RESUMO
Articular chondrocytes are the primary cells responsible for maintaining the integrity and functionality of articular cartilage, which is essential for smooth joint movement. A key aspect of their role involves mechanosensitive ion channels, which allow chondrocytes to detect and respond to mechanical forces encountered during joint activity; nonetheless, the variety of mechanosensitive ion channels involved in this process has not been fully resolved so far. Because some members of the two-pore domain potassium (K2P) channel family have been described as mechanosensors in other cell types, in this study, we investigate whether articular chondrocytes express such channels. RT-PCR analysis reveals the presence of TREK-1 and TREK-2 channels in these cells. Subsequent protein expression assessments, including Western blotting and immunohistochemistry, confirm the presence of TREK-1 in articular cartilage samples. Furthermore, whole-cell patch clamp assays demonstrate that freshly isolated chondrocytes exhibit currents attributable to TREK-1 channels, as evidenced by activation by arachidonic acid (AA) and ml335 and further inhibition by spadin. Additionally, exposure to hypo-osmolar shock activates currents, which can be attributed to the presence of TREK-1 channels, as indicated by their inhibition with spadin. Therefore, these findings highlight the expression of TREK channels in rat articular chondrocytes and suggest their potential involvement in regulating the integrity of cartilage extracellular matrix.
Assuntos
Cartilagem Articular , Condrócitos , Canais de Potássio de Domínios Poros em Tandem , Animais , Condrócitos/metabolismo , Canais de Potássio de Domínios Poros em Tandem/metabolismo , Canais de Potássio de Domínios Poros em Tandem/genética , Cartilagem Articular/metabolismo , Cartilagem Articular/citologia , Ratos , Células Cultivadas , Masculino , Mecanotransdução Celular , Técnicas de Patch-ClampRESUMO
The sodium pump, or Na+/K+-ATPase (NKA), is an essential enzyme found in the plasma membrane of all animal cells. Its primary role is to transport sodium (Na+) and potassium (K+) ions across the cell membrane, using energy from ATP hydrolysis. This transport creates and maintains an electrochemical gradient, which is crucial for various cellular processes, including cell volume regulation, electrical excitability, and secondary active transport. Although the role of NKA as a pump was discovered and demonstrated several decades ago, it remains the subject of intense research. Current studies aim to delve deeper into several aspects of this molecular entity, such as describing its structure and mode of operation in atomic detail, understanding its molecular and functional diversity, and examining the consequences of its malfunction due to structural alterations. Additionally, researchers are investigating the effects of various substances that amplify or decrease its pumping activity. Beyond its role as a pump, growing evidence indicates that in various cell types, NKA also functions as a receptor for cardiac glycosides like ouabain. This receptor activity triggers the activation of various signaling pathways, producing significant morphological and physiological effects. In this report, we present the results of a comprehensive review of the most outstanding studies of the past five years. We highlight the progress made regarding this new concept of NKA and the various cardiac glycosides that influence it. Furthermore, we emphasize NKA's role in epithelial physiology, particularly its function as a receptor for cardiac glycosides that trigger intracellular signals regulating cell-cell contacts, proliferation, differentiation, and adhesion. We also analyze the role of NKA ß-subunits as cell adhesion molecules in glia and epithelial cells.
