ARTICLE |
Correspondence to: Frank Thévenod, School of Biological Sciences, U. of Manchester, G.38 Stopford Bldg., Oxford Road, Manchester M13 9PT, UK. E-mail: frank.thevenod@man.ac.uk
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Summary |
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We have studied the expression and localization of several H+ and HCO3- transporters, whose presence in the rat pancreas is still unclear. The Cl-/HCO3- exchanger AE2, the Na+/H+ exchangers NHE1 and NHE4, and the 31-kD and 70-kD vacuolar H+-ATPase (V-ATPase) subunits were detected by immunoblotting and immunocytochemical techniques. Immunoblotting of plasma membranes with transporter-specific antibodies revealed protein bands at 160 kD for AE2, at
90 kD and
103 kD for NHE1 and NHE4, respectively, and at 31 kD and 70 kD for V-ATPase. NHE1 and NHE4 were further identified by amplification of isoform-specific cDNA using RT-PCR. Immunohistochemistry revealed a basolateral location of AE2, NHE1, and NHE4 in acinar cells. In ducts, NHE1 and NHE4 were basolaterally located but no AE2 expression was detected. V-ATPase was detected in zymogen granules (ZGs) by immunogold labeling, and basolaterally in duct cells by immunohistochemistry. The data indicate that NHE1 and NHE4 are co-expressed in rat pancreatic acini and ducts. Basolateral acinar AE2 could contribute to Cl- uptake and/or pH regulation. V-ATPase may be involved in ZG fusion/exocytosis and ductal HCO3- secretion. The molecular identity of the ductal Cl-/HCO3- exchanger remains unclear.
(J Histochem Cytochem 49:463474, 2001)
Key Words: bicarbonate transport, secretion, proton pump, exocrine glands
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Introduction |
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Secretion by the exocrine pancreas is carried out by two morphologically and functionally distinct epithelia: the acini and the ducts. Acinar cells secrete digestive enzymes together with a plasma-like primary fluid. This primary secretion is modified by the downstream duct cells to generate the HCO32-rich pancreatic juice. Several steps of this complex sequence of events are associated with pH changes of intra- and extracellular compartments that are mediated by primary or secondary active transporters for H1 and HCO32.In acinar cells, synthesis of digestive enzymes takes place in the rough endoplasmic reticulum (rER), while processing and sorting occur within the Golgi complex. After maturation of condensing vacuoles to zymogen granules (ZGs), the apically located ZGs exocytose the digestive enzymes into the lumen after hormonal stimulation. Whereas the pH in the rER is near neutral (6.5 using the pH-sensitive fluorescent dye BCECF (
The final electrolyte composition of the pancreatic juice is determined by duct secretion of NaHCO3. In the prevailing model, secretion depends on the luminal Cl- concentration. HCO3- is generated in the cytosol by a process involving Na+/K+-ATPase, an Na+/H+ exchanger, and NBC at the basolateral cell side, cytosolic carbonic anhydrase, and exit into the lumen via an Cl-/HCO3- exchanger. The activity of the Cl-/ HCO3- exchanger depends on a supply of luminal Cl- delivered to it from the cytosol by a recycling process utilizing an apical cAMP-activated Cl- channel, presumably CFTR. Na+ enters the lumen across cation-permeable intercellular junctions, driven by the lumen negativity (
To address some of the above unresolved issues and to clarify existing models for enzyme, fluid, and electrolyte secretion in rat pancreas, we have studied localization of the Cl-/HCO3- exchanger AE2, the Na+/H+ exchanger isoforms NHE1, and NHE4, and V-ATPase in the rat pancreas. Our results suggest that AE2, NHE1 and NHE4 are expressed in the basolateral plasma membranes of pancreatic acinar cells, where they may contribute to acidbase transport. V-ATPase is present in ZGs and basolaterally in duct cells. AE2 is not expressed in duct cells, whereas NHE1 and NHE4 are found localized in basolateral plasma membranes of duct cells. The presence of V-ATPase in both ZG membrane and duct cells indicates that V-ATPase may play a role in acinar secretion of digestive enzymes and ductal H+ extrusion.
