The dopamine D(3) preferring agonist
pramipexole (50ng) induced penile erection and
yawning when injected into the paraventricular
nucleus of the hypothalamus of male rats, like
the mixed D(1)/D(2)-like agonist apomorphine
(50ng), while the D(4) agonist PD 168,077
(100ng), induced penile erection only. These
responses lasted for 45-60min and occurred with
an increase of NO(2)- and NO(3)- concentrations
in the dialysate obtained from the
paraventricular nucleus by intracerebral
microdialysis. Pramipexole and apomorphine
responses were reduced by the D(2) preferring
antagonist L-741,626 (5?g), but not by the D(3)
preferring antagonist SB-277011A (10?g), or the
D(4) preferring antagonist L-745,870 (5?g),
injected into the PVN before the dopamine
agonist. In contrast, PD 168,077 responses were
reduced by L-745,870, but not by L-741,626 or
SB-277011A. Pramipexole, apomorphine and PD
168,077 effects were also reduced by the nitric
oxide synthase inhibitor
S-methyl-l-thiocitrulline (20?g) and the N-type
voltage-dependent Ca(2+) channels blocker
?-conotoxin (5ng), given into the
paraventricular nucleus, and by the oxytocin
antagonist
d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin (2?g),
given intracerebroventricularly but not into the
paraventricular nucleus before dopamine
agonists. These results suggest that stimulation
of D(2), but not D(3) or D(4) receptors, by
pramipexole or apomorphine increases Ca(2+)
influx in cell bodies of oxytocinergic neurons.
This increases the production of nitric oxide,
which activates oxytocinergic neurotransmission
in extra-hypothalamic brain areas and spinal
cord, leading to penile erection and yawning.
However, the stimulation of D(4) receptors by PD
168,077 also increases Ca(2+) influx/nitric
oxide production leading to penile erection, but
not yawning.
1. Introduction
Dopamine agonists induce penile erection and
yawning in laboratory animals and humans, these
responses being mediated by dopamine receptors
of the D2 but not of the D1 family
[1&endash;7]. The paraventricular
nucleus of the hypothalamus (PVN) is a brain
area where dopamine agonists act to induce
penile erection and yawning. Accordingly,
D2-like, but not D1-like agonists, induce these
responses when injected into the PVN of male
rats and D2-like, but not D1-like antagonists,
impair these responses when given into the PVN
before D2-like agonists
[1,2,8&endash;10]. Apparently, dopamine
agonists stimulate D2-like receptors located in
the cell bodies of oxytocinergic neurons
originating in the PVN and projecting to1.
Introduction Dopamine agonists induce penile
erection and yawning in laboratory animals and
humans, these responses being mediated by
dopamine receptors of the D2 but not of the D1
family [1&endash;7]. The paraventricular
nucleus of the hypothalamus (PVN) is a brain
area where dopamine agonists act to induce
penile erection and yawning. Accordingly,
D2-like, but not D1-like agonists, induce these
responses when injected into the PVN of male
rats and D2-like, but not D1-like antagonists,
impair these responses when given into the PVN
before D2-like agonists
[1,2,8&endash;10]. Apparently, dopamine
agonists stimulate D2-like receptors located in
the cell bodies of oxytocinergic neurons
originating in the PVN and projecting to
extra-hypothalamic brain areas and to the spinal
cord [2,8&endash;10]. Indeed, PVN
oxytocinergic cell bodies are innervated by
incertohypothalamic dopaminergic neurons
originating in the A13&endash;A14 cell groups of
Dahlstrom and Fuxe [11,12]. The
stimulation of PVN D2-like receptors increases
Ca2+ influx into the cell bodies of
oxytocinergic neurons, leading to the activation
of nitric oxide (NO) synthase. NO in turn
activates oxytocinergic neurons to release
oxytocin in the spinal cord and
extra-hypothalamic brain areas, leading to
penile erection and yawning [2,9,10].
Molecular biology studies allowed the
characterization of at least three D2-like
receptor subtypes, D2 (with the D2 short and D2
long splice variants), D3 and D4
[13&endash;15].
These receptor subtypes were cloned and
inserted into cultured cells allowing the
synthesis of selective agonists and antagonists.
