A dose of apomorphine or oxytocin that
induces penile erection and yawning increases
nitric oxide production in the paraventricular
nucleus of the hypothalamus, as determined by
the increase in NO2- and NO3- concentration
induced by these substances in the
paraventricular dialysate obtained from male
rats. All the above responses were prevented by
a dose of omega-conotoxin-GVIA as low as 5 ng.
This potent inhibitor of N-type Ca2+ channels
was injected into the paraventricular nucleus 15
min before apomorphine (50 ng) or oxytocin (10
ng). In contrast, omega-conotoxin was
ineffective when the above responses were
induced by N-methyl-D-aspartic acid (50 ng). The
peptide toxin (5 ng) was also ineffective on the
penile erection and yawning induced by the
nitric oxide donors sodium nitroprusside (50
microg) or hydroxylamine (50 microg), injected
into the paraventricular nucleus. The present
results suggest that omega-conotoxin-sensitive
Ca2+ channels are involved in the activation of
nitric oxide synthase, penile erection and
yawning induced by apomorphine and oxytocin, but
not by N-methyl-D-aspartic acid, at the
paraventricular level.
1. Introduction
Penile erection and yawning are two
different behavioural patterns that often occur
concomitantly under different physiological and
experimental conditions (Holmgren et al., 1985;
Argiolas and Gessa, 1991; Meisel and Sachs,
1994; Argiolas and Melis, 1995, 1998). While the
importance of penile erection in reproduction
does not need to be stressed, it is pertinent to
recall that yawning is considered an ancestral
vestige that subserves the purpose of arousal,
although its role is far from being clarified
(see Argiolas and Melis, 1998). Dopamine
receptor agonists, such as apomorphine, the
neurohypophyseal peptide, oxytocin and
N-methyl-D-aspartic acid (NMDA), a selective
agonist of the excitatory amino acid receptor of
the Ca 2+ NMDA subtype (Monaghan et al., 1989),
are among the most potent inducers of this
symptomatology in male rats. In particular, all
these substances induce penile erection and
yawning when injected in
nanogram amounts into the paraventricular
nucleus of the hypothalamus (for review see
Argiolas and Melis, 1995, 1998 and references
therein). Several lines of experimental evidence
suggest that these substances induce such
behavioural responses by activating
oxytocinergic neurons originating in the
paraventricular nucleus and projecting to
extrahypothalamic brain areas, i.e. the
hippocampus, the ventral medulla and the spinal
cord (see Argiolas and Melis, 1995, 1998). The
molecular mechanisms for apomorphine, oxytocin
and NMDA activation of oxytocinergic
transmission to induce these behavioural
responses, may involve Ca 2+ and pertussis
toxin-sensitive G proteins. First, the NMDA
effect is prevented by MK-801, a potent
non-competitive antagonist of the NMDA receptor
(Wong et al., 1986), which blocks Ca 2 influx
through the Ca 2
channel-coupled NMDA receptor. Second,
either organic Ca 2+ channel blockers or
nanogram amounts of w-conotoxin-GYIA, a potent
blocker of N-type Ca 2 channels (McCleskey et
al., 1987), prevent both apomorphine- and
oxytocin-induced penile erection and yawning
(Argiolas et al., 1989, 1990). Third, pertussis
toxin, which inhibits the activity of several G
proteins including the G0 protein
coupled to voltage-dependent Ca 2 channels
(see Dolphin, 1987), injected in the
paraventricular nucleus prevents both
apomorphine- and oxytocin-induced penile
erection and yawning (Stancampiano et al.,
1992).
We found that nitric oxide (No) is involved
at the paraventricular level in the control of
penile erection and yawning induced by
apomorphine, oxytocin and NMDA. First, the
microinjection of NGnitroLarginine methyl ester,
a potent and selective inhibitor of NO synthase
(Rees et al., 1990), the Ca
2-calmodulin-dependent enzyme that synthesizes
NO from L-arginine (see Snyder, 1992; Moncada
and Higgs, 1993; Southam and Garthwaite, 1993;
Schuman and Madison, 1994) into the
paraventricular nucleus inhibits the penile
erection and yawning induced by the above
substances (Melis et al., 1994a,b). Second, NO
donors microinjected into the paraventricular
nucleus induce penile erection and yawning
indistinguishable from those induced by the
above substances (Melis and Argiolas, 1995;
Melis et al., 1995). Third, NO donor-induced
penile erection and yawning are antagonized by
the oxytocin receptor antagonist [d(CH2
)5Tyr(Me)2-Orn8 ]vasotocm, given
intracerebroventricularly (i.c.v.) (Melis and
Argiolas, 1995; Melis et al., 1995). Since NO
synthase is present in high concentrations in
the paraventricular nucleus, including
oxytocinergic cell bodies (Bredt et al., 1990;
Vincent and Kimura, 1992; Sanchez et al., 1994;
Amir, 1995), these findings led us to suggest
that apomorphine, oxytocin and NMDA induce
penile erection and yawning by increasing the Ca
2 influx that, in turn, activates NO synthase in
the cell bodies of oxytocinergic neurons
mediating these behavioural responses (Argiolas
and Melis, 1995, 1998; Melis and Argiolas,
1997). The validity of this hypothesis was
supported by results of in vivo microdialysis
studies showing that dopamine receptor agonists,
oxytocin and NMDA, when given at doses that
induce penile erection and yawning, increase NO
production in the paraventricular dialysate
obtained from male rats (Melis et al., 1996,
1997a,b).
