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.     

Maternal-embryonic relationships in the goodeid teleost,Xenoophorus captivus

Summary The endodermal trophotaenial epithelium in goodeid embryos acts as a placental exchange site. Fine structural and cytochemical data indicate that the trophotaenial absorptive cells are endocytotically highly active. To test their micropinocytotic capacity and characterize the cellular mechanisms involved in membrane, solute and ligand movements, living embryos of Xenoophorus captivus were incubated in saline media containing horseradish peroxidase (HRP) and/or cationized ferritin (CF) in vitro, and the uptake of these tracer proteins examined by both time sequence analysis and pulse-chase procedures. In some embryos, the effects of prolonged exposure to CF injected into the ovarian cavity, was also investigated.Labelling of the free cell surface was detectable with CF only, but interiorization of both probes was quick from all incubation media. Adsorptive pinocytosis of CF and fluid-phase uptake of HRP sequentially labelled pinocytic vesicles, endosomes, and lysosome-like bodies. In addition, CF-molecules were sequestered within apical tubules and small vesicles. HRP was largely excluded from both organelles and ended up in the lysosomal compartment. For CF, two alternative pathways were indicated by the pulse-chase experiments; transcellular passage and regurgitation of tracer molecules to the apical cell surface. The latter procedure involves membrane and receptor recycling, in which apical tubules are thought to mediate.In double-tracer experiments, using an 81 excess of HRP, external labelling with CF was light or lacking after 1–3 min, and the initial uptake-phase produced pinocytic vesicles and endosomes that mainly contained HRP-reaction product. Prolonged incubation, however, resulted in densely CF-labelled plasmalemmal invaginations and pinocytic vesicles that predominantly carried ferritin granules. After 60 min, the vacuoles of the endosomal compartment contained either high concentrations of HRP-reaction product, both tracers side by side, or virtually exclusively CF.     

Interactions between Encephalitozoon cuniculi and macrophages. Parasitophorous vacuole growth and the absence of lysosomal fusion.

Encephalitozoon cuniculi grow within ever-increasing parasitophorous vacuoles (PV) in peritoneal macrophages. The PV boundary membrane conforms to a rich arrangement of blebs; similar, but free vesicles were observed within the PV space. An iron dextran-concanavalin A marker was used to express visually clustered distributions of Con A receptors on the PV boundary blebs and free vesicles; no marker was observed on other membrane surfaces within the PV. These results, combined with the observation that the PV grows while the host cytoplasm decreases in mass, implicate the PV boundary blebs of interiorizing into vesicles by a pinocytic mechanism. Phagocytic vacuoles, secondary lysosomes and pinocytic vesicles were labeled by incubating infected macrophages in minimum essential medium with ferritin. Ferritin readily accumulated in secondary lysosomes and phagocytic vacuoles; however, ferritin was excluded from parasitophorous vacuoles containing E. cuniculi. Acid phosphatase cytochemical reaction product was observed in lysosomes and phagocytic vacuoles; however, parasitophorous vacuoles with vegetative E. cuniculi were always negative.     

Osmotically induced microinjection of ricin bypasses a ricin internalization defect in a Chinese hamster ovary mutant cell line.

By osmotic lysis of pinocytic vesicles we were able to inject ricin or ricin A chain directly into the cytosol of Chinese hamster ovary cells. The lag time of 1 to 2 h before the onset of the inhibition of protein synthesis by ricin in intact cells was reduced to 15 to 30 min by this method. Preincubation of cells with a low concentration of nigericin, which was shown earlier to enhance the cytotoxicity of ricin, had no effect under this condition. Direct transfer of either intact ricin or the ricin A subunit by osmotic lysis of pinocytic vesicles into the cytosol of the ricin-resistant CHO mutant cell line 4-10 rendered the mutant 4-10 cells as sensitive to ricin as the CHO pro wild-type cells. Both the lag time and the rate of inhibition of protein synthesis in the wild-type and mutant cell lines after the introduction of ricin by osmotic lysis of pinocytic vesicles were the same. These results indicate that injection of ricin into the cytosol by osmotic lysis of pinosomes bypasses the internalization defect in the mutant cell line.     

