183 research outputs found

    Beneficial Renal and Pancreatic Phenotypes in a Mouse Deficient in FXYD2 Regulatory Subunit of Na,K-ATPase

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    The fundamental role of Na,K-ATPase in eukaryotic cells calls for complex and efficient regulation of its activity. Besides alterations in gene expression and trafficking, kinetic properties of the pump are modulated by reversible association with single span membrane proteins, the FXYDs. Seven members of the family are expressed in a tissue-specific manner, affecting pump kinetics in all possible permutations. This mini-review focuses on functional properties of FXYD2 studied in transfected cells, and on noteworthy and unexpected phenotypes discovered in a Fxyd2-/- mouse. FXYD2, the gamma subunit, reduces activity of Na,K-ATPase either by decreasing affinity for Na+, or reducing Vmax. FXYD2 mRNA splicing and editing provide another layer for regulation of Na,K-ATPase. In kidney of knockouts, there was elevated activity for Na,K-ATPase and for NCC and NKCC2 apical sodium transporters. That should lead to sodium retention and hypertension, however, the mice were in sodium balance and normotensive. Adult Fxyd2-/- mice also exhibited a mild pancreatic phenotype with enhanced glucose tolerance, elevation of circulating insulin, but no insulin resistance. There was an increase in beta cell proliferation and beta cell mass that correlated with activation of the PI3K-Akt pathway. The Fxyd2-/- mice are thus in a highly desirable state: the animals are resistant to Na+ retention, and showed improved glucose control, i.e. they display favorable metabolic adaptations to protect against development of salt-sensitive hypertension and diabetes. Investigation of the mechanisms of these adaptations in the mouse has the potential to unveil a novel therapeutic FXYD2-dependent strategy

    Immunoaffinity isolation of Na,K-ATPase α3 isoform from pig kidney

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    AbstractThe Na,K-ATPase α3 isoform of the catalytic subunit has been isolated from pig kidney microsomes. The procedure employs immunoaffinity chromatography on Sepharose 4B covalently coupled with monospecific antibodies a-II against the synthetic peptide including the putative α3 N-terminus. The structural analysis provides unambiguous proof that the isolated protein corresponds to the third transcript for the α3 isoform. The N-terminal amino acid sequence determined, Met-Gly-Asp-Lys-Lys-Asp-Asp, shows that unlike the α1 and α2 proteins, the mature Na,K-ATPase α3 isoform lacks post-translational proteolytic processing.ATPase isozyme, (Na+ + K+)-; Immunoaffinity chromatography; N-terminal sequence analysi

    Na+, K+-ATPase Subunit Composition in a Human Chondrocyte Cell Line; Evidence for the Presence of α1, α3, β1, β2 and β3 Isoforms

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    Membrane transport systems participate in fundamental activities such as cell cycle control, proliferation, survival, volume regulation, pH maintenance and regulation of extracellular matrix synthesis. Multiple isoforms of Na+, K+-ATPase are expressed in primary chondrocytes. Some of these isoforms have previously been reported to be expressed exclusively in electrically excitable cells (i.e., cardiomyocytes and neurons). Studying the distribution of Na+, K+-ATPase isoforms in chondrocytes makes it possible to document the diversity of isozyme pairing and to clarify issues concerning Na+, K+-ATPase isoform abundance and the physiological relevance of their expression. In this study, we investigated the expression of Na+, K+-ATPase in a human chondrocyte cell line (C-20/A4) using a combination of immunological and biochemical techniques. A panel of well-characterized antibodies revealed abundant expression of the α1, β1 and β2 isoforms. Western blot analysis of plasma membranes confirmed the above findings. Na+, K+-ATPase consists of multiple isozyme variants that endow chondrocytes with additional homeostatic control capabilities. In terms of Na+, K+-ATPase expression, the C-20/A4 cell line is phenotypically similar to primary and in situ chondrocytes. However, unlike freshly isolated chondrocytes, C-20/A4 cells are an easily accessible and convenient in vitro model for the study of Na+, K+-ATPase expression and regulation in chondrocytes

