EXPRESSION OF INDUCIBLE NITRIC-OXIDE SYNTHASE IN BOVINE CORNEAL ENDOTHELIAL-CELLS AND KERATOCYTES IN-VITRO AFTER LIPOPOLYSACCHARIDE AND CYTOKINES STIMULATION

Purpose. To determine whether bovine corneal endothelial (BCE) cells a nd keratocytes express the inducible form of nitric oxide synthase (NO S) after exposure to cytokines and lipopolysaccharide (LPS), and to st udy the regulation of NOS by growth factors. Methods. Cultures of bovi ne corneal endothelial cells and keratocytes were exposed to increasin g concentrations of LPS, interferon-gamma (IFN-gamma), and tumor necro sis factor-alpha (TNF-alpha). At selected intervals after exposure, ni trite levels in the supernatants were evaluated by the Griess reaction . Total RNA was extracted from the cell cultures, and messenger RNA le vels 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 l evels that was potentiate by the addition of TNF-alpha. Analysis by RT -PCR demonstrated that nitrite release was correlated to the expressio n of NOS-2 messenger RNA in BCE cells and keratocytes. Stereoselective inhibitors of NOS and cycloheximide inhibited LPS-IFN-gamma-induced n itrite 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 R NA accumulation in keratocytes but not in BCE cells. Conclusions. The results demonstrate that in vitro activation of keratocytes and BCE ce lls by LPS and cytokines induces NOS-2 expression and release of large amounts of NO. The high amounts of NO could be involved in inflammato ry corneal diseases in vivo.