RESUMEN
Mineralization of bones and teeth is tightly regulated by levels of extracellular inorganic phosphate (Pi) and pyrophosphate (PPi). Three regulators that control pericellular concentrations of Pi and PPi include tissue-nonspecific alkaline phosphatase (TNAP), progressive ankylosis protein (ANK), and ectonucleotide pyrophosphatase/phosphodiesterase 1 (ENPP1). Inactivation of these factors results in mineralization disorders affecting teeth and their supporting structures. This study for the first time analyzed the effect of decreased PPi on dental development in individuals with generalized arterial calcification of infancy (GACI) due to loss-of-function mutations in the ENPP1 gene. Four of the 5 subjects reported a history of infraocclusion, overretained primary teeth, ankylosis, and/or slow orthodontic tooth movement, suggesting altered mineral metabolism contributing to disrupted tooth movement and exfoliation. All subjects had radiographic evidence of unusually protruding cervical root morphology in primary and/or secondary dentitions. High-resolution micro-computed tomography (micro-CT) analyses of extracted primary teeth from 3 GACI subjects revealed 4-fold increased cervical cementum thickness ( P = 0.00007) and a 23% increase in cementum density ( P = 0.009) compared to age-matched healthy control teeth. There were no differences in enamel and dentin densities between GACI and control teeth. Histology revealed dramatically expanded cervical cementum in GACI teeth, including cementocyte-like cells and unusual patterns of cementum resorption and repair. Micro-CT analysis of Enpp1 mutant mouse molars revealed 4-fold increased acellular cementum thickness ( P = 0.002) and 5-fold increased cementum volume ( P = 0.002), with no changes in enamel or dentin. Immunohistochemistry identified elevated ENPP1 expression in cementoblasts of human and mouse control teeth. Collectively, these findings reveal a novel dental phenotype in GACI and identify ENPP1 genetic mutations associated with hypercementosis. The sensitivity of cementum to reduced PPi levels in both human and mouse teeth establishes this as a well-conserved and fundamental biological process directing cementogenesis across species (ClinicalTrials.gov NCT00369421).
Asunto(s)
Hipercementosis/diagnóstico por imagen , Hipercementosis/genética , Mutación con Pérdida de Función , Hidrolasas Diéster Fosfóricas/genética , Pirofosfatasas/genética , Calcificación Vascular/genética , Adulto , Animales , Niño , Femenino , Genotipo , Humanos , Masculino , Ratones , Linaje , Radiografía Panorámica , Diente Primario , Microtomografía por Rayos XRESUMEN
The tumor suppressor protein promyelocytic leukemia (PML) is a key regulator of inflammatory responses and tumorigenesis and functions through the assembly of subnuclear structures known as PML nuclear bodies (NBs). The inflammation-related cytokine tumor necrosis factor-α (TNFα) is known to induce PML protein accumulation and PML NB formation that mediate TNFα-induced cell death in cancer cells and inhibition of migration and capillary tube formation in endothelial cells (ECs). In this study, we uncover a novel mechanism of PML gene regulation in which the p38 MAPK and its downstream kinase MAP kinase-activated protein kinase 1 (MNK1) mediate TNFα-induced PML protein accumulation and PML NB formation. The mechanism includes the presence of an internal ribosome entry site (IRES) found within the well-conserved 100 nucleotides upstream of the PML initiation codon. The activity of the PML IRES is induced by TNFα in a manner that involves MNK1 activation. It is proposed that the p38-MNK1-PML network regulates TNFα-induced apoptosis in breast cancer cells and TNFα-mediated inhibition of migration and capillary tube formation in ECs.
