Expression and immunolocalization of aquaporin water channels in rat exocrine pancreas

Patricia T. Hurley1, Carole J. Ferguson1, Tae-Hwan Kwon2, Marie-Louise E. Andersen2, Alexander G. Norman1, Martin C. Steward1, Søren Nielsen2, and R. Maynard Case1

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


    ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP>-rich fluid secreted by the pancreatic duct cells, which helps to regulate the pH of the duodenal contents (2). Water flow across both the acinar and ductal epithelia is believed to be coupled osmotically to the active transepithelial transport of electrolytes. Both of these epithelia generate near-isotonic fluids, and consequently the transepithelial osmotic gradients driving the water flow must be small. It is therefore anticipated that the apical and basolateral water permeabilities of both acinar and duct cell membranes in the pancreas are relatively high. This is in contrast to the salivary glands, in which the ducts have a very low water permeability and electrolytes are reabsorbed without water to generate a hypotonic secretion (4).

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, alpha -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.


    METHODS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Table 1.   Aquaporin primer pairs used for RT-PCR

PCR products were analyzed by agarose gel electrophoresis and subsequent high-stringency Southern blotting by using 32P-labeled specific AQP probes. A positive control tissue was included for each primer set, and both an RT negative control and a PCR negative control reaction were performed. In addition to Southern blot analysis, PCR products were subcloned into TOPO 2.1 (Invitrogen, Gröningen, The Netherlands) and their identities were verified by sequencing.

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 from -80°C to -70°C, rinsed in pure methanol for 24 h while increasing the temperature from -70°C to -45°C, and infiltrated first with 1:1 Lowicryl HM20 and methanol, then a 2:1 mixture, and finally pure Lowicryl HM20 before ultraviolet polymerization for 2 days at -45°C and 2 days at 0°C. Immunolabeling was performed on ultrathin Lowicryl HM20 sections (60-80 nm). Sections were pretreated with a saturated solution of NaOH in absolute ethanol (2-3 s), rinsed, and preincubated for 10 min with 0.1% sodium borohydride and 50 mM glycine in 0.05 M Tris, pH 7.4, containing 0.1% Triton X-100. Sections were rinsed and incubated overnight at 4°C with anti-AQP8 diluted in 0.05 M Tris, pH 7.4, containing 0.1% Triton X-100 with 0.2% powdered milk (diluted 1:50). After being rinsed, sections were incubated for 1 h at room temperature with goat anti-rabbit IgG conjugated to 10-nm colloidal gold particles (GAR.EM10, 1:50; BioCell Research Laboratories, Cardiff, UK). The sections were stained with uranyl acetate and lead citrate before examination in a Philips CM100 or Philips 208 electron microscope.

Water 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.

Cell volume was measured by video microscopy on a Nikon Diaphot inverted microscope equipped with a charge-coupled device camera (902A; Watec, Las Vegas, NV) linked to a PC image capture board (LG-3; Scion, Frederick, MD). Cells were pipetted onto a glass coverslip at the base of a heated perfusion chamber and viewed under bright-field illumination. After the cells had become attached to the glass, the 220-µl chamber was perfused at 2.2 ml/min with a HEPES-buffered solution containing (in mM) 64 Na+, 5 K+, 1 Ca2+, 1 Mg2+, 67 Cl-, 10 HEPES, 10 glucose, and 145 sucrose, pH 7.4, gassed with 100% O2 at 37°C. The osmolarity of the perfusate was reduced from 290 mosM to 145 mosM by switching to an otherwise identical solution lacking sucrose. Cells pretreated with Hg2+ were exposed to 0.3 mM HgCl2 for 10 min before the experiment was begun, and this concentration was maintained in the perfusion solutions during the experiment.

