Effect of alterations in the size of the vacuolar compartment on pinocytosis in J774.2 macrophages

J774.2 macrophages cultured in medium containing 10 mg/ml sucrose accumulate the sugar by pinocytosis and become highly vacuolated, due to the sugar's osmotic effect within the vacuolar compartment. When such cells are incubated in medium containing 0.5 mg/ml invertase, the enzyme reaches the sucrose vacuoles by pinocytosis, then cleaves the sugar to more permeant monosaccharides. Within 4 hours, the vacuoles shrink to smaller, phase-dense organelles (Cohn and Ehrenreich, 1969, J. Exp. Med., 129:201). We have used this reversible expansion of the lysosomal compartment to address two questions: (1) Does the increased size of the lysosomal compartment affect pinocytic accumulation of solute, and (2) what is the fate of the vacuolar membrane and its soluble content during invertase-induced vacuole shrinkage? Using lucifer yellow (LY) as a probe for pinocytic fluid influx and efflux, we found that vacuolated cells accumulated 30–50% less LY than controls and returned to higher rates of pinocytosis after invertase-induced vacuole shrinkage. A similar reduction in LY accumulation was achieved after feeding cells latex beads to increase the size of the lysosomal compartment. Thus, treatments that increased the size of the lysosomal compartment reduced solute accumulation via pinocytosis. A dramatic shrinkage of LY-containing sucrose vacuoles followed pinocytosis of invertase. Despite this reduction in size of the LY-containing vacuoles, the overall rate of LY efflux did not increase significantly during invertase-induced vacuole collapse. Electron microscopy revealed that during shrinkage, the excess vacuolar membrane was compressed into whorled membranous organelles (residual bodies), with fluid markers (colloidal gold and, by inference, LY) trapped inside. The trapping of LY inside lysosomes as J774.2 macrophages returned to their normal dimensions indicates that nearly all of the surplus membrane contents were removed from circulation as well.     

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.     

The role of the mannose/N-acetylglucosamine receptor in the pinocytosis of horseradish peroxidase by mouse peritoneal macrophages

Saccharomyces cerevisiae mannan inhibits the pinocytosis of horseradish peroxidase (HRP) by resident, thioglycollate-,proteose peptone-, and Corynebacterium parvum-elicited macrophages from 30 to 70% when 1 mg/ml HRP is used, and 65 to 87% when 250 micrograms/ml HRP is used. In contrast, HRP uptake by J774 cells, a macrophage cell line reported to have little mannose receptor activity, is inhibited only about 25% by mannan. HRP uptake by resident and thioglycollate-elicited (thio) macrophages is also inhibited 34 and 66% by addition of EGTA to the medium and 55 and 79% by trypsin treatment of the macrophages, respectively. The inhibitory effect of EGTA can be reversed by 1 mM excess Ca2+. High extracellular concentrations of Ca2+, in the range of 10-20 mM, however, inhibit pinocytosis in resident macrophages by about 50%. Sucrose uptake by resident macrophages is not appreciably affected by mannan. These results support the hypothesis that HRP uptake is mediated by the macrophage mannose/N-acetylglucosamine receptor. PMA stimulates fluid-phase pinocytosis of HRP by thio macrophages but does not affect receptor-mediated uptake of HRP, while the combination of adenosine, homocysteine, and erythro-9-(2-hydroxy-3-nonyl) adenine (EHNA) selectively inhibits bulk-phase uptake by thio macrophages.     

The effects of acetylated low-density lipoproteins on fluid-phase pinocytosis by macrophages

A simple method has been set up to measure the rate of fluid-phase pinocytosis in resident mouse peritoneal macrophages in culture. The method uses 125I-labelled polyvinylpyrrolidone as a nondegradable marker of fluid-phase pinocytosis. The accumulation of 125I-labelled polyvinylpyrrolidone by the cells was directly proportional to its concentration in the culture medium up to at least 200 micrograms/ml. The estimates of the rate of fluid-phase pinocytosis were reproducible within each experiment (coefficient of variation 8.5%) but varied between individual experiments. Fluid-phase pinocytosis was undetectable at 4 degrees C and reduced greatly at 37 degrees C by metabolic inhibitors and 1 mM ZnSO4. High concentrations of human acetylated low-density lipoproteins, which are taken up rapidly by macrophages, decreased the rate of fluid-phase pinocytosis by up to about 70%. The inhibition was seen after only 2 h of incubation. Unmodified low-density lipoproteins, which are taken up only slowly by macrophages, did not usually inhibit fluid-phase pinocytosis (in fact, they sometimes increased it). Modified low-density lipoprotein uptake, leading to massive lipid accumulation in macrophages in the arterial wall, has been postulated to be involved in the pathogenesis of atherosclerosis. This study raises the possibility that the rate of fluid-phase pinocytosis in these lipid-laden arterial macrophages may be reduced.     

