Corneal deturgescence is part of a process in which the water content of the cornea is controlled by a dynamic process of pumping and leaking. The enzymes associated with pumping (corneal deturgescence) in fresh tissue and tissue cultures have been only partially defined and characterized. Two enzymes: Na+K+ATPase, in plasma membranes of corneal endothelial cells; and anion ATPase (ATP synthase), in the mitochondrial membranes of corneal endothelial cells, could be important in this process. For NA+K+ATPase, it is proposed to test varied levels of ATP, the cell's high energy compound, on its activity. In tissue cultures, activity of this enzyme will be assayed as a function of culture age and the number of culture passages. Furthermore, the influence of culturing with epithelial cells and/or keratocytes will be examined in regard to enzyme activity. This will be done such that the cells are separated from each other, but have culture media in common. Na+K+ATPase activity will also be measured in cultures maintained in low oxygen tension. In order to examine possible untoward effects of ocular drugs, the drugs will be included in tissue culture media at estimated in situ concentrations. Their possible effects on Na+K+ATPase activity will be investigated. Na+K+ATPase activity will be sub-localized in either baso-lateral or apical parts of the plasma membranes of corneal endothelial cells. This will be accomplished by subjecting membrane preparations to Percoll gradiant centrifugation. Mitochondrial, anion ATPase will be prepared as a complex and placed into liposomes with bacteriorhodopsin in order to form a biological model system where the enzyme functions as an ATP synthase (to generate ATP as in vivo). This system will be tested to determine whether it can be stimulated by bicarbonate. It may explain the participation of bicarbonate in deturgescence rather as a stimulator of an ATPase than as an ion which is transported by an ATPase. Together, all these studies may supply valuable basic information for a variety of chemically induced stresses that challange the process of deturgescence.
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