P2Y2 receptor of MDCK cells: cloning, expression, and cell-specific signaling

Alexander C. Zambon, Richard J. Hughes, J. Gary Meszaros, J. Julie Wu, Brian Torres, Laurence L. Brunton, and Paul A. Insel

Departments of Pharmacology and Medicine, Biomedical Sciences Graduate Program, University of California at San Diego, La Jolla, California 92093-0636


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Madin-Darby canine kidney (MDCK)-D1 cells, a canine renal epithelial cell line, co-express at least three different P2Y receptor subtypes: P2Y1, P2Y2, and P2Y11 (24). Stimulation of P2Y receptors in these cells results in the release of arachidonic acid (AA) and metabolites and the elevation of intracellular cAMP. To define in more precise terms the signaling contributed by the MDCK-D1 P2Y2 (cP2Y2) receptor, we have cloned and heterologously expressed it in CF2Th (canine thymocyte) cells, a P2Y2-null cell. Analysis by RT-PCR indicated that canine P2Y2 receptors are expressed in skeletal muscle, spleen, kidney, lung, and liver. When expressed in CF2Th cells, cP2Y2 receptors promoted phospholipase C-mediated phosphatidylinositol (PI) hydrolysis [uridine 5'-triphosphate >=  ATP > adenosine 5'-diphosphate > 2MT-ATP] and mobilization of intracellular Ca2+. In contrast to their actions in MDCK-D1 cells, cP2Y2 receptors did not stimulate formation of cAMP or AA release when expressed in CF2Th cells. The data indicate that cell setting plays an essential role in the ability of P2Y receptors to regulate AA release and cAMP formation. In particular, renal epithelial cells preferentially express components critical for cP2Y2-induced cAMP formation, including the expression of enzymes involved in the generation and metabolism of AA and receptors that respond to PGE2.

Madin-Darby canine kidney; epithelial cells; canine P2Y2 receptors; adenosine 3',5'-cyclic monophosphate; CF2Th thymocytes


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ATP AND OTHER NUCLEOTIDES act as potent signaling molecules via the activation of cell surface P2 receptors (10, 32). Two classes of P2 receptors have been described: guanine nucleotide binding protein (G protein)-coupled P2 receptors (P2Y) and intrinsic ion channels (P2X). Some tissues and cells express multiple P2Y receptor subtypes (5, 8, 24, 36, 40). The precise functional implications of this remain unclear. Expression of multiple P2Y receptor subtypes and the lack of subtype-specific ligands (both agonists and antagonists) have made it difficult to precisely characterize signaling mediated by a particular P2Y subtype.

Data from our laboratory indicate that the canine renal epithelial cell line, MDCK-D1, coexpresses multiple P2Y receptor subtypes: P2Y1, P2Y2, and P2Y11 (8, 24). Stimulation of MDCK-D1 cells with UTP, a quasi-selective P2Y2 agonist, promotes phospholipase C (PLC)-mediated hydrolysis of phosphoinositides, activation of phospholipase D (PLD) and cytoplasmic phospholipase A2 (PLA2), release of arachidonic acid (AA) and metabolites, and an increase in cAMP levels (1, 8, 23, 27, 40). The capacity of P2Y2 receptors to activate PLC, resulting in the formation of inositol phosphate (IP) and the mobilization of Ca2+, is widely recognized (10, 15, 19). However, the ability of P2Y2 receptors to stimulate adenylyl cyclase (AC), a Gs-coupled effector, is much more unusual, perhaps reflecting distinctive characteristics in the G protein-coupling ability of MDCK-D1 P2Y2 receptors.

