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* Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, La Jolla, California, USA; and
Department of Biology, University of Calgary, Alberta, Canada
1Correspondence: Division of Gastroenterology, Department of Medicine, University of California, San Diego, 9500 Gilman Dr. La Jolla, CA 92093-0063, USA. E-mail: h2dong{at}ucsd.edu
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Key Words: acid-stimulated 5-HT release • duodenal mucosal bicarbonate secretion • cytoplasmic Ca2+ signaling • Na+/Ca2+ exchanger
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. It has been estimated that 
60–90% of 5-HT in the body is stored in the GI tract and >90% of 5-HT in the GI tract is localized within enterochromaffin (EC) cells and enteric neurons (1
2
3
4)
. 5-HT plays a significant role in controlling GI motility, gastric acid secretion, intestinal Cl– transport, and duodenal mucosal bicarbonate secretion (DMBS) (5
6
7
8)
.
It has long been accepted that DMBS provides an important first line of defense against acid peptic injury. This significance is underscored in patients with duodenal ulcers in whom acid-stimulated DMBS is impaired (9
10
11
12)
. However, the precise mechanism(s) of DMBS are still largely unknown although several neural and humoral factors such as cholinergic agents, prostaglandin E2, vasoactive intestinal peptide, dopamine, somatostatin, and luminal acid have been demonstrated to be involved in regulating this physiological process (10
, 11
, 13)
. Luminal acidification may induce release of 5-HT from the duodenal mucosa, and we demonstrated recently that 5-HT is a potent stimulant of DMBS in vitro (7
, 8
, 14)
. In our previous studies we showed that 5-HT causes DMBS likely via direct actions on 5-HT receptors of duodenal epithelial cells and/or indirectly by stimulating acetylcholine (ACh) release from enteric neurons. We also provided preliminary evidence implying that 5-HT regulates DMBS perhaps via both cytoplasmic free Ca2+ ([Ca2+]cyt)- and cAMP-dependent pathways. Further RT-polymerase chain reaction (RT-PCR) analysis revealed that 5-HT4 receptors are localized to the duodenal mucosa and/or epithelial cells (7
, 8)
. However, relatively little is known about the precise mechanisms of 5-HT-mediated duodenal mucosal ion transport and DMBS. Moreover, whether 5-HT plays a physiological role in acid-stimulated DMBS remained largely unexplored.
Therefore, the aims of the present study were to determine 1) whether acid, a physiological stimulator, induces release of 5-HT from the murine duodenal mucosa; 2) whether 5-HT plays a physiological role in acid-stimulated DMBS; and 3) the precise mechanisms involved in this process, especially the role of the Ca2+ signaling pathway. We now provide evidence that, under physiological conditions, duodenal luminal acid stimulates 5-HT release. In turn, this induces duodenal mucosal ion transport and DMBS via a Ca2+ signaling pathway in which several plasma membrane ion channels and transporters are involved. Since 5-HT is an important enteric neurotransmitter, a full understanding of its physiological role and the mechanisms underlying regulation of DMBS by 5-HT may lead to novel approaches for treating disorders associated with damage to the duodenal mucosa, such as peptic ulcers.
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Measurement of acid-stimulated 5-HT release by enzyme immunoassay (EIA)
Acid-stimulated 5-HT release from duodenal tissues was measured as described previously (15
, 16)
. Briefly, a duodenal segment was removed from an anesthetized NIH Swiss mice mouse and divided into four equal segments. Each segment was inverted so that the mucosal surface faced to the outside, then tied at both ends with threads. After washing briefly with PBS, the segments were incubated in Earle balanced salt solution (EBSS) containing 0.1% BSA for 30 min at 37°C. Tissues were then washed twice in EBSS and incubated at 37°C for 20 min in EBSS containing 10 µM pargyline (a monoamine oxidase inhibitor) and alaproclate (a 5-HT uptake inhibitor). Each tissue was then placed into 0.5 ml of EBSS containing the same concentration of pargyline and alaproclate, adjusted by addition of HCl to pH 3, 4, 5, or control (no HCl added, pH 7.4), and incubated at 37°C for 5 min. The tissue was then removed, and the incubation medium was immediately neutralized and centrifuged twice for 2 min in a microcentrifuge to remove any cells or tissue fragments. Supernatants were stored at –70°C in the dark until assay and the tissues were gently blotted, then weighed. 5-HT levels in the supernatants were determined by EIA using a commercial kit (Beckman Coulter Inc., Somerset, NJ, USA) and by reference to a standard curve. Values are presented as pmoles 5-HT/mg tissue.
