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

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 350nm was used to estimate the apparent cytoplasmic [Mg2+] ([Mg2+]i). In Ca2+-free extracellular conditions (0.1mM EGTA) at 25°C, [Mg2+]i averaged 0.842±0.019mM. After the cells were loaded with Mg2+ by exposure to high extracellular [Mg2+] ([Mg2+]o), reduction of [Mg2+]o to 1mM (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 ∼10mM [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 (25μg/ml for 5min), 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 0.6–0.7mM in bathing solution [Mg2+] (i.e., [Mg2+]c of the skinned myocytes) of either 0.8mM or 4.0mM, 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 (∼0.9mM), with a half-maximal activation of ∼1.9mM [Mg2+]c. We conclude that [Mg2+]c is tightly regulated by an Mg2+ efflux that is dependent on extracellular [Na+].