Shimizu-Albergine Masami, Rybalkin Sergei D, Rybalkina Irina G, Feil Robert, Wolfsgruber Wiebke, Hofmann Franz, Beavo Joseph A
Department of Pharmacology, University of Washington, Seattle, Washington 98195-7280, USA.
J Neurosci. 2003 Jul 23;23(16):6452-9. doi: 10.1523/JNEUROSCI.23-16-06452.2003.
The nitric oxide (NO)-cGMP pathway has been implicated as playing a crucial role in the induction of cerebellar long-term depression (LTD). The amplitude and duration of the cGMP signal is controlled by cyclic nucleotide phosphodiesterases (PDEs). Here we identify PDE5 and PDE1B as the two major cGMP-hydrolyzing PDEs specifically and differentially expressed in the Purkinje neurons of mouse cerebellum. PDE5 was found in all Purkinje neurons, whereas PDE1B was detected only in a subset of these cells, suggesting that individual Purkinje cells may differentially regulate cGMP, depending on the PDE isozymes expressed. Although expression of guanylate cyclase and/or cGMP-dependent protein kinase (PKG) in Purkinje cells have been reported, neither cGMP accumulation nor PKG activation in these cells in vivo has been demonstrated. To determine if changes in PKG activation and PDE5 regulation occur in vivo we have examined the phosphorylation of PDE5 in mouse cerebellar Purkinje cells by immunocytochemistry and Western blot analyses using a phosphospecific PDE5 antibody. Injection of sodium nitroprusside or selective PKG activators into the lateral ventricle of mouse brain induced PDE5 phosphorylation in vivo, but was completely missing in Purkinje cell-specific PKG I knock-out mice. In cerebellar slices, treatment with sildenafil or IBMX led to different levels of phospho-PDE5 accumulation and activation of PDE5. These results suggest that phosphorylation of PDE5 in Purkinje neurons after cGMP-PKG activation performs a critical role in the termination of the cGMP signal during LTD progression; moreover, PDE5 phosphorylation may be used as an in vivo indicator for PKG activation.
一氧化氮（NO）-环鸟苷酸（cGMP）信号通路被认为在小脑长时程抑制（LTD）的诱导过程中起关键作用。cGMP信号的幅度和持续时间由环核苷酸磷酸二酯酶（PDEs）控制。在此，我们确定磷酸二酯酶5（PDE5）和磷酸二酯酶1B（PDE1B）是在小鼠小脑浦肯野神经元中特异性且差异表达的两种主要cGMP水解磷酸二酯酶。在所有浦肯野神经元中均发现了PDE5，而仅在这些细胞的一个亚群中检测到PDE1B，这表明单个浦肯野细胞可能根据所表达的磷酸二酯酶同工酶对cGMP进行差异调节。尽管已有报道称浦肯野细胞中存在鸟苷酸环化酶和/或cGMP依赖性蛋白激酶（PKG）的表达，但尚未证实这些细胞在体内有cGMP积累或PKG激活。为了确定PKG激活和PDE5调节在体内是否发生变化，我们使用磷酸特异性PDE5抗体，通过免疫细胞化学和蛋白质印迹分析检测了小鼠小脑浦肯野细胞中PDE5的磷酸化情况。向小鼠脑侧脑室注射硝普钠或选择性PKG激活剂可在体内诱导PDE5磷酸化，但在浦肯野细胞特异性PKG I基因敲除小鼠中则完全缺失。在小脑切片中，用西地那非或异丁基甲基黄嘌呤（IBMX）处理可导致不同水平的磷酸化PDE5积累和PDE5激活。这些结果表明，cGMP-PKG激活后浦肯野神经元中PDE5的磷酸化在LTD进展过程中cGMP信号的终止中起关键作用；此外，PDE5磷酸化可用作PKG激活的体内指标。
