RESUMO
BACKGROUND AND PURPOSE: Peptides from venomous animals have long been important for understanding pain mechanisms and for the discovery of pain treatments. Here, we hypothesized that Phα1ß, a peptide from the venom of the armed spider Phoneutria nigriventer, produces analgesia by blocking the TRPA1 channel. EXPERIMENTAL APPROACH: Cultured rat dorsal root ganglion (DRG) neurons, human fetal lung fibroblasts (IMR90) or HEK293 cells expressing the human TRPA1 (hTRPA1-HEK293), human TRPV1 (hTRPV1-HEK293) or human TRPV4 channels (hTRPV4-HEK293), were used for calcium imaging and electrophysiology. Nociceptive responses induced by TRPA1, TRPV1 or TRPV4 agonists or by bortezomib were investigated in mice. KEY RESULTS: Phα1ß selectively inhibited calcium responses and currents evoked by the TRPA1 agonist, allyl isothiocyanate (AITC), on hTRPA1-HEK293, IMR90 fibroblasts and DRG neurons. Phα1ß did not affect calcium responses evoked by selective TRPV1 (capsaicin) or TRPV4 (GSK 1016790A) agonists on the various cell types. Intrathecal (i.t.) and intraplantar (i.pl.) administration of low doses of Phα1ß (up to 300 pmol per paw) attenuated acute nociception and mechanical and cold hyperalgesia evoked by AITC (i.t. or i.pl.), without affecting responses produced by capsaicin or hypotonic solution. Notably, Phα1ß abated the TRPA1-dependent neuropathic pain-like responses induced by bortezomib. In vitro and in vivo inhibition of TRPA1 by Phα1ß was reproduced by a recombinant form of the peptide, CTK 01512-2. CONCLUSIONS AND IMPLICATIONS: Phα1ß and CTK 01512-2 selectively target TRPA1, but not other TRP channels. This specific action underlines the potential of Phα1ß and CTK 01512-2 for pain treatment.
Assuntos
Analgésicos/farmacologia , Proteínas do Tecido Nervoso/antagonistas & inibidores , Nociceptividade/efeitos dos fármacos , Venenos de Aranha/química , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Analgésicos/química , Animais , Canais de Cálcio/metabolismo , Células Cultivadas , Relação Dose-Resposta a Droga , Fibroblastos/efeitos dos fármacos , Gânglios Espinais/efeitos dos fármacos , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Proteínas do Tecido Nervoso/metabolismo , Neuralgia/tratamento farmacológico , Neurônios/efeitos dos fármacos , Ratos , Venenos de Aranha/farmacologia , Aranhas , Relação Estrutura-Atividade , Canal de Cátion TRPA1 , Canais de Potencial de Receptor Transitório/metabolismoRESUMO
Despite intense investigation, the mechanisms of the different forms of trigeminal neuropathic pain remain substantially unidentified. The transient receptor potential ankyrin 1 channel (encoded by TRPA1) has been reported to contribute to allodynia or hyperalgesia in some neuropathic pain models, including those produced by sciatic nerve constriction. However, the role of TRPA1 and the processes that cause trigeminal pain-like behaviours from nerve insult are poorly understood. The role of TRPA1, monocytes and macrophages, and oxidative stress in pain-like behaviour evoked by the constriction of the infraorbital nerve in mice were explored. C57BL/6 and wild-type (Trpa1(+/+)) mice that underwent constriction of the infraorbital nerve exhibited prolonged (20 days) non-evoked nociceptive behaviour and mechanical, cold and chemical hypersensitivity in comparison to sham-operated mice (P < 0.05-P < 0.001). Both genetic deletion of Trpa1 (Trpa1(-/-)) and pharmacological blockade (HC-030031 and A-967079) abrogated pain-like behaviours (both P < 0.001), which were abated by the antioxidant, α-lipoic acid, and the nicotinamide adenine dinucleotide phosphate oxidase inhibitor, apocynin (both P < 0.001). Nociception and hypersensitivity evoked by constriction of the infraorbital nerve was associated with intra- and perineural monocytic and macrophagic invasion and increased levels of oxidative stress by-products (hydrogen peroxide and 4-hydroxynonenal). Attenuation of monocyte/macrophage increase by systemic treatment with an antibody against the monocyte chemoattractant chemokine (C-C motif) ligand 2 (CCL2) or the macrophage-depleting agent, clodronate (both P < 0.05), was associated with reduced hydrogen peroxide and 4-hydroxynonenal perineural levels and pain-like behaviours (all P < 0.01), which were abated by perineural administration of HC-030031, α-lipoic acid or the anti-CCL2 antibody (all P < 0.001). The present findings propose that, in the constriction of the infraorbital nerve model of trigeminal neuropathic pain, pain-like behaviours are entirely mediated by the TRPA1 channel, targeted by increased oxidative stress by-products released from monocytes and macrophages clumping at the site of nerve injury.
