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 pla ys a significant role in macrophage killing of microorganisms. The sole pre cursor for NO is the amino acid arginine. To elucidate the mechanisms which regulate NO production in these cells we examined the effects of increasin g levels of dietary arginine on the induction of NO synthesis in macrophage s from channel catfish. Additionally, the effects of four different media c ompositions in 96-h cell incubations were evaluated. A 9-week feeding trial was conducted to ascertain the effects of different dietary arginine level s on NO production in peritoneal macrophages. Diets were formulated to cont ain 24% crude protein from casein and gelatin, and supplemented with crysta lline 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 maintai ned equal by replacing arginine with either glutamate or glycine. Diets wer e fed to apparent satiation to triplicate groups of channel catfish initial ly averaging 11.4 g/fish. At the end of the trial, peritoneal macrophages w ere elicited by injecting 1 mg lipopolysaccharide (LPS)/kg body weight. Mac rophages were collected, evaluated for viability and incubated in 24-well c ell culture plates for 96 h, using Krebs-Henseleit buffer with 0.5% bovine serum albumin as standard media. Arginine, citrulline, arginine plus glutam ine or arginine plus N-G-monomethy-L-arginine (N(G)MMA) were added to the c ulture medium to achieve a 0.5-mM concentration. Nitrate and nitrite produc tion were measured spectrophotometrically as indicators of NO generation. R esults 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 p lus 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 o f NO in channel catfish. Dietary arginine levels did not correlate signific antly with the amount of NO produced. In vivo, however, plasma arginine may partially regulate the intracellular availability of arginine and thus pro long the production of NO by macrophages. This would become physiologically relevant as disease-challenged fish deplete arginine from blood. Further r esearch to assess the effects of dietary arginine on channel catfish immuno competence in vivo appears warranted. (C) 1999 Elsevier Science B.V. All ri ghts reserved.