325

J. Physiol. (1978), 285, pp. 325-340 With 10 text-figure" Printed in Great Britain

ACID SECRETION BY ISOLATED CANINE GASTRIC MUCOSA

BY YUH-JYH KUO AND LINDA L. SHAXBOUR From the Department of Physiology, the University of Texas Medical School at Houston, Houston, Texas 77030, U.S.A.

(Received 31 March 1978) SUMMARY

1. An isolated gastric mucosal preparation from the dog stomach which is capable of acid secretion is described. Average values for normal resting potential difference (p.d.) was 49 + 2 mV (mucosal side negative with respect to the serosal side), shortcircuit current (I8c) was 172 + 4 ,uA and resistance (R) was 285 + 6 Q. cm2. Low rates of spontaneous acid secretion (0-0.58,uequiv/cm2.hr) were present initially but following short-circuiting of the tissue these values decreased to low levels (less than 0.1 uequiv/cm2. hr) within an hour. 2. Histamine in doses exceeding 10-6 M stimulated acid secretion, increased Ih, and decreased R. Concentrations ranging from 10-5 to 8 x 10-4 M produced maximal secretion. The maximal secretary rate achieved was 4-24 + 0 35 ,uequiv/cm2. hr. 3. Pentagastrin (10-8 M) and acetylcholine (10-6-10-5 M) also stimulated acid secretion with a lower maximal secretion as compared to histamine stimulation. These concentrations of pentagastrin and acetylcholine did not alter histamine stimulated acid secretion. Higher concentrations of pentagastrin (10-6 M) and acetylcholine (10-4 M) reversibly inhibited acid secretion of histamine stimulated mucosa. 4. These results demonstrate that there are many similarities between in vitro and in vivo findings on the dog stomach, indicating the great potential of the in vitro dog gastric mucosa for studies on the mechanism of action and interaction of gastric secretagogues. INTRODUCTION

Isolated preparations of gastric mucosa provide experimental advantages for the study of gastric function, as several complex factors affecting gastric secretion can be excluded. The amphibian isolated mucosal preparation has been used extensively and is well documented (Rehm, 1962; Kasbekar, 1967; Shoemaker, Hirschowitz & Sachs, 1967). However, only recently have successful and satisfactory preparations been developed for studies on the mammalian stomach. These include: the isolated rat stomach (Wan, Assem & Schild, 1974), guinea-pig stomach (Holton & Spencer, 1976), piglet mucosa (Forte, Forte & Machen, 1975), rabbit gastric mucosa (Fromm, Schwartz & Quijano, 1975), kitten fundic mucosa (Tepperman, Schofield & Tepperman, 1975) and mouse stomach (Wan, 1977). Most in vivo animal studies for gastric electrophysiological and pharmacological analyses have been performed on the dog. However, studies on isolated dog gastric musoca have been reported in only one abstract (Kitahara & Hogben, 1968), with the preparation and results not clearly described. Our early attempts to obtain acid

Y.-J. KUO AND L. L. SHANBOUR secretion in the isolated dog gastric mucosa were not successful, i.e. there was spontaneous secretion and the response to histamine was negligible under the conditions used in the experiments (Kuo, Shanbour & Sernka, 1974). The purpose of the present study was to develop a satisfactory preparation of the isolated dog gastric mucosa. The effects of secretagogues (histamine, pentagastrin and acetylcholine) on acid secretion and electrophysiological parameters are described. 326

METHODS

Mongrel dogs (15-20 kg) of either sex were fasted for 24-48 hr before experimentation with water available ad lib. This treatment resulted in stomach contents free of solid material. The dogs were anaesthetized with intravenous chloralose and ethylcarbamate (1 ml/kg of a solution containing 9-25 g chloralose and 92-5 g ethyl carbamate in 150 ml. normal saline). A midline laparotomy was performed and the viscera were exposed. A portion of the fundic area of the stomach was removed and rinsed in warm saline. The mucosal layer was carefully separated from the serosal muscle coat and connective tissue by scissors and forceps dissection (Sernka & Hogben, 1969). The resulting preparation was referred to as the gastric mucosa proper and the muscularis mucosa. The mucosa was mounted as a flat sheet between two lucite half-chambers

