The cannabinoid receptor antagonist SR-141716A induces penile erection in male rats: Involvement of paraventricular glutamic acid and nitric oxide

Abstract

The cannabinoid CB1 receptor antagonist SR141716A (0.5, 1 and 2 μg) induces penile erection when injected into the paraventricular nucleus of male rats. The pro-erectile effect of SR 141716A occurs concomitantly with an increase in the concentration of NO₂⁻ 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 μg) or HU 210 (5 μg), given into the paraventricular nucleus at doses unable to induce penile erection or to modify NO₂⁻ concentration. SR 141716A responses were also reduced by nitro-l-arginine methylester (20 μg), a non-selective NO synthase inhibitor, S-methyl-l-thiocitrulline (20 μg), a selective neuronal NO synthase inhibitor, the excitatory amino acid NMDA receptor antagonist dizocilpine ((+)MK 801) (1 μg), or the GABA_A receptor agonist muscimol (0.2 μg) injected into the PVN 15 min before SR 141716A. In contrast, the inducible NO synthase inhibitor l-N(6)-(1-iminoethyl)lysine (20 μg), the GABA_B receptor agonist baclofen (0.2 μg), the mixed dopamine receptor antagonist cis-flupenthixol (10 μg), and the oxytocin receptor antagonist d(CH₂)₅Tyr(Me)-Orn⁸-vasotocin (1 μg), were ineffective. Despite its inability to reduce penile erection and NO₂⁻ increase induced by SR 141716A when injected into the PVN, d(CH₂)₅Tyr(Me)-Orn⁸-vasotocin (1 μg) reduced almost completely penile erection without reducing paraventricular NO₂⁻ 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.

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 and Argiolas, 2003, Argiolas and Melis, 2004, Argiolas and Melis, 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, Argiolas and Melis, 2004, Argiolas and Melis, 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, Melis et al., 1997a, Melis et al., 1997b, Melis et al., 2003, Melis et al., 2004b, Succu et al., 2004, Succu et al., 2005), these neurons facilitate 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 Ca²⁺ influx in the cell bodies of these neurons, which causes in turn the activation of nitric oxide (NO) synthase, the Ca²⁺-calmodulin enzyme which converts l-arginine to NO (see Melis et al., 1998, Melis and Argiolas, 2003, Argiolas and Melis, 2004, Argiolas and Melis, 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 Ca²⁺ 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-Agró, 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 ((+)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 determining the concentration of NO₂⁻ and NO₃⁻ in the dialysate obtained from the PVN by means of intracerebral microdialysis. NO₂⁻ 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.