RESUMEN
AIMS: In hypertrophy and heart failure, the proarrhythmic persistent Na+ current (INaL ) is enhanced. We aimed to investigate the electrophysiological role of neuronal sodium channel NaV 1.8 in human hypertrophied myocardium. METHODS AND RESULTS: Myocardial tissue of 24 patients suffering from symptomatic severe aortic stenosis and concomitant significant afterload-induced hypertrophy with preserved ejection fraction was used and compared with 12 healthy controls. We performed quantitative real-time PCR and western blot and detected a significant up-regulation of NaV 1.8 mRNA (2.34-fold) and protein expression (1.96-fold) in human hypertrophied myocardium compared with healthy hearts. Interestingly, NaV 1.5 protein expression was significantly reduced in parallel (0.60-fold). Using whole-cell patch-clamp technique, we found that the prominent INaL was significantly reduced after addition of novel NaV 1.8-specific blockers either A-803467 (30 nM) or PF-01247324 (1 µM) in human hypertrophic cardiomyocytes. This clearly demonstrates the relevant contribution of NaV 1.8 to this proarrhythmic current. We observed a significant action potential duration shortening and performed confocal microscopy, demonstrating a 50% decrease in proarrhythmic diastolic sarcoplasmic reticulum (SR)-Ca2+ leak and SR-Ca2+ spark frequency after exposure to both NaV 1.8 inhibitors. CONCLUSIONS: We show for the first time that the neuronal sodium channel NaV 1.8 is up-regulated on mRNA and protein level in the human hypertrophied myocardium. Furthermore, inhibition of NaV 1.8 reduced augmented INaL , abbreviated the action potential duration, and decreased the SR-Ca2+ leak. The findings of our study suggest that NaV 1.8 could be a promising antiarrhythmic therapeutic target and merits further investigation.
Asunto(s)
Regulación de la Expresión Génica , Ventrículos Cardíacos/metabolismo , Hipertrofia Ventricular Izquierda/genética , Miocitos Cardíacos/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8/genética , ARN/genética , Potenciales de Acción , Anciano , Western Blotting , Diástole , Femenino , Ventrículos Cardíacos/patología , Ventrículos Cardíacos/fisiopatología , Humanos , Hipertrofia Ventricular Izquierda/diagnóstico , Hipertrofia Ventricular Izquierda/metabolismo , Masculino , Miocitos Cardíacos/patología , Canal de Sodio Activado por Voltaje NAV1.8/biosíntesis , Técnicas de Placa-Clamp , Reacción en Cadena en Tiempo Real de la Polimerasa , Retículo Sarcoplasmático/metabolismoRESUMEN
The somatosensory system allows us to detect a diverse range of physical and chemical stimuli including noxious ones, which can initiate protective reflexes to prevent tissue damage. However, the sensation of pain can - under pathological circumstances - outlive its usefulness and perpetrate ongoing suffering. Rodent model systems have been tremendously useful to help understand basic mechanisms of pain perception. Unfortunately, the translation of this knowledge into novel therapies has been challenging. We have investigated similarities and differences of human and mouse peptidergic (TRKA expressing) nociceptors using dual-color fluorescence in situ hybridization of dorsal root ganglia. By comparing the transcripts of a selected group of well-established nociceptive markers, we observed significant differences for some of them. We found co-expression of Trpv1, a key player for sensitization and inflammatory pain, with TrkA in a larger population in humans compared to mice. Similar results could be obtained for Nav1.8 and Nav1.9, two voltage gated sodium channels implicated in pathological forms of pain. Additionally, co-expression of Ret and TrkA was also found to be more abundant in human neurons. Moreover, the neurofilament heavy polypeptide was detected in all human sensory DRG neurons compared to a more selective expression pattern observed in rodents. To our knowledge, this is the first time that such detailed comparative analysis has been performed and we believe that our findings will direct future experimentation geared to understand the difficulties we face in translating findings from rodent models to humans.
