Na+/Ca2+ exchange regulates Ca2+-dependent duodenal mucosal ion transport and HCO3 secretion in mice

Hui Dong, Zachary M. Sellers, Anders Smith, Jimmy Y. C. Chow, and Kim E. Barrett

Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, San Diego, California

Submitted 24 August 2004 ; accepted in final form 12 October 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Stimulation of muscarinic receptors in duodenal mucosa raises intracellular Ca2+, which regulates ion transport, including HCO3 secretion. However, the underlying Ca2+ handling mechanisms are poorly understood. The aim of the present study was to determine whether Na+/Ca2+ exchanger (NCX) plays a role in the regulation of duodenal mucosal ion transport and HCO3 secretion by controlling Ca2+ homeostasis. Mouse duodenal mucosa was mounted in Ussing chambers. Net ion transport was assessed as short-circuit current (Isc), and HCO3 secretion was determined by pH-stat. Expression of NCX in duodenal mucosae was analyzed by Western blot, and cytosolic Ca2+ in duodenocytes was measured by fura 2. Carbachol (100 µM) increased Isc in a biphasic manner: an initial transient peak within 2 min and a later sustained plateau starting at 10 min. Carbachol-induced HCO3 secretion peaked at 10 min. 2-Aminoethoxydiphenylborate (2-APB, 100 µM) or LiCl (30 mM) significantly reduced the initial peak in Isc by 51 or 47%, respectively, and abolished the plateau phase of Isc without affecting HCO3 secretion induced by carbachol. Ryanodine (100 µM), caffeine (10 mM), and nifedipine (10 µM) had no effect on either response to carbachol. In contrast, nickel (5 mM) and KB-R7943 (10–30 µM) significantly inhibited carbachol-induced increases in duodenal mucosal Isc and HCO3 secretion. Western blot analysis showed expression of NCX1 proteins in duodenal mucosae, and functional NCX in duodenocytes was demonstrated in Ca2+ imaging experiments where Na+ depletion elicited Ca2+ entry via the reversed mode of NCX. These results indicate that NCX contributes to the regulation of Ca2+-dependent duodenal mucosal ion transport and HCO3 secretion that results from stimulation of muscarinic receptors.

sodium-calcium exchange


DUODENAL MUCOSAL BICARBONATE (HCO3) secretion is important in both health and disease, since it alkalinizes the adherent viscoelastic mucus gel, thereby providing an important line of mucosal defense. The mechanisms that govern duodenal mucosal HCO3 secretion include neurohumoral factors and luminal acid. Epithelial alkaline secretion in the duodenum is increased markedly by a low pH in the duodenal lumen (1, 9, 10, 14). Several transmitters, including ACh and vasoactive intestinal polypeptide, have been proposed as mediators of the efferent limb of the neural response (1, 9, 10). Similar to other secretory epithelia, cAMP-, cGMP-, and Ca2+-dependent mechanisms are believed to mediate the actions of the well-described secretagogues for duodenal mucosal HCO3 secretion (11, 28). ACh or its stable analog, carbachol, has been considered as Ca2+-dependent secretagogues and induce increases in free cytosolic Ca2+ concentration ([Ca2+]i) in epithelial cells that in turn stimulate duodenal mucosal HCO3 secretion (1, 810). However, although the Ca2+ signaling pathway is believed to play an important role in regulating duodenal mucosal HCO3 secretion, the precise mechanisms responsible for handling cytosolic Ca2+ in the duodenal mucosa are still unclear.

In rat colonic epithelium, it has generally been accepted that stimulation of muscarinic M3 receptors with the Ca2+-dependent secretagogue, ACh or carbachol, induces a cascade of reactions. Initially, the inositol 1,4,5-trisphoshate (IP3) pathway is activated, which leads to depletion of intracellular Ca2+ stores via stimulation of IP3 receptors (25). Depletion of intracellular Ca2+ stores stimulates influx of extracellular Ca2+, presumably through opening of store-operated channels (30). An increase in [Ca2+]i in turn induces Cl secretion from colonic epithelium (4, 34). In cultured human and rat duodenal enterocytes, it has also been demonstrated that carbachol induces a transient increase in [Ca2+]i via stimulation of muscarinic M3 receptors (8). However, it remains unknown how the increase in cytosolic Ca2+ occurs, what is the nature of the transporters and other proteins involved, and whether these regulate duodenal mucosal HCO3 secretion by controlling [Ca2+]i homeostasis.

Because epithelia do not express the voltage-operated Ca2+ channels that mediate Ca2+ influx in excitable cells, it is believed that store-operated Ca2+ channels fulfill this function (4, 12, 30). However, another candidate for mediating Ca2+ influx in epithelial cells is the Na+/Ca2+ exchanger (18, 34, 35). Na+/Ca2+ exchanger localized to the plasma membrane has been shown to play critical roles in regulating [Ca2+]i in several cell types, including cardiac myocytes, smooth muscle cells, and neurons (6, 26). Two families of plasma membrane Na+/Ca2+ exchanger proteins have been described in mammalian tissues, one in which Ca2+ movement is dependent only on Na+ (NCX family) and the other in which Ca2+ movement depends also on K+ (NCKX family). To maintain [Ca2+]i homeostasis, both NCX and NCKX exchangers can operate either in a forward (Ca2+ exit) mode or in a reversed (Ca2+ entry) mode, depending on the Na+ and Ca2+ (and K+) gradients and the potential across the plasma membrane (6). Although there is little information available on the relevance of Na+/Ca2+ exchange mechanisms to epithelial cells of the gastrointestinal tract, it has recently been shown that NCX1, NCKX3, and NCKX4 mRNA are all expressed in the rat small intestine (26). Additionally, functional studies from Kocks et al. (18) and Seip et al. (35) showed an interaction between store-operated, nonselective cation channels and Na+/Ca2+ exchanger during Cl secretion in the rat colon. However, the existence of Na+/Ca2+ exchanger in duodenal epithelial cells had not been examined. Thus the aim of this study was to explore whether Na+/Ca2+ exchange is molecularly and functionally expressed in duodenal epithelial cells and, if so, whether this transport system regulates duodenal mucosal HCO3 secretion by mediating Ca2+ influx in epithelial cells.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Animal preparation. This study was approved by the University of California, San Diego, Committee on Investigations Involving Animal Subjects. Experiments were performed on National Institutes of Health Swiss mice (6–10 wk of age). The mice were housed in a standard animal care room with a 12:12-h light-dark cycle and were allowed free access to food and water. Before each experiment, mice were deprived of food and water for 1 h.

