RESUMEN
Inward rectifying potassium channels sensitive to ATP levels (KATP) have been the subject of investigation for several decades. Modulators of KATP channels are well-established treatments for metabolic as well as cardiovascular diseases. Experimental studies have also shown the potential of KATP modulation in neurodegenerative disorders. However, to date, data regarding the effects of KATP antagonists/agonists in experiments related to neurodegeneration remain inconsistent. The main source of confusion in evaluating available data seems to be the choice of experimental models. The present study aims to provide a comprehensive understanding of the effects of both opening and blocking KATP channels in two forms of SH-SY5Y cells. Our results offer valuable insights into the significance of metabolic differences between differentiated and non-differentiated SH-SY5Y cells, particularly in the context of glibenclamide and diazoxide effects under normal conditions and during the initiation of pathological events simulating Parkinson's disease in vitro. We emphasize the analysis of mitochondrial functions and changes in mitochondrial network morphology. The heightened protein expression of KATP channels identified in non-differentiated SH-SY5Y cells seems to be a platform for a more significant impact of KATP modulators in this cell type. The efficiency of rotenone treatment in inducing morphological changes in the mitochondrial network depends on the differentiation status of SH-SY5Y cells.
Asunto(s)
Diferenciación Celular , Canales KATP , Mitocondrias , Humanos , Canales KATP/metabolismo , Mitocondrias/metabolismo , Enfermedad de Parkinson/metabolismo , Enfermedad de Parkinson/patología , Línea Celular Tumoral , Diazóxido/farmacologíaRESUMEN
BACKGROUND: Anti-citrullinated peptides antibodies (ACPA) are specific for rheumatoid arthritis and have been implicated in disease pathogenesis. ACPA examination is a new component of ACR/EULAR 2010 classification criteria for rheumatoid arthritis. ACPA positivity predicts a more erosive disease course with severe joint damage and extra-articular manifestations. OBJECTIVES: To evaluate the benefits of ACPA examination in patients with early undifferentiated arthritis and patients with rheumatoid arthritis. METHODS: We examined patients with arthritis and tested them for ACPA positivity. In every individual patient we evaluated if ACPA examination was necessary to establish the diagnosis of rheumatoid arthritis, or to change treatment, or if the diagnosis could have been established without ACPA examination (ACR/EULAR 2010 classification criteria was met without ACPA scoring). RESULTS AND CONCLUSIONS: The study was placed in Slovak Republic. We examined 833 patients with arthritis. There were 43 patients, or 62% of a subgroup of 69 who were ACPA positive whose ACPA examination was not needed-ACR/EULAR criteria was met without ACPA scoring. This number represents 5.1% of the total number examined. There were 15 patients, or 22% of the subgroup and 1.8% of the total whose diagnosis was revised to rheumatoid arthritis due to ACPA positivity-ACR/EULAR criteria were met solely with ACPA scoring. There were 11 patients (16% and 1.3%) whose medication was changed due to ACPA positivity. ACPA examination is useful in 3.1% of all examined patients. When we correlate data on ACPA positive patients, 38% of the patients profit from ACPA examinations. Considering the relatively low price of ACPA testing, this examination should not be excluded.
Asunto(s)
Anticuerpos Antiproteína Citrulinada/sangre , Artritis Reumatoide/clasificación , Autoanticuerpos/sangre , Péptidos Cíclicos/sangre , Factor Reumatoide/sangre , Adulto , Anciano , Artritis Reumatoide/sangre , Artritis Reumatoide/diagnóstico , Progresión de la Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Péptidos Cíclicos/inmunología , Eslovaquia/epidemiologíaRESUMEN
Heat shock proteins (HSPs) HSP27, HSP70 and HSP90 are molecular chaperones; their expression is increased after exposure of cells to conditions of environmental stress, including heat shock, heavy metals, oxidative stress, or pathologic conditions, such as ischemia, infection, and inflammation. Their protective function is to help the cell cope with lethal conditions. The HSPs are a class of proteins which, in normal cells, are responsible for maintaining homeostasis, interacting with diverse protein substrates to assist in their folding, and preventing the appearance of folding intermediates that lead to misfolded or damaged molecules. They have been shown to interact with different key apoptotic proteins and play a crucial role in regulating apoptosis. Several HSPs have been demonstrated to directly interact with various components of tightly regulated caspase-dependent programmed cell death. These proteins also affect caspase-independent apoptosis by interacting with apoptogenic factors. Heat shock proteins are aberrantly expressed in hematological malignancies. Because of their prognostic implications and functional role in leukemias, HSPs represent an interesting target for antileukemic therapy. This review will describe different molecules interacting with anti-apoptotic proteins HSP70 and HSP90, which can be used in cancer therapy based on their inhibition.
