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Am J Physiol Gastrointest Liver Physiol 291: G1031-G1040, 2006. First published June 15, 2006; doi:10.1152/ajpgi.00483.2005
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LIVER AND BILIARY TRACT

Expression of the Na+-HCO3– cotransporter and its role in pHi regulation in guinea pig salivary glands

Jingchao Li,1 Na-Youn Koo,1 Ik-Hyun Cho,1 Tae-Hwan Kwon,2 Se-Young Choi,1 Sung J. Lee,1 Seog B. Oh,1 Joong-Soo Kim,1 and Kyungpyo Park1

1Department of Physiology, College of Dentistry, Seoul National University and Dental Research Institute, Seoul; and 2Department of Biochemistry and Cell Biology, School of Medicine, Kyungpook National University, Taegu, Korea

Submitted 11 October 2005 ; accepted in final form 29 May 2006


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Patterns of salivary HCO3– secretion vary and depend on species and gland types. However, the identities of the transporters involved in HCO3– transport and the underlying mechanism of intracellular pH (pHi) regulation in salivary glands still remain unclear. In this study, we examined the expression of the Na+-HCO3– cotransporter (NBC) and its role in pHi regulation in guinea pig salivary glands, which can serve as an experimental model to study HCO3– transport in human salivary glands. RT-PCR, immunohistochemistry, and pHi measurements from BCECF-AM-loaded cells were performed. The amiloride-sensitive Na+/H+ exchanger (NHE) played a putative role in pHi regulation in salivary acinar cells and also appeared to be involved in regulation in salivary ducts. In addition to NHE, NBC also played a role in pHi regulation in both acini and ducts. In the parotid gland, NBC1 was functionally expressed in the basolateral membrane (BLM) of acinar cells and the luminal membrane (LM) of ducts. In the submandibular gland, NBC1 was expressed only in the BLM of ducts. NBC1 expressed in these two types of salivary glands takes up HCO3– and is involved in pHi regulation. Although NBC3 immunoreactivity was also detected in submandibular gland acinar cells and in the ducts of both glands, it is unlikely that NBC3 plays any role in pHi regulation. We conclude that NBC1 is functionally expressed and plays a role in pHi regulation in guinea pig salivary glands but that its localization and role are different depending on the type of salivary glands.

intracellular pH; Na+/H+ exchange

THE REGULATION of intracellular pH (pHi) in epithelial cells is critical for fluid and electrolyte absorption and secretion. Therefore, epithelial cells are equipped with diverse pHi regulatory mechanisms. However, there are some differences in the pHi regulatory mechanisms among species and gland types. In salivary acinar cells, the initial response to a fluid secretion stimulus is an acidification of the cytosol resulting from a HCO3– efflux into the lumen (10, 13, 19, 37), and the secreted HCO3– plays a role in buffering the acid produced by oral bacteria and in providing an optimal pH for salivary amylase activity (6).

It is known that intracellular HCO3– is generated by carbonic anhydrase, which converts the CO2 that has freely diffused into the cells from the blood into H+ and HCO3– (21). The H+ generated during this HCO3– production is then extruded outside the cells by the Na+/H+ exchanger (NHE) (19, 22, 35). However, relatively little is known about the transport mechanisms involved in HCO3– uptake or reabsorption in either acinar cells or the ducts. The rapid changes in HCO3– concentration over a wide range in human parotid glands (PGs) (2) suggests that additional or alternate pathways for HCO3– transport may exist in the acini and/or ducts.

After the cDNA encoding an electrogenic Na+-HCO3– cotransporter (NBC) in the kidney was cloned (29), several other subtypes from the NBC family, including pNBC1 (1), NBCn1 (4), and NBC3 (27), have been functionally characterized. Recent immunohistochemical studies on electrogenic NBC1 and electroneutral NBC3 in salivary glands (5, 23, 31) have suggested that these NBCs may also contribute to the supply of intracellular HCO3–. Guinea pigs have been used as an experimental model for studying HCO3– transport in the human pancreas (8). We also found that HCO3– concentrations of resting and stimulated whole saliva of guinea pigs are very similar to those of humans (2), which suggests that guinea pig salivary glands can also serve as an experimental model for studying HCO3– transport in human salivary secretion. Therefore, we examined the expression patterns and functions of NBC1 and NBC3 in guinea pig PGs and submandibular glands (SMGs).

