Introduction

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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 ₂-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 ₂-AR subtypes, _2A-ARs are broadly distributed throughout these regions, _2B-ARs are largely restricted to the thalamus, and _2C-ARs are concentrated in hippocampus, cortex, and, notably, striatum (Nicholas et al., 1997). Correspondingly, _2A-ARs (and _2C-ARs) are principally implicated in the above-specified functional roles of adrenergic pathways. Furthermore, _2A-ARs predominate as tonically active, inhibitory autoreceptors on adrenergic neurons, although a complementary role of _2C-AR autoreceptors has also been proposed (Kable et al., 2000; Millan et al., 2000a,b). _2A-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 ₂-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, ₂-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, ₂-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, ₂-AR antagonists facilitate antiparkinsonian actions of L-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 ₂-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 ₂-ARs (autoreceptors) may be favorable for its treatment. However, ₂-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 ₂-AR antagonists with L-dihydroxyphenylalanine or dopaminergic agents (Brefel-Courbon et al., 1998; Henry et al., 1999). Alternatively, ₂-AR antagonist and D₂ agonist properties might be incorporated into a single molecule. In fact, although data remain fragmentary, certain antiparkinsonian agents do interact with ₂-ARs. Notably, the ergot derivatives bromocriptine, cabergoline, and pergolide. However, they are also potent agonists at 5-hydroxytryptamine (serotonin) (5-HT)_2A and 5-HT_2C receptors, so any role of ₂-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 ₂-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-HT_1A 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 ₂-ARs have not been evaluated. The present study undertook, thus, a comprehensive in vitro and in vivo investigation of this issue.

	Materials and Methods

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Binding Studies. Affinities at native, rat D₂ and ₂-ARs, cloned h2A-, h_2B-, and h_2C-ARs, as well as other sites, were determined using conventional procedures described in detail elsewhere (Millan et al., 2000b,c). Conditions are summarized in Table 1. Isotherms were analyzed by nonlinear regression analysis and IC₅₀ values calculated using the program PRISM (GraphPad Software, San Diego, CA). IC₅₀ values were converted into K_i values in accordance with the equation K_i = IC₅₀/(1 + L/K_d), where L corresponds to the radioligand concentration and K_d is its dissociation constant.

Modulation of [³⁵S]GTPS Binding at Cloned, CHO-Expressed h₂-AR Subtypes. The procedure used has been documented in detail elsewhere (Millan et al., 2000b,c). Briefly, [³⁵S]GTPS (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 the K_b 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 ₂-ARs: [³⁵S]GTPS Autoradiography. [³⁵S]GTPS 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]GTPS, 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 to ³⁵S sensitive film. Binding densities were measured by computerized densitometry and ¹⁴C standard Microscales.

Actions at Porcine (p)_2A-AR Fusion Proteins. Fusion proteins were constructed and (transiently) expressed as detailed previously (Jackson et al., 1999). Briefly, Gi1 was coupled to the p_2A-AR (a generous gift of L. E. Limbird, Vanderbilt University, Nashville, TN) and spliced into the KpnI and EcoRI sites of the eukaryotic expression vector pcDNA to yield p_2A-AR-Gi1 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 Gi1 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 h₂-ARs. CHO cells expressing h_2A 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 pp42^mapk (extracellular signal receptor-activated kinase 2) and pp44^mapk (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 h_1A-ARs: Inhibition of NE-Induced [³H]Phosphatidylinositol (PI) Depletion. The influence of piribedil upon the activity of phospholipase C coupled to h_1A-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 of L-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 ₂-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.

	Results

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Binding Profile (Fig. 1; Table 1). Piribedil yielded pK_i values of 6.74 and 6.88, respectively, at striatal, rat D₂ receptors and cloned, CHO-transfected hD₂ receptors. At native, rat, cortical ₂-ARs, piribedil showed a pK_i of 6.36. Furthermore, the affinity of piribedil for cloned, h_2A-ARs (pK_i = 7.05) was slightly higher than its affinity at hD₂ receptors. Piribedil likewise manifested marked affinity for h_2C-ARs (7.16). However, it showed somewhat lower affinity for h_2B-AR (6.54). At native, cortical, rat ₁-ARs, the affinity of piribedil was weak (5.37), and its affinity was similarly modest at h_1A- and h_1B-ARs (6.09 and 5.21, respectively), although it showed higher affinity for h_1D-ARs (6.66). Piribedil manifested negligible (pK_i = <5.0) affinity for cloned h₁- and h₂-ARs, as well as for monoamine oxidases A and B and native, rat and cloned, human NE transporters.

View larger version (14K): [in this window] [in a new window]   Fig. 1.   Interaction of piribedil at native, rat (cortical) 2-ARs, compared with native, rat (striatal) D2 receptors and at cloned, hD2 compared with h2A-ARs. Data show isotherms for displacement of [3H]raclopride binding to D2 receptors and displacement of [3H]RX821,002 binding to 2-ARs. Data are representative of at least three experiments, each of which was performed in triplicate.