We report here the cloning and tissue expression of MDCK-D1 P2Y2 (cP2Y2) receptors. When heterologously expressed in canine thymocyte (CF2Th) cells, cP2Y2 receptors are capable of stimulating phosphoinositide hydrolysis and the mobilization of intracellular Ca2+ ([Ca2+]i). However, in contrast to native cP2Y2 receptors in MDCK-D1 cells, the heterologously expressed cP2Y2 receptor is capable of stimulating neither release of AA nor cAMP formation. The data thus indicate the key role of cell-specific post-receptor components in contributing to signaling properties of native vs. heterologously expressed cloned receptors and, in particular, of components distal to cP2Y2 receptors for AA release and cAMP formation by renal epithelial cells.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Materials. [3H]AA (10 Ci/mmol) and [3H]myo-D-inositol (80 Ci/ mmol) were purchased from DuPont-NEN, [5'-(3-O-thio) triphosphate (ATPgamma S)], and ADPbeta S were from Calbiochem; 2-methyl-thio ATP (2MT-ATP) and 2 MT-ADP were purchased from Research Biochemicals and all other nucleotides and bases were from Sigma-Aldrich, as was AA. Agarose was from FMC Bioproducts, Centricon Concentrator from Amicon, Ecoscint from National Diagnostics, TRIzol from GIBCO-BRL, indo 1-AM from Calbiochem, Moloney-murine leukemia virus (M-MLV) RT from GIBCO-BRL, random hexamers from Pharmacia Biotech, Amplitaq Gold Polymerase from Perkin Elmer, phenol-chloroform-isoamyl alcohol from Ambion, lambda ZAP cloning vector from Stratagene, chromogenic solution [enhanced chemiluminescence (ECL) reagent] from Amersham Pharmacia Biotech, autoradiography film from Kodak, and polyclonal anti-cPLA2 antibody and anti-rabbit antibody conjugated to alkaline phosphatase from Santa Cruz Biotechnology. The murine P2Y2 receptor cDNA was a gift of Dr. David Julius and colleagues. GraphPad Prism was a gift from Dr. Harvey Motulsky.

Cell culture. MDCK-D1 (a subclone of MDCK cells) and CF2Th cells were grown in DMEM supplemented with 10% heat-inactivated FCS as previously described (24). Two to three days before experimentation, cells were detached with a trypsin-EDTA solution and plated into 6-well or 12-well plates. All cells were ~75-90% confluent at the time of assays.

RNA and DNA isolation. Total RNA was isolated from MDCK-D1 and CF2Th cells grown in 175-cm2 flasks by using TRIzol. MDCK-D1 DNA was isolated from cells grown in 150-mm culture dishes. Cells were scraped into ice-cold PBS, collected by centrifugation (5 min at 500 g), and resuspended in 5 vol of digestion buffer (100 mM NaCl2, 10 mM Tris · HCl, 25 mM EDTA, 0.5% SDS, 1 mg/ml proteinase K, pH 8.0). After overnight incubation at 37°C, samples were extracted three times with phenol-chloroform-isoamyl alcohol and precipitated with ethanol (2 vol) and 3 M ammonium acetate (1/10 vol). Canine tissues were a gift from the laboratory of Dr. James Covell (UCSD). Tissues were excised immediately postmortem, immersed in liquid nitrogen, and stored at -80°C. Total RNA was isolated from pulverized tissues with TRIzol according to the manufacturer's instructions.

Canine P2Y2 receptor cloning. An MDCK-D1 cDNA library was prepared in the lambda ZAP cloning vector according to the manufacturer's instructions and screened under low-stringency conditions with a random-primed, full-length murine P2Y2 cDNA. A single clone was isolated from the library, digested with EcoR I, ligated into pBluescript, and sequenced. Nucleotide sequence alignment indicated the initially isolated clone was missing 21 nucleotides on the 5' end of the coding sequence. The missing fragment was isolated using a PCR strategy. An 83-bp P2Y2 fragment was amplified from genomic MDCK-D1 DNA by using a degenerate 5' primer, 5'-GGTC (A/C)(G/A)GGC(G/A)ATG-3', ending with the start site and a 3' primer, 5'-AAGCTT GAAGTCCTCATTGAAAC-3', containg a Hind III restriction site. The PCR product and library clone were digested with Nco I and Hind III and ligated together. The primary sequence of the cP2Y2 receptor is overall strikingly similar to previously cloned P2Y2 receptors (Fig. 1). The sequences diverge most in the intracellular COOH-terminal region where identity to the human, rat, and mouse sequences falls to ~70%.


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Fig. 1.   Sequence alignment of Madin-Darby canine kidney (MDCK)-D1, human, rat, and murine P2Y2 receptors. Shown here is a modified ClustalW alignment (31) of the inferred amino acid sequences of MDCK-D1, human, rat, and murine P2Y2 receptor clones. Putative transmembrane-spanning domains are underlined, potential G protein-coupled receptor kinase phosphorylation sites are denoted with a double asterisk (**), and potential protein kinase A/protein kinase C sites are denoted with an exclamation mark (!).

