Expression and function of 5-HT4 receptors in the mouse enteric ...

Am J Physiol Gastrointest Liver Physiol 289: G1148 –G1163, 2005. First published July 21, 2005; doi:10.1152/ajpgi.00245.2005.

Expression and function of 5-HT4 receptors in the mouse enteric nervous system Mintsai Liu, Matthew S. Geddis, Ying Wen, Wanda Setlik, and Michael D. Gershon Department of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia University, New York, New York Submitted 26 May 2005; accepted in final form 14 July 2005

Liu, Mintsai, Matthew S. Geddis, Ying Wen, Wanda Setlik, and Michael D. Gershon. Expression and function of 5-HT4 receptors in the mouse enteric nervous system. Am J Physiol Gastrointest Liver Physiol 289: G1148 –G1163, 2005. First published July 21, 2005; doi:10.1152/ajpgi.00245.2005.—The aim of the current study was to identify enteric 5-HT4 splice variants, locate enteric 5-HT4 receptors, determine the relationship, if any, of the 5-HT4 receptor to 5-HT1P activity, and to ascertain the function of 5-HT4 receptors in enteric neurophysiology. 5-HT4a, 5-HT4b, 5-HT4e, and 5-HT4f isoforms were found in mouse brain and gut. The ratio of 5-HT4 expression to that of the neural marker, synaptophysin, was higher in gut than in brain but was similar in small and large intestines. Submucosal 5-HT4 expression was higher than myenteric. Although transcripts encoding 5-HT4a and 5-HT4b isoforms were more abundant, those encoding 5-HT4e and 5-HT4f were myenteric plexus specific. In situ hybridization revealed the presence of transcripts encoding 5-HT4 receptors in subsets of enteric neurons, interstitial cells of Cajal, and smooth muscle cells. IgY antibodies to mouse 5-HT4 receptors were raised, affinity purified, and characterized. Nerve fibers in the circular muscle and the neuropil in ganglia of both plexuses were highly 5-HT4 immunoreactive, although only a small subset of neurons contained 5-HT4 immunoreactivity. No 5-HT4-immunoreactive nerves were detected in the mucosa. 5-HT and 5-HT1P agonists evoked a G protein-mediated long-lasting inward current that was neither mimicked by 5-HT4 agonists nor blocked by 5-HT4 antagonists. In contrast, the 5-HT4 agonists renzapride and tegaserod increased the amplitudes of nicotinic evoked excitatory postsynaptic currents. Enteric neuronal 5-HT4 receptors thus are presynaptic and probably exert their prokinetic effects by strengthening excitatory neurotransmission.

5-HT PLAYS MANY CRITICAL ROLES in the physiology of the bowel (43– 45). To understand these functions, it is thus necessary to identify the subtypes of 5-HT receptor that are expressed in the gut and to determine their locations and actions. The 5-HT receptors that are known to be expressed by enteric neurons include 5-HT1A (40, 60, 61), 5-HT2A (13, 35), 5-HT2B (14, 36), 5-HT3 (29, 67, 69, 102), and 5-HT4 (26, 27, 41, 59, 65, 79). In addition to these, another 5-HT receptor activity, called 5-HT1P, has been identified in the enteric nervous system (ENS) (15, 69). This receptor activity has thus far only been characterized operationally and with respect to its intracellular transduction coupling to Go (43, 44, 70, 82). The 5-HT1P receptor has been proposed by some authors to be identical to the 5-HT4 receptor (49, 62, 63) but has been thought to be unrelated to 5-HT4 by others (39, 41, 44). The 5-HT4 (47– 49, 55, 87, 92, 93) and 5-HT1P (44, 81) receptors are important

because each has been implicated in the initiation of peristaltic reflexes. The hypothesis that 5-HT4 receptors activate enteric neurons has not received any electrophysiological support but could be tested by determining the location and actions of 5-HT4 receptors in the ENS. The 5-HT4 receptor is also of considerable clinical significance because a 5-HT4 agonist, tegaserod, has proven to be a useful prokinetic agent with which to treat the constipation-predominant form of the irritable bowel syndrome (IBS-C) (20, 21, 50, 75, 77, 85, 90) and chronic constipation (56). Unfortunately, enteric 5-HT4 receptors have yet to be definitively localized, their functions in normal ENS physiology are not entirely clear, and the relationship of 5-HT4 receptors to the 5-HT1P receptor activity remains unclear. Several splice variants of the 5-HT4 receptor have been described since the sequences of “short” (5-HT4S) and “long” (5-HT4L) forms were first determined in the rat (42). These newer splice variants include the a, b, and e isoforms in rats (24), a, b, e, and f isoforms in mice (23–25), and a, b, c, d, g, n, h, and i isoforms in humans (3, 6, 8, 9, 16, 24, 73, 97, 98). The 5-HT4 splice variants differ from one another in the sequence of their COOH-terminal domain. Pharmacological properties of the 5-HT4 receptor in a given tissue may reflect the expression levels of the various 5-HT4 isoforms (6, 16, 24, 72, 98). The 5-HT4 receptor is thought to be coupled by Gs to the stimulation of adenylyl cyclase (AC), increase in cAMP, and activation of PKA (6, 10, 12, 68). This coupling suggests that 5-HT4 receptors may not be identical to the Go-coupled 5-HT1P activity of the ENS. Because most studies of 5-HT4 receptors have concerned the central nervous system (CNS) where they are widely distributed (11, 31, 64), the splice variants that characterize 5-HT4 expression in the ENS and other peripheral sites (51) must still be identified. The current study was designed to identify the 5-HT4 splice variants expressed in the gut, to locate enteric sites of 5-HT4 expression, to clarify the relationship of the 5-HT4 receptor to 5-HT1P activity, and to determine the function of 5-HT4 receptors in enteric neuronal signaling. Observations made in the current study suggest that 5-HT1P activity and 5-HT4 receptors are not related to one another. The enteric 5-HT4 receptor is presynaptic and acts to enhance the secretion of excitatory neurotransmitters, whereas 5-HT1P activity evokes a postsynaptic G protein-mediated slow sustained inward current. The data are compatible with the suggestion that 5-HT4 agonists are prokinetic because they strengthen neurotransmission in excitatory pathways.

Address for reprint requests and other correspondence: M. Liu, Dept. of Anatomy and Cell Biology, College of Physicians and Surgeons, Columbia Univ., P&S 12–513, 630 W. 168th St. New York, New York, 10032 (e-mail: [email protected]).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked “advertisement” in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

serotonin receptor subtypes; G proteins; patch-clamp recording; presynaptic receptors

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Animals. Experiments were carried out with CD-1 mice of either sex (1–2 mo of age). Procedures and care followed guidelines of the National Institutes of Health and were approved by the Animal Care and Use Committee of Columbia University. The small and large intestine were removed from the mice, and dissected laminar preparations of longitudinal muscle with adherent myenteric plexus (LMMP) and submucosa (containing the submucosal plexus) were prepared. Control material was also obtained from dissected preparations of hippocampus, frontal cortex, atrium, and liver. RNA isolation and RT-PCR. Total RNA (2 ␮g) was prepared from tissues by using a commercial reagent (RNA STAT-60; Tel-Test, Friendswood, TX) according to the directions of the manufacturer. cDNA was prepared from RNA by reverse transcription at 50°C (30 min) in the presence of random primers and Moloney Murine Leukemia Virus reverse transcriptase (SuperScript III; Invitrogen, Carlsbad, CA). Reverse transcriptase was omitted from control samples, which were employed for the detection of contamination of samples with genomic DNA. All experiments were carried out with 0.1% diethyl pyrocarbonate (DEPC; Sigma, St. Louis, MO)-treated distilled water. The cDNA was amplified by using PCR with Taq DNA polymerase (Promega, Madison, WI) in a Gene-Amp PCR System 9700 (Applied Biosystems, Foster City, CA). Initial denaturation was carried out at 94°C for 1 min. The touchdown method (30) was employed using a number of annealing temperatures in a single PCR (63–58°C for 10 cycles at 45 s/cycle). Final amplification was carried out with 20 cycles of denaturation at 94°C for 30 s, annealing at 58°C for 45 s, and elongation at 72°C for 1 min. After the final PCR cycle, the reaction was extended for an additional 7 min at 72°C after which the reaction products were cooled to 4°C. Primer pairs are listed in Table 1. PCR products were resolved by electrophoresis through 1% agarose gels containing ethidium bromide (0.3 ␮g/ml) in Tris/acetic acid/EDTA (TAE) electrophoresis buffer. The identity of the PCR products was verified by sequencing. For this purpose, the PCR products were cloned using a commercial kit (TOPO TA; Invitrogen). Clones with inserts were further processed to isolate plasmid DNA (Wizard Minipreps-Promega or QIAfilter Plasmid Midi kit-Qiagen). Sequencing of the plasmid DNA was accomplished by dye termination (ABI Automated Sequencer, Applied Biosystems) in the core facility of Columbia University. The final cDNA sequences were compared with those in the GenBank (BLAST search at National Center for Biotechnology Information, Bethesda, MD). The fluorescence of ethidium bromide in agarose gels was photographed, scanned, and digitized. The optical density of the digitized images was quantified by computer-assisted densitometry (Digital Science ID Image Analysis 1.51; Eastman Kodak, Rochester, NY). To assess relative quantities of transcripts encoding 5-HT4 receptor isoforms were normalized to those encoding synaptophysin, which was used as

