Role of mitogen-activated protein kinase pathway in acetylcholine-mediated in vitro relaxation of rat pulmonary artery

This study was designed to characterise the muscarinic receptor subtype responsible for acetylcholine-mediated in vitro pulmonary artery relaxation in rats and the importance of the presence of neostigmine (an anti-cholinesterase) during receptor characterisation. Cumulative administration of acetylcholine elicited concentration-dependent relaxation of phenylephrine (1 μM) precontracted preparations. Inclusion of neostigmine (10 μM) caused a parallel leftward shift with an increase of the p D 2 value (7.09 vs. 6.43) of the concentration–response curve of acetylcholine. The magnitude of maximum relaxation, however, was not affected. Using a range of conventional muscarinic receptor antagonists (atropine, pirenzepine, methoctramine, p-FHHSiD and tropicamide) and the highly selective Green Mamba muscarinic toxins (MT-3 and MT-7), it was found that muscarinic M 3 receptors are probably responsible for endothelium-dependent relaxation of the pulmonary artery upon acetylcholine challenge. Preincubation with N G-nitro- l-arginine methyl ester ( l-NAME, 20 μM, a nitric oxide synthase inhibitor), but not N G-nitro- d-arginine methyl ester ( d-NAME, 20 μM), abolished acetylcholine-elicited relaxation. Moreover, 6-anilino-5,8-quinolinedione (LY 83583, 1 μM) and methylene blue (1 μM) (both are guanylate cyclase inhibitors) markedly attenuated acetylcholine-elicited relaxation. However, the presence of indomethacin (3 μM, a cyclo-oxygenase inhibitor), (−)-perillic acid (30 μM, a p21 ras blocker), 2-[2′-amino-3′-methoxy-phenyl]-oxana-phthalen-4-one (PD 98059) (10 μM, a p42/p44 mitogen-activated protein kinase inhibitor), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1 H-imidazole (SB 203580) (1 μM, a p38 mitogen-activated protein kinase blocker), wortmannin (500 nM, a phosphatidylinositol-3 kinase inhibitor) and genistein (10 μM, a tyrosine kinase blocker) failed to alter acetylcholine-provoked pulmonary arterial relaxation. These results suggest that acetylcholine caused pulmonary arterial relaxation through the activation of muscarinic M 3 receptors in the endothelium. Moreover, the p21 ras/mitogen-activated protein kinase pathway seems to play no role in mediating acetylcholine-elicited relaxation.