The detection of the non-M2 muscarinic receptor subtype in the rat heart atria and ventricles

  • Jaromir Myslivecek
  • Martin Klein
  • Martina Novakova
  • Jan Ricny
Original Article


Mammal heart tissue has long been assumed to be the exclusive domain of the M2 subtype of muscarinic receptor, but data supporting the presence of other subtypes also exist. We have tested the hypothesis that muscarinic receptors other than the M2 subtype are present in the heart as minor populations. We used several approaches: a set of competition binding experiments with pirenzepine, AFDX-116, 4-DAMP, PD 102807, p-F-HHSiD, AQ-RA 741, DAU 5884, methoctramine and tripinamide, blockage of M1 muscarinic receptors using MT7 toxin, subtype-specific immunoprecipitation experiments and determination of phospholipase C activity. We also attempted to block M1–M4 receptors using co-treatment with MT7 and AQ-RA 741. Our results show that only the M2 subtype is present in the atria. In the ventricles, however, we were able to determine that 20% (on average) of the muscarinic receptors were subtypes other than M2, with the majority of these belonging to the M1 subtype. We were also able to detect a marginal fraction (6 ± 2%) of receptors that, based on other findings, belong mainly to the M5 muscarinic receptors. Co-treatment with MT7 and AQ-RA 741 was not a suitable tool for blocking of M1–M4 receptors and can not therefore be used as a method for M5 muscarinic receptor detection in substitution to crude venom. These results provide further evidence of the expression of the M1 muscarinic receptor subtype in the rat heart and also show that the heart contains at least one other, albeit minor, muscarinic receptor population, which most likely belongs to the M5 muscarinic receptors but not to that of the M3 receptors.


Heart Immunoprecipitation MT7 mamba toxin Muscarinic receptors Non-M2 muscarinic receptor PLC activity 



We would like to thank Prof. Lincoln Potter from University of Miami for his advice concerning MT7 and M5 muscarinic receptor detection, Dr. Maria-Laura Bolognesi from the University of Bologna for her advice with tripinamide properties, Prof. C. Melchiorre from the same University for his generous gift of this compound and Prof. K. Ladinsky from the Istituto De Angeli, S.p.A for his generous gift of compound AFDX-116. We appreciate the gift of AQ-RA 741 from Boehringer Ingelheim, Biberach, Germany (Dr. Doods and Dr. Mueller). The gift of PD 102807 from Parke-Davis is greatly appreciated. The work was supported mainly by grant GAUK 519/07 from Grant Agency of Charles University and in part by grant GAUK 289/07 from the same Grant Agency. Another support was obtained from grants MSMT CR LC554, MSM0021620849, GACR 304/08/0256 and by the Research project AV0Z 50110509. We gratefully acknowledge the technical assistance by Mrs. Dana Ungerová, Romana Ondřejová, Olga Martínková and Eva Šimáková.


