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(Circulation Research. 1996;79:86-93.)
© 1996 American Heart Association, Inc.

Articles

Molecular and Functional Identification of m₁ Muscarinic Acetylcholine Receptors in Rat Ventricular Myocytes

Virendra K. Sharma, Henry M. Colecraft, David X. Wang, Allan I. Levey, Elena V. Grigorenko, Hermes H. Yeh, Shey-Shing Sheu

the Department of Pharmacology and Physiology (V.K.S., H.M.C., D.X.W., S.-S.S.), School of Medicine and Dentistry, University of Rochester (NY); the Department of Neurology (A.I.L.), Emory University School of Medicine, Atlanta, Ga; and the Department of Physiology and Pharmacology (E.V.G., H.H.Y.), Bowman-Gray School of Medicine, Winston-Salem, NC.

Correspondence to Dr Shey-Shing Sheu, Department of Pharmacology and Physiology, University of Rochester Medical Center, 601 Elmwood Ave, Rochester, NY 14642. E-mail sheus@pharmacol.rochester.edu.

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±7 to 118±8 nmol/L and the peak Ca²⁺ transient level from 519±32 to 640±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 m₁ mAChR–selective antagonist. In contrast, the m₂ 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 m₁ mAChRs are expressed on adult rat ventricular myocytes in addition to m₂ mAChRs. The results further suggest that m₁ mAChRs mediate the stimulatory responses on Ca²⁺ transients to high concentrations of cholinergic agonists seen in these cells.

Key Words: muscarinic acetylcholine receptors • rat heart myocytes • Ca²⁺ transients • carbachol

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