RESUMEN
Astrocytes have multiple functions such as provision of nourishment and mechanical support to the nervous system, helping to clear extracellular metabolites of neurons and modulating synaptic transmission by releasing gliotransmitters. In excitable cells, voltage-gated K(+) (Kv) channels serve to repolarize during action potentials. Astrocytes are considered non-excitable cells since they are not able to generate action potentials. There is an abundant expression of various Kv channels in astrocytes but the functions of these Kv channels remain unclear. We examined whether these astrocyte Kv channels regulate astrocyte "excitability" in the form of cytosolic Ca(2+) signaling. Electrophysiological examination revealed that neonatal rat cortical astrocytes possessed both delayed rectifier type and A-type Kv channels. Pharmacological blockade of both delayed rectifier Kv channels by TEA and A-type Kv channels by quinidine significantly suppressed store-operated Ca(2+) influx; however, TEA alone or quinidine alone did not suffice to cause such suppression. TEA and quinidine together dramatically enhanced current injection-triggered membrane potential overshoot (depolarization); either drug alone caused much smaller enhancements. Taken together, the results suggest both delayed rectifier and A-type Kv channels regulate astrocyte Ca(2+) signaling via controlling membrane potential.
Asunto(s)
Astrocitos/metabolismo , Astrocitos/fisiología , Calcio/metabolismo , Canales de Potasio con Entrada de Voltaje/metabolismo , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Animales , Astrocitos/efectos de los fármacos , Células Cultivadas , Electrofisiología/métodos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Neuronas/efectos de los fármacos , Neuronas/metabolismo , Neuronas/fisiología , Bloqueadores de los Canales de Potasio/farmacología , Ratas , Ratas Sprague-Dawley , Transducción de Señal/efectos de los fármacos , Transducción de Señal/fisiologíaRESUMEN
BACKGROUND: Strong P2X7 receptor (P2X7R) activation causes Ca(2+) overload and consequent cell death. We previously showed that depletion of Ca(2+) stores and endoplasmic reticulum (ER) stress in differentiated NG108-15 neuronal cells contributed to P2X7R-mediated cytotoxicity. In this work, we assessed whether taurine (2-aminoethanesulfonic acid) could prevent this P2X7R-mediated cytotoxicity in this neuronal cell line. METHODS: Cytotoxicity markers were assessed by MTT assay and Western blotting. Cytosolic Ca(2+) and mitochondrial Ca(2+) concentrations were measured microfluorimetrically using fura-2 and rhod-2, respectively. Intracellular reactive oxygen species (ROS) production was assayed by the indicator 2',7'-dichlorodihydrofluorescein diacetate. RESULTS: Selective P2X7R agonist BzATP treatment causes neuronal cell death by causing cytosolic Ca(2+) overload, depletion of Ca(2+) stores, endoplasmic reticulum (ER) stress, and caspase-3 activation (cleaved caspase 3). Remarkably, taurine (10mM) pretreatment could prevent P2X7R-mediated neuronal cell death by blocking BzATP-mediated ER stress as determined by phosphorylated eukaryotic translation initiation factor 2α (peIF2α) and C/EBP-homologous protein (CHOP). However, taurine did not block BzATP-induced Ca(2+) overload and depletion of ER Ca(2+) stores. Interestingly, P2X7R activation did not result in mitochondrial Ca(2+) overload, nor did it affect mitochondrial membrane potential. BzATP-induced generation of intracellular reactive oxygen species (ROS) was prevented by taurine. CONCLUSIONS: The neuroprotective effect by taurine is attributed to the suppression of P2X7R-mediated ER stress and ROS formation.
