RESUMO
Aging is a physiological and immunological process involving the deterioration of human health, characterised by the progressive alteration of organs and their functions. The speed and extent of such decline are dependent on lifestyle, environment, and genetic factors. Moreover, with advancing age, humans become progressively more fragile and prone to acute and chronic diseases. Although the intestinal microbiota is predisposed to perturbations that accompany aging and frailty, it is generally accepted that the gut microbiota engages in multiple interactions that affect host health throughout the host life span. In the current study, an exhaustive in silico investigation of gut-associated bifidobacteria in healthy individuals from birth to old age revealed that Bifidobacterium longum subsp. longum is the most prevalent member, especially during infancy and in centenarians. Moreover, B. longum subsp. longum genome reconstruction and strain tracing among human gut microbiomes allowed the identification of prototypes of this taxon in the human gut microbiota of healthy elderly individuals. Such analyses guided culturomics attempts to isolate B. longum subsp. longum strains that matched the genomic content of B. longum subsp. longum prototypes from healthy elderly individuals. The molecular effects of selected B. longum subsp. longum strains on the human host were further investigated using in vitro microbe-host interactions, revealing differences in the host immune system transcriptome, with a reduction in gene expression of inflammation-related cytokines. These intriguing findings support the potential anti-aging effects of elderly associated prototypes of B. longum subsp. longum.
Assuntos
Bifidobacterium , Microbioma Gastrointestinal , Imunidade Inata , Humanos , Bifidobacterium/genética , Bifidobacterium/imunologia , Idoso de 80 Anos ou mais , Idoso , Lactente , Envelhecimento/imunologia , Pré-Escolar , Adulto , Adolescente , Adulto Jovem , Pessoa de Meia-Idade , Masculino , Feminino , Genoma Bacteriano/genética , Interações entre Hospedeiro e Microrganismos/imunologiaRESUMO
BACKGROUND: A subset of human hepatocellular carcinomas (HCC) exhibit mutations of ß-catenin gene CTNNB1 and overexpress Glutamine synthetase (GS). The CTNNB1-mutated HCC cell line HepG2 is sensitive to glutamine starvation induced in vitro with the antileukemic drug Crisantaspase and the GS inhibitor methionine-L-sulfoximine (MSO). METHODS: Immunodeficient mice with subcutaneous xenografts of the CTNNB1-mutated HCC cell lines HepG2 and HC-AFW1 were treated with Crisantaspase and/or MSO, and tumour growth was monitored. At the end of treatment, tumour weight and histology were assessed. Serum and tissue amino acids were determined by HPLC. Gene and protein expression were estimated with RT-PCR and western blot and GS activity with a colorimetric method. mTOR activity was evaluated from the phosphorylation of p70S6K1. RESULTS: Crisantaspase and MSO depleted serum glutamine, lowered glutamine in liver and tumour tissue, and inhibited liver GS activity. HepG2 tumour growth was significantly reduced by either Crisantaspase or MSO, and completely suppressed by the combined treatment. The combined treatment was also effective against xenografts of the HC-AFW1 cell line, which is Crisantaspase resistant in vitro. CONCLUSIONS: The combination of Crisantaspase and MSO reduces glutamine supply to CTNNB1-mutated HCC xenografts and hinders their growth.
Assuntos
Asparaginase/farmacologia , Asparaginase/uso terapêutico , Carcinoma Hepatocelular/tratamento farmacológico , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamina , Neoplasias Hepáticas/tratamento farmacológico , Carga Tumoral/efeitos dos fármacos , beta Catenina/genética , Animais , Antineoplásicos/uso terapêutico , Asparagina/sangue , Caderinas/análise , Carcinoma Hepatocelular/enzimologia , Carcinoma Hepatocelular/genética , Carcinoma Hepatocelular/patologia , Inibidores Enzimáticos/uso terapêutico , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Glutamina/análise , Glutamina/sangue , Células Hep G2 , Humanos , Antígeno Ki-67/análise , Neoplasias Hepáticas/enzimologia , Neoplasias Hepáticas/genética , Neoplasias Hepáticas/patologia , Masculino , Metionina Sulfoximina/uso terapêutico , Camundongos , Camundongos Nus , Mutação , Ensaios Antitumorais Modelo de Xenoenxerto , beta Catenina/análiseRESUMO
Hypoxia-inducible factor (HIF) prolyl hydroxylases (PHDs) are α-ketoglutarate (αKG)-dependent dioxygenases that function as cellular oxygen sensors. However, PHD activity also depends on factors other than oxygen, especially αKG, a key metabolic compound closely linked to amino-acid metabolism. We examined the connection between amino-acid availability and PHD activity. We found that amino-acid starvation leads to αKG depletion and to PHD inactivation but not to HIF stabilization. Furthermore, pharmacologic or genetic inhibition of PHDs induced autophagy and prevented mammalian target of rapamycin complex 1 (mTORC1) activation by amino acids in a HIF-independent manner. Therefore, PHDs sense not only oxygen but also respond to amino acids, constituting a broad intracellular nutrient-sensing network.
