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P2 Receptor Expression Profiles in Human Vascular Smooth Muscle and Endothelial Cells

Wang, Lingwei; Karlsson, Lena*; Moses, Sara; Hultgårdh-Nilsson, Anna; Andersson, Maria*; Borna, Catharina; Gudbjartsson, Tomas; Jern, Sverker*; Erlinge, David

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Journal of Cardiovascular Pharmacology: December 2002 - Volume 40 - Issue 6 - p 841-853
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Abstract

P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP and regulate several physiologic responses including cardiac function, vascular tone, smooth muscle cell proliferation, platelet aggregation, and release of endothelial factors (1–3). The P2 receptors can be divided into two classes on the basis of their signal transmission mechanisms and their characteristic molecular structures: ligand-gated intrinsic ion channels named P2X receptors and G-protein-coupled P2Y receptor subtypes. So far, the P2Y family is composed of six cloned and functionally defined subtypes (P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2Y12) (1,4). The P2X family is composed of seven cloned subtypes (P2X1-P2X7) (5,6).

In arteries, extracellular nucleotides cause vasoconstriction and increased blood pressure by activation of both P2X and P2Y receptors on smooth muscle cells (7). Furthermore, mitogenic effects on vascular smooth muscle cells (SMC) have been shown, which are mediated by several P2Y receptor subtypes (8). Most recently, UDP was shown to be a growth factor for vascular SMC by activation of P2Y6 receptors (9).

Extracellular nucleotides have several important effects mediated by activation of endothelial cells (EC): vasodilation and decreased blood pressure by release of prostaglandins, nitric oxide (NO) (10), and endothelium-derived hyperpolarizing factor, EDHF (11,12). This was recently demonstrated in humans since both UTP and ATP reduced forearm vascular resistance in a prostaglandin- and NO-independent way (13). Physiologic concentrations of UTP and ATP also caused a pronounced release of tissue-type plasminogen activator (t-PA), indicating a role as activators of the fibrinolytic system (13). The similar effect of UTP and ATP strongly suggests involvement of P2Y receptors, but further subclassification is not possible with the pharmacologic agents available currently.

In both the endothelium and vascular smooth muscle, P2 receptor characterization has been hampered by the lack of selective antagonists. Some progress has been made by using stable, selective agonists (14–17), but many questions remain. Previously, we measured mRNA expression in rat SMC, but accumulating evidence suggesting physiologic and clinical importance of the P2 receptors in cardiovascular disease calls for a better characterization in human tissue. We therefore decided to set up methods for the expression of human P2 receptors on the mRNA and protein levels. Using real-time PCR, we established a quantitative mRNA assay. The protein expression was studied using Western blotting with recently developed antibodies.

The aim of this study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and EC, and to compare the different expression profiles of P2 receptor in these tissues.

METHODS

The studies were approved by the local Ethics Committee of the Lund University and Göteborg University, and were conducted according to the principles of the Declaration of Helsinki.

Tissue preparation

Left internal mammary arteries with connecting tissue were removed from patients during coronary artery bypass grafting (CABG), immersed in cold oxygenated buffer solution (NaCl 119 m M, NaHCO3 15 m M, KCl 4.6 m M, CaCl2 × 2H2O 1.5 m M, NaH2PO4 × H2O 1.2 m M, MgCl2 × 6H2O 1.2 m M, glucose 5.6 m M), and transferred immediately to the laboratory. Vessels were dissected free of adhering tissue, stripped from the adventitia and outer media. The endothelial layer was removed by gentle rubbing of the intimal surface with a wooden stick under sterile conditions. The media were cleansed with cold oxygenated buffer solution and snap frozen in liquid nitrogen. Then the samples were frozen at −70°C for RNA and protein extraction.

Endothelial cells were obtained from human umbilical veins by collagenase treatment. In short, umbilical cords were obtained immediately after vaginal delivery (Department of Obstetrics, Sahlgrenska University Hospital/Östra, Göteborg, Sweden). After the vessel was rinsed with PBS, endothelial cells were isolated by incubation for 12 min at 37°C with 0.1% collagenase (type I). The cell pellet was washed and centrifuged for RNA extraction or for endothelial cells culture.

Smooth muscle cells were identified by immunofluorescence staining of α-actin filaments using a monoclonal antibody (mouse IgG) against α-actin and a second antimouse antibody labeled with Cy™3 (Jackson ImmunoResearch, West Grove, PA, U.S.A.). Endothelial cells were identified by immunofluorescence staining of von Willebrand factor using an antibody (rabbit IgG, DAKO) against von Willebrand factor and a second antirabbit antibody labeled with Cy™2 (Jackson ImmunoResearch).

