From the Instituto de Fisiología
Experimental, Consejo Nacional de Investigaciones Científicas y
Técnicas (CONICET), Universidad Nacional de Rosario,
Rosario, Santa Fe, Argentina 2000 and 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
Received for publication, October 16, 2000, and in revised form, January 11, 2001
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ABSTRACT |
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We previously found that water transport across
hepatocyte plasma membranes occurs mainly via a non-channel mediated
pathway. Recently, it has been reported that mRNA for the water
channel, aquaporin-8 (AQP8), is present in hepatocytes. To further
explore this issue, we studied protein expression, subcellular
localization, and regulation of AQP8 in rat hepatocytes. By subcellular
fractionation and immunoblot analysis, we detected an
N-glycosylated band of ~34 kDa corresponding to AQP8 in
hepatocyte plasma and intracellular microsomal membranes. Confocal
immunofluorescence microscopy for AQP8 in cultured hepatocytes showed a
predominant intracellular vesicular localization. Dibutyryl cAMP
(Bt2cAMP) stimulated the redistribution of AQP8 to plasma
membranes. Bt2cAMP also significantly increased hepatocyte
membrane water permeability, an effect that was prevented by the water
channel blocker dimethyl sulfoxide. The microtubule blocker colchicine
but not its inactive analog lumicolchicine inhibited the
Bt2cAMP effect on both AQP8 redistribution to cell surface
and hepatocyte membrane water permeability. Our data suggest that in
rat hepatocytes AQP8 is localized largely in intracellular vesicles and
can be redistributed to plasma membranes via a microtubule-depending,
cAMP-stimulated mechanism. These studies also suggest that aquaporins
contribute to water transport in cAMP-stimulated hepatocytes, a process
that could be relevant to regulated hepatocyte bile secretion.
Bile is formed primarily by hepatocytes and subsequently delivered
to the bile ducts where it is modified by cholangiocytes (i.e. the epithelial cells that line the bile ducts). Bile
secretion by hepatocytes involves the active transport of solutes
followed by the passive movement of water into the bile canaliculus in response to osmotic gradients created by these solutes (1, 2). Although
a substantial amount of data have been published about the molecular
identification of solute transporters and the mechanisms regulating
solute transport by hepatocytes (3), little attention has been focused
on the mechanistic and regulatory aspects involved in hepatocyte water transport.
Water can cross cellular plasma membranes through the lipid portion of
the bilayer by a diffusion mechanism or through aquaporin water
channels. Aquaporins, a family of recently identified integral membrane
proteins, increase cell membrane water permeability facilitating rapid
movement of water in response to osmotic gradients (4, 5).
We previously found based on biophysical and molecular biology studies
that water transport across hepatocyte plasma membranes occurs mainly
via a non-channel mediated pathway (6). As this observation seems to be
in contradiction with the recent identification of transcript for the
water channel aquaporin-8
(AQP8)1 in hepatocytes
(7-9), we further explored this issue by studying the protein
expression, subcellular localization, and possible regulation of AQP8
water channels in isolated rat hepatocytes.
Isolation and Incubation of Hepatocytes--
Hepatocytes were
isolated from livers of male Wistar rats by collagenase perfusion and
mechanical disruption as described previously (10). Hepatocytes
suspended in Krebs-Ringer-Hepes buffer, pH 7.4, were incubated at
37 °C according to these protocols: (a) for 10 min in the
presence of 0 or 100 µM dibutyryl cAMP
(Bt2cAMP; Sigma); (b) for 1 h in the
presence of 50 µM colchicine or lumicolchicine (Sigma)
and then for an additional 10 min in the presence of 0 or 100 µM Bt2cAMP. Subcellular fractionation and
water transport studies were carried out after these treatments. Cell
viability (assessed by trypan blue exclusion) was always >85% and not
affected by the treatments.
