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

目标。据报道，暴露于缺氧可激活离体心室肌细胞中ATP敏感的钾（K^+_<ATP>）通道。我们的目的是研究缺氧诱导的K^+_<ATP>通道激活的机制，使用专门设计的“气密灌注室”。采用胶原酶消化法分离豚鼠心室肌细胞。膜片钳全细胞模式下记录动作电位和膜电流。缺氧暴露在一个半封闭的密闭室中进行，以防止大气中的氧气扩散到缺氧的灌注液中。暴露于无糖缺氧使动作电位持续时间（APD）缩短至低于对照组的20%。[) 3分钟。随后的复氧迅速完全恢复APD。在缺氧过程中产生了与时间无关的大外向电流，并被再氧或格列本脲（K<@D1+@>D1<@ d2atp @>D2通道阻滞剂）的应用完全抑制。细胞外葡萄糖的存在并没有阻止缺氧时APD的缩短，尽管它显著降低了缩短的速度。缺氧时应用葡萄糖不能恢复无葡萄糖缺氧时缩短的动作电位。长期缺氧后，再氧诱导的APD恢复受到抑制。此外，反复暴露于缺氧/再氧会逐渐损害再氧过程中APD的恢复。K^+_<ATP>通道的激活发生在缺氧期间。调节通道活性的ATP的主要来源似乎是氧化磷酸化。由厌氧糖酵解产生的ATP（通过增加细胞外葡萄糖获得）仅在缺氧时氧化磷酸化严重受损时才会部分抑制通道活性。

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.