Yasuda Yoh, Matsuura Hiroshi, Ito Makoto, Matsumoto Tetsuya, Ding Wei-Guang, Horie Minoru
Department of Cardiovascular and Respiratory Medicine, Shiga University of Medical Science, Otsu, Shiga 520-2192, Japan.
Br J Pharmacol. 2005 May;145(2):156-65. doi: 10.1038/sj.bjp.0706191.
1 The present study was designed to examine the functional role of membrane phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P(2)) in the regulation of the muscarinic K(+) channel (I(K,ACh)) by extracellular ATP and adenosine in guinea-pig atrial myocytes, using the whole-cell patch-clamp method. 2 Bath application of ATP in micromolar concentrations typically evoked a transient activation of I(K,ACh); a rapid activation phase was consistently followed by a progressive decline even to the baseline level despite the continued presence of ATP. This progressive decline of I(K,ACh) was significantly attenuated either by blockade of phospholipase C (PLC) with compound 48/80 (100 microM) or by addition of PtdIns(4,5)P(2) (50 microM) to the cell inside, suggesting that depletion of membrane PtdIns(4,5)P(2) via PLC activation is mainly, if not totally, responsible for the progressive decline of I(K,ACh) during the presence of ATP. 3 When atrial myocytes were exposed to wortmannin (50 microM) following ATP (50 microM) application to impair the resynthesis of PtdIns(4,5)P(2), the activation of I(K,ACh) evoked by subsequently applied ATP (50 microM) was greatly reduced. Activation of I(K,ACh) by adenosine (100 microM) was partially reduced by pretreatment of atrial myocytes with ATP (100 microM) and was largely abolished by a further addition of wortmannin (50 microM) in the presence of ATP (100 microM). These results support the view that the activation of I(K,ACh) by ATP and adenosine depends on membrane PtdIns(4,5)P(2) that is subject to reduction by extracellular ATP. 4 The present study thus provides functional evidence to suggest that extracellular ATP activates PLC and thereby depletes membrane PtdIns(4,5)P(2) that is critically involved in the activation process of I(K,ACh) by its agonists ATP and adenosine in guinea-pig atrial myocytes.
1 本研究旨在利用全细胞膜片钳方法，研究豚鼠心房肌细胞中膜磷脂酰肌醇4,5-二磷酸（PtdIns(4,5)P(2)）在细胞外ATP和腺苷对毒蕈碱钾通道（I(K,ACh)）调节中的功能作用。2 微摩尔浓度的ATP浴灌流通常会引起I(K,ACh)的短暂激活；即使ATP持续存在，快速激活阶段之后也会持续下降至基线水平。I(K,ACh)的这种持续下降可通过用化合物48/80（100微摩尔）阻断磷脂酶C（PLC）或向细胞内添加PtdIns(4,5)P(2)（50微摩尔）而显著减弱，这表明通过PLC激活导致膜PtdIns(4,5)P(2)耗竭主要（如果不是完全）是ATP存在期间I(K,ACh)持续下降的原因。3 当在应用ATP（50微摩尔）后将心房肌细胞暴露于渥曼青霉素（50微摩尔）以损害PtdIns(4,5)P(2)的再合成时，随后应用的ATP（50微摩尔）引起的I(K,ACh)激活大大降低。用ATP（100微摩尔）预处理心房肌细胞可部分降低腺苷（100微摩尔）对I(K,ACh)的激活，在ATP（100微摩尔）存在下进一步添加渥曼青霉素（50微摩尔）则基本消除这种激活。这些结果支持这样的观点，即ATP和腺苷对I(K,ACh)的激活取决于膜PtdIns(4,5)P(2)，而细胞外ATP会使其减少。4 因此，本研究提供了功能证据表明，细胞外ATP激活PLC，从而耗尽膜PtdIns(4,5)P(2)，而膜PtdIns(4,5)P(2)在豚鼠心房肌细胞中对I(K,ACh)激动剂ATP和腺苷的激活过程至关重要。
