INTRODUCTION

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THE LARGEST STORE of serotonin (5-HT) in the body is found in the gastrointestinal tract (17). Most of this 5-HT is contained in the enterochromaffin (EC) cells of the mucosal epithelium (25). A smaller store of 5-HT is present in the enteric nervous system, where 5-HT is the neurotransmitter of a population of interneurons (25, 49). The function(s) of EC cells and the 5-HT they contain have yet to be definitively established. One hypothesis is that these cells are sensory transducers, which respond to increases in intraluminal pressure (or distortion of villi) by secreting 5-HT, which activates the mucosal processes of sensory neurons in the underlying lamina propria (7-11, 18, 44, 48). A second hypothesis is that EC cells play a paracrine role, regulating the rate of proliferation of neighboring epithelial cells by secreting 5-HT (46, 47). Both of these hypotheses envision a local action of 5-HT exerted within the immediate vicinity of the secretory source with the mucosa. Whether the target of mucosally secreted 5-HT is an epithelial cell or a nerve process, any postulate that 5-HT plays a role as a mucosal signaling molecule must account for the mucosal inactivation of 5-HT. In the absence of an adequate inactivating mechanism, receptors for 5-HT would be likely to desensitize.

5-HT is catabolized by the actions of monoamine oxidase (6) and, in the bowel, also by glucuronyl transferase (23, 27). Both of these enzymes, however, are intracellular and thus require that 5-HT be internalized by the cells that express them before these enzymes can catalyze the inactivation of 5-HT. At physiological pH, 5-HT, which has an acid dissociation constant of 10, is highly charged; therefore, unless a transporter is present in the plasma membrane, cells take up 5-HT poorly. In the central and peripheral nervous systems, 5-HT is inactivated primarily by reuptake into the serotonergic neurons that secrete it (19, 23, 26, 42, 43). This reuptake is mediated by a highly selective plasmalemmal 5-HT transporter (5-HTT or SERT) (4, 32).

The 5-HT transporter is sodium- and chloride-dependent and is inhibited specifically by fluoxetine and other serotonin-selective reuptake inhibitors (SSRIs) (5, 19). The neuronal 5-HT transporters of mice (12), rats (4, 32), and humans (41) have been cloned. Because there are no serotonergic neurons in the gastrointestinal mucosa (21, 25), it is unlikely that uptake by nerves can serve as a mucosal-inactivating mechanism for 5-HT. In fact, radioautographic studies of the uptake of [³H]5-HT have not detected any mucosal nerves with the ability to take up 5-HT (16, 26, 28, 42). We have recently demonstrated that mucosal crypt epithelial cells of the rat intestine express mRNA encoding the 5-HT transporter and can be immunostained with antibodies to the transporter (48). These cells also take up [³H]5-HT by a Na⁺- and fluoxetine-sensitive mechanism. These observations suggest that uptake by crypt epithelial cells could serve as the means by which mucosal 5-HT is inactivated.

Although the studies that have found evidence for the expression of the 5-HT transporter by mucosal epithelial cells have been carried out in rats (48), most of the physiological and pharmacological studies on the role of mucosal 5-HT in intestinal reflexes have been conducted in guinea pigs (7-11, 14, 44, 48). Unfortunately, probes prepared from the rat 5-HT transporter do not hybridize well with mRNA encoding the guinea pig 5-HT transporter (48); moreover, most antibodies to the rat 5-HT transporter (40) do not recognize that of the guinea pig.

Fluoxetine has been found initially to potentiate the peristaltic reflex in the guinea pig distal colon but then to block it (48). When the peristaltic reflex is blocked, the guinea pig colon no longer responds to 5-HT, although it retains responsivity to nicotine and electrical stimulation. These observations are compatible with the idea that inhibition of the 5-HT transporter by high concentrations of fluoxetine causes 5-HT receptors to become desensitized. The data support the hypotheses that the release of 5-HT by EC cells activates the mucosal processes of intrinsic sensory nerves to initiate the peristaltic reflex and that mucosal 5-HT transport represents the physiological mechanism that inactivates 5-HT. At the time fluoxetine was studied, however, it was not possible to confirm that the 5-HT transporter is expressed by epithelial cells of the guinea pig gut because probes that are effective in guinea pig tissues were not then available. In the absence of this confirmation, it was premature to conclude that the effect of fluoxetine or other SSRIs on the peristaltic reflex is due to inhibition of a mucosal 5-HT transporter.

In the current study, we sought to determine whether the 5-HT transporter is expressed by epithelial cells of the guinea pig bowel, as it is in rats. We also wished to determine whether the transporter may play a role in sensory reception of mucosal stimuli. We have thus cloned and sequenced a full-length cDNA encoding the 5-HT transporter from the guinea pig intestinal mucosa. When this cDNA is expressed in HeLa cells, the transfected cells take up 5-HT by a mechanism that is Na⁺ dependent and inhibited specifically by SSRIs. The affinities of the expressed transporter for various uptake inhibitors are similar to those of the 5-HT transporters that have been cloned from other species. In Northern blots, the guinea pig mucosal cDNA hybridizes well with a single species of RNA (3.7 kb) extracted from the guinea pig intestinal mucosa or brain. In situ hybridization revealed that a cRNA probe synthesized from the cDNA encoding the mucosal guinea pig 5-HT transporter hybridizes with mRNA in serotonergic neurons of the guinea pig gut and brain. The probe also hybridizes with guinea pig intestinal epithelial cells, although the distribution of such cells in the guinea pig is substantially different from that of cells containing mRNA encoding the 5-HT transporter in the rat (48). Immunocytochemical observations also suggest that the guinea pig 5-HT transporter is expressed by enterocytes throughout the crypt-villus axis. Inhibition of the mucosal 5-HT transporter with fluoxetine potentiates responses of submucosal neurons to mucosal stimuli. These observations indicate that the 5-HT transporter is expressed by guinea pig intestinal epithelial cells in addition to enteric and central serotonergic neurons and provide strong support for the hypothesis that 5-HT is a gastrointestinal mucosal signaling molecule.

	MATERIALS AND METHODS

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Animals and tissue preparation. Guinea pigs (250-350 g, obtained from Kingstar, Kingston, NH) were anesthetized and exsanguinated. These procedures have been approved by the Animal Care and Use Committee of Columbia University. The small intestine and brain were removed from the animals. The small intestine was opened, the lumen was cleaned, and the mucosa was removed by scraping the luminal surface with the edge of a spatula. For in situ hybridization, guinea pigs were perfused intracardially with heparinized saline, followed by perfusion for 5-10 min with 4% formaldehyde (freshly prepared from paraformaldehyde) in 0.1 M phosphate buffer (pH 7.5). The brain and a segment of small intestine were then removed from the animal and postfixed with the same fixative for an additional 4 h at room temperature or 12 h at 4°C. After fixation, tissues were rinsed with PBS, cryoprotected overnight with 30% sucrose (wt/vol) in PBS at 4°C, embedded in optimal cutting temperature compound (Miles, Elkhart, IN), and then stored at 80°C until used. Sections of small intestine and brain (10-15 µm) were cut with a cryostat-microtome and thaw-mounted onto 3-aminopropyltriethoxysilane-coated glass slides.

