1 School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom; and 2 Department of Cell Biology, Institute of Anatomy, University of Aarhus, DK-8000 Aarhus, Denmark
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ABSTRACT |
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Both the acinar and ductal cells of the pancreas secrete a near-isotonic fluid and may thus be sites of aquaporin (AQP) water channel expression. Northern blot analysis of mRNA from whole rat pancreas revealed high levels of AQP1 and AQP8 expression, whereas lower levels of AQP4 and AQP5 expression were just detectable by RT-PCR Southern blot analysis. Immunohistochemistry showed that AQP1 is localized in the microvasculature, whereas AQP8 is confined to the apical pole of the acinar cells. No labeling of acinar, ductal, or vascular tissue was detected with antibodies to AQP2-7. With immunoelectron microscopy, AQP8 labeling was observed not only at the apical membrane of the acinar cells but also among small intracellular vesicles in the subapical cytoplasm, suggesting that there may be regulated trafficking of AQP8 to the apical plasma membrane. To evaluate the contribution of AQPs to the membrane water permeability, video microscopy was used to measure the swelling of acinar cells in response to hypotonic stress. Osmotic water permeability was reduced by 90% following exposure to Hg2+. Since AQP8 is confined to the apical membrane, the marked effect of Hg2+ suggests that other water channels may be expressed in the basolateral membrane.
exocrine gland; fluid secretion; intracellular trafficking; water permeability
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INTRODUCTION |
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THE EXOCRINE
PANCREAS has a large capacity for fluid secretion. In humans,
~2.5 l of pancreatic juice are secreted into the duodenum each day.
At first approximation, the juice comprises two components: a
Cl-rich fluid secreted by the acinar cells, which
facilitates the transport of digestive enzymes to the duodenum, and a
copious HCO
Aquaporins (AQPs), a family of membrane proteins that functions as
water channels, have been identified in the plasma membranes of many
fluid-transporting epithelia and endothelia (1). The AQPs
are small integral membrane proteins with six hydrophobic, -helical,
membrane-spanning domains surrounding a highly selective aqueous pore
(1, 34). Currently, at least 10 AQPs have been identified
in mammals, each with a distinctive tissue distribution pattern. Of
these, AQP3, AQP4, AQP5, and AQP8 appear to be associated with exocrine
glands and also with the fluid-secreting epithelia of the eye and lung
(7, 19, 27).
AQP5, which was cloned from the rat submandibular gland (30), is expressed in the apical and canalicular membranes of salivary and lacrimal acinar cells (8, 11, 14, 24, 27). The importance of this water channel in salivary secretion is evidenced by the marked impairment of saliva production in AQP5 knockout mice (18). So far, no AQP has been identified in the basolateral membranes of salivary acinar cells. Basolateral channels have, however, been identified in lacrimal acinar cells and in the secretory epithelia of the airways and associated glands in which AQP5 is present in the apical membrane and AQP3 and/or AQP4 are found in the basolateral membrane (7, 25, 27).
Despite the existence of four likely locations for AQP water channels in the exocrine pancreas, namely the apical and basolateral membranes of both the acinar and ductal cells, none of the known AQPs had until recently been positively identified in this gland. AQP8, however, was cloned from rat pancreas and liver by Koyama et al. (15), and in situ hybridization studies suggest that this channel may be expressed in the acinar cells of the pancreas (15).
The aims of the present study were to systematically screen the rat pancreas for known AQPs by using Northern blot hybridization and RT-PCR as well as to determine their cellular and subcellular localization by immunohistochemistry and immunoelectron microscopy. Since many of the AQPs, including AQP8, are blocked by mercurial compounds, we also used video microscopy to examine the effect of Hg2+ on the rate of swelling of acinar cells in response to a hypotonic challenge. This has enabled us to evaluate the contribution of AQPs to the osmotic water permeability of the acinar cell plasma membrane.
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METHODS |
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RNA extraction.
Adult male Sprague-Dawley rats (Charles River Laboratories, Manston,
UK) were killed by cervical dislocation, and the relevant tissues were immediately dissected, frozen in liquid nitrogen, and
stored at 80°C. Total cellular RNA was extracted by the single-step guanidinium thiocyanate/acid phenol method (3). In the
case of the pancreas, the frozen tissue was first ground to a fine powder under liquid nitrogen using a pestle and mortar to minimize the
risk of RNA degradation from the activity of pancreatic RNases. Messenger RNA was isolated from the total cellular RNA using
oligo(dT)-coated Dynabeads according to the manufacturer's
instructions (Dynal).