Assuntos
ATPase Trocadora de Sódio-Potássio , ATPase Trocadora de Sódio-Potássio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , Animais , Humanos , Membrana Celular/metabolismo , Transdução de Sinais , Ouabaína/farmacologia , Ouabaína/metabolismo , Glicosídeos Cardíacos/metabolismo , Glicosídeos Cardíacos/farmacologia , Sódio/metabolismoRESUMO
Ouabain, a substance originally obtained from plants, is now classified as a hormone because it is produced endogenously in certain animals, including humans. However, its precise effects on the body remain largely unknown. Previous studies have shown that ouabain can influence the phenotype of epithelial cells by affecting the expression of cell-cell molecular components and voltage-gated potassium channels. In this study, we conducted whole-cell clamp assays to determine whether ouabain affects the activity and/or expression of TRPV4 channels. Our findings indicate that ouabain has a statistically significant effect on the density of TRPV4 currents (dITRPV4), with an EC50 of 1.89 nM. Regarding treatment duration, dITRPV4 reaches its peak at around 1 h, followed by a subsequent decline and then a resurgence after 6 h, suggesting a short-term modulatory effect related to on TRPV4 channel activity and a long-term effect related to the promotion of synthesis of new TRPV4 channel units. The enhancement of dITRPV4 induced by ouabain was significantly lower in cells seeded at low density than in cells in a confluent monolayer, indicating that the action of ouabain depends on intercellular contacts. Furthermore, the fact that U73122 and neomycin suppress the effect caused by ouabain in the short term suggests that the short-term induced enhancement of dITRPV4 is due to the depletion of PIP2 stores. In contrast, the fact that the long-term effect is inhibited by PP2, wortmannin, PD, FR18, and IKK16 suggests that cSrc, PI3K, Erk1/2, and NF-kB are among the components included in the signaling pathways.
Assuntos
Ouabaína , Canais de Cátion TRPV , Humanos , Animais , Ouabaína/farmacologia , Canais de Cátion TRPV/genética , Canais de Cátion TRPV/metabolismo , Transdução de Sinais , Células Epiteliais/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
The ß2 subunit of Na+, K+-ATPase was originally identified as the adhesion molecule on glia (AMOG) that mediates the adhesion of astrocytes to neurons in the central nervous system and that is implicated in the regulation of neurite outgrowth and neuronal migration. While ß1 isoform have been shown to trans-interact in a species-specific mode with the ß1 subunit on the epithelial neighboring cell, the ß2 subunit has been shown to act as a recognition molecule on the glia. Nevertheless, none of the works have identified the binding partner of ß2 or described its adhesion mechanism. Until now, the interactions pronounced for ß2/AMOG are heterophilic cis-interactions. In the present report we designed experiments that would clarify whether ß2 is a cell-cell homophilic adhesion molecule. For this purpose, we performed protein docking analysis, cell-cell aggregation, and protein-protein interaction assays. We observed that the glycosylated extracellular domain of ß2/AMOG can make an energetically stable trans-interacting dimer. We show that CHO (Chinese Hamster Ovary) fibroblasts transfected with the human ß2 subunit become more adhesive and make large aggregates. The treatment with Tunicamycin in vivo reduced cell aggregation, suggesting the participation of N-glycans in that process. Protein-protein interaction assay in vivo with MDCK (Madin-Darby canine kidney) or CHO cells expressing a recombinant ß2 subunit show that the ß2 subunits on the cell surface of the transfected cell lines interact with each other. Overall, our results suggest that the human ß2 subunit can form trans-dimers between neighboring cells when expressed in non-astrocytic cells, such as fibroblasts (CHO) and epithelial cells (MDCK).
