Pollock J S, Nakane M, Buttery L D, Martinez A, Springall D, Polak J M, Förstermann U, Murad F
Vascular Biology Project, Abbott Laboratories, Abbott Park, Illinois 60064.
Am J Physiol. 1993 Nov;265(5 Pt 1):C1379-87. doi: 10.1152/ajpcell.1993.265.5.C1379.
We have produced specific monoclonal antibodies (MAb) against particulate bovine aortic endothelial nitric oxide synthase. In Western blots, native and cultured bovine aortic endothelial cells as well as cultured bovine microvascular endothelial cells possess immunoreactive NO synthase. In dot blots, MAb H210 and H32 detect 1 ng and 100 pg of purified endothelial NO synthase, respectively. Both antibodies are specific to the endothelial NO synthase and do not cross-react with other known isoforms of NO synthase, namely from the brain, from cytokine/endotoxin-induced macrophages, or from cytokine/endotoxin-induced vascular smooth muscle cells. Immunohistochemical studies demonstrated the specificity of endothelial NO synthase for endothelial cells in various bovine and human tissues. Many types of endothelial cells, macrovascular, microvascular, arterial, and venous were found to possess this specific isoform of NO synthase. Electron microscopy showed the enzyme to be associated with the plasma membrane, membranes of cytoplasmic vesicles, and in the cytoplasm in human umbilical vein endothelial cells. The results demonstrate that particulate endothelial NO synthase is present in a site to act rapidly to produce NO for release into the blood or toward the smooth muscle in many vascular beds.
我们制备了针对颗粒状牛主动脉内皮型一氧化氮合酶的特异性单克隆抗体（MAb）。在蛋白质免疫印迹法中，天然和培养的牛主动脉内皮细胞以及培养的牛微血管内皮细胞均具有免疫反应性一氧化氮合酶。在斑点印迹法中，单克隆抗体H210和H32分别可检测到1 ng和100 pg的纯化内皮型一氧化氮合酶。这两种抗体对内皮型一氧化氮合酶具有特异性，且不与其他已知的一氧化氮合酶同工型发生交叉反应，这些同工型分别来自脑、细胞因子/内毒素诱导的巨噬细胞或细胞因子/内毒素诱导的血管平滑肌细胞。免疫组织化学研究证实了内皮型一氧化氮合酶在各种牛和人类组织中对内皮细胞的特异性。发现许多类型的内皮细胞，包括大血管、微血管、动脉和静脉内皮细胞，均具有这种特异性的一氧化氮合酶同工型。电子显微镜显示，在人脐静脉内皮细胞中，该酶与质膜、细胞质囊泡膜以及细胞质相关。结果表明，颗粒状内皮型一氧化氮合酶存在于一个能迅速发挥作用的位点，可产生一氧化氮并释放到血液中或作用于许多血管床中的平滑肌。
