Ca2+ influx is usually considered a central trigger of neuronal plasticity, especially in mechanisms related to transmitter release and synaptic modification. However, plasticity may also involve mechanisms that do not require Ca2+ influx into neurons. We tested whether short-term plasticity can occur in the CA1 region of the mouse hippocampus under Ca2+-free conditions. In Ca2+-free artificial cerebrospinal fluid, in which synaptic transmission was not detected, local stimulation evoked a population potential in CA1, and theta-burst stimulation led to short-term depression during the stimulation protocol. This depression included three separable components: depression within bursts, cumulative depression of the first response across bursts, and enhancement of within-burst depression during repeated bursts. Whole-cell current-clamp recordings showed that the TBS stimulation protocol also changed action-potential kinetics at the level of individual neurons. These changes were expressed in CA1 pyramidal neurons as a reduction of maximum rise rate and slowing of repolarization, whereas stratum oriens interneurons did not show within-burst depression nor enhancement. Together, these data show that theta-burst stimulation can induce Ca2+-influx-independent short-term plasticity of action potentials in hippocampal CA1 pyramidal cells.

Changes in the Na+ and K+ intracellular concentrations affect expression of the FOS gene. Here, we obtained a genetic construct coding for the TurboGFP-dest1 protein under control of the human FOS promoter (-549 to +155) and studied its expression in HEK293T cells exposed to monovalent metal cations. Amplification of the FOS promoter sequence from genomic DNA was efficient only in the presence of Li+ ions. Incubation of cells with ouabain or in a medium containing Li+ ions instead of Na+ ions caused intracellular accumulation of Na+ and Li+ ions, respectively. In addition, both stimuli increased the levels of endogenous FOS mRNA and the average fluorescence intensity of TurboGFP-dest1 in transfected cells. The mRNA levels of TurboGFP-dest1 were significantly higher than the FOS mRNA levels and were little affected by the stimuli.

Data on the structure of G-quadruplexes, noncanonical nucleic acid forms, supporting an idea of their potential participation in regulation of gene expression in response to the change in intracellular Na-i(+)/K-i(+) ratio are considered in the review. Structural variety of G-quadruplexes, role of monovalent cations in formation of this structure, and thermodynamic stability of G-quadruplexes are described. Data on the methods of their identification in the cells and biological functions of these structures are presented. Analysis of information about specific interactions of G-quadruplexes with some proteins was conducted, and their potential participation in the development of some pathological conditions, in particular, cancer and neurodegenerative diseases, is considered. Special attention is given to the plausible role of G-quadruplexes as sensors of intracellular Na-i(+)/K-i(+) ratio, because alteration of this parameter affects folding of G-quadruplexes changing their stability and, thereby, organization of the regulatory elements of nucleic acids. The data presented in the conclusion section demonstrate significant change in the expression of some early response genes under certain physiological conditions of cells and tissues depending on the intracellular Na-i(+)/K-i(+) ratio.

Data on the structure of G-quadruplexes, noncanonical nucleic acid forms, supporting an idea of their potential participation in regulation of gene expression in response to the change in intracellular Na+i/K+i ratio are considered in the review. Structural variety of G-quadruplexes, role of monovalent cations in formation of this structure, and thermodynamic stability of G-quadruplexes are described. Data on the methods of their identification in the cells and biological functions of these structures are presented. Analysis of information about specific interactions of G-quadruplexes with some proteins was conducted, and their potential participation in the development of some pathological conditions, in particular, cancer and neurodegenerative diseases, is considered. Special attention is given to the plausible role of G-quadruplexes as sensors of intracellular Na+i/K+i ratio, because alteration of this parameter affects folding of G-quadruplexes changing their stability and, thereby, organization of the regulatory elements of nucleic acids. The data presented in the conclusion section demonstrate significant change in the expression of some early response genes under certain physiological conditions of cells and tissues depending on the intracellular Na+i/K+i ratio.

Changes in intracellular concentrations of Na+ and K+ are shown to alter gene expression. Another monovalent cation, Li+, is well known as a medicine for treatment of psychiatric disorders, but mechanism of its action is obscure. Thus, it is important to evaluate the effect of Li+ on gene expression in endothelial cells. Here we studied influence of the increased intracellular Na+ or Li+ concentrations on transcription of Na+i/K+i-sensitive genes. Treatment of the human endothelial cells (HUVEC) with LiCl for 1.5 h resulted in accumulation of Li+ in the cells. This was followed by increase in the FOS and EGR1 mRNAs levels and decrease in the JUN and MYC mRNA levels. Treatment of HUVEC with the Na+-ionophore monensin led to accumulation of Na+ and loss of K+ ions. However, monensin had no significant effect on gene expression. Incubation of HUVEC with elevated extracellular NaCl concentration increased intracellular K+ concentration and transcription of the ATF3 gene, while transcription of the JUN gene decreased. These results indicate that Na+ and Li+ ions have different effects on the gene expression profile in the cells that is likely associated with the fact that they affect differently the intracellular monovalent cations ratio.

