l-365260 and big-gastrin

This study evaluated the role played by cholecystokinin (CCK) receptors' occupation in the control of somatostatin (SS) secretion in RIN-14B cells.. The presence of the CCK receptors 1 and 2 was confirmed by immunofluorescence, and SS secretion was evaluated by enzyme-linked immunosorbent assay.. By immunofluorescence, 95% of the cell population was composed of SS cells bearing both CCK-R subtypes with 5% of beta cells (data not shown). Cerulein (Cae), a CCK-1R agonist, and pentagastrin, a CCK-2R agonist, dose-dependently increased SS release, 3-fold at 1 mumol/L Cae, 2.5-fold at 10 mumol/L pentagastrin, with occupation of both CCKRs confirmed by L-364,178 and L-365,260 inhibition of CCK receptors 1 and 2. The occupation of high-affinity CCK-1R by Cae was confirmed on SS release with JMV-180, a high-affinity CCK-1R agonist, and absence of SS release inhibition at high Cae concentration occupying the low-affinity CCK-1R. These cells release more than 60% of their SS content by constitutive secretion, confirmed by cycloheximide and brefeldin inhibiting SS synthesis and intracellular trafficking, respectively.. Both CCKR subtypes occupy RIN-14B cells and initiate SS secretion through constitutive secretion controlled at SS synthesis level. Somatostatin secretion via the CCK-1R occupation mobilizes its high-affinity sites.

Topics: Animals; Benzodiazepinones; Brefeldin A; Cell Line; Ceruletide; Cholecystokinin; Cycloheximide; Devazepide; Dose-Response Relationship, Drug; Enzyme-Linked Immunosorbent Assay; Fluorescent Antibody Technique; Gastrins; Islets of Langerhans; Pentagastrin; Phenylurea Compounds; Protein Precursors; Protein Synthesis Inhibitors; Protein Transport; Rats; Receptor, Cholecystokinin A; Receptor, Cholecystokinin B; Sincalide; Somatostatin

Biologically active amidated gastrin is synthesized by carboxyl-terminal alpha-amidation of a glycine-extended progastrin post-translational processing intermediate (G-Gly). Although plasma levels of G-Gly are equivalent to those of gastrin, G-Gly has essentially no acute effect on gastric acid secretion. However, we have observed that inhibition of gastrin amidation leads to increased plasma concentrations of G-Gly and enhanced gastric acid secretion. We hypothesized, therefore, that G-Gly might have a chronic effect to increase H+,K(+)-ATPase expression in gastric parietal cells. In the present studies, we observed that a 2-day preincubation with G-Gly significantly enhanced histamine-stimulated [14C]aminopyrine uptake by isolated canine gastric parietal cells but acutely administered G-Gly had no effect. On Northern blot analysis, both G-Gly and gastrin dose-dependently increased H+,K(+)-ATPase alpha-subunit gene expression with maximal induction (225 +/- 35 and 170 +/- 29% of basal, mean +/- S.E.) achieved at concentrations of 10(-9) M G-Gly and 10(-8) M gastrin, respectively. Using an H+,K(+)-ATPase alpha-subunit gene-luciferase chimeric reporter construct transfected into primary cultured parietal cells, we observed that both G-Gly and gastrin increased luciferase activity in a manner similar to that obtained by Northern blot analysis. L365,260, a specific gastrin/CCKB receptor antagonist, completely reversed the stimulation of luciferase activity induced by gastrin but had no effect on G-Gly-stimulated activity. Gastrin increased [Ca2+]i, although G-Gly did not, however, genistein (a tyrosine kinase inhibitor) significantly reduced induction of luciferase activity by both G-Gly and gastrin. Specific binding of 125I-Leu15-G2-17-Gly to gastric parietal cells was dose-dependently displaced by G2-17-Gly but not by gastrin nor L365,260. Gastrin peptides truncated at the carboxyl- (G1-13) and amino terminus (G5-17-Gly) both induced H+,K(+)-ATPase alpha-subunit gene expression and inhibited 125I-Leu15-G2-17-Gly binding, but were less potent than G2-17-Gly. These data indicate that G-Gly may have a functional role in potentiating gastric acid secretagogue action via enhanced expression of the gene responsible for H+ generation through action at a novel receptor that can be distinguished from the gastrin/CCKB receptor. Thus, both the substrate and product of the terminal progastrin processing reaction appear to have complementary functions in regulation of

Topics: Aminopyrine; Animals; Base Sequence; Benzodiazepinones; beta-Galactosidase; Biological Transport; Blotting, Northern; Calcium; Cells, Cultured; Dogs; Dose-Response Relationship, Drug; Enzyme Induction; Gastrins; Gene Expression; Glycine; H(+)-K(+)-Exchanging ATPase; Histamine; Humans; Kinetics; Luciferases; Macromolecular Substances; Molecular Sequence Data; Parietal Cells, Gastric; Phenylurea Compounds; Promoter Regions, Genetic; Protein Precursors; Protein Processing, Post-Translational; Receptors, Cholecystokinin; Recombinant Fusion Proteins; Transfection