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British Journal of Pharmacology logoLink to British Journal of Pharmacology
. 2019 Jul 6;176(16):2894–2904. doi: 10.1111/bph.14715

Characterisation of P2Y2 receptors in human vascular endothelial cells using AR‐C118925XX, a competitive and selective P2Y2 antagonist

Markie O Muoboghare 1, Robert M Drummond 1, Charles Kennedy 1,✉
PMCID: PMC6637037  PMID: 31116875

Abstract

Background and Purpose

There is a lack of potent, selective antagonists at most subtypes of P2Y receptor. The aims of this study were to characterise the pharmacological properties of the proposed P2Y2 receptor antagonist, AR‐C118925XX, and then to use it to determine the role of P2Y2 receptors in the action of the P2Y2 agonist, UTP, in human vascular endothelial cells.

Experimental Approach

Cell lines expressing native or recombinant P2Y receptors were superfused constantly, and agonist‐induced changes in intracellular Ca2+ levels monitored using the Ca2+‐sensitive fluorescent indicator, Cal‐520. This set‐up enabled full agonist concentration–response curves to be constructed on a single population of cells.

Key Results

UTP evoked a concentration‐dependent rise in intracellular Ca2+ in 1321N1‐hP2Y2 cells. AR‐C118925XX (10 nM to 1 μM) had no effect per se on intracellular Ca2+ but shifted the UTP concentration–response curve progressively rightwards, with no change in maximum. The inhibition was fully reversible on washout. AR‐C118925XX (1 μM) had no effect at native or recombinant hP2Y1, hP2Y4, rP2Y6, or hP2Y11 receptors. Finally, in EAhy926 immortalised human vascular endothelial cells, AR‐C118925XX (30 nM) shifted the UTP concentration–response curve rightwards, with no decrease in maximum.

Conclusions and Implications

AR‐C118925XX is a potent, selective and reversible, competitive P2Y2 receptor antagonist, which inhibited responses mediated by endogenous P2Y2 receptors in human vascular endothelial cells. As the only P2Y2‐selective antagonist currently available, it will greatly enhance our ability to identify the functions of native P2Y2 receptors and their contribution to disease and dysfunction.

Abbreviations

1321N1‐hP2Y1

1321N1 cells stably expressing the human P2Y1 receptor

1321N1‐hP2Y11

1321N1 cells stably expressing the human P2Y11 receptor

1321N1‐hP2Y2

1321N1 cells stably expressing the human P2Y2 receptor

1321N1‐hP2Y4

1321N1 cells stably expressing the human P2Y4 receptor

1321N1‐rP2Y6 cells

1321N1 cells stably expressing the rat P2Y6 receptor

95% cl

95% confidence limits

AU

arbitrary units

CRC

concentration–response curve

NAM

negative allosteric modulator

What is already known

  • P2Y receptors are expressed throughout the body, but their functions are largely unclear.

  • There is a lack of potent, selective antagonists at most subtypes of P2Y receptor.

What this study adds

  • AR‐C118925XX is a potent, selective and reversible, competitive P2Y2 receptor antagonist.

  • AR‐C118925XX inhibited responses evoked by UTP in EAhy926 immortalised human vascular endothelial cells.

What is the clinical significance

  • AR‐C118925XX is the only potent, selective, and competitive P2Y2 antagonist currently available.

  • AR‐C118925XX will help identify the functions of native P2Y2 receptors and their contribution to disease.

1. INTRODUCTION

P2Y receptors are a family of eight GPCRs that mediate the actions of the endogenous nucleotides, ATP, ADP, UTP, and UDP (Abbracchio et al., 2006; Kennedy, Chootip, Mitchell, Syed, & Tengah, 2013; Rafehi & Müller, 2018). They are expressed in cells and tissues throughout the body, but their physiological functions are largely unclear. In part, this is because the endogenous agonists all act at multiple P2Y subtypes, but the major factor is the low potency and selectivity of many of the available antagonists. Currently, potent, selective antagonists have only been developed for P2Y1 (e.g., MRS2179, MRS2279, and MRS2500) and P2Y12 (e.g., ticagrelor, cangrelor, and clopidogrel) receptors (see Abbracchio et al., 2006; Kennedy et al., 2013). These played a major role in identifying physiological roles, such as that of P2Y1 receptors in peristalsis in the gut (see Kennedy, 2015) and of P2Y1 and P2Y12 receptors in platelet aggregation (Abbracchio et al., 2006; von Kügelgen, 2017). Clearly, the development of potent and selective antagonists at the other P2Y subtypes would greatly enhance our ability to determine their functions in health and disease.

