possibility that the "gastric hormone" may not as yet have been extracted investigation of any part of the stomach other than the pyloric mucosa.

Transcription

1 234 6I I :547x78I.5 HISTAMINE IN CANINE GASTRIC TISSUES. BY GERTRUDE GAVIN, E. W. McHENRY AmD M. J. WILSON. (From the Department of Physiological Hygiene, School of Hygiene, University of Toronto.) IN 1905 and 1906 Edkins reported that aqueous extracts of pyloric, when injected subcutaneously, caused secretion of gastric juice in the dog. Extracts of fundic did not stimulate gastric secretion, although they were stated to be more potent in lowering blood-pressure than the pyloric extracts. As a result of this work, the second or chemical phase of gastric secretion is now believed by many to be due to the formation in the pylqric mucous membrane of a specific substance called "gastrin," which when absorbed into the blood stimulates the gastric glands. In 1920 Popielski demonstrated that histamine caused gastric secretion, and since then there has been considerable speculation as to whether histamine and "gastrin" are identical. In 1932 Sacks, Ivy, Burgess and Vandolah reported the isolation of histamine from hog pyloric. They showed that, after incubation with histaminase, saline extracts of this tissue no longer produced gastric secretion. They concluded that if a "gastric hormone" has been extracted from the pyloric the active principle is histamine. These workers consider the possibility that the "gastric hormone" may not as yet have been extracted from the. In these studies no reference was made to the investigation of any part of the stomach other than the pyloric. In view of this similarity between "gastrin" and histamine it seemed advisable to study the distribution of the amine in various parts of normal stomachs. For this purpose dogs, after 16 hours' starvation or in some cases after a meat meal, were used. The animals were killed instantly by means of a captive-bolt pistol, the stomach was removed immediately, wiped as clean as possible with dry gauze, and then dissected into pyloric and muscle, and fundic and muscle. Each section was then finely minced, and weighed portions used for histamine determina-

2 HISTAMINE IN CANINE GASTRIC TISSUES. 235 tions. Extracts were prepared by our customary hydrochloric acid procedure [Best and Mc Henry, 1930]. The minced tissue was thoroughly mixed with hydrochloric acid within 30 mi. after the death of the dog, and it is unlikely that any change occurred in the histamine content of the tissue in the interval, especially since the gastric tissues of the dog do not contain histaminase in amounts detectable by the methods available [B e st and Mc Henry, 1930]. The depressor material present was determined by assay against a standard histamine solution, using the blood-pressure method in an etherized cat. Table I shows the depressor contents calculated as milligrams of histamine per kilogram of tissue. TABLE I. Histamine content of dogs' stomachs (mg./kg.) Pyloric Fundic No. Condition Mucosa Muscle Mucosa Muscle hr. starvation ,, ,, ,, Average hr. after meat meal ,, ij,, , Average A number of depressor substances, in addition to histamine, may be present in these tissues. Extracts prepared by acid digestion are free from adenylic acid and related compounds, and from "callicrein," since such treatment destroys these substances. All extracts were assayed before and after atropinization of the cat. Since these two assays were always identical it may be stated that the solutions did not contain choline or acetylcholine in amounts sufficient to be detectable by this procedure. However, an etherized cat does not respond to injections of small amounts of choline, and more sensitive methods have shown this compound to be present. Assay of the solutions against acetylcholine on isolated rabbit intestine showed small amounts of choline compounds to be present, and this result was confirmed by assays after acetylation of the solutions. These assays were kindly performed for us by Mr 0. M. Solandt. Incubation of the solutions with added histaminase under standard conditions as to ph and temperature [McHenry and Gavin, 1932] caused a disappearance of all the depressor activity. It has not yet been demonstrated that this enzyme is specific for histamine, but it does not

