Tubular lysosomes accompany stimulated pinocytosis in macrophages

A network of tubular lysosomes extends through the cytoplasm of J774.2 macrophages and phorbol ester-treated mouse peritoneal macrophages. The presence of this network is dependent upon the integrity of cytoplasmic microtubules and correlates with high cellular rates of accumulation of Lucifer Yellow (LY), a marker of fluid phase pinocytosis. We tested the hypothesis that the efficiency of LY transfer between the pinosomal and lysosomal compartments is increased in the presence of tubular lysosomes by asking how conditions that deplete the tubular lysosome network affect pinocytic accumulation of LY. Tubular lysosomes were disassembled in cells treated with microtubule-depolymerizing drugs or in cells that had phagocytosed latex beads. In unstimulated peritoneal macrophages, which normally contain few tubular lysosomes and which exhibit relatively inefficient transfer of pinocytosed LY to lysosomes, such treatments had little effect on pinocytosis. However, in J774 macrophages and phorbol ester-stimulated peritoneal macrophages, these treatments markedly reduced the efficiency of pinocytic accumulation of LY. We conclude that a basal level of solute accumulation via pinocytosis proceeds independently of the tubular lysosomes, and that an extended tubular lysosomal network contributes to the elevated rates of solute accumulation that accompany macrophage stimulation. Moreover, we suggest that the transformed mouse macrophage cell line J774 exhibits this stimulated pinocytosis constitutively.     

Fluid-phase endocytosis by isolated rat adipocytes

We have developed an assay, which uses radiolabeled sucrose as the marker, to measure the rate of fluid-phase endocytosis in isolated rat adipocytes. In addition, the assay was adapted to allow measurement of the release of sucrose from previously loaded cells (fluid-phase exocytosis). Adipocytes take up sucrose at an approximately linear rate for at least 1.5 hours. A portion of the pinocytosed sucrose is rapidly (half-time about 20 minutes) returned to the medium. The minimal value for fluid uptake by endocytosis is 57 nl/10(6) cells-h at 37 degrees C; this value corresponds to the formation of 110,000 endocytic vesicles of 100-nm diameter per cell per hour and the internalization of about 20% of the plasma membrane per hour. Insulin caused a small and variable increase in the rate of sucrose uptake. The average increase of 31% from 11 experiments is statistically significant at the level of P less than 0.01. A small insulin effect upon the uptake of the calcium complex of [14C]EDTA was also observed. Since this complex was taken up at 2.5 times the rate of sucrose, it probably entered by a combination of fluid-phase and adsorptive pinocytosis. Insulin did not elicit a significant change in the rate of sucrose release from preloaded cells.     

A human T lymphoblastic cell line lacks lamins A and C

Lamins A, B and C, the three major proteins of nuclear envelope, constitute a class of intermediate filament polypeptides. We have compared the amount of these polypeptides in two human cell lines, epithelial HeLa cells and T lymphoblasts KE 37. It was found that the three lamins were present in roughly equimolar stoichiometry in HeLa cells, while lamin B was the unique lamin component in T lymphoblasts. Moreover, 3-kb mRNA of lamin A and 2.1-kb mRNA of lamin C were detected with a human cDNA probe in HeLa cells but not in T lymphoblasts. These results suggest that (i) lamin B can build up the lamina structure in actively dividing somatic cells by itself, and (ii) lamin expression in lymphoid cells may be subject to important quantitative variations. Comparison of the lamin composition of human cloned T lymphocytes and Epstein-Barr virus-transformed human B lymphocytes confirmed this statement. The lamin B level was nearly equivalent in both cells but the content of lamins A and C varied to a large extent, being low in T cells and high in B cells.     

