JBC Anatrace, Inc.

HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
QUICK SEARCH:   [advanced]


     

Originally published In Press as doi:10.1074/jbc.M400360200 on March 4, 2004

J. Biol. Chem., Vol. 279, Issue 19, 19790-19799, May 7, 2004
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow All Versions of this Article:
279/19/19790    most recent
M400360200v1
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Right arrow Citation Map
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Inbe, H.
Right arrow Articles by Encinas, J. A.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Inbe, H.
Right arrow Articles by Encinas, J. A.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati  
What's this?

Identification and Characterization of a Cell-Surface Receptor, P2Y15, for AMP and Adenosine*

Hisayo Inbe, Shinichi Watanabe, Miwa Miyawaki, Eri Tanabe, and Jeffrey A. Encinas{ddagger}

From the Bayer Yakuhin, Ltd., Research Center Kyoto, 6-5-1-3 Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan

Received for publication, January 13, 2004


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
AMP and adenosine are found in all cell types and can be released by cells or created extracellularly from the breakdown of ATP and ADP. We have identified an orphan G protein-coupled receptor with homology to the P2Y family of nucleotide receptors that can respond to both AMP and adenosine. Based on its ability to functionally bind the nucleotide AMP, we have named it P2Y15. Upon stimulation, P2Y15 induces both Ca2+ mobilization and cyclic AMP generation, suggesting coupling to at least two different G proteins. It is highly expressed in mast cells and is found predominantly in the tissues of the respiratory tract and kidneys, which are known to be affected by AMP, adenosine, and adenosine antagonists. Until now, the effects of AMP have been thought to depend on its dephosphorylation to adenosine but we demonstrate here that P2Y15 is a bona fide AMP receptor by showing that it binds [32P]AMP. Because AMP and adenosine have bronchoconstrictive effects that can be inhibited by theophylline, we tested whether theophylline and other adenosine receptor antagonists can block P2Y15. We found inhibition at a theophylline concentration well within the therapeutic dose range, indicating that P2Y15 may be a clinically important target of this drug.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenosine can mediate diverse physiological effects including bronchoconstriction, inhibition of platelet aggregation, inhibition of lipolysis, induction of sedation, vasodilation, suppression of cardiac rate and contractility, and stimulation of gluconeogenesis. To date, five cell-surface receptors have been identified for adenosine, namely adenosine receptors A1, A2A, A2B, and A3 (collectively referred to as P1 receptors) and the growth hormone secretagogue receptor (1, 2). AMP has a similarly diverse repertoire of effects (3–6) including bronchoconstriction, stimulation of DNA synthesis, mitogenesis, and stimulation of chloride secretion, but no receptor for AMP has previously been reported. Numerous cell types can release adenosine including mast cells (7), kidney brush border cells (8), and cardiac cells (9), whereas AMP can be released by such cell types as activated platelets (10), neutrophils (4), and eosinophils (11). AMP can also be generated extracellularly from the hydrolysis of ATP and ADP by ecto-ATPases (12) and ecto-ATP diphosphohydrolases (13) and can be further dephosphorylated by ecto-5'-nucleotidases to produce adenosine (14). A number of widely used drugs have been developed such as theophylline (15) and cromolyn (16) that can modulate what are thought to be the effects of adenosine in diseases such as asthma, but their mechanisms of action are not yet fully understood.

Other extracellular nucleotides similarly induce a wide variety of responses in many cell types including muscle contraction and relaxation, vasodilation, neurotransmission, platelet aggregation, ion transport regulation, and cell growth. The effects are exerted mainly through two types of cell surface molecules, P2Y-type G protein-coupled receptors (GPCRs)1 and P2X-type ligand-gated ion channels. Nine nucleotide-stimulated P2Y-type GPCRs have been characterized to date in humans: P2Y1, P2Y11, P2Y12, and P2Y13, which are activated by the adenine nucleotides ATP or ADP; P2Y4, P2Y6, P2Y14, and CYSLT1, which are activated by the uridine nucleotides UTP or UDP (or in the case of P2Y14, UDP-glucose); and P2Y2, which is activated by both adenine and uridine nucleotides (17–20). None of these receptors has been shown to be able to bind adenosine or AMP. Here we report on the characterization of a GPCR with close homology to the P2Y receptors that can bind and respond to both AMP and adenosine.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
P2Y15 cDNA Cloning—Protein sequences of known P2Y receptors were used to search for homologs in the GenBankTM data base of the National Center for Biotechnology Information (www.ncbi.nlm.nih.gov) using the program tblastn. The search identified an intronless genomic sequence subsequently also found by others and designated in GenBankTM as the orphan receptor GPR80 (21). To clone the gene, human genomic DNA was used as template and PCR was performed using primers 5'-GCCAAACTGAACTCTCTTGTTTTCTTGC-3' and 5'-GCCCTGGCTTTGGCACATGATTAC-3' and a blend of HotStarTaq (Qiagen) and Pfu Turbo (Stratagene, La Jolla, CA) polymerases. PCR products were cloned into pCRII-TOPO (Invitrogen), cycle-sequenced with an ABI Prism Dye Terminator Cycle sequencing reaction kit (Applied Biosystems, Foster City, CA), and analyzed on an ABI Prism 377 sequencing system (Applied Biosystems). For functional studies, the cDNA was subcloned into a pDisplay vector (Invitrogen) to append an N-terminal hemagglutinin epitope and Ig{kappa} signal sequence.

Expression Profiling—25 µg of total RNA from the following were used as template in reactions to synthesize first-strand cDNA for expression profiling: human total RNA panels I-V (Clontech Laboratories, Palo Alto, CA), normal human lung primary cell lines (BioWhittaker Clonetics, Walkersville, MD), several common cell lines (ATCC, Manassas, VA), and various cells purified from peripheral blood. First-strand cDNA was synthesized using oligo(dT) (Nippon Gene Research Laboratories, Sendai, Japan) and the SuperscriptTM first-strand synthesis system for RT-PCR (Invitrogen) according to the manufacturer's protocol. For these samples, 1/1250 of the synthesized first-strand cDNA was subsequently used as template for quantitative PCR. Additional samples were purchased as presynthesized cDNAs (Human Immune System MTC Panel and Human Blood Fractions MTC Panel, Clontech Laboratories), and for these, 10 ng of cDNA was used as template for quantitative PCR.

