RESUMO
PURPOSE: The present study aimed at evaluating possible synergistic effects between two risk factors for cognitive decline and neurodegenerative disorders, i.e. iron overload and exposure to a hypercaloric/hyperlipidic diet, on cognition, insulin resistance, and hippocampal GLUT1, GLUT3, Insr mRNA expression, and AKT phosporylation. METHODS: Male Wistar rats were treated with iron (30 mg/kg carbonyl iron) or vehicle (5% sorbitol in water) from 12 to 14th post-natal days. Iron-treated rats received a standard laboratory diet or a high fat diet from weaning to adulthood (9 months of age). Recognition and emotional memory, peripheral blood glucose and insulin levels were evaluated. Glucose transporters (GLUT 1 and GLUT3) and insulin signaling were analyzed in the hippocampus of rats. RESULTS: Both iron overload and exposure to a high fat diet induced memory deficits. Remarkably, the association of iron with the high fat diet induced more severe cognitive deficits. Iron overload in the neonatal period induced higher insulin levels associated with significantly higher HOMA-IR, an index of insulin resistance. Long-term exposure to a high fat diet resulted in higher fasting glucose levels. Iron treatment induced changes in Insr and GLUT1 expression in the hippocampus. At the level of intracellular signaling, both iron treatment and the high fat diet decreased AKT phosphorylation. CONCLUSION: The combination of iron overload with exposure to a high fat diet only led to synergistic deleterious effect on emotional memory, while the effects induced by iron and by the high fat diet on AKT phosphorylation were comparable. These findings indicate that there is, at least to some extent, an additive effect of iron combined with the diet. Further studies investigating the mechanisms associated to deleterious effects on cognition and susceptibility for the development of age-associated neurodegenerative disorders are warranted.
Assuntos
Animais Recém-Nascidos , Dieta Hiperlipídica , Transportador de Glucose Tipo 1 , Hipocampo , Resistência à Insulina , Sobrecarga de Ferro , Transtornos da Memória , Ratos Wistar , Animais , Masculino , Dieta Hiperlipídica/efeitos adversos , Sobrecarga de Ferro/complicações , Sobrecarga de Ferro/metabolismo , Transtornos da Memória/etiologia , Hipocampo/metabolismo , Hipocampo/efeitos dos fármacos , Ratos , Transportador de Glucose Tipo 1/metabolismo , Transportador de Glucose Tipo 1/genética , Transportador de Glucose Tipo 3/metabolismo , Transportador de Glucose Tipo 3/genética , Receptor de Insulina/metabolismo , Receptor de Insulina/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Glicemia/metabolismo , Insulina/sangue , Transdução de SinaisRESUMO
Glioblastoma (GBM) is the most lethal among malignant gliomas. The tumor invasiveness and therapy-resistance are important clinical hallmarks. Growing evidence emphasizes the purinergic signaling contributing to tumor growth. Here we exposed a potential role of extracellular ATPase activity as a key regulator of temozolomide cytotoxicity and the migration process in GBM cells. The inhibition of ATP hydrolysis was able to improve the impact of temozolomide, causing arrest mainly in S and G2 phases of the cell cycle, leading M059J and U251 cells to apoptosis. In addition to eradicating GBM cells, ATP hydrolysis exhibited a potential to modulate the invasive phenotype and the expression of proteins involved in cell migration and epithelial-to-mesenchymal-like transition in a 3D culture model. Finally, we suggest the ATPase activity as a key target to decline temozolomide resistance and the migratory phenotype in GBM cells.
Assuntos
Neoplasias Encefálicas , Glioblastoma , Adenosina Trifosfatases/genética , Trifosfato de Adenosina/farmacologia , Antineoplásicos Alquilantes/farmacologia , Antineoplásicos Alquilantes/uso terapêutico , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Proliferação de Células , Resistencia a Medicamentos Antineoplásicos , Glioblastoma/patologia , Humanos , Hidrólise , Fenótipo , Temozolomida/farmacologia , Temozolomida/uso terapêuticoRESUMO
Glioblastoma (GBM) is the most aggressive and lethal among the primary brain tumors, with a low survival rate and resistance to radio and chemotherapy. The P2Y12 is an adenosine diphosphate (ADP) purinergic chemoreceptor, found mainly in platelets. In cancer cells, its activation has been described to induce proliferation and metastasis. Bearing in mind the need to find new treatments for GBM, this study aimed to investigate the role of the P2Y12R in the proliferation and migration of GBM cells, as well as to evaluate the expression of this receptor in patients' data obtained from the TCGA data bank. Here, we used the P2Y12R antagonist, ticagrelor, which belongs to the antiplatelet agent's class. The different GBM cells (cell line and patient-derived cells) were treated with ticagrelor, with the agonist, ADP, or both, and the effects on cell proliferation, colony formation, ADP hydrolysis, cell cycle and death, migration, and cell adhesion were analyzed. The results showed that ticagrelor decreased the viability and the proliferation of GBM cells. P2Y12R antagonism also reduced colony formation and migration potentials, with alterations on the expression of metalloproteinases, and induced autophagy in GBM cells. Changes were observed at the cell cycle level, and only the U251 cell line showed a significant reduction in the ADP hydrolysis profile. TCGA data analysis showed a higher expression of P2Y12R in gliomas samples when compared to the other tumors. These data demonstrate the importance of the P2Y12 receptor in gliomas development and reinforce its potential as a pharmacological target for glioma treatment.
