HCO3 secretion in the esophageal submucosal glands

Solange Abdulnour-Nakhoul,1 Nazih L. Nakhoul,1 Scott A. Wheeler,1 Paul Wang,1 Eric R. Swenson,2 and Roy C. Orlando1

1Veterans Administration Medical Center, and the Departments of Medicine and Physiology, Tulane University School of Medicine, New Orleans, Louisiana; and 2Department Medicine and Physiology, University of Washington, Seattle, Washington

Submitted 2 February 2004 ; accepted in final form 22 November 2004


    ABSTRACT
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
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The mammalian esophagus has the capacity to secrete a HCO3 and mucin-rich fluid in the esophageal lumen. These secretions originate from the submucosal glands (SMG) and can contribute to esophageal protection against refluxed gastric acid. The cellular mechanisms by which glandular cells achieve these secretions are largely unknown. To study this phenomenon, we used the pH-stat technique to measure luminal alkali secretion in an isolated, perfused pig esophagus preparation. Immunohistochemistry was used to localize receptors and transporters involved in HCO3 transport. The SMG-bearing esophagus was found to have significant basal alkali secretion, predominantly HCO3, which averaged 0.21 ± 0.04 µeq·h–1·cm–2. This basal secretion was doubled when stimulated by carbachol but abolished by HCO3 or Cl removal. Basal- and carbachol-stimulated secretions were also blocked by serosal application of atropine, pirenzipine, DIDS, methazolamide, and ethoxzolamide. The membrane-impermeable carbonic anhydrase inhibitor benzolamide, applied to the serosal bath, partially inhibited basal HCO3 secretion and blocked the stimulation by carbachol. Immunohistochemistry using antibodies to M1 cholinergic receptor or carbonic anhydrase-II enzyme showed intense labeling of duct cells and serous demilunes but no labeling of mucous cells. Labeling with an antibody to Na+-(HCO3)n (rat kidney NBC) was positive in ducts and serous cells, whereas labeling for Cl/HCO3 exchanger (AE2) was positive in duct cells but less pronounced in serous cells. These data indicate that duct cells and serous demilunes of SMG play a role in HCO3 secretion, a process that involves M1 cholinergic receptor stimulation. HCO3 transport in these cells is dependent on cytosolic and serosal membrane-bound carbonic anhydrase. HCO3 secretion is also dependent on serosal Cl and is mediated by DIDS-sensitive transporters, possibly NBC and AE2.

sodium-bicarbonate cotransporter; chloride/bicarbonate exchange; M1 muscarinic receptor; pH stat; carbonic anhydrase


THE HUMAN ESOPHAGUS CONTAINS large numbers of widely distributed esophageal submucosal glands (SMG). These glands can potentially contribute to esophageal defense against acid reflux because of their ability to secrete HCO3, mucin, and other products (1, 8, 9, 20, 21, 28, 29, and for a review, see Ref. 23). This protection would be particularly valuable during sleep when esophageal clearance mechanisms, such as upright position, swallow-induced persistalsis, and salivary secretion, are inoperative (16, 17, 21, 32, 39). HCO3 secreted by human esophagus is solely the product of esophageal SMG, since squamous epithelia do not secrete HCO3 (20). In humans, esophageal SMG have the capacity to secrete HCO3 in amounts sufficient to neutralize residual volumes of acid left in the esophagus after bolus clearance (28). Quantitatively, this HCO3 secretion can approach the HCO3 output of salivary glands at rest (10, 21, 27, 28).

Although potentially of clinical importance, there is little information about the cellular mechanisms by which esophageal SMG secrete HCO3. This is because of the technical difficulties of isolating the human esophagus from salivary and gastric contamination for the accurate collection and quantitation of SMG HCO3 and because of the limited availability of healthy esophageal tissue from esophagectomy specimens. However, the esophagi of some other mammalian species, e.g., pig, opossum, and dog, bear SMG (19), and this affords the opportunity for exploration of the cellular mechanisms of HCO3 secretion. In the present investigation, we utilized the isolated perfused pig esophagus to quantitate HCO3 secretion by the SMG in the presence and absence of various agonists and antagonists and supplemented this with immunohistochemical techniques to help define the receptor subtypes and cells of origin. The results show that SMG HCO3 secretion can be measured in vitro in the isolated perfused esophagus and that this secretion is stimulated by cholinergic agonists in part through activation of M1 receptors located on both serous acinar cells and duct cells of the SMG. Furthermore, the secretion of HCO3 by these cells is dependent on the generation of HCO3 by the action of carbonic anhydrase (CA) II and is partially dependent on serosal membrane-bound extracellularly oriented CA. HCO3 secretion is inhibited by the removal of basolateral Cl and is DIDS sensitive. Two HCO3 transporters, Na+-(HCO3)n (NBC) and Cl/HCO3 (AE2), were identified in the duct and serous cells of the glands. Ducts and serous cells seem to play an important role in HCO3 secretion in the esophageal SMG.