Exocytosis of pinocytic contents by Chinese hamster ovary cells

The extent of exocytosis of pinocytic vesicle contents was studied in suspension-cultured Chinese hamster ovary (CHO) cells using horseradish peroxidase (HRP) as a pinocytic content marker. HRP was shown to be internalized via fluid-phase pinocytosis in CHO cells. After an HRP pulse of 2.5-10 min a rapid decrease of 30-50% in cell-associated HRP activity was observed within 10-20 min at 37 degrees C. During this time the loss of cell-associated HRP was accompanied by an equivalent increase in extracellular HRP. After this rapid exocytosis of HRP, the remaining peroxidase activity decreased with a t1/2 of 6-8 h, the known lysosomal half-life of HRP. In pulse-chase experiments HRP was chased into a nonexocytic compartment. Based on cell fractionation and electron microscopic experiments, this nonexocytic compartment was identified as a lysosome and the compartment from which exocytosis occurs as a pinosome. The occurrence of pinocytic content exocytosis in cultured fibroblasts suggests that exocytosis of pinocytic vesicle contents is a general phenomenon.     

Membrane flow during pinocytosis. A stereologic analysis

HRP has been used as a cytochemical marker for a sterelogic analysis of pinocytic vesicles and secondary lysosomes in cultivated macrophages and L cells. Evidence is presented that the diaminobenzidine technique (a) detects all vaculoes containing encyme and (b) distinguishes between incoming pinocytic vesicles and those which have fused with pre-existing lysosomes to form secondary lososomes. The HRP reactive pinocytic vesicle spaces fills completely within 5 min after exposure to enzyme, while the secondary lysosome compartment is saturated in 45--60 min. The size distribution of sectioned (profile) vaculoe diameters was measured at equilibrium and converted to actual (spherical) dimensions using a technique modified from Dr. S. D. Wicksell. The most important findings in this study have to do with the rate at which pinocytosed fluid and surface membrane move into the cell and on their subsequent fate. Each minute macrophages form at least 125 pinocytic vesicles having a fractional vol of 0.43% of the cell's volume and a fractional area of 3.1% of the cell's surface area. The fractional volume and surface area flux rates for L cells were 0.05% and 0.8% per minute respectively. Macrophages and L cells thus interiorize the equivalent of their cell surface area every 33 and 125 min. During a 3-period, the size of the secondary lysosome compartment remains constant and represents 2.5% of the cell volume and 18% of the surface area. Each hour, therefore, the volume and surface area of incoming vesicles is 10 times greater than the dimensions of the secondary lysosomes in both macrophages and L cells. This implies a rapid reduction in vesicle size during the formation of the secondary lysosome and the egress of pinocytosed fluid from the vacuole and the cell. In addition, we postulate that membrane components of the vacuole are subsequently recycled back to the cell surface.     

Magnetic separation of pinocytic vesicles of defined age from Entamoeba histolytica

We describe a rapid and simple method to isolate pinocytic vesicles of defined age (residing time within the cell) from Entamoeba histolytica. Amoebas are allowed to pinocytize for greater than 5 min a suspension of superparamagnetic iron oxide particles, washed, and resuspended for predetermined periods (up to 150 min) in iron oxide-free medium. Subsequently, the cells are homogenized and iron oxide-containing vesicles are separated magnetically. Recovery of vesicles (estimated with fluorescein isothiocyanate-dextran as a quantitative marker for pinocytosis) was 20-40%. Contamination with "older" vesicles or with plasma membrane (estimated with fluorescein isothiocyanate-dextran and with fluorescein isothiocyanate-conjugated, succinylated concanavalin A, respectively) was negligible. Using this method we obtained evidence that in E. histolytica, contrary to the situation in animal cells, pinocytic vesicles within 150 min after invagination neither shrunk nor fused with each other to any significant extent. The method should be generally applicable to protozoa for the isolation of pinocytic vesicles and digestive vacuoles.     

Exocytosis of pinocytosed fluid in cultured cells: kinetic evidence for rapid turnover and compartmentation

