Extracellular hydrolysis of formyl peptides and subsequent uptake of liberated amino acids by alveolar macrophages

The mechanism of accumulation of radioactive label from fNle-Leu-[³H]Phe by guinea pig alveolar macrophages was investigated. The binding of fNle-Leu-[³H]Phe to macrophages reached equilibrium within 5 min at 4°C, but equilibrium could not be achieved at temperatures where fNle-Leu-Phe stimulated superoxide anion production is observed (e.g., 21-23°C). At this temperature a rapid phase of initial binding of fNle-Leu-[³H]Phe to its receptor was followed by continued accumulation of cell-associated radioactivity which was linear and was dependent on the extracellular pH, i.e., the rate increased as the pH was lowered from pH 8 to pH 6. Examination for possible intracellular hydrolysis of fNle-Leu-[³H]Phe revealed the presence of extensive amounts of [³H]phenylalanine, both cell-associated and in the medium. The increases in cell-associated [³H]phenylalanine correlated in time and pH with cell-associated radioactivity that was accumulated after stimulation with fNle-Leu-[³H]Phe. The addition of 1 mM unlabelled phenylalanine blocked the long term accumulation of label from fNle-Leu-[³H]Phe by macrophages. 1 mM phenylalanine had no measureable effect on fNle-Leu-Phe stimulated O^-₂ production, fNle-Leu-[³H]Phe hydrolysis or on fNle-Leu-[³H]Phe binding to its receptor. These results indicated that the long term accumulation of radioactivity by alveolar macrophages was due to extracellular hydrolysis of fNle-Leu-[³H]Phe followed by transport of liberated [³H]phenylalanine into the cells. A high affinity (K_m = 3.56·10^-8 M) transport system for phenylalanine was measured in alveolar macrophages, which was not stimulated by the addition of fNle-Leu-Phe. The extracellular hydrolysis of fNle-Leu-[³H]Phe could not be attributed to release of macrophage enzymes into the medium. The responsible proteinase appears to be membrane bound and has a K_m for the hydrolysis of fNle-Leu-[³H]Phe of 2.6·10^-7 M which is similar to the K_d (1.5·10^-7 M) for fNle-Leu-Phe binding. Taken together, these data suggest that for the alveolar macrophage: (1) formyl peptides are not internalized by a receptor-mediated process; (2) a surface proteinase can catalyze the hydrolysis of formyl peptides; and (3) [³H]phenylalanine formed by fNle-Leu-[³H]Phe hydrolysis is transported into the interior of the macrophage.