Relationship between serum gastrin levels and gastric secretion in Heidenhain pouch dogs.

Relationship between serum gastrin levels and gastric secretion was studied in Heidenhain pouch dogs. Bethanecol and tetramethylammonium increased gastric secretion without any significant change in the serum immunoreactive gastrin (IRG) level. Histamine increased gastric secretion but decreased the serum IRG level. Tetragastrin evoked gastric secretion concomitantly with an elevation of the serum IRG level, and the relationship was significant. Food-intake promptly increased the serum IRG level which correlated with the increased gastric secretion. Except for the first 15-min value after food-intake, a better correlation was obtained and was almost the same as that with tetragastrin-stimulation. Hexamethonium reduced the foodinduced secretion rate in parallel with reduction of the serum IRG level. Correlation between the secretion rate and serum IRG level after dosing was almost the same as that of the control. Atropine and secretin induced a stronger inhibition on the secretion rate than on the serum IRG level. Prostaglandin E/reduced the secretion rate, but produced no inhibitory effect on the serum IRG level. These results suggest that the food-induced gastric secretion in Heidenhain pouch dogs is due to the action of en dogenous gastrin, and that hexamethonium and prostaglandin E1 affect respectively the gastrin and parietal cells. Atropine and secretin affect both parietal and gastrin cells. Food-induced gastric secretion from denervated fundic (Heidenhain) pouch is con sidered to be produced by an action of gastrin (1). Involvement of cholinergic nerve pathway in the gastrin release has also been postulated (2). These studies, however, have been done using gastrin bioassay. Thus, changes in response of oxyntic glands to gastrin may be involved in the observed changes in gastrin release. Recently developed radioimmunoassay of gastrin has made it possible to determine gastrin concentrations in the blood without the complexity mentioned above (3, 4, 5). Al though at least three components have been identified in "immunoreactive gastrin (IRG)" (4, 5), the hypothesis concerning the gastrin mechanism should be re-examined using gastrin radioimmunoassay. We investigated the relationship between secretion rate and serum IRG levels in Heidenhain pouch dogs challenged by several secretory stimulants and inhibitors. MATERIALS AND METHODS Male and female Beagle dogs, weighing 5-10 kg, were used. A Heidenhain pouch was prepared with the dog under pentobarbital anesthesia according to the conventional method (6). The pouch was drained by a stainless steel cannula. At least 4 weeks were allowed for recovery from surgery before the start of experiments. The dogs were deprived of all solid food but were given free access to water for 24 hr before each experiment. Tests were carried out once a week. The test animals were fed 160 g of canned beef or given s.c., histamine dihydrochloride (50 tag/kg), tetragastrin (8 lug/ kg) or bethanecol chloride (30 beg/kg) every 15 min. Under these conditions, dogs secrete a submaximal amount of gastric juice (2-7 ml/ 15 min). Gastric juice was collected continuously from the pouch in 15-min samples. Gastric juice volume was recorded, and acid concentration was determined by titration with 0.1 N NaOH up to pH 7 using an automatic titrator (Hiranuma, Japan). Blood samples for the determination of serum gastrin were collected from the leg vein once before each test, and 15, 30, 45, 60, 90 and 120 min after feeding or dosing. Each sample was allowed to clot, centrifuged at 3,000 rpm at 4'C for 10 min, and the serum was aspirated and frozen at -20'C until assay. Serum IRG level was determined by radioimmunoassay as described by Yalow and Berson (3). Antisera to gastrin were produced in rabbits by immunization with synthetic human gastrin I (SHG : 1-17) conjugated with bovine serum albumin. 1251-SHG (specific activity: approx. 600 mCi/mg) was obtained from Dainabbott Radioisotope Lab., Japan. Antibody-bound 1251-SHG was separated with activated Amberlite IRG-58 (100-400 mesh, Organo, Japan). SHG was used as a standard gastrin. Serum samples were assayed twice. The following drugs were used; atropine sulfate (E. Merck), hexamethonium bromide (Methobromin, Yamanouchi), synthetic human gastrin (SHG: 1-17, Imperial Chemical Industries), tetragastrin (Aoc-Try-Met-Asp-Phe-NH2, Protein Research Foundation, Minoh), caerulein (Sigma Co.), cholecystokinin-pancreozymin (Eisai), secretin (Eisai), bethanecol chloride (Fujita Pharm.), tetramethylammonium chloride (Nakarai) and hista mine dihydrochloride (Sigma). Prostaglandin E, was a gift from Upjohn Research Labs., U.S.A. RESULTS Standard curve for inm7unoassay Using 0-1000 pg/ml of SHG as the standard solutions, the percent of bound 1251-SHG in total 125I-SHG after incubation for 5 days was plotted against the concentration of SHG using various concentrations of antisera. The slope of the standard curve was steeper for the range of 0-500 pg/ml of SHG when 1:2000 final dilutions were used. Thus, 1:2000 dilution was used in the following study. The standard curve is shown in Fig. 1. Specificity of antisera