Increased endocytosis and formation of multivesicular bodies in phorbol ester-stimulated human monoblastic U-937 cells

Experimental

Cell Research

181 (1989) 551-565

Increased Endocytosis and Formation in Phorbol Ester-Stimulated Human MIKAEL

NILSSON,*”

*Department of Anatomy, Pathology,

KENNETH

NILSSON,?

of Multivesicular Bodies Monoblastic U-937 Cells and KLAS

University of Gothenburg, Gothenburg, Uppsala University Hospital, Uppsala,

FORSBECK?,’

and fDepartment Sweden

of

The phorbol ester 12-0tetradecanoylphorbol-13-acetate (TPA) is known to arrest mitotic activity and induce macrophage differentiation in the U-937 monoblastic cell line. The acute effect of TPA on ultrastructural morphology and endocytic activity of U-937 cells was studied. TPA induced within 15 min a marked enlargement of multivesicular bodies (MVBs), comprising both volume and number of inclusion vesicles (other organelles appeared unchanged). At this stage the MBVs frequently showed tubular cytoplasmic extensions. Inclusion vesicles accumulated in MBVs with prolonged incubation (60 min). Horseradish peroxidase (HRP) and cationized ferritin (CF) added to the medium were routed preferentially to MVBs in TPA-stimulated cells. In contrast to MVBs of unstimulated cells many of the TPA-induced MVBs showed a positive cytochemical reaction to acid phosphatase. The MVBs in cells incubated with ionomycin, a calcium ionophore, did not differ from those of unstimulated cells. Cellular uptake of ‘*%HRP was increased five times the control values already after 5 min of TPA stimulation. The uptake increased further with prolonged incubation (60 min), but at a slower rate. Together these indicate a TPA-induced transfer by endocytosis of portions of the plasma membrane to the lysosomal system via MVBs. Consideration of MVBs as part of the receptor-mediated endocytic pathway suggests that this effect of TPA might involve down-regulation of cell-surface receptors. The possibility of MVBs as a proton-sequestrating compartment, responsible for the cytoplasmic alkalinization previously reported for TPA-stimulated U-937 monoblastic Cells [12], is discussed. @ 1989 Academic press, Inc.

A well-established effect of phorbol esters is their interference with cell growth and differentiation. Some cell types are induced to proliferate [l, 21 whereas others are arrested in their mitotic activity and begin to express differentiated functions [3-8, IS]. Most, if not all, effects of phorbol esters are thought to be mediated by the activation of protein kinase C [9], to which phorbol esters like 12-O-tetradecanoylphorbol-13-acetate (TPA) bind with high affinity. However, the sequence of events following activation of PKC, which ultimately leads to proliferation of differentiation, is only partially known. In this context, a rapid change of cytoplasmic pH (pHJ, mostly alkalinization, has been discussed as an intracellular signal of importance [lo]. In general, phorbol ester-induced alkalinization of the cytoplasm is considered to be mediated by the activation of a Na+/H+ exchange system, located in the plasma membrane of the cell 1111. However, recently we found in the monoblastic U-937 cell line that TPA-induced increase of pHi was not due to the extrusion of protons into the medium, despite ’ To whom reprint requests should be addressed at Department Gothenburg, Box 330 31, S-40033 Gothenburg, Sweden. * Present address: Kabi, Stockholm, Sweden. 551

of Anatomy,

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Copyrrght @ 1989 by Academic Press, Inc. All rights of reproduction m any form reserved 0014-4827/89 SO3.M)

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the existence of a functioning Na+/H+ exchanger, which suggests that the observed alkalinization instead might be accounted for by an intracellular mechanism [ 121. Initial morphological studies revealed the presence of large multivesicular bodies (MVBs) in TPA-stimulated but not in resting U-937 cells [13]. As judged by their dilation upon chloroquine treatment the MVBs appeared to be acidic and might, therefore, be a possible candidate as a proton-sequestrating compartment. In this study we have further investigated the nature and possible origin of MVBs occurring in TPA-stimulated U-937 monoblastic cells.

MATERIALS

AND

METHODS

Chemicals Cationized fenitin, horseradish peroxidase type VI, diaminobenzidine-hydrochloride and 12-Otetradecanoyl-13-acetate were obtained from Sigma Chemical Co. (St. Louis, MO). Ionomycin was obtained from Calbiochem (La Jolla, CA). Sodium-/&glycerophosphate was purchased from Merck (Darmstadt, FRG).

