1Department of Pharmacology and 2Curriculum in Neurobiology, The University of North Carolina at Chapel Hill, Chapel Hill, North Carolina
Submitted 13 July 2004 ; accepted in final form 26 October 2004
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Madin-Darby canine kidney; 16HBE14o; Caco-2; confocal microscopy; polarized targeting
Although P2Y receptors regulate multiple physiological processes in a variety of cells and tissues, one of their major roles is in the regulation of ion transport and stress responses in epithelial cells (18, 26). Epithelial cells line the interstitial surfaces in the lung, kidney, and intestine and create a barrier between the external environment and the underlying cells and tissue. This paracellular barrier is created by a complex of proteins known as the tight junction, which forms an intercellular connection that creates a monolayer impermeable to water and ions. Tight junctions also serve to demarcate two distinct membrane surfaces in polarized epithelial cells: the apical surface, which lies above the tight junction and faces the lumen, and the basolateral surface, which lies below the tight junction and contacts underlying cells. The differential expression of membrane proteins, including P2Y receptors, at one of these two surfaces allows these cells to regulate a broad range of homeostatic functions, including the movement of water, ions, and nutrients between the lumen and underlying tissue (43).
G protein-coupled P2Y receptors serve an important role in autocrine and paracrine regulation of ion and nutrient transport in epithelial cells. The first indication that P2Y receptors served in this capacity was the observation that ATP and UTP, when added to the apical surface of airway cells, promoted a Ca2+-activated Cl current (28). Multiple subsequent studies showed that all five of the subtypes in the P2Y1 receptor family are expressed in epithelial cells from various tissues (8, 17, 26, 27, 34, 38, 42, 44, 45). Moreover, many of these epithelial cells express multiple subtypes of P2Y receptors (33). Although mRNAs encoding P2Y12 and P2Y14 receptors have been observed in tissues containing epithelial cells, direct demonstration of receptor expression in epithelial cells has not been reported. These studies demonstrated that all five Gq-coupled subtypes of P2Y receptors (and potentially the Gi-coupled subtypes) are expressed in epithelial cells and highlight the prominent role of extracellular nucleotides in regulation of epithelial cell function.
Many of the aforementioned studies suggested that P2Y receptors are localized to distinct membrane surfaces in polarized epithelial cells. However, with the exception of the canine P2Y11 receptor, in which a receptor-green fluorescent protein (GFP) fusion protein was shown to be targeted to the basolateral membrane of Madin-Darby canine kidney (MDCK) cells (44), most of these studies have addressed the question of P2Y receptor polarization in an indirect manner or with potentially nonspecific antibodies that lend uncertainty to the conclusions regarding polarized targeting. To avoid these mitigating factors and to define the targeting properties of the entire family of P2Y receptors in epithelial cells, we determined the steady-state localization of P2Y receptors by visualizing hemagglutinin (HA)-tagged receptors expressed in MDCK type II [MDCK(II)] cells (ATCC, Rockville, MD) using confocal microscopy. These studies were further supplemented by quantification of receptor distribution by using biotinylation and measurement of agonist-induced changes in short-circuit current (Isc). Remarkably, our data indicate that all but one of the eight P2Y receptors are localized exclusively to either the apical or basolateral membrane surfaces of MDCK(II) cells. Moreover, a targeting profile nearly identical to that of the Gq-coupled P2Y receptor family in MDCK cells was obtained in lung 16HBE14o and colonic Caco-2 cells, suggesting that targeting of P2Y receptors is not a function of the cell line in which they are expressed. This is the first study to define the targeting properties of the entire family of P2Y receptors in polarized epithelial cells.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Cell culture and expression of receptor constructs. MDCK(II) cells were subcultured in DMEM/F-12 (1:1) medium (Invitrogen, Carlsbad, CA) supplemented with 5% fetal bovine serum (FBS; Hyclone, Gaithersburg, MD) and 1x penicillin/streptomycin in a humidified incubator at 37°C with 5% CO2-95% air. 16HBE14o cells, from an immortalized human bronchial epithelial cell line (7), were grown on collagen-coated plates in MEM (Invitrogen) supplemented with 10% FBS, 1% sodium pyruvate, 1% nonessential amino acids, and 1x penicillin/streptomycin. Caco-2 cells (11), from an immortalized human colonic epithelial cell line, were grown in the same medium as 16HBE14o cells, except that the FBS concentration was increased to 20%.