Quantitative PCR was performed in a LightCycler (Roche Applied Science) with oligonucleotide primers 5'-TTCGGATCGAATCTCGCCTGCT-3' and 5'-TGCTTGCTCAAGGTTCCCGCTTA-3' in the presence of the DNA-binding fluorescent dye SYBR Green I. Results were then converted into copy numbers of the gene transcript per nanogram of template cDNA by fitting to a standard curve. The standard curve was derived by simultaneously performing the quantitative PCR reaction on PCR products of known concentrations amplified beforehand from the target gene.

To correct for differences in mRNA transcription levels per cell in the various tissue types, a normalization procedure was performed using similarly calculated expression levels of five different housekeeping genes: glyceraldehyde-3-phosphate dehydrogenase, hypoxanthine guanine phosphoribosyl transferase, {beta}-actin, porphobilinogen deaminase, and {beta}2M. Expression levels of the five housekeeping genes in all of the tissue samples were measured in three independent reactions per gene using the LightCycler and a constant amount (25 µg) of starting RNA.

Expression levels were also measured using CodeLinkTM microarrays (Amersham Biosciences). Total RNA was prepared from human umbilical cord blood-derived mast cells (kindly provided by Professor H. Nagai, Department of Pharmacology, Gifu Pharmaceutical University, Gifu, Japan; prepared as described by Saito et al. (22)) and purified leukocyte fractions and then was used to synthesize biotin-labeled cRNA using the Amersham cRNA synthesis kit. cRNA yield was quantified by measuring absorbance at 260 nm, and then the cRNA was fragmented in 40 mM Tris acetate, pH 7.9, 100 mM KOAc, and 31.5 mM MgOAc at 94 °C for 20 min. 10 µg of fragmented cRNA from each sample was used for hybridization to a CodeLink UniSet 20K human expression bioarray chip (Amersham Biosciences). cRNAs bound to the microarrays were stained with streptavidin-Cy5, and the processed slides were scanned with an Axon GenePix 4000B scanner. Images for each slide were analyzed using the CodeLink expression analysis software (Amersham Biosciences).

Determination of Ligand Specificity—Hemagglutinin-tagged P2Y15 was transfected into HEK293 cells (ATCC) using LipofectAMINE (Invitrogen). Expression on the cell surface was verified by staining cells with phycoerythrin-labeled anti-hemagglutinin antibody (Santa Cruz Biotechnology, Santa Cruz, CA) and measuring fluorescence on a FAC-Sort (BD Biosciences). Stably transfected clones were generated by selection in G418 (500 µg/ml) and reconfirmed for cell-surface expression of the P2Y15 protein. Ligand screening was performed in a Ca2+ mobilization assay as follows. Stably transfected P2Y15 GPCR-expressing cells were seeded into 96-well plates and incubated overnight at 37 °C. The culture medium was aspirated and replaced with 100 µl of loading buffer consisting of 0.1% bovine serum albumin, 20 mM HEPES, 1 mM probenecid, 0.01% pluronic F127, and 1 µM Fluo-3-AM (Molecular Probes, Eugene, OR) in Hanks' buffered salt solution and incubated for 1 h at room temperature. The cells were then washed gently three times with wash buffer consisting of 0.1% bovine serum albumin, 20 mM HEPES, and 1 mM probenecid in Hanks' buffered salt solution. The washed cells were placed in an FDSS6000 functional drug screening system (Hamamatsu Photonics, Hamamatsu, Japan), and changes in cellular fluorescence were measured after adding serial dilutions of potential ligands. A panel of approximately 130 potential ligands for testing was assembled by selecting known ligands of the GPCRs most closely related to P2Y15 GPCR as well as several naturally occurring chemical relatives of the ligands. The panel included various bioactive lipids, eicosanoids, peptides, cannabinoids, chemokines, nucleosides, nucleotides, and chemically related substances, which were generally purchased from either Sigma or R&D Systems (Minneapolis, MN).

Ligand confirmation analyses were performed by repeating the Ca2+ mobilization experiments using the identified ligands to stimulate P2Y15 transfectants, identically constructed P2Y8 transfectants, and nontransfected HEK293 cells, all of which had been cultured for 2 h with or without 1 µM pertussis toxin. Antagonist assays for inhibition of calcium responses to AMP and adenosine were performed essentially as above with the exception that serial dilutions of antagonist compounds were added 5 min prior to the addition of ligand. Agonist assays for stimulation of calcium responses were performed in the same manner as the ligand screen.

AMP Conversion Assays—Transfected and nontransfected cells in Dulbecco's modified Eagle's medium were seeded at 105 cells/well into 96-well plates and incubated for 3 h. The medium was then exchanged with fresh medium, and 1.85 µM [3H]AMP (Amersham Biosciences) was added to the wells. After incubation for 5 or 60 min, 6 µl of the medium together with AMP and adenosine standards were applied to thin layer chromatography sheets, separated with isobutyl alcohol/isoamyl alcohol/2-ethoxyethanol/ammonia/H2O (9:6:18:9:15) as solvent, and visualized under UV light as described by Yegutkin et al. (23). The spots corresponding to AMP and adenosine were cut from the sheets, and the amounts of each were quantified by scintillation counting.

Receptor Binding Assays—105 cells/well in 96-well plates were washed twice for 1 h with Dulbecco's modified Eagle's medium. Wheat germ agglutinin scintillation proximity assay beads (Amersham Biosciences) were then added at 1 mg/well followed 1 h later by the addition of increasing concentrations of [3H]adenosine or [3H]AMP in a constant volume of HBS (10 mM Hepes, 130 mM NaCl, 5 mM KCl, 1 mM MgCl2, 1 mM CaCl2, and 1 g/liter glucose (6 µl/well)). After incubating at 4 °C for 16 h, the plates were centrifuged for 10 min at 1500 rpm and then scintillation was measured on a TopCount automated scintillation counter. Nonspecific binding measurements and competitive binding experiments were carried out under the same conditions but with either an excess of cold ligand (2.5 mM) or increasing concentrations of cold ligand, respectively. Adenosine binding in competitive binding experiments was measured after incubation for 1 h at 4 °C. Binding of [32P]AMP was carried out under the same conditions with the exception that instead of using scintillation proximity assay beads, at the end of the incubation, cells were washed three times by vacuum filtration and 100 µl of scintillation fluid was added to the wells. Kd values were determined by nonlinear regression using the program Prism (GraphPad Software, San Diego, CA).

