Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 305, Issue 1, pp 41–50 | Cite as

Identification and quantification of β-adrenoceptor sites in red blood cells from rats

  • G. Kaiser
  • G. Wiemer
  • G. Kremer
  • J. Dietz
  • D. Palm


For the direct characterization of β-adrenoceptors in membrane preparations from the reticulocyte rich blood of rats treated with acetyl phenylhydrazide the β-adrenergic antagonist ligand (3H)(−)dihydroalprenolol (DHAP) was used.

  1. 1.

    Specific binding of DHAP demonstrated one type of binding site in these membrane preparations. There was no evidence for negative co-operativity between the sites. A mean K D -value amounting to 6.51\+-0.8 nM (n=15) was calculated from equilibrium experiments; a similar K D -value (8.25 nM) was evaluated from the ratio of the rate constants of the dissociation and association reactions. In preparations of reticulocyte rich blood (53% reticulocytes) a mean density of DHAP binding sites of 0.602\+-0.05 pmoles/mg protein (n=15) was determined. The respective value in membrane preparations from reticulocyte poor blood, i.e. from untreated animals (2% reticulocytes), amounted to only 0.224\+-0.03 pmoles/mg protein (n=5) whereas the mean K D -value remained unaltered (K D =6.84\+-2.2 nM; n=5).

  2. 2.

    Specific binding sites for DHAP in membranes from reticulocyte rich blood can be looked at as true \gb-adrenoceptors:

    Specific binding of DHAP was competitively inhibited by β-adrenoceptor agonists and antagonists according to the structural specificity and stereospecificity of these compounds. The K D -values for agonists increased in the order isoprenaline < adrenaline < noradrenaline < phenylephrine, the (−)enantiomers being significantly more potent than the respective (+)enantiomers. The same was true also for β-adrenoceptor antagonists.

  3. 3.

    Using the highly purified enantiomers of fenoterol, a compound with 2 asymmetric centres, it was demonstrated that the 1R/1\t'R isomer was highly active while the enantiomer 1S/1\t'S did not show any affinity for \gb-adrenoceptors.

  4. 4.

    When the K D -values evaluated from binding experiments were compared to the respective K a - and K i -values obtained from adenyl cyclase assays in the same membrane preparations there resulted direct correlations (K D \~-K a or K i ). The identity of these values for \gb-adrenoceptor agonists is in favour of an optimal coupling state of the \gb-adrenoceptor-adenyl cyclase-system in immature red blood cells from rats.