Asunto(s)
Proteínas de Choque Térmico/fisiología , Leucemia/patología , Apoptosis , Proteínas de Choque Térmico HSP27/fisiología , Proteínas HSP70 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP70 de Choque Térmico/química , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas HSP90 de Choque Térmico/antagonistas & inhibidores , Proteínas HSP90 de Choque Térmico/química , Proteínas HSP90 de Choque Térmico/fisiología , HumanosRESUMEN
BACKGROUND: Brain tumors are a heterogeneous group of malignancies characterized by inter- and intratumoral heterogeneity. Among them, the most aggressive and, despite advances in medicine, still incurable remains glioblastoma. One of the reasons is the high recurrence rate of the disease and resistance to temozolomide, a golden standard in chemotherapy of brain tumors. Therefore, mapping the pathways responsible for tumorigenesis at the transcriptional level may help to determine the causes and aggressive behavior among different glial tumors. PATIENTS AND METHODS: Biopsies from patients with astrocytoma (N = 6), glioblastoma (N = 22), and meningioma (N = 14) were included in the sample set. A control group consisted of RNA isolated from healthy human brain (N = 3). The reverse-transcribed cDNAs were analyzed using the Human Cancer PathwayFinder™ real-time PCR Array in a 96-well format. The expression of 84 genes belonging to 9 signaling pathways (angiogenesis, apoptosis, cell cycle and senescence, DNA damage and repair, epithelial-to-mesenchymal transition, hypoxia, overall metabolism, and telomere dynamics) was determined for each sample. RESULTS: By determining the relative expression of selected genes, we characterized the transcriptomic profile of individual brain malignancies in the context of signaling pathways involved in tumorigenesis. We observed deregulation in 50, 52.4 and 53.6% % of the genes in glioblastomas, meningiomas and astrocytomas, respectively. The most pronounced changes with statistical significance compared to control were observed in the genes associated with epithelial-to-mesenchymal transition (CDH2, FOXC2, GSC, SNAI2, and SOX10), cellular senescence (BMI1, ETS2, MAP2K1, and SOD1), DNA repair (DDB2, ERCC3, GADD45G, and LIG4), and dynamic of telomeres (TEP1, TERF2IP, TNKS, and TNKS2). CONCLUSION: Based on the obtained data, we can conclude that individual diagnoses differ in transcriptomic profile. An individual molecular approach is therefore necessary in order to provide comprehensive and targeted therapy on multiple metabolic pathways in the diagnosis of brain tumors.
Asunto(s)
Astrocitoma , Neoplasias Encefálicas , Glioblastoma , Tanquirasas , Humanos , Glioblastoma/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/patología , Astrocitoma/genética , Astrocitoma/patología , Encéfalo , Carcinogénesis , Transformación Celular NeoplásicaRESUMEN
New insights into cancer cells - specific biological pathways are urgently needed to promote development of exactly targeted therapeutics. The role of oncoproteins and tumor suppressor proteins in proliferative signaling, cell cycle regulation and altered adhesion is well established. Chemicals, viruses and radiation are also generally accepted as agents that commonly induce mutations in genes encoding these cancer-inducing proteins, thereby giving rise to cancer. More recent evidence indicates the importance of two additional key factors imposed on proliferating cells - hypoxia and/or lack of glucose. These two additional triggers can initiate and promote the process of malignant transformation, when a low percentage of cells escape cellular senescence. Disregulated cell proliferation leads to formation of cellular masses that extend beyond the resting vasculature, resulting in oxygen and nutrient deprivation. Resulting hypoxia triggers a number of critical adaptations that enable cancer cell survival. The process of apoptosis is suppressed and glucose metabolism is altered. Recent investigations suggest that oxygen depletion stimulates mitochondria to compensate increased reactive oxygen species (ROS). It activates signaling pathways, such as hypoxia-inducible factor 1, that promote cancer cell survival and tumor growth. During the last decade, mitochondria have become key organelles involved in chemotherapy-induced apoptosis. Therefore, the relationship between mitochondria, ROS signaling and activation of survival pathways under hypoxic conditions has been the subject of increased study. Insights into mechanisms involved in ROS signaling may offer novel ways to facilitate discovery of cancer-specific therapies.
