The long term objectives of this proposal are to delineate the processes occurring in the mammalian corneal endothelium responsible for the maintenance of corneal thickness, to determine the effects of phototoxic (superoxide) products on corneal endothelial structure and function, and determine the effects of surfactants on the cornea. To achieve these objectives, the specific aims are: a) to determine the relationship between ion and fluid movement in rabbit endothelium in order to differntiate between several current models of endothelial transport. This will be achieved by measuring the effects of drugs and ionic variations on both ion flux movements across the isolated endothelium, as well as by effects of these same perturbations on either swelling or deswelling rate of the cornea in the specular microscope. The effects of imposed transendothelial pH or PCO2 gradients on ion fluxes will be determined, as will the determination of the generation of changes in ambient pH and PCO2 that occur in sealed chambers on either side of the endothelium. The relationship between cell sixe and margin lengths and ion fluxes will be made in endothelium healing after a cryongenic injury to distinguish paracellular and transcellular ionic pathways as they may vary with transformation of healing endothelium from large to small cells. Intracellular electrical potential difference of endothelial cells will be examined in response to ionic (Na+, Cl-, K+, HCO3-) or gas (PCO2) changes of the bathing solution as well as in response to a variety of chemicals used to modify specific membrane events. The experiments are designed to allow distinction between a symport for HCO3- and Na+ at the basolateral cell border of the endothelium and a system of HCO3- transport with Na+ passing either via a paracellular pathway or in a parallel cellular system, as well as providing information on other ionic flux coupling. The results will be interpreted in light of prevailing models, or modifications thereof dependent upon the data; b) determine the in vivo response of the endothelium to hydrogen peroxide. A variety of studies will be made including pachometry, adenine uptake, ultrastructural studies, ouabin binding, and myeloperoxidase, catalase and superoxide dismutase assays; and c) surfactants such as the anionic sodium lauryl sulfate and the cationic benzalkonium chloride will be examined for effects on the ultrastructure of normal cornea and corneas in which the epithelium has been damaged either chemically (n- heptanol) or mechanically.
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