RESUMEN
In the present study, we investigated the possible role of oxidative stress and the modulation of phospholipid turnover in two related models of pericyte injury, i.e., treatment with high glucose or advanced glycation end products (AGEs). Growing microcapillary pericytes from bovine retinas in culture were incubated, for 3 weeks, with 20-50 mM glucose or 2-20 microM AGEs, and peroxidation parameters (malondialdehyde, conjugated diene, hydroperoxide, glutathione (GSH) levels and lactate dehydrogenase (LDH) release) were evaluated. Arachidonate (AA) and choline release from membrane phospholipids was determined in pericytes prelabeled with [1-(14)C]arachidonate and [Me-(3)H]choline, respectively, and stimulated with elevated glucose or AGEs for 30 min or 2 h. [1-(14)C]arachidonate and [Me-(3)H]choline incorporation into phospholipids, for 2 h and 3 h respectively, was also studied in conditioned and serum-starved cultures. Finally, lysates of treated and control cells were assayed for cytosolic phospholipase A(2) (cPLA(2)), acyl-CoA:1-acyl-sn-glycero-3-phosphocholine O-acyltransferase (AT), CTP:phosphocholine cytidylyltransferase (CT) and microsomal choline phosphotransferase (CPT) enzyme activities. We found that high glucose and AGEs caused neither significant production of reactive oxygen species nor cell toxicity or death, unlike other cell types. Both agents had no significant effect on the cellular ultrastructure, evaluated by light and electron microscopy, AA incorporation and release, cytosolic phospholipase A(2) (cPLA(2)) and AT activities. On the contrary, choline incorporation into phosphatidylcholine, CT and CPT activities were significantly reduced either by 50 mM glucose or 20 microM AGEs. Simultaneously, [Me-(3)H]choline release was significantly stimulated by both agents. We conclude that prolonged treatments with high glucose or AGEs are not able to induce oxidative injury in bovine retinal capillary pericytes. Nevertheless, they do induce phospholipid hydrolysis and phospholipid enzyme activity inhibition.
Asunto(s)
Glucosa/farmacología , Productos Finales de Glicación Avanzada/farmacología , Pericitos/efectos de los fármacos , Fosfolípidos/metabolismo , Vasos Retinianos/efectos de los fármacos , Animales , Ácido Araquidónico/metabolismo , Caspasa 3 , Caspasas/metabolismo , Bovinos , Supervivencia Celular/efectos de los fármacos , Células Cultivadas , Colina/metabolismo , Glucosa/toxicidad , Productos Finales de Glicación Avanzada/toxicidad , L-Lactato Deshidrogenasa/análisis , Peroxidación de Lípido , Microscopía Electrónica , Estrés Oxidativo , Pericitos/metabolismo , Pericitos/ultraestructura , Especies Reactivas de Oxígeno/metabolismo , Vasos Retinianos/metabolismo , Albúmina Sérica Bovina/farmacologíaRESUMEN
Free radicals induced by organic peroxides or oxidized low density lipoprotein (oxLDL) play a critical role in the development of atherosclerosis. In investigating this process, and the concomitant inflammatory response, the role of pericytes, cells supporting the endothelial ones in blood vessels, has received little attention. In this study we tested the hypothesis that tert-butyl hydroperoxide (t-BuOOH) and oxLDL, administered in sublethal doses to the culture medium of retinal pericytes, function as prooxidant signals to increase the stimulation of the peroxidation process induced by lipopolysaccharide (LPS). Confluent cell monolayers were exposed to t-BuOOH (25-400 microM), native LDL or oxLDL (3.4-340 nmol hydroperoxides/mg protein, 1-100 micro). LPS (1 microg/ml), t-BuOOH (200 microM), and oxLDL (100 microM), but not native LDL, incubated for 24 h with cells, markedly increased lipid peroxidation, cytosolic phospholipase A2 (cPLA2) activity and arachidonic acid (AA) release in a time- and dose-dependent manner. AACOCF(3), a potent cPLA2 inhibitor, and the antioxidant alpha-tocopherol strongly inhibited the prooxidant-stimulated AA release. Long-term exposure to maximal concentrations of t-BuOOH (400 microM) or oxLDL (100 microM) had a sharp cytotoxic effect on the cells, described by morphological and biochemical indices. The presence of t-BuOOH or oxLDL at the same time, synergistically increased phospholipid hydrolysis induced by LPS alone. 400 microM t-BuOOH or 100 microM oxLDL had no significant effect on the stimulation of an apoptosis process estimated by DNA laddering and light and electron microscopy. The results indicate that (i) pericytes may be the target of extensive oxidative damage; (ii) activation of cPLA2 mediates AA liberation; (iii) as long-term regulatory signals, organic peroxide and specific constituents of oxLDL increase the pericyte ability to degrade membrane phospholipids mediated by LPS which was used, in the present study, to simulate in vitro an inflammatory burst in the retinal capillaries.
