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 acetylc holine-induced vasodilatation via increasing intracellular guanosine 3 ',5'-cyclic monophosphate (cGMP) levels. The cellular mechanisms invol ved in NO .-mediated pulmonary vasodilatation are complex and include membrane hyperpolarization. Using the patch-clamp technique in cell-at tached and inside-out configurations, me examined the effect of NO . g as, 3-morpholinosydnomimine hydrochloride (SIN-1), and perfusate from ACh-stimulated human pulmonary arterial endothelial cells, or endothel ium-derived relaxing factors (EDRF), on the Ca2+-dependent K+ (K-Ca) c hannels in isolated cultured human pulmonary arterial smooth muscle ce lls (HPSMC). NO ., SIN-1, and EDRF caused similar increases in K-Ca ch annel activity. Inhibiting cGMP generation with methylene blue or inhi biting the effect(s) of cGMP with, the cGMP antagonist S-bromoguanosin e 3',5'-cyclic monophosphorothioate Rp isomer Rp-cGMPS presented the N O .- and SIN-1-mediated activation of K-Ca channels, respectively Trea ting the human pulmonary arterial endothelial cells with methylene blu e blocked the EDRF-mediated activation of K-Ca channels in HPSMC. The cGMP analogue 8-bromo-cGMP increased K-Ca channel activity in intact c ells and in excised inside-out HPSMC membrane patches. In the presence of cGMP and ATP, the alpha-isozyme of the cGMP-dependent protein kina se (1 alpha-cGMP-PK) significantly increased K-Ca channel activity, an d the channel activation was further increased on addition of the prot ein phosphatase inhibitors okadaic acid and calyculin A. Furthermore, the cGMP-mediated K-Ca channel activation was reduced by the cyclic nu cleotide-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.