Asunto(s)
Diferenciación Celular/efectos de los fármacos , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Receptores Purinérgicos P2X7/metabolismo , Taurina/farmacología , Adenosina Trifosfato/análogos & derivados , Adenosina Trifosfato/toxicidad , Animales , Calcio/metabolismo , Técnicas de Cultivo de Célula , Línea Celular , Supervivencia Celular/efectos de los fármacos , Estrés del Retículo Endoplásmico/efectos de los fármacos , Potencial de la Membrana Mitocondrial/efectos de los fármacos , Ratones , Neuronas/citología , Neuronas/metabolismo , Agonistas del Receptor Purinérgico P2X/toxicidad , Especies Reactivas de Oxígeno/metabolismoRESUMEN
Although nisoxetine has been shown to elicit cutaneous (peripheral) anesthesia, spinal (central) anesthesia with nisoxetine was not exposed. The aim of this study was to examine spinal anesthesia of nisoxetine and its influence on the antinociceptive action of mepivacaine. We compared nisoxetine with an established local anesthetic mepivacaine for spinal anesthesia after rats were intrathecally injected with drugs. The drugs were spinally administered alone as well as in combination, and their potencies were compared via dose-response curves and isobolographic analysis. We showed that nisoxetine, as well as mepivacaine elicited spinal anesthesia in dose-dependent manners. On a 50% effective dose (ED50) basis, the spinal block effect of nisoxetine in motor function, proprioception, and nociception [0.99 (0.91-1.10), 0.85 (0.76-0.95), 0.82 (0.74-0.89)] was more potent (P < 0.05) than that of mepivacaine [1.28 (1.21-1.34), 1.14 (1.07-1.22), 0.99 (0.93-1.05)], respectively. Furthermore, the nociceptive/sensory blockade (ED50) was greater than the motor blockade in both nisoxetine and mepivacaine groups (P < 0.05). Saline group (vehicle) produced no spinal anesthesia. Coadministration of nisoxetine with mepivacaine displayed an additive effect. Our data reported nisoxetine produced significant anesthesia at spinal level, and additive interaction with the local anesthetic, mepivacaine. Intrathecal nisoxetine elicited more potent spinal anesthesia than mepivacaine.
Asunto(s)
Anestésicos Locales/farmacología , Fluoxetina/análogos & derivados , Mepivacaína/farmacología , Analgésicos/farmacología , Anestesia Raquidea/métodos , Animales , Relación Dosis-Respuesta a Droga , Interacciones Farmacológicas , Fluoxetina/farmacología , Inyecciones Espinales , Masculino , Ratas , Ratas Sprague-DawleyRESUMEN
Although carbetapentane produces skin (peripheral) infiltrative analgesia, the underlying mechanism of carbetapentane in local anesthesia is not well understood. The purpose of the study was to examine the effect of carbetapentane on voltage-gated Na(+) channels and its efficacy on spinal (central) anesthesia. We evaluated the effects of carbetapentane on rat motor and pain behavior (when administered intrathecally) and on voltage-gated sodium channels in differentiated neuronal NG108-15 cells. Carbetapentane exhibited dose-dependent spinal blockade with a more sensory-selective action over motor blockade (P<0.05). Carbetapentane was more potent than lidocaine (P<0.05) in spinal anesthesia. Intrathecal 5% dextrose (vehicle) elicited no spinal anesthesia. Lidocaine, used as a positive control, demonstrated concentration- and state-dependent effects on tonic block of voltage-gated Na(+) currents (IC50 of 49.6 and 194.6 µM at holding potentials of -70 and -100 mV, respectively). Carbetapentane was more potent (IC50 of 36.3 and 62.2 µM at holding potentials of -70 and -100 mV, respectively). Carbetapentane showed a much stronger frequency-dependence of block than lidocaine: with high frequency stimulation (3.33 Hz), 50 µM lidocaine produced an additional 30% blockade, while the same concentration of carbetapentane produced 70% more block. These results revealed carbetapentane had a more potent and prolonged spinal blockade with a more sensory/nociceptive-selective action over motor blockade in comparison with lidocaine. Spinal anesthesia with carbetapentane could be through inhibition of voltage-gated Na(+) currents.
