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Am J Physiol Gastrointest Liver Physiol 286: G444-G451, 2004. First published October 23, 2003; doi:10.1152/ajpgi.00105.2003
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HORMONES AND SIGNALING

5-HT induces duodenal mucosal bicarbonate secretion via cAMP- and Ca2+-dependent signaling pathways and 5-HT4 receptors in mice

Bi-Guang Tuo, Zachary Sellers, Petra Paulus, Kim E. Barrett, and Jon I. Isenberg

Division of Gastroenterology, Department of Medicine, School of Medicine, University of California, San Diego, California 92103

Submitted 6 March 2003 ; accepted in final form 18 October 2003


   ABSTRACT
TOP
 ABSTRACT
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
In previous studies, we have found that 5-hydroxytryptamine (5-HT) is a potent stimulant of duodenal mucosal bicarbonate secretion (DMBS) in mice. The aim of the present study was to determine the intracellular signaling pathways and 5-HT receptor subtypes involved in 5-HT-induced DMBS. Bicarbonate secretion by murine duodenal mucosa was examined in vitro in Ussing chambers. 5-HT receptor involvement in DMBS was inferred from pharmacological studies by using selective 5-HT receptor antagonists and agonists. The expression of 5-HT4 receptor mRNA in duodenal mucosa and epithelial cells was analyzed by RT-PCR. cAMP-dependent signaling pathway inhibitors MDL-12330A, Rp-cAMP, and H-89 and Ca2+-dependent signaling pathway inhibitors verapamil and W-13 markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and short-circuit current (Isc), whereas cGMP-dependent signaling pathway inhibitors NS-2028 and KT-5823 failed to alter these responses. Both SB-204070 and high-dose ICS-205930 (selective 5-HT4 receptor antagonists) markedly inhibited 5-HT-stimulated bicarbonate secretion and Isc, whereas methiothepine (5-HT1 receptor antagonist), ketanserin (5-HT2 receptor antagonist), and a low concentration of ICS-205930 (5-HT3 receptor antagonist) had no effect. RS-67506 (partial 5-HT4 receptor agonist) concentration-dependently increased bicarbonate secretion and Isc, whereas 5-carboxamidotryptamine (5-HT1 receptor agonist), {alpha}-methyl-5-HT (5-HT2 receptor agonist), and phenylbiguanide (5-HT3 receptor agonist) did not significantly increase bicarbonate secretion or Isc. RT-PCR analysis confirmed the expression of 5-HT4 receptor mRNA in murine duodenal mucosa and epithelial cells. These results demonstrate that 5-HT regulates DMBS via both cAMP- and Ca2+-dependent signaling pathways and 5-HT4 receptors located in the duodenal mucosa and/or epithelial cells.

serotonin; duodenal transport

DUODENAL MUCOSAL BICARBONATE secretion plays an important role in duodenal mucosal protection against acid-peptic injury. A number of neural and humoral factors [such as prostaglandin E2, vasoactive intestinal peptide (48), dopamine (13), somatostatin (34), luminal acid (19), etc.] are known to be involved in regulation of this physiological process.

5-Hydroxytryptamine (5-HT) is widely distributed in the gastrointestinal tract. More than 90% of 5-HT is localized within the enterochromaffin (EC) cells of gastrointestinal mucosal epithelia and enteric neurons (15, 42). 5-HT is an important neurotransmitter and intercellular messenger. A variety of neural, humoral, and intraluminal stimuli have been shown to release 5-HT from EC cells (47). 5-HT has been shown to participate in the regulation of gastrointestinal motility (31), gastric acid secretion (27), pancreatic secretion (44), and intestinal chloride secretion (36). Moreover, we recently showed that 5-HT is a potent stimulant of duodenal bicarbonate secretion (46), but the signal transduction pathway(s) and the 5-HT receptor subtypes involved in the action of 5-HT on duodenal bicarbonate secretion were unknown. However, elucidation of such information is important given clinical usage of specific 5-HT receptor antagonists.