Assuntos
Glioblastoma , Humanos , Ticagrelor/metabolismo , Ticagrelor/farmacologia , Difosfato de Adenosina/metabolismo , Glioblastoma/tratamento farmacológico , Plaquetas , Autofagia , Proliferação de Células , Receptores Purinérgicos P2Y12/metabolismo , Antagonistas do Receptor Purinérgico P2Y/metabolismoRESUMO
PURPOSE: To investigate the effects of lipoic acid (LA) supplementation during adulthood combined with supplementation later in life or LA administration only at old age on age-induced cognitive dysfunction, mitochondrial DNA deletions, caspase 3 and antioxidant response enzymes expression in iron-treated rats. METHODS: Male rats were submitted to iron treatment (30 mg/kg body wt of Carbonyl iron) from 12 to 14th post-natal days. Iron-treated rats received LA supplementation (50 mg/kg, daily) in adulthood and old age or at old age only for 21 days. Memory, mitochondrial DNA (mtDNA) complex I deletions, caspase 3 mRNA expression and antioxidant response enzymes mRNA expression were analyzed in the hippocampus. RESULTS: LA administration in adulthood combined with treatment later in life was able to reverse age-induced effects on object recognition and inhibitory avoidance memory, as well as on mtDNA deletions, nuclear factor (erythroid-derived 2)-like 2 (Nrf2) expression, and antioxidant enzymes disruption induced by iron in aged rats. LA treatment only at old age reversed iron-induced effects to a lesser extent when compared to the combined treatment. CONCLUSION: The present findings support the view that LA supplementation may be considered as an adjuvant against mitochondrial damage and cognitive decline related to aging and neurodegenerative disorders.
Assuntos
Ácido Tióctico , Animais , Antioxidantes , DNA Mitocondrial , Suplementos Nutricionais , Ferro , Masculino , RatosRESUMO
Aquatic animals are vulnerable to arsenic (As) toxicity. However, rarely does a contaminant occur alone in the aquatic environment. For this reason, this study was conducted to evaluate whether titanium dioxide nanoparticles (nTiO2) can interfere with the effects induced by As in Litopenaeus vannamei. Arsenic accumulation and metabolic capacity; expression and enzymatic activity of GSTΩ (glutathione-S-transferase omega isoform); antioxidant responses such as GSH, GR, and GST (reduced glutathione levels, glutathione reductase, and glutathione-S-transferase activity, respectively); and lipid peroxidation in the gills and hepatopancreas of shrimp were evaluated. The results are summarized as follows: (1) higher accumulation of As occurred in both tissues after exposure to As alone; (2) co-exposure to nTiO2 affected the capacity to metabolize As; (3) GSTΩ gene expression was not modified, but its activity was decreased by co-exposure to both contaminants; (4) As alone increased the GSH levels in the hepatopancreas, and co-exposure to nTiO2 reduced these levels in both tissues; (5) a decrease in the GST activity in the gills occurred with all treatments; (6) in the gills, GR activity was increased by As, and nTiO2 reversed this increase, whereas in the hepatopancreas co-exposure inhibited enzyme activity; (7) only in the hepatopancreas lipid damage was observed when animals were exposed to As or nTiO2 but not in co-exposure. The results showed that the As induces toxic effects in both tissues of shrimp and that co-exposure to nTiO2 can potentiate these effects and decrease the capacity to metabolize As, favoring the accumulation of more toxic compounds.
