Mechanisms of ascorbic acid stimulation of norepinephrine synthesis in neuronal cells

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Abstract

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.

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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