1Center for Oral Biology, Aab Institute of Biomedical Sciences, University of Rochester Medical Center, Rochester, New York 14642;2Molecular Medicine and Renal Units, Beth Israel Deaconess Medical Center, and3Department of Medicine, Harvard Medical School, Boston, Massachusetts 02215
Submitted 4 April 2003 ; accepted in final form 30 August 2003
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ABSTRACT |
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secretion; carbonic anhydrase; exchanger; calcium regulation
Of the 10 members of the mammalian SLC4A gene family, at least three (AE1-3 or SLC4A1-3) act as Na+-independent, DIDS-sensitive exchangers (40). AE4 (SLC4A9) may also share these properties, despite its greater sequence similarity with the other SLC4A family members, which exhibit Na+-dependent
or OH- transport, sometimes in exchange for Cl-, whereas the other members of this family are likely
cotransporters (1, 13, 20, 34, 41). AE1 gene products are highly expressed in the kidney and in erythrocytes; the erythroid polypeptide has been termed the band 3 protein (39). AE2 is nearly ubiquitously expressed (1), whereas AE3 expression is expressed at highest levels in the brain, heart, retina, and gut epithelia (16, 55). AE4 expression is limited to kidney, spleen, and salivary glands; DIDS sensitive in the rat (15), AE4 of rabbit is DIDS resistant (47).
Anion exchanger (AE) activity is regulated by numerous signals in epithelial cells (5). However, little is known about the regulation of exchanger activity in salivary glands. One report suggests that the
exchanger activity in mouse submandibular duct cells is upregulated by cAMP through the activation of the cystic fibrosis transmembrane conductance regulator (17). However, we are unaware of studies in which the regulation of anion exchange has been investigated in salivary gland acinar cells. The current studies were therefore performed to provide a better understanding of the regulation and function of
exchangers in this cell type. The functional characteristics of the
exchanger activity in acinar cells from mouse parotid and sublingual glands were consistent with the properties associated with members of the AE gene family; i.e., electroneutral, Na+-independent, and DIDS-sensitive
exchange. Moreover, an antibody specific for
exchanger isoform 2 (AE2) labeled mouse parotid and sublingual acinar cells and detected an
170-kDa protein in plasma membranes isolated from both glands. Importantly, AE activity was sensitive to the carbonic anhydrase (CA) inhibitor acetazolamide, and we observed that muscarinic receptor stimulation, the primary signal for activating salivation in vivo, upregulated the AE through a Ca2+-dependent mechanism. Collectively, these results indicate that the CA-dependent, Ca2+-modulated
exchanger in salivary gland acinar cells plays a major role in fluid secretion.
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MATERIALS AND METHODS |
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The -buffered NaCl solution contained (in mM): 110 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.33 NaH2PO4, 0.8 MgSO4, 1.2 CaCl2, 10 glucose, 20 HEPES, and 25 NaHCO3. Chloride salts were replaced with gluconate in the Cl--free solution, and additional calcium was added to compensate for chelation. NaHCO3 was replaced with sodium gluconate in the
-free solutions. For the Na+-free solution, sodium was replaced with N-methyl-D-glucamine, and for the high K+-containing solution, 70 mM NaCl was replaced by KCl.
-containing solutions were thoroughly gassed with 5% CO2-95% O2 before adjusting the pH to 7.4 with NaOH.
Animals and cell preparation. C57BL/6J male mice from Taconic (Germantown, NY) were fed ad libitum on standard diet and water with a 12:12-h light-dark cycle. Experiments were performed on animals aged between 2 and 6 mo. Parotid and sublingual gland cells were prepared following a protocol approved by the Animal Resources Committee of the University of Rochester. Briefly, mice were rendered unconscious by exposure to CO2 and killed by exsanguination. The parotid and sublingual gland cells were isolated by collagenase digestion as previously described (10). The glands were removed and finely minced in digestion medium (EMEM; Biofluids, Rockville, MD) containing collagenase P (0.3 mg·7.5 ml-1·animal-1), 2 mM L-glutamine, and 0.1% BSA. The minced glands were incubated at 37°C in a shaking water bath for 20 min, dispersed by gentle pipetting, and centrifuged. The cells were resuspended in fresh 7.5-ml collagenase digestion medium for an additional 40 min, at the end of which time the cells were rinsed and collected by centrifugation. To prevent an increase in the intracellular Ca2+ concentration ([Ca2+]) during stimulation, acinar cells were loaded with the Ca2+ chelator BAPTA by incubation for 30 min at room temperature in BAPTA-AM (50 µM).
