Activation of CFTR by ASBT-mediated bile salt absorption

Marcel J. C. Bijvelds,1 Huub Jorna,1 Henkjan J. Verkade,3 Alice G. M. Bot,1 Franz Hofmann,4 Luis B. Agellon,5 Maarten Sinaasappel,2 and Hugo R. de Jonge1

1Department of Biochemistry and 2Sophia Children's Hospital, Erasmus MC, Rotterdam; 3Department of Pediatrics, University Medical Center, Groningen, The Netherlands; 4Institut für Pharmakologie und Toxikologie, Technische Universität München, München, Germany; and 5Canadian Institutes of Health Research Group in Molecular and Cell Biology of Lipids and Department of Biochemistry, University of Alberta, Edmonton, Alberta, Canada

Submitted 18 May 2005 ; accepted in final form 20 July 2005


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In cholangiocytes, bile salt (BS) uptake via the apical sodium-dependent bile acid transporter (ASBT) may evoke ductular flow by enhancing cAMP-mediated signaling to the cystic fibrosis transmembrane conductance regulator (CFTR) anion channel. We considered that ASBT-mediated BS uptake in the distal ileum might also modulate intestinal fluid secretion. Taurocholate (TC) induced a biphasic rise in the short circuit current across ileal tissue, reflecting transepithelial electrogenic ion transport. This response was sensitive to bumetanide and largely abrogated in Cftr-null mice, indicating that it predominantly reflects CFTR-mediated Cl secretion. The residual response in Cftr-null mice could be attributed to electrogenic ASBT activity, as it matched the TC-coupled absorptive Na+ flux. TC-evoked Cl secretion required ASBT-mediated TC uptake, because it was blocked by a selective ASBT inhibitor and was restricted to the distal ileum. Suppression of neurotransmitter or prostaglandin release, blocking of the histamine H1 receptor, or pretreatment with 5-hydroxytryptamine did not abrogate the TC response, suggesting that neurocrine or immune mediators of Cl secretion are not involved. Responses to TC were retained after carbachol treatment and after permeabilization of the basolateral membrane with nystatin, indicating that BS modulate CFTR channel gating rather than the driving force for Cl exit. TC-induced Cl secretion was maintained in cGMP-dependent protein kinase II-deficient mice and only partially inhibited by the cAMP-dependent protein kinase inhibitor H89, suggesting a mechanism of CFTR activation different from cAMP or cGMP signaling. We conclude that active BS absorption in the ileum triggers CFTR activation and, consequently, local salt and water secretion, which may serve to prevent intestinal obstruction in the postprandial state.

chloride channel; cystic fibrosis; intestinal fluid transport; intestinal obstruction; signal transduction


CYSTIC FIBROSIS (CF) is caused by mutations in the CF transmembrane conductance regulator (CFTR) gene coding for a cAMP/ATP-regulated anion channel permeable to Cl and HCO3. CFTR mediates electrolyte and fluid transport in secretory epithelia in the respiratory system, pancreas, sweat glands, and intestine. In the liver, CFTR is expressed in the cholangiocytes lining the bile ducts and seems involved in bile production (10, 21). When CFTR function is compromised, dilatation and plugging of bile ducts may occur, suggesting that CF-related liver disease initiates from an interruption of bile flow and/or changes in bile composition (13, 47).

In the rat, CFTR is expressed only in the cholangiocytes of larger bile ducts (3). It was shown that tauro(litho)cholate induced a secretory response in these CFTR-carrying cells but not in cholangiocytes from smaller ducts, and it was proposed that this response is triggered by uptake of bile salts (BS) via the apical sodium-dependent bile acid transporter (ASBT), which colocalizes with CFTR in the apical membrane of these cells (2, 3). ASBT-mediated uptake can initiate cholehepatic cycling of conjugated BS (1). According to this model, BS that pass through the ductular epithelium would be shuttled back to the sinusoids for (renewed) uptake, processing, and secretion by hepatocytes (29). This recycling would not only stimulate BS-dependent canalicular flow but may also induce ductular fluid secretion by activation of CFTR. Because CFTR is known to conduct HCO3 and modulate the activity of other ion transporters, including Cl/HCO3 exchangers, this cholehepatic shunt may contribute to the regulation of bile pH and electrolyte balance, constituting a mechanism for tuning bile flow and composition.

As in cholangiocytes, ASBT and CFTR colocalize in the apical membrane of villus cells lining the distal ileum. Here, ASBT fulfils an important function in the salvage of BS, and a defect in ileal BS absorption may underlie the high BS loss observed in CF (22, 40). CFTR constitutes the major Cl conductance pathway in the apical membrane of crypt cells, required for intestinal electrolyte and fluid secretion. In this capacity, it is essential for passage of the intestinal contents, as meconium ileus (MI), a life-threatening obstruction in newborns, and distal intestinal obstruction syndrome (DIOS), in later life, are frequent causes of morbidity in CF (20). Obstructions are thought to occur because CFTR dysfunction dramatically reduces the capacity for intestinal fluid secretion and, in addition, may alter mucin production (48). The resulting dehydration of the epithelium and increase in the viscosity of mucus are considered key factors leading to impaction of intestinal contents.