Antagonist Properties at CHO-Transfected h₂-ARs: Inhibition of NE-Stimulated [³⁵S]GTPS Binding (Figs. 2 and 3). NE elicited a marked (ca. 8-fold) increase in [³⁵S]GTPS binding at h_2A-ARs with a pEC₅₀ value 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]GTPS binding with a pK_b of 6.50. In addition, in the presence of incremental concentrations of piribedil, the concentration-response relationship for induction of [³⁵S]GTPS 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 pA₂ value of 6.54 close to its pK_i (7.05) and pK_b (6.50). At h_2C-ARs, NE elicited a 2-fold (pEC₅₀ = 6.52) enhancement of [³⁵S]GTPS binding, which was concentration dependently abolished by piribedil (pK_b = 6.87). Piribedil did not itself modulate [³⁵S]GTPS binding. At h_2B-ARs, NE elevated [³⁵S]GTPS binding by 7.6-fold with a pEC₅₀ of 6.30, whereas piribedil was inactive. At h_2B-ARs, in contrast to h_2A- and h_2C-ARs, piribedil only marginally attenuated the stimulatory influence of NE, in line with its relatively low affinity at these sites (vide supra).

View larger version (24K): [in this window] [in a new window]   Fig. 2.   Blockade by piribedil of NE-induced [35S]GTPS binding at CHO-transfected h2A-, h2B-, and h2C-ARs. Left, enhancement of [35S]GTPS 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.

View larger version (14K): [in this window] [in a new window]   Fig. 3.   Schild analysis of the concentration-dependent antagonism by piribedil of the induction of [35S]GTPS binding by NE at CHO-transfected h2A-ARs. Left, concentration-response curve for stimulation of [35S]GTPS binding by NE at h2A-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 p_2A-AR-Gi1 Fusion Proteins (Figs. 4 and 5). In HEK293 cells transiently expressing p_2A-AR-Gi1-C351C (wild-type), p_2A-AR-Gi1-C351G, or p_2A-AR-Gi1-C351I fusion proteins, the influence of piribedil upon high-affinity GTPase activity was compared with that of NE, epinephrine, and the prototypical ₂-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 p_2A-AR-Gi1-C351I fusion protein is illustrated in Fig. 5. At the p_2A-AR-Gi1-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 (10⁹-10⁴ M). At the "low-sensitivity" p_2A-AR-Gi1-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" p_2A-AR-Gi1-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. 

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Actions of piribedil at HEK293 cell-transfected p2A-AR-Gi1 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 p2A-AR-Gi1-Cys351C fusion protein. B, induction of high-affinity GTPase activity at a mutant p2A-AR-Gi1-Cys351G fusion protein. C, induction of high-affinity GTPase activity at a mutant p2A-AR-Gi1-Cys359I fusion protein. Data are from representative experiments performed in triplicate, which were repeated on two separate occasions with identical results.

View larger version (13K): [in this window] [in a new window]   Fig. 5.   Concentration-dependent influence of piribedil upon high-affinity GTPase activity at a mutated p2A-AR-Gi1-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 p_2AAR-Gi1 Fusion Proteins (Fig. 5). At the p_2A-AR-Gi1-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 h_2A-ARs (Figs. 6 and 7). In CHO cells stably expressing h_2A-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 ₂-AR antagonist atipamezole. Pertussis toxin also abolished the actions of NE and piribedil.

View larger version (31K): [in this window] [in a new window]   Fig. 6.   Influence of piribedil upon MAPK activity at cloned CHO-transfected h2A-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 2-AR antagonist atipamezole and (except for clonidine) by pertussis toxin is shown. The experiment was performed on three occasions, each yielding identical results.

View larger version (19K): [in this window] [in a new window]   Fig. 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]GTPS Binding at Cerebral ₂-ARs (Figs. 8 and 9). NE (10 µM) elicited a pronounced increase in [³⁵S]GTPS 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]GTPS binding. Indeed, it elicited a mild, although nonsignificant, depression of basal binding.

View larger version (91K): [in this window] [in a new window]   Fig. 8.   Influence of piribedil compared with NE upon [35S]GTPS binding at 2-ARs localized in the lateral septum. Upper left, basal binding of [35S]GTPS. Upper right, binding of [35S]GTPS in the presence of NE (10 µM). Lower left, binding of [35S]GTPS 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.

View larger version (27K): [in this window] [in a new window]   Fig. 9.   Inhibition by piribedil of the facilitatory influence of NE upon [35S]GTPS binding at 2-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]GTPS 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 pairs t test.

Antagonist Properties at h₁-ARs: Blockade of NE-Induced [³H]PI Depletion (Fig. 10). In CHO cells stably expressing h_1A-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 pK_b of 5.59 ± 0.13. 

View larger version (12K): [in this window] [in a new window]   Fig. 10.   Antagonism by piribedil of the depletion of [3H]PI evoked by NE at cloned h1A-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.