Asunto(s)
Lipoproteínas LDL/farmacología , Oxidantes/farmacología , Pericitos/metabolismo , Fosfolípidos/metabolismo , Retina/metabolismo , terc-Butilhidroperóxido/farmacología , Animales , Bovinos , Células Cultivadas , Colorantes , Fragmentación del ADN , Hidrólisis/efectos de los fármacos , Lipopolisacáridos , Microscopía Electrónica , Estrés Oxidativo , Pericitos/química , Fosfolípidos/química , Retina/química , Sales de Tetrazolio , TiazolesRESUMEN
Several trophic factors are known to regulate the survival and growth of neurons in brain and peripheral tissues. Several findings suggest that basic fibroblast growth factor-2 (FGF-2) plays an important role in the "self-repair" responses that follow injuries such as trauma and brain ischemia and that FGF-2 contributes to the repair of damaged tissue. Transforming growth factor-beta (TGF-beta) is a potent growth-regulatory protein secreted by virtually all cells. In the present study, we used immunohistochemical techniques to investigate whether FGF-2 and TGF-beta1 participate in the healing of damaged tissue following partial brain ischemia. The profile of the observed immunoreactivities indicated that TGF-beta1 and FGF-2 release varies between the different cerebral areas subjected to ischemic insult. Moreover, the sectorial heterogeneity of immunocytochemical response suggests that, during postischemic reperfusion, neuronal recovery may be due not only to neuron-glia interaction but also to neurochemical conditions involving inhibitory interneurons.
Asunto(s)
Isquemia Encefálica/metabolismo , Factor 2 de Crecimiento de Fibroblastos/metabolismo , Regeneración Nerviosa/fisiología , Recuperación de la Función/fisiología , Daño por Reperfusión/metabolismo , Factor de Crecimiento Transformador beta/metabolismo , Animales , Isquemia Encefálica/patología , Isquemia Encefálica/fisiopatología , Corteza Cerebelosa/metabolismo , Corteza Cerebelosa/patología , Corteza Cerebral/metabolismo , Corteza Cerebral/patología , Modelos Animales de Enfermedad , Hipocampo/metabolismo , Hipocampo/patología , Masculino , Neuronas/metabolismo , Neuronas/patología , Ratas , Ratas Wistar , Daño por Reperfusión/patología , Daño por Reperfusión/fisiopatología , Factores de TiempoRESUMEN
The genus Cistus includes many typical species of Mediterranean flora; Cistus species are used as antidiarrhetics, as general remedies for treatment of various skin diseases in folk medicine and as anti-inflammatory agents. These species contain flavonoids that are considered to be chain-breaking antioxidants. In this work, we have investigated the effects of crude aqueous extracts from Cistus incanus and Cistus monspeliensis on DNA cleavage and their free-radical scavenging capacity. In addition, their effect on lipid peroxidation in rat liver microsomes was evaluated. These extracts showed a protective effect on DNA cleavage and a dose-dependent free-radical scavenging capacity; Cistus monspeliensis was more active than Cistus incanus; these results were confirmed by a significant inhibition of lipid peroxidation in rat liver microsomes. The experimental evidence, therefore, suggests that because of their antioxidant activity these extracts may offer excellent photoprotection for skin and may be useful in the treatment of human diseases where oxidative stress plays a key role.