Considering the interplay between ASBT and CFTR in bile ducts and its probable consequences for CF pathology, we speculated that a similar connection might exist between CFTR and ASBT in the intestine. Indeed, some evidence suggests that uptake of BS in the ileum, as part of their enterohepatic cycling, triggers ion and fluid transport. In the rat ileum, it was shown that luminal taurocholate (TC) increased the transmural potential difference (PD) (54). This rise correlated with the magnitude of BS transport and was absent from jejunal sections expressing CFTR but not ASBT, suggesting that active, cellular uptake of BS is critical for this response. The authors proposed that the rise in PD reflected a net cation influx via an electrogenic BS transport mechanism, as, indeed, is predicted by the 2Na+:1BS stoichiometry of ASBT-mediated BS uptake (57). However, BS-induced activation of a secretory pathway may also have contributed to the increase in transmural PD observed (55). To investigate this possibility, we measured the TC-induced electrical responses in the intestine of mice with a genetically induced defect in CFTR function and in wild-type (WT) littermates. We show that the ileal response is blunted in CF mice compared with control mice and that, in the total absence of CFTR, the residual response corresponds with electrogenic 2Na+:1TC cotransport. The response to TC was restricted to the distal ileum and abolished upon inhibition of ASBT activity, suggesting that ASBT-mediated BS uptake is a prerequisite for CFTR activation.


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Animals. Homozygous F508del mice (Cftrtm1Eur, FVB; 129/Sv) (19), Cftr-null mice (Cftrtm1Cam, C57BL/6; 129/Sv) (43), and WT littermates were reared in an environmentally controlled facility at Erasmus MC (Rotterdam, The Netherlands). cGMP-dependent protein kinase type II (cGKII)-null mice (129/Sv) and control littermates were maintained at the animal facility of Technische Universität (Munich, Germany). Animals had free access to water and food. All experiments were performed on animals 12–24 wk of age. Experimental protocols were approved by the Ethical Committee for Animal Experiments of Erasmus MC.

BS-induced bioelectrics and fluxes in the intestine. Tissue from the small intestine was collected from mice anesthetized with hypnorm (1 µl/g) and Diazepam (10 µg/g). The intestine was flushed with cold saline, and segments of ~1 cm in length were excised from the distal ileum and jejunum (1 and 15 cm proximal to the cecum, respectively). After blunt dissection of the muscle layers, mucosal tissue sheets were mounted in an Ussing chamber, and tissue was bathed in modified Meyler solution [composed of (in mmol/l) 128 NaCl, 4.7 KCl, 1.3 CaCl2, 1.0 MgCl2, 0.3 Na2HPO4, 0.4 NaH2PO4, 20 NaHCO3, 10 4-(2-hydroxyethyl)piperazine-1-ethanesulfonic acid] gassed with humidified 95% O2-5% CO2 at pH 7.3 at 37°C. The serosal bathing solution was supplemented with glucose (10 mmol/l) and indomethacin (20 µmol/l). The transepithelial PD was clamped at 0 mV using a DVC-1000 Dual Voltage Clamp (World Precision Instruments; Sarasota, FL). The resulting short circuit current (Isc), reflecting transepithelial electrogenic ion transport, was digitized (Digidata 1320, Axon Instruments; Foster City, CA) and recorded using Axoscope software (Axon Instruments). BS were added from stock solutions made in water (taurocholate) or ethanol [chenodeoxycholate (CDC) and tauro(urso)deoxycholate]. A transepithelial NaCl gradient was generated by replacing NaCl in the serosal bathing medium isosmotically with mannitol. Nystatin was added to the serosal medium from freshly made stocks at 0.36 g/l.

For assessing BS fluxes, tauro[carbonyl-14C]cholate or [carbonyl-14C]chenodeoxycholate was added to the mucosal bathing solution (7 kBq/1.8 ml), and samples were collected for up to 2 h from both half-chambers. Radioactivity was quantified by liquid scintillation counting. Unidirectional absorptive BS fluxes were calculated from an essentially linear portion of the uptake curve between 15 and 60 min after the introduction of BS.

Preparation of epithelial cell homogenates. Epithelial cell homogenates were prepared from three contiguous 3-cm segments of the most distal part of the small intestine (18). Briefly, everted segments were first vibrated for 2 min in isotonic saline to discard adhering luminal content, and, subsequently, epithelial cells were collected by vibration for 30 min in saline containing 5 mmol/l EDTA. Cells were washed twice in isotonic mannitol solution, suspended in the presence of a protease inhibitor cocktail (Complete, Boehringer; Mannheim, Germany), and snap frozen in liquid nitrogen. After samples were slow thawed, the cell homogenate was sonicated (10 s), and protein concentration was determined with a commercial reagent kit (Bio-Rad; Hercules, CA) using BSA as a reference. Samples were heated to 95°C for 10 min in denaturing sample buffer before being stored at –70°C.

Protein immunoblotting. Cell homogenate proteins were separated on 10% polyacrylamide gels (SDS-PAGE). Gels were run with equal amounts of protein per lane, as was ascertained by Coomassie staining of gels run in parallel (not shown). After protein was transferred to nitrocellulose membranes, the membranes were blocked with nonfat milk powder and then probed with a polyclonal antibody raised against hamster ASBT (49). Membranes were washed, incubated with secondary antibody (donkey anti-rabbit IgG horseradish peroxidase conjugate, Amersham Biosciences; Freiburg, Germany), and washed again (all steps without milk powder added). Peroxidase activity was detected with a chemiluminescence reagent (Pierce; Rockford, IL).

Immunohistochemistry. Paraformaldehyde-fixed sections (5 µm) from ileal and jejunal tissue (excised 3 and 15 cm proximal to the cecum, respectively) were embedded in paraffin. Sections were probed with ASBT antibody (see above) or a polyclonal antibody reactive against rodent CFTR (24), followed by an anti-rabbit IgG horseradish peroxidase- or fluorescein-conjugated secondary antibody, respectively. Peroxidase-labeled immune complexes were visualized by incubation with 3,3'-diaminobenzidine tetrahydrochloride, and these sections were counterstained with hematoxylin. For visualization of the fluorescein label, sections were embedded in Vectashield (Vector Laboratories; Burlingame, CA).

