Abstract

Small molecules interfering with Rac1 activation are considered as potential drugs and are already studied in animal models. A widely used inhibitor without reported attenuation of RhoA activity is NSC23766 [(N⁶-[2-[[4-(diethylamino)-1-methylbutyl]amino]-6-methyl-4-pyrimidinyl]-2-methyl-4,6-quinolinediamine trihydrochloride]. We found that NSC23766 inhibits the M₂ muscarinic acetylcholine receptor (M₂ mAChR)-induced Rac1 activation in neonatal rat cardiac myocytes. Surprisingly, NSC27366 concomitantly suppressed the carbachol-induced RhoA activation and a M₂ mAChR-induced inotropic response in isolated neonatal rat hearts requiring the activation of Rho-dependent kinases. We therefore aimed to identify the mechanisms by which NSC23766 interferes with the differentially mediated, M₂ mAChR-induced responses. Interestingly, NSC23766 caused a rightward shift of the carbachol concentration response curve for the positive inotropic response without modifying carbachol efficacy. To analyze the specificity of NSC23766, we compared the carbachol and the similarly G_iβγ-mediated, adenosine-induced activation of G_i protein–regulated potassium channel (GIRK) channels in human atrial myocytes. Application of NSC23766 blocked the carbachol-induced K⁺ current but had no effect on the adenosine-induced GIRK current. Similarly, an adenosine A₁ receptor-induced positive inotropic response in neonatal rat hearts was not attenuated by NSC23766. To investigate its specificity toward the different mAChR types, we studied the carbachol-induced elevation of intracellular Ca²⁺ concentrations in human embryonic kidney 293 (HEK-293) cells expressing M₁, M₂, or M₃ mAChRs. NSC23766 caused a concentration-dependent rightward shift of the carbachol concentration response curves at all mAChRs. Thus, NSC23766 is not only an inhibitor of Rac1 activation, but it is within the same concentration range a competitive antagonist at mAChRs. Molecular docking analysis at M₂ and M₃ mAChR crystal structures confirmed this interpretation.