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The Journal of Neuroscience, August 15, 2001, 21(16):6348-6361
Maintenance of Serotonin in the Intestinal Mucosa and Ganglia of
Mice that Lack the High-Affinity Serotonin Transporter: Abnormal
Intestinal Motility and the Expression of Cation
Transporters
Jason J.
Chen1,
Zhishan
Li1,
Hui
Pan1,
Dennis L.
Murphy2,
Hadassah
Tamir1, 3,
Hermann
Koepsell4, and
Michael D.
Gershon1
1 Department of Anatomy and Cell Biology, Columbia
University, College of Physicians and Surgeons, New York, New York
10032, 2 Laboratory of Clinical Science, National Institute
of Mental Health, Bethesda, Maryland 20892, 3 New York
State Psychiatric Institute, New York, New York 10032, and
4 Anatomisches Institut der Bayerischen
Julius-Maximilians-Universitat, D-97070 Würzburg, Germany
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ABSTRACT |
The enteric serotonin reuptake transporter (SERT) has been proposed
to play a critical role in serotonergic neurotransmission and in the
initiation of peristaltic and secretory reflexes. We analyzed potential
compensatory mechanisms and enteric function in the bowels of mice with
a targeted deletion of SERT. The guts of these animals were found to
lack mRNA encoding SERT; moreover, high-affinity uptake of 5-HT into
epithelial cells, mast cells, and enteric neurons was present in the
SERT +/+ bowel but absent in the SERT 
/ bowel. However, both the
SERT +/+ gut and the / gut expressed molecules capable of
transporting 5-HT, but with affinities and selectivity much lower than
those of SERT. These included the dopamine transporter (DAT) and
polyspecific organic cation transporters OCT-1 and OCT-3. DAT and OCT
immunoreactivities were present in both the submucosal and myenteric
plexuses, and the OCTs were also located in the mucosal epithelium.
5-HT was found in all of its normal sites in the SERT / bowel,
which contained mRNA encoding tryptophan hydroxylase, but no 5-HT was present in the blood of SERT / animals. Stool water and colon motility were increased in most SERT / animals; however, the increase in motility (diarrhea) occasionally alternated irregularly with decreased motility (constipation). The watery diarrhea is probably
attributable to the potentiation of serotonergic signaling in
SERT / mice, whereas the transient constipation may be caused by
episodes of enhanced 5-HT release leading to 5-HT receptor desensitization.
Key words:
serotonin reuptake transporter; enteric nervous system; gastrointestinal motility; organic cation transporters; dopamine
transporter; SERT knock-out mice
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INTRODUCTION |
The gut is the only organ that
manifests complex integrative behaviors and reflexes in the absence of
input from the CNS (Trendelenburg, 1917
; Furness and Costa,
1987; Gershon et al., 1994; Gershon, 1999). This ability is conferred
on the bowel by its intrinsic innervation, the enteric nervous system
(ENS) (Meissner, 1857; Auerbach, 1862; Bayliss and Starling, 1899;
Bayliss and Starling, 1900a,b), which contains both the intrinsic
primary afferent neurons and the interneurons (Bülbring et al.,
1958; Kirchgessner et al., 1992, 1996; Kunze et al., 1995; Bertrand et
al., 2000; Pan and Gershon, 2000). These cells enable the gut to detect
and process sensory stimuli without extra-enteric assistance.
Regulation of digestive activity by the ENS requires that it monitor
the pressure and chemical content of the intestinal lumen;
nevertheless, no nerves, intrinsic or extrinsic, penetrate the mucosal
epithelium. Neuronal detection of luminal stimuli, therefore, is a
trans-epithelial phenomenon. This process is mediated
by subsets of enteroendocrine cells, which are specialized to act as
sensory transducers (Kirchgessner et al., 1992; Grider et al., 1996,
1998; Pan and Gershon, 2000). These cells respond to luminal stimuli by
releasing chemical transmitters that activate underlying primary
afferent nerve fibers. The most well characterized of these sensory
transmitters is serotonin (5-HT), which is produced and secreted by
enterochromaffin (EC) cells (Erspamer, 1966; Vialli, 1966).
EC cells do not present a fixed target with which afferent nerve fibers
can form a junction. All of the cells of the mucosal epithelium,
including the EC cells, move because they are continuously replaced
(Stappenbeck et al., 1998; Wong et al., 1999). New cells are generated
in a stem cell zone in crypts and mature as they translocate to villus
tips, in which they die and slough into the lumen. Epithelial cells
also migrate to restore the integrity of the mucosal lining after
injury (Heath, 1996). Replacement of EC cells is slower than that of
enterocytes (Tsubouchi and Leblond, 1979), but the anatomical
relationship between EC cells and primary afferent nerve fibers varies,
and the distance between them is large (Wade and Westfall, 1985). EC
cells thus signal by secreting massive amounts of 5-HT (Erspamer,
1966), which can be tolerated because 5-HT is efficiently inactivated
within the bowel (Wade et al., 1996; Chen et al., 1998).
Inactivation is critical for the termination of responses to 5-HT to
prevent enteric 5-HT from exerting toxic effects on distant targets
(Gershon and Ross, 1962) and from desensitizing its receptors (Chen et
al., 1998). Mucosally released 5-HT is rapidly inactivated by reuptake
into mucosal epithelial cells (Wade et al., 1996; Chen et al., 1998)
mediated by a plasmalemmal 5-HT transporter serotonin reuptake
transporter (SERT), the same molecule that is responsible for
5-HT reuptake into serotonergic neurons. Inhibition of mucosal SERT
potentiates responses of intrinsic primary afferent neurons to the 5-HT
secreted by EC cells and, if severe and prolonged, causes neuronal 5-HT
receptors to desensitize (Chen et al., 1998; Pan and Gershon, 2000).
Recently, mice that lack SERT activity have been generated by the
targeted elimination of the second exon of the gene encoding SERT
(Bengel et al., 1998). These mice survive well, although they are
unable to take up 5-HT with high affinity and are insensitive to
3,4-methylenedioxymethamphetamine ("ecstasy"), a compound that
releases 5-HT by a transporter-dependent mechanism. The current
experiments were undertaken to investigate gastrointestinal function in
mice that lack SERT and to determine whether compensatory mechanisms
develop in these animals that permit them to inactivate 5-HT released
in the gut.
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MATERIALS AND METHODS |
Animals and tissue preparation. Mice lacking SERT
(SERT / mice) were bred in the Laboratory of Clinical Science at
the National Institute of Mental Health and transferred to Columbia
University for analysis. In these animals, the second exon of the SERT
gene has been replaced by homologous recombination with a
phosphoglycerine kinase-neo gene cassette (Bengel et al., 1998).