Assuntos
Antioxidantes/metabolismo , Arsenitos/toxicidade , Nanopartículas Metálicas/toxicidade , Estresse Oxidativo/efeitos dos fármacos , Penaeidae/efeitos dos fármacos , Compostos de Sódio/toxicidade , Titânio/toxicidade , Poluentes Químicos da Água/toxicidade , Animais , Arsenitos/metabolismo , Brânquias/efeitos dos fármacos , Brânquias/metabolismo , Hepatopâncreas/efeitos dos fármacos , Hepatopâncreas/metabolismo , Peroxidação de Lipídeos/efeitos dos fármacos , Penaeidae/metabolismo , Compostos de Sódio/metabolismo , Distribuição Tecidual , Poluentes Químicos da Água/metabolismoRESUMO
Background: Several different mechanisms have been proposed to explain long-term cognitive impairment in sepsis survivors. The role of persisting mitochondrial dysfunction is not known. We thus sought to determine whether stimulation of mitochondrial dynamics improves mitochondrial function and long-term cognitive impairment in an experimental model of sepsis.Methods: Sepsis was induced in adult Wistar rats by cecal ligation and perforation (CLP). Animals received intracerebroventricular injections of either rosiglitazone (biogenesis activator), rilmenidine, rapamycin (autophagy activators), or n-saline (sham control) once a day on days 7-9 after the septic insult. Cognitive impairment was assessed by inhibitory avoidance and object recognition tests. Animals were killed 24 h, 3 and 10 days after sepsis with the hippocampus and prefrontal cortex removed to determine mitochondrial function.Results: Sepsis was associated with both acute (24 h) and late (10 days) brain mitochondrial dysfunction. Markers of mitochondrial biogenesis, autophagy and mitophagy were not up-regulated during these time points. Activation of biogenesis (rosiglitazone) or autophagy (rapamycin and rilmenidine) improved brain ATP levels and ex vivo oxygen consumption and the long-term cognitive impairment observed in sepsis survivors.Conclusion: Long-term impairment of brain function is temporally related to mitochondrial dysfunction. Activators of autophagy and mitochondrial biogenesis could rescue animals from cognitive impairment.
Assuntos
Disfunção Cognitiva/complicações , Disfunção Cognitiva/patologia , Mitocôndrias/patologia , Sepse/complicações , Sepse/patologia , Animais , Autofagia/efeitos dos fármacos , Encéfalo/efeitos dos fármacos , Encéfalo/patologia , Modelos Animais de Doenças , Masculino , Mitocôndrias/efeitos dos fármacos , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/efeitos dos fármacos , Ratos Wistar , Rilmenidina/farmacologia , Rosiglitazona/farmacologia , Sirolimo/farmacologia , Análise de Sobrevida , Regulação para Cima/efeitos dos fármacos , Regulação para Cima/genéticaRESUMO
Brain-derived neurotrophic factor (BDNF) plays a key role in neural development and physiology, as well as in pathological states. Post-mortem studies demonstrate that BDNF is reduced in the brains of patients affected by neurodegenerative diseases. Iron accumulation has also been associated to the pathogenesis of neurodegenerative diseases. In rats, iron overload induces persistent memory deficits, increases oxidative stress and apoptotic markers, and decreases the expression of the synaptic marker, synaptophysin. Deferiprone (DFP) is an oral iron chelator used for the treatment of systemic iron overload disorders, and has recently been tested for Parkinson's disease. Here, we investigated the effects of iron overload on BDNF levels and on mRNA expression of genes encoding TrkB, p75NTR, catalase (CAT) and NQO1. We also aimed at investigating the effects of DFP on iron-induced impairments. Rats received iron or vehicle at postnatal days 12-14 and when adults, received chronic DFP or water (vehicle). Recognition memory was tested 19 days after the beginning of chelation therapy. BDNF measurements and expression analyses in the hippocampus were performed 24 h after the last day of DFP treatment. DFP restored memory and increased hippocampal BDNF levels, ameliorating iron-induced effects. Iron overload in the neonatal period reduced, while treatment with DFP was able to rescue, the expression of antioxidant enzymes CAT and NQO1.
Assuntos
Antioxidantes/farmacologia , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Deferiprona/farmacologia , Modelos Animais de Doenças , Quelantes de Ferro/farmacologia , Transtornos da Memória/tratamento farmacológico , Estresse Oxidativo/efeitos dos fármacos , Animais , Antioxidantes/química , Fator Neurotrófico Derivado do Encéfalo/análise , Deferiprona/química , Feminino , Hipocampo/efeitos dos fármacos , Quelantes de Ferro/química , Ratos , Ratos WistarRESUMO
OBJECTIVE AND DESIGN: Experimental animal models and human clinical studies support a crucial role for TLRs in infectious diseases. The aim of this study was to test the ability of MSCs, which have immunomodulatory effects, of altering the mRNA expression of toll-like receptors during a experimental model of sepsis in different tissues. MATERIALS AND METHODS: Three experimental groups (male C57BL/6 mice) were formed for the test: control group, untreated septic group and septic group treated with MSCs (1 × 106 cells/animal). Lungs, cortex, kidney, liver and colon tissue were dissected after 12 h of sepsis induction and TLR2/3/4/9 mRNA were evaluated by RT-qPCR. RESULTS: We observed a decrease of TLR2 and 9 mRNA expression in the liver of the sepsis group, while TLR3 was decreased in the lung and liver. No change was found between the sepsis group and the sepsis + MSC group. CONCLUSIONS: In this model of experimental sepsis the MSCs were unable to modify the mRNA expression of the different toll-like receptors evaluated.
