Efforts to develop effective aqueous suppressants that offer decreased morbidity and mortality in comparison to those currently available for glaucoma therapy, will likely rely on the ability to alter the specific cellular events which underlie aqueous humor production by the ciliary epithelium. However, the secretory process which results in aqueous humor production is incompletely understood and the identification of precise cellular mechanisms which underlie this process remain to be established. In this application we present evidence that vacuolar H+-ATPase resides in cytoplasmic and plasma membrane domains of the ciliary epithelium and may act as an important ion motive force in aqueous humor production. Translocation of this enzyme to and from the plasma membrane appears to be an important mechanism by which aqueous humor production may be regulated by drugs, hormones and neurotransmitters. We propose biochemical and functional experiments to accomplish studying the role of the H+-ATPase on water and ion transport by the ciliary epithelium as a newly described mechanism by which aqueous humor is produced and regulated. These studies will include discerning the mechanism of action of H+-ATPase inhibitors such as bafilomycin, and polyanions such as oligonucleotides, on aqueous humor dynamics. Transepithelial measurements of isolated, intact iris-ciliary body will be performed in the presence of H+-ATPase inhibitors. Measurement of intracellular pH in ciliary epithelial cells and isolated ciliary epithelium will be performed. Immunoblotting will be performed to quantitate the total levels of H+-ATPase subunits in cytosolic and membrane fractions of rabbit ciliary epithelium and in cultured ciliary epithelial cells in response to several classes of drugs. Experiments incorporating 35S-methionine labeling and immunoprecipitation with selective antibodies for the 56kD subunit of the H+-ATPase, will assess the ATPase subunit synthesis following drug pretreatment and whether other proteins associate with vacuolar H+-ATPase in the ciliary epithelium that may mediate the effects of putative translocation. Quantitative PCR for H+-ATPase mRNA will assess the expression level of mRNA. Finally, in situ hybridization will evaluate the differential expression of H+-ATPase mRNA in ciliary body tip and crypt. Specific H+-ATPase activity and the proton translocation function of the native ciliary epithelial enzyme in response to exogenous drugs and inhibitors will be performed, in addition to searching for possible endogenous modulators of H+ATPase function. Lastly, an effort will be made to inactivate the H+-ATPase activity in cultured cells using antisense RNA technology. In summary, the identification of vacuolar proton ATPase in the ciliary epithelium now permits further investigation of its functional role in this secretory tissue. Studies of H+-ATPase function and regulation may provide a new target for pharmacologic manipulation of ciliary epithelial ion transport to suppress aqueous production and lower intraocular pressure in the treatment of glaucoma.
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