Materials. Tauro[carbonyl-14C]cholate and [carbonyl-14C]chenodeoxycholate were purchased from Amersham (Little Chalfont, UK) and NEN Life Science Products (Boston, MA), respectively. The dimeric BS analog S910960 (CAS 142974-51-4) was obtained from Aventis Pharma Deutschland (Frankfurt am Main, Germany). Analytical grade inorganic salts, mannitol, and pH-buffering substances were purchased from Merck (Darmstadt, Germany), whereas carbachol came from its subsidiary, Calbiochem. All other chemicals were obtained from Sigma (St. Louis, MO) except if explicitly stated otherwise.

Statistical analysis. Data are presented as means ± SE. Statistical significance of differences between means was assessed using Student's t-test (two-sided, paired when contiguous tissue sections were compared) and accepted at a value of P < 0.05.


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ASBT protein expression. ASBT protein in the distal intestine was detected by protein immunoblotting (Fig. 1A). Two immunoreactive bands, at ~90 and ~45 kDa, represent the dimeric and monomeric protein, respectively. Immunostaining was present only in the most distal part of the mouse ileum. Immunohistochemistry demonstrated that ASBT was present at the apical pole of epithelial cells along the length of the entire villus (Fig. 1B). No immunostaining was found in the crypts of Lieberkuhn, nor was staining observed in jejunal preparations (Fig. 1C), underlining the discrete ileal expression of ASBT and the immunospecificity of the antibody. CFTR immunostaining in ileal sections was detected most prominently along the apical membrane of epithelial cells in the crypts. At a higher antibody titer, staining also became evident at the apical pole of villus cells, indicating the presence of low levels of CFTR at the luminal surface of these cells (Fig. 1D).



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Fig. 1. Localization of the apical sodium-dependent bile acid transporter (ASBT) and cystic fibrosis transmembrane conductance regulator (CFTR) in the distal mouse intestine. A: protein immunoblot probed with ASBT antibody showing localization of ASBT along the longitudinal axis of the distal intestine of Cftr+/+ (C57BL/6, 129/Sv) mice. Pooled cell homogenates from 3 animals prepared from consecutive 3-cm segments of the distal small intestine were applied in lanes P, M, and D (proximal to distal; 20 µg protein/lane). B: ASBT immunostaining in histological sections from the distal ileum of Cftr+/+ mice and mutants. Staining is present at the apical pole of epithelial cells in the villus (solid arrowheads) but not in the crypts of Lieberkuhn (open arrowhead). C: ASBT immunostaining was absent from jejunal sections. D: probing with distinct antibody titers revealed that CFTR immunostaining in Cftr+/+ mice is most prominent at the apical pole of ileal epithelial cells in the crypts but that CFTR expression extends to the villus cells (arrowheads). E: CFTR immunostaining was absent from tissue sections of Cftr-null mice. Original magnification: x400.

 
Ileal electric responses to TC are CFTR dependent. The addition of TC to the mucosal bathing solution evoked a dose-dependent electrical response across the murine distal ileum (Fig. 2). Responses had a biphasic character, starting with a minor but sharp increase in Isc immediately after the TC addition, followed by a more gradual rise, reaching a plateau within a few minutes. The response was markedly blunted in both Cftr-null mice and in homozygous F508del mice. Because 1) the uptake of conjugated trihydroxy BS, such as TC, is mediated principally by ASBT, and 2) these CF mice display enhanced fecal BS excretion (6), we hypothesized that this attenuated response in CF mice reflects reduced activity of electrogenic ASBT-mediated TC uptake. To test this assumption, we assessed ASBT activity by measuring the mucosa to serosa (absorptive) flux of radioactively labeled TC across the distal ileum in parallel with Isc measurements. This showed that in all groups, except for the Cftr-null group, the electric response was substantially larger than would be predicted by ASBT-mediated TC uptake alone (Fig. 3). Thus luminal exposure to TC induces a CFTR-dependent ion conductance that cannot be attributed to electrogenic ASBT activity. TC fluxes of homozygous F508del mice and WT littermates were similar, whereas a considerable (~17%; Fig. 3) decrease in TC uptake was observed in null mice compared with WT littermates. CDC was absorbed much less efficiently in the distal ileum than TC. In Cftr+/+ mice (C57BL/6, 129/Sv), the CDC flux, tested at the same concentration of 0.5 mmol/l, amounted to 12% of the TC flux (0.051 ± 0.003 vs. 0.44 ± 0.02 µmol·cm–2·h–1, N = 6, P < 0.001). In Cftr-null mice, the CDC flux was 0.043 ± 0.003 µmol·cm–2·h–1, which was not significantly different from controls (P = 0.12).



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Fig. 2. Bile salt (BS)-induced electrogenic ion transport across the distal ileum of CF mice and wild-type (WT) littermates. A: traces showing the increase in short circuit current (Isc) across the distal ileum induced by the addition of taurocholate (TC; 0.5 mmol/l) to the mucosal bathing solution typical for Cftr–/– and Cftr+/+ mice. The arrow indicates a slight deflection in the rise in Isc, which immediately follows after the initial sharp increase in Isc. B: the TC-induced rise in Isc was dose dependent in homozygous F508del ({Delta}/{Delta}) and Cftr-null mice and Cftr+/+ littermates. Statistically significant difference compared with the Cftr+/+ group: *P < 0.05 and **P < 0.01. N = 6 mice/group.

