RESUMEN
Alzheimer's disease (AD) is one of the most debilitating neurodegenerative nerve diseases, seriously affecting one's ability to carry out daily activities. AD is both progressive and incurable, but molecular studies have begun to shed light on the mechanisms that underlie it. Immunochemical staining showed that cell bodies of Purkinje cells in the cerebellum were significantly reduced in AD rats compared with normal rats. Heat shock protein 70 (HSP70) was found to prevent polyglutamine aggregation in Huntington's disease and spinocerebellar ataxias (SCAs) and to relieve symptoms in SCAs and Parkinson's disease. Recently, AD-related phenotypes were found to be suppressed in HSP70 transgenic rats. However, the effects of other HSPs and the mechanisms of HSP-triggered changes in AD are unknown. In this study, we found that expression levels of HSP60, -70, and -90 were downregulated in the cerebella of rats with AD. Furthermore, heat shock factor 1 (HSF1), a key transcription factor for the expression of HSP genes, was found to be greatly decreased in the cerebella of AD rats. Even more interesting, injection of lentivirus vector-HSF1 into the cerebella of AD rats significantly increased HSF1 and HSP expression levels and induced an increase in the number of Purkinje cell bodies. Our findings provide novel evidence that low expression of HSPs in AD rats is dependent on the low expression of HSF1, and increased expression of HSF1 contributes to the reversal of cerebellar Purkinje cell deficiency in AD. Therefore, increasing HSF1 expression is a potential new strategy for the treatment of AD.
Asunto(s)
Enfermedad de Alzheimer/patología , Cerebelo/metabolismo , Cerebelo/patología , Proteínas de Unión al ADN/metabolismo , Células de Purkinje/patología , Factores de Transcripción/metabolismo , Enfermedad de Alzheimer/tratamiento farmacológico , Enfermedad de Alzheimer/metabolismo , Animales , Cerebelo/efectos de los fármacos , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/uso terapéutico , Modelos Animales de Enfermedad , Proteínas del Choque Térmico HSP72/genética , Proteínas del Choque Térmico HSP72/metabolismo , Factores de Transcripción del Choque Térmico , Lentivirus/genética , Lentivirus/metabolismo , Ratas , Factores de Transcripción/genética , Factores de Transcripción/uso terapéuticoRESUMEN
Intracellular second messengers play an important role in capsaicin- and analogous-induced sensitization and desensitization in pain. Fluorescence Ca²âº imaging, enzyme immunoassay and PKC assay kit were used to determine a novel mechanism of different Ca²âº dependency in the signal transduction of capsaicin-induced desensitization. On the average, capsaicin increased cAMP, cGMP concentration and SP release in bell-shaped concentration-dependent manner, with the maximal responses at concentrations around 1 µM, suggesting acute desensitization of TRPV1 receptor activation. Capsaicin-induced intracellular Ca²âº concentration ([Ca²âº](i)) increase depended on extracellular Ca²âº influx as an initial trigger. The Ca²âº influx by capsaicin increased PKC activation and SP release. These increases were completely abolished in Ca²âº-free solution, suggesting that the modulation of capsaicin on PKC and SP are Ca²âº-dependent. Interestingly, the maximal cAMP increase by TRPV1 activation was not blocked Ca²âº removal, suggesting at least in part a Ca²âº-independent pathway is involved. Further study showed that cAMP increase was totally abolished by G-protein and adenylate cyclase (AC) antagonist, suggesting a G-protein-dependent pathway in cAMP increase. However, SP release was blocked by inhibiting PKC, but not G-protein or AC, suggesting a G-protein independent pathway in SP release. These results suggest that both Ca²âº-dependent and independent mechanisms are involved in the regulation of capsaicin on second messengers systems, which could be a novel mechanism underlying distinct desensitization of capsaicin and might provide additional opportunities in the development of effective analgesics in pain treatment.
