Elsevier

Cellular Signalling

Volume 14, Issue 10, October 2002, Pages 829-837
Cellular Signalling

Ligand modulation of [35S]GTPγS binding at human α2A, α2B and α2C adrenoceptors

https://doi.org/10.1016/S0898-6568(02)00030-XGet rights and content

Abstract

Affinities and efficacies of chemically diverse ligands—some of them used as clinical agents—were examined, employing [3H]RX821,002 and [35S]GTPγS binding assays, respectively, at human (h) cloned, hα2A, hα2B and hα2C adrenoceptors (AR) expressed in Chinese hamster ovary (CHO) cells. As compared to noradrenaline (NA, efficacy defined as 100%), the majority of the 13 agonists tested generally behaved as partial agonists. Amongst 18 antagonists, pKB and pKi values, which were highly correlated for each α2-AR subtype, failed to reveal any strikingly selective agents. Inverse agonist properties were not detected for any antagonist, consistent with a lack of constitutive activity suggested by the monophasic inhibition of [35S]GTPγS binding by GTPγS. These data should facilitate interpretation of experimental and clinical actions of adrenergic agonists. Moreover, they emphasize the continuing need for α2-AR subtype-selective antagonists in order to define further the roles and therapeutic relevance of hα2A-, hα2B-, and hα2C-AR.

Introduction

Central and peripheral actions of noradrenaline (NA) or adrenaline are mediated by three families of adrenoceptors (AR), namely, α1, α2 and β-AR [1], [2]. α2-AR are involved in numerous physiological and pathophysiological functions such as regulation of monoaminergic transmission, thermoregulation, nociception, cognitive functions, regulation of blood pressure and locomotion [3], [4]. In addition, α2 agonists are of use as anaesthetic and antiglaucoma agents [4]. Three subtypes, α2A, α2B and α2C-AR, encoded by different genes, have been reported [5]. In vitro and in vivo pharmacological analysis of adrenergic ligands [6], [7], [8], as well as recent studies using animals overexpressing or lacking genes encoding each α2-AR, has suggested specific functional roles [9], [10], [11]. Subtype-selective α2-AR agonists and antagonists would help to assign adrenergic functions to a given subtype as well as to lead to their use as therapeutic agents. In this context, it is important to consider both potency and efficacy of adrenergic ligands at α2-AR subtypes.

All three α2-AR subtypes are coupled to diverse transduction pathways, although inhibitory coupling to adenylyl cyclase appears to be predominant [12], [13], [14], [15]. Indeed, α2A-, α2B- and α2C-AR inhibit adenylyl cyclase activity via pertussis toxin-sensitive Gi/o proteins [16], [17]. Agonist and antagonist activity at receptors negatively coupled to adenylyl cyclase can be evaluated through binding studies with the nonhydrolysable GTP analogue [35S]GTPγS [18], [19]. Several studies have been performed at α2-AR either using the [35S]GTPγS binding technique [20], [21], [22], [23] or the measure of GTP hydrolysis [24], [25]. Although numerous agonists have been tested, only a few clinical agents have been evaluated. Concerning antagonists, certain antagonists were found to be either “silent” [21], [24] or were shown to act as “inverse agonists” at rat or human α2A-AR [20], [26] or at constitutively mutated human α2A-AR [27].

Thus, in the present study, we characterised and compared the potencies and efficacies of diverse adrenergic agonists [13] and antagonists [18] at all three human α2-AR subtypes expressed in Chinese hamster ovary (CHO) cells.

Section snippets

Cell lines

CHO-hα2A, CHO-hα2B and CHO-hα2C cell lines expressing the human recombinant AR (provided by Pr. A.D. Strosberg, ICGM, Paris) were grown in Ham-F12 medium supplemented with 2 mM glutamine, 10% foetal bovine serum, 100 IU/ml penicillin, 100 μg/ml streptomycin and 400 μg/ml G418, in an atmosphere of 5% CO2 at 37 °C. Cells were passaged every 3–4 days. These CHO-hα2A, CHO-hα2B and CHO-hα2C cell lines expressed 1.8, 3 and 1.3 pmol, respectively, of receptors per milligram of proteins as determined

Effects of adrenergic agonists on [35S]GTPγS binding at CHO-hα2A, CHO-hα2B and CHO-hα2C membranes

NA and adrenaline were equipotent at α2A-AR, while NA was more potent than adrenaline at hα2B- and hα2C-AR (Table 1). As compared to NA, brimonidine acted as full agonist at hα2A- but not at hα2B- and hα2C-AR (Fig. 1A, Table 1). Conversely, dexmedetomidine was a full agonist at hα2B-, and to a lesser extent at hα2C-, but not at hα2A-AR receptors (Table 1). The two compounds exhibited 10-fold selectivity for hα2A- vs. hα2B- and hα2C-AR receptors. Guanabenz, guanfacine and oxymetazoline were most

Discussion

The present study describes the modulation of [35S]GTPγS binding to G-proteins in transfected CHO cells expressing the human hα2A-, hα2B- or hα2C-AR for a large panel of adrenergic agonists and antagonists.

References (50)

  • P. Young et al.

    Eur J Pharmacol

    (1989)
  • E. MacDonald et al.

    Trends Pharmacol Sci

    (1997)
  • C.M. Fraser et al.

    J Biol Chem

    (1989)
  • M.G. Eason et al.

    J Biol Chem

    (1992)
  • L. Hein et al.

