©1995 by The American Society for Biochemistry and Molecular Biology, Inc.
Cationic Amphiphilic Drugs Inhibit the Internalization of Cholera Toxin to the Golgi Apparatus and the Subsequent Elevation of Cyclic AMP (*)

Anat Sofer , Anthony H. Futerman (§)

From the (1) Department of Membrane Research and Biophysics, Weizmann Institute of Science, Rehovot 76100, Israel

ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES

ABSTRACT

Cholera toxin (CT) consists of a pentameric B subunit which binds with high affinity to ganglioside GM, and an A subunit which stimulates adenylate cyclase, resulting in the elevation of cAMP. We now examine the effect of cationic amphiphilic drugs (CADs) on the internalization of rhodamine (Rh)-CT in cultured hippocampal neurons. CADs have recently been shown to inhibit receptor recycling by disrupting the assembly-disassembly of clathrin at the plasma membrane and on endosomes (Wang, L.-H., Rothberg, K. G., and Anderson, R. G. W.(1993) J. Cell Biol. 123, 1107-1117). Rh-CT was internalized by an energy- and temperature-dependent (presumably vesicular) mechanism to the Golgi apparatus. Internalization to the Golgi apparatus was completely but reversibly blocked by CADs, and the ability of CT to stimulate the elevation of cAMP was significantly reduced. In control cells, cAMP levels were elevated 2.3-fold after 20 min of incubation with CT, but in CAD-treated cells cAMP levels were only elevated 1.3-fold. The effect of CADs on CT internalization was not due to a direct effect of CADs on the Golgi apparatus. Our data demonstrate that CADs inhibit vesicular transport of CT to the Golgi apparatus and imply that the sorting of CT to the Golgi apparatus occurs in the same endosomal compartment involved in sorting recycling receptors to the plasma membrane, since both pathways are inhibited by CADs.


INTRODUCTION

Gangliosides are major constituents of the neuronal plasma membrane (PM)() where they have been postulated to play a variety of functions (1) . A large amount of information is available about ganglioside metabolism (2) . In contrast, much less is known about the intracellular transport of gangliosides (3), due in large part to the lack of suitable probes to monitor ganglioside traffic. In the case of ganglioside GM, some information about the pathways and regulation of transport has been obtained using cholera toxin (CT). CT consists of a pentameric B subunit which binds with high affinity to ganglioside GM and an A-subunit comprising two peptides, A and A, linked by a disulfide bond. The A-subunit activates adenylate cyclase via a stimulatory G protein, G (4). Fluorescent (5) , horseradish peroxidase-conjugated (6) , radioactive (7, 8) , and gold derivatives (9, 10) of either CT or CT-B (the B-subunit of cholera toxin) have been used to trace the internalization of CT from the cell surface to intracellular locations by both biochemical techniques and by light and electron microscopy.

At the cell surface, CT is concentrated in non-coated membrane invaginations (9) recently identified as caveolae (10) . However, some controversy exists as to whether CT (and other molecules) are internalized from caveolae, since caveolae have long half-lives and have not been directly observed to ``pinch off'' from the PM (11) . Moreover, CT rapidly appears in the same endosomal compartment as that labeled by -macroglobulin (8) , a ligand that enters cells via clathrin-coated pits. CT is not excluded from clathrin-coated pits (9, 10) , and some evidence exists for the internalization of a biotinylated derivative of GM from clathrin-coated pits (12). Despite the lack of consensus on the mechanism of internalization of CT, it has recently been unambiguously shown that a functional Golgi apparatus is necessary for generation of the A peptide, which results in stimulation of adenylate cyclase and associated toxicity ((13, 14), see also Ref. 15)).

In order to probe further the mechanism of CT internalization, we have studied the effect of cationic amphiphilic drugs (CADs) such as chlorpromazine, imipramine, and sphingosine on the internalization of CT and on the elevation of cAMP in cultured hippocampal neurons. CADs have previously been shown to disrupt receptor recycling (16, 17) by stimulating the recruitment of the AP-2 adaptor protein (18) and of clathrin to an uncoated, late endosomal compartment (19) . We now demonstrate that CADs reversibly inhibit the internalization of CT to the Golgi apparatus and the CT-stimulated elevation of cAMP.