The backgrounds of the mice were CD-1 and C57BL/6J. Both strains were
investigated, although no strain-related differences were found in any
of the experiments. Control animals consisted of heterozygous and
homozygous (wild-type) littermates of the mice that lacked SERT. Mice
that lacked both SERT and the dopamine (DA) transporter (DAT) (Sora et
al., 1998) were also investigated. These mice and their heterozygous and homozygous (wild-type) littermates were obtained from Dr. F. Scott
Hall (National Institute on Drug Abuse, Bethesda, MD) and
dissected in the laboratory of Dr. Courtney DeVries (Johns Hopkins
University, Baltimore, MD). The gut was removed at Johns Hopkins
University, fixed (see below), and transported to Columbia University
for additional analysis. The backgrounds of the double knock-out mice
lacking SERT and DAT were C57BL/6J.
For molecular and histological analyses, the mice were rapidly killed
by cervical dislocation. This procedure was approved by the Animal Care
and Use Committee of Columbia University. The small intestines and
brains were removed from the animals. Small intestines were opened and
cleaned. For physiological studies, segments of gut were placed in iced
Krebs' solution and bubbled with a mixture of
95%O2-5% CO2 until
further investigated. For extraction of RNA, the tissue was transferred
to an iced solution of Trizol (Life Technologies, Gaithersburg, MD).
Brains were placed in Trizol for use as a positive control in studies
of RNA.
Tissues to be examined by immunocytochemistry were fixed by immersion
for 6-24 hr with 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M phosphate buffer, pH 7.5, at
4°C. After fixation, tissues were rinsed with PBS,
cryoprotected overnight with 30% sucrose (w/v) in PBS at 4°C,
embedded in O.C.T. (Miles Inc., Elkhart, IN), and then stored at
80°C until they were used. Sections were cut with a
cryostat-microtome and thaw-mounted onto Superfrost/plus positively
charged glass slides (Fisher Scientific, Pittsburgh, PA).
Reverse transcription-PCR. Specimens were
homogenized in Trizol (1 ml/100 mg tissue). Chloroform (10% of the
total volume) was added, and the sample was covered, shaken vigorously,
and placed on ice for 10 min before being subjected to centrifugation for 15 min (13,000 × g). The aqueous phase was
removed, and isopropanol (60% of the Trizol volume) was used to
precipitate the RNA. The RNA pellet was washed by resuspending it in
70% ethanol. The suspension was then centrifuged for 5 min
(10,000 × g), dried, and redissolved in
diethylpyrocarbonate-treated water. The extracted RNA (2.5-3.0 µg)
was used as a template for random hexamer-primed first strand cDNA
synthesis catalyzed by Maloney murine leukemia virus reverse transcriptase (Life Technologies). One-tenth of the resulting cDNA from
the original RNA was used for PCR amplification. Pairs of
oligonucleotide primers were designed from the cDNA sequence of the
mouse SERT (GenBank accession number Y08870; forward, 5'-CAA AAC CAA
GAA CCA AGA G-3', for nucleotides 98-116; reverse, 5'-CAT AGC CAA TGA
CAG ACA G-3', for nucleotides 454-436). Additional pairs of primers
were used to detect cDNA encoding organic cation transporters (OCT)
1-3 (Busch et al., 1996b; Gründemann et al., 1998; Kekuda et
al., 1998; Urakami et al., 1998; Wu et al., 1998) and tryptophan
hydroxylase (Stoll and Goldman, 1991). The following primers were used:
OCT-1 (GenBank accession number AB016257; forward, 5'-TCC ATG TTG CTC
TTT CGC C-3', for nucleotides 638-656; reverse, 5'-TCA CAT TCA ACC AAT
GCA GCT C-3', for nucleotides 1334-1313), OCT-2 (GenBank accession
number AJ006036; forward, 5'-CCA GTG CAT GAG GTA TGA G-3', for
nucleotides 353-371; reverse, 5'-CAG GAG CCC AAC AGT AAA G-3', for
nucleotides 842-824), OCT-3 (GenBank accession number AF082566;
forward, 5'-TGG AAG CCA CTA ATA CCA GC-3', for nucleotides 547-566;
reverse, 5'-GGG ACC ACC CAG TAA TAG AG-3', for nucleotides 1114-1095),
tryptophan hydroxylase (GenBank accession number J04758; forward,
5'-ATG AGA GAA TTT GCC AAG ACC-3', for nucleotides 1258-1278; reverse, 5'-CGT GAA CTA TAT TTC CCT CAG C-3', for nucleotides 1668-1647), and
DAT (GenBank accession number AF109072; forward, 5'-GGT CAA GGA GCA GAA
TGG AG-3', for nucleotides 179-198; reverse, 5'-CAA AAT ACT CAG CAG
CGG G-3', for nucleotides 729-711). Twenty to 35 cycles of PCR
amplification were performed as follows: 94°C for 1 min, 55°C for
50 sec, and 72°C for 40 sec. A PCR product of ~357 bp was obtained.
To confirm the identity of the PCR product, it was cloned into a pCRII
vector by using the TA-cloning kit (Invitrogen, San Diego, CA).
Inserts in two individual clones were sequenced by the
dideoxynucleotide-chain termination method (in the core facility of
Columbia University). The sequence of the PCR product obtained from
both brain and gut with the indicated primers was identical to that of
the corresponding sequence in mouse SERT.
Assay of 3H-5-HT transport. To
assay the uptake of 5-HT, the opened gut was pinned flat on a support
of balsa wood. The preparations were then carried on their supporting
frames through a succession of vials in Krebs' solution supplemented
with L-ascorbic acid (100 µM) and the monoamine oxidase (MAO) inhibitor
pargyline (100 µM). The solutions were
maintained at 37°C in an atmosphere of 95%
O2-5% CO2. The frames
were cut in such a way that the flat sheets of tissue were exposed on
both sides to the ambient medium. Preparations were equilibrated for 30 min and then transferred to a solution containing
3H-5-HT (20 nM)
(DuPont NEN, Boston, MA). Specimens were incubated with
3H-5-HT for 30 min. The uptake of
3H-5-HT was terminated by washing the
tissues twice in 250 ml of iced Krebs' solution in separate
containers. Controls consisted of equivalent specimens incubated with
3H-5-HT for 30 min in solutions that
lacked Na+ (replaced isosmotically with
choline chloride) or that contained the SERT antagonists fluoxetine (10 µM) or chlorimipramine (0.1 µM). Additional specimens were incubated with
3H-5-HT for 30 min in the presence of
quinine (10 µM) to inhibit OCT-1 (Busch et al.,
1996b). To determine how much 3H-5-HT had
entered intracellular compartments,
3H-5-HT was extracted with 70% ethanol
(Gershon and Altman, 1971) and assayed by liquid scintillation spectrometry.
