RESUMO
Correlational and causal comparative research link ceramide (Cer), the precursor of complex sphingolipids, to some psychiatric (e.g., depression, schizophrenia (SZ), alcohol use disorder, and morphine antinociceptive tolerance) and neurological (e.g., Alzheimer's disease (AD), Parkinson disease (PD)) disorders. Cer generation can occur through the de novo synthesis pathway, the sphingomyelinase pathways, and the salvage pathway. The discoveries that plasma Cer concentration increase during depressive episodes in patients and that tricyclic and tetracyclic antidepressants functionally inhibit acid sphingomyelinase (ASM), the enzyme that catalyzes the degradation of sphingomyelin to Cer, have initiated a series of studies on the role of the ASM-Cer system in depressive disorder. Disturbances in the metabolism of Cer or SM are associated with the occurrence of SZ and PD. In both PD and SZ patients, the elevated levels of Cer or SM in the brain regions were associated with the disease. AD patients showed also an abnormal metabolism of brain Cer at early stages of the disease which may suggest Cer as an AD biomarker. In plasma of AD patients and in AD transgenic mice, ASM activity was increased. In contrast, partial ASM inhibition of Aß deposition improved memory deficits. Furthermore, in clinical and preclinical research, ethanol enhanced activation of ASM followed by Cer production. Limited data have shown that Cer plays an important role in the development of morphine antinociceptive tolerance. In summary, clinical and preclinical findings provide evidence that targeting the Cer system should be considered as an innovative translational strategy for some brain disorders.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Antidepressivos/farmacologia , Encéfalo/efeitos dos fármacos , Ceramidas/farmacologia , Transtornos da Memória/tratamento farmacológico , Doença de Alzheimer/metabolismo , Encéfalo/metabolismo , Ceramidas/química , Transtornos da Memória/metabolismo , Esfingomielina Fosfodiesterase/efeitos dos fármacosRESUMO
Although a group of FDA-approved drugs were previously identified with activity against Ebola virus (EBOV), most of them are not clinically useful because their human blood concentrations are not high enough to inhibit EBOV infection. We screened 795 unique three-drug combinations in an EBOV entry assay. Two sets of three-drug combinations, toremifene-mefloquine-posaconazole and toremifene-clarithromycin-posaconazole, were identified that effectively blocked EBOV entry and were further validated for inhibition of live EBOV infection. The individual drug concentrations in the combinations were reduced to clinically relevant levels. We identified mechanisms of action of these drugs: functional inhibitions of Niemann-Pick C1, acid sphingomyelinase, and lysosomal calcium release. Our findings identify the drug combinations with potential to treat EBOV infection.
Assuntos
Antivirais/farmacologia , Ebolavirus/efeitos dos fármacos , Internalização do Vírus/efeitos dos fármacos , Animais , Linhagem Celular , Chlorocebus aethiops , Claritromicina/farmacologia , Combinação de Medicamentos , Sinergismo Farmacológico , Doença pelo Vírus Ebola/tratamento farmacológico , Doença pelo Vírus Ebola/virologia , Ensaios de Triagem em Larga Escala , Humanos , Mefloquina/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Toremifeno/farmacologia , Triazóis/farmacologia , Células VeroRESUMO
Cytoplasmic TDP-43 aggregation is a pathological hallmark of amyotrophic lateral sclerosis and frontotemporal lobar degeneration. Here we investigated the role of exosomes in the secretion and propagation of TDP-43 aggregates. TDP-43 was detected in secreted exosomes from Neuro2a cells and primary neurons but not from astrocytes or microglia. Evidence is presented that protein aggregation and autophagy inhibition are factors that promote exosomal secretion of TDP-43. We also report that levels of exosomal TDP-43 full length and C-terminal fragment species are upregulated in human amyotrophic lateral sclerosis brains. Exposure of Neuro2a cells to exosomes from amyotrophic lateral sclerosis brain, but not from control brain, caused cytoplasmic redistribution of TDP-43, suggesting that secreted exosomes might contribute to propagation of TDP-43 proteinopathy. Yet, inhibition of exosome secretion by inactivation of neutral sphingomyelinase 2 with GW4869 or by silencing RAB27A provoked formation of TDP-43 aggregates in Neuro2a cells. Moreover, administration of GW4869 exacerbated the disease phenotypes of transgenic mice expressing human TDP-43A315T mutant. Thus, even though results suggest that exosomes containing pathological TDP-43 may play a key role in the propagation of TDP-43 proteinopathy, a therapeutic strategy for amyotrophic lateral sclerosis based on inhibition of exosome production would seem inappropriate, as in vivo data suggest that exosome secretion plays an overall beneficial role in neuronal clearance of pathological TDP-43.
