Hughes S J, Chalk J G, Ashcroft S J
Nuffield Department of Clinical Biochemistry, John Radcliffe Hospital, Headington, Oxford, U.K.
Biochem J. 1990 Apr 1;267(1):227-32. doi: 10.1042/bj2670227.
We examined the contribution of signal-transduction pathways to acetylcholine-induced insulin release in the clonal beta-cell line HIT-T15. To assess the importance of changes in cytosolic free Ca2+ [( Ca2+]i), we studied time courses of the effects of glucose and acetylcholine on [Ca2+]i and insulin release in quin 2-loaded HIT cells. Incubation in the presence of glucose (2 mM) resulted in a sustained increase in [Ca2+]i in HIT cells from 98 +/- 7 nM to 195 +/- 12 nM measured after 9 min, whereas subsequent addition of acetylcholine (50 microM) produced a transient increase in [Ca2+]i which reached a peak after 30 s (at 274 +/- 10 nM), returning to pre-stimulus levels after 3 min. In contrast, incubation of HIT cells with acetylcholine in the presence of glucose produced a sustained increase in insulin release over and above that stimulated by glucose alone; after 10 min acetylcholine had potentiated glucose-stimulated insulin release by an additional increment of 135%. The transient increase in [Ca2+]i induced by acetylcholine was dose-dependent, and was prevented by omission of glucose or extracellular Ca2+ from the incubation medium. It was also inhibited by inclusion of 50 microM-verapamil in the incubation medium (by 87 +/- 3%) or by decreasing the Na+ concentration in the medium (by 73 +/- 6%). To evaluate the role of the protein kinase C pathway, we have pretreated HIT cells with the phorbol ester 12-O-tetradecanoylphorbol acetate (TPA), to deplete the protein kinase C activity, and have compared their secretory activity with that of control cells. Protein kinase C activity was decreased by 73% in HIT cells cultured in the presence of 200 nM-TPA for 22-24 h. TPA pre-treatment also significantly decreased the insulin content of HIT cells, but had no effect on cell number or the increases in [Ca2+]i induced by glucose or acetylcholine. TPA-pre-treated cells responded comparatively less well to secretagogues than did control cells: glucose-stimulated insulin release was decreased by 40%, whereas potentiation by TPA was significantly decreased by 50% in comparison with control cells (P less than 0.05, n = 24). Acetylcholine (50 microM) potentiated glucose-stimulated insulin release by 61% in control cells. This effect was abolished in HIT cells pre-treated with TPA, whereas these cells still retained their normal secretory response to stimulation by forskolin. These data suggest that an early increase in [Ca2+]i may be important for the initial increase in insulin release induced by acetylcholine in HIT cells.(ABSTRACT TRUNCATED AT 400 WORDS)
我们研究了信号转导通路对克隆β细胞系HIT-T15中乙酰胆碱诱导的胰岛素释放的作用。为了评估胞质游离钙（[Ca2+]i）变化的重要性，我们研究了葡萄糖和乙酰胆碱对用喹啉2负载的HIT细胞中[Ca2+]i和胰岛素释放影响的时间进程。在葡萄糖（2 mM）存在下孵育导致HIT细胞中[Ca2+]i持续增加，9分钟后测量从98±7 nM增加到195±12 nM，而随后添加乙酰胆碱（50 μM）使[Ca2+]i产生短暂增加，30秒后达到峰值（274±10 nM），3分钟后恢复到刺激前水平。相比之下，在葡萄糖存在下用乙酰胆碱孵育HIT细胞，胰岛素释放比单独葡萄糖刺激时有持续增加；10分钟后乙酰胆碱使葡萄糖刺激的胰岛素释放额外增加了135%。乙酰胆碱诱导的[Ca2+]i短暂增加是剂量依赖性的，并且通过从孵育培养基中省略葡萄糖或细胞外钙而被阻止。在孵育培养基中加入50 μM维拉帕米（降低87±3%）或降低培养基中Na+浓度（降低73±6%）也可抑制该增加。为了评估蛋白激酶C通路的作用，我们用佛波酯12-O-十四酰佛波醇乙酸酯（TPA）预处理HIT细胞以耗尽蛋白激酶C活性，并将它们的分泌活性与对照细胞进行比较。在200 nM TPA存在下培养22 - 24小时的HIT细胞中，蛋白激酶C活性降低了73%。TPA预处理也显著降低了HIT细胞的胰岛素含量，但对细胞数量或葡萄糖或乙酰胆碱诱导的[Ca2+]i增加没有影响。TPA预处理的细胞对促分泌剂的反应比对照细胞相对较差：葡萄糖刺激的胰岛素释放降低了40%，而与对照细胞相比，TPA的增强作用显著降低了50%（P<0.05，n = 24）。在对照细胞中，乙酰胆碱（50 μM）使葡萄糖刺激的胰岛素释放增强了61%。在用TPA预处理的HIT细胞中该作用被消除，而这些细胞对福斯高林刺激仍保留正常的分泌反应。这些数据表明，[Ca2+]i的早期增加可能对HIT细胞中乙酰胆碱诱导的胰岛素释放的初始增加很重要。（摘要截短为400字）
