Endocytosis of cholera toxin by human enterocytes is developmentally regulated

Lei Lu,1 Sameer Khan,2 Wayne Lencer,3 and W. Allan Walker1

1Developmental Gastroenterology Laboratory, Massachusetts General Hospital, Boston, Massachusetts; 2The Aga Khan University Hospital, Karachi, Pakistan; and 3Combined Program in Pediatric Gastroenterology and Nutrition, Children's Hospital, Boston, Massachusetts

Submitted 19 November 2004 ; accepted in final form 20 March 2005


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Many secretory diarrheas including cholera are more prevalent and fulminant in young infants than in older children and adults. Cholera toxin (CT) elicits a cAMP-dependent chloride secretory response in intestinal epithelia, which accounts for the fundamental pathogenesis of this toxigenic diarrhea. We have previously reported that the action of this bacterial enterotoxin is excessive in immature enterocytes and under developmental regulation. In this study, we tested the hypothesis that enhanced endocytosis by immature human enterocytes may, in part, account for the excessive secretory response to CT noted in the immature intestine and that enterocyte endocytosis of CT is developmentally regulated. To test this hypothesis, we used specific inhibitors to define endocytic pathways in mature and immature cell lines. We showed that internalization of CT in adult enterocytes is less and occurs via the caveolae/raft-mediated pathway in contrast to an enhanced immature human enterocyte CT uptake that occurs via a clathrin pathway. We also present evidence that this clathrin pathway is developmentally regulated as demonstrated by its response to corticosteroids, a known maturation factor that causes a decreased CT endocytosis by this pathway.

clathrin; caveolae; endocytosis; intestinal development


DIARRHEAL DISEASE CONSTITUTES one of the major causes of morbidity and mortality in infants and children on a global scale (39). Despite advances in our understanding of its epidemiology and pathogenesis, cholera toxin (CT) at the beginning of the 21st century still has the potential to cause endemic disease worldwide (33). In cholera-endemic areas, the highest attack rates and most severe cases are in infants and young children (2, 10), suggesting a developmental basis for the disease.

Pathological response to CT is dependent on a complex, highly coordinated sequence of events. First, it is released from the bacterium onto the intestinal surface. Then, it binds to enterocyte receptors and is endocytosed as a toxin-receptor complex leading to the accumulation of intracellular cAMP. Finally, stimulation of chloride channels actively secretes Cl (32).

CT is a pentavalent protein comprised of one active (A) and five binding (B) subunits that interact with an enterocyte microvillus glycolipid ganglioside (GM1). In polarized epithelial cells, activation of signal transduction by CT requires endocytosis of toxin-receptor complexes into the apical endosome, translocation by retrograde transport into the Golgi cisternae or endoplasmic reticulum, and finally movement of the toxin to its site of action in the basolateral membrane (reviewed in Refs. 18, 23, and 32). In most cell types, a large fraction of the CT receptor GM1 clusters in caveolae, and therefore GM1 has been used as a marker for caveolae (glycolipid/raft domains). Previous studies by Lencer and colleagues (45, 46) and others (30, 38) have demonstrated that attachment to GM1 partitions CT into detergent-insoluble glycosphingolipid-rich (DIG) membranes that are critical to the toxin's function. Glycolipid rafts are DIG membranes that can be isolated from most mammalian cell types by virtue of their insolubility in a nonionic detergent (e.g., Triton X-100) and their floatation characteristics on low-density sucrose gradients (46).

Endocytosis of CT can occur in dynamin- and caveolae-mediated pathways in multiple cell types including enterocytes (17). However, CT can also be taken up by a clathrin-dependent pathway (10, 13). There has been compelling evidence that in some cell types, CT can be taken via clathrin- and caveolae-independent pathways (26, 41), and these different endocytic pathways can function simultaneously. In studies using mature enterocytes (e.g., T84 cells), CT has been shown to interact and be endocytosed by caveolae/lipid raft domains (45). T84, a crypt-like adult epithelial cell line traditionally used for these studies, was selected because other intestinal epithelial cell lines (Caco-2, HT-29, etc.) have less well-defined secretory and endocytic pathways (11, 48). The difference has also been shown in studies in this laboratory (data not shown). To distinguish endocytosis by the caveolae pathway from other endocytic pathways, particularly from the clathrin-dependent pathway, agents [filipin and methyl-{beta}-cyclodextrin (M{beta}CD)] that gradually deplete membrane cholesterol have been previously used as caveolae/lipid raft endocytosis inhibitors (37, 45).

During development, the immature intestine passes through a cellular phase in which enterocytes are highly endocytic (43). The existence of an enhanced endocytic capacity by the developing intestine may play a critical role in growth and differentiation due to an increased capacity to engulf intact hormones and growth factors (16, 42). However, the immature intestine is not able to distinguish between substances necessary for growth and development from those that can be potentially harmful. This enhanced capacity to endocytose microorganisms and protein antigens may help to explain the increased susceptibility of preterm as well as term infants to enteric infection and gastrointestinal allergic disorders (15).

In previous animal studies (5, 6, 36) and in a recent human study (24) from this laboratory, we have shown that at lower doses of CT, the immature intestine responds more actively than the mature intestine by increasing cAMP accumulation and an excessive chloride secretion. To further explain this increased response to CT by the immature gut and with the realization that the immature enterocyte has an increased endocytic capacity (7, 8, 42, 43), we have begun to examine endocytosis of CT as a potential contributing factor in this process.

Accordingly, we hypothesize that an age-dependent difference in CT uptake in immature vs. mature enterocytes may, in part, be due to differences in binding to the surface GM1 receptor and enhanced CT endocytosis leading to a subsequent excessive elevation of cAMP and enhanced secretion.

In this study, we utilized H4 and T84 cell lines as models for the study of the developmental response to CT in humans. H4 cells are a nontransformed human fetal small intestinal cell line and have morphologic and functional characteristics typical of intestinal crypt-like enterocytes. They are poorly differentiated (34) and therefore represent an appropriate cellular model for defining the CT response in the immature intestine.

T84 is a well-characterized and highly differentiated human colonic carcinoma cell line that functions as a mature crypt-like cell and is a useful model for studies of Cl secretion as well as endo- and transcytosis of CT (20, 22, 44). When grown on collagen-coated permeable supports, T84 cells form monolayers with increased high transepithelial resistance and consist of highly polarized columnar cells with structural similarity to adult intestinal crypt cells (25). It has been demonstrated that T84 cells have all the necessary cellular components to produce pathophysiological components to produce a pathophysiological response to CT (19, 20, 22).