Assuntos
Compostos de Anilina/farmacologia , Comportamento Animal/efeitos dos fármacos , Compostos de Benzilideno/farmacologia , Proteínas de Ligação a DNA/metabolismo , Exossomos/metabolismo , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Proteinopatias TDP-43/metabolismo , Animais , Linhagem Celular , Modelos Animais de Doenças , Humanos , Camundongos , Camundongos Transgênicos , Proteinopatias TDP-43/tratamento farmacológicoRESUMO
OBJECTIVE: To investigate the effect of the jianpi-jiedu formula (JPJD) on the expression of angiogenesis-relevant genes in colon cancer.â© Methods: Crude extract was obtained from JPJD by water extract method. The effect of JPJD crude extract on colon cancer cell proliferation capacity was determined by MTT assays. The IC50 value was calculated by GraphPad Prism5 software. Affymetrix gene expression profiling chip was used to detect significant differences in expressions of genes after JPJD intervention, and pathway enrichment analysis was performed to analyze the differentially expressed genes. Ingenuity Pathway Analysis software was applied to analyze differentially expressed genes relevant to tumor angiogenesis based on mammalian target of rapamycin (mTOR) signaling pathway and then the network diagram was built. Western blot was used to verify the protein levels of key genes related to tumor angiogenesis.â© Results: JPJD crud extract inhibited the proliferation capacity in colon cancer cells. The IC50 values in 24, 48, and 72 hours after treatment were 13.060, 9.646 and 8.448 mg/mL, respectively. The results of chip showed that 218 genes significantly upgraded, and 252 genes significantly downgraded after JPJD treatment. Most of the genes were related to the function of biosynthesis, metabolism, cell apoptosis, antigen extraction, angiogenesis and so on. There were 12 differentially expressed angiogenesis genes. IPA software analysis showed that the JPJD downregulated expression of sphingomyelin phosphodiesterase 3 (SMPD3), VEGF, vascular endothelial growth factor A (VEGFA), integrin subunit alpha 1 (ITGA1), cathepsin B (CTSB), and cathepsin S (CTSS) genes, while upregulated expressions of GAB2 and plasminogen activator, urokinase receptor (PLAUR) genes in the colorectal cancer cell. Western blot results demonstrated that JPJD obviously downregulated expressions of phospho-mTOR (P-mTOR), signal transducer and activator of transcription 3 (STAT3), hypoxia inducible factor-1α (HIF-1α), and VEGF proteins, while obviously upregulated the level of phospho-P53 (P-P53) protein.â© Conclusion: JPJD may inhibit colorectal tumor angiogenesis through regulation of the mTOR-HIF-1α-VEGF signal pathway.
Assuntos
Linhagem Celular Tumoral/efeitos dos fármacos , Neoplasias Colorretais/genética , Medicamentos de Ervas Chinesas/farmacologia , Animais , Western Blotting , Catepsina B/efeitos dos fármacos , Catepsina B/metabolismo , Catepsinas/efeitos dos fármacos , Catepsinas/metabolismo , Neoplasias Colorretais/irrigação sanguínea , Regulação para Baixo , Perfilação da Expressão Gênica/métodos , Humanos , Subunidade alfa do Fator 1 Induzível por Hipóxia/efeitos dos fármacos , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Cadeias alfa de Integrinas/efeitos dos fármacos , Cadeias alfa de Integrinas/metabolismo , Neovascularização Patológica/genética , Receptores de Ativador de Plasminogênio Tipo Uroquinase/efeitos dos fármacos , Receptores de Ativador de Plasminogênio Tipo Uroquinase/metabolismo , Fator de Transcrição STAT3/efeitos dos fármacos , Fator de Transcrição STAT3/metabolismo , Transdução de Sinais , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Serina-Treonina Quinases TOR/efeitos dos fármacos , Serina-Treonina Quinases TOR/metabolismo , Proteína Supressora de Tumor p53/efeitos dos fármacos , Proteína Supressora de Tumor p53/metabolismo , Regulação para Cima , Fator A de Crescimento do Endotélio Vascular/efeitos dos fármacos , Fator A de Crescimento do Endotélio Vascular/metabolismoRESUMO
Sphingolipids are involved in the pathogenesis of inflammatory diseases. The central molecule is ceramide, which can be converted into ceramide-1-phosphate (C1P). Although C1P can exert anti- and pro-inflammatory effects, its influence on cigarette smoke (CS)-induced lung inflammation is unknown. We aimed to clarify the role of C1P in the pathogenesis of CS-triggered pulmonary inflammation and emphysema in humans and mice. The effects of C1P were addressed on CS-induced lung inflammation in C57BL/6 mice, CS extract-triggered activation of human airway epithelial cells (AECs) and neutrophils from patients with chronic obstructive pulmonary disease. Differential cell counts in bronchoalveolar lavage fluid were determined by flow cytometry and pro-inflammatory cytokines were measured by ELISA. Expression and DNA binding of nuclear factor (NF)-κB and neutral sphingomyelinase (nSMase) were quantified by PCR, electrophoretic mobility shift and fluorometric assays. C1P reduced CS-induced acute and chronic lung inflammation and development of emphysema in mice, which was associated with a reduction in nSMase and NF-κB activity in the lungs. nSMase activity in human serum correlated negatively with forced expiratory volume in 1â s % predicted. In human AECs and neutrophils, C1P inhibited CS-induced activation of NF-κB and nSMase, and reduced pro-inflammatory cytokine release. Our results suggest that C1P is a potential target for anti-inflammatory treatment in CS-induced lung inflammation.