RT-PCR. Total RNA was extracted with Trizol (Life Technologies, Gaithersburg, MD) from the mucosa of the guinea pig small intestine and brain. Both tissues were homogenized in Trizol (1 ml/100 mg of tissue). Chloroform (10% of the total volume) was added and the sample was covered, shaken vigorously, and placed on ice for 10 min, and then centrifuged for 15 min (13,000 g). The aqueous phase was removed, and RNA was precipitated with isopropanol (60% of the Trizol volume). The RNA pellet was washed by resuspension in 70% ethanol. The suspension was centrifuged for 5 min (10,000 g), dried briefly, and dissolved in diethylpyrocarbonate-treated water. The extracted RNA (2.0 µg) was then used as a template for random hexamer-primed first-strand cDNA synthesis catalyzed by Maloney murine leukemia virus RT (Life Technologies). One sixth of the resulting cDNA from mucosal RNA was used for PCR amplification. Four pairs of oligonucleotide primers, designed from the cDNA sequence of the rat 5-HT transporter (4), were employed. These sets of primers were those used as internal primers for the double-strand sequencing of the 5-HT transporter cloned from the rat gut (48). Thirty cycles of PCR amplification were carried out as follows: 94°C for 1 min, 50°C for 1.3 min, and 72°C for 1 min. A PCR product of about 400 bp was obtained when forward primer no. 3 (5'-TACATGGCGGAGATGA-3') was paired with reverse primer no. 1 (5'-CCATAGAACCAAGACA-3'). This product corresponded to nucleotides 1268-1283 (sense) and 1650-1665 (antisense) of the rat 5-HT transporter (4) (GenBank database accession no. X63253). The mucosal PCR product was cloned into the 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 (Sequenase 2.0, United States Biochemicals, Cleveland, OH) and was found to be 398 bp in length.

cDNA cloning and sequencing. To obtain a full-size cDNA clone of the guinea pig mucosal 5-HT transporter, 5'-end PCR and 3'-end PCR were carried out. For 5'-end PCR, the consensus sequence of the untranslated region 5' to the ATG start codon of the human and rat 5-HT transporter was used to design an oligonucleotide primer (5'-TGGGATCCTTGGCAGATGG-3'), which was paired with the reverse primer no. 1 (see above) used to obtain the original guinea pig mucosal PCR product. PCR was performed by denaturation at 94°C for 2 min and amplification for 30 cycles (1 min each) at 94°C, 57°C, and 72°C. For 3'-end PCR, oligo(dT)₁₈ was paired with the forward primer no. 3 (see above) used to obtain the original guinea pig mucosal PCR product. The amplification reaction was carried out by denaturing at 94°C for 2 min and amplifying for 1 cycle of 94°C for 1 min, 50°C for 2 min, and 72°C for 2 min, followed by 30 cycles of 94°C for 1 min, 55°C for 1.5 min, and 72°C for 1.5 min. An aliquot of the PCR products was loaded into agarose gels for electrophoresis. The small 398-bp clone was used to generate a ³²P-labeled probe for Southern blotting by using random priming (Prime-a-Gene labeling system; Promega, Madison, WI). The ³²P-labeled probe was used to analyze the 3'-end PCR products by Southern hybridization to identify the product containing the sequence of the small clone. A 1.2-kb fragment that hybridized with the small clone was extracted from the agarose gel by using a commercial gel extraction kit (Qiagen, Santa Clarita, CA). The extracted cDNA was ligated into a pCRII vector (Invitrogen, Carlsbad, CA) and partially sequenced. The full-length cDNA of the guinea pig mucosal 5-HT transporter was finally obtained by PCR using sequences contained in the large 5'-end and 3'-end PCR products. The 5' primer incorporated a sequence (5'-TGGGATCCTTGGCAGATGG-3') in the untranslated region upstream from the start codon and the 3' primer incorporated a sequence (5'-ACATAGTGATAATGTCCCAGAG-3') downstream from the stop codon. The full-length cDNA was cloned into the TA cloning vector pCRII (Invitrogen, San Diego, CA) for double-strand sequencing. The insert was then cut from the vector with Xho I and Hind III and subcloned into a eukaryotic expression vector [pcDNA3.1(); Invitrogen].

Transfection. HeLa cells were cultured in a medium containing DMEM (Life Technologies) supplemented with 10% fetal bovine serum, penicillin and streptomycin. Plasmid DNA was purified using a commercial kit (Qiagen-tip 20). HeLa cells were grown in 24-well plates (5 × 10⁴ cells/well) and transfected with the full-length cDNA encoding the 5-HT transporter cloned from the guinea pig small intestine. The plasmid was added to the cells (1 µg/well) in the presence of lipofectamine (Life Technologies) according to the directions supplied by the manufacturer. The transfected cells were maintained in medium containing 0.5 µg/ml of G418 (Life Technologies) for selection for about 3 wk (22). Individual cells were selected and used to generate stably transfected clonal lines. Multiple lines of transfected cells exhibited an active uptake of [³H]5-HT, which could be inhibited by chlorimpramine. The clone that displayed the strongest 5-HT uptake was expanded and used for studies of the pharmacological properties of the 5-HT transporter.

Assay of [³H]5-HT transport. The transport assays were carried in 24-well plates (5). Typically, 5 × 10⁴ transfected cells/well were incubated with [³H]5-HT (13.3 nM; DuPont-NEN, Boston, MA) for 15 min at 37°C in Krebs solution, buffered with HEPES to pH 7.4, and supplemented with pargyline (100 µM) and L-ascorbic acid (100 µM). Nonspecific uptake of [³H]5-HT was taken as that manifested by HeLa cells transfected with the expression vector lacking the cDNA insert. Specific uptake of [³H]5-HT was considered to be the total uptake measured in cells transfected with the plasmid containing the cDNA insert minus the nonspecific uptake. The Na⁺ dependence of 5-HT transport was determined by studying the specific uptake of [³H]5-HT in Krebs solution that was modified by isotonically replacing NaCl with choline chloride. After incubation, the cells were washed three times with 1.0 ml of iced Krebs solution and lysed with 0.5 ml of 1.0% SDS. The released radioactivity was measured by liquid scintillation spectrometry. To obtain kinetic constants [Michaelis constant (K_m) and maximum velocity (V_max)], the velocity of [³H]5-HT uptake was plotted as a function of the concentration of 5-HT. Curves were obtained by nonlinear weighted least-square fits of the data (SigmaPlot version 5 for the Macintosh, Jandel Scientific Software, San Rafael, CA). A single population of noninteracting sites obeying Michaelis-Menton kinetics was assumed. All experiments in which the uptake of [³H]5-HT was measured were repeated three times. Rates were calculated as moles of 5-HT accumulated per cell per minute. The data are presented as means ± SE. The significance of differences between means was evaluated by ANOVA (Statview IV for the Macintosh; Abacus Concepts).

