RESUMO
Previous studies have demonstrated that acute colonic inflammation leads to an increase in dorsal root ganglia (DRG) neuronal excitability. However, the signaling elements implicated in this hyperexcitability have yet to be fully unraveled. Extracellular adenosine 5'-triphosphate (ATP) is a well-recognized sensory signaling molecule that enhances the nociceptive response after inflammation through activation of P2X3 receptors, which are expressed mainly by peripheral sensory neurons. The aim of this study is to continue investigating how P2X3 affects neuronal hypersensitivity in an acute colitis animal model. To achieve this, DNBS (Dinitrobenzene sulfonic acid; 200 mg/kg) was intrarectally administered to C57BL/6 mice, and inflammation severity was assessed according to the following parameters: weight loss, macroscopic and microscopic scores. Perforated patch clamp technique was used to evaluate neuronal excitability via measuring changes in rheobase and action potential firing in T8-L1 DRG neurons. A-317491, a well-established potent and selective P2X3 receptor antagonist, served to dissect their contribution to recorded responses. Protein expression of P2X3 receptors in DRG was evaluated by western blotting and immunofluorescence. Four days post-DNBS administration, colons were processed for histological analyses of ulceration, crypt morphology, goblet cell density, and immune cell infiltration. DRG neurons from DNBS-treated mice were significantly more excitable compared with controls; these changes correlated with increased P2X3 receptor expression. Furthermore, TNF-α mRNA expression was also significantly higher in inflamed colons compared to controls. Incubation of control DRG neurons with TNF-α resulted in similar cell hyperexcitability as measured in DNBS-derived neurons. The selective P2X3 receptor antagonist, A-317491, blocked the TNF-α-induced effect. These results support the hypothesis that TNF-α enhances colon-innervating DRG neuron excitability via modulation of P2X3 receptor activity.
Assuntos
Colite , Gânglios Espinais , Trifosfato de Adenosina , Animais , Inflamação , Camundongos , Camundongos Endogâmicos C57BL , Antagonistas do Receptor Purinérgico P2X , Receptores Purinérgicos P2X3 , Células Receptoras Sensoriais , Fator de Necrose Tumoral alfaRESUMO
Primary afferent fibers express extrasynaptic GABAA and GABAB receptors in the axons and soma. However, whether these receptors are tonically activated by ambient GABA and the source of the neurotransmitter is presently unknown. Here, we show that GABA release from dorsal root ganglia (DRG) does not depend on extracellular calcium, but depends upon calcium released from intracellular stores, and is mediated by Best1 channels. Using a preparation consisting of the spinal nerve in continuity with the DRG and the dorsal root, we found that endogenous GABA tonically activates GABA receptors, depressing the excitability of the primary afferents. In addition, using HPLC we found that GABA is released in the DRG, and by immunofluorescence microscopy we show the presence of GABA, the Best1 channel, and some enzymes of the putrescine pathway of GABA biosynthesis, in glutamine synthase- and GFAP-positive satellite glial cells. Last, we found that the blockade of the Best1 channel activity reduced the excitability of primary afferents and prevented the activation of the GABA receptors. These results suggest that satellite glial cells may be the source of endogenous GABA released in the DRG via Best1 channels, which tonically activates extrasynaptic GABA receptors.
Assuntos
Neurônios Aferentes , Ácido gama-Aminobutírico , Axônios , Gânglios Espinais , Neuroglia , Receptores de GABA-ARESUMO
Nociceptive stimuli attributes are codified in the periphery; at this level, D2-like dopamine (DA) receptor activation decreases the high voltage-gated Ca2+ current predominantly in mechanonociceptive neurons, which explains the presynaptic action mechanism of the antinociception produced by quinpirole when it is intrathecally administered in rats. However, the identity of D2-like DA receptor subtype that mediates this effect remains unknown. To answer this question, we used Fluo-4-based Ca2+ microfluorometry to study the depolarization-elicited [Ca2+]i increase in small non-peptidergic DRG neurons (identified by its binding to the Isolectin B4), and to test the effect of D2-like DA receptor activation by quinpirole in presence of selective antagonists for D2, D3, and D4 DA receptors. The results showed a significantly greater contribution of the D4 DA receptor in the down-modulation of depolarization-elicited [Ca2+]i increase in small non-peptidergic DRG neurons compared to the other receptors. Although the D2 and D3 receptor antagonists also slightly inhibited the effect of quinpirole, their effects were significantly weaker than those of the D4 receptor antagonist. Furthermore, we showed that quinpirole selectively inhibits the CaV2.2 Ca2+ channels. Our results suggest that the activation of the D4 DA receptors is a promising strategy for pain management at the spinal cord level.
