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J. Biol. Chem., Vol. 277, Issue 10, 7761-7765, March 8, 2002

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P2Y₁₁ Receptors Activate Adenylyl Cyclase and Contribute to Nucleotide-promoted cAMP Formation in MDCK-D₁ Cells

A MECHANISM FOR NUCLEOTIDE-MEDIATED AUTOCRINE-PARACRINE REGULATION^*

Brian Torres, Alexander C. Zambon, and Paul A. Insel

From the Department of Pharmacology, University of California, San Diego, La Jolla, California 92093-0636

Received for publication, October 28, 2001, and in revised form, January 9, 2002

	ABSTRACT

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Extracellular nucleotides activate P2Y receptors, thereby increasing cAMP formation in Madin-Darby canine kidney (MDCK-D₁) cells, which express P2Y₁, P2Y₂, and P2Y₁₁ receptors (Post, S. R., Rump, L. C., Zambon, A., Hughes, R. J., Buda, M. D., Jacobson, J. P., Kao, C. C., and Insel, P. A. (1998) J. Biol. Chem. 273, 23093-23097). The cyclooxygenase inhibitor indomethacin (indo) eliminates UTP-promoted cAMP formation (i.e. via P2Y₂ receptors) but only partially blocks ATP-promoted cAMP formation. The latter response is completely blocked by the nonselective P2Y receptor antagonist suramin. We have sought to identify the mechanism for this P2Y receptor-mediated, indo-resistant cAMP formation. The agonist rank order potencies for cAMP formation were: ADPS  MT-ADP > 2-MT-ATP > ADP, ATP, ATPS > UTP, AMP, adenosine. We found a similar rank order in MDCK-D₁ cells overexpressing cloned green fluorescent protein-tagged P2Y₁₁ receptors, but the potency of the agonists was enhanced, consistent with a P2Y₁₁ receptor-mediated effect. cAMP generation by the P2Y₁ and P2Y₁₁ receptor agonist ADPS was not inhibited by several P2Y₁-selective antagonists (PPADS, A2P5P, and MRS 2179). Forskolin synergistically enhanced cAMP generation in response to ADPS or PGE₂, implying that, like PGE₂, ADPS activates adenylyl cyclase via G_s, a conclusion supported by results showing ADPS and MT-ADP promoted activation of adenylyl cyclase activity in MDCK-D₁ membranes. We conclude that nucleotide-promoted, indo-resistant cAMP formation in MDCK-D₁ cells occurs via G_s-linked P2Y₁₁ receptors. These data describing adenylyl cyclase activity via endogenous P2Y₁₁ receptors define a mechanism by which released nucleotides can increase cAMP in MDCK-D₁ and other P2Y₁₁-containing cells.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cells commonly co-express multiple receptor subtypes that recognize the same physiological agonist, but it is difficult to define which among such receptor subtypes mediates a particular response. This can be a particularly vexing problem if subtype-selective agonists and antagonists are not available. One such example is P2Y receptors, which respond to ATP and other nucleotides, are expressed in a variety of tissues and cell types, and for which few subtype-selective antagonists exist (2-4). Our laboratory has undertaken a series of studies related to signal transduction by P2Y receptors in the renal epithelial cell line, MDCK¹-D₁ (reviewed in Refs. 5 and 6). P2Y receptors in MDCK-D₁ cells modulate membrane potential and short circuit current, and the receptors regulate phospholipases, intracellular [Ca²⁺], prostaglandin E₂ (PGE₂) formation, and cAMP accumulation (1, 7-14).

Cyclooxygenase (COX) inhibitors, such as indomethacin (indo), have proven useful for studying P2Y receptors in MDCK-D₁ cells (1, 12). Agonist-stimulated cAMP accumulation in MDCK-D₁ cells occurs via both indo-sensitive and -insensitive pathways. Response to the P2Y₂ agonist UTP is entirely indo-sensitive, whereas response to ATP is partially sensitive and to 2-methylthio-ATP (MT-ATP) is insensitive (1). These findings suggest that UTP, and ATP in part, stimulate P2Y₂ receptors to cause COX-mediated (perhaps by both COX1 and COX2, see Ref. 15) formation of arachidonic acid metabolites (e.g. PGE₂), which activate EP receptors to stimulate cAMP formation, while ATP and MT-ATP can also enhance cAMP formation via an indo-insensitive P2Y receptor pathway (1).