Asunto(s)
Apoptosis , Células Endoteliales de la Vena Umbilical Humana/fisiología , Proteínas Nucleares/genética , Biosíntesis de Proteínas , Factores de Transcripción/genética , Factor de Necrosis Tumoral alfa/fisiología , Proteínas Supresoras de Tumor/genética , Regiones no Traducidas 5' , Neoplasias de la Mama , Movimiento Celular , Proliferación Celular , Quimiocina CCL2/genética , Quimiocina CCL2/metabolismo , Regulación Neoplásica de la Expresión Génica , Células HL-60 , Humanos , Sitios Internos de Entrada al Ribosoma , Péptidos y Proteínas de Señalización Intracelular/metabolismo , Células MCF-7 , Metaloproteinasa 10 de la Matriz/genética , Metaloproteinasa 10 de la Matriz/metabolismo , Neovascularización Fisiológica , Proteínas Nucleares/metabolismo , Proteína de la Leucemia Promielocítica , Proteínas Serina-Treonina Quinasas/metabolismo , Factores de Transcripción/metabolismo , Proteínas Supresoras de Tumor/metabolismo , Proteínas Quinasas p38 Activadas por Mitógenos/metabolismoRESUMEN
BACKGROUND AND PURPOSE: Little information exists on the mechanisms that precipitate brain stem death, the legal definition of death in many developed countries. We investigated the role of tropomyocin receptor kinase B (TrkB) and its downstream signalling pathways in the rostral ventrolateral medulla (RVLM) during experimental brain stem death. EXPERIMENTAL APPROACH: An experimental model of brain stem death that employed microinjection of the organophosphate insecticide mevinphos bilaterally into the RVLM of Sprague-Dawley rats was used, in conjunction with cardiovascular, pharmacological and biochemical evaluations. KEY RESULTS: A significant increase in TrkB protein, phosphorylation of TrkB at Tyr(516) (pTrkB(Y516) ), Shc at Tyr(317) (pShc(Y317) ) or ERK at Thr(202) /Tyr(204) , or Ras activity in RVLM occurred preferentially during the pro-life phase of experimental brain stem death. Microinjection bilaterally into RVLM of a specific TrkB inhibitor, K252a, antagonized those increases. Pretreatment with anti-pShc(Y317) antiserum, Src homology 3 binding peptide (Grb2/SOS inhibitor), farnesylthioacetic acid (Ras inhibitor), manumycin A (Ras inhibitor) or GW5074 (Raf-1 inhibitor) blunted the preferential augmentation of Ras activity or ERK phosphorylation in RVLM and blocked the up-regulated NOS I/protein kinase G (PKG) signalling, the pro-life cascade that sustains central cardiovascular regulation during experimental brain stem death. CONCLUSIONS AND IMPLICATIONS: Activation of TrkB, followed by recruitment of Shc/Grb2/SOS adaptor proteins, leading to activation of Ras/Raf-1/ERK signalling pathway plays a crucial role in ameliorating central cardiovascular regulatory dysfunction via up-regulation of NOS I/PKG signalling cascade in the RVLM in brain stem death. These findings provide novel information for developing therapeutic strategies against this fatal eventuality.
Asunto(s)
Muerte Encefálica , Sistema Cardiovascular/efectos de los fármacos , Inhibidores de la Colinesterasa/toxicidad , Mevinfos/toxicidad , Receptor trkB/metabolismo , Animales , Western Blotting , Sistema Cardiovascular/fisiopatología , Activación Enzimática , Ensayo de Inmunoadsorción Enzimática , Masculino , Microinyecciones , Fosforilación , Ratas , Ratas Sprague-DawleyRESUMEN
We demonstrate a chip-scale compact optical curvature sensor. It consists of a low threshold InGaAsP microdisk laser on a flexible polydimethylsiloxane polymer substrate. The curvature dependence of lasing wavelength was characterized by bending the cavity at different bending radii. The measurements showed that the lasing wavelength decreases monotonously with an increasing bending curvature. A good agreement between experiment and three-dimensional finite-difference time-domain simulation was also obtained. The sensitivity of the compact device to the bending curvature is -23.7 nm/mm from the experiment.
RESUMEN
Compact microdisk cavities were fabricated on a polydimethylsiloxane substrate. The lasing of the flexible compact cavity was achieved with a low threshold power. The whispering-gallery mode of the microdisk was also characterized with three-dimensional finite-difference time-domain simulation. The curvature dependence in output power and threshold was also demonstrated by bending the microdisk cavity.
RESUMEN
Reversal of long term potentiation (LTP) may function to increase the flexibility and storage capacity of neuronal circuits; however, the underlying mechanisms remain incompletely understood. We show that depotentiation induced by low frequency stimulation (LFS) (2 Hz, 10 min, 1200 pulses) was input-specific and dependent on N-methyl-d-aspartate (NMDA) receptor activation. The ability of LFS to reverse LTP was mimicked by a brief application of NMDA. This NMDA-induced depotentiation was blocked by adenosine A(1) receptor antagonist. However, the reversal of LTP by LFS was unaffected by metabotropic glutamate receptor antagonism. This LFS-induced depotentiation was specifically prevented by protein phosphatase (PP)1 inhibitors, okadaic acid, and calyculin A but not by the PP2A or PP2B inhibitors. Furthermore, by using phosphorylation site-specific antibodies, we found that LFS-induced depotentiation is associated with a persistent dephosphorylation of the GluR1 subunit of amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor at serine 831, a protein kinase C and calcium/calmodulin-dependent protein kinase II (CaMKII) substrate, but not at serine 845, a substrate of cAMP-dependent protein kinase. This effect was mimicked by bath-applied adenosine or NMDA and was specifically prevented by okadaic acid. Also, the increased phosphorylation of CaMKII at threonine 286 and the decreased PP activity seen with LTP were overcome by LFS, adenosine, or NMDA application. These results suggest that LFS erases LTP through an NMDA receptor-mediated activation of PP1 to dephosphorylate amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptors and CaMKII in the CA1 region of the hippocampus.