Cyclic AMP Production Assay—Cyclic AMP production after stimulation of cells was measured with the Tropix cAMP screen (Applied Biosystems) according to the manufacturer's protocol. Stable transfectants and control cells (1 x 105 cells/well) were cultured for 2 h with or without 1 µM pertussis toxin and then treated for 30 min with 10 µM forskolin and serial dilutions of AMP or adenosine. The cells were then lysed, and the cAMP produced was measured by a cAMP-specific enzyme-linked immunosorbent assay. Concentrations of cAMP produced were calculated by comparing against cAMP standards measured simultaneously. The effect of adenosine deaminase (ADA) on ligand-stimulated cyclic AMP production was measured essentially as above with the exception that pertussis toxin was excluded and cells were treated for 30 min with 10 µM forskolin prior to the addition of 25 µM adenosine or 100 µM AMP. Serial dilutions of ADA (Roche Diagnostics) were added to the cultures 10 min prior to the addition of ligand.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
Cloning and Sequencing—In an effort to find new receptors for extracellular nucleosides and nucleotides, we searched for homologs of known P2Y nucleotide receptors in the GenBankTM data base of the National Center for Biotechnology Information using the program tblastn. P2Y receptors are receptors for diphosphate and triphosphate adenine and uridine nucleotides, although none respond to AMP or adenosine. Our search identified an intronless genomic sequence subsequently also found by others and designated as the orphan receptor GPR80 (21) or GPR99 (24). We are now renaming it P2Y15 as a new member of the P2Y family. A putative mouse ortholog of the gene has recently appeared in GenBankTM under accession number XP_139267 [GenBank] . We subsequently found the rat ortholog by using the mouse protein sequence in a tblastn query against rat genome sequences. An alignment of the human, mouse, and rat protein sequences is shown in Fig. 1.



View larger version (69K):
[in this window]
[in a new window]
  		FIG. 1.
Alignment of human, mouse, and rat P2Y15 amino acid sequences. Identical residues are shown with white backgrounds. Similar residues are shown with gray backgrounds, and dissimilar residues are shown with black backgrounds. Identity between either of the rodent sequences and the human sequence is 86%, and identity between the mouse and rat is 96%. Seven transmembrane regions as predicted by the computer program TMpred (42) are indicated with heavy overlines and are numbered TM1–7. The GenBankTM accession number of the rat P2Y15 sequence is AY191367 [GenBank] .

 
The conceptually translated protein product of the human P2Y15 gene shows 36% identity over its full-length with the nucleotide receptor P2Y1, increasing to an overall sequence similarity of 58% when amino acids with related physicochemical properties are included. Homology of the protein sequence with other P2Y nucleotide receptors P2Y2, P2Y4, P2Y6, and P2Y11 likewise shows an overall identity ranging from 25 to 35% and a similarity ranging from 43 to 57%. A similar but slightly lower level of homology is seen among the mouse and rat orthologs (excluding P2Y11, which has not yet been found in rodents). The human gene transcript encodes a polypeptide of 337 amino acids with a calculated molecular mass of 38.3 kDa. A phylogenetic analysis comparing the protein sequence with other GPCRs places the molecule among a cluster of other P2Y receptors distant from the known receptors for adenosine (Fig. 2). The gene sequence is found on the genomic contig NT_009952 [GenBank] , which has been localized to human chromosome 13q32.



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 2.
Unrooted phylogenetic analysis comparing the P2Y15 protein sequence with adenosine receptors, other P2Y receptors, and closely related GPCRs. Phylogenetic analysis was performed with the Neighbor Joining algorithm and the dendrogram drawn with the computer program Vector NTI. Known ligands are indicated.

 
Tissue Distribution of P2Y15—As a first step to investigating the function of P2Y15, we examined the distribution of P2Y15 messenger RNA expression in several different human tissues, cell types, and commonly used cell lines. We designed oligonucleotide primers near the 3' end of the coding region that could specifically amplify P2Y15 cDNA and used these in a quantitative RT-PCR analysis to measure relative transcript levels. Among the tissues tested, trachea, salivary glands, kidney, fetal brain, and lung showed the highest expression levels (Fig. 3a). The high expression in the respiratory tract prompted us to look in-depth at the cell types within the trachea and lung that might be responsible for the predominant expression there. Little or no expression, however, could be detected in any of the tested primary cell populations or transformed cell lines derived from lung tissues or from immune cell subtypes (Fig. 3b).



View larger version (11K):
[in this window]
[in a new window]
  		FIG. 3.
Tissue and cellular distribution of P2Y15. Expression in various human tissues (a) and cells (b) was assessed by quantitative RT-PCR. The x axis represents the approximate number of copies of messenger RNA transcript per 10 ng of total RNA after normalization to a set of five housekeeping genes. c, expression of P2Y15 and the P1 adenosine receptors (gene names ADORA1, ADORA2A, ADORA2B, and ADORA3) in various leukocyte subsets was analyzed by hybridization of cRNAs generated from cellular total RNAs to microarrays containing ~20,000 gene-specific probes. The x axis represents the relative fluorescence intensity of cRNAs bound to each genespecific probe. PBL, peripheral blood leukocytes; PBMN, peripheral blood mononuclear cells; PWM, pokeweed mitogen; LAK, lymphokine-activated killer cells; IANK, interleukin-2-activated natural killer cells.

 
Therefore, we investigated the possibility that the gene is expressed in a minor population of cells in the respiratory tract such as mast cells or eosinophils. Because mast cells and eosinophils typically yield only small amounts of mRNA, we analyzed the expression of P2Y15 in these cells by microarray analysis, which allows the analysis of more genes than quantitative RT-PCR and provides multiple controls to verify the quality of the sample preparations. Analysis of gene expression in CD4 and CD8 T cells, tonsil B cells, neutrophils, eosinophils, and mast cells showed that the P2Y15 gene is expressed specifically and at very high levels in mast cells (Fig. 3b). Indeed, the high level of P2Y15 expression in mast cells places it among the top 1% of the ~20,000 different gene probes included in the microarray. Similar specificity and high level expression in these cell types were not seen with any of the other P1 adenosine receptors (Fig. 3c) or P2 nucleotide receptors (data not shown).

