Molecular cloning and functional analysis of a novel P2 nucleotide receptor.

The cDNA encoding a novel P2 receptor was isolated from rat aortic smooth muscle cell library and functionally characterized. The cloned P2 receptor exhibits structural features characteristic of the G protein-coupled receptor family and shows 44 and 38% amino acid identity with previously cloned rat P2U and chicken P2Y receptors, respectively. The cloned P2 receptor is functionally coupled to phospholipase C but not to adenylate cyclase in C6 rat glioma cells transfected with the cloned P2 expression vector. The rank order of agonist potency as judged by intracellular Ca2+ mobilization responses is UTP > ADP = 2-methylthioATP > ADPβS > ATP = ATPγS, which is not compatible with any of the previously characterized P2 receptor subtypes. The nonselective P2 antagonists, suramin and reactive blue-2, inhibit nucleotide-induced phospholipase C activation in cells expressing the cloned P2 receptor. The cloned P2 receptor mRNA is abundantly expressed in various rat tissues including lung, stomach, intestine, spleen, mesentery, heart, and, most prominently, aorta. The results indicate that the novel metabotropic P2 receptor has pharmacological characteristics distinct from any of P2 receptor subtypes thus far identified and suggest the existence of a novel regulatory system by extracellular nucleotides of potential significance.

The cDNA encoding a novel P 2 receptor was isolated from rat aortic smooth muscle cell library and functionally characterized. The cloned P 2 receptor exhibits structural features characteristic of the G protein-coupled receptor family and shows 44 and 38% amino acid identity with previously cloned rat P 2U and chicken P 2Y receptors, respectively. The cloned P 2 receptor is functionally coupled to phospholipase C but not to adenylate cyclase in C6 rat glioma cells transfected with the cloned P 2 expression vector. The rank order of agonist potency as judged by intracellular Ca 2؉ mobilization responses is UTP > ADP ‫؍‬ 2-methylthioATP > ADP␤S > ATP ‫؍‬ ATP␥S, which is not compatible with any of the previously characterized P 2 receptor subtypes. The nonselective P 2 antagonists, suramin and reactive blue-2, inhibit nucleotide-induced phospholipase C activation in cells expressing the cloned P 2 receptor. The cloned P 2 receptor mRNA is abundantly expressed in various rat tissues including lung, stomach, intestine, spleen, mesentery, heart, and, most prominently, aorta. The results indicate that the novel metabotropic P 2 receptor has pharmacological characteristics distinct from any of P 2 receptor subtypes thus far identified and suggest the existence of a novel regulatory system by extracellular nucleotides of potential significance. P 2 nucleotide receptors mediate a wide variety of physiological responses to extracellular nucleotides, including vascular smooth muscle contraction and relaxation, neurotransmission, and endocrine and exocrine secretion (1,2). In the vascular system, nucleotides activate P 2 receptors on vascular smooth muscle cells to cause contraction (3). On the other hand, when nucleotides activate P 2 receptors on vascular endothelial cells, it stimulates release of the vasorelaxants, prostacyclin and NO, to cause vasorelaxation (4 -6). Nucleotide-induced vasoconstriction and relaxation were initially suggested to be mediated via two distinct subtypes of P 2 receptors, P 2X and P 2Y purinoceptors, respectively (7). However, recent studies on the agonist specificity and potency rank order have provided evidence for the existence of more than a single class of P 2 receptors in both vascular smooth muscle and endothelium. For example, the pyrimidine UTP as well as the P 2X selective agonist ␣,␤-methylene ATP evokes vasoconstriction in various vascular beds, leading to the suggestion that the third P 2 receptor that can interact with UTP, P 2U , mediates vasoconstriction, because UTP does not serve as a ligand for P 2X or P 2Y receptors (8 -12). Furthermore, activation of P 2 receptors with UTP and ␣,␤methylene ATP in smooth muscle cells was demonstrated to lead to activation of different downstream effector molecules (i.e. phospholipase C (2) and a cation channel intrinsic to P 2X receptors (13), respectively). Several studies also demonstrated that depending on vascular beds and animal species, P 2U receptors mediate nucleotide-induced endothelium-dependent relaxation (11,14,15). However, because of unavailability of a radioactive P 2 receptor ligand and an agonist and antagonist selective for each of the P 2 receptor subtypes, definitive identification of P 2 receptor subtypes that are expressed in vascular smooth muscle and endothelium has been hampered.
