Immunohistochemical and functional studies for M3 muscarinic receptors and cyclo-oxygenase-2 expressed in the mouse atrium

1. In mouse atrium, M₂ and M₃ muscarinic receptors (M₂R and M₃R) are involved in biphasic (negative and positive) inotropic actions of muscarinic agonists, and the positive inotropic action is reduced by indomethacin. The aim of our study was to determine the localization of M₂R, M₃R and cyclo-oxygenase (COX) in mouse atrium and to characterize muscarinic receptor-mediated positive inotropy.
2. M₂R immunoreactivity was found only on atrial myocardium, but M₃R immunoreactivity was localized on both the myocardium and endocardial endothelium. COX-1 and COX-2 immunoreactivities were identified in both myocardial and endocardial endothelium.
3. In electrically stimulated left atria, carbachol caused M₂R-mediated negative inotropy followed by M₃R-mediated positive inotropy. Removal of atrial endothelium reduced the positive inotropy without affecting the negative inotropy, suggesting that stimulation of endothelial M₃R mediates the positive inotropy.
4. N-[2-(cyclohexyloxy)-4-nitrophenyl]-methanesulfonamide (NS398, COX-2 inhibitor) decreased the carbachol-induced positive inotropy; however, 5-(4-chlorophenyl)-1-(4-methoxyphenyl)-3-trifluoromethylpyrazole (SC560, COX-1 inhibitor), 1-[[4,5-bis(4-methoxyphenyl)-2-thiazolyl]carbonyl]-4-methylpiperazine (FR122047, COX-1 inhibitor) and l-nitroarginine methylester did not affect the inotropic response.
5. M₃R activation caused positive chronotropy in spontaneously beating right atria when M₂R-mediated negative chronotropy was suppressed and rate of contraction was low, <350 beats min⁻¹.
6. Our results indicate that although M₃Rs are located on both myocardial cells and endocardial endothelial cells, only endothelial M₃Rs mediate positive inotropy in response to muscarinic agonists via activation of COX-2 in the mouse atrium. M₃R-mediated positive chronotropy counteracting M₂R-mediated negative chronotropy was also demonstrated.