Effects of amiloride on Na+ content and pinocytosis in Entamoeba histolytica

Amiloride, a blocker of Na+ leak and Na+-H+ exchange in animal cells, caused cells of Entamoeba histolytica to release Na+ (up to 40% of their original Na+ content within 90 min, at an amiloride concentration of 3 mM); K+ content was not affected. By comparing the unidirectional uptake of 22Na+ with that of the fluid-phase marker 125I-labeled poly(vinylpyrrolidone) we established that the amiloride-induced Na+ loss was due to inhibition of pinocytic Na+ uptake rather than to blockage of an amiloride-sensitive transport system in the plasma membrane. Amiloride penetrated the cells, and both its intracellular concentration and its effect on pinocytosis increased with pH. The permeant weak base quinacrine similarly inhibited pinocytosis in a pH-dependent manner. We conclude that the effect of amiloride on pinocytosis and, consequently, on Na+ content was due to its properties as a permeant weak base.     

Evidence for both prelysosomal and lysosomal intermediates in endocytic pathways

Horseradish peroxidase (HRP), an enzyme internalized by fluid phase pinocytosis, has been used to study the process by which pinosome contents are delivered to lysosomes in Chinese hamster ovary cells. Pinosome contents were labeled by allowing cells to internalize HRP for 3-5 min. Following various chase times, cells were either processed for HRP and acid phosphatase (AcPase) cytochemistry or homogenized and fractionated in Percoll gradients. In Percoll gradients, pinosomes labeled by a 3-5 min HRP pulse behaved as a vesicle population more dense than plasma membrane and less dense than lysosomes. In pulse- chase experiments, internalized HRP was chased rapidly (3-6 min chase) to a density position intermediate between the "initial" pinocytic vesicle population and lysosomes. With longer chase periods, a progressive accumulation of HRP in more dense vesicles was observed. Correspondence between the HRP distribution and lysosomal marker distribution was reached after a approximately 1-h chase. By electron microscope cytochemistry of intact cells, the predominant class of HRP- positive vesicles after pulse uptakes or a 3-min chase period was characterized by a peripheral rim of reaction product and was AcPase negative. After 10-120-min chase periods, the predominant class of HRP- positive vesicles was characterized by luminal deposits and HRP activity was frequently observed in multivesicular bodies. HRP-positive vesicles after a 10- or 30-min chase were AcPase-positive. No HRP activity was detected in Golgi apparatus. Together these observations indicate that progressive processing of vesicular components of the vacuolar apparatus occurs at both a prelysosomal and lysosomal stage.     

An electron microscopic study of the initial stages of infection of isolated tomato fruit protoplasts by tobacco mosaic virus

Summary Protoplasts were isolated from tomato fruit locule tissue and incubated with tobacco mosaic virus. Electron microscope observations on sections of suitably fixed and embedded material revealed that virus particles readily became attached to the plasmalemma, particularly in small invaginations in the surface of the protoplast. Virus particles were later observed in vesicles within the cytoplasm and it was clear that these vesicles were being formed as a result of pinocytic activity at the surface of the protoplast. Later, virus particles were observed near the nucleus. It is suggested that an initial attachment of the virus to the plasmalemma followed by a pinocytic uptake may represent the initial stages of virus infection of plant cells and that the pinocytic vesicle, containing virus, serves as the vehicle of cellular infection.     

