Involvement of M3 muscarinic receptors in ACh-induced increase in membrane-associated RhoA of rat bronchial smooth muscle.
It is known that RhoA is translocated from cytoplasm to cell membrane in bronchial smooth muscle when activated by acetylcholine (ACh) stimulation. In the present study, the effects of selective muscarinic receptor antagonist methoctramine, AF-DX116 (for M(2)) and 4-diphenylacetoxy N-methylpiperidine (4-DAMP; for M(3)) on the ACh-induced rat bronchial smooth muscle contraction and increase in membrane-associated RhoA were investigated to elucidate the muscarinic receptor subtype participating in these responses. To evaluate ACh-induced contraction of bronchial smooth muscle, bronchial ring of rat was prepared, suspended in an organ bath and the tension was measured isometrically. To quantify the ACh-induced increase in membrane-associated RhoA protein, western blot analysis was performed by using homogenates of membrane and cytosolic fractions of the rat bronchi. The muscarinic M(2) and M(3) receptors were detected by using RT-PCR in rat bronchial smooth muscle. Both the ACh-induced smooth muscle contraction and increase in membrane-associated RhoA were markedly inhibited by 4-DAMP, but not by methoctramine or AF-DX116. In conclusion, these results indicated contraction for the first time that the activation of RhoA occurs via M(3) receptor in rat bronchial smooth muscle.
Sakai
Hiroyasu
H
Department of Pharmacology, School of Pharmacy, Hoshi University, Tokyo, Japan. sakai@hoshi.ac.jp
Shirai
Tsuyoshi
T
Yamamoto
Maki
M
Chiba
Yoshihiko
Y
Misawa
Miwa
M
D000109:Acetylcholine / Q000494:pharmacology*; D000818:Animals; D001980:Bronchi / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D002462:Cell Membrane / Q000502:physiology; D018678:Cholinergic Agents / Q000494:pharmacology; D008297:Male; D009119:Muscle Contraction / Q000187:drug effects; D009130:Muscle, Smooth / Q000187:drug effects* / Q000378:metabolism / Q000502:physiology; D051381:Rats; D017208:Rats, Wistar; D043585:Receptor, Muscarinic M2 / Q000502:physiology; D043587:Receptor, Muscarinic M3 / Q000502:physiology*; D020742:rhoA GTP-Binding Protein / Q000502:physiology*
D018678:Cholinergic Agents; D043585:Receptor, Muscarinic M2; D043587:Receptor, Muscarinic M3; D020742:rhoA GTP-Binding Protein; D000109:Acetylcholine