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PubMed1991

Evidence that desferrioxamine cannot enter cells by passive diffusion.

Accumulation of [14C]desferrioxamine by rat visceral yolk sac in vitro has been compared with that of [14C]sucrose, a probe for fluid-phase pinocytosis. Kinetic parameters for both substrates are closely similar, as are the effects of inhibitors. It is concluded from these data, and from theoretical considerations, that desferrioxamine cannot enter cells other than by pinocytosis and that, once internalized, it will remain in the lysosomes. The results indicate the need for a re-evaluation of the pharmacokinetic mechanisms traditionally accepted for the drug's ability to deplete iron from cells and tissues.

Animals↗
PubMed1987

The blocked pinocytic activity of mitotic cells is restored in mitotic-interphase hybrids.

During mitosis there is an abrupt inhibition of a wide range of membrane functions, including fluid-phase and adsorptive pinocytosis. We have used cell hybrids formed between mitotic and interphase cells to approach the mechanism of this inhibition. We report that fluid pinocytosis is reactivated in the mitotic partner of hybrids formed between mitotic and interphase Chinese hamster ovary (CHO) cells. It thus appears that the interphase cell provides some necessary element(s) for membrane activity during mitosis. This dominance of interphase membrane properties stands in contrast with earlier evidence that mitotic nuclear properties dominate in similar mitotic-interphase hybrids.

Animals↗
PubMed1990

Fluid-phase endocytosis by primary cultures of bovine brain microvessel endothelial cell monolayers.

Blood-brain barrier (BBB) fluid-phase endocytosis was examined in primary cultures of bovine brain microvessel endothelial cell (BMEC) monolayers. By fluorescence spectroscopy. Lucifer yellow (LY, a fluorescent, soluble molecule used as a marker for pinocytosis) accumulation by BMEC was observed to be linear over a concentration range of 0.05 to 1.0 mg/ml. Time-dependent uptake of LY exhibited curvilinear kinetics composed of an initially rapid uptake rate of 1338 ng of LY/mg protein per hour at 0.5 mg/ml LY. Within 20 min, the rate of LY accumulation slowed to a steady-state rate of 23 ng of LY/mg protein per hour. Accumulation of LY was inhibited in the presence of metabolic inhibitors, potassium cyanide or 2-deoxyglucose, and was decreased, but not completely inhibited, at 4 degrees. Pulse-chase experiments revealed that efflux of LY was very rapid with at least 80% of the accumulated LY being lost within 2 min and was not sensitive to low temperature. Only 3-5% of the LY initially accumulated by BMEC remained cell-associated after a 30-min chase. The calculated turnover of the endocytic compartment's total volume (per hour) is 8- to 20-fold less than values for fibroblasts and macrophages, respectively. We have interpreted these data to suggest that the efflux of most of the LY involves loss from a rapidly recycled compartment of finite volume, possibly caveolae, that had sequestered marker during accumulation and suggest that these results are consistent with the present understanding of BBB pinocytosis in vivo.

Animals↗
PubMed1984

Activation of macrophages by lymphokines from T-cell clones: evidence for different macrophage-activating factors.

The data reported in this paper demonstrate that macrophage-activating factors (MAFs) are a heterogeneous group of T-cell-derived lymphokines. Two long-term T-cell clones, Cl 96 and PK 7.1.2E8, were potent sources of MAFs (MAF96 and MAF7.1.2E8). These MAFs could be distinguished by differential activation of macrophages. Activation of resident murine macrophages with MAF7.1.2E8 enhanced RNA and glycoprotein synthesis, hexosemonophosphate shunt (HMPS) activity, release of oxygen metabolites (O-2 and H2O2), pinocytosis and tumor cytostasis, whereas no effect on schistosomula killing and tumor cytolysis could be observed. In contrast, MAF96 enhanced glycoprotein synthesis, HMPS activity, release of oxygen metabolites and prostaglandin E, schistosomula killing, and tumor cytostasis and cytolysis, while RNA synthesis and pinocytosis were decreased. These findings show that MAFs from both T-cell clones share some properties but markedly differ in others. In addition, the macrophage-activating properties of MAF96 but not of MAF7.1.2E8 could selectively be inhibited by a rabbit anti-lymphokine antiserum. This demonstrates a serological difference between MAF activities from both clones. Although at optimal concns both MAFs were active in the absence of lipopolysaccharide (LPS), the activity of suboptimal doses of MAF96 but not of MAF7.1.2E8 could be enhanced by LPS. These findings show that different MAFs from T-cell clones may be useful to clarify molecular mechanisms of macrophage activation.