Cell culture

The endothelial cells pellet was seeded in 25-cm2 culture flasks precoated with 2% gelatin. Endothelial cells were maintained and subcultured in complete culture medium containing M199 (with glutamax-1), 20% FBS (Life Technologies, Invitrogen Corp., Carlsbad, CA, U.S.A.), penicillin 100 U/ml, streptomycin 100 μg/ml, heparin 2.5 U/ml, and ECGF diluted according to the manufacturer's recommendation. Culture flasks were incubated at 37°C in an atmosphere of 5% CO2 in air. After initial cell attachment and spreading (24 hours), medium was exchanged and cells refed with fresh complete medium. Routinely, cultures were fed three times per week and subcultured by detachment with trypsin/EDTA at confluence. At harvest, cells were detached by using trypsin/EDTA, washed once with PBS, pelleted, snap frozen in liquid nitrogen, and stored in −70°C until RNA and protein extraction.

RNA and protein extraction

Total cellular RNA and protein were extracted using TRIzol reagent (Gibco BRL, Invitrogen Corp.), according to the supplier's instructions. The resulting RNA pellets were cleaned up according to RNeasy Mini Cleanup Protocol (Qiagen, Hilden, Germany). Ribonucleic acid was dissolved in diethylpyrocarbonate (DEPC)-treated water. The RNA concentration was determined spectrophotometrically, with a ratio of optical density (OD) 260:280 exceeding 1.6 considered pure. Ribonucleic acid samples were stored at −70°C until used. The protein pellet was finally washed with 100% ethanol, vacuum dried, and resolved in 100 to 200 μl of 1% sodium dodecyl sulfate solution. The DC Protein Assay (Bio-Rad Laboratories AB, Hercules, CA, U.S.A.) was used to detect the protein concentration. Protein samples were stored at −20°C until used.

Reverse transcription

Reverse transcription of 1-μg total RNA was carried out in a 20-μl total volume of reaction mixture consisting of 5 m M MgCl2, 1 m M of each dNTP, 1 × PCR buffer, 2.5 of μM random hexamer, 1 U/μl of RNA-guard, and 2.5 U/μl of MuLV reverse transcription. Samples were incubated at 20°C for 10 min, at 42°C for 30 min, at 99°C for 5 min, and finally at 5°C for 5 min.

Design of primers and probes

Oligonucleotide primers and TaqMan probes were designed using the Primer Express software (Perkin-Elmer Applied Biosystems, Foster City, CA, U.S.A.), based on sequences from the GenBank database (Table 1). Each primer pair was selected so that the amplicon spanned an exon junction, if present, to avoid amplification of genomic DNA. Constitutively expressed GAPDH was selected as an endogenous control to correct for potential variation in RNA loading.

TABLE 1
TABLE 1:
Oligonucleotide primers and probes used for real-time quantitative polymerase chain reaction

Real-time PCR

Relative quantification of mRNA was performed on an ABI PRISM 7700 Sequence Detector (Perkin-Elmer Applied Biosystems; Applied Biosystems), as described previously (18). Briefly, the assay uses the 5″ nuclease activity of Taq polymerase to cleave a reporter dye from a nonextendable hybridization probe during the extension phase of the PCR. The fluorogenic probe is labeled with a reporter dye (FAM; 6-carboxy-fluorescein; emission maximum, 518 nm) at its 5″ end and a quencher dye (TAMRA; 6-carbxy-tetramethyl-rhodamine; emission maximum, 582 nm) at its 3″ end via a linker arm nucleotide (LAN). When the probe is intact, reporter dye emission is quenched due to the physical proximity of the reporter and quencher dyes. During the extension phase, the reporter dye is released, and the increase in dye emission is monitored in real time.

The threshold cycle (CT) is defined as the fractional cycle number at which the reporter fluorescence reaches a certain level (set to 10 times the SD of the baseline). As shown by Higuchi et al. (19), there is a linear relationship between CT and the log of the initial input amount. For amplicons designed and optimized according to the manufacturer's guidelines, amplification efficiency is typically close to 1. That is, product accumulation increases twofold until the plateau phase is reached.

Polymerase chain reaction was carried out in a 50-μl reaction mixture that contained 1× TaqMan Universal PCR Master Mix (Applied Biosystems), 15 pmol of both forward and reverse primers, 5 pmol of probe, and 1 μl of the cDNA templates. Thermal cycling conditions included the following steps. After 2 min at 52°C to activate ampUNG, the reaction mixture was preheated for 10 min at 95°C to activate Taq polymerase. Then, a 50-cycle two-step PCR was performed, consisting of 15 s at 95°C and 1 min at 60°C. Samples were amplified simultaneously in triplicate in a one-assay run.

