INTRODUCTION

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CA²⁺ signaling plays an important role in agonist- or hemodynamic stress-mediated regulation of endothelial cell (EC) functions. An increase in intracellular Ca²⁺ concentrations ([Ca²⁺]_i) has been observed in ECs stimulated with agonists such as ATP, histamine, bradykinin, and thrombin (8, 10) and also in ECs exposed to hemodynamic stresses such as shear stress and cyclic stretch (1, 18, 25). When [Ca²⁺]_i increases, Ca²⁺-binding proteins (such as the specific receptor calmodulin) bind to Ca²⁺, and the Ca²⁺-protein complexes then interact with other proteins in the cell to alter their functions. This results in changes in EC functions such as increased nitric oxide and prostacyclin production, which cause vasodilatation. Recent studies (2, 4) indicate that differences in the amplitude and duration of a [Ca²⁺]_i increase contribute to the differential activation of various transcription factors.

The pattern of an increase in [Ca²⁺]_i induced by ATP usually consists of a peak and a sustained phase (8). The peak is caused by the Ca²⁺ release from intracellular Ca²⁺ stores, and the sustained phase is due to the influx of extracellular Ca²⁺ across the cell membrane. The Ca²⁺ release is known to be mediated by P2 purinoceptors such as P2Y₁ and P2Y₂ (20). Binding of extracellular ATP to these receptors activates phospholipase C via GTP-binding protein and generates D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃], which triggers Ca²⁺ release from intracellular Ca²⁺ stores. The molecular mechanism for ATP-induced Ca²⁺ influx remains unclear, but the involvement of D-myo-inositol 1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P₄]-sensitive cation channels (17) or Ca²⁺-release-activated channels (11) has been suggested.

The recently discovered family of ligand-gated channels activated by extracellular ATP, the P2X receptors, is widely distributed in visceral and vascular smooth muscle cell types, as well as in numerous neuronal and glial cell types (3, 5). Seven different genes encoding P2X receptors have been identified in rat (rP2X₁, rP2X₂, rP2X₃, rP2X₄, rP2X₅, rP2X₆, and rP2X₇) and five human homologue receptors (hP2X₁, hP2X₃, hP2X₄, hP2X₅, and hP2X₇) have been characterized. Each P2X receptor subunit appears to have a common three-dimensional structure: two hydrophobic putative transmembrane domains with an intervening hydrophilic loop of almost 300 amino acids lying on the extracellular surface and intracellular NH₂ and COOH terminals. The P2X receptors, however, have never been reported to be expressed in vascular ECs. In this study, we investigated whether human vascular ECs express P2X purinoceptors, and if so, which subtype is predominantly expressed. Furthermore, to examine the role of P2X purinoceptors in the mechanism for the ATP-induced Ca²⁺ response, we inhibited P2X gene expression with antisense oligonucleotides.

	MATERIALS AND METHODS

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All animals were handled in accordance with the guidelines approved by the Animal Research Committee, University of Tokyo, which follow the guidelines outlined by the American Physiological Society.

Cell culture. Primary cultures of human umbilical vein ECs (HUVECs) were obtained from human umbilical cord veins by collagenase treatment, and ECs from aorta (HAECs), pulmonary artery (HPAECs), and microvessel (HMVECs) were purchased from Clontech. All ECs were cultured on a 1% gelatin-coated flask in medium-199 (ICN Biomedicals) containing 15% fetal bovine serum (GIBCO-BRL), 2 mM L-glutamine (GIBCO-BRL), 50 U/ml penicillin, 50 µg/ml streptomycin (ICN Biomedicals), 50 µg/ml heparin (Sigma), and 30 µg/ml EC growth factor (Becton-Dickinson) in an atmosphere of 5% CO₂ at 37°C. Cells were routinely passaged by trypsinization (ICN Biomedicals); those used for the present experiments were obtained during the fourth and tenth passages.

