distension of the stomach wall.

Transcription

1 J. Phy8ii. (1967), 19, pp With 1 text-figure Printed in Great Britain ACCELERATED MOBILIZATION AND FORMATION OF HISTAMINE IN THE GASTRIC MUCOSA EVOKED BY VAGAL EXCITATION BY G. KAHLSON, ELSA ROSENGREN AND R. THUNBERG From the Institute of Physiology, University of Lund, Sweden (Received 28 June 1966) SUMMARY 1. The changes in the rate of histamine formation and in the histamine content of the parietal cell containing region of the gastric mucosa have been studied in rats under the influence of agents which evoke or abolish vagal excitation. 2. The hypoglycaemia producing agents, insulin and 2-deoxyglucose (2-DG), raised the mucosal histamine-forming capacity (HFC) in a way similar to that previously observed on re-feeding, gastrin injection, and distension of the stomach wall. 3. In cats, insulin injection elicited an elevation of mucosal HFC similar to the corresponding effect of insulin in rats. 4. Hoechst 998, which inhibits post-ganglionic cholinergic transmission, counteracted the elevation of mucosal HFC following vagal excitation, but did not inhibit changes produced by gastrin, thus indicating the absence of a cholinergic intermediary link between gastrin and changes in mucosal histamine. 5. It is emphasized that although re-feeding, vagus excitation, gastrin and distension all produce similar changes in mucosal histamine, the clarification of the precise role of histamine as a natural stimulant for the parietal cells may require a fresh kind of approach. INTRODUCTION Re-feeding of fasted rats, mice and frogs evokes a mobilization of histamine and a concurrent acceleration of the rate of histamine formation in the parietal cell containing region of the gastric mucosa. The changes in mucosal histamine pertaining to feeding can be reproduced by injecting a purified gastrin preparation. These changes in mucosal histamine are not merely a product of secretory activity since they do not occur when acid secretion is excited by injection of histamine. Nor do they occur in the 29-2

2 456 G. KAHLSON, ELSA ROSENGREN AND R. THUNBERG non-parietal cell containing region in the rat, the only species so far studied in this particular respect (Kahlson, Rosengren, Svahn & Thunberg, 1964). In the course of the above-mentioned work a feed-back relation between mucosal content of histamine and level of histidine decarboxylase was disclosed whereby lessening of end-product repression (histamine release) resulted in increased enzyme activity and, conversely, increasing the concentration of the end-product by injecting histamine depressed the level of enzyme activity. The nature of the enzyme histidine decarboxylase responsible for mucosal HFC has been studied in its essential features by Kahlson, Rosengren & Thunberg (1963). These authors found that the enzyme responsible for histamine formation in vivo in the species studied is strongly inhibited by a-methylhistidine, and weakly inhibited by ac-methyldopa, and they assessed quantitatively in vitro and in vivo the significance of pyridoxal phosphate as a co-factor. On re-feeding, three distinct, but interrelated, excitatory processes come into operation, vagal excitation, gastrin release, and distension of the stomach. Having investigated the changes in mucosal histamine evoked by gastrin and distension it appeared essential to study changes which might result from vagal excitation. Because of observations on acid secretion suggesting that histamine acts through nerves (Clark, Curnow, Murray, Stephens & Wyllie, 1964), and further, that gastrin stimulates secretion by acting on nerves to release acetylcholine (Bennett, 1965) the effect of a compound blocking cholinergic transmission was also studied. METHODS Animals. Female rats of the Sprague-Dawley strain, weighing g, were used throughout. Young cats of two different litters, weighing 55-8 g, and g, respectively, were employed. Food was withheld for 15 hr in all animals before injections. Drugs. In a first series of experiments in rats the two vagus nerves to the stomach were dissected below the diaphragm and stimulated electrically. Motor responses were obtained but acid secretion was scanty. This mode of vagal excitation was abandoned and replaced by injection of either insulin, 5 i.u./kg, or 2-DG, 1 mg/kg (Hirschowitz & Sachs, 1965). Post-ganglionic cholinergic influences were antagonized by the compound Hoechst 998 (piperidino-ethyl-diphenyl-acetamide),.1 mg/kg (for references see Emmelin & Henriksson, 1953). Purified gastrin 11 was obtained from Professor Gregory, Liverpool, and injected in a dose of 1,ug/kg. Controls received NaCl solution (-9 g/1 ml.), 1 ml./kg. All injections were given subcutaneously. The animals were killed by a blow on the head and bled by the carotid arteries 3j hr after injection of insulin or 2-DG, and 1 hr after gastrin injection; Hoechst 998 was given 4 hr before killing the animal. Tissue preparation. The gastric mucosa and submucosa of the parietal cell containing region were removed by scraping with a scalpel after the stomach had been opened along the minor curvature, washed in ice-cold NaCl solution and pinned flat. The tissue was minced with scissors and examined for histamine content and histidine decarboxylase activity.