Immunoreactivities of caerulein, chlecystokinin-pancreozymin (PZ-CCK), secretin and tetragastrin were compared (Fig. 2). Cross-reactivity was found when more than 100 pg/ml of caerulein, 1000 pg/ml of tetragastrin and 0.01 Harper U/ml of PZ-CCK were added. The cross-reactivities of caerulein and tetragastrin were respectively one-tenth and one hundredth that of SHG. No significant cross-reactivity was found under 5 Harper U/ml of secretin. FIG. 1. Standard curve for radioimmunoassay of gastrin. Vertical bars represent ranges of values in duplicate determinations. FIG. 2. Cross-reactivity of some peptides with gastrin antisera Relationship between secretion and acid output There was a significant linear correlation between the secretion volume and acid output from Heidenhain pouch in dogs given food, tetragastrin, histamine, bethanecol or tetra methylammonium. The correlation was almost the same irrespective of the secretogogues used (Table 1). Based on these findings we refer hereinafter to the secretion volume in order to avoid complexity. Effect of secretory stimulants on serum gastrin level and secretion (i) Food The mean serum IRG level of 43 pg/ml increased to a peak of 211 pg/ml at 15 min after feeding, dropped slightly, leveled off between 30 and 45 min, and thereafter decreased gradually to 85 pg/ml at 120 min (Fig. 3). TABLE 1. Relationship between secretion volume and acid output induced by various secretory stimulants FIG. 3. Effect of feeding on the serum gastrin level and gastric secretion in Heidenhain pouch dogs. The arrow indicates the feed ing time (160 g of canned beef). N=number of animals. FIG. 4. Effect of bethanecol and tetramethyl ammonium on the serum gastrin level and gastric secretion in Heidenhain pouch dogs. Arrows indicate bethanecol and tetramethylammonium (T.M.A.) s.c. in jection. N=number of animals. Gastric secretion was insignificant when the animals were fasted. The secretion in creased gradually after feeding and reached a maximum of 3.6 ml/15 min at 30-45 min and decreased gradually to 0.5 ml/15 min at 120 min. (ii) Drugs Subcutaneous injection of bethanecol (30 /ig/kg) and tetramethylammonium (300 /ig/ kg) every 15 min provoked gastric secretion. The serum IRG level tended to increase during the first 30 min after the commencement of injection, and then decreased. (Fig. 4) Gastric secretion was initiated by intermittent subcutaneous injections of tetragastrin and increased gradually to a plateau of 2.1-2.6 ml/15 min 60 min after the first injection (Fig. 5). Tetragastrin also prompted an increase in the serum IRG level and the time course of increase paralleled that of secretion. Gastric secretion commenced at the first 15 min after the subcutaneous injection of histamine and leveled off at about 7 ml/15 nun 60 min after the first injection (Fig. 5). Serum IRG level decreased inversely with the increase in secretion, and fell to 22 pg/ml 120 min after the first injection. (iii) Relationship between gastrin level and secretion There was a significant relationship (r=0.540) between the increased serum IRG level and secretion volume prompted by food-intake. A better correlation was obtained when the first 15 min value was excluded, the correlation coefficient being 0.927 and almost identical with that for tetragastrin stimulation (Fig. 6). FIG. 5. Effect of teiragastrin and histamine on the serum gastrin level and gastric secretion in Heidenhain pouch dogs. See Fig. 4. FIG. 6. Relationship between the serum gas trin level and gastric secretion stimulated by food, tetragastrin and histamine. The equations of regression were calculated according to the least square method. The value 15 min after feeding is excluded from the calculation. There was also a significant relationship between IRG level and secretion volume induced by histamine. However, the secretion volume was inversely correlated with the IRG level (r= -0.937), which was in marked contrast to the results with tetragastrin and food were used as stimulants. No correlation was seen between gastric secretion induced by bethanecol and tetra methylammonium and serum IRG level. Effect of secretory inhibitors on food-induced serum gastrin level and secretion (i) Drugs Atropine (16 pg/kg) almost completely reduced gastric secretion induced by food (Fig. 7). Gastrin response, though depressed slightly, was still apparent even after admin istration of the drug. Hexamethonium (1 mg/kg) reduced both secretion and gastrin response to feeding, and the duration and degree of inhibition of the former paralleled those of the latter. Fic. 7. Effect of atropine and hexamethonium on food-induced serum gastrin level and gastric secretion. Solid lines represent the values without drug application. Broken lines and hatched areas represent the value with drug application. The arrow indicates the feeding time (160 g of canned beef). Drugs were given i.v. immediately before feeding. N number of animals. FIG. 8. Effect of secretin and prostaglandin E, on food-induced serum gastrin level and gastric secretion. See Fig. 7. Secretin (8 U/kg) reduced the food-induced secretion by approximately 50% for the first 120 min after feeding (Fig. 8). The gastrin response was inhibited slightly immediately