Ussing chamber experiments. Ussing chamber experiments were performed as previously described (15). After mice were anesthetized by halothane, the abdomen was opened by a midline incision. The proximal duodenum was removed and immediately placed in ice-cold isoosmolar mannitol and indomethacin (10 µM) solution (to suppress trauma-induced prostaglandin release). The duodenal tissue from each animal was stripped of seromuscular layers, divided, and examined in three chambers (window area, 0.1 cm2). Experiments were performed under continuous short-circuited conditions (Voltage-Current Clamp, VCC 600; Physiologic Instruments, San Diego, CA), and luminal pH was maintained at 7.40 by the continuous infusion of 5 mM HCl under the automatic control of a pH-stat system (ETS 822; Radiometer America, Westlake, OH). The volume of the titrant infused per unit time was used to quantitate HCO3 secretion. Measurements were recorded at 5-min intervals, and mean values for consecutive 5- or 10-min periods were averaged. The rate of luminal HCO3 secretion is expressed as micromoles per centimeter per hour. The short-circuit current (Isc) was measured in microamperes and converted to microequivalents per centimeter per hour.

After a 30-min period when basal parameters were measured, inhibitors were added to the tissues for 10~20 min, as dictated by the experimental design, followed by addition of carbachol (100 µM) to the serosal side of tissue. Electrophysiological parameters were then recorded for 60 min. Net changes in duodenal HCO3 secretion and Isc during the 60-min period ensuing after the addition of carbachol were also calculated.

Western blot analysis. Segments of duodenal tissue (~25 mg) were stripped of seromuscular layers as described above for Ussing chamber experiments. Tissue samples were then frozen immediately in liquid nitrogen. Protein was extracted by homogenization on ice in 500 µl lysis buffer containing (in mM): 20 Tris·HCl (pH 7.5), 150 NaCl, 1 disodium EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1 {beta}-glycerophosphate, 1 sodium orthovanadate, 1% Triton X-100, and complete protease inhibitor cocktail (Sigma, St. Louis, MO). Equal amounts of protein, as determined by Lowry assay (Dc assay; Bio-Rad, Hercules, CA), were combined with 2x Laemmli sample buffer and boiled for 5 min. Proteins were separated by electrophoresis on 7.5% SDS-PAGE and transblotted to nitrocellulose membranes. The protein-bound nitrocellulose sheets were first incubated overnight at 4°C in blocking buffer containing 5% nonfat dry milk in distilled water. Nitrocellulose sheets were incubated with R3F1 monoclonal antibody to NCX1 (Swant, Bellinzona, Switzerland) diluted in blocking buffer (1:5,000) for 1 h at room temperature and then rinsed for 1 h with a wash buffer containing 20 mM Tris, pH 7.5, 500 mM NaCl, and 1% Tween 20. The membranes were then incubated with horseradish peroxidase-conjugated donkey anti-mouse IgG antibody for 30 min at room temperature and washed for 1 h with agitation, changing the wash buffer every 15 min. Protein bands were visualized with ECL Plus detection reagents (Amersham and Pharmacia, Piscataway, NJ), with NCX1 bands occurring at 120 and 70 kDa.

Analysis of NCX function in mouse duodenocytes by Ca2+ imaging. Duodenal epithelial cells were isolated by a Ca2+ chelation procedure modified from Isenberg et al. (16). Murine proximal duodenum was excised similar to Ussing chamber experiments described above. After removal, the lumen was rinsed and exposed to a citrate solution for 5 min to remove the mucus layer. Final concentrations were (in mM): 134.2 Na+, 9.5 K+, 97.5 Cl, 5.6 HPO42–, 8.0 H2PO, 27.0 citrate, and 10 glucose. pH was adjusted to 7.4, and 10 µM indomethacin was added. The luminal surface was then exposed to an EDTA-containing solution for 20 min at 37°C of the following composition (in mM): 153.3 Na+, 4.7 K+, 140.2 Cl, 8.2 HPO, 1.5 H2PO, 1.0 EDTA, and 10 glucose. pH was adjusted to 7.4, and 10 µM indomethacin was added. Indomethacin was included during cell isolation to prevent stimulation of HCO3 secretion secondary to release of prostaglandins caused by tissue manipulation. Epithelial cells were separated from the structural components of the duodenum by gentle manipulation and then washed by addition of RPMI and centrifugation (500 g) for 3 min at 4°C. Aliquots (200 µl) of the cell suspension in RPMI were kept at 2°C until use (within 30–120 min).

Ca2+ transport into mouse duodenocytes was measured by fura 2 fluorescence ratio digital imaging. Freshly isolated duodenocytes plated on cell-TAK cell and tissue adhesive (BD Bioscience, Bedford, MA)-precoated coverslips were loaded with 5 µM fura 2-AM at room temperature for 30 min and then mounted in a perfusion chamber on a Nikon microscope stage. The ratio of fura 2 fluorescence with excitation at 340 or 380 nm was followed over time and captured using an intensified CCD camera (ICCD200) and an Image Master System (Photon Technology International, Lawrenceville, NJ). Cells were initially perfused with Na+-containing solution (140 mM NaCl, 5 mM KCl, 1 mM CaCl2, 10 mM D-glucose, and 10 mM HEPES-trimethylamine, pH 7.4) for 10 min, which was then alternated with a solution in which the NaCl was replaced with 140 mM N-methyl-D-glucamine (NMDG).

