IDENTIFICATION OF AN ENDOTHELIAL-LIKE TYPE-III NO SYNTHASE IN LLC-PK1 KIDNEY EPITHELIAL-CELLS

被引:65
作者
TRACEY, WR
POLLOCK, JS
MURAD, F
NAKANE, M
FORSTERMANN, U
机构
来源
AMERICAN JOURNAL OF PHYSIOLOGY | 1994年 / 266卷 / 01期
关键词
EPITHELIUM; ENDOTHELIUM; NITRIC OXIDE; ENDOTHELIUM-DERIVED RELAXING FACTOR; CELL CULTURE; ACTIVE TRANSPORT; KIDNEY; NITRIC OXIDE SYNTHASE;
D O I
10.1152/ajpcell.1994.266.1.C22
中图分类号
Q4 [生理学];
学科分类号
071003 ;
摘要
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, N-G-methyl-L-arginine (100 mu M) and N-G-nitro-L-arginine (100 mu M), as well as the calmodulin antagonists, trifluoperazine (100 mu M) and calmidazolium (30 mu M) 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 similar to 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.
引用
收藏
页码:C22 / C28
页数:7
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