GASTROENTEROLOGY Copyright 1969 by The Williams & Wilkins Co. Vol. 56, No.4 Printed in U.S.A. GASTRC MUCOSAL BLOOD FLOW DURNG SECRETORY NHBTON BY GASTRN PENTAPEPTDE AND GASTRONE DAVD J. COWLEY, M.D., CHARLES F. CODE, M.D., PH.D., AND RENE FASSE, M.D. Section of Physiology, Mayo Clinic and Mayo Foundation (Dr. Code), and Mayo Graduate School of Medicine, University of Minnesota (Dr. Cowley), Rochester, Minnesota, and Gastroenterology Research Laboratory, New York Medical College, New York, New York (Dr. Fiasse) The aminopyrine clearance technique was used in dogs to quantitate the gastric mucosal blood flow during secretory inhibition by a single, rapid intravenous injection of gastrin pentapeptide (lc 5,123) and of gastrone, the inhibitor substance from human achlorhydric gastric juice. A dose of 4 or 5 J.lg of gastrin produced a 68% reduction of blood flow and a 72% reduction of secretion. Reduction of blood flow appeared to precede reduction of secretion, and blood flow subsequently recovered more rapidly than did secretion. A dose of 15 J.lg of gastrone produced a 55% reduction of blood flow and a 66% reduction of secretion, but there was a lag of 1 hr after the injection of gastrone before secretion or blood flow was affected. Changes in blood flow and secretion were closely related during secretory inhibition by both gastrin (r =.95) and gastrone (r =.92). Recently, Jacobson and co-workers l - 3 developed a method of estimating gastric mucosal blood flow in the conscious dog by measuring the clearance of aminopyrine from plasma to gastric juice. A linear relationship was found between volume rate of secretion and mucosal blood flow in Heidenhain pouches of dogs secreting in response to histamine and gastrin. A direct relationship was also present during the reduction of secretion induced by the administration of Pitressin, secretin, and epinephrine. We undertook to determine, by this technique, whether gastrin and gastrone reduce blood flow while they inhibit gastric secretion. Some of the findings of the study were published in a preliminary communication. 4 Received June 2, 1968. Accepted November 6, 1968. Address requests for reprints to: Dr. Charles F. Code, Mayo Clinic, Rochester, Minnesota 5591. This investigation was supported in part by Research Grant AM-4391 from the National nstitutes of Health, United States Public Health Service. 659 Material and Methods The secretory inhibitors used in these studies were synthetic gastrin pentapeptide (lc 5,123, kindly supplied by Mr. J. D. Fitzgerald of the Pharmaceutical Division, C, Ltd., Cheshire, England) and the most highly purified gastrone from human achlorhydric gastric juice yet prepared in our laboratories (gastrone of ')' globulin-like electrophoretic mobility).5 Vagally denervated (Heidenhain) gastric fundic pouches were prepared in 3 female mongrel dogs weighing from 1. to 19.5 kg. No tests were performed for at least 3 weeks after the operation, during which period the animals regained their preoperative weight. All were in good health during the study. The maximal secretory capacity of each pouch when stimulated by intravenous infusion of histamine was determined on two separate occasions in each dog by the method of Code et a1. 6 Blood flow was determined by the technique described by Jacobson and co-workers.' The dog was fasted overnight (16 hr) and then was placed in a Pavlov support. A loading dose of aminopyrine was injected via an indwelling venous cannula and the blood level of aminopyrine was maintained by a continuous infusion
66 COWLEY ET AL. Vol. 56, No. 4 delivered by a solution-metering pump. A continuous infusion of histamine acid phosphate was delivered, via a second venous cannula, at a rate which established a secretory plateau of 5% or more of the pouch's maximal secretory capacity under histamine stimulation. Gastric juice was collected at 15-min intervals in the experiments with gastrin and at 3-min intervals in those with gastrone. The volume of each sample was measured, and the hydrogen ion concentration was determined by titration to ph 7 with a glass electrode ph meter and an automatic burette (Auto-Burette, Radiometer). Jugular venous blood was sampled at 1/2-hr intervals for estimation of plasma aminopyrine concentration. After a steady state of secretion had been obtained for four consecutive control periods, 4 or 5 lg of gastrin penta peptide or 15 lg of gastrone was injected rapidly, intravenously, in 5 ml of isotonic saline. Six further collections were made before the experiment was stopped. n the experiments with gastrone the dog's rectal temperature was taken at 3-min intervals using a standard clinical rectal thermometer inserted to a depth of 2 inches. The method for determination of mucosal blood flow was developed by Jacobson and collaborators l from observations made by Shore and co-workers. 7 Aminopyrine concentration was determined in aliquots of plasma and gastric juice by the method of Brodie and Axelrod. 