Identification of AMP and Adenosine as Functional Ligands for P2Y15—To identify the ligand of P2Y15, we generated stable transfectants with HEK293 cells and then tested for calcium mobilization in response to a panel of ligands. Among the potential ligands tested, only AMP and adenosine were able to induce a response in the transfectants while not inducing a similar response in either nontransfected HEK293 cells or HEK293 cells stably transfected with the control orphan GPCR P2Y8 in an identical vector construct. We detected a calcium response with an EC50 of 920 nM for AMP and 670 nM for adenosine (Fig. 4a). The calcium response to either ligand was not significantly affected by pertussis toxin (data not shown). Both stable transfectants and nontransfected cells mobilized calcium in response to ATP, ADP, and UTP, consistent with previous reports of HEK293 endogenously expressing P2Y1 and P2Y2 receptors (25). A further analysis by RT-PCR showed that the nucleotide receptors P2Y4, P2Y12, and P2Y13 and the adenosine receptors A2A and A2B (previously reported by Sunahara et al. (26)) are also expressed in HEK293 cells (data not shown). However, despite the endogenous expression of the adenosine receptors calcium, mobilization responses to adenosine in nontransfected cells could only be detected at very high adenosine concentrations and showed only a very weak response (Fig. 4a).



View larger version (16K):
[in this window]
[in a new window]
  		FIG. 4.
P2Y15 is a functional receptor for AMP and adenosine. AMP (filled symbols) and adenosine (open symbols) stimulated calcium mobilization (a) and cyclic AMP generation (b) in a dose-dependent manner in transfected HEK293 cells expressing P2Y15 (circles). Nontransfected HEK293 cells (squares), by contrast, showed little calcium mobilization in response to either ligand, and showed cyclic AMP generation in response to adenosine but not to AMP. Calcium mobilization was measured in 10-well replicates in an FDSS6000 functional drug screening system and plotted as the integral of the ratio of signal to background over a 60-s time period. Cyclic AMP generation in the presence of 10 µM forskolin was measured in duplicate with the Tropix cAMP screen. The data for cyclic AMP generation are representative of three separate experiments. c, no differences could be seen between conversion rates of AMP to adenosine by nontransfected HEK293 cells (white bars) and P2Y15-expressing cells (black bars). 1.85 µM [2-3H]AMP was added to triplicate cultures of nontransfectants and P2Y15 transfectants and then recovered from the medium after incubation times of 5 and 60 min. Thin layer chromatography analysis showed only two bands, which migrated together with AMP and adenosine standards, indicating minimal conversion to other products such as IMP, ADP, or ATP.

 
To determine the effect of P2Y15 stimulation on adenylate cyclase activity, we measured cyclic AMP accumulation in our P2Y15-HEK293 stable transfectants in response to AMP and adenosine. Stimulation with either ligand alone gave only minimal responses barely above the detection limit. In the presence of 10 µM forskolin, however, both AMP and adenosine induced the generation of cyclic AMP in a dose-dependent manner with an EC50 of 214 nM for AMP and 327 nM for adenosine (Fig. 4b). Nontransfected HEK293 cells similarly generated cyclic AMP in response to adenosine, probably because of the stimulation of endogenously expressed adenosine receptors, but did not respond strongly to AMP. The production of cyclic AMP in response to either ligand was not affected by pretreatment of the cells for 2 h with 1 µM pertussis toxin (data not shown), indicating that P2Y15 does not couple with an adenylate cyclase-inhibiting G protein. The responsiveness of the P2Y15 transfectants to AMP in the cyclic AMP assay did not appear to be due to an increased conversion rate of AMP to adenosine, because transfected and nontransfected cells showed similarly low rates of endogenous nucleotidase activity (Fig. 4c).

Saturation binding analysis of the ligands to the P2Y15 receptor in stable transfectants gave Kd values of 12.0 µM for [2-3H]adenosine (Fig. 5a) and 18.6 µM for [2-3H]AMP (data not shown). Because AMP can be dephosphorylated to adenosine by ectonucleotidases, we repeated the binding analysis with adenosine 5'-[32P]monophosphate to confirm that the binding being measured was AMP and not adenosine. This resulted in a similar binding curve with a Kd of 18.8 µM (Fig. 5b), indicating that AMP itself and not a breakdown product was binding to the receptor. We then performed competitive binding assays to determine whether one ligand could antagonize the binding of the other ligand to the P2Y15 transfectants. Although unlabeled AMP was able to block a large proportion of the binding of 10 µM [2-3H]adenosine to transfectants (Ki = 32.0 µM), it did not block the binding as completely as unlabeled adenosine and had little effect in blocking [3H]adenosine binding to nontransfected HEK293 cells (Fig. 5c). On the other hand, unlabeled adenosine was able to block the binding of 10 µM [2-3H]AMP to transfectants (Ki = 39.8 µM) with a potency similar to that of unlabeled AMP (Fig. 5d).Whereas these results provide evidence that both AMP and adenosine bind to P2Y15, the results also demonstrate a lack of specific AMP binding sites on the nontransfected cells because neither AMP nor adenosine could antagonize the binding of [2-3H]AMP to nontransfected HEK293 cells beyond the background level.



View larger version (24K):
[in this window]
[in a new window]
  		FIG. 5.
P2Y15 can bind both AMP and adenosine. Specific binding of increasing concentrations of [2-3H]adenosine (a) and [32P]AMP (b) to transfected HEK293 cells expressing P2Y15 (circles) and nontransfected HEK293 cells (squares) is shown. The binding of [2-3H]adenosine (c) or [2-3H]AMP (d) to P2Y15-transfected cells and nontransfected cells could be competed in both cases by unlabeled AMP (filled symbols) or adenosine (open symbols). All of the binding assays were performed in quadruplicate.

 
To further clarify whether AMP itself without its conversion to adenosine is able to induce a response in P2Y15 transfectants, we measured AMP- and adenosine-induced cyclic AMP production in the presence of ADA, an enzyme that breaks down adenosine to inosine. As expected, ADA inhibited the adenosine-induced response in a dose-dependent manner but showed little if any effect against the AMP-induced response, indicating that adenosine is not a mediator of the AMP response (Fig. 6).