Animals↗
PubMed1981

Pinocytic uptake and intracellular degradation of N-(2-hydroxypropyl)methacrylamide copolymers. A potential drug delivery system.

Synthetic 125I-labelled N-(2-hydroxypropyl)methacrylamide copolymers containing four different, potentially degradable peptidyl side chains were incubated with rat visceral yolk sacs cultured in vitro. All copolymers were captured by fluid-phase pinocytosis and three of the side chains were susceptible to lysosomal hydrolysis, resulting in release of [125I]iodotyrosine back into the culture medium. Uptake and degradation was completely inhibited by 2,4-dinitrophenol. The thiol-proteinase inhibitor leupeptin did not affect the rate of pinocytosis, but caused different degrees of inhibition of hydrolysis depending on side chain composition.

2,4-Dinitrophenol↗
PubMed1984

Tyrosinamide residues enhance pinocytic capture of N-(2-hydroxypropyl)methacrylamide copolymers.

N-(2-Hydroxypropyl)methacrylamide ( HPMA ) copolymers have been proposed as a potential lysosomotropic drug delivery system. HPMA copolymers bearing tyrosinamide residues, bound either directly to the polymer backbone or via a glycylglycine spacer, were radiolabelled with [125I]iodide and the effect of tyrosinamide content on their rate of pinocytic uptake by rat visceral yolk sacs cultured in vitro was measured. Incorporation of tyrosinamide enhanced uptake of the copolymer, most markedly at substitutions above 10 mol%. 2,4-Dinitrophenol, an inhibitor of pinocytosis, was used to confirm that tissue association of 125I-radiolabelled copolymer was due to pinocytic uptake. The side-chain -Gly-Gly-Tyr-NH2 was degraded following the internalization of copolymers containing this spacer and degradation was partially sensitive to the lysosomal thiol-proteinase inhibitor leupeptin. It is postulated that the effect of tyrosinamide residues is to increase the hydrophobicity of poly( HPMA ) and thus to increase its capacity for nonspecific adsorptive pinocytosis.

2,4-Dinitrophenol↗
PubMed1987

The axenic mutations and endocytosis in Dictyostelium.

The axenic mutations of Dictyostelium offer a unique opportunity to explore the relationship between phagocytosis and pinocytosis and to examine the mechanism by which a cell shifts from one mode of feeding to the other. The axenic mutations also provide a means of exploring the relationships between endocytosis and other forms of cell motility. This chapter has described the known mutations that affect axenic growth, methods for culturing wild-type and axenic cells and measuring their growth, and methods for monitoring the effects of the axenic mutations on endocytosis and cell movement. The importance has been emphasized of distinguishing effects of the axenic genotype that are expressed constitutively (i.e., during growth on either bacteria or liquid medium) from those that are a function of axenic growth conditions. The methods described in this chapter, applied to wild-type cells and to cells carrying a full complement of the axenic mutations, have shown that the axenic mutations have constitutive effects on cell-substratum interactions, and inducible effects on cell locomotion and pinocytosis.

Cell Movement↗
PubMed2001

Phenotypic and functional comparison of monocytes from cord blood and granulocyte colony-stimulating factor-mobilized apheresis products.