By carefully examining the different endocytic processes in immature vs. mature intestinal epithelia, we provide evidence to support a developmental difference in endocytosis of CT as a partial explanation for its accentuated secretory response in the immature gut.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials. CT was purchased from Calbiochem (San Diego, CA) and CT B subunit (CTB) conjugated to biotin (CTB-B) was from List Biological Laboratory (Campbell, CA). Brefeldin A (BFA) was obtained from Biomol (Plymouth Meeting, PA). Mouse monoclonal anti-GAPDH was obtained from Research Diagnostic (Flanders, NJ). Rabbit polyclonal anti-CT A subunit (anti-CTA) and anti-CTB were generated and characterized in Dr. Wayne Lencer's laboratory (19, 20). All horseradish peroxidase-conjugated secondary antibodies were purchased from Pierce (Rockford, IL). The enzyme immunoassay (EIA) kit for cAMP was obtained from Amersham Pharmaceutical. The complete protease inhibitor tablet (complete mini) was obtained from Roche Molecular Biochemicals (Indianapolis, IN). All other chemicals, if not specified, were purchased from Sigma (St. Louis, MO).

Cell cultures. T84 cells were obtained from the American Type Culture Collection (Manassas, VA) and grown on collagen-coated Transwell inserts (Costar, Cambridge, MA) or tissue culture plates as previously described (22). Cell passages 51–63 were used for all experiments. H4 cells are nontransformed human fetal small intestinal cells generated in our laboratory (obtained from a 16-wk-old therapeutically aborted fetus) (34) with the approval from the committee for human studies in Massachusetts General Hospital, which are comparable to the intestinal epithelial cell line (IEC)-6 rodent crypt cell line developed by Quaroni et al. (31). The cells were maintained in DMEM supplemented with 10% FBS, 20 mM L-glutamine, human recombinant insulin (0.5 U/ml), and standard antibiotics. Cell passages 10–22 were used for these experiments. Cells were plated on six-well tissue culture plates and grown to confluence before they were used in experiments.

Endocytic inhibitors. Cells were treated with various endocytic inhibitors to distinguish clathrin-dependent from -independent endocytosis using the dose previously shown to have an optimal effect in cell lines (29, 38, 41, 45). For clathrin-dependent endocytosis, cells were preincubated with 20 µg/ml chlorpromazine (CPZ). Other cells were pretreated with 1 µg/ml filipin, or 1 mM M{beta}CD for disruption inhibition of caveolar endocytosis. BFA (1 µg/ml) was used to block CT activity. Specificity of each treatment modality was evaluated by incubating cells with CT (20 nM) at 4°C to let binding take place or 37°C for 2 h, at which temperature the endocytosis of CT occurs, and by monitoring the internalization of CT by immunoblotting and fluorescence staining.

Trophic factors. Hydrocortisone was selected as a known maturation factor for this study (13, 28). H4 cells, and in some cases T84 cells, were grown to near confluence and then incubated with 1 µM hydrocortisone for 1 wk. After incubation, corticosteroids were washed away, and cells were then subjected to various stimuli and further analysis.

Animals. Four-week-old homozygous SCID mice (Jackson Laboratory, Bar Harbor, ME) were housed in a specific pathogen-free facility and fed with rodent chow and water ad libitum. Female Sprague-Dawley rats were purchased from Taconic (Germantown, NY). The animals were kept in an institutional animal facility with a 12:12-h light-dark cycle. They were fed rat chow and water ad libitum. This study has been approved by the Subcommittee on Research Animal Care at Massachusetts General Hospital. Experiments were performed when the litters were 2 wk of age for the preweaned group and 5 wk of age for the postweaned group. Adult animals were killed at 10 wk or later.

CT-response in human fetal small intestine xenografts. Human fetal small intestine xenografts were supplied by the Xenograft Core Facility in our laboratory. Acquisition of the fetal tissue complied with the American Physiological Society publications policy on the publication of research on human fetuses, fetal tissue, embryos, and embryonic cells. Fetal tissue was obtained with the approval from the Partners Human Study Committee and with signed permission. Briefly, fetal small intestines from 10- to 14-wk-old fetuses were transplanted subcutaneously into SCID mice and kept in a pathogen-free environment for 3 mo as previously reported (35). We have shown in several recent publications that fetal tissue transplanted into SCID mice reepithelializes and matures in a manner similar to that which occurs in the intrauterine environment as documented by expression of microvillous disaccharides and other enzymes (35). Mice were injected subcutaneously with hydrocortisone (50 mg/100 g body wt) or an equal volume of normal saline 1 wk before CT exposure. Tissue was obtained by carefully removing all the serosal and muscle tissues surrounding the intestinal mucosa and cutting them into small pieces using a surgical blade while immersed in HBSS containing 0.1% BSA. After exposure to CT or media under conditions previously used for cells at 4°C, tissues were washed with ice-cold HBSS to remove unbound CT, half of the samples were placed in media at 4°C to measure the surface binding of CT, and the other half were placed in media at 37°C to determine internalization. After incubation, tissues were washed with either HBSS or acidified HBSS in some samples to remove surface CT before being snap frozen in liquid nitrogen and stored at –70°C for further analysis.

Isolation of enterocytes from rat small intestine. Pre- and postweaned and adult rat small intestinal enterocytes were isolated by a method previously described (3). Briefly, a 30–40 cm length of small intestine (jejunum and ileum) was excised and washed with ice-cold HBSS (in mM: 140 NaCl, 5 KCl, 10 HEPES, 1.3 CaCl2, 0.5 MgCl2, 0.36 K2PO4, 5.5 D-glucose, and 4.2 NaCO3, pH 7.2, with 2.5 M Tris) and inverted. The preparation was then immersed in ice-cold Ca2–-free isolation buffer (in mM: 30 EDTA, 52 NaCl, 5 KCl, 10 HEPES, 2 DTT, and 60 HCl, pH 7.1, with 2.5 M Tris) and shaken for 5 min to disaggregate mucus. After discarding the first wash, intestine was washed twice in the same buffer and shaken for 10 min each time. The successive fractions were collected after 5 min centrifugation at 100 g. Pellets were rinsed once with HBSS and resuspended in DMEM containing 0.1% BSA. Cells were maintained at 4°C and used within 6 h of enterocyte isolation. The CT internalization in these cells was carried out as described in the previous section.

