FIG. 4.

Impact of NOS-2 inhibition on PGHS activity. (A) Resting rat alveolar macrophages were incubated with LPS (10 μg/ml) for 8 h to allow PGHS-2 induction without interference by pharmacological compounds, new medium containing LPS and the NOS-2 selective inhibitor AMT was then added for additional 8 h. The concentration-dependent decline in NO₂⁻ levels by AMT inversely correlated with a rise in PGHS activity. (B) In parallel to the direct assessment of PGHS in cell homogenates, formation of TxA₂ and PGE₂ by intact cells was detected by an enzyme immunoassay. (C) For an evaluation of PGHS-2 nitration, the enzyme was immunoprecipitated from cell homogenates and then stained with an anti-3-NT antibody. (D) In the same experimental setup, the SOD-mimetic CuDips was used instead of AMT, immunoprecipitated PGHS-2 was subjected to the Western blot analysis and stained with an anti-3-NT antibody. (E) The PGHS activity was determined in CuDips-treated cells by lysis, addition of ¹⁴C-labeled substrate AA (¹⁴C-AA), and separation of labeled prostanoids by thin layer chromatography. Values represent means±SD of three independent macrophage isolations. *p<0.05 versus t=0.