RESUMO
BACKGROUND: Alzheimer's disease is the most frequent age-related dementia, and is currently without treatment. To identify possible targets for early therapeutic intervention we focused on glutamate excitotoxicity, a major early pathogenic factor, and the effects of candesartan, an angiotensin receptor blocker of neuroprotective efficacy in cell cultures and rodent models of Alzheimer's disease. The overall goal of the study was to determine whether gene analysis of drug effects in a primary neuronal culture correlate with alterations in gene expression in Alzheimer's disease, thus providing further preclinical evidence of beneficial therapeutic effects. METHODS: Primary neuronal cultures were treated with candesartan at neuroprotective concentrations followed by excitotoxic glutamate amounts. We performed genome-wide expression profile analysis and data evaluation by ingenuity pathway analysis and gene set enrichment analysis, compared with alterations in gene expression from two independent published datasets identified by microarray analysis of postmortem hippocampus from Alzheimer's disease patients. Preferential expression in cerebrovascular endothelial cells or neurons was analyzed by comparison to published gene expression in these cells isolated from human cortex by laser capture microdissection. RESULTS: Candesartan prevented glutamate upregulation or downregulation of several hundred genes in our cultures. Ingenuity pathway analysis and gene set enrichment analysis revealed that inflammation, cardiovascular disease and diabetes signal transduction pathways and amyloid ß metabolism were major components of the neuronal response to glutamate excitotoxicity. Further analysis showed associations of glutamate-induced changes in the expression of several hundred genes, normalized by candesartan, with similar alterations observed in hippocampus from Alzheimer's disease patients. Gene analysis of neurons and cerebrovascular endothelial cells obtained by laser capture microdissection revealed that genes up- and downregulated by glutamate were preferentially expressed in endothelial cells and neurons, respectively. CONCLUSIONS: Our data may be interpreted as evidence of direct candesartan neuroprotection beyond its effects on blood pressure, revealing common and novel disease mechanisms that may underlie the in vitro gene alterations reported here and glutamate-induced cell injury in Alzheimer's disease. Our observations provide novel evidence for candesartan neuroprotection through early molecular mechanisms of injury in Alzheimer's disease, supporting testing this compound in controlled clinical studies in the early stages of the illness.
Assuntos
Doença de Alzheimer/genética , Benzimidazóis/farmacologia , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Fármacos Neuroprotetores/farmacologia , Tetrazóis/farmacologia , Transcriptoma/efeitos dos fármacos , Idoso , Idoso de 80 Anos ou mais , Doença de Alzheimer/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Apoptose/efeitos dos fármacos , Compostos de Bifenilo , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Bases de Dados Factuais , Modelos Animais de Doenças , Perfilação da Expressão Gênica , Ácido Glutâmico/toxicidade , Hipocampo/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Ratos Sprague-Dawley , Transdução de Sinais/efeitos dos fármacosRESUMO
Sartans (Angiotensin II AT(1) Receptor Blockers, ARBs) are powerful neuroprotective agents in vivo and protect against IL-1ß neurotoxicity in vitro. The purpose of this research was to determine the extent of sartan neuroprotection against glutamate excitotoxicity, a common cause of neuronal injury and apoptosis. The results show that sartans are neuroprotective, significantly reducing glutamate-induced neuronal injury and apoptosis in cultured rat primary cerebellar granule cells (CGCs). Telmisartan was the most potent sartan studied, with an order of potency telmisartan > candesartan > losartan > valsartan. Mechanisms involved reduction of pro-apoptotic caspase-3 activation, protection of the survival PI3K/Akt/GSK-3ß pathway and prevention of glutamate-induced ERK1/2 activation. NMDA receptor stimulation was essential for glutamate-induced cell injury and apoptosis. Participation of AT(1A) receptor was supported by glutamate-induced upregulation of AT(1A) gene expression and AT(1) receptor binding. Conversely, AT(1B) or AT(2) receptors played no role. Glutamate-induced neuronal injury and the neuroprotective effect of telmisartan were decreased, but not abolished, in CGCs obtained from AT(1A) knock-out mice. This indicates that although AT(1) receptors are necessary for glutamate to exert its full neurotoxic potential, part of the neuroprotective effect of telmisartan is independent of AT(1) receptor blockade. PPARγ activation was also involved in the neuroprotective effects of telmisartan, as telmisartan enhanced PPARγ nuclear translocation and the PPARγ antagonist GW9662 partially reversed the neuroprotective effects of telmisartan. The present results substantiate the therapeutic use of sartans, in particular telmisartan, in neurodegenerative diseases and traumatic brain disorders where glutamate neurotoxicity plays a significant role.
