1 Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University and 2 Department of Neuroscience, New York State Psychiatric Institute, New York, New York 10032; and 3 Laboratory of Clinical Science, National Institute of Mental Health, Bethesda, Maryland 20892
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ABSTRACT |
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The actions of
enteric 5-HT are terminated by 5-HT transporter (SERT)-mediated uptake,
and gastrointestinal motility is abnormal in SERT /
mice. We tested
the hypothesis that adaptive changes in enteric 5-HT3
receptors help SERT
/
mice survive despite inefficient 5-HT
inactivation. Expression of mRNA encoding enteric 5-HT3A
subunits was similar in SERT +/+ and
/
mice, but that of
5-HT3B subunits was fourfold less in SERT
/
mice.
5-HT3B mRNA was found, by in situ hybridization, in
epithelial cells and enteric neurons. 5-HT evoked a fast inward current
in myenteric neurons that was pharmacologically identified as
5-HT3 mediated. The EC50 of the 5-HT response
was lower in SERT +/+ (18 µM) than in SERT
/
(36 µM) mice and
desensitized rapidly in a greater proportion of SERT
/
neurons;
however, peak amplitudes, steady-state current, and decay time
constants were not different. Adaptive changes thus occur in the
subunit composition of enteric 5-HT3 receptors of SERT
/
mice that are reflected in 5-HT3 receptor affinity
and desensitization.
serotonin receptors; small intestine; enteric nervous system; electrophysiology
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INTRODUCTION |
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5-HT IS UTILIZED IN THE BOWEL as a neurotransmitter (24) and a paracrine messenger from enterochromaffin (EC) cells (17) to initiate peristaltic and secretory reflexes (8, 14, 42, 43, 54). Released 5-HT must be inactivated to prevent its actions from becoming excessive and receptors from desensitizing. To be inactivated, 5-HT must enter cells, because its catabolic enzymes are intracellular (25, 26). 5-HT is charged at physiological pH; therefore, its uptake across plasma membranes requires a high-affinity 5-HT transporter (5-HTT; SERT), which is identical in the brain (3, 4, 10), enteric nervous system (ENS), and gastrointestinal mucosa (11, 12, 65).
Transgenic mice that lack SERT have been generated by deletion of
the second exon by homologous recombination (1).
High-affinity uptake of 5-HT is not detectable in either the brain
(1) or the gut (12) of these animals. SERT
/
mice are viable into adulthood. In part, they survive because
transporters other than SERT mediate 5-HT uptake (12, 58).
In the gut, alternative transporters include the dopamine transporter
and organic cation transporters-1 and -3 (12). The
affinity of the dopamine transporter and the organic cation
transporters for 5-HT is much lower than that of SERT; however, they
have a high capacity and prevent 5-HT from accumulating to toxic
levels. A number of presumably adaptive changes have been found in the
brains of SERT
/
mice, including desensitization of presynaptic
5-HT1A receptors (18, 28, 44) and a
differential regulation of adenosine A1 and A2A
receptors (50). The alternative transporters and other
adaptive changes, however, compensate incompletely for the absence of
SERT. Developmental defects occur in SERT
/
mice, including a lack
of barrel formation by thalamocortical afferents in the cerebral cortex
(55), increased stool water, and a colorectal motility
that alternates between excessive (diarrhea) and inadequate
(constipation) (12).
5-HT3 receptors are expressed in the ENS (36, 47, 60) and play important roles in enteric physiology. They rapidly depolarize neurons (20, 47) by invoking a fast inward current (70, 71), which is responsible for 5-HT-mediated fast neurotransmission (71). A combination of 5-HT3 and 5-HT4 antagonists inhibits the peristaltic reflex (39, 51, 52). 5-HT3 antagonists decrease the frequency of migrating motor complexes in the isolated murine terminal ileum and colon (9). 5-HT3 receptors are also the physiological mediators of sensory nerve excitation by 5-HT from EC cells (2, 31, 34), which enables 5-HT3 antagonists to prevent the nausea associated with cancer chemotherapy (30).
The many roles played by 5-HT3 receptors in the bowel
suggest that the ability of the gut to function in SERT /
mice
might involve adaptive changes in 5-HT3 receptors.
Experiments were therefore carried out to examine the expression and
function of 5-HT3 receptors in the ENS of SERT
/
mice.
The transcription of mRNA encoding the 5-HT3A
(46) and 5-HT3B (15) subunits was
analyzed by RT-PCR and quantified. The previously unknown distribution
of 5-HT3B subunits was studied by in situ hybridization (ISH). 5-HT3 receptor functions were analyzed by whole cell
patch-clamp studies in cultured myenteric neurons. The data indicate
that the expression of 5-HT3B, but not that of
5-HT3A, is reduced in SERT
/
mice. This change is
associated with adaptive changes in receptor affinity and resistance to
desensitization that may help the ENS of SERT
/
mice to function
despite their abnormal 5-HT inactivation.
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MATERIALS AND METHODS |
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Animals.
Experiments were carried out with adult mice of either sex (6-12
mo of age; 30-40 g body wt). The genotypes of the animals were
SERT +/+ and SERT /
mice on backgrounds that were either CD-1
(n = 20) or C57BL/6J (n = 16).
Comparisons were always made between littermates. Genotypes of the mice
were determined as described by Bengel et al. (1). All of
the procedures involving animals and their care followed National
Institutes of Health guidelines and were approved by the Animal Care
and Use Committee of Columbia University.
RNA isolation and RT-PCR.
Total RNA (2 µg) was extracted from the ileum, colon, brain, and
spleen and was prepared by using a commercial kit (RNA STAT-60) according to the manufacturer's instructions (Tel-Test, Friendswood, TX). cDNA was prepared from this RNA by reverse transcription at 42°C
(30 min) in the presence of random primers and murine leukemia virus
reverse transcriptase (GeneAmp RNA PCR kit; Applied Biosystems, Foster
City, CA). The reverse transcriptase was omitted in controls and
permitted the detection of contamination of samples with genomic DNA.
DNA contamination was removed by treating RNA with RNase-free DNase I
(Promega, Madison, WI) for 15 min at 37°C before reverse
transcription. All experiments were carried out with 0.1% diethyl
pyrocarbonate (Sigma, St. Louis, MO)-treated distilled water. The cDNA
was amplified by using the PCR with Taq DNA polymerase
(Applied Biosystems). Initial denaturation was carried out at 95°C
for 2 min, the denaturation that accompanied each cycle was carried out
at 95°C for 45 s, and elongation was carried out at 72°C for 2 min (except in the case of -actin, for which the time for elongation
was 40 s). Primers, Mg2+ concentration, annealing time
and temperature, and number of cycles are listed in Table
1. After the final PCR cycle, the reaction was extended for an additional 10 min at 72°C, and the reaction products were then cooled to 4°C. PCR products were resolved by electrophoresis through 1% agarose gels with ethidium bromide (0.3 µg/ml) in Tris-borate-EDTA electrophoresis buffer. The identity of
the PCR products was verified by sequencing. For this purpose, PCR
products from the ileum were subcloned into a pCR II vector digested
with EcoR I by using a commercial kit (TA Cloning Kit; Invitrogen, Carlsbad, CA). Clones with inserts were further processed to isolate the plasmid DNA (Wizard Minipreps; Promega) and sequenced by
dye termination (ABI Automated Sequencer; Perkin Elmer) in the core
facility of Columbia University. The final cDNA sequences were compared
with those in GenBank (BLAST search at National Center for
Biotechnology Information, Bethesda, MD). All experiments were carried
out with RNA prepared from three separate littermate pairs of SERT +/+
and
/
mice.
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Competitive PCR.