Ussing chamber experiments
Ussing chamber experiments were performed as described previously (7
, 17)
. Briefly, after mice were anesthetized with halothane and euthanized by cervical dislocation, the abdomen was opened by a midline incision. The proximal duodenum was removed and immediately placed in ice-cold iso-osmolar 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; Physiological Instruments, San Diego, CA, USA) with luminal pH maintained at 7.40 by continuous infusion of 5 mM HCl under the automatic control of a pH-stat system (ETS 822; Radiometer America, Westlake, OH, USA). The volume of titrant infused per unit time was used to quantitate bicarbonate secretion. Measurements were recorded at 5 min intervals and mean values for consecutive 5 or 10 min periods were averaged. The rate of luminal bicarbonate secretion was expressed as µmol·cm–2·h–1. The short circuit current (Isc) was measured in microamperes and converted into µEq·cm–2·h–1. After a 30 min period during which basal parameters were measured, inhibitors were added to both sides of the tissues for another 30 min, as dictated by the experimental design, followed by addition of 5-HT to the serosal side. Electrophysiological parameters and bicarbonate secretion were then recorded for additional 60 min.
Acid-stimulated bicarbonate secretion in vivo
In vivo experiments were performed using a well-validated technique as described previously (18)
, with the bicarbonate concentration of samples being measured via a CO2-sensitive electrode rather than back titration. Mice were anesthetized by i.p. injection of a Hypnorm/Midazolam cocktail (25% Hypnorm plus 25% Midazolam) at a dose of 10 mg/kg. Anesthesia was maintained throughout the experiment via administration of smaller doses (20% of initial) of the same cocktail as needed, as determined by careful monitoring of both respiratory rate and response to tow-pinch. After anesthetization, the abdomen was opened and the duodenum accessed through two small incisions, one just below the ribcage on the left side and the other just below the sternum. Through the first incision the stomach was located and a small incision made just proximal to the pyloric sphincter. Through this incision, a soft polyethylene catheter was inserted into the stomach, gently pushed through the pyloric sphincter, and tied firmly into position with silk suture thread around the outside of the pyloric sphincter, isolating the proximal duodenum (5–10 mm) from the stomach. The junction of the pancreatic duct and the duodenum was located through the incision below the sternum. A small incision was made in the duodenum, and a second polyethylene catheter was inserted and tied into place just proximal to the junction with the pancreatic duct without interrupting the duodenal blood supply. Thus, pancreatic secretions were excluded from the test duodenal segment while the blood supply remained intact. Throughout the duration of the experiment, the duodenum maintained a healthy pink color and was kept moist within the abdominal cavity.
After surgery, the proximal duodenum was perfused (0.15 cc/min) with isotonic saline for 20 min. After this initial washout and recovery period, basal bicarbonate secretion was measured for 20 min. The mouse was then given an i.p. injection of drugs as indicated by the experimental design or control vehicle (DMSO), and bicarbonate secretion was measured for 6 min. The duodenal segment was then perfused with 10 mM HCl in isotonic saline for 5 min. After acidification, the segment was gently flushed to remove any residual acid and allowed a 5 min washout period. Bicarbonate secretion was then measured for an additional 42 min. After each experiment the length of the duodenal test segment was measured in situ to the nearest millimeter. As shown previously, animals could be sustained under these experimental conditions for >2 h. Sample volumes were measured by weight to the nearest 0.01 mg. The amount of bicarbonate in the effluents was quantitated through use of a CO2-sensitive electrode (Thermo Orion, Beverly, MA, USA). The electrode was calibrated prior to each day’s use by constructing a semilogarithmic standard curve using known bicarbonate concentrations. A 1 ml aliquot from each 6 min perfusion period was individually sampled, yielding a reading in mV. This reading was then converted back to an HCO3– concentration as dictated by the previously generated standard curve. In this way, bicarbonate outputs were determined for each 6 min period and expressed as micromoles per centimeter per hour. Stimulated bicarbonate outputs are presented as bicarbonate output over time and as net bicarbonate output (peak minus average basal output).
