Kupffer cell: Hepatocyte cocultures release nitric oxide in response to bacterial endotoxin

Nitric oxide (NO{radical dot}) is a short-lived intermediate in a biochemical pathway where l-arginine is converted to l-citrulline and nitrite/nitrate (NO₂^-/NO₃^-). This highly reactive molecule is the biologically active component of this inducible pathway in macrophages. Using a rat Kupffer cell:hepatocyte (KC:HC) coculture model, we have previously shown that this combination of cells produces large quantities of both citrulline and NO₂^-/NO₃^-) if exposed to lipopolysaccharides (LPS) but we did not determine whether nitric oxide was produced or released. We had also shown that this l-arginine metabolism was associated with a profound decrease in total protein synthesis. In these experiments, we show that KC:HC cocultures release nitric oxide into the culture supernatant if exposed to LPS. NO{radical dot} production by these cells requires l-arginine and is inhibited by N^G-monomethyl-l-arginine. In addition, the time course for NO{radical dot} release by KC:HC cocultures parallels the previously reported time course for NO₂^-/NO₃^- synthesis and the decrease in protein synthesis, supporting the hypothesis that NO{radical dot} is the reactive nitrogen intermediate of the pathway responsible for this inhibition of protein synthesis. Finally, we show that KC:HC cocultures release more NO{radical dot} than KC alone in response to LPS, and we propose that the combination of KC and HC acts as a functional unit capable of generating large amounts of NO{radical dot} from l-arginine in gram-negative sepsis.