Methodological validation

Serial cDNA template dilutions from 1:1 to 1:32 were performed separately for target genes P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4, and P2X7, and for endogenous control gene GAPDH (Fig. 1). This analysis showed that amplification efficiencies were almost identical for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4, P2X7, and GAPDH, as shown graphically in Figure 2. The slope for each line in this figure is less than 0.1, as recommended by the manufacturer. This confirms that the comparative CT method can be used for the relative quantification of target without running standard curves on the same plate (Perkin-Elmer Applied Biosystems; User Bulletin No. 2, December 1997). According to this method, the target gene normalized to GAPDH is expressed as ΔCT (CT of target gene minus CT of GAPDH). P2Y4 was arbitrarily chosen to be the calibrator in the comparative analysis and expressed as ΔCTP2Y4 (CT of target minus CT of GAPDH for P2Y4). According to Bulletin No. 2, the normalized calibrated value is given by the equation 2-ΔΔCT, in which ΔΔCT is ΔCT-ΔCTP2Y4. To further verify the specificity of PCR assays, the PCR was performed with non–reverse-transcribed total cellular RNA and samples lacking the DNA template. No significant amplifications were obtained in any of these samples (data not shown).

FIG. 1.
FIG. 1.:
Validation of amplification efficiency for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4, P2X7, and GAPDH. This diagram shows similar amplification efficiencies (slope) for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4, P2X7, and GAPDH, as demonstrated by the linear relationship between the PCR threshold cycles (CT value) of the target and the endogenous control plotted against the relative amount of loaded cDNA in serial dilutions from 1:1 through 1:32.
FIG. 2.
FIG. 2.:
Relative efficiency plot for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4, P2X7, and GAPDH. A validation experiment demonstrates that the efficiencies of the target and reference genes are approximately equal. All the absolute values of the slopes of log dilution versus ΔCT are smaller than 0.1.

Antibodies

Anti-P2X1 antibody (Alomone Labs, Jerusalem, Israel) is a polyclonal antibody raised in rabbit against highly purified peptide (CS) DPVATSSTLGLQENMRTS, corresponding to residues 382–399 of rat P2X1, with additional N-terminal cysteine and serine, which has been previously validated in human tissue (20). The antibody was affinity purified on immobilized P2X1. The epitope is specific for P2X1 and is not present in any other known protein. The human P2X1 has 15/15 identical residues.

Anti-P2Y1 antibody (Alomone Labs) is an antibody raised in rabbit against highly purified peptide (C) RALIYKDLDNSPLRRKS, corresponding to residues 242–258 of human P2Y1. The antibody was affinity purified on immobilized P2Y1, and has been used previously to detect the P2Y1 receptors in human platelets in Western blotting (Alomone Labs, see product certificate).

Anti-P2Y2 antibody (Alomone Labs) is an antibody raised in rabbit against highly purified peptide (C) KPAYGTTGLPRAKRKSVR, corresponding to residues 227–244 of rat P2Y2. Human P2Y2 has 17/19 identical residues.

Anti-P2Y4 and anti-P2Y6 antibodies (Molecular Pharmacology, GlaxoSmithKline, Glaxo Wellcome UK, Middlesex, U.K.) were raised in rabbit against the N-terminus of the receptor. Anti-P2Y11 antibody was raised in rabbit against the C-terminus of the receptor. For negative control, control antigen peptides supplied together with the antibodies were used.

SDS–PAGE and Western blotting

SDS-PAGE and Western blotting were performed to verify the specificity of the antibodies. Protein electrophoresis was performed on 10% Tri-HCl polyacrylamide ready gels (Bio-Rad Laboratories) in running buffer (25 m M Tris, 192 m M glycine, and 0.1% (w/v) SDS, pH 8.3) at a constant voltage of 75 V for 90 min and 200 V for 15 min, according to the manual of Mini-PROTEAN 3 Cell Kit (Bio-Rad Laboratories). Protein loadings were 15 μg in each well, and were diluted with 4x SDS-reducing sample buffer. Proteins were electrophoretically transferred to Hybond-C extranitrocellulose membranes (0.45 μg; Amersham Pharmacia Biotech, Uppsala, Sweden) at 300 mA, 100 V for 90 min in transfer buffer containing 25 m M of Tris-HCl, 192 of m M glycine, and 20% methanol, according to the instruction manual for the Mini Trans-Blot Electrophoretic Transfer Cell Kit (Bio-Rad Laboratories).

After transfer, the membrane was blocked for 1 h in TBS containing 0.1% Tween 20 + 5% dried skimmed milk. The membranes were incubated with rabbit antihuman P2Y1 (diluted 1:200), P2Y2 (diluted 1:400), P2Y4 (diluted 1:400), P2Y6 (diluted 1:250), P2Y11 (diluted 1:500), P2X1 (diluted 1:200), and negative control (peptide antigen preincubated with the same amount of antibody for 1 h at room temperature) overnight. At the end of the incubation, the membrane was washed once for 15 min and twice for 5 min with fresh changes of washing buffer (0.1% Tween 20 in TBS) at room temperature. Thereafter, the membranes were incubated with a secondary antibody (antirabbit Ig, horseradish peroxidase [HRP]-linked, diluted 1:1500) for 60 min, then washed again. Proteins were visualized by chemiluminescence using the ECL Western blotting RPN 2108 system (Amersham Pharmacia Biotech), and the signals were detected by autoradiography with Hyperfilm ECL (Amersham Pharmacia Biotech).