Recombinant retroviral particles were produced by calcium phosphate-mediated transfection of PA317 cells with pLXSN vectors containing HA-tagged human P2Y (hP2Y) receptor constructs as previously described (6) and were used to infect the various cell lines. Geneticin-resistant cells were selected after 710 days with 1 mg/ml G418 and maintained in medium containing 0.4 mg/ml G418.
Confocal microscopy. MDCK(II), 16HBE14o, and Caco-2 cells stably expressing HA-tagged hP2Y receptor constructs were seeded (6 x 105 cells/well) in 12-mm polyester Transwell inserts (0.4 µM; Corning Life Sciences, Acton, MA). All cells were allowed to polarize for 57 days with daily medium changes. Cell monolayers were washed with cold PBS++ (phosphate-buffered saline containing 2 mM Ca2+ and Mg2+), fixed and permeabilized with 20°C methanol for 4 min, and blocked with PBS containing 1% nonfat dry milk for 30 min at room temperature. Receptors were labeled with anti-HA mouse monoclonal antibody HA.11 (Covance, Berkeley, CA), and tight junctions were labeled with a rabbit polyclonal antibody to zonula occludens-1 (ZO-1; Zymed, South San Francisco, CA). Cells were washed three times with cold PBS++ and then labeled with goat anti-mouse A-488 (for P2Y receptors) and goat anti-rabbit A-594 (for ZO-1) secondary antibodies (Molecular Probes, Eugene, OR). The fixed and stained monolayers were washed several times with cold PBS++, excised from the Transwell inserts, and mounted on glass microscope slides with Slowfade mounting medium (Molecular Probes).
Confocal microscopy was performed on an Olympus Fluoview 300 laser scanning confocal imaging system (Melville, NY) configured with an IX70 fluorescence microscope fitted with a PlanApo x60 oil objective. Multiple XY (horizontal to the monolayer) and XZ (vertical to the monolayer) scans were acquired for each monolayer.
Quantitation of cell surface HA-tagged P2Y receptors. MDCK(II) cells stably expressing HA-tagged hP2Y receptors were seeded in duplicate in 24-mm Transwell inserts and allowed to polarize as described in Confocal microscopy. Monolayers were placed on ice and kept at 4°C for the duration of the experiment. Cells were washed with cold PBS++ three times for 5 min each and then labeled with 1 mg/ml sulfo-NHS-SS-biotin (Pierce, Rockford, IL) in cold PBS++ buffer, pH 8, for 40 min. The biotin solution was removed, and the reaction was quenched with 100 mM glycine in PBS++ for 10 min. The cells were washed and then incubated for 5 min with 0.7 ml Tris-Triton buffer (50 mM Tris·HCl, pH 7.4, 100 mM NaCl, 5 mM EDTA, and 1% Triton X-100) containing a protease inhibitor cocktail. The cells were passed 710 times through a 25-gauge needle and then incubated for 1.5 h with rocking. The cell lysate was centrifuged at 20,000 g for 30 min, and the supernatant was incubated with 50 µl of immobilized Neutravidin (Pierce) for 1.5 h. The resin was washed twice with Tris-Triton buffer, and biotin-labeled proteins were eluted from the Neutravidin resin by incubation with 35 µl of SDS-PAGE sample buffer containing 100 mM dithiothreitol for 10 min at 37°C. The dithiothreitol cleaves the disulfide within the biotin spacer and releases the proteins from Neutravidin under mild conditions.