Asunto(s)
Mitocondrias/fisiología , Terapia Molecular Dirigida , Neoplasias/terapia , Metabolismo Energético , Glucólisis , Humanos , Neoplasias/metabolismo , Fosforilación OxidativaRESUMEN
BACKGROUNDS: Translational medicine is a medical field encompassing basic research and development of new diagnostic and therapeutic strategies for clinical practice. The present scientific paper focuses on our previous experience in the field of chemoresistance testing in patients with oncological diseases. MATERIAL AND METHODS: Since 2005, we sampled 71 patients with a leukaemia (AML, ALL and CML) and 92 patients with a solid tumour (lung and gastrointestinal tract cancer). Malignant cell in vitro drug resistance testing was carried out using cytotoxic methyl-thiazol tetrazolium (MTT) assay. RESULTS: Based on the LC50 (lethal concentration of a drug killing 50% of cell population), we found that patients with acute myeloblastic leukaemia exhibit a greater degree of resistance than patients with acute lymphoblastic leukaemia. In patients with bronchogenic carcinomas, primary resistance to cisplatin was identified in 28% of tested samples, paclitaxel 36%, vincristine 50%, etoposide 56%, vinorelbine 57%, topotecan 62%, gemcitabine 77% and dacarbazine 86%. CONCLUSION: In vitro tests with gastrointestinal tract cancers also suggested high effectiveness of cisplatin (with the exception of gastric carcinoma) that was comparable with 5-fluorouracil. Even though the MTT assay has some limitations (insufficient number of vital cells, possible contamination by non-malignant cells, etc.), this in vitro method proved very effective in testing malignant cell resistance to clinically used cytostatics.
Asunto(s)
Ensayos de Selección de Medicamentos Antitumorales , Neoplasias Gastrointestinales/tratamiento farmacológico , Leucemia/tratamiento farmacológico , Neoplasias Pulmonares/tratamiento farmacológico , Adolescente , Adulto , Anciano , Anciano de 80 o más Años , Femenino , Humanos , Masculino , Persona de Mediana Edad , Adulto JovenRESUMEN
Acute myocardial infarction (AMI) is one of the leading causes of death among adults in older age. Understanding mechanisms how organism responds to ischemia is essential for the ischemic patient's prevention and treatment. Despite the great prevalence and incidence only a small number of studies utilize a metabolomic approach to describe AMI condition. Recent studies have shown the impact of metabolites on epigenetic changes, in these studies plasma metabolites were related to neurological outcome of the patients making metabolomic studies increasingly interesting. The aim of this study was to describe metabolomic response of an organism to ischemic stress through the changes in energetic metabolites and aminoacids in blood plasma in patients overcoming acute myocardial infarction. Blood plasma from patients in the first 12 h after onset of chest pain was collected and compared with volunteers without any history of ischemic diseases via NMR spectroscopy. Lowered plasma levels of pyruvate, alanine, glutamine and neurotransmitter precursors tyrosine and tryptophan were found. Further, we observed increased plasma levels of 3-hydroxybutyrate and acetoacetate in balance with decreased level of lipoproteins fraction, suggesting the ongoing ketonic state of an organism. Discriminatory analysis showed very promising performance where compounds: lipoproteins, alanine, pyruvate, glutamine, tryptophan and 3-hydroxybutyrate were of the highest discriminatory power with feasibility of successful statistical discrimination.
Asunto(s)
Dolor en el Pecho/sangre , Espectroscopía de Resonancia Magnética/métodos , Infarto del Miocardio/sangre , Ácido 3-Hidroxibutírico/sangre , Acetoacetatos/sangre , Biomarcadores/sangre , Dolor en el Pecho/fisiopatología , Femenino , Humanos , Lipoproteínas/sangre , Masculino , Metaboloma , Persona de Mediana Edad , Infarto del Miocardio/diagnóstico , Curva ROCRESUMEN
Dysfunction of mitochondria induced by ischemia is considered to be a key event triggering neuronal cell death after brain ischemia. Here we report the effect of ischemia-reperfusion on mitochondrial protein synthesis and activity of cytochrome c oxidase (EC 1.9.3.1, COX). By performing 4-vessel occlusion model of global brain ischemia, we have observed that 15 min of global ischemia led to the inhibition of COX subunit I (COXI) synthesis to 56 % of control. After 1, 3 and 24 h of reperfusion, COXI synthesis was inhibited to 46, 50 and 72 % of control, respectively. Depressed synthesis of COXI was not a result of either diminished transcription of COXI gene or increased proteolytic degradation of COXI, since both Northern hybridization and Western blotting did not show significant changes in COXI mRNA and protein level. Thus, ischemia-reperfusion affects directly mitochondrial translation machinery. In addition, ischemia in duration of 15 min and consequent 1, 3 and 24 h of reperfusion led to the inhibition of COX activity to 90.3, 80.3, 81.9 and 83.5 % of control, respectively. Based on our data, we suggest that inhibition of COX activity is rather caused by ischemia-induced modification of COX polypeptides than by inhibition of mitochondrial translation.