Cell lysis and fractionation. H4 and T84 cells were grown to confluence, washed with HBSS, and incubated with 20 nM CT for 1 h at 4°C. After reaching a steady state of CT binding, cells were washed with HBSS to remove unbound CT and incubated either at 4 or 37°C for 1 h. Surface-bound toxin was washed away with acidified HBSS (pH 2.5) in specific additional experiments as detailed elsewhere (46). Cells were then scraped into Tris-buffered solution (4°C) containing 1% Triton X-100 (1% TTBS, 10 mM Tris, 150 mM NaCl, 1% Triton X-100), 1 mM PMSF and a complete protease inhibitor tablet. Triton-soluble proteins were collected and membrane-associated proteins were solublized in 1% TTBS containing 0.5% SDS. Isolated mucosal tissues from xenografts were homogenized in 1% TTBS. Triton-soluble and insoluble fractions were collected as described above. Both fractions were analyzed by SDS-PAGE and Western blotting.

Sucrose equilibrium density centrifugation. Confluent monolayers of polarized or nonpolarized T84 and H4 cells were used for isolation of DIGs. All steps were completed at 4°C as described in detail elsewhere (1, 46). Briefly, cells were scraped into 2 ml of 1% TTBS and homogenized with seven strokes in a tight-fitting Dounce homogenizer on ice. The subsequent homogenate was adjusted to 40% sucrose with equal volume of 80% sucrose in 1% TTBS, layered under a linear 5–30% sucrose gradient, and centrifuged at 39,000 rpm for 16–20 h in a swinging bucket rotor (model SW 41; Beckman Instruments, Palo Alto, CA). Sequential 0.5- or 1-ml fractions were collected from the top of the gradient and a 20-µl sample from each fraction was analyzed by SDS-PAGE and Western blotting. Sucrose density was monitored by refractometry.

Measurement of cAMP. The content of cAMP in the cells treated with various agents and CT was measured by a cAMP EIA system from Amersham, as previously described (24).

Fluorescent microscopy of CT binding and internalization. H4 and T84 cells were plated on glass coverslips until confluence. After incubation with CTB-B at 4 or 37°C, cells were washed three times with HBSS at 4°C, and in some experiments, surface-bound CTB was disassociated and washed away with an acid wash. Cells were fixed with 4% ice-cold paraformaldehyde in PBS for 10 min at 4°C, blocked with 3% BSA in TBS (10 mM Tris, 150 mM NaCl pH 8.0) for 1 h before incubation with a Cy3-streptavidin conjugate for 1 h, and examined using a Zeiss Axiophot photomicroscope (Germany).

Immunoblotting. H4 and T84 cells were grown to confluence and subjected to various treatments. Cell lysates were collected and added onto 10–20% denaturing Tris·HCl polyacrylamide gels (Bio-Rad, Hercules, CA). Following electrophoresis, proteins were transferred to nitrocellulose membranes. After blocking with a 5% nonfat milk solution, membranes were probed with primary and secondary antibodies and visualized by enhanced chemiluminesence (Pierce, Rockford, IL). Quantification of the protein was measured by densitometry and expressed as the relative density unit ratio between the protein of interest and GAPDH (as a normal control for loading).

Statistics. Data were analyzed using StatView 4.1 software (Brainpower, Calabasas, CA).


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Difference in CT binding and internalization in H4 compared with T84 cells. Our previous study (24) measuring CT-stimulated cAMP accumulation in human IECs had shown that immature human enterocytes exhibited a significantly higher CT response compared with mature human enterocytes. In the present study, we extended this observation to determine in a time course experiment whether differences existed in CT binding and internalization between H4 and T84 cells as a possible explanation for the disparate cAMP responses. Cells were grown to near confluence before incubation with CT for varying time intervals at 4 and 37°C. We found that in H4 cells, CT binding to surface GM1 receptors was increased with exposure time and reached a plateau at 60 min, whereas the internalization of CT occurred almost immediately after exposure to CT. In contrast, in T84 cells there was a higher surface binding activity immediately after exposure to CT, but a 20- to 30-min time lag before detectable amounts of CT were internalized (Fig. 1A). Finally, there was a significantly higher proportion of internalization of surface-bound toxin in H4 (~50%) compared with T84 cells (~20–30%).



View larger version (61K):
[in this window]
[in a new window]
 
Fig. 1. A: cholera toxin (CT) bound to microvillus glycolipid ganglioside (GM1) surface receptors (4°C) and subsequent internalization (37°C) into H4 and T84 cells at different time intervals. Western blot analysis depicting the expected ~11-kDa band probed with an anti-CT B subunit (CTB) antibody in each cell type. CT binds to its surface receptor GM1 equally well in H4 and T84 cells, but there was an earlier and greater rate of internalization of CT by H4 cells compared with T84 cells. B: immunofluorescent depiction of CT bound to surface receptors in H4 and T84 cells. Cells were grown on coverslips and incubated with a CTB conjugated to biotin (CTB-B) at 4°C for CT binding. CT bindings in H4 and T84 cells were detected by incubation with a Cy3-streptavidin complex and examined by a fluorescent microscope at x40 magnification. Arrows point to surface staining of CTB.

 
To confirm this observation, we carried out parallel experiments using fluorescent microscopy to visualize toxin binding in both H4 and T84 cells. Our studies suggest that after 2-h exposure, there was no obvious difference in CT-binding between H4 and T84 cells (Fig. 1B).

Effect of endocytic inhibitors on the internalization of CT in H4 compared with T84 cells. We next investigated the pathways of CT internalization in immature (H4 cells) vs. mature human enterocytes (T84 cells). Using endocytic pathway-specific pharmacological inhibitors, we examined differences in endocytosis between these two cell lines. We first examined the effect of two classes of drugs on the uptake of CT by these cells. Cationic amphiphilic drugs, such as CPZ, act on the clathrin-dependent endocytic pathway and inhibit receptor-mediated endocytosis by reducing the number of coated pit-associated receptors. In contrast, cholesterol-binding agents, such as filipin or M{beta}CD, disrupt caveolae structure and function, thus blocking caveolae-mediated endocytosis. Cells were treated with CPZ, filipin, or M{beta}CD for 30 min and were exposed to CT at 4 or 37°C for 2 h. They were then washed and surface-bound toxin was removed by an acid wash (2 times) in selective experiments. CT internalization was monitored using anti-CTB antiserum and immunoblotting. We demonstrated that CT internalization in H4 cells was markedly inhibited by CPZ but was not significantly affected by filipin and M{beta}CD (Fig. 2A). In contrast, internalization of CT was significantly inhibited by filipin and M{beta}CD in T84 cells, whereas CPZ only partially inhibited CT uptake (Fig. 2B).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 2. Endocytic inhibitors affect endocytosis of CT in H4 and T84 cells. H4 and T84 cells were preincubated with various endocytic inhibitors such as chlorpromazine (CPZ; inhibits clathrin-mediated endocytosis), filipin, and methyl-{beta}-cyclodextrin (M{beta}CD) (cholesterol depleting agents that inhibit caveolae-mediated endocytosis) for 30 min before exposure to CT for 2 h. Western blot analysis depicts the expected 11-kDa band representing the CTB subunit on the cell surface (4°C; surface CT) or intracellular (37°C; intracellular CT) in H4 cells (A) and T84 cells (B). Densitometry data are presented as the CTB/GAPDH ratio. *P ≤ 0.05.