Images of individual acinar cells were captured at 10-s intervals during the experiment, and their image areas were measured subsequently by tracing around the perimeters of the cells on a graphics tablet using Scion Image software (Scion). Assuming spherical geometry, relative cell volume (VR) was calculated by normalizing the acinar cell image area (A) to the mean of the first three area measurements in each experiment (A0) and using
V<SUB>R</SUB><IT>=</IT>(<IT>A/A<SUB>0</SUB></IT>)<SUP><IT>1.5</IT></SUP>
The osmotic water permeability (Pf) of the acinar cell membrane was estimated from the initial rate of cell swelling (dVR/dt) measured when the perfusate osmolarity (Os) was reduced by withdrawal of 145 mM sucrose. The value of Pf was calculated using
P<SUB>f</SUB><IT>=</IT><FR><NU>V<SUB><IT>0</IT></SUB>dV<SUB>R</SUB><IT>/</IT>d<IT>t</IT></NU><DE><IT>S</IT><A><AC>V</AC><AC>&cjs1171;</AC></A><SUB>w</SUB>&sfgr;&Dgr;Os</DE></FR>
where V0 is the initial cell volume, S is the cell surface area, Vw is the partial molar volume of water, and sigma  is the osmotic reflection coefficient of sucrose (assumed to be 1.0).


    RESULTS
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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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Fig. 1.   Northern blot analysis of aquaporin (AQP) mRNA expression in rat pancreas. Brain, kidney, submandibular gland (SMG), and testis were included as positive controls. A: AQP8 mRNA (transcript size ~1.45 kb) was detected in pancreas and testis; B: AQP1 mRNA (~2.6 kb) was detected in submandibular gland, kidney, and pancreas; C: AQP5 mRNA (~1.6 kb) was detected in submandibular gland but not in pancreas or kidney; D: AQP4 mRNA (~5.5 kb) was detected in kidney but not pancreas or submandibular gland.

Membranes containing mRNA from rat pancreas and submandibular gland were also hybridized with a 32P-labeled probe for AQP1, together with kidney mRNA as a positive control. A ~2.6 kb transcript was seen in all of the tissues on the blot, although the signals for both the submandibular gland and the pancreas were much less intense than in the kidney (Fig. 1B). The weak signal seen in the submandibular gland is consistent with previous studies showing that AQP1 is expressed only in the capillary endothelial cells (17).

The same Northern blot was then stripped and reprobed with a 32P-labeled AQP5 probe (Fig. 1C). Although a transcript of the predicted size (~1.6 kb) was seen in the submandibular gland, included as a positive control for AQP5 (30), no signal was detected in the pancreas. The absence of AQP5 mRNA from the kidney was consistent with previous studies (35).

In the case of AQP4 (Fig. 1D), kidney mRNA yielded a faint signal at ~5.5 kb, which is consistent with the low expression of AQP4 in the inner medullary collecting duct principal cells (32, 35). No AQP4 signal, however, could be detected in mRNA from the pancreas.

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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Fig. 2.   RT-PCR Southern blot analysis of AQP mRNA expression in rat pancreas. A: AQP2 (827 bp) and AQP3 (859 bp) products were amplified from kidney (K+) but were not obtained with pancreas (P+) nor in the respective negative controls (K-, P-). B: an AQP4 product (890 bp) was amplified from brain (B+) and was also detectable in pancreas (P+), and a 793-bp band corresponding to AQP5 was amplified from both submandibular gland (SMG+) and pancreas (P+).

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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Fig. 3.   Immunohistochemical localization of AQP1 in rat pancreas using immunoperoxidase labeling of cryostat sections (10 µm; A and B). AQP1 labeling is associated with the microvasculature between the acini. Pancreatic acinar cells and ductal cells are not labeled. Magnification, ×1,000.



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Fig. 4.   Immunohistochemical localization of AQP8 in rat pancreas using immunoperoxidase labeling of cryostat sections (10 µm). A: Low-magnification view showing that AQP8 labeling is only seen in the pancreatic acinar cells. B-D: within the acini, the labeling is mainly confined to the apical pole of the cells (arrows), whereas no labeling is seen in the basal regions. E and F: immunolabeling controls performed by using antibodies preabsorbed with the immunizing peptide are negative. Magnifications, ×250 (A), ×650 (B, C, E) and ×1,000 (D, F).

