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THE JOURNAL OF Browcic~~ CHEMISTRY 0 1994 by The American Society for Biochemistry and Molecular Biology, Inc.

VOl. 269, No. 28, Issue of July 15, PP. 18607-18615,1994 Printed in U.S.A.

Selective Regulation of Apical Endocytosis in Polarized Madin-Darby Canine Kidney Cells by Mastoparan and CAMP*

(Received for publication, March 15, 1994, and in revised form, May 3, 1994)

Per EkerS, Pernille Kaae Holm$, Bo van Dews§, and Kirsten SandvigSfl From the $Institute for Cancer Research a t The Norwegian Radium Hospital, Montebello, 0310 Oslo, Norway and the §Structural Cell Biology Unit, Department of Medical Anatomy, The Panum Institute, University of Copenhagen, DK-2200 Copenhagen N , Denmark

We here demonstrate that mastoparan, fluoride, forskolin, cholera toxin, and b-bromoadenosine-3’-5’-cyclic monophosphate all selectively stimulate apical endocytosis of the protein toxin ricin without increasing the uptake at the basolateral side of polarized Madin-Darby canine kidney cells. Activation of adenylyl cyclase and an increased level of CAMP seem to increase ricin endocytosis by a clathrin-independent mechanism since stimulation of endocytosis by cholera toxin and d-bromoadenosine-3‘-5’-cyclic monophosphate occurred even when the clathrin-dependent pathway was blocked by low cytosolic pH. The data suggest that mastoparan stimulates apical endocytosis by interacting with heterotrimeric G proteins, and also this stimulation of endocytosis appears to be clathrin independent since the uptake of transferrin at the apical side was strongly inhibited by mastoparan after brefeldin A-induced missorting of the transferrin receptor to this pole of the cell. In addition, mastoparan stimulated apical endocytosis when clathrin-mediated endocytosis was blocked by acidification of the cytosol. Furthermore, we provide evidence for the existence of clathrin-independent endocytosis on both the apical and the basolateral surface of control Madin-Darby canine kidney cells. Our results suggest that endocytosis at the apical pole of epithelial cells can be regulated selectively by a physiological signal.

Endocytosis in many cell types occurs both by clathrin-dependent and clathrin-independent mechanisms (for review, see Refs. 1-3). The various steps in clathrin-mediated endocytosis seem to be dependent on GTP-binding proteins (4), and heterotrimeric G proteins are known to be involved in regulation of vesicle formation and transport in general (5-10). Additionally, clathrin-independent endocytosis has been found to be a regulated process (2, 11-14). Moreover, in polarized epithelial cells apical and basolateral endocytosis could be under differential control. We have recently found that brefeldin A, which inter- feres with binding of coat proteins involved in vesicle budding and vesicular transport (15, 16), selectively stimulates apical endocytosis and the formation of very large vacuoles in polarized MDCK’ cells (17), and Gottlieb et aE. (18) found that high

* This work was supported by the Norwegian and the Danish Cancer Societies, the Norwegian Research Counsil for Science and the Humani- ties, The Danish Medical Research Counsil, The Novo Nordisk Foun- dation, The Carlsberg Foundation, and NATO Collaborative Research Grant CRG 900517. The costs of publication of this article were de- frayed in part by the payment of page charges. This article must therefore be hereby marked “aduertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

7 To whom correspondence should be addressed. Tel.: 47-22506050;

The abbreviations used are: MDCK, Madin-Darby canine kidney; Fax: 47-22508692.

concentrations of cytochalasin D selectively inhibit apical endocytosis without affecting basolateral endocytosis. In this con- text we decided to extend our studies of regulation of endocytosis and transport of the cytotoxic protein ricin in polarized epithelia, using MDCK cells as a model system (17, 19). There is a considerable interest for ricin due to its potential impor- tance in the construction of potent immunotoxins and other toxin conjugates. In addition, ricin binds to a large number of cell surface molecules (1-3) and can therefore be used as a general marker to study membrane traffic in polarized epithelia. The question has been raised whether polarized cells have clathrin-independent endocytosis, and we now present evidence for the existence of such an endocytic mechanism on both the apical and the basolateral pole of MDCK cells growing as polarized monolayers on filters. Our results also demonstrate that incubation of polarized MDCK cells with mastoparan, cholera toxin, forskolin, fluoride, and 8-Br-CAMP selectively stimulates apical internalization of ricin, and the data suggest that this increased uptake is due to clathrin-independent endocytosis.