The 5-HT receptor population is comprised of several subtypes. Through pharmacological studies and molecular cloning (21, 22, 37), at least seven families of 5-HT receptor subtypes, including 5-HT1, 5-HT2, 5-HT3, 5-HT4, 5-HT5, 5-HT6, and 5-HT7, have been discovered. 5-HT and its receptors are found both in the central and peripheral nervous systems, as well as in a number of nonneuronal tissues in the gastrointestinal tract, cardiovascular system, and blood. 5-HT5, 5-HT6, and 5-HT7 receptors are cloned novel receptors with as yet undefined physiological correlates. On the other hand, according to the current classification, four main subtypes of 5-HT receptors, 5-HT1, 5-HT2, 5-HT3, and 5-HT4, can be distinguished functionally (21), and these four 5-HT receptors are also recognized to exist in the gastrointestinal tract. Each subtype of 5-HT receptors is involved in various regulatory functions in different organs (21, 22).

The aim of the present study was to further characterize the signal transduction pathway(s) and 5-HT receptor(s) that mediate duodenal mucosal bicarbonate secretion in mice.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Chemicals and solutions. 5-HT, 5-carboxamidotryptamine (5-CT), {alpha}-methyl-5-HT, methiothepine, ketanserin, ICS-205930, SB-204070, MDL-12330A, H-89, Rp-cAMP, verapamil, W-13, and KN-62 were purchased from Sigma (St. Louis, MO). 1-Phenylbiguanide and RS-67506 were from Tocris (Ellisville, MO). KT-5823 and NS-2028 were from Calibochem (San Diego, CA). All other chemicals were obtained from Fisher Scientific (Santa Clara, CA). For Ussing chamber studies, the mucosal solution 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 (in mM) 140 Na+, 5.4 K+, 1.2 Ca2+, 1.2 Mg2+, 120 Cl-, 25 , 2.4 , 2.4 , 10 glucose, and 0.001 indomethacin. The osmolalities for both solutions were ~284 osmol/kg H2O.

Animal preparation. Experiments were performed on White Swiss mice (6-10 wk of age). All studies were approved by the University of California-San Diego Committee on Investigations Involving Animals. 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 experiments, the mice were deprived of food and water for at least 1 h. After anesthesia with a cocktail of hypnorm and midazolam (10 ml/kg ip; Janssen Pharmaceutica, Beerse, Belgium), the abdomen was opened by a midline incision. The proximal duodenum (a portion stretching approximately from 2 mm distal to the pylorus to the common bile duct ampulla) was removed and immediately placed in ice-cold isosmolar mannitol and indomethacin (1 µM) solution (to suppress trauma-induced prostaglandin release). The anesthetized mice were then killed by cervical dislocation. The duodenum was opened along the mesenteric border and stripped of external serosal and muscle layers by sharp dissection in the abovementioned ice-cold isosmolar mannitol and indomethacin solution.

Ussing chamber experiments. Mucosa was mounted between two Lucite half-chambers with an exposed area of 0.1 cm2 and placed in an Ussing chamber. Duodenal tissue from each animal was randomly divided among three or four chambers for experiments. The mucosal side was bathed with unbuffered bicarbonate-free modified Ringer solution circulated by a gas lift with 100% O2. The serosal side was bathed with modified buffered Ringer solution (pH 7.4) containing 25 mM and gassed with 95% O2-5% CO2. Each bath contained 3.0 ml of the respective solution maintained at 37°C by a heated water jacket. Experiments were performed under continuous short-circuited conditions (voltage-current clamp model VCC 600; Physiologic Instruments, San Diego, CA) to maintain the electrical potential difference at zero, except for a brief period (<2 s) at each time point when the open-circuit potential difference was measured. Luminal pH was maintained at 7.40 by the continuous infusion of 5 mM HCl under the automatic control of a pH-stat system (model ETS 822; Radiometer America, Westlake, OH). The volume of the titrant infused per unit time was used to quantitate bicarbonate secretion. These measurements were recorded at 5-min intervals, and mean values for consecutive 10-min periods were calculated. The rate of luminal bicarbonate secretion is expressed as micromoles per square centimeter per hour. Short-circuit current (Isc) was measured in microamperes (µA) and converted into microequivalents per square centimeter per hour, and potential difference was measured in millivolts.