Assuntos
Canais de Cálcio Tipo N/efeitos dos fármacos , Agonistas de Dopamina/farmacologia , Neurônios/metabolismo , Quimpirol/farmacologia , Receptores de Dopamina D4/metabolismo , Animais , Cálcio/metabolismo , Canais de Cálcio Tipo N/metabolismo , Células Cultivadas , Feminino , Gânglios Espinais/metabolismo , Masculino , Neurônios/efeitos dos fármacos , Ratos , Ratos WistarRESUMO
Clinical and preclinical studies have shown that patients with Diabetic Neuropathy Pain (DNP) present with increased tumor necrosis factor alpha (TNF-α) serum concentration, whereas studies with diabetic animals have shown that TNF-α induces an increase in NaV1.7 sodium channel expression. This is expected to result in sensitization of nociceptor neuron terminals, and therefore the development of DNP. For further study of this mechanism, dissociated dorsal root ganglion (DRG) neurons were exposed to TNF-α for 6 h, at a concentration equivalent to that measured in STZ-induced diabetic rats that developed hyperalgesia. Tetrodotoxin sensitive (TTXs), resistant (TTXr) and total sodium current was studied in these DRG neurons. Total sodium current was also studied in DRG neurons expressing the collapsin response mediator protein 2 (CRMP2) SUMO-incompetent mutant protein (CRMP2-K374A), which causes a significant reduction in NaV1.7 membrane cell expression levels. Our results show that TNF-α exposure increased the density of the total, TTXs and TTXr sodium current in DRG neurons. Furthermore, TNF-α shifted the steady state activation and inactivation curves of the total and TTXs sodium current. DRG neurons expressing the CRMP2-K374A mutant also exhibited total sodium current increases after exposure to TNF-α, indicating that these effects were independent of SUMOylation of CRMP2. In conclusion, TNF-α sensitizes DRG neurons via augmentation of whole cell sodium current. This may underlie the pronociceptive effects of TNF-α and suggests a molecular mechanism responsible for pain hypersensitivity in diabetic neuropathy patients.
Assuntos
Gânglios Espinais/citologia , Peptídeos e Proteínas de Sinalização Intercelular/metabolismo , Canal de Sódio Disparado por Voltagem NAV1.7/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurônios/metabolismo , Sumoilação , Fator de Necrose Tumoral alfa/metabolismo , Regulação para Cima , Animais , Comportamento Animal , Membrana Celular/metabolismo , Diabetes Mellitus Experimental/sangue , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Glucose/metabolismo , Hiperalgesia/sangue , Hiperalgesia/complicações , Ativação do Canal Iônico , Masculino , Proteínas Mutantes/metabolismo , Ratos Sprague-Dawley , Ratos Wistar , Fator de Necrose Tumoral alfa/sangueRESUMO
Bactridine 2 (Bact-2) is an antibacterial toxin from Tityus discrepans venom which modifies isoforms 1.2, 1.4 and 1.6 voltage-dependent sodium (Nav) channels. Bactridine-induced Na+ outflow in Yersinia enterocolitica was blocked by amiloride, suggesting that Bact-2 effect was mediated by an amiloride sensitive sodium channel. In this study we show that Bact-2 increases also an outward rectifying current in rat dorsal root ganglia (DRG) sensory neurons; therefore, the nature of the outward rectifying currents was characterized and then the effect of Bact-2 on these currents was studied. These currents are enhanced by amiloride, are decreased by Na+ when an outward pH gradient is present and its reversal potential coincides with that of a Cl-/H+ exchanger, suggesting that rectifying currents are produced by the electrogenic Cl-/H+ exchanger modulated by the Na+/H+ antiporter. Bact-2 also leads to an increase of the outward currents in a similar way to the produced by the inhibition of the Na+/H+ exchanger. Additionally, the subsequent application of Bact-2 after blocking the Na+/H+ exchanger does not produce any further effect, suggesting that Bact-2 modifies the outward current by modulating the activity of the Na+/H+ exchanger. The effect of Bact-2 on pHi regulation was determined using the pH indicator BCECF. The results show that the Na+/H+ exchanger is blocked by amiloride and Na+ free solutions and is modulated by Bact-2 in a similar way as cariporide. This study validates that besides Nav channels, Bact-2 modulates the activity of the Na+/H+ exchanger.
Assuntos
Gânglios Espinais/citologia , Neurônios/efeitos dos fármacos , Venenos de Escorpião/química , Trocadores de Sódio-Hidrogênio/antagonistas & inibidores , Trocadores de Sódio-Hidrogênio/metabolismo , Amilorida , Animais , Antiporters/metabolismo , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Masculino , Ratos , Ratos Sprague-Dawley , Escorpiões/fisiologia , Sódio , ZincoRESUMO
It is well known that the CaVα2δ auxiliary subunit regulates the density of high voltage-activated Ca2+ channels in the plasma membrane and that alterations in their functional expression might have implications in the pathophysiology of diverse human diseases such as neuropathic pain. However, little is known concerning the transcriptional regulation of this protein. We previously characterized the promoter of CaVα2δ, and here we report its regulation by the transcription factor Egr-1. Using the neuroblastoma N1E-115 cells, we found that Egr-1 interacts specifically with its binding site in the promoter, affecting the transcriptional regulation of CaVα2δ. Overexpression and knockdown analysis of Egr-1 showed significant changes in the transcriptional activity of the CaVα2δ promoter. Egr-1 also regulated the expression of CaVα2δ at the level of protein. Also, functional studies showed that Egr-1 knockdown significantly decreases Ca2+ currents in dorsal root ganglion (DRG) neurons, while overexpression of the transcription factor increased Ca2+ currents in the F11 cell line, a hybrid of DRG and N18TG2 neuroblastoma cells. Studying the effects of Egr-1 on the transcriptional expression of CaVα2δ could help to understand the regulatory mechanisms of this protein in both health and disease.