RESUMEN
BACKGROUND: Chloral hydrate is a sedative-hypnotic drug widely used for relieving fear and anxiety in pediatric patients. However, mechanisms underlying the chloral hydrate-mediated analgesic action remain unexplored. Therefore, we investigated the effect of 2',2',2'-trichloroethanol (TCE), the active metabolite of chloral hydrate, on tetrodotoxin-resistant (TTX-R) Na+ channels expressed in nociceptive sensory neurons. METHODS: The TTX-R Na+ current (INa) was recorded from acutely isolated rat trigeminal ganglion neurons using the whole-cell patch-clamp technique. RESULTS: Trichloroethanol decreased the peak amplitude of transient TTX-R INa in a concentration-dependent manner and potently inhibited persistent components of transient TTX-R INa and slow voltage-ramp-induced INa at clinically relevant concentrations. Trichloroethanol exerted multiple effects on various properties of TTX-R Na+ channels; it (1) induced a hyperpolarizing shift on the steady-state fast inactivation relationship, (2) increased use-dependent inhibition, (3) accelerated the onset of inactivation, and (4) retarded the recovery of inactivated TTX-R Na+ channels. Under current-clamp conditions, TCE increased the threshold for the generation of action potentials, as well as decreased the number of action potentials elicited by depolarizing current stimuli. CONCLUSIONS: Our findings suggest that chloral hydrate, through its active metabolite TCE, inhibits TTX-R INa and modulates various properties of these channels, resulting in the decreased excitability of nociceptive neurons. These pharmacological characteristics provide novel insights into the analgesic efficacy exerted by chloral hydrate.
Asunto(s)
Nociceptores , Canales de Sodio , Ratas , Animales , Tetrodotoxina/farmacología , Tetrodotoxina/metabolismo , Nociceptores/metabolismo , Canales de Sodio/metabolismo , Canales de Sodio/farmacología , Hidrato de Cloral/farmacología , Hidrato de Cloral/metabolismo , Potenciales de la Membrana/fisiología , Ratas Sprague-Dawley , Ganglios Espinales/metabolismoRESUMEN
Palmitoylation is a common lipid modification known to regulate the functional properties of various proteins and is a vital step in the biosynthesis of voltage-activated sodium (Nav) channels. We discovered a mutation in an intracellular loop of rNav1.2a (G1079C), which results in a higher apparent affinity for externally applied PaurTx3 and ProTx-II, two voltage sensor toxins isolated from tarantula venom. To explore whether palmitoylation of the introduced cysteine underlies this observation, we compared channel susceptibility to a range of animal toxins in the absence and presence of 2-Br-palmitate, a palmitate analog that prevents palmitate incorporation into proteins, and found that palmitoylation contributes to the increased affinity of PaurTx3 and ProTx-II for G1079C. Further investigations with 2-Br-palmitate revealed that palmitoylation can regulate the gating and pharmacology of wild-type (wt) rNav1.2a. To identify rNav1.2a palmitoylation sites contributing to these phenomena, we substituted three endogenous cysteines predicted to be palmitoylated and found that the gating behavior of this triple cysteine mutant is similar to wt rNav1.2a treated with 2-Br-palmitate. As with chemically depalmitoylated rNav1.2a channels, this mutant also exhibits an increased susceptibility for PaurTx3. Additional mutagenesis experiments showed that palmitoylation of one cysteine in particular (C1182) primarily influences PaurTx3 sensitivity and may enhance the inactivation process of wt rNav1.2a. Overall, our results demonstrate that lipid modifications are capable of altering the gating and pharmacological properties of rNav1.2a.