  1. Adem A, Asblom A, Johnasson G, Mbuga PM, Karlsson E (1988) Toxins from the venom of the green mamba dendroaspis angusticeps that inhibit the binding of quinuclinidil benzilate to muscarinic acetylcholine receptors. Biochim Biophys Acta 968:340–345PubMedCrossRefGoogle Scholar
  2. Akaike H (1974) A new look at the statistical model identification. IEEE Trans Autom Control 19:716–723CrossRefGoogle Scholar
  3. Birk E, Riemer RK (1992) Myocardial cholinergic signaling changes with age. Pediatr Res 31:601–605PubMedGoogle Scholar
  4. Bolognesi ML, Minarini A, Budriesi R, Cacciaguerra S, Chiarini A, Spampinato S, Tumiatti V, Melchiorre C (1998) Universal template approach to drug design: polyamines as selective muscarinic receptor Antagonists. J Med Chem 41:4150–4160PubMedCrossRefGoogle Scholar
  5. Borda ES, Perez Leiros C, Camusso JJ, Bacman S, Sterin-Borda L (1997) Differential cholinoceptor subtype-dependent activation of signal transduction pathways in neonatal versus adult rat atria. Biochem Pharmacol 53:959–967PubMedCrossRefGoogle Scholar
  6. Brodde O-E, Bruck H, Leineweber K, Seyfarth T (2001) Presence, distribution and physiological function of adrenergic and muscarinic receptor subtypes in the human heart. Basic Res Cardiol 96:528–538PubMedCrossRefGoogle Scholar
  7. Brown SL, Brown JH (1983) Muscarinic stimulation of phosphatidylinositol metabolism in atria. Mol Pharmacol 24:351–356PubMedGoogle Scholar
  8. Buckley NJ, Hulme EC, Birdsall NJ (1990) Use of clonal cell lines in the analysis of neurotransmitter receptor mechanisms and function. Biochim Biophys Acta 1055:43–53PubMedCrossRefGoogle Scholar
  9. Camusso JJ, Sterin-Borda L, Rodriguez M, Bacman S, Borda E (1995) Pharmacological evidence for the existence of different subtypes of muscarinic acetylcholine receptors for phosphoinositide hydrolysis in neonatal versus adult rat atria. J Lipid Mediat Cell Signal 12:1–10PubMedCrossRefGoogle Scholar
  10. Carsi J, Potter LT (2000) M1-toxin isotoxins from the green mamba (Dendroaspis Angusticeps) that selectively block M1 muscarinic receptors. Toxicon 38:187–198PubMedCrossRefGoogle Scholar
  11. Caulfield MP, Birdsal NJM (1998) International union of pharmacology. XVII. Classification of muscarinic acetylcholine receptors. Pharmacol Rev 50:279–290PubMedGoogle Scholar
  12. Choppin A, Stepan GJ, Loury DN, Watson N, Eglen RM (1999) Characterization of the muscarinic receptor in isolated uterus of sham operated and ovariectomized rats. Br J Pharmacol 127:1551–1558PubMedCrossRefGoogle Scholar
  13. Colecraft HM, Egamino JP, Sharma VK, Sheu S-S (1998) Signaling mechanisms underlying muscarinic receptor-mediated increase in contraction rate in cultured heart cells. J Biol Chem 73:32158–32166CrossRefGoogle Scholar
  14. Dhein S, van Koppen CJ, Brodde OE (2001) Muscarinic receptors in the mammalian heart. Pharmacol Res 44:161–182PubMedCrossRefGoogle Scholar
  15. Dobrev D, Knuschke D, Richter F, Wettwer E, Christ T, Knaut M, Ravens U (2002) Functional identification of m1 and m3 muscarinic acetylcholine receptors in human atrial myocytes: influence of chronic atrial fibrillation. Circulation 106(Suppl):11–154Google Scholar
  16. Doods HN, Willim KD, Boddeke HW, Entzeroth M (1993) Characterization of muscarinic receptors in guinea-pig uterus. Eur J Pharmacol 250:223–230PubMedCrossRefGoogle Scholar
