The chloride transporter Na+-K+-Cl- cotransporter isoform-1 contributes to intracellular chloride increases after in vitro ischemia

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) (an in vitro model 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 mM during 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-](i) rose again 45 min later. To investigate sources of Cl- accumulation, we examined the effects of Cl- transport inhibitors on the rises in [Cl-](i) during 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-](i) during reoxygenation. In contrast, picrotoxin, which blocks the GABA-gated Cl- channel, did not inhibit the secondary rise in [Cl-](i) after OGD. [Cl-](i) increases 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.