An analysis of the enhancement of delayed rectifier K^+ current by P_2-purinoceptor stimulation

细胞外三磷酸腺苷以浓度依赖的方式增加豚鼠心房肌细胞延迟整流钾电流(L_K)的幅度，半数浓度(EC_(50))为0.95um，最大增加约2倍(Matsuura et al，J Physiol，1996)。在本研究中，我们用全细胞膜片钳技术研究了三磷酸腺苷对心肌细胞L_K的刺激作用。我们首先探讨了细胞外三磷酸腺苷是否增强L_K的快(L)、慢(L)或两者兼有。尾部实验和E-4031药理实验表明，三磷酸腺苷对豚鼠心房肌细胞L有选择性的增强作用，而对L的作用不明显。在此基础上，我们研究了三磷酸腺苷对豚鼠心房和心室肌细胞L刺激作用的信号转导机制。大于或e…的激动剂效价顺序更多的腺苷5‘-O-(3-硫代三磷酸)(ATP-Gamma)&gt；ADP&gt；&gt；腺苷表明参与了P2-嘌呤受体。这种L效应可被细胞内5‘-O-(2-硫代二磷酸，1 mM)鸟苷减弱，但不受百日咳毒素(PTX)的影响，表明PTX不敏感的G蛋白参与了这一反应。三磷酸腺苷不可逆地增强了携带5 mM三磷酸腺苷-伽玛斯的细胞中的L；Ks&gt；，这一反应可能涉及一种蛋白激酶的蛋白磷酸化，该蛋白激酶可以利用三磷酸腺苷-丙二醛作为磷酸供体。异丙肾上腺素(1um)通过蛋白激酶A(PKA)或12-O-十四酰佛波醇-13-乙酸酯(TPA，100 nM)通过蛋白激酶C(PKC)最大限度地刺激LKS时，ATP使LKS进一步增加，表明PKA和PKC不是该反应的中介。三磷酸腺苷对L的这种兴奋作用可被染料木素(50微米)部分抑制，但不受相同浓度大豆苷元的影响，提示酪氨酸蛋白的磷酸化至少部分参与了这一反应。因此，本研究揭示了细胞外ATP通过刺激G蛋白偶联的P2-嘌呤受体(P_2-P_2-嘌呤受体)激活某种酪氨酸蛋白激酶来增强LKS。较少

Extracellular ATP has been demonstrated to increase the amplitude of the delayed rectifier K^+ current (l_k) in a concentration-dependent manner with a half-maximal concentration (EC_<50>) of 0.95 muM and maximal increase of about a factor of 2 in guinea-pig atrial myocytes (Matsuura et al, J Physiol, 1996). In the present research project we characterized this stimulatory effects of ATP on l_K in isolated cardiac myocytes using the whole-cell patch-clamp technique. We first addressed the question whether extracellular ATP potentiates the rapid (l_<Kr>), the slow (l_<Ks>), or both components of l_K. An envelope of tails test and pharmacological experiments using E-4031 revealed that ATP selectively potentiates l_<Ks>, with no measurable effects on l_<Ks>, in guinea-pig atrial myocytes. We then investigated the signal transduction mechanism mediating the stimulatory effects of ATP on l_<1Ks> in guinea-pig atrial and ventricular myocytes. An agonist potency order of ATP<greater than or e … More qual> adenosine 5'-O-(3-thiotriphosphate) (ATP-gammaS)>ADP>>adenosine indicates an involvement of a P2-purinoceptor. This l_<Ks> response to ATP was attenuated by intracellular loading of guanosine 5'-O-(2-thiodiphosphate (GDP-betaS, 1 mM), but was not affected by pertussis toxin (PTX)-pretreatment, indicating that a PTX-insensitive G protein was involved in the response. ATP irreversibly enhanced l_<Ks> in cells loaded with 5 mM ATP-gammaS, suggesting that the response involved a protein phosphorylation possibly by a protein kinase which can utilize ATP-gammaS as a phosphate donor. ATP produced a further increase in lKs stimulated maximally either by isoprenaline (1 muM) via protein kinase A (PKA) or by 12-O-tetradecanoylphorbol-13-acetate (TPA, 100 nM) through protein kinase C (PKC), indicating that PKA and PKC did not mediate the response. This stimulatory effect of ATP on l_<Ks> was partially suppressed by genistein (50 muM) but not influenced by the same concentration of daidzein, suggesting that a tyrosine protein phosphorylation was, at least in part, involved in the response. Thus, the present research project revealed that extracellular ATP potentiates lKs by activating some kind of tyrosine protein kinases through a stimulation of G-protein coupled P2-purinoceptor (P2gamma-purinoceptor). Less