View larger version (25K):
[in this window]
[in a new window]
  		FIG. 6.
ADA can inhibit adenosine-induced but not AMP-induced P2Y15 signaling. Cyclic AMP production induced by 25 µM adenosine in transfectants expressing P2Y15 was effectively inhibited in a dose-dependent manner by ADA. Cyclic AMP production in response to 100 µM AMP; however, showed little change even at ADA concentrations up to 4 units/ml. Cyclic AMP production was measured in 4-well replicates in the presence of 10 µM forskolin as described in Fig. 4.

 
Characterization of Antagonists and Agonists of the P2Y15 Receptor—To test whether any of the known antagonists or agonists of adenosine receptors could antagonize P2Y15, we performed calcium mobilization assays in the presence of varying concentrations of such compounds. Antagonists tested were the nonselective adenosine receptor antagonists theophylline, caffeine, 3-isobutyl-1 methylxanthine, and 8-(p-sulfophenyl) theophylline; selective A1 receptor antagonists 8-phenyltheophylline and 8-cyclopentyl-1,3-dipropylxanthine (DPCPX); the selective A2A receptor antagonist 8-(3-chlorostyryl)caffeine; and the selective A2B receptor antagonists enprofylline and alloxazine. With the exception of alloxazine and DPCPX, which were agonistic in both transfected and nontransfected cells, all of these compounds were able to block the calcium mobilization induced by AMP and adenosine with Ki values for blocking AMP ranging from 200 nM for 8-(p-sulfophenyl)theophylline to 3,309 nM for caffeine (Fig. 7a and Table I) and Ki values for blocking adenosine ranging from 962 nM for 8-theophylline to 64,060 nM for enprofylline (Fig. 7b and Table I). Because many of these compounds can also act as phosphodiesterase inhibitors and cause an increase in cyclic AMP levels that can potentially inhibit Ca2+ mobilization, the compounds were also tested for their ability to inhibit ADP-induced Ca2+ mobilization in the same cells. None of the compounds, however, had significant inhibitory effects at concentrations <1 mM (data not shown). Adenosine receptor agonists tested were the nonselective agonist 5'-(N-ethylcaboxamido)adenosine, the selective A1 receptor agonist N6-cyclopentyladenosine, the selective A2A receptor agonist CGS-21680 hydrochloride, and the selective A3 receptor agonist 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide (Chloro-IB-MECA). Among these compounds, only 5'-(N-ethylcaboxamido)adenosine and N6-cyclopentyladenosine showed measurable agonist effects (Table II).



View larger version (26K):
[in this window]
[in a new window]
  		FIG. 7.
Antagonists of adenosine block AMP- and adenosine-induced P2Y15 signaling. Calcium mobilization induced by 10 µM AMP (a) or 10 µM adenosine (b) was effectively blocked in a dose-dependent manner by the indicated adenosine receptor antagonists. Curves for alloxazine and DPCPX, which showed agonistic effects in both transfectants and nontransfectants, are excluded. Calcium mobilization was measured in 6-well replicates as described in Fig. 4. The data are representative of three separate experiments.

 


View this table:
[in this window]
[in a new window]
  		TABLE I
Ki values for antagonists of P2Y15

Ki values for antagonists of AMP- and adenosine-stimulated Ca2+ mobilization derived by non-linear regression of the curves shown in Fig. 7, a and b. Abbreviations: IBMX, 3-isobutyl-1-methylxanthine; 8-SPT, 8-(p-sulfophenyl)theophylline; 8-PT, 8-phenyltheophylline; CSC, 8-(3-chlorostyryl)caffeine; —, no detectable antagonistic effect.

 


View this table:
[in this window]
[in a new window]
  		TABLE II
EC50 values for P2Y15 agonist-stimulated Ca2+ mobilization

Abbreviations: NECA, 5'-(N-ethylcaboxamido)adenosine; —, no detectable agonistic effect.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We describe here the identification of a cell-surface receptor that can respond to both AMP and adenosine. Although the responses to both ligands are pharmacologically similar, we consider the receptor a new member of the P2Y family because the primary structure of the receptor protein more closely resembles the P2Y nucleotide receptors than the P1 adenosine receptors. Moreover, the sequence contains the conserved basic residue arginine at position 268 that has been reported to be essential for binding the phosphate moiety of nucleotide ligands by P2Y receptors (27). The ability of the receptor to bind the nucleoside adenosine, however, is unique among the P2Ys and may require a broadening of the criteria for the P2Y classification.

Functionally, the receptor is able to respond to stimulation by inducing the mobilization of calcium ions and causing the generation of cyclic AMP. This dual functionality is similar to that of the adenosine receptor A2B (28, 29) and the nucleotide receptor P2Y11 (19), both of which show evidence of being dually coupled to activators of phospholipase C (calcium flux) and adenylate cyclase (cyclic AMP production). For both A2B and P2Y11, the activation of phospholipase C and adenylate cyclase is thought to be achieved through Gq class and Gs G proteins, respectively. Because P2Y15-induced calcium flux and cyclic AMP generation are both insensitive to pertussis toxin, which inactivates Go and Gi classes of G proteins, P2Y15 is probably similarly coupled to the pertussis toxin-insensitive Gq class and Gs G proteins. However, the nature of the P2Y15 coupling to adenylate cyclase-stimulatory Gs proteins in our system is not completely straightforward because detectable levels of cyclic AMP production could only be seen in the presence of the adenylate cyclase activator forskolin. This apparent requirement for forskolin, which synergistically potentiates the cyclic AMP production, may be specific to the HEK293 host cells used but in any case is reminiscent of the forskolin potentiation that has similarly been reported for {beta}-adrenergic receptors (30).

The similarity to other receptors, particularly A2B, which can respond to adenosine as a ligand and which is expressed on the HEK293 host cell used in the experiments presented here, was a potentially confounding factor in our analysis and therefore had to be taken into account when interpreting our results. In calcium mobilization experiments, despite previous reports of HEK293 calcium responses generated upon stimulation with adenosine (28, 29), the response of endogenous receptor to AMP and adenosine in our HEK293 cells was so slight as to be negligible. The transfection procedure itself also appeared to have no influence on the calcium response because transfectants generated with P2Y8 instead of P2Y15 also showed negligible responses to AMP and adenosine. On the other hand, in cyclic AMP production assays, nontransfected cells gave a response to adenosine that was indistinguishable from that seen in the transfectants. Although it was clear that AMP could cause a distinct increase in cyclic AMP production in the P2Y15 transfectants, we cannot say with certainty that adenosine stimulation of P2Y15 had the same effect. Confirmation of the ability of adenosine to stimulate P2Y15 to activate adenylate cyclase must therefore await the successful functional expression of P2Y15 in a cell type that does not express A2 adenosine receptors.