Cloning and sequencing of the P2X₄ receptor. Human lung total RNA (Clontech) was amplified by RT-PCR using sense and antisense primers for the P2X₄ receptor (Table 1) (6). The P2X₄ cDNA fragment was radiolabeled with [-³²P]dCTP using a random primer labeling kit (Takara). An HUVEC cDNA library (Clontech) was screened by lifting 1.2 × 10⁶ phages onto a Hybond-N nylon membrane (Amersham). After 12 h of prehybridization in 5× SSC (1× SSC is 750 mM sodium chloride and 75 mM sodium citrate, pH 7.0) (24), 5× Denhardt solution, 0.5% SDS, 10% dextran sulfate, and 0.25 mg/ml salmon testis DNA at 65°C, hybridization was carried out in the same solution with a radiolabeled P2X₄ probe for 20 h at 65°C. The positive clones were identified by autoradiography on X-ray film. The DNA from the positive clones was isolated, digested with EcoR I, and subcloned into a pBluescript II vector (Stratagene). The complete nucleotide sequence was determined using DNA sequencer 373S-36 (Applied Biosystems). The sequence is the same as was previously reported from other tissues, and the GenBank accession number is AF000234.

Northern blot analysis. mRNA was obtained from HUVECs, HAECs, HPAECs, and HMVECs using the MACS mRNA Isolation Kit (Miltenyi Biotec). Briefly, cells were lysed with a high-salt buffer containing 1% SDS. Colloidal super-paramagnetic MicroBeads conjugated to oligo(dT) were added to the lysed cells, and the lysate was passed through the magnetic field of the MACS separator column. mRNA that was hybridized to the oligo(dT) MicroBeads remained in the column. After the column was washed to remove protein, DNA, and rRNA, the pure mRNA was eluted using elution buffer.

RT-PCR analysis. Competitive PCR was used to compare mRNA levels of P2X subtypes. Heterologous competitors for the P2X₁, P2X₃, P2X₄, P2X₅, P2X₇, P2Y₁, and P2Y₂ genes were generated using the Competitive DNA Construction Kit (Takara). Both sense and antisense primers were synthesized. The sense primers consisted of the gene-specific sense sequence (Table 1) with an additional SP6 promoter sequence at the 5' end and a composite sense primer at the 3' end used only for competitor construction. The antisense primers were made up of each gene-specific antisense sequence, linked at the 3' end to a composite antisense primer. With the use of these primers, DNA competitor fragments were obtained by PCR amplification (30 cycles of 30 s at 94°C, 30 s at 60°C, and 45 s at 70°C). The DNA fragments were then transcribed into RNA fragments using SP6 RNA polymerase (Competitive RNA Transcription Kit, Takara). The RNA competitor fragments were extracted with phenol, chloroform, and isoamyl alcohol and precipitated with ethanol. The RNA concentration was determined spectrophotometrically and diluted to 800 amol/µl. Initially, competitive PCR was performed by adding 2 µl of the mRNA samples obtained from HUVECs or HAECs to 2 µl of different 10-fold dilutions of the RNA competitor fragments ranging from 0.00008 to 800 amol/µl (13). The mixture was kept at 37°C for 1 h and heated to 99°C for 5 min in the presence of Moloney murine leukemia virus RT (GIBCO-BRL), oligo(dT)_12-18, RNase inhibitor, each dNTP mixture, and dithiothreitol in a first-strand buffer. After reverse transcription, the PCR reactions were carried out using each target gene-specific primer (Table 1) in a solution containing ExTaq DNA polymerase (Takara) and [-³²P]dCTP. Each temperature cycle consisted of 95°C for 30 s, 60°C for 30 s, and 72°C for 1 min. The sequences of the PCR products showed that each mRNA was correctly amplified by those primers. A second series of competitive PCR assays was then carried out with consecutive 1:2 dilutions of competitors mixed with a constant amount of each target mRNA. The PCR products were separated by electrophoresis in a 5% polyacrylamide gel. The radioactivity of both target mRNA bands and competitor bands (known concentrations) was measured with a GS363 Molecular Imager System. The logarithm of the ratio of target bands to competitor bands was plotted as a function of the logarithm of the known amounts of competitor. The concentration of target mRNA molecules present in ECs corresponds to that of competitor at the competition equivalence point [log(target/competitor) = 0]. The -actin gene, which is a housekeeping gene, was used as a control for variation in RNA quality and quantity.

