Evaluation of reactive blue 2 derivatives as selective antagonists for P2Y receptors

www.elsevier.com/locate/vph

Vascular Pharmacology 39 (2003) 309–315

Evaluation of reactive blue 2 derivatives as selective antagonists

for P2Y receptors

Julia Browna,b,*, Colin A. Browna

aBiomedical Sciences Division, School of Applied Sciences, University of Wolverhampton, Wulfruna Street, Wolverhampton WV1 1SB, UKbSchool of Life Sciences, University of Keele, Keele, Staffordshire ST5 5BJ, UK

Received 1 November 2002; received in revised form 2 January 2003; accepted 1 April 2003

Abstract

P2Y receptor pharmacology is hampered by a lack of subtype selective antagonists. However, a recent study evaluated series of

compounds, structurally related to the dye reactive blue 2, for their antagonist selectivity at P2X vs. P2Y receptors. Acid blue 129, acid blue

80, acid blue 25 and acid violet 34 were found to be the most potent of the antagonists studied, at P2Y receptors [Naunyn Schmiedeberg’s

Arch. Pharmacol. 357 (1998) 111]. In this study, we have determined the ability of these four agents to selectively antagonize inositol

phosphate turnover mediated by P2Y1 and P2Y2 receptors that are natively expressed in bovine aortic endothelial (BAE) cells. Acid blue 129,

acid blue 80, and acid violet 34 shifted the dose–response curve of the P2Y1 agonist 2-methylthio adenosine trisphosphate (2MeSATP) to the

right. Acid blue 129 and acid blue 80 were also very weak antagonists of the P2Y2 agonist uridine 50-triphosphate (UTP). At 30 and 100 mM,

acid violet 34 failed to have any significant effect on the dose–response to UTP. However, at 10 mM, acid violet 34 enhanced the UTP

responses. Acid blue 80, acid blue 129 and acid violet 34 are P2Y vs. P2X selective, but show poor selectivity between P2Y1 and P2Y2

receptors and are therefore of limited use in the field of P2Y receptor pharmacology. Furthermore, contrary to previous reports, acid blue 25 is

not a P2Y-selective antagonist.

D 2003 Elsevier Science Inc. All rights reserved.

Keywords: Bovine aortic endothelial cells; P2Y1; P2Y2; Antagonists; Reactive blue 2; Acid blue 129

1. Introduction

P2Y receptors are a major class of receptors ubiquitously

located throughout the body. In particular, these receptors

play an important role in vascular regulation, being located on

endothelial and smooth muscle cells. The potential, however,

of these receptors as a target for clinical intervention in cir-

culatory disorders has not been realized largely due to a lack

of subtype-selective ligands. Furthermore, pharmacological

classification of natively expressed G-protein-coupled P2Y

nucleotide receptors has also been made difficult by a lack of

subtype-selective ligands, especially antagonists.

1537-1891/03/$ – see front matter D 2003 Elsevier Science Inc. All rights reserv

doi:10.1016/S1537-1891(03)00030-2

Abbreviations: ADPbS, adenosine 50-O-(2-thiodiphosphate) trilithium;

ATP, adenosine 50-triphosphate; BAE cells, bovine aortic endothelial cells;

2MeSADP, 2-methylthio adenosine diphosphate; 2MeSATP, 2-methylthio

adenosine trisphosphate; UTP, uridine 50-triphosphate.

* Corresponding author. School of Life Sciences, University of Keele,

Keele, Staffordshire ST5 5BJ, UK. Tel.: +44-1782-583670; fax: +44-1782-

583516.

E-mail address: [email protected] (J. Brown).

