DIRECT ACTIVATION OF K-CA CHANNEL IN AIRWAY SMOOTH-MUSCLE BY NITRIC-OXIDE - INVOLVEMENT OF A NITROTHIOSYLATION MECHANISM

Clinically, nitric oxide (NO.) is widely used as a pulmonary vaso- and bronchodilator agent. However, the precise molecular mechanisms by wh ich NO. induces smooth muscle relaxation are not well established. It has been suggested that NO. relaxes airway smooth muscle (ASM) via a 3 ',5'-cyclic guanosine monophosphate (cGMP)-dependent pathway, and our previous work has shown that Ca2+-activated K+ (K-Ca) channels are sus ceptible to cGMP-dependent protein kinase (PKG)-dependent phosphorylat ion (A. Alioua, J. P. Huggins, and E. Rousseau. Am. J. Physiol. 1995;2 68:L1057-L1063). To assess whether K-Ca channels are also directly act ivated by NO. or one of its derivatives such as peroxynitrite, the act ivity of these channels was measured upon fusion of sarcolemmal vesicl es derived from bovine tracheal smooth muscle cells into planar lipid bilayers (PLB). It was found that in the absence of adenosine triphosp hate (ATP), cGMP, and cGMP-dependent protein kinase, NO. donors such a s panamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) or 3-morpholinosydnonimine hydrochloride (SIN-1) in the presence of supe roxide dismutase (SOD), added on either side of the bilayer, caused a concentration-dependent increase in the open probability (Po) of K-Ca channels without altering their unitary conductance. Release of NO., w hich was measured by chemiluminescence analysis in parallel experiment s, affected the Sating behavior of K-Ca channels in the presence of SO D and ethyleneglycol-bis-(beta-aminoethyl ether)N,N'-tetraacetic acid (EGTA) by reducing the mean closed times and increasing the number and duration of short open events. PAPA NONOate, a true NO. donor, had si milar effects in the presence of ethylenediaminetetraacetic acid (EDTA ), a heavy-metal chelator, and K-urate, a peroxynitrite scavenger. Add ition of either 5 mM dithiothreitol (DTT) or 5 mM reduced glutathione (GSH), as well as 5 mM N-ethylmaleimide (NEM)-an alkylating agent-to t he trans (intracellular) side of an experimental chamber slightly incr eased channel Po but prevented further channel activation by NO. donor s. However, neither DTT nor GSH was able to reverse the effect of NO.. In contrast to SIN-1, DTT had no effect when added to the cis (extrac ellular) side of the chamber. This suggests that the effect of NO. is most Likely due to a chemical modification (nitrothiosylation) of intr acellular sulfhydryl group(s). Neither PAPA NONOate (NO.), nor SIN-1 h ad any effect on sarcolemmal Cl- channels reconstituted from the same membrane preparations. Pharmacomechanical measurements made on epithel ium-denuded rat bronchus showed that 100 nM charybdotoxin decreased th e sensitivity of bronchial smooth muscle to SIN-1-induced relaxations. Altogether, our data suggest that NO-induced bronchorelaxation occurs partly via a direct activation of K-Ca channels, possibly through a c ovalent interaction with the cytoplasmic side of their alpha subunit.