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Materials and Methods |
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Antibodies
An affinity-purified rabbit polyclonal antibody to the COOH-terminal amino acids 12241237 of mouse AE2 has been previously described (
Preparation of Tissue Homogenate and Isolation of Plasma Membranes
Four to six male Wistar rats were anesthetized with ether and perfused via the left ventricle with PBS until the organs was thoroughly blanched. Pancreas or kidney cortex homogenate was prepared by grinding the tissue with 50 strokes of a motor-driven Potter homogenizer in 10 ml of ice-cold homogenizing buffer containing (in mM) 280 mannitol, 10 HEPES, 10 KCl, 1 MgCl2, adjusted to pH 7.0, and a protease inhibitor "cocktail" (10 µM leupeptin, 2 mM benzamidine, and 0.1 mM Pefabloc SC). The homogenate was centrifuged at 50 x g for 5 min and the supernatant was collected. The pellet containing unbroken cells was resuspended in 10 ml of the same buffer and homogenized once more. After centrifugation at 50 x g for 5 min, both supernatants were combined.
To obtain pancreatic plasma membrane fractions, the cleared homogenate was centrifuged for 12 min at 1000 x g and the supernatant was centrifuged at 11,000 x g for 15 min. The 11,000 x g pellet was composed of a whitish fluffy upper layer and a yellowish bottom layer, which were separated. The 11,000 x g fluffy layer was mixed with 2.0 M sucrose buffer to a concentration of 1.25 M, which was layered on 2.0 M sucrose and overlaid with 0.3 M sucrose. The gradient was centrifuged at 140,000 x g for 90 min, and the whitish band enriched in plasma membranes (PMs) at the upper surface between the 0.3 M and 1.25 M sucrose density layers was collected (
For detection of the 70-kD V-ATPase subunit in pancreas, a modified preparation of tissue homogenate was applied. Pancreas was excised and immediately frozen in liquid N2. Tissue was ground under liquid N2 and suspended in MES (2-[N-morpholino]ethanesulfonic acid) buffer (pH 4.6). Protein concentration was determined according to
Isolation of ZGs and Purification of ZG Membranes
ZGs from rat exocrine pancreas were isolated as described elsewhere (
For purification of ZG membranes, the protocol described by
SDS-PAGE and Immunoblotting
Electrophoresis and blotting procedures were performed essentially as described earlier (
Amplification and Cloning of NHE1- and NHE4-specific cDNA
Degenerate oligonucleotides encoding NHE1 or NHE4 peptide sequences were synthesized and used for the RT-PCR. As a template we used cDNA obtained from standard reverse-transcriptase reactions (
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Tissue Preparation for Light Microscopy
Male Wistar rats were anesthetized with pentobarbital (Nembutal) (65 mg/kg IP) and perfusion-fixed with 2% paraformaldehyde/75 mM lysine/10 mM sodium periodate (PLP) as described by
Immunofluorescence Light Microscopy on Cryosections
Tissue blocks were cryoprotected in 30% sucrose for at least 1 hr and frozen in liquid N2. Indirect immunohistochemistry was performed on 5-µm cryosections. Sections were rehydrated in PBS for 5 min, treated with 1%SDS for 5 min (
Immunoperoxidase Light Microscopy on Paraffin Sections
For localization of the vacuolar-type H+-ATPase, immunoperoxidase light microscopy was applied as previously described (
Pre-embedding Immunogold Electron Microscopy
Electron microscopy on isolated rat pancreatic ZGs was performed as described earlier (
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Results |
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AE2 in Rat Pancreas
Fig 1A and Fig 1B illustrate the immunofluorescence labeling pattern observed with the antibody against aa 12241237 of mouse AE2 (1:800 dilution) on cryosections of PLP-fixed pancreas with epitope unmasking by SDS. Acinar cells (Fig 1A, A) revealed sharp AE2 staining of strong intensity at the basolateral cell sides. In contrast, AE2 immunoreactivity was absent in all cells lining the pancreatic duct segments, i.e., intercalated, intralobular (Fig 1B, ILD), interlobular, and main duct cells. Acinar staining was abolished by pre-incubation of the antibody with an excess of the AE2 peptide antigen (24 µg/ml) (Fig 1C).
Because the anti-AE2 aa 12241237 also crossreacts with the COOH-terminal sequence of AE1, competition experiments with AE1 antigen were also necessary to determine labeling specificity. Fig 1D shows that pre-incubation of the antibody with 12 µg/ml AE1 antigen (aa 917929) did not eliminate acinar basolateral immunostaining, indicating that the acinar labeling is indeed AE2.
AE2 polypeptide expression in rat pancreas was further evaluated by immunoblots on homogenate of whole rat pancreas (Ho) and a plasma membrane fraction (PM). The anti-AE2 antibody (1:10,000) recognized bands at 160 kD (Fig 1E, arrow),
110 kD, and
90 kD in the plasma membrane fraction (Fig 1E, Lane 2), whereas these bands were not detectable in the tissue homogenate (Fig 1E, Lane 1). In addition, a faint band at
75 kD was present in both homogenate and plasma membrane fractions. Labeling of all these bands in the PM fraction was abolished by co-incubation of the antibody with 24 µg/ml peptide antigen, as shown in Fig 1E, Lane 3.