Short-term Culture of Hepatocytes--
Freshly isolated
hepatocytes were suspended in Leibovitz 15 medium (L-15; Life
Technologies, Inc.), cultured on collagen-coated coverslips, and
maintained at 37 °C for 4 h in an air atmosphere. Incubations
were performed according to these protocols: (a) for 10 min
in the presence of 0 or 100 µM Bt2cAMP;
(b) for 1 h in the presence of 50 µM
colchicine or lumicolchicine and then for an additional 10 min in the
presence of 0 or 100 µM Bt2cAMP. Confocal immunofluorescence microscopy was carried out after these treatments.
Preparation of Subcellular Membrane Fractions--
Membrane
fractions enriched in plasma or intracellular microsomal membranes were
prepared from hepatocytes by differential centrifugation as previously
described by us (11). Briefly, cells were washed and sonicated in 0.3 M sucrose containing 0.1 mM
phenylmethanesulfonyl fluoride and 0.1 mM leupeptin
(Sigma). The plasma membrane fraction was obtained by centrifugation at 200,000 × g for 60 min on a discontinuous 1.3 M sucrose gradient. After removing the plasma membrane
band, the sucrose gradient was sonicated, diluted to 0.3 M,
and centrifuged at 17,000 × g for 30 min. The
resulting supernatant was centrifuged at 200,000 × g
for 60 min to yield the intracellular microsomal membrane fraction.
Fractions enriched in plasma or intracellular microsomal membranes were
also prepared from homogenates of colon mucosa and total liver
following the same procedure. For experiments in Fig. 1, hepatocytes as
well as total liver, colon mucosa, kidney, and lung tissues from normal
rats were homogenized in 0.3 M sucrose containing protease
inhibitors, subjected to low-speed centrifugation to obtain post
nuclear supernatants, and then subjected to centrifugation at
200,000 × g for 60 min to yield "total membranes."
Proteins in membrane fractions were assayed according to Lowry et
al. (12) using bovine serum albumin as standard. Alkaline
phosphatase activity (a plasma membrane marker) was assessed using a
commercially available enzyme kit (Sigma).
Immunoblotting--
Solubilized membrane fractions were
subjected to 12% SDS-polyacrylamide gel electrophoresis and
transferred to polyvinyl difluoride membranes (PerkinElmer Life
Sciences). After blocking, blots were incubated overnight at
4 °C with rabbit affinity-purified antibodies against AQP8 (1 µg/ml; Alpha Diagnostics International, San Antonio, TX). The blots
were then washed and incubated with horseradish peroxidase-conjugated
goat anti-rabbit immunoglobulin (Promega), and bands were detected by
enhanced chemiluminescence detection system (ECL; Amersham Pharmacia
Biotech). Autoradiographs were obtained by exposing polyvinyl
difluoride membranes to Kodak XAR film, and the bands were quantitated
by densitometry (Shimadzu CS-9000). To control for non-specific
reactions, blocking experiments were performed by using an antibody
preabsorbed with a 10-fold molar excess of AQP8 immunizing peptide
(Alpha Diagnostics International). Selected samples were digested with
10 µg/ml peptide/N-glycosidase F (PNGase F; New England Biolabs).
Immunofluorescence and Confocal Microscopy--
After culturing
and treatment (see above), hepatocytes were fixed with 2%
paraformaldehyde for 10 min at room temperature, permeabilized with
0.2% Triton X-100 for 2 min, and incubated overnight at 4 °C with
rabbit affinity-purified AQP8 antibodies (10 µg/ml; Alpha Diagnostics
International). After washing, coverslips were incubated with Texas
Red-conjugated goat anti-rabbit secondary antibody (Molecular Probes)
for an additional hour and mounted with ProLong (Molecular Probes).
Fluorescence localization was detected by confocal microscopy with a
laser scanning microscope (Carl Zeiss LSM-510). Images were collected
with the same confocal settings in a particular set of experiments.
With these settings no autofluorescence was detected. Controls using
omission of primary or secondary antibodies revealed no labeling.
Images were processed using Adobe Photoshop software.