Binding studies with these molecules allowed the
identification of dopamine receptor subtypes
across the brain, and suggested their
involvement in specific central functions and
pathologies, from schizophrenia to autism
[15]. Recently, a few D4 agonists were
found capable of inducing penile erection when
given systemically or into the PVN
[16&endash;19]. This led to compare the
effect of selective D2, D3 and D4 agonists and
antagonists on penile erection and yawning with
that of the D3-preferring agonist pramipexole
and apomorphine given systemically and/or into
the PVN. While some of these studies supported a
main role for D3 receptors in penile erection
and yawning induced by dopamine agonists (see
[20&endash;22]), other studies supported
a main role for D2 receptors (see
[23,24]). However, at variance from
[24] the above studies [21,23]
failed to confirm a pro-erectile role of D4
receptors. In order to further characterize the
role of PVN D2, D3 and D4 receptors, which have
been recently found to co-exist in the cell
bodies of PVN oxytocinergic neurons
[25,26], and the mechanism by means of
which pramipexole induces penile erection and
yawning, the effects of pramipexole, apomorphine
and the D4 preferring agonist PD 168,077 given
into the PVN on penile erection, yawning and on
the production of NO, measured by the
concentration of NO2 - and NO3 - in the PVN
dialysate obtained by intracerebral
microdialysis, were compared. The effect of D2,
D3 and D4 preferring receptor antagonists given
into the PVN before dopamine agonists on these
responses was also studied.
4. Discussion
The present results show for the first time
that pramipexole, a D3 preferring receptor
agonist [30&endash;32], when injected
into the PVN of male rats at a dose that induces
penile erection and yawning, increases the
production of NO in the PVN as determined by the
increase of the concentration of NO2 - and NO3 -
in the paraventricular dialysate obtained by
intracerebral microdialysis. These pramipexole
responses were similar to those obtained with
apomorphine, a classical mixed D1/D2-like
receptor agonist, which also induces penile
erection and yawning when injected into the PVN
and increases NO production in the
paraventricular dialysate [8,10,27].
Indeed, at the dose used (50 ng for both drugs),
the efficacy of the two compounds (e.g., the
maximal number of penile erections/rat and the
maximal increase in NO production elicited in
the first hour after treatment), did not differ
significantly. PD 168,077, a selective D4
receptor agonist [33], also induces
penile erection when injected into the PVN,
which occurs concomitantly to an increase in NO
production in the paraventricular dialysate,
although less effectively than pramipexole and
apomorphine. Indeed, at the dose of 100 ng, PD
168,077 increased the number of penile erection
episodes and the concentration of NO2 - and NO3
- by about 60&endash;65% only of the values
obtained with 50 ng of pramipexole and
apomorphine [18,34]. However, at
variance from pramipexole and apomorphine, this
D4 receptor agonist, at least at the dose used
in this study, was found unable to induce
yawning, as if D4 receptors in the PVN were not
involved in such response. Although it cannot be
completely ruled out that different responses
might have been obtained with higher doses of
these dopamine agonists, this is unlikely since
doses of pramipexole, apomorphine and PD 168,077
higher than 50 ng for pramipexole and
apomorphine and 100 ng for PD 168,077 up to 200
ng were found unable to increase further the
number of penile erection episodes when injected
into the PVN [24].
The present results also show that the
effects of pramipexole and apomorphine on penile
erection, yawning and paraventricular NO
production and of PD 168,077 on penile erection
and paraventricular NO production when given
directly into the PVN, are differentially
influenced by dopamine preferring antagonists of
the three dopamine receptor subtypes, D2, D3 or
D4, given into the PVN before the dopamine
agonists. Accordingly, penile erection, yawning
and the increase of NO production in the
paraventricular dialysate induced by
pramipexole, were reduced almost completely by
L-741,626, a D2 preferring receptor antagonist
[35,36], but not by SB-277011A, a D3
preferring receptor antagonist [37,38]
or L- 745,870, a D4 preferring receptor
antagonist [39]. Similar results were
obtained when penile erection, yawning and the
increase of NO production in the paraventricular
dialysate were induced by apomorphine, with the
only difference that these responses (but not
yawning) were found to be also decreased
slightly (by about 25%) by L-745,870, despite
the inability of this D4 receptor antagonist to
reduce pramipexole responses. In contrast,
penile erection and the increase in NO
production induced by PD 168,077 were
antagonized by L-745,870, which blocks D4
receptors, as expected [17,18,34], but
were not reduced by L-741,626 or SB-277011A,
which block D2 and D3 receptors, respectively.
