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1.
Biochim Biophys Acta ; 1852(5): 759-67, 2015 May.
Artículo en Inglés | MEDLINE | ID: mdl-25583115

RESUMEN

Ethylmalonic acid (EMA) accumulation occurs in various metabolic diseases with neurological manifestation, including short acyl-CoA dehydrogenase deficiency (SCADD) and ethylmalonic encephalopathy (EE). Since pathophysiological mechanisms responsible for brain damage in these disorders are still poorly understood, we investigated the ex vivo effects of acute intrastriatal administration of EMA on important parameters of energy and redox homeostasis in striatum from young rats. We evaluated CO(2) production from glucose, glucose utilization and lactate production, as well as the activities of the citric acid cycle (CAC) enzymes, the electron transfer chain (ETC) complexes II-IV (oxidative phosphorylation, OXPHOS) and synaptic Na(+),K(+)-ATPase. We also tested the effect of EMA on malondialdehyde (MDA) levels (marker of lipid oxidation) and reduced glutathione (GSH) levels. EMA significantly reduced CO(2) production, increased glucose utilization and lactate production, and reduced the activities of citrate synthase and of complexes II and II-III of the ETC, suggesting an impairment of CAC and OXPHOS. EMA injection also reduced Na(+),K(+)-ATPase activity and GSH concentrations, whereas MDA levels were increased. Furthermore, EMA-induced diminution of Na(+),K(+)-ATPase activity and reduction of GSH levels were prevented, respectively, by the antioxidants melatonin and N-acetylcysteine, indicating that reactive species were involved in these effects. Considering the importance of CAC and ETC for energy production and Na(+),K(+)-ATPase for the maintenance of the cell membrane potential, the present data indicate that EMA compromises mitochondrial homeostasis and neurotransmission in striatum. We presume that these pathomechanisms may be involved to a certain extent in the neurological damage found in patients affected by SCADD and EE.


Asunto(s)
Metabolismo Energético/efectos de los fármacos , Homeostasis/efectos de los fármacos , Malonatos/farmacología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Acetilcisteína/farmacología , Animales , Antioxidantes/farmacología , Dióxido de Carbono/metabolismo , Cuerpo Estriado/efectos de los fármacos , Cuerpo Estriado/metabolismo , Complejo II de Transporte de Electrones/metabolismo , Complejo III de Transporte de Electrones/metabolismo , Glucosa/metabolismo , Glutatión/metabolismo , Inyecciones Intraventriculares , Lactatos/metabolismo , Masculino , Malonatos/administración & dosificación , Malondialdehído/metabolismo , Melatonina/farmacología , Oxidación-Reducción/efectos de los fármacos , Ratas Wistar , Sinapsis/efectos de los fármacos , Sinapsis/enzimología
2.
J Bioenerg Biomembr ; 45(1-2): 137-44, 2013 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-23151916

RESUMEN

Refsum disease is an autosomal recessive disorder of peroxisomal metabolism biochemically characterized by highly elevated concentrations of phytanic acid (Phyt) in a variety of tissues including the cerebellum. Reduction of plasma Phyt levels by dietary restriction intake ameliorates ataxia, a common clinical manifestation of this disorder, suggesting a neurotoxic role for this branched-chain fatty acid. Therefore, considering that the underlying mechanisms of cerebellum damage in Refsum disease are poorly known, in the present study we tested the effects of Phyt on important parameters of bioenergetics, such as the activities of the respiratory chain complexes I to IV, creatine kinase and Na(+), K(+)- ATPase in cerebellum preparations from young rats. The activities of complexes I, II, I-III and II-III and Na(+), K(+)- ATPase were markedly inhibited (65-85%) in a dose-dependent manner by Phyt. In contrast, creatine kinase and complex IV activities were not altered by this fatty acid. Therefore, it is presumed that impairment of the electron flow through the respiratory chain and inhibition of Na(+), K(+)- ATPase that is crucial for synaptic function may be involved in the pathophysiology of the cerebellar abnormalities manifested as ataxia in Refsum disease and in other peroxisomal disorders in which brain Phyt accumulates.


