Program of Neurosciences and Department of Neurobiology, Institute of Biology, Fluminense Federal University, Niterói, 24020-971, Brazil.
J Biol Chem. 2012 Nov 9;287(46):38680-94. doi: 10.1074/jbc.M112.353961. Epub 2012 Sep 19.
In the retina information decoding is dependent on excitatory neurotransmission and is critically modulated by AMPA glutamate receptors. The Src-tyrosine kinase has been implicated in modulating neurotransmission in CNS. Thus, our main goal was to correlate AMPA-mediated excitatory neurotransmission with the modulation of Src activity in retinal neurons. Cultured retinal cells were used to access the effects of AMPA stimulation on nitric oxide (NO) production and Src phosphorylation. 4-Amino-5-methylamino-2',7'-difluorofluorescein diacetate fluorescence mainly determined NO production, and immunocytochemistry and Western blotting evaluated Src activation. AMPA receptors activation rapidly up-regulated Src phosphorylation at tyrosine 416 (stimulatory site) and down-regulated phosphotyrosine 527 (inhibitory site) in retinal cells, an effect mainly mediated by calcium-permeable AMPA receptors. Interestingly, experiments confirmed that neuronal NOS was activated in response to calcium-permeable AMPA receptor stimulation. Moreover, data suggest NO pathway as a key regulatory signaling in AMPA-induced Src activation in neurons but not in glial cells. The NO donor SNAP (S-nitroso-N-acetyl-DL-penicillamine) and a soluble guanylyl cyclase agonist (YC-1) mimicked AMPA effect in Src Tyr-416 phosphorylation, reinforcing that Src activation is indeed modulated by the NO pathway. Gain and loss-of-function data demonstrated that ERK is a downstream target of AMPA-induced Src activation and NO signaling. Furthermore, AMPA stimulated NO production in organotypic retinal cultures and increased Src activity in the in vivo retina. Additionally, AMPA-induced apoptotic retinal cell death was regulated by both NOS and Src activity. Because Src activity is pivotal in several CNS regions, the data presented herein highlight that Src modulation is a critical step in excitatory retinal cell death.
在视网膜中，信息解码依赖于兴奋性神经递质传递，并受到 AMPA 谷氨酸受体的严格调节。Src 酪氨酸激酶已被牵涉到调节中枢神经系统中的神经递质传递。因此，我们的主要目标是将 AMPA 介导的兴奋性神经递质传递与视网膜神经元中 Src 活性的调节相关联。我们使用培养的视网膜细胞来研究 AMPA 刺激对一氧化氮（NO）产生和 Src 磷酸化的影响。4-氨基-5-甲基氨基-2'，7'-二氟荧光素二乙酸酯荧光主要用于测定 NO 的产生，免疫细胞化学和 Western blot 用于评估 Src 的激活。AMPA 受体的激活可迅速上调视网膜细胞中 Src 酪氨酸 416 位（激活位）的磷酸化，下调磷酸化酪氨酸 527 位（抑制位），这种作用主要是由钙通透性 AMPA 受体介导的。有趣的是，实验证实神经元型一氧化氮合酶（nNOS）在钙通透性 AMPA 受体刺激下被激活。此外，数据表明 NO 途径是 AMPA 诱导的神经元中 Src 激活的关键调节信号，但不是在神经胶质细胞中。NO 供体 SNAP（S-亚硝基-N-乙酰-DL-青霉胺）和可溶性鸟苷酸环化酶激动剂（YC-1）模拟了 AMPA 对 Src Tyr-416 磷酸化的作用，这进一步证实了 Src 的激活确实受到 NO 途径的调节。获得和丧失功能的数据表明，ERK 是 AMPA 诱导的 Src 激活和 NO 信号的下游靶标。此外，AMPA 在器官型视网膜培养物中刺激 NO 的产生，并增加体内视网膜中 Src 的活性。此外，AMPA 诱导的视网膜细胞凋亡死亡受到 NOS 和 Src 活性的调节。由于 Src 活性在几个中枢神经系统区域中至关重要，因此本文的数据强调了 Src 调节是兴奋性视网膜细胞死亡的关键步骤。