Asunto(s)
Estimulación Eléctrica , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Sinapsis/fisiología , Adenosina/metabolismo , Animales , Hipocampo/enzimología , Técnicas In Vitro , Masculino , Fosfoproteínas Fosfatasas/metabolismo , Ratas , Ratas Sprague-Dawley , Receptores AMPA/metabolismo , Receptores de Glutamato Metabotrópico/metabolismo , Receptores de N-Metil-D-Aspartato/metabolismo , Transducción de SeñalRESUMEN
Using mouse hippocampal slices, we studied the induction of depotentiation of long-term potentiation (LTP) at the mossy fiber synapses onto CA3 pyramidal neurons. A long train of low-frequency (1 Hz/900 pulses) stimulation (LFS) induced a long-term depression of baseline synaptic transmission or depotentiation of previously established LTP, which was reversible and was independent of NMDA receptor activation. This LFS-induced depotentiation was observed when the stimulus was delivered 1 or 10 min after LTP induction. However, when LFS was applied at 30 min after induction, significantly less depotentiation was found. The induction of depotentiation on one input was associated with a heterosynaptic reverse of the LTP induced previously on a separate pathway. In addition, this LFS-induced depotentiation appeared to be mediated by the activation of group 2 metabotropic glutamate receptors (mGluRs), because it was mimicked by the bath-applied group 2 agonist (2S,2'R,3'R)-2-(2', 3'-dicarboxycyclopropyl) glycine and was specifically inhibited by the group 2 antagonists (S)-alpha-methyl-4-carboxyphenylglycine and (alphaS)-alpha-amino-alpha-(1S,2S)-2-carboxycyclopropyl-9H-xanthine-9-propanic acid. Moreover, the induction of depotentiation was entirely normal when synaptic transmission is blocked by glutamate receptor antagonist kynurenic acid and was associated with a reversal of paired-pulse facilitation attenuation during LTP expression. Pretreatment of the hippocampal slices with G(i/o)-protein inhibitor pertussis toxin (PTX) prevented the LFS-induced depotentiation. These results suggest that the activation of presynaptic group 2 mGluRs and in turn triggering a PTX-sensitive G(i/o)-protein-coupled signaling cascade may contribute to the LFS-induced depotentiation at the mossy fiber-CA3 synapses.
Asunto(s)
Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Sinapsis/fisiología , Animales , Estimulación Eléctrica/métodos , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/antagonistas & inhibidores , Hipocampo/efectos de los fármacos , Técnicas In Vitro , Ácido Quinurénico/farmacología , Potenciación a Largo Plazo/efectos de los fármacos , Ratones , Ratones Endogámicos ICR , Fibras Musgosas del Hipocampo/efectos de los fármacos , Fibras Musgosas del Hipocampo/fisiología , Toxina del Pertussis , Células Piramidales/efectos de los fármacos , Células Piramidales/metabolismo , Receptores de Glutamato Metabotrópico/agonistas , Receptores de Glutamato Metabotrópico/antagonistas & inhibidores , Receptores de Glutamato Metabotrópico/metabolismo , Transducción de Señal/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Factores de Virulencia de Bordetella/farmacologíaRESUMEN
The striatum is a crucial site of action for the motor effects of cannabinoids (CBs). However, the electrophysiological consequences of activation of CB receptors on the striatal neurons have not been established. Here we report for the first time that the cannabimimetic aminoalkylindole WIN 55,212-2 and the endogenous cannabinoid anandamide substantially depress corticostriatal glutamatergic synaptic transmission onto striatal neurons in the brain slice preparation. The selective CB1 receptor antagonist SR 141716 effectively reversed this inhibition. WIN 55,212-2 significantly increased the paired-pulse facilitation of synaptically evoked EPSCs, while having no effect on the sensitivity of postsynaptic neurons to [alpha]-amino-3-hydroxy-5-methylisoxazole-4-propionic acid. WIN 55,212-2 also reduced the frequency of spontaneous, action potential-dependent EPSCs (sEPSCs) without altering their amplitude distribution. Superfusion of WIN 55,212-2 elicited a membrane hyperpolarization accompanied by a decrease in input resistance. Both effects were blocked by intracellular caesium. In contrast, intracellular caesium failed to affect WIN 55,212-2-mediated synaptic inhibition. The WIN 55,212-2-mediated synaptic inhibition was blocked by the Gi/o protein inhibitor pertussis toxin (PTX), but not by the GABA(A) receptor antagonist bicuculline or GABA(B) receptor antagonist SCH 50911. Pretreatment with the N-type Ca2+ channel antagonist [omega]-conotoxin GVIA selectively abolished the WIN-55,212-2-mediated synaptic inhibition. These results suggest that cannabinoids depress the corticostriatal glutamatergic synaptic transmission through the activation of presynaptic CB1 receptors to inhibit N-type Ca2+ channel activity, which in turn reduces glutamate release. The presynaptic action of cannabinoids is mediated by a PTX-sensitive Gi/o protein-coupled signalling pathway.