Cells and Culture Conditions The parental U-937 cell line originates from a human histiocytic lymphoma and its phenotypic properties conform with those of immature monocytic cells [14]. TPA induces macrophage differentiation in these cells with a concombinant inhibition of growth [15]. The experiments presented in this paper were performed with the subline U-937 GTB, which have wild-type properties. The mycoplasma-free cell line was kept under standard culture conditions [16] in Ham’s F-10 medium supplemented with 10% newborn calf serum (NCS; GIBCO, Paisley, Scotland). The experiments were also carried out in this medium.

Experimental Procedures The cells were, unless otherwise stated, incubated in 1 ml medium in Ellerman tubes (2x lo6 cells per test tube). Incubations at 4°C were performed in an ice bath, and incubations at 37°C were in a thermostated water-bath. Incubations were terminated by fixation in ice-cold glutaraldehyde. Incubation with TPA. Cells (three experiments) were incubated with or without lo-’ M TPA at 37°C for various times (5, 15, 30, and 60 mitt; lo-’ M TPA was used throughout all experiments in this study). Cells were then processed for conventional electron microscopy as well as for demonstration of acid phosphatase activity (see below). Incubation with ionomycin. Cells (three experiments) were incubated with 10 mM ionomycin for 15 min at 37°C and then processed for morphological examination. TPA and uptake of horseradish peroxidase (HRP). ‘*‘I-Labeled HRP (sp act 25~10~ cpm/mg; iodinated by the Iodogen method [17]) was added to U-937 GTB cells (10-20x lo6 cells/ml; 1~10~ cpm/ml) in ice-cold medium. TPA were also added at this stage (omitted in controls). After removal of time 0 samples the cells were rapidly warmed to 37°C and incubated for up to 60 min. Aliquots of 2-4x lo6 cells (200 ul) were removed at different time intervals, rapidly chilled, and then pelleted by centrifugation through a cushion of phthalate oil (density 1.025 g/ml [18]). The bottoms of the centritugate tubes containing the cells were cut off and cell-associated radioactivity was determined by counting in a Beckman 8OOOg y-counter. Specific accumulation of lZ51-HRP was calculated by subtraction of the label at time 0 and the values were normalized to the accumulation of radioactivity in control cells after 60 min at 37°C (lOO%, average 600 cpm in three independent experiments; <0.5% of the total cpm). Cells were also incubated with HRP (1 mg/ml) at 37°C for 15 and 60 min, with or without simultaneous stimulation with TPA, for morphological examination (three experiments). The cells were fixed in 4% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4 for 1 h (which destroys endogenous peroxidase activity). The fixed cells were washed several times in ice-cold 0.05 M Tris-HCl, pH 7.6, and then preincubated with 0.05% diaminobenzidine (DAB) in 0.05 M Tris-HCl, pH 7.6, for 30 min. HzOz was then added to a tinal concentration of 0.001% and the cells were kept under gentle shaking for 1 h at room temperature. Hz02 was omitted or 1 m&f resorcinol was added in

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control incubations. The cells were finally washed in 0.15 M sodium cacodylate, postfixed in 2.5 % glutaraldehyde followed by 1% osmium tetroxide, and further processed for electron microscopy. TPA and uptake of cationizedferritin (CF). U-937 GTB cells (three experiments) were preincubated with CF (50 &ml) for 30 mm at 4°C in order to label cell-surface anionic sites. After being washed once in ice-cold buffer (to remove excess of tracer) the cells were incubated with or without TPA for 10, 20, and 40 min at 37°C and then processed for electron microscopy.

Acid Phosphatase Cytochemistry Unstimulated cells and cells exposed to TPA for 15 and 60 min at 37°C were fixed in 2.5% glutaraldehyde and then processed for acid phosphatase cytochemistry as modified from Gomori [ 191 using p-glycerophosphate as substrate and lead as the capturing ion. The reaction was carried out for 30 min at 37°C. /SGlycerophosphate was omitted in controls. After incubations the cells were rinsed, posttixed in 1% osmium tetroxide, and further processed for electron microscopy. Unstained as well as stained (with uranyl acetate and lead citrate) sections were examined.