Asunto(s)
Cerebelo/enzimología , Proteínas del Complejo de Cadena de Transporte de Electrón/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Ácido Fitánico/farmacología , Enfermedad de Refsum/enzimología , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Sinapsis/enzimología , Animales , Cerebelo/patología , Cerebelo/fisiopatología , Transporte de Electrón/efectos de los fármacos , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Humanos , Proteínas del Tejido Nervioso/metabolismo , Ratas , Ratas Wistar , Enfermedad de Refsum/patología , Enfermedad de Refsum/fisiopatología , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sinapsis/patología
3.
Neurosci Lett ; 520(1): 98-103, 2012 Jun 27.
Artículo en Inglés | MEDLINE | ID: mdl-22640895

RESUMEN

Acylpeptide hydrolase (ACPH), a serine protease present in the central nervous system (CNS), is believed to have a function in modulating synaptic plasticity, cleavage of beta amyloid peptide and degradation of aggregated oxidized proteins. In this report, we demonstrate for the first time the presence of ACPH in the synapse and its preferential localization at the pre-synaptic side. We isolated subcellular fractions from the rat telencephalon enriched in pre- versus post-synaptic components by using differential centrifugation steps to evaluate ACPH catalytic activity and expression level. Relative ACPH levels were determined by Western blot techniques while antibodies against synaptophysin and PSD-95 were used as positive pre- and post-synaptic markers, respectively. Our results show that ACPH protein levels are significantly increased at the synapse, which correlates with a 56% increase in ACPH activity. Furthermore, Western blot experiments show that ACPH is preferentially located at the pre-synaptic side and this is consistent with the increase of its enzymatic activity in fractions enriched in pre-synaptic components. These results give new insights regarding the localization and a putative role of ACPH in the CNS.


Asunto(s)
Péptido Hidrolasas/metabolismo , Telencéfalo/enzimología , Acetilcolinesterasa/metabolismo , Animales , Masculino , Ratas , Ratas Sprague-Dawley , Sinapsis/enzimología
4.
Cell Mol Neurobiol ; 32(2): 297-307, 2012 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-21993987

RESUMEN

Deficiency of 3-methylcrotonyl-CoA carboxylase activity is an inherited metabolic disease biochemically characterized by accumulation and high urinary excretion of 3-methylcrotonylglycine (3MCG), and also of 3-hydroisovalerate in lesser amounts. Affected patients usually have neurologic dysfunction, brain abnormalities and cardiomyopathy, whose pathogenesis is still unknown. The present study investigated the in vitro effects of 3MCG on important parameters of energy metabolism, including CO(2) production from labeled acetate, enzyme activities of the citric acid cycle, as well as of the respiratory chain complexes I-IV (oxidative phosphorylation), creatine kinase (intracellular ATP transfer), and synaptic Na(+),K(+)-ATPase (neurotransmission) in brain cortex of young rats. 3MCG significantly reduced CO(2) production, implying that this compound compromises citric acid cycle activity. Furthermore, 3MCG diminished the activities of complex II-III of the respiratory chain, mitochondrial creatine kinase and synaptic membrane Na(+),K(+)-ATPase. Furthermore, antioxidants were able to attenuate or fully prevent the inhibitory effect of 3MCG on creatine kinase and synaptic membrane Na(+),K(+)-ATPase activities. We also observed that lipid peroxidation was elicited by 3MCG, suggesting the involvement of free radicals on 3MCG-induced effects. Considering the importance of the citric acid cycle and the electron flow through the respiratory chain for brain energy production, creatine kinase for intracellular energy transfer, and Na(+),K(+)-ATPase for the maintenance of the cell membrane potential, the present data indicate that 3MCG potentially impairs mitochondrial brain energy homeostasis and neurotransmission. It is presumed that these pathomechanisms may be involved in the neurological damage found in patients affected by 3-methylcrotonyl-CoA carboxylase deficiency.


Asunto(s)
Encéfalo/enzimología , Metabolismo Energético/efectos de los fármacos , Glicina/análogos & derivados , Homeostasis/efectos de los fármacos , Mitocondrias/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , Sinapsis/enzimología , Animales , Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Encéfalo/patología , Dióxido de Carbono/metabolismo , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/enzimología , Corteza Cerebral/patología , Creatina Quinasa/metabolismo , Citosol/efectos de los fármacos , Citosol/enzimología , Proteínas del Complejo de Cadena de Transporte de Electrón/metabolismo , Glicina/farmacología , Mitocondrias/efectos de los fármacos , Mitocondrias/enzimología , Ratas , Ratas Wistar , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sinapsis/efectos de los fármacos , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo
5.
Biochim Biophys Acta ; 1798(3): 624-36, 2010 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-20026046

