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.
TRPV4 (transient receptor potential vanilloid 4) is a cation channel activated by hypotonicity, moderate heat, or shear stress. We describe the expression of TRPV4 during the differentiation of a corneal epithelial cell model, RCE1(5T5) cells. TRPV4 is a late differentiation feature that is concentrated in the apical membrane of the outmost cell layer of the stratified epithelia. Ca imaging experiments showed that TRPV4 activation with GSK1016790A produced an influx of calcium that was blunted by the specific TRPV4 blocker RN-1734. We analyzed the involvement of TRPV4 in RCE1(5T5) epithelial differentiation by measuring the development of transepithelial electrical resistance (TER) as an indicator of the tight junction (TJ) assembly. We showed that TRPV4 activity was necessary to establish the TJ. In differentiated epithelia, activation of TRPV4 increases the TER and the accumulation of claudin-4 in cell-cell contacts. Epidermal Growth Factor (EGF) up-regulates the TER of corneal epithelial cultures, and we show here that TRPV4 activation mimicked this EGF effect. Conversely, TRPV4 inhibition or knock down by specific shRNA prevented the increase in TER. Moreover, TRPP2, an EGF-activated channel that forms heteromeric complexes with TRPV4, is also concentrated in the outmost cell layer of differentiated RCE1(5T5) sheets. This suggests that the EGF regulation of the TJ may involve a heterotetrameric TRPV4-TRPP2 channel. These results demonstrated TRPV4 activity was necessary for the correct establishment of TJ in corneal epithelia and as well as the regulation of both the barrier function of TJ and its ability to respond to EGF. J. Cell. Physiol. 232: 1794-1807, 2017. © 2016 Wiley Periodicals, Inc.
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.
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 β-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.
Chronic hyperglycemia increases the risk of developing severe COVID-19 symptoms, but the related mechanisms are unclear. A mean glucose level upon hospital admission >166 mg/dl correlates positively with acute respiratory distress syndrome in patients with hyperglycemia. The objective of this study was to evaluate the relationship between sustained hyperglycemia and the outcome of hospitalized patients with severe COVID-19. We also evaluated the effect of high glucose concentrations on the expression of angiotensin-converting enzyme 2 (ACE2). We carried out a case-control study with hospitalized patients with severe COVID-19 with and without sustained hyperglycemia. In a second stage, we performed in vitro assays evaluating the effects of high glucose concentrations on ACE2 gene expression. Fifty hospitalized patients with severe COVID-19 were included, of which 28 (56%) died and 22 (44%) recovered. Patients who died due to COVID-19 and COVID-19 survivors had a high prevalence of hyperglycemia (96.4% versus 90.9%), with elevated central glucose upon admission (197.7 mg/dl versus 155.9 mg/dl, p = 0.089) and at discharge (185.2 mg/dl versus 134 mg/dl, p = 0.038). The mean hypoxemia level upon hospital admission was 81% in patients who died due to COVID-19 complications and 88% in patients who survived (p = 0.026); at the time of discharge, hypoxemia levels were also different between the groups (68% versus 92%, p ≤ 0.001). In vitro assays showed that the viability of A549 cells decreased (76.41%) as the glucose concentration increased, and the ACE2 gene was overexpressed 9.91-fold after 72 h (p ≤ 0.001). The relationship between hyperglycemia and COVID-19 in hospitalized patients with COVID-19 plays an important role in COVID-19-related complications and the outcome for these patients. In patients with chronic and/or sustained hyperglycemia, the upregulation of ACE2, and its potential glycation and malfunction, could be related to complications observed in patients with COVID-19.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.