Introduction Penile erection and
yawning are two different behavioral patterns
that often occur concomitantly in 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 alone or associated with
stretching is considered a vestigial behavior
that has survived through evolution and which
subserves the purpose of arousal. Among
substances that induce both these behavioral
responses, the best known are dopamine receptor
agonists, adrenocorticotropin (ACTH) and related
peptides and 5-HT receptor agonists that act
mainly on the 5-HT, receptor subtype.
Several lines of evidence suggest that the
paraventricular nucleus of the hypothalamus
plays a key role in the expression of the
behavioral responses induced by dopamine
receptor agonies, NMDA and oxytocin, but not by
ACTH or 5-HT receptor agonists. In particular,
it seems that dopamine receptor agonists,
NMDA and oxytocin itself induce the behavioral
responses by activating oxytocinergic
transmission. Accordingly, dopamine receptor
agonists, oxytocin and NMDA induce penile
erection and yawning when injected in this
hypothalamic nucleus, while ACTH and 5-HT
receptor agonists are ineffective; (2) dopamine
receptor agonist-, NMDA- and oxytocin- but not
ACTH- or 5-HT1c receptor agonist-induced penile
ercetion and yawning are prevented by the
central administration of oxytocin receptor
antagonists and by electrolytic lesions of the
paraventricular nucleus of the hypothalamus,
which induce an almost complete depletion of the
oxytocin content in extra-hypothalamic brain
areas and spinal cord.
Recently, we provided experimental evidence
that nitric oxide, the novel
transmitter/modulator present in several tissues
including brain, plays an important role in the
expression of penile erection and yawning
induced by apomorphine, a classical dopamine
receptor agonist, NMDA, oxytocin and 5-HT1c
receptor agonists.
Accordingly, (1) inhibitors of nitric
oxide-synthase, a calmodulin-depenilent
iron-containing enzyme that produces nitric
oxide from L-arginine, prevent the behavioral
responses induced by apomorphine, oxytocin, NMDA
and 5-HT1c receptor agonists when injected into
the lateral ventricles (i.c.v.); (2) the central
administration of nitroglycerin, a putative
nitric oxide donor, induces penile erection and
yawning apparently by increasing central
oxytocinergic transmission and (3) the
paraventricular nucleus of the hypothalamus
seems to be one of the areas where nitric oxide
controls the behavioral responses induced by all
the above agents except 5-HT1c receptor
agonists.
In the present study we further investigated
the involvement of nitric oxide in the control
of these behavioral responses by studying the
central effect of other classic nitric oxide
donors, such as sodium nitroprusside,
S-nitroso-N-acetyl-penicillamine, hydroxylamine
and isoamyl nitrite, on spontaneous penile
crection and yawning. Furthermore, since nitric
oxide is thought to act by activating guanylate
cyclase, the effect of methylene blue and LY
83583, putative guanylate cyclase inhibitors and
of hemoglobin, a nitric oxide scavenger on
nitric oxide donor-induced penile erection and
yawning as well as the effect of
8-bromo-guanosine 3':5'-cyclic monophosphate
(8-Br-cGMP), a metabolically stable analog of
cGMP, on spontaneous penile erection and yawning
were studied. Finally, the effect of a potent
nonapeptide oxytocin receptor antagonist,
d(CH2)5Tyr(Me)-Orn8-vasotocin, on nitric oxide
donor-induced penile erection and yawning is
also reported. [...]
Discussion : The present results show
that the putative nitric oxide donors sodium
nitroprusside, hydroxylamine and isoamyl nitrite
induce penile erection and yawning when injected
in the central nervous system, in particular in
the paraventricular nucleus of the hypothalamus.