Generation of the P2X₄ antibody. An antiserum against human P2X₄ receptor protein was generated in rabbits injected with a synthetic peptide (NH₂-RLYYREKKYKYVEDYC-COOH) comprising amino acid residues 364-378 of the sequences for the intracellular COOH-terminal domains of the cloned human P2X₄ receptor. Peptide was covalently linked to keyhole limpet hemocyanin, and rabbits were immunized by injection with the conjugated peptide every 2 wk for 8 wk. The anti-P2X₄ receptor antiserum was then affinity purified using a synthetic peptide (P2X₄ residues 364-378) immobilized on Sepharose 4B (Asahi Techno Glass).

Western blot analysis. HUVECs were washed with cold PBS and solubilized in 500 µl radioimmunoprecipitation assay (RIPA) buffer (1% Nonidet P-40, 20 mM Tris · HCl, 0.15 M NaCl, 0.5% sodium deoxycholate, 2 mM EDTA, 2 mM EGTA, 0.1% SDS, 0.2 mM Na₂MoO₄, 10 mM NaF, 1 mM Na₃VO₄, 1 mM phenylmethylsulfonyl fluoride, 5 µg/ml leupeptin, 5 µg/ml antipain, 5 µg/ml pepstatin A, and 0.2 U/ml aprotinin; pH 7.4). Lysates were centrifuged at 26,000 g for 30 min. The supernatants were immunoprecipitated using the anti-P2X₄ receptor antiserum, which was prebound to protein A-Sepharose beads (Millipore, Bedford, MA). After four washes with RIPA buffer, immunoprecipitated proteins were solubilized in SDS sample buffer (0.2 M Tris · HCl, 18% glycerol, 4% SDS, 0.01% bromphenol blue, and 10% -mercaptoethanol; pH 8.8) for SDS-PAGE. Gels were transferred to Immobilon polyvinylidene difluoride membranes (Millipore). Membranes were blocked in Tris-buffered saline with 5% skim milk and 0.1% Tween 20 and then incubated for 1 h with the anti-P2X₄ antiserum (3 µg/ml). Membranes were washed in PBS and incubated with anti-rabbit IgG horseradish peroxidase-conjugated antibody. The blots were developed using an enhanced chemiluminescence kit (Amersham) and analyzed by the GS363 Molecular Imager (Bio-Rad).

Antisense oligonucleotides. Antisense oligonucleotides (AS-oligos) targeted to the P2X₄ receptor and scrambled control oligos (S-oligos) were designed and synthesized by Biognostik (Göttingen, Germany). Sequences of phosphorothioated AS-oligos and S-oligos were 5'-CCTGAAATTGTAGCC-3' and 5'-TAATCGCTTCAGACG-3', respectively, and were FITC labeled at the 5' end. AS-oligos or S-oligos were transfected into cells using LipofectAMINE PLUS (GIBCO-BRL). Cellular uptake of AS-oligos was checked by the observation of FITC using a fluorescence microscope.

[Ca²⁺]_i determination. ECs, which were cultured on a 40-mm-diameter round coverslip coated with 1% gelatin, were loaded with indo 1-acetoxymethylester (Dojindo). The coverslip was placed in the FCS2, a parallel plate type of flow chamber (Bioptechs, Butler, PA), on the stage of an inverted microscope (Diaphot 300, Nikon). Agonists in Hanks' balanced salt solution were perfused through the chamber (using a peristaltic pump) at a flow rate of 3 ml/min to stimulate cells at 37°C.

Statistical analysis. Differences in [Ca²⁺]_i between the control and antisense-treated cells were evaluated by ANOVA, followed by Bonferroni modification of the t-test by using SPSS. Significance was assumed at P < 0.05.

