Departments of 1 Pediatrics and 3 Cell Biology, University of Miami School of Medicine, Miami, Florida 33101; and 2 Department of Medicine (Gastroenterology), University of Tennessee, Memphis, Tennessee 38104
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ABSTRACT |
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The cystic fibrosis transmembrane conductance regulator (CFTR) channel is regulated by cAMP-dependent vesicle traffic and exocytosis to the apical membrane in some cell types, but this has not been demonstrated in the intestinal crypt. The distribution of CFTR, lactase (control), and fluid secretion were determined in rat jejunum after cAMP activation in the presence of nocodazole and primaquine to disrupt vesicle traffic. CFTR and lactase were localized by immunofluorescence, and surface proteins were detected by biotinylation of enterocytes. Immunoprecipitates from biotinylated and nonbiotinylated cells were analyzed by streptavidin detection and immunoblots. Immunolocalization confirmed a cAMP-dependent shift of CFTR but not lactase from a subapical compartment to the apical surface associated with fluid secretion that was reduced in the presence of primaquine and nocodazole. Analysis of immunoblots from immunoprecipitates after biotinylation revealed a 3.8 ± 1.7-fold (P < 0.005) increase of surface-exposed CFTR after vasoactive intestinal peptide (VIP). These measurements provide independent corroboration supporting a role for vesicle traffic in regulating CFTR and cAMP-induced fluid transport in the intestine.
cystic fibrosis transmembrane conductance regulator; intestine
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INTRODUCTION |
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THE CYSTIC FIBROSIS transmembrane conductance regulator (CFTR) is an apical membrane chloride channel critical to the regulation of fluid, chloride, and bicarbonate transport in the intestine (15, 17, 32, 43). Mutations in the gene encoding CFTR result in the disease cystic fibrosis (CF) and are associated with the absence or dysfunction of CFTR on the apical membrane of epithelial cells, decreased cAMP-mediated fluid secretion, increased mucus viscosity, and intestinal obstruction (15). On the other hand, overstimulation of the secretory pathway and activation of luminal CFTR in the small intestine are implicated in the pathogenesis of toxigenic secretory diarrhea (11, 39).
Although cAMP- and protein kinase A-dependent phosphorylation is recognized as a major signal transduction pathway for CFTR activation, other second messenger pathways include PKC, Ca2+/calmodulin-dependent kinase, and cGMP-dependent kinase signal CFTR activation (12, 20). In some cell types, cAMP-dependent vesicle traffic also regulates CFTR exocytosis and insertion into the apical plasma membrane; however, this remains a highly controversial issue (6, 18, 25, 37, 38, 40).
Although cultured cell models proved useful in identifying CFTR signal transduction pathways, the applicability of these pathways to physiological events is not assured because of inherent differences in the properties of transformed cells and those of tissues expressing endogenous CFTR (8, 39). In the intestine, CFTR channel gating is regulated by cAMP- and cGMP-dependent phosphorylation of protein kinases A and G (12). Whether vesicle traffic regulates CFTR and fluid secretion in the intestine is unclear. Published studies using a variety of techniques to examine this question with T84 cells (a widely used chloride-secreting colonic cell line expressing CFTR) resulted in conflicting conclusions (9, 28, 39), and there are no in vivo studies addressing this issue except for our previous analysis (4) of CFTR high expresser (CHE) cells in rat small intestine.
In a recent study of isolated rat colonic crypts, membrane capacitance was used to determine cAMP-stimulated exocytosis of CFTR; however, the authors could not demonstrate an increase in membrane surface area on cAMP activation and therefore concluded that activation of CFTR by cAMP does not involve detectable exocytosis (13). However, cAMP-dependent chloride secretion in the rat colon has been shown to be dependent on intact microtubules, the molecular motors of vesicle transport (14). This latter evidence suggests a role for cAMP-dependent vesicle traffic in regulating chloride secretion in the rat intestine.
Although CHE cells are a predominantly villus cell population with undefined ion transport properties, the number of CFTR channels expressed on the surface of these cells was demonstrated to be regulated by cAMP-dependent exocytosis. The observation that CHE cells are also present in the human small intestine suggests that cAMP-dependent exocytosis of CFTR is also a potentially important regulatory mechanism in the human intestine (3, 34).
