RESUMO
Exposure to intense sound can cause damage to the delicate sensory and neuronal components of the cochlea leading to hearing loss. Such damage often causes the dendrites of the spiral ganglion neurons (SGN), the neurons that provide the afferent innervation of the hair cells, to swell and degenerate thus damaging the synapse. In models of neuropathic pain, axotomy, another form of afferent nerve damage, is accompanied by altered voltage-gated sodium channel (VGSC) expression, leading to neuronal hyperactivity. In this study, adult Wistar rats were exposed to noise which produced a mild, 20 dB hearing threshold elevation and their VGSC expression was investigated. Quantitative PCR showed decreased Na(V)1.1 and Na(V)1.6 mRNA expression in the SGN following noise exposure (29% and 56% decrease respectively) while Na(V)1.7 mRNA expression increased by approximately 20% when compared to control rats. Immunohistochemistry extended these findings, revealing increased staining for Na(V)1.1 along the SGN dendrites and Na(V)1.7 in the cell bodies after noise. These results provide the first evidence for selective changes in VGSC expression following moderate noise-induced hearing loss and could contribute to elevated hearing thresholds and to the generation of perceptual anomalies commonly associated with cochlear damage, such as tinnitus and hyperacusis.
Assuntos
Células Ciliadas Auditivas/fisiologia , Perda Auditiva Provocada por Ruído/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Plasticidade Neuronal/fisiologia , Neurônios Aferentes/fisiologia , Canais de Sódio/metabolismo , Animais , Limiar Auditivo/fisiologia , Cóclea/citologia , Cóclea/patologia , Cóclea/fisiologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Células Ciliadas Auditivas/citologia , Perda Auditiva Provocada por Ruído/patologia , Humanos , Masculino , Canal de Sódio Disparado por Voltagem NAV1.1 , Canal de Sódio Disparado por Voltagem NAV1.6 , Canal de Sódio Disparado por Voltagem NAV1.7 , Proteínas do Tecido Nervoso/genética , Neurônios Aferentes/citologia , Ratos , Ratos Wistar , Canais de Sódio/genética , Gânglio Espiral da Cóclea/citologiaRESUMO
We used a mouse with deletion of exons 4, 5, and 6 of the SCN11A (sodium channel, voltage-gated, type XI, alpha) gene that encodes the voltage-gated sodium channel Na(v)1.9 to assess its contribution to pain. Na(v)1.9 is present in nociceptor sensory neurons that express TRPV1, bradykinin B2, and purinergic P2X3 receptors. In Na(v)1.9-/- mice, the non-inactivating persistent tetrodotoxin-resistant sodium TTXr-Per current is absent, whereas TTXr-Slow is unchanged. TTXs currents are unaffected by the mutation of Na(v)1.9. Pain hypersensitivity elicited by intraplantar administration of prostaglandin E2, bradykinin, interleukin-1beta, capsaicin, and P2X3 and P2Y receptor agonists, but not NGF, is either reduced or absent in Na(v)1.9-/- mice, whereas basal thermal and mechanical pain sensitivity is unchanged. Thermal, but not mechanical, hypersensitivity produced by peripheral inflammation (intraplanatar complete Freund's adjuvant) is substantially diminished in the null allele mutant mice, whereas hypersensitivity in two neuropathic pain models is unchanged in the Na(v)1.9-/- mice. Na(v)1.9 is, we conclude, an effector of the hypersensitivity produced by multiple inflammatory mediators on nociceptor peripheral terminals and therefore plays a key role in mediating peripheral sensitization.
