Introduction

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Although treatment of Parkinson's disease has long centered on administration of the dopamine precursor L-dihydroxyphenylacetyl acid (L-DOPA), there is increasing interest in the therapeutic use of dopaminergic agonists, both in association with L-DOPA and as monotherapy (Hughes, 1997). Inasmuch as dopaminergic agents currently used as antiparkinson agents interact principally with "D₂-like" receptors, an important question concerns their comparative actions at D₂ receptors (of which functionally distinct short D_2S and long D_2L isoforms exist), D₃ receptors, and D₄ receptors. D_2S versus D_2L receptor isoforms differ in both their localization and their functional roles. The D_2S isoform is principally responsible for presynaptic control of dopamine release, whereas postsynaptic D_2S and D_2L receptors in the basal ganglia, via contrasting patterns of interaction with D₁ sites, differentially influence motor function; notably, blockade of D_2L sites underlies the extrapyramidal motor effects of dopaminergic antagonists (Wang et al., 2000). As shown in the accompanying article (Millan et al., 2002), therapeutically used antiparkinson agents recognize D_2S and D_2L isoforms with similar affinity, and many antiparkinson agents also interact with dopamine D₃ receptors. Although the density of striatal D₃ receptors is reduced upon degeneration of nigrostriatal dopaminergic pathways, exposure to L-DOPA may induce their up-regulation, reflecting complex regulatory mechanisms involving dopamine D₁ receptors and brain-derived neurotrophic factor (Quik et al., 2000; Guillin et al., 2001; Joyce, 2001). Nevertheless, the precise nature of functional interrelationships among D₃, D₂, and D₁ receptors, and the implication of D₃ receptors in the therapeutic compared with dyskinetic effects of antiparkinson agents, remain to be clarified (Joyce, 2001). The majority of antiparkinson agents also show significant affinity for D₄ receptors (Millan et al., 2002), but their engagement does not improve motor function; furthermore, antagonist properties at D₄ receptors may minimize psychiatric side effects and improve cognitive function (Newman-Tancredi et al., 1997; Arnsten et al., 2000).

Several antiparkinson drugs display pronounced affinities for h_2A-, h_2B-, and h_2C-ARs (Millan et al., 2002). This is of note in light of the importance of adrenergic mechanisms in the etiology and treatment of Parkinson's disease (Brefel-Courbon et al., 1998; Bezard et al., 2001). In addition to their postsynaptic localization, _2A-ARs are expressed as inhibitory autoreceptors on adrenergic neurons (Nicholas et al., 1997; Millan et al., 2000a,b). Furthermore, _2A-ARs exert an inhibitory influence upon ascending serotonergic pathways, frontocortical and subcortical dopaminergic pathways (Kable et al., 2000; Millan et al., 2000a,b) as well as corticolimbic cholinergic and glutamatergic pathways (Horn et al., 1982; Tellez et al., 1997; Boehm, 1999). Correspondingly, _2A-ARs fulfill an important role in the control of motor function, mood, and cognition (Arnsten, 1997; Kable et al., 2000; Millan et al., 2000b). Furthermore, _2B-ARs are enriched in the thalamus, a structure interlinked with the basal ganglia and involved in the disruption of motor function in Parkinson's disease, whereas _2C-ARs are concentrated in the striatum itself (Nicholas et al., 1997; Bezard et al., 2001). Gene knockout studies have indicated a modulatory influence of central _2C-ARs, complementary to _2A-ARs, upon motor and cognitive function (Kable et al., 1999; Bjorklund et al., 2000). Although the precise significance of individual ₂-AR subtypes remains unclear, there is evidence that ₂-AR antagonist properties may be useful in the management of Parkinson's disease. First, in rats sustaining unilateral 6-hydroxydopamine lesions of the substantia nigra pars compacta (SNPC), ₂-AR agonists and antagonists, respectively, inhibit and enhance amphetamine-induced rotation (Mavridis et al., 1991). Second, in primates displaying Parkinson's disease-like symptoms after treatment with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine, ₂-AR antagonists increase locomotor activity and reduce dyskinesias induced by L-DOPA (Brefel-Courbon et al., 1998; Bezard et al., 2001). Third, after enhancement of adrenergic transmission by blockade of _2A-AR autoreceptors, noradrenaline (NA) may (independently of ₂-ARs) exert neuroprotective actions at dopaminergic neurons in the SNPC (Troadec et al., 2001). Fourth, small-scale clinical studies indicate that the ₂-AR antagonist idazoxan improves motor performance in Parkinson's disease patients receiving L-DOPA (Brefel-Courbon et al., 1998).