Assuntos
Células-Tronco Mesenquimais , Sepse/genética , Receptores Toll-Like/genética , Animais , Células Cultivadas , Córtex Cerebral/metabolismo , Colo/metabolismo , Rim/metabolismo , Fígado/metabolismo , Pulmão/metabolismo , Masculino , Camundongos Endogâmicos C57BL , RNA Mensageiro/metabolismo , Sepse/metabolismoRESUMO
The gastrin-releasing peptide (GRP) and its receptor (GRPR) are important components of itch transmission. Upstream, but not downstream, aspects of GRPR signaling have been investigated extensively. We hypothesize that GRPR signals in part through the PI3Kγ/Akt pathway. We used pharmacological, electrophysiological, and behavioral approaches to further evaluate GRPR downstream signaling pathways. Our data show that GRP directly activates small-size capsaicin-sensitive DRG neurons, an effect that translates into transient calcium flux and membrane depolarization (⼠20 mV). GRPR activation also induces Akt phosphorylation, a proxy for PI3Kγ activity, in ex vivo naive mouse spinal cords and in GRPR transiently expressing HEK293 cells. The intrathecal injection of GRP led to intense scratching, an effect largely reduced by either GRPR antagonists or PI3Kγ inhibitor. Scratching behavior was also induced by the intrathecal injection of an Akt activator. In a dry skin model of itch, we show that GRPR blockade or PI3Kγ inhibition reversed the scratching behavior. Altogether, these findings are highly suggestive that GRPR is expressed by the central terminals of DRG nociceptive afferents, which transmit itch via the PI3Kγ/Akt pathway. SIGNIFICANCE STATEMENT: Itch is the most common symptom of the skin and is related to noncutaneous diseases. It severely impairs patients' quality of life when it becomes chronic and there is no specific or effective available therapy, mainly because itch pathophysiology is not completely elucidated. Our findings indicate that the enzyme PI3Kγ is a key central mediator of itch transmission. Therefore, we suggest PI3Kγ as an attractive target for the development of new anti-pruritic drugs. With this study, we take a step forward in our understanding of the mechanisms underlying the central transmission of itch sensation.
Assuntos
Sistema Nervoso Central/metabolismo , Peptídeo Liberador de Gastrina/metabolismo , Fosfatidilinositol 3-Quinase/metabolismo , Prurido/patologia , Receptores da Bombesina/metabolismo , Transmissão Sináptica/fisiologia , Potenciais de Ação/efeitos dos fármacos , Animais , Anticarcinógenos/uso terapêutico , Bombesina/análogos & derivados , Bombesina/uso terapêutico , Capsaicina/toxicidade , Sistema Nervoso Central/efeitos dos fármacos , Modelos Animais de Doenças , Inibidores Enzimáticos/farmacologia , Gânglios Espinais/citologia , Indóis/farmacologia , Masculino , Camundongos , Neurônios/efeitos dos fármacos , Neurônios/fisiologia , Limiar da Dor/efeitos dos fármacos , Fragmentos de Peptídeos/uso terapêutico , Prurido/induzido quimicamente , Prurido/complicações , Prurido/tratamento farmacológico , Quinoxalinas/farmacologia , Tempo de Reação/fisiologia , Transmissão Sináptica/efeitos dos fármacos , Tiazolidinedionas/farmacologia , p-Metoxi-N-metilfenetilamina/toxicidadeRESUMO
Hyperglycemia is the main feature for the diagnosis of diabetes mellitus (DM). Some studies have demonstrated the relationship between DM and dysfunction on neurotransmission systems, such as the purinergic system. In this study, we evaluated the extracellular nucleotide hydrolysis and adenosine deamination activities from encephalic membranes of hyperglycemic zebrafish. A significant decrease in ATP, ADP, and AMP hydrolyses was observed at 111-mM glucose-treated group, which returned to normal levels after 7 days of glucose withdrawal. A significant increase in ecto-adenosine deaminase activity was observed in 111-mM glucose group, which remain elevated after 7 days of glucose withdrawal. The soluble-adenosine deaminase activity was significantly increased just after 7 days of glucose withdrawal. We also evaluated the gene expressions of ecto-nucleoside triphosphate diphosphohydrolases (E-NTPDases), ecto-5'-nucleotidase, ADA, and adenosine receptors from encephala of adult zebrafish. The entpd 2a.1, entpd 2a.2, entpd 3, and entpd 8 mRNA levels from encephala of adult zebrafish were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expressions of adenosine receptors (adora 1 , adora 2aa , adora 2ab , and adora 2b ) were decreased in 111-mM glucose-treated and glucose withdrawal groups. The gene expression of ADA (ada 2a.1) was decreased in glucose withdrawal group. Maltodextrin, used as a control, did not affect the expression of adenosine receptors, ADA and E-NTPDases 2, 3, and 8, while the expression of ecto-5'-nucleotidase was slightly increased and the E-NTPDases 1 decreased. These findings demonstrated that hyperglycemia might affect the ecto-nucleotidase and adenosine deaminase activities and gene expression in zebrafish, probably through a mechanism involving the osmotic effect, suggesting that the modifications caused on purinergic system may also contribute to the diabetes-induced progressive cognitive impairment.
