CHLOROMETHYL KETONES BLOCK INDUCTION OF NITRIC-OXIDE SYNTHASE IN MURINE MACROPHAGES BY PREVENTING ACTIVATION OF NUCLEAR FACTOR-KAPPA-B

N-alpha-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and N-alpha-t osyl-L-lysine chloromethyl ketone (TLCK), serine protease inhibitors, block many cytotoxic functions of immune cells including superoxide an ion production, cytokine release, cell-mediated cytolysis, and nitric oxide (NO)-related macrophage functions. IFN-gamma/LPS-induced NO prod uction from murine peritoneal macrophages was inhibited by TPCK and TL CK in a dose-dependent manner (EC(50)s: similar to 20 mu M for TPCK an d similar to 30 mu M for TLCK). Viability exceeded 91% with 25 mu M TP CK and with 80 mu M TLCK. When TPCK treatment was delayed until 1 h of activation, the inhibitory effect was markedly reduced. After 2 h of the activation, TPCK was not effective anymore. Addition of either TNF -alpha or conditioned media from IFN-gamma/LPS-activated macrophage cu lture did not prevent the inhibitory effect of TPCK. Neither TPCK nor TLCK reduced enzymatic NO production from macrophage lysates. Lysates from TPCK-treated cells did not generate NO even after supplementing n ecessary cofactors for NO synthase. Immunoblotting analysis showed tha t simultaneous treatment of TPCK with IFN-gamma/LPS abolished the NO s ynthase expression, whereas delayed addition of TPCK was either partia lly effective or not effective at all. Furthermore, TPCK treatment red uced the concentration of mRNA for NO synthase without decreasing mRNA stability. Thus, the serine protease inhibitors directly blocked an e arly event in expression of NO synthase. Electrophoretic mobility shif t assay indicated that TPCK blocked the activation of nuclear factor-k appa B, a transcription factor necessary for NO synthase induction. TP CK also blocked disappearance of I kappa B from cytosol fraction, and nuclear translocation of NF-kappa B subunits p50 and p65. Delaying the addition of TPCK by 10 min partially prevented the inhibition of the NF-kappa B activation process and allowed partial resuming of NO produ ction. Thus, TPCK inhibited NO synthase induction by blocking NF-kappa B activation.