JOURNAL OF SURGICAL RESEARCH 48, 349-353 (1990) Kupffer CekHepatocyte Cocultures Release Nitric Oxide in Response to Bacterial Endotoxin’ T. R. BILLIAR, M.D., R. D. CURRAN, M.D., F. K. FERRARI, D. L. WILLIAMS, AND R. L. SIMMONS, M.D. Department of Surgery, 497 Scaife Hall, University of Pittsburgh, Pittsburgh, Pennsylmnia 15261 Presented at the Annual Meeting of the Association for Academic Surgery, Louisville, Kentucky, November 15-18, 1989 Nitric oxide (NO’) is a short-lived intermediate in a biochemical pathway where L-arginine is converted to L-citrulline and nitrite/nitrate (NOJNO,). 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 NOJNO, 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 metab- olism 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 re- ported time course for NOJNOi synthesis and the de- crease in protein synthesis, supporting the hypothesis that NO’ is the reactive nitrogen intermediate of the pathway responsible for this inhibition of protein syn- thesis. Finally, we show that KC:HC cocultures release more NO’ than KC alone in response to LPS, and we pro- pose that the combination of KC and HC acts as a func- tional unit capable of generating large amounts of NO from L-arginine in gram-negative sepsis. 0 1990 Academic Press, Inc. INTRODUCTION Nitric oxide (NO’) is a highly reactive molecule with a number of important biologic actions [ 11. By activating soluble guanylate cyclase, NO’ acts as an intracellular and intercellular messenger. It is through this mechanism that endothelial cell NO’ mediates relaxation of adjacent vas- cular smooth muscle cells [2,3] and inhibition of platelet aggregation [4] and adherence [5]. Also through this 1 Supported by National Institutes of Health Grants GM 37753 and AI 16869. mechanism, neutrophil NO’ may participate in cell che- motaxis [6], and NO’ from cerebellar neurons may play a role in signal transduction within the central nervous system [7]. NO’ can also inactivate a number of mito- chondrial enzymes [8, 91 and it is believed that it is through this mechanism that macrophage NO’ may inhibit the growth of some tumor cells [8-lo] and certain micro- organisms [ll]. NO’ is actually a short-lived intermediate in a bio- chemical pathway where L-arginine is converted to L-cit- rulline in a process during which one of the guanido ni- trogens of L-arginine is oxidized to NO’ (Fig. 1). In the presence of Hz0 and 02, NO’ is converted within seconds to the inactive end products nitrite (NO,) and nitrate (NO,). Stuehr and Marletta [la] were the first to show that a specific mammalian cell type, macrophages, had the capacity to synthesize NO, if exposed to lipopolysac- charide (LPS) and interferon-y (IFN7). Hibbs et al. [lo, 131 and Iyengar et al. [ 141 went on to show that L-arginine was the substrate for macrophage NO, and NO; synthesis before it was known that NO’ was the biologically active intermediate of the pathway. It has since been shown that all of the cell types mentioned above actually synthesize NO’ from L-arginine [2, 3, 7, 15-191. We have shown in a Kupffer cell (KC):hepatocyte (HC) coculture model that these two cell types when placed together have the capacity to metabolize large quantities of L-arginine to citrulline and NO,/NO, [20, 211 if ex- posed to LPS. The net effect of this L-arginine metabolism is a profound decrease in protein synthesis with almost no cell death under standard culture conditions [22]. We have also shown that if cultured alone, KC but not HC produce moderate levels of L-arginine metabolites in re- sponse to LPS. In other studies, we have demonstrated that HC do produce nitrogen oxides if exposed to condi- tioned medium from KC [23]. Even though we had dem- onstrated production of the stable end products, NO,/NO, , by liver cells in response to LPS, we had not measured release of authentic NO’ by KC:HC cocultures or KC. In these studies, experiments were performed to determine if NO’ was released by these cell types using a recently described method [15] which detects authentic NO’ in the culture supernatant. 349 002%4804/90 $1.50 Copyright 0 1990 by Academic Press, Inc. All rights of reproduction in any form reserved.