1 Internal Medicine II, Nagoya University School of Medicine, Showa-ku, Nagoya 466-8550, and 2 National Institute for Physiological Sciences, Myodaiji, Okazaki 444-8585, Japan
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ABSTRACT |
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The aquaporin (AQP) family of
water channels is distributed ubiquitously in many epithelia and plays
a fundamental role in transmembrane water transport. The aim of this
study is to identify the water transport pathway in pancreatic duct
cells where most of the HCO
isolated pancreatic duct; mercury-sensitive water channel; water permeability; fluid secretion
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INTRODUCTION |
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THE EXOCRINE PANCREAS
SECRETES a large volume of HCO
Discovery of aquaporins (AQPs) and their ubiquitous distribution in many secretory or absorptive epithelia (2, 21, 28, 34) strongly suggest that AQP water channels may also be responsible for water transport in pancreatic ducts. So far 10 AQPs (AQP0-AQP9) have been identified in mammals, but none of them have been identified in pancreatic duct cells. AQP1 immunoreactivity was present in the capillary endothelium of the rat pancreas (9, 27) and the apical and basolateral membranes of human acinar cells (8). The latter finding, however, was not confirmed in other species. In rat acinar cells, the presence of AQP8 was demonstrated by both in situ hybridization (18) and immunohistochemistry (9). These observations suggest that expression of known AQPs in duct cells may be too low to be detected by the present techniques because duct cells are scarce (16).
Invention of a technique for isolating rat interlobular ducts by Argent et al. (1) now allows investigation of the function of pancreatic duct cells (3). After a short-term culture, both ends of the isolated interlobular ducts seal spontaneously and the luminal space is separated from the bathing solution by ductal epithelial cells. A combination of microfluorometry, digital videomicroscopy, and micropuncture techniques enables us to continuously monitor the net ion and fluid transport into the duct lumen as well as the localization of channels and transporters (12-15, 17). In this study, using isolated rat pancreatic duct preparations, we have measured osmotic water permeability of duct membranes and have obtained evidence for water channels sensitive to mercuric chloride, an agent known to block AQP water channels (2, 10, 29, 33, 36). Furthermore, we have extracted cellular mRNA from isolated ducts to minimize mRNA from extraductal cells and have confirmed the expression of AQP1 mRNA and protein in rat interlobular ducts.
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MATERIALS AND METHODS |
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Animals. Male Sprague-Dawley rats were obtained from Japan SLC (Hamamatsu, Japan). All protocols were approved by the Animal Use Committees of Nagoya University and the National Institute for Physiological Sciences.
Materials. Reagents used and their manufacturers were: Cell-Tak (Becton Dickinson Labware, Bedford, MA), TRIzol reagent (Life Technologies, Rockville, MD), first-strand cDNA synthesis kit (Boehringer Mannheim), glyceraldehyde 3-phosphate dehydrogenase (GAPDH)-specific primers (Clontech, Palo Alto, CA), rabbit anti-rat AQP1 antibody and control antigenic peptide (Alpha Diagnostic, San Antonio, TX), rabbit anti-rat AQP5 antibody (5) (a generous gift from Dr T. Yamamoto, Niigata University, Japan), biotinylated goat anti-rabbit IgG (Jackson Immunoresearch, West Grove, PA), ENVISION/horseradish peroxidase (HRP)-labeled anti-rabbit IgG (DAKO), elite ABC kit (Vector, Burlingame, CA), diaminobenzidine tetrahydrochloride (DAB; Dojindo, Japan), seaplaque agarose (FMC Bioproducts, Rockland, ME), and secretin (Peptide Institute, Minoh, Japan). All other chemicals were purchased from Sigma (St. Louis, MO) unless otherwise indicated.
Isolation of rat pancreatic duct. Rats (200-250 g) were killed by cervical dislocation. The body and the tail of the pancreas were removed, and interlobular ducts (diameter 100-150 µm) were isolated by collagenase digestion and microdissection (1, 15). Ducts were cultured at 37°C in 5% CO2 in air for 12-18 h, during which time both ends sealed spontaneously.
