The other importance of these two studies l i e s i n the demonstration that these agents, and i n p a r t i c u l a r 16DM, while i n h i b i t i n g a c i d secretion have other secretory e f f e c t s which have been masked by the potency of a c i d i n h i b i t i o n . While these e f f e c t s may not be important p h y s i o l o g i c a l l y or even pharmacologically, f a i l u r e to recognize them has l e d to m i s i n t e r p r e t a t i o n of the e f f e c t of 16DM on the g a s t r i c mucosal b a r r i e r . Although 16DM i s the most potent prostaglandin both as an i n h i b i t o r of a c i d secretion and as a p r o t e c t i v e agent, i t has been considered dangerous f o r c l i n i c a l evaluation because of i t s apparent damaging e f f e c t on the g a s t r i c mucosal b a r r i e r . On the basis of these studies t h i s view of 16DM would appear to be unwarranted and i t may soon become a v a i l a b l e for c l i n i c a l evaluation. In the t h i r d study the a b i l i t y of PGE 2 and 15M to reverse established b a r r i e r damage was studied and compared with the e f f e c t of metiamide. 16DM was not studied because at the time t h i s study was planned 16DM was considered a b a r r i e r breaker. The r e s u l t s have been calculated as NIF and permeability factors for Na + and H +. The c a l c u l a t i o n of (K) from the computer c a l c u l a t e d slope of 71 the regression l i n e f o r the log of the concentration of the ions i s more accurate than the net ion f l u x calculated from the f i r s t and l a s t values, as i t involves the use of a l i n e based on seven points. As there i s no s i g n i f i c a n t d i f f e r e n c e between the volumes of f l u i d produced by the pouches i n the f i v e treatment groups and the groups with which they are compared, (Table XVI) and act i v e secretion does not occur with the agents used, t h i s method of c a l c u l a t i o n i s considered v a l i d . The conclusions are based on the (K) r e s u l t s - the NIF data i s used for support. After demonstrating the v a l i d i t y of the model i t was necessary to compare the degree of damage i n the Damage Period i n a l l the groups as t h i s can be used as an i n d i c a t i o n of the degree of damage present i n the Recovery Period to which the various therapeutic agents are applied. and i n the Damage Period p r i o r to treatment with t o p i c a l 15M were s i g n i f i c a n t l y lower than i n the untreated group i n d i c a t i n g that the degree of damage produced was l e s s . In the other four treatment groups the degree of damage was comparable with the untreated groups. Topical PGE„ had no e f f e c t on K + and K + i n d i c a t i n g no e f f e c t on the 2 H Na damaged b a r r i e r . Topical 15M caused a s i g n i f i c a n t reduction i n + but as the degree of damage was i n s u f f i c i e n t no conclusion can be drawn. I t i s l i k e l y that t h i s agent had no e f f e c t when applied t o p i c a l l y , but further tests i n which comparable degrees of damage were f i r s t produced would be necessary to e s t a b l i s h t h i s conclusively. Given intravenously, both PGE 2 and 15M caused a s i g n i f i c a n t reduction i n K + and K + i n d i c a t i n g that i n the ^ Na H * presence of e x i s t i n g g a s t r i c mucosal damage these agents had a b e n e f i c i a l e f f e c t . The permeability factors measured during the recovery period on VOLUMES PRODUCED (ml/1 hour) EXPERIMENT DAMAGE PERIOD RECOVERY PERIOD CONTROL 13.28 + 0.96 13.18 + 1.62 ASA 13.63 + 1.28 13.37 + 1.83 TOP. PGE 2 15.22 + 0.86 17.72 + 1.77 TOP. 15M 16.85 + 1.46 18.40 + 2.92 I.V. PGE 2 14.30 + 0.89 11.20 + 0.67 I.V. 15M 16.45 + 1.23 13.10 + 0.94 I.V. METIAMIDE 12.99 + 0.85 13.94 + 1.65 TABLE XVI: Volumes produced by the pouches i n the various experimental groups (Mean ± S.E.) 73 treatment with the two agents were the same as i n the o r i