Lack of manifestations of diazoxide/5-hydroxydecanoatesensitive KATP channel in rat brain nonsynaptosomal mitochondria
Lack of manifestations of diazoxide/5-hydroxydecanoatesensitive KATP channel in rat brain nonsynaptosomal mitochondria
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DOI：
10.1113/jphysiol.2005.091199
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发表时间：
2005-10-01
影响因子：
5.5
通讯作者：
Brustovetsky, N
中科院分区：
文献类型：
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作者：
Brustovetsky, T;Shalbuyeva, N;Brustovetsky, N
对二氮嗪和5-羟基癸酸（5-HD）敏感的线粒体ATP敏感性K+通道（mitoKATP）的药理学调节代表了保护细胞免受缺血/再灌注和卒中相关损伤的有吸引力的策略。为了重新评估mitoK（ATP）在脑中的功能作用，我们使用Percol梯度纯化的脑非突触体线粒体进行吸光度测定、基质体积的放射性同位素测量以及呼吸、膜电位（Delta Psi）和去极化诱导的K+流出的测量。透射电镜观察线粒体形态学变化。针对已知磺酰脲受体亚基的某些片段，SUR 1和SUR 2，以及针对质膜ATP敏感性K+通道（cellK（ATP））的K+内向整流亚基Kir 6.1和Kir 6.2的不同表位的多克隆抗体，被用来检测脑线粒体中的类似亚基。应用了推定的mitoK（ATP）的各种看似合理的阻滞剂（ATP、5-羟基癸酸盐、格列本脲、四苯基鏻阳离子）和开放剂（二氮嗪、吡那地尔、铬卡林、米诺地尔、睾酮），以显示该通道的作用在调节基质体积、呼吸以及线粒体内膜的Delta Psi和K+通量中。在各种试验中，应用于脑线粒体的药理学试剂都没有明确指出与mitoK（ATP）活性相关的过程。此外，免疫印迹分析没有提供明确的证据证明脑线粒体中存在类似于celIK（ATP）的mitoK（ATP）。腺苷酸转位酶（ANT）的抑制剂羧甲苷（carboxyloxide）可重新激活ATP抑制的去极化诱发的K+释放。此外，另一种ANT抑制剂米松酸（bongkrekic acid）也与ATP类似地抑制K+外流。这些观察结果暗示ANT在脑线粒体ATP敏感的K+转运。
Pharmacological modulation of the mitochondrial ATP-sensitive K+ channel (mitoKATP) sensitive to diazoxide and 5-hydroxydecanoate (5-HD) represents an attractive strategy to protect cells against ischaemia/reperfusion- and stroke-related injury. To re-evaluate a functional role for the mitoK(ATP) in brain, we used Percoll-gradient-purified brain nonsynaptosomal mitochondria in a light absorbance assay, in radioisotope measurements of matrix volume, and in measurements of respiration, membrane potential (Delta Psi) and depolarization-induced K+ efflux. The changes in mitochondrial morphology were evaluated by transmission electron microscopy (TEM). Polyclonal antibodies raised against certain fragments of known sulphonylurea receptor subunits, SUR1 and SUR2, and against different epitopes of K+ inward rectifier subunits Kir 6.1 and Kir 6.2 of the ATP-sensitive K+ channel of the plasma membrane (cellK(ATP)), were employed to detect similar subunits in brain mitochondria. A variety of plausible blockers (ATP, 5-hydroxydecanoate, glibenclamide, tetraphenylphosphonium cation) and openers (diazoxide, pinacidil, chromakalim, minoxidil, testosterone) of the putative mitoK(ATP) were applied to show the role of the channel in regulating matrix volume, respiration, and Delta Psi and K+ fluxes across the inner mitochondrial membrane. None of the pharmacological agents applied to brain mitochondria in the various assays pinpointed processes that could be unequivocally associated with mitoK(ATP) activity. In addition, immunoblotting analysis did not provide explicit evidence for the presence of the mitoK(ATP), similar to the celIK(ATP), in brain mitochondria. On the other hand, the depolarization-evoked release of K+ suppressed by ATP could be re-activated by carboxyatractyloside, an inhibitor of the adenine nucleotide translocase (ANT). Moreover, bongkrekic acid, another inhibitor of the ANT, inhibited K+ efflux similarly to ATP. These observations implicate the ANT in ATP-sensitive K+ transport in brain mitochondria.