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PubMed2004

The Rab5 effector Rabankyrin-5 regulates and coordinates different endocytic mechanisms.

The small GTPase Rab5 is a key regulator of clathrin-mediated endocytosis. On early endosomes, within a spatially restricted domain enriched in phosphatydilinositol-3-phosphate [PI(3)P], Rab5 coordinates a complex network of effectors that functionally cooperate in membrane tethering, fusion, and organelle motility. Here we discovered a novel PI(3)P-binding Rab5 effector, Rabankyrin-5, which localises to early endosomes and stimulates their fusion activity. In addition to early endosomes, however, Rabankyrin-5 localises to large vacuolar structures that correspond to macropinosomes in epithelial cells and fibroblasts. Overexpression of Rabankyrin-5 increases the number of macropinosomes and stimulates fluid-phase uptake, whereas its downregulation inhibits these processes. In polarised epithelial cells, this function is primarily restricted to the apical membrane. Rabankyrin-5 localises to large pinocytic structures underneath the apical surface of kidney proximal tubule cells, and its overexpression in polarised Madin-Darby canine kidney cells stimulates apical but not basolateral, non-clathrin-mediated pinocytosis. In demonstrating a regulatory role in endosome fusion and (macro)pinocytosis, our studies suggest that Rab5 regulates and coordinates different endocytic mechanisms through its effector Rabankyrin-5. Furthermore, its active role in apical pinocytosis in epithelial cells suggests an important function of Rabankyrin-5 in the physiology of polarised cells.

Adenoviridae↗
PubMed1975

Metabolism of sulfated glycosaminoglycans in cultivated bovine arterial cells. II. Quantitative studies on the uptake of 35SO4-labeled proteoglycans.

Cultured arterial fibroblasts were used for a quantitative study on adsorption, uptake and degradation of [35S]proteoglycans derived from secretions of cultured arterial or skin fibroblasts. The following results were obtained: 1) Proteoglycans added to the culture medium are integrated into the pool of cell membrane-associated (trypsin-removable) glycosaminoglycans by a saturable process, which depends on time and temperature. 2) Up to 17% of the added proteoglycans are taken up by the cells within 24 h. The uptake exhibits saturation kinetics, characteristic for adsorptive pinocytosis. Proteoglycan concentrations required for half-maximum uptake are higher than for half-maximum saturation of the glycosaminoglycan pool associated with the cell membrane. 3) After a lag phase, inorganic 35SO4 appears in the culture medium as a degradation product of the internalized proteoglycans. Pinocytosed proteoglycans are catabolized more rapidly than proteoglycans which remain inside the cell after their biosynthesis. 4) Pinocytosis exhibits specificity, the individual proteoglycans being internalized at different rates. The highest rate of uptake was measured for a dermatan-sulfate-rich proteoglycan. No competition of uptake between a dermatan-sulfate-rich and a heparan-sulfate-rich proteoglycan was observed. 5) Optimum pinocytosis requires an intact protein moiety and, presumably, undegraded carbohydrate chains of the proteoglycans.

Animals↗
PubMed1980

Action of prostaglandin E1 and F2 alpha on the pinocytic activity of horseradish peroxidase in bone-marrow derived macrophages.

The influence of prostaglandin E1 and F2 alpha on the pinocytic activity of bone-marrow macrophages was studied biochemically and morphometrically. The results show that prostaglandin E1 induces 44% inhibition of pinocytosis of horseradish peroxidase; prostaglandin F2 alpha has no significant effect. Morphometrical analysis of electron micrographs demonstrates that inhibition of pinocytosis is connected with diminished average size and raised number of pinocytic vesicles. The influence of prostaglandin E1 on pinocytosis is fully reversible. Further experiments are needed to explore the molecular mechanism of the prostaglandin E1 induced alteration of membrane function.

Animals↗
PubMed1991

Diversity in motile responses of human neutrophil granulocytes: functional meaning and cytoskeletal basis.