Drugs

Unless otherwise stated, all reagents and drugs were purchased from Sigma (St. Louis, MO, U.S.A.). Polymerase chain reaction consumables were purchased from Life Technologies or Perkin-Elmer Applied Biosystems. Some Western blotting reagents were from Amersham Pharmacia Biotech, United Kingdom or Bio-Rad Laboratories.

Statistical methods

Data are expressed as mean and standard error of the mean (SEM) unless otherwise stated. The number of vessels (from different subjects) tested are indicated by n. Statistical analysis of the normalized CT values (ΔCT) was performed with a one-way ANOVA, followed by a multiple comparison posttest (Tukey test) using GraphPad InStat, Version 3.00 (GraphPad Software, San Diego, CA, U.S.A.). Significant differences were considered at p value < 0.05 (two-tailed test).

RESULTS

P2 receptor mRNA quantification in SMC

In SMC, all the target genes P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4, and P2X7 could be detected (n = 5). To illustrate the expression of the P2 receptors relative to each other, the P2Y4 receptor was used as the calibrator for the others. That is, the other receptors were expressed as a ratio of the P2Y4. The P2Y4 receptor was used as the calibrator since it had the lowest expression in both cultured and precultured EC and the second lowest expression in SMC. Among the P2Y receptor subtypes, the P2Y2 had the highest expression, followed by the P2Y11 and P2Y6 receptors (Fig. 3a). The P2Y2 receptor had significantly higher expression than P2Y1 (p < 0.05). The lowest expressed P2Y receptor in SMC was P2Y1 (Fig. 3a).

FIG. 3.
FIG. 3.:
Bar graphs show expression of P2Y receptors normalized to GAPDH in smooth muscle cells (SMC) (A), cultured endothelial cells (EC) (B), and precultured endothelial cells (PEC) (C). P2Y4 was chosen to be the calibrator.

The most abundant P2 receptor in SMC was the P2X1 receptor. As shown in Figure 4a, the expression of P2X1 was 16.2 times higher than that of P2Y4. The expression of P2X1 was significantly higher than the expression of P2X4 (p < 0.01) and P2X7 (p < 0.001).

FIG. 4.
FIG. 4.:
Bar graphs show expression of P2X receptors normalized to GAPDH in smooth muscle cells (SMC) (A), cultured endothelial cells (EC) (B), and precultured endothelial cells (PEC) (C). P2Y4 was chosen to be the calibrator.

P2 receptor mRNA quantification in EC

In cultured endothelial cells, the expression of P2X1 was barely detectable at all in one half of the samples (i.e., did not reach the threshold for a CT value in 50 cycles' reaction). In the remaining half, CT values were almost 40, which means only one or two copies in the reaction wells. When the ΔCT value of the P2X1 receptor in endothelial cells was compared with that in SMC, the expression in SMC cells was found to be 1 million times higher.

Again, the P2Y4 expression was used as the calibrator for the others. That is, the other receptors were expressed as a ratio of the P2Y4. Among the P2Y receptors, the P2Y11 had the highest expression, followed by P2Y1 and P2Y2 (Fig. 3b). As shown in Figure 4b (n = 5), P2X4 was by far the highest expressed P2 receptor in cultured endothelial cells.

Since culture of cells in vitro might change the receptor expression, we also examined freshly harvested EC (precultured, n = 4)(Figs. 3c and 4c). For the P2Y receptors, the results were similar to those for the cultured EC. However, a minor difference was seen for the P2X receptors, where the P2X1 receptor was detectable, possibly due to contamination of SMC to the freshly harvested EC.

P2X4 was the highest expressed receptor in endothelial cells. Among the P2Y receptors, the expression of P2Y11 was higher than the expression of P2Y4 (p < 0.001) and P2Y6 (p < 0.001). P2Y1 and P2Y2 were higher than P2Y4 (p<0.001) and P2Y6 (p<0.001).

Western blotting for P2 receptors

All the different receptor Western blotting experiments were performed on at least three different individuals with nearly identical results.

The distribution of the P2X1 receptors in smooth muscle cells and endothelial cells is shown in Western blotting in Figure 5. For smooth muscle cells, there were three bands (260, 180, and 55 kd) in the P2X1 membrane, and two bands (260 and 180 kd) in the peptide control membrane (anti-P2X1 antibody preincubated with the same amount of control peptide antigen). This indicated that the 55-kd band represents the P2X1 receptor. For endothelial cells, no band could be found in either the P2X1 membrane or the peptide control membrane.