Asunto(s)
Antioxidantes/farmacología , Daño del ADN/efectos de los fármacos , Extractos Vegetales/farmacología , Plantas Medicinales , Animales , Antioxidantes/metabolismo , Depuradores de Radicales Libres/metabolismo , Depuradores de Radicales Libres/farmacología , Peróxido de Hidrógeno/farmacología , Peroxidación de Lípido/efectos de los fármacos , Peroxidación de Lípido/fisiología , Hígado/metabolismo , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Oxidantes/farmacología , Extractos Vegetales/metabolismo , Ratas , Piel/metabolismo , Rayos UltravioletaRESUMEN
Evidence is accumulating that intermediates of oxygen reduction may be associated with the development of alcoholic disease. In addition, free radical-induced perturbation of the oxidant/antioxidant balance in cells is widely recognized as the main causative factor of age-related disorders. In the present work, we investigated the effects of 25 months of ethanol consumption on the antioxidant defense system in different organs of rat in comparison with normal aging, in the absence and presence of treatment with metadoxine, an ion pair composed of pirrolidone carboxylate and pyridoxine. We demonstrate that aged rats underwent a significant perturbation of the antioxidant defense system, as indicated by depletion of reduced glutathione (GSH) content, and increases in oxidized GSH and free radical-induced luminescence associated with a decrease of GSH reductase and an increase of GSH transferase activities. These modifications, observed particularly in the liver and brain with respect to other organs, were enhanced by long-term alcohol exposure, and interestingly, significantly reduced after metadoxine supplementation. Our results indicate that increased GSH transferase activity and decreased GSH reductase activity, followed by thiol depletion, are important factors sustaining a pathogenic role for oxidative stress in aging and in all situations where age-correlated changes occur. Administration of metadoxine greatly reduces these metabolic abnormalities. This evidence supports the pharmacological potential of metadoxine in the management of alcoholic disturbances.
Asunto(s)
Antioxidantes/metabolismo , Etanol/toxicidad , Glutatión/metabolismo , Piridoxina/farmacología , Ácido Pirrolidona Carboxílico/farmacología , Envejecimiento/fisiología , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Combinación de Medicamentos , Etanol/administración & dosificación , Etanol/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Masculino , Oxidación-Reducción/efectos de los fármacos , Estrés Oxidativo , Ratas , Ratas WistarRESUMEN
Numerous experimental evidence sustains a pathogenic role for oxidative stress in aging. Acute and chronic ethanol metabolism is also known to be associated with oxidative perturbation of cellular oxidant/antioxidant balance. In the present work we investigated the effects of 25 months of ethanol consumption on the antioxidant defense system in different organs of rats, in comparison with normal and aged animals. We show that aged rats underwent a significant perturbation of the antioxidant defense system, as indicated by depletion of reduced glutathione content, increases in oxidized glutathione and free radical-induced urinary luminescence associated with a decrease of glutathione reductase and increase of glutathione transferase activities. These modifications, observed particularly in the liver and brain, were enhanced by long-term alcohol exposure. Our results indicate that increased glutathione transferase activity and decreased glutathione reductase activity, followed by thiol depletion, are important factors sustaining a pathogenic role for oxidative stress in aging and in all situations where age-correlated changes occur. They also reinforce the oxidative potential of toxic compounds, such as ethanol intoxication.
Asunto(s)
Etanol/administración & dosificación , Orina/química , Administración Oral , Envejecimiento , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Esquema de Medicación , Eritrocitos/metabolismo , Glutatión/sangre , Glutatión/metabolismo , Corazón/efectos de los fármacos , Intubación Gastrointestinal , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/efectos de los fármacos , Hígado/metabolismo , Mediciones Luminiscentes , Masculino , Especificidad de Órganos/efectos de los fármacos , Oxidación-Reducción/efectos de los fármacos , Ratas , Ratas WistarRESUMEN
In the present study we analyzed the age-dependent changes of mRNA levels for cytochrome c oxidase and FoF1-ATP synthase subunits in rat cerebral cortex and cerebellum. To establish whether the regulation of expression is transcriptional or post-transcriptional, the results were compared to those related to protein subunits levels, of the same enzymatic complexes, previously observed. The different patterns of age-related changes of mRNA subunits, in particular the lower increments, compared with those related to protein subunits, indicate that post-transcriptional mechanisms of regulation might be involved in the coordinated expression of the various subunits of each complex. Northern blotting analyses of RNA from the cerebellum of rats at the various ages, showed also differences in age-dependent patterns of transcription between cerebral cortex and cerebellum. Moreover, the major age-dependent changes of mitochondrial-encoded subunits, compared with the nuclear-encoded ones, previously observed at proteins level, occur also during transcription.
