Antiparkinsonian Agent Piribedil Displays Antagonist Properties at ...

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-
� 2-Adrenoceptors and Parkinson’s Disease 883
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
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