a neuron-specific product. Data were derived from three separate RNA preparations. Relative abundance of 5-HT transcripts was compared by one-way repeated-measures ANOVA. Data are reported as means ⫾ SE (unless otherwise noted). In situ hybridization. Transcripts encoding 5-HT4 receptors were located in tissues by in situ hybridization as described previously (67). Small segments of ileum were fixed for 3 h with 4% (wt/vol) formaldehyde (from paraformaldehyde) in PBS (pH 7.4). The tissue was cryoprotected, embedded in Neg-50 Frozen Section Medium (Richard-Allen Scientific), frozen in liquid N2, and sectioned (8 ␮m) at ⫺20°C using a cryostat-microtome. All solutions were made with 0.1% DEPC-treated distilled water. Digoxigenin-labeled cRNA probes were prepared from mouse cDNA encoding a region of the mouse 5-HT4 receptor between the third and the seventh transmembrane domains. PCR was used to obtain the cDNA (sense primer: 5⬘-TTCCCTGGACAGGTATTACGC-3⬘, antisense primer: 5⬘-ATAGCCAAGCCAGAGGAAAG-3⬘). Antisense and sense probes were linearized from plasmid DNA using HindIII (antisense) or XbaI (sense) and purified. The T7 and SP6 RNA polymerases were then used to transcribe the riboprobes, which were quantified by dot blotting (DIG-RNA labeling kit, Roche, Indianapolis, IN). Frozen sections were cut with a cryostat-microtome and incubated with sense or antisense probes (0.5 ng/␮l) in a moist chamber at 60°C for 16 h. Bound digoxigenin was detected with alkaline phosphatase-coupled sheep antibodies to digoxigenin (diluted 1:1,500; Roche). Alkaline phosphatase activity was demonstrated and visualized with nitroblue tetrazolium chloride and 5-bromo-4-chloro3-indolyl-phosphate (Roche) in a buffer containing NaCl (100 mM), MgCl2 (50 mM), Tween-20 (1%), and Tris 䡠 HCl (100 mM), pH 9.5. Levamisole (0.25 mg/ml; Sigma) was added to inhibit endogenous neuronal intestinal alkaline phosphatase. The blue reaction product representing the sites in the tissue sections containing mRNA encoding the 5-HT4 receptors were visualized by bright-field microscopy (Leica, Allendale, NJ). Antibody production. A peptide sequence found within the second extracellular loop of the mouse 5-HT4 receptor was selected for use as an immunogen. This peptide, with an added NH2-terminal cysteine and an aminocaproic acid spacer “Z” (CZDVIEKRKFSHNS) was synthesized and conjugated to maleimide-activated keyhole limpet hemacyanin. Two hens (at Aves Labs, Tigard, OR) were immunized intramuscularly with the conjugated peptide suspension. The IgY fractions were then isolated from the yolks of eggs collected from the immunized hens. IgY fractions were further purified by affinity chromatography using Sepharose beads coupled to the immunizing peptide. The depleted IgY fraction served as a negative control. DNA constructs and transfection of COS-7 cells. Antibody specificity was tested with transfected cells that expressed 5-HT4 receptors. The 5-HT4b isoform, which is the longest of the 5-HT4 splice variants

Table 1. Primers used for PCR Product

Accession Number

5-HT4a

Y09587

5-HT4b

Y09585

5-HT4c

Y09588

5-HT4f

AJ011369

Synaptophysin

X95818

␤-Actin

X03765

S or AS

Primer Pairs

Size, bp

S AS S AS S AS S AS S AS S AS

5⬘-CCAAGGCAGCCAAGACTTTA-3⬘ 5⬘-GCAAAACTGTATACCTTAGTACATGGGTG-3⬘ 5⬘-CCAAGGCAGCCAAGACTTTA-3⬘ 5⬘-AGCAGCCACCAAAGGAGAAG-3⬘ 5⬘-CCAAGGCAGCCAAGACTTTA-3⬘ 5⬘-GGTCTATTGCGGAAGAGCAGGGGGAAACTT-3⬘ 5⬘-CCAAGGCAGCCAAGACTTTA-3⬘ 5⬘-GTTAGACGGGAACAGGTCTTAGTACATG-3⬘ 5⬘-ACACATGCAAGGAACTGAGG-3⬘ 5⬘-TAGGATATGGGGATGGGAAG-3⬘ 5⬘-GTGGGCCGCTCTAGGCACCAA-3⬘ 5⬘-CTCTTTGATGTCACGCACGATTTC-3⬘

327

S, sense; AS, antisense. AJP-Gastrointest Liver Physiol • VOL

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and contains sequences found in all other 5-HT4 isoforms, was used for this purpose. The full-length 5-HT4b sequence, with added PstI and SalI sites, was amplified by PCR. The resulting PCR product was digested with PstI and SalI for ligation into a gWIZ-green fluorescent protein (GFP) vector (Gene Therapy Systems, San Diego, CA) using T4 DNA ligase (Roche). Plasmids were sequenced before use to verify that they contained the correct inserts. COS-7 cells (ATCC, Manassas, VA) were transfected with Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions and cultured on acid-washed glass coverslips for an additional 24 – 48 h. In most experiments, the cells were then fixed with methanol for 10 min at ⫺20°C or 4% formaldehyde (from paraformaldehyde) with PBS for 15 min at room temperature. Alternatively, to determine whether cell surface 5-HT4b expression could be detected without permeabilization, living cells were exposed to antibodies to the 5-HT4 receptor or to the control depleted IgY fraction. The fixed cells were washed three times with PBS, permeabilized with 0.05% (vol/vol) Triton X-100 (Sigma), and blocked with a fish serum BlokHen II (diluted 1:10; Aves Labs) and 10% normal horse serum in PBS for 30 min. For immunostaining, cells were incubated with the affinity-purified anti5-HT4 receptor IgY antibodies (0.35 ␮g/ml) in a solution containing 0.05% Triton X-100 (Sigma) and 10% normal horse serum in PBS at room temperature for 3 h. When live cells were examined, the primary antibody concentration was 1 ␮g/ml (4°C, 30 min, without agitation) and Triton X-100 was omitted. After being washed with PBS, the sites of bound primary antibody were detected by incubation with goat anti-chicken secondary antibody coupled to Alexa 594 (diluted 1:600, room temperature for fixed cells and 1:250, 4°C for live cells; Molecular Probes, Eugene, OR) for 30 min. Live cells were fixed with methanol at ⫺20°C for 10 min after exposure to secondary antibodies. Detection of GFP expression was enhanced in some experiments by immunostaining preparations with Alexa 488-labeled rabbit antibodies to GFP (diluted 1:1,000; Molecular Probes). Control preparations were treated similarly but were incubated with the depleted IgY from which the immunogen had been adsorbed. All preparations were washed extensively in PBS after immunostaining and mounted in Vectashield (Vector, Burlingame, CA). Immunofluorescence was observed with a vertical fluorescence microscope (DMR RXA2, Leica) equipped with a cooled charge-coupled device camera (Retiga EXi; Qimaging, Burnaby BC, Canada) and a computer-assisted videoimaging system software (Openlab 3.1; Improvision. Lexington, MA). Photomicrographs of preparations were also obtained with a laserscanning confocal microscope (LSM 510, Axiovert IM100; Carl Zeiss, Thornwood, NY). Confocal images were obtained at 512 ⫻ 512 pixels. Contrast of images was adjusted using Adobe Photoshop 7 software (Adobe Systems, San Jose, CA), which was also used to arrange images in figures. No other image manipulations were used. Western blotting. Membrane proteins were extracted from brain (hippocampus and frontal cortex) and small intestinal LMMP with 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS; Pierce, Rockford, IL) (32). Tissues from six mice were pooled and homogenized (Dounce glass homogenizer) in 10 volumes (vol/wt) of ice-cold 50 mM Tris 䡠 HCl with 0.1 mM PMSF (Sigma) buffer, pH 7.4, containing a cocktail of protease inhibitors (Roche). The lysate was centrifuged at 40,000 g for 20 min at 4°C. The pellet was then washed twice by resuspension and centrifugation in 10 volumes of the same buffer. The final washed pellet was resuspended in 2 volumes of homogenizing solution. Nine volumes of the final suspension were mixed with one volume of 0.1 M CHAPS (in 50 mM Tris 䡠 HCl, pH 7.4), sonicated for 10 s (20 W), and left to stand at 4°C for 60 min. The suspension was again sonicated and centrifuged at 100,000 g for 60 min at 4°C. The clear supernatant was dialyzed overnight at 4°C in PBS with 0.1 mM PMSF buffer (pH 7.4) to remove residual CHAPS. The protein concentration of the final mixture was measured (Bio-Rad protein assay; Bio-Rad, Richmond, CA), and the preparation was stored at ⫺80°C until used as the source of solubilized membrane protein. AJP-Gastrointest Liver Physiol • VOL