The method used to quantify mRNA encoding the 5-HT3B
subunits was based on the technique of competitive PCR
(63). Competitor DNA fragments for quantitative PCR were
obtained by using mouse-specific primers to amplify DNA from chicks, a
species that is a distant evolutionary relative of the mouse, under
low-stringency annealing conditions (45°C for 1 min; 30 cycles).
These artificially created fragments contain the mouse primer-specific
ends and thus can be used to quantify mouse DNA amplified by these
primers. The competitor DNA fragments differ in size from the
corresponding mouse target DNA and are selected by agarose gel
electrophoresis, which enables both fragments to be visually
distinguished. After electrophoresis, fragments of appropriate size
were excised and subcloned for sequencing as described above to verify
that the competitor DNA did not overlap the PCR product from the target DNA. To compare constant amounts of cDNA from the tissues, the cDNAs
were adjusted to equal amounts by using the -actin DNA for
calibration. Serial dilutions of competitor DNA were added to equal
amounts of cDNA of each sample before applying PCR with primer pairs
designed to amplify DNA encoding the 5-HT3B subunit (Table
1). For quantitative analysis, the ethidium bromide agarose gels were
photographed by using a UV illuminator, scanned, and digitized. The
optical density of the digitized images was analyzed by using Kodak
Digital Science 1D Image Analysis v.1.51 software (Eastman Kodak,
Rochester, NY).
Western blotting. The small intestines were rapidly removed, and the longitudinal muscle was removed with the adherent myenteric plexus (LMMP). The resulting LMMP sheets of tissue were homogenized in a cold lysis buffer (0.02 M Tris, 0.1% Triton, 1 mM EDTA) containing a commercial cocktail of protease inhibitors [diluted 1:10; a mixture of 4-(2-aminoethyl)benzenesulfonyl fluoride, aprotinin, leupeptin, bestatin, and pepstatin A; Sigma]. The lysate was centrifuged at a low speed to remove debris, and the protein concentration of the supernatant was measured by Bio-Rad protein assay (Bio-Rad Laboratories, Richmond, CA). The supernatant was then boiled for 5 min at 95°C in Laemmli solution (Bio-Rad) containing 0.35 M dithiothreitol. An aliquot containing 20 µg of protein was subjected to SDS-PAGE (10% polyacrylamide). The separated proteins were electrophoretically blotted onto a nitrocellulose sheet for Western blot analysis. The blots were bleached for 5 min with 6% H2O2 and blocked by incubation for 2 h in TBST buffer (0.05 M Tris, 0.15 M Tris, 0.05% Tween) containing 5% fat-free milk. The blots were probed with purified rabbit antibodies to the 5-HT3 receptor (diluted 1:1,000; Oncogene, Boston, MA). Immunoreactivity was identified with goat anti-rabbit secondary antibodies conjugated to horseradish peroxidase (diluted 1:5,000; Jackson Labs, West Grove, PA). Horseradish peroxidase activity was visualized with 4-chloro-1-naphthol (4-CN kit; Bio-Rad).
ISH.
Small segments of ileum were fixed for 3 h with 4% (wt/vol)
formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M PBS, pH
7.4. The tissue was cryoprotected by incubation overnight (at 4°C) in
PBS containing 30% (wt/vol) sucrose. Tissues were then embedded in a
sectioning compound (TissueTek OCT; Miles, Elkhart, IN), frozen in
liquid N2, and sectioned (10 µm) at 20°C by using a
cryostat-microtome. Cultured neurons were fixed in the same fixative
solution but kept for 1 h at room temperature. Fixed cultures were
washed with PBS for 30 min and stored at 4°C until used. All
experiments were carried out with 0.1% diethyl pyrocarbonate-treated
distilled water.
Immunocytochemistry. For immunocytochemistry (ICC), fixed preparations were permeabilized and blocked with 0.5% Triton X-100 (vol/vol) and 4% normal horse serum in PBS for 30 min. The preparations were then incubated overnight (at 4°C) with rabbit antibodies (1 µg/ml) raised against the rat 5-HT3 receptor (Oncogene). After being washed with PBS, the sites of bound primary antibody were detected by incubation with donkey anti-rabbit secondary antibodies coupled to FITC (diluted 1:400, 3 h at room temperature; Jackson Labs). The preparations were finally washed again with PBS and then were mounted in Vectashield (Vector Labs, Burlingame, CA). No immunostaining was observed when the primary antibody was omitted.
Combination of ISH and ICC. Double labeling was used to locate mRNA encoding the 5-HT3B subunit and 5-HT3 receptor protein simultaneously. ISH was always carried out before ICC. The digoxigenin-labeled probe used for ISH was detected by using antibodies to digoxigenin coupled to 5-carboxy-tetramethyl-rhodamine-N-hydroxy-succinimide ester (TAMRA, diluted 1:20; Roche). The red fluorescence of TAMRA provided a good contrast for the green fluorescence of FITC to detect antibodies bound to 5-HT3 receptors by ICC. Immunocytochemical preparations were examined by using a LSM 410 laser scanning confocal microscope (Zeiss, Thornwood, NY) equipped with a krypton/argon laser and attached to a Zeiss Axiovert 100TV inverted microscope. The fluorescence of FITC (excitation 488 nm, dichroic BP 515-540 nm) and TAMRA (excitation 568 nm, dichroic LP 590 nm) (Chroma optical filter sets) were viewed with dichroic mirrors and filters that permitted no cross-detection of fluorescence. Optical sections (10-15) were taken at 1.0-µm intervals. Images of 512 × 512 pixels were obtained with a confocal microscope and processed by using Adobe Photoshop 6.0.
Cell culture. Mice were anesthetized with CO2 and decapitated, following procedures approved by the Columbia University Animal Care and Use Committee. The small intestine was removed, cleaned, and placed in iced sterile-filtered Krebs solution of the following composition (in mM): 121.3 NaCl, 5.95 KCl, 14.3 NaHCO3, 1.34 NaH2PO4, 1.2 MgCl2, 2.5 CaCl2, and 12.7 glucose. The procedure used for the isolation of myenteric neurons for culture procedure has previously been described (41). Briefly, the LMMP was removed from the entire length of small intestine and minced into small pieces. The resulting tissue was suspended in 10 ml of oxygenated (95% O2-5% CO2) Krebs solution containing collagenase (type 1A; 1.3 mg/ml), protease (type IX; 1 mg/ml), DNase (type 1; 0.3 mg/ml), and bovine serum albumin fraction V (0.3 mg/ml). Tissue digestion was carried out with gentle stirring for 1 h at 37°C and terminated by centrifugation three times for 2 min at 2,000 rpm (Eppendorf microcentrifuge, model 5415C). The pellet was resuspended in culture medium consisting of DMEM and F-12K (Kaighn's modification) (1:1 mixture) supplemented with 10% heat-inactivated fetal bovine serum, gentamicin (50 µg/ml), penicillin-streptomycin (100 U/ml and 100 µg/ml), and amphotericin B (0.25 µg/ml). The suspended cells were then plated on glass coverslips coated with poly-L-ornithine (0.1 mg/ml; Sigma) and laminin (10 µg/ml) or Matrigel (1:4 dilution; Becton Dickinson Biosciences, Bedford, MA) and maintained in an incubator at 37°C in a humidified atmosphere of 5% CO2 for up to 2 wk. After 2 days in culture, 10 µM cytosine arabinoside (Sigma) was added to the culture medium to limit the proliferation of nonneuronal cells, and the medium was changed twice weekly thereafter.
Whole cell patch-clamp recording and drug application.