The uptake and fate of pinocytosed fluid were investigated in monolayers of pulmonary alveolar macrophages and fetal lung fibroblasts using the fluid-phase marker, [14C]sucrose. Initial experiments revealed that cellular accumulation of chromatographically repurified [14C]sucrose was not linear with incubation time. Deviation from linearity was shown to be due to constant exocytosis of accumulating marker. Chromatographic analysis revealed that the cells were unable to metabolize sucrose and were releasing it intact by a process that was temperature-sensitive but not dependent on extracellular calcium and magnesium. A detailed analysis of the kinetics of exocytosis was undertaken by preloading cells with [14C]sucrose for various lengths of time and then monitoring the appearance of radioactivity into isotope- free medium. Results indicated that modeling the process of fluid-phase pinocytosis and subsequent exocytosis required at least two intracellular compartments in series, one compartment being of small size and turning over very rapidly (t1/2 = 5 min in macrophages, 6--8 min in fibroblasts) and the other compartment being apparently larger in size and turning over very slowly (t1/2 = 180 min in macrophages, 430--620 min in fibroblasts). Computer-simulation based on this model confirmed that the kinetics of efflux faithfully reflected the kinetics of influx and that the rate of efflux completely accounted for the deviation from linearity of accumulation kinetics. Moreover, the sizes of the compartments and magnitude of the intercompartment fluxes were such that the majority of fluid internalized in pinocytic vesicles was rapidly returned to the extracellular space via exocytosis. This result provides direct experimental evidence for a process previously thought necessary based solely on morphological and theoretical considerations. Furthermore, the turnover of pinocytosed fluid was so dynamic that accumulation deviated from linearity even within the first few minutes of incubation. We were able to show that the kinetics of exocytosis allowed calculation of the actual pinocytic rate, a rate that was nearly 50% greater than the apparent initial rate obtained from the slope of the uptake curve over the first 10 min.     

PINOCYTOSIS IN FIBROBLASTS : Quantitative Studies In Vitro

Horseradish peroxidase (HRP) was used as a marker to determine the rate of ongoing pinocytosis in several fibroblast cell lines. The enzyme was interiorized in the fluid phase without evidence of adsorption to the cell surface. Cytochemical reaction product was not found on the cell surface and was visualized only within intracellular vesicles and granules. Uptake was directly proportional to the administered concentration of HRP and to the duration of exposure. The rate of HRP uptake was 0.0032–0.0035% of the administered load per 10⁶ cells per hour for all cells studied with one exception: L cells, after reaching confluence, progressively increased their pinocytic activity two- to fourfold. After uptake of HRP, L cells inactivated HRP with a half-life of 6–8 h. Certain metabolic requirements of pinocytosis were then studied in detail in L cells. Raising the environmental temperature increased pinocytosis over a range of 2–38°C. The Q₁₀ was 2.7 and the activation energy, 17.6 kcal/mol. Studies on the levels of cellular ATP in the presence of various metabolic inhibitors (fluoride, 2-desoxyglycose, azide, and cyanide) showed that L cells synthesized ATP by both glycolytic and respiratory pathways. A combination of a glycolytic and a respiratory inhibitor was needed to depress cellular ATP levels as well as pinocytic activity to 10–20% of control values, whereas drugs administered individually had only partial effects. In spite of the availability of an accurate quantitative assay for fluid and solute uptake, the function of pinocytosis in tissue culture cells remains unknown.     

Analysis of the effect of amines on inhibition of receptor-mediated and fluid-phase pinocytosis in rabbit alveolar macrophages

Incubation of rabbit alveolar macrophages in vitro with methyl amine led to a time- and concentration-dependent inhibition of uptake of alpha macroglobulin-125I-trypsin complexes (alpha M-125I-T). Upon addition of methyl amine (50 mM) to cells prelabeled with alpha M-125I-T there was a rapid inhibition of lysosomal catabolism of internalized ligand. In the absence of ligand, incubation of cells with 50 mM methyl amine led to a 40-70% decrease in surface-receptor number. The methyl amine-induced decrease in surface-receptor number only occurred in metabolically active cells since cells incubated at 0 degrees C, or treated with N-ethyl maleimide and incubated at 37 degrees C, did not show the effect. Incubation of cells at 37 degrees C with methyl amine also effected a 40-70% decrease in fluid-phase pinocytosis. Although there was a decline in surface-receptor number, the remaining population of receptors were capable of mediating (at least) one round of ligand internalization. However, further ligand uptake was prevented. Data demonstrate that although receptors were present on cell surfaces, they were incapable of mediating ligand internalization. Incubation of macrophages with chloroquine at 37 degrees C for 60 min also led to a disappearance of receptors, and a concomitant reduction in fluid-phase pinocytosis.     