    METHODS
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 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
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The Isolated Perfused Esophagus Preparation

Perfusion of isolated pig esophagus in vitro. Pig esophagi were obtained from the slaughterhouse. Immediately upon death of the animal, the tissue was isolated and placed in an ice-cold HEPES-Ringer solution and transferred to the laboratory. The muscularis externa was sharply dissected, leaving behind an intact cylindrical tube of mucosa and submucosa. The SMG are embedded in the submucosa and could easily be visualized under a stereoscope (x60). The esophageal tube was flushed with saline, cannulated at both ends, and mounted in a chamber where it was entirely submerged in physiological buffer, HCO3-Ringer (solution 1 in Table 1) at 37°C, and bubbled with 95% O2-5% CO2 (Fig. 1). This design permits isolation and independent manipulation of both luminal and serosal perfusates. A cross section of esophageal tissue stained with hematoxylin and eosin shows the SMG (Fig. 2).


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Table 1. Composition of solutions

 


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Fig. 1. Perfused esophagus preparation for the measurement of HCO3 secretion. The esophageal tube was cannulated at both ends and mounted in a chamber where luminal and basolateral sides of the esophagus were perfused independently. Unbuffered saline solution bubbled with N2 (CO2 free) is circulated through the lumen using a peristaltic pump, whereas the basolateral side was bathed in HCO3 Ringer and maintained at 37°C. Total alkaline secretion was recorded continuously by means of an automatic pH-stat system whose pH electrode and acid (0.01 N HCl) titrant were in contact with the luminal perfusate.

 


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Fig. 2. Photomicrograph of a cross section of the esophageal tissue stained with hematoxylin and eosin showing, from top to bottom, the esophageal lumen, the stratified squamous epithelium, the lamina propria, the muscularis mucosa, and the submucosa containing submucosal glands. The muscularis externa was removed. The total thickness of the tissue averaged 2 mm (magnification x25).

 
Measurement of HCO3 secretion. The lumen was perfused with 100 ml of an unbuffered isotonic saline solution (150 mM NaCl) titrated to pH 7.4 with 0.01 N NaOH and continuously bubbled with CO2-free N2. The solution was recirculated in the esophageal tube with a peristaltic pump at 10 ml/min. Total alkaline secretion was recorded continuously by means of an automatic pH-stat system whose pH electrode and acid (0.01 N HCl) titrant are in contact with the luminal perfusate (20). Total alkaline secretion was calculated per unit of time from the amount (volume and concentration) of HCl titrant added to the luminal bath to maintain pH 7.4. Alkaline secretion was noted every 10 min and was averaged over the course of 45–60 min in control conditions and in the presence of the agonist or the inhibitors. All the agonists and antagonists were added to the serosal side of the esophageal tissue because luminal addition of the antagonist may not effectively deliver the agent to the acinar or duct cells. This is because of the fact that the glands are deeply embedded in the submucosa (Fig. 2) and because fluid movement out of the duct, into the lumen, would limit the diffusion of antagonist to the apical membranes of these cells. Esophageal alkaline secretion resulting from HCO3 secretion was calculated using a modified method of Helm et al. (20, 21). Briefly, 100 ml of an isotonic saline solution (pH 7.4) were recirculated for ~1 h through the esophagus and were not titrated with the pH stat technique. Total alkaline secretion was determined by titrating the perfusate to pH 4.35, the pH at which 99% of HCO3 are titrated, and subtracting the contribution of the saline vehicle. The contribution of the saline vehicle was measured by titrating 100 ml of nonperfused saline solution (pH 7.4) to pH 4.35. Non-HCO3 alkaline secretion was then measured as follows: the perfusate titrated to pH 4.35 was bubbled with CO2-free N2 for 15 min to remove CO2 formed by the reaction of HCO3 with H+. The pH of this solution was then raised to its original value by the addition of 0.01 N NaOH and again titrated with 0.01 N HCl to pH 4.35. This second titration, after subtracting the contribution of the saline vehicle treated similarly, yields the non-HCO3 component of secretion. Alkaline secretion in the pig esophagus was found to be dominated (82 ± 6%, n = 3) by HCO3 secretion.