  17. Doods HN, Entzeroth M, Ziegler H, Mayer N, Holzer P (1994) Pharmacological profile of selective muscarinic receptor antagonists on guinea-pig ileal smooth muscle. Eur J Pharmacol 253:275–281PubMedCrossRefGoogle Scholar
  18. Dwivedi Y, Pandey GN (1999) Repeated administration of dexamethasone increases phosphoinositide-specific phospholipase C activity and mRNA and protein expression of the phospholipase C beta 1 isozyme in rat brain. J Neurochem 73:780–790PubMedCrossRefGoogle Scholar
  19. Eglen RM, Nahorski SR (2000) The muscarinic M(5) receptor: a silent or emerging subtype? Br J Pharmacol 130:13–21PubMedCrossRefGoogle Scholar
  20. Fisher JT, Vincent SG, Gomeza J, Yamada M, Wess J (2004) Loss of vagally mediated bradycardia and bronchoconstriction in mice lacking M2 or M3 muscarinic acetylcholine receptors. FASEB J 18:711–713PubMedGoogle Scholar
  21. Ford APDW, Eglen RM, Whiting RL (1992) Analysis of muscarinic cholinoceptors mediating phosphoinositide hydrolysis in Guinea pig cardiac muscle. Eur J Pharmacol Mol Pharmacol Section 225:105–112CrossRefGoogle Scholar
  22. Gallo MP, Alloatti G, Carola E, Oberto A, Cesare Levi R (1993) —1 muscarinic receptors increase calcium current and phosphoinositide turnover in Guinea-pig ventricular cardiocytes. J Physiol (London) 471:41–60Google Scholar
  23. Gomeza J, Shannon H, Konstenis E, Felder C, Zhang L, Brodkin J, Grinberg A, Sheng H, Wess J (1999) Pronounced pharmacologic deficits in M2 muscarinic acetylcholine receptor knockout mice. Proc Natl Acad Sci USA 96:1692–1697PubMedCrossRefGoogle Scholar
  24. Hardouin SN, Richmond KN, Zimmerman A, Hamilton SE, Feigl EO, Nathanson NM (2002) Altered cardiovascular responses in mice lacking the M1 muscarinic acetylcholine receptor. J Pharmacol Exp Ther 301:129–137PubMedCrossRefGoogle Scholar
  25. Hassal CJS, Stanford SC, Burnstok G, Buckley NJ (1993) Co-expression of four muscarinic receptor genes by the intrinsic neurons of the rat and Guinea-pig heart. Neuroscience 56:1041–1048CrossRefGoogle Scholar
  26. Hellgren I, Mustafa A, Riazi M, Sulliman I, Sylvén C, Adem A (2000) Muscarinic M3 receptor subtype gene expression in the human heart. Cell Mol Life Sci 57:175–180PubMedCrossRefGoogle Scholar
  27. Hoower DB, Baisden RH, Ximoy SX (1994) Localization of muscarinic receptor MRNAs in rat heart and intrinsic cardiac ganglia by in situ hybridization. Circ Res 75:813–820Google Scholar
  28. Krejčí A, Tuček S (2002) Quantitation of MRNAs for M1 to M5 subtypes of muscarinic receptors in rat heart and brain cortex. Mol Pharmacol 61:1267–1272PubMedCrossRefGoogle Scholar
  29. Lazareno S, Gharagozloo P, Kuonen D, Popham A, Birdsall NJ (1998) Subtype-selective positive cooperative interactions between brucine analogues and acetylcholine at muscarinic receptors: radioligand binding studies. Mol Pharmacol 53:573–589PubMedGoogle Scholar
  30. Levey AI, Stormann TM, Brann MR (1990) Bacterial expression of human muscarinic receptor fusion proteins and generation of subtype-specific antisera. FEBS Lett 275:65–69PubMedCrossRefGoogle Scholar
  31. Levey AI, Kitt CA, Simonds WF, Price DL, Brann MR (1991) Identification and localization of muscarinic acetylcholine receptor proteins in brain with subtype-specific antibodies. J Neurosci 11:3218–3226PubMedGoogle Scholar
  32. Li M, Yasuda RP, Wall SJ, Wellstein A, Wolfe BB (1991) Distribution of M2 muscarinic receptors in rat brain using antisera selective for M3 receptors. Mol Pharmacol 40:28–35PubMedGoogle Scholar