Assuntos
Benzimidazóis/farmacologia , Benzoatos/farmacologia , Ácido Glutâmico/toxicidade , Neurônios/efeitos dos fármacos , Fármacos Neuroprotetores/farmacologia , PPAR gama/metabolismo , Receptor Tipo 1 de Angiotensina/metabolismo , Bloqueadores do Receptor Tipo 1 de Angiotensina II/farmacologia , Animais , Apoptose/efeitos dos fármacos , Apoptose/fisiologia , Células Cultivadas , Cerebelo/efeitos dos fármacos , Cerebelo/metabolismo , Feminino , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Camundongos Transgênicos , Neurônios/fisiologia , PPAR gama/antagonistas & inibidores , Ratos , Ratos Sprague-Dawley , Receptor Tipo 1 de Angiotensina/genética , TelmisartanRESUMO
Commercially available angiotensin II At2 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, we characterized three commercially available At2 receptor antibodies: 2818-1 from Epitomics, sc-9040 from Santa Cruz Biotechnology, Inc., and AAR-012 from Alomone Labs. Using western blot analysis the immunostaining patterns observed were different for every antibody tested, and in most cases consisted of multiple immunoreactive bands. Identical immunoreactive patterns were present in wild-type and At2 receptor knockout mice not expressing the target protein. In the mouse brain, immunocytochemical studies revealed very different cellular immunoreactivity for each antibody tested. While the 2818-1 antibody reacted only with endothelial cells in small parenchymal arteries, the sc-9040 antibody reacted only with ependymal cells lining the cerebral ventricles, and the AAR-012 antibody reacted only with multiple neuronal cell bodies in the cerebral cortex. Moreover, the immunoreactivities were identical in brain tissue from wild-type or At2 receptor knockout mice. Furthermore, in both mice and rat tissue extracts, there was no correlation between the observed immunoreactivity and the presence or absence of At2 receptor binding or gene expression. We conclude that none of these commercially available At2 receptor antibodies tested met the criteria for specificity. In the absence of full antibody characterization, competitive radioligand binding and determination of mRNA expression remain the only reliable approaches to study At2 receptor expression.
Assuntos
Anticorpos/análise , Anticorpos/imunologia , Especificidade de Anticorpos , Receptores de Angiotensina/análise , Receptores de Angiotensina/imunologia , Animais , Western Blotting , Encéfalo/imunologia , Química Encefálica , Imuno-Histoquímica , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , RNA Mensageiro/genética , Ratos , Ratos Sprague-Dawley , Receptores de Angiotensina/genéticaRESUMO
Commercially available Angiotensin II AT1 receptor antibodies are widely employed for receptor localization and quantification, but they have not been adequately validated. In this study, six commercially available AT1 receptor antibodies were characterized by established criteria: sc-1173 and sc-579 from Santa Cruz Biotechnology, Inc., AAR-011 from Alomone Labs, Ltd., AB15552 from Millipore, and ab18801 and ab9391 from Abcam. The immunostaining patterns observed were different for every antibody tested, and were unrelated to the presence or absence of AT1 receptors. The antibodies detected a 43 kDa band in western blots, corresponding to the predicted size of the native AT1 receptor. However, identical bands were observed in wild-type mice and in AT1A knock-out mice not expressing the target protein. Moreover, immunoreactivity detected in rat hypothalamic 4B cells not expressing AT1 receptors or transfected with AT1A receptor construct was identical, as revealed by western blotting and immunocytochemistry in cultured 4B cells. Additional prominent immunoreactive bands above and below 43 kDa were observed by western blotting in extracts from tissues of AT1A knock-out and wild-type mice and in 4B cells with or without AT1 receptor expression. In all cases, the patterns of immunoreactivity were independent of the AT1 receptor expression and different for each antibody studied. We conclude that, in our experimental setup, none of the commercially available AT1 receptor antibodies tested met the criteria for specificity and that competitive radioligand binding remains the only reliable approach to study AT1 receptor physiology in the absence of full antibody characterization.
Assuntos
Anticorpos/metabolismo , Receptor Tipo 1 de Angiotensina/imunologia , Receptor Tipo 1 de Angiotensina/metabolismo , Animais , Sítios de Ligação de Anticorpos/imunologia , Células Cultivadas , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Camundongos Knockout , Ligação Proteica/imunologia , Ratos , Ratos Wistar , Receptor Tipo 1 de Angiotensina/biossínteseRESUMO
UNLABELLED: Cell swelling-induced insulin secretion represents an alternative pathway of stimulation of insulin secretion. INS-1E rat tumor beta cells do not release insulin in response to cell swelling in presence of Ca(2+) despite a good response to glucose challenge and appropriate increase in cell volume. Surprisingly, perifusion with Ca(2+)-depleted medium showed distinct secretory response of INS-1E cells to hypotonicity. Objective of this study was further characterization of the role of Ca(2+) in secretory process in INS-1 and INS-1E cell lines. Ca(2+) depleted hypotonic medium with 10 muM BAPTA/AM (intracellular chelator) induced insulin secretion from both types of cells. We demonstrated expression of L-type Ca(2+) channel Ca(v)1.2 and non-L-type Ca(2+) channels Ca(v)2.1 (P/Q-type), Ca(v)2.2 (N-type), and Ca(v)3.1 (T-type) in both cell lines. Inhibition of L type channel with nifedipine and/or P/Q type with omega-agatoxin IVA enabled distinct response to hypotonic medium also in INS-1E cells. Tetanus toxin (TeTx) in medium containing Ca(2+) and a group of calcium channel blockers inhibited hypotonicity-induced insulin secretion from INS-1 cells but not from INS-1E cells. CONCLUSION: Hypotonicity-induced insulin secretion from INS-1E cells is inhibited by extracellular Ca(2+), does not require intracellular Ca(2+) and is TeTx resistant.
