The beta-lactamase inhibitor clavulanic acid
induced penile erection and yawning in a dose
dependent manner when given intraperitoneally
(IP, 0.05-5mg/kg), perorally (OS, 0.1-5mg/kg)
and intracereboventricularly (ICV, 0.01-5?g/rat)
to male rats. The effect resembles that of the
dopamine receptor agonist apomorphine given
subcutaneously (SC) (0.02-0.25mg/kg), although
the responses of the latter followed a U
inverted dose-response curve, disappearing at
doses higher than 0.1mg/kg. Clavulanic acid
responses were reduced by about 55% by
haloperidol, a dopamine D2 receptor antagonist
(0.1mg/kg IP), and by
d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin, an
oxytocin receptor antagonist (2?g/rat ICV), both
given 15min before clavulanic acid. A higher
reduction of clavulanic acid responses (more
than 80%) was also found with morphine, an
opioid receptor agonist (5mg/kg IP), and with
mianserin, a serotonin 5HT(2c) receptor
antagonist (0.2mg/kg SC). In contrast, no
reduction was found with naloxone, an opioid
receptor antagonist (1mg/kg IP). The ability of
haloperidol,
d(CH(2))(5)Tyr(Me)(2)-Orn(8)-vasotocin and
morphine to reduce clavulanic acid induced
penile erection and yawning suggests that
clavulanic acid induces these responses, at
least in part, by increasing central
dopaminergic neurotransmission.
Dopamine in turn activates oxytocinergic
neurotransmission and centrally released
oxytocin induces penile erection and yawning.
However, since both penile erection and yawning
episodes were reduced not only by the blockade
of central dopamine and oxytocin receptors and
by the stimulation of opioid receptors, which
inhibits oxytocinergic neurotransmission, but
also by mianserin, an increase of central
serotonin neurotransmission is also likely to
participate in these clavulanic acid
responses.
1. Introduction
Clavulanic acid is a beta-lactamase
inhibitor produced by Streptomyces clavuligenus,
with a chemical structure similar to that of
penicillin. The drug is used clinically and
veterinary often in combination with
penicillins, e.g. amoxicillin, in order to
potentiate the antibacterial action of the
latter. The compound crosses readily the blood
brain barrier and has been recently reported to
possess anxiolytic properties in non human
primates (e.g., cotton-top tamarins, Saguinus
oedipus) (Kim et al., 2009). Clavulanic acid was
also found capable of exerting neuroprotective
effects in animal models of Parkinson's disease
(Huh et al., 2010; Kost et al., 2012). These
central effects may be secondary at least in
part to the ability of clavulanic acid to
increase dopamine and serotonin release from
dopamine and serotonin neurons (Kim et al.,
2009). Accordingly, the drug was recently found
to be able to increase dopamine release from two
dopaminergic neuronal cell lines, PC12 cells
(derived from adrenal gland pheochromocytoma)
and SH-SY5Y cells (derived fromhuman
neuroblastoma) in vitro by amechanism involving
vesicle trafficking and fusion through the
binding and regulation of Munc18-1 and Rab4
proteins (Kost et al., 2011).
In one of the above experiments with
cotton-top tamarins, it was also found that the
drug increased sexual arousal as indicated by
the increased rate of penile erections (see Kim
et al., 2009). This sexual effect was confirmed
in male rats by experiments showing that chronic
treatment with clavulanic acid given orally at
doses ranging from 0.01 to 1.0 mg/kg for
7&endash;14 days decreases the latency to the
first ejaculation and increases the number of
ejaculations in a classic test of male rat
copulatory activity (Chan et al., 2009).
Interestingly, at central level dopamine is one
of the most studied neurotransmitters involved
in the control of penile erection and sexual
behaviour. Accordingly, dopamine receptor
agonists increase penile erection and facilitate
copulatory behaviour in rodents (rats, mice and
others) and also in other mammals including non
human primates (seeMelis and Argiolas,
1995;McKenna, 2000; Andersson, 2001; Giuliano
and Rampin, 2001; Hull et al., 2002). Dopamine
receptor agonist-induced penile erection usually
(but not always) occurs concomitantly with
yawning (see Argiolas and Melis, 1998; Melis and
Argiolas, 1995 and references therein). Both
these effects are secondary to the stimulation
of dopamine receptors of the D2 family, mainly
D2 and D4 receptor subtypes, although a role for
the D3 receptor subtype cannot be completely
ruled out (see Brioni et al., 2004; Collins et
al., 2007, 2009; Depoortère et al., 2009;
Sanna et al., 2011, 2012a and references
therein). Different brain areas, such as the
medial preoptic area, the paraventricular
nucleus of the hypothalamus, the ventral
tegmental area, the nucleus accumbens and the
hippocampus are involved in these responses (see
Melis and Argiolas, 1995; Hull et al., 1995;
Melis et al., 2003; Andersen et al., 2003;
Andersen and Tufik, 2005; Succu et al., 2007;
Richards et al., 2009).
