Proinflammatory Cytokines and Lipopolysaccharide Synergize to Induce L-arginine Transport and Nitric Oxide Synthase in Vascular Smooth Muscle Cells

1. The interactions between proinflammatory cytokines and bacterial lipopolysaccharide (LPS) on L-arginine transporter and inducible nitric oxide synthase (iNOS) activities were examined in rat cultured aortic smooth muscle cells. 2. LPS induced a concentration (0.01-100 μg ml-1) and time-(8-24 h) dependent stimulation of nitrite production which was accompanied by a parallel increase in L-arginine transport. 3. Unlike LPS, activation of smooth muscle cells with either interferon-γ (IFN-γ, 100 U ml-1), tumour necrosis factor-α (TNF-α, 300 U ml-1) or interleukin-1α (IL-1α, 100 U ml-1) failed to stimulate L-arginine transport or increase nitrite accumulation. 4. When applied in combination with LPS (100 μg ml-1) both IFN-γ and TNF-α, but not IL-1α, enhanced the effects observed with LPS alone. Furthermore, activation of cells with LPS and IFN-γ had no effect on uptake of the neutral amino acid L-citrulline but selectively increased the V max for L-arginine transport 2.8-fold and nitrite levels from 24 ± 7 to 188 ± 14 pmol μg protein-1 24 h-1. 5. The substrate specificity, Na + and pH independence of saturable L-arginine transport in both unactivated (K m = 44 μM, V max = 3 pmol μg protein-1 min-1) and activated (K m = 75 μM, V max = 8.3 pmol μg protein-1 min-1) smooth muscle cells were characteristic of the cationic amino acid transport system y +. 6. Cycloheximide (1 μM) abolished induction of L-arginine transport and nitrite accumulation in response to LPS and IFN-γ. In contrast, the glucocorticoid dexamethasone (10 μM, 24 h) selectively blocked nitrite production. 7. Our results demonstrate that pro-inflammatory mediators selectively enhance transport of L-arginine under conditions of sustained NO synthesis by vascular smooth muscle cells. In addition, the differential inhibition of iNOS and L-arginine 3 transporter activity by dexamethasone suggests that distinct signalling pathways mediate induction of the cationic transport protein and iNOS. The close coupling between substrate supply and NO production may have important implications in the pathogenesis of several disease states including endotoxin shock.