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Zhongren Ding

Publications and source records attributed to Zhongren Ding.

8 recordsLinked to original sources
PubMed2005

Identification of a potent inverse agonist at a constitutively active mutant of human P2Y12 receptor.

Human platelets express two P2Y receptors: G(q)-coupled P2Y(1), and G(i)-coupled P2Y(12). Both P2Y(1) and P2Y(12) are ADP receptors on human platelets and are essential for ADP-induced platelet aggregation that plays pivotal roles in thrombosis and hemostasis. Numerous constitutively active G protein-coupled receptors have been described in natural or recombinant systems, but in the P2Y receptors, to date, no constitutive activity has been reported. In our effort to identify G protein coupling domains of the human platelet ADP receptor, we constructed a chimeric hemagglutinin-tagged human P2Y(12) receptor with its C terminus replaced by the corresponding part of human P2Y(1) receptor and stably expressed it in Chinese hamster ovary-K1 cells. It is interesting that the chimeric P2Y(12) mutant exhibited a high level of constitutive activity, as evidenced by decreased cAMP levels in the absence of agonists. The constitutive activation of the chimeric P2Y(12) mutant was dramatically inhibited by pertussis toxin, a G(i) inhibitor. The constitutively active P2Y(12) mutant retained normal responses to 2-methylthio-ADP, with an EC(50) of 0.15 +/- 0.04 nM. The constitutively active P2Y(12) mutant caused Akt phosphorylation that was abolished by the addition of pertussis toxin. Pharmacological evaluation of several P2Y(12) antagonists revealed (E)-N-[1-[7-(hexylamino)-5-(propylthio)-3H-1,2,3-triazolo-[4,5-d]-pyrimidin-3-yl]-1,5,6-trideoxy-beta-d-ribo-hept-5-enofuranuronoyl]-l-aspartic acid (AR-C78511) as a potent P2Y(12) inverse agonist and 5'-adenylic acid, N-[2-(methylthio)ethyl]-2-[(3,3,3-trifluoropropyl)thio]-, monoanhydride with (dichloromethylene)bis[phosphonic acid] (AR-C69931MX) as a neutral antagonist. In conclusion, this is the first report of a cell line stably expressing a constitutively active mutant of human platelet P2Y(12) receptor and the identification of potent inverse agonist.

Animals↗
PubMed2004

Arg333 and Arg334 in the COOH terminus of the human P2Y1 receptor are crucial for Gq coupling.

The P2Y(1) ADP receptor activates G(q) and causes increases in intracellular Ca(2+) concentration through stimulation of PLC. In this study, we investigated the role of the amino acid residues in the COOH terminus of the human P2Y(1) receptor in G(q) activation. Stimulation of Chinese hamster ovary (CHO-K1) cells stably expressing the wild-type human P2Y(1) receptor (P2Y(1)-WT cells), P2Y(1)-DeltaR340-L373, or P2Y(1)-DeltaD356-L373 with 2-methylthio-ADP (2-MeSADP) caused inositol phosphate production. In contrast, cells expressing P2Y(1)-DeltaT330-L373, a mutant lacking the entire COOH terminus, completely lost their response to 2-MeSADP. Similar data were obtained by using these cell lines and measuring Ca(2+) mobilization upon stimulation with 2-MeSADP, indicating that the 10 amino acids (330TFRRRLSRAT339) in the COOH terminus of the human P2Y(1) receptor are essential for G(q) coupling. Radioligand binding demonstrated that both the P2Y(1)-WT and P2Y(1)-DeltaT330-L373-expressing cells have almost equal binding of [(3)H]MRS2279, a P2Y(1) receptor antagonist, indicating that COOH-terminal truncation did not drastically affect the conformation of the receptor. CHO-K1 cells expressing a chimeric P2Y(12) receptor with the P2Y(1) COOH terminus failed to elicit G(q) functional responses, indicating that the P2Y(1) COOH terminus is essential but not sufficient for G(q) activation. Finally, cells expressing a double-mutant P2Y(1) receptor (R333A/R334A) in the conserved BBXXB region of the COOH terminus of the G(q)-activating P2Y receptors completely lost their functional ability to activate G(q). We conclude that the two arginine residues (R333R334) in the COOH terminus of the human P2Y(1) receptor are essential for G(q) coupling.

Adenosine Diphosphate↗
PubMed2004

Distribution of PAI-1 promoter 4G/5G polymorphism in healthy Chinese and functional characterization using a luciferase reporter vector.