Several antiparkinson agents also interact with h_1A-, h_1B-, and h_1D-ARs (Millan et al., 2002). Although the relevance of ₁-ARs to management of Parkinson's disease is less apparent than for their ₂-AR counterparts, they modulate ascending serotoninergic and dopaminergic transmission and play an important role in motor control (Millan et al., 2000a; Spreng et al., 2001; Stone et al., 2001). Indeed, ₁-AR antagonists interfere with the induction of rotation by antiparkinson agents in rats sustaining unilateral lesions of the SNPC (Mavridis et al., 1991). Furthermore, frontocortical ₁-ARs are implicated in the control of cognitive function (Arnsten, 1997). The perturbation of cardiovascular function associated with pronounced activation or blockade of ₁-ARs should also be accentuated (Guimarães and Moura, 2001).

The above-mentioned observations exemplify the importance of characterizing functional actions of antiparkinson agents at subtypes of "hD₂-like" receptor and h_1/2-AR. However, studies have been restricted to a few ligands at poorly characterized native sites compared with defined classes of (cloned) human receptor (see Discussion). Knowledge of the comparative agonist/antagonist profiles of antiparkinson agents remains, thus, fragmentary. The present study expanded, therefore, the multivariate analyses of binding profiles presented in the preceding article (Millan et al., 2002) in evaluating efficacies of diverse antiparkinson agents at cloned hD_2S, hD_2L, hD₃, and hD₄ dopamine receptors, and at h_2A-, h_2B- h_2C-, and h_1A-ARs, stably expressed in a common cellular system, Chinese hamster ovary (CHO) cells.

	Materials and Methods

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Determination of Drug Efficacies at hD₂-Like Receptors and at h₂-AR Subtypes by [³⁵S]GTPS Binding. Efficacies at CHO-expressed recombinant hD_2S, hD_2L, hD₃, and hD₄ (hD_4.4 isoform) receptors, and at CHO-expressed h_2A-, h_2B-, and h_2C-ARs were determined by measuring the influence of drugs alone and, where appropriate, in interaction with DA or NA upon [³⁵S]GTPS binding. The protocols used have been described in detail previously (Newman-Tancredi et al., 1997, 1999a,b; Millan et al., 2001). Briefly, the concentration of [³⁵S]GTPS was 0.1 nM (hD_2S, hD_2L, and hD₄), 0.2 nM (h_2A-AR, h_2B-AR, and h_2C-AR) or 1.0 nM (hD₃). The pH was 7.4 in each case and the temperature 22°C. Incubation time was 40 min for hD_2S, hD_2L, and hD₃ sites, 20 min for hD₄ sites, and 60 min for h₂-AR subtypes. The buffer contained 20 mM HEPES, 100 or 150 mM NaCl, 3 µM GDP, and 3 or 10 mM MgCl₂. Membranes were incubated with the antiparkinson agent alone and/or with DA (3 µM-hD_2S, 10 µM-hD_2L, and 1 µM-hD₄) or NA (10 µM for each subtype) for 15 min before the addition of [³⁵S]GTPS. Agonist efficacies were expressed as a percentage of the effect observed with maximally effective concentrations of DA (10 µM) or NA (10 µM). Experiments were terminated by rapid filtration through GF/B filters (Whatman, Maidstone, UK) using a 96-well cell harvester (Packard Instrument Company, Inc., Downers Grove, IL), and radioactivity was determined by liquid scintillation counting.

