RESUMO
Ouabain, an organic compound with the ability to strengthen the contraction of the heart muscle, was originally derived from plants. It has been observed that certain mammalian species, including humans, naturally produce ouabain, leading to its classification as a new type of hormone. When ouabain binds to Na+/K+-ATPase, it elicits various physiological effects, although these effects are not well characterized. Previous studies have demonstrated that ouabain, within the concentration range found naturally in the body (10 nmol/L), affects the polarity of epithelial cells and their intercellular contacts, such as tight junctions, adherens junctions, and gap junctional communication. This is achieved by activating signaling pathways involving cSrc and Erk1/2. To further investigate the effects of ouabain within the hormonally relevant concentration range (10 nmol/L), mRNA-seq, a high-throughput sequencing technique, was employed to identify differentially expressed transcripts. The discovery that the transcript encoding MYO9A was among the genes affected prompted an exploration of whether RhoA and its downstream effector ROCK were involved in the signaling pathways through which ouabain influences cell-to-cell contacts in epithelial cells. Supporting this hypothesis, this study reveals the following: (1) Ouabain increases the activation of RhoA. (2) Treatment with inhibitors of RhoA activation (Y27) and ROCK (C3) eliminates the enhancing effect of ouabain on the tight junction seal and intercellular communication via gap junctions. These findings further support the notion that ouabain acts as a hormone to emphasize the epithelial phenotype.
RESUMO
Ouabain is a cardiac glycoside, initially isolated from plants, and currently thought to be a hormone since some mammals synthesize it endogenously. It has been shown that in epithelial cells, it induces changes in properties and components related to apical-basolateral polarity and cell-cell contacts. In this work, we used a whole-cell patch clamp to test whether ouabain affects the properties of the voltage-gated potassium currents (Ik) of epithelial cells (MDCK). We found that: (1) in cells arranged as mature monolayers, ouabain induced changes in the properties of Ik; (2) it also accelerated the recovery of Ik in cells previously trypsinized and re-seeded at confluence; (3) in cell-cell contact-lacking cells, ouabain did not produce a significant change; (4) Na+/K+ ATPase might be the receptor that mediates the effect of ouabain on Ik; (5) the ouabain-induced changes in Ik required the synthesis of new nucleotides and proteins, as well as Golgi processing and exocytosis, as evidenced by treatment with drugs inhibiting those processes; and (5) the signaling cascade included the participation of cSrC, PI3K, Erk1/2, NF-κB and ß-catenin. These results reveal a new role for ouabain as a modulator of the expression of voltage-gated potassium channels, which require cells to be in contact with themselves.
Assuntos
Ouabaína , Canais de Potássio de Abertura Dependente da Tensão da Membrana , Animais , Ouabaína/farmacologia , Potássio/metabolismo , Canais de Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Células Epiteliais/metabolismo , Mamíferos/metabolismoRESUMO
Prostaglandins are a group of lipids that produce diverse physiological and pathological effects. Among them, prostaglandin E2 (PGE2) stands out for the wide variety of functions in which it participates. To date, there is little information about the influence of PGE2 on gap junctional intercellular communication (GJIC) in any type of tissue, including epithelia. In this work, we set out to determine whether PGE2 influences GJIC in epithelial cells (MDCK cells). To this end, we performed dye (Lucifer yellow) transfer assays to compare GJIC of MDCK cells treated with PGE2 and untreated cells. Our results indicated that (1) PGE2 induces a statistically significant increase in GJIC from 100 nM and from 15 min after its addition to the medium, (2) such effect does not require the synthesis of new mRNA or proteins subunits but rather trafficking of subunits already synthesized, and (3) such effect is mediated by the E2 receptor, which, in turn, triggers a signaling pathway that includes activation of adenylyl cyclase and protein kinase A (PKA). These results widen the knowledge regarding modulation of gap junctional intercellular communication by prostaglandins.
