1 The Water and Salt Research Center and 2 Department of Biochemistry, University of Aarhus, DK-8000 Aarhus C, Denmark; 3 Department of Oral Biology, Semmelweis University of Budapest, Budapest 1089, Hungary; 4 Division of Nephrology, University of California, Los Angeles, California 90095; and 5 School of Biological Sciences, University of Manchester, Manchester M13 9PT, United Kingdom
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ABSTRACT |
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Patterns of salivary HCO
bicarbonate transport; intracellular pH regulation
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INTRODUCTION |
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BICARBONATE TRANSPORTERS
PLAY VITAL ROLES in the epithelia of the kidney and
gastrointestinal tract (stomach, intestine, pancreas, salivary glands,
etc.). A primary function is in the regulation of intracellular pH,
which is important for normal cell function and is determined by the
balance between the activities of various acid extruders and acid
loaders. Many of the same transporters also play important roles in the
net secretion or absorption of H+ and
HCO
Several members of the Na+-HCO
Since the molecular identity of the first electrogenic NBC (NBC1) was
established in the kidney (26, 27), several other NBC1
variants and NBC1-like proteins have been identified (1, 3, 6,
11, 24). In addition, a number of homologous, but less closely
related, electroneutral NBCs has recently been cloned. These include
NBC3 from human skeletal muscle (23) and NBCn1 from rat
aortic smooth muscle (5) and are considered by some to be
splice variants of the retinal NBC2 isoform (30). In rat
kidney, we have demonstrated that NBC3 is present at the collecting
duct intercalated cells (13) and NBCn1 is mainly present
at the medullary thick ascending limb cells (33), and these are generally expected to mediate HCO
Saliva secretion in salivary glands is believed to occur in two stages:
the production of an isotonic, plasmalike primary fluid by the acinar
cells, and the subsequent net reabsorption of electrolytes, but not
water, in the ductal system to produce a hypotonic saliva
(8). The concentration of HCO. In the parotid glands of humans and other
primates, on the other hand, the concentration of
HCO
The role of NBCs in these processes is largely unknown. There is
functional evidence of NBC activity in the acinar cells of bovine
parotid (16), sheep parotid (22, 31), and
mouse submandibular gland (17). Furthermore,
immunohistochemical studies have indicated that NBC1 is present at the
basolateral membrane of the acinar cells in rat parotid
(28), human parotid (19), and mouse
submandibular gland (17). Basolaterally located NBCs such
as these could, together with acid extruders such as
Na+/H+ exchanger 1 (NHE1), contribute to the
supply of intracellular HCO
Recent functional and immunohistochemical data suggest that
electroneutral NBC isoforms are expressed at the apical membranes of
both acinar and ductal cells in the mouse submandibular gland (17). These apically located NBCs could, together with
NHE2 and NHE3, contribute either to ductal HCO
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MATERIALS AND METHODS |
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Tissue samples. Human parotid and submandibular salivary tissue was taken from adult patients undergoing surgery for various primary maxillofacial interventions. Patients gave informed consent, and the procedures were approved by the Semmelweis University of Budapest Regional Committee of Science and Research Ethics. Human kidney material was obtained as described in previous studies (18). The tissues used did not contain atypical cells when assessed microscopically. Rat tissue was derived from male Wistar rats weighing 250-300 g (M & B, Ry, Denmark) that were maintained on a standard rodent diet (Altromin, Lage, Germany) with free access to water.
RT-PCR.
For RT-PCR, samples were frozen in liquid nitrogen immediately after
surgery and stored at 70°C until use. Total RNA was extracted from
kidney and salivary gland tissue by the single-step guanidium
thiocyanate method. Total RNA (1 µg) was reverse transcribed at
42°C for 50 min using oligo(dT)15 primers and Expand RT
(Boehringer Mannheim). RT products were stored at
20°C until
required. Rat
-actin and human glyceraldehyde-3-phosphate
dehydrogenase (GAPDH) were amplified to assess the cDNA yield, and both
RT-negative and PCR-negative control reactions were performed.
