Ganglion mediated hydrogen ion and pepsin secretion in Gallus domesticus

Comp. Biochem. PhysioL, 1975, VoL 51A,pp. 633 to 637. Pergamon Press. Printed in Great Britain

GANGLION MEDIATED HYDROGEN ION AND PEPSIN SECRETION IN GALLUS DOMESTICUS RENE G. GIBSON, HARRY W. COLVIN,JR.* AND RAYE. BUROVat Department of Animal Physiology, University of California, Davis, California 95616, U.S.A. (Received 8 April 1974)

Almtract--1. Gastric secretion in White Leghorn cocks was studied following the intravenous administration of gastrin pentapeptide and histamine acid phosphate. 2. Both hydrogen ion and pepsin secretion were inhibited by hexamethonium when gastrin pentapeptid¢ was administered. Only hydrogen ion secretion was inhibited by hexamethonium when histamine acid phosphate was administered. 3. Gastric secretion stimulated by histamine acid phosphate was not inhibited by atropine administration. 4. Gallus domesticus may be capable of a separation of acid and pepsin secretion from the same cell type. The intramural ganglia may play an important role in the control of gastric secretion in this species.

INTRODUCTION THE DOMF.,STICfowl differs from mammalian species in that both acid and pepsin are produced from a single cell type (Menzies & Fisk, 1963; Toner, 1963). The kinetics of gastric secretion in response to various agents have been extensively described in the fowl (Burhol & Hirschowitz, 1970, 1972). No information is available as to the location or type of receptors upon which drugs may act. This is an investigation of possible receptor characteristics and location for two compounds, histamine acid phosphate and gastrin pentapeptide. MATERIALS AND METHODS Single-comb White Leghorn cocks (1.8-2.8 kg body weigh0, which had been fasted for 24 hr, were anesthetized with percutaneous pentobarbital and restrained on their backs; the brachial vein was cannulated for the administration of further anesthetic. The heart rate and blood pressure were monitored from the left internal carotid artery. Body temperature was maintained between 40-5 and 42-0°C. The thoracic and abdominal cavities were opened via a midventral incision and blunt dissection of the supracora~ideus muscle away from the sternal carina. The fascia covering the abdominal cavity was cut from the caudal left margin of the sternum to the junction of the keel and the posterior xiphistemal process. Unidirectional ventilation was established after rupturing the air sacs (Fedde & Burger, 1962). The proventriculuswas exposed by cutting through the posthepatic septum and retraction of the liver. A 4-cm long polyethylene cannula of 6 mm dia. with a 1-cm flange was inserted through an incision cranially into the caudal * To whom correspondence should be sent.

margin of the proventriculus. The cranial portion of the proventricuius was cannulated by the insertion of a 25-cm long polyethylene cannula of 3 mm dia.; it was introduced into the esophagus caudal to the crop and anchored in place by a ligature just cranial to the proventriculus. Gastric secretions were collected in the following manner: the caudal cannula was connected to the graduated centrifuge tubes used as collection vessels by a 20-cm long polyethylene tube of 3 mm dia with a fitted stopper equipped with an air release vent. The proventriculus was cleared of all particulate matter by the perfusion of 20 ml 0.9% saline at 37°C through the cranial cannula by means of a syringe. Gastric collections were made at 15-min periods by gently peffusing 10 ml of saline solution followed by 30 ml of air through the proventriculus. The volume of the collected perfusate was recorded to the nearest 0.1 ml and two 3-ml aliquots were immediately removed for titration with 0-01 N NaOH to pH 7. The pH of the perfusing saline solution was 6.9-7-I. In twenty-eight of thirty trials, 98 per cent of the volume of saline solution perfused through the anterior cannula was recovered. In all experiments, the basal rate of gastric acid secretion was below 0.1 m-equiv. H+/15min. Peptic activity of the gastric secretions was determined via a modification of the hemoglobin method (Bucher et aL, 1945). After collecting basal secretions for two consecutive 15-rain periods, either gastrin pentapeptide (Calbiochem, La Jolla, California) or histamine acid phosphate (Sigma Chemical Co., St. Louis, Missouri) was injected as a single intravenous dose. For dose-response relationships, each animal received only one compound doubled at hourly intervals but no more than three doses of any compound. The doses tested were 5, 10, 20, 60 and 125 ftg/kg gastrin and 50, 100, 200, 400, 800 and 1600 pg/kg histamine.