 
Differential effects of endocytic inhibitors on the CT response in H4 compared with T84 cells. It was necessary to determine whether endocytic inhibitors affected CT activation of cAMP, because only minute amounts of internalized toxin are required to exert its action on the cells. Accordingly, H4 and T84 cells, either grown on collagen-coated inserts or on a plastic surface, were pretreated for 30 min with CPZ, filipin, or M{beta}CD. BFA, an agent that blocks transcytosis of CT through Golgi apparatus and thus blocks CT function, was used as a general inhibitor of CT activation. Cells were then exposed to 20 nM of CT for 60 min. H4 cells treated with CPZ exhibited a complete inhibition of CT stimulation of intracellular cAMP accumulation, whereas filipin and M{beta}CD had only a minimal effect on CT-induced cAMP elevation in these cells (Fig. 3A). In contrast, in T84 cells, filipin and M{beta}CD treatment significantly reduced cAMP accumulation compared with untreated cells, yet CPZ had only a slight effect (Fig. 3B). As anticipated, BFA inhibited CT-induced cAMP accumulation equally well in both cell lines (Fig. 3). The inhibition of CT activity was not due to the direct inhibition of adenylate cyclase because the increased level of cAMP stimulated by forskolin was similar in untreated and treated cells (Fig. 3).



View larger version (25K):
[in this window]
[in a new window]
 
Fig. 3. Endocytic inhibitors affect CT-induced cAMP elevation in H4 cells compared with T84 cells. The human fetal small intestinal cell line H4 as well as T84 cells were incubated with various endocytic inhibitors such as CPZ, filipin, and M{beta}CD for 30 min before exposure to either CT (20 nM) or forskolin (1 µM) for 1 h. Cellular cAMP levels in H4 (A) and T84 cells (B) were measured and expressed as picomoles per milligram of total protein. Findings are presented as means ± SE, n = 6. *P < 0.05, **P < 0.01. BFA, brefeldin A.

 
Fluorescent microscopy experiments further confirmed the contrasting effects of these drugs on toxin internalization by H4 cells. Cells were treated with or without the endocytic inhibitors described above before exposure to CTB-B at 4 or 37°C. As expected, BFA exhibited complete disruption of CT intracellular trafficking (Fig. 4). Pretreatment with CPZ completely inhibited endocytosis of CT, whereas filipin and M{beta}CD had little effect on internalization of CT. These results were in agreement with CT internalization and cAMP activation data shown in Figs. 2 and 3, respectively. Pretreatment with the endocytic inhibitors had no effect on CT binding (data not shown).



View larger version (93K):
[in this window]
[in a new window]
 
Fig. 4. Immunofluorescent depiction of endocytic inhibitors affect CT intracellular trafficking in H4 cells. Cells were grown on coverslips and pretreated with various endocytic inhibitors such as CPZ (inhibits clathrin-mediated endocytosis), filipin, and M{beta}CD (cholesterol depleting agents that inhibit caveolae-mediated endocytosis) or BFA (agent that generally blocks CT transcytosis) for 30 min before exposure to CT. In some experiments cells were incubated with CTB-B at 4°C for CT binding. In other experiments, cells were incubated with CTB-B at 37°C for CT internalization, in which case, cell surface-bound CT was removed by an acidified HBSS wash. CT binding and internalization were detected by incubation with a Cy3-streptavidin complex and examined by a fluorescent microscope at x40 magnification. A: CTB at 4°C, B: CTB at 37°C, C: CPZ/CTB at 37°C, D: filipin/CTB at 37°C, E: M{beta}CD/CTB at 37°C, F: BFA/CTB at 37°C.

 
Effect of corticosteroids on CT endocytosis in immature human enterocytes (H4 cells). Corticosteroids, a documented trophic factor (13), have been known to influence the epithelial structure and function of immature enterocytes through several mechanisms including changes in membrane lipid composition (4). Epithelial function has in turn been modulated by changes in membrane lipids. Following a maturational stimulus, such as occurs with glucocorticoid exposure, remodeling of membrane phospholipids leads to changes in the activity of membrane transport (47). We therefore examined the effect of hydrocortisone on CT internalization in H4 cells. H4 cells were pretreated with hydrocortisone for 1 wk as mentioned in CT response in human fetal small intestine xenographs before exposure to CT. In other experiments, H4 cells were incubated with endocytic inhibitors after pretreatment with hydrocortisone or with media as a control before exposure to CT. Immunoblotting with anti-CTA/CTB was used to monitor the internalization of CT. We found that pretreatment with hydrocortisone had little effect on surface binding of CT to its receptor GM1, but decreased CT internalization compared with untreated cells (Fig. 5A). However, we found an increased CT-GM1 association with lipid rafts in H4 cells pretreated with hydrocortisone compared with untreated cells as demonstrated by DIGs on a sucrose density gradient. After hydrocortisone treatment for 1 wk as shown in Fig. 5B, H4 cells showed a higher lipid raft association of the surface-bound CT (49%) compared with that of untreated H4 cells (34%). Furthermore, in contrast to the untreated H4 cells as seen in Fig. 2A, pretreatment with hydrocortisone significantly diminished the inhibitory effect of CPZ on CT internalization and increased M{beta}CD inhibition of CT uptake in H4 cells (Fig. 5C), suggesting a shift of the endocytic process from a predominantly clathrin-dependent pathway to a caveolae/raft-mediated pathway.