In situ hybridization. mRNA encoding the guinea pig 5-HT transporter was located in the small intestine and brain by in situ hybridization. Probes were prepared from the full-length cDNA clone in the pCRII cloning vector, which has both the Sp6 and T7 promoters to permit in vitro transcription in the sense and antisense orientations. Both sense and antisense riboprobes were synthesized with ³⁵S-labeled UTP (Dupont-NEN) incorporated into cRNA transcribed from the cDNA encoding the guinea pig 5-HT transporter. Sense and antisense riboprobes were also synthesized with digoxigenin-labeled UTP (Boehringer-Mannheim, Indianapolis, IN) and were used for nonradioactive in situ hybridization. To locate the hybridizing ³⁵S- or digoxigenin-labeled probes, sections were removed from the freezer and postfixed with 4% formaldehyde (freshly prepared from paraformaldehyde). Preparations were acetylated with 0.1 M acetic anhydride and washed in 0.2× saline-sodium citrate (SSC). Tissues were first prehybridized for 2 h at room temperature in a mixture containing 50% formamide, 600 mM NaCl, 10 mM Tris (pH 7.5), 1× Denhardt reagent, 1.0 mM EDTA, 0.05% sheared DNA, 0.05% yeast total RNA, and 0.005% yeast tRNA. The sections were then hybridized at 50°C for 16-18 h in the presence of 600 mM NaCl, 10 mM Tris (pH 7.5), 1× Denhardt's reagent, 0.5 mM EDTA, 0.01% sheared DNA, 0.05% yeast total RNA, 0.005% yeast tRNA, 10% dextran sulfate, 10 mM dithiothreitol, and 0.1% SDS. Heat-denatured probes (5 × 10⁵ counts · min¹ · section¹) were added to the hybridization solution. After hybridization, the sections were washed, first for 30 min in 50% formamide, 1× SSC, and 10 mM dithiothreitol at 50°C, and then for 30 min with 0.5× SSC at room temperature. The washed sections were treated with 0.1 mg/ml RNase at room temperature to destroy nonhybridized single-stranded RNA. After they were washed with 500 mM NaCl, 10 mM Tris (pH 7.5), and 1.0 mM EDTA, the slides were washed for 2 h at 55°C in 0.2× SSC. To locate bound ³⁵S-labeled probes, sections were dehydrated in the presence of ammonium acetate (0.3 M) and coated with liquid photographic emulsion (Ilford, L2) for radioautography. Slides were exposed for 7 days in light-tight boxes at 4°C containing a drying agent and developed with Kodak D19. Processed sections were stained with hematoxylin and eosin and visualized by using indirect dark-field optics. To locate digoxigenin-labeled probes, sections were incubated overnight at 4°C with monoclonal antibodies to digoxigenin (diluted 1:1,000; Boehringer Mannheim). Sites of bound antibodies to digoxigenin were then visualized with goat anti-mouse secondary antibodies coupled to Cy3 (Jackson ImmunoResearch Laboratories, West Grove, PA). Slides were examined by vertical fluorescence microscopy (DMRB, Leica, Malvern, PA).

Immunocytochemistry. Primary antibodies generated against sequences found in the rat 5-HT transporter (see Table 2) were generously donated by Dr. Randy D. Blakely (Department of Pharmacology, Vanderbilt University, School of Medicine). These antibodies were used to attempt to immunostain the guinea pig 5-HT transporter expressed in HeLa cells or in situ in the intestine and brain. The same antibodies were also used to immunostain denatured proteins separated by PAGE and blotted onto nitrocellulose sheets (Western blots) (40). Each antibody reacted with a single 200-kDa band in blots prepared from HeLa cells transfected with cDNA encoding the guinea pig 5-HT transporter, as well as the guinea pig and rat brain stem and intestinal mucosa. Primary antibodies were applied at a dilution of 1:2,000 (which has previously been shown to be effective for immunostaining the rat 5-HT transporter). The antibodies were applied to fixed preparations of transfected and nontransfected (control) HeLa cells, dissected whole mounts of guinea pig small intestinal mucosa, submucosa, or longitudinal muscle with adherent myenteric plexus, and frozen sections of guinea pig intestine and brain stem. Cells and tissues to be examined were fixed with 4% formaldehyde, freshly prepared from paraformaldehyde (pH 7.4) for 1 (cells) or 4 h (tissues). Secondary antibodies (diluted 1:200; Vector Laboratories, Burlingame, CA) were labeled with biotin and visualized either with streptavidin-horseradish peroxidase (Kirkegaard & Perry Laboratories, Gaithersburg, MD) or avidinCy3 (Jackson ImmunoResearch Laboratories). Horseradish peroxidase was demonstrated with 3,3'-diaminobenzidine and H₂O₂.

Electrophysiological recording from submucosal neurons. A segment of ileum was excised from male guinea pigs, 10-20 cm proximal to the ileocecal junction, and placed in oxygenated (95% O₂-5% CO₂) Krebs solution of the following composition (in mM): 121.3 NaCl, 5.95 KCl, 2.5 CaCl_2, 14.3 NaHCO_3, 1.34 NaH₂PO_4, 1.2 MgCl₂ and 11.5 glucose. A 1.5-cm segment of ileum was cut open along the mesenteric border and pinned out flat (mucosal surface up) in a petri dish lined with a silicone elastomer. The mucosa and submucosa were separated from the circular muscle layer of the gut by dissection with a fine forceps and scissors under microscopic control. The mucosa was then removed from either side of the segment of tissue, leaving a central strip of intact mucosa flanked by exposed regions of submucosa within which individual submucosal ganglia could easily be visualized. After the dissection preparations were transferred to a small recording chamber (volume 1.0 ml) that had been coated with a silicone elastomer to which the tissue was pinned. Preparations were superfused (3.5 ml/min, 36°C) with Krebs solution oxygenated with a mixture of 95% O₂-5% CO₂.

