Glutamine is essential for nitric oxide synthesis by murine macrophages

Background. Recent studies suggest an interaction between L-arginine (Arg) and L-glutamine (Gln) in the control of nitric oxide (NO) synthesis. Endoto xemia enhances Gln demand and NO production. This study was initiated to in vestigate the effects of altered Gln availability on the capacity of macrop hages to produce NO and the interaction of Gln with L-citrulline (Cit) and Arg in the regulation of endotoxin-stimulated NO synthesis. Methods. Cultures of RAW 264.7 macrophages in MEM containing Gln (0 to 100 mM) or Arg (0 or 0.6 mM) and supplemented or not with Cit (0.31 to 10 mM) w ere exposed to Escherichia coli lipopolysaccharide (LPS) at 0.001 and 1 mu g/ml. After 24-h incubation, supernatants were evaluated for nitrite concen trations by Greiss reaction as a measure of NO synthesis. Results. LPS stimulated nitrite synthesis in a dose-dependent fashion. Macr ophages cultured in Gln-free medium containing Arg (0.6 mM) did not produce NO when stimulated with LPS. In contrast, in the presence of Arg and 0.001 mu g/ml LPS, adding as little as 0.31 mM Gln resulted in a 23-fold increas e in NO production (from 0.13 +/- 0.02 to 2.92 +/- 0.06 nmol/ml) (P < 0.000 1). Furthermore, a dose-dependent increase in LPS-stimulated nitrite releas e was observed with increasing amounts of Gln to as much as 1 mM. LPS-stimu lated macrophages cultured in Arg-free medium containing Gln (0.31-10 mM) d id not produce significant amounts of nitrite. However, in the absence of A rg, increasing extracellular Gln levels to 100 mM in the culture medium res ulted in nitrite synthesis (2.39 +/- 0.11 nmol/ml). Detectable levels of ni trite (2.84 +/- 0.21 nmol/ml) were also documented when stimulated macropha ges were incubated in culture medium lacking Arg but containing Cit (0.31 m M) and Gln (2 mM). Increasing Cit levels (0.63 to 10 mM) significantly augm ented nitrite release (P < 0.05). Once again, no detectable levels of nitri te were observed when macrophages were cultured in Gln-free medium, even wh en Arg and Cit were present. Conclusion. These results suggest that Gln is an essential amino acid far N O synthesis by macrophages and raise the strong possibility that Gln acts w ith nitric oxide synthase to catalyze the conversion of Arg to NO. The cons umption of Gln during sepsis may represent NO production. (C) 1999 Academic Press.