EXPERIMENTAL PROCEDURES Materials-Horseradish peroxidase, type 11 and VI-A, o-dianisidine,

diaminobenzidine, BSA (fraction VI, mastoparan (M-5280), 8-Br-cAMP, forskolin, cholera toxin, HEPES, and Tris were obtained from Sigma. Rp-8-Br-CAMPS was obtained from Biolog Life Science Institute, Bremen, Federal Republic of Germany, and H89 was purchased from Seikagaku Corp., Tokyo, Japan. NaLZ5I was from the Radiochemical Centre, Amersham, United Kingdom. Ricin and transferrin were iodi- nated by the iodogen method (20). Ricin-horseradish peroxidase was prepared by the N-succinimidyl-3-(2-pyridylthio)propionate method, and monovalent (1:l) fractions were thereafter collected by gel filtration as earlier described (21). Mastoparan analogues mas7 and mas17 were obtained from Peninsula Laboratories, Inc., Belmont, CA.

Cells-MDCK (strain I) cells were grown in Costar 3000 flasks (Costar, BadhoevedorpPThe Netherlands) or T-25 flasks (NUNC, Roskilde, Denmark). MDCK cells were also seeded on polycarbonate filters (Costar Transwell, pore size 0.4 pm, diameter 24.5 mm) at a density of 106/filter and used for experiments 3 or 4 days later (19). All filters used for experiments had a transepithelial resistance of at least 2000 ohm x cm2 as measured with the Millicell-ERS equipment (Milli- pore Corp., Bedford, MA), also at the end of the experiments. The medium used was DMEM (3.7 glliter sodium bicarbonate; Flow Labo- ratories, Imine, Scotland) containing 5% fetal calf serum, nonessential amino acids (Life Technologies Inc., Paisly, Scotland), and 2 mM ~-glutamine (Life Technologies Inc.). Endocytic uptake measurements of ricin and horseradish peroxidase in MDCK cells were performed in HEPES- (20 mM) buffered DMEM with 2 mM glutamine, without sodium bicarbonate (DMEM-H, Flow), supplemented with 0.2% BSA.

Measurements of Endocytosis and fianscytosis-Endocytosis of lZ5I- labeled ricin (100-200 ng/ml, 30,00040,000 cpdng) was measured as

BFA, brefeldin A , 8-Br-cAMP, 8-bromoadenosine-3’,5’-cyclic monophosphate; Rp-8-Br-cAMPS, 8-bromoadenosine-3‘,5’-cyclic monophospho- thionate Rp-isomer; BSA, bovine serum albumin; DMEM, Dulbecco’s

ered saline; M V B , multivesicular body; EM, electron microscopy. modified Eagle’s medium; cpm, counts/minute; PBS, phosphate-buff-

18607

18608 Selective Regulation of Apical Endocytosis in MDCK Cells

the amount of toxin that could not be removed by 5 x 15 min washes with 0.1 M lactose, 0.2% BSA in cold PBS and also as the amount of lactose-resistant toxin after 5 min at 37 "C as previously described (17, 22). The amount of transcytosed ricin was measured as the amount of 1251-labeled ricin that was released in the medium at the opposite to where ricin was added, plus the amount of ricin that came off the cells during a 5-min incubation with lactose at 37 "C (19). This last incubation was required to release toxin that was still surface bound after transcytosis. '251-Transferrin endocytosis was measured as described previously (23). Briefly, cells were incubated with '251-transferrin (50- 150 ng/ml; 10,000-20,000 cpdng) a t 37 "C for the indicated time, washed three times with ice-cold PBS, and then treated for 1 h a t 0 "C with serum-free medium containing 0.3% (w/v) Pronase. Then the cells and the medium were transferred to Eppendorf tubes and centrifuged for 2 min, and the radioactivity in the pellet and in the supernatant was measured. Uptake of horseradish peroxidase added apically was measured as earlier described (17). After endocytic uptake of horseradish peroxidase, the polycarbonate filters were cooled to 0 "C and washed 5 x 15 min with cold PBS, 0.2% BSA. The cells were subsequently lysed with 1% Triton X-100 and 0.05% SDS, and horseradish peroxidase in the lysate was quantitated by the o-dianisidine reaction as described (24).

Measurement ofcAMP-The content of CAMP in cells was measured by a cyclic-AMP 3H assay system from Amersham. In principle, 2 x lo6 cells growing in Petri dishes (diameter 5 cm) were washed twice in PBS and then dissolved in 750 pl of ice-cold HCl (10 mM) in ethanol (96%). After 5 min of incubation at 0 "C, the cells were removed with a rubber policeman, and the cell suspension was centrifuged for 10 min in an Eppendorf centrifuge. The supernatant was freeze-dried and the pellet was dissolved in 2 ml of KOH (0.2 M). The absorbance (280 nm) of this solution was used as a measurement of the amount of cells used. The freeze-dried supernatant was dissolved in 250 p1 of sodium acetate (0.5 M, pH 6.2). This solution was then used in the Amersham CAMP kit to measure the concentration of CAMP in the cells.

