Intracellular and extracellular concentrations of Na⁺ modulate Mg²⁺ transport in rat ventricular myocytes

¹ Tokyo Medical University

^* To whom correspondence should be addressed. E-mail: mkonishi{at}tokyo-med.ac.jp.

Submitted on June 16, 2005
Revised on July 13, 2005
Accepted on 18 July 2005

	Abstract

Apparent free cytoplasmic concentrations of Mg²⁺ ([Mg²⁺]_i) and Na⁺ ([Na⁺]_i) were estimated in rat ventricular myocytes using fluorescent indicators, furaptra (mag-fura-2) for Mg²⁺ and SBFI (sodium-binding benzofuran isophthalate) for Na⁺, at 25^oC in Ca²⁺-free conditions. Analysis included corrections for the influence of Na⁺ on furaptra fluorescence found in vitro and in vivo. The myocytes were loaded with Mg²⁺ in a solution containing 24 mM Mg²⁺ either in the presence of 106 mM Na⁺ plus 1 mM ouabain (Na⁺ loading) or in the presence of only 1.6 mM Na⁺ to deplete the cells of Na⁺ (Na⁺ depletion). The initial rate of decrease in [Mg²⁺]_i from the Mg²⁺-loaded cells was estimated in the presence of 140 mM Na⁺ and 1 mM Mg²⁺ as an index of the rate of extracellular Na⁺-dependent Mg²⁺ efflux. Average [Na⁺]_i, when estimated from SBFI fluorescence in separate experiments, increased from 12 mM to 31 mM and 47 mM after Na⁺ loading for 1 h and 3 h, respectively, and decreased to 0 mM after 3 h of Na⁺ depletion. The intracellular Na⁺ loading significantly reduced the initial rate of decrease in [Mg²⁺]_i, on average, by 40% at 1 h and by 64% at 3 h, suggesting that the Mg²⁺ efflux was inhibited by intracellular Na⁺ with 50% inhibition at 40 mM. Reduction of the rate of Mg²⁺ efflux was also observed when Na⁺ was introduced into the cells from a patch pipette that contained 130 mM Na⁺ through the amphotericin B-perforated cell membrane (perforated patch-clamp technique). When the cells were heavily loaded with Na⁺ with ouabain in combination with intracellular perfusion from the patch pipette containing 130 mM Na⁺, removal of extracellular Na+ caused an increase in [Mg²⁺]_i, albeit with very limited rate, which could be interpreted as reversal of the Mg²⁺ transport, i.e., Mg²⁺ influx driven by reversed Na⁺ gradient. Extracellular Na⁺-dependence of the rate of Mg²⁺ efflux revealed that the Mg²⁺ efflux was activated by extracellular Na⁺ with half-maximal activation at 55 mM. These results contribute to a quantitative characterization of the Na⁺-Mg²⁺ exchange in cardiac myocytes.

Key Words: Mg flux, SBFI, cardiac muscle, intracellular Mg, intracellular Na, mag-fura-2

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