Assuntos
Adenosina Trifosfatases/metabolismo , Proteínas de Transporte de Cátions/metabolismo , Moléculas de Adesão Celular Neuronais/metabolismo , Moléculas de Adesão Celular , ATPase Trocadora de Sódio-Potássio , Animais , Células CHO , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Cricetinae , Cricetulus , Cães , Humanos , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismoRESUMO
Tight junctions (TJs) are cellcell adhesion structures frequently altered by oncogenic transformation. In the present study the role of human papillomavirus (HPV) 16 E7 oncoprotein on the sealing of TJs was investigated and also the expression level of claudins in mouse cervix and in epithelial MadinDarby Canine Kidney (MDCK) cells. It was found that there was reduced expression of claudins 1 and 10 in the cervix of 7monthold transgenic K14E7 mice treated with 17ßestradiol (E2), with invasive cancer. In addition, there was also a transient increase in claudin1 expression in the cervix of 2monthold K14E7 mice, and claudin10 accumulated at the border of cells in the upper layer of the cervix in FvB mice treated with E2, and in K14E7 mice treated with or without E2. These changes were accompanied by an augmented paracellular permeability of the cervix in 2 and 7monthold FvB mice treated with E2, which became more pronounced in K14E7 mice treated with or without E2. In MDCK cells the stable expression of E7 increased the space between adjacent cells and altered the architecture of the monolayers, induced the development of an acute peak of transepithelial electrical resistance accompanied by a reduced expression of claudins 1, 2 and 10, and an increase in claudin4. Moreover, E7 enhances the ability of MDCK cells to migrate through a 3D matrix and induces cell stiffening and stress fiber formation. These observations revealed that cell transformation induced by HPV16 E7 oncoprotein was accompanied by changes in the pattern of expression of claudins and the degree of sealing of epithelial TJs.
Assuntos
Claudinas/biossíntese , Papillomavirus Humano 16/metabolismo , Proteínas E7 de Papillomavirus/metabolismo , Infecções por Papillomavirus/metabolismo , Junções Íntimas/metabolismo , Neoplasias do Colo do Útero/virologia , Animais , Células Cultivadas , Claudinas/genética , Claudinas/metabolismo , Modelos Animais de Doenças , Cães , Feminino , Papillomavirus Humano 16/isolamento & purificação , Humanos , Camundongos , Camundongos Transgênicos , Infecções por Papillomavirus/patologia , Infecções por Papillomavirus/virologia , Neoplasias do Colo do Útero/metabolismo , Neoplasias do Colo do Útero/patologiaRESUMO
The Na+, K+-ATPase transports Na+ and K+ across the membrane of all animal cells. In addition to its ion transporting function, the Na+, K+-ATPase acts as a homotypic epithelial cell adhesion molecule via its ß1 subunit. The extracellular region of the Na+, K+-ATPase ß1 subunit includes a single globular immunoglobulin-like domain. We performed Molecular Dynamics simulations of the ectodomain of the ß1 subunit and a refined protein-protein docking prediction. Our results show that the ß1 subunit Ig-like domain maintains an independent structure and dimerizes in an antiparallel fashion. Analysis of the putative interface identified segment Lys221-Tyr229. We generated triple mutations on YFP-ß1 subunit fusion proteins to assess the contribution of these residues. CHO fibroblasts transfected with mutant ß1 subunits showed a significantly decreased cell-cell adhesion. Association of ß1 subunits in vitro was also reduced, as determined by pull-down assays. Altogether, we conclude that two Na+, K+-ATPase molecules recognize each other by a large interface spanning residues 221-229 and 198-207 on their ß1 subunits.
Assuntos
Mutagênese Sítio-Dirigida , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Motivos de Aminoácidos , Animais , Células CHO , Cricetulus , Modelos Moleculares , Simulação de Acoplamento Molecular , Simulação de Dinâmica Molecular , Ligação Proteica , Conformação Proteica , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na+,K+-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine ß1 subunit become adhesive, and those homotypic interactions amongst ß1 subunits of the Na+,K+-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the α subunit of the Na+,K+-ATPase, inhibits the pump activity and induces the detachment of epithelial cells when used at concentrations above 300 nM. At nanomolar non-inhibiting concentrations, ouabain affects the adhesive properties of epithelial cells by inducing the expression of cell adhesion molecules through the activation of signaling pathways associated with the α subunit. In this study, we investigated whether the adhesion between ß1 subunits was also affected by ouabain. We used CHO fibroblasts stably expressing the ß1 subunit of the Na+,K+-ATPase (CHO ß1), and studied the effect of ouabain on cell adhesion. Aggregation assays showed that ouabain increased the adhesion between CHO ß1 cells. Immunofluorescence and biotinylation assays showed that ouabain (50 nM) increases the expression of the ß1 subunit of the Na+,K+-ATPase at the cell membrane. We also examined the effect of ouabain on the activation of signaling pathways in CHO ß1 cells, and their subsequent effect on cell adhesion. We found that cSrc is activated by ouabain and, therefore, that it likely regulates the adhesive properties of CHO ß1 cells. Collectively, our findings suggest that the ß1 subunit adhesion is modulated by the expression levels of the Na+,K+-ATPase at the plasma membrane, which is regulated by ouabain.