Electron Microscopy-Filter-grown MDCK cells were rinsed twice with DMEM-H and incubated with horseradish peroxidase (10 mg/ml) or ricin-horseradish peroxidase as described in the text. Fixation was carried out using 2% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.3. Following fixation, cells were processed for 3,3'-diaminobenzidine cyto- chemistry immediately and then contrasted en bloc with uranyl acetate and embedded in Epon as previously described (25). Sections were ex- amined in a Jeol 100 CX microscope.

RESULTS

Effect of Mastoparan on Ricin Endocytosis in Polarized MDCK Cells-Mastoparan, a peptide found in wasp venom, is a well known stimulator of heterotrimeric G proteins (26, 27). We therefore tested whether this compound had any effect on apical and basolateral endocytosis of the protein toxin ricin. As shown in Fig. lA, mastoparan strongly stimulated apical endocytosis of ricin, whereas there was rather a slight decrease in the uptake of ricin at the basolateral side. The uptake at the apical side was stimulated almost to the maximal level even when ricin uptake was measured after only 5 min (Fig. lB), suggesting that the increased toxin accumulation is due mainly to stimulated endocytosis, and not to reduced recycling. Control experiments showed that preincubation of the cells with mastoparan at 37 "C had no effect on the binding of ricin to the cells at 0 "C (data not shown). Furthermore, 50 VM mastoparan had no cytotoxic effect on these cells. During a 1-h incubation this concentration of mastoparan did not affect the protein synthesis, nor did it strongly reduce the electrical resistance across the monolayer (data not shown).

Mastoparan is believed to form an a-helix upon interaction with membranes (28). To examine the specificity of this peptide on the increased endocytosis, we tested the effect of two analogues, mas7, which like mastoparan can form an a-helix, and mas17 which is unable to obtain such a conformation. Mas7 was like mastoparan able to increase the ricin endocytosis, whereas mas17 was completely inactive in this system (Fig. 1D). Thus, the increased endocytosis seems to be dependent on the conformational change that is also required for activation of heterotrimeric G proteins.

A

, I #

240

u 0 1 0 2 0 3 0 4 0 5 0

C

FIG. 1. Effect of mastoparan on ricin (A, B, and D ) and transferrin ( C ) endocytosis in MDCK cells. A, effect of mastoparan on apical and basolateral endocytosis of ricin. MDCK cells growing on filters were incubated with increasing concentrations of mastoparan for 30 min at 37 "C. Then lZ5I-ricin was added either at the apical or at the basolateral side of the cells. The amount of endocytosed ricin was measured 15 min later. Apical control, 3239 cpm; basolateral control, 8911 cpm). B, apical ricin endocytosis in mastoparan-treated cells (50 p ~ , 30 rnin), increasing incubation time with toxin. MDCK cells growing on filters were incubated with and without mastoparan, and then lZ5I-ricin was added apically. The amount of endocytosed toxin was measured after increasing periods of time. Control values were: 5 min, 1171 cpm; 10 min, 2148 cpm; 15 min, 3014 cpm; 30 min, 6260 cpm. C , left panel: effect of mastoparan on transferrin uptake in plastic-grown MDCK cells. The cells were incubated with increasing concentrations of mastoparan for 30 min, and then '251-transferrin was added. 5 min later transferrin endocytosis was measured (cell-associated transferrin in the control, 3862 cpm; with 10 p~ mastoparan, 3462; with 25 PM mastoparan, 4663 cpm; with 50 PM mastoparan, 4305 cpm; with 100 PM masto-

cytosis of transferrin. MDCK cells grown on filters were treated with paran, 4674 cpm). C , right panel: effect of mastoparan on apical endo-

BFA (2 pg/ml) for 20 min a t 37 "C to induce appearance of transferrin receptors at the apical side. Then mastoparan (50 PM) was added to some of the cells, and 20 min later 1251-transferrin was added. Endocy- tosis of transferrin was measured 5 min later. The background value represents transferrin which was not removed from cells incubated with the ligand a t 0 "C. Total cell-associated transferrin in the control, 2382 cpm; with mastoparan, 2247 cpm; background (bound to cells on ice),

in polarized MDCK cells. The cells were incubated for 30 min a t 37 "C 1993 cpm. D, effect of mastoparan analogues on apical ricin endocytosis

with and without 50 PM mastoparan, mas7, or masl7. Then '"I-ricin was added to the apical side, and the amount of endocytosed ricin was measured 15 min later (control value, 2392 cpm). The bars in A represent S.D. (n = 5-10). In C and D, the bars represent deviations between parallels.