 


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Fig. 3. Comparison of unidirectional absorptive TC flux and TC-induced electrogenic ion transport across the distal ileum of CF mice and WT littermates. Mass transport and electric responses (hatched bars) were measured simultaneously after luminal TC exposure (0.5 mmol/l). *P < 0.05 and #P < 0.05, Cftr–/– vs. Cftr+/+. NS, no statistically significant difference. N = 6 mice/group.

 
BS-evoked Cl secretion depends on ASBT activity. In the ileum, S910960, a dimeric BS analog that blocks the transporter site of ASBT (34), reduced the electrical response by 95 ± 4% (N = 4, P < 0.001; Fig. 4A). S910960 did not affect the response elicited by forskolin (Fig. 4B), indicating that this BS analog did not exert an inhibitory effect on anion secretion in general but specifically inhibited the TC-induced CFTR-dependent secretory response by preventing uptake of BS via ASBT. ASBT substrates like taurine-conjugated deoxycholate and ursodeoxycholate induced similar biphasic rises in Isc as TC (Fig. 4D). These responses were sensitive to bumetanide, a selective inhibitor of Na+,K+,2Cl cotransporter type 1 (NKCC1), which generates the driving force needed for Cl secretion (Fig. 4C). Bumetanide (50 µmol/l) reduced a TC-induced rise in Isc by 51 ± 4% (N = 7, P < 0.001). Bumetanide was significantly (P < 0.05) more effective in inhibiting the forskolin-induced response (67 ± 6%, N = 7), reinforcing the notion that the TC response contains a bumetanide-insensitive nonsecretory component, i.e., electrogenic ASBT activity. The unconjugated dihydroxy bile acid CDC, which is a poor ASBT substrate (see Ileal electric responses to TC are CFTR dependent), induced a comparatively minor response (Fig. 4D). In the jejunum of Cftr+/+ (C57BL/6, 129/Sv) mice, the mucosa to serosa TC flux was 19-fold lower than that in the distal ileum, underlining the significance of ASBT for TC uptake (0.023 ± 0.006 µmol·cm–2·h–1, N = 3, vs. 0.44 ± 0.02 µmol·cm–2·h–1, N = 6, P < 0.001). Importantly, luminal TC did not evoke an electric response across the jejunal epithelium despite the fact that CFTR is abundantly expressed in jejunal enterocytes and can be activated by a forskolin-induced rise in cAMP levels (Fig. 5). The proximal colon was equally unresponsive (not shown).



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Fig. 4. ASBT-mediated BS absorption in the distal ileum triggers a secretory response in Cftr+/+ mice. A and B: traces representative of 4 similar experiments showing that inhibition of ASBT-mediated BS uptake by S910960 (0.1 mmol/l) abrogates the response to luminal TC (0.5 mmol/l) exposure (A) but does not block the response after a forskolin-evoked increase in cellular cAMP (B). C: trace representative of 7 similar experiments showing attenuation of the TC-evoked response by bumetanide (0.05 mmol/l). D: peak changes in Isc induced by luminal exposure (0.5 mmol/l) to taurodeoxycholate (TDC), tauro(urso)deoxycholate (TUDC), and chenodeoxycholate (CDC). Numbers of observations are shown inside the bars.

 


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Fig. 5. Luminal TC exposure does not induce electrogenic ion transport in the jejunum of Cftr+/+ mice. A: representative trace showing that forskolin (0.01 mmol/l), by increasing cellular cAMP, but not TC (0.5 mmol/l), induces a secretory response. B: peak changes in Isc elicited by TC and forskolin. N = 6 mice/group.

 
BS-evoked Cl secretion in the ileum and colon proceeds through distinct mechanisms. For the mouse colon, it has been shown that CDC, but not the more hydrophilic ursodeoxycholate, triggers a secretory response (26). In Ussing chamber experiments, it was shown that serosal CDC exposure was most effective and that this response was triggered by histamine release from mast cells in the submucosal layers. We reasoned that in the mouse ileum, BS might activate Cl secretion via a similar mechanism and that serosal administration would therefore increase their efficacy. Contrary to our supposition, serosal administration of TC (0.5 mmol/l) elicited only a weak secretory response in Cftr+/+ (FVB, 129/Sv) mice (12.8 ± 4.7 vs. 50.9 ± 9.3 µA/cm2 for serosal and mucosal exposure, respectively, N = 5, P < 0.01). This suggested to us that 1) BS entering the epithelial cell via ASBT directly activate a secretory pathway or 2) a putative accumulation of BS in subepithelial tissue triggers the release of endogenous secretagogues. Therefore, the involvement of neurocrine and immune mediators of Cl secretion was investigated. Blockade of neurotransmitter release from enteric nerve endings with tetrodotoxin (1 µmol/l) and blockade of prostaglandin synthesis with indomethacin (added routinely in our assays) and the lipoxygenase inhibitor nordihydroguaiaretic acid (NDGA; 20 µmol/l) did not affect the efficacy of TC (not shown). In addition, we tested for the involvement of the paracrine mediators histamine and 5-hydroxytryptamine. 5-Hydroxytryptamine induced a gradually declining secretory response but did not desensitize the epithelium to a subsequent TC challenge (Fig. 6A). Responsiveness to histamine abruptly declined in the distal intestine. In the section 4–5 cm proximal to the cecum, histamine evoked a robust transient response, similar in character to the response to carbachol and consistent with activation of H1 receptors (14, 25, 56). However, in the most distal part of the intestine, routinely used for our measurements, histamine had little effect on Isc, whereas the responsiveness to carbachol and TC were retained (Fig. 6B). Pretreatment with pyrilamine, a blocker of the histamine H1 receptor, while decreasing the basal current, did not abrogate the response to TC (Fig. 6C). Collectively, these findings argue against a principal effect of TC on paracrine mediators of Cl secretion.



