Duarte-Silva Aline T, Domith Ivan, Gonçalves-da-Silva Isabele, Paes-de-Carvalho Roberto
Program of Neurosciences, Institute of Biology, Fluminense Federal University, Niterói 24210-346, RJ, Brazil.
Instituto D'Or de Pesquisa e Ensino (IDOR), Rio de Janeiro 22281-100, RJ, Brazil.
Brain Sci. 2025 Apr 2;15(4):369. doi: 10.3390/brainsci15040369.
In addition to its known antioxidant function, the reduced form of vitamin C, ascorbate, also acts as a neuromodulator in the nervous system. Previous work showed a reciprocal interaction of ascorbate with glutamate in chicken embryo retinal cultures. Ascorbate modulates extracellular glutamate levels by inhibiting excitatory amino acid transporter 3 and promoting the activation of NMDA receptors and the consequent activation of intracellular signaling pathways involved in transcription and survival. In the present work, we investigated the regulation of AKT phosphorylation by ascorbate in chicken embryo retina cultures. Cultures of chicken embryo retina cells were tested using Western blot, immunocytochemistry, fluorescent probe transfection, and cellular imaging techniques. Our results show that ascorbate induces a concentration and time-dependent increase in AKT phosphorylation via the accumulation of extracellular glutamate, the activation of glutamate receptors, and the activation of the PI3K pathway. Ascorbate produces an increase in intracellular calcium accumulation and, accordingly, AKT phosphorylation by ascorbate is blocked by the calcium chelator BAPTA-AM. Moreover, AKT phosphorylation is also blocked by the nitric oxide synthase inhibitor 7-nitroindazole, indicating that it is mediated by calcium and nitric oxide-dependent mechanisms. We demonstrate that ascorbate modulates the PI3K/AKT pathway in retinal cultures through the activation of glutamate receptors and NO production in a calcium-dependent manner. Given that previous research has shown that glutamate induces ascorbate release in retinal cultures, our findings emphasize the significance of the reciprocal interactions between ascorbate and glutamate in retinal development. These findings provide further evidence supporting the role of ascorbate as a neuromodulator in retinal development.
除了其已知的抗氧化功能外，维生素C的还原形式抗坏血酸盐在神经系统中还充当神经调节剂。先前的研究表明，在鸡胚视网膜培养物中，抗坏血酸盐与谷氨酸之间存在相互作用。抗坏血酸盐通过抑制兴奋性氨基酸转运体3、促进NMDA受体的激活以及随后激活参与转录和存活的细胞内信号通路来调节细胞外谷氨酸水平。在本研究中，我们研究了抗坏血酸盐对鸡胚视网膜培养物中AKT磷酸化的调节作用。使用蛋白质免疫印迹、免疫细胞化学、荧光探针转染和细胞成像技术对鸡胚视网膜细胞培养物进行了检测。我们的结果表明，抗坏血酸盐通过细胞外谷氨酸的积累、谷氨酸受体的激活和PI3K途径的激活，诱导AKT磷酸化呈浓度和时间依赖性增加。抗坏血酸盐导致细胞内钙积累增加，因此，抗坏血酸盐诱导的AKT磷酸化被钙螯合剂BAPTA-AM阻断。此外，AKT磷酸化也被一氧化氮合酶抑制剂7-硝基吲唑阻断，表明它是由钙和一氧化氮依赖性机制介导的。我们证明，抗坏血酸盐通过激活谷氨酸受体和以钙依赖性方式产生一氧化氮，调节视网膜培养物中的PI3K/AKT途径。鉴于先前的研究表明谷氨酸在视网膜培养物中诱导抗坏血酸盐释放，我们的发现强调了抗坏血酸盐与谷氨酸之间相互作用在视网膜发育中的重要性。这些发现提供了进一步的证据，支持抗坏血酸盐作为神经调节剂在视网膜发育中的作用。
