Biology subjects

Vereninov, A. A.

Publications and source records attributed to Vereninov, A. A..

5 recordsLinked to original sources

bioRxiv2021

Role of cation-chloride cotransporters, Na/K-pump, and channels in cell water/ionic balance regulation under hyperosmolar conditions: in silico and experimental studies of opposite RVI and AVD responses of U937 cells to hyperosmolar media

The work provides a modern mathematical description of animal cell electrochemical system under a balanced state and during the transition caused by an increase in external osmolarity, considering all the main ionic pathways in the cell membrane: the sodium pump, K+, Na+, Cl- electroconductive channels and cotransporters NC, KC, and NKCC. The description is applied to experimental data obtained on U937 cells cultured in suspension, which allows the required assays to be performed, including determination of cell water content using buoyant density, cell ion content using flame photometry, and optical methods using flow cytometry. The study of these cells can serve as a useful model for understanding the general mechanisms of regulation of cellular water and ionic balance, which cannot be properly analyzed in many important practical cases, such as ischemic disturbance of cellular ionic and water balance, when cells cannot be isolated. An essential part of the results is the developed software supplied with an executable file, which allows researchers with no programming experience to calculate unidirectional fluxes of monovalent ions through separate pathways and ion-electrochemical gradients that move ions through them, which is important for studying the functional expression of channels and transporters. It is shown how the developed approach is used to reveal changes in channels and transporters underlying the RVI and AVD responses to the hyperosmolar medium in the studied living U937 cells.

cell biology↗

bioRxiv2021

Cation-chloride cotransporters, Na/K pump, and channels in cell water and ions regulation: in silico and experimental studies of the U937 cells under stopping the pump and during RVD

Cation-coupled chloride cotransporters play a key role in generating the Cl electrochemical gradient on the cell membrane which is important for regulation of many cellular processes. However, the cooperation of transporters and channels of the plasma membrane in holding the ionic homeostasis of the whole cell remains poorly characterized because of the lack of a suitable tool for its computation. Our software successfully predicted in real-time changes in the ion homeostasis of U937 cells after stopping the Na/K pump, but so far considered the model with only NC cotransporter. Here the model with all main types of cotransporters is used in computation of the rearrangements of ionic homeostasis due to stopping the pump and associated with the regulatory volume decrease (RVD) of cells swollen in hypoosmolar medium. The parameters obtained for the real U937 cells are used. Successful prediction of changes in ion homeostasis in real-time after stopping the pump using the model with all major cotransporters indicates that the model is reliable. Using this model for analysis RVD showed that there is a "physical" RVD, associated with the time-dependent changes in electrochemical ion gradients, but not with alteration of channels and transporters of the plasma membrane that should be considered in studies of truly active regulatory processes mediated by the intracellular signaling network. The developed software can be useful for calculation of the balance of the partial unidirectional fluxes of monovalent ions across the cell membrane of various cells under various conditions.

cell biology↗

bioRxiv2020

Comparison of cellular Na+ assays by flow fluorometry with ANG-2 and flame emission: Pitfalls in using ionophores for calibrating fluorescent probes

Monovalent ions, sodium in particular, are involved in fundamental cell functions, such as water balance and electric processes, intra- and intercellular signaling, cell movement, pH regulation and metabolite transport into and out of cells. Fluorescent probes are indispensable tools for monitoring intracellular sodium levels in single living cells in heterogeneous cell populations and tissues. Since the fluorescence of sodium-sensitive dyes in cells is significantly different from that in an aqueous solution, the fluorescence signal is calibrated in situ by changing the concentration of extracellular sodium in the presence of ionophores, making the membrane permeable to sodium and equilibrating its intra- and extracellular concentrations. The reliability of this calibration method has not been well studied. Here, we compare the determinations of the intracellular sodium concentration by flame emission photometry and flow cytometry using the Na+-sensitive probe Asante Natrium Green-2 (ANG). The intracellular Na+ concentration was altered using known ionophores or, alternatively, by blocking the sodium pump with ouabain or by causing cell apoptosis with staurosporine. The use of U937 cells cultured in suspension allowed the fluorometry of single cells by flow cytometry and flame emission analysis of samples checked for uniform cell populations. It is revealed that the ANG fluorescence of cells treated with ionophores is approximately two times lower than that in cells with the same Na+ concentration but not treated with ionophores. Although the mechanism is still unknown, this effect should be taken into account when a quantitative assessment of the concentration of intracellular sodium is required. Sodium sensitive fluorescent dyes are widely used at present, and the problem is practically significant.

cell biology↗

bioRxiv2019

Fluorometric intracellular Na+ measurement as compared with flame emission assay: An unexpected problem with gramicidin-based calibration

Fluorescent probes are a popular and indispensable tool for monitoring sodium concentration in living cells in situ. Calibration of fluorescent probes inside cells commonly uses ionophores to equilibrate intracellular and external ion concentrations. Here we test this calibration method using in parallel classical flame emission assay. Suspension human lymphoma cells allow both flow cytometry fluorometric study and flame emission assay. The most sensitive Na+ fluorescent probe ANG-2 and the most common ionophores were tested. Cellular Na+ was altered for calibration in three different ways: by stopping the sodium pump with ouabain, by inducing of apoptosis with staurosporine, and by gramicidin or amphotericin B treatment. We found that ANG-2 fluorescence in cells treated with gramicidin or amphotericin was about two fold lower than in the cells with the same sodium concentration but without ionophores. The equal fluorescence measured in the absence and in the presence of ionophores corresponds to different cell sodium concentrations. No effect of gramicidin on hydrolyzed ANG was observed in vitro. The mechanism, by which gramicidin decreases ANG fluorescence in cells is unlikely to be physical quenching and remains obscure. We conclude that ANG fluorescence does not display realistic cell Na+ if fluorescence in cell is measured in ionophore absence while calibrated in its presence.

physiology↗

bioRxiv2018

A tool for computation of changes in Na+, K+, Cl- channels and transporters due to apoptosis by data on cell ion and water content alteration

The study aims to know how the apoptotic alteration of cell ionic balance follows from the quantitatively characterised time dependent decrease in the sodium pump rate constant and changes in permeability coefficients of Cl-, K+, and Na+ channels. New experimental data on changes in cell K+, Na+, Cl-, water contents, and the Na+/ K+-ATPase-mediated K+ influx during the first 4 h of the staurosporine (STS) induced apoptosis are used as a basis for quantitative characterisation of channels and transporters responsible for apoptotic cell ion balance alteration. New computational tool is developed. It is found that the dynamics of alteration of ion and water balance in the studied U937 cells were associated with the decrease in the Na+/K+-ATPase rate coefficient by 2.2 times for 4 h, and a time-dependent increase in potassium channel permeabilitry, and a decrease in the sodium channel permeability, whereas the early decrease in [Cl-]i and cell volume were associated with an approximately 5-fold increase in the chloride channel permeability. The developed approach and the provided executable file can be used to identify the channels and transporters responsible for alterations of cell ion and water balance not only during apoptosis but in other physiological scenarios.

physiology↗