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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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.