View larger version (45K):
[in this window]
[in a new window]
 
Fig. 5. Hydrocortisone (HC) affects the endocytosis of CT in H4 cells. A: H4 cells were maintained in media alone or in media containing 1 µM HC for 1 wk before being challenged with CT (20 nM). Surface binding and internalization of CT in these cells were monitored by immunoblotting with an anti-CTB antibody. Western blot analysis depicts the expected ~11-kDa band representing CTB. GAPDH was used to control for equal loading. B: H4 cells pretreated with HC or media control were exposed to 20 nM CT at 4°C for 2 h. The surface-bound CT was applied onto 5–30% sucrose gradient, and fractions were probed with anti-CTB antibody. Sucrose fractions 1 to 5 contain most of the lipid rafts. CT appears shifted from a nonraft fraction to a raft fraction in HC-treated H4 cells (49%) compared with untreated H4 cells (34%). C: H4 cells preincubated with HC (1 µM) for 1 wk were then pretreated with endocytic inhibitors such as CPZ (inhibits clathrin-mediated endocytosis), filipin, and M{beta}CD (cholesterol depleting agents that inhibit caveolae-mediated endocytosis) for 30 min before exposure to CT at either 4°C (surface CT) or 37°C (intracellular CT). Western blot analysis depicts the expected ~11-kDa band representing CTB as probed with an anti-CTB antibody. GAPDH was used to control for protein equal loading.

 
Corticosteroids modulate CT-stimulated activation of cAMP in immature human enterocytes. Tissues from human fetal small intestinal xenografts were used to validate our findings in cell lines. In this study, we investigated whether an intestinal trophic factor, corticosteroids, could modulate CT-induced cAMP accumulation in immature enterocytes. SCID mice that carried xenografted human fetal small intestine transplants were treated with subcutaneous injections of 50 mg/100 g body wt hydrocortisone or equal volume of normal saline as a control for 1 wk before exposure to 50 nM CT for 2 h at 37°C. Tissues were harvested, and CT activation of adenylate cyclase was determined by measuring intracellular cAMP accumulation. We found that hydrocortisone significantly diminished CT-stimulated cAMP elevation in human enterocytes compared with controls (Fig. 6A).



View larger version (36K):
[in this window]
[in a new window]
 
Fig. 6. The effect of HC on the internalization of CT and CT-induced intracellular cAMP production in human fetal small intestinal xenografts. SCID mice with fetal small intestinal xenotransplants were injected subcutaneously with normal saline or HC (50 mg/100 g body wt) for 1 wk. Xenograft mucosa were harvested on day 7 and stimulated with CT in organ culture for 2 h. A: HC decreased cAMP elevation in response to CT compared with the normal saline-injected group. Findings are presented as means ± SE. *P < 0.05. B: HC decreased CT internalization compared with the normal saline-injected group as depicted by Western blot analysis with an anti-CT A subunit (anti-CTA) antibody. There is little change in the surface-bound CT, but the internalization of CT was reduced in cells pretreated with HC. GAPDH was used to control for equal loading. C: histology of the xenografted tissues after normal saline (left) or cortisone injection (right) at x10 magnification.

 
To further characterize the effect of hydrocortisone on the CT response in immature enterocytes, we used immunoblotting to monitor CT uptake in xenografted human small intestinal epithelia. SCID mice with xenografts were treated as before. Tissues were harvested and lysed for SDS-PAGE. We found that hydrocortisone inhibited total CT internalization compared with untreated controls (Fig. 6B). We have previously shown that morphologic changes and hydrolase changes occur under these conditions with cortisone injection (27, 28) (Fig. 6C).

Age-related differences of CT uptake in pre- and postweaned and adult rat small intestinal epithelia. To further validate the differences found in CT uptake between immature and mature IECs, we investigated whether there was a difference in CT internalization in enterocytes from pre- and postweaned and adult rat small intestinal epithelia. Western blot analysis from pre- and postweaned and adult rat enterocytes demonstrated an age-dependent decrease in CT uptake, and preweaned rat enterocytes exhibited the highest rate of internalization of CT compared with postweaned and adult rats (Fig. 7A). Since the investigations of H4 and T84 cells with pathway-specific endocytic inhibitors suggested different CT-uptake processes in these cells, we further investigated the endocytosis of CT in pre- and postweaned rat enterocytes. The isolated small intestinal enterocytes from pre- and postweaned rats were pretreated with CPZ, filipin, and M{beta}CD for 45 min before incubation with CT. Our preliminary study suggested a parallel pattern of endocytosis of CT presented in pre- and postweaned rat enterocytes to that in H4 vs. T84 cells. As depicted in Fig. 7B, CPZ (20 µg/ml) demonstrated a greater inhibition of CT internalization in preweaned rat enterocytes, whereas filipin (1 µg/ml) and M{beta}CD (1 mM) showed greater inhibition of CT internalization in the postweaned rat intestine.



View larger version (32K):
[in this window]
[in a new window]
 
Fig. 7. Age-dependent decrease of CT internalization in pre- and postweaned and adult rat small intestinal enterocytes. Enterocytes isolated from pre- and postweaned and adult Sprague-Dawley rats were incubated with CT (50 nM) for 2 h. Total cell lysates were used in SDS-PAGE probed with an anti-CTA antibody or anti-GAPDH antibody. A: rat enterocytes were incubated with CT at 37°C and probed with an anti-CTA antibody. Western blot analysis depicts the expected band representing the CTA or GAPDH. The densitometry data demonstrated a marked decrease with age in CT uptake by enterocytes. B: enterocytes from 2- or 5-wk-old rats were pretreated with CPZ (20 µg/ml), filipin (1 µg/ml), and M{beta}CD (1 mM) for 45 min before exposure to CT. Western blot analysis depicts the CTA subunit or GAPDH. The densitometry data demonstrated an age-dependent regulation in CT internalization.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
In this study, we have investigated pathways for the endocytosis of CT and the intracellular activation of cAMP by CT in fetal and adult human enterocytes. For the first time, we provide preliminary experimental evidence that endocytosis of CT is developmentally regulated and may, in part, explain the excessive CT-induced cAMP accumulation in immature human enterocytes previously reported. We demonstrated that an adult IEC line, T84 cells, exhibited a largely caveolae-dependent endocytosis of CT, whereas the human fetal intestinal cell line H4 cells showed an enhanced endocytosis of CT via a clathrin-dependent pathway. In addition, this clathrin dependence in H4 cells can be modulated by treatment with a known trophic factor, glucocorticoids, suggesting that it is developmentally regulated.