In receptor binding assays, whereas AMP clearly showed saturatable binding kinetics only in P2Y15 transfectants, adenosine was able to bind specifically to receptors on both nontransfectants and P2Y15 transfectants, albeit at a higher level in the transfectants. However, competitive binding experiments using cold AMP to compete against labeled adenosine binding showed that on P2Y15 transfectants adenosine binds to sites that can be competed with AMP, presumably P2Y15 receptors, but on the nontransfectants adenosine binds only to sites that cannot be competed with AMP such as other adenosine receptors, giving compelling evidence that adenosine binds to P2Y15. Although the affinity of the receptor for AMP and adenosine is relatively low, the ligand binding results we obtained are in line with the range of Kd values that have been reported so far for some of the other adenosine receptors and P2Y receptors. For example, agonist binding to the A2B adenosine receptor is typically in the double digit micromolar range (1) and [35S]ATP[{gamma}S] binding to P2Y receptors on tracheal gland cells has been reported as 2.5 and 20 µM (31). Furthermore, the lack of antagonism of the P2Y15 receptor by DPCPX and alloxazine, specific antagonists of A1 and A2B receptors, respectively, and the lack of significant agonism of the P2Y15 receptor by N6-cyclopentyladenosine, CGS-21680 hydrochloride, and Chloro-IB-MECA, specific agonists of A1, A2A, and A3, respectively, strongly suggest that the signaling responses measured were generated through activation of P2Y15 and not another adenosine receptor.

Nevertheless, a concern when conducting experiments with AMP is the potential for enzymatic breakdown of the molecule to adenosine. In most reports that have demonstrated the activity of AMP, for example, as an inducer of bronchoconstriction in the lungs of patients with asthma (3, 32) or as a paracrine activator of intestinal chloride ion secretion produced by neutrophils and eosinophils (11, 33) because of the absence of a receptor for AMP to which this activity could be attributed, it has been assumed that AMP is converted to adenosine before the resultant effects are produced. Our analysis of AMP to adenosine conversion rates did not show any evidence of enhanced AMP breakdown in P2Y15-expressing cells. Therefore, to determine whether AMP itself can bind P2Y15 or must first be converted to adenosine, we performed receptor binding assays using 32P-labeled AMP and found specific saturatable binding to the transfectants. Because the radiolabel on the molecule would be lost upon dephosphorylation and conversion to adenosine, our binding assay shows that AMP can bind to P2Y15 without conversion.

The expression of P2Y15 in mast cells, the respiratory tract, and kidney is consistent with effects that have been reported for AMP, adenosine, and adenosine antagonists in these tissues. In the respiratory tract, an immediate bronchoconstriction is typically experienced by patients with asthma upon the inhalation of AMP or adenosine (3) and adenosine has been found to be increased in the bronchoalveolar lavage fluid from airways of patients with chronic inflammatory conditions of the lung such as asthma and chronic obstructive pulmonary disease (34). Recently, adenosine or, more commonly, AMP, which is more easily solubilized, has been utilized in bronchoprovocation tests for the diagnosis and monitoring of asthma (3, 32) because of the better disease specificity of the test and correlation with inflammation state than other bronchoprovocators. Conversely, adenosine receptor antagonists, such as theophylline, have been used for over 50 years as effective bronchodilators (15). Evidence on the mechanism of adenosine and AMP-mediated bronchoconstriction has indicated an extracellular site of action and the stimulation or potentiation of mast cell mediator release (35). Recent studies to determine which adenosine receptor is responsible for the bronchoconstriction response, however, have failed to conclusively implicate a particular receptor, and on the contrary, experiments in rats have ruled out the role of any of the known P1 adenosine receptors, leading to the conclusion that an unknown receptor must be involved (36). Although it is still possible that the effects of AMP in the human respiratory tract are dependent upon its breakdown to adenosine and the subsequent stimulation of P1 adenosine receptors with the evidence provided here to show the existence of an AMP receptor, the alternative possibility of a direct effect by AMP must now also be considered. Regarding the expression in the kidneys, adenosine antagonists such as theophylline and caffeine are well known to possess diuretic effects. These effects are thought to be mediated primarily through the A1 and A2A adenosine receptors, but the pharmacology of these two receptors cannot satisfactorily account for the effects seen (37). The existence of a third receptor in the kidneys that can be affected by adenosine antagonists may help to explain the effects.

Adenosine receptor antagonists such as theophylline, enprofylline, and caffeine are among the world's most widely used drugs. However, their molecular mechanism remains undefined as do their sites of action, which include adenosine receptors, phosphodiesterases, histone deacetylases, and other sites that have yet to be found (38, 39). To determine whether P2Y15 is a target of such antagonists, several adenosine receptor antagonists were tested and all but two showed antagonist activity against P2Y15. The concentration of theophylline at which calcium responses could be inhibited (Ki of 0.7 µM against AMP; 5.6 µM against adenosine) is well below that considered to be the therapeutically optimal plasma concentration (usually 55–110 µM) (15) for this drug. Similarly, plasma concentrations of caffeine as low as 5 µM have been shown to relieve histamine-induced bronchoconstriction (40) and, at this concentration, caffeine can effectively inhibit AMP-induced calcium responses (Ki of 3.3 µM against AMP). Unexpectedly, however, the antagonists often showed a large difference in the concentrations required to block AMP-mediated signaling compared with those required to block adenosine-mediated signaling, even though AMP and adenosine show similar EC50 values for signaling and similar Kd values for binding. The underlying reason for this is unclear, but the discrepancy suggests that the mechanism of action of the antagonists is more complex than can be explained by simple affinity-based competition.

The identification of a receptor that binds and responds to both AMP and adenosine and is susceptible to blocking by adenosine receptor antagonists can help us to better understand the complex physiological effects of AMP and adenosine. Because the safety and effectiveness of adenosine antagonists in treating respiratory diseases and other ailments has been limited by toxicity in the central nervous system and heart (41) where most adenosine receptors are found, it will be of considerable interest to determine whether P2Y15 can be specifically targeted in order to develop better treatments.


