| 1. |
- Amisten, Stefan, et al.
(author)
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Increased risk of acute myocardial infarction and elevated levels of C-reactive protein in carriersof the Thr-87 variant of the ATP receptor P2Y11.
- 2007
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In: European Heart Journal. - : Oxford University Press (OUP). - 1522-9645 .- 0195-668X. ; 28:1, s. 13-18
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Journal article (peer-reviewed)abstract
- Aims Extracellular ATP acting on the P2Y(11) receptor regulates inflammatory cells. We hypothesized that polymorphisms in the receptor could influence the risk of acute myocardial infarction (AMI). Methods and results In the Malmo diet and cancer AMI case-control study (n = 3732) the P2Y(11) gene Thr-87 polymorphism was present in 19.8% of the controls and 22.9% in AMI patients (OR 1.21; P = 0.03). Stronger associations were found in patients with family history (FH) of AMI, 1.32; early-onset (EO) AMI, 1.43; or EO AMI combined with FH, 1.50; supporting a genetic mechanism. The Thr-87 homozygotes had an even greater risk of AMI, 1.94 (P = 0.04); and 2.48 in the EO AMI subgroup, suggesting a genetic dosage effect. In the cardiovascular risk factor group (n = 6055), 21.3% carried the Thr-87 allele. C-reactive protein was elevated in Thr-87 carriers: 1.6 mg/L vs. 1.3 mg/L (P = 0.001). No difference was seen for blood pressure, lipids, body mass index, smoking, or diabetes mellitus. Conclusion The common Ala-87-Thr polymorphism of the P2Y(11) receptor is associated with AMI and increased levels of C-reactive protein. We hypothesize that an inflammatory mechanism might be involved. The P2Y(11) receptor is a promising new drug target in the prevention of AMI.
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| 2. |
- Balogh, Johanna, et al.
(author)
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Phospholipase C and cAMP-dependent positive inotropic effects of ATP in mouse cardiomyocytes via P2Y(11)-like receptors.
- 2005
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In: Journal of Molecular and Cellular Cardiology. - : Elsevier BV. - 1095-8584 .- 0022-2828. ; 39:2, s. 223-230
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Journal article (peer-reviewed)abstract
- ATP is released as a cotransmitter together with catecholamines from sympathetic nerves. In the heart ATP has been shown to cause a pronounced positive inotropic effect and may also act in synergy with β-adrenergic agonists to augment cardiomyocyte contractility. The aim of the present study was to investigate the inotropic effects mediated by purinergic P2 receptors using isolated mouse cardiomyocytes. Stable adenine nucleotide analogs were used and the agonist rank order for adenine nucleotide stimulation of the mouse cardiomyocytes was AR-C67085 > ATPγS > 2-MeSATP >>> 2-MeSADP = 0, that fits the agonist profile of the P2Y11 receptor. ATPγS induced a positive inotropic response in single mouse cardiomyocytes. The response was similar to that for the β1 receptor agonist isoproterenol. The most potent response was obtained using AR-C67085, a P2Y11 receptor agonist. This agonist also potentiated contractions in isolated trabecular preparations. The adenylyl cyclase blocker (SQ22563) and phospholipase C (PLC) blocker (U73122) demonstrated that both pathways were required for the inotropic response of AR-C67085. A cAMP enzyme immunoassay confirmed that AR-C67085 increased cAMP in the cardiomyocytes. These findings are in agreement with the P2Y11 receptor, coupled both to activation of IP3 and cAMP, being a major receptor for ATP induced inotropy. Analyzing cardiomyocytes from desmin deficient mice, Des–/–, with a congenital cardiomyopathy, we found a lower sensitivity to AR-C67085, suggesting a down-regulation of P2Y11 receptor function in heart failure. The prominent action of the P2Y11 receptor in controling cardiomyocyte contractility and possible alterations in its function during cardiomyopathy may suggest this receptor as a potential therapeutic target. It is possible that agonists for the P2Y11 receptor could be used to improve cardiac output in patients with circulatory shock and that P2Y11 receptor antagonist could be beneficial in patients with congestive heart failure (CHF).
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| 3. |
- Bergdahl, Andreas, et al.
(author)
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Plasticity of TRPC expression in arterial smooth muscle: correlation with store-operated Ca2+ entry.
- 2005
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In: American Journal of Physiology: Cell Physiology. - : American Physiological Society. - 1522-1563 .- 0363-6143. ; 288:4, s. 872-880
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Journal article (peer-reviewed)abstract
- Loss of the smooth muscle contractile phenotype is critical in atherosclerosis and in restenosis after angioplasty, but its early signals are incompletely understood. In this study, we have explored the role of transient receptor potential canonical (TRPC) proteins, which have been suggested to mediate store-operated Ca2+ entry (SOCE). 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 Ca2+ 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. Ca2+ 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 approximately 50%. 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 Ca2+ handling, and that TRPC1 is a subunit of upregulated store-operated Ca2+ channels.
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| 4. |
- Braun, Oscar, et al.
