Identification of an endothelial-like type III NO synthase in LLC-PK1 kidney epithelial cells.
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.
Tracey
W R
WR
Vascular Biology Project, Abbott Laboratories, Abbott Park, Illinois 60064-3500.
Pollock
J S
JS
Murad
F
F
Nakane
M
M
Förstermann
U
U
D000594:Amino Acid Oxidoreductases / Q000145:classification / Q000378:metabolism*; D000818:Animals; D001483:Base Sequence; D002460:Cell Line; D004730:Endothelium, Vascular / Q000201:enzymology*; D004847:Epithelial Cells; D004848:Epithelium / Q000201:enzymology; D007527:Isoenzymes / Q000378:metabolism*; D007668:Kidney / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D019001:Nitric Oxide Synthase; D015345:Oligonucleotide Probes / Q000235:genetics; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D013552:Swine
D007527:Isoenzymes; D015345:Oligonucleotide Probes; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase; D000594:Amino Acid Oxidoreductases