3 236 G. GAVIN, E. W. MOHENRY AND M. J. WILSON. affect several related amines and iminazole compounds or choline. Incubation without histaminase did not alter the depressor effects of the gastric extracts. Previous work in this laboratory showed that histamine was not formed by the acid digestion procedure. Pure egg albumin and pure casein, 2 g. in each case, were carried through the process and the resultant extracts showed no depressor action. Solutions of l-histidine dichloride were similarly treated with the same result. After adding l-histidine dichloride to minced stomach, the mixture was extracted, and no increase was found in the depressor content of the final extract when compared with controls from the same lot of tissue. However, this procedure might have liberated the amine from a loosely bound complex in which it already existed in the decarboxylated form. Simple saline extracts of gastric tissues were found to contain a depressor substance which was similar to that present in the extracts resulting from acid digestion, and these saline extracts likewise lost their depressor effect when incubated with histaminase. Although these extracts of gastric tissues contained small amounts of choline compounds, the evidence cited in the above three paragraphs justifies the conclusion that all the depressor activity measured by the etherized cat procedure was due to histamine. Table II gives the average total amounts of histamine present in each type of tissue. Not only does fundic contain more histamine per TABLE II. Histamine content of a fasting dog's stomach. Amount of Weight histamine P.c. of Tissue g. mg. total Pyloric Pyloric muscle Fundic Fundic muscle Total 5.1 kilogram than does pyloric, but there is much more of the former tissue in the stomach. A surprisingly large amount, 80 p.c. of the total histamine content of the stomach, was present in the fundic, while only 12 p.c. was found in the pyloric. If the suggestion of Sacks, Ivy, Burgess and Vandolah is correct, that "gastrin" is histamine, then the fundic is probably a much more important source than the pyloric section. Muscle from either part of the stomach contains little histamine.

4 HISTAMINE IN CANINE GASTRIC TISSUES. 237 Since Edkins has stated so definitely that fundic extracts do not produce secretion, and this has received general acceptance, it seemed essential to carry out experiments on this point. For this work a dog with a gastric fistula was prepared for us by Dr Best. This dog has been given a number of solutions subcutaneously, and the stomach contents have been collected at 5-min. intervals after the injection, by means of a catheter inserted through the fistula. The results of these experiments are summarized in Table III. Simple saline extracts of fundic, TABLE III. Secretagogue effects of gastric extracts. Injected solution Volume of gastric juice Histamine collected in content 30 min. Volume and nature mg. c.c c.c. histamine standard c.c. histamine standard c.c. solution prepared by HCl digestion of fundic c.c. saline extract of fundic c.c. saline extract of pyloric c.c. saline extract of fundic c.c. solution from same lot incubated with histaminase 0 (formerly 0 4) None 70 c.c. solution from same lot incubated without histaminase c.c. of same lot after 6 days in refrigerator c.c. saline extract of pyloric 0 None incubated with histami- (formerly 0 2) nase 60 c.c. saline 0 None and solutions prepared from 8imilar tissue by acid digestion, definitely caused gastric secretion. Their power to do so was destroyed by incubation with histaminase, while a control experiment showed that the enzyme, and not simply incubation, was the inactivating factor. Histaminase similarly inactivated an extract of the pyloric, thus confirming Ivy's results. In all cases disappearance of secretagogue powers was coincident with a loss of depressor activity. As indicated above, this latter effect was due to the presence of histamine. Since the possibility existed that the dog might have developed a conditioned reflex, an injection of saline was given, but this failed to evoke secretion.

5 238 G. GAVIN, E. W. McHENRY AND M. J. WILSON. SUMMARY. 1. Histamine is contained in relatively large amounts in the mucous membrane of the stomach of the normal dog and in smaller quantities in the gastric muscle. Of the total amount of this substance contained in a stomach, approximately 80 p.c. is present in the fundic. 2. Extracts of dog's fundic, as well as those from pyloric tissue, have secretagogue powers which are lost after incubation of the solutions with histaminase. The results of Sacks, Ivy, Burgess and Vandolah on pyloric are thus confirmed and, in addition, it is shown that the fundic contains more secretagogue material than the pyloric. This result does not support the findings of Edkins. This investigation was carried out in the laboratory of, and under the direction of, Prof. C. H. Best, to whom and to Dr J. G. FitzGerald, Director of the School of Hygiene, we are greatly indebted. REFERENCES. Best, C. H. and McHenry, E. W. (1930). J. Phy8iol. 70, 349. Edkins, J. S. (1905). Proc. Roy. Soc. B, 76, 376. Edkins, J. S. (1906). J. Phy8iol. 34, 133. McHenry, E. W. and Gavin, Gertrude (1932). Biochem. J. 26, Popielski, L. (1920). PJfilger Arch. 178, 214. Sacks, J., Ivy, A. C., Burgess, J. P. and Vandolah, J. E. (1932). Amer. J. Phy8iol. 101, 331.