From the
Center for Basic Research in Digestive Diseases, Departments of Internal Medicine and Biochemistry and Molecular Biology, Mayo Medical School, Clinic and Foundation, Rochester, Minnesota 55905,
Departamento de Fisiologia, Facultad de Ciencias Bioquimicas, Suipacha 570, 2000 Rosario, Santa Fe, Argentina, ¶Duke University Medical Center, Durham, North Carolina, and ||Harvard Medical School, Beth Israel Deaconess Medical Center, Boston, Massachusetts 02215
Received for publication, February 27, 2003 , and in revised form, March 26, 2003.
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ABSTRACT |
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INTRODUCTION |
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In the studies described here, we found that cholangiocytes contain a subpopulation of vesicles in which the water channel AQP1, the chloride channel CFTR, and the chloride/bicarbonate exchanger, AE2, are sequestered in the basal state and that these proteins move together in a microtubule-dependent manner from an intracellular location to the apical cholangiocyte plasma membrane in response to choleretic stimuli. By a variety of independent and complementary biochemical and morphologic approaches, the data support the notion that a novel organelle containing proteins that accomplish both water and ion transport exists in cholangiocytes and participates in hormone-induced bile secretion. This concept is supported by a body of evidence on the physiological implications of colocalization of functionally related molecules in the kidney, including aquaporins with ion transporters, and membrane relocalization after treatment with agonists (9, 10, 11).
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EXPERIMENTAL PROCEDURES |
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Immunoisolation of AQP1-, CFTR-, and AE2-containing Vesicles Purified cholangiocytes were sonicated, and a postnuclear supernatant was prepared. The postnuclear supernatant was further purified on a linear sucrose gradient (8.640%), and the resulting microsomal fraction sedimented at 200,000 x g for 60 min to remove any remaining cytosol. This microsome-rich fraction is referred to as "starting material."
Immunoisolation was performed by a modification of the method of Howell et al. (14). Briefly, tosylated superparamagnetic beads (Dynabeads M-500, Dynal Inc., Oslo, Norway) were incubated overnight with a goat anti-rabbit IgG (Fc) linker secondary antibody (Biodesign International, Kennebunk, ME) at a concentration of 10 µg of antibody/mg of beads in borate buffer (100 mM H3BO3, pH 9.5). In this and all subsequent steps, beads were collected with a magnetic device (MPC; Dynal, Inc.). The amount of linker antibody that bound to the beads was reproducibly 5 µg/mg of beads. Coated Dynabeads were rotated for 12 h with a primary polyclonal antibody to AQP1 (10 µg/mg of beads) and 500 µg of purified microsome starting material. Control experiments were performed using Dynabeads coated with secondary anti-body alone. Beads were collected and rinsed with incubation buffer, and this immunoisolated fraction then represented the AQP1-containing vesicles specifically bound to the beads. Supernatant that remained after separation from the immunoisolated bound fraction was centrifuged at 16,000 x g for 10 min, and the pellet was resuspended in 0.25 M sucrose, representing the unbound fraction. For simplicity, we refer to the microsome-rich fraction as "starting material," the fraction containing the immunoisolated AQP1 vesicles attached to the beads as "bound," and the supernatant containing non-AQP1-containing vesicles as "unbound." These three fractions were subsequently analyzed biochemically and by immunoblotting.
In a separate set of experiments aimed at determining the reversibility and specificity of the vesicle isolation technique, we used polyclonal antibodies to either CFTR or AE2 as the linking antibody to the coated immunomagnetic beads and thereby immunoisolated a population of CFTR-containing vesicles or AE2-containing vesicles, respectively, which were then analyzed biochemically and by immunoblotting.
Characterization of Isolated VesiclesBiochemical characterization using specific organelle assays was performed to determine the purity of the starting material prior to the immunoisolation protocol. To determine the specificity of the immunoisolation technique, the same assays were performed on both the bound and the unbound fractions. N-acetyl--glucosaminidase (lysosomes) activity (15), malate dehydrogenase (mitochondria) activity (16), lactate dehydrogenase (cytosol) activity (17), and alkaline phosphodiesterase 1 (plasma membrane) were assayed as previously reported (18). Microsome activity was determined by two methods: microsomal esterase, assessed by the method of Beaufay and Berthet (18), and glucose-6-phosphatase using a commercially available spectrophotometric kit from Sigma. Golgi activity was determined fluorometrically with the substrate, 4-methylumbelliferyl-
-D-mannopyranoside, by the method of Farquhar (19). Protein concentration was determined by the fluorescamine method using bovine serum albumin as a standard (20).