g i n a l c o n t r o l experiments when the pouches were not exposed to a s p i r i n . Therefore i n the presence of e x i s t i n g g a s t r i c mucosal b a r r i e r damage these agents were able to restore g a s t r i c mucosal permeability to normal l e v e l s . This e f f e c t was not produced by metiamide. The f a i l u r e of metiamide to influence g a s t r i c mucosal permeability under these circumstances indicates that t h i s e f f e c t of the prostaglandins, which could be regarded as therapeutic rather than simply p r o t e c t i v e i s independent of a c i d i n h i b i t i o n . The a c i d i n h i b i t o r y p o t e n t i a l of the batch of metiamide used was confirmed (Appendix 1). A reduction i n mucosal blood flow could reduce i o n i c fluxes and account 64 65 for the findings i n the Recovery Period. However studies i n the dog ' show that the reduction i n mucosal blood flow associated with the i n h i b i t i o n of stimulated a c i d secretion by PGE^ i s the r e s u l t and not the cause of acid i n h i b i t i o n , and studies i n r a t s 6 6 i n d i c a t e that under basal conditions PGE^ has a vaso d i l a t o r e f f e c t on the g a s t r i c mucosa. The e f f e c t of 15M on basal blood flow has not been studied. There i s evidence that a s p i r i n 6 ^ and indomethacin 6^ can damage the sodium pump leading to the accumulation of i n t r a c e l l u l a r sodium and water. Such an e f f e c t could u l t i m a t e l y lead to c e l l l y s i s . 16DM has the opposite e f f e c t stimulating the sodium pump.6^ It i s pos s i b l e that an increase i n g a s t r i c mucosal permeability r e s u l t s from the interference with the normal homeostasis of mucosal c e l l s and that 74 in these experiments biochemical but not structural damage was induced by aspirin and then reversed by the infusion of PGE2 and 15M assuming these agents have the same cellular effects as 16DM. The rapid restoration of cellular homeostasis would return the mucosal epithelium to normal and lead to a return to normal permeability. If this explanation i s correct one would expect 16DM to have the same effect. Now that the confusion over the permeability effect of 16DM has been c l a r i f i e d this could be studied. Such a mechanism would explain why the protective action of the prosta glandins i s unrelated to acid secretory inhibition. The evidence provided here of the reversal of barrier damage in addition to the known acid inhibitory action of the prostaglandins could make them ideal therapeutic agents i n acute gastric mucosal lesions. It i s interesting to note that despite early . . 69 reports of the value of the receptor antagonists in erosive g a s t r i t i s , their value in stress induced upper gastrointestinal tract haemorrhage has 70 recently been questioned. 7 5 CONCLUSIONS The conclusions that can be drawn from the f i r s t experiment are that the three prostaglandins applied t o p i c a l l y to the r a t stomach stimulate a non p a r i e t a l c e l l s e c r e t i o n and 16DM and PGE 2 stimulate mucus production leading to an increase i n the free component of mucus, but have no e f f e c t on the b a r r i e r f r a c t i o n . Given intravenously a l l three agents cause an increase i n free mucus but without an increase on the b a r r i e r f r a c t i o n . Nonparietal c e l l secretion was not stimulated by intravenous administration. The second experiment indicated a species v a r i a t i o n i n the e f f e c t of the E 2 prostaglandins on nonparietal c e l l secretion. In the dogs 16DM both t o p i c a l l y and intravenously caused an increase i n the f l u i d secreted which contained sodium, chloride and bicarbonate. P G E 2 a n c * d i d not stimulate nonparietal c e l l bicarbonate secretion i n the dog. The f i n a l experiment demonstrates that the intravenous administration of PGE 2 and 15M can reverse established g a s t r i c mucosal b a r r i e r damage. This property i s not exhibited by t o p i c a l a p p l i c a t i o n of the prostaglandins, and i t i s not possessed by metiamide. 