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The cannabinoid receptor antagonist SR-141716A induces penile erection in male rats: Involvement of paraventricular glutamic acid and nitric oxide
Neuropharmacology 50 (2006) 219e228 www.elsevier.com/locate/neuropharm
The cannabinoid receptor antagonist SR-141716A induces penile erection in male rats: Involvement of paraventricular glutamic acid and nitric oxide Maria Rosaria Melis*, Salvatora Succu, Maria Stefania Mascia, Fabrizio Sanna, Tiziana Melis, Maria Paola Castelli, Antonio Argiolas Bernard B Brodie Department of Neuroscience and Centre of Excellence for The Neurobiology of Addictions, University of Cagliari, 09042 Monserrato, Italy Received 11 July 2005; received in revised form 22 September 2005; accepted 23 September 2005
Abstract The cannabinoid CB1 receptor antagonist SR141716A (0.5, 1 and 2 mg) induces penile erection when injected into the paraventricular nu cleus of male rats. The pro-erectile effect of SR 141716A occurs concomitantly with an increase in the concentration of NO 2 and NO3 in the paraventricular dialysate obtained by means of intracerebral microdialysis. Both penile erection and NO2 increase induced by SR 141716A were reduced by the prior injection into the PVN of the cannabinoid CB1 agonists WIN 55,212-2 (5 mg) or HU 210 (5 mg), given into the paraventricular nucleus at doses unable to induce penile erection or to modify NO 2 concentration. SR 141716A responses were also reduced by nitroL-arginine methylester (20 mg), a non-selective NO synthase inhibitor, S-methyl-L-thiocitrulline (20 mg), a selective neuronal NO synthase inhibitor, the excitatory amino acid NMDA receptor antagonist dizocilpine ((C)MK 801) (1 mg), or the GABAA receptor agonist muscimol (0.2 mg) injected into the PVN 15 min before SR 141716A. In contrast, the inducible NO synthase inhibitor L-N(6)-(1-iminoethyl)lysine (20 mg), the GABAB receptor agonist baclofen (0.2 mg), the mixed dopamine receptor antagonist cis-flupenthixol (10 mg), and the oxytocin receptor antagonist d(CH2)5Tyr(Me)-Orn8-vasotocin (1 mg), were ineffective. Despite its inability to reduce penile erection and NO 2 increase induced by SR 141716A when injected into the PVN, d(CH2)5Tyr(Me)-Orn8-vasotocin (1 mg) reduced almost completely penile erection without reducing paraventricular NO 2 increase when injected into the lateral ventricles 15 min before SR 141716A. The present results show that SR 141716 induces penile erection by a mechanism (possibly activation of excitatory amino acid neurotransmission), which causes the activation of neuronal NO synthase in paraventricular oxytocinergic neurons mediating penile erection. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Cannabinoid CB1 receptor agonists and antagonists; Excitatory amino acids; Oxytocin; Nitric oxide; Penile erection; Paraventricular nucleus; Rat
1. Introduction The paraventricular nucleus of the hypothalamus (PVN) is considered a sort of integration center between the central and the peripheral autonomous nervous system and is involved in the control of numerous functions, including male erectile function and sexual behavior (see Hull et al., 2002; Melis * Corresponding author. Bernard B. Brodie Department of Neuroscience, University of Cagliari, S.P. Sestu-Monserrato, Km 0.700, 09042 Monserrato (CA), Italy. Tel.: C39 070 6754317; fax: C39 070 6754320. E-mail address: [email protected] (M.R. Melis). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.09.009
and Argiolas, 2003; Argiolas and Melis, 2004, 2005 and references therein). Several lines of evidence show that a group of oxytocinergic neurons originating in the PVN and projecting to extra-hypothalamic brain areas, such as the hippocampus, the medulla oblongata and the spinal cord, plays an important role in the control of erectile function and copulatory behavior (see Argiolas and Melis, 1995, 2004, 2005; Giuliano and Rampin, 2000; McKenna, 2000; Andersson, 2001; Melis and Argiolas, 2003). Briefly, when activated, for instance by dopamine, excitatory amino acids, hexarelin analogues, pro-VGF peptides and oxytocin itself (Melis et al., 1996, 1997a,b, 2003, 2004b; Succu et al., 2004, 2005), these neurons facilitate
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erection and sexual activity, while reduced erectile function and sexual activity are found when these neurons are inhibited, for instance by GABA and by opioid peptides and opiates (Melis et al., 1999; Melis and Argiolas, 2002). Apparently, the activation of oxytocinergic neurons mediating erectile function is secondary to an increase in Ca2C influx in the cell bodies of these neurons, which causes in turn the activation of nitric oxide (NO) synthase, the Ca2C-calmodulin enzyme which converts L-arginine to NO (see Melis et al., 1998; Melis and Argiolas, 2003; Argiolas and Melis, 2004, 2005), and which is present in high concentrations in the PVN, including the cell bodies of oxytocinergic neurons (Torres et al., 1993; Sanchez et al., 1994; Benelli et al., 1995). Among the numerous receptor types found in the PVN are cannabinoid receptors of the CB1 subtype (Herkenham et al., 1991). Interestingly, cannabinoid CB1 receptors are found to be coupled in different neural tissues to the inhibition of voltage-dependent Ca2C channels (Mackie and Hille, 1992; Twitchell et al., 1997; Pan et al., 1998; Freund et al., 2003), which usually causes the inhibition of neurotransmitter release from synapses bearing these receptors. The systemic administration of endogenous (endocannabinoids) and exogenous cannabinoids induces motor disturbances, hypothermia, analgesia and endocrine effects (see Chaperon and Thiebot, 1999; Maccarrone and Finazzi-Agro´, 2002; Freund et al., 2003; Howlett et al., 2004; Pertwee, 2005). The latter are mediated mainly by the hypothalamus, as suggested by the increase in c-fos protein content found in different hypothalamic nuclei, including the PVN, after systemic cannabinoid treatment (McGregor et al., 1998; Patel et al., 1998). Interestingly, endogenous and exogenous cannabinoids have also been reported to exert inhibitory effects on penile erection and male sexual behavior (see Shrenker and Bartke, 1985; Ferrari et al., 2000; Da Silva et al., 2003; Melis et al., 2004a). In line with this hypothesis, SR 141716A, a potent CB1 receptor antagonist (Rinaldi-Carmona et al., 1994), given intraperitoneally was recently found capable of potentiating penile erection induced by apomorphine in male rats (Da Silva et al., 2003), and of inducing penile erection when injected into the PVN of male rats (Melis et al., 2004a). The latter effect was reduced by WIN 55,212-2, and CP 55,940, two potent CB1 receptor agonists, given into the PVN (Melis et al., 2004a). The proerectile effect of SR141716A was also reduced by the NO synthase inhibitor nitro-L-arginine methylester (L-NAME), the excitatory amino acid N-methyl-D-aspartic acid (NMDA) receptor antagonist dizocilpine ((C)MK 801), injected into the PVN before SR 141716A. In contrast, the mixed dopamine receptor antagonist cis-flupenthixol and the oxytocin receptor antagonist d(CH2)5Tyr(Me)-Orn8-vasotocin were ineffective. Despite its inability to reduce penile erection induced by SR 141716A when injected into the PVN, d(CH2)5Tyr(Me)Orn8-vasotocin was found capable of reducing almost completely penile erection when injected into the lateral ventricles (i.c.v.) before SR 141716A (Melis et al., 2004a). All together these results suggest that SR 141716A induces penile erection when injected into the PVN by activating NO synthase located inside the cell bodies of oxytocinergic neurons mediating
penile erection, which originate in the PVN and project to extra-hypothalamic brain areas, as shown for other compounds that induce penile erection by acting in the PVN (see above). In order to provide further support to the hypotheses discussed above, the effect of pro-erectile doses of SR 141716A injected into the PVN on NO production in the PVN was studied. NO production was measured by determin ing the concentration of NO 2 and NO3 in the dialysate obtained from the PVN by means of intracerebral microdialysis. NO 2 and NO3 are the metabolites of the reaction of newly synthesized NO with oxygen present in extracellular fluids and represent a reliable, although indirect tool for measuring NO synthase activity in biological tissues when blood is absent (Ignarro, 1990). The effect of several compounds that interfere with NO synthase activity, oxytocin neurons and erectile function at the PVN level, on SR 141716A-induced penile erection and NO production is also reported. 2. Materials and methods 2.1. Animals Male SpragueeDawley rats (220e250 mg; Charles River, Como, Italy) were used in all experiments. Animals were housed individually at a temperature of 24 C and with 60% humidity under a reversed 12 h light/dark cycle (lights on from 21:00 to 09:00 h). The experiments were performed between 09:00 and 13:00 h. All experiments were carried out in accordance with the guidelines of the European Communities Directive of 24 November 1986 (86/609/EEC) and the Italian Legislation (D.P.R. 116/92).
2.2. Drugs and peptides SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-4-methyl-1Hpyrazole-3-carboxyamide] was kindly provided by Sanofi-Synthelabo (Montpellier, F), WIN 55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one], HU 210 ((6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol), and (C)MK-801 were purchased from Research Biochemical International (Natick, MA, USA). L-NAME, S-methyl-L-thiocitrulline acetate (SMTC), N(6)-(1-iminoethyl)lysine-HCl (l-NIL), muscimol, baclofen and cis-flupenthixol-HCl were purchased from Sigma (S. Louis, MO, USA). d(CH2)5Tyr(Me)-Orn8-vasotocin was purchased from Bachem AG (CH).
