Antiparkinsonian Agent Piribedil Displays Antagonist Properties at ...

0022-3565/01/2973-876–887$3.00 

THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 297, No. 3 

Copyright © 2001 by The American Society for Pharmacology and Experimental Therapeutics 3648/903084 

JPET 297:876–887, 2001 Printed in U.S.A. 

Antiparkinsonian Agent Piribedil Displays Antagonist Properties 

at Native, Rat, and Cloned, Human � 2-Adrenoceptors: Cellular 

and Functional Characterization 

MARK J. MILLAN, DIDIER CUSSAC, GRAEME MILLIGAN, CRAIG CARR, VALÉRIE AUDINOT, ALAIN GOBERT, 

FRANĆOISE LEJEUNE, JEAN-MICHEL RIVET, MAURICETTE BROCCO, DELPHINE DUQUEYROIX, JEAN-PAUL NICOLAS, 

JEAN A. BOUTIN, and ADRIAN NEWMAN-TANCREDI 

Departments of Psychopharmacology (M.J.M., D.C., A.G., F.L., J.-M.R., M.B., D.D., A.N.-T.) and Molecular and Cellular Pharmacology (V.A., 

J.-P.N., J.A.B.), Institut de Recherches Servier, Centre de Recherches de Croissy, Paris, France; and Division of Biochemistry and Molecular 

Biology, Institute of Biomedical and Life Sciences, University of Glasgow, Glasgow, Scotland, United Kingdom (G.M., C.C.) 

Received December 11, 2000; accepted February 1, 2001 This paper is available online at http://jpet.aspetjournals.org 

ABSTRACT 

Compared with cloned, human (h)D 2 receptors (pK i � 6.9), the 

antiparkinsonian agent piribedil showed comparable affinity for 

h� 2A- (7.1) and h� 2C- (7.2) adrenoceptors (ARs), whereas its 

affinity for h� 2B-ARs was less marked (6.5). At h� 2A- and h� 2C- 

ARs, piribedil antagonized induction of [ 35 S]guanosine-5�-O-(3thio)triphosphate 

(GTP�S) binding by norepinephrine (NE) with 

pK b values of 6.5 and 6.9, respectively. Furthermore, Schild 

analysis of the actions of piribedil at h� 2A-ARs indicated competitive 

antagonism, yielding a pA 2 of 6.5. At a porcine � 2A-AR- 

Gi1�-Cys351C (wild-type) fusion protein, piribedil competitively 

abolished (pA 2 � 6.5) GTPase activity induced by epinephrine. 

However, at a � 2A-AR-Gi1�-Cys351I (mutant) fusion protein of 

amplified sensitivity, although still acting as a competitive antagonist 

(pA 2 � 6.2) of epinephrine, piribedil itself manifested 

weak partial agonist properties. Similarly, piribedil weakly induced 

mitogen-activated protein kinase phosphorylation via 

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 Ldihydroxyphenylalanine, 

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 

wild-type h� 2A-ARs, although attenuating its phosphorylation 

by NE. As demonstrated by functional [ 35 S]GTP�S autoradiography 

in rats, piribedil antagonized activation by NE of � 2-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 � 2-AR agonist xylazine. Finally, piribedil 

showed only modest affinity for rat � 1-ARs (5.9) and weakly 

antagonized NE-induced activation of phospholipase C via 

h� 1A-ARs (pK b � 5.6). In conclusion, piribedil displays essentially 

antagonist properties at cloned, human and cerebral, rat 

� 2-ARs. Blockade of � 2-ARs may, thus, contribute to its clinical 

antiparkinsonian profile. 

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, 

ABBREVIATIONS: DA, dopamine; AR, adrenoceptor; NE, norepinephrine; MPTP, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine; 5-HT, 5-hydroxytryptamine 

(serotonin); [ 35 S]GTP�S, 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. 

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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 abovespecified 

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 D 2 agonist actions offers a more realistic 

prospect for improved treatment. This might be 

achieved by adjunctive use of � 2-AR antagonists with Ldihydroxyphenylalanine 

or dopaminergic agents (Brefel- 

Courbon et al., 1998; Henry et al., 1999). Alternatively, 

� 2-AR antagonist and D 2 agonist properties might be incorporated 

into a single molecule. In fact, although data remain 

� 2-Adrenoceptors and Parkinson’s Disease 877 

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 

Binding Studies. Affinities at native, rat D 2 and � 2-ARs, cloned 

h�2A-, 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 IC 50 values 

calculated using the program PRISM (GraphPad Software, San Diego, 

CA). IC 50 values were converted into K i values in accordance 

with the equation K i � IC 50/(1 � L/K d), where L corresponds to the 

radioligand concentration and K d is its dissociation constant. 