Assuntos
Aminoácidos/metabolismo , Subunidade alfa do Fator 1 Induzível por Hipóxia/metabolismo , Pró-Colágeno-Prolina Dioxigenase/metabolismo , Animais , Linhagem Celular , Ativação Enzimática , Humanos , Ácidos Cetoglutáricos/metabolismo , Alvo Mecanístico do Complexo 1 de Rapamicina , Camundongos , Modelos Biológicos , Complexos Multiproteicos/antagonistas & inibidores , Complexos Multiproteicos/metabolismo , Pró-Colágeno-Prolina Dioxigenase/antagonistas & inibidores , Serina-Treonina Quinases TOR/antagonistas & inibidores , Serina-Treonina Quinases TOR/metabolismoRESUMO
Glutamate transport in early, undifferentiated oligodendrocytic precursors has not been characterized thus far. Here we show that human oligodendroglioma Hs683 cells are not endowed with EAAT-dependent anionic amino acid transport. However, in these cells, but not in U373 human glioblastoma cells, valproic acid (VPA), an inhibitor of histone deacetylases, markedly induces SLC1A1 mRNA, which encodes for the glutamate transporter EAAT3. The effect is detectable after 8h and persists up to 120h of treatment. EAAT3 protein increase becomes detectable after 24h of treatment and reaches its maximum after 72-96h, when it is eightfold more abundant than control. The initial influx of d-aspartate increases in parallel, exhibiting the typical features of an EAAT3-mediated process. SLC1A1 mRNA induction is associated with the increased expression of PDGFRA mRNA (+150%), a marker of early oligodendrocyte precursor cells, while the expression of GFAP, CNP and TUBB3 remains unchanged. Short term experiments have indicated that the VPA effect is shared by trichostatin A, another inhibitor of histone deacetylases. On the contrary, EAAT3 induction is neither prevented by inhibitors of mitogen-activated protein kinases nor triggered by a prolonged incubation with lithium, thus excluding a role for the GSK3ß/ß-catenin pathway. Thus, the VPA-dependent induction of the glutamate transporter EAAT3 in human oligodendroglioma cells likely occurs through an epigenetic mechanism and may represent an early indicator of commitment to oligodendrocytic differentiation.
Assuntos
Anticonvulsivantes/farmacologia , Transportador 3 de Aminoácido Excitatório/genética , Transportador 3 de Aminoácido Excitatório/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Ácido Valproico/farmacologia , 2',3'-Nucleotídeo Cíclico 3'-Fosfodiesterase/metabolismo , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Linhagem Celular Tumoral , Inibidores Enzimáticos/farmacologia , Proteína Glial Fibrilar Ácida/metabolismo , Humanos , Oligodendroglioma/patologia , Dibutirato de 12,13-Forbol/farmacologia , Transporte Proteico/efeitos dos fármacos , RNA Mensageiro/metabolismo , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/genética , Receptor alfa de Fator de Crescimento Derivado de Plaquetas/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de Tempo , Trítio/metabolismo , Tubulina (Proteína)/metabolismoRESUMO
Selected oncogenic mutations support unregulated growth enhancing glutamine availability but increasing the dependence of tumor cells on the amino acid. Data from literature indicate that a subset of HepatoCellular Carcinomas (HCC) is characterized by mutations of ß-catenin and overexpression of Glutamine Synthetase (GS). To assess if this phenotype may constitute an example of glutamine addiction, we treated four human HCC lines with the enzyme L-Asparaginase (ASNase), a glutaminolytic drug. ASNase had a significant antiproliferative effect only in the ß-catenin mutated HepG2 cells, which were partially rescued by the anaplerotic intermediates pyruvate and α-ketoglutarate. The enzyme severely depleted cell glutamine, caused eIF2α phosphorylation, inhibited mTOR activity, and increased autophagy in both HepG2 and in the ß-catenin wild type cell line Huh-7. When used with ASNase, the GS inhibitor methionine sulfoximine (MSO) emptied cell glutamine pool, arresting proliferation in ASNase-insensitive Huh-7 cells and activating caspase-3 and apoptosis in HepG2 cells. Compared with Huh-7 cells, HepG2 cells accumulated much higher levels of glutamine and MSO, due to the higher expression and activity of SNAT2, a concentrative transporter for neutral amino acids, but were much more sensitive to glutamine withdrawal from the medium. In the presence of ASNase, MSO caused a paradoxical maintenance of rapamycin-sensitive mTOR activity in both HepG2 and Huh-7 cells. ß-catenin silencing lowered ASNase sensitivity of HepG2 cells and of Huh-6 cells, another ß-catenin-mutated cell line, which also exhibited high sensitivity to ASNase. Thus, ß-catenin mutated HCC cells are more sensitive to glutamine depletion and accumulate higher levels of GS inhibitors. These results indicate that glutamine deprivation may constitute a targeted therapy for ß-catenin-mutated HCC cells addicted to the amino acid.