Antibodies. Antibodies were as follows. First, electroneutral NBC3: a synthetic peptide corresponding to amino acids 1197-1214 of the COOH terminus of human NBC3 (GenBank accession no. AF047033) (23) was used to generate a polyclonal antibody (13). This region of NBC3 is nearly identical in human, rat, and rabbit, and the antibody shows cross-reactivity among species (13). Second, electroneutral NBCn1: anti-NBCn1 was raised in rabbits against a synthetic peptide corresponding to amino acids 1204-1218 of the COOH terminus of NBCn1 (GenBank accession no. AF070475) (33). Note that, because the COOH terminus of all three rat NBCn1 splice variants are identical (5), the antibody does not distinguish among NBCn1B, NBCn1C, and NBCn1D.
Membrane fractionation and immunoblotting. Tissues were homogenized in an Ultra-Turrax T8 homogenizer (IKA Labortechnik, Staufen, Germany) for 15 s on ice in dissection buffer containing 0.3 M sucrose, 25 mM imidazole, 1 nM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 8.5 µM leupeptin (pH 7.2). The homogenate was centrifuged in an Eppendorf 5403 centrifuge (Eppendorf, Germany) at 4,000 g for 15 min at 4°C to remove whole cells and nuclei. The supernatant was then centrifuged at 200,000 g for 1 h to produce a pellet enriched for membranes. From the resultant pellets, gel samples were made containing 2% SDS Laemmli sample buffer. Prepared samples were loaded and run under reducing conditions on 6 or 10% homogenous Tris-glycine minigels (Novex, San Diego, CA). After transfer by electroelution to nitrocellulose membranes, blots were blocked for 1 h with 5% milk in PBS-Tris [80 mM Na2HPO4, 20 mM NaH2PO4, 100 mM NaCl, 0.1% Tween 20 (pH 7,5)]. They were then incubated overnight at 4°C with anti-NBC3 (diluted 1:1,000) or anti-NBCn1 (diluted 1:100). The labeling was visualized with horseradish peroxidase-conjugated secondary antibodies (P217, DAKO, Glostrup, Denmark) with an enhanced chemiluminescence system (Amersham Pharmacia Biotech, Buckinghamshire, UK). Immunolabeling controls were performed using antibodies preabsorbed with immunizing peptide.
Immunohistochemistry. Human salivary gland tissues destined for immunohistochemistry were cut into small pieces and immediately immersed in cold fixative (4% paraformaldehyde in 0.1 M PBS, pH 7.4) for at least 2-4 h before washing in buffer. Human kidney tissues were processed as described previously (18). Male Wistar rats were anesthetized with halothane inhalation, and salivary glands were fixed by transcardiac perfusion, or kidneys were fixed by retrograde aortic perfusion, with 4% paraformaldehyde in 0.1 M sodium cacodylate buffer. The tissues were removed and postfixed in the same fixative solution for 1 h. The tissue blocks were rinsed and dehydrated in ethanol followed by xylene and embedded in paraffin. For immunoperoxidase and immunofluorescence microscopy, the paraffin-embedded tissues were cut at 2-µm thickness on a rotary microtome (Leica, Germany). The staining was carried out using indirect immunoperoxidase or indirect immunofluorescence. The tissues were dewaxed and rehydrated.
For immunoperoxidase labeling, endogenous peroxidase was blocked by 0.5% H2O2 in absolute methanol for 30 min at room temperature. For antigen retrieval, sections were put in 1 mM Tris solution (pH 9.0) supplemented with 0.5 mM EGTA (3.6-dioxaoctamethylenedinitrilo-tetraacetic acid) and were heated using a microwave oven for 10 min. Nonspecific binding of immunoglobulin was prevented by incubating the sections in 50 mM NH4Cl in 0.01 M PBS (pH 7.4) for 30 min, followed by blocking in PBS supplemented with 1% BSA, 0.05% saponin, and 0.2% gelatin. Sections were incubated overnight at 4°C with primary antibodies diluted in PBS containing 0.1% BSA and 0.3% Triton X-100. The sections were then rinsed three times in PBS containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin for 10 min. The labeling was visualized with horseradish peroxidase-conjugated goat anti-rabbit secondary antibodies (P448, DAKO; diluted 1:200 in PBS containing 0.1% BSA and 0.3% Triton X-100) followed by incubation with 3,3-diaminobenzidine for 10 min. The sections were washed three times for 10 min in PBS and counterstained for 2 min in Mayers heematoxylin, rinsed for 20 min under running tap water, dehydrated, and mounted with Eukitt mounting medium (O. Kindler). The microscopy was carried out using a Leica DMRE microscope. For immunofluorescence microscopy, the labeling was visualized with incubation with FITC-conjugated goat anti-rabbit secondary antibodies (Alexa 488, Molecular Probes Europe, Leiden, The Netherlands). The sections were then rinsed in PBS three times for 10 min and mounted with glycerol supplied with antifading reagent (N-propyl galate). ![]() |
RESULTS |
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RT-PCR analyses.
