Localization and thiol dependancy of endogenous nitro compounds-mediating urethral photo-relaxation.

Using a nitric oxide (NO)-specific fluorescent probe, we have examined the location of NO generation in the urethra from sheep and rat when induced by either electrical field- or light-stimulation (EFS and LS, respectively). In addition, we studied the effect of specific glutathione (GSH) modifiers, acting upon different cellular GSH pools, on NO release and on urethral relaxation. Both EFS and LS led to fluorescence emission from a fiber network associated with neuronal NO synthase (nNOS) immunoreactive nerves. Both the relaxation and the fluorescence elicited by EFS were blocked by specific nNOS inhibitors, but these parameters were not significantly modified by endogenous GSH depletion. In contrast, the opposite was found for LS-induced responses. Moreover, when the mitochondrial pool was effectively reduced by incubation with ethacrynic acid, the responses to LS were further reduced until they disappeared after intensive LS. Our results confirm that while NO is released by nNOS activation, the photolytic breakdown of an endogenous nitro-compound, probably S-nitroso-glutathione, in nitrergic nerves (and in the vascular endothelium) is the only factor responsible for photo-relaxation. The possible role of this mechanism in NO inactivation and as a protective mechanism in NO-generating structures is further discussed.

Stereological study of the external urethral sphincter in the female urethra of the lamb: a new model for studies on urinary continence.

Some of the morphometrical features and functional properties of the lamb external urethral sphincter (EUS) were determined by means of ATPase histochemistry. In this study, eight female urethras of 2- to 3-month-old lambs were used. The samples were studied by using an unbiased stereological design and were statistically evaluated with a Kolmogorov-Smirnov test. Additionally, the mechanical activity of the different fibre types was analysed by measuring the isometric changes in tension induced by electrical field stimulation (EFS) of intrinsic nerves. The lamb EUS has an average fibre density of 1142.37 fibres/mm(2) (SD = 279.33). Our data showed the predominance of fast-twitch fibres or type II fibres, with a proportion of 30% (type I) and 70% (type II) in all cases. These percentages remained the same along the length of the EUS. However, a clear tendency to increase the number of both fibre types in the mid-EUS was observed. Interestingly, type I and II fibres are intermingled throughout the whole lamb EUS. Dynamic parameters of the contraction induced in lamb EUS preparations by EFS of intramural nerves, were intermediate between fast- and slow-twitch muscles. Furthermore, contractile activity showed no decrease in amplitude upon repetitive stimulation, suggesting the presence of a mixture of fibre types, in which fast but fatigue-resistant fibres (probably type IIA) seem to predominate. Our results indicate that lambs could be suggested as a new biological model to study those pathological processes, which involve the sphincteric regulation of the EUS.

Effects of superoxide anion generators and thiol modulators on nitrergic transmission and relaxation to exogenous nitric oxide in the sheep urethra.

The effects of superoxide anion generators, the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoine-1-oxyl 3-oxide (carboxy-PTIO), the specific guanylate cyclase inhibitor 1H-[1,2,4]-oxadiazole-[4,3-a]-quinoxalin-1-one (ODQ), and thiol modulating agents were investigated on relaxations induced by nitrergic stimulation and exogenous NO addition in the sheep urethra. Methylene blue (MB, 10 microM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml(-1)) inhibited NO-mediated relaxations, without affecting those induced by nitrergic stimulation. This resistance was not diminished following inhibition of endogenous Cu/Zn superoxide dismutase (Cu/Zn SOD) with diethyldithiocarbamic acid (DETCA, 3 mM), which almost abolished tissue SOD activity. Carboxy-PTIO (0.1 - 0.5 mM) inhibited NO-mediated relaxations but had no effect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml(-1)). Relaxations to NO were reduced, but not abolished, by ODQ (10 microM), while nitrergic responses were completely blocked. The thiol modulators, ethacrynic acid (0.1 mM), diamide (1.5 mM), or 5,5'-dithio-bis (2-nitrobenzoic acid) (DTNB, 0. 5 mM), and subsequent treatment with dithiothreitol (DTT, 2 mM) had no effect on responses to nitrergic stimulation or NO. In contrast, N-ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. L-cysteine (L-cys, 0.1 mM) had no effect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD-independent manner. Our results do not support the view that the urethral nitrergic transmitter is free NO, and the possibility that another compound is acting as mediator still remains open. British Journal of Pharmacology (2000) 129, 53 - 62

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

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.

Endothelium-dependent relaxation to acetylcholine in bovine oviductal arteries: mediation by nitric oxide and changes in apamin-sensitive K+ conductance.