Assuntos
5'-Nucleotidase/metabolismo , Adenosina Desaminase/metabolismo , Adenosina Trifosfatases/metabolismo , Encéfalo/enzimologia , Hiperglicemia/enzimologia , Receptores Purinérgicos P1/metabolismo , Animais , Modelos Animais de Doenças , Feminino , Perfilação da Expressão Gênica , Masculino , Reação em Cadeia da Polimerase , Transcriptoma , Peixe-ZebraRESUMO
Fish production ponds and natural water body areas located in close proximity to agricultural fields receive water with variable amounts of agrochemicals, and consequently, compounds that produce adverse effects may reach nontarget organisms. The aim of this study was to investigate whether waterborne methyl-parathion-based insecticide (MPBI) affected gene expression patterns of brain glucocorticoid receptor (GR), steroidogenic acute regulatory protein (StAR), and heat shock protein 70 (hsp70) in adult zebrafish (Danio rerio) exposed to this chemical for 96 h. Treated fish exposed to MPBI-contaminated water showed an inhibition of brain StAR and hsp70 gene expression. Data demonstrated that MPBI produced a decrease brain StAR and hsp70 gene expression.
Assuntos
Química Encefálica/efeitos dos fármacos , Proteínas de Choque Térmico HSP70/biossíntese , Inseticidas/toxicidade , Metil Paration/toxicidade , Fosfoproteínas/biossíntese , Agroquímicos/toxicidade , Animais , Expressão Gênica/efeitos dos fármacos , Masculino , Receptores de Glucocorticoides/biossíntese , Receptores de Glucocorticoides/efeitos dos fármacos , Poluentes Químicos da Água/toxicidade , Peixe-ZebraRESUMO
Copper is an essential micronutrient and a key catalytic cofactor in a wide range of enzymes. As a trace element, copper levels are tightly regulated and both its deficit and excess are deleterious to the organism. Under inflammatory conditions, serum copper levels are increased and trigger oxidative stress responses that activate inflammatory responses. Interestingly, copper dyshomeostasis, oxidative stress and inflammation are commonly present in several chronic diseases. Copper exposure can be easily modeled in zebrafish; a consolidated model in toxicology with increasing interest in immunity-related research. As a result of developmental, economical and genetic advantages, this freshwater teleost is uniquely suitable for chemical and genetic large-scale screenings, representing a powerful experimental tool for a whole-organism approach, mechanistic studies, disease modeling and beyond. Copper toxicological and more recently pro-inflammatory effects have been investigated in both larval and adult zebrafish with breakthrough findings. Here, we provide an overview of copper metabolism in health and disease and its effects on oxidative stress and inflammation responses in zebrafish models. Copper-induced inflammation is highlighted owing to its potential to easily mimic pro-oxidative and pro-inflammatory features that combined with zebrafish genetic tractability could help further in the understanding of copper metabolism, inflammatory responses and related diseases. Copyright © 2016 John Wiley & Sons, Ltd.
Assuntos
Cobre/toxicidade , Inflamação/fisiopatologia , Estresse Oxidativo/efeitos dos fármacos , Animais , Cobre/sangue , Modelos Animais de Doenças , Inflamação/induzido quimicamente , Larva/efeitos dos fármacos , Peixe-Zebra/genéticaRESUMO
Maple syrup urine disease (MSUD) is caused by an inborn error in metabolism resulting from a deficiency in the branched-chain α-keto acid dehydrogenase complex activity. This blockage leads to accumulation of the branched-chain amino acids (BCAA) leucine, isoleucine and valine, as well as their corresponding α-keto acids and α-hydroxy acids. High levels of BCAAs are associated with neurological dysfunction and the role of pro- and mature brain-derived neurotrophic factor (BDNF) in the neurological dysfunction of MSUD is still unclear. Thus, in the present study we investigated the effect of an acute BCAA pool administration on BDNF levels and on the pro-BDNF cleavage-related proteins S100A10 and tissue plasminogen activator (tPA) in rat brains. Our results demonstrated that acute Hyper-BCAA (H-BCAA) exposure during the early postnatal period increases pro-BDNF and total-BDNF levels in the hippocampus and striatum. Moreover, tPA levels were significantly decreased, without modifications in the tPA transcript levels in the hippocampus and striatum. On the other hand, the S100A10 mRNA and S100A10 protein levels were not changed in the hippocampus and striatum. In the 30-day-old rats, we observed increased pro-BDNF, total-BDNF and tPA levels only in the striatum, whereas the tPA and S100A10 mRNA expression and the immunocontent of S100A10 were not altered. In conclusion, we demonstrated that acute H-BCAA administration increases the pro-BDNF/total-BDNF ratio and decreases the tPA levels in animals, suggesting that the BCAA effect may depend, at least in part, on changes in BDNF post-translational processing.