The difficulties in pharmacological classification of P2

receptors have increased due to the number of receptors that

have recently been cloned. Currently, there are seven sub-

types in the P2X family; an eighth subtype has recently been

proposed, which may be a P2X5 orthologue (Bo et al.,

2000), and 12 subtypes in the P2Y family only six of which

are functional mammalian receptors (Burnstock and King,

1996; Boarder and Hourani, 1998; Communi et al., 1999;

Hollopeter et al., 2001). In addition, agonists previously

thought to be selective have been shown to activate more

than one P2Y receptor; for example, 2-methylthio adenosine

trisphosphate (2MeSATP) can activate P2Y1 and P2Y11, and

uridine 50-triphosphate (UTP) can activate P2Y2, P2Y4 and

P2Y6 receptors (Communi et al., 1997; Harden et al., 1998).

Of the antagonists currently available, many interact with

both P2X (ligand-gated ion channel receptors) and P2Y

receptors (Bultmann and Starke, 1994; Brown et al., 1995)

or are not subtype selective for P2Y receptors (Charlton et

al., 1996). However, a number of selective antagonists have

been designed for P2Y1 receptors, the most selective being

MRS2279 (Boyer et al., 2002). Whilst at the platelet ADP

ed.

J. Brown, C.A. Brown / Vascular Pharmacology 39 (2003) 309–315310

receptor, P2Y12, AR-C67085 is a selective antagonist (Ingall

et al., 1999). There are however, currently no selective anta-

gonists available for P2Y2,4,6,11 receptors.

The ability of dyes to interact with proteins in a specific

and reversible manner has resulted in their use as phar-

macological tools. Reactive blue 2 is also known as ciba-

cron blue 3GA; however, reactive blue 2 is a mixture of

isomers having the sulphonic acid residues in the meta or

para position whilst cibacron blue 3GA is the isomer with

the sulphonic acid residue in the ortho position. Reactive

blue 2 and cibacron blue 3GA have a high affinity for

protein sites associated with binding of nucleotides (Burton

et al., 1988). Reactive blue 2 is among the most widely used

P2 receptor antagonists. The ability of reactive blue 2 to

block natively expressed P2X receptors in rat urinary

bladder (Bo et al., 1994) and rat vas deferens (Bultmann

and Starke, 1994) has previously been demonstrated. In

addition, reactive blue 2 or cibacron blue 3GA blocks

responses in cloned P2X1, P2X2, P2X4, P2Y1, P2Y3,

P2Y4, P2Y6 and P2Y11 receptors (Brake et al., 1994; Simon

et al., 1995; Chang et al., 1995; Communi et al., 1996,

1999; Michel et al., 1996; Seguela et al., 1996; Webb et al.,

1996).

In a recent study, 12 compounds structurally related to

reactive blue 2 were investigated for their ability to block P2

receptor subtypes (Tuluc et al., 1998). Four derivatives of

reactive blue 2: acid blue 25, acid blue 80, acid blue 129 and

acid violet 34 (see Fig. 1 for structures) were the most potent

Fig. 1. Structure of (a) reactive blue 2 and derivatives of reactive blue 2: (b)

acid blue 80, (c) acid blue 129, (d) acid violet 34 and (e) acid blue 25.

antagonists at the P2Y receptors of guinea pig taenia coli

with an apparent Kd value of 0.5–1.4 mM. The most

promising P2Y antagonist was acid blue 129, which was

shown to be P2Y vs. P2X selective. We report here for the

first time the selectivity of these four derivatives of reactive

blue 2 at P2Y1 and P2Y2 receptors natively coexpressed in

BAE cells. The P2Y receptor pharmacology of bovine aortic

endothelial (BAE) cells has been extensively studied. These

cells accumulate inositol phosphates in response to aden-

osine 50-triphosphate (ATP), 2MeSATP and UTP (see, e.g.,

Allsop and Boarder, 1990; Motte et al., 1993, Pirotton et al.,

1996; Duchene and Takeda, 1997) and show responses that

are differentially sensitive to the antagonist suramin (Wil-

kinson et al., 1994). Effects of the putative antagonists were

assessed for their ability to antagonize 2MeSATP and UTP

induction of inositol phosphate turnover by P2Y1 and P2Y2

receptors, respectively. The aim of this investigation was to

identify a compound that was P2Y2-selective as there is

currently no P2Y2-selective antagonist available.