RESUMEN

Detergent-resistant membranes (DRMs) are a class of specialized microdomains that compartmentalize several signal transduction processes. In this work, DRMs were isolated from cerebral cortex synaptic endings (Syn) on the basis of their relative insolubility in cold Triton X-100 (1%). The lipid composition and marker protein content were analyzed in DRMs obtained from adult and aged animals. Both DRM preparations were enriched in Caveolin, Flotillin-1 and c-Src and also presented significantly higher sphingomyelin (SM) and cholesterol content than purified Syn. Total phospholipid-fatty acid composition presented an increase in 16:0 (35%), and a decrease in 20:4n-6 (67%) and 22:6n-3 (68%) content in DRM from adults when compared to entire synaptic endings. A more dramatic decrease was observed in the 20:4n-6 and 22:6n-3 content in DRMs from aged animals (80%) with respect to the results found in adults. The coexistence of phosphatidylcholine-specific-phospholipase C (PC-PLC) and phospholipase D (PLD) in Syn was previously reported. The presence of these signaling pathways was also investigated in DRMs isolated from adult and aged rats. Both PC-PLC and PLD pathways generate the lipid messenger diacylglycerol (DAG) by catalyzing PC hydrolysis. PC-PLC and PLD1 localization were increased in the DRM fraction. The increase in DAG generation (60%) in the presence of ethanol, confirmed that PC-PLC was also activated when compartmentalized in DRMs. Conversely, PLD2 was excluded from the DRM fraction. Our results show an age-related differential fatty acid composition and a selective localization of PC-derived signaling in synaptic DRMs obtained from adult and aged rats.


Asunto(s)
Detergentes/farmacología , Fosfatidilcolinas/metabolismo , Transducción de Señal/efectos de los fármacos , Sinapsis/efectos de los fármacos , Sinapsis/metabolismo , Envejecimiento/metabolismo , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Diglicéridos/metabolismo , Ácidos Grasos/metabolismo , Membranas/efectos de los fármacos , Membranas/enzimología , Ratas , Ratas Wistar , Sinapsis/enzimología , Fosfolipasas de Tipo C/metabolismo
6.
J Neurosci ; 27(19): 5190-9, 2007 May 09.
Artículo en Inglés | MEDLINE | ID: mdl-17494705

RESUMEN

Long-term potentiation (LTP) is an activity-dependent strengthening of synapses that is thought to underlie memory storage. Ca2+/calmodulin-dependent protein kinase II (CaMKII) has been a leading candidate as a memory molecule because it is persistently activated after LTP induction and can enhance transmission. Furthermore, a mutation that blocks persistent activation blocks LTP and forms of learning. However, direct evidence for a role of the kinase in maintaining synaptic strength has been lacking. Here, we show that a newly developed noncompetitive inhibitor of CaMKII strongly reduces synaptic transmission in the CA1 region of the hippocampal slice. This occurs through both presynaptic and postsynaptic action. To study the role of CaMKII in the maintenance of LTP, inhibitor was applied after LTP induction and then removed. Inhibition occurred in both LTP and control pathways but only partially recovered. The nonrecovering component was attributable primarily to a postsynaptic change. To test whether nonrecovery was attributable to a persistent reversal of LTP, we first saturated LTP and then transiently applied inhibitor. This procedure allowed additional LTP to be induced, indicating a reversal of an LTP maintenance mechanism. This is the first procedure that can reverse LTP by chemical means and suggests that a component of synaptic memory is attributable to CaMKII. The procedure also enhanced the LTP that could be induced in the control pathway, consistent with the idea that CaMKII is involved in controlling basal synaptic strength, perhaps as a result of LTP that occurred in vivo.