OBJECTIVE: It is well established that T cells are effector cells in graft-vs-host disease (GVHD), yet the contribution of graft monocytes is less well characterized. Therefore, monocytes in cord blood (CB) and granulocyte colony-stimulating factor-mobilized apheresis products (G-AP), two stem cell grafts associated with reduction of acute and chronic GVHD and relative reduction of acute GVHD, respectively, were compared phenotypically and functionally. MATERIALS AND METHODS: The frequencies, phenotypes, and pinocytosis activities of monocytes from CB and G-AP were determined by flow cytometry and their allostimulatory potential in a primary mixed leukocyte reaction. RESULTS: G-AP contained significantly more monocytes than CB (24.9% +/- 7.1% vs 8.8% +/- 1.5% CD14+ and 62.4 +/- 27.5 x 10(6) vs 0.9 +/- 0.2 x 10(6) CD14+ cells/mL). Monocytes from both sources revealed similar phenotypes. They expressed CD4, CD11a, CD11b, CD11c, CD18, CD32, CD33, CD45R0, CD48, CD50, CD54, CD58, CD64, CD86, CD102, CD116, CD123, and HLA-DR; showed no expression of CD1a and CD83; and weak expression of CD16, CD45RA, and CD80. The levels of CD80 and CD86 expression were comparable; however, in contrast to G-AP monocytes, CB monocytes lacked CD40. There was no difference in pinocytosis activity and allostimulatory capacity of CB and G-AP monocytes. CONCLUSIONS: Monocytes in CB and G-AP are phenotypically and functionally comparable. The only difference observed is the lack of CD40 on CB monocytes.

Antigen Presentation↗
PubMed2000

Rac is required for constitutive macropinocytosis by dendritic cells but does not control its downregulation.

BACKGROUND: Dendritic cells use constitutive macropinocytosis to capture exogenous antigens for presentation on MHC molecules. Upon exposure to inflammatory stimuli or bacterial products such as lipopolysaccharide (LPS), macropinocytosis is dramatically downregulated as part of a developmental programme leading to dendritic cell maturation, migration and activation of T cells. It is not known, however, how macropinocytosis is sustained in dendritic cells in the absence of exogenous stimuli, nor how it is downregulated upon maturation. We have tested the possibility that one or more members of the Rho family of GTPases are involved in and control pinocytosis in dendritic cells. RESULTS: We established dendritic cell populations that show constitutive macropinocytosis that was downregulated by LPS treatment. Microinjection of immature cells with dominant-negative Rac (N17Rac1) or treatment with Clostridium difficile toxin B, the phosphoinositide 3-kinase (PI3-K) inhibitor wortmannin, or LPS all inhibited the formation of macropinosomes but, surprisingly, did not eliminate membrane ruffling. Microinjection of N17Cdc42 or the Rho inhibitor C3 transferase eliminated actin plaques/podosomes and actin cables, respectively, but had little effect on the formation of macropinosomes. Surprisingly, dendritic cells matured with LPS had equivalent or even somewhat higher levels of active Rac than immature cells. Moreover, microinjection of a constitutively active form of Rac (V12Rac1) into mature dendritic cells did not reactivate macropinocytosis. CONCLUSIONS: Rac has an important role in the constitutive formation of macropinosomes in dendritic cells but may be required downstream of membrane ruffling. Furthermore, regulation of Rac activity does not appear to be the control point in the physiological downregulation of dendritic cell pinocytosis. Instead, one or more downstream effectors may be modulated to allow Rac to continue to regulate other cellular functions.

Animals↗
PubMed1985

Ingestion of erythrocytic stroma by Plasmodium chabaudi trophozoites: ultrastructural study by serial sectioning and 3-dimensional reconstruction.

An ultrastructural study of numerous serial sections of Plasmodium chabaudi trophozoites at various growth stages, followed by 3-dimensional reconstruction, allowed us to describe more precisely the internalization process of the erythrocytic stroma, both in space and in time. Two endocytotic processes are apparent. (1) Pinocytosis--as soon as the merozoite has become a young trophozoite (ring stage), small double membrane-bound vesicles can be seen budding off around the whole periphery of the parasite. After the inner membrane of the vesicle has disappeared, the contents alter and a pigment crystal appears. (2) Cytostomal system--this phenomenon coexists with, and eventually replaces, pinocytosis. It consists of invagination of the membrane of the parasitophorous vacuole and of the plasmalemma, through a typical cytostome, in order to form a cytostomal vacuole. This extends into a long tube, the cytostomal tube, which eventually digitates. When the tube reaches its maximal size, the cytostome disappears and the tube remains open to the erythrocytic stroma by a simple aperture. A new cytostome can form elsewhere on the parasite surface and another tube can extend. Two or three such tubes can coexist in a trophozoite although only one cytostome is functional at one time. At the end of the tubes vesicles bud off, the contents of which become modified as described previously. The residual product of haemoglobin degradation is the malarial pigment.