Assuntos
Comunicação Celular/efeitos dos fármacos , Dinoprostona/farmacologia , Células Epiteliais/efeitos dos fármacos , Junções Comunicantes/efeitos dos fármacos , Adenilil Ciclases/metabolismo , Animais , AMP Cíclico/metabolismo , Proteínas Quinases Dependentes de AMP Cíclico/metabolismo , Cães , Relação Dose-Resposta a Droga , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Junções Comunicantes/metabolismo , Células Madin Darby de Rim Canino , Receptores de Prostaglandina E Subtipo EP2/metabolismo , Transdução de Sinais/efeitos dos fármacos , Fatores de TempoRESUMO
Cannabidiol (CBD) has been used to treat a variety of cancers and inflammatory conditions with controversial results. In previous work, we have shown that breast cancer MCF-7 cells, selected by their response to inflammatory IL-1ß cytokine, acquire a malignant phenotype (6D cells) through an epithelial-mesenchymal transition (EMT). We evaluated CBD as a potential inhibitor of this transition and inducer of reversion to a non-invasive phenotype. It decreased 6D cell viability, downregulating expression of receptor CB1. The CBD blocked migration and progression of the IL-1ß-induced signaling pathway IL-1ß/IL-1RI/ß-catenin, the driver of EMT. Cannabidiol reestablished the epithelial organization lost by dispersion of the cells and re-localized E-cadherin and ß-catenin at the adherens junctions. It also prevented ß-catenin nuclear translocation and decreased over-expression of genes for ∆Np63α, BIRC3, and ID1 proteins, induced by IL-1ß for acquisition of malignant features. Cannabidiol inhibited the protein kinase B (AKT) activation, a crucial effector in the IL-1ß/IL-1RI/ß-catenin pathway, indicating that at this point there is crosstalk between IL-1ß and CBD signaling which results in phenotype reversion. Our 6D cell system allowed step-by-step analysis of the phenotype transition and better understanding of mechanisms by which CBD blocks and reverts the effects of inflammatory IL-1ß in the EMT.
Assuntos
Neoplasias da Mama/metabolismo , Canabidiol/farmacologia , Citocinas/metabolismo , Interleucina-1beta/metabolismo , Neoplasias da Mama/genética , Caderinas/metabolismo , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Regulação para Baixo , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Células MCF-7 , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Cicatrização , beta Catenina/metabolismoRESUMO
BACKGROUND/AIMS: Ouabain, a well-known plant-derived toxin, is also a hormone found in mammals at nanomolar levels that binds to a site located in the a-subunit of Naâº,Kâº-ATPase. Our main goal was to understand the physiological roles of ouabain. Previously, we found that ouabain increases the degree of tight junction sealing, GAP junction-mediated communication and ciliogenesis. Considering our previous results, we investigated the effect of ouabain on adherens junctions. METHODS: We used immunofluorescence and immunoblot methods to measure the effect of 10 nM ouabain on the cellular and nuclear content of E-cadherin, ß-catenin and γ-catenin in cultured monolayers of Marin Darby canine renal cells (MDCK). We also studied the effect of ouabain on adherens junction biogenesis through sequential Ca²âº removal and replenishment. Then, we investigated whether c-Src and ERK1/2 kinases are involved in these responses. RESULTS: Ouabain enhanced the cellular content of the adherens junction proteins E-cadherin, ß-catenin and γ-catenin and displaced ß-catenin and γ-catenin from the plasma membrane into the nucleus. Ouabain also increased the expression levels of E-cadherin and ß-catenin in the plasma membrane after Ca²âº replenishment. These effects on adherens junctions were sensitive to PP2 and PD98059, suggesting that they depend on c-Src and ERK1/2 signaling. The translocation of ß-catenin and γ-catenin into the nucleus was specific because ouabain did not change the localization of the tight junction proteins ZO-1 and ZO-2. Moreover, in ouabain-resistant MDCK cells, which express a Naâº,Kâº-ATPase α1-subunit with low affinity for ouabain, this hormone was unable to regulate adherens junctions, indicating that the ouabain receptor that regulates adherens junctions is Naâº,Kâº-ATPase. CONCLUSION: Ouabain (10 nM) upregulated adherens junctions. This novel result supports the proposition that one of the physiological roles of this hormone is the modulation of cell contacts.