    Protein Phosphatase 2A Interacts with the Na+,K+-ATPase and Modulates Its Trafficking by Inhibition of Its Association with Arrestin

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    Background: The P-type ATPase family constitutes a collection of ion pumps that form phosphorylated intermediates during ion transport. One of the best known members of this family is the Na +,K +-ATPase. The catalytic subunit of the Na +,K +-ATPase includes several functional domains that determine its enzymatic and trafficking properties. Methodology/Principal Findings: Using the yeast two-hybrid system we found that protein phosphatase 2A (PP2A) catalytic C-subunit is a specific Na +,K +-ATPase interacting protein. PP-2A C-subunit interacted with the Na +,K +-ATPase, but not with the homologous sequences of the H +,K +-ATPase. We confirmed that the Na +,K +-ATPase interacts with a complex of A- and C-subunits in native rat kidney. Arrestins and G-protein coupled receptor kinases (GRKs) are important regulators of G-protein coupled receptor (GPCR) signaling, and they also regulate Na +,K +-ATPase trafficking through direct association. PP2A inhibits association between the Na +,K +-ATPase and arrestin, and diminishes the effect of arrestin on Na +,K +-ATPase trafficking. GRK phosphorylates the Na +,K +-ATPase and PP2A can at least partially reverse this phosphorylation. Conclusions/Significance: Taken together, these data demonstrate that the sodium pump belongs to a growing list of io

    Evaluation of the toxicity of ammonia polluted waters for Carassius auratus gibelio (Bloch, 1782) by the «time sampling» method

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    Purpose. To determine the possibility of performing an express-evaluation of the toxicity of ammonia polluted waters based on ethological parameters of Carassius auratus gibelio (Bloch,1782) with the use «time sampling» method. Methodology. For biotesting in August 2017, water samples were taken from the Denishevsky reservoir of the Teteriv river and water supply network of the water service company, which were placed in aquaria (10 dm3) and a control and experimental groups of fish were formed according to the following scheme: control group – samples of settled (24 hours) clean tap water; experimental group D-1 – water samples from the Denishivsky reservoir with the total ammonia concentration of 1.5 mg/dm3; experimental group D-2 – samples of settled (24 hours) tap water with the total ammonia concentration of 1.5 mg/dm3. Test-objects were females of C. auratus gibelio, in which the parameters of typical and non-typical motor activity and feeding behavior were determined. Fish were exposed for 12 hours. For both experimental groups, water toxicity indexes were calculated in comparison with the control. Findings. In the studies, we showed the possibility of using the «time sampling» method for rapid assessment of the toxicity of polluted waters with ammonium concentration of 1.5 mg/dm3 based the ethological parameters of Prussian carp (C. auratus gibelio), which included the instantaneous fixation of the number of individuals preferring one or another act of behavior. It was shown that C. auratus gibelio females reacted on ammonia content by impairing the motor and feeding behavior. Settled tap water artificially pollution with ammonia (group D-2) appeared to be more toxic for fish than water polluted by natural way. Immobilization of individual fish in the group D-1 was 6.60% lower, while their survival rate was 11.11% hogher compared to the group D-1. Unlike fish of the experience groups, fish in the control group exhibited only single untypical ethological acts. The calculation based on the behavioral parameters of Prussian carp females demonstrated mainly medium (group D-1) and high (group D-2) levels of acute toxicity of experimental waters. Originality. For the first time, the express-evaluation of the toxicity of water with elevated ammonium content (1.5 mg/dm3) was carried out by observing the motor activity and feeding behavior of Prussian carp (C. auratus gibelio) by the «time sampling» method. Practical use. The results of the study are used for the rapid assessment of the toxicity of water of surface water sources of the Teteriv river

    Influence of ammonia on forming the toxicity of waters from the surface sources of water supply determined on Carassius auratus gibelio (Bloch, 1782)

