Peripheral sensitization increases opioid receptor expression and activation by Crotalphine in rats

Inflammation enhances the peripheral analgesic efficacy of opioid drugs, but the mechanisms involved in this phenomenon have not been fully elucidated. Crotalphine (CRP), a peptide that was first isolated from South American rattlesnake C.d. terrificus venom, induces a potent and long-lasting anti-nociceptive effect that is mediated by the activation of peripheral opioid receptors. Because the high efficacy of CRP is only observed in the presence of inflammation, we aimed to elucidate the mechanisms involved in the CRP anti-nociceptive effect induced by inflammation. Using real-time RT-PCR, western blot analysis and ELISA assays, we demonstrate that the intraplantar injection of prostaglandin E2 (PGE2) increases the mRNA and protein levels of the µ- and κ-opioid receptors in the dorsal root ganglia (DRG) and paw tissue of rats within 3 h of the injection. Using conformation state-sensitive antibodies that recognize activated opioid receptors, we show that PGE2, alone does not increase the activation of these opioid receptors but that in the presence of PGE2, the activation of specific opioid receptors by CRP and selective µ- and κ-opioid receptor agonists (positive controls) increases. Furthermore, PGE2 down-regulated the expression and activation of the δ-opioid receptor. CRP increased the level of activated mitogen-activated protein kinases in cultured DRG neurons, and this increase was dependent on the activation of protein kinase Cζ. This CRP effect was much more prominent when the cells were pretreated with PGE2. These results indicate that the expression and activation of peripheral opioid receptors by opioid-like drugs can be up- or down-regulated in the presence of an acute injury and that acute tissue injury enhances the efficacy of peripheral opioids.Fundação de Amparo a Pesquisa do Estado de São Paulo, Brazil (FAPESP, grant numbers 07/03404-4, 07/00135-2)Instituto Nacional de Ciencia e Tecnologia em Toxinologia (INCTTOX PROGRAM)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq, grant number 573790/2008-6)FAPESP (grant number 2008/57898-0)CAPES/CNPq, grant numbers 301508/2008-9 and 305345/2011-

Antinociceptive effect of crotalphine in bone pain induced by Walker 256 tumor cells into femoral cavity of rats.

Neste estudo padronizamos modelo de dor óssea induzida pela administração de células do tumor de Walker 256 em fêmur de ratos e avaliamos o efeito analgésico da crotalfina, um peptídeo com atividade analgésica. Análises radiográficas, tomográficas e cintilográficas demonstraram a presença de processos osteolíticos difusos, destruição do tecido cortical ósseo e intensa atividade osteogênica. Foi detectado aumento de partes moles adjacentes ao osso, fenômeno observado em pacientes com câncer ósseo. Análises histopatológicas mostraram a presença de células tumorais no pulmão, baço e fígado dos animais, indicando disseminação das células tumorais. Foi detectada a presença de hiperalgesia, alodinia e dor manifesta, a partir do 1º dia após a inoculação das células, persistindo por pelo menos 14 dias. Crotalfina (8 mg/kg, p.o) administrada no 14º dia, bloqueou estes fenômenos. Esse efeito antinociceptivo é de longa duração (48 h) e mediado por receptores opióides do tipo k e d. Não foi observado desenvolvimento de tolerância após o tratamento prolongado com crotalfina.The aim of the present work was to standardize a new rat model of bone pain induced by the injection of Walker 256 carcinoma cells into the femoral cavity of rats, and to evaluate the analgesic effect of crotalphine, an analgesic peptide. Radiographic, tomographic and scintigraphic analysis showed the presence of diffuse osteolytic processes, destruction of cortical bone tissue and intense osteogenic activity. Increase of soft tissue adjacent to the bone was also detected, being this phenomenon observed in patients with bone cancer. Hystopathological analysis showed the presence of tumor cells in lungs, spleen and liver, indicating the occurrence of metastasis. Tumor cell inoculation induced the presence of hyperalgesia, allodynia and spontaneous pain. Crotalphine (8 mg/kg, p.o.), administered on day 14, blocked these phenomena. This antinociceptive effect is long-lasting effect (2 days) and mediated by peripheral k and d- opioid receptors. Prolonged treatment with crotalphine (14 days) did not cause the development of tolerance to its antinociceptive effect

