Antiparkinsonian Agent Piribedil Displays Antagonist Properties at Native, Rat, and Cloned, Human α2-Adrenoceptors: Cellular and Functional Characterization

  1. Mark J. Millan1,
  2. Didier Cussac1,
  3. Graeme Milligan3,
  4. Craig Carr3,
  5. Valérie Audinot2,
  6. Alain Gobert1,
  7. Franćoise Lejeune1,
  8. Jean-Michel Rivet1,
  9. Mauricette Brocco1,
  10. Delphine Duqueyroix1,
  11. Jean-Paul Nicolas2,
  12. Jean A. Boutin2 and
  13. Adrian Newman-Tancredi1
  1. Departments of 1Psychopharmacology (M.J.M., D.C., A.G., F.L., J.-M.R., M.B., D.D., A.N.-T.) and 2Molecular and Cellular Pharmacology (V.A., J.-P.N., J.A.B.), Institut de Recherches Servier, Centre de Recherches de Croissy, Paris, France; and 3Division of Biochemistry and Molecular Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (G.M., C.C.)
     
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    Abstract

    Compared with cloned, human (h)D2 receptors (pKi = 6.9), the antiparkinsonian agent piribedil showed comparable affinity for hα2A- (7.1) and hα2C- (7.2) adrenoceptors (ARs), whereas its affinity for hα2B-ARs was less marked (6.5). At hα2A- and hα2C-ARs, piribedil antagonized induction of [35S]guanosine-5′-O-(3-thio)triphosphate (GTPγS) binding by norepinephrine (NE) with pKb values of 6.5 and 6.9, respectively. Furthermore, Schild analysis of the actions of piribedil at hα2A-ARs indicated competitive antagonism, yielding a pA2 of 6.5. At a porcine α2A-AR-Gi1α-Cys351C (wild-type) fusion protein, piribedil competitively abolished (pA2 = 6.5) GTPase activity induced by epinephrine. However, at a α2A-AR-Gi1α-Cys351I (mutant) fusion protein of amplified sensitivity, although still acting as a competitive antagonist (pA2 = 6.2) of epinephrine, piribedil itself manifested weak partial agonist properties. Similarly, piribedil weakly induced mitogen-activated protein kinase phosphorylation via wild-type hα2A-ARs, although attenuating its phosphorylation by NE. As demonstrated by functional [35S]GTPγS autoradiography in rats, piribedil antagonized activation by NE of α2-ARs in cortex, amygdala, and septum. Antagonist properties were also expressed in a dose-dependent enhancement of the firing rate of adrenergic neurons in locus ceruleus (0.125–4.0 mg/kg i.v.). Furthermore, piribedil (2.5–4.0 mg/kg s.c.) accelerated hippocampal NE synthesis, elevated dialysis levels of NE in hippocampus and frontal cortex, and blocked hypnotic-sedative properties of the α2-AR agonist xylazine. Finally, piribedil showed only modest affinity for rat α1-ARs (5.9) and weakly antagonized NE-induced activation of phospholipase C via hα1A-ARs (pKb = 5.6). In conclusion, piribedil displays essentially antagonist properties at cloned, human and cerebral, rat α2-ARs. Blockade of α2-ARs may, thus, contribute to its clinical antiparkinsonian profile.

    Parkinson's disease is characterized by massive degeneration of dopaminergic cell bodies in the substantia nigra pars compacta and a profound depletion of dopamine (DA) in the striatum (Hornykiewicz and Kish, 1986; Sian et al., 1999). This loss of nigrostriatal dopaminergic innervation elicits a spectrum of motor symptoms, including bradykinesia, rigidity, tremor, impaired gait, and postural instability. Parkinsonian patients also display depressed mood and cognitive deficits (Sian et al., 1999). Symptomatic treatment with l-dihydroxyphenylalanine, which is metabolized into DA, still provides the mainstay of management (Jenner, 1995; Montastruc et al., 1996). Unfortunately, however, upon prolonged exposure, its efficacy fluctuates and it is poorly effective against certain symptoms, such as cognitive dysfunction (Hurtig, 1997). Moreover, l-dihydroxyphenylalanine may be neurotoxic through transformation to 6-hydroxydopamine and elicits both autonomic side effects and dyskinesia (Jenner, 1995; Hurtig, 1997). Direct dopaminergic agonists provide advantages in terms of potential neuroprotective properties and a lesser propensity to elicit dyskinesia (Jenner, 1995; Montastruc et al., 1996). However, they elicit psychiatric side effects and efficacy upon long-term monotherapy remains under evaluation (Hurtig, 1997; Rascol et al., 2000). These observations justify efforts to identify strategies other than restitution of dopaminergic activity for relief of Parkinson's disease. In this regard, there is much interest in adrenergic mechanisms and α2-ARs.

    First, reflecting their innervation of corticolimbic structures, the thalamus and basal ganglia, adrenergic pathways play an important role in the control of motor behavior, mood, cognition, and attention (Arnsten et al., 1998; Brefel-Courbon et al., 1998; Millan et al., 2000a,b,c). Regarding α2-AR subtypes, α2A-ARs are broadly distributed throughout these regions, α2B-ARs are largely restricted to the thalamus, and α2C-ARs are concentrated in hippocampus, cortex, and, notably, striatum (Nicholas et al., 1997). Correspondingly, α2A-ARs (and α2C-ARs) are principally implicated in the above-specified functional roles of adrenergic pathways. Furthermore, α2A-ARs predominate as tonically active, inhibitory autoreceptors on adrenergic neurons, although a complementary role of α2C-AR autoreceptors has also been proposed (Kable et al., 2000; Millan et al., 2000a,b). α2A-ARs are also implicated in the inhibition of frontocortical and, possibly, subcortical dopaminergic pathways (Grenhoff and Svensson, 1988; Briley and Marien, 1994; De Villiers et al., 1995; Millan et al., 2000a,b,c), as well as corticolimbic serotonergic projections (Millan et al., 2000a,b,c). Modulation of dopaminergic and serotonergic transmission may also, thus, contribute to the control of motor behavior, mood, and cognition by α2-ARs.

