Fig. 1.

Time-dependent effects of ascorbate on eNOS activity and endothelial BH4 levels. (A) EA.hy926 cells were treated with 100 μM ascorbate for 0.5–24 h. Then an l-[¹⁴C]arginine/l-[¹⁴C]citrulline conversion assay was performed as described. l-[¹⁴C]Citrulline production was normalized to the untreated control (^⁎⁎⁎p<0.001; ns, not significant; mean±SEM, n=3). (B) HUVECs were incubated with 100 μM ascorbate for 0.5–24 h, and eNOS activity was determined as for (A) (^⁎⁎⁎p<0.001; ns, not significant; mean±SEM, n=3). (C) EA.hy926 cells were treated for 2 h with the indicated concentrations of ascorbate and eNOS activity was determined as for (A) (^⁎⁎p<0.01; ^⁎⁎⁎p<0.001; ns, not significant; mean±SEM, n=3). For (A–C), note the start of the y axis at 0.9 to better visualize the effect of ascorbate. (D) EA.hy926 cells were treated with 100 μM ascorbate for 0–24 h. Then intracellular BH4 levels were assessed as described under Materials and methods. The values obtained (pmol BH4/μg cellular protein) for each treatment condition were normalized to the cellular BH4 level at time point 0 (^⁎⁎p<0.01; mean±SEM, n=3).