Modulation of Mg2+ efflux from rat ventricular myocytes studied with the fluorescent indicator furaptra.
The fluorescent Mg(2+) 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 [Mg(2+)] ([Mg(2+)](i)). In Ca(2+)-free extracellular conditions (0.1 mM EGTA) at 25 degrees C, [Mg(2+)](i) averaged 0.842 +/- 0.019 mM. After the cells were loaded with Mg(2+) by exposure to high extracellular [Mg(2+)] ([Mg(2+)](o)), reduction of [Mg(2+)](o) to 1 mM (in the presence of extracellular Na(+)) induced a decrease in [Mg(2+)](i). The rate of decrease in [Mg(2+)](i) was higher at higher [Mg(2+)](i), whereas raising [Mg(2+)](o) slowed the decrease in [Mg(2+)](i) with 50% reduction of the rate at approximately 10 mM [Mg(2+)](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 microg/ml for 5 min), furaptra fluorescence intensity at 350-nm excitation decreased to 22%; thus approximately 78% of furaptra fluorescence appeared to represent cytoplasmic [Mg(2+)] ([Mg(2+)](c)), whereas the residual 22% likely represented [Mg(2+)] in organelles (primarily mitochondria as revealed by fluorescence imaging). [Mg(2+)] calibrated from the residual furaptra fluorescence ([Mg(2+)](r)) was 0.6-0.7 mM in bathing solution [Mg(2+)] (i.e., [Mg(2+)](c) of the skinned myocytes) of either 0.8 mM or 4.0 mM, suggesting that [Mg(2+)](r) was lower than and virtually insensitive to [Mg(2+)](c). We therefore corrected furaptra fluorescence signals measured in intact myocytes for this insensitive fraction of fluorescence to estimate [Mg(2+)](c). In addition, by utilizing concentration and dissociation constant values of known cytoplasmic Mg(2+) buffers, we calculated changes in total Mg concentration to obtain quantitative information on Mg(2+) flux across the cell membrane. The calculations indicate that, in the presence of extracellular Na(+), Mg(2+) efflux is markedly activated by [Mg(2+)](c) above the normal basal level (approximately 0.9 mM), with a half-maximal activation of approximately 1.9 mM [Mg(2+)](c). We conclude that [Mg(2+)](c) is tightly regulated by an Mg(2+) efflux that is dependent on extracellular [Na(+)].
Tursun
Pulat
P
Department of Physiology, Tokyo Medical University, 6-1-1 Shinjuku-ku, Tokyo 160-8402, Japan.
Tashiro
Michiko
M
Konishi
Masato
M
D000818:Animals; D002462:Cell Membrane / Q000378:metabolism*; D002478:Cells, Cultured; D003198:Computer Simulation; D005456:Fluorescent Dyes; D016257:Fura-2 / Q000031:analogs & derivatives*; D006352:Heart Ventricles / Q000166:cytology / Q000378:metabolism; D008274:Magnesium / Q000378:metabolism*; D008297:Male; D008657:Metabolic Clearance Rate; D008954:Models, Biological; D032383:Myocytes, Cardiac / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013050:Spectrometry, Fluorescence / Q000379:methods*
D005456:Fluorescent Dyes; C058776:2-(2-(5-carboxy)oxazole)-5-hydroxy-6-aminobenzofuran-N,N,O-triacetic acid; D008274:Magnesium; D016257:Fura-2