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mise à jour du
20 janvier 2008
Eur J Pharmacol
2000;404(1-2):137-143

EP 60761 and EP 50885, two hexarelin analogues, induce penile erection in rats
Melis MR, Succu S, Spano MS, Locatelli V, Torsello A, Muller EE, Deghenghi R, Argiolas A.
Bernard B. Brodie Department of Neuroscience, University of Cagliari, Italy
Tous les travaux de MR Melis & A Argiolas 
Tous les travaux de M Eguibar & G Holmgren

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The effect of hexarelin and four related peptide analogues, EP 40904, EP 40737, EP 50885 and EP 60761, injected into the paraventricular nucleus of the hypothalamus of male rats in doses between 2 and 2000 ng on spontaneous penile erection was studied. Of these peptides, EP 60761 and EP 50885, but not hexarelin, EP 40904 or EP 40737, increased dose-dependently the number of spontaneous penile erections. EP 60761 was active already at the dose of 20 ng, which induced the sexual response in 70% of the treated rats. The maximal response was induced by 200 ng of the peptide. EP 50885 was less potent than EP 60761, with 1000 ng being the minimal effective dose and 2000 ng as the dose required to induce the maximal response. At the doses used, both peptides also increased slightly the number of spontaneous yawning episodes. EP 60761- and EP 50885-induced penile erection was prevented by the oxytocin receptor antagonist [d(CH(2))(5)Tyr(Me)(2)-Orn(8)]vasotocin (0.1-1 microg) given intracerebroventricularly (i.c.v.), but not into the paraventricular nucleus (0.1-1 microg), by the competitive nitric oxide (NO) inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) given either into the paraventricular nucleus (10-20 microg) or i.c.v. (75-150 microg), by the N-type Ca(2+) channel blocker omega-conotoxin-GVIA (2-5 ng) or by the opiate morphine (1-10 microg), but not by the dopamine receptor antagonist (Z)-4-[3-[2-(trifluoromethyl)-9H-thioxanthen-9-ylidene]propyl]-1-p ipe razine-ethanol (cis-flupenthixol) (10 microg) or by the N-methyl-D-aspartic acid (NMDA) receptor antagonist (5R, 10S)-(+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5, 10-imine ((+)-MK-801) (1 microg), all given into the paraventricular nucleus before either peptide. The present results show that EP 60761 and EP 50885 induced penile erection by increasing central oxytocin transmission, possibly by activating NO synthase in the cell bodies of oxytocinergic neurons located in the paraventricular nucleus that control penile erection.
 
1. Introduction
 
Several neurotransmitters and neuropeptides are involved at the central level in the control of penile erection. Among these, the best known are adrenocorticotropin, oxytocin, opioid peptides, dopamine, serotonin, excitatory amino acids and nitric oxide (No). These compounds influence this sexual response by acting in different brain areas, namely the medial preoptic area, the paraventricular nucleus of the hypothalamus, the bed nucleus of the stria terminalis, the amygdala, the hippocampus, the medulla oblongata and the spinal cord (for a review on the central control of penile erection, see Meisel and Sachs, 1994; Argiolas and Melis, 1995; Andersson and Wagner, 1995; Argiolas, 1999). Recent studies suggest that oxytocinergic neurons, originating in the paraventricular nucleus and projecting to extrahypothalamic brain areas (i.e. the hippocampus, the ventral medulla and the spinal cord), are involved in the control of penile erection in in copula and ex copula contexts in the male rat (see Argiolas and Melis, 1995; Melis et al., 1999a,b; Veronneau-Longueville et al., 1999). Accordingly, the activation of these neurons by dopamine, excitatory amino acids and oxytocin itself, or by the presence of an inaccessible receptive female rat facilitates penile erection, while their inhibition by morphine and, possibly, by opioid peptides impairs this sexual response. The activation and the inhibition of these oxytocinergic neurons, together with the facilitation and the inhibition of penile erection, is apparently mediated by an increased and a decreased production of NO in the paraventricular nucleus, respectively, as determined by in vivo microdialysis (Melis et al., 1996, 1997a,b,c,d, 1998, 1 999a,b).
 
