Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes
Ionic mechanism of the effects of hydrogen peroxide in rat ventricular myocytes
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DOI：
10.1113/jphysiol.1997.sp022048
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发表时间：
1997-05-01
影响因子：
5.5
通讯作者：
Giles, WR
中科院分区：
文献类型：
--
作者：
Ward, CA;Giles, WR
1. 全细胞和两性霉素穿孔膜片钳技术已用于研究过氧化氢（H2O2）对成年大鼠心室单个肌细胞动作电位和潜在离子电流的影响。2．所获得的结果根据所使用的记录方法而显着不同。传统的全细胞记录，即用移液器的内容物透析肌质，未能显示出 H2O2 对动作电位或细胞缩短的任何显着影响。相反，当用两性霉素穿孔贴片法记录动作电位时，H2O2（50-200μM）产生动作电位的显着延长和细胞缩短的增加。3．两性霉素穿孔贴片法的电压钳记录显示，H2O2 没有引起显着变化，无论是独立于 Ca2+ 的瞬态外向 K+ 电流 (I-to) 或内向整流 K+ 电流 (I-K1)。4．应用Na+通道阻滞剂河豚毒素(TTX; 8 x 10(-6) M)可很大程度上抑制H2O2对动作电位的影响。此外，anthopleurin A (4 x 10(-7) M) 通过减缓 Na+ 电流 (I-Na) 的失活来增强它，模拟了 H2O2 对心室肌细胞动作电位的影响。这些对 I-Na 的影响也几乎被 TTX.5 完全阻断。 H2O2 可以通过减慢 I-Na 失活动力学来增强 I-Na 的假设，直接使用细胞附着的宏斑块的整体记录进行了测试。这些结果表明，当记录移液管中包含 H2O2 (200 μM) 时，晚开放事件显着增强。观察到 I-Na 整体失活相应减慢。6．使用阻断剂bisindolylmaelimide (BIS; 10(-7) M) 检查了蛋白激酶C (PKC) 是观察到的H2O2 效应的细胞内第二信使的可能性。在H2O2 暴露之前浴应用BIS 显着延迟并减弱了动作电位延长的发展。7．这些结果表明 H2O2 对大鼠心室有显着的电生理作用。这些效应对记录方法的依赖性表明细胞内第二信使的参与，并且 PKC 抑制剂 BIS 的结果支持了这种可能性。 H2O2 对动作电位下的离子电流最显着的影响是减缓 TTX 敏感的 I-Na 的失活。最近的分子研究表明，大鼠心脏 Na+ 通道异构体上存在 PKC 磷酸化位点，并且还表明 PKC 激活可以减缓 I-Na 的失活。
1. Whole-cell and amphotericin-perforated patch-clamp techniques have been used to study the effects of hydrogen peroxide (H2O2) on action potentials and underlying ionic currents in single myocytes from the ventricles of adult rat hearts.2. The results obtained differed markedly depending on the recording method utilized. Conventional whole-cell recordings, in which the myoplasm is dialysed with the contents of the pipette, failed to show any significant effects of H2O2 on the action potential or cell shortening. In contrast, when action potentials were recorded with the amphotericin-perforated patch method, H2O2 (50-200 mu M) produced a marked prolongation of the action potential and an increase in cell shortening.3. Voltage-clamp recordings with the amphotericin-perforated patch method showed that H2O2 caused no significant changes is either the Ca2+-independent transient outward K+ current (I-to) or the inwardly rectifying K+ current (I-K1).4. Application of tetrodotoxin (TTX; 8 x 10(-6) M), a Na+ channel blocker, largely inhibited the effects of H2O2 on the action potential. Moreover, anthopleurin A (4 x 10(-7) M), which augments Na+ current (I-Na) by slowing its inactivation, mimicked the effects of H2O2 on the action potential of ventricular myocytes. These effects on I-Na were also blocked almost completely by TTX.5. The hypothesis that H2O2 can augment I-Na by slowing its kinetics of inactivation was tested directly using ensemble recordings from cell-attached macropatches. These results demonstrated a significant enhancement of late opening events when H2O2 (200 mu M) was included in the recording pipette. A corresponding slowing of inactivation of the ensemble I-Na was observed.6. The possibility that protein kinase C (PKC) is an intracellular second messenger for the observed effects of H2O2 was examined using the blocker bisindolylmaelimide (BIS; 10(-7) M). Bath application of BIS prior to H2O2 exposure significantly delayed and also attenuated the development of the action potential prolongation.7. These results demonstrate marked electrophysiological effects of H2O2 in rat ventricle. The dependence of these effects on recording methods suggests involvement of an intracellular second messenger, and the results with the PKC inhibitor, BIS, support this possibility. The most prominent effect of H2O2 on the ionic current which underlie the action potential is a slowing of inactivation of the TTX-sensitive I-Na. Recent molecular studies have demonstrated a PKC phosphorylation site on the rat cardiac Na+ channel isoform and have also shown that PKC activation can slow inactivation of I-Na.