RT-PCR. Ten micrograms of total RNA were reverse transcribed in 25 µl with 5 µl 5× RT buffer (250 mM Tris · HCl, (pH) 8.3, 375 mM KCl, and 15 mM MgCl2) together with 0.002 optical density (OD) units of random hexamers, 10 mM dithiothreitol, 800 µM dNTP, and 200 U M-MLV RT. After a 1-h incubation at 37°C, each reaction was stopped by boiling for 4 min and then diluted to 100 µl in RNase-free water. Control reactions omitted RT enzyme (data not shown). One microgram of reverse transcribed RNA or 10 ng genomic DNA were added to a solution of 20 µM (each) forward and reverse P2Y2-specific primer (forward: 5'-AGT CCC CCG TGC TCT ACT TT-3'; reverse: 5'-GTC AGT CCT GTC CCA CCT GT-3'), 2.5 mM MgCl2 buffer, 1× PCR buffer (50 mM KCl, 10 mM Tris · HCl pH 8.3), 0.2 mM dNTP, 5 U of Amplitaq Gold polymerase, and H2O in a total volume of 50 µl. Temperature cycling proceeded as follows: once at 95°C for 10 min to activate the enzyme, 95°C for 30 s, 60°C for 90 s, and 72°C for 90 s, for 40 cycles, followed by 72°C for 10 min. PCR products were then subjected to gel electrophoresis on a 1% agarose gel, followed by extraction of each band by using a Qiaquick gel extraction kit. The DNA was resuspended in Tris-EDTA buffer (10 mM Tris-Cl, 1 mM EDTA, pH 8.0), and 1 vol of the gel-extracted PCR product was purified by using a Centricon concentrator. Purified fragments were sequenced on an ABI automated DNA sequencer, model 377, by using the same forward primers used to generate the PCR fragments.

Retroviral expression. The MDCK-D1 P2Y2 coding sequence was digested from pBluescript by using Kpn I and BamH I and ligated into a BamH I-linearized retroviral vector pLRNL. Replication-deficient vesicular stomatitis virus G (VSV-G) pseudotyped retrovirus (rdVSV-G rv) containing the MDCK-D1 P2Y2 clone was generated at the UCSD human gene therapy vector development laboratory under the supervision of Dr. Atsushi Miyanohara, as previously described (2, 39). The use of rdVSV-G rv facilitates a wider range of host cell infectability by allowing the retroviral particles to attach to components of the host cell membrane, such as phosphatidylserine, as opposed to the standard retrovirus-derived envelope proteins that must interact with protein receptors on the cell surface (38). Control cells were infected with pseudotyped retrovirus containing the LacZ reporter gene.

beta -Galactosidase activity. LacZ-expressing control cells were plated to ~50% confluency and washed twice with 2 ml PBS. Cells were then fixed in 2 ml of glutaraldehyde/PBS, 1:100, and incubated for 15 min. Fixative was removed, and cells were washed three times with PBS and incubated in 1 ml of 5-bromo-4-chloro-3-indoyl-beta -D-galactoside (X-Gal) solution [2 mM MgCl2, 5 mM K4Fe(CN)6 3H2O, 5 mM K3Fe(CN)6, 0.2% X-Gal in dimethylformamide] for 37°C for 2 h.

[Ca2+]i measurements. CF2Th cells infected with retrovirus containing either MDCK-D1 P2Y2 receptor or LacZ gene were grown overnight on 22-mm glass coverslips as described above. Cells were washed twice in HEPES-buffered saline [HBS containing (in mM) 130 NaCl, 5 KCl, 10 glucose, 1 CaCl2, 1 MgCl2, and 25 Na+-HEPES, pH 7.4] and incubated in 2 ml HBS containing 1 µM indo-1-AM at 37°C for 30 min. Cells were viewed by using an inverted Nikon Diaphot microscope. Fluorometric measurements in fields of 6-10 cells were collected by using the DX-100 system (Solamere Technology, Salt Lake City, UT), where the field was excited at 385 nm and the emission ratio was collected at 405 and 495 nm, and analyzed and plotted with Maclab software. Nanomolar Ca2+ values were calculated by using the following formula:
[Ca<SUP>2+</SUP>]<SUB>i</SUB><IT>=</IT><IT>K</IT><SUB>d</SUB>(R<IT>−</IT>R<SUB>min</SUB>)/(R<SUB>max</SUB><IT>−</IT>R) (<IT>&bgr;</IT>)
where R was the ratio at any time, and Rmin, Rmax, and beta  were determined by the ratio of fluorescence under Ca2+-depleted (+EGTA) and Ca2+-saturated (+ionomycin) conditions. The Ca2+ dissociation constant, Kd, was determined to be 250 nM for this dye and these optics at 37°C. Agonists were administered from 1,000× stocks to maintain a constant volume of 2 ml.