Rapid stimulation of fluid-phase endocytosis and exocytosis by insulin, insulin-like growth factor-I, and epidermal growth factor in KB cells

Effects of growth factors on fluid-phase endocytosis and exocytosis in human epidermoid carcinoma KB cells were examined by measuring horseradish peroxidase (HRP) as a marker. Insulin, insulin-like growth factor-I (IGF-I), and epidermal growth factor (EGF) promoted HRP accumulation. They also stimulated the efflux of the preloaded HRP from the cells. From these results it follows that these growth factors stimulate the influx as well as the efflux of HRP, because the accumulation rate is the sum of the influx rate and the efflux rate. The stimulation of both HRP accumulation and HRP efflux was rapidly induced within 2-4 min of the addition of growth factors and persisted for at least 60 min. The concentrations eliciting half-maximal stimulatory effects of insulin, IGF-I, and EGF were about 5 X 10(-7), 1 X 10(-9), and 5 X 10(-10) M, respectively. aIR-3 (anti-type I IGF receptor antibody) completely blocked the stimulation of HRP accumulation by IGF-I but very slightly inhibited the stimulation by insulin. The 528 IgG (anti-EGF receptor antibody) inhibited the stimulation of HRP accumulation by EGF. These results indicated that each of these growth factors stimulates the HRP accumulation mediated by the corresponding (homologous) growth factor receptors. The rapid stimulation of fluid-phase influx and efflux may constitute one of the common early cellular responses to growth factors.     

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.     

Uptake of Lucifer Yellow by plant cells in the presence of endocytotic inhibitors

Summary Lucifer Yellow (LY), a membrane-impermeant anion, was able to enterArabidopsis thaliana cells. LY was taken up by fluid-phase endocytosis and a plasmalemmal anionic carrier mechanism. Both mechanisms were shown to be concentration-dependent. At 0.1 mg/ml, LY was mainly taken up via fluid-phase endocytosis and concentrated in vesicular-like structures. At a ten-fold higher concentration (1 mg/ml), a plasmalemmal anionic carrier system allowed LY uptake and its accumulation in the central vacuole by a vacuolar anionic transporter. Chloroquine, cytochalasin B, monensin, and phorbol-12-myristate-13-acetate (PMA) hindered LY endocytosis. Brefeldin A did not modify LY uptake. The probenecidsensitive carrier uptake machinery showed sensitivity to chloroquine and PMA. Therefore the probenecid-sensitive transport mechanism seems to be complex and involve both acidification of a compartment and protein kinase C activity.Abbreviations CH carbohydrazide - DMSO dimethylsulfoxide - LY Lucifer Yellow - MES 2-[N-morpholino]-ethanesulfonic acid - MS Murashige and Skoog's medium - PMA phorbol-12-myristate-13-acetate - NAA naphthalene acetic acid     

Kinetics of fluid-phase pinocytosis in Dictyostelium discoideum amoebae

Kinetics of pinocytosis in Dictyostelium discoideum were investigated over an extended period of time (up to 6 hours) using fluorescein isothiocyanate (FITC)-dextran as a fluid-phase marker. FITC-dextran added to the medium accumulated rapidly inside the cells with a rate of influx equivalent to 9 microns3 of fluid/cell x min. After a period of about 90 min of uptake, the intracellular FITC-dextran level reached a plateau which corresponded to a strict balance between pinocytosis and exocytosis as shown both by efflux measurements and pulse experiments with (3H) dextran. At equilibrium, the amount of internalized marker reached a value equivalent to 790 microns3 of fluid taken up per amoeba, i.e. a volume paradoxically higher than the total aqueous space of the cell (520 microns3 ). FITC-dextran was thus markedly concentrated intracellularly. The endocytic compartment in which the intracellular FITC-dextran was concentrated could be completely washed out when FITC-dextran was removed from the external medium.     

