Mechanisms of ascorbic acid stimulation of norepinephrine synthesis in neuronal cells.

Ascorbic acid is well known to acutely stimulate norepinephrine synthesis in neurosecretory cells, but it has also been shown over several days to increase tyrosine hydroxylase mRNA and norepinephrine synthesis in cultured neurons. Since tyrosine hydroxylase is the rate-limiting step in catecholamine synthesis, an effect of ascorbate to increase tyrosine hydroxylase protein could contribute to its ability to increase or sustain catecholamine synthesis. Therefore, we evaluated whether tyrosine hydroxylase protein expression and function is increased in SH-SY5Y neuroblastoma cells by physiologically relevant intracellular ascorbate concentrations. SH-SY5Y neuroblastoma cells did not contain ascorbate and had only very low levels of norepinephrine in culture with L-tyrosine, the substrate for tyrosine hydroxylase. However, treatment of cells with ascorbate for 6 h or more markedly increased norepinephrine synthesis, such that intracellular ascorbate and norepinephrine increased in parallel with half maximal intracellular concentrations of about 1 mM ascorbate and 150 µM norepinephrine. This increase was enhanced by supplementing tetrahydrobiopterin, but was not mimicked by several antioxidants or by catalase or superoxide dismutase. Tyrosine hydroxylase protein expression increased at intracellular ascorbate concentrations above 1.5 mM. This contributed to norepinephrine generation, which was decreased 50-60% by inhibition of protein synthesis with cycloheximide at high intracellular ascorbate. These results suggest that expected physiologic neuronal ascorbate concentrations enhance norepinephrine synthesis both by maintaining tetrahydrobiopterin and increasing tyrosine hydroxylase expression.

Uptake, recycling, and antioxidant actions of alpha-lipoic acid in endothelial cells.

Alpha-lipoic acid, which becomes a powerful antioxidant in its reduced form, has been suggested as a dietary supplement to treat diseases associated with excessive oxidant stress. Because the vascular endothelium is dysfunctional in many of these conditions, we studied the uptake, reduction, and antioxidant effects of alpha-lipoic acid in cultured human endothelial cells (EA.hy926). Using a new assay for dihydrolipoic acid, we found that EA.hy926 cells rapidly take up and reduce alpha-lipoic acid to dihydrolipoic acid, most of which is released into the incubation medium. Nonetheless, the cells maintain dihydrolipoic acid following overnight culture, probably by recycling it from alpha-lipoic acid. Acute reduction of alpha-lipoic acid activates the pentose phosphate cycle and consumes nicotinamide adenine dinucleotide phosphate (NADPH). Lysates of EA.hy926 cells reduce alpha-lipoic acid using both NADPH and nicotinamide adenine dinucleotide (NADH) as electron donors, although NADPH-dependent reduction is about twice that due to NADH. NADPH-dependent alpha-lipoic acid reduction is mostly due to thioredoxin reductase. Pre-incubation of cells with alpha-lipoic acid increases their capacity to reduce extracellular ferricyanide, to recycle intracellular dehydroascorbic acid to ascorbate, to decrease reactive oxygen species generated by redox cycling of menadione, and to generate nitric oxide. These results show that alpha-lipoic acid enhances both the antioxidant defenses and the function of endothelial cells.