We have demonstrated that pHi is differently regulated depending on the type of salivary glands, even in the same species, namely, the guinea pig. NHE was the exclusive pHi regulator in SMG acinar cells. However, in addition to NHE, 5-(N-ethyl-N-isopropyl)amiloride (EIPA)-insensitive, Na+- and HCO3–-dependent NBC1 substantially contributed to pHi regulation in PG acinar cells and in PG and SMG duct cells in guinea pigs.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Materials and solutions. Collagenase (CLSPA type) was purchased from Worthington Biomedical (Lakewood, NJ), and 2',7'-bis-(2-carboxyethyl)-5-(and-6)-carboxyfluorescein (BCECF)-AM was obtained from Molecular Probes (Eugene, OR). All other reagents, including EIPA and DIDS, were obtained from Sigma (St. Louis, MO). The physiological salt solution (PSS) consisted of (in mM) 135 NaCl, 5.4 KCl, 1.2 CaCl2, 0.8 MgSO4, 0.33 NaH2PO4, 0.4 KH2PO4, 10 glucose, and 20 HEPES (pH 7.4 with NaOH). The HCO3–-free solution [HEPES-buffered solution (HBS)] contained (in mM) 140 NaCl, 5 KCl, 1 MgCl2, 1 CaCl2, 10 HEPES (pH 7.4 with NaOH), and 10 glucose. In the HCO3–-containing solution [HCO3–-buffered solution (BBS)], 25 mM NaCl was replaced with an equal concentration of NaHCO3. BBS was gassed with 5% CO2-95% O2. Na+-free solutions were prepared by replacing Na+ with N-methyl-D-glucamine (NMDG). NH4+-containing solutions were prepared by replacing 20 mM Na+ with 20 mM NH4+. Cl–-free solutions were prepared by replacing Cl– with an equimolar amount of gluconic acid. Additional Ca2+ was added to compensate for chelation by gluconate. NBC1 and NBC3 antibodies were purchased from Chemicon (Temacula, CA).

Saliva collection and determination of HCO3– concentrations. All animals were used according to the protocol of the Animal Care and Use Committee of Seoul National University. Male guinea pigs weighing 400–500 g were anesthetized intraperitoneally using pentobarbital sodium (50 mg/kg). Resting whole saliva of the guinea pigs was collected over two 5-min intervals by aspiration. The collection of stimulated saliva was done by a subcutaneous injection of pilocarpine (80 mg/kg). Resting and stimulated human whole saliva were obtained from five healthy volunteers. In human subjects, stimulated saliva was collected using 3% citric acid to stimulation the tongue. At the end of the saliva collection, the concentration of HCO3– was measured using an ion analyzer (Stat Profile pHOx Plus, Nova Biomedical).

RT-PCR of NBCs. Total RNA from guinea pig PGs was extracted by procedures of Qiagen RNeasy Mini or Micro Kits (Qiagen, Valencia, CA) and subjected to RT-PCR using an oligo-dT reverse transcriptase primer and Moloney murine leukemia virus reverse transcriptase (Invitrogen). Degenerate oligonucleotide primers derived from conserved NBC sequences of the rat (GenBank Accession No. AF124441), mouse (GenBank Accession No. AF141934), and human (GenBank Accession No. AF011390) were used to amplify cDNA clones of two different NBC isoforms (kNBC1 and pNBC1). Guinea pig beta-actin (GenBank Accession No. AF508792) was amplified to assess the cDNA yield. Primers were designed to amplify, specifically, the following transcripts pf guinea pig beta-actin (435 bp): 5'-TGCGTGACATCAAGGAGAAG-3' (sense) and 5'-GCTGGAAGGTGGAGAGTGAG-3' (antisense). The degenerate primers for the NBC1s (kNBC1 and pNBC1) were 5'-GAGAAAGGATCCATCATGC-3', 5'-AGCATGACAGCCTGCTGTA-3', 5'-TGTGCCCACAAGGTTCTTGTTC-3', and 5'-TTCTCTCCACCTGAGTACATATT-3'. The degenerative primers for NBC3 (GenBank Accession No. AF047033) were 5'-AGGAACACAAATTGAAGAAAGGAG-3' and 5'-ACTCCCATATAAAGGAAAACACCA-3'. The cycling parameters were as follows: 38 cycles of 94°C for 55 s, 57°C for 55 s, and 72°C for 2 min. Expected PCR products of ~437, 182, and 316 bp were generated, and DNA sequencing was analyzed using dye terminator methods with BaseStation (MJ Research). These PCR fragments showed a 95% homology to human NBC1 (GenBank Accession No. AF011390) and NBC3 (GenBank Accession No. AF047033). Subsequently, we designed the following specific primers for guinea pig NBC1: 5'-GTGCAAGTAGGATGTTCAA-3' and 5'-ATCTCATGGTACGACTTGTCTT-3'. The expected PCR product was ~320 bp.

Acinar cell and intralobular duct preparations. The guinea pig PG and SMG were dispersed into individual ducts (intralobular) and acini by a modification of the methods described previously (10). Briefly, guinea pigs were anesthetized by an inhalation of diethyl ether and killed by cervical dislocation. The PG or SMG was quickly removed and finely minced in PSS supplemented with 0.1% sodium pyruvate, 0.02% trypsin inhibitor, and 0.1% BSA. The tissues were then digested in the same solution containing collagenase (100 U/ml) at 37°C for 60 min with continuous agitation. During the incubation, cells were periodically dispersed by trituration at 20, 40, and 60 min.