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Lipoilación , Proteínas del Tejido Nervioso/farmacología , Proteínas del Tejido Nervioso/fisiología , Canales de Sodio/farmacología , Canales de Sodio/fisiología , Animales , Colesterol/farmacología , Cisteína/metabolismo , Espacio Intracelular/efectos de los fármacos , Espacio Intracelular/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Lipoilación/efectos de los fármacos , Proteínas Mutantes/metabolismo , Mutación/genética , Canal de Sodio Activado por Voltaje NAV1.2 , Proteínas del Tejido Nervioso/genética , Ratas , Canales de Sodio/genética , Venenos de Araña/toxicidad , XenopusRESUMEN
AIMS: Although programmed cell death protein 1 (PD-1) typically serves as a target for immunotherapies, a few recent studies have found that PD-1 is expressed in the nervous system and that neuronal PD-1 might play a crucial role in regulating neuronal excitability. However, whether brain-localized PD-1 is involved in seizures and epileptogenesis is still unknown and worthy of in-depth exploration. METHODS: The existence of PD-1 in human neurons was confirmed by immunohistochemistry, and PD-1 expression levels were measured by real-time quantitative PCR (RT-qPCR) and western blotting. Chemoconvulsants, pentylenetetrazol (PTZ) and cyclothiazide (CTZ), were applied for the establishment of in vivo (rodents) and in vitro (primary hippocampal neurons) models of seizure, respectively. SHR-1210 (a PD-1 monoclonal antibody) and sodium stibogluconate (SSG, a validated inhibitor of SH2-containing protein tyrosine phosphatase-1 [SHP-1]) were administrated to investigate the impact of PD-1 pathway blockade on epileptic behaviors of rodents and epileptiform discharges of neurons. A miRNA strategy was applied to determine the impact of PD-1 knockdown on neuronal excitability. The electrical activities and sodium channel function of neurons were determined by whole-cell patch-clamp recordings. The interaction between PD-1 and α-6 subunit of human voltage-gated sodium channel (Nav1.6) was validated by performing co-immunostaining and co-immunoprecipitation (co-IP) experiments. RESULTS: Our results reveal that PD-1 protein and mRNA levels were upregulated in lesion cores compared with perifocal tissues of surgically resected specimens from patients with intractable epilepsy. Furthermore, we show that anti-PD-1 treatment has anti-seizure effects both in vivo and in vitro. Then, we reveal that PD-1 blockade can alter the electrophysiological properties of sodium channels. Moreover, we reveal that PD-1 acts together with downstream SHP-1 to regulate sodium channel function and hence neuronal excitability. Further investigation suggests that there is a direct interaction between neuronal PD-1 and Nav1.6. CONCLUSION: Our study reveals that neuronal PD-1 plays an important role in epilepsy and that anti-PD-1 treatment protects against seizures by suppressing sodium channel function, identifying anti-PD-1 treatment as a novel therapeutic strategy for epilepsy.
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Epilepsia , Receptor de Muerte Celular Programada 1 , Humanos , Receptor de Muerte Celular Programada 1/metabolismo , Epilepsia/metabolismo , Hipocampo/metabolismo , Canales de Sodio/genética , Canales de Sodio/metabolismo , Canales de Sodio/farmacología , Convulsiones/inducido químicamente , Convulsiones/tratamiento farmacológico , Convulsiones/prevención & controlRESUMEN
BACKGROUND: In Brugada syndrome (BrS), phase 2 re-excitation/re-entry (P2R) induced by the transient outward potassium current (Ito) is a proposed arrhythmia mechanism; yet, the most common genetic defects are loss-of-function sodium channel mutations. OBJECTIVES: The authors used computer simulations to investigate how sodium channel dysfunction affects P2R-mediated arrhythmogenesis in the presence and absence of Ito. METHODS: Computer simulations were carried out in 1-dimensional cables and 2-dimensional tissue using guinea pig and human ventricular action potential models. RESULTS: In the presence of Ito sufficient to generate robust P2R, reducing sodium current (INa) peak amplitude alone only slightly potentiated P2R. When INa inactivation kinetics were also altered to simulate reported effects of BrS mutations and sodium channel blockers, however, P2R occurred even in the absence of Ito. These effects could be potentiated by delaying L-type calcium channel activation or increasing ATP-sensitive potassium current, consistent with experimental and clinical findings. INa-mediated P2R also accounted for sex-related, day and night-related, and fever-related differences in arrhythmia risk in BrS patients. CONCLUSIONS: Altered INa kinetics synergize powerfully with reduced INa amplitude to promote P2R-induced arrhythmias in BrS in the absence of Ito, establishing a robust mechanistic link between altered INa kinetics and the P2R-mediated arrhythmia mechanism.
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Síndrome de Brugada , Humanos , Animales , Cobayas , Síndrome de Brugada/genética , Arritmias Cardíacas/genética , Potenciales de Acción , Canales de Sodio/genética , Canales de Sodio/farmacología , Potasio/farmacologíaRESUMEN
There are two kinds of toxins in sea anemones: neurotoxins and pore forming toxins. As a representative of the sodium channel toxin, the neurotoxin ATX II in neurotoxin mainly affects the process of action potential and the release of transmitter to affect the inactivation of the sodium channel. As the representatives of potassium channel toxins, BgK and ShK mainly affect the potassium channel current. EqTx and Sticholysins are representative of pore forming toxins, which can form specific ion channels in cell membranes and change the concentration of internal and external ions, eventually causing hemolytic effects. Based on the above mechanism, toxins such as ATX II can also cause toxic effects in tissues and organs such as heart, lung and muscle. As an applied aspect it was shown that sea anemone toxins often have strong toxic effects on tumor cells, induce cancer cells to enter the pathway of apoptosis, and can also bind to monoclonal antibodies or directly inhibit relevant channels for the treatment of autoimmune diseases.