For radioautography, the specimens that had been exposed to
3H-5-HT were fixed overnight in a solution
containing 2.5% glutaraldehyde in 0.1 M phosphate buffer,
pH 7.4, at 4°C, to which 3% sucrose was added to prevent the
translocation of 3H-5-HT during fixation
(Fischman and Gershon, 1964; Gershon and Ross, 1966b). The fixed
preparations were washed, dehydrated through an ascending series of
ethanols, cleared in propylene oxide, and embedded in an epoxy resin
(Epon 812). Sections were cut at 1.0 µm and mounted on slides that
had been coated with chromium alum gelatin. The slides were ultimately
layered with Ilford L4 photographic emulsion (Ilford, Paramus, NJ) by
dipping in the melted emulsion. The emulsion-coated slides were exposed
for 3 weeks at room temperature in an atmosphere of dry 100%
CO2. The specimens were then developed with a
Kodak D19 developer (Eastman Kodak, Rochester, NY), fixed, washed, and
counterstained with toluidine blue. Slides were examined microscopically using both bright-field and reflected dark-field illumination.
Immunocytochemistry. Primary antibodies generated against
sequences found in the rat 5-HT transporter were generously donated by
Dr. Randy D. Blakely (Department of Pharmacology, Vanderbilt University
School of Medicine, Nashville, TN). These antibodies were used to
attempt to immunostain the murine transporter in the adult and
developing intestine. The same antibodies have been characterized
previously and used to immunostain guinea pig SERT (Chen et al., 1998).
Studies were also performed with rabbit polyclonal antibodies to OCT-1,
DAT, DA, and 5-HT. The antibodies against OCT-1 have been characterized
previously (Meyer-Wentrup et al., 1998; Karbach et al., 2000).
Antibodies to DAT and DA (murine monoclonal) were obtained from
Chemicon (Temecula, CA); antibodies to DA (polyclonal) and 5-HT were
supplied by Incstar Corporation (Stillwater, MN). The antibodies were
applied overnight in a humidified chamber at 4°C to fixed cryostat
sections of gut. Preparations that were not exposed to primary
antibodies were always processed simultaneously as routine controls.
Secondary antibodies (diluted 1:200) (Vector Laboratories, Burlingame,
CA) were labeled with biotin and visualized with avidin-Cy3 (Jackson
ImmunoResearch, West Grove, PA).
Visualization of submucosal neurons activated by mucosal
stimulation. Mice were killed as described above. The mucosa and submucosa were dissected from the circular muscle as an intact sheet of
tissue ~1.0 cm in length and pinned to silicone elastomer-coated dishes. Preparations were superfused with Krebs' solution that was
oxygenated with a mixture of 95% O2 and 5%
CO2. One half of each preparation was left
unstimulated and served as the control, whereas the other half was
subjected to experimental stimulation. Preparations were
stimulated by stroking villus tips for 5 min in the oral to anal
direction with a soft sponge. Activated neurons were visualized by
stimulation in the presence of
N-(3-(triethylammoniumpropyl)-4-(4-dibutylamino)styryl)pyridinium dibromide (FM2-10) (100 µM; Molecular Probes,
Eugene, OR). The enteric neuronal uptake of FM2-10 in vitro
is known to be stimulation dependent and thus prevented by the addition
of tetrodotoxin (0.5 µM) (Kirchgessner et al.,
1996; Chen et al., 1998). Stimulated preparations were washed for
10-15 min with iced Krebs' solution. The mucosa was then removed,
taking care to keep the tissue cold. Preparations were immediately
examined by fluorescence microscopy (exciting filter bandpass, 530-560
nm; dichroic mirror reflection short pass, 580 nm; edge wavelength, 580 nm). Neurons that had taken up FM2-10 were identified and counted in
both the control and experimental halves of each preparation. In some
controls, fluoxetine (1.0 µM) was added to the
superfusing solution 30 min before stimulation. Comparisons were made
between the number of neurons activated in the control and experimental
sides of each tissue.
Measurement of 5-HT. 5-HT was measured in the bowels
(Kalivas and Miller, 1985) and blood (Anderson et al., 1981) of
wild-type and SERT / mice. Mice were killed as described above;
blood was collected in 14 ml tubes, to which 1% EDTA (1:10 v/v) was added. Sodium metabisulfite (50 µl of a 2% stock solution; to prevent oxidation) and 100 µl of 3.4 M
perchloric acid (to precipitate protein) were added to 500 µl of
blood, vortex mixed, and placed on ice for 10-15 min. The mixture was
then centrifuged at 13,000 × g for 10 min. The
resulting supernatant was transferred to fresh microcentrifuge tubes
and stored at 80°C until analysis. Segments of ileum or colon
(~0.5 gm, wet weight) were collected in 2 ml of an isocratic mobile
phase buffer [0.1 M trichloroacetic acid, 0.01 M sodium acetate, 0.01 mM
EDTA, and 18% (v/v) methanol] at a pH of 3.8. Two hundred microliters
of 1% cysteine was added, and the tissues were immediately
homogenized. The homogenate was then centrifuged at 11,000 × g for 5 min. The resulting supernatant was transferred to
fresh microcentrifuge tubes and stored at 80°C. The supernatants
from blood and gut were used for the assay of 5-HT and 5-hydroxyindole
acetic acid by HPLC (Waters Associates, Milford, MA) with
electrochemical detection (Tamir et al., 1994; Liu et al., 2000).
Colonic transit. The motility of the colon was evaluated in
separate sets of control and SERT / littermates. Animals were lightly anesthetized with ether, and a glass microbead (3 mm in diameter) was inserted through the anus and pushed, with a polished glass rod, into the colon for a distance of 2 cm (Osinki et al., 1999).
The time from the completion of insertion to the expulsion of the bead
was measured to the nearest 0.1 min to provide an estimate of the rate
of transit in the colon.
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RESULTS |
The gut of SERT / mice lacks mRNA encoding SERT
Reverse transcription (RT)-PCR was used to analyze expression of
SERT in +/+, +/, and / mice to confirm that the targeted deletion
eliminates SERT expression in the gut, as well as in the brain. All of
the animals were littermates. The brains of the mice were analyzed as a
positive control. The PCR primers (see Materials and Methods) were
designed on the basis of sequences present in the presumptively deleted
second exon of the mouse gene that encodes SERT. mRNA was detected in
the brains of SERT +/+ and +/ animals but not in the brains of SERT
/ mice (Fig. 1). Similarly, mRNA
encoding SERT was detectable in the colons of SERT +/+ and +/ animals
but not in the colons of SERT / mice.
