1 Programme in Cell Biology and 2 Department of Pathology in the Research Institute, Hospital for Sick Children, Toronto, Ontario, Canada M5G 1X8
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ABSTRACT |
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The chloride channel ClC-2 has been implicated in neonatal airway chloride secretion. To assess its role in secretion by the small intestine, we assessed its subcellular expression in ileal segments obtained from mice and studied the chloride transport properties of this tissue. Chloride secretion across the mucosa of murine ileal segments was assessed in Ussing chambers as negative short-circuit current (Isc). If ClC-2 contributed to chloride secretion, we predicted on the basis of previous studies that negative Isc would be stimulated by dilution of the mucosal bath and that this response would depend on chloride ion and would be blocked by the chloride channel blocker 5-nitro-2-(3-phenylpropylamino) benzoic acid but not by DIDS. In fact, mucosal hypotonicity did stimulate a chloride-dependent change in Isc that exhibited pharmacological properties consistent with those of ClC-2. This secretory response is unlikely to be mediated by the cystic fibrosis transmembrane conductance regulator (CFTR) channel because it was also observed in CFTR knockout animals. Assessment of the native expression pattern of ClC-2 protein in the murine intestinal epithelium by confocal and electron microscopy showed that ClC-2 exhibits a novel distribution, a distribution pattern somewhat unexpected for a channel involved in chloride secretion. Immunolabeled ClC-2 was detected predominantly at the tight junction complex between adjacent intestinal epithelial cells.
tight junction; immunofluorescence; hypotonic shock; chloride efflux
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INTRODUCTION |
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THE MAJOR CHLORIDE CHANNEL thought, to date, to mediate chloride secretion in the small intestine is the cystic fibrosis transmembrane conductance regulator (CFTR) (1). However, it remains unclear whether CFTR is the only chloride channel capable of mediating secretion in this tissue (3, 10, 12). A previous study in rat ileum showed that a secretory chloride current can be activated by hypotonicity, but the molecular basis for the chloride conductance path was not determined (3). It has been suggested that the hypotonicity-activated chloride channel, ClC-2, may be capable of mediating chloride secretion in rodent neonatal airways because ClC-2 protein can be detected in the apical membrane of rodent neonatal airway epithelial cells (17). In addition, ClC-2 has been suggested to play a role in gastric chloride secretion (15). So far, only ClC-2 message expression has been studied in the intestinal epithelium and has been detected in the human intestinal cell line T84, in rat intestinal tissue (22) as well as in the murine duodenum (12). Evaluation of the potential role for ClC-2 in intestinal secretion requires an assessment of subcellular ClC-2 protein localization in this tissue.
In the present study, using wild-type (WT) and CFTR knockout mice, we showed that in the murine ileum, hypotonic shock elicits chloride secretion through a non-CFTR chloride channel that exhibits the pharmacological properties expected for ClC-2-mediated currents. However, assessment of native localization of ClC-2 in this tissue by confocal and electron microscopy revealed that ClC-2 exhibits a distribution pattern that is unexpected for a channel involved in chloride secretion. We detected ClC-2 protein predominantly at the tight junction complex between adjacent intestinal epithelial cells with only diffuse labeling of the apical brush-border membrane.
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MATERIALS AND METHODS |
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Mice. Experiments were conducted on male and female WT and cystic fibrosis (CF) mice, matched for genetic background (BalbC/129), age (6-8 wk old), and diet (normal chow). The phenotype of CF mice, or CFTR knockout (CftrmHSC/CftrmHSC) mice, used in this study was described previously (18). Briefly, all but 30% of animals with this genetic background die before 6 wk of age due to intestinal obstruction. In the present study, we investigated the molecular basis of the swelling-activated chloride conductance in ileal segments obtained from surviving CF mice and their WT siblings.
