Abstract
The triated adrenergic antagonists Prazosin ([3H]PRZ) and Idazoxan ([3H]IDA, or RX-781094) bind specifically and with high affinity to α1 and α2-adrenoceptors respectively, in membrane preparations from cerebral cortex. Saturation experiments performed to determine the density of receptors and the dissociation constant (K d) were analyzed by the methods of Eadie Hofstee, iterative modelling, and the procedure of Hill, while the specificity of the labelling was verified by displacement experiments. Since receptors are proteins, we examined the role of disulfide (−SS−) bridges and sulfhydryl (−SH) groups in the specific combination of [3H]PRZ and [3H]IDA to the α1 and α2-adrenoceptors. Pretreatment of the membranes with the −SS− reactive DL-dithiothreitol (DTT) or the alkylating agent N-ethylmaleimide (NEM), alone or in combination, decreased specific binding of both ligands, with only minor changes in the non-specific counts. The [3H]IDA binding (α2-sites) was more sensitive to both DTT and NEM than the [3H]PRZ sites (α2-adrenoceptors), and the initial changes induced by alkylation of the α2-site were due to an important decrease in the affinity for [3H]IDA, as judged by the increase in theK d. This modulation in the affinity caused by alkylation of a thiol group could explain the higher potency of the blocking agent tetramine disulfide benextramine at the α2-site. The results provide evidence for the participation of −SS− and −SH groups in the binding site of α1 and α2-adrenoceptors in the cerebral cortex.
Similar content being viewed by others
References
Aronstam, R. S., Abood, L. G., andHoss, W. 1978. Influence of sulfhydryl reagents and heavy metals on the functional state of the muscarinic acetylcholine receptor in rat brain. Mol. Pharmacol. 14:575–586.
Barlow, R. B. 1983. Biodata Handling with Microcomputers. Elsevier Science Publishers, Amsterdam.
Barrantes, J. F. 1980. Modulation of acetylcholine receptor states by thiol modification. Biochemistry 19:2957–2965.
Benfey, B. G., Brasili, L., Melchiorre, C., andBelleau, B. 1980. Potentiation and inhibition of nicotinic effects on striated muscle by the tetramine disulfide benextramine. Can. J. Physiol. Pharmacol. 58:984–988.
Cheng, Y.-C., andPrusoff, W. H. 1973. Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50 percent inhibition (I50) of an enzymatic reaction. Biochem. Pharmacol. 22:3099–3108.
Cleland, W. W. 1964. Dithiothreitol, a new protective reagent for SH groups. Biochemistry 3:480–482.
Curro, F. A., andGreenberg, S. 1983. Characterization of postsynaptic alpha1 and alpha2 adrenergic receptors in canine vascular smooth muscle. Can. J. Physiol. Pharmacol. 61:893–904.
Dausse, J. P., Guicheney, P., Diop, L., andMeyer, P. 1984. Caractérisation biochimique des récepteurs α-adrénergiques centraux. J. Pharmacol. (Paris) 15, (Suppl. 1):23–33.
Del Castillo, J., Escobar, I., andGijon, E. 1971. Effects of the electrophoretic application of sulfhydryi reagents to the end-plate receptors. Int. J. Neurosci. 1:199–209.
De Robertis, E., andFiszer De Plazas, S. 1970. Acetylcholinesterase and acetylcholine proteolipid receptor: two different component of Electroplax membranes. Biochim. Biophys. Acta 219:388–397.
Diop, L., Dausse, J.-P., andMeyer, P. 1983. Specific binding of [3H]rauwolscine to α2-adrenoceptors in rat cerebral cortex: comparison between crude and synaptosomal plasma membranes. J. Neurochem. 41:710–715.
Eadie, G. S. 1952. On the evaluation of the constantsV m andK M in enzyme reactions. Science 116:688.
Ehlert, F. J. Roeske, W. R., andYamamura, H. I. 1980. Regulation of muscarinic receptor binding by guanine nucleotides, ions, and N-ethylmaleimide. J. Supramol. Struct. 14:149–162.
Freedman, J. E., andAghajanian, G. K. 1984. Idazoxan (RX 781094)) antagonizes α2-adrenoceptors on the rat central neurons. Eur. J. Pharmacol. 105:265–272.
Gadie, B., Lane, A. C., McCarthy, P. S., Tulloch, I. F., andWalter, D. S. 1984. 2-Alkyl analogues of idazoxan (RX 781094) with enhanced antagonist potency and selectivity at central α2-adrenoceptors in the rat. Br. J. Pharc. 83:707–712.
Glossmann, H., andPresek, P. 1979. Alpha-noradrenergic receptors in brain membranes: sodium, magnesium and guanylnucleotides modulate agonist binding. Naunyn-Schmiedeberg's Arch. Pharmacol. 306:67–73.
Hill, A. V. 1910. The possible effects of the aggregation of the molecules of haemoglobin on its dissociation curves. J. Physiol. (Lond.) 40:iv-vii.
Hofstee, B. H. 1952. On the evaluation of the constants Vm andK M in enzyme reactions. Science 116:329–331.
Hornung, R., Presek, P., andGlossmann, H. 1979. Alpha adrenoceptors in rat brain: direct identification with prazosin. Naunyn-Schmiedeberg's Arch. Pharmac. 308:223–230.
Karlin, A., andBartels, E. 1966. Effects of blocking sulfhydryl groups and of reducing disulfide bonds on the acetylcholine-activated permeability system of the electroplax. Biochim. Biophys. Acta 126:525–535.
Klotz, I. M. 1982. Numbers of receptor sites from Scatchard graphs: facts and fantasies. Science 217:1247–1249.
