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Naunyn-Schmiedeberg's Archives of Pharmacology

, Volume 338, Issue 5, pp 523–528 | Cite as

Effect of thiols on β2-adrenoceptors in human mononuclear leucocytes

  • Bernhard Liebl
  • Thomas Anhäupl
  • Ekkehard Haen
  • Jörg Remien
Article

Summary

The effect of the disulfide reducing agent dithiothreitol (DTT) and other thiols on binding of the gb-adrenoceptor antagonist (−)-125iodocyanopindolol (125ICYP) to human mononuclear leucocytes (MNL) was investigated. Saturation experiments and dissociation kinetics revealed two classes of specific 125ICYP binding sites, one of high and the other of low affinity, respectively. In intact MNL DTT caused a decrease in specific binding. This was due almost selectively to a decrease in the affinity of high affinity binding sites, which decreased gradually in a concentration-dependent manner to the affinity of low affinity binding sites. In MNL membranes DTT decreased not only the affinity but also the number of high affinity binding sites. The DTT effect was completely reversible by simple reoxidation on air. The structural isomers (±)-DTT, (−)-DTT and dithioerythritol revealed identical effects on specific binding, whereas the monothiols mercaptoethanol and α-monothioglycerol, having a lower redox potential, were considerably less effective. In the same concentration range that influenced specific binding, DTT stimulated intracellular cAMP production. These results suggest functionally important disulfide bridges which regulate the affinity of β-adrenoceptor binding sites in human MNL. They stabilize the receptor in a high affinity state; their reduction causes the conversion of the high affinity state into a low affinity state in a process associated with stimulation of adenylate cyclase. Available evidence indicates that a similar transformation is made by β-adrenoceptor agonists. Consequently low affinity 125ICYP binding sites preexistent in untreated cells could represent a reduced receptor state resulting from agonist-receptor interaction in vivo.

Key words

Beta-adrenoceptors Lymphocytes 125Iodo-cyanopindolol Adenosine cyclic monophosphate Dithiothreitol 