Asunto(s)
Isquemia Encefálica/complicaciones , Corteza Cerebral/enzimología , Complejo IV de Transporte de Electrones/metabolismo , Mitocondrias/enzimología , Proteínas Mitocondriales/biosíntesis , Daño por Reperfusión/enzimología , Animales , Isquemia Encefálica/enzimología , Modelos Animales de Enfermedad , Regulación hacia Abajo , Complejo IV de Transporte de Electrones/biosíntesis , Complejo IV de Transporte de Electrones/genética , Regulación Enzimológica de la Expresión Génica , Masculino , Proteínas Mitocondriales/genética , Procesamiento Proteico-Postraduccional , ARN Mensajero/metabolismo , Ratas , Ratas Wistar , Daño por Reperfusión/etiología , Factores de Tiempo , Transcripción GenéticaRESUMEN
Oxidative stress and decline in cellular redox regulation have been hypothesized to play a key role in cardiovascular aging; however, data on antioxidant and redox regulating systems in the aging heart are controversial. The aim of the present study was to examine the effect of aging on critical antioxidant enzymes and two major redox-regulatory systems glutathione (GSH) and thioredoxin (Trx) system in hearts from adult (6-month-old), old (15-month-old), and senescent (26-month-old) rats. Aging was associated with a non-uniform array of changes, including decline in contents of reduced GSH and total mercaptans in the senescent heart. The activities of Mn-superoxide dismutase (SOD2), glutathione peroxidase (GPx), glutathione reductase (GR), and thioredoxin reductase (TrxR) exhibited an age-related decline, whereas catalase was unchanged and Cu,Zn-superoxide dismutase (SOD1) displayed only slight decrease in old heart and was unchanged in the senescent heart. GR, Trx, and peroxiredoxin levels were significantly reduced in old and/or senescent hearts, indicating a diminished expression of these proteins. In contrast, SOD2 level was unchanged in the old heart and was slightly elevated in the senescent heart. Decline in GPx activity was accompanied by a loss of GPx level only in old rats, the level in senescent heart was unchanged. These results indicate age-related posttranslational protein modification of SOD2 and GPx. In summary, our data suggest that changes are more pronounced in senescent than in old rat hearts and support the view that aging is associated with disturbed redox balance that could alter cellular signaling and regulation.
Asunto(s)
Envejecimiento/metabolismo , Antioxidantes/metabolismo , Miocardio/enzimología , Animales , Antioxidantes/análisis , Glutatión Peroxidasa/análisis , Glutatión Peroxidasa/metabolismo , Masculino , Miocardio/química , Oxidación-Reducción , Ratas , Ratas Wistar , Superóxido Dismutasa/metabolismoRESUMEN
Our understanding of the role played by reactive oxygen and nitrogen species in disease pathology and ageing is still insufficient. Reactive oxygen species and reactive nitrogen species can initiate protein and lipid oxidative damage that may be the most important contribution to ageing and age-related heart diseases. In the present study, we investigated the effect of ageing on oxidative damage of protein amino acid residues and lipids in heart homogenate and mitochondria of 4- and 26-month-old Wistar rats. Levels of dityrosine and levels of lysine conjugates increased in heart homogenate during ageing, although levels of conjugated dienes did not change. We observed significantly oxidative modification of tryptophan in heart mitochondria and increased levels of dityrosine with advancing age. However, levels of lysine conjugates, conjugated dienes as well as relative level of cytochrome c oxidase were unchanged in heart mitochondria during ageing. The results of this study suggest a different mechanism of oxidative modification in heart compartments during ageing and moreover, mitochondria and other cellular compartments are targets for oxidative modifications.
