title: Serine and cysteine proteinase inhibitors prevent nitric oxide production by activated macrophages by interfering with transcription of the inducible NO synthase gene.
abstract: The objective of this study was to ascertain the mechanism by which serine and cysteine proteinase inhibitors interfere with production of NO by LPS-activated rat alveolar macrophages. Macrophages were incubated in the presence of LPS+ test agent for 24 hr. Culture media were analyzed for NOX- accumulation, harvested cells were assayed for iNOS activity, and cellular RNA was extracted for determination of iNOS mRNA by Northern blot analysis. TPCK, TLCK, calpain inhibitor 1 (CPI-1) and calpain inhibitor 2 (CPI-2) each inhibited NOX- production and inducible iNOS expression in a concentration-dependent manner at 1-100 microM. TPCK and CPI-1 were about 10-fold more potent than TLCK and CPI-2, respectively. These data suggest that a chymotrypsin-like serine or cysteine proteinase is required for the LPS-inducible expression of the iNOS gene, perhaps by mechanisms involving activation of transcription factor NF-kappa B. Accordingly, a potent inhibitor of NF-kappa B activation whose action is attributed to inhibition of the chymotrypsin-like activity of the multicatalytic proteinase complex (MPC) was tested. Z-IE(O-t-Bu)A-Leucinal abolished NOX- production and inducible iNOS expression at 1 microM and showed over 50% inhibition at 10 nM. These observations indicate that inhibitors of MPC interfere with iNOS induction and provide strong evidence that MPC functions importantly in iNOS induction in macrophages.
mesh_terms: D000818:Animals; D015152:Blotting, Northern; D002460:Cell Line; D015853:Cysteine Proteinase Inhibitors / Q000494:pharmacology*; D004790:Enzyme Induction; D015870:Gene Expression / Q000187:drug effects; D006023:Glycoproteins / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D008262:Macrophage Activation; D016676:Macrophages, Alveolar / Q000187:drug effects / Q000201:enzymology*; D009569:Nitric Oxide / Q000037:antagonists & inhibitors / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis*; D051381:Rats; D015842:Serine Proteinase Inhibitors / Q000494:pharmacology*; D014107:Tosyllysine Chloromethyl Ketone / Q000494:pharmacology; D014108:Tosylphenylalanyl Chloromethyl Ketone / Q000494:pharmacology; D014158:Transcription, Genetic / Q000187:drug effects*
chemical_list: D015853:Cysteine Proteinase Inhibitors; D006023:Glycoproteins; D008070:Lipopolysaccharides; D015842:Serine Proteinase Inhibitors; C039304:calpain inhibitors; D014107:Tosyllysine Chloromethyl Ketone; D009569:Nitric Oxide; D014108:Tosylphenylalanyl Chloromethyl Ketone; D019001:Nitric Oxide Synthase