Asunto(s)
Calcio/metabolismo , Capsaicina/farmacología , Neuronas/metabolismo , Sistemas de Mensajero Secundario/efectos de los fármacos , Canales Catiónicos TRPV/fisiología , Animales , Capsaicina/metabolismo , Células Cultivadas , AMP Cíclico/metabolismo , GMP Cíclico/metabolismo , Masculino , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Transducción de Señal/fisiología , Sustancia P/metabolismoRESUMEN
Abnormal hyperexcitability of primary sensory neurons contributes to neuropathic pain development after nerve injury. Nerve injury profoundly reduces the expression of big conductance Ca(2+) -activated K(+) (BK) channels in the dorsal root ganglion (DRG). However, little is known about how nerve injury affects BK channel activity in DRG neurons. In this study, we determined the changes in BK channel activity in DRG neurons in a rat model of neuropathic pain and the contribution of brain-derived neurotrophic factor (BDNF) to reduced BK channel activity. The BK channel activity was present predominantly in small and medium DRG neurons, and ligation of L5 and L6 spinal nerves profoundly decreased the BK current density in these neurons. Blocking BK channels significantly increased neuronal excitability in sham control, but not in nerve-injured, rats. The BDNF concentration in the DRG was significantly greater in nerve-injured rats than in control rats. BDNF treatment largely reduced BK currents in DRG neurons in control rats, which was blocked by either anti-BDNF antibody or K252a, a Trk receptor inhibitor. Furthermore, either anti-BDNF antibody or K252a reversed reduction in BK currents in injured DRG neurons. BDNF treatment reduced the mRNA levels of BKα1 subunit in DRG neurons, and anti-BDNF antibody attenuated the reduction in the BKα1 mRNA level in injured DRG neurons. These findings suggest that nerve injury primarily diminishes the BK channel activity in small and medium DRG neurons. Increased BDNF levels contribute to reduced BK channel activity in DRG neurons through epigenetic and transcriptional mechanisms in neuropathic pain.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/biosíntesis , Canales de Potasio de Gran Conductancia Activados por el Calcio/metabolismo , Neuralgia/metabolismo , Células Receptoras Sensoriales/metabolismo , Nervios Espinales/lesiones , Animales , Epigénesis Genética , Ganglios Espinales/metabolismo , Masculino , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Reacción en Cadena en Tiempo Real de la Polimerasa , Reacción en Cadena de la Polimerasa de Transcriptasa InversaRESUMEN
High voltage-activated calcium channels (HVACCs) are essential for synaptic and nociceptive transmission. Although blocking HVACCs can effectively reduce pain, this treatment strategy is associated with intolerable adverse effects. Neuronal HVACCs are typically composed of α(1), ß (Cavß), and α(2)δ subunits. The Cavß subunit plays a crucial role in the membrane expression and gating properties of the pore-forming α(1) subunit. However, little is known about how nerve injury affects the expression and function of Cavß subunits in primary sensory neurons. In this study, we found that Cavß(3) and Cavß(4) are the most prominent subtypes expressed in the rat dorsal root ganglion (DRG) and dorsal spinal cord. Spinal nerve ligation (SNL) in rats significantly increased mRNA and protein levels of the Cavß(3), but not Cavß(4), subunit in the DRG. SNL also significantly increased HVACC currents in small DRG neurons and monosynaptic excitatory postsynaptic currents of spinal dorsal horn neurons evoked from the dorsal root. Intrathecal injection of Cavß(3)-specific siRNA significantly reduced HVACC currents in small DRG neurons and the amplitude of monosynaptic excitatory postsynaptic currents of dorsal horn neurons in SNL rats. Furthermore, intrathecal treatment with Cavß(3)-specific siRNA normalized mechanical hyperalgesia and tactile allodynia caused by SNL but had no significant effect on the normal nociceptive threshold. Our findings provide novel evidence that increased expression of the Cavß(3) subunit augments HVACC activity in primary sensory neurons and nociceptive input to dorsal horn neurons in neuropathic pain. Targeting the Cavß(3) subunit at the spinal level represents an effective strategy for treating neuropathic pain.