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Fig. 6. Role of paracrine mediators in BS-evoked Cl secretion in the distal ileum of Cftr+/+ mice. A: to test the effect of pretreatment with 5-hydroxytryptamine (5-HT; 0.05 mmol/l) on the TC-induced rise in Isc, contiguous tissue segments were compared. B: histamine did not evoke a response in the ileal segment 1 cm proximal to the cecum. C: pyrilamine (0.1 mmol/l) reduced basal Isc across the distal ileum but did not abrogate the responses to TC or carbachol. The traces shown are representative of experiments performed on tissue from at least 3 different animals.

 
ASBT-mediated BS uptake triggers CFTR activation. Cl secretion does not solely depend on activation of apical Cl channels such as CFTR but also relies on several basolaterally situated ion transport systems, i.e., NKCC1, various classes of K+ channels, and Na+,K+-ATPase. The concerted action of these ion transport systems generates the driving force for downhill Cl secretion (5). To assess whether BS target CFTR or one of these basolateral transport systems, we applied the ionophore nystatin to the serosal bathing medium to render the basolateral membrane permeable to monovalent ions. When tissue was bathed in Meyler solution, nystatin abolished the TC-evoked secretory response, underlining the role of basolateral ion transport in maintaining a high intracellular Cl potential and confirming that nystatin was effective in permeabilizing the basolateral membrane of epithelial cells (Fig. 7A). Next, a transepithelial NaCl gradient was applied, providing a driving force for both Na+-driven BS uptake and Cl influx across nystatin-treated cells. Imposing this gradient led to an increase in Isc, attributable to paracellular, diffusive Na+ transport. Under these conditions, before nystatin treatment, TC still triggered Cl secretion, despite an unfavorable transepithelial Cl gradient (Fig. 7B). When nystatin was subsequently added, transcellular Cl absorption across basolaterally permeabilized cells led to a rapid reversal of the current (Fig. 7B). In some tissue samples but not all, nystatin also reversed the basal current (compare Fig. 7, C with D). We propose that this variable response reflects distinct basal levels of apical Cl conductance. After nystatin treatment, the addition of TC continued to induce an apical Cl conductance in Cftr+/+ mice (Fig. 7, C–E) but not in Cftr–/– animals (Fig. 7F), indicating that activation of CFTR occurs. Incidentally, in instances where nystatin treatment revealed putative basal Cl secretory activity, we found that the capacity of the tissue to respond to subsequent addition of secretagogues (such as forskolin) was limited (compare Fig. 7, C with D).



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Fig. 7. Effect of TC on apical membrane Cl conductance in the distal ileum of Cftr+/+ mice. The basolateral membrane electrical resistance was clamped by nystatin treatment. A: under these conditions, in the absence of a transepithelial Cl gradient, TC did not evoke an electric response. B: In the presence of a mucosa to serosa NaCl gradient, the ionophore reversed the TC-evoked ion current, signifying an inward Cl current. Whereas nystatin, in some (C) but not all (D) tissue sections, also appeared to reverse the basal Cl conductance, the addition of TC invariably led to the induction of a further apical Cl conductance. Forskolin (0.01 mmol/l), but not carbachol (0.2 mmol/l), also induced an apical Cl conductance after (D) or before (E) TC exposure. F: in the nystatin-treated ileum of Cftr–/– mice, TC and forskolin had no effect on Isc. The traces shown are representative of experiments performed on tissue from at least 3 different animals.

 
Whereas TC and forskolin still triggered an apical Cl conductance after the basolateral membrane was exposed to the ionophore, this invariably abolished carbachol-evoked Cl transport (Fig. 7, D and E). This indicates that BS and Ca2+-dependent secretagogues activate Cl secretion through separate routes and is consistent with the fact that carbachol treatment did not desensitize tissue to a TC challenge (e.g., Fig. 6B). These findings also substantiate the general notion that Ca2+-dependent agonists, like carbachol and histamine, principally stimulate opening of basolateral K+ channels and that the ensuing cell hyperpolarization promotes Cl secretion but has no direct effect on CFTR activity (5, 56).

BS do not target CFTR through activation of cAMP- or cGMP-dependent protein kinases. Because our data show that BS target CFTR, we postulated that ASBT-mediated TC uptake leads to activation of a protein kinase known to phosphorylate and activate CFTR. In rat cholangiocytes, it was shown that ASBT-mediated uptake of some BS enhances (secretin induced) cAMP production, suggesting that a secretory response ensues from activation of PKA (2). Enhanced cAMP signaling in response to BS uptake may also occur in human gallbladder cells, as it was proposed that taurochenodeoxycholate and tauroursodeoxycholate induce a PKC-dependent activation of certain adenylate cyclase isoforms (9). However, in our studies, direct activation of PKC by exposure of ileal tissue to the phorbol ester phorbol-12-myristate-13-acetate did not mimic the secretory response evoked by TC, nor did the TC response show sensitivity to the specific PKC inhibitor GF109203X (not shown). These findings strongly argue against the involvement of PKC in TC-induced CFTR activation in the distal ileum.

