Biochemical and Biophysical Research Communications
Mechanisms of ascorbic acid stimulation of norepinephrine synthesis in neuronal cells
Highlights
► Ascorbic acid enhances norepinephrine synthesis in SH-SY5Y neuroblastoma cells. ► Norepinephrine synthesis increases linearly with intracellular ascorbate. ► Ascorbate enhances tetrahydrobiopterin-stimulated norepinephrine synthesis. ► Ascorbate increases protein expression of tyrosine hydroxylase. ► Ascorbate increases tyrosine hydroxylase expression at physiologic concentrations.
Introduction
Neuronal catecholamine synthesis is acutely enhanced by ascorbic acid at two steps in the pathway [1]. First, ascorbate helps to maintain the activity of tyrosine hydroxylase by recycling its essential co-factor, tetrahydrobiopterin. Ascorbate does this in a one-electron reduction of the trihydrobiopterin radical that is generated on hydroxylation of l-tyrosine by tyrosine hydroxylase. Ascorbate is much more efficient in this recycling than cellular thiol antioxidants, including reduced glutathione and cysteine [2]. Second, ascorbate directly contributes an electron to dopamine β-hydroxylase in neurosecretory vesicles to allow it to hydroxylate dopamine to form norepinephrine. Without ascorbate, the activity of dopamine β-hydroxylase is low, possibly maintained by an electron from dopamine itself [3], [4]. Both of these reactions are acute and depend on existing levels of the enzymes and co-factors involved.
A third effect of ascorbate to stimulate catecholamine synthesis may also be to enhance the transcription of tyrosine hydroxylase. Seitz et al. [5] reported that culture of SK-N-SH neuroblastoma cells with 200 μM ascorbate for 5 days more than doubled both intracellular norepinephrine levels and tyrosine hydroxylase mRNA, although tyrosine hydroxylase protein was not measured. In that work, a 2 h treatment with ascorbate increased dopamine and norepinephrine synthesis from l-tyrosine, but did not affect tyrosine hydroxylase mRNA.
In this work we examined the mechanism by which ascorbate stimulates norepinephrine generation from l-tyrosine in SH-SY5Y neuroblastoma cells, finding that optimal stimulation by ascorbate required at least 6 h, occurred at what are likely physiologic intracellular ascorbate concentrations, and was associated with increased tyrosine hydroxylase protein expression.
Section snippets
Materials
Sigma/Aldrich Chemical Co. (St. Louis, MO) supplied the analytical reagents, including l-ascorbic acid, bovine liver catalase, l-3,4-dihydroxyphenylalanine (l-DOPA), dopamine, norepinephrine, N-2-hydroxyethylpiperazine N′-2-ethanesulfonic acid (Hepes), sepiapterin, bovine liver superoxide dismutase, Tempol (4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl), and Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid).
Cell culture
SH-SY5Y neuroblastoma cells were obtained from the American Type Culture
Results
In the absence of added ascorbate, SH-SY5Y cells in culture contained very low amounts of norepinephrine (7.0 ± 3.1 μM, Fig. 1A, circle at zero hours) and no ascorbate (Fig. 1B, circle at zero hours). When cells were incubated with 100 μM ascorbate for various times, the norepinephrine content (Fig. 1A) of the cells increased in concert with intracellular ascorbate (Fig. 1B), both requiring 6 h to become maximal. Dopamine and l-DOPA levels were very low in these cells (results not shown), so
Discussion
SH-SY5Y cells contained very little norepinephrine and no detectable ascorbate in culture with 100 μM l-tyrosine, the substrate for tyrosine hydroxylase. However, treatment of the cells for 6 h with ascorbate increased norepinephrine to maximal levels. This effect was not mimicked by several other cell-penetrant antioxidants, including the one-electron-donor Tempol, a water-soluble vitamin E analog (Trolox), and two different thiol reagents (N-acetyl cysteine and dithiothreitol). It is also
Acknowledgments
This work was supported by NIH Grant NS 057674 and by the Vanderbilt Diabetes Research and Training Center (DK 020593).
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