Ding W G, Kitasato H, Matsuura H
Department of Physiology, Shiga University of Medical Science, Shiga 520-2192, Japan.
Exp Physiol. 2001 May;86(3):331-9. doi: 10.1113/eph8602173.
The present investigation was designed to examine whether calmodulin is involved in the inhibition of the ATP-sensitive K+ (K(ATP)) channel by glucagon-like peptide 1(7-36) amide (GLP-1) in mouse pancreatic beta-cells. Membrane potential, single channel and whole-cell currents through the K(ATP) channels, and intracellular free Ca2+ concentration ([Ca2+]i) were measured in single mouse pancreatic beta-cells. Whole-cell patch-clamp experiments with amphotericin-perforated patches revealed that membrane conductance at around the resting potential is predominantly supplied by the K(ATP) channels in mouse pancreatic beta-cells. The addition of 20 nM GLP-1 in the presence of 5 mM glucose significantly reduced the membrane K(ATP) conductance, accompanied by membrane depolarization and the generation of electrical activity. A calmodulin inhibitor N-(6-aminohexyl)-5-chloro-1-naphthalenesulphonamide (W-7, 20 microM) completely reversed the inhibitory actions of GLP-1 on the membrane K(ATP) conductance and resultant membrane depolarization. Cell-attached patch recordings confirmed the inhibition of the K(ATP) channel activity by 20 nM GLP-1 and its restoration by 20 microM W-7 or 10 microM calmidazolium at the single channel level. Bath application of 20 microM W-7 also consistently abolished the GLP-1-evoked increase in [Ca2+]i in the presence of 5 mM glucose. These results strongly suggest that the mechanisms by which GLP-1 inhibits the K(ATP) channel activity accompanied by the initiation of electrical activity in mouse pancreatic beta-cells include a calmodulin-dependent mechanism in addition to the well-documented activation of the cyclic AMP-protein kinase A system.
本研究旨在探讨在小鼠胰腺β细胞中，钙调蛋白是否参与胰高血糖素样肽1(7 - 36)酰胺（GLP - 1）对ATP敏感性钾通道（K(ATP)通道）的抑制作用。在单个小鼠胰腺β细胞中测量了膜电位、通过K(ATP)通道的单通道电流和全细胞电流，以及细胞内游离Ca2+浓度（[Ca2+]i）。用两性霉素穿孔膜片进行的全细胞膜片钳实验表明，在小鼠胰腺β细胞静息电位附近，膜电导主要由K(ATP)通道提供。在5 mM葡萄糖存在的情况下，加入20 nM GLP - 1可显著降低膜K(ATP)电导，同时伴有膜去极化和电活动的产生。钙调蛋白抑制剂N-(6 - 氨基己基)-5 - 氯-1 - 萘磺酰胺（W - 7，20 μM）完全逆转了GLP - 1对膜K(ATP)电导的抑制作用以及由此导致的膜去极化。细胞贴附式膜片记录证实，在单通道水平上，20 nM GLP - 1可抑制K(ATP)通道活性，而20 μM W - 7或10 μM钙调蛋白拮抗剂可使其恢复。在5 mM葡萄糖存在的情况下，浴槽中加入20 μM W - 7也始终消除了GLP - 1引起的[Ca2+]i升高。这些结果强烈表明，在小鼠胰腺β细胞中，GLP - 1抑制K(ATP)通道活性并引发电活动的机制，除了已充分证明的环磷酸腺苷 - 蛋白激酶A系统激活外，还包括一种钙调蛋白依赖性机制。