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Four birds were each given a single intravenous injection of 125/~g/kg gastrin and the time course of secretion determined. In a second series of nine birds, either 30 or 50 mg/kg hexamethonium bromide (Sigma Chemical Co., St. Louis, Missouri) were administered intravenously 30 rain prior to the injection of gastrin. Similar methods were employed in conjunction with histamine injections. Five birds, after an initial period of basal collection, were each injected intravenously with histamine (800/~g/kg). Twelve birds were given either 1 mg/kg atropine sulfate (Eli Lilly and Co., Indianapolis, Indiana), hexamethonium (30 mg/kg) or both atropine and hexarnethonium intravenously prior to histamine administration. Hexamethonium, a mammalian nicotinic receptor blocking agent (Voile & Koelle, 1970), was tested for ganglionic blockade in the fowl. In six birds, the left cervical vagus was isolated and dissected free from the surrounding fascia. The vagus was then cut and the distal end placed across bipolar silver stimulating electrodes. Electrical stimulation was produced by a Grass S-5 rectangular wave stimulator. The vagus of each animal was then subjected to a series of varying voltages and frequencies to determine the stimulation parameters which would produce maximal heart rate and blood pressure depresssion. The vagus of each bird was stimulated three times for 30 sec at 5-rain intervals. Hexamethouium (30 mg/kg) was then administered as previously described, and vagal stimulation repeated as before. All statistical analyses were performed according to the techniques considered in Steel & Torrie (1960).

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RESULTS Peak outputs of both acid (Fig. 1) and pepsin (Fig. 2) occurred together within the first 15 min after the intravenous injection of gastrin. The maximal secretions of both acid and pepsin, 1.47 + 0.20 m-equiv/15 min, and 70,000 + 1300 peptic units (P.U.)/15 min., respectively, occurred in response to 125 btg/kg gastrin. Both acid (Fig. 3) and pepsin (Fig. 4) secretion were significantly (P<0-005) inhibited by hexamethonium pretreatment at either 30 or 50 mg/kg in the first 15 min following the intravenous administration of 125/~g/kg gastrin. There was no significant difference between the inhibition produced by the two doses of hexamethonium. The marked increase in the ratio of pepsin to acid output following gastrin injection in animals pretreated with hexamethonium clearly shows that the secretion of acid was inhibited to a greater extent than was the secretion of pepsin (Fig. 5). The time--secretion relationship for acid and pepsin in response to various intravenous concentrations of histamine are shown in Figs 6 and 7, respectively. The peak secretion of acid (1.30+ 0.20 m-equiv/15 rain) was found in response to 800--1600pg/kg histamine, while the maximal pepsin secretion (116,200+ 12,500 P.U./15 rain) was found to occur in response to 1600 pg/kg histamine. The secretion of acid in response to histamine (Fig. 8) was unaffected by atropine pretreatment,

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intravenous injection of gastrin (Hirschowitz, 1968), while a comparable or smaller response maintained by histamine'was almost unaffected by the same atropine dose. In the domestic fowl both acid and pepsin secretion induced by gastrin are substantially inhibited by a single intravenous injection of hexamethonium (Figs 3 and 4). We propose that gastrin stimulates acid and pepsin secretion in the chicken through an

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action of the peptide on acetylcholine releasing structures in the intramural plexus of the proventriculus. The demonstration that hexamethonium effectively blocks vagal cardiac effects promotes the assumption that in the domestic fowl hexamethonium is an antagonist of ganglionic nicotinic receptors (Fig. 11). Urecholine, a musearinic receptor agonist, was unaffected by hexamethonium inhibition (Fig. I1) suggesting that hexamethonium is without effect on musearinic receptors. Sodium pentobarbital anesthesia has been previously demonstrated to effectively depress vagal activity to the proventriculus (Friedman, 1939); therefore, in the present study, the response to hexamethonium does not appear to be the result o f a greatly depressed vagal excitation. We suggest that stimulation of gastric secretion by gastrin in the domestic fowl is more complex than by a direct action of the hormone on effector cells. The ability of hexamethonium to inhibit the acid stimulatory effects o f histamine is consistent with previous findings in dogs (Nakajima & Magee, 1970) and rats (I-Iakanson & Liedberg, 1971). In the fowl histamine appears to stimulate one pathway for acid and a different one for pepsin secretion. The pathway for acid secretion is hexamethonium sensitive (Fig. 8) and mediated via nicotinic receptors but not musearinic receptors, while the action of