76 RATS 1 2 3 4 5 6 7 8 9 10 MEAN ± S .E . VOLUME in ml. 4.1 4.0 3.8 4.0 4.3 4.0 4.1 4.0 4.1 4.1 4.05 ±0 .03 ACID OUTPUT yeq H /3 Hr 107 68 76 68 60 24 131 88 41 41 ALCIAN BLUE BOUND MUCOSA CONTENTS 70.4 ± 1 0 . 2 0.70 0.81 0.92 0.70 0.92 1.17 0.88 0.44 0.88 0.59 0.80 ±0 .06 0.32 0.94 0.89 0.83 0.78 0.48 0.37 0.71 0.12 0.24 0.57 ± 0 . 0 9 (in mg.) TOTAL 1.02 1.75 1.81 1.53 1.70 1.65 1.25 1.15 1.00 0.83 1.37 ± 0 . 1 1 TABLE XVII: Experiment 1: Topical prostaglandins: Controls 77 RATS 1 2 3 4 5 6 7 8 9 10 MEAN ± S.E. VOLUME i n ml. 6.4 6.2 4.2 6.2 6.6 5.3 4.8 4.5 5.1 5.2 5.45 ±0.27 ACID OUTPUT yeq H /3 Hr 45.0 6.2 0 18.6 52.8 42.0 19.0 27.0 20.0 0 23.1 ±5.9 ALCIAN BLUE BOUND ( i n mg.) MUCOSA CONTENTS TOTAL 0.16 1.16 1.32 0.16 1.29 1.45 0.22 1.09 1.31 0.38 1.13 1.51 0.54 1.72 2.26 0.88 2.99 3.87 0.88 1.14 2.02 0.44 1.87 2.31 0.51 1.06 1.57 0.37 0.93 1.30 0.45 ±0.08 1.44 ±0.19 1.89 ±0.25 TABLE XVIII: Experiment 1: Topical prostaglandins: 15M 10 yg/ml 78 RATS 1 2 3 4 5 6 7 8 9 10 MEAN ± S.E. VOLUME i n ml. 5.4 5.2 5.0 5.4 5.3 5.0 4.8 5.1 4.8 4.6 5.07 ±0.08 ACID OUTPUT yeq H /3 Hr 5.4 16.0 8.0 38.0 16.0 20.0 24.0 20.0 34.0 23.0 20.44 ±3.22 ALCIAN BLUE BOUND (in mg.) MUCOSA CONTENTS TOTAL 1.35 0.14 1.49 0.49 1.79 2.28 0.81 1.95 2.76 1.25 3.36 4.61 1.03 2.75 3.78 0.37 1.19 1.56 0.37 1.14 1.51 0.37 1.66 2.03 0.44 0.57 1.01 0.22 2.05 2.67 0.67 ±0.13 1.66 ±0.30 2.33 ±0.35 TABLE XIX: Experiment 1: To p i c a l Prostaglandins: 16DM 10 yg/ml. 79 ALCIAN BLUE BOUND (in mg.) VOLUME ACID OUTPUT RATS i n ml. yeq H /3 Hr MUCOSA CONTENTS TOTAL 1 7.9 40.0 0.88 1.54 2.42 2 6.9 41.0 0.32 0.16 0.48 3 8.3 0 0.16 4.74 4.90 4 8.3 0 1.76 1.29 3.05 5 7.2 0 1.60 4.11 5.71 6 5.8 11.6 0.44 3.10 3.54 7 5.2 0 0.22 2.05 2.27 8 6.7 0 0.66 1.59 2.25 9 DIED DURING THE COURSE OF EXPERIMENT 10 5.0 22.0 0.44 0.89 1.33 MEAN 6.82 12.7 0.72 2.16 2.88 ± S.E. ±0.41 ±5.82 ±0.19 ±0.51 ±0.55 TABLE XX: Experiment 1: Topical Prostaglandins: PGE 100 yg/ml 80 ALCIAN BLUE BOUND (in mg.) VOLUME ACID OUTPUT RATS i n ml. yeq H /3 Hr MUCOSA CONTENTS TOTAL 1 4.0 64.0 0.60 1.28 1.88 2 DIED DURING COURSE OF EXPERIMENT 3 3.5 35.0 0.70 1.93 2.63 4 4.0 32.0 0.85 1.92 2.77 5 4.0 32.0 0.70 1.28 1.98 MEAN 3.88 40.75 0.71 1.60 2.31 ± S.E. ±0.12 ±7.78 ±0.04 ±0.18 ±0.22 TABLE XXI: Experiment 1: Intravenous Prostaglandins: Controls 81 ALCIAN BLUE BOUND (in mg.) VOLUME ACID OUTPUT RATS i n ml. yeq H /3 Hr MUCOSA CONTENTS TOTAL 1 4.4 70.4 1.05 4.62 5.67 2 4.3 112.0 1.00 4.82 5.82 3 INTRAVENOUS CANNULA DISLODGED 4 4.3 95.0 0.90 4.97 5.87 5 4.2 52.0 0.95 4.95 5.90 MEAN 4.22 82.4 0.98 4.72 5.82 ± S.E. +0.02 ±13.24 ±0.03 ±0.11 +0.04 TABLE XXII: Experiment 1: Intravenous Prostaglandins: 15M 0.6 yg/Kg/Hr. 82 ALCIAN BLUE BOUND (in mg.) VOLUME ACID OUTPUT RATS i n ml. yeq H /3 Hr MUCOSA CONTENTS TOTAL 1 4.1 16.4 0.57 1.48 2.05 2 4.0 56.0 0.70 3.83 4.53 3 4.1 45.1 0.44 2.06 2.50 4 4.0 60.0 0.60 3.74 4.34 5 4.0 48.0 0.72 2.97 3.69 MEAN 4.04 45.1 0.61 2.82 3.42 ± S.E. ±0.02 ±7.7 ±0.05 ±0.46 ±0.49 TABLE XXIII: Experiment 1: Intravenous Prostaglandins: 16DM 0.6 yg/Kg/Hr. 83 ALCIAN BLUE BOUND (in mg.) VOLUME ACID OUTPUT RATS i n ml. yeq 1^/3 Hr MUCOSA CONTENTS TOTAL 1 4.3 30.1 1.20 3.20 4.40 2 4.4 35.2 1.33 5.20 6.53 3 4.3 43.0 0.76 2.40 3.16 4 4.4 35.2 0.85 3.77 4.62 5 4.1 36.9 0.70 4.12 4.82 MEAN 4.3 36.1 0.97 3.74 4.70 ± S.E. ±0.05 ±2.07 ±0.12 ±0.47 ±0.54 TABLE XXIV: Experiment 1: Intravenous Prostaglandins: PGE 0.6 mg/Kg/Hr. 84 ALCIAN BLUE BOUND (in mg.) VOLUME ACID OUTPUT RATS i n ml. yeq H /3 Hr MUCOSA CONTENTS TOTAL 1 4.1 164.1 0.60 0.37 0.97 2 4.0 96.0 0.15 0.32 0.47 3 4.1 176.3 0.40 0.39 0.79 4 4.3 90.3 0.60 0.60 1.20 5 4.2 210.0 0.80 0.59 1.39 MEAN 4.14 147.34 0.51 0.45 0.96 ± S.E. ±0.01 ±23.38 ±0.11 ±0.06 ±0.16 TABLE XXV: Experiment 1: Subcutaneous Histamine: Controls. 