Assuntos
Ceramidas/farmacologia , Citocinas/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Pulmão/efeitos dos fármacos , Enfisema Pulmonar/imunologia , RNA Mensageiro/efeitos dos fármacos , Mucosa Respiratória/efeitos dos fármacos , Adulto , Idoso , Animais , Líquido da Lavagem Broncoalveolar/citologia , Líquido da Lavagem Broncoalveolar/imunologia , Células Cultivadas , Estudos Transversais , Citocinas/imunologia , Modelos Animais de Doenças , Células Epiteliais/imunologia , Células Epiteliais/patologia , Feminino , Humanos , Inflamação , Pulmão/imunologia , Pulmão/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Pessoa de Meia-Idade , NF-kappa B/efeitos dos fármacos , NF-kappa B/genética , NF-kappa B/metabolismo , Neutrófilos/imunologia , Doença Pulmonar Obstrutiva Crônica/imunologia , Doença Pulmonar Obstrutiva Crônica/patologia , Enfisema Pulmonar/patologia , RNA Mensageiro/metabolismo , Mucosa Respiratória/imunologia , Mucosa Respiratória/patologia , Fumaça , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/genética , Esfingomielina Fosfodiesterase/metabolismo , NicotianaRESUMO
BACKGROUND: Outcomes for organ transplantation are constantly improving because of advances in organ preservation, surgical techniques, immune clinical monitoring, and immunosuppressive treatment preventing acute transplant rejection. However, chronic rejection including transplant vasculopathy still limits long-term patient survival. Transplant vasculopathy is characterized by progressive neointimal hyperplasia leading to arterial stenosis and ischemic failure of the allograft. This work sought to decipher the manner in which the humoral immune response, mimicked by W6/32 anti-HLA antibody, contributes to transplant vasculopathy. METHODS AND RESULTS: Studies were performed in vitro on cultured human smooth muscle cells, ex vivo on human arterial segments, and in vivo in a model consisting of human arterial segments grafted into severe combined immunodeficiency/beige mice injected weekly with anti-HLA antibodies. We report that anti-HLA antibodies are mitogenic for smooth muscle cells through a signaling mechanism implicating matrix metalloproteinases (MMPs) (membrane type 1 MMP and MMP2) and neutral sphingomyelinase-2. This mitogenic signaling and subsequent DNA synthesis are blocked in smooth muscle cells silenced for MMP2 or for neutral sphingomyelinase-2 by small interfering RNAs, in smooth muscle cells transfected with a vector coding for a dominant-negative form of membrane type 1 MMP, and after treatment by pharmacological inhibitors of MMPs (Ro28-2653) or neutral sphingomyelinase-2 (GW4869). In vivo, Ro28-2653 and GW4869 reduced the intimal thickening induced by anti-HLA antibodies in human mesenteric arteries grafted into severe combined immunodeficiency/beige mice. CONCLUSIONS: These data highlight a crucial role for MMP2 and neutral sphingomyelinase-2 in vasculopathy triggered by a humoral immune response and open new perspectives for preventing transplant vasculopathy with the use of MMP and neutral sphingomyelinase inhibitors, in addition to conventional immunosuppression.
Assuntos
Anticorpos Anti-Idiotípicos/farmacologia , Artérias/transplante , Antígenos HLA/imunologia , Metaloproteinase 14 da Matriz/metabolismo , Metaloproteinase 2 da Matriz/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Doenças Vasculares/fisiopatologia , Compostos de Anilina/farmacologia , Animais , Anticorpos Anti-Idiotípicos/efeitos adversos , Artérias/patologia , Artérias/fisiopatologia , Compostos de Benzilideno/farmacologia , Células Cultivadas , Constrição Patológica/etiologia , Constrição Patológica/fisiopatologia , Modelos Animais de Doenças , Humanos , Hiperplasia/etiologia , Hiperplasia/fisiopatologia , Técnicas In Vitro , Metaloproteinase 14 da Matriz/efeitos dos fármacos , Metaloproteinase 2 da Matriz/efeitos dos fármacos , Inibidores de Metaloproteinases de Matriz , Camundongos , Camundongos SCID , Modelos Animais , Músculo Liso Vascular/citologia , Músculo Liso Vascular/efeitos dos fármacos , Músculo Liso Vascular/metabolismo , Neointima/patologia , Neointima/fisiopatologia , Piperazinas/farmacologia , Pirimidinas/farmacologia , RNA Interferente Pequeno/farmacologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Doenças Vasculares/etiologia , Enxerto VascularRESUMO
Sphingomyelinase (SMase) activity was measured in Entamoeba histolytica particulate and soluble subcellular fractions. The effects on SMase of incubation time, total protein concentration, pH, and several divalent cations were determined. SMase-C and other unidentified esterase activity were detected in soluble and particulate fractions. SMase-C was 94.5-96.0% higher than the unidentified esterase activity. Soluble and insoluble SMase-C specific activities increased with protein dose and incubation time. Soluble and insoluble SMase-C activities were maximum at pH 7.5 and were dependent on Mg(2+), Mn(2+), or Co(2+), and inhibited by Zn(2+), Hg(2+), Ca(2+), and EDTA. SMase-C was active in the pH range of 3-10 and its maximum activity was at pH 7.5. The soluble and insoluble SMases have remarkably similar physicochemical properties, strongly suggesting that E. histolytica has just one isoform of neutral SMase-C that had not been described before and might be essential for E. histolytica metabolism or virulence.
