Department of Medicine, Vanderbilt University School of Medicine, Nashville, TN 37232-6303, USA.
Biochem Biophys Res Commun. 2012 Sep 14;426(1):148-52. doi: 10.1016/j.bbrc.2012.08.054. Epub 2012 Aug 19.
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.
抗坏血酸是众所周知的急性刺激神经分泌细胞去甲肾上腺素合成，但它也已被证明在几天内增加培养神经元的酪氨酸羟化酶 mRNA 和去甲肾上腺素合成。由于酪氨酸羟化酶是儿茶酚胺合成的限速步骤，抗坏血酸增加酪氨酸羟化酶蛋白的作用可能有助于其增加或维持儿茶酚胺合成的能力。因此，我们评估了生理相关的细胞内抗坏血酸浓度是否会增加 SH-SY5Y 神经母细胞瘤细胞中的酪氨酸羟化酶蛋白表达和功能。SH-SY5Y 神经母细胞瘤细胞不含抗坏血酸，并且在用 L-酪氨酸（酪氨酸羟化酶的底物）培养时只有非常低水平的去甲肾上腺素。然而，用抗坏血酸处理细胞 6 小时或更长时间会显著增加去甲肾上腺素的合成，使得细胞内抗坏血酸和去甲肾上腺素的浓度与半最大细胞内浓度平行增加，约为 1mM 抗坏血酸和 150μM 去甲肾上腺素。四氢生物蝶呤的补充增强了这种增加，但几种抗氧化剂或过氧化氢酶或超氧化物歧化酶不能模拟这种增加。在细胞内抗坏血酸浓度高于 1.5mM 时，酪氨酸羟化酶蛋白表达增加。这有助于去甲肾上腺素的产生，当用环己酰亚胺在高细胞内抗坏血酸下抑制蛋白质合成时，去甲肾上腺素的产生减少了 50-60%。这些结果表明，预期的生理神经元抗坏血酸浓度通过维持四氢生物蝶呤和增加酪氨酸羟化酶表达来增强去甲肾上腺素的合成。