Assuntos
Antineoplásicos/farmacologia , Asparaginase/farmacologia , Carcinoma Hepatocelular/tratamento farmacológico , Inibidores Enzimáticos/farmacologia , Glutamato-Amônia Ligase/antagonistas & inibidores , Glutamina/metabolismo , beta Catenina/metabolismo , Antineoplásicos/metabolismo , Apoptose/efeitos dos fármacos , Asparaginase/metabolismo , Autofagia/efeitos dos fármacos , Carcinoma Hepatocelular/metabolismo , Carcinoma Hepatocelular/patologia , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Resistencia a Medicamentos Antineoplásicos , Fator de Iniciação 2B em Eucariotos/metabolismo , Inativação Gênica , Glutamato-Amônia Ligase/genética , Glutamato-Amônia Ligase/metabolismo , Humanos , Neoplasias Hepáticas/tratamento farmacológico , Neoplasias Hepáticas/metabolismo , Neoplasias Hepáticas/patologia , Terapia de Alvo Molecular , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Proteínas de Neoplasias/antagonistas & inibidores , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação/efeitos dos fármacos , Processamento de Proteína Pós-Traducional/efeitos dos fármacos , Proteínas Recombinantes/antagonistas & inibidores , Proteínas Recombinantes/metabolismo , beta Catenina/genéticaRESUMO
Excitatory amino acid carrier 1 (EAAC1) belongs to the family of the Na(+)-dependent glutamate carriers. Although the association between defective EAAC1 function and neurologic disease has been repeatedly studied, EAAC1 regulation is not yet fully understood. We have reported that in C6 glioma cells both the activity and membrane targeting of EAAC1 require the integrity of actin cytoskeleton. Here we show that, in the same model, EAAC1 partially co-localizes with actin filaments at the level of cell processes. Moreover, perinuclear spots in which EAAC1 co-localizes with the actin binding protein alpha-adducin are observed in some cells and, consistently, faint co-immunoprecipitation bands between EAAC1 and alpha-adducin are detected. Co-localization and partial co-immunoprecipitation of EAAC1 and adducin are still detectable after cell treatment with phorbol esters, a condition that leads to a protein kinase C (PKC)-dependent increase of EAAC1 expression on the membrane and to the phosphorylation of adducin. A co-immunoprecipitation band was also detected in protein extracts of rat hippocampus. The amount of adducin co-immunoprecipitated with EAAC1 increases after the treatment of C6 cells with retinoic acid, a differentiating agent that induces EAAC1 overexpression in this cell model. Moreover, in clones of C6 cells transfected with a hemagglutinin (HA)-tagged adducin, the bands of EAAC1 immunoprecipitated by an anti-HA antiserum were proportional to EAAC1 expression. These results suggest the existence of a pool of EAAC1 transporters associated with the actin binding protein alpha-adducin in a PKC-insensitive manner.
Assuntos
Proteínas de Ligação a Calmodulina/metabolismo , Proteínas do Citoesqueleto/metabolismo , Transportador 3 de Aminoácido Excitatório/metabolismo , Actinas/metabolismo , Animais , Linhagem Celular Tumoral , Hemaglutininas/metabolismo , Hipocampo/metabolismo , Imunoprecipitação , Fosforilação , Ligação Proteica , Ratos , Extratos de Tecidos/metabolismo , Tretinoína/farmacologiaRESUMO
Although carbon nanotubes (CNTs) are increasingly used, their biological effects are only incompletely characterized. However, experimental evidence suggests that the intratracheal instillation of CNTs causes the formation of interstitial granulomas and progressive pulmonary fibrosis in rodents. Using human epithelial Calu-3 cells as a model of airway epithelium in vitro, we have recently reported that the exposure to commercial multi-walled CNTs (MWCNTs) causes a progressive decrease of the transepithelial electrical resistance (TEER), pointing to a CNT-dependent impairment of the epithelial barrier function. To characterize better this behavior, we compared the effects of two types of MWCNTs and single-walled CNTs (SWCNTs) of different lengths on the TEER of Calu-3 monolayers. All the materials were used at a dose of 100 microg/mL corresponding to an exposure of 73 microg/cm(2) of cell monolayer. Only the longer MWCNTs and SWCNTs cause a significant decrease in TEER. To elucidate the mechanism underlying the changes in barrier function, the expression of the junction proteins occludin and ZO-1 has been also assessed. No significant decrease in the mRNA for either protein is detectable after the exposure to any type of CNTs. It is concluded that the impairment of barrier function in Calu-3 monolayers is a peculiar effect of CNTs endowed with clear cut fiber properties and is not referable to marked changes in the expression of junction proteins.
