Molecular and Cellular Biochemistry

, Volume 163, Issue 1, pp 113–123 | Cite as

β2-Adrenoceptor activation by zinterol causes protein phosphorylation, contractile effects and relaxant effects through a cAMP pathway in human atrium

  • Alberto J. Kaumann
  • Louise Sanders
  • James A. Lynham
  • Sabine Bartel
  • Meike Kuschel
  • Peter Karczewski
  • Ernst-Georg Krause
Part I: Cardiac Development and Regulation

Abstract

Evidence from ventricular preparations of cat, sheep, rat and dog suggests that both β1-adrenoceptors (β1AR) and β2-adrenoceptors (β2AR) mediate positive inotropic effects but that only β1AR do it through activation of a cAMP pathway. On the other hand, our evidence has shown that both β1 AR and β2 AR hasten relaxation of isolated human myocardium consistent with a common cAMP pathway. We have now investigated in the isolated human right atrial appendage, a tissue whose β-AR comprise around 2/3 of β1AR and 1/3 of β2AR, whether or not β2AR-mediated effects occur via activation of a cAMP pathway. We carried out experiments on atria obtained from patients without advanced heart failure undergoing open heart surgery. To activate β2AR, we used the β2AR-selective ligand zinterol. Experiments were carried out on paced atrial strips (1 Hz) and tissue homogenates and membrane particles. Zinterol caused positive inotropic and lusitropic (i.e. reduction of t1:2 of relaxation) effects with EC50 values of 3 and 2 nM, respectively. The zinterol-evoked effects were unaffected by the β AR-selective antagonist CGP 20712A (300 nM) but blocked surmountably by the β2AR-selective antagonist ICI 118551 (50 nM) which reduced both EC50 values to 1 μM. Zinterol stimulated adenylyl cyclase activity with an EC50 of 30 nM and intrinsic activity of 0.75 with respect to (−)-isoprenaline (600 μM); the effects were resistant to blockade by CGP 20712A (300 nM) but antagonised surmountably by ICI 118551 (50 nM). Zinterol bound to membrane PAR labelled with (−)-[125I] cyanopindolol with higher affinity for β2AR than for β- 1 AR; the binding to β2AR but not to β- BAR was reduced by GTPyS (10 μM). In the presence of CGP 20712A (300 nM) (−)-isoprenaline (400 μM); (to activate both β1AR and β2AR maximally) and zinterol (10 μM); increased contractile force 3.4-fold and 2.5-fold respectively and reduced relaxation tut by 32% and 18% respectively. These effects of (−)-isoprenaline and zinterol were associated (5 min incubation) with phosphorylation (pmol P/mg supernatant protein) of troponin I and C-protein to values of 8.4 ± 2.0 vs 12.4 ± 2.3 and 10.1 ± 2.5 vs 8.6 ± 1.6 respectively. (−)-Isoprenaline and zinterol also caused phosphorylation of phospholamban (1.8 ± 0.3 vs 0.4 ± 0.1 pmol P/mg respectively) specifically at serine residues. We conclude that in human atrial myocardium activation of both β1AR and β2AR leads to cAMP-dependent phosphorylation of proteins involved in augmenting both contractility and relaxation.

Key words

human atrium β2-adrenoceptors receptor binding zinterol adenylyl cyclase stimulation atrial relaxation and contraction protein phosphorylation troponin I C-protein phospholamban 