Effect of cAMP-, Ca2+-, and cGMP-dependent signaling pathway inhibitors on 5-HT-stimulated duodenal mucosal bicarbonate secretion and Isc. To explore the signaling pathways involved in the action of 5-HT, after a 20-min measurement of basal parameters, cAMP-dependent signaling pathway inhibitors MDL-12330A (10-5 M), Rp-cAMP (10-4 M), or H-89 (10-5 M); Ca2+-dependent signaling pathway inhibitors verapamil (5x10-5 M), W-13 (5x10-5 M), or KN-62 (10-5 M); or cGMP-dependent signaling pathway inhibitors NS-2028 (10-5 M) or KT-5823 (5x10-6 M) were added to the serosal side. Thirty minutes later, 5-HT was added to the serosal side. Duodenal bicarbonate secretion and Isc during the 60-min period after the addition of 5-HT were then determined.

Effect of 5-HT receptor antagonists on 5-HT-stimulated duodenal mucosal bicarbonate secretion and Isc. These studies were performed to determine 5-HT receptor subtype(s) involved in the action of 5-HT. After a 20-min measurement of basal parameters, one of the 5-HT receptor antagonists, methiothepine (10-5 M), ketanserin (10-6 M), ICS-205930 (10-7 or 10-5 M), SB-204070 (10-5 M), or vehicular control, was added to the serosal side at 30 min before the addition of 5-HT (10-4 M).

Effect of 5-HT receptor agonists on duodenal mucosal bicarbonate secretion and Isc. These studies were performed to examine the actions of 5-HT receptor agonists on duodenal bicarbonate secretion and Isc. After a 20-min measurement of basal parameters, one of the 5-HT receptor agonists, 5-CT (10-4 M), {alpha}-methyl-5-HT (10-4 M), 1-phenylbiguanide (10-4 M), RS-67506 (10-4 M), or vehicular control was added to the serosal side. Recordings were then made over the subsequent 60-min test period as described above for 5-HT.

RNA extraction and RT-PCR. Expression of 5-HT4 receptor mRNA in duodenal mucosa and epithelial cells was studied by RT-PCR. Segments of duodenal mucosae (~10 mg) were dissected free of seromuscular layers as described above for Ussing chamber experiments. For some studies, duodenal epithelial cells were further isolated according to a previously validated method (1). In brief, a 7-mm segment of proximal duodenum was excised. The lumen was rinsed to remove the mucous layer. The luminal surface was then exposed to an EDTA-containing solution and vortexed briefly to facilitate cell detachment. Duodenal mucosa and isolated duodenal epithelial cells were homogenized and lysed separately. Total RNA was extracted by using the RNeasy mini kit (Qiagen). RNA was treated with RNase-free DNase to remove any contaminating genomic DNA. Total RNA from duodenal mucosa or epithelial cells was converted into single-stranded cDNA by using Sensiscript reverse transcriptase (Qiagen) with oligo(dT)12-18 primer (0.5 µg/µl). The PCR reaction was carried out in a 50-µl reaction mixture by using Taq PCR Core Kit (Qiagen). The primer pairs for 5-HT4 receptor were sense 1 (5'-ATG GTC AAC AAG CCC TAT GC-3') and antisense 1 (AGG AAG GCA CGT CTG AAA GA-3'), corresponding, respectively, to bases 561-580 and 954-973 of the mus musculus 5-HT4 receptor cDNA (GenBank accession no. NM_008313 [GenBank] ). The final concentration of the primers was 0.2 µM. After denaturation at 94°C for 3 min, 35 cycles of PCR amplification were performed (94°C, 40 s; 55°C, 60 s; 72°C, 80 s). The last cycle included 10 min of final extension at 72°C. Two types of negative control (without template DNA and where reverse transcriptase was omitted) were included in every experiment, in which no PCR product was detected. Ten microliters of each PCR product was electrophoresed on a 1.2% agarose gel containing ethidium bromide. Resulting gel bands were visualized in a UV transilluminator, and images were captured by using a camera. Identification of 5-HT4 receptor expression was based on observation of an RT-PCR product of appropriate size (~412 bp).

Statistics. All results are expressed as means ± SE. Net peak bicarbonate and net peak Isc both refer to stimulated peak responses minus basal levels. Data were analyzed by one-way ANOVA followed by Newman-Keuls post hoc test. P < 0.05 was considered statistically significant.


