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PubMed1990

Morphologic changes in human carcinoma cells (A-431) stimulated by epidermal growth factor: effect of cholesterol and low-density lipoproteins on the ruffling response.

Stimulation of A-431 carcinoma cells with epidermal growth factor (EGF) causes dramatic morphologic responses including ruffling, rounding, and bulk-phase pinocytosis. In attempts to explore the mechanisms responsible for changes in plasmalemma topography, we have investigated the effects of exogenous sterols thought to alter membrane fluidity. Light and scanning electron microscopy revealed a time- and concentration-dependent inhibition of ruffling (greater than 90%) by cholesterol. This effect could be duplicated by preincubation of the cells with comparable levels of low-density lipoproteins (LDL). EGF-stimulated bulk-phase endocytosis also is inhibited by treatment with cholesterol. No alteration of EGF binding, kinase stimulation, or internalization was detected in cells incubated in cholesterol-enriched medium (175 micrograms/ml in 0.5% ethanol), nor did cholesterol or LDL have any effect on EGF-stimulated rounding. Morphometry of electron micrographs from cholesterol-treated cells revealed a selective depletion of interdigitating lateral surface membrane that normally appears to be recruited to generate apical ruffles. Thus, the sterol inhibition of ruffling may be due to redistribution of plasmalemma rather than to changes in membrane viscosity. Together with previous observations, these data suggest that EGF-stimulated ruffling and bulk-phase pinocytosis are related phenomena, whereas EGF-stimulated cell rounding is an independent process.

Carcinoma, Squamous Cell↗
PubMed1980

The ultrastructure of the protonephridial tubules of the freshwater planarian Bdellocephala brunnea.

A systematic electron microscopic investigation was undertaken to elucidate the regional differentiation of the protonephridial tubules of the freshwater planarian Bdellocephala brunnea. The excretory system consists of many independent protonephridia, each made up of the repeatedly branching "proximal tubules," and highly tortuous "distal tubule." The proximal tubules are composed of a simple ciliated epithelium except in the trunk which lacks ciliation. The cytoplasmic features of the ciliated cell closely resemble those of the flame cell, indicating highly active protein reabsorption by pinocytosis. Pinocytosis seems also active in the trunk of the proximal tubules and in the distal tubule, resulting in cytoplasmic vesicles of typical appearance. Salient features of the distal tubule cell are the increased density of the cytoplasmic matrix and the presence of many basal compartments of cytoplasm with mitochondria, a pattern common to many epithelia with osmoregulatory function. The morphology and possible functions of the planarian protonephridium are discussed in comparison with the vertebrate nephron.

Animals↗
PubMed1990

Uptake of horseradish peroxidase by the middle ear mucosa in experimentally induced otitis media.

Electron microscopy was used to study the characteristics of uptake of the protein tracer, horseradish peroxidase (HRP), by the middle ear mucosa of chinchillas. Two types of experimental otitis media were used: serous otitis media (SOM) produced by eustachian tube obstruction and purulent otitis media (POM) produced by inoculation of Streptococcus pneumoniae type 7F. HRP was placed in each tympanic bulla for either 1 min or 10 min. In the 1-min installation of HRP, the degree of HRP uptake was higher in SOM and POM animals than in control animals. Following the 10-min installation of HRP, the degree of HRP uptake was higher in SOM and POM animals than in control animals. Following the 10-min installation of HRP, the degree of tracer uptake was almost the same as among the control, SOM and POM animals. The mechanism of uptake was pinocytosis. In another experiment, 5-hydroxydopamine (mol. wt. 256) was used as a small tracer placed into the bulla to examine the permeability of epithelial tight junctions. No labelling of reaction product was observed in the tight junctions of the control, SOM or POM animals. These findings suggest that otitis media increases the uptake of HRP by pinocytosis and that epithelial tight junctions remain intact in inflammation of the middle ear cavity.

Animals↗
PubMed1985

Membrane specializations and their relation to HRP transport in the medial habenular nucleus of the rat.

