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mise à jour du
12 février 2003
Eur J Pharmacol
1998;359(1):19-26
lexique
Different effects of -conotoxin
on penile erection, yawning
and paraventricular nitric oxide in male rats
Succu S, MS Spano, et al

Chat-logomini

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.
conotoxin
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 -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 -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 -conotoxin on penile erection and yawning induced by the NO donors, sodium nitroprusside and hydroxylamine. [...]
 
Discussion : The present results showed that -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 -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 -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 -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 -conotoxin -sensitive Ca 2+ channels or to a still unidentified transduction system, which leads to the opening of -conotoxin-sensitive Ca 2+ channels. Were the latter hypothesis correct, opening of -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 -conotoxin-sensitive Ca 2+ channels are located in the same neurons. However -conotoxin might prevent apomorphine and oxytocin responses by inhibiting -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, -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 -conotoxin to prevent NMDA-induced NO production was found, for instance, in cultured striatal neurons. The inability of -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 -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 -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 -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.