3 VAGUS AND GASTRIC MUCOSAL HISTAMINE 457 Determination of histamine content was carried out by a modification of the origina. fluorometriemethod of Shore, Burkhalter& Cohn(1959). Briefly, themodification, as employed by Kahlson et al. (1963), involves isolation of histamine by ion exchange chromatography on Dowex 5 instead of extraction into butanol. A similar procedure has subsequently been used in fluorometric determination of histamine by Green & Erickson (1964). The advantage of the procedure is to improve specificity. It should be mentioned here that the fluorometric method, as employed in this laboratory, is also suitable for determination of free histamine in urine (R. Thunberg, to be published). In thirty-nine instances parallel determinations were made on the same samples with the fluorometric method and the conventional extraction procedure followed by biological assay on the guinea-pig's gut. In some instances additional determinations were carried out by a method involving reaction with dinitrofluorobenzene and thinlayer chromatography as devised by White (1966). Good agreement was found between the three methods as will be recorded in a separate report. Determination of HFC in vitro. The isotopic method originally elaborated by Schayer, and modified in this laboratory, was employed (Kahlson et al. 1963). This involved incubation of tissue samples with [14C]histidine and measurement of the [14C]histamine formed. In this method the concentration of [14C]histidine present in the incubation mixture is very low. Consequently the enzyme with a high affinity for this substrate is measured, whereas other enzymes with low affinity for histidine, i.e. DOPA-decarboxylase, and similar enzymes referred to as non-specific histidine decarboxylases, do not significantly contribute to the formation of ['4C]histamine. The enzyme measured should thus be designated simply as histidine decarboxylase. As already pointed out different amino acid decarboxylating enzymes can be differentiated by the use of inhibitors such as a-methylhistidine and a-methyldopa (Kahlson et al. 1963). RESULTS The changes in levels of mucosal HFC and histamine content were investigated in rats under the influence of vagal excitation produced by insulin or 2-DG. The cholinergic blocking compound Hoechst 998 was employed in order to antagonize changes thus produced. This compound was also used in experiments with the view to see whether a cholinergic mechanism is essential to the pertinent changes produced by gastrin. The results with these various influences and compounds are illustrated in Fig. 1 and detailed in Table 1. In the figure each column depicts the mean value and standard deviation of determinations of HFC and histamine content in five rats. Insulin. The HFC becomes elevated and the histamine content falls after injection of insulin. The change in HFC is largely reduced by pretreatment with Hoechst 998. The connexion between corresponding alterations in histamine content and HFC will presently be discussed in relation to observations on cats. 2-deoxyglucose (2-DG). This compound, alleged to excite gastric secretion via the vagus nerves (Hirschowitz & Sachs, 1965), likewise induced a substantial elevation of mucosal HFC and a fall in histamine content of low significance (P about -5). It was not investigated whether larger doses of