Assuntos
Junções Aderentes/efeitos dos fármacos , Ouabaína/farmacologia , Junções Aderentes/metabolismo , Animais , Proteína Tirosina Quinase CSK , Caderinas/metabolismo , Cálcio/metabolismo , Núcleo Celular/metabolismo , Cães , Células Madin Darby de Rim Canino , Microscopia de Fluorescência , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transdução de Sinais/efeitos dos fármacos , ATPase Trocadora de Sódio-Potássio/metabolismo , beta Catenina/metabolismo , gama Catenina/metabolismo , Quinases da Família src/metabolismoRESUMO
BACKGROUND/AIMS: The fact that ouabain has been identified as an endogenous substance, led us to inquire its physiological role in epithelial cells. Based on previous observations, we hypothesized that it influences processes related to cell contacts. Previously we have shown that nanomolar concentrations of ouabain up-regulate tight junctions, accelerate ciliogenesis, and increase gap junctional intercellular communication (GJIC). Given that silencing assays indicated that connexin 43 (Cnx43) is involved in the GJIC response, in the present work we study whether ouabain affects Cnx43 expression and distribution. METHODS: We seeded confluent monolayers of epithelial renal MDCK cells and incubated them with 10 nM ouabain during 1 h. Then we measured, by densitometric analysis of Western blot assays, the amount of Cnx43 in cells and in fractions enriched of plasma membrane. We also studied its localization with immunofluorescence and confocal microscopy. RESULTS: Cnx43 is remarkably displayed, outlining the borders of cells gathered in clusters, randomly scattered throughout the monolayer. Ouabain increases the density of such clusters, as well as the average number of cells per cluster, without inducing the synthesis of new Cnx43. It also promotes relocation towards the membrane, of subunits already available. The fact that such changes are inhibited by PP2 and PD98059 indicates that a signaling pathway, that includes c-Src and ERK1/2, is involved in this response. CONCLUSION: Ouabain induces the translocation of Cnx43 from the cytoplasm to the plasma membrane. These findings support our hypothesis that one of the physiological roles of ouabain is the modulation of physiological processes that depend on cell to cell contacts.
Assuntos
Conexina 43/genética , Inibidores Enzimáticos/farmacologia , Junções Comunicantes/efeitos dos fármacos , Ouabaína/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Proteína Tirosina Quinase CSK , Comunicação Celular/efeitos dos fármacos , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Conexina 43/metabolismo , Cães , Flavonoides/farmacologia , Junções Comunicantes/metabolismo , Regulação da Expressão Gênica , Células Madin Darby de Rim Canino , Proteína Quinase 1 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 1 Ativada por Mitógeno/genética , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 3 Ativada por Mitógeno/genética , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Transporte Proteico , Pirimidinas/farmacologia , Transdução de Sinais , Junções Íntimas/metabolismo , Quinases da Família src/antagonistas & inibidores , Quinases da Família src/genética , Quinases da Família src/metabolismoRESUMO
In addition to being a very well-known ion pump, Na(+), K(+)-ATPase is a cell-cell adhesion molecule and the receptor of digitalis, which transduces regulatory signals for cell adhesion, growth, apoptosis, motility and differentiation. Prolonged ouabain (OUA) blockage of activity of Na(+), K(+)-ATPase leads to cell detachment from one another and from substrates. Here, we investigated the cellular mechanisms involved in tight junction (TJ) disassembly upon exposure to toxic levels of OUA (≥300 nM) in epithelial renal canine cells (MDCK). OUA induces a progressive decrease in the transepithelial electrical resistance (TER); inhibitors of the epidermal growth factor receptor (EGFR, PD153035), cSrc (SU6656 and PP2) and ERK1/2 kinases (PD98059) delay this decrease. We have determined that the TER decrease depends upon internalization and degradation of the TJs proteins claudin (CLDN) 2, CLDN-4, occludin (OCLN) and zonula occludens-1 (ZO-1). OUA-induced degradation of proteins is either sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition. In agreement with the protein degradation findings, OUA decreases the cellular content of ZO-1 and CLDN-2 mRNAs but surprisingly, increases the mRNA of CLDN-4 and OCLN. Changes in the mRNA levels are sensitive (CLDN-4, OCLN and ZO-1) or insensitive (CLDN-2) to ERK1/2 inhibition as well. Thus, toxic levels of OUA activate the EGFR-cSrc-ERK1/2 pathway to induce endocytosis, internalization and degradation of TJ proteins. We also observed decreases in the levels of CLDN-2 protein and mRNA, which were independent of the EGFR-cSrc-ERK1/2 pathway.
