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Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 317, Issue 4, pp 294–301 | Cite as

Desensitization of the β-adrenoceptor-adenylate cyclase system of immature erythrocytes by in-vivo treatment of rats with isoprenaline

  • G. Wiemer
  • G. Kaiser
  • J. Dietz
  • M. Reinhardt
  • A. Wellstein
  • D. Palm
Article

Summary

Rats pretreated with 1-acetyl-2-phenylhydrazide, to induce reticulocytosis, were treated with (±)isoprenaline (4×30 mg/kg in 6 h intervals within 24 h before blood sampling) in order to desensitize the β-adrenoceptor-adenylate cyclase-system of circulating red cells.

  1. 1.

    In membrane preparations Vmax-values of (-)isoprenaline-sensitive adenylate cyclase activity declined by about 50% without significant alterations of the apparent Km-values. Basal activity, as well as enzyme activity stimulated maximally by guanylyl-imidodiphosphate and by fluoride were also decreased to the same extent.

     
  2. 2.

    In intact cells, maximal synthesis of cAMP stimulated by 10−5 M (-)isoprenaline was decreased by about 50% compared to cell suspensions from control animals in the presence of Ro 20-1724 or papaverine, inhibitors of phosphodiesterase. Without inhibition of phosphodiesterase, (-)isoprenaline stimulated cAMP synthesis in cell suspensions from desensitized animals exceeded that from control animals more than 10-fold. A 20–30% decrease of phosphodiesterase activity, measured in membrane and cytoplasmic fractions explains this unexpected result.

     
  3. 3.

    In membrane preparations from untreated animals, Bmax-values for the antagonist ligand [3H] dihydroalprenolol ([3H] DHA) were (0.98±0.16 pmoles/mg protein (n=7). For the agonist-ligand [3H] hydroxybenzylisoprenaline ([3H]-HBI) only 0.23±0.024 pmoles/mg protein (n=12) were obtained. After pretreatment of the animals with (±)isoprenaline, the Bmax-value for [3H] HBI was decreased by about 50%; whereas, that for [3H] DHA was decreased only by about 10%. No change in KD-values for both ligands occurred.

     
  4. 4.

    It is proposed that β-adrenergic desensitization in immature red cells, i.e. the decrease of (-)isoprenalinesensitive adenylate cyclase activity, results from loss of high affinity β-adrenoceptor sites. This might be induced by uncoupling the adrenoceptors from the nucleotide binding protein. A functional impairment or loss of this regulatory protein may be responsible for the apparent loss of adenylate cyclase activity. Since [3H] HBI, in contrast to [3H] DHA, labels predominantly β-adrenoceptors in the high affinity state it is a more sensitive marker to detect alterations at the adrenoceptor level following desensitization.

     

Key words

Desensitization β-Adrenoceptors Adenylate cyclase Agonist binding Rat reticulocytes 