Different agonists induce motility and shape changes, but only a specific polarized shape is correlated with directed migration. An intact and dynamic actin network appears to be important for motility and migration. Motility is usually associated with an increased level of F-actin, and a specific location of F-actin into surface protrusions. For locomotion, a specific location of F-actin, rather than a large net increase in F-actin appears to be of importance. Three major groups of responses can be distinguished on the basis of the type of shape changes, functional activity and organization of F-actin. 1. Agents capable of polarizing cells, such as chemotactic peptides, and microtubule-disassembling agents elicit, at appropriate concentrations, a marked chemokinetic response, but little if any fluid pinocytosis. F-actin shows a polar location, being concentrated mainly in the protrusions at the leading front. Chemotactic peptide also induces an increase in the level of F-actin and cytoskeleton-associated actin. It is, however, not clear if front-tail polarity and locomotion, induced by chemotactic peptide after longer time of stimulation, correlate with an actual increase in the level of cytoskeleton-associated actin. 2. Activators of protein kinase C such as PMA and diacylglycerols, induce nonpolar cells with surface projections. PMA and diacylglycerols stimulate pinocytosis substantially. All three agents tend to inhibit locomotion or chemotaxis as an immediate response. They also increase the percentage of cytoskeletal actin, and induce an enrichment of F-actin in surface projections. 3. Circus movement may occur in response to D20. These cells show little or no stimulation of locomotion or pinocytosis. Thus the functional significance of this motor response remains to be elucidated. We conclude that different agonists can induce motility and shape changes, but not necessarily chemotaxis. Only a polarized shape is correlated with directed locomotion. An intact and dynamic actin network appears to be important for motility including locomotion. Motility is usually associated with an increased level of F-actin, and a specific location of F-actin into surface protrusions. The actin-associated proteins alpha-Actinin, myosin and actin-binding protein appear also to be important for pseudopod formation. For locomotion, a specific location of F-actin, rather than a large net increase in F-actin may be of importance.

Actins↗
PubMed1991

Predicting the behaviour and selectivity of fluorescent probes for lysosomes and related structures by means of structure-activity models.

Cultured rat fibroblasts were exposed to 50 fluorescent probes of varied physicochemical characteristics. Probe concentrations, fluorochrome excitation wavelength and period of illumination, and cell-probe contact time were varied. Structure-activity relationships defining a number of classes of fluorescent probes for lysosomes and related processes and compartments were demonstrated. Numerical specifications are now available for several familiar classes of probes: (a) acidotropic weak bases, used as markers for low pH compartments; (b) markers of adsorptive pinocytosis, involving non-specific protein binding; (c) markers for fluid phase pinocytosis; and (d) viability stains involving intralysosomal enzymic activity. Two novel classes of probes have also been specified numerically: (a) acid-precipitated weak acids, as markers for low pH compartments; and (b) lipid-binding markers of adsorptive pinocytosis. Overall, these structure-activity models provide a tool for predicting whether or not compounds enter cells; and whether they accumulate in lysosomes and related compartments. Pathways of entry are also predicted. This tool should permit design and selection of improved probes, and provide a better understanding of existing reagents. Moreover these models are expected to be applicable to interactions between any non-polymeric xenobiotic with lysosomes and related compartments.

Animals↗
PubMed1981

Cerebrovascular permeability in mechanically induced hypertension.

