Cell Ion Homeostasis Research Articles

Quantification of metallothionein-III in brain tissues using liquid chromatography tandem mass spectrometry

Metallothioneins (MTs) are crucial for metal ion homeostasis in mammalian cells. Specialized mass spectrometry methods have been developed to detect MTs in tissue extracts, though facile methods with scalable throughput are lacking. To improve analytical throughput and repeatability, we developed a standardised liquid chromatography tandem mass spectrometry (LC-MS/MS) method for robust determination of metallothionein-3 (MT3) that is amenable to microplate processing. This method uses standard protein digestion conditions with commercially available reagents and commonly practiced reversed-phase chromatography, detecting MT3 at low ng/mL levels in human brain tissue extracts. We found that trypsin digestion largely underestimated MT3 levels, whereas endopeptidase Lys-C yielded vastly higher signals with low replicate variance. The choice of target peptide was critical for accurate MT3 detection – a peptide in the α-domain yielded the most robust signals. We demonstrate the utility of this method by comparing the expression of MT3 in post-mortem brain tissues of a cohort of Alzheimer's disease (AD) individuals and age-matched controls.

The Transient Receptor Potential Vanilloid Type 2 (TRPV2) Channel-A New Druggable Ca2+ Pathway in Red Cells, Implications for Red Cell Ion Homeostasis.

The Transient Receptor Potential Vanilloid Type 2 (TRPV2) Channel-A New Druggable Ca2+ Pathway in Red Cells, Implications for Red Cell Ion Homeostasis.

Human adenylate kinase 6 regulates WNK1 (with no lysine kinase-1) phosphorylation states and affects ion homeostasis in NT2 cells

Adenylate kinase 6 (AK6), a nucleus localized phosphotransferase in mammalians, shows ubiquitously expression and broad substrate activity in different tissues and cell types. Although the function of AK6 has been extensively studied in different cancer cell lines, its role in mammalian germline is still unknown. Here we showed that knockdown of AK6 inhibits cell proliferation and promotes cell apoptosis in human testicular carcinoma (NT2 cells). Co-immunoprecipitation experiment and in vitro pull down assay identified WNK1 (with no lysine kinase-1) as one of the AK6 interacting proteins in NT2 cells. Moreover, we found that AK6 regulates the phosphorylation states of WNK1 (Thr60) and affects phosphorylation level of Akt (Ser473) upon hypotonic condition, probably affecting chloride channel and regulating ion transport and homeostasis in NT2 cells and consequently contributing to the decreased cell proliferation rate. In conclusion, AK6 regulates WNK1 phosphorylation states and affects ion homeostasis in NT2 cells. These findings provide new insights into the function of AK6 and WNK1 in human testicular carcinoma. This work also provides foundation for further mechanism study of AK6 in spermatogenesis.

Cadmium exposure induces TNF-α-mediated necroptosis via FPR2/TGF-β/NF-κB pathway in swine myocardium

Cadmium (Cd) is one common environmental pollutant with systemic toxicity. Lipoxin A4 (LXA4) can regulate transforming growth factor-β (TGF-β) pathway and alleviate tissue injury via binding to formyl peptide receptor 2 (FPR2). The activation of nuclear factor-κB (NF-κB) pathway can promote the occurence of necroptosis. However, whether Cd exposure induces necroptosis in swine myocardium and the role of FPR2/TGF-β/NF-κB pathway in this process are unclear. Hence, we established Cd-exposed swine myocardial injury model by feeding a CdCl2 added diet (20 mg Cd/kg diet). Hematoxylin-eosin (H&E) staining was used to observe the morphological changes, and inductively coupled plasma mass spectrometry (ICP-MS) was performed to detect the levels of ion elements in myocardium. We further detected LXA4 and its receptor FPR2, TGF-β, Nrf2, NF-κB pathway and necroptosis related-genes expressions by RT-PCR and western blot. The results showed that Cd exposure induced necrotic cell death and ion homeostasis imbalance in swine myocardium. Moreover, Cd exposure increased the LXA4 content, inhibited the FPR2 expression, activated TGF-β pathway and suppressed Nrf2 pathway, activating the NF-κB pathway. In addition, Cd exposure increased the expressions of necroptosis related-genes TNF-α, TNFR1, RIP1, RIP3 and MLKL. It indicated Cd exposure induced necroptosis via FPR2/TGF-β/NF-κB pathway, revealing the potential mechanism of Cd-induced cardiotoxicity in swine myocardium.

