Purinergic Receptor Modulation of Lipopolysaccharide Signaling and Inducible Nitric-oxide Synthase Expression in RAW 264.7 Macrophages

Hu, Yun; Fisette, Philip L.; Denlinger, Loren C.; Guadarrama, Arturo G.; Sommer, Julie A.; Proctor, Richard A.; Bertics, Paul J.

doi:10.1074/jbc.273.42.27170

Cited by 132 publications

(145 citation statements)

References 37 publications

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“…The unique long C-terminal intracellular domain of P2X 7 is crucial for P2X 7 -induced pore formation, transduction, and signaling [1,72,80]. Three loss-of function SNPs (T357S, E496A, and I568N) and one gain-of-function SNP (Q460R) are located in the long intracellular domain [54][55][56]58].…”

Section: Discussionmentioning

confidence: 99%

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Sun

Chu

Singh

et al. 2009

Purinergic Signalling

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The P2X 7 receptor exhibits significant allelic polymorphism in humans, with both loss and gain of function variants potentially impacting on a variety of infectious and inflammatory disorders. At least five loss-offunction polymorphisms (G150R, R307Q, T357S, E496A, and I568N) and two gain-of-function polymorphisms (H155Y and Q460R) have been identified and characterized to date. In this study, we used RT-PCR cloning to isolate and characterize P2X 7 cDNA clones from human PBMCs and THP-1 cells. A previously unreported variant with substitutions of V80M and A166G was identified. When expressed in HEK293 cells, this variant exhibited heightened sensitivity to the P2X 7 agonist (BzATP) relative to the most frequent allele, as shown by pore formation measured by fluorescent dye uptake into cells. Mutational analyses showed that A166G alteration was critical for the gain-offunction change, while V80M was not. Full-length variants with multiple previously identified nonsynonymous SNPs (H155Y, H270R, A348T, and E496A) were also identified. Distinct functional phenotypes of the P2X 7 variants or mutants constructed with multiple polymorphisms were observed. Gain-of-function variations (A166G or H155Y) could not rescue the loss-of-function E496A polymorphism. Synergistic effects of the gain-of-function variations were also observed. We also identified the A348T alteration as a weak gain-of-function variant. Thus, these results identify the new gain-of-function variant A166G and demonstrate that multiple-gene polymorphisms contribute to functional phenotypes of the human P2X 7 receptor.Furthermore, the results demonstrate that the C-terminal of the cysteine-rich domain 1 of P2X 7 is critical for regulation of P2X 7 -mediated pore formation.

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Section: Discussionmentioning

confidence: 99%

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Sun

Chu

Singh

et al. 2009

Purinergic Signalling

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“…1, A and B). Because oATP is known to inhibit activation of the P2X7 nucleotide receptor, similar results in other macrophage types have led to the suggestion that autocrine signaling through P2 receptors accompanies and potentiates endotoxin signaling in macrophages (5)(6)(7)(8)(9). If so, then endotoxin activation should elicit ATP release from the cells.…”

Section: Endotoxin Signaling In Bac12f5 Macrophagesmentioning

confidence: 94%

“…Activation of the c-Jun N-terminal kinase cascade and production of IL-1␤ in response to endotoxin have also been observed in this particular macrophage line (33) (R. D. Beigi, S. B. Kertesy, and G. R. Dubyak, unpublished observations). Most experiments and results described below were replicated using RAW 264.7 macrophages, another murine cell line widely used for studies of endotoxin signaling (6,7,(17)(18)(19)34).…”

Section: Endotoxin Signaling In Bac12f5 Macrophagesmentioning

confidence: 99%

“…This hypothesis is based on observations that treatment of macrophages with ATP receptor antagonists, including periodate-oxidized ATP (oATP) and pyridoxal-phosphate-6-azophenyl-2Ј,4Ј-disulfonic acid (6), represses several of the short and long term macrophage responses to endotoxin, including ERK activation, NF-B translocation, and the release of TNF-␣, IL-1␤, NO, arachidonic acid, and cyclooxygenase-dependent products of arachidonic acid metabolism (5)(6)(7)(8)(9)(10)(11). In further support of this model, macrophages are known to express both G protein-coupled (P2Y) and ionotropic (P2X) nucleotide receptors, including the P2X7 pore-forming receptor (12)(13)(14).…”

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confidence: 99%

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Endotoxin Activation of Macrophages Does Not Induce ATP Release and Autocrine Stimulation of P2 Nucleotide Receptors