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

{ddagger} To whom correspondence should be addressed: Bayer Yakuhin, Ltd., Research Center Kyoto, 6-5-1-3 Kunimidai, Kizu-cho, Soraku-gun, Kyoto 619-0216, Japan, Tel.: 81-774-75-2468; Fax: 81-774-75-2506; E-mail: jencinas{at}post.harvard.edu.

1 The abbreviations used are: GPCR, G protein-coupled receptor; RT, reverse transcription; ADA, adenosine deaminase; DPCPX, 8-cyclopentyl-1,3-dipropylxanthine; Chloro-IB-MECA, 2-chloro-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide; RT, reverse transcription; HEK, human embryonic kidney. Back


   ACKNOWLEDGMENTS
 
We thank Eiko Okazaki for technical support.



   REFERENCES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 

  1. Fredholm, B. B., AP, I. J., Jacobson, K. A., Klotz, K. N., and Linden, J. (2001) Pharmacol. Rev. 53, 527–552[Abstract/Free Full Text]
  2. Tullin, S., Hansen, B. S., Ankersen, M., Moller, J., Von Cappelen, K. A., and Thim, L. (2000) Endocrinology 141, 3397–3402[Abstract/Free Full Text]
  3. Polosa, R., and Holgate, S. T. (1997) Thorax 52, 919–923[Medline] [Order article via Infotrieve]
  4. Madara, J. L., Patapoff, T. W., Gillece-Castro, B., Colgan, S. P., Parkos, C. A., Delp, C., and Mrsny, R. J. (1993) J. Clin. Investig. 91, 2320–2325[Medline] [Order article via Infotrieve]
  5. Wu, S. N., Lin, Y. T., and Chen, S. S. (1992) Jpn. J. Physiol. 42, 35–47[CrossRef][Medline] [Order article via Infotrieve]
  6. Lewis, M. D., Webster, J., Ham, J., Davies, J. S., and Scanlon, M. F. (1996) J. Clin. Endocrinol. Metab. 81, 1296–1298[Abstract]
  7. Marquardt, D. L., Gruber, H. E., and Wasserman, S. I. (1984) Proc. Natl. Acad. Sci. U. S. A. 81, 6192–6196[Abstract/Free Full Text]
  8. Williams, T. C., and Jarvis, S. M. (1991) Biochem. J. 274, 27–33[Medline] [Order article via Infotrieve]
  9. Decking, U. K., Schlieper, G., Kroll, K., and Schrader, J. (1997) Circ. Res. 81, 154–164[Abstract/Free Full Text]
  10. Jarvis, G. E., Evans, R. J., and Heath, M. F. (1996) Eur. J. Pharmacol. 315, 203–212[CrossRef][Medline] [Order article via Infotrieve]
  11. Resnick, M. B., Colgan, S. P., Patapoff, T. W., Mrsny, R. J., Awtrey, C. S., Delp-Archer, C., Weller, P. F., and Madara, J. L. (1993) J. Immunol. 151, 5716–5723[Abstract]
  12. Yegutkin, G. G., and Burnstock, G. (2000) Biochim. Biophys. Acta 1466, 234–244[Medline] [Order article via Infotrieve]
  13. Kaczmarek, E., Koziak, K., Sevigny, J., Siegel, J. B., Anrather, J., Beaudoin, A. R., Bach, F. H., and Robson, S. C. (1996) J. Biol. Chem. 271, 33116–33122[Abstract/Free Full Text]
  14. Misumi, Y., Ogata, S., Hirose, S., and Ikehara, Y. (1990) J. Biol. Chem. 265, 2178–2183[Abstract/Free Full Text]
  15. Weinberger, M., and Hendeles, L. (1996) N. Engl. J. Med. 334, 1380–1388[Free Full Text]
  16. Joad, J. P. (1990) Am. J. Respir. Cell Mol. Biol. 2, 193–198[Medline] [Order article via Infotrieve]
  17. Di Virgilio, F., Chiozzi, P., Ferrari, D., Falzoni, S., Sanz, J. M., Morelli, A., Torboli, M., Bolognesi, G., and Baricordi, O. R. (2001) Blood 97, 587–600[Abstract/Free Full Text]
  18. Hollopeter, G., Jantzen, H. M., Vincent, D., Li, G., England, L., Ramakrishnan, V., Yang, R. B., Nurden, P., Nurden, A., Julius, D., and Conley, P. B. (2001) Nature 409, 202–207[CrossRef][Medline] [Order article via Infotrieve]
  19. Communi, D., Gonzalez, N. S., Detheux, M., Brezillon, S., Lannoy, V., Parmentier, M., and Boeynaems, J. M. (2001) J. Biol. Chem. 276, 41479–41485[Abstract/Free Full Text]
  20. Abbracchio, M. P., Boeynaems, J. M., Barnard, E. A., Boyer, J. L., Kennedy, C., Miras-Portugal, M. T., King, B. F., Gachet, C., Jacobson, K. A., Weisman, G. A., and Burnstock, G. (2003) Trends Pharmacol. Sci. 24, 52–55[CrossRef][Medline] [Order article via Infotrieve]
  21. Lee, D. K., Nguyen, T., Lynch, K. R., Cheng, R., Vanti, W. B., Arkhitko, O., Lewis, T., Evans, J. F., George, S. R., and O'Dowd, B. F. (2001) Gene (Amst.) 275, 83–91[CrossRef][Medline] [Order article via Infotrieve]
  22. Saito, H., Ebisawa, M., Tachimoto, H., Shichijo, M., Fukagawa, K., Matsumoto, K., Iikura, Y., Awaji, T., Tsujimoto, G., Yanagida, M., Uzumaki, H., Takahashi, G., Tsuji, K., and Nakahata, T. (1996) J. Immunol. 157, 343–350[Abstract]
  23. Yegutkin, G. G., and Burnstock, G. (1999) J. Recept. Signal Transduct. Res. 19, 437–448[Medline] [Order article via Infotrieve]
  24. Wittenberger, T., Hellebrand, S., Munck, A., Kreienkamp, H. J., Chica Schaller, H., and Hampe, W. (2002) BMC Genomics 3, 17[Medline] [Order article via Infotrieve]
  25. Schachter, J. B., Sromek, S. M., Nicholas, R. A., and Harden, T. K. (1997) Neuropharmacology 36, 1181–1187[CrossRef][Medline] [Order article via Infotrieve]
  26. Sunahara, R. K., Dessauer, C. W., and Gilman, A. G. (1996) Annu. Rev. Pharmacol. Toxicol. 36, 461–480[CrossRef][Medline] [Order article via Infotrieve]
  27. Moro, S., Hoffmann, C., and Jacobson, K. A. (1999) Biochemistry 38, 3498–3507[CrossRef][Medline] [Order article via Infotrieve]
  28. Gao, Z., Chen, T., Weber, M. J., and Linden, J. (1999) J. Biol. Chem. 274, 5972–5980[Abstract/Free Full Text]
  29. Linden, J., Thai, T., Figler, H., Jin, X., and Robeva, A. S. (1999) Mol. Pharmacol. 56, 705–713[Abstract/Free Full Text]
  30. Morin, D., Sapena, R., Tillement, J. P., and Urien, S. (2000) Pharmacol. Res. 41, 435–443[CrossRef][Medline] [Order article via Infotrieve]
  31. Merten, M. D., Saleh, A., Kammouni, W., Marchand, S., and Figarella, C. (1998) Eur. J. Biochem. 251, 19–24[Medline] [Order article via Infotrieve]
  32. van den Berge, M., Kerstjens, H. A., and Postma, D. S. (2002) Clin. Exp. Allergy 32, 824–830[CrossRef][Medline] [Order article via Infotrieve]
  33. Lennon, P. F., Taylor, C. T., Stahl, G. L., and Colgan, S. P. (1998) J. Exp. Med. 188, 1433–1443[Abstract/Free Full Text]
  34. Driver, A. G., Kukoly, C. A., Ali, S., and Mustafa, S. J. (1993) Am. Rev. Respir. Dis. 148, 91–97[Medline] [Order article via Infotrieve]
  35. Marquardt, D. L. (1997) in Asthma (Barnes, P. J., Grunstein, M. M., Leff, A. R., and Woolcock, A. J., eds) pp. 585–591, Lippincott Williams & Wilkins, Philadelphia
  36. Hannon, J. P., Tigani, B., Wolber, C., Williams, I., Mazzoni, L., Howes, C., and Fozard, J. R. (2002) Br. J. Pharmacol. 135, 685–696[CrossRef][Medline] [Order article via Infotrieve]
  37. Weaver, D. R., and Reppert, S. M. (1992) Am. J. Physiol. 263, F991–F995[Medline] [Order article via Infotrieve]
  38. Howell, R. E. (1990) J. Pharmacol. Exp. Ther. 255, 1008–1014[Abstract/Free Full Text]
  39. Ito, K., Lim, S., Caramori, G., Cosio, B., Chung, K. F., Adcock, I. M., and Barnes, P. J. (2002) Proc. Natl. Acad. Sci. U. S. A. 99, 8921–8926[Abstract/Free Full Text]
  40. Henderson, J. C., O'Connell, F., and Fuller, R. W. (1993) Thorax 48, 824–826[Abstract]
  41. Melamed, J., and Beaucher, W. N. (1995) Ann. Allergy Asthma Immunol. 75, 516–520[Medline] [Order article via Infotrieve]
  42. Hofmann, K., and Stoffel, W. (1993) TMpred, Prediction of Transmembrane Regions and Orientation, www.ch.embnet.org/software/TMPRED_form.html

