Department of 1Pediatrics and 2Cell Biology, University of Pittsburgh School of Medicine, Pittsburgh, Pennsylvania
Submitted 9 November 2004 ; accepted in final form 20 April 2005
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ABSTRACT |
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small intestine; cystic fibrosis transmembrane conductance regulator; membrane traffic; phosphorylation
The jejunum is a major site of STa-elicited anion and fluid secretion that is mediated by CFTR (37). STa signals cGMP-dependent pathways that result in CFTR activation and inhibition of Na+ absorption on the apical membrane of jejunal enterocytes that results in net fluid secretion (19, 37, 38). STa binds to its receptor guanylyl cyclase C (GCC) on the apical surface of enterocytes, signals intracellular cGMP and a cGMP-dependent kinase (cGKII) to phosphorylate CFTR on the apical membrane. However, cGMP can also activate CFTR by cross activation of PKA, either directly or by inhibiting a cAMP-specific phosphodiesterase. STa exhibits marked tissue specificity that parallels the distribution of GCC and the cGMP-dependent kinase cGKII. Along the proximal-distal axis of rat intestine, the highest levels of GCC and cGKII protein are found in the proximal small intestine, and cGKII is most abundant on the apical membranes of villus epithelial cells of the jejunum (22). It is unknown whether STa or cGMP regulate CFTR in villus enterocytes in the jejunum. However, CFTR is present in the apical domain of villus cells in the proximal small intestine (1, 33) and both cAMP and cholera toxin have been shown to activate chloride secretion in the villus epithelium (18, 30).
Because proteins critical to STa-elicited anion secretion are enriched in the villus epithelium of rat jejunum (NHE3, GCC, and cGKII) and CFTR is the only known substrate for cGKII (7, 19, 22, 26), we hypothesized that STa and cGMP would regulate surface levels of CFTR in villus enterocytes by movement of CFTR from a subapical location to the cell surface similar to what we observed after cAMP stimulation in rat jejunum (35). Furthermore, while the role of PKA and cGKII phosphorylation in activating CFTR on the plasma membrane has been established, the physiological role of PKA or cGKII in regulating CFTR trafficking to the cell surface has not been established. In the current study, we sought evidence that STa and cGMP regulate trafficking of CFTR to the cell surface of villus enterocytes in rat jejunum and examined its relevance to STa-elicited fluid secretion. We also examined the physiological role that PKA- and PKG-dependent phosphorylation plays in regulating both cAMP- and cGMP-dependent trafficking of CFTR to the cell surface of jejunal enterocytes in vivo.
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MATERIALS AND METHODS |
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Reagents. The following reagents were used in this study: EZ-Link Sulfo-NHS-SS Biotin, streptavidin (Pierce Biotechnology, Rockford, IL), [14C]polyethylene glycol (PEG) 4000 (Amersham Biosciences, Piscataway, NJ), thiobutabarbital (Inactin), H8, H7, 8-bromoguanosine 3',5'-cyclic monophosphate sodium, cAMP, carnitine palmitoyl transferase (CPT)-cAMP, STa (Sigma, St. Louis, MO), and H89-dihydrochloride (EMD Biosciences, San Diego, CA).
Animal studies. The study was approved by the Animal Research Committee of the University of Pittsburgh School of Medicine. Male Sprague-Dawley rats (250300 g wt, Charles River Laboratories) were fasted and anesthetized with Inactin (120 mg/kg ip) injection. The jejunum was identified, intestinal loops (5 cm length) created with ligatures, and each loop instilled with either 0.5 ml of freshly prepared STa (0.5 µM) or saline. The abdomen was closed, the animal was kept warm, and the jejunal loops were examined for fluid accumulation 30 min later, with the use of a method described in our previous study (3). To examine the role of PKA or cGKII, protein kinase inhibitors H8 (10 µM), H89 (10 µM), H7 (50 µM), and staurosporine (1 µM) were administered in the presence or absence of STa (0.5 µM), dibutryl-cAMP (1 mM), CPT-cAMP (1 mM), or 8-bromo-cGMP (8-BrcGMP) (0.2 mM). To examine the effects of STa reversal on CFTR traffic, jejunal loops were treated with STa, the abdomen was closed, and the loops were reexamined 4 h after STa administration. At the end of the observation period, the animals were euthanized by administration of intraperitoneal injection of Inactin (200 mg/kg).