Melittin, a peptide from bee venom, was found to be able to interact with many proteins, including calmodulin target proteins and ion-transporting P-type ATPases. It is assumed that melittin mimics a protein module involved in protein-protein interactions within cells. Previously, a Na + /K + -ATPase containing the α1 isoform of the catalytic subunit was found to co-precipitate with a protein with a molecular weight of about 70 kDa that interacts with antibodies against melittin by cross immunoprecipitation. In the presence of a specific Na + /K + -ATPase inhibitor (ouabain), the amount of protein with a molecular weight of 70 kDa interacting with Na + /K + -ATPase increases. In order to identify melittin-like protein from murine kidney homogenate, a fraction of melittin-like proteins with a molecular weight of approximately 70 kDa was obtained using affinity chromatography with immobilized antibodies specific to melittin. By mass spectrometry analysis, the obtained protein fraction was found to contain three molecular chaperones of Hsp70 superfamily: mitochondrial mtHsp70 (mortalin), Hsp73, Grp78 (BiP) of endoplasmic reticulum. These data suggest that chaperones from the HSP-70 superfamily contain a melittin-like module.

Background/Aims: Cultured skeletal muscle cells subjected to electrical pulse stimulation (EPS) are widely employed as an in vitro model of exercising skeletal muscle. Numerous studies demonstrated that sustained excitation of skeletal muscle results in the dissipation of the transmembrane gradient of monovalent cations. During exercises the impending loss of excitability has to be counterbalanced by rapid restoration of the Na+i/K+i ratio. To understand mechanisms of the maintenance of muscle contractility, it is necessary to know which transporters are participated in the dissipation of Na+i and K+i gradients and how to activate Na,K-ATPase for its regeneration. This study was aimed at the identification of ion transporters involved in the dissipation of the transmembrane gradients of Na+ and K+induced by EPS, and Na,K-ATPase isoforms involved in its restoration. Methods: The differentiated C2C12 myotubes were subjected to electrical pulse stimulation in the presence or absence of different ion transport systems inhibitors followed by measurement of intracellular monovalent cations by flame atomic absorption spectrometry. Results: Electrical pulse stimulation of C2C12 myotubes results in the dissipation of Na+i/K+i gradient, which is maintained by α2-Na,K-ATPase. Na-K-2Cl cotransporter (NKCC), voltage-gated Na+ (Nav), and large conductance Ca2+-activated K+ channels (BKCa), and Na/H exchanger (NHE) are involved in the dissipation of this gradient. Suppression of calmodulin-dependent protein kinase II (CaMKII) increases Na+ efflux in EPS-treated C2C12 myotubes. Conclusion: NKCC, Nav, BKCa, and NHE are involved in the dissipation of Na+i/K+i gradient in EPS-treated C2C12 myotubes.

Cardiotonic steroids (CTS) are a group of steroid compounds derived from certain plants and animals. CTS are selective inhibitors of Na,K-ATPase, so for a long time they were widely used for medical purposes to treat heart failure and some other diseases. However, over time, doctors gradually began to refuse this group of drugs due to their narrow therapeutic range and a number of serious side effects. However, in parallel with the rise and fall of interest in CTS as a drug, interest in them as a single class of high-affinity Na,K-ATPase inhibitors has only increased. Numerous data on the effect of CTS on signaling cascades and cell viability made impelling a search for a rationale for the existence of such a subtle biological regulator. A large number of studies devoted to the potential use of CTS in the treatment of neurodegenerative, oncological, immune and other diseases "revitalized" the already "written off" group of the drugs. If initially the role of exogenous (mainly plant origin) CTS was studied, then by the 90s of the XX century the study of the role of endogenous CTS and their regulatory effects became a popular trend in biology and physiology. The role of endogenous CTS was studied in details in the pathogenesis of cardiovascular diseases [1]. A large number of researchers have unanimously stated that endogenous CTS are very complex regulators of the development of pathological processes; however, the exact cause-and-effect relationship that would unambiguously explain the processes of synthesis, utilization, and the role of endogenous CTS has not yet been established. The effects of CTS in the frame of various regulatory schemes have been described, but the predominant number of studies was devoted to arterial hypertension, heart and renal failure, cancer [2–4], aging [5,6], and neuroinflammation. The effects of both endo- and exogenous CTS on blood cells and have been studied to a much lesser extent.