Immunoblot assays were carried out as previously described (67). Briefly, membrane proteins (30 ␮g) were mixed with SDS sample buffer (Bio-Rad) containing dithiothreitol (350 mM), incubated at 70°C for 15 min, and then resolved on a 10% polyacrylamide gel (SDS-PAGE). Proteins were electrophoretically transferred from the gel to a nitrocellulose membrane (Bio-Rad). The blots were blocked by incubation for 1 h in fish serum (diluted 1:10; BlokHen II, Aves Labs) containing Tris-buffered saline (TBS), and then incubated in buffer containing 25 mM Tris, 150 mM NaCl, and 0.05% Tween-20 (TBST), with 5% (wt/vol) nonfat milk power (Bio-Rad) at room temperature for another hour. The blots were probed with purified chicken antibodies to the 5-HT4 receptor (70 ng/ml) or with the same concentration of the depleted IgY fraction overnight at 4°C. Immunoreactivity was identified with goat anti-chicken secondary antibodies conjugated to horseradish peroxidase (HRP; diluted 1:5,000; Aves Labs). HRP activity was detected using enhanced chemiluminescence (Pierce). All immunoblot assays were repeated in three separate preparations. Immunocytochemistry. Acetone fixation (at ⫺20°C for 15 min) or fixation by perfusion with 2% acrolein in PBS was used for immunocytochemistry because formaldehyde and glutaraldehyde were both found to eliminate immunoreactivity. Frozen sections of acetone-fixed duodenum, ileum, proximal, and distal colon were obtained with a cryostat-microtome (8-␮m sections) and incubated with fish serum (diluted 1:10 in PBS; BlokHen II, Aves Labs) for 30 min (at room temperature). Tissues were then permeabilized with 0.05% Triton X-100 and blocked with 10% normal horse serum in PBS for another hour. The preparations were exposed overnight to chicken anti-5-HT4 receptor antibodies (0.35 ␮g/ml; room temperature) and/or rat anti-cKit (ACK2) antibodies (5 ␮g/ml; at room temperature; eBioscience, San Diego, CA) in a solution that contained Triton X-100 and normal horse serum. After being washed with PBS, the sites of bound primary antibody were detected by incubation with goat anti-chicken secondary antibodies coupled to Alexa 488 (diluted 1:600; Molecular Probes) and/or goat anti-rat secondary antibodies coupled to Alexa 594 (diluted 1:600; Molecular Probes) for 3 h at room temperature. The preparations were washed again with PBS and mounted in Vectashield (Vector). Acrolein-fixed distal colon was frozen and sectioned as described above. Residual acrolein in the fixed tissues was destroyed by quenching sections with 1% (wt/vol) sodium borohydride in PBS at room temperature for 1 h. The sections were blocked and immunostained as described above, except that 0.1% saponin, rather than Triton X-100, was used for permeabilization. Sites of primary antibody binding were detected by incubation with donkey secondary antibodies to chicken IgY coupled to HRP (diluted 1:100; 3 h; room temperature; Jackson ImmunoResearch, West Grove, PA). Immunoreactivity was visualized with 3,3⬘-diaminobenzidine (DAB; Vector Laboratories DAB kit used according the manufacturer’s instructions). Control preparations were identically immunostained except that they were exposed to the depleted IgY fraction instead of antibodies to the 5-HT4 receptors. Alternatively, for ACK2, control sections were incubated only with secondary antibodies. Electron microscopy. Small pieces (1 cm3) of LMMP from the small intestine were fixed for electron microscopy (EM) in a mixture of 4% formaldehyde (from paraformaldehyde) and 0.01% glutaraldehyde (Sigma) for 3 h at room temperature. After being rinsed in PBS buffer and dehydration in cold ethanol, the tissues were embedded in LR Gold resin (Electron Microscopy Sciences, Fort Washington, PA) and polymerized under ultraviolet light (365 nm) at ⫺20°C. Ultrathin sections were collected on Formvar-coated nickel grids and treated with 10% fish serum (BlokHen II; Aves Labs) and 10% donkey serum with TBST for 30 min at room temperature to block nonspecific binding. The sections were incubated with chicken anti-5-HT4 receptor antibodies (0.35 ␮g/ml) in 4% donkey serum in TBST for 60 min at room temperature. Incubation with the antibody-depleted IgY fraction was used as a control. After being rinsed with TBS, the grids were incubated for 60 min at room temperature with donkey anti289 • DECEMBER 2005 •

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chicken IgY labeled with 12-nm gold particles (1:40; Jackson ImmunoResearch) in TBST. The grids were washed with TBS and postfixation with 2% glutaraldehyde and then washed with distilled water, stained with osmium tetroxide, uranyl acetate, and lead citrate (Electron Microscopy Sciences) and observed and photographed using a JEOL 1200EX microscope (JEOL, Peabody, MA). Electrophysiological acquisition and data analysis. Myenteric neurons from mice approximately 1 mo of age were cultured as described previously (67). Neurons were investigated 2–5 days after plating. Whole cell patch-clamp recordings were obtained as previously described (67). Briefly, patch pipettes (⬃5 M⍀) were filled with an internal pipette solution containing (in mM): 140 potassium gluconate, 0.1 CaCl2, 2 MgCl2, 1 EGTA, 2 Na2ATP, 0.2 Na3GTP, and 10 HEPES, pH 7.25 with KOH. The bath solution contained (in mM): 145 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgCl2, 11 glucose, and 10 HEPES, pH 7.35, with NaOH. A HEKA EPC-9B amplifier (HEKA Instruments, Southboro, MA) was used to voltage clamp neurons at ⫺60 mV. Recordings were carried out at room temperature (20 –22°C) on the stage of an inverted microscope (Axiovert IM35, Zeiss). Electrophysiological data were filtered at 5 kHz and digitized at 10 kHz and collected using the Pulse 8.54 program (HEKA). Only cells with series resistance ⬍15 M⍀ were analyzed. The liquid junction potential was ⬃11 mV, which was not corrected. Data for continuous gap-free recordings were digitized at 5 kHz by using a Digidata 1322A interface and AxoScope 8.1 program (Axon Instruments, Union City, CA). The bath solution was continuously perfused through the recording chamber by gravity. Current-voltage relationships were determined from a holding potential of ⫺60 mV by applying 400-ms test pulses (⫺120 to ⫹20 mV) in 10-mV increments. Experimental compounds were applied by changing gravity-fed perfusion lines of a rapid-exchange Y-tube system controlled with solenoid valves by means of the Pulse software (HEKA). The nonhydrolyzable GTP analog GTP␥S was included in the patch pipettes to increase the sensitivity of detection of G proteinmediated currents. The presence of GTP␥S enhances and prolongs the active state of G proteins (4). The tips of the recording pipettes were filled by capillary action to a depth of ⬃1 mm with the internal pipette solution to facilitate the formation of gigaohm seals with target neurons; the pipettes were then backfilled with the same solution supplemented with GTP␥S (600 ␮M). To help confirm the identity of G proteins activated by extracellular applications of 5-HT, antibodies to G␣o and G␣s subunits (Chemicon, Temecula, CA) were included in the internal solution within patch pipettes. The antibodies were dialyzed overnight at 4°C in PBS, pH 7.4, before use. In control experiments, dialyzed antibodies to G␣o and G␣s subunits were inactivated by exposure to heat (60°C) and then used in patch pipettes at the same dilution. Excitatory postsynaptic currents (EPSCs) were evoked with bipolar tungsten electrodes placed on a neurite visualized at a distance of 300 –500 ␮m from the patched neuron. Two tungsten electrodes (0.5 M⍀; World Precision Instruments, Sarasota, FL) were used to fabricate the stimulating electrodes by putting them together with a tip separation of 500 ␮m. Focal stimulation (0.5-ms pulses; 0.3 Hz) was applied with a Grass S48 stimulator (Astro-Med, West Warwick, RI). The minimal stimulating current necessary to evoke EPSCs that were stable in amplitude on successive stimuli was determined and used in all studies of presynaptic drug action. Evoked EPSCs were thus submaximal in amplitude. To confirm that evoked EPSCs were cholinergic, the nicotinic antagonist hexamethonium (C6; 300 ␮M) was added to the external solution. The fast EPSCs were considered to be cholinergic synaptic events if they were blocked reversibly by C6. Data are reported as means ⫾ SE, and the significance of differences was evaluated by paired Student’s t-tests (unless otherwise noted). 5-HT or 5-HT4 agonists were applied for 10 s at intervals of 5–10 min to avoid receptor desensitization. All experiments were carried out with submaximal concentrations of agonists. When antagonists were studied, they were applied for 60 s before the coapplication of an AJP-Gastrointest Liver Physiol • VOL