EXPERIMENTAL PROCEDURES

Materials

Timed pregnant rats (Wistar) were obtained from the Weizmann Institute Breeding Center. Fumonisin B was purchased from the Division of Food Science and Technology, CSIR, Pretoria, South Africa. CT was from Calbiochem, La Jolla, CA. N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl}-D-erythro-sphingosine (C-NBD-ceramide) and N-[5-(5,7-dimethylBodipy)-1-pentanoyl]-D-erythro-sphingosine (C-Bodipy-ceramide) were from Molecular Probes Inc., Eugene, OR. Chlorpromazine, imipramine, sphingosine, H7, and 3-isobutyl-1-methylxanthine were from Sigma. Ganglioside GM was supplied by Fidia Research Laboratories, Abano T., Italy. Protein A and an anti-cAMP antibody were from BioMakor, Rehovot, Israel. Acetic anhydride and triethylamine were obtained from BDH, Dorset, United Kingdom. Adenosine 3`,5`-cyclic 3-[I]iodotyrosine methyl (2000 Ci/mmol) was obtained from Amersham International plc, Amersham, U.K.

Incubation of Hippocampal Neurons with a Fluorescent Analog of CT

Hippocampal neurons cultured at low density (12,500-25,000 cells/13-mm glass coverslip) (20, 21) were incubated with Rhodamine (Rh)-conjugated cholera toxin (Rh-CT) (containing the A- and B-subunits) or the B-subunit of CT (Rh-CT-B) (prepared as in Ref. 5), for 20-30 min at 13-16 °C in HEPES-buffered medium (minimal essential medium containing 50 mM HEPES (pH 7.3), 4 mM NaHCO, 11 mg/ml pyruvic acid, 1 mM glutamine, 0.6% (w/v) glucose) and 0.1% (w/v) bovine serum albumin, and then warmed to 37 °C for various times. In one experiment (see Fig. 1C), neurons were incubated with exogenous ganglioside GM (5 nM). GM was dissolved in ethanol, dried under N, and dissolved in phosphate-buffered saline followed by sonication.


Figure 1: Rh-CT labels cell surface GM in cultured hippocampal neurons. Neurons were incubated with 10 µM fumonisin B immediately after transferring the coverslips to multiwell dishes containing cocultures of glial cells. After 3 days in culture, coverslips were removed and incubated with 5 nM Rh-CT in HEPES-buffered medium for 30 min at 16 °C. Rh-CT is barely detectable at the cell surface after fumonisin B-treatment (B), but is detected on the cell body, dendrites and axons of control cells (A). Addition of exogenous GM (5 nM) to the medium 18 h before incubation with Rh-CT restores the binding of Rh-CT at the cell surface (C). The bar corresponds to 20 µm.



Incubation of Hippocampal Neurons with C-NBD-ceramide and C-Bodipy-ceramide

Neurons were washed in HEPES-buffered medium, cooled to 13-16 °C and incubated with either a bovine serum albumin/C-NBD-ceramide or a bovine serum albumin/C-Bodipy-ceramide complex (molar ratio 1:1, in 10 mM HEPES (pH 7.3)) for 30-40 min, washed, and further incubated for various times at 37 °C; both ceramide analogs specifically label the Golgi apparatus (22, 23) . For long incubations, neurons were placed in Multiwell dishes containing glial cocultures in the CO incubator. Prior to observation, neurons were washed and ``back-exchanged'' with 0.34% or 2% (w/v) defatted-bovine serum albumin (3 10 min, 37 °C) for C-NBD-ceramide and C-Bodipy-ceramide, respectively.

Determination of cAMP

Neurons cultured at high density (230,000 cells/24-mm glass coverslip) (24) were used for cAMP determination. 5-6-day-old neurons were washed in HEPES-buffered medium for 15 min at 37 °C. Neurons were subsequently incubated for 5 min at 13-16 °C in HEPES-buffered medium containing 0.2 mM 3-isobutyl-1-methylxanthine and 0.01% (w/v) bovine serum albumin prior to addition of 5 nM CT for 30 min at 13-16 °C. Drugs were added to the medium for 5 min prior to warming to 37 °C. After various times, cells were removed from the coverslips by scraping with a rubber policeman into 7 mM sodium acetate buffer (pH 6.5) containing 0.2 mM 3-isobutyl-1-methylxanthine, lyophilized, and resuspended in distilled water to give a final concentration of 70 mM sodium acetate (pH 6.5). Cells were frozen in liquid nitrogen and thawed four times to completely release cAMP into the buffer; samples were stored at -80 °C. cAMP was determined by radioimmunoassay as described (25) .

Microscopy

Fluorescence microscopy was performed using Plan Apochromat 63X/1.4 and Plan Neofluar 40X/1.3 oil objectives of a Zeiss Axiovert 35 microscope equipped with filters for rhodamine or Bodipy fluorescence. Cells were photographed using a Contax 167MT camera and Kodak Tmax p3200 film.