4 458 G. KAHLSON, ELSA ROSENGREN AND R. THUNBERG this compound would produce greater changes. The elevation of HFC evoked by 2-DG was abolished by Hoechst 998. Gastrin. The elevation of HFC and the fall in histamine content observed agree with previous results (Kahlson et al. 1964). These changes are not Control Insulin 2-DG Gastrin 4 3 Fig. 1. HFC (pg/g.3 hr) and histamine content (pug/g) of rat gastric mucosa, in controls and under the influence of insulin, 2-DO and gastrin. The open and the hatched colulmns stand for values obtained in animals injected with NaCl solution and Hoechst 998, respectively, before the injection of NaCl solution (control), insulin, 2-DOS or gastrin. Each column depicts the mean value and standard deviation of determinations in five rats. abolished by treatment with Hoechst 998. Rather, the increase in HFO is even greater. No attempts were made to explain this seemingly paradoxical result. Hoechst 998. This drug was employed in place of atropine because it was found that atropine by itself elevated mucosal HFC, a disturbing phenomenon possibly related to its character as a histamine releaser. In search for a substitute, Hoechst 998 was chosen, in doses sufficient to antagonize the elevation of HFC by insulin, because it did not by itself significantly alter the level of HFO, as shown in Table 1 and in the extreme left section of Fig. 1. Observations in cats. Eight young cats from two litters were examined for mucosal HFC: and histamine content. Each litter was divided into two groups, the one serving as controls injected with NaOl solution, the other injected with insulin. Results listed in Table 2 show that in cats insulin produces changes of HFC which are similar to those seen in rats. The prerequisites for obtaining maxrimum elevation of mucosal HFC under

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6 46 G. KAHLSON, ELSA ROSENGREN AND R. THUNBERG the influence of insulin were not explored. A cat of litter 1 (figures in brackets in Table 2) intended to serve as a control, had escaped from the cage during the period of fasting. This cat did not have access to food but the antral part of the stomach was found stuffed with wooden shavings TABLE 2. HFC (ng/g. 3 hr) and histamine content (,glg) of gastric mucosa in eight young cats injected with NaCl solution (controls) and insulin, respectively. Figures in brackets refer to a cat with distended stomach HFC Content Controls Insulin Controls Insulin Litter 1 (86-93 g) Litter 2 (55-8 g) 38, (8) 44, 7 82, 92 82, 86 32, (23) 26, 27 23, 18 31, 2 and contained a highly acid juice, this in contrast to the other controls in which the stomach was seemingly devoid of juice, and the surface of the mucosa covered with mucus of about neutral reaction. In this cat the mucosal HFC was 8 ng/g. 3 hr. This finding is in agreement with previous observations in rats on the effect of distension of the stomach (Kahlson et al. 1964). With regard to alterations in histamine content it should be borne in mind that in cats the gastric mucosal histamine content in the normal state varies within a wide range among individuals, 5-39,ug/g in sixteen kittens (Haeger, Kahlson & Westling, 1953), 8-4 /tg/g in fifteen adult cats (Haeger & Kahlson, 1952). The number of experiments in Table 2 is too small for any conclusion to be drawn with respect to changes in histamine content. Further, it should be appreciated that a minor fall in content, which functionally might be significant, may not be detectable by the methods employed. Notwithstanding this, an initial but undetectable fall in histamine content might induce an elevation of HFC resulting in rapid replenishment of histamine content. Consequently, an occasional absence of a detectable fall in histamine content in the presence of an elevated HFC should not be taken as evidence against the operation of a feed-back coupling between tissue content of histamine and HFC. It should be noted (see Discussion) that insulin injection, i.e. vagus stimulation, is followed by an elevation of mucosal HFC. DISCUSSION The gastric mucosa of all species studied in this laboratory, man, dog, cat, rat, guinea-pig, hamster, mouse and frog, is rich in histidine decarboxylase. In the rat, mouse and cat mucosal HFC is much higher than in other tissues, exceeded only by the high levels of histidine decarboxylase discovered in embryonic tissues (for references see Rosengren, 1963, 1966). Extirpation of the whole stomach in the female rat is followed by a fall in