Recent studies have shown that mastoparan inhibits uptake of transferrin in permeabilized A431 cells (4). As shown in Fig. 1C (left panel ), the uptake of transferrin in MDCK cells grown on plastic was strongly reduced by mastoparan. Since the transferrin receptor is missorted to the apical side of BFA- treated polarized MDCK cells (29), we used BFA-treated cells to measure the effect of mastoparan on apical uptake of transferrin in MDCK cells grown on filters. The results showed that

Selective Regulation of Apical Endocytosis in MDCK Cells 18609

c -

U f: c- W

A Apical

300 ~

P

Basolateral

P

C

Aplcal Basolateral

cells. Filter-grown cells were incubated with and without NaF (10 mM) andAIC1, (50 p ~ ) for 30 min (A) , forskolin (100 p ~ ) also for 30 min (B) , and FIG. 2. Effect of fluoride and aluminum chloride (A), forskolin (B) , and cholera toxin (C) on ricin endocytosis in polarized MUCK

cholera toxin or cholera toxin B subunit (1 pg/ml) for 4 h (C). Then '251-ricin was added to the apical or the basolateral side of the filter-grown cells,

side, 1,364 cpm; Basolateral side, 3,904 cpm; C: Apical side, 2,482 cpm; Basolateral side, 22,470 cpm. Different batches of lZ5I-ricin were used in and the amount of endocytosed toxin was measured 15 min later (Control values: A , Apical side, 1,494 cpm; Basolateral side, 8,571 cpm; B: Apical

these experiments.). The error bars represent S.D. ( n = 3-81,

also uptake of transferrin at the apical side was inhibited by mastoparan (Fig. 1C (right panel)), suggesting that the increased ricin uptake occurring upon mastoparan treatment (Fig. 1, A and B ) is mediated via a clathrin-independent mechanism (see below).

Effects of Aluminum Fluoride, Forskolin, Cholera Toxin, and 8-Br-CAMP on Ricin Endocytosis in Polarized MDCK Cells- Another stimulator of heterotrimeric G proteins is aluminum fluoride (30). This compound also stimulated apical endocytosis of ricin and had no stimulating effect on the basolateral uptake (Fig. 2 A ) . The aluminum ion as such had no effect. However, F- alone also stimulated the apical endocytosis (Fig. 2 A ) . Neither aluminum fluoride nor fluoride alone had any effect on the amount of ricin bound to cells a t 0 "C (data not shown). Re- cently, F- was shown to stimulate some heterotrimeric G proteins (311, and F- has previously been shown to increase the level of CAMP in cell lysates containing adenylyl cyclase (32, 33). As shown in Fig. 3, in MDCK cells fluoride also increased the level of CAMP.

To test whether the effect on apical endocytosis could be mediated by an activation of adenylyl cyclase, we measured endocytosis of ricin after addition of forskolin, an activator of adenylyl cyclase, and cholera toxin, which ADP-ribosylates the a, subunit of heterotrimeric G proteins (34, 35) and thereby activates adenylyl cyclase. As shown in Fig. 3 both compounds did, also in MDCK cells, increase the level of CAMP, and, as shown in Fig. 2, B and C, these compounds stimulated apical endocytosis selectively. It turns out that even a slight stimulation in the CAMP level caused by low concentrations of forskolin (Fig. 3) has an effect on the endocytosis (compare Fig. 3 and Fig. 10). Interestingly, as also shown in Fig. 3, mastoparan did not increase the level of CAMP in the cells, and this compound therefore seems to affect endocytosis by a different mechanism. As described above for mastoparan and fluoride, neither cholera toxin nor forskolin affected the binding of ricin to the cells (data not shown).

The results described above suggested that the stimulation of apical endocytosis after incubation with fluoride, cholera toxin, and forskolin was mediated by an activation of adenylyl

paran on the level of CAMP in MDCK cells. MDCK cells were FIG. 3. Effect of cholera toxin, fluoride, forskolin, and masto-

incubated with and without cholera toxin (2 pg/ml) for 2 h, and with and without fluoride (10 mM), mastoparan (50 PM), or the indicated concentrations of forskolin for 30 min before the level of CAMP was determined as described under "Experimental Procedures." The bars represent S.D. ( n = 3 4 ) .

cyclase. We therefore tested whether a direct addition of a membrane permeant CAMP analogue, 8-Br-cAMP, could stimulate apical endocytosis. As shown in Fig. 4, this was indeed the case. Even when ricin uptake was measured after only 5 min of incubation with the toxin, there was a similar large effect of 8-Br-CAMP (data not shown), suggesting that the rate of uptake from the cell surface is increased. As in the experiments described above, 8-Br-CAMP had no effect on basolateral endocytosis (Fig. 4). The stimulatory effect on the apical endocytosis did not require protein synthesis. When the protein synthesis was blocked by addition of cycloheximide prior to addition of 8-Br-cAMP, endocytosis was stimulated to the same extent as in the absence of cycloheximide (data not shown). Interestingly, after longer periods of incubation, 8-Br-CAMP had a strong stimulatory effect on transcytosis of lZ5I-ricin in both directions,


220 A Apical 6 Basolateral

FIG. 4. Effect of 8-Br-CAMP on ricin endocytosis in polarized MDCK cells. Filter-grown MDCK cells were incubated with increasing concentrations of 8-Br-CAMP for 30 min at 37 “C. Then 1251-ricin was added to the apical side or to the basolateral side, and the amount of endocytosed toxin was measured 15 min later. Control values: A, 1425 cpm; B, 5439 cpm. The bars represent deviations between parallels.

although the stimulation was strongest from the basolateral side (Fig. 5).

