Skip to main content
PMC home page
Proceedings of the National Academy of Sciences of the United States of America logoLink to Proceedings of the National Academy of Sciences of the United States of America
. 1975 Apr;72(4):1564–1568. doi: 10.1073/pnas.72.4.1564

Identification of cardiac beta-adrenergic receptors by (minus) [3H]alprenolol binding.

R W Alexander, L T Williams, R J Lefkowitz
PMCID: PMC432578  PMID: 1055427

Abstract

(Minus) [3-H] alprenolol, a potent beta-adrenergic antagonist, was used to identify binding sites in a fraction of canine cyocardium. Beta adrenergic agonists and antagonists compete for these binding sites in a manner which directly parallels their known affinity for the cardiac beta-adrenergic receptor. Thus, binding was highly stereo-specific, with the (minus) isomers of beta-adrenergic agonists or antagonists being at least two orders of magnitude more potent than were the (plus) isomers in competing for these sites. The order of potency for inhibition of binding by beta-adrenergic agonists was (minus) isoproterenol greater than (minus) epinephrine greater than (minus) norepinephrine. The dissociation constant (KD) of (minus) alprenolol for the beta-adrenergic receptors was 7-11 nM as determined independently by direct binding studies or by inhibition of isoproterenol-stimulated adenylate cyclase [ATP pyrophosphate-lyase (cyclizing), EC 4.6.1.1]. The beta-adrenergic antagonist (minus) propranolol also had high affinity for the binding sites (KD equals 12 nM). The physiologically inactive catechol-containing compounds pyrocatechol and (plus or minus) dihydroxymandelic acid, as well as the metabolite (plus or minus) normetanephrine, and the alpha-adrenergic antagonist phentolamine did not compete for the binding sites at a concentration of 160 muM. Binding was rapid (t1/2 less than 30 sec) and was rapidly reversible (t1/2 less than 15 sec). The binding sites were saturable and bound 0.35 pmol of (minus) [3-H] alprenolol per mg of membrane protein. These characteristics suggest that these binding sites represent the cardiac beta-adrenergic receptors.

