Membrane transport by guinea pig peritoneal exudate leukocytes: effect of phagocytosis on hexose and amino acid transport

Short term, carrier mediated transport of D-glucose, L-leucine and L-lysine by guinea pig peritoneal macrophages was characterized. Analysis of the amino acid transport demonstrated two-limbed double reciprocal plots suggesting two transport systems for each amino acid. The low concentration limb of the curves established a Km of 0.1 mM for L-leucine and 0.05 mM for L-lysine; Vmax values were 2.0 and 2.85 nmole/mg protein/90 seconds, respectively. Leucine and lysine were shown to be competitive inhibitors of each other. Further competition studies revealed that other amino acids also had affinity for these carriers. Amino acid transport was found to be sensitive to sulfhydryl active compounds. Colchicine treatment of peritoneal macrophages did not inhibit the transport of the amino acids tested. Preloading macrophages with latex beads or heat-killed staphylococci by phagocytosis stimulated 2-deoxy-D-glucose (2-dOG) uptake markedly, but had no measurable effect on amino acid transport. Although total transport of 2-dOG increased in post-phagocytic macrophages, the kinetics of the system were not altered significantly. The Km for both pre- and post-phagocytic transport of 2-dOG was shown to be 1.2 mM and the Vmax was shown to increase from a pre-phagocytic value of 20 nmoles/mg protein/90 seconds to a post-phagocytic 27 nmoles/mg protein/90 seconds. Phagocytosis of heat-killed staphylococci by guinea pig polymorphonuclear leukocytes (PMNs), however, did not cause an augmentation in hexose transport in the cells. The presence of colchicine during phagocytosis did not alter subsequent uptake of amino acids by the macrophages.     

Chlamydia species-dependent differences in the growth requirement for lysosomes

Genome reduction is a hallmark of obligate intracellular pathogens such as Chlamydia, where adaptation to intracellular growth has resulted in the elimination of genes encoding biosynthetic enzymes. Accordingly, chlamydiae rely heavily on the host cell for nutrients yet their specific source is unclear. Interestingly, chlamydiae grow within a pathogen-defined vacuole that is in close apposition to lysosomes. Metabolically-labeled uninfected host cell proteins were provided as an exogenous nutrient source to chlamydiae-infected cells, and uptake and subsequent labeling of chlamydiae suggested lysosomal degradation as a source of amino acids for the pathogen. Indeed, Bafilomycin A1 (BafA1), an inhibitor of the vacuolar H(+)/ATPase that blocks lysosomal acidification and functions, impairs the growth of C. trachomatis and C. pneumoniae, and these effects are especially profound in C. pneumoniae. BafA1 induced the marked accumulation of material within the lysosomal lumen, which was due to the inhibition of proteolytic activities, and this response inhibits chlamydiae rather than changes in lysosomal acidification per se, as cathepsin inhibitors also inhibit the growth of chlamydiae. Finally, the addition of cycloheximide, an inhibitor of eukaryotic protein synthesis, compromises the ability of lysosomal inhibitors to block chlamydial growth, suggesting chlamydiae directly access free amino acids in the host cytosol as a preferred source of these nutrients. Thus, chlamydiae co-opt the functions of lysosomes to acquire essential amino acids.     

Nanoscale amphiphilic macromolecules as lipoprotein inhibitors: the role of charge and architecture

A series of novel amphiphilic macromolecules composed of alkyl chains as the hydrophobic block and poly(ethylene glycol) as the hydrophilic block were designed to inhibit highly oxidized low density lipoprotein (hoxLDL) uptake by synthesizing macromolecules with negatively charged moieties (ie, carboxylic acids) located in the two different blocks. The macromolecules have molecular weights around 5,500 g/mol, form micelles in aqueous solution with an average size of 20-35 nm, and display critical micelle concentration values as low as 10(-7) M. Their charge densities and hydrodynamic size in physiological buffer solutions correlated with the hydrophobic/ hydrophilic block location and quantity of the carboxylate groups. Generally, carboxylate groups located in the hydrophobic block destabilize micelle formation more than carboxylate groups in the hydrophilic block. Although all amphiphilic macromolecules inhibited unregulated uptake of hoxLDL by macrophages, inhibition efficiency was influenced by the quantity and location of the negatively charged-carboxylate on the macromolecules. Notably, negative charge is not the sole factor in reducing hoxLDL uptake. The combination of smaller size, micellar stability and charge density is critical for inhibiting hoxLDL uptake by macrophages.     

