Leukotoxin-activated human pulmonary artery endothelial cell produces nitric oxide and superoxide anion.
To provide evidence that pulmonary endothelial cells exposed to 9,10-epoxy-12-octadecenoate (Lx) produce nitric oxide (NO) and superoxide anion (O(2)(*-), we measured NO production, using a NO chemiluminescence analyzer, and nitric oxide synthase (NOS) activity, monitoring the conversion of L- [14C] arginine to L- [14C] citrulline, and O(2)(*-) by a fluorescence assay using a fluorescence spectrophotometer with hydroethidine (HE) in human pulmonary artery endothelial cells (HPAEC). NO production and eNOS were increased significantly when HPAEC were incubated with 10 microM Lx, and this effect was inhibited by L-NMMA or in the absence of extracellular Ca2+. Addition of 10 mM HE to the cell suspension spontaneously and continuously caused a subtle increase in fluorescence intensity, due to intracellular oxidation of HE to ethidium bromide (EB). Treatment of the cell suspension with Lx after the addition of HE exerted a dose-dependent increase in intracellular EB fluorescence. Pre-treatment with allopurinol, a xanthine oxidase inhibitor, decreased the intracellular EB fluorescence by 54% in HPAEC incubated with 100 microM Lx. These results show that Lx induces NO production via activation of eNOS and O(2)(*-) production in endothelial cells via activation of cellular xanthine oxidase. Thus, Lx is a bioactive lipid.
Okamura
Seitaro
S
Department of Internal Medicine, Fukui Medical University, Japan. seitao@dj8.so-net.ne.jp
Ameshima
Shingo
S
Demura
Yoshiki
Y
Ishizaki
Takeshi
T
Matsukawa
Shigeru
S
Miyamori
Isamu
I
D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000187:drug effects* / Q000378:metabolism; D004789:Enzyme Activation / Q000187:drug effects; D005098:Exotoxins / Q000494:pharmacology*; D006801:Humans; D007166:Immunosuppressive Agents / Q000494:pharmacology*; D009569:Nitric Oxide / Q000096:biosynthesis*; D019001:Nitric Oxide Synthase / Q000378:metabolism*; D011651:Pulmonary Artery / Q000187:drug effects / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*; D014969:Xanthine Oxidase / Q000378:metabolism
D005098:Exotoxins; D007166:Immunosuppressive Agents; C018579:leukotoxin; D013481:Superoxides; D009569:Nitric Oxide; D019001:Nitric Oxide Synthase; D014969:Xanthine Oxidase