Determination of Drug Efficacies at h_1A-ARs by [³H]Phosphatidylinositol ([³H]PI) Depletion. The efficacies of drugs alone, and in interaction with NA, were determined in CHO-expressed h_1A-ARs as described previously (Millan et al., 2001). Briefly, cells were labeled with 2 µCi/ml of [³H]myoinositol (10-20 Ci/mmol) for 24 h. Cells were washed and then incubated at 37°C for 30 min with the drug alone in Krebs-LiCl buffer: 15.6 mM NaH₂PO₄ pH 7, 120 mM NaCl, 4.8 mM KCl, 1.2 mM MgSO₄, 1.2 mM CaCl₂, 0.6% (w/v) glucose, 0.04% (w/v) bovine serum albumin, and 10 mM LiCl. In the absence of NA, ~40,000 dpm was typically detected, compared with ~25,000 in the presence of a maximally effective concentration of NA (30 µM). Drug efficacies were expressed as a percentage of the effect observed with a maximally effective concentration of NA (30 µM). For antagonist studies, cells were preincubated for 15 min with drug before the addition of NA (10 µM) and incubation continued for 30 min. Membranes were recovered by rapid filtration through GF/B filters (Whatman) using a 96-well cell harvester (Packard Instrument Company, Inc.), and the [³H]PI content was determined by scintillation counting (Millan et al., 2001).

Data Analyses. [³⁵S]GTPS and [³H]PI isotherms were analyzed by nonlinear regression using the program PRISM (GraphPad Software, San Diego, CA). K_B values for inhibition of DA- or NA-stimulated [³⁵S]GTPS binding at hD₂-like or h₂-ARs, and of NA-induced [³H]PI depletion at h_1A-ARs, were calculated as described previously (Lazareno and Birdsall, 1993; Newman-Tancredi et al., 1999a,b) according to the equation K_B = IC₅₀/{[(2 + (agonist/EC₅₀)^n_H)^n_H1]  1}, where IC₅₀ is the inhibitory concentration₅₀ of the antagonist, agonist is DA or NA concentration, EC₅₀ is effective concentration₅₀ of DA or NA alone, and n_H is Hill coefficient of the DA or NA stimulation isotherm. EC₅₀ values for NA at h_2A-, h_2B-, h_2C-, and h_1A-ARs were 354, 316, 302, and 329 nM, respectively. EC₅₀ values for DA at hD_2S, hD_2L, hD₃, and hD₄ receptors were 350, 340, 11, and 100 nM, respectively. Protein concentrations were determined by use of a bichinconic acid kit (Sigma, St. Quentin Fallavier, France). Pearson product-moment correlation coefficients were calculated for pEC₅₀ values determined herein compared with pK_i values determined in the accompanying article (Millan et al., 2002).

Drugs. Pramipexole dihydrochloride, piribedil hydrochloride, and ropinirole were synthesized by Servier Institut de Recherches (Paris, France). Lisuride maleate and terguride were donated by Schering (Berlin, Germany). Bromocriptine, ()-quinpirole HCl, pergolide methanesulfonate, and TL99 (6,7-dihydroxy-N,N-dimethyl-2-aminotetralin) were purchased from Sigma/RBI (Natick, MA). Apomorphine hydrochloride was purchased from Sigma. Roxindole methanesulfonate was donated by Merck (Darmstadt, Germany) and talipexole by Boehringer Ingelheim GmbH (Ingelheim, Germany). Cabergoline was obtained from Farmitalia Carlo Erba (Rueil-Malmaison, France). Quinelorane dihydrochloride was a gift from Eli Lilly & Co. (Indianapolis, IN).

	Results

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Drug Actions at hD_2S Receptors. At hD_2S receptors (B_max = 1.4 pmol/mg), at a maximally effective concentration (10 µM), DA enhanced [³⁵S]GTPS binding by ~2.5-fold; it displayed a pEC₅₀ value of 6.5 (Fig. 1; Table 1). Quinpirole, pramipexole, quinelorane, pergolide, and cabergoline behaved as highly efficacious agonists at hD_2S receptors in stimulating G protein activation ([³⁵S]GTPS binding) to a degree at least as marked as that of DA (E_max defined as 100%). TL99, talipexole, and apomorphine also showed high efficacies, whereas other ligands displayed less pronounced efficacies, ranging from 40% for terguride to 55% for lisuride. Roxindole showed very low efficacy. Drug potencies for stimulation of [³⁵S]GTPS binding (pEC₅₀ values) at hD_2S receptors correlated well (r = 0.82, P < 0.05) with their pK_i values determined in competition binding experiments (data not shown; Millan et al., 2002).