85 ALCIAN BLUE BOUND (in mg.) VOLUME ACID OUTPUT RATS i n ml. yeq H /3 Hr MUCOSA CONTENTS TOTAL 6 5.2 343.2 0.30 0.40 0.70 7 4.3 116.1 0.40 0.69 1.09 8 4.3 64.5 0.20 0.56 0.76 9 4.2 134.4 0.20 0.42 0.62 10 6.0 576.0 0.20 0.36 0.56 MEAN 4.80 246.84 0.26 0.49 0.75 ± S.E. ±0.34 ±95.03 ±0.04 ±0.06 ±0.09 TABLE XXVI: Experiment 1: Subcutaneous Histamine: Histamine 6 mg/Kg/Hr. 86 EXPERIMENT BICARBONATE SECRETED yeq/3 Hr DOG 3 DOG 13 DOG 16 DOG 23 MEAN ± S.E. CONTROL 15M 10 ug/ml 16DM 10 yg/ml PGE 2 100 yg/ml 183.73 0 161.73 227.84 121.99 ± 31.96 0 70.73 212.4 119.28 224.0 167.25 247.73 111.64 231.15 ± 23.93 222.75 254.05 305.36 316.47 960.57 115.80 735.50 396.45 483.37 ± 110.99 821.25 332.65 330.98 173.78 178.59 0 55.89 118.32 0 65.07 29.71 0 55.95 ± 22.79 TABLE XXVII: Experiment 2: Topical Prostaglandin: Bicarbonate Secretion yeq/3 Hr. VOLUME PRODUCE (in ml/3 Hr) EXPERIMENT CONTROL 15M 10 yg/ml 16DM 10 yg/ml PGE 2 100 yg/ml DOG 3 DOG 13 38.09 20.56 13.84 6.98 20.50 20.25 14.00 10.69 38.39 22.40 55.75 31.03 23.04 7.88 24.03 20.17 DOG 16 DOG 23 18.41 21.46 11.65 14.32 19.55 20.32 20.02 22.04 38.05 43.79 38.05 34.01 23.91 35.31 20.39 23.69 MEAN ± S.E. 18.16 ± 3.31 18.42 ± 1.39 37.68 ± 3.42 22.30 ± 2.65 TABLE XXVIII: Experiment 2: Topical Prostaglandins: Volume Secreted ml/3 Hr. 88 .+ + + C l L i Na K DOG NIF K NIF K NIF K NIF K 13 157.48 0.98 0.21 0.02 262.68 1.70 30.56 11.37 -96.61 -0.61 -5.49 -1.30 -25.15 -0.16 18.45 13.05 49.53 0.31 -4.60 -1.05 83.58 0.59 29.48 19.79 16 55.22 0.34 -5.53 -1.53 57.34 0.38 17.24 10.11 149.52 0.98 -3.35 -9.83 176.74 1.14 33.50 20.19 9.59 0.07 -4.89 -1.22 9.42 0.007 20.50 8.13 23 203.09 1.31 -0.73 -0.19 262.14 1.69 33.70 15.94 3 435.67 3.09 -1.40 -0.46 467.22 3.30 32.09 20.51 MEAN 119.60 0.80 -3.22 -0.82 161.74 1.08 26.94 14.88 ± S.E. ±56.30 ±0.38 ±0.80 ±0.17 ±58.08 ±0.38 ±2.47 ±1.73 TABLE XXIX: Experiment 2: Topical Prostaglandins: Permeability Data: Controls. 89 C l L i Na K DOG NIF K NIF K NIF K NIF K 13 1.00 0.09 1.31 0.35 173.66 1.25 14.59 14.81 87.17 0.58 -2.03 -0.53 137.50 1.01 20.19 13.84 16 200.60 1.33 -1.41 -0.37 201.81 1.47 19.37 16.28 190.97 1.34 -0.88 -0.16 238.47 1.77 20.05 13.51 3 246.49 1.57 3.97 0.99 384.85 2.73 16.52 18.09 38.45 0.22 -2.44 -0.64 154.27 1.10 12.25 13.53 23 101.16 0.67 -5.33 -1.47 263.48 1.79 35.41 19.45 138.22 1.02 -4.94 -1.40 284.32 2.12 30.36 17.43 MEAN 125.50 0.85 -1.46 -0.40 229.79 1.65 21.09 15.80 ± S.E. ±29.84 +0.17 ±1.08 ±0.28 ±28.77 ±0.20 ±2.79 ±0.80 TABLE XXX: Experiment 2: Topical Prostaglandin: 15M 10 yg/ml Permeability Data: 90 - .+ + + Cl Lx Na K DOG NIF K NIF K NIF K NIF K 13 268.97 1.86 7.53 1.86 344.43 2.45 23.42 12.84 255.09 1.86 -1.57 -0.38 358.39 2.54 22.58 17.22 16 729.64 5.45 -6.63 -3.03 850.81 6.11 49.92 19.50 290.54 2.09 -3.65 -1.18 465.53 3.34 26.81 15.78 3 703.91 5.04 -4.05 -1.85 950.60 6.72 32.56 19.02 872.21 6.43 -2.17 -0.48 927.97 6.53 34.82 20.58 23 734.99 5.33 1.10 0.26 750.87 5.50 40.50 25.33 570.91 4.11 2.24 0.81 682.83 4.90 37.28 22.21 MEAN 553.28 4.02 -0.90 -0.49 666.42 4.76 33.48 19.06 ± S.E. ±87.38 ±0.64 ±1.56 ±0.53 ±87.57 ±0.61 ±3.27 ±1.36 TABLE XXXI: Experiment 2: Topical Prostaglandins: Permeability Data: 16DM 10 pg/ml. 91 + + + C l L i Na K DOG NIF K NIF K NIF K NIF K 13 238.91 1.49 -0.66 -0.23 263.25 1.76 35.57 18.97 -136.70 -0.86 -6.48 -1.54 -62.63 -0.43 15.40 10.15 16 184.78 1.18 -4.99 -1.53 208.86 1.43 27.71 15.41 367.55 2.39 -1.09 -0.20 366.41 2.55 27.37 21.03 3 22.07 0.18 -0.62 -0.16 303.97 2.08 20.97 21.08 647.92 3.91 1.55 0.35 458.23 3.21 18.09 21.83 23 230.81 1.61 -5.95 -1.77 267.69 1.86 28.28 13.41 336.50 2.13 -6.94 -2.36 336.31 2.46 72.40 26.39 MEAN 236.48 1.50 -3.14 -0.93 267.76 1.86 30.72 18.53 ± S.E. ±82.85 ±0.50 ±1.15 ±0.34 ±54.22 ±0.37 ±6.37 ±1.84 TABLE XXXII: Experiment 2: Topical Prostaglandin: Permeability Data: PGE 2 100 yg/ml. 92 BICARBONATE SECRETION yeq/3 Hr EXPERIMENT CONTROL 15M 0.6 yg/Kg/Hr 16DM 0.6 yg/Kg/Hr PGE 0.6 mg/Kg/Hr DOG 4 DOG 12 24.44 92.14 137.73 88.26 0 75.10 0 47.35 462.84 580.59 363.48 418.11 45.23 