Recordings were carried out at room temperature on the stage of an
inverted microscope (Axiovert IM35, Zeiss) 2-14 days after plating
cells. The procedure was similar to that described previously (38). The culture medium was replaced with an external
solution containing (in mM) 145 NaCl, 4.7 KCl, 2.5 CaCl2,
1.2 MgCl2, 11 glucose, and 10 HEPES, pH 7.35, with NaOH.
The concentrations of K+ and Ca2+ were chosen
to match those in the Krebs solution used in prior studies with sharp
microelectrodes to make the data comparable. The recording chamber was
continually perfused at a rate of 0.5 ml/min. Recording electrodes were
made from borosilicate glass (inner diameter 0.86 mm, outer diameter
1.5 mm; Warner, Hamden, CT), pulled on a P-80/PC Brown-Flaming
micropipette puller (Sutter, Novato, CA), and had tip resistances of
5-8 M. The internal pipette solution contained (in mM) 140 potassium gluconate, 0.1 CaCl2, 2 MgCl2, 1 EGTA, 2 Na2ATP, 0.2 Na3GTP, and 10 HEPES, pH
7.25, with KOH. Whole cell currents were recorded using an Axoclamp 2A
amplifier (Axon Instruments, Union City, CA) running in continuous, single-electrode, voltage-clamp mode; membrane potential was recorded in current-clamp bridge mode. The holding potential was set at
60 mV.
Data for continuous gap-free recordings were digitized at 5 kHz by
using a Digidata 1322A interface and AxoScope 8.1 (Axon Instruments).
Data from the whole cell recordings were filtered at 2 kHz, digitized
at 10 kHz (ITC-16; Instrutech, Port Washington, NY), and collected by
using Pulse Control 5.0 (Instrutech) and IgorPro 3.1 (Wavemetrics, Lake
Oswego, OR). Compensation for passive and leak conductance was made by
adding a scaled average of four hyperpolarizing pulses (
5 mV)
delivered following each episode of data acquisition (Subtraction
Pulses Global).
Compounds used. Enzymes for digestion of tissue were obtained from Sigma. Tissue culture reagents were obtained from GIBCO Life Technologies (Grand Island, NY). 5-HT creatinine sulfate, 2-Me-5-HT, mCPBG, tropisetron, TTX, hexamethonium, and cytosine arabinoside were purchased from Sigma/RBI (Natick, MA). Ondansetron and alosetron were supplied by Glaxo Wellcome (Research Triangle Park, NC).
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RESULTS |
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mRNA encoding the 5-HT3A and 5-HT3B
subunits is present in the mouse gut.
mRNA encoding 5-HT3 subunits was detected in the bowel of
SERT +/+ and /
mice by using RT-PCR (Fig.
1). The mouse brain and spleen were
studied at the same time as the gut as positive and negative controls,
respectively. mRNA encoding
-actin was used to provide a
semiquantitative reference to which to compare the products of
amplification. PCR products of the expected size (Table 1) were
obtained when cDNA from the small intestine and colon was amplified
with primers corresponding to sequences found in the 5-HT3A
and 5-HT3B receptor subunits (Fig. 1A).
Subcloning and sequencing confirmed the identities of all of the PCR
products. As expected, mRNA encoding 5-HT3A and
5-HT3B receptor subunits was also detected in the brain of
SERT +/+ and
/
mice but not in the spleen.
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mRNA encoding the 5-HT3B subunit is decreased in the
gut of SERT /
mice.
The suggestion that expression of mRNA encoding 5-HT3B
subunit might be reduced in SERT
/
mice was investigated by using competitive PCR to quantify 5-HT3B expression in the small
intestine and colon of SERT +/+ and
/
mice. The cDNA obtained by
reverse transcription from the tissues of SERT +/+ and
/
mice were
compared (Fig. 2) by using
-actin as a
standard to calibrate the amount of cDNA. Equal amounts of target DNA
were then amplified simultaneously with known amounts of a competitor
DNA fragment (5-HT3B-c.f.), which was distinguished from
that of murine DNA by size. This competitor (358 bp) did not overlap
the PCR product from the mouse 5-HT3B subunit DNA (567 bp;
Fig. 2, insets). The amount of amplified DNA encoding the
5-HT3B subunits (5-HT3B-cDNA) was quantified by
comparing it to that of the serially diluted competitor
(5-HT3B-c.f.) by optical density. The
5-HT3B-c.f. (0.003-300 fmol/l) was coamplified with a
fixed amount (1 µg) of target DNA from small intestine (Fig.
2A) or colon (Fig. 2B) of SERT +/+ and
/
mice. The small intestine (Fig. 2A) and the colon (Fig.
2B) of SERT +/+ mice each contained about fourfold more mRNA
encoding the 5-HT3B subunit than did the small intestine
and colon of their SERT
/
littermates.
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mRNA encoding the 5-HT3B subunit is found in epithelial
cells and enteric neurons.
Although mRNA encoding the 5-HT3A receptor has been found
to be present in both submucosal and myenteric ganglia of the rat small
intestine (36), that encoding the 5-HT3B
subunit of the receptor has not previously been located in the bowel of
any species. The studies described above, however, indicated that it is
the expression of the 5-HT3B subunit, and not that of the
5-HT3A, that is affected by the absence of SERT in the gut
of SERT /
mice. ISH was thus used to identify the enteric cells in
which the 5-HT3B subunit is expressed. mRNA encoding the
5-HT3B subunit was observed in neurons of both the
myenteric and submucosal plexuses in SERT +/+ mice (Fig.
3A). mRNA encoding the
5-HT3B subunit was also present in mucosal epithelial cells
of the intestinal crypts of the SERT +/+ animals (Fig. 3B).
These cells are most likely to be EC cells, which are known to contain
5-HT3 receptors (23, 40, 57). mRNA encoding
the 5-HT3B subunit was also found in myenteric and
submucosal neurons in SERT
/
mice, and the degree to which the
riboprobe hybridized with cells in these animals appeared to be much
less than that in their SERT +/+ counterparts (Fig. 3C).
Because the intensity of labeling was so severely reduced in the SERT
/
animals, few labeled cell bodies could be discerned in the
ganglia of these animals, and no mRNA encoding the 5-HT3B subunit was detected in the epithelial cells of SERT
/
mice. No
labeling of control tissue was observed when sections of gut from
either SERT +/+ or
/
mice were hybridized with a sense riboprobe
(Fig. 3D).
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The 5-HT3B subunit is specifically expressed in a
subset of enteric neurons in SERT +/+ mice.
The polyclonal antibodies to the rat 5-HT3 receptor used in
the current study have previously been characterized in the rat central
nervous system and the guinea pig bowel (40, 49). The
sequence of amino acids (438-450) recognized by these
antibodies is identical to that of the corresponding domain of the
mouse 5-HT3A receptor (GenBank accession no. NP038589),
with the exception of only a single mismatch. This sequence is not
found in the 5-HT3B subunit. When probed with the
5-HT3 antibodies, a single protein band (~58 kDa),
corresponding in size to that of the 5-HT3A subunit, was
found in extracts of the murine LMMP (Fig.
4A); therefore, we used this
antibody to locate the 5-HT3A subunits. Myenteric neurons
were isolated from the small intestine of SERT +/+ mice and cultured.
Punctate 5-HT3A receptor immunoreactivity was found in a
subset of neurons, labeling both varicose neurites (Fig. 4B
and Fig. 5) and cell bodies (Fig.
5A). In similar cultures, mRNA encoding the
5-HT3B subunits was detected by ISH in a subset of
34.7 ± 7.8% of the total population of neurons (Fig. 4,
C and E). No labeling was observed when control
cells were hybridized with a sense riboprobe for the 5-HT3B
subunit (Fig. 4, D and F and Fig. 5B).
Double labeling to detect 5-HT3A receptor immunoreactivity and mRNA encoding the 5-HT3B subunit was carried out
simultaneously to determine whether both subunits of the
5-HT3 receptor are likely to be found in the same myenteric
neurons (Fig. 5A). Antibodies to the 5-HT3B
subunits are not available; therefore, mRNA encoding the
5-HT3B subunits was used as a surrogate marker for cells
containing 5-HT3B subunits, although one cannot be certain
that the 5-HT3B transcripts are translated. mRNA encoding
the 5-HT3B subunit was restricted to nerve cell bodies,
whereas the immunoreactivity of the 5-HT3A subunits was
present both in cell bodies and varicose neurites.