Amino acid transport and metabolism in nitrogen-starved cells of Saccharomyces cerevisiae.

Nitrogen-starved yeast derepress a general amino acid permease which transports basic and hydrophobic amino acids. Although both groups of amino acids are metabolized, the derivatives of the basic amino acids are retained by the cells, whereas those of the hydrophobic amino acids are released as acidic and neutral deaminated derivatives. The release of the deaminated derivatives of the hydrophobic amino acids only occurs in the presence of glucose, which presumably produces amino acceptors. The accumulation of intracellular amino acids results in trans-inhibition of the uptake of exogenous amino acids whether the intracellular amino acid is a basic amino acid or the product of intracellular transamination from a hydrophobic amino acid. Variation of permease and transaminase activity was measured during growth under repressed (ammonia-grown) and derepressed (proline-grown) conditions. Maximum levels for both activities occurs at the mid-exponential phase.     

Beryllium uptake and related biological effects studied in THP-1 differentiated macrophages

Investigation of cellular uptake of metal compounds is important in understanding metal-related toxicity and diseases. Inhalation of beryllium aerosols can cause chronic beryllium disease, a progressive, granulomatous fibrosis of the lung. Studies in laboratory animals and cultured animal cells indicate that alveolar macrophages take up beryllium compounds and participate in a hypersensitivity immune response to a beryllium-containing antigen. In the present work, human monocyte cell line THP-1 was induced with phorbol myristate acetate to differentiate into a macrophage. This cell with characteristics of human alveolar macrophages was employed to study cellular beryllium uptake and related biological effects. Morphological changes, phagocytosis of fluorescent latex beads, and cell surface CD14 expression were used to verify the successful differentiation of THP-1 monocytes into macrophages. An improved mass spectrometry method for quantitative analysis of intracellular beryllium as opposed to the traditional radioisotopic approach was developed using ICP-MS. The influence of the solubility of beryllium compounds, exposure duration, and beryllium concentration on the incorporation of beryllium was studied. Our data indicated that the uptake of particulate BeO was much more significant than that of soluble BeSO(4), suggesting the major cellular uptake pathway is phagocytosis. Nevertheless, subsequent DAPI nuclear staining and PARP cleavage study indicated that beryllium uptake had a negligible effect on the apoptosis of THP-1 macrophages compared to the unstimulated macrophage control. Meanwhile, no substantial variation of tumour necrosis factor-alpha production was observed for THP-1 macrophages upon beryllium exposure. These data imply alveolar macrophages could have some level of tolerance to beryllium and this may explain why most Be-exposed individuals remain healthy throughout life.     

Azithromycin,a lysosomotropic antibiotic,has distinct effects on fluid-phase and receptor-mediated endocytosis,but does not impair phagocytosis in J774 macrophages

Pretreatment of J774 mouse macrophages by the dicationic macrolide antibiotic, azithromycin (AZ), selectively inhibited fluid-phase endocytosis of horseradish peroxidase and lucifer yellow, but not phagocytosis of latex beads. AZ delayed sequestration of receptor-bound transferrin and peroxidase-anti-peroxidase immune complexes into cell-surface endocytic pits and vesicles, but did not slow down the subsequent rate of receptor-mediated endocytosis. AZ down-regulated cell surface transferrin receptors, but not Fc gamma receptors, by causing a major delay in the accessibility of internalized transferrin receptors to the recycling route, without slowing down subsequent efflux, resulting in redistribution of the surface pool to an intracellular pool. Acidotropic accumulation of AZ was associated with an extensive vacuolation of late endosomes/lysosomes, and these compartments became inaccessible to horseradish peroxidase and immune complexes, but not to latex beads. The inhibitory profile of AZ cannot be solely accounted for by vacuolation and interference with acidification. AZ may help in dissecting various steps of the endocytic apparatus such as lateral mobility of receptors at the plasma membrane, formation of clathrin-independent endocytic vesicles, orientation of transferrin receptors into the recycling route, and fusogenicity with lysosomes.     

