Summary
The tritiated adrenergic antagonists prazosin ([3H]PRZ) and idazoxan ([3H]IDA, or RX-781094) bind specifically and with high affinity in membrane preparations from cerebral cortex to alpha-1- and alpha-2-adrenoceptors respectively. Saturation experiments, performed to determine the density of receptors (Bmax; maximum binding capacity) and the dissociation constant (Kd 25 °C), were analyzed by the methods of Eadie and Hofstee, iterative modelling, and the procedure of Hill. The pharmacologic properties and specificity of the labelling was verified by displacement experiments using alpha-adrenergic antagonists and agonists. The antagonist drugs showed the following order of potency to displace [3H]prazosin: prazosin ≫ phentolamine ≫ corynanthine > pyrextramine ≫ yohimbine ≫ piperoxan > benextramine > idazoxan; for the agonists: clonidine ≫ (−)-noradrenaline ≫ (−)-adrenaline ≫ phenylephrine, while other drugs, such as (−)-propranolol, dopamine, (−)-isoproterenol and serotonin only competed with the alpha-1-ligand at concentrations above 20 μM. The alpha2-sites labelled by [3H]idazoxan were characterized by the antagonist displacement sequence idazoxan ≫ phentolamine > yohimbine = > piperoxan ≫ pyrextramine ≫ benextramine ≫ prazosin ≫ corynanthine. The agonists order of potency to compete with [3H]idazoxan was clonidine ≫ phenylephrine = > (−)-adrenaline > (−)-noradrenaline, and for other related drugs it was (−)-propranolol ≫ dopamine ≫ serotonin > (−)-isoproterenol. These competition experiments clearly showed two pharmacologically distinct sites, but question the relative specificity of some of the adrenergic drugs.
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Abbreviations
- [3H]PRZ:
-
[3H]prazosin
- [3H]IDA:
-
[3H]idazoxan
- Bmax :
-
maximum binding capacity
- Kd :
-
dissociation constant
- IC 50:
-
inhibitory concentration that reduces binding by 50%
- Ki :
-
inhibition-dissociation constant
- nH:
-
Hill coefficient
- CMC:
-
coefficient of multiple correlation
- fmol/mg p:
-
fentomoles per mg of protein
- nM:
-
nanomolar
References
Ahlquist RP (1948) A study of adrenergic receptors. Am J Physiol 153: 586–600
Barlow RB (1983) Biodata handling with microcomputers. Elsevier Science Publishers, Amsterdam
Brasili L, Angeli P, Brancia E, Melchiorre C (1984) N, N″-(Dithio-2, 1-ethanediyl)bis (N′-[pyrrol-2-ylmethyl]-1, 6-hexanediamine) (pyrextramine), a new irreversible α1-adrenoreceptor blocking agent of the tetramine disulfide class. Eur J Pharmacol 103: 181–184
Brasili L, Cassinelli A, Angeli P, Melchiorre C (1986) Thiol group may be involved in the irreversible blockade of presynaptic α2-adrenoceptors by pyrextramine and benextramine in the isolated guinea pig ileum. Life Sci 38: 1633–1640
Brière R, Sherwin AL, Robitaille Y, Olivier A, Quesney LF, Reader TA, (1986) α-1 adrenoceptors are decreased in human epileptic foci. Ann Neurol 19: 26–30
Bylund DB (1985) Heterogeneity of alpha-2 adrenergic receptors. Pharmacol Biochem Behav 22: 835–843
Bylund BD, U'Prichard DC (1983) Characterization of α1- and α2-adrenergic receptors. Int Rev Neurobiol 24: 343–431
Cavero I, Roach AG (1980) The pharmacology of prazosin, a novel antihypertensive agent. Life Sci 27: 1525–1540
Chapleo CB, Doxey JC, Myers PL, Roach AG (1981) RX 781094, a new potent, selective antagonist at α2-adrenoceptors. Br J Pharmacol 74: 942 P
Cheng YC, Prusoff WH (1973) Relationship between the inhibition constant (Ki) and the concentration of inhibitor which causes 50% inhibition (I 50) of an enzymatic reaction. Biochem Pharmacol 22: 3099–3108
Cheung YD, Barnett DB, Nahorski SR (1982) [3H]-Rauwolscine and [3H]-yohimbine binding to rat cerebral and human platelet membranes: Possible heterogeneity of α2-adrenoceptors. Eur J Biochem 84: 79–85
Chou T-C (1974) Relationships between inhibition constants and fractional inhibition in enzyme-catalyzed reactions with different numbers of reactants, different reaction mechanisms, and different types and mechanisms of inhibition. Mol Pharmacol 10: 235–247
Dausse JP, Guicheney P, Diop L, Meyer P (1984) Caractérisation biochimique des récepteurs α-adrénergiques centraux. J Pharmacol 15 [suppl I] 15: 23–33
Diop L, Dausse JP, Meyer 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
Docherty JR, McGrath JC (1980) A comparison of pre- and post-junctional potencies of several alpha-adrenoceptor agonists in the cardiovascular system and anococcygeus muscles of the rat. Naunyn-Schmiedebergs Arch Pharmacol 312: 107–116
Drew GM (1976) Effects of α-adrenoceptor agonists and antagonists on pre- and postsynaptically located α-adrenoceptors. Eur J Pharmacol 36: 313–320
Eadie GS (1952) On the evaluation of the constants Vm and Km in enzyme reactions. Science 116: 688