The present studies were designed to characterize more fully the nature of the latter pathway. Our working hypothesis, based in part on initial results obtained with MDCK-D₁ cells (12), was that the indo-resistant response might represent a P2Y₁ receptor effect. The current data show results not consistent with this hypothesis but instead suggest a key role for another receptor, the P2Y₁₁ receptor, in indo-resistant cAMP formation. The findings directly document a role for P2Y₁₁ receptors in stimulation of adenylyl cyclase activity and in potentially contributing to autocrine-paracrine regulation by nucleotides.

	EXPERIMENTAL PROCEDURES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

Cell Culture-- MDCK-D₁ cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% mixed serum (85% horse serum, 15% fetal bovine serum). Cells were used in assays at 60-80% confluency. GFP-tagged cP2Y₁₁ receptor-overexpressing MDCK-D₁ cells were cultured from the stable cell line prepared by Zambon et al. (14).

Measurement of cAMP Accumulation in Normal and GFP-tagged cP2Y₁₁ Receptor-overexpressing MDCK-D₁ Intact Cells-- Prior to the treatment of the cells, the growth medium was removed, and cells were equilibrated for 30 min at 37 °C in serum-free 20 mM HEPES-buffered Dulbecco's modified Eagle's medium (DMEH, pH 7.4). Subsequently, cells were incubated in fresh DMEH and various agents as shown in the figures. Incubations with the agonists ADPS, MT-ADP, ATPS, ATP, MT-ATP, ADP, AMP, adenosine, UTP, PGE₂, and forskolin and the antagonists PPADS, A2P5P, and MRS 2179 were conducted for 7 min at 37 °C in the presence of 200 µM IBMX, a phosphodiesterase inhibitor, and terminated by placing on ice and replacing the medium with 7.5% trichloroacetic acid. Trichloroacetic acid extracts were frozen (20 °C) until assay. Intracellular cAMP levels were determined by radioimmunoassay (Calbiochem) of trichloroacetic acid extracts following acetylation according to the manufacturer's protocol. Production of cAMP was normalized to the amount of acid-insoluble protein (Lowry and Bradford methods).

Preparation of Membranes-- Membranes were prepared from MDCK-D₁ cells as follows: cells were grown to confluency on 15-cm plates and washed twice with phosphate-buffered saline, the second wash containing 0 .01% EDTA. Cells were then incubated for 15 min at 37 °C in phosphate-buffered saline containing 0.01% EDTA. Detached cells were centrifuged at 1,000 rpm, and the resulting pellet was suspended in 10 ml of 4 °C lysis buffer (20 mM Tris-HCl, pH 7.4, 10 mM MgCl₂, 2 mM EDTA, 2 mM EGTA, 1 mM dithiothreitol) containing protease inhibitors (10 µM leupeptin, 500 nM pepstatin, 200 µM benzamidine, 200 µM 4-(2-aminoethyl) benzenesulfonylfluoride-HCl (AEBSF). Suspended cells were then placed under 450 p.s.i for 10 min to lyse the cells. The resulting lysed cell mixture was centrifuged at 2,000 rpm for 15 min at 4 °C to remove cell nuclei. The supernatant was separated from the nuclear pellet and centrifuged for 90 min at 15,000 rpm at 4 °C. The resulting pellet was suspended in buffer A (20 mM Tris-HCl, pH 7.4, 5 mM MgCl₂, 1 mM EDTA, 1 mM dithiothreitol), which included protease inhibitors as indicated previously. A protein assay (Bio-Rad) was run to determine membrane protein concentration. Membranes were stored at 80 °C.

Membrane Adenylyl Cyclase Assays-- 75 µg of membrane were placed in 1.5-ml Eppendorf tubes on ice. The volume was brought up to 250 µl using substrate (30 µM AMP-PNP), agonist, and buffer B (50 mM HEPES, pH 7.4, 1 mM EDTA, 5 mM MgCl₂, 1 mM IBMX, 1 mM dithiothreitol, 10 µM GTP, and protease inhibitors). Reaction tubes were then incubated in a 37 °C water bath for 30 min. The reaction was stopped by boiling for 1 min, and samples were then centrifuged for 10 min at 4 °C at 13,000 rpm. Supernatant cAMP levels were then determined using the radioimmunoassay protocol described above.

Materials-- Forskolin, PGE₂, AMP-PNP, PPADS, and anti-cAMP antibody were purchased from Calbiochem. ¹²⁵I-cAMP was purchased from PerkinElmer Life Sciences. ADPS, ATPS, MT-ADP, MT-ATP, ATP, ADP, AMP, adenosine, UTP, PGE₂, A2P5P, IBMX, and indomethacin were purchased from Sigma. MRS 2179 was generously provided by Dr. Kenneth Jacobson, National Institutes of Health.