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Neurotoxinas , Anémonas de Mar , Animales , Neurotoxinas/toxicidad , Neurotoxinas/metabolismo , Anémonas de Mar/metabolismo , Canales de Sodio/metabolismo , Canales de Sodio/farmacología , Canales de Potasio/metabolismo , Canales de Potasio/farmacología , Membrana Celular/metabolismoRESUMEN
HBI-3000 (sulcardine sulfate) has been shown to suppress various ventricular arrhythmias in animal models. The electrophysiological properties of HBI-3000 were investigated using standard microelectrode and patch-clamp techniques in single human ventricular myocytes. HBI-3000 led to concentration-dependent suppression of dofetilide-induced early afterdepolarizations in single nonfailing human ventricular myocytes and early afterdepolarizations seen in failing ventricular myocytes. The concentration-dependent prolongation of action potential duration (APD) by HBI-3000 was bell shaped with maximum response occurring around 10 µM. Interestingly, HBI-3000 at the concentration of 10 µM modestly prolonged the APD at all 3 basic cycle lengths. The slope of APD-cycle length curve of HBI-3000 was only slightly steeper than that of control (88.8 ± 7.7 ms/s vs. 78.9 ± 5.2 ms/s in control, n = 8, P > 0.05). HBI-3000 only showed a minimal use-dependent prolongation of the APD in human ventricular myocytes. HBI-3000 inhibited fast sodium current (INa-F), late sodium channel (INa-L), L-type calcium current (ICa-L), and rapidly activating delayed rectifier K current (IKr) in single human ventricular myocytes. The estimated half-maximal inhibitory concentration values of INa-F, INa-L, ICa-L, and IKr were 48.3 ± 3.8, 16.5 ± 1.4, 32.2 ± 2.9, and 22.7 ± 2.5 µM, respectively. The ion channel profile and electrophysiological properties of HBI-3000 are similar to those of ranolazine and chronic amiodarone (reduced INa-F, INa-L, ICa-L, and IKr). HBI-3000 may be a promising antiarrhythmic agent with low proarrhythmic risk.
Asunto(s)
Antiarrítmicos/farmacología , Fenómenos Electrofisiológicos , Ventrículos Cardíacos/metabolismo , Miocitos Cardíacos/fisiología , Acetanilidas , Potenciales de Acción/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Evaluación Preclínica de Medicamentos , Ventrículos Cardíacos/fisiopatología , Humanos , Células Musculares/metabolismo , Miocitos Cardíacos/metabolismo , Técnicas de Placa-Clamp/métodos , Fenetilaminas , Piperazinas , Ranolazina , Sodio/metabolismo , Sodio/farmacología , Canales de Sodio/farmacología , Canales de Sodio/fisiología , Sulfonamidas , Ésteres del Ácido SulfúricoRESUMEN
BACKGROUND: The ability to differentiate patient-specific human induced pluripotent stem cells in cardiac myocytes (hiPSC-CM) offers novel perspectives for cardiovascular research. A number of studies, that reported mainly on current-voltage curves used hiPSC-CM to model voltage-gated Na+ channel (Nav) dysfunction. However, the expression patterns and precise biophysical and pharmacological properties of Nav channels from hiPSC-CM remain unknown. Our objective was to study the characteristics of Nav channels from hiPSC-CM and assess the appropriateness of this novel cell model. METHODS: We generated hiPSC-CM using the recently described monolayer-based differentiation protocol. RESULTS: hiPSC-CM expressed cardiac-specific markers, exhibited spontaneous electrical and contractile activities, and expressed distinct Nav channels subtypes. Electrophysiological, pharmacological, and molecular characterizations revealed that, in addition to the main Nav1.5 channel, the neuronal tetrodotoxin (TTX)-sensitive Nav1.7 channel was also significantly expressed in hiPSC-CM. Most of the Na+ currents were resistant to TTX block. Therapeutic concentrations of lidocaine, a class I antiarrhythmic drug, also inhibited Na+ currents in a use-dependent manner. Nav1.5 and Nav1.7 expression and maturation patterns of hiPSC-CM and native human cardiac tissues appeared to be similar. The 4 Navß regulatory subunits were expressed in hiPSC-CM, with ß3 being the preponderant subtype. CONCLUSIONS: The findings indicated that hiPSC-CM robustly express Nav1.5 channels, which exhibited molecular and pharmacological properties similar to those in native cardiac tissues. Interestingly, neuronal Nav1.7 channels were also expressed in hiPSC-CM and are likely to be responsible for the TTX-sensitive Nav current.
Asunto(s)
Células Madre Pluripotentes Inducidas/metabolismo , Miocitos Cardíacos/metabolismo , Canales de Sodio/farmacología , Canales de Sodio Activados por Voltaje/metabolismo , Fenómenos Bioquímicos , Fenómenos Biofísicos , Western Blotting , Humanos , Inmunohistoquímica , Células Madre Pluripotentes Inducidas/citología , Células Madre Pluripotentes Inducidas/efectos de los fármacos , Miocitos Cardíacos/citología , Técnicas de Placa-Clamp , Canales de Sodio/metabolismo , Canales de Sodio Activados por Voltaje/farmacologíaRESUMEN
The peak sodium current underlies excitability and conduction in heart muscle, but a late sodium current flowing after the peak contributes to maintaining and prolonging the action potential plateau, and also to intracellular sodium loading, which in turn increases intracellular calcium with consequent effects on arrhythmia and diastolic function. Late sodium current is pathologically increased in both genetic and acquired heart disease, making it an attractive target for therapy to treat arrhythmia, heart failure, and angina. This review provides an overview of the underlying bases for the clinical implications of late sodium current block.
