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(Circulation Research. 1995;76:191-198.)
© 1995 American Heart Association, Inc.

Articles

Coexpression of P_2Y and P_2U Receptors on Aortic Endothelial Cells

Comparison of Cell Localization and Signaling Pathways

Didier Communi, Eric Raspe, Sabine Pirotton, Jean-Marie Boeynaems

From the Institute of Interdisciplinary Research, School of Medicine, Université Libre de Bruxelles (Belgium).

Correspondence to Dr D. Communi, Institute of Interdisciplinary Research, Université Libre de Bruxelles, Campus Erasme, Building C, 808 Route de Lennik, 1070 Brussels, Belgium.

	Abstract

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Abstract Depending on the vascular bed considered, the actions of ATP on the endothelium are mediated by either P_2Y or P_2U receptors. The two types of receptors seem to coexist on bovine aortic endothelial cells, where they are both coupled to phospholipase C. In this study, we have investigated whether they are truly coexpressed on the same cells and whether their signaling pathways diverge beyond phospholipase C activation. Measurements of [Ca²⁺]_i in single cells showed that almost all bovine aortic endothelial cells are responsive to both 2-methylthio-ATP (2MeSATP), an agonist of P_2Y receptors, and UTP, an agonist of P_2U receptors. UTP stimulated the release of prostacyclin from freshly isolated bovine aortic endothelial cells, even when they were exposed to cycloheximide at the time of their collection: this indicates that P_2U receptors must already be expressed on endothelial cells in situ and do not appear during cell culture. The time course of inositol phosphate (InsP) accumulation and the relative proportion of Ins(1,4,5)P₃, Ins(1,3,4,5)P₄, and Ins(1,3,4)P₃ were similar in cells stimulated by 2MeSATP or UTP. UTP and 2MeSATP both stimulated the hydrolysis of phosphatidylcholine by phospholipase D, as reflected by the release of [³H]choline from prelabeled cells. The responses to both agents were blocked after downregulation of protein kinase C, resulting from a prolonged exposure to phorbol 12-myristate 13-acetate: this blockade occurred at a step distal to phospholipase C activation. A single difference between the two pathways has been identified: the effect of 2MeSATP on InsP₃ was significantly more inhibited after a short exposure to phorbol 12-myristate 13-acetate than that of UTP. This discrepancy is consistent with the involvement of distinct G proteins in the activation of phospholipase C by P_2Y and P_2U receptors.

Key Words: ATP • P₂ receptors • endothelial cells • inositol phosphates • choline

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
ATP stimulates the release of prostaglandins, prostacyclin (PGI₂) as well as PGE₂, and nitric oxide from the vascular endothelium, as a consequence of phospholipase C activation and rise in [Ca²⁺]_i.¹ It was originally believed that these responses were mediated by P_2Y receptors.² However, several observations did not fit with this simple concept: in some cases, the P_2Y-specific agonist 2-methylthio-ATP (2MeSATP), although more potent than ATP, had a lower maximal effect.³ On the other hand, some endothelial responses to ATP are mimicked by UTP.^{3 4 5 6 7 8 9 10 11 12} O'Connor et al¹³ formulated the concept that two subtypes of P₂ receptors can be expressed on endothelial cells: P_2Y receptors, characterized by their high affinity for 2MeSATP and nucleotide, or P_2U receptors, which recognize both ATP and UTP. The relative contribution of P_2Y and P_2U receptors to the action of ATP is variable from one type of endothelial cells to the other. In microvascular endothelial cells from bovine adrenal medulla,^{5 9} rabbit myocardium,¹⁰ and rat brain,¹¹ the inositol phosphate (InsP) response to ATP seems to be mediated exclusively by P_2U receptors. On the contrary, the bovine aortic endothelial cell (BAEC) line AG4762 expresses only P_2Y receptors.⁵ The two types of receptors coexist on BAECs.^{4 12} The arguments supporting the involvement of both P_2U and P_2Y receptors in the InsP response of BAECs to ATP are the following: (1) The effects of 2MeSATP and UTP were additive, whereas the effects of ATP and either UTP or 2MeSATP were not. (2) ATP desensitized the responses to both UTP and 2MeSATP, whereas there was only a minimal cross desensitization between 2MeSATP and UTP. (3) Pertussis toxin inhibited the action of UTP but had no effect on the response to 2MeSATP. Since the role of UTP as an intercellular messenger is far from being established, P_2U receptors are primarily considered to be ATP receptors. Therefore, it is unclear why the same cells have two distinct types of ATP receptors, both coupled to the same effector mechanism. In the present study, we have investigated the possibilities that P_2Y and P_2U receptors are segregated on subpopulations of BAECs and that the signaling pathways of the two receptors diverge beyond the common initial event of phospholipase C activation.