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References

  1. 1.
    Kaumann AJ, Hall JA, Murray KJ, Wells FC, Brown MJ: A comparison of the effects of adrenaline and noradrenaline on human hearts: the role of β1- and β2-adrenoceptors in the stimulation of adenylate cyclase and contractile force. Eur Heart J 10 (Suppl B): 29–37, 1989Google Scholar
  2. 2.
    Buxton BF, Jones CR, Molenaar P, Summers RJ: Characterization and radioautographic localization of β-adrenoceptor subtypes in human cardiac tissues. Br J Pharmacol 92: 299–310, 1987PubMedGoogle Scholar
  3. 3.
    Kaumann AJ, Lynham JA, Sanders L, Brown AM, Molenaar P: Contribution of differential efficacy to the pharmacology of human β1 — and β2-adrenoceptors. Pharmacol Commun 6: 215–222, 1995Google Scholar
  4. 4.
    Gille E, Lemoine H, Ehle B, Kaumann AJ: The affinity of (−)-propranolol for β1- and β2-adrenoceptors of human heart. Differential antagonism of the positive inotropic effects and adenylate cyclase stimulation by (−)-noradrenaline and (−)-adrenaline. NaunynSchmiedeberg's Arch Pharmacol 331: 60–70, 1985Google Scholar
  5. 5.
    Kaumann AJ, Lemoine H: β2-adrenoceptor-mediated positive inotropic effects of adrenaline in human ventricular myocardium. Quantitative discrepancies between binding and adenylate cyclase stimulation. Naunyn-Schmiedeberg's Arch Pharmacol 335: 403–411, 1987Google Scholar
  6. 6.
    Kaumann AJ: Adrenaline and noradrenaline increase contractile force of human ventricle through both β1- and β2-adrenoceptors. Biomed Biochem Acta 46: S 411-S 416, 1987Google Scholar
  7. 7.
    Bristow MR, Hershberger RE, Port JD, Minobe W, Rasmussen R: β1 — and β2-adrenergic receptor-mediated adenylate cyclase stimulation in nonfailing and failing human ventricular myocardium. Mol Pharmacol 35:295–303, 1989PubMedGoogle Scholar
  8. 8.
    Green SA, Holt BD, Liggett SB: β1- and β2-adrenergic receptors display subtype-selective coupling to Gs. Mol Pharmacol 41: 889–893, 1992PubMedGoogle Scholar
  9. 9.
    Levy FO, Zhu X, Kaumann AJ, Birnbaumer L: Efficacy of β1-adrenergic receptors is lower than that of β2 adrenergic receptors. Proc Natl Acad Sci USA 90: 10798–10802, 1993PubMedGoogle Scholar
  10. 10.
    Hall JA, Kaumann AJ, Brown MJ Selective β1-adrenoceptor blockade enhances positive inotropic effects of endogenous catecholamines through β2-adrenoceptors in human atrium. Circ Res 66: 1610–1623, 1990PubMedGoogle Scholar
  11. 11.
    Lemoine H, Kaumann AJ: Regional differences of β1- and β2-adrenoceptor-mediated functions in feline heart. A β2-adrenoceptor-mediated positive inotropic effect possibly unrelated to cyclic AMP. Naunyn-Schmiedeberg's Arch Pharmacol 344: 56–69, 1991CrossRefGoogle Scholar
  12. 12.
    Reuter H: Calcium channel modulation by neurotransmitters, enzymes and drugs. Nature 301: 569–574, 1983PubMedGoogle Scholar
  13. 13.
    Katz AM: Interplay between inotropic and lusitropic effects of cyclic adenosine monophosphate on the myocardial cell. Circulation 82; (suppl 1): I-7-I-11, 1990Google Scholar
  14. 14.
    Kaumann AJ, Lemoine H, Schwederski-Menke U, Ehle B: Relations between β- adrenoceptor occupancy and increases of contractile force and adenylate cyclase activity induced by catecholamines in human ventricular myocardium. Acute desensitization and comparison with feline ventricle. Naunyn-Schmiedeberg's Arch Pharmacol 339: 99–112, 1989Google Scholar
  15. 15.
    Borea PA, Amerini S, Masini I, Cerbai E, Ledda F, Mantelli L, Varani K, Mugelli A: β1- and β2-adrenoceptors in sheep cardiac ventricular muscle. J Mol Cell Cardiol 24: 753–764, 1992CrossRefPubMedGoogle Scholar
  16. 16.
    Xiao RP, Lakatta EG: β1-Adrenoceptor stimulation and β2-adrenoceptor stimulation differ in their effects on contraction,cytosolic Ca2+, and Cat. current in single rat ventricular cells. Circ Res 73: 286–300, 1993PubMedGoogle Scholar
  17. 17.
