We have reported previously that peroxynitrite stimulates L-arginine release from astrocytes, but the mechanism responsible for such an effect remains elusive. To explore this issue, we studied the regulation of L-[(3)H]arginine transport by either exogenous or endogenous peroxynitrite in glial cells. A 2-fold peroxynitrite-mediated stimulation of l-arginine release in C6 cells was found to be Na(+)-independent, was prevented by 5 mm L-arginine and, although only in the presence of Na(+), was blocked by 5 mm L-alanine or L-leucine. Peroxynitrite-mediated stimulation of L-arginine uptake was trans-stimulated by 10 mm L-arginine and was inhibited in a dose-dependent fashion (k(i) of approximately 40 microm) by the system y(+) inhibitor N-ethylmaleimide in C6 cells. Endogenous production of peroxynitrite in lipopolysaccharide-treated astrocytes triggered an increased L-arginine transport activity without affecting Cat1 l-arginine transporter mRNA levels. However, Western blot analyses of peroxynitrite-treated astrocytes and C6 glial cells revealed a 3-nitrotyrosinated anti-Cat1-immunopositive band, strongly suggesting peroxynitrite-mediated Cat1 nitration. Furthermore, peroxynitrite stimulation of L-arginine release was abolished in fibroblast cells homozygous for a targeted inactivation of the Cat1 gene. Finally, peroxynitrite-triggered L-arginine released from astrocytes was efficiently taken up by neurons in an insert-based co-culture system. These results strongly suggest that peroxynitrite-mediated activation of the Cat1 transporter in glial cells may serve as a mechanism focused to replenish L-arginine in the neighboring neurons.