having exposed areas of 1 cms (Hogben, 1972). The chamber was maintained at constant temperature (36-37 'C) with a thermoregulator. A 10 ml. volume of warmed bathing solution was introduced into the chamber and immediately bubbled with 95 % 0 2-5 °% CO2. Recordings of electrical p.d. were subsequently initiated. With experience the preparative procedures, from the time the piece of fundic stomach was removed until the chamber was assembled and operational, took less than 15 min. The Ringer solution bathing the serosal (nutrient) side of the mucosa contained (M ): 135-0 Na+, 5-0 K+, 1-0 Ca'+, 1-0 Mg2+, 115-0 Cl-, 25-0 HCO3-, 1-0 SO42-, 1-0 HPO42- and 25-0 glucose, while the unbuffered mucosal (luminal) solution contained 135-0 Na+, 5-0 K+, 1-0 Mg2+, 115-0 Cl-, 13-5 SO42- and 39-5 mannitol. The concentrations of Na+, K+ and Cl- and the osmolality of both mucosal and serosal solutions were the same. Both solutions were agitated with the 95 % 0 2 and 5 % CO 2 gassing mixture which was first bubbled through water to minimize evaporative water loss. The pH of the serosal Ringer solution was between 7-3 and 7-4 whereas the pH of the unbuffered luminal solution was between 4-9 and 5 0 when gassed with 95 % 0 2-5 % CO 2. The serosal fluid was replaced with fresh warm Ringer

solution every 1-1-5 hr. The bridges for measurement of transmucosal electrical p.d. and for the passage of electric current through the mucosa were made of 154 mm-NaCl in 3 % agar. The p.d. bridges were located within 2-3 mm of the mucosa and connected through calomel electrodes (Radiometer K401) to a recording potentiometer of an automatic voltage clamp system (Shanbour, 1974). The potentiometer has 100 mV sensitivity full scale and less than 0-5 mV error. The observed p.d. was corrected for the small p.d. (less than 1 mV) between calomel electrodes plus agar saline bridge pairs. External current was delivered through Ag-AgCl electrodes in agar saline separated from the bathing solutions by diffusion barriers. By sending sufficient external current, the p.d. across the mucosa could be reduced to zero. The current required to maintain zero potential is equivalent to the current continuously generated by the mucosa and is referred to as the short-circuit current (I,,). After the p.d. reached a steady state (approximately one-half hour following mounting), the mucosa was short-circuited throughout the course of the experiments. With the automatic voltage clamp system. (Shanbour, 1974), the I,, was interrupted periodically for determination of the spontaneous transmucosal p.d. Resistance (R) was calculated as the ratio of open-circuit p.d. to I. (Rehm, 1962). Isolated gastric mucosse were generally allowed to equilibrate for 1 hr following short circuiting and before addition of secretagogues. The rate of H+ secretion was measured by means of the pH stat method introduced by Durbin & Heinz (1958). The radiometer of a Copenhagen pH stat (pH meter 28, Titrator 11 and autoburette 11) was used. The pH of the mucosal solution was maintained at a constant value between pH 4-7 and 5.0 by the addition of standardized 0-005 or 0-02 N-NaOH. The titrator was calibrated by the addition of known amounts of standard HCl to the mucosal chamber while the two chambers were separated by a piece of paraffin paper and 95 % 0 -575% CO2 was used to stir the fluid. Titrant additions into the mucosal side or

ACID SECRETION BY ISOLATED GASTRIC MUCOSA

327

secretagogue additions into the serosal side were usually small (less than 0X5 ml. during the entire experiment) and did not appreciably increase the volume of the chamber solution. Using the pH settings described above, the error in calculation of H+ secretion according to theoretical considerations by Sanders, Hayne & Rehm (1973) was negligible. The appearance of lactate and pyruvate on the luminal and serosal sides of the mucosa was determined 1 hr before and 1-2 hr after histamine was added into the serosal solution. The collected samples were assayed immediately by a lactate dehydrogenase method (Gutman & Wahlefeld, 1974). The secretagogues used in the experiments were: histamine phosphate (Eli Lilly and Company), pentagastrin (Ayerst Lab. Inc.) and acetylcholine chloride (Sigma Chemical Company). All secretagogues were made in concentrated solutions and added to the serosal solution in volumes of 0 1 ml. or less. Acetylcholine chloride was made fresh with distilled water for each experiment. Pentagastrin was dissolved in 0.1 N-ammonium hydroxide solution and neutralized with 0'1 NHCl. This solution was kept in the freezer at -40 'C. and used within a month. Its activity was tested by intravenous infusion of pentagastrin in amounts sufficient to stimulate acid secretion in the in vivo chambered canine stomach preparation (Kuo & Shanbour, 1976). Histamine phosphate was supplied in 1 mg histamine base per 1 ml. ampoule. The pH of the serosal solution did not change following the addition of secretagogues. The kits for analyses of lactate and pyruvate were obtained from Sigma Chemical Company.