V-ATPase in Rat Pancreas
Fig 2A shows the immunofluorescence labeling pattern of V-ATPase in rat pancreas using the antibody raised against the 70-kD V-ATPase subunit (1:200 dilution). Acinar cells (Fig 2A, A) exhibited strong, but diffuse V-ATPase immunoreactivity at the apical cell sides. At higher magnification (Fig 2B), labeling appeared localized at the area occupied by the ZGs (arrow). Identical V-ATPase distribution of weaker intensity was observed using the antibody against the 31-kD V-ATPase subunit (not shown). We performed ultrastructural examination to ensure that the distribution observed by light microscopy indeed represents labeling of ZGs. Fig 2C shows the results obtained by electron microscopy with the antibody against the 70-kD V-ATPase subunit on purified rat pancreatic ZGs using a pre-embedding protocol, as described in Materials and Methods. Silver-enhanced immunogold labeling was found with both antibodies, the particles being located at the membrane surface of the granules, which confirms the results obtained by light microscopy. No ZG labeling was observed in control experiments in which primary antibody was omitted, as shown in Fig 2D.
Pancreatic duct cells also exhibited labeling with both antibodies against the 31-kD and the 70-kD V-ATPase subunit. Fig 2E and Fig 2F illustrate V-ATPase distribution in pancreatic ducts by immunoperoxidase light microscopy on paraffin sections from pancreas fixed in Bouin's fixative. Intralobular duct cells (Fig 2E, ILD) showed V-ATPase staining of strong intensity at their basolateral cell sides. Occasionally, single cells exhibited staining at the apical cell side, as shown in Fig 2E and Fig 2F (arrows). Labeling in intralobular ducts was similar to that found in interlobular ducts, whereas main duct cells revealed basolateral staining of moderate intensity, with certain cells showing diffuse intracellular V-ATPase distribution (not shown). Labeling in duct cells was absent in the control experiments in which the primary antibody was either substituted by pre-immune serum (not shown), or preincubated with 0.5 mM peptide antigen (Fig 2G).
Functional studies (
Fig 2H shows an immunoblot analysis of whole rat pancreas homogenate (pHo), plasma membranes (PM), and ZG membranes (ZGM) with the antibody against the 31-kD V-ATPase subunit (1:2500). Rat kidney cortex homogenate (kHo) (Fig 2H, Lane 4) was used as a positive control. Similarly to kidney cortex homogenate, a major immunoreactive band at 31 kD (Fig 2H, arrow) was present in pHo (Fig 2H, Lane 1), PM (Fig 2H, Lane 2), and ZGM (Fig 2H, Lane 3). In pHo and PM, weaker bands of higher apparent molecular mass were also present. With the antibody against the 70-kD V-ATPase subunit, a 70-kD band was detected in kHo but no labeling was observed in pHo and ZGM (data not shown). This result was unexpected because both immunohistochemistry and immunogold procedures had revealed labeling with antibodies against both V-ATPase subunits at the ZGM (Fig 2A and Fig 2C) and immunohistochemical staining in duct cells (Fig 2E and Fig 2F). We suspected that the protein was degraded by proteolytic enzymes in pancreatic tissue during the preparation of ZGM, which lasted for several hours. Although both peptide sequences against which the antibodies are raised have multiple cleavage sites for the pancreatic enzymes trypsin and chymotrypsin, it seemed possible that the tertiary structure of the 70-kD V-ATPase subunit makes the cleavage sides particularly accessible to proteolytic enzymes. Indeed, modification of the tissue homogenization protocol (described in Materials and Methods), combining both tissue homogenization under liquid N2 and the use of acidic buffers to prevent activation of proteolytic enzymes, enabled detection of a single immunoreactive band at 70 kD in pHo with the antibody against the E V-ATPase subunit, as shown in Fig 2H, Lane 6. However, this modified protocol using liquid N2 cannot be applied for isolation of ZGs because it does not preserve the integrity of cellular organelles.