Visualization of submucosal neurons activated by mucosal stimulation. Guinea pigs were killed as described previously. In this case, the mucosa and submucosa were dissected from the circular muscle as an intact sheet of tissue about 2.0 cm in length. The sheets were then pinned to silicone elastomer-coated dishes and superfused with Krebs solution oxygenated with a mixture of 95% O₂-5% CO₂. One centimeter of tissue was considered the control piece, whereas the other centimeter served as the experimental unit of the preparation. The experimental unit was stimulated by applying 5 min of gentle stroking in the oral to anal direction of villus tips with the edge of a glass coverslip. The control piece of the segment was left unstimulated. To determine the effect of mucosal stroking on the activation of submucosal neurons, stimulation was carried out in the presence of FM2-10 (100 µM) (35), which was added to the superfusing solution at the time of stimulation and was present for 5 min. The uptake of FM2-10 has been demonstrated previously to be stimulation-dependent and totally blocked by tetrodotoxin (0.5 µM). After stimulation the preparations were washed with iced Krebs solution for 10-15 min. After the washout of free FM2-10, the mucosa was removed, with care taken to keep the tissue cold. Tissues were then examined by fluorescence microscopy (exciting filter band pass 530-560 nm; dichroic mirror reflection short pass 580 nm; edge wavelength 580 nm), the neurons that had taken up FM2-10 were identified, and the total number of labeled neurons in control and experimental halves of each preparation was counted. To determine the effects of inhibition of the mucosal 5-HT transporter, fluoxetine (10.0 nM and 1.0 µM) was added to the superfusing solution 30 min before stimulation. Comparisons were made between the number of neurons activated in control and experimental pieces of the tissue and between tissues stimulated in the presence or absence of fluoxetine.

	RESULTS

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cDNA encoding a putative 5-HT transporter was cloned from the guinea pig intestinal mucosa. RT-PCR was employed to investigate the expression of the 5-HT transporter in the mucosa of the guinea pig intestine. Total RNA was obtained from the small intestinal mucosa, which was scraped from the intestinal wall. After reverse transcription, the resulting cDNA was amplified with primers designed from consensus sequences in the rat and human 5-HT transporters. The brain stem, which includes the serotonergic neurons of the nuclei of the median raphe, was examined similarly as a positive control. PCR products of identical length were obtained from both the mucosa and the brain stem (Fig. 1). The size of the PCR products (~0.4 kb) was that predicted from the relative locations of the sequences of the primers.

View larger version (42K): [in this window] [in a new window]   Fig. 1.   RT-PCR products amplified from small intestinal mucosa and brain stem of guinea pig and rat with primers designed from sequences in cDNA encoding the rat serotonin (5-HT) transporter. Ethidium bromide-stained 1.2% agarose gel. Single 0.4-kb product is obtained from each tissue. gpM, guinea pig mucosa; gpB, guinea pig brain stem; rM, rat mucosa; rB, rat brain stem. Arrow, 564 bp.

View larger version (57K): [in this window] [in a new window]   Fig. 2.   Nucleotide and deduced amino acid sequences of 5-HT transporters of guinea pig (gp5-HTT), human (h5-HTT), and rat (r5-HTT). Underlined sequences indicate 12 stretches of hydrophobic amino acids presumed to span plasma membrane. Amino acids of human and rat 5-HT transporter sequences that are not conserved with those of guinea pigs are indicated below guinea pig sequence. * Position of stop codon. Sequence of 398-bp PCR fragment originally obtained from guinea pig small intestinal mucosa lies between arrows.

Sequence of putative 5-HT transporter from guinea pig intestinal mucosa is similar to those of humans and rats. The sequence of the full-length cDNA encoding the putative guinea pig 5-HT transporter was compared with those of the 5'-end and 3'-end PCR products. Over the corresponding regions, the sequences were found to be identical. At the nucleotide level, the sequence of the full-length cDNA encoding the putative guinea pig mucosal 5-HT transporter (Fig. 2) was found to be 85.6% identical to that of the rat 5-HT transporter and 86.5% identical to that of the 5-HT transporter of humans. At the amino acid level, the sequences were 89.2% (68 different/630 amino acids) identical for rats and 90% (63 different/630 amino acids) identical for humans. The greatest amount of amino acid diversity appears in the NH₂-terminal region of the molecules, before the first putative transmembrane domain. The highest degree of conservation of amino acids between species appears in the sixth and eighth putative transmembrane domains and in the intracellular loop between the second and third transmembrane domains.

Cells transfected with cDNA encoding putative 5-HT transporter from guinea pig mucosa exhibit a Na⁺-dependent uptake of [³H]5-HT. To verify that the full-length cDNA putatively encoding the guinea pig 5-HT transporter actually does so, HeLa cells were transfected with the cDNA and their ability to take up [³H]5-HT was investigated (Fig. 3). Almost no specific uptake of [³H]5-HT was detected in control HeLa cells that were not transfected with this cDNA. Similarly, HeLa cells that were transfected with the plasmid [pcDNA3.1()], which lacked the insert encoding the putative 5-HT transporter, failed to take up [³H]5-HT. In contrast, HeLa cells that had been transfected with a plasmid containing cDNA encoding the putative 5-HT transporter avidly took up [³H]5-HT. This uptake was linear for about 20 min. The HeLa cells transfected with cDNA encoding the putative 5-HT transporter, however, did not take up [³H]5-HT when they were incubated in Na⁺-free media. These observations suggest that the guinea pig cDNA encodes a transporter protein that, when expressed in transfected cells, is incorporated into the plasma membrane and transports 5-HT.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Time, cDNA, and Na+ dependence of uptake of [3H]5-HT by HeLa cells transfected with full-length cDNA encoding putative 5-HT transporter cloned from the guinea pig small intestinal mucosa (gpSERT). Cells were transfected with either a plasmid containing gpSERT cDNA insert (, ) or same plasmid, cDNA3.1(), without insert (). Cells were assayed for uptake of [3H]5-HT in presence (, ) or absence of Na+ (). Uptake of [3H]5-HT, which is gpSERT and Na+ dependent, is linear for about 20 min.

Uptake of [³H]5-HT by transfected cells is saturable and of high affinity. The properties of the transporter expressed by transfected HeLa cells were characterized further. The transport of [³H]5-HT was analyzed after 15 min of incubation, during the period when uptake was still a linear function of time. The measured uptake was thus proportional to the initial uptake. The uptake of [³H]5-HT was observed to be concentration dependent and saturable (Fig. 4). A single high-affinity interaction was found when the data were subjected to an Eadie-Hofstee transformation (Fig. 4, inset). The K_m was 618 nM and the V_max was 2.4 × 10¹⁷ mol · cell¹ · min¹, each measured by a nonlinear least-square analysis of the isotherm relating the velocity of [³H]5-HT uptake to the concentration of 5-HT. The measured value of K_m for the uptake of [³H]5-HT by transfected HeLa cells is extremely close to that (700 nM) reported for the uptake of [³H]5-HT by isolated strips of guinea pig myenteric neurons attached to strips of longitudinal muscle (24).

