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W. Lu, D. Reigada, J. Sévigny, C.H. Mitchell; ATPS Enhances Expression of the Ecto–ATPase NTPDase1: Relevance to Sustained Purinergic Signaling in RPE Cells . Invest. Ophthalmol. Vis. Sci. 2006;47(13):882.
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© ARVO (1962-2015); The Authors (2016-present)
Stimulation of both ATP and adenosine receptors causes important physiologic changes in RPE cells that may influence their relationship to the adjacent photoreceptors. The enzyme ecto–nucleoside triphosphate diphosphohydrolase 1 (NTPDase1; aka CD39) catalyzes the dual dephosphorylation of ATP and ADP to AMP, but it is unclear how its expression and activity are regulated. Classic negative feedback theory predicts enhanced enzyme activity upon increased exposure to substrate. This study thus asked whether prolonged exposure to ATP increased activity and how this increase was mediated.
Experiments were performed using cultured human ARPE–19 cells exposed to the slowly hydrolysable ATP analog ATPγS for various intervals. Quantification of ATP degradation provided a functional assessment of enzyme activity. NTPDase1 expression was determined using traditional RT–PCR, real–time PCR, and Western blots with the BU61 antibody.
Under control conditions, levels of NTPDase1 mRNA message and protein were both very low. Incubation of cells with ATPγS led to an increase in mRNA levels. This increase in message was accompanied by a time dependant rise in protein levels over 12 – 48 hrs, with a rise in the 78 kD band corresponding to the functional monomeric form of the enzyme. The ATPase activity of cells showed a similar time–dependent increase, indicating the upregulation was functionally relevant.
Prolonged exposure to extracellular ATPγS enhanced the ability of cells to degrade ATP by increasing levels of NTPDase1 mRNA and protein. This upregulation likely reflects a feedback circuit to maintain low levels of extracellular ATP. Enhanced expression of NTPDase1 can thus be used as an index of increased extracellular ATP levels under pathologic conditions. This is the first time such regulation of NTPDase1 has been reported. It will be interesting to determine whether such control is specific for RPE cells.
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