Regulation of Ca2+-activated K+ channels in pulmonary vascular smooth muscle cells: Role of nitric oxide

Nitric oxide (NO) is believed to mediate nitrovasodilators and acetylcholine-induced vasodilatation via increasing intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. The cellular mechanisms involved in NO .-mediated pulmonary vasodilatation are complex and include membrane hyperpolarization. Using the patch-clamp technique in cell-attached and inside-out configurations, me examined the effect of NO . gas, 3-morpholinosydnomimine hydrochloride (SIN-1), and perfusate from ACh-stimulated human pulmonary arterial endothelial cells, or endothelium-derived relaxing factors (EDRF), on the Ca2+-dependent K+ (K-Ca) channels in isolated cultured human pulmonary arterial smooth muscle cells (HPSMC). NO ., SIN-1, and EDRF caused similar increases in K-Ca channel activity. Inhibiting cGMP generation with methylene blue or inhibiting the effect(s) of cGMP with, the cGMP antagonist S-bromoguanosine 3',5'-cyclic monophosphorothioate Rp isomer Rp-cGMPS presented the NO .- and SIN-1-mediated activation of K-Ca channels, respectively Treating the human pulmonary arterial endothelial cells with methylene blue blocked the EDRF-mediated activation of K-Ca channels in HPSMC. The cGMP analogue 8-bromo-cGMP increased K-Ca channel activity in intact cells and in excised inside-out HPSMC membrane patches. In the presence of cGMP and ATP, the alpha-isozyme of the cGMP-dependent protein kinase (1 alpha-cGMP-PK) significantly increased K-Ca channel activity, and the channel activation was further increased on addition of the protein phosphatase inhibitors okadaic acid and calyculin A. Furthermore, the cGMP-mediated K-Ca channel activation was reduced by the cyclic nucleotide-dependent protein kinase inhibitor N-[2-(methylamino)ethyl]-5-isoquinlinesulfonamide (H-8). Thus, in HPSMC, the mechanism of NO .- and native EDRF-induced K-Ca channel activation appears to be mediated via cGMP-I alpha-cGMP-PK phosphorylation of K-Ca channels.