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

The objective of this study was to ascertain the mechanism by which se rine and cysteine proteinase inhibitors interfere with production of N O by LPS-activated rat alveolar macrophages. Macrophages were incubate d in the presence of LPS + test agent for 24 hr. Culture media were an alyzed for NOX- accumulation, harvested cells were assayed for iNOS ac tivity, 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 induci ble 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, re spectively. These data suggested that a chymotrypsin-like serine or cy steine 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 acti vation whose action is attributed to inhibition of the chymotrypsin-li ke activity of the multicatalytic proteinase complex (MPC) was tested. Z-IE(O-t-Bu)A-Leucinal abolished NOX- production and inducible iNOS e xpression at 1 mu M and showed over 50% inhibition at 10 nM. These obs ervations indicate that inhibitors of MPC interfere with iNOS inductio n and provide strong evidence that MPC functions importantly in iNOS i nduction in macrophages. (C) 1995 Academic Press, Inc.