Together these findings are in line with the
idea that penile erection, yawning and the
increase of NO production induced by pramipexole
and apomorphine, are mediated mainly by the
stimulation of dopamine receptors of the D2
subtype, and that D3 and D4 receptors play only
a minor role, if any, in the pro-erectile effect
of these dopamine receptor agonists and in their
ability to increase NO production in the PVN, as
already discussed in detail for
pramipexole-induced penile erection (see
[24] and below). Briefly, it is unlikely
that different results should have been obtained
with higher doses of the dopamine antagonists
used in this study. Accordingly, the doses of
L-741,626, SB 277011A and L-745,870 used in this
study were chosen because of the results of
dose&endash;response curves showing that while 5
g of L-741,626 injected into the PVN (the same
used in this study) reduced by more than 80% the
increase of penile erection episodes induced by
pramipexole given into the PVN, doses up to 5 g
of SB-277011A (half the dose used in this study)
and of FAUC 365 (another D3 preferring
antagonist with a binding affinity for D3
receptors 20 times higher than that of
SB-277011A [40]), and of L-745,870 up to
5 g, were completely ineffective against this
dopamine agonist [24].
The present findings also confirm that the
stimulation of D4 receptors in the PVN seems
sufficient per se to induce penile erection,
which also occurs concomitantly with an increase
of NO production in the PVN. Nonetheless, in
spite of these similarities with pramipexole and
apomorphine in increasing penile erection
episodes and NO production, this study shows
that PD 168,077 does not increase yawning when
injected into the PVN, at least at the dose used
in this study. Although it cannot be ruled out
that much higher doses of this compound may be
found capable of triggering yawning when
injected into the PVN, the finding is in line
with other studies showing that PD 168,077 and
other D4 agonists, such as ABT 724 and
RO-10-5824, are unable to induce yawning when
given systemically [20,21,23], as if D4
receptors, including those in oxytocinergic
neurons in the PVN [25,26] were not
involved in the facilitatory effect of D2-like
dopamine agonists on this behavioral response.
Whatever mechanism is responsible for the
inability of PD 168,077 to induce yawning, the
present results are in line with those of early
studies showing that D2-like agonists injected
into the PVN induce penile erection and yawning
by acting on dopamine receptors located in the
cell bodies of paraventricular oxytocinergic
neurons [25,26] rich in NO synthase and
projecting to the spinal cord and
extra-hypothalamic brain areas (see
[10,41] and references therein). The
stimulation of these receptors causes an
increase in Ca2+ influx into the cell bodies of
these oxytocinergic neurons leading to the
activation of NO synthase, a
Ca2+&endash;calmodulin dependent enzyme, which
increases NO production. NO in turn activates
oxytocinergic neurons to release oxytocin in
extra-hypothalamic brain areas controlling
penile erection and yawning, apparently by a
cGMP-independent mechanism [41,42]. In
line with this hypothesis, the increase of
penile erection and yawning and of NO production
in the PVN dialysate induced by pramipexole, are
strongly reduced by the prior injection into the
PVN of S-methyl-l-thiocitrulline, which inhibits
NO synthase [43] and by -conotoxin, a
potent blocker of N-type-voltage dependent Ca2+
channels [44], while
d(CH2)5Tyr(Me)2-Orn8-vasotocin, which blocks
oxytocin receptors [45], reduces penile
erection and yawning without affecting the
increase in NO production when injected into the
lateral ventricles but not into the PVN. These
results are similar to those found with
apomorphine and PD 168,077, as expected, since
pramipexole, like apomorphine and PD 168,077,
injected into the PVN acts on dopamine receptors
localized in the cell bodies of oxytocinergic
neurons [25,26] and not on oxytocin
receptors to activate oxytocinergic neurons
mediating penile erection and yawning, as
discussed extensively in early studies
[10,41]. Perhaps even more importantly
for this work, G-protein-coupled D2-like
receptors that may be linked to an increase in
Ca2+ influx in cell preparations have been
identified in cloned versions of D2-like
receptors, mainly of the D4 subtype (see
[16,46]). In this regard, it is
noteworthy that PD 168,077 injected into the
PVN, induces not only penile erection and
increases NO production, but also increases c-
Fos and extra-cellular signal regulated (ERK)
expression in the PVN [47], effects that
are all antagonized by D4 receptor antagonists
[18,47]. The increased c-Fos expression
induced by PD 168,077 in the PVN resembles the
ability of pro-erectile doses of apomorphine to
increase c-Fos content in discrete brain areas,
including the PVN, when given systemically
[48]. Unfortunately, it has still to be
confirmed that the increased c-Fos expression
induced by PD 168,077 occurs in PVN
oxytocinergic neurons and if this increase is
related to the pro-erectile effect of this D4
agonist [26].
