The Chloride Transporter Na+-K+-Cl−Cotransporter Isoform-1 Contributes to Intracellular Chloride Increases afterIn VitroIschemia

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Abstract
Ischemic episodes in the CNS cause significant disturbances in neuronal ionic homeostasis. To directly measure changes in intracellular Cl−concentration ([Cl−]i) during and after ischemia, we used Clomeleon, a novel ratiometric optical indicator for Cl−. Hippocampal slices from adult transgenic mice expressing Clomeleon in hippocampal neurons were subjected to 8 min of oxygen-glucose deprivation (OGD) (anin vitromodel for ischemia) and reoxygenated in the presence of glucose. This produced mild neuronal damage 3 h later that was prevented when the extracellular [Cl−] was maintained at 10 mmduring reoxygenation. OGD induced a transient decrease in fluorescence resonance energy transfer within Clomeleon, indicating an increase in [Cl−]i. During reoxygenation, there was a partial recovery in [Cl−]i, but [Cl−]irose again 45 min later. To investigate sources of Cl−accumulation, we examined the effects of Cl−transport inhibitors on the rises in [Cl−]iduring and after OGD. Bumetanide and furosemide, which inhibit Cl−influx through the Na+-K+-Cl−cotransporter isoform-1 (NKCC-1) and efflux through the K+-Cl−cotransporter isoform-2, were unable to inhibit the first rise in [Cl−]i, yet entirely prevented the secondary rise in [Cl−]iduring reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl−channel, did not inhibit the secondary rise in [Cl−]iafter OGD. [Cl−]iincreases during reoxygenation were accompanied by an increase in phosphorylation of NKCC-1, an indication of increased NKCC-1 activity after OGD. We conclude that NKCC-1 plays an important role in OGD-induced Cl−accumulation and subsequent neuronal damage.

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