Pinocytosis and degradation of exogenous proteins by cystinotic fibroblasts

Lysosomes of cystinotic human fibroblasts contain over 100-times the normal concentration of cystine. The high cystine concentration (probably in the millimolar range) might be expected to inhibit intralysosomal protein breakdown. A comparison of pinocytosis and degradation of five ¹²⁵I-labelled proteins (bovine serum albumin, formaldehyde-denatured bovine serum albumin, bovine pancreatic ribonuclease A and porcine lactate dehydrogenase isoenzymes H4 and M4) by human fibroblasts has been made, using one cystinotic and two normal cell-lines. The proteins each entered fibroblasts by adsorptive pinocytosis and were then degraded within the lysosomes by enzymes susceptible to leupeptin, the thiol-proteinase inhibitor. Each protein was captured by the fibroblasts at a characteristic rate, which was not different in cystinotic cells. Normal and cystinotic fibroblasts did not differ in their proteolytic capacity, as measured in extracts of disrupted cells. In intact fibroblasts, four of the five proteins were rapidly and fully digested following pinocytosis, in both cystinotic and normal cells. However, with formaldehyde-denatured albumin, the most resistant to degradation of the proteins tested, or with some other proteins in the presence of leupeptin, when the proteolytic capacity of lysosomes is diminished, intralysosomal degradation of pinocytosed protein was incomplete. Moreover, under these conditions, cystinotic cells demonstrated a lower rate of protein digestion than normal cells. It is concluded that pinocytic capture, rather than intralysosomal proteolysis, is commonly the rate-limiting step in the overall process of uptake and degradation of proteins by fibroblasts, and that intralysosomal cystine inhibits digestion of pinocytosed protein only in circumstances when degradation becomes the rate-limiting step.     

Selective iodination and polypeptide composition of pinocytic vesicles

We describe a method for the specific radioiodination of pinocytic vesicles (PVs) based upon the simultaneous endocytosis of lactoperoxidase (LPO) and glucose oxidase (GO). Initial experiments indicated that LPO was interiorized by the macrophage cell line J774 by fluid phase pinocytosis and without detectable binding to the plasma membrane (PM). Interiorization varied linearly with enzyme concentration and exposure time, was temperature dependent, and was undetectable at 4 degrees C. Employing EM cytochemistry, LPO activity was restricted to PVs after a 3- to 5-min pulse at 37 degrees C. These results formed the basis of the method for iodinating the luminal surface of PVs: 5-min exposure to both LPO and GO at 37 degrees C followed by washes and iodination (addition of 125I and glucose) at 4 degrees C. Enzyme-dependent incorporation of iodide into the polypeptides of both PV membrane and contents occurred. Several lines of evidence indicated that there was selective labeling of PV as opposed to PM. Iodination did not occur if the pinocytic uptake of LPO ad GO was inhibited by low temperature. EM autoradiography showed a cytoplasmic localization of grains, whereas a clear PM association was evident with surface labeling. LPO was iodinated only after PV labeling and was present within organelles demonstrating latency. After PV iodination, > 75% of several labeled membrane antigens could be immunoprecipitated by monoclonal antibodies only after cell lysis. In contrast, all labeled antigens were accessible to antibody on intact cells after surface labeling. The polypeptide compositions of PM and PV membrane were compared by SDS polyacrylamide gel electrophoresis and by quantitative immune precipitation using a panel of anti-J774 monoclonal antibodies. The electrophoretic profiles of iodinated proteins (15-20 bands) were strikingly similar in NP-40 lysates of both PV and PM iodinated cells. In addition, eight membrane antigens examined by immune precipitation, including the trypsin-resistant immunoglobulin (Fc) receptor and the H-2Dd histocompatibility antigen, were found to be iodinated to the same relative extents by both labeling procedures. We conclude that PV membrane is formed from a representative sample of PM polypeptide components.     