View larger version (24K): [in this window] [in a new window]   Fig. 1.   Influence of antiparkinson agents upon G protein coupling at hD2S (A), hD2L (B), and hD3 (C) receptors expressed in CHO cells. [35S]GTPS binding was carried out as described in Table 1. Binding is expressed as a percentage of that observed with a maximally effective concentration (10 µM) of dopamine (defined as 100%). Values shown are from representative experiments performed in triplicate and repeated on at least three occasions.

View this table: [in this window] [in a new window]   TABLE 1 Efficacies (Emax values) and potencies (pEC50 or pKb values) of antiparkinson agents at recombinant hD2S, hD2L, hD3, and hD4 receptors Efficacy (Emax) and potency (pEC50 or pKb) values at hD2S, hD2L, hD3, and hD4 receptors were determined by [35S]GTPS binding. pEC50 values for stimulation are indicated in normal case and pKb values for inhibition are indicated in bold. Emax values are percentages of the stimulation observed with a maximally efficacious (Emax = 100%) concentration of dopamine (see Materials and Methods) and are expressed as means ± S.E.M. values of at least three independent determinations performed in triplicate. pEC50 values are means of at least three independent determinations: S.E.M.s values (not shown) were less than 0.2 log units. Dopamine exhibited pEC50 values of 6.46, 6.49, 7.95, and 7.00 at hD2S, hD2L, hD3, and hD4 receptors, respectively. For piribedil, the pKb at hD4 receptors is indicated in the table. It exhibited an agonist pEC50 value of 6.4 at these sites. For roxindole, the pKb at hD2S receptors is given in the table. It exhibited an agonist pEC50 value of 8.11 at these sites.

Drug Actions at hD_2L Receptors. At hD_2L receptors (B_max = 2.2 pmol/mg), at a maximally effective concentration (10 µM), DA enhanced [³⁵S]GTPS binding by ~1.9-fold; it displayed a pEC₅₀ value of 6.5 (Fig. 1; Table 1). At hD_2L receptors, efficacies for all ligands were markedly lower than at hD_2S sites. Indeed, all ligands, except quinelorane, behaved as partial agonists. Roxindole and terguride induced no stimulation of [³⁵S]GTPS binding and displayed antagonist properties. The correlation coefficient for efficacies at hD_2L versus hD_2S receptors was 0.79 (P < 0.05). Drug potencies for stimulation of [³⁵S]GTPS binding (pEC₅₀ values) at hD_2L receptors correlated well (r = 0.93, P < 0.05) with their pK_i values determined in competition binding experiments (data not shown; Millan et al., 2002).

Drug Actions at hD₃ Receptors. At hD₃ receptors (B_max = 15.6 pmol/mg), at a maximally effective concentration (10 µM), DA enhanced [³⁵S]GTPS binding by ~1.6-fold; it displayed a pEC₅₀ value of 7.8 (Fig. 1; Table 1). All drugs behaved as agonists at hD₃ receptors, with efficacies varying from 30% (roxindole) and 34% (piribedil) to 88% (talipexole) and 94% (TL99). The lower efficacies of quinelorane and quinpirole at hD₃ versus hD_2S and hD_2L sites are of note, whereas roxindole and bromocriptine showed higher efficacies at hD₃ than hD_2S and hD_2L sites. Indeed, there was no consistent pattern of drug efficacies at hD₃ relative to hD_2S and hD_2L receptors. Accordingly, correlation coefficients for efficacies at hD₃ compared with hD_2L and hD_2S sites, although significant (P < 0.05), were only 0.59 and 0.49, respectively. Drug potencies for stimulation of [³⁵S]GTPS binding (pEC₅₀ values) at hD₃ receptors correlated significantly (r = 0.62, P < 0.05) with pK_i values determined in competition binding experiments (data not shown; Millan et al., 2002).