Some of the above neurotransmitters exert an opposite role on sexual function and eating. The most clear examples are those of oxytocin, which induces penile erection and facilitates sexual behaviour (see above) and decreases eating (see Arletti et al., 1989), and of opioid peptides, which impair penile erection and sexual behaviour (see above) and increase eating (see Morley, 1987). As the paraventricular nucleus is also involved in the control of feeding (see Morley, 1987), this raises the possibility that the two behaviours might be controlled in an opposite fashion by some of these neurotransmitters at the paraventricular level. Recently, a new class of peptide molecules that release growth hormone (GH) in experimental animals and humans with a efficacy higher than that of the endogenous GH-releasing hormone (GHRH) has been characterised (see Deghenghi, 1996; Muller et al., 1999 and references therein). These GH-releasing peptides, like GHRH, also increase eating in laboratory animals when injected intracerebroventricularly (i.c.v.) (Locke et al., 1995; Okada et al., 1996; Torsello et al., 1998). The eating effect is not strictly related to the ability of these peptides to release GH (Torsello et al., 1998), since they act on receptors different from those activated by GHRH (Codd et al., 1989; Pong et al., 1986; Howard et al., 1996) and are linked to intracellular messenger pathways different from those utilised by GHRH (Cheng et al., 1989; Akman et al., 1993). Here, we report that EP 60761 and EP 50885, two analogues of the GH-releasing peptide hexarelin (Deghenghi et al., 1994), induce in rats penile erection episodes indistinguishable from those elicited by dopamine receptor agonists, oxytocin or N-methyl-D-aspartic acid (NMDA), when injected into the paraventricular nucleus and irrespective of their effect on GH release and eating. A possible mechanism underlying the action of the two peptides on penile erection is also reported.
 
4. Discussion
 
The present results show that the hexarelin analogues EP 60761 and, with a lower potency, EP 50885, injected into the paraventricular nucleus induced penile erection episodes indistinguishable from those induced by oxytocin, dopamine receptor agonists and NMDA, which act in this hypothalamic nucleus to elicit this male sexual response. The facilitative effect of EP 60761 and of EP 50885 on penile erection was prevented by [d(CH2)5Tyr(Me)20m8 ]vasotocin, a potent and selective oxytocin receptor antagonist (Bankowski et al., 1980), by L-NAME, a potent NO synthase inhibitor (Rees et al., 1990), and by w-conotoxin-GYIA, a potent blocker of Ca 2 channels of the N-type (McCleskey et al., 1987). Hence, it is likely that EP 60761 and EP 50885 induce this sexual response by acting in the paraventricular nucleus with a mechanism similar to that suggested for oxytocin, dopamine receptor agonists and NMDA, e.g. by stimulating oxytocinergic neurons that originate in the paraventricular nucleus and project to extra-hypothalamic brain areas (see Argiolas and Melis, 1995). In agreement with this hypothesis, penile erection induced either by EP 60761 or by EP 50885 was prevented by [d(CH2)5Tyr(Me)2-Orn8]vasotocin when given i.c.v., but not into the paraventricular nucleus. This finding suggests that EP 60761 and EP 50885 induce penile erection by releasing oxytocin at sites distant from the paraventricular nucleus. A similar explanation was provided for the ability of [d(CH2 )5Tyr(Me)2-Orn8 ]vasotocin, when given i.c.v. but not into the paraventricular nucleus, to prevent dopamine receptor agonist- and NMDA-induced penile erection (Melis et al., 1992, 1994). The ability of L-NAME given either i.c.v. or into the paraventricular nucleus to prevent EP 60761- and EP 50885-induced penile erection suggests that the activation of oxytocinergic transmission by EP 60761 and EP 50885 is apparently mediated by an increased NO production in the paraventricular nucleus, as found for oxytocin-, dopamine receptor agonist- and NMDA-induced penile erection (Melis et al., 1996, 1997b,c). Activation of NO synthase in the paraventricular nucleus by EP 60761 or EP50885 might be secondary to an increased Ca 2+ influx, possibly in the cell bodies of oxytocinergic neurons mediating penile erection, because EP 60761- or EP 50885-induced penile erection was prevented by the blockade of N-type Ca 2 + channels in the paraventricular nucleus by w-conotoxin-GVIA. In line with this hypothesis, w-conotoxin-GVIA injected into the paraventricular nucleus prevents dopamine receptor agonistand oxytocin-induced penile erection (Argiolas et al., 1990; Succu et al., 1998). Likewise, NMDA-induced penile erection is prevented by (+)-MK-801 (Melis et al., 1994; Succu et al., 1998), which blocks NMDA receptor-coupled Ca 2 channels (Monaghan et al., 1989). Interestingly, an increased Ca 2+ influx has been shown to modulate the GH release induced by GH-releasing peptides, including hexarelin, in the pituitary gland (Akman et al., 1993; Sartor et al., 1985). Finally, favouring the view that EP 60761 and EP 50885 induce penile erection via oxytocinergic transmission was the ability of morphine to prevent this response when injected into the paraventricular nucleus. Indeed, morphine reduces oxytocin-, dopamine receptor agonist- and NMDA-induced penile erection, apparently by preventing the activation of NO synthase induced by these substances at the paraventricular level (Melis et al., 1997a,d).
 