As shown in Fig. 1, RNA samples from rat
kidney, parotid, and submandibular glands were assessed by RT-PCR for
transcripts of NBCn1B (473 bp), NBCn1C (389 bp), and NBCn1D (2.9 kb),
using specific primers designed to the available nucleotide sequences. Samples of human kidney, parotid, and submandibular glands were also
examined for NBC3 (983 bp; Fig. 1). Transcripts of the
housekeeping genes for -actin (189 bp) and GAPDH (605 bp) were used
as references for the rat and human tissue samples, respectively. As
shown in Fig. 1, RT-PCR yielded products that ran as single bands of
the expected size as detected by ethidium bromide-stained agarose gel
electrophoresis. Sequencing of all the products showed 100% identity
with the published nucleotide sequences.
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Immunoblotting analyses.
Immunoblotting was performed with antibodies to NBC3 and NBCn1, which
cross-react between rat and human, by use of membrane fractions
prepared from human and rat salivary glands as well as from human and
rat kidney as positive controls (Fig. 2).
Both isoforms were detected in the salivary glands of both species. The
NBC3 antibody revealed an ~200-kDa band in both human and rat
salivary glands as well as in kidney (Fig. 2A). The NBCn1 antibody, which does not distinguish between the B, C, and D splice variants, labeled an ~180-kDa band in both human salivary glands, and
a weak signal was also obtained in the rat submandibular gland (Fig.
2B). There was only a very faint band, however, in the rat parotid gland. Immunolabeling controls using peptide-preabsorbed antibodies showed absence of labeling in each case (data not shown).
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Immunohistochemical localization of NBCs in human and rat salivary
glands.
Immunohistochemistry was performed to examine the cellular and
subcellular localization of the electroneutral isoforms NBC3 (Fig.
3) and NBCn1 (Fig.
4) in human and rat parotid and
submandibular glands and in kidney as a positive control.
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NBC3. As shown in Fig. 3, sections of rat (Fig. 3A) and human (Fig. 3B) kidney used as a positive control revealed NBC3 labeling in collecting duct intercalated cells, consistent with previous studies (13). In the rat parotid gland (Fig. 3C), there was strong apical labeling of acinar cells (arrow) and intralobular ducts (arrowhead), in particular, striated ducts. In the rat submandibular gland (Fig. 3D), there was also apical labeling in the intralobular ducts (arrowheads), whereas staining in the acini was less prominent.
In the human parotid gland (Fig. 3E) and submandibular gland (Fig. 3F), very strong apical labeling of the NBC3 was observed in the striated ducts. This is also seen at higher magnification in Fig. 3G. Apical labeling was also evident in the intercalated ducts (arrowhead, ID in Fig. 3G) and main excretory ducts of both glands (arrowheads in Fig. 3, I and J). The purely serous acinar cell population of the human parotid gland was also labeled at the apical membrane (arrow, SA in Fig. 3G), whereas labeling in the mixed submandibular gland was confined to the apical membrane of the serous acini and serous demilunes (Fig. 3, F and H) and absent from the mucous acini of the human submandibular gland (MA in Fig. 3, F and H).NBCn1. As shown in Fig. 4, NBCn1 labeling in rat (Fig. 4A) and human (Fig. 4, B and C) kidney was associated with the basolateral membranes of the medullary thick ascending limbs and intercalated cells of the collecting ducts. This is consistent with previous observations in the rat kidney (33). In both human parotid (Fig. 4, F and G) and submandibular (Fig. 4, H and I) glands, very strong NBCn1 labeling was seen in the striated ducts. This was confined to the extensively folded basolateral plasma membrane domains (arrows in Fig. 4, F-I) whereas there was no labeling observed in the acini (Fig. 4, F-I). The immunolabeling controls were negative (insets in Fig. 4, F and H).