    Trends Cardiovasc Med

    (1997)
  • K. Pohjanoksa et al.

    Eur J Pharmacol

    (1997)
  • S. Lazareno et al.

    Life Sci

    (1993)
  • J.R. Jasper et al.

    Biochem Pharmacol

    (1998)
  • J.M. Peltonen et al.

    Eur J Pharmacol

    (1998)
  • S. Gessi et al.

    Life Sci

    (1999)
  • S. Virolainen et al.

    Eur J Pharmacol

    (1997)
  • C.C. Jansson et al.

    Eur J Pharmacol

    (1999)
  • F.P. Tolentino-Silva et al.

    Brain Res

    (2000)
  • G. Bricca et al.

    Eur J Pharmacol

    (1989)
  • H.E. Shannon et al.

    Pain

    (2000)
  • C.A. Fairbanks et al.

    Pain

    (2000)
  • V. Audinot et al.

    Neuropharmacology

    (2001)
  • L.C. Murrin et al.

    Eur J Pharmacol

    (2000)
  • D.B. Bylund et al.

    Pharmacol Rev

    (1994)
  • J.P. Hieble et al.

    J Med Chem

    (1995)
  • R.R. Ruffolo et al.

    Annu Rev Pharmacol Toxicol

    (1993)
  • R.R. Ruffolo et al.

    J Med Chem

    (1995)
  • R. Aantaa et al.

    Ann Med

    (1995)
  • A. Renouard et al.

    J Pharmacol Exp Ther

    (1994)
  • M.J. Millan et al.

    J Pharmacol Exp Ther

    (1994)
  • Cited by (24)

    • Yokukansan inhibits morphine tolerance and physical dependence in mice: The role of α<inf>2A</inf>-adrenoceptor

      2012, Neuroscience
      Citation Excerpt :

      Various concentrations of YKS, UH, GR, the seven UH-derived components, and the eight GR-derived components were prepared in 50% dimethylsulfoxide. A [35S]GTPγS binding assay was carried out to evaluate the α2A-AR agonistic or antagonistic activity of each test substance, with slight modifications of the methods described by Audinot et al. (2002). In brief, to confirm the agonistic activity, 0.42 μl of each test substance or vehicle (50% dimethylsulfoxide) was pre-incubated for 20 min at 30 °C with a membrane solution prepared from Sf9 insect cells expressing recombinant human α2A-AR (50 μl; 25–30 μg protein/ml) and 10 μM GDP in 20 mM HEPES buffer, pH 7.4, containing 100 mM NaCl, 10 mM MgCl2, 1 mM dithiothreitol, and 1 mM EDTA (25 μl).

    • Characterization of α<inf>2B</inf>-adrenoceptor ligand binding in the presence of Muscarinic toxin α and delineation of structural features of receptor binding selectivity

      2012, European Journal of Pharmacology
      Citation Excerpt :

      In the case of antagonists, many widely used compounds like yohimbine, rauwolscine, atipamezole and idazoxan readily discriminate between α1- and α2-adrenoceptors, but they show poor selectivity among α2-adrenoceptor subtypes. This is true also for a large number of other α2-adrenoceptor ligands (Audinot et al., 2002; Gentili et al., 2007). However, some recent advances on this front have opened up new possibilities in α2-adrenoceptor research.

    • Favourable involvement of α <inf>2A</inf>-adrenoreceptor antagonism in the I <inf>2</inf>-imidazoline binding sites-mediated morphine analgesia enhancement

      2012, Bioorganic and Medicinal Chemistry
      Citation Excerpt :

      The increased tail-flick latency was still observed 120 min after the administration. On the contrary, confirming previous data,15 the known α2-AR non subtype selective antagonists idazoxan or yohimbine,7,16 used in this test as reference compounds, were devoid of this effect. These results, supporting Scheinin and co-worker’s hypothesis,13 suggested the favourable role played by the inhibition of the sole α2A-AR subtype in morphine analgesia enhancement induced by 2.

    • From the cell to the clinic: A comparative review of the partial D<inf>2</inf>/D<inf>3</inf> receptor agonist and α<inf>2</inf>-adrenoceptor antagonist, piribedil, in the treatment of Parkinson's disease

      2010, Pharmacology and Therapeutics
      Citation Excerpt :

      By contrast, talipexole behaved as a (partial) agonist, with about half the efficacy of NA itself (Fig. 5) (Millan et al., 2001; Newman-Tancredi et al., 2002a). This pattern of effects could be reproduced employing antibodies specifically directed against Gαi3 or Gαo, major G-protein isoforms activated by α2-ARs (Wise et al., 1997; Audinot et al., 2002). Piribedil displayed marginal efficacy alone at Gαi3 and blocked its activation by hα2A- and hα2C-ARs, while behaving as a pure antagonist with zero efficacy at Gαo (Fig. 6).

    View all citing articles on Scopus
    1

    Present address: EntoMed SA, Rue Tobias Stimmer, 67400 Illkirch, France.

    2

    Present address: Sanofi-Synthelabo Recherche Département Cardiovasculaire Thrombose 1, Avenue Pierre Brossolette, 91385 Chilly-Mazarin Cedex, France.

    3

    Present address: Sanofi-Synthelabo Recherche Département de Génomique Cardiovasculaire 1, Avenue Pierre Brossolette, 91385 Chilly-Mazarin Cedex, France.

    4

    Present address: Aventis, centre de Vitry, 13, quai Jules Guesde, 94403 Vitry-sur-Seine Cedex, France.

    View full text