Electron Microscopy Cells were fixed in 2.5% glutaraldehyde in 0.1 M sodium cacodylate, pH 7.4 for 1 h and, after being washed in 0.15 M sodium cacodylate, postfixed in 1% osmium tetroxide in 0.1 M sodium cacodylate for 1 h. After dehydration in ethanol and propylene oxide the pelleted cells were embedded in epoxy resin (Agar 100; Agar Aids, Stanstead, England). Sections, contrasted with uranyl acetate and lead citrate, were examined and photographed in a Philips 400 T electron microscope.

Electron Microscopic Morphometry Morphometry was performed on cells incubated with or without TPA for 15 min as well as on cells exposed to TPA alone or simultaneously with CF for 10,20, and 40 min. Randomly chosen cells were photographed at a magnification of x 10,000. Changes in the morphology of MVBs were evaluated on paper copies. The number of inclusion vesicles per cell profile was counted to estimate the relative amount of membrane in MVBs. The relative area of MVBs in each cell profile was evaluated by pointcounting [20].

RESULTS Ultrastructural

Morphology

of the U-937 Cells

The ultrastructural appearance of the U-937 GTB cells has been described earlier [14]. The organelles of the cells displayed a polarized distribution (Fig. 1). Mitochondria, some very large with areas devoid of cristae, were frequently encountered in one portion of the cytoplasm. There were few cytoplasmic vesicles and vacuoles in this region of the cell. The cytoplasm on the opposite side of the nucleus was dominated by an extensive Golgi apparatus and a heterogenous population of vesicles and vacuoles, including occasional small multivesicular bodies (MVBs). The MVBs were mainly located close to the Golgi complex (Fig. 1). In general, the inclusion vesicles of the MBVs were few and the major portion of the MVB profiles was empty of electron dense content. Mitochondria were occasionally encountered also in this portion of the cytoplasm. Ribosomes and narrow cisternae of rough endoplasmic reticulum were distributed in all parts of the cytoplasm. Incubation with TPA or Zonomycin

As recently reported [13] incubation with TPA caused pronounced changes in the appearance of the MVBs in U-937 GTB cells. The total section area of the

554 Nilsson,

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Fig. 1. Unstimulated U-937 GTB cell. One part of the cell contains numerous mitochondria (M) whereas the other part is occupied by an extensive Golgi apparatus (G). Small MVBs (arrows) are present in the cytoplasm near the Golgi apparatus. N, nucleus. x8000.

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Graph 1. Histogram reflecting the distribution profile of MVB area/cell profile (data from three experiments). Upper panel; unstimulated U-937 cells (n=128); lower panel; U-937 cells exposed to TPA for 15 min (n=137).

MVBs (Graph 1) as well as the number of inclusion vesicles in MVBs per cell profile (Graph 2) were markedly increased after 15 min of stimulation with TPA (no changes were observed after 5 min of stimulation). At this time the limiting membrane of the MVBs often showed tubular extensions into the surrounding cytoplasm (Figs 2 and 3) as well as invaginations into the MVB lumen (Fig. 3), the latter probably representing inclusion vesicles that were partly pinched off. With prolonged incubation time the number of inclusion vesicles increased gradually, and the MVBs assumed a more spherical shape, lacking tubular extensions, and appeared to be completely filled with inclusion vesicles (Fig. 4). The ultrastructural appearance of organelles other than MVBs were not obviously changed in TPA-stimulated cells. U-937 GTB cells were also incubated with ionomycin. There was no morphological change in ionomycin-treated cells as compared to unstimulated cells. The Fig. 2. Part of a U-937 GTB cell stimulated with TPA for 1.5min. One small and one large MVB are present. The limiting membrane of the large MVB expresses tubular cytoplasmic extensions (arrows). G, Golgi apparatus; N, nucleus. ~30,000. Fig. 3. Two MVBs in a cell stimulated with TPA for 15 min. Cytoplasmic extensions (arrows) as well as luminal invaginations (arrowheads) are observed in their limiting membrane. ~41,000.

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Graph 2. Histogram of the number of inclusion vesicles/cell Upper panel; unstimulated U-937 cells (n= 125); lower panel;

profile (data from three experiments). U-937 cells exposed to TPA for 15 min

(n= 135).