Assuntos
Entamoeba histolytica/enzimologia , Esterases/metabolismo , Esfingomielina Fosfodiesterase/metabolismo , Animais , Cálcio/farmacologia , Cobalto/farmacologia , Cricetinae , Relação Dose-Resposta a Droga , Entamoeba histolytica/patogenicidade , Concentração de Íons de Hidrogênio , Magnésio/farmacologia , Masculino , Manganês/farmacologia , Mercúrio/farmacologia , Mesocricetus , Proteínas de Protozoários/metabolismo , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Fatores de Tempo , Virulência , Zinco/farmacologiaRESUMO
BACKGROUND: Boswellic acid is a type of triterpenoids with antiinflammatory and antiproliferative properties. Sphingomyelin metabolism generates multiple lipid signals affecting cell proliferation, inflammation, and apoptosis. Upregulation of acid sphingomyelinase (SMase) has been found in several inflammation-related diseases such as inflammatory bowel diseases, atherosclerosis, and diabetes. METHODS: The present study is to examine the effect of 3-acetyl-11-keto-beta-boswellic acids (AKBA), a potent boswellic acid, on acid SMase activity and expression in intestinal cells. Both transformed Caco-2 cells and non-transformed Int407 cells were incubated with AKBA. After incubation, the change of acid SMase activity was assayed biochemically, the enzyme protein was examined by Western blot, and acid SMase mRNA was quantified by qPCR. RESULTS: We found that AKBA decreased acid SMase activity in both intestinal cell lines in dose and time dependent manners without affecting the secretion of the enzyme to the cell culture medium. The effect of AKBA was more effective in the fetal bovine serum-free culture medium. Among different types of boswellic acid, AKBA was the most potent one. The inhibitory effect on acid SMase activity occurred only in the intact cells but not in cell-free extract in the test tubes. At low concentration, AKBA only decreased the acid SMase activity but not the quantity of the enzyme protein. However, at high concentration, AKBA decreased both the mass of acid SMase protein and the mRNA levels of acid SMase in the cells, as demonstrated by Western blot and qPCR, respectively. Under the concentrations decreasing acid SMase activity, AKBA significantly inhibited cell proliferation. CONCLUSION: We identified a novel inhibitory effect of boswellic acids on acid SMase expression, which may have implications in human diseases and health.
Assuntos
Regulação Enzimológica da Expressão Gênica/efeitos dos fármacos , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Triterpenos/farmacologia , Anti-Inflamatórios não Esteroides/farmacologia , Células CACO-2 , Linhagem Celular , Humanos , Inflamação/etiologia , Intestinos/citologia , Intestinos/enzimologia , Esfingomielina Fosfodiesterase/biossínteseRESUMO
Major depression is a severe mood disorder with a lifetime prevalence of more than 10%. The pharmacokinetic hypothesis claims that a slow accumulation of antidepressant drugs by acid trapping mainly into lysosomes is responsible for the therapeutic latency and that a lysosomal target mediates the antidepressant effects. The lysosomal lipid metabolizing enzyme acid sphingomyelinase (ASM) cleaves sphingomyelin into ceramide and phosphorylcholine. In a pilot study, the activity of this enzyme was increased in peripheral blood cells of patients with major depressive disorder (MDD), making the ASM an interesting molecular target of antidepressant drugs. Indeed, several antidepressant drugs functionally inhibit ASM. The ASM/ceramide pathway might be a missing link unifying independent findings in neurobiology and the treatment of MDD such as therapeutic latency, oxidative stress, immune activation and increased risk of cardiovascular disease.
Assuntos
Ceramidas/fisiologia , Transtorno Depressivo Maior/fisiopatologia , Antidepressivos/farmacocinética , Encéfalo/metabolismo , Transtorno Depressivo Maior/tratamento farmacológico , Transtorno Depressivo Maior/psicologia , Método Duplo-Cego , Humanos , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Sphingolipids such as sphingosine-1-phosphate (S1P), ceramide, or sphingomyelin are essential constituents of plasma membranes and regulate many (patho)physiological cellular responses inducing apoptosis and cell survival, vascular permeability, mast cell activation, and airway smooth muscle functions. The complexity of sphingolipid biology is generated by a great variety of compounds, diverse receptors, and often antagonistic functions of different sphingolipids. For instance, apoptosis is promoted by ceramide and prevented by S1P, and pulmonary vascular permeability is increased by S1P2/3 receptors and by ceramide, whereas S1P1 receptors stabilize barrier integrity. Several enzymes of the sphingolipid metabolism respond to external stimuli such as sphingomyelinase isoenzymes that are activated by many stress stimuli and the sphingosine kinase isoenzymes that are activated by allergens. The past years have provided increasing evidence that these processes contribute to pulmonary disorders including asthma, chronic obstructive pulmonary disease, acute lung injury, and cystic fibrosis. Sphingolipid metabolism offers several novel therapeutic targets for the treatment of lung diseases such as emphysema, asthma, cystic fibrosis, respiratory tract infection, sepsis, and acute lung injury.