Asunto(s)
Envejecimiento/metabolismo , Encéfalo/enzimología , Complejo IV de Transporte de Electrones/genética , Regulación de la Expresión Génica , ATPasas de Translocación de Protón/genética , Animales , Northern Blotting , Cerebelo/enzimología , Corteza Cerebral/enzimología , Complejo IV de Transporte de Electrones/metabolismo , ATPasas de Translocación de Protón/metabolismo , ARN Mensajero/metabolismo , Ratas , Ratas Endogámicas WKY , Transcripción GenéticaRESUMEN
It is generally accepted that reactive oxygen species have a major role in the mediation of cell damage and that free sulphydryl (SH) groups are vital in cellular defence against endogenous or exogenous oxidants. Modification of cellular oxidant/antioxidant balance has been involved in the neuropathogenesis of several diseases, e.g., stroke, Parkinson's disease, Alzheimer's disease and physiological ageing. An increasingly important area of antioxidant defence is based on sulphydryl chemistry, owing to the role of SH groups in the function of macromolecular structures such as enzymes and cellular membranes. Thiols, however, may themselves generate deleterious free radicals, and thionyl radicals, which have been demonstrated to originate in biological systems through enzymatic reactions of different peroxidases, by reacting with molecular oxygen or hydrogen peroxide are able to promote reactions of oxidatives stress. In the present study we provide experimental evidence suggesting a selective effect of cysteine in promoting reactions of oxidative stress in the brain areas of substantia nigra and septum, but not in other areas. In contrast, exogenous administration of reduced glutathione led to a significant decrease of lipoperoxidation in the brain areas of cortex and hippocampus, associated to selective changes in the endogenous pool of thiols.
Asunto(s)
Cisteína/toxicidad , Glutatión/farmacología , Peroxidación de Lípido/efectos de los fármacos , Estrés Oxidativo/efectos de los fármacos , Sustancia Negra/efectos de los fármacos , Animales , Corteza Cerebral/efectos de los fármacos , Corteza Cerebral/metabolismo , Cisteína/administración & dosificación , Glutatión/administración & dosificación , Hipocampo/efectos de los fármacos , Hipocampo/metabolismo , Masculino , Malondialdehído/metabolismo , Distribución Aleatoria , Ratas , Ratas Wistar , Tabique Pelúcido/efectos de los fármacos , Tabique Pelúcido/metabolismo , Sustancia Negra/metabolismo , Compuestos de Sulfhidrilo/sangre , Compuestos de Sulfhidrilo/farmacología , Reactivos de Sulfhidrilo/toxicidadRESUMEN
The aim of the study was to determine whether a combination of pyridoxine and pyrrolidone carboxylate (PCA) could influence the antioxidant defence system in hepatic and extrahepatic tissues during acute ethanol administration. The results showed that ethanol treatment led to a significant depletion of reduced glutathione and increase in oxidized glutathione (GSSG) in the different organs, associated with decreased activity of glutathione reductase. Pretreatment of animals with Metadoxine (PCA + pyridoxine) one hour before ethanol administration produced a significant protection against glutathione (GSH) depletion in the different organs examined. This was consistent with an increase in glutathione reductase activity. In view of the fact that free thiol compounds such as glutathione are vital in cellular defence against oxidants and that decreases in reduced glutathione precede lethal cell injury resulting from free radical accumulation, our study supports the effectiveness of Metadoxine as a useful therapeutic agent effective in the management of all those pathological conditions where a severe imbalance of cellular redox state seems to take place as a result of the generation of free radical species.