2. Methods

2.1. Cell culture

BAE cells were cultured in minimal essential medium

with D-valine (PAA Laboratories, Yeovil, Somerset, UK)

supplemented with 10% fetal bovine serum (FBS), 100 U/

ml penicillin, 100 mg/ml streptomycin and 2 mM L-gluta-

mine.

2.2. Measurement of [3H]inositol polyphosphate turnover

Measurement of [3H]inositol polyphosphate turnover

was according to Brown et al. (2000). Cells were labelled

for 24 h with 0.5 mCi [3H]myo-inositol (17.0 Ci/mmol)

(Amersham) per well in serum-free M199 medium (PAA

Laboratories). Lithium chloride was added to cells at a final

concentration of 10 mM for 10 min prior to stimulations.

Dyes were then added for 10 or 30 min as indicated prior to

agonists. The 15-min agonist stimulations were terminated

by removal of M199 medium and the immediate addition of

ice-cold 0.5 M trichloroacetic acid (TCA). Cells were

incubated on ice for 1 h and acid extracts washed with

3�4 volumes of water-saturated diethyl ether and buffered

to pH 7.0 with 0.6 M NaHCO3. The fraction containing

[3H]IP1–IP4 was recovered from 1 ml Dowex 1X8-400

(Sigma Chemical Company, Poole, Dorset, UK) columns,

by extraction with 1 M ammonium formate.

2.3. Data analysis

Concentration effect curves, curve fitting and EC50

values were determined using the GRAPH PAD prism

analysis program (San Diego, CA, USA). Data are ex-

pressed as arithmetical mean values±S.E.M. Means were

J. Brown, C.A. Brown / Vascular Pharmacology 39 (2003) 309–315 311

tested for significant difference using Student’s t test for

paired data. Values were considered significantly different at

a probability of P<.05 or less.

2.4. Materials

2-Methylthio adenosine diphosphate (2MeSADP),

2MeSATP and UTP were supplied by Sigma. Acid blue 25,

acid blue 80, acid blue 129 and acid violet 34 were supplied

by Aldrich, Gillingham, Dorset, UK. Tissue culture medium

and supplements were supplied by PAA Laboratories.

Fig. 3. Dose responses to (a) 2MeSATP and (b) UTP in the absence of acid

blue 25 (&), and in the presence of 100 mM acid blue 25 (~). Data are

mean±S.E.M. from three experiments performed in triplicate.

3. Results

3.1. P2Y receptor pharmacology of BAE cells

Fig. 2 shows accumulation of inositol phosphates in

response to 2MeSADP and 2MeSATP with mean EC50

values of (2.51±0.17)�10�6 M and (2.77±0.21)�10�6 M

(n=4), respectively. Thus, 2MeSADP and 2MeSATP were

equipotent for the P2Y1 receptor expressed by these cells,

these data agree with previous findings (Henderson et al.,

1995). 2MeSATP is an agonist at both the P2Y1 and P2Y11

receptor; however, 2MeSATP at concentrations up to100 mMfailed to elicit a cAMP response in BAE cells (data not

shown) suggesting that 2MeSATP responses are due to

activation of the P2Y1 receptor. Therefore, in this study,

2MeSATP was used to study the effects of derivatives of

reactive blue 2 on the P2Y1 receptor expressed by BAE cells.

Further studies using the uridine nucleotides UTP and UDP

showed that while stimulation of the cells with UTP showed

robust agonist responses (e.g., Fig. 3b) hexokinase-treated

UDP, up to a concentration of 100 mM, was without effect.

Fig. 2. Dose-dependent accumulation of inositol phosphates in response to

2MeSADP (&) and 2MeSATP (~) in BAE cells. Data are mean±S.E.M.

from three experiments performed in triplicate.

Therefore, responses mediated by UTP would appear to be

via P2Y2 receptors and not P2Y4 and/or P2Y6 receptors.