TABrE 1. Potential difference, short-circuit current, resistance, spontaneous secretion and wet weight of isolated dog gastric mucosa P.d. (mV) I,, (#A) R (0.cm2) H+ (puequiv/cmin.hr) Wet weight (mg/cm2)

X + s.E. 49+ 2 172 + 4

285+6 0-32 + 0-04 69-05 + 5-32

Range 31-65 108-220 204-375 0-0-58 38-08-110-4

n

36 36 36 17 16

P.d., I,, and R were the values at steady state and before adding secretagogues. Spontaneous secretion (H+) was the total secretion in 1 hr after the membrane circuited. Mean + standard error (x + s.E.); and number of tissues(n).

was

short-

RESULTS

Re8ting mUco8a. P.d., ,,c' R, spontaneous H+ secretion and tissue wet weight in the isolated dog gastric mucosa are shown in Table 1. Most of the tissues displayed some spontaneous secretion when bathed in the nutrient solution and short circuited but decreased to low levels during the first hour. Simultaneously, p.d., I., and R reached a steady state. A typical experiment is illustrated in Fig. 1. Due to the above characteristics, isolated dog gastric mucosae were generally allowed to equilibrate for 1 hr following short circuiting and before adding secretagogues. 'Resting' gastric mucosal preparations were thus obtained. The mucosa was responsive to stimuli for at least 3 hr following short circuiting of the tissue.

Sensitivity to aecretagoguea A Histamine. typical response of the isolated dog gastric mucosa to the addition and removal of histamine is shown in Fig. 2. Before the addition of histamine, the resting mucosa reached steady state with no spontaneous acid secretion, exhibited a p.d. of 41 mV, I&, of 124 ,uA and a resistance of 332 Q. cm2. Following the addition of 10-6 M histamine to the serosal solution (arrow labelled H), short-circuit current

328 Y.-J. KUO AND L. L. SHANBOUR increased, resistance decreased, acid secretion commenced with no change in p.d. The initiation of acid secretion was observed within 3-10 min following histamine addition in thirteen experiments. After histamine was removed from the nutrient solution by washing the serosal side with fresh solution (at least three times), resistance returned to near-resting levels. Both p.d. and Ih, increased to levels slightly greater than resting levels. Acid secretion was abolished within 10 min. The mucosa was still responsive to histamine 1 hr after washing. 7

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A typical experiment illustrated in Fig. 3 shows the long-term response of the mucosa to histamine. The addition of histamine to the serosal fluid resulted in the establishment of secretion, an initial increase in p.d. and 'Sc with a decrease in resistance. Within 50 min following the addition of histamine, the rate of acid secretion reached maximum, followed by a slight decrease that remained stable for over 3 hr. There was a tendency for the p.d. and 'sc to decline and for the resistance to recover to a level that was greater than before histamine. In each of a total of nine experiments, there was an initial increase in I~c and a decrease in R. The initial change in p.d. was not consistent, showing either an increase, no change or a decrease. When the secretory rate reached a steady state (40-60 mmn after histamine), the

329 ACID SECRETION BY ISOLATED GASTRIC MUCOSA average p.d. and R were significantly lower than in the resting state (Table 2). However, the decrease in I'c in this state was not significant. The average maximal secretary rate was 4-24 + 0- 35 ,uequiv/cm2 .hr. After this event, potential difference and IC continued to decrease, although in two out of nine experiments, the p.d. and I., were still greater than the values before histamine. There was considerable variation in the change in resistance following steady state. In five out of nine experiments, resistance remained at a stable value. In three experiments, resistance

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TAI3I!