Fluorescence measurement of intracellular pH. Intracellular pH (pHi) was monitored as previously described (31). Briefly, isolated acinar cells were loaded with the pH-sensitive fluoroprobe BCECF by incubation for 30 min at room temperature in BCECF-AM (2 µM). BCECF-loaded cells adhered to the base of a superfusion chamber mounted on a Nikon Diaphot 200 microscope interfaced with an Axon imaging workbench system (Axon Instruments, Foster City, CA). BCECF was excited at 490 and 440 nm, and the emitted fluorescence was collected at 530 nm. pHi was estimated by in situ calibration of the fluorescence ratio F490/F440 performed using the nigericin-high-K+ method (46). The high-K+ solution contained (in mM): 120 KCl, 20 NaCl, 0.8 MgCl2, 20 HEPES, and 0.005 nigericin, and the pH was adjusted from 5.6 to 8. The relationship between F490/F440 and pHi was linear over a pH range of 6.4-7.6 (n = 5). Figures are presented as data averaged from multiple acinar units from single representative experiments. Values quoted are the means ± SE for the number of aggregates examined using preparations of tissue isolated from three or more individual mice. The intrinsic buffering capacity was calculated following step changes in the concentration of the weak base NH4Cl in Na+- and -free solutions containing 100 µM of the CA inhibitor acetazolamide. To characterize the major AE in salivary acinar cells (>80%), parallel experiments were performed in the presence of DIDS. Data were then expressed as the DIDS-sensitive component of the alkalinization activated by Cl- removal by subtracting the DIDS-insensitive activity.
Membrane preparation. Mice were anesthetized with chloryl hydrate (400 mg/kg body wt) and injected intraperitoneally with heparin (13.6 units/g body wt). Animals were then perfused through the left ventricle with PBS (0.9% NaCl in 10 mM sodium phosphate buffer) until the salivary glands were thoroughly cleared of blood. Parotid and sublingual glands were dissected, and plasma membranes were prepared as previously described by Moore-Hoon and Turner (26). Briefly, glands were minced in homogenizing buffer containing 10 mM HEPES (pH 7.4 with Tris), 10% sucrose, 0.1 mM PMSF, and complete protease inhibitor cocktail (1 Tablet/50 ml working solution; Roche Applied Science, Indianapolis, IN). Tissue was homogenized using an Ultra-Turrax T8 polytron (IKA-WERKE, Germany) and centrifuged at 2,500 g for 5 min. The supernatant was removed and saved, and the pellet was resuspended in the same volume of homogenizing buffer and centrifuged as before. This process was repeated, and the collected supernatants were combined and centrifuged at 22,000 g for 20 min. The supernatant was discarded, and the resulting pellet was suspended at 10 ml/g of starting gland wet weight in suspension buffer (PBS containing complete protease inhibitor cocktail) and centrifuged at 43,700 g for 20 min. The final pellet was suspended in 1 ml suspension buffer/g of the initial wet weight and was then sequentially passed through 25- and 30-gauge needles. Aliquots were frozen in liquid nitrogen until use.
SDS-PAGE and Western blotting. Proteins (50 µg protein/lane) were separated by SDS-PAGE on 10% Tricine gels and then transferred onto polyvinylidene membranes using buffer containing 10% methanol and 10 mM 3-[cyclohexylamino]-1-propanesulphonic acid (pH 11; adjusted with Tris base). Membranes were blocked for 1 h at room temperature in Tris-buffered saline containing 4% nonfat dry milk and 0.04% Tween-20 and were then incubated overnight at 4°C with primary antibody (1:5,000 dilution for affinity-purified anti-AE2). After incubation with horseradish peroxidase-conjugated goat anti-rabbit IgG (1:10,000 dilution; Jackson ImmunoResearch Laboratories, West Grove, PA) for 1 h at room temperature, the labeled proteins were visualized using enhanced chemiluminescence (ECL detection kit, Amersham Biosciences UK, Little Chalfont, UK). Highrange prestained standards were from Invitrogen. In control experiments, the anti-AE2 antibody was preabsorbed (incubation at 37°C for 1 h) with the immunizing peptide (24 µg/ml).