Northern blot hybridization. Messenger RNA samples (3 µg/lane as determined by spectrophotometry) were subjected to electrophoresis on a 1.2% agarose-formaldehyde gel, transferred onto a Duralon nylon membrane (Stratagene, La Jolla, CA), and hybridized at 42°C overnight with 32P-labeled cDNA probes (Rediprime kit; Amersham Pharmacia Biotech, Little Chalfont, UK) corresponding to the coding regions of specific AQPs. Membranes were subsequently washed at high stringency and exposed to X-ray film overnight.
RT-PCR analysis.
First-strand cDNA was generated from 1-5 µg of total RNA using
SuperScript II RT (Life Technologies, Paisley, UK) according to the
manufacturer's instructions. The cDNA was then used for PCR with
various primer sets designed to amplify specific rat AQPs (Table
1). All primer sets were designed from
published sequence data and spanned introns to eliminate amplification
of genomic DNA. The PCR protocol consisted of an initial denaturation of 4 min at 94°C followed by 30 cycles of denaturation for 1 min at
94°C, annealing for 1 min at 55°C (50°C for AQP1), extension for
1 min at 72°C, and a final extension period of 8 min.
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Immunohistochemistry and immunoelectron microscopy. Previously characterized, affinity-purified polyclonal primary antibodies were used for AQP1-6 as follows: AQP1, LL266AP (33); AQP2, LL127AP (5, 23); AQP3, LL178AP (6); AQP4, LL182AP (32); AQP5 (14, 27); and AQP6 (36).
For AQP7, antibodies were raised in rabbits against a peptide corresponding to amino acids 259-269 in the COOH terminus of rat AQP7 (9) with a cysteine added to the amino terminal (NH2-CLIHAGIPPQGS-COOH). For AQP8, antibodies (RA 2277/1262AP) were raised against amino acids 249-263 in the COOH terminus of rat AQP8 (10, 15) with a cysteine added to the amino terminal (NH2-CLFIGDEKTRLILKSR-COOH). Pancreas was fixed for light microscopy (n = 3) by retrograde perfusion via the aorta with periodate-lysine-paraformaldehyde (0.01 M NaIO4, 0.075 M lysine, and 2% paraformaldehyde in 0.0375 M Na2HPO4 buffer, pH 6.2) or for immunoelectron microscopy (n = 3) with 4% paraformaldehyde in 0.1 M cacodylate buffer. For preparation of cryostat sections, tissues were cryoprotected in 25% sucrose (16, 36). Cryostat sections of pancreas (10 µm) were incubated overnight at 4°C with primary antibodies (see above), and labeling was visualized with a horseradish peroxidase-conjugated secondary antibody (P448, 1:100; DAKO, Glostrup, Denmark) (16, 36). Immunolabeling controls were performed using preabsorption of the antibodies with the immunizing peptide. For immunoelectron microscopic localization of AQP8, the frozen samples were freeze substituted in a Reichert AFS freeze substitution unit (16, 28, 29, 36). The samples were sequentially equilibrated over 3 days in methanol containing 0.5% uranyl acetate at a temperature that was gradually raised fromWater permeability measurements.
Adult male Sprague-Dawley rats were killed by halothane inhalation. The
pancreas was chopped into small pieces and incubated for 30 min at
37°C in a HEPES-buffered physiological saline solution (in mM: 104 Na+, 5 K+, 1 Mg2+, 111 Cl, 25 HEPES, and 15 glucose, pH 7.4) to which were added
70 U/ml collagenase (Type IV, Worthington), 0.12 mg/ml trypsin
inhibitor (type II-S, Sigma), 0.5 mM Ca2+, and 1% BSA
(fraction V, Sigma). After two washings, the fragments were shaken for
3 min at 37°C in Ca2+-free HEPES buffer supplemented with
3 mM EDTA. After being washed and resuspended in HEPES buffer
containing 2 mM Ca2+ and 1% BSA, the cells were
dissociated by repeated trituration through pipette tips of decreasing
size and filtered through a 75-µm nylon mesh. The cell suspension was
washed twice in HEPES buffer containing 2 mM Ca2+ and 4%
BSA, resuspended in 0.2 mM Ca2+ and 0.1% BSA, and stored
on ice until required.
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RESULTS |
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Northern blot analysis of AQP expression.