Our previous studies of cerebrovascular permeability in angiotensin-induced acute hypertension demonstrated that the principal mechanism resulting in increased permeability is enhanced pinocytosis. In order to exclude the possibility that the enhanced pinocytosis was a direct effect of exogenous angiotensin, cerebrovascular permeability alterations were studied in nonpharmacologically induced acute hypertension. Rats receiving horseradish peroxidase (HRP) intravenously, were sacrificed 2 1/2 minutes after the onset of hypertension induced by placing a clip on the abdominal aorta. These animals showed the same pattern of permeability alterations as had been observed previously in animals with angiotensin-induced acute hypertension. Focal segments of penetrating arterioles in the temporal and parietal cortex showed increased permeability to HRP. Permeable vessels showed increased numbers of pinocytotic vesicles and the interendothelial junctions revealed no alterations. Enhanced pinocytosis appears to be the principal mechanism resulting in increased cerebrovascular permeability in this model as well as suggesting that the alterations of cerebrovascular permeability observed previously in angio-tensin-induced acute hypertension occur due to the hypertensive state and are not a direct drug effect of exogenous angiotensin.

Animals↗
PubMed1996

Regulation of receptor-mediated endocytosis by Rho and Rac.

Pinocytosis and membrane ruffling are among the earliest and most dramatic cellular responses to stimulation by growth factors or other mitogens. The small Ras-related G proteins Rho and Rac have a regulatory role in membrane ruffling and activated Rho has been shown to stimulate pinocytosis when microinjected into Xenopus oocytes. In contrast to these well established effects of Rho and Rac on plasma membrane morphology and bulk pinocytosis, there has been no evidence for their involvement in the regulation of receptor-mediated endocytosis in clathrin-coated pits. Here we show that activated Rho and Rac inhibit transferrin-receptor-mediated endocytosis when expressed in intact cells. Furthermore, we have reconstituted these effects in a cell-free system and established that Rho and Rac can regulate clathrin-coated vesicle formation.

Cell Line↗
PubMed1997

Loss of binding and entry of liposome-DNA complexes decreases transfection efficiency in differentiated airway epithelial cells.

The target cells for gene therapy of cystic fibrosis lung disease are the well differentiated cells that line airway lumens. Employing cultures of airway epithelial cells that grow like "islands" and exhibit a continuum of cellular differentiation, we studied the mechanisms that render well differentiated cells more difficult to transfect with cationic liposomes than poorly differentiated cells. The poorly differentiated cells at the edge of the islands were transfectable with liposome-DNA complexes (pCMVbeta:LipofectACE = 1:5 (w/w)), whereas the more differentiated cells in the center of the islands were not. Evaluation of the steps leading to lipid-mediated transfection revealed that edge cells bound more liposome-DNA complexes, in part due to a more negative surface charge (as measured by cationized ferritin binding), and that edge cells internalized more liposome-DNA complexes than central cells. Edge cells exhibited receptor-mediated endocytosis of LDL, pinocytosis of 10-nm microspheres, and phagocytosis of 2-microm microspheres, whereas central cells were only capable of receptor-mediated endocytosis. Cytochalasin B, which inhibited pinocytosis by 65% and phagocytosis by 93%, decreased edge cell liposome-DNA complex entry by 50%. Potassium depletion, which decreased phagocytosis by >90% but had no effect on pinocytosis, inhibited edge cell liposome-DNA complex entry by 71%. These results indicate that liposome-DNA complexes enter edge cells via phagocytosis and that this pathway is not detectable in central cells. In conclusion, both reduced negative surface charge and absence of phagocytosis internalization pathways in relatively differentiated cells may explain differentiation-dependent decrements in cationic liposome-mediated gene transfer in airway epithelia.

Animals↗
PubMed1977

Binding, internalization, and degradation of high density lipoprotein by cultured normal human fibroblasts.