In the medial habenular nucleus of the rat, ependymal and endothelial membrane specializations were studied with TEM and freeze-fracturing. They comprise ependymal adherent junctions - not manifest in freeze-fracture replicas-, gap junctions, and membrane-associated orthogonal particle complexes ("assemblies") - not identifiable in thin-sectioned material. Ependymal tight junctions being absent, no brain-liquor barrier exists. The capillary endothelium is provided with tight junctions only. Intraventricularly injected HRP was transported in large amounts through the ependyma, mainly through the intercellular spaces and additionally by way of massive pinocytosis through the cytoplasm of particular ependymal cells only, and finally through the parenchymal intercellular compartments towards habenular capillaries. Following intravenous injection of HRP, considerable transport of the enzyme took place by means of transendothelial pinocytosis, followed by some pinocytotic transport through diverse parenchymal elements and markedly profuse incorporation and lysis within pericytes. The habenular blood-brain barrier appeared to be considerably leaky with respect to HRP.

Animals↗
PubMed1993

The shape and distribution of lysosomes and endocytosis in the ciliary epithelial cells of rats.

The shape and distribution of lysosomes in the ciliary epithelium of rat eyes were examined by electron microscopy combined with acid phosphatase (ACPase) cytochemistry and three-dimensional observation of 2 microns-thick sections. ACPase activity was cytochemically localized in lysosomes and trans Golgi cisternae in the non-pigmented epithelial (NPE) and pigmented epithelial (PE) cells. In NPE cells, it was shown three-dimensionally, that most lysosomes had an elongate form, up to 5 microns in length, and a diameter of 70-100 nm. These elongate lysosomes (nematolysosomes) were predominantly located in the basal region of the cells. In contrast, PE cells had spherical lysosomes distributed at random throughout the cytoplasm. However, no nematolysosomes were seen in the PE cells. When the isolated ciliary processes were incubated in a medium containing horseradish peroxidase (HRP), HRP was incorporated into the nematolysosome-like structures by pinocytosis from the basal surface of the NPE cells. These findings suggest that nematolysosomes are associated with the pinocytotic activity of NPE cells. The pinocytosis-nematolysosomal route may be involved in the uptake and degradation of macromolecules from the aqueous humor in the posterior chamber.

Acid Phosphatase↗
PubMed1979

REgional functional differentiation in the gut of the grasscarp, Ctenopharyngodon idella (Val.).

A regional differentiation--reflecting structural differences--of the intestine of larval and juvenile grasscarps can be illustrated by studying the activity of alkaline phosphatase and the uptake of orally administered horseradish peroxidase. Pinocytosis takes place in a welldefined area of about 23% of the length of the gut (segment II). Neither the rostral +/- 68% (segment I) nor the caudal +/- 9% (segment III) shows absorption of the enzyme. Alkaline phosphatase activity, mainly localized at the microvilli of the enterocytes is high in the first segment of the gut and low in the second segment. In larvae, the activity decreases sharply at the transition from segment I to segment II. The activity is weak or absent in the caudal third segment. Quantitative histochemical data are confirmed by biochemical analyses. Alkaline phosphatase activity is found all over the mucosal folds of the first segment, with relatively weak activity at the base and at the tip of the folds. This may be related to a renewal of the epithelium. Our results suggest that active absorption of digested food takes place mainly in the rostral first segment, while the uptake of macromolecules by pinocytosis is a function of the second segment. Comparison of the results with information available in literature leads to a rejection of the hypothesis that the uptake of protein macromolecules in Cyprinids is to be attributed to the absence of a stomach and therefore to an inefficient digestion of proteins.

Aging↗
PubMed1990

Mechanism of cystine reaccumulation by cystinotic fibroblasts in vitro.