Ussing chamber analysis. Short-circuit current (Isc) measurements were performed on freshly excised WT and CF murine intestinal tissues from the ileum, defined as the segment 1-5 cm proximal to the cecum. Tissues were mounted in Ussing chambers with an aperture of 0.28 cm2. The buffer bathing the tissues was composed of 112 mM NaCl, 28 mM mannitol, 10 mM KHCO3, 1.2 mM K2HPO4, 2 mM CaCl2, 1.2 mM MgCl2, and 5 mM glucose in the apical bath and 5 mM mannitol in the serosal bath and was gassed with 95% O2 and heated to 37°C. After 5-8 min of measurement of basal current, the apical solution was changed to one lacking mannitol (28 mM) for 20% hypotonic shock [or 80% isotonicity, determined with a Wescor 5500 vapor pressure osmometer (Johns Scientific, Toronto, ON, Canada)]. The low-chloride buffer was prepared as above, except that 112 mM NaCl was replaced with 112 mM sodium gluconate and 5.8 mM CaCl2 to account for the calcium-chelating effect of gluconate (3). Stock solutions of 5-nitro-2-(3-phenylpropylamino) benzoic acid (NPPB) and 4,4'-diisothiocyanato-stilbene-2,2'-disulfonic acid (DIDS) were prepared in DMSO and used at a final concentration of 0.5 mM, applied into the apical bath.
RT-PCR analysis.
Total RNA was isolated from murine intestinal tissues with the use of
the Trizol extraction protocol (GIBCO-BRL) followed by DNA removal with
(1 unit) DNase 1 (Ambion, Austin, TX). Reverse transcription with
oligo(dT) primers (using the Superscript preamplification kit;
GIBCO-BRL) was followed by PCR analysis in a 20-µl volume containing
0.5 µM of each primer (murine -actin or murine ClC-2: 5'-AGT TCC
TAG AAT ATG GAC AGA GCC-3' and 5'-AAA GAG GGA GAG GAA CT-3' spanning
498 bp), 0.2 mM of each deoxynucleoside, 1 µl of cDNA, and 0.6 units
of Ultratherm thermophilic DNA polymerase (Eclipse Molecular
Biologicals, Ontario, ON, Canada). The ClC-2 fragment was amplified
with a GeneAmp PCR System 9700 (Perkin Elmer/Applied Biosystems) with
35 cycles of 30 s at 94°C, 63°C, and 72°C. The PCR products
were ligated into pCR2.1 vector (Invitrogen) with Taq
polymerase (GIBCO-BRL), and the sequence identity was confirmed by
BLAST sequence database search.
Anti-ClC-2 antibody. The polyclonal antibody used for detecting and localizing ClC-2 protein in the murine intestinal epithelium was generated against a glutathione-S-transferase (GST)-fusion peptide containing amino acids (residues 31-74) of rat ClC-2 (rClC-2 cDNA; kindly provided by T. Jentsch). The ClC-2-specific antibody was affinity purified as previously described (24).
Western analysis. Intestinal epithelial scrapings from three WT mice were added to 15 ml of lysis buffer containing 15 mM Tris, 10 mM KCl, 1.5 mM MgCl2, and protease inhibitors (10 µg/ml leupeptin, 10 µg/ml aprotinin, 1 mM benzamidine, 10 µM E64, and 2 mM dithiothreitol). The suspension was vortexed and centrifuged for 10 min at 4°C at 4,000 rpm (JA-17, Beckman) to remove organelles. The supernatant was centrifuged for 2 h at 4°C at 100,000 g to isolate crude membrane preparation. Of this preparation, 50 µg were analyzed by SDS-PAGE (8% gel) with the use of anti-ClC-2 antibody at a concentration of 1 µg/ml. Immunoreactive protein was detected using the enhanced chemiluminescence system (Life Science, Chicago, IL).
Immunoperoxidase staining. Immunoperoxidase staining was performed on 5-µm paraffin sections of Formalin-fixed tissues. The ClC-2 antibody was applied at a concentration of 116 µg/ml and was detected with a peroxidase-conjugated streptavidin system (Vectastain Elite ABC kit; Vector Laboratories, Peterborough, UK) and counterstained with hematoxylin. Specificity of the antibody for ClC-2 was assessed by competition using a 100-fold excess of antigenic ClC-2 fusion protein. Slides were viewed with the use of a ×10 objective on an Olympus Bx microscope, and images were captured using the CoolSnap program (Roper Scientific).
Immunofluorescence labeling. Immunofluorescence labeling was performed on 5-µm cryosections of intestinal tissues fixed in methanol at 4°C. After 2.5 h of incubation at 25°C with the zonula occludens-1 (ZO-1)-specific monoclonal antibody (2.5 ug/ml; Chemical International, Temecula, CA) and the affinity-purified polyclonal antibody against ClC-2 (58 µg/ml), sections were washed with PBS and incubated with FITC-conjugated anti-mouse or anti-rabbit secondary antibodies (0.02 mg/ml; Molecular Probes, Eugene, OR). Slides were viewed with a ×100 objective on an Olympus Vanox AHBT3 microscope, and with the use of epifluorescence, images were captured with the Image-Pro program (Media Cybernetics). For confocal microscopy, sections were viewed with a ×100 objective on a Leica TCS 4D microscope, and with the use of epifluorescence, images were captured using the SCANware 5.01 program.
