Glaucoma is the premier cause of blindness in African Americans and the second leading cause of blindness in the United States. While the mainstay of treatment is beta-adrenergic receptor (beta-AR) antagonists which suppress aqueous secretion, the signaling pathways regulating the ion channels and transporters coupled to aqueous secretion are appreciated primarily at a protein level. There is little understanding of the beta-AR receptors in the aqueous pathway at the molecular level. Much remains to be learned about """"""""upstream (e.g., transcriptional, translational, and post-translational events) and """"""""downstream"""""""" (e.g., distribution, phosphorylation, and sequestration) pathways relative to any of the G protein-coupled receptors known at the membrane level of the ciliary epithelial bilayer. The physician scientist candidate's long-term objective is to understand the molecular and cellular biology of G protein-coupled receptors mediating aqueous secretion. She proposes to learn the tools of these disciplines and to accomplish the following four specific aims: (I) The potential heterogeneous expression of the beta-AR (i.e., beta1, beta2, and beta3) in nonpigmented ciliary epithelium (NPE) will be studied by ribonuclease protection assay. (2) The correlation of transcript heterogeneity in these cells with protein expression will be assessed by radioligand binding and adenylate cyclase assays. These molecular, pharmacological, and biochemical studies will be conducted in 5V40- transformed human NPE and confirmed in nontransformed NPE cultures. (3) One of the principal mechanisms of steroid hormone action is modulating gene transcription; the 5'-flanking regions of the beta1- and beta2-AR genes have great homology to a glucocorticoid responsive element consensus sequence. A similar beta-AR regulatory process is hypothesized in NPE. Transcriptional up-regulation of these receptors may contribute to the pathophysiology of steroid-induced glaucoma. beta-AR transcript levels and protein expression will be assessed in NPE-cells exposed to dexamethasone. (4) Since the localization of the beta-AR has not yet been determined, in situ hybridization will be used to identify the cell types expressing beta-AR transcripts within the ciliary epithelial bilayer and other regions of the anterior segment. At the level of the plasma membrane, the cellular distribution of the beta-AR is expected to be polarized given the secretory nature of the ciliary bilayer. This receptor """"""""trafficking"""""""" pattern will be examined by confocal microscopy in cells and epithelial bilayer explants labeled with fluorescein-conjugated ligands, epitope- labeled receptors, or subtype specific antibodies; such studies reflect the physiological significance in native tissue. These proposed studies will lead to a better appreciation for the fundamental mechanisms that regulate aqueous production and that may play a role in the pathophysiology of glaucoma.
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