The venom of Phoneutria nigriventer
has been the focus of intensive research in recent years since it is of interest for discovering novel pharmaceutical bioactive peptides. This venom has a potent neurotoxic effect and many of its toxins have been already isolated and studied in detail [1
]. One of the best characterized toxins, δ-CNTX-Pn1c, also known as PnTx2-6 [2
], exerts interesting pharmacological effects and it was originally studied as a modulator of voltage-gated sodium channels [1
]. Recently, this toxin has been studied as a potentiator of erectile function. δ-CNTX-Pn1c improves erectile function of normotensive and DOCA-salt hypertensive rats [3
] and it also ameliorates the erectile function of rats with bilateral cavernous nerve crush injury [4
The synthetic peptide PnPP-19 comprehends 19 amino acid residues and it represents part of the primary structure of the spider toxin δ-CNTX-Pn1c. This peptide has been suggested to be a promising drug candidate for the treatment of both erectile dysfunction and pain. Through histopathological experiments [5
], it was shown that PnPP-19 does not induce any sign of toxicity in different tissues (brain, heart, lung, liver and kidney) and it no longer modulates Nav channels [5
]. Furthermore, it does not cause death or hypersensitivity reactions and it induces only low immunogenicity in mice. However, similar to the native toxin δ-CNTX-Pn1c, PnPP-19 does potentiate erectile function. The exact molecular target through which PnPP-19 improves erectile function still awaits elucidation [5
]. Regarding the pain pathway, PnPP-19 induces both peripheral and central antinociception. This antinociceptive effect elicited by the peptide seems to involve the activation of opioid and cannabinoid receptors along with the activation of the NO/cGMP/KATP
Millions of people suffer from acute or chronic pain every year, which makes pain a serious global public health problem. Chronic pain, for instance, may cause an enormous socioeconomic impact with associated costs in treatment and reduced levels of productivity [9
]. Nowadays, there is an urge for the development of novel potent and more selective analgesic drugs that elicit less undesirable side effects [10
The opioid receptors belong to the superfamily of GPCRs and they are coupled to Gi
proteins. The activation of these receptors may contribute to cellular hyperpolarization, and might impair neurotransmitters release, by suppressing calcium influx and stimulating potassium channels. The activation of the three different opioid receptor subtypes (μ-, δ- and κ-) might inhibit different calcium channels in various mammalian tissues [11
]. Therefore, measuring calcium currents could be a complementary way for verifying opioid activation. In addition, the direct inhibition of calcium channels by exogenous substances may also induce per se antinociception. This is the case, for example, of two antinociceptive P. nigriventer
toxins, PnTx3-3 and PnTx3-6 [12
], and the well-known Food and Drug Administration (FDA) approved analgesic drug Prialt®
]. Regarding the potassium channels, it has been shown that opioid receptor activation leads to opening of different potassium channels, among which are inward rectifying potassium channels (GIRK) [11
]. As such, measuring the alteration of potassium flux through the cell membrane might also be an alternative way of investigating opioid receptor activation.
Since the peripheral and central antinociception induced by PnPP-19 involves opioid activation [6
], the aim of this work was to identify whether this synthetic peptide could directly activate opioid receptors and investigate the possible subtype selectivity for μ-, δ- and/or κ-opioid receptors co-expressed with GIRK1/GIRK2 and RGS4. Furthermore, we also studied the modulation of calcium influx driven by PnPP-19 in dorsal root ganglion (DRG) neurons, and analyzed whether this modulation was opioid-mediated. Our data show that PnPP-19 may selectively activate μ-opioid receptors, however with low potency. Interestingly, activation of opioid receptors induced by the PnPP-19 does not stimulate the recruitment of β-arrestin2. However, it does induce indirectly inhibition of calcium channels and, consequently, impairs calcium influx in DRG neurons.
Previous literature data, obtained using behavioral tests, suggested that the peripheral and central antinociceptive effect induced by PnPP-19 is partially because of μ- and δ-opioid receptors activation [6
]. Therefore, we verified whether this synthetic peptide could indeed directly bind and activate the different isoforms of opioid receptors (μ-, δ- and κ-). Moreover, since it was already described that activation of opioid receptors suppresses calcium influx through inhibition of different voltage-gated calcium channels, we also investigated the modulation of calcium influx induced by PnPP-19 in DRG neurons. Our data demonstrates that, among the μ-, δ- and κ-opioid receptor subtypes, PnPP-19 may selectively activate, with relatively low potency, the μ-opioid receptor subtype. Remarkably, it seems that the peptide does not induce the recruitment of β-arrestin2 by activating opioid receptors and, most likely, PnPP-19 binds to a different binding site of the opioid receptor than DAMGO (a selective opioid agonist). In addition, PnPP-19 induced an inhibition of calcium channels, very likely through activation of opioid receptors, in the whole-cell patch-clamp assay; it also diminished the calcium influx observed by fluorescence microscopy.