Assuntos
Cálcio/farmacologia , Insulina/metabolismo , Toxina Tetânica/farmacologia , Animais , Bloqueadores dos Canais de Cálcio/farmacologia , Canais de Cálcio Tipo L/química , Canais de Cálcio Tipo L/genética , Canais de Cálcio Tipo L/metabolismo , Canais de Cálcio Tipo P/química , Canais de Cálcio Tipo P/genética , Canais de Cálcio Tipo P/metabolismo , Canais de Cálcio Tipo Q/química , Canais de Cálcio Tipo Q/genética , Canais de Cálcio Tipo Q/metabolismo , Canais de Cálcio Tipo R/química , Canais de Cálcio Tipo R/genética , Canais de Cálcio Tipo R/metabolismo , Canais de Cálcio Tipo T/química , Canais de Cálcio Tipo T/genética , Canais de Cálcio Tipo T/metabolismo , Linhagem Celular Tumoral , Tamanho Celular , Ácido Egtázico/análogos & derivados , Ácido Egtázico/farmacologia , Soluções Hipotônicas/química , Secreção de Insulina , Masculino , Nifedipino/farmacologia , Ratos , Proteínas SNARE/metabolismo , Proteínas SNARE/fisiologia , ômega-Agatoxina IVA/farmacologiaRESUMO
UNLABELLED: Alcohol causes reactive hypoglycemia by attenuating the release of counter regulatory hormones, redistribution of pancreatic blood flow and direct stimulation of insulin secretion. Objective of this study was characterization of ethanol-induced insulin secretion. Signaling of ethanol- and glucose-induced insulin release from INS-1 and INS-1E cells was compared. Both cell lines responded similarly to all experimental interventions. In contrast to glucose, ethanol-induced insulin secretion was not hindered in calcium depleted medium or by addition of 10 microM BAPTA/AM (intracellular chelator). Inhibitor of protein kinase C Bisindolylmaleimide (3 microM) abolished glucose- but not ethanol-induced insulin secretion. Tetanus toxin (20 nM), inhibitor of SNARE proteins complex formation, blocked ethanol-induced insulin secretion. Both 5 mM N-ethylamaleimide and 10 microM ZnCl(2) (inhibitor of protein tyrosine phosphatases), which block disassembly of SNARE complexes and their further participation in exocytosis, increased basal insulin secretion. In contrast to glucose, already high insulin secretion was further increased after ethanol stimulation in either treatment. CONCLUSION: Signaling of ethanol-induced insulin secretion from INS-1 and INS-1E cell lines bypasses calcium and PKC involving steps, is sensitive to tetanus toxin but resistant to N-ethymaleimide and ZnCl(2). An extra pool of secretory vesicles not available for glucose is exploited for exocytosis after ethanol stimulation.
Assuntos
Etanol/farmacologia , Insulina/metabolismo , Animais , Cálcio/metabolismo , Linhagem Celular Tumoral , Cloretos/farmacologia , Glucose/farmacologia , Indóis/farmacologia , Secreção de Insulina , Maleimidas/farmacologia , Proteína Quinase C/metabolismo , Ratos , Proteínas SNARE/metabolismo , Toxina Tetânica/farmacologia , Compostos de Zinco/farmacologiaRESUMO
BACKGROUND: This study was undertaken to examine putative mechanisms of calcium independent signal transduction pathway of cell swelling-induced insulin secretion. METHODS: The role of phospholipase A(2), G proteins, and soluble N-ethylmaleimide-sensitive-factor attachment protein receptor (SNARE) in insulin secretion induced by 30% hypotonic medium was studied using isolated rat pancreatic islets. RESULTS: In contrast to glucose stimulation, osmotically induced insulin secretion from pancreatic islets was not inhibited by 10 micromol/l bromoenol lactone, an iPLA(2) (Ca(2+) independent phospholipase) inhibitor. Similarly, preincubation of islets for 20 hours with 25 microg/ml mycophenolic acid to inhibit GTP synthesis fully abolished glucose-induced insulin secretion but was without effect on hypotonicity stimulated insulin release. Glucose-induced insulin secretion was prevented by preincubation with 20 nmol/l tetanus toxin (TeTx), a metalloprotease inactivating soluble SNARE. Cell swelling-induced insulin secretion was inhibited by TeTx in the presence of calcium ions but not in calcium depleted medium. The presence of N-ethylmaleimide (NEM, 5 mmol/l, another inhibitor of SNARE proteins) in the medium resulted in high basal insulin secretion and lacking response to glucose stimulation. In contrast, high basal insulin secretion from NEM treated islets further increased after hypotonic stimulation. CONCLUSION: G proteins and iPLA(2) - putative mediators of Ca(2+) independent signaling pathway participate in glucose but not in hypotonicity-induced insulin secretion. Hypotonicity-induced insulin secretion is sensitive to clostridial neurotoxin TeTx but is resistant to NEM.