Animals↗
PubMed1974

Glucose transport in Entamoeba histolytica.

That the uptake of glucose by the parasitic amoeba Entamoeba histolytica occurs by an equilibrative transport system is supported by the following observations. 1. The rate of glucose uptake is several orders of magnitude greater than the uptake by pinocytosis. 2. The uptake of glucose exhibits saturation kinetics, with K(m)=1.6mm and V(max.) ranging from 2 to 5mumol/min per ml of cells at 37 degrees C. 3. The glucose analogues 2-deoxyglucose, 3-O-methylglucose and d-xylose are transported by the glucose system although with much less affinity. Competitive inhibition was observed between pairs of substrates, with K(i) values for any sugar closely coincident with the corresponding K(m). 4. d-Xylose, a sugar not metabolized by the cells, equilibrated with 80% of the amoebal cell water. 5. Cells equilibrated with xylose exhibited countertransport of this sugar against its concentration gradient when another substrate was added to the medium. 6. Blocking of glycolysis by iodoacetate or F(-) has no immediate effect on transport. The presence of a glucose-transport system in E. histolytica contrasts with the situation found in the non-parasitic amoeba, where pinocytosis seems to be the only mechanism of solute uptake.

Binding, Competitive↗
PubMed1983

Biosynthesis and turnover of the phosphomannosyl receptor in human fibroblasts.

The phosphomannosyl receptor mediates intracellular targeting of newly synthesized acid hydrolases to lysosomes, and is also expressed as a pinocytosis receptor on the cell surface of fibroblasts. We have purified the phosphomannosyl receptor from bovine liver and produced rabbit antibodies to the bovine receptor. The antibodies partially blocked pinocytosis of human spleen beta-glucuronidase by fibroblasts, a process mediated by the phosphomannosyl receptor. Affinity-purified antibodies to the phosphomannosyl receptor were used to study the biosynthesis and turnover of the receptor in human fibroblasts. Phosphomannosyl receptor immunoprecipitated after a 15 min pulse-labelling of fibroblasts with [35S]methionine exhibited an identical mobility on sodium dodecyl sulphate/polyacrylamide gels as purified bovine liver phosphomannosyl receptor. Pulse-chase experiments for up to 3 days provided no evidence for changes in molecular weight attributable to post-translational processing of the phosphomannosyl receptor. Turnover studies determined that the half-life of the phosphomannosyl receptor in normal human fibroblasts was 24-29 h. The half-life of the receptor was slightly longer (32 h) in I-cell disease fibroblasts and normal fibroblasts exposed to leupeptin (32 h), slightly shorter in fibroblasts exposed to NH4Cl (23 h) and saturating amounts of ligand (21 h) and unaffected in cells exposed to mannose 6-phosphate (24 h). These studies show that the turnover of the phosphomannosyl receptor in fibroblasts is very slow, in contrast with its rate of internalization in endocytosis, and that its rate of degradation is not greatly altered by a variety of agents that affect lysosomal protein turnover and/or receptor-mediated endocytosis. These results suggest that the degradative activities of the lysosomes do not play an important role in phosphomannosyl receptor turnover in cultured fibroblasts.

Antibodies↗
PubMed1981

Expanding the definition of the blood-brain barrier to protein.

Tight junctions between cerebral endothelial cells and the near absence of pinocytosis and vesicular transport of blood-borne protein into and across these cells are believed to constitute the mammalian blood-brain barrier. In the present investigation evidence is provided to indicate that the capillary endothelium of the mouse brain pinocytosis the enzymatic tracer horseradish peroxidase (EC 1.11.1.17) from cerebral blood under normal conditions. This protein and the internalized endothelial surface membrane associated with it are directed, for the most part, to acid hydrolase-positive lysosomes for degradation. Although peroxidase was never seen in the perivascular clefts, the lysosomes of pericytes were peroxidase-positive. Pericytes are macrophage-like cells located on the abluminal surfaces of cerebral microvasculature; these cells may serve as the first line of defense once the blood-brain barrier is breached. The definition of the blood-brain barrier should be expanded to include consideration of the lysosomal system of organelles in endothelial cells and pericytes.