SCBN cell culture and siRNA treatment
SCBN is a duodenal epithelial cell line with a canine genotype. It grows as polarized confluent monolayers and expresses calcium-dependent chloride secretion (19
, 20)
. SCBN cells were grown according to published methods (20)
. Briefly, cells of passages 23–33 were grown to confluence (5 days) in 75 cm2 flasks. Cells were fed with fresh Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% FBS, L-glutamine, and streptomycin every 2–3 days. After the cells had grown to confluence, they were replated onto 12 mm round coverslips (Warner Instruments Inc., Hamden, CT, USA) and incubated for at least 24 h before use.
For siRNA experiments, a 19-mer siRNA was designed to target NCX1 mRNA specifically (GenBank Accession NM_019268) with a sense strand (5'-3'): GGACCAAGAUGACGAGGAA. This siRNA was screened against the GenBank database and no other significant matches were found. A scrambled sequence siRNA (Neg-siRNA) that had no sequence homology to any known genes was used as a control. siRNA or Neg-siRNA was mixed with Lipofectamine 2000 transfection reagent in serum-free Opti-MEM medium (Invitrogen, San Diego, CA, USA) according to the manufacturer’s protocol and incubated for 20 min at room temperature. The oligomer-Lipofectamine 2000 complexes were then added to cells in culture dishes with complete medium so that the final concentration of the oligomer was 50 nM. Cells were incubated with oligomer complexes in a 5% CO2 incubator at 37°C for 48 h before use.
Western blot analysis of NCX
Proteins were extracted from SCBN cells by homogenization on ice in 500 µl of lysis buffer containing (in mM): 20 Tris·HCl (pH 7.5), 150 NaCl, 1 disodium EDTA, 1 EGTA, 2.5 sodium pyrophosphate, 1ß-glycerophosphate, 1 sodium orthovanadate, 1% Triton X-100, and complete protease inhibitor cocktail (Sigma, St. Louis, MO, USA). Equal amounts of protein, as determined by Lowry assay (Dc assay; Bio-Rad, Hercules, CA, USA), were combined with 2x Laemmli sample buffer and boiled for 5 min. Proteins were separated by electrophoresis on 4–15% SDS-PAGE and transblotted to nitrocellulose membranes. The protein-bound nitrocellulose sheets were first incubated for 30 min at room temperature in blocking buffer containing 2% nonfat dry milk in distilled water. Nitrocellulose sheets were then incubated overnight at 4°C with R3F1 monoclonal antibody (mAb) to NCX1 (Swant, Bellinzona, Switzerland) diluted in blocking buffer (1:5,000), 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 (Ab) 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 enhanced chemiluminescence (ECL) Plus detection reagents (Amersham and Pharmacia, Piscataway, NJ, USA), with NCX1 bands occurring at 120 and 70 kDa.
Analysis of NCX function by digital Ca2+ imaging
[Ca2+]cyt levels in SCBN cells were measured by Fura-2 fluorescence ratio digital imaging as described previously (17)
. Briefly, SCBN cells, grown on coverslips, were loaded with 5 µM Fura-2 acetoxymethyl ester (AM) (dissolved in 0.01% Pluronic F-127 plus 0.1% DMSO in physiological salt solution described below) at room temperature for 50 min, then washed in normal physiological salt solution for at least 20 min. Thereafter, the coverslips with SCBN cells were mounted in a perfusion chamber on a Nikon microscope stage. Cells were initially perfused with a normal physiological salt solution for 5 min, then perfused with the same solution containing 5-HT (100 µM). After that, 5-HT was washed out, followed by a Na+-free solution in which NaCl was replaced with 140 mM LiCl (to maintain isoosmolality). The ratio of Fura-2 fluorescence with excitation at 340 or 380 nm (F340/380) was followed over time and captured using an intensified CCD camera (ICCD200) and a MetaFluor Imaging System (Universal Imaging, Corporation, Downingtown, PA, USA).
