STa and cGMP stimulate CFTR translocation to the surface of villus enterocytes in rat jejunum and is regulated by protein kinase G

Franca Golin-Bisello,1 Neil Bradbury,2 and Nadia Ameen1,2

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


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
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 REFERENCES
 
The cystic fibrosis transmembrane conductance regulator (CFTR) is critical to cAMP- and cGMP-activated intestinal anion secretion and the pathogenesis of secretory diarrhea. Enterotoxins released by Vibrio cholerae (cholera toxin) and Escherichia coli (heat stable enterotoxin, or STa) activate intracellular cAMP and cGMP and signal CFTR on the apical plasma membrane of small intestinal enterocytes to elicit chloride and fluid secretion. cAMP activates PKA, whereas cGMP signals a cGMP-dependent protein kinase (cGKII) to phosphorylate CFTR in the intestine. In the jejunum, cAMP also regulates CFTR and fluid secretion by insertion of CFTR from subapical vesicles to the surface of enterocytes. It is unknown whether cGMP signaling or phosphorylation regulates the insertion of CFTR associated vesicles from the cytoplasm to the surface of enterocytes. We used STa, cell-permeant cGMP, and cAMP agonists in conjunction with PKG and PKA inhibitors, respectively, in rat jejunum to examine whether 1) cGMP and cGK II regulate the translocation of CFTR to the apical membrane and its relevance to fluid secretion, and 2) PKA regulates cAMP-dependent translocation of CFTR because this intestinal segment is a primary target for toxigenic diarrhea. STa and cGMP induced a greater than fourfold increase in surface CFTR in enterocytes in association with fluid secretion that was inhibited by PKG inhibitors. cAMP agonists induced a translocation of CFTR to the cell surface of enterocytes that was prevented by PKA inhibitors. We conclude that cAMP and cGMP-dependent phosphorylation regulates fluid secretion and CFTR trafficking to the surface of enterocytes in rat jejunum.

small intestine; cystic fibrosis transmembrane conductance regulator; membrane traffic; phosphorylation


TOXIGENIC SECRETORY DIARRHEA remains the primary cause of infant death in developing countries, where it is responsible for 5 million deaths and 1 billion episodes of diarrhea each year in children <5 yr old (34). Morbidity and mortality results from massive intestinal fluid secretion that leads to dehydration, hypovolemia, and death. The proximal small intestine is the primary target for the two major infectious agents implicated in toxigenic secretory diarrhea. Escherichia coli and Vibrio cholerae elaborate enterotoxins that signal cAMP and cGMP pathways to activate the cystic fibrosis transmembrane conductance regulator (CFTR) chloride channel, and produce massive fluid and anion secretion (12, 13, 38). Enterotoxigenic strains of E. coli elaborate two classes of enterotoxins: heat-labile and heat-stable enterotoxins (STa). Heat-labile enterotoxins are homologous to cholera toxin and produce chloride secretion by elevation of intracellular cAMP. STa causes elevation of intracellular cGMP to evoke chloride secretion and diarrhea. STa is a small peptide that is the major pathogen in humans (15). Cholera toxin and cAMP agonists activate PKA-dependent phosphorylation of CFTR (14). Cholera toxin and cAMP also stimulate the recruitment of CFTR from subapical vesicles to increase the number of CFTR transporters on the apical plasma membrane of both crypt and villus enterocytes, although the precise mechanisms responsible for CFTR recruitment from subapical vesicles to the plasma membrane remain unclear (3–5).

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 (3–5). 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.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Antibodies. AME-4991 is a rabbit polyclonal antibody raised against a synthetic 13-residue peptide of the carboxy terminus of rat CFTR. The peptide was synthesized and purified by W. M. Keck Biotechnology Resource Center (New Haven, CT). Pooled anti-sera were affinity purified on a peptide column as previously described (21). The previously characterized antibody to lactase (YBB2/61) was kindly provided by Dr. Andrea Quaroni (Department of Physiology, Cornell University, Ithaca, NY) (29). The polyclonal antibody against cGKII was a gift from Dr. Hugo de Jonge (Erasmus University, Rotterdam, The Netherlands). Polyclonal antibodies raised against NKCC1 (T84) were provided by Dr. Christopher Lytle (UCSD, Riverside, CA). All fluorescent secondary antibodies were purchased from Molecular Probes (Eugene, OR). Goat anti-rabbit IgG Horseradish peroxidase was purchased from BD Biosciences (San Jose, CA).

