MEASUREMENT OF NA+-K+ PUMP CURRENT IN ISOLATED RABBIT VENTRICULAR MYOCYTES USING THE WHOLE-CELL VOLTAGE-CLAMP TECHNIQUE - INHIBITION OF THE PUMP BY OXIDANT STRESS
MEASUREMENT OF NA+-K+ PUMP CURRENT IN ISOLATED RABBIT VENTRICULAR MYOCYTES USING THE WHOLE-CELL VOLTAGE-CLAMP TECHNIQUE - INHIBITION OF THE PUMP BY OXIDANT STRESS
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DOI：
10.1161/01.res.72.1.91
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发表时间：
1993-01-01
影响因子：
20.1
通讯作者：
MATSUURA, H
中科院分区：
文献类型：
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作者：
SHATTOCK, MJ;MATSUURA, H
自由基诱导的氧化应激与心脏缺血和再灌注损伤有关。许多研究报道，氧化应激降低了分离的Na+，K+-ATP酶的活性。用全细胞电压钳技术研究了氧化应激对离体兔心室肌细胞Na ~+-K ~+泵电流的影响。通过虎红（50 nM）的光活化产生单线态氧和超氧化物。台氏液和移液器溶液的组成设计为阻断通道电流和生电Na+-Ca 2+交换。用含有30 mM钠的移液管溶液通过宽尖端（1-2-MOMEGA）电极透析细胞，并在电压斜坡方案期间记录外向Na+-K+泵电流。使用这样的斜坡协议的有效性得到了证实，通过比较与稳态Na+-K+泵电流测量在200毫秒的平方钳步骤结束。活性电流被无钾的台氏溶液或哇巴因（100 μ M）消除，Na+-K+泵电流被定义为记录的电流的K（o）-敏感部分。细胞内钠和细胞外钾对Na+-K+泵电流的激活揭示了Na（i）和K（o）的半数最大激活所需的钾浓度分别为18.7 mM和1.88 mM。在所有电压下，氧化强制降解均抑制Na+-K+泵电流，因此在暴露于光活化虎红10分钟后，在0 mV下测量的Na+-K+泵电流降低约50%。Na+-K+泵电流的电压依赖性，但是，没有深刻的影响，氧化应激。被动膜电流记录在所有主要产电离子通道，交换器，或泵的情况下，不受氧化应激。这一观察结果表明，在Na+-K+泵抑制和钙超载发生的时间过程中，氧化应激不会引起非特异性膜损伤和脂质双分子层被动电阻的变化。氧化应激对Na ~+-K ~+泵活性的抑制可能参与了缺血/再灌注损伤和再灌注诱导的细胞钙超载。
Free radical-induced oxidant stress has been implicated in ischemia and reperfusion-induced injury in the heart. A number of studies have reported that oxidant stress reduces the activity of isolated Na+,K+-ATPase enzyme. We have studied the effects of oxidant stress on the Na+-K+ pump current recorded in isolated rabbit ventricular myocytes using the whole-cell voltage-clamp technique. Singlet oxygen and superoxide were generated by the photoactivation of rose bengal (50 nM). The compositions of Tyrode's and pipette solutions were designed to block channel currents and electrogenic Na+-Ca2+ exchange. Cells were dialyzed with a pipette solution containing 30 mM sodium via wide-tipped (1-2-MOMEGA) electrodes, and outward Na+-K+ pump current was recorded during a voltage-ramp protocol. The validity of using such a ramp protocol was confirmed by comparison with steady-state Na+-K+ pump current measurements made at the end of 200-msec square-clamp steps. Active currents were abolished by potassium-free Tyrode's solution or ouabain (100 muM), and Na+-K+ pump current was defined as the K(o)-sensitive fraction of recorded currents. The activation of Na+-K+ pump current by intracellular sodium and extracellular potassium revealed a concentration of potassium necessary for half-maximal activation of 18.7 mM for Na(i) and 1.88 mM for K(o). Oxidant stress inhibited Na+-K+ pump current at all voltages, such that after a 10-minute exposure to photoactivated rose bengal, Na+-K+ pump current measured at 0 mV was reduced by approximately 50%. The voltage dependence of Na+-K+ pump current was, however, not profoundly affected by oxidant stress. Passive membrane currents recorded in the absence of all major electrogenic ion channels, exchangers, or pumps were unaffected by oxidant stress. This observation suggests that, over the time course during which Na+-K+ pump inhibition and calcium overload occur, oxidant stress does not cause nonspecific membrane damage and changes in the passive resistance of the lipid bilayer. The inhibition of Na+-K+ pump activity by oxidant stress may contribute to ischemia/reperfusion injury and reperfusion-induced cellular calcium overload.