DIFFERENTIAL REGULATION OF L-ARGININE TRANSPORT AND INDUCIBLE NOS IN CULTURED VASCULAR SMOOTH-MUSCLE CELLS

Experiments were performed to characterize the uptake of L-arginine in rat aortic smooth muscle cells (SMC) and to examine whether inducers of nitric oxide synthase (NOS) could regulate the transport of L-argin ine into these cells. L-Arginine transport by SMC was saturable, Na+ i ndependent, strongly inhibited in the presence of other cationic amino acids, and could be stimulated by preloading the cells with cationic amino acids. Kinetic studies revealed the presence of both a high [Mic haelis constant (K-m) similar to 125 mu M] and low (K-m similar to 1.4 mM) affinity L-arginine transporter. Treatment of vascular SMC with i nterleukin-1 beta (IL-1 beta) and tumor necrosis factor-alpha (TNF-alp ha) resulted in parallel increases in L-arginine transport and nitric oxide (NO) synthesis, as measured by nitrite production. Increasing th e concentration of IL-1 beta and TNF-alpha caused a progressive elevat ion in nitrite production but did not further stimulate L-arginine upt ake. Treatment of SMC with the combination of TNF-alpha and interferon -gamma (IFN-gamma) synergistically enhanced nitrite release without ha ving any additional effect on L-arginine transport. Both induction of L-arginine transport and NOS activity by these cytokines were blocked by cycloheximide. Finally, treatment of SMC with interferon-gamma and N-6,2'-O-dibutyryl adenosine 3',5'-cyclic monophosphate selectively st imulated the formation of nitrite by SMC but had no effect on L-argini ne transport. These results demonstrate that L-arginine transport by c ultured vascular SMC is mediated by the system y(+) carrier and that i nducers of NOS differentially regulate the activity of this transporte r. The limited capacity of inducers of NOS to stimulate L-arginine tra nsport by SMC may provide an important regulatory mechanism in control ling the release of NO at sites of vascular injury.