Hyperosmotic stimulation of endothelial cells often leads to its dysfunction accompanied, among other things, by proinflammatory response. The mechanisms of this phenomenon are not fully understood. It may arise due to increase in the plasma Na + concentration, due to increase in the extracellular osmolarity, increase in the intracellular Na + i /K + i ratio, and/or change in the cell stiffness. In the present study we investigated the effects of short-term increase in osmolarity of extracellular medium on the mRNA content of some genes important for endothelial function (including Na + i /K + i -sensitive ones) and the equivalent elasticity constant of human umbilical vein endothelial cells membranes. Hyperosmotic stimulation of these cells with NaCl but not mannitol resulted in accumulation of Na + ions inside the cells despite the Na,K-ATPase activation, and was also accompanied by the decrease in their equivalent elasticity constant. The amount of IL1α mRNA decreased with increasing osmolarity of the extracellular medium, whereas the amount of ATF3 , PAR2 , and PTGS2 mRNAs increased only in response to the increasing NaCl concentration. At the same time, under the conditions of our experiments, we did not detect changes in the expression of the osmoprotective transcription factor NFAT5. The obtained data indicate that the increase of extracellular Na + concentration in the physiological range is an independent factor that affects intracellular Na + i /K + i ratio and regulates expression of some genes (in particular, ATF3 , PAR2 , PTGS2 ) in endothelial cells.

— Since the discovery of Na + ,K + -ATPase by Jens Skou in 1957, this enzyme has been considered exclusively as a transporter that ensures the active transport of Na + and K + ions across the cell plasma membrane; therefore, its structure and mechanism of functioning, as well as its involvement in secondary ion transport systems have been studied in detail. In the present review, the data on the structure and functioning of the enzyme are briefly reviewed. The role of Na + ,K + -ATPase as a receptor for cardiotonic steroids (CTS), whose binding to the enzyme initiates a variety of signaling pathways through protein–protein interactions modified also by changes in the intracellular concentration of Na + and K + ions by inhibiting the Na + ,K + -ATPase transport function and Ca 2+ , by mediating changes in Na/Ca-exchange activity, was described in more detail. All these provide a variety of CTS effects, including their effect on gene expression, the state of tight junctions, cell adhesion, induction of myocardial hypertrophy, stimulation of free-radical oxygen species generation, and initiation of cell death or survival depending on tissue type. Data on the discovery of endogenous CTS are presented, as well as an analysis of published data indicating that concentrations of endogenous CTS are so low that they are unlikely to cause inhibition of Na + ,K + -ATPase. In this connection, the data on the enzyme activation by low doses of CTS are presented, and the idea of a possible summation of the concentrations of various steroids is suggested. Possible directions for the study of multiple functions of Na + ,K + -ATPase are discussed in the conclusion.

The maintenance of an uneven distribution of Na+ and K+ ions between the cytoplasm and extracellular medium is the basis for the functioning of any animal cell. Changes in the intracellular ratio of these cations occur in response to numerous stimuli and are important for the cell activity regulation. Numerous experimental data have shown that gene transcription in mammalian cells can be regulated by changes in the intracellular [Na+]i/[K+]i ratio. Here, we discuss possible mechanisms of such regulation in various cell types, with special attention to the [Ca2+]-independent signaling pathways that suggest the presence of an intracellular sensor of monovalent cations. As such sensor, we propose the secondary structures of nucleic acids called G-quadruplexes. They are widely represented in mammalian genomes and are often found in the promoters of genes encoding transcription factors.

Cardiotonic steroids (CTSs) are specific inhibitors of Na,K-ATPase (NKA). They induce diverse physiological effects and were investigated as potential drugs in heart diseases, hypertension, neuroinflammation, antiviral and cancer therapy. Here, we compared the inhibition mode and binding of CTSs, such as ouabain, digoxin and marinobufagenin to NKA from pig and rat kidneys, containing CTSs-sensitive (α1S) and -resistant (α1R) α1-subunit, respectively. Marinobufagenin in contrast to ouabain and digoxin interacted with α1S-NKA reversibly, and its binding constant was reduced due to the decrease in the deepening in the CTSs-binding site and a lower number of contacts between the site and the inhibitor. The formation of a hydrogen bond between Arg111 and Asp122 in α1R-NKA induced the reduction in CTSs' steroid core deepening that led to the reversible inhibition of α1R-NKA by ouabain and digoxin and the absence of marinobufagenin's effect on α1R-NKA activity. Our results elucidate that the difference in signaling, and cytotoxic effects of CTSs may be due to the distinction in the deepening of CTSs into the binding side that, in turn, is a result of a bent-in inhibitor steroid core (marinobufagenin in α1S-NKA) or the change of the width of CTSs-binding cavity (all CTSs in α1R-NKA).

