Northington F J, Koehler R C, Traystman R J, Martin L J
Department of Pediatrics, Johns Hopkins Medical Institutions, Baltimore, MD 21287, USA.
Brain Res Dev Brain Res. 1996 Aug 20;95(1):1-14. doi: 10.1016/0165-3806(96)00051-x.
Two constitutively expressed isoforms of nitric oxide synthase (NOS) have been identified, Nos1 and Nos3. Nos1 was originally identified in neuronal cells and Nos3 in endothelial cells. Because the biochemical activity of NOS is developmentally regulated, we tested the hypothesis that protein expression is also developmentally regulated. Antibodies to Nos1 and Nos3 were evaluated for specificity by immunoblotting and then used for immunohistochemistry. In fetal and adult sheep brain homogenates, Nos1 antibodies identified one immunoreactive band of proteins at 155 kDa. The Nos3 antibody detected one immunoreactive band at 145 kDa that comigrated with a reactive band in endothelial cell lystates. Immunoblots of developing neocortex demonstrated that Nos1 was enriched at early gestational ages, whereas Nos3 expression was relatively constant throughout development. By immunohistochemistry, distinct isoform-specific patterns of immunoreactivity were detected. At 60 days, Nos1 immunoreactivity is primarily localized in neuropil, but by midgestation, nonpyramidal neurons are labeled in the cortical plate. Developing neurites are Nos1-positive at 60 and 71 days, decreasing in abundance by 93 days. By 93 days the striatum is fully populated by Nos1-expressing nonprincipal neurons. In hippocampus and subthalamic nucleus, Nos1 immunoreactivity is greatest at 60 and 71 days gestation, decreasing thereafter. Immunoreactivity for Nos3 delineates cerebrovasculature maturation from a primarily radial to a highly complex branching arrangement. Hindbrain structures achieve mature organization of the cerebrovasculature before forebrain. We conclude that constitutive NOS protein expression is developmentally regulated and that distinct isoforms of NOS are regulated differentially during brain development. Expression of Nos3 parallels maturation of the cerebrovasculature, whereas the transient, region- and cell type-dependent enrichment of Nos1 in the developing brain may indicate a temporally and spatially restricted role for this enzyme in the maturation of specific neuronal populations.
已鉴定出一氧化氮合酶（NOS）的两种组成型表达同工型，即Nos1和Nos3。Nos1最初在神经元细胞中被鉴定出来，Nos3则在内皮细胞中被鉴定出来。由于NOS的生化活性受发育调控，我们检验了蛋白质表达也受发育调控这一假设。通过免疫印迹评估了针对Nos1和Nos3的抗体的特异性，然后将其用于免疫组织化学。在胎儿和成年绵羊脑匀浆中，Nos1抗体识别出一条155 kDa的蛋白质免疫反应条带。Nos3抗体检测到一条145 kDa的免疫反应条带，该条带与内皮细胞裂解物中的一条反应条带迁移位置相同。发育中的新皮层的免疫印迹显示，Nos1在妊娠早期富集，而Nos3的表达在整个发育过程中相对恒定。通过免疫组织化学，检测到了不同的同工型特异性免疫反应模式。在60天时，Nos1免疫反应主要定位于神经毡，但到妊娠中期，皮质板中的非锥体神经元被标记。发育中的神经突在60天和71天时呈Nos1阳性，到93天时丰度降低。到93天时，纹状体中完全布满了表达Nos1的非主要神经元。在海马体和丘脑下核中，Nos1免疫反应在妊娠60天和71天时最强，此后降低。Nos3的免疫反应描绘了脑血管从主要的放射状到高度复杂的分支排列的成熟过程。后脑结构在 forebrain 之前实现脑血管的成熟组织。我们得出结论，组成型NOS蛋白表达受发育调控，并且在脑发育过程中，NOS的不同同工型受到不同的调控。Nos3的表达与脑血管的成熟平行，而Nos1在发育中的大脑中短暂的、区域和细胞类型依赖性富集可能表明该酶在特定神经元群体成熟中具有时间和空间上受限的作用。 （注：原文中“forebrain”未翻译，因为不确定其准确含义，可根据具体专业内容补充完整）