Among all the toxins isolated from the venom of Phoneutria nigriventer
and its derivatives, only two of them are able to elicit antinociception via activation of opioid receptors. The antinociceptive effect of the toxin PnTx4(6-1), also known as δ-Ctenitoxin-Pn1a [2
], is partially blocked when selective antagonists of both μ- and δ-opioid receptors are administered [15
]. Likewise, antinociception of PnPP-19, a δ-CNTX-Pn1c derivative [5
], occurs also through activation of those very same opioid receptors [6
]. On the other hand, it was never investigated whether these peptides may directly bind and activate opioid receptors. Our present data show that PnPP-19 might selectively activate, with low potency, only the μ-opioid receptor subtype. However, through behavioral experiments, it was shown that the antinociception induced by PnPP-19 also involves activation of δ-opioid receptors. Here, we demonstrated that PnPP-19 is incapable of activating directly δ-opioid receptors. Therefore, activation of this specific subtype of receptor in vivo may occur via an indirectly pathway, as previously suggested by Freitas and collaborators [6
PnPP-19 is the first synthetic peptide, derived from a spider toxin, proven to act directly on opioid receptors, and more specifically, on μ-opioid receptor subtype. Novel ligands of the μ-opioid receptor are of clinical and social importance since the common used analgesic drugs, such as morphine, fentanyl and oxymorphone, elicit both their beneficial pharmacological effect and undesirable side effects through activation of opioid receptors [16
]. One of the very serious and life-threatening conditions developed following the use of the usual opioid agonist medicines is respiratory paralysis [16
]. It has been demonstrated that the induction of respiratory paralysis, as well as other side effects, after the use of opioids may be linked with the recruitment of the β-arrestin pathway, which is stimulated downstream following activation of μ-opioid receptor [18
]. Since opioid receptors are still one of the most relevant targets for pain treatment, great effort is being put in the development of new opioid agonists that elicit fewer negative side effects [22
]. In this way, the lack of β-arrestin2 recruitment by PnPP-19 underlines the potential of this peptide as a possible lead compound in the development of improved opioid agonists. Recently, a very selective and potent μ-opioid agonist was developed and named PZM21. Despite its great potency and selectiveness against μ-opioid receptors, administration of PZM21 induced minimal β-arrestin2 recruitment. Therefore, the use of PZM21 induced a long-lasting analgesia along with decreased respiratory depression and constipation when compared to morphine [10
]. For this reason, studies concerning the exact mechanism of action of PnPP-19 in the pain pathway are of interest since PnPP-19 showed no induction of β-arrestin2 recruitment in cell culture. It thus seems that the peptide has a site of interaction different from the DAMGO binding site, since in our experiments the presence of PnPP-19 has no influence on DAMGO-induced β-arrestin2 recruitment. However, one other hypothesis for the lack of β-arrestin2 recruitment by PnPP-19 could be the low potency of which PnPP-19 might bind to μ-opioid receptors. Therefore, further investigation is required in order to elucidate the exact mechanism of PnPP-19 interaction with the opioid receptors. Moreover, a better characterization of the target of this synthetic peptide in erectile function is required in order to develop a PnPP-19 derived drug without unwanted side effects.
The interaction between opioid receptors and ion channels has been a subject of much interest during decades. Various studies suggest that activation of opioid receptors causes hyperpolarization of the cell and consequently prevents neurotransmitter release by inducing an inhibition of calcium channels [11
] and activation potassium channels [11
]. According to in vitro studies, incubation of a selective agonist of μ-opioid receptors with HEK293 cells co-expressing μ-opioid receptors together with voltage-gated N-type calcium channels (Cav2.2) or R-type (Cav2.3) channels induced an inhibition of both calcium channels tested [29
]. Moreover, experiments, conducted with primary culture of vestibular afferent neurons and DRG neurons, suggest that selective stimulation of μ-opioid receptors may inhibit T-, L- and N-type calcium channels supposedly through activation of a Gαi/o
]. Accordingly, our results demonstrate that PnPP-19 inhibits calcium influx in DRG neurons and that this inhibition is suppressed by the unspecific opioid antagonist naloxone. These data show that inhibition of calcium influx induced by PnPP-19 is mediated by activation of opioid receptors. Recently, it was demonstrated that DAMGO (selective μ-opioid agonist) induces inhibition of calcium influx and action potential-evoked Ca2+
fluorescent transients in individual peripheral nociceptive fiber free nerve endings from trigeminal ganglion. The authors have shown that activation of “big conductance” Ca2+
) mediates this inhibition of calcium influx induced by DAMGO. Furthermore, the activation of this subtype of potassium channel plays a major role on μ-opioid induced antinociception in a behavioral test for trigeminal nociception. Therefore, it is likely that PnPP-19 might also modulate potassium channels, since this synthetic peptide may act as an opioid agonist. However, further experiments are needed to investigate whether PnPP-19 indeed interferes with potassium channel activity.
In conclusion, the data we present here shows for the first time a spider toxin derivative that may act as a selective μ-opioid agonist. PnPP-19 directly binds and activates, albeit with low potency, only the μ-opioid receptor subtype. In DRG neurons, the activation of μ-opioid receptors induced by PnPP-19 generates an inhibition of calcium channels, consequently reducing or even eliminating calcium influx. This modulation of calcium channels appears to follow activation of opioid receptors, confirming once again the role of PnPP-19 as an opioid agonist. Interestingly, notwithstanding the activation of opioid receptors, PnPP-19 does not induce β-arrestin2 recruitment. This could be due its low potency; however, it may also be a consequence of a differential opioid activation mechanism in which β-arrestin2 recruitment is not stimulated. Further studies with PnPP-19 could lead to the development of new and more potent opioid agonists that in turn could elicit antinociception with possibly less side effects by not inducing recruitment of β-arrestin2.