	RESULTS

TOP ABSTRACT INTRODUCTION MATERIALS AND METHODS RESULTS DISCUSSION REFERENCES

Vascular ECs express the P2X₄ receptor. Northern blot analysis using the P2X₄ cDNA probe demonstrated that P2X₄ mRNA was expressed in HUVECs, HAECs, HPAECs, and HMVECs (Fig. 1). However, another P2X subtype P2X₁ was not expressed in any of these EC lines.

View larger version (71K): [in this window] [in a new window]   Fig. 1.   Northern blot analysis of human P2X4 and P2X1 mRNA. Left: distribution of P2X4 mRNA in cultured human endothelial cells (ECs) of microvessel (HMVECs), pulmonary artery (HPAECs), aorta (HAECs), and umbilical vein (HUVECs). Right: distribution of P2X1 mRNA. Poly (A)+ RNA (2 µg) was loaded in each lane; size markers are shown on the left. Human lung tissue RNA was used as a positive control. Expression of -actin mRNA was also determined in the same samples.

View larger version (34K): [in this window] [in a new window]   Fig. 2.   Competitive PCR analysis of P2X subtype mRNA levels in HAECs and HUVECs. Top: representative examples of Molecular Imager-processed signals from PCR products of the P2X4 (left) or P2X5 (right) templates in HUVECs and increasing quantities of competitor after separation on a polyacrylamide gel. Bottom: quantitative analysis of mRNA for five receptor subtypes in HAECs and HUVECs. At the competition equivalence point, the original number of target RNA molecules corresponds to the initial number of competitor molecules present. The level of mRNA of each subtype is expressed as a percentage of P2X4 mRNA levels in HAECs.

AS-oligo knockout of P2X₄ receptor expression in ECs. To assess the physiological role of P2X₄ receptors in ECs, we used AS-oligos to specifically knock out P2X₄ receptor function. When HUVECs were treated with the AS-oligos, P2X₄ receptor mRNA and protein levels decreased to ~25% of control levels, although neither changed after S-oligo treatment (Fig. 3). The AS-oligos had no effect on the levels of P2X₁, P2X₃, P2X₅, P2X₇, P2Y₁, or P2Y₂ mRNA expression (Fig. 4), indicating that the AS-oligos specifically knock out the expression of P2X₄ receptors.

View larger version (28K): [in this window] [in a new window]   Fig. 3.   Changes in P2X4 mRNA and protein levels induced by antisense oligonucleotides (AS-oligos). Results from RT-PCR (left) and Western blot (right) are shown for control, nontreated; sense oligonucleotides (S-oligos), S-oligo treated; and AS-oligos, AS-oligo-treated HUVECs. Top: representative signal bands (GAPDH, glyceraldehydes-3-phosphate dehydrogenase). Bottom: quantitative analyses using densitometry. The y-axis indicates the percentage of control. Data are means ± SD of five separate experiments. P2X4 mRNA and protein levels in AS-oligo-treated HUVECs decreased to ~25% of the control levels.

View larger version (24K): [in this window] [in a new window]   Fig. 4.   Effects of AS-oligos on the mRNA levels of five P2X subtypes, P2Y1, and P2Y2. Two days after treatment of HUVECs with S-oligos and AS-oligos, total RNA was extracted, and competitive PCR was performed. Open, hatched, and solid bars represent controls, S-oligo-treated, and AS-oligo-treated HUVECs, respectively. The y-axis indicates mRNA levels relative to the P2X4 mRNA control level taken as 100%. AS-oligos only affected P2X4 expression.

P2X₄ receptors mediate ATP-induced Ca²⁺ influx in ECs. When HUVECs were stimulated with ATP (4 µM), they showed an initial peak and subsequent sustained phase in [Ca²⁺]_i (Fig. 5A). HUVECs treated with S-oligos showed almost the same Ca²⁺ response pattern as observed in the control cells (Fig. 5B). On the other hand, HUVECs treated with AS-oligos showed an initial peak but no subsequent sustained phase in [Ca²⁺]_i (Fig. 5C). This Ca²⁺ response pattern was quite similar to that seen in the absence of extracellular Ca²⁺ (Fig. 5, D-F), indicating that Ca²⁺ influx was inhibited in AS-oligo-treated cells. This was confirmed by a quantitative analysis of the ATP-induced Ca²⁺ responses (Fig. 6). The same phenomenon was also observed in HAECs and HPAECs (data not shown). These findings indicate that the P2X₄ receptor mediates ATP-induced Ca²⁺ influx in human ECs.