Modulation of Ion Transport Across Plant Membranes by Polyamines: Understanding Specific Modes of Action Under Stress.

This work critically discusses the direct and indirect effects of natural polyamines and their catabolites such as reactive oxygen species and γ-aminobutyric acid on the activity of key plant ion-transporting proteins such as plasma membrane H+ and Ca2+ ATPases and K+-selective and cation channels in the plasma membrane and tonoplast, in the context of their involvement in stress responses. Docking analysis predicts a distinct binding for putrescine and longer polyamines within the pore of the vacuolar TPC1/SV channel, one of the key determinants of the cell ionic homeostasis and signaling under stress conditions, and an additional site for spermine, which overlaps with the cytosolic regulatory Ca2+-binding site. Several unresolved problems are summarized, including the correct estimates of the subcellular levels of polyamines and their catabolites, their unexplored effects on nucleotide-gated and glutamate receptor channels of cell membranes and Ca2+-permeable and K+-selective channels in the membranes of plant mitochondria and chloroplasts, and pleiotropic mechanisms of polyamines’ action on H+ and Ca2+ pumps.

Physiological and molecular responses of wheat plants to mycorrhizal and epibrassinolide interactions under salinity

Soil salinity is a major constraint on plant growth and metabolism. This work was carried out to study the possible responses of wheat (Triticum aestivum L.) plants to 24-epibrassinolide (EBL) and the arbuscular mycorrhizal (AM) fungus, Glomus mosseae, supplied alone or in combination under salt stress. Plants were leaf sprayed with 5 µM EBL, and then were treated with 0 and 150 mM NaCl for 10 days. Then plants were harvested for biochemical and gene expression (Real-time PCR) analyses involved. All data were analyzed by SPSS v.18 and the treatment means were compared by using Duncan’s test at P < 0.05 level of significance. The findings showed improved protein biosynthesis, K+ accumulation and K+/Na+ ratio in shoots as well as reduced Na+ levels in shoots and roots when EBL and G. mosseae were applied together under saline conditions. In addition, the expression of the NHX1 gene was up-regulated in EBL and AM fungi treatments and their interactions compared to control plants under salinity, however, no differences were found when compared with single applications. These results point to the possibility that BRs and AM fungi may have potential in alleviating salt stress by maintaining ion homeostasis in cells and modulating Na+/H+ antiporters.

Role of the Cation-Chloride-Cotransporters in Cardiovascular Disease.

The SLC12 family of cation-chloride-cotransporters (CCCs) is comprised of potassium chloride cotransporters (KCCs), which mediate Cl− extrusion and sodium-potassium chloride cotransporters (N[K]CCs), which mediate Cl− loading. The CCCs play vital roles in cell volume regulation and ion homeostasis. The functions of CCCs influence a variety of physiological processes, many of which overlap with the pathophysiology of cardiovascular disease. Although not all of the cotransporters have been linked to Mendelian genetic disorders, recent studies have provided new insights into their functional role in vascular and renal cells in addition to their contribution to cardiovascular diseases. Particularly, an imbalance in potassium levels promotes the pathogenesis of atherosclerosis and disturbances in sodium homeostasis are one of the causes of hypertension. Recent findings suggest hypothalamic signaling as a key signaling pathway in the pathophysiology of hypertension. In this review, we summarize and discuss the role of CCCs in cardiovascular disease with particular emphasis on knowledge gained in recent years on NKCCs and KCCs.

Subcellular mechanism of microbial inactivation during water disinfection by cold atmospheric-pressure plasma