Assuntos
Hiperalgesia/metabolismo , Neuropeptídeos/biossíntese , Nervos Periféricos/metabolismo , Canais de Sódio/biossíntese , Animais , Feminino , Hiperalgesia/genética , Inflamação/genética , Inflamação/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Canal de Sódio Disparado por Voltagem NAV1.9 , Neurônios Aferentes/metabolismo , Neuropeptídeos/genética , Dor/genética , Dor/metabolismo , Medição da Dor/métodos , Canais de Sódio/genéticaRESUMO
The P2X(7) purinoceptor is a ligand-gated cation channel, expressed predominantly by cells of immune origin, with a unique phenotype which includes release of biologically active inflammatory cytokine, interleukin (IL)-1beta following activation, and unique ion channel biophysics observed only in this receptor family. Here we demonstrate that in mice lacking this receptor, inflammatory (in an adjuvant-induced model) and neuropathic (in a partial nerve ligation model) hypersensitivity is completely absent to both mechanical and thermal stimuli, whilst normal nociceptive processing is preserved. The knockout animals were unimpaired in their ability to produce mRNA for pro-IL-1beta, and cytometric analysis of paw and systemic cytokines from knockout and wild-type animals following adjuvant insult suggests a selective effect of the gene deletion on release of IL-1beta and IL-10, with systemic reductions in adjuvant-induced increases in IL-6 and MCP-1. In addition, we show that this receptor is upregulated in human dorsal root ganglia and injured nerves obtained from chronic neuropathic pain patients. We hypothesise that the P2X(7) receptor, via regulation of mature IL-1beta production, plays a common upstream transductional role in the development of pain of neuropathic and inflammatory origin. Drugs which block this target may have the potential to deliver broad-spectrum analgesia.
Assuntos
Neuralgia/imunologia , Neuralgia/fisiopatologia , Receptores Purinérgicos P2/genética , Animais , Western Blotting , Contagem de Células , Doença Crônica , Feminino , Gânglios Espinais/citologia , Expressão Gênica/imunologia , Humanos , Hiperalgesia/imunologia , Hiperalgesia/fisiopatologia , Interleucina-1/genética , Ligadura , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos DBA , Camundongos Knockout , Nociceptores/fisiologia , Precursores de Proteínas/genética , Receptores Purinérgicos P2/metabolismo , Receptores Purinérgicos P2X7RESUMO
This study compared the actions of members of five different chemical classes of vanilloid agonists at the recombinant rat vanilloid VR1 receptor expressed in HEK293 cells, and at endogenous vanilloid receptors on dorsal root ganglion cells and sensory nerves in the rat isolated mesenteric arterial bed. In mesenteric beds, vanilloids elicited dose-dependent vasorelaxation with the rank order of potency: resiniferatoxin>>capsaicin=olvanil>phorbol 12-phenyl-acetate 13-acetate 20-homovanillate (PPAHV)>isovelleral. Scutigeral was inactive. Responses were abolished by capsaicin pretreatment and inhibited by ruthenium red. In VR1-HEK293 cells and dorsal root ganglion neurones, Ca(2+) responses were induced by resiniferatoxin>capsaicin=olvanil>PPAHV; all four were full agonists. Isovelleral and scutigeral were inactive. The resiniferatoxin-induced Ca(2+) response had a distinct kinetic profile. Olvanil had a Hill coefficient of approximately 1 whilst capsaicin, resiniferatoxin and PPAHV had Hill coefficients of approximately 2 in VR1-HEK293 cells. The capsaicin-induced Ca(2+) response was inhibited in a concentration-dependent manner by ruthenium red>capsazepine>isovelleral. These data show that resiniferatoxin, capsaicin, olvanil and PPAHV, but not scutigeral and isovelleral, are agonists at recombinant rat VR1 receptors and endogenous vanilloid receptors on dorsal root ganglion neurones and in the rat mesenteric arterial bed. The vanilloids display the same relative potencies (resiniferatoxin>capsaicin=olvanil>PPAHV) in all of the bioassays.
Assuntos
Alcaloides/farmacologia , Capsaicina/análogos & derivados , Artérias Mesentéricas/efeitos dos fármacos , Receptores de Droga/metabolismo , Anti-Inflamatórios não Esteroides/farmacologia , Capsaicina/farmacologia , Células Cultivadas , Diterpenos/farmacologia , Interações Medicamentosas , Humanos , Indicadores e Reagentes , Fenóis/farmacologia , Ésteres de Forbol/farmacologia , Sesquiterpenos Policíclicos , Receptores de Droga/efeitos dos fármacos , Proteínas Recombinantes/efeitos dos fármacos , Proteínas Recombinantes/metabolismo , Rutênio Vermelho/farmacologia , Células Receptoras Sensoriais/efeitos dos fármacos , Sesquiterpenos/farmacologiaRESUMO
The development of new therapeutics for management of pain is likely to become much more mechanism based, and therefore, we need a more thorough understanding of the different pain development pathways. The afferent fibers of sensory neurons, with their cell bodies in the dorsal root ganglia (DRG), are thought to be key in pain mechanisms. DRG neurons can be prepared from embryonic, postnatal, or adult tissue. Embryonic preparations have the advantage of higher cell yields and greater proportion of neurons, but they are dependent on neurotrophins for the first week of culture. In contrast, dissociated postnatal and adult DRG sensory neurons offer the possibility to study mature neurons that may better resemble the in vivo characteristics of these cells. Here, we describe the dissociation of DRG sensory neurons from postnatal and adult rats. DRG are dissected and dissociated using a prolonged trypsin/collagenase treatment, followed by mechanical separation of the neurons. We have routinely prepared them for electrophysiological studies by the methods outlined in this chapter and describe some of the pitfalls that we have encountered, with hints of how to overcome them.