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 (250–300 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 (250–300 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.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Localization of cGKII and CFTR in rat jejunum. Previous studies (37) in rat intestine identified cGKII, a specific intestinal brush border isoform of cGMP kinase, as the principal regulator of CFTR and cGMP-activated anion secretion. The distribution of cGKII in cryostat sections of rat jejunum is shown in Fig. 1, and is consistent with previous localization studies indicating that cGKII is preferentially expressed in the villus epithelium of rat jejunum (Fig. 1, C and D) (22).



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Fig. 1. Distribution of cGMP-dependent protein kinase II (cGKII) in rat jejunum. Cryostat sections (5 µm) of rat jejunum were double labeled to detect cGKII (green fluorescence) and F-actin (red fluorescence) using anti-cGKII antibodies and Texas red phalloidin. A: control section labeled in the absence of primary antibody. B: distribution of apical F-actin staining. C: cGKII decorates the apical domain of the villus epithelium (arrows). D: colocalization (yellow) of cGKII distribution with F-actin confirms enrichment of cGKII in the apical domain of the villus epithelium although some cGKII is detected in the crypt (double-headed arrow). E: phase-contrast image of immunolabeled section (magnification x250).

 
We confirmed that our CFTR antibody AME-4991 recapitulated the staining pattern for CFTR in rat intestinal sections similar to other well-characterized antibodies used in our previous studies (1, 2, 4). In cryostat sections of rat jejunum, the antibody AME-4991 detects very high levels of CFTR in the subpopulation of CFTR high-expresser (CHE) cells (2). Low levels of CFTR are detected on the apical domain of non-CHE villus enterocytes and CFTR is also identified in the apical domain of crypt cells (Fig. 2A).



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Fig. 2. Heat stable enterotoxin (STa) induces apical recruitment of cystic fibrosis transmembrane conductance regulator (CFTR) in the villus epithelium. Cryostat sections were double labeled to detect CFTR using AME-4991 (green fluorescence) and lactase (red fluorescence) using the antibody YBB 2/61. A: in unstimulated saline-treated jejunum, AME-4991 detects very high levels of CFTR in CFTR high-expressor (CHE) cells (long arrow), lower levels of CFTR on the apical domain of crypt cells (double-headed arrow), and lowest levels of CFTR are on the apical domain of non-CHE cells on the villus (short arrows). B: distribution of lactase in saline-treated jejunum on the apical membranes of villus enterocytes and the superficial crypts. C: co-localization of CFTR and lactase distribution in saline treated jejunum. D: high-magnification view of CFTR distribution in villus section of control saline-treated jejunum. E: distribution of CFTR 30 min after STa reveals prominent CFTR fluorescence along the apical membranes of the villus (arrows), a CHE cell is indicated (small arrow). F: distribution of lactase does not change in STa-treated jejunum. G: colocalization of lactase and CFTR in STa-treated jejunum. H: high-magnification view of CFTR distribution in villus section of STa-treated jejunum confirms recruitment to the apical membranes of villus enterocytes (arrows). I: control section labeled in the absence of primary antibody (magnification, AG; x250; H and I, x600).

 
STa stimulates recruitment of CFTR to apical membrane of villus cells in rat jejunum. We examined the distribution of CFTR in jejunal sections 30 min after STa administration, the time interval associated with maximal fluid secretion in this intestinal segment (10). Similar to our approach in previous studies, we compared changes in CFTR distribution in jejunal sections with that of lactase, an absorptive hydrolase and an integral membrane protein that is not regulated by cGMP- or cAMP-dependent vesicle trafficking, but is enriched on the apical membranes of villus epithelial cells (3, 29). The distribution of lactase and CFTR in saline and STa-treated jejunum is shown in Fig. 2. Under the same conditions of immunolabeling, labeling for CFTR on the apical domain of the villus (except for CHE cells) was barely detectable above the background in saline-treated sections, similar to our previous observations (1) under steady-state conditions (Fig. 2, A and D). Thirty minutes after STa administration, CFTR was prominent along the apical membranes of villus enterocytes (Fig. 2, E and H) compared with that of saline controls, whereas the distribution of lactase did not change (Fig. 2, B, C, F, and G). Quantification of CFTR fluorescence in cryostat sections double labeled to detect CFTR and apical F-actin revealed a fourfold increase in apical CFTR in STa-treated sections compared with saline-treated controls (Fig. 3A). The STa-induced redistribution of CFTR to the apical membrane of villus enterocytes was associated with fluid accumulation in jejunal loops (Fig. 3B).



