Identification of an endothelial-like type III NO synthase in LLC-PK1 kidney epithelial cells.-猫眼课题宝

LLC-PK1 肾上皮细胞中内皮样 III 型 NO 合酶的鉴定。

DOI：

发表时间：

1994

期刊：

American Journal of Physiology

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中科院分区：

文献类型：

作者：

W. R. Tracey;J. Pollock;F. Murad;M. Nakane;U. Förstermann

猪肾小管上皮细胞 (LLC-PK1) 在用各种激动剂刺激后产生一氧化氮或相关化合物（例如亚硝基硫醇）。我们现在报告了 LLC-PK1 细胞中组成型颗粒一氧化氮 (NO) 合酶的鉴定和表征。在腺苷 2',5'-二磷酸-Sepharose 上部分纯化后，颗粒状 NO 合酶活性在接近总内含体积的情况下从 Superose 6 凝胶渗透柱中异常洗脱，与内皮（III 型）NO 合酶观察到的情况类似。上皮和内皮NO合酶的底物/辅因子要求是相同的，即依赖于L-精氨酸、(6R)-5,6,7,8-四氢生物蝶呤、FAD、钙和钙调蛋白。精氨酸类似物 NG-甲基-L-精氨酸 (100 µM) 和 NG-硝基-L-精氨酸 (100 µM) 以及钙调蛋白拮抗剂三氟拉嗪 (100 µM) 和卡米达唑 (30 µM) 抑制上皮酶活性。通过蛋白质印迹分析，抗 III 型 (H32)，而非抗 I 型（脑，6763-5）或抗 II 型（巨噬细胞，8196）NO 合酶抗体，在 LLC-PK1 颗粒部分中检测到约 140 kDa 的单个免疫反应带。最后，使用聚合酶链式反应证明了 LLC-PK1 细胞中 III 型 NO 合酶 mRNA 的存在。这些数据表明 LLC-PK1 肾上皮细胞含有 III 型 NO 合酶，该酶通常与血管内皮相关。

Porcine kidney tubular epithelial cells (LLC-PK1) produce nitric oxide or a related compound (e.g., a nitrosothiol) after stimulation with various agonists. We now report the identification and characterization of a constitutive, particulate nitric oxide (NO) synthase from LLC-PK1 cells. After partial purification on adenosine 2',5'-bisphosphate-Sepharose, the particulate NO synthase activity eluted anomalously from Superose 6 gel permeation columns near the total included volume, similar to that observed for the endothelial (type III) NO synthase. Substrate/cofactor requirements of the epithelial and endothelial NO synthases were identical, i.e., dependency on L-arginine, (6R)-5,6,7,8-tetrahydrobiopterin, FAD, calcium and calmodulin. The epithelial enzyme activity was inhibited by the arginine analogues, NG-methyl-L-arginine (100 microM) and NG-nitro-L-arginine (100 microM), as well as the calmodulin antagonists, trifluoperazine (100 microM) and calmidazolium (30 microM). Anti-type III (H32), but not anti-type I (brain, 6763-5) or anti-type II (macrophage, 8196) NO synthase antibodies, detected a single immunoreactive band in the LLC-PK1 particulate fraction of approximately 140 kDa by Western blot analysis. Finally, the presence of type III NO synthase mRNA in LLC-PK1 cells was demonstrated using the polymerase chain reaction. These data indicate that LLC-PK1 kidney epithelial cells contain type III NO synthase, which has been classically associated with the vascular endothelium.