	Materials and Methods

Top Abstract Introduction Materials and Methods Results Discussion References

 
Materials
Collagenase type Ia, ATP, UTP, phorbol 12-myristate 13-acetate (PMA), and BrA23187 were from Sigma Chemical Co; 2MeSATP was from Research Biochemicals Inc; trypsin was from Flow Laboratories; and the culture media, reagents, and fetal calf serum were purchased from GIBCO. The radioactive products D-myo-[2-³H]inositol (17.7 Ci/mmol), [2-³H]methylcholine chloride (85 Ci/mmol), and 6-keto[³H]prostaglandin F₁ (120 Ci/mmol) were from Amersham. Dowex AG1X8 (formate form) was from Bio-Rad Laboratories. Pluronic F-127 and fura 2-AM were from Molecular Probes. The µBondapack C18 column (3.9x300 mm) was purchased from Waters Millipore. The solvents for high-performance liquid chromatography (HPLC) were obtained from Romil Chemicals Limited.

Cell Culture
BAECs were obtained by collagenase digestion of the aorta excised from a freshly slaughtered cow and cultured in a medium composed of minimum essential medium (MEM) D-valine (90% [vol/vol]), fetal calf serum (10% [vol/vol]), 2 mmol/L glutamine, 100 U penicillin per milliliter, 100 µg streptomycin per milliliter, and 2.5 µg amphotericin B per milliliter. MEM D-valine was used to prevent survival of contaminating smooth muscle cells. The cells were incubated at 37°C in a humidified air–CO₂ (19:1) incubator. When the primary culture formed a confluent monolayer, the cells were harvested with trypsin (0.1% [wt/vol]) in a Ca²⁺- and Mg²⁺-free Hanks' buffer and subcultured in 35-mm-diameter Petri dishes. For the second and the following passages, the medium was replaced by Dulbecco's modified Eagle's medium (DMEM, 70% [vol/vol]), Ham's F-12 medium (20% [vol/vol]), fetal calf serum (10% [vol/vol]), antibiotics, amphotericin B, and glutamine at the same concentrations. The cells were used between the second and fourth passages.

Measurement of Cytosolic Calcium in Single Cells
[Ca²⁺]_i was measured in BAECs by using the fluorescent Ca²⁺ indicator fura 2 as described initially by Grynkiewicz et al.¹⁴ Practically, the cells were cultured on circular glass coverslips, in the same culture medium as described above (except with the omission of amphotericin B). The cells were seeded either at low or high density to measure the [Ca²⁺]_i on individual cells or on a cell population. After 1 day of culture, the cells were incubated at room temperature in the dark for 30 minutes in a HEPES buffer (mmol/L: NaCl 120, KCl 5, MgCl₂ 1, CaCl₂ 1, glucose 10, and HEPES 20, pH 7.4) supplemented with pluronic F-127 (0.025%, 1/1000 dilution from a stock in dimethyl sulfoxide) and with fura 2-AM (1 µmol/L). After loading, the cells were quickly washed with the HEPES buffer and allowed to equilibrate at room temperature for 15 to 30 minutes in the dark in the same HEPES buffer. Then the coverslips were mounted in a homebuilt device placed on the stage of a Nikon Diaphot inverted microscope coupled to a Spex CM-1 fluorometer and incubated at room temperature in the HEPES buffer described above. The total volume of the device chamber was 250 µL. Fresh medium could be either perfused from the bottom of the chamber or added from its top. Excess medium was retrieved by a peristaltic pump through a tube placed in such a way that the remaining volume was kept constantly at 100 µL. The cells were challenged by adding 100 µL of a twofold concentrated solution from the top of the chamber. The cells were observed through a Nikon x100 oil immersion (numerical aperture, 1, 3) objective. Individual cells (with no contact with other cells as assessed by transmission microscopy) were isolated through a diaphragm for fluorescence measurements. They were excited alternatively at 340 and 380 nm through a Nikon DM400 dichroic mirror by using the excitation source of the Spex CM-1 fluorometer. The emission light was collected through a Nikon BA520 barrier filter with a photomultiplier tube connected to the Spex CM-1 fluorometer. The ratio between the fluorescence intensities recorded after excitation at 340 and 380 nm is related to the [Ca²⁺]_i.¹⁴ The BAEC [Ca²⁺]_i values were calculated accordingly from those fluorescence ratio values; after that the ratios of minimal and maximal fluorescence were obtained; at the end of the experiment, the experimental buffer was replaced by an EGTA buffer (mmol/L: NaCl 120, KCl 5, MgCl₂ 1, HEPES 20, and EGTA 50, pH 7.4), and the Ca²⁺-free fluorescence values were obtained after the addition of 25 µmol/L 4-BrA23187. The Ca²⁺-saturated fluorescence values were obtained after washing of the EGTA buffer and after exposure of the cells to a high-Ca²⁺ buffer (mmol/L: choline chloride 120, KCl 5, MgCl₂ 1, CaCl₂ 50, caffeine 10, and HEPES 20, pH 7.4).