Water permeability of isolated pancreatic duct segments. The sealed ducts were stored at 4°C in the isotonic HEPES-buffered solution before use. The isotonic HEPES-buffered solution contained (in mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and 10 HEPES (290 mosM). The hypotonic HEPES-buffered solution contained (in mM): 67.5 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 D-glucose, and 10 HEPES (145 mosM). The solutions were equilibrated with 100% O2 and were adjusted to pH 7.4 with HCl at room temperature. The luminal volume change after exposure to the hypotonic solution was measured by a modification of the methods described previously (14, 31). The ducts were attached to the glass coverslips precoated with Cell-Tak and were superfused at 22°C on the stage of an inverted microscope. The bright-field images of the duct were obtained at 5-s intervals using a charge-coupled device camera.
The initial values of the length (Lo), diameter (2Ro), and image area (Ao) of the duct lumen were measured in the first image. Lo and Ro of all the ducts used for experiments were 396.3 ± 16.8 and 57.6 ± 2.4 µm (n = 30), respectively. The luminal surface area of the epithelium was taken to be 2
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Measurement of the fluid secretory rate.
Sealed ducts were superfused with the standard
HCO1 · mm
2).
RT-PCR. Total cellular RNAs were extracted from the whole pancreas and freshly isolated pancreatic duct segments by homogenization in TRIzol reagent. cDNA was reverse-transcribed from total cellular RNA using the first-strand cDNA synthesis kit according to the manufacturer's instructions. PCR amplification was performed using the following primers:
AQP1 sense, 5'-GGACAATGTGAAGGTGTCACTGGC-3'; antisense, 5'-CCAGAAAATCCAGTGGTTTGAGAA-3' (500 bp); AQP2 sense, 5'-GGGCCAGCTCCCCACCCTCTG-3'; antisense, 5'-ATCATCAAACTTGCCAGTGAC-3' (500 bp); AQP3 sense, 5'-GTTCCGTGGCTCAAGTGGTGC-3'; antisense, 5'-GCCCATGGAGGTCCCAATGAC-3' (500 bp); AQP4 sense, 5'-CCATTAACTGGGGTGGCTCAG-3'; antisense, 5'-AACTGCAGGGCCAAAGGATCG-3' (500 bp); AQP5 sense, 5'-CAGCACTCAAGTGGCCCTCGGCTC-3'; antisense, 5'-AAAGATCGGGCTGGGTTCATGGAA-3' (500 bp); AQP6 sense, 5'-ACGTTTTCTTTGGTGTGGGCT-3'; antisense, 5'-AGGGCCGAAGGAGCGAGCTGG-3' (500 bp); AQP7 sense, 5'-TTGGCCTTGGTTCCGTGGCTC-3'; antisense, 5'-GGACACCCCAAGAACGCAAAC-3' (500 bp); AQP8 sense, 5'-GAGCAGTACATACAACCGTGTGTG-3'; antisense, 5'-CCAGTAGATCCAATGGAAGTCCCA-3' (600 bp); AQP9 sense, 5'-TTATAATGATTGTCCTTGGAT-3'; antisense, 5'-GATCAGGAGGCTAATGACAAC-3' (500 bp). The PCR protocol was: 94°C, 30 s; 65°C, 30 s; 72°C, 30 s; 35 cycles. Primers were derived from published AQP sequences with GenBank accession nos. L07268 (AQP1), D13906 (AQP2), D17695 (AQP3), U14007 (AQP4), U16254 (AQP5), AF083879 (AQP6), AB05507 (AQP7), AB005547 (AQP8), and AB013112 (AQP9). Templates for positive controls were cDNAs prepared from the kidney (AP1, AQP2, AQP3, and AQP6), brain (AQP4), submandibular gland (AQP5), testis (AQP7 and AQP8), and liver (AQP9). GAPDH specific primers (452 bp) were used for the positive controls. PCR products were subjected to electrophoresis on 2% agarose gel.Immunohistochemistry. Under deep anesthesia by pentobarbital, 6-wk-old rats were killed by intracardiac perfusion with Ringer solution followed by 4% paraformaldehyde containing 0.2% picric acid in 0.1 M phosphate buffer (pH 7.2). Ducts isolated as described above were immersed in 4% paraformaldehyde in 0.1 M phosphate buffer immediately after isolation or after overnight culture and fixed for 4 h at 4°C. After being washed with PBS, the ducts were embedded in 3.5% agarose/PBS. Specimens were immersed in 10, 20, and then 30% sucrose in PBS at 4°C for 4-18 h, and then frozen as described previously (6). Sections of 10-µm thickness were cut with a cryostat (model 2800E, Yung Frigocut) and mounted on albumin-coated glass slides. Sections were then incubated with 1% H2O2 in methanol for 30 min at room temperature to block endogenous peroxidase activity, washed with PBS three times, and preincubated with 10% normal goat serum-0.05% NaN3 in PBS for 30 min at room temperature. Then sections were incubated with rabbit anti-AQP1 (0.5-1 µg/ml) or anti-AQP5 (1:750 dilution) antibody in PBS containing 1% BSA-0.05% NaN3 at 4°C overnight. After being washed three times with PBS, sections were incubated with ENVISION/HRP-labeled anti-rabbit IgG for 2-3 h at room temperature or biotinylated anti-rabbit IgG (5 µg/ml of 1% BSA-0.05% NaN3, in PBS) for 2 h followed with ABC complex for 2 h. The peroxidase reaction was developed with 0.02% DAB in 0.05 M Tris · HCl (pH 7.6) containing 0.01% H2O2 for 5 min. After ethanol dehydration, sections were mounted and examined with a microscope equipped with Normarsky optics (model AX70, Olympus). For the control, sections were incubated without primary antibody or incubated with antibody preabsorbed with 2-5 µg/ml of antigenic synthetic peptides.
