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(+)/Ca(2+) exchanger (NCX) contribute to intracellular Na(+) and Ca(2+) overload in astrocytes following oxygen-glucose deprivation (OGD) and reoxygenation (REOX). Here, we further investigated whether NKCC1 and NCX play a role in mitochondrial Ca(2+) (Ca(m)(2+)) overload and dysfunction. OGD/REOX caused a doubling of mitochondrial-releasable Ca(2+) (P < 0.05). When NKCC1 was inhibited with bumetanide, the mitochondrial-releasable Ca(2+) was reduced by approximately 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 Ca(2+) 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.
Kintner
Douglas B
DB
Dept. of Neurological Surgery, University of Wisconsin School of Medicine, Madison, WI 53792, USA.
Luo
Jing
J
Gerdts
Josiah
J
Ballard
Andy J
AJ
Shull
Gary E
GE
Sun
Dandan
D
D000818:Animals; D001253:Astrocytes / Q000378:metabolism*; D015687:Cell Hypoxia / Q000502:physiology; D002478:Cells, Cultured; D002540:Cerebral Cortex / Q000502:physiology*; D053078:Membrane Potential, Mitochondrial / Q000502:physiology; D008564:Membrane Potentials / Q000502:physiology; D051379:Mice; D018345:Mice, Knockout; D008928:Mitochondria / Q000502:physiology*; D015427:Reperfusion Injury / Q000503:physiopathology; D019831:Sodium-Calcium Exchanger / Q000378:metabolism*; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism*; D064506:Solute Carrier Family 12, Member 2
C578878:Slc12a2 protein, mouse; D019831:Sodium-Calcium Exchanger; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2