The L-arginine-nitric oxide-cGMP pathway is involved in the peripheral anti-nociceptive effect of CRP and opioid agonists. The activation of opioid receptors regulates a variety of intracellular signaling cascades, including the PI3Kc/AKT signaling pathway, AMD-070 as well as the activation of mitogenactivated protein kinases and protein kinase C. The high efficacy and long-lasting peripheral anti-nociceptive effects of crotalphine have only been observed in the presence of inflammation, and the mechanisms involved in the enhanced effects of CRP in the presence of inflammation are currently unknown. In the present study, we used prostaglandin E2 induced hyperalgesia in a rat model to characterize the antinociceptive effect caused by the peripheral administration of CRP and compared this response with the responses induced by select opioid receptor agonists. Furthermore, we also investigated the intracellular signaling pathways activated by CRP and the opioid receptor agonists. Previously, we demonstrated that the anti-nociceptive effect of CRP administered orally in this hyperalgesia model is mediated by the activation of the peripheral k-opioid receptor. To determine whether opioid receptors are involved in the local anti-nociceptive effect of this peptide, rats were administered selective antagonists of the opioid receptors by i.pl. injection. The anti-nociceptive effect of CRP was abolished in the paw injected with nor-BNI, an antagonist of the k-opioid receptors, but not by CTOP or ICI 174,864, m- and d-opioid receptor antagonists, respectively. These results indicate that the peripheral anti-nociceptive effect of CRP is mediated by the activation of the peripheral k-opioid receptor. It has been well established that GPCRs undergo conformational changes in the N-terminus following receptor activation. To determine whether PGE2 activates opioid receptors, we performed an ELISA assay using conformation state-sensitive antibodies that recognize activated opioid receptors according to the methods published Trastuzumab. To determine the total expression level of the opioid receptors, the same antibodies used for the immunoblot assays were also used in these assays. Consistent with the immunoblot assay results, the intraplantar injection of PGE2 increased the expression levels of the m- and kopioid receptors in the plantar tissue and the DRG and decreased the expression level of the d opioid receptor in the plantar tissue. No changes in opioid receptor activation, however, were detected using the conformation state-sensitive antibodies compared with the levels observed in the tissues from naı ¨ve animals. This result indicates that PGE2 does not activate opioid receptors. Because PGE2 altered the expression profiles of the opioid receptors without altering their activation, we investigated whether CRP or opioid receptor agonists increase the activation of the opioid receptors in PGE2-sensitized plantar tissue. We previously reported that CRP, a 14-aminoacid peptide isolated from C. d. terrificus venom, has a potent, long-lasting and peripheral opioid receptor-mediated anti-nociceptive effect. In this study, we demonstrated that the potency and longlasting anti-nociceptive effect of CRP depends on tissue inflammation.