Ar. Baydoun et Dml. Morgan, Inhibition of ornithine decarboxylase potentiates nitric oxide production in LPS-activated J774 cells, BR J PHARM, 125(7), 1998, pp. 1511-1516
I We have examined whether modulation of the polyamine biosynthetic pathway
, through inhibition by alpha-difluoromethylornithine (DFMO) of the rate li
miting enzyme, ornithine decarboxylase (ODC), modulates NO synthesis in J77
4 macrophages.
2 DFMO potentiated LPS-stimulated nitrite production in both a concentratio
n- and time-dependent manner, increasing nitrite levels by 48+/-5% at 10 mM
. This effect was observed in cells pre-treated with DFMO for 24 h prior to
stimulation with LPS. Addition of DFMO 12 h after LPS failed to potentiate
LPS-induced nitrite production.
3 Supplementation of the culture medium with horse serum (10%) in place of
foetal calf serum (10%) caused no significant change in either LPS-induced
nitrite production or in the ability of DFMO (10 mM) to potentiate LPS-indu
ced NO synthesis.
4 Metabolism of L-[H-3]arginine to L-[H-3]citrulline by partially purified
inducible nitric oxide synthase (iNOS) was not significantly altered by eit
her DFMO (1-10 mM) or by putrescine (0.001-1 mM), spermidine (0.001-1 mM) o
r spermine (0.001-1 mM). iNOS activity was also unaffected by 1 mM EGTA but
was markedly attenuated (70+/-0.07%) by L-NMMA (100 mu M).
5 Pre-incubation of cells with DFMO (10 mM; 24 h) prior to activation with
LPS resulted in enhanced (similar to 2 fold) iNOS protein expression.
6 These results show that DFMO potentiates LPS-induced nitrite production i
n the murine macrophage cell line J774. Since the only known mechanism of a
ction of DFMO is inhibition of ODC, and thus polyamine biosynthesis, we con
clude that expression of iNOS can be critically regulated by endogenous pol
yamines.