Asunto(s)
Ganglios Espinales/citología , Ganglios Espinales/metabolismo , Nociceptores/metabolismo , Anciano , Animales , Femenino , Humanos , Hibridación in Situ , Masculino , Ratones , Persona de Mediana Edad , Canal de Sodio Activado por Voltaje NAV1.8/biosíntesis , Canal de Sodio Activado por Voltaje NAV1.9/biosíntesis , Proteínas de Neurofilamentos/biosíntesis , Dolor/metabolismo , Proteínas Proto-Oncogénicas c-ret/biosíntesis , Receptor trkA/biosíntesis , Especificidad de la Especie , Canales Catiónicos TRPV/biosíntesisRESUMEN
BACKGROUND: Pain is one critical hallmark of inflammatory responses. A large number of studies have demonstrated that stromal cell-derived factor 1 (SDF1, also named as CXCL12) and its cognate receptor C-X-C chemokine receptor type 4 (CXCR4) play an important role in immune reaction and inflammatory processes. However, whether and how SDF1-CXCR4 signaling is involved in inflammatory pain remains unclear. METHODS: Under the intraplantar (i.pl.) bee venom (BV) injection-induced persistent inflammatory pain state, the changes of SDF1 and CXCR4 expression and cellular localization in the rat dorsal root ganglion (DRG) were detected by immunofluorescent staining. The role of SDF1 and CXCR4 in the hyperexcitability of primary nociceptor neurons was assessed by electrophysiological recording. Western blot analysis was used to quantify the DRG Nav1.8 and phosphorylation of ERK (pERK) expression. Behavioral tests were conducted to evaluate the roles of CXCR4 as well as extracellular signal-regulated kinase (ERK) and Nav1.8 in the BV-induced persistent pain and hypersensitivity. RESULTS: We showed that both SDF1 and CXCR4 were dramatically up-regulated in the DRG in i.pl. BV-induced inflammatory pain model. Double immunofluorescent staining showed that CXCR4 was localized in all sizes (large, medium, and small) of DRG neuronal soma, while SDF1 was exclusively expressed in satellite glial cells (SGCs). Electrophysiological recording showed that bath application with AMD3100, a potent and selective CXCR4 inhibitor, could reverse the hyperexcitability of medium- and small-sized DRG neurons harvested from rats following i.pl. BV injection. Furthermore, we demonstrated that the BV-induced ERK activation and Nav1.8 up-regulation in the DRG could be blocked by pre-antagonism against CXCR4 in the periphery with AMD3100 as well as by blockade of ERK activation by intrathecal (i.t.) or intraplantar (i.pl.) U0126. At behavioral level, the BV-induced persistent spontaneous pain as well as primary mechanical and thermal hypersensitivity could also be significantly suppressed by blocking CXCR4 and Nav1.8 in the periphery as well as by inhibition of ERK activation at the DRG level. CONCLUSIONS: The present results suggest that peripheral inflammatory pain state can trigger over release of SDF1 from the activated SGCs in the DRG by which SGC-neuronal cross-talk is mediated by SDF1-CXCR4 coupling that result in subsequent ERK-dependent Nav1.8 up-regulation, leading to hyperexcitability of tonic type of the primary nociceptor cells and development and maintenance of persistent spontaneous pain and hypersensitivity.
Asunto(s)
Quimiocina CXCL12/biosíntesis , Hiperalgesia/metabolismo , Sistema de Señalización de MAP Quinasas/fisiología , Canal de Sodio Activado por Voltaje NAV1.8/biosíntesis , Nociceptores/metabolismo , Receptores CXCR4/biosíntesis , Animales , Masculino , Neuronas/metabolismo , Dolor/metabolismo , Ratas , Ratas Sprague-Dawley , Regulación hacia Arriba/fisiologíaRESUMEN
Fabry disease (FD) is one of the X-linked lysosomal storage disorders caused by deficient functioning of the alpha-galactosidase A (α-GalA) enzyme. The α-GalA deficiency leads to multi-systemic clinical manifestations caused by the preferential accumulation of globotriaosylceramide in the endothelium and vascular smooth muscles. A hallmark symptom of FD patients is peripheral pain that appears in the early stage of the disease. Pain in FD patients is a peripheral small-fiber idiopathic neuropathy, with intra-epidermal fiber density and integrity being used for diagnosing FD in humans. However, the molecular correlates underlying pain sensation in FD remain elusive. Here, we have employed the α-GalA gene KO mouse as a model of FD in rodents to investigate molecular changes in their peripheral nervous system that may account for their algesic symptoms. The α-GalA null mice display neuropathic pain as evidenced by thermal hyperalgesia and mechanical allodynia, with histological analyses showing alterations in cutaneous innervation. Additionally, KO mice showed a decreased and scattered pattern of neuronal terminations consistent with the reduction in neuronal terminations in skin biopsies of patients with small fiber neuropathies. At the molecular level KO animals showed an increase in the expression of TRPV1 and Nav1.8, and a decrease in the expression of TRPM8. Notably, these alterations are observed in young animals. Taken together, our findings imply that the α-GalA KO mouse is a good model in which to study the peripheral small fiber neuropathy exhibited by FD patients, and provides molecular evidence for a hyperexcitability of small nociceptors in FD.