    Second, parkinsonian patients show a loss of locus ceruleus localized adrenergic neurons (Hornykiewicz and Kish, 1986; Sandyk and Iacono, 1990; Brefel-Courbon et al., 1998). This depletion of NE, which is seen in the cortex (notably in the motor cortex), in limbic structures (for example, in the nucleus accumbens), and in the spinal cord, aggravates the motor, emotional, cognitive, and sensory deficits of Parkinson's disease (preceding citations).

    Third, α2-AR antagonists potentiate induction of rotation by dopaminergic agonists in rats bearing unilateral lesions of the substantia nigra (Mavridis et al., 1991a; Chopin et al., 1999). They also enhance the ability of dopaminergic agonists to alleviate perturbation of motor functions provoked by reserpine and haloperidol (Brefel-Courbon et al., 1998; M. Brocco, unpublished observations). Furthermore, in primates, α2-AR antagonists attenuate motor symptoms elicited by the neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) (Colpaert et al., 1990; Bezard et al., 1999), whereas lesions of the locus ceruleus exacerbate pathological changes and delay recovery (Mavridis et al., 1991b; Bing et al., 1994). Moreover, α2-AR antagonists facilitate antiparkinsonian actions ofl-dihydroxyphenylalanine in this model while simultaneously suppressing its dyskinetic side effects (Bezard et al., 1999; Henry et al., 1999; Grondin et al., 2000).

    Fourth, small-scale clinical studies in parkinsonian patients suggest a modest improvement upon administration of α2-AR antagonists (Peyro-Saint-Paul et al., 1997; Ruzicka et al., 1997). Moreover, Rascol et al. (1997) reported that coadministration of idazoxan improves l-dihydroxyphenylalanine-elicited dyskinesia.

    Collectively, the above-mentioned data indicate that a deficiency of adrenergic transmission may contribute to motor, cognitive, and/or emotional symptoms of Parkinson's disease, and that blockade of α2-ARs (autoreceptors) may be favorable for its treatment. However, α2-AR antagonist properties alone may be insufficient to control Parkinson's disease, and their association with D2 agonist actions offers a more realistic prospect for improved treatment. This might be achieved by adjunctive use of α2-AR antagonists withl-dihydroxyphenylalanine or dopaminergic agents (Brefel-Courbon et al., 1998; Henry et al., 1999). Alternatively, α2-AR antagonist and D2agonist properties might be incorporated into a single molecule. In fact, although data remain fragmentary, certain antiparkinsonian agents do interact with α2-ARs. Notably, the ergot derivatives bromocriptine, cabergoline, and pergolide. However, they are also potent agonists at 5-hydroxytryptamine (serotonin) (5-HT)2A and 5-HT2Creceptors, so any role of α2-ARs in their functional profiles remains unclear (DeMarinis and Hieble, 1989;Seyfried and Boettcher, 1990). Furthermore, other agents, such as talipexole, are efficacious agonists at α2-ARs (Meltzer et al., 1989; Gessi et al., 1999; A. Newman-Tancredi, unpublished observations).

    The dopaminergic agonist piribedil (Trivastal), which is used clinically for the treatment of Parkinson's disease (Rondot and Ziegler, 1992; Smith et al., 2000), is of particular interest inasmuch as its arylpiperazine structure differs markedly from other antiparkinsonian agents. Moreover, with the exception of weak partial agonist activity at h5-HT1A receptors, piribedil possesses negligible affinity for serotonergic receptors and other sites (Dourish, 1983; DeMarinis and Hieble, 1989; Seyfried and Boettcher, 1990; A. Newman-Tancredi, unpublished observations). To date, however, potential actions of piribedil at α2-ARs have not been evaluated. The present study undertook, thus, a comprehensive in vitro and in vivo investigation of this issue.

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    Materials and Methods

    Binding Studies.

    Affinities at native, rat D2 and α2-ARs, cloned hα2A-, hα2B-, and hα2C-ARs, as well as other sites, were determined using conventional procedures described in detail elsewhere (Millan et al., 2000b,c). Conditions are summarized in Table1. Isotherms were analyzed by nonlinear regression analysis and IC50 values calculated using the program PRISM (GraphPad Software, San Diego, CA). IC50 values were converted intoKi values in accordance with the equation Ki = IC50/(1 +L/Kd), where Lcorresponds to the radioligand concentration andKd is its dissociation constant.

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    Table 1

    Affinities (pKis values) of piribedil at diverse adrenoceptor subtypes and related sites

    Modulation of [35S]GTPγS Binding at Cloned, CHO-Expressed hα2-AR Subtypes.

    The procedure used has been documented in detail elsewhere (Millan et al., 2000b,c). Briefly, [35S]GTPγS (1000 Ci/mmol; Amersham Pharmacia Biotech, Les Ulis, France) was used at a concentration of 0.1 nM. Samples (containing 50 μg of protein) were incubated for 60 min at 22°C. The buffer composition was as follows: 20 mM HEPES (pH 7.4), 100 mM NaCl, 3 μM GDP, and 3 mM MgSO4. Incubations were terminated by rapid filtration through Whatman GF/B filters using a Filter Harvester (Packard, Meriden, CT). Radioactivity retained on the filters was quantified by liquid scintillation counting. Antagonist properties of piribedil against fixed concentrations of NE, IC50 values were determined, and theKb calculated as described previously (Newman-Tancredi et al., 1998). In additional antagonist studies, the concentration-response curve for NE was performed in the presence of incremental concentrations of piribedil and Schild Analysis performed to yield pA2 values.

    Actions at Cerebral α2-ARs: [35S]GTPγS Autoradiography.

    [35S]GTPγS autoradiography was carried out as described by Newman-Tancredi et al. (2000). Briefly, slides with three to four brain sections were incubated for 60 min in 50 mM HEPES (pH 7.5), 150 mM NaCl, 0.2 mM EGTA, 0.2 mM dithiothreitol, 2.5 mM GDP, 10 mM MgCl2, 0.05 nM [35S]GTPγS, plus agonist/antagonist ligands. Following incubation, sections were washed with ice-cold buffer and then dipped into ice-cold deionized distilled water. The slides were dried and placed in X-ray cassettes apposed to35S sensitive film. Binding densities were measured by computerized densitometry and 14C standard Microscales.

    Actions at Porcine (p)α2A-AR Fusion Proteins.