Asunto(s)
Angina de Pecho , Arritmias Cardíacas , Insuficiencia Cardíaca , Canales de Sodio , Potenciales de Acción/fisiología , Angina de Pecho/tratamiento farmacológico , Angina de Pecho/metabolismo , Angina de Pecho/fisiopatología , Arritmias Cardíacas/tratamiento farmacológico , Arritmias Cardíacas/metabolismo , Arritmias Cardíacas/fisiopatología , Fármacos Cardiovasculares/farmacología , Técnicas Electrofisiológicas Cardíacas/métodos , Insuficiencia Cardíaca/tratamiento farmacológico , Insuficiencia Cardíaca/metabolismo , Insuficiencia Cardíaca/fisiopatología , Humanos , Canales de Sodio/farmacología , Canales de Sodio/fisiologíaRESUMEN
Neuropathic pain has been hypothesized to be the result of aberrant expression and function of sodium channels at the site of injury. To investigate the effects of NaV1. 8 antisense oligonucleotide on the expression of sodium channel mRNA in dorsal root ganglion (DRG) neurons in chronic neuropathic pain. 24 Sprague-Dawley rats weighing 200-260 g were anesthetized with the intraperitoneal injection of 300 mg x kg(-1) choral hydrate. The CCI model was made by loose ligation of sciatic nerve trunk by 4-0 chromic gut. The mechanical and thermal pain threshold were measured before operation and 1, 3, 5, 7, 9, 11, 13 days after operation. A PE-10 catheter was implanted in subarachnoid space at lumbar region. On the 7th postoperative day the animals were randomly divided into 4 groups. The drugs were injected intrathecally twice a day for 5 consecutive days in group 2-4. The animals were decapitated 14 days after the surgery. The L4-L6 DRG of the operated side was removed and crushed, and total RNA was extracted with Trizol reagent. The contralateral side was used as control. The change of NaV1. 8 sodium channel transcripts was determined by RT-PCR. Pain threshold was significantly lowered after CCI as compared with that in control group and was elevated 3 days after antisense oligonucleotide injection. Sensory neuron specific TTX-R sodium channel NaV1. 8 transcript was down-regulated after antisense oligonucleotide injection at the dosage of 45 microg as compared with that in CCI group (P < 0.01), and it was even greater at the dosage of 90 microg. The intrathecally injected NaV1. 8 antisense oligonucleotide can reduce the mechanical allodynia and thermal hyperalgesia partially by downregulating the SNS transcript expression.
Asunto(s)
Ganglios Espinales/metabolismo , Proteínas del Tejido Nervioso/farmacología , Neuralgia/metabolismo , Oligonucleótidos Antisentido/farmacología , Canales de Sodio/biosíntesis , Animales , Inyecciones Espinales , Masculino , Canal de Sodio Activado por Voltaje NAV1.8 , Neuralgia/etiología , Nociceptores/fisiopatología , Umbral del Dolor , ARN Mensajero/biosíntesis , ARN Mensajero/genética , Distribución Aleatoria , Ratas , Ratas Sprague-Dawley , Nervio Ciático/lesiones , Nervio Ciático/fisiopatología , Canales de Sodio/genética , Canales de Sodio/farmacologíaRESUMEN
Two models of plasma membrane oscillators may explain the regulation of calcium homeostasis in frog melanotrophs. In the majority (70%) of cells a high frequency and small amplitude fluctuations characterize the spontaneous calcium level. In the 30% of remaining cells a low frequency and high amplitude oscillations were observed. Utilization of EGTA, U73122 and ryanodine suggested that calcium homeostasis in frog melanotrophs is dependent on extra- but not on intracellular calcium pools. EGTA was able to block calcium oscillations and to decrease basal calcium level in non-oscillatory cells. omega-Conotoxin, N-type calcium channels antagonist, stopped calcium oscillations but not modified calcium level in non-oscillatory cells. Nifedipine, antagonist of L-type calcium channels, had no effect either on calcium waves formation or on basal level of calcium in non-oscillatory cells. omega-Conotoxin and nifedipine were able to decrease the spontaneous alpha-MSH release from whole NILs while only omega-conotoxin had inhibitory effect on hormonal output from dispersed melanotrophs. Nickel (Ni2+) provoked dose-dependent effect. At 2 mM concentration Ni2+ blocked either calcium oscillations or alpha-MSH release. In contrast, a 0.5 mM concentration had stimulatory effect on both the phenomenons. Similarly, mibefradil (antagonist of T-type calcium channel), was able to induce an increase in [Ca2+](i) after modification of calcium fluctuations in non-oscillatory cells. Utilization of veratridine and TTX, agonist and antagonist of Na channels, respectively, indicated that mobilization of extracellular sodium, by TTX-sensitive and TTX-resistant Na channels, stimulates a hormonal output resulting from increase of [Ca2+](i). In the presence of TTX, veratridine was able to generate a calcium oscillations, which were also observed after inactivation of TTX-sensitive channel. Bepridil (antagonist of Na-Na exchange of the Na+/Ca2+ exchanger) and Na-free medium had powerful effect on increase of [Ca2+](i). The same observations obtained after administration of ouabain, antagonist of Na+/K+ dependent ATPase, confirmed dependence of calcium homeostasis on sodium distribution. Furthermore, dibutyryl-cAMP induced calcium oscillations suggesting implication of intracellular phosphorylation in the generation of calcium waves. Taken together, our results suggest that each type of calcium homeostasis is controlled by different mechanisms. Calcium fluctuations may be ascribed to the high frequency activity of T-type calcium channel, TTX-sensitive and TTX-resistant sodium channels. Calcium oscillations may be generated by the destabilization of the steady-state Na+/Ca2+ gradient provoked by intracellular inactivation of TTX-sensitive Na channel. This ionic unbalance would increase Ca-Ca exchange of Na+/Ca2+ exchanger, which by local depolarization promotes opening of N-type calcium channel responsible for calcium wave. In both types of homeostasis, the calcium and sodium overload is avoided by opening of K+ voltage- and Ca-dependent channels, and by increase in activities of Na+/K+ ATPase and forward mode of Na+/Ca2+ exchanger.
