Effects of carbachol on gastrin and somatostatin release in rat antral tissue culture

Recent studies have demonstrated that somatostatin-containing cells are in close anatomic proximity to gastrin-producing cells in antral mucosa, suggesting a potential local regulatory role for somatostatin. The purpose of this study was to examine further the relationships between gastrin and somatostatin and the effects of the cholinergic agonist carbachol on content and release of gastrin and somatostatin using rat antral mucosa in tissue culture. Antral mucosa was cultured at 37 degrees C in Krebs-Henseleit buffer, pH 7.4, gassed with 95% O2-5% CO2. After 1 h, the culture medium was decanted and the tissue was boiled to extract mucosal gastrin and somatostatin. Inclusion of carbachol 2.5 X 10(-6) M in the culture medium decreased medium somatostatin from 1.91 +/- 0.28 (SEM) ng/mg tissue protein to 0.62 +/- 0.12 ng/mg (p less than 0.01), extracted mucosal somatostatin from 2.60 +/- 0.30 to 1.52 +/- 0.16 ng/mg (p less than 0.001), and percentage of somatostatin released from 42% +/- 2.6% to 27% +/- 2.2% (p less than 0.01). Carbachol also increased culture media gastrin from 14 +/- 2.5 to 27 +/- 3.0 ng/mg protein (p less than 0.01). Tissue content and release of gastrin and somatostatin were also examined during culture of rat antral mucosa in culture media containing antibodies to somatostatin in the presence and in the absence of carbachol. Incubation with somatostatin antisera, both with and without carbachol, markedly increased culture media concentrations of somatostatin, all of which was effectively bound by antibodies present in the media. Antibody binding of somatostatin was accompanied by significant increases in culture media gastrin concentrations, both in the presence and in the absence of carbachol. Results of these studies support the hypothesis that antral somatostatin exerts a local regulatory effect on gastrin release and that cholinergic stimulation of gastrin release is mediated, at least in part, through inhibition of somatostatin synthesis and release.     

Effects of exogenous and endogenous bombesin on gastrin secretion from rat antral mucosa in tissue culture

Effects of exogenous and endogenous bombesin on gastrin secretion were examined using rat antral mucosa in tissue culture. Gastrin secretion was significantly stimulated by exogenous bombesin at a dose of 10(-8) M. Atropine 10(-6) M, which abolished the action of the cholinergic agent carbachol to stimulate gastrin secretion, had no effect on bombesin-stimulated gastrin secretion. In addition, gastrin secretion was significantly inhibited by anti-bombesin antiserum used to block the effect of endogenous bombesin by immunoneutralization. These findings suggest that the stimulation of gastrin secretion by bombesin does not involve cholinergic neural pathways and that endogenous bombesin exerts a continuous stimulation on gastrin secretion in the basal state.     

Effects of bombesin on the release of glycine-extended progastrin (gastrin G) in rat antral tissue culture

Recently, glycine-extended processing intermediates of progastrin were identified in porcine stomach using a radioimmunoassay with conventional polyclonal antisera developed against a synthetic peptide analogue for progastrin processing intermediates, gastrin 6-G(Tyr-Gly-Trp-Met-Asp-Phe-Gly). We developed monoclonal antibodies specific for glycine-extended processing intermediates of progastrin (gastrin G). Monoclonal antibody 109-21 appeared to require the carboxyl-terminal pentapeptide structure of gastrin 6-G for maximal binding. Cross-reactivities of 109-21 against gastrin 17 I, gastrin 17 II, cholecystokinin-octapeptide, des(SO3) cholecystokinin-octapeptide, and gastrin 6-G-R-R were respectively 1%, less than 0.1%, less than 0.1%, 0.1%, and 0.5%. With this monoclonal antibody and a polyclonal gastrin antibody we examined the concentrations of gastrin and gastrin G in tissue and the effects of bombesin on the release of gastrin and gastrin G from rat antral mucosa in tissue culture. The gastrin G to gastrin ratio was 2.2 in rat antral mucosa and 0.66 in rat duodenal mucosa. In tissue culture, bombesin significantly stimulated gastrin and gastrin-G secretion at doses of 10(-8) and 3 X 10(-8) M. Atropine (10(-6) M) abolished the actions of carbachol to stimulate gastrin and gastrin-G secretion but had no effect on bombesin-stimulated gastrin and gastrin-G secretion. These results suggest that gastrin G is cosecreted with gastrin in response to carbachol and bombesin, and the stimulation of gastrin and gastrin-G secretion by bombesin does not involve cholinergic neural pathways and may reflect a direct action on gastrin cells.     