Water Transport Studies--
Following incubation as described
above, hepatocytes were suspended in Hepes (25 mM)-buffered
sucrose (300 mosM, pH 7.4), cooled at 10 °C, and
subjected to hypotonic challenge (100 mosM). The size of
hepatocytes was determined by quantitative phase contrast microscopy, a
methodology previously validated by us (6). The osmotic membrane water
permeability (Pf) was calculated from the initial rate of cell swelling as described previously (6). In some
experiments, before assessing water permeability, hepatocytes were
incubated for 5 min with 500 mM dimethyl sulfoxide
(Me2SO), an established aquaporin water channel
blocker (13, 14). At the concentration used, Me2SO did not
affect hepatocyte viability assessed by trypan blue exclusion. The
described AQP8 inhibitor, mercuric chloride (7-9), could not be used
in our studies because hepatocyte viability was significantly affected.
Aquaporin-8 Protein Expression in Hepatocytes
As shown in Fig. 1A,
immunoblotting analysis for AQP8 detected a band of ~34 kDa in total
liver and hepatocyte membranes. An identical band in location was
obtained for colon mucosa (another rat AQP8 message-expression tissue)
(7, 8), whereas no band was detected for kidney or lung tissues
(negative controls for rat AQP8 expression) (7, 8). The 34 kDa band was
absent when AQP8 antibody was preabsorbed with a molar excess of the immunizing peptide (not shown). After incubation with
N-glycosidase, the 34 kDa band shifted to the predicted
28 kDa molecular mass (Fig. 1B) indicating the
presence of N-glycosylation in the AQP8 molecule.
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
MATERIALS AND METHODS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
RESULTS
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES
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Fig. 1.
Expression and
N-glycosylation of AQP8 protein in hepatocytes.
A, anti-AQP8 immunoblot of total membranes from hepatocytes
or indicated tissues. Lanes were loaded with 30 µg of protein for
hepatocyte and liver and 100 µg of protein for colon, kidney, and
lung (see "Materials and Methods" for details). B,
anti-AQP8 immunoblot of total hepatocyte membranes (30 µg
protein/lane) before ( ) or after (+) digestion with
peptide/N-glycosidase F.
Subcellular Localization of Aquaporin-8 in Hepatocytes
Effect of Bt2cAMP--
Subcellular fractionation
followed by immunoblot analysis showed the AQP8 band in hepatocyte
plasma and intracellular microsomal membranes (Fig.
2). Similar results were obtained with
total liver, whereas in colon mucosa the immunoreactive band was only
present in plasma membranes (not shown). Exposure of cells to
Bt2cAMP resulted in an increase of AQP8 in hepatocyte
plasma membranes (125%, p < 0.05) and a simultaneous
decrease of AQP8 in microsomal membranes (58%, p < 0.05) (Fig. 2). Based on these data and the total proteins in each
hepatocyte membrane fraction, we estimated that under basal
(non-stimulated) conditions most of the hepatocyte AQP8 (~75%) would
reside in intracellular microsomal membranes; 25% would be in plasma
membranes. After Bt2cAMP treatment, AQP8 became predominant
in plasma membranes (i.e. ~60% of total).
Bt2cAMP did not alter the yields of total membrane
proteins, the specific activity of plasma membrane alkaline phosphatase
in hepatocyte membrane fractions, or the amount of AQP8 in total
hepatocyte membranes (data not shown). Confocal immunofluorescence
microscopy in cultured hepatocytes was in agreement with the
quantitative immunoblot analysis. Thus, AQP8 labeling was observed
mostly in intracellular vesicular structures throughout the cytosol;
plasma membranes exhibited very low labeling (Fig.
3A). Bt2cAMP
caused a decrease in AQP8 intracellular labeling and a
simultaneous increase in plasma membrane. (Fig.
3B).
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Together, these data are consistent with a predominant intracellular vesicular location of AQP8 in hepatocytes, and a cAMP-induced translocation of the water channel to the cell surface.
