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 Kappa B Activation and Late Inducible NOS Expression

Previous results have indicated that lipopolysaccharide (LPS) plus interferon-gamma (IFNgamma) 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 A sub(2) (PLA sub(2)), cytosolic PLA sub(2) (cPLA sub(2)) 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/IFNgamma itself is a potential stimulus for NOS-I. Indeed, LPS/IFNgamma 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/IFNgamma promotes NO formation, sensitive to exogenous AA, in cells in which cPLA sub(2) is pharmacologically inhibited or genetically depleted. Because NO suppresses the NF Kappa B-dependent NOS-II expression, inactivation of NOS-I by the LPS/IFNgamma-induced AA pathway provides optimal conditions for NF Kappa B activation and subsequent NOS-II expression. Inhibition of cPLA sub(2) activity, while reducing the availability of AA, consistently inhibited NF Kappa 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/IFNgamma-dependent tyrosine phosphorylation of NOS-I and inhibition of its activity are mediated by endogenous AA.