Very recently, the cDNAs encoding for mouse (16), human (17), and rat (18) P 2U receptors and chicken brain P 2Y receptor (19) have been isolated. It is, however, unknown whether these P 2 receptor subtypes are expressed in the vascular tissues or in fact mediate vascular responses to nucleotides. It was reported that the cloned chicken brain P 2Y receptor, when expressed in Xenopus oocytes, displays a significantly different agonist potency rank order from that for endothelium-dependent relaxation (19). Hence, it is still possible that another P 2 receptor subtype may be responsible for nucleotide-induced vascular responses. In the present report we demonstrate the isolation of a cDNA encoding a novel P 2 receptor subtype from rat aortic smooth muscle cell cDNA library, which is coupled to the Ca 2ϩ phospholipase C messenger system and shows a distinct agonist specificity from any other known P 2 receptor. This receptor is predominantly expressed in rat aorta and several other tissues.

MATERIALS AND METHODS
Cell Culture-Primary cultures of rat aortic smooth muscle (RASM) 1 cells were obtained by the explant method (20). C6 -15 rat glioma cells were donated by Dr. Hama (Department of Pharmacology, University of Tsukuba). Both cells were grown in Dulbecco's modified Eagle's minimal essential medium containing 10% (v/v) fetal bovine serum (Commonwealth Serum Laboratory, Australia), 100 g/ml streptomycin, and 100 units/ml penicillin G under the atmosphere of 95% air plus 5% CO 2 .
RNA Preparation and cDNA Library Construction-Total RNA was isolated from rat tissues and RASM cells for Northern analysis by the acid guanidine isothiocyanate/phenol/chloroform extraction method as described by Chomczynski and Sacchi (21). For cDNA synthesis, total RNA was isolated from rat kidney by the LiCl-urea method (22) and from RASM cells by the guanidine isothiocyanate/CsCl 2 procedure (23). Poly(A) ϩ RNA was purified by passing twice through an oligo(dT)cellulose (Type III, Collaborative Research) column. Double-stranded * This work was supported by grants from the Ministry of Education, Science, and Culture of Japan and the Tsumura Foundation for Cardiovascular Research and by funds from the Japan Heart Foundation and the Japan Research Foundation for Clinical Pharmacology. 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.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBank TM /EMBL Data Bank with accession number(s) D63665.
Screening of cDNA Library and DNA Sequencing-About 6 ϫ 10 5 clones from the kidney gt10 cDNA library were screened with the 45-mer degenerate oligodeoxynucleotide 5Ј-GTGCACGCTIATGCAIGT-GAGGAAIAGGATGCTGC(AC)GTAGAGGTT (I indicates inosine) corresponding to the third transmembrane domain conserved between mouse P 2U receptor and chicken P 2Y receptor that was end-labeled with [␥-32 P]ATP (DuPont NEN) and T4 polynucleotide kinase (New England Biolabs Inc.). Hybridization was performed at 42°C in 0.87 M NaCl, 20% formamide, 0.5% SDS, and 167 g/ml salmon sperm DNA. The filters were washed in 2 ϫ SSC (0.15 M NaCl, 0.015 M sodium citrate)/1% SDS at 30°C followed by autoradiography. phages found to hybridize to the probe were plaque-purified. We further screened 1.2 ϫ 10 6 clones from the RASM cDNA library under high stringency conditions with the 0.8-kbp AccI-PstI fragment of the coding region of the clone isolated from the kidney library that was labeled with [␣-32 P]dCTP (DuPont NEN) by the random priming method. We also cloned rat P 2U from the RASM cDNA library with the same 45 mer degenerate oligodeoxynucleotide as a probe. Sequence analysis of the coding region of the isolated clone revealed its identity to rat P 2U (18). The cloned cDNA inserts were subcloned into pUC118, and both strands were sequenced by the dideoxy chain termination method with T7 DNA polymerase using ALFred DNA sequencer (Pharmacia). Nucleotide and amino acid sequences were analyzed with the GENETYX program (Software Development, Japan).