Effects of Microparticle Size and Fc Density on Macrophage Phagocytosis

Controlled induction of phagocytosis in macrophages offers the ability to therapeutically regulate the immune system as well as improve delivery of chemicals or biologicals for immune processing. Maximizing particle uptake by macrophages through Fc receptor-mediated phagocytosis could lead to new delivery mechanisms in drug or vaccine development. Fc ligand density and particle size were examined independently and in combination in order to optimize and tune the phagocytosis of opsonized microparticles. We show the internalization efficiency of small polystyrene particles (0.5 µm to 2 µm) is significantly affected by changes in Fc ligand density, while particles greater than 2 µm show little correlation between internalization and Fc density. We found that while macrophages can efficiently phagocytose a large number of smaller particles, the total volume of phagocytosed particles is maximized through the non-specific uptake of larger microparticles. Therefore, larger microparticles may be more efficient at delivering a greater therapeutic payload to macrophages, but smaller opsonized microparticles can deliver bio-active substances to a greater percentage of the macrophage population. This study is the first to treat as independent variables the physical and biological properties of Fc density and microparticle size that initiate macrophage phagocytosis. Defining the physical and biological parameters that affect phagocytosis efficiency will lead to improved methods of microparticle delivery to macrophages.     

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of 125I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronectin receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.     

Definition of fibronectin-mediated uptake of gelatinized latex by liver slices and macrophages

These studies show that both liver slices and macrophages carried out fibronectin concentration-dependent uptake of ¹²⁵I-labeled gelatin-coated latex (test latex). Lack of phagocytosis of test latex by liver slices was shown directly by electron microscopy and indirectly by trypsin treatment, which caused the release of all test latex taken up in response to fibronectin. Inhibitors of phagocytosis did not alter this uptake. On the other hand, trypsin released only a portion of test latex from macrophages. Inhibitors of phagocytosis did not effect the released radioactive particles from macrophages but greatly reduced the trypsin-resistant radioactivity, taken as representing phagocytized particles. Opsonization of test latex with fibronectin did not require heparin but its association with liver slices occurred only in the presence of heparin. Macrophages, however, readily bound and internalized the opsonized test latex and heparin only potentiated these reactions. Gelatin competed with test latex for fibronectin for opsonization, but did not inhibit binding and phagocytosis of fibronectin-test latex complexes. Finally, soluble fibronectin-gelatin complexes did not compete for binding and phagocytosis of fibronectin-test latex complexes. Thus, fibronectin concentrated on the surface of latex is preferred for interaction with the fibronection receptor of macrophages. Gelatin, however, was not essential for this reaction, because fibronectin directly coupled to latex was also readily taken up.     