2.3. Microinjections and microdialysis in the paraventricular nucleus Microinjections and microdialysis were performed in the PVN of the same male rat by using microdialysis probes (approximately 1 mm of free surface for dialysis), glued with an epoxy resin to a microinjection cannula made with fused capillary silica tubing ending adjacently to the U-shaped dialysis membrane, prepared as previously described (Melis et al., 1996, 1997a,b, 1998). The probes were implanted stereotaxically (Stoelting, Co., Wood Dale, IL, USA) into the PVN under chloral hydrate anesthesia 2 days before the experiments (co-ordinates: 0.2 mm anterior to bregma, 0.4 mm lateral to midline and 7.2 mm ventral to dura) (Pellegrino and Cushman, 1971). The animals were given 2 days to recover from surgery, and each rat was used only once. For microinjections into the PVN, the microinjection cannula was connected by polyethylene tubing to a 1000-ml Hamilton syringe driven by a Stoelting microsyringe pump. The probe was perfused with Ringer’s solution, containing 147 mM NaCl, 3 mM KCl and 1.2 mM CaCl2, pH 6.5, at a constant flow rate of 2 ml/min by using a Stoelting 200 microsyringe pump. After a 2-h equilibration period, dialysate was collected every 20 min
M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228 in fractions of 40 ml in polyethylene tubes kept at 10e15 C for the determi nation of NO 2 and NO3 as described below. After the collection of three dialysate aliquots, SR 141716 was dissolved in ethanol and diluted with distilled water (final ethanol concentration 3%) and injected into the PVN in a volume of 0.3 ml. The animals were observed for 100 min, during which four additional dialysate fractions of 40 ml each were collected every 20 min and penile erection episodes were counted. In those experiments in which WIN 55,212-2 or HU 210 were used, these compounds were dissolved in 10% Tween 80 or in ethanol then diluted with distilled water (final ethanol concentration 3%), respectively, and microinjected into the PVN in a volume of 0.3 ml over a period of 2 min, 15 min before SR 141716A. In those experiments in which L-NAME, SMTC, l-NIL, (C)MK 801, cis-flupenthixol, d(CH2)5Tyr(Me)-Orn8-vasotocin, muscimol or baclofen was used, these compounds were dissolved in Ringer’s solution and microinjected into the PVN in a volume of 0.3 ml over a period of 2 min, 15 min before SR 141716A. When d(CH2)5Tyr(Me)2-Orn8-vasotocin was injected i.c.v., the peptide was dissolved in Ringer’s solution and injected in a volume of 10 ml in 2 min, 15 min before SR 141716A through a microinjection cannula similar to that used for PVN injection, but that was shortened to reach the lateral ventricle (3.0 mm below the surface of the skull) instead of the PVN. Rats injected with appropriate vehicle solutions (alcohol diluted with water to the 3% concentration; 10% Tween 80 or Ringer’s solution) depending on the injected substance(s) were used as controls.
221
2.6. Histology At the end of the experiments the animals were killed by decapitation, the brain was immediately removed and stored in 2% aqueous formaldehyde for 12e5 days. To localize the position of the probe tip, 50 mm transverse brain sections were prepared by means of a freezing microtome, stained with Neutral Red and inspected on a phase contrast microscope. The site of the probe tip was localized by following the probe tract through a series of brain sections. Only those animals found to have the probe tip positioned correctly in the PVN were considered for the statistical evaluation of the results (Fig. 1).
2.7. Statistics For the statistical evaluation of the results, the area under the curves (AUC) obtained by plotting penile erection, NO 2 and NO3 values versus time in each animal was first calculated with the classical trapezoidal rule. The AUCs were then statistically compared between groups with the ManneWhitney U-test with the significance level set at 0.01, in order to show significant differences between the groups that received a different pharmacological treatment.
3. Results 2.4. Determination of dialysate
NO 2
and
NO 3
in the paraventricular
The concentration of NO 2 and NO3 was measured in the paraventricular dialysate by a modification of the Griess reaction, as already described in detail (Melis et al., 1996, 1997a,b; 1998). Briefly, NO 2 in the dialysate was used for the diazotization of sulfanilamide and subsequent coupling to N-(1-naphthyl)-ethylene-diammine. The azo dye was then quantified by high-pressure liquid chromatography (HPLC) from its absorbance at 546 nm. The sensitivity of the assay was 0.1 mM and the response was found to be linear, with increas ing concentrations of NO 2 up to 25 mM. For the determination of NO3 in the dialysate, NO 3 was previously reduced to NO2 with copper-cadmium, as already described (Melis et al., 1996). Total NO 2 was then measured as described above and the NO 3 content was calculated by subtracting that of NO 2 found in the dialysate without copper-cadmium reduction. The sensitivity of the method was 3 mM, and the response was linear with NO 3 up to 30 mM.
2.5. Behavioral studies Rats were placed individually in Plexiglas cages (30 ! 30 ! 30 cm). After a 30 min habituation period, the microdialysis probe was connected via polyethylene tubing to a 1000 ml Hamilton microsyringe driven by a Stoelting microsyringe pump on one end and to the polyethylene collecting loop on the other hand. The cannula for PVN injections was also connected to a 10 ml Hamilton microsyringe driven by a microinfusion pump via polyethylene tubing. After a 2-h equilibration period of perfusion of the dialysis probe with Ringer’s solution, SR 141716A was given in the PVN over a 2 min period. When CB1 receptor agonists, L-NAME, SMTC, l-NIL, (C)MK 801, cis-flupenthixol, d(CH2)5Tyr(Me)2-Orn8-vasotocin, muscimol or baclofen was used, these compounds were injected into the PVN 15 min before the CB1 receptor agonist. The doses of the compounds used in this study were similar to those found able to interfere with erectile function when injected into the PVN in previous studies. In those experiments in which d(CH2)5Tyr(Me)2-Orn8-vasotocin was injected i.c.v., this compound was microinjected i.c.v. 15 min before the injection of SR 141716A into the PVN. After treatments, rats were observed for the entire duration of the experiment in order to replace filled loops with empty ones every 20 min and to count penile erection episodes. Penile erections were scored when the penis emerged from the penile sheath, which was usually accompanied by penile grooming and hip flexions.
3.1. Effect of SR 141716A injected into the PVN on penile erection and on the concentration of NO 2 and NO 3 in the dialysate obtained from the PVN of male rats: doseeresponse curves SR141716A (0.5, 1 and 2 mg) injected into the PVN induces penile erection episodes that occur concomitantly with an increase in the concentration of NO 2 and NO3 in the paraventricular dialysate (Fig. 2). The minimal effective dose was 0.5 mg, which increased penile erection episodes from 0.3 G 0.03 to 1.12 G 0.12 and the concentration of NO 2 and NO3 from 0.61 G 0.17 mM and 4.15 G 0.69 mM, to 0.95 G 0.10 mM and 6.01 G 0.79 mM, respectively. The maximal effect was found with the dose of 2 mg SR141716A, which increased penile erections episodes from 0.3 G 0.03 to 3.25 G 0.45, and the concentration of NO 2 and NO 3 from 0.61 G 0.17 mM and 4.15 G 0.69 mM, to 1.35 G 0.21 mM and 10.10 G 0.13 mM, respectively. Penile erections started within 10 min after treatment and lasted for at least 60 min. The increase in NO 2 and NO3 was already observed 20 min after treatment in the first 20-ml aliquot of collected dialysate and disappeared 80 min later. Accordingly, the AUCs of rats treated with the different doses of SR 141716A for penile erection, NO 2 and NO3 concentration were significantly higher than that of Ringer’s solution-treated rats (P ! 0.001) (Fig. 2). 3.2. Effect of WIN 55,212-2 and HU 210 injected into the PVN on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A WIN 55,212-2 (5 mg) and HU 210 (5 mg) when injected into the PVN 15 min before SR 141716A (1 mg) reduced both penile erection and the NO 2 increase induced by the CB1 antagonist given into the PVN (Fig. 3). Accordingly, the AUCs
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Fig. 1. Schematic representation of a coronal section of the rat brain showing the track of the microdialysis probe from the cortex to the PVN of a male rat treated with SR 141716A, which showed penile erection episodes and in which an increased NO production was measured. The portion of the Neutral Red-stained section showing the tip of the microdialysis probe in the PVN is magnified in the insert. Abbreviations: PVN, paraventricular nucleus of the hypothalamus; V, third ventricle.
of WIN 55,212-2 C SR 141716A-, and HU 210 C SR 141716A-treated rats for NO 2 and penile erection were significantly lower than that of SR 141716A-treated rats (P ! 0.001) (Fig. 3). At the dose used, WIN 55,212-2 and HU 210 failed to modify basal penile erection values and NO 2 levels in the paraventricular dialysate (results not shown).
3.3. Effect of L-NAME, SMTC and l-NIL injected into the PVN on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A The non-selective NO synthase inhibitor L-NAME (20 mg) and the selective neuronal NO synthase inhibitor SMTC (20 mg) (Furfine et al., 1994; Narayanan and Griffith, 1994) given into the PVN 15 min before SR 141716A (1 mg) reduced both penile erection and the concomitant increase of NO 2 in the paraventricular dialysate induced by the CB1 antagonist (Fig. 4). In contrast, the inducible NO synthase inhibitor lNIL (20 mg) (Moore et al., 1994), injected into the PVN 15 min before SR 141716A was ineffective on the increase of penile erection and of NO 2 concentration induced by the CB1 antagonist (Fig. 4). Accordingly, the AUCs of LNAME C SR 141716A- and SMTC C SR 141716A-treated rats for NO 2 and penile erection were significantly lower than that of SR 141716A-treated rats (P ! 0.001), while there was no difference between the AUCs of l-NIL C SR 141716A- and SR141716A-treated rats for NO 2 and penile erection (P O 0.1) (Fig. 4).
