Hool L C, Middleton L M, Harvey R D
Department of Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106-4970, USA.
Circ Res. 1998 Jul 13;83(1):33-42. doi: 10.1161/01.res.83.1.33.
The whole-cell patch-clamp technique was used to monitor the effects of genistein, a tyrosine kinase inhibitor, on membrane currents recorded from isolated guinea pig ventricular myocytes. Under control conditions, genistein (50 micromol/L) did not activate the latent cAMP-regulated Cl- current (ICl). However, in the presence of a subthreshold concentration (1 nmol/L) of the beta-adrenergic agonist isoproterenol (Iso), genistein caused a near-maximal activation of this current. In the absence of genistein, Iso activated ICl with an EC50 of 5 nmol/L. In the presence of genistein, Iso activated ICl with an EC50 of 0.3 nmol/L. This facilitatory effect was not observed in the presence of daidzein (50 micromol/L), an analogue of genistein that only weakly inhibits tyrosine kinase activity. Furthermore, peroxovanadate, a potent inhibitor of phosphotyrosine phosphatase activity, inhibited ICl activated by Iso alone, and it blocked the stimulatory effect of genistein in the presence of Iso. To determine whether the stimulatory effect of genistein was specific for ICl, we also studied its action on the cAMP-regulated delayed rectifier K+ current (IK) and L-type Ca2+ current (ICa-L) present in these cells. Basal IK and ICa-L were partially (approximately 30% to 40%) inhibited by genistein. However, this inhibitory effect was mimicked by daidzein, suggesting that inhibition of tyrosine kinase activity is not involved. In addition to the nonspecific inhibitory effect, genistein also caused a significant increase in the beta-adrenergic sensitivity of the unblocked cationic currents. In the absence of genistein, 1 nmol/L Iso had no effect on either IK or ICa-L. However, in the presence of genistein, 1 nmol/L Iso significantly increased the magnitude of both currents. These results suggest that tyrosine kinase activity may play an important role in regulating beta-adrenergic responsiveness of the heart.
采用全细胞膜片钳技术监测酪氨酸激酶抑制剂染料木黄酮对豚鼠离体心室肌细胞记录的膜电流的影响。在对照条件下，染料木黄酮（50 μmol/L）未激活潜在的cAMP调节氯离子电流（ICl）。然而，在存在亚阈值浓度（1 nmol/L）的β-肾上腺素能激动剂异丙肾上腺素（Iso）的情况下，染料木黄酮使该电流几乎达到最大激活。在不存在染料木黄酮时，Iso激活ICl的半数有效浓度（EC50）为5 nmol/L。在存在染料木黄酮时，Iso激活ICl的EC50为0.3 nmol/L。在存在染料木黄酮类似物大豆苷元（50 μmol/L）时未观察到这种促进作用，大豆苷元仅微弱抑制酪氨酸激酶活性。此外，过氧钒酸盐是一种有效的磷酸酪氨酸磷酸酶活性抑制剂，它抑制单独由Iso激活的ICl，并在存在Iso时阻断染料木黄酮的刺激作用。为了确定染料木黄酮的刺激作用是否对ICl具有特异性，我们还研究了其对这些细胞中存在的cAMP调节延迟整流钾电流（IK）和L型钙电流（ICa-L）的作用。基础IK和ICa-L被染料木黄酮部分抑制（约30%至40%）。然而，大豆苷元模拟了这种抑制作用，表明抑制酪氨酸激酶活性与之无关。除了非特异性抑制作用外，染料木黄酮还导致未被阻断的阳离子电流的β-肾上腺素能敏感性显著增加。在不存在染料木黄酮时，1 nmol/L Iso对IK或ICa-L均无影响。然而，在存在染料木黄酮时，1 nmol/L Iso显著增加了两种电流的幅度。这些结果表明，酪氨酸激酶活性可能在调节心脏的β-肾上腺素能反应性中起重要作用。