Immunohistochemistry

Sections of esophageal tissue were stained by the immunoperoxidase technique for labeling with specific antibodies. Multiple tissues from at least four different animals were used for labeling with each antibody. The primary antibodies used in this study were the following: rabbit anti-CA II against human erythrocyte polyclonal antibody, rabbit anti-M1 muscarinic ACh receptor affinity purified polyclonal antibody (Chemicon, Temecula, CA), rabbit anti-rat kidney NBC [rkNBC (38); generously provided by Dr. Walter Boron], and rabbit anti-SA6 antibody against the COOH-terminal of mouse AE2 amino acids 1224–1237 (generously provided by Dr. Seth Alper). The sections were fixed overnight in 10% formalin, dehydrated, and embedded in paraffin blocks. Thin sections (5 µm) were cut and mounted on slides. Slides were baked overnight at 50°C. For staining with NBC antibody (38), additional specimens were fixed in periodate-lysine-paraformaldehyde (PLP) and were cryoprotected overnight in 30% sucrose in PBS and frozen in liquid nitrogen. Cryosections (5 µm thickness) were cut on a Reichert cryostat and mounted on gelatin-coated slides. The tissues were exposed to different concentrations of the primary antibody to determine the optimal concentration needed. For negative controls, the primary antibodies were reacted with the fusion protein for the individual antibody or were incubated without the primary antibody.

Immunoperoxidase method. Paraffin sections were dewaxed in xylene and hydrated in a series of graded alcohol solutions followed by PBS. Cryosections were hydrated in PBS. All sections were incubated in 0.3% hydrogen peroxide in methanol to block endogenous peroxidase and then washed in PBS. For labeling with anti-AE2CT, sections were treated with 1% SDS in PBS to enhance the staining (11). Sections were then incubated with normal serum to block nonspecific binding. They were then incubated with the primary antibody followed by incubation with biotinylated secondary antibody against the IgG of the species providing the primary antibody. The sections were then treated with avidin-biotinylated enzyme complex (Vector Laboratories, Burlingame, CA). Peroxidase activity was detected using SIGMA FAST 3,3'-diaminobenzidine (Sigma, St. Louis, MO) as a substrate. Specimens were counterstained with hematoxylin, dehydrated, and mounted for study by light microscopy using a Zeiss Axioplan 2 or an Olympus IMT2 microscope. Pictures were taken using a digital charge-coupled device camera or an Olympus camera using Kodak 100 ASA film.

Solutions

The composition of the Ringer solutions is given in Table 1. Benzolamide (BNZ) was provided by Dr. E. R. Swenson. DIDS and all other chemicals were obtained from Sigma. Inhibitors insoluble in aqueous solutions were dissolved in a small volume of DMSO and added to the solution. The concentrations used were based on the concentrations required to achieve maximal inhibition of the transporters or of CA, as reported previously in other preparations. The concentration of DMSO never exceeded 0.1% of the final solution.

Statistical Analysis

Data are presented as means ± SE. Data were analyzed using the two-tailed paired Student's t-test unless otherwise indicated; n is the no. of experiments.


    RESULTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Measurement of HCO3 Secretion

Pig esophagi were perfused using a recirculated unbuffered luminal solution connected to a pH-stat system as described in METHODS. After being mounted and equilibrated, basal HCO3 secretion recorded in 19 esophagi averaged 0.21 ± 0.04 µeq·h–1·cm–2 with a range of 0.01 to 0.65 µeq·h–1·cm–2. When the cholinergic agonist carbachol, 10 µM, was added to the serosal bathing solution, it resulted in approximately a doubling of the rate of HCO3 secretion over the course of 15–20 min from 0.14 ± 0.05 to 0.27 ± 0.04 µeq·h–1·cm–2 (n = 5, P < 0.01). Subsequent addition of atropine (10 µM), a general cholinergic antagonist, to the serosal bath abolished both basal and carbachol-stimulated HCO3 secretion (0.015 ± 0.01 µeq·h–1·cm–2, n = 5; Fig. 3).



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Fig. 3. Basal HCO3 secretion in the esophagus and the effect of carbachol. Carbachol (10 µM) increased HCO3 secretion by twofold, whereas atropine inhibited basal and carbachol-stimulated secretion; n = 5, P < 0.01. *Statistical difference from previous condition.