FIG. 5.
FIG. 5.:
Western blotting of P2X1 subtype in smooth muscle cells and endothelial cells. For smooth muscle cells (SMC), there were three bands (260, 180, and 55 kd) in the anti-P2X1 membrane and two bands (260 kd and 180 kd) in the peptide control membrane (anti-P2X1 antibody preincubated with the control peptide antigen). This indicated that the 55-kd band is P2X1. For the endothelial cells (EC), no band could be found in either the P2X1 membrane or the negative control membrane. Data are representative of four independent experiments.

There were three P2Y1 bands (45, 90, and 180 kd) in smooth muscle cells and endothelial cells, with an additional possible band approximately 250 kd (Fig. 6). The band approximately 90 kd in EC was denser than that in SMC, and the band approximately 45 kd was found only in SMC. In the peptide control membrane (anti-P2Y1 antibody preincubated with control peptide antigen), no band was found, indicating that all the bands represent the P2Y1 receptor. The predicted molecular mass of the P2Y1 receptor has been reported as 42 kd (21). We tried to separate possible protein associations in the molecule by treating samples with Triton X-100 (0.1%), EDTA (4 m M), and DTT (10 m M), or by prolonging incubation time (30 min at 95°C), in addition to β-mercaptoethanol (5%), but neither of these protocols changed the band size (data not shown).

FIG. 6.
FIG. 6.:
Western blotting of P2Y1 subtype in smooth muscle cells and endothelial cells. In the anti-P2Y1 membrane, there were three bands (45, 90, and 180 kd) in smooth muscle cells (SMC) and two bands (90 kd and 250 kd) in endothelial cells (EC). It showed that the band approximately 90 kd in EC was somewhat denser than that in SMC, and that the 45kd band appeared only in SMC. In the peptide control membrane (anti-P2Y1 antibody preincubated with the same amount of control peptide antigen), no band was found. Data are representative of five independent experiments.

Another possible explanation for increased molecular mass is glycosylation of the receptor in its extracellular domain. To remove N-linked glycosylations during denaturing conditions, N-glycosidase F deglycosylation kit (Roche Diagnostics GmbH, Roche Applied Science, Mannheim, Germany) was used according to the supplier's instructions. We found a decrease in the 180-kd band of approximately 150 kd after treatment with the deglycosylation kit (data not shown).

A difference in band size was noted for P2Y2 (Fig. 7). There was a band approximately 33 kd in smooth muscle cells and bands approximately 36 kd and 50 kd in endothelial cells. No band was found in peptide control membrane, which indicates that all the bands represent the P2Y2 receptor.

FIG. 7.
FIG. 7.:
Western blotting of P2Y2 subtype in smooth muscle cells and endothelial cells. There was a band approximately 33 kd in smooth muscle cells (SMC) and bands approximately 36 kd and 50 kd in endothelial cells (EC). In the peptide control membrane (anti-P2Y2 antibody preincubated with the same amount of control peptide antigen), only a very weak band was found. Data are representative of three independent experiments.

The results for the P2Y4 receptor were similar to those for the P2Y1 receptor (Fig. 8). There was a band approximately 33 kd in smooth muscle cells and a band approximately 100 kd in endothelial cells. No band was found in peptide control membrane, which indicates that all the bands represent the P2Y4 receptor.

FIG. 8.
FIG. 8.:
Western blotting of P2Y4 subtype in smooth muscle cells and endothelial cells. There was a band approximately 33 kd in smooth muscle cells (SMC) and a band approximately 100 kd in endothelial cells (EC). In the peptide control membrane (anti-P2Y4 antibody preincubated with the same amount of control peptide antigen), no band was found. Data are representative of three independent experiments.

The P2Y6 receptor was found in both SMC and endothelial cells with a band of 45 kd. A band of higher molecular weight was found in SMC at 68 kd and in endothelial cells at 75 kd (Fig. 9). No band was found in peptide control membrane, which indicates that all the bands represent the P2Y6 receptor.

FIG. 9.
FIG. 9.:
Western blotting of P2Y6 subtype in smooth muscle cells and endothelial cells. There were bands approximately 45 kd and 68 kd in smooth muscle cells (SMC) and 45 kd and 75 kd in endothelial cells (EC). In the peptide control membrane (anti-P2Y6 antibody preincubated with the same amount of control peptide antigen), no band was found. Data are representative of three independent experiments.

For the P2Y11 receptor, there was a strong band at 60 kd in endothelial cells, but no detectable band in SMC (Fig. 10). No band was found in peptide control membrane, which indicates that the 60-kd band represents the P2Y11 receptor.