   RESULTS
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
Effect of cAMP-, Ca2+-, and cGMP-dependent signaling pathway inhibitors on 5-HT-stimulated duodenal mucosal bicarbonate secretion and Isc. We first sought to identify signaling pathway(s) that might underlie the effect of 5-HT on duodenal mucosal bicarbonate secretion. To accomplish this, a pharmacological approach was taken. Thus the adenylyl cyclase inhibitor MDL-12330A (10-5 M), the cAMP antagonist Rp-cAMP (10-4 M), and the cAMP-dependent PKA inhibitor H-89 (10-5M) all markedly inhibited 5-HT-stimulated duodenal bicarbonate secretion and Isc (P < 0.001) (Fig. 1). Like-wise, the Ca2+ channel blocker verapamil (5 x 10-5 M) and the calmodulin antagonist W-13 (5 x 10-5 M) also markedly inhibited 5-HT-stimulated duodenal bicarbonate secretion and Isc (P < 0.01), whereas the Ca2+/camodulin-dependent protein kinase (CaM-PK) inhibitor KN-62 did not reduce 5-HT-stimulated duodenal bicarbonate secretion or Isc (Fig. 2). In contrast, neither the guanylyl cyclase inhibitor NS-2028 (10-5 M) nor the cGMP-dependent PKG inhibitor KT-5823 (5 x 10-6 M), which can effectively inhibit guanylyl cyclase and PKG, respectively, at these concentrations (16, 43), reduced 5-HT-stimulated duodenal bicarbonate secretion or Isc (Fig. 3).



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  		Fig. 1. Role of cAMP-dependent signaling pathways in 5-hydroxytryptamine (5-HT)-stimulated duodenal bicarbonate secretion (A) and short-circuit current (Isc) (B) in murine duodenum. Adenylyl cyclase inhibitor MDL-12330A (10-5 M), cAMP antagonist Rp-cAMP (10-4 M), PKA inhibitor H-89 (10-5 M), or control vehicle was added to the serosal side 30 min before 5-HT (10-4 M). Values are expressed as means ± SE; n >= 10 in each series. These drugs markedly inhibited 5-HT-stimulated bicarbonate secretion and Isc. ***P < 0.001 (compared with control) by one-way ANOVA with Student-Newman-Keuls post hoc test.

 


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  		Fig. 2. Role of Ca2+-dependent signaling pathways in 5-HT-stimulated duodenal bicarbonate secretion (A) and Isc (B) in murine duodenum. The Ca2+ channel blocker verapamil (5 x 10-5 M), calmodulin inhibitor W-13 (5 x 10-5 M), CaM-PK inhibitor KN-62 (10-5 M), or control vehicle was added to the serosal side 30 min before 5-HT (10-4 M). Values are expressed as means ± SE; n >= 10 in each series. Verapamil and W-13, but not KN-62, markedly inhibited 5-HT-stimulated bicarbonate secretion and Isc. **P < 0.01, ***P < 0.001 (compared with control) by one-way ANOVA with Student-Newman-Keuls post hoc test.

 


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  		Fig. 3. Role of cGMP-dependent signaling pathways in 5-HT-stimulated duodenal bicarbonate secretion (A) and Isc (B) in murine duodenum. The guanylyl cyclase inhibitor NS-2028 (10-5 M), PKG inhibitor KT-5823 (5 x 10-6 M), or control vehicle was added to the serosal side 30 min before 5-HT (10-4 M). Values are expressed as means ± SE; n >= 10 in each series. Neither NS-2028 nor KT-5823 altered 5-HT-stimulated bicarbonate secretion and Isc.

 

