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NEUROPHARMACOLOGY

Stimulation of the P2Y₁ Receptor Up-Regulates Nucleoside-Triphosphate Diphosphohydrolase-1 in Human Retinal Pigment Epithelial Cells

Department of Physiology, University of Pennsylvania School of Medicine, Philadelphia, Pennsylvania (W.L., D.R., C.H.M.); and Centre de Recherche en Rhumatologie et Immunologie, Centre Hospitalier Universitaire de Québec, Université Laval, Quebec, Canada (J.S.)

Stimulation of receptors for either ATP or adenosine leads to physiologic changes in retinal pigment epithelial (RPE) cells that may influence their relationship with the adjacent photoreceptors. The ectoenzyme nucleoside-triphosphate diphosphohydrolase-1 (NTPDase1) catalyzes the dual dephosphorylation of ATP and ADP to AMP. Although NTPDase1 can consequently control the balance between ATP and adenosine, it is unclear how its expression and activity are regulated. Classic negative feedback theory predicts an increase in enzyme activity in response to enhanced exposure to substrate. This study asked whether exposure to ATP increases NTPDase1 activity in RPE cells. Although levels of NTPDase1 mRNA and protein in cultured human ARPE-19 cells were generally low under control conditions, exposure to slowly hydrolyzable ATPS led to a time-dependent increase in NTPDase1 mRNA that was accompanied by a rise in levels of the functional 78-kDa protein. Neither NTPDase2 nor NTPDase3 mRNA message was elevated by ATPS. The ATPase activity of cells increased in parallel, indicating the up-regulation of NTPDase1 was functionally relevant. The up-regulation of NTPDase1 protein was partially blocked by P2Y₁ receptor inhibitors MRS2179 (N⁶-methyl-2'-deoxyadenosine-3',5'-bisphosphate) and MRS2500 [2-iodo-N⁶-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate] and increased by P2Y₁ receptor agonist MRS2365 [(N)-methanocarba-2MeSADP]. In conclusion, prolonged exposure to extracellular ATPS increased NTPDase1 message and protein levels and increased ecto-ATPase activity. This up-regulation reflects a feedback circuit, mediated at least in part by the P2Y₁ receptor, to regulate levels of extracellular purines in subretinal space. NTPDase1 levels may thus serve as an index for increased extracellular ATP levels under certain pathologic conditions, although other mechanisms could also contribute.

Address correspondence to: Dr. Claire H. Mitchell, Department of Physiology, University of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085. E-mail: chm{at}mail.med.upenn.edu