View larger version (19K): [in this window] [in a new window]   Fig. 4.   Substrate dependence of transport of 5-HT by HeLa cells transfected with full-length cDNA encoding putative 5-HT transporter cloned from the guinea pig small intestinal mucosa. Assays of transport of 5-HT were carried out with 14 nM [3H]5-HT and various concentrations of nonradioactive 5-HT. When the rate of transport was plotted as a function of concentration of 5-HT, account was taken of changes in specificity of substrate. Inset, Eadie-Hoftsee transformation of data. Michaelis constant (618 nM) and maximum velocity (2.4 × 1017 mol · cell1 · min1) were determined from computer-assisted nonlinear weighted least-square fits of nontransformed data. V, velocity; S, substrate concentration.

Uptake of [³H]5-HT by transfected cells is inhibited by compounds known to antagonize transporter-mediated uptake of 5-HT. The identity of the cloned transporter as the 5-HT transporter of guinea pigs was confirmed by evaluating the ability of known uptake inhibitors (tricyclic antidepressants and SSRIs) to inhibit the uptake of [³H]5-HT by transfected HeLa cells. These compounds were found to antagonize effectively the uptake of [³H]5-HT by transfected HeLa cells (Fig. 5, Table 1). The rank order of potency of the tricyclic antidepressants against the uptake of [³H]5-HT by transfected cells, chlorimipramine >> imipramine >> desipramine, was observed to be the same as that reported for these compounds against the uptake of [³H]5-HT by central or enteric serotonergic neurons (19, 24, 26). Desipramine, however, which is thought to be relatively more selective for the norepinephrine than the 5-HT transporter, was a more potent inhibitor of the uptake of [³H]5-HT by cells transfected with cDNA encoding the guinea pig transporter than would have been anticipated from its reported ability to inhibit the transport of the rat 5-HT transporter (4). As would be expected for an effect mediated by a 5-HT transporter, the SSRI fluoxetine potently inhibited the uptake of [³H]5-HT by the HeLa transfected cells transfected with the cDNA encoding the guinea pig transporter and was more potent than zimelidine. The measured inhibitory constant (K_i) values (Table 1) for both the tricyclic antidepressants and the SSRIs against the uptake of [³H]5-HT by transfected HeLa cells were all lower than that observed for 5-HT itself.

View larger version (25K): [in this window] [in a new window]   Fig. 5.   Antagonism of 5-HT transport by HeLa cells transfected with cDNA encoding putative 5-HT transporter cloned from guinea pig small intestinal mucosa. Percent inhibition of uptake of [3H]5-HT is plotted as a function of concentration of inhibitor that was tested. Data are expressed relative to control assays carried out in absence of antagonists.

Northern analysis reveals single hybridizing species of mRNA in guinea pig intestinal mucosa and brain stem. The distribution of mRNA encoding the putative guinea pig mucosal 5-HT transporter was investigated, first by Northern analysis and then by in situ hybridization. It was reasoned that if the transporter cloned from the guinea pig intestinal mucosa was actually the guinea pig 5-HT transporter, then cDNA encoding the mucosal transporter would be expected to hybridize in Northern blots with a single species of mRNA from the guinea pig brain stem, which contains central serotonergic neurons. A probe prepared from the mucosal transporter would also be expected to label neurons of the nuclei of the median raphe and myenteric plexus. Northern analysis was carried out with total RNA extracted from the guinea pig brain stem and intestinal mucosa. For comparison, total RNA extracted from the rat brain stem and intestinal mucosa was also analyzed. Blots were hybridized with ³²P-labeled probe prepared from bp 1200-1597 of the guinea pig intestinal mucosal cDNA. A single species of hybridizing mRNA (~3.7 kb) was found both in the guinea pig brain stem and the intestinal mucosa; however, no hybridizing mRNA could be detected either in the rat brain stem or the rat intestinal mucosa (Fig. 6). The size of the hybridizing guinea pig mRNA is similar to those reported for mRNA encoding the 5-HT transporters of rat (3.7 kb) (4), human (3.7 kb) (41), and mouse (3.4 kb) (12).

View larger version (56K): [in this window] [in a new window]   Fig. 6.   Tissue expression of mRNA hybridizing with cDNA encoding putative 5-HT transporter cloned from the guinea pig small intestinal mucosa. Single species of mRNA (3.7 kb) hybridizes in Northern blots with this cDNA. The guinea pig probe does not hybridize with mRNA from rat tissues. Lane A, guinea pig brain stem; lane B, guinea pig mucosa; lane C, rat brain stem; lane D, rat mucosa. Markers show positions of 28S and 18S ribosomal RNA.

mRNA encoding guinea pig 5-HT transporter is located in nuclei of median raphe and intestinal epithelium. In situ hybridization was used to locate cells that expressed the mRNA encoding the guinea pig 5-HT transporter. Tissue sections (Fig. 7) were hybridized with a ³⁵S-riboprobe prepared from the full-length cDNA cloned from the guinea pig intestinal mucosa. Sites of bound probe were located by radioautography. Labeling was considered specific if it appeared on sections hybridized with the antisense probe but not on sections hybridized with the sense probe (control). Although the goal was to locate mRNA encoding the 5-HT transporter in the gut, the dorsal nucleus of the median raphe, which is rich in serotonergic neurons, was also examined as a positive control. Intense labeling was obtained in both the dorsal raphe nucleus and the small intestine (Fig. 7, A and C) when sections were hybridized with the antisense probe. In contrast, no labeling of any structures was found in alternate serial sections that were hybridized with the sense probe (Fig. 7, B and D). The labeling of the raphe and the intestine was thus considered specific and indicative of the presence of mRNA that hybridized with the antisense probe in both locations. In the dorsal raphe nucleus (Fig. 7A) labeling was confined to neurons situated within the confines of the nucleus. In the sections of intestine, labeling was confined to the mucosa, where it was restricted to the epithelium (Fig. 7C). In contrast to the rat, where mRNA encoding the 5-HT transporter appears to be expressed only by crypt epithelial cells (48), both crypt and villus epithelium were labeled in the guinea pig.