Drug Actions at hD₄ Receptors. At hD₄ receptors (B_max = 1.4 pmol/mg), at a maximally effective concentration (10 µM), DA enhanced [³⁵S]GTPS binding by ~2.2-fold; it displayed a pEC₅₀ value of 7.0 (Table 1). Although bromocriptine did not interact with hD₄ receptors, agonist efficacies of the other drugs varied widely. Thus, although TL99, quinelorane, and quinpirole showed relatively high efficacies (~70%), pergolide, talipexole, cabergoline, apomorphine, roxindole, pramipexole, and lisuride showed less marked efficacies of 32 to 56%. Piribedil displayed very low efficacy (7%) and antagonized the stimulation by DA of [³⁵S]GTPS binding. Terguride, which was inactive alone, similarly blocked the action of DA. On the other hand, roxindole was more efficacious at hD₄ than at hD_2S and hD_2L receptors. Thus, there was no consistent pattern of drug efficacies at hD₄ versus hD_2S, hD_2L, and hD₃ receptors and correlation coefficients, although significant (P < 0.05), were modest: hD_2S, r = 0.57; hD_2L, r = 0.55; and hD₃, r = 0.44. Drug potencies for stimulation of [³⁵S]GTPS binding (pEC₅₀ values) at hD₄ receptors correlated well (r = 0.78, P < 0.05) with their pK_i values determined in competition binding experiments (data not shown; Millan et al., 2002).

Drug Actions at h_2A-, h_2B-, and h_2C-ARs. At h_2A-, h_2B-, and h_2C-ARs, a maximally effective concentration of NA (10 µM) increased [³⁵S]GTPS binding by 7.2-, 6.6-, and 2.7-fold, respectively; pEC₅₀ values were 6.2, 6.5, and 6.5, respectively (Figs. 2, 3 and 4; Table 2). Antiparkinson agents differed markedly concerning their functional activities at h_2A-, h_2B-, and h_2C-ARs. TL99 behaved as a high-efficacy agonist at each subtype of h₂-AR, whereas talipexole behaved as a partial agonist at each subtype. On the other hand, pergolide showed pronounced efficacy at h_2B-ARs, intermediate efficacy at h_2A-ARs, and low efficacy at h_2C-ARs. Pramipexole revealed partial agonist properties at h_2A-ARs; actions were not evaluated at h_2B- and h_2C-ARs owing to its low affinities at these sites (Millan et al., 2002). Apomorphine showed low efficacy only at h_2A-ARs, whereas high concentrations of quinelorane and quinpirole revealed weak partial agonist actions at h_2A- and h_2B-ARs, respectively. Agonist pEC₅₀ values for stimulation of [³⁵S]GTPS binding corresponded well to their respective pK_i values as defined in competition binding assays (Millan et al., 2002). In view of its high affinity and low efficacy at h_2A-AR subtypes, apomorphine was further evaluated in interaction with NA and shown to behave as an antagonist. Furthermore, several other drugs also reversed NA-stimulated [³⁵S]GTPS binding. For drugs behaving as antagonists, pK_B values correlated well (P < 0.05) with their respective pK_i values derived from competition binding studies (data not shown; Millan et al., 2002): h_2A-ARs, r = 0.94; h_2B-ARs, r = 0.86; and h_2C-ARs, r = 0.87. 

