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mise à jour du
25 mars 2004
Brain Research Bull
1997; 44; 6; 689-694
Prevention by morphine of N-Methyl-D-Aspartic Acid induced penile erection and yawning: involvement of Nitric Oxide  
MR Melis, S Succu, A Argiolas
Bernard B. Brodie Department of Neuroscience, University of Cagliari, Italy

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Introduction : Penile erection and yawning are two different behavioral patterns that often occur concoinitantly in different experimental and physiological conditions. Among substances, that induce both penile erection and yawning, the best known are dopamine receptor agonists (i.e., apomorphine), oxytocin, adrenocorticotropin, and related peptides, 5-HT receptor agonists that act mainly ou the 5-HT1c receptor subtype and N-methyl-D-aspartic acid (NMDA). lnterestinlgly, NMDA-induced penile erection and yawning are apparently mediated by the stimulation of the excitatory aminoacid receptors of the NMDA subtype because they are prevemed by dizolcipine (MK-801), a noncompetitive antagonist of these receptors, and are not induced by (-)-a-(amino)-3-hydroxy-5-methylisoxazole-4propionic acid (AMPA) or by trans-l-amino-1,3-cyclopentanedicarboxylic acid (ACPD), agonists of the AMPA and metabotropic receptor subtype, respectively. NMDA, like dopamine agonists and oxytocin, induces penile erection and yawning by acting in the paraventricular nucleus of the hypothalamus (PVN), apparently by activaling central oxytocinergic transmission, these being responses prevented by the central administration of oxytocin receptor antagonists.
 
Experimental evidence also suggest that NMDA, like dopamine agonists and oxytocin, induces penile erection and yawning by activating nitric oxide (NO) synthase in the PVN, which in turn leads to an increase of central oxytocinergic transmission. This supports the involvement of the recently discovered neurotransniitter/neuromodulator NO in the expression of penile erection and yawning induced by NMDA.
 
Accordingly, NO donors injected into the PVN induce penile erection and yawning that are indistinguishable from those induced by NMDA and that are prevented by the potent oxytocin receptor antagonist d(CH2)5Tyr(Me)-Orn8-vasotocin -given into the lateral ventricles (ICV). The involvement of NO in the induction of penile crection and yawning by NMDA has been recently substantiated by measuring the production of NO in vivo in the PVN of male rats by microdialysis. Indeed, NMDA-induced penile erection and yawning were found to be associated to an increase of the concentration of NO2 and NO3, the reaction products of newly synthetized NO with O2 that are an indirect but reliable indicator of NO production by biological tissues in vivo, in the dialysate collected from a vertical inicrodialysis probe implanted in the PVN.
Furthermore, this NMDA-induced NO2 and NO3 increase was prevented not only by MK-801 as expected, but also by NO synthase inhibitors given ICV or into the PVN, which prevented also NMDA-induced penile crection and yawning. This report shows that these NMDA-induced behavioral responses are also prevented by the opiate morphine injected in the PVN and that this effect of the opiate on penile erection and yawning induced by NMDA is related to a concomitant prevention of the NO production induced by NMDA in tnePVN of male rats as determined by in vivo microdialysis.
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Discussion : The present results show that morphine injected in the PVN antagonizes dose dependently penile erection and yawning induced by NMDA injected ni the paraventricular nucleus of the hypothalamus. Most important, this inhibitory effect of morphine on NMDA-induced penile crection and yawning was related to a concomitant prevention of the NMDA-induced increase of the NO2 and NO3 concentration in the paraventricular dialysate. This finding not only adds further support to the hypothesis that NMDA induces penile erection and yawning by stimulating NO synthase in the PVN, as found in several other brain areas, but also suggests that morphine prevents NMDA responses by preventing NMDA-induced NO synthase activation. Indeed. the concentration of NO2 and NO3- in the extracellular fluids represents a reliable indicator of the production of NO in biological ttissues, that is of the activity of NO synthase, which converts L-arginine to citrulline and NO at least when blood is absent. In this condition. the newly formed NO would be oxidized in few seconds by O2, mainly to NO2 and, to a lesser extent, to NO31. The effect of morphine on NMDA-induced increase of NO production, penile erection. and yawning seems to be mediated by the stimulation of opioid receptors, because these effects are prevented by the prior blockade of opioid receptors by naloxone. The reccptor involved in mediating morphine effect seems to be of the µ subtype, because U-69,593, an opioid agonist 500 times more selective than morphine on the opioid receptors of the k subtype is ineffective on NMDA responses.
 
