G-Quadruplexes as Sensors of Intracellular Na+/K+ Ratio: Potential Role in Regulation of Transcription and Translation.

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.

Sodium Ions as Regulators of Transcription in Mammalian Cells.

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.

Increased Extracellular Sodium Concentration as a Factor Regulating Gene Expression in Endothelium.

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.

Na,K-ATPase as a target for endogenous cardiotonic steroids: What's the evidence?

With an exception of few reports, the plasma concentration of ouabain and marinobufagenin, mostly studied cardiotonic steroids (CTS) assessed by immunoassay techniques, is less than 1 nM. During the last 3 decades, the implication of these endogenous CTS in the pathogenesis of hypertension and other volume-expanded disorders is widely disputed. The threshold for inhibition by CTS of human and rodent α1-Na,K-ATPase is ∼1 and 1000 nM, respectively, that rules out the functioning of endogenous CTS (ECTS) as natriuretic hormones and regulators of cell adhesion, cell-to-cell communication, gene transcription and translation, which are mediated by dissipation of the transmembrane gradients of monovalent cations. In several types of cells ouabain and marinobufagenin at concentrations corresponding to its plasma level activate Na,K-ATPase, decrease the [Na+]i/[K+]i-ratio and increase cell proliferation. Possible physiological significance and mechanism of non-canonical Na+ i/K+ i-dependent and Na+ i/K+ i-independent cell responses to CTS are discussed.

Effect of Dynamic and Static Load on the Concentration of Myokines in the Blood Plasma and Content of Sodium and Potassium in Mouse Skeletal Muscles.

Modulation of cytokine production by physical activity is of considerable interest, since it might be a promising strategy for correcting metabolic processes at both cellular and systemic levels. The content of IL-6, IL-8, and IL-15 in the plasma and the concentration of monovalent cations in the skeletal muscles of trained and untrained mice were studied at different periods after static and dynamic exercises. Dynamic loads caused an increase in the IL-6 content and decrease in the IL-15 content in the plasma of untrained mice, but produced no effect on the concentration of IL-8. In trained mice, the effect of a single load on the concentration of IL-6 and IL-15 in the plasma was enhanced, while the concentration of IL-8 decreased. Static loads produced a similar, but more pronounced effect on the plasma concentration of IL-6 and IL-15 compared the dynamic exercises; however, the concentration of IL-8 in response to the static exercise increased significantly. Prior training reinforced the described response for all the myokines studied. Dynamic load (swimming) increased the intracellular content of sodium but decreased the content of potassium in the mouse musculus soleus. Similar response was observed after the static load (grid hanging) in the musculus biceps; but no correlation of this response with the prior training was found. Possible mechanisms involved in the regulation of cytokine secretion after exercise are discussed, including triggering of gene transcription in response to changes in the [Na+]i/[K+]I ratio.

Transcriptomic Changes in Endothelial Cells Triggered by Na,K-ATPase Inhibition: A Search for Upstream Na+i/K+i Sensitive Genes.

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.

Elevation of Intracellular Na+ Contributes to Expression of Early Response Genes Triggered by Endothelial Cell Shrinkage.

BACKGROUND/AIMS: Prolonged hyperosmotic shrinkage evokes expression of osmoprotective genes via nuclear factor NFAT5-mediated pathway and activates Na+ influx via hypertonicity-induced cation channels (HICC). In human umbilical vein endothelial cells (HUVEC) elevation of intracellular sodium concentration ([Na+]i) triggers transcription of dozens of early response genes (ERG). This study examined the role of monovalent cations in the expression of Na+i-sensitive ERGs in iso- and hyperosmotically shrunken HUVEC. METHODS: Cell volume was measured by 3D reconstruction of cell shape and as 14C-urea available space. Intracellular Na+ and K+ content was measured by flame atomic absorption spectrometry. ERG transcription was estimated by RT-PCR. RESULTS: Elevation of medium osmolality by 150 mM mannitol or cell transfer from hypo- to isosmotic medium decreased cell volume by 40-50%. Hyperosmotic medium increased [Na+]i by 2-fold whereas isosmotic shrinkage had no impact on this parameter. Hyperosmotic but not isosmotic shrinkage increased up-to 5-fold the content of EGR1, FOS, ATF3, ZFP36 and JUN mRNAs. Expression of these ERGs triggered by hyperosmotic shrinkage and Na+,K+-ATPase inhibition by 0.1 µM ouabain exhibited positive correlation (R2=0.9383, p=0.0005). Isosmotic substitution of NaCl by N-methyl-D-glucamine abolished an increment of [Na+]i and ERG expression triggered by mannitol addition. CONCLUSION: Augmented expression of ERGs in hyperosmotically shrunken HUVEC is mediated by elevation of [Na+]i.

Ubiquitous and cell type-specific transcriptomic changes triggered by dissipation of monovalent cation gradients in rodent cells: Physiological and pathophysiological implications.

