Role of Na+-K+-Cl- cotransport and Na+/Ca2+ exchange in mitochondrial dysfunction in astrocytes following in vitro ischemia

Na+-K+-Cl-cotransporter isoform 1 (NKCC1) and reverse mode operation of the Na+/Ca2(+) exchanger (NCX) contribute to intracellular Na+ and Ca2+ overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca2+ (Ca-m(2+)) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca2(+) (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca2+ was reduced by similar to 42% (P < 0.05). Genetic ablation of NKCC1 also reduced Ca-m(2+) accumulation. Moreover, OGD/REOX in NKCC1(+/+) astrocytes caused dissipation of the mitochondrial membrane potential (Psi(m)) to 42 +/- 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of Psi(m) was attenuated significantly (66 +/- 10% of controls, P < 0.05). Cells were also subjected to severe in vitro hypoxia by superfusion with a hypoxic, acidic, ion-shifted Ringer buffer (HAIR). HAIR/REOX triggered a secondary, sustained rise in intracellular Ca2+ that was attenuated by reversal NCX inhibitor KB-R7943. The hypoxia-mediated increase in Ca-m(2+) was accompanied by loss of Psi(m) and cytochrome c release in NKCC1(+/+) astrocytes. Bumetanide or genetic ablation of NKCC1 attenuated mitochondrial dysfunction and astrocyte death following ischemia. Our study suggests that NKCC1 acting in concert with NCX causes a perturbation of Ca-m(2+) homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia.