Molecular and Functional Identification of m1 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-transcriptase PCR, bands were obtained corresponding to the expected sizes for the m1 and m2 but not for the m3 to m5 mAChRs. The identity of the m1 and m2 bands was confirmed by single-cell PCR, restriction digest mapping, and Southern blot analysis. The presence of m1 and m2, but not m3, mAChR protein in these cells was shown by indirect immunofluorescence studies using subtype-specific antibodies. It was further investigated whether the identified m1 mAChR was responsible for the stimulatory effects on Ca2+ transients by high concentrations of carbachol (>10 μmol/L) known to occur in these cells. In pertussis toxin–treated ventricular myocytes electrically stimulated at 1 Hz, carbachol (300 μmol/L) increased the basal Ca2+ level from 96±...