NHE1 and NHE4 in Rat Pancreas
Fig 3A and Fig 3B show the staining pattern using the anti-NHE1 antibody on PLP-fixed cryosections of rat pancreas. Acinar cells (Fig 3A, A) revealed sharp NHE1 staining of moderate intensity at the basolateral cell sides. NHE1 immunoreactivity was also present in pancreatic ducts: Fig 3B shows the NHE1 labeling pattern in intralobular (ILD) duct cells, showing uniform NHE1 staining at their basolateral membranes. An identical staining pattern was also observed in interlobular and main duct cells (not shown), thus confirming the data reported by
No NHE4 labeling was detected with the anti-NHE4 monoclonal antibody 11H11 on PLP-fixed cryosections that had not been treated with SDS. However, by use of an antigen unmasking protocol by treating the cryosections with 1% SDS for 5 min before antibody incubation, NHE4 immunolabeling was observed in rat pancreas (Fig 3C and Fig 3D). Acinar cells (Fig 3C and Fig 3D, A) showed specific NHE4 staining in their basolateral cell sides. Similarly, basolateral NHE4 labeling was also observed in intralobular (Fig 3D, ILD), interlobular and main duct segments. The results of immunoblotting experiments with the polyclonal antibodies against NHE1 (1:2500) and NHE4 (1:2500) on a rat pancreas plasma membrane fraction (PM) are shown in Fig 3E, Lanes 1 and 2. The anti-NHE1 antibody crossreacted with proteins from plasma membranes of sizes 115 kD,
90 kD,
76 kD, and
58 kD, whereas the anti-NHE4 antibody recognized a major immunoreactive band at
103 kD.
Fig 4 shows NHE1 and NHE4 mRNA expression in whole rat pancreas and kidney tissue cDNA used as a positive control, as detected by RT-PCR. With NHE1-specific degenerate primers (
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Discussion |
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AE2
The anion exchanger (AE) gene family includes products of three genes, AE1, AE2, and AE3 (90 kD,
110 kD, and
160 kD (Fig 1E). The
160-kD band is similar to the apparent molecular mass (Mr) of AE2a/b (
90-kD and
110-kD bands correspond to the Mr of AE1, the lower band likely reflects residual erythrocytes in the tissue sample. The identity of the
110-kD band remains uncertain, but it resembles polypeptides previously noted in several secretory cell types, alveolar Type II cells (
Immunohistochemistry showed basolateral AE2 labeling in pancreatic acinar cells (Fig 1A and Fig 1B), which is in agreement with previous functional studies (
V-ATPase
Zymogen Granules.
Our results show the presence of the 31-kD and 70-kD subunits of V-ATPase in ZG membranes (Fig 2B and Fig 2C). Active H+ pumping by V-ATPases is generally held responsible for acidification of intracellular compartments. Our data suggest that the acidic intragranular pH previously reported (
Ducts.
When expressed in plasma membranes of epithelial cells, V-ATPases may also contribute to generation of intracellular HCO3- by extruding H+ out of cells (
NHE1 and NHE4
In the present study we have demonstrated NHE1 and NHE4 expression in basolateral membranes of acinar and duct cells (Fig 3A3D). The immunohistochemical data are extended by immunoblotting of plasma membranes with anti-NHE1 antibody, which revealed immunoreactive bands at 115 kD,
90 kD, and
76 kD. The
115-kD band likely corresponds to the mature processed form of NHE1 (91 kD). The
90-kD (and perhaps also the
76 kD) band may represent a precursor form of NHE1, as previously reported (
103 kD, which is in agreement with the results of previous studies on fibroblasts transfected with NHE4 cDNA (
The failure to obtain PCR products of the carboxy-terminus of pancreatic NHE4 appears in contrast to the detection of NHE4 with antibodies raised against C-terminal NHE4 sequences. Degradation or absence of the 3'-end of the pancreatic cDNA in the present study could be ruled out because the reverse transcription was not random but was oligo dT-primed. One possible explanation is that the epitope recognized by the antibody directed against the 40 C-terminal NHE4 aa is located outside of the sequence covered by the primers used (in aa 677690 or aa 696704). The 3'-reverse primers "C-stop", "C-rev1", and "C-rev2" covered the sequences corresponding to aa 711717, aa 704710, and aa 690696, respectively. However, the polyclonal antibody used in the present study was raised against aa 677717 of NHE4. Alternatively, differences between the 3'-terminal sequences of the primers derived from the stomach NHE sequence published by
Pancreatic duct cells are highly specialized bicarbonate secretory cells. They appear to be equipped with several H+ extrusion mechanisms at their basolateral membranes, such as Na+/H+ exchangers (
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Acknowledgments |
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Supported by a grant from Deutsche Mukoviszidose e.V. (FT, ER) and by National Institute of Diabetes and Digestive and Kidney Diseases grants DK-43495 (SLA) and DK 34854 (Harvard Digestive Diseases Center).
Received for publication September 7, 2000; accepted December 7, 2000.
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