RELAXATION MECHANISMS INDUCED BY STIMULATION OF NERVES AND BY NITRIC-OXIDE IN SHEEP URETHRAL MUSCLE

1. Isolated transverse and longitudinally oriented preparations of sheep urethra precontracted with noradrenaline responded to electrical field stimulation (EE'S) 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 (HbO(2)) each shifted the concentration-response curve for NO to the right without affecting EPS-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. 4. 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. 5. 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. 6. In urethral preparations prelabelled with [H-3]myoinositol, exposure to NA caused an accumulation of [H-3]inositol phosphates, which was unaffected by pretreatment with 8-Br-cGMP or dibutyryl-cAMP. 7. EPS failed to induce a relaxant response in excess [K+](0)-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. 8. Relaxation elicited by SNP was not accompanied by any change in cGMP or cAMP levels, and was unaffected by MB, HbO(2), K+ channel blockers (TEA and glibenclamide), ouabain or high [K+](0) solution. This suggested that relaxation was caused by a 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.