Eluted proteins were separated by SDS-PAGE on a 10% gel and transferred overnight to nitrocellulose membranes. Membranes were blotted via a standard Western blotting protocol with the anti-HA monoclonal antibody conjugated to horseradish peroxidase (3F10; Roche Biochemicals, Indianapolis, IN). The blots were developed with SuperSignal West Pico chemiluminescent substrate (Pierce), and the resulting bands were imaged on a Bio-Rad Fluor-S system and quantitated with Bio-Rad QuantityOne software (Bio-Rad, Hercules, CA).
Radioligand binding assay. A binding assay for membranes was performed as previously described (41). Briefly, various concentrations of MDCK(II) membranes were incubated with an approximate Ki concentration (8 nM) of [3H]MRS2279 for 30 min at 4°C. Binding reactions were terminated by the addition of 4 ml of cold assay buffer (20 mM Tris·HCl, pH 7.5, 145 mM NaCl, and 5 mM MgCl2) and filtered through GF/A filters to retain membrane-bound [3H]MRS2279. Filters were washed once with cold assay buffer and placed in scintillation fluid for measurement of radioactivity. Specific binding of 8 nM [3H]MRS2279 to MDCK membranes was determined as total radioligand bound minus the radioligand bound in the presence of 30 µM MRS2179, a P2Y1 receptor-selective antagonist (2).
Ussing chamber measurement of Isc. MDCK(II) cells stably expressing HA-tagged hP2Y receptors were seeded in 12-mm polyester Snapwell inserts (Corning Life Sciences) and allowed to polarize for 57 days as described in Confocal microscopy. The inserts were placed in Ussing chambers and monitored for changes in Isc in response to cumulative concentrations of the appropriate nucleotides added to either the mucosal (apical) or serosal (basolateral) surface. The maximal response at each concentration was plotted as a cumulative increase in Isc vs. nucleotide concentration.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Figure 1 shows XY and XZ cross sections of wild-type MDCK(II) cells and MDCK(II) cells expressing each of the eight P2Y receptor subtypes. Wild-type MDCK(II) cells showed staining of the tight junctions but no staining with the anti-HA antibody, demonstrating the specificity of both antibodies in MDCK(II) cells. Confocal micrographs of MDCK(II) cells expressing each P2Y receptor revealed that seven of the eight receptor subtypes were localized at steady state to either the apical or basolateral membrane surface. P2Y1, P2Y11, P2Y12, and P2Y14 receptors were expressed heavily along the lateral regions of the cell below the tight junction with a low level of expression at the basal membrane. Essentially no visible staining for these receptors was observed in the apical membrane. In contrast, P2Y2, P2Y4, and P2Y6 receptors were expressed exclusively at the apical membrane, with little to no staining below the tight junction (Fig. 1). The only receptor that was not localized was the P2Y13 receptor. Thus the family of P2Y receptors shows a distinct pattern of polarized expression in MDCK(II) cells.
|
|
|
|
The functional activity of the exogenous P2Y1 receptor also was examined in polarized MDCK(II) cells by measuring Isc in Ussing chambers across monolayers of wild-type cells or cells expressing the P2Y1 receptor (Fig. 4). Isc is the summation of the flow of both cations and anions through multiple channels across a monolayer of cells. Increasing concentrations of 2-methylthio-ADP (2-MeSADP) were added cumulatively to either the apical or basolateral compartments, and Isc was measured. Interpretation of these experiments was complicated by the endogenous expression in MDCK cells of the canine homologs of P2Y1, P2Y2, and P2Y11 receptors (34, 44), which give rise to increases in Isc in the absence of exogenous expression of human P2Y receptors. Thus we relied on the observation that concentration-response curves for agonists shift to the left as a function of increases in GPCR expression (21). This procedure has been utilized previously by Zambon et al. (44) in their studies with the canine P2Y11 receptor.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The marked polarized distribution of seven of the eight P2Y receptor subtypes was striking. In contrast, only two of the five muscarinic receptor subtypes, M2 and M3, are targeted to distinct membrane domains in MDCK(II) epithelial cells (31). Polarization of P2Y receptors may result from the fact that all five of the Gq-coupled receptors (17, 25, 42, 44) [and potentially 2 of the 3 Gi-coupled receptors (3, 15)] are natively expressed in polarized cell types, i.e., epithelial and endothelial cells, where targeting of receptors to distinct membrane surfaces is critical for proper function. These data suggest that the seven polarized receptors contain targeting signals that direct the protein to either the apical or basolateral surface. Therefore, receptors not known to be expressed endogenously in epithelial cells, such as the P2Y13 receptor, might lack the proper targeting information to ensure a polarized distribution and by default have an unsorted phenotype. Our data are consistent with this idea.
