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Abstract
We previously demonstrated that lipopolysaccharide (LPS) increases expression of the prostaglandin H synthase-2 (PGHS-2) gene (Hempel, S.L., Monick, M.M., and Hunninghake, G.W. (1994) J. Clin. Invest. 93, 391-396). In this study, the expression of the PGHS-2 gene in response to changes in cell oxidant tone was studied. During LPS exposure, inhibition of synthesis of the free radical, NO., resulted in a small decrease in prostaglandin E2 synthesis that did not reach statistical significance. There was no effect on enzyme mass or mRNA. In contrast, incubation of alveolar macrophages in the presence of LPS plus the antioxidant pyrrolidine dithiocarbamate, the spin trap 5,5-dimethyl-1-pyrroline-N-oxide, or hypoxia, resulted in near complete inhibition of prostaglandin E2 synthesis, PGHS-2 enzyme synthesis, and gene transcription of PGHS-2 mRNA. There was no evidence of cytotoxicity. These results demonstrate that synthesis of PGHS-2 in response to LPS is inhibited by agents that decrease cell oxidant tone.
Highlights
From the Department of Medicine and Departmentof Veterans Affairs Medical Center, The University of Zowa, Iowa City, Iowa 52242
ClinI.nvest. 93,391496).In While this suggests that oxidant tone may regulatPeGHS-2 this study,theexpression of the PGHS-2gene in re- enzyme activity,it is likelythat the PGHS-2 gene is regusponse to changes in cell oxidant tone was studied
N F K B is inhibited by reducing Tbtoernrahamcheseryta,epotifeown,xLactiPsuhaSb,enaosprtpelieuosifnusnfletttocrehftadepaolaniv5nn,eet5onin-loazdexyraiimdmrmeaetacnhocmtyrmlpoa-ppyslslrhe-raotpoegrym lreidisrRnioinhNnleiiAbtn.hdieotIeii-fntoNhppn-cirroooeoxcnssai---dre,ctcsaoihtifineacsck(t2eaon5nr)sPNaG(nF2HdK1S,rBe-2s2ci2etg,enet(2n44se0t))uc..odIInittetiasshiniannossdt bibckoeantethonewatndnhe,AemhPhoou-1nwmssetairvtnaeetPrie,faGdncHhdtaShan-a2NgteFcstoKhinneBtaglandin E, synthesis, PGHS-2 enzyme synthesis, and oxidant tone can regulate expression of the human PGHS-2 gene transcriptionof PGHS-2 mRNA There was noevi- gene andlor if this change is mediated sbpyecific oxidants, such dence of cytotoxicity
Summary
Macrophages were cultured 24 h in the prrsence o f 10 pg o f Ll'S'ml andthe NO. I'GE, synthesis was measured by RIA ofthe culture medium. Values on they axis represent total synthesis over 24 h. Viahility o f thc cultured cells was dctrrmined using trypan blur exclusion antl lactate dehydrogenasr release. (,'uIturr during Ifypn.uin-Frrshlyisolatrdmacrophagrson6-well plates wrre placed in a sr:tled acrylic continuous-flow chamher withina dry incubator at 37 "C. The paired controls consisted o f cells from t h e samr preparation placed in a room air incubator with a gas mixturrof 95'7 room air, 5"; CO,. Irnmrrnohlot Drtrrtion n f PGHS-2-Cell pellets from nlvrolar macrophageswere lysed in 150 pl o f solubilization hufTer a t 2 5 ' C tl"r. An aliquot of the supernatant wasused to drterminr protein hy the Coomassir Blur " " "
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