Asunto(s)
Envejecimiento/metabolismo , Mitocondrias Cardíacas/metabolismo , Miocardio/metabolismo , Naftalenosulfonatos de Anilina , Animales , Complejo IV de Transporte de Electrones/metabolismo , Colorantes Fluorescentes , Peroxidación de Lípido , Lisina/metabolismo , Masculino , Oxidación-Reducción , Ratas , Ratas Wistar , Sustancias Reactivas al Ácido Tiobarbitúrico/metabolismo , Triptófano/metabolismo , Tirosina/análogos & derivados , Tirosina/metabolismoRESUMEN
OBJECTIVE: To determine clinical benefits of mRNA aromatase expression in entopic endometrium as a diagnostic marker of endometriosis. DESIGN: Prospective clinical trial. SETTING: Department of Obstetrics and Gynaecology of Jessenius Medical Faculty and Faculty Hospital, Martin. METHODS: The expression of mRNA aromatase of eutopic endometrium was determined among women who underwent laparoscopy or laparotomy due to pelvic pain, infertility or benign pelvic tumor. Endometriosis was confirmed histologicaly and classified by rAFS. RESULTS: On the basis of entering criteria 23 women were enrolled in this study and divided into two subgroups: 12 endometriotic and 11 without endometriosis. Sensitivity of aromatase expression was 75% and specificity 54.5% at the cut-off value of at least minimal aromatase activity. By the presence of estrogen-dependent diseases- endometriosis, myomas or endometrial hyperplasia 18 women were compared to 5 disease free women. In this case, sensitivity of aromatase expression was 72.2 and specificity 80%. CONCLUSION: Aromatase expression in eutopic endometrium is a good diagnostic marker for endometriosis.
Asunto(s)
Aromatasa/genética , Hiperplasia Endometrial/diagnóstico , Endometriosis/diagnóstico , Estrógenos/fisiología , Leiomioma/diagnóstico , ARN Mensajero/análisis , Neoplasias Uterinas/diagnóstico , Adulto , Aromatasa/metabolismo , Biomarcadores/análisis , Hiperplasia Endometrial/enzimología , Endometriosis/enzimología , Endometrio/enzimología , Femenino , Humanos , Leiomioma/enzimología , Reacción en Cadena de la Polimerasa , Neoplasias Uterinas/enzimologíaRESUMEN
Protein p53 is the tumor suppressor involved in cell cycle control and apoptosis. As a transcription factor p53 controls many cell processes and helps in prevention of cancer development. The p53 gene is polymorphic. Polymorphisms can affect the important regions involved in protein tumor suppressor activity. The well-known polymorphisms are the polymorphisms BstUI in exon 4 and MspI in intron 6. Both are supposed to be associated with cancer development. The purpose of this study was to investigate the genotype frequencies and associations of these polymorphisms with breast cancer in Slovak population. We observed the prevalence of BstUIPro (27.47%) and MspIA1 (17.58%) alleles and BstUIPro/Pro (8.79%) and MspIA1/A1 (5.49%) genotypes in breast cancer patients in comparison with controls 23.40%, 14.10%, 5.77%, 1.92% respectively. However the differences were not significant. After division of the cases and controls according to the age the prevalence of the risk alleles and genotypes in women at the age 50 years or less was higher as compared to women older than 50 years. In the younger women group, the p53 BstUI polymorphism genotype frequencies were 6.2% for BstUIPro/Pro, 31.0% for BstUIArg/Pro and 62.8% for BstUIArg/Arg in controls and 11.11 %, 40.74% and 48.15% in cases respectively. The risk of disease for BstUIPro/Pro genotype was more than two-fold higher in comparison with the BstUIArg/Arg (OR=2.34, 95% CI=0.53-10.24). In p53 MspI the genotype frequencies were 1.77% for MspIA1/A1, 24.78% for MspIA1/A2 and 73.45% for MspIA2/A2 in controls and 11.11%, 18.52% and 70.37% in cases respectively. The risk of disease for MspIA1/A1 genotype was more than six-fold higher in comparison with the MspIA2/A2 (OR=6.55, 95% CI=1.02-41.98). When we evaluated the association of both polymorphisms together with the breast cancer risk we observed that the highest risk was connected with the genotype BstUIPro/Pro / MspIA1/A1 (OR=2.99, 95% CI=0.69-13.06). Our results indicate that both BstUI and MspI p53 polymormphisms might play the role in the breast cancer development especially in women younger than 50 years.