Characteristics of taurine transport in rat liver lysosomes

Taurine (2-aminoethanesulfonic acid) is a unique sulfur amino acid derivative that has putative nutritional, osmoregulatory, and neuroregulatory roles and is highly concentrated within a variety of cells. The permeability of Percoll density gradient purified rat liver lysosomes to taurine was examined. Intralysosomal amino acid analysis showed trace levels of taurine compared to most other amino acids. Taurine uptake was Na(+)-independent, with an overshoot between 5-10 minutes. Trichloroacetic acid extraction studies and detergent lysis confirmed that free taurine accumulated in the lysosomal space. Kinetic studies revealed heterogeneous uptake with values for Km1 = 31 +/- 1.82 and Km2 greater than 198 +/- 10.2 mM. The uptake had a pH optimal of 6.5 and was stimulated by the potassium specific ionophore valinomycin. The exodus rate was fairly rapid, with a t1/2 of 5 minutes at 37 degrees C. Analog inhibition studies indicated substrate specificity similar to the plasma membrane beta-alanine carrier system, with inhibition by beta-alanine, hypotaurine, and taurine. alpha-Alanine, 2-methylaminoisobutyric acid (MeAIB), and threonine were poor inhibitors. No effects were observed with sucrose and the photoaffinity derivative of taurine NAP-taurine [N-(4-azido-2-nitrophenyl)-2-aminoethanesulfonate]. In summary, rat liver lysosomes possess a high Km system for taurine transport that is sensitive to changes in K+ gradient and perhaps valinomycin induced diffusional membrane potential. These features may enable lysosomes to adapt to changing intracellular concentrations of this osmotic regulatory substance.     

Langerhans cells: target cells in immune complex reactions.

Skin of normal, cobra venom extract-treated, and C₄-deficient guinea pigs was injected with ferritin-antiferritin or with peroxidase-antiperoxidase immune complexes. Skin and draining lymph nodes were studied to compare the phagocytosis of these immune complexes by Langerhans cells (LC) and by macrophages. When complement was present, immune complexes were damaging to LC, and uptake of the immune complexes, although present, was limited. When components of complement were absent or diminished, increased numbers of LC in lymph nodes were seen, but damage to LC was absent or decreased. However no detectable change in the amount of phagocytosis by LC was noted. Since some LC can carry antigen from skin to lymph nodes and may be involved in the presentation of antigen to lymphoid cells in some cell-mediated immune responses, impairment or abolition of LC function by immune complexes could represent a mechanism through which the local presence of antibodies might interfere with the induction and elicitation of cellular immunity by antigen. Moreover, damage to LC and subsequent release of intracellular (lysosomal?) substances may constitute a general mechanism of response in the skin to injury and may be an integral part of inflammatory and allergic skin reactions.     

Cholesterol-rich membrane rafts and Lyn are involved in phagocytosis during Pseudomonas aeruginosa infection

The mechanism of phagocytosis of pathogens remains to be fully characterized. We report a novel phagocytosis pathway for Pseudomonas aeruginosa, which is initiated by cholesterol-rich membrane rafts and is dependent on Lyn, primarily an immune regulator with both positive and negative roles. Blocking of Lyn or blocking of cholesterol synthesis significantly inhibited phagocytosis by alveolar macrophages. We found that Lyn, via Src homology 2 and 3 domains, bound to and then activated PI3K and Akt to regulate intracellular routing of the engulfed P. aeruginosa. Further analysis indicates that Lyn and raft components entered in phagosomes and late lysosomes. Finally, respiratory burst was dependent on Lyn and membrane rafts, as confirmed by small interfering RNA and dominant-negative strategies. Our investigations demonstrate that Lyn along with membrane rafts plays a fundamental role in phagocytosis by alveolar macrophages during infection.     

Characterization of the binding site on thioglycolate-stimulated mouse peritoneal macrophages that mediates the uptake of very low density lipoproteins

Isolated mouse peritoneal macrophages that had been stimulated with thioglycolate were shown to take up and degrade normal human 125I-very low density lipoproteins (VLDL). Uptake occurred via a specific cell surface receptor which was shown to be 1) temperature-dependent, 2) calcium-dependent, and 3) susceptible to proteolytic digestion. The receptor-mediated uptake and degradation of VLDL markedly stimulated the synthesis and accumulation of triglyceride and cholesteryl ester within macrophages. The degradation of the protein and lipid portions of VLDL occurred within lysosomes. Competition studies showed that the binding site for VLDL was different from the receptor for normal low density lipoproteins or for acetylated low density lipoproteins but that there was cross competition with beta-VLDL. In addition, positive charges appeared to play an important role in the recognition of VLDL by their receptors since polyamines were able to markedly inhibit VLDL binding, degradation, and lipid accumulation while negatively charged compounds were without effects. These studies indicate that 1) stimulated mouse peritoneal macrophages possess specific receptors which recognize normal human VLDL and 2) the receptor-mediated uptake of VLDL results in the accumulation of triglyceride and cholesteryl ester within macrophages.     