Chemicals and solutions. Carbachol, atropine, nifedipine, ryanodine, RPMI, and indomethacin were purchased from Sigma Chemical. KB-R7943 and 2-aminoethoxydiphenylborate (2-APB) were from Tocris (Ellisville, MO). Fura 2-AM was from Molecular Probes (Eugene, OR). All other chemicals were obtained from Fisher Scientific (Santa Clara, CA). The mucosal solution used in Ussing chamber experiments contained the following (in mM): 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl, 25 gluconate, and 10 mannitol. The serosal solution contained the following (in mM): 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl, 25 HCO3, 1.4 HPO, 2.4 H2PO4, 10 glucose, and 0.01 indomethacin. The osmolalities for both solutions were ~284 mosmol/kg H2O.

Statistical analysis. Results are expressed as means ± SE. Differences between means were considered to be statistically significant at P < 0.05 using Student's t-test for paired or unpaired values, as appropriate.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Carbachol induces increases in Isc and HCO3 secretion. After duodenal mucosal tissues were equilibrated in Ussing chambers for 30 min, a stable 30-min measurement of basal Isc and HCO3 secretion was recorded. Subsequently, carbachol (100 µM) or its vehicle (double-distilled water) was added to the serosal side of the tissue. Serosal addition of the vehicle did not induce any change in Isc or HCO3 secretion. However, addition of carbachol induced a significant increase in Isc in a biphasic manner: an initial transient peak and a second sustained plateau (Fig. 1). The initial peak of Isc reached its maximum in 2 min and then declined slowly to a plateau value, still above baseline, ~10 min after carbachol addition (Fig. 1A). The second plateau phase persisted for at least 50 min (Fig. 1A). Carbachol also induced a significant increase in HCO3 secretion in a monophasic manner, which reached a peak value at 10 min after addition and then persisted for at least 40 min (Fig. 1B). In Ca2+-free solutions, carbachol did not induce significant changes in Isc or HCO3 secretion (Fig. 1). The duodenal mucosal response to carbachol was also specifically mediated by muscarinic receptors, since atropine (10 µM) totally abolished both the initial peak (2.6 ± 0.5 µeq·cm–2·h–1 vs. baseline 2.4 ± 0.4 µeq·cm–2·h–1, n = 5, P > 0.05) and second plateau of Isc induced by carbachol (2.6 ± 0.4 µeq·cm–2·h–1 vs. baseline 2.4 ± 0.4 µeq·cm–2·h–1, n = 5, P > 0.05).



View larger version (18K):
[in this window]
[in a new window]
 
Fig. 1. Time course of carbachol-stimulated murine duodenal mucosal short-circuit current (Isc; A) and HCO3 secretion (B) in the presence and nominal absence of extracellular Ca2+. Carbachol (100 µM) was added serosally at the time indicated by the arrow. Values are means ± SE; n = 6 experiments in each series. Responses differ significantly from basal values before carbachol addition: *P < 0.05 and ***P < 0.001 by Student's t-test for paired samples.

 
Involvement of intracellular Ca2+ stores in carbachol-induced increases in Isc and HCO3 secretion. Intracellular Ca2+ release from IP3-sensitive Ca2+ stores, after stimulation of muscarinic M3 receptors by carbachol, has been considered to play a critical role in the regulation of Cl secretion across colonic epithelium (12, 18, 34). However, it was still unclear whether this also applied to duodenal mucosal ion transport, and specifically to HCO3 secretion. Thus, to investigate the involvement of IP3-sensitive Ca2+ stores in carbachol-induced increases in duodenal mucosal Isc and HCO3 secretion, the effect of 2-APB, a cell-permeable IP3 receptor antagonist, was tested in Ussing chamber experiments. Addition of 2-APB (100 µM) to both the mucosal and serosal sides of duodenal tissues significantly inhibited the carbachol-induced increase in Isc (Fig. 2A). The initial peak was decreased by 51% (P < 0.001), and the second plateau was totally abolished (Fig. 2A). However, 2-APB did not significantly inhibit carbachol-induced HCO3 secretion (Fig. 2B). To further investigate the involvement of IP3-sensitive Ca2+ stores in the increase in duodenal Isc and HCO3 secretion induced by carbachol, the effect of LiCl was tested in another set of Ussing chamber experiments. LiCl is presumed to block IP3 recycling by inhibiting the hydrolysis of inositol phosphates, which results in loss of the IP3 signal (34). Addition of LiCl (30 mM) to both the mucosal and serosal sides of duodenal tissues significantly inhibited the carbachol-induced increase in Isc. The initial peak was decreased by 47% (P < 0.001), and the second plateau was totally abolished (Fig. 3A). However, similar to 2-APB, LiCl did not significantly inhibit carbachol-induced HCO3 secretion (Fig. 3B).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Effect of 2-aminoethoxydiphenylborate (2-APB) on the ability of carbachol to stimulate murine duodenal mucosal Isc (A) and net peak HCO3 secretion (B). 2-APB (100 µM) was added to both sides, and carbachol (100 µM) was added serosally at the times indicated by the arrows. Values are expressed as means ± SE; n = 7 experiments in each series. Values differ significantly from those in the absence of 2-APB: *P < 0.05 and ***P < 0.001 by Student's t-test.

 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 3. Effect of LiCl on the ability of carbachol to stimulate murine duodenal mucosal Isc (A) and net peak HCO3 secretion (B). LiCl (30 mM) was added to both sides, and carbachol (100 µM) was added serosally at the times indicated by the arrows. Values are expressed as means ± SE; n = 5 experiments in each series. Values differ significantly from those in the absence of LiCl: *P < 0.05 and ***P < 0.001 by Student's t-test.

