Xu M, Ng Y K, Leong S K
Department of Anatomy, Faculty of Medicine, National University of Singapore, MD10, 4 Medical Drive, 117597, Singapore.
Nitric Oxide. 2000 Oct;4(5):483-95. doi: 10.1006/niox.2000.0301.
We previously reported on the differential expression of neuronal nitric oxide synthase (nNOS) in neurons of the nucleus dorsalis (ND) and red nucleus (RN), as well as differential roles of nitric oxide (NO) in these two distinct groups' neurons characterized with different nNOS phenotypes after lower thoracic spinal cord hemisection. To further understand the enzyme, nNOS expression was studied at the subcellular and mRNA levels by using electron microscopic immunohistochemistry (EM-IHC) and in situ hybridization respectively. Possible transcriptional regulation by c-Jun or CREB in the differential nNOS expression in both ND and RN neurons was also studied. nNOS mRNA was not found in the normal ND neurons, but was shown in the normal RN neurons. After spinal cord hemisection, nNOS mRNA was induced in the ipsilateral ND, while upregulated on both sides of the RN, which preceded protein induction or upregulation. By EM-IHC, nNOS immunoreaction products were predominantly bound to the membrane of the mitochondria, rough endoplasmic reticulum (rER), Golgi apparatus, and nuclear envelope in the RN neurons of normal rats as well as rats subjected to spinal cord hemisection. In contrast, nNOS-immunoreactive deposits in the experimental ND neurons were found to be mainly granular, being dispersed throughout the cytoplasmic matrix. It is speculated that the differential subcellular localizationof nNOS indicates that axotomy may trigger different nNOS transcripts and lead to different nNOS isoform expression in the normally non-nNOS- and normally nNOS-containing neurons. c-Jun was induced in the ipsilateral ND neuronsand upregulated only in the contralateral RN neurons. Activation of CREB by phosphorylation was occasionally detectable in the ND neurons, but not in the RN neurons. Double-labeling data showed a large proportion of c-Jun and nNOS colocalization in neurons of the ipsilateral ND and contralateral RN after spinal cord hemisection. However, dissociation of nNOS expression kinetics with c-Jun was observed in the ipsilateral RN. The results implied that nNOS expression might not be under the direct transcriptional regulation by c-Jun, although it seemed to be closely related to the c-Jun expression.
我们之前报道了背核（ND）和红核（RN）神经元中神经元型一氧化氮合酶（nNOS）的差异表达，以及一氧化氮（NO）在胸段脊髓半切术后具有不同nNOS表型的这两组不同神经元中的不同作用。为了进一步了解该酶，分别使用电子显微镜免疫组织化学（EM-IHC）和原位杂交在亚细胞和mRNA水平研究了nNOS表达。还研究了c-Jun或CREB在ND和RN神经元nNOS差异表达中可能的转录调控作用。正常ND神经元中未发现nNOS mRNA，但在正常RN神经元中可见。脊髓半切术后，同侧ND中诱导出nNOS mRNA，而RN两侧均上调，且先于蛋白质的诱导或上调。通过EM-IHC，正常大鼠以及脊髓半切术后大鼠的RN神经元中，nNOS免疫反应产物主要结合在线粒体、粗面内质网（rER）、高尔基体和核膜上。相比之下，实验性ND神经元中的nNOS免疫反应性沉积物主要呈颗粒状，分散在整个细胞质基质中。据推测，nNOS在亚细胞水平的差异定位表明，轴突切断可能触发不同的nNOS转录本，并导致正常不含nNOS和正常含nNOS的神经元中不同nNOS亚型的表达。c-Jun在同侧ND神经元中被诱导，且仅在对侧RN神经元中上调。在ND神经元中偶尔可检测到磷酸化激活的CREB，但在RN神经元中未检测到。双标数据显示，脊髓半切术后同侧ND和对侧RN神经元中有很大比例的c-Jun和nNOS共定位。然而，在同侧RN中观察到nNOS表达动力学与c-Jun的解离。结果表明，nNOS表达可能不受c-Jun的直接转录调控，尽管它似乎与c-Jun表达密切相关。