Beigi

Dubyak

2000

The Journal of Immunology

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Receptors for extracellular nucleotides (P2, or purinergic receptors) have previously been implicated in the transduction of endotoxin signaling in macrophages. The most compelling evidence has been the observation that inhibitors of ionotropic nucleotide (P2X) receptors, including periodate-oxidized ATP (oATP), attenuate a subset of endotoxin-induced effects such as activation of NF-κB and up-regulation of inducible NO synthase. We investigated whether endotoxin induces ATP release from a murine macrophage cell line (BAC1.2F5) using sensitive on-line assays for extracellular ATP. These cells constitutively released ATP, producing steady-state extracellular concentrations of ∼1 nM when assayed as monolayers of 106 adherent cells bathed in 1 ml of medium. However, the macrophages did not release additional ATP during either acute or prolonged endotoxin stimulation. In addition, cellular ecto-ATPase activities were measured following prolonged endotoxin activation and were found not to be significantly altered. Although oATP treatment significantly attenuated the endotoxin-induced production of NO, this inhibitory effect was not reproduced when the cells were coincubated with apyrase, a highly effective ATP scavenger. These results indicate that activation of macrophages by endotoxin does not induce autocrine stimulation of P2 nucleotide receptors by endogenous ATP released to extracellular compartments. Moreover, the data suggest that the ability of oATP to interfere with endotoxin signaling is due to its interaction with molecular species other than ATP-binding P2 receptors.

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“…Its activation is associated with apoptosis in the immune system and release of inflammatory cytokines, such as IL-1β (8,9,11). The P2X 7 receptor is distinct from the other P2X subunits in that at high μM concentrations of agonists, it forms or activates a large pore in addition to a cation channel (12,13).…”

Section: Introductionmentioning

confidence: 99%

Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

et al. 2002

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Chemically funtionalized analogues of antagonists of the P2X 7 receptor, an ATP-gated cation channel, were synthesized as tools for biophysical studies of the receptor. These functionalized congeners were intended for use in chemical conjugation with retention of biological potency. The antagonists were L-tyrosine derivatives, related to [N-benzyloxycarbonyl-O-(4-arylsulfonyl)-Ltyrosyl]benzoylpiperazine (such as MRS2409, 2). The analogues were demonstrated to be antagonists in an assay of human P2X 7 receptor function, consisting of inhibition of ATP-induced K + efflux in HEK293 cells expressing the recombinant receptor. The analogues were of the general structure R 1 -Tyr(OR 2 )-piperazinyl-R 3 , in which three positions (R 1 -R 3 ) were systematically varied in structure through introduction of chemically reactive groups. Each of the three positions was designed to incorporate a 3-or 4-nitrophenyl group. The nitro groups were reduced using NaBH 4 -copper(II) acetylacetonate to amines, which were either converted to the isothiocyanate groups, as potential affinity labels for the receptor, or acylated, as models for conjugation. An alternate route to N α -3-aminobenzyloxycarbonyl functionalization was devised.The various positions of functionalization were compared for effects on biological potency, and the R 2 and R 3 positions were found to be most amenable to derivatization with retention of high potency. Four dimeric permutations of the antagonists were synthesized by coupling each of the isothiocyanate derivatives to either the precursor amine or to other amine congeners. Only dimers linked at the R 2 -position were potent antagonists. In concentration-response studies, two derivatives, a 3-nitrobenzyloxycarbonyl derivative 18 and a 4-nitrotoluenesulfonate 26b, displayed IC 50 values of roughly 100 nM as antagonists of P2X 7 receptor-mediated K + flux.

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Purinergic Receptor Modulation of Lipopolysaccharide Signaling and Inducible Nitric-oxide Synthase Expression in RAW 264.7 Macrophages

Cited by 132 publications

References 37 publications

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Endotoxin Activation of Macrophages Does Not Induce ATP Release and Autocrine Stimulation of P2 Nucleotide Receptors

Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

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Purinergic Receptor Modulation of Lipopolysaccharide Signaling and Inducible Nitric-oxide Synthase Expression in RAW 264.7 Macrophages

Cited by 132 publications

References 37 publications

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Identification and characterization of a novel variant of the human P2X7 receptor resulting in gain of function

Endotoxin Activation of Macrophages Does Not Induce ATP Release and Autocrine Stimulation of P2 Nucleotide Receptors

Functionalized Congeners of Tyrosine-Based P2X7 Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization

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Product

Resources

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Functionalized Congeners of Tyrosine-Based P2X₇ Receptor Antagonists: Probing Multiple Sites for Linking and Dimerization