Direct Activation of K_Ca Channel in Airway Smooth Muscle by Nitric Oxide: Involvement of a Nitrothiosylation Mechanism?

Abstract

Clinically, nitric oxide (NO*) is widely used as a pulmonary vaso- and bronchodilator agent. However, the precise molecular mechanisms by which 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+ (KCa) channels are susceptible to cGMP-dependent protein kinase (PKG)-dependent phosphorylation (A. Alioua, J. P. Huggins, and E. Rousseau. Am. J. Physiol. 1995;268:L1057-L1063). To assess whether KCa channels are also directly activated by NO. or one of its derivatives such as peroxynitrite, the activity of these channels was measured upon fusion of sarcolemmal vesicles derived from bovine tracheal smooth muscle cells into planar lipid bilayers (PLB). It was found that in the absence of adenosine triphosphate (ATP), cGMP, and cGMP-dependent protein kinase, NO* donors such as 1-propanamine-3-(2-hydroxy-2-nitroso-1-propylhydrazine) (PAPA NONOate) or 3-morpholinosydnonimine hydrochloride (SIN-1) in the presence of superoxide dismutase (SOD), added on either side of the bilayer, caused a concentration- dependent increase in the open probability (Po) of KCa channels without altering their unitary conductance. Release of NO*, which was measured by chemiluminescence analysis in parallel experiments, affected the gating behavior of KCa channels in the presence of SOD 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 similar effects in the presence of ethylenediaminetetraacetic acid (EDTA), a heavy-metal chelator, and K-urate, a peroxynitrite scavenger. Addition of either 5 mM dithiothreitol (DTT) or 5 mM reduced glutathione (GSH), as well as 5 mM N-ethylmaleimide (NEM)-an alkylating agent-to the trans (intracellular) side of an experimental chamber slightly increased channel Po but prevented further channel activation by NO* donors. 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 (extracellular) side of the chamber. This suggests that the effect of NO* is most likely due to a chemical modification (nitrothiosylation) of intracellular sulfhydryl group(s). Neither PAPA NONOate (NO*), nor SIN-1 had any effect on sarcolemmal Cl- channels reconstituted from the same membrane preparations. Pharmacomechanical measurements made on epithelium-denuded rat bronchus showed that 100 nM charybdotoxin decreased the 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 KCa channels, possibly through a covalent interaction with the cytoplasmic side of their alpha subunit.

A Catalytically Inactive Mutant of Type I cGMP-dependent Protein Kinase Prevents Enhancement of Large Conductance, Calcium-sensitive K+ Channels by Sodium Nitroprusside and cGMP

“…These results are further consistent with the observed phenotype of cGKI-deficient knockout mice, which display impaired endothelium and NO-dependent relaxation of smooth muscle, resulting in vascular and intestinal dysfunction (45,47). The question of whether NO and NO donors may also be able to directly activate BK Ca channels is not supported by our data and remains controversial (55)(56)(57)(58); it is possible that such a phenomenon may depend upon the specific preparation in use, along with the types and concentrations of agents under study.…”

Protein kinase G regulates the basal tension and plays a major role in nitrovasodilator‐induced relaxation of porcine coronary veins

“…This postulation is further supported by a similar increase in basal tension of the veins induced by ODQ and the fact that the difference in basal cGMP content between coronary veins with and without endothelium was eliminated not only by nitro-L-arginine but also by ODQ. Some studies have shown that nitric oxide may exert its effect through cGMP-independent mechanisms such as nitrosylation of the target protein (Abderrahmane et al, 1998;Janssen et al, 2000). However, in the coronary veins, nitric oxide appears to work solely via the cGMP pathway.…”

“…However, nitric oxide may also cause relaxation through cGMP-independent as well as PKG-independent mechanisms (Abderrahmane et al, 1998;Janssen et al, 2000;Francis et al, 2005). The contribution of PKG to the EDNO-mediated regulation of the basal tension and of responses induced by nitrovasodilators is not well understood.…”

Effect of PDE5 inhibition combined with free oxygen radical scavenger therapy on erectile function in a diabetic animal model

“…Additionally, NO can directly activate calcium-dependent potassium channels inducing hyperpolarization of the cell membrane. 9,10 These cellular changes induce a cascade of physiologic events culminating in smooth muscle relaxation, increased vascular flow and ultimately penile erection.…”