Coordinate regulation of L-arginine uptake and nitric oxide synthase activity in cultured endothelial cells.
Despite intracellular L-arginine concentrations that should saturate endothelial nitric oxide synthase (eNOS), nitric oxide production depends on extracellular L-arginine. We addressed this 'arginine paradox' in bovine aortic endothelial cells by simultaneously comparing the substrate dependence of L-arginine uptake and intracellular eNOS activity, the latter measured as L-[3H]arginine conversion to L-[3H]citrulline. Whereas the Km of eNOS for L-arginine was 2 microM in cell extracts, the L-arginine concentration of half-maximal eNOS stimulation was increased to 29 microM in intact cells. This increase likely reflects limitation by L-arginine uptake, which had a Km of 108 microM. The effects of inhibitors of endothelial nitric oxide synthesis also suggested that extracellular L-arginine availability limits intracellular eNOS activity. Treatment of intact cells with the calcium ionophore A23187 reduced the L-arginine concentration of half-maximal eNOS activity, which is consistent with a measured increase in L-arginine uptake. Increases in eNOS activity induced by several agents were closely correlated with enhanced L-arginine uptake into cells (r = 0.89). The 'arginine paradox' may be explained in part by regulated L-arginine uptake into a compartment, probably represented by caveolae, that contains eNOS and that is distinct from the bulk cytosolic L-arginine.
Hardy
Thomas A
TA
Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-6303, USA. james.may@mcmail.vanderbilt.edu
May
James M
JM
D000818:Animals; D001011:Aorta / Q000166:cytology; D001120:Arginine / Q000378:metabolism*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D000001:Calcimycin / Q000494:pharmacology; D002417:Cattle; D021941:Caveolae / Q000201:enzymology*; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000201:enzymology*; D005110:Extracellular Space / Q000737:chemistry; D007476:Ionophores / Q000494:pharmacology; D007700:Kinetics; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D052250:Nitric Oxide Synthase Type III
D007476:Ionophores; D000001:Calcimycin; D001120:Arginine; D019001:Nitric Oxide Synthase; D052250:Nitric Oxide Synthase Type III