As additional evidence of vesicle purity, solubilized fractions from the immunoisolation protocol were immunoblotted with specific antibodies to (i) calnexin (Affinity Bioreagents, Inc., Golden, CO), an integral membrane protein of the endoplasmic reticulum; (ii) TGN-38, a trans-Golgi network protein; and (iii) AQP4 (Alpha Diagnostics International, Inc., San Antonio, TX), a protein found in cholangiocytes exclusively in the basolateral plasma membrane.
Vesicle suspensions were prepared for transmission electron microscopy using a primary fixative of 2.5% glutaraldehyde, 0.25 M sucrose for 1 h at 4 °C. A second and poststaining fixative consisting of 1% OsO4 and 1% uranyl acetate, respectively, was followed by dehydration in alcohol and embedding in Quetol as previously described (21). Electron micrographs were generated on a Phillips 201 electron microscope.
Immunoblotting for AQP1, CFTR, and AE2To determine the presence of AQP1, CFTR, and AE2 in the immunoisolated fractions, we performed immunoblotting using specific antibodies to AQP1 (Alpha Diagnostics International), CFTR (22), and AE2.
In a set of experiments designed to assess the specificity of the antibody for CFTR used for immunoblotting, we performed immunoblots for CFTR and AQP1 on cell lysates from cultured mouse cells obtained from a line of CFTR (-/-) knockout mice. In these immunoblots, the cell lysate was positive for AQP1 and negative for CFTR, demonstrating the specificity of the CFTR antibody (data not shown).
Dual Labeled Immunogold for AQP1, CFTR, and AE2Immortalized, nonmalignant mouse cholangiocytes (23) were grown on type 1 collagen (Biocoat; Becton Dickinson, Bedford, MA) in culture media of minimal essential media (Invitrogen) with 10% fetal bovine serum and 1x penicillin/streptomycin. Confluent, polarized cholangiocytes were treated with media alone (basal) or media with the choleretic agonist, dibutyryl-cyclic AMP (100 µM), forskolin (3 µM), and isobutylmethylxanthine (100 µM) (to prevent the degradation of cAMP) for 30 min at 37 °C. The cells were then processed for fixation and immunogold labeling according to the method of McCaffery and Farquhar (24) and then exposed to primary antibodies to AQP1 (rabbit polyclonal) and CFTR (mouse monoclonal). Grids were then incubated with a mixture of donkey anti-rabbit IgG linked to 6-nm gold (AQP1) and donkey anti-mouse 12-nm gold (CFTR) and examined with a Joel 1200 electron microscope at 80 kV.
Electron micrographs from both basal and agonist groups were printed at a final magnification of x30,000, and pictures were randomized for counting. Vesicles were quantified in four groups: (i) vesicles containing only 6-nm gold particles, representing specific labeling for AQP1; (ii) vesicles containing only 12-nm gold particles, labeled for CFTR; (iii) vesicles labeling for both AQP1 and CFTR; and (iv) vesicles without any labeling. In a parallel experiment, vesicles were exposed to 5-nm gold particles labeled for AE2 and 10-nm gold particles labeled for CFTR. The same criteria used to quantitate the associated gold particles for AQP1 and CFTR was used as described for AE2 and CFTR. The micrographs represent photographs from six grids from each of three different vesicle preparations. Micrographs were counted randomly in each group and counted three times to obtain means ± S.E. Data are expressed as the number of gold particles in each group as a percentage of the total.
Animal Model for in Vivo ExperimentsIn vivo experiments were performed using rats in which the common bile duct was ligated for 1 week (8). After the biliary obstruction was released, 1.0 ml of 10-7 M secretin (Peninsula Laboratories, Belmont, CA) or vehicle (phosphate-buffered saline, pH 7.4) was administered via the femoral vein.
In separate experiments, BDL rats were injected intravenously with 0.5 µmol/100 g of body weight colchicine or -lumicolchicine in phosphate-buffered saline 2 h prior to administration of secretin. Bile was collected, and cholangiocytes were isolated immediately after secretin administration as previously described (6).
For immunoblotting experiments, apical and basolateral plasma membranes were prepared from isolated cholangiocytes derived from whole liver as previously described by us (21).