3.4. Effect of (C)MK 801 and cis-flupenthixol injected into the PVN on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A The excitatory amino acid NMDA receptor antagonist (C)MK 801 (1 mg) given into the PVN 15 min before SR 141716A (1 mg) reduced both penile erection and the concomitant increase of NO 2 in the paraventricular dialysate induced by the CB1 antagonist (Fig. 5). In contrast, the dopamine receptor antagonist cis-flupenthixol (10 mg), injected into the PVN 15 min before SR 141716A was ineffective on the increase of penile erection and of NO 2 concentration induced by the CB1 antagonist (Fig. 4). Accordingly, the AUCs of (C)MK 801 C SR 141716A-treated rats for NO 2 and penile erection were significantly lower than that of SR 141716Atreated rats (P ! 0.001), while there was no difference between the AUCs of cis-flupenthixol C SR 141716A- and SR141716A-treated rats for NO and penile erection 2 (P O 0.1) (Fig. 5). 3.5. Effect of d(CH2)5Tyr(Me)2-Orn8-vasotocin given into the PVN or i.c.v. on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A The oxytocin receptor antagonist d(CH2)5Tyr(Me)-Orn8vasotocin (1 mg) given into the PVN 15 min before SR 141716A (1 mg) failed to reduce both penile erection and the NO 2 increase induced by the CB1 antagonist (Fig. 6). Accordingly, there was no difference between the AUCs of d(CH2)5Tyr(Me)-Orn8-vasotocin C SR 141716A-and SR
M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228 Veh, AUC=84.4
Veh, AUC=88.20
1.4
SR 0.5, AUC=104
NO2-(µM)
1.5
*
SR 1.0, AUC=110.6 SR 2.0, AUC=120
+
+*
1.0
NO2-(µM)
2.0
223
*
#
+
Veh+SR, AUC=139.0 WIN+SR, AUC=95.2
*
*
HU210+SR, AUC=102.6
1.0 #
+
0.6
#+
* #+
0.5 0.2
0.0
1.5 AUC=580 AUC=680
*
NO3-(µM)
AUC=810
10
Penile erections/rat
15
AUC=854
* +
5
+
#
*
+
#
AUC=44.4 AUC=82.8
*
AUC=44.0
1.0
AUC=45.0
*
0.5
#+
* +
#+
40
60
#
0.0 0 -40
Penile erections/rat
2.0
*
AUC=83.0
*
1.0
+ +
#
*
40
60
+
0.5
0.0
-20
20
80 min
Fig. 3. Effect of WIN 52,522-2, CP 55,490 and HU 210 injected into the PVN on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. WIN 52,522-2 (5 mg) dissolved in 10% Tween 80 and HU 210 (5 mg) dissolved in ethanol and diluted with distilled water (final ethanol concentration 3%), were injected into the PVN in a volume of 0.3 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicles alone (Veh). The other experimental conditions were identical to those reported in the legend of Fig. 2. Values are the means G SEM of 6 rats. *(Veh C SR) P ! 0.001 with respect to vehicle-treated rats; #(Win C SR), C(HU C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
AUC=87.6
-40
0
SR 141716A
AUC=44.6 AUC=58.6
1.5
-20
0
20
80 min
SR 141716A Fig. 2. Effect of SR141716A injected into the PVN on penile erection and NO 2 and NO3 concentration in the paraventricular dialysate obtained from the PVN by means of intracerebral microdialysis. SR 141716A (0.5, 1 and 2 mg), dissolved in ethanol and diluted with distilled water (final ethanol concentration 3%), was injected into the PVN in a volume of 0.3 ml in 2 min. Controls rats were injected with vehicle alone. Vehicle (Veh) or SR 141716A was injected into the PVN at time 0, after 120 min equilibration time. After treatment, rats were put individually into Plexiglas cages and observed for 80 min in order to count penile erection episodes and to replace filled loops with emp ty ones every 20 min for the determination of NO 2 and NO3 concentration in the dialysate. The areas under the curve (AUC), obtained by plotting penile erections, NO 2 and NO3 concentrations versus time for each group shown in the figure, were calculated with the trapezoidal rule as described in Section 2. Values are the means G SEM of 6 rats. #(SR 0.5), C(SR 1), *(SR 2) P ! 0.001 with respect to vehicle-treated rats (ManneWhitney U-test).
141716A-treated rats (P O 0.1) (Fig. 5). Despite its inability to reduce SR 141716A-induced penile erection and the NO 2 increase when injected into the PVN, d(CH2)5Tyr(Me)-Orn8vasotocin (1 mg) reduced SR 141716A-induced penile erection
but not the NO 2 increase when injected i.c.v. (Fig. 6). Accordingly, the AUC of i.c.v. d(CH2)5Tyr(Me)-Orn8-vasotocin C SR 141716A-treated rats for penile erection was significantly lower than that of SR 141716A-treated rats (P ! 0.001), while the AUCs for NO 2 were not (P O 0.1) (Fig. 6). 3.6. Effect of muscimol and baclofen given into the PVN on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A The GABAA receptor agonist muscimol (0.2 mg) given into the PVN 15 min before SR 141716A (1 mg) reduced both penile erection and the NO 2 increase induced by the CB1 antagonist (Fig. 7). Accordingly, the AUCs of muscimol C SR 141716A-treated rats were much lower than those of SR 141716A-treated rats (P ! 0.001) (Fig. 7). In contrast, the GABAB receptor agonist baclofen (0.2 mg) injected into the PVN 15 min before the CB1 antagonist was unable to reduce SR 141716A-induced penile erection and the NO 2 increase (Fig. 7). Accordingly, there was no difference between the
M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228
224
SMTC+SR, AUC=81.0 l-NIL+SR, AUC=103.0
0.8
0.4
Penile erections/rat
1.6
*
Veh+SR, AUC=109.6 L-NAME+SR, AUC=84.0
+
* +
#
#
§
§
1.2
1.2
*
+
#
* *
0.8
+
# 0.4
§
+
#
*+
# §
§
cis-Flu+SR, AUC=106.0
+ 0.8
1.6
AUC=88.2 AUC=46.6 AUC=83.0
*
MK-801+SR, AUC=81.0
* #
+
+
#
*
#
0.4
§
AUC=44.4 AUC=50.0
Veh, AUC=85.2 Veh+SR, AUC=106.6
NO2-(µM)
NO2-(µM)
1.2
1.6
Veh, AUC=87.2
Penile erections/rat
1.6
AUC=44.4 AUC=85.2
*
AUC=45.6
1.2
AUC=87.8
+ 0.8 #
*
+ #
+ #
*
0.4 0.0
0.0
-40
-20
0
20
40
60
80 min
SR 141716A
-40
-20
0
20
40
60
80 min
SR 141716A
Fig. 4. Effect of L-NAME, SMTC and l-NIL injected into the PVN on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. L-NAME (20 mg), SMTC (20 mg) and l-NIL (20 mg), dissolved in Ringer’s solution were injected into the PVN in a volume of 0.3 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicle alone (Veh). The other experimental conditions were identical to those reported in the legend of Figs. 2 and 3. Values are the means G SEM of 5 rats. *(Veh C SR), C(l-NILCSR) P ! 0.001 with respect to vehicle-treated rats; x(L-NAME C SR), #(SMTC C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
Fig. 5. Effect of (C)MK 801 and cis-flupenthixol injected into the PVN on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. (C)MK 801 (1 mg) and cis-flupenthixol (10 mg), dissolved in Ringer’s solution were injected into the PVN in a volume of 0.3 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicle alone (Veh). The other experimental conditions were identical to those reported in the legend of Figs. 2 and 3. Values are the means G SEM of 5 rats. *(Veh C SR), C(cis-Flu C SR) P ! 0.001 with respect to vehicle-treated rats; #(MK-801 C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
AUCs of baclofen C SR 141716A- and SR 141716A-treated rats for penile erection and NO 2 (P O 0.1) (Fig. 7).