Immunogold electron microscopy. Subcellular localization of immunogold-labeled ClC-2 in villus tip cells was detected by transmission electron microscopy. Paraformaldehyde- and glutaraldehyde-fixed tissues from the ileum of a WT mouse were infused with sucrose, frozen, and then substituted in absolute methanol containing uranyl acetate. Samples were then warmed, infiltrated in Lowicryl HM20 resin, embedded in gelatin capsules, and polymerized under ultraviolet light. Ultrathin sections were mounted on Formvar-coated nickel grids, incubated with affinity-purified ClC-2 antibody (580 µg/ml) followed by goat anti-rabbit IgG 10-nm gold complex, and stained with saturated aqueous uranyl acetate and lead citrate. Controls included omission of the primary antibody or competition with a 100-fold excess of the GST-ClC-2 fusion peptide or a 100-fold excess of GST alone.
Gold particle density determination. Random images of immunogold-labeled sections were captured with the use of a charge-coupled device camera (AMT, Danvers, MA) in the transmission electron microscope (JEOL 1200EXII; JEOL USA, Peabody, MA). A minimum of 3 fields from 25 epithelial cells were examined from 5 samples. Particle density was determined using NIH Image 1.51 (National Institutes of Health, Bethesda, MD). Density of labeling was determined for the apical cell junctions (particles/µm), apical membrane (particles/µm), and cytoplasm (particles/µm2). Data were expressed as means ± SE.
Statistics. The significance of differences between paired experimental groups was assessed using the Student's t-test for paired data.
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RESULTS |
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A non-CFTR chloride channel contributes to swelling-activated chloride secretion in the murine ileum. A previous study showed that hyposmotic media elicits chloride secretion in the rat ileum through an unidentified chloride channel presumably expressed in the apical membrane (3). To determine whether a similar swelling-induced chloride conductance is expressed in the apical membrane of the murine ileum, we applied hypotonic shock to the apical epithelial membrane of the ileum while monitoring the transepithelial Isc.
As previously reported by others (9), a luminally directed negative Isc was spontaneously generated by the intestinal epithelia of WT mice before hypotonic shock stimulation (i.e.,
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ClC-2 message and protein are expressed in the murine small
intestinal epithelium.
To determine whether ClC-2 expression is consistent with chloride
secretion in the ileum, we first assessed ClC-2 mRNA and protein
expression in the intestine of WT and CFTR knockout mice. Using RT-PCR
analysis with sequence-specific primers to murine ClC-2 (see
MATERIALS AND METHODS), we detected ClC-2 message in the
ileum of adult WT and CFTR knockout mice (Fig.
2A). Therefore, ClC-2 message
is expressed in the small intestine of both WT and CFTR knockout
animals.
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ClC-2 protein localizes primarily to the tight junction complex and
diffusely to the apical membrane.
As previously mentioned, our immunofluorescence studies show that ClC-2
is predominantly situated in proximity to the tight junction complex.
Similarly, electron microscopy revealed that ClC-2 decorated by
immunogold was located predominantly, but not exclusively, in clusters
at the apical aspect of the tight junction complex (Fig.
5). Gold particles were counted in a
minimum of 3 fields from 25 epithelial cells from 5 different
intestinal sections. In proximity to the tight junction, we found
4.2 ± 1.3 particles of gold per micrometer of membrane. On the
other hand, in the brush border, we found 0.04 ± 0.03 particles
of gold per micromillimeter of membrane, similar to the number of
grains detected per square micrometer of cytoplasm (0.03 ± 0.01).
This labeling pattern was effectively competed with the GST-ClC-2
peptide against which the ClC-2 antibody was raised (Fig.
5B), but not by GST alone (data not shown).
