Objective The authors determined the endothelial arginine transport mechanism and the potential role of a tumor necrosis factor (TNF)-alpha-mediated signal transduction pathway involving protein kinase C (PKC) in regulating this transport in cultured endothelial cells. Summary Background Data The vascular endothelium metabolizes arginine to generate nitric oxide (NO), and an increase in NO production can be stimulated by several cytokines. The mechanism(s) responsible for the accelerated arginine transport are poorly understood. Methods Arginine transport was assayed in confluent human umbilical vein endothelial cells in the presence of TNF +/- the PKC inhibitor chelerythrine chloride. Results Carrier-mediated arginine transport was accomplished by two Na+-independent transporters, System y(+) (80% of total transport) and System b(0,+) (20% of transport). Tumor necrosis factor (0.1-2 ng/mL) increased System y(+)-mediated arginine transport in a time- and dose-dependent manner by augmenting System y(+) transport maximal capacity (control V-max,, = 1325 +/- 60 pmol/mg protein/minute vs. TNF V-max = 3015 +/- 110 pmol/mg protein/minute, p < 0.01) without affecting transporter affinity (control Km = 30 +/- 1.4 mu M vs. 34 +/- 1.3 mu M arginine, p = NS). Stimulation was maximal at the 8-hour time point and was inhibited by both actinomycin D and cycloheximide. In addition, inhibition of PKC with chelerythrine abrogated the TNF-augmented arginine transport. Similarly, incubation of cells with the direct PKC activator TPA (phorbol ester 12-myristate 13-acetate) stimulated System y(+)-mediated arginine transport nearly fivefold, secondary to an increase in transporter V-max (TPA V-max = 5349 +/- 310 pmol/mg protein/minute, p < 0.001 vs. control), with no change in Km. This TPA-induced stimulation of arginine transport also was blocked by cheierythrine Cl, actinomycin D, and cycloheximide. Incubation of TNF-stimulated cells with two NO synthase inhibitors did not reduce transport activity, suggesting that the arginine transporter and the NO synthase enzyme may, in part, be independently regulated. Tumor necrosis factor and TPA only slightly increased System b(0,+)-mediated arginine transport. Treatment of cells with dcAMP (dibutyryl cyclic adenosine monophosphate, a protein kinase A activator) did not alter arginine transport activity, indicating that PKA activation was not required for the response to occur. Conclusions These data indicate that TNF stimulates arginine transport in human umbilical vein endothelial cells via a process that requires activation of the intracellular messenger PKC, which in turn signals de novo protein synthesis, possibly of the yi arginine transporter protein itself.