Rapid acidification of endocytic vesicles containing alpha 2-macroglobulin

We have used fluorescein-labeled alpha 2-macroglobulin (F-alpha 2M) to measure pH changes in the microenvironment of internalized ligands following receptor-mediated endocytosis. Fluorescence intensities of single BALB/c 3T3 mouse fibroblasts were measured by using a microscope spectrofluorometer with narrow bandpass excitation filters. The pH was determined from the ratio of fluorescein fluorescence intensities with 450 nm and 490 nm excitation. A standard pH curve was obtained by incubating cells with F-alpha 2M for 30 min at 37 degrees C followed by fixation and incubation in buffers of varying pH. To measure the pH of endocytic vesicles, cells were incubated with F-alpha 2M for 15 min at 37 degrees C. Fluorescence intensities were measured on living cells within 5 min of rinsing. Under these conditions, the pH of the F-alpha 2M microenvironment was 5.0 +/- 0.2. Using colloidal gold-alpha 2M for electron microscopic localizations we have verified that, under these conditions, alpha 2M is predominantly in uncoated vesicles that are negative for acid phosphatase activity. With further incubation for 1/2 hr, we obtained a pH of 5.0 +/- 0.2 for the F-alpha 2M. Using fluorescein dextran, we obtained a lysosomal pH of 4.6 +/- 0.2. These results indicate that endocytic vesicles become acidic prior to fusion with lysosomes.     

Acid intracellular vesicles and the cytolysis of mammalian target cells by Entamoeba histolytica trophozoites

Entamoeba histolytica kills mammalian target cells in a multi-step sequential process with separate adherence, cytolytic, and phagocytic events. In the studies reported here, we used fluorescein isothiocyanate linked to dextran to label the endocytic vesicles of the HM1 strain of E. histolytica and measure vesicle pH (5.1 +/- 0.2 by spectrofluorimetry). Concentrations of NH4Cl (1.0-10.0 mM) sufficient to increase vesicle pH to greater than or equal to 5.7 inhibited amebic killing of target Chinese hamster ovary (CHO) cells as assayed by trypan blue staining, by the release of 3H-thymidine previously incorporated into CHO cell monolayers, and by the release of 111indium oxine from radiolabeled CHO cells. Similar effects were also observed with two other weak bases, primaquine and chloroquine (both 50 microM). In contrast, NH4Cl (10 mM) did not affect either the adherence or phagocytic events, as measured by amebic adherence to CHO cells at 4 degrees C and by the binding and ingestion of 3H-leucine-labeled bacteria. In the presence of NH4Cl and the carbohydrate ligand asialofetuin, there was no evidence of intracellular trapping of the amebic galactose-inhibitable lectin; inhibition of adherence by cycloheximide (10 micrograms/ml for 3 h) suggested rapid turnover of the surface lectin. Prolonged exposure to NH4Cl for 48 h (which had no effect on amebic protein synthesis) or shorter exposure to cycloheximide (10 micrograms for 3 h) produced persistent inhibition of cytolysis. These results indicate that an uninterrupted acid pH in intracellular endocytic vesicles is necessary for the cytolysis of target cells by E. histolytica trophozoites.     

Adenovirus-induced leakage of co-endocytosed macromolecules into the cytosol

The early interaction between KB cells and adenovirus was studied by examining the uptake of an extracellular fluorescent macromolecule, FITC (fluorescein isothiocyanate)-labeled dextran (FD). When cells in suspension were incubated with both adenovirus and FD, cell-associated FD increased 2- to 3-fold the value obtained without adenovirus. Under fluorescence microscopy, cells incubated with adenovirus showed bright, whole-cellular fluorescence; whereas, those incubated without adenovirus, or with heat-inactivated virus, showed weaker fluorescence, mainly of the pinocytic vesicles. The increased uptake of the FD by adenovirus was inhibited by treating KB cells with the drugs chloroquine, ammonium chloride and monensin that raise the pH of the acidic compartment. Entry of adenovirus into the KB cell's nucleus also was inhibited by these drugs. The conclusion is that entry of adenovirus into the cell involves its passage of an acidic compartment (probably the endocytic vesicle) and that co-endocytosed macromolecules are released into the cytosol on entry.     