AR‐C118925XX was developed by AstraZeneca around 20 years ago as a P2Y2 antagonist, but only a conference abstract was published at the time (Meghani, 2002), which did not include crucial pharmacological properties, such as its KB or pA2. Several studies have since been published that used AR‐C118925XX to investigate the role of native P2Y2 receptors in the actions of P2Y agonists in various cell types (Cosentino et al., 2012; Gabl et al., 2016; Hochhauser et al., 2013; Kemp, Sugar, & Jackson, 2004; Magni, Merli, Verderio, Abbracchio, & Ceruti, 2015; Onnheim et al., 2014; Wang et al., 2015; see also review by Rafehi & Müller, 2018). They did not, however, report the KB or pA2 of AR‐C118925XX or relate the concentrations used (mostly 1 and 10 μM) to its potency or selectivity. Inhibition of other P2Y subtypes could not, therefore, be ruled out based on the data published at this time.

Accurate values of antagonist potency are essential for effective experimental use. Recently, AR‐C118925XX became commercially available, so the aims here were to quantify the pA2 of AR‐C118925XX at recombinant P2Y2 receptors stably expressed in a cell line. Selectivity was then determined by studying its effects at other P2Y subtypes. Finally, AR‐C118925XX was used to investigate native P2Y2 receptors in human vascular endothelial cells. Using a system that enabled full agonist concentration–response curves (CRCs) to be constructed in the absence and presence of AR‐C118925XX on a single population of cells, we found that AR‐C118925XX is a very potent, selective, and reversible P2Y2 receptor antagonist. Furthermore, it inhibited responses evoked by UTP in human vascular endothelial cells, indicating expression of endogenous P2Y2 receptors. Thus, the development of AR‐C118925XX and characterisation of its pharmacological properties remove a substantial barrier to our ability to identify the functions of native P2Y2 receptors.

2. METHODS

2.1. Cell culture

1321N1 (ECACC Cat# 86030402, RRID:CVCL_0110) is a human astrocytoma cell line that does not endogenously express any of the eight P2Y receptor subtypes or respond to the naturally occurring nucleotide agonists, such as UTP and ATP (Abbracchio et al., 2006; Filtz, Li, Boyer, Nicholas, & Harden, 1994; Parr et al., 1994). 1321N1 cells stably expressing recombinant human P2Y1 (1321N1‐hP2Y1), hP2Y2 (1321N1‐hP2Y2), P2Y4 (1321N1‐hP2Y4), P2Y11 (1321N1‐hP2Y11) or rat P2Y6 (1321N1‐rP2Y6) receptors, tSA201 (ECACC Cat# 96121229, RRID:CVCL_2737) and EAhy926 cells (ATCC Cat# CRL‐2922, RRID:CVCL_3901) were used. They were maintained in 5% CO2, 95% O2 in a humidified incubator at 37°C, in DMEM (Life Technologies, Paisley, UK, catalogue numbers 21969‐035‐1321N1, tSA201 cells, 41965‐039—EAhy926 cells), supplemented with 10% fetal calf serum, 1% non‐essential amino acids, and 1% penicillin (10,000 U·ml−1) and streptomycin (10 mg·ml−1). Prior to recording intracellular Ca2+, the cells were plated onto 13‐mm glass coverslips coated with poly‐l‐lysine (0.1 mg·ml−1) and experiments performed once a confluent monolayer of cells had developed. Experiments were performed unblinded and unrandomised, as the experimenter (M. M.) carried out all cell culture and was aware of which cell line was being used.

2.2. Ca2+ imaging

Cells were bathed in a buffer composed of (mM): NaCl, 122; KCl, 5; HEPES, 10; KH2PO4, 0.5; NaH2PO4, 0.5; MgCl2, 1; glucose, 11; and CaCl2, 1.8, titrated to pH 7.3 with NaOH. Intracellular Ca2+ was monitored using the Ca2+‐sensitive fluorescent indicator, Cal‐520. Cells on a coverslip were incubated for 1 hr at 37°C in the dark in buffer containing Cal‐520‐AM ester (5 μM) and Pluronic™ F‐127 (0.05% w/v in DMSO). The coverslip was then placed vertically in the recording chamber of a Perkin Elmer LS50B luminescence spectrophotometer and the cells superfused continuously with buffer, applied under gravity at 4 ml·min−1 at room temperature.

Cal‐520 fluorescence, measured as arbitrary units (AU) in a population of cells, was sampled at 10 Hz following stimulation at 490 ± 15 nm and the emission recorded at 525 ± 15 nm using FL Winlab software (V4.00.02). Resting Ca2+ levels were stable over the course of the experiment. Agonists were added in the superfusate until the response reached a peak (60–90 s) at 10‐min intervals. For each drug addition, the data were exported to GraphPad Prism v7.01 (GraphPad, San Diego, CA, USA), where peak response amplitude was determined by the experimenter (M. M.) manually placing a cursor on the baseline and peak. All measurements were inspected and confirmed by C. K.

2.3. Experimental protocols

The experimental protocols and design adhere to the recommendations of Curtis et al. (2018).

