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, 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
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
Ouabain is a cardiac glycoside that has been described as a hormone, with interesting effects on epithelial physiology. We have shown previously that ouabain induces gap junctional intercellular communication (GJIC) in wild, sensitive cells (MDCK-S), but not in cells that have become insensitive (MDCK-I) by modifying their Na+-K+-ATPase. We have also demonstrated that prostaglandin E2 (PGE2) is able to induce increased GJIC by a mechanism other than ouabain, that does not depend on Na+-K+-ATPase. In this work we show, by dye transfer assays, that when MDCK-S and MDCK-I are randomly mixed, to form monolayers, the latter stablish GJIC, because of stimulation by a compound released to the extracellular media, by MDCK-S cells, after treatment with ouabain, as evidenced by the fact that monolayers of only MDCK-I cells, treated with a conditioned medium (CM) that is obtained after incubation of MDCK-S monolayers with ouabain, significantly increase their GJIC. The further finding that either (1) pre-treatment with COX-2 inhibitors or (2) addition to CM of antagonists of EP2 receptor abolish CM's ability to induce GJIC in MDCK-I monolayers indicate that PGE2 is the GJIC-inducing compound. Therefore, these results indicate that, in addition to direct stimulation, mediated by Na+-K+-ATPase, ouabain enhances GJIC indirectly through the paracrine production of PGE2.
Assuntos
Cardiotônicos/farmacologia , Dinoprostona/metabolismo , Junções Comunicantes/fisiologia , Ouabaína/farmacologia , Comunicação Parácrina , Animais , Cães , Células Madin Darby de Rim Canino , Transdução de SinaisRESUMO
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
Gap junctions are molecular structures that allow communication between neighboring cells. It has been shown that gap junctional intercellular communication (GJIC) is notoriously reduced in cancer cells compared to their normal counterparts. Ouabain, a plant derived substance, widely known for its therapeutic properties on the heart, has been shown to play a role in several types of cancer, although its mechanism of action is not yet fully understood. Since we have previously shown that ouabain enhances GJIC in epithelial cells (MDCK), here we probed whether ouabain affects GJIC in a variety of cancer cell lines, including cervico-uterine (CasKi, SiHa and Hela), breast (MDA-MB-321 and MCF7), lung (A549), colon (SW480) and pancreas (HPAF-II). For this purpose, we conducted dye transfer assays to measure and compare GJIC in monolayers of cells with and without treatment with ouabain (0.1, 1, 10, 50 and 500 nM). We found that ouabain induces a statistically significant enhancement of GJIC in all of these cancer cell lines, albeit with distinct sensitivity. Additionally, we show that synthesis of new nucleotides or protein subunits is not required, and that Csrc, ErK1/2 and ROCK-Rho mediate the signaling mechanisms. These results may contribute to explaining how ouabain influences cancer.
Assuntos
Cardiotônicos/farmacologia , Comunicação Celular , Junções Comunicantes/efeitos dos fármacos , Neoplasias/patologia , Ouabaína/farmacologia , Apoptose , Proliferação de Células , Humanos , Neoplasias/tratamento farmacológico , Neoplasias/metabolismo , Transdução de Sinais , Células Tumorais CultivadasRESUMO
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
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
Development of tight junctions and cell polarity in epithelial cells requires a complex cellular machinery to execute an internal program in response to ambient cues. Tight junctions, a product of this machinery, can act as gates of the paracellular pathway, fences that keep the identity of plasma membrane domains, bridges that communicate neighboring cells. The polarization internal program and machinery are conserved in yeast, worms, flies and mammals, and in cell types as different as epithelia, neurons and lymphocytes. Polarization and tight junctions are dynamic features that change during development, in response to physiological and pharmacological challenges and in pathological situations like infection.
Assuntos
Polaridade Celular/fisiologia , Junções Íntimas/fisiologia , Animais , Blastocisto/fisiologia , Blastocisto/ultraestrutura , Caenorhabditis elegans/fisiologia , Caenorhabditis elegans/ultraestrutura , Adesão Celular , Drosophila/fisiologia , Drosophila/ultraestrutura , Células Epiteliais/fisiologia , Células Epiteliais/ultraestrutura , Humanos , Proteínas de Membrana/química , Proteínas de Membrana/fisiologia , Modelos Biológicos , Complexos Multiproteicos , Neurônios/fisiologia , Neurônios/ultraestrutura , Neutrófilos/fisiologia , Neutrófilos/ultraestrutura , Fenótipo , Saccharomyces cerevisiae/fisiologia , Saccharomyces cerevisiae/ultraestrutura , ATPase Trocadora de Sódio-Potássio/fisiologia , Linfócitos T/fisiologia , Linfócitos T/ultraestruturaRESUMO
Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on sequence similarity searches and immunological and functional data, this protein is the canine homologue of human GEF-H1 and mouse Lfc, two previously identified Rho-specific exchange factors known to associate with microtubules in nonpolarized cells. In agreement with these observations, immunofluorescence of proliferating MDCK cells revealed that the endogenous canine GEF-H1/Lfc associates with mitotic spindles. Functional analysis based on overexpression and RNA interference in polarized MDCK cells revealed that this exchange factor for Rho regulates paracellular permeability of small hydrophilic tracers. Although overexpression resulted in increased size-selective paracellular permeability, such cell lines exhibited a normal overall morphology and formed fully assembled TJs as determined by measuring transepithelial resistance and by immunofluorescence and freeze-fracture analysis. These data indicate that GEF-H1/Lfc is a component of TJs and functions in the regulation of epithelial permeability.