Lowry, O. H., Rosebrough, N. J., Farr, A. L., andRandall, R. J. 1951. Protein measurements with Folin phenol reagent. J. Biol. Chem. 193:265–275.
Lucas, M., Hanoune, J., andBockaert, J. 1978. Chemical modification of the β-adrenergic receptors coupled with adenylate cyclase by disulfide bridge-reducing agents. Molec. Pharmacol. 14:227–236.
Mattens, E., Bottari, S., Vokaer, A., andVauquelin, G. 1985. Arginine and cysteine residues in the ligand binding site of alpha 2-adrenergic receptors. Life Sci. 36:355–362.
Melchiorre, C. 1980. Selectivity of α1 and α2 adrenergic agonists and antagonists. I1 Farmaco (Ed. Sci.) 35:535–550.
Miach, P. J., Dausse, J.-P., andMeyer, P. 1978. Direct biochemical demonstration of two types of alpha-adrenoceptors in rat brain. Nature 274:492–494.
Parker, R. B., andWaud, D. R. 1971. Pharmacological estimation of drug-receptor dissociation constants. Statistical evaluation. I. Agonists. J. Pharmacol. Exp. Ther. 177:1–12.
Pimoule, C., Scatton, B., andLanger, S. Z. 1983. [3H]RX 781094: A new antagonist ligand labels α2-adrenoceptors in the rat brain cortex. Eur. J. Pharmacol. 95:79–85.
Quennedy, M.-C., Bockaert, J., andRuout, B. 1984. Direct and indirect effects of sulfhydryl blocking agents on agonist and antagonist binding to central 26-2- and 26-3. Biochem. Pharmacol. 33:3923–3928.
Reader, T. A., andBrière, R. 1982. Modulation by sodium, lithium and calcium of specific3H-prazosin binding in neocortex. Soc. Neurosci. Abstr. 8:659.
Reader, T. A., andBrière, R. 1983. Selective noradrenergic denervation and [3H]-prazosin binding sites in rat neocortex. Brain Res. Bull. 10:155–158.
Reader, T. A., andBrière, R. 1983. Long-term unilateral noradrenergic denervation: monoamine content and3H-prazosin binding sites to rat neocortex. Brain Res. Bull. 11:687–692.
Reader, T. A., andBrière, R. 1985. Evidence for the participation of disulfide and sulfhydryl groups in the specific binding of [3H]prazosin in cerebral cortex. Neurochem. Res. 10:107–122.
Reader, T. A., andDe Robertis, E. 1974. The response of artificial lipid membranes containing a cholinergic hydrophobic protein from Electrophorus electricus. Biochim. Biophys. Acta 352:192–201.
Reader, T. A., andJasper, H. H. 1984. Interactions between monoamines and other transmitters in cerebral cortex. Pages 195–225,in Descarries, L., Reader, T. A., andJasper, H. H. (eds.), Monoamine Innervation of Cerebral Cortex. Alan R. Liss, New York.
Rouot, B., U'Prichard, D. C., andSnyder, S. H. 1980. Multiple alpha2-adrenergic receptor sites in rat brain. Selective regulation of high affinity [3H]clonidine binding by guanine nucleotides and divalent cations. J. Neurochem. 34:374–384.
Salman, K. N., Chai, H. S., Miller, D. D., andPatil, P. N. 1976. Influence of group selective reagents in tissues containing α- and β-adrenoceptors. Eur. J. Pharmacol. 36:41–48.
Smith, J. R., andSimon, E. J. 1980. Selective protection of stereospecific enkephalin and opiate binding againts inactivation by N-ethylmaleimide: evidence for two classes of opiate receptors. Proc. Natl. Acad. Sci. U.S.A. 77:281–284.
Starke, K. 1981. α-Adrenoceptor subclassification. Rev. Physiol. Biochem. Pharmacol. 88:199–236.
Strauss, W. L. 1984. Sulfhydryl groups and disulfide bonds: modification of amino acid residues in studies of receptor structure and function. Pages 85–97,in Venter, J. C., andHarrison, L. C. (eds.), Membranes, Detergents and Receptor Solubilization. Alan R. Liss, Inc., New York.
Suen, E. T., Stefanini, E., andClement-Cormier, Y. C. 1978. Evidence for the essential thiol groups and disulfide bonds in agonist and antagonist binding to the dopamine receptor. Biochem. Biophys. Res. Commun. 96:953–960.
U'Prichard, D. C. 1984. Biochemical characteristics and regulation of brain α2-adrenoceptors. Pages 55–75,in Salama, A. I. (ed.)), Presynaptic modulation of postsynaptic receptors in mental diseases, Ann. N.Y. Acad. Sci. Vol. 430, Plenum Press, New York.
Vohra, M. M., andChaudry, A. 1984. Antagonism by the tetramine disulfide benextramine of the inhibitory effects mediated by prejunctional alpha2-adrenoceptors and by postjunctional histamine H2 receptors in the mouse vas deferens. Can. J. Physiol. Pharmacol. 62:1147–1151.
Woodcock, E. A., andMurley, B. 1982. Increased central α2-adrenergic receptors measured with [3H]-yohimbine in the presence of sodium ion and guanylnucleotides. Biochem. Biophys. Res. Commun. 105:252–258.
Zitwin, J. A., andWaud, D. R. 1982. How to analyze binding, enzyme and uptake data: the simplest case, a single phase. Life Sci. 30:1407–1422.
Author information
Authors and Affiliations
Rights and permissions
About this article
Cite this article
Reader, T.A., Brière, R. & Grondin, L. Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Role of disulfide and sulfhydryl groups. Neurochem Res 11, 9–27 (1986). https://doi.org/10.1007/BF00965161
Accepted:
Issue Date:
DOI: https://doi.org/10.1007/BF00965161