Asunto(s)
Intoxicación Alcohólica/tratamiento farmacológico , Glutatión/metabolismo , Hígado/efectos de los fármacos , Piridoxina/farmacología , Ácido Pirrolidona Carboxílico/farmacología , Intoxicación Alcohólica/metabolismo , Animales , Encéfalo/efectos de los fármacos , Encéfalo/metabolismo , Combinación de Medicamentos , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Riñón/efectos de los fármacos , Riñón/metabolismo , Hígado/enzimología , Masculino , Ratas , Ratas WistarRESUMEN
The levels of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of F(0)F(1)-ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, 26 months of age were analyzed by Western blot. Age-related changes in the content of subunits, encoded either in mitochondrial or nuclear DNA, were observed.
RESUMEN
It is generally accepted that reactive oxygen species have a major role in the mediation of cell damage and that free sulfhydryl groups are vital in cellular defence against endogenous or exogenous oxidants. Modification of cellular oxidant/antioxidant balance has been involved in the neuropathogenesis of several diseases, e.g., stroke, Parkinson's disease, Alzheimer's disease and physiological ageing. An increasingly important area of antioxidant defence is based on sulfhydryl chemistry, owing to the role of -SH groups in the function of macromolecular structures such as enzymes and cellular membranes. Thiols, however, may themselves generate deleterious free radicals. In the present study we provide experimental evidence suggesting a selective effect of cysteine in promoting reactions of oxidative stress in the brain areas of substantia nigra and septum, but not in other different areas which were associated with corresponding changes in the activity of antioxidant enzymes.
Asunto(s)
Antioxidantes/metabolismo , Cisteína/farmacología , Peroxidación de Lípido/efectos de los fármacos , Especies Reactivas de Oxígeno/metabolismo , Sustancia Negra/efectos de los fármacos , Animales , Evaluación Preclínica de Medicamentos , Glutatión Peroxidasa/metabolismo , Glutatión Reductasa/metabolismo , Glutatión Transferasa/metabolismo , Masculino , Ratas , Ratas Wistar , Sustancia Negra/metabolismoRESUMEN
The contents of subunits I, II/III, and IV of cytochrome c oxidase and of subunits alpha, beta and gamma of FoF1 ATP synthase in inner mitochondrial membrane proteins purified from cerebral cortex of rat at 2, 6, 12, 18, 24, and 26 months of age were analyzed by western blot. Age-related changes in the content of subunits, either of mitochondrial or nuclear origin, were observed. All the cytochrome c oxidase (COX) subunits examined showed an age-related increase from 2-month-old rats up to 24 months with a decrease at the oldest age (26 months). The same pattern of age-dependent changes was observed for gamma ATP synthase, while the alpha and beta subunits increased progressively up to 26 months.
Asunto(s)
Envejecimiento/metabolismo , Corteza Cerebral/enzimología , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/enzimología , ATPasas de Translocación de Protón/metabolismo , Animales , Western Blotting , Corteza Cerebral/ultraestructura , Masculino , Ratas , Ratas Endogámicas WKYRESUMEN
The levels of mRNAs for the subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA)-selective glutamate receptors (GluR-1, -2, -3, -4) in the rat hippocampus during aging were measured by Northern blotting. The distribution of these receptors was also examined at the protein level by immunoblotting using antibodies to GluR-1 and to an epitope common to GluR-2 and GluR-3 (denoted GluR-2/3). During aging a significant decrease of GluR-1 protein, but no change in the corresponding mRNA level, was observed. No differences in the level of GluR-2/3 protein and GluR-2, -3, and -4 mRNAs at the various ages examined (4, 12, and 24 months) were detected. Our results show that AMPA receptors are only slightly influenced by the aging process in the rat hippocampus. The slight decrease in GluR-1 protein content, not accompanied by a parallel decrease in the GluR-1 mRNA level, might be explained by a decreased translational efficiency or an increased protein degradation of the GluR-1 subunit.
