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18 mars 2004
European J Pharmacology
1995; 294; 1-9
Nitric oxide donors induce penile erection and yawning when injected in the central nervous system of male rats
MR Melis, A Argiolas
Bernard B. Brodie Department ofNeuroscience, Cagliari, Italy
Tous les travaux de MR Melis & A Argiolas 
Tous les travaux de M Eguibar & G Holmgren

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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.

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