Corneal transparency is determined by the hydration of the tissue which, in turn, is determined by the equilibrium between fluid entry into the tissue (leak) and its removal by Na+-K+ ATPase-dependent fluid transport out of the tissue (pump) by the endothelial cells. The chief theme of this proposal is how these pump and leak mechanisms are regulated. The first study will examine the hypothesis that the fluid pump is regulated by A2 adenosine receptor- and cyclic nucleotide-mediated mechanisms. Activated receptors may cause changes in intracellular second messenger systems, which would then influence fluid movement by modulating transport enzymes, exchangers or channels. Physiological (fluid transport) and biochemical (levels of cyclic nucleotide and enzyme activities) responses to modulators of adenosine A2 receptors will be compared, and the ionic dependence of these effects will be determined along with binding studies of specific agonists/antagonists. The second study will examine the Na+-K+ ATPase itself, assessing its distribution and trafficking with the endothelial cells. Fluid transport is driven by the enzyme sites located specifically on the plasma membrane; modification of turnover rates of these sites may provide a means of altering endothelial fluid movement. Turnover of the ATPase will be assessed by both immunocytochemical and biochemical methods, using brefeldin A to inhibit trafficking in experiments designed to evaluate this process in the recovery of enzyme activity which is observed in intact corneas following short-term ouabain exposure. The third study will determine what aspects of endothelial cell function are critically dependent upon the supply of ATP and the metabolism of glucose. In the absence of glucose, the fluid pump of the endothelium begins to fail, prior to any loss of ATP, probably due to loss of protection against oxidation. Cellular antioxidants will be measured before and during swelling and will be added exogenously in attempts to prevent or delay swelling. In ATP depleted corneas, swelling rates greatly exceed those found after complete inhibition of the fluid pump with ouabain, suggesting that ATP is critical for maintaining the permeability barrier. Permeability, 86Rb uptake, transendothelial resistance and morphology will be measured in corneas at different stages of ATP depletion. The three Aims of the proposed research examine the several roles of ATP in endothelial cell function; as precursor of cyclic nucleotide, as energy source for active transport, and as regulator of cell and tissue integrity.
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