Degradation of extracellular ATP by the retinal pigment epithelium

David Reigada,1 Wennan Lu,2 Xiulan Zhang,2,3 Constantin Friedman,2 Klara Pendrak,2 Alice McGlinn,2 Richard A. Stone,2 Alan M. Laties,2 and Claire H. Mitchell1

Departments of Physiology1 and 2Ophthalmology, School of Medicine, University of Pennsylvania, Philadelphia, Pennsylvania; and 3Zhongshan Ophthalmic Center, Sun Yat-Sen University, Guangzhou, People's Republic of China

Submitted 7 November 2004 ; accepted in final form 19 April 2005


    ABSTRACT
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 DISCUSSION
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Stimulation of ATP or adenosine receptors causes important physiological changes in retinal pigment epithelial (RPE) cells that may influence their relationship to the adjacent photoreceptors. While RPE cells have been shown to release ATP, the regulation of extracellular ATP levels and the production of dephosphorylated purines is not clear. This study examined the degradation of ATP by RPE cells and the physiological effects of the adenosine diphosphate (ADP) that result. ATP was readily broken down by both cultured human ARPE-19 cells and the apical membrane of fresh bovine RPE cells. The compounds ARL67156and {beta}{gamma}-mATP inhibited this degradation in both cell types. RT-PCR analysis of ARPE-19 cells found mRNA message for multiple extracellular degradative enzymes; ectonucleotide pyrophosphatase/phosphodiesterase eNPP1, eNPP2, and eNPP3; the ectoATPase ectonucleoside triphosphate diphosphohydrolase NTPDase2, NTPDase3, and some message for NTPDase1. Considerable levels of ADP bathed RPE cells, consistent with a role for NTPDase2. ADP and ATP increased levels of intracellular Ca2+. Both responses were inhibited by thapsigargin and P2Y1 receptor inhibitor MRS 2179. Message for both P2Y1 and P2Y12 receptors was detected in ARPE-19 cells. These results suggest that extracellular degradation of ATP in subretinal space can result in the production of ADP. This ADP can stimulate P2Y receptors and augment Ca2+ signaling in the RPE.

ectoapyrase; PC-1; CD39; CD39L1; P2Y1; P2Y12; ADP; ATP release; photoreceptors; retinal detachment


THE RETINAL PIGMENT EPITHELIUM (RPE) has an intimate anatomic relationship with the photoreceptor outer segments. This juxtaposition underlies a close functional relationship, and the RPE performs a variety of roles that maximize photoreceptor health. For example, the distal tips of the outer segments are regularly phagocytosed by the RPE cells to enable continual resynthesis of these outer segments (37). Transport mechanisms on the apical membrane of the RPE regulate the ionic composition of the subretinal space located between the RPE cells and the outer segments, controlling the ionic driving forces on the photoreceptor outer segments (15). In addition, the transport of fluid and ions from the apical membrane facing the photoreceptors to the basolateral membrane of the RPE is thought to be one of the main forces keeping the retina attached (21).

The exogenous addition of purines can trigger responses capable of modifying the interaction between the RPE and photoreceptors. Stimulation of ATP receptors increases the rate of ion and fluid transport from subretinal space toward the choroid (30). Agonists for P2Y2 receptors can increase Ca2+ levels in RPE cells and consequently enhance the rate of fluid absorption across monolayers of bovine and fetal human RPE cells. This stimulation may have important clinical implications because agonists facilitate retinal reattachment in rat (20) and rabbit models of retinal detachment (25). The dephosphorylated nucleoside adenosine can also modify the relationship between the RPE and photoreceptors. Stimulation of A2 adenosine receptors inhibits the phagocytosis of rod outer segments (9). The adenosine agonist 2Cl adenosine reduces the detrimental effects of glucose on RPE cells (18), while adenosine augments the c-wave of the electroretinogram in chicks (23).