Statistics-- Data shown generally represent mean ± S.E. of multiple determinations. Curves shown in Figs. 1 and 3 were obtained using the sigmoidal dose response function of Graph Pad Prizm (Graphpad Software Inc., San Diego, CA); analysis using this program yielded EC₅₀ values shown in text and tables.

	RESULTS

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

As a first step toward investigating the basis for indo-resistant cAMP formation in MDCK-D₁ cells, we determined the ability of different P2 agonists to elicit this response. Stimulation of cells with UTP was maximally able to increase cAMP levels ~3-fold over basal levels, but consistent with previous results (1, 12), pretreatment with 1 µM indo abolished this response (Fig. 1A). Treatment of cells with indo also partially decreased ATP-stimulated cAMP accumulation but was less effective than the inhibition of the response to UTP, particularly at higher concentrations of ATP (Fig. 1B). The methylthio- derivative of ATP, MT-ATP, produced a largely indo-resistant increase in cAMP levels (Fig. 1C). ATP can be sequentially dephosphorylated to ADP, AMP, and adenosine by ecto-ATPases and nucleotidases (16, 17). To assess whether ATP was responsible for the observed increase in cAMP levels or whether an ATP metabolite might be responsible, we treated cells with an ATPase-resistant form of ATP, ATPS (16), and obtained results comparable with those observed with ATP (Fig. 1D), suggesting that ATP need not be metabolized to raise cAMP levels.

View larger version (19K): [in this window] [in a new window]   Fig. 1.   The effect of indo on ADPS cAMP generation is minimal compared with its effect on cAMP generation by nucleotides in MDCK-D1 cells. Cells were treated as described under "Experimental Procedures." Cells were incubated for 30 min with or without indomethacin (1 µM), then for 7 min with IBMX (200 µM) and the indicated agonist. Cells were then assayed for cAMP. Data are mean ± S.E. of triplicate determinations and are representative of results obtained in at least three separate experiments.

Like ATP, ADP elevated indo-resistant cAMP levels in a concentration-dependent manner (Fig. 1E). ADPS, a nonhydrolyzable analog of ADP, and MT-ADP both increased indo-resistant cAMP levels as much or more than did ADP, ATP, or ATPS (Fig. 1, F and G). The ADPS-stimulated cAMP increase was entirely indo-resistant. Stimulation of MDCK-D₁ cells with AMP and adenosine, both metabolic breakdown products of ADP, did not elevate cAMP (Ref. 12 and data not shown).

The observed pattern of response for the different agonists, in particular with reference to ATP, ADP, ADPS, and MT-ATP, suggested that indo-resistant cAMP accumulation might be the result of an action at the P2Y₁ receptor (2-4). We thus tested several P2Y₁ antagonists to determine whether this receptor might mediate indo-resistant cAMP accumulation. The P2Y-nonselective antagonist suramin blocked this response to 10 µM ADPS in MDCK-D₁ cells (Fig. 2), but several other putative P2Y₁-selective antagonists proved ineffective, including PPADS and A2P5P (2, 18, 19). Additionally, the highly selective P2Y₁ antagonist MRS 2179 failed to inhibit indo-resistant cAMP accumulation in MDCK-D₁ cells even though in parallel control studies MRS 2179 inhibited canine P2Y₁-mediated inositol trisphosphate formation in P2Y₁-transfected 1231N1 cells (Ref. 20 and data not shown). These findings suggested that the receptor responsible for the indo-insensitive effect is not the P2Y₁ receptor but instead is another receptor, perhaps the P2Y₁₁ receptor, which in MDCK-D₁ cells also responds to adenine bisphosphates (14, 21).

View larger version (13K): [in this window] [in a new window]   Fig. 2.   . The P2Y antagonist suramin, but not the P2Y1 antagonists PPADS, A2P5P, and MRS 2179, inhibits indo-insensitive cAMP generation in ADPS (10 µM)-stimulated MDCK-D1 cells. Cells were incubated with indomethacin (1 µM, 30 min) and antagonist (PPADS, 10 min; suramin, 5 min; A2P5P, 5 min; MRS 2179, 5 min) prior to incubation with IBMX (200 µM) and ADPS for 7 min. Data are normalized to cAMP levels obtained with ADPS (10 µM) without antagonist and are mean ± S.E. of triplicate determinations representative of values obtained in at least two separate experiments.