Asunto(s)
Canales de Calcio/genética , Canales de Calcio/metabolismo , Neuralgia/metabolismo , Neuralgia/patología , Nocicepción , Células Receptoras Sensoriales/metabolismo , Regulación hacia Arriba , Animales , Secuencia de Bases , Regulación hacia Abajo , Ganglios Espinales/lesiones , Ganglios Espinales/metabolismo , Ganglios Espinales/patología , Ganglios Espinales/fisiopatología , Ácido Glutámico/metabolismo , Masculino , Neuralgia/genética , Neuralgia/fisiopatología , Células del Asta Posterior/metabolismo , Células del Asta Posterior/patología , Transporte de Proteínas , Ratas , Ratas Sprague-Dawley , Células Receptoras Sensoriales/patología , Células Receptoras Sensoriales/fisiologíaRESUMEN
Painful neuropathy is one of the most serious complications of diabetes and remains difficult to treat. The muscarinic acetylcholine receptor (mAChR) agonists have a profound analgesic effect on painful diabetic neuropathy. Here we determined changes in T-type and high voltage-activated Ca(2+) channels (HVACCs) and their regulation by mAChRs in dorsal root ganglion (DRG) neurons in a rat model of diabetic neuropathy. The HVACC currents in large neurons, T-type currents in medium and large neurons, the percentage of small DRG neurons with T-type currents, and the Cav3.2 mRNA level were significantly increased in diabetic rats compared with those in control rats. The mAChR agonist oxotremorine-M significantly inhibited HVACCs in a greater proportion of DRG neurons with and without T-type currents in diabetic than in control rats. In contrast, oxotremorine-M had no effect on HVACCs in small and large neurons with T-type currents and in most medium neurons with T-type currents from control rats. The M(2) and M(4) antagonist himbacine abolished the effect of oxotremorine-M on HVACCs in both groups. The selective M(4) antagonist muscarinic toxin-3 caused a greater attenuation of the effect of oxotremorine-M on HVACCs in small and medium DRG neurons in diabetic than in control rats. Additionally, the mRNA and protein levels of M(4), but not M(2), in the DRG were significantly greater in diabetic than in control rats. Our findings suggest that diabetic neuropathy potentiates the activity of T-type and HVACCs in primary sensory neurons. M(4) mAChRs are up-regulated in DRG neurons and probably account for increased muscarinic analgesic effects in diabetic neuropathic pain.
Asunto(s)
Canales de Calcio Tipo T/metabolismo , Diabetes Mellitus Experimental/metabolismo , Neuropatías Diabéticas/metabolismo , Neuropatías Diabéticas/patología , Receptor Muscarínico M4/fisiología , Células Receptoras Sensoriales/metabolismo , Células Receptoras Sensoriales/patología , Animales , Canales de Calcio Tipo T/biosíntesis , Canales de Calcio Tipo T/genética , Canales de Calcio Tipo T/fisiología , Diabetes Mellitus Experimental/genética , Diabetes Mellitus Experimental/patología , Neuropatías Diabéticas/genética , Modelos Animales de Enfermedad , Masculino , Neuralgia/etiología , Neuralgia/patología , Neuralgia/prevención & control , ARN Mensajero/biosíntesis , Ratas , Ratas Sprague-Dawley , Receptor Muscarínico M4/biosíntesis , Receptor Muscarínico M4/genética , Regulación hacia Arriba/genéticaRESUMEN
ω-3 polyunsaturated fatty acid docosahexaenoic acid (DHA) enhances cognitive functions; however, the underlying molecular mechanism remains unclear. Compelling evidence suggests that the endocannabinoid/endovanilloid systems play a pivotal role in regulating cognitive function. Thus, to correlate the effect of DHA on cognitive performance with the expression of endocannabinoid and endovanilloid receptors, we supplemented the diet of rats with DHA and performed in vitro experiments that focused on the endocannabinoid/endovanilloid receptors. We found that in vivo supplementation with an appropriate dose of DHA (150 or 300mg/kg/d) significantly improved learning and memory but that a higher intake (600mg/kg/d) increased the risk of memory impairment. In addition, we found that some subtypes of endocannabinoid/endovanilloid receptors (cannabinoid [CB] and transient receptor potential vanilloid [TRPV] receptors) were regulated in vitro by different concentrations of DHA in primary hippocampal neuron culture medium. Real-time polymerase chain reaction and western blot analysis showed that expression of both CB1 and TRPV1 was upregulated in a dose-dependent manner and reached a maximum level at 30µmol/L (CB1) and 60µmol/L (TRPV1) DHA. However, TRPV2 expression was downregulated in a dose-dependent fashion, and the peak of TRPV2 suppression was observed at 60µmol/L. The dose-dependent effects of DHA on the expression of these receptors were well correlated with DHA's effect on spatial memory. Meanwhile, CB2, TRPV3, and TRPV4 expressions were not altered at diverse concentrations of DHA. We concluded that some subtypes of endocannabinoid/endovanilloid receptors might be involved in enhanced spatial memory induced by DHA supplementation.