Northern Blot Analysis-Poly(A) ϩ RNA or total RNA prepared from rat tissues and cells was separated by formaldehyde/1.0% agarose gel electrophoresis and transferred to a nylon membrane (Hybond N, Amersham Corp). The membranes were hybridized with the randomly labeled cDNA probe (0.8-kbp AccI-PstI fragment of the cloned P 2 cDNA or 1.0-kbp P 2U cDNA fragment encoding about the half of the second transmembrane domain and its entire remaining carboxyl-terminal side of the P 2U receptor) at 42°C in the presence of 50% (v/v) formamide. The membranes were washed in 0.1 ϫ SSC/0.1% SDS at 50°C and autoradiographed.
Transfection and Measurements of Intracellular Free Ca 2ϩ Concentration, Inositol Phosphates, and Cyclic AMP Contents-The 2.1-kbp BamHI-EcoRV fragment of the cDNA cloned from the kidney library was made blunt-ended with the Klenow fragment of Escherichia coli DNA polymerase I and adaptor-ligated into the BstXI site of the eukaryotic expression vector pME18S (24). C6 -15 cells were transfected with the plasmid DNA by the calcium phosphate precipitation method (23). For the measurement of inositol phosphates, 24 h after the transfection the cells were labeled with 1 Ci/ml of myo-[2-3 H]inositol in Dulbecco's modified Eagle's minimal essential medium supplemented with 0.2% bovine serum albumin for 24 h. The cells were stimulated with various nucleotides at 100 M for 60 min in the presence of 10 mM LiCl, and the reaction was quenched by adding 10% (v/v) perchloric acid. The fraction containing total inositol phosphates (inositol monophosphate, inositol bisphosphate, and inositol trisphosphate) was separated as described previously (25,26), and the radioactivity was quantitated by scintillation counting. For the measurement of cyclic AMP, 48 h after the transfection, cells were stimulated with various nucleotides at 100 M for 5 min in the presence of 0.2 mM 3-isobutyl-1-methylxanthine and 0.2 M forskolin. The reaction was terminated by adding HCl (a final concentration of 0.1 M), and the amount of cyclic AMP in the acid extracts was measured by radioimmunoassay using a Yamasa cyclic AMP kit (Choshi, Japan) (27).
The intracellular free Ca 2ϩ concentration ([Ca 2ϩ ] i ) was measured in trypsinized cells stably expressing the cloned receptor by using a fluorescent Ca 2ϩ indicator fura-2 (Dojin, Kumamoto, Japan). For obtaining cells that stably express the cloned receptor, C6 -15 cells were cotransfected with the receptor expression plasmid and neomycin-resistant gene expression vector pKM3 (Dr. H. Nojima, Osaka University) by the calcium phosphate precipitation method and after 48 h were selected with G418 (Life Technologies, Inc.) (600 g/ml). The fluorescence of fura-2-loaded cells was measured with a CAF-110 spectrofluorimeter (Japan Spectroscopy, Inc., Tokyo, Japan) with excitation at 340 and 380 nm and emission at 500 nm as described previously (28). The [Ca 2ϩ ] i was calculated from the measurements of the ratio of fluorescence intensities as described by Grynkiewicz et al. (29).

Cloning and Amino Acid Sequence of a Novel G Proteincoupled Receptor-
We screened a cDNA library of rat kidney with a synthetic oligonucleotide encoding a conserved region of the third transmembrane domain between mouse P 2U and chicken P 2Y receptors, and isolated a single hybridization-positive clone with an insert of 3.8 kbp. The partial sequence of this clone revealed a 984-bp open reading frame encoding a novel 328-amino acid protein. The predicted amino acid sequence was 44 and 38% (in overall) identical to that of rat P 2U receptor (18) and chicken P 2Y receptor (19), respectively, which is in the expected range for receptors within the same family. Because Northern analysis using the coding region of this clone as a probe revealed a major band of approximately 2.2 kb in mRNA from various rat tissues and a very faint band of 3.8 kb, which is observed only in mRNA from aorta (see Fig. 3), we screened a cDNA library of RASM cells with the coding region of the 3.8-kbp clone as a probe and isolated a clone with a 2.0-kbp insert. Sequence analysis of the 2.0-kbp clone revealed the 984-bp open reading frame identical to the 3.8-kbp clone and a shorter 3Ј-untranslated region than the 3.8-kbp clone.
