STIMULATION OF GLUCAGON-LIKE PEPTIDE-1 SECRETION BY MUSCARINIC AGONIST IN A MURINE INTESTINAL ENDOCRINE CELL-LINE
STIMULATION OF GLUCAGON-LIKE PEPTIDE-1 SECRETION BY MUSCARINIC AGONIST IN A MURINE INTESTINAL ENDOCRINE CELL-LINE
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DOI：
10.1210/en.134.5.2011
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发表时间：
1994-05-01
期刊：
影响因子：
4.8
通讯作者：
CHAYVIALLE, JA
中科院分区：
文献类型：
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作者：
ABELLO, J;YE, F;CHAYVIALLE, JA
对肠道L细胞胆碱能调节的研究主要集中在肠高血糖素的释放上，而对其信号转导途径尚未明确。本文以从内分泌细胞系STC-1释放的免疫反应类高血糖素样多肽-1(GLP-1)为指标进行了研究，该细胞系已被证明含有胰高血糖素原mRNA转录本。免疫细胞化学和放射免疫分析显示STC-1细胞内有丰富的GLP-1免疫反应。用特异性识别GLP-1 C端酰胺化形式的抗血清199D测定，细胞含量为4927+/-689pg/10(6)个细胞。培养2 h后，GLP-1的分泌量占GLP-1细胞总分泌量的1.4+/-0.3%，10亩M forsklin和100 nM 12-O-十四酰佛波醇13-醋酸酯分别显著增加到对照的206%和574%。胆碱能激动剂卡巴胆碱以浓度依赖的方式刺激GLP-1的分泌，在1 mM卡巴胆碱时达到最大释放量(控制值的228%)。M受体拮抗剂[N-甲基]东莨菪碱([H-3]NMS)与细胞匀浆的结合具有时间依赖性、特异性和可饱和性。Scatchard分析揭示了一类受体(K-d，14 pm；结合容量，20fmol/mg蛋白)。卡巴胆碱(0.1 mM~1 mM)剂量依赖性地移位[H-3]NMS结合，在不改变腺苷环化酶活力的情况下增加细胞内钙离子浓度。不同拮抗剂抑制[H-3]NMS结合、卡巴胆碱诱导的细胞内钙升高和卡巴胆碱刺激的GLP-1分泌的效力顺序为：阿托品(非选择性)>；4-二苯基乙酰氧基-N-甲基哌啶甲碘(M3)和GT；吡仑西平(M1)和GT；AF-DX 116(M2)。因此，本研究结果表明，胆碱能激动剂诱导的GLP-1的分泌依赖于内分泌肠道细胞株STC-1上的M_3-亚型受体。该系统可用于研究GLP-1分泌的细胞机制。
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.