Endocytosis, intracellular transport, and turnover of anionic and cationic proteins in cultured mouse peritoneal macrophages

It was previously shown that cultured mouse peritoneal macrophages ingest anionic derivatives of horseradish peroxidase (HRP) and ferritin by fluid-phase endocytosis and accumulate them in lysosomes. Cationic derivatives were taken up by adsorptive endocytosis and transported to lysosomes but subsequently appeared also in stacked cisternae, tubules, and vesicles of the Golgi complex. In the present investigation, the effect of molecular net charge on the rate of cellular inactivation of a protein was studied. The results demonstrate that anionized HRP was inactivated at a higher initial rate than cationized HRP. This is in agreement with the finding that the cationic protein partly escaped from the digestive compartment of the cells, that means the lysosomes. The effects of phorbol myristate acetate (PMA)--a diterpene ester and a tumor promoter--and monensin--a carboxylic ionophore and a perturbant of the Golgi complex--on fluid-phase endocytosis of HRP and intracellular transport of cationized ferritin (CF) were also studied. PMA stimulated fluid-phase endocytosis of HRP but did not interfere with transport of CF to the Golgi complex. Contrarily, monensin inhibited uptake of HRP and almost totally blocked transport of CF to the Golgi complex. The findings support the idea that membrane and content of endocytic vesicles are treated separately. The content is emptied into lysosomes where macromolecular material normally is degraded. The membrane becomes part of the lysosomal envelope in connection with endocytic vesicle-lysosome fusion. Subsequently, membrane patches are detached from the lysosomes and reutilized. This is at least partly mediated via the Golgi complex and particularly its tubular and vesicular parts. Since the cationic tracers do not bind to the membrane in a stable way, it is not possible to extend the conclusions to individual membrane constituents.     

Insulin stimulates fluid-phase endocytosis and exocytosis in 3T3-L1 adipocytes

Fluid phase endocytosis by monolayers of 3T3-L1 adipocytes has been followed by measuring [14C]sucrose uptake, a well characterized pinocytic marker. Insulin, at a maximal stimulatory concentration, increased the pinocytic rate by 2-fold within 5 min of its addition; this activation persisted for at least 2 h. The dose-response curve for the enhancement of fluid-phase endocytosis by insulin was identical with that for the stimulation of hexose transport, as measured by the uptake of 2-deoxyglucose. The concentration of insulin eliciting half-maximal effects was 6 nM. These results suggest that activation of endocytosis and hexose uptake by insulin are triggered by the same signalling event. Insulin-activated pinocytosis was not dependent upon the increased metabolism of D-glucose that occurs in response to the hormone, since the stimulation of fluid-phase endocytosis occurred in the absence of 5 nM glucose. Fluid-phase exocytosis was examined by loading cells with [14C]sucrose for various times and then measuring tracer efflux. The rate of sucrose release was biphasic; a portion of the internalized sucrose was rapidly released from the cell (t1/2 approximately 5 min), whereas the remainder was released slowly (t1/2 approximately to 5 h). These results are consistent with a sequential two-compartment model in which the [14C] sucrose first enters a compartment from which about 70% of the sucrose is rapidly released back into the medium and the remaining 30% is transferred to a second compartment. Therefore, the true rate of endocytosis is much greater than the observed accumulation rates, except after short uptake times. Insulin increases the rate of sucrose efflux from both compartments as well as the rate of transfer from the first compartment to the second compartment by about 2-fold. Furthermore, insulin increased the apparent size of the first and second compartments by 1.6- and 3-fold, respectively. The lysosomotropic agent chloroquine (200 muM) had only a small effect on fluid movements in these cells. The rapid and prolonged stimulation of fluid-phase endocytosis and exocytosis by insulin are hitherto unrecognized effects of this hormone.     

