RESUMEN
Cyclic nucleotide-gated (CNG) channels, which are directly activated by cAMP and cGMP, have long been known to play a key role in retinal and olfactory signal transduction. Emerging evidence indicates that CNG channels are also involved in signaling pathways important for pain processing. Here, we found that the expression of the channel subunits CNGA2, CNGA3, CNGA4 and CNGB1 in dorsal root ganglia, and of CNGA2 in the spinal cord, is transiently altered after peripheral nerve injury in mice. Specifically, we show using in situ hybridization and quantitative real-time RT-PCR that CNG channels containing the CNGB1b subunit are localized to populations of sensory neurons and predominantly excitatory interneurons in the spinal dorsal horn. In CNGB1 knockout (CNGB1-/-) mice, neuropathic pain behavior is considerably attenuated whereas inflammatory pain behavior is normal. Finally, we provide evidence to support CNGB1 as a downstream mediator of cAMP signaling in pain pathways. Altogether, our data suggest that CNGB1-positive CNG channels specifically contribute to neuropathic pain processing after peripheral nerve injury.
Asunto(s)
AMP Cíclico , Canales Catiónicos Regulados por Nucleótidos Cíclicos/genética , Proteínas del Tejido Nervioso/genética , Neuralgia/psicología , Dolor/inducido químicamente , Dolor/psicología , Animales , Canales Catiónicos Regulados por Nucleótidos Cíclicos/biosíntesis , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Inflamación/inducido químicamente , Inflamación/patología , Inyecciones Espinales , Ratones Endogámicos C57BL , Ratones Noqueados , Neuralgia/patología , Dolor/patología , Equilibrio Postural/efectos de los fármacos , Transducción de Señal/efectos de los fármacos , Traumatismos de la Médula Espinal/metabolismo , Traumatismos de la Médula Espinal/patologíaRESUMEN
A large body of evidence indicates that nitric oxide (NO)/cGMP signaling essentially contributes to the processing of chronic pain. In general, NO-induced cGMP formation is catalyzed by 2 isoforms of guanylyl cyclase, NO-sensitive guanylyl cyclase 1 (NO-GC1) and 2 (NO-GC2). However, the specific functions of the 2 isoforms in pain processing remain elusive. Here, we investigated the distribution of NO-GC1 and NO-GC2 in the spinal cord and dorsal root ganglia, and we characterized the behavior of mice lacking either isoform in animal models of pain. Using immunohistochemistry and in situ hybridization, we demonstrate that both isoforms are localized to interneurons in the spinal dorsal horn with NO-GC1 being enriched in inhibitory interneurons. In dorsal root ganglia, the distribution of NO-GC1 and NO-GC2 is restricted to non-neuronal cells with NO-GC2 being the major isoform in satellite glial cells. Mice lacking NO-GC1 demonstrated reduced hypersensitivity in models of neuropathic pain, whereas their behavior in models of inflammatory pain was normal. By contrast, mice lacking NO-GC2 exhibited increased hypersensitivity in models of inflammatory pain, but their neuropathic pain behavior was unaltered. Cre-mediated deletion of NO-GC1 or NO-GC2 in spinal dorsal horn neurons recapitulated the behavioral phenotypes observed in the global knockout. Together, these results indicate that cGMP produced by NO-GC1 or NO-GC2 in spinal dorsal horn neurons exert distinct, and partly opposing, functions in chronic pain processing.
Asunto(s)
Inflamación/enzimología , Neuralgia/enzimología , Isoformas de Proteínas/metabolismo , Guanilil Ciclasa Soluble/metabolismo , Animales , Modelos Animales de Enfermedad , Adyuvante de Freund/toxicidad , Ganglios Espinales/enzimología , Inflamación/inducido químicamente , Ratones , Ratones Endogámicos C57BL , Ratones Transgénicos , Proteínas Musculares/genética , Proteínas Musculares/metabolismo , Neuralgia/etiología , Dimensión del Dolor , Isoformas de Proteínas/genética , ARN Mensajero/metabolismo , Guanilil Ciclasa Soluble/genética , Médula Espinal/enzimología , Proteína 2 de Transporte Vesicular de Glutamato/metabolismo , Proteínas del Transporte Vesicular de Aminoácidos Inhibidores/metabolismoRESUMEN
Impaired NO-cGMP signaling has been linked to several neurological disorders. NO-sensitive guanylyl cyclase (NO-GC), of which two isoforms-NO-GC1 and NO-GC2-are known, represents a promising drug target to increase cGMP in the brain. Drug-like small molecules have been discovered that work synergistically with NO to stimulate NO-GC activity. However, the effects of NO-GC stimulators in the brain are not well understood. In the present study, we used Förster/fluorescence resonance energy transfer (FRET)-based real-time imaging of cGMP in acute brain slices and primary neurons of cGMP sensor mice to comparatively assess the activity of two structurally different NO-GC stimulators, IWP-051 and BAY 41-2272, in the cerebellum, striatum and hippocampus. BAY 41-2272 potentiated an elevation of cGMP induced by the NO donor DEA/NO in all tested brain regions. Interestingly, IWP-051 potentiated DEA/NO-induced cGMP increases in the cerebellum and striatum, but not in the hippocampal CA1 area or primary hippocampal neurons. The brain-region-selective activity of IWP-051 suggested that it might act in a NO-GC isoform-selective manner. Results of mRNA in situ hybridization indicated that the cerebellum and striatum express NO-GC1 and NO-GC2, while the hippocampal CA1 area expresses mainly NO-GC2. IWP-051-potentiated DEA/NO-induced cGMP signals in the striatum of NO-GC2 knockout mice but was ineffective in the striatum of NO-GC1 knockout mice. These results indicate that IWP-051 preferentially stimulates NO-GC1 signaling in brain slices. Interestingly, no evidence for an isoform-specific effect of IWP-051 was observed when the cGMP-forming activity of whole brain homogenates was measured. This apparent discrepancy suggests that the method and conditions of cGMP measurement can influence results with NO-GC stimulators. Nevertheless, it is clear that NO-GC stimulators enhance cGMP signaling in the brain and should be further developed for the treatment of neurological diseases.
