Impact of manganese accumulation on Na,K-ATPase expression and function in the cerebellum and striatum of C57Bl/6 mice.

Overexposure to Mn causes a neurological disorder-manganism-with motor symptoms that overlap closely with disorders associated with haploinsufficiency in the gene encoding for α3 isoform of Na+,K+-ATPase (NKA). The present study was designed to test the hypothesis that behavioral changes in the mouse model of manganism may be associated with changes in the expression and activity of α3 NKA in the cerebellum (CB) and striatum (STR)-the key brain structures responsible for motor control in adult mice. C57Bl/6 mice were exposed to MnCl2 at 0.5 g/L (in drinking water) for up to eight weeks. After four weeks of Mn consumption, Mn levels were increased in the CB only. Behavioral tests demonstrated decreased performance of Mn-treated mice in the shuttle box test (third through sixth weeks), and the inclined grid walking test (first through sixth weeks), suggesting the development of learning impairment, decreased locomotion, and motor discoordination. The activity of NKA significantly decreased, and the expression of α1-α3 isoforms of NKA increased in the second week in the CB only. Thus, signs of learning and motor disturbances developing in this model of manganism are unlikely to be directly linked to disturbances in the expression or activity of NKA in the CB or STR. Whether these early changes may contribute to the pathogenesis of later behavioral deficits remains to be determined.

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.

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.

Transcriptomic changes triggered by ouabain in rat cerebellum granule cells: Role of α3- and α1-Na+,K+-ATPase-mediated signaling.

It was shown previously that inhibition of the ubiquitous α1 isoform of Na+,K+-ATPase by ouabain sharply affects gene expression profile via elevation of intracellular [Na+]i/[K+]i ratio. Unlike other cells, neurons are abundant in the α3 isoform of Na+,K+-ATPase, whose affinity in rodents to ouabain is 104-fold higher compared to the α1 isoform. With these sharp differences in mind, we compared transcriptomic changes in rat cerebellum granule cells triggered by inhibition of α1- and α3-Na+,K+-ATPase isoforms. Inhibition of α1- and α3-Na+,K+-ATPase isoforms by 1 mM ouabain resulted in dissipation of transmembrane Na+ and K+ gradients and differential expression of 994 transcripts, whereas selective inhibition of α3-Na+,K+-ATPase isoform by 100 nM ouabain affected expression of 144 transcripts without any impact on the [Na+]i/[K+]i ratio. The list of genes whose expression was affected by 1 mM ouabain by more than 2-fold was abundant in intermediates of intracellular signaling and transcription regulators, including augmented content of Npas4, Fos, Junb, Atf3, and Klf4 mRNAs, whose upregulated expression was demonstrated in neurons subjected to electrical and glutamatergic stimulation. The role [Na+]i/[K+]i-mediated signaling in transcriptomic changes involved in memory formation and storage should be examined further.

Anti-fibrotic effects of tannic acid through regulation of a sustained TGF-beta receptor signaling.

BACKGROUND: Pulmonary fibrosis is a progressive disease characterized by structural distortion of the lungs. Transforming growth factor-beta (TGF-beta) is a key cytokine implicated in the pathogenesis of pulmonary fibrosis. TGF-beta-induced myofibroblast differentiation characterized by expression of smooth muscle alpha-actin and extracellular matrix proteins is a key process in pathogenesis of fibrotic disease. Tannic acid is a natural polyphenol with diverse applications. In this study, we investigated the effect of tannic acid on myofibroblast differentiation and pulmonary fibrosis in cultured cells and in bleomycin model of the disease. METHODS: Primary cultured human lung fibroblasts (HLF) were used. The relative levels of proteins were determined by Western blotting. HLF contraction was measured by traction microscopy. Bleomycin-induced pulmonary fibrosis in mice was used as the disease model. RESULTS: Tannic acid inhibited TGF-beta-induced expression of collagen-1 and smooth muscle alpha-actin (SMA) as well as force generation by HLF. Tannic acid did not affect initial phosphorylation of Smad2 in response to TGF-beta, but significantly inhibited sustained Smad2 phosphorylation, which we recently described to be critical for TGF-beta-induced myofibroblast differentiation. Accordingly, tannic acid inhibited Smad-dependent gene transcription in response to TGF-beta, as assessed using luciferase reporter for the activity of Smad-binding elements. Finally, in mouse model of bleomycin-induced pulmonary fibrosis, therapeutic application of tannic acid resulted in a significant reduction of lung fibrosis, decrease in collagen-1 content and of Smad2 phosphorylation in the lungs. CONCLUSIONS: This study demonstrates the anti-fibrotic effect of tannic acid in vitro and in vivo through a regulation of sustained Smad2 phosphorylation.

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.

Control of lung myofibroblast transformation by monovalent ion transporters.