To further investigate the relationship
between Ca 2 + ions and NO in the control of
penile erection and yawning induced by
apomorphine, oxytocin and NMDA, we first used in
vivo microdialysis in studies on the effect of
w-conotoxin injected into the paraventricular
nucleus on the increase of paraventricular NO
production induced by a dose of the above
substances that induces penile erection and
yawning. Second, we tested the effect of
w-conotoxin on penile erection and yawning
induced by the NO donors, sodium nitroprusside
and hydroxylamine.
4. Discussion
The present results showed that w-conotoxin,
a potent and selective blocker of N-type Ca 2
channels (McCleskey et al., 1987), injected into
the paraventricular nucleus prevents the
apomorphine- and oxytocin-induced increase of
N05 and NO concentration in the paraventricular
dialysate, and the penile erection and yawning.
This agrees with the results of previous studies
showing that penile erection and yawning induced
by apomorphine or oxytocin were prevented by
w-conotoxin given either i.c.v. or directly into
the paraventricular nucleus (Argiolas et al.,
1990). Since N05 and NO are the products of the
reaction of newly synthetized NO with 02
(Ignano, 1990; Luo et al., 1993; Ohta et al.,
1994; Melis et al., 1996), and NO is synthesized
by the Ca 2-calmodulin-dependent NO synthase
(Snyder, 1992; Moncada and Higgs, 1993; Southam
and Garthwaite, 1993; Schuman and Madison, 1994)
these results provide further support for the
hypothesis that apomorphine and oxytocin
increase the Ca 2 + concentration in the cell
bodies of paraventricular oxytocinergic neurons
mediating penile erection and yawning. The
increased Ca 2+ concentration, in turn,
activates NO synthase in these oxytocinergic
neurons (see Section 1). Most important, the
potency of w-conotoxin to prevent the increase
in NO production, penile erection and yawning
induced by apomorphine or by oxytocin confirms
that Ca 2 influx through N-type Ca 2+ channels
plays an important role in the activation of NO
synthase and consequent behavioural responses
induced by these compounds, as previously
suggested (Melis et al., 1997a,b). As to the
mechanism by means of which the activation of
dopamine or oxytocin receptors may cause the
opening of w-conotoxin-sensitive Ca 2 channels,
one possibility is that both dopamine and
oxytocin receptors in the paraventricular
nucleus are coupled through a G protein,
directly to w conotoxin-sensitive Ca 2 channels
or to a still unidentified transduction system,
which leads to the opening of
w-conotoxin-sensitive Ca 2 + channels. Were the
latter hypothesis correct, opening of
w-conotoxin-sensitive Ca 2+ channels would be
mediated by changes in the content of second
messengers (e.g. diacylglycerol or
inosytol-triphospate) as found in other tissues
(for a review on the transduction systems
coupled to dopamine receptors see Baldessarini,
1996 and for those coupled to oxytocin receptors
see Argiolas and Gessa, 1991 and Lambert et al.,
1994). In line with this possibility, pertussis
toxin, which inhibits several G proteins (for a
review see Dolphin, 1987), injected into the
paraventricular nucleus prevents both
apomorphine- and oxytocin-induced penile
erection and yawning (Stancampiano et al.,
1992).
The above explanation is based mainly on the
assumption that both apomorphine and oxytocin
act directly on receptors located in the cell
bodies of oxytocinergic neurons mediating penile
erection and yawning and that w
conotoxin-sensitive Ca 2+ channels are located
in the same neurons. However w-conotoxin might
prevent apomorphine and oxytocin responses by
inhibiting w-conotoxinsensitive Ca 2 channels
located presynaptically, that is by inhibiting
the release of other neurotransmitters and/or
neuropeptides that activate oxytocinergic
neurons to induce penile erection and yawning,
i.e. dopamine, excitatory amino acids or
oxytocin itself. Were this the case, apomorphine
would increase the release of oxytocin or
excitatory amino acids, to induce the above
responses, while oxytocin would increase the
release of dopamine or excitatory amino acids.
However, several findings argue against this
possibility. First, MK-801, which blocks NMDA
receptors (Wong et al., 1986), injected into the
paraventricular nucleus is unable to prevent
penile erection and yawning induced by
apomorphine or by oxytocin, despite preventing
NMDA-induced penile erection, yawning and the
concomitant increase in paraventricular NO
production (Melis et al., 1997b). Second,
haloperidol, which blocks dopamine receptors, is
unable to prevent penile erection and yawning
induced by oxytocin or by NMDA, despite its
ability to prevent apomorphine-induced penile
erection, yawning and the concomitant increase
in paraventricular NO production (Melis et al.,
1996, 1 997a,b). Third, [d(CH2
)5Tyr(Me)2-Orn8 ]vasotocin, which blocks
oxytocinergic receptors (Bankowski et al.,
1980), is unable to prevent apomorphine- and
NMDA-induced penile erection, yawning and the
concomitant increase in paraventricular NO
production, when injected into the
paraventricular nucleus (Melis et al., 1996,
1997b), despite its ability to prevent oxytocin
responses (Melis et al., 1997a).