Statistical AnalysisDensitometric scanning was performed using the software Molecular Analyst (Bio-Rad). All values are expressed as mean ± S.E. Significance was determined using Student's t test; p < 0.05 was considered statistically significant.
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RESULTS |
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Fig. 1B displays results of immunoblots for proteins of the endoplasmic reticulum (calnexin), Golgi (TGN-38), plasma membrane (AQP4), and AQP1. Calnexin, TGN-38, and AQP4 were detected at their expected molecular weights and in their respective positive controls but were absent from the bound fraction. AQP1 was present at 28 kDa in the positive control of rat RBC plasma membrane and in the microsome and bound fractions.
Table I shows a biochemical analysis of the microsomal starting material, the fraction bound to the immunomagnetic beads, and the unbound fraction. As expected, the microsomal derived starting material showed an enrichment in activity of the microsomal markers, microsomal esterase, and glucose-6-phosphatase; enzyme activities for lysosomes, mitochondria, and plasma membranes were undetectable, demonstrating that the starting material did not contain these organelles. The amounts of measurable lactate dehydrogenase (cytosolic marker) and -D-mannopyranoside (Golgi marker) were negligible.
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Immunoblotting of Isolated Subcellular FractionsThe immunoblot shown in Fig. 2A illustrates that AQP1, CFTR, and AE2 were all significantly (p < 0.05) enriched in the bead-associated AQP1-immunoisolated vesicles compared with the microsomal preparation: AQP1, 2.4-fold; CFTR, 1.7-fold; AE2, 2.8-fold. There was virtually no detectable AQP1, CFTR, or AE2 in the lanes loaded with an equal amount of protein from the unbound fraction.
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Fig. 2, B and C, shows the results of immunoblotting for AQP1, CFTR, and AE2 on fractions from the reverse immunoisolations using antibodies to CFTR to isolate CFTR-containing vesicles (B) or antibodies to AE2 to isolate AE2-containing vesicles (C). In vesicles isolated with the antibody to CFTR (B), the bound fractions were significantly (p < 0.05) enriched 3.2-fold over the starting material for CFTR, 2.5-fold for AQP1, and 2.7-fold for AE2. In vesicles isolated with the antibody to AE2 (C), the bound fractions were significantly (p < 0.05) enriched 3.4-fold over the starting material for AE2, 2.6-fold for AQP1, and 2.5-fold for CFTR.
Fig. 3, A and B, illustrates colocalization in two sets of dual labeled immunogold experimental combinations (AQP1 with CFTR (top) and AE2 with CFTR (bottom)). Our observations revealed populations of small beads alone (6-nm gold for AQP1 and AE2), large beads alone (12-nm gold for CFTR), and clusters of colocalized small and large beads together shown in the enlarged insets. Quantitation revealed that AQP1 and CFTR and AE2 and CFTR colocalized in 25 and 34%, respectively, of the total vesicles counted (Fig 3, A1 and B1). Following agonist stimulation (Fig. 4), we observed relocation of the gold particles to the apical plasma membrane (Fig. 4, A1 and B1) compared with the basal state (Fig 4, A and B), consistent with our previous biochemical and morphologic data. Since the normal mouse cholangiocytes were grown in a polarized confluent manner prior to fixation, we concluded that the agonist-induced migration is domain-specific. Quantitation of the gold particles (Fig. 4, C and D) revealed a significant 2-fold or greater increase in the apical membrane-associated redistribution. In addition, the apical membrane-associated labeling contains numerous clusters of small and large gold particles, implying that the transporters may remain associated after agonist-induced redistribution to the apical membrane. Statistical analysis was performed on the quantitation of gold particles in the basal state according to the Kullback-Liebler hypothesized model of randomness (26, 27) to assess "degree of association" between AQP1 and CFTR and between AE2 and CFTR in an effort to minimize the possibility that the apparent colocalization of the immunogold beads was a random event. Hypothetically, the proportions of vesicles with small gold beads only, large beads only, and both small and large beads should all be equal to 1/3. To measure the departure from randomness for each reading from a given micrograph, the Kullback-Liebler distance was computed. A calculated value of <0.05 indicates a significant departure from randomness. The measured
value of the colocalized AQP1/CFTR and AE2/CFTR was 0.031 and 0.030, respectively, supporting our interpretation that the observed colocalization of small and large beads was not a random event.
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Apical Versus Basolateral Insertion of AQP1, CFTR, and AE2 in Secretin-treated BDL RatsShown in Fig. 5A are data from in vivo experiments demonstrating the secretin-induced relocation of AQP1, CFTR, and AE2 to the apical plasma membrane of rat cholangiocytes.