Assuntos
Brônquios/fisiopatologia , Nanotubos de Carbono , Sequência de Bases , Western Blotting , Brônquios/citologia , Linhagem Celular Tumoral , Primers do DNA , Células Epiteliais , Humanos , Técnicas In Vitro , Potenciais da Membrana , Reação em Cadeia da PolimeraseRESUMO
The transport of excitatory amino acids (EAA) in CNS is performed by a family of high affinity, sodium dependent carriers. One of these transporters, excitatory amino acid carrier 1 (EAAC1), is known to be regulated by several mechanisms that modify carrier abundance on the plasma membrane. Much less is known on EAAC1 regulation at the level of gene expression. Here we report that, in C6 rat glioma cells, a line recently described to contain neural stem-like cells, EAAC1 is markedly induced by all trans-retinoic acid (ATRA), a well known differentiating agent. Consistently, ATRA stimulates EAA transport, with the maximal effect observed at concentrations>or=1 microM. After 4 days of treatment with 10 microM ATRA, the transport Vmax is fivefold enhanced, Slc1a1 mRNA is increased by 400% compared with control, EAAC1 carrier is sixfold overexpressed and the C6 culture is greatly enriched of cells with bipolar morphology strongly positive for EAAC1 immunoreactivity. Compared with untreated cells, ATRA-treated C6 cells express less Slc1a3 mRNA, for the transporter GLAST, but significantly higher levels of Slc1a2 mRNA, for the transporter GLT-1, although no expression of either protein is detected with Western blot in both untreated and ATRA-treated cells. Consistently, the inhibition pattern of aspartate transport and its stimulation by phorbol esters are indicative of a transport process due to EAAC1 operation. Under the conditions adopted, ATRA treatment causes the induction of proteolipid protein, an oligodendrocytic marker. These results indicate that, in C6 cells, ATRA stimulates the expression of EAAC1, possibly as a step toward oligodendrocytic differentiation, and constitute the first demonstration of the induction of this transporter by a differentiating agent.
Assuntos
Antineoplásicos/farmacologia , Diferenciação Celular/efeitos dos fármacos , Transportador 3 de Aminoácido Excitatório/metabolismo , Regulação Neoplásica da Expressão Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Animais , Ácido Aspártico/metabolismo , Ácido Aspártico/farmacologia , Linhagem Celular Tumoral , Relação Dose-Resposta a Droga , Transportador 3 de Aminoácido Excitatório/antagonistas & inibidores , Aminoácidos Excitatórios/farmacologia , Glioma/patologia , Proteínas do Tecido Nervoso/metabolismo , RatosRESUMO
PURPOSE: To evaluate the activity of the antitumor enzyme L: -asparaginase (ASNase) on tumor cells of mesenchymal origin and the contribution of glutamine synthetase (GS) to the adaptation to the metabolic stress caused by the anti-tumor enzyme. METHODS: We studied the effects of ASNase in six human sarcoma cell lines: HT1080 (fibrosarcoma); RD (rhabdomyosarcoma); SW872 (liposarcoma); HOS, SAOS-2, and U2OS (osteosarcoma) in the absence or in the presence of the GS inhibitor methionine L: -sulfoximine (MSO). RESULTS: HT1080 and SW872 cells were highly sensitive to ASNase-dependent cytotoxicity. In contrast, RD, SAOS-2, HOS, and U2OS cells exhibited only a partial growth suppression upon treatment with the anti-tumor enzyme. In these cell lines ASNase treatment was associated with increased levels of GS. When ASNase was used together with MSO, the proliferation of the poorly sensitive cell lines was completely blocked and a significant decrease in the IC(50) for ASNase was observed. Moreover, when ASNase treatment was carried on in the presence of MSO, HOS and U2OS osteosarcoma cells exhibited a marked cytotoxicity, with increased apoptosis. CONCLUSIONS: In human sarcoma cells (1) GS markedly contributes to the metabolic adaptation of tumor cells to ASNase and (2) the inhibition of GS activity enhances the antiproliferative and cytotoxic effects of ASNase. The two-step interference with glutamine metabolism, obtained through the combined treatment with ASNase and MSO, may provide a novel therapeutic approach that should be further investigated in human tumors of mesenchymal origin.