Immunohistochemistry. Mice were anesthetized with chloryl hydrate (400 mg/kg body wt) and injected intraperitoneally with heparin (13.6 U/g body wt). Animals were perfused with PBS through the left ventricle until the glands were completely cleared of blood. The parotid and sublingual glands were removed, and acinar cell aggregates were prepared as described above. Acinar cells were fixed with 3% paraformaldehyde for 30 min, washed with PBS, and treated with 1% SDS for 10 min. Cells were then blocked as previously described (18), incubated overnight at 4°C in a 1:800 dilution of affinity-purified rabbit polyclonal anti-AE antibody, and then incubated with donkey anti-rabbit IgG coupled to Cy3 (1:2,000 dilution in PBS + 0.2% BSA; Jackson ImmunoResearch Laboratory) for 1 h at room temperature. Images were recorded in 1-µm z steps using a confocal microscope (Leica Microsystems, Bannockburn, IL).
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RESULTS |
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Mammalian AEs are electroneutral, including the Na+-dependent exchanger (13). Figure 2A demonstrates that the rate of alkalinization in sublingual acini was unaffected by the removal of external Na+, demonstrating that the
exchanger activity is Na+ independent in salivary gland acinar cells. Furthermore, the AE is electroneutral, because depolarization of the plasma membrane caused by exposure to high extracellular K+ (28) did not affect the rate of alkalinization on Cl- removal (Fig. 2B). Summaries of the results from multiple experiments such as those shown in Fig. 2, A and B, are given in Fig. 2C along with comparable results from parotid acinar cells (Fig. 2D). The intrinsic buffer capacity was comparable for sublingual (31.40 ± 3.75 mM) and parotid (29.35 ± 7.26 mM) acinar cells. In both cell types, the change in pH after a step change in weak base was essentially instantaneous, suggesting that Na+-dependent transport processes contribute little, if any, to the intrinsic buffering capacity. Nevertheless, we concede the possibility that Na+-dependent changes in buffer capacity might be reflected to a limited extent in the measurement.
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exchange is dependent on CA activity. The DIDS-sensitive,
-dependent AE activity described above was Na+ independent and electroneutral, properties consistent with expression of the AE1-AE4 members of the SLC4A gene family (15, 20). Recent evidence suggests that the
exchange associated with the expression of human AE1, -2, and -3 in HEK293 cells is functionally linked to CA activity (43, 44). To evaluate this relationship in native salivary gland acinar cells, the effects of the CA inhibitor acetazolamide (100 µM) were tested on AE activity. Acetazolamide dramatically and reversibly inhibited
exchange in sublingual acinar cells (Fig. 3A). Figure 3B summarizes the results from multiple experiments such as those shown in Fig. 3A for sublingual acini in the absence, presence, and following washout of acetazolamide. Similar effects were observed in parotid acini, although the magnitude of the inhibition by acetazolamide was less (Fig. 3B). It is not clear whether this difference reflects the expression of different AEs, different levels of CA, or some other mechanism. In contrast, Na+/H+ exchanger activity measured in CO2/bicarbonate in sublingual and parotid acinar cells was insensitive to acetazolamide (data not shown). The lack of acetazolamide inhibition of acinar Na+/H+ exchanger activity suggests that the acetazolamide inhibition of anion exchange is not a general phenomenon of all pH regulatory transporters.
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Muscarinic regulation of exchanger activity. Stimulation of M3 muscarinic receptors is coupled to a rise in the intracellular [Ca2+] (7, 27, 48), the primary signal for activating fluid secretion from salivary gland acinar cells.
exchangers play a significant part in this process by serving as a basolateral Cl- uptake pathway; thus muscarinic stimulation might be expected to upregulate the activity of this AE. To test this possibility, acini from parotid and sublingual glands were stimulated with the muscarinic receptor agonist carbachol (CCh) before removal of extracellular Cl-. CCh (10 µM) increased the rate of
exchange in sublingual (
35%) and parotid (
90%) acinar cells. The results shown in Fig. 4 summarize the magnitude of the increase in the DIDS-sensitive
exchanger activity during CCh stimulation in parotid and sublingual acinar cells (A and B, respectively).
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To investigate this effect further, we examined the Ca2+ dependency of the muscarinic receptor-induced upregulation of the AE. Thapsigargin, an inhibitor of intracellular Ca2+-ATPase, was used to increase the intracellular [Ca2+] in the absence of muscarinic receptor activation. Preincubation with thapsigargin before extracellular Cl- removal mimicked the effects of CCh. These results are consistent with an increase in Ca2+ being required for the muscarinic-induced upregulation of the exchanger in parotid and sublingual acinar cells. The results in Fig. 5A summarize the magnitude of the increase in the DIDS-sensitive
exchanger activity during thapsigargin stimulation in parotid (
65%) and sublingual (
50%) acinar cells. Furthermore, chelation of intracellular Ca2+ with BAPTA prevented the activation of anion exchange by CCh (Fig. 5B). Collectively, these results indicate that muscarinic receptor stimulation increased
exchanger activity through a Ca2+-dependent process.