Asunto(s)
Cuerpo Estriado/citología , Neuronas/fisiología , Terminales Presinápticos/metabolismo , Receptores de Droga/metabolismo , Transmisión Sináptica/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Analgésicos/farmacología , Animales , Benzoxazinas , Cloruro de Cadmio/farmacología , Bloqueadores de los Canales de Calcio/farmacología , Canales de Calcio Tipo N/metabolismo , Agonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Subunidades alfa de la Proteína de Unión al GTP Gi-Go/metabolismo , Ácido Glutámico/metabolismo , Masculino , Morfolinas/farmacología , Naftalenos/farmacología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Neuronas/efectos de los fármacos , Técnicas de Cultivo de Órganos , Toxina del Pertussis , Piperidinas/farmacología , Pirazoles/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de Cannabinoides , Receptores de Droga/antagonistas & inhibidores , Receptores de GABA-A/metabolismo , Receptores de GABA-B/metabolismo , Rimonabant , Transmisión Sináptica/efectos de los fármacos , Tetrodotoxina/farmacología , Factores de Virulencia de Bordetella/farmacología , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Over the past two decades there has been a progressive understanding of the properties and mechanisms underlying long-term potentiation (LTP) of synaptic efficacy, a putative mechanism for learning and memory storage in the brain. Although LTP is remarkable for its stability, recent work has provided evidence that various manipulations can disrupt LTP if applied shortly after its induction. This kind of reversal of synaptic strength from the potentiated state to pre-LTP levels is termed depotentiation. Depotentiation of LTP is effectively induced by low-frequency afferent stimulation (1-5 Hz), brief periods of hypoxia, application of adenosine receptor agonists and brief cooling shocks. The examples of depotentiation described to date are input specific, and not differently expressed during development. The mechanisms responsible for this phenomenon remain to be fully characterized, although some possibilities are dependent on NMDA receptor activation, the increases in intracellular Ca2+, and altered states of protein kinases or phosphatases. In this review, we summarize the recent data concerning putative depotentiation mechanisms and the implications of this phenomenon in the mechanisms of "forgetting", and discuss the prevention of saturation of the storage capacity of a neuronal network.
Asunto(s)
Hipocampo/fisiología , Aprendizaje/fisiología , Potenciación a Largo Plazo/fisiología , Memoria/fisiología , Neuronas/fisiología , Adenosina/metabolismo , Animales , Señalización del Calcio/fisiología , Estimulación Eléctrica/efectos adversos , Hipocampo/citología , Humanos , Neuronas/citología , Receptores de Glutamato/efectos de los fármacos , Receptores de Glutamato/metabolismo , Transmisión Sináptica/fisiologíaRESUMEN
The effects of extracellular acidification on the synaptic function and neuronal excitability were investigated on the hippocampal CA1 neurons. A decrease of extracellular pH from 7.4 to 6.7 did not alter either the resting membrane potential or the neuronal membrane input resistance. Extracellularly recorded field excitatory postsynaptic potentials (fEPSPs) and population spikes (PSs) were significantly reduced by acidosis. Additionally, the amplitude of presynaptic fiber volley was also reduced. The sensitivity of postsynaptic neurons to N-methyl-D-aspartate, but not to alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid, was depressed by acidosis. Lowering of extracellular pH did not significantly affect the magnitude of paired-pulse facilitation (PPF) of synaptic transmission. Acidosis also reversibly limited the sustained repetitive firing (RF) of Na(+)-dependent action potentials elicited by injection of depolarizing current pulses into the pyramidal cells. The limitation of RF by extracellular acidification was accompanied by the reduction of the maximal rate of rise (;V(max)) of the action potentials and the amplitude of afterhyperpolarization. Neither the Na (+)/H (+) antiporter blocker 5-(N -ethyl -N -isopropyl)-amiloride nor the selective adenosine A (1) receptor antagonist 1,3-dipropyl -8-cyclopentylxanthine, however, affected the acidosis -induced synaptic depression. It was also found that acidosis did not affect either the induction r maintenance of long -term potentiation (LTP) at Schaffer collateral -CA 1 synapses. These results suggest that the extracellular acidosis -induced synaptic depression is likely to result from an inhibition of presynaptic Na (+) conductance, thereby decreasing the amplitude of action potentials in individual afferent fibers or the number of afferent fiber activation to stimuli and then indirectly affecting the signaling processes contributing to trigger neurotransmitter release.
