Background and purpose:
The P2Y11 receptor, a member of the group of metabotropic nucleotide receptors, shows a stereospecific ligand recognition of Pα-substituted ATP derivatives (ATP-α-S isomers). These compounds are suitable candidates for the development of selective P2Y11 receptor agonists that might be used as immune modulators. We have analysed the binding mode of ATP at the P2Y11 receptor by molecular modeling and site-directed mutagenesis. Based on our recent findings, we decided to decipher the molecular determinants of stereoselective recognition at the P2Y11 receptor.Experimental approach:
Two amino acid residues [Glu186 in the extracellular loop 2 and Arg268 in the transmembrane domain 6 (TM6)], which are part of the nucleotide-binding pocket, were selected and studied by mutational analyses. We expected these residues to be involved in determining the stereospecificity of the P2Y11 receptor.Key results:
After mutation of Arg268 to alanine or glutamine, the stereospecific recognition of the ATP-α-S isomers at the P2Y11 receptor was lost. In contrast, at the Glu186Ala receptor mutant, the stereoselective differentiation between these isomers was increased. On the Arg268Gln/Glu186Ala double mutant we observed no further effect, except for additivity in the decrease in potency of both isomers, as compared with the single-point mutants.Conclusions and implications:
Our results show that the stereospecificity of the P2Y11 receptor for Pα-substituted ATP derivatives is largely determined by the basic residue Arg268 in TM6. This will allow the design of receptor-subtype selective ligands. 相似文献BACKGROUND AND PURPOSE
P2Y1 is a purine receptor that triggers platelet aggregation. Its inhibition was studied in patients with stable coronary artery disease (CAD) receiving standard anti-platelet therapy.EXPERIMENTAL APPROACH
Blood samples from 10 patients on aspirin therapy (ASA, 80 mg·day−1) were withdrawn before and 24 h after the administration of 450 mg clopidogrel (ASA/C) and were anti-coagulated with citrate or hirudin/PPACK in the presence or absence of the P2Y1 inhibitor MRS2179 (M, 100 µM). Platelet responses to ADP (2.5 µM) and TRAP (2.5 µM), and collagen-induced thrombosis under flow conditions were analysed.KEY RESULTS
Compared with ASA, ASA + M strongly inhibited ADP-induced peak platelet aggregation (88%), late aggregation (84%), P-selectin expression (85%) and αIIbβ3 activation (62%) (28%, 65%, 70% and 51% inhibition, respectively, for ASA/C vs. ASA). ASA + M also inhibited platelet/monocyte and platelet/neutrophil conjugate formation by 69% and 71% (57% and 59% for ASA/C vs. ASA). In TRAP-activated blood, ASA + M unexpectedly inhibited αIIbb3 activation by 30%. In blood perfused in collagen-coated glass capillaries (shear rate of 1500 s−1), ASA/C prevented thrombus growth beyond 5 min in relation to thrombus fragments embolization. ASA + M with or without clopidogrel completely prevented thrombus formation. Finally, ex vivo addition of MRS2179 and ASA to the blood of healthy donors markedly blocked thrombus formation on collagen in flow conditions, in contrast to ASA plus the P2Y12 inhibitor 2-MeSAMP.CONCLUSIONS AND IMPLICATIONS
Through particularly efficient complementarities with ASA to inhibit platelet activation and thrombus formation, the inhibition of P2Y1 in the blood of patients with CAD appears to play a more important role than previously anticipated. 相似文献Areas covered: We searched articles about P2Y12 receptor inhibitors and stroke in PubMed published until December 2014. This is a comprehensive review of the role of P2Y12 receptor inhibitors alone and in combination with aspirin in the secondary prevention of noncardioembolic stroke.