RESULTS

The Internalization of Rh-CT

Initial experiments were performed to characterize the mechanism of internalization of CT in cultured hippocampal neurons. These neurons are a particularly suitable model to analyze CT internalization since they contain high levels of ganglioside GM. Moreover, the rate of internalization of CT to the Golgi apparatus is rapid on early days in culture and decreases as neurons mature.()

The specificity of CT binding to GM was determined by incubating neurons with fumonisin B, a specific inhibitor of sphingolipid synthesis (20, 26) . After 3 days of incubation with fumonisin B, levels of Rh-CT binding to the PM were significantly reduced (Fig. 1B), whereas in control cells, Rh-CT labeled the somatodendritic and axonal PMs (Fig. 1A). Addition of exogenous GM (5 nM) to fumonisin B-treated cells (Fig. 1C) restored the binding of CT. Identical results were obtained using either intact Rh-CT or Rh-CT-B. Preincubation with a 10-fold excess of unlabeled CT also significantly reduced Rh-CT labeling to the cell surface.

When 3-day-old neurons were labeled with Rh-CT for 30 min at 16 °C, the cell surface was brightly labeled (see Fig. 1A). No intracellular fluorescence was observed if the temperature was maintained at 16 °C. However, upon subsequent warming to 37 °C for 30 min, Rh-CT accumulated in a perinuclear area (Fig. 2A) that corresponded to the Golgi apparatus, as demonstrated by double-labeling studies using C-NBD-ceramide (not shown). Preincubation with sodium azide (5 mM) and 2-deoxyglucose (50 mM) abolished internalization to the Golgi apparatus (Fig. 2B) at 37 °C. Together, these results demonstrate that Rh-CT is internalized when bound to GM via an energy- and temperature-dependent, presumably vesicular mechanism to the Golgi apparatus in cultured hippocampal neurons.


Figure 2: Rh-CT is internalized by an energy-dependent mechanism to the Golgi apparatus. Six-day-old neurons were preincubated for 15 min at 37 °C with sodium azide (5 mM) and 2-deoxyglucose (50 mM) in HEPES-buffered medium minus glucose. Neurons were subsequently cooled to 16 °C for 5 min, incubated with 5 nM Rh-CT for 30 min, and then warmed to 37 °C for 30 min. The metabolic inhibitors were present throughout the incubation with Rh-CT. In control cells (A), Rh-CT is internalized to the Golgi apparatus, but is not internalized from the PM in cells treated with metabolic inhibitors (B). The bar corresponds to 10 µm.



The Effect of CADs on Rh-CT Internalization

Neurons were incubated with Rh-CT for 30 min at 13-16 °C prior to addition of chlorpromazine for 5 min at 13-16 °C, followed by warming to 37 °C for 20 min. In chlorpromazine-treated cells, Rh-CT was detected in punctate, vesicular structures (Fig. 3, C and D), whereas in untreated cells, intense Golgi apparatus-labeling was detected (Fig. 3, A and B). Golgi apparatus labeling could be restored in chlorpromazine-treated cells by washing and addition of fresh medium that did not contain chlorpromazine (Fig. 3, E and F). Two other CADs, sphingosine (Fig. 3, G and H) and imipramine (not shown) had identical effects to chlorpromazine inasmuch as Rh-CT internalization to the Golgi apparatus was inhibited, and Rh-CT accumulated in punctate structures. In contrast, the protein kinase C inhibitors, H7 (Fig. 3, I and J) and staurosporine (not shown) had no effect on Rh-CT internalization. Similarly, these compounds had no effect on the distribution of clathrin and the AP-2 adaptor in cultured fibroblasts (19).


Figure 3: CADs inhibit the internalization of Rh-CT to the Golgi apparatus. Six-day-old neurons were incubated with 5 nM Rh-CT in HEPES-buffered medium for 30 min at 13-16 °C. Neurons were then incubated with 25 µM chlorpromazine (C and D), 5 µM sphingosine (G and H), or 25 µM H7 (I and J) for 5 min at 13-16 °C, prior to incubation at 37 °C for 20 min. Untreated cells are shown in panels A and B. In panels E and F, cells were incubated with 25 µM chlorpromazine as above, washed to remove chlorpromazine, and placed in multiwell dishes containing a glial monolayer in the CO incubator for an additional 90 min. Neurons were fixed with 4% paraformaldehyde for 20 min at 37 °C prior to observation. The left-hand panels show phase contrast micrographs, and the right-hand panels show immunofluorescence. The bar corresponds to 20 µm.