 
Identification of Cholinergic M1 Receptors

Because the stimulation of SMG secretion by carbachol and its complete inhibition by atropine indicated that cholinergic receptors were important in both basal and stimulated secretion, we sought to identify the receptor subtype by performing similar experiments using pirenzipine, a blocker of the cholinergic M1 receptor subtype (12). Pirenzipine (0.1 mM), added serosally, decreased HCO3 secretion from a control value of 0.22 ± 0.04 µeq·h–1·cm–2 to a value of 0.15 ± 0.06 µeq·h–1·cm–2 (Fig. 4, n = 6, P < 0.05). Subsequent addition of 10 µM carbachol to the serosal bathing solution failed to increase HCO3 secretion, which remained at 0.09 ± 0.05 µeq·h–1·cm–2 (n = 6). These experiments suggest that most of the cholinergic receptors are of the M1 subtype. Even though pirenzipine did not totally abolish basal HCO3 secretion like atropine, carbachol did not cause any increase in HCO3 secretion in the presence of pirenzipine. Confirmation and localization of the M1 receptors in the SMG was obtained by immunohistochemistry using a rabbit anti-M1 muscarinic ACh receptor affinity-purified polyclonal antibody. As shown in Fig. 5, both the serous demilunes and the ductal epithelium stained positive, indicating the presence of M1 receptors on these structures. This suggests that these components of the SMG are important in the regulation of HCO3 secretion. When the primary antibody was omitted or reacted with the specific binding protein (1.5 µg/ml antibody to 5 µg/ml binding protein), tissue sections stained negative, indicating the specificity of the staining to the M1 receptor protein.



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Fig. 4. Effect of pirenzipine, an antagonist of M1 muscarinic subtype receptor, on esophageal HCO3 secretion. Pirenzipine inhibited basal HCO3 secretion by ~50% (n = 6, P < 0.05) and also inhibited the stimulation by carbachol (10 µM).

 


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Fig. 5. Photomicrographs showing immunolocalization of the M1 muscarinic receptor using the immunoperoxidase technique in the serous cells or demilunes (A, arrow) and in the ducts (B, arrow). m, Mucous acinus where the labeling was negative. Rabbit anti-M1 muscarinic ACh receptor affinity purified polyclonal antibody was used on formalin-fixed paraffin-embedded tissue sections. The incubating medium of the primary antibody contained 0.05% Tween and 0.3% Triton. The antibody was used at a dilution of 1.5 µg/ml.

 
Identification of HCO3 Transport

To define the nature of the membrane transporters involved in HCO3 secretion by SMG, we first established that HCO3 was responsible for the alkaline secretion detected in our system. This was done by serosal replacement of HCO3 Ringer with a HCO3-free HEPES-buffered Ringer (solution 3 in Table 1), a maneuver that gradually abolished alkaline secretion; mean value declined from 0.18 ± 0.05 to 0.006 ± 0.004 µeq·h–1·cm–2 (n = 4, P < 0.05). Subsequent addition of carbachol (10 µM) did not cause any increase in HCO3 secretion. This suggests that one or more HCO3 transporters are present in the SMG. Among the possibilities are the NBC family of transporters (7) and AE (4). Because transporters belonging to both families are irreversibly inhibited by stilbene derivatives, we added 0.1 mM DIDS to the serosal solution while measuring basal HCO3 secretion. In these experiments, DIDS inhibited HCO3 secretion from a control value of 0.22 ± 0.04 to 0.09 ± 0.04 µeq·h–1·cm–2 (n = 4, P < 0.02). Furthermore, when 10 µM carbachol was added serosally to the preparation, there was no increase in HCO3 secretion. (Fig. 6). These data suggest the presence of DIDS-sensitive membrane transport system(s) involved in the secretion of HCO3 from SMG.



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Fig. 6. Effect of DIDS, an inhibitor of HCO3 transport mechanisms, including Na+-HCO3 cotransporter (NBC) and Cl/HCO3 exchange (AE2), on esophageal HCO3 secretion. The addition of DIDS (0.1 mM) to the serosal bath inhibited basal HCO3 secretion and the increase in HCO3 secretion observed with the addition of carbachol (10 µM; n = 4, P < 0.02).

 
The data obtained with HCO3-free Ringer and with DIDS support the possibility that either NBC, Cl/HCO3 exchanger, or both are involved in HCO3 secretion. To investigate the presence of these transporters in the SMG, we immunostained the esophageal tissue for NBC using a rabbit polyclonal antibody to rkNBC (K1A immune serum), which recognizes the COOH-terminal portion (last 46 residues) of rkNBC, rat pancreas NBC, and rat brain NBC (36, 38). We also immunostained the tissue for AE2 using a rabbit polyclonal column affinity purified anti-SA6 antibody against the COOH-terminal of mouse AE2 amino acids 1224–1237 (5).