Evidence for Clathrin-independent Endocytosis in Polarized MDCK Cells-It has previously been shown that non-polarized cells have different endocytic mechanisms, both clathrin-dependent and clathrin-independent endocytosis (for review, see Refs. 1-3). Moreover, our results with mastoparan suggested that clathrin-independent endocytosis can occur also in polarized MDCK cells. To study whether this is the case in un- treated (control) MDCK cells as well, we used two different methods that both perturb the uptake from clathrin-coated pits, and investigated whether ricin, which is bound all over the cell-surface, is still endocytosed. As shown in Fig. 6, acidification of the cytosol which in other cell types studied blocks the formation of coated vesicles from coated pits (36, 371, will also in MDCK cells block the uptake of transferrin. The endocytic uptake of transferrin was inhibited not only in unpolarized MDCK cells (Fig. 6A), but also on the basolateral side of polarized MDCK cells from where transferrin is normally endocytosed (Fig. 6B). In addition, after missorting of the transferrin receptor to the apical side by addition of BFA (291, uptake of transferrin added to this pole of the cells was inhibited by low cytosolic pH (Fig. 6C), suggesting that the formation of coated vesicles even at this side is blocked under these conditions. Without BFA treatment there was no measurable binding and endocytosis of transferrin at the apical side (data not shown). As shown in Fig. 6D, although there is a reduced uptake, ricin is still endocytosed both from the apical and the basolateral side when clathrin-mediated endocytosis is blocked, suggesting that also in the polarized MDCK cells there is clathrin-independent endocytosis. The effect of acidification on endocytosis of ricin is expressed in percent to make it easier to compare directly the effect on apical and basolateral uptake. Absolute values for ricin binding and endocytosis have previously been published (19). As seen in Fig. 6D, the block in clathrin-mediated endocytosis has a stronger effect on the uptake of ricin from the apical side than from the basolateral side, suggesting that a larger fraction of the nonperturbed endocytic uptake is mediated via clathrin-coated pits and vesicles on the apical than on the basolateral side. We have previously shown by EM that ricin-horseradish peroxidase is internalized and trans- ported intracellularly like native ricin (21,381. Fig. 6 , E and F, show that also in acidified MDCK cells ricin is endocytosed from the apical surface and delivered to endosomes. It should,

A Apical

I addition

+

B: Basoiateral addition

- + FIG. 5. Effect of 8-Br-CAMP on transcytosis of ricin. Filter-grown

cells were incubated with and without 8-Br-CAMP (2 mM) for 30 min at 37 “C. Then lZ5I-ricin was added either to the apical (A) or the basolateral surface ( B ) , and the cells were incubated for 1 h at 37 “C. The amount of transcytosed ricin was then measured as described under “Experimental Procedures.” A: control transcytosed, 4,238 cpm; endocytosed, 29,207 cpm; with 8-Br-CAMP: transcytosed, 14823 cpm; endocytosed, 59,771 cpm. B: control transcytosed, 1,550 cpm; endocytosed, 11,0736 cpm; with 8-Br-CAMP: transcytosed, 4,093 cpm; endocytosed, 110,135 cpm). The burs represent S.D. of three experiments

however, be stressed that we cannot exclude that non-clathrin- mediated uptake is also somewhat sensitive and could respond differentially to acidification on the two sides. The small mastoparan-induced decrease in ricin endocytosis at the basolateral side (see Fig. IA) is probably due to inhibition of uptake via coated pits. Thus, when the cytosol was acidified to block this pathway, mastoparan did not give any additional decrease in the ricin uptake (data not shown).

Another way to interfere with clathrin-mediated endocytosis is to remove the clathrin coats from the cell surface by potassium depletion of the cytosol (3942). EM studies revealed that also in MDCK cells this method strongly decreased the number of clathrin coats at the cell surface (data not shown), and as shown in Fig. 7, transferrin endocytosis at the basolateral side of the cells was inhibited. However, in this case ricin was still endocytosed both from the apical and the basolateral side (Fig. 7). In agreement with the data described above, inhibition of the clathrin-mediated pathway with potassium-depletion reduced the uptake of ricin to a larger extent at the apical than at the basolateral side.

Regulation of Endocytosis in Cells with Low Cytosolic pH-As shown above, low cytosolic pH is able to inhibit formation of clathrin-coated vesicles in MDCK cells. Addition of cholera toxin, 8-Br-cAMP, and mastoparan did not abolish the inhibition of transferrin uptake at the basolateral side of filter- grown MDCK cells (Fig. 8). Similar results were obtained with uptake of transferrin at the apical pole after BFA-induced missorting of the transferrin receptor to this pole (data not shown). In spite of the block in transferrin endocytosis at low cytosolic pH, cholera toxin, 8-Br-cAMP, and mastoparan were all able to stimulate apical ricin endocytosis (Fig. 9A), suggesting that the stimulated uptake is clathrin-independent. Fig. 9B reveals endocytic uptake of ricin in acidified cells treated with 8-Br- CAMP.

