The retinal pigment epithelium controls a number of functions critical for the visual process, including the transport of fluid and ions out of subretinal space, and the periodic phagocytosis of disc membranes. shed from rod outer segments. Recent evidence suggests that the addition of purines ATP and adenosine to RPE cells can modify both of these functions. The goal of the current proposal is to determine the source of these purines and how their availability can be modified to affect phagocytosis and fluid flow. There are three Specific Aims. First, physiologic triggers for ATP release will be identified. In particular, the ability of neurochemicals showing circadian fluctuation to stimulate ATP release from fresh bovine RPE cells will be determine with the luciferin/luciferase assay system. The pathway for ATP exit will be probed using blockers for putative ATP transporters, including the cystic fibrosis transmembrane conductance regulator expressed in these cells.
The second aim i s to determine if this released ATP is converted extracellularly into adenosine. Adenosine levels will be measured directly in response to triggering the release of ATP. The activity of the enzymes responsible for the extracellular conversion of ATP to adenosine will be measured and altered pharmacologically. The specific isoforms of these enzymes will be identified using the PCR reaction. The specific isoforms of these enzymes will be identified using the PCR reaction. Third, physiologic ramifications produced by this route will be examined. Effects on phagocytosis will be monitored directly by examining the uptake of fluorescent rod outer segments and beads by RPE cells. Changes in the rate of C1-transport will be assessed by measured the C1-dependent short circuit current across the tissue. This research will provide information about the endogenous coordination of purinergic signals in the sub-retinal space by identifying the source of ATP and adenosine capable of stimulating purinergic receptors and the consequent effects of this stimulation on ion flow and phagocytosis.
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