title: Role of mitogen-activated protein kinase pathway in acetylcholine-mediated in vitro relaxation of rat pulmonary artery.
abstract: 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 microM) precontracted preparations. Inclusion of neostigmine (10 microM) caused a parallel leftward shift with an increase of the pD(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 microM, a nitric oxide synthase inhibitor), but not N(G)-nitro-D-arginine methyl ester (D-NAME, 20 microM), abolished acetylcholine-elicited relaxation. Moreover, 6-anilino-5,8-quinolinedione (LY 83583, 1 microM) and methylene blue (1 microM) (both are guanylate cyclase inhibitors) markedly attenuated acetylcholine-elicited relaxation. However, the presence of indomethacin (3 microM, a cyclo-oxygenase inhibitor), (-)-perillic acid (30 microM, a p21(ras) blocker), 2-[2'-amino-3'-methoxy-phenyl]-oxana-phthalen-4-one (PD 98059) (10 microM, a p42/p44 mitogen-activated protein kinase inhibitor), 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole (SB 203580) (1 microM, a p38 mitogen-activated protein kinase blocker), wortmannin (500 nM, a phosphatidylinositol-3 kinase inhibitor) and genistein (10 microM, 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.
authors.affiliation: Department of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, PR China.
authors.forename: Wai Yee
authors.initials: WY
authors.lastname: Choy
authors.forename: Yung Fat
authors.initials: YF
authors.lastname: Wong
authors.forename: Yiu Wa
authors.initials: YW
authors.lastname: Kwan
authors.forename: Alice Lai Shan
authors.initials: AL
authors.lastname: Au
authors.forename: Wing Hung
authors.initials: WH
authors.lastname: Lau
authors.forename: Kenneth
authors.initials: K
authors.lastname: Raymond
authors.forename: Joan Zhong
authors.initials: JZ
authors.lastname: Zuo
mesh_terms: D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D002217:Carbachol / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004730:Endothelium, Vascular / Q000502:physiology; D006162:Guanylate Cyclase / Q000502:physiology; D066298:In Vitro Techniques; D007213:Indomethacin / Q000494:pharmacology; D008297:Male; D020928:Mitogen-Activated Protein Kinases / Q000502:physiology*; D009388:Neostigmine / Q000494:pharmacology; D009569:Nitric Oxide / Q000502:physiology; D019869:Phosphatidylinositol 3-Kinases / Q000502:physiology; D016283:Proto-Oncogene Proteins p21(ras) / Q000502:physiology; D011651:Pulmonary Artery / Q000187:drug effects* / Q000502:physiology; D051381:Rats; D017207:Rats, Sprague-Dawley; D011976:Receptors, Muscarinic / Q000145:classification; D014664:Vasodilation / Q000187:drug effects*
chemical_list: D011976:Receptors, Muscarinic; D009569:Nitric Oxide; D009388:Neostigmine; D002217:Carbachol; D019869:Phosphatidylinositol 3-Kinases; D020928:Mitogen-Activated Protein Kinases; D016283:Proto-Oncogene Proteins p21(ras); D006162:Guanylate Cyclase; D000109:Acetylcholine; D007213:Indomethacin