Asunto(s)
Hipocampo/fisiología , ARN Mensajero/genética , Receptores de Glutamato/clasificación , Ácido alfa-Amino-3-hidroxi-5-metil-4-isoxazol Propiónico/farmacología , Factores de Edad , Envejecimiento , Animales , Autorradiografía , Expresión Génica , Masculino , Ratas , Ratas Sprague-Dawley , Receptores de Glutamato/fisiologíaRESUMEN
Rats subjected to chronic ethanol intake for a period of 28 days showed significant elevation in blood ethanol levels, a marked decrease in hepatic reduced glutathione (GSH) content and a decrease in liver tryptophan pyrrolase (TPO) activity. A daily intraperitoneal injection of a combined solution of pyrrolidone carboxylate (PCA) and vitamin B6 (pyridoxine hydrochloride) (0.3 mmoles/kg) into ethanol-treated rats resulted in the blood ethanol levels becoming significantly reduced, while the hepatic GSH content and TPO activity were markedly elevated. Our results support the view that PCA and pyridoxine operate to restore the redox imbalance of the hepatocytes caused by chronic alcohol intake.
Asunto(s)
Alcoholismo/tratamiento farmacológico , Hígado/metabolismo , Piridoxina/uso terapéutico , Ácido Pirrolidona Carboxílico/uso terapéutico , Alcoholismo/metabolismo , Animales , Quimioterapia Combinada , Glutatión/metabolismo , Masculino , Nucleótidos/metabolismo , Piridinas/metabolismo , Ratas , Ratas Sprague-Dawley , Triptófano Oxigenasa/metabolismoRESUMEN
Free fatty acids (FFA) and fatty acid ethyl esters (FAEE) were extracted from different organs of rats administered ethanol, which was found to have induced FAEE formation, which reached its highest levels in the heart, followed by kidney, brain and liver; the ethanol administration resulted also in a marked increase of total FFA content, particularly in brain, kidney, heart and liver. Pretreatment of animals with Metadoxine one hour before ethanol administration inhibited significantly both FAEE and FFA accumulation in all organs examined. These effects were concomitant with the decreased levels of ethanol in blood found in alcohol-intoxicated rats pretreated with Metadoxine. Our results point to the role of fatty acid ethyl esters as possible mediators in the production of alcohol-dependent syndromes, especially in organs lacking oxidative pathways. Administration of Metadoxine, through an increment in alcohol metabolism and turnover, greatly reduces this metabolic abnormality, warranting its potential usefulness as a pharmacological tool in alcoholism management.
Asunto(s)
Ésteres/metabolismo , Etanol/farmacología , Ácidos Grasos/metabolismo , Piridoxina/farmacología , Ácido Pirrolidona Carboxílico/farmacología , Animales , Combinación de Medicamentos , Masculino , Ratas , Ratas Wistar , Distribución TisularAsunto(s)
Astrocitos/metabolismo , Encéfalo/metabolismo , ADN/biosíntesis , Sustancias de Crecimiento/farmacología , Factor I del Crecimiento Similar a la Insulina/farmacología , Proteínas del Tejido Nervioso/biosíntesis , ARN/biosíntesis , Aminoácidos/metabolismo , Animales , Animales Recién Nacidos , Astrocitos/efectos de los fármacos , Células Cultivadas , Factor de Crecimiento Epidérmico/farmacología , Factor 2 de Crecimiento de Fibroblastos/farmacología , Insulina/farmacología , Cinética , Ratas , Timidina/metabolismo , Transferrina/farmacología , Tritio , Uridina/metabolismoRESUMEN
Several age-dependent modifications of inner mitochondrial membrane and synaptosomal plasma membrane proteins from different brain regions of 4-, 12-, 18- and 24-month-old male Wistar rats, were observed. Some proteins, identified by immunoblotting assay as various subunits of mitochondrial respiratory chain complexes and calmodulin, were particularly impaired. Chronic treatment with CDP-choline at a dose of 20 mg/kg body weight per day for 28 days caused significant changes in the amounts of several of the above mentioned proteins. Most of the proteins, which decreased during aging, showed a significant increase after CDP-choline treatment compared with the corresponding control values at the same age. The effect of CDP-choline might be due to: the increased availability of cytidylic nucleotides, which in the brain are present in limited amounts compared to the other nucleotides; the increased content of total adenine nucleotides; the improvement of brain energy metabolism.