Asunto(s)
Acidosis/metabolismo , Espacio Extracelular/metabolismo , Hipocampo/metabolismo , Potenciación a Largo Plazo , Transmisión Sináptica , Acidosis/inducido químicamente , Potenciales de Acción/efectos de los fármacos , Adenosina/metabolismo , Adenosina/farmacología , Animales , Dióxido de Carbono/metabolismo , Dióxido de Carbono/farmacología , Membrana Celular/metabolismo , Líquido Cefalorraquídeo/metabolismo , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipocampo/citología , Ácido Clorhídrico , Concentración de Iones de Hidrógeno , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , N-Metilaspartato/metabolismo , N-Metilaspartato/farmacología , Ratas , Ratas Sprague-Dawley , Bicarbonato de Sodio , Intercambiadores de Sodio-Hidrógeno/metabolismo , Ácidos Sulfúricos , Transmisión Sináptica/efectos de los fármacos , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/metabolismo , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacologíaRESUMEN
Previous work has shown that seizure-like activity can disrupt the induction of long-term potentiation (LTP). However, how seizure-like event disrupts the LTP induction remains unknown. To understand the cellular and molecular mechanisms underlying this process better, a set of studies was implemented in area CA1 of rat hippocampal slices using extracellular recording methods. We showed here that prior transient seizure-like activity generated by perfused slices with Mg(2+)-free artificial cerebrospinal fluid (ACSF) exhibited a persistent suppression of LTP induction. This effect lasted between 2 and 3 h after normal ACSF replacement and was specifically inhibited by N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphovaleric acid (D-APV) and L-type voltage-operated Ca(2+) channel (VOCC) blocker nimodipine, but not by non-NMDA receptor antagonist 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX). In addition, this suppressive effect was specifically blocked by the selective protein kinase C (PKC) inhibitor NPC-15437. However, neither Ca(2+)/calmodulin-dependent protein kinase II inhibitor KN-62 nor cAMP-dependent protein kinase inhibitor Rp-adenosine 3', 5'-cyclic monophosphothioate (Rp-cAMPS) affected this suppressive effect. This persistent suppression of LTP was not secondary to the long-lasting changes in NMDA receptor activation, because the isolated NMDA receptor-mediated responses did not show a long-term enhancement in response to a 30-min Mg(2+)-free ACSF application. Additionally, in prior Mg(2+)-free ACSF-treated slices, the entire frequency-response curve of LTP and long-term depression (LTD) is shifted systematically to favor LTD. These results suggest that the increase of Ca(2+) influx through NMDA channels and L-type VOCCs in turn triggering a PKC-dependent signaling cascade is a possible cellular basis underlying this seizure-like activity-induced inhibition of LTP.
Asunto(s)
Hipocampo/metabolismo , Potenciación a Largo Plazo/fisiología , Magnesio/metabolismo , Proteína Quinasa C/metabolismo , Sinapsis/metabolismo , 2-Amino-5-fosfonovalerato/farmacología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Líquido Cefalorraquídeo/metabolismo , Medios de Cultivo/farmacología , Estimulación Eléctrica , Antagonistas de Aminoácidos Excitadores/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Espacio Extracelular/metabolismo , Antagonistas del GABA/farmacología , Hipocampo/citología , Técnicas In Vitro , Magnesio/farmacología , Deficiencia de Magnesio/metabolismo , Masculino , Potenciales de la Membrana/efectos de los fármacos , Plasticidad Neuronal/efectos de los fármacos , Plasticidad Neuronal/fisiología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/antagonistas & inhibidores , Receptores de N-Metil-D-Aspartato/metabolismoRESUMEN
We evaluated the anticonvulsant effect of Chai-Hu-Long-Ku-Mu-Li-Tan (TW-001), a Chinese herbal medicine, and its mechanisms in several standard rodent models of generalized seizure. TW-001 (4 g/kg, p.o.) significantly increased the threshold for tonic electroconvulsions and the threshold for tonic seizures in response to i.v. infusion of pentylenetetrazole (PTZ). In the s.c. PTZ seizure test, both the incidence and severity of seizures were decreased by TW-001. TW-001 (1-10 mg/ml) did not alter resting membrane potential or input resistance of the hippocampal CA1 neurons, but elicited a reversible suppression of stimulus-triggered epileptiform activity in area CA1 and spontaneously occuring epileptiform burst discharges in area CA3 elicited by picrotoxin. Both field excitatory postsynaptic potentials and population spikes were reversibly depressed by TW-001 (0.5-15 mg/ml) in a concentration-dependent manner. The sensitivity of postsynaptic neurons to a glutamate-receptor agonist, alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid or N-methyl-D-aspartate, was not altered by TW-001 (10 mg/ml). However, TW-001 (5 mg/ml) clearly increased the magnitude of paired-pulse facilitation. TW-001 (5-10 mg/ml) reversibly limited the repetitive firing and reduced the maximal rate of rise of action potentials elicited by injection of depolarizing current pulses (0.4 nA, 200 ms) into the pyramidal cells. TW-001 (1-10 mg/ml) exerted a concentration-dependent reduction of the tetrodotoxin-sensitive sodium currents and high voltage-activated calcium currents. These results suggest that TW-001 is an interesting new anticonvulsant agent that exerts its anticonvulsant activity through inhibition of sodium and calcium channels, stabilizing neuronal membrane excitability and inhibiting glutamate release.