NBC. Immunoperoxidase staining with anti-NBC was done in both PLP fixed cryosections and in sections from formalin-fixed paraffin-embedded tissues. The results from the immunostaining with anti-NBC in paraffin sections are shown in Fig. 7 and were not noticeably different from those obtained in the cryosections. There was a strong labeling of the serous demilunes (Fig. 7A) and of the intralobular (Fig. 7B) and interlobular ducts, and this staining looked more pronounced on the luminal side of the cells. There was very faint labeling in the mucous acinar cells. When the primary antibody was omitted or reacted with the fusion protein (maltose-binding fusion protein MBP-K1A, at 15 µg/ml) before its application to the tissue, the labeling was negative (Fig. 7C). These data suggest that an NBC transporter is present in the serous and in the duct cells of SMG and support the functional data above that NBC is likely to play an important role in HCO3 secretion in pig esophagus.



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Fig. 7. Photomicrographs showing immunoperoxidase localization of NBC using a rabbit polyclonal antibody to rat kidney NBC (rkNBC, K1A immune serum), which recognizes the COOH-terminal portion (last 46 residues) of rkNBC, rat pancreas NBC, and rat brain NBC. NBC immunostaining was strong in the serous cells or demilunes (A, arrow) and in the ducts (B, arrow) and faint in the mucous cells (m in A and B). This experiment was performed on sections from formalin-fixed paraffin-embedded tissues. Tween (0.1%) was added to the incubating medium of the primary antibody. When the primary antibody was omitted or reacted with the fusion protein (maltose-binding fusion protein MBP-K1A, at 15 µg/ml) before its application to the tissue, the labeling was negative (C). Magnification x150.

 
AE2CT. Immunoperoxidase staining with anti-SA6 (AE2 antibody) was done in sections from formalin-fixed paraffin-embedded tissues. The tissues were pretreated with 1% SDS as described in METHODS. Inter- and intralobular ducts stained strongly positive to anti-SA6 antibody (Fig. 8, A and B, respectively). The staining of the serous cells to anti-SA6 antibody was faint, whereas the acinar mucous cells did not stain at all (Fig. 8B). When the antibody was reacted with the SA6 (AE2CT) fusion protein, the staining was negative (Fig. 8C). Competition of the antibody with SA35 (AE2 NT) unrelated in sequence to SA6 yielded positive staining and was used as a positive control in competition experiments.



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Fig. 8. Photomicrographs showing immunolocalization of AE2 using the immunoperoxidase technique and a rabbit polyclonal column affinity purified anti-SA6 antibody against the COOH-terminal of mouse AE2 amino acids 1224–1237. The staining was done in sections from formalin-fixed paraffin-embedded tissues. The tissues were pretreated with 1% SDS as described in METHODS. The interlobular (arrow in A) and intralobular (arrow in B) ducts stained strongly positive to the antibody. The serous cells stained very weakly, whereas staining in acinar mucous cells ("m" in B) was absent. When the antibody was reacted with the SA6 (AE2CT) fusion protein, the staining was negative (C). Magnification x150.

 
Role of Serosal Cl

The presence of Cl/HCO3, revealed by immunohistochemistry, indicates a possible role for Cl in HCO3 secretion. To investigate the role of serosal Cl in HCO3 secretion, we deleted Cl from the serosal bathing solution (Cl was replaced with cyclamate, solution 2 in Table 1). In six tissues, removal of Cl caused HCO3 secretion to decline from 0.10 ± 0.01 to 0.02 ± 0.01 µeq·h–1·cm–2 (n = 6, P < 0.05). Figure 9 shows a plot of the changes in HCO3 secretion upon removal of serosal Cl. It is to be noted that, upon removal of Cl, there was an initial slight and transient increase in HCO3 secretion followed by a sharp decrease in secretion. The transient increase in HCO3 secretion is consistent with the presence of a Cl/HCO3 exchanger on the basolateral side of the cell, which is initially reversed by the removal of serosal Cl. This leads to the accumulation of HCO3 in the cell, which can transiently increase secretion. Prolonged exposure to a Cl-free solution (~80 min) eventually leads to the inhibition of HCO3 secretion, as observed, most likely by depletion of intracellular Cl and inhibiting the exchanger and other Cl-dependent mechanisms. Other factors, such as changes in intracellular pH (an expected increase) or cell volume changes, may also play a role. Further removal of Cl from the esophageal lumen (NaCl was replaced with sodium cyclamate) caused HCO3 secretion to stop totally. The addition of carbachol in the absence of serosal Cl did not cause a significant increase in HCO3 secretion.