2.4. Determining the effects of UTP and AR‐C118925XX at hP2Y2 receptors

This experimental set‐up enabled full agonist CRCs to be constructed on a single population of cells. All coverslips of 1321N1‐hP2Y2 cells were first exposed to UTP (1 μM) twice to confirm cell viability. CRCs were then generated by superfusing cells with increasing concentrations of UTP at 10‐min intervals; hence, drug addition was not randomised. Reproducibility of these responses was determined by then generating a second CRC on the same population of cells. To facilitate comparison of the two curves, the data were normalised by calculating each response in AU as a percentage of that to UTP (1 μM) in the first CRC. This concentration is close to but not quite at the top of the UTP CRC. The second CRC also served as a time‐matched control for the effects of AR‐C118925XX, as described next. EC50 and maximum values calculated by fitting the Hill equation to the two sets of data were compared using Student's paired t test.

The effects of AR‐C118925XX at hP2Y2 receptors were determined by generating two UTP CRCs for each coverslip of 1321N1‐hP2Y2 cells. The first, to UTP alone, served as the control. The cells were then superfused with a given concentration of AR‐C118925XX for 5 min. Thereafter, the second UTP CRC was constructed in the continuous presence of that concentration. Again, the data were normalised by calculating each response in AU as a percentage of that to UTP (1 μM) in the first CRC. The dose ratio for the rightwards shift induced by AR‐C118925XX was calculated from the EC50 values of the two curves. The data generated using a range of concentrations of AR‐C118925XX were pooled and used to construct a Schild plot. To determine if the maximum responses to ADP were reduced by AR‐C118925XX, the maximum values calculated by fitting the Hill equation to the second CRC were compared with those from the time‐matched controls described in the previous paragraph, using one‐way ANOVA with Tukey's comparison.

Reversibility of the inhibitory effects of AR‐C118925XX on washout was investigated by first exposing cells to UTP (1 μM) twice to confirm cell viability and then repeatedly adding UTP (100 nM), a concentration that is just above the EC50, at 10‐min intervals. Under these conditions, UTP (100 nM) elicits highly reproducible responses. Once a control response to UTP was obtained, AR‐C118925XX was applied to the cells for 5 min, before co‐administration with UTP. The antagonist was then washed out and the recovery of the UTP response monitored over the next 20–70 min, as appropriate. Since the point of this experiment was to determine if the responses fully recovered, the data were normalised by calculating the response in AU as a percentage of the control response to UTP. No statistical tests were applied to these data.

2.5. Selectivity of AR‐C118925XX

In preliminary experiments, CRCs for an appropriate agonist were constructed in cells expressing the other P2Y subtypes that couple to Ca2+ mobilisation and from these two concentrations were chosen that evoked responses that were (a) close to the top of the CRC (reference concentration) and (b) 50–75% of the maximum response (test concentration), as follows: 1321N1‐hP2Y1—ADP (1 μM/100 nM), 1321N1‐hP2Y4—UTP (10 μM/1 μM), 1321N1‐rP2Y6—UDP (1 μM/100 nM), 1321N1‐hP2Y11—ATP (10 μM/2 μM), and tSA201 cells—ADP (10 μM/1 μM). All coverslips were first exposed to the higher concentration of agonist twice to confirm cell viability. The response also served as a reference for statistical analysis, as described in the next paragraph. The lower, test concentration was then applied repeatedly at 10‐min intervals, and once a control response was obtained, AR‐C118925XX (1 μM) was applied to the cells for 5 min before co‐administration with the agonist.

Since the aim of this experiment was to determine if AR‐C118925XX acts as an antagonist at the other P2Y subtypes, the data are presented as a percentage of the agonist control response. To enable parametric statistical analysis, however, responses in a given cell line to the agonist test concentration were calculated as a percentage of the initial response to the agonist reference concentration. The values obtained in the absence and then presence of AR‐C118925XX were then compared using Student's paired t test.

2.6. Investigating the presence of P2Y2 receptors in EAhy926 cells

Ca 2+ imaging UTP CRCs were constructed in the absence and presence of AR‐C118925XX and analysed statistically in the same manner as described above for 1321N1‐hP2Y2 cells, except that (a) cells were first exposed to UTP (10 μM) twice to confirm cell viability and (b) data were normalised by calculating each response in AU as a percentage of that to UTP (10 μM) in the first CRC.

