Metabolic Inhibition Strongly Inhibits Na +-Dependent Mg 2+ Efflux in Rat Ventricular Myocytes

Abstract

We measured intracellular Mg 2+ concentration ([Mg 2+] i) in rat ventricular myocytes using the fluorescent indicator furaptra (25°C). In normally energized cells loaded with Mg 2+, the introduction of extracellular Na + induced a rapid decrease in [Mg 2+] i: the initial rate of decrease in [Mg 2+] i (initial Δ[Mg 2+] i/Δ t) is thought to represent the rate of Na +-dependent Mg 2+ efflux (putative Na +/Mg 2+ exchange). To determine whether Mg 2+ efflux depends directly on energy derived from cellular metabolism, in addition to the transmembrane Na + gradient, we estimated the initial Δ[Mg 2+] i/Δ t after metabolic inhibition. In the absence of extracellular Na + and Ca 2+, treatment of the cells with 1 μM carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone, an uncoupler of mitochondria, caused a large increase in [Mg 2+] i from ∼0.9 mM to ∼2.5 mM in a period of 5–8 min (probably because of breakdown of MgATP and release of Mg 2+) and cell shortening to ∼50% of the initial length (probably because of formation of rigor cross-bridges). Similar increases in [Mg 2+] i and cell shortening were observed after application of 5 mM potassium cyanide (KCN) (an inhibitor of respiration) for ≥90 min. The initial Δ[Mg 2+] i/Δ 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 Δ[Mg 2+] i/Δ 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 Δ[Mg 2+] i/Δ t measurements, which is well below the [Na +] i level for half inhibition of the Mg 2+ efflux (∼40 mM). Normalization of intracellular pH using 10 μM nigericin, a H + ionophore, did not reverse the inhibition of the Mg 2+ efflux. From these results, it seems likely that a decrease in ATP below the threshold of rigor cross-bridge formation (∼0.4 mM estimated indirectly in the this study), rather than elevation of [Na +] i or intracellular acidosis, inhibits the Mg 2+ efflux, suggesting the absolute necessity of ATP for the Na +/Mg 2+ exchange.