Assuntos
Asparaginase/farmacologia , Glutamato-Amônia Ligase/antagonistas & inibidores , Sarcoma/tratamento farmacológico , Sarcoma/enzimologia , Antineoplásicos/farmacologia , Caspases/metabolismo , Relação Dose-Resposta a Droga , Ensaios de Seleção de Medicamentos Antitumorais , Glutamato-Amônia Ligase/metabolismo , Humanos , Metionina Sulfoximina/farmacologia , Células Tumorais CultivadasRESUMO
Sodium-dependent neutral amino acid transporter-2 (SNAT2), the ubiquitous member of SLC38 family, accounts for the activity of transport system A for neutral amino acids in most mammalian tissues. As the transport process performed by SNAT2 is highly energized, system A substrates, such as glutamine, glycine, proline and alanine, reach high transmembrane gradients and constitute major components of the intracellular amino acid pool. Moreover, through a complex array of exchange fluxes, involving other amino acid transporters, and of metabolic reactions, such as the synthesis of glutamate from glutamine, SNAT2 activity influences the cell content of most amino acids, thus determining the overall size and the composition of the intracellular amino acid pool. As amino acids represent a large fraction of cell organic osmolytes, changes of SNAT2 activity are followed by modifications in both cell amino acids and cell volume. This mechanism is utilized by many cell types to perform an effective regulatory volume increase (RVI) upon hypertonic exposure. Under these conditions, the expression of SNAT2 gene is induced and newly synthesized SNAT2 proteins are preferentially targeted to the cell membrane, leading to a significant increase of system A transport Vmax. In cultured human fibroblasts incubated under hypertonic conditions, the specific silencing of SNAT2 expression, obtained with anti-SNAT2 siRNAs, prevents the increase in system A transport activity, hinders the expansion of intracellular amino acid pool, and significantly delays cell volume recovery. These results demonstrate the pivotal role played by SNAT2 induction in the short-term hypertonic RVI and suggest that neutral amino acids behave as compatible osmolytes in hypertonically stressed cells.
Assuntos
Sistema A de Transporte de Aminoácidos/fisiologia , Aminoácidos Neutros/metabolismo , Adaptação Fisiológica , Transporte Biológico , Membrana Celular/metabolismo , Tamanho Celular , Células Cultivadas , Fibroblastos/metabolismo , Regulação da Expressão Gênica , Humanos , Soluções Hipertônicas , Interferência de RNA , RNA Interferente Pequeno , Equilíbrio HidroeletrolíticoRESUMO
A0, a Cu(II) thioxotriazole complex, produces severe cytotoxic effects on HT1080 human fibrosarcoma cells with a potency comparable to that exhibited by cisplatin. A0 induced a characteristic series of changes, hallmarked by the formation of eosin- and Sudan Black-B-negative vacuoles. No evidence of nuclear fragmentation or caspase-3 activation was detected in cells treated with A0 which, rather, inhibited cisplatin-stimulated caspase-3 activity. Membrane functional integrity, assessed with calcein and propidium iodide, was spared until the late stages of the death process induced by the copper complex. Vacuoles were negative to the autophagy marker monodansylcadaverine and their formation was not blocked by 3-methyladenine, an inhibitor of autophagic processes. Negativity to the extracellular marker pyranine excluded vacuole derivation from the extracellular fluid. Ultrastructural analysis indicated that A0 caused the appearance of many electronlight cytoplasmic vesicles, possibly related to the endoplasmic reticulum, which progressively enlarge and coalesce to form large vacuolar structures that eventually fill the cytoplasm. It is concluded that A0 triggers a non-apoptotic, type 3B programmed cell death (Clarke in Anat Embryol (Berl) 181:195-213, 1990), characterized by an extensive cytoplasmic vacuolization. This peculiar cytotoxicity pattern may render the employment of A0 to be of particular interest in apoptosis-resistant cell models.