Although the identification of effective reactive oxygen species (ROS) generated by plasma has been extensively studied, yet the subcellular mechanism of microbial inactivation has never been clearly elucidated in plasma disinfection processes. In this study, subcellular mechanism of yeast cell inactivation during plasma-liquid interaction was revealed in terms of comprehensive factors including cell morphology, membrane permeability, lipid peroxidation, membrane potential, intracellular redox homeostasis (intracellular ROS and H2O2, and antioxidant system (SOD, CAT and GSH)), intracellular ionic equilibrium (intracellular H+ and K+) and energy metabolism (mitochondrial membrane potential, intracellular Ca2+ and ATP level). The ROS analysis show that ·OH, 1O2, ·O2−and H2O2 were generated in this plasma-liquid interaction system and ·O2−served as the precursor of 1O2. Additionally, the solution pH was reduced. Plasma can effectively inactivate yeast cells mainly via apoptosis by damaging cell membrane, intracellular redox and ion homeostasis and energy metabolism as well as causing DNA fragmentation. ROS scavengers (l-His, d-Man and SOD) and pH buffer (phosphate buffer solution, PBS) were employed to investigate the role of five antimicrobial factors (·OH, 1O2, ·O2−, H2O2 and low pH) in plasma sterilization. Results show that they have different influences on the aforementioned cell physiological activities. The ·OH and 1O2 contributed most to the yeast inactivation. The ·OH mainly attacked cell membrane and increased cell membrane permeability. The disturb of cell energy metabolism was mainly attributed to 1O2. The damage of cell membrane as well as extracellular low pH could break the intracellular ionic equilibrium and further reduce cell membrane potential. The remarkable increase of intracellular H2O2 was mainly due to the influx of extracellular H2O2 via destroyed cell membrane, which played a little role in yeast inactivation during 10-min plasma treatment. These findings provide comprehensive insights into the antimicrobial mechanism of plasma, which can promote the development of plasma as an alternative water disinfection strategy.

Mechanism of Salt Stress Tolerance in Plants: Role of Cation/H+ Antiporters

Salinity is an important adverse environmental problem that caused a loss in the sense of reducing yield per plant, morphological, and physiological functions of crop plants. The plants compete with environmental stress conditions to withstand following normal growth and development. The exchange of cations or protons (H+) takes place across the cell membrane to maintain the osmotic pressure of cells under salt stress conditions. There is a huge number of cation/H+ antiporter 1 protein-producing gene by plant cells under salt stress conditions has been identified. However, a few have been characterized and sequenced which contributes to ion homeostasis and osmotic adjustment of cells. These cation/H+ antiporters are produced and stored in the vacuoles, endosomal forms and in the cytoplasm. The cation/H+ antiporters are involved in the homeostasis of K+, Na+, and pH of the cell under salinity stress conditions. The cation/H+ antiporters help plants cells to regulate all physiological functions under salt stress conditions.

Resistance to Bipyridyls Mediated by the TtgABC Efflux System in Pseudomonas putida KT2440.

Resistance-nodulation-division (RND) transporters are involved in antibiotic resistance and have a broad substrate specificity. However, the physiological significance of these efflux pumps is not fully understood. Here, we have investigated the role of the RND system TtgABC in resistance to metal ion chelators in the soil bacterium Pseudomonas putida KT2440. We observed that the combined action of an RND inhibitor and the chelator 2,2'-bipyridyl inhibited bacterial growth. In addition, the deletion of ttgB made the strain susceptible to 2,2'-bipyridyl and natural bipyridyl derivatives such as caerulomycin A, indicating that TtgABC is required for detoxification of compounds of the bipyridyl family. Searching for the basis of growth inhibition by bipyridyls, we found reduced adenosine triphosphate (ATP) levels in the ttgB mutant compared to the wild type. Furthermore, the expression of genes related to iron acquisition and the synthesis of the siderophore pyoverdine were reduced in the mutant compared to the wild type. Investigating the possibility that 2,2'-bipyridyl in the ttgB mutant mediates iron accumulation in cells (which would cause the upregulation of genes involved in oxidative stress via the Fenton reaction), we measured the expression of genes coding for proteins involved in intracellular iron storage and the response to oxidative stress. However, none of the genes was significantly upregulated. In a further search for a possible link between 2,2'-bipyridyl and the observed phenotypes, we considered the possibility that the ion chelator limits the intracellular availability of metabolically important metal ions. In this context, we found that the addition of copper restores the growth of the ttgB mutant and the production of pyoverdine, suggesting a relationship between copper availability and iron acquisition. Taken together, the results suggest that detoxification of metal chelating compounds of the bipyridyl family produced by other bacteria or higher ordered organisms is one of the native functions of the RND efflux pump TtgABC. Without the efflux pump, these compounds may interfere with cell ion homeostasis with adverse effects on cell metabolism, including siderophore production. Finally, our results suggest that TtgABC is involved in resistance to bile salts and deoxycholate.