Azithromycin, a lysosomotropic antibiotic, impairs fluid-phase pinocytosis in cultured fibroblasts

The dicationic macrolide antibiotic azithromycin inhibits the uptake of horseradish peroxidase (HRP) by fluid-phase pinocytosis in fibroblasts in a time- and concentration-dependent fashion without affecting its decay (regurgitation and/or degradation). The azithromycin effect is additive to that of nocodazole, known to impair endocytic uptake and transport of solutes along the endocytic pathway. Cytochemistry (light and electron microscopy) shows a major reduction by azithromycin in the number of HRP-labeled endocytic vesicles at 5 min (endosomes) and 2 h (lysosomes). Within 3 h of exposure, azithromycin also causes the appearance of large and light-lucentlelectron-lucent vacuoles, most of which can be labeled by lucifer yellow when this tracer is added to culture prior to azithromycin exposure. Three days of treatment with azithromycin result in the accumulation of very large vesicles filled with pleiomorphic content, consistent with phospholipidosis. These vesicles are accessible to fluorescein-labeled bovine serum albumin (FITC-BSA) and intensively stained with filipin, indicating a mixed storage with cholesterol. The impairment of HRP pinocytosis directly correlates with the amount of azithromycin accumulated by the cells, but not with the phospholipidosis induced by the drug. The proton ionophore monensin, which completely suppresses azithromycin accumulation, also prevents inhibition of HRP uptake. Erythromycylamine, another dicationic macrolide, also inhibits HRP pinocytosis in direct correlation with its cellular accumulation and is as potent as azithromycin at equimolar cellular concentrations. We suggest that dicationic macrolides inhibit fluid-phase pinocytosis by impairing the formation of pinocytic vacuoles and endosomes.     

Fluid-Phase Pinocytosis of Native Low Density Lipoprotein Promotes Murine M-CSF Differentiated Macrophage Foam Cell Formation

During atherosclerosis, low-density lipoprotein (LDL)-derived cholesterol accumulates in macrophages to form foam cells. Macrophage uptake of LDL promotes foam cell formation but the mechanism mediating this process is not clear. The present study investigates the mechanism of LDL uptake for macrophage colony-stimulating factor (M-CSF)-differentiated murine bone marrow-derived macrophages. LDL receptor-null (LDLR−/−) macrophages incubated with LDL showed non-saturable accumulation of cholesterol that did not down-regulate for the 24 h examined. Incubation of LDLR−/− macrophages with increasing concentrations of ¹²⁵I-LDL showed non-saturable macrophage LDL uptake. A 20-fold excess of unlabeled LDL had no effect on ¹²⁵I-LDL uptake by wild-type macrophages and genetic deletion of the macrophage scavenger receptors CD36 and SRA did not affect ¹²⁵I-LDL uptake, showing that LDL uptake occurred by fluid-phase pinocytosis independently of receptors. Cholesterol accumulation was inhibited approximately 50% in wild-type and LDLR−/− mice treated with {"type":"entrez-nucleotide","attrs":{"text":"LY294002","term_id":"1257998346","term_text":"LY294002"}}LY294002 or wortmannin, inhibitors of all classes of phosphoinositide 3-kinases (PI3K). Time-lapse, phase-contrast microscopy showed that macropinocytosis, an important fluid-phase uptake pathway in macrophages, was blocked almost completely by PI3K inhibition with wortmannin. Pharmacological inhibition of the class I PI3K isoforms alpha, beta, gamma or delta did not affect macrophage LDL-derived cholesterol accumulation or macropinocytosis. Furthermore, macrophages from mice expressing kinase-dead class I PI3K beta, gamma or delta isoforms showed no decrease in cholesterol accumulation or macropinocytosis when compared with wild-type macrophages. Thus, non-class I PI3K isoforms mediated macropinocytosis in these macrophages. Further characterization of the components necessary for LDL uptake, cholesterol accumulation, and macropinocytosis identified dynamin, microtubules, actin, and vacuolar type H(+)-ATPase as contributing to uptake. However, Pak1, Rac1, and Src-family kinases, which mediate fluid-phase pinocytosis in certain other cell types, were unnecessary. In conclusion, our findings provide evidence that targeting those components mediating macrophage macropinocytosis with inhibitors may be an effective strategy to limit macrophage accumulation of LDL-derived cholesterol in arteries.     