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    Purpose. Determination of the influence of ammonia in waters from surface sources of water supply of Zhytomyr city on forming the toxicity of these waters determined by test-reactions of atypical motor activity of Carassius auratus gibelio (Bloch, 1782) with the use of the «time sampling» method during 2012–2014. Methodology. Biotesting was performed at the Municipal Enterprise "Zhytomyrvodokanal". Water samples were taken once a month time from the Teteriv river reservoirs and tap water network and then placed into aquaria (8 dm3 on a group). Control and experimental groups of fish were formed according to the following scheme: control group — samples of settled (24 hours) tap water; experimental group D-1 — water samples from the Denyshivske reservoir; experimental group D-2 — water samples from the Vidsichne water intake. Test specimens were females of C. auratus gibelio. Biotesting was conducted using the «time sampling» method by keeping fish (n=30) in water for 12 hours. The toxicity indexes of waters were calculated on the basis of the following test-reactions: spiral-like and vector movements, jumping out from water, immobilization and death of fish. Statistical processing of study results were performed using cross-correlation and regression analysis in MS Excel 2007 and Statistica-6. Findings. The study showed an effect of ammonia on the toxicity of waters from reservoirs of the Teteriv river that was determined by atypical motor activity with the use of the «time sampling» method, which consisted in the instantaneous fixation of the number of individuals that favored one or another act of behavior. It was shown that females not adapted to the action of ammonia reacted to its concentration in water of more than 0.55 mg/dm3 by disorders in movements. Unlike fish of experience groups, only single pathological acts were observed in the control group. A positive moderate relationship, which had a tendency to an increase, was found between the ammonium content water of surface sources of water supply and their toxicity in 2012, and it reached high degree in 2014 (r=0.6773 in Denyshivske reservoir and r=0.6685 in Vidsichne water intake). The linear regression equations built from obtained data allow forecasting the toxicity of surface waters by ammonium concentration separately for the reservoir and water intake. Originality. Dependence of the toxicity of waters from the surface sources of water supply of Zhytomyr city on ammonium content in these waters was first determined by test-reaction of atypical motor activity of C. auratus gibelio with the use of the «time sampling» method. The linear regression equations allowed determining the dangerous threshold of ammonia concentration (0.58 mg/dm3) which resulted in 50% water toxicity for C. auratus gibelio

    EXPRESS-ASSESSMENT OF TOXISITY OF WATERS FROM SURFACE WATER SUPPLY SOURCES WITH THE USE OF DANIO RERIO

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    Purpose. An increase in the informativeness and shortening of the timing required for biotesting of water from surface water sources in the city of Zhytomyr with the use of Danio rerio Hamilton-Buchanan instead of the traditional test object Poecilia reticulata Peters and observation of their ethology using the «time sampling» method. Methods. Water samples were collected from reservoirs of the Teteriv river in April 2016. They were used placed in aquaria (10 dm3), where control and experimental groups were formed according to the following scheme: control group – samples with settled (24 hours) tap water; experimental group D-1 - water samples from the Denishivsky reservoir; experimental group D-2 – from water intake Vidsichne. Test specimens were females of P. reticulata and D. rerio, in which the parameters of typical and non-typical motor activity and feeding behavior were determined. Fish were exposed to water for 12 hours. Water toxicity indexes were determined for both experimental and control groups. Findings. The studies showed the possibility of using the "time sampling" method for rapid assessment of the water toxicity of surface water sources of Zhytomyr with the use of P. reticulata and D. rerio, which consists in the instantaneous fixation of the number of individuals preferring different behavior acts. D. rerio were more susceptible to the combined action of pollutants in experimental waters than the traditional test object guppies regarding the test reactions of increasing the vector movement and reducing the activity of consumed feed (in 12 hours after the start of biotesting on average by 8,43% in the group D1 and by 11,71% in the group D2). The calculation of toxicity indexes based on the above-mentioned ethological acts, which exceeded 50%, detected an average level of pollution of the experimental water. Originality. For the first time, the rapid assessment of the water toxicity of surface water sources in the city Zhytomyr was carried out by observing the motor activity and feeding behavior of D. rerio. Practical value. The results of the research are used for the rapid assessment of the toxicity of water of surface water sources in the city Zhytomyr

    The Mechanism of Na-K Interaction on Na,K-ATPase

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