Modulation of Mg²⁺ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra

¹ Tokyo Medical University
² Tokyo Medical Univeristy

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

Submitted on November 2, 2004
Revised on December 3, 2004
Accepted on 22 December 2004

	Abstract

The fluorescent Mg²⁺ indicator furaptra (mag-fura-2) was introduced into single ventricular myocytes by incubation with its acetoxy-methyl ester form. The ratio of furaptra's fluorescence intensity at 382 and 350 nm was used to estimate the apparent cytoplasmic [Mg²⁺] ([Mg²⁺]_i). In Ca²⁺-free extracellular conditions (0.1 mM EGTA) at 25^oC, [Mg²⁺]_i averaged 0.842 ± 0.019 mM. After the cells were loaded with Mg²⁺ by exposure to high extracellular [Mg²⁺] ([Mg²⁺]_o), reduction of [Mg²⁺]_o to 1 mM (in the presence of extracellular Na⁺) induced a decrease in [Mg²⁺]_i. The rate of decrease in [Mg²⁺]_i was higher at higher [Mg²⁺]_i, while raising [Mg²⁺]_o slowed the decrease in [Mg²⁺]_i with 50% reduction of the rate at 10 mM [Mg²⁺]_o. Because a part of furaptra molecules were likely trapped inside intracellular organelles, we assessed possible contribution of the indicator fluorescence emitted from the organelles. When the cell membranes of furaptra-loaded myocytes were permeabilized with saponin (25 µg/ml for 5 min), furaptra fluorescence intensity at 350 nm excitation decreased to 22%; thus about 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg²⁺] ([Mg²⁺]_c) while the residual 22% likely represented [Mg²⁺] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg²⁺] calibrated from the residual furaptra fluorescence ([Mg²⁺]_r) was 0.6-0.7 mM in bathing solution [Mg²⁺] (i.e., [Mg²⁺]_c of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg²⁺]_r was lower than and virtually insensitive to [Mg²⁺]_c. We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg²⁺]_c. In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg²⁺_c buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg²⁺ flux across the cell membrane by utilizing concentration and dissociation constant values of known cytoplasmic Mg²⁺ buffers. The calculations indicate that, in the presence of extracellular Na⁺, Mg²⁺ efflux is markedly activated by [Mg²⁺]_c above the normal basal level (0.9 mM), with a half maximal activation of 1.9 mM [Mg²⁺]_c. We conclude that [Mg²⁺]_c is tightly regulated by a Mg²⁺ efflux that is strongly dependent on extracellular [Na⁺].

Key Words: Mg buffers, Mg transport, cardiac muscle, intracellular Mg, mag-fura-2, mitochondria

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