Assuntos
Endocitose/efeitos dos fármacos , Sistema de Sinalização das MAP Quinases/efeitos dos fármacos , Proteína Quinase 1 Ativada por Mitógeno/metabolismo , Proteína Quinase 3 Ativada por Mitógeno/metabolismo , Ouabaína/farmacologia , Proteólise/efeitos dos fármacos , Proteínas de Junções Íntimas/metabolismo , Animais , Células Cultivadas , Cães , Células Madin Darby de Rim CaninoRESUMO
BACKGROUND/AIMS: The finding that endogenous ouabain acts as a hormone prompted efforts to elucidate its physiological function. In previous studies, we have shown that 10 nM ouabain (i.e., a concentration within the physiological range) modulates cell-cell contacts such as tight junctions and apical/basolateral polarity. In this study, we examined whether 10 nM ouabain affects another important cell-cell feature: gap junction communication (GJC). METHODS: We employed two different approaches: 1) analysis of the cell-to-cell diffusion of neurobiotin injected into a particular MDCK cell (epithelial cells from dog kidneys) in a confluent monolayer by counting the number of neighboring cells reached by the probe and 2) measurement of the electrical capacitance. RESULTS: We found that 10 nM ouabain increase GJC by 475% within 1 hour. The Na+-K+-ATPase acts as a receptor of ouabain. In previous works we have shown that ouabain activates c-Src and ERK1/2 in 1 hour; in the present study we show that the inhibition of these proteins block the effect of ouabain on GJC. This increase in GJC does not require synthesis of new protein components, because the inhibitors cycloheximide and actinomycin D did not affect this phenomenon. Using silencing assays we also demonstrate that this ouabain-induced enhancement of GJC involves connexins 32 and 43. CONCLUSION: Ouabain 10 nM increases GJC in MDCK cells.
Assuntos
Comunicação Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Junções Comunicantes/efeitos dos fármacos , Ouabaína/administração & dosagem , Animais , Cães , Células Epiteliais/efeitos dos fármacos , Células Madin Darby de Rim CaninoRESUMO
The exchange of substances between higher organisms and the environment occurs across transporting epithelia whose basic features are tight junctions (TJs) that seal the intercellular space, and polarity, which enables cells to transport substances vectorially. In a previous study, we demonstrated that 10 nM ouabain modulates TJs, and we now show that it controls polarity as well. We gauge polarity through the development of a cilium at the apical domain of Madin-Darby canine kidney cells (MDCK, epithelial dog kidney). Ouabain accelerates ciliogenesis in an ERK1/2-dependent manner. Claudin-2, a molecule responsible for the Na(+) and H(2)O permeability of the TJs, is also present at the cilium, as it colocalizes and coprecipitates with acetylated α-tubulin. Ouabain modulates claudin-2 localization at the cilium through ERK1/2. Comparing wild-type and ouabain-resistant MDCK cells, we show that ouabain acts through Na(+),K(+)-ATPase. Taken together, our previous and present results support the possibility that ouabain constitutes a hormone that modulates the transporting epithelial phenotype, thereby playing a crucial role in metazoan life.
Assuntos
Cílios/metabolismo , Células Epiteliais/metabolismo , Ouabaína/química , Animais , Caderinas/metabolismo , Adesão Celular , Comunicação Celular , Linhagem Celular , Proliferação de Células , Claudinas/metabolismo , Cães , Imunoprecipitação , Espectroscopia de Ressonância Magnética/métodos , Ouabaína/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Esteroides/metabolismo , Junções Íntimas , Fatores de TempoRESUMO
Epithelial cells treated with high concentrations of ouabain (e.g., 1 microM) retrieve molecules involved in cell contacts from the plasma membrane and detach from one another and their substrates. On the basis of this observation, we suggested that ouabain might also modulate cell contacts at low, nontoxic levels (10 or 50 nM). To test this possibility, we analyzed its effect on a particular type of cell-cell contact: the tight junction (TJ). We demonstrate that at concentrations that neither inhibit K(+) pumping nor disturb the K(+) balance of the cell, ouabain modulates the degree of sealing of the TJ as measured by transepithelial electrical resistance (TER) and the flux of neutral 3 kDa dextran (J(DEX)). This modulation is accompanied by changes in the levels and distribution patterns of claudins 1, 2, and 4. Interestingly, changes in TER, J(DEX), and claudins behavior are mediated through signal pathways containing ERK1/2 and c-Src, which have distinct effects on each physiological parameter and claudin type. These observations support the theory that at low concentrations, ouabain acts as a modulator of cell-cell contacts.
Assuntos
Células Epiteliais/efeitos dos fármacos , Ouabaína/farmacologia , Junções Íntimas/efeitos dos fármacos , Animais , Proteína Tirosina Quinase CSK , Dextranos/química , Cães , Relação Dose-Resposta a Droga , Inibidores Enzimáticos/farmacologia , Células Epiteliais/citologia , MAP Quinases Reguladas por Sinal Extracelular/metabolismo , Íons , Modelos Biológicos , Potássio/química , Proteínas Tirosina Quinases/metabolismo , Transdução de Sinais , ATPase Trocadora de Sódio-Potássio/metabolismo , Quinases da Família srcRESUMO
Although nanotechnologies are increasingly present in numerous sectors of the economy, training resources available to workers exposed to them are still rare. In the European Union (EU), some initiatives exist that inform workers about exposure and risks, but they lack two key dimensions: the involvement of workers themselves in designing and implementing training materials and the key role played by safety representatives in improving occupational health and safety in EU member states. Making workers actors of their own training, rather than recipients of it, and empowering them, so that they can collectively question unsafe situations and ask for changes in their working conditions, is how training can positively impact their health and safety. This article describes a training package (materials, infographics, interactive web applet) designed specifically to achieve this objective. Developed under the NanoDiode project, it focuses on six key themes: types of nanomaterials, uses at work, risks, presence in the workplace, exposure, and experience sharing.