Assuntos
Aminoácidos de Cadeia Ramificada/administração & dosagem , Fator Neurotrófico Derivado do Encéfalo/biossíntese , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Neostriado/efeitos dos fármacos , Neostriado/metabolismo , Precursores de Proteínas/biossíntese , Animais , Injeções Subcutâneas , Masculino , Ratos , Ratos WistarRESUMO
Anxiety is characterized by unpleasant bodily sensations, such as pounding heart and intense fear. The therapy involves the administration of benzodiazepine drugs. Purinergic signaling participates in the induction of several behavioral patterns and their actions are inactivated by ectonucleotidases and adenosine deaminase (ADA). Since there is evidence about the involvement of purinergic system in the actions mediated by benzodiazepines, we evaluated the effects in vitro and in vivo of administration of diazepam and midazolam on nucleoside triphosphate diphosphohydrolases, ecto-5'-nucleotidase, and ADA activities in zebrafish brain, followed by the analysis of gene expression pattern of these enzymes and adenosine receptors (A1, A2a1, A2a2, A2b). The in vitro studies demonstrated that diazepam decreased ATP (66 % for 500 µM) and ADP hydrolysis (40-54 % for 10-500 µM, respectively). Midazolam decreased ATP (16-71 % for 10-500 µM, respectively) and ADP (48-73.5 % for 250-500 µM, respectively) hydrolysis as well as the ecto-ADA activity (26-27.5 % for 10-500 µM, respectively). AMP hydrolysis was decreased in animals treated with of 0.5 and 1 mg/L midazolam (32 and 36 %, respectively). Diazepam and midazolam decreased the ecto-ADA activity at 1.25 mg/L and 1 mg/L (31 and 33 %, respectively), but only 0.1 mg/L midazolam induced an increase (40 %) in cytosolic ADA. The gene expression analysis demonstrated changes on ecto-5'-nucleotidase, A1, A2a1, A2a2, and A2b mRNA transcript levels after acute treatment with benzodiazepines. These findings demonstrated that benzodiazepine exposure induces a modulation of extracellular nucleotide and nucleoside metabolism, suggesting the purinergic signaling may be, at least in part, related to benzodiazepine effects.
Assuntos
Ansiolíticos/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/metabolismo , Diazepam/farmacologia , Midazolam/farmacologia , 5'-Nucleotidase/metabolismo , Adenosina Desaminase/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Trifosfato de Adenosina/metabolismo , Animais , Relação Dose-Resposta a Droga , Feminino , Expressão Gênica/efeitos dos fármacos , Masculino , Modelos Animais , RNA Mensageiro/metabolismo , Receptores Purinérgicos P1/metabolismo , Peixe-ZebraRESUMO
Extracellular ATP may act as a danger signalling molecule, inducing inflammation and immune responses in infection sites. The ectonucleotidases NTPDase and ecto-5'-nucleotidase are enzymes that modulate extracellular nucleotide levels; these enzymes have been previously characterised in Trichomonas vaginalis. Iron plays an important role in the complex trichomonal pathogenesis. Herein, the effects of iron on growth, nucleotide hydrolysis and NTPDase gene expression in T. vaginalis isolates from female and male patients were evaluated. Iron from different sources sustained T. vaginalis growth. Importantly, iron from haemoglobin (HB) and haemin (HM) enhanced NTPDase activity in isolates from female patients and conversely reduced the enzyme activity in isolates from male patients. Iron treatments could not alter the NTPDase transcript levels in T. vaginalis. Furthermore, our results reveal a distinct ATP, ADP and AMP hydrolysis profile between isolates from female and male patients influenced by iron from HB and HM. Our data indicate the participation of NTPDase and ecto-5'-nucleotidase in the establishment of trichomonas infection through ATP degradation and adenosine production influenced by iron.
