Zhu Z, Li Y L, Li D P, He R R
Department of Physiology, Institute of Basic Medicine, Hebei Medical University, Shijiazhuang, People's Republic of China.
Pflugers Arch. 2000 Apr;439(6):808-13. doi: 10.1007/s004249900200.
Ischemic or hypoxic preconditioning in experimental animals and humans is described. The mechanism of preconditioning may involve several endogenous substances released from ischemic or hypoxic tissues (such as adenosine, noradrenaline and bradykinin) that stimulate protein kinase C (PKC), which then phosphorylates ATP-sensitive potassium channels (K(ATP) channels). However, the effect of hypoxic preconditioning on K(ATP) channels in guinea-pig ventricular myocytes is unclear. The uncoupler carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) has been shown to activate K(ATP) channels in isolated cardiac cells. In the present study we tested whether anoxic preconditioning (APC) could affect the opening of K(ATP) channels activated by metabolic inhibition (MI) induced by FCCP in cell-attached and inside-out patches from guinea-pig ventricular myocytes. We measured the channel activity as NP(o)i and calculated it using the formula Po=I/(Ni), where Po is open-state probability, I is the mean patch current carried by all K(ATP) channels activated in a particular patch for a certain period of time, N is the number of functioning channels in the patch, and i is the unitary current of the K(ATP) channels. In cell-attached membrane patches, after about 5 min of initiating MI, K(ATP) channels were activated at a holding potential of +40 mV (NP(o)i=3.70+/-0.9 pA); APC pretreatment (3 min of anoxia followed by 7 min of reoxygenation) before MI (APC+MI group) shortened the time to activate K(ATP) channels by MI (2.3+/-0.5 min) and increased the activity of K(ATP)currents (NP(o)i=8.4+/-0.5 pA). This effect of APC was eliminated by administration of a PKC blocker, chelerythrine (5 microM), for 5 min before the APC pretreatment. In the inside-out patches, the IC50 of intracellular ATP against the K(ATP) channels in the APC+MI group was significantly increased to 642 microM compared to that in the MI group (IC50 of intracellular ATP =252 microM). Chelerythrine inhibited the effect of APC on the sensitivity of K(ATP) channels to the intracellular ATP concentration (IC50 of [ATP]i=301 microM). Our results demonstrate that APC can increase and accelerate the opening of K(ATP) channels induced by MI, and decrease the sensitivity of K(ATP) channels to [ATP]i, which is mediated by promoting the activation of PKC induced by APC.
本文描述了实验动物和人类中的缺血或缺氧预处理。预处理的机制可能涉及缺血或缺氧组织释放的几种内源性物质（如腺苷、去甲肾上腺素和缓激肽），这些物质刺激蛋白激酶C（PKC），然后PKC使ATP敏感性钾通道（K(ATP)通道）磷酸化。然而，缺氧预处理对豚鼠心室肌细胞中K(ATP)通道的影响尚不清楚。解偶联剂羰基氰化物对-(三氟甲氧基)苯腙（FCCP）已被证明可激活分离的心肌细胞中的K(ATP)通道。在本研究中，我们测试了缺氧预处理（APC）是否会影响由FCCP诱导的代谢抑制（MI）激活的K(ATP)通道在豚鼠心室肌细胞的细胞贴附式和内面向外膜片中的开放。我们将通道活性测量为NP(o)i，并使用公式Po = I/(Ni)进行计算，其中Po是开放概率，I是在特定膜片中所有K(ATP)通道在一定时间内激活所携带的平均膜片电流，N是膜片中功能通道的数量，i是K(ATP)通道的单位电流。在细胞贴附式膜片中，在开始MI约5分钟后，K(ATP)通道在+40 mV的钳制电位下被激活（NP(o)i = 3.70±0.9 pA）；在MI之前进行APC预处理（3分钟缺氧随后7分钟复氧）（APC + MI组）缩短了MI激活K(ATP)通道的时间（2.3±0.5分钟）并增加了K(ATP)电流的活性（NP(o)i = 8.4±0.5 pA）。在APC预处理前5分钟给予PKC阻断剂白屈菜红碱（5 microM）可消除APC的这种作用。在内面向外膜片中，与MI组相比，APC + MI组中细胞内ATP对K(ATP)通道的IC50显著增加至642 microM（细胞内ATP的IC50 = 252 microM）。白屈菜红碱抑制了APC对K(ATP)通道对细胞内ATP浓度敏感性的影响（[ATP]i的IC50 = 301 microM）。我们的结果表明，APC可以增加并加速MI诱导的K(ATP)通道的开放，并降低K(ATP)通道对[ATP]i的敏感性，这是通过促进APC诱导的PKC激活介导的。
