L-ascorbic acid potentiates nitric oxide synthesis in endothelial cells

Ascorbic acid has been shown to enhance impaired endothelium-dependent vaso dilation in patients with atherosclerosis by a mechanism that is thought to involve protection of nitric oxide (NO) from inactivation by free oxygen r adicals. The present study in human endothelial cells from umbilical veins and coronary arteries investigates whether L-ascorbic acid additionally aff ects cellular NO synthesis. Endothelial cells were incubated for 24 h with 0.1-100 mu M ascorbic acid and were subsequently stimulated for 15 min with ionomycin (2 mu M) Or thrombin (1 unit/ml) in the absence of extracellular ascorbate. Ascorbate pretreatment led to a 3-fold increase of the cellular production of NO measured as the formation of its co-product citrulline an d as the accumulation of its effector molecule cGMP. The effect was saturat ed at 100 mu M and followed a similar kinetics as seen for the uptake of as corbate into the cells. The investigation of the precursor molecule L-gulon olactone and of different ascorbic acid derivatives suggests that the enedi ol structure of ascorbate is essential for its effect on NO synthesis. Asco rbic acid did not induce the expression of the NO synthase (NOS) protein no r enhance the uptake of the NOS substrate L-arginine into endothelial cells . The ascorbic acid effect was minimal when the citrulline formation was me asured in cell lysates from ascorbate-pretreated cells in the presence of k nown cofactors for NOS activity. However, when the cofactor tetrahydrobiopt erin was omitted from the assay, a similar potentiating effect of ascorbate pretreatment as seen in intact cells was demonstrated, suggesting that asc orbic acid may either enhance the availability of tetrahydrobiopterin or in crease its affinity for the endothelial NOS. Our data suggest that intracel lular ascorbic acid enhances NO synthesis in endothelial cells and that thi s may explain, in part, the beneficial vascular effects of ascorbic acid.