Assuntos
Hemina/química , Hemoglobinas/química , Ferro/fisiologia , Nucleotídeos/metabolismo , Trichomonas vaginalis/enzimologia , 5'-Nucleotidase/metabolismo , Difosfato de Adenosina/metabolismo , Monofosfato de Adenosina/metabolismo , Adenosina Trifosfatases/metabolismo , Trifosfato de Adenosina/metabolismo , Técnicas de Cultura de Células , Feminino , Expressão Gênica/efeitos dos fármacos , Humanos , Hidrólise , Ferro/administração & dosagem , Masculino , RNA de Protozoário , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Tricomoníase/enzimologia , Trichomonas vaginalis/efeitos dos fármacos , Trichomonas vaginalis/crescimento & desenvolvimentoRESUMO
Antipsychotic agents are used for the treatment of psychotic symptoms in patients with several brain disorders, such as schizophrenia. Atypical and typical antipsychotics differ regarding their clinical and side-effects profile. Haloperidol is a representative typical antipsychotic drug and has potent dopamine receptor antagonistic functions; however, atypical antipsychotics have been developed and characterized an important advance in the treatment of schizophrenia and other psychotic disorders. Purine nucleotides and nucleosides, such as ATP and adenosine, constitute a ubiquitous class of extracellular signaling molecules crucial for normal functioning of the nervous system. Indirect findings suggest that changes in the purinergic system, more specifically in adenosinergic activity, could be involved in the pathophysiology of schizophrenia. We investigated the effects of typical and atypical antipsychotics on ectonucleotidase and adenosine deaminase (ADA) activities, followed by an analysis of gene expression patterns in zebrafish brain. Haloperidol treatment (9 µM) was able to decrease ATP hydrolysis (35%), whereas there were no changes in hydrolysis of ADP and AMP in brain membranes after antipsychotic exposure. Adenosine deamination in membrane fractions was inhibited (38%) after haloperidol treatment when compared to the control; however, no changes were observed in ADA soluble fractions after haloperidol exposure. Sulpiride (250 µM) and olanzapine (100 µM) did not alter ectonucleotidase and ADA activities. Haloperidol also led to a decrease in entpd2_mq, entpd3 and adal mRNA transcripts. These findings demonstrate that haloperidol is an inhibitor of NTPDase and ADA activities in zebrafish brain, suggesting that purinergic signaling may also be a target of pharmacological effects promoted by this drug.
Assuntos
Adenosina Desaminase/metabolismo , Adenosina Trifosfatases/metabolismo , Antipsicóticos/farmacologia , Encéfalo/efeitos dos fármacos , Encéfalo/enzimologia , Peixe-Zebra/fisiologia , Animais , Benzodiazepinas/farmacologia , Feminino , Haloperidol/farmacologia , Hidrólise , Masculino , Olanzapina , Sulpirida/farmacologia , Proteínas de Peixe-Zebra/metabolismoRESUMO
Gliomas are the most common malignant brain tumors in adults. Bradykinin (BK) displays an important role in cancer, although the exact role of kinin receptors in the glioma biology remains unclear. This study investigated the role of kinin B1 and B2 receptors (B1R and B2R) on cell proliferation in human glioblastoma cell lineages. The mRNA expression of B1R and B2R was verified by RT-qPCR, whereas the effects of kinin agonists (des-Arg(9)-BK and BK) were analyzed by cell counting, MTT assay and annexin-V/PI determination. The PI3K/Akt and ERK1/2 signaling activation was assessed by flow cytometry. Our results demonstrated that both human glioblastoma cell lines U-138MG and U-251MG express functional B1R and B2R. The proliferative effects induced by the incubation of des-Arg(9)-BK and BK are likely related to the activation of PI3K/Akt and ERK 1/2 pathways. Moreover, the pre-incubation of the selective PI3Kγ blocker AS252424 markedly prevented kinin-induced AKT phosphorylation. Noteworthy, the selective B1R and B2R antagonists SSR240612 and HOE-140 were able to induce cell death of either lineages, with mixed apoptosis/necrosis characteristics. Taken together, the present results show that activation of B1R and B2R might contribute to glioblastoma progression in vitro. Furthermore, PI3K/Akt and ERK 1/2 signaling may be a target for adjuvant treatment of glioblastoma with a possible impact on tumor proliferation.
Assuntos
Proliferação de Células , Glioma/metabolismo , Glioma/patologia , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Fosfatidilinositol 3-Quinases/metabolismo , Proteínas Proto-Oncogênicas c-akt/metabolismo , Receptor B1 da Bradicinina/metabolismo , Receptor B2 da Bradicinina/metabolismo , Apoptose , Western Blotting , Bradicinina/análogos & derivados , Bradicinina/farmacologia , Antagonistas de Receptor B1 da Bradicinina/farmacologia , Antagonistas de Receptor B2 da Bradicinina/farmacologia , Dioxóis/farmacologia , Citometria de Fluxo , Glioma/tratamento farmacológico , Humanos , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/genética , Fosfatidilinositol 3-Quinases/genética , Fosforilação/efeitos dos fármacos , Proteínas Proto-Oncogênicas c-akt/genética , RNA Mensageiro/genética , Reação em Cadeia da Polimerase em Tempo Real , Receptor B1 da Bradicinina/química , Receptor B1 da Bradicinina/genética , Receptor B2 da Bradicinina/química , Receptor B2 da Bradicinina/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Sulfonamidas/farmacologia , Células Tumorais CultivadasRESUMO
Tritrichomonas foetus is a protist that causes bovine trichomoniasis and presents a well-developed Golgi. There are very few studies concerning the Golgi in trichomonads. In this work, monoclonal antibodies were raised against Golgi of T. foetus and used as a tool on morphologic and biochemical studies of this organelle. Among the antibodies produced, one was named mAb anti-Golgi 20.3, which recognized specifically the Golgi complex by fluorescence and electron microscopy. By immunoblotting this antibody recognized two proteins with 60 and 66 kDa that were identified as putative beta-tubulin and adenosine triphosphatase, respectively. The mAb 20.3 also recognized the Golgi complex of the Trichomonas vaginalis, a human parasite. In addition, the nucleotide coding sequences of these proteins were identified and included in the T. foetus database, and the 3D structure of the proteins was predicted. In conclusion, this study indicated: (1) adenosine triphosphatase is present in the Golgi, (2) ATPase is conserved between T. foetus and T. vaginalis, (3) there is new information concerning the nucleic acid sequences and protein structures of adenosine triphosphatase and beta-tubulin from T. foetus and (4) the mAb anti-Golgi 20.3 is a good Golgi marker and can be used in future studies.
