Inhibition of nitric-oxide synthase-I (NOS-I)-dependent nitric oxide production by lipopolysaccharide plus interferon-gamma is mediated by arachidonic acid. Effects on NF-κB activation and late inducible NOS expression

Previous results have indicated that lipopolysaccharide (LPS) plus interferon-_ (IFN_) inhibits nitric-oxide synthase (NOS)-I activity in glial cells. We report here that arachidonic acid (AA) plays a pivotal role in this response, which was consistently reproduced in different glial cell lines and in primary rat astrocytes. This notion was established using pharmacological inhibitors of phospholipase A2 (PLA2), cytosolic PLA2 (cPLA2) antisense oligonucleotides, and AA add-back experiments. This approach not only allowed the demonstration that AA promotes inhibition of NOS-I activity but also produced novel experimental evidence that LPS/ IFN_ itself is a potential stimulus for NOS-I. Indeed, LPS/IFN_ fails to generate nitric oxide (NO) via NOS-I activation simply because it activates the AA-dependent signal that impedes NOS-I activity. Otherwise, LPS/ IFN_ promotes NO formation, sensitive to exogenous AA, in cells in which cPLA2 is pharmacologically inhibited or genetically depleted. Because NO suppresses the NF_B-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFN_-induced AA pathway provides optimal conditions for NF_B activation and subsequent NOS-II expression. Inhibition of cPLA2 activity, while reducing the availability of AA, consistently inhibited NF_B activation and NOS-II mRNA induction and delayed NO formation. These responses were promptly reestablished by addition of exogenous AA. Finally, we have demonstrated that the LPS/IFN_-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.