Isolation of interlobular ducts and microperfusion via the ductal lumen. Microdissection of the ducts was performed under a stereomicroscope by a previously described method, which was originally developed for isolating interlobular ducts from the guinea pig pancreas (8). After the PG and SMG were removed, they were injected with digestion buffer and coarsely chopped. The digestion buffer consisted of DMEM containing 80 U/ml collagenase, 400 U/ml hyaluronidase, and 2 mg/ml BSA. Chopped tissues were put into the digestion buffer, gassed with 5% CO2-95% O2, shaken in a 37°C water bath for 35 min, and then changed with fresh digestion buffer for another 30 min. After digestion, the tissues were washed three times with DMEM and resuspended in a storage buffer consisting of DMEM with 3% (wt/vol) BSA. The interlobular ducts were then microdissected under the stereomicroscope. Dissected interlobular ducts were placed in storage buffer on ice until the pHi was measured. We constructed the cannula using a yellow tip to make its diameter small enough to cannulate the interlobular excretory duct under the stereomicroscope. The microdissected interlobular duct was first cannulated by a modified yellow tip, and its tip was then connected again with the polyethylene cannula (inner diameter 0.28 mm and outer diameter 0.61 mm) for the microperfusion study. A DAD-12 (ALA Scientific Instruments, Westbury, NY) system with a controlled pressure of N2 gas (30–80 psi) was used for microperfusion at a flow rate of 4 µl/10 min.

pHi measurements. Isolated acini or intralobular ducts were incubated with 2 µM BCECF-AM for 30 min at room temperature and washed once with PSS containing 0.1% BSA. They were kept on ice until use. For the microperfusion study, 5 µM BCECF-AM was loaded via the ductal lumen. The fluorescence of isolated acini or ducts was measured by photon counting using a Photon Technology system (South Brunswick, NJ). BCECF fluorescence was recorded at excitation wavelengths of 440 and 490 nm with an emission wavelength of 530 nm. The 490-to-440-nm fluorescence ratios were calibrated using the high-potassium nigericin procedure described previously (34). Values are reported means ± SE of the numbers of acinar aggregates or ducts examined. Acidification of the cells was induced by an exposure to 20 mM NH4Cl for 1 min. We then observed the pHi recovery for the two different types of salivary glands. During the pHi measurements, bath solution was superfused at a flow rate of 3 ml/min.

Immunohistochemistry of NBCs. Male guinea pigs were anesthetized via inhalation of diethyl ether. The PG and SMG were removed and postfixed in cold fitative (3% paraformaldehyde in 0.01 M phosphate buffer, pH 7.4) for 3 h. The tissues were rinsed and dehydrated in ethanol followed by xylene, embedded in paraffin, and cut at a thickness of 2 µm. For immunohistochemistry, sections were dewaxed, rehydrated, and incubated for 30 min with 3% H2O2 in 100% methanol to remove endogenous peroxidase activity. For antigen retrieval, sections were put in Tris-EGTA buffer (1 mM Tris solution supplemented with 0.5 mM EGTA, pH 9.0) and heated using a microwave for 10 min. Nonspecific binding of immunoglobulin was prevented by incubating sections in 50 mM NH4Cl in 0.01 M PBS (pH 7.4) for 30 min, followed by blocking with 0.01M PBS containing 1% BSA, 0.05% saponin, and 0.2% gelatin for 1 h at room temperature. Sections were then incubated overnight at 4°C with primary antibodies diluted in 0.01 M PBS containing 0.1% BSA and 0.3% Triton X-100. After being washed three times in washing buffer (0.01 M PBS containing 0.1% BSA, 0.05% saponin, and 0.2% gelatin), sections were incubated with a biotinylated secondary antibody (Vector Laboratories, Burlingame, CA) at a dilution of 1:200 for 1 h at room temperature followed by an incubation with avidin and biotinylated horseradish peroxidase complex (Vector Laboratories) at 1:100 for 1 h at room temperature. Sections were then visualized with 3,3'-diaminobenzidine (Sigma). The rat kidney was used as a positive control. After being counterstained with hematoxylin, sections were dehydrated and coverslipped. Slides were examined and photographed with a light microscope. Polyclonal antibodies to the subunits of NBC1/pNBC and NBC3 were obtained from Chemicon. The immunogens of the guinea pig anti-NBC1 (NBC1/pNBC) and rabbit anti-NBC3 were amino acids 928–1035 of the COOH terminus of human NBC1 and a 19-amino acid peptide sequence with the cytoplasmic COOH terminus of human NBC3, respectively.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 GRANTS
 REFERENCES
 
HCO3– concentrations in resting or stimulated whole saliva of guinea pigs and humans. The saliva collected from guinea pigs and humans was analyzed for HCO3– concentrations. We used whole saliva, which is a mixture of salivas from three sets of major glands and many minor glands. Resting and stimulated whole saliva of the guinea pigs contained 4.0 ± 1.0 (n = 5) and 16.2 ± 0.8 mM (n = 5) HCO3–, respectively, whereas, in humans, resting and stimulated saliva contained 2.4 ± 0.3 (n = 5) and 16.4 ± 3.1 mM (n = 5) HCO3–, respectively. The results indicate that HCO3– concentrations of guinea pig saliva, in either a resting or stimulated state, are very similar to those of humans.