Asunto(s)
Anestesia Raquidea , Ciclopentanos/farmacología , Fenómenos Electrofisiológicos/efectos de los fármacos , Sodio/metabolismo , Animales , Conducta Animal/efectos de los fármacos , Conducta Animal/fisiología , Línea Celular , Masculino , Actividad Motora/efectos de los fármacos , Actividad Motora/fisiología , Ratas , Ratas Sprague-Dawley , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
BACKGROUND: Although nisoxetine has been shown to elicit infiltrative cutaneous local anesthesia, the inhibition of voltage-gated Na(+) channels by nisoxetine has not been reported. The aim of this study was to evaluate the effect of nisoxetine on Na(+) currents and its efficacy on spinal anesthesia. METHODS: In in vitro studies, the voltage-clamp method was employed to examine whether nisoxetine blocked Na(+) currents in mouse neuroblastoma N2A cells. RESULTS: Mepivacaine showed concentration- and state-dependent effect on tonic blockade of voltage-gated Na(+) currents (IC50 of 3.7 and 74.2 µM at holding potentials of -70 and -100 mV, respectively). Nisoxetine was more potent (IC50 of 1.6 and 28.6 µM at holding potentials of -70 and -100 mV, respectively). In in vivo studies, after rats were intrathecally injected with nisoxetine and mepivacaine, the dose-response curves were constructed. Nisoxetine acted like local anesthetic mepivacaine and induced spinal anesthesia with a more sensory-selective action (p < 0.05) over motor blockade in a dose-related fashion. Intrathecal 5% dextrose (vehicle) produced no spinal anesthesia. On the 50% effective dose (ED50) basis, nisoxetine elicited more potent spinal anesthesia than did mepivacaine (p < 0.05). CONCLUSIONS: Our results showed that nisoxetine displayed a more potent and prolonged spinal anesthesia with a more sensory/nociceptive-selective action over motor blockade, compared with mepivacaine. The local anesthetic effect of nisoxetine could be probably due to the suppression of Na(+) currents.
Asunto(s)
Anestesia Raquidea/métodos , Anestésicos Locales/farmacología , Fluoxetina/análogos & derivados , Bloqueadores del Canal de Sodio Activado por Voltaje/farmacología , Anestésicos Locales/administración & dosificación , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Fluoxetina/administración & dosificación , Fluoxetina/farmacología , Concentración 50 Inhibidora , Inyecciones Espinales , Masculino , Mepivacaína/administración & dosificación , Mepivacaína/farmacología , Ratones , Neuroblastoma/metabolismo , Técnicas de Placa-Clamp , Ratas , Ratas Sprague-Dawley , Bloqueadores del Canal de Sodio Activado por Voltaje/administración & dosificación , Canales de Sodio Activados por Voltaje/efectos de los fármacos , Canales de Sodio Activados por Voltaje/metabolismoRESUMEN
BACKGROUND: Although diphenidol has long been deployed as an anti-emetic and anti-vertigo drug, its mechanism of action remains unclear. In particular, little is known as to how diphenidol affects neuronal ion channels. Recently, we showed that diphenidol blocked neuronal voltage-gated Na(+) channels, causing spinal blockade of motor function, proprioception and nociception in rats. In this work, we investigated whether diphenidol could also affect voltage-gated K(+) and Ca(2+) channels. METHODS: Electrophysiological experiments were performed to study ion channel activities in two neuronal cell lines, namely, neuroblastoma N2A cells and differentiated NG108-15 cells. RESULTS: Diphenidol inhibited voltage-gated K(+) channels and Ca(2+) channels, but did not affect store-operated Ca(2+) channels. CONCLUSION: Diphenidol is a non-specific inhibitor of voltage-gated ion channels in neuronal cells.
