-
- 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.
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- 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.
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- 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).
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- 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.
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