The PKA inhibitor H89 caused a minor, but significant, attenuation of the TC response (–22 ± 5%, N = 10, P < 0.01), compatible with a causative or permissive role of PKA (Fig. 8). However, H89 was substantially more effective in inhibiting the fully cAMP/PKA-mediated response to forskolin (Fig. 8), suggesting that TC uptake affects additional signaling pathways. In line with these results, in the distal ileum of Cftr+/+ (C57BL/6, 129/Sv) mice, maximal activation of PKA by forskolin-induced cAMP production did not abrogate the TC-induced response, although it was reduced by approximately one-third compared with the TC response of adjacent tissue sections that were not pretreated with forskolin (26.4 ± 4.8 vs. 39.5 ± 6.5 µA/cm2, N = 5, P = 0.17). Similarly, prior TC exposure significantly attenuated the secretory response elicited by forskolin (24.7 ± 2.0 µA/cm2, N = 24, vs. 68.9 ± 4.8 µA/cm2, N = 13, for TC-exposed and untreated tissue, respectively, P < 0.001). The moderate attenuation of the TC response after forskolin pretreatment most probably indicates that, under these conditions, CFTR-mediated Cl efflux is maximally activated and/or that the capacity for basolateral Cl entry starts to limit the rate of transepithelial Cl transport. Therefore, although these results indicate that forskolin and BS share a common target, viz. CFTR, it cannot be deduced from these experiments that they operate through the same signaling cascade.



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Fig. 8. Role of cAMP/PKA-mediated signal transduction in TC-induced CFTR activation. Ileal tissue of Cftr+/+ mice was incubated for 20 min in the presence or absence of the PKA inhibitor H89 (20 µmol/l added bilaterally) before luminal exposure to TC (0.5 mmol/l). The effectiveness of H89 in blocking cAMP/PKA-mediated anion secretion was tested by the subsequent addition of forskolin (10 µmol/l) to the serosal bathing medium. A: comparison of the secretory response to TC and forskolin in the absence or presence of H89 performed on contiguous tissue segments. B: maximal change in Isc induced by these secretagogues tested in the absence or presence of H89 shown by comparing contiguous tissue segments. **P < 0.01. N = 10 mice/group.

 
Next, we tested the involvement of cGKII. This membrane-associated kinase is known to phosphorylate and activate intestinal CFTR and is targeted by the intestinal endogenous secretagogue guanylin and by heat-stable bacterial enterotoxins that provoke Cl secretion by activating a guanylyl cyclase-associated receptor in the luminal membrane of epithelial cells (23, 50). Ursodeoxycholate and CDC may increase cGMP levels in rabbit (42) and human (15) colonocytes, respectively, suggesting that luminal BS exposure could trigger guanylyl cyclase activity. However, in cGKII-null mice, i.e., in the total absence of kinase activity, TC still induced CFTR activation in the distal ileum, leading to an increase in Isc that was not significantly different from the one observed in cGKII+/+ littermates (28.5 ± 4.7 vs. 33.8 ± 3.3 µA/cm2 for cGKII–/– and cGKII+/+ mice, respectively, N = 4, P = 0.40). Therefore, we concluded that TC does not target the guanylin receptor or cGKII in the mouse distal ileum.


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In this study, we showed that luminal exposure to low, physiologically relevant levels of hydrophilic BS elicits electrogenic ion transport in the murine distal ileum. This response was bumetanide sensitive and largely abolished in Cftr-null mice, indicating that it is dependent on CFTR and reflects Cl and HCO3 secretion. No response to TC was found in the colon or jejunum, consistent with previous observations made in the rat intestine (55). Because the TC-evoked response seemed restricted to the unique site of ASBT-mediated active BS absorption, which is especially important for the salvage of such conjugated trihydroxy BS, otherwise absorbed ineffectively (4, 33, 36), we inferred that ASBT-mediated TC uptake might be crucial for this response. Indeed, we found that inhibition of ASBT activity by the dimeric BS analog S910960 also blocked a rise in Isc and that CDC, a relatively poor ASBT substrate, induced marginal responses, even though this dihydroxy bile acid is a potent activator of Cl secretion in the colon (26). In Cftr-null mice, only the first component of the normally biphasic response was retained, and this CFTR-independent response corresponded with electrogenic, ASBT-mediated 2Na+,1TC cotransport. This indicates that the ensuing more gradual response seen in other genotypes depends completely on CFTR and most probably does not involve additional apical Cl channels. Homozygous F508del mice showed an intermediate response, in line with partial retention of F508del-CFTR channel function (24).

Our measurements showed that ileal TC uptake was reduced by 17% in Cftr-null mice. Because the distal ileum is the discrete site of active BS uptake and has a pivotal role in the near-complete recovery (~95%) of the intestinal BS load (16, 30), this reduction may well explain the high level of fecal BS excretion reported previously for our Cftr-null mice (6). However, for homozygous F508del mice, we did not find a reduction in ileal BS uptake, despite the fact that fecal BS loss is increased to a similar extent as in null mice (6). This discrepancy suggests that the scatter intrinsic to in vitro flux measurements may have obscured such a modest reduction in transport activity in homozygous F508del mice or that the total capacity for BS absorption, rather than the absorption rate in the distal ileum, is slightly impaired.

The effects of BS on intestinal fluid secretion have been studied mostly in the colon, in relation to the secretory diarrhea that is caused by incomplete absorption of BS in the small intestine. Initially, it was proposed that BS induce a cAMP-dependent response in epithelial cells, which may have been triggered by prostaglandin release from cells in the lamina propria (7, 59). While this mechanism might well be relevant for colonic secretion, we performed all our experiments in the presence of the cycloxygenase inhibitor indomethacin, and we did not detect an effect of lipoxygenase suppression by NDGA, in effect excluding a role for prostaglandin production in the ileal response to BS exposure.