Assuntos
Capacitação em Serviço/organização & administração , Indústria Manufatureira/organização & administração , Nanoestruturas , Exposição Ocupacional/prevenção & controle , Gestão da Segurança/organização & administração , Humanos , Indústria Manufatureira/normas , Doenças Profissionais/prevenção & controle , Saúde Ocupacional , Cultura Organizacional , Gestão da Segurança/normas , Engajamento no Trabalho , Local de Trabalho/organização & administraçãoRESUMO
Cardiac glycosides are a group of compounds widely known for their action in cardiac tissue, some of which have been found to be endogenously produced (ECG). We have previously studied the effect of ouabain, an endogenous cardiac glycoside, on the physiology of epithelial cells, and we have shown that in concentrations in the nanomolar range, it affects key properties of epithelial cells, such as tight junction, apical basolateral polarization, gap junctional intercellular communication (GJIC), and adherent junctions. In this work, we study the influence of digoxin and marinobufagenin, two other endogenously expressed cardiac glycosides, on GJIC as well as the degree of transepithelial tightness due to tight junction integrity (TJ). We evaluated GJIC by dye transfer assays and tight junction integrity by transepithelial electrical resistance (TER) measurements, as well as immunohistochemistry and western blot assays of expression of claudins 2 and 4. We found that both digoxin and marinobufagenin improve GJIC and significantly enhance the tightness of the tight junctions, as evaluated from TER measurements. Immunofluorescence assays show that both compounds promote enhanced basolateral localization of claudin-4 but not claudin 2, while densitometric analysis of western blot assays indicate a significantly increased expression of claudin 4. These changes, induced by digoxin and marinobufagenin on GJIC and TER, were not observed on MDCK-R, a modified MDCK cell line that has a genetically induced insensitive α1 subunit, indicating that Na-K-ATPase acts as a receptor mediating the actions of both ECG. Plus, the fact that the effect of both cardiac glycosides was suppressed by incubation with PP2, an inhibitor of c-Src kinase, PD98059, an inhibitor of mitogen extracellular kinase-1 and Y-27632, a selective inhibitor of ROCK, and a Rho-associated protein kinase, indicate altogether that the signaling pathways involved include c-Src and ERK1/2, as well as Rho-ROCK. These results widen and strengthen our general hypothesis that a very important physiological role of ECG is the control of the epithelial phenotype and the regulation of cell-cell contacts.
RESUMO
HEK293 cells are widely used as a host for expression of heterologous proteins; yet, little care has been taken to characterize their endogenous membrane components, including ion channels. In this work, we aimed to describe the biophysical and pharmacological properties of endogenous, voltage-dependent potassium currents (IKv). We also examined how its expression depends on culture conditions. We used the electrophysiological technique of whole-cell patch clamp to record ion currents from HEK293 cells. We found that HEK cells express endogenous, voltage-dependent potassium currents. We also found that diverse culture conditions, such as the passage number, the cell density, the type of serum that complements the culture media and the substratum, affect the magnitude and shape of IKv, resulting from the relative contribution of fast, slow, and noninactivating component currents. Incubation of cells in mature monolayers with trypsin-EDTA, notoriously reduces the magnitude and modifies the shape of voltage-dependent potassium endogenous currents; nonetheless HEK cells recover IKv's magnitude and shape within 6 h after replating, with a process that requires synthesis of new mRNA and protein subunits, as evidenced by the fact that actinomycin D and cycloheximide, inhibitors of synthesis of mRNA and protein, respectively, impair the recovery of IKv after trypsinization. In addition to be useful as a model expression system, HEK293 may be useful to understand how cells regulate the density of ion channels on the membrane.