View larger version (18K): [in this window] [in a new window]   Fig. 2.   Influence of antiparkinson agents upon G protein coupling at h2A-adrenoceptors expressed in CHO cells. [35S]GTPS binding was carried out as described in Table 1. Binding is expressed as a percentage of that observed with a maximally effective concentration (10 µM) of noradrenaline (defined as 100%). Values shown are from representative experiments performed in triplicate and repeated on at least three occasions.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Influence of antiparkinson agents upon G protein coupling at h2B-adrenoceptors expressed in CHO cells. [35S]GTPS binding was carried out as described in Table 1. [35S]GTPS binding is expressed as a percentage of that observed with a maximally effective concentration (10 µM) of noradrenaline (defined as 100%). Values shown are from representative experiments performed in triplicate and repeated on at least three occasions.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Influence of antiparkinson agents upon G protein coupling at h2C-adrenoceptors expressed in CHO cells. [35S]GTPS binding was carried out as described in Table 1. [35S]GTPS binding is expressed as a percentage of that observed with a maximally effective concentration (10 µM) of noradrenaline defined as 100%). Values shown are from representative experiments performed in triplicate and repeated on at least three occasions.

View this table: [in this window] [in a new window]   TABLE 2 Efficacies (Emax values) and potencies (pEC50 or pKb values) of antiparkinson agents at recombinant h2A-, h2B-, and h2C-adrenoceptors Efficacy (Emax) and potency (pEC50 or pKb) values were determined by [35S]GTPS binding. pEC50 values for stimulation are indicated in normal case, and pKb values for inhibition are indicated in bold. Emax values are percentages of the stimulation observed with a maximally efficacious (Emax = 100%) concentration of noradrenaline (10 µM) and are expressed as means ± S.E.M. values of at least three independent determinations performed in triplicate. pEC50 or pKb values are means of at least three independent determinations: S.E.M. values (not shown) were less than 0.2 log units. The Bmax at h2A-, h2B-, and h2C-ARs was 1.8, 1.0, and 1.3 pmol/mg, respectively. Noradrenaline exhibited pEC50 values of 6.45, 6.50, and 6.52 at h2A-, h2B-, and h2C-ARs, respectively. Apomorphine displayed a pKb of 6.58 at h2A-ARs, and pergolide displayed a pKb of 6.96 at h2C-ARs.

Drug Actions at h_1A-ARs. Ligands that exhibited significant binding affinity at h_1A-ARs (pK_i values 6.0; Millan et al., 2002) were evaluated in a functional test of phospholipase C activation, depletion of membrane-bound [³H]PI (Fig. 5; Table 3). In this procedure, NA itself revealed a pEC₅₀ value of 6.51. No compound stimulated phospholipase C activity when tested alone, indicating an absence of agonist properties. In contrast, in order of decreasing potency, roxindole, bromocriptine, lisuride, terguride, cabergoline, and piribedil all reversed the stimulation of [³H]PI hydrolysis induced by noradrenaline (10 µM), demonstrating antagonist properties. pK_B values at h_1A-ARs correlated well (r = 0.96, P < 0.05) with pK_i values obtained from competition binding assays (data not shown; Millan et al., 2002).

View larger version (16K): [in this window] [in a new window]   Fig. 5.   Influence of antiparkinson agents upon stimulation of phospholipase C activity by noradrenaline at h1A-receptors expressed in CHO cells. [3H]PI depletion studies were carried out as described under Materials and Methods. The antagonist actions of drugs were examined against noradrenaline (10 µM). Values shown are from representative experiments performed in triplicate and repeated on at least three occasions.

View this table: [in this window] [in a new window]   TABLE 3 Efficacies and potencies (pKb values) of antiparkinson agents at recombinant h1A-ARs Efficacies (Emax) are for drugs alone compared with a maximally effective (100%) concentration of NA (30 µM). Potencies (pKb) at h1A-ARs (Bmax = 2.5 pmol/mg) were determined by blockade of NA-stimulated [3H]PI depletion. Noradrenaline exhibited a pEC50 value of 6.48. Pramipexole, ropinirole, quinelorane, apomorphine, pergolide, talipexole, TL99, and quinpirole, which have low affinities at h1A-ARs (pKi values of <6.0, see accompanying paper) were not tested. pKb values are means of at least three independent determinations: S.E.M. values were less than 0.2 log units. Experiments were carried out in triplicate.

	Discussion

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This comprehensive comparison of the actions of 14 antiparkinson agents at eight classes of cloned "hD₂-like" and h₁/h₂-AR revealed marked differences in efficacies, observations of significance to their contrasting functional profiles in experimental models and in human.