Effects of 5-HT receptor antagonists on 5-HT-stimulated duodenal mucosal bicarbonate secretion and Isc. The effects of 5-HT receptor antagonists on 5-HT-stimulated duodenal bicarbonate and Isc are shown in Fig. 4. The 5-HT1 receptor antagonist methiothepine (10-5 M), which can effectively inhibit 5-HT1 receptors at this concentration (33), the 5-HT2 receptor antagonist ketanserin (10-6 M), which can effectively inhibit 5-HT2 receptors at this concentration (33), and a low concentration of ICS-205930 (10-7 M), which acts selectively as a 5-HT3 receptor antagonist when employed at a final concentration of 10-7 M (15, 17), had no effect on 5-HT-stimulated duodenal bicarbonate secretion or Isc. On the other hand, a high concentration of ICS-205930 (10-5 M), at which this drug is known to antagonize the 5-HT4 as well as the 5-HT3 receptor (18), markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and Isc (P < 0.001). Similarly, SB-204070 (10-5 M), a highly selective 5-HT4 receptor antagonist, also markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and Isc (P < 0.001). Moreover, the inhibitory effect of SB-204070 on 5-HT-stimulated duodenal bicarbonate secretion and Isc was concentration dependent (P < 0.0001) (Fig. 5). SB-204070 (10-6 M) produced a significant inhibitory effect on the action of 5-HT (P < 0.05). At the highest concentration of SB-204070 (10-4 M), it reduced 5-HT-stimulated duodenal bicarbonate secretion by 73.8% and Isc by 76.9%. The concentration of SB-204070 required to inhibit bicarbonate secretion and Isc was therefore essentially equivalent. The IC50s for bicarbonate secretion and Isc were ~1.1 x 10-6 M and 0.23 x 10-6 M, respectively. In addition, none of the antagonists studied altered basal duodenal bicarbonate secretion or Isc by themselves (data not shown).



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  		Fig. 4. Effects of 5-HT receptor antagonists on 5-HT-stimulated duodenal bicarbonate secretion (A) and Isc (B) in murine duodenum. Methiothepine (10-5 M), ketanserin (10-6 M), ICS-205930 (10-7 M or 10-5 M), or SB-204070 (10-5 M) was added into the serosal side 30 min before 5-HT (10-4 M). Values are expressed as means ± SE; n = 10 in each series. SB-204070 and ICS-205930 (10-5 M) markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and Isc. Neither ISC-205930 (10-7 M), ketanserin, nor methiothepine significantly altered 5-HT-stimulated duodenal bicarbonate secretion and Isc. ***P < 0.001 (compared with 5-HT only) by one-way ANOVA with Student-Newman-Keuls post hoc test.

 


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  		Fig. 5. Effects of graded concentrations of the 5-HT4 receptor antagonist SB-204070 on 5-HT-stimulated duodenal bicarbonate secretion (A) and Isc (B) in murine duodenum. Each concentration was tested independently in a separate tissue and added to the serosal side 30 min before 5-HT (10-4 M). The control bar represents the response to 5-HT alone. Values are expressed as means ± SE; n = 10 in each series. SB-204070 reduced 5-HT-stimulated duodenal bicarbonate secretion and Isc in a concentration-dependent manner (P < 0.0001). SB-204070 (10-6 M) produced a significant inhibitory effect on the action of 5-HT. *P < 0.05, **P < 0.01, ***P < 0.001 (compared with control) by one-way ANOVA with Student-Newman-Keuls post hoc test.

 

Effect of 5-HT receptor agonists on duodenal mucosal bicarbonate secretion and Isc. To examine fully the receptors involved in mediating 5-HT-induced bicarbonate secretion, we studied the effect of 5-HT receptor agonists on duodenal bicarbonate secretion. The effect of 5-HT receptor agonists on duodenal bicarbonate secretion and Isc is shown in Fig. 6. The 5-HT1 receptor agonist 5-CT (10-4M), 5-HT2 receptor agonist {alpha}-methyl-5-HT (10-4 M), and 5-HT3 receptor agonist phenylbiguanide (10-4 M) did not significantly stimulate either duodenal bicarbonate secretion or Isc (P > 0.05, compared with control). These agonists have previously been shown to activate relevant 5-HT receptors when used at the concentrations employed here (33, 45). On the other hand, RS-67506 (10-4 M), a partial agonist of 5-HT4 receptor, markedly stimulated duodenal bicarbonate secretion and Isc (P < 0.001). The effect of RS-67506 on duodenal bicarbonate secretion and Isc was concentration dependent with EC50 of 9.2 x 10-6 M and 23.5 x 10-6 M, respectively, for elevating bicarbonate secretion and Isc, respectively (Fig. 7). RS-67506 was less efficacious than 5-HT. The net maximal increases in duodenal bicarbonate secretion and Isc induced by RS-67506 (10-4 M) were equivalent to 67.4 and 49.6% of those evoked by the same concentration of 5-HT, respectively (Fig. 6).



