SERINE AND CYSTEINE PROTEINASE-INHIBITORS PREVENT NITRIC-OXIDE PRODUCTION BY ACTIVATED MACROPHAGES BY INTERFERING WITH TRANSCRIPTION OF THEINDUCIBLE NO SYNTHASE GENE
Jm. Griscavage et al., SERINE AND CYSTEINE PROTEINASE-INHIBITORS PREVENT NITRIC-OXIDE PRODUCTION BY ACTIVATED MACROPHAGES BY INTERFERING WITH TRANSCRIPTION OF THEINDUCIBLE NO SYNTHASE GENE, Biochemical and biophysical research communications, 215(2), 1995, pp. 721-729
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.