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AE2 expression in salivary glands. The functional properties of the exchanger activity in salivary acinar cells are comparable with those previously described for members of the SLC4A gene family. The housekeeping AE2 has an extensive tissue distribution (2), including salivary glands, suggesting that its expression may correlate with the activity seen in salivary gland acinar cells. Previous immunohistochemical studies demonstrated that AE2 is expressed in the basolateral membranes of rat parotid and submandibular gland acinar cells (14, 37). To investigate the distribution of AE2 in mouse parotid and sublingual glands, we performed similar studies using the same antibody as used by He et al. (14). The confocal image in Fig. 6A demonstrates that AE2 is present in the basolateral plasma membrane, but it is not clear whether staining extends into the apical and canalicular membranes. AE2 is also present in intracellular regions of parotid acinar cells but is excluded from much of the apical and central areas of the cytosol where secretory granules are stored. Figure 6B is a Nomarski image of the same cells as in Fig. 6A. Parotid acinar cell staining was abolished when the primary antibody was preabsorbed with an AE2 peptide antigen (Fig. 6C). A Nomarski image of the same cluster of acinar cells in Fig. 6C is shown in Fig. 6D. Similar to the parotid, both intracellular and plasma membrane staining was detected in sublingual gland acinar cells (data not shown).
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Immunohistochemistry with an AE2-specific antibody labeled protein in both plasma membrane and intracellular compartments (Fig. 6). Plasma membranes were isolated (26) to further examine the protein labeled by this antibody in salivary gland cells. Western blot analysis of the plasma membrane isolated from parotid and sublingual glands identified a band at 170 kDa, consistent with the predicted molecular mass of AE2 (Fig. 7A). Plasma membranes prepared from glands that were not perfused with PBS to eliminate red blood cells displayed an additional band of lower molecular weight (data not shown) that likely represents erythroid band 3 protein (AE1). The amount of the 170-kDa protein was approximately twofold greater in sublingual glands, consistent with the greater activity in acinar cells from this gland (see Fig. 1). All labeling was abolished when the antibody was preabsorbed with the AE2 peptide fragment used to generate the antibody (24 µg/ml), indicating the immunospecificity of the immunoblot results (Fig. 7B).
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DISCUSSION |
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At least two mammalian gene families, SLC26A and SLC4A, include genes that encode AE proteins (21, 40). Analysis of the amino acid sequences reveals no detectable relationship between these families, suggesting that they do not share a common ancestral protein. The properties of the AE activity found in acinar cells were similar to those previously described for AE1-AE4, members of the SLC4A gene family (3, 15, 16, 22). Of these, AE2 is expressed in most tissues (1), whereas the expression patterns of AE1 (39), AE3 (16, 55), and AE4 (15, 47) are restricted. Of these latter three AE proteins, only AE4 has to date been reported in mouse salivary glands where its expression is localized to the basolateral membrane of submandibular duct cells (15). However, this study did not examine AE4 expression in parotid or sublingual salivary glands. The DIDS-sensitive exchange in mouse parotid and sublingual acinar cells was independent of extracellular Na+ and the membrane potential. In contrast, the SLC4A8 and SLC4A10 gene products exhibit sodium-dependent anion transport (13, 54). The relatively high concentration of DIDS necessary to inhibit exchange in salivary acinar cells is consistent with AE2 expression. Moreover, similar to other AE proteins, extracellular
was required, suggesting that OH- is a poor transport substrate in mouse salivary gland acinar cells. Similarly, short-chain fatty acids such as formate and acetate were unsuitable transport substrates (data not shown), in contrast to the AE expressed in ovine parotid acini (30) and the SLC26A4 AE pendrin (42). Pendrin and some other members of the SLC26A gene family can transport a wider spectrum of anions, including OH-, I-, and
(42). Nevertheless, the functional properties and inhibitor sensitivities of the various members of the SLC26A family have not been defined under the experimental conditions used in this study; therefore, it is not possible to rule out a role for these AEs in salivary acinar cells.