Immunostaining The immuno‐related procedures used comply with the recommendations made by the British Journal of Pharmacology. P2Y2 receptor protein expression was studied using a modified version of the methods we used recently to demonstrate P2Y2 expression in rat isolated carotid arteries (Lee et al., 2018). Briefly, cells were fixed in 4% paraformaldehyde for 20 min at room temperature, washed three times in 100‐μM glycine solution, then three times with a buffer composed of (mM): NaCl, 137; KCl, 2.7; NaH2PO4, 1.5; and Na2HPO4, 15.2, pH 7.4. Cells were permeabilised with 0.2% Triton X‐100 in 5‐mM NH4Cl solution for 10 min and washed three times with buffer. They were then incubated in antibody buffer solution containing 5% BSA for 1 hr at room temperature, followed by three washes with antibody wash solution containing 5% BSA. Next, they were incubated with a rabbit anti‐P2Y2 receptor (H‐70) polyclonal IgG antibody raised against an epitope corresponding to amino acids 308–370 of the C‐terminus (cat no. sc‐20124, batch no. A1303, RRID:AB_2156139, 1:100, Santa Cruz, Dallas, TX, USA) in buffer containing 5% BSA and 2% donkey serum, overnight at 4°C. They were then washed three times with antibody wash solution containing 5% BSA, followed by incubation with Alexa Fluor 488 donkey anti‐rabbit polyclonal IgG antibody raised against rabbit γ immunoglobin (cat no. R37118, batch no. A21206, RRID AB_2556546, 1:1,000, Life Technologies) in antibody buffer solution for 1 hr at room temperature, followed by three washes with antibody wash solution without BSA and finally incubated in buffer before imaging. Negative controls were performed in the absence of the primary or secondary antibody. To visualise their nucleus, cells were incubated in buffer containing 0.5 mg·ml−1 DAPI. Each solution was used once only.

The Alexa Fluor 488 antibody was excited using 488‐nm wide‐field epifluorescence illumination provided by a LED (CoolLED pE‐300ultra, CoolLED Ltd, Andover, UK) and visualised using a back‐illuminated electron‐multiplying charge coupled device camera (iXon Life 888; Andor, Belfast, UK; 13‐μm pixel size) through a 40× (oil immersion; numerical aperture 1.3; Nikon S Fluor) objective lens. Fluorescence emission was recorded at 10 Hz. Fluorescence illumination was controlled, and images (16‐bit depth) were captured, using ImageJ (National Institutes of Health, Bethesda, MD, USA). DAPI was excited at 365 nm and fluorescence recorded and visualised in the same way. Images (8‐bit depth) were then analysed and prepared for publication using ImageJ.

2.7. Data and statistical analysis

The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology (Curtis et al., 2015). Experiments were designed to have an equal n per group within each protocol based on previous studies on the individual cell lines (Kennedy, Herold, Qi, Harden, & Nicholas, 2000; Morrow, Nicholas, & Kennedy, 2014) and where n = individual coverslips of cells. Coverslips were excluded if the initial responses to the high concentration of agonist were too small for responses to lower concentrations to be measured accurately and with confidence. Values in the text and figures refer to mean ± SEM or geometric mean with 95% confidence limits (95% cl) for EC50 values. When appropriate, CRCs were fitted to the data by logistic (Hill equation), non‐linear regression analysis (GraphPad Prism v7.01), and EC50 and maximum values were calculated. The Gaddum–Schild equation was used to calculate the dissociation constant (KB) of AR‐C118925XX in EAhy926 cells. Statistical analysis was performed using Student's paired or unpaired t test, or one‐way ANOVA, as appropriate and as described in each experimental protocol above. Differences were considered significant when P < .05. ANOVA post hoc tests were conducted only if F was significant and there was no variance in homogeneity.

2.8. Materials

ATP (Na2 salt, cat. no. A7699), ADP (Na salt, cat. no. A2754), UTP (Na3(H2O)2 salt, cat. no. 94370), and UDP (Na salt, cat. no. U4125; Sigma‐Aldrich Co, Gillingham, Dorset, UK) were dissolved in deionised water as 10‐mM stock solution. AR‐C118925XX (Tocris, Bristol, UK) was dissolved in DMSO as a 10‐mM stock. All were frozen immediately and stored at −20°C and then diluted in buffer on the day of use. This was performed unblinded, as the experimenter was aware of which compound was being diluted. Cal‐520‐AM ester (Life Technologies) was dissolved in DMSO as a 1‐mM stock solution, frozen immediately, and stored at −20°C. On the day of use, it was diluted in buffer, as described above. Pluronic™ F‐127 (Life Technologies) was supplied as a 20% w/v solution in DMSO and stored at room temperature. DAPI was obtained from Fisher Scientific UK (Loughborough, UK). Common chemicals were supplied by Sigma‐Aldrich Co, Fisher Scientific UK, and VWR International (Lutterworth, UK) and were of the highest purity available; 0.1% DMSO has no effect on Ca2+ levels or nucleotide‐evoked responses in 1321N1 cells expressing recombinant P2Y receptors (Kennedy, unpublished observations).

2.9. Nomenclature of targets and ligands

Key protein targets and ligands in this article are hyperlinked to corresponding entries in http://www.guidetopharmacology.org, the common portal for data from the IUPHAR/BPS Guide to PHARMACOLOGY (Harding et al., 2018), and are permanently archived in the Concise Guide to PHARMACOLOGY 2017/18 (Alexander et al., 2017).