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Fig. 3. STa induces a fourfold increase in apical CFTR fluorescence and fluid accumulation in jejunal loops. A: CFTR fluorescence intensities (expressed in pixels) were determined in cryostat sections double labeled for CFTR and apical F-actin from STa and saline-treated tissues, as described in MATERIALS AND METHODS. NS, not significant. *P < 0.001, means ± SE (n = minimum 10 sections from each tissue, 4 animals). B: fluid accumulation in jejunal loops after STa or saline. Jejunal loops (5 cm) were injected with either 400 µl of warm saline or 0.5 µM STa in the same volume saline and loop weight and length measured 30 min later. *P < 0.005, data are expressed as loop weight/length of intestine after 30 min and represents means ± SE (n = 6 experiments).

 
Because apical CFTR anion secretion in intestinal epithelia is dependent on the Na+/K+/2Cl (NKCC1) cotransporter on the basolateral membranes (6), we examined the distribution of NKCC1 in cryostat sections of rat jejunum (Fig. 4). Consistent with the described crypt-villus gradient of expression for CFTR in the small intestine (33), NKCC1 was more prominent in the basolateral membranes of crypts and its abundance decreased toward the villus tip, but it was clearly detected on the basolateral membranes of villus enterocytes, supporting the observations by others that villus cells participate in anion secretion (23, 27, 32).



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Fig. 4. Distribution of Na+-K+-2Cl cotransporter (NKCC1) and lactase in rat jejunum. Cryostat sections (5 µm) were double labeled to detect NKCC1 using the antibody T84 (green fluorescence) and apical F-actin (red fluorescence) was detected using Texas red phalloidin. A: distribution of NKCC1 on the basolateral membranes of crypt cells. B: NKCC1 is also distributed on the basolateral membranes of villus cells. C: distribution of apical F-actin staining in the absence of NKCC1 antibody. D: control section labeled in the absence of antibodies (magnification x200).

 
Surface biotinylation confirms STa-dependent insertion and increase of CFTR in apical membranes of isolated enterocytes. The morphological observations that STa induced a recruitment of CFTR to the apical domain of villus enterocytes prompted us to confirm that STa indeed stimulated trafficking of CFTR to the surface of jejunal enterocytes. Because confocal microscopy lacks the sensitivity to distinguish cell surface proteins from that in close proximity to but beneath the membrane, surface biotinylation techniques were employed to detect plasma membrane proteins in enterocytes, and biotinylated proteins were analyzed with Western blots using specific antibodies to determine a shift in the distribution of surface proteins after STa treatment. We used freshly isolated villus enterocytes in the same method as our previous study (3). With the use of this cell isolation method, we could effectively and rapidly isolate fractions of crypt and villus enterocytes while preserving enterocyte polarity. Figure 5 shows the distribution of several apical markers in a fraction of predominantly villus enterocytes that was immunolabeled to detect lactase, CFTR, and apical F-actin (Fig. 5, AE).



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Fig. 5. Apical polarity and membrane integrity are preserved in freshly isolated villus enterocytes. Fractions of freshly isolated enterocytes from rat jejunum were air dried on poly-L-lysine-coated glass slides and indirect immunofluorescence labeling performed to detect lactase (green fluorescence), F-actin (red fluorescence), and cell nuclei (blue fluorescence). A: lactase decorates the apical membranes of villus enterocytes. B: F-actin labeling of apical domain of villus enterocytes. C: co-localization of lactase and F-actin (yellow) delineates the apical domain in villus cell fraction, nuclei are stained blue. D: lactase decorates the brush border in a villus enterocyte, nucleus is indicated in blue. E: co-localization (yellow) of distribution of lactase and apical F-actin on the apical domain of an isolated villus enterocyte A-C. Magnification x200 (D) and x600 (E). F: actin is detected in Western blots of cell lysates (Lys) (10 µg protein loaded) but not surface biotinylated samples (20 µg protein) from STa or saline treated enterocytes.

 
As we showed before (3), isolated villus cells using this method maintain polarity and a well preserved apical brush border that express apical markers, such as lactase and apical F-actin. We further confirmed that the biotin reagent did not leak into the enterocytes and label intracellular proteins after isolation. Western blots of cell lysates and of samples after surface biotinylation were prepared from fractions of enterocytes and analyzed to detect the intracellular protein F-actin (Fig. 5F). Actin was detected in Western blots of total cell lysates, but not in samples of biotinylated enterocytes from the same preparation.

