Cytokine regulation of nitric oxide synthase in mouse retinal pigment epithelial cells in culture

Janet R. Sparrow*, Carl Nathan, Yoram Vodovotz

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

38 Scopus citations


Nitric oxide is an intercellular signaling molecule whose numerous functions include regulation of vascular tone, mediation of the cytotoxic effects of macrophages and potentiation of synaptic transmission. For some cellular functions, nitric oxide synthesis is mediated by the inducible form of nitric oxide synthase. We now show that cultured mouse retinal pigment epithelial cells exposed to interferon-γ and lipopolysaccharide, express inducible nitric oxide synthase. The latter was detected immunocytochemically in interferon-γ-lipopolysaccharide-treated retinal pigment epithelium using rabbit antiserum to a synthetic peptide of mouse nitric oxide synthase. Untreated cultures of retinal pigment epithelium or cultures treated with either interferon-γ or lipopolysaccharide alone were not immunoreactive. Induction of iNOS in γ-interferon-lipopolysaccharide-stimulated retinal pigment epithelial cells was also evidenced by the presence of nitric oxide synthase enzyme activity in lysates of stimulated but not unstimulated retinal pigment epithelial cells. On immunoblots of lysates of stimulated murine retinal pigment epithelial cells, rabbit antiserum to iNOS recognized a 130-kDa protein which comigrated with the inducible nitric oxide synthase of macrophages and which was not detectable in lysates of unstimulated retinal pigment epithelial cells nor in lysates of cells treated with only interferon-γ or lipopolysaccharide alone. Nitrite, a stable endproduct of NO formation by cells, was detectable in the culture supernatants after 18-24 hr of exposure to interferon-γ and lipopolysaccharide, and continued to accumulate in a linear fashion for at least 96 hr. Treatment of cultured retinal pigment epithelium with interferon-γ, lipopolysaccharide and either basic fibroblast growth factor or epidermal growth factor as third signals augmented inducible nitric oxide synthase expression as evidenced by intensified signals on immunoblots, enhanced accumulation of nitrite and increased iNOS enzyme activity. Conversely, when transforming growth factor-β was present in the culture medium, γ-interferon-LPS-induced expression of nitric oxide synthase and NO release were reduced. We conclude that interferon-γ synergizes with lipopolysaccharide to induce synthesis of inducible nitric oxide synthase and production of nitric oxide by murine retinal pigment epithelium and that this induction can be modulated by basic fibroblast growth factor, epidermal growth factor or transforming growth factor-β.

Original languageEnglish
Pages (from-to)129-139
Number of pages11
JournalExperimental Eye Research
Issue number2
StatePublished - Aug 1994
Externally publishedYes


  • Basic fibroblast growth factor
  • Epidermal growth factor
  • Inducible nitric oxide synthase
  • Interferon-gamma
  • Lipopolysaccharide
  • Nitric oxide
  • Retinal pigment epithelium
  • Transforming growth factor-beta


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