 
To investigate the involvement of Ca2+ release from the endoplasmic reticulum through ryanodine receptors in the carbachol-induced increase in Isc and HCO3 secretion across duodenal mucosa, both ryanodine and caffeine were studied in Ussing chamber experiments. Addition of ryanodine (100 µM) to both the mucosal and serosal sides of duodenal tissues did not affect the carbachol-induced increase in either the initial peak (8.0 ± 0.7 µeq·cm–2·h–1 for control vs. 9.6 ± 0.7 µeq·cm–2·h–1 in the presence of ryanodine, n = 5, P > 0.05) or second plateau (3.2 ± 0.6 µeq·cm–2·h–1 for control vs. 3.4 ± 0.7 µeq·cm–2·h–1 in the presence of ryanodine, n = 5, P > 0.05) of Isc. The carbachol-induced increase in HCO3 secretion was also not affected by ryanodine (net peak HCO3 secretion: 0.62 ± 0.07 µmol·cm–2·h–1 for control vs. 0.68 ± 0.18 µmol·cm–2·h–1 in the presence of ryanodine, n = 5, P > 0.05). Similarly, caffeine (10 mM) did not prevent the ability of carbachol to cause a subsequent increase in duodenal mucosal Isc (initial peak: 5.9 ± 0.5 µeq·cm–2·h–1 for control vs. 5.8 ± 0.8 µeq·cm–2·h–1 in the presence of caffeine, n = 5, P > 0.05). These data likely rule out the possibility that ryanodine receptors in the endoplasmic reticulum are involved in the carbachol-induced increase in Isc and HCO3 secretion across duodenal mucosa.

Role of NCX in carbachol-induced increases in Isc and HCO3 secretion. Influx of extracellular Ca2+ in cells is the major pathway by which [Ca2+]i is increased in a sustained fashion. This influx mechanism encompasses voltage-operated Ca2+ channels, store-operated nonselective cation channels, and the reversed mode of the Na+/Ca2+ exchanger. To investigate the Ca2+ influx mechanism(s) involved in the ability of carbachol to increase Isc and/or HCO3 secretion across duodenal mucosa, different plasma membrane Ca2+ influx pathways were assessed pharmacologically in Ussing chamber experiments. First, nifedipine was used to block voltage-operated Ca2+ channels. Addition of nifedipine (10 µM) to both mucosal and serosal sides of duodenal tissues did not prevent the carbachol-induced increase in Isc. The initial peak was 6.8 ± 0.7 µeq·cm–2·h–1 for control and 6.4 ± 0.9 µeq·cm–2·h–1 in the presence of nifedipine (n = 6, P > 0.05). The second plateau of Isc was 2.9 ± 0.2 µeq·cm–2·h–1 for control and 3.1 ± 0.8 µeq·cm–2·h–1 in the presence of nifedipine (n = 6, P > 0.05). Thus both the initial peak and plateau phases of the increase in Isc were identical whether or not nifedipine was present. Second, nickel was used as a blocker for both store-operated nonselective cation channels and the Na+/Ca2+ exchanger. Mucosal plus serosal addition of nickel (5 mM) significantly inhibited the carbachol-induced increase in duodenal mucosal Isc. The initial peak was decreased by 66% (P < 0.01), and the second plateau was totally abolished (Fig. 4A). The carbachol-induced increase in HCO3 secretion was also abolished by nickel (Fig. 4B). Third, KB-R7943, a selective inhibitor for the reversed mode of the Na+/Ca2+ exchanger, was used to test more definitively the involvement of NCX in carbachol-induced increases in Isc and HCO3 secretion. Mucosal plus serosal addition of KB-R7943 (10 µM) did not alter the initial peak in Isc evoked by carbachol across duodenal mucosa, but it prevented the second plateau phase of Isc in duodenal mucosa treated with carbachol (Fig. 5A). Moreover, the ability of carbachol to increase HCO3 secretion was also prevented by KB-R7943 (10 µM; Fig. 5B). No further inhibition was observed when KB-R7943 was added at a concentration of 30 µM for either parameter (Fig. 5). KB-R7943 at 10 µM is accepted to be a selective inhibitor for the reversed mode of NCX (17, 36), and thus our data suggest that NCX is specifically implicated in both the plateau phase of Isc and HCO3 secretory increases in duodenal mucosa.



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 4. Effect of nickel on the ability of carbachol to stimulate murine duodenal mucosal Isc (A) and net peak HCO3 secretion (B). NiCl2 (5 mM) was added to both sides, and carbachol (100 µM) was added serosally at the times indicated by the arrows. Values are expressed as means ± SE; n = 7 experiments in each series. Values differ significantly from those in the absence of NiCl2: *P < 0.05 and ***P < 0.001 by Student's t-test.

 


View larger version (21K):
[in this window]
[in a new window]
 
Fig. 5. Effect of KB-R7943 on the ability of carbachol to stimulate murine duodenal mucosal Isc (A) and HCO3 secretion (B). KB-R7943 (10 or 30 µM) was added to both sides, and carbachol (100 µM) was added serosally at the times indicated by the arrows. Values are expressed as means ± SE; n = 7 experiments in each series. Values differ significantly from those in the absence of KB-R7943: *P < 0.05 by Student's t-test.