Asunto(s)
Neoplasias de la Mama/genética , Polimorfismo Genético , Proteína p53 Supresora de Tumor/genética , Adulto , Anciano , Anciano de 80 o más Años , Alelos , Estudios de Casos y Controles , ADN de Neoplasias/genética , Desoxirribonucleasa HpaII/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Femenino , Genotipo , Humanos , Persona de Mediana Edad , Mapeo Restrictivo , Factores de RiesgoRESUMEN
The Ca(2+)-binding proteins parvalbumin (PV) and calbindin D-28k (CB) are key players in the intracellular Ca(2+)-buffering in specific cells including neurons and have profound effects on spatiotemporal aspects of Ca(2+) transients. The previously observed increase in mitochondrial volume density in fast-twitch muscle of PV-/- mice is viewed as a specific compensation mechanism to maintain Ca(2+) homeostasis. Since cerebellar Purkinje cells (PC) are characterized by high expression levels of the Ca(2+) buffers PV and CB, the question was raised, whether homeostatic mechanisms are induced in PC lacking these buffers. Mitochondrial volume density, i.e. relative mitochondrial mass was increased by 40% in the soma of PV-/- PC. Upregulation of mitochondrial volume density was not homogenous throughout the soma, but was selectively restricted to a peripheral region of 1.5 microm width underneath the plasma membrane. Accompanied was a decreased surface of subplasmalemmal smooth endoplasmic reticulum (sPL-sER) in a shell of 0.5 microm thickness underneath the plasma membrane. These alterations were specific for the absence of the "slow-onset" buffer PV, since in CB-/- mice neither changes in peripheral mitochondria nor in sPL-sER were observed. This implicates that the morphological alterations are aimed to specifically substitute the function of the slow buffer PV. We propose a novel concept that homeostatic mechanisms of components involved in Ca(2+) homeostasis do not always occur at the level of similar or closely related molecules. Rather the cell attempts to restore spatiotemporal aspects of Ca(2+) signals prevailing in the undisturbed (wildtype) situation by subtly fine tuning existing components involved in the regulation of Ca(2+) fluxes.
Asunto(s)
Retículo Endoplásmico/patología , Retículo Endoplásmico/ultraestructura , Mitocondrias/patología , Parvalbúminas/deficiencia , Células de Purkinje/ultraestructura , Animales , Western Blotting/métodos , Calbindinas , Calcio/metabolismo , Corteza Cerebelosa/citología , Electroforesis en Gel Bidimensional/métodos , Técnica del Anticuerpo Fluorescente/métodos , Ratones , Ratones Endogámicos C57BL , Ratones Noqueados , Microscopía Electrónica de Transmisión/métodos , Mitocondrias/ultraestructura , ATPasas Transportadoras de Calcio de la Membrana Plasmática/metabolismo , Células de Purkinje/patología , Proteína G de Unión al Calcio S100/genética , Estadísticas no ParamétricasRESUMEN
The incubation of the gerbil forebrain microsomes in the presence of ferrous sulphate and EDTA for either 30 min or for 60 min at a temperature of 37 degrees C led to the inhibition of Ca2+-ATPase in both a concentration- and time-dependent manner. The concentrations of Fe2+ which led to the inhibition of 50% of the Ca2+-ATPase activity (IC50-value) at these times were 0.59 mM and 0.07 mM, respectively. The preincubation of microsomes with 0.1 mM of stobadine prevented the inhibition of Ca2+-ATPase, however, the effectivity of prevention was dependent on the Fe2+ concentration. The net effect of stobadine was an increase in IC50-value to 0.76 mM. Unlike stobadine, reduced glutathione is a naturally occurring water soluble antioxidant. Glutathione at the concentration of 0.1 mM had no significant protective effect on the inhibition of Ca2+-ATPase. The protective effect of a stobadine-glutathione mixture was also investigated; 0.1 mM of stobadine in combination with 0.1 mM of glutathione was more potent in prevention of Fe2+-induced inhibition of Ca2+-ATPase than stobadine alone (IC50=1. 31 mM). In addition, we have investigated the effect of various stobadine-glutathione molar ratios (the total concentration of both antioxidants being 0.2 mM) on Fe2+-induced inhibition of Ca2+-ATPase. The results indicated that the best stobadine-glutathione ratio was close to 1 : 1. The effect of 0.04 mM stobadine in combination with 0.16 mM glutathione was comparable to the effect of 0.2 mM of stobadine alone, whereas 0.2 mM glutathione was almost ineffective. These results may suggest a possible role of membrane in Fe2+-induced inhibition of Ca2+-ATPase.