Asunto(s)
Anticonvulsivantes/farmacología , Medicamentos Herbarios Chinos/farmacología , Convulsiones/tratamiento farmacológico , Potenciales de Acción/efectos de los fármacos , Animales , Canales de Calcio/efectos de los fármacos , Relación Dosis-Respuesta a Droga , Medicamentos Herbarios Chinos/toxicidad , Hipocampo/efectos de los fármacos , Hipocampo/fisiología , Masculino , Potenciales de la Membrana/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Canales de Sodio/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacosRESUMEN
Long-term potentiation (LTP) and long-term depression (LTD) are two main forms of activity-dependent synaptic plasticity that have been extensively studied as the putative mechanisms underlying learning and memory. Current studies have demonstrated that prior synaptic activity can influence the subsequent induction of LTP and LTD at Schaffer collateral-CA1 synapses. Here, we show that prior short-term synaptic disinhibition induced by type A gamma-aminobutyric acid (GABA) receptor antagonist picrotoxin exhibited a facilitation of LTP induction and an inhibition of LTD induction. This effect lasted between 10 and 30 min after washout of picrotoxin and was specifically inhibited by the L-type voltage-operated Ca2+ channel (VOCC) blocker nimodipine, but not by the N-methyl-D-aspartate (NMDA) receptor antagonist D-2-amino-5-phosphopentanoic acid (D-APV). Moreover, this picrotoxin-induced priming effect was mimicked by forskolin, an activator of cyclic adenosine monophosphate (cAMP)-dependent protein kinase (PKA), and was blocked by the adenylyl cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536) and the PKA inhibitor Rp-adenosine 3',5'-cyclic monophosphothioate (Rp-cAMPS). It was also found that following picrotoxin application, CA1 neurons have a higher probability of synchronous discharge in response to a population of excitatory postsynaptic potential (EPSP) of fixed slope (EPSP/spike potentiation). However, picrotoxin treatment did not significantly affect paired-pulse facilitation (PPF). These findings suggest that a brief of GABAergic disinhibition can act as a priming stimulus for the subsequent induction of LTP and LTD at Schaffer collateral-CA1 synapses. The increase in Ca2+ influx through L-type VOCCs in turn triggering a cAMP/PKA signalling pathway is a possible molecular mechanism underlying this priming effect.
Asunto(s)
Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Adenina/análogos & derivados , Adenina/farmacología , Inhibidores de Adenilato Ciclasa , Animales , Colforsina/análogos & derivados , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Estimulación Eléctrica , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Antagonistas de Receptores de GABA-A , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Picrotoxina/farmacología , Piperidinas/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de GABA-A/fisiología , Tionucleótidos/farmacologíaRESUMEN
Hypotonicity activates volume-sensitive Cl- currents, which are implicated in the regulatory volume decrease (RVD) responses and transport of taurine in human cervical cancer HT-3 cells. In this study, the role of cytoskeleton in the regulation of volume-sensitive Cl- channels and RVD responses in HT-3 cells was studied. Cells were incubated with various compounds, which depolymerized or polymerized cytoskeletal elements, i.e. actin filaments and microtubules. The hypotonicity-induced changes in Cl- conductance and in cell volume were measured by whole-cell voltage clamping and cell size monitoring, respectively. Our results show that in HT-3 cells hypotonicity activated an outward rectified Cl- current that was abrogated by Cl- channel blockers. Cytochalasin B, an actin-depolymerizing compound, induced a substantial increase in Cl- conductance under isotonic condition and potentiated the expression of Cl- currents in hypotonic stress. Phorbol 12-myristate 13-acetate (PMA) significantly inhibited the cytochalasin B-induced activation of Cl- conductance under isotonic condition. On the other hand, treatment with cytochalasin B significantly prolonged the RVD responses. Phalloidin, a stabilizer of actin polymerization, did not change the basal currents under isotonic condition, but completely abolished the increase in whole-cell Cl- conductance elicited by hypotonicity and retarded the cell volume recovery. Colchicine, a microtubule-assembly inhibitor, had no effect on either basal Cl- conductance or volume-sensitive Cl- current and was unable to inhibit the RVD responses. Taxol, a microtubule-stabilizing compound, did not alter the basal Cl- conductance, but inhibited the activation of volume-sensitive Cl- channels as well as the process of RVD in a dose-dependent manner. These data support the notion that functional integrity of actin filaments and microtubules plays critical roles in maintaining the RVD responses and activation of Cl- channels in human cervical cancer HT-3 cells.