8 Clearance was calculated from the formula C = GV/P in which C = clearance of aminopyrine (milliliters per minute), G = concentration of aminopyrine in gastric juice (milligrams per milliliter), P = concentration of aminopyrine in plasma (milligrams per milliliter), and V = volume rate of secretion (milliliters per minute). The concentration ratio R = G/P was also calculated. To allow combination of data from pouches of different sizes, the results were standardized by expressing the changes in secretory volume, mucosal blood flow, and concentration ratio R which occurred in each test as percentage change from the mean of the control values of each test (control in each experiment equaled 1). The results from all the dogs, in each series of tests, could then be pooled. Standard errors of the means were derived, and the probable significances of the differences from the control values were estimated by Fisher's t-test. 9 Results Preliminary experiments. The aminopyrine clearance was measured while the dose rate of a continuous infusion of histamine was doubled hourly and then successively halved after maximal secretory rates had been obtained. When the aminopyrine plasma clearance was plotted against the volume rate of secretion, a linear relationship was found. n two tests in one dog, r was.92; in one test in another dog, r was.85.." -J Q:... <: l) 12 8 - -1-,-+- Gaston i.v. 4- SOPO t Blood Flow '" '" <: <t :t l) *" 4 3 6 9 12 15 1. mg Histamine.5 Base hr i.v. o MNUTES FG. 1. Effect of rapid intravenous injection of 4 or 5 Lg of gastrin pentapeptide on acid output and mucosal blood flow of gastric pouch. Data are shown as mean values of three tests on each of 3 dogs with vagally denervated pouches secreting at 5% of maximal capacity (histamine stimulation). The asterisks indicate that mean values after gastrin are significantly different from control values before gastrin (P >.2).
April 1969 GASTRC MUCOSAL BLOOD FLOW 661 nhibition by a single, rapid dose of gastrin. Preliminary experiments were performed in all of the dogs to determine the minimal dose of gastrin which would invariably inhibit histamine-induced secretion. A lo-g dose sometimes produced inhibition, but more often it increased secretion. Doses of 4 to 5 g always produced inhibition. Since the effects of 4 to 5 g were very similar, the results they produced were pooled for analysis. Nine experiments (three on each of the 3 dogs) were done using 4 or 5 g (fig. 1). Control values for the individual dogs are shown in table 1. Prompt reduction of the secretory rate always occurred (mean maximal inhibition, -72%) 3 min after injection of gastrin. Progressive recovery followed with return to control values at 9 min after injection of gastrin. The mucosai blood flow was reduced in a parallel fashion, with mean maximal inhibition of - 68% at 3 min. Recovery of blood flow was more rapid than recovery of secretion, with return to control values at 6 min and a significant increase above control values at 6 to 75 min after gastrin administration. To determine whether a relationship existed between secretion and blood flow in the mucosa, the percentage changes of secretory volume were plotted against the percentage changes of blood flow (control values equaled 1) (fig. 2). The data obtained during the period of reduction of secretion were plotted separately from those obtained during recovery. A correlation was found (r =.95, P =.1) between secretion and blood flow while secretion was decreasing; during recovery the variability was great and the correlation was less striking (r =.74, P <.1). The concentration ratio R attained a plateau value during the control period of each test. t was reduced by 1% during the first 15-min period after the injection of gastrin. A pronounced increase then occurred with maximal values usually reached at 6 min followed by a return toward control values. No side effects were observed after the injections of gastrin. nhibition by gastrone. Five experiments were performed on 2 dogs (two on dog A and three on dog C) (fig. 3). A latent interval of 1 hr, during which no changes occurred, followed the injection of gastrone, then secretion and mucosal blood flow were progressively reduced until mean maximal inhibitions of - 66 and -56%, respectively, were reached at 3 hr. When the experiment was stopped, 21 min after injection of gastrone, recoveries of secretion and blood flow were just beginning. A linear relationship (fig. 4) between secretory rate and mucosal blood flow was present throughout the entire period of observation after gastrone administration. The control values for each dog are shown in table 2. They reflect the relationship between secretory output and mucosal blood flow and also the difference in size of the pouches. The pouches of dogs A and B were removed under anesthesia after the study was completed. The wet weight of the pouches and their mucosal lining was determined. When the rate of secretion stimulated by histamine was steady at close to half maximal, the mucosal blood Dog T ABL E 1. M ean control values in experiments with gastrina Volume m1 Secretory output per 3 min Hel meq A............ 5.1 ±.75.746 ±.158 B......... 5.2 ±.6.776 ±.89 C............ 2.4 ±.12.295 ±.45 a Means ± SD in t hree tests on each of 3 dogs. b R, concent ration rat io, G/ P. Pouch mucosal aminopyrine clearance Secretory rate 1_ J?b ml/15 min % max 19. 1 ± 27.9 55.6 37.7 227.3 ± 27.45 59. 3 4 4.6 78.5 ± 17.85 5. 33.