Although these responses were observed after the addition of purine agonists, the source and regulation of the endogenous stimuli for these receptors is unclear. RPE cells can release ATP into the extracellular space (6, 26, 32), and preliminary evidence suggests that extracellular dephosphorylating enzymes may be involved in the conversion of ATP to adenosine in the subretinal space (26). The particular enzymes contributing to this dephosphorylation can determine extracellular levels of intermediary ADP. ADP is capable of stimulating several P2Y receptors, and binding of ADP to the P2Y1 receptor can elevate intracellular Ca2+ in many cell types (35). The present study was focused on the degradation of ATP by the RPE. Using pharmacological, molecular, and biochemical analysis, we have examined the enzymes responsible for this degradation and whether the residual balance of purines has a physiological impact on signaling for RPE cells. Portions of this work were presented previously in abstract form (8, 19, 27, 28).


    METHODS
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Cell culture. The human ARPE-19 cell line (5) was obtained from the American Type Culture Collection (Manassas, VA) and grown in a 1:1 mixture of Dulbecco's modified Eagle's medium and Ham's F-12 medium with 3 mM L-glutamine (GIBCO-BRL, Grand Island, NY) and 10% fetal bovine serum (FBS; HyClone Laboratories, Logan, UT). Cells were incubated at 37°C in 5% CO2 and subcultured with 0.25% trypsin and 0.02% EDTA.

ATP measurements. ARPE-19 cells were grown to confluence in 96-well plates. Growth medium was removed, and wells were washed with a control isotonic solution composed of (in mM) 105 NaCl, 5 KCl, 4 Na+-HEPES, 6 HEPES acid, 5 Na2HCO3, 60 mannitol, 5 glucose, 1.3 CaCl2, and 0.5 MgCl2. ATP was dissolved into this control solution, and 90 µl of this ATP mixture was added to each well 5–20 s before recording began. The bioluminescent luciferin/luciferase assay was used to measure ATP levels. Luciferin/luciferase was stored frozen as a stock solution with 450 µl of control solution/50 µl of H2O per vial and diluted a further 12.5-fold in control solution before being added to each well. Measurements were made by adding 10 µl of this assay stock to 90 µl of solution in each well at time 0. In some trials, the inhibitors 6-N,N-diethyl-D-{beta}{gamma}-dibromomethylene ATP (ARL67156, {beta}{gamma}-methylene ATP ({beta}{gamma}-mATP), pyridoxal-phosphate-6-azophenyl-2',4'-disulfonate (PPADS), or levamisole were added to the wells with the ATP. None of the compounds affected the luciferase assay when tested with 10 nM ATP in a cell-free trial (n = 6). ATP was quantified using the Luminoskan Ascent luminometer (Thermo Labsystems, Franklin, MA) as previously described (32). Readings from each well were integrated over 100 ms and sampled in succession every 20 s.

Bovine eyes were bisected at the ora serrata and the retinas were removed; this is defined as the bovine RPE eyecup and has the apical membrane facing the cup interior (32). After the eyecup was rinsed with control solution, 100 nM ATP was added in a total volume of 1 ml. ARL67156or {beta}{gamma}-mATP was added with the ATP as indicated. After 20 min, a 600-µl sample was removed and frozen at –20°C. The ATP content was measured by combining 90 µl of the defrosted sample with 10 µl of luciferin/luciferase assay stock in a single well of a 96-well plate, and luminescence was recorded as described above. Experiments using both bovine and ARPE-19 cells were performed at room temperature (21–25°C).

High-performance liquid chromatography. Purine levels were determined from samples of bovine RPE eyecup obtained as described above and from aliquots of extracellular solution bathing wells of confluent ARPE-19 cells. Samples were obtained 20 min after addition of 100 nM ATP to enhance detection. High-performance liquid chromatography (HPLC) was performed using a BAS PM80 pump (Bioanalytical Systems, West Lafayette, IN) and a Discovery HS C18 25 cm x 2.1 mm, 5 µM column (Supelco, Bellefonte, PA) fit with a Beckman 160 UV detector (Beckman Instruments, Berkeley, CA). A flow rate 0.25 ml/min and a mobile phase of 0.05 M tetraethylammonium-phosphoric acid buffer were used. All purines were detected using absorbance at 260 nm. The elution time for each purine was defined separately and in combination to confirm identification. The area under the curve was converted into purine concentration with samples of known concentration. To control for biological and experimental variability between samples obtained on different days, the relative amounts of ATP, ADP, AMP, and adenosine were normalized to the total purine levels for each trial.