Assuntos
Pneumopatias/fisiopatologia , Pulmão/fisiopatologia , Esfingolipídeos/fisiologia , Animais , Membrana Celular/fisiologia , Ceramidases/efeitos dos fármacos , Ceramidases/fisiologia , Modelos Animais de Doenças , Humanos , Pneumopatias/tratamento farmacológico , Camundongos , Fosfotransferases (Aceptor do Grupo Álcool)/efeitos dos fármacos , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/fisiologiaRESUMO
Ceramide has been shown to be capable to trigger apoptosis in almost any cell, including tumor cells. Ceramide is generated by a de novo pathway or by sphingomyelinases. Sphingomyelinases hydrolyze sphingomyelin in biological membranes to release ceramide and they are named acid, neutral and alkaline sphingomyelinase depending on their maximum activity at acid, neutral and alkaline pH values, respectively. Stimuli that trigger a release of ceramide to mediate apoptosis include CD95, TNF-receptor, DR5, gamma-irradiation, cytotoxic drugs, UV-light, bacteria, viruses, some forms of developmental death, anti-CD20 and disruption of the cell's contact with its matrix, to name a few. Here, we will focus on the role of acid sphingomyelinase in malignant tumors, which function in apoptosis is best characterized and documented by genetic models. We will discuss concepts that unify the biological actions of ceramide and describe the role of ceramide in important anti-tumor treatment modalities, such as gamma-irradiation and chemotherapy.
Assuntos
Apoptose/efeitos dos fármacos , Ceramidas/uso terapêutico , Neoplasias/tratamento farmacológico , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Animais , Antineoplásicos/uso terapêutico , Ceramidas/metabolismo , Humanos , Microdomínios da Membrana/efeitos dos fármacos , Microdomínios da Membrana/metabolismo , Neoplasias/metabolismo , Neoplasias/radioterapia , Transdução de Sinais/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismoRESUMO
BACKGROUND AND PURPOSE: The age-related decline in vasorelaxation is largely due to ceramide-induced induction of phosphatase 2A (PP2A), which limits nitric oxide synthase (eNOS) phosphorylation at stimulatory sites. We hypothesized that ceramide accumulation was from an age-related loss of endothelial glutathione (GSH) and subsequent activation of neutral sphingomyelinase (nSMase), an enzyme whose activity increases when GSH is limited. EXPERIMENTAL APPROACH: Old (30-32 mo) F344xBN rats were given (R)-alpha-lipoic acid (LA), an agent known to induce GSH synthesis. Vasorelaxation was measured in aortic rings; GSH and ceramide levels, activity of nSMase and eNOS phosphorylation (by Western blot) was measured in aortic endothelial cells, isolated from the same aortas. KEY RESULTS: In old animals, endothelium-dependent relaxation in aortic rings was decreased, GSH levels and its redox state in aortic endothelia were over 30% lower and nSMase activity and endothelial ceramide levels were three-fold increased, relative to young (2-4 mo) rats. LA treatment of old animals improved relaxation in aortic rings, reversed the changes in endothelial GSH, in nSMase activities and in ceramide levels. Similar effects on GSH levels and nSMase activity in old rats were also induced by treatment with GSH monoethylester. Activation (by phosphorylation) of eNOS was decreased by about 50% in old rats and this age-related decrease was partially reversed by LA treatment. CONCLUSIONS AND IMPLICATIONS: Decreased endothelial GSH was partly responsible for the age-related loss of vascular endothelial function and LA might be therapeutically evaluated to treat endothelial dysfunction.
Assuntos
Envelhecimento , Endotélio Vascular/efeitos dos fármacos , Ácido Tióctico/farmacologia , Vasodilatação/efeitos dos fármacos , Animais , Aorta Torácica/efeitos dos fármacos , Aorta Torácica/metabolismo , Western Blotting , Ceramidas/metabolismo , Endotélio Vascular/metabolismo , Glutationa/efeitos dos fármacos , Glutationa/metabolismo , Masculino , Óxido Nítrico Sintase Tipo III/efeitos dos fármacos , Óxido Nítrico Sintase Tipo III/metabolismo , Fosforilação/efeitos dos fármacos , Ratos , Ratos Endogâmicos BN , Ratos Endogâmicos F344 , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Vasodilatação/fisiologiaRESUMO
Beta-cell mass is regulated by a balance between beta-cell growth and beta-cell death, due to apoptosis. We previously reported that apoptosis of INS-1 insulinoma cells due to thapsigargin-induced ER stress was suppressed by inhibition of the group VIA Ca2+-independent phospholipase A2 (iPLA2beta), associated with an increased level of ceramide generation, and that the effects of ER stress were amplified in INS-1 cells in which iPLA2beta was overexpressed (OE INS-1 cells). These findings suggested that iPLA2beta and ceramides participate in ER stress-induced INS-1 cell apoptosis. Here, we address this possibility and also the source of the ceramides by examining the effects of ER stress in empty vector (V)-transfected and iPLA2beta-OE INS-1 cells using apoptosis assays and immunoblotting, quantitative PCR, and mass spectrometry analyses. ER stress induced expression of ER stress factors GRP78 and CHOP, cleavage of apoptotic factor PARP, and apoptosis in V and OE INS-1 cells. Accumulation of ceramide during ER stress was not associated with changes in mRNA levels of serine palmitoyltransferase (SPT), the rate-limiting enzyme in de novo synthesis of ceramides, but both message and protein levels of neutral sphingomyelinase (NSMase), which hydrolyzes sphingomyelins to generate ceramides, were temporally increased in the INS-1 cells. The increases in the level of NSMase expression in the ER-stressed INS-1 cells were associated with corresponding temporal elevations in ER-associated iPLA2beta protein and catalytic activity. Pretreatment with BEL inactivated iPLA2beta and prevented induction of NSMase message and protein in ER-stressed INS-1 cells. Relative to that in V INS-1 cells, the effects of ER stress were accelerated and/or amplified in the OE INS-1 cells. However, inhibition of iPLA2beta or NSMase (chemically or with siRNA) suppressed induction of NSMase message, ceramide generation, sphingomyelin hydrolysis, and apoptosis in both V and OE INS-1 cells during ER stress. In contrast, inhibition of SPT did not suppress ceramide generation or apoptosis in either V or OE INS-1 cells. These findings indicate that iPLA2beta activation participates in ER stress-induced INS-1 cell apoptosis by promoting ceramide generation via NSMase-catalyzed hydrolysis of sphingomyelins, raising the possibility that this pathway contributes to beta-cell apoptosis due to ER stress.