that do not modify this sexual response and basal NO 2 and NO concentration (Melis et al., 2004a and this study), it is 3 likely that these SR 141716A responses are mediated by the blockade of paraventricular cannabinoid CB1 receptors. The blockade of cannabinoid CB1 receptors by SR 141716A causes in turn the activation of NO synthase localized in the cell bodies of oxytocinergic neurons originating in the PVN and projecting to extra-hypothalamic brain areas and to the spinal cord. This causes the activation of oxytocinergic neurons, which release oxytocin in brain areas distant from the PVN, facilitating penile erection (see Melis and Argiolas, 2003; Argiolas and Melis, 1995, 2004, 2005 and references therein). Accordingly, SR 141716A-induced penile erection is prevented by d(CH2)5Tyr(Me)2-Orn8-vasotocin, a potent oxytocin receptor antagonist, when given into the lateral ventricles, but not into the PVN, at doses capable of preventing penile erection induced by other agents, which induce this sexual response by acting in the PVN (see above). The localization of the cannabinoid CB1 receptors and the mechanism by means of which their blockade increases penile erection by increasing paraventricular NO synthase activity
4. Discussion The present study confirms that SR 141716A, a potent cannabinoid CB1 receptor antagonist (Rinaldi-Carmona et al., 1994), injected into the PVN of male rats increases the number of spontaneous penile erection episodes (Melis et al., 2004a), and shows that this effect occurs concomitantly with an increase in the activity mainly of neuronal NO synthase, mea sured by the increase in the concentration of NO 2 and NO3 found in the paraventricular dialysate obtained by intracerebral microdialysis, as already shown for other compounds that induce penile erection when injected into the PVN (e.g., dopamine agonists, NMDA, oxytocin, hexarelin analogues, pro-VGF peptides) (see Section 1 and references therein). As both the pro-erectile effect and the increase in NO 2 and NO concentration are reduced by WIN 55,212-2 and HU 3 210, two potent cannabinoid CB1 receptor agonists, at doses
M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228 1.6
Veh+veh, AUC=86 Veh+SR, AUC=111
NO2-(µM)
1.2
* +§
*+
§
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1.2
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* +
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#
#
0.4
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AUC=44.57 AUC=86.30
*
AUC=84.15 AUC=51.08
0.8
+
*
+ # #
*
Penile erections/rat
Penile erections/rat
1.6
*
Mus+SR, AUC=90
+
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Veh, AUC=86 Veh+SR, AUC=110
§
PVNOXYant+SR, AUC=109
NO2-(µM)
1.6
225
+ #
0.4
0.0
AUC=45
*
AUC=88
1.2
AUC=48 AUC=83
*
0.8
+
+
#
#
20
40
0.4
*
+
#
0.0
-40
-20
0
20
40
60
80 min
SR 141716A
-40
-20
0
60
80 min
SR 141716A
8
Fig. 6. Effect of d(CH2)5Tyr(Me)-Orn -vasotocin injected into the PVN or i.c.v. on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. d(CH2)5Tyr(Me)-Orn8-vasotocin (1 mg), dissolved in Ringer’s solution was injected into the PVN in a volume of 0.3 ml in 2 min or i.c.v. in a volume of 10 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicle alone (Veh). The other experimental conditions were identical to those reported in the legend of Figs. 2 and 3. Values are the means G SEM of 5 rats. *(Veh C SR), C(PVN Oxy ant C SR), P ! 0.001 with respect to vehicle-treated rats; #(ICV Oxy ant C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
and oxytocinergic neurotransmission are unknown at present. The available data support the hypothesis that cannabinoid CB1 receptors are located in excitatory amino acid-containing (possibly glutamic acid) synapses impinging on the cell bodies of oxytocinergic neurons mediating penile erection, rather than to be located directly in the cell bodies of oxytocinergic neurons themselves. It is likely that these cannabinoid CB1 receptors exert a tonic inhibition on the release of this excitatory amino acid, which activates oxytocinergic neurons mediating penile erection through the activation of NO synthase localized in these neurons (see Melis and Argiolas, 2003; Argiolas and Melis, 1995, 2004, 2005). Accordingly, (1) cannabinoid CB1 receptor agonists (WIN 55,212-2, CP 55,940 and HU 210) when injected into the PVN at doses that do not influence spontaneous penile erection (Melis et al., 2004a), and do not modify basal NO production (this study), reduce SR 141716-induced penile erection and activation of NO synthase; (2) SR 141716A-induced penile erection and the concomitant increase in NO production are reduced by the injection into the PVN of (C)MK-801, which blocks
Fig. 7. Effect of muscimol and baclofen injected into the PVN or into the lateral ventricles on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. Muscimol (0.2 mg) or baclofen (0.2 mg) dissolved in Ringer’s solution was injected into the PVN in a volume of 0.3 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicle alone (Veh). The other experimental conditions were identical to those reported in the legend of Figs. 2 and 3. Values are the means G SEM of 5 rats. *(Veh C SR), C(Bac C SR) P ! 0.001 with respect to vehicle-treated rats; #(Mus C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
NMDA receptors, and of L-NAME and by SMTC, which inhibit the first all NO synthase isoforms and the second neuronal NO synthase selectively (Furfine et al., 1994; Narayanan and Griffith, 1994), but not by l-NIL, an inhibitor of inducible NO synthase (Moore et al., 1994) (this study and Melis et al., 2004a); and (3) NMDA injected into the PVN induces penile erection and increases NO production, which are also antagonized by (C)MK-801 and by L-NAME (Melis et al., 1997b; Melis and Argiolas, 2003; Argiolas and Melis, 2004; 2005). In line with this hypothesis, glutamic acid concentration increases in the dialysate obtained from the PVN during sexual activity (Melis et al., 2004b) and the stimulation of cannabinoid CB1 receptors has been shown to inhibit the release of glutamic acid in different brain tissues (Robbe et al., 2001; Pistis et al., 2002; Brown et al., 2003; Breivogel et al., 2004; Freund et al., 2003; Howlett et al., 2004 and references therein). In line with the interpretation given above, the inability of WIN 55,212-2, CP 55,940 and HU 210 to influence spontaneous and drug-induced penile erection when injected into the PVN (this paper and Melis et al., 2004a), suggests that the tonic activation of cannabinoid receptors controlling
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excitatory synapses in the PVN is already maximal. Indeed, in this case any further stimulation of cannabinoid CB1 receptors would be unable to exert any additional inhibition on excitatory neurons impinging on the cell bodies of oxytocinergic neurons mediating penile erection. Alternatively, cannabinoid CB1 receptors might be located in inhibitory, possibly GABAergic synapses, which act on glutamic acid-containing neurons impinging on the cell bodies of oxytocinergic neurons mediating penile erection. The stimulation of these cannabinoid CB1 receptors might enhance the release of GABA, while their blockade might decrease its release, inducing in turn an increase in the release of glutamic acid that activates oxytocinergic neurons. Accordingly, (1) the stimulation of GABAA by muscimol, but not of GABAB receptors by baclofen, injected into the PVN reduces penile erection and the increase in NO production induced by SR 141716A as well as by NMDA (Melis and Argiolas, 2002); and (2) cannabinoid CB1 receptor agonists have been also found capable of enhancing GABAergic neurotransmission in several circumstances (De Miguel et al., 1998). However, this second interpretation is complicated by at least two lines of experimental evidence. First, muscimol can inhibit NO synthase and oxytocinergic neurons by acting directly on GABAA receptors localized in the cell bodies of oxytocinergic neurons, as GABAergic synapses impinging on oxytocinergic cell bodies known to inhibit oxytocinergic activity in several circumstances have been well characterized (Bisset et al., 1990; Roland and Sawchenko, 1993; Voisin et al., 1995; Boubada et al., 1996; Jourdain et al., 1999); and second, muscimol can directly inhibit excitatory amino acidergic neurons impinging on oxytocinergic neurons mediating penile erection (see Melis and Argiolas, 2002). Nevertheless, the two interpretations may not be mutually exclusive and both mechanisms may contribute to the pro-erectile effect of SR 141716A and its ability to increase NO production when injected into the PVN. The above explanation is based mainly on the assumption that SR 141716A induces penile erection by acting into the PVN by increasing excitatory amino acid neurotransmission acting directly on excitatory amino acidergic synapses or indirectly by decreasing inhibitory GABAergic input to these excitatory synapses, and that the increase in excitatory amino acid neurotransmission in turn activates neuronal NO synthase in the cell bodies of oxytocinergic neurons leading to an increased NO production. NO in turn activates oxytocinergic neurons to release oxytocin in sites distant from the PVN leading to penile erection. However, other explanations cannot be ruled out. For instance SR 141716A might activate neuronal NO synthase and oxytocinergic neurons mediating erectile response by interacting directly with cannabinoid receptors located in their cell bodies, or by increasing the activity of other neurotransmitters and/or neuropeptides present in the PVN and already known to induce penile erection by activating NO synthase and oxytocinergic neurons, such as dopamine and oxytocin itself, or by inhibiting the activity of endogenous opioids, which reduce, like GABA, the activity of oxytocinergic neurons and penile erection by acting on opioid receptors of the subtype m located in the cell bodies of oxytocinergic
neurons (see Melis et al., 1999 and references therein). Although further experiments are necessary to verify the above possibilities in detail, from the available data it is unlikely that SR 141716A induces penile erection when injected into the PVN by increasing dopamine or oxytocin release in the PVN, since both cis-flupenthixol, a potent antagonist of dopamine receptors, and d(CH2)5Tyr(Me)2-Orn8-vasotocin, a potent antagonist of oxytocin receptors, are unable to reduce SR 141716A-induced penile erection and the increase in NO production when injected into the PVN. Indeed, at the doses used in this study, cis-flupenthixol and d(CH2)5Tyr(Me)2-Orn8-vasotocin are able to prevent penile erection induced by the dopamine agonist apomorphine and by oxytocin given in the PVN, respectively (Melis and Argiolas, 2003; Argiolas and Melis, 2004, 2005 and references therein). In conclusion, the present study show that SR 141716A, a cannabinoid CB1 receptor antagonist, induces penile erection when injected into the PVN, apparently by increasing neuronal NO synthase activity and oxytocinergic neurotransmission, as shown for other compounds that induce penile erection when injected into the PVN. This effect is apparently mediated by an increase in excitatory amino acid, possibly glutamic acid neurotransmission, which may be due to the inhibition of cannabinoid CB1 receptors located in excitatory amino acidergic synapses and controlling glutamic acid release, or of cannabinoid CB1 receptors located on GABAergic synapses impinging on the above excitatory synapses to inhibit glutamic acid release. Released glutamic acid stimulates in turn its receptors located in the cell bodies of oxytocinergic neurons increasing intracellular Ca2C concentration inside these cell bodies. This stimulates neuronal NO synthase to produce NO, which activates oxytocinergic neurons to release oxytocin in sites distant from the PVN, leading to penile erection. The ability of SR 141716A to induce penile erection when injected into the PVN is in line with the reported inhibitory effects of cannabinoids given systemically on sexual behavior (Shrenker and Bartke, 1985; Ferrari et al., 2000 and references therein) and suggests that cannabinoid CB1 receptors in the PVN can influence erectile function and sexual behavior. However, further studies with cannabinoid CB1 agonists and antagonists not only on spontaneous penile erections, but also on reflex erections and non-contact erections (which occur in male rats exposed to inaccessible receptive female rats), are necessary to clarify in detail the role of paraventricular cannabinoid CB1 receptors in erectile function. Acknowledgements This work was partially supported by a MIUR grant to AA and MRM, and a grant from Fondazione Banco di Sardegna to MPC. References Andersson, K.E., 2001. Pharmacology of penile erection. Pharmacological Reviews 53, 417e450.