Assuntos
Adesão Celular/efeitos dos fármacos , Fibroblastos/efeitos dos fármacos , Fibroblastos/metabolismo , Ouabaína/farmacologia , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Células CHO , Membrana Celular/metabolismo , Cricetulus , Expressão Gênica , Ligação Proteica , Subunidades Proteicas/genética , Proteínas Proto-Oncogênicas c-akt/metabolismo , Transdução de Sinais , Sódio/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/genética , Quinases da Família src/metabolismoRESUMO
Na+, K+-ATPase, or the Na+ pump, is a key component in the maintenance of the epithelial phenotype. In most epithelia, the pump is located in the basolateral domain. Studies from our laboratory have shown that the ß1 subunit of Na+, K+-ATPase plays an important role in this mechanism because homotypic ß1-ß1 interactions between neighboring cells stabilize the pump in the lateral membrane. However, in the retinal pigment epithelium (RPE), the Na+ pump is located in the apical domain. The mechanism of polarization in this epithelium is unclear. We hypothesized that the apical polarization of the pump in RPE cells depends on the expression of its ß2 subunit. ARPE-19 cells cultured for up to 8 weeks on inserts did not polarize, and Na+, K+-ATPase was expressed in the basolateral membrane. In the presence of insulin, transferrin and selenic acid (ITS), ARPE-19 cells cultured for 4 weeks acquired an RPE phenotype, and the Na+ pump was visible in the apical domain. Under these conditions, Western blot analysis was employed to detect the ß2 isoform and immunofluorescence analysis revealed an apparent apical distribution of the ß2 subunit. qPCR results showed a time-dependent increase in the level of ß2 isoform mRNA, suggesting regulation at the transcriptional level. Moreover, silencing the expression of the ß2 isoform in ARPE-19 cells resulted in a decrease in the apical localization of the pump, as assessed by the mislocalization of the α2 subunit in that domain. Our results demonstrate that the apical polarization of Na+, K+-ATPase in RPE cells depends on the expression of the ß2 subunit.
RESUMO
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
The very existence of higher metazoans depends on the vectorial transport of substances across epithelia. A crucial element of this transport is the membrane enzyme Na(+),K(+)-ATPase. Not only is this enzyme distributed in a polarized manner in a restricted domain of the plasma membrane but also it creates the ionic gradients that drive the net movement of glucose, amino acids, and ions across the entire epithelium. In a previous work, we have shown that Na(+),K(+)-ATPase polarity depends on interactions between the beta subunits of Na(+),K(+)-ATPases located on neighboring cells and that these interactions anchor the entire enzyme at the borders of the intercellular space. In the present study, we used fluorescence resonance energy transfer and coprecipitation methods to demonstrate that these beta subunits have sufficient proximity and affinity to permit a direct interaction, without requiring any additional extracellular molecules to span the distance.