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Fig. 11 (a). Typical blood pressure decline during vagal stimulation. Double arrows delineate stimulation periods (30 sec stimulationevery 5 rain). Single arrow indicates the administration of hexamethonium bromide (30 mg/kg). (b). Period of blood pressure decline following the administration of hexamethonium bromide. Note the inhibition of the vagal effect on blood pressure during the application of electrical stimuli (double arrows). (c). Influence of intravenouslyadministered urecholine chloride (20 pg/kg) (single arrows) on blood pressure during hexamethonium bromide blockade. histamine on pepsin secretion appears to have neither nicotinic nor muscarinic receptors (Fig.9) and differs from that for acid secretion. Thus, the domestic fowl may be capable of a separation of acid and pepsin secretion even though both originate from the same cell type. In conclusion, it appears that in the domestic fowl the intramural ganglia may play an important role in the control of gastric secretion. SUMMARY Gastric secretion was studied in White Leghorn cocks in which proventricular cannulas had been installed. Hexamethonium inhibited the gastrin pentapeptide stimulated acid and pepsin secretion; however, the secretion of acid was inhibited to a greater extent than pepsin secretion. The secretion of acid stimulated by the administration of histamine acid phosphate was tmeffected by atropine pretreatment but was inhibited by bexamethonium. Pepsin secretion stimulated by histamine acid phosphate was uneffected by hexamethonium. These findings indicate that the domestic fowl may be capable of a separation of acid and pepsin secretion from the same cell type. The intramural ganglia may play an important role in the control of gastric secretion in this species. Acknowledgement--This work was supported in part by the Training Grant in Physiology, GM 01934, University of California, Davis, California. REFERENCES BUCHER G. R., GnOSSMAN M. I. & IvY A. C. (1945)

A pepsin method: the role of dilution in the determinationofpepticactivity. GastroenterologyS, 501-511.

BURHOL P. G. & HIRSCHOWlTZB. I. (1970) Single subcutaneous doses of histamine and pentagastrin in gastric fistula chickens. Am. J. Physiol. 218, 1671-1675. BURHOL P. G. & HIRSCHOWITZ B. 1. (1972) Doseresponses with subcutaneous infusion of histamine in gastric fistula chickens. Am. J. Physiol. 222, 308-313. FEDDE M. R. & BURGERR. E. (1962) A gas heating and humidifyingaccessory for the unidirectionalrespirator. Poult. Sci. 41, 679. FRIEDMANM. H. F. (1939) Gastric secretion in birds. J. cell. comp. Physiol. 13, 219-234. HAKANSONR. & LmDBEROG. (1971) Evidence against histamine as final chemostimulator of gastric acid secretion. Am.J. Physiol. 221, 641--643. HIRSCHOWrrz B. I. (1968) Gastrin I, pentagastrin and histamine in the fistula dog. Fedn Proc. Fedn Am. Socs exp. Biol. 27, 1318-1321. MENZlESG. & FLSKA. (1963) Observations on the oxynrico-peptic cells in the proventricular mucosa of Gallus domesticus. Q. Jl microsc. Sci. 104, 207-215. NAKA~n~AS. & MAGEED. F. (1970) The effect of hexamethonium on gastric acid and pepsin response to pcntagastrin, histamine and AHR602. Europ. J. Pharmac. 10, 277-282. STEEL R. G. D. & TORRIE J. H. (1960) Principles and Procedures of Statistics. McGraw-Hill, New York. TONER P. G. (1963) The fine structure of resting and active cells in the submucosal glands of the fowl proventriculus. J. Anat. 97, 575-583. VOLLER. L. & KOELLEG. B. (1970) Ganglionic stimulating and blocking agents. In The Pharmacological Basis of Therapeutics; edited by GOODMANL. S. & GILMAN A. 4th Edn, Chapt. 27, pp. 585-600. Macmillan, New York. Key Word Index---Gastric acid secretion; pepsin secretion; gastrin pentapeptide; histamine acid phosphate; hexamethonium bromide; atropine sulfate; urecholine chloride; Callus domesticus.