Expert opinion: The potential benefit of more potent antiplatelet therapy for secondary stroke prevention must be weighed against the risk of bleeding in patients with IS. Short-term (≤ 3 months) dual antiplatelet therapy with clopidogrel and aspirin that is initiated early after IS or transient ischemic attack due to large artery atherosclerosis appears most efficient. 相似文献
- The mitogen-activated protein (MAP) kinase signalling pathway can be activated by a variety of heterotrimeric Gi/Go protein-coupled and Gq/G11 protein-coupled receptors. The aims of the current study were: (i) to investigate whether the Gi/Go protein-coupled adenosine A1 receptor activates the MAP kinase pathway in transfected Chinese hamster ovary cells (CHO-A1) and (ii) to determine whether adenosine A1 receptor activation would modulate the MAP kinase response elicited by the endogenous P2Y2 purinoceptor.
- The selective adenosine A1 receptor agonist N6-cyclopentyladenosine (CPA) stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50 7.1±0.4 nM). CPA-mediated increases in MAP kinase activity were blocked by PD 98059 (50 μM; 89±4% inhibition), an inhibitor of MAP kinase kinase 1 (MEKI) activation, and by pre-treating cells with pertussis toxin (to block Gi/Go-dependent pathways).
- Adenosine A1 receptor-mediated activation of MAP kinase was abolished by pre-treatment with the protein tyrosine inhibitor, genistein (100 μM; 6±10% of control). In contrast, daidzein (100 μM), the inactive analogue of genistein had no significant effect (96±12 of control). MAP kinase responses to CPA (1 μM) were also sensitive to the phosphatidylinositol 3-kinase inhibitors wortmannin (100 nM; 55±8% inhibition) and LY 294002 (30 μM; 40±5% inhibition) but not to the protein kinase C (PKC) inhibitor Ro 31-8220 (10 μM).
- Activation of the endogenous P2Y2 purinoceptor with UTP also stimulated time and concentration-dependent increases in MAP kinase activity in CHO-A1 cells (EC50=1.6±0.3 μM). The MAP kinase response to UTP was partially blocked by pertussis toxin (67±3% inhibition) and by the PKC inhibitor Ro 31-8220 (10 μM; 45±5% inhibition), indicating the possible involvement of both Gi/Go protein and Gq protein-dependent pathways in the overall response to UTP.
- CPA and UTP stimulated concentration-dependent increases in the phosphorylation state of the 42 kDa and 44 kDa forms of MAP kinase as demonstrated by Western blotting.
- Co-activation of CHO-A1 cells with CPA (10 nM) and UTP (1 μM) produced synergistic increases in MAP kinase activity which were not blocked by the PKC inhibitor Ro 31-8220 (10 μM).
- Adenosine A1 and P2Y2 purinoceptor activation increased the expression of luciferase in CHO cells transfected with a luciferase reporter gene containing the c-fos promoter. However, co-activating these two receptors produced only additive increases in luciferase expression.
- In conclusion, our studies have shown that the transfected adenosine A1 receptor and the endogenous P2Y2 purinoceptor couple to the MAP kinase signalling pathway in CHO-A1 cells. Furthermore, co-stimulation of the adenosine A1 receptor and the P2Y2 purinoceptor produced synergistic increases in MAP kinase activity but not c-fos mediated luciferase expression.