0 47.44 0 DOG 16 DOG 23 115.46 0 69.29 0 44.99 0 52.41 22.12 113.18 289.15 314.90 0 86.14 272.1 48.05 0 MEAN ± S.E. 65.91 ± 18.52 30.13 ± 10.11 317.78 ± 66.27 62.37 ± 31.88 TABLE XXXIII: Experiment 2: Intravenous Prostaglandin: Bicarbonate Secretion yeq/3 Hr. 93 VOLUME PRODUCE (in ml/3 Hr) EXPERIMENT CONTROL 15M 10 yg/ml 16DM 0.6 yg/Kg/Hr PGE 0.6 mg/Kg/Hr DOG 4 DOG 12 13.37 11.20 10.41 8.63 8.87 13.90 11.59 11.85 25.08 24.23 30.63 29.01 11.94 12.25 9.77 14.34 DOG 16 DOG 23 22.23 8.47 10.69 22.50 9.49 14.16 16.91 8.94 17.77 19.28 27.48 22.33 7.57 18.92 12.55 9.48 MEAN ± S.E. 13.43 ± 2.01 11.91 ± 1.01 24.47 ± 1.60 12.10 ± 1.22 TABLE XXXIV: Experiment 2: Intravenous Prostaglandin: Volume secreted ml/3 Hr. 94 + + + C l L i Na K DOG NIF K NIF K NIF K NIF K 16 -50.47 -0.36 -3.09 -0.81 -18.05 -0.13 14.06 10.59 128.57 0.96 -3.21 -0.81 145.01 1.15 24.10 14.57 23 71.17 0.49 -5.88 -1.74 103.63 0.73 20.39 21.16 128.53 0.92 -2.41 -0.66 167.47 1.22 11.81 12.92 4 -86.11 -0.60 -7.70 -2.00 -86.79 -0.61 15.02 14.58 101.63 0.71 -3.01 -0.78 109.79 0.79 22.74 16.87 12 -108.39 -0.77 -10.81 -2.96 -107.74 -0.79 13.70 11.00 69.00 0.49 -5.94 -1.43 30.01 0.23 15.50 16.43 MEAN 31.74 0.23 -5.25 -1.39 42.91 0.32 17.16 14.76 ± S.E. ±34.55 ±0.25 ±0.28 ±37.20 ±37.20 ±0.26 ±1.61 ±1.21 TABLE XXXV: Experiment 2: Intravenous Prostaglandins: Permeability Data: Controls. 95 C l DOG NIF K 4 -42.11 -0.30 -7.57 -0.06 23 -67.78 -0.46 -4.52 -0.05 16 71.01 0.52 -114.45 -0.81 12 -41.20 -0.30 -47.68 -0.34 MEAN -31.78 -0.22 ± S.E. ±19.12 ±0.10 L i + N NIF K NIF -3.46 -0.88 -58.37 -4.38 -1.19 -75.33 -9.04 -2.45 -63.29 -5.75 -1.69 20.20 -3.40 -0.92 49.99 -7.71 -2.00 -105.21 -3.04 -0.83 -40.02 -2.04 -0.51 -50.84 -4.85 -1.30 -40.35 ±0.86 ±0.22 ±18.03 K NIF K -0.42 11.71 14.67 -0.55 12.15 14.81 -0.44 28.16 19.96 0.12 15.66 5.95 0.37 27.70 15.11 -0.75 32.36 25.20 -0.30 4.36 5.42 -0.36 6.67 6.82 -0.29 17.34 13.11 ±0.10 ±3.76 ±2.38 TABLE XXXVI: Experiment 2: Intravenous Prostaglandins: Permeability Data: 15M 0.6 yg/Kg/Hr. 96 - + + + C l L i Na K DOG NIF K NIF K NIF K NIF K 12 433.92 3.06 -4.85 -1.46 519.54 3.75 19.67 16.98 515.99 3.61 -2.22 -0.57 644.89 4.34 23.46 20.14 23 271.77 1.94 -2.85 -0.83 330.32 2.35 27.19 22.85 217.28 1.52 -3.96 -1.12 257.10 1.83 20.78 9.56 4 481.15 3.42 -0.76 -0.35 567.71 4.12 22.93 17.22 394.88 2.74 -2.17 -0.64 531.32 3.71 24.69 18.49 16 213.79 1.49 -4.55 -1.11 74.03 0.73 24.52 10.95 376.97 2.64 1.52 0.22 449.92 3.34 36.61 22.00 MEAN 363.21 2.55 -2.48 -0.73 421.85 3.02 24.98 17.27 ± S.E. ±41.28 ±0.28 ±0.74 ±0.17 ±66.79 ±0.43 ±1.85 ±1.70 TABLE XXXVII: Experiemnt 2: Intravenous Prostaglandins: Permeability Data: 16DM 0.6 yg/Kg/Hr. 97 C l DOG NIF K 4 -12.75 -0.10 -44.81 -0.31 12 68.90 0.47 70.68 0.51 23 176.29 1.27 -54.44 -0.37 16 5.17 0.03 109.27 0.76 MEAN 39.66 0.28 ±.S.E. ±28.46 ±0.20 L i ' NIF K -4.77 -1.24 -6.22 -1.66 -4.66 -1.16 -3.62 -0.90 -4.62 -1.20 -9.68 -2.44 -1.34 -0.32 -2.44 -0.66 -4.66 -1.19 ±0.88 ±0.22 + Na NIF K -8.98 -0.06 -52.79 -0.39 40.98 0.30 11.10 0.10 212.77 1.52 0.76 0.10 23.66 0.17 18.41 0.14 30.73 0.22 ±27.80 ±0.17 NIF K 21.76 19.69 21.71 17.32 22.14 18.09 21.37 17.50 31.07 17.44 19.48 16.63 13.65 10.96 11.56 12.71 20.34 16.29 ±2.09 ±1.03 TABLE XXXVIII: Experiment 2: Intravenous Prostaglandins: Permeability Data: PGE 2 0.6 yg/Kg/Hr. 98 DAMAGE PERIOD NIF ueq/min K RECOVERY PERIOD NIF ueq/min K Na 13.38 3.28 11.58 9.21 16.11 13.69 6.29 10.65 25.04 10.60 22.87 19.01 25.67 27.20 14.29 17.23 12.71 9.52 8.41 11.16 14.39 ±1.47 H -1.51 -5.24 -2.47 -5.50 -7.89 -8.23 2.45 6.78 -9.08 -3.90 -5.91 16.04 -2.52 2.13 -3.79 -13.12 Na 0.30 0.08 0.27 0.22 0.37 0.32 0.14 0.19 0.54 0.23 -7.75 0.48 3.42 0.31 0.51 0.41 0.33 0.37 0.24 0.26 -2.95 0.20 -0.79 0.25 -2.49 ±1.44 0.30 ±0.03 H -0.11 -0.06 -0.11 -0.10 -0.16 -0.14 -0.05 -0.09 -0.23 -0.13 -0.21 -0.15 -0.22 -0.19 -0.13 -0.15 -0.17 -0.16 -0.07 -0.09 -0.13 ±0.01 Volume ml/Hr 17.45 8.03 9.60 9.23 11.85 11.26 11.87 15.15 13.06 11.07 12.72 15.09 16.81 27.20 11.92 14.04 17.74 8.87 9.10 13.7 2 13.28 ±0.96 + + Na H + + Na H 14.81 -4.59 0.25 -0.15 5.25 -16.48 0.19 -0.14 11.12 -3.00 0.27 -0.11 14.27 -0.56 0.27 -0.12 ±2.08 ±2.53 ±0.03 ±0.02 Volume ml/Hr 15.81 11.77 0.23 -0.12 20.40 10.74 2.40 0.23 -0.07 10.64 13.19 7.18 0.22 -0.10 14.83 6.24 -5.84 0.18 -0.08 7.53 16.49 1.88 0.35 -0.02 12.58 23.39 -4.31 0.46 -0.02 13.39 25.71 5.3 0.38 -0.23 23.42 9.92 8.36 10.79 13.18 ±1.62 XXXIX: Experiment 3: Permeability Data And Volumes For Each Period: Controls. 