Aim. A comparative review of the rabeprazole properties vs. other PPIs, its efficacy and safety in treatment for aciddependent diseases.Key points. Rabeprazole provides a rapid proton pump blockade in parietal cells due to its high dissociation constant (pKa). A lower rabeprazole metabolic dependence on cytochrome P-450 enzyme system renders its antisecretory effect predictable and reduces the risk of interactions with other drugs metabolised through this system. A faster antisecretory effect and higher acid-suppressive activity of rabeprazole determine its better clinical efficacy in treatment for such acid-dependent diseases as gastroesophageal reflux disease and peptic ulcer. This makes rabeprazole (Pariet) a preferred drug in course and maintenance therapies for acid-dependent diseases, as well as in H. pylori eradication.Conclusion. The rabeprazole properties of high acid suppression potential, persistent antisecretory effect from first day of therapy, non-enzymatic metabolism and pleiotropic action determine its high efficacy in treatment for a wide range of acid-dependent diseases at a minimal risk of drug interaction.

High-salt consumption contributes to the development of hypertension and is considered an independent risk factor for vascular remodelling, cardiac hypertrophy and stroke incidence. Alterations in NO production, inflammation and endothelial cell stiffening are considered now as plausible mediators of cardiovascular dysfunction. We studied early responses of endothelial cells (HUVEC) caused by a moderate increase in extracellular sodium concentration. Exposure of HUVEC to elevated sodium within the physiological range up to 24 h is accompanied by changes in monovalent cations fluxes and Na,K-ATPase activation, and, in turn, results in a significant decrease in the content of PTGS2, IL6 and IL1LR1 mRNAs. The expression of NOS3 and FOS genes, as well as the abundance of cytosolic and nuclear NFAT5 protein, remained unchanged. We assessed the mechanical properties of endothelial cells by estimating Young's modulus and equivalent elastic constant using atomic force and interference microscopy, respectively. These parameters were unaffected by elevated-salt exposure for 24 h. The data obtained suggest that even small and short-term elevations of extracellular sodium concentration affect the expression of genes involved in the control of endothelial function through the Na+i/K+i-dependent mechanism(s).

Aim. Presentation of the Forum “Deprescribing and optimal selection of proton pump inhibitors” held in Moscow on 29 September 2020 during the 26th United Russian Gastroenterology Week.Key points. The Forum was aimed at discussing issues associated with improving the proton pump inhibitor (PPIs) therapy in treatment and prevention of acid-related diseases and upper gastrointestinal tract (GIT) disorders induced by non-steroidal anti-inflammatory drugs (NSAIDs) and antiplatelet medications. Deprescribing is considered to be an effective strategy of a motivated reduction of the PPI dosage, duration of therapy and the patient’s transfer from a regular to on-demand intake regimen. The choice of PPI may condition an optimal therapy for acid-related diseases.Conclusion. PPIs prevail in therapies for acid-related diseases and NSAID-induced upper GIT lesions. PPI deprescribing should be a strategy of choice if medically indicated. A non-enzymatic metabolism, high acid suppression, stable antisecretory effect from day 1 of therapy and cytoprotective action justify the application of rabeprazole (Pariet®) for optimising therapies for acid-related diseases and implementing the deprescribing strategy.