Acid Phosphatase↗
PubMed1987

Tubular lysosome morphology and distribution within macrophages depend on the integrity of cytoplasmic microtubules.

Pinocytosis of the fluorescent dye lucifer yellow labels elongated, membrane-bound tubular organelles in several cell types, including cultured human monocytes, thioglycolate-elicited mouse peritoneal macrophages, and the macrophage-like cell line J774.2. These tubular structures can be identified as lysosomes by acid phosphatase histochemistry and immunofluorescence localization of cathepsin L. The abundance of tubular lysosomes is markedly increased by treatment with phorbol 12-myristate 13-acetate (10 ng/ml). When labeled by pinocytosis of microperoxidase and examined by electron microscopic histochemistry, the tubular lysosomes have an outside diameter of approximately 75 nm and a length of several micrometers; they radiate from the cell's centrosphere in alignment with cytoplasmic microtubules and intermediate filaments. Incubation of phorbol myristate acetate-treated macrophages at 4 degrees C or in medium containing 5 microM colchicine or nocodazole at 37 degrees C leads to disassembly of microtubules and fragmentation of the tubular lysosomes. Return of the cultures to 37 degrees C or removal of nocodazole from the medium leads to reassembly of microtubules and the reappearance of tubular lysosomes within 10-20 min. We conclude that microtubules are essential for the maintenance of tubular lysosome morphology and that, in macrophages, a significant proportion of the lysosomal compartment is contained within these tubular structures.

Animals↗
PubMed1957

Microkinetospheres and VP satellites of pinocytic cells observed in tissue cultures of Gey's strain HeLa with phase contrast cinematographic techniques.

By tissue culture methods and with the use of phase contrast, interference color contrast, and time-lapse cinematographic equipment, the activity of a cytoplasmic organoid, termed the microkinetosphere, has been followed and correlated with pinocytosis. A transformation of several microkinetospheres by coherence and coalescence into the solitary VP satellite was observed in cells undergoing pinocytosis in serum nutrients. A correlation of both of these structures to cytoplasmic organoids described by others, notably with the electron microscope, and a hypothesis on the nature of the microkinetosphere and its transformation was presented.

HeLa Cells↗
PubMed1959

An electron microscopic study of the intestinal villus. II. The pathway of fat absorption.

The intestinal pathway for absorbed fat was traced in thin sections of intestinal villi from rats fed corn oil by stomach tube after a fast of 24 to 40 hours. For electron microscopy the tissues were fixed in chilled buffered osmium tetroxide and embedded in methacrylate. For light microscopy, other specimens from the same animals were fixed in formal-calcium, mordanted in K(2)Cr(2)O(7), and embedded in gelatin. Frozen sections were stained with Sudan black B or Sudan IV. About 20 minutes after feeding, small fat droplets (65 mmicro maximal diameter) appear in the striated border between microvilli. At the same time fat particles are seen within pinocytotic vesicles in the immediately subjacent terminal web. In later specimens the fat droplets are generally larger (50 to 240 mmicro) and lie deeper in the apical cytoplasm. All intracellular fat droplets are loosely enveloped in a thin membrane, the outer surface of which is sometimes studded with the fine particulate component of the cytoplasm. This envelope, apparently derived from the cell surface by pinocytosis, has at this stage evidently become a part of the endoplasmic reticulum. Just above the nucleus numerous fat droplets lie clustered within the dilated cisternae of the Golgi complex. As absorption progresses fat droplets appear in the intercellular spaces of the epithelium, in the interstitial connective tissue spaces of the lamina propria, and in the lumen of the lacteals. All of these extracellular fat droplets are devoid of a membranous envelope. The picture of fat absorption as reconstructed from these studies involves a stream of fat droplets filtering through the striated border, entering the epithelial cell by pinocytosis at the bases of the intermicrovillous spaces, and coursing through the endoplasmic reticulum to be discharged at the sides of the epithelial cell into extracellular spaces. From the epithelial spaces, the droplets move into the lamina propria and thence into the lymph. If the lumen of the endoplasmic reticulum is considered as continuous with the extracellular phase, then the entire pathway of fat absorption may be regarded as extracellular. However, it is impossible to evaluate from the electron microscopic evidence thus far available the quantitative importance of particulate fat absorption by the mechanism described.