7 VAGUS AND GASTRIC MUCOSAL HISTAMINE 461 the urinary excretion of free histamine to considerably less than half of normal (High, Shepherd & Woodcock, 1965); a similar observation was also made in this laboratory (E. Rosengren, unpublished). These findings imply that presumably more than half of the whole-body histamine formation takes place in the stomach wall. Even in the fasting state the glandular mucosa produces histamine at high rates, hence the amine is continuously discharged into the blood stream, and partly excreted. Yet, interdigestive acid secretion is reported to be absent in some species, for example dog and man, and scanty in the rat and cat (Lane, Ivy & Ivy, 1957). A logical conclusion would be that histamine set free from the mucosa is a poor, or ineffective, stimulant of acid secretion. The disappearance of histamine from the gastric mucosa is accelerated by feeding, vagal excitation and injection of gastrin. Smith (1954, 196) reported that a crude gastrin preparation released histamine from the stomach wall and also from other tissues. The mucosal histamine, in particular, has been found susceptible to release by these means. Schayer & Ivy (1957, 1958) discovered that [14C]histamine, laid down in the gastric mucosa of rats following injection of [14C]histidine, was released on feeding, and appeared in the urine. Similarly, vagal excitation and injection of purified gastrin induce release of mucosal histamine (Kim & Shore, 1963; Haverback, Tecimer, Dyce, Cohen, Stubrin & Santa Ana, 1964; Kahlson et al. 1964). The observation that the mucosal histamine content falls under the influence of factors producing excitation of hydrochloric acid secretion has been taken by some authors to support the view originally expressed by Babkin (1938) that histamine constitutes the final link in the chain of events which results in excitation of the parietal cells to secrete (for references see Gregory, 1962; Code, 1965; Haverback, Stubrin & Dyce, 1965; Shore, 1965). Other workers in this field may feel that the mere observation that histamine disappears from the mucosa, whilst secretion subsequent to a meal proceeds for several hours, would be difficult to reconcile with the view as postulated. A different picture presents itself in face of the demonstration that feeding, as well as the principal excitatory components operating subsequent to feeding, i.e. vagal excitation, gastrin and distension of the stomach wall, induce an accelerated rate of histamine formation which persists for several hours. The elevation of histidine decarboxylase is presumed to be brought about by an initial lowering of the mucosal content of preformed histamine, the levels of the enzyme and the end-product being interrelated by the feed-back coupling already referred to. The newly formed histamine moves freely through the stomach wall, as evidenced by its appearance in the urine (Kahlson et al. 1964). It appears difficult to visualize an intramural compartmental arrangement so set as

8 462 G. KAHLSON, ELSA ROSENGREN AND R. THUNBERG to prevent functional contact between the newly formed histamine and the parietal cells, including the blood vessels draining the mucosa. Histamine newly formed at high rates thus appears potentially designed to provide secretory stimulation and, concomitantly, for the vasodilation prerequisite to secretion. This view appears acceptable only on the assumption that during the interdigestive phase the histamine formed at considerable rates either escapes functional contact with the parietal cells, or that the intramural concentration of newly formed histamine is subthreshold for stimulation. Both these assumptions appear perilous and at present do not lend themselves to the test of experiment. In this connexion it should be emphasized that the mucosal histamine content, i.e. the amount of preformed loosely held histamine, lacks stimulatory potency, as evidenced from the fact that the mucosal histamine content is higher in the nonsecreting state and lower during the course of secretion. Having elaborated means to inhibit histamine formation in vivo, Kahlson et al. (1963, 1964) found in rats, in which the mucosal histamine content had been lowered to about 5 % of normal by inhibiting histamine formation, that injection of gastrin still produced the typical elevation of mucosal HFC and an acid secretory response of normal magnitude. These authors conclude that the excitatory mechanism, whatever its precise nature, operates at the terminal link with a wide safety margin. By contrast, Levine (1965) reported that in rats in which histamine formation had been inhibited by a hydrazino analogue of histidine resulting in about 5 % fall in stomach and urinary histamine, the acid secretory response to insulin or gastrin was blocked. It has previously been shown that feeding, injection of gastrin and distension of the stomach induce acceleration of histamine formation by elevation of mucosal histidine decarboxylase activity. The present work reveals a similar change in mucosal histamine under the influence of agents producing vagal excitation. As the ultimate outcome of the various studies it appears clear that the three individual excitatory influences operating on feeding have a common functional feature, namely the power to accelerate greatly formation and turn-over rate of histamine in the parietal cell containing region of the gastric mucosa. The three influences appear to have a common goal, the full elucidation of which, owing to difficulties inherent in the problem, may well be far ahead. This work was supported by Research Grant 5 R1 HD255-6 from the National Institute of Health, Bethesda, Md.

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