Key words

Red Blood Cells Rats β-Adrenoceptors Adenyl cyclase (3H) (−)Dihydroalprenolol 


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  1. Ahlquist, R. P.: Adrenergic beta-blocking agents. In: E. Jucker (ed.): Prog. Drug Res. 20, 27–43. Basel-Stuttgart: Birkhäuser 1976Google Scholar
  2. Aurbach, G. D., Fedak, S. A., Woodard, C. J., Palmers, J. S., Hauser, D., Troxler, F.: β-Adrenergic receptors: Stereospecific interaction of an iodinated β-blocking agent with high affinity site. Science 186, 1223–1224 (1974)Google Scholar
  3. Bartlett, G. R.: Phosphate compounds in rat erythrocytes and reticulocytes. Biochem. Biophys. Res. Commun. 70, 1055–1062 (1976)Google Scholar
  4. Bartsch, W., Dietmann, K., Leinert, H., Sponer, G.: Der Einfluß von Carazolol und Methypranol auf das Herz im Vergleich zu anderen β-Rezeptorenblockern. Arzneim.-Forsch. 27, 1022–1026 (1977)Google Scholar
  5. Belleau, B.: Steric effects in catecholamine interactions with enzymes and receptors. Pharmacol. Rev. 18, 131–140 (1966)Google Scholar
  6. Bilezikian, J. P., Spiegel, A. M., Brown, E. M., Aurbach, G. D.: Identification and persistence of beta adrenergic receptors during maturation of the rat reticulocyte. Mol. Pharmacol. 13, 775–785 (1977)Google Scholar
  7. Boeynaems, J. N., Dumond, J. E.: Quantitative analysis of the binding of ligands to their receptors. J. Cyclic Nucleotide Res. 1, 123–142 (1975)Google Scholar
  8. Brown, E. M., Aurbach, G. D., Hauser, D., Troxler, F.: β-Adrenergic receptor interactions: Characterisation of iodohydroxybenzylpindolol as a specific ligand. J. Biol. Chem. 251, 1232–1238 (1976a)Google Scholar
  9. Brown, E. M., Fedak, S. A., Woodard, C. J., Aurbach, G. D.: β-Adrenergic receptor interactions: Direct comparison of receptor interaction and biological activity. J. Biol. Chem. 251, 1239–1246 (1976b)Google Scholar
  10. Bylund, D. B., Snyder, S. H.: Beta-adrenergic receptor binding in membrane preparations from mammalian brain. Mol. Pharmacol. 12, 568–580 (1976)Google Scholar
  11. Charness, M. E., Bylund, D. B., Beckmann, B. S., Hollenberg, M. D., Snyder, S. H.: Independent variation of β-adrenergic receptor binding and catecholamine-stimulated adenylate cyclase activity in rat erythrocytes. Life Sci. 19, 243–280 (1976)Google Scholar
  12. Cheng, Y.-C., Prusoff, W. H.: Relationship between inhibition constant (K l) and the concentration of inhibitor which causes 50 per cent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22, 3099–3108 (1973)Google Scholar
  13. Cuatrecasas, P.: Hormone receptors — Their function in cell membranes and some problems related to methodology. In: G. I. Drummond, P. Greengard, G. A. Robison (eds.): Adv. Cyclic Nucleotide Res. 5, 79–104 (1975)Google Scholar
  14. Dixon, M.: from M. Dixon, E. C. Webb: Enzymes. London: Longmans 1964Google Scholar
  15. Engelhardt, A., Traunecker, W.: Berotec, bronchospasmolytische und kardiovaskuläre Wirkungen im Tierexperiment. In: Berotec-Symposium 1971. Int. J. Clin. Pharmacol. Ther. Toxicol., Suppl. 4, 6–13 (1972)Google Scholar
  16. Furchgott, R. F.: The classification of adrenoceptors (adrenergic receptors). An evaluation from the standpoint of receptor theory. In: H. Blaschko, E. Muscholl (eds.) Catecholamines, pp. 283–335. Berlin-Heidelberg-New York: Springer 1972Google Scholar
  17. Gauger, D., Palm, D., Kaiser, G., Quiring, K.: Adenyl cyclase activities in rat erythrocytes during stress erythropoiesis: Localization of the enzyme in the reticulocytes. Life Sci. 13, 31–40 (1973)Google Scholar
  18. Gauger, D., Kaiser, G., Quiring, K., Palm, D.: The β-adrenergic receptor-adenyl-cyclase system of rat reticulocytes. Naunyn-Schmiedeberg's Arch. Pharmacol. 289, 379–389 (1975)Google Scholar
  19. Gibson, D. G.: Pharmacodynamic properties of β-adrenergic receptor blocking drugs in man. Drugs 7, 8–38 (1974)Google Scholar
  20. Harden, T. K., Wolfe, B. B., Molinoff, P. D.: Binding of iodinated beta adrenergic antagonists to proteins derived from rat heart. Mol. Pharmacol. 12, 1–15 (1976)Google Scholar
  21. Helmreich, E. J. M.: Hormone-receptor interactions. FEBS Letters 61, 1–5 (1976)Google Scholar
  22. Hill, A. V.: A new mathematical treatment of ionic concentration in muscle and nerve under the action of electric currents, with a theory as to their mode of excitation. J. Physiol. (Lond.) 40, 190–224 (1910)Google Scholar
  23. Kaiser, G., Wiemer, G., Dietz, J., Hellwich, M., Palm, D.: Characterisation by ligand binding of β-adrenergic receptors in membranes from red blood cells from rats. Naunyn-Schmiedeberg's Arch. Pharmacol. 293, R5 (1976)Google Scholar
  24. Kaiser, G., Palm, D., Quiring, K., Gauger, D.: The adrenergic β-receptor-effector system of the premature erythrocyte: Indication for adrenergic control of the erythron? Pharmacol. Res. Commun. 9, 93–103 (1977)Google Scholar
