J. Phy8iol. (1976), 255, pp. 465-479 With 5 text-figuree Printed in Great Britain

465

ACID SECRETION BY GUINEA-PIG ISOLATED STOMACH

By PAMELA HOLTON AND JAN SPENCER From the Department of Physiology, St Mary's Hospital Medical School, Paddington, London, W2 1PG (Received 5 June 1975) SUMMARY

1. An isolated stomach preparation from the guinea-pig is described. 2. Both histamine acid phosphate (1-4 jig/ml.) and theophylline hydrate (0.2-3-2 mg/ml.) separately stimulated hydrochloric acid, HCl, secretion from the guinea-pig stomach preparation. A linear dose-response relationship was obtained for theophylline. 3. Addition of theophylline (0.2 and 1-6 mg/ml.) during maximal response to histamine increased the secretion further, whereas addition of histamine during maximal response to theophylline did not cause further secretion. 4. The secretary activities of Noz-MeH (0.3-5.0 /LM), NO-cMe2H (1.29.5 /M) and 5-MeH (1-5-12 #M) were compared with histamine (0.9-13 /LM) on a threshold background secretion induced by theophylline (0-2 mg/ml.). Linear log.-dose response relationships were obtained for each test drug. The results confirm that Na-MeH is a more potent secretagogue than histamine. 5. Pentagastrin (0.3-1 0 ajg/ml.) stimulated HCl secretion in approximately half the experiments. The response was often transitory. In the other experiments, pentagastrin had no effect on HCl secretion although subsequent administration of histamine did stimulate HC1 secretion.

INTRODUCTION

Investigations in which drugs are used to study the mechanisms involved in the gastric acid secretary process necessitate the availability of in vitro preparations where possible complicating factors such as transport, metabolism and excretion of the drug are minimized. The amphibian isolated mucosa preparation has been used extensively and has been considered far superior to mammalian preparations in terms of survival and experimental reproducibility. The early work on mammalian preparations suggested that special

PAMELA HOLTON AND JAN SPENCER conditions were necessary for satisfactory secretion. Davenport (1947) developed an excised mouse stomach preparation for studying metabolism which Davenport & Jensen (1948) used to study acid secretion. However, in 1950, Davenport & Chavre improved the rate of acid secretion from this preparation by using hyperbaric oxygen and adopted this as a routine. Dikstein & Sulman (1965) described an everted rat stomach preparation. The test substances were added to the mucosal Ringer which was buffered and contained an anti-foaming agent. Hydrochloric acid, HCl, secretion was measured as a fall in pH of the mucosal solution. Shoemaker, Sachs & Hirschowitz (1966) studied the electrical and secretary properties of the guinea-pig isolated mucosa but could not stimulate secretion. They observed a very low electrical potential which suggested damage to the transport mechanism and their results were not reproducible. However, recently some of these practical problems have been overcome and more satisfactory preparations have been described. These include: the rat isolated stomach (Wan, Assem & Schild, 1974); the rat isolated mucosa (Hearst & Main, 1976); the piglet mucosa (Forte, Forte & Machen, 1975); and the rat whole stomach (Parsons, 1975). This paper describes the guinea-pig isolated stomach preparation which we have developed and used to demonstrate stimulation and inhibition of gastric HCl secretion with a variety of agents. 466