Murine bone marrow-derived macrophages differentiated with GM-CSF become foam cells by PI3Kγ-dependent fluid-phase pinocytosis of native LDL

Accumulation of cholesterol by macrophage uptake of LDL is a key event in the formation of atherosclerotic plaques. Previous research has shown that granulocyte-macrophage colony-stimulating factor (GM-CSF) is present in atherosclerotic plaques and promotes aortic lipid accumulation. However, it has not been determined whether murine GM-CSF-differentiated macrophages take up LDL to become foam cells. GM-CSF-differentiated macrophages from LDL receptor-null mice were incubated with LDL, resulting in massive macrophage cholesterol accumulation. Incubation of LDL receptor-null or wild-type macrophages with increasing concentrations of ¹²⁵I-LDL showed nonsaturable macrophage LDL uptake that was linearly related to the amount of LDL added, indicating that LDL uptake was mediated by fluid-phase pinocytosis. Previous studies suggest that phosphoinositide 3-kinases (PI3K) mediate macrophage fluid-phase pinocytosis, although the isoform mediating this process has not been determined. Because PI3Kγ is known to promote aortic lipid accumulation, we investigated its role in mediating macrophage fluid-phase pinocytosis of LDL. Wild-type macrophages incubated with LDL and the PI3Kγ inhibitor AS605240 or PI3Kγ-null macrophages incubated with LDL showed an ∼50% reduction in LDL uptake and cholesterol accumulation compared with wild-type macrophages incubated with LDL only. These results show that GM-CSF-differentiated murine macrophages become foam cells by fluid-phase pinocytosis of LDL and identify PI3Kγ as contributing to this process.     

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.     

Phorbol myristate acetate stimulates pinocytosis and membrane spreading in mouse peritoneal macrophages

Phorbol myristate acetate (PMA) at a concentration of 0.01 microgram/ml causes an approximately threefold increase in surface area of resident, proteose-peptone-elicited, and thioglycolate-broth-elicited mouse peritoneal macrophages. Resident and proteose-peptone-elicited macrophages, cultured for 24 h in the presence of PMA, increase their pinocytic rate twofold in response to addition of PMA (0.01 microgram/ml) to the medium. Thioglycolate-broth-elicited macrophages, cultured for 24 h in the absence of PMA, immediately increase their pinocytic rate 2- to 3.5-fold in response to a single challenge with PMA (0.01 microgram/ml). Cytochalasin B, colchicine, and podophyllotoxin have only modest inhibitory effects on the basal rate of pinocytosis and on PMA-induced cellular spreading, but completely block the stimulatory effects of PMA on pinocytosis in thioglycolate- broth-elicited macrophages. Cytochalasin D markedly inhibits both basal and PMA-stimulated pinocytosis in these cells. Thus, PMA is a useful tool for studying mechanisms of macrophage spreading and for enhancing the overall rate of pinosome formation.     

Mathematical modeling of marker influx and efflux in cells

The tumor promoter, phorbol 12-myristate 13-acetate (PMA), affects the processing of fluid that enters a cell from the ambient medium. Previous work showed that marker accumulates to a higher level in PMA-treated than in untreated cells. Since PMA also affects the physical activity of the membrane and stimulates the normal process of taking up extracellular fluid, called endocytosis, it is important to learn whether the perturbations in fluid processing can be attributed entirely to a change in the cell’s limiting membrane. To this end, a model for fluid uptake and processing was developed and applied to experiments in which a marker for extracellular fluid was added to cells. From previous work on marker accumulation, it was deduced that there were at least two functional compartments involved in fluid movement. Compartment I is a rapidly filling and rapidly recycling compartment. Compartment II is a slowly filling and emptying compartment. Three routes of vesicle traffic must be considered, one mediating influx from the ambient medium into compartment I, a second, efflux from compartment I to the medium, and a third efflux from compartment I into compartment II. Using earlier models for processing, workers found it difficult to estimate rates of movement through either of the latter routes, as well as the volume of compartment I. The difficulty arises from the fact that only one kinetic constant can be estimated directly from data, namely the instantaneous uptake rate. The remaining data depend on measuring the total mass of marker in the cells. Since the concentration of marker in the cell changes continuously, it is advantageous to employ differential equations to simulate the tracer movement. By applying the model to experimental values, we found estimates for all three rates of fluid movement and the volume of compartment I. It is thought that the model will enable us to determine whether apparent alterations in the time course of uptake arise solely from altered properties of the limiting membrane. This revised version submitted December, 2000. Research supported in part by a grant from the National Institutes of Health, R15 CA78322-01.     

