Neuroepithelial bodies in mammalian lung express functional serotonin type 3 receptor

X. W. Fu*, D. Wang*, J. Pan, S. M. Farragher, V. Wong, and E. Cutz

Division of Pathology, Department of Pediatric Laboratory Medicine, The Research Institute, The Hospital for Sick Children and University of Toronto, Toronto, Ontario, Canada M5G 1X8


    ABSTRACT
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ABSTRACT
INTRODUCTION
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Serotonin (5-HT) type 3 receptor (5-HT3-R) is a ligand-gated ion channel found primarily in the central and peripheral nervous system. We report expression and functional characterization of 5-HT3-R in pulmonary neuroepithelial body (NEB) cells. Using nonisotopic in situ hybridization, we demonstrate expression of 5-HT3-R mRNA in NEB cells in the lungs of different mammals (hamster, rabbit, mouse, and human). Dual immunocytochemistry (for 5-HT and 5-HT3-R) and confocal microscopy localized 5-HT3-R on NEB cell plasma membrane from rabbit. The electrophysiological characteristics of 5-HT3-R in NEB cells were studied in fresh slices of neonatal hamster lung using the whole cell patch-clamp technique. Application of the 5-HT (5-150 µM) and 5-HT3-R agonist 2-methyl-5-HT (5-150 µM) induced inward currents in a concentration-dependent manner. The 5-HT-induced current was blocked (76.5 ± 5.9%) by the specific 5-HT3-R antagonist ICS-205-930 (50 µM), whereas katanserin and p-4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylbenzamide had minimal effects. Forskolin had no effect on desensitization and amplitude of the 5-HT-induced current. The reduction of Ca2+ and Mg2+ in the extracellular solution enhanced the amplitude of the 5-HT-induced current because of slower desensitization. Our studies suggest that 5-HT3-R in NEB cells may function as an autoreceptor and may potentially be involved in modulation of hypoxia signaling.

airway chemoreceptor; serotonin receptor ligand-gated ion channel; whole cell patch clamp; in situ hybridization; neuropeithelial body


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
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DISCUSSION
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SEROTONIN (5-HT) is a multifunctional amine with diverse responses elicited through the activation of different 5-HT receptor subtypes widely distributed in neural and nonneural tissues, including the lung (6). The serotonin receptor 3 subtype (5-HT3-R) belongs to a family of ligand-gated ion channels identified in certain neuronal-derived cell lines and in peripheral and central neurons (4, 5, 13). The cloning of functional 5-HT3-R from the neuroblastoma cell line NCB-20 has confirmed that the 5-HT3-R is a member of the superfamily of ligand-gated ion channels and is structurally related to the nicotinic gamma -aminobutyric acid A and glutamate ionophore receptors (21).

Radioligand binding studies have identified 5-HT3-R binding sites in the central nervous system (CNS) of rodents and primates (15, 36). The presence of the 5-HT3-R in the CNS has also been demonstrated by electrophysiological recordings from dissociated mouse hippocampus neurons (41, 42). In situ hybridization studies showed that the pattern of 5-HT3-R mRNA expression within the brain suggests several possible roles for this receptor, including cell proliferation, differentiation, or migration of CNS neurons (33). In the peripheral nervous system, 5-HT3-R mRNA transcripts were observed within cranial nerve sensory ganglia and olfactory neuroepithelia (33). Electrophysiological and immunohistochemial studies have identified 5-HT3-R in guinea pig myenteric ganglia (46) and in rat petrosal ganglia (47).

Pulmonary neuroepithelial bodies (NEB) are innervated clusters of amine- and peptide-producing cells widely distributed throughout the airway mucosa of mammalian lungs, including human lungs (2, 18). NEB together with solitary pulmonary neuroendocrine cells (PNEC) constitute a multifunctional pulmonary neuroendocrine system with potential role(s) during lung development, neonatal adaptation, and in a variety of perinatal pulmonary disorders (2).

The function of PNEC/NEB is modulated via their amine and neuropeptide mediators (2). The principal amine produced by PNEC/NEB is 5-HT, and it has been identified in lungs of all animal species examined, including lower vertebrates (26, 45). The precise role of 5-HT originating from these specialized lung cells is presently unknown. The postulated role(s) include local effects, i.e., bronchoconstriction, vasomotor tone, and/or growth factor-like properties (29). Because NEB exhibit many features of an airway chemoreceptor, including innervation via the vagal afferent pathway (19), and release 5-HT in response to acute hypoxia (3), a possible role in modulation of hypoxia signaling has been suggested (2).

Previous studies in our laboratory have identified and partially characterized O2-sensing mechanisms in NEB cells using cultures of isolated NEB (44) or NEB in situ using a fresh lung slice preparation (7). These studies have shown that NEB cells express an O2 sensor protein (identified as a multicomponent NADPH oxidase) linked to an O2-sensitive K+ current (39). According to the "membrane" model of O2 sensing, hypoxia affects the function of the oxidase, resulting in reduced reactive oxygen species production, including H2O2, leading to closure of the O2-sensitive K+ channel followed by membrane depolarization, opening of voltage-activated Ca2+ channels, influx of extracellular Ca2+, and neurotransmitter release (20). Evidence for 5-HT release from NEB cells exposed to acute hypoxia has been documented both in vivo (17) and in vitro (3). However, the precise target(s) for NEB-derived 5-HT has not yet been characterized. In many respects, NEB resemble glomus cells of the carotid body (CB), a well-defined arterial chemoreceptor (8). The involvement of amine mechanisms in modulation of CB chemoreceptor function has been well characterized (8). In contrast, the role of 5-HT in NEB cell function is largely unknown.

