Modulation of Inducible Nitric Oxide Synthase Expression by an Uremic Catabolyte, Methylguanidine, in Lps-stimulated J774 Macrophages

1. We have investigated whether methylguanidine (MG), an uremic toxin, modulates the expression of the inducible nitric oxide synthase (iNOS) and nitric oxide (NO) release in vitro in LPS-induced J774 macrophages. 2. Addition of MG (0.01-1mM) or L-NAME (0.01-1mM), 30 min before LPS challenge, inhibited significantly (37.77 % inhibition, P<0.001; n=12) but only at the highest concentration tested LPS-induced nitrite production. 3.When J774 macrophages were pretreated with MG or L-NAME (0.01-1mM) 18h prior stimulation with LPS, MG significantly (P<0.001) inhibited, in a concentration related manner, LPS-stimulated nitrite production reducing nitrite levels by 40.26 % (n=12), 31.85 % (n=9) and 26.68 % (n=6) for MG 1, 0.1 and 0.01 mM respectively. 4. MG, added to the culture medium of J774 macrophages 24 h after LPS challenge, also reduced significantly and in a concentration-related manner nitrite production inhibiting NO release by 50.0 % (P<0.001, n=32), 26.0 % (P<0.05, n=23) and 3.18 % for MG 1, 0.1 and 0.01 mM respectively. In this condition when the culture medium was supplemented with Larginine (10mM; L-ARG) the inhibitory effect of MG (1mM) on nitrite production was reversed to 10.86 % significantly different (P<0.05) from MG-inhibitory effect observed in normal medium. 5. Preincubation of cells with MG (1mM; 24 h) 30 min and 18 h prior to activation with LPS resulted in inhibited iNOS protein expression by 44.16±5.7 % (P<0.01; n=3) and 30.47±3.9 % (P<0.01; n=3). In the same conditions L-NAME (1mM) did not modify significantly iNOS expression. 7. MG (1mM) or L-NAME (1mM) added to the culture medium 24 h after LPS challenge did not modify iNOS protein expression. 8. Our results show that MG inhibited LPS-induced nitrite production in the murine macrophages cell line J774. This inhibitory effect could be partly due to the antagonistic