RT-PCR. RNA was extracted from ARPE-19 cells using the TRIzol reagent (Invitrogen, Carlsbad, CA). Reverse transcription was performed with 1–2 µg of total RNA using the SuperScript first-strand synthesis system (Invitrogen), with a negative control reaction performed without the SuperScript reverse transcriptase. PCR was performed on a thermocycler with the AmpliTaq gold polymerase system (Applied Biosystems, Foster City, CA) using 2–5 µl of cDNA, 1.5–3 mM MgCl2, 0.5 µl of Taq, and 0.2–0.6 µM primers in a 50-µl reaction. Published primers were used for eNPP1 (7) and NTPDase2 (36). The successful NTPDase1 primers were designed with the MacVector program (Oxford Molecular Group/Accelrys, Burlington, MA). All other primers were designed with the Prime3 software program (http://frodo.wi.mit.edu/cgi-bin/primer3/primer3_www.cgi). All primers used are listed in Table 1, with corresponding GenBank accession numbers listed in Table 2. The successful amplification of NTPDase1 was performed at 95°C for 1 min, 55°C for 1 min, and 72°C for 2 min for 35 cycles. The band was visualized on a 2% agarose gel, photographed, and purified from a 2% LMP agarose gel using the QIAEX II gel extraction kit (Qiagen, Valencia, CA). The gel-purified product was reamplified, cloned using a TOPO TA cloning kit with Top 10 F' cells (Invitrogen), and purified using the Wizard Plus Minipreps kit (Promega). NTPDase2 message was amplified after 94°C for 1 min, 64°C for 0.5 min, and 72°C for 1 min for 35 cycles, and then the product was reamplified and run on a 1% agarose gel, photographed, and purified using the Wizard Miniprep kit (Promega, Madison, WI). The eNPP1 message was amplified after 94°C for 1 min, 58°C for 1 min, and 72°C for 1 min for 35 cycles, run on a 1% agarose gel, photographed, and purified using the QIAquick PCR purification kit (Qiagen, Valencia, CA). All other sequences were amplified after 95°C for 1 min, 58°C for 1 min, 72°C for 1 min for 35 cycles, and handled in the same manner as for eNPP1. All purified products were sequenced with the University of Pennsylvania Cell Center Sequencing Facility. The resulting sequence was identified using the BLAST nucleotide database (http://www.ncbi.nlm.nih.gov).


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Table 1. Primers used in PCR amplification

 

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Table 2. Confirmation of PCR product identity

 
Intracellular Ca2+ measurements. Levels of intracellular Ca2+ were determined in two ways. Experiments examining the time course of Ca2+ responses (see Fig. 4, A and B) were performed using techniques described in detail previously (32). In brief, ARPE-19 cells grown on 12-mm coverslips for 3–6 days were loaded with 10 µM fura-2 AM and 0.2% Pluronic acid F-127 (Molecular Probes/Invitrogen, Eugene, OR) for 30 min at room temperature, rinsed, and mounted on a Nikon Diaphot microscope. Cells were visualized using a x20 magnification fluorescence objective, and the field was alternatively excited at 340 and 380 nm. The fluorescence emitted at 510 nm was recorded at 1 Hz using a photomultiplier tube and analyzed using the Felix software suite (Photon Technology International, Princeton, NJ). The ratio of light excited at 340 nm to that at 380 nm was converted to Ca2+ concentration using standard calibration techniques performed on the coverslip at the end of each experiment (32). All experiments were performed at room temperature.