 
Molecular and functional identification of NCX in mouse duodenal mucosa. Our data presented above indicate that NCX is involved in the regulation of Ca2+-dependent duodenal mucosal ion transport and HCO3 secretion. To test this hypothesis further, we screened for the expression of NCX proteins in mouse duodenal mucosa. R3F1, an anti-NCX monoclonal antibody, recognized two proteins with molecular masses of 120 and 70 kDa, corresponding to previous reports of the native NCX1 protein (Refs. 24 and 37 and Fig. 6A). The 70-kDa protein represents a short from of NCX1, which may be either a proteolytic cleavage product or a functional, truncated form of NCX1 (37). We next sought to obtain direct evidence that functional NCX activity is present in duodenal epithelial cells. For this purpose, Ca2+ imaging experiments were conducted in which cellular Na+ concentrations were altered to elicit Ca2+ entry via the reversed mode of NCX. Reduction of extracellular Na+ from 140 to 0 mM (replaced by 140 mM NMDG to maintain isoosmolality) caused a significant increase in cytosolic Ca2+, which was reduced by KB-R7943 (10 µM; Fig. 6, B and C). Thus our data showing expression of NCX1 proteins and direct measurements of cytosolic Ca2+ are consistent with functional expression of the reversed mode of NCX1 in mouse duodenocytes.



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 6. Evidence for Na+/Ca2+ exchanger (NCX1) protein and function in freshly isolated murine duodenocytes. A: 30 µg of proteins derived from total homogenates of duodenal mucosa separated by SDS-PAGE, transferred to nitrocellulose, and incubated with anti-NCX antibody. The molecular masses of the bands are shown as standards. The blot shown is representative of 3 similar blots. B and C: Ca2+-sensitive fura 2 fluorescence ratios (F340/380) recorded in duodenocytes. The cells were kept in a normal physiological solution in the presence or absence of 10 µM KB-R7943. Extracellular Na+ was then reduced to 0 mM as indicated. In C, values are expressed as means ± SE; n = 12 experiments in each series. KB-R7943 significantly reduced the rise in Ca2+ induced by Na+ removal: ***P < 0.001 by Student's t-test.

 

    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
It is generally accepted that epithelial HCO3 secretion is an important factor in protection of the duodenal mucosa against acid-induced injury. A number of intracellular signaling pathways, such as cAMP-, cGMP-, and Ca2+-dependent mechanisms, are believed to mediate this important physiological process (11, 28). Ca2+ acts as a universal second messenger to regulate many different cellular functions in a variety of cells, including epithelial cells. For example, intracellular Ca2+ is considered to be an important regulator of intestinal ion transport (8–10). In excitable cells, it is well known that Ca2+ entry is mainly mediated via voltage-operated Ca2+ channels. However, less is known about Ca2+ entry pathways in nonexcitable cells, such as epithelial cells that do not express voltage-operated Ca2+ channels (30). T84 cells and native colonic epithelial cells are a major focus of the investigations on Ca2+ handling mechanisms and the regulation of Cl transport by Ca2+ in the gastrointestinal tract (12, 18, 31, 35). However, Ca2+ handling mechanisms in duodenal mucosal epithelia and associated regulation of HCO3 secretion were unclear. There was essentially no information available about the specific Ca2+ entry pathways existing in native duodenal mucosal epithelia. Therefore, in the present study, we examined the potential role of the reversed mode of NCX in regulating duodenal mucosal ion transport and HCO3 secretion, secondary to an effect on intracellular Ca2+ homeostasis.

In colonic epithelial cells, Ca2+-dependent secretagogues induce a biphasic increase in [Ca2+]i: an initial peak, caused by the release of Ca2+ from intracellular IP3-sensitive stores, followed by Ca2+ influx from the extracellular medium (25). Ca2+ influx during the second phase is generally thought to be caused by the opening of store-operated nonselective cation channels in the plasma membrane, which are activated by depletion of the intracellular Ca2+ stores (30). In either case, an increase in [Ca2+]i activates Ca2+-sensitive basolateral K+ channels in colonic epithelial cells. The resulting hyperpolarization favors the exit of Cl resulting from an increase in the driving force, which induces Cl secretion (7, 34). Although there is no information available about the molecular identification of NCX, functional studies have suggested that NCX exists and there is an interaction between store-operated nonselective cation channels and NCX in rat colonic epithelium (18, 34, 35).

NCX1 was the first sarcolemmal Na+/Ca2+ exchanger to be cloned from cardiac muscle. This exchanger is clearly essential for cardiac excitation-contraction coupling (6, 36). NCX1 is abundantly expressed in many tissues, such as the heart, brain, kidney, smooth muscle, and skeletal muscle (6). NCX1 mRNA has also been found in the rat small intestine by Northern blotting analysis (26). Functional studies using radioactive 45Ca2+ transport measurements also demonstrated the possible existence of NCX in the basolateral membrane of mammalian enterocytes (13, 19). However, the relevance of these findings for the proximal duodenum, an intestinal segment with distinct transport properties, was unknown. In the present study, we present clear evidence for the expression of NCX1 protein in mouse duodenal mucosae. Immunoblotting of duodenal mucosal lysates revealed a pattern of proteins typically observed in NCX-expressing cells, including bands at 120 and 70 kDa corresponding to native full-length and truncated forms of NCX1 described in other cell types (24, 32, 37). Moreover, we obtained convincing evidence for an Na+-dependent Ca2+ transport system in duodenocytes, which was sensitive to inhibition by the classical NCX inhibitor KB-R7943 at a concentration specific for inhibition of the reversed mode of NCX function. Thus our immunoblotting and functional experiments provided strong evidence for expression of a functional NCX protein in duodenocytes and suggested that this protein could play an important role in the regulation of Ca2+ entry upon stimulation of muscarinic M3 receptors (8).