Investigation into whether the P2Y₁₁ receptor can cause indo-resistant cAMP elevation in MDCK-D₁ cells is hampered by the lack of antagonists to this receptor. Therefore, as an alternative strategy, we used P2Y₁₁ receptors that we had cloned from MDCK-D₁ cells expressed as P2Y₁₁-GFP receptors in MDCK-D₁ cells (14) and assessed cAMP accumulation. In the cells overexpressing P2Y₁₁-GFP receptors, we found a 5- to 10-fold increase in sensitivity of indo-resistant cAMP formation in response to several nucleotides, compared with responses observed with native MDCK-D₁ cells (Table I and Fig. 3). The relative potency of the different nucleotides in P2Y₁₁-GFP-overexpressing MDCK-D₁ cells was similar to that for native MDCK-D₁ cells, although compared with other agonists, ADP had a somewhat greater enhancement in apparent potency in the P2Y₁₁-overexpressing cells (Table I). UTP treatment gave no response; thus overexpression of P2Y₁₁ receptors did not alter P2Y₂ response (data not shown). Taken together with data in the native cells (Figs. 1 and 2), this enhancement of the indo-resistant cAMP formation in P2Y₁₁-overexpressing MDCK-D₁ cells is consistent with the notion that P2Y₁₁, and not P2Y₁, receptors mediate this response.

View larger version (16K): [in this window] [in a new window]   Fig. 3.   Overexpression of the P2Y11 receptor in native MDCK cells enhances indo-resistant cAMP accumulation. Native MDCK cells (top panel) or MDCK cells overexpressing P2Y11-GFP receptors (bottom panel) were treated as described under "Experimental Procedures." Cells were incubated for 30 min with indo (1 µM) and then for 7 min with IBMX (200 µM) and the indicated agonist. Data are plotted as a percentage of maximal stimulation mean ± S.E. of triplicate determinations and are representative values of those obtained in at least two separate experiments. 100% cAMP values (cell type, agonist, fmol of cAMP/µg protein ± S.E.): MDCK, ATP, 47.7 ± 0.77; MDCK, MT-ATP, 174 ± 1.14; MDCK, ADPS, 359 ± 12.2; MDCK Y11, ATP, 518 ± 20.6; MDCK Y11, MT-ATP, 504 ± 118; and MDCK Y11, ADPS, 678 ± 25.1.

To define the molecular mechanism underlying the indo-resistant cAMP elevation in MDCK-D₁ cells, we assessed whether this response results from the coupling of a P2Y receptor, presumably the P2Y₁₁ receptor, to an increase in adenylyl cyclase activity. In previous studies it has been difficult to ascertain whether P2Y₁₁ receptors increase cAMP via a receptor/G_s-mediated activation of adenylyl cyclase activity or indirectly via alterations in calcium or other regulators of cAMP formation (14, 22-24). We used two approaches to assess this possibility. The diterpene forskolin enhances coupling of G_s-linked receptors to adenylyl cyclase, thereby enhancing the ability of G_s receptors to raise cellular cAMP levels (12, 25-27). Co-incubation of indo-pretreated MDCK-D₁ cells with 10 µM ADPS and 0.1 µM forskolin led to a cAMP elevation that was greater than the sum of the individual responses for forskolin and ADPS. Co-incubation of forskolin and PGE₂, a known G_s activator in MDCK-D₁ cells (12), showed an even greater enhancement of cAMP levels than was caused by co-incubation with ADPS and forskolin (Fig. 4). Nevertheless, the synergistic effect obtained with forskolin and agonists, when expressed as -fold increase over the sum of separate forskolin- and agonist-stimulated cAMP accumulations, was similar or even greater for ADPS-stimulated cAMP generation than for PGE₂ response (Fig. 4, inset). These results suggest that ADPS, like PGE₂, activates G_s to promote cAMP formation in MDCK-D₁ cells.

View larger version (14K): [in this window] [in a new window]   Fig. 4.   Co-incubation of ADPS or PGE2 with forskolin in the presence of indo yields synergistic cAMP generation in MDCK-D1 cells. Cells were incubated with 1 µM indo (30 min) and then IBMX (200 µM) with 10 µM ADPS or PGE2 ± 0.1 µM forskolin (7 min). The first bar in each group represents the sum of separate agonist and forskolin responses. The second bar represents treatment of cells with agonist and forskolin together. The Inset shows -fold increase between the first and second bars of each group. Data, represented as -fold cAMP production relative to basal levels, are mean ± S.E. of triplicate determinations averaged from two separate experiments. Basal value: 8.41 ± 3.31 fmol of cAMP/µg of protein.