In the mouse colon, CDC evoked Cl secretion by activation of mast cells, triggering a histaminergic response (26). Such dihydroxy bile acids, with both hydroxyl groups in the {alpha}-configuration, are considered most potent activators of fluid secretion in the human colon (8, 39). They may even directly trigger secretory pathways in epithelial cells, as it was shown subsequently that low levels of taurodeoxycholate evoke Cl efflux from T84 human colonic tumor cells via activation of inositol (1,4,5)trisphosphate-mediated Ca2+ release (17). Importantly, TC, however, did not elicit a secretory response in T84 cells (17) or trigger a release of endogenous secretagogues in the human (39) and rabbit (53) colon or, as we found, in the mouse colon. Indeed, it has been shown that taurine-conjugated cholate and urso(deoxy)cholate, as well as unconjugated ursodeoxycholate (a dihydroxy bile acid, but with one hydroxyl group in the {beta}-configuration), induce minimal histamine release from cultured murine mast cells, even at concentrations up to 10 mmol/l (41). Consistent with these findings, serosal exposure of the rat distal ileal epithelium to this high concentration of TC did not evoke a secretory response (55), whereas, in our hands, the serosal addition of TC to the mouse ileum induced a minor response that is potentially due to leakage of TC to the luminal compartment. In contrast, luminal exposure to such relatively hydrophilic conjugated BS, at levels well below their critical micellar concentration (~11 mmol/l for TC) (44), evoked a robust and sustained secretory response in the distal ileum of mice [and rats (54, 55)].

Previously, the involvement of a histamine H1 receptor-mediated response in ileal BS-induced Cl secretion was deduced from experiments showing that TC evoked a rise in Isc across the mouse ileum that was lowered by ~30% in the presence of pyrilamine (mepyramine) (28). Yet, in the mouse colon, pyrilamine caused a much more pronounced inhibition (by ~70%) of the response elicited by CDC (26). This suggests that, whereas this histaminergic pathway plays a prominent role in BS-induced colonic secretion induced by dihydroxy bile acids, it is of minor importance for the ileal response to conjugated trihydroxy BS. Accordingly, we found that pyrilamine markedly reduced basal Isc but did not prevent a TC-evoked increase in Isc. Because we used a substantially lower luminal TC concentration (0.5 mmol/l) than was applied previously (2.5 mmol/l) (28) for testing the effects of pyrilamine, our results do not discount the possibility that at higher levels, TC could induce the release of endogenous secretagogues. Suppression of basal Isc by pyrilamine was previously reported for the rabbit ileum, and it was shown that histamine induced a highly transient and variable response in this tissue, leading the authors to conclude that an endogenous histamine release may affect the response to exogenous stimulation (25). Similarly, we found the ileal response to histamine to be highly variable and, furthermore, found the most distal part of the ileum to be refractory to histamine stimulation, suggesting that the pyrilamine-sensitive component we detected signals a basal histamine release that desensitizes the tissue to further histaminergic stimulation. In addition, we found that another secretagogue produced endogenously, 5-hydroxytryptamine, resulted in prolonged desensitization but, like histamine, did not attenuate the response to TC.

So far, our data lead us to conclude that BS do not trigger secretion induced by secretagogues released from neuronal or immune cells in the submucosa. Therefore, most plausibly, BS accumulation in ASBT-carrying ileocytes activates CFTR directly, via a relay mechanism within the same cell or via paracrine signaling between epithelial cells. For cholangiocarcinoma cells (46) and T84 colonic tumor cells (17), it was shown that certain BS may evoke Cl secretion by enhancing Ca2+ release from intracellular stores. In the intestinal epithelium, Ca2+ agonists induce secretion principally by activation of basolaterally situated K+ channels. To assess whether TC uptake in ileal enterocytes evokes secretion by activation of such channels, we used nystatin to clamp the K+ gradient across the basolateral membrane. This action completely abolished the response to carbachol but did not prevent the response to TC or forskolin, which activates CFTR by inducing PKA-mediated phosphorylation. These findings indicate that basolaterally situated Ca2+-dependent K+ channels are not the prime target of TC. To further substantiate this conclusion, we tested the effect of carbachol pretreatment on the TC response. Carbachol not only activates prosecretory Ca2+-dependent K+ channels but also triggers sustained activation of Ca2+-independent, antisecretory signaling cascades. Thus carbachol typically induces a transient secretory response and blocks subsequent responses to other secretagogues known to increase intracellular Ca2+, like histamine and thapsigargin (31, 32, 52), but it does not antagonize the action of secretagogues known to induce phosphorylation/activation of CFTR. In line with our observations on permeabilized cells, we found that pretreatment with carbachol did not blunt the response to TC. Collectively, these data argue against a TC-induced Ca2+-mediated secretory response and strongly suggest that BS act at the apical pole of the cell.

