While interstitial cells of Cajal (ICC) in the urethra respond to nitric oxide (NO) donors by increasing cGMP, it remains unclear whether urethral ICC are functionally innervated by nitrergic nerves. We have addressed this issue in the rat and sheep urethra, where cGMP production and relaxation were compared in preparations subjected to electrical field stimulation (EFS; 2 Hz, 4 min) of nitrergic nerves or to exogenous S-nitroso-L-cysteine (SNC; 0.1 mM, 4 min). Upon EFS, cGMP immunoreactivity (cGMP-ir) was observed in both smooth muscle cells (SMC) and in spindle-shaped cells that contained c-kit and vimentin, features of ICC. Similarly, cGMP-ir was preferentially, but inconsistently, found in ICC of the outer muscle layer on exposure to SNC. We found separate functional groups of ICC within the urethra. Thus only ICC present in the muscle layers (ICC-M) but not those in the serosa (ICC-SR) and lamina propia (ICC-LP) seem to be specifically influenced by activation of neuronal NO synthase (nNOS). Thus the increase in cGMP-ir in the ICC-M induced by EFS was prevented by Nomega-nitro-L-arginine and ODQ. Urethral ICC did not express nNOS, although they were closely associated with nitrergic nerves. cGMP-ir was also present in the urothelium (in the rat but not in the sheep) and the vascular endothelium but not in neural structures, such as the nerve trunks and nerve terminals. Together, these results suggest a model of parallel innervation in which both SMC and ICC-M are effectors of nerve-released NO in the urethra.
1. 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. 2. 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 potency: 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. 3. Methylene blue (MB) and oxyhaemoglobin (HbO2) mechanism independent of NO generation. 9. A dense network of NADPH diaphorase-positive fibres associated with both the circular and longitudinal smooth muscle layers of sheep urethra was found. 10. The possibility that a nitrosyl compound which releases NO in the target tissue is the NANC transmitter in sheep urethra is discussed. The data also suggest a role for increased cGMP levels in the relaxation mechanisms, whereas changes in cAMP levels, phosphoinositide hydrolysis or K+ channel activation do not seem to be involved.
Isolated smooth muscle preparations from the sheep urethra responded to electrical field stimulation with contraction when basal tension was low (5-6 mN), but with relaxation when the preparations were contracted with noradrenaline (NA), clonidine, or prostaglandin F2a. No relaxant response could be elicited in high K+ (124 mM) contracted preparations. Electrically induced relaxations had a threshold of less than 1 Hz and a maximum at 8 Hz. Both contractant and relaxant responses were abolished by tetrodotoxin, indicating that they were caused by transmitters released from nerves. The amplitude of the relaxant responses showed a highly significant correlation to the tension induced by noradrenaline. A coefficient (R/T) was calculated relating relaxation to noradrenaline-induced tension. In this way it is possible to separate the effect of drugs on muscle tension (non-specific effect) from their action on the electrically induced relaxation (specific effect). Chemical sympathectomy with 6-OHDA did not significantly modify the relaxant response to 6 Hz in noradrenaline contracted strips, as evaluated by the R/T coefficient. The electrically induced relaxation was not affected by hexamethonium, propranolol, phentolamine, muscarinic receptor blockade, cocaine, indomethacin, or methysergide. Both nifedipine and Bay K 8644 inhibited significantly the response induced by electrical stimulation, decreasing its maximum. Nifedipine, but not Bay K 8644, significantly reduced the level of tension induced by noradrenaline, and its effect, evaluated by the R/T coefficient, was an increase in the electrically induced relaxation, whereas Bay K 8644 had a significant inhibitory effect.(ABSTRACT TRUNCATED AT 250 WORDS)
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.
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