The targeting profile of the family of P2Y receptors revealed an unexpected pattern. P2Y receptors activated solely by adenine nucleotides, i.e., the P2Y1, P2Y11, and P2Y12 receptors, are localized to the basolateral membrane of MDCK(II) epithelia, whereas those P2Y receptors activated by uridine nucleotides, i.e., P2Y2, P2Y4, and P2Y6 receptors, are localized to the apical membrane (Fig. 5). The P2Y14 receptor, which is activated by UDP sugars such as UDP-glucose, is also localized to the basolateral membrane. However, the targeting of this receptor may be more a function of its high homology to the adenine nucleotide-selective Gi-coupled P2Y12 receptor than to its ligand. The significance of this unusual localization pattern is unclear, but one intriguing possibility may be that the distribution of P2Y receptors has evolved to complement the preferential release of adenine nucleotides at the basolateral membrane and uridine nucleotides at the apical membrane. However, it is well documented that both ATP and UTP are released from the apical surface of epithelial cells in response to mechanical stimulation and hypotonic challenge (16, 24). In addition, Lazarowski and Harden (23) demonstrated a general release of UTP from primary epithelial cells, although the relative amounts released from the two membrane surfaces is unknown and difficult to measure because of the complex nature of nucleotide metabolism and conversion that occurs within the interstitial space. Thus the significance, if any, of this differential targeting of P2Y receptors remains unclear.
|
The only receptor that deviated somewhat from its targeting profile obtained in MDCK(II) cells was the P2Y2 receptor, which in addition to its primarily apical localization was also expressed at lower levels along the lateral membranes of 16HBE14o cells. Interestingly, this low level of lateral staining also was observed in another human epithelial cell line derived from lung, BEAS-2B (37) (data not shown), but not in Caco-2 cells (Fig. 3), suggesting that the small amount of lateral staining of the P2Y2 receptor may be a property of airway cells in particular. Consistent with this observation, Boucher and colleagues (16, 32) demonstrated that UTP promoted intracellular Ca2+ mobilization when added to the basolateral surface of nasal epithelium derived from wild-type mice. These responses were not observed in nasal epithelium derived from P2Y2 receptor (/) mice, demonstrating that the responses are due to activation of basolateral P2Y2 receptors. The physiological relevance of this observation is not clear, but our results suggest that the mechanisms utilized by epithelial cells to target the P2Y2 receptor to the apical membrane are not as stringent in epithelial cells from lung compared with those from other tissues.
Although the polarized targeting of the Gq-coupled P2Y receptors is consistent with the majority of results based on functional activity (10, 26, 29, 44), our results conflict with several reports on the polarized expression of P2Y receptors in epithelial cells. For example, Sage and Marcus (39) suggested a basolateral localization for P2Y2 in vestibular dark epithelia on the basis of immunostaining with a commercial antibody. However, results based on commercial P2Y2 receptor antibody staining alone should be viewed with caution, because these antibodies exhibit questionable specificity for P2Y2 receptors. A study by Dranoff et al. (9) utilized indirect pharmacological assays to suggest a polarization of P2Y1, P2Y2, P2Y4, and P2Y6 receptors at the apical membrane of rat bile duct epithelia. However, it is difficult to determine unequivocally which P2Y receptor subtypes are present at the apical membrane because of the complexities of tissues with unknown metabolizing and interconverting enzyme activities (19, 22). Thus, without better reagents, including subtype-selective agonists and antagonists and antibodies with rigorously demonstrated receptor specificity, it is extremely difficult to show polarized targeting of P2Y receptors in complex tissues.