Statistical analysis. Data are presented as means ± SE. Statistical analysis was carried out by analysis of variance followed by Dunnet's procedure for multiple comparisons. P < 0.05 was considered significant.
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RESULTS |
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Osmotic water permeability of isolated interlobular ducts.
To examine the osmotic water permeability of the ductal epithelium, the
isolated duct segments were initially superfused with the isotonic
HEPES-buffered solution (290 mosM) at 22°C. Figure 1A shows the changes in the
luminal area after switching the bathing solution to the hypotonic
HEPES-buffered solution (145 mosM). The luminal area rapidly increased
by 17.6 ± 1.7% (n = 5) in the first 30 s
after the exposure to the hypotonic solution, indicating that the NaCl
gradient caused water influx into the luminal space. Pf in the initial 5 s was 161.6 ± 21.1 µm/s. The luminal area reached a plateau after 30 s.
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Effects of basolateral HgCl2 on osmotic water permeability. To examine the effects of the basolateral HgCl2 on the osmotic water movement, the ducts were preincubated in the standard isotonic bath solution containing 0.3 or 3 mM HgCl2 for 10 min. The increase of the luminal area 30 s after the exposure to the hypotonic solution was reduced by 34.7% with 0.3 mM HgCl2 and by 78.4% with 3 mM HgCl2 (Fig. 1A). Pf was significantly reduced to 90.6 ± 19.7 µm/s (n = 5) with 0.3 mM HgCl2 and to 34.8 ± 20.2 µm/s (n = 5) with 3 mM HgCl2 (Fig. 1B).
Effects of the luminal HgCl2 on osmotic water
permeability.
To examine the effects of the luminal HgCl2 on the osmotic
water movement, the duct lumen was micropunctured and the luminal fluid
was replaced with the isotonic HEPES-buffered solution with or without
HgCl2. To avoid the external contact of the basolateral membrane with HgCl2, the ducts were continuously superfused
with the standard isotonic solution during the procedure. After a
10-min preincubation period, the bathing solution was switched to the hypotonic solution. The increase of the luminal area at 30 s was reduced by 66.4% with 0.3 mM HgCl2 in the lumen and by
87.6% with 3 mM HgCl2 (Fig.
2A). Pf
(232.4 ± 47.5 µm/s) was significantly reduced to 49.2 ± 10.9 and 16.0 ± 15.9 µm/s by 0.3 and 3 mM HgCl2 in
the lumen, respectively (Fig. 2B).
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Effects of basolateral and luminal HgCl2 on fluid
secretion.
In the presence of HCO1 · mm
2
(n = 4, P < 0.05). Basolateral
applications of HgCl2 (3 mM) abolished the
secretin-stimulated fluid secretion (0.02 ± 0.08 nl · min
1 · mm
2,
P < 0.05) (Fig. 3). When
the duct lumen was filled with the HCO
1 · mm
2, n = 4).
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AQPs in whole pancreas and isolated interlobular duct cells.
As shown in Fig. 4, amplified DNA
fragments for AQP1 and AQP8 and smaller amounts of AQP5 and AQP7
transcripts were clearly detected in the whole pancreas. These
transcripts were identical in size to those obtained from the kidney
(AQP1), submandibular gland (AQP5), and testis (AQP7, AQP8) RNAs as
templates. Other AQP (AQP2, AQP3, AQP4, AQP6, and AQP9) transcripts
were not detected. A large amount of AQP1 and smaller amounts of AQP5
were detected in the isolated interlobular ducts, suggesting that
interlobular duct cells contain AQP1 and AQP5 transcripts.