  33. Luthin GR, Harkness J, Artymyshyn RP, Wolfe BR (1988) Antibodies to a synthetic peptide can be used to distinguish between muscarinic acetylcholine receptor binding sites in brain and heart. Mol Pharmacol 34:327–333PubMedGoogle Scholar
  34. Max SI, Liang JS, Potter LT (1993) Stable allosteric binding of m1-toxin to m1 muscarinic receptors. Mol Pharmacol 44:1171–1175PubMedGoogle Scholar
  35. Mayanil CSK, Richardson RM, Hosey MM (1991) Subtype-specific antibodies for muscarinic receptors. I. Characterization using transfected cells and avian and mammalian cardiac membranes. Mol Pharmacol 40:900–907PubMedGoogle Scholar
  36. Meyer T, Wellner-Kienitz MC, Biewald A, Bender K, Eickel A, Pott L (2001) Depletion of phosphatidylinositol 4,5-bisphosphate by activation of phospholipase C-coupled receptors causes slow inhibition but not desensitization of G protein-gated inward rectifier K+ current in atrial myocytes. J Biol Chem 276:5650–5658PubMedCrossRefGoogle Scholar
  37. Moscona-Amir E, Henis YI, Sokolovsky M (1989) Aging of rat heart myocytes disrupts muscarinic receptor coupling that leads to inhibition of CAMP accumulation and alters the pathway of muscarinic-stimulated phosphoinositide hydrolysis. Biochemistry 28:7130–7137PubMedCrossRefGoogle Scholar
  38. Mysliveček J, Říčný J, Kolář F, Tuček S (2003) The effects of hydrocortisone treatment on the rat heart muscarinic and adrenergic a1, b1 and b2 receptors, propranolol-resistant binding sites and on some consequent steps in intracellular signaling. NS Arch Pharmacol 368:366–376CrossRefGoogle Scholar
  39. Mysliveček J, Říčný J, Palkovits M, Kvetňanský R (2004) The effects of short-term immobilization stress on muscarinic receptors, beta-adrenoceptors and adenylyl cyclase In different heart regions. Ann NY Acad Sci 1018:315–322PubMedCrossRefGoogle Scholar
  40. Myslivecek J, Novakova M, Palkovits M, Krizanova O, Kvetnansky R (2006) Distribution of mRNA and binding sites of adrenoceptors and muscarinic receptors in the rat heart. Life Sci 79:112–120PubMedCrossRefGoogle Scholar
  41. Nadler E, Barnea O, Vidne B, Isakov A, Shavit G (1993) Positive inotropic effect in the heart produced by acetylcholine. J Basic Clin Physiol Pharmacol 4:229–248PubMedGoogle Scholar
  42. Oberhauser V, Schwertfeger E, Rutz T, Beyersdorf F, Rump LC (2001) Acetylcholine release in human heart atrium. Influence of muscarinic acetylcholine autoreceptors, diabetes, and age. Circulation 103:1638–1643PubMedGoogle Scholar
  43. Olinas MC, Maullu C, Adem A, Mulugeta E, Karlsson E, Onali P (2000) Inhibition of acetylcholine muscarinic M1 receptor function by the M1-selective ligand muscarinic toxin 7 (MT-7). Br J Pharmacol 131:447–452CrossRefGoogle Scholar
  44. Perez CC, Tobar ID, Jimenez E, Castaneda D, Rivero MB, Concepcion JL, Chiurillo MA, Bonfante-Cabarcas R (2006) Kinetic and molecular evidences that human cardiac muscle express non-M2 muscarinic receptor subtypes that are able to interact themselves. Pharmacol Res 54:345–355PubMedCrossRefGoogle Scholar
  45. Ponicke K, Heinroth-Hoffmann I, Brodde OE (2003) Demonstration of functional M3-muscarinic receptors in ventricular cardiomyocytes of adult rats. Br J Pharmacol 138:156–160PubMedCrossRefGoogle Scholar
  46. Reever CM, Ferrari-DiLeo G, Flynn DD (1997) The M5 (m5) receptor subtype: fact or fiction? Life Sci 60:1105–1112PubMedCrossRefGoogle Scholar
  47. Sharma VK, Colecraft HM, Wang DX, Levey AI, Grigorenko EV, Yeh HH, Sheu SS (1996) Molecular and functional identification of M1 muscarinic acetylcholine receptors in rat ventricular myocytes. Circ Res 79:86–93PubMedGoogle Scholar