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The cannabinoid receptor antagonist SR-141716A induces penile erection in male rats: Involvement of paraventricular glutamic acid and nitric oxide Maria Rosaria Melis*, Salvatora Succu, Maria Stefania Mascia, Fabrizio Sanna, Tiziana Melis, Maria Paola Castelli, Antonio Argiolas Bernard B Brodie Department of Neuroscience and Centre of Excellence for The Neurobiology of Addictions, University of Cagliari, 09042 Monserrato, Italy Received 11 July 2005; received in revised form 22 September 2005; accepted 23 September 2005
Abstract The cannabinoid CB1 receptor antagonist SR141716A (0.5, 1 and 2 mg) induces penile erection when injected into the paraventricular nu cleus of male rats. The pro-erectile effect of SR 141716A occurs concomitantly with an increase in the concentration of NO 2 and NO3 in the paraventricular dialysate obtained by means of intracerebral microdialysis. Both penile erection and NO2 increase induced by SR 141716A were reduced by the prior injection into the PVN of the cannabinoid CB1 agonists WIN 55,212-2 (5 mg) or HU 210 (5 mg), given into the paraventricular nucleus at doses unable to induce penile erection or to modify NO 2 concentration. SR 141716A responses were also reduced by nitroL-arginine methylester (20 mg), a non-selective NO synthase inhibitor, S-methyl-L-thiocitrulline (20 mg), a selective neuronal NO synthase inhibitor, the excitatory amino acid NMDA receptor antagonist dizocilpine ((C)MK 801) (1 mg), or the GABAA receptor agonist muscimol (0.2 mg) injected into the PVN 15 min before SR 141716A. In contrast, the inducible NO synthase inhibitor L-N(6)-(1-iminoethyl)lysine (20 mg), the GABAB receptor agonist baclofen (0.2 mg), the mixed dopamine receptor antagonist cis-flupenthixol (10 mg), and the oxytocin receptor antagonist d(CH2)5Tyr(Me)-Orn8-vasotocin (1 mg), were ineffective. Despite its inability to reduce penile erection and NO 2 increase induced by SR 141716A when injected into the PVN, d(CH2)5Tyr(Me)-Orn8-vasotocin (1 mg) reduced almost completely penile erection without reducing paraventricular NO 2 increase when injected into the lateral ventricles 15 min before SR 141716A. The present results show that SR 141716 induces penile erection by a mechanism (possibly activation of excitatory amino acid neurotransmission), which causes the activation of neuronal NO synthase in paraventricular oxytocinergic neurons mediating penile erection. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Cannabinoid CB1 receptor agonists and antagonists; Excitatory amino acids; Oxytocin; Nitric oxide; Penile erection; Paraventricular nucleus; Rat
1. Introduction The paraventricular nucleus of the hypothalamus (PVN) is considered a sort of integration center between the central and the peripheral autonomous nervous system and is involved in the control of numerous functions, including male erectile function and sexual behavior (see Hull et al., 2002; Melis * Corresponding author. Bernard B. Brodie Department of Neuroscience, University of Cagliari, S.P. Sestu-Monserrato, Km 0.700, 09042 Monserrato (CA), Italy. Tel.: C39 070 6754317; fax: C39 070 6754320. E-mail address: [email protected] (M.R. Melis). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.09.009
and Argiolas, 2003; Argiolas and Melis, 2004, 2005 and references therein). Several lines of evidence show that a group of oxytocinergic neurons originating in the PVN and projecting to extra-hypothalamic brain areas, such as the hippocampus, the medulla oblongata and the spinal cord, plays an important role in the control of erectile function and copulatory behavior (see Argiolas and Melis, 1995, 2004, 2005; Giuliano and Rampin, 2000; McKenna, 2000; Andersson, 2001; Melis and Argiolas, 2003). Briefly, when activated, for instance by dopamine, excitatory amino acids, hexarelin analogues, pro-VGF peptides and oxytocin itself (Melis et al., 1996, 1997a,b, 2003, 2004b; Succu et al., 2004, 2005), these neurons facilitate
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erection and sexual activity, while reduced erectile function and sexual activity are found when these neurons are inhibited, for instance by GABA and by opioid peptides and opiates (Melis et al., 1999; Melis and Argiolas, 2002). Apparently, the activation of oxytocinergic neurons mediating erectile function is secondary to an increase in Ca2C influx in the cell bodies of these neurons, which causes in turn the activation of nitric oxide (NO) synthase, the Ca2C-calmodulin enzyme which converts L-arginine to NO (see Melis et al., 1998; Melis and Argiolas, 2003; Argiolas and Melis, 2004, 2005), and which is present in high concentrations in the PVN, including the cell bodies of oxytocinergic neurons (Torres et al., 1993; Sanchez et al., 1994; Benelli et al., 1995). Among the numerous receptor types found in the PVN are cannabinoid receptors of the CB1 subtype (Herkenham et al., 1991). Interestingly, cannabinoid CB1 receptors are found to be coupled in different neural tissues to the inhibition of voltage-dependent Ca2C channels (Mackie and Hille, 1992; Twitchell et al., 1997; Pan et al., 1998; Freund et al., 2003), which usually causes the inhibition of neurotransmitter release from synapses bearing these receptors. The systemic administration of endogenous (endocannabinoids) and exogenous cannabinoids induces motor disturbances, hypothermia, analgesia and endocrine effects (see Chaperon and Thiebot, 1999; Maccarrone and Finazzi-Agro´, 2002; Freund et al., 2003; Howlett et al., 2004; Pertwee, 2005). The latter are mediated mainly by the hypothalamus, as suggested by the increase in c-fos protein content found in different hypothalamic nuclei, including the PVN, after systemic cannabinoid treatment (McGregor et al., 1998; Patel et al., 1998). Interestingly, endogenous and exogenous cannabinoids have also been reported to exert inhibitory effects on penile erection and male sexual behavior (see Shrenker and Bartke, 1985; Ferrari et al., 2000; Da Silva et al., 2003; Melis et al., 2004a). In line with this hypothesis, SR 141716A, a potent CB1 receptor antagonist (Rinaldi-Carmona et al., 1994), given intraperitoneally was recently found capable of potentiating penile erection induced by apomorphine in male rats (Da Silva et al., 2003), and of inducing penile erection when injected into the PVN of male rats (Melis et al., 2004a). The latter effect was reduced by WIN 55,212-2, and CP 55,940, two potent CB1 receptor agonists, given into the PVN (Melis et al., 2004a). The proerectile effect of SR141716A was also reduced by the NO synthase inhibitor nitro-L-arginine methylester (L-NAME), the excitatory amino acid N-methyl-D-aspartic acid (NMDA) receptor antagonist dizocilpine ((C)MK 801), injected into the PVN before SR 141716A. In contrast, the mixed dopamine receptor antagonist cis-flupenthixol and the oxytocin receptor antagonist d(CH2)5Tyr(Me)-Orn8-vasotocin were ineffective. Despite its inability to reduce penile erection induced by SR 141716A when injected into the PVN, d(CH2)5Tyr(Me)Orn8-vasotocin was found capable of reducing almost completely penile erection when injected into the lateral ventricles (i.c.v.) before SR 141716A (Melis et al., 2004a). All together these results suggest that SR 141716A induces penile erection when injected into the PVN by activating NO synthase located inside the cell bodies of oxytocinergic neurons mediating
penile erection, which originate in the PVN and project to extra-hypothalamic brain areas, as shown for other compounds that induce penile erection by acting in the PVN (see above). In order to provide further support to the hypotheses discussed above, the effect of pro-erectile doses of SR 141716A injected into the PVN on NO production in the PVN was studied. NO production was measured by determin ing the concentration of NO 2 and NO3 in the dialysate obtained from the PVN by means of intracerebral microdialysis. NO 2 and NO3 are the metabolites of the reaction of newly synthesized NO with oxygen present in extracellular fluids and represent a reliable, although indirect tool for measuring NO synthase activity in biological tissues when blood is absent (Ignarro, 1990). The effect of several compounds that interfere with NO synthase activity, oxytocin neurons and erectile function at the PVN level, on SR 141716A-induced penile erection and NO production is also reported. 2. Materials and methods 2.1. Animals Male SpragueeDawley rats (220e250 mg; Charles River, Como, Italy) were used in all experiments. Animals were housed individually at a temperature of 24 C and with 60% humidity under a reversed 12 h light/dark cycle (lights on from 21:00 to 09:00 h). The experiments were performed between 09:00 and 13:00 h. All experiments were carried out in accordance with the guidelines of the European Communities Directive of 24 November 1986 (86/609/EEC) and the Italian Legislation (D.P.R. 116/92).
2.2. Drugs and peptides SR 141716A [N-(piperidin-1-yl)-5-(4-chlorophenyl)-4-methyl-1Hpyrazole-3-carboxyamide] was kindly provided by Sanofi-Synthelabo (Montpellier, F), WIN 55,212-2 [4,5-dihydro-2-methyl-4(4-morpholinylmethyl)-1-(1-naphthalenyl-carbonyl)-6H-pyrrolo[3,2,1-i,j]quinolin-6-one], HU 210 ((6aR)-trans-3-(1,1-dimethylheptyl)-6a,7,10,10a-tetrahydro-1hydroxy-6,6-dimethyl-6H-dibenzo[b,d]pyran-9-methanol), and (C)MK-801 were purchased from Research Biochemical International (Natick, MA, USA). L-NAME, S-methyl-L-thiocitrulline acetate (SMTC), N(6)-(1-iminoethyl)lysine-HCl (l-NIL), muscimol, baclofen and cis-flupenthixol-HCl were purchased from Sigma (S. Louis, MO, USA). d(CH2)5Tyr(Me)-Orn8-vasotocin was purchased from Bachem AG (CH).