  25. Kaiser, G., Wiemer, G., Kremer, G., Dietz, J., Hellwich, M., Palm, D.: Correlation between isoprenaline-stimulated synthesis of cyclic AMP and occurrence of adrenergic β-receptors in immature erythrocytes from rats. Eur. J. Pharmacol. 48, 255–262 (1978)Google Scholar
  26. Lefkowitz, R. J., Limbird, L. E., Mukherjee, C., Caron, M. G.: The β-adrenergic receptor and adenylcyclase. Biochim. Biophys. Acta 457, 1–39 (1976)Google Scholar
  27. Limbird, L. E., Lefkowitz, R. J.: Negative cooperativity among β-adrenergic receptors in frog erythrocyte membranes. J. Biol. Chem. 251, 5007–5014 (1976)Google Scholar
  28. Lowry, O. H., Rosebrough, N. J., Farr, A. L., Randall, R. J.: Protein measurement with Folin phenol reagent. J. Biol. Chem. 193, 265–275 (1951)Google Scholar
  29. Lucas, M., Bockaert, J.: Use of (−)-(3H)dihydroalprenolol to study beta adrenergic receptor-adenylate cyclase coupling in C6 glioma cells: Role of 5′-guanylylimidodiphosphate. Mol. Pharmacol. 13, 314–329 (1976)Google Scholar
  30. Maguire, M. E., Wiklund, R. A., Anderson, H. J., Gilman, A. G.: Binding of [125I] iodohydroxybenzylpindolol to putative β-adrenergic receptors of rat glioma cells and other cell clones. J. Biol. Chem. 251, 1221–1231 (1976)Google Scholar
  31. Maguire, M. E., Ross, M., Gilman, A. G.: β-Adrenergic receptor: Ligand binding and the interaction with adenyl cyclase. In: P. Greengard, G. A. Robison (eds.): Adv. Cyclic Nucleotide Res. 8, 1–83 (1977)Google Scholar
  32. Malchoff, C. D., Marinetti, G. V.: Hormone action at the membrane level. VI. Binding of (−)-(3H) dihydroalprenolol and (±)-(3H) isoproterenol to turkey erythrocytes and correlation with adenyl cyclase activity. Biochim. Biophys. Acta 538, 541–554 (1978)Google Scholar
  33. Mukherjee, C., Caron, M. G., Coverstone, M., Lefkowitz, R. J.: Identification of adenylate cyclase-coupled β-adrenergic receptors in frog erythrocytes with (−)-(3H)-alprenolol. J. Biol. Chem. 250, 4869–4876 (1975)Google Scholar
  34. Palm, D.: Adrenerge Rezeptoren und Rezeptorenblocker. In: R. Dietz, D. Ganten, K. G. Hofbauer, J. B. Lüth (eds.): Essentieller Hochdruck und seine Behandlung, S. 94–111. Stuttgart-New York: Schattauer 1977Google Scholar
  35. Palm, D., Kaiser, G., Dietz, J., Wiemer, G., Hellwich, M.: Adrenerge β-Rezeptoren in der Cytoplasmamembran unreifer Ratten-Erythrozyten. Quantifizierung durch Ligandenbindung nach hämolytischer Anämie. 19. Jahres-Kongreß Dtsch. Ges. Hämat., Freiburg 1976. Blut 33, 225 (1976)Google Scholar
  36. Palm, D., Kaiser, G., Wiemer, G., Dietz, J., Hellwich, M., Kremer, G.: Adrenergic receptor sites, adenylcyclase activity and cAMP in red blood cells from rats. Abstr. 6th Congress of the Polish Pharmacological Society, Katowice 1977, pp. 111–112Google Scholar
  37. Patil, P. N., LaPidus, J. P.: Stereoisomerism of adrenergic drugs. Rev. Physiol. 66, 213–260 (1972)Google Scholar
  38. Patil, P. N., Miller, D. D., Trendelenburg, U.: Molecular geometry and adrenergic drug activity. Pharmacol. Rev. 26, 323–392 (1974)Google Scholar
  39. Pfeuffer, T., Helmreich, E. J. M.: Activation of pigeon erythrocyte membrane adenylate cyclase by guanyl nucleotide analogues and separation of a nucleotide binding protein. J. Biol. Chem. 250, 867–876 (1975)Google Scholar
  40. Quiring, K., Gauger, D., Kaiser, G., Palm, D.: Adenyl cyclase activity in non-nucleated red blood cells: Evidence for its localization in the reticulocytes. Experientia 29, 526–527 (1973a)Google Scholar
  41. Quiring, K., Kaiser, G., Gauger, D., Palm, D.: Catecholamine-inactivating enzymes in rat reticulocytes. Naunyn-Schmiedeberg's Arch. Pharmacol. 279, 93–97 (1973b)Google Scholar
  42. Randall, M. H., Altman, L. J., Lefkowitz, R. J.: Structure and biological activity of (−)-(3H) dihydroalprenolol, a radioligand for studies of β-adrenergic receptors. J. Med. Chem. 20, 1090–1094 (1977)Google Scholar
  43. Triggle, D. J.: Neurotransmitter-receptor interactions. London-New York: Academic Press 1971Google Scholar
  44. Williams, T. L., Snyderman, R., Lefkowitz, R. J.: Identification of β-adrenergic receptors in human lymphocytes by (−)-(3H) alprenolol binding. J. Clin. Invest. 57, 149–155 (1976)Google Scholar
  45. Wolfe, B. B., Harden, T. K., Molinoff, P. B.: In vitro study of β-adrenergic receptors. Ann. Rev. Pharmacol. Toxicol. 17, 575–604 (1977)Google Scholar
  46. Zatz, M., Kebabian, J. W., Romero, J. A., Lefkowitz, R. J., Axelrod, J.: Pineal beta adrenergic receptor: Correlation of binding of 3H-l-alprenolol with stimulation of adenylate cyclase. J. Pharmacol. Exp. Ther. 196, 714–722 (1976)Google Scholar

Copyright information

© Springer-Verlag 1978

Authors and Affiliations

  • G. Kaiser
    • 1
  • G. Wiemer
    • 1
  • G. Kremer
    • 1
  • J. Dietz
    • 1
  • D. Palm
    • 1
  1. 1.Zentrum der PharmakologieKlinikum der Johann Wolfgang Goethe-UniversitätFrankfurt am Main 70Federal Republic of Germany

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