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Fig. 4. A: ADP and ATP can raise intracellular Ca2+ in ARPE-19 cells. A 20-s exposure to ADP led to a clear elevation in Ca2+ that was approximately one-half the amplitude of the response to ATP. A 4-min interval between purine applications allowed for repeat responses and indicates that the difference in amplitudes is consistent. The trace is representative of three independent trials. B: a more sustained application of 100 µM ADP leads to a sustained increase in intracellular Ca2+ levels. The trace is representative of four independent trials. C: the peak Ca2+ response to 100 µM ATP was inhibited by depleting internal stores with 1 µM thapsigargin (Thaps.) and by the P2Y1 receptor blocker MRS 2179 (100 µM; MRS). Removing adenosine with adenosine deaminase (1 U/ml; ADA) had no effect, and the effect of extracellular Ca2+ removal (0 Ca2+) was not significant. *P < 0.05, one-way ranked ANOVA and Dunn's posttest. D: the peak Ca2+ response to 100 µM ADP was also inhibited by thapsigargin and MRS 2179, while adenosine deaminase had no effect. Labels as in C. *P < 0.05, one-way ranked ANOVA and Dunn's posttest. E: expression of P2Y receptors for ADP in ARPE-19 RPE cells. P2Y1 (lane 2) and P2Y12 (lane 4) were analyzed by performing RT-PCR with specific primer sequences, and appropriately sized bands were detected. Negative controls (–RT reaction) are shown in lanes 1 and 2. Molecular weight standard (lane M). All markers bands in lanes (M) are 100 bp with large band at 600 bp.

 
Experiments involved with the pharmacology of the response (Fig. 4, C and D) were performed on ARPE-19 cells grown to confluence on 96-well plates. The growth medium was removed, and cells were washed with 200 µl of isotonic solution. Dye was loaded into cells by incubating each well for 30 min with 50 µl of a control Ringer solution containing 10 µM fura-2 AM and 0.02% Pluronic acid solution. Wells were washed and 90 µl of control Ringer solution, or the appropriate antagonist solution was placed in each well. (Cells were pretreated with thapsigargin for 30 min before this point.) The Ca2+-free solution had no EGTA. Cells were excited alternatively at 340 and 380 nm for 10 ms each, and the fluorescent emitted >510 nm was measured in a microplate fluorometer (Fluoroskan Ascent; Labsystems, Franklin, MA). After a baseline reading obtained at 5 min, 10 µl of ATP or ADP were injected into each well using the internal injector system placed in the fluorometer to produce a final concentration of 100 µM. Because some procedures such as the removal of extracellular Ca2+ or the addition of thapsigargin complicated the calibration, the analysis was based on the ratio of light emitted at 340 vs. 380 nm. The increase in this ratio was compared with prestimulated baseline levels, with peak levels used for comparison. This elevation was expressed as a percentage of the mean control response to enable comparison between ATP and ADP effects on different days.

Data analysis and materials. Data are expressed as means ± SE. An unpaired Student's t-test was used to test for significance when two variables were present. The effect of drugs on the Ca2+ response was determined using a one-way ranked ANOVA and Dunn's posttest with SigmaPlot software (SPSS, Chicago, IL). Decay time constants used in Fig. 1 were obtained by fitting a single exponential y = y0 + ae–bx ({tau} = 1/b) to the mean decay curves using the least-squares method with SigmaPlot software. The percentage block of degradation of 100 nM ATP produced by ecto-ATPase inhibitors was defined as 100 x [1(–b/a)], where a is the mean difference in luminescence levels from t = 0–1 min compared with t = 59–60 min and b is the same difference in the presence of the inhibitor. All reagents were obtained from Sigma Chemical (St. Louis, MO) unless otherwise noted.



