STIMULATION OF GLUCAGON-LIKE PEPTIDE-1 SECRETION BY MUSCARINIC AGONIST IN A MURINE INTESTINAL ENDOCRINE CELL-LINE

Studies on the cholinergic regulation of intestinal L-cells have been focused on the release of enteroglucagon, but the signal transduction pathways were not defined. These were here investigated by using as index the release of immunoreactive glucagon-like peptide-1 (GLP-1) from the endocrine cell line STC-1, that has been shown to contain proglucagon mRNA transcripts. Abundant GLP-1 immunoreactivity was revealed in STC-1 cells at immunocytochemistry and by RIA. The cell content was 4927 +/- 689 pg/10(6) cells, as measured with antiserum 199D that recognizes specifically the C-terminal amidated forms of GLP-1. The secretion of GLP-1 over a 2-h incubation period amounted to 1.4 +/- 0.3% of the total GLP-1 cell content and was significantly increased by 10 mu M forskolin and 100 nM 12-O-tetradecanoylphorbol 13-acetate to 206% and 574% of control values, respectively. The cholinergic agonist carbachol stimulated GLP-1 secretion in a concentration-dependent manner; maximal release was observed at 1 mM carbachol (228% of the control value). Binding of the muscarinic antagonist [N-methyl-]scopolamine ([H-3]NMS) on cell homogenates was time dependent, specific, and saturable. Scatchard analysis revealed one class of receptors (K-d, 14 pM; binding capacity, 20 fmol/mg protein). Carbachol(0.1 mu M to 1 mM) dose dependently displaced [H-3] NMS binding and increased the intracellular calcium concentration without modification of adenylate cyclase activity. The order of potency of different antagonists, showing a preferential affinity for M1, M2, and M3 muscarinic receptor subtypes, to inhibit [H-3]NMS binding, the carbachol-induced increase in intracellular calcium, and carbachol-stimulated GLP-1 secretion, was as follows: atropine (nonselective) > 4-diphenylacetoxi-N-methylpiperidine methiodide (M3) > pirenzepine (M1) > AF-DX 116 (M2). The results of the present study, therefore, demonstrate that secretion of GLP-1 induced by cholinergic agonist depends on muscarinic M3-subtype receptors in the endocrine intestinal cell line STC-1. This system may prove useful to study the cellular mechanisms of GLP-1 secretion.