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J. Sanderson, P. Sidaway, N. Niyadurupola, D. C. Broadway; Purinoceptor Activation Regulates IL-1β Expression in Retinal Pigment Epithelial Cells. Invest. Ophthalmol. Vis. Sci. 2008;49(13):5546.
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We have previously shown that human RPE cells express multiple purinoceptor subtypes that signal via Ca2+. The purpose of these experiments was to investigate downstream signalling of purinoceptor activation, specifically investigating regulation of IL-1β which may have a role to play in the pathophysiology of ocular diseases including glaucoma.
ARPE-19 cells were maintained at 35oC in DMEM/HamF12 (pH7.4) supplemented with 10% FCS. Ca2+ measurements were made using ratiometric digital imaging techniques with the Ca2+-indicator Fura-2. To investigate IL-1β expression, ARPE-19 cells were exposed to agonists for 24 hours in serum free medium. Expression was measured by real time quantitative (Taqman) RT-PCR.
The purinoceptor agonists ATP and BzATP (30s exposure) both caused a dose dependent increase in intracellular Ca2+ in ARPE-19 cells. ATP caused a biphasic increase with an initial peak indicative of rapid release from intracellular stores, followed by a lower sustained influx component lasting approximately 6 minutes. In contrast, the BzATP response was not biphasic, increasing rapidly, but then decreasing at a steady rate over an approximate 6 minute period. Exposure to the agonists for 24 hours caused a dose dependant increase in expression of IL-1β. ATP (100uM) and BzATP (100uM) resulted in a 3 and 3.5 fold increase in IL-1β mRNA respectively.
Purinoceptor stimulation causes an increase in expression of IL-1β in RPE cells. This may be via the classical P2X7 Ca2+-mediated pathway, but a contribution by P2Y signalling cannot be ruled out.
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