FIG. 10.
FIG. 10.:
Western blotting of P2Y11 subtype in smooth muscle cells and endothelial cells. There was a band approximately 60 kd in endothelial cells (EC). There was no band in smooth muscle cells (SMC). In peptide control membrane (anti-P2Y11 antibody preincubated with the same amount of control peptide antigen), no band was found. Data are representative of four independent experiments.

DISCUSSION

In this study, we set up methods to study the expression of P2 receptors in human vascular tissue at the mRNA and protein levels using real-time PCR and Western blotting. The main findings showed a clearly different expression pattern between endothelial cells and SMC. The P2X1 receptor was exclusively expressed in SMC, and the P2X4 receptor was most abundantly expressed in endothelial cells. The P2Y2 receptor showed high expression in both cell types, with weak expression for the P2Y4 receptor. As expected, P2Y1 had high expression in endothelial cells. The most surprising finding was the high expression of the P2Y11 receptors in the endothelium.

We chose the left internal mammary artery as a clinically relevant representative for human arteries, but also because of its availability in relatively large quantities. Furthermore, SMC proliferation plays an important role in intimal thickening in atherosclerosis and restenosis, and is crucial for long-term patency of bypass grafts (22). Internal mammary arteries (IMA) are common materials in coronary bypass. Knowledge of P2 receptor expressions in IMA may contribute to the understanding of spasm during harvesting or after grafting, and the mechanisms of restenosis after coronary artery bypass graft (CABG). In the experiments, the adventitia and the endothelium were always removed. Successful removal of the endothelium was confirmed in vasomotor experiments by absence of dilatory responses to acetylcholine. Thus, the remaining material should represent SMC without contamination of endothelial cells. Since it was not possible to get large enough quantities of endothelial cells from the IMA, endothelial cells were harvested by collagenase treatment of human umbilical veins. Unfortunately, we found a small amount of SMC contamination in the freshly isolated EC. Therefore, we also used EC cultured for two to three passages in which SMC contamination could be excluded.

mRNA quantification

Quantification of mRNA expression has been approached in many ways. Northern blotting is the most straightforward, but the sensitivity is low. Regular PCR does not give quantification. Competitive RT-PCR is a highly sensitive method, but the synthesis of the competitor RNA is time consuming. Furthermore, relatively high amounts of RNA are needed of the samples. The recent development of real-time PCR has solved several of these problems. However, it requires validating of the amplification efficiencies for the genes studied. We found that amplification efficiencies were almost identical for P2Y1, P2Y2, P2Y4, P2Y6, P2Y11, P2X1, P2X4, P2X7, and the reference gene GAPDH.

The P2X1 receptor

The first evidence of the presence of P2 receptor subtypes suggested P2X receptors to be present in vascular SMC and P2Y receptors in EC (16). Although, this was revised later as seven subtypes of the P2X and six of the P2Y receptor were cloned. Despite this, it is remarkable how well this first hypothesis holds true in our findings. The P2X receptor was defined by Kennedy and Burnstock (16) as sensitive and rapidly desensitized by αβ-MeATP, which fits the cloned P2X1 receptor (23). Immunohistochemistry in rat blood vessels has shown this to be the dominating subtype in SMC (24). In our mRNA quantification, the difference between SMC and EC was impressive. Despite the high sensitivity of PCR, the P2X1 receptor was only barely detectable in the cultured EC samples, with a million times higher expression in SMC. In the freshly harvested EC, P2X1 receptors were detectable at a low concentration, probably due to SMC contamination. As compared with the P2Y receptors, the P2X1 receptor had 6 (P2Y2) to 26 (P2Y1) times higher expression in SMC. This is in agreement with pharmacologic studies, in which the P2X1 receptor has long been regarded as an important contractile receptor in blood vessels. However, it is different from our own findings in rat aorta (8), wherein we found two- to threefold higher expression of P2Y receptor subtypes. The difference is probably dependent on methodology. The competitive RT-PCR used in the rat study is highly dependent on the quality and quantification of the competitor. This method is good for comparisons of one gene in different samples, but not for comparisons between different genes. By contrast, the real-time PCR method enables quantitative between-gene comparisons when each assay is fully optimized, as in the current study. Furthermore, expression of each target gene was normalized to the endogenous expression of GAPDH. A potential problem in quantifying mRNA of P2Y receptors is that they, in contrast to the P2X1 receptor, lack introns. To exclude the possibility of genomic DNA contamination in the P2Y receptor assays, amplification of non–reverse-transcribed samples was performed. These negative control assays confirmed no or extremely low product accumulation levels. Furthermore, if DNA contamination were a factor, it would have increased the P2Y receptor levels, not the P2X1 levels.

