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Vasorelaxant effects of oleamide in rat small mesenteric artery indicate action at a novel cannabinoid receptor
Oleamide (cis-9-octadecenoamide), which was identified in the CSF of sleep-deprived cats (Cravatt et al., 1995), exhibits some cannabimimetic responses including hypotension (Lichtman et al., 2002) despite its low affinities at the currently known CB1 and CB2 cannabinoid receptors (Bradshaw & Walker., 2005). Here we have investigated whether or not oleamide is also a vasorelaxant in rat small mesenteric arteries. Male Wistar rats (300-400g) were killed with sodium pentobarbitone (120mg kg-1, i.p.). Third generation mesenteric arteries (2mm long) were mounted in a wire myograph under normalised tension in oxygenated Krebs-Henseleit solution with 10 µM indomethacin (White et al., 2001). After precontraction with methoxamine (10 µM), relaxations to cumulative addition of oleamide were determined. Results are expressed as mean ± s.e.mean. Inhibitors were incubated with tissues for 30 min before determination of a concentration/relaxation curve. Statistical analysis was by two-way analysis of variance of the whole concentration/response curve; when significant interaction between concentration and treatment occurred this was followed by a Bonferroni post-hoc test with P<0.05 taken as the level of significance. Oleamide elicited vasorelaxation (EC50 = 1.2 ± 0.2 µM, Rmax = 99.1 ± 3.9%, n = 8) which was markedly reduced by removal of the endothelium. The nitric oxide synthase inhibitor L-nitroarginine methyl ester (300 µM) significantly reduced the oleamide-induced relaxation (E 50 = 5.3 ± 1.6 µM, Rmax = 59.2 ± 7.7%, n = 7; P < 0.01) as did blockade of Ca2+-sensitive K+ channels by combination of apamin (50 nM) with charybdotoxin (50 nM) (EC50 = 2.1 ± 0.2 µM, Rmax = 58.4 ± 1.9%, n = 5; P < 0.05). Functional desensitization of the vanilloid receptor system by pre-treatment with capsaicin (10 µM for 30 min) caused an approximate 30-fold shift to the right in the oleamide concentration/response curve (n = 7; P < 0.01). Pertussis toxin (400 ng/ml) caused a significant 2-fold shift in the response curve(EC50 = 2.2 ± 0.4 µM, Rmax = 66.8 ± 4.5%, n = 6; P < 0.01). The cannabinoid CB1 receptor antagonist rimonabant (SR 141716A; 3 µM) significantly inhibited relaxation induced by oleamide (EC50 = 3.5 ± 0.3 µM, Rmax = 75.1 ± 1.9%; n = 8; P < 0.05). In contrast, neither the more selective CB1 receptor antagonist, AM 251 (1 µM) nor the CB2 receptor antagonist, SR 144528 (1 µM) had any significant effect on the relaxation induced by oleamide. However, O-1918 (10 µM), a putative antagonist at the novel endothelial cannabinoid receptor (abnormal-cannabinoid site; Offertáler et al., 2003), markedly reduced the relaxation to oleamide (n = 7; P < 0.01). The response induced by oleamide was markedly reduced by pre-incubating the vessels with oleamide itself (they were exposed to 10 µM for 30 min, and then washed out before the construction of the test concentration-dose response curve; n = 5; P < 0.01). Similarly, the relaxation induced by AEA was also markedly reduced (n = 6; P < 0.01). In contrast, pre‑incubation with oleamide produced no significant reduction in the potency of abnormal-cannabidiol. The present study shows that oleamide relaxes the rat isolated small mesenteric artery through multiple mechanisms. The response is partly dependent on the presence of a functional endothelium, and the activation of KCa channels, as well as involving perivascular capsaicin-sensitive sensory nerves. It also suggests that oleamide shares a receptor with anandamide in the rat mesenteric artery which is distinct from both the currently known cannabinoid receptors as well as the novel endothelial abn-cbd site . This receptor is sensitive to both SR 141716A and O-1918, and is possibly coupled to KCa and Gi/o.
Bradshaw H.B. & Walker, J.M. (2005). Br. J. Pharmacol.. 144, 459-465. |
