1 Liver Center, Yale University School of Medicine, New Haven, Connecticut 06520; and 2 Departments of Medicine and Physiology, Cardiovascular Institute, University of California San Francisco, San Francisco, California 94143
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ABSTRACT |
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The
mechanisms by which fluid moves across the luminal membrane of
cholangiocyte epithelia are uncertain. Previous studies suggested that
aquaporin-1 (AQP1) is an important determinant of water movement in rat
cholangiocytes and that cyclic AMP mediates the movement of these water
channels from cytoplasm to apical membrane, thereby increasing the
osmotic water permeability. To test this possibility we measured
agonist-stimulated fluid secretion and osmotically driven water
transport in isolated bile duct units (IBDUs) from AQP1 wild-type (+/+)
and null (/
) mice. AQP1 expression was confirmed in a mouse
cholangiocyte cell line and +/+ liver. Forskolin-induced fluid
secretion, measured from the kinetics of IBDU luminal expansion, was
0.05 fl/min and was not impaired in
/
mice. Osmotic water
permeability (Pf), measured from the initial rate of IBDU
swelling in response to a 70-mosM osmotic gradient, was 11.1 × 10
4 cm/s in +/+ mice and 11.5 × 10
4
cm/s in
/
mice. Pf values increased by ~50% in both
+/+ and
/
mice following preincubation with forskolin. These
findings provide direct evidence that AQP1 is not rate limiting for
water movement in mouse cholangiocytes and does not appear to be
regulated by cyclic AMP in this species.
aquaporins; cholangiocytes; bile formation; adenosine 3',5'-cyclic monophosphate
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INTRODUCTION |
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BILE DUCT EPITHELIAL CELLS (cholangiocytes) play an important role in bile formation by modifying the primary secretions from hepatocytes by a number of secretory and absorptive transport mechanisms (4, 5). The primary secretory product of cholangiocytes is a bicarbonate-rich fluid that is secreted in response to secretin (3, 24), bombesin (9), vasoactive intestinal peptide (8), and other agonists (4). Absorption of fluid and solutes from the biliary lumen is facilitated by the action of the sodium/hydrogen exchanger (NHE)3 (26), bile salt transporter (ISBT) (2, 13), and glucose transporter (SGLT-1) (14). Water moves into bile from cholangiocytes in response to a small osmotic gradient created by these transport processes. However, it remains unclear whether water moves into and out of the biliary lumen through the cell membrane, via paracellular routes, or through specific ~30-kDa protein channels known as aquaporins (AQP) (1, 17).
In epithelia in which there is a high rate of water flux, such as the kidney and gastrointestinal tract, water movement is facilitated by one or more of the ten AQPs identified in mammals (1, 17). Several AQPs have been described recently in rat hepatocytes (AQPs 0, 3, 8, and 9) (10, 29), although water is thought to cross hepatocyte cell membranes primarily by pathways other than water channels (32). AQP homologs have also been described in rat cholangiocytes including AQP1, which is localized in the cytoplasm and the apical membrane (20, 27), and AQP4, which is restricted to the basolateral membrane (21). A recent preliminary report of RT-PCR studies describes eight different AQP homologs in cholangiocytes (29). Unlike all other cell types in which AQP1 is constitutively expressed, studies by Marinelli et al. (20, 21) suggest that the expression of AQP1 on the apical luminal membrane of the cholangiocyte is regulated by the insertion of AQP1-containing vesicles in response to increases in cellular cAMP. These findings are based on studies in which osmotic water permeability coefficients have been obtained in isolated rat cholangiocytes prepared from bile duct-ligated rats. In those studies, the distribution of AQP1 has been determined by Western blot analysis of apical and basolateral membrane preparations obtained from rat cholangiocytes preparations before and after the exposure of rat cholangiocytes to the hormone secretin, a stimulant of cAMP in this tissue. Immunohistochemical studies for AQP1 in liver sections from rats treated with secretin also showed intensified staining in apical regions of cholangiocyte (22).
In many tissues, AQPs are not rate limiting for transcellular water movement (23), and, despite prior studies, it remains unclear whether AQPs are rate limiting for transcellular water movement in the biliary epithelium. In biliary epithelium, the net flux of water is very small compared with epithelial tissues such as the kidney or salivary gland, in which these channels are necessary to facilitate the large fluxes of water that move across these epithelia (16, 28). In the present study, we have assessed water movement across isolated bile duct units (IBDUs) in the rat and mouse and used AQP1 knockout mice to define the contribution of AQP1 to active fluid secretion and osmotically driven water transport. IBDUs have been a useful in vitro model to study the mechanisms of fluid secretion and absorption from intact polarized cholangiocytes (25, 26).