3.2. Effects of acid blue 25 on inositol phosphate turnover

Acid blue 25 (100 mM) had no significant effect on either

P2Y1 or P2Y2 responses to 2MeSATP and UTP, respect-

ively (Fig. 3a, b). Furthermore, increasing the incubation

time with acid blue 25 from 10 to 30 min prior to addition of

agonists was also without effect in these cells (data not

shown).


The P2Y1 agonist, 2MeSATP concentration dependently

increased inositol phosphate turnover in BAE cells with a

mean EC50 value of (8.10±1.56)�10�7 M (n=4). Fig. 4a

shows that [3H]InsP1 – 4 accumulation in response to

2MeSATP was inhibited by increasing concentrations of

acid blue 129. Acid blue 129 at 10, 30 and 100 mM caused

significant rightward shifts in the response to 2MeSATP

relative to 2MeSATP in the absence of antagonist, with the


(a) 2MeSATP and (b) UTP in the absence of acid blue 129 (&) and in the

presence of acid blue 129 at 10 (~), 30 (!) and 100 mM (^). Data are

mean±S.E.M. from four experiments performed in triplicate.


(a) 2MeSATP and (b) UTP in the absence of acid blue 80 (&) and in the

presence of acid blue 80 at 10 (~), 30 (!) and 100 mM (^). Data are

mean±S.E.M. from four experiments performed in triplicate.


EC50 values of 2MeSATP increasing to (3.93±0.60)�10�6

M (P<.05 vs. control), (5.49±0.54)�10�6 M (P<0.001 vs.

control) and (4.67±0.89)�10�6 M (P<0.001 vs. control),

respectively. In addition, the presence of acid blue 129

(100 mM) also resulted in decreased maximal effects of

2MeSATP.

The P2Y agonist UTP also dose dependently increased

inositol phosphate turnover in BAE cells with a mean EC50

of (2.05±0.36)�10�5 M (n=4). In contrast to its effect at

P2Y1 receptors, acid blue 129 only acted as a very weak

antagonist of the UTP response. The rightward shifts at 10,

30 and 100 mM were not significant, with mean EC50 values

of UTP of (1.25±0.30)�10�5 M, (4.63±1.31)�10�5 M and

(3.37±0.82)�10�5 M, respectively (n=4) (Fig. 4b).


Responses to 2MeSATP and UTP in the presence of

increasing concentrations of acid blue 80 displayed similar

kinetics to the effects of acid blue 129 (Fig. 5a, b). Dose–

response curves for 2MeSATP were significantly shifted to

the right compared to responses for 2MeSATP in the

absence of antagonist. EC50 values for the control, 10

mM, 30 mM and 100 mM, were (7.39±0.98)�10�7 M,

(3.71±1.03)�10�6 M (P<0.001 vs. control), (4.10±0.89)�10�6 M (P<0.001 vs. control) and (1.86±0.54)�10�6 M

(P<0.001 vs. control) (n=4), respectively. The presence of

acid blue 80 (100 mM) also resulted in decreased maximal

effects.

Acid blue 80 also acted as a weak antagonist of the UTP

response; however, the rightward shifts observed were not

significantly different from responses to UTP alone. The

apparent EC50 values for the control, 10 mM, 30 mM and 100

mM were (2.65±0.94)�10�5 M, (3.66±1.09)�10�5 M,

(4.30±0.58)�10�5 M and (6.74±1.78)�10�5 M (n=4),

respectively.