Comparative studies were made of the metabolism of plasma high density lipoprotein (HDL) and low density lipoprotein (LDL) by cultured normal human fibroblasts. On a molar basis, the surface binding of (125)I-HDL was only slightly less than that of (125)I-LDL, whereas the rates of internalization and degradation of (125)I-HDL were very low relative to those of (125)I-LDL. The relationships of internalization and degradation to binding suggested the presence of a saturable uptake mechanism for LDL functionally related to high-affinity binding. This was confirmed by the finding that the total uptake of (125)I-LDL (internalized plus degraded) at 5 micro g LDL protein/ml was 100-fold greater than that attributable to fluid or bulk pinocytosis, quantified with [(14)C]sucrose, and 10-fold greater than that attributable to the sum of fluid endocytosis and adsorptive endocytosis. In contrast, (125)I-HDL uptake could be almost completely accounted for by the uptake of medium during pinocytosis and by invagination of surface membrane (bearing bound lipoprotein) during pinocytosis. These findings imply that, at most, only a small fraction of bound HDL binds to the high-affinity LDL receptor and/or that HDL binding there is internalized very slowly. The rate of (125)I-HDL degradation by cultured fibroblasts (per unit cell mass) exceeded an estimate of the turnover rate of HDL in vivo, suggesting that peripheral tissues may contribute to HDL catabolism. In accordance with their differing rates of uptake and cholesterol content, LDL increased the cholesterol content of fibroblasts and selectively inhibited sterol biosynthesis, whereas HDL had neither effect.

Binding Sites↗
PubMed1995

Kinetic modelling of liposome degradation in peritoneal macrophages.

The objective of this study was to quantify and model the degradation process of liposomes in peritoneal macrophages (PMs). Iodinated albumin (125I-alb) was chosen to be the marker of liposome degradation. The time course of the degradation of free 125I-alb after pinocytosis by PMs followed first-order kinetics with a half-life of 23 min. The degradation of liposomally encapsulated 125I-alb was also quantified. Kinetic modelling of liposome degradation indicated the existence of two kinetically different processes, one with a half-life of 13 min and the other with a half-life of 7.5 h. Comparing the degradation of liposomal and free 125I-alb suggested that 125I-alb was delivered to lysosomes much faster through phagocytosis than pinocytosis. These results indicate that the intracellular degradation kinetics of pinosomes and phagosomes is different. This method can quantify the rate and extent of liposomal degradation in macrophages and provide kinetic information on the intracellular destiny of liposomally encapsulated compounds.

Albumins↗
PubMed1996

Different cytokines regulate antigen uptake and presentation of a precursor dendritic cell line.

Langerhans cells (LC) and dendritic cells (DC) need to be activated in order to perform their antigen-presenting function. In this study, we explored the influence of cytokines on the uptake and presentation of protein antigens by the retrovirally immortalized myeloid cell line FSDC. This cell line was generated from mouse fetal skin and was previously shown to have the characteristics of early DC precursors. Both FSDC and bone marrow-derived DC (BM-DC) were more effective in the pinocytosis of FITC-conjugated ovalbumin (FITC-OVA) and dextran (FITC-DX) than B cells or macrophages. Pretreatment of FSDC with granulocyte/macrophage colony-stimulating factor (GM-CSF) +/- interleukin (IL)-4 enhanced the pinocytic uptake of FITC-OVA and FITC-DX, but did not induce antigen-presenting capacity. In contrast, untreated FSDC or FSDC pre-incubated with GM-CSF +/- IL-4 suppressed T cell responses. Treatment of FSDC with IFN-gamma reduced pinocytosis but increased the expression of MHC and co-stimulatory/adhesion molecules and promoted efficient presentation of OVA protein or peptide to the specific DO11.10 T cell hybridoma or to naive CD4+ T cells from DO11.10 TCR-transgenic mice. The results suggest that antigen uptake and antigen presentation in DC are regulated by different cytokine signals provided by the surrounding tissue.

Adjuvants, Immunologic↗
PubMed1990

Caffeine, an inhibitor of endocytosis in Dictyostelium discoideum amoebae.