To exclude the possibility that CADs abolished Golgi apparatus labeling by affecting the integrity of the Golgi apparatus, 3-day-old neurons were first incubated with either Rh-CT (Fig. 4, A and B) or C-Bodipy-ceramide (Fig. 4, C and D), and then treated with chlorpromazine for 20 min (Fig. 4, B and D). No changes in Golgi apparatus morphology were observed upon treatment with chlorpromazine; similar results were seen in 6-day-old neurons. These data confirm that chlorpromazine inhibits Rh-CT accumulation in the Golgi apparatus by disrupting a step of Rh-CT endocytosis between the PM and the Golgi apparatus.


Figure 4: CADs have no effect on Golgi apparatus morphology. Three-day old neurons were incubated with 5 nM Rh-CT (A and B) or 5 µM C-Bodipy-ceramide (C and D) for 30 min at 13-16 °C, followed by washing and warming to 37 °C for 2.5 or 15 h, respectively. Cells were subsequently incubated in the absence (A and C) or presence (B and D) of 25 µM chlorpromazine for 20 min at 37 °C. Neurons labeled with Rh-CT were fixed with 4% paraformaldehyde for 20 min at 37 °C prior to observation. The bar corresponds to 10 µm.



The Effect of CADs on CT-stimulated Elevation of cAMP

CT mediates its cytotoxic effects by stimulating adenylate cyclase, resulting in intracellular accumulation of cAMP (4) . Initial experiments determined that Rh-CT and CT have identical effects on the elevation of cAMP, whereas CT-B was totally ineffective. To determine the effect of CADs on this process, neurons were incubated with CT at 13-16 °C prior to incubation with CADs or other drugs at 37 °C. No elevation in cAMP levels were observed in either control or CAD-treated cells for 10 min after warming, similar to the lag period observed in the elevation of cAMP in other cells (5, 27) . However, after 20 min, cAMP levels were elevated 2.3-fold in control cells (). In contrast, cAMP levels were only elevated 1.3-fold in chlorpromazine-treated cells. After 30 min, cAMP levels were elevated 3.1- and 1.7-fold, respectively, in control and chlorpromazine-treated cells (). Likewise, 20 and 30 min after imipramine-treatment (100 µM), cAMP levels were only elevated 1.4- and 2.2-fold, respectively, and after sphingosine treatment (5 µM), cAMP levels were elevated 1.7- and 2.1-fold. The effect of chlorpromazine on cAMP was reversible (). H7, an inhibitor of protein kinase C () and staurosporine (not shown) had no effect on CT stimulation of cAMP. Thus, in addition to inhibiting the vesicular transport of Rh-CT to the Golgi apparatus, CADs also inhibit the ability of CT to elevate cAMP.

DISCUSSION

The major finding of the current study is that internalization of CT to the Golgi apparatus, and the subsequent elevation of cAMP, is inhibited by three CADs, namely chlorpromazine, imipramine, and sphingosine. CADs have recently been shown to effect endocytosis by disrupting the assembly-disassembly of clathrin from coated pits and endosomes (19) . Clathrin binding to coated pits is mediated by the AP-2 adaptor protein. AP-2 binding to the membrane is itself mediated via an integral membrane protein, the AP-2 receptor (18) , that exists in two states, one that binds AP-2 with high affinity and one that does not bind AP-2 (28) . Switching between these two states is believed to regulate the assembly-disassembly of clathrin coats. Thus, soon after formation of clathrin-coated vesicles, the AP-2 receptor switches to its non-binding state, resulting in the release of AP-2 and clathrin into the cytosol and the generation of non-coated vesicles. In cells treated with CADs, the AP-2 receptor becomes activated on a non-coated endosomal compartment (19, 28) , leading to the recruitment of AP-2 and clathrin. The recycling of receptors for low density lipoprotein (19) , transferrin (16) , and -macroglobulin and epidermal growth factor (17) , is inhibited, although the receptors do not accumulate in the same endosomal compartment that acquires clathrin (19). Our current data suggest that the sorting of CT to the Golgi apparatus occurs in the same endosomal compartment involved in sorting recycling receptors to the plasma membrane, since both processes are inhibited by CADs.