r Pharmacology 39 (2003) 309–315 313

3.5. Effects of acid violet 34 on inositol phosphate turnover

Acid violet 34 at 10 mM had no significant effect on the

dose response to 2MeSATP (Fig. 6a). In contrast, 30 and 100

mM acid violet 34 caused significant rightward shifts in the

dose–response curves to 2MeSATP. In addition, acid violet

34 (100 mM) decreased the maximal effect of 2MeSATP in a

manner similar to the effects seen for acid blue 129 and acid

blue 80. Mean EC50 values for control, 10 mM, 30 mM and

100 mM were (9.29±0.87)�10�7 M, (1.03±0.42)�10�6 M

(NS), (2.51±0.38)�10�6 M (P<0.001 vs. control) and

(2.43±0.67)�10�6 M (P<0.001 vs. control) (n=4), respect-

ively. In contrast to these effects, 10 mM acid violet 34 en-

hanced the response to UTP, this increase was significant at

300 mM UTP (P<.01 vs. UTP alone). There was no signific-

ant difference between the dose response to UTP in the

presence and absence of 30 mM or 100 mM acid violet 34

(Fig. 6b). Mean EC50 values for control, 10 mM, 30 mM and

100 mM were (2.07±0.87)�10�5 M, (3.38±1.06)� 10�5 M,

J. Brown, C.A. Brown / Vascula


(a) 2MeSATP and (b) UTP in the absence of acid violet 34 (&) and in

the presence of acid violet 34 at 10 (~), 30 (!) and 100 mM (^) in

BAE cells. Data are mean±S.E.M. from four experiments performed

in triplicate.

(2.78±1.01)�10�5 M and (6.50±2.07)�10�5 M (n=4),

respectively.

3.6. Receptor specificity of dyes

In addition to P2Y receptors, BAE cells also exhibit

robust responses to the nonpurinergic agent, bradykinin.

None of the dyes tested were able to inhibit responses to

bradykinin at concentrations up to 100 mM (data not

shown).

4. Discussion

A recent study reported that four derivatives of reactive

blue 2: acid blue 129, acid blue 80, acid blue 25, and acid

violet 34 were potent antagonists of P2Y receptors, with

acid blue 129 being the most interesting in light of its

apparent substantial P2Y vs. P2X selectivity (Tuluc et al.,

1998). In this study, we have extended this work to

determine the effects of these four derivatives of reactive

blue 2 on P2Y1 and P2Y2 receptors that are natively coex-

pressed on BAE cells. Our findings reveal that only three of

these compounds are functional antagonists at P2Y recep-

tors. Initial experiments confirmed that none of the com-

pounds displayed agonist-like properties at concentrations

up to 100 mM (data not shown), although at low concen-

trations (10 mM) acid violet 34 enhanced the agonist-

induced responses to UTP (Fig. 6b).

Stimulation of P2Y1 and P2Y2 receptors by 2MeSATP

and UTP, respectively, resulted in inositol phosphate turn-

over in BAE cells (Brown et al., 1995). One of the

compounds tested, acid blue 25, was unable to antagonize

either P2Y1 or P2Y2 receptor-mediated responses in these

cells at concentrations up to 100 mM (Fig. 3a, b). This was

surprising since a previous report found this compound to be

one of the most potent of a range of putative P2Yantagonists

studied (Tuluc et al., 1998). The differences observed with

acid blue 25 between this study and that of Tuluc et al.

(1998) might be attributable to the presence of different

receptor populations in the two test systems, as they used

adenosine 50-O-(2-thiodiphosphate) trilithium (ADPbS) thathas effects at both P2Y1 and P2Y11 receptors. Acid blue 129

and acid blue 80 displayed similar kinetics in their ability to

block the P2Y1 and P2Y2 responses in BAE cells, with each

antagonist being more effective at P2Y1 sites compared to

P2Y2 sites. The precise nature of the antagonism is unclear,

but does not appear to be truly competitive. This is also in

contrast with the report of Tuluc et al. (1998) who found

that acid blue 129 and acid blue 80 blocked relaxation of

guinea pig taenia coli caused by ADPbS in a competitive

manner. From our data, it was not possible to determine

pA2 values for either of these compounds since shifts in the

dose–response curves were not truly parallel, supporting the

notion that the nature of the antagonism is not purely

competitive. Furthermore, although previous findings


showed that acid blue 129 was the most promising deriv-

ative of reactive blue 2 in selectively blocking P2Y vs. P2X

responses (Tuluc et al., 1998), we show that despite some

degree of selectivity for P2Y1 receptors at low concentra-

tions this compound is a relatively weak antagonist at P2Y

receptors.