Asunto(s)
Ácidos Docosahexaenoicos/farmacología , Hipocampo/efectos de los fármacos , Aprendizaje por Laberinto/efectos de los fármacos , Receptor Cannabinoide CB1/metabolismo , Canales Catiónicos TRPV/metabolismo , Animales , Relación Dosis-Respuesta a Droga , Hipocampo/metabolismo , Masculino , Aprendizaje por Laberinto/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptor Cannabinoide CB1/genética , Canales Catiónicos TRPV/genéticaRESUMEN
Nitric oxide (NO) is involved in many physiological functions, but its role in pain signaling remains uncertain. Surprisingly, little is known about how endogenous NO affects excitatory and inhibitory synaptic transmission at the spinal level. Here we determined how NO affects excitatory and inhibitory synaptic inputs to dorsal horn neurons using whole-cell recordings in rat spinal cord slices. The NO precursor L-arginine or the NO donor SNAP significantly increased the frequency of glycinergic spontaneous and miniature inhibitory postsynaptic currents (IPSCs) of lamina II neurons. However, neither L-arginine nor SNAP had any effect on GABAergic IPSCs. L-arginine and SNAP significantly reduced the amplitude of monosynaptic excitatory postsynaptic currents (EPSCs) evoked from the dorsal root with an increase in paired-pulse ratio. Inhibition of the soluble guanylyl cyclase abolished the effect of L-arginine on glycinergic IPSCs but not on evoked monosynaptic EPSCs. Also, inhibition of protein kinase G blocked the increase in glycinergic sIPSCs by the cGMP analog 8-bromo-cGMP. The inhibitory effects of L-arginine on evoked EPSCs and high voltage-activated Ca(2+) channels expressed in HEK293 cells and dorsal root ganglion neurons were abolished by blocking the S-nitrosylation reaction with N-ethylmaleimide. Intrathecal injection of L-arginine and SNAP significantly increased mechanical nociceptive thresholds. Our findings suggest that spinal endogenous NO enhances inhibitory glycinergic input to dorsal horn neurons through sGC-cGMP-protein kinase G. Furthermore, NO reduces glutamate release from primary afferent terminals through S-nitrosylation of voltage-activated Ca(2+) channels. Both of these actions probably contribute to inhibition of nociceptive transmission by NO at the spinal level.
Asunto(s)
Ácido Glutámico/metabolismo , Glicina/metabolismo , Óxido Nítrico/farmacología , Nociceptores/metabolismo , Células del Asta Posterior/efectos de los fármacos , Células del Asta Posterior/metabolismo , Transmisión Sináptica/efectos de los fármacos , Animales , Canales de Calcio/metabolismo , Línea Celular , GMP Cíclico/metabolismo , Proteínas Quinasas Dependientes de GMP Cíclico/metabolismo , Ganglios Espinales/efectos de los fármacos , Ganglios Espinales/metabolismo , Guanilato Ciclasa/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Masculino , Nitrosación/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Ácido gamma-Aminobutírico/metabolismoRESUMEN
Abnormal hyperexcitability of primary sensory neurons plays an important role in neuropathic pain. Voltage-gated potassium (Kv) channels regulate neuronal excitability by affecting the resting membrane potential and influencing the repolarization and frequency of the action potential. In this study, we determined changes in Kv channels in dorsal root ganglion (DRG) neurons in a rat model of diabetic neuropathic pain. The densities of total Kv, A-type (IA) and sustained delayed (IK) currents were markedly reduced in medium- and large-, but not in small-, diameter DRG neurons in diabetic rats. Quantitative RT-PCR analysis revealed that the mRNA levels of IA subunits, including Kv1.4, Kv3.4, Kv4.2, and Kv4.3, in the DRG were reduced approximately 50% in diabetic rats compared with those in control rats. However, there were no significant differences in the mRNA levels of IK subunits (Kv1.1, Kv1.2, Kv2.1, and Kv2.2) in the DRG between the two groups. Incubation with brain-derived neurotrophic factor (BDNF) caused a large reduction in Kv currents, especially IA currents, in medium and large DRG neurons from control rats. Furthermore, the reductions in Kv currents and mRNA levels of IA subunits in diabetic rats were normalized by pre-treatment with anti-BDNF antibody or K252a, a TrkB tyrosine kinase inhibitor. In addition, the number of medium and large DRG neurons with BDNF immunoreactivity was greater in diabetic than control rats. Collectively, our findings suggest that diabetes primarily reduces Kv channel activity in medium and large DRG neurons. Increased BDNF activity in these neurons likely contributes to the reduction in Kv channel function through TrkB receptor stimulation in painful diabetic neuropathy.