Shown in Fig. 1 are nucleotide and deduced amino acid sequences of this clone. An in-frame initiating codon (nucleotides 440 -442 in Fig. 1) is in the context of the Kozak translation initiation consensus sequence (30) and is preceded by an in-frame stop codon. The predicted molecular mass of 36.7 kDa of this protein is substantially low among G protein-coupled receptors and close to that of A1 adenosine receptor (31) and several odorant receptors (32) so far reported to have the smallest molecular weight. Hydropathy analysis (33) of the clone reveals seven stretches of hydrophobic amino acids, predicted to represent membrane-spanning domains characteristic of the G protein-coupled receptors. The amino-terminal region preceding the putative first transmembrane domain contains a single potential asparagine-linked glycosylation site, and the third intracellular loop and cytoplasmic tail have two recognition sites (Ser-235 and Thr-320) for phosphorylation by protein kinase C (34,35). This protein also possesses a number of residues conserved in most of the G protein-coupled receptors such as Asp in the second transmembrane domain, Leu in the second and the seventh transmembrane domains, Arg-Tyr immediately behind the third transmembrane domain, and Pro in the fifth, the sixth, and the seventh transmembrane domains (36,37). A Cys in the carboxyl-terminal region conserved in many of the G protein-coupled receptors, which may be a membrane-anchoring palmitoylation site, and a Asp in the third transmembrane domain conserved in the G protein-coupled receptors for charged amines are absent in this protein (37). All these characteristics suggest that this protein represents a G protein-coupled receptor.
Shown in Fig. 2 is alignment of the amino acid sequences of this protein and two previously cloned P 2 receptors, rat P 2U and chicken P 2Y . 25% of the residues are conserved among these three proteins. The observed similarities are mostly confined to the region between the first and the seventh transmembrane domains, whereas the amino acid sequences and the length of the amino and carboxyl termini are variant. The next most similar proteins found from a data base search are rat G10D orphan receptor (29% identity in a 294-amino acid overlap), rat thrombin receptor (28% identity in a 294-amino acid overlap), and rat type 1b angiotensin II receptor (27% identity in a 300-amino acid overlap).
Tissue Distribution of the mRNA of the Cloned Receptor-In order to assess the tissue expression of the cloned receptor gene and the size of the transcript, poly(A) ϩ RNA isolated from various rat tissues was subjected to Northern blot analysis (Fig. 3). A major 2.2-kb transcript is most abundant in the aorta, where a faint 3.8-kb transcript is also observed. The 2.2-kb transcript is abundantly expressed in the lung, stomach, intestine, spleen, and mesentery and is present in smaller amounts in the heart and kidney. It is barely detected in the brain. The 2.2-kb transcript is also readily detected in total RNA extracts from primary cultures of RASM cells. The distribution of the cloned receptor is clearly different from that of the P 2U receptor, which is expressed predominantly in the skeletal muscle, kidney, liver, and heart, and of the P 2Y , which is expressed in the brain, digestive tract, and skeletal muscle.
Because previous pharmacological studies demonstrated in vascular tissues the expression of P 2 receptors that respond to UTP (8 -12), we compared the expression of P 2U receptor and the cloned new P 2 receptor in the large capacitance vessel aorta, the mesentery, which includes lots of resistance vessels of smaller calibers, and cultured RASM cells. The 2.8-kb P 2U transcript is abundantly expressed in the aorta like the case of the cloned P 2 receptor (Fig. 4). The P 2U transcript is also readily detected in the mesentery and RASM cells. The signal intensities of both P 2U and cloned new P 2 receptors transcripts in each tissue and cell appear to be roughly similar (compare Figs. 3 and 4). However, strict quantitative comparison of expression levels of P 2U and cloned P 2 receptor transcripts in Figs. 3 and 4 may not be possible, although the lengths and specific radioactivities of employed cDNA probes and exposure times of membranes to films in the Northern analyses were similar.
