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Fetal yawning assessed by 3D and 4D sonography
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Behavioural Pharmacology
2009;20(4):303-311
Penile erection and yawning induced
by dopamine D2-like receptor agonists in rats:
influence of strain and contribution of dopamine D2,
but not D3 and D4 receptors
Ronan Depoortère, Laurent Bardin, Michael Rodrigues, Erika Abrial, Monique Aliaga and Adrian Newman-Tancredi
 
Centre de Recherche Pierre Fabre, Castres France

Chat-logomini

 
Dopamine (DA) is implicated in penile erection (PE) and yawning (YA) in rats through activation of D2-like receptors. However, the exact role of each subtype (D2, D3 and D4) of this receptor family in PE/YA is still not clearly elucidated. We recorded concomitantly PE and YA after treatment with agonists with various levels of selectivity for the different subtypes of D2-like receptors. In addition, we investigated the efficacy of antagonists with selective or preferential affinity for each of the three receptor subtypes to prevent apomorphine-induced PE and YA.
 
Wistar rats were more sensitive than Long-Evans rats to the erectogenic activity of the nonselective DA agonist apomorphine (0.01-0.08 mg/kg), whereas Sprague-Dawley rats were insensitive. However, all the three strains were equally sensitive to apomorphine-induced YA. In Wistar rats, apomorphine (0.01-0.63mg/kg), the D2/D3 agonists quinelorane and (+)7-OH-DPAT (0.000625-10mg/kg) or PD 128,907 (0.01-10mg/kg), but not the D4 agonists PD-1 68,077, RO-1 0-5824 and ABT-724 (0.04-0.63 mg/kg), produced PE and YA with bell-shaped dose-response curves. Similarly, ABT-724 and CP226-269 (another D4 agonist) failed to elicit PE and YA in Sprague-Dawley rats.
 
Furthermore, in Wistar rats, PE and YA elicited by apomorphine (0.08 mg/kg) were not modified by selective D3 (S33084 and SB-277011, 0.63-10 mg/kg) or D4 (L-745,870 and RBI-257, 0.63-2.5 mg/kg) antagonists, but were prevented by the preferential D2 blocker L-741,626 (near-full antagonism at 2.5 mg/kg). The present data do not support a major implication of either DA D3 or D4 receptors in the control of PE and YA in rats, but indicate a preponderant role of DA D2 receptors.
 
 
Introduction
 
Dopamine (DA) has been implicated in the mechanism of penile erection (PE). Apomorphine, a direct DA receptor agonist, with marked affinity for DA D2-like (D2/D3/D4) receptors, but more modest affinity for DA Dl-like (D,/D_5) receptors (Millan et al., 2002) consistently facilitates PE in rodents and humans (Benassi-Benelli et al., 1979; Lal et al., 1984, 1987; Gower et al., 1986; Segraves et al., 1991). This proerectile activity has been suggested to rely on activation of DA D2-like receptors (Benassi-Benelli et al., 1979). However, the usefulness of apomorphine in the clinic has been limited because of its lower efficacy than PDE5 inhibitors, such as sildenafil, and troublesome side-effects, such as nausea (related to activation of DA D2 receptors in the area postrema) (Carson, 2008).
 
This has spurred the investigation of the role of members of the DA D2-like receptors family other than D2 in PE, with a particular focus on D4 receptors. This was based on the assumption that targeting DA D4 receptors for PE dysfunction could offer advantages over nonselective activation of D2/D3/D4 receptors, as activation of DA D4 receptors is not emetogenic (Osinski et al., 2005). Studies on rats have pointed towards a role of DA D4 receptors in the central control of PE: selective agonists at this receptor, such as A-412887, PD-168,077, PiP3EA, CP226-269, ABT-724 and ABT-670 (Brioni et al., 2004; Cowart et al., 2004; Hsieh et al., 2004; Moreland et al., 2005; Melis et d, 2006; Patel et al., 2006) dosedependently promote PE, and the effects of PD-168,077 and PiP3EA can be reversed by the DA D4 receptor antagonist L-745,870 (Melis et al., 2006).
 
