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 (PGI2) in porcine aortic endothelial cells cultured and superfused on microcarriers. 2. Saturable L-arginine transport (Km = 0.14 +/- 0.03 mM; Vmax = 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 NG-monomethyl-L-arginine (L-NMMA), but not N omega-nitro-L-arginine methyl ester (L-NAME), trans-stimulated efflux of L-[3H]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 PGI2 was still detectable. 7. Basal rates of L-arginine transport and NO release were unaffected during superfusion of cells with a nominally Ca(2+)-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.
Bogle
R G
RG
Vascular Biology Research Centre, King's College, London, UK.
Baydoun
A R
AR
Pearson
J D
JD
Mann
G E
GE
D000818:Animals; D001011:Aorta / Q000378:metabolism*; D001120:Arginine / Q000378:metabolism*; D002478:Cells, Cultured; D004727:Endothelium / Q000378:metabolism; D007700:Kinetics; D008353:Mannitol / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D013552:Swine
D009569:Nitric Oxide; D008353:Mannitol; D001120:Arginine