330 Y.-J. KUO AND L. L. SHANBOUR increased to above the pre-histamine level, while a sustained decrease in resistance was observed in one other experiment. The histamine concentration in the nutrient solution in the above experiments was 4-5 x 10-5 M and it is possible that this concentration exceeds the optimal concentration for the production of the maximal secretary rate. It is also possible that the response in electrophysiological parameters is a pharmacological rather than

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physiological effect of histamine. With much higher doses of histamine, secretion could be inhibited. In order to test these possibilities, the effects of different histamine concentrations were determined (Fig. 4). Histamine, 10- and 10-7 M, did not produce any significant changes in acid secretion or p.d. while I., decreased slighty and resistance increased with 10-7 M histamine. Addition of 10-6 M histamine produced an increase in Isc, an initiation of acid secretion, a decrease in resistance and a slight decrease in p.d. Administration of 10-5 M-histamine was followed by an increase in secretary rate and relatively little change in the electrophysiological parameters. When increasing concentrations of histamine (ten and eighty fold) were added to the serosal solution, secretary rate remained at a plateau, short-circuit

2

331 ACID SECRETION BY ISOLATED GASTRIC MUCOSA current and p.d. decreased with a recovery in resistance. Two additional experiments were performed and the results were essentially the same as shown in Fig. 4. The final concentration of histamine in the nutrient fluid was calculated on the assumption that histamine was not absorbed by the mucosa. Obviously, especially at the lower concentrations, the actual final concentration might be much less than the calculated value. In the experiment shown, the threshold of histamine concentration for stimulating acid production was approximately 10-6 M and maximal secretary rates were attained between 10-5and 10M SI.

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experiments, histamine-stimulated secretion was also reduced by this large dose of pentagastrin, while 10-8 M-pentagastrin increased acid secretion, i.e. secretary rate increased from 4-89 to 5-88 ,uequiv/cm2.hr. Inhibition of histamine stimulated acid secretion with the high concentration of pentagastrin was reversible when the pentagastrin was washed out of the serosal solution. When the serosal side of the mucosa was exposed to a high concentration of pentagastrin (10-6 M) there was an initial decrease in p.d. and Isc, and an initial increase in R (Fig. 8). After this initial response (approximately 10-20 min), potential difference and I.c tended to increase and resistance decreased. Acid secretion remained at

334 Y.-J. KUO AND L. L. SHANBOUR a low value (less than 0 2 #sequiv/cm2.hr). In the presence of this high level of pentagastrin in the serosal solution, administration of histamine into the serosal side produced only a low secretary rate (0.5 /tequiv/cm2. hr.) Potential difference and 'Sc exhibited a slight initial increase then decreased and resistance decreased. As acid secretion started to decrease, p.d., I,, and R increased. AL

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The effects of low and high doses of acetylcholine on the histamine stimulated gastric mucosa were similar to those of pentagsatrin except for the differences in the concentrations. A low concentration (10-6 M) of acetylcholine produced no change in acid secretion while a high concentration (10-4 M) inhibited acid secretion and increased p.d. and R (Fig. 9). Although all three secretagogues stimulated acid secretion in the isolated dog gastric mucosa, the potency varied. A comparison of dose-response curves for the three secretagogues is shown in Fig. 10. The following may be concluded from this data: (1) the maximal secretary rate achieved was histamine > pentagastrin > acetylcholine, (2) on a molar basis, pentagastrin > acetylcholine > histamine in their effects on inducing acid secretion; (3) the concentrations of pentagastrin, acetylcholine and histamine to elicit maximal secretary rate were 1 x 10-8, 2 x 10-6 and 1 x 10-5 M, respectively; (4) there was an optimal concentration to stimulate

335 ACID SECRETION BY ISOLATED GASTRIC MUCOSA acid secretion for pentagastrin and acetylcholine, however, a concentration of histamine as high as 8 x 10-4 M did not reverse the effects on acid secretion. Organic acid production. The rate of appearance of pyruvate in the luminal and serosal bathing solutions before and after histamine addition was less than 002 ,uequiv/cm2 . hr. Simultaneously, the rate of appearance of lactate was less than 0*2 /tequiv/cm2.hr. Secretion of both organic acids was negligible compared to the rates of luminal acid.

