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10th International Congress "Cell Volume Regulation: Novel Therapeutic Targets and Pharmacological Approaches"
GLUTATIONYLATION OF a-SUBUNIT OF NA,K-ATPASE FROM RAT HEART RESULTS IN THE ENZYME INHIBITION
Meng, X.1, Petrushanko, I.Yu2, Klimanova, E.A.1, Dergousova, E.A.1, and Lopina, O.D.1
1 Department of Biochemistry, School of Biology, M. V. Lomonosov Moscow State University, Moscow, Russian Federation
2 Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Moscow, Russian Federation
Na,K-ATPase (Na-pump) is known to participate in the regulation of cell volume, therefore the change of Na-pump activity may be important for cell volume adjustment. We focused in this study on the regulation of Na,K-ATPase under hypoxia conditions when concentration of oxidized glutathione is increased. Using specific antibodies we have shown that partially purified preparation of Na,K-ATPase from rat heart contains al- and a2-isoforms. Both enzyme subunits were found in S-glutathionylated state. Glu-tathionylation of al- (but not a2-subunits) is partially removed if purification of Na,K-ATPase was made in the presence of dithiothreitol. Addition of oxidized glutathione
in vitro inhibits both isoenzymes and increases the level of S-glutathionylation of both subunits. Na,K-ATPase activity of a2-isoform was more sensitive to the action of oxidized glutathione than the activity of al-isoform. Inhibition of isoenzyme containing al-subunit is presented by two phases, inhibition constants are equal to 3821 and 246 M-1min-1 for fast and slow phases, respectively. Adenylic nucleotides protect Na,K-ATPase activity from the inhibition by oxidized glutathione, their protecting activity falls in the range: ATP, ADP, AMP.
Study was supported by RFBR grant №12-04-00103a and State Agreement № 16.512.11.2280.
THE USE OF TRANSMISSION-THROUGH-DYE MICROSCOPY FOR STUDYING APOPTOSIS AND VOLUME CHANGES IN ADHERENT CELLS
Model, M.A.
Department of Biological Sciences, Kent State University, Kent, Ohio, USA
In transmission-through-dye (TTD) microscopy, a nontoxic and cell-impermeant dye Acid Blue 9 is added to the cell culture medium, and the sample is placed in a shallow gap that is just slightly deeper than the cells. In the simplest case, such a gap can be formed between a coverslip and a slide kept apart by a small amount of grease. Alternatively, an attachment to a condenser with a horizontal watertight window at the bottom can be built for an inverted microscope; it is lowered into a Petri dish to bring the window close to the cells, thus creating a temporary narrow gap between the cells and the window. As the cells displace the dye, the depth of the absorbing layer becomes complementary to cell thickness; when such a sample is imaged in transmitted light at the wavelength of maximal dye absorption (630 nm), thicker cells appear brighter. By applying logarithmic transformation to the image and correcting it for the background level, the image contrast can be quantitatively converted to cell thickness and volume [1]. The accuracy of measurements by TTD imaging has been verified using spherical beads and confocal scanning. Dead cells with leaky plasma
membranes are darker than the background and are easily distinguishable from intact cells.
TTD can be realized on any widefield microscope, with only a single bandpass filter added anywhere in the optical path, or on a laser scanning microscope in transmission mode. It is well suited for prolonged time-lapse experiments because the cells are not exposed to high-intensity illumination and the results are not affected by possible instability in the light source. TTD is also compatible with fluorescence imaging, making possible simultaneous measurements of the cell volume and various fluorescent markers [2]. We are currently using this method to investigate cell volume changes during apoptosis and the osmotic properties of normal and apoptotic cells.
References
1. Model, M.A. 2012. Imaging the cell's third dimension. Microsc. Today, 20, pp. 32-37.
2. Kasim, N.R., Kuzelova, K., Holoubek, A., and Model, M.A. Live fluorescence and transmission-through-dye microscopic study of actinomycin-induced apoptosis and apoptotic volume decrease. Apoptosis, 2013, doi: 10.1007/s10495-013-0804-z
Бюллетень сибирской медицины, 2013, том 12, № 4, с. 24-68
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