Effect of environmental hyperthermia on gastrin, somatostatin and motilin in rat ulcerated antral mucosa

AIM: To study the effect of environmental hyperthermia on gastrin, somatostatin and motilin in rat ulcerated antral mucosa.METHODS: Forty-two Wistar rats were equally divided into six groups, according to the room temperature (high and normal) and the treatment (acetic acid, normal saline and no treatment). Levels of gastrin, somatostatin and motilin in rat ulcerated antral mucosa were measured with a radioimmunoassay method.RESULTS: The average temperature and humidity were 32.5℃ and 66.7% for the high temperature group, and 21.1℃ and 49.3% for the normal temperature group,respectively. Gastric ulcer model was successfully induced in rat injected with 0.05 mL acetic acid into the antrum. In rats with gastric ulcers, the levels of gastrin and motilin increased, whereas the somatostatin level declined in antral mucosa, compared with those in rats treated with normal saline and the controls. However, the change extent in the levels of gastrin, motilin and somatostatin in antral mucosa was less in the high temperature group than in the normal temperature group.CONCLUSION: The levels of gastrin, somatostatin and motilin in rat ulcerated antral mucosal tissue remain relatively stable in a high temperature environment, which may relate to the equilibration of the dynamic system.     

Participation of antral somatostatin in the local regulation of gastrin release

In anaesthetized pigs gastrin release was stimulated by irrigation of the antrum with bicarbonate and by instillation of a meat extract. The concentration of gastrin and somatostatin was measured by radioimmunoassay both in the antral and peripheral venous blood. The increase in gastrin was coupled to a significant decrease in somatostatin immunoreactivity as measured in the antral venous blood both during instillation of alkali and meat extract. In peripheral blood, the differences were much less evident and not statistically significant. It is speculated that the decrease in release of antral somatostatin during alkalinization and instillation of meat extract is the primary event which is followed by a diminished inhibition of gastrin liberation. Thus, the present data support the hypothesis that antral somatostatin participates in the local regulation of gastrin release.     

Relationship between gastrin cell number, serum, antral mucosa and luminal gastrin concentration and gastric acidity in antral atrophic gastritis.

The aim of our study was to investigate the relationship between gastrin producing cell density with antral mucosa, luminal and serum gastrin concentration in antral atrophic gastritis. Our study group consisted of 17 patients: six with mild atrophic gastritis, seven with moderate atrophic gastritis and four with severe atrophic gastritis. None of the patients had type-A atrophic gastritis but the body mucosa was affected by superficial gastritis at various extent in some. A group of 15 healthy subjects served as control. All subjects underwent gastroscopic examination with multiple bioptic sampling. Radioimmunoassay was used for gastrin determination and photomicroscopy for gastrin producing cell density assessment. Electron microscopy was used to assess the gastrin producing granule density index. Patients with moderate and severe atrophic gastritis showed a lower gastric acidity and acid output as compared to control. Serum gastrin did not show significant differences among the groups. In moderate and severe atrophic gastritis, gastrin producing cell granule density index, gastrin producing cell density and antral mucosa gastrin concentration were significantly lower when compared with control and decreased with advancing of the severity of atrophic gastritis. In atrophic gastritis, however, the latter two measurements were not correlated. In moderate and severe atrophic gastritis luminal gastrin concentration significantly increased, compared with control, after the severity of atrophic gastritis. Gastrin producing cell granule density index and luminal gastrin concentration showed a significant correlation with gastric pH. These data suggest that in antral atrophic gastritis with reduced gastric acidity, the decrement of gastrin producing cells is followed by gastrin producing cell hyperfunction with increased luminal release of gastrin.     

