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mise à jour du
11 mars 2004
Neuroscience letters
1994; 179; 9-12
Nitric oxide synthase inhibitors
prevent N-methyl-D-aspartic acid
induced penile erection and ya wning in male rats
MR Melis, R Stancampiano, A Argiolas

Bernard B. Brodie Department of Neuroscience, University of Cagliari, Italy


Penile erection and yawning are two différent behavioral responses that often occur concomitantly in experimental animals. Among agents that induce both penile erection and yawning in male rats the best known are dopaminergic agonists ), oxytocin and ACTH-related peptides. Recently, N-methyl-D-aspartic acid (NMDA) also was found to induce penile erection, yawning and grooming when injected in the paraventricular nucleus of the hypothalamus (PVN). NMDA-induced penile erection and yawning are apparently mediated by the stimulation of excitatory amino acid receptors of the NMDA subtype, that in turn activates oxytocinergic transmission, being prevented by MK-801, a non-competitive antagonist of this receptor subtype and by the oxytocin receptor antagonist d(CH2),Tyr(Me)-Orn'-vasotocin. Interestingly, NMDA receptor stimulation induces a calcium influx that has been associated with the activation of nitric oxide (NO) synthase to produce NO in several tissues, including brain neurons, which in turn causes the stimulation of guanylate cyclase. This raises the possibility that NMDA induces penile erection and yawning by activating NO synthase in the PVN. In order to test this hypothesis, the effect of NO-synthase inhibitors, methylene blue, an inhibitor of guanylate cyclase and methemoglobin, a NO scavenge, on NMDA-induced penile erection and yawning was studied in male rats.
Male Sprague Dawley rats (200-220 g) were used in all the experiments. Animals were caged in groups of 4-6 at 24°C, humidity 60%, with water and standard laboratory food ad libitum. Stainless-steel guide cannulas (22 gauge) aimed unilaterally at the PVN were stereotaxically implanted under chloral hydrate anaesthesia 5 days befère the experiments (coordinates: 0.2 mm anterior to bregma, 0.4 lateral to midline and 2.0 mm ventral to dura). The animals were given 5 days to recover from surgery; each rat was used only once. The same guide cannula was used for intracerebroventricular (i.c.v.) and PVN injections. Drugs were dissolved in saline and injected i.c.v. in 10 jul in 1 min, and into the PVN in 0.3 ul in 2 min via an internal cannula (28 gauge), which extended 1.0 mm and 5.3 mm below the tip of the guide cannula for i.c.v. and PVN injections, respectively. The internai cannula was connected by polyethylene tubing to a 10µl Hamilton syringe driven by a Stoelting,microinfusion pump. After injection, the tip of the cannula was left in the injection site for 30 s in order to allow the spread of the injected solution. N-NitrO-L-arginine methylester (NAME), N-monomethyl-L-arginine (NMMA), N-monomethyl-Darginine (D-NMMA), Methylene blue and bovine methemoglobin (ail from Sigma, St. Louis, MOI USA), (5R,IOS)-(+)-5-methyl-10, 1 1-dihydro-5H-dibenzo[a,d] cyclo-hepten-5,10-imine hydrogen maleate «+)-MK801) or saline were given i.c.v. or in the PVN 15 min before NMDA. NMDA was given in the PVN. Shortly after NMDA treatment, animais were placed individually into Plexiglas cages (30 x 30 x 30 cm) and observed for 60 min, during which penile erection and yawning episodes were counted. Only those animais that were found to have the cannula tip positioned correctly in the lateral ventricles or in the PVN by histological examination (see [ 18,19] for details) were considered for the statistical evaluation of the results by means of Oneway ANOVA, followed by Duncan's multiple range test. A P < 0.05 was considered significant.