It is well established that when cystine-depleted cystinotic cells are cultured in cystine-containing medium, they reaccumulate cystine within their lysosomes more rapidly than when cultured in cystine-free medium. This has been a puzzling result, since the lysosome membrane of cystinotic cells is impermeable to cystine. To probe the mechanism of cystine reaccumulation, we have measured reaccumulation in the presence of colchicine, an inhibitor of pinocytosis, or of glutamate, a competitive inhibitor of cystine transport into human fibroblasts. Colchicine had no effect, thus eliminating pinocytosis as a putative mechanism for cystine translocation from the culture medium to the lysosomes. Glutamate, however, strongly inhibited cystine reaccumulation. It is concluded that the true mechanism is as follows. 1. Exogenous cystine crosses the plasma membrane on the cystine-glutamate porter. 2. Cystine is reduced in the cytoplasm by GSH. 3. The cysteine that is generated enters the lysosome, where it becomes cystine by participating in the reduction of cystine residues during intralysosomal proteolysis, or by autoxidation.

Biological Transport↗
PubMed2001

Inclusions in novel perivascular macrophages (Mato's fluorescent granular perithelial cells) and neurons in the cerebral cortex of Hex A- and Hex B-deficient mice.

Beta-hexosaminidases are important enzymes for lipid and saccharide metabolism in the brain. In mice deficient in these enzymes, indigestible metabolic intermediates deposit in neurons. Inclusions such as membranous cytoplasmic bodies (MCB) and zebra bodies were seen in neurons of Tay-Sachs (TS) model mice, Sandhoff's disease (SD) model mice, and double knockout (DKO) mice. However, the cerebral perivascular macrophages discovered by Mato are active in the uptake of waste products and regarded as scavenger cells under steady-state conditions. We observed that indigestible components derived from neurons were taken up by the perivascular macrophages of TS mice by pinocytosis, but those of SD and DKO mice contained only pale inclusions and had marked vacuolations, and pinocytosis was rarely observed. Histochemically, the inclusions in the perivascular macrophages of TS mice were positive for the PAS stain, but those of SD and DKO mice were negative. In addition, the perivascular cells of TS mice expressed clear positive immunoreactivity against BM-8 and F4/80, but those of DKO mice had very weak BM-8 and F4/80 immunoreactivity. These differences between TS, SD, and DKO mice are based on their metabolism of oligosaccharides and glycosaminoglycans (GAG). Thus, hexosaminidase B is more important for keeping normal morphology and function of perivascular macrophages than hexosaminidase A. The foamy cells that appeared along the cerebral microvessels in lipidosis and saccharidosis were identified as perivascular macrophages (Mato's fluorescent granular perithelial cells: FGP cells).

Animals↗
PubMed1983

Blood-spinal cord barrier response to transection.

The integrity of the blood-spinal cord barrier (BSB) distal to a surgical transection was evaluated at the light and electron microscopic levels, using the tracers Evans blue albumin (EBA) and horseradish peroxidase (HRP). At the light microscopic level, the orange fluorescence of the EBA was detected as far as 0.45 mm distal to the transection. The tracer was not only diffusely distributed in the neuropil but also was apparently taken up by ventral horn neurons and glial cells. A feathery halo of EBA extending beyond their walls indicated that there had occurred some leakage of EBA from intrinsic vessels into the surrounding cord tissue. The microvasculature was evaluated at the ultrastructural level for evidence of changes in permeability to HRP. Pinocytosis and vesicular transport of the protein were apparent in some endothelial cells. In addition, there was limited evidence for leakage across tight junctions. After 1 day the barrier selectivity appeared to be restored and vesicular transport of HRP was not found. Endothelial pinocytosis, however, appeared enhanced for as long as 7 days following injury.

Animals↗
PubMed1993

A possible signal-coupling role for cyclic AMP during endocytosis in Amoeba proteus.