Assuntos
Técnicas de Cultura de Células , Canais de Potássio de Abertura Dependente da Tensão da Membrana/metabolismo , Meios de Cultura , Células HEK293 , Humanos , Ativação do Canal Iônico/fisiologia , Técnicas de Patch-Clamp , Canais de Potássio de Abertura Dependente da Tensão da Membrana/genéticaRESUMO
Inflammation and oxidative stress play major roles in endothelial dysfunction, and are key factors in the progression of cardiovascular diseases. The aim of this study was to evaluate in vitro the effect of three subfractions (SFs) from the Cucumis sativus aqueous fraction to reduce inflammatory factors and oxidative stress induced by angiotensin II (Ang II) in human microvascular endothelial cells-1 (HMEC-1) cells. The cells were cultured with different concentrations of Ang II and 0.08 or 10 µg/mL of SF1, SF2, or SF3, or 10 µmol of losartan as a control. IL-6 (Interleukin 6) concentration was quantified. To identify the most effective SF combinations, HMEC-1 cells were cultured as described above in the presence of four combinations of SF1 and SF3. Then, the effects of the most effective combination on the expression of adhesion molecules, the production of reactive oxygen species (ROS), and the bioavailability of nitric oxide (NO) were evaluated. Finally, a mass spectrometry analysis was performed. Both SF1 and SF3 subfractions decreased the induction of IL-6 by Ang II, and C4 (SF1 and SF3, 10 µg/mL each) was the most effective combination to inhibit the production of IL-6. Additionally, C4 prevented the expression of adhesion molecules, reduced the production of ROS, and increased the bioavailability of NO. Glycine, arginine, asparagine, lysine, and aspartic acid were the main components of both subfractions. These results demonstrate that C4 has anti-inflammatory and antioxidant effects.
Assuntos
Aminoácidos/farmacologia , Angiotensina II/toxicidade , Anti-Inflamatórios/farmacologia , Antioxidantes/farmacologia , Cucumis sativus , Células Endoteliais/efeitos dos fármacos , Inflamação/prevenção & controle , Estresse Oxidativo/efeitos dos fármacos , Extratos Vegetais/farmacologia , Aminoácidos/isolamento & purificação , Anti-Inflamatórios/isolamento & purificação , Antioxidantes/isolamento & purificação , Moléculas de Adesão Celular/metabolismo , Linhagem Celular , Cucumis sativus/química , Relação Dose-Resposta a Droga , Células Endoteliais/metabolismo , Humanos , Inflamação/induzido quimicamente , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Interleucina-6/metabolismo , Óxido Nítrico/metabolismo , Fitoterapia , Extratos Vegetais/isolamento & purificação , Plantas Medicinais , Espécies Reativas de Oxigênio/metabolismoRESUMO
This paper introduces Teslaphoresis, the directed motion and self-assembly of matter by a Tesla coil, and studies this electrokinetic phenomenon using single-walled carbon nanotubes (CNTs). Conventional directed self-assembly of matter using electric fields has been restricted to small scale structures, but with Teslaphoresis, we exceed this limitation by using the Tesla coil's antenna to create a gradient high-voltage force field that projects into free space. CNTs placed within the Teslaphoretic (TEP) field polarize and self-assemble into wires that span from the nanoscale to the macroscale, the longest thus far being 15 cm. We show that the TEP field not only directs the self-assembly of long nanotube wires at remote distances (>30 cm) but can also wirelessly power nanotube-based LED circuits. Furthermore, individualized CNTs self-organize to form long parallel arrays with high fidelity alignment to the TEP field. Thus, Teslaphoresis is effective for directed self-assembly from the bottom-up to the macroscale.
RESUMO
BACKGROUND: Interleukin 1 beta (IL-1beta) and other inflammatory cytokines are reported to induce phenotypic changes in epithelial breast cancer tumor cells related to increased invasiveness. Mechanisms involved in the process are not well understood. METHODS: The noninvasive breast cancer epithelial cell line MCF-7 was used to investigate the IL-1beta-induced phenotype. Live cells expressing EGFP-actin were monitored for cell morphology changes and actin cytoskeleton dynamics by time-lapse video microscopy in the presence of IL-1beta and specific inhibitors of actin signaling pathways. Chemotaxis, invasion of Matrigel, MMP activity and expression of S100A4 in cells treated with IL-1beta were assessed by migration assays, zymograms and immunoblots. RESULTS: Exposure to IL-1beta specifically induced a change in MCF-7 cells from a typical epithelial morphology into elongated cells, showing numerous dynamic actin-rich lamellae and peripheral ruffles characteristic of fibroblasts. These cells could scatter from compact cell colonies and respond to chemoattractants such as the homing-associated chemokine CXCL-12. Pharmacological blockage of actin signaling pathways and negative mutants of RhoGTPases revealed that actin reorganization and enhanced motility are regulated via PI3K/Rac 1 activation. IL-1beta-stimulated cells expressed the metastasis promoter S100A4, increased secretion of active MMP-9 and MMP-2 and invasion of extracellular matrix proteins. CONCLUSIONS: IL-1beta induces a PI3K/Rac 1-regulated reorganization of the actin cytoskeleton of MCF-7 cells that is required for cell scattering, elongation and migration. The enhanced motility is accompanied by expression of protein markers correlated with invasive behavior.