The affinity of rodent Na+,K+-ATPase α1-subunit to cardiotonic steroids (CTS) is known to be approximately 1000-fold less than the affinity of Na+,K+-ATPase α1-subunit from other mammals. The CTS-resistant isoform of Na+,K+-ATPase α1-subunit (α1R) is expressed in rodent cells, in contrast to the CTS-sensitive isoform of the α1-subunit (α1S), which is expressed in cells of other mammals. Earlier we have established that incubation with ouabain in concentrations that completely suppressed the activity of Na+,K+-ATPase α1-isoform (α1S-Na+,K+-ATPase) led to a death of human endothelial and smooth muscle cells but did not affect the survival of rat cells expressing α1R-Na+,K+-ATPase. Conformational transitions that are induced by CTS binding to Na+,K+-ATPase play a key role in the process of cell survival. To reveal differences in the CTS-induced conformational changes of α1R- and α1S-Na+,K+-ATPase isoforms, we used three different CTS (two cardenelids, ouabain and digoxin, and one bufadienolid, marinobufagenin) and analyzed the trypsinolysis products of α1-subunits in two main conformations of Na+,K+-ATPase (E1 and E2-P). The treatment of the pig kidney α1S-Na,K-ATPase in E1 conformation by trypsin results in a significant decrease of the amount of α1S-subunit and in the appearance of protein fragments with molecular masses of about 40, 35, 23, and 19 kDa. Preincubation of Na+,K+-ATPase in E1 conformation with ouabain or with digoxin (1 mM) leads to a decrease of the amount of α1S-subunit, increase of the amount of polypeptide fragment with molecular mass of about 40 kDa, and a significant rise of the amount of fragment with molecular mass of about 35.5 kDa, which was not found after the preincubation of the Na+,K+-ATPase in E1 conformation with marinobufagenin (1 mM). In the absence of CTS the trypsinolysis of α1S-Na+,K+-ATPase in E2-P conformation results in a decrease of the amount of α1S-subunit and an increase of the amount of proteolytic products with molecular mass of about 40 and 35.5 kDa. Preincubation of the Na+,K+-ATPase in E2-P conformation in the presence of any of the CTS studied induces the appearance of big amount of an additional peptide fragment with molecular mass of about 45 kDa. Preincubation of α1R-Na+,K+-ATPase from rat kidney with any of the CTS does not change the composition of proteolytic products and their molecular masses in either E1 or E2-P conformation. The results suggest that the structure of the CTS-binding site and a conformational response of α1-Na+,K+-ATPase to binding of CTS is mainly determined by the primary structure of the CTS-resistant and CTS-sensitive α1-subunits of the Na+,K+-AТРase.

Stimulus-dependent elevation of intracellular Ca2+ affects gene expression via well-documented calmodulin-mediated signaling pathways. Recently, we found that the addition of extra- and intracellular Ca2+ chelators increased, rather than decreased, the number of genes expressed, and that this is affected by the elevation of [Na+]i/[K+]i-ratio. This assumes the existence of a novel Na+i/K+i-mediated Ca2+i-independent mechanism of excitation-transcription coupling. To identify upstream Na+i/K+i-sensitive genes, we examined the kinetics of transcriptomic changes in human umbilical vein endothelial cells (HUVEC) subjected to Na,K-ATPase inhibition by ouabain or K+-free medium. According to our data, microRNAs, transcription factors, and proteins involved in immune response and inflammation might be considered as key components of Na+i/K+i-mediated excitation-transcription coupling. Special attention was focused on the FOS gene and the possible mechanism of transcription regulation via G-quadruplexes, non-canonical secondary structures of nucleic acids, whose stability depends on [Na+]i/[K+]i-ratio. Verification of the [Na+]i/[K+]i-sensitive transcription regulation mechanism should be continued in forthcoming studies.

Ouabain is of cardiotonic steroids (CTS) family that is plant-derived compounds and is known for many years as therapeutic and cytotoxic agents. They are specific inhibitors of Na,K-ATPase, the enzyme, which pumps Na+ and K+ across plasma membrane of animal cells. Treatment of cells by CTS affects various cellular functions connected with the maintenance of the transmembrane gradient of Na+ and K+. Numerous studies demonstrated that binding of CTS to Na,K-ATPase not only suppresses its activity but also induces some signal pathways. This review is focused on different mechanisms of two ouabain effects: their ability (1) to protect rodent cells from apoptosis through the expression of [Na+]i-sensitive genes and (2) to trigger death of non-rodents cells (so-called «oncosis»), possessing combined markers of «classic» necrosis and «classic» apoptosis. Detailed study of oncosis demonstrated that the elevation of the [Na+]i/[K+]i ratio is not a sufficient for its triggering. Non-rodent cell death is determined by the characteristic property of “sensitive” to ouabain α1-subunit of Na,K-ATPase. In this case, ouabain binding leads to enzyme conformational changes triggering the activation of p38 mitogen-activated protein kinases (MAPK) signaling. The survival of rodent cells with ouabain-«resistant» α1-subunit is connected with another conformational transition induced by ouabain binding that results in the activation of ERK 1/2 signaling pathway.