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Fig. 8. Effects of histamine (H, 4-5 x 10-5 M) on pentagastrin (P) affected dog gastric mucosa in vitro. DISCUSSION

Characteristics of the isolated dog gastric mucosa. The isolated dog gastric mucosa has a more stable p.d. than the isolated stomach wall preparation (Kitahara, Fox & Hogben, 1969). Potential difference was maintained at a high value for over 3 hr in the isolated mucosa whereas p.d. decreased by about 50 % in the isolated stomach wall preparation, indicating that the latter deteriorated with time. The deterioration

Y.-J. KUO AND L. L. SHANBOUR

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3337 ACID SECRETION BY ISOLATED GASTRIC MUCOSA may have been the result of inadequate oxygenation due to the thickness of the tissue since the isolated gastric mucosa is much thinner than the whole stomach wall (69.05 + 5-32 mg wet weight/cm2 compared to 295 + 30 mg wet weight/cm2). The comparable p.d. values (49 mV) between the isolated mucosa and the in vivo situation also demonstrates that the epithelial integrity is not damaged when the muscle coats are separated from the mucosa. Previous studies on acid secretion with the isolated dog gastric mucosa have reported low acid secretary rates (Kuo et al. 1974), possibly because of the different nutrient solution employed. In early studies, TES buffer (composition (mM): NaCl, 133-0; KCI, 5-0; MgSO4, 1-0; CaCl2, 1P0; Na2HPO4, 1P0; N-tris (hydroxymethyl) methyl-2-aminoethane sulphonic acid (TES), 10-0; glucose, 25-0; and NaOH, approximate 4-0; pH = 7-4.) was used as the nutrient solution and was gassed with 100% 02; however, a bicarbonate-Ringer and 95 % 02-5 % CO2 gas mixture was used in the present studies. Sanders et al. (1973) have shown that exogenous CO2 is essential for acid secretion in the isolated frog gastric mucosa. The CO2 may also be a critical factor for the production of acid in the in vitro dog gastric mucosa. The isolated dog gastric mucosa has essentially a low rate of spontaneous secretion as compared to frog (Rehm, 1962), bullfrog (Kasbekar, 1967), rat (Sernka & Hogben, 1969), guinea-pig (Shoemaker, Sachs & Hirschowitz, 1966; Holton & Spencer, 1976), piglet (Forte et al. 1975) and rabbit (Fromm et al. 1975). Furthermore, the spontaneous secretion continued to decrease after electrophysiological parameters stabilized. Since spontaneous secretion of acid may be followed by relative insensitivity to added secretagogues (Shoemaker et al. 1966; Sernka & Hogben, 1969), non-secreting resting mucosa are preferable for in vitro studies. For example, investigators have attempted to obtain resting gastric mucosal preparations in amphibians by two methods. The first method has been to incubate the spontaneously secreting mucosa in histamine-free nutrient media for more than 16 h to obtain a zero secretory state (Kasbekar, 1967). In the second, spontaneously secreting epithelia, after 60-120 min of incubation in the absence of both exogenous substrate and secretagogue, have been treated with 0- 1-1-0 mM-burimamide until secretion was abolished (Shoemaker, Buckner, Spenny & Sachs, 1974). In contrast, the isolated dog gastric mucosa is a resting mucosa without any drug treatment, and the mucosa is sensitive to secretagogue stimulation. Therefore, physiologically induced changes in transport can be related to changes in the electrical and metabolic parameters of the mucosa. Davenport & Jensen (1948) have shown that there are significant releases of pyruvic and lactic acids in the isolated mouse stomach. Similar findings have been obtained in the isolated guinea-pig stomach (Holton & Spencer, 1975). However, in the isolated rabbit gastric mucosa, Fromm et al. (1975) could not demonstrate that pyruvate and lactate contributed significantly to the acid production. In the present studies, luminal appearance rates of pyruvate and lactate were less than 0-5 % and 5-0 % of the rate of acid secretion, respectively, indicating that both organic acids could not account for the luminal acid secretion. Additionally, the observation that consecutive settings of the pH at 5-8 and 4-8 made no essential difference in the secretary rate provides further evidence that the contribution of organic acids to luminal acidification is negligible. If the source of H+ ions was an organic acid that entered the secretary fluid in its undissociated form, then the