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DISCUSSION |
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In this work we described a non-CFTR chloride secretory current in mouse small intestine that is activated by hypotonic solutions. This chloride secretory current shares properties previously reported for a secretory current described by Diener et al. (3) in the rat small intestine, namely, its activation by hypotonicity, inhibition by NPPB, and lack of sensitivity to DIDS. We suggest that this activity is mediated by ClC-2 because ClC-2 is known to be activated by hypotonic solutions in heterologous expression systems (22, 24). Furthermore, we found that ClC-2 protein is expressed endogenously in the epithelium of intestinal villi. While the classic model of secretion postulates that chloride secretion occurs primarily from the intestinal crypts (23), there are several reports suggesting that chloride secretion can also occur from the intestinal villi (2, 4, 20). Using a combination of fluorescence and electron microscopy, we determined that ClC-2 protein in the murine intestinal epithelium is concentrated close to the membranes of the tight junction complex. Therefore, ClC-2 has a novel subcellular distribution pattern that has not been described previously for chloride channels involved in anion secretion. Hence, further study will be required to determine the role of ClC-2 in chloride secretion by the intestinal epithelium.
As previously mentioned, ClC-2 is known to be activated by hypotonicity and, hence, may mediate the secretory chloride response observed in CFTR knockout mice. However, other non-CFTR chloride channels may possibly participate in this response. Our pharmacological analyses suggest that the response is relatively insensitive to the chloride channel blocker DIDS. Because DIDS, in concentrations comparable to those employed in the present study, is known to inhibit calcium-activated currents in intestinal epithelia (possibly mediated by murine calcium-sensitive chloride channel) (1, 10), the ubiquitous swelling-activated volume-sensitive organic osmolyte/anion channel, and ClC-3 (5, 21), these channels are not likely to play a role in the response. Ultimately, it will be necessary to generate a ClC-2 knockout mouse to define the relative contribution of ClC-2 to the secretion observed.
The subcellular distribution of ClC-2 is unique for an anion channel because it is concentrated close to the tight junction complex. The tight junction, a dynamic structure in epithelial tissue, plays a key role in regulating paracellular transport and the barrier functions of the intestine. Hence, we plan to directly assess the role for ClC-2 in these functions in our future studies. Furthermore, it remains to be determined whether specific interactions exist between ClC-2 and scaffolding and/or cytoskeletal proteins that reside in the tight junction complex, i.e., the ZO family of proteins, which participate in the targeting or retention of ClC-2 at this site in the small intestine (6-8, 16). We suggest that such interactions may be relatively specific for the small intestine; as Murray et al. (17) localized ClC-2 to the apical membrane of airway epithelial cells and in the mouse colon, ClC-2 is localized at the basolateral membrane (Gyömörey K and Bear CE, unpublished observations).
The physiological stimulus that activates ClC-2 in native
tissue is not known. In heterologous expression systems, ClC-2 currents appear to require activation by stressors such as hypotonic shock, low pH, or hyperpolarization to 100 mV (11). On the
other hand, ClC-2 endogenously expressed in neuronal cells is
basally active at resting membrane potentials (19),
suggesting that ClC-2 regulation may vary with the host cell type. We
observed that the ileal epithelium of CFTR knockout mice is capable of
supporting NPPB-sensitive, DIDS-insensitive chloride secretion under
resting conditions and suggest that ClC-2 may be partially active under
basal conditions in this tissue, as in neuronal cells
(19). In our studies, luminal hypotonic shock appeared to
stimulate ClC-2 activity. Osmotic change may in fact be a physiological
regulator of ClC-2 in the intestine because absorptive epithelia
experience local changes in osmolarity as a result of nutrient
transport (14). In fact, expression of ClC-2 at the villus
tip enterocytes of the jejunum overlaps with the expression of the
sodium-glucose cotransporter SGLT1 at this site (25). It
also has been suggested that phosphorylation by protein kinase A and
protein kinase C can modulate ClC-2 currents (15,
19). The role of these signaling pathways in the
regulation of ClC-2 in native epithelial cells will be the subject of
future studies.
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ACKNOWLEDGEMENTS |
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CFTR knockout animals and WT siblings were generously provided by Lap-Chee Tsui and Richard Rozmahel (Genetics Department, Research Institute).
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FOOTNOTES |
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This research was supported by operating grants to C. E. Bear from the Canadian Cystic Fibrosis Foundation (CCFF), Medical Research Council (MRC), and National Institutes of Health Specialized Center of Research Grant 01 P50 DK-490096-06. K. Gyömörey was supported by a CCFF Studentship Award, and C. E. Bear was supported by an MRC Scientist Award.
Address for reprint requests and other correspondence: C. E. Bear, Division of Cell Biology, Research Institute, Hospital for Sick Children, Dept. of Physiology, Univ. of Toronto, 555 Univ. Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: bear{at}sickkids.on.ca).
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.
Received 5 May 2000; accepted in final form 27 July 2000.
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