Chemicals and solutions
5-HT, pargyline, alaproclate, SB204070, thapsigargin, indomethacin, and glybenclamide were purchased from Sigma. KB-R7943 mesylate and 2-aminoethoxydiphenyl borate (2-APB) were purchased from Tocris Bioscience (Ellisville, MO, USA). Fura-2 AM was from Molecular Probes (Eugene, OR, USA). The other chemicals were obtained from Fisher Scientific (Santa Clara, CA, USA). The mucosal solution used in Ussing chamber experiments contained the following (in mmol/L): Na+ 140, K+ 5.4, Ca2+ 1.2, Mg2+ 1.2, Cl– 120, gluconate 25, and mannitol 10. The serosal solution contained the following (in mmol/L): Na+ 140, K+ 5.4, Ca2+ 1.2, Mg2+ 1.2, Cl– 120, HCO3– 25, HPO42– 2.4, H2PO4– 2.4, glucose (Glc) 10, and indomethacin 0.01.
The physiological salt solution used in digital Ca2+ measurement contained the following (in mmol/L): Na+ 140, K+ 5.0, Ca2+ 2, Cl– 147, HEPES 10, and Glc 10. For the Na+-free solution, Na+ was replaced by Li+. For the Ca2+-free solution, Ca2+ was omitted and 0.5 mM EGTA was added to prevent possible Ca2+ contamination. The osmolalities for all solutions were 284 mOsm/kg.
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 or 1-way ANOVA followed by Newman-Keuls post hoc test, as appropriate.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 8)
. However, the physiological role of 5-HT in the regulation of duodenal ion transport was elusive. It was therefore important to determine whether physiological stimulators can induce 5-HT release from the murine duodenum. Accordingly, we first sought to assess whether acidification of murine duodenal tissue results in 5-HT release. The mucosal side of mouse duodenal segments was exposed for 5 min to acidified Earle balanced salt solution with different pH values and 5-HT release was measured by EIA. As shown in Fig. 1
, acid significantly stimulates 5-HT release from duodenal tissues. However, this response was not concentration dependent, since equivalent levels of 5-HT release were stimulated by solutions with a pH of 3, 4, or 5. This corresponds to the fact that acid-stimulated DMBS is known to be a threshold phenomenon (10
, 11
, 13)
.
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Inhibition of acid-stimulated DMBS in vivo by a selective 5-HT4 receptor antagonist
In our previous Ussing chamber study (7)
we demonstrated that 5-HT-mediated duodenal mucosal ion transport and DMBS were attenuated in a dose-dependent manner by SB204070, a selective 5-HT4 receptor antagonist. The objective of the next experiments was to determine whether acid-stimulated 5-HT release has a physiologically significant role in DMBS in vivo.
Acid-stimulated duodenal HCO3– secretion was measured in whole animal experiments. As shown in Fig. 2A, in control animals, duodenal luminal perfusion with 10 mM HCl resulted in a robust increase in DMBS. Bicarbonate secretion reached a maximum 30 min after HCl stimulation, then declined to near basal levels within the next 10 min. The effects of SB204070 on acid-induced DMBS are also shown in Fig. 2
. Injection of SB204070 (10 mg/kg, i.p.) prior to luminal duodenal acidification abolished the acid-induced increase in DMBS (Fig. 2A
). Net peak HCO3– secretion, calculated from the difference between the baseline and the peak value at 30 min, was used to describe acid-stimulated HCO3– secretion (Fig. 2B
). Although SB204070 did not affect HCO3– secretion at 1 mg/kg, at concentrations of 5 and 10 mg/kg it significantly and dose-dependently attenuated net peak HCO3– secretion. Thus, our findings suggest that 5-HT4 receptors play a physiological role in acid-stimulated duodenal HCO3– secretion in vivo.
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The role of Ca2+ in 5-HT-mediated Isc and DMBS
Although 5-HT has been demonstrated to stimulate intestinal Cl– secretion and DMBS, the underlying mechanisms are still largely unknown. In our previous studies (7)
we provided preliminary data showing that 5-HT-mediated duodenal ion transport was inhibited by verapamil, a Ca2+ channel blocker, suggesting that a Ca2+ signaling pathway might be involved in this response. However, since verapamil at such a high concentration could exert nonspecific effects in addition to blocking plasma membrane Ca2+ channels, additional studies were needed. Moreover, [Ca2+]cyt handling mechanisms in duodenocytes are poorly understood.