3. RESULTS

3.1. Determination of the pA2 of AR‐C118925XX

The first aim of this study was to calculate the pA2 value of AR‐C118925XX at human P2Y2 receptors. Initial experiments determined the potency of the P2Y2 agonist, UTP, in 1321N1‐hP2Y2 cells and the reproducibility of its action. UTP (10 nM to 3 μM) evoked a concentration‐dependent rise in intracellular Ca2+ with an EC50 = 82 nM (95% cl = 52–112 nM; Figure 1a,b). There was no significant change in the EC50 of a second CRC generated on the same population of cells (107 nM, 95% cl = 51–163 nM), but the maximum response according to the fit of the Hill equation was significantly decreased from 103 ± 1% to 97 ± 1% of the response to UTP (1 μM) in the first CRC (Figure 1b).

Figure 1.

Figure 1

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UTP elicits reproducible CRC in 1321N1‐hP2Y2 cells. (a) The superimposed traces show changes in Cal‐520 fluorescence evoked by superfusion of cells with UTP (10 nM to 3 μM), as indicated by the horizontal bar. All records are from the same population of cells. (b) The mean peak amplitude of responses evoked by UTP is shown (n = 6). Two consecutive CRCs were constructed per coverslip of cells. The data are expressed as a percentage of the response to UTP (1 μM) in the first CRC. Vertical lines show SEM. For some points, the error bars are shorter than the height of the symbol. The curves represent the fit of the Hill equation to the data

Preincubation with AR‐C118925XX (10 nM to 1 μM) had no effect per se on intracellular Ca2+ levels but produced a progressive rightwards shift in the UTP CRC (Figure 2a). The inhibition was surmountable, and there were no differences in the maxima of the second CRCs obtained in the absence and presence of AR‐C118925XX. A Schild plot of these data has an X‐intercept = −8.30 and slope = 0.985 ± 0.028 (Figure 2b), giving a pA2 = 8.43. Thus, AR‐C118925XX appears to be a potent, competitive antagonist at hP2Y2 receptors.

Figure 2.

Figure 2

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AR‐C118925XX is a competitive antagonist at hP2Y2 receptors. (a) The mean peak amplitude of responses evoked by UTP (10 nM to 300 μM) in 1321N1‐hP2Y2 cells in the absence and presence of AR‐C118925XX (10 nM to 1 μM) is shown (n = 6 each). The data are expressed as a percentage of the response to UTP (1 μM) in the control CRC for each coverslip of cells. Vertical lines show SEM. For some points, the error bars are shorter than the height of the symbol. The curves represent the fit of the Hill equation to the data. (b) A Schild plot constructed from the data in panel (a) is shown. The straight line represents the fit of the data by linear regression (r 2 = .0996). The horizontal lines indicate the mean of the values

3.2. Reversibility of the actions of AR‐C118925XX

To determine if the inhibitory effects of AR‐C118925XX are reversible on washout, UTP (100 nM), which is just above its EC50, was applied repeatedly at 10‐min intervals and evoked highly reproducible responses in the absence of AR‐C118925XX (Figure 3). Coapplication of AR‐C118925XX (30 nM to 1 μM) abolished the effect of UTP, but this recovered to time‐matched control values when UTP was reapplied after AR‐C118925XX washout. Thus, the inhibitory actions of AR‐C118925XX reverse fully on washout.

Figure 3.

Figure 3

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The inhibitory actions of AR‐C118925XX are reversible. The graph shows the time course of responses evoked by repeated addition of UTP (100 nM) to 1321N1‐hP2Y2 cells at 10‐min intervals in the absence (0 min), presence (10 min), and following washout of AR‐C118925XX (30 nM to 1 μM; 20–80 min; n = 5 each). AR‐C118925XX was added for 5 min as indicated by the horizontal bar. The data are expressed as a percentage of the control response to UTP, obtained before AR‐C118925XX addition (0 min). The open circles and dashed line show the time‐matched control when AR‐C118925XX was not applied to the cells. Vertical lines show SEM

3.3. Selectivity of AR‐C118925XX

The selectivity of AR‐C118925XX was then investigated by determining the effects of 1 μM, a concentration that is 270 times greater than its KB at P2Y2 receptors (3.7 nM), at the other P2Y subtypes that couple to Ca2+ mobilisation. This high concentration had no effect on basal intracellular Ca2+ levels in any of the cell lines used, nor did it affect the rise evoked by ADP (1 μM) in tSA201 cells, a modified HEK‐293 cell line that expresses endogenous P2Y1 receptors (Shakya Shrestha, Parmar, Kennedy, & Bushell, 2010; Figure 4a,b). Likewise, AR‐C118925XX did not inhibit responses mediated via recombinant hP2Y1, hP2Y4, rP2Y6, or hP2Y11 receptors stably expressed in 1321N1 cells (Figure 4b). Thus, AR‐C118925XX displays a high degree of selectivity for P2Y2 receptors.

Figure 4.