Asunto(s)
Actinas/fisiología , Tamaño de la Célula/fisiología , Canales de Cloruro/metabolismo , Microtúbulos/fisiología , Actinas/efectos de los fármacos , Tamaño de la Célula/efectos de los fármacos , Canales de Cloruro/antagonistas & inhibidores , Canales de Cloruro/efectos de los fármacos , Colchicina/farmacología , Citocalasina B/farmacología , Femenino , Humanos , Soluciones Hipotónicas , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Microtúbulos/efectos de los fármacos , Presión Osmótica , Paclitaxel/farmacología , Técnicas de Placa-Clamp , Faloidina/farmacología , Acetato de Tetradecanoilforbol/farmacología , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/patologíaRESUMEN
The involvement of adenosine on the development of time-dependent reversal of long-term potentiation (LTP) by low-frequency stimulation (LFS) was investigated at Schaffer collateral-CA1 synapses of rat hippocampal slices. A train of LFS (2 Hz, 10 min, 1200 pulses) had no long-term effects on synaptic transmission but produced lasting depression of previously potentiated responses. This reversal of LTP (depotentiation) was observed when the stimulus was delivered
Asunto(s)
Adenosina/fisiología , Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Neuronas/fisiología , Células Piramidales/fisiología , Sinapsis/fisiología , Animales , Buspirona/farmacología , Colforsina/farmacología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores , Técnicas In Vitro , Análisis de los Mínimos Cuadrados , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Neuronas/efectos de los fármacos , Piperazinas/farmacología , Antagonistas de Receptores Purinérgicos P1 , Células Piramidales/efectos de los fármacos , Ratas , Ratas Sprague-Dawley , Receptores de Serotonina/fisiología , Receptores de Serotonina 5-HT1 , Antagonistas de la Serotonina/farmacología , Agonistas de Receptores de Serotonina/farmacología , Sinapsis/efectos de los fármacos , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología , Teobromina/análogos & derivados , Teobromina/farmacología , Tionucleótidos/farmacología , Factores de Tiempo , Xantinas/farmacologíaRESUMEN
1. Protein tyrosine phosphorylation is thought to play an important role in the regulation of neuronal function. Previous work has shown that protein tyrosine kinase (PTK) inhibitors can inhibit the induction of long-term potentiation (LTP), a candidate synaptic mechanism involved in memory formation. However, how PTK activity might contribute to LTP induction remains elusive. To understand the role of PTK pathways in the development of LTP better, a set of studies was implemented in area CA1 of rat hippocampal slices using both intra- and extracellular recordings. We show here that bath application or injection into postsynaptic cells of the PTK inhibitors genistein and lavendustin A blocked the induction of LTP produced by high-frequency tetanic stimulation. 2. Application of lavendustin A 10 min before or 3 min after induction effectively blocked LTP. However, application at 10 or 30 min after induction had no detectable effect on potentiation. 3. PTK inhibitor pretreatment did not affect the long-lasting enhancement of synaptic response produced by phorbol 12,13-dibutyrate (PDBu), forskolin plus 3-isobutyl-L-methylxanthine (IBMX), or tetraethylammonium (TEA). In contrast, PTK inhibitors significantly blocked postanoxic LTP. 4. EPQ(pY)EEIPIA, an activator of Src family PTKs, produced a gradual and robust increase in the synaptic response and occluded LTP. 5. These results suggest that Src family kinases are potential candidates for the PTKs contributing to the molecular mechanism of LTP induction at Schaffer collateral-CA1 synapses.
Asunto(s)
Hipocampo/fisiología , Potenciación a Largo Plazo/fisiología , Proteínas Tirosina Quinasas/fisiología , 1-Metil-3-Isobutilxantina/farmacología , Animales , Colforsina/farmacología , Estimulación Eléctrica , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Hipoxia/fisiopatología , Técnicas In Vitro , Potenciación a Largo Plazo/efectos de los fármacos , Masculino , Fenoles/farmacología , Forbol 12,13-Dibutirato/farmacología , Inhibidores de Fosfodiesterasa/farmacología , Proteínas Tirosina Quinasas/antagonistas & inhibidores , Ratas , Ratas Sprague-Dawley , Receptores AMPA/fisiología , Receptores de N-Metil-D-Aspartato/fisiología , Tetraetilamonio/farmacología , Familia-src Quinasas/fisiologíaRESUMEN
The aim of this study was to study the possible intracellular mechanisms underlying the anoxia-induced long-term potentiation (anoxic LTP) in the CA1 neurons of rat hippocampal slices using extra- and intracellular recording techniques. Superfusion of the hippocampal slices with the protein kinase C (PKC) inhibitors NPC-15437 (20 microM) or H-7 (20 microM) specifically prevented the induction of anoxic LTP. Moreover, the anoxic LTP was completely abolished in neurons intracellularly recorded with the selective PKC inhibitor PKCI 19-36 (50 microM). The specific cAMP-dependent protein kinase (PKA) inhibitor Rp-cyclic adenosine 3',5'-monophosphate (Rp-cAMPS, 25 microM) had no effect on the anoxic LTP. It is concluded that induction of anoxic LTP requires the activation of postsynaptic PKC.
Asunto(s)
Hipocampo/citología , Potenciación a Largo Plazo/fisiología , Neuronas/enzimología , Proteína Quinasa C/antagonistas & inhibidores , 1-(5-Isoquinolinesulfonil)-2-Metilpiperazina/farmacología , Animales , Hipoxia de la Célula/fisiología , AMP Cíclico/análogos & derivados , AMP Cíclico/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/antagonistas & inhibidores , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Masculino , Neuronas/citología , Técnicas de Cultivo de Órganos , Piperidinas/farmacología , Proteína Quinasa C/metabolismo , Ratas , Ratas Sprague-Dawley , Tionucleótidos/farmacologíaRESUMEN
1. The present study was carried out to identify the specific protein kinase C (PKC) isoform involved in regulatory volume decrease (RVD) responses, and to investigate the signal transduction pathways underlying the activation of volume-sensitive chloride channels in human cervical cancer HT-3 cells. The role of Ca2+ in RVD and in the activation of chloride currents was also studied. 2. The time course of RVDs was prolonged by microinjection of PKC-alpha antibody but not by PKC-beta or PKC-gamma antibody, and also by exposure to Ca2+-free medium, in particular when combined with microinjection of EDTA. Immunofluorescence staining showed that hypotonic superfusion evoked the translocation of PKC-alpha to the cell membrane, whereas PKC-beta or PKC-gamma remained unaffected. The translocation of PKC-alpha was observed a few minutes after hypotonic stress, reaching peak intensity at 30 min, and returned to the cytoplasm 60 min after hypotonic exposure. Western blot analyses showed an increased PKC-alpha level in terms of intensity and phosphorylation in the cell membrane, while neither PKC-beta nor PKC-gamma was activated upon hyposmotic challenge. 3. Whole-cell patch-clamp studies demonstrated that neomycin and PKC blockers such as staurosporine and H7 inhibited volume-sensitive chloride currents. The inhibitory effect of neomycin on chloride currents can be reversed by the PKC activator phorbol 12-myristate, 13-acetate (PMA). Moreover, the PKC inhibitor and PKC-alpha antibody, but not PKC-beta or PKC-gamma antibody, significantly attenuated the chloride currents. The activation of volume-sensitive chloride currents were insensitive to the changes of intracellular Ca2+ but required the presence of extracellular Ca2+. 4. Our results suggest the involvement of PKC-alpha and extracellular Ca2+ in RVD responses and the activation of volume-sensitive chloride channels in HT-3 cells.
