CHARACTERIZATION AND MODULATION OF EDHF-MEDIATED RELAXATIONS IN THE RAT ISOLATED SUPERIOR MESENTERIC ARTERIAL BED

1 We have used the isolated, buffer-perfused, mesenteric arterial bed of the rat (preconstricted with methoxamine or 60 mM K+) to characteri ze nitric oxide (NO)-independent vasorelaxation which is thought to be mediated by the endothelium-derived hyperpolarizing factor (EDHF). 2 The muscarinic agonists carbachol, acetylcholine (ACh) and methacholin e 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 N-G-n itro-L-arginine methyl ester (L-NAME, 100 mu M) 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; met hacholine, 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%) i n the presence of L-NAME. The vasorelaxant effects of carbachol were n ot significantly altered by the cyclo-oxygenase inhibitor indomethacin (10 mu M; n=4). 3 The K+ channel blocker, tetraethylammonium (TEA, 10 mM) also significantly (P<0.001) reduced both the potency of carbacho l (ED50=1.97 +/- 0.14 nmol in presence of TEA) and the maximum relaxat ion (R-max=74.6 +/- 3.2% in presence of TEA, P<0.05, n=3). When TEA wa s added in the presence of L-NAME (n=4), there was a further significa nt (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 R-max was also significantly (P<0.05) reduced (74.6 +/- 4.2%). The ATP-sensitive K+ channel inhibitor, glibenclamide (10 mu M), had no effect on carba chol-induced relaxation (n=9). 4 High extracellular K+ (60 mM) signifi cantly (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 R- max was also significantly (P<0.01) reduced (control, 83.4 +/- 2.7%; h igh K+, 40.3 +/- 9.2%). The residual vasorelaxation to carbachol in th e presence of high K+ was abolished by L-NAME (100 mu M; n=5). In prep arations preconstricted with high K+, the potency of sodium nitropruss ide was not significantly different from that in preparations precontr acted with methoxamine, though the maximal response was reduced (62.4 +/- 3.4% high K+, n=7; 83.1 +/- 3.1% control, n=7). 5 In the presence of the cytochrome P450 inhibitor, clotrimazole (1 mu M, n=5 and 10 mu M, n=4), the dose-response curve to carbachol was significantly shifte d to the right 2 fold (P<0.05) and 4 fold (P<0.001) respectively, an e ffect which was further enhanced in the presence of L-NAME. R-max was significantly (P<0.01) reduced by the presence of 10 mu M clotrimazole alone, being 86.9 +/- 2.5% in its absence and 61.8 +/- 7.8% in its pr esence (n=6). 6 In the presence of the cell permeable analogue of cycl ic GMP, 8-bromo cyclic GMP (6 mu M), the inhibitory effects of L-NAME on carbachol-induced relaxation were substantially enhanced (ED50: L-N AME alone, 0.52 +/- 0.11 nmol, n=5; L-NAME + 8-bromo cyclic GMP, 1.42 +/- 0.28 nmol, n=7. R-max: L-NAME alone, 82.2 +/- 2.4%; L-NAME + 8-bro mo cyclic GMP, 59.1 +/- 1.8%. P<0.001). These results suggest that the magnitude of the NO-independent component of vasorelaxation is reduce d when functional cyclic GMP levels are maintained, suggesting that ba sal NO (via cyclic GMP) may modulate EDHF activity and, therefore, on loss of basal NO production the EDHF component of endothelium-dependen t relaxations becomes functionally greater. 7 The present investigatio n demonstrates that muscaranic receptor-induced vasorelaxation in the rat mesenteric arterial bed is mediated by both NO-dependent and indep endent mechanisms. The L-NAME-insensitive mechanism, most probably occ urs via activation of a K+ conductance and shows the characteristics o f EDHF-mediated responses. Finally, the results demonstrate that EDHF activity may become upregulated on inhibition of NO production and thi s may compensate for the loss of NO.