Phosphoinositide hydrolysis assay. CF2Th cells were grown in six-well plates to ~80% confluency in 2 ml of DMEM/well containing 10% FCS. Cells were loaded in myo-D-[2-3H]inositol (2.5 µCi/ml) for 12 h. Thirty minutes before agonist stimulation, 10 mM LiCl was added. Cells were then stimulated for 10 min with various agonists, and reactions were stopped by aspiration of medium and addition of ice-cold methanol-HCl (50% MeOH:1 N HCl). Total inositol phosphates were separated from [3H]inositol by chromatography over Dowex-1-formate. [3H]IP content was assessed by liquid scintillation spectrometry.

AA and metabolite release. MDCK-D1 cells or cP2Y2-expressing CF2Th cells were incubated overnight in 12-well plates with 0.33 µCi/ml of [3H]AA. The following day the medium was changed, and cells were equilibrated for 1.5 h. Cells were then stimulated with various concentrations of UTP for 30 min. Medium was then removed and counted in 10 ml of scintillation fluid.

Western blot analysis of cPLA2. MDCK and CF2Th cells were grown to confluence then collected, after rinsing with and being scraped into ice-cold PBS, by centrifugation (600 g for 5 min at 4°C) and resuspended in lysis buffer [10 mM Tris · HCl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% (wt/vol) Triton X-100, 1% (wt/vol) sodium deoxycholate, 1 mM phenylmethylsulfonyl fluoride]. Protein concentration was measured by the method of Peterson (21). One hundred micrograms of whole-cell lysate from each cell type were separated by SDS-PAGE on a 10% polyacrylamide gel. Proteins were transferred to Immobilon-P membrane by electroblotting. The membrane was blocked with a solution of nonfat milk (2 h at room temperature) then rinsed three times with PBS-T (PBS with 0.1% Tween 20) and incubated for 2 h at room temperature with anti-cPLA2 antibody (polyclonal diluted 1:1,000). The membrane was rinsed five times with PBS-T and incubated for a further 2 h with goat anti-rabbit antibody conjugated to alkaline phosphatase. After further rinsing five times with PBS-T, the membranes were incubated with chromogenic ECL solution, and XAR-5 autoradiography film was exposed by contact with the membrane for 1 min.

Measurement of cAMP accumulation. Cells were grown in 24-well plates to 70-85% confluency. Before treatment of cells, growth medium was removed and cells were equilibrated for 30 min at 37°C in serum-free DMEM containing 20-mM HEPES buffer (DMEH; pH 7.4). Subsequently, cells were incubated in fresh HEPES-buffered DMEM and various agents. Unless otherwise indicated, incubations with the agonists were conducted for 10 min at 37°C in the presence of 200 µM IBMX, a cyclic nucleotide phosphodiesterase inhibitor, and terminated by placing on ice, followed by aspiration of medium and the addition of 7.5% TCA. Intracellular cAMP levels were determined by radioimmunoassay of TCA extracts following acetylation, according to the manufacturer's protocol (Calbiochem). Production of cAMP was normalized to the amount of acid-insoluble protein assayed by Bio-Rad protein assay.

