Contribution of epoxyeicosatrienoic acids to the hypoxia-induced activation of Ca²⁺-activated K⁺ channel current in cultured rat hippocampal astrocytes

Brief hypoxia differentially regulates the activities of Ca²⁺-activated K⁺ channels (K_Ca) in a variety of cell types. We investigated the effects of hypoxia (<2% O₂) on K_Ca channel currents and on the activities of cytochrome P450 2C11 epoxygenase (CYP epoxygenase) in cultured rat hippocampal astrocytes. Exposure of astrocytes to hypoxia enhanced macroscopic outward K_Ca current, increased the open state probability (NPo) of 71 pS and 161 pS single-channel K_Ca currents in cell-attached patches, but failed to increase the NPo of both the 71 pS and 161 pS K_Ca channel currents recorded from excised inside-out patches. The hypoxia-induced enhancement of macroscopic K_Ca current was attenuated by pretreatment with tetraethylammonium (TEA, 1 mM) or during recording using low-Ca²⁺ external bath solution. Exposure of astrocytes to hypoxia was associated with generation of superoxide as detected by staining of cells with the intracellular superoxide detection probe hydroethidine (HE), attenuation of the hypoxia-induced activation of unitary K_Ca channel currents by superoxide dismutation with tempol, and as quantitated by high-pressure liquid chromatography/fluorescence assay using HE as a probe. In cultured astrocytes in which endogenous CYP epoxygenase activity has been inhibited with either miconazole or N-methylsulfonyl-6-(2-propargyloxyphenyl) hexanamide (MSPPOH) hypoxia failed to increase the NPo of both the 71 pS and 161 pS K_Ca currents and generation of superoxide. Hypoxia increased the level of P450 epoxygenase protein and production of epoxyeicosatrienoic acids (EETs) from cultured astrocytes, as determined by immunohistochemical staining and LC/MS analysis, respectively. Exogenous 11,12-EET increased the NPo of both the 71 pS and 161 pS K_Ca single-channel currents only in cell-attached but not in excised inside-out patches of cultured astrocytes. These findings indicate that hypoxia enhances the activities of two types of unitary K_Ca currents in astrocytes by a mechanism that appears to involve CYP epoxygenase-dependent generation of superoxide and increased production or release of EETs.