Myofibroblast differentiation is a critical process in the pathogenesis of tissue fibrosis. We focus our mini-review on recent data showing an implication of monovalent ion transporters in fibroblast to myofibroblast transformation of human lung fibroblasts (HLF). In cultured HLF, cardiotonic steroids (CTS) known as potent inhibitors of Na+,K+-ATPase suppress myofibroblast differentiation in parallel with up- and down-regulated expression of cyclooxygenase-2 (COX-2) and TGF-ß receptor subunit TGFBR2, respectively. K+-free medium mimics antifibrotic action of CTS indicating a key role of elevated intracellular [Na+]i/[K+]i ratio. Augmented expression of COX-2 is abolished by inhibition of Na+/Ca2+ exchanger. Side-by-side with CTS acting via elevation of the [Na+]i/[K+]i ratio fibroblast to myofibroblast transformation is also suppressed by potent inhibitors of Ca2+-activated chloride channels tannic acid and K+,Cl- cotransporter DIOA. The relative impact of [Formula: see text] -mediated and -independent signaling triggered by elevated [Na+]i/[K+]i ratio and altered intracellular anion handling in transcriptomic changes involved in myofibroblast differentiation should be examined further.

Na+,K+-ATPase as a Target for Treatment of Tissue Fibrosis.

Myofibroblast activation is a critical process in the pathogenesis of tissue fibrosis accounting for 45% of all deaths. No effective therapies are available for the treatment of fibrotic diseases. We focus our mini-review on recent data showing that cardiotonic steroids (CTS) that are known as potent inhibitors of Na+,K+-ATPase affect myofibroblast differentiation in a cell type-specific manner. In cultured human lung fibroblasts (HLF), epithelial cells, and cancer-associated fibroblasts, CTS blocked myofibroblast differentiation triggered by profibrotic cytokine TGF-ß. In contrast, in the absence of TGF-ß, CTS augmented myofibroblast differentiation of cultured cardiac fibroblasts. The cell type-specific action of CTS in myofibroblast differentiation is consistent with data obtained in in vivo studies. Thus, infusion of ouabain via osmotic mini-pumps attenuated the development of lung fibrosis in bleomycintreated mice, whereas marinobufagenin stimulated renal and cardiac fibrosis in rats with experimental renal injury. In TGF-ß-treated HLF, suppression of myofibroblast differentiation by ouabain is mediated by elevation of the [Na+]i/[K+]i ratio and is accompanied by upregulation of cyclooxygenase COX-2 and downregulation of TGF-ß receptor TGFBR2. Augmented expression of COX-2 is abolished by inhibition of Na+/Ca2+ exchanger, suggesting a key role of [Ca2+]i-mediated signaling. What is the relative impact in tissue fibrosis of [Na+]i,[K+]iindependent signaling documented in several types of CTS-treated cells? Do the different conformational transitions of Na+,K+-ATPase α1 subunit in the presence of ouabain and marinobufagenin contribute to their distinct involvement in myofibroblast differentiation? Additional experiments should be done to answer these questions and to develop novel pharmacological approaches for the treatment of fibrosis-related disorders.

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.

Na⁺i,K⁺i-Dependent and -Independent Signaling Triggered by Cardiotonic Steroids: Facts and Artifacts.

Na⁺,K⁺-ATPase is the only known receptor of cardiotonic steroids (CTS) whose interaction with catalytic α-subunits leads to inhibition of this enzyme. As predicted, CTS affect numerous cellular functions related to the maintenance of the transmembrane gradient of monovalent cations, such as electrical membrane potential, cell volume, transepithelial movement of salt and osmotically-obliged water, symport of Na⁺ with inorganic phosphate, glucose, amino acids, nucleotides, etc. During the last two decades, it was shown that side-by-side with these canonical Na⁺i/K⁺i-dependent cellular responses, long-term exposure to CTS affects transcription, translation, tight junction, cell adhesion and exhibits tissue-specific impact on cell survival and death. It was also shown that CTS trigger diverse signaling cascades via conformational transitions of the Na⁺,K⁺-ATPase α-subunit that, in turn, results in the activation of membrane-associated non-receptor tyrosine kinase Src, phosphatidylinositol 3-kinase and the inositol 1,4,5-triphosphate receptor. These findings allowed researchers to propose that endogenous CTS might be considered as a novel class of steroid hormones. We focus our review on the analysis of the relative impact Na⁺i,K⁺i-mediated and -independent pathways in cellular responses evoked by CTS.

Downregulation of TGF-ß Receptor-2 Expression and Signaling through Inhibition of Na/K-ATPase.

Transforming growth factor-beta (TGF-ß) is a multi-functional cytokine implicated in the control of cell growth and differentiation. TGF-ß signals through a complex of TGF-ß receptors 1 and 2 (TGFßR1 and TGFßR2) that phosphorylate and activate Smad2/3 transcription factors driving transcription of the Smad-target genes. The Na+/K+-ATPase is an integral plasma membrane protein critical for maintaining the electro-chemical gradient of Na+ and K+ in the cell. We found that inhibition of the Na+/K+ ATPase by ouabain results in a dramatic decrease in the expression of TGFßR2 in human lung fibrobalsts (HLF) at the mRNA and protein levels. This was accompanied by inhibition of TGF-ß-induced Smad phosphorylation and the expression of TGF-ß target genes, such as fibronectin and smooth muscle alpha-actin. Inhibition of Na+/K+ ATPase by an alternative approach (removal of extracellular potassium) had a similar effect in HLF. Finally, treatment of lung alveolar epithelial cells (A549) with ouabain also resulted in the downregulation of TGFßR2, the inhibition of TGF-ß-induced Smad phosphorylation and of the expression of mesenchymal markers, vimentin and fibronectin. Together, these data demonstrate a critical role of Na+/K+-ATPase in the control of TGFßR2 expression, TGF-ß signaling and cell responses to TGF-ß.

