Antiparkinsonian Agent Piribedil Displays Antagonist Properties at ...

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
� 2-Adrenoceptors and Parkinson’s Disease 881
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
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