Characterization of magnesium transport in rat ventricular myocytes

Abstract

The fluorescent Mg²⁺ indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with acetoxy-methyl ester of the indicator. Intracellular free Mg²⁺ concentration ([Mg²⁺]_i) was estimated from the fluorescence intensity ratio at 382 nm and 350 nm excitation in Ca²⁺-free conditions (0.1 mM EGTA) at 25°C. After the cells were loaded with Mg²⁺ by incubation in high extracellular Mg²⁺ concentration ([Mg²⁺]_o), reduction of [Mg²⁺]_o to 1 mM induced a decrease in [Mg²⁺]_i. The decrease in [Mg²⁺]_i was accelerated by initial [Mg²⁺]_i above the basal level (~0.8 mM) with a half maximum [Mg²⁺]_i of ~1.5 mM. On the other hand, raising [Mg²⁺]_o slowed the decrease in [Mg²⁺]_i with 50% reduction of the rate at ~10 mM [Mg²⁺]_o. Since it was likely that a part of furaptra molecules were distributed in intracellular organelles, we assessed the fluorescence signal emitted from the organelles. The cell membrane of the indicator-loaded myocytes were permeabilized with saponin (50 μg/ml) in "the internal solution" that contained 0.8 mM free Mg²⁺ and 5 mM ATP (pH 7.1). While the indicator fluorescence intensity at 350 nm excitation was quickly dissipated by ~80% after saponin treatment, the intensity ratio was essentially unchanged. Raising free Mg²⁺ concentration of the internal solution to 4 mM only caused a small change in the fluorescence ratio that corresponded to an increase in [Mg²⁺]_i of ~0.15 mM. We conclude that the decrease in [Mg²⁺]_i observed in the present study primarily reflects Mg²⁺ efflux, which is modulated by Mg²⁺ gradient across the cell membrane. [Jpn J Physiol 54 Suppl:S69 (2004)]