Enhancement of nitric oxide production by methylecgonidine in cultured neonatal rat cardiomyocytes

1 In the present experiments, we investigated the effects of methylecgonidine (MEG) on nitric oxide (NO) production in cultured neonatal rat cardiomyocytes. Incubation of cultured cardiomyocytes with carbachol or MEG for 48 h significantly enhanced NO production. No release was increased from 1.48+/-0.13 muM (mg protein)(-1) for control to 5.73+/-0.19 muM (mg protein)(-1) for 1 Aim carbachol treated cells (P<0.001). In addition, incubation with 1 Aim MEG enhanced NO production to 5.55+/-0.28 muM (mg protein)(-1). The effects of MEG on NO production were concentration-dependent. The muscarinic antagonist atropine prevented the enhancement of NO production induced by carbachol or MEG. Compared to MEG-induced NO production, cocaine was much less potent. 2 The enhancement of NO production by carbachol or MEG was even greater in cultured cardiomyocytes transfected with the M-2 cDNA. After 48-h incubation with 1 muM carbachol or 1 muM MEG, NO production was increased by 6.5 and 6.7 fold, respectively, in cardiomyocytes overexpressing M, receptors. Coincubation with atropine or N-G-vitro-L-arginine methyl ester abolished the enhancement of NO production. In contrast, NO production enhanced by carbachol or MEG in M-1- or M-2-transfected cardiomyocytes was similar to the level in non-transfected cells. 3 Western blot analysis showed that the protein levels of M-1, M-2, and M-3 were significantly increased in cardiomyocytes transfected with the receptor cDNAs, but MEG had no effect on the expressions. It is interesting that both carbachol and MEG caused a significant increase in constitutive endothelial NO synthase (eNOS) only in M-2-transfected cardiomyocytes, not in nontransfected, M-1- or M-2-transfected cells. Again, atropine blocked the MEG-produced induction of eNOS. 4 Our data demonstrate that MEG significantly enhanced NO production in cultured cardiomyocytes and that the enhancement of NO production may result from MEG stimulation of muscarinic M-2 receptors.