RT-PCR analysis for NBC1 and NBC3 expression in guinea pig salivary glands. RT-PCR was performed to determine if electrogenic NBC1 and electroneutral NBC3 mRNA are expressed in the PG and SMG of guinea pigs. NBC1 transcripts with a predicted molecular size of 437 bp (NH2 terminal) and of 182 bp (middle portion) were observed from whole PG tissue (Fig. 1A). We confirmed that our RT-PCR product was NBC1 by comparing its partial sequence with the sequence of human NBC1 (1) and the guinea pig NBC1 gene (11), the results of which are shown in our companion study. It was found that NBC1 in our experiments is the pancreatic type, pNBC1 (11), which showed a 95% sequence homology with human pNBC1 (GenBank Accession No. AF011390). Subsequently, using the specific pNBC1 primer for the guinea pig (with an expected product size of 320 bp), we then characterized NBC1 mRNA expression in acini and ducts from two different glands, the PG and SMG (Fig. 1B). Figure 1B, top, shows that NBC1 transcripts are expressed in both acini and ducts of the PG but only in ducts of the SMG. Figure 1B, middle, shows NBC3 transcripts with a predicted molecular size of 316 bp. In PGs, the mRNA of NBC3 was detected only in ducts, but NBC3 mRNA was expressed both in acinar and ducts in SMGs.


Figure 1
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  		Fig. 1. RT-PCR analysis of Na+-HCO3– cotransporter (NBC) isoforms in the parotid gland (PG) and submandibular gland (SMG) of the guinea pig. A: NBC1 transcripts with a predicted molecular size of 437 (NH2 terminal) and 182 bp (middle portion) were generated using degenerative oligonucleotide primers derived from conserved NBC1 sequences of the rat, mouse, and human. B: NBC1 transcripts in acini and ducts (top). In this case, we used specific primers (NH2 terminal) for guinea pig NBC1. NBC1 transcripts were detected from both acini and ducts in the PG but only from ducts in the SMG. Middle, NBC3 transcripts in acini and ducts with a predicted molecular size of 316 bp. NBC3 mRNA was expressed in PG ducts, SMG ducts, and acini. Bottom, beta-actin transcripts in PG and SMG acini and ducts.

 
Immunohistochemical localization of NBC1 and NBC3. Immunohistochemistry was performed to examine the cellular and subcellular localization of NBCs in guinea pig salivary glands. The rat kidney (32) was used as a positive control, which revealed NBC1 expression (indicated by arrows) at the basolateral membrane (BLM) of proximal tubules (Fig. 2A). Figure 2, B–D, shows staining of NBC1 in guinea pig PGs. Strong labelings were observed in the BLM (Fig. 2B) of acinar cells. In intralobular (Fig. 2C) and interlobular ducts (Fig. 2D), strong stainings of NBC1 were shown in the luminal membrane (LM). In SMGs, NBC1 staining was evident in the BLM of intralobular (Fig. 2E) and interlobular ducts (Fig. 2F) but not in acini. Figure 3 shows NBC3 expression (indicated by arrows). Staining in intercalated cells of the cortical collecting duct in the rat kidney was used as a positive control (12) (Fig. 3A). In the SMG, NBC3 staining was strong in the LM of acinar cells (Fig. 3, B–D) and the LM of intralobular (Fig. 3, B and C) and interlobular ducts (Fig. 3D). In PGs, NBC3 staining was observed in the BLM of intralobular (Fig. 3E) and interlobular ducts (Fig. 3F) but not in acini.


Figure 2
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  		Fig. 2. Labeling of NBC1 (indicated by arrows). A: rat kidney used as a positive control, which revealed NBC1 labeling at the basolateral membrane (BLM) side of proximal tubules. B: NBC1 antibody-stained BLM of PG acinar cells. C and D: strong labelings of NBC1 in the luminal membrane (LM) of intralobular ducts (C) and interlobular excretory ducts (D). E: strong labelings of BLM were observed in SMG intralobular duct cells, but there was no NBC1 staining in SMG acinar cells. F: strong labelings of NBC1 in the BLM of interlobular ducts of the SMG. Magnification: x400 in A–F.

 

Figure 3
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  		Fig. 3. Immunoperoxidase labeling of NBC3 (indicated by arrows) in the rat kidney (A), SMG (B–D), and PG (E and F) from the guinea pig. A: NBC3 immunolabeling was seen at intercalated cells of the cortical collecting duct in the rat kidney, which was used as a positive control. B–D: in SMGs, NBC3 labeling was associated with the apical plasma membrane of acini (B–D), intralobular ducts (C), and interlobular ducts (D). E and F: in PGs, NBC3 labeling was associated with the basolateral plasma membrane of intralobular ducts (E) and interlobular ducts (F), whereas no labeling was seen in acini (E). Magnification: x630 in A–F.

