Matsubayashi T, Matsuura H, Ehara T
Department of Physiology, Saga Medical School, Saga 849-8501, Japan.
Pflugers Arch. 1999 Apr;437(5):635-42. doi: 10.1007/PL00008090.
The effects of extracellular adenosine 5'-triphosphate (ATP) on the delayed rectifier K+ current (IK) were studied in guinea-pig ventricular myocytes using the whole-cell voltage-clamp technique. ATP increased IK concentration dependently with a concentration eliciting a half-maximal response of 1.86 microM and a maximal increase of about 1.8-fold. The enhancement of IK developed slowly, the effect reaching a maximum in about 1.6 min after application of ATP. The rank order of agonist potency in enhancing IK was 2-methylthio-ATP>/= ATP>>alpha,beta-methylene-ATP. The ATP response was attenuated in guanosine 5'-O-(2-thiodiphosphate) (GDPbetaS)- loaded cells, but was not affected by pertussis toxin (PTX)-pre-treatment, indicating that a PTX-insensitive G protein is involved in the response. These features are consistent with operation of P2Y-type purinoceptors. ATP produced a further increase in IK stimulated maximally either by isoprenaline (1 microM) through protein kinase A (PKA) or by 12-O-tetradecanoylphorbol 13-acetate (TPA, 100 nM) through protein kinase C (PKC), while 1-(5-isoquinolinesulfonyl)-2-methylpiperazine dihydrochloride (H-7, 10 microM) did not affect the ATP response, suggesting that PKA and PKC do not mediate the response. ATP irreversibly enhanced IK in cells loaded with adenosine 5'-O-(3-thiotriphosphate) (ATPgammaS, 5 mM) or okadaic acid (10 microM), a phosphatase inhibitor, suggesting that a phosphorylation step is present after the receptor stimulation. Genistein, an inhibitor of tyrosine phosphorylation, suppressed the ATP response significantly, while daidzein, an inactive analogue of genistein, had little effect on it, although both genistein or daidzein alone decreased IK. It is hypothesized that tyrosine phosphorylation plays a role in the signalling pathway involved in the enhancement of cardiac IK by P2Y-purinergic stimulation.
采用全细胞膜片钳技术，在豚鼠心室肌细胞中研究了细胞外5'-三磷酸腺苷（ATP）对延迟整流钾电流（IK）的影响。ATP浓度依赖性地增加IK，引起半数最大反应的浓度为1.86微摩尔，最大增加约1.8倍。IK的增强发展缓慢，在应用ATP后约1.6分钟达到最大值。增强IK的激动剂效力顺序为2-甲硫基-ATP≥ATP>>α,β-亚甲基-ATP。在加载5'-O-(2-硫代二磷酸)鸟苷（GDPβS）的细胞中，ATP反应减弱，但不受百日咳毒素（PTX）预处理的影响，表明一种对PTX不敏感的G蛋白参与了该反应。这些特征与P2Y型嘌呤受体的作用一致。ATP使通过蛋白激酶A（PKA）由异丙肾上腺素（1微摩尔）或通过蛋白激酶C（PKC）由12-O-十四烷酰佛波醇-13-乙酸酯（TPA，100纳摩尔）最大刺激的IK进一步增加，而1-(5-异喹啉磺酰基)-2-甲基哌嗪二盐酸盐（H-7，10微摩尔）不影响ATP反应，提示PKA和PKC不介导该反应。ATP在加载5'-O-(3-硫代三磷酸)腺苷（ATPγS，5毫摩尔）或磷酸酶抑制剂冈田酸（10微摩尔）的细胞中不可逆地增强IK，提示在受体刺激后存在一个磷酸化步骤。酪氨酸磷酸化抑制剂染料木黄酮显著抑制ATP反应，而染料木黄酮的无活性类似物大豆苷元对其影响很小，尽管单独的染料木黄酮或大豆苷元都降低IK。据推测，酪氨酸磷酸化在P2Y嘌呤能刺激增强心脏IK的信号通路中起作用。