Cytoplasmic levels of cAMP in Amoeba proteus were measured utilizing radioimmunoassays under control conditions and when stimulated by inducers of either pinocytosis or phagocytosis. In control cells, cytoplasmic cAMP levels were approximately 0.39 pM/mg cells. When exposed to either chemotactic peptide or mannose which stimulate phagocytosis in the amoeba, there is a rapid doubling of the cAMP level within 45 sec of stimulation which then returns to the control level within 3-5 min. Theophylline prolongs the elevation of cytoplasmic cAMP in stimulated cells and is also capable of eliciting food vacuole formation in the amoeba. In addition isoproterenol also causes food vacuole formation in the amoeba as well as a large and prolonged increase in cytoplasmic cAMP levels. Inducers of pinocytosis (BSA and Na Cl) also elicit changes in cytoplasmic cAMP in the amoeba, but the response appears to differ from that elicited by inducers of phagocytosis in that the peak cAMP levels are broader and biphasic. It is concluded that cAMP plays a signal-coupling role during the early phases of both forms of endocytosis in Amoeba proteus.

Amoeba↗
PubMed2005

The ABC transporter PGP-2 from Caenorhabditis elegans is expressed in the sensory neuron pair AWA and contributes to lysosome formation and lipid storage within the intestine.

The functional role of the ABC transporter PGP-2 from the nematode Caenorhabditis elegans has been studied by combining phenotype analyses of pgp-2 deletion mutants or pgp-2 RNAi treated worms with reporter gene studies using a pgp-2::GFP construct. pgp-2 mutants showed a strong reduction of lipid stores. In addition, we found that in the case of the pgp-2 mutant or after pgp-2 RNAi the worms were unable to perform pinocytosis and to acidify intestinal lysosomes. Especially under cholesterol-restricted conditions, the viability of the mutant was reduced. Surprisingly, the chemosensory AWA neurons in the head region were identified as expression sites by reporter gene studies. These neurons are known to be involved in attraction behaviour towards odorants associated with potential food bacteria. Our results imply that PGP-2 is involved in a signalling process that connects sensory inputs to intestinal functions, possibly by influencing acidification of intestinal lysosomes, which in turn may affect pinocytosis and lipid storage.

ATP Binding Cassette Transporter, Subfamily B↗
PubMed2000

Macrophage-mediated activation of camptothecin analogue T-2513-carboxymethyl dextran conjugate (T-0128): possible cellular mechanism for antitumor activity.

Camptothecin (CPT) analogue T-2513-carboxymethyl (CM) dextran conjugate (T-0128) suppressed human tumor xenografts that were refractory to CPTs. This improvement was explained by its altered pharmacokinetics, but the cellular mechanism of action is still not clear. For this reason, in the present study we examined the determinants of T-0128 action at the cellular level. In vitro tests showed that T-0128 was inactive, indicating that the requirement for its activity lies in the release of linked T-2513, accompanied by the cellular uptake of the conjugate. The accumulation varied between cell lines: tumor cells, including Walker-256 carcinoma and B16 melanoma, showed only a marginal uptake and an undetectable drug release in the medium. In contrast, macrophage-like cells, such as J774.1, internalized T-0128 very efficiently, and liberated T-2513. With regard to the mode of accumulation, fluid-phase pinocytosis seems to be a key factor based on the followings: a similar cell-specificity existed in the uptake of FITC dextran, a marker of fluid-phase pinocytosis. Also, the macrophage uptake of T-0128 increased almost linearly with its medium concentration and was insensitive to dextran sulfate, a ligand for macrophage scavenger receptor. Comparative efficacy studies of T-0128 in the presence and absence of macrophages demonstrated that macrophages increased the efficacy of T-0128. The enhancement could be explained in terms of increases in the amount of released T-2513. Overall, these results lead us to the conclusion that T-0128 acts like a Trojan horse with the help of macrophages: T-0128 is taken up by macrophages in tumor tissues, and the liberated T-2513 kills tumor cells.

Animals↗
PubMed1980

Hyperpolarisation of rat peritoneal macrophages phagocytosing latex particles.

The changes that take place in the plasma membrane during endocytosis are still poorly understood. Studies of the electrical properties and related permeabilities of cells during pinocytosis and phagocytosis should provide insight into these changes. This approach has not been much exploited, although there have been a few relevant reports. For example, induction of pinocytosis in amoebae produces a marked decrease in plasma membrane resistance, and exposure of polymorphonuclear leukocytes to phagocytosable particles can engender alterations in ionic permeabilities. The present report demonstrates that the induction of phagocytosis in rat peritoneal macrophages is accompanied by a sustained hyperpolarisation of the cells.