662 COWLEY ET AL. Vol. 56, No. 4 y=4j.78+ r =.74.. Control = 1%.................... SECRETON (%).. 5.. Progre ssive Recovery of Secretion.. O. 756x 15.... BLOOD FLOW(%).. 6 Progressive Reduction 5 l. of Secretion y=7.j+.87x r =.95 FG. 2. Relationship between gastric mucosal blood flow and gastric acid output after rapid intravenous injection of 4 or 5 /log of gastrin pentapeptide in 3 dogs with vagally denervated gastric pouches. 6., represent values during first 3 min after gastrin injection (period of progressive reduction of secretion);., represent values during subsequent 6 min (period of progressive recovery of secretion). o Collection R Gostrone 15p.g 12 t O,-+-,-t- -+-,- -, -.J - +- Secretory Volume.. a:: f.- 8 <J '"' <:l 4 1. <>: mg Histamine :x: <J.5 Base / hr * ;. v. 6 12 18 24 3 MNUTE S FG. 3. Effect of intravenous injection of 15 /log of gastrone prepared from a single collection of achlorhydric juice (collection R) on acid output and mucosal blood flow of gastric pouch. Data are shown as mean values of three tests in 1 dog and two in another dog, both with vagally denervated pouches secreting at 5% of maximal capacity (histamine stimulation). The asterisks indicate that mean values after gastrone are significantly different from control values before gastrone (P >.2).
April 1969 GASTRC MUCOSAL BLOOD FLOW 663 flow was 1.2 and 1. 7 ml per min per g wet mucosa in these 2 dogs (table 3). Dog B was resistant to the inhibitory effect of gastrone (fig. 5): 15 lg produced very little change of secretion or of blood flow and 35 lg also had little effect. A mild febrile response occurred after administration of gastrone, with a range of.4 to 1.6 F above the control rectal temperature. Aminopyrine did not suppress this fever. The greatest increases of body temperature, of 1.4 and 1.6 F, occurred in the resistant dog B which showed very little change in secretion or mucosal blood flow after gastrone. Discussion The aminopyrine clearance technique was used in the study because it allows 48 CO> 36.., "- e " CO> 24 -J "- /!! 2 " l<j r ::. 84 ::; CO> () () _J '' y 21. 3 + 33.6x SECRETORY VOLUME Y 767.8 + 39.9x r ::.986 1m! / 3 m in) FG. 4. Relationship between gastric mucosal blood flow and gastric acid output after injection of 15 p.g of gastrone in 2 dogs with vagally denervated gastric pouches of different sizes., values for one dog;., values for other dog. quantitation of mucosal blood flow in the conscious, secreting dog. Furthermore, it allows simultaneous estimation of secretion and blood flow over a prolonged period. Our preliminary findings showed a close correlation between volume rate of secretion and gastric plasma aminopyrine clearance during successive doubling of the dose of intravenously infused histamine. This result confirms similar observations by Jacobson and his associates.! UvnHs 1 in 1943 and Gillespie and Grossman ll in 1963 demonstrated that a single, rapid intravenous injection of gastrin inhibits the secretory response of a gastric pouch in a dog to either histamine or to gastrin itself. Furthermore, Gillespie 12 showed that the inhibition was produced irrespective of the secretory rate of the pouch and that it occurred only when the gastrin was injected rapidly. Gregory and Traci: 1 showed that pure human gastrin has the same effect. The phenomenon appears to be peculiar to the dog and has not been found in man!2 or in the anesthetized!4 or unanesthetized! 