Retention of pinocytized solute by CHO cell lysosomes correlates with molecular weight

We compared the exocytosis by Chinese hamster ovary (CHO) cells of a set of fluid-phase pinocytic tracers. The tracers were horseradish peroxidase (HRP), a glycoprotein of approximately 40 kDa, lucifer yellow (LuY), a 457 dalton, membrane-impermeant fluorescent dye, and glucose polymers ranging from sucrose through higher molecular weight, fluorescein isothiocyanate (FITC) dextrans. After a long term uptake (16-20 h), each of these tracers was localized to lysosomes. Exocytosis of the majority of the small molecule tracers, LuY and [14C] sucrose, was observed over a period of a few to several h. There was no significant exocytosis of 42 kDa FITC dextran or HRP during an 18-20 h chase, while lower molecular weight dextrans were exocytosed. After co-accumulation of LuY and HRP in lysosomes, only the low molecular weight marker was exocytosed. These observations suggest retention of endocytized solutes within lysosomes is dependent on molecular size and may be limited by the rate of diffusion of molecules into shuttle vesicles.     

Mobilization of iron from endocytic vesicles. The effects of acidification and reduction

The factors necessary to dissociate iron from transferrin in endocytic vesicles and to mobilize the iron across the vesicle membrane were studied in a preparation of endocytic vesicles markedly enriched in transferrin-transferrin receptor complexes isolated from rabbit reticulocytes. Vesicles were prepared with essentially fully saturated transferrin by incubating the reticulocytes with the protonophore carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone prior to incubation with 59Fe, 125I-transferrin with or without fluorescein isothiocyanate labeling. Initiation of acidification by the addition of ATP was sufficient to achieve dissociation of 59Fe from transferrin with a rate constant of 0.054 +/- 0.06 s-1. Mobilization of 59Fe out of the vesicles required, besides ATP, the addition of a reductant with 1 mM ascorbate, allowing approximately 60% mobilization at 10 min with a rate constant of 0.0038 +/- 0.0006 s-1. An NADH:ferricyanide reductase activity could be demonstrated in the vesicles with an activity of 7.1 x 10(-9) mol of NADH reduced per min/mg of vesicle protein. Both dissociation and mobilization were inhibited by N-ethylmaleimide, carbonyl cyanide 4-(trifluoromethoxy)phenylhydrazone, and monensin. Mobilization, but not dissociation, was inhibited by the permeant Fe(II) chelator alpha,alpha'-dipyridyl. The Fe(III) chelators deferoxamine, diethylenetriaminepentaacetic acid, and apotransferrin did not promote mobilization of dissociated iron in the absence of a reductant. This study establishes the basis for the cellular incorporation of iron through the endocytic pathway in which the endocytic vesicle membrane utilizes, in a sequential way, an acidification system, an iron reduction system, and an Fe(II) transporter system.     

The fine structure of the digestive tubules of the Marine Bivalve Cardium edule

The epithelium lining the digestive tubules of Cardium edule consists of three cell types, namely mature digestive cells, mature secretory cells and immature flagellated cells. Both the secretory and flagellated cells exhibit a pronounced basiphilia and occur in well-defined crypts. The secretory cells are pyramidal in shape and characterized by the possession of a well-developed granular endoplasmic reticulum and Golgi apparatus. Golgi vesicles derived from the latter migrate to the apical region of the cell where they release their contents into the lumen of the tubules. It is possible that the secretion contains enzymes and although it is likely that such enzymes would function primarily in the lumen of the tubules they may also be the source of the weak proteolytic activity which has been recorded in the gastric fluid of many bivalves. The immature flagellated cells are columnar in shape and possess a poorly developed endoplasmic reticulum and numerous free ribosomes. Although no evidence for this was obtained it is suggested that they may serve to replace either or both of the mature cell types. The digestive cells vary from cuboidal to columnar, possess distinctive Golgi elements with characteristic intracisternal membranous elements, and are capable of ingesting exogenous material from the lumen of the tubule. The process of ingestion was examined following feeding experiments with (a) a mixture of iron oxide and colloidal graphite (Aquadag), (b) whole blood from pigeon and (c) ferritin. Individual particles of graphite were enclosed in phagosomes by a process of phagocytosis, while the proteins haemoglobin and ferritin were ingested by a process of pinocytosis; the membrane enclosing the pinocytic vesicles possesses a characteristic outer granular coat. The contents of both the phagocytic and pinocytic vesicles were transferred to larger bodies considered to be primarily phagosomes in the sub-apical regions of the cell. These possess an interconnecting system of membrane-bound channels which ramifies through the apical cytoplasm. Phagolysosomes deeper in the cytoplasm of the cell were identified by the presence of exogenous material and a positive reaction to tests for acid phosphatase activity. They showed changes in appearance which could be put into a series suggestive of the progressive intracellular digestion of the ingested material.     

