*Article* **PKCζ-Mitogen-Activated Protein Kinase Signaling Mediates Crotalphine-Induced Antinociception**

**Bárbara G. de Freitas † , Natália G. Hösch † , Leandro M. Pereira, Tereza C. Barbosa, Gisele Picolo, Yara Cury and Vanessa O. Zambelli \***

> Laboratory of Pain and Signaling, Butantan Institute, São Paulo 05503-900, Brazil; bguimaf@gmail.com (B.G.d.F.); natalia.hosh@esib.butantan.gov.br (N.G.H.); le.marciopereira@yahoo.com.br (L.M.P.); tereza.barbosa@butantan.gov.br (T.C.B.); gisele.picolo@butantan.gov.br (G.P.); yara.cury@esib.butantan.gov.br (Y.C.)

**\*** Correspondence: vanessa.zambelli@butantan.gov.br; Tel.: +55-11-2627-9765

† These authors have contributed equally to this work.

**Abstract:** Crotalphine (CRP) is a structural analogue to a peptide that was first identified in the crude venom from the South American rattlesnake *Crotalus durissus terrificus*. This peptide induces a potent and long-lasting antinociceptive effect that is mediated by the activation of peripheral opioid receptors. The opioid receptor activation regulates a variety of intracellular signaling, including the mitogen-activated protein kinase (MAPK) pathway. Using primary cultures of sensory neurons, it was demonstrated that crotalphine increases the level of activated ERK1/2 and JNK-MAPKs and this increase is dependent on the activation of protein kinase Cζ (PKCζ). However, whether PKCζ-MAPK signaling is critical for crotalphine-induced antinociception is unknown. Here, we biochemically demonstrated that the systemic crotalphine activates ERK1/2 and JNK and decreases the phosphorylation of p38 in the lumbar spinal cord. The in vivo pharmacological inhibition of spinal ERK1/2 and JNK, but not of p38, blocks the antinociceptive effect of crotalphine. Of interest, the administration of a PKCζ pseudosubstrate (PKCζ inhibitor) prevents crotalphine-induced ERK activation in the spinal cord, followed by the abolishment of crotalphine-induced analgesia. Together, our results demonstrate that the PKCζ-ERK signaling pathway is involved in crotalphine-induced analgesia. Our study opens a perspective for the PKCζ-MAPK axis as a target for pain control.

**Keywords:** analgesic peptide; protein kinase C; hyperalgesia; cell-signaling

**Key Contribution:** Crotalphine-induced analgesia is mediated by down-regulation of p38-MAPK and up-regulation of PKCζ-MAPK signaling pathway.