The molecular mechanism(s) subserving EP 60761 and EP 50885 activation of oxytocinergic neurons in the paraventricular nucleus is unknown at present. It may be hypothesised that the two peptides stimulate specific receptors, possibly located in the cell bodies of oxytocinergic neurons and coupled to Ca 2+ influx. In this regard, it is pertinent to recall that specific receptors for GH-releasing peptides have been identified in the pituitary gland, the hypothalamus and other brain regions (Codd et al., 1989; Pong et al., 1986; Howard et al., 1996, Muccioli et al., 1998). Perhaps more relevant to this work, activation of these receptors triggers GH release from the pituitary by increasing Ca 2 influx (Akman et al., 1993; Sartor et al., 1985). If this hypothesis is correct, since hexarelin was inactive, whereas EP 50885 and EP 60761 did induce penile erection when injected into the paraventricular nucleus, the existence of a GH-releasing peptide receptor mediating penile erection with an affinity for EP 60761 and EP 50885 higher than that for hexarelin may be envisaged. Along this line, it is noteworthy that EP 50885 and hexarelin are both capable of releasing GH and of increasing eating behaviour, EP 40737 is active on GH release but is inactive on eating, EP 40904 is instead inactive on GH release but active on eating, and EP 60761 has no activity either on GH release or eating (Torsello et al., 1998). From the foregoing, the GH-releasing peptide receptors mediating penile erection would be different from those mediating GH release or eating behaviour. This would also explain the surprising inability of hexarelin to induce penile erection. Indeed, this finding might simply indicate that receptors mediating penile erection have binding properties for GH-releasing peptides different from those mediating GH release or eating behaviour. The existence of a specific receptor mediating a distinct effect of these peptides, i.e. penile erection, would not be so speculative, since previous experimental evidence supports the existence of different sub-populations of GH-releasing peptide receptors. First, the effects of hexarelin and its analogues on GH release are divorced from the eating effects (Torsello et al., 1998). Second, receptors for hexarelin and other GH-releasing peptides, whose activation induces effects independent of the GH-releasing properties of the peptides, have been detected in other tissues, e.g. in the heart (Bisi et al., 1999; Bodart et al., 1999; Locatelli et al., 1999). Finally, cloning studies have revealed the existence of several forms of receptors for GH-releasing peptides (Smith et al., 1999). Alternatively, other possible mechanisms of actions for EP 60761 and EP 50885 cannot be ruled out at present. For instance, these peptides might induce penile erection by releasing neurotransmitters that influence penile erection in the paraventricular nucleus, e.g. dopamine, oxytocin and/or excitatory amino acids. However, this hypothesis is unlikely, since (+)-MK-801, an antagonist of NMDA receptors,
 
cis-flupenthixol, which blocks dopamine receptors, and [d(CH2)5Tyr(Me)2-Orn8]vasotocin, an antagonist of oxytocin receptors, failed to prevent EP 60761- and EP 50885-induced penile erection when injected into the paraventricular nucleus. This finding suggests that the erectile response induced by the two peptides is not mediated by the release of dopamine or excitatory amino acids or oxytocin in the paraventricular nucleus. If this was the case, the above compounds would have been capable of preventing EP 60761- and EP 50885-induced penile erection, as was found for dopamine receptor agonist-, NMDAor oxytocin-induced penile erection (Argiolas and Melis, 1995).
 
In conclusion, EP 60761 and EP 50885, two peptides related to the GH-releasing peptide hexarelin, induced penile erection when injected into the paraventricular nucleus, apparently via pathways unrelated to those subserving the release of GH or increase of eating behaviour. Further studies are mandatory to clarify the molecular mechanism by which EP 60761 and EP 50885 induce this sexual response. However, the finding that these peptides induce penile erection when injected into the paraventricular nucleus, a kind of integrative centre between the central and peripheral nervous system (Swanson and Sawchensko, 1983) that plays an important role in the control of sexual function (see Argiolas and Melis, 1995), suggests that peptide molecules of this kind may be useful for the treatment of erectile dysfunction of central origin in men.