Chinese hamster ovary cell lysosomes rapidly exchange contents

We have used cell fusion to address the question of whether macromolecules are rapidly exchanged between lysosomes. Donor cell lysosomes were labeled by the long-term internalization of the fluid- phase pinocytic markers, invertase (sucrase), Lucifer Yellow, FITC- conjugated dextran, or Texas red-conjugated dextran. Recipient cells contained lysosomes swollen by long-term internalization of dilute sucrose or marked by an overnight FITC-dextran uptake. Cells were incubated for 1 or 2 h in marker-free media before cell fusion to clear any marker from an endosomal compartment. Recipient cells were infected with vesicular stomatitis virus as a fusogen. Donor and recipient cells were co-cultured for 1 or 2 h and then fused by a brief exposure to pH 5. In all cases, extensive exchange of content between donor and recipient cell lysosomes was observed at 37 degrees C. Incubation of cell syncytia at 17 degrees C blocked lysosome/lysosome exchange, although a "priming" process(es) appeared to occur at 17 degrees C. The kinetics of lysosome/lysosome exchange in fusions between cells containing invertase-positive lysosomes and sucrose-positive lysosomes indicated that lysosome/lysosome exchange was as rapid, if not more rapid, than endosome/lysosome exchange. These experiments suggest that in vivo the lysosome is a rapidly intermixing organellar compartment.     

Quantitation of fluid phase uptake in paramecium. 1. Kinetics in the cells blocked in phagocytic activity

Uptake of horseradish peroxidase (HRP) was quantified in the unicellular eukaryote Paramecium aurelia. About 80% of the total HRP accumulated within 20 min entered the cells via a fluid phase pathway as demonstrated measuring the HRP internalization in the presence of yeast mannan. The rate of HRP accumulation was concentration-dependent and was found to be linear over the range of 50–500 μg HRP per ml of the extracellular medium. During the first 10 min of exposure to HRP, the mannan-uninhibitable uptake was found to reach 1.2–1.65 ng HRP per mg protein (depending on the concentration of the marker in the medium), which corresponds to 0.68 fl per cell/min. Accumulation of HRP reached a plateau within the next 10–15 min and its intracellular uptake was 2–2.55 ng HRP per mg protein. When the phagocytic activity of the cells was blocked with 1-propranolol, the amount of cell-accumulated HRP was 1.8–2.1-fold higher than in the control and the rate of the marker uptake within the first 10 min of incubation reached 1.114 fl per cell/min.     

Serum-free culture of resting, PHA-stimulated, and transformed lymphoid cells, including hybridomas

A chemically-defined medium containing casein, insulin, testosterone, transferrin, and linoleic acid (CITTL) has been developed for the culture of lymphoid cells. Resting human mononuclear leukocytes can be maintained in CITTL for up to 8 weeks with excellent viability. In the absence of mitogen, undifferentiated monocytes become the predominant cell type by 7–11 days as assessed by morphology, population distribution of cell sizes, and fluorescence-activated cell sorter analysis. Phytohemagglutinin (PHA)-induced proliferation is retained in these cultures for 7–10 days and results in a population of 85% T lymphoblasts. T lymphoblasts survive for 2 weeks in CITTL following mitogenic stimulation and proliferate for up to 5 weeks in CITTL if supplemented with 2-mercaptoethanol and conditioned medium containing crude T-cell growth factor. In addition to supporting proliferation of normal lymphoid cells, CITTL also sustains growth of human and murine hybridomas, as well as a variety of transformed lymphoid cell lines. The ability of a minimum essential medium supplemented with 5 or 6 factors to support growth of normal and malignant lymphoid cells suggests that only a limited number of extracellular signals are required for mitogenesis of these cells.     

Fluid phase endocytosis and galactosyl receptor-mediated endocytosis employ different early endosomes.