Asunto(s)
Envejecimiento/metabolismo , Química Encefálica/efectos de los fármacos , Citidina Difosfato Colina/farmacología , Mitocondrias/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Sinaptosomas/metabolismo , Animales , Membrana Celular/metabolismo , Electroforesis en Gel de Poliacrilamida , Hipoxia Encefálica/metabolismo , Immunoblotting , Masculino , Mitocondrias/efectos de los fármacos , Ratas , Ratas Wistar , Sinaptosomas/efectos de los fármacosRESUMEN
The age-dependent modifications of synaptosomal plasma membrane protein composition in three different rat brain regions (cerebral cortex, cerebellum and striatum) at various ages (4, 12 and 24 months) were studied. The proteins were separated by gel-electrophoresis and the quantity of the different polypeptides was determined densitometrically from the stained gels. In the three brain regions examined several age-related modifications in the amount of the synaptosomal plasma membrane proteins were observed. In particular a significant decrease in the content of some synaptosomal plasma membrane proteins at 24 months of age was found. The age-related modifications in the protein composition of synaptosomal plasma membrane may cause changes in many brain functions, such as neurotransmission, ionic transport and enzyme activities. Particularly interesting is the decrease of a protein with 18 kDa mol. wt. This protein has been identified as calmodulin by immunoblotting assay. The decrease in the amount of this protein may be correlated to the impairment of several Ca(2+)-requiring processes in the aging brain.
Asunto(s)
Envejecimiento/metabolismo , Proteínas del Tejido Nervioso/metabolismo , Membranas Sinápticas/metabolismo , Sinaptosomas/metabolismo , Animales , Especificidad de Anticuerpos , Encéfalo/anatomía & histología , Encéfalo/metabolismo , Electroforesis en Gel de Poliacrilamida , Immunoblotting , Masculino , Peso Molecular , Ratas , Ratas WistarRESUMEN
The effect of hypoxia on the protein composition of mitochondria from cerebral cortex of rats at 4, 12, and 24 months of age was investigated. The proteins were separated by electrophoresis on SDS polyacrylamide gels and the percent content was evaluated by measuring the optical density of the stained gels. The results demonstrate that hypoxic treatment causes a decrease in the amount of some proteins as follows: the 90 and the 16 kDa Mw proteins at 4 months; the 82 and the 79 kDa Mw proteins at 24 months; the 52-49, 35 and 20 kDa at all ages investigated; the 44 kDa protein at 4 and 12 months and the 28 kDa protein at 4 and 24 months of age. Our results show that hypoxic conditions affect mitochondrial protein composition to a greater extent than aging alone.
Asunto(s)
Corteza Cerebral/metabolismo , Hipoxia/metabolismo , Mitocondrias/metabolismo , Proteínas/metabolismo , Envejecimiento , Animales , Corteza Cerebral/crecimiento & desarrollo , Electroforesis en Gel de Poliacrilamida , Masculino , Peso Molecular , Proteínas/aislamiento & purificación , Ratas , Ratas Endogámicas , Valores de ReferenciaRESUMEN
The effect of hypoxia on the protein composition of synaptic plasma membranes (SPM) isolated from cerebral cortex of rats at 4, 12, and 24 months of age was investigated. The proteins were separated by SDS polyacrylamide gel electrophoresis and the percent content was evaluated by measuring the optical density of the stained gels. After hypoxic treatment various proteins showed significant changes. Some proteins were only affected at 4 and 12 months of age and not at 24 months. The various modified proteins may be identified according to their molecular weight, as follows: the 18 kDa protein with calmodulin; the 23 kDa protein with D3 subunits; the 28 kDa protein could contain the delta subunit of the Ca2+ channel. The changes in the amount of some SPM proteins during hypoxia is consistent with the alteration in membrane polarization and neurotransmission observed in this condition. The effect of aging at the synaptosomal level seems to be a selective process; after hypoxia the age-related changes of many proteins are more pronounced.