More recently, there has been a comprehensive analysis of the function of DA D3 receptors in the control of PE in rats (Collins et al., 2007b, 2009). These authors concluded that the proerectile activity of apomorphine and pramipexole (a DA D2/D3 receptor agonist) are mediated by activation of DA D3 receptors, based on interaction studies that used preferential DA D3 or D2 receptor antagonists. SB-27701 1 and PG01037, compounds with over a 100-fold in-vitro selectivity for rat DA D3 over D2 receptors (10.7 vs. 2800nmol/l and 0.7 versus 93nmolIl, respectively: Reavill et al., 2000; Grundt et al., 2005), dosedependently antagonized pramipexole-induced PE. However, the doses at which antagonism was observed with these two DA D3 receptor antagonists were rather high (total blockade at 32mg/kg for both), in light of the high (SB-277011) and very high (PG01037) affinities for the target receptor. In addition, the authors justified the selectivity of these two DA D3 receptor antagonists, based on the fact that these compounds do not produce catalepsy over these dose ranges (Collins et al., 2007b, 2009). However, as argued in more detail in the Discussion, catalepsy necessitates at least 80% occupancy of DA D2 receptors (Wadenberg et al., 2000), and is obtained at doses higher than those necessary to block other DA D2 receptormediated behaviours. Hence, the possibility remains that at such doses, especially for PG01037, these compounds may interact in vivo with receptors other than DA D3 receptors. In addition, these authors reported that DA D4 receptor agonists, contrary to what had been reported earlier (vide supra), did not produce PE. Nonetheless, these data from Collins et al. (2007b, 2009) suggest that PE in rodents might provide a useful in-vivo model sensitive to DA D3 receptor ligands.
 
DA D2-like receptor agonists have also been reported to produce yawning (YA) in rodents (Mogilnicka and Klimek, 1977; Smith et al., 1997) and humans (Lal et al., 2000). Whereas data indicate that the DA D4-subtype is not implicated in YA behaviour (Collins et al., 2007a), the exact role played by DA D2 and D3 receptors is still controversial (Millan et al., 2000; Collins et al., 2005, 2007a, b).
 
 
The aims of this study are three-fold:
 
(1) To determine the strain of rat that is maximally sensitive to the PE promoting-effects of dopaminergic receptor agonists. Therefore, we chose to assess the effects of apomorphine (chosen because of its well-known PE-promoting effects) in SpragueDawley, Wistar and Long-Evans rats.
 
(2) To investigate, in the most sensitive strain, the efficacy of agonists selective for DA D2/D3 or D4 receptors to induce PE and YA.
 
(3) To assess, in the selected strain, the contribution of D2, D3 or D4 receptors in the proerectile activity of apomorphine using selective or preferential antagonists. Care was taken, in particular for the DA D3 receptor in view of the controversy surrounding its implication in PE and YA (vide supra), to use doses of 'selective' antagonists that can be considered to retain selectivity for the targeted receptor in vivo (for justification of doses, see Methods).
 
Discussion
 
There was a marked difference in the erectogenic effects of the nonselective DA receptor agonist apomorphine in the three strains studied, with Wistar rats showing the highest sensitivity. In this strain, apomorphine produced dose-dependent (bell-shaped) PE and YA. This effect was mimicked by other DA D2/D3 receptor agonists, but not by D4 receptor agonists. Furthermore, under the present experimental conditions, apomorphine-induced PE and YA were not modified by selective DA D3 or D4 receptor antagonists, at the doses tested, but were prevented by the preferential DA D2 receptor antagonist L-741,626.
 
The three strains studied differed in their sensitivity to the effects of apomorphine on PE, but not on yawning Over the dose range of 0.01-0.08 mg/kg, apomorphine produced similar effects on YA in the three rat strains studied (Wistar, Long-Evans and Sprague-Dawley). Concerning the proerectile activity, Sprague-Dawley rats were basically nonresponsive to the effects of apomorphine, despite being sensitive to its YA-inducing effects. This latter observation rules out the hypothesis that this strain might have been less sensitive to the pharmacodynamic effects of apomorphine, or that this compound might have a particular pharmacokinetic profile in this strain. However, it might have been the case that higher doses of apomorphine would have produced a greater number of PE in Long-Evans and Sprague-Dawley rats. Several laboratories have reported apomorphine-induced PE in Sprague-Dawley and Long-Evans rats (especially in the former, more commonly used for this type of assay: for example, Bernabé et d, 1999; Melis et iii., 2006). However, there could be differences in the genotype! phenotype and pharmacological sensitivity, for rats of a given strain but from different breeders.
 
DA D2/D3 receptor agonists differed in their profile of induction of spontaneous PE and YA in Wistar rats In Wistar rats, all four DA D2/D3 receptor agonists produced bell-shaped curves for YA and PE, although that of PD-128,907 for the latter behaviour was much flatter than those for the other compounds. One interesting effect observed here is that at some doses, DA D2/D3 receptor agonists produced one behaviour (PE or YA) preferentially over the other. For example, apomorphine and quinelorane, at one (former) or two (latter) doses produced significantly more PE than YA, with little, if any, YA. In contrast, PD-128,907 preferentially generated YA at two out of six doses, with no significant PE at any dose tested (though there was a strong tendency at a single dose). In contrast, (+ )7-OH-DPAT produced PE and YA at the same doses, with clearly superimposable bell-shaped curves. Interestingly, a rather similar pattern of differential effect on PE or YA was observed in the study of Collins et al. (2009), most strikingly with the preferential DA D receptor agonist suminarole. These different effects on PE or YA could result from differences in localization of DA D2/D3 receptors implicated in either behaviour, with the various agonists displaying brain regional selectivity. Alternatively, it might be that PE-inducing and YA-inducing DA receptors are differentially sensitive (differential levels of occupancy by the endogenous agonist, coupling to different G proteins, etc.), therefore pharmacological activation by agonists results in mixed profiles.
 