Effect of cAMP on the Osmotic Water Transport in
Hepatocytes--
The time course of relative hepatocyte volume in
response to an inwardly directed sucrose gradient is shown in Fig.
4A. The osmotic gradient
caused water influx and cell swelling. The rate of the swelling
response was significantly increased by Bt2cAMP. The water
channel blocker Me2SO inhibited Bt2cAMP-induced
increases in hepatocyte water transport. Corresponding
Pf values are shown in Fig. 4B.
Average hepatocyte volume (in isotonic media) was not affected by
Bt2cAMP.
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These data support the notion that cAMP promotes osmotic membrane water transport in hepatocytes via insertion of aquaporin water channels.
Effect of Colchicine on cAMP-induced Redistribution of
Aquaporin-8--
Colchicine but not its inactive analog
lumicolchicine markedly inhibited
the Bt2cAMP action on AQP8 redistribution to cell surface
(Figs. 5 and 6) or hepatocyte
Pf (Fig. 7).
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Together, these data suggest that the
cAMP-dependent redistribution of AQP8 from intracellular
vesicles to the cell surface depends on microtubules.
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DISCUSSION |
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To our knowledge, this is the first study on the functional expression and regulation of AQP8 protein in mammalian cells. The major findings reported here relate to mechanistic and regulatory aspects involved in hepatocyte water transport. Our data, using isolated rat hepatocytes, suggest that the water channel AQP8 is localized largely in intracellular vesicles and can be redistributed to plasma membrane via a microtubule-dependent, cAMP-stimulated mechanism. Thus, hepatocytes would be able to regulate their membrane water permeability.
AQP8 has been identified recently (7-9), and its transcript has been found to be present in rat liver (7, 8). In situ hybridization studies suggest that AQP8 mRNA is present in hepatocytes; nevertheless, studies on protein expression were not undertaken (7). In the present study, we consistently found AQP8 protein expression in rat hepatocytes. Digestion with N-glycosidase indicated that AQP8 molecule is N-glycosylated in agreement with the predicted consensus site for N-linked glycosylation in the AQP8 deduced amino acid sequence (8).
Our biochemical and immunofluorescence studies indicate that hepatocyte AQP8 is primarily located within the cell, presumably in a vesicular compartment. AQP8 expression on cell surface is very low under basal (non-stimulated) conditions, suggesting a small contribution of this aquaporin to total membrane water permeability. This agrees with our previous observation that, in the basal state, water transport across hepatocyte plasma membranes occurs mainly via a non-channel-mediated pathway because AQP8 is sequestered within an intracellular compartment (6). Upon Bt2cAMP stimulation, intracellular AQP8 is re-localized to plasma membrane, thereby raising the cell surface water permeability and facilitating osmotic water transport.
Bt2cAMP is well known to activate protein kinase A. The
AQP8 molecule lacks consensus protein kinase A phosphorylation sites (8), which suggests that the effect of cAMP is not directed toward AQP8
protein itself but rather to protein mediators involved in vesicle
trafficking. Moreover, Bt2cAMP has also been found to
stimulate the vesicle trafficking to the hepatocyte plasma membrane of
several other transporters, i.e.
Cl/HCO3
exchanger (15),
canalicular bile acid transporter (16), organic anion transporter, mrp2
(17, 18), multidrug resistance protein transporter (19), and
basolateral Na+/taurocholate cotransporter (20). Our
experiments also show that Bt2cAMP-induced AQP8 vesicle
trafficking seems to be dependent on the integrity of microtubules as
observed for some of the mentioned solute transporters (15-18). Thus,
whether AQP8 is packaged along with solute transporters in the same
population of vesicles as has been observed for aquaporin-1 water
channels and cystic fibrosis transmembrane regulator Cl
channels in cholangiocytes (21) or are in separate vesicles that
respond to the same signal needs to be determined.
Cyclic AMP stimulates microtubule assembly by phosphorylation of microtubule-associated proteins via cAMP-dependent kinases (22). Thus, the effect of Bt2cAMP on AQP8 trafficking may be mediated by stimulation of microtubule-associated proteins.
