Nakano Kyoko, Suga Sechiko, Takeo Teruko, Ogawa Yoshiji, Suda Toshihiro, Kanno Takahiro, Wakui Makoto
Department of Physiology, Hirosaki University School of Medicine, Hirosaki 036-8562, Japan.
Endocrinology. 2002 Feb;143(2):569-76. doi: 10.1210/endo.143.2.8625.
We investigated the mechanism by which acetylcholine (ACh) regulates insulin secretion from rat pancreatic beta-cells. In an extracellular solution with 5.5 mM glucose, ACh increased the rate of insulin secretion from rat islets. In islets treated with bisindolylmaleimide (BIM), a PKC inhibitor, ACh still increased insulin secretion, but the increment was lower than that without BIM. In the presence of nifedipine, an L-type Ca(2+) channel blocker, on the other hand, ACh did not increase insulin secretion. In isolated rat pancreatic beta-cells, ACh caused depolarization followed by action potentials. This ACh effect was observed even in cells treated with BIM. In the presence of nifedipine, ACh caused only depolarization. These ACh effects were prevented by atropine. In the perforated whole-cell configuration, ramp pulses from -90 to -50 mV induced membrane currents mostly through ATP-sensitive K(+) channels (K(ATP)). These currents were reduced in size by ACh in cells either treated or untreated with BIM; whereas the loading of cells with U-73122 (a phospholipase C inhibitor) or BAPTA/AM (a Ca(2+) chelator) abolished the ACh effect. In the standard whole-cell configuration, ACh reduced the currents through K(ATP) with 0.5 mM EGTA, but not with 10 mM EGTA, in the pipette solution. Intracellular application of GDPbetaS or heparin also inhibited the ACh effect. In the inside-out single-channel recordings, elevation of the Ca(2+) concentration inside the membrane from 10 nM-10 microM decreased K(ATP) activity only in the presence of ATP. The affinity of ATP to K(ATP) became 4.5 times higher with the higher concentration of Ca(2+). These results suggest that Ca(2+) from ACh receptor signaling modulates the sensitivity of K(ATP) to ATP. A positive-feedback mechanism of intracellular Ca(2+)-dependent Ca(2+) influx was also demonstrated.
我们研究了乙酰胆碱（ACh）调节大鼠胰腺β细胞胰岛素分泌的机制。在含有5.5 mM葡萄糖的细胞外溶液中，ACh增加了大鼠胰岛的胰岛素分泌速率。在用双吲哚马来酰亚胺（BIM，一种蛋白激酶C抑制剂）处理的胰岛中，ACh仍能增加胰岛素分泌，但增加幅度低于未用BIM处理的情况。另一方面，在存在L型钙通道阻滞剂硝苯地平的情况下，ACh不会增加胰岛素分泌。在分离的大鼠胰腺β细胞中，ACh引起去极化，随后产生动作电位。即使在用BIM处理的细胞中也观察到这种ACh效应。在存在硝苯地平的情况下，ACh仅引起去极化。这些ACh效应可被阿托品阻断。在穿孔全细胞模式下，从-90 mV到-50 mV的斜坡脉冲诱导的膜电流主要通过ATP敏感性钾通道（K(ATP)）。在用或未用BIM处理的细胞中，这些电流的大小都因ACh而减小；而用U-73122（一种磷脂酶C抑制剂）或BAPTA/AM（一种钙螯合剂）处理细胞则消除了ACh效应。在标准全细胞模式下，在移液器溶液中含有0.5 mM乙二醇双四乙酸（EGTA）时，ACh会减小通过K(ATP)的电流，但含有10 mM EGTA时则不会。细胞内应用二磷酸鸟苷β硫酯（GDPβS）或肝素也会抑制ACh效应。在膜内面向外的单通道记录中，仅在有ATP存在的情况下，膜内钙离子浓度从10 nM升高到10 μM会降低K(ATP)的活性。随着钙离子浓度升高，ATP对K(ATP)的亲和力提高了4.5倍。这些结果表明，来自ACh受体信号的钙离子调节K(ATP)对ATP的敏感性。还证明了细胞内钙依赖性钙内流的正反馈机制。