Data analysis. Phosphoinositide hydrolysis assays were conducted in six-well plates, and concentration points were run in duplicate. Basal counts were subtracted and all points were divided by maximal signal obtained by stimulation of P2Y2-expressing cells with 10 mM UTP, a value of ~4,000 counts/min. Curves were determined by nonlinear regression using GraphPad Prism software fit to a sigmoidal dose-response formula. Fits are represented as means ± range of duplicate determinations from a representative experiment with similar results obtained in at least three separate experiments. cAMP levels and AA release were also analyzed with GraphPad Prism software. Data shown are the means ± SE of triplicate samples from a representative experiment. Each experiment was replicated at least three times with similar results.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

MDCK-D1 P2Y2 receptor cloning. We obtained a clone of the cP2Y2 receptor that comprised all but the first 21 nucleotides of the coding sequence by screening an MDCK-D1 cDNA library with a murine P2Y2 receptor probe. The missing 5' coding sequence was obtained by PCR by using a degenerate 5' primer anchored at its 3' end with the ATG start codon and a downstream 3' primer that flanked a unique Acc I restriction site within the coding sequence. The cDNA clone was digested with Acc I and EcoR I, the PCR fragment with Kpn I and Acc I, and pBluescript SK (+) with Kpn I and EcoR I. These fragments were ligated together, yielding the full-length clone. The deduced amino acid sequence of the MDCK-D1 receptor was greater than 90% identical to the human (19), rat (3), and mouse (15) sequences between the NH2 terminal and the end of the seventh putative transmembrane-spanning domain. After the seventh putative transmembrane-spanning domain, however, the sequence homology drops off considerably. Thus the amino acid identity among MDCK-D1, human, mouse, and rat clones falls to ~70% in the last 65 amino acids, and the homology falls to 80% (Fig. 1). Possible differences in sites for phosphorylation are outlined in the figure legend. The two rodent clones are, as would be expected, the most similar to one another.

RT-PCR. To define the pattern of tissue expression of cloned cP2Y2 receptors, RT-PCR was used to assess the tissue distribution of cP2Y2 transcripts. Total RNA was isolated by using TRIzol reagent from canine tissues stored at -80°C that were excised immediately postmortem. Primers were designed to amplify a 525-bp fragment (494-1019) of the cP2Y2 receptor. RT-PCR yielded a single 525-bp band from multiple tissues, including liver, lung, skeletal muscle, spleen, and kidney (Fig. 2). Products were confirmed by sequencing.


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Fig. 2.   Localization of P2Y2 transcripts by RT-PCR. Total RNA was isolated from canine tissues stored at -80°C that were excised immediately postmortem. One microgram of total RNA was reverse transcribed and used as a template for PCR with primers amplifying a 525-bp fragment of the MDCK-D1 P2Y2 receptor corresponding to bases 494-1019 of the coding region. Control RT reactions, carried out in the absence of RT to confirm the absence of DNA contamination, resulted in no PCR products (data not shown). All fragments were confirmed by sequencing.

Retroviral expression of MDCK-D1 P2Y2 receptor in CF2Th cells. The cP2Y2-receptor coding sequence was cloned into a retroviral vector pLRNL, which was then used to generate replication-deficient rdVSV-G rv to facilitate expression in eukaryotic cells. CF2Th cells, a canine thymocyte P2Y2-null cell line, were infected with this rdVSV-G rv containing either a LacZ-reporter gene or the MDCK-D1 P2Y2 receptor construct. We chose to use CF2Th cells because they are a canine cell line, they lack P2Y2 receptor mRNA, and in preliminary studies they showed no response [Ca2+ mobilization, inositol phosphate (IP) formation] to stimulation with P2Y agonists. LacZ-infected CF2Th cells showed a multiplicity of infection >98% when assayed for beta -galactosidase activity (data not shown), demonstrating that CF2Th cells contain the necessary membrane components for rdVSV-G rv attachment and infection.

IP production. Infection with the retroviral MDCK-D1 P2Y2 receptor conferred on CF2Th cells the ability to respond to a variety of P2Y agonists with an increase in IP production. The rank order of potency of these agonists, based on EC50 values, was UTP >=  ATP > ATPgamma S > ADP > UDP > 2MT-ATP (Fig. 3). This profile is consistent with that of previously cloned P2Y2 receptors (15, 19). CF2Th cells were also challenged with angiotensin, endothelin, histamine, carbachol, vasopressin, phenylephrine, lysophosphatidic acid, and thrombin; none of these drugs was able to stimulate IP formation (data not shown). Pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium, a P2X and P2Y1 antagonist (26), had no effect on ATP-stimulated IP formation by the canine P2Y2 receptor.