Consistent with the idea that nonexcitable cells do not express voltage-operated Ca2+ channels, we found that the voltage-operated Ca2+ channel blocker nifedipine had no effect on carbachol-induced increases in Isc across duodenal mucosae. On the other hand, store-operated nonselective cation channels are thought to be responsible for Ca2+ entry in some nonexcitable cells and might be an alternative pathway for Ca2+ entry upon the stimulation of muscarinic M3 receptors (30). However, there was no information available about this pathway in the native duodenal epithelium. We cannot rule out a role for such channels in carbachol-stimulated ion transport in duodenocytes. However, the selective NCX inhibitor KB-R7943 was as effective in blocking the effects of carbachol on Isc and HCO3 secretion as was nickel, which blocks both NCX and store-operated cation channels. This implies that the role of the latter channels in regulating duodenal ion transport, if it exists, may be relatively minor. Alternatively, store-operated channels may in fact interact functionally with NCX, as has been described in colonic epithelia (18, 35).

Vagally mediated stimulation of duodenal alkaline secretion has been demonstrated in all species studied. Several studies have shown that the nonselective muscarinic antagonist atropine inhibits the stimulation of muscosal HCO3 secretion by carbachol in rats (23, 33) and guinea pigs (29) in vivo, as well as in rabbit duodenal mucosa in vitro (15). In our study, atropine abolished carbachol-induced increases in mouse duodenal mucosal Isc and HCO3 secretion, suggesting that these are specific responses likely occurring via stimulation of muscarinic receptors on mouse duodenocytes. Chew et al. (8) demonstrated that carbachol induced increases in [Ca2+]i via stimulation of M3 muscarinic receptor subtype in individual rat and human duodenocytes. It is well known that increases in [Ca2+]i play a key regulatory role in duodenal mucosal ion transport and HCO3 secretion. Our study shows that carbachol stimulates both duodenal mucosal ion transport and HCO3 secretion in a manner dependent on the combined effect of Ca2+ release from IP3-sensitive intracellular stores as well as extracellular Ca2+ entry. However, compared with overall duodenal mucosal ion transport, carbachol-induced HCO3 secretion appears to be more dependent on extracellular Ca2+ entry than on intracellular Ca2+ release, based on the following observations: 1) carbachol evoked an initial peak of Isc to maximal levels within 2 min, but its effect on HCO3 secretion was more delayed in the presence of normal extracellular Ca2+ concentrations (Fig. 1); 2) in Ca2+-free solutions, carbachol was unable to stimulate HCO3 secretion but still caused a small, but reproducible, initial peak in Isc (Fig. 1A); 3) 2-APB, an IP3 receptor antagonist (27, 38), and LiCl, an inhibitor of IP3 recycling that eventually results in loss of the IP3 signal (3, 4), clearly inhibited carbachol-induced Isc responses in duodenal mucosa but did not significantly affect HCO3 secretion (Figs. 2 and 3); and 4) in contrast, KB-R7943, a selective inhibitor for the reversed mode of NCX (17, 36), did not inhibit the carbachol-induced initial peak in Isc but significantly inhibited both the second phase of Isc and HCO3 secretion produced by carbachol (Fig. 5). Taken together, these data suggest that Ca2+ entry via the reversed mode of NCX plays a key regulatory role specifically in duodenal mucosal HCO3 secretion.

Mammalian cells maintain a low cytoplasmic concentration of Na+ (~10–15 mM) compared with the extracellular concentration of Na+ (~140 mM) because of the activity of the Na+-K+-ATPase. Under this physiological condition, one can ask whether it is energetically feasible for Na+/Ca2+ exchange to operate in the reversed mode to induce Ca2+ entry. In this regard, Blaustein et al. (2, 5, 6) and van Breemen et al. (20–22) have recently proposed working models for Ca2+ entry via the reversed mode of Na+/Ca2+ exchange, although they have not yet been generally accepted. Briefly, upon the stimulation of G protein-coupled receptors, the opening of IP3 receptor channels induces emptying of Ca2+ from the sarcoplasmic reticulum (SR) store. This leads to opening of the putative store-operated nonselective cationic channel (NSCC), which is believed to be much more permeable to Na+ than Ca2+. Under physiological conditions, opening of this NSCC should result mainly in Na+ influx into the restricted plasma membrane-SR junctional space. This inward cationic current causes membrane depolarization. Both the increase in Na+ and depolarization can then drive Na+/Ca2+ exchange into its reversed mode of operation, bringing Ca2+ into the cell. More recently, it has also been suggested that there is a functional and physical interaction of transient receptor potential (TRP) cation channels with NCX proteins. Our data obtained from the measurement of Isc across duodenal mucosae are consistent with those from colonic epithelial cells (12, 18), providing further support for Blaustein and van Breemen et al.'s working models for the reversed mode of Na+/Ca2+ exchange. Because Cl makes a major contribution to Isc across the intestinal mucosa, Cl secretion is most likely regulated by this IP3 cascade. However, our data do not support the involvement of the IP3 cascade in the regulation of duodenal mucosal HCO3 secretion. Whether mechanisms other than the IP3 cascade link stimulation of muscarinic receptors to the activation of the reversed mode of Na+/Ca2+ exchanger, such as nicotinic acid adenine dinucleotide phosphate, protein kinase C, diacylglycerol, or direct coupling of muscarinic receptors to TRP channels or NCX, will require further study.

The localization of Na+/Ca2+ exchangers in the intestinal tract remains unclear. This transport process was reported to be localized to the basolateral membrane (13, 19), but there is also evidence for apical localization in the colonic epithelium (34). In our preliminary study, serosal addition alone of KB-R7943 did not significantly affect duodenal mucosal Isc or HCO3 secretion. Recently, we performed an immunohistochemical analysis demonstrating that NCX1 may be localized on both the basolateral and apical membranes of duodenal mucosal epithelial cells (unpublished observations). The polarity of NCX expression and its relevance for duodenal epithelial cell function will remain a subject of active investigation.