Assuntos
Actinas/metabolismo , Neoplasias da Mama/metabolismo , Neoplasias da Mama/patologia , Interleucina-1beta/farmacologia , Linhagem Celular Tumoral , Movimento Celular/efeitos dos fármacos , Forma Celular/efeitos dos fármacos , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/metabolismo , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Células Epiteliais/patologia , Feminino , Humanos , Interleucina-1beta/metabolismo , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Mesoderma/patologia , Microscopia de Vídeo , Invasividade Neoplásica/patologia , Fenótipo , Fosfatidilinositol 3-Quinases/metabolismo , Receptores CXCR4/metabolismo , Receptores Tipo I de Interleucina-1/metabolismo , Proteínas Recombinantes/farmacologia , Transdução de Sinais , Proteínas rac1 de Ligação ao GTP/metabolismoRESUMO
Permeability alterations of microvascular endothelia may be a factor in the plasma leakage produced by dengue virus infection. Confluent monolayers of the human dermal microvascular endothelial cell line HMEC-1 were utilized as an experimental model to study the cellular responses induced by the virus. Infected monolayers showed increased permeability for [(3)H]mannitol, but no changes were observed for 4-70 kDa dextrans at 48 h post-infection (p.i.), a time at which viral titres reached maximal values and 40 % of the cells expressed viral proteins. A further increase in permeability occurred at 72 h, still without evident cytopathic effects on the monolayer. Coinciding with this, actin was reorganized in the infected cells and the tight junction protein occludin was displaced to the cytoplasm. Increments in the thickness of stress fibres and focal adhesions were observed in uninfected cells neighbouring infected cells. Culture medium from infected monolayers induced permeability changes and thickening of actin-containing structures in control cultures that resembled those observed 48 h p.i. Interleukin (IL) 8 was found in culture medium at concentrations ranging from 20 to 100 pg ml(-1). Neutralizing antibodies against IL8 partially inhibited the changes produced by the culture medium as well as those induced by addition of IL8. Genistein inhibited the effect of the culture medium and the phosphorylation of proteins associated with focal adhesions and indicated the participation of tyrosine kinases. These findings suggest that IL8 production by infected monolayers contributes to the virus-induced effect on the cytoskeleton and tight junctions and thereby modifies transendothelial permeability.
Assuntos
Permeabilidade da Membrana Celular/fisiologia , Citoesqueleto/fisiologia , Vírus da Dengue/fisiologia , Dengue/fisiopatologia , Endotélio Vascular/virologia , Interleucina-8/metabolismo , Microcirculação/virologia , Junções Íntimas/fisiologia , Linhagem Celular , Permeabilidade da Membrana Celular/efeitos dos fármacos , Meios de Cultura , Citoesqueleto/efeitos dos fármacos , Citoesqueleto/ultraestrutura , Endotélio Vascular/imunologia , Endotélio Vascular/fisiologia , Humanos , Interleucina-8/farmacologia , Fosforilação , Proteínas Recombinantes/farmacologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/ultraestrutura , Proteínas Virais/análiseRESUMO
Membrane-cytoskeleton interactions have been shown to be crucial to modulate polarity, cell shape and the paracellular pathway in epithelial MDCK cell monolayers. In particular, actin organization and myosin-dependent contractility play an important role in the regulation of these functions. Participation of myosin in vectorial transport, expressed as formation of domes, was investigated in confluent monolayers of high transepithelial electrical resistance (TER) plated on non-permeable supports. Cells exposed to 2,3-butanedione monoxime, a selective inhibitor of myosin ATPase, showed a remarkable increase in the number of domes. Replacement of extracellular Na+ and Cl- and inhibition of Na+-K+-ATPase blocked the induction of domes. The monoxime also caused a reduction of the TER leading to an increase in the paracellular flux of small molecular weight dextran. However, immunofluorescence microscopy of drug-treated cells showed that the localization and staining pattern of tight junction proteins ZO-1, occludin, and claudin 1, or the actin-myosin ring at the zonula adherens, were not modified. Treatment with the drug produced striking re-arrangements of actin filaments at the microvilli and at the basal level of the cells. Our data show that disruption of actin-myosin interaction at several cellular sites contributed importantly to the increased transport activity and the formation of the domes. These results point to the relevant role or actin-myosin dynamics and actin organization in the regulation of ion and water channel activity in these cells.
