P2 receptors mediate the actions of the extracellular nucleotides ATP, ADP, UTP, and UDP, regulating several physiologic responses including cardiac function, vascular tone, smooth muscle cell (SMC) proliferation, platelet aggregation, and the release of endothelial factors. P2 receptor characterization has been hampered by the lack of selective antagonists. The aim of the current study was to investigate the mRNA and protein expression of P2X and P2Y receptors in human SMC and in endothelial cells (EC). Smooth muscle cells were obtained from human mammary artery and EC from human umbilical vein. Using real-time PCR, the authors established quantitative mRNA assays. Protein expression was studied using Western blotting with recently developed antibodies. The P2X1 receptor was highly specific for human SMC, while the P2X4 was the highest expressed receptor in EC. The P2Y2 receptor was present in both SMC and EC. UTP-mediated effects in these cells are likely to be mediated by P2Y2 and not P2Y4 receptors since the latter had considerably lower expression. The P2Y6 receptor was expressed in both SMC and EC. The P2Y1 and surprisingly the P2Y11 receptors were the most abundantly expressed P2Y receptors in the endothelium. Overall, Western blotting confirmed the mRNA findings in most aspects, and most interestingly, indicated oligomerization of the P2Y1 receptor that may be important for its function. In conclusion, P2X1, P2Y2, and P2Y6 are the most expressed P2 receptors in SMC and are thus probably mediating the contractile and mitogenic actions of extracellular nucleotides. The P2X4, P2Y11, P2Y1, and P2Y2 are the most expressed P2 receptors in EC, and are most likely mediating release of nitric oxide, endothelium-dependent hyperpolarizing factor (EDHF), and t-PA induced by extracellular nucleotides. These findings will help to direct future cardiovascular drug development against the large P2 receptor family.

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...

Previous studies have suggested that plasminogen activator inhibitor 1 (PAI-1) released from platelets convey resistance of platelet-rich blood clots to thrombolysis. However, the majority of PAI-1 in platelets is inactive and therefore its role in clot stabilization is unclear. Because platelets retain mRNA and capacity for synthesis of some proteins, we investigated if platelets can de novo synthesize PAI-1 with an active configuration. PAI-1 mRNA was quantified with real-time polymerase chain reaction and considerable amounts of PAI-1 mRNA were detected in all platelet samples. Over 24 hours, the amount of PAI-1 protein as determined by an enzyme-linked immunosorbent assay increased by 25% (P ‫؍‬ .001). Metabolic radiolabeling with 35 S-methionine followed by immunoprecipitation confirmed an ongoing PAI-1 synthesis, which could be further stimulated by thrombin and inhibited by puromycin. The activity of the newly formed PAI-1 was investigated by incubating platelets in the presence of tissue-type plasminogen activator (tPA).This functional assay showed that the majority of the new protein was in an active configuration and could complexbind tPA. Thus, there is a continuous production of large amounts of active PAI-1 in platelets, which could be a mechanism by which platelets contribute to stabilization of blood clots. (Blood. 2004; 104:3943-3948)

Abstract-The aim of this study was to examine a possible role for extracellular pyrimidines as inotropic factors for the heart. First, nucleotide plasma levels were measured to evaluate whether UTP is released in patients with coronary heart disease. Then, inotropic effects of pyrimidines were examined in isolated mouse cardiomyocytes. Finally, expression of pyrimidine-selective receptors (a subgroup of the P2 receptors) was studied in human and mouse heart, using real time polymerase chain reaction, Western blot, and immunohistochemistry. Venous plasma levels of UTP were increased (57%) in patients with myocardial infarction. In electrically stimulated cardiomyocytes the stable P2Y 2/4 agonist UTP␥S increased contraction by 52%, similar to ␤ 1 -adrenergic stimulation with isoproterenol (65%). The P2Y 6 -agonist UDP␤S also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y 6 -blocker MRS2578. The phospholipase C inhibitor U73122 inhibited both the UDP␤S and the UTP␥S-induced inotropic effect, indicating an IP 3 -mediated effect via P2Y 6 receptors. The P2Y 14 agonist UDP-glucose was without effect. Quantification of mRNA with real time polymerase chain reaction revealed P2Y 2 as the most abundant pyrimidine receptor expressed in cardiomyocytes from man. Presence of P2Y 6 receptor mRNA was detected in both species and confirmed at protein level with Western blot and immunohistochemistry in man. In conclusion, UTP levels are increased in humans during myocardial infarction, giving the first evidence for UTP release in man. UTP is a cardiac inotropic factor most likely by activation of P2Y 2 receptors in man.