Abstract
Epoxyeicosatrienoic acids (EETs) are arachidonic acid metabolites of cytochrome P450 monooxygenase, which are released from endothelial cells and dilate arteries. Dilation seems to be caused by activation of large-conductance Ca2+-activated K+ channels (BKCa) leading to membrane hyperpolarization. Previous studies suggest that EETs activate BKCa channels via ADP-ribosylation of the G protein Gαs with a subsequent membrane-delimited action on the channel [Circ Res 78:415–423, 1996; 80:877–884, 1997;85:349–356, 1999]. The present study examined whether this pathway is present in human embryonic kidney (HEK) 293 cells when the BKCa α-subunit (cslo-α) is expressed without the β-subunit. 11,12-EET increased outward K+ current in whole-cell recordings of HEK293 cells. In cell-attached patches, 11,12-EET also increased the activity of cslo-α channels without affecting unitary conductance. This action was mimicked by cholera toxin. The ADP-ribosyltransferase inhibitors 3-aminobenzamide and m-iodobenxylguanidine blocked the stimulatory effect of 11,12-EET. In inside-out patches 11,12-EET was without effect on channel activity unless GTP was included in the bathing solution. GTP and GTPγS alone also activatedcslo-α channels. Dialysis of cells with anti-Gαs antibody completely blocked the activation of cslo-α channels by 11,12-EET, whereas anti-Gαi/o and anti-Gβγ antibodies were without effect. The protein kinase A inhibitor KT5720 and the adenylate cyclase inhibitor SQ22536 did not reduce the stimulatory effect of 11,12-EET on cslo-α channels in cell-attached patches. These data suggest that EET leads to Gαs-dependent activation of the cslo-α subunits expressed in HEK293 cells and that the cslo-β subunit is not required.
Footnotes
- Received May 4, 2000.
- Accepted September 19, 2000.
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Send reprint requests to: Kathleen Keef, Ph.D., Department of Physiology & Cell Biology, University of Nevada, Reno, NV 89557. E-mail: kathy{at}physio.unr.edu
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This work was supported by the Banyu Fellowships in Lipid Metabolism and Atherosclerosis, which are sponsored by Banyu Pharmaceutical Co., Ltd.; The Merck Company foundation (M.F.); National Institutes of Health Grant HL40399 (K.D.K.); and National Institute of Diabetes and Digestive and Kidney Diseases Grant 41315 (B.H.).
- The American Society for Pharmacology and Experimental Therapeutics
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