The endocytosis of CT in T84 cells has been previously well characterized to be mainly a caveolae/raft-mediated process (46). To compare the caveolae-mediated endocytosis for CT with other endocytic pathways, we first had to define the endocytic pathways of CT in H4 cells. H4 cells are nontransformed human fetal small intestinal cells and comparable in structure and function to the primary IEC-6 rodent crypt cell line developed by Quaroni et al. (31). H4 cells have morphologic and functional characteristics typical of intestinal enterocytes, but they are poorly differentiated (34) and represent an appropriate cellular model for defining the CT response in the immature intestine. These studies demonstrate that CT binds to the H4 cellular surface where a small fraction of surface-bound CT is associated with sphingolipid/cholesterol-enriched detergent-insoluble domain/raft, e.g., the caveolae-mediated endocytic pathway. However, the initial step of CT internalization cannot be blocked by drugs that deplete cholesterol, such as M{beta}CD or filipin, which inhibits caveolae-mediated endocytosis but can be blocked by CPZ, which causes aberrant endosomal accumulation of clathrin on internalized vesicles (e.g., inhibits the clathrin endocytic pathway). These findings are indistinguishable from internalization of the transferrin receptor, previously reported by Shogomori and Futerman (38), which is taken up by enterocytes via an exclusively clathrin-dependent mechanism, suggesting that in H4 cells, the endocytosis of CT is mediated principally by a clathrin-dependent mechanism. This endocytic pathway results in a more rapid and efficient internalization of the toxin-receptor complex compared with the caveolae-mediated pathway operational in adult enterocytes.

T84 cells, an established model for the adult enterocyte, have also been used as a model for the study of CT endocytosis and action on intestinal epithelia (1, 21). T84 cells when grown on collagen-coated permeable supports form morphologically well-differentiated monolayers and express the necessary ion channel symporters and pumps in the appropriate membrane domains for vectorial transport (22, 29). It has been demonstrated that T84 cells have all the necessary cellular components to produce a pathophysiologic response to CT. We confirmed that T84 cells exhibit a caveolae-mediated endocytosis of CT as previously reported by other studies (1, 46). In contrast to the internalization of CT in H4 cells, this process can be blocked by the cholesterol depletion agents filipin and M{beta}CD, confirming its caveolae-associated nature.

A central dogma of the caveolae/glycolipid raft endocytosis has been that the "flask shaped" invagination of caveolae is a specific consequence of the association of caveolin-1 with selected raft domains (reviewed in Ref. 30). Caveolin-1 is essential for the formation and stability of caveolae. Few to no caveolae invaginations are present in cells in which caveolin-1 expression levels are reduced or absent. Since the levels of caveolin-1 present in H4 and T84 cells are comparable (unpublished data), we have reasoned that there are caveolae on the H4 cell plasma membrane and the surface-bound CT-GM1 complex is at least partially caveolae/raft-associated as suggested by its presence in DIGs. While the different endocytic mechanisms used by T84 and H4 cells may simply reflect different cell types, we propose the possibility that endocytosis of CT by human enterocytes is developmentally regulated, and the sensitivity of different membrane components such as lipids, glycolipid-binding toxins, and their intracellular signal responses to intracellular trafficking machinery depend on the maturation of these cells.

To support this hypothesis, experiments using glucocorticoids as a maturational stimulus show that CT internalization in H4 cells shifts from a clathrin-dependent to a clathrin-independent mechanism, and a significantly larger amount of surface-bound CT becomes lipid raft-associated. In addition, studies by Badizadegan et al. (1) comparing isolated primary mature human enterocytes to T84 cells have shown a similar capacity for CT signal transduction via specific association of the toxin-GM1 complex with DIGs. In these studies, the internalization of CT was indistinguishable in human small intestinal epithelia from that in T84 cells, suggesting that T84 cells are an appropriate model for studying the cellular mechanism of the CT response.

In like manner, among different age groups using isolated rat enterocytes from pre- and postweaned and adult small intestine, we showed an age-dependent decrease in the internalization of CT but no significant difference in CT binding to its surface receptor. The animal model further validated our findings in cell lines and strengthened our argument that the difference in the endocytosis of CT between H4 and T84 cells is not merely a cell-type difference but represents a developmentally regulated process. Our preliminary data suggest that a clathrin-dependent endocytosis of CT exists in preweaned rat enterocytes compared with a cholesterol-dependent endocytosis of CT in postweaned rat enterocytes (Fig. 7B). However, further studies are needed to characterize this change in endocytosis during gut development.

The developing intestine expresses an enhanced capacity for endocytosis of macromolecules present in the luminal compartment. During development of the intestinal epithelium, a transition occurs from a stratified to a simple columnar epithelium (9, 12). After the conversion, enterocytes assemble an extensive endocytic complex in the apical cytoplasm. This apical endocytic complex is located just beneath the microvillus membrane and is comprised of an elaborate array of membrane tubules and vesicles (40). In the human fetus, this complex is present by the 10th wk and persists at least until the 22nd wk of gestational age (7). Clathrin-coated vesicles have long been considered the most important pathway for internalization of ligand-receptor complexes during intestinal development and are thought to provide a rapid clearance of proteins from fetal amniotic fluid that contains abundant amounts of growth factors and hormones (42). Thus it is presumed that this developmental endocytotic transfer of biologically active molecules may play an important role in the pre- and postnatal maturation of the intestine.

Unfortunately premature infants are also more susceptible to intestinal infection by a variety of infectious agents and their toxins. This may also be due to the same enhanced endocytic capacity and/or increased transepithelial transport capacity in the developing intestine. Our results suggest a role for the clathrin-dependent process in immature human enterocytes leading to a more rapid and efficient uptake of CT, which subsequently results in increased activation of Gs{alpha} and adenylyl cyclase leading to a CT-stimulated cAMP accumulation and an excessive secretory response. In animal studies (14), cortisone administration could alter the diffuse pattern of anionic sites on the microvillus membrane of newborn rats. This change parallels a premature reduction in the endocytic apparatus of the cell. In our cell line studies, we demonstrate that corticosteroid treatment induces a conversion from a clathrin-dependent pathway to favor the more mature caveolae/raft-mediated pathway. To support this conclusion, we have shown that corticosteroid treatment also inhibits the excessive CT-induced cAMP-mediated secretory response noted in immature human enterocytes. We also provide additional experiments using hydrocortisone treatment in human fetal small intestinal xenografts and noted that cortisone treatment decreased uptake of CT receptor complex and caused a shift in endocytosis to a caveolae/raft-mediated pathway as well as a decreased activation of cAMP. These studies in cell lines confirmed by pre- and postweaned and adult rats as well as human xenograft studies suggest that the excessive secretory response to CT by immature enterocytes appears to be due to an immature, excessive endocytosis of the CT-receptor complex.