View larger version (22K): [in this window] [in a new window]   Fig. 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 precursor L-dihydroxyphenylalanine in rats pretreated with the decarboxylase inhibitor NSD1015. Absolute levels of L-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).

View larger version (30K): [in this window] [in a new window]   Fig. 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 ₂-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.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

Binding Profile at ₂-AR Subtypes Compared with D₂ Receptors. Although certain agents differentiate rat _2A- from h_2A-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 h_2A- and h_2C-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 h_2B-ARs. Although this difference was not marked and any functional significance remains to be elucidated, relatively modest (antagonist) activity at h_2B- versus h_2A/2C-ARs was also indicated by [³⁵S]GTPyS studies discussed below. Inasmuch as agonist properties of piribedil at dopamine D₂ receptors 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 ₂-ARs were similar to affinities at D₂ sites.

Antagonism of NE-Induced [³⁵S]GTPS Binding at ₂-ARs. Pertussis toxin-sensitive coupling of ₂-ARs to Gi proteins can be quantified by binding of [³⁵S]GTPS, which recognizes the -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]GTPS binding by NE at h_2A- and h_2C-ARs. These antagonist properties were expressed competitively inasmuch as piribedil displaced the concentration-response curve for NE at h_2A-ARs in parallel to the right. Autoradiographical techniques allowing visualization of ₂-AR-coupled G proteins in cerebral tissue have recently been developed (Happe et al., 2000). This approach demonstrated that, like the selective ₂-AR antagonist atipamezole (Newman-Tancredi et al., 2000), piribedil antagonizes induction of [³⁵S]GTPS binding by NE in insular cortex, amygdala, and septum. Inasmuch as these structures possess a high density of _2A-ARs (Nicholas et al., 1997), their blockade likely participates to this action of piribedil, although a contribution of _2C-ARs should not be discounted. In this light, studies of the striatum, which is enriched in _2C-ARs, as well as the locus ceruleus, which primarily bears _2A-AR autoreceptors, would be of interest (Nicholas et al., 1997). Furthermore, it is unclear to what extent pre- versus postsynaptic ₂-ARs contribute to enhancement of [³⁵S]GTPS binding by NE (Happe et al., 2000; Newman-Tancredi et al., 2000). The tendency of piribedil to suppress basal [³⁵S]GTPS binding might be considered indicative of inverse agonist properties at constitutively active ₂-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 h_2A-ARs. Thus, this modest inhibitory influence of piribedil upon basal [³⁵S]GTPS binding likely reflects residual NE.

Interaction with p_2A-AR-Gi1 Fusion Proteins. Porcine _2A-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 p_2A-AR-Gi1-Cys351C (wild-type) fusion protein, underpinning the [³⁵S]GTPS 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 ₂-AR "antagonists" at mutated ₂-ARs (Hieble et al., 1995; Pauwels et al., 2000). Piribedil might, in theory, stimulate [³⁵S]GTPS binding at wild-type _2A-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 h₂-AR-Mediated MAPK Activity. In line with the latter possibility, partial agonist properties of piribedil at wild-type h_2A-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]GTPS/GTPase measures of efficacy at wild-type h_2A-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]GTPS autoradiography (vide supra) and functional studies (vide infra), piribedil displays robust antagonist properties at cerebral ₂-ARs, including highly sensitive _2A-AR autoreceptors.

Interaction with ₁-ARs. Although piribedil displayed antagonist properties at h_1A-ARs, this action was expressed weakly. Furthermore, in contrast to ₁-AR antagonists, which interact with excitatory ₁-ARs on raphe serotoninergic neurons (Millan et al., 2000a), piribedil failed to suppress dialysate levels of 5-HT (data not shown). Blockade of ₁-ARs is, thus, unlikely to play an important role in the functional actions of piribedil. Indeed, antagonism of ₁-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 ₂-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 ₂-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 _2A-ARs in modulation of adrenergic transmission, although _2C-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 _2A- and _2C-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-HT_1A 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-HT_1A receptors (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 -ARs, NE transporters and monoamine oxidases influence extracellular levels of NE (Millan et al., 2000a), piribedil showed negligible affinity for these sites.

Influence upon ₂-AR-Mediated Sedation. Engagement of ₂-AR autoreceptors elicits sedation (Hayashi and Maze, 1993; Millan et al., 1994, 2000b; Kable et al., 2000) and, in analogy to other ₂-AR antagonists, piribedil suppressed induction of LRR by xylazine. In distinction, ₁-AR antagonists enhance sedative actions of ₂-AR agonists (Hayashi and Maze, 1993; Millan et al., 2000b). Correspondingly, these data emphasize that ₂-AR antagonist properties of piribedil outweigh its weak blockade of ₁-ARs. Activation of D₂/D₃ receptors 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.

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 ₂-ARs (Hieble et al., 1995). Importantly, further, piribedil shows similar affinity for ₂-ARs and D₂ receptors. Together with agonist actions at D₂ receptors, blockade of ₂-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 h_2B-ARs, the relative role of (cerebral) _2A- compared with _2C-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 ₂-AR blockade in the management of Parkinson's disease.