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Vol. 59, Issue 5, 1029-1036, May 2001
Research Center, Montreal Heart Institute, Montreal, Quebec, Canada
(H.W., H.H., L.Z., H.S., G.S., S.N., Z.W.); Department of Medicine,
Montreal Heart Institute and University of Montreal, Montreal, Quebec,
Canada (S.N., Z.W.); and Department of Pharmacology and Therapeutics,
McGill University, Montreal, Quebec, Canada (S.N.)
Five isoforms of the muscarinic acetylcholine receptor (mAChR) have
been identified by molecular cloning and designated
m1-m5, of which four correspond to the
functional subtypes M1, M2, M3, and
M4 in primary tissues. The presence of M5
receptors in tissues remains uncertain. The present study was designed
to explore the diversity and cellular distribution of various mAChR
subtypes in human hearts. Competition binding of
[N-methyl-3H]-scopolamine methyl
chloride with various mAChR antagonists yielded data consistent
with the presence of multiple subtypes (M1/M2/M3/M5) of mAChRs
in both human atrial (HA) and ventricular (HV) tissues. Expression of
mRNAs encoding all five subtypes was readily detected by reverse
transcription-polymerase chain reaction in both HA and HV samples.
Immunoblotting with subtype-specific antibodies confirmed the presence
of M1, M2, M3, and M5,
but not M4, proteins in membrane preparations from both HA
and HV. The protein levels of M1 and M2 were
comparable between HA and HV. Although the density of M3
appeared ~10-fold higher in HV than HA, that of M5 was
~5 times lower in HV than in HA. Positive immunostaining of single
ventricular myocytes by M1, M2, M3,
and M5 antibodies, respectively, was consistently detected.
Under confocal microscopy, M5 showed characteristic
localization to the intercalated discs, whereas other subtypes were
more evenly distributed throughout the surface membrane. Our results
provide the first molecular evidence for the presence of multiple
subtypes of mAChR, including endogenous M5 receptors, in
human hearts and suggest that different subtypes have different tissue
distributions and cellular localization.
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