5-HT3A-immunoreactive neurons were found also to contain mRNA encoding the 5-HT3B subunit (Fig. 5A), a
distribution consistent with the idea that both subunits are expressed
by the same cells. No signal was detected in the cytoplasm when control
cultures were hybridized under identical conditions with a sense
riboprobe for the 5-HT3B subunit (Fig. 5B).
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Current responses recorded from mouse enteric neurons.
We evaluated the functional consequences of the downregulation of the
5-HT3B subunits in the myenteric neurons of SERT /
mice
by using whole cell patch-clamp recording. The myenteric neurons of
mice have not previously been investigated by means of the patch-clamp
technique; however, the properties of the murine myenteric neurons
investigated in the current study were found to be similar to those
reported for cultured myenteric neurons of guinea pigs (32,
64). In current-clamp mode, all of the neurons patched in this
study had a resting membrane potential that was more negative than
40
mV, fired action potentials when injected with pulses of
depolarizing current, and showed action potential frequency adaptation
when injected with long (400 ms) depolarizing current pulses. The
resting membrane potentials for SERT +/+ and
/
myenteric neurons,
48.69 ± 1.16 mV (n = 23) and
45.80 ± 1.05 mV (n = 20), respectively, did not differ
significantly, nor did their input resistance of 160.8 ± 25.2 M
and 236.6 ± 58.8 M
for SERT +/+ and
/
enteric neurons
(means ± SE, n = 8). In voltage-clamp mode,
neurons were identified as those cells that generated
Na+-dependent fast inward currents at the onset of 400-ms
depolarizing voltage steps (from a holding potential of
60 mV); TTX
(1 µM) was used to block the Na+-dependent fast inward
currents, and hexamethonium (300 µM) was used to block cholinergic
synapses in cultured myenteric neurons (data not shown). Prior studies
of guinea pig myenteric neurons (32, 64) used
KCl-containing patch pipettes instead of the potassium gluconate
employed in the current study; therefore, we verified that the
potassium gluconate in the internal solution did not affect the
recorded current-voltage relationship by comparing the slopes of
current responses to depolarizing voltage ramps that changed the
holding potential from
80 to 0 mV in 160 ms. The input resistance was
156.79 ± 21.86 M
(n = 4) when recorded with
pipettes filled with KCl and 173.88 ± 20.83 M
(n = 4) when recorded with pipettes filled with
potassium gluconate. These values do not differ significantly.
5-HT and 5-HT3 agonists evoke fast inward currents in
myenteric neurons.
Fast inward currents were evoked in myenteric neurons by local
applications (10 s) of 50 µM 5-HT, mCPBG, or 2-Me-5-HT (Fig. 6A) in 66.7%
(n = 42/63) of enteric neurons from SERT +/+ mice and
in about the same proportion (63.5%; n = 33/52) of
those from SERT /
animals. The peak amplitude and the time to peak
response of the fast inward current for all of the tested compounds
were directly related to their concentrations. In contrast, the rate at
which the response decayed was an inverse function of the
concentrations of these agonists (Fig. 6B). In small subsets
of neurons, 5-HT evoked a prolonged sustained inward current or an
outward current (~5-20 pA; Fig. 6C). These responses
appeared to be independent of the fast inward current elicited by 5-HT
because they were observed in neurons that did or did not display the
fast response and thus are likely mediated by other G protein-coupled
subtypes of 5-HT receptors on subpopulations of myenteric neurons,
which appeared to involve other subtypes of 5-HT receptors and were not
analyzed with data derived from responses mediated by 5-HT3 receptors.
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5-HT3 antagonists block 5-HT-induced fast inward
currents.
The 5-HT-evoked fast inward currents were abolished by the
5-HT3-selective antagonists tropisetron (1 µM),
ondansetron (1 µM), or alosetron (0.15-3 µM). Alosetron (0.15 µM) completely abolished the fast inward current, and, in the subset
of neurons in which the fast inward current was followed by a sustained
current, alosetron also abolished the sustained current (Fig.
7A). Ondansetron (1 µM) also
antagonized the fast inward current evoked by 5-HT (Fig.
7B). Ondansetron (1 µM) reproducibly abolished the fast inward current, but, in contrast to alosetron, it did not block the
small sustained currents in the subset of cells in which they were
elicited; these cells were excluded from the final analysis (Fig.
7C). Recovery following the washout of antagonists was slow and usually incomplete during the 30-min recording period. Similar effects of ondansetron and alosetron have been obtained in studies of
cultured myenteric neurons of guinea pigs (70, 71). Our observations confirm the identification of 5-HT-evoked fast inward currents as 5-HT3-mediated events.
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5-HT3 receptor sensitivity is decreased in SERT /
mice.
To determine the effect of the knockout of SERT on the
5-HT3 receptor sensitivity, the peak of the fast inward
current induced by 5-HT was measured as a function of the 5-HT
concentration (Fig. 8). In both SERT +/+
and
/
mice, the threshold response was obtained at ~1 µM 5-HT
and then increased in a concentration-dependent manner. The Hill
coefficient measured in neurons from SERT +/+ mice
(nH = 0.87) was similar to that measured in
those from SERT
/
animals (nH = 0.89) and in
both was slightly less than unity; however, the concentration-effect
curve for the 5-HT-induced fast inward current was shifted to the right
in the SERT
/
animals (P < 0.01, Wilcoxon
signed-rank test; n = 8). The EC50 was
17.65 µM in neurons from SERT +/+ mice vs. 35.88 µM in SERT
/
mice. The downregulation of 5-HT3B subunits in SERT
/
mice is thus associated with a reduction in the affinity of
5-HT3 receptors for 5-HT.
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Peak currents activated by 5-HT3 agonists.
The amplitudes of the fast inward currents evoked by a 50-µM
concentration of three 5-HT3 agonists, 5-HT, mCPBG, and
2-Me-5-HT, were compared in myenteric neurons from SERT +/+ and /
mice. The amplitudes of the inward current evoked by these compounds were found to differ in neurons from both SERT +/+ and
/
mice so
that 5-HT > mCPBG > 2-Me-5-HT (Table
2); however, differences between SERT +/+
and
/
mice were not significant.
|
Time course of 5-HT-mediated currents.
The maximal amplitude of the 5-HT-induced inward current was reached
quickly. The current decayed over a slower time course that could be
defined by two exponential time constants (fast and
slow). Three types of response, rapidly desensitizing,
mixed, and slowly desensitizing, were distinguished on the basis of the time course of the decay (Fig. 9; Table
3). A
rapidly desensitizing response was defined as one in which the
transient inward current fully decayed within 1 s (Fig.
9A). A slowly desensitizing response was one in which the
inward current did not fully decay during the 10-s application of 5-HT
(50 µM; Fig. 9C); the steady-state current that followed
the deactivation phase disappeared within 12 s. A mixed response
was defined as one in which the time to decay of the inward current was
>1 but <10 s (Fig. 9B). For each type of response (rapidly
desensitizing, mixed, and slowly desensitizing), the peak amplitude,
the decay time constants (
fast and
slow), and the steady-state current (Cs) were compared in
myenteric neurons from SERT +/+ and
/
mice (Table 3).