To study the mechanisms of 5-HT-mediated Isc and DMBS, a set of experiments was conducted in Ussing chambers in the presence or absence of extracellular Ca2+. As shown in Fig. 3
, in normal Ca2+-containing solutions, 5-HT (100 µM) increased Isc in a biphasic manner: the first initial peak of Isc reached its maximum 2–3 min after the addition of 5-HT, then declined slowly, while a second plateau of increased Isc was sustained for at least 50 min after the addition of 5-HT (Fig. 3A
). However, 5-HT induced DMBS in a monophasic fashion; this response slowly reached a peak value 20 min after the addition of 5-HT, which was then sustained for at least 40 min (Fig. 3B
). However, both 5-HT-mediated Isc and DMBS in Ussing chambers were reduced in Ca2+-free solutions (Fig. 3)
, indicating that extracellular Ca2+ likely plays an important role in 5-HT-mediated Isc and DMBS. To assess tissue viability in Ca2+-free solutions, glucose (25 mM) was added to the mucosal reservoir of tissues in some experiments. As expected, stimulation of Na+-glucose transport by glucose addition increased Isc (data not shown), indicating that the viability of tissues was not influenced by Ca2+-free solutions.
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Having observed that [Ca2+]cyt is important in 5-HT-mediated duodenal ion transport, we focused on this signaling pathway and further studied the [Ca2+]cyt handling mechanisms that might be involved in this response. In an earlier study we demonstrated that plasma membrane Na+/Ca2+ exchanger (NCX) proteins are expressed in the duodenal mucosa and play an important role in carbachol-mediated DMBS by controlling [Ca2+]cyt homeostasis (17)
. Therefore, to determine the involvement of NCX in 5-HT-mediated duodenal ion transport, KB-R7943, a selective inhibitor of the reversed mode of NCX (21)
, was tested in Ussing chamber experiments. As shown in Fig. 4
, the first phase of 5-HT-induced Isc was not affected by KB-R7943 (10 µM). However, the second phase of 5-HT-induced Isc, as well as DMBS, was abolished, strongly suggesting involvement of the reversed mode of NCX in these responses. Next, to determine whether intracellular Ca2+ release via IP3 receptors is involved in 5-HT-mediated duodenal ion transport, a plasma membrane-permeable, selective inhibitor of IP3 receptors, 2-APB, was tested. As shown in Fig. 5
, 2-APB (10 µM) inhibited both phases of 5-HT-induced Isc and net peak DMBS, suggesting the involvement of intracellular Ca2+ release from the endoplasmic reticulum (ER) in these responses.
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Membrane transporters involved in response to 5-HT
To determine whether intermediate Ca2+-activated K+ channels (IKCa) and CFTR are downstream molecular targets for the increase in [Ca2+]cyt induced by 5-HT, the effects of TRAM-34 and glybenclamide, selective inhibitors for IKCa channels and CFTR, were tested in Ussing chamber experiments. As shown in Fig. 6
, TRAM-34 (10 µM) significantly inhibited both phases of 5-HT-induced Isc and abolished DMBS. On the other hand, glybenclamide (300 µM) inhibited the first, but not the second phase of 5-HT-induced Isc, but also significantly reduced DMBS. These data suggest the involvement of IKCa channels and CFTR in 5-HT-induced ion transport in general and DMBS specifically.
