Clapp L H
Rayne Institute, St Thomas's Hospital, London, UK.
Cardiovasc Res. 1995 Sep;30(3):460-8.
Although the existence of ATP-sensitive K+ (KATP) channels in vascular muscle is widely accepted, there appears to be little consensus as to what the primary regulator of these channels is under physiological or pathophysiological conditions. Recent evidence has suggested that nucleotide diphosphates (NDPs) may play a more important role than ATP. However, since the properties of vascular KATP channels are quite diverse, and the effects of these nucleotides are poorly understood, the aim of this study was to test the hypothesis that intracellular ATP can regulate whole-cell KATP current (IK,ATP) in the absence of NDPs.
Single cells were isolated from rabbit main pulmonary artery by enzymatic treatment with papain. Whole-cell patch clamp experiments were performed in cells dialysed with different nucleotides. The effect of the KATP channel activator, levcromakalim (10 microM), was investigated at a holding potential of -60 mV. The contribution of IK,ATP to the holding current was defined as the current which was blocked by glibenclamide following washout of levcromakalim.
Lowering the intracellular ATP concentration ([ATP]i) from 1 to 0.1 mM, in the presence or absence of GTP, enhanced the levcromakalim-induced current (Ilev) by approximately 2.5 fold and increased a glibenclamide-sensitive background K+ current (Iglib). However, Iglib was larger with GTP and the total glibenclamide-sensitive current (Ilev+Iglib) increased with time. Significant activation of Iglib failed to occur when the pipette contained no nucleotides and the responses to levcromakalim were generally much smaller than seen with ATP. GDP (0.5 mM), in the absence of pipette ATP, activated a large background K+ current which had similar properties to Ilev. Consistent with this was the observation that Ilev became substantially reduced in the presence of GDP, presumably because a significant amount of IK,ATP was already activated.
The response to levcromakalim in isolated cells from pulmonary artery was, as expected for an agent activating KATP channels, modulated by changes in the pipette [ATP]. This effect was not dependent on the presence of other pipette nucleotides, although the possibility cannot be excluded that metabolites from the cellular breakdown of ATP are essential for normal channel regulation. GDP could also activate IK,ATP under conditions where the channel is probably in a low phosphorylation state. The time-dependent effects of GTP require further work to determine the precise mechanism, but may suggest that GTP and/or G-proteins are involved in the regulation of KATP channels.
尽管血管平滑肌中存在ATP敏感性钾（KATP）通道已被广泛认可，但对于这些通道在生理或病理生理条件下的主要调节因子是什么，似乎尚未达成共识。最近的证据表明，核苷二磷酸（NDPs）可能比ATP发挥更重要的作用。然而，由于血管KATP通道的特性差异很大，且对这些核苷酸的作用了解甚少，本研究的目的是检验在不存在NDPs的情况下，细胞内ATP能否调节全细胞KATP电流（IK,ATP）这一假设。
用木瓜蛋白酶酶解处理从兔主肺动脉分离单个细胞。在用不同核苷酸透析的细胞中进行全细胞膜片钳实验。在-60 mV的钳制电位下研究KATP通道激活剂左卡尼汀（10 μM）的作用。IK,ATP对钳制电流的贡献定义为在洗脱左卡尼汀后被格列本脲阻断的电流。
在存在或不存在GTP的情况下，将细胞内ATP浓度（[ATP]i）从1 mM降至0.1 mM，可使左卡尼汀诱导的电流（Ilev）增强约2.5倍，并增加格列本脲敏感的背景钾电流（Iglib）。然而，GTP存在时Iglib更大，且总的格列本脲敏感电流（Ilev+Iglib）随时间增加。当移液管中不含核苷酸时，Iglib未出现明显激活，且对左卡尼汀的反应通常比ATP存在时小得多。在移液管中无ATP的情况下，GDP（0.5 mM）激活了一种与Ilev性质相似的大的背景钾电流。与此一致的是，在GDP存在时Ilev显著降低，推测是因为大量的IK,ATP已被激活。
正如激活KATP通道的药物所预期的那样，肺动脉分离细胞对左卡尼汀的反应受移液管[ATP]变化的调节。这种效应不依赖于移液管中其他核苷酸的存在，尽管不能排除ATP细胞分解代谢产物对正常通道调节至关重要的可能性。在通道可能处于低磷酸化状态的条件下，GDP也可激活IK,ATP。GTP的时间依赖性效应需要进一步研究以确定确切机制，但可能提示GTP和/或G蛋白参与KATP通道的调节。