2.3. Microinjections and microdialysis in the paraventricular nucleus Microinjections and microdialysis were performed in the PVN of the same male rat by using microdialysis probes (approximately 1 mm of free surface for dialysis), glued with an epoxy resin to a microinjection cannula made with fused capillary silica tubing ending adjacently to the U-shaped dialysis membrane, prepared as previously described (Melis et al., 1996, 1997a,b, 1998). The probes were implanted stereotaxically (Stoelting, Co., Wood Dale, IL, USA) into the PVN under chloral hydrate anesthesia 2 days before the experiments (co-ordinates: 0.2 mm anterior to bregma, 0.4 mm lateral to midline and 7.2 mm ventral to dura) (Pellegrino and Cushman, 1971). The animals were given 2 days to recover from surgery, and each rat was used only once. For microinjections into the PVN, the microinjection cannula was connected by polyethylene tubing to a 1000-ml Hamilton syringe driven by a Stoelting microsyringe pump. The probe was perfused with Ringer’s solution, containing 147 mM NaCl, 3 mM KCl and 1.2 mM CaCl2, pH 6.5, at a constant flow rate of 2 ml/min by using a Stoelting 200 microsyringe pump. After a 2-h equilibration period, dialysate was collected every 20 min
M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228 in fractions of 40 ml in polyethylene tubes kept at 10e15 C for the determi nation of NO 2 and NO3 as described below. After the collection of three dialysate aliquots, SR 141716 was dissolved in ethanol and diluted with distilled water (final ethanol concentration 3%) and injected into the PVN in a volume of 0.3 ml. The animals were observed for 100 min, during which four additional dialysate fractions of 40 ml each were collected every 20 min and penile erection episodes were counted. In those experiments in which WIN 55,212-2 or HU 210 were used, these compounds were dissolved in 10% Tween 80 or in ethanol then diluted with distilled water (final ethanol concentration 3%), respectively, and microinjected into the PVN in a volume of 0.3 ml over a period of 2 min, 15 min before SR 141716A. In those experiments in which L-NAME, SMTC, l-NIL, (C)MK 801, cis-flupenthixol, d(CH2)5Tyr(Me)-Orn8-vasotocin, muscimol or baclofen was used, these compounds were dissolved in Ringer’s solution and microinjected into the PVN in a volume of 0.3 ml over a period of 2 min, 15 min before SR 141716A. When d(CH2)5Tyr(Me)2-Orn8-vasotocin was injected i.c.v., the peptide was dissolved in Ringer’s solution and injected in a volume of 10 ml in 2 min, 15 min before SR 141716A through a microinjection cannula similar to that used for PVN injection, but that was shortened to reach the lateral ventricle (3.0 mm below the surface of the skull) instead of the PVN. Rats injected with appropriate vehicle solutions (alcohol diluted with water to the 3% concentration; 10% Tween 80 or Ringer’s solution) depending on the injected substance(s) were used as controls.
221
2.6. Histology At the end of the experiments the animals were killed by decapitation, the brain was immediately removed and stored in 2% aqueous formaldehyde for 12e5 days. To localize the position of the probe tip, 50 mm transverse brain sections were prepared by means of a freezing microtome, stained with Neutral Red and inspected on a phase contrast microscope. The site of the probe tip was localized by following the probe tract through a series of brain sections. Only those animals found to have the probe tip positioned correctly in the PVN were considered for the statistical evaluation of the results (Fig. 1).
2.7. Statistics For the statistical evaluation of the results, the area under the curves (AUC) obtained by plotting penile erection, NO 2 and NO3 values versus time in each animal was first calculated with the classical trapezoidal rule. The AUCs were then statistically compared between groups with the ManneWhitney U-test with the significance level set at 0.01, in order to show significant differences between the groups that received a different pharmacological treatment.
3. Results 2.4. Determination of dialysate
NO 2
and
NO 3
in the paraventricular
The concentration of NO 2 and NO3 was measured in the paraventricular dialysate by a modification of the Griess reaction, as already described in detail (Melis et al., 1996, 1997a,b; 1998). Briefly, NO 2 in the dialysate was used for the diazotization of sulfanilamide and subsequent coupling to N-(1-naphthyl)-ethylene-diammine. The azo dye was then quantified by high-pressure liquid chromatography (HPLC) from its absorbance at 546 nm. The sensitivity of the assay was 0.1 mM and the response was found to be linear, with increas ing concentrations of NO 2 up to 25 mM. For the determination of NO3 in the dialysate, NO 3 was previously reduced to NO2 with copper-cadmium, as already described (Melis et al., 1996). Total NO 2 was then measured as described above and the NO 3 content was calculated by subtracting that of NO 2 found in the dialysate without copper-cadmium reduction. The sensitivity of the method was 3 mM, and the response was linear with NO 3 up to 30 mM.
2.5. Behavioral studies Rats were placed individually in Plexiglas cages (30 ! 30 ! 30 cm). After a 30 min habituation period, the microdialysis probe was connected via polyethylene tubing to a 1000 ml Hamilton microsyringe driven by a Stoelting microsyringe pump on one end and to the polyethylene collecting loop on the other hand. The cannula for PVN injections was also connected to a 10 ml Hamilton microsyringe driven by a microinfusion pump via polyethylene tubing. After a 2-h equilibration period of perfusion of the dialysis probe with Ringer’s solution, SR 141716A was given in the PVN over a 2 min period. When CB1 receptor agonists, L-NAME, SMTC, l-NIL, (C)MK 801, cis-flupenthixol, d(CH2)5Tyr(Me)2-Orn8-vasotocin, muscimol or baclofen was used, these compounds were injected into the PVN 15 min before the CB1 receptor agonist. The doses of the compounds used in this study were similar to those found able to interfere with erectile function when injected into the PVN in previous studies. In those experiments in which d(CH2)5Tyr(Me)2-Orn8-vasotocin was injected i.c.v., this compound was microinjected i.c.v. 15 min before the injection of SR 141716A into the PVN. After treatments, rats were observed for the entire duration of the experiment in order to replace filled loops with empty ones every 20 min and to count penile erection episodes. Penile erections were scored when the penis emerged from the penile sheath, which was usually accompanied by penile grooming and hip flexions.
3.1. Effect of SR 141716A injected into the PVN on penile erection and on the concentration of NO 2 and NO 3 in the dialysate obtained from the PVN of male rats: doseeresponse curves SR141716A (0.5, 1 and 2 mg) injected into the PVN induces penile erection episodes that occur concomitantly with an increase in the concentration of NO 2 and NO3 in the paraventricular dialysate (Fig. 2). The minimal effective dose was 0.5 mg, which increased penile erection episodes from 0.3 G 0.03 to 1.12 G 0.12 and the concentration of NO 2 and NO3 from 0.61 G 0.17 mM and 4.15 G 0.69 mM, to 0.95 G 0.10 mM and 6.01 G 0.79 mM, respectively. The maximal effect was found with the dose of 2 mg SR141716A, which increased penile erections episodes from 0.3 G 0.03 to 3.25 G 0.45, and the concentration of NO 2 and NO 3 from 0.61 G 0.17 mM and 4.15 G 0.69 mM, to 1.35 G 0.21 mM and 10.10 G 0.13 mM, respectively. Penile erections started within 10 min after treatment and lasted for at least 60 min. The increase in NO 2 and NO3 was already observed 20 min after treatment in the first 20-ml aliquot of collected dialysate and disappeared 80 min later. Accordingly, the AUCs of rats treated with the different doses of SR 141716A for penile erection, NO 2 and NO3 concentration were significantly higher than that of Ringer’s solution-treated rats (P ! 0.001) (Fig. 2). 3.2. Effect of WIN 55,212-2 and HU 210 injected into the PVN on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A WIN 55,212-2 (5 mg) and HU 210 (5 mg) when injected into the PVN 15 min before SR 141716A (1 mg) reduced both penile erection and the NO 2 increase induced by the CB1 antagonist given into the PVN (Fig. 3). Accordingly, the AUCs
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M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228
Fig. 1. Schematic representation of a coronal section of the rat brain showing the track of the microdialysis probe from the cortex to the PVN of a male rat treated with SR 141716A, which showed penile erection episodes and in which an increased NO production was measured. The portion of the Neutral Red-stained section showing the tip of the microdialysis probe in the PVN is magnified in the insert. Abbreviations: PVN, paraventricular nucleus of the hypothalamus; V, third ventricle.
of WIN 55,212-2 C SR 141716A-, and HU 210 C SR 141716A-treated rats for NO 2 and penile erection were significantly lower than that of SR 141716A-treated rats (P ! 0.001) (Fig. 3). At the dose used, WIN 55,212-2 and HU 210 failed to modify basal penile erection values and NO 2 levels in the paraventricular dialysate (results not shown).
3.3. Effect of L-NAME, SMTC and l-NIL injected into the PVN on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A The non-selective NO synthase inhibitor L-NAME (20 mg) and the selective neuronal NO synthase inhibitor SMTC (20 mg) (Furfine et al., 1994; Narayanan and Griffith, 1994) given into the PVN 15 min before SR 141716A (1 mg) reduced both penile erection and the concomitant increase of NO 2 in the paraventricular dialysate induced by the CB1 antagonist (Fig. 4). In contrast, the inducible NO synthase inhibitor lNIL (20 mg) (Moore et al., 1994), injected into the PVN 15 min before SR 141716A was ineffective on the increase of penile erection and of NO 2 concentration induced by the CB1 antagonist (Fig. 4). Accordingly, the AUCs of LNAME C SR 141716A- and SMTC C SR 141716A-treated rats for NO 2 and penile erection were significantly lower than that of SR 141716A-treated rats (P ! 0.001), while there was no difference between the AUCs of l-NIL C SR 141716A- and SR141716A-treated rats for NO 2 and penile erection (P O 0.1) (Fig. 4).
