Role of nitric oxide production through M2-cholinergic receptors in cultured rat ventricular myocytes.

We previously reported that carbachol (CCh) caused the enhancement of NO production coinciding with negative chronotropy in cultured rat ventricular myocytes. In this study, we examined which subtype of muscarinic cholinergic receptor mediated these effects of CCh by measuring the NOx production with an HPLC-Griess reaction system and monitoring the beating with a Fotonic Sensor. The enhancement of NO production and negative chronotropy by 10(-4) M CCh stimulation were significantly inhibited by 10(-6) M atropine, 10(-6) M methoctramine, 3 x 10(-4) M L-NMMA, and 10(-5) M methylene blue. On the other hand, 10(-6) M pirenzepine and 10(-6) M HHSiD had no influence on the negative chronotropy by 10(-4) M CCh stimulation. Both 10(-6) M pirenzepine and 10(-6) M HHSiD suppressed the enhancement of NO production by 10(-4) M CCh stimulation slightly though not statistically. In addition, the m2 cholinergic receptor gene was expressed in our cell preparations, as demonstrated by reverse-transcriptase/PCR analysis. We concluded that M2-cholinergic receptor-mediated negative chronotropy may be due in part to activation of the NO-signaling pathway in cultured rat ventricular myocytes.