Effect of iron on adherence and cytotoxicity of Entamoeba histolytica to CHO cell monolayers

Iron is an essential element for almost all living organisms. The possible role of iron for growth, adherence and cytotoxicity of Entamoeba histolytica was evaluated in this study. The absence of iron from TYI-S-33 medium stopped amebic growth in vitro. However, iron concentrations in the culture media of 21.4-285.6 microM did not affect the growth of the amebae. Although growth was not retarded at these concentrations, the adhesive abilities of E. histolytica and their cytotoxicities to CHO cell monolayer were correlated with iron concentration. Amebic adhesion to CHO cell monolayers was significantly reduced by low-iron (24.6 +/- 2.1%) compared with 62.7 +/- 2.8 and 63.1 +/- 1.4% of amebae grown in a normal-iron and high-iron media, respectively. E. histolytica cultured in the normal- and high-iron media destroyed 69.1 +/- 4.3% and 72.6 +/- 5.7% of cultured CHO cell monolayers, but amebae grown in the low-iron medium showed a significantly reduced level of cytotoxicity to CHO cells (2.8 +/- 0.2%). Addition of divalent cations other than iron to amebic trophozoites grown in the low-iron medium failed to restore levels of the cytotoxicity. However, when E. histolytica grown in low-iron medium were transferred to normal-iron medium, the amebae showed completely restored cytotoxicity within 7 days. The result suggests that iron is an important factor in the adherence and cytotoxicity of E. histolytica to CHO cell monolayer.     

Introduction of macromolecules into cultured mammalian cells by osmotic lysis of pinocytic vesicles

We have developed a new procedure for introducing macromolecules into cultured mammalian cells based on osmotic lysis of pinocytic vesicles. Cells are first incubated in culture medium containing 0.5 M sucrose, 10% polyethylene glycol 1000 and the macromolecule to be transferred. Cells are then placed in medium diluted with 0.66 parts water. Most pinocytic vesicles formed in the presence of sucrose burst in hypotonic medium, thereby releasing the enclosed macromolecule. L929 cells remain fully viable after a single hypertonic sucrose treatment, and a majority survives four successive rounds of osmotic lysis. This procedure, termed osmotic lysis of pinosomes, has been used to transfer substantial amounts of horseradish peroxidase, antiricin antibodies and dextran 70,000 into the cytosol of L929 cells. Direct comparison of the degree of ricin resistance conferred by transfer of antiricin antibodies revealed pinosome lysis to be equal, if not superior, to injection mediated by red blood cells.     

Flux measurement in single cells by fluorescence microphotolysis

Fluorescence microphotolysis — widely employed for diffusion studies — can be used to measure transfer (flux) of fluorescent solutes through membranes in single cells and organelles. This article analyses the methodological basis of flux measurements, provides experimental tests, and discusses potential applications. The principle of the method is to equilibrate cells, organelles or vesicles with a fluorescent solute, to deplete the interior of individual cells etc. of fluorescene by the pulse of a high-intensity microbeam, and to monitor influx of solute by microfluorometry. Simple equations are given and a computer curve fitting program is described by which rate constants of influx and membrane permeability coefficients can be derived from fluorescence measurements. The permeability of individual leaky human erythrocyte ghosts to fluorescein-isothiocyanate-labelled bovine serum albumin has been measured under various conditions. Multiple exposure to the high-intensity microbeam had no effect on permeability within experimental error. Flux measurements have been also performed on individual vesicles of 1–2 m radius which had been derived from ghosts. The potential application of the method to sub-lightmicroscopic vesicles and to organelles within living cells is discussed.Abbreviation FITC-BSA fluorescein isothiocyanate-labeled bovine serum albumin     

Identification of free coated pinocytic vesicles in Swiss 3T3 cells.