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Señalización del Calcio/efectos de los fármacos , Hipófisis/citología , Ranidae/fisiología , Canales de Sodio/farmacología , Animales , Calcio/metabolismo , Calcio/farmacología , Células Cultivadas , Citosol/química , Homeostasis/efectos de los fármacos , Masculino , Microscopía Fluorescente , Perfusión/métodos , Canales de Potasio/farmacología , Sodio/farmacología , Canales de Sodio/efectos de los fármacos , Canales de Sodio/fisiología , Tetrodotoxina/farmacologíaRESUMEN
A site-directed antibody was used immunocytochemically to measure the distribution of sodium channels in the tissues of a spider mechanoreceptor organ. The VS-3 slit sense organ contains 7-8 pairs of bipolar sensory neurons; these neurons are representative of a wide range of arthropod mechanoreceptors. Sensory transduction is thought to occur at the tips of the dendrites and to cause action potentials that are regeneratively conducted to the cell bodies, although it has not been possible to confirm this by direct intracellular recordings from the dendrites. Wholemount preparations were labelled by immunofluorescence and thin sections were immunogold labelled, using an antibody to the highly conserved SP19 sequence of the voltage-activated sodium channel. Labelling for sodium channels was found in the neurons and in their surrounding glial cells. Both cytoplasm and membranes were labelled, but immunogold particles were clearly aligned along cell membranes, indicating that the majority of labelling represented membrane-bound sodium channels. Channel density in the dendrites was similar to the axons and higher than in the cell bodies, supporting the idea of active conduction in the sensory dendrites. Labelling in glial cell membranes was indistinguishable from the neighboring neurons, suggesting a significant role for sodium channels in the functions of these supporting cells.
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Mecanorreceptores/metabolismo , Canales de Sodio/metabolismo , Arañas/metabolismo , Potenciales de Acción/fisiología , Secuencia de Aminoácidos , Animales , Dendritas/metabolismo , Dendritas/fisiología , Dendritas/ultraestructura , Femenino , Inmunohistoquímica , Mecanorreceptores/ultraestructura , Microscopía Electrónica , Datos de Secuencia Molecular , Neuroglía/fisiología , Neuronas Aferentes/metabolismo , Neuronas Aferentes/fisiología , Neuronas Aferentes/ultraestructura , Neuropéptidos/farmacología , Canales de Sodio/farmacología , Canales de Sodio/ultraestructuraRESUMEN
The amiloride-sensitive epithelial Na(+) channel (ENaC), which is made of three different but homologous subunits, controls the rate of transepithelial Na(+) absorption in a variety of epithelia. The present study investigated the functional role of its subunits in regulating ENaC activity, measured as amiloride sensitive short-circuit current (I(SC)), in the mouse endometrial epithelium under different culture conditions. The treatment of the cultured epithelia with aldosterone (1 microM) or culturing cells on filters coated with concentrated Matrigel resulted in an increase in the amiloride-sensitive I(SC). Semiquantitative RT-PCR demonstrated that the expression of alpha and beta subunits was not significantly altered by these treatments, but an increase in the gamma subunit expression was observed. An 11-fold increase, induced by aldosterone, in the expression of the gamma subunit, but not in the alpha and beta subunits, was confirmed by capillary electrophoresis with laser-induced fluorescence (CE-LIF). The treatment of endometrial cells with antisense against the gammaENaC subunit abolished the aldosterone-enhanced amiloride-sensitive I(SC). The results indicated an important role of gammaENaC subunit in determining ENaC activity, and a possible role of the gammaENaC subunit in interacting with CFTR was also discussed.