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Figure 1.
mRNA encoding SERT can be demonstrated in the
murine gut by means of RT-PCR. The expected PCR product is 357 bp. mRNA
encoding SERT is not found in the brains (positive control) or in the
colons of SERT / mice. In contrast, in wild-type (SERT +/+) mice,
both the brain and colon contain mRNA encoding SERT. The
lanes marked + illustrate the results
obtained when RT was present. The lanes marked
were obtained when RT was absent (control indicating
that the preparations were not contaminated with DNA).
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Enteric nerves take up 3H-5-HT in SERT +/+ but not in
SERT / mice
The uptake of 3H-5-HT in the gut was
investigated in SERT +/+ and / mice to determine whether there is a
functional deficit in the gut of the knock-out animals that corresponds
to their genetic abnormality. Radioautography was used so that the
sites responsible for 3H-5-HT uptake in
the bowel wall could be identified. The tissue was incubated for 30 min
at 37°C with 20 nM 3H-5-HT,
a concentration that is below the measured
Km of enteric SERT (0.6-0.7
µM) (Gershon and Altman, 1971; Chen et al.,
1998). This concentration was selected to limit the uptake of
3H-5-HT to that mediated by a
high-affinity transporter, such as SERT, or if there were to be one, a
functionally similar molecule. After incubation, the tissue was washed
and fixed. 3H-5-HT is retained and can be
fixed in the bowel with glutaraldehyde, whereas radioactive metabolites
wash out of the tissue and are not fixed (Gershon and Ross, 1966a,b).
Radioautographic labeling, therefore, is attributable to the presence
of 3H-5-HT itself. In SERT +/+ mice,
3H-5-HT was found to be taken up by axons
of the myenteric plexus (Fig.
2A,C,D)
and also by occasional myenteric nerve cell bodies (Fig.
2C,D). In addition,
3H-5-HT was taken up by epithelial cells
(Fig. 2A) and by mast cells (Fig.
2D, inset). The degree of radioautographic
labeling was far more intense in the neuronal elements and mast
cells than in the epithelium. Mast cells, however, were infrequently
encountered in tissue sections. No differences were noted in the
pattern of labeling in small and large intestines. The uptake of
3H-5-HT was abolished by fluoxetine (10 µM) (Fig. 2B) but was not affected by the addition of quinine (10 µM)
(data not shown), an inhibitor of the polyspecific organic cation
transporter OCT-1 (Busch et al., 1996b).
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Figure 2.
Radioautographic labeling of high-affinity
3H-5-HT uptake sites in the bowel wall of SERT +/+ and SERT
/ mice. A, SERT +/+ small intestine. The
arrow points to a myenteric ganglion labeled by
3H-5-HT. Note the low level of labeling (but greater than
that of background) present over the intestinal epithelium.
B, SERT +/+ small intestine. Fluoxetine was present
during incubation with 3H-5-HT. The arrow
points to a neuron in a myenteric ganglion. Neither the ganglion nor
the epithelium is labeled by 3H-5-HT. C,
D, SERT +/+ colon. A tangential section is cut through the intestinal muscle that
includes an interganglionic connective (arrowheads) and
a neuron (arrow) of the myenteric plexus. Note the
intense labeling of both neural elements. The smooth muscle is not
labeled and thus does not take up 3H-5-HT. The same field
has been visualized by means of incident (vertical) dark-field
microscopy (C) and with a combination of incident
dark-field and transmitted bright-field microscopy
(D). D, Inset, A mast cell
is double labeled with toluidine blue (to demonstrate metachromasia)
and 3H-5-HT. The cell was visualized as in
C. E, F, SERT / colon.
Only a background level of labeling is evident. The same field has been
visualized with a dark field (E) and with
interference contrast optics to show the unstained structures in the
section (F). The arrowheads in
F outline the location of a myenteric ganglion.
cm, Circular muscle; lm, longitudinal
muscle. A mast cell is shown that is stained with toluidine blue
(F, inset), but no radioautographic
labeling can be detected in the corresponding dark field
(E, inset). G,
H, SERT / colon. The section passes through the
mucosa. No radioautographic labeling can be discerned. The section was
photographed to show the same field in dark field
(G) and interference contrast views
(H). The locations of intestinal crypts,
epithelia (ep), and a goblet cell
(g) are indicated in H.
Scale bars, 25 µm. The scale bars in D,
F, and H also apply to the corresponding
dark-field images in C, E, and
G.
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In contrast to the uptake of 3H-5-HT in
the gut wall of SERT +/+ mice, no sites were observed under any
conditions to become radioautographically labeled by
3H-5-HT in the bowels of SERT /
animals (Fig. 2E-H). Specifically, only a
background density of silver grains was found in the SERT / gut
over the myenteric plexus (Fig. 2, compare E,
F), the epithelium (Fig. 2, compare G,
H), or mast cells (Fig. 2, compare inset
E, inset F). These observations
confirm previous observations about the locations of
3H-5-HT uptake sites in the normal bowel
wall (Gershon and Ross, 1966b; Wade et al., 1996) and indicate that
high-affinity reuptake sites are completely lacking in SERT / mice.
These data, and the sensitivity of 3H-5-HT
uptake to inhibition by fluoxetine and not by quinine, thus support the
idea that high-affinity uptake of 3H-5-HT
in the bowel wall is exclusively mediated by SERT.
mRNA encoding DAT is present in the bowels of SERT
/ mice
Because the SERT / mice are able to survive and eat relatively
normally, we investigated the possibility that they might be able to
express one or more other transporters that could at least
partially compensate for the absence of SERT. Although
the affinity of DAT for 5-HT is much less than that of SERT (Blakely et
al., 1991; Giros et al., 1992; Chen et al., 1998), DAT can transport
5-HT if the 5-HT concentration is sufficiently high (Jackson and
Wightman, 1995; Cases et al., 1998). The enteric expression of DAT was
thus investigated by RT-PCR to determine whether DAT, which might be
able to compensate for the absence of SERT, is expressed in SERT /
mice. The expression of DAT in the brains of the same animals was
studied as a positive control. mRNA encoding DAT was detected in both
the brains and ileum of wild-type (CD-1) mice (Fig.
3A). DAT expression was then
studied in the intestines of SERT and DAT single and double knock-out animals. As expected, no mRNA encoding DAT was detected in the bowels
of mice with a DAT / genotype (Fig. 3B), confirming that the DAT gene expressed in the gut is identical to that cloned originally from the brain. However, mRNA encoding DAT was found in the
intestines of SERT / mice, as well as in those of SERT +/+ and +/
animals. These data are consistent with the possibility that DAT
expression in the SERT / bowel partially compensates in these
animals for the absence of SERT. To do so, however, DAT would have to
be expressed in sites that are proximate to those in which 5-HT is
secreted.