Measurement of PGI₂ Release
Endothelial cells were isolated from bovine aorta as described above, seeded in 16-mm-diameter wells of 24-well plastic trays, and incubated for 5 hours in complete medium supplemented or not with cycloheximide (2 µg/mL). Cells were then washed with DMEM and equilibrated in this medium for 20 minutes. The agonists (ATP, UTP, and 2MeSATP) or water were added for an additional 20 minutes. The production of PGI₂ was measured by the radioimmunoassay of its stable degradation product 6-ketoprostaglandin F₁ directly in the incubation medium as described previously.¹⁵

Measurement of InsPs
Cell Labeling and InsP Extraction
Subconfluent cells were incubated for 24 hours in a medium containing inositol-free MEM, fetal calf serum (5% [vol/vol]), antibiotics, and amphotericin B as described above, supplemented with D-myo-[³H]inositol (10 µCi/mL). Cells were then washed twice with DMEM and incubated in this medium for 30 minutes before the addition of the agonist; the incubation was stopped after 15 seconds by rapidly replacing the medium with 1 mL HClO₄ (3% [vol/vol]). The dishes were rinsed with 1 mL HClO₄ (1% [vol/vol]), and the lysates were neutralized with KOH (0.765 mol/L) and HEPES (0.375 mol/L) to a final pH of 8. After 30 minutes at 0°C, KClO₄ was eliminated by centrifugation (1500g for 10 minutes at 4°C). The supernatant was collected, and the pellet was washed with 1 mL of a mixture of Na₂B₄O₇ (5 mmol/L) and EDTA (0.5 mmol/L) and recentrifuged as before. Five milliliters of this mixture (5 mmol/L Na₂B₄O₇ and 0.5 mmol/L EDTA) was added to the pool of the two supernatants.

InsP Separation
This separation was achieved by chromatography on Dowex AG1X8 (formate form). Inositol, glycerophosphoinositol, InsP₁, InsP₂, and InsP₃ were eluted sequentially with 20 mL of water, 8 mL of 60 mmol/L ammonium formate/5 mmol/L Na₂B₄O₇, 20 mL of 150 mmol/L ammonium formate/5 mmol/L Na₂B₄O₇, 18 mL of 0.4 mol/L ammonium formate/0.1 mol/L formic acid, and 12 mL of 0.7 mol/L ammonium formate/0.1 mol/L formic acid, respectively. A 4-mL aliquot of each fraction was counted by liquid scintillation.

Separation of InsP₃ and InsP₄ Isomers by HPLC
The cells were incubated for 48 hours in a medium containing inositol-free MEM, fetal calf serum (5% [vol/vol]), antibiotics, and amphotericin B as described above, supplemented with D-myo-[³H]inositol (10 µCi/mL). Cells were then washed twice with inositol-free MEM and incubated in this medium for 30 minutes, agonists were added for 15 seconds, and reactions were stopped by replacing the medium with 0.5 mL of 20% trichloroacetic acid (TCA) [vol/vol], 10 mmol/L EDTA, and 0.2 mg/mL phytic acid hydrolysate.¹⁶ The cells were scraped, and the dishes were rinsed with an equal volume of water. The proteins were eliminated by centrifugation, and the TCA contained in the supernatant was removed after five extractions with 3 mL diethyl ether. The samples were adjusted to pH 4 with 0.1 mol/L KOH and concentrated by using a Speed-Vac. They were resuspended in 400 µL of 50 mmol/L tributylammonium (pH 4). InsP₃ and InsP₄ isomers were separated on a µBondapack C18 column eluted isocratically in 5 mmol/L tributylammonium, 10% methanol, and 100 mmol/L KH₂PO₄ (pH 4) at a 1.2-mL/min flow rate. Fractions of 0.10 and 0.25 minutes were collected for InsP₃ and InsP₄, respectively. The samples were coinjected with 300 cpm [³²P]Ins(1,3,4,5)P₄. [³²P]Ins(1,3,4,5)P₄ was prepared from Ins(1,4,5)P₃ and [-³²P]ATP with a purified Ins(1,3,4)P₃ 3 kinase.