In summary, this study represents a preliminary observation that in human fetal cells CT-GM1-associated endocytosis is developmentally regulated and involves a clathrin-dependent mechanism. The enhanced internalization of CT plays a crucial role in CT-induced Gs{alpha} activation and cAMP elevation and may help to explain our previous observation that at lower doses, CT is more active in the immature gut. The use of CT as a probe to examine developmental regulation of endocytic processes in human fetal enterocytes may present a powerful tool for our understanding of the association between signal transduction and intracellular trafficking that is crucial to cell function. Moreover, these studies may lead to a better understanding of the development of mucosal barrier function and provide new insight into reducing the excessive CT response in the immature intestine leading to more frequent and severe age-related secretory diarrhea.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by grants from the American Gastroenterological Association/AstraZeneca Fellowship/Faculty Transition Award (to L. Lu) and National Institute of Diabetes and Digestive and Kidney Diseases Grants RO1-DK-70260, PO1-DK-33506, and P-30-DK-40561 (to W. A. Walker).


    ACKNOWLEDGMENTS
 
The authors thank Dr. Jim Casanova for valuable suggestions and insights.


    FOOTNOTES
 

Address for reprint requests and other correspondence: W. Allan Walker, Developmental Gastroenterology Laboratory, Massachusetts General Hospital, 114 16th St. (114–3503), Charlestown, MA 02129-4404 (e-mail: wwalker{at}partners.org)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Badizadegan K, Dickinson BL, Wheeler HE, Blumberg RS, Holmes RK, and Lencer WI. Heterogeneity of detergent-insoluble membranes from human intestine containing caveolin-1 and ganglioside GM1. Am J Physiol Gastrointest Liver Physiol 278: G895–G904, 2000.[Abstract/Free Full Text]
  2. Bhattacharya SK, Datta D, Bhattacharya MK, Garg S, Ramamurthy T, Manna B, Nair GB, Nag A, and Moitra A. Cholera in young children in an endemic area. Lancet 340: 8834–8835, 1992.
  3. Bjerknes M and Cheng H. Methods for the isolation of intact epithelium from the mouse intestine. Anat Rec 199: 565–574, 1981.[CrossRef][ISI][Medline]
  4. Brasitus T, Dudeja P, Dahiya R, and Halline A. Dexamethasone-induced alterations in lipid composition and fluidity of rat proximal-small-intestinal brush-border membranes. Biochem J 248: 455–461, 1987.[ISI][Medline]
  5. Chu S, Ely I, and Walker WA. Age and cortisone alter host responsiveness to cholera toxin in the developing gut. Am J Physiol Gastrointest Liver Physiol 256: G220–G226, 1989.[Abstract/Free Full Text]
  6. Cohen M, Moyer M, Luttrell M, and Giannella R. The immature rat small intestine exhibits an increased sensitivity and response to Escherichia coli heat-stable enterotoxin. Pediatr Res 20: 555–560, 1986.[Abstract]
  7. Colony MP and Trier J. Development of villus absorptive cells in the human fetal small intestine: a morphological and morphometric study. Anat Rec 195: 463–482, 1979.[CrossRef][Medline]
  8. Colony PC and Specian RD. Endocytosis and vesicular traffic in fetal and adult colonic goblet cells. Anat Rec 218: 365–372, 1987.[CrossRef][ISI][Medline]
  9. Dunn J. The fine structure of the absorptive epithelial cells of the developing small intestine of the rat. J Anat 101: 57–68, 1967.[ISI][Medline]
  10. Glass RI, Becker S, Huq MI, Stoll BJ, Khan MU, Merson MH, Lee JV, and Black RE. Endemic cholera in rural Bangladesh, 1966–1980. Am J Epidemiol 116: 959–970, 1982.[Abstract]
  11. Grasset E, Pinto M, Dussaulz E, Zweibaum A, and Desjeux J. Epithelial properties of human colonic carcinoma cell line Caco-2: electrical parameters. Am J Physiol Cell Physiol 247: C260–C267, 1984.[Abstract]
  12. Hayward A. Changes in fine structure of developing intestinal epithelium associated with pinocytosis. J Anat 102: 57–70, 1967.[ISI]
  13. Henning S, Rubin D, and Shulman R. Ontogeny of the intestinal mucosa. In: Physiology of the Gastrointestinal Tract, edited by Johnson L. New York: Raven, 1994, p. 571–610.
  14. Jersild R. Restricted mobility and endocytosis of anionic sites on newborn rat jejunal brush border membranes. Anat Rec 202: 61–71, 1982.[CrossRef][ISI][Medline]
  15. Kliegman R, Walker WA, and Yolken R. Necortizing enterocolitis: research agenda for a disease of unknown etiology and pathogenesis. Pediatr Res 34: 701–708, 1993.[Abstract]
  16. Koldovsky O. Hormonally active peptides in human milk. Acta Paediatr Suppl 402: 89–93, 1994.
  17. Le PU and Nabi IR. Distinct caveolae-mediated endocytic pathways target the Golgi apparatus and the endoplasmic reticulum. J Cell Sci 116: 1059–1071, 2002.[CrossRef][ISI]
  18. Lencer WI. Microbes and Microbial Toxins: Paradigms for Microbial-Mucosal Interactions. V. Cholera: invasion of the intestinal epithelial barrier by a stably folded protein toxin. Am J Physiol Gastrointest Liver Physiol 280: G781–G786, 2001.
  19. Lencer WI, Constable C, Moe S, Jobling MG, Webb HM, Ruston S, Madara JL, Hirst TR, and Holmes RK. Targeting of cholera toxin and Escherichia coli heat labile toxin in polarized epithelia: role of COOH-terminal KDEL. J Cell Biol 131: 951–962, 1995.[Abstract]
  20. Lencer WI, Moe S, Rufo PA, and Madara JL. Transcytosis of cholera toxin subunits across model human intestinal epithelia. Proc Natl Acad Sci USA 92: 10094–10098, 1995.[Abstract/Free Full Text]
  21. Lencer WI, Delp C, Neutra MR, and Madara JL. Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesiclar traffic. J Cell Biol 117: 1197–1209, 1992.[Abstract]
  22. Lencer WI, Delp C, Neutra MR, and Madara JL. Mechanism of cholera toxin action on a polarized human intestinal epithelial cell line: role of vesicular traffic. J Cell Biol 117: 1197–1209, 1992.[Abstract]