View larger version (32K): [in this window] [in a new window]   Fig. 5.   ATP-induced Ca2+ response in indo 1-acetoxymethlyester (AM)-loaded HUVECs. A, D: control HUVECs; B, E: HUVECs treated with S-oligos; C, F: HUVECs treated with AS-oligos. A-C: Ca2+ responses in the absence of EGTA. D-F: Ca2+ responses in the presence of EGTA. Extracellular Ca2+ was chelated by preincubating HUVECs with 1 mM EGTA for 1 min. Each graph represents eight intracellular Ca2+ concentration ([Ca2+]i) responses of 8 single cells. Control and S-oligo-treated HUVECs showed an initial peak and subsequent sustained phase in [Ca2+]i in response to 4 µM ATP. AS-oligo-treated cells showed an initial peak but no sustained phase in [Ca2+]i. In the presence of EGTA, only an initial peak was observed in control, S-oligo-treated, and AS-oligo-treated HUVECs. F, flourescence. F405/F480, ratio of fluorescence at 405 nm to that at 480 nm.

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Quantitative analysis of ATP-induced [Ca2+]i responses in indo 1-AM-loaded HUVECs. Ca2+ responses to 4 µM ATP were quantitatively analyzed in S-oligo-treated HUVECs without EGTA and with EGTA and in AS-oligo-treated cells. Mean F405/F480 ratios at each of 12 points on a Ca2+-response curve are shown as means ± SD of 27 cells. The Ca2+-response curve for AS-oligo-treated cells is almost identical to that for S-oligo-treated cells under an extracellular Ca2+-free condition. *Statistically significant difference (P < 0.01) between S-oligo-treated and AS-oligo-treated HUVECs; this indicates that under the condition of 4 µM ATP stimulation, Ca2+ influx is markedly inhibited in AS-oligo-treated cells, and P2X4 mediates ATP-induced Ca2+ influx.

View larger version (25K): [in this window] [in a new window]   Fig. 7.   Ca2+ responses to different ATP concentration ([ATP]) in indo 1-AM-loaded HUVECs. Changes in [Ca2+]i were measured when HUVECs treated with S-oligos (A) or AS-oligos (B) were stimulated with 1 to 20 µM ATP. The initial peak and sustained phase in [Ca2+]i increased in accord with increased [ATP]. In AS-oligo-treated cells, the sustained phase in [Ca2+]i was almost abolished at <4 µM ATP but reappeared at higher [ATP] (10 µM).

P2X₄ receptors are not involved in histamine-induced Ca²+ influx. Histamine induced an initial peak and subsequent sustained phase in [Ca²⁺]_i, which is quite similar to that seen in ATP stimulation (Fig. 8). Treatment of HUVECs with the P2X₄ receptor AS-oligos, however, did not affect the histamine-induced Ca²⁺ response. This suggests that Ca²⁺-permeable cation channels other than P2X₄ receptors are involved in the histamine-induced Ca²⁺ influx.

View larger version (25K): [in this window] [in a new window]   Fig. 8.   Histamine-induced Ca2+ response in indo 1-AM-loaded HUVECs. Ca2+ responses to 4 µM histamine were quantitatively analyzed (as described in Fig. 6) in S-oligo-treated HUVECs without EGTA and with EGTA and in AS-oligo-treated cells. Data are means ± SD from 30 cells. AS-oligos had no effect on the histamine-induced Ca2+ response in HUVECs.

