*2.4. Spinal PKCζ Is Involved in the Antinociceptive Effect of Crotalphine*

The activation of the κ-opioid receptor increases the phosphorylation of MAPKs (ERK1/2 and JNK) in neuronal and non-neuronal cells [6,9]. In addition, pretreatment of sensory neurons (DRG cells) with a PKCζ pseudosubstrate abolishes crotalphine-mediated ERK1/2 and JNK phosphorylation [8]. Based on this data and on the results showing that spinal ERK1/2 and JNK are involved in crotalphine-induced analgesia, we further investigated whether PKCζ plays a role in crotalphine effects. To examine the PKCζ participation on the mechanical nociceptive threshold, we used the PKCζ pseudosubstrate, which selectively inhibits the atypical PKCζ isozyme [8,10]. The PKCζ pseudosubstrate was injected by an intrathecal route (3 μg) [11] before the crotalphine administration (Figure 5A). As shown in Figure 5B, the analgesia induced by crotalphine was completely abolished by the PKCζ pseudosubstrate.

**Figure 4.** ERK1/2 and JNK inhibitors prevent crotalphine-induced analgesia. (**A**) Schematic representation of the experimental procedure. (**B**) Nociceptive threshold was assessed in the rat paw pressure test before (0) and 3 h after systemic administration of crotalphine (1 μg/kg) with concomitant intrathecal injection of the ERK inhibitor (ERK-I, 30 μg/30 μL), JNK inhibitor (JNK-I, 30 μg/30 μL) or p38 inhibitor (p38-I, 30 μg/30 μL). Data represents mean values ± SEM. \* significantly different from baseline (dotted line), *n* = 6 per group (*p* < 0.05). The observer was blinded to the experimental conditions.

**Figure 5.** PKCζ mediates crotalphine-induced ERK1/2 activation and antinociception. (**A**) Schematic representation of paw pressure test and experimental procedure. (**B**) PKCζ pseudosubstrate (ζΨ substrate; 3 μg/30 μL) was intrathecally injected 15 min before systemic crotalphine administration (1 μg/kg) and nociceptive threshold was assessed before PKCζ pseudosubstrate injection and 1 h after crotalphine treatment. Data represents mean values ± SEM. \*significantly different from baseline (dotted line), *n* = 6 per group (*p* < 0.05). (**C**) Representative blots showing the phosphorylated and total ERK1/2 and JNK levels in the total lysate of the spinal cord. ERK1/2 (**D**) and JNK (**E**) MAPKs were determined by Western blot analysis in lumbar spinal cord extracts from rats treated with crotalphine and the PKCζ pseudosubstrate. Graphs represent the ratio between the phosphorylated protein and the total amount of the targeted protein. Data are presented as mean ± SEM and expressed as % of control (TAT + saline) animals. \* significantly different from mean values of TAT + saline treated animals, *n* = 6 per group (*p* < 0.05). \*\* significantly different from mean values of TAT + saline treated animals, *n* = 6 per group (*p* < 0.01). The observer was blinded to the experimental conditions.

Finally, to examine the direct role that PKCζ plays in the activation of the MAPKs by crotalphine, we used the PKCζ pseudosubstrate and performed the biochemical studies. Our results show that PKCζ inhibition prevents crotalphine-induced ERK1/2 activation (Figure 5D), without interfering with the JNK (Figure 5E). These data suggest that spinal PKCζ activation mediates crotalphine-induced ERK1/2 activation that culminates in analgesia.

#### **3. Discussion**

Crotalphine, a 14-amino acid peptide isolated from *C. d. terrificus* venom, has a potent, long-lasting and KOR-mediated antinociceptive effect [1,8,12,13]. In the present study, we showed that a single systemic administration of crotalphine induces analgesia for 5 h in non-sensitized rats. Moreover, when the peptide is administered in PGE2 sensitized rats, a potent antinociceptive effect is detected for 5 days. Importantly, the peptide did not induce delayed hypersensitivity, which is a decrease in the nociceptive threshold that follows the analgesic effect, a side effect frequently produced by systemic morphine [7,14]. Together, these results confirm the previous finding showing that a peripheral sensitization enhances the antinociceptive effects of opioid-like drugs [8].

