Differential mechanisms of urethral smooth muscle relaxation by several NO donors and nitric oxide

Abstract

We have examined the mechanisms of action of a broad spectrum of nitric oxide (NO) donors, including several S-nitrosothiols, sodium nitroprusside (SNP) and nitroglycerine (GTN), in relation to their relaxant activity of urethral smooth muscle. For all the compounds examined, NO release (in solution and in the presence of urethral tissue), relaxation responses, elevations in cGMP levels and the effect of thiol modulators were evaluated and compared with the effect of NO itself. Whilst all NO donors, except GTN, released NO in solution due to photolysis or chemical catalysis, this release was not correlated with their relaxant activity in sheep urethral preparations, which were furthermore not affected by the NO scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl 3-oxide (cPTIO; 0.3 mM). A substantial NO-generating activity was found for S-nitroso-L-cysteine (CysNO) and S-nitroso-N-acetyl-D,L-penicillamine (SNAP) in the presence of urethral cytosolic fractions, suggesting metabolic activation to NO in the cytosol of the target tissue. In contrast, NO generation from S-nitroso-N-acetyl-L-cysteine (N-ac-CysNO), S-nitrosoglutathione (GSNO) and SNP were reduced by the presence of urethral homogenate and/or subcellular fractions, suggesting direct NO transfer to tissue constituents. NO donors and NO gas induced dissimilar degrees of cGMP accumulation in urethral tissue, while they were essentially equipotent as urethral relaxants. Furthermore, 1H-[1,2,4] -oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ; 10 microM) inhibited both relaxation and cGMP accumulations, but with different potency for the different compounds. Oxidation of sarcolemmal thiol groups with 5-5'-dithio-bis[2-nitrobenzoic acid] (DTNB; 0.5 mM) enhanced relaxations to GSNO, an effect that was reversed by dithiotreitol (DTT; 1 mM), suggesting a direct effect through nitrosylation/oxidation reactions at the cell membrane, while relaxations to NO and to all the other compounds were not affected by these treatments. Finally, photodegradation of SNP induced the formation of a stable intermediate that still evoked NO-cGMP-mediated relaxations. This indicates that the assumption that SNP is fully depleted of NO by exposure to light should be revised. It can be concluded that important differences exist in the mechanisms by which distinct NO donors relax urethral smooth muscle and they cannot be regarded simply as NO-releasing prodrugs.

Elevated Levels of S -Nitrosoalbumin in Preeclampsia Plasma

“…26 The assay is based on a combination of a commonly used decomposition of S-nitrosothiols by UV irradiation 19,29,30 and subsequent capture of released NO by a fluorogenic reagent, DAF-2. 25 We found that the use of UV irradiation is particularly appropriate for measurements in plasma, where the traditional use of Cu 1ϩ or Hg 1ϩ to decompose S-nitrosothiols is complicated by the high metal-binding capacity of plasma proteins, including albumin.…”

“…We found that the intensity of DAF-2T fluorescence was strongly dependent on the concentration of added Cu in the range of 0.1 to 1.0 mmol/L and that a saturation of the DAF-2T fluorescence response from a plasma sample (50 L of undiluted plasma) was only achieved at Cu concentrations Ͼ1.0 mmol/L (data not shown). Therefore, to determine the content of S-nitrosothiols in plasma samples, we used a sensitive procedure based on UVinduced decomposition of S-nitrosothiols 19,29 and subsequent detection of released NO with a specific reagent, DAF-2, 25 that produces DAF-2T with a characteristic fluorescence emission spectra ( Figure 1). The method permits reliable quantitative measurements of S-nitrosothiols at concentrations in plasma as low as 50 nmol/L.…”

The Relaxant Activity of 4,7-Dimethyl-1,2,5-oxadiazolo[3,4-d]-pyridazine 1,5,6-Trioxide in the Mouse Corpus Cavernosum

“…In addition, N-ethylmaleimide significantly inhibited the relaxations to sodium nitroprusside, whereas diamide had no significant effect. When the level of thiols in the tissue drops below some critical point, nitric oxide becomes highly susceptible to oxidation/degradation; thus, its effective concentration is decreased along with soluble guanylate cyclase activation/cGMP synthesis/relaxation (Ignarro et al, 1981;Aleryani et al, 1999;Garcia-Pascual, 1999. Reversal of the effect of N-ethylmaleimide in the presence of exogenously added glutathione and L-cysteine may confirm this suggestion.…”

“…Previously, it was shown that thiols and thiol-modifying compounds can influence FPTO-dependent activation of sGC in a crude extract of rat lung but not using a highly purified enzyme preparation or intact tissue (Kots et al, 2000). Because it is known that redox state of endogenous thiol groups in corpus cavernosum plays an important role in vascular reactivity (Göçmen et al, 1997(Göçmen et al, , 1998Mateo and de Artinano, 2000), we also examined the effect of FPTO in the absence or presence of N-ethylmaleimide (nonspecific thiolalkylating agent), diamide (thiol-modifying agent), cysteine, glutathione, and dithiothreitol (thiol-specific reducing agent) (Murphy et al, 1991;Campbell et al, 1996;Garcia-Pascual et al, 1999Resim et al, 2002). To further investigate the mechanism of FPTO-dependent relaxation, we compared the effects of thiols and thiol-modifying substances on FPTOand sodium nitroprusside-induced activation of highly purified sGC and in organ bath experiments as well as production of nitric oxide from FPTO.…”

Nicorandil, a Potassium Channel Opener and Nitric Oxide Donor, Improves the Frequent Urination without Changing the Blood Pressure in Rats with Partial Bladder Outlet Obstruction

“…Neuroanatomic studies have demonstrated a rich nitrergic innervation and NOS enzyme activity in the urethra but sparse innervation in the detrusor in rats. 2,21,22) Exogenously applied NO-donors have been shown to relax pre-contracted urethral smooth mus- 23) leading to decrease in intra-urethral pressure. 15) Sublingual administration of ISDN to humans was shown to decrease in the bladder outlet resistance.…”