In the rat salivary glands, faint marginal labeling of NBCn1 was detected in the granular ducts of the submandibular glands (Fig. 4E) but not in the acini. No labeling was seen in either the acini or the ducts of the rat parotid glands (Fig. 4D), although there was some weak staining of blood vessels (not shown). ![]() |
DISCUSSION |
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Fluid secretion by salivary acinar cells is driven by secondary
active transport of anions, principally Cl ions in
rodents. The cellular and molecular mechanisms responsible for acinar
Cl
secretion and its subsequent reabsorption in the
ductal system are relatively well established (8, 32).
However, most salivary glands also secrete significant amounts of
HCO
Until recently, it has been widely assumed that intracellular
HCO
Given the marked differences in salivary HCO
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Although it has been suggested that the acinar cells of the human
parotid might be the main source of salivary HCO reabsorption, it is possible that some of the
Cl
reabsorbed at the luminal membrane of the striated
duct cells is exchanged for HCO
reabsorption. On the other hand, the basolateral
Na+/H+ exchanger working in conjunction with
carbonic anhydrase activity would also generate intracellular
HCO
From a comparative viewpoint, it is interesting to note that NBCn1 was
only weakly expressed in the striated ducts of the rat submandibular
gland and, apart from some expression in blood vessels, not at all in
the secretory tissue of the rat parotid gland. These glands,
particularly the rat parotid, secrete significantly less
HCO
In contrast to the NBCn1 isoform, the other electroneutral cotransporter, NBC3, appears to be uniformly expressed at the apical membranes of both the acinar and duct cells in all four glands examined in this study. This finding is probably consistent with recent observations in the mouse submandibular gland (17). In that study, however, the apical labeling obtained with the NBC3 antibody was attributed to the NBCn1 splice variants, which share many sequence similarities with NBC3 at the COOH terminus. Our results, however, suggest that NBC3 and NBCn1 are immunologically distinct, and we would therefore predict that the luminal membrane isoform in the mouse is actually NBC3. Although NBCn1B fragments were successfully amplified from mouse duct cell preparations by RT-PCR (17), this isoform may actually be expressed at the basolateral membrane, as appears to be the case for NBCn1 in the human glands.
On the basis of data from mouse, rat, and human, the presence of NBC3
at the apical membrane of acinar and ductal cells appears to be a
general feature of salivary glands. The presence of this and other
Na+-dependent HCO
Clearly, electroneutral NBC3 and NBCn1 have different cellular distribution patterns in the human and rat salivary glands. We have shown previously that the NBC3 and NBCn1 antibodies also show markedly different labeling patterns in the kidney1 (13, 33). Thus, although the amino acid sequence of human NBC3 is 89-92% identical to that of the rat NBCn1 clones and they may well be splice variants of the corresponding gene in the two species (5, 30), it seems likely that their physiological roles are quite different.
The presence of different isoforms, or splice variants, of the same
transporter in the apical and basolateral domains of epithelial cells
has been extensively observed in the kidney (7, 29). In
some cases, one may be involved in transepithelial electrolyte transport, whereas the other has a housekeeping role in maintaining normal cytosolic ion concentrations. In the salivary glands, we would
suggest that NBC3 and NBCn1 are both involved in transepithelial transport but under quite different physiological conditions. The
basolateral NBCn1 may participate in ductal HCO
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ACKNOWLEDGEMENTS |
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We thank Dr. I. Bögi for access to human tissue, and I. M. Paulsen and M. Pedersen for expert technical assistance.
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FOOTNOTES |
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The Water and Salt Research Center at the University of Aarhus was established by support from Danmarks Grundforskningsfond. Support for this study was provided by the Karen Elise Jensen Foundation, the Novo Nordic Foundation, the Danish Medical Research Council, the University of Aarhus Research Foundation, the University of Aarhus, the Wellcome Trust, and the Commission of the European Union (K.A. 3.1.2 and K.A. 3.1.3. Programmes).
Address for reprint requests and other correspondence: S. Nielsen, The Water and Salt Research Center, Institute of Anatomy, Univ. of Aarhus, DK-8000 Aarhus C, Denmark (E-mail: sn{at}ana.au.dk).
1 NBCn1 immunolabeling in rat kidney is present in the basolateral plasma membranes of thick ascending limb cells in the outer medulla as well as of intercalated cells in inner medulla. NBC3 is exclusively associated with intercalated cells in connecting tubules and in cortical, outer medullary, and initial inner medullary collecting ducts.
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.
March 6, 2002;10.1152/ajpgi.00421.2001
Received 31 October 2001; accepted in final form 22 February 2002.
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