  23. Lencer WI and Tsai B. The intracellular voyage of cholera toxin: going retro. Trends Biochem Sci 28: 639–645, 2003.[CrossRef][ISI][Medline]
  24. Lu L, Baldeon ME, Savidge T, Pothoulakis C, and Walker WA. Development of microbial-human enterocyte interaction: cholera toxin. Pediatr Res 54: 212–218, 2003.[Abstract/Free Full Text]
  25. Madara JL, Stafford J, Dharmsathaphorn K, and Carlson S. Structural analysis of a human intestinal epithelial cell line. Gastroenterology 92: 1133–1145, 1987.[ISI][Medline]
  26. Massol RH, Larsen JE, Fujinaga Y, Lencer WI, and Kirchhausen T. Cholera toxin toxicity does not require functional Arf6- and dynamin-dependent endocytic pathways. Mol Biol Cell 15: 3631–3641, 2004.[Abstract/Free Full Text]
  27. Nanthakumar NN, Young C, Ko JS, Meng D, Chen J, Buie T, and Walker WA. Glucocorticoid responsiveness in developing human intestine: possible role in prevention of necrotizing enterocolitis. Am J Physiol Gastrointest Liver Physiol 288: G85–G92, 2005.[Abstract/Free Full Text]
  28. Nanthakumar NN, Klocic CE, and Walker WA. Normal and glucocorticoid-induced development of human small intestinal xenograft. Am J Physiol Regul Integr Comp Physiol 285: R1–R9, 2003.[Abstract/Free Full Text]
  29. Orlandi PA and Fishman PH. Filipin-dependent inhibition of cholera toxin: evidence for toxin internalization and activation through caveolae-like domains. J Cell Biol 141: 905–915, 1998.[Abstract/Free Full Text]
  30. Pang H, Le PU, and Nabi IR. Gangloside GM1 levels are a determinant of the extent of caveolae/raft-dependent endocytosis of cholera toxin to the Golgi apparatus. J Cell Sci 117: 1421–1430, 2004.[Abstract/Free Full Text]
  31. Quaroni A, Wands J, Trelstad R, and Isselbacher K. Epithelial cell cultures from rat small intestine. J Cell Biol 80: 245–265, 1979.
  32. Raufman JP. Cholera. Am J Med 104: 386–394, 1997.[CrossRef][ISI]
  33. Sack DA, Sack RB, Nair GB, and Siddique AK. Cholera. Lancet 363: 223–233, 2004.[CrossRef][ISI][Medline]
  34. Sanderson I, Ezzell R, Kedinger M, Erlanger M, Xu Z, Pringault E, Leon-Robine S, Louvard D, and Walker WA. Human fetal enterocytes in vitro: modulation of the phenotype by extracellular matrix. Proc Natl Acad Sci USA 93: 7717–7722, 1996.[Abstract/Free Full Text]
  35. Savidge T, Morey A, Ferguson D, Leming K, Shmakov A, and Phillips A. Human intestinal development in a severe-combined immunodeficient xenograft model. Differentiation 56: 361–371, 1995.[CrossRef]
  36. Seo J, Chu S, and Walker WA. Development of intestinal host defense: an increased sensitivity in the adenylate cyclase response to cholera toxin in suckling rats. Pediatr Res 25: 225–227, 1989.[Abstract]
  37. Shogomori H and Futerman AH. Cholesterol depletion by methyl-{beta}-cyclodextrin blocks cholera toxin transport from endosomes to the Golgi apparatus in hippocampal neurons. J Neurochem 78: 991–999, 2001.[CrossRef][ISI][Medline]
  38. Shogomori H and Futerman AH. Cholera toxin is found in detergent-insoluble rafts/domains at the cell surface of hippocampal neurons but is internalized via a raft-independent mechanism. J Biol Chem 276: 9182–9188, 2001.[Abstract/Free Full Text]
  39. Snyder JD and Merson MH. The magnitude of the global problem of acute diarrheal disease: a review of active surveillance data. Bull World Health Organ 60: 605–613, 1982.[ISI][Medline]
  40. Staley T, Corley L, Bush L, and Jones E. The ultrastructure neonatal calf intestine and absorption of heterologous proteins. Anat Rec 172: 559–580, 1972.[CrossRef][ISI][Medline]
  41. Torgersen ML, Skretting G, van Deurs B, and Sandvig K. Internalization of cholera toxin by different endocytic mechanisms. J Cell Sci 114: 3737–3741, 2001.[ISI][Medline]
  42. Weaver L, Freigberg E, Israel E, and Walker WA. Epidermal growth factor in human amniotic fluid (Abstract). Gastroenterology 95: 1436, 1988.[ISI][Medline]
  43. Wilson JM and Casanova JE. Development of endocytosis in the intestinal epithelium. In: Development of the Gastrointestinal Tract, edited by Sanderson IR and Walker WA. Hamilton, ON, Canada: BC Decker, 2000, p. 71–81.
  44. Wolf AA, Jobling MG, Wimer-Mackin S, Ferguson-Maltzman M, Madara JL, Holmes RK, Lencer WI. Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia. J Cell Biol 141: 917–927, 1998.[Abstract/Free Full Text]
  45. Wolf AA, Fujinaga Y, and Lencer WI. Uncoupling of the cholera toxin-GM1 ganglioside receptor complex from endocytosis, retrograde golgi trafficking, and downstream signal transduction by depletion of membrane cholesterol. J Biol Chem 277: 16249–16256, 2002.[Abstract/Free Full Text]
  46. Wolf AA, Jobling MG, Wimer-Mackin S, Ferguson-Maltzman M, Madara JL, Holmes RK, and Lencer WI. Ganglioside structure dictates signal transduction by cholera toxin and association with caveolae-like membrane domains in polarized epithelia. J Cell Biol 141: 917–927, 1998.[Abstract/Free Full Text]
  47. Yorio T and Frazier L. Phopholipids and electrolyte transport. Proc Soc Exp Biol Med 195: 293–303, 1990.[Abstract]
  48. Zweibaum A, Pinto M, Chevalier G, ED, Triadou N, Lacroix B, Haffen K, Brun JL, and Rousset M. Enterocytic differentiation of a subpopulation of the human colon tumor cell line HT-29 selected for growth in sugar-free medium and its inhibition by glucose. J Cell Physiol 122: 21–29, 1985.[CrossRef][ISI][Medline]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
289/2/G332    most recent
00521.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Lu, L.
Articles by Walker, W. A.
Articles citing this Article
PubMed
PubMed Citation
Articles by Lu, L.
Articles by Walker, W. A.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.