View larger version (173K): [in this window] [in a new window]   Fig. 7.   Cells that contain mRNA encoding the guinea pig 5-HT transporter were located by in situ hybridization. Dorsal nucleus of median raphe hybridized with 35S-labeled antisense (A) or sense (B) riboprobes prepared from cDNA encoding putative guinea pig 5-HT transporter. After exposure to probes, bound radioactivity was located in sections of brain by radioautography. Tissue was stained with hematoxylin and eosin and examined by using combination of bright-field and vertical dark-field illumination. Radioautographic silver grains appear white. Many neurons are labeled in sections hybridized with antisense but not sense riboprobes. The guinea pig small intestine was analyzed with same 35S-labeled antisense (C) and sense (D) riboprobes used to examine brain stem (shown in A and B). Epithelial lining of both crypts and villi (arrows) are heavily labeled in sections hybridized with antisense but not sense riboprobes. Dissected preparations of longitudinal muscle with adherent myenteric plexus were analyzed as whole mounts of tissue. Preparations were subjected to hybridization with same probes as were used to investigate distribution of mRNA encoding putative guinea pig 5-HT transporter in tissue sections. Outlines of ganglia are indicated by arrowheads. Relatively few cells were labeled by 35S-labeled antisense riboprobe (E) and none were labeled by 35S-labeled sense probe (F). Labeling was generally confined to cell bodies of neurons and it was rare to find more than a single labeled neuron in any one ganglion; however, in some neurons, such as that shown in E (arrow), long dendritic processes were also labeled. mRNA encoding putative guinea pig 5-HT transporter was located by nonradioactive in situ hybridization. Antisense (G and H) and sense (I) riboprobes were labeled with digoxigenin. Digoxigenin-labeled probes were visualized in tissues by immunocytochemistry, using antibodies to digoxigenin coupled to Cy3. Sections were examined by fluorescence microscopy. Labeled cells (arrows) were found in sections of gut (G) and raphe nuclei (H) hybridized with antisense probe. No cells were labeled when sections of gut or brain stem (I) were hybridized with sense probe. Nonspecific fluorescence of lipofuscin can be seen in sections of brain. Markers in A-F, as well as H and I, 100 µm; marker in G 50 µm.

mRNA encoding guinea pig 5-HT transporter is located in enteric neurons. Because serotonergic neurons represent only a small subset of myenteric neurons (25), they are only rarely encountered in random sections of the intestine. Sections pass through only a small number of ganglia and thus through only a minority of the ~100 neurons of an average myenteric ganglion (34). To properly analyze the expression of mRNA encoding a gene product expected to be found in serotonergic neurons, therefore, it is necessary to examine whole mounts of tissue. The myenteric plexus of the guinea pig can readily be dissected, as it is attached to the underlying longitudinal muscle in a flat sheet of tissue. When this tissue is defatted and infiltrated with liquid photographic emulsion, it can be analyzed by radioautography even when the radioisotope to be localized is a weak -emitter (30). Intense labeling of scattered neurons was seen in whole mounts of the longitudinal muscle with attached myenteric plexus (Fig. 7E) when tissues were hybridized with a ³⁵S-antisense probe. The hybridizing mRNA was found in the cell bodies and often also in processes, presumably dendrites, that trailed away from the labeled neuron but remained within ganglia. Neither the glial cells within ganglia nor the extraganglionic smooth muscle or connective tissue was ever labeled. No labeling of any structures was found in similar preparations that were hybridized with the sense probe (Fig. 7F).

Guinea pig 5-HT transporter can be located by immunocytochemistry. Previous attempts to immunostain the 5-HT transporter in guinea pig tissues were carried out with antibodies directed at epitopes of the rat 5-HT transporter that are poorly conserved among other Na⁺-dependent transporters (48). These antibodies were raised by immunizing rabbits either with a synthetic peptide that incorporated a sequence in the fourth putative extracellular loop or a fusion protein that incorporated the final 34 amino acids (597-630) of the COOH terminus (40). The antibodies to the extracellular loop were not found to be useful for immunocytochemistry either in rat or in guinea pig, but the antibodies to the COOH-terminal domain were very effective in rats (48). These antibodies labeled crypt epithelial cells in the rat intestinal mucosa and serotonergic neurons in the rat brain and rat myenteric plexus. The same antibodies, however, failed to label either epithelial cells or neurons specifically in guinea pigs.

View larger version (166K): [in this window] [in a new window]   Fig. 8.   Expression of 5-HT transporter immunoreactivity was demonstrated with antibody A-SERT-50. A: HeLa cells transfected with cDNA encoding the guinea pig 5-HT transporter. Cultured cells are examined as whole mount. Diffuse immunoreactivity of cell cytoplasm and punctate regions of concentrated 5-HT transporter immunoreactivity are evident. Marker, 25 µm. B: guinea pig brain stem in region of median forebrain bundle. Many axons are immunoreactive. Marker, 50 µm. C and D: ganglia of myenteric plexus of guinea pig small intestine. Tissue was frozen and sectioned at 5 µm. Arrows, immunoreactive process that surrounds nonimmunoreactive nerve cell bodies. D, inset: rare 5-HT transporter-immunoreactive myenteric neuron. Markers, 25 µm. E: ganglion of submucosal plexus of guinea pig small intestine. Arrow, immunoreactive process that surrounds nonimmunoreactive nerve cell bodies. Some immunofluorescence of crypt epithelial cells is evident in upper left corner. Marker, 25 µm. F and G: villous surface of the guinea pig small intestine. Note immunoreactivity of enterocytes and its concentration in Golgi region of cells (arrow in F) and absence of immunoreactivity in goblet cells (arrowheads in F). Immunoreactivity of tips of microvilli is well seen in G (arrow), forming punctate line at some distance from what appears to be apical surface of cells. Basolateral surface and cytoplasm of enterocytes are also immunoreactive. H: crypt epithelium of rat duodenum. Pattern of 5-HT transporter immunostaining is different from that seen in guinea pigs. Immunoreactivity is concentrated in particular crypt epithelial cells (arrow) as previously reported (48).

Inhibition of 5-HT transporter potentiates effects of mucosal stimulation. Physiological studies were carried out to test the hypotheses that 5-HT-containing EC cells are sensory transducers that activate the mucosal processes of intrinsic sensory neurons (7-11, 18, 44, 48) and that the reuptake of 5-HT by mucosal epithelial cells is important in this process as a mechanism that inactivates 5-HT (48). Two methods were used to cause mucosal sensory nerves to become excited. First, 5-HT or electrical stimuli were applied to the mucosal surface of the bowel, and responses were recorded intracellularly in submucosal neurons. Second, the mucosal surface of the gut was stimulated mechanically (by stroking), and the submucosal neurons activated by this stimulus were visualized with the fluorescent probe FM2-10 (35) and quantified. The myenteric plexus was removed in both types of experiments to be certain that submucosal and not myenteric sensory neurons were responsible for observed responses.

View larger version (12K): [in this window] [in a new window]   Fig. 9.   Fluoxetine potentiates fast excitatory postsynaptic potentials (EPSPs) that are evoked in submucosal neurons by focal electrical stimulation applied to the mucosa. Consecutive records from a single impaled neuron are shown. Con (control), fast EPSP follows stimulus artifact. Fluox, fast EPSP was again evoked after addition of fluoxetine (10 µM). Fluox + C6, fast EPSP is abolished by hexamethonium (100 µM). Wash, fast EPSP returns 5 min after washout of hexamethonium. TTX, fast EPSP is abolished by tetrodotoxin (0.3 µM).

View larger version (88K): [in this window] [in a new window]   Fig. 10.   Uptake of FM2-10 allows submucosal neurons activated by mucosal stimulation (stroking) to be visualized. This field shows a group of excited neurons after stroking of the mucosa in the presence of fluoxetine (1.0 µM). Numbers of such cells on control and stimulated sides of preparations in absence or presence of fluoxetine are shown in Fig. 11. Marker, 50 µm.