The extent to which vesicle traffic regulates fluid secretion and the number of CFTR channels expressed on the surface of the small intestinal lumen (the predominant site of CFTR-mediated fluid secretion) is unknown. In previous studies (2), we used immunoelectron microscopy to examine and quantify the subcellular distribution of CFTR. These observations confirmed that CFTR was associated with subapical vesicles and the plasma membrane of both crypt and CHE cells. Furthermore, quantification of the subcellular distribution in these cells supported a role for CFTR regulation by vesicle traffic in the rat small intestine (2). On the basis of these observations, we used independent morphological and biochemical methodologies in conjunction with fluid secretion measurements in the current study to determine whether cAMP and vesicle traffic regulate the exocytosis of CFTR to the apical membrane of the crypts and the whole mucosa of rat proximal small intestine.
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MATERIALS AND METHODS |
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Animal preparation and fluid secretion measurements.
The study was approved by the Animal Research Committee of the
University of Miami School of Medicine. Male Sprague-Dawley rats
(Charles River Laboratories) weighing 250-300 g were fed standard
chow. Rats were fasted overnight and anesthetized with 45 mg/kg
pentobarbital sodium administered intraperitoneally. After anesthesia,
a tracheotomy was performed to maintain a patent airway. Rectal
temperature of 38.1°C was maintained with a thermostatically controlled heating lamp. Rats were subjected to a laparotomy via a
midline incision, a 30-cm length of jejunum was identified and cannulated proximally and distally, and the lumen was washed with warm
0.9% NaCl. The saline was removed from the loop by blowing through the
proximal cannula. The distal cannula was removed, and ligatures were
placed to create four intestinal loops each ~4 cm in length with a
2-cm length of intestine separating each loop. Equal volumes (~0.5
ml) of drug [0.1 mM primaquine + 1.0 mM 8-bromo-cAMP (8-BrcAMP),
10 µg/ml nocodazole + 1.0 mM 8-BrcAMP, 1.0 mM 8-BrcAMP, or PBS]
were delivered into each loop by a fluid-filled syringe that was
weighed before (A, g) and after (B, g) delivery into each loop. The abdomen was closed, and the animal was observed for
2 h. At the end of the study period, the abdomen was opened and
intestinal loops were excised, blotted free of excess fluid, and
weighed (C, g). The loops were then cut open, drained,
blotted, and reweighed (D, g). The amount of fluid recovered
was obtained by subtraction (C D, g). The
net movement of fluid into or from the loop was calculated as
[(C
D)
(A
B)] (g). Fluid absorption was reflected by a net loss of
fluid from the loop, (A
B) > (C
D), and secretion occurred if there
was a net gain of fluid into the loop, (C
D) > (A
B). Because the
densities of the solutions weighed are all approximately equal to that
of water, the fluid weights are assumed to be identical with fluid
volumes. Fluid movements in or out of the loops were calculated as
micrograms per centimeter of jejunal length per minute. At the end of
the study period, intestinal tissues from each loop were embedded in
OCT embedding medium, frozen in liquid nitrogen-cooled isopentane, and
prepared for immunocytochemistry as described previously (3, 4).
Reagents. All chemicals were obtained from Sigma (St. Louis, MO) except where stated. R3194 and R3195 are affinity-purified polyclonal antibodies raised against rodent CFTR and were provided by C. R. Marino. The specificity of both antibodies has been documented in rats (1, 2, 4, 44). The previously characterized antibody to lactase, YBB 2/61, was a gift from Dr. A. Quaroni (Cornell University, Ithaca, NY; Ref. 29). The antibody to AKAP149 was purchased from Alomone Laboratories (Jerusalem, Israel).
Isolation of intestinal enterocytes. Segments of rat jejunum were removed 30 min after VIP administration, and villus and crypt enterocytes were isolated as described previously (24). Suspensions of freshly isolated enterocytes were subject to Trypan blue exclusion, immunofluorescence immunocytochemistry, and surface biotinylation studies.
