Inhibitors of cation-chloride-cotransporters affect hypoxic/hypoglycemic injury in hippocampal slices

Abstract

Electroneutral cation–chloride cotransporters are abundantly expressed in the brain and are involved in the regulation of the intracellular Cl − concentration and thus γ-aminobutyric acid-dependent inhibition of neuronal excitability. As yet there is little evidence whether or not Na +–K +–2Cl − or K +–Cl − cotransporters are involved in neuronal hyperexcitability and death in cerebral ischemia. In this study, by measuring propidium iodide staining in organotypic hippocampal slice cultures from young rats and population spike recovery in acutely isolated hippocampal slices from adult rats after a hypoxic/hypoglycemic insult, we were able to assess if cation–chloride cotransport inhibitors reduce neuronal injury. The Na +–K +–2Cl − cotransport inhibitor bumetanide in the range of 1–10 μM reduced neuronal damage in the slice cultures by 25%, but did not affect population spike recovery in acutely isolated slices. In contrast the K +–Cl − cotransport inhibitor [(dihydroindenyl)oxy] alkanoic acid (DIOA, 100 μM) significantly diminished the restitution of the population spikes from 33% before to 8% after hypoxia/hypoglycemia and increased the damage in the slice cultures by 60%. Consequently, our data suggest that the Na +–K +–2Cl − cotransporter may contribute to neuronal injury and that the activity of the K +–Cl − cotransporters is an intrinsic protective mechanism of neurons against ischemic damage.