3.4. Effect of (C)MK 801 and cis-flupenthixol injected into the PVN on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A The excitatory amino acid NMDA receptor antagonist (C)MK 801 (1 mg) given into the PVN 15 min before SR 141716A (1 mg) reduced both penile erection and the concomitant increase of NO 2 in the paraventricular dialysate induced by the CB1 antagonist (Fig. 5). In contrast, the dopamine receptor antagonist cis-flupenthixol (10 mg), injected into the PVN 15 min before SR 141716A was ineffective on the increase of penile erection and of NO 2 concentration induced by the CB1 antagonist (Fig. 4). Accordingly, the AUCs of (C)MK 801 C SR 141716A-treated rats for NO 2 and penile erection were significantly lower than that of SR 141716Atreated rats (P ! 0.001), while there was no difference between the AUCs of cis-flupenthixol C SR 141716A- and SR141716A-treated rats for NO and penile erection 2 (P O 0.1) (Fig. 5). 3.5. Effect of d(CH2)5Tyr(Me)2-Orn8-vasotocin given into the PVN or i.c.v. on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A The oxytocin receptor antagonist d(CH2)5Tyr(Me)-Orn8vasotocin (1 mg) given into the PVN 15 min before SR 141716A (1 mg) failed to reduce both penile erection and the NO 2 increase induced by the CB1 antagonist (Fig. 6). Accordingly, there was no difference between the AUCs of d(CH2)5Tyr(Me)-Orn8-vasotocin C SR 141716A-and SR
M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228 Veh, AUC=84.4
Veh, AUC=88.20
1.4
SR 0.5, AUC=104
NO2-(µM)
1.5
*
SR 1.0, AUC=110.6 SR 2.0, AUC=120
+
+*
1.0
NO2-(µM)
2.0
223
*
#
+
Veh+SR, AUC=139.0 WIN+SR, AUC=95.2
*
*
HU210+SR, AUC=102.6
1.0 #
+
0.6
#+
* #+
0.5 0.2
0.0
1.5 AUC=580 AUC=680
*
NO3-(µM)
AUC=810
10
Penile erections/rat
15
AUC=854
* +
5
+
#
*
+
#
AUC=44.4 AUC=82.8
*
AUC=44.0
1.0
AUC=45.0
*
0.5
#+
* +
#+
40
60
#
0.0 0 -40
Penile erections/rat
2.0
*
AUC=83.0
*
1.0
+ +
#
*
40
60
+
0.5
0.0
-20
20
80 min
Fig. 3. Effect of WIN 52,522-2, CP 55,490 and HU 210 injected into the PVN on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. WIN 52,522-2 (5 mg) dissolved in 10% Tween 80 and HU 210 (5 mg) dissolved in ethanol and diluted with distilled water (final ethanol concentration 3%), were injected into the PVN in a volume of 0.3 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicles alone (Veh). The other experimental conditions were identical to those reported in the legend of Fig. 2. Values are the means G SEM of 6 rats. *(Veh C SR) P ! 0.001 with respect to vehicle-treated rats; #(Win C SR), C(HU C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
AUC=87.6
-40
0
SR 141716A
AUC=44.6 AUC=58.6
1.5
-20
0
20
80 min
SR 141716A Fig. 2. Effect of SR141716A injected into the PVN on penile erection and NO 2 and NO3 concentration in the paraventricular dialysate obtained from the PVN by means of intracerebral microdialysis. SR 141716A (0.5, 1 and 2 mg), dissolved in ethanol and diluted with distilled water (final ethanol concentration 3%), was injected into the PVN in a volume of 0.3 ml in 2 min. Controls rats were injected with vehicle alone. Vehicle (Veh) or SR 141716A was injected into the PVN at time 0, after 120 min equilibration time. After treatment, rats were put individually into Plexiglas cages and observed for 80 min in order to count penile erection episodes and to replace filled loops with emp ty ones every 20 min for the determination of NO 2 and NO3 concentration in the dialysate. The areas under the curve (AUC), obtained by plotting penile erections, NO 2 and NO3 concentrations versus time for each group shown in the figure, were calculated with the trapezoidal rule as described in Section 2. Values are the means G SEM of 6 rats. #(SR 0.5), C(SR 1), *(SR 2) P ! 0.001 with respect to vehicle-treated rats (ManneWhitney U-test).
141716A-treated rats (P O 0.1) (Fig. 5). Despite its inability to reduce SR 141716A-induced penile erection and the NO 2 increase when injected into the PVN, d(CH2)5Tyr(Me)-Orn8vasotocin (1 mg) reduced SR 141716A-induced penile erection
but not the NO 2 increase when injected i.c.v. (Fig. 6). Accordingly, the AUC of i.c.v. d(CH2)5Tyr(Me)-Orn8-vasotocin C SR 141716A-treated rats for penile erection was significantly lower than that of SR 141716A-treated rats (P ! 0.001), while the AUCs for NO 2 were not (P O 0.1) (Fig. 6). 3.6. Effect of muscimol and baclofen given into the PVN on penile erection and NO 2 increase occurring in the paraventricular dialysate after SR 141716A The GABAA receptor agonist muscimol (0.2 mg) given into the PVN 15 min before SR 141716A (1 mg) reduced both penile erection and the NO 2 increase induced by the CB1 antagonist (Fig. 7). Accordingly, the AUCs of muscimol C SR 141716A-treated rats were much lower than those of SR 141716A-treated rats (P ! 0.001) (Fig. 7). In contrast, the GABAB receptor agonist baclofen (0.2 mg) injected into the PVN 15 min before the CB1 antagonist was unable to reduce SR 141716A-induced penile erection and the NO 2 increase (Fig. 7). Accordingly, there was no difference between the
M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228
224
SMTC+SR, AUC=81.0 l-NIL+SR, AUC=103.0
0.8
0.4
Penile erections/rat
1.6
*
Veh+SR, AUC=109.6 L-NAME+SR, AUC=84.0
+
* +
#
#
§
§
1.2
1.2
*
+
#
* *
0.8
+
# 0.4
§
+
#
*+
# §
§
cis-Flu+SR, AUC=106.0
+ 0.8
1.6
AUC=88.2 AUC=46.6 AUC=83.0
*
MK-801+SR, AUC=81.0
* #
+
+
#
*
#
0.4
§
AUC=44.4 AUC=50.0
Veh, AUC=85.2 Veh+SR, AUC=106.6
NO2-(µM)
NO2-(µM)
1.2
1.6
Veh, AUC=87.2
Penile erections/rat
1.6
AUC=44.4 AUC=85.2
*
AUC=45.6
1.2
AUC=87.8
+ 0.8 #
*
+ #
+ #
*
0.4 0.0
0.0
-40
-20
0
20
40
60
80 min
SR 141716A
-40
-20
0
20
40
60
80 min
SR 141716A
Fig. 4. Effect of L-NAME, SMTC and l-NIL injected into the PVN on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. L-NAME (20 mg), SMTC (20 mg) and l-NIL (20 mg), dissolved in Ringer’s solution were injected into the PVN in a volume of 0.3 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicle alone (Veh). The other experimental conditions were identical to those reported in the legend of Figs. 2 and 3. Values are the means G SEM of 5 rats. *(Veh C SR), C(l-NILCSR) P ! 0.001 with respect to vehicle-treated rats; x(L-NAME C SR), #(SMTC C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
Fig. 5. Effect of (C)MK 801 and cis-flupenthixol injected into the PVN on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. (C)MK 801 (1 mg) and cis-flupenthixol (10 mg), dissolved in Ringer’s solution were injected into the PVN in a volume of 0.3 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicle alone (Veh). The other experimental conditions were identical to those reported in the legend of Figs. 2 and 3. Values are the means G SEM of 5 rats. *(Veh C SR), C(cis-Flu C SR) P ! 0.001 with respect to vehicle-treated rats; #(MK-801 C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
AUCs of baclofen C SR 141716A- and SR 141716A-treated rats for penile erection and NO 2 (P O 0.1) (Fig. 7).