Endocytosis may originate both in coated pits and in uncoated regions of the plasma membrane. In hepatocytes it has been shown that fluid phase endocytosis (here defined as 'pinocytosis') is unaffected by treatments that arrest coated pit-mediated endocytosis, indicating that pinocytosis is primarily a clathrin-independent process. In this study we have tried to determine possible connections between pinocytosis and clathrin-dependent endocytosis in rat hepatocytes by means of subcellular fractionation, electron microscopy, and by assessing the influence of inhibitors of clathrin-dependent endocytosis on pinocytosis. As marker for clathrin-dependent endocytosis was used asialoorosomucoid (AOM) labelled with [(125)I]tyramine cellobiose ([(125)I]TC). [(125)I]TC-labelled bovine serum albumin ([(125)I]TC-BSA) was found to be a useful marker for pinocytosis. Its uptake in the cells is not saturable, and any remnants of [(125)I]TC-BSA associated with the cell surface could be removed by incubating the cells with 0.3% pronase at 0 degrees C for 60 min. The data obtained by electron microscopy and by subcellular fractionation suggested that early after initiation of uptake (<15 min) [(125)I]TC-BSA and [(125)I]TC-AOM were present in different endocytic vesicles. The two probes probably join prior to their entrance in the lysosomal compartment. The relation between endocytosis via coated pits and pinocytosis was also studied with techniques that induced a selective density shift either in the clathrin-dependent pathway (by AOM-HRP) or in the pinocytic pathway (by allowing uptake of AuBSA). Both treatments indicated that the two probes ([(125)I]TC-AOM and [(125)I]TC-BSA) were early after uptake, at least partly, in separate endocytic compartments. The different distribution of the fluid phase marker and the ligand (internalised via coated pits) was not due to a difference in the rate at which they enter a later compartment, since a lowering of the incubation temperature to 18 degrees C, which should keep the probes in the early endosomes, did not affect their early density distribution. Incubation of cells in a hypertonic medium reduced uptake both of [(125)I]TC-AOM and [(125)I]TC-BSA; the uptake of [(125)I]TC-AOM was, however, reduced much more than that of the fluid phase marker. This finding supports the notion that the two probes enter the cells via different routes.     

Convergent potency of internalized gelonin immunotoxins across varied cell lines, antigens, and targeting moieties

Gelonin-based immunotoxins vary widely in their cytotoxic potency as a function of antigen density, target cell internalization and trafficking kinetics, and conjugate properties. We have synthesized novel gelonin immunotoxins using two different binding scaffold types (single-chain antibody variable fragments and fibronectin domains) targeting two different tumor antigens (carcinoembryonic antigen and EGF receptor). Constructs were characterized using an antigen-negative cell line (HT-1080), cell lines positive for each antigen (HT-1080(CEA) for carcinoembryonic antigen and A431 for EGF receptor), and a cell line positive for both antigens (HT-29). Immunotoxins exhibited K(d) values between 8 and 15 nm and showed 20-2000-fold enhanced cytotoxicity compared with gelonin (IC(50) ～ 0.25-30 nM versus 500 nM). Using quantitative fluorescence flow cytometry, we measured internalization of gelonin (via pinocytosis) and gelonin-based immunotoxins (via antigen-dependent, receptor-mediated endocytosis). Results were matched with cytotoxicity measurements made at equivalent concentration and exposures. Unexpectedly, when matched internalization and cytotoxicity data were combined, a conserved internalized cytotoxicity curve was generated that was common across experimental conditions. Considerable variations in antigen expression, trafficking kinetics, extracellular immunotoxin concentration, and exposure time were all found to collapse to a single potency curve on the basis of internalized immunotoxin. Fifty percent cytotoxicity occurred when ～ 5 × 10(6) toxin molecules were internalized regardless of the mechanism of uptake. Cytotoxicity observed at a threshold internalization was consistent with the hypothesis that endosomal escape is a common, highly inefficient, rate-limiting step following internalization by any means tested. Methods designed to enhance endosomal escape might be utilized to improve the potency of gelonin-based immunotoxins.     

The effects of incubation of marrow in tissue fragments in vitro on the growth of adherent cells from this marrow

Femoral marrow was either cultured as a single cell suspension immediately following collection from the donor mouse or following 4 day incubation in vitro of the whole marrow shaft. Several parameters of growth of adherent, i.e. composed of fibroblastoid cells and macrophages, colonies were determined following 14 day culture in Dulbecco medium at 37 degrees C. These included: number and diameter of macroscopic colonies, number of macrophages per fibroblastoid cell inside the colonies and per eyefield in intercolony spaces, number of cells in supernatant from the culture. The 4 day incubation of marrow fragments in vitro (Dulbecco medium, 37 degrees C) doubled the number of adherent colonies grown from this marrow and, moreover, the colonies formed were increased in size. Other parameters of cell growth in these cultures were unchanged. These data suggest that under conditions of in vitro incubation of marrow shaft (close cell-to-cell contact) marrow fibroblastoid colony forming units (MF-CFU) are stimulated to self-renewal.     

