The utilization of dipeptides containing L-arginine by chicken macrophages

Abstract

L-Arginine is the precursor of NO, a cytotoxic agent of macrophages. Studies were carried out to determine whether dipeptides containing arginine can be utilized by lipopolysaccharide (LPS)-activated avian macrophages for NO production. A chicken macrophage cell line, the HD11 cell, was used in all experiments. Peptidase activities were observed in fetal bovine serum (FBS) and macrophage serum free medium (Mac-SFM). Therefore, the utilization of dipeptides by macrophages was examined using Dulbecco's modified Eagle medium (D-MEM), a chemically defined medium, in short-term culture without FBS. Nitrite accumulation in the culture medium was used as the indicator of NO production. At concentrations of 0.15 mM in the culture media, L-leucinyl-L-arginine was 89% as effective as L-arginine in providing substrate for NO production. L-Argininyl-L-leucine was 38% as effective as L-arginine. The effectiveness increased to 93 and 58%, respectively, when the concentrations of dipeptides and arginine were 1.0 mM. Both values were slightly higher in a second experiment (97 and 70%, respectively). L-Lysine (10 mM) inhibited nitrite formation from all three sources of L-arginine. In studies of initial rates of transport by HD11 cells in Hanks Balanced Salts solution (HBSS), both L-argininyl-L-leucine and L-leucinyl-L-arginine inhibited arginine uptake. As lysine and arginine share a common transporter for cationic amino acids and are known to compete for transport, these studies suggest that the peptides were hydrolyzed extracellularly, yielding arginine that was transported into the cell where it served as a substrate for NO synthesis.