Stimulation of 1 Adrenergic Receptors Downregulates Ecto–5' Nucleotidase Activity on the Apical Membrane of the RPE

Abstract

Abstract: : Purpose: The rate of rod outer segment (ROS) phagocytosis by the RPE is maximal shortly after light onset. Adenosine decreases ROS phagocytosis by RPE cells, and our previous work has shown that adenosine is produced in subretinal space by the dephosphorylation of 5’AMP by ecto–5’ nucleotidase. As epinephrine is released from the retina at onset of light, we asked whether catecholamines could modify ecto–5’ nucleotidase activity and adenosine production. Methods: Activity of ecto–5’ nucleotidase was determined with the P_iPer Phosphate Assay Kit, in which phosphate is proportional to absorbance at 565 nm. The extracellular solution bathing intact ARPE–19 cells was assayed after 30 min exposure to ecto–5’nucleotidase substrate. Activity was also measured from the solution bathing the apical membrane of the bovine RPE eyecup. Adenosine levels were determined with HPLC. Results: Enzymes on the surface of ARPE–19 cells dephosphorylated 5’AMP but did not act on 2’AMP. The production of phosphate from 5’AMP was inhibited by αßmAMP, implying ecto–5’ nucleotidase activity was responsible. Degradation of 5’AMP was inhibited by both epinephrine and norepinephrine. Phenylephrine inhibited the reaction in a concentration–dependent manner, implying involvement of the alpha–1 adrenergic receptor. Phosphate measurements were supported by HPLC showing that norepinephrine reduced levels of adenosine and increased levels of 5’AMP after addition of ATP to the bath. In the bovine–RPE eyecup, norepinephrine reduced the production of phosphate from 5’AMP, indicating both receptor and enzyme face sub–retinal space. Conclusions: Stimulation of alpha–1 adrenergic receptors on the apical membrane of the RPE can downregulate ecto–5’ nucleotidase activity. As epinephrine is released at light onset, the corresponding decrease in subretinal adenosine levels may contribute to the enhanced ROS phagocytosis that occurs at this time.

Keywords: adenosine • retinal pigment epithelium • neurotransmitters/neurotransmitter systems