Assuntos
Animais Recém-Nascidos , Técnicas de Cultura de Células/métodos , Separação Celular/métodos , Gânglios Espinais/citologia , Células Receptoras Sensoriais/citologia , Animais , Dor/fisiopatologia , Ratos , Células Receptoras Sensoriais/fisiologiaRESUMO
Vanilloid receptor-1 (TRPV1) is a nonselective cation channel, predominantly expressed by sensory neurons, which plays a key role in the detection of noxious painful stimuli such as capsaicin, acid, and heat. TRPV1 antagonists may represent novel therapeutic agents for the treatment of a range of conditions including chronic pain, migraine, and gastrointestinal disorders. Here we describe the in vitro pharmacology of N-(2-bromophenyl)-N'-[((R)-1-(5-trifluoromethyl-2-pyridyl)pyrrolidin-3-yl)]urea (SB-705498), a novel TRPV1 antagonist identified by lead optimization of N-(2-bromophenyl)-N'-[2-[ethyl(3-methylphenyl)amino]ethyl]urea (SB-452533), which has now entered clinical trials. Using a Ca(2+)-based fluorometric imaging plate reader (FLIPR) assay, SB-705498 was shown to be a potent competitive antagonist of the capsaicin-mediated activation of the human TRPV1 receptor (pK(i) = 7.6) with activity at rat (pK(i) = 7.5) and guinea pig (pK(i) = 7.3) orthologs. Whole-cell patch-clamp electrophysiology was used to confirm and extend these findings, demonstrating that SB-705498 can potently inhibit the multiple modes of receptor activation that may be relevant to the pathophysiological role of TRPV1 in vivo: SB-705498 caused rapid and reversible inhibition of the capsaicin (IC(50) = 3 nM)-, acid (pH 5.3)-, or heat (50 degrees C; IC(50) = 6 nM)-mediated activation of human TRPV1 (at -70 mV). Interestingly, SB-705498 also showed a degree of voltage dependence, suggesting an effective enhancement of antagonist action at negative potentials such as those that might be encountered in neurons in vivo. The selectivity of SB-705498 was defined by broad receptor profiling and other cellular assays in which it showed little or no activity versus a wide range of ion channels, receptors, and enzymes. SB-705498 therefore represents a potent and selective multimodal TRPV1 antagonist, a pharmacological profile that has contributed to its definition as a suitable drug candidate for clinical development.
Assuntos
Ácidos/farmacologia , Capsaicina/farmacologia , Temperatura Alta , Pirrolidinas/farmacologia , Canais de Cátion TRPV/antagonistas & inibidores , Ureia/análogos & derivados , Animais , Ligação Competitiva/efeitos dos fármacos , Sinalização do Cálcio/efeitos dos fármacos , Linhagem Celular , Células Cultivadas , Relação Dose-Resposta a Droga , Eletrofisiologia , Cobaias , Humanos , Concentração de Íons de Hidrogênio , Potenciais da Membrana/efeitos dos fármacos , Estrutura Molecular , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Neurônios/fisiologia , Técnicas de Patch-Clamp , Pirrolidinas/química , Ratos , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/fisiologia , Transfecção , Ureia/química , Ureia/farmacologiaRESUMO
Small molecule antagonists of the vanilloid receptor 1 (TRPV1, also known as VR1) are disclosed. Ureas such as 5 (SB-452533) were used to explore the structure activity relationship with several potent analogues identified. Pharmacological studies using electrophysiological and FLIPR Ca(2+) based assays showed compound 5 was an antagonist versus capsaicin, noxious heat and acid mediated activation of TRPV1. Study of a quaternary salt of 5 supports a mode of action in which compounds from this series cause inhibition via an extracellularly accessible binding site on the TRPV1 receptor.