 
pHi recovery in PG and SMG acinar cells. Throughout the experiments, we used a NH4Cl prepulse technique to acidify the cells. When cells are exposed to NH4Cl for 1 min (indicated by the solid bars), they are transiently alkalinized and then become acidified later. Using this technique, we measured the pHi recovery rate from cell acidosis and investigated the involved transporters using specific blockers. Figure 4 shows pHi recovery patterns of acinar cells from two different types of guinea pig salivary glands, the SMG (A and B) and PG (C and D). Figure 4, A and C, shows the pHi recovery in HBS, whereas Fig. 4, B and D, shows the pHi recovery in BBS. The pHi of acinar cells in both salivary glands did not recover after the NH4Cl pulse in the Na+-free solution (indicated by the open bars), suggesting that the pHi recovery was Na+ dependent. We first examined the effect of an amiloride derivative, EIPA, which is a NHE inhibitor, on pHi recovery. As shown in Fig. 4, A and C, 1 µM EIPA completely inhibited pHi recoveries in both types of acinar cells in HBS. The experiment was repeated in a different bath solution, BBS (Fig. 4, B and D). Although the pHi recovery was also completely inhibited by 1 µM EIPA in SMG acinar cells (Fig. 4B), 1 µM EIPA only partly inhibited the pHi recovery in PG acinar cells (Fig. 4D).


Figure 4
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  		Fig. 4. Intracellular pH (pHi) recovery patterns from NH4Cl (solid bars)-induced acidification in guinea pig salivary glands and the effects of 5-(N-ethyl-N-isopropyl)amiloride (EIPA). A: normal pHi recovery in SMG acinar cells and complete inhibition by 1 µM EIPA (hatched bar) in HEPES-buffered solution (HBS). B: complete inhibition of pHi recovery in SMG acinar cells in the presence of 1 µM EIPA in HCO3–-buffered solution (BBS). C: normal pHi recovery and complete inhibition by 1µM EIPA in PG acinar cells in HBS. D: partial inhibition of pHi recovery in PG acinar cells by 1 µM EIPA in BBS. Shown are representative experiments from 5–12 experiments.

 
Functional expression of NBC1 in PG acinar cells. Figure 5A summarizes the results of the experiments shown in Fig. 4. The pHi recovery rate of SMG acinar cells was not much different between the two bath solutions, HBS and BBS. Furthermore, the complete inhibition of pHi recovery in SMG acinar cells in the presence of 1 µM EIPA in both HBS and BBS suggests that NHE1 is the exclusive pHi regulator in this tissue. In PG acinar cells, the rate of pHi recovery in BBS (0.687 ± 0.091 pH units/min, n = 5) was much faster (P < 0.01) than in HBS (0.288 ± 0.028 pH units/min, n = 12). In the presence of HCO3–, EIPA did not completely inhibit the pHi recovery (0.151 ± 0.034 pH units/min, n = 11), suggesting that in PG acinar cells, an EIPA-insensitive, HCO3–- and Na+-dependent mechanism, in addition to NHE, is involved in pHi recovery. Thus, we further examined whether the residual component of pHi recovery is inhibited by DIDS, a specific NBC1 blocker. Figure 5B shows a representation of four experiments in BBS. The EIPA-insensitive pHi recovery component in PG acinar cells was completely inhibited by 1 mM DIDS.


Figure 5
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  		Fig. 5. A: summarized result of pHi recoveries of acinar cells (n = 5–12) and the effects of DIDS. The pHi recovery rates of SMG acinar cells were not significantly different (P > 0.1) between HBS (–) and BBS (+). By contrast, in PG acinar cells, the pHi recovery rate in BBS was much faster (**P < 0.01) than those in HBS. The pHi recovery of PG acinar cells in BBS was partly blocked by 1 µM EIPA compared with those in HBS (*P < 0.05). B: complete inhibition of pHi recoveries in PG acinar cells in the presence of 1 µM EIPA (hatched bars) and 1 mM DIDS (shaded bar) in BBS. The EIPA-insensitive pHi recovery component (0.218 ± 0.044 pH units/min, n = 4) in PG acinar cells was completely blocked by 1 mM DIDS.

 
pHi recovery and NBC1 expression in intralobular ducts. We next examined pHi regulation in intralobular striated ducts from two different types of salivary glands, the PG and SMG. Figure 6, A and B, shows superimposed raw traces for pHi recoveries from NH4Cl-induced acidosis at various EIPA concentrations in HBS. The degree of inhibition by EIPA was in proportion to its concentration in both ducts. The 1 µM EIPA concentration that had completely inhibited pHi recovery in acinar cells (see Fig. 4, A and C) partly inhibited the pHi recovery of PG and SMG ducts by 59 ± 9% (n = 4) and 73 ± 11% (n = 3), respectively, compared with their full recovery rates. Only the higher concentrations of EIPA, 100 and 10 µM, could almost completely inhibit pHi recovery by 97 ± 8% (n = 3) and 93 ± 7% (n = 4), respectively, in PG and SMG ducts. The IC50 was estimated as 0.33 ± 0.12 (n = 4) and 0.18 ± 0.17 µM (n = 3) in PG and SMG ducts, respectively (Fig. 6C). By contrast, in BBS (Fig. 6, D and E), the higher concentrations (100 and 10 µM) of EIPA inhibited pHi recovery by only 42 ± 6% (n = 4) and 49 ± 9% (n = 3) in PG and SMG ducts, respectively, compared with the normal recovery. These residual pHi recoveries were further inhibited by 93 ± 4% (n = 3) and 91 ± 8% (n = 3) in PG and SMG ducts by 1 mM DIDS, suggesting that, in addition to NHEs, NBC1 is functionally expressed in the two intralobular duct types. On the basis of the immunohistochemistry data, intralobular NBC1 appears to be expressed in the LM of PG ducts and the BLM of SMG ducts. However, the immunohistochemical data alone may not be sufficient to verify the functional expression of NBC1. Therefore, we went on further to perform a microperfusion study in interlobular excretory ducts, which enabled separated perfusions, one via the bath and the other via the ductal lumen.