Assuntos
Adenosina Trifosfatases/metabolismo , Anticorpos Antiprotozoários/imunologia , Antígenos de Protozoários/imunologia , Complexo de Golgi/ultraestrutura , Infecções Protozoárias em Animais/parasitologia , Tritrichomonas foetus/ultraestrutura , Adenosina Trifosfatases/química , Adenosina Trifosfatases/imunologia , Sequência de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sequência de Bases , Bovinos , Feminino , Complexo de Golgi/química , Complexo de Golgi/metabolismo , Camundongos , Camundongos Endogâmicos BALB C , Microscopia Eletrônica de Transmissão/veterinária , Microscopia de Fluorescência/veterinária , Modelos Moleculares , Proteínas de Protozoários/química , Proteínas de Protozoários/imunologia , Proteínas de Protozoários/metabolismo , Alinhamento de Sequência/veterinária , Análise de Sequência de DNA/veterinária , Trichomonas vaginalis/enzimologia , Trichomonas vaginalis/imunologia , Tritrichomonas foetus/enzimologia , Tritrichomonas foetus/genética , Tritrichomonas foetus/imunologiaRESUMO
The danger of ionizing radiation exposure to human health is a concern. Since its wide use in medicine and industry, the development of radioprotectors has been very significant. Adenosine exerts anti-inflammatory actions and promotes tissue protection and repair, by activating the P1 receptors (A1, A2A, A2B, and A3). Zebrafish (Danio rerio) is an appropriate tool in the fields of toxicology and pharmacology, including the evaluation of radiobiological outcomes and in the search for radioprotector agents. This study aims to evaluate the effect of adenosine in the toxicity induced by radiation in zebrafish. Embryos were treated with 1, 10, or 100 µM adenosine, 30 min before the exposure to 15 Gy of gamma radiation. Adenosine potentiated the effects of radiation in heart rate, body length, and pericardial edema. We evaluated oxidative stress, tissue remodeling and inflammatory. It was seen that 100 µM adenosine reversed the inflammation induced by radiation, and that A2A2 and A2B receptors are involved in these anti-inflammatory effects. Our results indicate that P1R activation could be a promising pharmacological strategy for radioprotection.
Assuntos
Adenosina , Peixe-Zebra , Humanos , Animais , Adenosina/farmacologia , Raios gama/efeitos adversos , Frequência Cardíaca , Anti-InflamatóriosRESUMO
Huntington's disease (HD) is a progressive neurodegenerative disease characterized by neuropsychiatric disturbance, cognitive impairment, and locomotor dysfunction. In the early stage (chorea) of HD, expression of dopamine D2 receptors (D2R) is reduced, whereas dopamine (DA) levels are increased. Contrary, in the late stage (bradykinesia), DA levels and the expression of D2R and dopamine D1 receptors (D1R) are reduced. 3-Nitropropionic acid (3-NPA) is a toxin that may replicate HD behavioral phenotypes and biochemical aspects. This study assessed the neurotransmitter levels, dopamine receptor gene expression, and the effect of acute exposure to quinpirole (D2R agonist) and eticlopride (D2R antagonist) in an HD model induced by 3-NPA in adult zebrafish. Quinpirole and eticlopride were acutely applied by i.p. injection in adult zebrafish after chronic treatment of 3-NPA (60 mg/kg). 3-NPA treatment caused a reduction in DA, glutamate, and serotonin levels. Quinpirole reversed the bradykinesia and memory loss induced by 3-NPA. Together, these data showed that 3-NPA acts on the dopaminergic system and causes biochemical alterations similar to late-stage HD. These data reinforce the hypothesis that DA levels are linked with locomotor and memory deficits. Thus, these findings may suggest that the use of DA agonists could be a pharmacological strategy to improve the bradykinesia and memory deficits in the late-stage HD.