AJP - Gastrointestinal and Liver Physiology, Vol 267, Issue 3 423-G432, Copyright © 1994 by American Physiological Society

ARTICLES

Rat parietal cell receptors for GLP-1-(7-36) amide: northern blot, cross-linking, and radioligand binding

J. Schmidtler, K. Dehne, H. D. Allescher, V. Schusdziarra, M. Classen, J. J. Holst, A. Polack and W. Schepp
Department of Internal Medicine II, Technical University of Munich, Germany.

The intestinal peptide hormone glucagon-like peptide-1 (GLP-1) (7-36) amide is a potent stimulus of H+ production in isolated rat parietal cells, suggesting the presence of specific GLP-1-receptors on this cell type. Our aim was to characterize these receptors. Enzymatically isolated rat gastric mucosal cells (F0) were fractionated by counterflow elutriation, resulting in five fractions (F1-F5) according to increasing cell diameter and parietal cell content (3, 5, 4, 27, 81%). Additional 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). Northern blot of total cellular RNA from F0-F7 with a probe specific for the 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 125I-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 125I-GLP-1-(7-36) amide was correlated with parietal cell content (r = 0.99; P < 0.01) and H+ production ([14C]aminopyrine 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-affinity binding sites (KD = 2.8 +/- 0.6 x 10(-10) M, Bmax = 6.8 +/- 1.4 fmol/10(6) cells, 4,096 +/- 793 receptors/parietal cells). The following 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 units less potent displacers while gastric inhibitory peptide, vasoactive intestinal peptide, and secretin were ineffective. We conclude that rat parietal cells are equipped with specific high-affinity receptors for GLP-1-(7-36) amide, which, in addition, are present in as yet unidentified small cells (F1, F2) but not in chief cells.

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