99 DAMAGE PERIOD DOG 14 NIF yeq/min Na 12.49 12.16 H -8.70 -9.00 Na 0.29 0.30 K H -0.17 -0.16 RECOVERY PERIOD NIF yeq/min K Volume ml/Hr Na H Na H ^'ft 13.51 -7.63 0.35 -0.13 8 .68 Volume ml/Hr 8.94 14.58 11.19 2.75 -4.29 0.20 0.20 -0.17 -0.14 19.87 15.17 14.88 0.21 0.26 -0.13 12.88 15 14.81 12.80 -0.78 -9.99 0.32 0.31 -0.17 -0.16 17.58 8.03 15.40 -12.59 0.40 -0.17 10.76 11.78 13.90 -15.35 -14.30 0.31 0.37 -0.17 -0.18 12.13 13.38 10.41 -5.07 0.26 -0.12 10.76 21 28.67 35.02 -28.37 -16.90 0.57 0.60 -0.41 -0.43 14.62 25.86 29.96 -30.35 0.65 -0.36 13.66 37.13 49.39 -27.74 -37.82 0.64 0.71 -0.49 -0.61 9.10 18.73 43.81 -21.36 0.64 -0.46 19.73 22 27.89 20.45 -16.22 -21.16 0.55 0.50 -0.39 -0.26 21.69 9.73 23.17 10.66 0.43 -0.19 26.81 28.97 34.46 -33.07 -34.50 0.52 0.59 -0.44 -0.50 12.29 10.38 25.10 -8.14 0.41 -0.24 12.70 17 25.60 12.14 -4.79 -12.77 0.42 0.29 -0.31 -0.20 19.85 10.15 12.94 -9.62 0.34 -0.13 7.37 32.92 13.68 -18.41 -20.42 0.27 0.36 -0.19 -0.20 12.57 10.97 13.88 -8.19 0.36 -0.13 10.08 MEAN 22.50 -16.64 0.42 -0.29 13.63 20.30 ± S.E. ±2.50 ±2.54 ±0.03 ±0.04 ±1.28 ±3.28 -9.20 0.41 ±3.51 ±0.05 -0.21 ±0.04 13.37 ±1.83 TABLE XL: Experiment 3: Permeability Data And Volumes for Each Period: A s p i r i n Damage. 100 DOG 14 15 DAMAGE PERIOD NIF yeq/min K Na 7.76 31.97 20.52 H 1.62 -1.23 -7.84 Na 0.08 0.60 0.47 H -0.14 -0.30 -0.21 Volume ml/Hr 21.43 12.59 RECOVERY PERIOD NIF yeq/min K Na + + H Na H 25.91 3.01 0.43 -0.18 Volume ml/Hr 14.55 13.68 5.49 0.26 -0.10 17.31 18.01 13.40 18.25 -4.60 -8.36 0.20 0.27 -0.17 -0.20 13.60 17.69 9.22 -1.81 0.12 -0.06 8.26 18.10 14.26 -6.40 -17.55 0.35 0.33 -0.23 -0.25 18.43 8.71 29.26 -4.08 0.48 -0.22 20.86 21 22 36.51 -26.57 13.92 21.89 3.74 14.24 -16.34 -6.22 -8.54 -27.62 0.77 0.27 0.31 0.05 0.41 -0.44 17.11 -0.21 -0.23 -0.24 -0.29 12.93 20.51 16.16 8.96 37.32 0.45 0.61 -0.29 23.08 1.88 -11.52 -0.01 -0.15 10.54 25.18 -10.79 0.44 -0.29 15.09 17 23.09 16.73 -14.32 -6.57 0.25 0.26 -0.29 -0.18 19.37 13.86 32.63 0.13 0.25 -0.29 27.62 26.35 23.92 -11.12 -11.16 0.34 0.35 -0.32 -0.25 17.29 16.32 28.38 -5.45 0.31 -0.26 18.23 20.77 21.66 -15.65 -15.94 0.43 0.44 -0.31 -0.3 0 12.29 12.34 34.62 -0.62 0.47 -0.35 18.24 MEAN 19.28 -11.35 0.35 -0.25 15.22 23.80 -3.41 0.34 -0.22 17.74 ± S.E. ±1.85 ±1.83 ±0.04 ±0.01 ±0.86 ±3.69 ±1.86 ±0.06 ±0.30 ±1.77 TABLE XLI: Permeability Data And Volumes for Each Period: T o p i c a l PGE 10 yg/ml. 101 DAMAGE PERIOD NIF ueq/min DOG Na 14 + 13.86 6.89 11.21 8.59 15 37.36 10.92 34.15 23.26 21 16.05 7.85 17 29.65 11.28 4.87 19.04 24.52 13.84 MEAN 17.08 ± S.E. ±2.49 H -8.98 -9.12 0.64 -13.63 -10.24 -20.68 -3.40 -11.63 -13.56 -4.57 -15.94 -6.59 K Na 0.21 0.13 0.12 0.19 0.53 0.23 0.39 0.34 9.88 0.08 -2.19 0.09 0.45 0.17 -5.29 0.04 0.51 0.26 0.34 0.24 -7.14 0.24 ±1.86 ±0.03 H -0.17 -0.11 -0.15 -0.14 -0.37 -0.19 -0.36 -0.26 -0.15 -0.11 -0.34 -0.12 -0.13 -0.18 -0.36 -0.13 -0.21 ±0.02 Volume 13.72 13.94 13.92 9.90 23.56 12.72 30.29 24.34 18.24 12.90 21.18 20.57 9.82 14.98 18.66 10.80 RECOVERY PERIOD NIF ueq/min K H Na H ml/Hr Na 17.82 6.44 0.22 -0.13 18.84 9.27 3.11 0.11 -0.08 12.05 24.82 -12.80 0.36 -0.27 17.63 18.11 -11.71 0.24 -0.24 15.12 33.63 14.47 7.82 16.85 17.63 ±1.46 ±2.95 -2.97 0.18 -0.17 16.58 0.29 -0.25 3.77 0.22 -5.59 0.13 18.99 17.37 37.54 -0.09 12.79 -0.09 11.96 -0.39 0.22 -0.17 18.40 ±3.48 ±0.03 ±0.03 ±2.93 TABLE XLII: Experiment 3: Permeability Data And Volumes For Each Period: Topical 15M 10 yg/ml. 102 DOG 14 15 21 22 DAMAGE PERIOD NIF ueq/min K MEAN Na 16.85 