    Fusion proteins were constructed and (transiently) expressed as detailed previously (Jackson et al., 1999). Briefly, Gi1α was coupled to the pα2A-AR (a generous gift of L. E. Limbird, Vanderbilt University, Nashville, TN) and spliced into theKpnI and EcoRI sites of the eukaryotic expression vector pcDNA to yield pα2A-AR-Gi1α fusion proteins in pCDNA3. HEK293 cells were grown to confluency (18–24 h) before transfection with pcDNA3 (2.5–2.8 μg). Two days following transfection, cells were harvested. Three different Gi1α sequences were used: the wild-type (cysteine) (Cys351C) form, a Cys351G (glycine) mutant, and a Cys351I (isoleucine) mutant. Cells expressing the two mutant forms were treated for 24 h before harvesting with pertussis toxin (50 ng/ml). Cells were maintained at −80°C and high-affinity GTPase assays performed on membrane-containing particulate fractions (Jackson et al., 1999). Nonspecific GTPase activity was evaluated in parallel with assays containing GTP (100 μM). Experiments were performed three times on membranes derived from individual cell transfections.

    Influence upon Mitogen-Activated Protein Kinase (MAPK) Activity Coupled to hα2-ARs.

    CHO cells expressing hα2A receptors were grown as previously described (Millan et al., 2000b,c). For MAPK determinations, the procedure was essentially as described in Cussac et al. (1999). Cells were grown in six-well plates until confluent. The cells were then washed twice with serum-free medium and incubated overnight in this medium. Drugs were diluted in the serum-free medium and added to cells to obtain the appropriate final concentration. For antagonist studies, cells were preincubated for 10 min with atipamezole and then stimulated with either NE or piribedil for 5 min. To study the antagonist actions of piribedil, it was added together with NE for a period of 5 min. At the end of incubation periods, 0.25 ml/well of Laemmi sample buffer containing 200 mM dithiothreitol was added. Whole cell lysates were boiled for 3 min at 95°C. In experiments with pertussis toxin, cells were treated overnight in serum-free medium with a concentration of 100 ng/ml pertussis toxin. Cell extracts (14 μl) were loaded on 15-well 10% polyacrylamide gels and “fully” activated MAPK was revealed using a monoclonal antibody specifically raised against the phosphorylated pp42mapk (extracellular signal receptor-activated kinase 2) and pp44mapk(extracellular signal receptor-activated kinase 1) forms on both threonine and tyrosine residues (NanoTools, Denzlingen, Germany), followed by enhanced chemiluminescence detection with horseradish peroxidase as a secondary antibody (Amersham Pharmacia Biotech). All autoradiograms were analyzed by computerized densitometry using AIS software, (Imaging Research, St. Catherine's, ON, Canada).

    Antagonist Properties at hα1A-ARs: Inhibition of NE-Induced [3H]Phosphatidylinositol (PI) Depletion.

    The influence of piribedil upon the activity of phospholipase C coupled to hα1A-ARs was determined using [3H]PI depletion. CHO cells were loaded with [3H]myoinositol and incubated in 96-well plates at 37°C for 30 min with NE or piribedil in Krebs-LiCl buffer. For antagonist studies, cells were preincubated (5 min) with piribedil prior to NE (30 μM). Assays were stopped with 0.4 ml of methanol/HCl (88 ml of 100% methanol + 12 ml of 1 N HCl). Cells were stored at −20°C for 2 h to facilitate cell lysis. Plates were sonicated for 2 min and membranes recovered with a Filtermate harvester (Packard) through GF/B filters impregnated with 0.1% v/v polyethyleneimine followed by three washes with distilled, deionized water. Radioactivity was determined using a Top-Count microplate (Packard). In the absence of NE, ∼40,000 dpm was typically detected compared with ∼25,000 in its presence (30 μM).

    Animals.

    Unless otherwise specified, these studies used male Wistar rats of 200 to 250 g housed in sawdust-lined cages with unrestricted access to standard chow and water. There was a 12-h light/dark cycle with lights on at 7.30 AM. Laboratory temperature and humidity were 21 ± 0.5°C and 60 ± 5%, respectively. Animals were adapted to laboratory conditions for at least a week prior to testing. All animal use procedures conformed to international European ethical standards (86/609-EEC) and the French National Committee (décret 87/848) for the care and use of laboratory animals.

    Influence upon Electrical Activity Cell Bodies in Locus Ceruleus.

    As described previously (Millan et al., 2000b,c), following anesthesia with chloral hydrate (400 mg/kg i.p.), rats were placed in a stereotaxic apparatus and a tungsten microelectrode lowered into the locus ceruleus. Coordinates were as follows: AP, −1.2 from zero; L, 1.2; and DV, −5.5/−6.5 from dura. Neurons were characterized by 1) their distinctive waveform (with a notch on the final ascending component), and 2) induction upon contralateral paw pinch of an acceleration in firing rate followed by a short silence. Following baseline recording (≥5 min), vehicle or piribedil was administered i.v. (in a volume of 0.5 ml/kg) in cumulative doses every 2 to 3 min. Drug effects were quantified over the 60-s bin corresponding to their time of peak action. Spike2 software (CED, Cambridge, England) was used for data acquisition and analysis. Data are expressed as a percentage of change from baseline firing rate (defined as 0%). Data were analyzed by two-way ANOVA followed by Newman-Keuls test for paired data and the ID50 values [95% confidence limits (CL)] calculated.

    Influence upon Extracellular Levels of NE and 5-HT.

    As previously described (Millan et al., 2000b,c), the guide cannula CMA11 was implanted 1 week prior to experimentation under pentobarbital anesthesia (60.0 mg/kg i.p.) at the following coordinates: FCX: AP, +2.2 from bregma; L, ±0.6; and DV, −0.2 from dura; and dorsal hippocampus: AP, −3.6 from bregma; L, ±1.2; and DV, −2.3 from dura. A cuprophane CMA/11 probe (4 mm in length for the FCX and 2 mm in length for the hippocampus, and 0.24-mm outer diameter) was lowered into position. Two hours after implantation, three basal samples of 20 min each were taken. Piribedil or vehicle was administered and samples were taken for a further 3 h. Levels of NE and 5-HT were quantified by high-performance liquid chromatography followed by coulometric detection (Millan et al., 2000b,c). The assay limit of sensitivity was 0.1 to 0.2 pg/sample for NE and 5-HT. Data were analyzed by ANOVA with sampling time as the repeated within-subject factor.