We propose the following scheme to describe CT endocytosis (Fig. 5A) and the effects of CADs (Fig. 5B). CT binds to GM on the external leaflet of the PM via a high affinity binding site on the B-subunit of CT. At low temperature, CT accumulates in caveolae on the cell surface (9, 10) , although it is not excluded from clathrin-coated pits (10) . No internalization of CT occurs at low temperature (i.e. 13-16 °C, see Fig. 1A). Upon warming to 37 °C, CT is internalized from either clathrin-coated pits (Fig. 5, Ai) or from caveolae (Fig. 5, Aii). Since these two pathways converge (8, 29) (Fig. 5, Aiv) after removal of clathrin from clathrin-coated vesicles (Fig. 5, Aiii), the exact site of internalization has no bearing on the subsequent fate of CT in the endocytic pathway. Non-coated vesicles move along the endocytic pathway to a sorting compartment (Fig. 5, Av). CT is sorted away (Fig. 5, Avii) from recycling membrane components (Fig. 5, Avi) and components targeted to lysosomes (not shown), and targeted to the Golgi apparatus (Fig. 5, Aviii) where it is processed to produce the A-subunit (13, 15) . The accumulation of coated endosomes upon CAD treatment disrupts the further processing of CT to the Golgi apparatus (Fig. 5, Bv). Since CADs have no effect on the initial uncoating of coated vesicles (19) (Fig. 5, Biii), cells are able to internalize CT (Fig. 5, Bi) at least as far as early endosomes (Fig. 5, Biii and iv).


Figure 5: Schematic representation of the pathways of internalization of CT in the absence (A) and presence (B) of CADs. For details, see ``Discussion.'' CT is represented by the symbol T, and clathrin coats by thick lines.



Although no labeling of the Golgi apparatus by CT was observed after CAD treatment (see Fig. 3, D and H), there was a small increase in CT-stimulated cAMP levels (). It is possible that small amounts of CT, below the limit of resolution of the light microscope, are nevertheless delivered to the Golgi apparatus in the presence of CADs. Alternatively, CT could elevate cAMP levels in a compartment prior to the Golgi apparatus. Biochemical studies have indicated that activation of adenylate cyclase requires CT internalization and processing in an acidic-endosomal compartment (7) . However, the fraction used in this study was a ``Golgi-endosome'' fraction that is often used as a source of enriched Golgi apparatus membranes (30) . Moreover, in Caco-2 and SK-N-MC cells, chloroquine had no effect on CT stimulation of cAMP accumulation (14) although it did inhibit cAMP generation in hepatocytes (7) . In PtK1 cells, in which the Golgi apparatus but not endosomes are resistant to Brefeldin A, no changes were observed in the ability of CT to stimulate adenylate cyclase (13) . Together, these data suggest that transport to the Golgi apparatus is normally necessary for CT to mediate its effects on adenylate cyclase.

In summary, we demonstrate that the internalization of CT to the Golgi apparatus proceeds via an endosomal compartment that can acquire clathrin coats upon treatment with CADs. It will be of interest to study the effect of CADs on the internalization of other molecules to the Golgi apparatus, particularly other toxins (i.e. Shiga toxin (31) ) that bind to glycosphingolipids on the cell surface.

  
Table: CT-stimulated elevation of cAMP after chlorpromazine or H7 treatment

Five- to six-day-old neurons were incubated with 5 nM CT for 30 min at 13-16 °C prior to incubation with either 25 µM chlorpromazine or 25 µM H7 at 37 °C. At the indicated times, cells were removed from the coverslips by scraping, and levels of cAMP determined as described under ``Experimental Procedures'' in both cells and medium. cAMP levels in the medium were less than 6-7% of that measured in cells and are not included in the table. Results are shown as fmol/coverslip. The relative stimulation of cAMP by CT is compared to time = 0 min and shown in parenthesis. Results are mean ± S.D., n = 6 for control cells and n = 3 for chlorpromazine and H7-treated cells.



FOOTNOTES

*
This work was supported by a grant from the Basic Research Foundation of the Israel Academy of Sciences and Humanities, and by Grant 91-00278 from the United States-Israel Binational Science Foundation, Jerusalem, Israel. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore by hereby marked ``advertisement'' in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

§
Incumbent of the Recanati Career Development Chair in Cancer Research. To whom correspondence should be addressed. Tel.: 972-8-342704; Fax: 972-8-344112; E mail: BMFUTER@WEIZMANN.WEIZMANN.AC.IL.

The abbreviations used are: PM, plasma membrane; C-Bodipy-ceramide, N-[5-(5,7-dimethylBodipy)-1-pentanoyl]-D-erythro-sphingosine; CAD, cationic amphiphilic drug; C-NBD-ceramide, N-{6-[(7-nitrobenzo-2-oxa-1,3-diazol-4-yl)amino]caproyl}-D-erythro-sphingosine; CT, cholera toxin; CT-B, B-subunit of cholera toxin; Rh, rhodamine.

A. Sofer and A. H. Futerman, unpublished observations.


ACKNOWLEDGEMENTS

We thank Rivi Zisling for expert help in preparing hippocampal cultures and Professor Nava Dekel for help with the cAMP assay.


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