No abstract

We have used the isolated, buffer‐perfused, mesenteric arterial bed of the rat (preconstricted with methoxamine or 60 mm K+) to characterize nitric oxide (NO)‐independent vasorelaxation which is thought to be mediated by the endothelium‐derived hyperpolarizing factor (EDHF). The muscarinic agonists carbachol, acetylcholine (ACh) and methacholine caused dose‐related relaxations in preconstricted preparations with ED50 values of 0.18±0.04 nmol (n=8), 0.05±0.02 nmol (n=6) and 0.26±0.16 nmol (n=5), respectively. In the same preparations NG‐nitro‐l‐arginine methyl ester (l‐NAME, 100 μm) significantly (P<0.05) decreased the potency of all the agents (ED50 values in the presence of l‐NAME: carbachol, 0.66±0.11 nmol; ACh, 0.28±0.10 nmol; methacholine, 1.97±1.01 nmol). The maximal relaxation to ACh was also significantly (P<0.05) reduced (from 85.3±0.9 to 73.2±3.7%) in the presence of l‐NAME. The vasorelaxant effects of carbachol were not significantly altered by the cyclo‐oxygenase inhibitor indomethacin (10 μm; n=4). The K+ channel blocker, tetraethylammonium (TEA, 10 mm) also significantly (P<0.001) reduced both the potency of carbachol (ED50=1.97±0.14 nmol in presence of TEA) and the maximum relaxation (Rmax=74.6±3.2% in presence of TEA, P<0.05, n=3). When TEA was added in the presence of l‐NAME (n=4), there was a further significant (P<0.001) decrease in the potency of carbachol (ED50=22.4±13.5 nmol) relative to that in the presence of l‐NAME alone, and Rmax was also significantly (P<0.05) reduced (74.6±4.2%). The ATP‐sensitive K+ channel inhibitor, glibenclamide (10 μm), had no effect on carbachol‐induced relaxation (n=9). High extracellular K+ (60 mm) significantly (P<0.01) reduced the potency of carbachol (n=5) by 5 fold (ED50: control, 0.16±0.04 nmol; high K+, 0.88±0.25 nmol) and the Rmax was also significantly (P<0.01) reduced (control, 83.4±2.7%; high K+, 40.3±9.2%). The residual vasorelaxation to carbachol in the presence of high K+ was abolished by l‐NAME (100 μm; n=5). In preparations preconstricted with high K+, the potency of sodium nitroprusside was not significantly different from that in preparations precontracted with methoxamine, though the maximal response was reduced (62.4±3.4% high K+, n=7; 83.1±3.1% control, n=7). In the presence of the cytochrome P450 inhibitor, clotrimazole (1 μm, n=5 and 10 μm, n=4), the dose‐response curve to carbachol was significantly shifted to the right 2 fold (P<0.05) and 4 fold (P<0.001) respectively, an effect which was further enhanced in the presence of l‐NAME. Rmax was significantly (P<0.01) reduced by the presence of 10 μm clotrimazole alone, being 86.9±2.5% in its absence and 61.8±7.8% in its presence (n=6). In the presence of the cell permeable analogue of cyclic GMP, 8‐bromo cyclic GMP (6 μm), the inhibitory effects of l‐NAME on carbachol‐induced relaxation were substantially enhanced (ED50: l‐NAME alone, 0.52±0.11 nmol, n=5; l‐NAME+8‐bromo cyclic GMP, 1.42±0.28 nmol, n=7. Rmax: l‐NAME alone, 82.2±2.4%; l‐NAME+8‐bromo cyclic GMP, 59.1±1.8%. P<0.001). These resu...

The endogenous cannabinoid anandamide has recently been identified as a vasorelaxant but the underlying mechanisms are controversial. The vasorelaxant responses to anandamide have now been examined in the rat mesenteric arterial bed. Anandamide caused potent vasorelaxations (pD2= 6.24 ± 0.06; Rmax= 89.4 ± 2.2 %) which were unaffected by inhibition of nitric oxide synthase with NG‐nitro‐l‐arginine methyl ester (l‐NAME; 300 μm). The responses were also predominantly endothelium independent and were unaffected by the cannabinoid CB1 receptor antagonist SR141716A (1 μm), although at higher concentrations (3 and 10 μm) SR141716A was inhibitory. Both 1 mm ouabain (pD2= 5.90 ± 0.07; Rmax= 50.4 ± 6.5 %) and 100 μm 18α‐glycyrrhetinic acid (pD2= 6.04 ± 0.14; Rmax= 40.9 ± 5.8 %) opposed anandamide‐induced vasorelaxation. However, the gap junction inhibitors carbenoxolone (100 μm) and palmitoleic acid (50 μm) did not affect vasorelaxation to anandamide. Relaxation to anandamide was significantly attenuated by both capsaicin pretreatment to deplete the sensory nerves of neurotransmitters (pD2= 5.86 ± 0.18; Rmax= 56.3 ± 5.2 %) and the vanilloid antagonist ruthenium red (10 μm; pD2= 5.64 ± 0.09; Rmax= 33.7 ± 3.9 %). However, these inhibitory effects were prevented by the additional presence of l‐NAME, when the relaxation to anandamide was unaffected (pD2= 6.19 ± 0.07; Rmax= 81.9 ± 2.8 %). The inhibitor of neuronal nitric oxide synthase, 7‐nitroindazole, also prevented capsaicin from inhibiting the responses to anandamide. The results of this study point to anandamide acting via several mechanisms, which include the involvement of sensory nerves, but only in the presence of nitric oxide.