The effect of the trimethylxanthine, caffeine, was examined on the growth and endocytosis pathways of the vegetative amoebae of the cellular slime mold Dictyostelium discoideum. Caffeine at concentrations of 1.5-3 mM was found to inhibit axenic growth, fluid-phase pinocytosis, and secretion of lysosomal enzymes. Cell viability was unaffected by incubation for 16 hours with 5 mM caffeine but decreased markedly thereafter. Phagocytosis of the bacterium Escherichia coli by Dictyostelium amoebae was also inhibited by caffeine, although at concentrations twofold to threefold higher. Caffeine rapidly entered into amoebae to reach an equilibrium between extracellular and intracellular concentrations, and it was not appreciably metabolized by Dictyostelium. Inhibition of growth and endocytosis was reversible upon removal of the drug and was partially counteracted by 10 mM adenosine. As caffeine discharged intracellular calcium stores in Dictyostelium (Abe et al., 1988), its inhibitory effect on endocytosis could result from the perturbation of calcium homeostasis. In agreement with this hypothesis, the cation La3+ (10 microM), a Ca2(+)-transport inhibitor, also strongly reduced fluid-phase pinocytosis.

Acid Phosphatase↗
PubMed1989

3-deaza-adenosine inhibition of stimulus-response coupling in human polymorphonuclear leukocytes.

In an effort to define better the functional role of S-adenosyl-methionine-mediated methylation reactions in modulating polymorphonuclear (PMN) functional responses to chemotactic stimuli, we investigated the effects of 3-deaza-adenosine (3-DZA), a known inhibitor of methylation reactions in phagocytic cells, on formyl methionyl-leucyl-phenylalanine (FMLP)-induced responses in human PMN leukocytes. Using the fluorescent cyanine dye 3,3'-dipropylthiocarbocyanine (di-S-C3-(5)) as an optical probe of membrane potential we observed that 3-DZA at concentrations that inhibit FMLP-induced O2- production does not significantly alter FMLP-induced changes in transmembrane potential. Additional studies showed an inhibitory effect of 3-DZA on FMLP-induced PMN pinocytosis and to a lesser degree on FMLP-induced degranulation. However, pretreatment of PMNs with 3-DZA did not alter FMLP-induced changes in Quin-2 fluorescence, an indicator of changes in intracellular calcium levels. These findings demonstrate a dissociation between chemotactic factor-induced cell membrane depolarization, changes in intracellular calcium, and specific neutrophil functional responses and suggest that chemotactic factor-induced changes in transmembrane potential and intracellular calcium are independent of chemotactic factor-induced methylation reactions. Furthermore, 3-DZA did not alter phorbol myristate acetate induced O2- production or fluid pinocytosis indicating a stimulus specificity for the inhibitory effects of this agent on O2- production.

Aminoglycosides↗
PubMed2001

Dietary vitamin E and rainbow trout (Oncorhynchus mykiss) phagocyte functions: effect on gut and on head kidney leucocytes.

The effects of vitamin E (deficiency or supplementation) on the non-specific immune system in rainbow trout, Oncorhynchus mykiss, were evaluated. Rainbow trout were fed daily a semi-purified diet supplemented with vitamin E at 0, 28 and 295 mg x kg(-1) of diet. After 80 days of experimental feeding, the phagocytic function (respiratory burst evaluated by the CL response, phagocytosis) from gut leucocytes and head kidney enriched macrophages was measured; head kidney cell pinocytosis and serum lysozyme activity were also analysed. The results showed that some phagocyte functions were influenced by dietary vitamin E. When fish were fed the high dietary dose of vitamin E an enhancement of phagocytosis was found, but only significantly for the leucocytes isolated from the gut of rainbow trout; moreover, an impaired response was also observed in the fish fed no vitamin E for 80 days. However, no significant differences were noticed on the oxidative burst (CL) response of both gut and head kidney cells according to the dietary dose of vitamin E. Pinocytosis evaluated on head kidney cells was not influenced by dietary vitamin E. Fish fed vitamin E at 295 mg x kg(-1) had a lower serum lysozyme activity than those fed with vitamin E at 28 mg x kg(-1) and the fish fed no vitamin E for 80 days had an impaired activity. Thus, the present results demonstrate that altered dietary levels of vitamin E modulates the phagocytic functions of gut leucocytes in rainbow trout; moreover, the vitamin E diet effect seems to be greater on the local intestinal response as compared to systemic (head kidney). Taken together, this study confirms the crucial role of gut phagocytes in mucosal non-lymphoid defences in fish.