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Fig. 1. ATP degradation by ARPE-19 cells. A: on-line measurement of the ATP degradation by ARPE-19 cells after the addition of 3 nM ({bullet}), 30 nM ({circ}), 100 nM ({blacktriangleup}), 300 nM ({square}), and 1,000 nM ATP ({lozenge}). An identifying symbol is indicated at t = –3 min for each trace. Luminescence levels reflect the conversion of ATP into photons. The graph represents the mean of 24 experiments, with SE represented by the dotted lines; the SE is frequently smaller than the symbol. The mean response found in the absence of cells for a given concentration was subtracted from each record. B: data shown in A fit with exponential decay curves, and the calculated time constants are plotted as a function of starting ATP concentration ({circ}). The time constant increased with substrate levels, with an exponential rise fitting equation y = 60.62(1 – e–0.031x) with R2 = 0.78 (solid line). C: on-line measurement showing the ATP-mediated ATP release. At time 0, 10 µM ATP was added to the cells. While the initial portion of the records was dominated by degradation, an increase in the luminescence beginning 10 min after addition of ATP indicates a release of ATP into the bath. The symbols represent the means and the dots indicate SE of 16 trials. ALU, arbitrary light units.

 

    RESULTS
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ATP degradation. The ability of cultured human ARPE-19 cells to degrade ATP was examined after the addition of ATP to the solution bathing intact cells. ATP levels were clearly decreased after the purine was added to the bath at concentrations of 3–1,000 nM (Fig. 1A). ATP levels took only 15 min to fall to baseline after the addition of 3 nM ATP, while levels remained somewhat elevated 60 min after the addition of 1,000 nM ATP. Although these experiments were performed in the presence of the luciferine/luciferase assay mixture, the minimal decrease in luminescence signal in the absence of cells was used as a control and subtracted, so that the degradation observed in Fig. 1A is due primarily to enzymes present on the extracellular surface of the cells. The time constant of this decay increased with the concentration of ATP, with the data fit reasonably well by an exponential rise at lower concentrations (Fig. 1B). Increasing the amount of ATP to 10 µM triggered a secondary release of ATP (Fig. 1C). While this likely represents an ATP-mediated ATP release, this release complicated the analysis and limited the quantification of enzyme kinetics.

To further characterize the enzymes responsible for the extracellular degradation of ATP by RPE cells, the effects of a variety of ecto-ATPase inhibitors were examined (Fig. 2A). Under control conditions, luminescent levels fell by 80% after the addition of 100 nM ATP during the 60 min of the investigation. ARL67152blocked this ATP degradation by 66% when added with the ATP. The methylene ATP analog {beta}{gamma}-mATP blocked 78% of the ATP degradation from ARPE-19 cells. The inhibitory effects of {beta}{gamma}-mATP and ARL67156were additive, together blocking 99% of the decrease in luminescence. The ectoalkaline phosphatase inhibitor levamisole had no effect on the degradation of ATP at 100 µM (Fig. 2B). Higher concentrations of levamisole interfered with the assay. The compound PPADS has been reported to inhibit both ectonucleoside triphosphate diphosphohydrolases NTPDase1 and NTPDase2 and eNPP1 (11) in addition to its actions as a P2X antagonist (29). However, the effects of PPADS on ATP degradation in our experiments were complex; the rate of degradation was reduced, but the initial ATP levels were considerably smaller. An assessment of the inhibitory effect of PPADS was not undertaken.



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Fig. 2. Inhibition of ATP degradation from ARPE-19 cells. A: on-line measurements of ATP (100 nM) degradation under control conditions ({bullet}) and after the addition of 100 µM ARL67156({square}), 100 µM {beta}{gamma}-mATP ({blacklozenge}), and 100 µM ARL67156+ 100 µM {beta}{gamma}-mATP ({triangleup}). Each symbol represents the mean of five consecutive data points from six experiments for each condition, and the SE are represented by dots. In many cases, the SE values are smaller than the symbols. An identifying symbol is shown to the right of each trace. B: the alkaline phosphatase inhibitor levamisole (100 µM) had no effect on the degradation of 1 µM ATP. {circ}, control (n = 32); {triangleup}, levamisole (n = 8). SE are the same as in A. A small rise in luminescence is observed, consistent with a small release of ATP at this concentration.