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Figure 3.
mRNA encoding DAT is present in the mouse gut and
brain. The expected PCR product is 550 bp. A, Control
mice. mRNA encoding DAT can be detected in the brain (positive control)
and ileum. The smaller PCR product that is evident on gels lacks 132 bp
in the coding region and creates an in-frame deletion.
B, mRNA encoding DAT is not detectable in the ileum of
mice with a DAT / genotype but is present in DAT +/+ and +/
animals and is detectable in the bowels of mice lacking SERT.
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DAT immunoreactivity is present in the myenteric and
submucosal plexuses
Studies of dopamine metabolites and the sensitivity of enteric
axons to the neurotoxin 6-hydroxydopamine first suggested that the
murine bowel might contain dopaminergic neurons (Eaker et al., 1988).
These observations, the presence of mRNA encoding DAT in the gut of
SERT +/+ and SERT / mice (Fig. 3), and the more recent
demonstration that colonic motility is abnormally slow in DAT /
mice (Walker et al., 2000) imply that DAT is likely to be expressed by
enteric neurons in the mouse. If DAT were to be expressed by neurons
and processes close to serotonergic nerve terminals, then 5-HT uptake
into neurites that express DAT might be able to compensate in SERT
/ mice for the absence of SERT and limit the action of neuronally
secreted 5-HT. Immunocytochemistry was thus used to locate sites of DAT
expression in the bowels of SERT +/+ and SERT / mice. DAT
immunoreactivity was found in both the myenteric (Fig.
4A,B)
and submucosal (Fig. 4C,D) plexuses of both SERT
+/+ (Fig. 4A,C) and SERT /
(Fig. 4B,D) mice.
DAT-immunoreactive neuronal perikaryal, as well as DAT-immunoreactive
neurites, were located in myenteric ganglia. Occasional neurites
extended into the tertiary plexus (Fig.
4A,B). These observations are
compatible with the possibility that DAT expressed in the myenteric and
submucosal plexuses partially compensates in SERT / mice for the
absence of SERT. However, DAT immunoreactivity was not found in the
mucosa (data not shown). Therefore, DAT might be able to mediate 5-HT uptake in the ENS of SERT / mice, but it cannot compensate for the
absence of SERT in the mucosa.
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Figure 4.
DAT immunoreactivity is present in the
ENS of SERT +/+ and SERT / mice. All four panels
illustrate DAT immunoreactivity. A, Myenteric plexus of
the colon of a mouse with a +/+ genotype for both SERT and DAT.
B, Myenteric plexus of the colon of a mouse with a /
genotype for SERT and a +/ genotype for DAT. The
arrows indicate the location of DAT-immunoreactive nerve
cell bodies; the arrowheads indicate the location of
neurites entering the tertiary plexus. C, Submucosal
plexus of the small intestine of a mouse with a +/+ genotype for both
SERT and DAT. D, Submucosal plexus of the small
intestine of a mouse with a / genotype for SERT and a +/+ genotype
for DAT.
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mRNA encoding polyspecific organic cation transporters is expressed
in the gut of wild-type and SERT / mice
Polyspecific OCT-1 was first isolated from a rat kidney library
and was reported also to be expressed in the rat intestine and liver
(Gründemann et al., 1994). OCT-1 has only ~
the
affinity of SERT for 5-HT, but it has a high capacity and is able to
transport 5-HT and other monoamines across plasma membranes (Busch et
al., 1996a). Therefore, we investigated the expression of OCT-1 in the
brains and intestines of SERT +/+ and / mice (Fig.
5A-C). mRNA encoding OCT-1
was found in the colons but not in the brains of SERT +/+ mice,
confirming that the expression of OCT-1 in mice is similar to that in
rats (Gründemann et al., 1994). In contrast, mRNA encoding OCT-1
was present in both the colons and the brains of the SERT /
animals. OCT-1 was expressed in the small intestine, as well as the
colon, and was present in SERT +/+, +/, and / mice (Fig.
5B,C). These data are consistent with the possibility that OCT-1 contributes to the inactivation of 5-HT
when SERT is absent. The presence of mRNA encoding OCT-1 in the brains
of SERT / mice, when it is not present in the brains of +/+
animals, also suggests that the expression of OCT-1 is upregulated in
the CNS when SERT is absent. Semiquantitative RT-PCR suggested that the
expression of OCT-1 might be similarly upregulated in the small
intestines but not in the colons of SERT / mice.
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Figure 5.
mRNA encoding OCT-1 and OCT-3 can be
demonstrated in the murine gut by means of RT-PCR. The expected PCR
products are 697 bp for OCT-1 and 568 bp for OCT-3. A,
mRNA encoding OCT-1 is found in both the brains and colons of SERT
/ mice. In contrast, only the colon and not the brain contains mRNA
encoding OCT-1 in wild-type (SERT +/+) mice. mRNA encoding  -actin
was simultaneously demonstrated by RT-PCR. B, mRNA
encoding OCT-1 is found in both the small intestines and the colons of
SERT +/+, +/, and / mice. Expression of OCT-1 appears to be
greater relative to that of -actin in the small intestines of SERT
/ mice compared with the small intestines of SERT +/ or +/+ mice.
A similar apparent upregulation of OCT-1 is not seen in the SERT /
colon. C, mRNA encoding OCT-1 appears to be upregulated
in the small intestines of SERT / mice, but the knock-out of DAT
does not appear to affect the expression of OCT-1. D,
mRNA encoding OCT-3 is found in both the small intestines and the
colons of SERT +/+, +/, and / mice. The knock-out of DAT does not
affect the expression of OCT-3.
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OCT-3 is another polyspecific cation transporter that, similar to
OCT-1, is capable of transporting 5-HT (Kekuda et al., 1998; Wu et al.,
1998). Although OCT-3 is most abundantly expressed in the placenta, it
has also been reported to be expressed in the intestine and the brain.
In fact, OCT-3 has been demonstrated recently to be identical to the
extraneuronal monoamine transporter (or uptake2),
which was originally identified because of its ability to take up
catecholamines (Iversen, 1967). As expected, mRNA encoding OCT-3 was
detected in the small and large intestines of SERT +/+ mice and was
also found in these organs in SERT +/ and / animals (Fig.
5D). The expression of OCT-1 (Fig. 5C) or OCT-3
(Fig. 5D) in the gut was not affected by the targeted
deletion of DAT. In contrast to transcripts encoding OCT-1 and OCT-3,
mRNA encoding OCT-2 could not reproducibly be detected in the
intestines of SERT +/ or / mice (data not shown). mRNA encoding
OCT-2, however, was detected in the brains and kidneys of the same
animals, suggesting that little OCT-2 is expressed in the bowel.