agonist. The amplitudes of four consecutive evoked EPSCs were averaged and recorded at 60-s intervals. Experiments were completed ⬍1 h after recordings were begun to minimize time-dependent changes in response properties or deterioration of cells. Off line data analysis was carried out with software programs AxoGraph 4.8 (Axon Instruments) or MiniAnalysis 5.8 (Synaptosoft, Decatur, GA). Compounds used. All drugs were prepared as stock solutions in distilled water, ethanol, 1-methyl-2-pyrrolidinone, or DMSO at greater than 103 times the highest experimental concentration according to the instruction of manufacturer and stored at ⫺20°C until use. The stock solution was subsequently diluted in the external solution for the experimental use. 5-HT creatinine sulfate, 5-methoxytryptamine (5MeOT), 1-(m-chlorophenyl)-biguanide, guanosine-5⬘-O-(3-thiotriphosphate; GTP␥S), guanosine-5⬘-O-(2-thiodiphosphate; GDP␤S), hexamethonium, tetrodotoxin, 1-methyl-2-pyrrolidinone, and DMSO were purchased from Sigma/RBI (Natick, MA). WAY 100325 was obtained from Wyeth (Madison, NJ). 5-HTP-DP, N-acetyl-5-hydroxytryptophyl-5-hydroxytryptophan amide, was obtained from Dr. H. Tamir (New York State Psychiatric Institute, New York, NY). Endo-N-(8-methyl-8azabicyclo[3,2,1]oct-3-yl)-2,3-dihydro-3-ethyl-2-oxo-1H-benzimidazole1-carboxamide (BIMU 1) was obtained from Boehringer-Ingleheim (Milan, Italy). Renzapride {BRL 24924; (⫾)-endo-4-amino-5-chloro-2methoxy-N-(1-azabicyclo[3,3,1]non-4-yl)benzamide monhydrochloride}, [1-[2-(methylsulphonylamino)ethyl]-4-piperidinyl]methyl 1-methyl1H-indole-3-carboxylate maleate (GR 113808), and 8-amino-7chloro-(N-butyl-4-piperidye)methylbenzo-1,4-dioxan-5-carboxylate hydrochloride (SB 204070) were obtained from GlaxoSmithKline (Harlow, UK). 5-Hydroxyindalpine (5-OHIP) was obtained from Rhone-Poulenc Sante (Gennevilliers, France). Tegaserod [3-(5-methoxy-1H-indol-3-ylmethylene)-N-pentylcarbazimidamide hydrogen maleate] was obtained from Novartis (Basel, Switzerland). RESULTS

Transcripts encoding four 5-HT4 receptor isoforms are present in the mouse gut. RT-PCR (touchdown method of amplification) was used with primers corresponding to sequences found in the mouse 5-HT4 receptor to identify the subtypes of 5-HT4 receptor that are transcribed in the gut. Each primer pair was selected so as to amplify selectively a single cDNA corresponding to one of the 5-HT4 isoforms. cDNA, extracted from the brain and atrium, which are known to express 5-HT4 receptors, were investigated as positive controls, whereas the liver, which is thought not to express 5-HT4 receptors, served as a negative control. As an additional negative control, to rule out potential contamination from genomic DNA, reverse transcriptase was omitted before amplification. Loading of gels was standardized so that an approximately equal amount of ␤-actin PCR product was present in each lane. Subcloning and sequencing were carried out to confirm the identities of the PCR products. Transcripts encoding each of the known isoforms of mouse 5-HT4 receptor (a, b, e, and f) were found in the gut, brain, and atrium but not in the liver (Fig. 1). Transcripts encoding synaptophysin were also amplified and used to semiquantitatively normalize the data to the relative amount of mRNA encoding a synapse-specific gene product (Fig. 1). Notably, transcripts encoding the 5-HT4e and 5-HT4f isoforms, which have previously been considered to be brain-specific (24), were detected in the mouse gut. Transcripts encoding 5-HT4 receptors, normalized to those encoding synaptophysin, were highly abundant in the gut, even compared with the brain and the atrium (Fig. 2). Expression of the 5-HT4a and 5-HT4b receptors was dominant in all regions of the bowel and more pronounced in the submucosal than in 289 • DECEMBER 2005 •

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Fig. 1. Transcripts encoding splice variants (a, b, e, and f) of 5-HT4 receptors are present in the mouse gut. 5-HT4 receptors were detected in the indicated tissues by means of RT-PCR. The expected size of the PCR products is shown to the right of the gels. Transcripts encoding 5-HT4a and 5-HT4b receptors were detected in the brain and atrium (positive controls) and in the longitudinal muscle with adherent myenteric plexus (LMMP) and submucosa [containing the submucosal plexus (SMP)] of the small intestine (SI), proximal colon (PC), and distal colon (DC), but not in the liver (negative control). Transcripts encoding 5-HT4e and 5-HT4f receptors were largely restricted to the brain and the LMMP of the SI and PC; very little was found in the SMP. The amount of cDNA added to each lane was normalized to that encoding ␤-actin amplified from each tissue. Reverse transcriptase (RTase) was omitted in controls to rule out contamination by genomic DNA. Transcripts encoding the synaptic protein synaptophysin (SYP) were simultaneously amplified to provide an indicator of the cDNA from each tissue that is derived from neurons.

the LMMP preparation. The apparent expression of mRNA encoding 5-HT4e and 5-HT4f receptors were myenteric plexus specific (Fig. 2). No significant difference was observed in the expression pattern of 5-HT4 transcripts in the small and large intestines. Enteric neurons contain transcripts encoding the 5-HT4 receptor. In situ hybridization was used to identify the cells that contained transcripts encoding 5-HT4 receptors in the mouse gut. mRNA encoding 5-HT4 receptors was detected in enteric neurons and smooth muscle. A cRNA probe specifically labeled subsets of neurons in both the myenteric and submucosal plexuses (Fig. 3, A and B). The same cRNA probe also labeled a subset of smooth muscle cells (Fig. 3C). No labeling of any epithelial cells was detected (Fig. 3C). As a control, sections were hybridized with a riboprobe in the sense configuration; in contrast to the experimental antisense cRNA, no labeling with the sense probe was obtained (Fig. 3D). Preparation and characterization of antibodies to the 5-HT4 receptor. Polyclonal antibodies to the mouse 5-HT4 receptor were generated in chickens to ascertain that not just mRNA, AJP-Gastrointest Liver Physiol • VOL

Fig. 2. Semiquantitative PCR was used to evaluate the relative expression of mRNA encoding splice variants of the 5-HT4 receptors. The optical density of PCR products obtained by amplifying cDNA with 5-HT4 isoform-specific primers was expressed as a ratio to that of cDNA simultaneously amplified with SYP-specific primers (see Fig. 1). Within the gut, the ratios to SYP of transcripts encoding the 5-HT4a and 5-HT4b receptors were higher in preparations containing the submucosal than the myenteric plexus, higher in the gut than in the brain, and least abundant in the atrium. Ratios to SYP of transcripts encoding the encoding 5-HT4e and 5-HT4f receptors were not as great as those of the 5-HT4a and 5-HT4b receptors but were brain and myenteric plexus specific. Data points are means ⫾ SE of 3 measurements.

but also 5-HT4 receptor protein is expressed in the bowel and to make it possible to identify the cells in which this protein is expressed. A synthetic peptide corresponding in sequence to amino acids (168 –179; mouse 5-HT4 receptor; GenBank accession no. NP 032339) was used as the immunogen. This sequence is found in the second extracellular domain of the 5-HT4 receptor. The IgY fraction of the resulting antiserum

Fig. 3. Transcripts encoding 5-HT4 receptors are present in a subset of enteric neurons and smooth muscle cells. mRNA encoding the 5-HT4 receptor was located in frozen sections of mouse ileum by means of in situ hybridization. The arrows point to myenteric (A) and submucosal (B) neurons that contain transcripts encoding 5-HT4 receptors. The arrowheads (C) point to labeled smooth muscle cells. D: control sections, hybridized with a sense riboprobe, lacked reaction product. Scale bar ⫽ 25 ␮m. 289 • DECEMBER 2005 •

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Fig. 4. Affinity purified IgY antibodies to 5-HT4 receptors immunostain 5-HT4-expressing cells. COS-7 cells were transfected with cDNA encoding the 5-HT4 receptor tagged with green fluorescent protein (GFP). A and B: anti-5-HT4 IgY (A) immunostains transfected 5-HT4-GFP-expressing cells (B). C and D: control. Adsorbed anti-5-HT4 IgY (C) fails to immunostain transfected 5-HT4-GFP-expressing cells (D). E and F. Control: COS-7 cells transfected only with a GFP-expressing vector. Anti-5-HT4 IgY (E) fails to immunostain GFP-expressing cells (F). G: anti-5-HT4 IgY (G) immunostains the surfaces of living nonpermeabilized transfected 5-HT4-GFP-expressing cells. H: adsorbed anti-5-HT4 IgY fails to immunostain the surfaces of living nonpermeabilized transfected 5-HT4-GFP-expressing cells. Scale bars ⫽ 20 ␮m.