METHODS Standard procedure Male guinea-pigs (300-500 g) were given a low residue diet for 48 h or for 24 h. In some experiments food was subsequently removed for 24 h. Water was freely available. This treatment resulted in stomachs without solid contents. The guineapigs were stunned by a blow on the head and killed by exsanguination. The stomach was rapidly removed, cut along the lesser curvature, inverted, washed and placed in ice-cold serosal saline. The composition of this solution was (mM): 110, NaCl; 5, KCl; 3-6, CaCl2; 1-2, MgCl2; 26, NaHCO3 and 16-7, glucose (Sernka & Hogben, 1969). The acid secretary portion of the stomach was then cut along the greater curvature into half-stomach preparations. Each preparation was slightly stretched and tied over the end of a short Perspex tube (diameter 1-2 cm), mucosal side inwards and set up at 340 C in a bath containing 100 ml. serosal saline bubbled with 95% 02 and 5% C02. 4-5 ml. mucosal saline solution (in mM: 136, NaCl; 5, KCl; 3-6, CaCl2; 1-2, MgCl2 and 16-7, glucose), bubbled with 100% 02, was added to the mucosal side, so that the levels of fluid were the same on either side of the preparation (Fig. 1). The mucosal solution was removed and replaced every 15 min, the samples were titrated first to pH 5-2, then to pH 7-0 against 10-3 MNaOH. The pH was measured with an EIL glass electrode and an EIL pH meter. The serosal solution was renewed at least every hour; drugs were administered to the serosal side of the preparation. The following drugs were used: histamine acid phosphate (B.D.H.); Na-methylhistamine dichloride, Na-N0-dimethylhistamine dichloride and 5-methyl histamine

HCl SECRETED BY GUINEA-PIG ISOLATED STOMACH 467 dichloride (institute of Pharmacology and Institute of Pharmaceutical Chemistry, Parma, Italy); pentagastrin (Peptavlon, I.C.I.); theophylline hydrate (B.D.H.); sodium thiocyanate (Fisons); and burimamide and metiamide (Smith, Kline & French Laboratories).

100% °

\0

5%

C02+95 % 02

Water 34 IC out

Serosal saline Mucosal saline Water

Tissue

Fig. 1. Experimental arrangement for studying secretion by the guineapig stomach in vitro. See text for details of dimensions of the apparatus and composition of serosal and mucosal salines.

Stimulation of acid secretion. The secretagogues were: pentagastrin, 0 3-I -0 ,ug/mI.; histamine acid phosphate, 1-4 #sgfml.; and theophylline hydrate, 0-2-3-2 mg/ml. A range of doses of histamine and the dichloride derivatives of Nm-methylhistamine, Na-Na-dimethylhistamine and 5-methylhistamine were administered on a secretary plateau induced by 0-2 mg/ml. theophylline. The test drugs were administered for 60-90 min, removed from the serosal bath by washing the preparation twice and the base line secretary level regained before applying another dose. Usually three or four tests could be completed on each preparation. Inhibition of acid secretion. Once stable secretary plateaux had been established, inhibitors of acid secretion were added to the serosal solution for 30 min periods. Inhibitors were removed by washing the preparation twice. The inhibitors tested were burimamide 50 ,ug/ml. and metiamide, 10 ,ug/ml. (H2-receptor antagonists: Black, Duncan, Durant, Ganellin & Parsons, 1972; Black & Spencer, 1973) and sodium thiocyanate, 10 mm, which acts on the hydrogen ion pump (Forte, 1968). Calculation and expression of results. In order to obtain the acid content of each mucosal sample in #smol H+/cm2.h, the titre was multiplied by 4 and divided by the area of the preparation (1.13 cm2). The increase in acid secretion due to administration of stimulant was expressed in units of secretion (Armol H+/Cm2 . h + S.E. of the mean) above a control period secretary plateau. Results obtained using inhibitors were expressed as the percentage decrease of a secretary plateau induced by a named stimulator. The level of resting acid production (a) was subtracted from the level of the stimulated plateau (b). The minimum level of secretion obtained with the inhibitor (c) was also subtracted from the level of stimulated secretion. Therefore, percentage inhibition = b - c/b - a x 100.