D_2S and D_2L Receptors. In the only previous comparison of antiparkinson agonists at hD_2S versus hD_2L receptors (ropinirole, talipexole, pergolide, lisuride, and bromocriptine), no marked differences in efficacy were apparent (Gardner et al., 1997; Gardner and Strange, 1998). In the present, more extensive work, however, drug efficacies were invariably higher at hD_2S sites. The reasons underlying this difference require elucidation, but it may be of pertinence that hD_2S and hD_2L receptors differentially interact with distinct subtypes of G protein, as implicated in their contrasting patterns of coupling to calcium channels (Wolfe and Morris, 1999). Furthermore, the expression level (2.2 pmol/mg) of hD_2L sites herein was higher than that of hD_2S sites (1.4 pmol/mg), whereas the inverse was true for Gardner et al. (1997) (1.3 versus 2.7 pmol/mg, respectively). In the light of these comments, it should briefly be pointed out that receptor density can play an important role in determining drug efficacy (Newman-Tancredi et al., 2001). Although receptor density (B_max) can easily be determined at pure populations of transfected receptors (see Results), equivalent information for defined networks of neurons is not available because B_max estimations in native tissue almost inevitably incorporate neurons not expressing the receptor in question. Thus, although the receptor densities of hD_2S and hD_2L sites here were in the same range as previous studies (Coldwall et al., 1999; Perachon et al., 1999), they cannot be compared with certainty to cerebral populations. Furthermore, it is important to note differences in density between pre- versus postsynaptic populations of D₂ (and D₃) receptors (Seyfried and Boettcher, 1990), as well as the up-regulation of postsynaptic sites after damage to dopaminergic innervation (Kostrzewa, 1995; Newman-Tancredi et al., 2001), an experimental manipulation that mimics the pathology of Parkinson's disease (see below).

hD₃ Receptors. Although hD₃ receptors couple less efficiently to G proteins in CHO cells than their hD_2S/hD_2L counterparts, DA stimulated [³⁵S]GTPS binding in the high-expressing cell line used herein (Newman-Tancredi et al., 1999b). The substantial affinities of apomorphine, quinpirole, pramipexole, talipexole, bromocriptine, and pergolide corroborate studies of their actions in models of microphysiometry and mitogenesis (Mierau et al., 1995; Coldwell et al., 1999; Perachon et al., 1999). The high efficacy of TL99 at hD₃ receptors is of note in view of its marked efficacy at hD_2S/hD_2L and hD₄ sites, whereas the modest efficacies of terguride and roxindole at hD₃ sites mimic their low efficacies at hD_2L and (terguride) hD₄ sites. As concerns piribedil, its intermediate efficacy at hD₃ receptors resembles its actions at hD_2S/hD_2L receptors and is consistent with agonist properties in vivo at D₃ autoreceptors (Millan et al., 1995). As discussed elsewhere (Joyce, 2001), the role of D₃ sites in the expression of beneficial and deleterious actions of antiparkinson agents remains unclear, a question of particular importance because, as shown herein, all antiparkinson agents activated D₃ receptors.

hD₄ Receptors. In line with studies of CHO cells expressing the hD_4.2 isoform (Gilliland and Alper, 2000) and of cloned, rat D₄ sites (Gazi et al., 2000), quinpirole showed substantial efficacy at hD₄ (hD_4.4) receptors. This characteristic was shared by quinerolane and TL99. The agonist properties of pergolide, apomorphine, talipexole, and pramipexole at hD₄ sites complement work using other measures of drug efficacy and/or other hD₄ isoforms (Mieurau et al., 1995; Coldwell et al., 1999; Gazi et al., 2000; Gilliland and Alper, 2000). Like pergolide, two other ergolines, cabergoline and lisuride, similarly showed agonist properties at hD₄ sites. In contrast, piribedil displayed low efficacy at hD₄ receptors, whereas bromocriptine was inactive. Because bromocriptine and piribedil are clinically efficacious antiparkinson agents, these data support the argument that activation of D₄ receptors is not necessary for therapeutic efficacy (Newman-Tancredi et al., 1997). Moreover, the essentially D₄ antagonist properties of piribedil may limit psychiatric side effects and contribute to its improvement of cognitive function (Arnsten et al., 2000; Nagaraja and Jayashree, 2001).