Assuntos
Apoptose , Ceramidas/metabolismo , Fosfolipases A2 do Grupo VI/metabolismo , Insulinoma/enzimologia , Neoplasias Pancreáticas/enzimologia , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Apoptose/fisiologia , Linhagem Celular Tumoral , Retículo Endoplasmático/efeitos dos fármacos , Retículo Endoplasmático/enzimologia , Chaperona BiP do Retículo Endoplasmático , Regulação Enzimológica da Expressão Gênica , Humanos , Hidrólise , Inibidores de Fosfodiesterase/farmacologia , RNA Interferente Pequeno , Retroviridae/genética , Espectrometria de Massas por Ionização por Electrospray , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Tapsigargina/farmacologia , TransfecçãoRESUMO
Curcumin has been shown to inhibit cell growth and induce apoptosis in colon cancer cells. The metabolism of sphingomyelin has implications in the development of colon cancert. We examined whether curcumin affects the enzymes that hydrolyse sphingomyelin in Caco-2 cells. The cells were cultured in both monolayer and polarized conditions and stimulated with curcumin. The activities of sphingomyelinases were determined. Sphingomyelin and its hydrolytic products were analysed by thin layer chromatography. The changes of acid sphingomyelinase protein were examined by Western blotting. We found that curcumin reduced the hydrolytic capacity of the cells against choline-labelled sphingomyelin, associated with a mild increase of cellular sphingomyelin in the cells. Analysis of the hydrolytic products revealed that the activity was derived from acid sphingomyelinase not from phospholipase D. The curcumin-induced reduction of acid SMase required more than 8 h stimulation. Western blotting showed reduced acid sphingomyelinase protein after curcumin stimulation. The inhibitory effect was more potent in monolayer cells than in polarised cells. No changes of other sphingomyelinases were identified. In the concentrations inhibiting acid sphingomyelinase, curcumin inhibited DNA synthesis and induced cell death. In conclusion, curcumin inhibits acid sphingomyelinase and the effect might be involved in its antiproliferative property against colon cancer cells.
Assuntos
Antineoplásicos Fitogênicos/farmacologia , Curcuma , Curcumina/farmacologia , Fitoterapia , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Antineoplásicos Fitogênicos/administração & dosagem , Antineoplásicos Fitogênicos/uso terapêutico , Apoptose/efeitos dos fármacos , Células CACO-2/efeitos dos fármacos , Células CACO-2/enzimologia , Proliferação de Células/efeitos dos fármacos , Curcumina/administração & dosagem , Curcumina/uso terapêutico , Humanos , Extratos Vegetais/administração & dosagem , Extratos Vegetais/farmacologia , Extratos Vegetais/uso terapêutico , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Hydrophobic bile salts activate NADPH oxidase through a ceramide- and PKCzeta-dependent pathway as an important upstream event of bile salt-induced hepatocyte apoptosis. The mechanisms underlying bile salt-induced ceramide formation have remained unclear to date and thus were studied in rat hepatocytes. Proapoptotic bile salts, such as taurolithocholylsulfate (TLCS), lowered the apparent pHves within seconds from 6.0 to 5.6 in an FITC-dextran-accessible endosomal compartment that also contains acidic sphingomyelinase. Simultaneously, a rapid decrease in N-(ethoxycarbonylmethyl)-6-methoxyquinolinium bromide (MQAE) fluorescence was observed, suggestive of an increase in cytosolic [Cl-], which is known to activate vacuolar-type H+-ATPase. No vesicular acidification or increase in cytosolic [Cl-] was found in response to the non-apoptotic bile salt taurocholate or the anti-apoptotic bile salt tauroursodesoxycholate. Inhibition of TLCS-induced endosomal acidification by bafilomycin or 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid largely abolished the TLCS-induced ceramide-formation and downstream ceramide-dependent processes, such as p47phox-serine phosphorylation, NADPH oxidase activation, CD95 activation and apoptosis. These responses were also abolished after knockdown of acidic sphingomyelinase in rat hepatocytes. In conclusion, hydrophobic, proapoptotic bile salts stimulate ceramide formation through chloride-dependent acidification of endosomes, with subsequent activation of acidic sphingomyelinase. Our data suggest that changes in ion homeostasis underlie the stimulation of ceramide formation in response to hydrophobic bile acids as an important upstream event of bile salt-induced apoptosis.