5 cat. Our results confirm Gillespie's observation that synthetic gastrin pentapeptide and gastrin extracts have the same inhibitory effect. n addition, we have demonstrated that the reduction in secretory rate is accompanied by a parallel reduction in mucosal blood flow as defined by aminopyrine clearance. We did not measure systemic blood pressure but there is evidence showing that the rapid intravenous injection of a large dose of gastrin pentapeptide has no significant effect on the systemic arterial blood pressure of conscious dogs (E. D. Jacobson, personal communication). Dog TABLE 2. A ean control values in experiments with gastrone" Secretory output per 3 min Pouch mucosal Secretory aminopyrine clearance rate Volume Hel J(b ml A (2 tests).......... 9.8 ±.53 C (3 tests). '".... 4.4 ±.14 " Means ± SD in five experiments. b R, concentration ratio, G/P. meq 1.461 ±.8.531 ±.31 ml/3 min % max 47.7 ± 29.4 55. 41.4 155.4 ± 31.5 46. 35.2
664 COWLEY ET AL. Vol. 56, No.4 TABLE 3. Pouch weight, histamine stimulation, and blood flow Dog Pouch wet weight 1 Rate Mus- Sub- Mu- of cularis mucosa cosa secretion Histamine stimulation Pouch mucosal blood flow Ml pe! mm Ml per mm per g wet mucosaa --._--- - - g % max A.... 7. 4.5 1.9 56 1.2 12 8 B..... 8.1 4.3 9. 1 59 1 15..2 1 1.7 a Mean for the 2 dogs, 1.4 ml per min per g wet mucosa. --J Q: >-- <: <> 12 8 4 lu '" <: 12 ::t "" <> * 8 4 - - L _ J Collection R Gastrone 35p9 r - 6 12 18 MNUTES 24 mg His/amine Bose hr (i.v. ) 1. L _ J -- Blood. Flow 3 FG. 5. Lack of effect of intravenous injection of 15 and 35 lg of the same preparation of gastrone as used in tests illustrated in figure 3 on mucosal blood flow and gastric acid output of vagally denervated pouch secreting at 5% of maximum (histamine stimulation) in dog B..5 1..5 Our results indicate that there is a relationship between secretion and blood flow during secretory inhibition by gastrin and gastrone, but they do not demonstrate which is cause and which is effect. However, the concentration ratio R in the experiments with gastrin sheds some light on this question. R may be regarded as the volume of blood which must flow through the pouch mucosa in order to produce 1 ml of pouch secretion. The values for R during the control periods in these experiments ranged from 33 to 45, which agree well with the value of 4 found by Shore and coworkers. 7 The decrease in this ratio during the first 15 min after injection of gastrin indicates that an initial decrease of the mucosal blood flow occurred relative to the secretory rate. From 15 to 9() min, R increased above control values, suggesting that the blood flow during this period was high with respect to secretion and that blood flow was returning to control values more rapidly than was secretion. This sequence of changes in the blood flowsecretion relationship gives some support to the interpretation that a sudden decrease of blood flow caused the inhibition. Brunschwig and associates 6 demonstrated in 1939 that the achlorhydric gastric juice of patients with pernicious anemia contains a potent inhibitor of gastric secretion. t was subsequently namt:d "gastrone" by Code.17 Similar inhibitory activity has been found in acidic gastric juice, in saliva, and in the juice from canine antral pouches. t is known to inhibit the secretory response to histamine and to a meat meal, and also to inhibit secretion in the pylorus-ligated rat. ts mode of action is unknown. n 1965, Rudick and co-workers l 8 showed that the inhibitor substance from canine antral pouches reduced the total blood flow to the stomach, as measured by an ultrasonic flowmeter. Our results are in agreement. Rudick's method has been criticized by Jacobson l9 on the grounds that it did not quantitate blood flow and that total blood flow to the stomach does not necessarily parallel that through the mucosa. Rudick's results were similar to ours, however, in showing a time lag before reduction of blood flow occurred. We have demonstrated also that when secretory inhibition is produced by gastrone, a corresponding reduction in gastric mucosal
