Dinucleoside polyphosphates act as agonists on purinergic P2Y receptors to mediate a variety of cellular processes. Symmetrical, naturally occurring purine dinucleotides are found in most living cells and their actions are generally known. Unsymmetrical purine dinucleotides and all pyrimidine containing dinucleotides, however, are not as common and therefore their actions are not well understood. To carry out a thorough examination of the activities and specificities of these dinucleotides, a robust method of synthesis was developed to allow manipulation of either nucleoside of the dinucleotide as well as the phosphate chain lengths. Adenosine containing dinucleotides exhibit some level of activity on P2Y 1 while uridine containing dinucleotides have some level of agonist response on P2Y 2 and P2Y 6 . The length of the linking phosphate chain determines a different specificity; diphosphates are most accurately mimicked by dinucleoside triphosphates and triphosphates most resemble dinucleoside tetraphosphates. The pharmacological activities and relative metabolic stabilities of these dinucleotides are reported with their potential therapeutic applications being discussed.

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1 The contractile and relaxant eects of the dierent P2 receptors were characterized in the rat isolated mesenteric artery by use of extracellular nucleotides, including the stable pyrimidines uridine 5'-O-thiodiphosphate (UDPbS) and uridine 5'-O-3-thiotriphosphate (UTPgS). 2 The selective P2X receptor agonist, ab-methylene-adenosine triphosphate (ab-MeATP) stimulated a potent (pEC 50 =6.0) but relatively weak contraction (E max =57% of 60 mM K + ). The contractile concentration-response curve of adenosine triphosphate (ATP) was biphasic when added in single concentrations. The ®rst part of the response could be desensitized by ab-MeATP, indicating involvement of P2X receptors, while the second part might be mediated by P2Y receptors. 3 The contractile P2Y receptors were further characterized after P2X receptor desensitization with 10 mM ab-MeATP. Uridine diphosphate (UDP), uridine triphosphate (UTP) and ATP stimulated contraction only in high concentrations (1 ± 10 mM). The selective P2Y 6 agonist, UDPbS, and the P2Y 2 /P2Y 4 -receptor agonists UTPgS and adenosine 5'-O-3-thiotriphosphate (ATPgS) were considerably more potent and ecacious (E max &250% of 60 mM K + ). Adenosine 5'-O-thiodiphosphate (ADPbS) was inactive, excluding contractile P2Y 1 receptors. 4 After precontraction with 1 mM noradrenaline, UTP, ADP and ATP induced relaxations with similar potencies (pEC 50 &5.0). UTPgS, ADPbS and ATPgS were approximately one log unit more potent indicating the presence of endothelial P2Y 1 and P2Y 2 /P2Y 4 receptors. The P2Y 6 receptor agonist, UDPbS, had no eect. 5 UDPbS and UTPgS are useful tools when studying P2 receptors in tissue preparations with ectonucleotidase activity. Contractile responses can be elicited by stimulation of P2Y 6 and, slightly less potently, P2Y 2 /P2Y 4 receptors. The P2X response was relatively weak, and there was no P2Y 1 response. Stimulation of P2Y 1 and P2Y 2 /P2Y 4 receptors elicited relaxation, while P2Y 6 did not contribute.

linge. UDP acts as a growth factor for vascular smooth muscle cells by activation of P2Y 6 receptors. Am J Physiol Heart Circ Physiol 282: H784-H792, 2002; 10.1152/ajpheart.00997.2000.-Mitogenic effects of the extracellular nucleotides ATP and UTP are mediated by P2Y 1, P2Y2, and P2Y4 receptors. However, it has not been possible to examine the highly expressed UDP-sensitive P2Y6 receptor because of the lack of stable, selective agonists. In rat aorta smooth muscle cells (vascular smooth muscle cells; VSMC), UDP and UTP stimulated 3 H-labeled thymidine incorporation with similar pEC50 values (5.96 and 5.69). Addition of hexokinase did not reduce the mitogenic effect of UDP. In cells transfected with P2Y receptors the stable pyrimidine agonist uridine 5'-O-(2-thiodiphosphate) (UDP␤S) was specific for P2Y 6 with no effect on P2Y1, P2Y2, or P2Y4 receptors. UDP␤S stimulated [3 H]thymidine and [ 3 H]leucine incorporation and increased cell number in VSMC. Flow cytometry demonstrated that UDP stimulated cell cycle progression to both the S and G 2 phases. The intracellular signal pathways were dependent on phospholipase C, possibly protein kinase C-␦, and a tyrosine kinase pathway but independent of G i proteins, eicosanoids, and protein kinase A. The half-life of P2Y6 receptor mRNA was Ͻ1 h by competitive RT-PCR. The mitogen-activated protein kinase kinase inhibitor PD-098059 significantly suppressed, whereas ATP and interleukin-1␤ upregulated, expression of P2Y6 receptor mRNA. The results demonstrate that UDP stimulates mitogenesis through activation of P2Y 6 receptors and that the receptor is regulated by factors important in the development of vascular disease. uridine 5Ј-diphosphate; gene expression THE CARDIOVASCULAR EFFECTS of extracellular nucleotides have received increasing attention since the beneficial effects of the platelet inhibitory ADP receptor antagonist clopidogrel were demonstrated in atherosclerotic disease (2). Several other receptors for extracellular nucleotides (P2 receptors) have been cloned and found to be involved in other processes in cardiovascular regulation. Previous studies demonstrated that the extracellular nucleotides ATP and UTP act as growth factors for vascular smooth muscle cells (VSMC) by activation of P2Y 1 , P2Y 2 , and P2Y 4 receptors (10, 14). However, it has not been possible to examine the UDPactivated P2Y 6 receptor, which has the highest mRNA expression in the media of the rat aorta (11), because of the lack of selective agonists and antagonists.UTP is present in all cells at ϳ10% of ATP levels and is formed through both salvage and de novo pathways. UTP can be released from both platelets and endothelial cells under physiologically relevant stimuli (1,20,30,34). Released uridine nucleotides are catabolized by ectonucleotidases that are present on a variety of tissues and cells, suggesting that the concentration of extracellular UTP also reflects the concentration of extracellular UDP (9). So far, there is no assay for UDP quantification, so we do not know in what concen...