Y.-J KUO AND L. L. SHANBOUR measured secretary rate would be a function of the pK of the organic acid, i.e. altering the secretory rate when the setting of the pH stat was changed. The maximal acid secretion of the isolated dog gastric mucosa was 4-24 sequiv/ cm2. hr. This value is only slightly less than the secretary rate obtained from the piglet gastric mucosa (Forte et al. 1975) and is comparable to other species, including frog, bullfrog, Necturus, rat, guinea-pig, rabbit and kitten. Stimulation of acid secretion with histamine in the isolated dog gastric mucosa resulted in an initial increase in short-circuit current and a decrease in resistance. This was probably due to stimulation of not only H+ secretion, but also of other ion transport mechanisms, such as Cl- transport. The changes in the electrical parameters were also not as great as the changes in acid secretion following pentagastrin and acetylcholine administration, indicating changes in other ion transport processes. Ion flux studies are necessary to further support or refute this conclusion. Comparison with in vivo dog studies. The dog is one of the few mammalian species whose stomach does not secrete acid spontaneously in vivo (Mao, Jacobson & Shanbour, 1973). No spontaneous acid secretion after electrical parameters stabilized in vitro confirm the in vivo findings, indicating a specific characteristic of the dog stomach. Histamine, gastrin and acetylcholine may act in vivo directly on the fundic glands to provoke acid secretion (Grossman, 1967). Maximal responses to histamine and gastrin are equal in dogs with vagally innervated main stomach (gastric fistulas) when the drugs are given by continuous intravenous infusion (Passaro & Grossman, 1964). However, the maximal acid response of the vagally denervated Heidenhain pouch to gastrin is only 45 % of that to histamine (Gillespie & Grossman, 1963). This discrepancy in the in vivo results has not been explained but is probably due to the numerous factors involved, i.e. different surgical procedures and complex neural and systemic hormonal influences. The present studies with isolated gastric mucosal preparations without these complex factors suggests that the potency of the direct effect of secretagogues on the parietal cell in the dog is histamine > pentagastrin > acetylcholine. No inhibitory effects of histamine on the isolated dog gastric mucosa were observed at histamine concentrations up to 8 x 10-4 M. In vivo, supramaximal doses of histamine infusion have been demonstrated to produce a comparable or a slightly lower secretary rate than maximal acid secretion in the gastric fistula preparation (Marks, Komarov & Shay, 1960) and in the Heidenhain pouch (Gillespie & Grossman, 1963). Intravenous infusions of gastrin (Gillespie & Grossman, 1963) and acetylcholine into the arteries of the gastric fundus of the dog (Pevsner & Grossman, 1955) have been demonstrated to elicit stimulation of acid secretion at low dosage rates and suppression of acid secretion at high rates. The present studies demonstrate that there are optimal concentrations for gastrin and acetylcholine to stimulate acid secretion. Pentagastrin and acetylcholine may have failed to stimulate acid secretion in other isolated mammalian gastric mucosae (Holton & Spencer, 1976; Forte et al. 1975) because of incorrect selection of doses in addition to species differences. The inhibitory action of high doses of pentagastrin and acetylcholine on histamine stimulated acid secretion in vitro is also similar to that observed in vivo. Gillespie & Grossman (1963) have shown that a high dose of gastrin, when administered as a 338

ACID SECRETION BY ISOLATED GASTRIC MUCOSA 339 single rapid intravenous injection to dogs secreting in response to a continuous injection of gastrin or of histamine, produced marked inhibition of acid secretion. The inhibitory effects of large doses of cholinomimetic drugs on histamine stimulated acid secretion have also been demonstrated by Gray & Ivy (1937). Grossman and his colleagues (Anderson & Grossman, 1965; Gillespie & Grossman, 1964; Johnson & Grossman, 1969; Passaro, Gillespie & Grossman, 1963) have reported that in the innervated or denervated dog stomach potentiation of gastric acid secretion occurs with urecholine and gastrin, urecholine and histamine and gastrin and histamine. However, Hirschowitz, Sachs & Hutchison (1973) did not observe potentiation or synergism between histamine and pentagastrin in the gastric fistula dog preparation. Potentiation was also not observed in the present study. Dr Shanbour is the recipient of Research Scientist Development Award 5 K02 AA-70463-05. This work was supported in part by N.I.H. grant 2 RO1 AA-00194-06.

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