The marked difference in P2X1 receptor levels was confirmed at the protein level. Western blot analysis demonstrated three bands in SMC, but addition of a control peptide revealed that only the strong 55 kd band was specific for the P2X1 receptor antibody. In endothelial cells, no bands were detected.

The main focus in this study was on the P2Y and the P2X1 receptors since reports of other P2X receptor subtypes in SMC and EC are scarce. However, the P2X7 receptor has been found in human saphenous vein SMC and the P2X4 in human EC, which prompted us to set up quantitative real-time PCR for those receptors as well (25,26). However, the mRNA expression of P2X4 and P2X7 receptors was low in SMC.

In conclusion, the P2X1 receptor is expressed only in SMC, with no expression in EC, at neither the mRNA nor the protein level. It could be used as a specific SMC marker and as a target for the development of blood pressure–reducing drugs.

P2Y receptors in SMC

The presence of a contractile P2Y receptor was first shown by von Kugelgen et al. (27), who demonstrated contractile responses to nucleotides that could not be desensitized by αβ-MeATP. These authors also suggested the existence of contractile pyrimidine (UTP and UDP)-sensitive receptors. Molecular cloning later revealed an ADP-sensitive receptor (P2Y1), an ATP- and UTP-sensitive receptor (P2Y2), a UTP-sensitive receptor (P2Y4), a UDP-sensitive receptor (P2Y6), and an ATP-sensitive receptor (P2Y11) (reviewed in Ralevic and Burnstock [7]).

The selective P2Y1 receptor agonists 2-MeSADP and ADPβS have no contractile effects in arteries, but are very potent endothelium-dependent vasodilators (7,14). In agreement with this, the P2Y1 receptor had the lowest expression of the studied receptors in the SMC, and was one of the two most abundantly expressed receptors in EC, supporting the original hypothesis by Kennedy and Burnstock (16). Surprisingly, however, the sizes of the bands in the Western blot analyses differed between EC and SMC. The larger bands were stronger in EC, but the 45-kd band, which fits the size of the receptor protein better, was only in SMC. One explanation for this could be oligomerization of the receptors. P2X receptors have been reported to form complex homo- or heterotrimeric receptors whose activity differs with each oligomer (28,29). A similar complexity may also apply to P2Y receptors in view of the recent demonstration that G protein–coupled GABAB receptors form heterodimers with specific and different properties, as compared with the individual homomeric receptors (30,31). Moore et al. (32) found that there was a 63-kd band in human and rat brain membranes, and Moran-Jimenez and Matute (33) also found a major band of 42 kd in Western blotting of tissue homogenates from rat and bovine brain. In the cardiovascular system, we found a similar 180-kd band in heart, coronary artery SMC, umbilical vein SMC, and platelets (data not shown). If the 180-kd band, which could be a tetramer, is the functional band of P2Y1 in the human cardiovascular system, the 90-kd and 45-kd bands in the current study may represent dimers and monomers. If this holds true, why does it exist as a monomer only in SMC, and does this explain the lack of P2Y1-mediated effects in SMC? The data suggest that the P2Y1 receptor needs to be oligomerized to be active. It is also possible that P2Y receptors form heteromers with other receptor types or other proteins (25). Heteromeric association has been shown for the P2Y1 receptor and the adenosine (A1) receptor, wherein the P2Y1 agonist ADPβS still activated the oligomeric receptor association, but instead of the usual P2Y1-associated Gq protein, it activated the A1 receptor-associated Gi/o protein, resulting in inhibition of cAMP (25). These complexes were not SDS resistant. However, when we tried to dissociate the protein detected in our Western blottings by treating samples with Triton X-100, EDTA, and DTT, and prolonging incubation time, it did not have any effect on band size, indicating that P2Y1 could form SDS-resistant oligomerization similar to that in other G-protein-coupled receptors (34). Another possibility for the increased molecular mass is glycosylation of the receptor in the extracellular domain. We used N-glycosidase F to digest samples and found a reduction in band size by approximately 30 kd between treated and untreated samples. Thus, glycosylation cannot explain the large differences in size.

In summary, we have tried several approaches to dissociate the band, with no success. The results indicate the possibility of P2Y1 receptor oligomerization. Firmly proving oligomerization sequencing of the band would be necessary, which is expensive and difficult, yet we have started exploring this possibility.

Dissection of the vascular actions of the pyrimidine-sensitive receptor has not been possible until synthesis and characterization of the stable analogues UTPγS and UDPβS (9,17). UTPγS is selective for P2Y2 and P2Y4 receptors, but cannot discriminate between them (17). UDPβS is selective for P2Y6 receptors, with no effects on the other P2 receptors (9). Using these selective agonists, it was recently demonstrated that both P2Y2/4 and P2Y6 receptors mediate strong arterial contractions. P2Y2/4 also mediated prominent vasodilation while P2Y6 did not have any vasodilator effects in rat (14). In agreement with this, we found high expression of P2Y2 receptors in both SMC and EC at both the mRNA and protein levels.