Assuntos
Ceramidas/biossíntese , Endossomos/metabolismo , Hepatócitos/metabolismo , Ácido Taurolitocólico/análogos & derivados , Animais , Apoptose/efeitos dos fármacos , Células Cultivadas , Ceramidas/química , Cloretos/química , Cloretos/metabolismo , Endossomos/química , Endossomos/efeitos dos fármacos , Fluorescência , Hepatócitos/efeitos dos fármacos , Concentração de Íons de Hidrogênio , Interações Hidrofóbicas e Hidrofílicas , Masculino , NADPH Oxidases/efeitos dos fármacos , NADPH Oxidases/metabolismo , Fosforilação , Ratos , Ratos Wistar , Serina/metabolismo , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Relação Estrutura-Atividade , Ácido Taurolitocólico/química , Ácido Taurolitocólico/metabolismo , Ácido Taurolitocólico/farmacologia , Fatores de TempoRESUMO
The molecular mechanisms of hepatic ischemia/reperfusion (I/R) damage are incompletely understood. We investigated the role of ceramide in a murine model of warm hepatic I/R injury. This sphingolipid induces cell death and participates in tumor necrosis factor (TNF) signaling. Hepatic ceramide levels transiently increased after the reperfusion phase of the ischemic liver in mice, because of an early activation of acidic sphingomyelinase (ASMase) followed by acid ceramidase stimulation. In vivo administration of an ASMase inhibitor, imipramine, or ASMase knockdown by siRNA decreased ceramide generation during I/R, and attenuated serum ALT levels, hepatocellular necrosis, cytochrome c release, and caspase-3 activation. ASMase-induced ceramide generation activated JNK resulting in BimL phosphorylation and translocation to mitochondria, as the inhibition of ASMase by imipramine prevented these events. In contrast, blockade of ceramide catabolism by N-oleyolethanolamine (NOE), a ceramidase inhibitor, enhanced ceramide levels and potentiated I/R injury compared with vehicle-treated mice. Pentoxifylline treatment prevented TNF upregulation and ASMase activation. Furthermore, 9 of 11 mice treated with imipramine survived 7 days after total liver ischemia, compared with 4 of 12 vehicle-treated mice, whereas 8 of 8 NOE-treated mice died within 2 days of total liver ischemia. In conclusion, ceramide generated from ASMase plays a key role in I/R-induced liver damage, and its modulation may be of therapeutic relevance.
Assuntos
Regulação para Baixo , Fígado/irrigação sanguínea , RNA Mensageiro/genética , Traumatismo por Reperfusão/enzimologia , Esfingomielina Fosfodiesterase/genética , Animais , Western Blotting , Modelos Animais de Doenças , Endocanabinoides , Inibidores Enzimáticos/farmacologia , Etanolaminas/farmacologia , Seguimentos , Galactosilgalactosilglucosilceramidase/metabolismo , Fígado/efeitos dos fármacos , Fígado/enzimologia , Camundongos , Camundongos Endogâmicos C57BL , Ácidos Oleicos , Reação em Cadeia da Polimerase , Traumatismo por Reperfusão/patologia , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismoRESUMO
Ceramide is involved in the pathogenesis of insulin resistance in skeletal muscles of humans and rodents. However, there are conflicting reports in the literature on the effect of thiazolidinediones (a new class of insulin sensitizing drugs) on skeletal muscle ceramide content. Therefore, the aim of our study was to examine the effect of pioglitazone on the level of ceramide and its metabolites and on the activity of the key enzymes of ceramide metabolism in different skeletal muscle types of the rat. The experiments were carried out on rats fed either a standard chow or a high-fat diet for 21 days. Each group was divided into two subgroups: control and treated with pioglitazone for 14 days. High-fat diet increased the content of ceramide in the soleus and in the red section of the gastrocnemius, but not in the white section of the latter. The activity of neutral Mg(2+)-dependent sphingomyelinase and acid sphingomyelinase was simultaneously reduced in all examined muscles. Administration of pioglitazone decreased ceramide level in the soleus and in the red section of the gastrocnemius in rats fed either diet. This effect could not be attributed to decreased rate of ceramide formation from sphingomyelin or to its augmented deacylation to sphingosine. Pioglitazone treatment reduced the concentration of plasma free fatty acids in rats fed on either diet. Therefore, we conclude that the drug decreased the muscle content of ceramide by reducing its de novo synthesis. The results of our study indicate that reduction in ceramide level may be one of the mechanisms by which pioglitazone improves skeletal muscle insulin sensitivity.