Figure 4

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AR‐C118925XX is selective for hP2Y2 receptors. (a) The superimposed traces show changes in Cal‐520 fluorescence evoked by superfusion of tSA201 cells with ADP (1 μM), as indicated by the horizontal bar, in the absence (black line) and presence (blue line) of AR‐C118925XX (1 μM). All records are from the same population of cells. (b) The peak amplitude of responses evoked by ADP (1 μM) in tSA201 cells, ADP (100 nM) in 1321N1‐hP2Y1 cells, UTP (1 μM) in 1321N1‐hP2Y4 cells, UDP (100 nM) in 1321N1‐rP2Y6 cells, and ATP (2 μM) in 1321N1‐hP2Y11 cells in the presence of AR‐C118925XX (1 μM) is shown (n = 5 each). They are expressed as a percentage of the agonist control response, obtained before AR‐C118925XX addition. The horizontal and vertical lines indicate mean and SEM

3.4. The presence of functional P2Y2 receptors in EAhy926 endothelial cells

The final aim of this study was to determine the role of native P2Y2 receptors in the actions of UTP. EAhy926 cells are an immortalised human vascular endothelial cell line (Edgell, McDonald, & Graham, 1983) that we previously showed to be responsive to UTP (Graham, McLees, Kennedy, Gould, & Plevin, 1996; Paul et al., 2000). UTP (100 nM to 30 μM) increased intracellular Ca2+ in EAhy926 endothelial cells in a concentration‐dependent manner (EC50 = 670 nM, 95% cl = 535–837 nM; Figure 5a,b). There was no significant change in the EC50 value when a second CRC was then constructed on the same population of cells (EC50 = 680 nM, 95% cl = 506–912 nM), but the maximum response was significantly decreased from 108 ± 3% to 96 ± 4% of the response to UTP (10 μM) in the first CRC (Figure 5b).

Figure 5.

Figure 5

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UTP acts at P2Y2 receptors in EAhy926 endothelial cells. (a) The superimposed traces show changes in Cal‐520 fluorescence evoked by superfusion of EAhy926 cells with UTP (100 nM to 30 μM), as indicated by the horizontal bar. All records are from the same population of cells. (b) The mean peak amplitude of responses evoked by UTP (100 nM to 30 μM) when two consecutive CRCs were constructed per coverslip of cells is shown (n = 5). Panel (c) shows the same when the 2nd curve was generated in the presence of AR‐C118925XX (30 nM; n = 5). The data are expressed as a percentage of the response to UTP (10 μM) in the first CRC. Vertical lines show SEM. For some points, the error bars are shorter than the height of the symbol. The curves represent the fit of the Hill equation to the data

Preincubation with AR‐C118925XX (30 nM), a concentration that is less than 10‐fold higher than its KB at P2Y2 receptors and substantially lower than the concentration that we showed aboveto be inactive at other P2Y‐subtypes, had no effect on the basal intracellular Ca2+ level, but shifted the UTP curve rightwards (EC50 = 7.6 µM, 95% cl. = 4.4–13.2 µM), with no decrease in maximum response (92 ± 8% of the response to UTP (10 µM) in the first CRC) relative to that of the time‐matched control (96 ± 4%) (Figure 5C). Gaddum–Schild analysis gave a KB = 3.0 nM.

3.5. Expression of P2Y2 receptors in EAhy926 endothelial cells

To support these pharmacological data, P2Y2 receptor expression in EAhy926 cells was visualised using an anti‐P2Y2 antibody that was previously used to identify an immunoreactive band of the predicted MW of the P2Y2 receptor in Western blots of EA926hy cell lysates (Raqeeb, Sheng, Ao, & Braun, 2011). Furthermore, we recently demonstrated the same antibody displayed immunoreactivity in 1321N1‐hP2Y2, but not wild‐type 1321N1 cells (Lee et al., 2018). Figure 6a shows P2Y2 receptor‐like immunoreactivity in EAhy926 cells. Negative controls show a lack of staining when either the secondary or primary antibody was omitted (Figure 6b,c).

Figure 6.

Figure 6

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P2Y2 receptor immunostaining in EAhy926 endothelial cells. Representative images show nuclear staining by DAPI (blue, left‐hand column), P2Y2 receptor‐like immunoreactivity (green, middle column), and overlay of both (right‐hand column), when cells were incubated with (a) both primary (1°) and secondary (2°) antibodies, (b) the 1° antibody only, and (c) the 2° antibody only. Scale bars = 50 μm

4. DISCUSSION

A major impediment to determining the functions of the majority of P2Y receptor subtypes is the lack of useful antagonists. By measuring changes in Ca2+ levels in cell lines expressing recombinant or native P2Y receptors, we demonstrated that AR‐C118925XX is a very potent, selective, and apparently competitive antagonist at the P2Y2 subtype. Furthermore, by recording from a single population of constantly superfused cells, we were able to show that the inhibitory effects of AR‐C118925XX reversed fully on washout. Finally, combining our knowledge of the pharmacological profile of AR‐C118925XX, with demonstration of P2Y2‐like immunoreactivity, revealed functional expression of endogenous P2Y2 receptors in human vascular endothelial cells. Thus, AR‐C118925XX is a powerful new tool for determining the functions of P2Y2 receptors in health and disease and identifying new therapeutic targets.