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In the unstimulated state, any AQP1 in the plasma membrane is present principally in the apical domain. Following intravenous infusion of 10-7 M secretin into BDL rats, which causes a 24-fold increase in bile secretion (Fig. 5C), the total amount of AQP1 is increased in both the band representing total plasma membrane and in the apical domain by 2.4- and 2.2-fold, respectively, relative to membranes isolated from rats infused with vehicle alone. Immunoblots for CFTR and AE2 were performed on the same fractions of total plasma membranes as well as fractions enriched in the apical and basolateral plasma membrane domains. In the absence of an intravenous infusion of secretin (i.e. vehicle infusion), total plasma membrane CFTR and AE2 are predominantly apical in their location. Following IV infusion of secretin, the amount of CFTR and AE2 in plasma membranes, like AQP1, also increases 3.8-fold in the total plasma membrane and 2.4-fold in the apical plasma membrane.
We next employed the microtubule blocker colchicine, which we had previously shown inhibits secretin-induced bile flow (6). These data are shown again in Fig. 5C for the purpose of correlating secretin-induced changes in bile flow with the blocked increase in movement to the plasma membrane of AQP1, CFTR, and AE2. As shown in Fig. 5B, pretreatment of rats with colchicine, followed by administration of secretin, nearly abolished the increase of AQP1, CFTR, and AE2 in cholangiocyte plasma membranes. The inactive analog, -lumicolchicine, did not affect the action of secretin on AQP1, CFTR, and AE2 membrane distribution or bile flow compared with rats receiving no treatment.
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DISCUSSION |
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We used immunoisolation to prepare a subpopulation of vesicles from cholangiocytes that were enriched in AQP1, CFTR, and AE2. The degree of enrichment we observed in the immunoisolated fraction seems reasonable given that our starting material is a purified microsomal fraction rather than a postnuclear supernatant. Moreover, using three different antibodies, we achieved the same relative degree of enrichment of the three proteins in the immunoisolated fraction. In comparably designed studies to immunoisolate endothelial plasmalemmal vesicles, the caveolin fraction that bound to the magnetic beads was enriched 45-fold compared with a more crude starting material of whole rat lung (25). In addition, the antibodies and proteins in the isolation and wash buffer (required for the immunopurification) adhere to the beads and cause an important underestimation of actual enrichment.
The concept that transporters for water and ions reside in the same vesicle in cholangiocytes is teleologically appealing, since water moves passively in response to osmotic gradients established by active Cl--driven secretion, the currently accepted paradigm of ductal bile formation. These transient gradients are thought to be established by the transport of bicarbonate via the
exchanger located in the apical cholangiocyte membrane and operating in parallel with the CFTR chloride channel. Recently published observations, (3, 5, 6, 28) and those reported here support the notion that a secretin-induced increase in cholangiocyte cAMP both triggers the exocytic insertion of AQP1 into the cholangiocyte apical membrane and activates apical CFTR chloride channels. Previously published data from our laboratory have demonstrated that exposure of highly purified isolated rat cholangiocytes to secretin caused significant, dose-dependent increases in osmotic membrane water permeability (Pf) (e.g. increased by 60% with 10-7 M secretin), which was reversibly inhibited by the water channel blocker, mercuric chloride (5).
Several lines of evidence exist that support the colocalization of water with ion transporters or other regulators of exocytosis, based on data generated in the tubule cells of the kidney using techniques of immunblotting and immunostaining. These include SNAP-25-associated Hrs-2 colocalizing with AQP2 (11), AQP2 with VAMP2 (10), and AQP2 with CFTR and the amiloride-sensitive epithelial Na+ channel (EnaC) (9). Evidence for the cAMP-mediated stimulation of apical targeting of vesicles containing flux molecules has been shown not only by us for AQP1 in cholangiocytes but also in a number of other epithelia, including in T84 colonocytes for CFTR and in kidney tubule epithelia for AQP2 (28, 29, 30, 31, 32). Moreover, it has been reported that secretin stimulates exchange in isolated cholangiocytes (33) and increases CFTR in the apical membrane of pancreatic duct cells via cAMP (34). The last three amino acids in the COOH terminus of CFTR (TRL) comprise a PDZ-interacting domain that appears to be required for the targeting of CFTR to the apical plasma membrane in human airway and kidney epithelial cells (35). In addition, it has been proposed that CFTR and its PDZ proteins in concert with ezrin affect other epithelial ion channels in their movement to a subapical compartment (35). Since AQP1 does not contain a known apical targeting signal, colocalization of AQP1 in a vesicle with CFTR would provide a plausible mechanism explaining how AQP1 can redistribute to the apical cholangiocyte plasma membrane in response to a secretory agonist. In kidney collecting tubule cells, AQP2 colocalizes with VAMP1 and -2, and these proteins are all trafficked to the apical plasma membrane in response to vasopressin (36). Thus, the model of agonist-driven, polarized targeting and insertion of flux proteins into specific plasma membrane domains of secretory epithelia has precedent.