Asunto(s)
Factor Neurotrófico Derivado del Encéfalo/fisiología , Diabetes Mellitus Experimental/metabolismo , Neuropatías Diabéticas/metabolismo , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Canales de Potasio con Entrada de Voltaje/metabolismo , Células Receptoras Sensoriales/patología , Potenciales de Acción/fisiología , Animales , Carbazoles/administración & dosificación , Diabetes Mellitus Experimental/enzimología , Diabetes Mellitus Experimental/patología , Neuropatías Diabéticas/enzimología , Neuropatías Diabéticas/patología , Alcaloides Indólicos/administración & dosificación , Masculino , Canales de Potasio con Entrada de Voltaje/fisiología , Ratas , Ratas Sprague-Dawley , Receptor trkB/fisiología , Células Receptoras Sensoriales/enzimología , Células Receptoras Sensoriales/metabolismoRESUMEN
OBJECTIVES: This study was carried out to investigate the effects of WIN55,212-2, a potential cannabinoid receptor agonist, on voltage-gated sodium currents I(Na) in cultured trigeminal ganglion neurons of rats, and to investigate whether the anti-nociceptive effects of cannabinoid receptor subtype 1 (CB1) were produced through its modulation on I(Na). METHODS: Whole cell patch clamp techniques were used to record I(Na) before and after WIN55,212-2 was perfused in cultured trigeminal ganglion neurons of rats. RESULTS: WIN55,212-2 (0.01 micromol/l) could enhance I(Na) slightly by 11.5 +/- 4.7% (n=7, p<0.05), and this effect could not be blocked by AM251, the CB1 receptor antagonist. However, WIN55,212-2 could inhibit I(Na) in concentration dependent manner at concentrations from 0.1 to 100 micromol/l. The inhibitory rates were 17.4 +/- 6.0, 22.5 +/- 7.8, 43.9 +/- 9.4 and 73.9 +/- 6.7% respectively by 0.1, 1, 10, 100 micromol/l WIN55,212-2, and the EC(50) was 17.8 micromol/l (n=7, p<0.05 or p<0.01). This inhibitory effect could be blocked partly by 1 micromol/l AM251 (n=7, p<0.05). WIN55,212-2 (0.01 micromol/l) shifted the active curve of I(Na) leftward slightly (n=7, p<0.05), but had no effect on its stable inactive curve (n=7, p>0.05). WIN55,212-2 (10 micromol/l) did not affect the active and stable inactive curves of I(Na) (n=7, p>0.05). CONCLUSION: WIN55,212-2 had bidirectional (two phases) effects on I(Na) in trigeminal ganglion neurons. It might act on different receptors, and the CB1 receptor participated in its modulation on I(Na).
Asunto(s)
Benzoxazinas/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Morfolinas/farmacología , Naftalenos/farmacología , Neuronas/efectos de los fármacos , Canales de Sodio/fisiología , Ganglio del Trigémino/citología , Animales , Células Cultivadas , Relación Dosis-Respuesta a Droga , Relación Dosis-Respuesta en la Radiación , Interacciones Farmacológicas , Estimulación Eléctrica/métodos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/efectos de la radiación , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Potenciales de la Membrana/efectos de la radiación , Técnicas de Placa-Clamp/métodos , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Sprague-DawleyRESUMEN
AIM: To investigate the effect of capsaicin on IA and IK in cultured rat trigeminal ganglion (TG) neurons. METHODS: Whole-cell patch clamp technique was used to record the IA and IK before and after capsaicin perfusion at different concentrations. RESULTS: In capsaicin-sensitive (CS) neurons, capsaicin was shown to selectively inhibit IA in dose-dependent manner, the IC50 was 0.99 micromol x L(-1). Yet capsaicin showed no inhibitory effect on IK, capsaicin (10 micromol x L(-1)) only slightly inhibited IK by 13.2%. In capsaicin-insensitive (CIS) neurons, capsaicin (1 micromol x L(-1)) showed no significant inhibitory effect on IA and IK, capsaicin (10 micromol x L(-1)) only slightly inhibited IA and IK by 16.8% and 15.3%, respectively. Neither 1 micromol x L(-1) nor 10 micromol x L(-1) capsaicin showed effect on the G-V curve of IA and IK. CONCLUSION: Capsaicin was found to selectively inhibit the IA current in CS neurons, and this effect may contribute to hyperalgesia when capsaicin was first used.