Assay of Choline Release
BAECs were labeled during a 24-hour incubation in 1 mL of complete culture medium containing 10 µCi [methyl-³H]choline (80 µCi/mmol). The cells were then washed twice with DMEM and incubated in 1 mL of DMEM in the presence of the tested agents. Aliquots of the incubation media (100 µL) were collected at various times for liquid scintillation counting.

Protein Phosphorylation
After labeling with [³²P]phosphate, the phosphorylated proteins from BAECs were resolved by two-dimensional gel electrophoresis, as previously described.¹⁷

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Fluorometric Measurement of [Ca²⁺]_i
The fluorometric measurement of [Ca²⁺]_i following fura 2 loading was used to evaluate the behavior of single BAECs. As shown in Fig 1, most BAECs responded to both 2MeSATP (1 µmol/L) and UTP (10 µmol/L) with a rise in [Ca²⁺]_i. This result was obtained in confluent monolayers but also with cells completely isolated from each other. Of the 29 cells from four independent cultures that have been studied individually, 26 had a significant response to both UTP and 2MeSATP; only 3 did not respond to 2MeSATP after being first challenged with UTP. Similar results were obtained when the agonists were used at a submaximal concentration (0.1 µmol/L 2MeSATP and 1 µmol/L UTP) to exclude cross-reaction of 2MeSATP with P_2U receptors and vice versa (8 cells of 11 responded to both agonists; data not shown).

View larger version (17K): [in this window] [in a new window]   Figure 1. Graphs showing the effects of 2-methylthio-ATP (2MeSATP), UTP, and ATP on the [Ca2+]i of individual bovine aortic endothelial cells (BAECs). BAEC [Ca2+]i was measured on the single cell level as described in "Materials and Methods." The results presented were obtained with two cells from the same culture. The agents were added at the time indicated by the arrow (2MeSATP, 1 µmol/L; UTP, 10 µmol/L; and ATP, 100 µmol/L). At the end of each stimulation, the incubation buffer was rapidly perfused to wash the agonist out until the [Ca2+]i stabilized. At this time, buffer was added as control; thereafter, the second or third agent under investigation was added as indicated. W indicates wash; B, buffer.

Prostacyclin Release From Freshly Isolated BAECs
UTP stimulates the release of PGI₂ from porcine aortic endothelial cells⁴ but does not induce endothelium-dependent relaxation in the porcine aorta.¹⁸ One possible explanation of this discrepancy would be that the P_2U receptors are not present on the aortic endothelium in situ but are induced in cell culture. To test this hypothesis, we measured the release of PGI₂ from ATP- and UTP-stimulated BAECs shortly after their isolation, as described in "Materials and Methods." Fig 2 shows that both UTP and ATP stimulated the release of PGI₂ from these freshly isolated cells. To exclude the possibility that the response to UTP could be mediated by receptors rapidly neosynthesized as a consequence of the cell isolation procedure, cycloheximide was included in the incubation fluid during the 5-hour period between collagenase digestion and testing (Fig 2) or even in the collagenase-containing harvesting medium (data not shown). The UTP response was maintained in the presence of cycloheximide at a concentration (2 µg/mL=7 µmol/L) known to produce a 95% inhibition of protein synthesis in BAECs.¹⁵ The stimulation induced by UTP was 6.9-fold in the absence of cycloheximide and 5.3-fold in its presence (mean of three independent experiments).

View larger version (39K): [in this window] [in a new window]   Figure 2. Bar graph comparing the effects of ATP and UTP on the release of prostacyclin (PGI2) from freshly isolated bovine aortic endothelial cells (BAECs). BAECs were obtained by collagenase digestion of the bovine aorta and incubated in complete medium with or without 2 µg/mL cycloheximide (Cx) for 5 hours before the stimulation (20 minutes) with ATP (100 µmol/L) or UTP (100 µmol/L). The incubation medium was removed for the measurement of PGI2 production, and results are expressed as mean±SD of triplicate determinations in one representative experiment of three. CONT indicates control; 6-keto-PGF1, 6-ketoprostaglandin F1.

