Corneal hydration and transparency are dependent on the ion and fluid transport properties of the corneal endothelium. Fluid transport from stroma to aqueous humor is apparently coupled to the net transport of Na and HCO3 and is sensitive to the pH of the bathing solution. A complete model for ion coupled fluid transport however, is not available. Since extra- and intracellular [HCO3] are determined by extra- and intracellular pH (pHo and pHi), it is reasonable to suggest that any model of HCO3 transport will require an understanding of pHi regulation. This notion is supported by work showing that additions of drugs which are known to influence pHi regulation (amiloride and DIDS) also inhibit endothelial fluid transport. This study proposes to identify and characterize the pHi regulatory and HCO3- transport mechanisms present in fresh and cultured bovine corneal endothelial cells by utilizing the pH sensitive intracellular fluorescent probe, BCECF. Cells will also be grown on filter supports to allow independent access to apical and basal surfaces in order to determine the locations of the HCO3 transport mechanisms and establish a complete model for transendothelial HCO3- transport. Transendothelial fluid secretion can also be measured across filter cultures so that the HCO3- transport mechanisms essential for fluid transport and the transport mechanism-that is rate limiting for fluid flux can be identified. A long term goal is to identify agents or conditions that stimulate HCO3- transport and test their ability to stimulate fluid secretion as well. Lastly, post-surgical specimens of Fuch's Endothelial Dystrophy will be obtained and the pH regulatory and HCO3- transport capacities will be tested in order to determine if and how pump function is deranged in this disease. Once the transport deficit is identified, the long term goal is to test drugs which stimulate it or drugs that inhibit an opposing transport mechanism in order to enhance fluid secretion in this diseased endothelia.
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