Abstract : 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 µg) or
hydroxylamine (50 µg), 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.
Introduction : Penile erection and
yawning are two different behavioural patterns
that often occur concomitantly under different
physiological and experimental conditions. 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. 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+ channel-coupled NMDA subtype, 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. 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. 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,
which blocks Ca 2+ influx through the Ca 2+
channel-coupled NMDA receptor. Second, either
organic Ca 2+ channel blockers or nanogram
amounts of ½-conotoxin-GVIA, a potent blocker of
N-type Ca 2+ channels, prevent both apomorphine-
and oxytocin-induced penile erection and
yawning. Third, pertussis toxin, which inhibits
the activity of several G proteins including the
G. protein coupled to voltage-depenilent Ca
channels, injected in the paraventricular
nucleus prevents both apomorphine- and
oxytocin-induced penile erection and
yawning.
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 NG -nitro-L-arginine methyl
ester, a potent and selective inhibitor of NO
synthase, the Ca2+ calmudoin-depenilent enzyme
that synthesizes NO from L-arginine into the
paraventricular nucleus inhibits the penile
erection and yawning induced by the above
substances.
Second, NO donors microinjected into the
paraventricular nucleus induce penile erection
and yawning indistinguishable from those induced
by the above substances.
Third, NO donor-induced penile erection and
yawning are antagonized by the oxytocin receptor
antagonist (d(CH2),Tyr(Me)2-orn8)vasotocin,
given intracerebroventricularly (i.c.v.). Since
NO synthase is present in high concentrations in
the paraventricular nucleus, including
oxytocinergic cell bodies, these findings led us
to suggest that apomorphine, oxytocin and NMDA
induce peinile erection and yawning by
increasing the Ca influx that, in turn,
activates NO synthase in the cell bodies of
oxytocinergic neurons mediating these
behavioural responses. 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.
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
½-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
½-conotoxin on penile erection and yawning
induced by the NO donors, sodium nitroprusside
and hydroxylamine. [...]
Discussion : The present results
showed that ½-conotoxin, a potent and selective
blocker of N-type Ca 2+ channels, injected into
the paraventricular nucleus prevents the
apomorphine- and oxytocin-induced increase of
N02 and N03 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
½-conotoxin given either i.c.v. or directly into
the paraventricular nucleus.
Since N02 and N03 are the products of the
reaction of newly synthetized NO with O2, and NO
is synthesized by the Ca2+ calmodulin dependent
NO synthase 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 Ca2+ concentration, in turn,
activates NO synthase in these oxytocinergic
neurons. Most important, the potency of
½-conotoxin to prevent the increase in NO
production, penile erection and yawning induced
by apomorphine or by oxytocin confirins 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.
As to the mechanism by means of which the
activation of dopamine or oxytocin receptors may
cause the opening of ½-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 ½-conotoxin -sensitive Ca 2+
channels or to a still unidentified transduction
system, which leads to the opening of
½-conotoxin-sensitive Ca 2+ channels. Were the
latter hypothesis correct, opening of
½-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.
In line with this possibility, pertussis toxin,
which inhibits several G proteins, injected into
the paraventricular nucleus prevents both
apomorphine- and oxytocin-induced penile
erection and yawning.
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
½-conotoxin-sensitive Ca 2+ channels are located
in the same neurons. However ½-conotoxin might
prevent apomorphine and oxytocin responses by
inhibiting ½-conotoxin sensitive 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 arnino 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,
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. 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 concornitant increase in paraventricular NO
production. Third, (d(CH2),Tyr(Me)2-orn8)
vasotocin, which blocks oxytocinergic receptors,
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, despite its ability to
prevent oxytocin responses.
In contrast, ½-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, the finding
suggests that ù)-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 ½-conotoxin to prevent NMDA-induced
NO production was found, for instance, in
cultured striatal neurons. The inability of
½-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.
The present results also showed that
½-conotoxin does not prevent penile erection and
yawning induced by the NO donors sodium
nitroprusside and hydroxylarnine 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, rather than by modulating
Ca 2+ influx through ½-conotoxin-sensitive Ca 2+
channels. Interestingly, NO donors modulate NMDA
receptor-coupled Ca 2+ channels 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. However, since MK-801,
which blocks NMDA receptor-coupled Ca 2+
channels, 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 ammo 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 nor
haloperidol, which blocks dopamine receptors, it
is unlikely that NO donors modulate apomorphine
or oxytocin receptors or facilitate the release
of endogenous dopan-àne 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.
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 in
different cell lines and brain tissues.
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 ½-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.