Involvement of Protein Kinase A in Regulation of Endo- cytosis-In order to test whether the effect on endocytosis of cholera toxin, Br-CAMP, forskolin, and mastoparan were all mediated via protein kinase A, we tested the effect of the protein kinase A inhibitor H89 (43) on the stimulation of ricin endocytosis. This inhibitor strongly counteracted the effect of


FIG. 6. Effect of cytosolic acidification on transferrin and ricin endocytosis. MDCK cells grown on plastic (A) or on filters (B-F) were incubated a t pH 5.5 without or with either the indicated concentrations of acetic acid or with 10 mM acetic acid (B-F). After 5 min, '2511-transferrin, "'I-ricin, or ricin-horseradish peroxidase were added, and after 5 min further incubation with transferrin and after 15 min of incubation with ricin, the binding and endocytosis were measured as described under "Experimental Proce- dures." In A, cell-associated transferrin in the control was 15,818 cpm; and with increasing concentrations of HAC: 13,737, 16,694, 19,531, and 19,653 cpm; B, similar experiment in counts, see Fig. 8; C, plotted in counts; D, control apical side: 8,732 cpm; control basolateral side, 17,412 cpm. The columns marked with background in B and D show the radioactivity associated with cells incubated with transferrin on ice and then treated with Pronase as in the endocytosis assay. In E is shown small vesicles with ricin-horseradish peroxidase internalized from from the apical surface (arrows), and in F an unlabeled multivesicular endosome (open arrow) as well as a labeled one (arrow) are seen. Bar, 0.5 PM.

5 10 15 20 Acetic acid (mM)

1 2 3 4 5

1 2 3

Acetic acid (mu)

Acetic acid (10 mM) Rkin-HRP, 15 min apical

Acetic mid (10 mM) Ricin-HRP, 15 min apical

cholera toxin, 8-Br-cAMP, and forskolin (Fig. 10, A, B, and D ) , and forskolin as expected increased the level of cAMP in the whereas there was no effect on the apical stimulation after MDCK cells, whereas no increase in the cAMP level could be addition of mastoparan (Fig. lOC), further supporting the no- measured after addition of mastoparan (Fig. 3). Furthermore, tion that this compound might act via a different pathway. This the inactive CAMP analogue Rp-8-Br-CAMPS counteracted the hypothesis was also supported by the finding that cholera toxin increase in ricin endocytosis caused by 8-Br-cAMP, whereas


there was no reduction in the effect of mastoparan, supporting the view that the effect of this compound on endocytosis is not mediated via CAMP (data not shown).

Effects of Mastoparan and 8-Br-CAMP on Endocytosis of the Fluid-phase Marker Horseradish Peroxidase-Finally, we found it relevant to investigate whether the increase in apical endocytosis of membrane-bound ricin caused by mastoparan and 8-Br-CAMP could also be demonstrated for a protein internalized in the fluid phase. For this purpose we used horseradish peroxidase. Biochemical measurements revealed that the uptake of horseradish peroxidase from the apical side was increased to the same extent as the uptake of ricin by addition of

FIG. 7. Effect of potassium depletion on transferrin and ricin endocytosis. MDCK cells growing on filters were exposed for 5 min a t 37 "C to a mixture of 50% water and 50% of a potassium-free buffer containing 0.14 M NaCI, 20 mM HEPES, 2 mM CaCI,, and 1 mg/ml glucose. Then the cells were washed in the potassium-free buffer and incubated for 20 min more in this solution, or in a buffer containing also 5 mM KC1. 1251-Transferrin was then added to the basolateral side (A), and 5 min later binding and endocytosis were measured as described under "Experimental Procedures." Cell-associated transferrin in the control, 4,053 cpm; K+-depleted cells, 2,345 cpm. Ricin was added either a t the apical or at the basolateral side (B) , and binding and endocytosis were measured as described under "Experimental Procedures" 15 min later. Ricin endocytosed a t the apical side: control, 16,890 cpm; K+- depleted cells, 6,911 cpm. Basolateral side: control, 43,100; K+-depleted cells, 31,660 cpm. The bars indicate deviations between parallels.

8-Br-CAMP (data not shown), and quantitative analysis of EM preparations revealed a clear increase in the number of apical endosomes labeled by endocytosed horseradish peroxidase following treatment either with 8-Br-CAMP or mastoparan (Fig. 11). Moreover, since we have previously shown that BFA in- duces formation of a special population of very large vesicles at the apical side of MDCK cells concomitantly with increased endocytosis (171, we wanted to see whether mastoparan and CAMP induced a similar morphological change. However, EM clearly showed that the stimulated endocytosis in cells treated with mastoparan and CAMP was associated with the formation of typical multivesicular body (MVB)-like endosomes (25), similar in size and morphology to those in control cells (Fig. 11).

