Study of intracellular modulation mechanisms of cardiac ATP-sensitive K^+ channels and their pathophysiological implications.

对ATP敏感的K~+通道(K_&lt；ATP&gt；)具有被[ATP]_i强烈抑制的特点，但对处于工作状态的K~+通道也是必需的。调节通道功能的因素很多，但很难将其在心肌缺血时开放的真正作用归因于这些因素。我们以前曾证明，镁三磷酸腺苷对After Rundown的重新激活是由水解引起的。由于在肌动蛋白细胞骨架组装过程中也利用了ATP水解酶，因此我们研究了K_lt；ATP&>活性和肌动蛋白聚合状态之间的联系。应用肌动蛋白干扰物可导致快速衰弱。肌动蛋白稳定剂在部分收缩通道中恢复通道活动，而在完全收缩或完全激活的通道中无效。镁-三磷酸腺苷加F-肌动蛋白使通道完全恢复。因此，肌动蛋白细胞骨架的组装和拆解对K；…起着调制作用。接下来，我们考察了K；当[ATP]_I浓度为1~2 mM时，胰酶处理的心肌细胞膜片可开放更多的通道，而ADP+Mg~(2+)和低pH值可使通道开放概率增加，说明心肌缺血早期，细胞内ATP水平不会显著下降。这种开放可以在与缺血相关的辅助因素如ADP和低pH值的存在下实现。另一种缺血产物，腺苷，被认为是心肌缺血时K_&lt；ATP和Gt；的激活剂，但其在全细胞条件下的实际作用尚未得到证实。我们使用了制霉菌素穿孔方法，这种方法不会干扰细胞条件。利用这一技术，腺苷被证明在代谢抑制期间缩短K&lt；ATP&Gt；开放，这是通过刺激A2受体介导的。PKC的激活也涉及K_&lt；ATP和PKC的开放，但腺苷和PKC并不像其他组所报道的那样协同作用。A2受体和PKC激活之间的细胞内机制正在进行广泛的研究。较少

The ATP-sensitive K^+ channels (K_<ATP>) are characterized by a strong inhibition by [ATP]_I but ATP is also necessary for the channels in an operative states. There are numerous factors to modulate the channel functions, which are difficult to attribute to real role for their openings during myocardial ischemia. We previously demonstrated that reactivation of after rundown by MgATP was caused by hydrolysis. Since ATP hydrolysis is also utilized in the process of actin cytoskeletal assembly, we examined the linkage between the K_<ATP> activity and the status of actin polymerization. Application of actin disrupters induced quick rundown. Actin stabilizers restored the channel activity in partially rundown channels, while they were ineffective in complete rundown or fully activated channels. MgATP plus F-actin restored the completely rundown channels. Therefore, the assembly and disassembly of actin cytoskeleton play a modulatory role for K_<ATP> Next, we examined whether or not K_<ATP> … More could be opened in the presence of milimor order of [ATP]_I. Using the trypsin-treated patches which showed least rundown, the presence of ADP plus Mg^<2+>, low pH increased the channel open probability in the presence of 1-2 mM [ATP]_I. From these results, we can conclude that K_<ATP> will be opened during early phase of myocardial ischemia where cellular ATP levels do not drop dramatically. This openings can be achieved with the presence of co-factors such as ADP and low pH,which are associated with ischemia. Another ischemic product, adenosine, is supposed to be an activator of K_<ATP> during myocardial ischemis, but its actual contribution in the whole-cell condition has not been demonstrated. We used a nystatin-perforated method which did not disturb the cellular condition. Using this technique, adenosine was shown to shorten K_<ATP> openings during metabolic inhibition, which was mediated through A2-receptor stimulation. The activation of PKC was also involved the K_<ATP> openings, but adenosine and PKC did not work synergistically as reported by the other groups. The intracellular mechanism between A2-receptor and PKC activation is under the extensive investigation. Less