Discrimination between citrulline and arginine transport in activated murine macrophages: inefficient synthesis of NO from recycling of citrulline to arginine

Open Access
Publisher Website
Google Scholar
Abstract
1 The kinetics, specificity, pH- and Na+-dependency of l-citrulline transport were examined in unstimulated and lipopolysaccharide (LPS)-activated murine macrophage J774 cells. The dependency of nitric oxide production on extracellular arginine or citrulline was investigated in cells activated with LPS (1 μg ml−1) for 24 h. 2 In unstimulated J774 cells, transport of citrulline was saturable (Kt = 0.16 mm and Vmax = 32 pmol μg−1 protein min−1), pH-insensitive and partially Na+-dependent. In contrast to arginine, transport of citrulline was unchanged in LPS-activated (1 μg ml−1, 24 h) cells. 3 Kinetic inhibition experiments revealed that arginine was a relatively poor inhibitor of citrulline transport, whilst citrulline was a more potent inhibitor (Ki = 3.4 mm) of arginine transport but only in the presence of extracellular Na+. Neutral amino acids inhibited citrulline transport (Ki = 0.2–0.3 mm), but were poor inhibitors of arginine transport. 4 Activated J774 cells did not release nitrite in the absence of exogenous arginine. Addition of citrulline (0.01 − 10 mm), in the absence of exogenous arginine, could only partially restore the ability of cells to synthesize nitrite, which was abolished by 100 μm NG-nitro-l-arginine methyl ester or NG-iminoethyl-l-ornithine. 5 Intracellular metabolism of l-[14C]-citrulline to l-[14C]-arginine was detected in unstimulated J774 cells and was increased further in cells activated with LPS and interferon-γ. 6 We conclude that J774 macrophage cells transport citrulline via a saturable but nonselective neutral carrier which is insensitive to induction by LPS. In contrast, transport of arginine via the cationic amino acid system y+ is induced in J774 cells activated with LPS. 7 Our findings also confirm that citrulline can be recycled to arginine in activated J774 macrophage cells. Although this pathway provides a mechanism for enhanced arginine generation required for NO production under conditions of limited arginine availability, it cannot sustain maximal rates of NO synthesis.