Structural determinants of the hyperalgesic activity of myotoxic Lys49-phospholipase A2

Abstract Background Bothropstoxin-I (BthTx-I) is a Lys49-phospholipase A2 (Lys49-PLA2) from the venom of Bothrops jararacussu, which despite of the lack of catalytic activity induces myotoxicity, inflammation and pain. The C-terminal region of the Lys49-PLA2s is important for these effects; however, the amino acid residues that determine hyperalgesia and edema are unknown. The aim of this study was to characterize the structural determinants for the Lys49-PLA2-induced nociception and inflammation. Methods Scanning alanine mutagenesis in the active-site and C-terminal regions of BthTx-I has been used to study the structural determinants of toxin activities. The R118A mutant was employed as this substitution decreases PLA2 myotoxicity. In addition, K115A and K116A mutants – which contribute to decrease cytotoxicity – and the K122A mutant – which decreases both myotoxicity and cytotoxicity – were also used. The H48Q mutant – which does not interfere with membrane damage or myotoxic activity – was used to evaluate if the PLA2 catalytic site is relevant for the non-catalytic PLA2-induced pain and inflammation. Wistar male rats received intraplantar injections with mutant PLA2. Subsequently, hyperalgesia and edema were evaluated by the paw pressure test and by a plethysmometer. Native and recombinant BthTx-I were used as controls. Results Native and recombinant BthTx-I induced hyperalgesia and edema, which peaked at 2 h. The R118A mutant did not induce nociception or edema. The mutations K115A and K116A abolished hyperalgesia without interfering with edema. Finally, the K122A mutant did not induce hyperalgesia and presented a decreased inflammatory response. Conclusions The results obtained with the BthTx-I mutants suggest, for the first time, that there are distinct residues responsible for the hyperalgesia and edema induced by BthTx-I. In addition, we also showed that cytolytic activity is essential for the hyperalgesic effect but not for edematogenic activity, corroborating previous data showing that edema and hyperalgesia can occur in a non-dependent manner. Understanding the structure-activity relationship in BthTx-I has opened new possibilities to discover the target for PLA2-induced pain

Peripheral kappa and delta opioid receptors are involved in the antinociceptive effect of crotalphine in a rat model of cancer pain

Cancer pain is an important clinical problem and may not respond satisfactorily to the current analgesic therapy. We have characterized a novel and potent analgesic peptide, crotalphine, from the venom of the South American rattlesnake Crotalus durissus terrificus. In the present work, the antinociceptive effect of crotalphine was evaluated in a rat model of cancer pain induced by intraplantar injection of Walker 256 carcinoma cells. Intraplantar injection of tumor cells caused the development of hyperalgesia and allodynia, detected on day 5 after tumor cell inoculation. Crotalphine (6 μg/kg), administered p.o., blocked both phenomena. The antinociceptive effect was detected 1 h after treatment and lasted for up to 48 h. Intraplantar injection of nor-binaltorphimine (50 g/paw), a selective antagonist of κ-opioid receptors, antagonized the antinociceptive effect of the peptide, whereas N,N-diallyl-Tyr-Aib-Phe-Leu (ICI 174,864, 10 μg/paw), a selective antagonist of δ-opioid receptors, partially reversed this effect. On the other hand, D-Phe-Cys-Tyr-D-Trp-Orn-Thr-Pen-Thr amide (CTOP, 20 g/paw), an antagonist of μ-opioid receptors, did not modify crotalphine-induced antinociception. These data indicate that crotalphine induces a potent and long lasting opioid-mediated antinociception in cancer pain. © 2013 Elsevier Inc

Peripheral 5-HT3 Receptors Are Involved in the Antinociceptive Effect of Bunodosine 391