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The current study was undertaken to determine whether adaptation
occurs in 5-HT3 receptors in SERT /
mice to compensate for the inefficient inactivation of 5-HT in these animals. 5-HT receptors in SERT
/
mice are likely to be exposed to concentrations of 5-HT that are higher and more prolonged than normal. At the time its
primary structure was first deduced (46), the
5-HT3 receptor was thought to be composed of a single
protein, now known as the 5-HT3A subunit. Since the
properties of native 5-HT3 receptors are largely mimicked
by the functional expression of 5-HT3A subunits, the role
of 5-HT3B subunits has not been entirely clear. The
5-HT3B subunit has now been cloned and sequenced from
tissues of humans (15, 16), rats, and mice
(33). Rodent neurons normally express heteromeric
5-HT3A/B subunits (33).The 5-HT3B
subunit cannot, by itself, constitute an effective ligand-gated ion
channel and thus functions only in conjunction with 5-HT3A
subunits (15, 16, 59). Although the pharmacology of
expressed homomeric and heteromeric 5-HT3 receptors is
similar (6), biophysical properties of the two types of
5-HT3 receptor complexes differ (15, 16, 33).
The single-channel conductance of activated heteromeric
5-HT3A/B receptors is greater than that of homomeric 5-HT3A receptors; moreover, heteromeric receptor complexes
are less permeable than homomeric 5-HT3A receptors to
Ca2+ and less sensitive to inhibition by tubocurarine.
Adaptive changes in expression of 5-HT3B subunits in SERT
/
mice, therefore, might allow 5-HT3-mediated responses
to be modulated.
Both the A and the B subunits of the 5-HT3 receptors were
found to be expressed in the murine bowel by RT-PCR, but the level of
expression of the 5-HT3B subunit was higher in SERT +/+
than in SERT /
animals. In contrast, no difference between SERT +/+ and
/
mice was detected by means of semiquantitative RT-PCR in the
expression of the 5-HT3A subunits (5-HT3A and
5-HT3AL/S). In SERT +/+ mice, mRNA encoding the
5-HT3B subunit was found, by ISH, to be located in
epithelial cells and in both submucosal and myenteric neurons. This
mRNA was also detectable in the SERT
/
animals, although
quantitative measurements (competitive PCR) indicated that the level of
5-HT3B expression was about fourfold higher in the gut of
SERT +/+ mice than in their SERT
/
littermates. These observations
suggest that there is probably more 5-HT3B subunit protein
available for incorporation into enteric 5-HT3 receptors in
SERT +/+ than in SERT
/
animals. If so, then the ratio of
heteromeric (5-HT3A/B) to homomeric (5-HT3A)
receptor complexes would be greater in SERT +/+ mice.
The concentration-effect curve for 5-HT-induced fast inward currents
was found, in whole cell patch-clamp studies of cultured myenteric
neurons, to shift to the right in neurons of SERT /
mice. The
observations that the 5-HT3 antagonists tropisetron, ondansetron, and alosetron blocked the 5-HT-induced fast inward currents confirmed that they were 5-HT3 mediated. The
rightward shift of the concentration-effect curve suggests that the
affinities of the 5-HT3 receptors of SERT
/
mice for
5-HT are lower than those of SERT +/+ animals. Other investigators have
also reported that the affinities of homomeric and heteromeric
5-HT3 receptors expressed differ, although the nature of
the difference appears to depend on intrinsic properties of the
heterologous cells in which the receptors are expressed (15, 16,
33); nevertheless, the whole cell recordings and measurements of
Ca2+ influx have revealed that coexpression of
5-HT3B subunits with 5-HT3A subunits can
enhance the affinity of 5-HT3 receptor complexes for 5-HT
in heterologous cells (16). The decreased sensitivity of
the enteric neuronal 5-HT3 receptors of SERT
/
mice for
5-HT may thus be due to the associated decrease in expression of
5-HT3B subunits that occurs in these animals.
The proportion of neurons that exhibited rapidly desensitizing
5-HT3-mediated fast inward currents increased in SERT /
mice. The rate of desensitization of homomeric (5-HT3A)
receptor complexes has been reported to be accelerated by the
extracellular presence of Ca2+ (37, 69).
Homomeric (5-HT3A) subunits are both Ca2+
permeant and inhibited by Ca2+ (7, 53, 56). In
contrast, heteromeric (5-HT3A/B) receptors are relatively
impermeable to Ca2+; therefore, native 5-HT3
receptors, which have much lower permeabilities to divalent cations
than those reported for 5-HT3A homomeric receptors, are
likely to contain 5-HT3B subunits (15, 16). In
the current study, 5-HT3-mediated currents were recorded
with the extracellular [Ca2+] set at 2.5 mM; therefore,
Ca2+ could play a role in the desensitization of
5-HT3 receptors if those of SERT
/
mice were to be
hyperpermeable to Ca2+. The relative increase in the
proportion of 5-HT3A subunits that occurs in enteric
neurons of SERT
/
mice may be associated with an increase in the
permeability of 5-HT3 receptor complexes to Ca2+, which in turn could contribute to the acceleration of
desensitization of the receptors in affected neurons. Conceivably, the
rapid desensitization of 5-HT3 receptors might also be
regulated by receptor phosphorylation. Phosphorylation has been
reported to occur in studies of transfected cells that express
5-HT3A receptors (5, 27, 35, 68). There is no
reason, however, to assume that the mechanism by which 5-HT3 receptor desensitization is regulated in cell lines
is the same as that in the native myenteric neurons of SERT
/
mice confronted with slow inactivation of 5-HT. Cells that express only
5-HT3A subunits, moreover, obviously cannot downregulate 5-HT3B expression. The increase in the proportion of
rapidly desensitizing neurons, however achieved, would be expected to
protect against overstimulation by the prolonged presence of 5-HT in
contact with 5-HT3 receptors in SERT
/
mice.
It is not clear why the change in rate of desensitization is seen in
only some neurons and not all of those studied. The most likely
explanation is that the partial nature of the phenomenon is a
reflection of the means by which the rate of desensitization is
measured. Obviously, for any given neuron, it is impossible to record
the rate of desensitization of 5-HT3 receptors before and
after the knockout of SERT. Only populations of neurons can be examined
in mice that do or do not express SERT. A small change in the rate of
desensitization, therefore, is evident only at the extremes of the
population, an increase in the proportion of rapidly desensitizing
neurons at the expense of those which desensitize slowly. Changes in
rates of 5-HT3 receptor desensitization in the mass of
neurons in between are missed because there is no way to determine how
any single neuron of a SERT /
mouse might have behaved if SERT had
been present. Still to be determined, moreover, is the mechanism that
links the presumed excess of 5-HT in the environment of neurons of SERT
/
mice to the observed alteration in the expression of the gene
encoding the 5-HT3B subunit.
The electrophysiological properties of the inward current evoked by
5-HT in enteric neurons were found to vary from cell to cell in both
SERT +/+ and /
mice even though an effort was made to maintain
constant recording conditions. Conceivably, the proportions of
5-HT3A and 5-HT3B subunits may vary between
cells and even within heteromeric receptor complexes. Because the
respective sensitivities of the techniques of ISH and ICC are unknown,
the proportions of 5-HT3A and 5-HT3B subunits
in individual receptor complexes and cells cannot be determined by ICC
or ISH. An attempt was made to assay mRNA encoding the
5-HT3B subunits in cytoplasm aspirated from cells used for
patch-clamp recording; however, consistent measurements could not be
obtained in each cell from which a recording was obtained. It thus
remains possible that a varying subunit composition of
5-HT3 receptor complexes contributes to the
electrophysiological differences between 5-HT3-mediated responses recorded in different neurons. In addition, distortions might
have been encountered in individual neurons due to their extension of
neurites and consequent space-clamp problems. We compared the kinetic
responses of the whole-cell currents from two populations of cultured
neurons (SERT +/+ and
/
). Differences between populations of
neurons in response to 5-HT3 agonists were studied rather
than change in Ca2+ or K+ currents in
individual neurons. Distortions due to uncontrolled space-clamp
effects, therefore, are likely to contribute to the overall variability
but are unlikely to systematically affect either population and thus
should not affect significant differences between the two populations.