 
RT-PCR. The presence of mRNA message for enzymes capable of degrading extracellular ATP in other epithelial cells was examined in ARPE-19 cells using standard RT-PCR techniques. Message was readily found for NTPDase2 and NTPDase3 (Fig. 3A). Message was also detected for eNPP1, eNPP2, and eNPP3 (Fig. 3B). Although three sets of primer pairs were unable to detect NTPDase1, a fourth set did reveal a band of expected size (Fig. 3A). All six products were sequenced, and the sequence was found to be similar to the appropriate gene (Table 2). In all cases, no product was detected when reverse transcriptase was omitted from the reaction.



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Fig. 3. RT-PCR for degradative ectoenzymes from ARPE-19 cells. A: expression of NTPDases in human RPE cells. NTPDase1(lane 1), NTPDase2 (lane 4), and NTPDase3 (lane 6) transcripts were analyzed by RT-PCR with specific primer sequences and appropriately sized bands were detected. Negative controls (–RT reaction) are shown in lanes 2, 3, and 5. Molecular weight standard (M); all 100-bp steps with large band at 500 bp for NTPDase1 and at 600 bp for NTPDase 2 and 3. B: detection of eNPPases in human RPE cells. eNPP1(lane 2), eNPP2 (lane 4), and eNPP3 transcripts were analyzed by RT-PCR with specific primer sequences and appropriately sized bands were detected. Negative controls (–RT reaction) are shown in lanes 1, 3, and 5. Molecular weight standard, (M); 100-bp steps with large band at 600 bp.

 
Effect of ADP on intracellular Ca2+. The product of NTPDase2 activity is ADP, while the other enzymes identified convert ATP to AMP (39). The balance of extracellular purines bathing RPE cells can indicate the relative enzyme activities. The relative distribution of purines surrounding ARPE-19 cells was 0.45/0.09/0.42/0.03 for ATP/ADP/AMP and adenosine, respectively, when assessed after the addition of 100 nM ATP (n = 6). The presence of ADP in the solution bathing these cells led us to ask whether ADP could trigger a response in ARPE-19 cells. Because ADP can act at several G protein-coupled P2Y receptors (13, 22, 35) ATP and ADP were tested for their relative effects on intracellular Ca2+ levels. To limit receptor desensitization and depletion of intracellular Ca2+ stores, agonists were applied for 20 s, followed by a 240-s wash. This protocol allowed multiple applications of agonist to be compared within the same experiment. As shown in Fig. 4A, both ADP and ATP elevated intracellular Ca2+ levels in ARPE-19 cells. However, the response to 100 µM ADP was considerably less than the response to 100 µM ATP. In all, ADP increased levels to 750 ± 30 nM, while ATP raised them to 1,460 ± 90 nM (P < 0.0005; n = 6 for both). When applied for a longer duration, ADP was capable of triggering an initial peak, followed by an undershoot and then a longer-term rise in Ca2+ (Fig. 4B). In four experiments, Ca2+ levels rose to 183 ± 27 nM when measured 400 s after addition of 100 µM ADP from a baseline of 147 ± 16 nM.

The mechanism underlying this elevation of intracellular Ca2+ by ATP and ADP was examined (Fig. 4, C and D). Depletion of Ca2+ stores in the endoplasmic reticulum with 1 µM thapsigargin greatly reduced the ability of both ATP and ADP to raise the Ca2+ level. MRS 2179, an inhibitor of the P2Y1 receptor (35), reduced the response to both ATP and ADP. Removing extracellular adenosine with adenosine deaminase (1 U/ml) had no significant effect on the response to either agonist.

This characterization suggested that the response to ADP was mediated primarily by stimulation of P2Y1 receptors. Molecular analysis found evidence for the P2Y1 receptor in ARPE-19 cells using RT-PCR (Fig. 4E). Message for the ADP-sensitive P2Y12 receptor was also detected. In both cases, the PCR products showed a high degree of similarity with the relevant sequence (Table 2).