MVB area/cell profile and the number of inclusion vesicles/cell profile in ionomytin-treated cells (not shown) were distributed essentially as presented in Graphs 1 and 2 for unstimulated cells. Acid Phosphatase Cytochemistry

In unstimulated U-937 GTB cells a reaction for acid phosphatase activity was present in small vesicles and large irregularly shaped vacuoles (not shown), probably representing primary lysosomes and phagolysosomes, respectively [26, 39411. Small MVBs were only occasionally reactive (not shown). In cells stimulated with TPA for 15 min the enlarged MVBs often contained precipitate (Figs. 5 and 6). The precipitate was distributed in the matrix of MVBs, outside the inclusion vesicles. MVBs free of precipitate were encountered in the same cell as positively stained MVBs (Figs. 5 and 6). Positively stained MVBs were more frequent after 60 min than after 15 min of stimulation with TPA. Endocytosis of HRP

HRP was used as a marker of fluid phase pinocytosis [21]. Unstimulated U-937 GTB cells accumulated ‘*‘I-HRP with time in an almost linear manner (Fig. 7).

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Fig. 4. Stimulation with TPA for 60 min. Two spherical MVBs (arrows) are present on either side of an extensive Golgi apparatus (G). The MVBs appear to be completely filled with inclusion vesicles. The limiting membrane of the MVB to the right is not distinct, due to tangential sectioning. N, nucleus. X23,000. Figs. 5 and 6. Acid phosphatase cytochemistry of cells stimulated with TPA for 60 min. Positively stained MVBs (arrows) are present together with MVBs essentially free of precipitates (arrowheads). N, nucleus. ~30,000 (Fig. 5); x39,000 (Fig. 6).

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SPECIFIC ACCUMULATION

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Fig. 7. Effect of TPA on the uptake of mI-HRP in U-937 GTB cells. Cells were mixed with ice-cold reagents, and accumulation was started by quickly raising the temperature to 37°C. Aliquots (0.2 ml) of the incubations were removed at the indicated times (horizontal axis). Cell-associated radioactivity was determined after centrifugation of the cells through cushion of phthalate. Specific accumulation was calculated by subtraction of the cpm values obtained in ice-cold incubations, and the values were normalized to the cpm of control cells after 60 min at 37°C [lOO%; mean values +SEM from 11 (60 min), 7 (15 min), and 2 (5 and 30 min) measurements, respectively; three independent experiments]. n , Control; +, TPA.

TPA treatment caused a rapid increase in cell-associated ‘251-HRP, which at 5 min amounted to more than 50% of the uptake in control cells at 60 min. The difference in uptake compared to unstimulated cells at 5 min was five times greater. Over the whole 60-min period of incubation, the increased uptake of 1251HRP in TPA-stimulated cells was approximately 2.5 times the control values. Unstimulated cells exposed to HRP for 15 and 60 min and then processed for peroxidase cytochemistry had a dense reaction product in a heterogeneous population of vesicles and irregularly shaped vacuoles throughout cytoplasm (not shown). Small MVBs were occasionally labeled (not shown). In TPA-stimulated cells simultaneously exposed to HRP the enlarged MVBs contained large amounts of dense reaction product (Figs. 8 and 9). The dense reaction product was distributed between the inclusion vesicles (Fig. 8). In MVBs Fig. 8. Simultaneous incubation with HRP and TPA for 60 min, followed by peroxidase cytochemistry. Numerous MVBs contain large amounts of dense reaction product (arrows). Also other, irregularly shaped vacuoles (arrowheads) as well as small vesicles (small arrows) are positively stained. The confhrence of small vesicles with an MVB is suggested. N, nucleus. ~25,000. Fig. 9. Incubation with HRP and TPA for 60 min. Inclusion vesicles are present in the lumen periphery of an MVB. A dense reaction product is distributed along the inner surface of the MVB limiting membrane as well as the outer membrane surface of the inclusion vesicles. ~20,000. Fig. 10. TPA stimulation for 40 min after labeling of the cell surface with CF. The large MVB contains numerous aggregates of CF particles (arrows). The CF appears to be associated with the surface of inclusion vesicles. X40,000. Fig. 11. CF labeling of the cell surface and subsequent stimulation with TPA for 40 min. CF are present at the cell surface (arrowheads) as well as in a large MVB (arrows). Other structures in the cytoplasm are absent of CF. ~33,000.