Animals↗
PubMed1979

Putrescine uptake by the cellular slime mould dictyostelium discoideum.

1. Rapid labelling occurs when myxamoebae of Dictyostelium discoideum strain AX2 are incubated with [1,4-14C]putrescine. Labelling is energy-dependent. 2. The label enters a pool from which rapid exchange with extracellular putrescine does not occur, and labelling is believed to represent uptake into the cells. 3. The concentration-dependence of putrescine uptake indicates that a number of systems are involved, at least one of which is saturable, with a Km of 9.1 micro M-putrescine. At high putrescine concentrations the overall uptake process is non-saturable. 4. Significant metabolism of putrescine and loss of intracellular putrescine to the medium only occurred when cells were incubated with millimolar concentrations of extracellular putrescine. 5. Putrescine uptake was inhibited by diamines, polyamines, bivalent metal ions and omega-aminocarboxylic acids. 6. The ability to take up putrescine at low concentrations decreased during starvation of myxamoebae. 7. The results are interpreted in terms of a model for putrescine uptake involving adsorptive pinocytosis at low concentrations and fluid-phase pinocytosis at high concentrations.

Carboxylic Acids↗
PubMed1983

The fate of homologous 125I-labelled immunoglobulin G within rat visceral yolk sacs incubated in vitro.

When 125I-labelled rat IgG (immunoglobulin G) is incubated in vitro with visceral yolk sacs from 17.5-day-pregnant rats, the protein is readily degraded. The major radioactive digestion product that accumulates in the medium is [125I]iodo-L-tyrosine. When rotenone (10 microM) is also present in the incubation medium, the rate of digestion of IgG is inhibited to the same extent as the rate of pinocytosis of 125I-labelled polyvinylpyrrolidone. Proteolysis is likewise inhibited when either NH4Cl (30 mM) or leupeptin (30 micrograms/ml) is present in the medium. The above findings strongly suggest that the observed proteolysis occurs within lysosomes. Normally, yolk sacs that have been exposed in vitro to radiolabelled substrates release radioactivity slowly when the tissue is re-incubated, unless the substrate can be degraded within lysosomes and released in the form of low-molecular-weight hydrolysis products. However, in such experiments 125I-labelled rat IgG shows quite exceptional behaviour in being rapidly released in an apparently intact form (as well as being degraded). If an agent that inhibits pinocytosis (e.g. rotenone or 2,4-dinitrophenol) is present in the incubation medium during exposure of the tissue to 125I-labelled rat IgG, it abolishes release of macromolecular radioactivity on re-incubation of the tissue. Enhanced tissue accumulation of 125I-labelled rat IgG, induced by the presence of leupeptin in the medium during the uptake phase, resulted in no concomitant increase in the amount of 125I-labelled IgG released in macromolecular form on re-incubation of the tissue. These findings indicate that the observed rapid release of 125I-labelled IgG is unlikely to represent release from lysosomes and is more compatible with release from a separate class of vesicle that does not fuse with lysosomes.

2,4-Dinitrophenol↗
PubMed2004

Ultraviolet B radiation suppresses endocytosis, subsequent maturation, and migration activity of langerhans cell-like dendritic cells.