SERT and OCT-1 proteins are expressed in the ENS
SERT immunoreactivity has been demonstrated previously in the
submucosal and myenteric plexuses of rats (Wade et al., 1996) and
guinea pigs (Chen et al., 1998). We now report that SERT
immunoreactivity is similarly present in the submucosal and myenteric
plexuses of mice; it can be seen in both SERT +/+ (data not shown) and SERT +/ animals (Fig.
6A,B).
The distribution of SERT immunoreactivity in the ENS is similar to that
of 5-HT (Costa et al., 1982; Furness and Costa, 1982) (see Fig. 9);
SERT-immunoreactive axons are abundant in both plexuses, but nerve cell
bodies are found only in the myenteric plexus. As in guinea pigs and
rats (Wade et al., 1996; Chen et al., 1998), SERT immunoreactivity can
also be found in the mucosal epithelia of mice (Fig.
7A). The distribution of SERT immunoreactivity in the murine mucosa is found throughout the crypt-villus axis (Fig. 7A). This distribution is similar
to that of the guinea pig (Chen et al., 1998) but differs from that of mucosal SERT in the rat (Wade et al., 1996), in which it is primarily expressed only in intestinal crypts.
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Figure 6.
SERT and OCT-1 immunoreactivities are found in
both the submucosal and myenteric plexuses. A,
B, SERT immunoreactivity. Scale bars, 50 µm.
C-F, OCT-1 immunoreactivity. Scale bar, 50 µm. SERT
immunoreactivity is abundant in the neurites of the neuropil of both
the submucosal (A) and myenteric
(B) plexuses of a SERT +/ mouse. Only the
myenteric plexus contains SERT-immunoreactive nerve cell bodies
(arrow). OCT-1-immunoreactive nerve cell bodies
(arrows) are abundant in both the submucosal
(C, E) and myenteric (D,
F) plexuses of SERT +/+ and SERT /
mice.
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Figure 7.
The enteric distributions of SERT and OCT-1
immunoreactivities are similar but not identical. Sections of mouse
intestine were obtained with a cryostat-microtome and immunostained
with antibodies to SERT (A) or OCT-1
(B, C). A, SERT
immunoreactivity in the SERT +/+ small intestine; note the presence of
SERT immunoreactivity in the mucosal epithelium (Ep), as
well as in nerves (n). Neuronal perikarya can be
found in myenteric (My) but not submucosal ganglia.
B, OCT-1 immunoreactivity in the SERT / small
intestine. OCT-1 immunoreactivity is present in the mucosal
epithelium (Ep), as well as in the ENS. In
contrast to the immunoreactivity of SERT, that of OCT-1 is also present
in neurons of the submucosal plexus (SmP).
OCT-1-immunoreactive bundles of nerve fibers are prominent in the
mucosa (n). C, OCT-1
immunoreactivity in the SERT +/+ colon. In the colon, as in the small
intestine, OCT-1 immunoreactivity is present in both the epithelium
(Ep) and nerves. Note that OCT-1 immunoreactivity is
found in nerve fibers within the thick colonic circular muscle. Scale
bar: A-C, 50 µm.
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The immunoreactivity of OCT-1, similar to that of SERT, was found in
both the submucosal (Fig. 6C,D) and myenteric
(Fig. 6E,F) plexuses. In
contrast to SERT immunoreactivity, that of OCT-1 was observed in many
nerve cell bodies in both plexuses. OCT-1-immunoreactive neuronal
perikarya in the myenteric plexus, moreover, were far more abundant
than those that displayed the immunoreactivities of SERT or 5-HT. The
enteric distribution of OCT-1 immunoreactivity also resembled that of
SERT in that it was located in the mucosal epithelium, as well as in
nerves (Fig. 7B,C). The expression
of OCT-1 immunoreactivity in SERT +/+ mice (Figs.
6C,E, 7B) was indistinguishable from
that seen in SERT / animals (Figs.
6D,F, 7C). The location of OCT-1 suggests that its ENS expression is not limited to
serotonergic neurons and suggests that it is expressed by far more
neurons than is SERT. OCT-1, however, is present in locations in which it would have access to 5-HT secreted by either axon terminals within
the enteric plexuses or EC cells of the intestinal mucosa.
The blood, but not the bowel, is depleted of 5-HT in SERT
/ mice
The 5-HT levels of the blood and intestines of SERT /
mice were determined and compared with those of their SERT +/+
littermates. 5-HT was measured in whole blood so as to include the
stores within both platelets and plasma. Surprisingly, although the
5-HT level of whole blood was substantial in SERT +/+ mice, virtually
no 5-HT could be detected in the blood of the SERT / littermates of
the same animals (Fig.
8A). In contrast to the
blood, 5-HT levels in the intestines of the SERT / mice were not
significantly different from those of the SERT +/+ animals (Fig.
8B). These data suggest that SERT biosynthesis or
reuptake, mediated by an alternative transporter, can compensate for
the loss of the SERT-mediated ability to retrieve secreted 5-HT in the
gut of SERT / animals. In contrast, platelets, which do not
synthesize 5-HT, are totally dependent on uptake to obtain 5-HT
(Erspamer, 1966). Platelets thus do not appear to express a transporter
other than SERT that can compensate for the absence of SERT in their
plasma membrane. The virtual absence of 5-HT from the blood of SERT
/ mice suggests that plasma 5-HT is negligible when platelets do
not take up or contain 5-HT. The overflow of 5-HT secreted from the
bowel must therefore be removed, despite the absence of SERT in the
knock-out mice, before reaching the systemic circulation.
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Figure 8.
5-HT levels are depleted in the blood but not in
the gut of SERT / mice. A, Whole-blood 5-HT,
measured by HPLC with electrochemical detection, in the blood of SERT
+/+ and SERT / mice. Note that the amount of 5-HT in the blood of
SERT / mice is below the level of sensitivity of the technique
(~1 pmol/ml). B, The 5-HT content of the full
thickness of the bowel wall was measured in the small intestines and
colons of SERT +/+ and SERT / mice. The tissue 5-HT concentration
is higher in the colon than in small intestine in both types of mice
(p < 0.01); however, in neither region of
the gut is there a significant difference between SERT +/+ and SERT
/ mice in the level of enteric 5-HT.