InsP Accumulation in BAECs Stimulated by ATP, UTP, and 2MeSATP
As shown in Fig 3, the time courses of the accumulation of InsP₃ in response to ATP, UTP, and 2MeSATP were fundamentally similar with a rapid and transient action; a maximum stimulation was reached after 10 to 15 seconds of incubation in the presence of the agonist, and the level had almost returned to the baseline after 1 minute (Fig 3). This rapid time course had been observed previously in ATP-stimulated BAECs.^{5 19} If we consider the stimulations at 15 seconds, the responses to 2MeSATP (20 µmol/L) and to UTP (100 µmol/L) represented 63±17% and 94±23%, respectively, of the response to ATP (100 µmol/L) (mean±SD of four independent experiments).

View larger version (24K): [in this window] [in a new window]   Figure 3. Time course of inositol-1,4,5-trisphosphate (InsP3) production in bovine aortic endothelial cells ( BAECs) in response to ATP, 2-methylthio-ATP (2MeSATP), and UTP. [3H]Inositol-labeled cells were incubated for different periods of time with 100 µmol/L ATP (), 100 µmol/L UTP (), and 20 µmol/L 2MeSATP () ( indicates control cells). InsP3 fraction was isolated as described in "Materials and Methods." Results are expressed as mean±SD of triplicate values from one representative experiment of four.

InsP₃ and InsP₄ Isomer Characterization by Ion-Pairing Reverse-Phase HPLC
The time courses of production of Ins(1,4,5)P₃, Ins(1,3,4,5)P₄, and Ins(1,3,4)P₃ were comparable in UTP and in 2MeSATP-stimulated cells (Fig 4) and were also similar to that previously reported for the effect of ATP²⁰ : (1) The accumulation of Ins(1,4,5)P₃ was abundant, rapid in onset (maximum in 15 to 30 seconds), and transient (return to baseline in 1 minute). (2) Ins(1,3,4,5)P₄ also rapidly increased after the addition of the agonist (maximum level was obtained within the minute) but to a lesser extent than Ins(1,4,5)P₃, and its level had only slightly decreased after 10 minutes. (3) Significant increase of the Ins(1,3,4)P₃ level was observed after only 30 seconds of stimulation, and the maximal production obtained after 1 minute was maintained for at least 10 minutes. The ratio between the three isomers was the same after stimulation of the cells with UTP or with 2MeSATP (Fig 4).

View larger version (19K): [in this window] [in a new window]   Figure 4. Time course of accumulation of inositol-1,4,5-trisphosphate [Ins(1,4,5)P3], inositol-1,3,4,5-tetrakisphosphate [Ins(1,3,4,5)P4], and inositol-1,3,4-trisphosphate [Ins(1,3,4)P3] in bovine aortic endothelial cells (BAECs) stimulated with UTP and 2-methylthio-ATP (2MeSATP). BAECs labeled with [3H]inositol were incubated in the presence of 100 µmol/L UTP () or 20 µmol/L 2MeSATP () for different times between 15 seconds and 10 minutes. represents unstimulated cells. Inositol phosphates were extracted and separated on a µBondapack C18 column as described in "Materials and Methods." Results are expressed as mean±SD of triplicate determinations in one representative experiment of two.