Asunto(s)
Antioxidantes/farmacología , ATPasas Transportadoras de Calcio/antagonistas & inhibidores , Carbolinas/farmacología , Compuestos Ferrosos/farmacología , Glutatión/farmacología , Microsomas/enzimología , Prosencéfalo/enzimología , Animales , Combinación de Medicamentos , Activación Enzimática/efectos de los fármacos , Gerbillinae , Microsomas/efectos de los fármacosRESUMEN
Treatment with FeSO(4)/EDTA (0.2 micromol Fe(II) per mg of protein) was used to study the effect of oxidative stress on lipid peroxidation and structural properties of endoplasmic reticulum (ER) membranes isolated from rabbit brain. Oxidative stress resulted in conjugated diene formation and a decrease of 1-anilino-8-naphthalenesulfonate (ANS) fluorescence in a time-dependent manner. In contrast, fluorescence anisotropy of 1, 6-diphenyl-1,3,5-hexatriene was increased early after the initiation of lipid peroxidation and no further increase was observed after 1, 2 and 3 h of peroxidation. FeSO(4)/EDTA treatment was accompanied by formation of conjugates of lipid peroxidation products with membrane proteins, as detected by the increase in fluorescence excitation (350-360 nm) and emission (440-450 nm) maximum. Oxidative stress also induced a marked decrease of the intrinsic fluorescence of aromatic amino acids, suggesting modification or changes in the environment of these amino acid residue(s). The lipid antioxidant, stobadine, completely prevented the changes of ANS fluorescence and production of peroxidized lipid-protein conjugates whereas tryptophan fluorescence was only partially protected. These results suggest that Fe(II) induces both lipid-mediated- and lipid peroxidation independent-modification of ER membrane proteins. The study also demonstrates that stobadine is a potent inhibitor of Fe(II)-induced protein modification.
Asunto(s)
Antioxidantes/farmacología , Carbolinas/farmacología , Retículo Endoplásmico/metabolismo , Hierro/farmacología , Peroxidación de Lípido/efectos de los fármacos , Proteínas de la Membrana/metabolismo , Aminoácidos/química , Naftalenosulfonatos de Anilina/química , Animales , Encéfalo/citología , Encéfalo/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Fluorescencia , Colorantes Fluorescentes/química , Radicales Libres/metabolismo , Membranas Intracelulares/efectos de los fármacos , Membranas Intracelulares/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/efectos de los fármacos , Oxidación-Reducción , Estrés Oxidativo , ConejosRESUMEN
Non-species isoform-specific antibodies against three isoforms of the plasma membrane Ca2+ pump (PMCA) were used for immuno-localization of PMCA by Western blot analysis in membrane preparations isolated from different regions of gerbil brain. All three gene products were detected in the membranes from hippocampus, cerebral cortex and cerebellum. However, they showed a distinct distribution pattern. Two proteins were revealed in the case of PMCA1 with molecular masses 129 and 135 kDa. The antibody against PMCA2 recognized three proteins of about 130-137 kDa. Only one protein was detected with the anti-PMCA3 antibody. Levels of immuno-signal for the PMCA isoforms varied significantly among the different brain regions. The PMCA1 is the most abundant in the cerebro-cortical and hippocampal membrane preparations. The PMCA2 was detected in a lesser amount comparing to PMCA1 and was highest in the membrane preparations from cerebellum and in a slightly lesser amount from cerebral cortex. Anti-PMCA3 antibody stained weakly and was localized in the cerebellar and hippocampal membrane preparations. Transient forebrain ischemia (10 min) and reperfusion (for a prolonged period up to 10 d) leads to a significant decrease of PMCA immuno-signal. This decrease could be ascribed to the loss of PMCA1 signal, especially in hippocampal membrane preparations.
Asunto(s)
Encéfalo/metabolismo , ATPasas Transportadoras de Calcio/metabolismo , Proteínas de la Membrana/metabolismo , Isoformas de Proteínas/metabolismo , Daño por Reperfusión/metabolismo , Animales , Gerbillinae , MasculinoRESUMEN
The effect of oxidative stress in vitro induced by radical generating systems (RGS) (Fe2+-EDTA and Fe2+-EDTA plus H2O2) on synaptosomal and microsomal ion transport systems as well as on the membrane fluidity was investigated. Oxidative insult reduced Na+, K+-ATPase activity by 50.7% and Na+-dependent Ca2+ uptake measured in choline media by 46.7%. Membrane fluidity was also significantly reduced as observed with the fluorescent probe. Stobadine (ST) prevented the decrease in membrane fluidity and Na+-dependent Ca2+ uptake, however Na+, K+-ATPase activity was only partially protected, indicating a more complex mechanism of inhibition. Incubation of microsomes with RGS led to the loss of ability of membranes to sequester Ca2+, as well as to the decrease of Ca2+-ATPase activity and to the increase of Ca2+ permeability to 125.1%. The relative potency of the two RGS to decrease membrane fluidity correlated well with the system's potencies to induce lipid peroxidation. The extent of protection against depression of Ca2+ uptake values and Ca2+-ATPase activity by membrane soluble antioxidants (U-74500A, U-83836E, t-butylated hydroxytoluene-BHT and ST) was dependent on the experimental conditions and on the dose and nature of antioxidant used. ST seems to be at least as affective as BHT and 21-aminosteroids, and more potent than tocopherol acetate. Water soluble glutathione had no significant effect on the RGS induced inhibition of Ca2+-ATPase activity. Combination of ST with glutathione enhanced ST antioxidant efficacy, so drug combination might be beneficial therapeutically.