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‘Graph 3. The number of inclusion vesicles/cell profile in U-937 cells exposed to TPA alone or TPA together with CF (one experiment; mean of 42 cell profiles in each situation).

containing few inclusion vesicles, mainly present in the lumen periphery of the MVBs, the dense reaction product was limited to the outer membrane surface of the inclusion vesicles and the inner surface of the MVB limiting membrane (Fig. 9). Also structures other than MVBs, similar to those observed in unstimulated cells, were labeled (Figs. 8 and 9). Endocytosis

of CF

CF was used as a marker of plasma membrane internalization [22]. After being labeled at 4°C for 30 min CF was found along the surface of the cells (not shown). Raising the temperature to 37°C caused internalization of CF. After 20 as well as 40 min of incubation intracellular CF particles were mainly located in irregularly shaped vacuoles and only occasionally in small MVBs (not shown). The number of intracellular structures labeled with CF was much less than that of cells labeled with HRP (described above). In TPA-stimulated cells internalized CF was more abundant than in controls. Intracellular CF was predominantly located in large MVBs (Figs. 9, 10, and 11). The CF particles were associated, in the form of small aggregates, with the outer surface on inclusion vesicles (Figs. 9 and 10). In one experiment, where the surface of the cells was labeled with CF to a very high extent, the TPA-induced increment of inclusion vesicles was markedly retarded (Graph 3). DISCUSSION Possible Origin and Function Monoblastic Cells

of Multivesicular

The observations in this study confirm tion of large MVBs in U-937 GTB cells size of the MVBs as well as their content that of the MVBs present in unstimulated min and increased further with prolonged

Bodies in U-937

and extend a previous report of formaacutely stimulated with TPA [13]. The of inclusion vesicles markedly exceeded cells. This was evident already after 15 stimulation with TPA. Parallel incuba-

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tions with i2’I-HRP also demonstrated a TPA-induced increase in the endocytic activity of U-937 cells. Interestingly, the largest increment in HRP uptake was obtained within 5 min of incubation, thus preceding the first morphological signs of MVB enlargement. Because internalized HRP (as well as CF) in TPA-stimulated cells was present to a much larger extent in MVBs than in controls this indicates that the TPA-induced enhancement of endocytosis, at least partly, is routed to this organelle. Phorbol ester has previously been shown to stimulate endocytosis in peritoneal macrophages [50, 511. In other cells MVBs have been demonstrated as prelysosomal compartments involved in receptor-mediated endocytosis [23-261. In this respect, these structures also have been referred to as multivesicular endosomes [25]. Typical morphological signs of vacuoles involved in receptor-mediated endocytosis are tubular extensions of their limiting membrane into the surrounding cytoplasm [27, 281. The membrane of such extensions appears to be enriched in receptors that, subsequent to dissociation from ligand, return to the cell surface (receptor recycling), hence the designation CURL (compartment of uncoupling receptor and ligand) for this type of organelle [28]. Because in the present study we observed that the MVBs at 15 min of TPA stimulation frequently expressed similar cytoplasmic extensions, which were not encountered in resting cells and less frequent in cells exposed to TPA for 60 min, it is probable that the MVBs are part of the receptor-mediated endocytic pathway also in U-937 cells. Following this, it is highly suggestive that the TPA-induced increase in endocytic activity, which apparently is directed mainly toward the MVBs, might involve internalization of cell-surface receptors. Indeed, rapid down-regulation of certain cellsurface receptors, i.e. the receptors for insulin [30], insulin-like growth factors [31], transferrin [32], and low-density lipoprotein [33], has been demonstrated in U-937 cells upon TPA stimulation without the presence of specific ligands. In other cells the same effect of TPA has been shown for e.g. the receptors of epidermal growth factor [34], C3b [35] and tumor necrosis factor [36]. MVBs could serve as a deposition site for down-regulated cell-surface receptors similar to the transfenin receptor no longer needed by the hemoglobinized reticulocytes [37]. The transferrin receptors are withdrawn from the cell surface by endocytic vesicles and, after fusion and fission events at the MVB limiting membrane, integrated into the membrane of inclusion vesicles. Finally the inclusion vesicles are released from the cell by fusion of the MVB with the plasma membrane [37]. Another example of sequestration of cell-surface receptors in the membrane of inclusion vesicles is ligand-induced internalization of EFG receptors in epidermoid carcinoma (A431) cells [38]. It is likely that the inclusion vesicles appearing in the MVBs of U-937 cells also are derived from the plasma membrane. CF, previously adsorbed to the surface of the cells, was routed almost exclusively to MVBs upon TPA stimulation (Fig. 12), which is in contrast to the situation with endocytosed HRP, where the tracer was found to a large extent also in other intracellular structures. This qualitative difference in the endocytic pattern between HRP and CF most likely depends on their different physicochemical properties, making them unspecific markers of, respectively,