We report here expression of 5-HT3-R mRNA in NEB cells in lungs from different mammals using the nonisotopic in situ hybridization (NISH) method. Using a double-immunolabeling method and confocal microscopy on cultures of NEB isolated from rabbit fetal lung, we demonstrate colocalization of 5-HT and 5-HT3-R immunoreactivity in the same NEB cells. We also present data on electrophysiological and pharmacological characterization of functional 5-HT3-R in NEB cells using a fresh lung slice preparation from hamster neonatal lung. Our studies demonstrate expression of functional 5-HT3-R in NEB cells of mammalian lungs. In NEB cells, 5-HT3-R may function as an autoreceptor, since these cells are a known source of 5-HT in the lung.


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Animal lung tissues were obtained from fetal or neonatal Syrian golden hamsters, New Zealand White rabbits, and mice (black C57BL/6J). The samples of human neonatal lungs were obtained at autopsy from cases without pulmonary disease. All lung tissues were fixed in 10% neutral buffered formalin embedded in paraffin, and 5-µm sections were placed on sialized slides. Subsequently, the sections were deparaffinized and processed for microscopy studies.

Immunohistochemistry

For identification of NEB in lungs of different mammals, a variety of immunomarkers were used. We used a monoclonal anti-5-HT antibody (Sera-Lab, Crawley Down, Sussex, UK) for rabbit lung, polyclonal anti-calcitonin gene-related peptide (CGRP) antibody (Cambridge Research Biochemicals, Wilmington, DE) for hamster and mouse lungs, and polyclonal anti-bombesin/gastrin-releasing peptide (GRP) antibody (Chemicon International, Temecula, CA) for human lung samples. Immunohistochemical methods and individual protocols for localization of 5-HT and various peptides were as previously reported (39).

For immunocytochemical colocalization of 5-HT and 5-HT3-R in NEB cells, we used cultures of NEB isolated from late fetal rabbit lung as previously reported (44). For dual-immunofluorescence staining and confocal microscopy to colocalize the cytoplasmic neuromarker (i.e., 5-HT) and a cell membrane epitope (i.e., 5-HT3-R), we used methods and protocols similar to those recently developed in our laboratory (unpublished observations). Isolated NEB cells were grown on Lab-Tech chamber slides (Nalgen; Nunc, Naperville, IL) and fixed in 3.8% paraformaldehyde for 10 min. To permeabilize the cells, we used 0.5% Triton X-100. To block nonspecific background staining, the slides were incubated with 20% normal donkey serum and 4% BSA followed by washes with PBS (pH 7.4).

To overcome potential limitations of the use of rabbit polyclonal antibody on rabbit tissues, we used the method of Gu et al. (9). The primary rabbit polyclonal antibody against 5-HT3-R (1:3,000; Oncogene, Boston, MA) and biotinylated anti-rabbit IgG secondary antibody (1:300; Jackson ImmunoResearch, Mississauga, ON) were complexed in a microfuge tube for 3 h at room temperature, and then primary rat monoclonal anti-5-HT antibody (1:100; DiaSorin, Stillwater, MN) was added to the complex. The antibody mixture was applied on cultures and incubated for 48 h at 4°C in a humidified chamber. As a positive control for 5-HT3-R antibody, we used frozen sections of rat brain fixed in 4% paraformaldehyde and an immunostaining protocol as recommended by the manufacturer.

To visualize 5-HT (cytoplasmic epitope), the slides were incubated with FITC-labeled donkey anti-rat IgG secondary antibody (1:100; Jackson ImmunoResearch) for 2 h at room temperature. To visualize 5-HT3-R (cell membrane epitope), the slides were incubated with egg white avidin (10 µg/ml; Molecular Probes, Eugene, OR) in PBS. Excess avidin was removed by washing in PBS, and signal detection was mediated by incubation with 5,6-tetramethylrhodamine biocytin (biocytinTMR; 0.1 µg/ml; Molecular Probes) in PBS for 1 h at room temperature.

Fluorescent images (dual labeling; FITC-biocytinTMR) of 5-HT, NEB cell marker, and 5-HT3-R were acquired with a Leica confocal laser-scanning microscope, and images were analyzed with Scanware software. The cells were optically sectioned by scanning the depth of focus (in steps from 1 µm). FITC (green signal) and biocytinTMR (red signal) images were merged into a composite image to establish a colocalization. Further image processing was done using Abode Photoshop 4.0 software.