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MATERIALS AND METHODS |
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Collagenase B was purchased from Roche Diagnostics
(Indianapolis, IN). DNase, hyaluronidase, protease inhibitors, BSA,
saponin, and forskolin were obtained from Sigma (St. Louis, MO).
Pronase was from Calbiochem (La Jolla, CA), Matrigel was from
Collaborative Biomedical, and MEM, -MEM, gentamycin, and Trizol were
from Gibco (Grand Island, NY).
Isolation of bile duct units.
AQP1 wild-type (+/+) and null (/
) mice (CD1 genetic background)
were generated as described previously (18) and maintained in the Yale Univ. Medical School animal care facility before use. All
mice were coded, and the genotype was unknown to investigators until
completion of the study. In addition, in selected experiments, IBDUs
were isolated from Sprague-Dawley rats (Charles River, Wilmington, MA).
Experimental protocols were approved by the Yale Animal Care and Use Committee.
Western blot analysis. Genotypes were confirmed by Western blot analysis from microsomal membrane preparations of mouse kidney with protease inhibitors by standard methods using an anti-AQP1 primary antibody diluted 1:2,500 overnight (rabbit polyclonal, Chemicon International, Temecula, CA) and an enhanced chemiluminescence (Amersham, Arlington Heights, IL) detection method. Western blot analyses were also determined in homogenates from a mouse cholangiocyte cell line, kindly provided by Dr. Y. Uneo (Sendai, Japan) (19).
Immunofluorescence.
Immunofluorescence microscopy was used to assess the presence of AQP1
in rat and mouse liver, rat kidney, and a mouse cholangiocyte cell
line. Once removed, livers were cut into small cubes and quick frozen
in liquid nitrogen-cooled freon before cryosectioning at 5 µm. Tissue
sections as well as the cholangiocyte cell line grown on glass
coverslips were fixed in 20°C acetone for 10 min, washed in PBS
containing 0.05% saponin (PBS/S), then blocked with 1% BSA for 30 min. Rabbit anti-AQP1 antibody was incubated on the tissue or cells for
2 h at 5 µg/ml, washed in PBS/S for 1 h, then incubated in
Cy2 or Cy3 anti-rabbit IgG secondary antibody (Jackson ImmunoResearch
Laboratories, West Grove, PA) for 1 h. After the slides were
washed in PBS, they were mounted with Vectashield (Vector Laboratories,
Burlingame, CA) and photographed using a Nikon E800 epifluorescence
microscope and Hamamatsu charge-coupled device camera.
RT-PCR. RT-PCR was done on a mouse cholangiocyte cell line. The strong expression of AQP1 in liver endothelial cells prevented reliable interpretation of similar data from IBDUs and isolated cholangiocyte preparations. RNA was isolated using the Trizol method, and first-strand cDNA was amplified using the Superscript system (Gibco-BRL). The AQP1 specific primers 5'-CATGGCCAGTGAAATCAAGAAGAAGC-3' (forward) and 5'-TAGATGCCCAGGC CAAGCCTCCTC-3' (reverse) were synthesized from the Yale Univ. Keck oligonucleotide facility. PCR was performed using 1.25 µM primers, 200 µM dNTP mix, 5 U Taq DNA polymerase, 1.5 mM MgCl2, 2 µl of first-strand DNA, and PCR buffer (20 mM Tris · HCl, 50 mM KCl). Samples were placed in a programmable thermal controller (M. J. Research), where they were heated to 94°C for 3 min to denature and annealed at 62°C and repeated 35 cycles. PCR products were confirmed by sequencing in the Keck Biotechnology Resource Laboratory, Yale Univ.
Experimental protocols.
After 48 h in culture, bile duct fragments were transferred on
glass coverslips to the stage of an inverted video microscope, where
they were perifused at a rate of l ml/min at 37°C in a 36-µl thermostatically controlled chamber (Warner Instruments, Hamden, CT) to
facilitate rapid perfusate exchange. Equilibration of perfusate under
these conditions was achieved within 5 s (98%). IBDUs from AQP1
+/+ and /
mice and normal rats were perifused with isotonic Krebs-Ringer bicarbonate (KRB; 300 mosM) for 10 min before
initiating the specific protocol.