The aim of this study was to analyze the distribution of plasminogen activator inhibitor-1 (PAI-1) promoter 4G/5G polymorphism in healthy Chinese and compare the transcription activity of the two PAI-1 promoters using luciferase reporter vector pGL(3). PAI-1 promoter 4G/5G polymorphism was analyzed in 77 healthy Chinese using allele-specific polymerase chain reaction (PCR). The PAI-1 promoters from individuals homozygous for the insertion (5G) and deletion (4G) were cloned into luciferase reporter vector pGL(3), respectively. The transcription activities of the two promoters (PAI-1-4G and PAI-1-5G) were then compared using the luciferase reporter vectors expressed in ECV304 and HepG(2) cell lines. Among the 77 healthy Chinese the genotype frequency for 4G/4G, 4G/5G, and 5G/5G was 49.1, 44.1, and 6.5%, respectively. Both of the two PAI-1 promoters cloned into luciferase reporter vector pGL(3), expressed very well both in ECV304 and HepG(2), and have similar basic transcription activity. Thrombin (10-20 U/ml) has no effects on the transcription activity of the two promoters expressed in ECV304, while in the HepG(2) cell line interleukin-1 (IL-1) 158 U/ml increased the transcription activity of PAI-1-4G by 67% (p<0.05, n=5) without effects on PAI-1-5G transcription activity. The distribution of PAI-1 promoter 4G/5G polymorphism in Chinese is obviously different from that in Caucasians with the 4G allele more frequently found in Chinese. PAI-1 promoter 4G/5G polymorphism does not alter basic transcription activity and 4G promoter has increased response to IL-1.

Cell Line, Tumor↗
PubMed2004

Role of G protein-gated inwardly rectifying potassium channels in P2Y12 receptor-mediated platelet functional responses.

The role of the G(i)-coupled platelet P2Y(12) receptor in platelet function has been well established. However, the functional effector or effectors contributing directly to alphaIIbbeta3 activation in human platelets has not been delineated. As the P2Y(12) receptor has been shown to activate G protein-gated, inwardly rectifying potassium (GIRK) channels, we investigated whether GIRK channels mediate any of the functional responses of the platelet P2Y(12) receptor. Western blot analysis revealed that platelets express GIRK1, GIRK2, and GIRK4. In aspirin-treated and washed human platelets, 2 structurally distinct GIRK inhibitors, SCH23390 (R(+)-7-chloro-8-hydroxy-3-methyl-1-phenyl-2,3,4,5-tetrahydro-1H-3-benzazepine hydrochloride) and U50488H (trans-(+/-)-3,4-dichloro-N-methyl-N-[2-(pyrrolidinyl)cyclohexyl] benzeneacetamide methanesulfonate), inhibited adenosine diphosphate (ADP)-, 2-methylthioADP (2-MeSADP)-, U46619-, and low-dose thrombin-mediated platelet aggregation. However, the GIRK channel inhibitors did not affect platelet aggregation induced by high concentrations of thrombin, AYPGKF, or convulxin. Furthermore, the GIRK channel inhibitors reversed SFLLRN-induced platelet aggregation, inhibited the P2Y(12)-mediated potentiation of dense granule secretion and Akt phosphorylation, and did not affect the agonist-induced G(q)-mediated platelet shape change and intracellular calcium mobilization. Unlike AR-C 69931MX, a P2Y(12) receptor-selective antagonist, the GIRK channel blockers did not affect the ADP-induced adenlylyl cyclase inhibition, indicating that they do not directly antagonize the P2Y(12) receptor. We conclude that GIRK channels are important functional effectors of the P2Y(12) receptor in human platelets.

3,4-Dichloro-N-methyl-N-(2-(1-pyrrolidinyl)-cycloh↗
PubMed2003

Molecular mechanism of nucleotide-induced primary granule release in human neutrophils: role for the P2Y2 receptor.

Nucleotides are released during vascular injury from activated platelets and broken cells, which could stimulate human neutrophils. In this study, we characterized the P2Y receptors and investigated the functional effects of extracellular nucleotides on human neutrophils. Pharmacological characterization using selective agonists and pertussis toxin revealed that human neutrophils express only functional P2Y2 receptors. However, P2Y2 receptor agonists ATP or uridine triphosphate (UTP) caused intracellular Ca2+ increases in isolated human neutrophils with an EC50 of 1 microM but failed to cause release of primary granules from human neutrophils. ATP and UTP were equally potent in causing elastase release from human neutrophils in the presence of exogenous soluble fibrinogen, whereas ADP and UDP were without effect. We investigated whether nucleotides depend on generated arachidonic acid metabolites to cause degranulation. However, phenidone and MK-886, inhibitors of the 5-lipoxygenase pathway, failed to block nucleotide-induced intracellular calcium mobilization and elastase release. ATP and UTP caused activation of p38 MAPK and ERK1/2 in human neutrophils. In addition, the inhibitors of the MAPK pathway, SB-203580 and U-0126, inhibited nucleotide-induced elastase release. We conclude that fibrinogen is required for nucleotide-induced primary granule release from human neutrophils through the P2Y2 receptor without a role for arachidonic acid metabolites. Both ERK1/2 and p38 MAPK play an important role in nucleotide-induced primary granule release from human neutrophils.