In fact, these behavioral responses were not
seen when nitric oxide donors, with the
exception of isoamyl nitrite, were injected into
the lateral ventricles, because these responses
might have been masked by the often dramatic and
different behavioral effects that were probably
due to the different affinity and/or metabolic
biotransformation of these agents in specific
brain tissues. In fact, in agreement with
previous studies, although both
S-nitroso-N-acetylpenicillamine and sodium
nitroprusside injected i.c.v. induced
hypermotility, the latter induced also
convulsions followed by death, probably due to
the formation of highly toxic cyanide ions. It
is likely that the formation of cyanide ions
that diffuse into the brain is responsible also
for the death of about 50% of the rats injected
in the paraventricular nucleus with the highest
dose of sodium nitroprusside 12 h after
treatment, Likewise, hydroxylamine was
ineffective when injected i.c.v. because of its
potent convulsant effect but was very active
when injected locally in the paraventricular
nucleus. The ineffectiveness of
Snitroso-N-acetyl-penicillamine, in spite of its
ability to release nitric oxide spontaneously
even more effectively than sodium nitroprusside
and hydroxylamine is difficult to explain.
However, one possible explanation is that the
dose injected in the paraventricular nucleus (10
µg) was too low to induce the behavioral
responses. In fact higher doses were not
injected because this compound was poorly
soluble even in 95% ethanol and effective nitric
oxide donors were active only at doses higher
than 20µg.
The present results confirm and extend
previous findings showing that nitroglycerin,
another nitric oxide donor, induces penile
erection and yawning when injected i.c.v. or in
the paraventricular nucleus and provide further
evidence that paraventricular nitric oxide plays
a key role in the control of penile erection and
yawning. Indeed, (1) nitric oxide-synthase
inhibitors prevent apomorphine-, oxytocin- and
NMDA-induced penile erection and yawning when
injected in the paraventricular nucleus; (2) the
prevention by nitric oxide-synthase
înhibitors of apomorphine-, oxytocinand
NMDA-induced penile erection and yawning is
reversed by L-arginine; (3) the paraventricular
nucleus is one of the brain areas richest in
nitric oxide-synthase and (4) nitric
oxide-synthase is localized in paraventricular
oxytocinergic neurons. It is conceivable that
the nitric oxide donors induce these behavioral
responses by producing nitric oxide in the
paraventricular nucleus, which would in turn
activate central oxytocinergic transmission,
since the penile erection and yawning induced by
sodium nitroprusside, hydroxylamine and isoamyl
nitrite as well as nitroglycerin were prevented
by the i.c.v. injection of
d(CH2)5Tyr(Me)-Orn8-vasotocin, a potent oxytocin
receptor antagonist. However, since
d(CH2),Tyr(Me)-Orn8vasotocin was ineffective
when given in the paraventricular nucleus, it is
likely that drug-derived nitric oxide, as
proposed for endogenous nitric oxide, activates
oxytocinergic neurons originating in the
paraventricular nucleus and projecting to extra-
hypothalamic areas. The hippocampus or the
ventral medulla/spinal cord might be among these
areas, since both receive an oxytocinergic
innervation and are involved in the expression
of these behavioral responses to oxylocin.
The molecular mechanism by which endogenous
or drug-derived nitric oxide in the
paraventricular nucleus activates oxytocinergic
neurons to induce penile crection and yawning is
unknown. One possibility is that nitric oxide
activates guanylate cyclase, one of the most
common targets of this modulator in several
tissues including the brain, which would in turn
increase the concentration of the second
messenger cGMP, Accordingly, methylene blue and
LY 83583, two guanylate cyclase inhibitors,
given into the lateral ventricles prevent nitric
oxide donor-induced penile erection and yawning.
However, the involvement of guanylate cyclase
and cGMP, especially at the level of the
paraventricular nucleus, in these behavioral
responses must be considered with caution for at
least three reasons. First, methylene blue and
LY 8-3583 did not prevent nitric oxide
donor-induced penile erection and yawning when
injected into the paraventricular nucleus;
second, hemoglobin, a potent nitric oxide
scavenger, was ineffective in preventing nitric
oxide donor-induced responses when injected into
the paraventricular nucleus; and third, perhaps
most important, 8-BrcGMP, a stable cGMP analog
that can mimic the effect of endogenous cGMP
produced by nitric oxide activation of guanylate
cyclase, was unable to induce penile erection
and yawning when injected into the
paraventricular nucleus. Although the inability
of hemoglobin to prevent nitric oxide
donor-induced responses when injected directly
in the paraventricular nucleus might be
explained by the inability of this compouild to
cross cellular membranes because of its high
molecular weight, all together the above
findings suggest that these substances induce
penile erection and yawning after their
injection in the paraventricular nucleus by a
mechanism not related to the stimulation of
guanylate cyclase, that is by a
cGMP-indepenilent mechanism. Accordingly, if one
assumes that nitric oxide donor-induced
responses are mediated by nitric oxide produced
in target neurons, nitric oxide might interact
with numerous other enzymes that, like guanylate
cyclase, bind metal ions such as iron, as
described for instance in fibroblasts.