Assuntos
Hiperalgesia/fisiopatologia , Macrófagos/efeitos dos fármacos , Monócitos/efeitos dos fármacos , Neuralgia/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Canais de Potencial de Receptor Transitório/fisiologia , Acetanilidas/antagonistas & inibidores , Acetanilidas/farmacologia , Acetofenonas/farmacologia , Animais , Quimiocina CCL2/antagonistas & inibidores , Ácido Clodrônico/farmacologia , Hiperalgesia/metabolismo , Macrófagos/metabolismo , Masculino , Camundongos , Camundongos Knockout , Monócitos/metabolismo , Neuralgia/metabolismo , Oximas/antagonistas & inibidores , Oximas/farmacologia , Purinas/antagonistas & inibidores , Purinas/farmacologia , Canal de Cátion TRPA1 , Ácido Tióctico/farmacologia , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Canais de Potencial de Receptor Transitório/genéticaRESUMO
Acute gout attacks produce severe joint pain and inflammation associated with monosodium urate (MSU) crystals leading to oxidative stress production. The transient potential receptor ankyrin 1 (TRPA1) is expressed by a subpopulation of peptidergic nociceptors and, via its activation by endogenous reactive oxygen species, including hydrogen peroxide (H2O2), contributes to pain and neurogenic inflammation. The aim of this study was to investigate the role of TRPA1 in hyperalgesia and inflammation in a model of acute gout attack in rodents. Inflammatory parameters and mechanical hyperalgesia were measured in male Wistar rats and in wild-type (Trpa1(+/+)) or TRPA1-deficient (Trpa1(-/-)) male mice. Animals received intra-articular (ia, ankle) injection of MSU. The role of TRPA1 was assessed by receptor antagonism, gene deletion or expression, sensory fiber defunctionalization, and calcitonin gene-related peptide (CGRP) release. We found that nociceptor defunctionalization, TRPA1 antagonist treatment (via ia or oral administration), and Trpa1 gene ablation abated hyperalgesia and inflammatory responses (edema, H2O2 generation, interleukin-1ß release, and neutrophil infiltration) induced by ia MSU injection. In addition, we showed that MSU evoked generation of H2O2 in synovial tissue, which stimulated TRPA1 producing CGRP release and plasma protein extravasation. The MSU-elicited responses were also reduced by the H2O2-detoxifying enzyme catalase and the reducing agent dithiothreitol. TRPA1 activation by MSU challenge-generated H2O2 mediates the entire inflammatory response in an acute gout attack rodent model, thus strengthening the role of the TRPA1 receptor and H2O2 production as potential targets for treatment of acute gout attacks.