What is the physiological significance of NCX expressed in duodenal mucosal epithelium? Because NCX has been shown to be functionally expressed on the basolateral membrane and the main site of intestinal Ca2+ absorption is in the small intestine, it was proposed that NCX participates in intestinal Ca2+ absorption (6, 13, 19). In fish enterocytes, NCX appears to be the main mechanism by which transcellular fluxes of Ca2+ are extruded from the cells at the serosal surface. However, whether this is also the case in mammals is less clear (6). In contrast, we have shown that NCX proteins and function are present in mouse duodenal mucosal epithelium, where this transporter plays a key regulatory role in duodenal mucosal ion transport and HCO3 secretion. As mentioned above, duodenal HCO3 secretion plays an important role in preventing gastric acid-induced duodenal mucosal injury. Therefore, NCX proteins in the gastrointestinal tract might be potential therapeutic targets for the treatment of gastrointestinal diseases, such as duodenal ulcer. However, more work needs to be performed to address the detailed physiological and pathological roles of duodenal NCX in acid-stimulated duodenal HCO3 secretion.

In summary, this study shows that NCX plays a key role in the regulation of Ca2+-dependent duodenal mucosal ion transport and HCO3 secretion in mice. In future studies, we may define relative roles of the subfamilies of K+-dependent (NCKX) and K+-independent (NCX) exchangers in the duodenal mucosa, since mRNAs for both subfamilies have been shown to be expressed in the small intestine (26) but they could not readily be distinguished on functional grounds in the present study. A better understanding of the regulation of duodenal HCO3 secretion by NCX may lead to novel approaches for treating gastroduodenal disorders arising from acid-peptic damage to the duodenal mucosa.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by a National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33491–18 (to K. E. Barrett). H. Dong serves as a coinvestigator on Grant DK-33491.


    ACKNOWLEDGMENTS
 
We thank Dr. Jonathan Lytton, Department of Biochemistry and Molecular Biology, University of Calgary, Canada, for kindly providing the R3F1 antibody.


