van Staveren W C G, Glick J, Markerink-van Ittersum M, Shimizu M, Beavo J A, Steinbusch H W M, de Vente J
Department of Psychiatry and Neuropsychology, Division Cellular Neuroscience, Maastricht University, European Graduate School of Neuroscience (EURON), P.O. Box 616, 6200 MD Maastricht, The Netherlands.
J Neurocytol. 2002 Sep-Nov;31(8-9):729-41. doi: 10.1023/a:1025704031210.
In this study, we report the cloning of the rat cGMP-specific phosphodiesterase type 9 (PDE9A) and its localization in rat and mouse brain by non-radioactive in situ hybridization. Rat PDE9A was 97.6% identical to mouse PDE9A1 and showed 92.1% similarity on the amino acid level to the human homologue. PDE9A mRNA was widely distributed throughout the rat and mouse brain, with the highest expression observed in cerebellar Purkinje cells. Furthermore, strong staining was detected in areas such as cortical layer V, olfactory tubercle, caudate putamen and hippocampal pyramidal and granule cells. Comparison of PDE9A mRNA expression by double staining with the cellular markers NeuN and glial fibrillary acidic protein demonstrated that PDE9A expression was mainly detected in neurons and in a subpopulation of astrocytes. Using cGMP-immunocytochemistry, the localization of cGMP was investigated in the cerebellum in which the highest PDE9 expression was demonstrated. Strong cGMP immunoreactivity was detected in the molecular layer in the presence of the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (IBMX). After treatment with soluble guanylyl cyclase activators the granular layer also showed cGMP staining, whereas no clear immunostaining was detected in Purkinje cells under all conditions investigated, which might be due to the presence of the IBMX-insensitive PDE9A in these cells. The present findings indicate that PDE9A is highly conserved between species and is widely distributed throughout the rodent brain. PDE9A is probably involved in maintenance of low cGMP levels in cells and might play an important role in a variety of brain functions involving cGMP-mediated signal transduction.
在本研究中，我们报告了大鼠9型环磷酸鸟苷特异性磷酸二酯酶（PDE9A）的克隆及其通过非放射性原位杂交在大鼠和小鼠脑中的定位。大鼠PDE9A与小鼠PDE9A1的同源性为97.6%，在氨基酸水平上与人类同源物的相似性为92.1%。PDE9A mRNA广泛分布于大鼠和小鼠脑内，在小脑浦肯野细胞中表达最高。此外，在诸如皮质第V层、嗅结节、尾状壳核以及海马锥体细胞和颗粒细胞等区域检测到强染色。通过与细胞标记物NeuN和胶质纤维酸性蛋白进行双重染色比较PDE9A mRNA表达，结果表明PDE9A表达主要在神经元和星形胶质细胞亚群中检测到。利用环磷酸鸟苷免疫细胞化学方法，在PDE9表达最高的小脑中研究了环磷酸鸟苷的定位。在存在非选择性磷酸二酯酶抑制剂3 - 异丁基 - 1 - 甲基黄嘌呤（IBMX）的情况下，在分子层检测到强环磷酸鸟苷免疫反应性。用可溶性鸟苷酸环化酶激活剂处理后，颗粒层也显示出环磷酸鸟苷染色，而在所有研究条件下，浦肯野细胞中均未检测到明显的免疫染色，这可能是由于这些细胞中存在对IBMX不敏感的PDE9A。本研究结果表明，PDE9A在物种间高度保守，广泛分布于啮齿动物脑内。PDE9A可能参与维持细胞内环磷酸鸟苷的低水平，并可能在涉及环磷酸鸟苷介导信号转导的多种脑功能中发挥重要作用。
