Pinocytosis and Intracellular Degradation Protein: Modulation by Amino Acids of Exogenous

Intracellular degradation of exogenous (serum) proteins provides a source of amino acids for cellular protein synthesis. Pinocytosis serves as the mechanism for delivering exogenous protein to the lysosomes, the major site of intracellular degradation of exogenous protein. To determine whether the availability of extracellular free amino acids altered pinocytic function, we incubated monolayers of pulmonary alveolar macrophages with the fluid-phase marker, [14C]sucrose, and we dissected the pinocytic process by kinetic analysis. Additionally, intracellular degradation of endogenous and exogenous protein was monitored by measuring phenylalanine released from the cell monolayers in the presence of cycloheximide. Results revealed that in response to a subphysiological level of essential amino acids or to amino acid deprivation, (a) the rate of fluid-phase pinocytosis increased in such a manner as to preferentially increase both delivery to and size of an intracellular compartment believed to be the lysosomes, (b) the degradation of exogenously supplied albumin increased, and (c) the fraction of phenylalanine derived from degradation of exogenous albumin and reutilized for de novo protein synthesis increased. Thus, modulation of the pinosome-lysosome pathway may represent a homeostatic mechanism sensitive to the availability of extracellular free amino acids. There are four sources of essential amino acids available for cellular protein synthesis: intraceUular free amino acids, amino acids derived from degradation of cellular protein, extracellular free amino adds, and amino acids derived from degradation of extracellular protein. Work in our laboratories (1, 18, 27, 29) and those of others (see references 28 and 34 for review) has demonstrated the intracellular compartmentation of amino acids from these sources. Historically, degradation of serum protein has not been considered a significant source of amino acids for de novo protein synthesis, based almost solely on the work of Eagle and Piez (11). However, new, though indirect, experimental evidence strongly suggests that alveolar macrophages can use exogenous (serum) proteins, specifically albumin, as a source of amino acids for protein synthesis in the presence of physiological (27) and subphysiological (17) levels of extracellular free amino acids. It is also known that serum proteins are internalized by both fluid-phase and adsorptive pinocytosis (12, 13, 32) and are degraded in the lysosomes (10, 12, 14, 25). Therefore, since pinocytosed serum proteins might serve as an alternative source of amino acids (to free extracellular amino adds), we hypothesized that: (a) the rate of pinocytosis might be regulated by the availability of extracellular free amino acids, and (b) under conditions of amino acid deprivation, intraceUular degradation of exogenous protein would increase. We tested these hypotheses using monolayers of alveolar macrophages and employing a kinetic analysis that we have described recently (4). MATERIALS AND METHODS Cells: Pulmonary alveolar macrophages were lavaged from lungs of male rabbits (or guinea pigs) with calciumand magnesium-free phosphate-buffered saline (PBS) as described previously (22). After being washed and counted, the ccUs were suspended in Dulbecco's PBS supplemented with 5.6 mM glucose, pH 7.4 (PBS~), plated at 2-4 x 106 macrophages per 35-ram Falcon tissue culture dish (Falcon Labware, Oxnard, CA), and allowed to form adherent monolayers for 1 h at 37°C before experiments were begun. The medium used for all experiments was Dulbccco's PBSc supplemented with amino acids and vitamins, as specified. HeLa cells were grown in Eagle's minimal essential medium (MEM) suppleI-HE ]OURNAL OF CELL BIOLOGY • VOLUME 96 JUNE 1983 1586-1591 1586 © The Rockefeller University Press • 0021-9525/83/06/1586/06 $1.00 on Jne 6, 2017 D ow nladed fom Published June 1, 1983