Surface biotinylation of isolated enterocytes. Suspensions of freshly isolated cells from rat small intestine after VIP or diluent infusion were biotinylated by rotating in the cold for 30 min in freshly prepared 1.0 mM sulpho-NHS biotin [bicinchoninic acid (BCA) protein assay kit; Pierce Laboratories, Rockford, IL] in PBS-CM (PBS supplemented with 1.3mM Ca2Cl and 1.0 mM Mg2Cl). Control cells were incubated with PBS-CM. After biotinylation, cells were washed in the cold with 50 mM NH4Cl in PBS-CM to quench unreacted free biotin and immunoprecipitation was performed. Surface biotinylation of CFTR from the lumen of the intact intestine was also attempted; however, complete diffusion into the deep crypts (a major site of CFTR expression) could not be ensured. Surface biotinylation was therefore performed in isolated cells because the yield of isolated crypt and villus enterocytes was high in our hands and polarity and membrane preservation could be ensured in the majority of cells. Indeed, it has been shown that isolated enterocytes remain well polarized for hours (45), a fact that we tested further in our experiments (see Fig. 3E).
Immunoprecipitation of CFTR. Enterocytes were lysed in immunoprecipiation buffer (IP) containing 0.5% Triton X-100, 0.1% SDS, and 0.5% sodium deoxycholate in PBS pH 7.4 supplemented with a protease inhibitor cocktail (Sigma). Samples were homogenized and sonicated and then centrifuged for 15 min in the cold at 14,000 rpm. Protein content was measured by UV absorption (Pierce Laboratories), and immunoprecipitation was performed with a minimum of 1 mg/ml protein in supernatants. Supernatants were precleared with protein A-Sepharose beads (Amersham Pharmacia, Piscataway, NJ) for 1 h in the cold. Immunoprecipitations were performed with the anti-CFTR antibody R3194, antibody to lactase YBB2/61, or nonspecific rabbit IgG. Antibody or serum was added to supernatants and incubated for 2 h at 4°C. Protein A agarose (5 mg/sample) was added to samples and resuspended in IP buffer containing 1% (wt/vol) globulin-free bovine serum albumin (BSA), and then samples were rotated overnight in the cold. Samples were then centrifuged (14,000 rpm), supernatants were discarded, and the beads were washed in IP buffer supplemented with 0.5 M KCl. The immunoprecipitates were eluted in 1% SDS, 4 M urea, and 1 mM Tris · HCl, pH 6.8, precipitated on ice in trichloroacetic acid, acetone extracted, and air dried. The dried pellets were resuspended in sample buffer before analysis by Western blot.
Western blotting.
Immunoprecipitates were analyzed by SDS-PAGE using a 7.5% gel and
proteins transferred to polyvinylidene difluoride (PVDF) membranes by a
semi-dry transfer method. After transfer of proteins, membranes were
washed in deionized water, nonspecific proteins were blocked in
PBS-0.05% Tween 20 containing 5% nonfat dry milk for 2 h, and
biotinylated proteins were detected by streptavidin peroxidase binding
(Sigma). Western blots of nonbiotinylated immunoprecipitates were
analyzed with antibodies to CFTR (R3194) and lactase (YBB2/61). Western
blots of CFTR or IgG immunoprecipitates were also analyzed with a
commercial antibody to AKAP 149 (Alomone Labs). Detection of primary
antibodies was accomplished by using goat anti-rabbit (1:10,000) or
anti-mouse (1:8,000) peroxidase secondary antibodies (Sigma). After
immunodetection, membranes were exposed to chemiluminescence (preflashed Hyperfilm ECL, Amersham Pharmacia). Densitometric analysis
of protein bands was performed with a Kodak Image Station 440 CF and
IS440CF image analysis software. Bands were selected, and signal was
weighted as number of pixels × (average pixel intensity in the
band average pixel intensity in the background).
Immunocytochemistry. Intestinal tissues from all experiments were prepared for immunocytochemical localization studies with indirect immunofluorescent immunolabeling of cryostat sections from jejunum as described previously (1, 4), and labeled sections were examined on a Leica epifluorescent microscope. Confocal microscopy and image analysis of CFTR fluorescence intensities were performed as described previously (4) with a Zeiss LSM 510 microscope equipped with image analysis software. The apical domain of CFTR in labeled sections was determined from images of perpendicular parallel sections labeled with fluorescent phalloidin and measured ~1.5 µm in length from the luminal surface. The CFTR signal below that depth was considered the subapical compartment. Acquisition of parameters were adjusted with the software so that the pixel intensity of the brightest fluorescence was not saturated (>255 pixels). Data was collected from an average of 30 cells in random sections (average 10 sections) from each tissue examined.