In conclusion, we utilized three different approaches, including the direct method of confocal microscopy, to show the highly polarized expression pattern of the entire family of P2Y receptors. These data are for the most part consistent with previous reports and extend our knowledge of the localization of P2Y receptors in epithelial cells. Because the polarization of cell surface proteins to either the apical or basolateral membrane of epithelial cells is achieved by the presence of targeting signals within the primary protein sequence, our data suggest that seven of the eight hP2Y receptors contain targeting signals that direct their expression to one of the two membrane surfaces of MDCK cells. Studies to identify these targeting signals in P2Y receptors and to understand how these signals function are currently in progress.
![]() |
GRANTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
FOOTNOTES |
---|
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.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2. Boyer JL, Mohanram A, Camaioni E, Jacobson KA, and Harden TK. Competitive and selective antagonism of P2Y1 receptors by N6-methyl 2'-deoxyadenosine 3',5'-bisphosphate. Br J Pharmacol 124: 13, 1998.[CrossRef][ISI][Medline]
3. Chambers JK, Macdonald LE, Sarau HM, Ames RS, Freeman K, Foley JJ, Zhu Y, McLaughlin MM, Murdock P, McMillan L, Trill J, Swift A, Aiyar N, Taylor P, Vawter L, Naheed S, Szekeres P, Hervieu G, Scott C, Watson JM, Murphy AJ, Duzic E, Klein C, Bergsma DJ, Wilson S, and Livi GP. A G protein-coupled receptor for UDP-glucose. J Biol Chem 275: 1076710771, 2000.
4. Communi D, Gonzalez NS, Detheux M, Brezillon S, Lannoy V, Parmentier M, and Boeynaems JM. Identification of a novel human ADP receptor coupled to Gi. J Biol Chem 276: 4147941485, 2001.
5. Communi D, Govaerts C, Parmentier M, and Boeynaems JM. Cloning of a human purinergic P2Y receptor coupled to phospholipase C and adenylyl cyclase. J Biol Chem 272: 3196931973, 1997.
6. Comstock KE, Watson NF, and Olsen JC. Design of retroviral expression vectors. In: Methods in Molecular Biology: Recombinant Gene Expression Protocols, edited by Tuan R. Totowa, NJ: Humana, 1997, vol. 62, p. 207222.
7. Cozens AL, Yezzi MJ, Kunzelmann K, Ohrui T, Chin L, Eng K, Finkbeiner WE, Widdicombe JH, and Gruenert DC. CFTR expression and chloride secretion in polarized immortal human bronchial epithelial cells. Am J Respir Cell Mol Biol 10: 3847, 1994.[Abstract]
8. Cressman VL, Lazarowski E, Homolya L, Boucher RC, Koller BH, and Grubb BR. Effect of loss of P2Y2 receptor gene expression on nucleotide regulation of murine epithelial Cl transport. J Biol Chem 274: 2646126468, 1999.
9. Dranoff JA, Masyuk AI, Kruglov EA, LaRusso NF, and Nathanson MH. Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia. Am J Physiol Gastrointest Liver Physiol 281: G1059G1067, 2001.
10. Dubyak GR. Knock-out mice reveal tissue-specific roles of P2Y receptor subtypes in different epithelia. Mol Pharmacol 63: 773776, 2003.
11. Fogh J, Fogh JM, and Orfeo T. One hundred and twenty-seven cultured human tumor cell lines producing tumors in nude mice. J Natl Cancer Inst 59: 221226, 1977.[ISI][Medline]
12. Folsch H, Ohno H, Bonifacino JS, and Mellman I. A novel clathrin adaptor complex mediates basolateral targeting in polarized epithelial cells. Cell 99: 189198, 1999.[CrossRef][ISI][Medline]
13. Forbes II. Human airway epithelial cell lines for in vitro drug transport and metabolism studies. Pharm Sci Technol Today 3: 1827, 2000.[CrossRef][Medline]
14. Harden TK. The G-protein-coupled P2Y receptors. In: Cardiovascular Biology of Purines, edited by Burnstock G, Dobson JGJ, Liang BT, and Linden J. London: Kluwer Academic, 1998, p. 181205.