DISCUSSION

The present results indicate that endocytosis in polarized MDCK cells occurs by clathrin-dependent as well as clathrin- independent mechanisms. The finding that methods known to inhibit uptake form clathrin-coated pits reduce endocytic uptake of ricin to a larger extent on the apical than on the basolateral side suggests that clathrin-mediated endocytosis con- tributes more to the total endocytic uptake on the apical than on the basolateral side. However, we cannot exclude that clathrin-independent endocytosis also might be somewhat sensitive to the treatments used and that non-clathrin-mediated endocytosis on the apical side might be more affected than on the basolateral side.

The results also show that endocytosis in polarized MDCK cells can be regulated selectively at the apical side by increased levels of CAMP and by mastoparan. Although mastoparan has been reported to increase the level of CAMP in human platelets (441, we found no evidence for involvement of CAMP in the mastoparan-induced endocytosis shown here. Additionally, it should be noted that mastoparan is a more potent stimulator of Gi than of G, (28). Our experiments with mastoparan analogues suggest that mastoparan-induced endocytosis is mediated by activation of heterotrimeric G proteins. Only the analogue that like mastoparan can form an a-helix upon interaction with membranes, a structure required for interaction with the heterotrimeric G proteins (28), is able to stimulate endocytosis, suggesting that the effect may be mediated via these G proteins. The concentrations of mastoparan that increase apical endocytosis when added to intact MDCK cells are similar to

FIG. 8. 8-Br-cAMP (A), cholera toxin (B) , and mastoparan (C) do not affect the ability of low cytosolic pH to inhibit uptake of transferrin at the basolateral side of polarized MDCK cells. MDCK cells growing on filters were incubated with and without cholera toxin (1 pg/ml) for 4 h at 37 "C, or with and without 8-Br-CAMP (2 m) or mastoparan (50 p ~ ) for 30 min a t 37 "C. Then the cells were exposed to a HEPES-buffered medium adjusted to pH 5.5, and in some cases the buffer contained 10 mM acetic acid to acidify the cytosol. After 5 min further incubation, '251-labeled transferrin was added basolaterally, and binding and endocytosis of transferrin were measured 5 min later as described under "Experimental Procedures." '251-transferrin was also added to some filters at 0 "C to get background values for the endocytosis assay. The burs represent deviations between parallels.


FIG. 9. Ability of cholera toxin, 8-Br- CAMP, and mastoparan to increase apical ricin endocytosis in acidified cells. A, MDCK cells growing on filters were incubated with and without cholera toxin (1 pg/ml) for 4 h at 37 “C, or with and without 8-Br-CAMP (2 mh1) or mastoparan (50 PM) for 30 min. Then the cells were exposed to a HEPES-buffered medium adjusted to pH 5.5, and in some cases the buffer contained 10 mM acetic acid to acidify the cytosol. After 5 min further incubation, ‘2”I-labeled ricin was added apically (A), and the amount of endocytosed toxin was measured as described under “Experimental Procedures” 15 min later. The burs represent deviations between parallels. Control values for ricin endocytosed, 3,700-11,500 cpm in the different experiments. B, MDCK cells growing on filters were treated as indicated in A, column 6 (8-Br-cAMP + HAC). The cells were then incubated for

added to the apical side (As). A labeled 15 min with ricin-horseradish peroxidase

multivesicular endosome (M) is shown. Arrows outline the intercellular space between adjacent cells. Bar, 0.5 pm. HAC - + - + - + - +

Choleratox. - - + + - - - - &Br-cAMP - - - - + + ” Mastoparan - - - .) - - + +

FIG. 10. Effect of the protein kinase inhibitor H89 on apical stimulation of ricin endocytosis. MDCK cells grown on filters were incubated for 2 h a t 37 “C in the absence and presence of the indicated concentrations of H89. Then cholera toxin (2 pg/ml), 8-Br-CAMP (0.2 mhfl), mastoparan (50 p ~ ) , and the indicated concentrations of forskolin were added. 2 h after addition of cholera toxin, and 30 min after addition of 8-Br-cAMP, mastoparan, and forskolin, ricin endocytosis was

“Experimental Procedures.” Ricin endocytosed: control values, 3526 measured at the apical side during a 15-min interval as described under

cpm (A); 460 cpm, (B); 1500 cpm (C); 1005 cpm (D).

