Modulation of intracellular na(+) and ca(2+) induced by the cardiac na(+)-ca(2+) exchanger in Guinea pig ventricular myocytes.

摘要

The Na(+)-Ca(2+) exchanger current was measured in single guinea pig ventricular myocytes, using the whole-cell voltage-clamp technique, and intracellular free calcium concentration ([Ca(2+)](i)) was monitored simultaneously with the fluorescent probe Indo-1 applied intracellularly through a perfused patch pipette. In external solutions, which have levels of Ca(2+) ( approximately 66 microM Ca(2+)) thought low enough to inhibit exchanger turnover, the removal of external Na(+) (by replacement with Li(+)) induced both an outward shift of the holding current and an increase in [Ca(2+)](i), even though the recording pipette contained 30 mM bis(O-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), sufficient to completely block phasic contractions. The effects of Na(+) removal were blocked either by the extracellular application of 2 mM Ni(2+) or by chelating extracellular Ca(2+) with 1 mM EGTA. In the presence of 10 microM Ryanodine, the effects of external Na(+) substitution with Li(+) on both membrane current and [Ca(2+)](i) were attenuated markedly in amplitude and at a much slower time course. Reversal potentials were estimated by using ramp pulses and by defining exchange currents as the Ni(2+)-sensitive components. The experimental values of the reversal potential and [Ca(2+)](i) were used to calculate cytosolic Na(+) ([Na(+)](i)) by assuming an exchanger stoichiometry of 3Na(+) : 1Ca(2+). These calculations suggested that in the nominal absence of external Ca(2+) ( approximately 66 microM under our experimental conditions), the exchanger operates at -40 mV as though approximately 40 mM Na(+) had accumulated in the vicinity of the intracellular binding sites. We conclude that under the conditions of low extracellular Ca(2+) and high intracellular Ca(2+) buffering, the Na(+)-Ca(2+) exchanger can still generate sufficient Ca(2+) influx on the removal of external Na(+) to markedly increase cytosolic free Ca(2+).