Adenosine Triphosphate↗
PubMed2003

Inactivation of the human P2Y12 receptor by thiol reagents requires interaction with both extracellular cysteine residues, Cys17 and Cys270.

Human platelets express 2 G protein-coupled nucleotide receptors: the platelet adenosine diphosphate (ADP) receptor coupled to stimulation of phospholipase C (P2Y(1)) via heterotrimeric guanosine 5-triphosphate (GTP)-binding protein G(q), and the platelet ADP receptor coupled to inhibition of adenylyl cyclase (P2Y(12)) via heterotrimeric GTP-binding protein G(i). Although these 2 receptors are encoded on the same chromosome and have similar pharmacologic profiles, they have different reactivities toward thiol reagents. The thiol agent p-chloromercuribenzene sulfonic acid (pCMBS) and the active metabolites from antiplatelet drugs, clopidogrel and CS-747, inactivate the P2Y(12) receptor and are predicted to interact with the extracellular cysteine residues on the P2Y(12) receptor. In this study we identified the reactive cysteine residues on the human P2Y(12) receptor by site-directed mutagenesis using pCMBS as the thiol reagent. Cys97Ser and Cys175Ser mutants of the P2Y(12) receptor did not express when transfected into Chinese hamster ovary (CHO-K1) cells, indicating the essential nature of a disulfide bridge between these residues. The Cys17Ser, Cys270Ser, and Cys17Ser/Cys270Ser double mutants had similar median effective concentration (EC(50)) values for ADP and 2-methylthio-ADP (2-MeSADP) when compared with the wild-type P2Y(12). Similarly, the median inhibitory concentration (IC(50)) values for BzATP (2',3'-O-(4- benzoylbenzoyl) adenosine 5'-triphosphate), an antagonist of the P2Y(12) receptor, also did not differ dramatically among these mutants and the wild-type P2Y(12) receptor. pCMBS inactivated the wild-type P2Y(12) receptor in a concentration-dependent manner, whereas it had no effect on the P2Y(1) receptor. Finally, pCMBS partially affected the G(i) coupling of Cys17Ser or Cys270Ser receptor mutants, but had no effect on Cys17Ser/Cys270Ser P2Y(12) receptor-mediated inhibition of adenylyl cyclase. These results indicate that, unlike the P2Y(1) receptor, which has 2 essential disulfide bridges linking its extracellular domains, the P2Y(12) receptor has 2 free cysteines in its extracellular domains (Cys17 and Cys270), both of which are targets of thiol reagents. We speculate that the active metabolites of clopidogrel and CS-747 form disulfide bridges with both Cys17 and Cys270 in the P2Y(12) receptor, and thereby inactivate the receptor.

4-Chloromercuribenzenesulfonate↗
PubMed2003

ADP receptors--targets for developing antithrombotic agents.

Platelet P2 receptors--P2Y1, P2Y12, and P2X1--constitute the means by which adenine nucleotides can activate platelets. Coactivation of the Galphaq-coupled P2Y1 and Galphai2-coupled P2Y12 receptors is necessary for ADP-mediated platelet activation, which forms the basis of using P2 antagonists as antithrombotic drugs. P2Y1 receptor antagonists inhibit platelet activation, while P2Y1 knockout mice show longer bleeding times than normal mice but few other problems; however, its ubiquitous expression in other tissues renders P2Y1 questionable as an antithrombotic target. The P2Y12 receptor is expressed nearly exclusively in platelets and brain, making it an attractive antithrombotic target. Antagonists for the P2Y12 receptor have been developed that either require metabolic activation to covalently inhibit P2Y12 and are irreversible, or simply are competitive in nature and thus reversible. Ticlopidine and clopidogrel are irreversible P2Y12 antagonists and have been repeatedly proven as clinical antithrombotic agents. In addition, a recently reported P2Y12 antagonist, CS-747, shows promise as a future antithrombotic drug. The AR-C series of compounds represent reversible P2Y12 antagonists and have been used extensively to characterize the function of P2Y12 in platelets. Clinical studies show that AR-C69931MX is as effective as clopidogrel; furthermore, the combination of AR-C69931MX (cangrelor) and clopidogrel confers greater antagonism of P2Y12 than either antagonist alone. The P2X1 receptor is a calcium channel that functions to potentiate agonist-induced platelet shape change, and its inhibition or loss has little if any effect on hemostasis. A combination of P2Y1 and P2Y12 antagonists may represent an additional course of antithrombotic treatment.

Animals↗