Freedman JE, Aghajanian GK (1984) Idazoxan (RX 781094) selectively antagonizes α2-adrenoceptors on rat central neurons. Eur J Pharmacol 105: 265–272
Gadie B, Lane AC, McCarthy PS, Tulloch IF, Walter DS (1984) 2-alkyl analogues of idazoxan (RX 781094) with enhanced antagonist potency and selectivity at central α2-adrenoceptors in the rat. Br J Pharmacol 83: 707–712
Hill AV (1910) The possible effects of the aggregation of the molecules of hemoglobin on its dissociation curves. J Physiol (Lond) 1: iv-vii
Hofstee BHJ (1952) On the evaluation of the constants Vm and Km in enzyme reactions. Science 116: 329–331
Hornung R, Presek P, Glossmann H (1979) Alpha adrenoceptors in rat brain: direct identification with prazosin. Naunyn-Schmiedebergs Arch Pharmacol 308: 223–230
Howlett DR, Taylor P, Walter DS (1982) α-adrenoceptor selectivity studies with RX 781094 using radioligand binding to cerebral cortex. Br J Pharmacol 76: 294 P
Kawahara RS, Bylund DB (1985) Solubilization and characterization of putative alpha-2 adrenergic isoceptors from the human platelet and the rat cerebral cortex. J Pharmac Exp Ther 233: 603–610
Klotz IM (1982) Numbers of receptor sites from Scatchard graphs: facts and fantasies. Science 217: 1247–1249
Kobinger W (1984) New concepts on α-adrenoceptors in pharmacology. J Pharmacol [suppl I] 15: 5–22
Lavin TN, Hoffman BB, Lefkowitz RG (1981) Identification of alphaadrenergic receptors in uterine smooth muscle membranes by [3H]-dihydroergocryptine binding. Mol Pharmacol 20: 28–34
Lippert B, Belleau B (1973) Recent advances in the active site chemistry of the adrenergic alpha-adrenoceptor. In: Usdin E, Snyder SH (eds) Frontiers in catecholamine research. Pergamon Press, New York, pp 369–371
Lowry OH, Rosebrough NJ, Farr AL, Randall RJ (1951) Protein measurements with Folin phenol reagent. J Biol Chem 193: 265–275
Melchiorre C (1980) Selectivity of α1- and α2-adrenergic agonists and antagonists. Farmaco Ed Sci 35: 535–550
Miach PJ, Dausse JP, Meyer P (1978) Direct biochemical demonstration of two types of alpha-adrenoceptors in rat brain. Nature 274: 492–494
Nagatomo T, Tsuchihashi H, Sasaki S, Nakagawa Y, Nakahara H, Imai S (1985) Displacement by α-adrenergic agonists and antagonists of3H-prazosin bound to the α-adrenoceptors of the dog aorta and the rat brain. Jpn J Pharmacol 37: 181–187
Parker RB, Waud DR (1971) Pharmacological estimation of drug-receptor dissociation constants. Statistical evaluation. I. Agonists. J Pharmacol Exp Ther 177: 1–12
Pimoule C, Scatton B, Langer SZ (1983) [3H] RX 781094: a new antagonist ligand labels α2-adrenoceptors in the rat brain cortex. Eur J Pharmacol 95: 79–85
Reader TA, Brière R (1983 a) Selective noradrenergic denervation and3H-prazosin binding sites in rat neocortex. Brain Res Bull 10: 155–158
Reader TA, Brière R (1983 b) Long-term unilateral noradrenergic denervation: monoamine content and3H-prazosin binding sites in rat neocortex. Brain Res Bull 11: 687–692
Reader TA, Brière 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 TA, Brière R (1986) Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: role of disulfide and sulfhydryl groups. Neurochem Res 11: 9–27
Reader TA, Jasper HH (1984) Interactions between monoamines and other transmitters in cerebral cortex. In: Descarries L, Reader TA, Jasper HH (eds) Monoamine innervation of cerebral cortex. Alan R Liss, New York, pp 195–225
Starke K (1981) α-Adrenoceptor subclassification. Rev Physiol Biochem Pharmacol 88: 199–236
Summers RJ, Jarrott B, Louis WJ (1980) Selectivity of a series of clonidine-like drugs for α1-and α2-adrenoceptors in rat brain. Neurosci Lett 20: 347–350
Timmermans PBMWM, van Zwieten PA (1979) The interaction between prazosin and clonidine at α-adrenoceptors in rats and cats. Eur J Pharmacol 55: 57–66
Vohra MM, Chaudry A (1984) Antagonism by the tetramine disulfide benextramine of the inhibitory effects mediated by prejunctional alpha-adrenoceptors and by postjunctional histamine H2 receptors in the mouse vas deferens. Can J Physiol Pharmacol 62: 1147–1151
Wood CL, Arnett CD, Clarke WR, Tsai BS, Lefkowitz RJ (1979) Subclassification of alpha-adrenergic receptors by direct binding studies. Biochem Pharmacol 28: 1277–1282
Zitwin JA, Waud DR (1982) How to analyse binding, enzyme and uptake data: the simplest case, a single phase. Life Sci 30: 1407–1422
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Reader, T.A., Brière, R. & Grondin, L. Alpha-1 and alpha-2 adrenoceptor binding in cerebral cortex: Competition studies with [3H]prazosin and [3H]idazoxan. J. Neural Transmission 68, 79–95 (1987). https://doi.org/10.1007/BF01244641
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DOI: https://doi.org/10.1007/BF01244641