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.

1 To define further the role of nitric oxide (NO) in urinary tract function, we have measured the presence of nitric oxide synthase (NOS) activity, and its relationship with functional NO-mediated responses to electrical field stimulation (EFS) in the urethra, the detrusor and the ureter from sheep. NOS activity was assayed by the conversion of L-['4C]-arginine to L-['4C]-citrulline. Endogenous production of citrulline was confirmed by thin layer chromatography. 2 NOS enzymatic activity was detected in the cytosolic fraction from tissue homogenates with the following regional distribution (pmol citrulline mg-' protein min-'): urethra (33 + 3.3), detrusor (13.1 + 1.1) and ureter (1.5 + 0.2). No activity was detected in the particulate fraction of any region. 3 NOS activity was dependent on Ca2+-calmodulin and required exogenously added NADPH and tetrahydrobyopterin (BH4) for maximal activity. Exclusion of calmodulin from the incubation mixture did not modify NOS activity, but it was significantly reduced in the presence of the calmodulin antagonist, calmidazolium, suggesting the presence of enough endogenous calmodulin to sustain the observed NOS activity. 4 NOS activity was inhibited to a greater extent by NG-nitro-L-arginine (L-NOARG) and its methyl ester (L-NAME) than by NG-monomethyl-L-arginine (L-NMMA), while 7-nitroindazole (7-NI) was a weak inhibitor and L-cannavine had no effect. 5 Citrulline formation could be inhibited by superoxide dismutase in an oxyhaemoglobin-sensitive manner, suggesting feedback inhibition of NOS by NO. 6 EFS induced prominent NO-mediated relaxations in the urethra while minor or no responses were observed in the detrusor and the ureter, respectively. Urethral relaxations to EFS were inhibited by NOS inhibitors with the rank order of potency: L-NOARG = L-NAME>7-NI> L-NMMA. 7In conclusion, we have demonstrated the presence of NO-synthesizing enzymatic activity in the sheep urinary tract which shows similar characteristics to the constitutive NOS isoform found in brain. We suggest that the enzymatic activity measured in the urethral muscle layer may account for the NOmediated urethral relaxation during micturition whereas regulation of detrusor and ureteral motor function by NOS containing nerves is less likely. Keywords: Nitric oxide; nitric oxide synthase; urethra; detrusor; ureter; nitrergic relaxation; urinary tract Introduction It has been suggested that nitric oxide (NO) has important regulatory functions in the urinary tract acting as a nonadrenergic, non-cholinergic (NANC)-relaxant transmitter. Specially relevant is the proposed role of NO in mediating the decrease in outlet resistance which accompanies micturition (Andersson, 1993). A rich supply of nitrergic nerves has been identified by NADPH-diaphorase histochemistry and nitric oxide synthase (NOS) immunohistochemistry in the outflow region (urethra, bladder neck and bladder base or trigone) of several species including rat (McNeill et al., 1992), pig (Persson et al., 1993), sheep (Triguero et al., 1993 and human (Sme...

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.

1 The eects of superoxide anion generators, the nitric oxide (NO) scavenger 2-(4-carboxyphenyl)-4,4,5,5-tetramethylimidazoine-1-oxyl 3-oxide (carboxy-PTIO), the speci®c 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. 2 Methylene blue (MB, 10 mM), pyrogallol (0.1 mM) and xanthine (X, 0.1 mM)/xanthine oxidase (XO, 0.1 u ml 71) inhibited NO-mediated relaxations, without aecting 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. 3 Carboxy-PTIO (0.1 ± 0.5 mM) inhibited NO-mediated relaxations but had no eect on responses to nitrergic stimulation, which were not changed by treatment with ascorbate oxidase (2 u ml 71 ). 4 Relaxations to NO were reduced, but not abolished, by ODQ (10 mM), while nitrergic responses were completely blocked. 5 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 eect on responses to nitrergic stimulation or NO. In contrast, N-ethylmaleimide (NEM, 0.2 mM) markedly inhibited both relaxations. 6 L-cysteine (L-cys, 0.1 mM) had no eect on responses to NO, while it inhibited those to nitrergic stimulation, in a Cu/Zn SOD-independent manner. 7 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.

1 Mechamisms underlying the relaxant response to acetylcholine (ACh) were examined in bovine oviductal arteries (o.d. 300-500 pm and i.d. 150-300 pm) in vitro. Vascular rings were treated with indomethacin (10 pM) 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 gM-1 mM), an inhibitor of nitric oxide (NO) formation. The inhibition caused by L-NOARG (10 pM) 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 pM).5 In NA (10 gM)-contracted rings, ACh induced sustained and concentration-dependent increases in cyclic GMP, which were reduced below basal values by L-NOARG (100 pM), 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 pM), 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 pM), tetraethylammonium (TEA, 1 mM) or charybdotoxin (50 nM), but was selectively blocked by apamin (0.1-1 pM). 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.

In the present work we have characterized the relaxant response induced by light stimulation (LS) in the lower urinary tract from sheep, pig and rat, establishing its relationship with nitrergic neurotransmission. Urethral, but not detrusor, preparations showed pronounced photo‐relaxation (PR) which declined progressively following repetitive LS. Sheep urethral PR was again restored either spontaneously or (to a greater extent) by exogenous nitric oxide (NO) addition and by electrical field stimulation (EFS) of intrinsic nitrergic nerves. Greater NO generation was detected from sheep urethral than from detrusor homogenates following illumination. Sheep urethral PR was inhibited by oxyhaemoglobin, but not by methaemoglobin, carboxy‐PTIO, extracellular superoxide anion generators or superoxide dismutase. Guanylyl cyclase but not adenylyl cyclase activation mediates urethral relaxation to LS. Urethral PR was more resistant to inhibition by L‐thiocitrulline than EFS‐induced responses, although this agent prevented PR restoration by high‐frequency EFS. Urethral PR was TTX insensitive and partially modified in high‐K+ solutions. Cold storage for 24 h greatly impaired urethral PR, although it was restored by high‐frequency EFS. Repetitive exposure to LS, EFS or exogenous NO induced changes in the shape of the EFS‐induced nitrergic relaxation, possibly by pre‐synaptic mechanisms. In conclusion, we suggest the presence of an endogenous, photo‐labile, nitro‐compound store in the urethra, which seems to be replenished by neural nitric oxide synthase activity, indicating a close functional relationship with the nitrergic neurotransmitter.

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1 In vitro experiments were designed to characterize postjunctional a-adrenoceptor subtypes in ring segments (1 mm length; outer diameter 300-500pm) from arteries supplying the oviduct of the heifer. 2 Noradrenaline, adrenaline and phenylephrine evoked concentration-dependent contractile responses. The pD2 values were 5.67, 5.89 and 5.93, respectively. Medetomidine, clonidine and B-HT 920 (2-amino-6-allyl-5,6,7,8-tetra-hydro-4H-(thiazo)-4,5-d-azepine) were ineffective.3 The a-adrenoceptor selective antagonists, prazosin (1 nM-0.1 pM) and rauwolscine