Increased [Mg²⁺]_oreduces Ca²⁺ influx and disruption of mitochondrial membrane potential during reoxygenation

Abstract

Increase in extracellular Mg2+ concentration ([Mg2+]o) reduces Ca2+ accumulation during reoxygenation of hypoxic cardiomyocytes and exerts protective effects. The aims of the present study were to investigate the effect of increased [Mg(2+)](o) on Ca2+ influx and efflux, free cytosolic Ca2+ ([Ca2+]i) and Mg2+ concentrations ([Mg2+]i), Ca2+ accumulation in the presence of inhibitors of mitochondrial or sarcoplasmatic reticulum Ca2+ transport, and finally mitochondrial membrane potential (Delta(psi)m). Isolated adult rat cardiomyocytes were exposed to 1 h of hypoxia and subsequent reoxygenation. Cell Ca2+ was determined by 45Ca2+ uptake, and the levels of [Mg2+]i and [Ca2+]i were determined by flow cytometry as the fluorescence of magnesium green and fluo 3, respectively. Ca2+ influx rate was significantly reduced by approximately 40%, whereas Ca2+ efflux was not affected by increased [Mg2+]o (5 mM) during reoxygenation. [Ca2+]i and [Mg2+]i were increased at the end of hypoxia, fell after reoxygenation, and were unaffected by increased [Mg2+]o. Clonazepam, a selective mitochondrial Na+/Ca2+ exchange inhibitor (100 microM), significantly reduced Ca2+ accumulation by 70% and in combination with increased [Mg2+]o by 90%. Increased [Mg2+]o, clonazepam, 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 Delta(psi)m and cell Ca2+ in reoxygenated cells treated with increased [Mg2+]o and clonazepam. In conclusion, increased [Mg2+]o (5 mM) inhibits Ca2+ accumulation by reducing Ca2+ influx and preserves Delta(psi)m without affecting [Ca2+]i and [Mg2+]i during reoxygenation. Preservation of mitochondria may be an important effect whereby increased [Mg2+]o protects the postischemic heart.

Mitochondrial signals initiate the activation of c‐Jun N‐terminal kinase (JNK) by hypoxia‐reoxygenation

“…Calcium accumulates slowly during hypoxia but rapidly during reoxygenation, when electron flow is resumed and mitochondrial membranes are repolarized (53)(54)(55). Intracellular calcium levels have been reported to increase Ͼfivefold during reoxygenation with between two-and threefold increases of mitochondrial calcium (56). Our results suggest that this increase of calcium is a component of the signal that activates the JNK pathway.…”

Anoxia induces Ca2+ influx and loss of cell membrane integrity in rat extensor digitorum longus muscle

“…Nifedipine caused no reduction in 45 Ca uptake, suggesting that the L‐type Ca 2+ channels are not involved. In rat cardiomyocytes, increased extracellular Mg 2+ concentration (5 m m ) has been shown to reduce [Ca 2+ ] i by 40% and LDH release by 90% after 60 min of anoxia followed by 120 min of reoxygenation (Sharikabad et al 2001 a , b ). In our experiments, around 40% of the anoxia‐induced increase in 45 Ca uptake was inhibited by 11.2 m m Mg 2+ .…”

PKC-α and TAK-1 are intermediates in the activation ofc-Jun NH2-terminal kinase by hypoxia-reoxygenation

“…Calcium accumulates slowly during hypoxia but rapidly during reoxygenation when electron flow is resumed (11,31). Intracellular calcium levels have been reported to increase approximately fivefold during hypoxia-reoxygenation (43). Our results suggest that this increase of intracellular calcium is a component of the signal that activates the Pyk2-JNK pathway, possibly by contributing to the activation of PKC-␣.…”