Functional Properties of the Cloned Receptor-Functional characterization of P 2 receptors has not been easy, because many of the cells often used for transfection assay, such as COS7, CHO, and Ltk cells, express endogenous P 2 receptors, and specific radioactive ligands for P 2 receptors have not been available. For transfection we employed a subline of C6 rat glioma cells (C6 -15) that we found not to express endogenous P 2 receptors at a significant level as evaluated with the measurement of intracellular Ca 2ϩ mobilization response to nucleotides. As shown in Fig. 5A (left), in the presence of 1.25 mM extracellular Ca 2ϩ the addition of ADP (300 M) induces a rapid rise in [Ca 2ϩ ] i , which peaks within 20 s and then gradually declines to the second plateau in transfected C6 -15 cells. Addition of Co 2ϩ , a Ca 2ϩ channel blocker, causes complete inhibition of the plateau phase of the [Ca 2ϩ ] i response. In contrast, in the absence of extracellular Ca 2ϩ ADP still elicits an initial transient increase in the [Ca 2ϩ ] i , although the amplitude of the peak [Ca 2ϩ ] i is smaller than in the presence of extracellular Ca 2ϩ but not the second plateau of the [Ca 2ϩ ] i response (Fig. 5A, right). The profile of the [Ca 2ϩ ] i response is similar between ADP and other effective nucleotides, although the amplitude of the [Ca 2ϩ ] i response is varied (see below). These results indicate that the cloned receptor functions as a P 2 receptor for nucleotides, which is coupled to mobilization of Ca 2ϩ from both intra-and extracellular pools.
To characterize the agonist specificity of the cloned P 2 receptor, C6 -15 cells expressing the cloned P 2 receptor were stimulated with various concentrations of nucleotides, and the [Ca 2ϩ ] i response was monitored. As shown in Fig. 5B, UTP is the most potent with the EC 50 value of approximately 1 M, which is similar to the reported EC 50 values for UTP in mouse and human P 2U receptors. Unlike the P 2U receptor, however, ATP (EC 50 ϭ 500 M) is more than two orders less potent than UTP, and ADP and 2-methylthioATP (EC 50 ϭ 50 M for both nucleotides) are more potent for the cloned P 2 receptor than ATP. AMP and adenosine are both totally ineffective (data not shown). The P 2X -selective agonist ␣,␤-methylene ATP is also ineffective. Thus, the agonist specificity of the cloned new P 2 receptor does not exactly fit with any of previously cloned P 2 receptors.
To explore whether nucleotide-induced Ca 2ϩ mobilization is linked to phospholipase C activation in C6 -15 cells expressing the cloned P 2 receptor, the production of inositol phosphates was measured in [ 3 H]inositol-prelabeled cells. As shown in Fig.  6, in cells transfected with the cloned P 2 receptor-expression vector, the addition of 100 M ADP stimulates the production of total inositol phosphates about 4.3-fold. In contrast, ADP is without effect on cells transfected with vector alone. The results indicate that the cloned P 2 receptor is coupled to phospholipase C. To examine whether the receptor is coupled to phospholipase C via a pertussis toxin-sensitive G protein, we studied the effect of pertussis toxin pretreatment on ADPinduced inositol phosphate production. Pretreatment of pertussis toxin (10 ng/ml) for 24 h does not affect ADP (100 M)induced inositol phosphate production at all (Table I). Thus, the cloned P 2 receptor appears to be coupled to phospholipase C via a pertussis toxin-insensitive G protein.
To examine potential coupling of the cloned P 2 receptor to adenylate cyclase, the effects of nucleotides on cellular cyclic AMP contents were examined in C6 -15 cells transfected with vector alone or the cloned P 2 receptor-expression vector (Table  II). Forskolin increases cyclic AMP contents 3.5-4.2-fold in cells transfected with vector alone or the cloned P 2 -expression vector. Either ADP, ATP, or UTP at 100 M does not change cyclic AMP contents in forskolin-stimulated cells transfected with either construct. The results indicate that the cloned P 2 receptor is not coupled positively or negatively to adenylate cyclase in C6 -15 cells.