Fresh bovine RPE. While ARPE-19 cells provide a useful model for examining the degradation of purines, it is important to determine whether these processes also occur in fresh cells and to determine the polarity of the response. The degradation was thus examined in the fresh bovine RPE eyecup preparation. Because the apical membrane of RPE cells faces the interior of the eyecup from which samples were obtained, this preparation also provides information about the functional polarity of the response. To determine whether enzymes capable of degrading ATP were located on the apical membrane of fresh cells, 100 nM ATP was added to the center of a bovine RPE eyecup devoid of retina. ATP levels in the eyecup were reduced to 83.8 ± 5.8% of control levels when sampled 20 min after addition of 100 nM ATP (n = 14). However, the reduction of ATP levels in the eyecup was inhibited considerably by both 100 µM ARL67156and 100 µM {beta}{gamma}-mATP (Fig. 5, A and B). The relative distribution of ATP/ADP/AMP and adenosine present in the bovine eyecup was 0.54/0.10/0.23/0.14, respectively, when assessed after the addition of ATP (n = 6).



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Fig. 5. Bovine eyecup data. ATP levels were determined from the solution inside the bovine RPE eyecup 20 min after the addition of 100 nM ATP using the luciferase system. A: addition of 100 µM ARL67156slowed this degradation, leading to increased levels of ATP (closed bar) when compared with levels in control solutions (open bar). n = 4 for each. *P = 0.044, unpaired Student's t-test. Values were normalized to the mean control for each day's experiment. B: presence of 100 µM {beta}{gamma}-mATP (closed bar) in the bath increased the amount of ATP remaining in the bovine RPE eyecup treated with 100 nM ATP compared with control (open bar), indicating the {beta}{gamma}-mATP also inhibited the degradation of ATP. n = 9 for each. *P = 0.009, unpaired Student's t-test.

 

    DISCUSSION
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The present study has demonstrated that ATP is readily degraded by both fresh and cultured RPE cells. Degradation by both cell types was inhibited by ARL67156and {beta}{gamma}-mATP, implying the activity of multiple enzymes. Message for multiple degradative enzymes was detected in ARPE-19 cells using RT-PCR. These enzymes included NTPDase2, which produces ADP, and analysis of extracellular purine levels suggested ADP was present. Intracellular Ca2+ levels were increased by both ATP and ADP. This increase was blocked by thapsigargin and the P2Y1 receptor inhibitor MRS-2179. The presence of the P2Y1 receptor was confirmed with RT-PCR. Together these findings support a role for multiple degradative enzymes and suggest that the balance of purines in subretinal space produced by these enzymes on the apical membrane of the RPE can effect cellular signaling.

ATP degradation is likely due to the actions of both eNPPs and NTPDases. A recent study by Joseph et al. (17) indicated that {beta}{gamma}-mATP has a much greater inhibitory effect on ATP degradation by cells expressing eNPPs than by cells expressing NTPDases. This implies that the inhibition of ATP breakdown by {beta}{gamma}-mATP in RPE cells is due to action on eNPPs. While the relative effectiveness of ARL67156at these different ecto-ATPases is unknown, its ability to inhibit only 66% of the degradation by RPE cells in the present study is consistent with its partial block of the ATP degradation by astrocytes (17) and blood cells (4). The relatively low AMP-to-ADP ratios in solutions surrounding ARPE-19 and fresh bovine RPE cells suggest NTPDase2 activity on the RPE membranes. The action of NTPDase1 results in an AMP-to-ADP ratio >10, NTPDase2 leads to a ratio of ~3, and eNPPs convert ATP directly into AMP + pyrophosphate (10, 12). These observations suggest that NTPDase2 makes a contribution to the degradation of purines by RPE cells. The contribution of ectoalkaline phosphatase is likely to be minimal because the inhibitor levamisole had no effect on degradation at 100 µM. Interference with our assay precluded use of the higher concentrations frequently used, but the compound has a Ki of 50 µM at HL-60 cells (34), and 100 µM blocked 75% of alkaline phosphatase activity in the kidney (3). This suggests that a 100 µM concentration should have produced some block if alkaline phosphatase had been active.