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References

  1. Anhäupl T, Liebl B, Remien J (1988) Kinetic and equilibrium studies of 125iodocyanopindolol binding to β-adrenoceptors on human lymphocytes: evidence for the existence of two classes of binding sites. J Recept Res 8(1–4):47–57CrossRefGoogle Scholar
  2. Böyum A (1968) Isolation of mononuclear cells and granulocytes from human blood. Scand J Clin Invest 21:77–89CrossRefGoogle Scholar
  3. Brodde OE, Engel G, Hoyer D, Bock KD, Weber F (1981) The β-adrenergic receptor in human lymphocytes: subclassification by the use of a new radioligand, (±) 125iodocyanopindolol. Life Sci 11:2189–2198CrossRefGoogle Scholar
  4. Cleland WW (1964) Dithiothreitol, a new protective reagent for SH-groups. Biochemistry 3:480–482CrossRefGoogle Scholar
  5. Fitzgerald DJ, Doyle V, O'Brian ET, Kelly JG, O'Malley K (1983) Beta-adrenoceptor density and responsiveness in borderline hypertension. J Hypertension 1:260–262Google Scholar
  6. Haen E (1987) The peripheral lymphocyte as clinical model for receptor disturbances: asthmatic diseases. Bull Europ Respir 22:539–541Google Scholar
  7. Heidenreich KA, Weiland GA, Molinoff PB (1982) Effects of magnesium and N-ethylmaleimide on the binding of 3H-hydroxybenzyl-isoproterenol to β-adrenergic receptors. J Biol Chem 257:804–810PubMedGoogle Scholar
  8. Konigsberg W (1972) Reduction of disulfide bonds in proteins with dithiothreitol. Methods Enzymol 25 (Part B):387–392CrossRefGoogle Scholar
  9. Korner M, Gilon C, Schramm M (1982) Locking of hormone in the β-adrenergic receptor by attack on a sulfhydryl in an associated component. J Biol Chem 257:3389–3396PubMedGoogle Scholar
  10. Kühl PW (1985) A redox cycling model for the action of β-adrenoceptor agonists. Experientia 41:1118–1122CrossRefGoogle Scholar
  11. Landmann R, Bürgisser E, Wesp M, Bühler FR (1984) Betaadrenergic receptors are different in subpopulations of human circulating lymphocytes. J Recept Res 4:37–50CrossRefGoogle Scholar
  12. Lucas M, Hanoune J, Bockaert J (1978) Chemical modification of the beta-adrenergic receptors coupled with adenylate cyclase by disulfide bridge-reducing agents. Mol Pharmacol 14:227–236PubMedGoogle Scholar
  13. Mahler HR, Cordes EH (1968) Basic biological chemistry. Harper and Row,NY, 24:346Google Scholar
  14. Motulsky HJ, Insel PA (1982) Adrenergic receptors in man. Direct identification, physiologic regulation, and clinical alterations. N Engl J Med 307:18–29CrossRefGoogle Scholar
  15. Mukherjee C, Lefkowitz RJ (1977) Regulation of beta adrenergic receptors in isolated frog erythrocyte plasma membranes. Mol Pharmacol 13:291–303PubMedGoogle Scholar
  16. Pedersen SE, Ross EM (1986) Functional activation of β-adrenergic receptors by thiols in the presence and absence of agonists. J Biol Chem 260:14150–14157Google Scholar
  17. Prior TI, Patel V, Drummond GI (1985) Inactivation of the β-adrenergic receptor in cardiac muscle by dithiols. Can J Physiol Pharmacol 63:932–936CrossRefGoogle Scholar
  18. Remien J (1984) β-Adrenoceptor recognition sites in living lymphocytes: a model for receptor properties in human disease? Eur J Respir Dis 65 (135):208–214Google Scholar
  19. Scatchard G (1949) The attraction of proteins for small molecules and ions. Ann NY Acad Sci 51:660–672CrossRefGoogle Scholar
  20. Vauquelin G, Bottari S, Kanarek L, Strosberg AD (1979) Evidence for essential disulfide bonds in β 1-adrenergic receptors of turkey erythrocyte membranes. Inactivation by dithiothreitol. J Biol ] Chem 254:4462–4469Google Scholar
  21. Vauquelin G, Bottari S, Strosberg AD (1980) Inactivation of β-adrenergic receptors by N-ethylmaleimide: permissive role of β-adrenergic agents in relation to adenylate cyclase activation. Mol Pharmacol 17:163–171PubMedGoogle Scholar
  22. Vauquelin G, Severne Y, Bottaxi S, Andre C (1984) Agonist-mediated conformational modification of β-adrenergic receptors. In: Liss AR (ed) Catecholamines: basic and peripheral mechanisms. Liss Inc., New York, pp 271–278Google Scholar
  23. Wong A, Hwang SM, Cheng HY, Crooke ST (1987) Structureactivity relationships of β-adrenergic receptor-coupled adenylate cyclase: implications of a redox mechanism for the action of agonists at β-adrenergic receptors. Mol Pharmacol 31:368–376PubMedGoogle Scholar
  24. Wright M, Drummond GI (1983) Inactivation of the β-adrenergic receptor in skeletal muscle by dithiols. Biochem Pharmacol 32:509–515CrossRefGoogle Scholar
  25. Yamaoka K, Tanigawara Y, Nakagawa T, Uno T (1981) A pharmacokinetic analysis program (MULTI) for microcomputer. J Pharm Dyn 4:879–885CrossRefGoogle Scholar

Copyright information

© Springer-Verlag 1988

Authors and Affiliations

  • Bernhard Liebl
    • 1
  • Thomas Anhäupl
    • 1
  • Ekkehard Haen
    • 1
  • Jörg Remien
    • 1
  1. 1.Walther-Straub-Institut für Pharmakologie und ToxikologieMünchenGermany

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