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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Fig. 5.   Immunoelectron microscopy of rat pancreatic acinar cells in ultrathin Lowicryl HM20 sections. A: survey view of exocrine pancreas. Rectangle indicates the area presented at higher magnification in B. B: AQP8 Immunogold labeling is associated with the apical plasma membrane (arrowhead) and subapical intracellular vesicles (arrows). L, lumen. Magnifications, ×5,600 (A) and ×30,000 (B).



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Fig. 6.   Immunoelectron microscopy of the apical and subapical parts of the pancreatic acinar cell in ultrathin Lowicryl HM20 sections. AQP8 labeling is associated with the apical plasma membrane (arrowheads) and with subapical intracellular vesicles (arrows), but not zymogen granules (z). L, lumen; M, mitochondria. Magnification, ×42,000.

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) × 10-3 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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Fig. 7.   Swelling of isolated rat pancreatic acinar cells in response to a 50% reduction in extracellular osmolarity. Relative cell volume was calculated from video microscope images captured at 10-s intervals. Data are means ± SE of 10 control experiments () and 7 experiments in which the cells were pretreated for 10 min with 0.3 mM HgCl2 (open circle ).

Acinar cells pretreated with 0.3 mM HgCl2 for 10 min before hypotonic stress showed a dramatically reduced rate of swelling (Fig. 7). In response to the same osmolarity change, cell volume increased by only 8 ± 2% (n = 7) in 2 min with little further change thereafter. From the initial rate of swelling, Pf was estimated to be (0.10 ± 0.03) × 10-3 cm/s, indicating a 90% reduction of the membrane water permeability by HgCl2.


    DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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<UP><SUB>3</SUB><SUP>−</SUP></UP>-rich isotonic fluid in response to stimulation by the hormone secretin (2). Although the ductal epithelial cells represent <5% of the cellular mass of the gland, they generate at least as much fluid as the total acinar cell population. The fluid secretory rate per unit area of epithelium is therefore considerably greater in the ductal system, and it would be surprising if water channels are not expressed in these cells. Interestingly, the epithelium of the biliary ductal system, which shows many structural and functional similarities to the pancreatic ducts, is known to express AQP1 in both the apical and basolateral membranes (20, 31). Furthermore, AQP1 expression in these cells is regulated by secretin (21, 22). However, we could find no evidence of AQP1 expression in the pancreatic ducts of the rat, and we may conclude either that the major fluid secretory function of the exocrine pancreas occurs without the involvement of AQP water channels or that the ducts express water channels that have not yet been characterized.

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.


    ACKNOWLEDGEMENTS

We thank Zhila Nikrozi and Inger Merete Paulsen for expert technical assistance.


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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12.   Ishikawa, Y, Eguchi T, Skowronski T, and Ishida H. Acetylcholine acts on M3 muscarinic receptors and induces the translocation of aquaporin 5 water channel via cytosolic Ca2+ elevation in rat parotid glands. Biochem Biophys Res Commun 245: 835-840, 1998[ISI][Medline].

13.   Ishikawa, Y, Skowronski MT, Inoue N, and Ishida H. alpha (1)-Adrenoceptor-induced trafficking of aquaporin-5 to the apical plasma membrane of rat parotid cells. Biochem Biophys Res Commun 265: 94-100, 1999[ISI][Medline].

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17.   Li, J, Nielsen S, Dai YS, Lazowski KW, Christensen EI, Tabak LA, and Baum BJ. Examination of rat salivary glands for the presence of the aquaporin chip. Pflügers Arch 428: 455-460, 1994[ISI][Medline].

18.   Ma, TH, Song YL, Gillespie A, Carlson EJ, Epstein CJ, and Verkman AS. Defective secretion of saliva in transgenic mice lacking aquaporin-5 water channels. J Biol Chem 274: 20071-20074, 1999[Abstract/Free Full Text].

19.   Ma, TH, and Verkman AS. Aquaporin water channels in gastrointestinal physiology. J Physiol (Lond) 517: 317-326, 1999[Abstract/Free Full Text].

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21.   Marinelli, RA, Pham L, Agre P, and LaRusso NF. Secretin promotes osmotic water transport in rat cholangiocytes by increasing aquaporin-1 water channels in plasma membrane---evidence for a secretin-induced vesicular translocation of aquaporin-1. J Biol Chem 272: 12984-12988, 1997[Abstract/Free Full Text].

