FIG. 1.

Nitration of PGHS-2 and its inhibition in an in vivo lung inflammation model. (A) Rats were treated with LPS (200 μg in 200 μl PBS, intranasally) for 12, 24, or for 24 together with the NOS-2 inhibitor AMT (1 mM in 200 μl) or the SOD-mimetic CuDips (1 mM in 200 μl). Following lavage from isolated lungs, alveolar macrophages were separated from other cell types and protein expression was analyzed by Western blots, stained for PGHS-2 or NOS-2. Additionally, PGHS-2 was separated from the crude protein extracts by immunoprecipitation and then analyzed by Western blot using and antibody against 3-NT. (B) The PGHS activity was detected in cell lysates of the LPS-challenged, freshly isolated, macrophages after addition of the substrate AA (¹⁴C-AA) for 20 min. (C) Cell number of macrophages and neutrophils in crude lavages of control and LPS-challenged rats were detected by the differential cell count (•=one animal). For the detection of HO-1 mRNA (D) and protein levels (E), as well as for the analysis of total HO activity and intracellular levels of heme (F), rats were challenged for 0, 12, or 24 with LPS. Values represent means±SD of three independent isolations. *p<0.05 versus t=0. 3-NT, 3-nitrotyrosine; AA, arachidonic acid; AMT, 5,6-dihydro-6-methyl-4H-1,3-thiazin-2-amine; CuDips, Cu(II)2(3,5-diisopropylsalicylate); HO-1, heme oxygenase-1; LPS, lipopolysaccharide; NOS-2, nitric oxide synthase-2; PBS, phosphate-buffered saline; PGHS-2, prostaglandin endoperoxide H₂ synthase-2; SD, standard deviation; SOD, superoxide dismutase.