15. 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]
16. Homolya L, Steinberg TH, and Boucher RC. Cell to cell communication in response to mechanical stress via bilateral release of ATP and UTP in polarized epithelia. J Cell Biol 150: 13491360, 2000.
17. Homolya L, Watt WC, Lazarowski ER, Koller BH, and Boucher RC. Nucleotide-regulated calcium signaling in lung fibroblasts and epithelial cells from normal and P2Y2 receptor (/) mice. J Biol Chem 274: 2645426460, 1999.
18. Insel PA, Ostrom RS, Zambon AC, Hughes RJ, Balboa MA, Shehnaz D, Gregorian C, Torres B, Firestein BL, Xing M, and Post SR. P2Y receptors of MDCK cells: epithelial cell regulation by extracellular nucleotides. Clin Exp Pharmacol Physiol 28: 351354, 2001.[CrossRef][ISI][Medline]
19. 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]
20. Keefer JR and Limbird LE. The 2A-adrenergic receptor is targeted directly to the basolateral membrane domain of Madin-Darby canine kidney cells independent of coupling to pertussis toxin-sensitive GTP-binding proteins. J Biol Chem 268: 1134011347, 1993.
21. Kenakin T. Pharmacologic Analysis of Drug-Receptor Interaction. Philadelphia, PA: Lippincott-Raven, 1997.
22. Lazarowski ER, Boucher RC, and Harden TK. Mechanisms of release of nucleotides and integration of their action as P2X- and P2Y-receptor activating molecules. Mol Pharmacol 64: 785795, 2003.
23. Lazarowski ER and Harden TK. Quantitation of extracellular UTP using a sensitive enzymatic assay. Br J Pharmacol 127: 12721278, 1999.[CrossRef][ISI][Medline]
24. Lazarowski ER, Homolya L, Boucher RC, and Harden TK. Direct demonstration of mechanically induced release of cellular UTP and its implication for uridine nucleotide receptor activation. J Biol Chem 272: 2434824354, 1997.
25. Lazarowski ER, Paradiso AM, Watt WC, Harden TK, and Boucher RC. UDP activates a mucosal-restricted receptor on human nasal epithelial cells that is distinct from the P2Y2 receptor. Proc Natl Acad Sci USA 94: 25992603, 1997.
26. Leipziger J. Control of epithelial transport via luminal P2 receptors. Am J Physiol Renal Physiol 284: F419F432, 2003.
27. Marcus DC and Scofield MA. Apical P2Y4 purinergic receptor controls K+ secretion by vestibular dark cell epithelium. Am J Physiol Cell Physiol 281: C282C289, 2001.
28. Mason SJ, Paradiso AM, and Boucher RC. Regulation of transepithelial ion transport and intracellular calcium by extracellular ATP in human normal and cystic fibrosis airway epithelium. Br J Pharmacol 103: 16491656, 1991.[ISI][Medline]
29. McAlroy HL, Ahmed S, Day SM, Baines DL, Wong HY, Yip CY, Ko WH, Wilson SM, and Collett A. Multiple P2Y receptor subtypes in the apical membranes of polarized epithelial cells. Br J Pharmacol 131: 16511658, 2000.[CrossRef][ISI][Medline]
30. Mostov KE, Verges M, and Altschuler Y. Membrane traffic in polarized epithelial cells. Curr Opin Cell Biol 12: 483490, 2000.[CrossRef][ISI][Medline]
31. Nadler LS, Kumar G, and Nathanson NM. Identification of a basolateral sorting signal for the M3 muscarinic acetylcholine receptor in Madin-Darby canine kidney cells. J Biol Chem 276: 1053910547, 2001.