Transcriptome Analysis of ppdnmt2 and Identification of Superoxide Dismutase as a Novel Interactor of DNMT2 in the Moss Physcomitrella patens.

DNMT2 is a DNA/tRNA cytosine methyltransferase that is highly conserved in structure and function in eukaryotes. In plants however, limited information is available on the function of this methyltransferase. We have previously reported that in the moss Physcomitrella patens, DNMT2 plays a crucial role in stress recovery and tRNAAsp transcription/stability under salt stress. To further investigate the role of PpDNMT2 at genome level, in this study we have performed RNA sequencing of ppdnmt2. Transcriptome analysis reveals a number of genes and pathways to function differentially and suggests a close link between PpDNMT2 function and osmotic and ionic stress tolerance. We propose PpDNMT2 to play a pivotal role in regulating salt tolerance by affecting molecular networks involved in stress perception and signal transduction that underlie maintenance of ion homeostasis in cells. We also examined interactome of PpDNMT2 using affinity purification (AP) coupled to mass spectrometry (AP-MS). Quantitative proteomic analysis reveals several chloroplast proteins involved in light reactions and carbon assimilation and proteins involved in stress response and some not implicated in stress to co-immunoprecipitate with PpDNMT2. Comparison between transcriptome and interactome datasets has revealed novel association between PpDNMT2 activity and the antioxidant enzyme Superoxide dismutase (SOD), protein turnover mediated by the Ubiquitin-proteasome system and epigenetic gene regulation. PpDNMT2 possibly exists in complex with CuZn-SODs in vivo and the two proteins also directly interact in the yeast nucleus as observed by yeast two-hybrid assay. Taken together, the work presented in this study sheds light on diverse roles of PpDNMT2 in maintaining molecular and physiological homeostasis in P. patens. This is a first report describing transcriptome and interactome of DNMT2 in any land plant.

Title: Excess Zn-induced changes in physiological parameters and expression levels of TaZips in two wheat genotypes

Zinc (Zn) is an important higher plant micronutrient; however, high amounts of Zn in soil may negatively affect plant growth. In this study, two wheat (Triticum aestivum L.) genotypes [Aikang58 (AK) and Zhengmai0856 (ZM)] were grown in nutrient solutions containing various Zn concentrations and were characterized using physiological and molecular approaches. Compared to sufficient Zn supply (0.25 mg L−1), excess Zn (2.5 mg L−1) significantly decreased the biomass and root morphological features (root length, volume, and surface area) of AK, whereas only a slight decrease was observed in ZM. ZM maintained higher Zn concentration and translocation factor, and lower net photosynthetic rate than AK under excess Zn supply. Excess Zn decreased Cu, Fe, and Mn concentrations in roots of both wheat genotypes, whereas Mn concentrations in shoots increased. In addition, the expression of TaZIPs indicated that TaZIP5, −6, and −13 in roots are primarily involved in Zn uptake when the medium has elevated Zn levels. The translocation of more Zn from the roots to shoots facilitates in maintaining the efficiency of photosynthesis by using lower amounts of photosynthetic pigments, and TaZIP genes involved in cell ion homeostasis contributes to greater Zn tolerance in ZM than AK.

Transcriptome analysis suggests mechanisms for a novel flowering type: Cleistogamous wheat

Wheat is one of the most important staple crops worldwide. Fusarium head blight severely reduces wheat yield and quality. Cultivation of a novel type of cleistogamous wheat mutant, ZK001, which was created by static magnetic field treatment, is a new strategy for controlling Fusarium head blight. However, little is known about the mechanism of cleistogamy in wheat. The present study demonstrated that anthers of ZK001 were retained on the glumes at all flowering stages, whereas those of YM18 were extruded from the paleae and lemmae. There was a clear difference in the morphological characteristics of lodicules between YM18 and ZK001. Lodicule calcium and potassium contents were significantly higher in YM18 than in ZK001 from white to yellow anther stages. In Fusarium head blight resistance, the diseased kernel rate and deoxynivalenol content of ZK001 were markedly lower than those of YM18 and QM725. Comparative transcriptome analysis of YM18 and ZK001 was performed to identify regulatory mechanisms of cleistogamy. The main differentially expressed genes identified in the spikelets of YM18 and ZK001 at the green anther stage were associated with cell walls, carbohydrates, phytohormones, water channel, and ion binding, transport, and homeostasis. These differentially expressed genes may play an important role in regulating cellular homeostasis, osmotic pressure, and lodicule development. The results indicate that ZK001 lost the ability to push the lemmae and paleae apart during the flowering stage because of the thin lodicules. ZK001 was speculated to provide structural barriers for Fusarium head blight during the flowering stage. The thin lodicule of ZK001 results from low levels of soluble sugar, calcium ions, and potassium ions in the lodicules. These levels are regulated by differentially expressed genes.