Asunto(s)
Proteínas Quinasas Dependientes de Calcio-Calmodulina/metabolismo , Hipocampo/enzimología , Potenciación a Largo Plazo/fisiología , Trastornos de la Memoria/enzimología , Memoria/fisiología , Péptidos/farmacología , Sinapsis/enzimología , Animales , Proteína Quinasa Tipo 2 Dependiente de Calcio Calmodulina , Proteínas Quinasas Dependientes de Calcio-Calmodulina/antagonistas & inhibidores , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Hipocampo/efectos de los fármacos , Hipocampo/fisiopatología , Potenciación a Largo Plazo/efectos de los fármacos , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Memoria/efectos de los fármacos , Trastornos de la Memoria/inducido químicamente , Trastornos de la Memoria/fisiopatología , Inhibición Neural/efectos de los fármacos , Inhibición Neural/fisiología , Técnicas de Cultivo de Órganos , Técnicas de Placa-Clamp , Fragmentos de Péptidos/farmacología , Ratas , Ratas Long-Evans , Sinapsis/efectos de los fármacos , Membranas Sinápticas/efectos de los fármacos , Membranas Sinápticas/metabolismo , Transmisión Sináptica/efectos de los fármacos , Transmisión Sináptica/fisiología
7.
Metab Brain Dis ; 22(1): 77-88, 2007 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-17295076

RESUMEN

The objective of the present study was to investigate the in vitro effects of the branched-chain alpha-keto acids accumulating in maple syrup urine disease, namely L-2-ketoisocaproic acid, L-2-keto-3-methylvaleric acid and L-2-ketoisovaleric acid on Na(+), K(+)-ATPase activity in synaptic plasma membranes from cerebral cortex of 35-day-old rats. All keto acids significantly inhibited Na(+), K(+)-ATPase activity at concentrations similar (1 mM) or even lower (0.5 mM) than those found in blood and cerebrospinal fluid of maple syrup urine disease patients. We also tested the effects of alanine on this enzyme activity. Alanine per se did not alter Na(+), K(+)-ATPase activity, but totally prevented the branched-chain alpha-keto acids-induced Na(+), K(+)-ATPase inhibition, indicating that alanine and the keto acids may possibly bind to the same site on the enzyme. We also observed that the branched-chain amino acids leucine, isoleucine and valine also inhibited Na(+) K(+)-ATPase activity to a similar degree as that of the branched-chain alpha-keto acids and that alanine was able to fully prevent these effects. Considering that Na(+), K(+)-ATPase is a critical enzyme for normal brain development and functioning, it is presumed that these findings may be involved in the pathophysiology of the neurological dysfunction of maple syrup urine disease.


Asunto(s)
Corteza Cerebral/metabolismo , Cetoácidos/metabolismo , Enfermedad de la Orina de Jarabe de Arce/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sinapsis/enzimología , Alanina/farmacología , Animales , Membrana Celular/enzimología , Activación Enzimática/efectos de los fármacos , Activación Enzimática/fisiología , Hemiterpenos , Fenilalanina/farmacología , Ratas , Ratas Wistar
8.
Neurosci Res ; 41(1): 67-70, 2001 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-11535295

RESUMEN

The superficial layers of the rat superior colliculus (sSC) receive innervation from retina and include nitric oxide synthase (NOS)-immunoreactive neurons. We used electron microscopic immunocytochemistry to assess the subcellular localization of neuronal NOS (nNOS) in the sSC. nNOS immunoreactivity was detected on the external membrane of mitochondria, endoplasmic reticulum, in pre- and postsynaptic profiles and also diffusely distributed in the cytosol. Postsynaptic labeled regions were often associated with presumptive retinal unlabeled terminals. Microtubules also appeared intensely labeled. These results show that NOS immunoreactive neurons may be innervated by retinal terminals and suggest an association of nNOS with cytoskeletal elements.


Asunto(s)
Compartimento Celular/fisiología , Neuronas/enzimología , Óxido Nítrico Sintasa/metabolismo , Células Ganglionares de la Retina/enzimología , Colículos Superiores/enzimología , Sinapsis/enzimología , Vías Visuales/enzimología , Animales , Inmunohistoquímica , Membranas Intracelulares/enzimología , Membranas Intracelulares/ultraestructura , Microscopía Electrónica , Microtúbulos/enzimología , Microtúbulos/ultraestructura , Neuronas/ultraestructura , Óxido Nítrico/metabolismo , Orgánulos/enzimología , Orgánulos/ultraestructura , Ratas , Células Ganglionares de la Retina/ultraestructura , Colículos Superiores/ultraestructura , Sinapsis/ultraestructura , Vías Visuales/ultraestructura
9.
Neuroscience ; 103(2): 457-64, 2001.
Artículo en Inglés | MEDLINE | ID: mdl-11246160