Assuntos
Antineoplásicos/farmacologia , Autofagia , Fibrossarcoma/ultraestrutura , Compostos Organometálicos/farmacologia , Triazóis/farmacologia , Apoptose , Linhagem Celular Tumoral , Cisplatino/farmacologia , Humanos , Vacúolos/ultraestruturaRESUMO
We report here that chlorpromazine, a first generation antipsychotic drug, inhibits anionic amino acid transport mediated by system X(-) (AG) (EAAT transporters) in cultured human fibroblasts. With 30 microM chlorpromazine, transport inhibition is detectable after 3 h of treatment, maximal after 48 h (>60%), and referable to a decrease in V(max). Chlorpromazine effect is not dependent upon changes of membrane potential and is selective for system X(-) (AG) since transport systems A and y(+) are not affected. Among antipsychotic drugs, the inhibitory effect of chlorpromazine is shared by two dibenzodiazepines, clozapine and olanzapine, while other compounds, such as risperidon, zuclopentixol, sertindol and haloperidol, are not effective. Transport inhibition by clozapine and olanzapine, but not by chlorpromazine, is reversible, suggesting that the mechanisms involved are distinct. These results indicate that a subset of antipsychotic drugs inhibits EAAT transporters in non-nervous tissues and prompt further investigation on possible alterations of glutamate transport in peripheral tissues of schizophrenic patients.
Assuntos
Sistemas de Transporte de Aminoácidos Acídicos/antagonistas & inibidores , Aminoácidos/metabolismo , Antipsicóticos/farmacologia , Benzodiazepinas/farmacologia , Clorpromazina/farmacologia , Clozapina/farmacologia , Fibroblastos/efeitos dos fármacos , Transporte Biológico , Células Cultivadas , Fibroblastos/metabolismo , Humanos , OlanzapinaRESUMO
Engineered nanoparticles (NP) comprise various classes of technological materials with innovative properties. Although inhalation is less likely for engineered nanomaterials (NM) compared with ambient or mineral dust particles, this can happen during bulk manufacture and handling of freely dispersible NP. In this mini-review we summarize recent data on NP and CNT (carbon nanotubes) hazards, with particular emphasis on toxic effect on lung and in cell culture of lung origin. Owing to the highest deposition efficiency in the alveolar area, primary interactions of NM occur with epithelial and alveolar macrophages (AM). Scarce data are available to date on the cell mechanisms underlying NM permeability across the airway epithelium, but the absorption of NP through airways does not seem to require epithelial mediation, suggesting rather the involvement of alternative mechanisms such as AM-dependent dissemination. The relationship between toxicity and particle characteristics may be complex, involving size, surface area and surface chemistry. Some NM act according to an oxidative stress paradigm, but possible NM interactions with biological systems may result in additional forms of injury. In particular, CNT, a man-made forms of crystalline carbon, are currently attracting intense research efforts because of their unique properties, which make them suitable for many uses in biomedicine and pharmacology. Although CNT stimulate cytokine production and induce inflammatory reactions, they could behave also as conventional fibers, showing the ability to cause lung granulomas and fibrotic reactions in experimental animals. Production and marketing of NM is advancing much more rapidly than research on NM safety. This phenomenon will have a strong impact on the approach of occupational physicians to health risks from NP. In literature increasing evidence suggests that NM are potentially hazardous to humans and that strict industrial hygiene measures should be taken to limit exposure during their manipulation. Moreover, given the uncertainty about the NM features endowed with pathogenetic relevance, the toxicological properties of a specific NP should be evaluated on an individual basis by new screening strategies based on current acquisitions.
Assuntos
Pulmão/efeitos dos fármacos , Nanoestruturas/toxicidade , Saúde Ocupacional , Medição de Risco , Citocinas/biossíntese , Dano ao DNA , Humanos , Pulmão/metabolismo , Pulmão/patologia , Permeabilidade , Fibrose Pulmonar/induzido quimicamente , Espécies Reativas de Oxigênio/metabolismoRESUMO
When grown on permeable supports, pancreatic duct adenocarcinoma CAPAN-1 cells establish very high values of transepithelial resistance (TER). The addition of ethanol produced a dose-related, reversible drop in the TER of these cells, ranging from 15% (with 1% ethanol) to 65% (with 10% ethanol). The ethanol effect was rapid and reversible. The resistance decrease was associated with an increase in monolayer permeability to mannitol. No significant decrease in cell ATP was detected for ethanol concentrations lower than 7%. Confocal vertical sections of calcein-loaded monolayers of CAPAN-1 cells, grown on plasticware, showed a progressive deflation of domes detectable after 5 min of treatment with 2% ethanol. Incubation in an ethanol-free medium caused a progressive dome restoration. Immunocytochemical analysis of ethanol-treated cells indicated that ZO-1 and occludin exhibited clear cut distribution changes while the perijunctional actin pattern was slightly modified. Electron microscopy showed that a discrete intercellular space was detectable between adjacent ethanol-treated cells but not between control cells. These data indicate that ethanol is a tight junction barrier opener in pancreatic duct cells.
