Translational control of inducible nitric oxide synthase expression by arginine can explain the arginine paradox

L-Arginine is the only endogenous nitrogen-containing substrate of NO synthase (NOS), and it thus governs the production of NO during nervous system development as well as in disease states such as stroke, multiple sclerosis, Parkinson's disease, and HIV dementia. The "arginine paradox" refers to the dependence of cellular NO production on exogenous L-arginine concentration despite the theoretical saturation of NOS enzymes with intracellular L-arginine. Herein, we report that decreased availability of L-arginine blocked induction of NO production in cytokine-stimulated astrocytes, owing to inhibition of inducible NOS (iNOS) protein expression. However, activity of the promoter of the NOS gene, induction of NOS mRNA, and stability of NOS protein were not inhibited under these conditions. Our results indicate that inhibition of NOS activity by arginine depletion in stimulated astrocyte cultures occurs via inhibition of translation of iNOS mRNA. After stimulation by cytokines, uptake of L-arginine negatively regulates the phosphorylation status of the eukaryotic initiation factor (eIF2alpha), which, in turn, regulates translation of NOS mRNA. eIF2alpha phosphorylation correlates with phosphorylation of the mammalian homolog of yeast GCN2 eIF2alpha kinase. As the kinase activity of GCN2 is activated by phosphorylation, these findings suggest that GCN2 activity represents a proximal step in the MOS translational regulation by availability of L-arginine. These results provide an explanation for the arginine paradox for NOS and define a distinct mechanism by which a substrate can regulate the activity of its associated enzyme.