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
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ABSTRACT |
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ectoapyrase; PC-1; CD39; CD39L1; P2Y1; P2Y12; ADP; ATP release; photoreceptors; retinal detachment
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).
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METHODS |
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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 520 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--dibromomethylene ATP (ARL67156,
-methylene ATP (
-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 -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 (2125°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 12 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 25 µl of cDNA, 1.53 mM MgCl2, 0.5 µl of Taq, and 0.20.6 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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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 + aebx ( = 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 = 01 min compared with t = 5960 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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RESULTS |
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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 -mATP blocked 78% of the ATP degradation from ARPE-19 cells. The inhibitory effects of
-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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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 -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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DISCUSSION |
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ATP degradation is likely due to the actions of both eNPPs and NTPDases. A recent study by Joseph et al. (17) indicated that -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
-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 eNPP1eNPP3 and NTPDase1 but not NTPDase2, whereas nasal epithelial cells display eNPP1eNPP3 and NTPDase2 but not NTPDase1 (31). A differential expression also was found by Joseph et al. (17), with PC12 cells expressing NTPDase1NTPDase3 but not eNPP1eNPP3, 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 70400 µM (24, 38, 39), their contribution to the extracellular purine levels is difficult to predict. However, the ability of ARL67156and -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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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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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REFERENCES |
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2. Bodor ET, Waldo GL, Hooks SB, Corbitt J, Boyer JL, and Harden TK. Purification and functional reconstitution of the human P2Y12 receptor. Mol Pharmacol 64: 12101216, 2003.
3. Calhau C, Martel F, Hipólito-Reis C, and Azevedo I. Differences between duodenal and jejunal rat alkaline phosphatase. Clin Biochem 33: 571577, 2000.[CrossRef][ISI][Medline]
4. Crack BE, Pollard CE, Beukers MW, Roberts SM, Hunt SF, Ingall AH, McKechnie KC, IJzerman AP, and Leff P. Pharmacological and biochemical analysis of FPL 67156, a novel, selective inhibitor of ecto-ATPase. Br J Pharmacol 114: 475481, 1995.[ISI][Medline]
5. Dunn KC, Marmorstein AD, Bonilha VL, Rodriguez-Boulan E, Giordano F, and Hjelmeland LM. Use of the ARPE-19 cell line as a model of RPE polarity: basolateral secretion of FGF5. Invest Ophthalmol Vis Sci 39: 27442749, 1998.[Abstract]
6. Eldred JA, Sanderson J, Wormstone M, Reddan JR, and Duncan G. Stress-induced ATP release from and growth modulation of human lens and retinal pigment epithelial cells. Biochem Soc Trans 31: 12131215, 2003.[ISI][Medline]
7. Farahbakhsh NA. Ectonucleotidases of the rabbit ciliary body nonpigmented epithelium. Invest Ophthalmol Vis Sci 44: 39523960, 2003.
8. Friedman C, Zhang X, Laties AM, and Mitchell CH. Ecto-nucleotidases on RPE cells: a potential source of extracellular adenosine (Abstract). Invest Ophthalmol Vis Sci 43: U197, 2002.
9. Gregory CY, Abrams TA, and Hall MO. Stimulation of A2 adenosine receptors inhibits the ingestion of photoreceptor outer segments by retinal pigment epithelium. Invest Ophthalmol Vis Sci 35: 819825, 1994.[Abstract]
10. Grobben B, Anciaux K, Roymans D, Stefan C, Bollen M, Esmans EL, and Slegers H. An ecto-nucleotide pyrophosphatase is one of the main enzymes involved in the extracellular metabolism of ATP in rat C6 glioma. J Neurochem 72: 826834, 1999.[CrossRef][ISI][Medline]
11. Grobben B, Claes P, Roymans D, Esmans EL, Van Onckelen H, and Slegers H. Ecto-nucleotide pyrophosphatase modulates the purinoceptor-mediated signal transduction and is inhibited by purinoceptor antagonists. Br J Pharmacol 130: 139145, 2000.[CrossRef][ISI][Medline]
12. Heine P, Braun N, Heilbronn A, and Zimmermann H. Functional characterization of rat ecto-ATPase and ecto-ATP diphosphohydrolase after heterologous expression in CHO cells. Eur J Biochem 262: 102107, 1999.
13. Hollopeter G, Jantzen HM, Vincent D, Li G, England L, Ramakrishnan V, Yang RB, Nurden P, Nurden A, Julius D, and Conley PB. Identification of the platelet ADP receptor targeted by antithrombotic drugs. Nature 409: 202207, 2001.[CrossRef][ISI][Medline]
14. Huang P, Lazarowski ER, Tarran R, Milgram SL, Boucher RC, and Stutts MJ. Compartmentalized autocrine signaling to cystic fibrosis transmembrane conductance regulator at the apical membrane of airway epithelial cells. Proc Natl Acad Sci USA 98: 1412014125, 2001.
15. Hughes BA, Gallemore RP, and Miller SS. Transport mechanisms in the retinal pigment epithelium. In: The Retinal Pigment Epithelium: Function and Disease, edited by Marmor MF and Wolfensberger TJ. New York: Oxford University Press, 1998, p. 103134.
16. Joseph SM, Buchakjian MR, and Dubyak GR. Colocalization of ATP release sites and ecto-ATPase activity at the extracellular surface of human astrocytes. J Biol Chem 278: 2333123342, 2003.