Modulation of [ 35 S]GTP�S Binding at Cloned, CHO-Expressed 

h� 2-AR Subtypes. The procedure used has been documented 

in detail elsewhere (Millan et al., 2000b,c). Briefly, 

[ 35 S]GTP�S (1000 Ci/mmol; Amersham Pharmacia Biotech, Les Ulis, 

France) was used at a concentration of 0.1 nM. Samples (containing 

50 �g 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 �M GDP, and 3 mM MgSO 4. 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, IC 50 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 pA 2 values. 

Actions at Cerebral � 2-ARs: [ 35 S]GTP�S Autoradiography. 

[ 35 S]GTP�S 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 MgCl 2, 0.05 nM [ 35 S]GTP�S, 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 35 S sensitive film. 

Binding densities were measured by computerized densitometry and 

14 C standard Microscales. 

Actions at Porcine (p)� 2A-AR Fusion Proteins. Fusion proteins 

were constructed and (transiently) expressed as detailed pre- 



878 Millan et al. 

TABLE 1 

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

Data are means � S.E.M. of three to four determinations. 

Receptor Species Tissue Radioligand Piribedil 

D2 Rat Striatum 

nM 

[ 3 H]Spiperone (0.2) 6.74 � 0.11 

D2 Human CHO [ 125 I]Iodosulpride (0.1) 6.88 � 0.03 

�2 Rat Cortex [ 3 H]RX821,002 (0.4) 6.36 � 0.07 

�2A Human CHO [ 3 H]RX821,002 (0.8) 7.05 � 0.19 

�2B Human CHO [ 3 H]RX821,002 (4.0) 6.54 � 0.02 

�2C Human CHO [ 3 H]RX821,002 (0.6) 7.16 � 0.12 

�1 Rat Frontal cortex [ 3 H]Prazosin (0.2) 5.37 � 0.06 

�1A Human CHO [ 3 H]Prazosin (0.1) 6.09 � 0.08 

�1B Human CHO [ 3 H]Prazosin (0.1) 5.21 � 0.07 

�1D Human CHO [ 3 H]Prazosin (0.1) 6.66 � 0.11 

�1 Human Sf9 [ 3 H]CGP12177 (0.15) �5.0 

�2 Human Sf9 [ 3 H]CGP12177 (0.15) �5.0 

NET Rat Brain [ 3 H]Nisoxetine (2.0) �5.0 

NET Human CHO [ 3 H]Nisoxetine (2.0) �5.0 

MAO A Rat Brain [ 3 H]RO41-1049 (5.0) �5.0 

MAO B Rat Brain [ 3 H]RO19-6327 (15) �5.0 

NET, NE transporter; MAO, monoamine oxidase. 

viously (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 �g). 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 

�M). Experiments were performed three times on membranes derived 

from individual cell transfections. 

Influence upon Mitogen-Activated Protein Kinase (MAPK) 

Activity Coupled to h� 2-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 �l) 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 

[ 3 H]Phosphatidylinositol (PI) Depletion. The influence 

of piribedil upon the activity of phospholipase C coupled to h� 1A-ARs 

was determined using [ 3 H]PI depletion. CHO cells were loaded with 

[ 3 H]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 �M). 

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 2hto 

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 �M). 

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 ID 50 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 Ldihydroxyphenylalanine 

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 ED 50 (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 

Binding Profile (Fig. 1; Table 1). Piribedil yielded pK i 

values of 6.74 and 6.88, respectively, at striatal, rat D 2 receptors 

and cloned, CHO-transfected hD 2 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 hD 2 

Fig. 1. Interaction of piribedil at native, rat (cortical) � 2-ARs, compared 

with native, rat (striatal) D 2 receptors and at cloned, hD 2 compared with 

h� 2A-ARs. Data show isotherms for displacement of [ 3 H]raclopride binding 

to D 2 receptors and displacement of [ 3 H]RX821,002 binding to � 2-ARs. 

Data are representative of at least three experiments, each of which was 

performed in triplicate. 


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 h� 1- and h� 2-ARs, as well as for monoamine oxidases 

A and B and native, rat and cloned, human NE transporters. 

