Abstract
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 gastric acid secretion.
Highlights
From the :j:J)epartment of Internal Medicine, §Pediatrics, and IlPhysiology, University of Michigan Medical Center, Ann Arbor, Michigan 48109-0368
Using an H+,K+-ATPase a-subunit gene-Iuciferase 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
We explored the nature of the receptor/ligand interaction mediating the observed effects of gastrin and G-Gly on H+,K+-ATPase a-subunit gene expression
Summary
Materials-Human gastrin heptadecapeptide (G17-NH2) and sulfated cholecystokinin octapeptide (CCKS) were purchased from Peninsula Laboratories Inc. After washing with Hanks' balanced salt solution, parietal cells were enriched by centrifugal elutriation. Northern Blot Analysis-Purified parietal cells suspended in Earle's balanced salt solution (EBSS)' containing 0.1% bovine serum albumin °were incubated with the peptides under study for 1 h at 37°C in 95% 2 ,5% CO2 , Total RNA was isolated by a modification of the method of Chomczynski [12]. Aliquots of RNA (10 fJ-g) were electrophoresed on a 1.25% formaldehyde-agarose gel, blotted to a nylon membrane (maximum strength NYTRAN, Schleicher & Schuell), and hybridized to a.
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