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P. Sidaway, N. Niyadurupola, D. C. Broadway, J. Sanderson; Functional Evidence for Purinoceptor Expression in RGC-5 Cells. Invest. Ophthalmol. Vis. Sci. 2009;50(13):82.
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There is known to be a release of purines from the posterior of the eye caused by a range of pathological insults. Responses of retinal ganglion cells to extracellular purines may be implicated in the pathogenesis of glaucoma.
A rat retinal ganglion cell line (RGC-5) was routinely cultured in DMEM, supplemented with 10% FCS. Prior to use, RGC-5 cells were differentiated by exposure to 1µM staurosporine (SS) for 24 hours. Cells were loaded with the ratiometric dye Fura-2 and used for calcium imaging up to 2 days post differentiation.
The purinoceptor agonists ATP, UTP, BzATP, ADP and UDP stimulate a concentration-dependant increase in intracellular calcium (Ca2+i) in RGC-5 cells. ATP and UTP had the greater affinity for RGC-5 purinoceptors and were equipotent (EC50~ 30µM). This compares to EC50s for BzATP, ADP and UDP of ~80µM, ~100µM and ~180µM respectively. ATP, UTP, and BzATP gave the highest maximal responses. Removal of extracellular calcium had no effect on ATP, UTP, BzATP, ADP or UDP-mediated calcium increase. ATP and UTP mediated responses were both inhibited by the P2 antagonists PPADS and suramin to a similar extent, and were also proven to be thapsigargin sensitive.
Differentiated RGC-5 cells can respond to a range of different purinoceptor agonists. Functional data suggests that these agonists are signaling through P2Y receptors. There was no evidence for activation of P2X receptors using these techniques. The equipotent data suggest that P2Y2 receptors are the dominant subtype.
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