RAT PARIETAL-CELL RECEPTORS FOR GLP-1-(7-36) AMIDE - NORTHERN BLOT, CROSS-LINKING, AND RADIOLIGAND BINDING

The intestinal peptide hormone glucagon-like peptide-1 (GLP-1) (7-36) amide is a potent stimulus of H+ production in isolated rat parietal c ells, suggesting the presence of specific GLP-1-receptors on this cell type. Our aim was to characterize these receptors. Enzymatically isol ated rat gastric mucosal cells (F0) were fractionated by counterflow e lutriation, resulting in five fractions (F1-F5) according to increasin g cell diameter and parietal cell content (3, 5, 4, 27, 81%). Addition al density gradient centrifugation of F4 yielded enriched chief cells (74%; parietal cells: 1%; F6), whereas density gradient centrifugation of F5 almost purified parietal cells (97%; chief cells: 1%; F7). Nort hern blot of total cellular RNA from F0-F7 with a probe specific for t he GLP-1-(7-36) amide receptor revealed two RNA species of 2.7 and 3.6 kb. These messages were present to some extent in small cells (F1, F2 ), much more pronounced in F5, abundant in F7, barely detectable in F3 and F4, and absent from F6. Cross-linking of I-125-labeled GLP-1(7-36 ) amide to parietal cell membranes revealed a single 59-kDa band that was abolished by unlabeled GLP-1-(7-36) amide. Throughout fractions F1 -F7 specific binding of I-125-GLP-1-(7-36) amide was correlated with p arietal cell content (r = 0.99; P < 0.01) and H+ production ([C-14]ami nopyrine accumulation) in response to GLP-1-(7-36) amide or histamine (r = 0.96; P < 0.01). Binding was maximal in purified parietal cells ( F7), whereas almost no binding was detectable in enriched chief cells (F6). In F7, Scatchard analysis revealed a single class of high-affini ty binding sites (K-D = 2.8 +/- 0.6 x 10(-10) M, B-max = 6.8 +/- 1.4 f mol/10(6) cells, 4,096 +/- 793 receptors/parietal cells). The followin g half-maximal inhibition values were found for GLP-1-(7-36) amide and (1-37) and (1-36) amide: 6.6 +/- 0.9 x 10(-10), 1.4 +/- 0.7 x 10(-7), and 2.6 +/- 0.4 x 10(-7) M, respectively. Pancreatic glucagon, GLP-2, and oxyntomodulin, products of the proglucagon gene, were 3-4 log uni ts less potent displacers while gastric inhibitory peptide, vasoactive intestinal peptide, and secretin were ineffective. We conclude that r at parietal cells are equipped with specific high-affinity receptors f or GLP-1-(7-36) amide, which, in addition, are present in as yet unide ntified small cells (F1, F2) but not in chief cells.