The results of the nucleotide selectivity of the cloned P 2 receptor described above in the present study indicate that the pharmacological feature of the cloned P 2 receptor is distinct from that of either mouse and human P 2U receptor or chicken P 2Y receptor. We further tried to characterize the pharmacological property of the cloned P 2 receptor by examining the susceptibility of the cloned P 2 receptor to known P 2 receptor antagonists (38,39). As shown in Fig. 7, suramin (100 M), an antagonist for P 2X , P 2Y , and P 2T receptors, slightly (20%) but significantly inhibited ADP-induced inositol phosphate production in cells expressing the cloned P 2 receptor. Reactive blue-2 (100 M), an antagonist for P 2X and P 2Y receptors, more strongly (77%) inhibited ADP-induced inositol phosphate production. Thus, the cloned P 2 receptor is sensitive to known P 2 receptor antagonists. FIG. 2. Alignment of the amino acid sequence of the cloned P 2 receptor with those of rat P 2U receptor and chicken P 2Y receptor. The approximate positions of the transmembrane domains are indicated by boxes, and conserved residues are shaded. Gaps (Ϫ) are introduced so as to maximize the alignment. Rat P 2U and chicken P 2Y were recommended to be renamed as P 2Y2 and P 2Y1 , respectively, by Bernard et al. (42).

FIG. 3. Northern blot analysis of mRNA isolated from rat tissues and cells with the cloned P 2 receptor cDNA as a probe.
Poly(A) ϩ RNA (5 g each except aorta where 10 g was loaded) from rat tissues and total RNA (20 g) from cultured RASM cells were separated by formaldehyde/1.0% agarose gel electrophoresis. Hybridization was performed as described under "Materials and Methods," followed by autoradiography.
FIG. 4. Northern blot analysis of mRNA isolated from rat aorta and mesentery and rat aortic smooth muscle cells with P 2U receptor cDNA as a probe. Poly(A) ϩ RNA (10 g) from rat tissues and total RNA (20 g) from RASM cells were separated by formaldehyde/1.0% agarose gel electrophoresis. Hybridization was performed as described under "Materials and Methods," followed by autoradiography. DISCUSSION Based on their pharmacological properties and signaling mechanisms, mammalian P 2 receptors have been classified into five subtypes, P 2X , P 2Y , P 2Z , P 2T , and P 2U (1, 2). Among them, P 2T subtype is expressed in very limited cell types including platelets (1,2). P 2X and P 2Z have the properties of ligand-gated ion channels. P 2X receptor cDNAs have recently been cloned (40,41) and found to have only two transmembrane domains and a pore-forming motif. On the other hand, P 2Y and P 2U are a metabotropic type of P 2 receptors and widely expressed in various tissues. Recent molecular cloning of mouse, human, and rat P 2U and chicken P 2Y receptors revealed that they belong to G protein-coupled receptors and are linked to intracellular Ca 2ϩ mobilization (16 -19). In the present study we demonstrate the isolation of a cDNA clone encoding a novel member of P 2 receptors. Evaluation of functional properties of the new receptor has revealed that it is not categorized into any of the classical P 2 receptor subtypes mentioned above.