Figure 6
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  		Fig. 6. pHi regulation in intralobular ducts from two different types of salivary glands: the PG and SMG. A and B: superimposed raw traces for pHi recoveries from a NH4Cl-induced acid load in the presence of various concentrations of EIPA in PG ducts (A) and SMG ducts (B). C: average IC50 values of EIPA in HBS from the two ducts. D: pHi recovery in PG ducts (0.721 ± 0.047 pH units/min, n = 4) was only partly inhibited by 100 µM EIPA (0.416 ± 0.045 pH units/min, n = 4) in BBS, and the residual pHi recovery component was further inhibited by 1 mM DIDS (0.050 ± 0.026 pH units/min, n = 3). E: pHi recovery pattern in SMG ducts using the same protocol as for D. In this experiment, 10 µM EIPA was used. The normal pHi recovery (0.729 ± 0.027 pH units/min, n = 4) was partly inhibited by 10 µM EIPA (0.369 ± 0.064 pH units/min, n = 3), and the residual pHi recovery component was further inhibited by 1 mM DIDS (0.066 ± 0.055 pH units/min, n = 3). D and E each show representative curves from 3 experiments.

 
Subcellular localization and NBC1 expression in interlobular ducts. Figure 7 shows a series of microperfusion studies in interlobular ducts of the PG and SMG. When we studied the pHi recovery in the LM of the ducts, the bath was superfused with Na+-free solution to inhibit the pHi recovery that may occur in the BLM of ducts. The addition of Na+ (140 mM) into the ductal lumen caused pHi recovery from the NH4Cl-induced acidification, and 1 µM EIPA completely inhibited the pHi recovery in HBS in PG interlobular ducts (Fig. 7A). However, the same concentration of EIPA did not block the pHi recovery in BBS, and the residual pHi recovery component was completely blocked by the addition of 1 mM DIDS (Fig. 7B). This result suggests that NBC1 is functionally expressed in the LM of the interlobular duct. We also wanted to know whether Cl–/HCO3– exchange is expressed in the LM. Figure 7C shows one of three typical experiments. When we omitted Cl– (indicated by the solid bar) from the ductal lumen, pHi was dramatically increased, and the increased pHi was restored to the prestimulus level by the readdition of Cl–, suggesting that Cl–/HCO3– exchange is also functionally expressed in the LM. We then added 1 mM DIDS into the ductal lumen to examine whether NBC1 functions in a resting state. The application of DIDS for 5 min slowly decreased the pHi from 7.45 to 7.2, suggesting that HCO3– in the ductal lumen is reabsorbed in the resting state. However, the pHi recovery pattern in the LM of SMG interlobular ducts was different. Figure 7D shows superimposed traces of pHi recovery under different EIPA concentrations in HBS. We found that 5 µM EIPA only partly (71 ± 4%, n = 3) inhibited pHi recovery and that 50 µM EIPA inhibited pHi recovery by 94 ± 6% (n = 3) compared with normal recovery. This result indicates that the LM of SMG interlobular ducts expresses EIPA-resistant NHE isoforms, probably NHE3. Furthermore, Fig. 7E shows that the inhibitory effect of the highest concentration of EIPA, 50 µM, on the pHi recovery was not significantly different (P > 0.1) between HBS (94 ± 6%) and BBS (92 ± 5%, n = 3), suggesting that NBC1 is not functionally expressed in the LM of SMGs.


Figure 7
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  		Fig. 7. pHi recovery patterns in the LM of PG (A–C) and SMG interlobular ducts (D and E) by the microperfusion technique via the ductal lumen. A: normal pHi recovery (0.097 ± 0.003 pH units/min, n = 3) was completely inhibited by 1 µM EIPA in PG ducts in HBS. B: pHi recovery pattern in the presence of 1 µM EIPA in BBS (0.101 ± 0.011 pH units/min, n = 3). EIPA only partly inhibited pHi recovery, and the residual pHi recovery component was further completely inhibited by 1 mM DIDS. C: rapid increase of pHi induced by Cl– removal from the ductal lumen. The decreased pHi was recovered to the prestimulus level by the readdition of Cl–. Application of 1 mM DIDS slowly decreased pHi from 7.45 to 7.2 over 5 min. D: superimposed raw traces of pHi recoveries in various concentrations of EIPA (0, 5, and 50 µM) in HBS. The degree of inhibition of the pHi recovery by EIPA was in proportion to its concentration. The highest concentration of EIPA, 50 µM, was needed to almost completely inhibit pHi recovery in the LM. E: pHi recovery pattern in the presence of the highest concentration of EIPA, 50 µM, in BBS (0.037 ± 0.022 pH units/min, n = 3). There was no significant difference (P > 0.1) between BBS and HBS (0.025 ± 0.009 pH units/min, n = 3). A–C and E are representative experiments from 3 or 4 experiments.