Different effects of peptides and their amino acids on antibody production and phagocytic activity of the neutrophils in mice

The effects of some natural polypeptides fragments and amino acids which were incorporated in them on the thymus-dependent SRBC immune response and on the phagocytosis of Staphylococcus by murine peritoneal neutrophils were compared. It was found that the peptides LGIP and PYIK which consisted of those amino acids that stimulated phagocytosis but did influence the immune response (K, P, Y, L) did not change the immune response as well as phagocytosis. The peptides TKPR, TTKD and LGIPE which included those amino acids that were able to stimulate both immune response and phagocytosis (T, E, D) enhanced both activities. Nevertheless the peptide PYIKV containing valine (that stimulated the antibody production as well as phagocytosis) did not effect the immune response but enhanced the phagocytic index. The existence of two immunoregulatory systems--the peptide system and the amino acid one--was suggested.     

Effect of antilysosomal sera on the phagocytosis of corpuscular antigens in a macrophage culture]

A study was made of the effect of the antilysosomal sera on various phases of phagocytosis Macrophages fluorochromized with acridine orange served as a model for the assessment of the effect of the antilysosomal sera on the phagocytosis: the fluorescent-microscopic method permitted to evaluate quantitatively the activity of the antilysosomal sera and to study the intracellular changes in the phagocytized antigen associated with its interaction with the lysosomes. The data obtained showed the antisera to the enzymes and the lysosome membranes to inhibit the phagocytosis both in the presence of a complement and without it. It was also demonstrated that the antilysosomal sera influenced the activity of the acid phosphatase and its distribution in the macrophages.     

In vitro activation of tumoricidal properties in mouse macrophages using the chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) incorporated in liposomes

Summary We investigated the ability of free or liposome-incorporated synthetic chemotactic peptide N-formyl-methionyl-leucyl-phenylalanine (FMLP) to generate tumoricidal properties in mouse macrophages. As FMLP contains three hydrophobic amino acid residues, it can readily be incorporated into multilamellar vesicles (MLV) consisting of phosphatidylcholine (PC) and phosphatidylserine (PS). The incorporation of FMLP into MLV with a PC:PS ratio of 7:3 mol at FMLP concentrations of up to 10^–4 M did not affect the phagocytosis of liposomes by mouse peritoneal macrophages. Studies with radioactive FMLP revealed that higher levels of FMLP can be delivered to macrophages by liposomes than in the free, nonencapsulated form. Treatment of mouse macrophages with liposome-encapsulated FMLP, but not with free FMLP, generated tumoricidal properties in the macrophages. The mechanism appears to involve an intracellular site since 100-fold concentrations of free FMLP or free N-acetyl-methionyl-leucyl-phenylalanine, the FMLP agonist, failed to competitively inhibit the macrophage's tumoricidal properties generated by liposome-encapsulated FMLP.     

Surfactant protein A binds to IgG and enhances phagocytosis of IgG-opsonized erythrocytes

Surfactant protein (SP)-A and SP-D, immunoglobulins, and complement all modulate inflammation within the lung by regulating pathogen clearance. For example, SP-A binds to and opsonizes a variety of bacteria and viruses, thereby enhancing their phagocytosis by innate immune cells such as alveolar macrophages. Immunoglobulins, which bind to antigen and facilitate Fc receptor-mediated phagocytosis, can also activate complement, a family of soluble proteins with multiple host defense functions. Previous studies showed that SP-A and complement protein C1q interact. Since complement protein C1q binds to IgG and IgM immune complexes, the hypothesis tested in this study was that SP-A, which is structurally homologous to C1q, also binds to IgG and affects its functions. SP-A binds to the Fc, rather than the Fab, region of IgG. Binding is calcium dependent but not inhibited by saccharides known to bind to SP-A's carbohydrate recognition domain. The binding of SP-A does not inhibit the formation of immune complexes or the binding of IgG to C1q. In contrast, SP-A enhances the uptake of IgG-coated erythrocytes, suggesting that SP-A might be influencing Fc receptor-mediated uptake. In summary, this study shows a novel interaction between SP-A and IgG and a functional consequence of the binding.     