17. Joseph SM, Pifer MA, Przybylski RJ, and Dubyak GR. Methylene ATP analogs as modulators of extracellular ATP metabolism and accumulation. Br J Pharmacol 142: 10021014, 2004.[CrossRef][ISI][Medline]
18. Kato K, Thomas TP, Stevens MJ, Greene DA, and Nakamura J. 2-Chloroadenosine reverses hyperglycemia-induced inhibition of phosphoinositide synthesis in cultured human retinal pigment epithelial cells and prevents reduced nerve conduction velocity in diabetic rats. Metabolism 48: 827833, 1999.[CrossRef][ISI][Medline]
19. Lu W, Reigada D, Pendrak K, McGlinn A, Sévigny J, Laties AM, Stone RA, and Mitchell CH. RPE cells express both NTPDase1 and NTPDase2: resulting ADP can increase cell calcium (Abstract). Invest Ophthalmol Vis Sci 45: 3685, 2004.
20. Maminishkis A, Jalickee S, Blaug SA, Rymer J, Yerxa BR, Peterson WM, and Miller SS. The P2Y2 receptor agonist INS37217 stimulates RPE fluid transport in vitro and retinal reattachment in rat. Invest Ophthalmol Vis Sci 43: 35553566, 2002.
21. Marmor MF. Mechanisms of retinal adhesiveness. In: The Retinal Pigment Epithelium: Function and Disease, edited by Marmor MF and Wolfensberger TJ. New York: Oxford University Press, 1998, p. 392405.
22. Marteau F, Le Poul E, Communi D, Labouret C, Savi P, Boeynaems JM, and Suarez Gonzalez N. Pharmacological characterization of the human P2Y13 receptor. Mol Pharmacol 64: 104112, 2003.
23. Maruiwa F, Nao-i N, Nakazaki S, and Sawada A. Effects of adenosine on chick retinal pigment epithelium: membrane potentials and light-evoked responses. Curr Eye Res 14: 685691, 1995.[ISI][Medline]
24. Mateo J, Harden TK, and Boyer JL. Functional expression of a cDNA encoding a human ecto-ATPase. Br J Pharmacol 128: 396402, 1999.[CrossRef][ISI][Medline]
25. Meyer CH, Hotta K, Peterson WM, Toth CA, and Jaffe GJ. Effect of INS37217, a P2Y2 receptor agonist, on experimental retinal detachment and electroretinogram in adult rabbits. Invest Ophthalmol Vis Sci 43: 35673574, 2002.
26. Mitchell CH. Release of ATP by a human retinal pigment epithelial cell line: potential for autocrine stimulation through subretinal space. J Physiol 534: 193202, 2001.
27. Mitchell CH, Zhang X, Pendrak K, and Stone RA. Ecto ATDPase and ecto-5' nucleotidase activity on the apical membrane of the RPE (Abstract). Invest Ophthalmol Vis Sci 44: U383, 2003.
28. Mitchell CH, Zhang XL, Pendrak K, Friedman C, and Stone RA. Extracellular formation of adenosine from ATP in the retinal pigment-epithelium (Abstract). FASEB J 17: A471, 2003.
29. North RA and Surprenant A. Pharmacology of cloned P2X receptors. Annu Rev Pharmacol Toxicol 40: 563580, 2000.[CrossRef][ISI][Medline]
30. Peterson WM, Meggyesy C, Yu K, and Miller SS. Extracellular ATP activates calcium signaling, ion, and fluid transport in retinal pigment epithelium. J Neurosci 17: 23242337, 1997.
31. Picher M and Boucher RC. Metabolism of extracellular nucleotides in human airways by a multienzyme system. Drug Dev Res 52: 6675, 2001.[CrossRef][ISI]
32. Reigada D and Mitchell CH. Release of ATP from RPE cells involves both CFTR and vesicular transport. Am J Physiol Cell Physiol 288: C132C140, 2005.
33. Ryan JS, Baldridge WH, and Kelly ME. Purinergic regulation of cation conductances and intracellular Ca2+ in cultured rat retinal pigment epithelial cells. J Physiol 520: 745759, 1999.
34. Scheibe RJ, Kuehl H, Krautwald S, Meissner JD, and Mueller WH. Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid. J Cell Biochem 76: 420436, 2000.[CrossRef][ISI][Medline]
35. Von Kugelgen I and Wetter A. Molecular pharmacology of P2Y-receptors. Naunyn Schmiedebergs Arch Pharmacol 362: 310323, 2000.[CrossRef][ISI][Medline]
36. Wood E, Broekman MJ, Kirley TL, Diani-Moore S, Tickner M, Drosopoulos JHF, Islam N, Park JI, Marcus AJ, and Rifkind AB. Cell-type specificity of ectonucleotidase expression and upregulation by 2,3,7,8-tetrachlorodibenzo-p-dioxin. Arch Biochem Biophys 407: 4962, 2002.[CrossRef][ISI][Medline]
37. Young RW and Bok D. Participation of the retinal pigment epithelium in the rod outer segment renewal process. J Cell Biol 42: 392403, 1969.
38. Zimmermann H. Ectonucleotidases. In: Purinergic and Pyrimidinergic Signalling: Molecular, Nervous, and Urogenitary System Function, edited by Abbracchio MP and Williams M. New York: Springer-Verlag, 2001, vol. I, p. 209250.
39. Zimmermann H. Extracellular metabolism of ATP and other nucleotides. Naunyn Schmiedebergs Arch Pharmacol 362: 299309, 2000.[CrossRef][ISI][Medline]
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