RESUMEN

This study was performed using intracellular and multiunit extracellular recording techniques in order to characterize the role of nitric oxide in the afferent synaptic transmission of the vestibular system of the axolotl (Ambystoma tigrinum). Bath application of nitric oxide synthase inhibitors N(G)-nitro-L-arginine (0.01microM to 10microM) and N-nitro-L-arginine methyl ester hydrochloride (0.1microM to 1000microM) elicited a dose-dependent decrease in the basal discharge of the semicircular canal afferent fibers. N(G)-Nitro-L-arginine also diminished the response to mechanical stimuli. Moreover, N(G)-nitro-L-arginine (1microM) produced a hyperpolarization associated with a decrease in the spike discharge and diminished the frequency of the excitatory postsynaptic potentials on afferent fibers recorded intracellularly. Nitric oxide donors were also tested: (i) S-nitroso-N-acetyl-DL-penicillamine (0.1microM to 100microM) increased the basal discharge and the response to mechanical stimuli. At the maximum effective concentration (100microM) this drug affected neither the amplitude nor the frequency of the excitatory postsynaptic potentials. However, it slightly depolarized the afferent neurons and decreased their input resistance. (ii) 3-Morpholino-sydnonimine hydrochloride did not significantly affect the basal discharge or the mechanically evoked peak response of afferent neurons at any of the concentrations used (1microM to 1000microM). However, after 10min of perfusion in the bath, 1microM and 10microM 3-morpholino-sydnonimine hydrochloride significantly modified the baseline of the mechanically evoked response, producing an increase in the mean spike discharge of the afferent fibers. These results indicate that nitric oxide may have a facilitatory role on the basal discharge and on the response to mechanical stimuli of the vestibular afferent fibers. Thus, nitric oxide probably participates in the sensory coding and adaptative changes of vestibular input in normal and pathological conditions.


Asunto(s)
Células Ciliadas Vestibulares/metabolismo , Ácido Nítrico/metabolismo , Transmisión Sináptica/fisiología , Potenciales de Acción/efectos de los fármacos , Potenciales de Acción/fisiología , Ambystoma , Animales , Inhibidores Enzimáticos/farmacología , Potenciales Postsinápticos Excitadores/efectos de los fármacos , Potenciales Postsinápticos Excitadores/fisiología , Molsidomina/análogos & derivados , Molsidomina/farmacología , NG-Nitroarginina Metil Éster/farmacología , Donantes de Óxido Nítrico/farmacología , Óxido Nítrico Sintasa/metabolismo , Nitroarginina/farmacología , Penicilamina/análogos & derivados , Penicilamina/farmacología , S-Nitroso-N-Acetilpenicilamina , Sinapsis/enzimología
10.
Salud ment ; Salud ment;22(6): 42-7, dic.-ene. 1999. ilus
Artículo en Español | LILACS | ID: lil-266813

RESUMEN

La comunicaicón química interneuronal está basada en la transmisión y decodificación de señales químicas a través de la liberación de neurotransmisores y péptidos bioactivos. Asimismo, durante más de dos décadas también se ha estudiado el papael funcional de componentes protéicos de alta masa molecular que son libradas al espacio sináptico donde ejercen acciones biológicas en las neuronas posinápticas. Estas protéinas son sintetizadas en el rER y segregadas dentro de sistemas intracelulares de transporte para su neurosecreción por mecanismo molecualres definidos. Adicionalmente esta macromoléculas también parecen tener relevancia funcional en el mantenimiento estructural de la sinápsis, mediante la interacción dual neuron-glía, además de modular eventos regulatorios en neuronas blanco durante su transporte transneuronal en sistemas de transmisión específicos


Asunto(s)
Sinapsis/enzimología , Sinapsis/química , Canales de Calcio , Interneuronas/química , Comunicación Celular/fisiología , Proteínas/biosíntesis , Proteínas/química , Proteínas , Neurotransmisores
11.
Salud ment ; Salud ment;22(4): 56-63, jul.-ago. 1999. ilus, tab
Artículo en Español | LILACS | ID: lil-254600

