| Summary: | In view of the rapidly growing interest in P2Y receptors and the lack of subtype selective ligands, especially antagonists, the aim of this study was to evaluate novel ligands of P2Y receptors. This was performed using putative P2Y-selective antagonists reviewed in the literature and by designing and synthesising novel peptide ligands. Bovine aortic endothelial (BAE) cells that co-express P2Yl and P2Y2 receptors and ECV304, a human cell line were used for this study. ECV304 were evaluated as a suitable model for studying P2Y receptor pharmacology using currently available agonists and antagonists in assays of second messengers. Results showed that ECV304 cells express two P2Y receptors, a P2Y2-like receptor and a P2Y11-like receptor. During the study doubt was cast as to the origin of the human cell line ECV304. It was thought that these cells had spontaneously transformed from human umbilical vein endothelial cells. In this study it was clearly demonstrated that ECV304 cells shared the same DNA fingerprint as T24/83 bladder cancer epithelial cells and were indeed not endothelial in origin. However, ECV304 cells are human cells natively expressing P2Y receptors and are a very useful research tool for studying P2Y receptor pharmacology. Reactive blue 2 is a P2 receptor antagonist, but it is not subtype-selective, having effects at both P2X and P2Y receptors. A recent study showed that derivatives of reactive blue 2: acid blue 129, acid blue 80, acid blue 25 and acid violet 34, are P2Y- versus P2Xselective. These four derivatives have been investigated in this study for their relative selectivity at P2Y1 versus P2Y2 receptors using stimulation of inositol phosphate turnover in BAE cells as a measure of activity. Acid blue 25 failed to antagonise either the P2Y1 or the P2Y2 receptor. The other three compounds were shown to be weak antagonists that were not subtype-selective and had activity that was not truly competitiveNovel peptide ligands have been designed to mimic extracellular domains of the human P2Y2 receptor. Surprisingly, these novel mimetic peptides had "agonist-like" properties. Peptides alone directly activated second messenger production in bovine aortic endothelial cells and ECV304, and also augmented agonist responses in ECV304 cells. Interestingly, analogues of mimetic peptides were also capable of enhancing sub-maximal doses of natural agonists in ECV304 cells. These findings represent a unique action of mimetic peptides as they have effects at nonpeptide P2Y receptors. These observations indicate an important role of extracellular domains, particularly the third extracellular loop, in signal transduction by P2Y2 receptors. Furthermore, the "agonist-like" activity of P2Y2 receptor mimetic peptides has important implications for the study of P2Y receptor activation and may have therapeutic potential e. g. in the treatment of cystic fibrosis. Finally, these findings may be equally applicable to the design of allosteric modulators of other G protein-coupled receptors
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