In contrast, w-conotoxin does not prevent
NMDA-induced NO production, penile erection and
yawning, while it prevents these responses when
they are induced by apomorphine and oxytocin.
Since these NMDA responses are easily explained
if NMDA increases the Ca 2+ concentration in the
cell bodies of oxytocinergic neurons mediating
penile erection and yawning by opening NMDA
receptor-coupled Ca 2 channels (see Melis et
al., 1997b), the finding suggests that
w-conotoxin-sensitive Ca 2 channels play only a
minor role, if any, in the NMDA-induced
activation of NO synthase of paraventricular
oxytocinergic neurons mediating the above
behavioural responses. A similar failure of
w-conotoxin to prevent NMDA-induced NO
production was found, for instance, in cultured
striatal neurons (Rodriguez-Alvarez et al.,
1997). The inability of w-conotoxin to prevent
NO synthase activation by NMDA is in line with
the hypothesis that NMDA does not act by
modulating the release of other
neurotransmitters and/or neuropeptides present
in the paraventricular nucleus, such as
excitatory amino acids (Van den Pol, 1991),
dopamine (Buijs et al., 1984; Lindvall et al.,
1984) or oxytocin itself (see Argiolas and
Gessa, 1991).
The present results also showed that
w-conotoxin does not prevent penile erection and
yawning induced by the NO donors sodium
nitroprusside and hydroxylamine when they are
injected into the paraventricular nucleus. The
finding is in line with the hypothesis that NO
formed by these compounds (and endogenous NO as
well) acts as an intracellular messenger inside
the oxytocinergic neurons mediating penile
erection and yawning (see Argiolas and Melis,
1995, 1998; Melis and Argiolas, 1997), rather
than by modulating Ca 2 influx through
co-conotoxin- sensitive Ca 2 channels.
Interestingly, NO donors modulate NMDA
receptor-coupled Ca 2 channels (Kiedrowski et
al., 1992; Hoyt et al., 1992) and other ionic
channels as well, through cyclic guanosine 3,5'
monophosphate (c-GMP)-dependent and -independent
mechanisms in brain tissues and neuronal cell
lines (Chen and Schofiels, 1995; Clementi et
al., 1995; Koh et al., 1995; Sawada et al.,
1995). However, since MK-801, which blocks NMDA
receptor-coupled Ca 2 channels (Wong et al.,
1986), is unable to prevent NO donor-induced
penile erection and yawning, it is unlikely that
NO donors modulate NMDA receptors in the
paraventricular nucleus to induce these
behavioural responses. Conversely, the inability
of MK-801 to prevent NO donor-induced penile
erection and yawning suggests that these
compounds do not release excitatory amino acids
in the paraventricular nucleus to induce these
behavioural responses. Since NO donor-induced
penile erection and yawning are prevented by
neither oxytocin receptors antagonists given
into the paraventricular nucleus (Melis and
Argiolas, 1995; Melis et al., 1995) nor
haloperidol, which blocks dopamine receptors
(Melis et al., 1995 and this study), it is
unlikely that NO donors modulate apomorphine or
oxytocin receptors or facilitate the release of
endogenous dopamine or oxytocin in the
paraventricular nucleus to induce these
behavioural responses.
Unfortunately the present results do not
clarify the mechanism(s) by means of which
endogenous or NO donor-derived NO activates
oxytocinergic transmission at the
paraventricular level to induce penile erection
and yawning. c-GMP is apparently not involved,
at least at the paraventricular level, in these
behavioural responses, although a role of
guanylate cyclase in sites distant from the
paraventricular nucleus cannot be ruled out
(Melis et al., 1994a,b, 1995, 1996, 1997a,b;
Melis and Argiolas, 1995). Since Ca 2 influx
plays a key role in the increase of NO
production, penile erection and yawning induced
by apomorphine, NMDA and oxytocin, it is
tempting to speculate that NO donors induce
changes in intracellular Ca 2 + similar to those
induced by the above compounds in the
paraventricular oxytocinergic neurons mediating
these behavioral responses. It is consistent
with this possibility that NO donors induce
complex changes in intracellular Ca 2+ in
different cell lines and brain tissues
(Kiedrowski et al., 1992; Hoyt et al., 1992;
Clementi et al., 1995; Yolk et al., 1997).
In conclusion the present results are
compatible with the hypothesis that apomorphine,
oxytocin and NMDA increase NO synthesis directly
in the oxytocinergic neurons mediating penile
erection and yawning, rather than by modulating
the release of other neurotransmitters and/or
neuropeptides in the paraventricular nucleus.
However, while w-conotoxin-sensitive Ca 2
channels play an important role when the above
responses are induced by apomorphine or by
oxytocin, these Ca 2+ channels seem to play only
a minor role, if any, when the responses are
induced by NMDA or by NO donors.