Differential Modulation of the Accumulation of InsP₃ by Protein Kinase C
We investigated the effect of protein kinase C (PKC) activation by PMA on the production of InsP₃ induced by ATP, UTP, and 2MeSATP. The response to 2MeSATP was rapidly and markedly inhibited after pretreatment of the cells with PMA (50 nmol/L) (Fig 5): the inhibitions were 74% after a pretreatment of 6 minutes, 92% after 12 minutes, and 100% after 30 minutes (mean of two independent experiments). The stimulation of InsP₃ accumulation by UTP was inhibited by PMA to a lower extent and with a slower time course (Fig 5): 16% after 6 minutes, 23% after 12 minutes, and 42% after 30 minutes (mean of two independent experiments). To exclude the possibility that the greater sensitivity to PMA of the 2MeSATP response was due to its lower magnitude compared with the UTP response, we investigated the effect of PMA (50 nmol/L) at different concentrations of UTP and 2MeSATP, producing stimulations of overlapping intensities. As shown on Fig 6, the inhibition by PMA was stronger for 2MeSATP (70% to 100%) than for UTP (10% to 30%) at all concentrations tested. In particular, at concentrations of agonists producing comparable effects on InsP₃, the inhibitions were, respectively, 28±8% for UTP (10 µmol/L) and 83±9% for 2MeSATP (1 µmol/mL) (mean±SD of three independent experiments). As shown in Fig 7, we observed that the inhibition of the response to ATP after a pretreatment of 10 minutes of the cells with PMA was intermediately between that of the UTP and 2MeSATP responses. The inhibitions observed were 46±19% for 100 µmol/L ATP, 25±22% for 100 µmol/L UTP, and 82±16% for 20 µmol/L 2MeSATP (mean±SD of seven independent experiments). This is consistent with the involvement of both P_2Y and P_2U receptors in the response to ATP. In contrast with its selective effect on the P_2Y-mediated generation of InsP₃, PMA reduced the rise in [Ca²⁺]_i induced by both 2MeSATP and UTP (data not shown). This result is not unexpected; in many cell types, PMA lowers [Ca²⁺]_i independently from InsP₃, at least partially by stimulating Ca²⁺ extrusion.^{21 22 23}

View larger version (32K): [in this window] [in a new window]   Figure 5. Time course (bar graph) of the effect of phorbol 12-myristate 13-acetate (PMA) on the UTP- and 2-methylthio-ATP (2MeSATP)-induced inositol trisphosphate (InsP3) production in cultured bovine aortic endothelial cells (BAECs). [3H]Inositol-labeled cells were challenged with PMA (50 nmol/L) for 6, 12, or 30 minutes and then incubated with no agents (CONT), 2MeSATP (20 µmol/L), or UTP (100 µmol/L) for 15 seconds. InsP3 fraction was isolated, and results are expressed as mean±SD of triplicate determinations in one representative experiment of two.

View larger version (14K): [in this window] [in a new window]   Figure 6. Effect of phorbol 12-myristate 13-acetate (PMA) on the UTP- and the 2-methylthio-ATP (2MeSATP)-induced inositol trisphosphate (InsP3) production in cultured bovine aortic endothelial cells (BAECs). Concentration-action curves of UTP and 2MeSATP are shown. [3H]InsP3-labeled BAECs were exposed 10 minutes to 50 nmol/L PMA before the stimulation (15 seconds) with increasing concentrations of UTP and 2MeSATP. CONT indicates control cells. InsP3 fraction was isolated, and results are expressed as mean±SD of triplicate determinations in one representative experiment of three.

View larger version (25K): [in this window] [in a new window]   Figure 7. Bar graph showing the effect of phorbol 12-myristate 13-acetate (PMA) on the ATP-, UTP-, and 2-methylthio-ATP (2MeSATP)-induced inositol trisphosphate (InsP3) production in bovine aortic endothelial cells. [3H]InsP3-labeled cells were challenged with PMA (50 nmol/L) for 10 minutes and then incubated with no agents (CONT), ATP (100 µmol/L), UTP (100 µmol/L), or 2MeSATP (20 µmol/L) for 15 seconds. InsP3 fraction was isolated as described in "Materials and Methods." Results are expressed as mean±SD of triplicate determinations in one representative experiment of seven.

Choline Release
In BAECs, ATP stimulates the phospholipase D hydrolysis of phosphatidylcholine into choline and phosphatidic acid.^{24 25} 2MeSATP and UTP also stimulated the release of [³H]choline metabolites from BAECs, and they produced very similar maximal responses (Fig 8): the maximal responses to UTP (100 µmol/L) and 2MeSATP (20 µmol/L) represented, respectively, 98±5% and 93±13% of the responses to ATP (100 µmol/L) (mean±SD of five independent experiments). Considering the importance of PKC in the action of ATP on the phosphatidylcholine metabolism in BAECs,²⁴ we investigated the effect of a 24-hour exposure of the cells to PMA (500 nmol/L) on the UTP and 2MeSATP responses. Such a long pretreatment is known to deplete the cells in PKC.²⁴ As shown on Fig 9, top, the release of choline induced by ATP, UTP, and 2MeSATP was completely abolished after prolonged PMA treatment, whereas the InsP₃ accumulation induced by the same agonists was not affected (Fig 9, bottom). These data indicate that PKC plays a crucial role in the activation of phospholipase D by both P_2Y and P_2U receptors, at a step distal to phospholipase C activation.