Somatostatin inhibition of basal and carbachol-stimulated gastrin release in rat antral organ culture

The effects of somatostatin on gastrin release and total gastrin immunoreactivity were examined under in vitro conditions in rat antral organ culture experiments. Basal antral gastrin release was inhibited by somatostatin. Total culture gastrin contents (culture medium gastrin plus extracted antral mucosal gastrin) at 6 h were reduced significantly by 10(-5) M (p less than 0.02) and 10(-4) M (P less than 0.01) somatostatin. Gastrin release into the culture media stimulated by the cholinergic agent, carbachol (10(-5) M), was suppressed by somatostatin: at 30 min and 6 h of culture 10(-8) M somatostatin inhibited carbachol-stimulated gastrin release by 66% and 54%, respectively, and 10(-5) M and 10(-4) M somatostatin completely abolished gastrin release. The rate of gastrin release stimulated by carbachol was suppressed significantly by each dose of somatostatin examined (10(-8) M to 10(-4) M). The present studies indicate that somatostatin inhibits both gastrin secretion by cultured rat antral mucosa and total antral culture gastrin contents, and demonstrate that cholinergically-mediated gastrin secretion is inhibited by somatostatin.     

Mucus, gastrin and somatostatin cells in cultured rat antral mucosa: immunofluorescence, ultrastructural and radioimmunological studies

Cells were isolated from the gastric antrum of newborn rats (7 and 10 days old) with the intent of studying mucus, gastrin (G), and somatostatin (D) cells. These cells were maintained in culture for 20 days. Their secretory properties were studied in vitro by cytochemical, immunocytochemical and radioimmunological methods. In vitro, mucus cells as well as G and D cells synthesized their secretory products intensely for the first 48 h, but beyond this point, their activity decreased. Mucus cells had a high rate of multiplication and formed sheets of epithelial cells in vitro. Their PAS-positive secretions were synthesized up until the 7th day of culture. During the first 3 days of culture, gastrin cells secreted detectable amounts of the hormone in the culture medium, but afterwards their secretion decreased. Somatostatin cells remained active until at least the 7th day of culture. They displayed long cytoplasmic processes which may serve as a means of communication with neighboring cells. Using ultrastructural techniques, mucus and endocrine cells were found to persist in culture. From a morphological point of view, they appeared similar to the cells found in the original antral tissue and this is an argument for the persistence of the secretory properties in cultivated cells. This experimental model appears to be reproducible and may be useful in the study of secretions of somatostatin, gastrin and mucus in the gastric antrum of the rat.     

Somatostatin and gastrin content of gastric antral mucosa in ulcer disease

Gastrin and somatostatin containing cells are abundant in the gastric antral mucosa suggesting a role for these peptides in gastric physiology, presumably acid secretion. The concentration of these peptides in antral mucosa in ulcer disease is controversial, some finding normal levels, others decreased somatostatin levels. Biopsies of antral mucosa from patients with ulcer disease and non-ulcer dyspepsia were obtained at endoscopy, and somatostatin and gastrin concentration were measured by specific radioimmunoassay. Levels were similar in non-ulcer, duodenal and gastric ulcer patients but prior treatment with H₂-receptor antagonists in duodenal ulcer patients led to a fall in somatostatin and a rise in gastrin mucosal levels. It is thus unlikely that a lack of somatostatin or an increase in gastrin are factors in the pathogenesis of duodenal ulcer, but the cells may behave abnormally in ulcer disease.     

Circadian rhythms in the number of gastrin cells, DNA synthesizing cells and labelling index of gastrin cells in the antral mucosa of the rat

Circadian variations of the gastrin cell (G-cell) number, the DNA synthesizing cell (S phase cell) number and labelling index of G-cell in antral mucosa were studied using the simultaneous double immunoenzymatic labelling method of gastrin and bromodeoxyuridine (BrdU) both in fed and 24 h fasted rats. No significant change was observed in the G-cell number. The S phase cell number and labelling index of G-cell showed significant circadian rhythms. Labelling index of G-cell markedly decreased in 24 h fasted rats in comparison to that in fed rats. These results suggest that DNA synthesis in G-cells has a circadian rhythm and that the activity is influenced by food ingestion.     