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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Fig. 6. STa induces trafficking of CFTR to the cell surface of villus enterocytes. Thirty minutes after STa or saline administration, jejunal enterocytes were lysed and equivalent protein loads analyzed to detect CFTR by SDS-PAGE. CFTR was detected using the antibody AME-4991. Western blots for CFTR (A) and actin (B) reveals no change in protein in cell lysates and STa administration. Enterocytes were isolated 30 min after STa, surface proteins were biotinylated and isolated on streptavidin agarose. Equivalent loads of biotinylated proteins were resolved by SDS-PAGE and analyzed by Western blot for CFTR (AME-4991) and lactase (YBB2/61). C: levels of surface biotinylated CFTR (band C) are >4-fold increased in STa-treated enterocytes over saline controls. D: levels of surface lactase remain unchanged after STa. E: surface levels of CFTR are reduced to near that of saline treated controls 4 h after STa administration. F: quantification of surface biotinylated CFTR in enterocytes after 30 min or 4 h after STa or saline. *P < 0.005, data represents means ± SE (n = 6 experiments).

 
PKA regulates cAMP-dependent translocation of CFTR and its distribution on plasma membrane. cAMP and PKA phosphorylation activates the CFTR channel on the apical membrane and PKA regulates CFTR endocytosis in CFTR over expression cell models (8, 20). However, the physiological role of PKA in regulating CFTR trafficking has not been examined. Although we previously observed a cAMP-dependent translocation and insertion of CFTR from subapical vesicles to the apical plasma membrane in the intestine (3, 4), the relevance of PKA in CFTR trafficking in enterocytes was unknown. We examined the role of PKA in cAMP-dependent translocation of CFTR in the jejunum. Surface biotinylated CFTR was determined in villus enterocytes 30 min after administration of the cAMP agonists dibutryl-cAMP and CPT-cAMP in vivo in the presence and absence of PKA inhibitors. Both dibutryl-cAMP (not shown) and CPT-cAMP stimulated an increase in surface levels of CFTR in enterocytes that was reduced by the PKA inhibitors H7 and H89 but not by the PKG inhibitor H8 (Fig. 7, A and B). Of the PKA inhibitors tested, H89 was more effective in inhibiting cAMP-dependent trafficking of CFTR to the cell surface.



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Fig. 7. PKA but not PKG regulates cAMP-stimulated traffic of CFTR to the cell surface. A: carnitine palmitoyl transferase (CPT)-cAMP induces an increase in surface expression for CFTR that is reduced by H7 and more effectively by H89. B: pretreatment with the PKG inhibitor H8 does not alter the increase of surface CFTR induced by CPT-cAMP in enterocytes. C: surface levels of CFTR in CPT-cAMP-treated enterocytes were 263 ± 18 vs. 95 ± 1.8 for saline controls. Pretreatment with H8 reduced surface CFTR to 225 ± 10 after CPTcAMP, P = 0.13, n = 4 experiments. D: fluid secretion determination in jejunum after CPT-cAMP, and pretreatment with H8 or saline as determined by [14C]polyethylene glycol (PEG) method. Pretreatment with H8 reduced CPT-cAMP-elicited fluid transport from 91 ± 9.0 to 64.2 µl·cm–1·30 min–1, n = 5 experiments, P = 0.11. Enterocytes were isolated 30 min after pretreatment with CPT-cAMP, in the presence/absence of PKA inhibitors (H89 and H7) or PKG inhibitor (H8) and biotinylated. Equivalent amounts of biotinylated plasma membrane proteins were resolved by SDS-PAGE and analyzed by Western blot analysis. CFTR was detected using AME-4991.

 
Quantification of surface levels of CFTR after administration of cAMP agonists in the presence of H8 confirmed that this PKG inhibitor did not have a significant effect on the cAMP-induced translocation of CFTR in enterocytes (Fig. 7, B and C). Mean surface levels of CFTR after CPT-cAMP were 260 ± 18 vs. 235 ± 10 (P = 0.13) in enterocytes pretreated with H8. Measurements of fluid secretion determined in jejunal loops using the [14C]PEG 4000 method similarly revealed that pretreatment with the PKG inhibitor H8 (Fig. 7D) had a minor effect in reducing the cAMP-induced fluid secretion from 91 ± 9.0 to 64.2 µl·cm–1·30 min–1 (n = 5 experiments, P = 0.11). The decrease in fluid transport and surface CFTR after pretreatment with H8 was not statistically significant. These results suggest that PKG is not important to cAMP-dependent trafficking of CFTR or fluid transport in the jejunum.