Assuntos
Membrana Celular/metabolismo , Células Epiteliais/metabolismo , Fatores de Troca do Nucleotídeo Guanina/isolamento & purificação , Proteínas Proto-Oncogênicas/isolamento & purificação , Junções Íntimas/metabolismo , Proteínas rho de Ligação ao GTP/metabolismo , Citoesqueleto de Actina/metabolismo , Citoesqueleto de Actina/ultraestrutura , Animais , Comunicação Celular/genética , Membrana Celular/ultraestrutura , Permeabilidade da Membrana Celular/genética , Células Cultivadas , Cães , Células Epiteliais/ultraestrutura , Matriz Extracelular/genética , Matriz Extracelular/metabolismo , Regulação da Expressão Gênica/fisiologia , Fatores de Troca do Nucleotídeo Guanina/genética , Humanos , Imuno-Histoquímica , Microscopia Eletrônica , Microtúbulos/metabolismo , Microtúbulos/ultraestrutura , Proteínas Proto-Oncogênicas/genética , Interferência de RNA/fisiologia , Fatores de Troca de Nucleotídeo Guanina Rho , Fuso Acromático/metabolismo , Fuso Acromático/ultraestrutura , Junções Íntimas/ultraestruturaRESUMO
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
The polarized distribution of Na+,K+-ATPase plays a paramount physiological role, because either directly or through coupling with co- and countertransporters, it is responsible for the net movement of, for example, glucose, amino acids, Ca2+, K+, Cl-, and CO3H- across the whole epithelium. We report here that the beta-subunit is a key factor in the polarized distribution of this enzyme. 1) Madin-Darby canine kidney (MDCK) cells (epithelial from dog kidney) express the Na+,K+-ATPase over the lateral side, but not on the basal and apical domains, as if the contact with a neighboring cell were crucial for the specific membrane location of this enzyme. 2) MDCK cells cocultured with other epithelial types (derived from human, cat, dog, pig, monkey, rabbit, mouse, hamster, and rat) express the enzyme in all (100%) homotypic MDCK/MDCK borders but rarely in heterotypic ones. 3) Although MDCK cells never express Na+,K+-ATPase at contacts with Chinese hamster ovary (CHO) cells, they do when CHO cells are transfected with beta1-subunit from the dog kidney (CHO-beta). 4) This may be attributed to the adhesive property of the beta1-subunit, because an aggregation assay using CHO (mock-transfected) and CHO-beta cells shows that the expression of dog beta1-subunit in the plasma membrane does increase adhesiveness. 5) This adhesiveness does not involve adherens or tight junctions. 6) Transfection of beta1-subunit forces CHO-beta cells to coexpress endogenous alpha-subunit. Together, our results indicate that MDCK cells express Na+,K+-ATPase at a given border provided the contacting cell expresses the dog beta1-subunit. The cell-cell interaction thus established would suffice to account for the polarized expression and positioning of Na+,K+-ATPase in epithelial cells.
Assuntos
Epitélio/enzimologia , ATPase Trocadora de Sódio-Potássio/biossíntese , Junções Aderentes , Animais , Western Blotting , Células CHO , Linhagem Celular , Membrana Celular/metabolismo , Técnicas de Cocultura , Cricetinae , Cães , Impedância Elétrica , Fibroblastos/metabolismo , Immunoblotting , Microscopia de Fluorescência , Modelos Biológicos , Plasmídeos/metabolismo , Ligação Proteica , Estrutura Terciária de Proteína , Sódio/química , Junções Íntimas , TransfecçãoRESUMO
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
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
Ouabain, a toxic of vegetal origin used for centuries to treat heart failure, has recently been demonstrated to have an endogenous counterpart, most probably ouabain itself, which behaves as a hormone. Therefore, the challenge now is to discover the physiological role of hormone ouabain. We have recently shown that it modulates cell contacts such as gap junctions, which communicate neighboring cells, as well as tight junctions (TJs), which are one of the two differentiated features of epithelial cells, the other being apical/basolateral polarity. The importance of cell contacts can be hardly overestimated, since the most complex object in the universe, the brain, assembles itself depending on what cells contacts what other(s) how, when, and how is the molecular composition and special arrangement of the contacts involved. In the present chapter, we detail the protocols used to demonstrate the effect of ouabain on the molecular structure and functional properties of one of those cell-cell contacts: the TJ.
Assuntos
Comunicação Celular/efeitos dos fármacos , Comunicação Celular/fisiologia , Diferenciação Celular/efeitos dos fármacos , Células Epiteliais/metabolismo , Junções Comunicantes/metabolismo , Ouabaína/farmacologia , Potenciometria/métodos , Junções Íntimas/metabolismo , Animais , Western Blotting , Adesão Celular/efeitos dos fármacos , Linhagem Celular , Claudina-1 , Conexinas/genética , Conexinas/metabolismo , Dextranos/análise , Cães , Impedância Elétrica , Eletroforese em Gel de Poliacrilamida , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Fluoresceína-5-Isotiocianato/análogos & derivados , Fluoresceína-5-Isotiocianato/análise , Junções Comunicantes/efeitos dos fármacos , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Plasmídeos , RNA Mensageiro/análise , RNA Mensageiro/biossíntese , Junções Íntimas/efeitos dos fármacos , TransfecçãoRESUMO
At 10 nM, ouabain elicits changes in cell contacts, which are independent and usually in opposite direction to effects occurring at µM levels, suggesting that these depend on entirely different mechanisms.1 However, this does not discard the possibility that in both instances ouabain would act on the same receptor. We demonstrate that such is the case by comparing the response of wild and ouabain-resistant MDCK cells on a very special type of cell contact, the tight junction (TJ).