    FOOTNOTES
 

Address for reprint requests and other correspondence: H. Dong, Division of Gastroenterology, Dept. of Medicine, UCSD Medical Center 8414, 200 West Arbor Drive, San Diego, CA 92103 (E-mail: h2dong{at}ucsd.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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Allen A, Flemstrom G, Garner A, and Kivilaakso E. Gastroduodenal mucosal protection. Physiol Rev 73: 823–857, 1993.[Abstract/Free Full Text]
  2. Arnon A, Hamlyn JM, and Blaustein MP. Na+ entry via store-operated channels modulates Ca2+ signaling in arterial myocytes. Am J Physiol Cell Physiol 278: C163–C173, 2000.[Abstract/Free Full Text]
  3. Berridge MJ, Downes CP, and Hanley MR. Neural and developmental actions of lithium: a unifying hypothesis. Cell 59: 411–419, 1989.[CrossRef][ISI][Medline]
  4. Binder HJ and Sandle GJ. Electrolyte transport in the mammalian colon. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York, NY: Raven, 1994, p. 2133–2171.
  5. Blaustein MP and Golovina VA. Structural complexity and functional diversity of endoplasmic reticulum Ca2+ stores. Trends Neurosci 24: 602–608, 2001.[CrossRef][ISI][Medline]
  6. Blaustein MP and Lederer WJ. Sodium/calcium exchange: its physiological implications. Physiol Rev 79: 763–854, 1999.[Abstract/Free Full Text]
  7. Bohme M, Diener M, and Rummel W. Calcium- and cyclic-AMP-mediated secretory responses in isolated colonic crypts. Pflügers Arch 419: 144–151, 1991.[CrossRef][ISI][Medline]
  8. Chew CS, Safsten B, and Flemstrom G. Calcium signaling in cultured human and rat duodenal enterocytes. Am J Physiol Gastrointest Liver Physiol 275: G296–G304, 1998.[Abstract/Free Full Text]
  9. Flemstrom G. Gastric and duodenal mucosal secretion of bicarbonate. In: Physiology of the Gastrointestinal Tract, edited by Johnson LR. New York, NY: Raven, 1994, p. 1285–1309.
  10. Flemstrom G and Isenberg JI. Gastroduodenal mucosal alkaline secretion and mucosal protection. News Physiol Sci 16: 23–28, 2001.[ISI][Medline]
  11. Flemstrom G and Jedstedt G. Stimulation of duodenal mucosal bicarbonate secretion in the rat by brain peptides. Gastroenterology 97: 412–420, 1989.[ISI][Medline]
  12. Frings M, Schultheiss G, and Diener M. Electrogenic Ca2+ entry in the rat colonic epithelium. Pflügers Arch 439: 39–48, 1999.[CrossRef][ISI][Medline]
  13. Ghijsen WE, De Jong MD, and Van Os CH. Kinetic properties of Na+/Ca2+ exchange in basolateral plasma membranes of rat small intestine. Biochim Biophys Acta 730: 85–94, 1983.[ISI][Medline]
  14. Hogan DL, Ainsworth MA, and Isenberg JI. Gastroduodenal bicarbonate secretion. Aliment Pharmacol Ther 8: 475–488, 1994.[ISI][Medline]
  15. Hogan DL, Yao B, Steinbach JH, and Isenberg JI. The enteric nervous system modulates mammalian duodenal mucosal bicarbonate secretion. Gastroenterology 105: 410–417, 1993.[ISI][Medline]
  16. Isenberg JI, Ljungstrom M, Safsten B, and Flemstrom G. Proximal duodenal enterocyte transport: evidence for Na+-H+ and Cl-HCO3 exchange and NaHCO3 cotransport. Am J Physiol Gastrointest Liver Physiol 265: G677–G685, 1993.[Abstract/Free Full Text]
  17. Iwamoto T and Kita S. Development and application of Na+/Ca2+ exchange inhibitors. Mol Cell Biochem 259: 157–161, 2004.[CrossRef][ISI][Medline]
  18. Kocks S, Schultheiss G, and Diener M. Ryanodine receptors and the mediation of Ca2+-dependent anion secretion across rat colon. Pflügers Arch 445: 390–397, 2002.[CrossRef][ISI][Medline]
  19. Kumar V and Prasad R. Thyroid hormones stimulate calcium transport systems in rat intestine. Biochim Biophys Acta 1639: 185–194, 2003.[ISI][Medline]
  20. Lee CH, Poburko D, Kuo KH, Seow CY, and van Breemen C. Ca2+ oscillations, gradients, and homeostasis in vascular smooth muscle. Am J Physiol Heart Circ Physiol 282: H1571–H1583, 2002.[Abstract/Free Full Text]
  21. Lee CH, Poburko D, Sahota P, Sandhu J, Ruehlmann DO, and van Breemen C. The mechanism of phenylephrine-mediated [Ca2+]i oscillations underlying tonic contraction in the rabbit inferior vena cava. J Physiol 534: 641–650, 2001.[Abstract/Free Full Text]
  22. Lee CH, Rahimian R, Szado T, Sandhu J, Poburko D, Behra T, Chan L, and van Breemen C. Sequential opening of IP3-sensitive Ca2+ channels and SOC during alpha-adrenergic activation of rabbit vena cava. Am J Physiol Heart Circ Physiol 282: H1768–H1777, 2002.[Abstract/Free Full Text]
  23. Lenz HJ, Vale WW, and Rivier JE. TRH-induced vagal stimulation of duodenal HCO3 mediated by VIP and muscarinic pathways. Am J Physiol Gastrointest Liver Physiol 257: G677–G682, 1989.[Abstract/Free Full Text]
  24. Li L, Guerini D, and Carafoli E. Calcineurin controls the transcription of Na+/Ca2+ exchanger isoforms in developing cerebellar neurons. J Biol Chem 275: 20903–20910, 2000.[Abstract/Free Full Text]
  25. Lindqvist SM, Sharp P, Johnson IT, Satoh Y, and Williams MR. Acetylcholine-induced calcium signaling along the rat colonic crypt axis. Gastroenterology 115: 1131–1143, 1998.[ISI][Medline]
  26. Lytton J, Li XF, Dong H, and Kraev A. K+-dependent Na+/Ca2+ exchangers in the brain. Ann NY Acad Sci 976: 382–393, 2002.[Abstract/Free Full Text]
  27. Ma HT, Patterson RL, van Rossum DB, Birnbaumer L, Mikoshiba K, and Gill DL. Requirement of the inositol trisphosphate receptor for activation of store-operated Ca2+ channels. Science 287: 1647–1651, 2000.[Abstract/Free Full Text]
  28. Montrose MH, Keely SJ, and Barrett KE. Electrolyte secretion and absorption: small intestine and colon. In: Textbook of Gastroenterology, edited by Yamada T AD, Owyang C, Powell DW, and Silverstein FE. Philadelphia, PA: Lippincott, 1999, p. 321–355.
  29. Odes HS, Muallem R, Reimer R, Beil W, Schwenk M, and Sewing KF. Cholinergic regulation of guinea pig duodenal bicarbonate secretion. Am J Physiol Gastrointest Liver Physiol 265: G270–G276, 1993.[Abstract/Free Full Text]
  30. Parekh AB and Penner R. Store depletion and calcium influx. Physiol Rev 77: 901–930, 1997.[Abstract/Free Full Text]
  31. Reinlib L, Mikkelsen R, Zahniser D, Dharmsathaphorn K, and Donowitz M. Carbachol-induced cytosolic free Ca2+ increases in T84 colonic cells seen by microfluorimetry. Am J Physiol Gastrointest Liver Physiol 257: G950–G960, 1989.[Abstract/Free Full Text]
  32. Rosker C, Graziani A, Lukas M, Eder P, Zhu MX, Romanin C, and Groschner K. Ca2+ signaling by TRPC3 involves Na+ entry and local coupling to the Na+/Ca2+ exchanger. J Biol Chem 279: 13696–13704, 2004.[Abstract/Free Full Text]
  33. Safsten B, Jedstedt G, and Flemstrom G. Cholinergic influence on duodenal mucosal bicarbonate secretion in the anesthetized rat. Am J Physiol Gastrointest Liver Physiol 267: G10–G17, 1994.[Abstract/Free Full Text]
  34. Schultheiss G, Ribeiro R, Schafer KH, and Diener M. Activation of apical K+ conductances by muscarinic receptor stimulation in rat distal colon: fast and slow components. J Membr Biol 195: 183–196, 2003.[CrossRef][ISI][Medline]
  35. Seip G, Schultheiss G, Kocks SL, and Diener M. Interaction between store-operated non-selective cation channels and the Na+-Ca2+ exchanger during secretion in the rat colon. Exp Physiol 86: 461–468, 2001.[Abstract/Free Full Text]
  36. Shigekawa M and Iwamoto T. Cardiac Na+-Ca2+ exchange: molecular and pharmacological aspects. Circ Res 88: 864–876, 2001.[Abstract/Free Full Text]
  37. Van Eylen F, Kamagate A, and Herchuelz A. A new Na+/Ca2+ exchanger splicing pattern identified in situ leads to a functionally active 70kDa NH(2)-terminal protein. Cell Calcium 30: 191–198, 2001.[CrossRef][ISI][Medline]
  38. van Rossum DB, Patterson RL, Ma HT, and Gill DL. Ca2+ entry mediated by store depletion, S-nitrosylation, and TRP3 channels. Comparison of coupling and function. J Biol Chem 275: 28562–28568, 2000.[Abstract/Free Full Text]




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
288/3/G457    most recent
00381.2004v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Google Scholar
Articles by Dong, H.
Articles by Barrett, K. E.
Articles citing this Article
PubMed
PubMed Citation
Articles by Dong, H.
Articles by Barrett, K. E.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2005 by the American Physiological Society.