Irreversible versus repairable membrane poration: differences in permeabilization elicited by Bordetella Adenylate Cyclase Toxin and its hemolysin domain in macrophages.

Rapid plasma membrane repair in response to pore-forming toxins is crucial for cell survival, but the molecular mechanisms employed by eukaryotic nucleated cells to maintain membrane integrity and the specificities of such pathways remain poorly understood. Here, we have explored the permeabilization elicited by the Bordetellapertussis adenylate cyclase toxin, a 200-kDa protein toxin with α-helical pore-forming domain that forms pores of tunable size, and evaluated the response of target macrophages to such toxin poration. We show here that the response and the fate of target macrophages depend on toxin pore width. We find that the toxin's hemolysin moiety induces a transient membrane permeabilization by forming wide enough pores allowing Ca2+ influx into the target cell cytosol. This activates a Ca2+ -dependent cellular response involving exocytosis and endocytosis steps eliminating toxin pores and restoring membrane integrity. In contrast, the full-length native toxin, at low concentrations, forms very small pores that cause insidious perturbation of cell ion homeostasis that escapes control by the macrophage membrane repair response, eventually leading to cell death. Our data reveal that permeability to Ca2+ and ATP are key elements in the membrane repair pathway for eliminating α-helical pores of bacterial origin.

Arbuscular mycorrhizas modulate root polyamine metabolism to enhance drought tolerance of trifoliate orange

Arbuscular mycorrhizas enhance the drought tolerance of host plants through several underlying mechanisms. Polyamines (PAs) are known to protect plant cells from the damages of drought by enhancing the antioxidant defense system, restricting ethylene synthesis, maintaining cell pH and ion homeostasis, preventing chlorophyll loss, and so on; concomitantly, the mechanism through which arbuscular mycorrhizal (AM) fungi modulate PA metabolism to enhance drought tolerance of plants remains unclear. A pot experiment was conducted to assess how an AM fungus, Funneliformis mosseae, affects root PA homeostasis, activities and gene expressions of PA-related synthesizing and degrading enzymes, hydrogen peroxide (H2O2, a PA metabolite) production, and antioxidant enzyme gene expressions in trifoliate orange (Poncirus trifoliata) exposed to drought stress (DS). AM seedlings showed higher growth traits, leaf water potential, two plasma membrane intrinsic protein aquaporin gene expressions, and chlorophyll concentrations than non-AM seedlings under well-watered (WW) and DS conditions. Mycorrhizal treatment induced higher putrescine and cadaverine but lower spermidine and spermine concentrations, with higher activity of PA catabolic enzymes (copper-containing diamine oxidase, CuAO; polyamine oxidase, PAO) and putrescine synthases (ornithine decarboxylaseby; arginine decarboxylase, ADC). Mycorrhizas up-regulated the expression of the spermine synthase gene, PtSPMS, under DS, and down-regulated the transcript levels of PA catabolic enzyme genes (PtCuAO1, PtCuAO2, PtCuAO6, and PtCuAO8) and PA synthase genes (PtADC1 and PtADC2) under DS. PtPAO1, PtPAO2, and PtPAO3 had higher expression levels in AMF-inoculated seedlings, as compared to non-AMF-inoculated seedlings, under DS, triggering reactive oxygen species-related signalling for stress responsiveness through low H2O2 levels by up-regulating the expression of PtMn-SOD, PtCu/Zn-SOD, and PtCAT1. This study demonstrated that mycorrhizas have the capacity to modulate PA metabolism to enhance the drought tolerance of plants.

The SAUR41 subfamily of SMALL AUXIN UP RNA genes is abscisic acid inducible to modulate cell expansion and salt tolerance in Arabidopsis thaliana seedlings.