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Fig. 9. Effect of removal of Cl from the serosal bath on HCO3 secretion. Upon Cl removal, there was a small transient increase followed by a sharp decrease in HCO3 secretion. Values are means ± SE from 6 tissues.

 
Role and Identification of CA

CA is an enzyme that catalyzes the reversible reaction CO2 + H2O {leftrightarrow} HCO3 + H+ and can therefore generate HCO3 for transport across the cell membrane. It has been reported that the cytoplasmic COOH-terminal domain of Cl/HCO3 contains a binding site for CA II. The interaction of CA and Cl/HCO3 forms a "transport metabolon," which is a membrane protein complex involved in regulation of HCO3 transport (41). It is therefore expected that, in the absence of CA, generation of HCO3 and Cl/HCO3 activity would be curtailed, leading to inhibition of HCO3 secretion. To determine the role of CA in the generation and/or transport of HCO3 in the esophageal glands, we measured HCO3 secretion in the perfused esophagus in the presence and absence of ethoxzolamide (ETXZ). ETXZ, (0.2 mM), a membrane-permeable inhibitor of CA, was added to the serosal bath. As shown in Fig. 10, ETXZ inhibited HCO3 secretion from a control value of 0.25 ± 0.02 µeq·h–1·cm–2 to a value of 0.06 ± 0.02 µeq·h–1·cm–2 (n = 3, P < 0.05). When carbachol was added in the presence of ETXZ, HCO3 secretion was not stimulated and continued to decline to 0.014 ± 0.002 µeq·h–1·cm–2. These observations suggest that secretion of HCO3 by SMG is strongly dependent on the activity of CA.



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Fig. 10. Effect of the carbonic anhydrase (CA) inhibitor ethoxzolamide (ETXZ) on esophageal HCO3 secretion. The addition of ETXZ (0.2 mM) to the serosal bath inhibited basal HCO3 secretion and the increase in HCO3 secretion observed with carbachol (10 µM); n = 3, P < 0.05.

 
It has also been reported that the cell surface-anchored CA IV interacts with the extracellular loop of Cl/HCO3 to accelerate HCO3 transport (40). A membrane-permeable CA inhibitor can inhibit extracellular and intracellular CA. To identify the role of extracellular CA, we measured HCO3 secretion in the perfused esophagus in the presence and absence of BNZ, a membrane-impermeable inhibitor of CA (43). Basal HCO3 secretion in five tissues averaged 0.07 ± 0.005 µeq·h–1·cm–2. The addition of BNZ to the serosal bath at a concentration of 10 µM decreased HCO3 secretion to 0.05 ± 0.01 µeq·h–1·cm–2 (n = 5, P < 0.02) over the course of ~1 h. Carbachol addition, which in control tissues almost doubles HCO3 secretion, did not have a significant effect and secretion stayed at 0.06 ± 0.01 µeq·h–1·cm–2. In comparison and because BNZ is considered "antipodal" in properties to methazolamide (MTZ; see Ref. 24), a set of four tissues was exposed to MTZ. Basal secretion was 0.22 ± 0.06 µeq·h–1·cm–2. Two tissues were exposed to 1 mM MTZ and two other tissues to 0.2 mM MTZ. In both sets of tissues, HCO3 secretion stopped totally in 20 min and subsequent carbachol addition did not cause any additional HCO3 secretion.

Cytosolic CA II, a cytoplasmic variety of the enzyme, has been previously identified in human esophageal SMG (14), in salivary glands of different mammalian species (6, 30, 31). We therefore examined its presence in the SMG tissue of pig esophagus by immunolabeling with an antibody to CA II (rabbit anti-CA II, human erythrocyte polyclonal antibody). The staining was done in formalin-fixed paraffin-embedded tissue sections. Serous demilunes (Fig. 11A), interlobular ducts, and intralobular ducts (Fig. 11B) stained intensely positive for CA II, whereas the mucous cells stained negative. Figure 11C shows a control section where only the primary antibody was omitted from the experiment.



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Fig. 11. Photomicrographs showing immunolocalization of CA II using the immunoperoxidase technique and an antibody to CA II (rabbit anti-CA II, human erythrocyte polyclonal antibody) at a concentration of 20 µg/ml. The staining was done in formalin-fixed paraffin-embedded tissue sections. Serous demilunes (arrow in A) and intralobular (arrow in B) and interlobular ducts stained intensely positive for CA II. C: control section in which the primary antibody was omitted from the experiment. Magnification x150.

