Zinc ion (Zn2+), the second most abundant transition metal after iron in the body, is essential for neuronal activity and also induces toxicity if the concentration is abnormally high. Our previous results show that exposure of cultured cortical neurons to dopamine elevates intracellular Zn2+ concentrations ([Zn2+]i) and induces autophagosome formation but the mechanism is not clear. In this study, we characterized the signaling pathway responsible for the dopamine-induced elevation of [Zn2+]i and the effect of [Zn2+]i in modulating the autophagy in cultured rat embryonic cortical neurons. N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN), a membrane-permeable Zn2+ chelator, could rescue the cell death and suppress the autophagosome puncta number induced by dopamine. Dopamine treatment increased the lipidation level of the endogenous microtubule-associated protein 1A/1B-light chain 3 (LC3 II), an autophagosome marker. TPEN added 1h before, but not after, dopamine treatment suppressed the dopamine-induced elevation of LC3 II level. Inhibitors of the dopamine D1-like receptor, protein kinase A (PKA), and NOS suppressed the dopamine-induced elevation of [Zn2+]i. PKA activators and NO generators directly increased [Zn2+]i in cultured neurons. Through cell fractionation, proteins with m.w. values between 5 and 10kD were found to release Zn2+ following NO stimulation. In addition, TPEN pretreatment and an inhibitor against PKA could suppress the LC3 II level increased by NO and dopamine, respectively. Therefore, our results demonstrate that dopamine-induced elevation of [Zn2+]i is mediated by the D1-like receptor-PKA-NO pathway and is important in modulating the cell death and autophagy.

中文翻译：

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多巴胺通过cAMP一氧化氮信号级联反应提高培养的大鼠胚胎皮层神经元中细胞内锌的浓度。

锌离子（Zn2 +）是人体中仅次于铁的第二高含量的过渡金属，对于神经元活动至关重要，如果浓度异常高，也会引起毒性。我们以前的结果表明，培养的皮质神经元暴露于多巴胺会提高细胞内Zn2 +浓度（[Zn2 +] i）并诱导自噬体形成，但机制尚不清楚。在这项研究中，我们表征了负责多巴胺诱导的[Zn2 +] i升高和[Zn2 +] i调节培养的大鼠胚胎皮层神经元自噬作用的信号通路。N，N，N'，N'-四（2-吡啶基甲基）乙二胺（TPEN）是一种可透过膜的Zn2 +螯合剂，可以挽救细胞死亡并抑制多巴胺诱导的自噬点数。多巴胺治疗可增加内吞性微管相关蛋白1A / 1B-轻链3（LC3 II）的脂质水平，该蛋白是自噬体标记。TPEN在多巴胺治疗之前（而非之后）添加了1h，从而抑制了多巴胺诱导的LC3 II水平升高。多巴胺D1样受体，蛋白激酶A（PKA）和NOS的抑制剂抑制了多巴胺诱导的[Zn2 +] i升高。PKA激活剂和NO生成器直接增加了培养的神经元中的[Zn2 +] i。通过细胞分级分离，发现mw值在5和10kD之间的蛋白质在NO刺激后释放出Zn2 +。此外，TPEN预处理和PKA抑制剂可以抑制NO和多巴胺分别增加的LC3 II水平。所以，