Inhibitors of the 5-lipoxygenase arachidonic acid pathway induce ATP release and ATP-dependent organic cation transport in macrophages

We have previously described that arachidonic acid (AA)-5-lipoxygenase (5-LO) metabolism inhibitors such as NDGA and MK886, inhibit cell death by apoptosis, but not by necrosis, induced by extracellular ATP (ATPe) binding to P2X7 receptors in macrophages. ATPe binding to P2X7 also induces large cationic and anionic organic molecules uptake in these cells, a process that involves at least two distinct transport mechanisms: one for cations and another for anions. Here we show that inhibitors of the AA-5-LO pathway do not inhibit P2X7 receptors, as judged by the maintenance of the ATPe-induced uptake of fluorescent anionic dyes. In addition, we describe two new transport phenomena induced by these inhibitors in macrophages: a cation-selective uptake of fluorescent dyes and the release of ATP. The cation uptake requires secreted ATPe, but, differently from the P2X7/ATPe-induced phenomena, it is also present in macrophages derived from mice deficient in the P2X7 gene. Inhibitors of phospholipase A2 and of the AA-cyclooxygenase pathway did not induce the cation uptake. The uptake of non-organic cations was investigated by measuring the free intracellular Ca^2 + concentration ([Ca^2 +]_i) by Fura-2 fluorescence. NDGA, but not MK886, induced an increase in [Ca^2 +]_i. Chelating Ca^2 + ions in the extracellular medium suppressed the intracellular Ca^2 + signal without interfering in the uptake of cationic dyes. We conclude that inhibitors of the AA-5-LO pathway do not block P2X7 receptors, trigger the release of ATP, and induce an ATP-dependent uptake of organic cations by a Ca^2 +- and P2X7-independent transport mechanism in macrophages.     

Influenza virus-induced immune complexes suppress alveolar macrophage phagocytosis

Immune complexes in the lungs are capable of inducing adverse responses. Herein we have detailed the formation of immune complexes in the lungs of influenza virus-infected mice and examined their effect on alveolar macrophage defenses. On days 3, 7, 10, 15, and 30 after aerosol infection with influenza A/PR8/34 virus, the acellular pulmonary lavage fluid was tested for viral antigen, specific viral antibody, and immune complexes by immunoassays. Whereas peak viral antigen (day 3) diminished to undetectable levels by day 10, specific viral antibody remained at a low concentration until day 10, after which it rapidly increased. Immune complex concentrations increased through day 7, peaked at day 10, and gradually returned to the control level by day 30. These data demonstrate that immune complexes of detectable size are induced by influenza virus infection during the interface between antigen excess and antibody excess conditions. Since alveolar macrophages are the pivotal phagocytic defense cells in the lung, the ability of normal alveolar macrophages to ingest opsonized erythrocytes was quantitated in the presence of immune complexes from lavage fluid. Immune complexes from day 10 virus-infected lungs caused a dose-dependent suppression of antibody-mediated phagocytosis to 30% of control values. In contrast, although these immune complexes also markedly decreased the phagocytosis of antibody-coated yeast cells, they did not significantly impair the antibody-independent ingestion of unopsonized yeast cells by macrophages. the suppressive effects of immune complexes on alveolar macrophages may, in part, explain the phagocytic dysfunction that occurs 7 to 10 days after influenza virus pneumonia.     

Pinocytosis and intracellular degradation of exogenous protein: modulation by amino acids

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.