Asunto(s)
Canales de Calcio/efectos de los fármacos , Neuroblastoma/metabolismo , Piperidinas/farmacología , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidores , Animales , Antieméticos/farmacología , Canales de Calcio/metabolismo , Diferenciación Celular , Línea Celular Tumoral , Fenómenos Electrofisiológicos , RatonesRESUMEN
Magnolol, a polyphenolic compound isolated from Houpu, a Chinese herb from the bark of Magnolia officinalis, has been reported to have in vitro and in vivo neuroprotective effects. In spite of these reported beneficial effects, studies on the direct impact of magnolol on neuronal ion channels have been scarce. Whether magnolol affects voltage-gated Na(+) channels (VGSC) and voltage-gated K(+) (Kv) channels is unknown. Using the whole-cell voltage-clamp method, we studied the effects of magnolol on voltage-gated ion channels in neuronal NG108-15 cells. Magnolol inhibited VGSC channels with mild state-dependence (IC(50) of 15 and 30 µM, at holding potentials of -70 and -100 mV, respectively). No frequency-dependence was observed in magnolol block. Magnolol caused a left-shift of 18 mV in the steady-state inactivation curve but did not affect the voltage-dependence of activation. Magnolol inhibited Kv channels with an IC(50) of 21 µM, and it caused a 20-mV left-shift in the steady-state inactivation curve without affecting the voltage-dependence of activation. In conclusion, magnolol is an inhibitor of both VGSC and Kv channels and these inhibitory effects may in part contribute to some of the reported neuroprotective effects of magnolol.
Asunto(s)
Compuestos de Bifenilo/farmacología , Lignanos/farmacología , Neuronas/efectos de los fármacos , Fármacos Neuroprotectores/farmacología , Bloqueadores de los Canales de Potasio/farmacología , Canales de Potasio con Entrada de Voltaje/metabolismo , Bloqueadores de los Canales de Sodio/farmacología , Canales de Sodio/metabolismo , Línea Celular Tumoral , Humanos , Activación del Canal Iónico/efectos de los fármacos , Neuronas/metabolismo , Canales de Potasio con Entrada de Voltaje/antagonistas & inhibidoresRESUMEN
ETHNOPHARMACOLOGICAL RELEVANCE: Apocynum venetum Linn. (Apocynaceae family), also called Luobuma, is a shrub which grows widely in the Xinjiang Autonomous Region of China. Its leaves are used in herbal tea for the treatment of hypertension, anxiety and depression. Animal studies have also shown that Apocynum venetum leaf extract (AVLE) also exerts anti-depressant and anti-anxiety activities. The effects of AVLE on neuronal tissues in vitro are not fully understood. MATERIALS AND METHODS: Using the whole-cell voltage-clamp method, we studied the effects of AVLE on ion channels in cultured mouse neuroblastoma N2A cells. RESULTS: AVLE inhibited voltage-gated inward Na(+) current in a reversible and concentration-dependent manner (half-inhibitory concentration was 18 µg/ml and maximum inhibition at 100 µg/ml). AVLE specifically promoted steady-state inactivation of Na(+) channels but did not affect voltage-dependence of activation. The inhibitory effect was not use-dependent and was not affected by 300µM L-NAME, suggesting that NO was not involved in the action of AVLE in neuronal cells. AVLE also had a mild inhibitory effect on voltage-gated K(+) channels, but did not affect ATP-sensitive K(+) channels. CONCLUSIONS: Since voltage-gated Na(+) and K(+) channels are associated with neuronal excitability and therefore affect neurotransmission, the modulation of neuronal ion channels by AVLE may exert neuropharmacological effects. In particular, the inhibition of voltage-gated Na(+) currents by AVLE may in part account for the psychopharmacological effects of this herbal remedy.
Asunto(s)
Ansiolíticos/farmacología , Antidepresivos/farmacología , Apocynum , Neurotransmisores/farmacología , Extractos Vegetales/farmacología , Canales de Sodio/efectos de los fármacos , Sodio/metabolismo , Animales , Línea Celular Tumoral , Relación Dosis-Respuesta a Droga , Inhibidores Enzimáticos/farmacología , Ratones , NG-Nitroarginina Metil Éster/farmacología , Neuroblastoma , Hojas de la Planta , Canales de Potasio con Entrada de Voltaje/efectos de los fármacos , Canales de Sodio/fisiologíaRESUMEN
Neuritogenesis is essential in establishing the neuronal circuitry. An important intracellular signal causing neuritogenesis is cAMP. In this report, we showed that an increase in intracellular cAMP stimulated neuritogenesis in neuroblastoma N2A cells via a PKA-dependent pathway. Two voltage-gated K(+) (Kv) channel blockers, 4-aminopyridine (4-AP) and tetraethylammonium (TEA), inhibited cAMP-stimulated neuritogenesis in N2A cells in a concentration-dependent manner that remarkably matched their ability to inhibit Kv currents in these cells. Consistently, siRNA knock down of Kv1.1, Kv1.4, and Kv2.1 expression reduced Kv currents and inhibited cAMP-stimulated neuritogenesis. Kv1.1, Kv1.4, and Kv2.1 channels were expressed in the cell bodies and neurites as shown by immunohistochemistry. Microfluorimetric imaging of intracellular [K(+)] demonstrated that [K(+)] in neurites was lower than that in the cell body. We also showed that cAMP-stimulated neuritogenesis may not involve voltage-gated Ca(2+) or Na(+) channels. Taken together, the results suggest a role of Kv channels and enhanced K(+) efflux in cAMP/PKA-stimulated neuritogenesis in N2A cells.