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Endometrio/fisiología , Canales de Sodio/farmacología , Canales de Sodio/fisiología , Sodio/farmacocinética , Absorción , Aldosterona/farmacología , Amilorida/farmacología , Animales , Técnicas de Cultivo de Célula , Diuréticos/farmacología , Electroforesis Capilar , Endometrio/citología , Células Epiteliales/fisiología , Canales Epiteliales de Sodio , Femenino , Regulación de la Expresión Génica , Ratones , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Regulación hacia ArribaRESUMEN
To study the mechanism of the increases in cytosolic Ca2+ ([Ca2+]i) induced by anoxia, the author examined the effect of removing extracellular Ca2+ on the [Ca2+]i response in cultured nodose ganglion neurons (control) and carotid body glomus cells prepared from newborn rabbits using a fura-2 microfluorimetry. The sensory neurons showed a small increase in [Ca2+]i during anoxia which was not affected by the removal of extracellular Ca2+ or D600. On the other hand, in the chemoreceptor glomus cells, Ca2+ removal eliminated the anoxia-induced large [Ca2+]i increase in 85% of the tested cells (n = 67), and depressed it in the rest. Interestingly, recovery from exposure to an anoxic Ca(2+)-free solution reversibly produced a large and transient rise in [Ca2+]i. The magnitude of this post-anoxic/Ca(2+)-free [Ca2+]i transient correlated with the intensity of the suppression of the [Ca2+]i response to anoxia in Ca(2+)-free solution. Also the [Ca2+]i transient was mostly inhibited by L-type Ca2+ channel blockers (nifedipine and D600), but was not affected by tetrodotoxin. These results suggest that the anoxia-induced large increase in [Ca2+]i were, in most glomus cells, coming from extracellular sources and, in a few cells, from both extracellular sources and from intracellular pools, whereas the slight increase in [Ca2+]i in sensory neurons was probably produced by releasing Ca2+ from intracellular stores. The post-anoxic/Ca(2+)-free [Ca2+]i transient, seen in the chemoreceptor cells alone, may have resulted from modification of the anoxic [Ca2+]i response by Ca2+ removal and involves mostly L-type Ca2+ channels and a few other Ca2+ entry pathways.
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Calcio/metabolismo , Cuerpo Carotídeo/metabolismo , Citosol/metabolismo , Neuronas Aferentes/metabolismo , Oxígeno/metabolismo , Animales , Animales Recién Nacidos , Bloqueadores de los Canales de Calcio/farmacología , Cuerpo Carotídeo/efectos de los fármacos , Células Cultivadas , Células Quimiorreceptoras/efectos de los fármacos , Células Quimiorreceptoras/metabolismo , Citofotometría , Transporte Iónico/efectos de los fármacos , Neuronas Aferentes/efectos de los fármacos , Ganglio Nudoso/efectos de los fármacos , Ganglio Nudoso/metabolismo , Conejos , Bloqueadores de los Canales de Sodio , Canales de Sodio/farmacologíaRESUMEN
Changrolin (2, 6-bis[pyrrolidin-1-ylmethyl]-4-[quinazolin-4-ylamino] phenol) is an anti-arrhythmic drug derived from ß-dichroine, an active component of the Chinese medicinal herb, Dichroa febrifuga Lour. To elucidate the mechanism underlying the anti-arrhythmic effect of changrolin, we used the whole-cell patch-clamp technique to characterize the electrophysiological actions of changrolin in isolated rat cardiomyocytes. In this study, changrolin inhibited delayed rectified K(+) currents (I(K)) in a concentration-dependent manner with inhibiting the current by 11.9%±4.7%, 27.8%±3.4%, 31.5%±3.6% and 40.8%±3.7% at 10, 30, 100 and 300 µM, respectively (n=7-8). Changrolin was less effective against transient outward K(+) currents (I(to)), and only showed significantly inhibitory effect at the highest concentration (300 µM). Changrolin also induced a concentration-dependent inhibition of sodium currents (I(Na)) with an IC(50) of 10.19 µM (Hill coefficient=-1.727, n=6-7). In addition, changrolin exerted a holding potential-dependent block on Na(+) channels, produced a hyperpolarizing shift in the steady-state inactivation curve, as well as exhibited a marked frequency-dependent component to the blockade of Na(+) channels. Finally, calcium currents (I(Ca)) was decreased by changrolin in a concentration-dependent manner with an estimated IC(50) of 74.73 µM (Hill coefficient=-0.9082, n=6). In conclusion, changrolin blocks Na(+) and Ca(2+) channels, and also blocks K(+) channels (I(to) and I(K)) to some extent. Notably, changrolin preferentially blocks the inactivated state of Na(+) channels. These effects lead to a modification of electromechanical function and likely contribute to the termination of arrhythmia.