RESUMEN

En los últimos 30 años un extenso cúmulo de información ha sido reportada sobre la identificación y caracterización fisiológica y molecular de las proteínas y las enzimas; cuya liberación espontánea e inducida a partir del tejido neuronal y de los tejidos extraneuronales, como las células cromafines de la glándula suprarrenal, han permitido no sólo la subsecuente clonación y aislamiento molecular de estas moléculas protéicas, sino que, además, mediante la aplicación de métodos de hibridación in situ, de la generación de anticuerpos selectivos, conjuntamente con el apoyo de técnicas inmunohistoquímicas, se ha podido detectar el origen, localización y expresión celular de enzimas como la acetilcolinesterasa específica (AChE), y la dopamina-betahidroxilasa (DBH), en diversas regiones del cerebro de distintas especies mamíferas, de donde se ha encontrado su capacidad para ejercer funciones neurotróficas. Asimismo, se han abordado similares planteamientos experimentales para identificar y localizar la expresión celular y subcelular de proteínas como las de la familia de los polipéptidos, representados por las cromograninas y las secretoneurinas, que son polipéptidos, cuya síntesis celular modificaciones postraduccionales, almacenamiento y segregación vesicular, como su exocitosis celular en el tejido neuronal y en los tejidos neuroendocrinos, han sido estudiadas in vivo e in vitro a partir de diferentes preparaciones biológicas, cuyos resultados han sido motivo de múltiples publicaciones. Más aún, estas proteínas parecen ser precursoras de varios péptidos bioactivos, en los que se han observado diversas funciones de tipo autocrino, paracrino, o endocrino, en el tejido neuronal y en los tejidos extraneuronales. Asimismo, una extensa familia de factores tróficos, conocidos como neurotrofinas, incluyendo el Factor de Crecimiento Neuronal (NGF), recientemente se aislaron, identificaron, y caracterizaron molecular y funcionalmente, in vivo e in vitro, con lo que se demostró su capacidad para regular y mantener la supervivencia de diferentes poblaciones de células neuronales en el sistema nervioso central y periférico de múltiples especies de vertebrados e invertebrados, lo que parece mediarse por la activación de diversos subgrupos de receptores membranales, recientemente identificados y localizados en discretas poblaciones neuronales


Asunto(s)
Sinapsis/enzimología , Sinapsis/química , Ganglios Basales , Interneuronas/enzimología , Interneuronas/fisiología , Interneuronas/química , Neuronas/enzimología , Neuronas , Neuronas/química , Neurosecreción/fisiología , Neurotransmisores/fisiología , Comunicación Celular
12.
Brain Res Mol Brain Res ; 65(1): 34-43, 1999 Feb 19.
Artículo en Inglés | MEDLINE | ID: mdl-10036305

RESUMEN

We evaluated the in vitro phosphorylation of the presynaptic substrate of protein kinase C (PKC), GAP-43/B-50 and the PKC activity in the striatum of rats submitted to a circling training (CT) test during postnatal development. Motor activity at 30 days of age, but not at other ages, produced a unilateral reduction (-29.5%; p<0.001) in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum with respect to the ipsilateral side, while non-trained control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of trained animals also showed a significant reduction (-29.3%; p<0. 001) in the incorporation of 32P-phosphate into GAP-43. This decreased in vitro GAP-43 phosphorylation was seen at 30 min, but not immediately after circling motor behavior. This contralateral change in GAP-43 phosphorylation correlated with the running speed developed by the animals [(r=0.9443, p=0.0046, n=6, relative to control group) and (r=0.8813, p=0.0203, n=6, with respect to the ipsilateral side of the exercised animals)]. On the contrary, GAP-43/B-50 immunoblots did not show changes in the amount of this phosphoprotein among the different experimental groups. Back phosphorylation assays, performed in the presence of bovine purified PKC, increased the level of GAP-43/B-50 phosphorylation in the striatum contralateral to the sense of turning [(+22%; p<0.05, with respect to ipsilateral side of the same trained group) and (+21%; p<0.05, relative to control group)]. Taken together, these results demonstrate that the activity developed in the CT test induces a reduction in the phosphorylation state of GAP-43/B-50 in the specific site for PKC. We conclude that general markers of activity-dependent neuronal plasticity are also altered in the same period that long-lasting changes in striatal neuroreceptors are triggered by circling motor behavior.