 

    DISCUSSION
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present study examined the cellular mechanisms of HCO3 secretion by esophageal SMG. For this purpose, experiments were conducted on the isolated, perfused pig esophagus, an organ that serves as a good model for human esophagus in many aspects (13). Like the human esophagus, the pig esophagus bears SMG and is capable of secreting significant amounts of alkali. These SMG are seromucous glands containing mucous acini with serous demilunes (18, 22). In addition, this preparation has several distinct advantages: 1) it allows accurate measurements of luminal secretion of alkali, 2) it allows independent manipulations of luminal and basolateral solutions in the intact esophagus, and 3) alkali secretion can be assessed independently of the influence of the central nervous system.

Our data indicate that there is a significant basal (nonstimulated) secretion of alkali by the SMG-bearing esophagus. The basal alkali secretion averaged 0.21 ± 0.04 µeq·h–1·cm–2, which was readily doubled by the addition of the cholinergic agonist, carbachol, to the serosal bathing solution. Alkali secretion in the esophagus is a product of the SMG, since the squamous epithelium does not secrete alkali (1, 20). Moreover, our data established that the major component of this alkali secretion is HCO3. This was confirmed by the following results. 1) Alkali secretion was dominated by HCO3 secretion (82 ± 6%). 2) Alkali secretion, basal and stimulated by carbachol, was abolished by the removal of HCO3 from the serosal bath. 3) Alkali secretion, basal and stimulated, was inhibited by DIDS, a known inhibitor of HCO3 transport.

To further investigate the pathway of alkali secretion, we documented that SMG HCO3 secretion was not only markedly stimulated by serosal carbachol but abolished by the addition of the cholinergic antagonist atropine. The inhibition of basal HCO3 secretion by atropine suggests that SMG secretion is strongly influenced by parasympathetic tone. Adding pirenzipine, a known cholinergic M1-receptor antagonist, to the serosal bath reduced basal secretion by ~50% and completely abolished carbachol-stimulated secretion. In addition, using immunohistochemistry, we identified significant immunolabeling of M1 receptors on cells lining the ducts within the SMG and on the cells comprising the serous demilunes. There was no staining of mucous cells, indicating that the regulation of mucin secretion by SMG likely differs from that of SMG HCO3 secretion. The presence of M1 receptors on SMG supports the ability of cholinergic agonists to act directly on the SMG rather than through an intermediate neural mechanism. The partial inhibition of basal secretion by pirenzipene suggests that other cholinergic receptor subtypes may be involved in basal HCO3 secretion.

The mechanisms by which the esophageal SMG secrete HCO3 are unknown. One possible model is that secretion in the SMG, like in the similarly structured salivary glands, occurs in two stages (25, 42, 46). The acini secrete a fluid isotonic to plasma, which is subsequently modified in the duct system by reabsorption of Na+ and Cl and secretion of K+ and HCO3. Inhibition of basal and carbachol-stimulated secretion by DIDS indicates a major role of HCO3 transporters in the generation of secreted HCO3. This was confirmed by the immunolocalization of NBC using an antibody that recognizes the COOH-terminal portion (last 46 residues) of rkNBC, rat pancreas NBC, and rat-brain NBC (38). This transporter was localized to the duct cells (intra- and interlobular) and to the serous demilunes. These experiments indicate the presence of at least one isoform of NBC in the esophageal SMG. Recent studies on the rat salivary glands have demonstrated the presence of NBC on the basolateral side of acinar and duct cells of the parotid gland. In the submandibular glands, NBC is present on the basolateral and in some instances apical side of the duct cells (37). Further experiments are needed to determine the homology between the esophageal isoform and the kidney, pancreas, or brain isoforms and to determine the apical or basolateral location of this transporter.

Another HCO3 transport mechanism known to play an important role in pH regulation and HCO3 transport in a variety of cells is AE2. This exchanger is present in the salivary glands (26, 44). Several isoforms of this exchanger have been identified (2). AE2 is the most ubiquitously expressed form of the transporter and in most epithelial cells is located on the basolateral side of the cell (3). Because the electrochemical gradient of the cell usually favors Cl entry in the cell, this exchanger allows the influx of Cl and the efflux of HCO3. Our experiments indicate that AE2 is strongly expressed in the cell membranes of interlobular and intralobular ducts. On the other hand, the serous demilunes stained faintly for AE2, whereas staining in the mucous cells was negative.

