title: Kinetic and molecular evidences that human cardiac muscle express non-M2 muscarinic receptor subtypes that are able to interact themselves.
abstract: In heart tissue five isoforms of the muscarinic acetylcholine receptor (mAChR) have been identified, designated m1-m5, of which only M1, M2 and M3 have functional evidences for their role in cardiac physiology. The present study was designed to explore the diversity of mAChR subtypes in human hearts and determine whether these subtypes are able to interact themselves. Expression of mRNAs encoding all five subtypes was readily detected by RT-PCR reaction in both atrial (A) and ventricle (V) samples. Immunoblotting, MABA and ELISA with subtype-specific antibodies confirmed the presence of M1, M2, M3, M4 and M5 proteins in membrane preparations from both A and V. Kinetic characterization using [(3)H]-QNB shown: (1) atrium had greater B(max) than did the ventricle, (2) [(3)H]-QNB behave as an allosteric modulator, inducing cooperativity at high and disclosing heterogeneity at low concentrations, (3) heterogenity was observed in pirenzepine, biperiden and tropicamide competition curves, being the high affinity sites compatible with M1 and M4 muscarinic receptor subtypes and (4) methoctramine competition curves in presence of selective muscarinic receptor subtypes antagonist displayed heterogeneity profile still maintaining cooperativity (n(H)>1), indicating muscarinic receptors subtypes are able to form homo- and hetero-oligomers. In conclusion, our results provide molecular and kinetic evidence for the presence of multiple subtypes of mAChR in human hearts, which are able to undergo discrete transitions from a non-cooperative kinetics of non-interacting monomers to a cooperative kinetics of interacting oligomers.
authors.affiliation: Unidad de Bioquímica Dr. José Antonio Moreno Yánes, Escuela de Medicina Pablo Acosta Ortiz, Universidad Centro Occidental Lisandro Alvarado, Avenida Libertador con Avenida Andrés Bello, Barquisimeto, Estado Lara, Código 3001, Venezuela.
authors.forename: Carmen Carlota Nello
authors.initials: CC
authors.lastname: Pérez
authors.forename: Iván Darío Bravo
authors.initials: ID
authors.lastname: Tobar
authors.forename: Eliézer
authors.initials: E
authors.lastname: Jiménez
authors.forename: Darwin
authors.initials: D
authors.lastname: Castañeda
authors.forename: Maria Belén
authors.initials: MB
authors.lastname: Rivero
authors.forename: Juan Luís
authors.initials: JL
authors.lastname: Concepción
authors.forename: Miguel Angel
authors.initials: MA
authors.lastname: Chiurillo
authors.forename: Rafael
authors.initials: R
authors.lastname: Bonfante-Cabarcas
mesh_terms: D000328:Adult; D001667:Binding, Competitive; D005786:Gene Expression Regulation / Q000187:drug effects; D006325:Heart Atria / Q000187:drug effects / Q000378:metabolism*; D006352:Heart Ventricles / Q000187:drug effects / Q000378:metabolism*; D006801:Humans; D066298:In Vitro Techniques; D008297:Male; D018721:Muscarinic Agonists / Q000494:pharmacology; D018727:Muscarinic Antagonists / Q000494:pharmacology; D009206:Myocardium / Q000378:metabolism*; D012333:RNA, Messenger / Q000378:metabolism; D011869:Radioligand Assay; D011976:Receptors, Muscarinic / Q000235:genetics / Q000378:metabolism*; D020133:Reverse Transcriptase Polymerase Chain Reaction
chemical_list: D018721:Muscarinic Agonists; D018727:Muscarinic Antagonists; D012333:RNA, Messenger; D011976:Receptors, Muscarinic