4.1. Mode of action of AR‐C118925XX

In this study, UTP evoked a concentration‐dependent rise in cytoplasmic Ca2+ levels in 1321N1‐hP2Y2 cells, with an EC50 of 82 nM, which is close the value (73 nM) we reported previously (Morrow et al., 2014). AR‐C118925XX did not affect basal Ca2+ levels on its own but progressively shifted the UTP CRC rightwards, in a parallel manner and with no decrease in the maximum response. The slope of the Schild plot constructed from these data was almost 1, and the pA2 was 8.43, equivalent to a KB of 3.7 nM. Thus, classical Schild analysis indicates that AR‐C118925XX is a competitive P2Y2 antagonist rather than a negative allosteric modulator (NAM). Thus far, however, the antagonist properties of AR‐C118925XX have only been determined using Ca2+ changes as a bioassay. This is relevant because it is now clear that the apparent mode of antagonism of NAMs can vary depending upon the agonist used, the signalling pathway studied, and the extent of signalling amplification. For example, BPTU, a P2Y1 NAM, caused a progressive rightwards parallel shift of the 2‐methylthioADP CRC, with no decrease in maximum, when inositol phosphate production or ERK1/2 stimulation was measured, but suppressed the maximum when β‐arrestin2‐mediated P2Y1 receptor internalisation was studied (Gao & Jacobson, 2017). Furthermore, BPTU had a different activity profile against another P2Y1 agonist. This biased functional antagonism suggests that each signalling event may be mediated via a specific receptor conformation.

In the present study, AR‐C118925XX had no effect on resting Ca2+. This is important because superfusion‐induced shear stress may induce release of ATP and UTP from endothelial cells, which can then act in an autocrine/paracrine manner to stimulate P2Y receptors in the same or neighbouring cells (Burnstock, 2017; Wang et al., 2015). The lack of effect indicates that either superfusion did not induce nucleotide release or, if it did, the flow rate was fast enough to wash the nucleotides away from the cell surface and so prevent P2Y2 receptor activation. This is consistent with other projects in our laboratory using constant superfusion. In no instance has an antagonist that inhibits an agonist‐induced rise in Ca2+, caused any change in resting Ca2+ level in cells expressing native or recombinant P2Y receptors (Kennedy, unpublished observations).

While this report was in preparation, Rafehi, Burbiel, Attah, Abdelrahman, and Müller (2017) published an improved procedure for synthesis of AR‐C118925XX and details of its pharmacological actions at hP2Y2 receptors expressed in 1321N1 cells. It is notable that the reported potency of AR‐C118925XX (pA2 = 7.43, KB = 37.2 nM) is an order of magnitude lower than ours. Furthermore, it appeared to increase the maximum response induced by UTP, and the Schild slope (0.816) was substantially less than 1. Interestingly, in a subsequent study, Rafehi et al. (2017), the IC50 of AR‐C118925XX against responses evoked by the EC80 of UTP was 62.9 nM. EC80 is theoretically four times EC50, which was 5.61 nM. Applying the Cheng–Prusoff equation generates a KB for AR‐C118925XX of approximately 13 nM, which is closer to the value calculated here. Shortly afterwards, a group from AstraZeneca described the original design and synthesis of AR‐C118925XX and reported pA2 and KB values of 7.8 and 15.8 nM, respectively, at hP2Y2 receptors expressed in Jurkat cells (Kindon et al., 2017). Neither the Schild plot nor the slope of the plot was included, however, so the mode of antagonism cannot be confirmed.

The biggest methodological difference between our study and those of Rafehi, Burbiel, et al. (2017), Rafehi, Neumann, et al. (2017), and Kindon et al. (2017) is that they used multi‐well plates and a microplate reader to record changes in cytoplasmic Ca2+ levels. To generate a CRC, multiple populations of cells were stimulated once only, with a single concentration of UTP. In contrast, we constantly superfused a single population of cells, first generating a control CRC by repeatedly applying UTP in increasing concentrations and then repeating the process in the presence of AR‐C118925XX. This is analogous to organ bath‐type studies, where multiple sets of data can be generated on a single tissue. Such systems have an inbuilt control and less variability than microplate readers, which may be why our Schild plot slope was close to 1. Disadvantages are that superfusion uses more drug and takes longer to generate data. Unsurprisingly, multi‐well plates and microplate readers are much more widely used, but their limitations do need to be noted and considered. Accurate determination of antagonist KB values is essential when considering an effective concentration of antagonist to use when studying native receptors in healthy and diseased cells and tissues.