 
We next examined the pHi recovery mechanisms in the BLM of interlobular ducts. The ductal lumen was perfused with Na+-free solution to inhibit the pHi recovery that may occur in the LM of ducts. Figure 8, A and B, shows pHi recovery patterns from PGs. The addition of Na+ to the bath solutions neither induced pHi recovery in HBS (Fig. 8A) nor BBS (Fig. 8B), although the addition of Na+ into the ductal lumen rapidly restored pHi. This result strongly suggests that any Na+-dependent pHi recovery mechanisms, such as NHE1, do not exist in the BLM of PG ducts. In contrast to the PG, the addition of Na+ to the bath solution rapidly restored pHi in SMG ducts, and the pHi recovery rate was further increased by the addition of Na+ into the ductal lumen in HBS from a rate of 0.094 ± 0.016 (n = 3) to 0.502 ± 0.023 pH units/min (n = 3; Fig. 8C). Figure 8D shows that 1 µM EIPA only partly inhibited pHi recovery by 38 ± 12% (n = 3) compared with the normal recovery in BBS, and the residual component was further inhibited by 90 ± 2% (n = 3) by the addition of 1 mM DIDS, suggesting that NBC1 is functionally expressed in the BLM. All our major findings are summarized in Table 1.


Figure 8
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  		Fig. 8. pHi recovery patterns in the BLM of PG (A and B) and SMG interlobular ducts (C and D) by the microperfusion technique. It is noteworthy that the addition of Na+ into the bath solution did not induced pHi recovery in HBS (A) or BBS (B) until the addition of Na+ into the ductal lumen had rapidly restored pHi. C: in SMG interlobular ducts, the addition of Na+ into the bath solution induced pHi recovery (0.094 ± 0.016 pH units/min, n = 3), which further increased by the addition of Na+ into the ductal lumen (0.502 ± 0.023 pH units/min, n = 3). The concentration of 1 µM EIPA almost completely inhibited this pHi recovery in the BLM (0.021 ± 0.004 pH units/min, n = 3). D: 1 µM EIPA only partly inhibited pHi recovery (0.078 ± 0.005 pH units/min, n = 3) in BBS, and the residual pHi recovery component was almost completely inhibited by 1 mM DIDS (0.013 ± 0.003 pH units/min, n = 3) in the BLM. A–D are representative experiments selected from 3 or 4 experiments.

 

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  		Table 1. Summary of NBC1 and NBC3 expression patterns in guinea pig salivary glands

 

   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
GRANTS
 REFERENCES
 
Guinea pig salivary glands as a HCO3– transport model. HCO3– is the most important buffering system in saliva. Although its concentration is very low in unstimulated saliva, it can be increased up to 60 mM at high flow rates in humans (2). However, it is still unclear what mechanisms mediate HCO3– secretion and absorption in salivary glands. The marked differences in salivary HCO3– secretion among species and among glands within species suggests that the expression patterns of the NBC may well differ both among species and glands. For example, with increasing salivary flow rates, HCO3– concentrations rise in human PGs but fall in rat PGs (33). Interestingly, we found that the value of the HCO3– concentration of whole saliva in guinea pigs was similar with that of humans, suggesting that guinea pig salivary glands may be used as an appropriate experimental model for studying HCO3– transport in humans.

Functional expression of NHEs in guinea pig salivary glands. We found that there was a significant difference in the threshold concentrations of EIPA required to inhibit NHE activity between two tissues, acini and ducts from two salivary glands (the PG and SMG). Amiloride-sensitive NHEs, probably NHE1 and/or NHE2, played a putative role in pHi regulation in salivary acinar cells, because 1 µM of EIPA, a specific blocker for NHE1 or NHE2, completely inhibited pHi recovery in HBS. However, the inhibition threshold of EIPA in duct cells was more than one order of magnitude higher than that in acinar cells. On the basis of the inhibitory efficiency of the known EIPA to different NHE isoforms (17), another type of NHE isoform, probably NHE3 located in the LM (24), appears to act together with NHE1 to regulate pHi in ducts. We did not further examine the molecular identities of the NHE isoforms expressed in these ducts, because our main aim in the present study was to elucidate a HCO3– transport mechanism in relation with the expression of NBCs. Thus, after determining the maximal concentration of EIPA needed to inhibit NHEs, we studied the activity of NBCs in the presence of the maximal concentration of EIPA.