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References

  1. Browning ET, Brostrom CO, Groppi VE (1976) Altered adenosine cyclic 3′,5′-monophosphate synthesis and degradation by C-6 astrocytoma cells following prolonged exposure to norepinephrine. Mol Pharmacol 12:32–40Google Scholar
  2. Chuang DM, Kinnier WJ, Farber L, Costa E (1980) A biochemical study of receptor internalization during β-adrenergic receptor desensitization in frog erythrocytes. Mol Pharmacol 18:348–355Google Scholar
  3. De Lean A, Stadel JM, Lefkowitz RJ (1980) A ternary complex model explains the agonist-specific binding properties of the adenylate cyclase coupled β-adrenergic receptor. J Biol Chem 255:7108–7117Google Scholar
  4. Downs RW, Spiegel AM, Singer M, Reen S, Aurbach GD (1980) Fluoride stimulation of adenylate cyclase is dependent on the guanine nucleotide regulatory protein. J Biol Chem 255:949–954Google Scholar
  5. Galant SP, Durisetti L, Underwood S, Insel PA (1978) Decreased betaadrenergic receptors on polymorphonuclear leucocytes after adrenergic therapy. N Engl J Med 17:933–936Google Scholar
  6. Gauger D, Kaiser G, Quiring K, Palm D (1975) The β-adrenergic receptor adenyl cyclase system of rat reticulocytes. Naunyn-Schmiedeberg's Arch Pharmacol 289:379–389Google Scholar
  7. Green AG, Clark RB (1981) Adenylate cyclase coupling proteins are not essential for agonist-specific desensitization of lymphoma cells. J Biol Chem 256:2105–2108Google Scholar
  8. Hanski E, Levitzki A (1978) The absence of desensitization in the beta adrenergic receptors of turkey reticulocytes and erythrocytes and its possible origin. Life Sci 22:53–60Google Scholar
  9. Harden TK, Cotton UC, Waldo GL, Lutton JK, Perkins JP (1980) Catecholamine-induced alterations in sedimentation of membrane bound β-adrenergic receptors. Science 210:441–443Google Scholar
  10. Harwood JP, Conti M, Conn PM, Dufau ML, Catt KJ (1978) Receptor regulation and target cell responses: Studies in the ovarian luteal cell. Mol Cell Endocrinol 11:121–135Google Scholar
  11. Harwood JP, Dufau ML, Catt KJ (1979) Differing specificities in the desensitization of ovarian adenylate cyclase by epinephrine and human chorionic gonadotropin. Mol Pharmacol 15:439–444Google Scholar
  12. Hoffman BB, Mullikin-Kilpatrick D, Lefkowitz RJ (1979) Desensitization of beta-adrenergic stimulated adenylate cyclase in turkey erythrocytes. J Cyclic Nucleotide Res 5:355–366Google Scholar
  13. Hoffman BB, Lefkowitz RJ (1980) Radioligand binding studies of adrenergic receptors: New insight into molecular and physiological regulation. Ann Rev Pharmacol Toxicol 20:581–608Google Scholar
  14. Johnson GL, Wolfe BB, Harden TK, Molinoff PB, Perkins JP (1978) Role of β-adrenergic receptors in catecholamine-induced desensitization of adenylate cyclase in human astrocytoma cells. J Biol Chem 253:1472–1480Google Scholar
  15. Kaiser G, Wiemer G, Kremer G, Dietz J, Hellwich M, Palm D (1978) Correlation between isoprenaline-stimulated synthesis of cyclic AMP and occurrence of β-adrenoceptors in immature erythrocytes from rats. Eur J Pharmacol 48:255–262Google Scholar
  16. Kalbhen DA, Koch HJ (1967) Methodische Untersuchungen zur quantitativen Mikrobestimmung von ATP in biologischem Material mit dem Firefly-Enzym-System. Z Klin Chem Klin Biochem 5:299–304Google Scholar
  17. Kebabian JW, Zatz M, Romero JA, Axelrod J (1975) Rapid changes in rat pineal β-adrenergic receptor: Alterations in l-[3H] alprenolol binding and adenylate cyclase. Proc Natl Acad Sci USA 72:3735–3739Google Scholar
  18. Lefkowitz RJ, Hoffman BB (1980) Adrenergic receptors. Adv Cyclic Nucleotide Res 12:37–47Google Scholar
  19. Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurement with Folin phenol reagent. J Biol Chem 193:265–275Google Scholar
  20. Mukherjee C, Caron MG, Lefkowitz RJ (1975) Catecholamine-induced subsensitivity of adenylate cyclase associated with loss of β-adrenergic receptor binding sites. Proc Natl Acad Sci USA 72:1945–1949Google Scholar
  21. Nemecek GM, Ray KP, Butcher RW (1979) Inhibition of cyclic nucleotide phosphodiesterase during exposure of WI-38 cells to prostaglandin E1. J Biol Chem 254:598–601Google Scholar
  22. Perkins JP, Johnson GL, Harden TK (1978) Drug-induced modification of the responsiveness of adenylate cyclase to hormones. Adv Cyclic Nucleotide Res 9:19–32Google Scholar
  23. Pike LJ, Lefkowitz RJ (1980) Activation and desensitization of β-adrenergic receptor-coupled GTPase and adenylate cyclase of frog and turkey erythrocyte membranes. J Biol Chem 255:6860–6867Google Scholar
  24. Rodbell M (1980) The role of hormone receptors and GTP-regulatory proteins in membrane transduction. Nature 284:17–22Google Scholar
  25. Sheppard H, Wiggan G (1971) Analogues of 4-(3,4-dimethoxybenzyl)-2-imidazolidinone as potent inhibitors of rat erythrocyte adenosine cyclic 3′, 5′-phosphate phosphodiesterase. Mol Pharmacol 7:111–115Google Scholar
  26. Simpson IA, Pfeuffer T (1980) Functional desensitization of β-adrenergic receptors of avian erythrocytes by catecholamines and adenosine 3′, 5′-phosphate. Eur J Biochem 111:111–116Google Scholar
  27. Su YT, Harden TK, Perkins JP (1980) Catecholamine-specific desensitization of adenylate cyclase. Evidence for a multiple process. J Biol Chem 255:7410–7419Google Scholar
  28. Terasaki WL, Brooker G, de Vellis J, Inglish D, Hsu CJ, Moylan RD (1978) Involvement of cyclic AMP and protein synthesis in catecholamine refractoriness. Adv Cyclic Nucleotide Res 9:33–52Google Scholar
  29. Tomeh JF, Cryer PE (1980) Biphasic adrenergic modulation of β-adrenergic receptors in man. Agonist induced early increment and late decrement in β-adrenergic receptor number. J Clin Invest 65:836–840Google Scholar
  30. Tovey KC, Oldham KG, Whelan JAM (1974) A simple and direct assay for cyclic AMP in plasma and other biological samples using an improved competitive binding technique. Clin Chim Acta 56:221–234Google Scholar
  31. Vallières J, Cote C, Bukowiecki L (1979) Regulation of β-adrenergic receptors in rat skeletal muscles by catecholamines in vivo. Gen Pharmacol 10:63–67Google Scholar
  32. Wessels RM, Mullikin D, Lefkowitz RJ (1979) Selective alteration in high affinity agonist binding: A mechanism of beta-agonist receptor desensitization. Mol Pharmacol 16:10–20Google Scholar
  33. Wiemer G, Palm D, Kremer G, Reinhardt M (1978a) β-Adrenoceptors and adenylcyclase activity in erythrocytes from rats pretreated with isoprenaline. Naunyn-Schmiedeberg's Arch Pharmacol 302 Suppl R 207Google Scholar
  34. Wiemer G, Kaiser G, Palm D (1978b) Effects of Mg2+, Mn2+ and Ca2+ on adenylcyclase activity. Naunyn-Schmiedeberg's Arch Pharmacol 303:145–152Google Scholar
  35. Wiemer G, Reinhardt M, Wellstein A, Palm D (1981) Differentiation between agonist binding and antagonist binding at β-adrenoceptors of rat erythrocytes. Naunyn-Schmiedeberg's Arch Pharmacol 316 Suppl R 232Google Scholar

Copyright information

© Springer-Verlag 1981

Authors and Affiliations

  • G. Wiemer
    • 1
  • G. Kaiser
    • 1
  • J. Dietz
    • 1
  • M. Reinhardt
    • 1
  • A. Wellstein
    • 1
  • D. Palm
    • 1
  1. 1.Zentrum der PharmakologieKlinikum der Johann Wolfgang Goethe-UniversitätFrankfurt/MainGermany

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