High-stringency Northern blot analysis of rat brain, kidney, pancreas,
submandibular gland, and testis with a 32P-labeled probe
for AQP8 (Fig. 1A) yielded a
strong signal of ~1.45-kb transcript size in pancreas, as shown
previously (15), and in testis which was included as a
positive control (9). No signals were seen in the other
tissues included on the Northern blot.
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RT-PCR analysis of AQP expression. For a more sensitive evaluation of AQP expression in the pancreas, a series of RT-PCR reactions was carried out using rat pancreas cDNA as the template. Specific primers were designed for AQP1-5 and AQP8 (Table 1), and cDNA samples from kidney, brain, submandibular gland, and testis were included in the PCR reactions as positive controls. All of the control reactions, in which DNA was replaced by water, proved negative.
As anticipated, AQP1 and AQP8 were readily detected in pancreas by RT-PCR (data not shown). Products of the expected size for AQP2 and AQP3 were amplified successfully from kidney, and their identities were confirmed by Southern blotting, but nothing was obtained from the pancreas cDNA using these primers (Fig. 2A). PCR products were obtained, however, by using the specific AQP4 and AQP5 primers. High-stringency Southern blot analysis (Fig. 2B) and subsequent sequence analysis of the subcloned PCR products indicated their identity.
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Localization of AQPs by immunohistochemistry.
Immunohistochemistry performed on cryostat sections of rat pancreas
revealed that AQP1 is strongly expressed in the microvasculature (Fig.
3, A and B). In
contrast, antibodies for AQP2-7 did not show any labeling of
acinar cells, ducts, or vascular structures at the light microscope
level (not shown). The antibody for AQP8, however, showed intense
staining of the apical domains of the pancreatic acinar cells (Fig.
4, A-D). The specificity
of the AQP8 antibody was confirmed by the negative result obtained when the antibody was preabsorbed with the immunizing peptide (Fig. 4,
E and F). There was no labeling of the pancreatic
ducts or vascular structures by the AQP8 antibody.
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Immunoelectron microscopic localization of AQP8.
To determine the subcellular localization of AQP8 in the pancreas,
immunoelectron microscopy was performed using Immunogold labeling of
sections prepared from pancreatic tissues from three normal Wistar
rats. The pancreatic tissues were embedded in Lowicryl HM20 by
cryosubstitution. AQP8 Immunogold labeling was observed in the
pancreatic acinar cells (Figs. 5 and
6) but not in other cell types. In the
acinar cells, AQP8 Immunogold labeling was associated with apical
plasma membrane domains (Figs. 5 and 6) and more abundantly with
intracellular vesicles in the subapical regions of the cell (Fig.
5B and Fig. 6). In contrast, no labeling of zymogen granules
was observed. Immunolabeling controls (n = 3) using
antibodies preabsorbed with excess immunizing peptide were all negative
(not shown).
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Water permeability of the acinar cell plasma membrane.
To determine the osmotic water permeability of the acinar cell plasma
membrane and examine its sensitivity to mercurial inhibition, the rate
of swelling of isolated acinar cells in response to hypotonic stress
was measured by video microscopy. When the extracellular fluid bathing
the cells was switched from an isotonic (290 mosM) solution containing
145 mM sucrose to a hypotonic (145 mosM) solution lacking the sucrose,
the volume of the acinar cells increased rapidly by 51 ± 4%
(mean ± SE; n = 10) over a period of 2 min and
more slowly thereafter (Fig. 7). From the
initial rate of increase in cell volume, and assuming an initial
osmotic gradient of 145 mosM, the water permeability of the acinar cell
membrane, including apical and basolateral domains, was estimated to be (1.06 ± 0.11) × 103 cm/s (n = 10). Increasing the flow rate through the chamber did not significantly
alter this value, so it can be assumed that the swelling rate was not
limited by the time required for the exchange of solutions in the bath.
Nonetheless, the presence of unstirred layers around the cells, which
were attached to a coverslip at the base of the chamber, will
inevitably mean that this value for Pf is to
some extent an underestimate.
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DISCUSSION |
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As judged by our molecular and immunohistochemical data, the predominant AQPs expressed in the exocrine pancreas of the rat are AQP8 and AQP1. Of the other known mammalian AQPs, only AQP4 and AQP5 were detectable by RT-PCR Southern blot analysis, albeit at very low expression levels. They were not, however, observed by immunohistochemistry.
