Glutamine metabolism to glucosamine is necessary for glutamine inhibition of endothelial nitric oxide synthesis

L-Glutamine is a physiological inhibitor of endothelial NO synthesis. The p resent study was conducted to test the hypothesis that metabolism of glutam ine to glucosamine is necessary for glutamine inhibition of endothelial NO generation. Bovine venular endothelial cells were cultured for 24 h in the presence of 0, 0.1, 0.5 or 2 mM D-glucosamine, or of 0.2 or 2 mM L-glutamin e with or without 20 muM 6-diazo-5-oxo-L-norleucine (DON) or with 100 muM a zaserine. Both DON and azaserine are inhibitors of L-glutamine:D-fructose-6 -phosphate transaminase (isomerizing) (EC 2.6.1.16), the first and rate con trolling enzyme in glucosamine synthesis. Glucosamine at 0.1, 0.5 and 2 mM decreased NO production by 34, 45 and 56% respectively compared with contro ls where glucosamine was lacking. DON (20 muM) and azaserine (100 muM) bloc ked glucosamine synthesis and prevented the inhibition of NO generation by glutamine. Neither glutamine nor glucosamine had an effect on NO synthase ( NOS) activity. arginine transport or cellular tetrahydrobiopterin and Ca2levels. However, both glutamine and glucosamine inhibited pentose cycle act ivity and decreased cellular NADPH concentrations; these effects of glutami ne were abolished by DON or azaserine. Restoration of cellular NADPH levels by the addition of 1 mM citrate also prevented the inhibiting effect of gl utamine or glucosamine on NO synthesis. A further increase in cellular NADP H levels by the addition of 5 mM citrate resulted in greater production of NO. Collectively, our results demonstrate that the metabolism of glutamine to glucosamine is necessary for the inhibition of endothelial NO generation by glutamine. Glucosamine reduces the cellular availability of NADPH (an e ssential cofactor for NOS) by inhibiting pentose cycle activity, and this m ay be a metabolic basis for the inhibition of endothelial NO synthesis by g lucosamine.