Inability of rat alveolar macrophages to recycle L-citrulline to L-arginine despite induction of argininosuccinate synthetase mRNA and protein, and inhibition of nitric oxide synthesis by exogenous L-citrulline

In the present study it was tested whether rat alveolar macrophages (AM Phi) convert L-citrulline to L-arginine to maintain nitric oxide (NO) synthesis under conditions of limited availability of L-arginine. Rat AM Phi (0.5 x 10(6) cells/well, cultured for 20 h in the absence or presence of 1 mu g/ml lipopolysaccharides, LPS), were incubated for 6 h in amino acid-free Krebs solution and nitrite accumulation was determined as a measure of NO synthesis. After culture in the absence of LPS, nitrite in the incubation media was at the detection limit, independent of the addition of L-arginine or L-citrulline. AM Phi, cultured in the presence of LPS, produced about 4 nmol per 106 cells and 6 h nitrite, and L-arginine enhanced nitrite accumulation in a concentration-dependent manner, maximally about threefold (EC50: 55 mu M). In LPS-treated AM Phi L-citrulline (up to 10 mM) failed to enhance nitrite accumulation, but rather inhibited it by about 50% in the presence of 100 mu M L-arginine, i.e. when NO synthesis was enhanced. L-Arginine in the culture medium was H-3-labelled and its metabolism analysed by HPLC. In medium of AM Phi exposed to LPS [H-3]-L-arginine was reduced by about 60% after a 20-h culture period and this was almost balanced by an almost equal increase in [H-3]-L-citrulline and [H-3]-L-ornithine, i.e. L-arginine was markedly consumed. When [C-14]-L-citrulline was added to the culture medium of AM Phi, no significant formation of [C-14]-L-arginine could be detected. On the other hand, argininosuccinate synthetase mRNA (by RT-PCR) and protein (by Western blot) was marginally detectable in control AM Phi, but clearly induced after exposure to LPS. Finally, L-citrulline was shown to inhibit L-arginine uptake in a concentration dependent manner, by about 50% at 10 mM. In conclusion, although the expression of argininosuccinate synthetase in rat AM Phi can be induced by LPS, AM Phi appear not to be able to recycle significant amounts of L-citrulline to L-arginine to maintain sustained NO synthesis. On the contrary, at high concentrations L-citrulline can reduce NO synthesis, and this effect appears to be caused by inhibitory effects on L-arginine uptake.