Our immunogold data provide additional evidence for the colocalization of AQP1, AE2, and CFTR and are reminiscent of the work of Nielsen et al. (36), who demonstrated dual labeling and colocalization of AQP2 with VAMP2. The apparent increase in membrane-associated vesicles containing only AQP1, which was greater than the individual increases of CFTR alone and AE2 alone, may indicate the presence of specific signal or target proteins on the plasma membrane that are selective to AQP1 and not CFTR or AE2. Alternatively, the vesicles containing both flux proteins may fuse with the plasma membrane where the proteins are subsequently sorted independently, causing a difference in the net number of gold particles remaining on the membrane at the time of fixation. Whether these functionally related flux proteins are also retrieved together upon withdrawal of the secretory stimulus or in response to a cholestatic stimulus (e.g. somatostatin) remains to be elucidated. Data generated using the model of isolated bile duct units from AQP1-ko mice were interpreted as suggesting that AQPs may not be rate-limiting for transcellular water movement in biliary epithelia; interpretation of these data should also include the possibility that up-regulation of other AQPs that we have shown are present in cholangiocytes may compensate for the absence of AQP1 in cholangiocytes (37).
Thus, although recycling as a regulatory mechanism for epithelial cell transport is an accepted paradigm, our work here substantially extends this concept by introducing the novel notion that a single intracellular hormone-responsive vesicular compartment might contain several proteins that are linked both anatomically and functionally in an integrated fashion to accomplish a cellular process, in this case coupled water and electrolyte secretion.
Our in vivo observation that administration of the microtubule depolymerizing agent, colchicine, inhibited the secretin-induced increase of AQP1, AE2, and CFTR in the cholangiocyte plasma membrane as well as secretin-induced ductal bile secretion is consistent with studies in kidney-proximal tubule cells, showing that microtubules are required for insertion of AQP1 into the plasma membrane (38). Moreover, the extension of our observations to this in vivo situation provides additional evidence in a more physiologically relevant model that the molecular events we describe are important in ductal bile secretion.
It seems plausible that defects or alterations in the coordinated trafficking of related proteins could be responsible for a variety of diseases. For example, it has been reported that both message and protein expression of the exchanger is diminished in patients with primary biliary cirrhosis, a disease in which the cholangiocyte is the target cell (39). The secretion of bicarbonate into bile is strongly linked to increased efflux of chloride into the lumen and the conductance of apical CFTR (40). Moreover, the aquaporin family of water channel proteins also include mutations and abnormalities in transport that may result in nephrogenic diabetes insipidis (AQP2) (41), cataracts (AQP0) (42), Sjögren's syndrome (AQP5) (43, 44), or an impaired urinary concentration ability (AQP1) (45).
In conclusion, the data presented in this study, together with our previous observations (5, 6), support our proposed molecular model of ductal bile secretion. The model is now extended by the proposed existence of a biochemically and morphologically characterized novel transporting organelle in cholangiocytes that appears to contain proteins for water and chloride transport, flux proteins that play integral roles in hormone-induced ductal bile secretion. Clearly, additional studies will be necessary to fully elucidate the quantitative relevance of our observations to total bile formation.
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FOOTNOTES |
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** To whom correspondence should be addressed: Center for Basic Research in Digestive Diseases Mayo Medical School, Clinic and Foundation, 200 First St., SW, Rochester, MN 55905. Tel.: 507-284-1006; Fax: 507-284-0762; E-mail: larusso.nicholas{at}mayo.edu.
1 The abbreviations used are: AQP, aquaporin; BDL, bile duct ligation; CFTR, cystic fibrosis trans-membrane regulator; AE2, chloride/bicarbonate exchanger.
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ACKNOWLEDGMENTS |
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REFERENCES |
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