Related Experiment Videos
Ouabain-resistant human lymphoblastoid lines altered in the (Na+ + K+)-dependent ATPase membrane transport system
Abstract:
Diploid human lymphoblastoid cells with altered response to ouabain inhibition of the (Na+ + K+)-dependent ATPase transport system, manifest both in whole cells and in purified plasma membrane vesicles, were selected for their resistance to 0.1 muM ouabain. Ouabain-resistant (OUA(R)) cells with normal growth at 50 times this dose were recovered at a frequency 1 X 10(-6). This frequency was increased 9-fold after exposure to ethyl methane sulphonate but was decreased by the frameshift mutagen ICR-191, under conditions where both increased the frequency of 8-azaguanine-resistant colonies. The ouabain resistance phenotype was stable after 200 population doublings in the absence of ouabain. OUA(R) clones show showed 30-50% of the wild type amount of 3H-ouabain bound per cell, with the same dissociation constant for ouabain, 0.1 muM at 0.5 mM K+, as observed in wild-type cells. Both the initial rate of uptake of 86Rb+ in OUA(R) cells and the (Na+ + K+)-dependent ATPase activity of OUA(R) plasma membranes showed decreased sensitivity to ouabain inhibition. However, growth and transport properties of OUA(R) cells in the absence of ouabain were unchanged compared with wild type cells.
Insights
Researchers developed ouabain-resistant (OUA(R)) human cells by altering the sodium-potassium pump. These OUA(R) cells exhibit stable resistance and reduced sensitivity to ouabain, impacting the (Na+ + K+)-dependent ATPase transport system.
Area of Science:
- Cell Biology
- Molecular Biology
- Biochemistry
Background:
- The (Na+ + K+)-dependent ATPase is crucial for cellular ion transport and maintaining membrane potential.
- Ouabain is a specific inhibitor of this ATPase, widely used to study its function.
- Understanding mechanisms of drug resistance is vital for therapeutic development.
Purpose of the Study:
- To select and characterize human lymphoblastoid cells resistant to ouabain.
- To investigate the stability and nature of the ouabain resistance phenotype.
- To analyze the impact of ouabain resistance on the (Na+ + K+)-dependent ATPase activity and ion transport.
Main Methods:
- Selection of ouabain-resistant (OUA(R)) diploid human lymphoblastoid cells.
- Mutation frequency analysis using ethyl methane sulphonate and ICR-191.
- Stability assessment of OUA(R) phenotype over cell doublings.
- Quantification of 3H-ouabain binding and dissociation constant determination.
- Measurement of 86Rb+ uptake and (Na+ + K+)-dependent ATPase activity in OUA(R) cells and membranes.
Main Results:
- OUA(R) cells were recovered at a frequency of 1 X 10(-6), with stable resistance observed after 200 population doublings.
- Ethyl methane sulphonate increased OUA(R) frequency, while ICR-191 decreased it.
- OUA(R) cells bound 30-50% less 3H-ouabain, with an unchanged dissociation constant.
- Reduced ouabain sensitivity was observed in 86Rb+ uptake and (Na+ + K+)-dependent ATPase activity in OUA(R) cells.
- Normal growth and transport properties were maintained in OUA(R) cells without ouabain.
Conclusions:
- A stable ouabain-resistant phenotype was established in human lymphoblastoid cells.
- The resistance is associated with reduced ouabain binding and decreased sensitivity of the (Na+ + K+)-dependent ATPase.
- These findings provide insights into the mechanisms of ouabain resistance and ATPase function.