Fluid secretion studies. For measurement of fluid accumulation in a closed-loop model, overnight fasted rats (250300 g) were anesthetized, and intestinal loops (5 cm) were isolated, ligatures placed, and loops injected with either saline alone or STa toxin prepared in saline (400 µl). Loop weight and length were measured after removal of mesentery and connective tissues as described (35) and fluid accumulation expressed as loop weight/length of intestine after the 30-min period. In vivo measurements of fluid secretion was also determined using [14C]PEG 4000 (1 µCi/ml). For these studies, the animals were prepared as above, the intestinal contents were removed through a small distal incision by gently rinsing the lumen with prewarmed isotonic saline, and residual saline was removed. [14C]PEG 4000(1 µCi/ml) was added to the agonist or saline and administered (0.5 ml) into loops for 30 min. Samples (20 µl) were taken after 5 min and at the end of the study period and analyzed in a scintillation counter as described previously (39). The amount of fluid absorbed/secreted in the 30-min period was determined from the [14C]PEG 4000 concentration difference between 5 and 30 min and the volume of fluid originally present.
Immunocytochemistry and confocal microscopy.
Intestinal tissues and isolated cells were prepared for immunofluorescence localization studies as described (1, 3, 4). Labeled sections and cells were examined on a Nikon Microphot FXL epifluorescent microscope equipped with Olympus digital camera. Confocal microscopy and image analysis was performed using a confocal microscope (model 510 Meta; Zeiss, Jena, Germany) with a x63, 1.4 numerical aperture oil-immersion objective. Images were collected at fixed pinhole, laser power, and PMT settings. Analysis of apical fluorescence intensities was performed using Metamorph (UIC, Downingtown, PA) and performed as described (3). In brief, the apical domain of CFTR in immunolabeled sections was determined from images of perpendicular parallel sections double-labeled to detect CFTR and phalloidin, and fluorescence intensity (in pixels) determined 1.5 µm from the luminal surface. The CFTR signal below that depth was considered the subapical compartment. Parameter acquisition was adjusted with the software so that the pixel intensity of the brightest fluorescence was not saturated. Data were collected from an average of 30 cells in random sections double labeled for CFTR or lactase and F-actin (average 10 sections) from each tissue (4 animals) examined and is expressed as the ratio of apical to subapical fluorescence intensity.
Isolation of intestinal enterocytes, surface biotinylation, and Western blot analysis. Jejunal segments were everted and threaded onto a glass spiral and vibrated at 50 Hz in an ice-cold isolation buffer (in mM/l) 112 NaCl, 30 Na2-EDTA, 0.5 DTT, 20 HEPES-Tris, pH 1) at timed intervals to release the villus and crypt enterocytes as described previously (3, 23). After isolation, the villus fractions of enterocytes were washed in PBS with calcium and magnesium and labeled with Sulpho NHS-SS-Biotin (Pierce Biotechnology, Rockford, IL) as before (3). After biotinylation, the cells were lysed with TGH buffer (25 mM HEPES, 10% glycerol, and 1% Triton X-100, pH 7.4), normalized for protein content, and biotinylated proteins were bound to Immunopure Immobilized Streptavidin Agarose (Pierce Biotechnology). The biotinylated apical membrane proteins CFTR and lactase were analyzed by SDS-PAGE and detected using goat anti-rabbit (1:10,000) or anti-mouse (1:8,000) peroxidase secondary antibodies (Sigma). Membranes were exposed to chemiluminescence and preflashed film (Hyperfilm ECL, Amersham Pharmacia) and protein bands were quantified with the use of a Bio-Rad Fluor S-Multi-imager and Quantity One Image Analysis Software.
Statistical analysis. Data are expressed as means ± SE. The significance of differences in mean values was determined by the two-tailed Student's t-test. P < 0.05 was considered significantly different.