Cyclic AMP is also able to induce synthesis of transporters in hepatocytes (23). Nevertheless, new aquaporin synthesis is unlikely to significantly contribute to cAMP-induced increase of plasma membrane AQP8 because total hepatocyte AQP8 was unaltered by Bt2cAMP. In addition, the stimulatory effect of cAMP was seen within a 10-min period; protein synthesis usually requires a longer time.
The fact that in colon mucosa AQP8 is not present in intracellular membranes suggests that this aquaporin is constitutively expressed in colon epithelia and that the subcellular distribution and regulation of AQP8 is tissue specific.
Bile duct epithelial cells or cholangiocytes express at least two other members of the aquaporin family of proteins, i.e. aquaporin-1, located mainly in the apical (luminal) membrane domain and in intracellular vesicles (11, 24, 25), and aquaporin-4, present only in the basolateral membrane domain (26). The cAMP-dependent hormone secretin regulates the subcellular distribution of aquaporin-1 by stimulating its exocytic insertion exclusively in the apical plasma membrane domain of cholangiocytes (25), a mechanism that seems to be involved in ductal bile formation. In contrast, aquaporin-4 in cholangiocytes is constitutively expressed in the basolateral plasma membrane domain (26). In hepatocytes, cyclic AMP-regulated targeting of membrane transporters has been reported to involve both the apical as well as the basolateral plasma membrane domains (15-20). Additional studies with different experimental approaches will be required to determine the polarized distribution and regulated vectorial trafficking of AQP8 in hepatocytes.
Rat hepatocytes also express another aquaporin water channel, i.e. aquaporin-9 (27). Whether this channel shares the subcellular location and regulation with AQP8 is a matter of further studies.
Immunohistochemistry studies for AQP8 in intact rat liver2 as well as membrane fractionation and immunoblotting of total liver homogenate showed that AQP8 associated mainly to an intracellular membrane compartment. These observations suggest that our findings in isolated cells are relevant to the in vivo situation. As mentioned, hepatic bile is thought to be formed by the passive movement of water from plasma to bile canaliculus in response to osmotic gradients established by the active secretion of solutes such as bile acids and glutathione (1, 2). Interestingly, hepatocyte bile secretion can be increased by cAMP and by hormones acting through the cAMP cascade, phenomena that are also inhibited by colchicine (28, 29). Thus, it is tempting to speculate that AQP8 could facilitate the osmotically driven water transport during cAMP-stimulated hepatocyte bile formation.
In conclusion, our data support the concept that rat hepatocytes are
able to modulate water permeability by inducing the
microtubule-dependent targeting of vesicles containing AQP8
water channels to the plasma membrane, a process that could be relevant
to regulated hepatocyte bile secretion.
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ACKNOWLEDGEMENTS |
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We thank X. M. Chen for assistance in confocal microscopy and E. Ochoa and J. M. Pellegrino for excellent technical help.
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FOOTNOTES |
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* This work was supported by Grant PICT 03589 from Agencia Nacional de Promoción Científica y Tecnológica (to R. A. M.), a grant from Consejo Nacional de Investigaciones Científicas y Técnicas (CONICET), Grant DK24031 from the National Institutes of Health (to N. F. L.), and a grant from the Mayo Foundation.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.
¶ To whom correspondence should be addressed: Instituto de Fisiología Experimental, Facultad de Ciencias Bioquímicas y Farmacéuticas Universidad Nacional de Rosario, Suipacha 570, 2000 Rosario, Santa Fe, Argentina. Tel.: 54-341-4305799; Fax: 54-341-4399473; E-mail: rmarinel@fbioyf.unr.edu.ar.
Published, JBC Papers in Press, January 17, 2001, DOI 10.1074/jbc.M009403200
2 P. Splinter and N. F. LaRusso, unpublished data.
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ABBREVIATIONS |
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The abbreviations used are: AQP8, aquaporin-8; Pf, osmotic membrane water permeability.
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