Mechanisms of anoxia-induced activation of ATP-sensitive K channels in isolated ventricular myocytes

目标。据报道，缺氧可激活心肌细胞上的ATP敏感性钾(K^+&lt；ATP&gt；)通道。我们的目的是利用专门设计的“密闭灌流室”来研究缺氧引起K~(++)通道激活的机制。采用胶原酶消化法分离豚鼠心肌细胞。膜片钳全细胞模式记录动作电位和膜电流。在半封闭密闭室中进行缺氧暴露，防止大气中的氧气扩散到缺氧灌流液中。暴露于无糖缺氧环境中，13例患者的动作电位时程缩短至对照组的20%以下。[)3分钟。随后的复氧迅速而完全地恢复了APD。在缺氧过程中形成了不随时间变化的大的外向电流，并被复氧或应用K&lt；@d1+@d1；d1；@D2ATP@D2通道阻断剂格列本脲完全抑制。细胞外葡萄糖的存在虽然显著降低了动作电位时程缩短的速度，但并不能阻止时程缩短。在缺氧状态下应用葡萄糖不能恢复因无糖缺氧而缩短的动作电位。在长期缺氧后，复氧诱导的时程恢复受到抑制。此外，反复缺氧/复氧使复氧过程中动作电位时程的恢复逐渐减弱。K^+_lt；ATP；通道在缺氧时被激活。调节通道活性的ATP的主要来源似乎是氧化磷酸化。来自厌氧糖酵解的ATP(通过增加胞外葡萄糖获得)仅在缺氧时氧化磷酸化严重受损时才能部分抑制通道活性。

Objective. Exposure to anoxia has been reported to activate ATP-sensitive potassium (K^+_<ATP>) channels in isolated ventricular myocytes. We aim to investigate the mechanisms underlying the anoxia-induced activation of K^+_<ATP> channels, using specially designed "air-tight perfusion chamber".Methods. Guinea pig ventricular myocytes were isolated using collagenase digestion. Action potentials and membrane currents were recorded in the whole-cell mode of patch clamp. Exposure to anoxia was performed in a semi-closed airtight chamber, which prevented the diffusion of atmospheric oxygen into anoxic perfusate.Results. Exposure to glucose-free anoxia shortened the action potential duration (APD) to less than 20% of control in 13 (]SY.+-。[) 3 min. Subsequent reoxygenation rapidly and completely restored the APD.The time-independent large outward current developed during anoxia, and was completely suppressed by reoxygenation or by the application of glibenclamide, a K<@D1+@>D1<@D2ATP@>D2 channel blocker. The presence of extracellular glucose did not prevent the APD from shortening during anoxia, although it significantly decreased the rate of shortening. Application of glucose during anoxia could not restore the action potential that was shortened by glucose-free anoxia. Reoxygenation-induced restoration of the APD was inhibited after a long-lasting anoxia. In addition, repeated exposure to anoxia/reoxygenation progressively impaired the recovery of APD during reoxygenation.Conclusions. Activation of K^+_<ATP> channels occurs during anoxia. The primary source of ATP that regulates the channel activity seems to be oxidative phosphorylation. ATP derived from anaerobic glycolysis (attained by the increase of extracellular glucose) was observed to partially suppress the channel activity only when oxidative phosphorylation was severely impaired during anoxia.