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  		Fig. 6. Effects of 5-HT receptor agonists on basal duodenal bicarbonate secretion (A) and Isc (B) in murine duodenum. After a 20-min measurement of basal values, 5-carboxamidotryptamine (5-CT; 10-4 M), {alpha}-methyl-5-HT (10-4 M), phenylbiguanide (10-4 M), RS-67506 (10-4 M), 5-HT (10-4 M), or the control vehicle was added to the serosal side of a separate tissue. Values are expressed as means ± SE; n = 10 in each series. RS-67506 markedly stimulated duodenal bicarbonate secretion and Isc, whereas 5-CT, {alpha}-methyl-5-HT, and phenylbiguanide did not significantly stimulate duodenal bicarbonate secretion and Isc. ***P < 0.001 (compared with control) by one-way ANOVA with Student-Newman-Keuls post hoc test.

 


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  		Fig. 7. Concentration-dependent effects of the 5-HT4 receptor agonist RS-67506 on duodenal bicarbonate secretion (A) and Isc (B) in murine duodenum. Each concentration was tested independently in a separate tissue. Values are expressed as means ± SE; n = 10 in each series. RS-67506 concentration-dependently stimulated duodenal bicarbonate secretion and Isc (P < 0.0001). RS-67506 (10-5 M) produced a significant response, and the maximal response occurred at 10-4 M RS-67506. *P < 0.05, ***P < 0.001 (compared with control) by one-way ANOVA with Student-Newman-Keuls post hoc test.

 

Expression of 5-HT4 receptor mRNA in murine duodenal mucosa and epithelial cells. The pharmacological studies described above indicated that 5-HT4 receptors mediate the effect of 5-HT on both duodenal mucosal bicarbonate secretion and Isc. To confirm that this receptor subtype is present in the murine duodenum, we assessed the expression of 5-HT4 receptor mRNA in duodenal mucosa and epithelial cells by using RT-PCR. Figure 8 shows a typical ethidium bromide-stained gel for 5-HT4 receptor RT-PCR products in duodenal mucosa and epithelial cells. Prominent bands are present for 5-HT4 receptors. The location of the bands for 5-HT4 receptors corresponds to the expected amplified cDNA fragment size based on the choice of oligonucleotide primers. 5-HT4 receptor RT-PCR product was detected, indicating that functional 5-HT4 receptors likely exist in murine duodenal mucosa and epithelial cells.



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  		Fig. 8. RT-PCR analysis of 5-HT4 receptor mRNA in murine duodenal mucosa and epithelial cells. Duodenal mucosa or isolated epithelial cells were homogenized. Total RNA was extracted and reverse transcribed into cDNA as described in MATERIALS AND METHODS. Specific primers for 5-HT4 receptor were used to amplify a DNA fragment. The DNA generated was analyzed on an agarose gel and stained with ethidium bromide. Lane 1: DNA size marker. Lane 2: 5-HT4 receptor product from duodenal mucosa. Lane 3: 5-HT4 receptor product from duodenal epithelial cells. Lane 4: negative control with no DNA template. Lane 5: negative control with no reverse transcriptase. The location of bands for the 5-HT4 receptor corresponds to expected amplified cDNA fragment size (~412 bp).

 