Functional expression of NBCs in guinea pig salivary glands. Table 1 summarizes our major findings for NBC1 and NBC3 expression, including all the RT-PCR, immunohistochemistry, and functional studies. Our results provided evidence for a heterogeneous distribution of NBCs in guinea pig salivary glands. NBC1 showed distinct membrane domain localization; it was expressed in the BLM of PG acinar cells and the LM of intralobular and interlobular PG ducts. Particularly, it is noteworthy that NBC1 was functionally expressed in the LM of PG ducts. Roussa (30) also reported NBC1 expression by immunohistochemistry in the LM of human PG striated ducts. We not only verified its localization by immunohistochemistry but also confirmed its function by pHi measurements using microperfusion. Although immunolabeling of NBC3 was also detected in PG and SMG ducts, we did not find any evidence of NBC3 involvement in pHi regulation. Throughout the experiments, 1 mM DIDS completely inhibited all of the residual EIPA-insensitive pHi recovery components in BBS. The absence of pHi recovery in the BLM of PG ducts suggests that NBC3, with its location confirmed by immunohistochemistry, does not play any role in pHi regulation. However, in the SMG duct, we cannot rule out the possibility that high concentrations of EIPA (>50 µM) inhibited NBC3 activity. Whether high concentrations of EIPA inhibit NBC3 activity (27) or not (25) is still controversial.

Physiological role of NBCs in guinea pig salivary glands. We found that when guinea pig PG acinar cells were acidified, the pHi recovered to the prestimulus level. In this process, in addition to NHE1 and/or NHE2, NBC1 in the BLM also contributed to pHi recovery by taking up HCO3–. The favorable electrical potential due to a membrane depolarization by Cl– and/or HCO3– efflux (9, 10, 26) can further provide a driving force for HCO3– influx via NBC1. The concentration of HCO3– varies with the flow rate. Unstimulated saliva contains very little HCO3–, whereas stimulated saliva contains a much higher concentration (2). One possible explanation for the very low HCO3– concentration in unstimulated saliva is due to the reabsorption of HCO3– in ducts. However, there is no candidate NBC reported, especially in PG ducts, that may mediate HCO3– reabsorption. To our knowledge, this is the first report of a functional expression of NBC1 in the LM of PG ducts in mammalian salivary glands. Our results strongly suggest that luminal NBC1 plays a dominant role in HCO3– reabsorption. Although NBC3 has been believed to play a HCO3– salvage role in mouse SMGs (16), we did not find any evidence of NBC3 expression in the LM of PG ducts. This discrepancy may be due to the fact that NBCs involved in the salvage of HCO3– depend on the species and type of salivary gland. As in acinar cells, duct cells are also depolarized by agonists. The depolarization can be induced by Cl– or HCO3– exit via the CFTR (7). This favorable electromotive force for the influx mode of NBC1 may further accelerate the reabsorption of HCO3– from the ductal lumen. This luminal NBC1 also appears to function in the resting state, because there was a slow decrease of ductal pHi in the presence of 1 mM DIDS. It is unlikely that other DIDS-sensitive NBCs, such as AE1–3 of the SLC4 family (28) and some electrogenic Cl–/HCO3– extrangers of the SLC26 family (20), are involved in this process. Although the direction of Cl– and HCO3– movement via the Cl–/HCO3– exchanger depends on local gradients, it is likely that the Cl–/HCO3– exchanger in our experiment generates net Cl– uptake and HCO3– efflux, because the Cl– concentration, 115 mM, of the microperfusion solution via the ductal lumen may be much higher than that of duct cells. Although any data for the intracellular Cl– concentration in guinea pig duct cells are not available, studies have reported a Cl– concentration of 38 ± 8 and 57.7 ± 4.1 mM in the rat (36) and rabbit SMG ducts (14), respectively. If we assume that the Cl– concentration of guinea pig duct cells is not much different from that of other rodents such as the rat or rabbit, the Cl–/HCO3– exchanger in our experiments should generate net Cl– uptake and HCO3– efflux in the resting state. To keep the HCO3– concentration of the saliva low would be advantageous to facilitate a sour taste in the resting state (18). The high HCO3– concentration in stimulated PG saliva can be explained partly by the limited time for the duct system to reabsorb HCO3– at high flow rates and partly by the activation of the luminal Cl–/HCO3– exchanger (15). HCO3– is also one of the main components in SMG saliva (3). The presence of NBC1 at the BLM of SMG ducts suggests that NBC1 may mediate HCO3– uptake into duct cells to compensate for the HCO3– secretion through the CFTR. In this tissue, a strong HCO3– reabsorption mechanism mediated by NBC1 observed in PG ducts may not be necessary, because the amount of HCO3– in primary saliva secreted from SMG acinar cells will be much less than that secreted by PGs by the absence of basolateral NBC1.

This study demonstrates that the mechanisms for pHi regulation in the guinea pig PG and SMG are different. NBC1, in addition to NHEs, is involved in regulating the pHi in PG acinar cells and in both ducts of the PG and SMG. We also provide new evidence that NBC1 is expressed in the LM of PG ducts and may play a role in HCO3– reabsorption.


   GRANTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
REFERENCES
 
This work was supported by Korea Research Foundation Grant KRF-2002-041-E00215.


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. Park, Dept. of Physiology, College of Dentistry, Seoul National Univ., Yeongeondong 28, Chongnoku, Seoul 110-749, South Korea (e-mail: kppark{at}snu.ac.kr)

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.


   REFERENCES
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

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