Prior patch-clamp studies of enteric 5-HT3 receptors used intracellular solutions that contained Cs+ or high concentrations of EGTA (70, 71). These additives block the Ca2+-activated K+ conductance modulated by the activity of G protein-coupled 5-HT receptors, which were thus not detected. We used an intracellular solution containing potassium gluconate to permit K+ channels to contribute to the recorded 5-HT-induced currents; therefore, we were able to test voltage responses in the current-clamp mode at the beginning of each experiment to compare the responses we obtained with patch electrodes with those obtained in prior studies in current-clamp mode with sharp microelectrodes. Very few previous current-clamp studies of murine myenteric neurons with sharp microelectrodes have been reported, and this is the first report of whole cell patch-clamp recordings from myenteric neurons. Small steady-state currents during the desensitizing phase of 5-HT3 receptor-mediated responses have previously been observed in guinea pig myenteric neurons with CsCl-containing patch pipettes that would have masked the effects of G protein-coupled receptors on K+ channels (71). We excluded responses to 5-HT or agonists that were associated with a delayed onset or a sustained inward and/or outward current in our final data analysis to be certain that the responses we measured were exclusively those mediated by 5-HT3 receptors. No hyperpolarization-activated cationic currents were observed in either voltage- or current-clamp mode, and 5-HT-evoked fast inward currents were invariably abolished by the 5-HT3-selective antagonists tropisetron, ondansetron, and alosetron; therefore, the small steady-state current recorded during the desensitizing phase of the fast inward current mediated by 5-HT could not have been influenced by metabotropic G protein-coupled 5-HT receptors, such as the 5-HT1P receptor (24). None of the G protein-coupled subtypes of 5-HT receptors are antagonized by ondansetron or alosetron in the concentrations of these drugs that were utilized.
Colorectal motility is most often greater than normal in animals that
lack SERT, leading to excessive stool water; however, some knockout
mice also undergo transient episodes of extremely slow colorectal
motility (12). These periods of constipation are thought
to be associated with 5-HT receptor desensitization. It is possible
that the increased tendency of 5-HT3 receptors to
desensitize rapidly in SERT /
mice contributes to the constipation. Chronic inhibition of 5-HT3 receptors in human subjects
with alosetron was also associated with an increased incidence of
constipation (19). It is possible that adaptive changes in
5-HT3 receptors similar to those found in SERT
/
mice
might also occur in the chronic administration of SERT inhibitors, such
as serotonin-selective reuptake inhibitors, tricyclic
antidepressants, and cocaine, for the treatment of depression or
for recreational use. Gastrointestinal side effects of these compounds
are frequent and often vexatious.
In summary, adaptive compensation occurs in enteric 5-HT3
receptors of SERT /
mice. These changes consist of the
downregulation of 5-HT3B subunits, which is associated
electrophysiologically with an increase in the EC50 for
receptor sensitivity and an increase in the proportion of neurons that
desensitize rapidly. Major electrical properties of
5-HT3-mediated fast inward currents, including the activation peak amplitude, the exponential time constants of the decay
curve, and the amplitude of the steady-state current are unchanged by
the knockout of SERT. The adaptive changes are thus unlikely to
interfere with 5-HT3-gated channel functions; however, their nature is such that the changes should be protective against excessive stimulation of neurons via 5-HT3 receptors in
animals in which extracellular 5-HT after release from stimulated EC
cells or enteric neurons is likely to be persistent. The mechanism by which SERT knockout feeds back to alter 5-HT3B subunit
expressions remains to be determined.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank Drs. Anne-Cecile Trillat, Christóvão Albuquerque, and Elena Fiorica-Howells for their advice and Dr. Allen Mangel (Glaxo Wellcome) for ondansetron and alosetron.
![]() |
FOOTNOTES |
---|
This work was supported by National Institutes of Health (NIH) grants to M. D. Gershon (NS-12969) and S. Rayport (DA-00356). The confocal microscopy facility was supported by NIH Grants RR-10506 and RR-13701.
Address for reprint requests and other correspondence: M. Liu, Dept. of Anatomy and Cell Biology, Columbia Univ., College of Physicians and Surgeons, New York, NY 10032 (E-mail: ml27{at}columbia.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.
September 4, 2002;10.1152/ajpgi.00203.2002
Received 29 May 2002; accepted in final form 9 August 2002.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Bengel, D,
Murphy DL,
Andrews AM,
Wichems CH,
Feltner D,
Heils A,
Mossner R,
Westphal H,
and
Lesch KP.
Altered brain serotonin homeostasis and locomotor insensitivity to 3,4-methylenedioxymethamphetamine ("Ecstasy") in serotonin transporter-deficient mice.
Mol Pharmacol
53:
649-655,
1998
2.
Blackshaw, LA,
and
Grundy D.
Effects of 5-hydroxytryptamine on discharge of vagal mucosal afferent fibres from the upper gastrointestinal tract of the ferret.
J Auton Nerv Syst
45:
41-50,
1993[ISI][Medline].
3.
Blakely, RD,
Berson HE,
Fremeau RT,
Caron MG,
Peek MM,
Prince HK,
and
Bradley CC.
Cloning and expression of a functional serotonin transporter from rat brain.
Nature
354:
66-70,
1991[ISI][Medline].
4.
Blakely, RD,
De Felice LJ,
and
Hartzell HC.
Molecular physiology of norepinephrine and serotonin transporters.
J Exp Biol
196:
263-281,
1994
5.
Boddeke, HWGM,
Meigel I,
Boeijinga P,
Arbuckle J,
and
Docherty RJ.
Modulation by calcineurin of 5-HT3 receptor function in NG108-15 neuroblastoma x glioma cells.
Br J Pharmacol
118:
1836-1840,
1996[Abstract].
6.
Brady, CA,
Stanford IM,
Ali I,
Lin L,
Williams JM,
Dubin AE,
Hope AG,
and
Barnes NM.
Pharmacological comparison of human homomeric 5-HT3A receptors versus heteromeric 5-HT3A/3B receptors.
Neuropharmacology
41:
282-284,
2001[ISI][Medline].
7.
Brown, AM,
Hope AG,
Lambert JJ,
and
Peters JA.
Ion permeation and conduction in a human recombinant 5-HT3 receptor subunit (h5-HT3A).
J Physiol
507:
653-665,
1998
8.
Bülbring, E,
and
Crema A.
The release of 5-hydroxytryptamine in relation to pressure exerted on the intestinal mucosa.
J Physiol
146:
18-28,
1959[ISI].
9.
Bush, TG,
Spencer NJ,
Watters N,
Sanders KM,
and
Smith TK.
Effects of alosetron on spontaneous migrating motor complexes in murine small and large bowel in vitro.
Am J Physiol Gastrointest Liver Physiol
281:
G974-G983,
2001
10.
Chang, AS,
Chang SM,
Starnes DM,
Schroeter S,
Bauman AL,
and
Blakely RD.
Cloning and expression of the mouse serotonin transporter.
Mol Brain Res
43:
185-192,
1996[ISI][Medline].
11.
Chen, JX,
Pan H,
Rothman TP,
Wade PR,
and
Gershon MD.
Guinea pig 5-HT transporter: cloning, expression, distribution, and function in intestinal sensory reception.
Am J Physiol Gastrointest Liver Physiol
275:
G433-G448,
1998
12.
Chen, JJ,
Zhishan L,
Pan H,
Murphy DL,
Tamir H,
Koepsell H,
and
Gershon MD.
Maintenance of serotonin in the intestinal mucosa and ganglia of mice that lack the high affinity serotonin transporter (SERT): abnormal intestinal motility and the expression of cation transporters.
J Neurosci
21:
6348-6361,
2001
13.
Conlon, RA,
and
Rossant J.
