Ecto ATDPase and Ecto-5’nucleotidase Activity on the Apical Membrane of the RPE

Abstract

Abstract: : Purpose: Stimulation of ATP receptors on the RPE apical membrane can modify fluid and ion transport, and stimulation of RPE adenosine receptors can modify outer segment phagocytosis. In other cells, ectoATDPase and ecto-5’-nucleotidase can convert extracellular ATP into adenosine, and our previous work has identified these enzymes on the RPE by molecular and immunohistochemical methods. This study investigates the functional activity of these enzymes to learn if extracellular ATP can be converted to adenosine by ecto-enzymes on the apical surface of RPE cells. Methods: ATP levels were measured with the luciferin/luciferase assay, adenosine was quantified by HPLC with UV detection, and 5’nucleotidase activity was determined by the 5’Nucleotidase assay (Sigma). Experiments were performed on bovine RPE eyecups with the retina removed and on ARPE-19 cultured human RPE cells. Results: The degradation of extracellular ATP by both cultured human cells and fresh bovine RPE was prevented by the ecto-ATPase inhibitor ARL-67156 (100 µM). ARL-67156 also prevented the production of adenosine by the bovine eyecup. The levels of extracellular ATP and adenosine were inversely related, suggesting they were derived from a common pool. The activity of ecto-5’nucleotidase in ARPE-19 cells was reduced 58% by 100 µM αßmADP, an inhibitor of ecto-5’nucleotidase, and preliminary results suggest that levels of adenosine in the bovine eyecup also were reduced in the presence of αßmADP. Conclusions: The RPE can produce adenosine from ATP via ecto-ATDPase and ecto-5’nucleotidase. The use of intact RPE eyecups localizes the enzyme activity to the apical membrane, suggesting it could affect purine levels in subretinal space. As RPE cells release ATP across the apical membrane, ecto-enzyme activity may modulate the relative stimulation of ATP vs. adenosine receptors by altering relative agonist concentrations in subretinal space.

Keywords: adenosine • retinal degenerations: cell biology • neurotransmitters/neurotransmitter systems