View larger version (30K): [in this window] [in a new window]   Fig. 11.   Fluoxetine increases the number of submucosal neurons that become excited by stroking the mucosa. Uptake of FM2-10 was quantified on control and mucosally stimulated sides of preparations in absence or presence of 2 concentrations of fluoxetine.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Studies were carried out to determine whether a 5-HT transporter identical to that of serotonergic neurons is expressed in the mucosa of the guinea pig small intestine. Although the 5-HT transporter has been found to be present in the mucosa of the rat gut (48), it had not previously been demonstrated to be expressed in that of guinea pigs. The activity of the 5-HT transporter is critical for the inactivation of 5-HT. Enzymes that are known to catabolize 5-HT, such as monoamine oxidase (6) and, in the bowel, glucuronyl transferase (23, 27), are intracellular. These enzymes thus are not by themselves able to remove 5-HT from the extracellular space, where it gains access to 5-HT receptors expressed on the plasma membranes of target cells. For monoamine oxidase or glucuronyl transferase to catabolize 5-HT, it is necessary for 5-HT first to enter the cells that contain these enzymes. Serotonergic axons are able to inactivate the 5-HT they secrete because they express the 5-HT transporter in their plasma membrane, which catalyzes the reuptake of released 5-HT (4, 19, 23, 26, 32, 42, 43).

The hypothesis has been advanced that 5-HT secreted by EC cells activates 5-HT receptors both on extrinsic (3, 31) and intrinsic sensory neurons (7-11, 44, 48) (Fig. 12). For example, experiments carried out with isolated segments of guinea pig gut have suggested that EC cells release 5-HT in response to mucosal pressure or distortion (9) and that this release is responsible for the initiation both of peristaltic (48) and secretory reflexes (14, 44). Furthermore, intrinsic sensory neurons of the submucosal plexus have been demonstrated directly to become active following the application of pressure to the guinea pig small intestinal mucosa, and their activation is blocked by 5-HT antagonists (35, 36). Despite these observations, a role for 5-HT as an EC cell-to-sensory nerve transmitter cannot be considered to have been definitively established unless a mechanism for terminating its action in the guinea pig intestinal mucosa can be identified. This hypothesis requires a mechanism for 5-HT inactivation in the mucosa. If the 5-HT transporter-mediated reuptake of 5-HT is this mechanism, then the mucosal expression of the 5-HT transporter should not be confined to the rat gut. The previous absence of definitive evidence that there is a 5-HT transporter in the guinea pig intestinal mucosa thus presented a problem for understanding the role of 5-HT in intestinal function.

View larger version (24K): [in this window] [in a new window]   Fig. 12.   Diagram depicting proposed mechanism of activating intrinsic sensory nerves in submucosal plexus. Myenteric plexus was dissected away from preparations used in the current study and thus does not affect results. Pressure (Pr), distortion, focal electrical stimulation, or 5-HT was applied to mucosa (left). These stimuli are presumed to cause 5-HT to be released from enterochromaffin (EC) cells. Secreted 5-HT stimulates mucosal processes of intrinsic sensory neurons, which are cholinergic and activate follower cells (interneurons or secretomotor neurons) by fast EPSPs mediated by nicotinic receptors. In the current studies, recordings were made in follower cells, which were impaled with a sharp microelectrode. As a result the fast EPSPs evoked in these cells were abolished by tetrodotoxin, as well as by hexamethonium. Mucosal action of 5-HT is terminated by its transporter-mediated uptake by enterocytes, which presumably catabolize the 5-HT they take up. Inhibition of mucosal transporter by fluoxetine potentiates stimulation of sensory neurons by 5-HT, recruiting more of such cells to be activated by a given stimulus. Effect of inhibiting 5-HT transport is to potentiate amplitude of compound cholinergic fast EPSPs in follower cells and to increase numbers of activated submucosal neurons.

Because rat cDNA and riboprobes hybridize poorly with mRNA encoding the guinea pig 5-HT transporter, and commercially available antibodies to the rat 5-HT transporter do not recognize that of guinea pigs, we postulated that the expression of the 5-HT transporter in the guinea pig bowel was not detected in earlier studies because appropriate reagents were not available. In the current study, therefore, we used RT-PCR to determine whether the guinea pig 5-HT transporter could be cloned from the intestinal mucosa. Because cDNA encoding the rat 5-HT transporter does hybridize, albeit weakly, with that of guinea pig (48), initial amplimers used for PCR were designed from the sequence of the rat 5-HT transporter. A 398-bp PCR fragment was obtained from the guinea pig small intestinal mucosa that was 89.5% identical at the nucleotide level to the sequence of the equivalent region of the rat and human 5-HT transporters. This fragment was employed to obtain a full-length cDNA by NH₂- and COOH-terminal extension.

The full-length guinea pig mucosal cDNA encoded a protein that was deduced to be 630 amino acids in length, the same length as the 5-HT transporters of rat (4), human (41), and mouse (12). The full-length guinea pig mucosal cDNA was 90.0% identical to the sequence of the human 5-HT transporter and 89.2% identical to the rat 5-HT transporter. In common with the 5-HT transporters of other species, moreover, hydropathy analysis of the deduced amino acid sequence of the guinea pig mucosal cDNA suggested that the molecule has 12 hydrophobic membrane-spanning domains. Also like the 5-HT transporters of other species, the guinea pig mucosal cDNA has two potential glycosylation sites between the third and fourth transmembrane domains, implying that this region of the molecule is extracellular. Further identities between the guinea pig mucosal cDNA and the 5-HT transporters of other species are conserved potential casein kinase II and PKC phosphorylation sites. The potential casein kinase II sites are COOH-terminal in location, whereas the PKC sites are found both in the NH₂- and COOH-terminal domains and in F, the linker region between the fourth and fifth membrane-spanning regions. It is conceivable that the guinea pig mucosal cDNA has an additional site that can be phosphorylated by PKC (Thr⁵⁹⁴), which is not present in the published sequences of the 5-HT transporters of other species. This site, however, is predicted to be the last amino acid included in the twelfth membrane-spanning domain. The strong similarity between the 5-HT transporters of other species and the guinea pig mucosal cDNA suggested that guinea pig cDNA encodes the guinea pig 5-HT transporter.