Assuntos
Carcinoma Ductal Pancreático/metabolismo , Etanol/farmacologia , Neoplasias Pancreáticas/metabolismo , Solventes/farmacologia , Junções Íntimas/metabolismo , Actinas/metabolismo , Carcinoma Ductal Pancreático/patologia , Carcinoma Ductal Pancreático/ultraestrutura , Linhagem Celular Tumoral , Humanos , Imuno-Histoquímica , Manitol/metabolismo , Proteínas de Membrana/metabolismo , Microscopia Eletrônica de Transmissão , Ocludina , Neoplasias Pancreáticas/patologia , Neoplasias Pancreáticas/ultraestrutura , Permeabilidade/efeitos dos fármacos , Fosfoproteínas/metabolismo , Junções Íntimas/efeitos dos fármacos , Proteína da Zônula de Oclusão-1RESUMO
Many epithelial cells cultured on plastic ware form domes, fluid-filled localized raisings of the cell monolayer. Domes are due to active vectorial ion transport and their presence demonstrates the maintenance of a differentiated polarized phenotype and of tight junctional complexes. Through a confocal laser microscope equipped with a special flow chamber, intact domes were evaluated in real time for prolonged experimental periods. Both in CAPAN-1 pancreatic duct adenocarcinoma cells and in renal tubular LLC-PK1 cells, vertical sections of calcein-loaded cultures provided a clear visualization of dome outlines during the slow deflation induced by specific agonists (respectively, 1 microM secretin or 10 microM vasopressin). Section series of calcein-loaded domes were used for three-dimensional reconstructions. In CAPAN-1 cultures, cell depolarization induced by secretin was detected with the potentiometric dye bis-oxonol. In the same cells pyranine, a fluid phase marker that is cell impermeant, visualized dome compartment and paracellular pathways, also providing an evaluation of dome fluid pH. Confocal laser scanning microscopy of domes represents a convenient device for the functional assessment of living epithelial cells.
Assuntos
Células Epiteliais/citologia , Microscopia Confocal/métodos , Sulfonatos de Arila/química , Linhagem Celular , Células Cultivadas , Células Epiteliais/fisiologia , Fluoresceínas/química , Corantes Fluorescentes/química , Potenciais da MembranaRESUMO
System A is a secondary active, sodium dependent transport system for neutral amino acids. Strictly coupled with Na,K-ATPase, its activity determines the size of the intracellular amino acid pool, through a complex network of metabolic reaction and exchange fluxes. Many hormones and drugs affect system A activity in specific cell models or tissues. In all the cell models tested thus far the activity of the system is stimulated by amino acid starvation, cell cycle progression, and the incubation under hypertonic conditions. These three conditions produce marked alterations of cell volume. The stimulation of system A activity plays an important role in cell volume restoration, through an expansion of the intracellular amino acid pool. Under normal conditions, system A substrates represent a major fraction of cell compatible osmolytes, organic compounds that exert a protein stabilizing effect. It is, therefore, likely that the activation of system A represents a portion of a more complex response triggered by exposure to stresses of various nature. Since system A transporters have been recently cloned, the molecular bases of these regulatory mechanisms will probably be elucidated in a short time.
Assuntos
Sistemas de Transporte de Aminoácidos/química , Aminoácidos/metabolismo , Sistemas de Transporte de Aminoácidos/metabolismo , Animais , Transporte Biológico , Membrana Celular/metabolismo , Humanos , Cinética , Modelos Biológicos , Osmose , Ligação Proteica , Isoformas de Proteínas , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The expression of the osmosensitive sodium/myo-inositol cotransporter (SMIT) is regulated by multiple tonicity-responsive enhancers (TonEs) in the 5'-flanking region of the gene. In response to hypertonicity, the nuclear abundance of the transcription factor TonE-binding protein (TonEBP) is increased, and the transcription of the SMIT gene is induced. Transport system A for neutral amino acids, another osmosensitive mechanism, is progressively stimulated if amino acid substrates are not present in the extracellular compartment. Under this condition, as in hypertonicity, cells shrink and mitogen-activated protein kinases are activated. We demonstrate here that a clear-cut nuclear redistribution of TonEBP, followed by SMIT expression increase and inositol transport activation, is observed after incubation of cultured human fibroblasts in Earle's balanced salts (EBSS), an isotonic, amino acid-free saline. EBSS-induced SMIT stimulation is prevented by substrates of system A, although these compounds do not compete with inositol for transport through SMIT. We conclude that the incubation in isotonic, amino acid-free saline triggers an osmotic stimulus and elicits TonEBP-dependent responses like hypertonic treatment.