View larger version (16K): [in this window] [in a new window]   Figure 8. Concentration-action curves showing the action of ATP, UTP, and 2-methylthio-ATP (2MeSATP) on the [3H]choline metabolite release from bovine aortic endothelial cells. [3H]Choline-labeled cells were incubated for 40 minutes with increasing concentrations of ATP (), UTP (), or 2MeSATP (). Results are expressed as mean±SD of triplicate determinations in one representative experiment of five.

View larger version (31K): [in this window] [in a new window]   Figure 9. Top, Bar graph showing the effect of protein kinase C depletion on the release of [3H]choline metabolites from bovine aortic endothelial cells (BAECs). [3H]Choline-labeled cells were treated with or without phorbol 12-myristate 13-acetate (PMA, 500 nmol/L) for 24 hours and then incubated 40 minutes in the absence of agents (CONT) or in the presence of ATP (100 µmol/L), UTP (100 µmol/L), 2-methylthio-ATP (2MeSATP, 20 µmol/L), or PMA (50 nmol/L). Results are expressed as mean±SD of triplicate determinations in one representative experiment of three. Bottom, Bar graph showing the effect of protein kinase C depletion on the production of inositol trisphosphate (InsP3) in cultured BAECs. [3H]InsP3-labeled BAECs were incubated in the presence or in the absence of 500 nmol/L PMA for 24 hours before the stimulation (15 seconds) with the agonist (100 µmol/L ATP, 100 µmol/L UTP, or 20 µmol/L 2MeSATP). InsP3 fraction was isolated, and results are expressed as mean±SD of triplicate determinations in one representative experiment of two.

Protein Phosphorylation
The phosphorylation of BAEC proteins has been analyzed previously by Demolle et al,¹⁷ who used two-dimensional gel electrophoresis; their study revealed an increased phosphorylation of 18-, 28-, and 36-kD substrates after a 10-minute stimulation by ATP. These various substrates were also phosphorylated in response to UTP and 2MeSATP (see arrows on Fig 10), and no consistent difference could be detected between the phosphorylation patterns induced by the two agonists.

View larger version (78K): [in this window] [in a new window]   Figure 10. Pattern of protein phosphorylation in bovine aortic endothelial cells in response to UTP and 2-methylthio-ATP (2MeSATP). Cells were labeled 6 hours with [32P]phosphate (500 µCi/mL) and then stimulated 10 minutes with UTP (100 µmol/L) and 2MeSATP (20 µmol/L). The phosphorylated proteins were resolved by two-dimensional gel electrophoresis, as previously described.17 MW indicates molecular weight; pHi, isoelectric pH; and CONT, absence of agonist.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

 
There are numerous examples of heterogeneity among endothelial cells, which in some cases can be detected inside a single culture dish. Takeda et al²⁶ observed an inwardly rectifying K⁺ current in 100% of the BAECs that they impaled. However, only one third of the cells exhibited a rapidly inactivating outward K⁺ current activated by depolarization (A-type current). Both classes of cells could be found in the same culture dish. A T-type transient Ca²⁺ current has been found in 100% of the bovine adrenal medullary endothelial cells investigated, but only 25% of them had an additional sustained current involving L-type channels.²⁷ In cultures of rat cerebral vascular endothelial cells, subpopulations of -glutamyltranspeptidase–positive cells have been identified; these cells were few in number and randomly distributed in the monolayers.²⁸ Therefore, the hypothesis that P_2Y and P_2U receptors might be segregated on distinct subpopulations of BAECs deserved to be tested. Our measurements of single-cell [Ca²⁺]_i have demonstrated that most BAECs respond to both 2MeSATP and UTP, indicating the colocalization of P_2Y and P_2U receptors on the same cells. Nevertheless, the existence of small subpopulations of cells responsive only to UTP (or 2MeSATP) cannot be excluded.

It is known that UTP stimulates the release of PGI₂ from cultured porcine aortic endothelial cells⁴ but does not induce an endothelium-dependent relaxation of the porcine aorta, unlike 2MeSATP, which behaves as a full agonist.¹⁸ Analogous with the observation that muscarinic receptor expression differs between freshly isolated and cultured endothelial cells,²⁹ one possible explanation for this discrepancy could be that P_2U receptors are not expressed on endothelial cells in situ in the aorta but appear in culture. This is not the case, since we have now shown that UTP mimics the stimulatory effect of ATP on PGI₂ release from BAECs shortly after their isolation by collagenase digestion. Furthermore, inclusion of cycloheximide in the incubation medium, or even in the collagenase-containing collection fluid, did not decrease the response to UTP. These results allow us to exclude the possibility that P_2U receptors were rapidly neosynthesized in response to the isolation procedure and indirectly support the view that they are expressed on endothelial cells in situ in the bovine aorta. This conclusion is consistent with the recent report that both P_2Y and P_2U receptors mediate an endothelium-dependent relaxation of bovine intercostal arteries.³⁰ The lack of P_2U-mediated endothelium relaxation in the porcine aorta¹⁸ remains thus unexplained.