468

PAMELA HOLTON AND JAN SPENCER

Preliminary experiments and comments on standard procedure Choice of guinea-pig. The rate at which oxygen can be supplied is thought to limit the rate of acid secretion in isolated stomachs (Davenport & Chavr6, 1950). The guinea-pig was therefore chosen because the whole stomach wall is only 0-55 mm thick. It was considered likely that the gastric secretary responses might vary during the oestrous cycle in female guinea-pigs. In order to rule out this potential source of error, male guinea-pigs were used. No seasonal variations were observed. Isolated stomach or isolated mucosa. As the muscle coat might be expected to interfere with diffusion to and from the secreting cell, a few experiments were carried out in which the muscle layer was removed by blunt dissection. The results obtained with these preparations did not differ significantly from those obtained with the whole stomach preparation. Removal of the muscle coat was not therefore adopted as a routine. Dietary pre-treatment. The stomachs of guinea-pigs on a normal diet were greatly distended with solid food. This had two disadvantages. Distension of the stomach itself causes acid secretion (Lim, Ivy & McCarthy, 1925), and also the stomach had to be handled a great deal in order to wash it out completely. In these preparations, a high rate of spontaneous acid secretion was often observed; the secretion fell gradually throughout the experiment and frequently the preparation did not respond to stimulation. This observation confirms those of R. L. Shoemaker (personal communication). A fasting period of 24 h (Wan, Assem & Schild 1974) was not sufficient to empty the stomach. These guinea-pig stomachs which contained different amounts of solid food gave varying results which were not readily reproducible. Often the preparations could not be stimulated at all. Longer periods of fasting (48-60 h) caused unacceptable weight loss and often ulceration of the mucosa. A procedure of controlled feeding was devised as described above, resulting in a stomach which contained only a small amount of liquid. Choice of temperature. The body temperature of the guinea-pig is 38.50 C. Earlier experiments were carried out at 370 C, but reducing the temperature to 340 C caused no appreciable change in acid production and has the advantage of decreasing metabolic activity and therefore the oxygen requirement. Gassing of the bathing solutions. The gastric mucosa requires more CO2 to secrete HC1 than is produced by tissue metabolism. Davies & Longmuir (1948) found that maximal stimulation of frog isolated gastric mucosa with histamine caused ulceration in the absence of external supplies of CO2. This ulceration was interpreted as being due to accumulation of alkali within the parietal cells, and did not occur when CO2 was supplied. Imamura (1967) confirmed earlier observations that hydrogen ion secretion was severely reduced in the frog isolated mucosa when the concentration of CO2 was below 5 %. Therefore, 500 CO2 was supplied in the gassing mixture to the serosal solution of the isolated guinea-pig stomach. In four experiments, the effect of increasing the concentration of CO2 to 7 % was investigated. The amount of bicarbonate in the serosal solution was also increased to 35-6 mm to maintain the pH at 7-4. No change in the rate of acid secretion was observed. The mucosal solution was unbuffered and gassed with 100 % 02, in order to minimize buffering the acid secreted. On a few occasions, once a stimulated plateau had been established, the rate of gassing of either the mucosal or serosal solution was decreased. A continuous supply of oxygen to the mucosal solution appeared to be more critical than gassing the serosal solution. Although in both cases reduction of the gassing rate caused the plateau level to fall and was reversed by returning to the normal rate of gassing,

HC1 SECRETED BY GUINEA-PIG ISOLATED STOMACH 469 the fall in secretion was larger, more rapid and more prolonged on reducing the supply of oxygen to the mucosal surface. Titration of 8amples. The mucosal samples were titrated to pH 5-2 then to pH 7-0 against 10-3 M-NaOH. The volume of NaOH required to titrate the sample from pH 5-2 to 7 0 represents the buffering capacity of the sample, which did not vary significantly during the experiment. Therefore, any change in total acid secretion represents stimulation or inhibition of HCl secretion. The results presented in this paper are calculated from the titre obtained by titration to pH 70.