   DISCUSSION
TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
In a previous study (46), we found that 5-HT is a potent stimulant of duodenal mucosal bicarbonate secretion. However, the intracellular signaling transduction pathways underlying the 5-HT-induced duodenal secretory response had not been investigated. Bicarbonate is secreted in response to a number of agonists via the activation of various signal transduction pathways. cAMP, Ca2+, and cGMP are three important intracellular modulators of duodenal mucosal bicarbonate secretion (12). We thus examined the role of cAMP-, Ca2+-, and cGMP-dependent signaling pathways in 5-HT-induced duodenal bicarbonate secretion. Our results demonstrated that an adenylyl cyclase inhibitor, a cAMP antagonist, and a PKA inhibitor markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and Isc. Likewise, a Ca2+ channel blocker and a calmodulin inhibitor, but not a CaM-PK inhibitor, also markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and Isc. However, a guanylyl cyclase inhibitor and a PKG inhibitor failed to alter 5-HT-stimulated bicarbonate secretion and Isc. These results indicated that 5-HT likely stimulates duodenal mucosal bicarbonate secretion via both cAMP- and Ca2+-dependent signaling pathways, but the Ca2+ signaling pathway is independent of CaM-PK. cAMP is a predominant signaling pathway in duodenal bicarbonate secretion, and indeed inhibition of this pathway had a greater effect on bicarbonate secretion than did modulation of calcium-related signaling. In a study of epididymal epithelial cells, Leung et al. (29) found that 5-HT increased intracellular cAMP concentrations and stimulated anion secretion via the activation of cAMP-dependent signal transduction pathways. 5-HT also increased cytosolic Ca2+ in rat heart endothelial cells (26) and stimulated net Ca2+ flux in the ventricular muscle of a mollusc (10). In addition, in our previous study in this model (46), we found that 5-HT stimulated the release of ACh from duodenal mucosa in mice. ACh is known to stimulate cell secretion mainly by increasing cellular Ca2+ levels (11, 32). We therefore conclude that 5-HT stimulates duodenal bicarbonate secretion via increases in duodenal cAMP and Ca2+ concentrations but acts independently of cGMP-dependent pathways. However, it remains unknown whether these signaling events occur at the level of the epithelium exclusively or also involve indirect pathways requiring additional cell types.

We further studied the 5-HT receptor subtypes involved in the action of 5-HT. This study demonstrated that the 5-HT1 receptor antagonist methiothepine or the 5-HT2 receptor antagonist ketanserin failed to inhibit 5-HT-induced duodenal bicarbonate secretion or Isc. Likewise, a low concentration of ICS-205930 (10-7 M), at which it is known to act as a 5-HT3 receptor antagonist (39, 41), had no effect on 5-HT-induced duodenal bicarbonate secretion or Isc. However, a high concentration of ICS-205930 (10-5 M), at which it is known to antagonize 5-HT4 receptors as well as 5-HT3 receptors (18, 23), markedly reduced 5-HT-stimulated duodenal bicarbonate and Isc. SB-204070 (10-5 M), a specific 5-HT4 receptor antagonist, also markedly reduced 5-HT-stimulated duodenal bicarbonate secretion and Isc. In addition, the 5-HT1 receptor agonist 5-CT, 5-HT2 receptor agonist {alpha}-methyl-5-HT, and 5-HT3 receptor agonist phenylbiguanide did not significantly stimulate duodenal bicarbonate secretion or Isc. In contrast, a partial 5-HT4 receptor agonist, RS-67506, concentration-dependently stimulated duodenal bicarbonate secretion and Isc. These findings indicated that 5-HT4 receptor likely mediates 5-HT-induced duodenal mucosal bicarbonate secretion and Isc.

Specific mechanism(s) whereby 5-HT binding to 5-HT4 receptors activates duodenal mucosal bicarbonate secretion are not yet clear. In our previous study using the same model of murine duodenum in vitro (46), we found that 5-HT-stimulated duodenal bicarbonate secretion was partially inhibited by the neurotoxin TTX and cholinergic receptor antagonist atropine, indicating that 5-HT-stimulated duodenal bicarbonate secretion in mice involves both neural and nonneural pathways. For the case of intestinal chloride secretion, some studies showed that 5-HT4 receptors mediate chloride secretion via a nonneural pathway (5-7), whereas the 5-HT3 receptor appears to be the primary receptor mediating effects dependent on the enteric nervous system (5). However, in the guinea pig isolated ileal mucosal preparation, 5-HT4 receptor mediated chloride secretion via TTX-sensitive mechanism (28). These findings suggest that secretory responses to 5-HT4 receptor ligation are mediated both neuronally and nonneuronally. In this study, we demonstrated that the 5-HT4 receptor alone appears to mediate 5-HT-stimulated duodenal bicarbonate secretion. Considering our previous results in this model (46), we can speculate that the 5-HT4 receptor mediates 5-HT-stimulated duodenal bicarbonate secretion in mice by both neural and nonneural pathways.