4.2. Selectivity of AR‐C118925XX

In this study, 1‐μM AR‐C118925XX, a concentration 270 times greater than its KB, had no effect at P2Y1, P2Y4, P2Y6, or P2Y11 receptors, so AR‐C118925XX is highly selective for P2Y2 receptors over the other P2Y subtypes that mobilise Ca2+. This is important, as the endogenous P2Y agonists have complex pharmacological profiles and each stimulates at least two of the eight subtypes. UTP activates not only P2Y2 but also P2Y4 and possibly P2Y6 receptors (Abbracchio et al., 2006; Bar et al., 2008; Guns et al., 2006; Haanes et al., 2016; Kennedy et al., 2013; Rafehi & Müller, 2018), so sensitivity of a cell or tissue to UTP is not proof of P2Y2 receptor expression. Furthermore, no P2Y4 antagonists are currently commercially available, and while the P2Y6 antagonist, MRS2578, has reasonably high potency, its action is non‐surmountable and irreversible (Mamedova, Joshi, Gao, Von Kügelgen, & Jacobson, 2004) and effects at sites other than P2Y6 receptors have been noted (Mitchell, Syed, Tengah, Gurney, & Kennedy, 2012). Kemp et al. (2004) reported that 10‐μM AR‐C118925XX had no effect at 37 other GPCR and ion channels. The only clear indication of an off‐target action of sub‐μM concentrations is at P2X3 receptors, with an IC50 of 819 nM (Rafehi, Burbiel, et al., 2017). The KB was not calculated, however. Nonetheless, AR‐C118925XX is clearly highly selective for P2Y2 receptors, and its introduction into the P2Y pharmacopoeia is a major advance in the purinergic field.

4.3. Native P2Y2 receptors in human endothelial cells

We showed here that AR‐C118925XX also inhibited the rise in Ca2+ evoked by UTP in human EAhy926 vascular endothelial cells, in a surmountable manner. The KB, 3.0 nM, is close to that seen at recombinant hP2Y2 receptors and 333‐fold lower than a concentration (1 μM) that is inactive at other P2Y subtypes. Thus, UTP appears to act via P2Y2 receptors to mobilise Ca2+ in EAhy926 cells. This is consistent with our demonstration of P2Y2‐like immunoreactivity and the detection of P2Y2 mRNA and protein in these cells (Raqeeb et al., 2011).

UTP has long been known to have multiple actions on endothelial cells, including inducing inositol phosphate metabolism, Ca2+ mobilisation, PGI2 and NO release, and vasodilation (Needham, Cusack, Pearson, & Gordon, 1987; O'Connor, Dainty, & Leff, 1991; Ralevic & Burnstock, 1991; Raqeeb et al., 2011; Lustig et al., 1992; Motte, Pirotton, & Boeynaems, 1993; Wilkinson, Purkiss, & Boarder, 1993), but its site of action was unclear. As noted above, UTP stimulates several P2Y subtypes, and mRNA and, to a lesser extent protein, for most P2Y subtypes are found in endothelial cells (Burnstock & Knight, 2004; Erlinge & Burnstock, 2008). Some insight has been provided by P2Y2 receptor knockout (Bar et al., 2008; Guns et al., 2006; Haanes et al., 2016) and knockdown (Raqeeb et al., 2011), but although these are powerful techniques, they have limitations. Potent, selective, competitive antagonists, like AR‐C118925XX, have the advantages of ease of use and applicability in humans and are a powerful, complimentary tool for studying receptor function. We recently used the dual approach of AR‐C118925XX and immunoreactivity to show that P2Y2 receptors are present in rat carotid artery endothelial cells and couple to Ca2+ mobilisation (Lee et al., 2018). Thus, P2Y2 receptors may be a major site of action of UTP in vascular endothelial cells in general.

4.4. Conclusion

P2Y2 receptors are expressed in many tissues and cell types in humans, but the lack of useful antagonists has hindered determination of their physiological and pathophysiological roles. Nonetheless, potential therapeutic targets have been proposed, including colorectal cancer (Gendron, Placet, & Arguin, 2017), atherosclerosis, nephrogenic diabetes insipidus, and osteoporosis (see Rafehi, Burbiel, et al., 2017; Rafehi & Müller, 2018). As the only potent, selective, and competitive P2Y2 antagonist currently available, AR‐C118925XX will be invaluable in identifying native P2Y2 receptor function and their relevance as a target for the development of novel therapeutic agents.

AUTHOR CONTRIBUTIONS

M.O.M., R.M.D. and C.K. designed the experiments. M.O.M. and C.K. conducted the experiments, performed data analysis and interpreted the data. C.K. drafted the manuscript, which was critically revised by M.O.M. and R.M.D.. All authors approved the final version.

CONFLICT OF INTEREST

The authors declare no conflicts of interest.

DECLARATION OF TRANSPARENCY AND SCIENTIFIC RIGOUR

This Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research as stated in the BJP guidelines for Design & Analysis, and Immunoblotting and Immunochemistry, and as recommended by funding agencies, publishers, and other organisations engaged with supporting research.

ACKNOWLEDGEMENTS

The authors thank Matthew Lee and Dr Calum Wilson for their help and advice in visualising hP2Y2 receptor expression.

Muoboghare MO, Drummond R, Kennedy C. Characterisation of P2Y2 receptors in human vascular endothelial cells using AR‐C118925XX, a competitive and selective P2Y2 antagonist. Br J Pharmacol. 2019;176:2894–2904. 10.1111/bph.14715

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