Some of the issues to be addressed are the relative contribution of NBC and AE2 to HCO3 secretion and whether they play a direct role in this process. The main questions in this respect are whether these transporters are located at the basolateral membrane or the luminal membrane and the specific stoichiometry of NBC. Considering NBC, the presence of the transporter on the luminal membrane in a 3:1 configuration (HCO3:Na+) leads to HCO3 efflux and therefore a direct role in HCO3 secretion in the duct. If NBC is localized to the basolateral membrane, then only a 2:1 isoform (or possibly the electroneutral form of NBC) could lead to HCO3 influx in the cell and ultimately secretion of HCO3. Alternatively, a basolateral 3:1 configuration of NBC favors HCO3 efflux, which does not point to a direct role in HCO3 secretion via this transporter. As for AE2, most studies indicate its presence at the basolateral membrane, where it transports HCO3 out of the cell. As such, AE2 does not directly secrete HCO3 in the duct, and its likely role in these cells would be in regulation of intracellular pH. Further functional and molecular studies are needed to elucidate these mechanisms.

CA II and IV (cytosolic and membrane bound, respectively) play a role in the regulation of cellular HCO3 and recently have been shown to bind to HCO3 transporters like AE2 and kNBC to form a transport metabolon that enhances the transporter activity by providing it with HCO3 (34, 35, 40, 41, 45). The inhibition of HCO3 secretion by ETXZ and MTZ, two permeable CA inhibitors, indicates a major role of CA in generating HCO3 in the esophageal SMG. However, the effect of the membrane-permeable CA inhibitors is not restricted to the cytosol but results in the inhibition of both cytosolic and extracellular CA. The role of extracellular CA was determined by the use of BNZ, a membrane-impermeable CA inhibitor that only partially inhibited basal HCO3 secretion but abolished the effect of carbachol. The very high permeability of MTZ corresponded to a complete inhibition of HCO3 secretion. ETXZ inhibited HCO3 secretion by 76%, whereas the less permeable BNZ only inhibited HCO3 secretion by 29%. It can be concluded from our experiments that basal and carbachol-stimulated HCO3 secretion are dependent on intracellular CA. Extracellular CA, possibly CA IV, which is present in several epithelial cells of the alimentary tract including the esophagus (15, 33), has a limited role in basal secretion but seems to be implicated in carbachol-stimulated secretion. The role of basolateral membrane-bound CA could be in generating HCO3 in the vicinity of a putative HCO3 transporter (NBC) bringing HCO3 from the serosal side to the cell.

The removal of serosal Cl caused an initial slight and transient increase in HCO3 secretion, consistent with the presence of Cl/HCO3 exchange on the basolateral side of the cell. This was followed by a sharp decrease and almost total inhibition of secretion, indicating a major role of Cl in generating HCO3 secretion or in the maintenance of ion gradients necessary for that secretion. The role of luminal Cl transport is more difficult to address and needs to be further elucidated. In our experiments, removal of luminal Cl would only deplete Cl from the esophageal lumen and will not guarantee its removal from the lumens of the glands. This is because the glands are embedded in the submucosa and are several hundred (~1,600) micrometers away from the luminal surface of the esophagus (Fig. 2).

The presence of M1 receptors, NBC, and CA in the same cells (of serous demilunes and in the ducts) indicates that secretion of HCO3 in the SMG occurs in these cells. This likely involves a cascade of events involving the activation of M1 receptor, the generation of HCO3 in the cell by CA, and the transport of HCO3 in and/or out of the cell by an NBC transporter.

In conclusion, this study established that alkali secretion by esophageal SMG is mostly the result of HCO3 generation and transport. HCO3 secretion is stimulated by cholinergic agonists and mediated by M1 receptors. The generation of HCO3 by CA is an important step in this process. DIDS-sensitive HCO3 transporter(s) NBC and/or AE2 contribute to HCO3 transport, which is also inhibited by the removal of serosal Cl. The ducts and serous cells of the glands seem to play an important role in HCO3 transport and secretion.


    GRANTS
 TOP
 ABSTRACT
 METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by the Office of Research and Development (Health Services Research and Development) Department of Veterans Affairs (to S. Abdulnour-Nakhoul) and by National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-36013 (to R. C. Orlando) and DK-62295 (to N. L. Nakhoul).


    ACKNOWLEDGMENTS
 
We thank Anita Verdun for generously donating the pig tissues. We thank Dr. Walter Boron for providing the NBC antibody and Dr. Seth Alper for the AE2 antibody.


    FOOTNOTES
 

Address for reprint requests and other correspondence: S. Abdulnour-Nakhoul, The VA Medical Center, Attn: Research, Rm. 5F151, 1601 Perdido St., New Orleans, LA 70112–2699 (E-mail: solange{at}tulane.edu)

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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