(author)
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Residual platelet ADP reactivity after clopidogrel treatment is dependent on activation of both the unblocked P2Y(1) and the P2Y (12) receptor and is correlated with protein expression of P2Y (12).
- 2007
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In: Purinergic Signalling. - : Springer Science and Business Media LLC. - 1573-9546 .- 1573-9538. ; 3:3, s. 195-201
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Journal article (peer-reviewed)abstract
- Two ADP receptors have been identified on human platelets: P2Y(1) and P2Y(12). The P2Y(12) receptor blocker clopidogrel is widely used to reduce the risks in acute coronary syndromes, but, currently, there is no P2Y(1) blocker in clinical use. Evidence for variable responses to clopidogrel has been described in several reports. The mechanistic explanation for this phenomenon is not fully understood. The aim of this study was to examine mechanisms responsible for variability of 2MeS-ADP, a stable ADP analogue, induced platelet reactivity in clopidogrel-treated patients. Platelet reactivity was assessed by flow cytometry measurements of P-selectin (CD62P) and activated GpIIb/IIIa complex (PAC-1). Residual 2MeS-ADP activation via the P2Y(12) and P2Y(1) receptors was determined by co-incubation with the selective antagonists AR-C69931 and MRS2179 in vitro. P2Y(1) and P2Y(12) receptor expression on both RNA and protein level were determined, as well as the P2Y(12) H1 or H2 haplotypes. Our data suggest that the residual platelet activation of 2MeS-ADP after clopidogrel treatment is partly due to an inadequate antagonistic effect of clopidogrel on the P2Y(12) receptor and partly due to activation of the P2Y(1) receptor, which is unaffected by clopidogrel. Moreover, a correlation between increased P2Y(12) protein expression on platelets and decreased response to clopidogrel was noticed, r(2)=0.43 (P<0.05). No correlation was found between P2Y(12) mRNA levels and clopidogrel resistance, indicating post-transcriptional mechanisms. To achieve additional ADP inhibition in platelets, antagonists directed at the P2Y(1) receptor could be more promising than the development of more potent P2Y(12) receptor antagonists.
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| 5. |
- Kim, HS, et al.
(author)
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Methanocarba modification of uracil and adenine nucleotides: High potency of northern ring conformation at P2Y(1), P2Y(2), P2Y(4), and P2Y(11) but not P2Y(6) receptors
- 2002
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In: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 1520-4804 .- 0022-2623. ; 45:1, s. 208-218
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Journal article (peer-reviewed)abstract
- The potency of nucleotide antagonists at P2Y(1) receptors was enhanced by replacing the ribose moiety with a constrained carbocyclic ring (Nandanan, et al. J. Med. Chem. 2000, 43, 829842). 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 P2Y(1) or human P2Y(1), P2Y(2), P2Y(4), P2Y(6), and P2Y(11) receptors stably expressed in astrocytoma cells was measured. At recombinant human P2Y(1) and P2Y(2) receptors, (N)-methanocarba-ATP was 138- and 41-fold, respectively, more potent than racemic (S)-methanocarba-ATP as an agonist. (N)methanocarba-ATP activated P2Y(11) 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 P2Y(4) receptors with an EC50 of 85 nM. (N)-Methanocarba-uridine 5'-diphosphate (UDP) was inactive at the hP2Y(6) 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 P2Y(4) response (pEC(50) = 6.1 +/- 0.2), while the diphosphate was inactive as either an agonist or antagonist in a P2Y(6) 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 P2Y(1), P2Y(2), P2Y(4), and P2Y(11) receptors.
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| 6. |
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| 7. |
- Wihlborg, Anna-Karin, et al.
(author)
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2,2 '-nitrophenylisatogen potentiates P2X(1) receptor mediated vascular contraction and blood pressure elevation
- 2003
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In: Drug Development Research (Proceedings of the Seventh International Symposium on Adenosine and Adenine Nucleotides - Part 2). - : Wiley. - 1098-2299 .- 0272-4391. ; 59:1, s. 82-87
-
Conference paper (peer-reviewed)abstract
- The objective of this research was to examine the effects of chemical compounds with possible P2 receptor modulating effects and to characterize the potentiating effects of 2,2'-nitrophenylisatogen (NPI) on P2X(1) receptors in vitro and in vivo. Chemical compounds were tested in an in vitro pharmacological assay using vascular segments from the rat mesenteric artery stimulated by P2 receptor-specific agonists. Contractions were expressed as a percentage of 60 mM K+-induced contractions. Blood pressure was evaluated in pithed rats. NPI (30 muM) added 15 min before addition of the P2X(1) receptor-specific agonist alphabeta-MeATP increased the maximum vasoconstriction from 23% to 49% (an increase of 113%). Furthermore, NPI prevented the desensitization of repeated UP-MeATP contractions. Related compounds were examined, and 2-(3-methoxy-phenyl)-1-oxy-indol-3-one (MPI) had similar effects as NPI, but several others lacked effect. NPI had no effect on ADPbetaS (P2Y(1)) or acetylcholine-mediated vasodilatation, nor on UTP (P2Y(2/4)), UDP (P2Y(6)), or noradrenaline-mediated contractions. In pithed rats, the blood pressure response to 50 nmol/kg-infusion of alphabeta-MeATP was increased from 50+/-6 to 63+/-5 mmHg (P < 0.05), but had no effect on basal blood pressure or on the cardiovascular response to preganglionic nerve stimulation. In conclusion, NPI and MPI potentiates P2X(1) receptor vascular contractions in vitro and (NPI) blood pressure effects in vivo. It is possible that the effect is mediated by an inhibition of P2X(1) receptor desensitization. Drug Dev. Res. (C) Wiley-Liss, Inc.