A role of dopamine receptors recently
identified in cavernous tissue on penile
erection has been also suggested, although the
available data are still very scarce (Senbel,
2011). Interestingly, a dopamine&endash;oxytocin
interaction is thought to occur in a
hypothetical complex neural circuit
interconnecting many of the above brain areas,
which controls not only the consummatory phase
of sexual behaviour (e.g., penile erection and
copulation), but also sexual motivation, sexual
arousal and sexual reward (see Melis and
Argiolas, 2011). Moreover, since yawning
inmammalsmay be an unconscious signal that
communicates one's own
physiological/psychological state to
othermembers of a social group, e.g., it may
represent a primitive, unconscious form of
empathy (see Palagi et al., 2009), this neural
circuitmay be also involved in other central
functions in which dopamine and oxytocin play a
role, mainly social and emotional behaviours
(Hurlemann et al., 2010; Lee et al., 2009;
Neumann, 2008; Palagi et al., 2009; Sanna et
al., 2012b). In order to provide direct support
for a role of dopamine in the facilitatory
effect of clavulanic acid on male sexual
response and the other central functions on
which this drug is supposed to exert an effect
(i.e., anxiety, neuroprotection) (see above), we
compared the effects of clavulanic acid on
penile erection and yawning in male rats with
those of apomorphine, a classic dopamine
receptor agonist known for its ability to induce
both these responses. The effects of drugs known
to interfere with spontaneous or drug-induced
penile erection and yawning are also reported 4.
Discussion
The present results show that the potassium
salt of clavulanic acid given IP induced penile
erection and yawning in male rats. The drug was
already active at doses as low as 0.1 mg/kg,
with the maximal responses found at the dose of
2.0 mg/kg. The drug induced penile erection and
yawning alsowhen given per OS or into the
cerebral ventricles, suggesting that clavulanic
acid induced these behavioural responses by
acting in the central nervous system. The effect
of clavulanic acid given IP resembles the effect
of apomorphine, a mixed dopamine D1/D2 agonist
that is extremely effective in inducing penile
erection and yawning. However, while the
efficacy of clavulanic acid in inducing penile
erection was comparable to that of the dopamine
receptor agonist, its efficacy in inducing
yawning was much lower than that of apomorphine,
especially when the drug was given per OS or
ICV. Indeed, yawning tended to disappear when
clavulanic acid was given by these routes of
administration. The reason for the tendency of
yawning to disappear is unknown, although it is
likely that this is related to the route of
administration. Indeed, it may be that the drug
given IP reaches brain areas which are involved
in yawning at a concentration that makes this
response likely to occur, while this does not
happen when the drug is given per OS or ICV.
Another difference between clavulanic acid and
apomorphine is that no U inverted dose response
curve was found with clavulanic acid at the
doses used in this study. This is at variance
from apomorphine, for which it is well
established that doses up to 0.08 mg/kg SC
increase penile erection and yawning in a
dose-dependent manner, and that these responses
tend back to basal values with doses higher than
0.1 mg/kg SC, as these induce marked
hyper-motility and stereotyped behaviour that
masks yawning and penile erection, as found also
in this study. Although it is possible that
doses of clavulanic acid higher than 5 mg/kg may
induce effects that mask the appearance of
penile erection and yawning, this is unlikely,
since this dose is already two&endash;three-fold
the dose found to induce the maximal response in
the present experiments, irrespective of the
route of administration, and 5-fold the dose
found capable of inducing penile erection in
cotton top tamarins (Kimet al., 2009) and of
facilitating male rat sexual behaviour (Chan et
al., 2009).
The mechanism by which clavulanic acid
induces penile erection and yawning by acting in
the central nervous system is unknown at the
moment. However, some explanation may be
suggested by the results showing that clavulanic
acid induced responses were markedly reduced by
drugs which are known for their ability to
reduce or abolish these responses when induced
by apomorphine. First, clavulanic acid-induced
penile erection and yawning were reduced by more
than 50% by haloperidol, a classic non selective
dopamine D2-like receptor antagonist, which is
also well known for its ability to reduce these
behavioural responses when induced by
apomorphine and other dopamine receptor agonists
(see Melis et al., 1987, 1996). Second,
clavulanic acid responses were also reduced by
more than 50% by d(CH2)5Tyr(Me)2-Orn8-vasotocin,
an oxytocin receptor antagonist (Bankowski et
al., 1980), given ICV at doses that reduced
penile erection and yawning induced also by
apomorphine, which induces these responses by
increasing central oxytocinergic
neurotransmission (see Melis and Argiolas, 2011;
Sanna et al., 2012b).
However, it must be noted that the reduction
of clavulanic acid responses by haloperidol and
d(CH2)5Tyr(Me)2-Orn8-vasotocin (about 55%) is
lower than that of apomorphine responses (about
85%). In spite of this difference, morphine, an
opioid receptor agonist, but not naloxone, an
opioid receptor antagonist, reduced almost
completely penile erection and yawning induced
by clavulanic acid when given at doses that
reduced almost completely also apomorphine- and
oxytocin-induced responses. These results may be
explained by the ability of opioid receptor
agonists to markedly decrease oxytocin
neurotransmission at central and posterior
pituitary level (see Melis et al., 1999 and
references therein). All together, the above
findings support the hypothesis that clavulanic
acid induces penile erection and yawning, at
least in part, by increasing dopamine
neurotransmission in the central nervous system.