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Fig. 12. Internalized CF in a cell stimulated with TPA for 40 min. The MVBs are the only cytoplasmic structures that contain CF (arrows). G, Golgi apparatus; N, nucleus. ~30,000.

fluid-phase endocytosis and plasma membrane internalization. In addition, in cells where the cell surface was heavily labeled with CF the TPA-induced formation of inclusion vesicles appeared to be less pronounced than in TPAstimulated cells incubated in parallel without CF. This further supports the notion that inclusion vesicle formation depends on events taking place at the level of the plasma membrane. Presumably the endocytic transfer of plasma membrane portions to the MVBs in some way was disturbed by the CF attached to the cell surface. Taken together, a plausible explanation for increased endocytosis and enlargement of MVBs in TPA-stimulated U-937 GTB cells might be that TPA initiates rapid internalization of cell-surface receptors, which then are transported to the MVBs (Fig. 13). The down-regulated molecules, sequestered in the membrane of inclusion vesicles, might then be subjected to hydrolytic and/or enzymatic digestion as fusion of MVBs with primary lysosomes was indicated by their acquisition of acid phosphatase activity. Lysosomal fusion with MVBs has been demonstrated to occur in other cells [26, 39411. The findings likely reflect early events taking place during TPA-induced differentiation of U-937 monoblastic cells toward macrophage-like cells [ 151.

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e TPA

Fig. 13. Model illustrating the development of large MVBs in TPA-stimulated U-937 monoblastic cells. The increased endocytosis upon TPA stimulation is mainly routed to the MVB. The enlarged MVBs obtain acid phosphatase activity probably by fusion with primary lysosomes (PLY). See text under Discussion for additional comments.

Cytoplasmic Alkalinization

and Multivesicular

Bodies

In a previous study on U-937 cells was found that TPA, as in many other cells, induced a rapid alkalinization of the cytoplasm [ 121. The achieved increase of about 0.09 pH units reached steady state after l&15 min of incubation. However, unlike the situation in other cell systems [ 111, the TPA-induced alkalinization was not due to the activation of the Na+/H+ exchanger located in the plasma membrane, suggesting that the protons probably were sequestered in some intracellular compartment. An intracellular mechanism has also been proposed for the alkalinization at fertilization in the sea urchin egg [29]. Several organelles could serve as acceptors of protons in such a process [24]. It is wellknown that endocytosis is accompanied by acidification of the involved structures [24]. For instance, uncoupling of receptor and ligand [24, 45, 463 and probably also membrane fusion between different endocytic compartments [47-49] are pHdependent processes. It thus seems likely that the TPA-mediated increase in endocytic activity as observed in this study on U-937 cells also involves compartmental acidification. That the MVBs of TPA-stimulated U-937 cells indeed are acidic, likely due to the action of a proton pump [42-44], is indicated by their swelling upon chloroquine treatment [13] and their positive reaction for acid phosphatase. As protons used in acidification of endocytic compartments in all probability are derived from the surrounding cytoplasm a net increase in the pHi should be expected. Thus, the rapid pHi alkalinization observed in TPA-stimulated U-937 GTB cells [12] might be secondary to acidification of the activated endocytic system, of which the MVBs are a central part. The reverse relationship, i.e., the enlargement of MVBs being dependent on a preceding alkalinization of the cytoplasm, is unlikely as the calcium-ionophore ionomycin, known to

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induce cytoplasmic alkalinization in U-937 GTB cells by activation the Na+/H+ exchanger [ 131, did not affect the appearance of MVBs compared to unstimulated cells. We thank Professor Lam E. Ericson for valuable comments on the manuscript. The skillful technical assistance of Gunnel Bokhede and Yvonne Josefsson is gratefully acknowledged. This study was supported by the Swedish Medical Research Council (12x-537) and the Swedish Cancer Society.

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Received July 27, 1988 Revised version received November 11, 1988