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 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.
Choy
Wai Yee
WY
Department of Pharmacy, Faculty of Medicine, The Chinese University of Hong Kong, Shatin, Hong Kong SAR, PR China.
Wong
Yung Fat
YF
Kwan
Yiu Wa
YW
Au
Alice Lai Shan
AL
Lau
Wing Hung
WH
Raymond
Kenneth
K
Zuo
Joan Zhong
JZ
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*
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