13.63 13.63 11.12 11.84 9.79 17.08 11.26 18.14 19.51 18.67 13.93 34.52 34.18 26.20 31.48 31.23 24.60 25.17 26.31 26.67 33.28 18.47 21.28 21.20 ±1.62 H 13.87 0.74 -0.60 3.14 4.16 -13.81 -12.22 -1.77 -2.04 -3.50 -8.48 -10.78 -34.63 -17.80 -19.20 -25.17 -35.03 -14.31 -26.13 -18.70 -34.33 -22.23 -16.42 -27.48 -13.44 ±2.72 Na 0.22 0.22 0.34 0.19 0.22 0.27 0.45 0.22 0.34 0.37 0.45 0.35 0.64 0.60 0.57 0.69 0.64 0.46 0.61 0.50 0.53 0.63 0.40 0.55 0.43 ±0.01 H -0.13 -0.16 -0.09 -0.15 -0.11 -0.16 -0.19 -0.12 -0.22 -0.20 -0.19 -0.17 -0.53 -0.40 -0.35 -0.41 -0.48 -0.29 -0.38 -0.34 -0.50 -0.39 -0.31 -0.35 -0.27 ±0.03 RECOVERY PERIOD NIF yeq/min K Volume ml/Hr Na 0.82 18.41 15.36 11.16 14.48 13.61 7.33 9.22 9.36 24.95 14.92 14.23 9.21 17.81 24.24 14.08 16.69 12.11 14.15 10.81 17.70 16.92 14.31 12.38 9.87 + H Na 0.70 H ml/Hr 6.71 0.06 -0.03 11.35 4.00 -0.05 -0.04 8.77 7.99 1.24 0.18 -0.06 10.52 13.00 2.92 0.26 -0.10 11.02 7.57 -4.33 0.22 -0.05 10.27 8.82 3.29 0.21 -0.04 10.94 16.52 -10.60 0.37 -0.16 9.13 15.50 -2.68 0.35 -0.14 16.47 12.90 1.67 14.48 -13.82 12.22 -9.23 15.89 -5.00 14.30 10.52 -2.14 ±0.89 ±1.58 ±1.88 0.25 -0.09 11.29 0.38 -0.18 14.94 0.30 -0.13 8.60 0.36 -0.19 11.20 0.23 -0.10 11.20 ±0.04 ±0.01 ±0.67 TABLE XLIII: Experiment 3: Permeability Data and Volumes For Each Period: I.V. PGE 2 0.3 mg/Kg/Hr. 103 DOG 14 DAMAGE PERIOD NIF ueq/min K RECOVERY PERIOD Na 3.96 5.82 13.46 12.71 H -10.23 -0.27 10.99 2.85 + Na 0.11 0.06 0.18 0.13 H -0.08 -0.08 -0.11 -0.14 NIF ueq/min Volume ml/Hr 5.45 9.49 11.58 18.72 15.78 K + + + Na H Na 11.78 0.08 8.98 8.84 0.16 -0.03 14.25 -0.03 12.31 18.07 9.28 11.55 -10.58 0.26 0.25 -0.14 -0.12 21.27 10.36 10.77 -0.35 0.24 -0.10 12.21 15 22.57 15.50 -10.05 13.10 0.33 0.17 -0.25 -0.07 19.29 18.01 10.46 2.96 0.21 -0.05 13.59 21 16.72 19.85 19.67 14.74 -1.55 2.24 -23.44 0.21 0.37 0.40 0.36 0.17 -0.18 -0.18 -0.31 -0.14 17.04 16.97 9.09 -13.73 0.31 8.05 18.24 -9.82 0.26 13.38 -0.10 -0.24 13.17 32.32 -1.74 0.58 -0.25 20.81 29.00 24.87 0.25 -0.14 26.27 2.47 12.92 -0.06 -0.01 13.94 39.13 31.25 -13.03 -21.54 0.65 0.60 -0.45 -0.39 23.31 16.28 18.73 -20.96 0.48 -0.23 14.64 22 26.92 37.11 MEAN 20.44 ± S.E. ±2.42 -32.24 -24.63 0.64 0.74 -4.63 0.35 ±3.53 ±0.05 -0.45 -0.47 -0.23 ±0.03 16.02 17.57 16.45 ±1.23 21.01 -2.96 0.37 12.13 -1.23 0.23 ±1.98 ±3.90 ±0.05 -0.22 17.20 -0.10 13.10 ±0.03 ±0.94 TABLE XLIV: Experiment 3: Permeability Data And Volumes For For Each Period: I.V. 15M 0.6 pg/Kg/Hr. 104 DAMAGE PERIOD RECOVERY PERIOD DOG 14 NIF ueq/min Na 16.30 11.44 H 3.93 -11.07 Na 0.29 0.28 K H -0.16 -0.15 Volume ml/Hr 20.24 11.52 NIF yeq/min Na H K Na 16.00 -8.73 0.37 -0.18 15.80 9.39 17.73 -16.56 -9.26 0.24 0.39 -0.18 -0.19 11.41 11.66 14.96 -10.24 0.33 -0.15 12.94 15 12.67 18.80 -9.21 -12.91 0.25 0.44 -0.18 -0.20 14.27 11.47 14.41 -6.62 0.33 -0.15 13.42 14.10 17.55 -16.10 -12.32 0.29 0.38 -0.24 -0.23 12.28 10.12 15.11 -4.59 0.32 -0.12 10.02 17 30.29 22.94 -12.37 -22.32 0.51 0.53 -0.35 -0.34 16.96 9.87 31.82 -0.55 0.53 -0.26 21.03 14.97 19.00 -8.00 -12.30 0.35 0.43 -0.17 0.23 13.33 12.80 15.65 -5.11 0.32 -0.16 10.48 MEAN 17.09 -11.53 0.36 -0.22 12.99 16.95 -5.97 0.37 -0.17 13.94 ± S.E. ±1.60 ±1.80 ±0.30 ±0.02 ±0.85 ±1.75 ±1.39 ±0.03 ±0.02 ±1.65 TABLE XLV: Experiment 3: Permeability Data And Volumes For Each Pouch: Metiamide 3 mg/Kg/Hr. 105 BIBLIOGRAPHY 1. 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E f f e c t of g a s t r i c secretory i n h i b i t o r s on the g a s t r i c mucosal b a r r i e r . Gut 16: 437-442, 1975. 49. Bolton JP, Cohen MM. Permeability e f f e c t s of E^ prostaglandins on canine g a s t r i c mucosa. Gastroenterology 70: 865, 1976. 50. Robert A, Nezamis JE, P h i l l i p s JP. E f f e c t of prostaglandin E^ on g a s t r i c secretion and ulcer formation i n the r a t . Gastroenterology 55: 481-487, 1968. 51. Fung WP, Lee SK, Karim SMM. E f f e c t of prostaglandin 15(R)-15-methyl- E^-methyl ester on the g a s t r i c mucosa i n patients with p e p t i c u l c e r a t i o n - an endoscopic and h i s t o l o g i c a l study. Prostaglandins 5: 465-472, 1974. 