Acid violet 34, at 30 and 100 mM, acted as an antagonist

of the P2Y1 response, but was without any significant effect

at P2Y2 sites. Thus, acid violet 34 appears to be more

selective for P2Y1 over P2Y2 receptors than acid blue 129

or acid blue 80. However, at a concentration of 10 mM, acid

violet 34 caused a small but significant enhancement of the

UTP response. The precise reason for this effect is not clear

and cannot be directly determined from the present study;

however, in this context it is worth noting similar effects

reported with other P2Y antagonists such as suramin and

PPADS (Chen et al., 1996) have been attributed to the

‘‘ectonucleotidase inhibition’’ properties of these com-

pounds. These potential ‘‘dual effects’’ of purinergic antag-

onists make interpretation of the degree of ‘‘true’’

antagonism difficult for such agents, but could be one

possible explanation for the apparent noncompetitive nature

of the antagonism observed with the dyes in this study.

All the dyes investigated in this study are derivatives of

anthraquinone, all have the 1-amino-anthraquinone-2-sulph-

onate core structure (see Fig. 1 for structures). Acid blue 25,

the smallest derivative of the parent compound reactive blue

2 consisting totally of the 1-amino-anthraquinone-2-sulph-

onate core, failed to antagonize the effects of either

2MeSATP or UTP. Our data are in contrast to those of

Tuluc et al. (1998), who attributed antagonism at P2Y

receptors to the affinity residues, which are largely or totally

in the 1-amino-anthraquinone-2-sulphonate core. Rather

surprisingly, acid blue 129, a derivative of acid blue 25

with a closely related structure to this compound, behaved

as an antagonist of both 2MeSATP and UTP responses.

Furthermore, acid blue 80, which has two separate aromatic

substituents at positions 1 and 4, also displayed similar

kinetics to acid blue 129. There is, however, structural

similarity between acid blue 80 and acid blue 129 at

position 4, which could explain the ability of both com-

pounds to block P2Y receptor responses. Acid violet 34,

which carries two separate aromatic substituents at positions

1 and 5, only blocked responses to 2MeSATP. In the light of

these data, it is not easy to resolve the inability of acid blue

25 to antagonize either the 2MeSATP or UTP response.

Thus, from our data, it is not possible to apply any clear

structure–activity relationships to the compounds tested.

5. Conclusions

The aim of this study was to search for P2Y receptor

subtype-selective antagonists. However, data from our

experiments showed that both acid blue 129 and acid blue

80 were weak antagonists with limited selectivity, moreover,

both compounds acted with kinetics that were not purely

competitive. These findings are similar to those previously

reported for other P2 antagonists (e.g., Bultmann et al.,

1996a,b; Wittenburg et al., 1996). Acid violet 34 displayed

selectivity for the P2Y1 receptor vs. the P2Y2 receptor and

may warrant further investigation for its potential ability to

selectively antagonise other members of the P2Y receptor

family. Acid blue 25 was not able to antagonise either P2Y1

or P2Y2 responses in BAE cells. This is contrary to the

findings of Tuluc et al. (1998) using guinea pig taenia coli.

One possibility for these differences is that there may be

other members of the P2Y receptor family being expressed

in guinea pig taenia coli that are responsive to ADPbS and

inhibited by acid blue 25.

Despite the emergence of a number of new antagonist

compounds at both P2X and P2Y receptors, it is clear that at

best these compounds are only partially selective with many

resulting in noncompetitive blockade of purinergic sites.

Several studies have demonstrated that many of these

compounds have other effects in addition to any putative

antagonism, such as inhibition of ectonucleotidase activity

(Chen et al., 1996) and blockade of other cellular mecha-

nisms (Shehnaz et al., 2000). Thus, while acid blue 80, acid

blue 129 and acid violet 34 are P2Y vs. P2X selective these

compounds are of limited use in the field of P2Y receptor

pharmacology and truly selective potent antagonists at P2Y

receptors remain a desideratum.

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Evaluation of reactive blue 2 derivatives as selective antagonists for P2Y receptors