DISCRIMINATION BETWEEN CITRULLINE AND ARGININE TRANSPORT IN ACTIVATED MURINE MACROPHAGES - INEFFICIENT SYNTHESIS OF NO FROM RECYCLING OF CITRULLINE TO ARGININE

1 The kinetics, specificity, pH- and Na+-dependency of L-citrulline transport were examined in unstimulated and lipopolysacchharide (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 mu g ml(-1)) for 24 h. 2 In unstimulated J774 cells, transport of citrulline was saturable (K-t = 0.16 mM and V-max = 32 pmol mu g(-1) protein min(-1)), pH-insensitive and partially Na+-dependent. In contrast to arginine, transport of citrulline was unchanged in LPS-activated (1 mu g 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 (K-i = 3.4 mM) of arginine transport but only in the presence of extracellular Na+. Neutral amino acids inhibited citrulline transport (K-i = 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 mu M N-G-nitro-L-arginine methyl ester or N-G-iminoethyl-L-ornithine. 5 Intracellular metabolism of L-[C-14]-citrulline to L-[C-14]-arginine was detected in unstimulated J774 cells and was increased further in cells activated with LPS and interferon-gamma. 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.