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The role of NCX1 in 5-HT-mediated Ca2+ signaling in SCBN cells
To explore more definitively the mechanism of the 5-HT-induced Ca2+ signaling pathway in duodenocytes, we turned to a cell culture model. Thus, we directly measured [Ca2+]cyt levels in 5-HT treated SCBN cells, a well-characterized nontransformed duodenal epithelial crypt cell line. As shown in Fig. 7A, addition of 5-HT (100 µM) evoked a [Ca2+]cyt signal in SCBN cells when the cells were stimulated in normal physiological salt solutions. We next sought to obtain direct evidence that functional NCX activity is present in SCBN 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. Reducing extracellular Na+ from 140 to 0 mM (to drive NCX into its reversed mode and bring extracellular Ca2+ into cells) caused a significant increase in [Ca2+]cyt, suggesting that NCX is functionally expressed in SCBN cells and may be involved in 5-HT-induced [Ca2+]cyt signaling. To test this hypothesis further, we screened for the expression of NCX proteins in SCBN cells. R3F1, an anti-NCX mAb, recognized two proteins with molecular masses of 120 and 70 kDa, corresponding to previous reports of the native NCX1 protein (22
, 23)
(Fig. 8
). The 70 kDa protein represents a short form of NCX1, which may be either a proteolytic cleavage product or a functional, truncated form of NCX1 (23)
. Finally, to assess the involvement of NCX in 5-HT-induced [Ca2+]cyt signaling, SCBN cells were treated with 50 nM of a negative control siRNA or one specific for NCX1 for 48 h. Thereafter, the expression of NCX1 protein was detected by Western blot analysis and the function of NCX1 was determined by digital Ca2+ imaging. As shown in Fig. 8
, the expression of NCX1 proteins was dramatically reduced by pretreatment with the specific NCX1 siRNA but not by the negative control siRNA. Moreover, [Ca2+]cyt signaling induced by either 5-HT or the removal of extracellular Na+ was substantially attenuated by pretreatment with the specific NCX1 siRNA (Fig. 7B, C
). Thus, these findings provide direct evidence not only for the expression and function of NCX1 proteins in duodenal epithelial cells, but also for their involvement in 5-HT-mediated duodenal ion transport via control of [Ca2+]cyt signaling.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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, 8)
we have shown that 5-HT likely releases ACh from the enteric cholinergic neurons and binds to 5-HT4 receptors on duodenal epithelial cells to mediate ion transport via both [Ca2+]cyt- and cAMP-dependent pathways. However, our previous conclusions rested heavily on the use of pharmacological agents. Moreover, the physiological role played by 5-HT in DMBS, and the underlying mechanisms, were largely unknown. Therefore, we conducted experiments at the level of cells, tissues, and intact animals to further explore these important issues. The present study demonstrates that duodenal luminal acidification stimulates the release of 5-HT, which may, at least in part, act on duodenal epithelial cells to mediate DMBS via a Ca2+-dependent signaling pathway.
Although luminal acid is widely considered to be the main physiological stimulus for DMBS, the precise mechanisms underlying acid-stimulated DMBS have not been well elucidated. The secretory response to luminal acid may involve a number of neural and non-neural factors such as ACh, prostaglandin E2 (PGE2), 5-HT, and vasoactive intestinal polypeptide (VIP) (10
, 11
, 24)
. 5-HT is primarily localized to enterochromaffin (EC) cells (4
, 6
7
8)
. Prior work has suggested that luminal acidification was followed by both the release of 5-HT and increased luminal bicarbonate secretion (14
, 16)
. However, it was not known before whether these responses were related in a cause-and-effect manner. Luminal acid may directly stimulate intestinal EC cells to release 5-HT or initially stimulate enteric cholinergic neurons to release ACh, which subsequently mediates 5-HT release from EC cells (25)
. The technique used to evaluate luminal acid-stimulated release of 5-HT into the duodenal lumen has been widely used (14
, 16
, 26)
. We have now demonstrated that luminal acid (pH 3–5) significantly stimulates release of 5-HT into the mucosal bathing solution. The action of acid is specific because the conductance and histology of duodenal tissues were not significantly different from normal tissues after exposure to luminal pHs of 3–6 for 45 min (16)
. More important, SB204070, a selective 5-HT4 receptor antagonist, significantly inhibited acid-stimulated DMBS in vivo. This finding not only supports our previous findings that 5-HT4 receptors are functionally expressed and linked to DMBS in the duodenal mucosa, but also indicates that 5-HT is a potent and physiologically relevant contributor to acid-induced DMBS. Some comment is necessary, however, regarding the relatively high doses of SB204070 that were needed for effect in the current experiments. In a previous study (27)
, SB204070 at 1 mg/kg completely inhibited increased defecation caused by 5-HT4 receptor activation in the mouse. However, in the present study, 10 mg/kg of SB204070 was needed to completely inhibit acid-stimulated DMBS in vivo (Fig. 2)
. This discrepancy may derive from 1) different administered routes of SB204070 (s.c. vs. i.p.), 2) different parameters measured (defecation vs. bicarbonate secretion), and 3) the possible involvement of factors other than 5-HT in acid-stimulated DMBS.
