SERINE AND CYSTEINE PROTEINASE-INHIBITORS PREVENT NITRIC-OXIDE PRODUCTION BY ACTIVATED MACROPHAGES BY INTERFERING WITH TRANSCRIPTION OF THE INDUCIBLE NO SYNTHASE GENE

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 mu M. TPCK and CPI-1 were about 10-fold more potent than TLCK and CPI-2, respectively. These data suggested 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 mu M 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. (C) 1995 Academic Press, Inc.