Regulation of L-arginine transport and nitric oxide release in superfused porcine aortic endothelial cells

1. We have investigated whether changes in extracellular ion composition and substrate deprivation modulate basal and/or bradykinin-stimulated L-arginine transport and release of nitric oxide (NO) and prostacyclin (PGI(2)) in porcine aortic endothelial cells cultured and superfused on microcarriers. 2. Saturable L-arginine transport (K-m = 0.14 +/- 0.03 mM; V-max = 2.08 +/- 0.54 nmol min(-1) (5 x 10(6) cells)(-1)) was pH insensitive and unaffected following removal of extracellular Na+ or Ca2+. 3. Cationic arginine analogues, including L-lysine and L-ornithine, inhibited L-arginine transport, whilst 2-methylaminoisobutyric acid, beta-2-amino-bicyclo[2,2.1]-heptane-2-carboxylic acid, L-phenylalanine, 6-diazo-5-oxo-norleucine, L-glutamine, L-cysteine and L-glutamate were poor inhibitors. 4. Deprivation of L-arginine (30 min to 24 h) reduced intracellular free L-arginine levels from 0.87 +/- 0.07 to 0.40 +/- 0.05 mM (P < 0.05) and resulted in a 40 % stimulation of L-arginine, L-lysine and L-ornithine transport. 5. L-arginine and N-G-monomethyl-L-arginine (L-NMMA), but not N-omega-nitro-L-arginine methyl ester (L-NAME), trans-stimulated efflux of L-[H-3]arginine. 6. Depolarization of endothelial cells with 70 mM K+ reduced L-arginine influx and prevented the stimulation of transport by 100 nM bradykinin, but agonist-induced release of NO and PGI(2) was still detectable. 7. Basal rates of L-arginine transport and NO release were unaffected during superfusion of cells with a nominally Ca2+-free solution. Bradykinin-stimulated L-arginine transport was insensitive to removal of Ca2+, whereas agonist-induced NO release was abolished. 8. Although bradykinin-stimulated NO release does not appear to be coupled directly to the transient increase in L-arginine transport, elevated rates of L-arginine influx via system y(+) in response to agonist-induced membrane hyperpolarization or substrate deprivation provide a mechanism for enhanced L-arginine supply to sustain NO generation.