In the present study, ATP, UTP, and 2MeSATP induced InsP₃ accumulation with a similar rapid time course, characteristic of aortic endothelial cells^{5 19 20} ; the time course is definitely slower in microvascular endothelial cells,^{5 10} probably as a result of higher 3-kinase activity.⁵ Our results differ from those of Purkiss et al,³¹ who reported that the response to UTP was more sustained than that to 2MeSATP. The reason for that discrepancy is unclear; it might be related to a difference in methodology, since these authors used a mass assay of InsP₃, whereas we used a radiolabeling procedure. The possibility of a receptor-specific regulation of InsP₃-kinase is supported by the observation that in human umbilical vein endothelial cells, histamine induces a larger accumulation of InsP₄ than thrombin, although the rate of Ins(1,4,5)P₃ formation is similar in response to the two agonists.³² Therefore, we compared the time course and relative magnitude of Ins(1,4,5)P₃, Ins(1,3,4)P₃, and InsP(1,3,4,5)P₄ accumulation in response to UTP versus 2MeSATP but also found no difference. Like ATP, UTP and 2MeSATP stimulated the release of choline from prelabeled cells, presumably as a consequence of phospholipase D activation.^{24 25} The effects of the three agonists were inhibited to the same extent after a prolonged incubation of the cells with PMA to downregulate protein kinase C, whereas under the same experimental conditions, their effect on InsP₃ accumulation was maintained. These results indicate that the activation of phospholipase D mediated by either P_2Y or P_2U receptors is dependent on PKC at a step distal to phospholipase C activation.

One difference that we have identified between P_2Y and P_2U receptor-mediated phospholipase C activation in BAECs is a differential sensitivity to desensitization by PMA. The effect of 2MeSATP on InsP₃ was more strongly and more rapidly inhibited by PMA than was the UTP effect. The difference between the two agonists was observed over the entire concentration-action curve and in particular at concentrations of these agonists that produced a comparable stimulation of InsP₃ formation. The inhibition of the ATP response was intermediately between the inhibition of the UTP and 2MeSATP effects. This differential sensitivity to PMA has been observed also by Purkiss et al³¹ and is reminiscent of the differential effect of pertussis toxin on the formation of InsP₃ induced by UTP or 2MeSATP¹ : pertussis toxin inhibited the response to UTP and to ATP in a similar way but had no effect on the response to 2MeSATP. In this respect, the P_2Y receptor behaves like the thrombin and bradykinin receptors, whose coupling to phospholipase C in endothelial cells is insensitive to pertussis toxin but inhibited by PMA.^{33 34 35 36} It is currently accepted that receptor-mediated phospholipase C activation can involve distinct G proteins: either a member of the pertussis toxin–insensitive G_q/G₁₁ family or a pertussis toxin–sensitive G_i protein.^{37 38} The ß₁ isoform of phospholipase C is activated by _q/₁₁, whereas the ß dimer of G_i2 stimulates the ß₂ isoform of phospholipase C.³⁸ Taken together, the data on endothelial cells are consistent with the hypothesis that the P_2Y receptors are coupled to G_q/G₁₁, whereas the P_2U receptors would be coupled to G_i2.

	Acknowledgments

 
This study was supported by the Belgian Programme on Interuniversity Poles of Attraction initiated by the Belgian State, Prime Minister's Office, Federal Service for Science, Technology, and Culture, by an Action de Recherche Concertée of the Communauté Française de Belgique, by the Fonds de la Recherche Scientifique Médicale, and by a special grant from the National Bank of Belgium. We are grateful to Dr J.E. Dumont for his continuous interest and generous support. Dr Communi is a fellow of the Institut pour l'Encouragement de la Recherche Scientifique Dans l'Industrie et l'Agriculture; Drs Raspé and Pirotton are Chargé de Recherche of the Fonds National de la Recherche Scientifique.

Received January 5, 1994; accepted October 31, 1994.

	References

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