Ouabain-induced changes in MAP kinase phosphorylation in primary culture of rat cerebellar cells.

Cardiotonic steroid (CTS) ouabain is a well-established inhibitor of Na,K-ATPase capable of inducing signalling processes including changes in the activity of the mitogen activated protein kinases (MAPK) in various cell types. With increasing evidence of endogenous CTS in the blood and cerebrospinal fluid, it is of particular interest to study ouabain-induced signalling in neurons, especially the activation of MAPK, because they are the key kinases activated in response to extracellular signals and regulating cell survival, proliferation and apoptosis. In this study we investigated the effect of ouabain on the level of phosphorylation of three MAPK (ERK1/2, JNK and p38) and on cell survival in the primary culture of rat cerebellar cells. Using Western blotting we described the time course and concentration dependence of phosphorylation for ERK1/2, JNK and p38 in response to ouabain. We discovered that ouabain at a concentration of 1 µM does not cause cell death in cultured neurons while it changes the phosphorylation level of the three MAPK: ERK1/2 is phosphorylated transiently, p38 shows sustained phosphorylation, and JNK is dephosphorylated after a long-term incubation. We showed that ERK1/2 phosphorylation increase does not depend on ouabain-induced calcium increase and p38 activation. Changes in p38 phosphorylation, which is independent from ERK1/2 activation, are calcium dependent. Changes in JNK phosphorylation are calcium dependent and also depend on ERK1/2 and p38 activation. Ten-micromolar ouabain leads to cell death, and we conclude that different effects of 1-µM and 10-µM ouabain depend on different ERK1/2 and p38 phosphorylation profiles. Copyright © 2016 John Wiley & Sons, Ltd.

Critical role of the α1-Na(+), K(+)-ATPase subunit in insensitivity of rodent cells to cytotoxic action of ouabain.

In rodents, ubiquitous α1-Na(+), K(+)-ATPase is inhibited by ouabain and other cardiotonic steroids (CTS) at ~10(3)-fold higher concentrations than those effective in other mammals. To examine the specific roles of the CTS-sensitive α1S- and CTS-resistant α1R-Na(+), K(+)-ATPase isoforms, we compared the effects of ouabain on intracellular Na(+) and K(+) content, cell survival, and mitogen-activated protein kinases (MAPK) in human and rat vascular smooth muscle cells (HASMC and RASMC), human and rat endothelial cells (HUVEC and RAEC), and human and rat brain astrocytes. 6-h exposure of HASMC and HUVEC to 3 µM ouabain dramatically increased the intracellular [Na(+)]/[K(+)] ratio to the same extend as in RASMC and RAEC treated with 3000 µM ouabain. In 24, 3 µM ouabain triggered the death of all types of human cells used in this study. Unlike human cells, we did not detect any effect of 3000-5000 µM ouabain on the survival of rat cells, or smooth muscle cells from mouse aorta (MASMC). Unlike in the wild-type α1(R/R) mouse, ouabain triggered death of MASMC from α1(S/S) mouse expressing human α1-Na(+), K(+)-ATPase. Furthermore, transfection of HUVEC with rat α1R-Na(+), K(+)-ATPase protected them from the ouabain-induced death. In HUVEC, ouabain led to phosphorylation of p38 MAPK, whereas in RAEC it stimulated phosphorylation of ERK1/2. Overall, our results, demonstrate that the drastic differences in cytotoxic action of ouabain on human and rodent cells are caused by unique features of α1S/α1R-Na(+), K(+)-ATPase, rather than by any downstream CTS-sensitive/resistant components of the cell death machinery.

Carnosine prevents necrotic and apoptotic death of rat thymocytes via ouabain-sensitive Na/K-ATPase.

It is known that ouabain, a selective inhibitor of Na/K-ATPase, not only can cause the activation of signal cascades, which regulate the cell viability, but also can cause the accumulation of free radicals, which can evoke the oxidative stress. We have shown that the nanomolar concentrations of ouabain result in the temporary increase in the level of intracellular free radicals, but the millimolar concentration of ouabain induces a stable intracellular accumulation of free radicals in rat thymocytes. The increasing level of free radicals resulting from both low and high concentrations of ouabain can be attenuated by the antioxidant, carnosine. Moreover, the long-term incubation with ouabain leads to the cell death by necrosis and apoptosis. Ouabain-mediated apoptosis and necrosis were also abolished by carnosine.