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| 8. |
- Wihlborg, Anna-Karin, et al.
(author)
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ADP Receptor P2Y12 Is Expressed in Vascular Smooth Muscle Cells and Stimulates Contraction in Human Blood Vessels.
- 2004
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In: Arteriosclerosis, Thrombosis and Vascular Biology. - 1524-4636. ; 24:10, s. 1810-1815
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Journal article (peer-reviewed)abstract
- 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 = 15 +/- 6% of 60 mmol/L K+ contraction, pEC(50) = 5.6 +/- 0.6, E-max = 21 +/- 1%, pEC(50) = 6.8 +/- 0.1, and E-max = 48 +/- 9%, pEC(50) = 6.6 +/- 0.4). 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.
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| 9. |
- Wihlborg, Anna-Karin, et al.
(author)
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Extracellular nucleotides induce vasodilatation in human arteries via prostaglandins, nitric oxide and endothelium-derived hyperpolarising factor.
- 2003
-
In: British Journal of Pharmacology. - : Wiley. - 1476-5381 .- 0007-1188. ; 138:8, s. 1451-1458
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Journal article (peer-reviewed)abstract
- 1. The present study was aimed at examining P2 receptor-mediated vasodilatation in human vessels. The isometric tension was recorded in isolated segments of the human left internal mammary artery branches precontracted with 1 muM noradrenaline. 2. Endothelial denudation abolished the dilator responses. 3. The selective P2Y1 agonist, 2-MeSADP, induced a potent vasodilatation (pEC50=6.9plusminus0.1). The P2Y1 antagonist of 10 muM, MRS 2216, shifted the 2-MeSADP concentration-response curve 1.1 log units to the right. The combined P2Y1 and P2X agonist, 2-MeSATP, stimulated a dilatation with a potency similar to that of 2-MeSADP. Furthermore, MRS 2216 had a similar antagonistic effect on both 2-MeSATP and 2-MeSADP indicating that P2X receptors do not mediate vasodilatation. 4. Both the P2Y2/4 agonist, UTPitalic gammaS and the P2Y6 agonist, UDPbetaS, stimulated potent dilatations (pEC50=7.8plusminus0.4 for UTPitalic gammaS and 8.4plusminus0.2 for UDPbetaS). 5. The 2-MeSADP-induced nitric oxide (NO)-mediated dilatation was studied in the presence of 10 muM indomethacin, 50 nM charybdotoxin and 1 muM apamin. The involvement of the endothelium-derived hyperpolarising factor (EDHF) was investigated in the presence of 0.1 mML-NOARG and indomethacin. The involvement of prostaglandins was investigated in the presence of L-NOARG, charybdotoxin and apamin. Both NO, EDHF and prostaglandins mediated 2-MeSADP dilatation with similar efficacy (Emax=25plusminus5% for NO, 25plusminus6% for EDHF and 27plusminus5% for prostaglandins). 6. In conclusion, extracellular nucleotides induce endothelium-derived vasodilatation in human vessels by stimulating P2Y1, P2Y2/4 and P2Y6 receptors, while P2X receptors are not involved. Endothelial P2Y receptors mediate dilatation by release of EDHF, NO and prostaglandins
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| 10. |
- Wihlborg, Anna-Karin, et al.
(author)
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Positive inotropic effects by uridine triphosphate (UTP) and uridine diphosphate (UDP) via P2Y(2) and P2Y(6) receptors on cardiomyocytes and release of UTP in man during myocardial infarction
- 2006
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In: Circulation Research. - 0009-7330. ; 98:7, s. 970-976
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Journal article (peer-reviewed)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 gamma S increased contraction by 52%, similar to beta(1)-adrenergic stimulation with isoproterenol (65%). The P2Y(6)-agonist UDP gamma S also increased cardiomyocyte contraction (35%), an effect abolished by the P2Y(6)-blocker MRS2578. The phospholipase C inhibitor U73122 inhibited both the UDP beta S and the UTP gamma S-induced inotropic effect, indicating an IP3-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. For the first time we demonstrate inotropic effects of UDP, mediated by P2Y(6) receptors via an IP3-dependent pathway. Thus, the extracellular pyrimidines ( UTP and UDP) could be important inotropic factors involved in the development of cardiac disease.
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