Langerhans cells capture exogenous antigens through fluid phase pinocytosis and receptor-mediated endocytosis and migrate to lymph nodes, where they present processed antigen to T cells. Ultraviolet B radiation impairs the antigen-presenting function of Langerhans cells, resulting in antigen-specific immunosuppression of contact hypersensitivity. We tested the notion that ultraviolet B radiation inhibits the endocytic activity of Langerhans cells, leading to impaired migration and maturation. Human monocyte-derived Langerhans cell-like dendritic cells that took up lucifer yellow or fluorescein isothiocyanate dextran exclusively migrated in response to 6Ckine/secondary lymphoid chemokine, and matured, as evidenced by an increase in CD54 and CD86 expression and potent stimulatory activity in allogeneic mixed lymphocyte reaction. Exposing Langerhans cell-like dendritic cells to 20-40 mJ per cm2 of ultraviolet B radiation reduced their endocytic activity in fluid phase pinocytosis (measured by uptake of lucifer yellow) and in receptor-mediated endocytosis (measured by uptake of fluorescein isothiocyanate dextran). Membrane ruffling and CD32 expression were also suppressed by ultraviolet B radiation. Ultraviolet B-irradiated, endocytosing Langerhans cell-like dendritic cells had less movement towards 6Ckine, expressed less CD54 and CD86, and had less effective stimulatory activity in allogeneic mixed lymphocyte reaction than nonirradiated, endocytosing Langerhans cell-like dendritic cells. Endocytosis upregulated tumor necrosis factor alpha production by Langerhans cell-like dendritic cells, but prior ultraviolet B radiation inhibited this enhancement. These data suggested that impaired endocytosis and subsequent inhibitory migration and maturation of Langerhans cells by ultraviolet B radiation could contribute to local immunosuppression of contact hypersensitivity.

Adult↗
PubMedDate not supplied

The cerebral endothelium during pregnancy: a potential role in the development of eclampsia.

The authors investigated the influence of pregnancy on cerebral endothelial cell permeability in response to an acute elevation in intravascular pressure that caused forced dilatation of myogenic tone. Third-order branches of the posterior cerebral artery (PCA) were dissected from nonpregnant (NP) and late-pregnant (LP, days 19 to 20) Sprague-Dawley rats and mounted on glass cannulas in an arteriograph chamber that allowed control over intravascular pressure and measurement of both diameter and permeability to fluorescent dextran (3000 Da). Permeability was determined at 75 mm Hg and after a step increase in pressure to 200 mm Hg. The extent of pinocytosis and transcellular transport in response to pressure was evaluated separately in the same groups of animals at 75 and 200 mm Hg using transmission electron microscopy. All arteries developed myogenic tone at 75 mm Hg that was lost when pressure was increased to 200 mm Hg to cause forced dilatation. The increased pressure caused a significant increase in permeability to dextran and enhanced pinocytosis in arteries from LP animals, but not in NP animals whose permeability remained constant at both pressures. These results suggest a pregnancy-specific effect on the cerebral endothelium that may promote enhanced vascular permeability during acute hypertension and may contribute to the edema formation and neurologic complications of eclampsia.

Animals↗
PubMed1990

The relationship between antigen concentration, antigen internalization, and antigenic complexes: modeling insights into antigen processing and presentation.

Native antigen is processed and subsequently presented on the surface of antigen-presenting cells, an important step in the elicitation of an immune response. The early events of antigen processing and presentation include: ingestion of a native antigen, intracellular degradation to expose an antigenic peptide fragment, binding of this fragment with an MHC class II molecule, and display of this newly formed complex on the cell surface. Through the development of a mathematical model, a set of mathematical equations which describes the time-dependent appearance, disappearance, and movement of individual molecules, quantitative insight can be gained into the pathways and rate-limiting steps of antigen presentation. The credibility of the model has been verified by comparison to literature data. For example, it has been shown experimentally that macrophages require 60 min for effective antigen presentation, whereas B cells require 6-8 h. The mathematical model predicts these presentation times and identifies the difference in the cell's respective pinocytic rates and sizes as important parameters. B cells capture antigen in their environment through nonspecific fluid-phase pinocytosis as well as by binding antigen to their surface immunoglobulin, allowing receptor-mediated uptake. Uptake of antigen via receptor-mediated endocytosis has been reported to require 1,000-fold less antigen than uptake via nonspecific pinocytosis. The mathematical model clearly predicts this decrease in concentration. The model also makes quantitative predictions for the number of MHC class II-antigen complexes needed to produce T cell stimulation.

Animals↗