April 1969 GASTRC MUCOSAL BLOOD FLOW 665 blood flow takes place. This interpretation is supported by the finding, in the resistant dog, that when gastrone fails to inhibit secretion it also has no effect on blood flow. The mean rate of mucosal blood flow during histamine stimulation in our tests was 1.4 ml per min per g wet mucosa, which is somewhat greater than that of 1. ml per min per g wet mucosa obtained by Delaney and Grim 2 in unanesthetized dogs. They used a different method and their tests were done when secretion was unstimulated. Considering these differences, our results support their earlier findings. REFERENCES 1. Jacobson, E. D., R. H. Linford, and M.. Grossman. 1966. Gastric secretion in relation to mucosal blood flow studied by a clearance technic. J. Clin. nvest. 45: 1-13. 2. Jacobson, E. D., M. M. Eisenberg, and K. G. Swan. 1966. Effects of histamine on gastric blood flow in conscious dogs. Gastroenterology 51 : 466-472. 3. Swan, K. G., and E. D. Jacobson. 1966. Effects of gastrin and pentapeptide on gastric blood flow in conscious dogs. Surg. Forum 17: 33-35. 4. Cowley, D. J., and C. F. Code. 1967. Gastric mucosal blood flow during secretory inhibition by gastrin pentapeptide (abstr.). Gastroenterology 52: 1131. 5. Glass, G. B. J., C. F. Code, K. Katsuhiko, and R. Fiasse. 1967. Fractionation of endogenous inhibitors of gastric secretion (gastrone) by physico-chemical means, p. 45-425. n T. K. Shnitka, J. A. L. Gilbert, and R. C. Harrison [eds.), Gastric secretion: mechanisms and control. Pergamon Press, New York. 6. Code, C. F., C. M. Blackburn, G. R. Livermore, and H. V. Ratke. 1949. A method for the quantitative determination of gastric secretory inhibition. Gastroenterology 13: 573-587. 7. Shore, P. A., B. B. Brodie, and C. A. M. Hogben. 1957. The gastric secretion of drugs: a ph partition hypothesis. J. Pharmacol. Exp. Ther. 119: 361-369. 8. Brodie. B. B., andj. Axelrod. 195. The fate of aminopyrine (Pyramid on) in man and methods for the estimation of aminopyrine and its metabolites in biological material. J. Pharmacol. Exp. Ther. 99: 171-184. 9. Fisher, R. A. 1958. Statistical methods for research workers, Ed. 13, 356 p. Hafner Publishing Co., nc., New York. 1. Uvniis, B. 1943. The gastric secretory excitant from the pyloric mucosa. Acta Physiol. Scand. 6: 97-17. 11. Gillespie,. E., and M.. Grossman. 1963. nhibition of gastric secretion by extracts containing gastrin. Gastroenterology 44: 31-31. 12. Gillespie,. E. 1966. nhibition of acid secretion by gastrin extracts, p. 229-253. n M.. Grossman [ed.), Gastrin. University of California Press, Los Angeles. 13. Gregory, R. A., and H. J. Tracy. 1964. The constitution and properties of two gastrins extracted from hog antral mucosa. Gut 5: 13-114. 14. Blair, E. L. 1964. The internal and external secretions of the stomach. Thesis. University of Newcastle Upon Tyne. 15. Emas, S., and M.. Grossman. 1967. Comparison of gastric secretion in conscious dogs and cats. Gastroenterology 52: 29-34. 16. Brunschwig, A., J. Van Prohaska, T. H. Clarke, and E. Kandel. 1939. A secretory depressant in gastric juice of patients with pernicious anemia. J. Clin. nvest. 18: 415-422. 17. Code, C. F. 1958. Discussion. Gastroenterology 34: 21. 18. Rudick, J., L. S. Semb, W. G. Guntheroth, G. L. Mullins, H. N. Harkins, and L. M. Nyhus. 1965. Gastric blood flow and acid secretion in the conscious dog under various physiological and pharmacological stimuli. Surgery 58: 45-56. 19. Jacobson, E. D. 1967. Progress in gastroenterology: the circulation of the gastrointestinal tract. Gastroenterology 52: 98-112. 2. Delaney, J. P., and E. Grim. 1964. Canine gastric blood flow and its distribution. Amer. J. Physiol. 27: 1195-122.