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati    What's this?


This article has been cited by other articles:


Home page
 J. Immunol.Home page
D. Myrtek, T. Muller, V. Geyer, N. Derr, D. Ferrari, G. Zissel, T. Durk, S. Sorichter, W. Luttmann, M. Kuepper, et al.
Activation of Human Alveolar Macrophages via P2 Receptors: Coupling to Intracellular Ca2+ Increases and Cytokine Secretion
J. Immunol., August 1, 2008; 181(3): 2181 - 2188.
[Abstract] [Full Text] [PDF]

Home page
 Pharmacol. Rev.Home page
M. P. Abbracchio, G. Burnstock, J.-M. Boeynaems, E. A. Barnard, J. L. Boyer, C. Kennedy, G. E. Knight, M. Fumagalli, C. Gachet, K. A. Jacobson, et al.
International Union of Pharmacology LVIII: Update on the P2Y G Protein-Coupled Nucleotide Receptors: From Molecular Mechanisms and Pathophysiology to Therapy
Pharmacol. Rev., September 1, 2006; 58(3): 281 - 341.
[Abstract] [Full Text] [PDF]

Home page
 J. Am. Soc. Nephrol.Home page
B. Kadereit, P. Fustier, K. Shojaati, B. M. Frey, F. J. Frey, and M. G. Mohaupt
Extracellular ATP Determines 11{beta}-Hydroxysteroid Dehydrogenase Type 2 Activity via Purinergic Receptors
J. Am. Soc. Nephrol., December 1, 2005; 16(12): 3507 - 3516.
[Abstract] [Full Text] [PDF]

Home page
 Proc Am Thorac SocHome page
P. J. Barnes
Theophylline in Chronic Obstructive Pulmonary Disease: New Horizons
Proceedings of the ATS, November 1, 2005; 2(4): 334 - 339.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
Z. Ding, F. Tuluc, K. R. Bandivadekar, L. Zhang, J. Jin, and S. P. Kunapuli
Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling
Am J Physiol Cell Physiol, March 1, 2005; 288(3): C559 - C567.
[Abstract] [Full Text] [PDF]

Home page
 Mol. Pharmacol.Home page
S. J. Ennion, A. D. Powell, and E. P. Seward
Identification of the P2Y12 Receptor in Nucleotide Inhibition of Exocytosis from Bovine Chromaffin Cells
Mol. Pharmacol., September 1, 2004; 66(3): 601 - 611.
[Abstract] [Full Text] [PDF]

Home page
 Am. J. Physiol. Cell Physiol.Home page
D. Erlinge
Extracellular ATP: a central player in the regulation of vascular smooth muscle phenotype. Focus on "Dual role of PKA in phenotype modulation of vascular smooth muscle cells by extracellular ATP"
Am J Physiol Cell Physiol, August 1, 2004; 287(2): C260 - C262.
[Full Text] [PDF]