Most primary auxin response genes are classified into three families: AUX/IAA, GH3 and SAUR genes. Few studies have been conducted on Arabidopsis thaliana SAUR genes, possibly due to genetic redundancy among different subfamily members. Data mining on arabidopsis transcriptional profiles indicates that the SAUR41 subfamily members of SMALL AUXIN UP RNA genes are, strikingly, induced by an inhibitory phytohormone, abscisic acid (ABA). We aimed to reveal the physiological roles of arabidopsis SAUR41 subfamily genes containing SAUR40, SAUR41, SAUR71 and SAUR72. Transcriptional responses of arabidopsis SAUR41 genes to phytohormones were determined by quantitative real-time PCR. Knock out of SAUR41 genes was carried out with the CRISPR/Cas9 (clustered regulatory interspaced short palindromic repeats/CRISPR-associated protein 9) genome editing technique. The saur41/40/71/72 quadruple mutants, SAUR41 overexpression lines and the wild type were subjected to ultrastructural observation, transcriptome analysis and physiological characterization. Transcription of arabidopsis SAUR41 subfamily genes is activated by ABA but not by gibberellic acids and brassinosteroids. Quadruple mutations in saur41/40/71/72 led to reduced cell expansion/elongation in cotyledons and hypocotyls, opposite to the overexpression of SAUR41; however, an irregular arrangement of cell size and shape was observed in both cases. The quadruple mutants had increased transcription of calcium homeostasis/signalling genes in seedling shoots, and the SAUR41 overexpression lines had decreased transcription of iron homeostasis genes in roots and increased ABA biosynthesis in shoots. Notably, both the quadruple mutants and the SAUR41 overexpression lines were hypersensitive to salt stress during seedling establishment, whereas specific expression of SAUR41 under the ABA-responsive RD29A (Responsive to Desiccation 29A) promoter in the quadruple mutants rescued the inhibitory effect of salt stress. The SAUR41 subfamily genes of arabidopsis are ABA inducible to modulate cell expansion, ion homeostasis and salt tolerance. Our work may provide new candidate genes for improvement of plant abiotic stress tolerance.

Salt-Induced Damage is Alleviated by Short-Term Pre-Cold Treatment in Bermudagrass (Cynodon dactylon).

Excess salinity is a major environmental stress that limits growth and development of plants. Improving salt stress tolerance of plants is important in order to enhance land utilization and crop yield. Cold priming has been reported to trigger the protective processes in plants that increase their stress tolerance. Bermudagrass (Cynodon dactylon) is one of the most widely used turfgrass species around the world. However, the effect of cold priming on salt tolerance of bermudagrass is largely unknown. In the present study, wild bermudagrass was pre-treated with 4 °C for 6 h before 150 mM NaCl treatment for one week. The results showed that the cell membrane stability, ion homeostasis and photosynthesis process which are usually negatively affected by salt stress in bermudagrass were alleviated by short-term pre-cold treatment. Additionally, the gene expression profile also corresponded to the change of physiological indexes in bermudagrass. The results suggest that cold priming plays a positive role in improving salt stress tolerance of bermudagrass.

Expression of potassium channels is relevant for cell survival and migration in a murine bone marrow stromal cell line.

S17 is a clonogenic bone marrow stromal (BMS) cell line derived from mouse that has been extensively used to assess both human and murine hematopoiesis support capacity. However, very little is known about the expression of potassium ion channels and their function in cell survival and migration in these cells. Thus, the present study was designed to characterize potassium ion channels using electrophysiological and molecular biological approaches in S17 BMS cells. The whole-cell configuration of the patch clamp technique has been applied to identify potassium ion currents and reverse transcription polymerase chain reaction (RT-PCR) used to determine their molecular identities. Based on gating kinetics and pharmacological modulation of the macroscopic currents we found the presence of four functional potassium ion channels in S17 BMS cells. These include a current rapidly activated and inactivated, tetraethylammonium-sensitive, (IKV ) in most (50%) cells; a fast activated and rapidly inactivating A-type K + current (IK A -like); a delayed rectifier K + current (IK DR ) and an inward rectifier potassium current (IK IR ), found in, respectively 4.5%, 26% and 24% of these cells. RT-PCR confirmed the presence of mRNA transcripts for the alpha subunit of the corresponding functional ion channels. Additionally, functional assays were performed to investigate the importance of potassium currents in cell survival and migration. 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide analyses revealed a reduction in cell viability, while wound healing assays revealed reduced migration potential in cells incubated with different potassium channel blockers. In conclusion, our data suggested that potassium currents might play a role in the maintenance of overall S17 cell ionic homeostasis directly affecting cell survival and migration.