    Influence upon Cerebral Turnover of NE.

    Using a procedure detailed previously (Millan et al., 2000c), NE turnover was determined in the hippocampus, a structure enriched in NE compared with DA. The influence of piribedil and vehicle was evaluated 60 min following their administration and 30 min following injection of the decarboxylase inhibitor NSD1015 (100 mg/kg s.c.). Tissue levels ofl-dihydroxyphenylalanine were determined by high-performance liquid chromatography and electrochemical detection as previously (Millan et al., 2000b). The influence of piribedil upon levels of l-dihydroxyphenylalanine was expressed relative to vehicle (defined as 100%). Data were analyzed by ANOVA followed by Dunnett's test.

    Influence upon α2-AR-Mediated Sedation: Loss of Righting Reflex (LRR) in Rats.

    The LRR in rats was evaluated according to a scoring system described previously (Millan et al., 1994, 2000b). Briefly, rats were placed on their backs on a lab surface covered with paper wadding and their ability to right themselves was assessed as follows: score 0, normal, complete righting reflex; score 1, attempted righting reflex, turn of at least 90 degrees; score 2, attempted righting reflex, turn of less than 90 degrees; and score 3, total LRR, no attempt to turn. Xylazine (40.0 mg/kg i.p.) or vehicle was administered 30 min prior to determination of the LRR, and piribedil or vehicle was injected 30 min before xylazine. Data were analyzed nonparametrically. For induction of LRR, the percentage of rats displaying a score of 1 or higher was determined. All rats receiving vehicle showed values of zero. For antagonist studies, the percentage of animals displaying a score of 2 or less was determined. All (N = 12) rats receiving xylazine yielded values of 3. The ED50 (95% CL) was calculated.

    Drugs.

    Piribedil, HCl, and xylazine were dissolved in sterile water and injected s.c. and i.p., respectively. All drugs were synthesized internally, except NE, which was purchased from Sigma (Quentin Fallavier, France). Drug doses are in terms of the base.

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    Results

    Binding Profile (Fig. 1; Table1).

    Piribedil yielded pKi values of 6.74 and 6.88, respectively, at striatal, rat D2 receptors and cloned, CHO-transfected hD2 receptors. At native, rat, cortical α2-ARs, piribedil showed a pKi of 6.36. Furthermore, the affinity of piribedil for cloned, hα2A-ARs (pKi = 7.05) was slightly higher than its affinity at hD2 receptors. Piribedil likewise manifested marked affinity for hα2C-ARs (7.16). However, it showed somewhat lower affinity for hα2B-AR (6.54). At native, cortical, rat α1-ARs, the affinity of piribedil was weak (5.37), and its affinity was similarly modest at hα1A- and hα1B-ARs (6.09 and 5.21, respectively), although it showed higher affinity for hα1D-ARs (6.66). Piribedil manifested negligible (pKi = <5.0) affinity for cloned hβ1- and hβ2-ARs, as well as for monoamine oxidases A and B and native, rat and cloned, human NE transporters.

    Figure 1
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    Figure 1

    Interaction of piribedil at native, rat (cortical) α2-ARs, compared with native, rat (striatal) D2 receptors and at cloned, hD2 compared with hα2A-ARs. Data show isotherms for displacement of [3H]raclopride binding to D2 receptors and displacement of [3H]RX821,002 binding to α2-ARs. Data are representative of at least three experiments, each of which was performed in triplicate.

    Antagonist Properties at CHO-Transfected hα2-ARs: Inhibition of NE-Stimulated [35S]GTPγS Binding (Figs.2 and3).

    NE elicited a marked (ca. 8-fold) increase in [35S]GTPγS binding at hα2A-ARs with a pEC50value of 6.21, whereas piribedil, evaluated over an extensive range of concentrations, was inactive. Indeed, piribedil concentration dependently and completely suppressed NE (10 μM) stimulated [35S]GTPγS binding with a pKb of 6.50. In addition, in the presence of incremental concentrations of piribedil, the concentration-response relationship for induction of [35S]GTPγS binding by NE was progressively shifted in parallel to the right consistent with competitive antagonism. Schild analysis yielded a slope (1.1 ± 0.1) not significantly different from unity and a pA2value of 6.54 close to its pKi (7.05) and pKb (6.50). At hα2C-ARs, NE elicited a 2-fold (pEC50 = 6.52) enhancement of [35S]GTPγS binding, which was concentration dependently abolished by piribedil (pKb = 6.87). Piribedil did not itself modulate [35S]GTPγS binding. At hα2B-ARs, NE elevated [35S]GTPγS binding by 7.6-fold with a pEC50 of 6.30, whereas piribedil was inactive. At hα2B-ARs, in contrast to hα2A- and hα2C-ARs, piribedil only marginally attenuated the stimulatory influence of NE, in line with its relatively low affinity at these sites (vide supra).

    Figure 2
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    Figure 2

    Blockade by piribedil of NE-induced [35S]GTPγS binding at CHO-transfected hα2A-, hα2B-, and hα2C-ARs. Left, enhancement of [35S]GTPγS binding by NE compared with piribedil. Right, concentration-dependent inhibition of the actions of NE by piribedil. Data are representative of at least three experiments, each of which was performed in triplicate.

    Figure 3
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    Figure 3

    Schild analysis of the concentration-dependent antagonism by piribedil of the induction of [35S]GTPγS binding by NE at CHO-transfected hα2A-ARs. Left, concentration-response curve for stimulation of [35S]GTPγS binding by NE at hα2A-AR in the presence of incremental concentrations of piribedil. Right, Schild transformation of data. Similar data were obtained in three experiments, each of which was performed in triplicate.

    Influence upon the High-Affinity GTPase Activity of pα2A-AR-Gi1α Fusion Proteins (Figs.4 and5).

