Molecular and functional identification of m(1) muscarinic acetylcholine receptors in rat ventricular myocytes

The expression of muscarinic acetylcholine receptor (mAChR) subtypes in freshly isolated adult rat ventricular myocytes was investigated by reverse transcription of cellular mRNA followed by amplification of cDNA using the polymerase chain reaction (PCR). After reverse-trans criptase PCR, bands were obtained corresponding to the expected sizes for the m(1) and m(1) but not for the m(3) to m(5) mAChRs. The identity of the m(1) and m(2) bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m(1) and m(2), but not m(3), mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified mi mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol (>10 mu mol/L) known to occur in these cells. In pertussis toxin-treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 mu mol/L) increased the basal Ca2+ level from 96+/-7 to 118+/-8 nmol/L and the peak Ca2+ transient level from 519+/-32 to 630+/-36 nmol/L (mean+/-SEM, P<.05 for both, n=8). These effects of carbachol on Ca2+ transients were antagonized by 10 nmol/L pirenzepine, an m(1) mAChR-selective antagonist. In contrast, the m(2) mAChR-selective antagonist methoctramine(up to 100 nmol/L) did not inhibit the response. These results are the first to use single-cell PCR to probe cardiomyocyte-specific gene expression and indicate that m(1) mAChRs are expressed on adult rat ventricular myocytes in addition to m(2) mAChRs. The results further suggest that m(1) mAChRs mediate the stimulatory responses on Ca2+ transients to high concentrations of cholinergic agonists seen in these cells.