Antagonist Properties at CHO-Transfected h� 2-ARs: 

Inhibition of NE-Stimulated [ 35 S]GTP�S Binding 

(Figs. 2 and 3). NE elicited a marked (ca. 8-fold) increase in 

[ 35 S]GTP�S binding at h� 2A-ARs with a pEC 50 value of 6.21, 

whereas piribedil, evaluated over an extensive range of concentrations, 

was inactive. Indeed, piribedil concentration dependently 

and completely suppressed NE (10 �M) stimulated 

[ 35 S]GTP�S 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 [ 35 S]GTP�S 

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 pA 2 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 (pEC 50 � 6.52) 

enhancement of [ 35 S]GTP�S binding, which was concentration 

dependently abolished by piribedil (pK b � 6.87). Piribedil 

did not itself modulate [ 35 S]GTP�S binding. At h� 2B-ARs, 

NE elevated [ 35 S]GTP�S binding by 7.6-fold with a pEC 50 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). 

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 GT- 

Pase 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 

pEC 50 of 6.24 � 0.12. Epinephrine similarly was a full agonist: 

pEC 50 � 6.89 � 0.10. In contrast, clonidine displayed a 

submaximal effect (35 � 1%, pEC 50 � 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” 

p� 2A-AR-Gi1�-C351G fusion protein, higher concentrations 

of NE and epinephrine also behaved as agonists (pEC 50 

� 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 (pEC 50 � 6.40 � 0.05) and epinephrine (pEC 50 � 

6.90 � 0.13) was marked and clonidine, although still a 

partial agonist, showed substantial activity (54 � 2%, pEC 50 

� 7.15 � 0.01). In this system, piribedil revealed mild (12 � 

1%) partial agonist activity in enhancing GTPase activity 

with a pEC 50 of 6.40 � 0.12. 




Fig. 2. Blockade by piribedil of NE-induced [ 35 S]GTP�S binding at CHO-transfected h� 2A-, h� 2B-, and h� 2C-ARs. Left, enhancement of [ 35 S]GTP�S 

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. 

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 pA 2 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 epi- 

nephrine at the wild-type C351C fusion protein (pA 2 � 

6.36 � 0.17) and the C351G mutant (pA 2 � 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 pEC 50 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 pEC 50 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. 

Inhibition of NE-Activated [ 35 S]GTP�S Binding at 

Cerebral � 2-ARs (Figs. 8 and 9). NE (10 �M) elicited a 

pronounced increase in [ 35 S]GTP�S binding as quantified in 

the insular cortex, amygdala, and lateral septum. This action 

of NE was blocked by coincubation with piribedil (100 �M). 

Applied alone, piribedil did not enhance [ 35 S]GTP�S binding. 

Indeed, it elicited a mild, although nonsignificant, depression 

of basal binding. 

Antagonist Properties at h� 1-ARs: Blockade of NE- 

Induced [ 3 H]PI Depletion (Fig. 10). In CHO cells stably 

expressing h� 1A-ARs, NE elicited a dose-dependent depletion 

of membrane-bound [ 3 H]PI, reflecting the positive coupling of 

these sites to phospholipase C. In contrast, piribedil did not 

modify [ 3 H]PI levels. Indeed, it concentration dependently, 

albeit weakly, attenuated the action of NE with a pK b of 

5.59 � 0.13. 

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. 

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 


Fig. 3. Schild analysis of the concentration-dependent antagonism by piribedil of the induction of [ 35 S]GTP�S binding by NE at CHO-transfected 

h� 2A-ARs. Left, concentration-response curve for stimulation of [ 35 S]GTP�S binding by NE at h� 2A-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. 

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). 

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 ED 50 (95% CL) of 32 (21–50) mg/kg s.c. 

Discussion 

Binding Profile at � 2-AR Subtypes Compared with 

D 2 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 [ 35 S]GTPyS studies discussed 

below. Inasmuch as agonist properties of piribedil at 

dopamine D 2 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 D 2 sites. 

Antagonism of NE-Induced [ 35 S]GTP�S Binding at 

� 2-ARs. Pertussis toxin-sensitive coupling of � 2-ARs to Gi 

proteins can be quantified by binding of [ 35 S]GTP�S, 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 [ 35 S]GTP�S 

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 




Fig. 4. Actions of piribedil at HEK293 cell-transfected p� 2A-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 p� 2A-AR-Gi1�-Cys351C 

fusion protein. B, induction of high-affinity GTPase activity at a mutant 

p� 2A-AR-Gi1�-Cys351G fusion protein. C, induction of high-affinity GT- 

Pase activity at a mutant p� 2A-AR-Gi1�-Cys359I fusion protein. Data are 

from representative experiments performed in triplicate, which were 

repeated on two separate occasions with identical results. 

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., 

Fig. 5. Concentration-dependent influence of piribedil upon high-affinity 

GTPase activity at a mutated p� 2A-AR-Gi1�-Cys351I fusion protein, and 

inhibition of the action of epinephrine (EPI). A, concentration-dependent 

facilitatory influence of piribedil. B, displacement of the concentrationresponse 

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. 

