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.