those that induce exocytosis in intact human platelets (44). Mastoparan-induced exocytosis of insulin in the P-cell line RINm5F was increased with increasing mastoparan concentration in the range 10-35 PM (45). The effect of higher concentrations were not shown. In this last cell line mastoparan (20 PM) had, like in the system described here, no effect on the level of CAMP. The various results obtained with the RINm5F cells suggest that heterotrimeric G proteins may be involved in the process (45). Additionally in solubilized systems concentrations in the same range have been found to give maximal stimulation of GTPase activity (28, and interestingly, the concentrations of mastoparan required to inhibit uptake of transferrin in intact MDCK cells are only slightly higher than those required to

inhibit coated vesicle budding in permeabilized A431 cells (4). In a number of the studies where heterotrimeric G proteins

are thought to influence membrane traffic, the mediator of the observed effect is not known ( 5 , 6,s-10). Our experiments do, however, not exclude that other interactions, with a similar specificity, could be involved. Mastoparan has also been shown to interact with the small GTP-binding proteins rho and rac (461, and rac was recently demonstrated to be important for growth factor-induced ruffling (47). However, as judged by EM, the mastoparan-induced endocytosis in the present study does not involve ruMing and macropinocytosis leading to large vesicles (47). As described, the stimulation of endocytosis is associated with an increased number of tracer-labeled endosomes with normal morphology.

The finding that mastoparan stimulates ricin endocytosis under conditions where transferrin endocytosis is strongly inhibited suggests that the increased uptake is mediated by a clathrin-independent mechanism. We have previously shown that acidification of the cytosol inhibits the formation of clathrin-coated vesicles in a number of cell types (3, 11, 361, and as shown here, the same is the case in MDCK cells. The finding that mastoparan, cholera toxin, and 8-Br-CAMP all stimulate apical endocytosis even when the cytosol is acidified and transferrin endocytosis is blocked, thus supports the idea that the regulation of endocytosis at the apical side is clathrin-independent.

Like mastoparan, CAMP has been found to affect a number of transport systems. In T84 cells CAMP was found to stimulate exocytosis, whereas endocytosis was inhibited (48). Similarly, phagocytosis in rat retinal pigment epithelia cells was inhibited by drugs linked to CAMP production (49). In addition, CAMP is a mediator for exocytosis of the vacuolar apical com- partment in MDCK cells (50) . The concentrations of 8-Br-CAMP which induce exocytosis are similar to those found to increase endocytosis in this study. In both systems quite low concentrations of 8-Br-CAMP are effective. CAMP also potentiates the calcium-induced release of insulin in p-cells (51). Furthermore,


. -

:. .

Mastoparan

D CAMP

. . ,...

*.

FIG. 11. EM studies of the effect of mastoparan and CAMP on horseradish peroxidase endocytosis. MDCK cells grown on filters were kept for 30 min a t 37 "C without ( A ) or with (I? and C ) 50 p~ mastoparan and then incubated from the apical side with horseradish peroxidase (10 mg/ml) for 15 min a t 37 "C before fixation and processing for EM. The pictures shown are representative samples. In the control ( A ) a single MVB-like endosome (M) is present. Arrows outline the interdigitating intercellular space between the two adjacent cells. Bar, 0.5 pm. In B is seen three MVB-like endosomes (M) and some smaller, horseradish peroxidase-labeled structures (open arrows) which were not included in the quantitation. In C is shown the lateral portions of two adjacent cells (small arrows indicate the intercellular space). In the left cell one MVB ( M ) is seen, in the right one three M V B s (M) are present. D shows the quantitative data from the mastoparan experiment (-, without and +, with 50 p~ mastoparan) shown in A-C, as well as from a similar experiment with 2 mM 8-Rr-CAMP. Sections were cut perpendicular to the filter, and from each experiment (2 experimentals + 2 controls) the number of MVB-like endosomes (like those in A-C) per cell profile of 50 cells were counted. Error bars are S.E.

in actively elongating axons movement of a class of phase- ment (55). By contrast, BFA might induce a special kind of dense organelles in the retrograde direction was less efficient macropinocytosis leading to the formation of unusually large when the concentration of CAMP was increased (52). In sensory vacuoles (17). However, like CAMP BFA also stimulated tran- neurons ofAplysia, CAMP induced formation of clathrin-coated scytosis from the basolateral side (17). Although the detailed pits and vesicles by increasing expression of clathrin light mechanism for the changes shown here has not yet been clari- chain (53). However, in the system here described, protein syn- fied, regulation of apical endocytosis and transcytosis may be thesis was not required for the CAMP-induced increase in api- an important physiological response to hormones acting on the cal endocytosis. Recently, Verreg and co-workers (54) found basolateral membrane of some epithelia. that membrane movement in polarizedA6 kidney cells was also regulated by aldosterone and vasopress~n/vasotoc~n~ in the Acknowledgments-We thank Anne-Grethe Myrann, Tove Lie Berle,

system here described, the detailed mechanisms of action are sen for expert technical assistance. Marianne Lund, Annemette Ohlsen, Keld Ottosen, and Kirsten Peder-

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