Whether or not free coated vesicles are involved during internalization of ligands bound to the receptors of coated pits is controversial. Free coated vesicles cannot be identified with certainty in random individual thin sections - reconstructions based on consecutive thin sections are required. The thickness of the sections determines the reliability of such reconstructions. In the present study, serial section electron microscopy was applied to Swiss 3T3 cells and the topographical resolution yielded by 80 nm and 20 nm sections was compared. Swiss 3T3 cells in monolayer at 37 degrees C were exposed for 5 min to cationized ferritin (CF) which is a marker of pinocytic vesicles. Subsequently the cells were fixed, pelleted and further processed for electron microscopy. The results showed that reconstructions of coated CF-labeled structures based on consecutive sections of an average thickness of approximately 80 nm could not be performed with certainty. A substantial fraction (25%) of the examined profiles appeared to be free vesicles, but narrow surface connections could easily have been missed in these thick sections. The series of the much thinner 20 nm sections provided a better resolution allowing the narrowest surface connections to be identified. Accordingly, the number of truly free, coated vesicles was much lower than the number of apparently free vesicles in the thick sections. However, free coated vesicles labeled with CF were identified in the consecutive 20 nm sections (4% of the examined profiles).     

Ultrastructural immunoperoxidase demonstration of autologous albumin in the alveolar capillary membrane and in the alveolar lining material in normal rats

The location of autologous serum albumin within the alveolar-capillary membrane was studied in the rat under physiological conditions using antialbumin antibodies labeled with peroxidase. Albumin was detected in the lung interstitium, and in numerous pinocytic vesicles within endothelial cells and type I alveolar epithelial cells. The immunoreaction was also positive at the level of plasmalemmal membranes of both cell types and in the alveolar lining material.     

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.     

Effect of a hypertonic sucrose solution and 5-(N,N-hexamethylene)-amiloride on receptor-mediated and liquid-phase endocytosis

Hypertonic sucrose and amiloride or derivatives of the latter are commonly used to selectively inhibit clathrin-dependent (usually considered as a synonim of receptor-mediated endocytosis) or clathrin-independent endocytosis. Though these approaches are widely used in experimental practice, their limitations have not been studied in detail. In the present work, an attempt was made to evaluate possible side effects and selectivity of these agents towards the type of endocytosis. It was found that the incubation of A431 cells in 0.4 M sucrose resulted in a decrease in both intracellular accumulation and surface binding of the RME marker 125I-EGF. However, while the binding drops by about 3 times, the internalization of EGF in low concentrations was inhibited by more than 30 times, and that of high EGF concentrations by 5-10 times. It may evidence that at high EGF concentrations about 10-20% of ligand-receptor complexes can enter the cells through clathrin-independent pathway. Nevertheless, these results cannot be interpreted so unambigously, because we found that at the incubation longer than 30 min a significant portion of cells became dead or damaged, yielding about 50% of the whole population. By immunofluorescent assay, 5-(N,N-hexametylen)-amiloride commonly used to inhibit fluid phase endocytosis was demonstrated to reduce the staining of fluorescein-containing pinocytic vesicles, but it did not inhibit totally the entering of this marker. Under simultaneous stimulation of fluorescein and EGF endocytosis in the presence of the amiloride derivative, such a residual fluorescence shifted with time to the juxtranuclear region, which is characteristic of the late steps of RME. We suppose that a significant portion of extracellular fluid phase can be included in clathrin-dependent vesicles, whose formation is not disturbed by amiloride. It means that conclusions about the degree of pinocytosis inhibition are to be corrected, taking into account the constitutive clathrin-dependent endocytosis.