In the tissue and whole animal studies, 5-HT-induced DMBS may occur via two different pathways: 1) an indirect action on enteric nerves to release other secretagogues, and/or 2) a direct action on 5-HT receptors of duodenocytes. In a previous study (8)
, we found that tetrodotoxin (TTX) or atropine partially reduced 5-HT-stimulated DMBS, and 5-HT evoked ACh release from duodenal mucosal preparations, suggesting that a cholinergic neural pathway contributes to the response to 5-HT. However, we assume that 5-HT may also mediate DMBS, at least in part, via a direct action on 5-hydroxytryptamine receptors of duodenocytes because 1) residual 5-hydroxytryptamine-evoked DMBS remained when neurotransmitter release from enteric nerve endings was blocked by TTX or atropine (8)
; 2) indomethacin was included in all Ussing chamber experiments, excluding a possible role for PGE2 in 5-hydroxytryptamine-evoked DMBS; 3) RT-PCR analysis demonstrated the expression of 5-hydroxytryptamine4 receptor mRNA in murine duodenal epithelial cells (7)
; and 4) 5-hydroxytryptamine increased [Ca2+]cyt concentrations in isolated duodenocytes (28)
and SCBN cells. Indeed, the various mechanisms whereby 5-hydroxytryptamine activates DMBS further underscore the physiological significance of this process. Day et al. (29)
have also provided evidence that a 5-hydroxytryptamine3 receptor is present at the mucosal level that mediates rat distal colonic Cl– secretion by a nonneural pathway, supporting our notion that 5-hydroxytryptamine may act directly on 5-hydroxytryptamine receptors of duodenocytes.
Since the precise mechanisms underlying 5-hydroxytryptamine-mediated intestinal Cl– and HCO3– secretion had not been elucidated, we designed studies to address this important issue. In our previous study we found that 5-hydroxytryptamine-mediated duodenal ion transport was inhibited by verapamil (7)
, suggesting that this response might be partially mediated by a [Ca2+]cyt signaling pathway. In the present study, extracellular Ca2+ depletion abolished 5-hydroxytryptamine-induced Isc and DMBS, indicating that [Ca2+]cyt is heavily involved in these responses. Therefore, the [Ca2+]cyt signaling pathway became the focus of our further studies. In colonocytes, stimulation with Cl– secretagogues increases [Ca2+]cyt via release of intracellular Ca2+ from the ER, as well as Ca2+ entry across the plasma membrane (30
31
32
33
34)
. Only the first of these Ca2+ sources is likely to be functionally significant to mediate Cl– secretion (35
36
37)
. By analogy, we tested the Ca2+ source involved in 5-hydroxytryptamine-mediated duodenal Isc and DMBS. First, we found that blockade of intracellular Ca2+ release via IP3 receptors from the ER by 2-APB significantly inhibited transport responses to 5-hydroxytryptamine. Second, inhibition of the reversed mode of NCX by KB-R7943 selectively attenuated the second phase of the Isc and DMBS responses to 5-hydroxytryptamine. However, the first phase of the Isc response to 5-hydroxytryptamine was not affected by KB-R7943, and may be ascribable to Cl– secretion (35
, 37)
. Taken together, our data suggest that 5-hydroxytryptamine may mediate Ca2+-dependent duodenal Cl– secretion by inducing release of intracellular Ca2+ from the ER, whereas its effect on DMBS may be mediated by the stimulation of extracellular Ca2+ entry via the reversed mode of NCX (17
, 33
, 35
, 37
, 38)
.