Indirect immunofluorescence labeling for apical membrane markers (lactase and CFTR) was performed on freshly isolated enterocytes from rat jejunum to confirm preservation of polarity. A drop of cell suspension was placed onto a poly L-lysine-coated slide and allowed to air dry. Briefly, cells were fixed in 2% paraformaldehyde for 10 min and washed in 50 mM ammonium chloride, and nonspecific proteins were blocked in PBS-BSA 1% for 30 min. Cells were exposed to primary antibody or PBS-BSA 1% for 1 h at room temperature in a moist chamber. Primary antibody was detected with FITC-conjugated secondary antibody diluted in PBS-BSA 1%. After immunolabeling, slides were examined with a Leica epifluorescent microscope. ![]() |
RESULTS |
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Morphological distribution of CFTR in rat jejunum after cAMP
stimulation.
Our previous light microscopic localization of CFTR in rat proximal
small intestinal crypts revealed a subapical distribution for CFTR,
suggesting the presence of CFTR in a vesicular compartment. Immunoelectron microscopic examination revealed that although CFTR was
detected on the apical membrane, the majority of CFTR was associated
with subapical vesicles, supporting a role for vesicle insertion in
regulating CFTR and anion secretion in the crypt (2). VIP,
a cAMP agonist, also induced a redistribution of CFTR from the
subapical compartment to the apical domain in villus CHE cells as
observed previously (4). On the basis of these
observations, we examined the distribution of CFTR in the crypt after
VIP (Fig. 1E). We compared the
distribution with that of lactase, an integral apical membrane protein
that is not regulated by cAMP-dependent vesicle traffic (Fig. 1,
A and B; Refs. 23, 26).
Although lactase is absent in proliferative undifferentiated crypt
cells and the highest levels of CFTR are found in this compartment, both lactase and CFTR are present on the apical pole of newly differentiated crypt cells that are more superficially located (Fig. 1,
A and D; Refs. 3, 29,
34). In fact, lactase is mostly found in a subapical
compartment in the crypts (Fig. 1, A and B),
whereas it is expressed in the brush border in the villus (29). Examination of crypt sections from rat jejunum
revealed that lactase did not redistribute to the cell surface after
VIP administration (Fig. 1, B compared with A).
CFTR, on the other hand, was distributed in a broad subapical region
under control conditions (Fig. 1D) and redistributed to the
apical surface in a narrow band (correlating with the region of
phalloidin label) after VIP (Fig. 1E). To further confirm
that the effect of VIP was mediated by cAMP, similar experiments were
conducted after luminal 8-BrcAMP stimulation. Examination of labeled
sections by confocal microscopy after administration of the
membrane-permeant agonist 8-BrcAMP similarly confirmed a redistribution
of CFTR from a predominant subapical compartment (Fig.
2A) to the region corresponding to the apical microvilli of crypt epithelial cells (Fig.
2B). The 8-BrcAMP-dependent shift of CFTR from the subapical to apical domain corresponded with an almost threefold increase in the
ratio of apical to subapical CFTR fluorescence (6.15 ± 3.08)
compared with unstimulated PBS controls (2.14 ± 1.03;
P < 0.001) and was associated with net fluid movement
into the lumen of the jejunum (Table 1).
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Detection of CFTR exocytosis by surface biotinylation in vivo. Although immunofluorescence examination of the distribution of CFTR in intestinal sections after cAMP agonist treatment suggested a shift of CFTR from a subapical compartment to the apical domain, we wished to independently confirm that cAMP indeed stimulated exocytosis of CFTR to the surface of intestinal cells. Immunolocalization in toto could not confirm this because our antibodies were raised against the cytoplasmic COOH terminus of CFTR. We therefore used the technique of surface biotinylation, a sensitive method that is widely used to quantify surface proteins in cells and to study membrane traffic of proteins and has been used in studies examining CFTR membrane traffic (28, 30).