Elevation of [Na+]i/[K+]i-ratio is considered as one of the major signals triggering transcriptomic changes in various cells types. In this study, we identified ubiquitous and cell type-specific [Formula: see text] -sensitive genes by comparative analysis of transcriptomic changes in ouabain-treated rat aorta smooth muscle cells and rat aorta endothelial cells (RASMC and RAEC, respectively), rat cerebellar granule cells (RCGC), and mouse C2C12 myoblasts. Exposure of the cells to ouabain increased intracellular Na+ content by ~14, 8, 7, and 6-fold and resulted in appearance of 7577, 2698, 2120, and 1146 differentially expressed transcripts in RAEC, RASMC, C2C12, and RCGC, respectively. Eighty-three genes were found as the intersection of the four sets of identified transcripts corresponding to each cell type and are classified as ubiquitous. Among the 10 top upregulated ubiquitous transcripts are the following: Dusp6, Plk3, Trib1, Ccl7, Mafk, Atf3, Ptgs2, Cxcl1, Spry4, and Coq10b. Unique transcripts whose expression is cell-specific include 4897, 1523, 789, and 494 transcripts for RAEC, RASMC, C2C12, and RCGC, respectively. The role of gene expression and signal pathways induced by dissipation of transmembrane gradient of monovalent cations in the development of various diseases is discussed with special attention to cardiovascular and pulmonary illnesses.

Augmented gene expression triggered by Na+,K+-ATPase inhibition: Role of Ca2+i-mediated and -independent excitation-transcription coupling.

In rat vascular smooth muscle cells (RVSMC), 3-h Na+,K+-ATPase inhibition by ouabain or in K+-free medium resulted in the inversion of the [Na+]i/[K+]i ratio and elevation up to 7-fold the content of Egr1, Atf3, Nr4a1 and Ptgs2 mRNAs. Ouabain increased the rate of 45Ca2+ influx by 2-fold that was abolished by L-type voltage-gated Ca2+ channel blocker nicardipine, but it was resistant to Na+/Ca2+ exchanger inhibitor KB-R7943. To study the role of Ca2+-mediated signaling in the expression of Na+i/K+i-sensitive genes we used intracellular Ca2+ chelator BAPTA and incubated RVSMC in Ca2+-free medium. The elevation of Nr4a1 and Ptgs2 expression triggered by ouabain was diminished in Ca2+-depeleted cells as well as in the presence of nicardipine and calmodulin antagonists A-7 and W-7. Ptgs2 expression was also suppressed by inhibitor of Ca2+/calmodulin-dependent protein kinase (CaMKII) KN-93 whereas increment of Nr4a1 content triggered by ouabain was attenuated by inhibitor of Ca2+/calmodulin-dependent protein phosphatase (calcineurin, CaN) cyclosporin A. Neither Ca2+ depletion nor above listed compounds had any impact on the augmented expression of Egr1 and Atf3 in ouabain-treated RVSMC. Our results strongly suggest that dissipation of transmembrane gradient of monovalent cations increases Ptgs2 and Nr4a1 transcription via augment Ca2+ influx through L-type Ca2+ channels that, in turn, leads to CaMKII-mediated phosphorylation of CREB and calcineurin-mediated dephosphorylation of NFAT, respectively. Additional experiments should be performed to identify intermediates of Na+i,K+i-mediated Ca2+-independent excitation-transcription coupling involved the regulation of Egr1 and Atf3 expression.

Time- and dose dependent actions of cardiotonic steroids on transcriptome and intracellular content of Na+ and K+: a comparative analysis.

Recent studies demonstrated that in addition to Na+,K+-ATPase inhibition cardiotonic steroids (CTSs) affect diverse intracellular signaling pathways. This study examines the relative impact of [Na+]i/[K+]i-mediated and -independent signaling in transcriptomic changes triggered by the endogenous CTSs ouabain and marinobufagenin (MBG) in human umbilical vein endothelial cells (HUVEC). We noted that prolongation of incubation increased the apparent affinity for ouabain estimated by the loss of [K+]i and gain of [Na+]i. Six hour exposure of HUVEC to 100 and 3,000 nM ouabain resulted in elevation of the [Na+]i/[K+]i ratio by ~15 and 80-fold and differential expression of 258 and 2185 transcripts, respectively. Neither [Na+]i/[K+]i ratio nor transcriptome were affected by 6-h incubation with 30 nM ouabain. The 96-h incubation with 3 nM ouabain or 30 nM MBG elevated the [Na+]i/[K+]i ratio by ~14 and 3-fold and led to differential expression of 880 and 484 transcripts, respectively. These parameters were not changed after 96-h incubation with 1 nM ouabain or 10 nM MBG. Thus, our results demonstrate that elevation of the [Na+]i/[K+]i ratio is an obligatory step for transcriptomic changes evoked by CTS in HUVEC. The molecular origin of upstream [Na+]i/[K+]i sensors involved in transcription regulation should be identified in forthcoming studies.

Deoxygenation Affects Composition of Membrane-Bound Proteins in Human Erythrocytes.

BACKGROUND/AIMS: ATP release from erythrocyte plays a key role in hypoxia-induced elevation of blood flow in systematic circulation. We have previously shown that hemolysis contributes to erythrocyte ATP release triggered by several stimuli, including hypoxia, but the molecular mechanisms of hypoxia-increased membrane fragility remain unknown. METHODS: In this study, we compared the action of hypoxia on hemolysis, ATP release and the composition of membrane-bound proteins in human erythrocytes. RESULTS: Twenty minutes incubation of human erythrocytes in the oxygen-free environment increased the content of extracellular hemoglobin by ∼1.5 fold. Paired measurements of hemoglobin and ATP content in the same samples, showed a positive correlation between hemolysis and ATP release. Comparative analysis of SDS-PAGE electrophoresis of erythrocyte ghosts obtained under control and deoxygenated conditions revealed a ∼2-fold elevation of the content of membrane-bound protein with Mr of ∼60 kDa. CONCLUSION: Deoxygenation of human erythrocytes affects composition of membrane-bound proteins. Additional experiments should be performed to identify the molecular origin of 60 kDa protein and its role in the attenuation of erythrocyte integrity and ATP release in hypoxic conditions.