Objective-ADP plays an important role in platelet aggregation by activating P2Y 12 receptors. We assessed the hypothesis that P2Y 12 receptors are expressed in vascular smooth muscle cells (VSMC). Methods and Results-P2Y 12 receptor mRNA was found to have a high expression among the P2 receptors in human VSMC, significantly higher than the other 2 ADP receptors (P2Y 1 and P2Y 13 , real-time polymerase chain reaction). Western blots gave a band of 50 kD, similar to that in platelets. To unmask a P2Y 12 receptor-mediated vasoconstriction by simulating the in vivo situation, vessels were precontracted to a submaximal level. 2-MeSADP stimulated contractions in vessel segments from internal mammary artery (IM), IM branches and small veins (E max ϭ15Ϯ6% of 60mmol/L K ϩ contraction, pEC 50 ϭ5.6Ϯ0.6, E max ϭ21Ϯ1%, pEC 50 ϭ6.8Ϯ0.1, and E max ϭ48Ϯ9%, pEC 50 ϭ6.6Ϯ0.4). The selective P2Y 12 antagonist AR-C67085 blocked 2-MeSADP contractions. The contraction was not reduced in patients using clopidogrel, a drug inhibiting ADP-induced platelet aggregation by blocking the P2Y 12 receptor. This may be explained by the high instability of the active clopidogrel metabolite that never reaches the systemic circulation. Conclusion-ADP acting on P2Y 12 receptors not only is important for platelet activation but also stimulates vasoconstriction. Stable drugs with antagonistic effects on P2Y 12 receptors, affecting both platelets and VSMC, could be of double therapeutic benefit in their prevention of both thrombosis and vasospasm. Key Words: vasoconstriction Ⅲ P2Y receptors Ⅲ platelets Ⅲ ADP E xtracellular nucleotides such as adenosine triphosphate (ATP), adenosine diphosphate (ADP), uridine triphosphate, and uridine diphosphate are released from sympathetic nerves, platelets, and endothelial and inflammatory cells. 1 Nucleotides induce vasoconstriction-stimulating P2 receptors on vascular smooth muscle cells (VSMC), regulating vascular tone and blood pressure. 1,2 Furthermore, extracellular nucleotides have been shown to mediate growth stimulation and migration in VSMC. 3,4 P2 receptors can be divided into 2 classes on the basis of their signal transmission mechanisms and their characteristic molecular structures: ligand-gated intrinsic ion channels, P2X receptors, and G-protein-coupled P2Y receptors. The P2Y family is composed of 8 cloned and functionally defined subtypes. [5][6][7][8][9] ADP is stored in high concentrations in platelet granula and is released as a positive feedback mechanism in response to most platelet activators. After release, ADP augments platelet aggregation by stimulation of P2Y 12 and P2Y 1 receptors. 10 The first clinical application of P2 receptor antagonists has been the use of thienopyridines as platelet aggregation inhibitors. Clopidogrel and ticlopidine are prodrugs that are converted in the liver into irreversible antagonists against P2Y 12 receptors. Clopidogrel is more efficient than aspirin in reducing the composite end point myocardial infarction, stroke, and death in patients with vascular disease...

. Contractility of rat cerebral arteries in organ culture is preserved for several days, whereas SOCE is increased. In correlation with this increase is that nifedipine-insensitive whole cell current, activated by depletion of intracellular Ca 2ϩ stores, was increased by 50% in cells isolated from arteries cultured for 3 days. TRPC1 and TRPC6 mRNA were more than fivefold increased in cells isolated after organ culture, whereas TRPC3 was decreased. Immunofluorescent staining and/or Western blotting of arteries and isolated cells showed upregulation of TRPC1 and TRPC6 proteins during organ culture. In intact arteries, TRPC4 expression correlated with the amount of endothelium present. Ca 2ϩ addition after store depletion caused a contraction in cultured, but not in freshly dissected, arteries. A polyclonal TRPC1 antibody directed against an extracellular epitope inhibited this contraction by ϳ50%. To investigate the basis of the TRPC upregulation and assess its possible clinical significance, segments of human internal mammary artery were organ cultured for 24 h and then exposed to balloon dilatation in vitro, followed by further culturing for up to 48 h. After dilatation, TRPC1 and TRPC6 mRNA were progressively increased compared with undilated control segments. The results of this study indicate that vascular injury enhances plasticity in TRPC expression, that TRPC expression correlates with cellular Ca 2ϩ handling, and that TRPC1 is a subunit of upregulated store-operated Ca 2ϩ channels.

The potency of nucleotide antagonists at P2Y1 receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829-842). We have now synthesized ring-constrained methanocarba analogues (in which a fused cyclopropane moiety constrains the pseudosugar ring) of adenine and uracil nucleotides, the endogenous activators of P2Y receptors. Methanocarba-adenosine 5'-triphosphate (ATP) was fixed in either a Northern (N) or a Southern (S) conformation, as defined in the pseudorotational cycle. (N)-Methanocarba-uridine was prepared from the 1-amino-pseudosugar ring by treatment with beta-ethoxyacryloyl cyanate and cyclization to form the uracil ring. Phosphorylation was carried out at the 5'-hydroxyl group through a multistep process: Reaction with phosphoramidite followed by oxidation provided the 5'-monophosphates, which then were treated with 1,1'-carbonyldiimidazole for condensation with additional phosphate groups. The ability of the analogues to stimulate phospholipase C through activation of turkey P2Y1 or human P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11 receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y1 and P2Y2 receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)-methanocarba-ATP activated P2Y11 receptors with a potency similar to ATP. (N)-Methanocarba-uridine 5'-triphosphate (UTP) was equipotent to UTP as an agonist at human P2Y2 receptors and also activated P2Y4 receptors with an EC(50) of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y6 receptor. The vascular effects of (N)-methanocarba-UTP and (N)-methanocarba-UDP were studied in a model of the rat mesenteric artery. The triphosphate was more potent than UTP in inducing a dilatory P2Y4 response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y6 receptor-mediated contractile response. Our results suggest that new nucleotide agonists may be designed on the basis of the (N) conformation that favors selectivity for P2Y1, P2Y2, P2Y4, and P2Y11 receptors.