采用全细胞电压钳技术测量分离的兔心室肌细胞中的钠钾泵电流。氧化应激对泵的抑制作用。

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.

作者信息

Shattock M J, Matsuura H

机构信息

Cardiovascular Research, Rayne Institute, St. Thomas' Hospital, London, UK.

出版信息

Circ Res. 1993 Jan;72(1):91-101. doi: 10.1161/01.res.72.1.91.

DOI:10.1161/01.res.72.1.91

PMID:8380265

Abstract

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-M omega) 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 microM), and Na(+)-K+ pump current was defined as the Ko-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 Nai and 1.88 mM for Ko. 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.

摘要

自由基诱导的氧化应激与心脏缺血及再灌注损伤有关。多项研究报道氧化应激会降低分离出的Na⁺,K⁺-ATP酶的活性。我们使用全细胞电压钳技术研究了氧化应激对分离的兔心室肌细胞中记录的Na⁺-K⁺泵电流的影响。孟加拉玫瑰红（50 nM）光激活产生单线态氧和超氧阴离子。设计台氏液和吸管内液的成分以阻断通道电流和电致Na⁺-Ca²⁺交换。通过宽尖端（1 - 2 MΩ）电极用含30 mM钠的吸管内液透析细胞，并在电压斜坡方案期间记录外向Na⁺-K⁺泵电流。通过与在200毫秒方波钳制步骤结束时进行的稳态Na⁺-K⁺泵电流测量相比较，证实了使用这种斜坡方案的有效性。无钾台氏液或哇巴因（100 μM）可消除活性电流，Na⁺-K⁺泵电流定义为记录电流中对钾敏感的部分。细胞内钠和细胞外钾对Na⁺-K⁺泵电流的激活显示，对于细胞内钠（Nai），半最大激活所需的钾浓度为18.7 mM，对于细胞外钾（Ko）为1.88 mM。氧化应激在所有电压下均抑制Na⁺-K⁺泵电流，使得在暴露于光激活的孟加拉玫瑰红10分钟后，在0 mV处测量的Na⁺-K⁺泵电流降低约50%。然而，氧化应激并未对Na⁺-K⁺泵电流的电压依赖性产生深远影响。在不存在所有主要电致离子通道、交换器或泵的情况下记录的被动膜电流不受氧化应激影响。这一观察结果表明，在发生Na⁺-K⁺泵抑制和钙超载的时间过程中，氧化应激不会导致非特异性膜损伤和脂质双层被动电阻的变化。氧化应激对Na⁺-K⁺泵活性的抑制可能导致缺血/再灌注损伤和再灌注诱导的细胞钙超载。