Asunto(s)
AMP Cíclico/farmacología , Neuritas/efectos de los fármacos , Neuritas/metabolismo , Neuroblastoma/metabolismo , Neurogénesis/efectos de los fármacos , Canales de Potasio con Entrada de Voltaje/metabolismo , 1-Metil-3-Isobutilxantina/farmacología , Animales , Canales de Calcio/metabolismo , Diferenciación Celular/efectos de los fármacos , Línea Celular Tumoral , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Silenciador del Gen/efectos de los fármacos , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Ratones , Bloqueadores de los Canales de Potasio/farmacología , Inhibidores de Proteínas Quinasas/farmacología , Canales de Sodio/metabolismo , Valinomicina/farmacologíaRESUMEN
Interaction between the selectivity filter and the adjacent pore helix of voltage-gated K(+) (Kv) channels controls pore stability during K(+) conduction. Kv channels, having their selectivity filter destabilized during depolarization, are said to undergo C-type inactivation. We examined the functionality of a residue at the pore helix of the Kv1.2 channel (V370), which reportedly affects C-type inactivation. A mutation into glycine (V370G) caused a shift in reversal potential from around -72 to -9 mV. The permeability ratios (P(Na)/P(K)) of the wild type and V370G mutant are 0.04 and 0.76, respectively. In the wild-type, P(Rb)/P(K), P(Cs)/P(K) and P(Li)/P(K) are 0.78, 0.10 and 0.05, respectively. Kv1.2 V370G channels had enhanced permeability to Rb(+) and Cs(+) (P(Rb)/P(K) and P(Cs)/P(K) are 1.63 and 1.18, respectively); however, Li(+) permeability was not significantly augmented (P(Li)/P(K) is 0.13). Therefore, in addition to its known effect on pore stability, V370 of Kv1.2 is also crucial in controlling ion selectivity.
Asunto(s)
Cationes Monovalentes/metabolismo , Activación del Canal Iónico/genética , Canal de Potasio Kv.1.2/genética , Sustitución de Aminoácidos , Cesio/metabolismo , Humanos , Litio/metabolismo , Modelos Moleculares , Neoplasias de la Boca/metabolismo , Estructura Secundaria de Proteína , Rubidio/metabolismo , Células Tumorales CultivadasRESUMEN
Lignans are natural phytochemicals which exhibit multiple pharmacological effects such as anti-inflammation, antivirus and anti-tumor activities. Whether they have effects on neural tissues and ion channels is still unknown. The effects of several arylnaphathalene lignans purified from Taiwania cryptomerioides on voltage-gated K(+) (Kv) channels in mouse neuroblastoma N2A cells were examined. These lignans included Taiwanin E, helioxanthin (HXT) and diphyllin. All lignans showed inhibitory effects on Kv channels and HXT was the most potent compound (IC(50)=1.7 µM). The mechanism of HXT block was further investigated. Its action was found to be extracellular but not intracellular. HXT accelerated current decay, caused a left-shift in steady-state inactivation curve but had no effect on voltage-dependence of activation. HXT block was unaffected by intracellular K(+) concentrations. Further, it did not affect ATP-sensitive K(+) channels. Our data therefore suggest that HXT is a potent and specific blocker of Kv channels, possibly with an inhibitory mechanism involving acceleration of slow inactivation.