Some studies have shown that the stimulation of 5-HT4 receptors facilitates the release of ACh from not only the central nervous system (8, 38) but also from enteric nerve terminals (24, 25). In our previous study in this model (46), we also found that 5-HT stimulates the release of ACh from duodenal mucosa in mice. ACh is an important neurotransmitter and plays an important role in the regulation of both duodenal bicarbonate secretion (20) and intestinal chloride secretion (9). Therefore, it is possible that the 5-HT4 receptor mediates duodenal mucosal bicarbonate by stimulating localized mucosal cholinergic neurons. On the other hand, the 5-HT4 receptor is a member of the superfamily of G proteincoupled receptors and is positively coupled to adenylate cyclase (4, 18). It has been demonstrated that the activation of 5-HT4 receptors augments adenylate cyclase activity and elevates cAMP levels in rat esophagus (14) and human colon (30). Likewise, in isolated mucosal cells from rat distal colon, the study of Albuquerque et al. (2) demonstrated that 5-HT acts at a 5-HT4 receptor to induce production of cAMP in rat distal crypt colonocytes. Our RT-PCR study demonstrated that 5-HT4 receptor mRNA are expressed not only in the duodenal mucosa as a whole but also likely in duodenal epithelial cells, indicating that functional 5-HT4 receptors may exist in duodenal mucosa and epithelial cells. Therefore, 5-HT may act directly at 5-HT4 receptors on epithelial cells to induce cAMP production and regulate duodenal bicarbonate secretion. Our signal transduction data are likewise consistent with a direct effect of 5-HT mediated by cAMP and by an indirect effect, involving the known Ca2+-dependent secretagogue ACh, mediated by calcium.

5-HT3 receptors are found on neurons of both central and peripheral origin. In the periphery, they are located on pre- and postganglionic autonomic neurons and on neurons of the sensory nervous system (22). In addition to its pronounced effect on the cardiovascular system, the 5-HT3 receptor also mediates the regulation of gastrointestinal motility and secretion. In the rat, 5-HT3 receptors have been shown to mediate colonic chloride secretion via neural pathways (5, 41). However, in the present study, we were unable to observe that the 5-HT3 receptor antagonist, low-dose ICS-205930, influenced 5-HT-stimulated duodenal bicarbonate secretion or that the 5-HT3 receptor agonist phenylbiguanide had any stimulatory effect on duodenal bicarbonate secretion, suggesting that 5-HT3 receptors may not participate in regulating duodenal bicarbonate secretion in mice. Likewise, the 5-HT2 receptor antagonist ketanserin had no inhibitory effect on 5-HT-induced duodenal bicarbonate secretion and the 5-HT2 receptor agonist {alpha}-methyl-5-HT had no stimulatory effect on duodenal bicarbonate secretion, suggesting that 5-HT2 receptor is not involved in duodenal bicarbonate secretion in mice either. Other reports showed that ketanserin inhibits 5-HT-induced secretion in rat colon (40) and rat jejunum (3). The differences among these results indicate that the function of 5-HT receptors depends on the species and anatomic region. In fact, the 5-HT1 receptor is comprised of five receptor subtypes. Some of these subtypes exist in the gastrointestinal tract (17, 35). However, at present, it has not been reported that the 5-HT1 receptor is involved in regulation of intestinal secretion. In this study, our results also demonstrated that the 5-HT1 receptor did not mediate duodenal bicarbonate secretion.

In conclusion, our results demonstrated that 5-HT stimulates duodenal bicarbonate secretion via a 5-HT4 receptor and both cAMP- and Ca2+-dependent signal pathways. RT-PCR confirmed that 5-HT4 receptor mRNAs are expressed in duodenal mucosa and epithelial cells, indicating that functional 5-HT4 receptors may exist in epithelial and perhaps also subepithelial compartments.


   ACKNOWLEDGMENTS
 
We thank Paul M. Quinton for advice and comments and Linda Thomas-Kroleski for help with manuscript preparation.

GRANTS

This work was supported by National Institute of Diabetes and Digestive and Kidney Diseases Grant DK-33491 (to J. I. Isenberg and K. E. Barrett).


   FOOTNOTES
 

Address for reprint requests and other correspondence: K. E. Barrett, Univ. of California, San Diego Medical Center, Div. of Gastroenterology, 8414, 200 W. Arbor Dr. San Diego, CA 92103-8414 (E-mail: kbarrett{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
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