32. Paradiso AM, Ribeiro CM, and Boucher RC. Polarized signaling via purinoceptors in normal and cystic fibrosis airway epithelia. J Gen Physiol 117: 5367, 2001.[CrossRef][ISI][Medline]
33. Post SR, Jacobson JP, and Insel PA. P2 purinergic receptor agonists enhance cAMP production in Madin-Darby canine kidney epithelial cells via an autocrine/paracrine mechanism. J Biol Chem 271: 20292032, 1996.
34. Post SR, Rump LC, Zambon A, Hughes RJ, Buda MD, Jacobson JP, Kao CC, and Insel PA. ATP activates cAMP production via multiple purinergic receptors in MDCK-D1 epithelial cells. Blockade of an autocrine/paracrine pathway to define receptor preference of an agonist. J Biol Chem 273: 2309323097, 1998.
35. Qi AD, Kennedy C, Harden TK, and Nicholas RA. Differential coupling of the human P2Y11 receptor to phospholipase C and adenylyl cyclase. Br J Pharmacol 132: 318326, 2001.[CrossRef][ISI][Medline]
36. Ralevic V and Burnstock G. Receptors for purines and pyrimidines. Pharmacol Rev 50: 413492, 1998.
37. Reddel RR, Ke Y, Gerwin BI, McMenamin MG, Lechner JF, Su RT, Brash DE, Park JB, Rhim JS, and Harris CC. Transformation of human bronchial epithelial cells by infection with SV40 or adenovirus-12 SV40 hybrid virus, or transfection via strontium phosphate coprecipitation with a plasmid containing SV40 early region genes. Cancer Res 48: 19041909, 1988.[Abstract]
38. Robaye B, Ghanem E, Wilkin F, Fokan D, Van Driessche W, Schurmans S, Boeynaems JM, and Beauwens R. Loss of nucleotide regulation of epithelial chloride transport in the jejunum of P2Y4-null mice. Mol Pharmacol 63: 777783, 2003.
39. Sage CL and Marcus DC. Immunolocalization of P2Y4 and P2Y2 purinergic receptors in strial marginal cells and vestibular dark cells. J Membr Biol 185: 103115, 2002.[CrossRef][ISI][Medline]
40. Torres B, Zambon AC, and Insel PA. P2Y11 receptors activate adenylyl cyclase and contribute to nucleotide-promoted cAMP formation in MDCK-D1 cells. A mechanism for nucleotide-mediated autocrine-paracrine regulation. J Biol Chem 277: 77617765, 2002.
41. Waldo GL, Corbitt J, Boyer JL, Ravi G, Kim HS, Ji XD, Lacy J, Jacobson KA, and Harden TK. Quantitation of the P2Y1 receptor with a high affinity radiolabeled antagonist. Mol Pharmacol 62: 12491257, 2002.
42. Wong CH and Ko WH. Stimulation of Cl secretion via membrane-restricted Ca2+ signaling mediated by P2Y receptors in polarized epithelia. J Biol Chem 277: 90169021, 2002.
43. Yeaman C, Grindstaff KK, and Nelson WJ. New perspectives on mechanisms involved in generating epithelial cell polarity. Physiol Rev 79: 7398, 1999.
44. Zambon AC, Brunton LL, Barrett KE, Hughes RJ, Torres B, and Insel PA. Cloning, expression, signaling mechanisms, and membrane targeting of P2Y11 receptors in Madin Darby canine kidney cells. Mol Pharmacol 60: 2635, 2001.
45. Zambon AC, Hughes RJ, Meszaros JG, Wu JJ, Torres B, Brunton LL, and Insel PA. P2Y2 receptor of MDCK cells: cloning, expression, and cell-specific signaling. Am J Physiol Renal Physiol 279: F1045F1052, 2000.
46. Zhang FL, Luo L, Gustafson E, Palmer K, Qiao X, Fan X, Yang S, Laz TM, Bayne M, and Monsma F Jr. P2Y13: identification and characterization of a novel Gi-coupled ADP receptor from human and mouse. J Pharmacol Exp Ther 301: 705713, 2002.