Depth of the Steroid Core Location Determines the Mode of Na,K-ATPase Inhibition by Cardiotonic Steroids.

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).

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.

In vitro assay for the efficacy assessment of AAV vectors expressing microdystrophin.

AAV-delivered microdystrophin genes hold great promise for Duchenne muscular dystrophy (DMD) treatment. It is anticipated that the optimization of engineered dystrophin genes will be required to increase the efficacy and reduce the immunogenicity of transgenic proteins. An in vitro system is required for the efficacy testing of genetically engineered dystrophin genes. We report here on the proof of concept for an in vitro assay based on the assessment of sarcolemma damage after repetitively applied electrical stimuli. The primary cell culture of myoblasts was established from wild-type C57BL/10ScSnJ and dystrophin-deficient mdx mice. The preparation parameters and the differentiation of contractile myotubes were optimized. DAPI and TO-PRO-3 dyes were used to assess myotubular membrane permeability in response to electrical pulse stimulation (EPS). Myotubes derived from mdx mice exhibited a greater increase in membrane damage, as assessed by TO-PRO-3-measured permeability after EPS, than was exhibited by the healthy control myotubes. AAV-DJ particles carrying the microdystrophin gene were used to transduce mdx-derived differentiated myotubes. Microdystrophin delivery ameliorated the disease phenotype and reduced the EPS-induced membrane damage to a level comparable to that of the healthy controls. Thus, the in vitro system was shown to be capable of supporting studies on DMD gene therapy.

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.

Transcriptomic changes in C2C12 myotubes triggered by electrical stimulation: Role of Ca2+i-mediated and Ca2+i-independent signaling and elevated [Na+]i/[K+]i ratio.

Elevation of Ca2+i and AMP-activated protein kinase (AMPK) are considered as major signals triggering transcriptomic changes in exercising skeletal muscle. Electrical pulse stimulation (EPS) of cultured myotubes is widely employed as an in vitro model of muscle contraction. This study examines the impact of Ca2+i-mediated and Ca2+i-independent signaling in transcriptomic changes in EPS-treated C2C12 myotubes. Electrical pulse stimulation (40 V, 1 Hz, 10 ms, 2 h) resulted in [Ca2+]i oscillations, gain of Na+i, loss of K+i, and differential expression of 3215 transcripts. Additions of 10 µM nicardipine abolished [Ca2+]i oscillations but did not affect elevation of the [Na+]i/[K+]i ratio seen in EPS-treated myotubes. Differential expression of 1018 transcripts was preserved in the presence of nicardipine, indicating a Ca2+i-independent mechanism of excitation-transcription coupling. Among nicardipine-resistant transcripts, we noted 113 transcripts whose expression was also affected by partial Na+,K+-ATPase inhibition with 30 µM ouabain providing the same elevation of the [Na+]i/[K+]i ratio as in EPS-treated cells. Electrical pulse stimulation increased phosphorylation of CREB, ATF-1, Akt, ERK, and p38 MAPK without any impact on phosphorylation of acetyl-CoA carboxylase and Unc-51 like autophagy activating kinase-1, i.e. downstream markers of AMPK activation. Unlike CREB, ATF-1, and MAPKs, an increment in Akt phosphorylation was abolished by nicardipine. Thus, our results show that Ca2+i-independent signaling plays a key role in altered expression of 30% of studied genes in EPS-treated myotubes. This signaling pathway is at least partially triggered by dissipation of transmembrane gradients of monovalent cations.

Search for Upstream Cell Volume Sensors: The Role of Plasma Membrane and Cytoplasmic Hydrogel.

The plasma membrane plays a prominent role in the regulation of cell volume by mediating selective transport of extra- and intracellular osmolytes. Recent studies show that upstream sensors of cell volume changes are mainly located within the cytoplasm that displays properties of a hydrogel and not in the plasma membrane. Cell volume changes occurring in anisosmotic medium as well as in isosmotic environment affect properties of cytoplasmic hydrogel that, in turn, trigger rapid regulatory volume increase and decrease (RVI and RVD). The downstream signaling pathways include reorganization of 2D cytoskeleton and altered composition of polyphosphoinositides located on the inner surface of the plasma membrane. In addition to its action on physico-chemical properties of cytoplasmic hydrogel, cell volume changes in anisosmotic conditions affect the ionic strength of the cytoplasm and the [Na+]i/[K+]i ratio. Elevated intracellular ionic strength evoked by long term exposure of cells to hypertonic environment resulted in the activation of TonEBP and augmented expression of genes controlling intracellular organic osmolyte levels. The role of Na+i/K+i -sensitive, Ca2+i -mediated and Ca2+i-independent mechanisms of excitation-transcription coupling in cell volume-adjustment remains unknown.

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.

Vasoconstriction triggered by hydrogen sulfide: Evidence for Na+,K+,2Cl-cotransport and L-type Ca2+ channel-mediated pathway.

OBJECTIVES: This study examined the dose-dependent actions of hydrogen sulfide donor sodium hydrosulphide (NaHS) on isometric contractions and ion transport in rat aorta smooth muscle cells (SMC). METHODS: Isometric contraction was measured in ring aortas segments from male Wistar rats. Activity of Na+/K+-pump and Na+,K+,2Cl-cotransport was measured in cultured endothelial and smooth muscle cells from the rat aorta as ouabain-sensitive and ouabain-resistant, bumetanide-sensitive components of the 86Rb influx, respectively. RESULTS: NaHS exhibited the bimodal action on contractions triggered by modest depolarization ([K+]o=30 mM). At 10-4 M, NaHS augmented contractions of intact and endothelium-denuded strips by ~ 15% and 25%, respectively, whereas at concentration of 10-3 M it decreased contractile responses by more than two-fold. Contractions evoked by 10-4 M NaHS were completely abolished by bumetanide, a potent inhibitor of Na+,K+,2Cl-cotransport, whereas the inhibition seen at 10-3 M NaHS was suppressed in the presence of K+ channel blocker TEA. In cultured SMC, 5×10-5 M NaHS increased Na+,K+,2Cl- - cotransport without any effect on the activity of this carrier in endothelial cells. In depolarized SMC, 45Ca influx was enhanced in the presence of 10-4 M NaHS and suppressed under elevation of [NaHS] up to 10-3 M. 45Ca influx triggered by 10-4 M NaHS was abolished by bumetanide and L-type Ca2+ channel blocker nicardipine. CONCLUSIONS: Our results strongly suggest that contractions of rat aortic rings triggered by low doses of NaHS are mediated by activation of Na+,K+,2Cl-cotransport and Ca2+ influx via L-type channels.

Electrical pulse stimulation decreases electrochemical Na+ and K+ gradients in C2C12 myotubes.

Electrical pulse stimulation (EPS)-treated cultured myotubes are widely employed as an in vitro model of muscle contraction. Here we examined time-dependent EPS action and dose-dependent ouabain action on [Na+]i and [K+]i in C2C12 myotubes. After 2 h of EPS (40 V, 1 Hz, 10 ms) [Na+]i increased by ∼150% whereas [K+]i declined by ∼20%. 3 µM ouabain had a negligible impact on [Na+]i and [K+]i in control cells but increased the [Na+]i/[K+]i ratio in EPS-treated myotubes by 85%. Thus, our results show for the first time that EPS results in dissipation of Na+ and K+ gradients in cultured myotubes and suggest that the augmented production of endogenous cardiotonic steroids may contribute to elevation of the [Na+]i/[K+]i ratio in exercising muscle.

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.