52. Dean ACB. Protective e f f e c t of carbenoxolone i n drug induced l e s i o n s of the stomach. Carbenoxolone Sodium. A Symposium, JM Robson; FM S u l l i v a n (Ed.) London; Butterworths, pp. 33-39, 1968. 53. Corne SJ, Morrissey SM, Woods RJ. A method of q u a n t i t a t i v e estimation of g a s t r i c b a r r i e r mucus. J . Ph y s i o l . (Lond.) 242: 116-117, 1974. 54. Whiteman P. The qua n t i t a t i v e determination of glycoaminoglycans i n mine with A l c i a n Blue 8GX. Biochem. J . 121: 251-257, 1973. 55. Chung RSK, F i e l d M, S i l e n W. Permeability of g a s t r i c mucosa of hydrogen and l i t h i u m . Gastroenterology 64: 593-598, 1973. 56. Ivey KJ. Gastric mucosal b a r r i e r . Gastroenterology 61: 247-257, 1971. 57. Piper DW, Whitecross D, Leonard P., Clarke A. A l c i a n Blue binding properties of g a s t r i c j u i c e . Gastroenterology 59: 534-538, 1970. Malawer SJ, Powell DW. An improved t u r b i d i m e t r i c analysis of polyethylene g l y c o l u t i l i z i n g an e m u l s i f i e r . Gastroenterology 53: 250-256, 1967. Hyd£n S. A tu r b i d i m e t r i c method for the determination of higher polyethylene glycols i n b i o l o g i c a l materials. Ann. Roy. Agr i c . C o l l . 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Main IHM, Whittle SJR. The e f f e c t s of E and A prostaglandins on g a s t r i c mucosal blood flow and a c i d secretion i n the r a t . B i . J . Pharmac. 49: 428-436, 1973. 67. Kuo Y-J, Shanbour LL. Mechanism of action of a s p i r i n on canine g a s t r i c mucosa. Amer. J . P h y s i o l . 230: 762-767, 1976. 68. Jacobson ED, Chaudhury TK, Thompson WJ. Mechanism of g a s t r i c mucosal cyto p r o t e c t i o n by prostaglandins. Gastroenterology 70: 897, 1976. 69. MacDonald AS, Steele BJ, Bottomley MC. Treatment of stress-induced upper g a s t r o i n t e s t i n a l haemorrhage with metiamide. Lancet i : 68-70, 1976. 70. Dudley HAF, F i e l d i n g LP, Glazer G. Gastroduodenal rebleeding on cimetidine Lancet i : 481, 1977. APPENDIX Confirmation of the Effectiveness of the Metiamide used i n Experiment 3 An experiment was performed i n two dogs to e s t a b l i s h that the batch of metiamide used was e f f e c t i v e as an i n h i b i t o r of acid secretion. An i n f u s i o n of histamine di-HCl, 50 ug/Kg was given to two dogs over 105 minutes, the mean 15 minute acid outputs are recored as the s o l i d l i n e i n Figure 8. In a subsequent experiment the same dogs were subjected to the same i n f u s i o n of histamine but metiamide 3 mg/Kg/hour was infused from 60-120 minutes. The broken l i n e i ndicates the mean 15 minute acid output i n t h i s experiment. The batch of metiamide was considered normal i n i t s antisecretory e f f e c t . Metiamide 3 mgs/kg/hr I.V. Time in minutes FIGURE 8: I l l u s t r a t e s the i n h i b i t o r y e f f e c t of intravenous metiamide on histamine stimulated acid output from a Heidenhain pouch. In both experiments a constant i n f u s i o n of histamine di-HCl 50 yg/Kg/Hr was given from 15 to 120 minutes. The dotted l i n e indicates the e f f e c t of metiamide 3 mgs/Kg/Hr from 60 to 120 minutes. 116 2. E f f e c t of D i l u t i o n of A l c i a n Blue Binding An experiment was performed i n which the e f f e c t of s e r i a l d i l u t i o n s of 1 ml. of g a s t r i c j u i c e on A l c i a n Blue binding was studied. The r e s u l t s (Table XLVI) i l l u s t r a t e that the d i f f e r i n g volumes of g a s t r i c j u i c e are not l i k e l y to have introduced a s i g n i f i c a n t e r r o r . ±1/ DILUTIONS 1 ml. 2 ml. 4 ml. 8 ml. ALCIAN BLUE BOUND mg/ml 0.14 0.07 0.04 0.01 TABLE XLVI: E f f e c t of s e r i a l d i l u t i o n s of 1 ml. of g a s t r i c j u i c e on A l c i a n Blue binding.

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