Characterization of plasma membrane calcium ATPase in the human lens

The crystallin lens is an avascu1ar transparent organ that transmits that focuses light on the retina. The lens has two types of cells: a mono layer of epithelial cells on the anterior surface and fiber cells. Although most cells in the lens are fibers, the lens epithelium is responsible for active ion transport that is crucial to lens calcium, sodium, and potassium homeostasis. Ca2+ is involved in many cellular and physiological events in the lens. Calcium-ATPases are essential to the maintenance of calcium homeostasis. There is a close association between Ca2+ content and lens opacity. Cataract is a complex, multifactorial, and degenerative disorder and one of the most common eye diseases. In all human cataracts, the total calcium content is elevated.; Since Ca2+-ATPase is a major determinant of lens calcium homeostasis; we examined the expression of Ca2+-ATPase isoforms in human lenses with age and cataract. We also investigated the modulation of Ca2+-ATPase by hydrogen peroxide and calcium. We used Western blot, RT-PCR, and quantitative real time RT-PCR techniques to accomplish these goals. We examined the expression of different PMCA isoforms in the cataractous lenses and compared it with that of the clear younger and older lenses. We showed that PMCA1 - 4 are all expressed in the epithelium of the clear younger, and older, as well as cataractous human lenses, but not in the cortex or nucleus of the cataractous or clear lenses. We demonstrated that in cataractous human lenses, only PMCA2 is expressed at higher protein and mRNA levels, while the expression of PMCA1, PMCA3 and PMCA4 are unchanged in cataractous or clear older and younger lenses. Our results showed that all PMCA isoforms are expressed in both human lens epithelial and cultured human lens epithelial cell line (HLE B-3); we did not detect any PMCA isoforms in cortex or nucleus. We also examined the effect(s) of oxidation on the lens PMCA and SERCA isoforms in HLE B-3 cells. We treated the HLE B-3 cells with various concentrations of H2O2 and observed that H 2O2 caused a significant increase in both protein and mRNA levels of PMCA1, PMCA2, SERCA3, and SERCA2b as shown by Western blot and quantitative real time RT-PCR experiments. We also examined the effects of thapsigargin on the expression of PMCA and SERCA pumps. We showed that both PMCA1 and SERCA3 isoform protein and mRNA are upregulated in thapsigargin-treated HLE B-3 cells in a time and dose-dependent manner. But thapsigargin had no significant effect on the protein or mRNA levels of PMCA2, 3, 4 or SERCA2b. Inhibiting SERCA by thapsigargin elevates the intracellular calcium levels and epithelial cells respond to this calcium elevation by upregulating the calcium regulatory machinery of the cells. This probably indicates a compensatory mechanism to restore the calcium concentration to the physiological resting levels considering the harmful effects of increased intracellular levels in the cells.