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Coordinate regulation of L-arginine uptake and nitric oxide synthase activity in cultured endothelial cells.

Hardy TA, May JM
Free Radic Biol Med. 2002 32 (2): 122-31

PMID: 11796200 · DOI:10.1016/s0891-5849(01)00781-x

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.

MeSH Terms (14)

Animals Aorta Arginine Biological Transport Calcimycin Cattle Caveolae Cells, Cultured Endothelium, Vascular Extracellular Space Ionophores Kinetics Nitric Oxide Synthase Nitric Oxide Synthase Type III

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