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Fig. 3.   P2Y2-induced inositol phosphates formation. CF2Th cells expressing MDCK-D1 P2Y2 receptors were labeled with [3H]inositol and incubated in the presence of varied concentrations of agonists for 10 min as indicated (See MATERIALS AND METHODS). The data are means ± SE of duplicate determinations from a representative experiment that was replicated at least 3 times. PPAD, pyridoxal-phosphate-6-azophenyl-2',4'-disulphonic acid 4-sodium; ATPgamma S, adenosine 5'-O-(3-thiotriphosphate); 2MT-ATP, 2-methyl-thio ATP.

[Ca2+]i measurements. CF2Th cells (expressing cP2Y2 receptor or LacZ gene) and MDCK-D1 cells were loaded with 1 µM indo 1-AM. After stimulation with the indicated concentrations of UTP, cytoplasmic free Ca2+ was measured by single-excitation spectrofluorometric analysis. LacZ-expressing CF2Th cells failed to show an increase in [Ca2+]i levels when stimulated by UTP (Fig.4C, inset). In contrast, the P2Y2-infected CF2Th cells showed a rapid concentration-dependent elevation of cytoplasmic free Ca2+ ([Ca2+]i), in part in an oscillatory manner, in response to stimulation with various concentrations of UTP. UTP also stimulated an increase in cytosolic Ca2+ in MDCK-D1 cells, although these cells were less responsive to low doses of UTP compared with CF2Th-expressing cells (Fig. 4, A vs. D). The concentration-response range for UTP stimulation of Ca2+ mobilization was similar to that observed for phosphoinositide hydrolysis (Fig. 3). [Ca2+]i levels in CF2Th cells were calculated to be (in nM): 80 (basal), 680 (0.1 µM UTP), 1,020 (1 µM UTP), and 1,130 (30 µM UTP). MDCK-D1 [Ca2+]i levels were comparable with values of (in nM): 80 (basal), 210 (0.1 µM UTP), 1,066 (1 µM UTP), and 1,170 (30 µM UTP). These values should be considered to be estimates because it is difficult to obtain precise calibration (Rmin and Rmax), a problem that is inherent in any cell-attached fluorimeter system.


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Fig. 4.   Intracellular Ca2+ release. P2Y2 expressing CF2Th cells (A-C) and MDCK-D1 cells (D-F) were assayed for elevations in intracellular Ca2+ with indo 1 as described in MATERIALS AND METHODS. Figure 4C (inset) shows the lack of response of control (no P2Y2 receptor) CF2Th cells to UTP (30 µM); effect of thapsigargin (TG; 5 µM) confirms storage of releasable Ca2+ in these cells. Estimated peak cytoplasmic free Ca2+ ([Ca2+]i) values are given in RESULTS. Intracellular free Ca2+ (ordinate) is represented as the ratio of emissions at 405 nm and 495 nm. Abscissa values are in min:s.

AA and metabolite release. cP2Y2-expressing CF2Th cells and MDCK-D1 cells were loaded overnight with [3H]AA and then assayed for their ability to release AA in response to stimulation with UTP. Unlike results in MDCK-D1 cells, cP2Y2 receptors expressed in CF2Th cells were unable to stimulate release of AA in response to stimulation with UTP (Fig. 5A).


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Fig. 5.   Arachidonic acid (AA) and metabolite release and phospholipase A2 (cPLA2) expression. A: MDCK-D1 cells and P2Y2-expressing CF2Th cells were loaded with [3H]arachidonate overnight and then incubated with various concentrations of UTP for 10 min as described in MATERIALS AND METHODS. Three micromolar mellitin (Mel) was used as a positive control for PLA2 activation in CF2Th cells (inset). The data are means ± SE of triplicate samples from a representative experiment that was replicated at least 3 times with similar results. B: protein (125 µg each) from MDCK and CF2Th whole cell lysate was analyzed by Western blot using anti-cPLA2 primary antibody and goat anti-rabbit secondary antibody conjugated to alkaline phosphatase and visualized by chemiluminscence.

