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Modulation of Mg²⁺ Efflux from Rat Ventricular Myocytes Studied with the Fluorescent Indicator Furaptra

Department of Physiology, Tokyo Medical University, Tokyo, Japan

Correspondence: Address reprint docrequests to Dr. Masato Konishi, Dept. of Physiology, Tokyo Medical University, 6-1-1 Shinjuku, Shinjuku-ku, Tokyo 160-8402, Japan. Tel.: 81-3-3351-6141; Fax: 81-3-5379-0658; E-mail: mkonishi{at}tokyo-med.ac.jp.

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°C, [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, whereas raising [Mg²⁺]_o slowed the decrease in [Mg²⁺]_i with 50% reduction of the rate at 10 mM [Mg²⁺]_o. Because a part of the 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 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg²⁺] ([Mg²⁺]_c), whereas 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⁺ buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg²⁺ flux across the cell membrane. 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 an Mg²⁺ efflux that is dependent on extracellular [Na⁺].

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