Assuntos
Polaridade Celular , Subunidades Proteicas/química , Subunidades Proteicas/metabolismo , ATPase Trocadora de Sódio-Potássio/química , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Linhagem Celular , Cricetinae , Cricetulus , Cães , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Transferência Ressonante de Energia de Fluorescência , Modelos Moleculares , Ligação Proteica , Conformação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Subunidades Proteicas/genética , Ratos , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , ATPase Trocadora de Sódio-Potássio/genéticaRESUMO
The space between neighboring epithelial cells is sealed by the tight junction (TJ). When this seal is leaky, such as in the proximal tubule of the kidney or the gallbladder, substances may cross the epithelium between the cells (paracellular pathway). Yet, when TJs are really hermetic, as is the case in the epithelium of the urinary bladder or the colon, substances can mainly cross the epithelium through the transcellular pathway. The structure of the TJ involves (so far) some 50-odd protein species. Failure of any of these components causes a variety of diseases, some of them so serious that fetuses are not viable. A fast-growing number of diseases are recognized to depend or involve alterations in the TJ. These include autoimmune diseases, in which intestinal TJs allow the passage of antigens from the intestinal flora, challenging the immune system to produce antibodies that may cross react with proteins in the brain, thyroid gland or pancreas. TJs are also involved in cancer development, infections, allergies, etc. The present article does not catalogue all TJ diseases known so far, but describes one of each type as illustration. It also depicts the efforts being made to find pharmaceutical agents that would seal faulty TJs or release their grip to allow for the passage of large molecules through the upper respiratory and digestive tracts, such as insulin, thyroid, appetite-regulatory peptide, etc.
Assuntos
Doenças Autoimunes/patologia , Permeabilidade da Membrana Celular , Epitélio/patologia , Doenças Genéticas Inatas/patologia , Infecções/patologia , Neoplasias/patologia , Junções Íntimas/patologia , Animais , Doenças Autoimunes/fisiopatologia , Permeabilidade da Membrana Celular/genética , Permeabilidade da Membrana Celular/fisiologia , Epitélio/fisiologia , Doenças Genéticas Inatas/fisiopatologia , Humanos , Infecções/fisiopatologia , Proteínas de Membrana/genética , Neoplasias/fisiopatologia , Junções Íntimas/efeitos dos fármacos , Junções Íntimas/genética , Junções Íntimas/fisiologiaRESUMO
The assembly and permanent sealing of tight junctions (TJs) depend crucially on cell-cell contacts containing E-cadherin. This poses a puzzling problem because, while TJs can be established between epithelial cells from different tissues and even different animal species ("heterotypic TJs"; Gonzalez-Mariscal et al. 1989, J Membr Biol 107:43), the cell-cell binding mediated by E-cadherin is a highly specific one (Takeichi 1995, Curr Opin Cell Biol 7:619). Yet the demonstration that TJs can be established at heterotypic borders is open to two distinct challenges. First, it is based on transepithelial electrical resistance (TER) and restriction to ruthenium red permeation only, which today are known to be just two of the many characteristics of TJs; and second some attributes of the TJs (e.g. the presence of specific molecules) have been found even in cells that do not establish these structures. This raised the question of whether heterotypic TJs were not true or full TJs. In the present work we demonstrate that heterotypic TJs in mixed monolayers of MDCK cells with a different cell type (LLC-PK1) are true TJs through several criteria, such as TER, the ability to stop the membrane diffusion of fluorescent sphingomyelin from the apical to the lateral domain, the presence of ZO-1, ZO-2, occludin, claudin-1 and claudin-2. We then turn to the presence of E-cadherin at heterotypic borders, and observe that it cannot be detected by the highly specific DECMA-1 antibody, in spite of the fact that this antibody does reveal the presence of E-cadherin at homotypic contacts of the same cell. Yet, ECCD-2, an antibody against another domain of E-cadherin, reveals that this molecule may be present at both types of borders. Thus, E-cadherin is present at heterotypic borders, yet it seems to be in a conformation unable to bind DECMA-1. Our results suggest: (1) that heterotypic borders can establish fully developed TJs; (2) that the sealing of these heterotypic TJs depends on E-cadherin; (3) but that this dependence is mediated through a cascade of chemical reactions involving two different G-proteins, PLC, PKC and calmodulin, which we have characterized elsewhere (Balda et al. 1991, J Membr Biol 122:193); and (4) hence molecules of E-cadherin that trigger junction formation can act from a distant homotypic contact.