In MDCK-D1 cells, activation of cPLA2 is a key mechanism for AA release (34, 35). Western blot analysis indicates that CF2Th cells possess cPLA2 (Fig. 5B), thus implying that these cells appear to lack other components required for AA release. Additional studies indicated that CF2Th cells are capable of AA release as treatment of cells with 3 µM mellitin lead to a 10-fold increase in AA release (Fig. 5A, inset).

cAMP formation. MDCK-D1 cells and CF2Th cells expressing either cP2Y2 receptor or LacZ gene were stimulated with various agonists to assess the ability of these agonists to stimulate cAMP formation. As indicated in Fig.6, stimulation of CF2Th cells (both Lac Z and P2Y2 expressing) with UTP, AA, or PGE2 did not result in an increase in the production of cAMP. By contrast, all three agents stimulated cAMP accumulation in MDCK-D1 cells. cAMP production in response to stimulation of beta -adrenergic receptors with isoproterenol was significantly potentiated by expression of cP2Y2 receptors in CF2Th cells, and this response was increased to an even greater extent when cP2Y2 receptor-expressing cells were coincubated with isoproterenol and UTP. We used the diterpene forskolin to enhance coupling of Gs to AC (6, 22) and assessed whether this approach would uncover coupling of cP2Y2 receptors to an increase in cAMP generation in cP2Y2-expressing CF2Th cells. Although forskolin increased the isoproterenol-mediated increase in cAMP formation in MDCK-D1 cells and CF2Th cells (LacZ or P2Y2 expressing), treatment with forskolin failed to reveal coupling of cP2Y2 receptors to an increase in cAMP in CF2Th cells.


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Fig. 6.   cAMP formation in MDCK-D1 cells and CF2Th cells. cAMP accumulation was assessed in MDCK-D1 cells and CF2Th cells (either P2Y2-expressing cells or LacZ control cells) as described in MATERIALS AND METHODS. Top: cells were incubated with 100 µM UTP, 100 µM AA, or 1 µM PGE2. Bottom: agonists and concentrations used were 100 µM UTP, 10 µM isoproterenol, 1 µM forskolin, or combinations thereof. The data are means ± SE of triplicate samples from a representative experiment that was replicated at least 3 times with similar results.


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

We have cloned a 1.2-kb coding sequence of a cP2Y2 receptor from an MDCK-D1 cell cDNA library that encodes a 374-residue protein. Hydropathy analysis of the deduced translation product is consistent with the presence of seven transmembrane-spanning regions, a motif that is typical of the G protein-coupled family of receptors. The deduced amino acid sequence of the MDCK-D1 receptor was very similar overall to human, rat, and mouse P2Y2 receptors. This is as would be expected for interspecies variations for a receptor that exhibits identical or near-identical pharmacology. Rather surprisingly, however, we have found that, belying the overall similarity, there is substantially greater divergence between the COOH-terminal region of the receptor in these different species, and some of these differences encompass targets for phosphorylation by regulatory kinases (see Fig. 1). The COOH-terminal region of P2Y2 receptors has clearly been shown to be an important determinant of regulation of these receptors (9). Thus this raises the possibility that, despite sharing identical pharmacology, P2Y2 receptors might be differently regulated among the species (28). RT-PCR analysis of a variety of canine tissues indicates that the MDCK-P2Y2 receptor is expressed in skeletal muscle, spleen, lung, and kidney.

We used CF2Th cells to express cloned P2Y2 receptors. Previous studies of P2Y receptors have utilized either 1321N1 (4, 15), COS (18), Chinese hamster ovary (CHO) (4), or Jurkat (33) cells for expression studies, because these cells have been documented to either lack or have very low levels of native P2Y-receptor expression. The use of canine CF2Th has allowed us to study cloned MDCK-D1-P2Y2 receptors in a cell originating from the same species. Stable expression by retroviral infection of MDCK-P2Y2 receptors in CF2Th cells conferred the ability of infected cells to respond to extracellular nucleotides through stimulation of IP formation and the release of Ca2+ from intracellular stores. The rank order of potency for stimulation of IP accumulation in these cells was UTP >=  ATP > ADP > 2MT-ATP, a pharmacological profile consistent with that of P2Y2 receptors (3, 15, 19).

