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Ouabain-resistant (Na+,K+)-ATPase enzyme activity in chemically induced ouabain-resistant C3H/10T1/2 cells
M L Shibuya1, T Miura, J R Lillehaug
1Department of Pathology, University of Southern California, Los Angeles.
Abstract:
To further understand the molecular nature of changes leading to chemically induced ouabain resistance in C3H/10T1/2 Cl 8 (10T1/2) cells, we isolated plasma membranes from wild-type and ouabain-resistant (Ouar) 10T1/2 cells and characterized (Na,K)-ATPase activity in the plasma membrane fraction. (Na+,K+)-ATPase enzyme activity in membrane fractions extracted from wild-type 10T1/2 cells was inhibited in a concentration-dependent manner by ouabain and was completely inhibited by 2.4 mM ouabain. Lineweaver-Burke and Eisenthal/Cornish-Bowden analysis indicated that the inhibition was uncompetitive. Ten to 45% of (Na+,K+)-ATPase enzyme activity extracted from three Ouar 10T1/2 cell lines cultured in 1 mM ouabain was resistant to 2.4 mM ouabain, depending on the cell line. Resistance of (Na+,K+)-ATPase activity in the plasma membrane fraction of Ouar cells to inhibition by ouabain and resistance of cultured Ouar cells to the cytotoxicity of ouabain occurred over similar concentrations of ouabain (0.1-3mM). Two ouabain-resistant cell lines, Ouar MNNG Cl 2 and Ouar MCA Cl 16-7, demonstrated the same total (Na+,K+)-ATPase specific activity as 10T1/2 cells, but the fraction of Ouar enzyme activity increased (from 18 to 40% in MNNG Cl 2 cells and from 10 to 25% in Sp Ouar Cl 16 cells) when the cells were cultured in ouabain. Thermal denaturation profiles and pH dependence profiles of (Na+,K+)-ATPase activity in plasma membranes from wild-type and Ouar 10T1/2 cells were identical. A 3.9-kb (Na,K)-ATPase alpha subunit mRNA transcript was found in 10T1/2 cells, and in the Ouar MNNG Cl 2 cell line cultured in the presence or absence of ouabain. There was no amplification of the gene coding for the alpha subunit of (Na+,K+)-ATPase in the chemically induced Ouar MNNG Cl 2 cell line, whether this cell line was cultured in the presence or absence of ouabain. These studies provide further evidence that the Ouar phenotype of chemically induced and spontaneous Ouar 10T1/2 cell lines derives from Ouar (Na+,K+)-ATPase activity, and that this Ouar (Na+,K+)-ATPase activity increases further in some cell lines cultured in the presence of ouabain.
Insights
Chemically induced ouabain resistance in cells stems from altered sodium-potassium ATPase activity. This ouabain-resistant enzyme activity increases further when cells are cultured with ouabain, confirming its role in cellular resistance.
Area of Science:
- Biochemistry
- Cell Biology
- Molecular Biology
Background:
- Ouabain is a cardiac glycoside that inhibits the sodium-potassium ATPase (Na,K-ATPase) enzyme.
- Understanding the molecular basis of ouabain resistance is crucial for studying cellular defense mechanisms and drug resistance.
- C3H/10T1/2 Cl 8 (10T1/2) cells provide a model system to investigate chemically induced ouabain resistance.
Purpose of the Study:
- To elucidate the molecular mechanisms underlying chemically induced ouabain resistance in 10T1/2 cells.
- To characterize the activity and properties of (Na,K)-ATPase in ouabain-resistant (Ouar) cells compared to wild-type cells.
- To determine if changes in (Na,K)-ATPase gene expression or amplification contribute to ouabain resistance.
Main Methods:
- Isolation of plasma membranes from wild-type and Ouar 10T1/2 cells.
- Characterization of (Na,K)-ATPase enzyme activity, including inhibition kinetics by ouabain.
- Analysis of enzyme activity under varying ouabain concentrations and cellular culture conditions.
- Investigation of (Na,K)-ATPase alpha subunit mRNA transcript levels and gene amplification.
Main Results:
- (Na,K)-ATPase activity in wild-type cells was inhibited uncompetitively by ouabain.
- A significant fraction (10-45%) of (Na,K)-ATPase activity in Ouar cells was resistant to ouabain inhibition.
- The concentration range for ouabain resistance in enzyme activity mirrored cellular resistance to ouabain cytotoxicity.
- Ouar cell lines showed increased fractions of ouabain-resistant (Na,K)-ATPase activity upon culture in ouabain, without changes in total activity or alpha subunit mRNA levels/gene amplification.
Conclusions:
- The ouabain-resistant phenotype in 10T1/2 cells is directly linked to ouabain-resistant (Na,K)-ATPase activity.
- Culturing cells in ouabain can further increase the proportion of resistant (Na,K)-ATPase activity in certain cell lines.
- The resistance mechanism does not appear to involve amplification of the (Na,K)-ATPase alpha subunit gene or significant alterations in mRNA levels.