Circadian rhythms in the number of gastrin cells,DNA synthesizing cells and labelling index of gastrin cells in the antral mucosa of the rat

Circadian variations of the gastrin cell (G-cell) number, the DNA synthesizing cell (S phase cell) number and labelling index of G-cell in antral mucosa were studied using the simultaneous double immunoenzymatic labelling method of gastrin and bromodeoxyuridine (BrdU) both in fed and 24 h fasted rats. No significant change was observed in the G-cell number. The S phase cell number and labelling index of G-cell showed significant circadian rhythms. Labelling index of G-cell markedly decreased in 24 h fasted rats in comparison to that in fed rats. These results suggest that DNA synthesis in G-cells has a circadian rhythm and that the activity is influenced by food ingestion.     

Relation of antral pH to gastrin release and fundal pH in dogs

Summary and conclusions Four dogs were prepared with Heidenhain pouches and cannulas placed in the fundus and antrum. Fasting pouch output was measured for 1 hr. Changes in pH in the fundus and pouch, and pouch acid production were then determined while antral pH was varied by perfusion in increasing and decreasing sequences. Results were virtually identical with ascending and descending antral pH values.With an antral pH below 1.8 no appreciable pouch secretory effect was found, the values being identical with those obtained under fasting conditions. A transition zone was encountered between antral pH 1.8–3.0 in which relatively small and variable amounts of pouch acid were produced. Above an antral pH of 3.0 a significant stimulatory effect was noted that did not increase with further increments of antral pH.Fundal and pouch pH were relatively high and had little relationship to one another when antral pH was less than 1.8. The values fell and became more closely associated at the antral transition zone of pH 1.8–3.0. Above an antral pH of 3.0, fundus and pouch pH decreased and fluctuated in a parallel manner. Pouch pH was consistently below fundal pH.Supported by a grant from Wyeth Laboratories, Inc., Radnor, Pa.     

Decreased sulfation of serum and tissue gastrin in hypergastrinemia of antral origin

The sulfation of gastrin in serum, antrum and duodenum was studied in 22 normo- and 20 hypergastrinemic patients. The ratio between gastrin-17 and gastrin-34 was measured in antrum and duodenum. The degree of sulfation was reduced in the antrum of hypergastrinemic patients (35.3 +/- 1.3%, mean +/- SEM) compared with 48.0 +/- 2.1% in normo-gastrinemic patients (p less than 0.001). The degree of sulfation in serum and duodenum was similar to that of the antral gastrins in all patients. The percentage of gastrin-34 in antrum was increased (7.3 +/- 0.7%) in hypergastrinemic compared with 4.9 +/- 0.3% in normogastrinemic patients (p less than 0.01). In the duodenum the percentage of gastrin-34 was similar in normo- and hypergastrinemia. When classified according to clinical diagnosis, sulfation of antral gastrin was normal in duodenal ulcer (47.6 +/- 4.5%) but decreased in gastric ulcer (36.7 +/- 1.6%, p less than 0.01) and pernicious anemia (31.3 +/- 1.9%, p less than 0.001) compared with 48.2 +/- 2.2% in control patients. In pernicious anemia a larger proportion of antral gastrins occurred as gastrin-34 (8.2 +/- 0.9%) compared with 4.8 +/- 0.4% in control patients (p less than 0.01). Our study suggests that both sulfation and proteolytic processing of the gastrin precursor is diminished in hypergastrinemia of antral origin.     

Effects of pirenzepine on omeprazole-induced gastrin gene expression in rat antral tissues

Pirenzepine has inhibitory effects on gastrin secretion bothin vivo andin vitro. The aim of this study was to determine the mechanism responsible for the suppression of omeprazole-induced hypergastrinemia that occurs with pirenzepine treatment. The effects were measured in rats treated with oral omeprazole plus intraperitoneal pirenzepine or saline once daily for seven days in the antrum. The serum gastrin level increased significantly by more than sixfold with omeprazole treatment; additional treatment with pirenzepine suppressed this increase by 48%. Pirenzepine treatment did not change the level of gastrin mRNA but significantly increased the level of somatostatin mRNA. Combination treatment with omeprazole plus pirenzepine significantly decreased the gastrin mRNA level to half and significantly increased the somatostatin mRNA level up to 1.4-fold of the levels achieved with omeprazole treatment alone. These results suggest that the stimulatory effect of omeprazole on gastrin synthesis is partially blocked by pirenzepine via mediation of somatostatin synthesis in the antrum.     