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RESULTS |
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Immunoblot, PCR, and immunofluorescence.
Immunoblots of the crude membrane fraction from kidneys of +/+
mice showed bands at the predicted molecular sizes of nonglycosylated AQP1 (28 kDa) and glycosylated AQP1 (43 kDa; Fig.
1A). These bands were not
detected in kidneys from AQP1 /
mice. AQP1 was strongly expressed
on endothelial cells both in rat and mouse liver (Fig. 2). Because these cells are present in
the IBDU preparations, they prevented a direct assessment of AQP1 on
cholangiocytes by Western blot analysis. However, Western blot of
membranes from a mouse cholangiocyte cell line showed AQP1 protein
expression (Fig. 1A, right) and transcript
expression (Fig. 1B).
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Agonist-induced secretion and Pf.
The effects of forskolin on volume expansion in the IBDU model in rat
and mice are shown in Fig. 3 and
summarized in Table 1. There was no
significant difference in the choleretic response to forskolin in IBDUs
from AQP1 +/+ vs. /
mice when adjusted for differences in the size
of luminal space before the forskolin infusion. However, both the AQP1
wild-type and null mice responded to forskolin with lower rates of
secretion compared with rat IBDUs [49 ± 9 fl · min
1 · pl
1 baseline
volume for +/+ (n = 61) and 39 ± 9 fl · min
1 · pl
1 for
/
(n = 29) vs. 78 ± 12 fl · min
1 · pl
1
(n = 10) for rat IBDUs]. These choleretic effects of
forskolin confirm previously published observations on the effects of
cyclic AMP-mediated secretion in rat and mice IBDUs (10,
25). The luminal diameters of IBDUs used for these studies
averaged 28.7 ± 0.9 µm (n = 155) at 48 h.
Given that the cross-sectional diameters of these ducts are nearly
twice that value, these bile duct units are derived from the
interlobular ducts that are known to be responsive to secretin and
cyclic AMP-mediated secretion (12).
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DISCUSSION |
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The importance of AQPs in transcellular fluid movement is tissue dependent. In general, AQPs are thought to facilitate fluid movement in tissues in which rapid osmotic fluid movement is required, such as in kidney tubules and secretory glands (1, 17, 31). Although prior studies have identified AQP1 in rat cholangiocytes (10, 11, 20, 27), the role of AQPs as a rate-limiting determinant of net fluid secretion in intact bile ducts has not been examined. In the present study, we have used AQP1 knockout mice to assess the role of AQP1 on water movement in mouse cholangiocyte IBDUs following agonist-induced secretion and osmotic gradients.
Our findings provide direct evidence that AQP1 is not an important
determinant of Pf and thus fluid secretion in mouse
cholangiocytes. First, the choleretic response to forskolin was not
impaired in AQP1 null mice (Fig. 3 and Table 1). Second, Pf
in IBDUs from AQP1 /
mice was not different from that in IBDUs from
+/+ mice (Fig. 5). This finding is consistent with the
equivocal-to-weak AQP1 immunostaining in cholangiocytes from +/+ mice,
with strongly positive control staining in kidney (Fig. 2). Possibly,
other AQPs might be upregulated in this knockout model in the
cholangiocyte, although this is unlikely given the low
Pf of ~10
3 cm/s, suggesting lipid-mediated
water transport, and because other AQPs are not upregulated in the
kidney and other tissues in AQP1 null mice (31).
Previous studies have indicated that the expression of AQP1 on the
luminal membrane is stimulated by cyclic AMP in rat cholangiocytes (22, 27). Regulated AQP1 vesicular transport is discrepant with studies of AQP1 regulation in other tissues where this water channel is constitutively expressed (17). In addition,
protein kinase A phosphorylation sites have not been demonstrated in
AQP1, in contrast to AQP2, which is highly regulated by cyclic AMP in other epithelia (17). Indeed in a transfected cell culture
model, an AQP2 chimera containing the AQP1 COOH terminus fails to
undergo regulated vesicular transport (30). For these
reasons, we examined the effects of forskolin and secretin pretreatment
in rat and mouse IBDUs on the response to rapid changes in water
movement induced by hypotonicity. Forskolin is a potent stimulant of
the catalytic subunit of adenyl cyclase and is the strongest agonist for cyclic AMP-mediated signal-transduction events. Previous studies from our laboratory indicate that forskolin is a potent stimulus of
cyclic AMP-dependent secretion in rat IBDUs (25). If
cyclic AMP stimulated cytoplasmic to apical membrane movement of water channels in cholangiocytes, initial rates of volume expansion following
hypotonic media-induced water movement should increase. However, we
could find only a small, albeit significant, effect of forskolin
pretreatment on these two parameters, and this was observed in both
AQP1 +/+ and /
mice but not rat. Because these differences in
Pf values are small, it is unlikely that cyclic AMP would
have a functionally significant effect on passive water movement across
this epithelium. Previous studies demonstrated that secretin increased
Pf in isolated rat cholangiocytes (20, 22).
