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

Abstract

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_m²⁺) 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 ∼42% ( P < 0.05). Genetic ablation of NKCC1 also reduced Ca_m²⁺accumulation. Moreover, OGD/REOX in NKCC1^+/+astrocytes caused dissipation of the mitochondrial membrane potential (Ψ_m) to 42 ± 3% of controls. In contrast, when NKCC1 was inhibited with bumetanide, depolarization of Ψ_mwas 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_m²⁺was accompanied by loss of Ψ_mand 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_m²⁺homeostasis and mitochondrial dysfunction and cell death following in vitro ischemia.