was obtained and affinity purified. The utility of the antibodies (anti-5-HT4) for the immunocytochemical detection of 5-HT4 receptors was evaluated with transfected COS-7 cells that expressed the 5-HT4b receptor tagged with GFP (Fig. 4). The chicken anti-5-HT4 reacted with the 5-HT4b GFP-expressing COS-7 cells (Fig. 4, A and B). In contrast, no immunofluorescence was seen when the 5-HT4b GFP-expressing cells were exposed to a depleted IgY control fraction (anti-5-HT4 adsorbed with the immunogen; Fig. 4, C and D) or when cells that expressed only GFP were exposed to anti-5-HT4 IgY (Fig. 4, E and F). When the COS-7 cells were permeabilized, 5-HT4 immunoreactivity was predominantly intracellular (Fig. 4A); however, when nonpermeabilized 5-HT4b GFP-expressing cells were exposed to anti-5-HT4 IgY (Fig. 4G), the immunofluorescence was prominent on the cell surface. No surface immunofluorescence was detected when nonpermeabilized 5-HT4b GFP-expressing cells were exposed to the depleted IgY fraction (Fig. 4H). These observations suggest that the anti-5HT4 reagent can be employed to locate 5-HT4 receptors by immunocytochemistry, that the receptors reach the cell surface even when they are expressed as an exogenous protein in a cell line, and that the antibodies react with the ectodomain of the 5-HT4 receptor and thus can detect 5-HT4-expresssing cells without prior permeabilization. An immunoblot was carried out with membrane proteins extracted from the small intestinal LMMP and from the brain to verify the specificity of the anti-5-HT4 reagent. The membrane proteins were separated by SDS-PAGE, and the resulting blots were probed with anti-5-HT4 and the depleted IgY control fraction. A single major immunoreactive band (46 kDa), corresponding in size to that of the mouse 5-HT4 receptor, was found in membrane extracts of the small intestine AJP-Gastrointest Liver Physiol • VOL

LMMP and brain. In contrast, no labeling was observed when the blots were probed with the depleted IgY fraction (Fig. 5). 5-HT4 receptor protein is specifically expressed in a subset of enteric neurons. Anti-5-HT4 was used to investigate the distribution of 5-HT4 immunoreactivity in the bowel wall. Tissue was fixed either with acetone (Fig. 6, A-D) or acrolein (Fig. 6, E-H). Subsets of neurons, and especially the ganglionic neuropil, were found to be 5-HT4-immunoreactive in both the myenteric (Fig. 6, A-F) and submucosal plexuses. 5-HT4 immunoreactivity was observed in all regions of the small intestine from the duodenum (Fig. 6A) through the terminal ileum (Fig. 6B) and was also seen in both the proximal (Fig. 6C) and

Fig. 5. Membrane proteins extracted from the LMMP of the small intestine and brain were separated by SDS-PAGE, blotted onto nitrocellulose, and probed with anti-5-HT4 IgY. A single major immunoreactive band (46 kDa) was found in both LMMP and brain. No immunoreactivity was detected when the blot was probed with a depleted IgY fraction from which the immunogen had been adsorbed. Markers for molecular weights (in kDa) are shown to the left of the immunoblots. 289 • DECEMBER 2005 •

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Fig. 6. Sites of 5-HT4 immunoreactivity were located in the bowel wall by immunocytochemistry with anti-5-HT4 IgY. A-D: acetone fixation, sites of bound IgY located with Alexa 488-labeled secondary antibodies and immunofluorescence; A: duodenum; B: ileum; C: proximal colon; D: distal colon. 5-HT4 immunoreactivity is very dense in the ganglionic neuropil of the myenteric plexus and surrounds non-5-HT4-immunoreactive nerve cell bodies (arrows). The dense labeling of fibers in the myenteric plexus makes immunoreactive nerve cell bodies difficult to distinguish. 5-HT4-immunoreactive nerve cell bodies are more obvious in the submucosal plexus (arrowheads). 5-HT4-immunoreactive nerve fibers are visible in the circular muscle (*). The smooth muscle also displays 5-HT4 immunoreactivity, which is most apparent in the longitudinal layer (**). E–H: acrolein fixation, distal colon; IgY located with horseradish peroxidase (HRP)-labeled secondary antibodies and 3,3⬘-diaminobenzidine (DAB). Acrolein fixation partially suppresses the immunoreactivity of the myenteric neuropil enabling 5-HT4-immunoreactive nerve cell bodies to be discerned (arrows). No 5-HT4-immunoreactive fibers can be detected in the mucosa (E, F) after either method of fixation. No immunostaining (H) is seen with deleted IgY from which antibodies were adsorbed with the immunogen. I–L: acetone fixation, ileum; ICCs are doubly labeled by antibodies to 5-HT4 receptors (I, K) and Kit (J, K). The merged images are illustrated in K. Because these interstitial cells of Cajal (ICCs) are associated with the myenteric plexus, they are IC-MY. No immunostaining (L) is seen when sections are incubated with deleted IgY antibodies and without antibodies to Kit (ACK2). Scale bars ⫽ 20 ␮m.

distal (Fig. 6, D–E) colon. Smooth muscle cells were 5-HT4 immunoreactive, and patches of immunoreactivity outlined the cells, suggesting that the receptors are concentrated in the plasma membranes. No structures were immunostained in the mucosa (Fig. 6F). Neither mucosal nerves nor epithelial cells were 5-HT4-immunoreactive. No immunostaining was obtained when tissue sections were incubated with the depleted IgY fraction instead of anti-5-HT4 (Fig. 6H). Sections of small intestine were coimmunostained with antibodies to Kit (ACK2) and 5-HT4 receptors to determine whether interstitial cells of Cajal (ICC) express 5-HT4 receptors. Kit is an ICC marker in the bowel (19, 99), and ICCs were most prominent bordering myenteric ganglia (IC-MY) and in the deep muscle plexus (IC-DMP) at the junction of the circular muscle layer and the submucosa (Fig. 6J). IC-DMP had tapering long processes that paralleled the long axis of smooth muscle fibers, and IC-MY often formed branches around neurons in the myenteric plexus. Coincident localization of 5-HT4 and Kit immunoreactivity was found in IC-MY (Fig. 6, I–K) and IC-DMP, although 5-HT4 immunoreactivity was very sparse in IC-DMP. No AJP-Gastrointest Liver Physiol • VOL

immunostaining was obtained when control tissue sections were incubated without ACK2 and with the depleted IgY fraction of anti-5-HT4 (Fig. 6L). At the electron microscopic level, 5-HT4 immunoreactivity was found in axons of the myenteric plexus and in smooth muscle cells (Fig. 7). Axonal immunoreactivity was concentrated in subsets of varicosities, which contained both large dense cored and small electron lucent synaptic vesicles (Fig. 7A). Within these axons, large, dense, cored granules were often labeled (arrows). In contrast to the immunoreactivity found when sections were exposed to anti-5-HT4 IgY, no labeling was obtained when sections were incubated with the depleted IgY fraction (Fig. 7B). In smooth muscle cells, 5-HT4 immunoreactivity was found in scattered elements of rough endoplasmic reticulum and in caveolae in or near the plasma membrane (Fig. 7A, inset). Only a subset of the axons in the ganglionic neuropil contained 5-HT4 immunoreactivity (Fig. 8). Within these axons, 5-HT4 immunoreactivity was most concentrated inside regions of varicose expansion (Fig. 8, A and B, insets). 289 • DECEMBER 2005 •

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Fig. 7. A 5-HT4-immunoreactive axonal terminal varicosity of the myenteric plexus is illustrated (A) and the plasma membrane of a smooth muscle cell is shown in the inset. Sites of bound IgY were detected with secondary antibodies coupled to colloidal gold (12 nm diameter). The varicosity contains both large, dense, cored, and small electron lucent synaptic vesicles. Note that immunogold particles are concentrated over large, dense, cored granules (arrows) and other vesicular structures. A caveolus of the smooth muscle cell (arrow; inset contains 5-HT4 immunoreactivity; scale bar ⫽ 100 nm). Note the close association of the immunogold particles with the membrane of the caveolus. Almost no labeling with gold (B) was seen when sections were exposed to deleted IgY from which antibodies were adsorbed with the immunogen instead of anti-5-HT4. Scale bars ⫽ 200 nm.

5-HT4 receptors facilitate EPSCs evoked in myenteric neurons. Although both fast and slow depolarizing responses to 5-HT have been observed when recordings have been made from enteric neurons impaled with sharp microelectrodes (39), only a fast inward current has previously been observed with the patch-clamp technique to be evoked by 5-HT (67, 101, 102). The fast responses, however recorded, are 5-HT3 mediated (67, 101, 102). The 5-HT3 receptor is a ligand-gated ion channel (29); all of the other subtypes of 5-HT receptor are G protein coupled (39, 53). It is possible that the dialysis of intracellular contents that occurs when neurons are patched obscures or interferes with the intracellular events of signal transduction from G protein-coupled receptors. If so, then the changes in transmembrane current resulting from G proteinAJP-Gastrointest Liver Physiol • VOL