Finally, this study provides further
evidence for a differential role of D2, D3 and
D4 receptors present in the PVN in mediating
penile erection and yawning induced by dopamine
D2-like receptor agonists. In particular, the
major role of D2 vs. D3 receptor in penile
erection and yawning induced by the D3
preferring agonist pramipexole suggested by this
study, is in line with the recent study by
Depoortère [23] and Sanna
[24] and their coworkers, but is in
contrast with those by Collins and coworkers
[20,21], which supports a major role for
D3 vs. D2 receptors. Briefly, while
Depoortère [23] and Sanna
[24] and their coworkers found that
apomorphine- and pramipexole-induced penile
erection and yawning are strongly reduced by
L-741,626, but not by SB- 277011-A and other D3
antagonists, Collins and coworkers
[20,21] found that pramipexole-induced
penile erection and yawning are significantly
reduced not only by L-741,626, but also by SB-
277011-A given at doses similar to that used by
Depoortère [23] and Sanna
[24] and their coworkers, and completely
abolished by a very high doses of this compound,
which is likely to act not only on D3 receptors
but also on D2 receptors (see [23,24]
and references therein). In spite of the above
similarities and differences with the results of
Sanna and coworkers [24] and this study,
the studies by Collins [21] and by
Depoortère [23] and coworkers
failed to confirm the pro-erectile effect of D4
receptor agonists, such as PD 168,077 (used also
in this study) and other D4 receptor agonists as
well (e.g., ABT 724, A 412997, etc.)
[16&endash;19,49,50]. The reasons of
these discrepancies are unknown, although they
may be due to methodological differences in the
experimental protocols (i.e., several doses of
each antagonist vs. a full dose&endash;response
curve for each agonist [20&endash;22],
against several doses of each antagonist vs. a
single dose of agonist [23,24],
different rat strains [23], different
nutritional state [51], etc.).
In this regard, it must be emphasized that
in the present study dopamine agonists and
antagonists were given directly into the PVN,
therefore the observed responses are the results
of the selective action of these drugs on
dopamine receptors in this specific brain area
only. This is at variance from the studies of
Collins [20,21] and Depoortère
[23] and their coworkers, in which drug
effects were studied only after systemic
administration. This allows administered drugs
to reach and act in every part of the central
nervous system and in the periphery as well.
Therefore, the observed behavioral effects are
the results of the algebraic sum of the effects
of these drugs at different brain and peripheral
sites, since dopamine can influence penile
erection and yawning by acting in different
brain areas (see [41] and references
therein). Moreover, the D2, D3 and D4
antagonists used in these studies have been
identified for their ability to act on the
different dopamine receptor subtypes mainly in
in vitro experiments with cell membranes or
cultured cells. It would be therefore considered
not surprising that drugs with a similar profile
in vitro, show different effects when
administered in vivo and that these effects may
be different when these drugs are given
systemically or directly into specific brain
areas, i.e., the PVN. Finally, although direct
injection into the PVN eliminates eventual
pharmacokinetic differences (ability to cross
blood brain barrier, different metabolism rate,
and so on), also in this case it is very
difficult to prove that D2, D3 and D4 agonists
and antagonists act on dopamine receptor
subtypes with a profile identical to that found
in in vitro experiments. Therefore, dopamine
receptor agonists and antagonists with
selectivities for the different dopamine
receptor subtypes much higher than those of the
drugs now available are necessary to define the
exact contribution of PVN D2, D3 and D4 receptor
subtypes in penile erection and yawning induced
by D2-like dopamine agonists.
In conclusion, the present results
show that pramipexole injected into the PVN of
male rats at a dose that induces penile
erection, induces also yawning and increases NO
production in this hypothalamic nucleus with
potency similar to that of apomorphine. PD
168,077 also injected into the PVN, induces
penile erection and increases NO production,
although with an efficacy lower than that of
pramipexole and apomorphine as expected, but was
unable to induce yawning. Since pramipexole
responses, like those of apomorphine, were
reduced almost completely by the prior injection
into the PVN of dopamine receptor antagonists,
which block preferentially D2 receptors, this
suggests a main role of receptors of the D2
subtype in these responses, at least when
induced by these dopamine agonists. However, the
stimulation of D4 receptors in the PVN is
sufficient per se to induce penile erection and
to increase NO production. As
pramipexole-induced penile erection and yawning
are also reduced by the prior injection into the
PVN of S-methyl-l-thiocitrulline, which inhibits
NO synthase, of -conotoxin, which blocks N-type
voltage dependent Ca2+ channels and of
d(CH2)5Tyr(Me)2-Orn8- vasotocin, which blocks
oxytocin receptors, these findings show that
this drug also, like apomorphine, acts in the
PVN to induce penile erection and yawning by
increasing NO production, thereby activating
central oxytocinergic neurotransmission.
