Characterization and modulation of EDHF-mediated relaxations in the rat isolated superior mesenteric arterial bed.

Abstract

1. 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). 2. 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 (1-NAME, 100 microM) 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 microM; n = 4). 3. 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 microM), had no effect on carbachol-induced relaxation (n = 9). 4. 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 microM; 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). 5. In the presence of the cytochrome P450 inhibitor, clotrimazole (1 microM, n = 5 and 10 microM, 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 microM clotrimazole alone, being 86.9 +/- 2.5% in its absence and 61.8 +/- 7.8% in its presence (n = 6). 6. In the presence of the cell permeable analogue of cyclic GMP, 8-bromo cyclic GMP (6 microM), 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 results suggest that the magnitude of the NO-independent component of vasorelaxation is reduced when functional cyclic GMP levels are maintained, suggesting that basal NO (via cyclic GMP) may modulate EDHF activity and, therefore, on loss of basal NO production the EDHF component of endothelium-dependent relaxations becomes functionally greater. 7. The present investigation demonstrates that muscaranic receptor-induced vasorelaxation in the rat mesenteric arterial bed is mediated by both NO-dependent and independent mechanisms. The L-NAME-insensitive mechanism, most probably occurs via activation of a K+ conductance and shows the characteristics of EDHF-mediated responses. Finally, the results demonstrate that EDHF activity may become upregulated on inhibition of NO production and this may compensate for the loss of NO.