Our data also have a bearing on the unsolved question whether the vascular effects of UTP/UTPγS are mediated by P2Y2 or P2Y4 receptors. In both SMC and EC, the P2Y4 receptor mRNA levels were much lower than the P2Y2 levels. Thus, in humans, the P2Y2 receptor seems to be the most important UTP-sensitive receptor on both SMC and EC. P2Y4 bands were detected in Western blotting. Unfortunately, it is not possible to make a quantitative comparison between the P2Y2 and P2Y4 Western blottings since the band intensities are dependent on the different antibodies, the incubation times, and autoradiography procedures. Similar to the P2Y1 receptor, we found variable band sizes for P2Y4, which could be explained by oligomerization. P2Y4 could exist as a trimer in EC, as compared with a monomer in SMC, just as it was recently found that P2X1, P2X2, and P2X3 exist as trimers in the tissue (28,29).

In animal studies, UDP-stimulated contractions mediated via P2Y6 receptors have been demonstrated, but so far, there are no reports of UDP-mediated endothelium-dependent dilation (14). Surprisingly, we found P2Y6 receptor expression at both the mRNA and protein levels, with no major differences between SMC and EC. In Western blottings, a 45-kd band was seen in both cell types with similar intensity. We also found a band of higher molecular weight at 68 kd in SMC and at 75 kd in EC, but this may be due to nonspecific binding of the polyclonal antibody since the bands disappeared when the antibody concentration was decreased (data not shown). The endothelial expression of P2Y6 receptors in this human material has led us to explore the dilatory effect of UDPβS on human blood vessel. Preliminary data show a clear UDPβS-mediated dilation (to be published), confirming the current mRNA and protein results and emphasizing the importance of human studies.

The P2Y11 receptor was detected in SMC at the mRNA level, but it was not detected in Western blotting, indicating a more important role as an endothelial receptor (see later).

Endothelial cells

As mentioned earlier, the P2Y1 receptor was highly expressed in EC, which is in agreement with previous pharmacologic characterizations. Totally unexpected was that the P2Y11 receptor was the most highly expressed P2Y receptor in EC. This was also unambiguously confirmed in the Western blottings, in which a strong 60-kd band was detected in the EC and no band in the SMC. The P2Y11 receptor has not previously been detected in the cardiovascular system (35), and since no specific agonists or antagonists are available, there is no pharmacologic evidence for any vascular effects. With these new data, one must ask whether not many of the previously described effects of ATP on the endothelium ascribed to the P2Y1 or the P2Y2 receptor instead have been mediated via the P2Y11 receptor. Thus, the P2Y11 receptor could be an important mediator of vasodilation and blood pressure control via prostaglandin, NO, or EDHF release. In addition, it could have a fibrinolytic role by releasing t-PA (13). Interestingly, the most expressed endothelial receptors, the P2Y11 and the P2Y1 receptors, are also most closely related in their amino acid sequence among the P2 receptors (35).

Studies of gene expression are often done in cultured EC. However, the lack of shear stress and other differences in the conditions of cultured cells in vitro might affect the receptor expression. Therefore, we also examined freshly harvested EC (Figs. 3c and 4c). For the P2Y receptors, the results were similar to those for the cultured EC.

Evidence for the existence of P2X receptors in EC was scarce until Yamamoto et al. (36) found high levels of P2X4 receptor expression in human EC. These authors found that P2X4 receptors mediate ATP-induced calcium influx in human vascular endothelial cells (36), and that the receptor expression can be altered by shear stress (37,38). We were able to confirm their findings that the P2X4 receptor is the most expressed P2X receptor in EC. Furthermore, we demonstrated that it is expressed in far higher mRNA levels than all the P2Y receptor subtypes, which by the original definition should be the dominating EC receptors. It seems that the ion-channel-coupled P2 receptors need a higher mRNA expression than the G-protein-coupled P2Y receptors to be of functional importance. This could depend on the efficacy of their intracellular mechanisms.

In conclusion, P2X1, P2Y2, and P2Y6 and are the most expressed P2 receptors in SMC and mediate the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and most likely mediate the release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development directed against the large P2 receptor family.

Acknowledgments:

The authors thank Diane J. Cousens and Marie-Ange Watson (Molecular Pharmacology group, GlaxoWellcome, UK) for the kind gift of the P2Y4, P2Y6, and P2Y11 antibodies and their control peptides; Roya Fatemeh Doroudi for designing the first primer and probe; and Terrence Heathfield for his suggestions regarding Western blot analysis.

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Keywords:

Endothelial receptors; P2 receptor; Real-time PCR; Smooth muscle; Western blotting

© 2002 Lippincott Williams & Wilkins, Inc.