mediated signal transduction might become apparent if G protein coupling could be potentiated or prolonged. To do so, the solution inside of the pipettes used to patch enteric neurons was layered so that an internal solution containing the nonhydrolyzable GTP␥S was layered over a non-GTP␥S-containing solution at the pipette tips. These pipettes allowed an initial recording of the response to 5-HT to be made without internal GTP␥S (Fig. 9A) while subsequent recordings from the same neurons of the response to 5-HT were obtained in its presence (Fig. 9B). As noted previously, 5-HT evoked only a fast inward current in the absence of GTP␥S (Fig. 9A); however, after 10 –15 min, when GTP␥S had diffused into the cells, the 5-HT-elicited fast inward current was now followed by a prolonged inward current that was sustained for ⬎15 s. The fast inward current evoked by 5-HT before the diffusion of GTP␥S into the cells was blocked by 1.0 ␮M granisetron, a 5-HT3 antagonist (data not illustrated). The 5-HT-induced slow sustained inward current obtained in the presence of GTP␥S (Fig. 9B) was granisetron resistant, averaged ⫺137.6 ⫾ 16.0 pA (n ⫽ 8), and was detected in ⬃20% of myenteric neurons (n ⫽ 70). To verify that the GTP␥S-dependent manifestation of the 5-HT-evoked slow sustained inward current is related to the effect of GTP␥S on G protein coupling, responses to 5-HT in neurons that were patched with pipettes containing GTP␥S were compared with those in neurons patched with pipettes containing GTP␤S. When GTP␥S was included in the patch pipette, 5-HT evoked a sustained inward current with a rapid onset (Fig. 9C). In contrast, when GDP␤S was included in the patch pipette, the inward current elicited by 5-HT decayed rapidly and was not sustained (Fig. 9D). The application of TTX (1 ␮M) affected neither the rapid onset nor the slow sustained component of the inward current associated with responses to 5-HT in the presence of internal GTP␥S (Fig. 9, E and F). The failure of TTX to affect either component of the inward current evoked by 5-HT suggests that the cells from which recordings were obtained respond directly to 5-HT rather than indirectly to a transmitter released from other cells in the cultures; however, agonists may release a neurotransmitter from terminals via a TTX-insensitive mechanism (89). To determine which G protein is involved in mediating the slow sustained component of the 5-HT-evoked inward current, antibodies to G␣o or G␣s were included in the patch pipettes. Antibodies to G␣o and G␣s were investigated because slow depolarizing responses of enteric neurons have been reported to be Go coupled (81), whereas 5-HT4-mediated responses are coupled to Gs (41). The human 5-HT4b receptor, however, couples to G␣i/o, in addition to G␣s, when transiently expressed as an exogenous receptor in HEK 293 cells (83). When antibodies to G␣o were included with GTP␥S in the patch pipette, the fast inward current elicited by 5-HT decayed rapidly, as with GDP␤S, and was followed by virtually no sustained current (Fig. 9G). The sustained current was still noticed when the heat-inactivated antibody to G␣o was included with GTP␥S in the patch pipette (data not shown). In contrast, when a dialyzed rabbit antibody to G␣s was included with GTP␥S in the patch pipette, the fast inward current did not decay as rapidly as with the antibody to G␣o, and a small current was sustained despite the presence of the antibody (Fig. 9H). The inclusion of the antibody to G␣s, however, did reduce the amplitude of the sustained current from that seen with GTP␥S alone. The specificity of antibodies to G␣o (40 kDa) (66) and 289 • DECEMBER 2005 •

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Fig. 8. A 5-HT4 immunoreactive axon courses diagonally through the neuropil of an ileal myenteric ganglion; within this axon, immunogold particles are concentrated in regions of varicose expansion (indicated by the arrows at A and B and in the corresponding insets). The other axons and cellular processes in the field are not labeled. Scale bar ⫽ 200 nm.

G␣s (42 and 45 kDa; 2 isoforms of G␣s) (88) was confirmed by immunoblotting with LMMP of small intestines (Fig. 9I). These observations suggest that one or more G protein-coupled receptors contribute to the sustained phase of the inward current evoked by 5-HT. In the presence of GTP␥S, WAY 100325 (50 ␮M), a specific 5-HT1P agonist (81), induced a slow sustained inward current (Fig. 10A), which was blocked by 5-HTP-DP (5 ␮M), a 5-HT1P antagonist (Fig. 10B). In the presence of GTP␥S, the 5-HT4 agonists 5-MeOT (50 ␮M; Fig. 10C) and BIMU 1 (50 ␮M; Fig. 10D) evoked only low-amplitude fast transient inward currents when first applied (respectively, ⫺30.5 ⫾ 4.5 or ⫺10.6 ⫾ 2.2 pA; n ⫽ 4). Neither of these compounds, nor another 5-HT4 agonist, renzapride (5-HT4 agonist/5-HT3 antagonist; 1.0 ␮M; n ⫽ 4; not illustrated), evoked a sustained inward current; moreover, no significant change in the currentvoltage relationship was detected in the presence or absence of 5-MeOT, BIMU 1, or renzapride (data not shown). The 5-HT4 antagonists SB 204070 (1 ␮M; Fig. 10F) and GR 113808 (1 ␮M; data not shown) failed to affect the GTP␥S-dependent slow sustained inward current evoked by 5-HT (50 ␮M; n ⫽ 3). In a subset of cells, many spontaneous low-amplitude transient fast currents (Fig. 11A), which resembled fast EPSCs, were superimposed on the slow sustained inward current evoked by the local application of 5-HT. Although the 5-HT4 AJP-Gastrointest Liver Physiol • VOL

antagonist GR 113808 (1 ␮M) did not block the slow sustained inward current, GR 113808 irreversibly abolished the superimposed low-amplitude transient fast currents (Fig. 11, B and C). The low-amplitude transient fast currents, therefore, are likely to have been EPSCs evoked presynaptically by 5-HT (see below; Fig. 12). The 5-HT1P agonist 5-OHIP (50 ␮M), similar to 5-HT, evoked a GTP␥S-dependent slow sustained inward current (⫺55.5 ⫾ 14.7 pA; n ⫽ 3; Fig. 11D). This effect was not shared by the 5-HT4-agonist tegaserod (1.0 ␮M) applied to the same cells (Fig. 11E). The slow sustained inward current evoked by 5-OHIP was blocked by 5-HTP-DP (5 ␮M; Fig. 11F). These observations confirm that the GTP␥S-dependent slow sustained inward current evoked by 5-HT is a 5-HT1P-mediated event. It is not related to 5-HT4 receptors. Experiments were carried out to test the hypothesis, suggested by the low-amplitude transient fast currents seen in the presence of 5-HT4 agonists (Figs. 10, C and D, and 11A), that 5-HT4 receptors act presynaptically to evoke or potentiate fast EPSCs. Most fast excitatory neurotransmission in the myenteric plexus is mediated by ACh and is nicotinic. We therefore investigated the ability of hexamethonium to block putative EPSCs. Stimulating fibers that innervated patched neurons evoked fast EPSCs. The resulting evoked EPSCs were reversibly blocked by the nicotinic antagonist hexamethonium (300 289 • DECEMBER 2005 •

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Fig. 9. The slow sustained inward current evoked by 5-HT is G protein mediated. A, B: neuron patched with a pipette containing GTP␥S layered over a non-GTP␥S-containing solution at the pipette tips. A: only a fast inward current is initially evoked by 5-HT (50 ␮M) in the absence of GTP␥S. B: after 20 min, when the overlying GTP␥S has equilibrated at the pipette tip, the fast inward current evoked by 5-HT (50 ␮M) is followed by a sustained inward current. C: neuron patched with a pipette containing GTP␥S. 5-HT is added continuously for 10 s (dashed line). 5-HT induces an inward current with a rapid onset; the response does not desensitize but is sustained in the continuous presence of 5-HT. D: neuron patched with a pipette containing GDP␤S. 5-HT is added continuously for 10 s (dashed line). 5-HT induces a fast inward current that decays rapidly despite the continuous presence of 5-HT; the fast response thus desensitizes readily. E and F: neuron patched with a pipette containing GTP␥S. 5-HT is added continuously for 10 s (dashed line). 5-HT evokes a sustained inward current that is not affected by TTX (1 ␮M; compare E with F). G: neuron patched with a pipette containing GTP␥S and antibodies to G␣o. 5-HT evokes only a fast inward current fast inward current that decays rapidly. As when GDP␤S is present in patch pipettes, antibodies to G␣o prevented the sustained inward current (compare with D). H: neuron patched with a pipette containing GTP␥S and antibodies to G␣s. The sustained component of the inward current evoked by 5-HT is partially inhibited (compare with D and G). I: immunoblots of membrane proteins extracted from small intestinal LMMP demonstrating the specificity of antibodies to G␣o and G␣s. A single immunoreactive band is seen at the expected molecular weight (40 kDa; arrow) when probed with antibodies to G␣o. Two immunoreactive bands are seen molecular weights expected for the two isoforms of G␣s (42 and 45 kDa; arrow). Markers for molecular weights (in kDa) are shown to the left of immunoblots.