Exogenous retinoic acid rapidly induces anterior ectopic expression of murine Hox-2 genes in vivo.
Development
116:
357-368,
1992
14.
Cooke, H, J,
Sidhu M,
and
Wang YZ.
5-HT activates neural reflexes regulating secretion in the guinea-pig colon.
Neurogastroenterol Motil
9:
181-186,
1997[ISI][Medline].
15.
Davies, PA,
Pistis M,
Hanna MC,
Peters JA,
Lambert JJ,
Hales TG,
and
Kirkness EFA
The 5-HT3B subunit is a major determinant of serotonin-receptor function.
Nature
397:
359-363,
1999[ISI][Medline].
16.
Dubin, AE,
Huvar R,
D'Andrea MR,
Pyati J,
Zhu JY,
Joy KC,
Wilson SJ,
Galindo JE,
Glass CA,
Luo L,
Jackson MR,
Lovenberg TW,
and
Erlander MG.
The pharmacological and functional characteristics of the serotonin 5-HT(3A) receptor are specifically modified by a 5-HT(3B) receptor subunit.
J Biol Chem
274:
30799-30810,
1999
17.
Erspamer, V.
Pharmacology of indolealklyamines.
Pharmacol Rev
6:
425-487,
1954[ISI].
18.
Fabre, V,
Beaufour C,
Evrard A,
Rioux A,
Hanoun N,
Lesch KP,
Murphy DL,
Lanfumey L,
Hamon M,
and
Martres MP.
Altered expression and functions of serotonin 5-HT1A and 5-HT1B receptors in knock-out mice lacking the 5-HT transporter.
Eur J Neurosci
12:
2299-2310,
2000[ISI][Medline].
19.
Friedel, D,
Thomas R,
and
Fisher RS.
Ischemic colitis during treatment with alosetron.
Gastroenterology
120:
557-560,
2001[ISI][Medline].
20.
Galligan, JJ.
Electrophysiological studies of 5-hydroxytryptamine receptors on enteric neurons.
Behav Brain Res
73:
199-201,
1996[ISI][Medline].
21.
Galligan, JJ.
5-HT receptors on single enteric nerves.
In: 5-Hydroxytryptamine in the Gastrointestinal Tract, edited by Gaginella TS,
and Galligan JJ.. Boca Raton, FL: CRC, 1995, p. 109-126.
22.
Galligan, JJ,
Tatsumi H,
Shen KZ,
Surprenant A,
and
North RA.
Cation current activated by hyperpolarization (IH) in guinea pig enteric neurons.
Am J Physiol Gastrointest Liver Physiol
259:
G966-G972,
1990
23.
Gebauer, A,
Merger M,
and
Kilbinger H.
Modulation by 5-HT3 and 5-HT4 receptors of the release of 5-hydroxytryptamine from the guinea pig small intestine.
Naunyn Schmiedebergs Arch Pharmacol
347:
137-140,
1993[ISI][Medline].
24.
Gershon, MD.
5-HT (serotonin) physiology and related drugs.
Curr Opin Gastroenterol
16:
113-120,
2000[ISI].
25.
Gershon, MD.
Biochemistry and physiology of serotonergic transmission.
In: Handbook of Physiology. The Nervous System. Cellular Biology of Neurons. Bethesda, MD: Am Physiol Soc, 1977, sect. 1, vol. I, pt. 1, chapt. 16, p. 573-623.
26.
Gershon, MD,
and
Ross LL.
Radioisotopic studies of the binding, exchange, and distribution of 5-hydroxytryptamine synthesized from its radioactive precursor.
J Physiol
186:
451-476,
1966[ISI][Medline].
27.
Glitsch, M,
Wischmeyer E,
and
Karschin A.
Functional characterization of two 5-HT3 receptor splice variants isolated from a mouse hippocampal cell line.
Pflügers Arch
432:
134-143,
1996[ISI][Medline].
28.
Gobbi, G,
Murphy DL,
Lesch K,
and
Blier P.
Modifications of the serotonergic system in mice lacking serotonin transporters: an in vivo electrophysiological study.
J Pharmacol Exp Ther
296:
987-995,
2001
29.
Greenfield, LJ, Jr,
and
Macdonald RL.
Whole-cell and single-channel 1
1
2s GABAA receptor currents elicited by a "multipuffer" drug application device.
Pflügers Arch
432:
1080-1090,
1996[ISI][Medline].
30.
Gregory, RE,
and
Ettinger DS.
5-HT3 receptor antagonists for the prevention of chemotherapy-induced nausea and vomiting. A comparison of their pharmacology and clinical efficacy.
Drugs
55:
173-189,
1998[ISI][Medline].
31.
Grundy, D,
Blackshaw LA,
and
Hillsley K.
Role of 5-hydroxytryptamine in gastrointestinal chemosensitivity.
Dig Dis Sci
39, Suppl 12:
44S-47S,
1994[Medline].
32.
Hanani, M,
Francke M,
Hartig W,
Grosche J,
Reichenbach A,
and
Pannicke T.
Patch-clamp study of neurons and glial cells in isolated myenteric ganglia.
Am J Physiol Gastrointest Liver Physiol
278:
G644-G651,
2000
33.
Hanna, MC,
Davies PA,
Hales TG,
and
Kirkness EF.
Evidence for expression of heteromeric serotonin 5-HT(3) receptors in rodents.
J Neurochem
75:
240-247,
2000[ISI][Medline].
34.
Hillsley, K,
Kirkup AJ,
and
Grundy D.
Direct and indirect actions of 5-hydroxytryptamine on the discharge of mesenteric afferent fibers innervating the rat jejunum.
J Physiol
506:
551-561,
1998
35.
Hubbard, PC,
Thompson AJ,
and
Lummis SC.
Functional differences between splice variants of the murine 5-HT(3A) receptor: possible role for phosphorylation.
Brain Res Mol Brain Res
81:
101-108,
2000[ISI][Medline].
36.
Johnson, DS,
and
Heinemann SF.
Detection of 5-HT3R-A, a 5-HT3 receptor subunit, in submucosal and myenteric ganglia of rat small intestine using in situ hybridization.
Neurosci Lett
184:
67-70,
1995[ISI][Medline].
37.
Jones, S,
and
Yakel JL.
Ca2+ influx through voltage-gated Ca2+ channels regulates 5-HT3 receptor channel desensitization in rat glioma x mouse neuroblastoma hybrid NG108-15 cells.
J Physiol
510:
361-370,
1998
38.
Joyce, MP,
and
Rayport S.
Mesoaccumbens dopamine neuron synapses reconstructed in vitro are glutamatergic.
Neuroscience
99:
445-456,
2000[ISI][Medline].
39.
Kadowaki, M,
Wade PR,
and
Gershon MD.
Participation of 5-HT3, 5-HT4, and nicotinic receptors in the peristaltic reflex of the guinea pig distal colon.
Am J Physiol Gastrointest Liver Physiol
271:
G849-G857,
1996
40.
Kirchgessner, AL.
Distribution of 5-HT3 receptors in the mucosa of the guinea pig bowel supports a role in intrinsic and extrinsic sensory transmission (Abstract).
Gastroenterology
120:
198,
2001.
41.
Kirchgessner, AL,
and
Liu M.
Orexin synthesis and response in the gut.
Neuron
24:
941-951,
1999[ISI][Medline].
42.
Kirchgessner, AL,
Liu MT,
and
Gershon MD.
In situ identification and visualization of neurons that mediate enteric and enteropancreatic reflexes.
J Comp Neurol
371:
270-286,
1996[ISI][Medline].
43.
Kirchgessner, AL,
Tamir H,
and
Gershon MD.
Identification and stimulation by serotonin of intrinsic sensory neurons of the submucosal plexus of the guinea pig gut: activity-induced expression of Fos immunoreactivity.
J Neurosci
12:
235-249,
1992[Abstract].