Morphological examination of fixed intestinal segments after isolation confirmed that we could successfully retrieve most cells for biotinylation and immunoprecipitation, including those from the crypts, within 30 min. Lack of damage to isolated enterocytes was confirmed by Trypan blue exclusion in cell suspensions. Furthermore, immunofluorescence labeling of freshly isolated cells confirmed preservation of polarity by the presence of apical markers (Fig. 3, E and F) in isolated cells. Immunoprecipitations were performed on freshly isolated cells with the CFTR antibody R3194 and nonspecific rabbit IgG as negative controls, and immunoprecipitates were analyzed for CFTR by Western blots using the same CFTR antibody. Western blot analysis of immunoprecipitates from VIP-stimulated or control cells with R3194 detected a broad band of mature CFTR (band C) of molecular mass of 170-185 kDa (Fig. 3A, lane 2) and a smaller band of immature CFTR of ~148 kDa in native rat tissues but not in IgG controls (Fig. 3A, lane 1) as shown previously (1, 9). These results confirmed the success of the immunoprecipitation.
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DISCUSSION |
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In the current study, two independent techniques were used to confirm that physiological cAMP stimulation and vesicle traffic regulate the number of CFTR channels on the surface of the rat small intestinal epithelium. This observation resolves the current controversy regarding the role of membrane traffic in regulating CFTR in the intestine. Although cAMP-dependent exocytosis of CFTR to the apical membrane has been demonstrated in villus CHE cells (4), the physiological relevance of that observation remains unknown. Our observation in this work that CFTR is regulated in vivo by cAMP-dependent vesicle traffic and channel insertion in both crypt and villus cells in association with fluid secretion, however, provides strong support for physiological membrane traffic regulation of CFTR and intestinal anion secretion.
Both receptor (VIP)- and non-receptor (8-BrcAMP)-mediated cAMP agonists induced a redistribution of CFTR from the subapical to the apical domain in the jejunum. The lack of change in the distribution of lactase in crypt cells after cAMP agonist stimulation confirmed that the cAMP-dependent redistribution of CFTR was specific, because lactase is not regulated by cAMP-dependent vesicle insertion (23, 26). To determine whether the cAMP-induced redistribution of CFTR from the subapical to the apical domain is regulated by vesicle traffic, we disrupted vesicle traffic in vivo with primaquine and nocodazole. In rat jejunum, primaquine (0.1 mM) was a potent inhibitor of the cAMP-dependent shift of CFTR from the subapical to the apical domain in the crypt and reduced the fluid secretory response of 8-BrcAMP (Table 1). The accumulation of CFTR in the subapical compartment in the crypt in the presence of primaquine is consistent with the observations by others of its effect in inhibiting receptor recycling and in producing an intracellular accumulation of endocytosed receptors, blocking the exit of receptors from the early endosomes and recycling to the plasma membrane (41). The reduction in the fluid secretory response to 8-BrcAMP in vivo in the presence of primaquine (~35%) supports the notion that insertion of new CFTR channels into the membrane contributes importantly to augmenting fluid secretion.
These experiments, however, may allow an alternative albeit nonexclusive interpretation. If CFTR is continuously recycling between the apical domain and the subapical compartment, primaquine may interrupt the cycle and accumulate CFTR in the early endosomal compartment. The fluorescence measurements in confocal images actually point to this scenario. In that case, we could conclude that the balance between exocytosis and endocytosis of CFTR is almost as important as channel gating as a regulatory factor. Our observations in the intestine support the results of recent studies in oocytes demonstrating that primaquine drastically reduced cAMP-dependent CFTR chloride currents and effectively blocked vesicle and protein traffic (40). Further studies will be necessary to assess the relative contributions of exocytosis and endocytosis to the steady-state levels of surface CFTR on cAMP stimulation.
Although the effects of microtubule disruption on CFTR distribution and fluid movement were less striking than those of primaquine, they also suggest a role of membrane traffic in regulating the number of CFTR channels on the apical surface and provide support for the previous observation that cAMP-dependent chloride secretion in rat intestine is regulated by microtubules (14).
Surface biotinylation, a well-established technique used to assess the delivery of proteins to the plasma membrane (30, 31), confirmed cAMP-stimulated CFTR exocytosis. However, the finding that other unidentified polypeptides were coimmunoprecipitating with CFTR and were also upregulated by cAMP, as shown in Fig. 3, was unexpected. Our first interpretation was that other proteins possessing at least one ectoplasmic domain capable of biotinylation may be nonspecific contaminants of the immunoprecipitation. This, however, was unlikely for the following reasons: 1) these other biotinylated proteins were CFTR antibody specific in the immunoprecipitation and did not appear in controls immunoprecipitated with nonimmune IgG (Fig. 3B, lane 3); 2) the conditions for immunoprecipitation were rather stringent, detergents were present in all washes, and one of the washes was performed in high salt (0.6 M KCl) conditions; and 3) the sucrase-isomaltase immunoprecipitation experiments (Fig. 3C) supported the notion that our immunoprecipitations were "clean" (in those cases no additional bands were observed).