Asunto(s)
Enfermedad de Fabry/genética , Neuralgia/genética , alfa-Galactosidasa/genética , Animales , Modelos Animales de Enfermedad , Enfermedad de Fabry/patología , Femenino , Regulación de la Expresión Génica , Humanos , Ratones , Ratones Noqueados , Canal de Sodio Activado por Voltaje NAV1.8/biosíntesis , Neuralgia/enzimología , Neuralgia/patología , Piel/enzimología , Piel/metabolismo , Piel/patología , Canales Catiónicos TRPV/biosíntesis , alfa-Galactosidasa/metabolismoRESUMEN
CCL2 [chemokine (C-C motif) ligand 2] contributes to the inflammation-induced neuropathic pain through activating VGSC (voltage-gated sodium channel)-mediated nerve impulse conduction, but the underlying mechanism is currently unknown. Our study aimed to investigate whether PKC (protein kinase C)-NF-κB (nuclear factor κB) is involved in CCL2-induced regulation of voltage-gated sodium Nav1.8 currents and expression. DRG (dorsal root ganglion) neurons were prepared from adult male Sprague-Dawley rats and incubated with various concentration of CCL2 for 24 h. Whole-cell patch-clamps were performed to record the Nav1.8 currents in response to the induction by CCL2. After being pretreated with 5 and10 nM CCL2 for 16 h, CCR2 [chemokine (C-C motif) receptor 2] and Nav1.8 expression significantly increased and the peak currents of Nav1.8 elevated from the baseline 46.53±4.53 pA/pF to 64.28±3.12 pA/pF following 10 nM CCL2 (P<0.05). Compared with the control, significant change in Nav1.8 current density was observed when the CCR2 inhibitor INCB3344 (10 nM) was applied. Furthermore, inhibition of PKC by AEB071 significantly eliminated CCL2-induced elevated Nav1.8 currents. In vitro PKC kinase assays and autoradiograms suggested that Nav1.8 within DRG neurons was a substrate of PKC and direct phosphorylation of the Nav1.8 channel by PKC regulates its function in these neurons. Moreover, p65 expression was significantly higher in CCL2-induced neurons (P<0.05), and was reversed by treatment with INCB3344 and AEB071. PKC-NF-κB are involved in CCL2-induced elevation of Nav1.8 current density by promoting the phosphorylation of Nav1.8 and its expression.
Asunto(s)
Quimiocina CCL2/metabolismo , Ganglios Espinales/metabolismo , Regulación de la Expresión Génica/fisiología , Canal de Sodio Activado por Voltaje NAV1.8/biosíntesis , Neuronas/metabolismo , Proteína Quinasa C/metabolismo , Factor de Transcripción ReIA/biosíntesis , Animales , Células Cultivadas , Transporte Iónico/fisiología , Masculino , Fosforilación/fisiología , Ratas , Ratas Sprague-DawleyRESUMEN
The pathogenesis of pain in irritable bowel syndrome (IBS) is poorly understood, and treatment remains difficult. We have previously reported that colon-specific dorsal root ganglion (DRG) neurons were hyperactive in a rat model of IBS induced by neonatal colonic inflammation (NCI). This study was designed to examine plasticity of voltage-gated Na(+) channel activities and roles for the endogenous hydrogen sulfide-producing enzyme cystathionine ß-synthetase (CBS) in chronic visceral hyperalgesia. Abdominal withdrawal reflex (AWR) scores were recorded in response to graded colorectal distention in adult male rats as a measure of visceral hypersensitivity. Colon-specific DRG neurons were labeled with 1,1'-dioleyl-3,3,3',3-tetramethylindocarbocyanine methanesulfonate and acutely dissociated for measuring Na(+) channel currents. Western blot analysis was employed to detect changes in expressions of voltage-gated Na(+) (Na(V)) channel subtype 1.7, Na(V)1.8, and CBS. NCI significantly increased AWR scores when compared with age-matched controls. NCI also led to an ~2.5-fold increase in Na(+) current density in colon-specific DRG neurons. Furthermore, NCI dramatically enhanced expression of Na(V)1.7, Na(V)1.8, and CBS in colon-related DRGs. CBS was colocalized with Na(V)1.7 or -1.8 in colon-specific DRG neurons. Administration of O-(carboxymethyl)hydroxylamine hemihydrochloride (AOAA), an inhibitor for CBS, remarkably suppressed Na(+) current density and reduced expression of Na(V)1.7 and Na(V)1.8. More importantly, intraperitoneal or intrathecal application of AOAA attenuated AWR scores in NCI rats in a dose-dependent manner. These data suggest that NCI enhances Na(+) channel activity of colon DRG neurons, which is most likely mediated by upregulation of CBS expression, thus identifying a potential target for treatment for chronic visceral pain in patients with IBS.