2000), piribedil antagonizes induction of [ 35 S]GTP�S 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 



Fig. 6. Influence of piribedil upon MAPK activity at cloned CHO-transfected h� 2A-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. 

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. 

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 [ 35 S]GTP�S binding by NE (Happe et al., 2000; 

Newman-Tancredi et al., 2000). The tendency of piribedil to 

suppress basal [ 35 S]GTP�S 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 [ 35 S]GTP�S 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 epineph- 


rine at a p� 2A-AR-Gi1�-Cys351C (wild-type) fusion protein, 

underpinning the [ 35 S]GTP�S 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 [ 35 S]GTP�S 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 h� 2-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 

[ 35 S]GTP�S/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 [ 35 S]GTP�S autoradiography (vide supra) 

and functional studies (vide infra), piribedil displays robust 




Fig. 8. Influence of piribedil compared with NE upon [ 35 S]GTP�S binding at � 2-ARs localized in the lateral septum. Upper left, basal binding of 

[ 35 S]GTP�S. Upper right, binding of [ 35 S]GTP�S in the presence of NE (10 �M). Lower left, binding of [ 35 S]GTP�S in the presence of piribedil (100 �M). 

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. 

Fig. 9. Inhibition by piribedil of the facilitatory influence of NE upon 

[ 35 S]GTP�S 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 [ 35 S]GTP�S 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 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 D 2/D 3 agonists do not influence frontocortical 

adrenergic pathways (Millan et al., 2000a), activation by 

piribedil of D 2/D 3 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 extracel- 



Fig. 10. Antagonism by piribedil of the depletion of [ 3 H]PI evoked by NE at cloned h� 1A-ARs transfected into CHO cells. Data are representative of 

three independent experiments, each performed in triplicate. 

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. 

lular 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 

D 2/D 3 receptors is unlikely to be involved since dopaminergic 

agonists only variably and submaximally attenuate hypnotic 

sedative actions of xylazine (M. Brocco, unpublished observations). 


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 �l 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. 

General Discussion. Several general points emerge from 

these studies. 

First, although piribedil is not a potent agent, its affinity at 




h� 2A- and h� 2C-ARs was comparable to that at D 2 receptors. 

This suggests that at therapeutically relevant doses activating 

D 2 receptors, piribedil also occupies � 2A- and � 2C-ARs. 

Thus, � 2-AR blockade by piribedil likely contributes to its 

functional actions, although the relative implication of � 2Aversus 

� 2C-ARs remains to be clarified. 

Second, certain other antiparkinsonian agents interact 

with � 2-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-HT 1A 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-HT 2A and 5-HT 2C receptors (DeMarinis and Hieble, 1989; 

Seyfried and Boettcher, 1990; A. Newman-Tancredi, unpublished 

observations). 

Third, activation of postsynaptic � 2-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 � 2-AR agonists impair 

performance in certain cognitive tasks in humans (Arnsten et 

al., 1998; Jäkälä et al., 1999). Activation of postsynaptic 

� 2-ARs also potentiated antiparkinsonian actions of a �-opioid 

agonist in rats (Hill and Brotchie, 1999). However, the 

“quality” of movement was “poor” and, in more conventional 

models, � 2-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 � 2-ARs 

is seen only following marked depletion of endogenous pools 

of NE. In any event, the motor-depressant (and hypotensive), 

autoreceptor-mediated actions of � 2-AR agonists are difficult 

to reconcile with their potential utilization in parkinsonian 

patients. Thus, � 2-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 

� 2-ARs are simultaneously antagonized, favorable actions 

will be mediated via “functionally intact” and colocalized, 

postsynaptic � 1- and �-ARs (Arnsten et al., 1998; Brefel- 

Courbon et al., 1998; Millan et al., 2000a). Furthermore, 

blockade of inhibitory � 2-ARs on dopaminergic and serotonergic 

pathways should likewise be favorable (Millan et al., 

2000a). 

Finally, � 2-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 Gi1� is 

implicated in the fusion protein-mediated activation of 

GTPase, the precise species of Gi transducing MAPK phosphorylation 

and [ 35 S]GTP�S 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 � 2-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 � 2-ARs (Hieble 

et al., 1995). Importantly, further, piribedil shows similar 

affinity for � 2-ARs and D 2 receptors. Together with agonist 

actions at D 2 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 D 2 

receptor activation and � 2-AR blockade in the management 

of Parkinson’s disease. 

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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Send reprint requests to: Dr. Mark J. Millan, Institut de Recherches Servier, 

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Seine, Paris, France. E-mail: mark.millan@fr.netgrs.com

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