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JA Eldred, J Sanderson, IM Wormstone, JR Reddan, G Duncan; Stress-induced ATP Release and Growth Modulation of Human Lens and Retinal Pigment Epithelial Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3544.
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Purpose: ATP release has been shown to occur following stimulation in several cellular systems. This study was undertaken to determine if lens and retinal epithelial cells release ATP in response to physiological stresses and to elucidate a possible role for ATP. Methods: Two cell lines, human lens epithelial cells (FHL 124) and retinal pigment epithelial cells (htert-RPE), were routinely cultured and seeded by placing a 25µl drop containing 5-10K cells onto a culture dish. The cells were allowed to adhere for 4 hours before flooding with serum-supplemented medium. Cells were serum-starved for 24 hours prior to exposure to experimental conditions. Growth was assessed over 4 days by the change in patch area stained with Coomassie blue and determined by image analysis. ATP levels in medium samples from cell cultures and aqueous humour samples from cataract patients were quantified using a luciferase detection kit (Roche). Extracellular proteins in cell culture samples were determined by the BCA assay (Pierce). Results: Analysis of aqueous humour samples showed a mean ATP level of 37.8±7.7nM (n=52). In order, to induce oxidative, osmotic and inflammatory associated stresses, 25µM H2O2, 50mM NaCl and 10ng/ml TGF ß2 respectively were added to FHL 124 and htert-RPE cultures maintained in serum-free medium. All treatments resulted in significantly increased levels of ATP in the medium within 10 minutes relative to untreated controls. In all cases external protein levels remained unaffected. The role of ATP on growth was then investigated. The patch areas of both lens and retinal epithelial cultures increased in serum-free medium and growth was further stimulated by addition of 10ng/ml EGF. Addition of ATP at concentrations as low as 0.1nM significantly inhibited growth of both cell types, under serum free or EGF-stimulated conditions. Conclusion: Aqueous humour from cataract patients contains detectable levels of ATP. Furthermore, lens and RPE cells release ATP in response to a range of different stresses. When added to the medium, low concentrations of ATP inhibit autocrine and EGF stimulated growth.
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