Assuntos
Actinas/metabolismo , Transporte Biológico Ativo/fisiologia , Diacetil/análogos & derivados , Diacetil/farmacologia , Inibidores Enzimáticos/farmacologia , Células Epiteliais/metabolismo , Miosinas/metabolismo , Equilíbrio Hidroeletrolítico/fisiologia , Actinas/antagonistas & inibidores , Junções Aderentes/efeitos dos fármacos , Junções Aderentes/metabolismo , Animais , Transporte Biológico Ativo/efeitos dos fármacos , Adesão Celular/efeitos dos fármacos , Adesão Celular/fisiologia , Agregação Celular/efeitos dos fármacos , Agregação Celular/fisiologia , Membrana Celular/efeitos dos fármacos , Membrana Celular/metabolismo , Células Cultivadas , Cloro/deficiência , Cães , Impedância Elétrica , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/ultraestrutura , Canais Iônicos/efeitos dos fármacos , Canais Iônicos/metabolismo , Miosinas/antagonistas & inibidores , Sódio/deficiência , ATPase Trocadora de Sódio-Potássio/antagonistas & inibidores , ATPase Trocadora de Sódio-Potássio/metabolismo , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/metabolismo , Equilíbrio Hidroeletrolítico/efeitos dos fármacosRESUMO
BACKGROUND: Melatonin, newly synthesized by the pineal gland, is rapidly released to general circulation reaching a nanomolar concentration. Cyclic production of melatonin synchronizes body rhythms with the photoperiod. Moreover, changes in urine production and osmolarity have been observed in the kidney during the night. However, the precise mechanisms by which plasma-circulating melatonin modifies renal physiology are not clearly understood. METHODS: Madin-Darby canine kidney (MDCK) cell monolayers transport water vectorially from the apical to the basolateral side forming blisters or domes. Transport in epithelial cells is regulated by tight junction sealing, ion pumps and channels, and cytoskeleton organization, among other processes. MDCK cells were used to study vectorial water transport to determine the role of microfilament organization and protein kinase C (PKC) in dome formation in culture conditions that mimic the cyclic pattern of melatonin circulation in plasma. RESULTS: Melatonin cyclically increased dome formation by 50% and caused enlargement and thickening of stress fibers in cells surrounding the domes. Optimal increase in dome formation was observed at nanomolar concentrations of melatonin after 6 hours, concomitantly with a 28% decrease in the transepithelial electrical resistance, which remained low for up to 12 hours, without apparent change in fluorescein isothiocyanate (FITC)-dextran flux. A blockage in dome formation elicited by melatonin was observed in monolayers preincubated with the Na+-K+-ATPase or PKC inhibitors. CONCLUSION: The results obtained indicate that melatonin cyclically modifies the transepithelial permeability in kidney-derived cells through PKC activation and microfilament reorganization, and supports the hypothesis that melatonin may synchronize daily body rhythms through cyclic cytoskeletal rearrangements.
Assuntos
Antioxidantes/farmacologia , Rim/citologia , Melatonina/farmacologia , Equilíbrio Hidroeletrolítico/efeitos dos fármacos , Actinas/metabolismo , Animais , Linhagem Celular , Ritmo Circadiano/fisiologia , Relação Dose-Resposta a Droga , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Proteína Quinase C/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Fibras de Estresse/metabolismo , Água/metabolismoRESUMO
Se estandarizó un ELISA para la detección de anticuerpos monoclonales a las proteínas E y NS1 del virus dengue. Se aplicó un ELISA indirecto utilizando como fuente de antígeno células C6/36 inoculadas con la cepa A-15 aislada durante la epidemia de dengue 2 en 1981. Estas células fueron fijadas en placas ELISA a una concentración de 200 000 células/pozo. Se utilizó un control celular en condiciones similares. Para normalizar el sistema se emplearon anticuerpos monoclonales específicos a ambas proteínas. Se realizaron estudios a diferentes tiempos de incubación para determinar el momento de mayor expresión de estas proteínas en la membrana celular. Los resultados muestran una respuesta máxima a las 72 horas posinoculación para ambas proteínas, se obtuvo una sensibilidad para la detección de NS1 de 14,7 ng/mL y para la E de 1,43 ng/mL. Este sistema permite el tamizaje primario de anticuerpos monoclonales a las proteínas E y NS1 del virus dengue 2.