Bunodosine 391 (BDS 391), a low molecular weight compound isolated from the sea anemone Bunodosoma cangicum, increases the nociceptive threshold and inhibits inflammatory hyperalgesia. Serotonin receptors are involved in those effects. In this study, we have expanded the characterization of the antinociceptive effect of BDS 391 demonstrating that, in rats: (a) the compound inhibits (1.2–12 ng/paw) overt pain, in the formalin test, and mechanical hyperalgesia (0.6–6.0 ng/paw) detected in a model of neuropathic pain; (b) intraplantar administration of ondansetron, a selective 5-HT3 receptor antagonist, blocks the effect of BDS 391, whereas ketanserin, a 5-HT2 receptor antagonist, partially reversed this effect, indicating the involvement of peripheral 5-HT2 and 5-HT3 receptors in BDS 391 antinociception; and (c) in binding assay studies, BDS 391 was not able to displace the selective 5-HT receptor antagonists, suggesting that this compound does not directly bind to these receptors. The effect of biguanide, a selective 5-HT3 receptor agonist, was also evaluated. The agonist inhibited the formalin’s nociceptive response, supporting an antinociceptive role for 5-HT3 receptors. Our study is the first one to show that a non-peptidic low molecular weight compound obtained from a sea anemone is able to induce antinociception and that activation of peripheral 5-HT3 receptors contributes to this effect

Effect of crotalphine on cyclic GMP levels in primary culture of DRG cells.

<p>Cells from DRG were obtained from naïve rats and incubated at 37°C in a 5% of CO₂, for two days. On the third day, the cells were incubated with Nor-BNI (1 µM) for 30 min and PGE₂ (1 µM) or vehicle (saline - Sal) for 15 min, and further incubated with 1 µM of crotalphine (CRP), U50,488, or saline, for 15 min. Cells were lysed and subjected to enzyme immunoassay.*Significantly different from mean values of control cells (p<0.05), # Significantly different from mean values of CRP alone (p<0.05), Significantly different from mean values of U 50,488 alone (p<0.05). cGMP levels were analysed by one-way analysis of variance with a post-hoc Tukey test. Data were analyzed by one-way analysis of variance (ANOVA) with <i>post-hoc</i> testing by Tukey.</p

PGE₂ improves agonist-mediated opioid receptor activation.

<p>Naïve or prostaglandin E2 (PGE₂, 100 ng/paw) injected rats were treated with DAMGO (5 µg/paw), U50,488 (10 µg/paw), DPDPE (20 µg/paw), (Panel A) or crotalphine (CRP, 0.6 ng/paw) (Panel B). The agonists, CRP, or vehicle (control) were administered 2 h after PGE2 injection. ELISA assay was performed in slices of plantar tissue, using conformation state-sensitive antibodies (μ, κ, δ activated). Tissues were collected 3 h after PGE₂ injection. *Significantly different from mean values of control animals (dotted line), n = 6 animals per group (p<0.05). #Significantly different from mean values of CRP or agonist alone (p<0.05). Data were analyzed by one-way analysis of variance (ANOVA) with <i>post-hoc</i> testing by Tukey.</p

Intraplantar injection effect of prostaglandin E₂ (PGE₂) on protein expression of μ (A), κ (B) and δ (C) opioid receptors.

<p>The changes in protein levels of opioid receptors were determined by immunobloting, in dorsal root ganglia (DRG) and plantar tissue obtained 3 h after PGE₂ injection (100 ng/paw). Data are presented as mean ± SEM and expressed as % of control (naïve) animals. *Significantly different from mean values of naïve animals, n = 6 per group (p<0.05). Data were analyzed by one-way analysis of variance (ANOVA) with <i>post-hoc</i> testing by Tukey.</p

Effect of crotalphine (CRP) on AKT phosphorylation in primary culture of DRG cells.

<p>Cells from DRG were obtained from naïve rats and incubated at 37°C in a 5% of CO₂, for two days. On the third day, the cells were incubated with PGE₂ (1 µM) or vehicle (saline - Sal) for 15 min. and further incubated with 1 µM of CRP, U50,488, or saline, for 15 min. Cells were scrapped and homogenized, and the total homogenate was subjected to western blotting. (A) The ratio of phospho-protein to total protein is expressed as percentage from control (Sal+Sal). Three separate experiments were carried out on different occasions.*Significantly different from mean values of control cells (p<0.05). AKT levels were analysed by one-way analysis of variance with a <i>post-hoc</i> Tukey test. (B) Representative blots showing the levels AKT in the total lisate of DRGs.</p