Assuntos
Aminoácidos/farmacologia , Proteínas de Transporte/metabolismo , Proteínas de Choque Térmico/metabolismo , Proteínas de Membrana , Pele/citologia , Pele/metabolismo , Simportadores , Transativadores/metabolismo , Adolescente , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Núcleo Celular/metabolismo , Tamanho Celular/efeitos dos fármacos , Tamanho Celular/fisiologia , Células Cultivadas , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Inositol/metabolismo , Soluções Isotônicas/farmacologia , Masculino , Pressão Osmótica , Solução Salina Hipertônica/farmacologia , Sódio/metabolismo , Fatores de TranscriçãoRESUMO
The activity of transport system A for neutral amino acids is adaptively stimulated upon amino acid starvation. In cultured human fibroblasts this treatment causes an increase in the expression of the ATA2 system A transporter gene. ATA2 mRNA increase and transport stimulation are suppressed by system A substrates, but they are unaffected by other amino acids. Supplementation of amino acid-starved cells with substrates of system A causes a decrease in both ATA2 mRNA and system A transport activity. These results suggest a direct relationship between ATA2 expression and system A transport activity.
Assuntos
Proteínas de Transporte/biossíntese , Sistemas de Transporte de Aminoácidos , Northern Blotting , Linhagem Celular , Meios de Cultura/metabolismo , Fibroblastos/metabolismo , Humanos , Prolina/metabolismo , Transporte Proteico , RNA Mensageiro/metabolismo , Fatores de TempoRESUMO
In lysinuric protein intolerance (LPI), impaired transport of cationic amino acids in kidney and intestine is due to mutations of the SLC7A7 gene. To assess the functional consequences of the LPI defect in nonepithelial cells, we have characterized cationic amino acid (CAA) transport in human fibroblasts obtained from LPI patients and a normal subject. In both cell types the bidirectional fluxes of arginine are due to the additive contributions of two Na(+)-independent, transstimulated transport systems. One of these mechanisms, inhibited by N-ethylmaleimide (NEM) and sensitive to the membrane potential, is identifiable with system y(+). The NEM- and potential-insensitive component, suppressed by L-leucine only in the presence of Na(+), is mostly due to the activity of system y(+)L. The inward and outward activities of the two systems are comparable in control and LPI fibroblasts. Both cell types express SLC7A1 (CAT1) and SLC7A2 (CAT2B and CAT2A) as well as SLC7A6 (y+LAT2) and SLC7A7 (y+LAT1). We conclude that LPI fibroblasts exhibit normal CAA transport through system y(+)L, probably referable to the activity of SLC7A6/y+LAT2.
Assuntos
Diamino Aminoácidos/metabolismo , Proteínas de Transporte de Ânions , Antiporters , Arginina/farmacocinética , Proteínas de Transporte/genética , Proteínas de Transporte/metabolismo , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Aminoacidúrias Renais/metabolismo , Pele/citologia , Pele/metabolismo , Adolescente , Sistemas de Transporte de Aminoácidos Básicos , Transporte Biológico/efeitos dos fármacos , Transporte Biológico/fisiologia , Cátions , Células Cultivadas , Primers do DNA , Inibidores Enzimáticos/farmacologia , Etilmaleimida/farmacologia , Fibroblastos/citologia , Fibroblastos/metabolismo , Humanos , Leucina/farmacocinética , Óxido Nítrico/metabolismo , Fenótipo , Aminoacidúrias Renais/genética , Proteínas SLC4A , Sódio/farmacologiaRESUMO
The effects of secretin, the physiological secretagogue for pancreatic ducts, were studied in CAPAN-1 pancreatic duct carcinoma cells. When grown to confluence on plastic dishes, CAPAN-1 cells form domes and exhibit marked increases in culture content of Na+ and urea distribution space (UDS). This parameter is measured as an index of both intracellular and dome compartments under the conditions adopted. Both Na increase and dome formation are inhibited by long term incubation with phorbols, DIDS, DPC, EIPA, H2DIDS, and brefeldin. Short term treatment with secretin or 8-Br-cAMP/teophylline causes dome collapse and a marked decrease in UDS and culture content of Na. Secretin-induced sodium decrease is not abolished by ion channel inhibitors, suggesting that diffusion routes other than ion channels are involved in hormone effects. This hypothesis is also in agreement with data obtained on CAPAN-1 cells cultured on permeable inserts, where no change in Na content or UDS is detected upon secretin treatment. Confluent monolayers exhibit a high transepithelial resistance (Rms) which is markedly and reversibly decreased by secretin. The hormone also decreases the transepithelial voltage (Vms) and raises the monolayer permeability to mannitol. It is concluded that secretin enhances the paracellular permeability of pancreatic duct cells. This effect of secretin, unknown thus far, may be involved in the mechanism of pancreatic secretion in vivo.