In a previous study we demonstrated that NCX proteins play a critical role in the regulation of Ca2+-dependent duodenal mucosal ion transport and DMBS resulting from stimulation of muscarinic receptors (17)
. To further evaluate 5-hydroxytryptamine-mediated [Ca2+]cyt signaling and the involvement of NCX, we employed digital Ca2+ imaging to directly measure [Ca2+]cyt levels in SCBN cells. We chose SCBN cells for our present study because 1) the cell line is a well-characterized nontransformed duodenal epithelial crypt cell line that forms electrically tight monolayers (19
, 20)
; 2) SCBN cells functionally express CFTR channels and have been widely used in the study of Ca2+-dependent Cl– secretion (20)
; and 3) SCBN cells secrete HCO3–, as demonstrated by us (unpublished observations) and others (20)
. All these features make SCBN a valuable cell model for our studies of 5-hydroxytryptamine-mediated [Ca2+]cyt signaling in duodenal epithelia. Consistent with results obtained from freshly isolated human and rat duodenal enterocytes (28)
, we have shown that 5-hydroxytryptamine increases [Ca2+]cyt in SCBN cells. Further, as we showed that NCX proteins are functionally expressed in SCBN cells, we hypothesized that NCX may be involved in this response. Indeed, we found that treatment with a specific NCX1 siRNA greatly decreased the [Ca2+]cyt response to both 5-hydroxytryptamine and to the removal of extracellular Na+, further supporting our hypothesis that the reversed mode of NCX1 is involved in 5-hydroxytryptamine-mediated [Ca2+]cyt signaling. Taken together, these data indicate that NCX plays an important role in 5-hydroxytryptamine-mediated [Ca2+]cyt signaling, and therefore Ca2+-dependent DMBS in response to luminal acidification. These results are consistent with our previous observations that NCX is involved in carbachol-induced Ca2+-mediated DMBS (17)
, providing further evidence for our notion that NCX proteins are crucial universal regulators of [Ca2+]cyt signaling pathways in duodenal epithelial cells.
Besides the [Ca2+]cyt signaling pathway, other signaling pathways or mechanisms may be involved in 5-hydroxytryptamine-mediated duodenal ion transport. As we demonstrated earlier, the cAMP signaling pathway may also play a role in the duodenal response to 5-hydroxytryptamine (7)
, but its involvement needs to be confirmed by direct measurement of cAMP concentrations in duodenocytes. 5-Hydroxytryptamine might also inhibit a Na+/H+ exchanger (NHE), which would subsequently activate DMBS. 5-Hydroxytryptamine has been shown to inhibit NHE activity in Caco-2 cells by acting on 5-hydroxytryptamine4 receptors (39)
, and inhibition of NHE is known to increase murine DMBS (40
, 41)
. However, there is currently no direct evidence to support the idea that 5-hydroxytryptamine mediates DMBS via inhibition of NHE in duodenocytes. All these proposed mechanisms merit further study, but are beyond the scope of the present work.
While [Ca2+]cyt has long been considered an important regulator of duodenal mucosal HCO3– secretion, the downstream molecular effectors of Ca2+-mediated DMBS have remained elusive. CFTR, IKCa, Cl–/HCO3– exchangers, and Ca2+-activated Cl– channels (CaCC) are candidates for the molecular targets by which elevated [Ca2+]cyt may mediate DMBS (6
, 10
, 42
, 43)
. We have shown in our previous Ussing chamber studies that Cl– removal does not alter 5-hydroxytryptamine-mediated DMBS, implying that CFTR, rather than a Cl–/HCO3– exchanger, is involved in this mechanism (8)
. In addition, activation of CFTR alone is insufficient to evoke colonic transepithelial Cl– secretion; activation of basolateral membrane K+ channels is a necessary component of this secretory response (44
, 45)
. We therefore studied whether IKCa channels and CFTR play roles in 5-hydroxytryptamine-mediated DMBS as they do in Ca2+-mediated colonic Cl– secretion (46
, 47)
. We provide evidence that 5-hydroxytryptamine induces increases in [Ca2+]cyt, which may activate basolateral IKCa and thereby stimulate DMBS through apical CFTR channels. However, the molecular identity of the pathways involved in 5-hydroxytryptamine-mediated DMBS requires additional studies.
In conclusion, the present study shows that 5-hydroxytryptamine released from the duodenal mucosa in response to luminal acid stimulation may act on 5-hydroxytryptamine4 receptors located on epithelial cells to evoke DMBS in a Ca2+-dependent fashion in addition to an indirect effect mediated by enteric nerves. The plasma membrane NCX transporter as well as IKCa and CFTR channels are likely involved in the molecular mechanisms of Ca2+-mediated DMBS evoked by 5-hydroxytryptamine. A fuller understanding of the physiological role of 5-hydroxytryptamine in duodenal ion transport may help to further illuminate the precise mechanisms of acid-stimulated DMBS in health, as well as ways to intervene in states where it is deficient, such as peptic ulcer disease and cystic fibrosis.
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ACKNOWLEDGMENTS |
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Received for publication April 25, 2006. Accepted for publication July 17, 2006.
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