Regulation of Ca(2+)-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, we 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 Ca(2+)-dependent K+ (KCa) channels in isolated cultured human pulmonary arterial smooth muscle cells (HPSMC). NO., SIN-1, and EDRF caused similar increases in KCa channel activity. Inhibiting cGMP generation with methylene blue or inhibiting the effect(s) of cGMP with the cGMP antagonist 8-bromoguanosine 3',5'-cyclic monophosphorothioate Rp isomer Rp-cGMPS prevented the NO.- and SIN-1-mediated activation of KCa channels, respectively. Treating the human pulmonary arterial endothelial cells with methylene blue blocked the EDRF-mediated activation of KCa channels in HPSMC. The cGMP analogue 8-bromo-cGMP increased KCa 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 (I alpha-cGMP-PK) significantly increased KCa 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 KCa 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 KCa channel activation appears to be mediated via cGMP-I alpha-cGMP-PK phosphorylation of KCa channels.
Department of Internal Medicine, Veterans Affairs Medical Center, Salt Lake City, Utah, USA.
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Peng
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Hoidal
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Farrukh
D002118:Calcium / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D008564:Membrane Potentials / Q000502:physiology; D008981:Molsidomine / Q000031:analogs & derivatives / Q000494:pharmacology; D009131:Muscle, Smooth, Vascular / Q000187:drug effects*; D009569:Nitric Oxide / Q000494:pharmacology*; D015221:Potassium Channels / Q000187:drug effects*; D011652:Pulmonary Circulation / Q000187:drug effects*
D004791:Enzyme Inhibitors; D015221:Potassium Channels; D009569:Nitric Oxide; C002385:linsidomine; D008981:Molsidomine; D002118:Calcium