Asunto(s)
Canales de Cloruro/fisiología , Isoenzimas/fisiología , Proteína Quinasa C/fisiología , Western Blotting , Señalización del Calcio/fisiología , Tamaño de la Célula , Estimulación Eléctrica , Electrofisiología , Femenino , Técnica del Anticuerpo Fluorescente Directa , Humanos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Proteína Quinasa C-alfa , Transducción de Señal/fisiología , Fracciones Subcelulares/fisiología , Células Tumorales Cultivadas , Neoplasias del Cuello Uterino/patologíaRESUMEN
Circular dichroism (CD) and 2-dimensional NMR were used to study the solution conformation of conantokin-T (Con-T), a small peptide toxin found in the venom of fish-hunting cone snails, and its Glu-substituted analog. Con-T lacks disulfide bonds but contains many gamma-carboxyglutamic acids (Gla), a post-translationally modified residue. Our results show that Con-T adopts an alpha-helical conformation in aqueous solution even in the absence of calcium. Glu replacements diminish both helicity and function of Con-T. The helical content of Con-T is higher than most natural helical peptides of this length in aqueous solution. The sequence of this small toxin incorporates several known elements that stabilize alpha-helical structure in peptides. Gla residues form several salt bridges that stabilize helical conformation of Con-T.
Asunto(s)
Ácido 1-Carboxiglutámico/química , Antagonistas de Aminoácidos Excitadores/química , Venenos de Moluscos/química , Péptidos/química , Estructura Secundaria de Proteína , Secuencia de Aminoácidos , Animales , Dicroismo Circular , Conotoxinas , Antagonistas de Aminoácidos Excitadores/farmacología , Hipocampo/efectos de los fármacos , Péptidos y Proteínas de Señalización Intercelular , Espectroscopía de Resonancia Magnética , Modelos Moleculares , Datos de Secuencia Molecular , Venenos de Moluscos/farmacología , Neuronas/efectos de los fármacos , Péptidos/farmacología , Péptidos Cíclicos/farmacología , Ratas , Ratas Sprague-Dawley , Receptores de N-Metil-D-Aspartato/efectos de los fármacosRESUMEN
The effects of L-deprenyl (selegiline), a highly selective monoamine oxidase type B (MAO-B) inhibitor, on cell excitability of rat hippocampal CA1 neurons were examined in slice preparations using intracellular recording techniques. Superfusion of L-deprenyl (10 and 20 microM) reversibly limited the repetitive firing (RF) of action potentials elicited by injection of depolarizing current pulses (100 ms) into the pyramidal cells. At a concentration of 1-50 microM, L-deprenyl did not alter resting membrane potential or input resistance of the hippocampal CA1 neurons. The limitation of RF by L-deprenyl (20 microM) was accompanied by the reduction of the maximal rate of rise (Vmax) of the action potentials in a non-voltage-dependent manner. In 80% of recorded cells, application of L-deprenyl (20 microM) produced an increase in the amplitude and duration of afterhyperpolarization (AHP). The limitation of L-deprenyl on RF was mimicked by other MAO-B inhibitors, pargyline and 4-phenylpyridine. In addition, the ability of L-deprenyl to limit RF was not observed in the hippocampal CA1 neurons taken from dopamine (DA)-depleted rats. Moreover, we also observed that the L-deprenyl-induced limitation of RF was specifically antagonized by (+/-)-7-bromo-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzaz epine (SKF-83566, 5 microM), a selective D1 dopaminergic receptor antagonist. However, the D2 dopaminergic receptor antagonist, sulpiride (5 microM), had no effect on L-deprenyl's action. These results indicate that the MAO-B inhibitory ability leading to an increase of the dopaminergic tone in the hippocampus is involved, at least in part, in the L-deprenyl-induced reduction of neuronal excitability in the CA1 region of rat hippocampus and that the D1 dopaminergic receptor is involved in L-deprenyl's action.