    In HEK293 cells transiently expressing pα2A-AR-Gi1α-C351C (wild-type), pα2A-AR-Gi1α-C351G, or pα2A-AR-Gi1α-C351I fusion proteins, the influence of piribedil upon high-affinity GTPase activity was compared with that of NE, epinephrine, and the prototypical α2-AR partial agonist clonidine. Their maximal effects at fixed concentrations are illustrated in Fig. 4, and the full concentration response for induction of GTPase activity by piribedil at the pα2A-AR-Gi1α-C351I fusion protein is illustrated in Fig. 5. At the pα2A-AR-Gi1α-C351C fusion protein, NE elicited a marked increase in high-affinity GTPase activity with a maximal effect defined as 100% and a pEC50 of 6.24 ± 0.12. Epinephrine similarly was a full agonist: pEC50 = 6.89 ± 0.10. In contrast, clonidine displayed a submaximal effect (35 ± 1%, pEC50 = 7.27 ± 0.18) lower than that of NE, whereas piribedil was inactive over a broad range of concentrations (10−9–10−4 M). At the “low-sensitivity” pα2A-AR-Gi1α-C351G fusion protein, higher concentrations of NE and epinephrine also behaved as agonists (pEC50 = 5.24 ± 0.03 and 5.74 ± 0.05, respectively), clonidine showed no virtually agonist activity (2 ± 1%), and piribedil was inactive. On the other hand, at a “high-sensitivity” pα2A-AR-Gi1α-C351I fusion protein, the maximal stimulation elicited by NE (pEC50 = 6.40 ± 0.05) and epinephrine (pEC50 = 6.90 ± 0.13) was marked and clonidine, although still a partial agonist, showed substantial activity (54 ± 2%, pEC50 = 7.15 ± 0.01). In this system, piribedil revealed mild (12 ± 1%) partial agonist activity in enhancing GTPase activity with a pEC50 of 6.40 ± 0.12.

    Figure 4
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    Figure 4

    Actions of piribedil at HEK293 cell-transfected pα2A-AR-Gi1α fusion proteins, as determined by a high-affinity GTPase assay. A, induction of high-affinity GTPase activity by piribedil compared with NE, epinephrine (EPI), and clonidine at a wild-type pα2A-AR-Gi1α-Cys351C fusion protein. B, induction of high-affinity GTPase activity at a mutant pα2A-AR-Gi1α-Cys351G fusion protein. C, induction of high-affinity GTPase activity at a mutant pα2A-AR-Gi1α-Cys359I fusion protein. Data are from representative experiments performed in triplicate, which were repeated on two separate occasions with identical results.

    Figure 5
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    Figure 5

    Concentration-dependent influence of piribedil upon high-affinity GTPase activity at a mutated pα2A-AR-Gi1α-Cys351I fusion protein, and inhibition of the action of epinephrine (EPI). A, concentration-dependent facilitatory influence of piribedil. B, displacement of the concentration-response curve for epinephrine to the right in the presence of incremental concentrations of piribedil. C, Schild transformation of data from B. Data are means ± S.E.M.s of three independent experiments performed in triplicate.

    Antagonism of High-Affinity GTPase Activity of pα2AAR-Gi1α Fusion Proteins (Fig. 5).

    At the pα2A-AR-Gi1α-C351I fusion protein, in the presence of incremental concentrations of piribedil, the concentration response for enhancement of GTPase activity by epinephrine was displaced in parallel to the right without any loss of maximal effect. Schild analysis of these data yielded a pA2 of 6.24 ± 0.02 and a slope (0.96 ± 0.07) not significantly different from unity, indicating competitive antagonist properties. Similar observations were obtained (data not shown) upon Schild analysis of the antagonist properties of piribedil versus epinephrine at the wild-type C351C fusion protein (pA2 = 6.36 ± 0.17) and the C351G mutant (pA2 = 6.50 ± 0.10).

    Influence upon MAPK Activity in CHO Cells Transfected with hα2A-ARs (Figs. 6 and7).

    In CHO cells stably expressing hα2A-ARs, NE concentration dependently activated (phosphorylated) MAPK with a pEC50 of 7.52 ± 0.16. Clonidine also stimulated MAPK with an efficacy similar to that of NE. Piribedil concentration dependently enhanced MAPK phosphorylation with a pEC50 of 6.41 ± 0.17, although its maximal effect was only 33 ± 7% compared with NE defined as 100%. Furthermore, piribedil concentration dependently (and partially) attenuated the stimulatory action of NE. The stimulation elicited by NE, clonidine, and piribedil was, in each case, abolished by the selective α2-AR antagonist atipamezole. Pertussis toxin also abolished the actions of NE and piribedil.

    Figure 6
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    Figure 6

    Influence of piribedil upon MAPK activity at cloned CHO-transfected hα2A-ARs. A, representative experiment is presented to illustrate the comparative influence of piribedil versus NE upon MAPK activity. B, quantification of their actions is displayed. Data are means ± S.E.M.s of three independent experiments. C and D, blockade of the actions of piribedil, clonidine, and NE by the selective α2-AR antagonist atipamezole and (except for clonidine) by pertussis toxin is shown. The experiment was performed on three occasions, each yielding identical results.

    Figure 7
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    Figure 7

    Inhibition by piribedil of the induction of MAPK by NE. A, representative experiment is presented to illustrate the inhibitory influence of piribedil upon the induction of MAPK activity by NE. B, quantification of this inhibitory influence upon the action of NE. Data are means ± S.E.M.s of three independent experiments.

    Inhibition of NE-Activated [35S]GTPγS Binding at Cerebral α2-ARs (Figs. 8and 9).

    NE (10 μM) elicited a pronounced increase in [35S]GTPγS binding as quantified in the insular cortex, amygdala, and lateral septum. This action of NE was blocked by coincubation with piribedil (100 μM). Applied alone, piribedil did not enhance [35S]GTPγS binding. Indeed, it elicited a mild, although nonsignificant, depression of basal binding.

    Figure 8
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    Figure 8

    Influence of piribedil compared with NE upon [35S]GTPγS binding at α2-ARs localized in the lateral septum. Upper left, basal binding of [35S]GTPγS. Upper right, binding of [35S]GTPγS in the presence of NE (10 μM). Lower left, binding of [35S]GTPγS in the presence of piribedil (100 μM). Lower right, inhibitory influence of piribedil upon enhancement of binding by NE. Data are representative of four independent experiments. See Fig. 9 for further analysis.