The supposition that the guinea pig mucosal cDNA encodes the guinea pig 5-HT transporter was confirmed by expressing the guinea pig protein in HeLa cells. The transfected HeLa cells, but not the nontransfected parent line, took up 5-HT by a high-affinity, saturable mechanism. The uptake of 5-HT by transfected cells was found to be Na⁺ dependent and antagonized by tricyclic antidepressants and SSRIs, which are known to inhibit the action of the 5-HT transporter. Whereas the rank order of potency of the tricyclic antidepressants, chlorimipramine >> imipramine >> desipramine, was similar to that reported for the 5-HT transporters of other species (2), each of these compounds was seen to be a more potent inhibitor of the expressed guinea pig 5-HT transporter than of the 5-HT transporters of rats or humans (Table 1). In contrast, the SSRI fluoxetine was found to be a less potent inhibitor of the expressed guinea pig 5-HT transporter than of the 5-HT transporters of rats or humans (Table 1). Earlier studies in which the uptake of [³H]5-HT by rat cortical synaptosomes was compared with those of guinea pigs also revealed that tricyclic antidepressant compounds are more potent inhibitors of [³H]5-HT uptake in guinea pigs, whereas SSRIs are more potent in rats (33). These observations led to the conclusion that the 5-HT uptake systems in rats and guinea pigs are similar but not identical. The current observations are consistent with this conclusion.

The rat and human 5-HT transporters have been shown to be markedly different in their affinity for tricyclic antidepressants (1). This difference has been traced to a single amino acid in the twelfth putative transmembrane domain, Phe⁵⁸⁶ in humans, and Val⁵⁸⁶ in the rat. The guinea pig 5-HT transporter resembles that of the rat in that it too has a Val⁵⁸⁶, rather than the Phe⁵⁸⁶ of the human 5-HT transporter. The K_i for inhibition by imipramine of [³H]5-HT uptake into transfected HeLa cells (2.6 ± 0.3 ×10⁸ M) lies between the K_i values reported for the rat (4.6 ± 0.9 × 10⁸ M) and human (8.2 ± 3.4 × 10⁹ M) 5-HT transporters, although it is closer to that of the rat (see Table 1). These observations thus support the idea that Phe⁵⁸⁶ is responsible for the very high affinity interactions of tricyclic antidepressants with the human 5-HT transporter.

It is of interest that in the COOH-terminal domain, only 4 of 34 amino acids distinguish the sequences of the guinea pig, rat, and human 5-HT transporters. This degree of similarity makes it difficult to explain why only one of three different antibodies generated against sequences in the COOH-terminal domain of the rat 5-HT transporter appears to be able to demonstrate the guinea pig 5-HT transporter by immunocytochemistry (Table 2). All three of these antibodies recognize the rat and human proteins (40), and all three appear to react with the denatured guinea pig 5-HT transporter in Western blots (data not illustrated). It is possible that antibodies to the rat sequences would fail to label the guinea pig 5-HT transporter in tissues, if the tertiary structure of the guinea pig 5-HT transporter were to be different from that of rat or human 5-HT transporters. The guinea pig 5-HT transporter contains a Cys⁶²⁶. The corresponding amino acid is Arg⁶²⁶ in rat and human. It seems plausible therefore that disulfide bonding involving the Cys⁶²⁶ of the guinea pig 5-HT transporter, perhaps with Cys⁵²², which is found in the adjacent intracellular domain, causes an internal loop to form in the guinea pig 5-HT transporter that precludes its recognition by some antibodies to the COOH-terminal domain of the rat 5-HT transporter. A-SERT-50 and A-SERT-48 were prepared against COOH-terminal peptides that differ from one another only in that the peptide used to generate A-SERT-48 was eight amino acids greater in length than A-SERT-50. It is possible that the sequence recognized by A-SERT-48 is masked in situ because it extends almost to the Cys⁶²⁶. The antibodies to external domains of the rat 5-HT transporter have not proven to be useful for the immunocytochemical localization of the 5-HT transporter in rats. It is therefore not surprising that they also do not appear to react with the 5-HT transporter in guinea pig tissues.

The distribution of cells found by in situ hybridization to contain mRNA encoding the guinea pig 5-HT transporter was similar, but not identical, to that of cells that express 5-HT transporter immunoreactivity. In particular, neurons of the nuclei of the guinea pig median raphe were heavily labeled by in situ hybridization, as was a small subset of neurons in the myenteric plexus of the guinea pig small intestine. It has been estimated that the proportion of guinea pig enteric neurons that are serotonergic is about 2-3% and they are thought to be restricted to the myenteric plexus (15, 20, 29). This proportion is about that found by in situ hybridization to express mRNA encoding the 5-HT transporter in the guinea pig bowel and corresponds well to the small number of guinea pig myenteric nerve cell bodies that expressed 5-HT transporter immunoreactivity. Moreover, both hybridizing and immunoreactive neuronal perikarya were observed only in the myenteric plexus, and the appearance of 5-HT transporter immunoreactivity in sectioned ganglia is very similar to that previously reported for the radioautographic labeling of the same structures following the incubation of the gut with [³H]5-HT (16, 28). Axons (both central and enteric) were the only neuronal structures that displayed 5-HT transporter immunoreactivity but did not contain mRNA encoding the guinea pig 5-HT transporter that could be demonstrated by in situ hybridization. This difference in localization, however, is not a discrepancy but is almost certainly due to the virtual absence of RNA in axons. The axonal expression of the 5-HT transporter is thus explained by axonal transport. In both the brain and gut a small number of serotonergic neurons evidently provide a very large number of axonal processes.

The results with in situ hybridization thus suggest that the same 5-HT transporter molecule is likely to be expressed in neurons of the brain and bowel. Because the 5-HT transporter was originally cloned from RNA extracted from the guinea pig mucosa, which contains no nerve cell bodies, it is apparent that the guinea pig 5-HT transporter is not exclusively a neuronal molecule. The idea that nonneuronal cells of the guinea pig intestinal mucosa and central neurons express the same 5-HT transporter was confirmed by Northern analysis, which revealed only a single hybridizing species of mRNA of identical size in brain and gut.

Data obtained with in situ hybridization and immunocytochemistry provide strong support for the idea that the 5-HT transporter is expressed by guinea pig mucosal epithelial cells. In contrast to the rat, however, in which the expression of the 5-HT transporter is restricted to crypt epithelial cells (48), in the guinea pig mRNA encoding the 5-HT transporter, as well as immunoreactive protein, is found in epithelial cells throughout the length of the crypt-villus axis. This distribution suggests that the function of the mucosal 5-HT transporter in guinea pigs is not limited to the region of the crypts, but instead extends to the tips of villi. The guinea pig mucosa is thicker than that of rats; therefore, 5-HT released near villus tips might not be inactivated rapidly enough by a transporter limited in its distribution to the crypts. 5-HT-containing EC cells in the guinea pig small intestine are more concentrated in crypts, but they are also present in the walls of the villi. Restricted stimuli, moreover, such as puffs of N₂ directed at the tips of villi, have been demonstrated to activate neurons in the guinea pig submucosal plexus by a 5-HT-dependent mechanism (36). It thus seems probable that the release of 5-HT from the wall of guinea pig villi, and not just the region of the crypts, can activate mucosal sensory nerves. The expression of the 5-HT transporter throughout the villus