Abstract
Purpose: :
Adenosine has previously been shown to inhibit cell proliferation in the human RPE cell line hTERT RPE. The aim of these experiments was to investigate growth and signalling via adenosine receptors in these cells. Also to determine the concentration of adenosine to which RPE cells could be exposed in vivo by analysis of subretinal fluid.
Methods: :
Cell growth was monitored by measuring the increase in area of cells over a 4 day period. ERK and JNK/SAPK were detected on Western blots using antibodies to both the phosphorylated and non–phosphorylated forms and quantified using densitometry analysis. Subretinal fluid was collected from patients during retinal detachment surgery. Adenosine was analysed by HPLC with mass detection and ATP by luciferin/luciferase assay. Gene expression was assessed by Affymetrix gene array analysis.
Results: :
Adenosine (100pM) inhibited growth of hTERT RPE cells by approximately 70% over a 4 day period. The signalling mechanisms underlying this decrease in growth were then investigated. Exposure to adenosine (100pM) caused a small but significant decrease in ERK1/2 phosphorylation at 10 and 30 minutes. Over this time period an increase in phosphorylation of JNK/SAPK was also observed. The A1 receptor agonist CPA (100pM) gave a total inhibition of cell growth over the 4 day period. CPA (100pM) also caused a decrease in ERK phosphorylation and an increase in SAPK phosphorylation. The presence of mRNA in these cells for the A1 receptor was detected by gene array. Analysis of subretinal fluid from patients with retinal detachment showed the presence of adenosine (range 12.96nM – 84.31nM; n=3). ATP was found at lower concentrations than adenosine (range 89pM – 681pM; n=3).
Conclusions: :
Signalling via the MAPK and JNK/SAPK pathways may mediate the decrease in proliferation of hTERT RPE cells by low concentrations of adenosine via the A1 receptor. These pathways may also mediate a stress response in RPE cells during pathological conditions such a retinal detachment.
Keywords: adenosine • receptors: pharmacology/physiology • retinal pigment epithelium