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RESULTS |
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Western blots of lysates normalized for protein content confirmed that treatment with STa for 30 min did not alter protein levels of CFTR or actin in villus enterocytes (Fig. 6, A and B). The distribution of surface-biotinylated CFTR in enterocytes was examined in relation to the apical membrane protein lactase. Thirty minutes after STa treatment, surface levels of CFTR are increased greater than fourfold over saline controls (Fig. 6, C and F), whereas surface levels of lactase remain unchanged (Fig. 6D). To determine whether the STa-induced trafficking of CFTR to the plasma membrane was reversible, we treated jejunal loops with saline or STa for 30 min, then examined the loops 4 h later (the time interval associated with complete reversal of STa-elicted fluid secretion) (10). There was no evidence of fluid accumulation by visual examination. The mean fluid accumulation [in wt (g)/cm] in STa-treated loops 4 h after administration was 0.13 ± 0.004 compared with 0.11 ± 0.001 (n = 7 loops from each condition, 4 animals examined) and after saline treatment. The difference between the two was not statistically significant. We isolated enterocytes from the STa and saline treated segments and analyzed surface levels of CFTR by biotinylation. Four hours after STa treatment, surface levels of CFTR were reduced to near that of control saline treated enterocytes (Fig. 6E). Surface levels of CFTR in enterocytes that were exposed to STa was 147 ± 7.2 compared with 122.5 ± 14.5 signal intensity in pixel value (Fig. 6F) for saline-treated enterocytes (n = 6 experiments). The difference between the two was not statistically significant.
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cGKII regulates STa and cGMP-dependent translocation of CFTR and its distribution on plasma membrane of villus enterocytes. Because H8 did not prevent the cAMP-induced trafficking of CFTR to the cell surface, we sought to examine the role of cGKII on cGMP-induced trafficking of CFTR to the cell surface of villus enterocytes. Both H8 and staurosporine have been shown to specifically inhibit cGKII in brush border membranes from rat intestine (37). We therefore examined surface levels of CFTR in enterocytes 30 min after STa or cell-permeant cGMP agonists in the presence and absence of H8 and staurosporine. STa and 8-BrcGMP induced a prompt increase in surface levels of CFTR in enterocytes 30 min after agonist treatment. Both H8 and staurosporine effectively reduced surface levels of CFTR that were elevated after STa or the cGMP agonist 8-BrcGMP (Fig. 8, A, C, and E). In these experiments, H8 reduced surface levels of CFTR after STa from 941 ± 90 to 199 ± 22 (P < 0.005) and staurosporine reduced surface levels of CFTR induced by 8-BrcGMP from 1,008 ± 63 to 302 ± 35 (P < 0.005, n = 4 experiments). Western blots of lysates prepared from isolated enterocytes and normalized for protein content confirmed that total levels of CFTR in samples remained unchanged (Fig. 8B). We also confirmed that treatment with the cell-permeant cGMP agonist 8-CPT-cGMP (0.2 mM) increased surface levels of CFTR as we observed for 8-BrcGMP (data not shown) and pretreatment with H8 also reduced surface levels of CFTR after 8-CPT-cGMP treatment.
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DISCUSSION |
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Because at least 50% of CFTR in native jejunal enterocytes reside in subapical vesicles in the cytoplasm (5), and cholera toxin and other cAMP agonists stimulate trafficking and insertion of CFTR from subapical vesicles to the plasma membrane of crypt and villus enterocytes in rat jejunum in association with anion secretion, regulated traffic of CFTR to the plasma membrane is likely to be an important contributor to cAMP-activated anion secretion in the intestine (35). However, the physiological role that PKA plays in regulating cAMP-dependent trafficking of CFTR to the plasma membrane of native enterocytes and its relevance to intestinal fluid transport was not known.
The observation that CFTR is present on the apical domain of villus enterocytes in rat jejunum in the same location as the STa receptor, GCC and cGKII, prompted us to examine (1) the role that cGMP and cGKII may play in regulating CFTR trafficking to the cell surface of villus enterocytes and (2) its relationship to fluid transport in the jejunum. We confirmed previous observations of cGKII localization in rat jejunum that this cGMP-specific kinase, critical to CFTR activation in the jejunum, is primarily found on the apical domain of villus epithelial cells, with low abundance in the crypt (22). Our CFTR antibody AME-4991 reliably reproduced the immunofluorescence staining pattern and the biochemical profile of CFTR by Western blots and surface biotinylation assays from rat intestinal tissues similar to other well-characterized antibodies (R3194, R3195) used in our previous studies (1, 3).