Whatever mechanism nitric oxide activates in
the paraventricular nucleus to induce penile
erection and yawning, the ineffectiveness of
hemoglobin, which because of its high molecular
weight is unable to cross cellular membranes to
prevent nitric oxide donor-induced responses
suggests also that nitric oxide acts
intracellularly, possibly in the same neurons in
which it is formed, to induce penile erection
and yawning. This implies that nitric oxide
released extracellular in the paraventricular
nucleus is not involved in the control of these
behavioral responses. This hypothesis is
complicated by the finding that methylene blue
or LY 83583 given i.c.v. prevented nitric oxide
donor-induced responses, which favours a
neurotransmitter role of nitric oxide in brain
areas distant from the paraventricular nucleus
rather than a local messenger role in the
paraventricular nucleus. A possible unifying
explanation for such discrepancy is that nitric
oxide activates those oxytocinergic neurons
where it is formed, releasing in turn oxytocin
which activates directly or indirectly a
methylene blue- or LY 83583-sensitive guanvlate
cyclase mechanism in extrahypothalamic brain
arcas. This hypothesis is supported by the above
discussed ability of
d(CH2)5Tyr(Me)Orn8-vasotocin given i.c.v. to
prevent nitric oxide donor-induced penile
erection and yawning. Unfortunately, the present
results do not provide definitive evidence for a
role of guanylate cyclase in extra-hypothalamic
brain areas in the control of penile crection
and yawning. In fact, methylene blue or LY 83583
given i.c.v. might prevent nitric oxide
donor-induced penile erection and yawning by
acting with a mechanism not related to the
inhibition of guanylate cyclase and secondly
8-Br-cGMP injected i.c.v. induced a marked
hyperactivity that masked the above responses
and resembled that induced by nitric oxide
donors. \vhich is partially reversed by
methylene blue .
The interpretation given above of the present
results is based on two main assumptions. The
first is the existence of central oxytocinergic
projections originating in the paraventricular
nucleus and projecting to extra-hypothalmamic
brain areas that mediate peinle erection and
yawning. These pathways are activated by several
neurotransmitters, such as dopamine, glutamic
acid and oxytocin itself, and this activation is
secondary to the activation of nitric oxide
synthase in the cell bodies of paraventricular
oxytocinergic neurons. The second assumption is
that nitric oxide donors act exclusively
increasing the nitric oxide concentration in the
injection site, in particular in the cell bodies
of oxytocinergic neurons, to cause penile
erection and yawning. Although the possibility
that nitrie oxide donors induce the above
responses by other mechanisms cannot be
completely ruled out, this assumption is
strongly supported by the ability of several
nitric oxide donors to induce similar and
dose-depenilent responses apparently mediated by
the same mechanisms.
In conclusion, nitric oxide donors induce
penile crection and yawning when injected into
the paraventricular nucleus by activating
oxytocinergic transmission. These responses are
apparently not mediated by the activation of
guanylate cyclase in this hypothalamic nucleus,
although a role of this enzyme and thus of uGMP
in extra-hypothalamic brain areas cannot be
ruled out. Interestingly nitric oxide donors,
e.g. nitroglycerin and sodium nitroprusside,
have been also found to induce penile erection
in impotent men when used as topical ointments
or after intracavernosal administration, and
isoamyl nitrite has been known for its
aphrodisiac effect for many years. Our results
show that nitric oxide plays a key role in the
control of penile erection not only at a penile
level but also in the central nervous
system.