The prevention by morphine of NMDA-induced paraventricular NO production, penile erection, and yawning resembles the ability of the opiate to prevent these responses when induced by apomorphine and by oxytocin injected in the PVN. Because NMDA-induced penile erection and yawning are also prevented hN the ICV administration of potent oxytocin receptor anta-onists 121 j~ which also prevent these behavioral responses when induced by apomorphine and oxytocin, the findings are in fine with the hypothesis that opioid receptors in the PVN control in an inhibitory fashion those oxytocinergic neurons projecting to extrahypothalainic brain areas whose activation by NMDA, dopamine or oxytocin leads to the expression of penile erection and yawning by activating NO synthase in the PVN. Accordingly, inhibitory µ opioid receptors are present in the cell bodies of paraventricular oxytocinergic neurons projecting to the neurohypophysis.
 
However, it must be noted that the prevention by morphine of the behavioral responses induced by NMDA was observed at doses of the opiate that did not prevent a completely NMDA-induced increase of the concentration of NO2 and NO3 in the paraventricular dialysate. A possible explanation for this discrepancy is that NMDA also activates NO synthase in neurons that are not involved in the expression of penile erection and yawing. In agreement with this possibility, NO synthase has been identified not only in oxytocinergic but also in vasopressinergic cell bodies, in the PVN. If one assumes that NMDA activation of NO synthase is secondary to an increased Ca 2+ influx through the NMDA receptor coupled Ca2+ channels, as suggested in the cerebellum and the hippocampus, a possible rnechanism by means of which morphine prevents the NMDA induced activation of NO synthase in the PVN, is that the opiate decreases Ca 2+ influx, which in turn causes a decrease in the activity of NO synthase, a calcium-calmodulin-dependent enzyme [ in the cell bodies of oxytocinergic neurons mediatig penile erection and yawning.
 
The decreased NO formation would, in turn, reduce the oxytocinergic transmission by a yet unknown mechanism, apparently unrelated to the activation of guanylate cyclase, thereby decreasing the behavioral responses. Accordingly, the inhibition of Ca2+ influx by stimulation of opioid receptors coupled to G proteins or directly to Ca2+ channels has been already reported in several tissues and cells, and the prevention by morphine of NMDA-induced penile erection and yawning resembles the prevention of these responses induced by apomorphine and oxytocin by the blockade of N-type voltage-dependent calcium channels by nanogram amounts of omega-conotoxin injected into the PVN.
However, the present results do not rule out other mechanisms by means of which morphine mlght interfere with NO synthase activation. For instance, morphine might alter NO synthase activity in oxytocinergic neurons by modulating the release of other neurotransmitters/neuromodulators in the PVN or the activity of adenylate cyclase, one of the best-knwon target of opioid peptides.
 
In contrast, a direct inhibition of NO synthase by morphine is unlikely, because (1) morphine alone does not cause any decrease in basal NO2 and NO3- levels (this study), and (2) acute or chionic morphine does not alter NO synthase activity ni brain homogenates or its expression, as measured by the amount of NO-synthase mRNA in several mouse brain areas.
In contrast with the results of this study and of other studies stimulatory effects of morphine ou NO synthase activity have been reported. This morphine effect bas been found in homogenates of mouse cerebellum but not of other brain areas in cultured human endothelial cells, and invertebrate microglia. The reason for this discrepancy is unknown. One possibility is that the receptors, activated by morphine in these tissues are linked to transduction systems different from those present in the PVN. Accordingly, in endothelial cells and invertebrate microglia morphine was known to induce a short-lasting NO release by acting on a so-called µ3 receptor, which is sensitive to morphine but not to opioid peptides and which has not been identified yet in brain.
In this regard it is noteworthy that morphine inhibits drug induced penile erection and yawning through opioid peptide-sensitive µ receptors, because stable enkephalin and P-endorphin analogs are as effective as morphine in preventing, these beliavioral response.
Interactions between opioids and excitalory aniino acids and opioids and NO have been shown to occur in other central functions. For instance, opioid antinociception, tolerance and dependence can be antagonized by excitatory amino acid receptor antagonists, suggesting that opioids and glutamic acid can act sinergistically at least in the control of the above functions. Likewise, NO seems to be involved in the expression of opioïd antinociception tolerance and dependence although with some discrepancy. In these studies either facilitatory or inhibitory effects of NO or morphine effects were find. In conclusion, the present results show that morphine interacts with glutamic acid and NO in opposite manner in the control of penile erection and yawing at the level. In particular, these findings raise the possibility that µ opioid receptors might decrease NO synthase activity through machanisms related to the closure/opening of ion channels thate infere withe Ca2+ influx. Although the mechanism of this inetraction has still to be elucidated, our results confirm and extend precious studies that µ opiod receptors exert potent inhibitory effects on penile erection and yawning, and that these effects are in part mediated by the inhibition of NO synthase in the PVN.