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5-HT immunoreactivity is present in enteric nerves of SERT
/ mice
Immunocytochemical studies were performed to locate 5-HT
immunoreactivity in the ENS of SERT / mice. Although tryptophan hydroxylase has been reported to be present in the ENS (Yu et al.,
1999), some investigators have suggested that enteric neurons do not
synthesize 5-HT but obtain it exclusively through SERT-mediated uptake
(Costa et al., 1996; Sang and Young, 1996). This idea is based on the
reports from several investigators that enteric neurons must be
preloaded with an exogenous amine (5-HT or 5,7-dihydroxytryptamine) that reacts with antibodies to 5-HT to demonstrate their 5-HT immunoreactivity (Costa et al., 1982). If enteric neurons do depend on
the activity of SERT to obtain 5-HT, no 5-HT would be expected to be
present in enteric neurons of SERT / mice. Enteric neurons would be
predicted to behave just as do blood platelets (see above). In the
current experiments, 5-HT immunoreactivity was found to be readily
demonstrable in the ENS of both SERT +/+ and SERT / mice (Fig.
9). In fact, the appearance of 5-HT
immunoreactivity in both the myenteric [Fig. 9, compare A
and C (SERT +/+) with B and D (SERT
/)] and the submucosal plexus could not be distinguished in the
two types of mice. However, the appearance of the 5-HT-immunoreactive neurites was not identical in SERT +/+ and SERT / mice. The 5-HT-immunoreactive varicose terminal axons in myenteric ganglia were
far more abundant in the SERT / animals than in SERT +/+ mice. In
contrast, there were fewer coarse nonvaricose tracts running through
the myenteric ganglia in SERT / mice than in their SERT +/+
littermates. The tertiary plexus also contained 5-HT-immunoreactive
varicose neurites in both SERT +/+ and SERT / mice; however, the
5-HT-immunoreactive axons of the tertiary plexus were more abundant in
SERT / mice. The 5-HT-immunoreactive neurites in the submucosal
plexus in both SERT +/+ (Fig. 9E) and SERT / (Fig.
9F) mice were mainly varicose terminal axons; no consistent morphological differences were detected in
5-HT-immunoreactive elements of the submucosal plexus in the two types
of mice. 5-HT-immunoreactive nerve cell bodies were observed in the
myenteric plexuses of both SERT +/+ (Fig.
9A,C) and SERT / (Fig.
9B) mice, but no significant differences were found in
either their numbers or their appearance in the two types of mice. In
the SERT +/+ or the SERT / mice, it was not necessary to preload
the tissue with an exogenous amine to visualize 5-HT-immunoreactive
neurons and neurites.
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Figure 9.
5-HT immunoreactivity is present in the
ENS of both SERT +/+ and SERT / mice. 5-HT immunoreactivity was
demonstrated in laminar preparations of the bowel wall containing
either the myenteric or submucosal plexus without
preloading the tissue with an exogenous 5-HT-immunoreactive molecule or
otherwise amplifying the endogenous 5-HT content of the bowel. Tissue
was kept on ice during dissection and fixed immediately thereafter to
minimize the potential for postmortem 5-HT uptake. Preparations from
SERT +/+ mice are illustrated in A, C,
and E. Preparations from SERT / mice are illustrated
in B, D, and F.
A, B, Stomach. C, Colon.
D, Ileum. E, F, Colon. The
locations of nerve cell bodies are indicated by the
arrows [A, B
(inset), C]. The locations of neurites
in the tertiary plexus are indicated by arrowheads
(B-D).
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mRNA encoding tryptophan hydroxylase is present in the gut of
mice lacking SERT, DAT, or both SERT and DAT
SERT / and DAT / mice were bred to obtain double knock-out
animals that lacked both transporters. The DAT / mice were of
interest because the motility of the colon has been found to be
abnormally slow in these animals (Walker et al., 2000). Expression of
mRNA encoding tryptophan hydroxylase was investigated in the bowels of
the resulting animals by RT-PCR. The genotypes of the mice examined and
their corresponding expression of tryptophan hydroxylase are presented
in Figure 10. Brains were investigated as a positive control. RT-PCR was made semiquantitative by using the
relative expression of -actin as an internal standard. mRNA encoding
tryptophan hydroxylase was detected in the bowels of all of the
animals, regardless of their SERT or DAT genotype; however, an apparent
upregulation of mRNA encoding tryptophan hydroxylase was observed in
each of the SERT / mice, regardless of their DAT genotype (Fig.
10). These observations confirm that 5-HT can be synthesized in the
bowels of SERT / mice and are compatible with the possibility that
the expression of tryptophan hydroxylase is upregulated to compensate
for the loss of the SERT-mediated ability of cells to take up 5-HT. In
contrast, the enteric expression of tryptophan hydroxylase appears to
be independent of that of DAT.
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Figure 10.
mRNA encoding tryptophan hydroxylase
(TrH) was detected in the intestines of the
offspring of SERT and DAT knock-out mice. The expected PCR product is
410 bp. The expression of tryptophan hydroxylase was investigated by
semiquantitative RT-PCR in the bowels of mice lacking SERT, DAT, or
both SERT and DAT. The brains of wild-type mice were also studied as a
positive control. The SERT and DAT genotypes of the mice that were
examined are listed above the lanes in the gels
illustrating the corresponding PCR products. Note that there is an
apparent increase in tryptophan hydroxylase expression in all of the
animals with a SERT / genotype (*), but that the level of
expression of tryptophan hydroxylase is independent of the DAT genotype
of the animals.
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Rectal motility is altered in SERT / mice
Stool weight and water content were measured in SERT +/+ and /
mice and compared. Littermates of each genotype were used in the study
and were examined without regard to sex. The animals were closely
matched in terms of body weight (Fig.
11A); however, the
wet weight of the stool collected over a period of 24 hr was significantly greater in SERT / mice compared with their SERT +/+
littermates (p < 0.001). The increased wet
weight of the stool of the SERT / mice was attributable to a
significant increase in stool water (p < 0.001)
(Fig. 11A). The motility of the distal colon and
rectum was assessed to test the hypothesis that stool water is
increased in SERT / mice because an excessively rapid rate of stool
transit in these animals provides inadequate time for normal water
resorption in their large intestine. Expulsion of glass beads from the
rectum was used to evaluate motility. The beads were inserted into the
colon at a uniform distance of 2 cm from the anal verge. The time to
expulsion was then measured for each SERT +/+ or / mouse
individually. These times were plotted as a histogram (Fig.
11B,C). The time required for the beads to be expelled was shorter in most of the SERT / mice than in
their SERT +/+ littermates; however, in a small subset of SERT /
individuals, the time to expulsion was actually greater than that of
any of the SERT +/+ animals. The longest time a SERT +/+ mouse took to
expel the bead was ~350 min (Fig. 11B), whereas some SERT / animals required 425-550 min (Fig. 11C).
