ATP LINKED EFFECTORS OF NA+, K+ ATPASE AND CATARACTS

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DESCRIPTION: The long term goal of this project is to determine the role of the lens Na,K-ATPases and their ATP-linked effectors (H+, Na+, K+, ATP, ADP, and digitalis alkaloids) in cataract formation. Historically, increased lens Na+ concentrations, common in human cataract lenses, were believed to result from the dysfunction of membrane Na+ transport systems of the lens epithelium. ATP hydrolysis by the Na, K-ATPase of the lens epithelium is altered in a majority of human cataract lenses. Furthermore, several of these changes in ATP hydrolysis correlate with the systemic diseases, diabetes and hypertension. More recent studies (presented in the progress report suggest a more complex picture. There are at least 3 types of Na, K-ATPase in the plasma membrane lens epithelium and one type of Na,K-ATPase in the plasma membrane of lens fibers. The Na,K-ATPase of the lens fibers is responsible for a majority of the lenticular Na+:K+ exchange. In human cataract lenses the lens fiber cell Na,K-ATPase is partially to completely inhibited. Na,K-ATPases are also present in cell nucleus and endoplasmic reticulum of cultured lens epithelial cells and in the cell nucleus and ER of rat liver. The primary focus of the proposed research is to characterize the Na,K-ATPases of the subcellular fractions of normal lenses. Hypothesis I. Na,K-ATPase subunits are active in the plasma membrane and subcellular organelles including the cell nucleus and endoplasmic reticulum. Aims I, II, and III are designed to test this hypothesis. Aim I is to determine the B subunit isoforms that are associated with the catalytic a subunit isoforms of the plasma membrane bound Na, K-ATPases of the mammalian lens. Aim II is to determine the subcellular locations of the a and B subunit isoforms in culture lens epithelial cells, in epithelium of fresh lens and in the superficial fibers of fresh lens using immunocytochemistry as well as antibody staining of Western blots of SDSPAGE separations of carefully prepared subcellular fractions. Aim III is to characterize the Na,K-ATPases in each subcellular fraction by the digitalis alkaloid binding studies, digitalis alkaloid sensitive ATP hydrolysis measurements and digitalis alkaloid sensitive phosphoenzyme formation. Hypothesis II. Chronic elevation of insulin or glucose change the interaction of the lens plasma membrane Na,K-ATPase with their ATP linked effectors, thus affecting changes in K+ transport. Aim IV is designed to test this hypothesis. Aim IV is to determine the effects the effects of chronic elevated glucose and insulin levels of K+ influx (86Rb+ as a tracer) in the epithelium and fibers of cultured lenses.