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V. Tovell, J. Sanderson; Potentiation of P2Y1 Mediated Ca2+ Responses by Adenosine Involves the MAP Kinase Pathway . Invest. Ophthalmol. Vis. Sci. 2006;47(13):2860.
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Adenosine potentiates the ATP induced calcium response in human cultured RPE cells. Previous studies from this laboratory have shown that ADP increases calcium in these cells via the P2Y1 receptor subtype whereas ATP appears to signal predominantly via the P2Y2 purinoceptor. The aim of this study was to investigate the effects of adenosine on ADP and ATP mediated calcium responses and to examine possible mechanisms of crosstalk.
RPE segments from human donor eyes were grown from explant cultures in Dulbecco’s Minimum Essential Medium (DMEM) supplemented with 10% foetal calf serum. Cells up to passage 3 were used in this study. Receptor–mediated calcium responses were monitored using the ratiometric dye fura–2. Measurements of cAMP were carried out using an enzyme–linked immunosorbent assay (ELISA).
Adenosine (10 µM) has no effect on calcium when applied on its own but potentiated sub–threshold responses to ADP (0.5–1 µM) in approximately 50 % of cells. Previous studies have shown that potentiation of the ATP induced calcium response can occur via cAMP mediated pathways. In the present experiments adenosine (10 µM), CPA (A1 receptor agonist, 100 nM) and CGS21680 (A2 receptor agonist, 10 µM), all applied for 5 minutes, had no significant effect on the levels of cAMP in cultured RPE cells. Furthermore, the PKA inhibitor H–89 (10 µM) has no effect on the potentiation of ADP or ATP. Pre–incubation with the MEK inhibitor u0126 (10 µM) fully inhibited the potentiation of ADP induced responses. Potentiation of the ATP induced response in these cells was not inhibited by u0126.
Cross–talk between the adenosine and P2Y1 receptor signalling pathways in RPE cells does not involve cAMP signalling but the MAP kinase pathway appears to play an important role. The cross–talk between adenosine and ATP mediated responses appears to be independent of cAMP and MAP kinase pathways.
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