Several preclinical studies have shown that opioids activate the MAPK pathway [9,15–17] and this activation is usually associated with cellular stress, inflammation and activation of the sensory neuron that ultimately contribute to nociception [18,19]. However, the use of MAPK inhibitors as analgesics in humans is controversial [20] and the clinical trials involve mainly p38 inhibitors [21,22]. Here, we revealed that spinal ERK1/2 and JNK are activated at the same time period that crotalphine induces analgesia. Importantly, the pharmacological disruption of these kinases is sufficient to blunt the antinociceptive effect. Although several studies have shown that the activation of opioid receptors leads to ERK1/2 and JNK activation [23,24], there are few studies correlating the MAPK pathway with analgesia. Recently, Abraham and co-workers (2018) showed that the ERK1/2 signaling is required by KOR agonists to induce analgesia, since the agonist efficacy was reduced in females with estrogen-induced ERK1/2 impairment [6]. Of interest, one systemic dose of morphine in rats promotes analgesia through spinal ERK activation [7], however, whether this effect is mediated by KOR is unknown. Together, these studies suggest that regardless of its up-stream signaling, the same MAPK can activate different signaling pathways simultaneously (essential and detrimental) and therefore affects the effectiveness of MAPK inhibitor as analgesics.

As mentioned before, crotalphine activates ERK1/2 and JNK in cultured sensory neurons mediated by KOR since the selective opioid receptor antagonist, Nor-BNI, prevents this effect [8]. In the present study, we did not check whether KOR is involved in crotalphine-induced ERK1/2 and JNK activation; however, crotalphine induces the release of dynorphin A that activates KOR in vivo [2].

Unlike what was observed for ERK and JNK, the basal levels of spinal p38 phosphorylation were decreased for at least 96 h after crotalphine administration. As expected, the p38 inhibitor did not interfere with the peptide effect. The phosphorylation of p38 is crucial for the activation of the transcription factors that are involved with the synthesis of pro-inflammatory cytokines and neuromediators, such as calcitonin gene-related peptide (CGRP) and substance P [25–27]. Aiming to investigate the mechanisms involved in the crotalphine long-lasting effect, we tracked the MAPK levels in the spinal cord for 96 h. Our biochemical data confirm the behavioral results showing that crotalphine may have a prolonged inhibitory effect that lasts up to 96 h. However, there is a limitation in our study since the phosphorylation levels in PGE2-sensitized animals was not determined. However, we can speculate from the results obtained with naïve rats that p38 inhibition may contribute to the long-lasting crotalphine-induced analgesia in sensitized rats. Further studies are needed to address this interesting effect.

PKC activation represents an early signaling element in the opioid pathways to ERK and the PKCζ isoform is responsible for mediating KOR-induced ERK phosphorylation [9,15,28]. Additionally, cell culture assays have shown that crotalphine-induced

phosphorylation of ERK1/2 and JNK requires PKCζ activation [8]. Thus, to confirm the contribution of PKCζ in crotalphine-induced antinociception, we injected the PKCζ pseudosubstrate intrathecally, which selectively inhibits the atypical PKCζ isozyme, in non-sensitized rats. Our results demonstrate that PKCζ activation is involved in crotalphine-induced analgesia since the PKCζ pseudosubstrate prevented the antinociception. Moreover, Western blot analyses showed that the PKCζ pseudosubstrate decreased the levels of ERK1/2 phosphorylation, but not of JNK, which was increased in the lumbar spinal cord of rats treated with crotalphine. These results are consistent with the previous data showing that crotalphine activates ERK via PKCζ in cultured sensory neurons [8]. The absence of effect on JNK phosphorylation does not exclude that this pathway is modulated by PKCζ, since in sensitized sensory neuron cultures this kinase is inhibited by crotalphine. Moreover, it is important to mention that, given the short half-life of the PKCζ pseudosubstrate, the crotalphine effects were assessed at 1h after treatment.

Additional studies are required to understand which molecules and/or signaling pathways are being modulated by these kinases. However, it is possible that crotalphine and maybe KOR-agonists use PKCζ/ERK1/2 signaling pathway to regulate transcription factors that are essential for the expression of receptors/channels and/or activation of others signaling pathways, which are involved with antinociception [19,23,29,30]. For example, MAPK phosphorylation activates transcription factors, such as cAMP responsive element binding protein (CREB), which regulates dynorphin gene expression [30,31].

A number of limitations to our study should be noted; for example, we did not investigate whether crotalphine crosses the blood–brain barrier and directly acts on the spinal cord. We also did not demonstrate whether the effect detected in spinal cord tissue is a consequence of a peripheral action, for example, the peptide acting on sensory fiber synapses with the spinal cord neurons. Further studies are necessary to address these questions.

Taken together, our findings show that crotalphine induces analgesia by downregulating p38 signaling pathways and activating ERK and JNK in the spinal cord via PKCζ. These results contribute to the understanding of the molecular mechanisms that are involved in the analgesic effect of drugs with opioid activity and opens a perspective for the PKCζ-MAPK axis as a target for pain control.

#### **4. Conclusions**

In conclusion, our findings demonstrate that a single analgesic dose of crotalphine results in activation of ERK and JNK, and this phenomenon is mediated by PKCζ activation. Targeting the PKCζ-MAPK axis may become an interesting therapeutic alternative to induce analgesia.
