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 (cNOS), 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 cNOS activity, the latter measured as L- [H-3]arginine conversion to L- [H-3]citrulline. Whereas the K-m of eNOS for L-arginine was 2 muM in cell extracts, the L-arginine concentration of half-maximal eNOS stimulation was increased to 29 muM in intact cells. This increase likely reflects limitation by L-arginine uptake, which had a K-m of 108 muM. 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. (C) 2002 Elsevier Science Inc.