Multiple pathways underlying endothelium‐dependent relaxation in the rabbit isolated femoral artery

1 In isolated segments of the rabbit femoral artery stimulated with noradrenaline, both acetylcholine (1nM‐10μ M ) and the calcium ionophore A23187 (1nM‐100 μ M ) evoked endothelium‐dependent smooth muscle relaxation and hyperpolarization while bradykinin (0.01–100 nM) had no effect. 2 The nitric oxide synthase inhibitors, N G ‐nitro‐ L ‐arginine (L‐NOARG; 100 μ M ; 20 min) or N G ‐nitro‐ L ‐arginine methyl ester (L‐NAME; 100 μ M ; 20 min) each abolished the hyperpolarization and the majority of the relaxation to acetylcholine (maximal response reduced from 96.8 ±2.3% to 2.0 ± 1.4%). 3 The potassium channel blocker, glibenclamide (10 μ M ; 10 min) also abolished the change in membrane potential to acetylcholine but did not modify the smooth muscle relaxation. 4 In contrast, neither L‐NAME nor glibenclamide modified the comparable responses of the femoral artery to A23187, which were also unaffected by the cyclo‐oxygenase inhibitor, indomethacin (10 μ M ). 5 In artery segments stimulated with potassium chloride (25 mM), the maximal change in tension and membrane potential evoked by A23187 (100 μ M ) was significantly reduced from 95.0 ±4.5% and 23.0±2.0mV to 69.0 ±10.1% and 12.0±1.5 mV, respectively. Under these conditions L‐NAME further reduced the relaxation but not the accompanying hyperpolarization to A23187. 6 Endothelium‐denuded arterial segments sandwiched with endothelium‐intact ‘donor’ segments gave qualitatively similar relaxant responses to those described above for acetylcholine and A23187. 7 Exogenous nitric oxide (0.5–10μ M ) stimulated a transient relaxation in precontracted artery segments, which at concentrations above 5 μ M was accompanied by smooth muscle hyperpolarization (maximum 8.5 ± 3.2 mV; n = 4). The hyperpolarization but not the relaxation to nitric oxide was abolished by either glibenclamide or 25 mM potassium. 8 These data indicate that in the femoral artery, acetylcholine‐induced relaxation can be attributed solely to the release of nitric oxide from the endothelium, which then stimulates relaxation independently of a change in smooth muscle membrane potential. In contrast, both the relaxation and hyperpolarization evoked by A23187 appear to be mediated predominantly by nitric oxide‐independent pathways which appear to involve a diffusible factor released from the endothelium. The results suggest that this diffusible hyperpolarizing factor can be released from endothelial cells in the femoral artery by A23187 but not by acetylcholine.