Abstract
Antigenic cross-linking of the high affinity IgE receptors on mast cells induced the synthesis of prostaglandin D(2) (PGD(2)). The production of PGD(2) in L9 cells, which overexpressed non-mitochondrial phospholipid glutathione peroxidase (PHGPx), was only one-third that in the control line of cells (S1 cells). The reduction in the formation of PGD(2) in L9 cells was reversed upon inhibition of PHGPx activity by buthionine sulfoximine. Experiments with inhibitors demonstrated that prostaglandin H synthase-2 (PGHS-2) was the isozyme responsible for the production of PGD(2) upon cross-linking of IgE receptors. The conversion of radiolabeled arachidonic acid to prostaglandin H(2) (PGH(2)) was strongly inhibited in L9 cells, whereas the rate of conversion of PGH(2) to PGD(2) was the same in L9 cells and S1 cells, indicating that PGHS was inactivated in L9 cells. The PGHS activity in L9 cells was about half that in S1 cells. However, PGHS activity in L9 cells increased to the level in S1 cells upon the addition of the hydroperoxide 15-hydroperoxyeicosatetraenoic acid or of 3-chloroperoxybenzoic acid. These results suggest that non-mitochondrial PHGPx might be involved in the inactivation of PGHS-2 in nucleus and endoplasmic reticulum via reductions in levels of the hydroperoxides that are required for full activation of PGHS. Therefore, it appears that PHGPx might function as a modulator of the production of prostanoids, in addition to its role as an antioxidant enzyme.
Highlights
The Reactive oxygen species (ROS)-mediated damage to intracellular molecules is limited by cellular antioxidant enzymes, such as phospholipid hydroperoxide glutathione peroxidase (PHGPx), classical glutathione peroxidase, superoxide dismutase, and catalase [5]
Production of Prostaglandin D2 in phospholipid glutathione peroxidase (PHGPx)-overexpressing RBL-2H3 Cells—The production of prostaglandins was examined in mock-transfected cells (S1 cells) and in cells that overexpressed non-mitochondrial PHGPx (L9 cells) by autoradiography after prior labeling with radioactive arachidonic acid (Fig. 1A)
We examined the effect of overexpression of non-mitochondrial PHGPx in RBL-2H3 cells, which generated prostaglandin D2 (PGD2) predominantly, together with small amounts of other prostanoid products such as PGE2, 6-keto-PGF1␣, and thromboxane B2 in response to immunoglobulin E (IgE)-DNP or A23187 [30]
Summary
Reagents—Antibodies, raised in rabbit, against PGHS-1 from rat were a kind gift from Dr M. After removal of the medium, cells were washed twice with PBS and incubated in PBS that contained 1 mM CaCl2, 0.5 mM MgSO4, and 20 M [1-14C]arachidonic acid (3.7 kBq; 170.7 MBq/mmol) for 30 min. Arachidonic acid for 24 h and activated by cross-linking their IgE receptors, by sequential treatment with IgE-DNP and DNP-hSA. Cells were washed, and receptor cross-linking was effected by incubation with 10 g/ml DNP-hSA for 1 h at 37 °C in culture medium. Assay of PGHS Activity—RBL-2H3 cells (5 ϫ 106 cells) were activated by cross-linking their IgE receptors by sequential treatment with IgE-DNP and DNP-hSA. After sequential treatment with IgE-DNP and DNP-hSA, cells were washed with PBS and incubated with 5 M DCFH-DA in PBS for 15 min. Quantitation of Proteins—Concentrations of proteins were determined with Protein Assay Reagent (Bio-Rad) with bovine serum albumin as the standard
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