Asunto(s)
Colitis/fisiopatología , Cistationina betasintasa/biosíntesis , Ganglios Espinales/fisiología , Canal de Sodio Activado por Voltaje NAV1.7/fisiología , Canal de Sodio Activado por Voltaje NAV1.8/fisiología , Ácido Acético , Ácido Aminooxiacético/farmacología , Animales , Animales Recién Nacidos , Carbocianinas , Colitis/inducido químicamente , Colorantes , Cistationina betasintasa/antagonistas & inhibidores , Hiperalgesia/fisiopatología , Síndrome del Colon Irritable/fisiopatología , Masculino , Canal de Sodio Activado por Voltaje NAV1.7/biosíntesis , Canal de Sodio Activado por Voltaje NAV1.8/biosíntesis , Ratas , Ratas Sprague-Dawley , Reflejo Abdominal/efectos de los fármacosRESUMEN
Irritable bowel syndrome (IBS) is a common gastrointestinal disorder characterized by abdominal pain in association with altered bowel movements. The underlying mechanisms of visceral hypersensitivity remain elusive. This study was designed to examine the role for sodium channels in a rat model of chronic visceral hyperalgesia induced by neonatal maternal deprivation (NMD). Abdominal withdrawal reflex (AWR) scores were performed on adult male rats. Colon-specific dorsal root ganglion (DRG) neurons were labeled with DiI and acutely dissociated for measuring excitability and sodium channel current under whole-cell patch-clamp configurations. The expression of Na(V)1.8 was analyzed by Western blot and quantitative real-time PCR. NMD significantly increased AWR scores, which lasted for ~6 wk in an association with hyperexcitability of colon DRG neurons. TTX-resistant but not TTX-sensitive sodium current density was greatly enhanced in colon DRG neurons in NMD rats. Compared with controls, activation curves showed a leftward shift in NMD rats whereas inactivation curves did not differ significantly. NMD markedly accelerated the activation time of peak current amplitude without any changes in inactivation time. Furthermore, NMD remarkably enhanced expression of Na(V)1.8 at protein levels but not at mRNA levels in colon-related DRGs. The expression of Na(V)1.9 was not altered after NMD. These data suggest that NMD enhances TTX-resistant sodium activity of colon DRG neurons, which is most likely mediated by a leftward shift of activation curve and by enhanced expression of Na(V)1.8 at protein levels, thus identifying a specific molecular mechanism underlying chronic visceral pain and sensitization in patients with IBS.