Various cell types have been shown to differentially express multiple enzymes capable of degrading ATP. Human bronchial epithelial cells contain eNPP1–eNPP3 and NTPDase1 but not NTPDase2, whereas nasal epithelial cells display eNPP1–eNPP3 and NTPDase2 but not NTPDase1 (31). A differential expression also was found by Joseph et al. (17), with PC12 cells expressing NTPDase1–NTPDase3 but not eNPP1–eNPP3, while C6 glioma cells expressed eNPP1 and eNPP3 but not NTPDases. In the eye, fresh rabbit ciliary epithelial cells possessed mRNA message for NTPDase1 and eNPP1 but not for NTPDase2 (7). Because the pigmented ciliary epithelial cells are developmentally akin to the RPE, this difference is particularly interesting.

The ATP-mediated ATP release shown in Fig. 1C complicates the analysis of enzyme activity, because this release can clearly overwhelm the degradative capacity of the enzymes. The increase in time constant with elevated ATP levels may be influenced by the ATP-mediated release, and it is possible that the jump in time constant found when 1 µM ATP was added to cells reflects this release. Because the Km for all three enzymes is typically 70–400 µM (24, 38, 39), their contribution to the extracellular purine levels is difficult to predict. However, the ability of ARL67156and {beta}{gamma}-mATP to inhibit the degradation of 100 nM ATP indicates that ectoenzymes are indeed acting at lower concentrations. The balance between ATP release and degradation may ultimately be determined by the local microenvironment. As implied in previous reports (14, 16), a differential clustering of release sites, degradation enzymes, and receptors may allow higher levels of ATP to act without triggering a secondary release of ATP.

The measurements shown in Fig. 4 indicate that ADP can elevate the intracellular Ca2+ of RPE cells. The effects of thapsigargin and MRS 2179 are consistent with a role for the P2Y1 receptor in the response (35), and the detection of message for the P2Y1 receptor supports this finding. Identification of the P2Y12 receptor suggests that ADP has additional roles in RPE physiology, because ATP is relatively ineffective at the human P2Y12 receptor (2, 13). The primary coupling of the P2Y12 receptor to the Gi protein makes a direct contribution to the fast Ca2+ peak unlikely, however. The relative amounts of purines found in the solution surrounding RPE cells, combined with the relative responses shown in Fig. 4A, suggest that ATP produces a larger total effect than ADP on cellular Ca2+. However, the ability of ADP to elevate Ca2+ and the presence of NTPDase2 on RPE cells suggest that ADP may contribute to the increased fluid flow that accompanies the response to ATP itself (30). This is supported by a report that ADP can activate ionic currents in rat RPE cells (33). The results from the bovine RPE eyecup preparation suggest that this degradation is mediated by enzymes on the apical membrane of the RPE that alter the balance of purines in subretinal space.


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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Eye Institute (NEI) Grants R01 EY-013434 (to C. H. Mitchell), EY-10009 (to A. M. Laties), and EY-07354 and EY-001583 (to R. A. Stone), and by grants from Research to Prevent Blindness (to R. A. Stone), the Jody Sack Fund, the Paul and Evanina Bell Mackall Foundation Trust (to R. A. Stone), and NEI Core Vision Grant EY-001583, and by Grant 30471850 from the Chinese National Scientific Research Fund and Grant 04009334 from the Guangdong Scientific Research Fund (to X. Zhang).


    ACKNOWLEDGMENTS
 
We thank Tejvir S. Khurana and Henry Shuman for help with molecular biology and Maurine Maguire for statistical assistance.


    FOOTNOTES
 

Address for reprint requests and other correspondence: C. H. Mitchell, Dept. of Physiology, Univ. of Pennsylvania, 3700 Hamilton Walk, Philadelphia, PA 19104-6085 (e-mail: chm{at}mail.med.upenn.edu)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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