A novel feature of cP2Y2 receptors expressed in MDCK-D1 cells is that UTP stimulates cAMP production, a response that can be blocked by the cyclooxygenase inhibitor indomethacin, indicating that the release of AA and its conversion to prostaglandins, in particular PGE2, is essential for the coupling of cP2Y2 receptors to the activation of adenylyl cyclase (23, 24). Our data show that the ability of cP2Y2 receptors to stimulate adenylyl cyclase is a cell-specific event and not due to the ability of cP2Y2 receptors to couple directly to Gs. The data indicate that cloned cP2Y2 receptors fail to mobilize AA in CF2Th cells and that even in control CF2Th cells, in contrast with MDCK-D1 cells, neither AA nor PGE2 promote cAMP formation. Even though CF2Th cells possess cPLA2, the cloned P2Y2 receptors are unable to activate this enzyme, as evidenced by the lack of AA release. However, absence of AA release is not the sole mechanism for the inability to produce cAMP.

This appears not to be due to differences in levels of receptor expression. Due to the lack of a commercially available antibody, cP2Y2 protein levels cannot be assessed; however, based on RT-PCR analysis (Fig. 2), P2Y2-overexpressing CF2Th cells and MDCK-D1 cells express similar levels of P2Y2 transcripts. Thus differences at multiple loci may contribute to the cell-specific differences in UTP/P2Y2-mediated cAMP formation. Perhaps less likely, however not unprecedented (25), is the possibility that cP2Y2 receptors might selectively activate one or more isoforms of AC that are uniquely expressed in certain target cells. Data for other receptor systems e.g., (14, 16, 17, 20, 37) have hypothesized the importance of cell-specific components in signal transduction. Our data suggest that renal epithelial cells, such as MDCK cells, possess the ability to release AA and respond to AA metabolites (PGE2) that are lacking in other cell types, such as CF2Th cells. The resultant elevations in cAMP can then act in a negative-feedback loop by attenuating subsequent AA release (35).

An unexpected aspect of the present results is that the expression of cP2Y2 receptors in CF2Th cells leads to a potentiation of the stimulation of cAMP levels by endogenously expressed beta -adrenergic receptors and adenosine receptors (data not shown). The potentiation is most apparent when cells are simultaneously incubated with UTP and isoproterenol. A precise mechanism for this potentiation is not known, although higher levels of Gq-coupled cP2Y2 receptors could lead to elevated basal stimulation of Gq that may serve to prime the cells for subsequent stimulation by Gs-linked receptors. Co-stimulation of Gq- and Gs-coupled receptors can lead to a potentiation of AC activity via several possible mechanisms, including the complex of Gbeta gamma subunits (29), Ca2+-calmodulin regulation (29, 30), and direct phosphorylation of specific AC isoforms by PKC (7, 11-13). The data indicate that the mere co-expression of a P2Y2 receptor in a cell could potentiate Gs-mediated signaling in that cell.

In summary, we report the cloning and species-specific heterologous expression of MDCK-D1-P2Y2 (cP2Y2) receptors. When expressed in CF2Th cells, the receptor is capable of mobilizing [Ca2+]i and stimulates inositol-phosphate formation with a rank order of potency (UTP >=  ATP > ADP > 2MT-ATP) similar to previously cloned P2Y2 receptors. In contrast to the case in MDCK-D1 cells, cP2Y2 receptors expressed in CF2Th cells are not able to stimulate either the release of AA or the production of cAMP, indicating the requirement of cell-specific effectors in P2Y2-mediated receptor signaling, even when expressed in a species-specific manner. Renal epithelial cells, such as MDCK cells, appear to express crucial post-receptor components (presumably components involved in prostaglandin formation and action) that are required for P2Y2-mediated release of AA and formation of cAMP.


    ACKNOWLEDGEMENTS

We thank Elizabeth Peters for initial isolation of a partial clone of cP2Y2 receptor from an MDCK-D1 cell cDNA library.


    FOOTNOTES

Training and research grants from the National Institutes of Health (HL-41307, GM-07752) and an American Society for Pharmacology and Experimental Therapeutics summer undergraduate fellowship (to J. J. Wu) supported this work.

Address for reprint requests and other correspondence: P. A. Insel, Dept. of Pharmacology 0636, UCSD School of Medicine, Jolla, CA 92093-0636 (E-mail: pinsel{at}ucsd.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.

Received 27 March 2000; accepted in final form 2 August 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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