Assuntos
Ceramidas/metabolismo , Hipoglicemiantes/farmacologia , Músculo Esquelético/metabolismo , Tiazolidinedionas/farmacologia , Amidoidrolases/metabolismo , Animais , Ceramidases , Gorduras na Dieta , Ácidos Graxos/sangue , Resistência à Insulina/fisiologia , Magnésio/metabolismo , Masculino , Pioglitazona , Ratos , Ratos Wistar , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Esfingosina/metabolismoRESUMO
We investigated the effects of UDCA on sphingomyelinase (SMase) in Caco 2 cells cultured in monolayer and polarized conditions. Alkaline SMase activity was high in polarized cells whereas, acid and neutral SMase activities were high in monolayer cells. In polarized cells, UDCA increased alkaline SMase expression and caspase 3 activity but had no effect on acid and neutral SMases. In monolayer cells, UDCA reduced both acid and neutral SMase activities, inhibited cell proliferation, but had little effect on alkaline SMase and caspase 3 activities. In conclusion, UDCA differentially affects SMase activity, cell proliferation, and apoptosis in colonic cells depending on the cell conditions.
Assuntos
Apoptose/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Colagogos e Coleréticos/farmacologia , Esfingomielina Fosfodiesterase/metabolismo , Ácido Ursodesoxicólico/farmacologia , Células CACO-2/efeitos dos fármacos , Células CACO-2/enzimologia , Caspase 3 , Caspases/metabolismo , Humanos , Esfingomielina Fosfodiesterase/efeitos dos fármacosRESUMO
Incubation of Schistosoma mansoni lung-stage larvae in 90% corn oil for 6 hr was shown to elicit exposure of their, otherwise masked, apical membrane antigens to binding of anti-schistosome antibodies in the indirect membrane immunofluorescence test (IF). The possibility that unsaturated fatty acids (FA) are responsible for this effect was herein supported by IF data on ex vivo lung-stage larvae of S. mansoni and S. haematobium incubated for 1/2-2 hr with 80% corn oil, 50% olive oil, or 10-20 microM arachidonic acid. Treatment with unsaturated FA followed by filipin staining for cholesterol visualization indicated that unsaturated FA do not induce exposure of schistosomular surface membrane antigens via extraction of surface membrane cholesterol. Evidence using inhibitors and stimulators of neutral sphingomyelinase suggested that unsaturated FA perhaps activate worm tegument-bound neutral sphingomyelinase, leading to sphingomyelin hydrolysis and changes in surface membrane fluidity. Larval apical membrane antigens are, thus, allowed to diffuse freely in the plane of the membrane and bind specific antibodies in IE Excessive sphingomyelin hydrolysis might explain why high FA concentrations or long incubation periods eventually lead to larval death. The significant decrease (P < 0.01) in S. mansoni and increase (P < 0.02) in S. haematobium worm recovery in BALB/c mice given unsaturated FA-high and -poor diets, respectively, indicated these findings have in vivo relevance and led to the proposal that unsaturated FA likely plays a role in natural attrition of S. mansoni and S. haematobium lung-stage larvae.
Assuntos
Gorduras Insaturadas na Dieta/administração & dosagem , Ácidos Graxos Insaturados/farmacologia , Pulmão/parasitologia , Schistosoma haematobium/efeitos dos fármacos , Schistosoma mansoni/efeitos dos fármacos , Animais , Antígenos de Helmintos/imunologia , Antígenos de Superfície/imunologia , Colesterol/análise , Cricetinae , Gorduras Insaturadas na Dieta/farmacologia , Ativação Enzimática/efeitos dos fármacos , Ácidos Graxos Insaturados/administração & dosagem , Filipina , Técnica Indireta de Fluorescência para Anticorpo , Larva/efeitos dos fármacos , Larva/enzimologia , Larva/imunologia , Masculino , Mesocricetus , Camundongos , Camundongos Endogâmicos BALB C , Distribuição Aleatória , Schistosoma haematobium/enzimologia , Schistosoma haematobium/imunologia , Schistosoma mansoni/enzimologia , Schistosoma mansoni/imunologia , Esquistossomose Urinária/dietoterapia , Esquistossomose Urinária/parasitologia , Esquistossomose mansoni/dietoterapia , Esquistossomose mansoni/parasitologia , Esfingomielina Fosfodiesterase/antagonistas & inibidores , Esfingomielina Fosfodiesterase/efeitos dos fármacos , Esfingomielina Fosfodiesterase/metabolismo , Esfingomielinas/metabolismo , Coloração e Rotulagem/métodosRESUMO
Mitochondria reduce Cr(VI) to Cr(V) with concomitant generation of reactive oxygen species, thereby exhibiting cytotoxic effects leading to apoptosis in various types of cells. To clarify the mechanism by which Cr(VI) induces apoptosis, we examined the effect of Cr(VI) on Chinese hamster ovary (CHO) cells. Cr(VI) increased cellular levels of ceramide by activating acid sphingomyelinase (ASMase) and inhibiting the phosphorylation of pleckstrin homology domain-containing protein kinase B (Akt). Cr(VI) also induced cyclosporin A- and trifluoperazine-sensitive depolarization of mitochondria and activated caspase-3, 8 and 9, thereby causing fragmentation of cellular DNA. The presence of desipramine, an inhibitor of ASMase, and membrane permeable pCPT-cAMP suppressed the Cr(VI)-induced activation of caspases and DNA fragmentation. These results suggested that accumulation of ceramide play an important role in the Cr(VI)-induced apoptosis of CHO cells through activation of mitochondrial membrane permeability transition.