Full text

PDF
1567

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Aurbach G. D., Fedak S. A., Woodard C. J., Palmer J. S., Hauser D., Troxler F. Beta-adrenergic receptor: stereospecific interaction of iodinated beta-blocking agent with high affinity site. Science. 1974 Dec 27;186(4170):1223–1224. doi: 10.1126/science.186.4170.1223. [DOI] [PubMed] [Google Scholar]
  2. Benfey B. G., Carolin T. Effect of phenylephrine on cardiac contractility and adenyl cyclase activity. Can J Physiol Pharmacol. 1971 Jun;49(6):508–512. doi: 10.1139/y71-065. [DOI] [PubMed] [Google Scholar]
  3. Bilezikian J. P., Aurbach G. D. A beta-adrenergic receptor of the turkey erythrocyte. I. Binding of catecholamine and relationship to adenylate cyclase activity. J Biol Chem. 1973 Aug 25;248(16):5577–5583. [PubMed] [Google Scholar]
  4. Bilezikian J. P., Aurbach G. D. A beta-adrenergic receptor of the turkey erythrocyte. II. Characterization and solubilization of the receptor. J Biol Chem. 1973 Aug 25;248(16):5584–5589. [PubMed] [Google Scholar]
  5. Birnbaumer L., Pohl S. L., Kaumann A. J. Receptors and acceptors: a necessary distinction in hormone binding studies. Adv Cyclic Nucleotide Res. 1974;4(0):239–281. [PubMed] [Google Scholar]
  6. Cuatrecasas P., Tell G. P., Sica V., Parikh I., Chang K. J. Noradrenaline binding and the search for catecholamine receptors. Nature. 1974 Jan 11;247(5436):92–97. doi: 10.1038/247092a0. [DOI] [PubMed] [Google Scholar]
  7. Dempsey P. J., Cooper T. Supersensitivity of the chronically denervated feline heart. Am J Physiol. 1968 Nov;215(5):1245–1249. doi: 10.1152/ajplegacy.1968.215.5.1245. [DOI] [PubMed] [Google Scholar]
  8. Furchgott R. F. The pharmacological differentiation of adrenergic receptors. Ann N Y Acad Sci. 1967 Feb 10;139(3):553–570. doi: 10.1111/j.1749-6632.1967.tb41229.x. [DOI] [PubMed] [Google Scholar]
  9. Hall Z. W. Release of neurotransmitters and their interaction with receptors. Annu Rev Biochem. 1972;41:925–952. doi: 10.1146/annurev.bi.41.070172.004425. [DOI] [PubMed] [Google Scholar]
  10. Kaumann A. J., Birnbaumer L. Studies on receptor-mediated activation of adenylyl cyclases. IV. Characteristics of the adrenergic receptor coupled to myocardial adenylyl cyclase: stereospecificity for ligands and determination of apparent affinity constants for beta-blockers. J Biol Chem. 1974 Dec 25;249(24):7874–7885. [PubMed] [Google Scholar]
  11. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  12. Lefkowitz R. J. Commentary. Molecular pharmacology of beta-adrenergic receptors--a status report. Biochem Pharmacol. 1974 Aug;23(15):2069–2076. doi: 10.1016/0006-2952(74)90571-1. [DOI] [PubMed] [Google Scholar]
  13. Lefkowitz R. J., Haber E. A fraction of the ventricular myocardium that has the specificity of the cardiac beta-adrenergic receptor. Proc Natl Acad Sci U S A. 1971 Aug;68(8):1773–1777. doi: 10.1073/pnas.68.8.1773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  14. Lefkowitz R. J., Haber E., O'Hara D. Identification of the cardiac beta-adrenergic receptor protein: solubilization and purification by affinity chromatography. Proc Natl Acad Sci U S A. 1972 Oct;69(10):2828–2832. doi: 10.1073/pnas.69.10.2828. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Lefkowitz R. J. Heterogeneity of adenylate cyclase-coupled beta-adrenergic receptors. Biochem Pharmacol. 1975 Mar 1;24(5):583–590. doi: 10.1016/0006-2952(75)90178-1. [DOI] [PubMed] [Google Scholar]
  16. Lefkowitz R. J., Mukherjee C., Coverstone M., Caron M. G. Stereospecific (3H)(minus)-alprenolol binding sites, beta-adrenergic receptors and adenylate cyclase. Biochem Biophys Res Commun. 1974 Sep 23;60(2):703–709. doi: 10.1016/0006-291x(74)90297-6. [DOI] [PubMed] [Google Scholar]
  17. Lefkowitz R. J., Sharp G. W., Haber E. Specific binding of -adrenergic catecholamines to a subcellular fraction from cardiac muscle. J Biol Chem. 1973 Jan 10;248(1):342–349. [PubMed] [Google Scholar]
  18. Lefkowitz R. J. Stimulation of catecholamine-sensitive adenylate cyclase by 5'-guanylyl-imidodiphosphate. J Biol Chem. 1974 Oct 10;249(19):6119–6124. [PubMed] [Google Scholar]
  19. Levitzki A., Atlas D., Steer M. L. The binding characteristics and number of beta-adrenergic receptors on the turkey erythrocyte. Proc Natl Acad Sci U S A. 1974 Jul;71(7):2773–2776. doi: 10.1073/pnas.71.7.2773. [DOI] [PMC free article] [PubMed] [Google Scholar]
  20. MURAD F., CHI Y. M., RALL T. W., SUTHERLAND E. W. Adenyl cyclase. III. The effect of catecholamines and choline esters on the formation of adenosine 3',5'-phosphate by preparations from cardiac muscle and liver. J Biol Chem. 1962 Apr;237:1233–1238. [PubMed] [Google Scholar]
  21. Marinetti G. V., Ray T. K., Tomasi V. Glucagon and epinephrine stimulation of adenyl cyclase in isolated rat liver plasma membranes. Biochem Biophys Res Commun. 1969 Jul 23;36(2):185–193. doi: 10.1016/0006-291x(69)90313-1. [DOI] [PubMed] [Google Scholar]
  22. Mayer S. E. Effects of adrenergic agonists and antagonists on adenylate cyclase activity of dog heart and liver. J Pharmacol Exp Ther. 1972 Apr;181(1):116–125. [PubMed] [Google Scholar]
  23. Potter L. T. Uptake of propranolol by isolated guinea-pig atria. J Pharmacol Exp Ther. 1967 Jan;155(1):91–100. [PubMed] [Google Scholar]
  24. Roth J. Peptide hormone binding to receptors: a review of direct studies in vitro. Metabolism. 1973 Aug;22(8):1059–1073. doi: 10.1016/0026-0495(73)90225-4. [DOI] [PubMed] [Google Scholar]
  25. Salomon Y., Londos C., Rodbell M. A highly sensitive adenylate cyclase assay. Anal Biochem. 1974 Apr;58(2):541–548. doi: 10.1016/0003-2697(74)90222-x. [DOI] [PubMed] [Google Scholar]
  26. Schramm M., Feinstein H., Naim E., Lang E., Lasser M. Epinephrine binding to the catecholamine receptor and activation of the adenylate cyclase in erythrocyte membranes (hormone receptor- -adrenergic receptor-cyclic AMP-turkey). Proc Natl Acad Sci U S A. 1972 Feb;69(2):523–527. doi: 10.1073/pnas.69.2.523. [DOI] [PMC free article] [PubMed] [Google Scholar]
  27. Sobel B. E., Henry P. D., Robison A., Bloor C., Ross J., Jr Depressed adenyl cyclase activity in the failing guinea pig heart. Circ Res. 1969 Apr;24(4):507–512. doi: 10.1161/01.res.24.4.507. [DOI] [PubMed] [Google Scholar]
  28. Tomasi V., Koretz S., Ray T. K., Dunnick J., Marinetti G. V. Hormone action at the membrane level. II. The binding of epinephrine and glucagon to the rat liver plasma membrane. Biochim Biophys Acta. 1970 Jul 7;211(1):31–42. doi: 10.1016/0005-2736(70)90120-3. [DOI] [PubMed] [Google Scholar]
  29. Vatner D. E., Lefkowitz R. J. (3H)-Propranolol binding sites in myocardial membranes: nonidentity with beta adrenergic receptors. Mol Pharmacol. 1974 May;10(3):450–456. [PubMed] [Google Scholar]
  30. Wildenthal K. Studies of fetal mouse hearts in organ culture: influence of prolonged exposure to triiodothyronine on cardiac responsiveness to isoproterenol, glucagon, theophylline, acetylcholine and dibutyryl cyclic 3',5'-adenosine monophosphate. J Pharmacol Exp Ther. 1974 Aug;190(2):272–279. [PubMed] [Google Scholar]

Articles from Proceedings of the National Academy of Sciences of the United States of America are provided here courtesy of National Academy of Sciences

RESOURCES