	DISCUSSION

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The present study is the first to demonstrate that cultured human vascular ECs express P2X₄ receptors, which are ATP-gated ion channels that mediate Ca²⁺ influx induced by ATP. P2X receptors are widely distributed in various tissues of mammals, including smooth muscle of the urinary bladder and arteries, kidney, pancreas, lung, cardiac myocytes, sensory and sympathetic ganglia, and brain and spinal cord. Five genes encoding the human P2X receptor subtypes (P2X₁, P2X₃, P2X₄, P2X₅, and P2X₇) have been cloned (6, 7, 14, 16, 23, 27). Each subtype seems to be preferentially expressed in different tissues. For instance, the rP2X₁ receptor is predominantly localized to smooth muscles, but the rP2X₂ receptor, which is not expressed in smooth muscles, is found predominantly in neurons (5). In this study, Northern blot analysis showed expression of P2X₄ mRNA in cultured human ECs from umbilical vein, aortic, pulmonary artery, and microvessel but failed to detect P2X₁ transcripts. Competitive PCR using sense and antisense primers for these P2X receptor subtypes revealed that the level of P2X₄ mRNA was significantly higher than that of any of the other subtypes in cultured human ECs. Western blot analysis and fluorescence immunostaining (data not shown) using an anti-P2X₄ polyclonal antibody showed that HUVECs also express P2X₄ at protein levels. These results suggest that HUVECs predominantly express P2X₄ receptors.

Because there are no specific inhibitors available, we used AS-oligos to selectively interfere with P2X₄ to assess its physiological role in ECs. Treatment of ECs with the AS-oligos markedly decreased P2X₄ mRNA levels but did not change the mRNA levels of other subtypes, including P2X₁, P2X₃, P2X₅, P2X₇, P2Y₁, and P2Y₂. Western blots using an anti-P2X₄ polyclonal antibody confirmed that protein levels of the P2X₄ receptor were also decreased in the AS-oligo-treated ECs. Treatment of ECs with S-oligos had no effect on the P2X₄ mRNA or protein levels. Attenuation of P2X₄ activity by AS-oligos was reversed by incubating AS-oligo-treated cells in normal media for 1 wk, indicating that the effect of AS-oligos was temporary (data not shown). The AS-oligos were not toxic to ECs and did not cause any change in cell morphology, as assessed by trypan blue dye exclusion test and phase-contrast microscopy. Taken together, AS-oligos may be used to effectively and selectively disrupt P2X₄ receptor function in ECs.

When P2X₄ expression was inhibited by the AS-oligos in HUVECs, the ATP-induced increase in [Ca²⁺]_i, especially the sustained phase, was markedly suppressed. This phenomenon was also observed in HAECs and HPAECs. These results indicate that P2X₄ receptors contribute to ATP-induced Ca²⁺ influx in ECs. The inhibitory effect of AS-oligos on the ATP-induced Ca²⁺ influx varied depending on the [ATP] used. At <4 µM ATP, the Ca²⁺ influx was almost abolished, and at >4 µM ATP, it was partially inhibited. Because AS-oligos do not completely knock out P2X₄ expression in ECs (as shown in Fig. 3), high doses of ATP may activate the remaining P2X₄ receptors to induce Ca²⁺ influx. High doses of ATP may also open other P2X subtypes, such as P2X₇, or activate unknown Ca²⁺-permeable channels other than P2X receptors. The Ca²⁺ influx seen at high [ATP] may also be due to store-operated Ca²⁺ influx (capacitative Ca²⁺ entry) triggered by P2Y-mediated Ca²⁺ release from Ca²⁺ stores (21). Recently the transient receptor potential protein has been found to form store-operated cation channels in human ECs (9, 28). Furthermore, high doses of ATP may inhibit Ca²⁺ removal from the cytoplasm or prolong Ca²⁺ release from the intracellular Ca²⁺ store. Histamine induces a [Ca²⁺]_i peak and a sustained phase in ECs that are similar to those seen with ATP stimulation. Because the P2X₄ AS-oligos had no effect on the histamine-induced Ca²⁺ response, cation channels other than the P2X₄ receptor seem to be involved in the histamine-induced Ca²⁺ influx. These results indicate that channels responsible for agonist-activated Ca²⁺ influx may vary with different agonists. Further studies are needed to determine how P2Y-mediated Ca²⁺ release and influx are related to P2X₄ receptor-mediated Ca²⁺ influx in ECs.