The presence of AQP1 in the microvasculature is not surprising. Fluid secretion requires substantial fluxes of water from plasma to interstitium, and the capillary endothelium, which is not fenestrated in the exocrine pancreas, might otherwise be a limiting barrier. AQP1 has a similar distribution in the vascular endothelial cells of rat submandibular and parotid salivary glands (17), where it presumably serves a similar function.
The localization of AQP8 in the apical membrane of the pancreatic
acinar cells suggests that its role in the pancreas is analogous to
that of AQP5 in salivary glands. It may thus be supposed to provide the
major route for water movement across the apical membrane during
secretion of the isotonic, Cl-rich fluid that is evoked
by physiological secretagogues such as CCK and ACh (2).
Interestingly, immunoelectron microscopy indicates that AQP8 is also
present intracellularly within subapical vesicles. Indeed, these
vesicles appear to account for much of the AQP8 expressed in these
cells. The same is true for AQP2 in renal collecting duct principal
cells in which, on stimulation with vasopressin, the AQP2 located in
intracellular vesicles is shuttled to the apical membrane to provide
the high level of water permeability necessary for water reabsorption
(26). There is also evidence from rat parotid glands that
Ca2+-mobilizing agonists stimulate the translocation of
AQP5 from intracellular membranes to the apical membrane as part of the fluid secretory response (12, 13). It is therefore
possible that AQP8 is subject to a similar trafficking mechanism in the pancreas. The absence of AQP8 from zymogen granule membranes suggests that insertion of AQP8 in the apical membrane is probably not directly
linked to the exocytosis of pancreatic enzymes.
The apparent absence of AQPs from the basolateral membrane of pancreatic acinar cells is surprising, although the same is possibly true of salivary acinar cells, in which none of the known mammalian AQPs has yet been identified (27). The mRNA for basolateral AQPs expressed in the acinar cells should be highly abundant in the pancreas and therefore readily detectable. However, when Koyama et al. (15) screened pancreatic mRNA by RT-PCR by using degenerate primers designed to identify other AQP homologs, most of the clones they obtained were of AQP8.
Our measurements of acinar cell swelling in response to hypotonic stress suggest that much of the water permeability of the cell membrane is blocked by Hg2+. Since this measurement does not distinguish between the apical and basolateral membrane domains, it is impossible to tell whether the measured water permeability can be entirely attributed to the apical membrane, where AQP8 is expressed, or whether there is also a contribution from mercurial-sensitive channel(s) in the basolateral membrane. Given the very small area of the apical membrane, and the fact that Hg2+ reduced the total water permeability by as much as 90%, it is tempting to speculate that there is an as yet unidentified water channel in the basolateral membrane. On the other hand, it is possible that the inherent low level of water permeability found in most epithelial membranes combined with the much larger surface area of the basolateral membrane is sufficient to accommodate the water movement that takes place across this surface of the cells. Thus the possible presence of AQPs in the basolateral plasma membranes of acinar cells is highly speculative at this stage.
Unlike the ducts of some other exocrine glands, whose main function is
to modify a primary secretion generated by the acinar cells, the
pancreatic ducts secrete large volumes of HCO
In summary, we have demonstrated mRNA and protein expression of AQP1 and AQP8 in rat pancreas. Immunohistochemistry revealed that AQP1 is localized in the capillary endothelia and that AQP8 is present at the apical pole of the pancreatic acinar cells. Immunoelectron microscopy further demonstrated that AQP8 is associated with both the apical membrane and subapical intracellular vesicular structures. This suggests that AQP8 may be regulated by trafficking from intracellular vesicles to the apical membrane as part of the fluid secretory response. We have also demonstrated mRNA expression of AQP4 and AQP5 in rat pancreas by RT-PCR Southern blot analysis, although they were not detectable by immunohistochemistry. Further studies are therefore needed to identify the AQPs expressed at both the basolateral membrane of the acinar cells and particularly in the secretory ducts.
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ACKNOWLEDGEMENTS |
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We thank Zhila Nikrozi and Inger Merete Paulsen for expert technical assistance.
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FOOTNOTES |
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Support for this study was provided by the Biotechnology and Biological Sciences Research Council (UK), the Karen Elise Jensen Foundation, the Novo Nordic Foundation, the Danish Medical Research Council, the University of Aarhus Research Foundation, the University of Aarhus, and the Commission of the European Union (EU-Biotech Program and EU-TMR Program).
Address for reprint requests and other correspondence: S. Nielsen, Dept. of Cell Biology, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).
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 31 July 2000; accepted in final form 16 November 2000.
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