Jurado Sandra, Sánchez-Prieto José, Torres Magdalena
Departamento de Bioquímica, Facultad de Veterinaria, Universidad Complutense, 28040-Madrid, Spain.
Neurochem Int. 2004 Nov;45(6):833-43. doi: 10.1016/j.neuint.2004.03.013.
It is known that the nitric oxide (NO)/cGMP pathway affects neuronal development and the expression of the different proteins is developmentally dependent in several brain areas. However, so far there are no data on the expression of the proteins involved in this signalling system during the development of the cerebellar granule cell, one of the most widely used models of neuronal development. This study was accordingly designed to analyse the developmental regulation of neuronal nitric oxide synthase (nNOS), soluble guanylyl cyclase subunits (alpha1, alpha2 and beta1) and cGMP-dependent protein kinases (cGK I and cGK II) in cerebellar granule cells through real time-polymerase chain reaction (RT-PCR) and Western blotting. We were able to detect guanylyl cyclase subunits and cGK I and cGK II in cerebellar granule cells at every stage of development examined (cells freshly isolated from 7-day-old rat pups, and cells cultured for 7 days or 14 days). Expression levels, nevertheless, varied significantly at each stage. nNOS, alpha2 and beta1 and cGK II levels increased during granule cell development, while alpha1 and cGK I showed an opposite behaviour pattern; the levels of these latter proteins diminished as the cells matured. The functionality of this pathway was assessed by stimulating cells kept in culture for 7 days with DEA/NO or with N-methyl-D-aspartate (NMDA). Cells responded by increasing intracellular cGMP and activating cGMP-dependent protein kinase activity, which effectively phosphorylated two well-known substrates of this activity, the vasodilator stimulated phosphoprotein (VASP) and the cAMP response element binding protein (CREB). In summary, through both functional and biochemical tests, this is the first demonstration of a complete NO/cGMP signalling transduction pathway in cerebellar granule cells. Our results also indicate the developmental regulation of the proteins in this system.
已知一氧化氮（NO）/环磷酸鸟苷（cGMP）信号通路影响神经元发育，并且在几个脑区中，不同蛋白质的表达具有发育依赖性。然而，迄今为止，在小脑颗粒细胞（神经元发育中最广泛使用的模型之一）发育过程中，尚无关于该信号系统中相关蛋白质表达的数据。因此，本研究旨在通过实时聚合酶链反应（RT-PCR）和蛋白质免疫印迹法，分析小脑颗粒细胞中神经元型一氧化氮合酶（nNOS）、可溶性鸟苷酸环化酶亚基（α1、α2和β1）以及cGMP依赖性蛋白激酶（cGK I和cGK II）的发育调控情况。我们能够在每个检测的发育阶段（从7日龄大鼠幼崽新鲜分离的细胞，以及培养7天或14天的细胞）的小脑颗粒细胞中检测到鸟苷酸环化酶亚基以及cGK I和cGK II。然而，每个阶段的表达水平差异显著。在颗粒细胞发育过程中，nNOS、α2和β1以及cGK II的水平升高，而α1和cGK I呈现相反的行为模式；随着细胞成熟，后两种蛋白质的水平降低。通过用二乙胺/NO（DEA/NO）或N-甲基-D-天冬氨酸（NMDA）刺激培养7天的细胞来评估该信号通路的功能。细胞通过增加细胞内cGMP并激活cGMP依赖性蛋白激酶活性做出反应，该活性有效地磷酸化了该活性的两个众所周知的底物，即血管舒张刺激磷蛋白（VASP）和环磷酸腺苷反应元件结合蛋白（CREB）。总之，通过功能和生化测试，这是首次在小脑颗粒细胞中证明完整的NO/cGMP信号转导通路。我们的结果还表明了该系统中蛋白质的发育调控。