Asunto(s)
Membrana Celular/metabolismo , Cuerpo Estriado/crecimiento & desarrollo , Cuerpo Estriado/metabolismo , Proteína GAP-43/metabolismo , Neuronas Motoras/enzimología , Proteína Quinasa C/metabolismo , Animales , Conducta Animal/fisiología , Western Blotting , Condicionamiento Psicológico/fisiología , Proteína GAP-43/análisis , Locomoción/fisiología , Masculino , Fosforilación , Ratas , Ratas Sprague-Dawley , Sinapsis/enzimología
13.
J Biol Chem ; 273(7): 4258-65, 1998 Feb 13.
Artículo en Inglés | MEDLINE | ID: mdl-9461624

RESUMEN

Collagen-tailed asymmetric acetylcholinesterase (AChE) forms are believed to be anchored to the synaptic basal lamina via electrostatic interactions involving proteoglycans. However, it was recently found that in avian and rat muscles, high ionic strength or polyanionic buffers could not detach AChE from cell-surface clusters and that these buffers solubilized intracellular non-junctional asymmetric AChE rather than synaptic forms of the enzyme. In the present study, asymmetric AChE forms were specifically solubilized by ionic buffers from synaptic basal lamina-enriched fractions, largely devoid of intracellular material, obtained from the electric organ of Torpedo californica and the end plate regions of rat diaphragm muscle. Furthermore, foci of AChE activity were seen to diminish in size, number, and staining intensity when the rat synaptic basal lamina-enriched preparations were treated with the extraction buffers. In the case of Torpedo, almost all the AChE activity was removed from the pure basal lamina sheets. We therefore conclude that a major portion of extracellular collagen-tailed AChE is extractable from rat and Torpedo synaptic basal lamina by high ionic strength and heparin buffers, although some non-extractable AChE activity remains associated with the junctional regions.


Asunto(s)
Acetilcolinesterasa/metabolismo , Membrana Basal/enzimología , Heparina/farmacología , Sales (Química)/farmacología , Acetilcolinesterasa/clasificación , Animales , Membrana Basal/citología , Membrana Basal/ultraestructura , Centrifugación por Gradiente de Densidad , Diafragma/enzimología , Órgano Eléctrico/enzimología , Histocitoquímica , Microscopía Electrónica , Placa Motora/citología , Placa Motora/enzimología , Concentración Osmolar , Ratas , Solubilidad , Sinapsis/enzimología , Torpedo
14.
Neurosci Lett ; 224(2): 131-5, 1997 Mar 14.
Artículo en Inglés | MEDLINE | ID: mdl-9086474

RESUMEN

Postsynaptic densities (PSDs) were isolated from rat brain cortex and hippocampus, purified and incorporated into giant (5-80 microns in diameter) liposomes. Gigaohm seals were obtained with a patch-clamp pipette, and a giant liposome PSD-containing membrane patch, was excised and recorded. The PSD was always oriented in an inside-out configuration. This allowed receptor agonists or antagonists to be added from the interior of the recording pipette, and also the addition of different substances, such as ATP, calcium, calmodulin and others to the 'intracellular' side of the PSD, i.e. to the bath. alpha-Amino-3-hydroxy-5-methylisoxazole propionic acid (AMPA) receptor agonists such as quisqualate or AMPA induced in the PSD a complex pattern of electrical activity, that was blocked by 10 microM 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX), but not by 2-aminophosphonovalerate (APV). The currents generated by 0.5-1 microM quisqualate were increased by about 100% when the PSDs were phosphorylated. Similar findings were obtained when the agonist was 0.2-2 microM kainate. These currents were also blocked by a non-N-methyl-D-aspartate (NMDA) receptor antagonist but not by APV, and were increased by about 70% by phosphorylation of the PSDs. Addition of 5-10 microM NMDA plus 1 microM glycine to the 'extracellular' side of the PSD, led to a characteristic pattern of activity, with the opening of multiple receptor ion channels. This was entirely blocked by 10 microM APV. Addition of extracellular Mg2+ (1-2 mM) induced a voltage-dependent block of the currents. Phosphorylation of the PSD led to an increase of Mg(2+)-blocked current of about 80%. The effect of phosphorylation on ion channel activity showed a markedly different requirement for calcium and for calmodulin among the AMPA, kainate and NMDA types of glutamate receptors, thus suggesting that each receptor type is coupled at the synapse with a unique complement of protein phosphokinases.