 
DA D4 receptor agonists failed to elicit spontaneous PE and YA, and DA D4 receptor selective antagonists did not prevent apomorphine-induced PE and YA.
 
In contrast to what has been reported by others (Brioni et al., 2004; Cowart et al., 2004; Hsieh et al., 2004; Moreland et d, 2005; Melis et d, 2006), we did not observe significant levels of PE after systemic administration of DA D4 receptor agonists (PD-168,077, RO-10-5824 and ABT-724) in Wistar rats. Although we did not explore the pharmacology of these compounds to the same depth in Sprague-Dawley rats (and only injected a small number of Long-Evans rats: data not shown), results collected in these latter two strains also indicate a lack of erectogenic effects of DA D4 receptors agonists. Interestingly, Collins et al. (2007b, 2009) also reported that ABT-724 and PD-168,077, over dose ranges similar to those used here, elicited very little, if any, PE in Wistar rats. Reasons for the discrepancy between these two sets of negative results, and those reporting positive effects of DA D4 receptor agonists on PE (see above) are not clear; for each agonist, we took care to test dose ranges encompassing doses shown to be active in these positive studies, pretreatment times and routes of administration were comparable, and rats were relatively young (as age has been shown to affect sensitivity of rats to pharmacological challenges with DA receptor agonists: Varrin and Heaton, 1992). In addition, our experimental conditions were adequate for detecting pharmacologically induced PE, as attested by the positive effects of apomorphine (especially in the Wistar strain). However, most other positive studies with DA D4 receptor agonists report levels of PE well below those reported here for apomorphine and other DA D2/D3 receptor agonists. Subtle variations in extraneous experimental conditions (season of testing, conditions of housing, sexual experience of subject, rat supplier, etc.) might affect the impact of DA D4 receptor agonists on PE in rats.
 
In accordance with published data (Melis et d, 2006; Collins et al., 2007b, 2009), L-745,870 failed to markedly affect apomorphine-induced PE in Wistar rats. Together with the lack of antagonism by RBI-257, another DA D4 receptor antagonist (Kula et al., 1997), and the absence of PE elicited by selective DA D4 receptor agonists observed here (vide supra) and elsewhere (Collins et al., 2007b, 2009), these data do not support a role for DA D4 receptor in PE in rats. Concerning YA behaviour, the absence of effects of DA D4 receptor agonists, and the lack of efficacy of DA D4 receptor antagonist to reverse apomorphine effects, are in line with earlier reports (Collins et al., 2007a, b, 2009).
 
DA D3 receptor selective antagonists did not prevent apomorphine-induced PE and YA
 
We did not observe significant antagonism by the DA D3 receptor antagonists S33084 and SB-277,011, both up to 10mg/kg, on apomorphine-induced and/or quineloraneinduced PE and YA. SB-277,011 had a nonsignificant tendency to increase the number of PE at the highest dose tested; this is in contrast with data from Collins et al. (2007a, b, 2009) on YA and PE (see below). In addition, at 10 mg/kg (a dose that can be considered to start occupying DA D2 receptors, see Methods) S33084 showed a nonsignificant tendency to reduce apomorphine-induced PE and YA. Millan et al. (2000) also failed to observe significant effects of S33084 and GR218,231 (another selective DA D3 receptor antagonist: Murray et al., 1996), both up to 10mg/kg, on 7-OH-DPATinduced YA (and PE for GR218,231). However, S33084 significantly attenuated 7-OH-DPAT-induced PE from 6.3 mg/kg; reasons for this slightly higher potency are unclear, but might relate to the nature of the DA receptor agonist used (7-OH-DPAT versus apomorphine) and/or the dose. All three antagonists have high affinity for rat DA D3 versus D2 receptors: K (affinity constant) = 1.9 versus l6Onmol/l for S33084; K=2.1 versus 245 nmol/l for GR218,231 (Millan et al., 2000) and K= 11 versus 2800nmol/l for SB-277011 (Reavill et al., 2000). Considering that S33084 (Dubuffet et al., 1999), GR218,231 (Depoortère et d, 2002) and SB-277011 (Reavill et al., 2000) have satisfactory brain penetration, such high affinities for DA D3 receptors would be expected to have resulted in minimal effective doses substantially below 10mg/kg for all three compounds. Indeed, in behavioural and neurochemical models (spontaneous deficit of social interaction and on cortical acetylcholine release) claimed to be selectively sensitive to DA D3 receptor antagonism (Millan et d, 2007), S33084 and SB-27701 1 were active at doses equal to or markedly less than 10mg/kg, starting from 0.16 and 0.63 mg/kg, respectively. Of note, L-741,626, an antagonist with slight (around 10-fold) preferential affinity for D2 over D3 receptors (Kulagowski et al., 1996; Millan et al., 2000) was without effect up to 10mg/kg s.c. (Millan et al., 2007), further reinforcing the hypothesis that these two models are sensitive to selective blocking of DA D3 receptors.
 