h₂-ARs. Striking differences in drug efficacies were seen at h₂-AR subtypes. In analogy to piribedil (Millan et al., 2001), lisuride, bromocriptine, and apomorphine displayed antagonist properties, observations amplifying functional studies of isolated organs and hippocampal NA release in rats (McPherson, 1984; Jackisch et al., 1985). In line with their high affinities for h₂-ARs (Millan et al., 2002), roxindole and two further ergot-related ligands, terguride and cabergoline, also manifested potent ₂-AR antagonist properties. In contrast, in line with in vivo studies (at undefined ₂-AR subtypes) in rodents (Horn et al., 1982; Meltzer et al., 1989; Sánchez and Arnt, 1992), TL99 displayed agonist, and talipexole partial agonist, properties at h_2A-, h_2B-, and h_2C-ARs. Extending observations of partial agonist properties at central ₂-ARs in rodents (Ferrari et al., 1993), pramipexole displayed modest efficacy at h_2A-ARs. Any potential significance of this (low-potency) action in vivo, however, remains to be clarified. On the other hand, in vivo studies in rodents have revealed agonist actions of pergolide at central ₂-ARs (Langtry and Clissold, 1990) and particularly pronounced agonist properties at h_2B-ARs were observed here.

Actions at h_1A-ARs. Roxindole and the ergot derivatives bromocriptine, lisuride, and terguride interact with cloned h₁-ARs (Millan et al., 2002), although, with the exception of a study of bromocripine at peripheral ₁-ARs (McPherson, 1984), no information on their functional activities is available. Thus, their potent antagonism of NA-induced [³H]PI depletion at cloned h_1A-ARs is of note, whereas cabergoline and piribedil showed weak antagonist properties in line with their low affinities at these sites (Millan et al., 2002). Potent blockade of h₁-ARs may be unfavorable inasmuch as ₁-AR antagonists interfere with antiparkinson properties in experimental models (Mavridis et al., 1991). Although blockade of ₁-ARs in the cortex and on pars reticulata GABAergic neurons may be involved, generalized sedative/motor-suppressive effects due to blockade of _1A-ARs in motor nuclei of the brainstem and the spinal cord may also be of significance (Stone et al., 2001). Blockade of segmental and peripheral _1A-ARs may also be deleterious in that it exacerbates the perturbation of cardiovascular function elicited via stimulation of spinal dopaminergic receptors (Guimarães and Moura, 2001). On the other hand, inasmuch as frontocortical ₁-ARs inhibit working memory, their blockade might improve cognitive function (Arnsten, 1997), although there is currently no clinical support for this possibility. Further investigations should determine the potential importance for antiparkinson agents of actions at h_1B- and h_1D-AR subtypes (Millan et al., 2002), which likewise modulate motor, cognitive, and cardiovascular function (Guimarães and Moura, 2001; Spreng et al., 2001; Stone et al., 2001).

	Conclusions

Top Abstract Introduction Materials and Methods Results Discussion Conclusions References

The present data reveal striking differences among antiparkinson agents concerning efficacies at multiple classes of hD₂-like receptor and ha₁/ha₂-AR. These observations amplify receptor-binding analyses of the accompanying article (Millan et al., 2002) in demonstrating that antiparkinson drugs are heterogeneous rather than a common group of "dopaminergic agonists". In this light, as discussed above, partial agonist and agonist properties at D_2S/D_2L receptors are favorable in the management of motor symptoms of Parkinson's disease, whereas blockade of _2A-ARs may improve cognitive-attentional function and mood. The present data provide a framework for additional studies of the significance of these and other subtypes of dopaminergic receptor and ₁/₂-AR in the etiology of Parkinson's disease, and in the beneficial and deleterious properties of antiparkinson agents.