Nitrergic relaxation in urethral smooth muscle: involvement of potassium channels and alternative redox forms of NO

Abstract

We examined the contribution of K+ channels to the relaxation responses induced by different redox forms of nitric oxide (NO., NO- and NO+) in comparison with those evoked by electrical field stimulation (EFS) of nitrergic nerves in the sheep urethra. K+ channel blockers with different selectivity profile were used. Sodium nitroprusside (SNP) and different S-nitrosothiols were used as NO+ donors, Angeli's salt as an NO- donor and nitroglycerin (GTN) was chosen as a representative compound known to require metabolic activation in the target tissue. Pure NO gas was used to prepare NO. solutions. Relaxation evoked by EFS of nitrergic nerves or by exogenous NO was not inhibited by any of the K+ channel blockers, but was enhanced by 4-aminopyridine [inhibitor of voltage-dependent K+ (KV) channels]. This suggests that, whereas K+ channel activation and hyperpolarization of the postsynaptic membrane do not contribute to relaxation, prejunctional modulation of the nitrergic neurotransmission by Kv channels may be relevant. Relaxation induced by NO+ or NO- donors was not affected by K+ channel blockade with the following exceptions: glybenclamide, a blocker of ATP-sensitive K+ channels (KATP), enhanced responses to SNP and Angeli's salt, 4-aminopyridine inhibited relaxation evoked by Angeli's salt and GTN, and charybdotoxin, a blocker of large-conductance, Ca2+-activated K+ channels (BKCa) inhibited those induced by the S-nitrosothiol S-nitrosoglutathione. These results do not suggest the existence of a general mechanism of action on K+ channels for compounds releasing either NO+ or NO- in the sheep urethra. None of the K+ channel blockers affected relaxation induced by the membrane-permeable analogue of cGMP, 8-bromo-cGMP. However, the fact that the addition of the phosphodiesterase inhibitor zaprinast (0.1 mM) enhanced the relaxation to Angeli's salt, while preventing the inhibition induced by 4-aminopyridine, suggests that involvement of guanylate cyclase activation in the action of NO donors on K+ channels can not be excluded. Accordingly, the guanylate cyclase inhibitors 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ, 10 microM) and 4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one (NS 2028, 10 microM) almost abolished relaxations to EFS and Angeli's salt. In contrast, ODQ only moderately inhibited relaxations to NO.. In addition, the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethyl imidazoline-1-oxyl 3-oxide (carboxy-PTIO) effectively inhibited responses to NO. whilst not affecting those to EFS or NO-, suggesting a close similarity between the nitrergic transmitter and nitroxyl ion. We conclude that nitrergic relaxation induced either by the endogenous transmitter or by exogenous NO donors in the ovine urethra is not mediated by postsynaptic alterations in the K+ conductance; only a prejunctional modulation through Kv channels seems to be significant. In addition, the production and/or release of alternative redox forms of NO, such as NO-, may be involved in neurotransmission processes in the urethra.

Mechanisms Underlying Activation of Soluble Guanylate Cyclase by the Nitroxyl Donor Angeli’s Salt

“…Our results with cultured endothelial cells clearly show that carboxy-PTIO inhibits cGMP accumulation induced by AS in the presence of extracellular SOD, demonstrating that the drug does scavenge AS-derived NO as expected. However, in line with several published studies (Li et al, 1999;Costa et al, 2001;Wanstall et al, 2001;Irvine et al, 2003Irvine et al, , 2007Hewett et al, 2005), carboxy-PTIO did not block the effect of AS in the absence of added SOD (Fig. 5B).…”

“…Contrasting the general view that AS-induced activation of sGC results from oxidation of HNO to NO (Zamora et al, 1995), several studies reported that vasodilation in response to AS was blocked by ODQ but not by the NO scavenger carboxy-PTIO, although the effects of NO donor compounds or endothelial NO synthase activation were inhibited as expected (Li et al, 1999;Costa et al, 2001;Wanstall et al, 2001;Irvine et al, 2003Irvine et al, , 2007. This discrepancy is still unresolved and is taken as evidence for NO-independent sGC activation by HNO (Irvine et al, 2008).…”

NO − Activates Soluble Guanylate Cyclase and K v Channels to Vasodilate Resistance Arteries

“…In addition, our findings concur with a previous study in a nonvascular preparation, the sheep isolated urethra, where relaxation responses to Angeli's salt were found to be impaired in part by 4-aminopyridine. 25 Our study, together with that of Costa and colleagues, 25 suggests an ability of NO Ϫ to activate K v channels. Whether NO Ϫ activates K v channels directly or through a cGMP-dependent mechanism remains to be determined.…”

Generation of nitroxyl by heme protein-mediated peroxidation of hydroxylamine but not N-hydroxy-L-arginine

“…Certainly, HNO donors have been shown to be effective pharmacology agents both in vitro and in vivo for a variety of conditions ranging from treatment of congestive heart failure to alcoholism (e.g. [1][2][3][4][5][6][7][8][9][58][59][60][61][62][63][64][65]), despite high levels of cytoplasmic GSH.…”