The experiments in this proposal are designed to analyze the regulation and function of the plasma membrane and intracellular mechanisms that determine ion and fluid transport across the bovine and human RPE. This should provide the basis for a pharmacology of the RPE and hopefully lead to the therapeutic alleviation of retina/RPE diseases that cause fluid accumulation in the subretinal space. Focus is on the extracellular signals, membrane receptors, and intracellular signalling pathways that help regulate: (1) ion, metabolite, and fluid movement across the RPE; and (2) the chemical composition of the cells and their environment. The choice of signals, receptors and pathways is restricted by functional considerations. For example, epinephrine is a potent stimulator of transepithelial fluid absorption (in vitro) and acetazolamide has been apparently successful in increasing fluid absorption from the subretinal space of patients with cystoid macular edema. In both cases, the membrane and cellular mechanisms that determine the efficacy of these drugs will to be determined. The preliminary data demonstrates that fresh explant tissue from human donors (adult or fetal) can be maintained and studied in-vitro for hours. The first step will be to determine the channels, cotransporters, exchangers and pumps at each membrane. The mechanisms that regulate pHi and [Ca++] will be determined. The hormones, neurotransmitters, growth factors and other substances that are known to affect human cultured RPE cells will be also studied with particular emphasis on those drugs or hormones already known to alter solute-linked fluid transport. Fetal tissue of varying gestational age will be used to follow the development of a key transport protein. The HCO3-dependent mechanisms seem to be absent in fetal RPE (~20 weeks). This could be because the proteins have not yet been assembled or if assembled have not yet been routed to the plasma membrane. Another possibility is that they are in place but not yet turned on. These possibilities will be studied by using polyclonal antibodies to a conserved COOH-terminal peptide of mouse band 3, which contains the catalytic site for anion exchange activity. Immunofluorescence microscopy will be used to localize this anion exchange- related polypeptide to intracellular or plasma membrane sites.
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