Asunto(s)
Antioxidantes/farmacología , Encéfalo/efectos de los fármacos , Carbolinas/farmacología , Fármacos Neuroprotectores/farmacología , Estrés Oxidativo/fisiología , Animales , Transporte Biológico Activo/efectos de los fármacos , Encéfalo/metabolismo , Calcio/metabolismo , Retículo Endoplásmico/efectos de los fármacos , Retículo Endoplásmico/enzimología , Retículo Endoplásmico/metabolismo , Gerbillinae , Técnicas In Vitro , Peroxidación de Lípido/efectos de los fármacos , Fluidez de la Membrana/efectos de los fármacos , Microsomas/efectos de los fármacos , Microsomas/metabolismo , Estrés Oxidativo/efectos de los fármacos , Prosencéfalo/metabolismo , Prosencéfalo/ultraestructura , Conejos , Especies Reactivas de Oxígeno/metabolismo , Formación Reticular/efectos de los fármacos , Formación Reticular/enzimología , Formación Reticular/metabolismo , Intercambiador de Sodio-Calcio/antagonistas & inhibidores , Intercambiador de Sodio-Calcio/metabolismo , ATPasa Intercambiadora de Sodio-Potasio/antagonistas & inhibidores , ATPasa Intercambiadora de Sodio-Potasio/metabolismo , Sinaptosomas/efectos de los fármacos , Sinaptosomas/enzimología , Sinaptosomas/metabolismoRESUMEN
In the myocardium, the sarcoplasmic reticulum (SR) plays an essential role in the regulation of cytosolic free Ca2+ ion concentration and, hence, in the contraction-relaxation cycle. The aim of this review is to summarize the role of the SR, particularly the main SR Ca2+ transport proteins, Ca2+-ATPase pump and Ca2+ release channel (ryanodine receptor), in contractile impairment during ischaemia and reperfusion. As suggested by most studies, SR dysfunction may contribute to contractile failure during ischaemia. However, SR function is largely restored during reperfusion and minor changes are unlikely to explain the severe postischaemic contractile dysfunction.
Asunto(s)
Contracción Miocárdica , Daño por Reperfusión Miocárdica/fisiopatología , Retículo Sarcoplasmático/fisiología , Animales , Calcio/metabolismo , Humanos , Aturdimiento MiocárdicoRESUMEN
Postsynaptic potential is only one aspect of extensive communication between neurons and their synapses. Besides generating of potential changes by activation of ionic channels, neurotransmitters may activate receptors linked with the transient concentration changes of one or several intracellular second messengers, including calcium ions (Ca2+). In the neuronal cells calcium triggers and controls specific processes. Transient changes of Ca2+ concentration within the cell play an important signal role by coupling electrical and chemical impulses generated on the plasma membrane with the intracellular systems of responses. Several proteins and/or protein complexes, whose functions are directly controlled by calcium, have been identified in the neuronal cells. Their biochemical properties and physiological importance as well as cellular localization are discussed in this paper.
Asunto(s)
Calcio/metabolismo , Neuronas/fisiología , Transducción de Señal , Sinapsis/fisiología , Animales , Encéfalo/fisiología , Proteínas de Unión al Calcio/metabolismo , Comunicación Celular , Membrana Celular/fisiología , Homeostasis , Mitocondrias/metabolismo , Modelos Neurológicos , Proteínas del Tejido Nervioso/metabolismo , Transmisión SinápticaRESUMEN
The intracellular free Ca2+ concentrations show complex fluctuations in time and space in response to a variety of stimuli, and act as a pluripotent signal for many neuronal functions. Activation of cells is associated with Ca2+ influx from the extracellular space through voltage-dependent and/or receptor-operated Ca2+ channels localized on the plasma membrane, and/or by release of Ca2+ from intracellular stores to reach Ca2+ concentrations of up to micromolar levels. During cell relaxation, calcium concentration decreases to resting levels via ATP-driven Ca2+ transport both to the extracellular space and into the intracellular stores. Thus, Ca2+ homeostasis in neuronal cells is maintained by several systems differing by their mechanisms, biochemical characteristics and intracellular localization. Their biochemical properties and physiological importance as well as cellular localization are discussed in this short review.