    Figure 9
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    Figure 9

    Inhibition by piribedil of the facilitatory influence of NE upon [35S]GTPγS binding at α2-ARs localized in lateral (lat) septum, insular cortex (ctx), and amygdala. Data are means ± S.E.M.s of four independent experiments for percentage [35S]GTPγS simulation relative to basal values, which were defined as 100%. These were 182 ± 8, 276 ± 37, and 244 ± 54 nCi/g tissue equivalent for insular cortex, lateral septum, and amygdala, respectively. The differences of NE to basal values and of piribedil/NE to NE values were significant (P < 0.05) in each structure in a matched pairst test.

    Antagonist Properties at hα1-ARs: Blockade of NE-Induced [3H]PI Depletion (Fig.10).

    In CHO cells stably expressing hα1A-ARs, NE elicited a dose-dependent depletion of membrane-bound [3H]PI, reflecting the positive coupling of these sites to phospholipase C. In contrast, piribedil did not modify [3H]PI levels. Indeed, it concentration dependently, albeit weakly, attenuated the action of NE with a pKb of 5.59 ± 0.13.

    Figure 10
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    Figure 10

    Antagonism by piribedil of the depletion of [3H]PI evoked by NE at cloned hα1A-ARs transfected into CHO cells. Data are representative of three independent experiments, each performed in triplicate.

    Activation of Locus Ceruleus-Adrenergic Neurons (Fig.11).

    In anesthetized rats, piribedil evoked a dose-dependent and pronounced increase in the electrical activity of locus ceruleus-localized, adrenergic cell bodies over a dose range of 0.125 to 4.0 mg/kg i.v. At its maximally effective dose (4.0), firing rate was approximately doubled relative to baseline values.

    Figure 11
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    Figure 11

    Influence of piribedil upon the electrical activity of adrenergic neurons in the locus ceruleus (LC). Top, representative neuron that illustrates the dose-dependent increase in firing rate elicited by piribedil. Bottom, dose-response relationship for activation of adrenergic neurons is shown. Data are means ± S.E.M, N = 5. ANOVA as follows.F(4,24) = 8.4, P < 0.01. *P < 0.05 to vehicle (VEH) values in Newman-Keuls test.

    Enhancement of Hippocampal Synthesis of NE.

    Piribedil dose dependently and significantly accelerated NE synthesis in the hippocampus, as quantified by determination of its precursorl-dihydroxyphenylalanine in rats pretreated with the decarboxylase inhibitor NSD1015. Absolute levels ofl-dihydroxyphenylalanine for vehicle were 0.69 ± 0.04 mg/tissue (=100.0 ± 5.3%). Expressed relative to these values, the effect of piribedil was as follows: piribedil (2.5 mg/kg s.c.) = 100.2 ± 7.1%; piribedil (10.0) = 120.7 ± 7.1%; and piribedil (40.0) = 145.3 ± 6.2%; F(3,32) = 9.0, P < 0.001.

    Elevation of Extracellular Levels of NE in Frontal Cortex and Hippocampus (Fig. 12).

    Piribedil evoked a dose-dependent (2.5–40.0 mg/kg s.c.) and marked increase in extracellular levels of NE in the FCX of freely moving rats. This action was selective inasmuch as levels of 5-HT in the same samples were not significantly elevated (data not shown). In the hippocampus, piribedil likewise elicited a dose-dependent (2.5–40.0 mg/kg s.c.) and significant increase in levels of NE without influencing those of 5-HT (data not shown).

    Figure 12
    View larger version:
    Figure 12

    Influence of piribedil upon extracellular levels of norepinephrine in the frontal cortex and dorsal hippocampus of freely moving rats. Top, frontal cortex. Bottom, dorsal hippocampus. Data are means ± S.E.M.s of NE levels expressed relative to basal, pretreatment values (defined as 100%). These were 1.25 ± 0.09 and 0.92 ± 0.09 pg/20 μl of dialysate for frontal cortex and dorsal hippocampus, respectively. N ≥ 5/value. ANOVA as follows. Frontal cortex: 2.5, F(1,12) = 0.1, P > 0.05; 5.0, F(1,15) = 5.3, P < 0.05; 10.0, F(1,14) = 19.9, P < 0.01; and 40.0,F(1,12) = 75.5, P < 0.01. Dorsal hippocampus: 2.5, F(1,14) = 0.1,P > 0.05; 10.0, F(1,14) = 10.7, P < 0.01; and 40.0,F(1,13) = 82.9, P < 0.01. Asterisks indicate significance of drug-treated groups versus vehicle-treated group. *P < 0.05.

    Inhibition of Sedative-Hypnotic Properties of α2-AR Agonist Xylazine.

    Xylazine elicited a complete LRR at a dose of 40.0 mg/kg (mean score = 3.0 ± 0.0), whereas piribedil was devoid of activity (80.0 mg/kg s.c., score = 0.0 ± 0.0). Indeed, piribedil completely (score = 0.0 ± 0.0 at 80.0 mg/kg, s.c.) and dose dependently blocked the action of xylazine with an ED50 (95% CL) of 32 (21–50) mg/kg s.c.

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    Discussion

    Binding Profile at α2-AR Subtypes Compared with D2 Receptors.

    Although certain agents differentiate rat α2A- from hα2A-ARs, like the majority of ligands, piribedil showed similar affinities for these species homologs (Renouard et al., 1994; Bylund, 1995; Hieble et al., 1995). Furthermore, although agents distinguishing hα2A- and hα2C-ARs have been documented, like most drugs (preceding citations), the affinity of piribedil for these sites was comparable. In fact, the affinity of piribedil was slightly less pronounced at hα2B-ARs. Although this difference was not marked and any functional significance remains to be elucidated, relatively modest (antagonist) activity at hα2B- versus hα2A/2C-ARs was also indicated by [35S]GTPyS studies discussed below. Inasmuch as agonist properties of piribedil at dopamine D2receptors are fundamental to its clinical, antiparkinsonian properties (Dourish, 1983; Rondot and Ziegler, 1992), it is of importance that its affinities for native and cloned, human α2-ARs were similar to affinities at D2 sites.

    Antagonism of NE-Induced [35S]GTPγS Binding at α2-ARs.