Fig. 1 shows that penile erection and yawning induced by NMDA (50 ng into the PVN) were prevented dosedepentlently by NAME (75-150,ug), NMMA (250-500 mg) and Methylene blue (200-400 ug) but not by DNMMA (250-500 /ig), the inactive isomer of NMMA, or by methemoglobin (50-100,ug) ail given i.c.v. 15 min before NMDA. As shown in Fig. 2, NAME (10-20,ug), like MK-801 (50-100 ng), prevented NMDA-induced penile erection and yawning when injected into the PVN 15 min befère NMDA. In contrast, both Methylene blue (10-20,ug) and methemoglobin (10-20,ug) injected into the PVN were ineffective.

The present results show that NMDA-induced penile erection and yawning is prevented by the NO-synthase inhibitors given i.c.v. with a potency that is parallel to their potency in inhibiting NO-synthase The most potent, NAME, prevented NMDA-induced responses also when injected in the PVN. Since these NMDA responses are prevented also by the selective antagonist of NMDA receptors MK-801 and by the nonapeptide oxytocin receptor antagonist d(CH,),-Tyr(Me)2 -Orn'vasotocin the findings suggest that NMDA induces penile erection and yawning by activating PVN NOsynthase, thereby increasing the content of NO, which would in turn activate oxytocinergic transmission. In agreement with this hypothesis, NO-synthase inhibitors prevent oxytocin-induced penile erection and yawning; the NO donor nitroglycerin induces penile erection and yawning when injected in the PVN by activating oxytocinergic transmission, the PVN is one of the brain areas that contains the highest number of NOsynthase-immunostaining neurons and NO-synthase is localized in paraventricular oxytocinergic neurons. It is likely that NMDA-induced NO-synthase activation is mediated by an increased calcium influx through NMDA receptor-coupled calcium channels, as proposed to explain the NMDA-induced increase of cyclic guanosine monophosphate (cGMP) in cerebellar granular cells in vitro and from rat cerebral cortex in vivo.

The mechanism(s) by means of which NO, formed by the stimulation of PVN NMDA receptors, would activate oxytocinergic transmission to induce penile erection and yawning is unknown. The prevention by i.c.v. Methylene blue, an inhibitor of guanylate cyclase, of the NMDA responses suggests that NMDA activates the NO-cGMP pathway.
In agreement with this hypothesis, NO would be released from those neurons in which is formed and activate guanylate cyclase in target cells as a neurotransmitter. However, a role of guanylate cyclase in these NMDA behavioral responses must be considered with caution for at least two reasons. The first is that both Methylene blue and methemoglobin, a NO scavenger, did not prevent NMDA responses when injected directly in the PVN. This raises the possibility that NMDA-induced penile erection and yawning are mediated by a mechanism not related to the stimulation of guanylate cyclase, that is by a cGMP-indepenilent mechanims. Accordingly, NO might interact with other enzymes that, like guanylate cyclase, bind metal ions such as iron, as described for instance in fibroblasts.
The second reason is that Methylene blue might inhibit directly NO-synthase. However, this is unlikely in our experiments because Methylene blue, unlike NAME, did not prevent penile erection and yawning when injected in the PVN. Whatever mechanism is activated by NO, the failure of methemoglobin, that does not cross cellular membranes [231, to prevent NMDA-induced penile erection and yawning when injected i.c.v. or in the PVN, suggests also that NO acts intracellularly in the neurons where it is formed, rather than as a neurotransmitter, in contrast to what suggested above. One possible unifying explanation for this discrepancy is that NO activates the neurons in which it is formed, thereby releasing a neurotransmitter, e.g. oxytocin (see above), which in turn activates a Methylene blue-sensitive guanylate cyclase in a brain area(s) distant from the PVN. The identification of this brain area(s) will allow to clarify the role of guanylate cyclase in NMDA-induced penile erection and yawning, for instance by studying the effect of stable analogs of cGMP injected in this area on these responses.

In conclusion NMDA induces penile erection and yawning in male rats apparently by increasing NO synthesis in the PVN. This provides further evidence that NO plays a key role in the control of penile erection not only in the corpus cavernosum, but also in the central nervous system.