1. Mechanisms underlying the relaxant response to acetylcholine (ACh) were examined in bovine oviductal arteries (o.d. 300-500 microns and i.d. 150-300 microns) in vitro. Vascular rings were treated with indomethacin (10 microM) to prevent the effects of prostaglandins. 2. ACh elicited a concentration-related relaxation in ring segments precontracted with noradrenaline (NA), which was abolished by endothelium denudation. 3. The ACh-induced relaxation was attenuated but not abolished by NG-nitro-L-arginine (L-NOARG, 1 microM-1 mM), an inhibitor of nitric oxide (NO) formation. The inhibition caused by L-NOARG (10 microM) was reversed by addition of excess of L-arginine but not D-arginine (1 mM). 4. In high K+ (40-60 mM)-contracted rings, ACh was a much less effective vasodilator and its relaxant response was completely abolished by L-NOARG (100 microM). 5. In NA (10 microM)-contracted rings, ACh induced sustained and concentration-dependent increases in cyclic GMP, which were reduced below basal values by L-NOARG (100 microM), while potent relaxation persisted. Similar increases in cyclic GMP were evoked by ACh in high K+ (50 mM)-treated arteries and under these conditions, both cyclic GMP accumulation and relaxation were L-NOARG-sensitive. 6. S-nitroso-L-cysteine (NC), a proposed endogenous precursor of endothelial NO, also induced cyclic GMP accumulation in NA-contracted oviductal arteries. 7. Methylene blue (MB, 10 microM), a proposed inhibitor of soluble guanylate cyclase, inhibited both endothelium-dependent relaxation to ACh and endothelium-independent response to exogenous NO, whereas relaxation to NC remained unaffected. 8. The L-NOARG-resistant response to ACh was not affected by either ouabain (0.5 mM), glibenclamide (3 microM), tetraethylammonium (TEA, 1 mM) or charybdotoxin (50 nM), but was selectively blocked by apamin (0.1-1 microM). However, apamin did not inhibit either relaxation to ACh in high K(+)-contracted rings or endothelium-independent relaxation to either NO or NC. 9. Apamin and MB inhibited ACh-induced relaxation in an additive fashion, suggesting the involvement of two separate modulating mechanisms. 10. These results suggest that ACh relaxes bovine oviductal arteries by the release of two distinct endothelial factors: a NO-like substance derived from L-arginine, which induces cyclic GMP accumulation in smooth muscle, and another non-prostanoid factor acting by hyperpolarization mechanisms through alterations in apamin-sensitive K+ conductance.

Relaxation mechanisms induced by stimulation of nerves and by nitric oxide in sheep urethral muscle.

Isolated transverse and longitudinally oriented preparations of sheep urethra precontracted with noradrenaline responded to electrical field stimulation (EFS) with stimulus-dependent non-adrenergic, non-cholinergic (NANC) relaxations. Exogenous nitric oxide (NO) (acidified NaNO2), S-nitroso-L-cysteine (NC), sodium nitroprusside (SNP), 8-Br-cGMP, dibutyryl-cAMP, forskolin and isoprenaline each relaxed precontracted transverse urethral preparations in a concentration-dependent manner in order of protency: NC > forskolin > isoprenaline = SNP > NO > 8-Br-cGMP = dibutyryl-cAMP. Longitudinally oriented preparations responded to NO and NC with concentration-dependent relaxation, no different from that observed in transverse strips. Methylene blue (MB) and oxyhaemoglobin (HbO2) each shifted the concentration-response curve for NO to the right without affecting EFS-induced relaxation. Similarly, concentration-dependent responses to NC were not affected by MB. The inhibition of relaxation to NO by MB was prevented by superoxide dismutase, suggesting the inhibition was caused by extracellular generation of superoxide anions. EFS-induced relaxation was accompanied by elevation of cGMP. However, for the same level of relaxation, exogenous NO and NC induced 15- and 23-times higher increases in cGMP values, respectively, than EFS. cAMP levels were not affected by EFS- or NO-induced relaxation, although a large increase accompanied relaxation induced by forskolin. Forskolin also increased cGMP content. Pretreatment with MB reduced basal levels of cGMP and inhibited both relaxation and rise in cGMP levels induced by NO. SNP-elicited relaxant responses, in the presence of MB, were accompanied by an accumulation of cGMP; cAMP levels were unaffected. MB reduced cGMP levels induced by NC, while the relaxant response was unchanged. In urethral preparations prelabelled with [3H]myoinositol, exposure to NA caused an accumulation of [3H]inositol phosphates, which was unaffected by pretreatment with 8-Br-cGMP or dibutyryl-cAMP. EFS failed to induce a relaxant response in excess [K+]o-contracted preparations, while relaxation with exogenous NO was unaffected. Ouabain abolished EFS-induced relaxation and reduced responses to NO. Neither TEA nor glibenclamide affected relaxation to either EFS or NO. Relaxation elicited by SNP was not accompanied by any change in cGMP or cAMP levels, and was unaffected by MB, HbO2, K+ channel blockers (TEA and glibenclamide), ouabain or high [K+]o solution. This suggested that relaxation was caused by a mechanism independent of NO generation. A dense network of NADPH diaphorase-positive fibres associated with both the circular and longitudinal smooth muscle layers of sheep urethra was found.(ABSTRACT TRUNCATED AT 400 WORDS)