Identification of Purinergic Receptor Subtypes in Human RPE Cells

Abstract

Abstract: : Evidence for multiple purinergic receptor subtypes in human RPE cells. Purpose: To investigate functional expression of purinergic (P2Y and adenosine) receptors in cultured human retinal pigment epithelial (RPE) cells. Methods: 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. Western blot techniques and quantataive (Taqman) PCR were used to determine receptor subtype expression in human cultured RPE cells. Results: ATP, UTP, ADP, 2MeS–ATP and UDP produced concentration–related increases in intracellular calcium ([Ca²⁺]_i) in cultured human RPE cells. The EC₅₀ for ATP and UTP was 5µM and 8µM respectively. This indicates the presence of P2Y₂ receptors in these cells. This was confirmed by Western blot techniques. The maximal responses for ADP and 2MeS–ATP were approximately half the amplitude of those produced by ATP and UTP and the EC₅₀s were lower (0.3µM for 2MeS–ATP and 2µM for ADP). This suggests the presence of the P2Y₁ receptor subtype. Pre–treatment with the P2Y₁ antagonist MRS–2179 (10µM) inhibited the ADP response further indicating the presence of the P2Y₁ receptor subtype. Adenosine has no effect on ([Ca²⁺]_i) when applied on its own, but potentiates a sub–threshold response to ATP in a concentration–dependant manner. Pharmacological studies suggest the involvement of more than one adenosine receptor subtype in potentiation. This was confirmed by Taqman PCR techniques which indicated the presence of A₁, A_2A and A_2B receptors, but not A₃.Conclusions:The data suggest that more than one P2 receptor subtype is present in human cultured RPE cells and three adenosine receptor subtypes are also present. Physiological responses via purinergic receptor activation are therefore likely to use multiple purinergic receptor subtypes.

Keywords: receptors • calcium • gene/expression