Sequence analysis of the cloned P 2 receptor demonstrates that the receptor possesses structural properties characteristic of the G protein-coupled receptor superfamily and has a con-   (Fig. 2). Functional analysis of the cloned P 2 receptor shows that this receptor is coupled to phospholipase C and Ca 2ϩ mobilization (Fig. 5A), like P 2U and P 2Y receptors. However, the agonist selectivity of the cloned P 2 receptor clearly differs from that of P 2U and P 2Y receptors; the rank order of agonist potency for the cloned P 2 receptor is UTP Ͼ 2-methylthioATP ϭ ADP Ͼ ADP␤S Ͼ ATP ϭ ATP␥S (Fig.  5B), whereas for the mouse P 2U receptor UTP ϭ ATP Ͼ ATP␥S Ͼ Ͼ 2-methylthioATP ϭ ADP, and for the chicken P 2Y receptor 2-methylthioATP Ͼ ATP Ͼ ADP Ͼ Ͼ UTP. Thus, the cloned P 2 receptor resembles the P 2U receptor in that it can respond to the pyrimidine UTP with a relatively high sensitivity (Fig. 5B). However, the cloned P 2 receptor also share some properties with classical P 2Y receptors in that the cloned P 2 and classical P 2Y receptors respond to ADP and 2-methylthioATP with a higher sensitivity than the P 2U receptor and that responses mediated by both cloned P 2 and classical P 2Y receptors are inhibited by the receptor antagonist, reactive blue-2 (37) (Fig.  7). All these results indicate that the cloned P 2 receptor is a novel metabotropic P 2 receptor, which, together with the recently cloned P 2U and P 2Y receptors, constitutes a distinct family of G protein-coupled P 2 receptors. Barnard et al. (42) and Fredholm et al. (31) in their recent reviews have recommended reclassifying cloned P 2 receptors into the two major families, the G protein-coupled receptor P 2Y and the intrinsic ion channel type receptor P 2X . They proposed terming newly discovered G protein-coupled P 2 receptors as P 2Y1 , P 2Y2 , P 2Y3 , etc., by consecutively numbering them. They designated the chicken brain P 2Y and its rat equivalent as P 2Y1 , mouse and human P 2U as P 2Y2 , and the third G protein-coupled P 2 receptor with a strong preference for ADP that they have recently cloned but not published yet as P 2Y3 (42). Our P 2 receptor clearly belongs to the G protein-coupled P 2 receptor family and should in future be named in a systematic way as proposed (31,42).
Detection of a strong signal of our cloned P 2 receptor transcript in rat aorta (Fig. 3) implicates its physiological role in vascular function. Because the cloned P 2 receptor transcript is detected in cultured rat aortic smooth muscle cells, the cloned P 2 receptor is likely expressed in the smooth muscle layer of the vascular wall, where it may be involved in the regulation of vascular tone. The cloned P 2 receptor transcript is also detected in the mesentery, indicating the expression of the cloned P 2 receptor in both large capacitance vessels and smaller resistance vessels (Fig. 3). Because activation of the cloned P 2 receptor stimulates phospholipase C to induce an increase in the [Ca 2ϩ ] i (Figs. 5 and 6), the cloned P 2 receptor on vascular smooth muscle cells most likely mediates vascular smooth muscle contractile response. In addition, it is an interesting possibility that the cloned P 2 receptor on vascular smooth muscle cells may also be involved in promoting vascular smooth muscle cell growth under physiological or pathological conditions, because it was shown that nucleotides induce stimulation of DNA synthesis in cultured rat vascular smooth muscle cells (43,44). The moderate levels of the cloned P 2 receptor transcript are detected in smooth muscle organs, including stomach and intestine, suggesting that the cloned P 2 receptor is involved in the functional regulation of nonvascular smooth muscle as well.
A number of recent reports demonstrated that the pyrimidine UTP causes potent vasoconstriction in a variety of vascular beds (8 -11). The present study may suggest that the cloned P 2 receptor mediates UTP-induced vasoconstriction. We found that the P 2U transcript is also detected in rat aorta, mesentery, and cultured RASM cells (Fig. 4). Therefore, both our novel P 2 and P 2U receptors may be involved in the reported UTP-induced vascular contraction. A previous study (45) demonstrated that the rank order of potency for nucleotide-induced Ca 2ϩ mobilization in RASM cells is more consistent with that of P 2U receptors (16,17). This may suggest that P 2U rather than our cloned P 2 receptor functionally predominates in RASM cells. However, it is possible that yet another P 2Y subtype different from our cloned P 2 or P 2U (P 2Y2 ) are also involved in nucleotide-induced vasoconstriction, because some studies suggested the involvement of a P 2Y subtype with pharmacological properties distinct from either our P 2 or P 2U in nucleotideinduced vascular contraction (10,12). Identification of appropriate radioactive ligands and selective antagonists for each P 2 receptor subtype will undoubtedly greatly help to promote understanding the physiology of P 2 receptors.