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AQP1 immunoreactivity in situ and in the isolated ducts.
To confirm the expression of AQP1 and AQP5 proteins in the rat
pancreatic duct cells, we then examined the distribution of AQP1 and
AQP5 with affinity-purified antibodies. The isolated ducts, including
the lumen, epithelial cells, and periductal connective tissues of
20-30 µm were ~65-110 µm in diameter. These correspond to the medium- to larger-size interlobular pancreatic ducts. In this
size of ducts, most of the epithelial cells were immunopositive to
anti-AQP1 antibody in situ. AQP1 immunoreactivity was observed in the
periphery of the ductal epithelial cells, especially in the basolateral
side (Fig. 5, A and
B). AQP1 immunoreactivity was also observed in the luminal
membrane of the duct cells. The intensity of the immunoreaction,
however, varied from cell to cell. The capillaries in the connective
tissue were intensely immunostained with anti-AQP1 antibody. In the
isolated ducts, immunoreactivity was observed in both the luminal and
basolateral sides of the duct cells (Fig. 5D). Capillaries
that remained in the connective tissues of the isolated ducts showed
intense immunostaining (Fig. 5, D and E, small
arrows). AQP1 immunoreactivity was similar both in the isolated ducts
fixed immediately after isolation and those fixed after overnight
culture (data not shown). The AQP1 immunoreactivity both in situ and in
isolated ducts was abolished with the preabsorption of the antibody
with 2-5 µg/ml of the synthetic antigenic peptide (Fig. 5,
C and F).
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DISCUSSION |
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The rat pancreas was chosen for the present investigation, because both the physiological function of pancreatic ducts and the AQP proteins are well characterized in this species. Using isolated interlobular ducts, we have succeeded for the first time in demonstrating that 1) AQP-type water channels are present in both the basolateral and luminal membranes of rat interlobular duct cells, 2) the mercury-sensitive water channel pathway accounts for ~90% of the water transport into the lumen, and 3) AQP1 of the known AQPs in the luminal and basolateral membranes appear to be the main water channels in interlobular ducts.
Calculated osmotic water permeability of the interlobular duct epithelium in this study (160-230 µm/s) was comparable with those reported in other epithelial tissues where AQPs were expressed. In cholangiocytes, where AQP1 and AQP4 were expressed, Pf was 50 µm/s (30). Pf in distal airways of the guinea pig was 40-50 µm/s (4). In isolated rat intrahepatic bile ducts, Pf ranged from 100 (outward) to 480 (inward) µm/s depending on the direction of the osmotic gradients (24). In contrast, the water permeability (10.6 µm/s) of acinar cell membrane (9), where AQP8 was expressed, was much smaller (4.6-6.6%) than that observed in the duct epithelium. Acinar cells and duct cells in the rat pancreas comprise 77 and 11% of the total cells, respectively (16). Assuming that 80% of fluid was derived from duct cells and the remaining 20% from acinar cells, water transport via a single acinar cell would be only 3.6% of that via a single duct cell. It seems, therefore, that the water permeabilities of the ducts and acini are in reasonably good agreement with water movements across the respective cell membranes.
Mercury-sensitive water channels appear to be present in both the basolateral and luminal membranes of duct cells, since both basolateral and luminal applications of HgCl2 induced a concentration-dependent inhibition of osmotic water permeability of the ducts (Figs. 1 and 2). This finding agrees with the present immunohistochemical observation that AQP1 immunoreactivity was present in the luminal and basolateral membranes of the isolated interlobular ducts (Fig. 5, D and E). A remarkable inhibition of Pf (>90%) by the luminal HgCl2 suggests that water movement in the duct epithelium is mainly transcellular via water channels. HgCl2 is known to block the water permeability by reacting with the Cys189 residue situated close to the narrowing of the water pore of the AQP1 molecule (25, 29, 36). Other AQPs, except AQP4 and AQP7, are also sensitive to mercury reagents (10). DMSO is another blocker of the water channel (33). Preincubation with a high concentration (500 mM) of DMSO inhibits the water permeability of CHIP28 (AQP1) expressed in ovary cells (19) and of AQP8 relocalized to the plasma membrane of hepatocytes by dibutyryl cAMP (7). The inhibitory mechanism of DMSO appears to be different from that of HgCl2, but the details are not known (33). AQP3 and AQP9, although they were not expressed in pancreatic duct cells, are also sensitive to phloretin, a classic inhibitor of urea transport (11, 32). Thus at present, the known inhibitors of the water and solute permeability are not specific and, therefore, the AQP molecules cannot be characterized by their inhibitors.