AE2 is considered important in several epithelial tissues, where its function is dependent on targeting. Localization of AE2 is restricted to the basolateral membranes of rat parotid (14, 37) and pancreatic acini (36) as well as the epithelial cells of human colon and intestine (4). Consistent with earlier reports, our localization studies detected basolateral targeting of AE2 in mouse parotid and sublingual acini, but we cannot rule out apical staining as well. We also detected considerable intracellular staining. Antibody preabsorbed with an AE2 peptide antigen abolished both plasma membrane and cytosolic staining, suggesting that AE2 may play an important functional role in both the plasma membrane and intracellular organelles. Examination of plasma membrane proteins by Western blotting revealed immunoreactive bands at 170 and
110 kDa in mouse parotid and sublingual glands. The 110-kDa band was similar in size to the red blood cell band-3 protein AE1 and disappeared in membranes isolated from glands that had been perfused with PBS to remove blood. The 170-kDa band was similar in size to the predicted molecular weight of AE2, suggesting that AE2 may be associated with the AE activity we detected in mouse salivary gland acinar cells.
Muscarinic receptor stimulation, the primary signal for activating salivation, significantly enhanced the activity of the AE in acinar cells. This result is consistent with exchange playing a critical role in the fluid secretion process. Muscarinic receptor stimulation activates phospholipase C, generating inositol 1,4,5-trisphosphate (IP3) and diacylglycerol, which activates protein kinase C and triggers protein secretion (38). The increase in the IP3 concentration raises the intracellular [Ca2+], which, in turn, activates the fluid secretion process (23, 24). Muscarinic receptor stimulation induces Cl- loss via channels and thereby reduces the Cl- gradient. This raises the possibility that the magnitude of the increase in AE activity observed during muscarinic stimulation may have been underestimated. When we increased the intracellular [Ca2+] with the Ca2+-ATPase inhibitor thapsigargin,
exchanger activity increased to a comparable magnitude with that observed during muscarinic stimulation. Moreover, chelation of intracellular [Ca2+] prevented the upregulation of the AE induced by muscarinic receptor stimulation. Together, these results clearly link in salivary acinar cells muscarinic receptor-induced upregulation of the AE to intracellular Ca2+ mobilization. It is interesting to note that an increase in the intracellular [Ca2+] is the primary signal in this cell type for activating most, if not all, of the other ion transporters involved in the fluid secretion process including the Na+/H+ exchanger (10, 35), Na+-K+-2Cl- cotransporter (12), K+ (29) and Cl- channels (6). The use of a common signal for activation of these various ion transporters provides a simple and rapidly reversible mechanism to regulate the magnitude and duration of fluid secretion.
We also found the exchange in mouse parotid and sublingual acinar cells to be strongly reduced by the CA inhibitor acetazolamide. Acetazolamide does not block erythroid AE1-mediated AE activity (9), unlike other CA inhibitors (8). Therefore, our results suggest that CA activity is important for
exchanger activity in native salivary acinar cells. The mechanism for this inhibition is unclear, but the simplest interpretation of our results is that acetazolamide inhibits the intracellular alkalinization by generally reducing CA activity throughout the cell. Alternatively, similar results to ours have been observed in HEK293 cells expressing AE1, -2, or -3 where a physical interaction necessary for maximal activity was observed between these AEs and CA (45, 53). The results of these latter studies indicate that the close association of CA with the
exchanger enhances activity by supplying substrate near the transport site of the exchanger. Importantly, NHE1, the dominant Na+/H+ exchanger in mouse parotid (10) and sublingual (31) acinar cells, can also physically associate with CA II (19). However, in contrast to the effects of acetazolamide on
exchange, acetazolamide had no effect on Na+/H+ exchanger activity measured in CO2/bicarbonate for salivary acinar cells (data not shown).
Collectively, our results are consistent with the hypothesis that AE2 mediates the major portion of the exchange across the basolateral membrane of mouse parotid and sublingual acinar cells. Nevertheless, our results do not allow us to state whether other AEs contribute to this process. Indeed, the difference in the acetazolamide sensitivity of the exchangers from these two glands suggests that different AEs may be expressed. AE activity was strongly dependent on CA activity and was upregulated by muscarinic receptor stimulation. Therefore, anion exchange may play a major role in Cl- uptake and thereby significantly contribute to salivary gland fluid secretion. Indeed, parotid glands in mice with targeted disruption of the Na+-K+-2Cl- cotransporter gene Nkcc1 continued to secrete, and this secretion was associated with enhanced AE2 expression (11). Mice lacking expression of the
exchanger AE2 will likely be required to determine the extent to which AE2 is directly involved in the salivary gland fluid secretion process.
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ACKNOWLEDGMENTS |
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GRANTS
This work was supported by National Institute of Health Grants DE-09692 and DE-13539 (to J. E. Melvin).
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FOOTNOTES |
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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.
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REFERENCES |
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