Nitric oxide production in activated macrophages from channel catfish (Ictalurus punctatus):: influence of dietary arginine and culture media

Recent studies have demonstrated that fish macrophages produce NO which plays a significant role in macrophage killing of microorganisms. The sole precursor for NO is the amino acid arginine. To elucidate the mechanisms which regulate NO production in these cells we examined the effects of increasing levels of dietary arginine on the induction of NO synthesis in macrophages from channel catfish. Additionally, the effects of four different media compositions in 96-h cell incubations were evaluated. A 9-week feeding trial was conducted to ascertain the effects of different dietary arginine levels on NO production in peritoneal macrophages. Diets were formulated to contain 24% crude protein from casein and gelatin, and supplemented with crystalline L-amino acids to meet the amino acid requirements of channel catfish. Four isocaloric dietary treatments provided arginine at either 0.52, 1.00, 1.50 or 2.00% of diet. The total amount of amino acid nitrogen was maintained equal by replacing arginine with either glutamate or glycine. Diets were fed to apparent satiation to triplicate groups of channel catfish initially averaging 11.4 g/fish. At the end of the trial, peritoneal macrophages were elicited by injecting 1 mg lipopolysaccharide (LPS)/kg body weight. Macrophages were collected, evaluated for viability and incubated in 24-well cell culture plates for 96 h, using Krebs-Henseleit buffer with 0.5% bovine serum albumin as standard media. Arginine, citrulline, arginine plus glutamine or arginine plus N-G-monomethy-L-arginine (N(G)MMA) were added to the culture medium to achieve a 0.5-mM concentration. Nitrate and nitrite production were measured spectrophotometrically as indicators of NO generation. Results indicate that cultured catfish macrophages synthesize NO in response to bacterial LPS at different rates depending upon composition of culture media. NO production was increased when arginine, citrulline and arginine plus glutamine were used as substrates; arginine plus glutamine had the most significant increase (5-fold). Macrophage NO generation was inhibited by N(G)MMA, suggesting an arginine-dependent metabolic pathway for production of NO in channel catfish. Dietary arginine levels did not correlate significantly with the amount of NO produced. In vivo, however, plasma arginine may partially regulate the intracellular availability of arginine and thus prolong the production of NO by macrophages. This would become physiologically relevant as disease-challenged fish deplete arginine from blood. Further research to assess the effects of dietary arginine on channel catfish immunocompetence in vivo appears warranted. (C) 1999 Elsevier Science B.V. All rights reserved.