44.
La Cour, CM,
Boni C,
Hanoun N,
Lesch KP,
Hamon M,
and
Lanfumey L.
Functional consequences of 5-HT transporter gene disruption on 5-HT(1a) receptor-mediated regulation of dorsal raphe and hippocampal cell activity.
J Neurosci
21:
2178-2185,
2001
45.
Lankiewicz, S,
Lobitz N,
Wetzel CH,
Rupprecht R,
Gisselmann G,
and
Hatt H.
Molecular cloning, functional expression, and pharmacological characterization of 5-hydroxytryptamine3 receptor cDNA and its splice variants from guinea pig.
Mol Pharmacol
53:
202-212,
1998
46.
Maricq, AV,
Peterson AS,
Brake AJ,
Myers RM,
and
Julius D.
Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel.
Science
254:
432-437,
1991[ISI][Medline].
47.
Mawe, GM,
Branchek TA,
and
Gershon MD.
Peripheral neural serotonin receptors: identification and characterization with specific antagonists and agonists.
Proc Natl Acad Sci USA
83:
9799-9803,
1986[Abstract].
48.
Miquel, MC,
Emerit MB,
Gingrich JA,
Nosjean A,
Hamon M,
and
Mestikawy SE.
Developmental changes in the differential expression of two serotonin 5-HT3 receptor splice variants in the rat.
J Neurochem
65:
475-483,
1995[ISI][Medline].
49.
Morales, M,
Battenberg E,
and
Bloom FE.
Distribution of neurons expressing immunoreactivity for the 5HT3 receptor subtype in the rat brain and spinal cord.
J Comp Neurol
402:
385-401,
1998[ISI][Medline].
50.
Mössner, R,
Albert D,
Persico AM,
Hennig T,
Bengel D,
Holtmann B,
Schmitt A,
Keller F,
Simantov R,
Murphy D,
Seif I,
Deckert J,
and
Lesch KP.
Differential regulation of adenosine A(1) and A(2A) receptors in serotonin transporter and monoamine oxidase A-deficient mice.
Eur Neuropsychopharmacol
10:
489-493,
2000[ISI][Medline].
51.
Nagakura, Y,
Kontoh A,
Tokita K,
Tomoi M,
Shimomura K,
and
Kadowaki M.
Combined blockade of 5-HT3- and 5-HT4-serotonin receptors inhibits colonic functions in conscious rats and mice.
J Pharmacol Exp Ther
281:
284-290,
1997
52.
Neya, T,
Mizutani M,
and
Yamasato T.
Role of 5-HT3 receptors in peristaltic reflex elicited by stroking the mucosa in the canine jejunum.
J Physiol
471:
159-173,
1993[Abstract].
53.
Niemeyer, MI,
and
Lummis SC.
The role of the agonist binding site in Ca(2+) inhibition of the recombinant 5-HT(3A) receptor.
Eur J Pharmacol
428:
153-161,
2001[ISI][Medline].
54.
Pan, H,
and
Gershon MD.
Activation of intrinsic afferent pathways in submucosal ganglia of the guinea pig small intestine.
J Neurosci
20:
3295-3309,
2000
55.
Persico, AM,
Mengual E,
Moessner R,
Hall FS,
Revay RS,
Sora I,
Arellano J,
DeFelipe J,
Gimenez-Amaya JM,
Conciatori M,
Marino R,
Baldi A,
Cabib S,
Pascucci T,
Uhl GR,
Murphy DL,
Lesch KP,
Keller F,
and
Hall SF.
Barrel pattern formation requires serotonin uptake by thalamocortical afferents, and not vesicular monoamine release.
J Neurosci
21:
6862-6873,
2001
56.
Peters, JA,
Hales TG,
and
Lambert JJ.
Divalent cations modulate 5-HT3 receptor-induced currents in N1E-115 neuroblastoma cells.
Eur J Pharmacol
151:
491-495,
1988[ISI][Medline].
57.
Racké, K,
Reimann A,
Schwörer H,
and
Kilbinger H.
Regulation of 5-HT release from enterochromaffin cells.
Behav Brain Res
73:
83-87,
1996[ISI][Medline].
58.
Ravary, A,
Muzerelle A,
Darmon M,
Murphy DL,
Moessner R,
Lesch KP,
and
Gaspar P.
Abnormal trafficking and subcellular localization of an N-terminally truncated serotonin transporter protein.
Eur J Neurosci
13:
1349-1362,
2001[ISI][Medline].
59.
Reeves, DC,
and
Lummis SC.
The molecular basis of the structure and function of the 5-HT3 receptor: a model ligand-gated ion channel.
Mol Membr Biol
19:
11-26,
2002[ISI][Medline].
60.
Richardson, BP,
Engel G,
Donatsch P,
and
Stadler PA.
Identification of serotonin M-receptor subtypes and their specific blockade by a new class of drugs.
Nature
316:
216-231,
1985[ISI].
61.
Rugiero, F,
Gola M,
Kunze WAA,
Reynaud JC,
Furness JB,
and
Clerc N.
Analysis of whole-cell currents by patch clamp of guinea-pig myenteric neurones in intact ganglia.
J Physiol
538:
447-463,
2002
62.
Sepulveda, MI,
Lummis SC,
and
Martin IL.
The agonist properties of m-chlorophenylbiguanide and 2-methyl-5-hydroxytryptamine on 5-HT3 receptors in N1E-115 neuroblastoma cells.
Br J Pharmacol
104:
536-540,
1991[Abstract].
63.
Überla, K,
Platzer C,
Diamantstein T,
and
Blankenstein T.
Generation of competitor DNA fragments for quantitative PCR.
PCR Methods Appl
1:
136-139,
1991[Medline].
64.
Vogalis, F,
Hillsley K,
and
Smith TK.
Diverse ionic currents and electrical activity of cultured myenteric neurons from the guinea pig proximal colon.
J Neurophysiol
83:
1253-1263,
2000
65.
Wade, PR,
Chen J,
Jaffe B,
Kassem IS,
Blakely RD,
and
Gershon MD.
Localization and function of a 5-HT transporter in crypt epithelia of the gastrointestinal tract.
J Neurosci
16:
2352-2364,
1996[Abstract].
66.
Werner, P,
Kawashima E,
Reid J,
Hussy N,
Lundstrom K,
Buell G,
Humbert Y,
and
Jones KA.
Organization of the mouse 5-HT3 receptor gene and functional expression of two splice variants.
Brain Res Mol Brain Res
26:
233-241,
1994[ISI][Medline].
67.
Wood, JD.
Electrical and synaptic behavior of enteric neurons.
In: Handbook of Physiology. The Gastrointestinal System. Motility and Circulation. Bethesda, MD: Am Physiol Soc, 1989, sect. 6, vol. I, pt. 1, chapt. 14, p. 465-517.
68.
Yakel, JL,
and
Jackson MB.
5-HT3 receptors mediate rapid responses in cultured hippocampus and a clonal cell line.
Neuron
1:
615-621,
1988[ISI][Medline].
69.
Yakel, JL,
Lagrutta A,
Adelman JP,
and
North RA.
Single amino acid substitution affects desensitization of the 5-hydroxytryptamine type 3 receptor expressed in Xenopus oocytes.
Proc Natl Acad Sci USA
90:
5030-5033,
1993[Abstract].
70.
Zhai, J,
Gershon MD,
Walsh J,
Wong H,
and
Kirchgessner AL.
Inward currents in neurons from newborn guinea pig intestine: mediation by 5-HT3 receptors.
J Pharmacol Exp Ther
291:
374-382,
1999
71.
Zhou, X,
and
Galligan JJ.
Synaptic activation and properties of 5-hydroxytryptamine3 receptors in myenteric neurons of guinea pig intestine.
J Pharmacol Exp Ther
290:
803-810,
1999