Assuntos
Gota/metabolismo , Peróxido de Hidrogênio/metabolismo , Hiperalgesia/metabolismo , Inflamação/metabolismo , Canais de Cátion TRPC/metabolismo , Animais , Antioxidantes/farmacologia , Western Blotting , Modelos Animais de Doenças , Ensaio de Imunoadsorção Enzimática , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Knockout , Ratos , Ratos Wistar , Reação em Cadeia da Polimerase em Tempo RealRESUMO
OBJECTIVE: Gout is a common cause of inflammatory arthritis and is provoked by the accumulation of monosodium urate (MSU) crystals. However, the underlying mechanisms of the pain associated with acute attacks of gout are poorly understood. The aim of this study was to evaluate the role of transient receptor potential ankyrin 1 (TRPA-1) and TRPA-1 stimulants, such as H2 O2 , in a rodent model of MSU-induced inflammation. METHODS: MSU or H2 O2 was injected into the hind paws of rodents or applied in cultured sensory neurons, and the intracellular calcium response was measured in vitro. Inflammatory or nociceptive responses in vivo were evaluated using pharmacologic, genetic, or biochemical tools and methods. RESULTS: TRPA-1 antagonism, TRPA-1 gene deletion, or pretreatment of peptidergic TRP-expressing primary sensory neurons with capsaicin markedly decreased MSU-induced nociception and edema. In addition to these neurogenic effects, MSU increased H2 O2 levels in the injected tissue, an effect that was abolished by the H2 O2 -detoxifying enzyme catalase. H2 O2 , but not MSU, directly stimulated sensory neurons through the activation of TRPA-1. The nociceptive responses evoked by MSU or H2 O2 injection were attenuated by the reducing agent dithiothreitol. In addition, MSU injection increased the expression of TRPA-1 and TRP vanilloid channel 1 (TRPV-1) and also enhanced cellular infiltration and interleukin-1ß levels, and these effects were blocked by TRPA-1 antagonism. CONCLUSION: Our results suggest that MSU injection increases tissue H2 O2 , thereby stimulating TRPA-1 on sensory nerve endings to produce inflammation and nociception. TRPV-1, by a previously unknown mechanism, also contributes to these responses.
Assuntos
Dor Aguda/metabolismo , Artrite Gotosa/metabolismo , Peróxido de Hidrogênio/metabolismo , Inflamação/metabolismo , Canais de Cátion TRPC/metabolismo , Ácido Úrico/metabolismo , Acetanilidas/farmacologia , Dor Aguda/induzido quimicamente , Dor Aguda/tratamento farmacológico , Animais , Artrite Gotosa/induzido quimicamente , Artrite Gotosa/tratamento farmacológico , Modelos Animais de Doenças , Peróxido de Hidrogênio/farmacologia , Inflamação/induzido quimicamente , Inflamação/tratamento farmacológico , Masculino , Camundongos , Camundongos Knockout , Oxidantes/metabolismo , Oxidantes/farmacologia , Purinas/farmacologia , Ratos , Ratos Wistar , Células Receptoras Sensoriais/metabolismo , Canal de Cátion TRPA1 , Canais de Cátion TRPC/agonistas , Canais de Cátion TRPC/antagonistas & inibidores , Ácido Úrico/farmacologiaRESUMO
Chemotherapy-induced peripheral neuropathy (CIPN) is a severe and painful adverse reaction of cancer treatment in patients that is little understood or treated. Cytotoxic drugs that cause CIPN exert their effects by increasing oxidative stress, which activates the ion channel TRPA1 expressed by nociceptors. In this study, we evaluated whether TRPA1 acted as a critical mediator of CIPN by bortezomib or oxaliplatin in a mouse model system. Bortezomib evoked a prolonged mechanical, cold, and selective chemical hypersensitivity (the latter against the TRPA1 agonist allyl isothiocyanate). This CIPN hypersensitivity phenotype that was stably established by bortezomib could be transiently reverted by systemic or local treatment with the TRPA1 antagonist HC-030031. A similar effect was produced by the oxidative stress scavenger α-lipoic acid. Notably, the CIPN phenotype was abolished completely in mice that were genetically deficient in TRPA1, highlighting its essential role. Administration of bortezomib or oxaliplatin, which also elicits TRPA1-dependent hypersensitivity, produced a rapid, transient increase in plasma of carboxy-methyl-lysine, a by-product of oxidative stress. Short-term systemic treatment with either HC-030031 or α-lipoic acid could completely prevent hypersensitivity if administered before the cytotoxic drug. Our findings highlight a key role for early activation/sensitization of TRPA1 by oxidative stress by-products in producing CIPN. Furthermore, they suggest prevention strategies for CIPN in patients through the use of early, short-term treatments with TRPA1 antagonists.