Mg2+ extrusion activated by hydrogen peroxide in rat ventricular myocytes.

Abstract

To investigate the effect of reactive oxygen species (ROS) on intracellular-free Mg2+ concentration ([Mg2+]i), we measured [Mg2+]i in rat ventricular myocytes using a fluorescent indicator, mag-fura-2. The treatment of hydrogen peroxide (H2O2, 500 μM) for 5 min decreased [Mg2+]i from 1.02 ± 0.08 (mean ±SD, n = 7) to 0.75 ± 0.18 mM (p = 0.018, by Wilcoxon signed-rank test) in ∼9 min (including the 5-min treatment and subsequent washout). [Mg2+]i was continuously decreased in ∼ 20 min, and then recovered to the basal level (∼1.0 mM) gradually in the Ca2+-free Tyrode's solution. The half maximal effective concentration of H2O2 on the Mg2+- decrease was estimated to be between 400 and 425 μM. The rate of decrease in [Mg2+]i by H2O2 in the Ca2+-free Tyrode's solution (−0.61± 0.42 μM/s, n = 36) was not significantly different from that obtained in the absence of extracellular Na+ or Mg2+. The addition of 1 mM Ca2+ extracellularly slowed the rate of Mg2+- decrease, on average, by ∼60% (p = 0.002, by Mann-Whitney U-test). The H2O2-induced Mg2+ decrease in Na+-free (substituted with N-methyl-d-glucamine) /Ca2+-free Tyrode's solution was inhibited by 200 μM imipramine, a known inhibitor of Na+/Mg2+ exchange. Rat hearts were perfused with the Ca2+-free Tyrode's solution containing 500 μM H2O2 for 5 min on the Langendorff apparatus. The Mg2+-concentration of the cardiac perfusate was significantly increased after H2O2 administration, suggesting that the H2O2-induced Mg2+-decrease was caused by Mg2+- extrusion. Pyocyanin, a generator of reactive oxygen species also reduced [Mg2+]i. The Mg2+-decrease by pyocyanin was inhibited by a pretreatment of n-acetyl cysteine, an anti-oxidant. In conclusion, it is suggested ROS disrupt intracellular Mg2+ homeostasis, and that the existence of a Na+-independent Mg2+- extrusion system activated by H2O2 in cardiomyocytes.