22.   Marinelli, RA, Tietz PS, Pham LD, Rueckert L, Agre P, and LaRusso NF. Secretin induces the apical insertion of aquaporin-1 water channels in rat cholangiocytes. Am J Physiol Gastrointest Liver Physiol 276: G280-G286, 1999[Abstract/Free Full Text].

23.   Marples, D, Christensen S, Christensen EI, Ottosen PD, and Nielsen S. Lithium-induced down-regulation of aquaporin-2 water channel expression in rat kidney medulla. J Clin Invest 95: 1838-1845, 1995[ISI][Medline].

24.   Matsuzaki, T, Suzuki T, Koyama H, Tanaka S, and Takata K. Aquaporin-5 (AQP5), a water channel protein, in the rat salivary and lacrimal glands: immunolocalization and effect of secretory stimulation. Cell Tissue Res 295: 513-521, 1999[ISI][Medline].

25.   Moore, M, Ma TH, Yang BX, and Verkman AS. Tear secretion by lacrimal glands in transgenic mice lacking water channels AQP1, AQP3, AQP4 and AQP5. Exp Eye Res 70: 557-562, 2000[ISI][Medline].

26.   Nielsen, S, Chou CL, Marples D, Christensen EI, Kishore BK, and Knepper MA. Vasopressin increases water permeability of kidney collecting duct by inducing translocation of aquaporin-CD water channels to plasma-membrane. Proc Natl Acad Sci USA 92: 1013-1017, 1995[Abstract].

27.   Nielsen, S, King LS, Christensen BM, and Agre P. Aquaporins in complex tissues. II. Subcellular distribution in respiratory and glandular tissues of rat. Am J Physiol Cell Physiol 273: C1549-C1561, 1997[ISI][Medline].

28.   Nielsen, S, Nagelhus EA, Amiry Moghaddam M, Bourque C, Agre P, and Ottersen OP. Specialized membrane domains for water transport in glial cells: high-resolution immunogold cytochemistry of aquaporin-4 in rat brain. J Neurosci 17: 171-180, 1997[Abstract/Free Full Text].

29.   Nielsen, S, Pallone T, Smith BL, Christensen EI, Agre P, and Maunsbach AB. Aquaporin-1 water channels in short and long loop descending thin limbs and in descending vasa recta in rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 268: F1023-F1037, 1995[Abstract/Free Full Text].

30.   Raina, S, Preston GM, Guggino WB, and Agre P. Molecular cloning and characterization of an aquaporin cDNA from salivary, lacrimal, and respiratory tissues. J Biol Chem 270: 1908-1912, 1995[Abstract/Free Full Text].

31.   Roberts, SK, Yano M, Ueno Y, Pham L, Alpini G, Agre P, and LaRusso NF. Cholangiocytes express the aquaporin chip and transport water via a channel-mediated mechanism. Proc Natl Acad Sci USA 91: 13009-13013, 1994[Abstract/Free Full Text].

32.   Terris, J, Ecelbarger CA, Marples D, Knepper MA, and Nielsen S. Distribution of aquaporin-4 water channel expression within rat kidney. Am J Physiol Renal Fluid Electrolyte Physiol 269: F775-F785, 1995[Abstract/Free Full Text].

33.   Terris, J, Ecelbarger CA, Nielsen S, and Knepper MA. Long-term regulation of four renal aquaporins in rats. Am J Physiol Renal Fluid Electrolyte Physiol 271: F414-F422, 1996[Abstract/Free Full Text].

34.   Verkman, AS, and Mitra AK. Structure and function of aquaporin water channels. Am J Physiol Renal Physiol 278: F13-F28, 2000[Abstract/Free Full Text].

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36.   Yasui, M, Kwon TH, Knepper MA, Nielsen S, and Agre P. Aquaporin-6: an intracellular vesicle water channel protein in renal epithelia. Proc Natl Acad Sci USA 96: 5808-5813, 1999[Abstract/Free Full Text].


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