Evidence that different mechanisms underlie smooth muscle relaxation to nitric oxide and nitric oxide donors in the rabbit isolated carotid artery

The endothelium‐dependent relaxants acetylcholine (ACh; 0.03–10 μ m ) and A23187 (0.03–10 μ m ), and nitric oxide (NO), applied either as authentic NO (0.01–10 μ m ) or as the NO donors 3‐morpholino‐sydnonimine (SIN‐1; 0.1–10 μ m ) and S‐nitroso‐N‐acetylpenicillamine (SNAP; 0.1–10 μ m ), each evoked concentration‐dependent relaxation in phenylephrine stimulated (1–3 μ m ; mean contraction and depolarization, 45.8±5.3 mV and 31.5±3.3 mN; n =10) segments of rabbit isolated carotid artery. In each case, relaxation closely correlated with repolarization of the smooth muscle membrane potential and stimulated a maximal reversal of around 95% and 98% of the phenylephrine‐induced depolarization and contraction, respectively. In tissues stimulated with 30 m m KCl rather than phenylephrine, smooth muscle hyperpolarization and relaxation to ACh, A23187, authentic NO and the NO donors were dissociated. Whereas the hyperpolarization was reduced by 75–80% to around a total of 10 mV, relaxation was only inhibited by 35% ( n =4–7 in each case; P <0.01). The responses which persisted to ACh and A23187 in the presence of 30 m m KCl were abolished by either the NO synthase inhibitor l ‐N G ‐nitroarginine methyl ester ( l ‐NAME; 100 μ m ) or the inhibitor of soluble guanylyl cyclase 1H‐[1,2,4]oxadiazolo[4,3‐a]quinoxalin‐1‐one (ODQ; 10 μ m ; 10 min; n =4 in each case; P <0.01). Exposure to ODQ significantly attenuated both repolarization and relaxation to ACh, A23187 and authentic NO, reducing the maximum changes in both membrane potential and tension to each relaxant to around 60% of control values ( n =4 in each case; P <0.01). In contrast, ODQ almost completely inhibited repolarization and relaxation to SIN‐1 and SNAP, reducing the maximum responses to around 8% in each case ( n =3–5; P <0.01). The potassium channel blockers glibenclamide (10 μ m ), iberiotoxin (100 n m ) and apamin (50 n m ), alone or in combination, had no significant effect on relaxation to ACh, A23187, authentic NO, or the NO donors SIN‐1 and SNAP ( n =4 in each case; P >0.05). Charybdotoxin (ChTX; 50 n m ) almost abolished repolarization to ACh ( n =4; P <0.01) and inhibited the maximum relaxation to ACh, A23187 and authentic NO each by 30% ( n =4–8; P <0.01). Application of ODQ (10 μ m ; 10 min) abolished the ChTX‐insensitive responses to ACh, A23187 and authentic NO ( n =4 in each case; P <0.01 When the concentration of phenylephrine was reduced (to 0.3–0.5 μ m ) to ensure the level of smooth muscle contraction was the same as in the absence of potassium channel blocker, ChTX had no effect on the subsequent relaxation to SIN‐1 ( n =4; P >0.05). However, in the presence of tone induced by 1–3 μ m phenylephrine (51.2±3.3 mN; n =4), ChTX significantly reduced relaxation to SIN‐1 by nearly 50% (maximum relaxation 53.2±6.3%, n =4; P <0.01). These data indicate that NO‐evoked relaxation of the rabbit isolated carotid artery can be mediated by three distinct mechanisms: (a) a cyclic GMP‐dependent, voltage‐independent pathway, (b) cyclic GMP‐mediated smooth muscle repolarization and (c) cyclic GMP‐independent, ChTX‐sensitive smooth muscle repolarization. Relaxation and repolarization to both authentic and endothelium‐derived NO in this large conduit artery appear to be mediated by parallel cyclic GMP‐dependent and ‐independent pathways. In contrast, relaxation to the NO‐donors SIN‐1 and SNAP appears to be mediated entirely via cyclic GMP‐dependent mechanisms.British Journal of Pharmacology (1998) 123 , 1351–1358; doi: 10.1038/sj.bjp.0701746