Elsevier

Vascular Pharmacology

Volume 39, Issue 6, December 2002, Pages 309-315
Vascular Pharmacology

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

https://doi.org/10.1016/S1537-1891(03)00030-2Get rights and content

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 5′-triphosphate (UTP). At 30 and 100 μM, acid violet 34 failed to have any significant effect on the dose–response to UTP. However, at 10 μM, 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.

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 circulatory 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.

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 subtypes 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 5′-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 Bültmann 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 receptor, P2Y12, AR-C67085 is a selective antagonist (Ingall et al., 1999). There are however, currently no selective antagonists 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 pharmacological tools. Reactive blue 2 is also known as cibacron 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 (Bültmann 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, Communi et al., 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 antagonists at the P2Y receptors of guinea pig taenia coli with an apparent Kd value of 0.5–1.4 μM. 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 adenosine 5′-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 (Wilkinson 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.

Section snippets

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 μg/ml streptomycin and 2 mM l-glutamine.

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 μCi [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

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 μM failed to elicit a cAMP response in BAE cells (data not shown)

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 coexpressed on BAE cells. Our findings

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., Bültmann et al., 1996a, Bültmann et al., 1996b, Wittenburg et al., 1996). Acid violet 34 displayed selectivity for

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