In the studies of Collins et al. (2005, 2007a, b, 2009), higher doses of SB-277011 (10-56 mg/kg, s.c.) than those used by Millan et d (2007) in their models claimed to be 'specific' for DA D3 receptor activation, were necessary to substantially or completely prevent YA or PE induced by PD-128,907 or pramipexole. In addition, SB-277011, given at 41 mg/kg orally (i.e. around 15mg/kg i.p./s.c., based on a 35-43% oral bioavailability index in rats: Stemp et al., 2000; Austin et al., 2001), significantly antagonized apomorphine-induced climbing in mice (Reavill et al., 2000), an effect positively and highly correlated with in-vitro affinity for DA D2 receptors (Bardin et al., 2006). These data suggest that doses of SB277011 around 10-15 mg/kg, in rodents, may occupy DA D2 receptors to a significant level. The level of occupancy is insufficient to produce catalepsy (which occurs at over 80% receptor occupancy: Wadenberg et d, 2000), but nonetheless probably sufficient to start antagonizing other in-vivo behavioural effects resulting from activation of DA D2 receptors. Consistent with this hypothesis, the efficacious dose for 50% effect (ED50) of haloperidol, in rats, for reversal of pramipexole-induced or apomorphineinduced PE and YA is around 0.03 mg/kg (Gower et al., 1984; Collins et al., 2007b, 2009), or 0.07 mg/kg in the conditioned avoidance test, another model sensitive to DA D receptors blockade (Bardin et al., 2007). These ED50, are around one order of magnitude lower than those eliciting catalepsy in rats (0.30 or 0.59 mg/kg, depending on the catalepsy model: Bardin et al., 2007). To summarize, the results of this interaction study with selective DA D3 receptor antagonist, at the doses used, are not in favour of a major implication of this subtype of dopaminergic receptor in the PE-eliciting and YAeliciting effects of apomorphine.
 
Apomorphine-induced PE and YA was blocked by preferential DA D2 receptor antagonism
 
PE and YA elicited by apomorphine (and quinelorane) could only be prevented by L-741,626, an antagonist with modest (around 10-fold) preferential affinity for D over D3 receptors (Kulagowski et al., 1996; Millan et al., 2000). Our data are entirely consistent with those of Millan et al. (2000) and Collins et al. (2007a, b, 2009), who reported that 2.5 or 3.2mg/kg L-741,626 nearly fully antagonized PE and YA produced by 7-OH-DPAT, PD-128,907 or pramipexole. Such in-vivo potencies are compatible with the in-vitro affinity of L-741,626 for rat D2 receptors (20-3Onmol/l: in-house unpublished data; Millan et al., 2000). To summarize, these positive data with DA D2 receptor antagonists, together with the negative results observed with DA D3 receptor antagonists discussed above (at doses that we consider to be selective for DA D3 receptors), suggest that D receptors are the most likely candidates for mediating PE and YA elicited by apomorphine in rats.
 
Conclusion
 
Our data do not support a role for DA D4 receptors in the control of either PE or YA in rats, an observation in contrast to those of others (Brioni et al., 2004; Cowart et al., 2004; Hsiehetd, 2004; Moreland et al., 2005; Melis et al., 2006), but in full agreement with more recent reports (Collins et al., 2007b, 2009). The profile of DA D3/D2 receptor agonists for eliciting YA and PE was remarkably similar to that shown by others (Millan etal., 2000; Collins et al., 2007b, 2009). However, under our experimental conditions, that is, with doses of antagonists that we believe to be selective for their respective targets, our data led us to conclude, similarly to some authors (Millan et cl., 2000), but contrary to others (Collins et cl., 2005, 2007a, b, 2009), that DA D2 receptors are more likely than their D3 counterparts to play a preponderant role in the control of PE or YA in rats. One should, however, remain cautious in generalizing these rodent findings to other species - in particular humans - and results from clinical trials for erectile dysfunctions with selective DA D4 (and perhaps D3) receptor agonists are eagerly anticipated to clarify the role of the various DA receptor subtypes in PE.