Animals↗
PubMed1982

Evidence for intracellular digestion of an exogenous protein by rat peritoneal macrophages.

The differential effects of lower incubation temperature on the digestion of formaldehyde-denatured 125I-labelled bovine serum albumin by intact rat peritoneal macrophages and by macrophage homogenates were studied. Digestion by intact macrophages was abolished at 20 degrees C and below, but a near-linear relationship between digestion rate and temperature between 37 degrees C and 5 degrees C was found with homogenates. Several known inhibitors of pinocytosis were found to inhibit the generation of digestion products from 125I-labelled albumin by macrophages. It is concluded that pinocytosis is an obligatory step in the catabolism of exogenous proteins by macrophages.

Animals↗
PubMed1975

Electron microscopical study of initial and final stages of fowl plague virus-replication in chick embryo cells.

Cellular uptake of fowl plague virus occurs 10-30 minutes after inoculation of chick embryo cells. The penetration of the virions is by pinocytosis (viropexis); fusion with the cellular membrane has not been observed. After pinocytosis the virions become gradually disintegrated. Budding of newly formed virions from the cellular membrane starts 3 hours post inoculation (p.i.) and reaches its maximum 8 hours p.i. At the same time budding takes place into electron microscopically empty and autophagic vacuoles. Eight hours p.i. about 3 per cent of the infected cells show budding of virions from the surface and into cytoplasmic vacuoles. Labelling of the cellular membrane with ruthenium red demonstrated that these cytoplasmic vacuoles are not simple cross-sections of invaginations of the cellular membrane. Cluster-like structures were found at 6 hours p.i. in the nuclei of infected cells; however, the suggestion that the clusters develop from nucleoli could not be confirmed.

Animals↗
PubMed1989

Endocytosis and inositol hexakisphosphate levels in ras transformants of Dictyostelium discoideum amoebae.

Fluid-phase pinocytosis kinetics and lysosomal enzyme secretion parameters were measured in Dictyostelium discoideum amoebae constructed from strain AX3 by transformation with a multicopy plasmid carrying either a normal ras gene (ras-Gly12), a mutated ras gene (ras-Thr12) or by the vector carrying the geneticin resistance gene only (pDNEO2). It was found that the pinocytosis rate and extent as well as the lysosomal enzyme secretion were slightly different in the three strains. These changes, however, were related to minor modifications of the cellular volumes. The overall concentration of inositol hexakisphosphate was similar in the three strains.

Dextrans↗
PubMed1984

Intracellular transport and esterification of exchangeable cholesterol in cultured human lung fibroblasts.

We have studied the intracellular fate of exchangeable cholesterol in a model system with lipid vesicles (cholesterol/phospholipid mole ratio 1:1) and cultured human lung fibroblasts. Exchangeable [3H]cholesterol in lipid vesicles was readily incorporated into cellular plasma membranes and transported to intracellular esterification sites. The formation of [3H]cholesteryl esters was not affected by cytoskeleton-disrupting drugs. A reduction of cellular pinocytosis by 75% did not reduce the formation of tracer-labelled esters, suggesting that membrane flow due to the energy-dependent pinocytosis is no major contributor to the intracellular transport of molecular cholesterol between plasma membranes and esterification sites. The formation of [3H]cholesteryl esters was not significantly affected by energy poisons (NaF and KCN) but was inhibited (to 50%) by chloroquine at 50 microM. This may indicate that membrane-derived cholesterol passes through the lysosomal compartment on its way to intracellular esterification sites.

Cells, Cultured↗