Dopamine in turn increases central oxytocinergic
neurotransmission, leading to penile erection
and yawning, as already suggested for
apomorphine and other dopamine receptor agonists
(see Melis et al., 1996; Melis and Argiolas,
2011). This interpretation is in line with
previous studies showing that clavulanic acid is
able to increase extracellular dopamine
concentration in the dialysate obtained fromthe
nucleus accumbens ofmale rats by intracerebral
microdialysis (Kim et al., 2009). As to the
mechanism by which clavulanic acid facilitates
central dopamine release, it is noteworthy that
the compound was found ineffective in binding
assays for 63 different signalling pathways and
mediators that included the classical
neurotransmitters, their receptors and
transporters, ion channels, hormones, brain/gut
peptides, prostaglandins, and enzymes (Kim et
al., 2009).
Nonetheless, it is tempting to speculate
that this mechanism may be similar to that
suggested to occur in neuronal cell lines, e.g.,
PC12 cells (derived from adrenal
pheochromocitoma), and SH-SY5Y cells (derived
from neuroblastoma) exposed to clavulanic acid.
In these cells, the drug was recently reported
to be able to increase dopamine release
apparently by a mechanism involving vesicle
trafficking and fusion through the binding and
regulation of Munc18-1 and Rab4 proteins (Kost
et al., 2011), although other mechanisms cannot
be ruled out. The above interpretation is
complicated by two findings of this study.
First, the reduction of clavulanic acid
responses by haloperidol and
d(CH2)5Tyr(Me)2-Orn8-vasotocin (about 55%) was
lower than that of apomorphine responses (about
85%), as if clavulanic acid responses were not
completely due to an increased dopaminergic
neurotransmission. Second, clavulanic
acid-induced penile erection and yawning were
markedly reduced by mianserin, a putative 5-HT2c
receptor antagonist (Berendsen and Gower, 1986;
Berendsen et al., 1990; Harvey et al., 1999), at
doses that reduced apomorphine-induced penile
erection, but not yawning. Indeed, these results
support a possible role of serotonin in
clavulanic acid responses, in addition to that
of dopamine. In particular this finding suggests
that clavulanic acid increases not only the
release of dopamine but also that of serotonin
(see also Kim et al., 2009), which in turn acts
on 5HT2c receptors to induce both penile
erection and yawning. Accordingly, 5HT2c
receptor agonists such as m-CPP
[1-(3-chlorophenyl)-piperazine] and
TFMPP
[N-(3-trifluoromethylphenyl)-piperazine],
were found capable of inducing penile erection
and yawning episodes in male rats, which were
indistinguishable from those induced by
apomorphine and which were antagonized by
mianserin given at doses that antagonized also
penile erection, but not yawning, when induced
by apomorphine and oxytocin (see Berendsen and
Gower, 1986; Berendsen et al., 1990;
Stancampiano et al., 1995).
These results led to suggest that 5HT2c
receptors located downstream to dopamine and
oxytocin are involved in the control of penile
erection (Kimura et al., 2008; Stancampiano et
al., 1995),while such a link between dopamine,
oxytocin and serotonin does not occur in yawning
(see Stancampiano et al., 1995). In view of
these findings, it is likely that centrally
released serotonin participates in clavulanic
acid-induced penile erection and yawning,
respectively, by acting either at 5HT2c
receptors located in sites downstreamto dopamine
and oxytocin for the former response, and by a
mechanism not involving dopamine or oxytocin for
the latter. Studies aimed at characterizing
further the role of dopamine and serotonin in
clavulanic acid-induced penile erection and
yawning (for instance through selective
monoamine depletion studies or with the direct
microinjection of dopamine and/or serotonin
receptor antagonists into brain areas relevant
for penile erection and yawning) are necessary
to clarify this point.
5. Conclusions
In conclusion, clavulanic acid given IP, ICV
or per OS induces penile erection and yawning in
male rats, apparently by activating central
dopaminergic neurotransmission, which leads to
an increase in central oxytocinergic
neurotransmission (Argiolas and Melis, 1998;
Melis and Argiolas, 2011). In line with this
hypothesis, clavulanic acid-induced penile
erection and yawning are impaired by dopamine
receptor antagonists, oxytocin receptor
antagonists and by other drugs such asmorphine,
which reduces oxytocin release. However, since
clavulanic acid induced penile erection and
yawning are also reduced bymianserin, which
interferes with these behavioural responses by
blocking 5HT2c receptors (Kimura et al., 2008;
Stancampiano et al., 1995), an increase of
central serotonin neurotransmission is also
likely to participate in these effects of
clavulanic acid. Whatever mechanism is
responsible for clavulanic acid-induced penile
erection, the results of this study are in line
with the recently reported facilitatory effect
of this compound on erectile function in
nonhuman primates (Kim et al., 2009) and on
copulatory behaviour in male rats (Chan et al.,
2009).