    Pertussis toxin-sensitive coupling of α2-ARs to Gi proteins can be quantified by binding of [35S]GTPγS, which recognizes the α-subunit of Gi and other G proteins (Jasper et al., 1998;Newman-Tancredi et al., 1998; Millan et al., 2000b). In line with its binding profile, piribedil concentration dependently abolished enhancement of [35S]GTPγS binding by NE at hα2A- and hα2C-ARs. These antagonist properties were expressed competitively inasmuch as piribedil displaced the concentration-response curve for NE at hα2A-ARs in parallel to the right. Autoradiographical techniques allowing visualization of α2-AR-coupled G proteins in cerebral tissue have recently been developed (Happe et al., 2000). This approach demonstrated that, like the selective α2-AR antagonist atipamezole (Newman-Tancredi et al., 2000), piribedil antagonizes induction of [35S]GTPγS binding by NE in insular cortex, amygdala, and septum. Inasmuch as these structures possess a high density of α2A-ARs (Nicholas et al., 1997), their blockade likely participates to this action of piribedil, although a contribution of α2C-ARs should not be discounted. In this light, studies of the striatum, which is enriched in α2C-ARs, as well as the locus ceruleus, which primarily bears α2A-AR autoreceptors, would be of interest (Nicholas et al., 1997). Furthermore, it is unclear to what extent pre- versus postsynaptic α2-ARs contribute to enhancement of [35S]GTPγS binding by NE (Happe et al., 2000; Newman-Tancredi et al., 2000). The tendency of piribedil to suppress basal [35S]GTPγS binding might be considered indicative of inverse agonist properties at constitutively active α2-ARs (Murrin et al., 2000; Pauwels et al., 2000). However, this action did not attain statistical significance and cellular models discussed below suggest that piribedil possesses weak partial agonist activity at hα2A-ARs. Thus, this modest inhibitory influence of piribedil upon basal [35S]GTPγS binding likely reflects residual NE.

    Interaction with pα2A-AR-Gi1α Fusion Proteins.

    Porcine α2A-ARs are homologous to their human counterparts (Bylund, 1995; Jackson et al., 1999) and piribedil (competitively) blocked enhancement of GTPase activity by epinephrine at a pα2A-AR-Gi1α-Cys351C (wild-type) fusion protein, underpinning the [35S]GTPγS binding studies. The pertussis toxin-sensitive Cys351 position is important in determining efficacy of coupling to the Gi protein and a decrease and increase in hydrophobicity upon replacement of cysteine by glycine and isoleucine discourages and favors this interaction, respectively (Jackson et al., 1999). Correspondingly, intrinsic efficacy of ligands is respectively blunted and amplified (Fig. 4; Jackson et al., 1999). It is, thus, intriguing that the Cys351I mutant revealed a modest enhancement in GTPase activity with piribedil, in analogy to partial agonist actions of α2-AR “antagonists” at mutated α2-ARs (Hieble et al., 1995; Pauwels et al., 2000). Piribedil might, in theory, stimulate [35S]GTPγS binding at wild-type α2A-ARs under certain conditions, such as high “receptor reserve” (Hieble et al., 1995). Since fusion proteins possess an “invariant” 1:1 receptor/G protein stoichiometry, this issue requires evaluation with other approaches.

    Modulation of hα2-AR-Mediated MAPK Activity.

    In line with the latter possibility, partial agonist properties of piribedil at wild-type hα2A-ARs were revealed by weak and pertussis toxin-sensitive phosphorylation of MAPK, a response for which clonidine behaved as a full agonist (Fig. 6) (Alblas et al., 1993; Kribben et al., 1997). This difference to [35S]GTPγS/GTPase measures of efficacy at wild-type hα2A-ARs likely reflects signal “amplification” downstream of receptor-G protein coupling. Nevertheless, in all cellular models, actions of NE and epinephrine were attenuated by piribedil. This is a crucial consideration inasmuch as NE is spontaneously released from adrenergic neurons. Indeed, as demonstrated both by [35S]GTPγS autoradiography (vide supra) and functional studies (vide infra), piribedil displays robust antagonist properties at cerebral α2-ARs, including highly sensitive α2A-AR autoreceptors.

    Interaction with α1-ARs.

    Although piribedil displayed antagonist properties at hα1A-ARs, this action was expressed weakly. Furthermore, in contrast to α1-AR antagonists, which interact with excitatory α1-ARs on raphe serotoninergic neurons (Millan et al., 2000a), piribedil failed to suppress dialysate levels of 5-HT (data not shown). Blockade of α1-ARs is, thus, unlikely to play an important role in the functional actions of piribedil. Indeed, antagonism of α1-ARs suppresses rather than facilitates motor function (Mavridis et al., 1991a; Hayashi and Maze, 1993; Millan et al., 2000b) (see below).

    Modulation of Ascending Adrenergic Transmission.

    Blockade of tonically active α2-AR autoreceptors increases electrical activity of adrenergic cell bodies and enhances NE release and synthesis in terminal structures (Trendelenburg et al., 1999;Millan et al., 2000a,b,c). Correspondingly, like α2-AR antagonists, piribedil excited locus ceruleus neurons, elevated extracellular levels of NE in FCX and hippocampus, and accelerated hippocampal NE synthesis. Collectively, anatomical, pharmacological, and genetic analyses indicate a key role of α2A-ARs in modulation of adrenergic transmission, although α2C-ARs may also contribute (Trendelenburg et al., 1999; Kable et al., 2000; Millan et al., 2000a,b). In view of antagonist actions of piribedil at both α2A- and α2C-sites, their relative importance remains to be elucidated. Inasmuch as selective D2/D3 agonists do not influence frontocortical adrenergic pathways (Millan et al., 2000a), activation by piribedil of D2/D3 sites cannot underlie its enhancement of adrenergic transmission. Stimulation of 5-HT1A autoreceptors, by reducing serotonergic transmission, disinhibits frontocortical adrenergic pathways (Millan et al., 2000a). However, this mechanism is also unlikely to be relevant since piribedil shows only low activity at 5-HT1Areceptors (Seyfried and Boettcher, 1990; A. Newman-Tancredi, unpublished observations) and failed to modify extracellular levels of 5-HT (data not shown). Finally, although actions at β-ARs, NE transporters and monoamine oxidases influence extracellular levels of NE (Millan et al., 2000a), piribedil showed negligible affinity for these sites.

