Stimulation of the P2Y1 receptor up-regulates nucleoside-triphosphate diphosphohydrolase-1 in human retinal pigment epithelial cells

Abstract

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 ATPgammaS 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 ATPgammaS. 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 ATPgammaS 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.

Fig. 1

RT-PCR for NTPDase1 from human ARPE-19 cells. A, PCR with a primer pair specific for NTPDase1 generated a 558-bp band in cDNA from cells treated with ATPγS for 48 h but not in untreated control cells. The band intensity increased with the starting volume of cDNA. B, densitometric values of the RT-PCR amplified NTPDase1 products increased linearly with an increasing amount of cDNA in cells exposed to 100 μM ATPγS (black circles). No product was detected from cells in control medium (white triangles). A first order linear regression is fit to the data. Similar increases in NTPDase1 message were found in three independent sets of cells exposed to ATPγS for 48 h, and in all cases, densitometric values increased with the amount of cDNA. C, PCR with β-actin primer pair generated single 244-bp bands in both control and ATPγS-treated cells. D, densitometric values of β-actin products increased linearly in both ATPγS-treated (black circles) and control cells (white triangles). Data are fit with a single order regression. E and F, time course of up-regulation of NTPDase1 mRNA. Cells were exposed to ATPγS continuously for the time indicated. Message for NTPDase1 was first detected after 12-h exposure to ATPγS, whereas levels of β-actin remained constant throughout. NTPDase1 data points are connected with a line. A similar time course was found in three independent trials.

Fig. 2

Quantitative PCR analysis of NTPDase1 expression in RPE cells. Amplification of NTPDase1 in RPE cells exposed to control or 100 μM ATPγS medium for 48 h was performed with SYBR Green real-time PCR. cDNA samples were diluted 1/10, and all reactions were performed in triplicate. The CT of housekeeping gene β-actin was similar for both control and ATPγS-treated cells (lines 1 and 2, respectively), whereas CT for the NTPDase1 message was attained after considerably fewer amplification cycles in ATPγS-treated cells (line 3) compared with untreated cells (line 4). No signal was detected from the no-template control (bottom line). Similar increases were found using message from three independent trials, each performed in triplicate. The figure indicates the mean of such triplicate reactions from one particular trial.

Fig. 3

Western blot analysis for NTPDase1 protein levels. A, immunoblotting with antibody BU61 demonstrates that incubation of cells with 100 μM ATPγS for 48 h led to detectable 78-kDa bands in protein from whole-cell lysates. B, bands (78 kDa) were also detected in protein purified from cell membranes after incubation with 100 μM ATPγS for 48 h. C, incubation of cells with 100 μM ATPγS for 12, 24, and 48 h led to detectable 78-kDa bands on immunoblots of increasing intensity from cell lysate material. D, quantification of staining intensity indicates that the largest increase in NTPDase1 protein occurred after 12 to 24 h of exposure to ATPγS. A similar increase in NTPDase1 protein levels after 24 h in ATPγS was found in 14 trials.

Fig. 4

Incubation with ATPγS increased ecto-ATPase activity. A, cells exposed to 100 μM ATPγS for 48 h (gray triangles) hydrolyzed 1 μM ATP more rapidly than untreated control cells (black circles). Intermediate preincubations of 15 and 24 h led to intermediate increases in hydrolysis but are not shown here for reasons of clarity. Results show the mean of five to eight wells from a trial representative of three independent experiments. Light levels represent the photons given off with the luciferin/luciferase reaction, an index of the level of ATP present in the bath [in arbitrary units (A.U.)]. B, time constant of decay decreased exponentially with preincubation time. *, p < 0.05 versus no preincubation, n = 5 to 8. In A, error bars are smaller than symbols.

Fig. 5

Involvement of P2Y₁ receptors in up-regulation of NTPDase1. P2 antagonists were given to cells 10 min before 100 μM ATPγS, and the 78-kDa band was detected with antibody BU61 from protein extracted from whole-cell lysates 24 h later. A, nonspecific P2 antagonist oATP (100 μM) decreased the net up-regulation of NTPDase1 by 25%. The upper trace is representative of four independent trials, with the mean ± S.E. given in the bars below. B, effect of RB2 (50 μM) was variable with some trials like that illustrated above indicating a drastic reduction, although the mean decrease was not significant (n = 8). C, P2Y₁ receptor antagonist MRS2179 (100 μM) led to a clear decrease in band intensity (n = 7). D, a second P2Y₁ antagonist, MRS2500 (10 nM), also decreased band intensity (n = 4). E, P2Y₁ agonist MRS2365 (100 nM) significantly increased NTPDase1 levels compared with control after 24-h exposure, whereas the rise with 100 μM 2MeSATP was not significant (n = 2–3). *, p < 0.05 versus ATPγS alone (A–D) or versus control (E).