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 degrees C, but equilibrium could not be achieved at temperatures where fNle-Leu-Phe stimulated superoxide anion production is observed (e.g., 21-23 degrees 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 O2- 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 (Km = 3.56 X 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 Km for the hydrolysis of fNle-Leu-[3H]Phe of 2.6 X 10(-7) M which is similar to the Kd (1.5 X 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.
Basilion
J P
JP
Stickle
D F
DF
Holian
A
A
D000596:Amino Acids / Q000378:metabolism*; D000818:Animals; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D066298:In Vitro Techniques; D007700:Kinetics; D008264:Macrophages / Q000378:metabolism*; D008297:Male; D009504:Neutrophils / Q000378:metabolism; D009842:Oligopeptides / Q000378:metabolism*; D010649:Phenylalanine / Q000378:metabolism / Q000494:pharmacology; D011650:Pulmonary Alveoli / Q000378:metabolism; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic / Q000502:physiology; D013481:Superoxides / Q000378:metabolism; D013696:Temperature; D011503:Transglutaminases / Q000032:analysis; D014316:Tritium
D000596:Amino Acids; D009842:Oligopeptides; D044042:Receptors, Formyl Peptide; D011971:Receptors, Immunologic; D014316:Tritium; D013481:Superoxides; D010649:Phenylalanine; C024998:N-formylnorleucyl-leucyl-4-phenylalanine; D011503:Transglutaminases