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

Nitric oxide (NO.) 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. production by these cells requires L-arginine and is inhibited by NG-mono-methyl-L-arginine. In addition, the time course for NO. 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. 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. 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. from L-arginine in gram-negative sepsis.