Increased [Mg2+](o) reduces Ca2+ influx and disruption of mitochondrial membrane potential during reoxygenation

Increase in extracellular Mg2+ concentration ([Mg2+](o)) reduces Ca2+ accum ulation during reoxygenation of hypoxic cardiomyocytes and exerts protectiv e effects. The aims of the present study were to investigate the effect of increased [Mg2+](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]( o)) and Mg2+ concentrations ([Mg2+](i)), Ca-2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta psi (m)). Isolated adu lt rat cardiomyocytes were exposed to 1h of hypoxia and subsequent reoxygen ation. Cell Ca2+ was determined by Ca-45(2+) uptake, and the levels of [Mg2 +](i) and [Ca2+](i) were determined by flow cytometry as the fluorescence o f magnesium green and fluo 3, respectively. Ca2+ influx rate was significan tly reduced by similar to 40%, whereas Ca2+ efflux was not affected by incr eased [Mg2+](o) (5 mM) during reoxygenation. [Ca2+](i) and [Mg2+](i) were i ncreased at the end of hypoxia, fell after reoxygenation, and were unaffect ed by increased [Mg2+](o). Clonazepam, a selective mitochondrial Na+/Ca2+ e xchange inhibitor (100 muM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+](o) by 90%. Increased [Mg2+](o), c lonazepam, and the combination of both attenuated the hypoxia-reoxygenation -induced reduction in Delta psi (m), determined with the cationic dye JC-1 by flow cytometry. A significant inverse correlation was observed between D elta psi (m) and cell Ca2+ in reoxygenated cells treated with increased [Mg 2+](o) and clonazepam. In conclusion, increased [Mg2+](o) (5 mM) inhibits C a2+ accumulation by reducing Ca2+ influx and preserves Delta psi (m) withou t affecting [Ca2+](i) and [Mg2+](i) during reoxygenation. Preservation of m itochondria may be an important effect whereby increased [Mg2+](o) protects the postischemic heart.