Stimulation of Gastrin Secretion and Synthesis in Antral Organ Culture

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Abstract

The purpose of the present study was to examine stimulation of gastrin release and the synthesis of gastrin directly by measurement of incorporation of [3H]tryptophan into gastrin in rat antral mucosal explants maintained in organ culture. Gastrin synthesis and secretion were assessed simultaneously at intervals over the 24-h duration of explant culture. Antral mucosal explants from fed female Wistar rats (4-5 wk, 100-150 g) were cultured at 37°C (95% O2/5% CO2) in medium containing 70% Trowell-T8 and 10% NCTC-135 without unlabeled tryptophan, 10% dialyzed fetal calf serum and [3H]tryptophan (100 μCi/ml). Antral tissue was harvested at regular intervals during 24-h culture periods. Incorporation of [3H]tryptophan into immunoreactive gastrin was determined by techniques utilizing double-antibody immunoprecipitation. Antral tissue protein synthesis was assessed by measurements of incorporation of [3H]tryptophan into tissue protein of cultured antral explants. In paired experiments, gastrin synthesis and secretion in the presence of dibutyryl cAMP (DBCAMP) were compared to those observed under control conditions. Gastrin and protein specific activity progressively increased with time. Gastrin specific activity at 30 min increased from 3.3±0.5 (SEM) to 55.2±10.6 fmol [3H]tryptophan/pmol gastrin (or from 1.57±0.48 to 26.28±5.05 pmol [3H]tryptophan/μg gastrin) at 24 h: specific activity of antral tissue protein at 30 min increased from 33.6±8.4 to 1,660±236 fmol [3H]tryptophan/μg protein at 16 h. Culturing of explants for 4 h in the presence of cycloheximide (100 μg/ml) inhibited both gastrin synthesis and protein synthesis by greater than 90 and 95%, respectively. DBCAMP (10 mM) significantly increased both the synthesis and secretion of antral gastrin when compared with control cultured explants. Results of these experiments provide direct demonstration of gastrin synthesis by rat antral mucosal explants in organ culture, indicate that both gastrin and total antral protein synthesis are inhibited by cycloheximide, and demonstrate DBCAMP-induced stimulation of both gastrin synthesis and secretion, suggesting the potentially important role of cyclic AMP in gastrin cell function.

Authors

Richard F. Harty, Jan C. van der Vijver, James E. McGuigan