Asunto(s)
Encéfalo/metabolismo , GMP Cíclico/análisis , Guanilato Ciclasa/metabolismo , Óxido Nítrico/metabolismo , Animales , Células Cultivadas , GMP Cíclico/metabolismo , Transferencia Resonante de Energía de Fluorescencia/métodos , Ratones Noqueados , Neuroimagen/métodos , Neuronas , Células de PurkinjeRESUMEN
Intermediate conductance calcium-activated potassium channels (KCa3.1) have been recently implicated in pain processing. However, the functional role and localization of KCa3.1 in the nociceptive system are largely unknown. We here characterized the behavior of mice lacking KCa3.1 (KCa3.1-/-) in various pain models and analyzed the expression pattern of KCa3.1 in dorsal root ganglia (DRG) and the spinal cord. KCa3.1-/- mice demonstrated normal behavioral responses in models of acute nociceptive, persistent inflammatory, and persistent neuropathic pain. However, their behavioral responses to noxious chemical stimuli such as formalin and capsaicin were increased. Accordingly, formalin-induced nociceptive behavior was increased in wild-type mice after administration of the KCa3.1 inhibitor TRAM-34. In situ hybridization experiments detected KCa3.1 in most DRG satellite glial cells, in a minority of DRG neurons, and in ependymal cells lining the central canal of the spinal cord. Together, our data point to a specific inhibitory role of KCa3.1 for the processing of noxious chemical stimuli.
Asunto(s)
Ganglios Espinales/metabolismo , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/metabolismo , Neuralgia/metabolismo , Dolor Nociceptivo/metabolismo , Animales , Péptido Relacionado con Gen de Calcitonina/metabolismo , Células Cultivadas , Epéndimo/efectos de los fármacos , Epéndimo/metabolismo , Epéndimo/patología , Femenino , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/patología , Inflamación/metabolismo , Inflamación/patología , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/antagonistas & inhibidores , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/deficiencia , Canales de Potasio de Conductancia Intermedia Activados por el Calcio/genética , Masculino , Ratones Endogámicos C57BL , Ratones Noqueados , Neuralgia/patología , Neuroglía/efectos de los fármacos , Neuroglía/metabolismo , Neuroglía/patología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/patología , Dolor Nociceptivo/patología , Umbral del Dolor/efectos de los fármacos , Umbral del Dolor/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Pirazoles/farmacología , Nervio Ciático/lesiones , Fármacos del Sistema Sensorial , Médula Espinal/efectos de los fármacos , Médula Espinal/metabolismo , Médula Espinal/patologíaRESUMEN
Chronic pain is accompanied by production of reactive oxygen species (ROS) in various cells that are important for nociceptive processing. Recent data indicate that ROS can trigger specific redox-dependent signaling processes, but the molecular targets of ROS signaling in the nociceptive system remain largely elusive. Here, we performed a proteome screen for pain-dependent redox regulation using an OxICAT approach, thereby identifying the small GTPase Rab7 as a redox-modified target during inflammatory pain in mice. Prevention of Rab7 oxidation by replacement of the redox-sensing thiols modulates its GTPase activity. Immunofluorescence studies revealed Rab7 expression to be enriched in central terminals of sensory neurons. Knockout mice lacking Rab7 in sensory neurons showed normal responses to noxious thermal and mechanical stimuli; however, their pain behavior during inflammatory pain and in response to ROS donors was reduced. The data suggest that redox-dependent changes in Rab7 activity modulate inflammatory pain sensitivity.