Expression of inducible nitric oxide synthase in bovine corneal endothelial cells and keratocytes in vitro after lipopolysaccharide and cytokines stimulation.
OBJECTIVE
To determine whether bovine corneal endothelial (BCE) cells and keratocytes express the inducible form of nitric oxide synthase (NOS) after exposure to cytokines and lipopolysaccharide (LPS), and to study the regulation of NOS by growth factors.


METHODS
Cultures of bovine corneal endothelial cells and keratocytes were exposed to increasing concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necrosis factor-alpha (TNF-alpha). At selected intervals after exposure, nitrite levels in the supernatants were evaluated by the Griess reaction. Total RNA was extracted from the cell cultures, and messenger RNA levels for inducible NOS (NOS-2) were measured by reverse transcription-polymerase chain reaction (RT-PCR).


RESULTS
Exposure of BCE cells and keratocytes to LPS and IFN-gamma resulted in an increase of nitrite levels that was potentiate by the addition of TNF-alpha. Analysis by RT-PCR demonstrated that nitrite release was correlated to the expression of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced nitrite release in both cells, whereas transforming growth factor-beta (TGF-beta) slightly potentiated it. Fibroblast growth factor-2 (FGF-2) inhibited LPS-IFN-gamma-induced nitrite release and NOS-2 messenger RNA accumulation in keratocytes but not in BCE cells.


CONCLUSIONS
The results demonstrate that in vitro activation of keratocytes and BCE cells by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammatory corneal diseases in vivo.
Dighiero
P
P
Dévelopment, Vieillissement et Pathologie de la Rétine, U450 Institut National de la Santé et de la Recherche Médicale, Association Claude Bernard, Paris, France.
Behar-Cohen
F
F
Courtois
Y
Y
Goureau
O
O
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003319:Corneal Stroma / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D016207:Cytokines / Q000494:pharmacology*; D017931:DNA Primers / Q000737:chemistry; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects* / Q000201:enzymology; D004790:Enzyme Induction; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006133:Growth Substances / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology*; D019001:Nitric Oxide Synthase / Q000096:biosynthesis* / Q000187:drug effects / Q000235:genetics; D009573:Nitrites / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism; D012486:Salmonella typhimurium
D016207:Cytokines; D017931:DNA Primers; D004791:Enzyme Inhibitors; D006133:Growth Substances; D008070:Lipopolysaccharides; D009573:Nitrites; D012333:RNA, Messenger; D019001:Nitric Oxide Synthase