    Xiao RP, Hohl C, Altschuld R, Jones LR, Livington B, Ziman B, Tantini B, Lakatta EG: β2-Adrenergic receptor-stimulated increase in cAMP in rat heart cells is not coupled to changes in Ca2+ dynamics, contractility, or phospholamban phosphorylation. J Biol Chem 269: 1–6, 1994PubMedGoogle Scholar
  18. 18.
    Xiao RP, Xiangwu J, Lakatta EG: Functional coupling of the β2 adrenoceptor to a pertussis toxin-sensitive G protein in cardiac myocytes. Mol Pharmacol 47: 322–329, 1995PubMedGoogle Scholar
  19. 19.
    Altschuld R, Starling RC, Hamlin RL, Billman GE, Hensley J, Castillo L, Fertel RH, Hohl CM, Robitaille P-ML, Jones L, Xiao RP, Lakatta EG: Response of failing canine and human heart cells to β2- adrenergic stimulation. Circulation 92: 1612–1618, 1995PubMedGoogle Scholar
  20. 20.
    Gwee MCE, Non MW, Raper C, Rodger IW: Pharmacological actions of a new β-adrenoceptor agonist MJ-9184- I, in anaesthetized cats. Br J Pharmacol 46: 375–385, 1972PubMedGoogle Scholar
  21. 21.
    Minneman KP, Hegstrand LR, Molinoff PB: The pharmacological specificity of beta-1 and beta-2 adrenergic receptors in rat heart and lung in vivo. Mol Pharmacol 16: 21–33, 1979PubMedGoogle Scholar
  22. 22.
    Brodde O-E, Karad K, Zerkowski H-R, Rohm N, Reidemeister JC: Coexistence of β1- and β2 adrenoceptors in human right atrium. Direct identification by (±)-[125I] iodocyanopindolol. Circ Res 53: 752–758, 1983PubMedGoogle Scholar
  23. 23.
    Bristow MR, Hershberger RE, Port JD, Gilbert EM, Sandoval A, Rasmussen R, Cates AE, Feldman AM: β-Adrenergic pathways in nonfailing and failing human ventricular myocardium. Circulation 82 (suppl 1) I-12-I-25, 1990Google Scholar
  24. 24.
    Brodde O-E, O'Hara N, Zerkowski H-R, Rohm N: Human cardiac β-adrenoceptors: both β1- and β2-adrenoceptors are functionally coupled to adenylate cyclase in right atrium. J Cardiovasc Pharmacol 6: 1184–1191, 1984PubMedGoogle Scholar
  25. 25.
    Dooley DJ, Bittiger H, Reymann NC: CGP 20712A; a useful tool for quantifying β1- and β2-adrenoceptors. Eur J Pharmacol 130: 137–140, 1986CrossRefPubMedGoogle Scholar
  26. 26.
    Kaumann AJ, Birnbaumer L: Characteristics of the adrenergic receptor coupled to myocardial adenylyl cyclase: stereospecificity for ligands and determination of apparent affinity constants for β- blockers. J Biol Chem 249: 7874–7885, 1974PubMedGoogle Scholar
  27. 27.
    Salomon Y, Londos C, Rodbell M: A highly sensitive adenylate cyclase assay. Anal Biochem 58: 541–548, 1974PubMedGoogle Scholar
  28. 28.
    Bradford M: A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding. Anal Biochem 72: 248–254, 1976CrossRefPubMedGoogle Scholar
  29. 29.
    Karczewski P, Bartel S, Krause E-G: Differential sensitivity to isoproterenol of troponin I and phospholamban phosphorylation in isolated rat heart. Biochem J 266: 115–122, 1990PubMedGoogle Scholar
  30. 30.
    Lowry OH, Rosebrough NJ, Farr AL, Randall RJ: Protein measurement with folin phenol reagent. J Biol Chem 193: 265–275, 1951PubMedGoogle Scholar
  31. 31.
    Drago, GA, Colyer J: Discrimination between two sites of phosphorylation on adjacent amino acids by phosphorylation site-specific antibodies to phospholamban. J Biol Chem 269: 25073–25077, 1994PubMedGoogle Scholar
  32. 32.
    Wegener AD, Simmerman HKB, Lindemann JP, Jones LR: Phospholamban phosphorylation in intact ventricles. Phosphorylation of serine 16 and threonine 17 in response to beta-adrenergic stimulation. J Biol Chem 264: 11468–11474, 1989PubMedGoogle Scholar
  33. 33.
    Karczewski P, Bartel S, Haase H, Krause E-G: Isoproterenol induces both CAMP-and calcium-dependent phosphorylation of phospholamban in canine heart in vivo. Biomed Biochem Acta 46: 433–439, 1987Google Scholar
  34. 34.
    Colyer J, Wang JH: Dependence of cardiac sarcoplasmic reticulum pump activity on the phosphorylation status of phospholamban. J Biol Chem 255:17486–17493, 1991Google Scholar
  35. 35.
    Xu A, Hawkins C, Narayanan N: Phosphorylation and activation of the Ca2+/calmodulin-dependent protein kinase. J Biol Chem 268: 8394–8397, 1993PubMedGoogle Scholar
  36. 36.