Effect of aging on gastric acid secretion,serum gastrin,and antral gastrin content in rats

The purpose of this study was to characterize the effects of aging on gastric acid secretion and on serum and antral concentrations of gastrin in rats. Young and old Fischer 344 rats were prepared with gastric fistulas. Twenty-four hours after surgery, graded doses of human synthetic gastrin-17 (SHG-17) (2, 5, 10, 20, and 40 g/kg) were given intravenously in random order. Gastric secretions were collected for gastric acid measurement before and at 15-min intervals after each dose of gastrin. In a separate study, blood was collected and the stomachs were removed for antral gastrin extraction from fed young and old rats. Serum and antral gastrin was measured by radioimmunoassay. The basal and gastrin-stimulated acid secretions were significantly decreased in aged rats compared to the young rats. The basal acid output was 0.4±0.2 eq/15 min in the aged rats and 1.5±0.5 eq/15 min in the young. The maximal acid output stimulated by gastrin was 11.1±1.8 eq/15 min in the aged rats and 24.2±2.8 eq/15 min in the young. Both serum and antral concentrations of gastrin were significantly decreased in aged rats. Serum gastrin concentration was 114.8±7.4 pg/ml in the aged rats and 192.0±14.4 pg/ml in the young. Antral gastrin concentration was 3.9±0.5 g/g tissue in the aged rats, which was significantly less than the concentration in the young (6.5±0.4 g/g tissue). Antral gastrin content did not change with aging. Gastric acid secretion in aged rats is significantly decreased compared to the young in both the basal condition and in response to fixed doses of exogenous gastrin. Diminished concentrations of circulating gastrin may well be responsible, at least in part, for the diminished acid secretion in the aged rats.Part of this work was presented at the special session on aging during the Digestive Disease Week held by the American Gastroenterology Association (AGA) in New York, May 14, 1985, and has been published in abstract form (Gastroenterology 88:1445, 1985).Supported by grants from the National Institutes of Health (RO1 DK 15241, PO1 DK 35608, RCDA CA 00854, CA 38651) and a grant from the American Cancer Society (PDT-220).     

The effect of bombesin, cholecystokinin, gastrin, and their antagonists on proliferation of pancreatic cancer cell lines

BACKGROUND: The role of cholecystokinin (CCK) and gastrin in the development and growth of pancreatic cancer cells is controversial. The aim of this study was to evaluate the role of CCK-8S, gastrin-17, bombesin, and their antagonists on cell lines from patients with pancreatic cancer. METHODS: Cell lines were established from pancreatic cancers operated on at our department. The cells were grown in 10% fetal calf serum (FCS). The effects of CCK-8S, gastrin-17, bombesin, and their antagonists in different concentrations and for different time intervals were studied. The cell number was evaluated with the XTT method. RESULTS: The cell line LN 36 responded with increased cell number to stimulation by gastrin-17 and decreased cell number to inhibition by the CCK-B receptor antagonist L-365,260. In contrast, LPC 1 responded with increased cell number to CCK-8S and decreased cell number to the CCK-A receptor antagonist devazepide. LPC 2, 6, and 7 were stimulated by CCK-8S, gastrin-17, and their antagonists. LPC 3 showed decreased cell number after inhibition by the antagonists, and LPC 5 and 10 showed increased cell number after stimulation by CCK-8S and gastrin-17. LPC 4 was stimulated by CCK-8S, and LPC 8 was stimulated by all substances except gastrin-17. Intermittent administration of the substances to LN 36 led to a greater effect on the cell number than administration every day, which was not the case with LPC 1 and LPC 3. Bombesin led to an increased growth in LPC 5 but not in LPC 3. CONCLUSION: CCK-8S and gastrin-17 led to an increased cell number in some cell lines. A blockade of the CCK-A and CCK-B receptors by their antagonists led to an increased, an unaffected, or a decreased cell number of the cell lines. The effect of bombesin on different cell lines also varied. This shows a great heterogenicity among pancreatic cancer cells from different patients.