The z scores for the expulsion times showed that they were normally
distributed in SERT +/+ mice; all scores fell within ±2 SDs of the
mean (Fig. 11B). In contrast, the z scores showed
that the distribution of expulsion times was highly skewed in the SERT
/ animals (Fig. 11C); the bulk of the times were faster
than the mean, but a small number were slower, and some were much
slower (>2 SDs greater than the mean). These observations suggest that
the motility of the colon and rectum is increased in most SERT /
mice but decreased in a small subset of these mice. The SERT /
animals can thus be divided into a larger group with diarrhea (~75%
of the animals; bead expulsion times below the lower 95% confidence
limit of the SERT +/+ mean) and a smaller remaining group with
constipation (Fig. 11D) (bead expulsion times above
the upper 95% confidence limit of the SERT +/+ mean). The colorectal
motility of the SERT / mice with diarrhea was significantly faster
than that of their SERT +/+ littermates (p < 0.001). In contrast, the motility of the colon and rectum of the small
subset of constipated SERT / mice was significantly slower than
that of either their SERT +/+ littermates (p < 0.002) or the SERT / mice with diarrhea (p < 0.001). When rectal motility of individual SERT / animals was
measured at weekly intervals, the glass bead expulsion times were found
to change so that animals that were constipated when first examined
developed diarrhea and mice with diarrhea became constipated (data not
shown).
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Figure 11.
Colorectal motility is abnormal in SERT
/ mice. A, SERT +/+ and / littermates are
compared with respect to body weight, the wet weight of stools (per 24 hr), and the water content of the 24 hr stools. Note that the mice do
not differ with respect to body weight, but that the SERT / animals
excrete heavier stools that contain more water than those of their SERT
+/+ littermates. B, C, Histograms and
corresponding z scores are shown for the distributions of time to
expulsion of a glass bead placed into the colon 2 cm from the anal
verge. Note that the distribution of expulsion times approximates that
of a normal distribution in SERT +/+ mice (B) but
is highly skewed in SERT / animals (C). Most
of the expulsion times of the SERT / mice are shorter than those of
controls, but the expulsion times are highly prolonged in a small
subset of the animals. D, The mean expulsion times for
the glass beads are compared between the SERT +/+ mice and the two
groups of SERT / animals, one with diarrhea and one with
constipation. Expulsion times of the SERT / mice with diarrhea are
significantly faster than those of SERT +/+ mice, whereas those of
constipated mice are significantly longer.
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DISCUSSION |
The local SERT-mediated uptake of 5-HT has been postulated to be
responsible for terminating serotonergic signal transmission from EC
cells to primary afferent nerves in the mucosa and neurotransmission in
the enteric plexuses (Wade et al., 1996; Chen et al., 1998; Pan and
Gershon, 2000). SERT-mediated reuptake also appears to inactivate 5-HT
(Hoffman et al., 1991), which is secreted by the mast cells of mice
during immune responses (Gershon et al., 1975; Askenase et al., 1980;
Tamir et al., 1982; Askenase et al., 1983). We have confirmed by RT-PCR
that the intestines of SERT / mice lack mRNA encoding SERT. The
targeted deletion of the second exon of the gene encoding SERT,
therefore, prevents the expression of SERT in the gut, as well as in
the brain (Bengel et al., 1998). In addition, radioautographic studies
with 3H-5-HT indicated that the
high-affinity uptake of 5-HT in the bowels of SERT / mice is
deficient in epithelial cells, mast cells, and nerve fibers. In the
face of these defects, enteric serotonergic signaling would be expected
to be abnormal in these animals. Because free 5-HT is sufficiently
toxic enough to mediate anaphylactic shock in mice (Gershon and Ross,
1962), its release from the large EC cell 5-HT pool in SERT /
animals might even be lethal.
SERT / mice gain weight and survive well (Bengel et al., 1998;
Rioux et al., 1999; Fabre et al., 2000). Regardless of whether or not
the physiology of the SERT / gut is normal, the bowel must function
well enough to support life; therefore, neither peristaltic
(Kirchgessner et al., 1992, 1996; Foxx-Orenstein et al., 1995; Grider
et al., 1996, 1998; Jin et al., 1999; Pan and Gershon, 2000) nor
secretory (Sidhu and Cooke, 1995; Cooke et al., 1997) reflexes, both of
which are thought to be initiated by 5-HT, can be absent. Conceivably,
these reflexes persist in SERT-deficient mice because 5-HT is
inactivated by means that do not involve SERT. Alternatively,
transmitters other than 5-HT might mediate the reflexes in SERT / animals.
MAO (MAO-A, MAO-B) and all of the other enzymes that catabolize 5-HT
are intracellular (Blashko and Levine, 1966; Gershon, 1977; Gershon et
al., 1990). Uptake into cells is therefore essential for 5-HT to be
inactivated. However, 5-HT is highly charged and hydrophilic at a
physiological pH and thus does not readily permeate plasma membranes. A
mechanism to transport 5-HT into cells is thus required to metabolize
5-HT after it has been secreted, as well as to reduce the 5-HT
concentration in the local environment of 5-HT receptors. The inability
of epithelial cells, mast cells, and neurites of SERT / mice to
take up 3H-5-HT at low concentrations (20 nM) suggests that SERT is the only transporter expressed in
the bowel with a high affinity for 5-HT; moreover, no other
transporters are able to compensate for the loss of SERT in mediating
high-affinity 5-HT transport. However, the bowel was found to express
other molecules that transport 5-HT, although they lack the selectivity
and affinity of SERT for 5-HT. mRNA encoding DAT, OCT-1, and OCT-3 and
the corresponding immunoreactivities of the respective proteins were
all found to be present in the bowels of SERT +/+ and SERT / mice.
DAT immunoreactivity was confined to the ENS, a location that would
enable DAT to contribute to the termination of the neurotransmitter
actions of 5-HT. DAT has been shown to mediate the uptake of 5-HT by
central dopaminergic neurons when 5-HT accumulates in the brains of
mice that lack MAO-A (Cases et al., 1998). The immunoreactivity
of OCT-1 was observed in the mucosal epithelium, as well as in the ENS;
therefore, OCT-1 could contribute to inactivation of the 5-HT secreted
by EC cells, mast cells, or enteric neurons. OCT-3 protein was not located, but as the extraneuronal monoamine transporter (or
uptake2) site (Iversen, 1967), it is known to be
widely expressed in non-neuronal tissue and thus likely to be located
in the intestinal mucosa. Between them, the presence of DAT and the
OCTs in the SERT / intestine might thus compensate, at least
partially, for the absence of SERT. These transporters might also
prevent released 5-HT from reaching toxic concentrations and, in the
absence of challenge to the bowel, allow serotonergic signaling to
occur. The apparent upregulation of OCT-1 in the brains and intestines
of SERT / mice is consistent with these suggestions.
The observation that essentially no 5-HT was found in the blood of SERT
TOP
ABSTRACT
INTRODUCTION