␮M; Fig. 12A). The 5-HT4 agonists renzapride (100 nM; Fig. 12B) and tegaserod (100 nM; Fig. 12C) each significantly increased the amplitude of evoked EPSCs (154.4 ⫾ 12.1 and 155.0 ⫾ 10.6% of control, respectively; n ⫽ 6; P ⬍ 0.05). Responses to both agonists desensitized rapidly, making it difficult to obtain responses to higher concentrations of either compound. The 5-HT4 antagonist GR 113808 (10 nM) did not affect EPSCs; however, GR 113808 blocked the facilitation of fast EPSCs elicited either by renzapride or tegaserod to 14.1 ⫾ 1.4 and 12.2 ⫾ 0.3% of the control, respectively (n ⫽ 3; Fig. 12D). These data confirm that 5-HT4 receptors act presynaptically to increase the amplitude of cholinergic (nicotinic) EPSCs in mouse myenteric neurons. These observations are consistent with those made on other species in which both facilitation of fast EPSPs (41, 65, 79) and release of ACh have been reported (47). AJP-Gastrointest Liver Physiol • VOL

DISCUSSION

The current study was designed to identify the 5-HT4 splice variants expressed in the gut, to locate enteric sites of 5-HT4 expression, to clarify the relationship of the 5-HT4 receptor to 5-HT1P activity, and to determine the function of 5-HT4 receptors in enteric neuronal signaling. With regard to the expression of splice variants of the 5-HT4 receptor, our data indicate that each of the four isoforms (a, b, e, and f) of the mouse 5-HT4 receptor that have been found in the brain (24) are also expressed in the gut. Expression of the 5-HT4a and 5-HT4b isoforms predominated in all regions of the bowel and in both plexuses; however, the level of expression of each of these isoforms, normalized to the expression of synaptophysin to correct for the proportion of transcripts derived from neurons, was greater in the submucosal than the myenteric plexus. The 289 • DECEMBER 2005 •

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Fig. 10. Slow sustained current induced by 5-HT is not mediated by 5-HT4 receptors. A and B: neuron patched with a pipette containing GTP␥S. The 5-HT1P agonist WAY 100325 (50 ␮M) induces a slow sustained inward current (A), which is blocked by the selective 5-HT1P receptor antagonist 5-HTP-DP (5 ␮M; B). C and D: neuron patched with pipettes containing GTP␥S. Neither 5–5-methoxytryptamine (5-MeOT; 50 ␮M; C) nor endo-N(8-methyl-8-azabicyclo[3,2,1]oct-3-yl)-2,3-dihydro-3-ethyl-2-oxo-1H-benzimidazole-1-carboxamide (BIMU 1; 50 ␮M; D), both of which are 5-HT4 agonists, evokes a sustained inward current. E and F: neuron patched with a pipette containing GTP␥S and exposed to 5-HT for 10 s (dashed line). E: 5-HT evokes a sustained inward current that is maintained in the presence of 5-HT. F: the 5-HT-evoked slow inward current is not affected by the presence of the 5-HT4 antagonist SB 204070 (1 ␮M).

relatively high concentration of the predominant 5-HT4 isoforms in the submucosal plexus is consistent with the possibility that 5-HT4 functions are particularly critical for events that occur in this region of the ENS (see below). Although expression of the 5-HT4e and 5-HT4f isoforms was far less than that of 5-HT4a or 5-HT4b, 5-HT4e and 5-HT4f expression was myenteric plexus specific. The 5-HT4e and 5-HT4f isoforms have previously been considered to be restricted to the brain (24). 5-HT4e and 5-HT4f expression in the bowel was probably missed in prior studies because the whole gut, in which these

transcripts are rare, and not dissected layers, in which they are more concentrated, was examined; nevertheless, although not brain specific, 5-HT4e and 5-HT4f are probably neuron specific, and their restriction to the myenteric plexus illustrates the resemblance of this plexus to the CNS. Transcripts encoding the 5-HT4 receptor were identified by in situ hybridization in subsets of enteric neurons of both plexuses, ICCs, and smooth muscle cells of the muscularis externa. Enteric 5-HT4 expression, therefore, is not restricted to the ENS. These data are consistent with prior pharmacological studies that have reported direct 5-HT4-mediated effects on gastrointestinal muscle as well as nerve. Reported neuronal 5-HT4 actions have all been excitatory. For example, 5-HT4 stimulation enhances the amplitude of electrically driven twitches of longitudinal smooth muscle because it increases the release of ACh from motoneurons (27, 59, 86, 92, 94). 5-HT4 stimulation, within the ENS is also excitatory (87) but has been analyzed mainly by using microelectrodes, which have detected an increase in the amplitude of fast EPSPs (41, 65, 79, 93). 5-HT4 receptor stimulation has also been reported to facilitate peristaltic reflexes by increasing the release of ACh and CGRP from activated submucosal intrinsic primary afferent neurons (IPANs) (37, 47– 49, 55). In contrast to the excitatory, prokinetic responses mediated by the neuronal actions of the 5-HT4 receptor, those that are mediated by 5-HT4 receptors on muscle cells, in rat esophagus, rat ileum, and human colon have all been reported to be inhibitory (5, 18, 63, 91, 95). It is not surprising that the Gs-coupled 5-HT4 receptor, which increases intracellular cAMP, should relax intestinal muscle cells. Virtually anything that increases the concentration of cAMP in smooth muscle relaxes these cells (17, 71, 100). The surprise is that the neuronal and muscular effects of the 5-HT4 receptor should be functionally antagonistic. The function of 5-HT4 receptors on ICCs and especially IC-MY, in which they are most numerous, is difficult to predict, because it has yet to be investigated. The ICC 5-HT4 receptors are probably coupled to Gs and associated with a rise in cAMP, as in other cells, but the consequences of increasing cAMP in ICC remain to be determined. There is evidence that ICCs are innervated by excitatory motor nerves (54); therefore, it is conceivable that 5-HT4 receptors on ICCs amplify excitatory signals and thus synergize with those on axons terminal. It is possible that smooth muscle (and ICC) 5-HT4 receptors are

Fig. 11. The slow sustained current induced by 5-HT is affected by compounds that target 5-HT1P but not 5-HT4 receptors. A-C. neuron patched with a pipette containing GTP␥S. A: 5-HT (50 ␮M) elicits a sustained inward current with superimposed low-amplitude transient fast currents that resemble excitatory postsynaptic currents (EPSCs). B and C: the 5-HT4 antagonist GR 113808 (1 ␮M) irreversibly abolishes the low-amplitude transient fast currents but fails to block the 5-HT-evoked slow sustained inward current. D-F: neuron patched with a pipette containing GTP␥S. D: 5-hydroxyindalpine (5-OHIP; 50 ␮M), a specific 5-HT1P agonist, evokes a sustained inward current. E: the selective 5-HT4 agonist tegaserod (1 ␮M) does not induce an inward current. F: the slow sustained inward current evoked by 5-OHIP is blocked by 5-HTP-DP (5 ␮M). AJP-Gastrointest Liver Physiol • VOL

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Fig. 12. 5-HT4 receptors act presynaptically in myenteric neurons. A: fast excitatory current is evoked by presynaptic electrical stimulation and blocked reversibly by hexamethonium (300 ␮M). The response is thus a cholinergic (nicotinic) stimulation-evoked EPSC. B and C: the 5-HT4 agonist renzapride (100 nM; B) and tegaserod (100 nM; C) reversibly increase the amplitude of the stimulation-evoked EPSC. D: the 5-HT4 antagonist GR-113808 (10 nM) does alter the stimulation-evoked EPSC; however, GR-113808 blocks the tegaserod-induced increase in the amplitude of the stimulationevoked EPSC (compare with C).

nonfunctional under physiological circumstances. Neither circular nor longitudinal muscle layers are located near a source of 5-HT. EC cells and serotonergic neurons are the major sources of 5-HT in the bowel. EC cells are located in the mucosa, and the wall of the bowel contains a barrier that inhibits the diffusion of 5-HT from the mucosa to the muscularis externa or myenteric plexus (57). Enteric serotonergic neurons are interneurons and do not innervate the enteric musculature (33, 38). Conceivably, therefore, the inhibitory 5-HT4 receptors of enteric smooth muscle do not normally oppose their excitatory counterparts in the ENS; however, they may do so under emergency circumstances, as for example during intestinal anaphylaxis, when a massive release of 5-HT floods the bowel (46). The presence of 5-HT4 receptors in muscle illustrates the importance of studying receptors in a physiological context. Antibodies were raised in chickens to locate 5-HT4 receptors in the ENS. The immunogen was a synthetic peptide with a sequence found in a 5-HT4 ectodomain common to all 5-HT4 isoforms. An ectodomain was chosen to enable receptors to be visualized on the surfaces of nonpermeabilized living cells, and the common domain was selected to permit all sites of 5-HT4 expression to be detected, regardless of isoform. The resulting antibody, of the IgY class, was affinity purified to enhance its specificity. The antibodies to the 5-HT4 receptor reacted with 5-HT4b receptors expressed by transiently transfected COS-7 cells, and as expected, the antibodies reacted with the surfaces of living cells without prior permeabilization. Western blots of protein extracted from the brain and the gut confirmed that the antibodies were specific for 5-HT4 receptor protein. A subset of enteric neurons and especially the neuropil of both submucosal and myenteric plexuses were 5-HT4 immunoreactive; 5-HT4 immunoreactivity was also seen in nerve fibers within the circular muscle layer. Smooth muscle cells were 5-HT4 immunoreactive, and the immunoreactivity appeared to be concentrated in caveolae of the plasma membrane. Caveolae are often sites where receptors and other signaling molecules AJP-Gastrointest Liver Physiol • VOL