Asunto(s)
Colon/metabolismo , Ganglios Espinales/fisiología , Privación Materna , Canal de Sodio Activado por Voltaje NAV1.8/biosíntesis , Animales , Hiperalgesia , Síndrome del Colon Irritable , Masculino , Canal de Sodio Activado por Voltaje NAV1.8/efectos de los fármacos , Canal de Sodio Activado por Voltaje NAV1.8/fisiología , Neuronas/metabolismo , Ratas , Reflejo Abdominal , Tetrodotoxina/farmacologíaRESUMEN
BACKGROUND: Inflammation or nerve injury-induced upregulation and release of chemokine CC chemokine ligand 2 (CCL2) within the dorsal root ganglion (DRG) is believed to enhance the activity of DRG nociceptive neurons and cause hyperalgesia. Transient receptor potential vanilloid receptor 1 (TRPV1) and tetrodotoxin (TTX)-resistant Na(v)1.8 sodium channels play an essential role in regulating the excitability and pain transmission of DRG nociceptive neurons. We therefore tested the hypothesis that CCL2 causes peripheral sensitization of nociceptive DRG neurons by upregulating the function and expression of TRPV1 and Nav1.8 channels. METHODS: DRG neuronal culture was prepared from 3-week-old Sprague-Dawley rats and incubated with various concentrations of CCL2 for 24 to 36 hours. Whole-cell voltage-clamp recordings were performed to record TRPV1 agonist capsaicin-evoked inward currents or TTX-insensitive Na(+) currents from control or CCL2-treated small DRG sensory neurons. The CCL2 effect on the mRNA expression of TRPV1 or Na(v)1.8 was measured by real-time quantitative RT-PCR assay. RESULTS: Pretreatment of CCL2 for 24 to 36 hours dose-dependently (EC(50) value = 0.6 ± 0.05 nM) increased the density of capsaicin-induced currents in small putative DRG nociceptive neurons. TRPV1 mRNA expression was greatly upregulated in DRG neurons preincubated with 5 nM CCL2. Pretreating small DRG sensory neurons with CCL2 also increased the density of TTX-resistant Na(+) currents with a concentration-dependent manner (EC(50) value = 0.7 ± 0.06 nM). The Na(v)1.8 mRNA level was significantly increased in DRG neurons pretreated with CCL2. In contrast, CCL2 preincubation failed to affect the mRNA level of TTX-resistant Nav1.9. In the presence of the specific phosphatidylinositol-3 kinase (PI3K) inhibitor LY294002 or Akt inhibitor IV, CCL2 pretreatment failed to increase the current density of capsaicin-evoked inward currents or TTX-insensitive Na(+) currents and the mRNA level of TRPV1 or Na(v)1.8. CONCLUSIONS: Our results showed that CCL2 increased the function and mRNA level of TRPV1 channels and Na(v)1.8 sodium channels in small DRG sensory neurons via activating the PI3K/Akt signaling pathway. These findings suggest that following tissue inflammation or peripheral nerve injury, upregulation and release of CCL2 within the DRG could facilitate pain transmission mediated by nociceptive DRG neurons and could induce hyperalgesia by upregulating the expression and function of TRPV1 and Na(v)1.8 channels in DRG nociceptive neurons.
Asunto(s)
Quimiocina CCL2/fisiología , Ganglios Espinales/metabolismo , Canal de Sodio Activado por Voltaje NAV1.8/biosíntesis , Neuronas/metabolismo , Canales Catiónicos TRPV/biosíntesis , Regulación hacia Arriba/genética , Potenciales de Acción/genética , Animales , Células Cultivadas , Ganglios Espinales/citología , Canal de Sodio Activado por Voltaje NAV1.8/genética , Neuronas/citología , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Canales Catiónicos TRPV/genéticaRESUMEN
In attempt to understand the underlying mechanisms of cancer-induced bone pain, we investigated the presence of two tetrodotoxin-resistant sodium channels, Nav1.8 (SNS/PN3) and Nav1.9 (SNS2/NaN), in dorsal root ganglia (DRG) neurons in an animal model of bone cancer pain. Thirty-six female Sprague-Dawley rats were randomized into three groups: Sham operation group (Sham), cancer-bearing animals killed after 7 days (C7) and cancer-bearing animals killed after 14 days group (C14). After establishment of bone cancer pain model, behavioral tests were carried out to determine the paw withdrawal threshold (PWT) of mechanical and thermal hyperalgesia, respectively. Real-time RT-PCR, Western bolt and Immunofluorescence were used to determine the mRNA and protein expression of Nav1.8 and Nav1.9 in ipsilateral lumbar 4-5 DRG. Compared to Sham group, PWT of mechanical and thermal hyperalgesia in C14 group displayed a significant decrease (P<0.01) from post-operation day (POD) 5 and POD7 to the end point of the observation, respectively. Compared to Sham group, the relative mRNA expression of Nav1.8 and Nav1.9 exhibited a significant up-regulation in C14 group (8.9 times and 9 times, respectively, P<0.01) but not C7 group (1.5 times and 2.4 times, respectively). Western blot and Immunofluorescence revealed an apparent increase of Nav1.8 (P<0.05) and Nav1.9 (P<0.05) protein in C14 group compared with Sham group. The up-regulation of mRNA and protein levels of Nav1.8 and Nav1.9 suggested their potential involvement in the development and maintenance of bone cancer pain.