The ultimate goal of this project is to contribute to our understanding of the mechanism of human senile cataract formation and to assist in the development of medical treatment strategies which will prevent or retard the process of cataractogenesis. Studies from this and other laboratories have shown that there are significant electrolyte and osmotic disturbances in the cataractous lens. From such observations, it is concluded that there is cell membrane dysfunction in most cortical cataracts and that these changes always include a loss of calcium homeostasis in the lens. We have hypothesized that derangement of calcium balance in the lens will lead to a cascade of events contributing substantially to cataract development. Calcium homeostasis is made possible through a delicate balance between lens cell membrane permeability and the energy dependent transport of calcium by Ca-ATPase at the level of the lens cell membrane (PM Ca-ATPase) and at the level of endoplasmic reticulum. (SERCA Ca-ATPase). It is hypothesized that these isoforms of Ca-ATPase are expressed differentially during aging, as a result of physiological and biochemical stress, and in the development of cataract. Thus, it is the objective of the proposed studies to assess the importance of Ca-ATPase isoforms in the clear lenses of differing ages and also in cataractous lenses. In order to better understand the normal modulation of Ca-ATPase isoform expression, we will use human cell culture methodology to explore factors that regulate isoform expression. It is anticipated that some of the changes seen in cell culture will parallel some of the changes observed in human lenses. A final objective is to examine the relative sensitivity of selected isoforms to post-translational modifications as a result of oxidative damage.
AIM 1 : Determine the changes in Ca-ATPase isoform expression that occur with normal aging and the development of cataract.
AIM 2 : Evaluate the modulation of Ca-ATPase isoform expression in cultured human epithelial cells. Isoform expression will be quantified using nuclease protection analysis.
AIM 3 : The functional properties of isolated isoforms of Ca-ATPase will be evaluated to show their relative sensitivity to oxidative inhibition, their dependence upon lipid environment, their activation requirements, and their transport function. We will apply techniques such as DNA recombinant transfection of a specific gene into immature cells to express an isoform of Ca-ATPase. The isoform will be purified and reconstituted into lipid vesicles where we can measure calcium transport kinetics under a variety of conditions. We will specifically examine the dependence of activity on lipid composition and oxidative stress. We hypothesize that isoforms will exhibit differing sensitivity to changing lipid environments and oxidative challenges.