Electron microscopy study of deoxyribonuclease penetration into chick embryo fibroblasts and the effect of the enzyme on the cells

Peroxidase labeled DNase (0.25 g/ml) penetrated into the chick fibroblast culture cells by means of pinocytosis to be localized in pinocytotic vacuoles. Within a 24 hour-incubation of the cell culture with DNase a relative volume of vacuoles in cells increased from 4.6 to 19.5 per cent, the majority of pinocytic vacuoles being filled with labeled DNase. The incubation of cells with non-labeled DNase (24 hours) did not damage DNA-containing cell structures. The data obtained permit to explain the selective effect of the enzyme on the viral DNA and the inhibition of reproduction of DNA-containing viruses by the interaction of the viral DNA and DNase inside pinocytic vacuoles which protect the cells from the enzyme damaging effect.     

Effect of suramin on pinocytosis by resident rat peritoneal macrophages: an analysis using four different substrates

The effect of suramin on pinocytosis and intralysosomal proteolysis by resident rat peritoneal macrophages cultured in vitro has been studied. Suramin had little effect on the rate of pinocytic uptake of two non-adsorptive substrates [14C]sucrose and [3H]dextran, but unexpectedly enhanced uptake of a third, 125I-labelled polyvinylpyrrolidone (PVP). Since this enhanced uptake was completely abolished by NaF at a concentration known to inhibit pinocytosis, it clearly represented an increased internalization of substrate and not merely a superficial binding to the cell surface. It was concluded that suramin (i) does not affect the rate of formation of pinocytic vesicles but (ii) acts as a bivalent ligand, binding to both the macrophage surface and the 125I-labelled polyvinylpyrrolidone, thus converting a non-adsorptive into an adsorptive substrate. Suramin (500 micrograms/ml) decreased both the rate of uptake of formaldehyde-denatured 125I-labelled bovine serum albumin (BSA) (an adsorptive substrate) and the rate of its subsequent intracellular degradation. Thus, depending on the substrate chosen to measure pinocytosis, the same modifier may stimulate or inhibit uptake or be without effect.     

Membrane cycling and macrovacuolation under the influence of cytochalasin: kinetic and morphometric studies

Fibroblasts exposed to higher doses of cytochalasin accumulate very big discrete endoplasmic vacuoles, the membrane of which is derived by internalization of plasmalemma. Morphometry confirms that the amount of surface interiorized is equal to the difference between the original cell surface area (before CD) and the reduced surface area measurable after CD-induced rounding. Correspondingly, there is a nearly two-fold increase in the activity of the ectoenzyme 5'-nucleotidase (a marker for plasma membrane) internally within the cytoplasm, after treatment with CD. Macrovacuolation increases cell volume by approximately 30%. Surface membrane is internalized as micropinocytotic vesicles at a rate measurable by the accumulation of HRP, a marker of fluid-phase pinocytosis. Uptake of HRP is shown to be enhanced at all times during exposure to CD, and is balanced by accelerated exocytic recycling of membrane except during a phase (approximately 4-8 hr) in which pinocytic uptake exceeds exocytosis. Vesicular membrane accumulated intracellularly in this period is retained in the endoplasm, and by successive fusions forms vacuoles in close approximation to microfilament aggregates. Once established, this new macrovacuolar membrane compartment is in dynamic equilibrium with the cell surface, and its membrane is cycled like the plasma membrane, in a mutual exchange of pinosomes between the several vacuoles and the cell surface. In drug-free medium vacuole membrane apparently reverts to the surface by pinocytotic recycling, and the cells recover normal characteristics 4-6 hr after withdrawal of cytochalasin.