Fluid secretion evoked by secretin, a physiological secretagogue, was
almost completely abolished by a basolateral or luminal application of
HgCl2 (Fig. 3), which is consistent with the notion that
water moves via water channels after an osmotic gradient created by
active bicarbonate transport. However, the interpretation of these data
requires caution, because HgCl2 may affect not only membrane water channels but also other ion channels and transporters involved in HCO
Previous immunohistochemical studies of the rat pancreas detected AQPs in acini (AQP8) and capillaries (AQP1) but not in duct cells (9, 18, 27). In this study, to identify the AQP transcripts expressed in duct cells, RT-PCR was carried out using the isolated interlobular ducts as template. AQP1 and AQP5, but not AQP7 and AQP8 present in the whole pancreas, were detected in the isolated ducts. In agreement with a previous immunohistochemical study (27), the capillaries in the surrounding connective tissues of interlobular ducts were intensely stained by AQP1 antibody. Hence, AQP1 transcripts in the isolated ducts were, in part, derived from the capillary endothelium.
Apart from endothelial cells, the epithelial cells in medium- to larger-size interlobular pancreatic ducts were the only cells in the pancreas that showed a positive reaction to AQP1 antibody (Fig. 5). In rats (27), unlike humans (8), acinar cells were immunonegative for AQP1. The dense concentration of AQP1 immunoreactivity in the periphery of duct cells indicates that AQP1 molecules are localized in the luminal and basolateral membranes. The apparent order of the intensities of AQP1 immunoreactivity was the endothelial cell membranes greater than the luminal and basolateral duct membranes, which probably reflects the order of amounts of AQP1 protein expressed and hence water movements across these membranes. Because the distribution of AQP1 immunoreactivity in the isolated condition was similar to that in situ, the water permeability characteristics observed in the isolated duct would be very close to those in vivo.
Although AQP5 transcript was detected in pancreatic ducts by RT-PCR (Fig. 4), the present affinitypurified antibody failed to detect AQP5 immunoreactivity in the duct epithelium. In agreement with previous studies (5, 26), pneumocytes and acini of the submandibular gland were clearly stained with this antibody. Therefore, expression of AQP5 protein in the pancreatic duct may be too low to be detected by the present immunohistochemical techniques.
Recently, Ma et al. (20) found that neither pancreatic fluid secretion nor biliary secretion was affected in AQP1 knockout mice, although amylase and lipase secretion stimulated with secretin and CCK were significantly reduced. This observation is at variance with our present finding on rat pancreatic ducts and earlier reports on the biliary system in rats and humans that AQP1 was expressed in the epithelium of these tissues (27, 30). The reason for the apparent discrepancy is not known, because it has not been studied whether AQP1 is normally expressed in the mouse biliary and pancreatic duct systems. It has been shown that there are species variations of AQPs expressed in the gastrointestinal system (21). Therefore, unlike rats, AQP1 may not be expressed in the mouse biliary and pancreatic ducts.
In summary, we have provided the first evidence for the presence of mercury-sensitive water channels in both luminal and basolateral membranes of rat pancreatic ducts from which most of the water in the pancreatic juice is derived. The calculated osmotic water permeability was consistent with the existence of functional water channels in duct cells. AQP1 molecules in both the luminal and basolateral membranes appear to be the main water pathway in rat interlobular ducts, although there remains the possibility that yet unidentified AQPs may be expressed.
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ACKNOWLEDGEMENTS |
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We thank Drs. V. Wray and K. Ishibashi for their suggestions and N. Yamaguchi and K. Tuchiya for their technical assistance.
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FOOTNOTES |
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This work was supported by grants from the Pancreas Research Foundation and the Ministry of Education, Science, Technology, Sports, and Culture of Japan.
Address for reprint requests and other correspondence: S. Naruse, Internal Medicine II, Nagoya Univ. School of Medicine, 65 Tsurumai-cho, Showa-ku, Nagoya 466-8550, Japan (E-mail snaruse{at}med.nagoya-u.ac.jp).
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.00198.2001
Received 10 May 2001; accepted in final form 5 November 2001.
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