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 (AMΦ) convert l-citrulline to l-arginine to maintain nitric oxide (NO) synthesis under conditions of limited availability of l-arginine. Rat AMΦ (0.5×10⁶ cells/well, cultured for 20 h in the absence or presence of 1 μg/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. AMΦ, 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 μM). In LPS-treated AMΦ l-citrulline (up to 10 mM) failed to enhance nitrite accumulation, but rather inhibited it by about 50% in the presence of 100 μM l-arginine, i.e. when NO synthesis was enhanced. l-Arginine in the culture medium was ³H-labelled and its metabolism analysed by HPLC. In medium of AMΦ exposed to LPS [³H]-l-arginine was reduced by about 60% after a 20-h culture period and this was almost balanced by an almost equal increase in [³H]-l-citrulline and [³H]-l-ornithine, i.e. l-arginine was markedly consumed. When [¹⁴C]-l-citrulline was added to the culture medium of AMΦ, no significant formation of [¹⁴C]-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 AMΦ, 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 AMΦ can be induced by LPS, AMΦ 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. Received: 21 April 1998 / Accepted: 2 October 1998