As a more direct test for the ability of a P2Y receptor to couple to an increase in adenylyl cyclase activity, we sought to assay enzyme activity, but we reasoned that such an assay would require use of a nucleotide as a substrate, which unlike ATP was not active at the P2Y receptors that increase cAMP levels in MDCK-D₁ cells. We chose as an alternate substrate AMP-PNP, which has previously been used as a substrate for adenylyl cyclase assays (28) and which we found in preliminary studies did not promote indo-resistant cAMP accumulation (following 30 min of treatment of cells with 30 µM AMP-PNP, data not shown). We also found that AMP-PNP yielded linear time- and protein-dependent cAMP generation in MDCK-D₁ membranes, whereas MT-ADP and ADPS, nucleotides that promoted cAMP accumulation in intact cells, did not (data not shown).

MT-ADP, ADPS, and PGE₂ all elevated cAMP levels in the presence of AMP-PNP, whereas UTP had no effect (Fig. 5). Thus, while MT-ADP and ADPS lack the ability to serve as substrates for adenylyl cyclase, in combination with AMP-PNP they, like PGE₂, are agonists that can increase adenylyl cyclase activity, consistent with activation of P2Y₁₁ receptors linked to G_s. In contrast, UTP, which stimulates G_q-coupled P2Y₂ receptors (2, 3, 13), did not lead to increased adenylyl cyclase activity. Taken together with other data shown here, these findings provide direct evidence for P2Y₁₁ receptor-mediated activation of adenylyl cyclase activity.

View larger version (12K): [in this window] [in a new window]   Fig. 5.   MT-ADP, ADBS, and PGE2 elevate adenylyl cyclase activity in MDCK membranes. MDCK membranes, prepared as described under "Experimental Procedures," were incubated with the substrate AMP-PNP (30 µM), IBMX (200 µM) for 30 min, and the indicated agonist. Data represent mean ± S.E. of triplicate determinations averaged from three experiments of -fold cAMP elevation over membrane treatment with AMP-PNP alone (basal). Basal value: 1.97 ± 0.34 fmol of cAMP/µg of protein.

	DISCUSSION

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

P2Y receptors are increasingly recognized as providing an important means by which extracellular nucleotides regulate a wide variety of cell types (2-4). Of the seven unique P2Y receptor subtypes (P2Y₁, P2Y₂, P2Y₄, P2Y₆, P2Y₁₁, P2Y₁₂, and P2Y₁₃) all, with the exception of P2Y₁₂ and P2Y₁₃ subtypes, couple via G_q/11-dependent mechanisms to the activation of phospholipase C and increases in cellular [Ca²⁺], but the various receptor subtypes have quite different abilities to regulate formation of cAMP. Certain of the receptors (e.g. P2Y₁₂, P2Y₁₃) are known to utilize G_i-dependent mechanisms to inhibit cAMP formation (29, 30), whereas other P2Y receptors may regulate adenylyl cyclase activity via effects on regulators of adenylyl cyclase, such as [Ca²⁺] or protein kinase C.

MDCK-D₁ cells, in common with several other cell types, express several different P2Y receptor subtypes (1, 6, 12, 31, 32). Early work on MDCK-D₁ cells, prior to the molecular cloning and precise identification of the different P2Y receptor subtypes, emphasized the ability of added nucleotides to alter membrane potential, ion flux, or short circuit current (7, 8). Recent studies have indicated that MDCK-D₁ cells release ATP and that this released nucleotide helps contribute to basal activity of signal transduction pathways (15, 33). Of the three different P2Y receptors that we have thus far detected on MDCK-D₁ cells (P2Y₁, P2Y₂, and P2Y₁₁, see Ref. 1), our previous work documented that P2Y₂ receptors mediate the indo-sensitive (i.e. COX-dependent) cAMP accumulation via the ability of the receptors to increase formation of PGE₂ and the subsequent activation of EP receptors linked to G_s and adenylyl cyclase activity (1, 12, 13).