Rapid stimulation of pinocytosis in human carcinoma cells A-431 by epidermal growth factor

Horseradish peroxidase (HRP) uptake was used to measure fluid-phase pinocytosis in monolayers of human epithelioid carcinoma cells (A-431). Histochemistry confirmed that cell-associated HRP was restricted to intracellular vesicles. Biochemical methods showed that HRP uptake in control cultures was directly proportional to the duration of exposure. The addition of low concentrations of epidermal growth factor (EGF) to the incubation media produced a 10-fold increase in the initial rate of pinocytosis. The EGF effect was rapid (within 30 s) but transient; the rate of pinocytosis returned to control levels within 15 min. Metabolic inhibitors reduced the EGF-stimulated rate of pinocytosis by greater than 90%. A conjugate of EGF and ferritin (F:EGF) was used to simultaneously compare the intracellular locations of EGF and HRP. Much of F:EGF was internalized in approximately 100-nm vesicles, while most of the HRP was located in much larger vesicles (range 0.1--1.2 micrometer) which also contained F:EGF. The tumor-promoter 12-0-tetradecanoyl-phorbol-13-acetate, which shares several biological activities with EGF, was also effective in stimulating an increase in the rate of pinocytosis.     

Effect of wortmannin and phorbol ester on Paramecium fluid-phase uptake in the presence of transferrin

The kinetics of the uptake of the fluid phase marker Lucifer Yellow (LY), and its alteration by wortmannin, an inhibitor of phosphatidylinositol-3 kinase (PI-3K), and the PKC modulators: GF 109203 X, an inhibitor, and phorbol ester, an activator was studied in eukaryotic model Paramecium aurelia. Spectrophotometric quantification of LY accumulation was performed in the presence or absence of transferrin, a marker of receptor-mediated endocytosis. Internalization of LY showed a curvilinear kinetics: the high initial rate of LY uptake (575 ng LY/mg protein/hr) decreased almost 5-fold within 15 min, reaching plateau at 126 ng/mg protein/hr. Transferrin induced a small increase (7.5%) in the fluid phase uptake rate (after 5 min) followed by a small decrease at longer incubation times. Lucifer Yellow and transferrin (visualized by streptavidin-FITC) were localized in Paramecium by 3-D reconstruction by confocal microscopy. LY showed a scattered, diffuse fluorescence typical of fluid phase uptake whereas transferrin accumulated in membrane-surrounded endosomes. Wortmannin did not affect LY accumulation but decreased it when transferrin was present in the incubation medium. This suggests an effect on the transferrin uptake pathway, presumably on the stage of internalization in "mixing" endosomes to which transferrin and LY were targeted. Phorbol ester diminished LY accumulation by 22% and this effect persisted up to 25 min of incubation. PKC inhibitor did not affect LY uptake. However, in the presence of transferrin, the LY uptake increased within the first 15 minutes followed by a rapid 20% decrease in comparison to the control. Such an effect of PKC modulators suggests that PMA action on fluid phase uptake is not directly mediated by PKC.     

Effect of hypo-osmotic incubation on membrane recycling

Incubation of alveolar macrophages in hypo-osmotic media causes a time-and temperature-dependent increase in the number of surface receptors for three different ligands. Exposure of cells to solutions of 210 mOsM or less, at 37 degrees C but not at 0 degree C, resulted in an increase in the number of surface receptors for diferric transferrin, alpha-macroglobulin-protease complexes, and mannose-terminated glycoproteins. Upon media dilution at 37 degrees C, surface receptor number reached a maximum within 5 min and returned to near-normal values by 30 min. The increase in surface receptor number was the result of a decrease in the rate of internalization of receptors, either occupied or unoccupied. The rate of receptor exteriorization was unaltered by hypo-osmotic incubation of cells. The rate of fluid-phase pinocytosis was also inhibited upon incubation in hypo-osmotic solution. In experiments in which both receptor-mediated endocytosis and fluid phase pinocytosis were measured on the same samples, inhibition of both processes occurred with the same kinetics and to a similar extent. The rate of receptor-mediated endocytosis recovered to normal rates after 60 min in hypo-osmotic solutions, whereas the rate of fluid phase pinocytosis did not recover to the same extent.