Because STa elicits maximal fluid secretion in rat jejunum within 30 min (10), we examined changes in the subcellular distribution of CFTR in enterocytes at this time interval to seek evidence of its regulation by trafficking because CFTR biosynthesis is not assured before 34 h in the intestine (40). STa induced a rapid and specific translocation of CFTR in villus enterocytes that was associated with fluid secretion. The fourfold increase in apical CFTR fluorescence in tissue sections after STa stimulation was consistent with changes in surface levels of CFTR, as detected by biotinylation. Similar to our previous approach, we confirmed the specificity of CFTR trafficking by comparing changes in CFTR distribution with lactase, an apical membrane hydrolase that is enriched in villus cells of the small intestine and is not regulated by membrane traffic (24, 25). The increase in surface levels of CFTR and fluid accumulation that we observed 30 min after STa were reduced to near that of saline-treated controls at 4 h, when STa-elicited fluid secretion in rat jejunum is reversed (9, 10). These observations support an increase in the number of CFTR transporters on the surface of enterocytes as a basis for STa-elicited fluid secretion in the jejunum.
Because STa and cGMP can also activate CFTR by PKA, we examined changes in surface levels of CFTR after cAMP agonists in the presence of both PKA and PKG inhibitors. Consistent with our previous observations, cAMP stimulated the translocation of CFTR to the cell surface of enterocytes (3). The PKA inhibitors H7 and H89 inhibited the cAMP-stimulated translocation of CFTR to the cell surface, although H89 was more effective than H7. While cAMP has been shown to regulate cell surface expression of CFTR in vitro, to our knowledge, the observations that PKA inhibition reversed the cAMP-induced redistribution of CFTR to the cell surface in jejunal enterocytes provides the first convincing evidence that PKA phosphorylation of CFTR regulates the distribution of CFTR in endosomal pools and that on the cell surface in vivo.
The observation that the PKG inhibitor H8 did not interfere with cAMP-induced translocation of CFTR to the cell surface nor with cAMP activated fluid secretion in vivo suggests that cGKII does not play a major role in regulating either cAMP-dependent traffic in villus enterocytes or fluid transport. Our observations are consistent with ion transport studies suggesting an insignificant contribution of cGMP cross-activation of PKA-dependent stimulation of the short circuit current in rat jejunum (37). However, the fact that H8 and staurosporine effectively inhibited both the STa-dependent translocation of CFTR in enterocytes and fluid secretion supports the notion that cGKII plays an important role in regulating cGMP-dependent translocation of CFTR and STa-dependent anion secretion in rat jejunum (37). The fact that H8 also blocked CFTR translocation in the presence of the cell-permeant agonist CPT-cGMP suggests that CFTR translocation was not due to cross activation of PKA by cGMP because this agonist directly activates cGMP and does not inhibit a cAMP-activated PDE. Although the specificity of H8 for inhibiting cGKII in the native intestine has been demonstrated, examination of intestinal tissues from cGKII knock out mice would prove useful to confirm a specific role for cGKII in regulating STa-dependent CFTR traffic (28). These studies are the first to our knowledge to demonstrate a role for cGMP and cGKII in regulating CFTR traffic and its relevance to fluid secretion and the first confirmation that PKA regulates cAMP-dependent translocation of CFTR to the apical membrane in native epithelial tissues.
The results of these studies provide a new paradigm for understanding the pathogenesis of toxigenic secretory diarrhea, and the critical role that cyclic nucleotide dependent trafficking of CFTR plays in this important global disease. We demonstrated for the first time an important role for STa and cGMP in regulated trafficking of CFTR to the cell surface, and confirmed a critical role for PKA and cGKII phosphorylation in regulating CFTR trafficking physiologically. The observation that cyclic nucleotide regulated trafficking of CFTR occurs in all CFTR expressing cells in the jejunum confirms that certainly in the intestine regulated trafficking of CFTR is not cell type specific. Our observations on CFTR trafficking in the native intestine would suggest that compounds that target cyclic nucleotide-dependent trafficking and insertion of CFTR to the cell surface are likely to prove effective in the treatment of toxigenic secretory diarrhea.
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GRANTS |
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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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.
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