However, secretin did not increase Pf significantly in rat
or mice IBDUs in our experiments compared with controls. In addition,
we found that the Pf for rat IBDUs was consistently lower
than values observed in mouse IBDUs. Although the reasons for these
differences is not known, they could be explained by different levels
of expression of AQPs between these two species. Indeed, net water
fluxes across cholangiocyte epithelium vary considerably among species
(6) but are particularly low in rodents. Bile is largely
secreted from hepatocytes, and less than 10% of total bile productions
or less than ~100 µl/g of liver tissue per hour originates from
cholangiocytes in the rat. Thus rapid changes in water permeability
across this epithelium would not be expected. Indeed, significant
changes in net bile production have not been observed in the living
anesthetized AQP1 null mouse (15). This contrasts markedly
to the kidney epithelia and endothelia where fluid movement is several
orders of magnitude larger (17) and where the loss of AQP1
results in rapid dehydration due to increased urine volumes.
Pf values in this study were significantly lower than
previously reported for isolated rat cholangiocytes (20,
27). For this reason, we also measured the Pf in
normal rat IBDUs when water movement was stimulated by exposure to
hypotonic media to assess whether there were species differences
between rat and mouse. However, we obtained a similar range of
Pf measurements in the rat IBDUs (12.7 × 104 cm/s) as observed in the mouse model.
Differences in technique might account for the lower Pf values that we have observed. Our studies assessed movement of water across the cell and into the closed space of the IBDU lumen over a 60-s time period in response to the change in extracellular osmolarity, a process requiring water to cross several membrane barriers. Other studies measured Pf in single-cell preparations of cholangiocytes purified on immunomagnetic beads and exposed these cells to larger osmotic gradients for 5-30 s, in which more than one water channel must be contributing to the cell volume changes. Although we cannot exclude an influence from basolateral water channels on our measurements, luminal volume changes in response to hypotonicity should occur only by movement of water across the apical membrane or through paracellular pathways. We think it is unlikely that alterations in paracellular permeability account for the lower water permeability in our preparations, because Texas-red Dextran-40 is excluded from the lumens of most IBDUs (25) and higher, rather then lower, Pf values would be expected if leaky paracellular junctions had affected water movements. Thus our measurements would seem to directly reflect changes in apical water movement. It is also unlikely that our Pf values are affected by delays in fluid exchange in the chamber, because 98% of the perfusate fluid equilibrated within 5 s after the media was changed to the hypotonic buffer.
Together, our findings indicate that AQP1 is not a significant
determinant of water permeability in the mouse cholangiocyte and is not
rate limiting for forskolin-stimulated secretion. Furthermore, AQP1
is not regulated by cAMP in the mouse cholangiocyte. These observations
are consistent with studies in other cell types. Nevertheless, we
cannot exclude the possibility that other AQP homologs might be
upregulated in the AQP1 /
mouse and compensate for the absence of
AQP1 in these experiments and mask a cyclic AMP effect. This
possibility also seems unlikely, however, given the absence of such
adaptations in other tissues such as the kidney.
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ACKNOWLEDGEMENTS |
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We thank L. Quain for breeding and genotyping the transgenic mice and Dr. T. Ma for critical discussions.
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FOOTNOTES |
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This work was supported by United States Public Health Service Grants DK-25636 (to J. L. Boyer), DK-35124 (to A. S. Verkman and the Yale Liver Center cell isolation, morphology, and molecular biology cores), and DK-PO-34989. The development of the mouse cholangiocyte line was supported by grants-in-aid for scientific research from the Ministry of Education, Science, and Culture of Japan (09770339).
Address for reprint requests and other correspondence: J. L. Boyer, Yale Univ. School of Medicine, P.O. Box 208019, 333 Cedar St., New Haven, CT 06520-80019 (E-mail: james.boyer{at}yale.edu).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
10.1152/ajpgi.00540.2001
Received 26 December 2001; accepted in final form 19 April 2002.
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