NO/PGI2-independent vasorelaxation and the cytochrome P450 pathway in rabbit carotid artery

1. The nature and cellular mechanisms that are responsible for endothelium-dependent relaxations resistant to indomethacin and N(G)-nitro-L-arginine methyl ester (L-NAME) were investigated in phenylephrine (PE) precontracted isolated carotid arteries from the rabbit. 2. In the presence of the cyclo-oxygenase inhibitor, indomethacin (10 μM), acetylcholine (ACh) induced a concentration- and endothelium-dependent relaxation of PE-induced tone which was more potent than the calcium ionophore A23187 with pD₂ values of 7.03 ± 0.12 (n = 8) and 6.37 ± 0.12 (n = 6), respectively. The ACh-induced response was abolished by removal of the endothelium, but was not altered when indomethacin was omitted (pD₂ value 7.00 ± 0.10 and maximal relaxation 99 ± 3%, n = 6). Bradykinin and histamine (0.01-100 μM) had no effect either upon resting or PE-induced tone (n = 5). 3. In the presence of indomethacin plus the NO synthase inhibitor, L-NAME (30 μM), the response to A23187 was abolished. However, the response to ACh was not abolished, although it was significantly inhibited with the pD₂ value and the maximal relaxation decreasing to 6.48 ± 0.10 and 67 ± 3%, respectively (for both P < 0.01, n = 8). The L-NAME/indomethacin insensitive vasorelaxation to ACh was completely abolished by preconstriction of the tissues with potassium chloride (40 mM, n = 8). 4. The Ca²⁺-activated K⁺ (K(Ca)) channel blockers, tetrabutylammonium (TBA, 1 mM, n = 5) and charybdotoxin (CTX, 0.1 μM, n = 5), completely inhibited the nitric oxide (NO) and prostacyclin (PGI₂)-independent relaxation response to ACh. However, iberiotoxin (ITX, 0.1 μM, n = 8) or apamin (1-3 μM, n = 6) only partially inhibited the relaxation. 5. Inhibitors of the cytochrome P450 mono-oxygenase, SKF-525A (1-10 μM, n = 6), clotrimazole (1 μM, n = 5) and 17-octadecynoic acid (17-ODYA, 3 μM, n = 7) also reduced the NO/PGI₂-independent relaxation response to ACh. 6. In endothelium-denuded rings of rabbit carotid arteries, the relaxation response to exogenous NO was not altered by either K(Ca) channel blockade with apamin (1 μM, n = 5) or CTX (0.1 μM, n = 5), or by the cytochrome P450 mono-oxygenase blockers SKF-525A (10 μM, n = 4) and clotrimazole (10 μM, n = 5). However, the NO-induced response was shifted to the right by LY83583 (10 μM, n = 4), a guanylyl cyclase inhibitor, with the pD₂ value decreasing from 6.95 ± 0.14 to 6.04 ± 0.09 (P < 0.01). 7. ACh (0.01-100 μM) induced a concentration-dependent relaxation of PE-induced tone in endothelium-denuded arterial segments sandwiched with endothelium-intact donor segments. This relaxation to ACh was largely unaffected by indomathacin (10 μM) plus L-NAME (30 μM), but abolished by the combination of indomethacin, L-NAME and TBA (1 mM, n = 5). 8 These data suggest that in the rabbit carotid artery: (a) ACh can induce the release of both NO and EDHF, whereas A23187 only evokes the release of NO from the endothelium, (b) the diffusible EDHF released by ACh may be a cytochrome P450-derived arachidonic acid metabolite, and (c) EDHF-induced relaxation involves the opening of at least two types of K(Ca) channels, whereas NO mediates vasorelaxation via a guanosine 3':5'-cyclic monophosphate (cyclic GMP)-mediated pathway, in which a cytochrome P450 pathway and K(Ca) channels do not seem to be involved.