Steven Jason Crellin and Dr. Steven Graves, Department of Chemistry and Biochemistry
Age-related cataractogenesis is a worldwide leading cause of blindness. The fact that the causes as well as the apparent symptoms of cataract are diverse has contributed to increased difficulty in the study of the ailment. However, a ubiquitous symptom in animal cataract is the presence of elevated sodium concentrations within the lens. Therefore, studies have emphasized the importance of the sodium pump, or Na+,K+-ATPase in the lens, particularly in the lens epithelial cells.1 The sodium pump is primarily responsible for extruding three Na+ ions from within the cell and taking in two K+ ions, thus establishing a net negative charge within the cell. Noting increased intracellular sodium concentrations and the responsibility of the sodium pump for the maintenance of the intracellular sodium concentrations, researchers have searched for sodium pump inhibitors in vivo. They have isolated a class of compounds called digitalis-like factors or cardiac glycosides that are common inhibitors of sodium pump activity. 2 Ouabain is one such inhibitor.
Past experiments in my lab have demonstrated that, during 24 hour ouabain treatment, there is a shift in abundances of sodium pump units from the cytosol of bovine lens epithelial cells to the membrane. We then attributed this shift in sodium pump units to some type of cellular compensatory mechanism that would attempt to restore normal intracellular and extracellular ionic concentrations as well as the electrochemical gradient to normal values. The purpose of the present study was to ascertain the effectiveness of the observed shift in sodium pump units.
Bovine eyes were obtained from a local slaughterhouse and consequently the lens was removed. The epithelial cells were then cultured by removing the lens capsule and agitating the capsule in culture medium. The cells were then placed in culture plates and allowed to grow. After reaching confluence, some plates were treated with 10-5 M ouabain. Consequently, test and control plates were harvested and prepared for a non-radioactive rubidium (Rb) uptake assay.3 This preparation consisted of washing the cells in a choline chloride buffer to remove extracellular Rb and other impurities followed by a 30 minute incubation in a RbCl-PBS buffered solution. A RbCl buffer was used both because of its ability to be monitored by spectroscopic methods as well as because of the fact that the sodium pump can also take up Rb+ in a manner similar to K+. Consequently, the cells were washed with the aforementioned washing buffer to remove excess extracellular Rb. The cells were then lysed and prepared for the graphite furnace atomic absorption spectrometer, where intracellular Rb concentrations were determined. These values were then normalized by comparing them to the protein concentrations of each sample as determined by the Bradford Protein Assay to account for differences in cell size.
After performing the above assays on both control and test samples, the results were analyzed. Comparing the protein concentration determined by the Bradford assay to the Rb concentrations determined by graphite furnace atomic absorption spectrometry showed that the intracellular concentration of Rb in each of the ouabain samples averaged 55.33± 15.29% of the Rb concentrations of the control samples. This statistically significant difference in Rb concentrations within the cells shows that the previously observed compensatory shift of sodium pump units from the cytosol to the membrane was ultimately ineffective in restoring the electrochemical gradients established by the sodium pump in situ.
The obtained results were consistent with the hypothesized results before the analysis. We expected that, despite the increase in sodium pump abundances in the membrane region of the cells, the ability of the sodium pump to function would remain impaired. and consequently the appropriate cellular concentrations of Na+ and K+. This hypothesis was indeed verified by the significantly lower concentrations of Rb observed in the ouabain treated samples compared to the control samples. Hence, the cellular compensation for sodium pump inhibition is ultimately ineffective.
References
- Delamere, N.A., Tamiya, S., Expression, regulation and function of Na,K-ATPase in the lens. Prog. Ret. Eye Res., 23, 593-615 (2004).
- Samuelov, S., Lichtstein, D., Digitalis-like compounds and Na,K-ATPase activity in bovine lens. Eur. J. of Physiol., 433, 435-441 (1997).
- Graves, S.W., Zhen, Y., A novel assay of cell rubidium uptake using graphite furnace atomic
absorption: application to rats on a magnesium-deficient diet. J. Nutr. Biochem.,16(5):291-6. (2005).