Metabolic Inhibition Strongly Inhibits Na+-Dependent Mg2+ Efflux in Rat Ventricular Myocytes

We measured intracellular Mg2+ concentration ([Mg2+](i)) in rat ventricular myocytes using the fluorescent indicator furaptra (25 degrees C). In normally energized cells loaded with Mg2+, the introduction of extracellular Na+ induced a rapid decrease in [Mg2+](i): the initial rate of decrease in [Mg2+](i) (initial Delta[Mg2+](i)/Delta t) is thought to represent the rate of Na+-dependent Mg2+ efflux (putative Na+/Mg2+ exchange). To determine whether Mg2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na+ gradient, we estimated the initial Delta[Mg2+](i)/Delta t after metabolic inhibition. In the absence of extracellular Na+ and Ca2+, treatment of the cells with 1 mu M carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg2+](i) from similar to 0.9 mM to similar to 2.5 mM in a period of 5-8 min (probably because of breakdown of MgATP and release of Mg2+) and cell shortening to similar to 50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg2+](i) and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for >= 90 min. The initial Delta[Mg2+](i)/Delta t was diminished, on average, by 90% in carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone-treated cells and 92% in KCN-treated cells. When the cells were treated with 5 mM KCN for shorter times (59-85 min), a significant decrease in the initial Delta[Mg2+](i)/Delta t (on average by 59%) was observed with only a slight shortening of the cell length. Intracellular Na+ concentration ([Na+](i)) estimated with a Na+ indicator sodium-binding benzofuran isophthalate was, on average, 5.0-10.5 mM during the time required for the initial Delta[Mg2+](i)/Delta t measurements, which is well below the [Na+](i) level for half inhibition of the Mg2+ efflux (similar to 40 mM). Normalization of intracellular pH using 10 mu M nigericin, a H+ ionophore, did not reverse the inhibition of the Mg2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (similar to 0.4 mM estimated indirectly in the this study), rather than elevation of [Na+](i) or intracellular acidosis, inhibits the Mg2+ efflux, suggesting the absolute necessity of ATP for the Na+/Mg2+ exchange.