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Antiarrítmicos/farmacología , Medicamentos Herbarios Chinos , Fenómenos Electrofisiológicos/efectos de los fármacos , Miocitos Cardíacos/efectos de los fármacos , Quinazolinas/farmacología , Potenciales de Acción/efectos de los fármacos , Animales , Antiarrítmicos/metabolismo , Canales de Calcio/efectos de los fármacos , Canales de Calcio/farmacología , Ventrículos Cardíacos/efectos de los fármacos , Masculino , Técnicas de Placa-Clamp , Canales de Potasio/efectos de los fármacos , Canales de Potasio/farmacología , Quinazolinas/metabolismo , Ratas , Ratas Sprague-Dawley , Canales de Sodio/efectos de los fármacos , Canales de Sodio/farmacologíaRESUMEN
In voltage-clamp experimental on slices of the rat brainstem the effects of 5-HT and GABA on serotonergic neurons of nucleus raphe magnus were investigated. Local applications of 5-HT induced an increase in IPCSs frequency and amplitude in 45% of serotonergic cells. The effect suppressed by the blocker of fast sodium channels tetradotoxin. Antagonist of GABA receptor gabazine blocked IPSCs in neurons both sensitive and non-sensitive to 5-HT action. Applications of GABA induced a membrane current (I(GABA)), which was completely blocked by gabazine. The data suggest self-control of the activity of serotonergic neurons in nucleus raphe magnus by negative feedback loop via local GABAergic interneurons.
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Neuronas/fisiología , Núcleos del Rafe/fisiología , Serotonina/fisiología , Ácido gamma-Aminobutírico/fisiología , Animales , Retroalimentación Fisiológica , Antagonistas de Receptores de GABA-A , Técnicas In Vitro , Activación del Canal Iónico , Masculino , Potenciales de la Membrana , Neuronas/efectos de los fármacos , Piridazinas/farmacología , Núcleos del Rafe/efectos de los fármacos , Ratas , Ratas Wistar , Serotonina/farmacología , Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Ácido gamma-Aminobutírico/farmacologíaRESUMEN
The oligodendrocyte is the myelin-forming cell in the central nervous system. Despite the close interaction between axons and oligodendrocytes, there is little evidence that neurons influence myelinogenesis. On the contrary, newly differentiated oligodendrocytes, which mature in culture in the total absence of neurons, synthesize the myelin-specific constituents of oligodendrocytes differentiated in vivo and even form myelin-like figures. Neuronal electrical activity may be required, however, for the appropriate formation of the myelin sheath. To investigate the role of electrical activity on myelin formation, we have used highly specific neurotoxins, which can either block (tetrodotoxin) or increase (alpha-scorpion toxin) the firing of neurons. We show that myelination can be inhibited by blocking the action potential of neighboring axons or enhanced by increasing their electrical activity, clearly linking neuronal electrical activity to myelinogenesis.
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Sistema Nervioso Central/crecimiento & desarrollo , Vaina de Mielina/fisiología , Potenciales de Acción , Animales , Células Cultivadas , Estimulación Eléctrica , Ratones , Microscopía Electrónica , Vaina de Mielina/efectos de los fármacos , Vaina de Mielina/ultraestructura , Nervio Óptico/crecimiento & desarrollo , Potasio/farmacología , Venenos de Escorpión/farmacología , Canales de Sodio/farmacología , Tetrodotoxina/farmacología , Factores de TiempoRESUMEN
Ion channels are important for many cellular functions and disease states including cystic fibrosis and multidrug resistance. Previous work in the Dunning rat model of prostate cancer has suggested a relationship between voltage-activated Na+ channels (VASCs) and the invasive phenotype in vitro. The objectives of this study were to 1) evaluate the expression of VASCs in the LNCaP and PC-3 human prostate cancer cell lines by Western blotting, flow cytometry, and whole-cell patch clamping, 2) determine their role in invasion in vitro using modified Boyden chambers with and without a specific blocker of VASCs (tetrodotoxin). A 260-kd protein representing VASCs was found only in the PC-3 cell line, and these were shown to be membrane expressed on flow cytometry. Patch clamping studies indicated that functional VASCs were present in 10% of PC-3 cells and blocking these by tetrodotoxin (600 nmol/L) reduced their invasiveness by 31% (P = 0.02) without affecting the invasiveness of the LNCaP cells. These results indicate that the reduction of invasion is a direct result of VASC blockade and not a nonspecific action of the drug. This is the first report of VASCs in a human prostatic cell line. VASCs are present in PC-3 but not LNCaP cells as determined by both protein and functional studies. Tetrodotoxin reduced the invasiveness of PC-3 but not LNCaP cells, and these data suggest that ion channels may play an important functional role in tumor invasion.