Kinetic studies of K-Cl cotransport in cultured rat vascular smooth muscle cells.

During aging, and development of atherosclerosis and cardiovascular disease (CVD), aortic vascular smooth muscle cells (VSMCs) transition from healthy contractile to diseased synthetic phenotypes. K-Cl cotransport (KCC) maintains cell volume and ion homeostasis in growth and differentiation, and hence is important for VSMC proliferation and migration. Therefore, KCC activity may play a role in the contractile-to-synthetic VSMC phenotypic transition. Early, medium, and late synthetic passage VSMCs were tested for specific cytoskeletal protein expression. KCC-mediated ouabain- and bumetanide-insensitive Rb+ (a K+ congener) influx was determined as Cl--dependent Rb+ influx at different external Rb+ and Cl- ion concentrations, [Rb+]o and [Cl-]o. Expressions of the cytoskeletal proteins α-actin, vimentin, and desmin fell from early through late synthetic VSMCs. KCC kinetic parameters, such as maximum velocity ( Vm), and apparent Cl- and Rb+ affinities ( Km), were calculated with Lineweaver-Burk, Hanes-Woolf, and Hill approximations. Vm values of both Rb+- and Cl--dependent influxes were of equal magnitude, commensurate with a KCC stoichiometry of unity, and rose threefold from early to late synthetic VSMCs. Hill coefficients for Rb+ and Cl- correlated with cell passage number, suggesting increased KCC ligand cooperativity. However, Km values for [Cl-]o were strikingly bimodal with 60-80 mM in early, ~20-30 mM in medium, and 60 mM in late passage cells. In contrast, Km values for [Rb+]o remained steady at ~17 mM. Since total KCC isoform expression was similar with cell passage, structure/function changes of the KCC signalosome may accompany the transition of aortic VSMCs from a healthy to a diseased phenotype.

Toxic Properties of Holothurian’s Triterpene Glycosides

Holothurians (or sea cucumbers) are echinoderms and are found in all areas of the world oceans. These animals produce special low-molecular metabolites - triterpene glycosides, which are a means of chemical protection of holothurians from predators. The content of triterpene glycosides in the tissues of holothurians can reach a significant amount of up to 1 g/kg, and especially many of these compounds are localized in the Cuvierian tubules - special protection organ presents in a number of tropical holothurians. Tritirepene glycosides of holothurians are quite toxic, they exhibit hemohttp://journal.ofhim.ru/index.php/vestnik/article/view/86lytic, cytotoxic and neurotoxic activity at a concentration range of 1×104–1×106 M. The toxic properties of glycosides are based on the ability of these compounds to interact with ∆ 5-sterols (mainly cholesterol) of plasma membranes and form ion-conducting complexes. In turn, this leads to a change in the ion permeability and selectivity of biomembranes, disruption of barrier properties, ion homeostasis and osmolarity of cells, and further to cell lysis and death. Contact with holoturias when diving in shallow water, which is fraught with damage to the eyes and mucous membranes by triterpene glycosides, may be of some danger to humans. Excessive consumption of commercial edible holothurians in food, especially without prior heat treatment, as is customary in some South-East Asia countries, can lead to diarrhea and dyspepsia. Triterpene glycosides in the bloodstream can lead to blood lysis and serious consequences up to death. The holothurians are animals commercially harvested in Russia. They are used in food and for the preparation of medicinal supplements and preparations. Excessive consumption of commercial edible holothurians for food, especially without long-term boiling, as is customary in Southeast Asia, can lead to diarrhea and dyspepsia. Triterpene glycosides in the bloodstream can lead to blood lysis and serious consequences up to death. The use of dietary supplements with an uncontrolled glycoside content is fraught with similar consequences, and the presence of immunosuppressants among glycosides that have fallen into such additives and drugs can worsen the condition of patients or sick animals. Control of the qualitative and quantitative content of glycosides in food and medicinal products from holothurians should be an integral part of measures to improve the biological safety of citizens of the Russian Federation