congregate; 5-HT2B receptors are also concentrated in caveolae of smooth muscle (35). No 5-HT4 immunoreactivity was detected in mucosal nerves. The presence of 5-HT4 immunoreactivity on smooth muscle cell plasma membranes is consistent with the prior pharmacological studies that suggested that 5-HT4 receptor stimulation directly relaxes smooth muscle (5, 18, 63, 91, 95) and with our demonstration by in situ hybridization (see above) that smooth muscle cells contain transcripts encoding the 5-HT4 receptor. The absence 5-HT4 receptor immunoreactivity from mucosal nerve fibers is problematic. Stimulation of 5-HT4 receptors by 5-HT secreted from EC cells has been proposed to be the event that initiates peristaltic reflexes (37, 47– 49). Because the processes of IPANs that interface with EC cells extend into the mucosa, this hypothesis predicts that 5-HT4 receptors should be present on mucosal nerve fibers. Our failure to observe such 5-HT4-immunoreactive nerve fibers thus suggests that 5-HT released from EC cells probably does not directly stimulate submucosal IPANs. Conceivably, 5-HT4 receptors acting at the level of the submucosal plexus, within which they are most highly concentrated, amplify or strengthen transmission from IPANs that have been excited by another receptor. 5-HT4 receptors have been reported to evoke duodenal bicarbonate secretion in vitro (96), an effect that has been thought to be mucosal. The investigation of bicarbonate secretion, however, was not accompanied by a demonstration of 5-HT4 immunoreactivity or mRNA by in situ hybridization. Stripped mucosal preparations, such as those used to measure bicarbonate secretion, probably contain submucosal ganglia, which in turn might have been the locus of the 5-HT4 activity that affects bicarbonate secretion. Some investigators have speculated that 5-HT1P activity in the bowel is actually due to stimulation of 5-HT4 receptors (49, 62, 63). The evidence for this speculation is largely derived from pharmacological studies of smooth muscle, a cell type that has never been claimed to express 5-HT1P activity. 5-HT4 agonists presynaptically increase the release of ACh from 289 • DECEMBER 2005 •

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electrically stimulated preparations of bowel; this effect is not blocked by the 5-HT1P antagonist 5-HTP-DP (26 –28, 59). 5-HT4 receptors also act presynaptically to increase the amplitude of fast EPSPs evoked by stimulating inputs to myenteric neurons (41, 65, 79, 80), an effect that is not shared by 5-HT1P agonists. Finally, 5-HT1P activity has been found to stimulate submucosal IPANs (81); moreover, this effect is blocked by 5-HTP-DP and resistant to 5-HT4 antagonists. Mucosal application of 5-HT and 5-HT1P agonists activate submucosal IPANs, whereas 5-HT4 agonists do not. If 5-HT1P receptor activity is not, as proposed, identical to the 5-HT4 receptor (49, 62, 63), then the 5-HT-induced initiation of peristaltic and secretory reflexes may be 5-HT1P and not 5-HT4 dependent (44, 81). The presynaptic localization of the 5-HT4 receptor and the physiological determination that it acts presynaptically thus correspond; moreover, because 5-HT4 receptors do not act postsynaptically, they are unlikely to be the primary activators of the submucosal IPANs that drive the peristaltic reflex. In the current study, postsynaptic effects of 5-HT were found but they could not be attributed to stimulation of 5-HT4 receptors. Inclusion of GTP␥S made it possible to observe responses to 5-HT mediated by G protein-coupled receptors. These responses consisted of a slowly developing and longlasting inward current that could neither be blocked by a 5-HT4 antagonist nor mimicked by a 5-HT4 agonist and thus was clearly not 5-HT4 mediated. The slow inward current, however, was antagonized by 5-HTP-DP and evoked by the 5-HT1P agonists 5-OHIP and WAY100325. The slow inward current, therefore, was 5-HT1P dependent. In contrast, 5-HT and 5-HT4 agonists (tegaserod and renzapride) acted presynaptically to enhance the amplitude of fast EPSCs. These EPSCs were blocked by hexamethonium and thus were cholinergic (nicotinic). 5-HT4 antagonism by GR113808 did not affect the evoked EPSCs but prevented their enhancement by 5-HT4 agonists. Interestingly, in some cells, exposure to 5-HT appeared to elicit low-amplitude transient fast currents, which could be superimposed on the 5-HT1P-mediated slow inward current. The low-amplitude transient fast currents resembled fast EPSCs. When this phenomenon occurred, the low-amplitude transient fast currents, but not the slow inward current, were blocked by GR113808. Presynaptic activation of 5-HT4 receptors may therefore sometimes be able to evoke the release of transmitter to give rise to an EPSC-like activity. These observations, taken as a whole, suggest that 5-HT4 receptors are predominantly, if not exclusively, presynaptic. Their stimulation enhances and may even provoke cholinergic neurotransmission. 5-HT4 receptors belong to the group of seven-transmembrane G protein-coupled receptors and have been reported to mediate a number of postsynaptic responses in the CNS and the heart. For example, 5-HT4 receptors activate cardiac L-type Ca2⫹ channels or pacemaker currents in atrial myocytes (58, 78, 84), facilitate the hyperpolarization-activated cation current and a cyclic nucleotide-gated channel in hippocampal neurons (7, 22, 52), reduce the Ca2⫹-activated K⫹ current responsible for the slow afterhyperpolarization in hippocampal neurons (1), and inhibit a voltage-activated K⫹ current in collicular neurons (2, 10, 34). It would thus not have been surprising to find postsynaptic 5-HT4-mediated effects in the ENS. The 5-HT4 receptor, however, is clearly not identical to 5-HT1P activity, which is postsynaptic. In intestinal muscle cells, AJP-Gastrointest Liver Physiol • VOL

5-HT4 receptor stimulation increases inositol 1,4,5-trisphophsphate, intracellular Ca2⫹ concentration, and cAMP by a mechanism that is insensitive to pertussis toxin (62, 63). The 5-HT1P effect on enteric neurons is due to the antagonism of a Ca2⫹activated K⫹ current; therefore, it would be counterproductive for the 5-HT1P-initiated action to be associated with an increase in intracellular Ca2⫹ concentration. The signal-transduction pathway of the 5-HT1P receptor is pertussis toxin sensitive and blocked by intracellular injection of antibodies to G␣o (81, 82). 5-HT1P activity involves the generation of diacylglycerol, which stimulates PKC to inactivate the Ca2⫹-activated K⫹ current and, in type II/AH neurons, inhibits the afterhyperpolarization (76, 81). PKC can also activate type II adenylate cyclase, which increases intracellular cAMP and thus stimulates PKA, which acts with PKC to close Ca2⫹activated K⫹ channels (81). The coupling of 5-HT4 receptors in enteric neurons to G␣s is not inhibited by pertussis toxin (41). In the current study, intracellular antibodies to G␣o were more effective than antibodies to G␣s in blocking the slow inward current evoked by 5-HT. These data are thus consistent with the idea that the slow inward current is 5-HT1P and not 5-HT4 mediated, although the modest effect of intracellular antibodies to G␣s raises the possibility that 5-HT stimulates another receptor Gs-coupled 5-HT receptor isoform, such as 5-HT7 (74), which may contribute to the increase in cAMP elicited in enteric neurons by 5-HT. It is not necessary for 5-HT4 agonists to evoke a postsynaptic effect on IPANs or other enteric neurons to exert a prokinetic action. They could do so nicely by increasing the strength of neurotransmission at cholinergic synapses, which our data and those of others have demonstrated that they do. Such an action is compatible with the observations that 5-HT4 agonists promote the release of neurotransmitters from mucosally activated submucosal IPANs and are necessary to evoke a normal peristaltic reflex (47). 5-HT4 receptors thus are not likely to be the initiators of the peristaltic reflex, but they would strengthen those reflexes when they are elicited by natural stimuli. ACKNOWLEDGMENTS We thank Drs. S. Rayport, R. Ambron, and Y. Sung for advice. GRANTS This work was supported by National Institutes of Health Grant NS-12969 and Novartis (to M. D. Gershon). REFERENCES 1. Andrade R and Chaput Y. 5-Hydroxytryptamine4-like receptors mediate the slow excitatory response to serotonin in the rat hippocampus. J Pharmacol Exp Ther 257: 930 –937, 1991. 2. Ansanay H, Dumuis A, Sebben M, Bockaert J, and Fagni L. cAMPdependent, long-lasting inhibition of a K⫹ current in mammalian neurons. Proc Natl Acad Sci USA 92: 6635– 6639, 1995. 3. Bach T, Syversveen T, Kvingedal AM, Krobert KA, Brattelid T, Kaumann AJ, and Levy FO. 5-HT4(a) and 5-HT4(b) receptors have nearly identical pharmacology and are both expressed in human atrium and ventricle. Naunyn Schmiedebergs Arch Pharmacol 363: 146 –160, 2001. 4. Baidan LV, Zholos AV, and Wood JD. Modulation of calcium currents by G-proteins and adenosine receptors in myenteric neurones cultured from adult guinea-pig small intestine. Br J Pharmacol 116: 1882–1886, 1995. 5. Baxter GS, Craig DA, and Clarke DE. 5-Hydroxytryptamine4 receptors mediated relaxation of the rat oesophageal tunica muscularis mucosae. Naunyn Schmiedebergs Arch Pharmacol 343: 439 – 446, 1991. 289 • DECEMBER 2005 •

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