BACKGROUND AND PURPOSE
Extracellular nucleotides are released at high concentrations from damaged cells and function through P2 receptor activation. Intestinal epithelial restitution, which is defined as cell migration independent of cell proliferation, is an important initial step in the process of wound healing. In this study, we investigated the role of extracellular nucleotides in intestinal epithelial migratory responses.EXPERIMENTAL APPROACH
Wound-healing and trans-well migration assays were performed with a rat intestinal epithelial cell line (IEC-6). The concentrations of extracellular nucleotides released from injured IEC-6 cells were measured by HPLC. TGF-β expression was assessed by RT-PCR and elisa.KEY RESULTS
Scratching the monolayer of IEC-6 cells induced cell migration. Pretreatment with apyrase or MRS2578, a selective P2Y6 antagonist, inhibited the wound-induced cell migration. Among the cellular nucleotides, only ATP and uridine 5''-diphosphate (UDP) were detected in the culture medium after cell wounding. Exogenously applied UDP dose-dependently enhanced the migration more effectively than ATP but did not induce proliferation. In addition, cell wounding and UDP increased the expression of TGF-β, and both the wound-induced and UDP-enhanced migration were inhibited by MRS2578 or ALK5Inhibitor (ALK5i), a TGF-β receptor blocker. Furthermore, cell wounding and UDP stimulation up-regulated the expression of P2Y6 receptor mRNA, and this effect was suppressed by MRS2578 or ALK5i.CONCLUSION AND IMPLICATIONS
Wound-induced UDP evokes intestinal epithelial restitution by activation of P2Y6 receptors, which mediates de novo synthesis of TGF-β. In addition, the expression of P2Y6 receptors is increased by cell wounding and UDP, which constitutes a positive-feedback loop for mucosal repair. 相似文献Background and purpose:
P2Y nucleotide receptors are involved in the regulation of vascular tone, smooth muscle cell (SMC) proliferation and inflammatory responses. The present study investigated whether they are involved in atherosclerosis.Experimental approach:
mRNA of P2Y receptors was quantified (RT-PCR) in atherosclerotic and plaque-free aorta segments of apolipoprotein E-deficient (apoE–/–) mice. Macrophage activation was assessed in J774 macrophages, and effects of non-selective purinoceptor antagonists on atherosclerosis were evaluated in cholesterol-fed apoE–/– mice.Key results:
P2Y6 receptor mRNA was consistently elevated in segments with atherosclerosis, whereas P2Y2 receptor expression remained unchanged. Expression of P2Y1 or P2Y4 receptor mRNA was low or undetectable, and not influenced by atherosclerosis. P2Y6 mRNA expression was higher in cultured J774 macrophages than in cultured aortic SMCs. Furthermore, immunohistochemical staining of plaques demonstrated P2Y6-positive macrophages, but few SMCs, suggesting that macrophage recruitment accounted for the increase in P2Y6 receptor mRNA during atherosclerosis. In contrast to ATP, the P2Y6-selective agonist UDP increased mRNA expression and activity of inducible nitric oxide synthase and interleukin-6 in J774 macrophages; this effect was blocked by suramin (100–300 µM) or pyridoxal-phosphate-6-azophenyl-2′-4′-disulphonic acid (PPADS, 10–30 µM). Finally, 4-week treatment of cholesterol-fed apoE–/– mice with suramin or PPADS (50 and 25 mg·kg−1·day−1 respectively) reduced plaque size, without changing plaque composition (relative SMC and macrophage content) or cell replication.Conclusions and implications:
These results suggest involvement of nucleotide receptors, particularly P2Y6 receptors, during atherosclerosis, and warrant further research with selective purinoceptor antagonists or P2Y6 receptor-deficient mice. 相似文献Areas covered: Basic science articles, clinical studies, and reviews from 1992–2017 were searched using Pubmed library to collet impactful literature. After an introduction to the purinergic receptor biology, this review summarizes current knowledge on P2Y12 receptor inhibitors. Furthermore, we describe the subsequent improvements of next-generation P2Y12 receptor inhibitors facing the ambivalent problem of bleeding events versus prevention of arterial thrombosis in a variety of clinical settings. Therefore, we summarize data from relevant preclinical and clinical trials of currently approved P2Y12 receptor inhibitors (clopidogrel, prasugrel, ticagrelor, cangrelor) and provide strategies of drug switching and management of bleeding events.