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Carcinoma/metabolismo , Carcinoma/patología , Neoplasias de la Próstata/metabolismo , Neoplasias de la Próstata/patología , Canales de Sodio/biosíntesis , Canales de Sodio/farmacología , Animales , Western Blotting , Encéfalo/metabolismo , Cámaras de Difusión de Cultivos , Electrofisiología , Citometría de Flujo , Humanos , Masculino , Invasividad Neoplásica , Técnicas de Placa-Clamp , Ratas , Tetrodotoxina/farmacología , Células Tumorales CultivadasRESUMEN
Aldosterone maintains total organism sodium balance in all higher vertebrates. The level of sodium reabsorption is primarily determined by the action of aldosterone on epithelial sodium channels (ENaC) in the distal nephron. Recent work shows that, in an aldosterone-sensitive renal cell line (A6), aldosterone regulates sodium reabsorption by short- and long-term processes. In the short term, aldosterone regulates sodium transport by inducing expression of the small G-protein, K-Ras2A, by stimulating the activity of methyl transferase and S-adenosyl-homocysteine hydrolase to activate Ras by methylation, and, possibly, by subsequent activation by K-Ras2A of phosphatidylinositol phosphate-5-kinase (PIP-5-K) and phosphatidylinositol-3-kinase (PI-3-K), which ultimately activates ENaC. In the long term, aldosterone regulates sodium transport by altering trafficking, assembly, and degradation of ENaC.
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Aldosterona/farmacología , Células Epiteliales/metabolismo , Canales de Sodio/farmacología , Sodio/farmacocinética , Aldosterona/metabolismo , Aldosterona/fisiología , Animales , Anuros , Transporte Biológico/fisiología , Membrana Celular/fisiología , Conductividad Eléctrica , Células Epiteliales/efectos de los fármacos , Canales Epiteliales de Sodio , Proteínas de Unión al GTP/genética , Proteínas de Unión al GTP/metabolismo , Técnicas de Placa-Clamp/métodos , Canales de Sodio/farmacocinética , ATPasa Intercambiadora de Sodio-Potasio/fisiologíaRESUMEN
BACKGROUND & AIMS: Visceral mechanoreceptors are critical for perceived sensations and autonomic reflex control of gastrointestinal function. However, the molecular mechanisms underlying visceral mechanosensation remain poorly defined. Degenerin/epithelial Na+ channel (DEG/ENaC) family ion channels are candidate mechanosensory molecules, and we hypothesized that they influence visceral mechanosensation. We examined the influence of the DEG/ENaC channel ASIC1 on gastrointestinal mechanosensory function, on gastric emptying, and on fecal output. We also compared its role in gastrointestinal and somatic sensory function. METHODS: To assess the role of ASIC1 we studied wild-type and ASIC1-/- mice. Reverse-transcription polymerase chain reaction (RT-PCR) and Western blot analysis determined expression of ASIC1 messenger RNA and protein in vagal and spinal sensory ganglia. Colonic, gastroesophageal, and cutaneous afferent fibers were characterized by functional subtype and their mechanical stimulus-response relationships were determined. Gastric emptying was determined by using a 13CO2 breath test. Behavioral tests assessed somatic mechanical and thermal sensitivity. RESULTS: ASIC1 was expressed in sensory ganglia and was lost after disruption of the ASIC1 gene. Loss of ASIC1 increased mechanosensitivity in all colonic and gastroesophageal mechanoreceptor subtypes. In addition, ASIC1-/- mice showed almost double the gastric emptying time of wild-type mice. In contrast, loss of ASIC1 did not affect function in any of the 5 types of cutaneous mechanoreceptors, nor did it affect paw withdrawal responses or fecal output. CONCLUSIONS: ASIC1 influences visceral but not cutaneous mechanoreceptor function, suggesting that different mechanisms underlie mechanosensory function in gut and skin. The role of ASIC1 is highlighted by prolonging gastric emptying of a meal in ASIC1-/- animals.
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Vaciamiento Gástrico/fisiología , Mecanorreceptores/fisiología , Proteínas de la Membrana/genética , Proteínas de la Membrana/farmacología , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/farmacología , Canales de Sodio/genética , Canales de Sodio/farmacología , Canales Iónicos Sensibles al Ácido , Animales , Pruebas Respiratorias , Dióxido de Carbono/análisis , Fenómenos Fisiológicos del Sistema Digestivo , Conducta Alimentaria , Regulación de la Expresión Génica , Humanos , Proteínas de la Membrana/biosíntesis , Ratones , Proteínas del Tejido Nervioso/biosíntesis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Canales de Sodio/biosíntesisRESUMEN
The regulatory role of actin filament organization on epithelial Na+ channel activity is reviewed in this report. The actin cytoskeleton, consisting of actin filaments and associated actin-binding proteins, is essential to various cellular events including the maintenance of cell shape, the onset of cell motility, and the distribution and stability of integral membrane proteins. Functional interactions between the actin cytoskeleton and specific membrane transport proteins are, however, not as well understood. Recent studies from our laboratory have determined that dynamic changes in the actin cytoskeletal organization may represent a novel signaling mechanism in the regulation of ion transport in epithelia. This report summarizes work conducted in our laboratory leading to an understanding of the molecular steps associated with the regulatory role of the actin-based cytoskeleton on epithelial Na+ channel function. The basis of this interaction lies on the regulation by actin-binding proteins and adjacent structures, of actin filament organization which in turn, modulates ion channel activity. The scope of this interaction may extend to such relevant cellular events as the vasopressin response in the kidney.