Asunto(s)
Corteza Cerebral/metabolismo , Hipocampo/metabolismo , Proteínas Quinasas/metabolismo , Receptores de Glutamato/metabolismo , Sinapsis/química , 2-Amino-5-fosfonovalerato/farmacología , 6-Ciano 7-nitroquinoxalina 2,3-diona/farmacología , Adenosina Trifosfato/farmacología , Animales , Calcio/farmacología , Calmodulina/farmacología , Corteza Cerebral/química , Ácido Egtácico/farmacología , Estimulación Eléctrica , Agonistas de Aminoácidos Excitadores/farmacología , Antagonistas de Aminoácidos Excitadores/farmacología , Glicina/farmacología , Hipocampo/química , Ácido Kaínico/farmacología , Potenciación a Largo Plazo/fisiología , Magnesio/farmacología , N-Metilaspartato/farmacología , Técnicas de Placa-Clamp , Fosforilación , Ácido Quiscuálico/farmacología , Ratas , Sinapsis/efectos de los fármacos , Sinapsis/enzimología
15.
Brain Res ; 737(1-2): 327-30, 1996 Oct 21.
Artículo en Inglés | MEDLINE | ID: mdl-8930385

RESUMEN

Miniature end-plate currents (MEPCS), from synaptic spots on the caudal muscles of Bufo marinus tadpoles, were analyzed in both pre- and postsynaptic domains, when protein kinase A (PKA) activity modificators were used. Sp-cAMPS diasteromer induced an increase in MEPC frequency, which was completely reversed by Rp-cAMPS. However, changes in the decay time of MEPCS were not detected. Dibutyryl-cAMP produced a similar presynaptic action, but its postsynaptic action was similar to butyrate. Presynaptic effect of forskolin (FSK), if any, is masked by the increase of MEPC frequency produced by dimethylsulfoxide (DMSO), the solvent used.


Asunto(s)
Bufo marinus/crecimiento & desarrollo , Proteínas Quinasas Dependientes de AMP Cíclico/metabolismo , Unión Neuromuscular/enzimología , Sinapsis/enzimología , Administración Tópica , Animales , Antiinflamatorios/farmacología , Bucladesina/farmacología , Colforsina/farmacología , Proteínas Quinasas Dependientes de AMP Cíclico/efectos de los fármacos , Dimetilsulfóxido/farmacología , Potenciales de la Membrana/efectos de los fármacos , Unión Neuromuscular/efectos de los fármacos , Técnicas de Placa-Clamp , Terminales Presinápticos/efectos de los fármacos , Terminales Presinápticos/fisiología , Sinapsis/efectos de los fármacos
16.
Rev. méd. IMSS ; 34(3): 195-9, mayo-jun. 1996. tab, ilus
Artículo en Español | LILACS | ID: lil-203001

RESUMEN

Se informan los hallazgos neurradiológicos de dos pacientes pediátricos con aminoaciduria glutárica tipo I (AG-I), a quienes se les realizó tomografía de cabeza y, sólo a uno, estudio de imagen de resonancia magnética. El diagnóstico bioquímico se llevo a cabo mediante la medición de ácido glutártico libre en orina, así como por cromatografía de gas para determinar la actividad enzimática del glutaril Co-A deshidrogenasa en los leucocitos. El retardo en la mielinización, con su consecuente disminución generalizada de la sustancia blanca, es característico de varias enfermedades metabólicas; las evidencias de las alteraciones de disgénesis cerebral se obtienen básicamente mediante estudios de neuroimagen. Se debe sospechar una probable enfermedad hereditaria del metabolismo como AG-I cuando se tenga un paciente pediátrico con retraso psicomotor, alteraciones motoras extrapiramidales o macrocrania, en el cual se obtengan los siguientes hallazgos neurorradiológicos: atrofia bilateral de la fosa temporal asociada con áreas hipodensas difusas en la sustancia blanca, ligera atrofia o pérdida del volumen de los ganglios basales, hipoplasia del vermis del cerebelo y ocasionalmente colección subdural de líquido.


Asunto(s)
Preescolar , Humanos , Masculino , Femenino , Sinapsis/enzimología , Orina/química , Encefalopatías Metabólicas/diagnóstico , Neurorradiografía , Neurotransmisores/biosíntesis , Glutaratos/análisis , Errores Innatos del Metabolismo , Ácido Glutámico/aislamiento & purificación , Enfermedades Metabólicas , Manifestaciones Neurológicas
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