Expert opinion: An enormous amount of pharmacologic and clinical data is available for the application of P2Y12 receptor inhibitors. Today prasugrel, ticagrelor and clopidogrel are the standard of care drugs during dual antiplatelet therapy for ACS patients, but have considerable rates of bleeding. Recent and future clinical trials will provide evidence for subsequent escalation and de-escalation strategies of P2Y12 receptor inhibition. These data may pave the way for an evidence-based, individualized P2Y12 receptor inhibitor therapy. 相似文献
Background and Purpose
The P2Y14 receptor is the newest member of the P2Y receptor family; it is Gi/o protein-coupled and is activated by UDP and selectively by UDP-glucose and MRS2690 (2-thiouridine-5′-diphosphoglucose) (7–10-fold more potent than UDP-glucose). This study investigated whether P2Y14 receptors were functionally expressed in porcine isolated pancreatic arteries.Experimental Approach
Pancreatic arteries were prepared for isometric tension recording and UDP-glucose, UDP and MRS2690 were applied cumulatively after preconstriction with U46619, a TxA2 mimetic. Levels of phosphorylated myosin light chain 2 (MLC2) were assessed with Western blotting. cAMP concentrations were assessed using a competitive enzyme immunoassay kit.Key Results
Concentration-dependent contractions with a rank order of potency of MRS2690 (10-fold) > UDP-glucose ≥ UDP were recorded. These contractions were reduced by PPTN {4-[4-(piperidin-4-yl)phenyl]-7-[4-(trifluoromethyl)phenyl]-2-naphthoic acid}, a selective antagonist of P2Y14 receptors, which did not affect responses to UTP. Contraction to UDP-glucose was not affected by MRS2578, a P2Y6 receptor selective antagonist. Raising cAMP levels and forskolin, in the presence of U46619, enhanced contractions to UDP-glucose. In addition, UDP-glucose and MRS2690 inhibited forskolin-stimulated cAMP levels. Removal of the endothelium and inhibition of endothelium-derived contractile agents (TxA2, PGF2α and endothelin-1) inhibited contractions to UDP glucose. Y-27632, nifedipine and thapsigargin also reduced contractions to the agonists. UDP-glucose and MRS2690 increased MLC2 phosphorylation, which was blocked by PPTN.Conclusions and Implications
P2Y14 receptors play a novel vasocontractile role in porcine pancreatic arteries, mediating contraction via cAMP-dependent mechanisms, elevation of intracellular Ca2+ levels, activation of RhoA/ROCK signalling and MLC2, along with release of TxA2, PGF2α and endothelin-1. 相似文献- Neuropeptide Y (NPY) and NPY receptors are most abundant in the hippocampal formation where they modulate cognitive functions. Expression of NPY receptors in rat cultured primary hippocampal cells was investigated in the present study by use of combined molecular, pharmacological and immunohistochemical approaches, including the cloning of the rat Y2 receptor described here for the first time.
- More than 70% of the hippocampal neurones were endowed with [125I]-[Leu31,Pro34]PYY Y1-like receptor silver grain accumulations and Y1 receptor immunostaining. These radio- and immuno-labelling signals were distributed over cell bodies and processes of bipolar, stellate and pyramidal-like neuronal cells, as confirmed by neurone-specific enolase and MAP-2 staining.
- Competition binding profiles revealed that specific [125I]-[Leu31,Pro34]PYY binding was competitively displaced according to a ligand selectivity pattern prototypical of the Y1 receptor sub-type with [Leu31,Pro34]substituted NPY/PYY analogues>>C-terminal fragments=pancreatic polypeptides, with the non-peptide antagonist BIBP3226 being most potent. This profile excludes the possible labelling by [125I]-[Leu31,Pro34]PYY of the newly cloned Y4, Y5 and Y6 receptors.
- The expression of the genuine Y1 receptor was confirmed by RT–PCR in hippocampal cultures. In contrast, negligible levels of Y2-like/[125I]-PYY3–36 binding were detected in these cultures in spite of the presence of its mRNA, as characterized by RT–PCR. The expression of both the Y1 and the Y2 receptor mRNAs was also noted in normal embryonic hippocampal tissues showing that signals expressed in cultured neurones were also present in utero.
- Taken together, these results suggest that the Y1 receptor subtype may be of critical importance in the normal functioning of the rat hippocampus, especially during brain development and maturation.