Antiparkinsonian Agent Piribedil Displays Antagonist Properties at ...

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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 Downloaded from jpet.aspetjournals.org by guest on February 13, 2013
884 Millan et al. 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- Downloaded from jpet.aspetjournals.org by guest on February 13, 2013
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Antiparkinsonian Agent Piribedil Displays Antagonist Properties at ...

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