Animals↗
PubMed1980

Internalization of macromolecules from the medium in Suctoria.

Takophrya infusionum like all other Suctoria lacks an oral cavity. Its feeding apparatus consists of tentacles, long narrow tubes through which the contents of the living prey are ingested. For normal growth, reproduction, and longevity of clones, Tokophrya needs supplements deriving from the medium in addition to living prey. Since Tokophrya lacks a mouth, these supplements can reach the cytoplasm only through the complex structure of the cortex, which is composed of a three-membraned pellicle and a dense epiplasm. In addition, external to the cortex, an extraneous coat covers the whole organism. Only the outer pellicular plasma membrane is continuous; the other two and the epiplasm are interrupted by the outer plasma membrane which invaginates at intervals forming the so-called pits. The invaginated plasma membrane dips down into the cytoplasm where it extends to form a saccule. Experiments with cationized ferritin and Thorotrast provide evidence that internalization of these macromolecules takes place through the pits by pinocytosis. The membrane of the saccules of the pits forms invaginations which pinch off giving rise to small, flattened vesicles containing the tracers. The tracers were never found free in the cytoplasm but exclusively in the flat vesicles. These vesicles are thus the vehicles transporting macromolecules from the medium to the cytoplasm. The saccules of the pits are the natural loci of pinocytosis and together with the flattened vesicles perform an important function in Suctoria, supplying the organisms with macromolecules from the medium.

Animals↗
PubMed1969

The fate of peptides pinocytosed by macrophages in vitro.

A series of small peptides, such as might arise in the course of intralysosomal protein digestion, were screened for the ability to escape, intact, from mouse peritoneal macrophage lysosomes. Inability to penetrate lysosomal membranes was inferred from a peptide's induction of lysosomal swelling, or vacuolization, in cultured macrophages. Two of the peptides tested, (D-Glu)(2) and (D-Ala)(3), induced vacuolization. Neither peptide was susceptible to hydrolysis by enzymes in macrophages or in the serum-containing culture medium. Their morphological effect was inhibited by parafluorophenylalanine, an inhibitor of pinocytosis. Once formed by either peptide, the vacuoles persisted for several hours in peptide-free medium. Quantitative studies of radioactively labeled (D-Glu)(2) confirmed the morphological evidence that (D-Glu)(2) is taken up by pinocytosis and stored, intact, in macrophage lysosomes. The majority of the peptides which failed to induce vacuolization-(L-Ala)(2), L-Ser.L-Ala, L-Val.L-Ala, L-Ala.L-Thr, Gly.D, L-Phe, L-Ala.D-His, (L-Ala)(3), (L-Glu)(2), and D-Leu.L-Tyr-were found to be susceptible to hydrolysis by cellular or serum peptidases. Their failure to induce vacuolization was attributed to their hydrolysis to subunits capable of penetrating lysosomal membranes. Some of the peptides which had failed to induce vacuolization-(D-Ala)(2), D-Ser.D-Ala, D-Val.D-Ala, Gly-D-Asn, D-Ala.D-Thr, and D-Arg.D-Val-were found to be indigestible. Except for the cytotoxic peptide D-Arg.D-Val, peptides in this category all had lower molecular weights and volumes than (Glu)(2) or (Ala)(3). It is inferred that these peptides are small enough to escape from macrophage lysosomes, while (Glu)(2) and (Ala)(3) are too large to escape intact. The implications of this inference for the mechanism of intracellular digestion of pinocytosed proteins are discussed.

Alanine↗