  48. Shi H, Wang H, Wang Z (1999) Identification and characterization of multiple subtypes of muscarinic acetylcholine receptors and their physiological functions in canine hearts. Mol Pharmacol 55:497–507PubMedGoogle Scholar
  49. Sterin-Borda L, Echague AV, Leiros CP, Genaro A, Borda E (1995) Endogenous nitric oxide signalling system and the cardiac muscarinic acetylcholine receptor-inotropic response. Br J Pharmacol 115:1525–1531PubMedGoogle Scholar
  50. Sun LS, Huber F, Robinson RB, Bilezikian JP, Steinberg SF, Vulliemoz Y (1996) Muscarinic receptor heterogeneity in neonatal rat ventricular myocytes in culture. J Cardiovasc Pharmacol 27:455–461PubMedCrossRefGoogle Scholar
  51. Trendelenburg AU, Gomeza J, Klebroff W, Zhou H, Wess J (2003) Heterogeneity of presynaptic muscarinic receptors mediating inhibition of sympathetic transmitter release: a study with M2- and M4-receptor-deficient mice. Br J Pharmacol 138:469–480PubMedCrossRefGoogle Scholar
  52. Trendelenburg AU, Meyer A, Wess J, Starke K (2005) Distinct mixtures of muscarinic receptor subtypes mediate inhibition of noradrenaline release in different mouse peripheral tissues, as studied with receptor knockout mice. Br J Pharmacol 145:1153–1159PubMedCrossRefGoogle Scholar
  53. Wall SJ, Yasuda RP, Hory F, Flagg S, Martin BM, Ginnis EI, Wolfe BB (1991a) Production of antisera selective for M1 muscarinic receptors using fusion proteins: distribution of M1 receptors in rat brain. Mol Pharmacol 39:643–649PubMedGoogle Scholar
  54. Wall SJ, Yasuda RP, Li M, Wolfe BB (1991b) Development of an antiserum against M3 muscarinic receptors: distribution of M3 receptors in rat tissues and clonal cell lines. Mol Pharmacol 40:783–789PubMedGoogle Scholar
  55. Wang HZ, Han H, Zhang LM, Si H, Schram G, Nattel S, Wang ZG (2001) Expression of multiple subtypes of muscarinic receptors and cellular distribution in the human geart. Mol Pharmacol 59:1029–1036PubMedGoogle Scholar
  56. Wang Z, Shi H, Wang H (2004) Functional M3 muscarinic acetylcholine receptors in mammalian hearts. Br J Pharmacol 142:395–408PubMedCrossRefGoogle Scholar
  57. Wang H, Lu Y, Wan Z (2007) Function of cardiac M3 receptors. Autonom Autacoid Pharmacol 27:1–11CrossRefGoogle Scholar
  58. Willmy-Matthes P, Leineweber K, Wangemann T, Silber RE, Brodde OE (2003) Existence of functional M3-muscarinic receptors in the human heart. Naunyn Schmiedebergs Arch Pharmacol 368:316–319PubMedCrossRefGoogle Scholar
  59. Yang CM, Chen F-F, Sung T-C, Hsu H-F, Wu D (1993) Pharmacological characterization of muscarinic receptors in neonatal rat cardiomyocytes. Am J Physiol 265:C666–C673PubMedGoogle Scholar
  60. Yasuda RP, Ciesla W, Flores LR, Wall SJ, Li M, Satkus SA, Weisstein JS, Spagnola BV, Wolfe BB (1992) Development of antisera selective for M4 and M5 muscarinic cholinergic receptors: distribution of M4 and M5 receptors in rat brain. Mol Pharmacol 43:149–157Google Scholar

Copyright information

© Springer-Verlag 2008

Authors and Affiliations

  • Jaromir Myslivecek
    • 1
  • Martin Klein
    • 1
  • Martina Novakova
    • 1
  • Jan Ricny
    • 2
    • 3
  1. 1.Institute of Physiology, 1st Faculty of MedicineCharles UniversityPragueCzech Republic
  2. 2.Prague Psychiatric CentrePragueCzech Republic
  3. 3.Institute of PhysiologyAcademy of Sciences of the Czech RepublicPragueCzech Republic

Personalised recommendations