    Talosi L, Edes I, Kranias EG: Intracellular mechanisms mediating reversal of beta-adrenergic stimulation in intact beating hearts. Am J Physiol 264: H791-H797, 1993PubMedGoogle Scholar
  37. 37.
    Karczewski P, Kuschel M, Baltas LG, Bartel S, Krause EG: Site-specific phosphorylation of a phospholamban peptide by cyclic nucleotide-and Ca 2+/calmodulin-dependent protein kinases of cardiac sarcoplasmic reticulum. Basic Res Cardiol (in press), 1996Google Scholar
  38. 38.
    Bartel S, Stein B, Eschenhagen T, Mende U, Neumann J, Schmitz W, Krause EAG, Karczewski P, Scholz H: Protein phosphorylation in isolated trabeculae from nonfailing and failing human heart. Mol Cell Biochem (in press), 1996Google Scholar
  39. 39.
    Koss KL, Ponniah S, Jones WK, Grupp IL, Kranias EG: Differential phospholamban gene expression in murine cardiac compartments. Circ Res 77: 342–353, 1995PubMedGoogle Scholar
  40. 40.
    Kranias EG: Regulation of Ca2+ transport by phosphoprotein phosphatase activity associated with sarcoplasmic reticulum. J Biol Chem 260:11006–11010, 1985PubMedGoogle Scholar
  41. 41.
    Garvey JL, Kranias EG, Solaro RJ: Phosphorylation of C-protein, troponin I and phospholamban in isolated rabbit hearts. Biochem J 249:709–714, 1988PubMedGoogle Scholar
  42. 42.
    Herzig JW, Riiegg JC: Investigations on glycerinated cardiac muscle fibres in relation to the problem of regulation of cardiac contractility — effects of Ca2+ and CAMP. Basic Res Cardiol 75: 26–33, 1980PubMedGoogle Scholar
  43. 43.
    Feldman AM, Ray PE, Silan CM, Mercer JA, Minrobe W. Bristow MR: Selective gene expression in failing human heart. Circulation 83:1866–1872, 1993Google Scholar
  44. 44.
    Arai M, Alpert NR, MacLennan DH, Barton P, Periasamy M: Alterations in sarcoplasmic reticulum gene expression in human heart failure: a possible mechanism for alterations in systolic and diastolic properties of the failing myocardium. Circ Res. 72: 463–469, 1993PubMedGoogle Scholar
  45. 45.
    Movsesian MA, Karimi M, Green K, Jones LR: Ca2+ transporting ATPase, phospholamban, and calsequestrin levels in nonfailing and failing human heart. Circulation 90: 653–657, 1994PubMedGoogle Scholar
  46. 46.
    Schwinger RHG, Böhm M, Schmidt U, Karczewski P, Bavendiek U, Flesch M, Krause EAG, Erdmann E: Unchanged protein levels of SERCAII and phospholamban but reduced Ca2+ uptake and Ca2+ ATPase activity of cardiac sarcoplasmic reticulum from patients with dilated cardiomyopathy compared to nonfailing patients. Circulation 92:3220–3228, 1995PubMedGoogle Scholar
  47. 47.
    Feldman AM, Cates AE, Veazey WB, Hershberger RE, Bristow MR, Baughman KL, Baumgartner WA, van Dop C: Increase in the 40,000mol wt pertussis toxin substrate (G-protein) in the failing human heart. J Clin Invest 82: 189–197, 1988PubMedGoogle Scholar
  48. 48.
    Eschenhagen, T: G proteins and the heart. Cell Biol Int 17: 723–749, 1993CrossRefGoogle Scholar
  49. 49.
    Kaumann AJ, Sanders L: Both β1- and β2-adrenoceptors mediate catecholamine- evoked arrhythmias in isolated human right atrium. Naunyn-Schmiedeberg's Arch Pharmacol 348: 536–540, 1993Google Scholar
  50. 50.
    Kaumann AJ, Sanders L, Hall JA, Murray KJ, Brown MJ: Stimulation of β1- and β2-adrenoceptors in human ventricular myocardium from failing hearts hastens the onset of relaxation. Br J Pharmacol 105: 283 P, 1992Google Scholar
  51. 51.
    DelMonte F, Kaumann AJ, Poole-Wilson PA, Wynne DG, Pepper J, Harding SE: Coexistence of functional β1- and β2 adrenoceptors in single myocytes from human ventricle. Circulation 88: 889–893, 1993Google Scholar

Copyright information

© Kluwer Academic Publishers 1996

Authors and Affiliations

  • Alberto J. Kaumann
    • 1
  • Louise Sanders
    • 1
  • James A. Lynham
    • 1
  • Sabine Bartel
    • 2
  • Meike Kuschel
    • 2
  • Peter Karczewski
    • 2
  • Ernst-Georg Krause
    • 2
  1. 1.Human Pharmacology LaboratoryThe Babraham InstituteCambridgeUK
  2. 2.Max-Delbrück-Centrum für Molekulare MedizinForschungsschwerpunkt KardiologieBerlinGermany

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