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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Fig. 8. PKG regulates cGMP-stimulated translocation of CFTR to the cell surface. A: in equivalent loads of surface biotinylated proteins from villus enterocytes, surface levels of CFTR are markedly increased over saline-treated controls 30 min after STa administration. Pretreatment with the PKG inhibitor H8 almost completely blocks the increase in surface CFTR that was induced by STa to levels near that of saline control. B: Western blots of lysates normalized for protein content reveals no change in total levels of CFTR in samples. C: similar to STa, the cGMP agonist 8-BrcGMP stimulates a robust increase in surface CFTR compared with saline controls, that is inhibited by staurosporine. D: distribution of surface biotinylated CFTR in enterocytes after STa, pretreatment with H89 or H8 and saline controls. E: surface levels of CFTR in STa-treated enterocytes were 941 ± 90, 719 ± 55 after pretreatment with H89, and 199 ± 22 after pretreatment with H8 vs. 129 ± 9 in saline controls, n = 4 experiments. *P = 0.057, difference between STa and pretreatment with H89 was not statistically significant. F: fluid secretion determination in jejunum after STa, pretreatment with H89, H8, or saline, as determined by [14C]PEG method. *P = 0.55, net fluid transport in STa-treated jejunum was 141 µl–1·cm·30 min–1 compared with 131 µl·cm–1·30 min–1 after pretreatment with H89, and 27 µl·cm–1·30 min–1 after pretreatment with H8, P = 0.002, n = 5 experiments.

 
Because the PKA inhibitor H89 was more effective than H7 in blocking cAMP induced trafficking of CFTR, we used H89 in control experiments to determine whether cross activation by PKA contributed to STa-induced traffic of CFTR. Although H89 reduced surface levels of CFTR that were increased after STa from 941 ± 90 to 719 ± 55 (P = 0.57, n = 4 experiments) the decrease was not statistically significant. Under the same conditions, however, H8 was very effective in reducing surface CFTR levels after STa down to 199 ± 22 (P < 0.005, n = 4 experiments) levels that approached those of unstimulated saline controls (129 ± 9) (Fig. 8E). The effect of pretreatment with H89 and H8 on surface levels of CFTR after STa were paralleled by a mild (but statistically insignificant) reduction in STa-elicited fluid secretion by H89 (141 vs. 131 µl·cm–1·30 min–1; P = 0.55, n = 4 experiments) but a significant reduction of fluid secretion in the presence of H8 (from 141 to 27 µl·cm–1·30 min–1; P = 0.002). These results support a major role for cGKII in regulating STa-dependent trafficking and anion transport function in the intestine.


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The importance of CFTR as the final mediator of cAMP and cGMP-activated anion secretion in the intestine is firmly established (16, 17, 31). The jejunum is a primary pathophysiological target site for enterotoxin-elicited anion secretion and is a major site of CFTR expression (10, 11, 36). Upregulation of CFTR function on the apical plasma membrane of small intestinal enterocytes underlies the pathophysiology of toxigenic diarrhea. Although CFTR-mediated anion secretion has been shown to be regulated by PKA- and cGKII-dependent phosphorylation in the intestine, whether anion secretion follows phosphorylation of CFTR that is already resident on the apical plasma membrane or is elicited after the insertion of vesicle-associated CFTR from subapical endosomes into the membrane to increase the number of transporters is unclear.

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 (3–5). 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 3–4 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.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-1K08DK02846 and Children's Hospital Pittsburgh Research Advisory Committee grants and Pennsylvania Department of Health, Tobacco Formula Funding (to N. Ameen).


    ACKNOWLEDGMENTS
 
The authors thank Drs. Dan Devor and David Perlmutter for helpful suggestions.


    FOOTNOTES
 

Address for reprint requests and other correspondence: N. Ameen, Dept. Cell Biology, Univ. of Pittsburgh School of Medicine, 3550 Terrace St., BST-S330, Pittsburgh, PA 15261 (e-mail: nadia.ameen{at}chp.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.


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 DISCUSSION
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