RESULTS

Resting acid production The non-stimulated (resting) acid production was 2-4 mol H+/cm2. h and remained stable for the duration of the experiment. This is similar to the basal acid production of 3 /tmol H+/cm2. h found in the guinea-pig by Shoemaker et al. (1966) and 2-7 Ismol H+/cm2.h in the rat (Sernka & Hogben, 1969). Sodium thiocyanate is an inhibitor of gastric acid secretion which is believed to act at the hydrogen-ion pump (Forte, 1968) and we have used it to define HCl secretion. In this paper, thiocyanate sensitive acid production is referred to as HCl secretion, and the portion remaining as non-HCl acid production. In the presence of NaSCN (10 mM), the total resting acid production was decreased to about half (1.01 6 ,mol H+/cm2.h). The resting HCl secretion is probably due to the presence of a small amount of liquid food normally found in the stomach. The substances contributing to the non-HCl acid production have not been fully identified. The following results show that CO2 does not contribute, but that lactic acid production may account for some of the total non-HCl acid production. Carbon dioxide could accumulate in the mucosal solution either from metabolism within the mucosa or by diffusion from the serosal solution. Although we considered that the vigorous bubbling of the mucosal solution would blow out any CO2 from these sources, we tested to what extent CO2 could be contributing to the acid production. In five experiments the mucosal samples were divided in half and one portion was bubbled with t00 % 02 for 5 min before titration. The other portion was titrated immediately. There was no difference in the volume of NaOH required to titrate the paired half samples to pH 7 0. We conclude that there was no dissolved CO2 in the mucosal samples. Lactic acid produced by cell metabolism might also contribute to the non-HCl acid production, although most of the lactate produced in the cell diffuses into the serosal solution (Durbin, 1968). The concentration of lactate required in the mucosal sample to give an acid production of 1.0-1 -6 ,umol H+/cm2. h is 30-48 fLM. Six samples

PAMELA HOLTON AND JAN SPENCER of mucosal fluid were analysed for lactate without extraordinary precautions to avoid contamination. The concentration of lactate varied, but did not seem to be related to the non-HCl secretion. The mean concentration was 29 + 74 /uM (+ S.E. of mean). Thus we have not excluded the possibility that lactate contributes significantly to the non-HCl acid production. Further investigation of this question did not seem to be justified. The gastric mucosa produces a buffer with a pH 6, which is probably mucin (Sernka, 1969). This observation was confirmed in our experiments by the finding that mucosal solution from unresponsive preparations (normally discarded) had a pH 6. We cannot exclude the possibility that this buffer is responsible for some of the non-HCl acid production. 470

B

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0

1

2

5 4 3 Time (h) Histamine

6

7

Fig. 2. Stimulation of HCl secretion by histamine and the effect of burimamide and metiamide on histamine-induced acid secretion. Acid secretion in ,smol/cm2.h is plotted on the ordinate against time in h on the abscissa. Histamine acid phosphate, 2 /zg/ml., induced HC1 secretion and this stimulation was antagonized by burimamide, 50 #sg/ml. (B) and metiamide 10 #g/ml. (M).

Stimulated HCl secretion Histamine.In eighteenexperiments, histamine acid phosphate 1-4/tg/ml. decreased the pH to 3 5-4-0 in the mucosal solution and increased HCl secretion as shown in Fig. 2. The effect was reversible and maintained while histamine was kept in the serosal solution (up to 7 h). The dose-response relationship for histamine, No,-methylhistamine (Na-MeH), Na-Na-dimethylhistamine (Na-Me2H) and 5-methyihistamine

HCI SECRETED BY GUINEA-PIG ISOLATED STOMACH 471 (5-MeH). A preliminary account of these results was given by Holton, Impicciatore & Spencer (1975). The secretary activities of Na-MeH, N'2-Me2H and 5-MeH were compared with histamine on a threshold background secretion induced by theophylline (0.2 mg/ml.). Four or five dose levels, differing by a factor of 2, of the four substances, were

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Acid secretion by guinea-pig isolated stomach.

J. Phy8iol. (1976), 255, pp. 465-479 With 5 text-figuree Printed in Great Britain 465 ACID SECRETION BY GUINEA-PIG ISOLATED STOMACH By PAMELA HOLTO...
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