    Influence upon α2-AR-Mediated Sedation.

    Engagement of α2-AR autoreceptors elicits sedation (Hayashi and Maze, 1993; Millan et al., 1994, 2000b; Kable et al., 2000) and, in analogy to other α2-AR antagonists, piribedil suppressed induction of LRR by xylazine. In distinction, α1-AR antagonists enhance sedative actions of α2-AR agonists (Hayashi and Maze, 1993; Millan et al., 2000b). Correspondingly, these data emphasize that α2-AR antagonist properties of piribedil outweigh its weak blockade of α1-ARs. Activation of D2/D3receptors is unlikely to be involved since dopaminergic agonists only variably and submaximally attenuate hypnotic sedative actions of xylazine (M. Brocco, unpublished observations).

    General Discussion.

    Several general points emerge from these studies.

    First, although piribedil is not a potent agent, its affinity at hα2A- and hα2C-ARs was comparable to that at D2 receptors. This suggests that at therapeutically relevant doses activating D2 receptors, piribedil also occupies α2A- and α2C-ARs. Thus, α2-AR blockade by piribedil likely contributes to its functional actions, although the relative implication of α2A-versus α2C-ARs remains to be clarified.

    Second, certain other antiparkinsonian agents interact with α2-ARs (Montastruc et al., 1996). However, piribedil behaves essentially as an antagonist, whereas several, such as talipexole, are efficacious agonists (Meltzer et al., 1989; Gessi et al., 1999). Moreover, apart from mild affinity at 5-HT1A sites, piribedil is devoid of activity at multiple serotonergic receptors. In contrast, other agents, such as bromocriptine, pergolide, and cabergoline, are potent agonists at 5-HT2A and 5-HT2C receptors (DeMarinis and Hieble, 1989; Seyfried and Boettcher, 1990; A. Newman-Tancredi, unpublished observations).

    Third, activation of postsynaptic α2-ARs facilitates working memory tasks integrated in FCX (Arnsten et al., 1998). This mechanism has, thus, been advocated for management of cognitive deficits in neuropsychiatric disorders. However, the active dose range is narrow and α2-AR agonists impair performance in certain cognitive tasks in humans (Arnsten et al., 1998;Jäkälä et al., 1999). Activation of postsynaptic α2-ARs also potentiated antiparkinsonian actions of a κ-opioid agonist in rats (Hill and Brotchie, 1999). However, the “quality” of movement was “poor” and, in more conventional models, α2-AR agonists interfere with antiparkinsonian actions of dopaminergic agonists in rats (Meltzer et al., 1989; Mavridis et al., 1991a; Chopin et al., 1999) and primates (Gomez-Mancilla and Bédard, 1993). Moreover, enhancement of motor function via activation of postsynaptic α2-ARs is seen only following marked depletion of endogenous pools of NE. In any event, the motor-depressant (and hypotensive), autoreceptor-mediated actions of α2-AR agonists are difficult to reconcile with their potential utilization in parkinsonian patients. Thus, α2-AR (autoreceptor) antagonism, leading to a reinforcement in (deficient) corticolimbic adrenergic transmission, represents a more realistic hypothesis for improved management of Parkinson's disease. Even if postsynaptic α2-ARs are simultaneously antagonized, favorable actions will be mediated via “functionally intact” and colocalized, postsynaptic α1- and β-ARs (Arnsten et al., 1998; Brefel-Courbon et al., 1998; Millan et al., 2000a). Furthermore, blockade of inhibitory α2-ARs on dopaminergic and serotonergic pathways should likewise be favorable (Millan et al., 2000a).

    Finally, α2-ARs engage diverse intracellular cascades via different subtypes of G protein (Bylund, 1995; Hieble et al., 1995; Brink et al., 2000). The present study focused on their principle mode of coupling via Gi. However, although Gi1α is implicated in the fusion protein-mediated activation of GTPase, the precise species of Gi transducing MAPK phosphorylation and [35S]GTPγS binding remains to be established. Thus, the influence of piribedil upon specific subclasses of Gi, and upon other G proteins (such as Gs) coupled to α2-ARs, would be of interest to evaluate further.

    Summary and Conclusions.

    Although piribedil differs structurally from imidazolines (such as idazoxan), from alkaloids (such as yohimbine), and from other prototypical antagonists, it shares their interaction with α2-ARs (Hieble et al., 1995). Importantly, further, piribedil shows similar affinity for α2-ARs and D2 receptors. Together with agonist actions at D2 receptors, blockade of α2-ARs may, thus, contribute to its functional profile: notably, its influence upon motor performance, mood, and cognitive function in Parkinson patients. This issue is currently under clinical investigation. In this regard, although piribedil shows only modest affinity at hα2B-ARs, the relative role of (cerebral) α2A- compared with α2C-ARs in its actions requires elucidation. In conclusion, piribedil provides a distinctive experimental and clinical tool for evaluation of the significance of combined D2 receptor activation and α2-AR blockade in the management of Parkinson's disease.

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    Acknowledgments

    We thank V. Pasteau, L. Verrielle, N. Fabry, L. Cistarelli, C. Melon, and H. Gressier for technical assistance. We thank M. Soubeyran for preparation of the manuscript.

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    Footnotes

    • Send reprint requests to: Dr. Mark J. Millan, Institut de Recherches Servier, Center de Recherches de Croissy, 125 chemin de Ronde, 78290 Croissy/Seine, Paris, France. E-mail:mark.millan{at}fr.netgrs.com

    • Abbreviations:
      DA
      dopamine
      AR
      adrenoceptor
      NE
      norepinephrine
      MPTP
      1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine
      5-HT
      5-hydroxytryptamine (serotonin)
      [35S]GTPγS
      guanosine-5′-O-(3-thio)triphosphate
      CHO
      Chinese hamster ovary
      HEK
      human embryonic kidney
      GTP
      guanosine triphosphate
      MAPK
      mitogen-activated-protein kinase
      PI
      phosphatidylinositol
      CL
      confidence limits
      FCX
      frontal cortex
      LRR
      loss of righting reflex
      p
      porcine
      h
      human
      • Received December 11, 2000.
      • Accepted February 1, 2001.
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