that do not modify this sexual response and basal NO 2 and NO concentration (Melis et al., 2004a and this study), it is 3 likely that these SR 141716A responses are mediated by the blockade of paraventricular cannabinoid CB1 receptors. The blockade of cannabinoid CB1 receptors by SR 141716A causes in turn the activation of NO synthase localized in the cell bodies of oxytocinergic neurons originating in the PVN and projecting to extra-hypothalamic brain areas and to the spinal cord. This causes the activation of oxytocinergic neurons, which release oxytocin in brain areas distant from the PVN, facilitating penile erection (see Melis and Argiolas, 2003; Argiolas and Melis, 1995, 2004, 2005 and references therein). Accordingly, SR 141716A-induced penile erection is prevented by d(CH2)5Tyr(Me)2-Orn8-vasotocin, a potent oxytocin receptor antagonist, when given into the lateral ventricles, but not into the PVN, at doses capable of preventing penile erection induced by other agents, which induce this sexual response by acting in the PVN (see above). The localization of the cannabinoid CB1 receptors and the mechanism by means of which their blockade increases penile erection by increasing paraventricular NO synthase activity
4. Discussion The present study confirms that SR 141716A, a potent cannabinoid CB1 receptor antagonist (Rinaldi-Carmona et al., 1994), injected into the PVN of male rats increases the number of spontaneous penile erection episodes (Melis et al., 2004a), and shows that this effect occurs concomitantly with an increase in the activity mainly of neuronal NO synthase, mea sured by the increase in the concentration of NO 2 and NO3 found in the paraventricular dialysate obtained by intracerebral microdialysis, as already shown for other compounds that induce penile erection when injected into the PVN (e.g., dopamine agonists, NMDA, oxytocin, hexarelin analogues, pro-VGF peptides) (see Section 1 and references therein). As both the pro-erectile effect and the increase in NO 2 and NO concentration are reduced by WIN 55,212-2 and HU 3 210, two potent cannabinoid CB1 receptor agonists, at doses
M.R. Melis et al. / Neuropharmacology 50 (2006) 219e228 1.6
Veh+veh, AUC=86 Veh+SR, AUC=111
NO2-(µM)
1.2
* +§
*+
§
0.8
0.4
1.2
1.2
Bac+SR, AUC=105
+
* +
0.8
#
#
#
0.4
1.6
AUC=44.57 AUC=86.30
*
AUC=84.15 AUC=51.08
0.8
+
*
+ # #
*
Penile erections/rat
Penile erections/rat
1.6
*
Mus+SR, AUC=90
+
*
ICVOXYant+SR, AUC=108
Veh, AUC=86 Veh+SR, AUC=110
§
PVNOXYant+SR, AUC=109
NO2-(µM)
1.6
225
+ #
0.4
0.0
AUC=45
*
AUC=88
1.2
AUC=48 AUC=83
*
0.8
+
+
#
#
20
40
0.4
*
+
#
0.0
-40
-20
0
20
40
60
80 min
SR 141716A
-40
-20
0
60
80 min
SR 141716A
8
Fig. 6. Effect of d(CH2)5Tyr(Me)-Orn -vasotocin injected into the PVN or i.c.v. on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. d(CH2)5Tyr(Me)-Orn8-vasotocin (1 mg), dissolved in Ringer’s solution was injected into the PVN in a volume of 0.3 ml in 2 min or i.c.v. in a volume of 10 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicle alone (Veh). The other experimental conditions were identical to those reported in the legend of Figs. 2 and 3. Values are the means G SEM of 5 rats. *(Veh C SR), C(PVN Oxy ant C SR), P ! 0.001 with respect to vehicle-treated rats; #(ICV Oxy ant C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
and oxytocinergic neurotransmission are unknown at present. The available data support the hypothesis that cannabinoid CB1 receptors are located in excitatory amino acid-containing (possibly glutamic acid) synapses impinging on the cell bodies of oxytocinergic neurons mediating penile erection, rather than to be located directly in the cell bodies of oxytocinergic neurons themselves. It is likely that these cannabinoid CB1 receptors exert a tonic inhibition on the release of this excitatory amino acid, which activates oxytocinergic neurons mediating penile erection through the activation of NO synthase localized in these neurons (see Melis and Argiolas, 2003; Argiolas and Melis, 1995, 2004, 2005). Accordingly, (1) cannabinoid CB1 receptor agonists (WIN 55,212-2, CP 55,940 and HU 210) when injected into the PVN at doses that do not influence spontaneous penile erection (Melis et al., 2004a), and do not modify basal NO production (this study), reduce SR 141716-induced penile erection and activation of NO synthase; (2) SR 141716A-induced penile erection and the concomitant increase in NO production are reduced by the injection into the PVN of (C)MK-801, which blocks
Fig. 7. Effect of muscimol and baclofen injected into the PVN or into the lateral ventricles on SR 141716A-induced penile erection and on the concomitant NO 2 increase in the paraventricular dialysate. Muscimol (0.2 mg) or baclofen (0.2 mg) dissolved in Ringer’s solution was injected into the PVN in a volume of 0.3 ml in 2 min 15 min before SR 141716A (1 mg). Controls rats were injected with vehicle alone (Veh). The other experimental conditions were identical to those reported in the legend of Figs. 2 and 3. Values are the means G SEM of 5 rats. *(Veh C SR), C(Bac C SR) P ! 0.001 with respect to vehicle-treated rats; #(Mus C SR) P ! 0.001 with respect to the Veh C SR-treated rats (ManneWhitney U-test).
NMDA receptors, and of L-NAME and by SMTC, which inhibit the first all NO synthase isoforms and the second neuronal NO synthase selectively (Furfine et al., 1994; Narayanan and Griffith, 1994), but not by l-NIL, an inhibitor of inducible NO synthase (Moore et al., 1994) (this study and Melis et al., 2004a); and (3) NMDA injected into the PVN induces penile erection and increases NO production, which are also antagonized by (C)MK-801 and by L-NAME (Melis et al., 1997b; Melis and Argiolas, 2003; Argiolas and Melis, 2004; 2005). In line with this hypothesis, glutamic acid concentration increases in the dialysate obtained from the PVN during sexual activity (Melis et al., 2004b) and the stimulation of cannabinoid CB1 receptors has been shown to inhibit the release of glutamic acid in different brain tissues (Robbe et al., 2001; Pistis et al., 2002; Brown et al., 2003; Breivogel et al., 2004; Freund et al., 2003; Howlett et al., 2004 and references therein). In line with the interpretation given above, the inability of WIN 55,212-2, CP 55,940 and HU 210 to influence spontaneous and drug-induced penile erection when injected into the PVN (this paper and Melis et al., 2004a), suggests that the tonic activation of cannabinoid receptors controlling
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excitatory synapses in the PVN is already maximal. Indeed, in this case any further stimulation of cannabinoid CB1 receptors would be unable to exert any additional inhibition on excitatory neurons impinging on the cell bodies of oxytocinergic neurons mediating penile erection. Alternatively, cannabinoid CB1 receptors might be located in inhibitory, possibly GABAergic synapses, which act on glutamic acid-containing neurons impinging on the cell bodies of oxytocinergic neurons mediating penile erection. The stimulation of these cannabinoid CB1 receptors might enhance the release of GABA, while their blockade might decrease its release, inducing in turn an increase in the release of glutamic acid that activates oxytocinergic neurons. Accordingly, (1) the stimulation of GABAA by muscimol, but not of GABAB receptors by baclofen, injected into the PVN reduces penile erection and the increase in NO production induced by SR 141716A as well as by NMDA (Melis and Argiolas, 2002); and (2) cannabinoid CB1 receptor agonists have been also found capable of enhancing GABAergic neurotransmission in several circumstances (De Miguel et al., 1998). However, this second interpretation is complicated by at least two lines of experimental evidence. First, muscimol can inhibit NO synthase and oxytocinergic neurons by acting directly on GABAA receptors localized in the cell bodies of oxytocinergic neurons, as GABAergic synapses impinging on oxytocinergic cell bodies known to inhibit oxytocinergic activity in several circumstances have been well characterized (Bisset et al., 1990; Roland and Sawchenko, 1993; Voisin et al., 1995; Boubada et al., 1996; Jourdain et al., 1999); and second, muscimol can directly inhibit excitatory amino acidergic neurons impinging on oxytocinergic neurons mediating penile erection (see Melis and Argiolas, 2002). Nevertheless, the two interpretations may not be mutually exclusive and both mechanisms may contribute to the pro-erectile effect of SR 141716A and its ability to increase NO production when injected into the PVN. The above explanation is based mainly on the assumption that SR 141716A induces penile erection by acting into the PVN by increasing excitatory amino acid neurotransmission acting directly on excitatory amino acidergic synapses or indirectly by decreasing inhibitory GABAergic input to these excitatory synapses, and that the increase in excitatory amino acid neurotransmission in turn activates neuronal NO synthase in the cell bodies of oxytocinergic neurons leading to an increased NO production. NO in turn activates oxytocinergic neurons to release oxytocin in sites distant from the PVN leading to penile erection. However, other explanations cannot be ruled out. For instance SR 141716A might activate neuronal NO synthase and oxytocinergic neurons mediating erectile response by interacting directly with cannabinoid receptors located in their cell bodies, or by increasing the activity of other neurotransmitters and/or neuropeptides present in the PVN and already known to induce penile erection by activating NO synthase and oxytocinergic neurons, such as dopamine and oxytocin itself, or by inhibiting the activity of endogenous opioids, which reduce, like GABA, the activity of oxytocinergic neurons and penile erection by acting on opioid receptors of the subtype m located in the cell bodies of oxytocinergic
neurons (see Melis et al., 1999 and references therein). Although further experiments are necessary to verify the above possibilities in detail, from the available data it is unlikely that SR 141716A induces penile erection when injected into the PVN by increasing dopamine or oxytocin release in the PVN, since both cis-flupenthixol, a potent antagonist of dopamine receptors, and d(CH2)5Tyr(Me)2-Orn8-vasotocin, a potent antagonist of oxytocin receptors, are unable to reduce SR 141716A-induced penile erection and the increase in NO production when injected into the PVN. Indeed, at the doses used in this study, cis-flupenthixol and d(CH2)5Tyr(Me)2-Orn8-vasotocin are able to prevent penile erection induced by the dopamine agonist apomorphine and by oxytocin given in the PVN, respectively (Melis and Argiolas, 2003; Argiolas and Melis, 2004, 2005 and references therein). In conclusion, the present study show that SR 141716A, a cannabinoid CB1 receptor antagonist, induces penile erection when injected into the PVN, apparently by increasing neuronal NO synthase activity and oxytocinergic neurotransmission, as shown for other compounds that induce penile erection when injected into the PVN. This effect is apparently mediated by an increase in excitatory amino acid, possibly glutamic acid neurotransmission, which may be due to the inhibition of cannabinoid CB1 receptors located in excitatory amino acidergic synapses and controlling glutamic acid release, or of cannabinoid CB1 receptors located on GABAergic synapses impinging on the above excitatory synapses to inhibit glutamic acid release. Released glutamic acid stimulates in turn its receptors located in the cell bodies of oxytocinergic neurons increasing intracellular Ca2C concentration inside these cell bodies. This stimulates neuronal NO synthase to produce NO, which activates oxytocinergic neurons to release oxytocin in sites distant from the PVN, leading to penile erection. The ability of SR 141716A to induce penile erection when injected into the PVN is in line with the reported inhibitory effects of cannabinoids given systemically on sexual behavior (Shrenker and Bartke, 1985; Ferrari et al., 2000 and references therein) and suggests that cannabinoid CB1 receptors in the PVN can influence erectile function and sexual behavior. However, further studies with cannabinoid CB1 agonists and antagonists not only on spontaneous penile erections, but also on reflex erections and non-contact erections (which occur in male rats exposed to inaccessible receptive female rats), are necessary to clarify in detail the role of paraventricular cannabinoid CB1 receptors in erectile function. Acknowledgements This work was partially supported by a MIUR grant to AA and MRM, and a grant from Fondazione Banco di Sardegna to MPC. References Andersson, K.E., 2001. Pharmacology of penile erection. Pharmacological Reviews 53, 417e450.
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