Another potential artifact that could explain biotinylation of multiple bands is damage to the plasma membrane during the isolation of enterocytes before biotinylation. This possibility was ruled out by verifying Trypan blue exclusion in parallel cell suspensions and by actually permeabilizing some cell suspensions with saponin. The latter resulted in an increase in the number of biotinylated bands, indicating that in the absence of saponin the plasma membrane was tight. The observation that AKAP coimmunoprecipitates with CFTR (Fig. 3D) in the intestine supports the notion that the physiological regulation of CFTR by PKA involves a physical and functional association with AKAP as demonstrated recently (19). The coimmunoprecipitation of AKAP with CFTR indicated that under the conditions of homogenization, detergent solubilization, and immunoprecipitation used here, the NHERF-ezrin insoluble scaffold that normally holds CFTR (35, 36) is at least partially preserved.
At least one other transmembrane protein, Na+/H+ exchanger (NHE), is known to be attached to this scaffold in addition to CFTR (42). Although the apparent molecular masses of the biotinylated bands that we found do not correspond to that of NHE-3 (97 kDa; Ref. 5), it is conceivable that other membrane proteins are also attached to the same scaffold. Furthermore, because some membrane proteins do not biotinylate and because we cannot assert that the scaffold is totally intact, the actual number of membrane proteins attached to the same scaffold of CFTR may be actually greater than three (CFTR and the 2 unknown proteins found in this work). On the other hand, the data presented here do not rule out the possibility that the additional unidentified proteins may be directly bound to CFTR and not to a NHERF-type scaffold. Identification of these proteins in future investigations will be important before any mechanistic model can be postulated.
In previous work from our laboratory (7) and others (27), it was found that cAMP stimulates exocytosis of apical membrane proteins at a post-Golgi step. The lack of effect of cAMP stimulation on lactase would suggest, however, that cAMP-dependent exocytosis is restricted to a subpopulation of apical membrane proteins. It has been generally accepted that cAMP operates by increasing membrane traffic (7, 27). If that is the case, the results in this work would suggest that at least two subpopulations of subapical vesicles must exist, one that carries CFTR and other proteins regulated by cAMP-dependent delivery and another cAMP-independent pathway that facilitates the transport of proteins such as lactase. Such a senario raises interesting questions regarding potentially different pathways that may regulate the formation and sorting of these two different subpopulations of vesicles.
Another alternative explanation that by no means excludes differences in vesicle traffic pathways is that cAMP may actually increase the number of binding sites available in the scaffold itself. Because the scaffold contains PKA and AKAP, it is conceivable that its binding capacity may be modulated by cAMP. In that scenario, cAMP stimulation would increase the number of surface molecules for all the membrane proteins that bind to the same scaffold, disregarding the vesicles that transport them to the cell surface. In other words, retention in the apical domain would be responsible for the increase of surface CFTR and some other proteins. In both cases, the results of this study suggest that the increase in the number of CFTR channels on the surface of intestinal epithelial cells on cAMP stimulation contributes substantially to regulating fluid secretion and is regulated by vesicle traffic. The observations in this study should provide the basis for a critical examination of membrane traffic in the pathogenesis of CFTR-mediated diseases in the intestine.
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ACKNOWLEDGEMENTS |
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We thank Dr. A. Quaroni for the generous gift of antibodies and Dr. G. McLaughlin and M. Hernandez for technical assistance.
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FOOTNOTES |
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Present address of N. A. Ameen: Pediatric Gastroenterology and Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, PA 15213.
Address for reprint requests and other correspondence: P. Salas, Dept. of Cell Biology and Anatomy, R-124, Univ. of Miami School of Medicine, PO Box 016960, Miami, FL 33101 (E-mail: psalas{at}miami.edu).
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.
10.1152/ajpcell.00261.2002
Received 11 June 2002; accepted in final form 27 August 2002.
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