Several lines of evidence from the current studies, designed to identify alternative mechanisms by which ATP might increase cellular cAMP levels, are consistent with the conclusion that MDCK-D₁ P2Y₁₁ receptors are responsible for the COX-independent (indo-resistant) increases in cAMP formation promoted by ATP and other nucleotides: 1) the rank order of potency of agonists in promoting this response; 2) the insensitivity to blockade by several P2Y₁-selective antagonists; 3) the increased potency of several agonists in P2Y₁₁-overexpressing MDCK-D₁ cells; 4) the synergistic enhancement in response by co-incubation of cells with nucleotides plus forskolin; and 5) the ability of two P2Y₁₁ agonists, MT-ADP and ADPS, to enhance adenylyl cyclase activity in MDCK-D₁ cell membranes. Taken together, we believe these results provide strong evidence for P2Y₁₁ receptor-promoted stimulation of adenylyl cyclase activity, presumably secondary to enhanced enzyme activity by receptor-mediated activation of G_s.

This is the first data of which we are aware to document that P2Y₁₁ receptors directly activate adenylyl cyclase activity. At the time of the initial cloning of P2Y₁₁ receptors, Communi et al. (22, 23) utilized two different cell types to heterologously express the cloned P2Y11 receptors and to conclude that these receptors couple to both G_s and G_q. However, this conclusion was open to alternative interpretations, given the use of different cell types to assess receptor coupling to the different pathways. Data by those workers and others who have reported on the ability of P2Y₁₁ receptors to stimulate cAMP formation (e.g. 24, 34) have involved studies with intact cells, for which the possibility of system-dependent influences has been noted (35, 36). A number of factors can influence cAMP formation, degradation, or export from cells. Some of those factors are likely to be altered by activation of the G_q-linked pathways, such as [Ca²⁺], activity of protein kinase C and other downstream kinases (e.g. MAP kinase), and phosphodiesterase activity, etc. Our results that show activation of adenylyl cyclase activity in MDCK-D₁ cell membranes provide definitive evidence for the notion that P2Y₁₁ receptors stimulate adenylyl cyclase activity, presumably by direct activation of G_s. The method that we used to draw this conclusion, involving use of AMP-PNP as the substrate for adenylyl cyclase assays, may prove useful to others interested in assessing effects of P2Y₁₁ receptors on adenylyl cyclase activity.

Overall, our results provide evidence that ATP and ADP, two physiologic nucleotides that can exist in the extracellular space, are able to raise cAMP levels in native cells via activation of P2Y₁₁ receptors. As such, these results provide a mechanism, in addition to activation of P2Y₂ or adenosine receptors, by which exogenous or endogenously released nucleotides can increase cellular levels of this important cyclic nucleotide. Given the evidence that MDCK-D₁ and a number of types of cells both release ATP and possess P2Y₁₁ receptors, nucleotide-mediated activation of P2Y₁₁ receptors provides a means for autocrine-paracrine regulation of epithelial and other cell types.

	ACKNOWLEDGEMENTS

We thank Ken Jacobson (National Institutes of Health) for providing MRS 2179, Richard Hughes for providing data on inositol trisphosphate formation in MRS 2179-treated 1321N1 cells, Larry Brunton, who co-mentored Alexander C. Zambon during his doctoral dissertation, Steven Post, who participated in early phases of work in this study, and Steve Burch for assistance in preparation and submission of this manuscript.

	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.

To whom correspondence should be addressed: Dept. of Pharmacology, University of California, San Diego, 9500 Gilman Dr., La Jolla, CA 92093-0636. Tel.: 858-534-2295; Fax: 858-822-1007; E-mail: inseloffice@ucsd.edu.

Published, JBC Papers in Press, January 11, 2002, DOI 10.1074/jbc.M110352200

	ABBREVIATIONS

The abbreviations used are: MDCK, Madin-Darby canine kidney cells; COX, cyclooxygenase; indo, indomethacin; ATPS, adenosine 5'-O-(thio)triphosphate; MT-ATP, 2-methylthio-ATP; MT-ADP, 2-methylthio-ADP; PPADS, pyridoxal-phosphate-6-azophenyl-2',4'disulfonic acid 4 sodium; AMP-PNP, adenosine 5'-(,-imino)triphosphate; IBMX, isobutylmethylxanthine; GFP, green fluorescent protein; PGE₂, prostaglandin E₂; A2P5P, adenosine 2',5'-diphosphate; ADPS, adenosine 5'-O-2-(thio)diphosphate; ATPS, adenosine 5'-O-3-(thio)triphosphate; MAP, mitogen-activated protein.

	REFERENCES

TOP ABSTRACT INTRODUCTION EXPERIMENTAL PROCEDURES RESULTS DISCUSSION REFERENCES

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