Immunohistochemistry indicates that ASBT is localized along the entire length of the villus, down to its base, but not in crypts. Conversely, CFTR is expressed predominantly in crypt cells, but its localization does extend into the villus. This pattern suggests that both proteins principally colocalize in the apical membrane of cells in the (lower) villus. Thus assuming a signal relay mechanism linking BS uptake and CFTR activation within the same cell, only a subset of the total CFTR pool would be accessible for activation. Nevertheless, our data indicate that TC, at 0.5–2.0 mmol/l, induced a secretory response that amounts to about 45–75% of the forskolin response, which presumably involves the entire CFTR pool in crypt and villus cells. It suggests that the villus compartment may have a higher secretory capacity than is generally projected. Of note, the apical surface of villus cells, carrying well-differentiated microvilli, is two to three orders of a magnitude increased compared with crypt cells. Therefore, although apical CFTR protein immunostaining decreases along the crypt-villus axis, the total amount of CFTR in the villus epithelium may, in fact, approximate the level found in the crypts. In accordance with this notion, it has been shown that, whereas immunohistochemistry shows that cGKII protein is present most abundantly in the villi and only at low levels in the upper part of the crypts (37), the cGKII-dependent secretory response in the mouse jejunum amounts to over 50% of the forskolin response (51). However, although our data indicate a major, if not exclusive, role of the villus epithelium in the BS-induced secretory response, we do not entirely exclude the possibility that the crypt compartment participates in the response. First, low levels of ASBT protein, undetectable by immunostaining, may be present in the crypt epithelium, promoting BS uptake and local CFTR activation. Second, we cannot rule out the remote possibility that BS uptake in villus cells leads, via a paracrine signaling route, to activation of the CFTR pool in crypt cells that do not (yet) express ASBT.

Although our results indicate that BS modulate the activity of CFTR, it cannot be deduced from these experiments whether BS directly interact with the channel or modulate the level of channel phosphorylation through effects on kinases and/or phosphatases. Our data indicate that of the major kinases known to activate CFTR by phosphorylation, only PKA may be implicated in the response to TC uptake. In fact, the PKA inhibitor H89 only slightly attenuated the TC response, significantly less effectively than it attenuates the fully cAMP/PKA-mediated forskolin response. Consequently, it is unlikely that BS induce a major cAMP/PKA-mediated response, a conclusion that is supported by the observation that prior activation of this signaling cascade by forskolin only moderately attenuated a subsequent response to TC. Therefore, we tentatively conclude that TC-induced CFTR activation involves modulation of an additional signaling cascade. Our future studies aim to delineate this activation mechanism at the molecular level.

Whereas anomalous accumulation of lipophilic BS in the colon provokes secretory diarrhea, ASBT-mediated uptake of hydrophilic BS in the distal ileum, a normal component of the enterohepatic cycle of BS, induces a secretory response that may be of physiological significance. As lipids are absorbed in the upper small intestine, hydrophilic (conjugated) BS released from mixed micelles will transit to the distal small intestine, because their absorption occurs almost exclusively through ASBT (16, 36). Even though solvable in aqueous solutions, distally accumulating BS may self-associate, and such micelle-like structures could incorporate into lipid bilayers, compromising epithelial integrity (44). Induction of fluid secretion upon ASBT-mediated BS uptake may protect the epithelium by diluting BS that accumulate at the cell surface. Correspondingly, in the bile duct epithelium, CFTR seems to play a comparable role in controlling luminal BS levels and protection of the epithelium against BS-induced cell injury, as CF-associated liver disease results from an interruption of bile flow and a cytotoxic accumulation of BS (47). ASBT-mediated uptake of BS in CFTR-expressing cholangiocytes would constitute a mechanism for sensing and adjusting biliary BS levels that links to ductular fluid secretion by potentiation of a secretin-inducable secretory pathway (2).

Intriguingly, several epidemiological studies (11, 12, 35, 38), if not all (58), indicate that an association exists between CF-related liver disease and the incidence of intestinal obstructions, suggesting an interrelated pathogenesis. In CF, intestinal obstructions occur predominantly in the distal intestine, both in newborns (MI) and in later life (DIOS). They may ensue from pancreatic insufficiency (observed in virtually all CF infants with MI), leading to incomplete enzymatic digestion of lipids and proteins and, consequently, distal accumulation of undigested food remnants. However, the absence of intestinal CFTR activity also plays a crucial role in the pathogenesis of these syndromes, as intestinal bicarbonate and fluid secretion aid the enzymatic breakdown of nutrients, and fluid production normally facilitates peristaltic propulsion of intestinal contents. The importance of intestinal CFTR function is emphasized by the severe intestinal phenotype of current CF mouse models, which show a high incidence of MI equivalent in the absence of overt pancreatic or hepatobiliary dysfunction (27, 45). Indeed, the strong reduction in BS-induced Cl secretion we observed in Cftr-null mice compared with the relatively mild effect in our homozygous F508del mice is in line with the higher incidence of intestinal obstructions in null mice compared with this particular F508del CF mouse model (Cftrtm1Eur), which shows residual CFTR-mediated Cl secretion (19, 45). Conceivably, because BS uptake in the distal intestine constitutes a physiological trigger for fluid secretion after postprandial stimulation of bile secretion, coupling of ASBT to CFTR activity assists in normal transit of the intestinal contents along the ileocecal trajectory.


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This study was supported by The Netherlands Organization for Scientific Research Grant 902-22-092 and by Stomach-Liver-Intestine Foundation Grant MWO 03-15. H. J. Verkade is a Fellow of the Royal Netherlands Academy of Arts and Sciences.


    ACKNOWLEDGMENTS
 
The authors are indebted to Dr. W. Kramer (Aventis Pharma Deutschland; Frankfurt am Main, Germany) for the generous donation of S910960 and to Prof. C. R. Marino (University of Tennessee; Memphis, TN) for kindly donating the anti-rodent CFTR antibody.


    FOOTNOTES
 

Address for reprint requests and other correspondence: M. J. C. Bijvelds, Dept. of Biochemistry, Erasmus MC, PO Box 1738, 3000 DR Rotterdam, The Netherlands (e-mail: m.bijvelds{at}erasmusmc.nl)

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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