Purinoceptor Expression in Human Retinal Pigment Epithelium

Abstract

Purpose:: Signalling via extracellular ATP has a role to play in RPE function. We have previously demonstrated the functional expression of P2Y₁, P2Y₂, A₁ and A₂ receptor subtypes in cultured human RPE cells using Ca²⁺ imaging techniques. The aim of this study was to investigate the protein and gene expression of P1 (Adenosine) and P2Y receptors in human native and human cultured RPE cells.

Methods:: Native RPE tissue was obtained from human eyes within 24 hours post mortem. Primary human RPE cells were grown from donor RPE using explant culture techniques. The cultures were maintained at 35^oC in DMEM (pH7.4) supplemented with 10% FCS. Ca²⁺ measurements were made using ratiometric digital imaging techniques with the Ca²⁺-indicator Fura-2. Expression at the protein and RNA level was measured using western blot and quantitative PCR techniques.

Results:: Human cultured RPE cells express the protein for the P2Y₁ and P2Y₂ receptor subtypes. This confirms Ca²⁺ imaging data which showed functional expression of these receptors in these cells. Expression of P2Y₁ and P2Y₂ protein was also found in human native RPE cells although the expression level was found to be lower when compared to cultured cells. In both native and cultured RPE, expression of P2Y₁ and P2Y₂ mRNA was found. In cultured RPE cells, exposure to UDP caused an increase in cell calcium, indicating the presence of P2Y₆ receptors. The presence of these receptors was confirmed at the protein and RNA level in cultured cells and also native RPE tissue. Expression of the P2Y₄ receptor subtype was found at the mRNA level but not at the protein level in both cultured and native samples. Ca²⁺ imaging data did not indicate functional expression of this receptor subtype. P2Y₁₂ was found to be present in native RPE tissue at the mRNA level, but was not detected by western blot. Cultured cells were negative for P2Y₁₂. All 4 adenosine receptor subtypes were found to be present at the mRNA level in native RPE cells and cultured RPE cells express the mRNA for the A₁, A_2A and A_2B receptor subtypes.

Conclusions:: These data confirm that human RPE cells express multiple purinoceptor subtypes.

Keywords: retinal pigment epithelium • receptors: pharmacology/physiology • adenosine