Preparation of 5-HT3-R cRNA Probes and NISH

To generate cRNA probes, we cut the full-length clone of 5-HT3-R derived from the NCB-20 cell line (gift from Dr. D. Julius; see Ref. 21) with restriction enzymes HindIII and EcoRV. Subsequently, we subcloned a 690-bp HindIII-EcoR V fragment into the pGEM4Z vector at HindIII-SmaI sites. The subclone was linearized by HindIII, and Sp6 RNA polymerase was used to synthesize antisense RNA probe in the presence of digoxigenin-II-UTP (37, 39). For the sense probe, a subclone was linearized by EcoRI, and T7 RNA polymerase was used to synthesize the probe under the same conditions as for the antisense probe. Our protocol for NISH with digoxigenin-labeled 5-HT3-R cRNA probe was as described previously (37). Detection of the signal was achieved by application of the Dig Nucleic Acid Detection Kit (Roche Molecular Biochemicals, Boehringer Mannheim, Mannheim, Germany). As a positive control and a test for 5-HT3-R RNA probe specificity, we used sections of rat brain. Sections incubated with sense probe served as negative controls. To cross-identify the cells expressing mRNA for 5-HT3-R in lung tissue, the sections of lungs from different animal species were first immunostained with respective antibodies (i.e., 5-HT for rabbit, CGRP for hamster and mouse, bombesin/GRP for humans) to localize NEB, followed by NISH using digoxigenin-labeled 5-HT3-R RNA antisense probe. The sense probe for 5-HT3-R was used as a negative control.

Lung Slice Preparation

For electrophysiological studies, lung tissues from Syrian golden hamsters of both sexes were used between 1 and 7 days of age. The hamsters were killed by an intraperitoneal Euthanyl (pentobarbital sodium, 100 mg/kg) injection. The lungs were perfused with Krebs solution and then were embedded in 2% agarose (FMC Bioproducts, Rockland, ME). Sectioning was performed with tissue immersed in ice-cold Krebs solution that had the following composition (in mM): 140 NaCl, 3 KCl, 1.8 CaCl2, 1 MgCl2, 10 HEPES, and 5 glucose at pH 7.3 adjusted with NaOH (7). Transverse lung slices (200-300 µm) were cut with a Vibratome (Ted Pella, Redding, CA).

Electrophysiological Techniques and Solutions

For electrophysiological recordings, the lung slices were transferred to a recording chamber mounted on the stage of a Nikon microscope (Optiphot-2UD; Nikon, Tokyo, Japan). The perfusing Krebs solution had the following composition (in mM): 130 NaCl, 3 KCl, 2.5 CaCl2, 1 MgCl2, 10 NaHCO3, 5 HEPES, and 10 glucose at pH 7.35~7.4. To identify NEB cells in a fresh lung tissue, the slices were incubated with the vital dye neutral red (0.02 mg/ml) for 15 min at 37°C, as previously described (44). To isolate inward currents, an internal pipette solution with the following composition was used (in mM): 130 CsCl, 1 CaCl2, 2 MgCl2, 10 EGTA, 10 HEPES, and 4 ATP-Na with pH adjusted to 7.2 with CsOH. The chamber, which had a volume of 0.2 ml, was perfused continuously with oxygenated (95% O2-5% CO2) Krebs solution at a rate of 6-7 ml/min. All recordings were made from submerged lung slices at a temperature 29 ± 2°C.

Drugs were applied to the perfusate, and their delivery to the cells was controlled by separate valves. 5-HT and forskolin were obtained from Sigma (Sigma, Oakville, Ontario, Canada). 2-Methyl-5-HT maleate, p-4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylbenzamide (p-MPPI hydrochloride), ketanserin, and 3-tropanyl-indole-3-carboxylate (ICS-205-930) were obtained from Research Biochemicals International (Natick, MA). Stock solutions (1-10 mM) of all the drugs were prepared on the day of the experiment in twice distilled water and were diluted with the Krebs solution to their final concentration before use.

An Axopatch 200B (Axon Instruments, Foster, CA) amplifier was used for agonist-evoked inward currents in the whole cell voltage-clamp mode. Whole cell patch recordings were performed as described by Hamill et al. (10). The data were filtered at 5 kHz. The level of the fluid over the slices was kept low to minimize stray capacitance. Voltage commands and data acquisition were done using pCLAMP6 software and a DigiData 1200 interface (Axon Instruments). All data are given as means ± SE. Statistical analysis was performed using the paired and unpaired Student's t-test. Differences were considered to be statistically significant at P < 0.05.


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Expression and Localization 5-HT3-R mRNA and Protein in Mammalian NEB cells

To verify the specificity of our 5-HT3-R probes, we performed NISH for 5-HT3-R mRNA on sections of rat brain, a previously documented expression site (33). Sections of rat brain incubated with antisense 5-HT3-R RNA probe showed strong signal in the cortex and pyramidal cell layer (Fig. 1a). The specific 5-HT3-R mRNA signal was localized in the cytoplasm of neurons in respective brain regions (Fig. 1b). An adjacent section of brain incubated with a sense probe generated no signal, confirming the specificity of the NISH reaction (Fig. 1c).


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Fig. 1.   Nonisotopic in situ hybridization (NISH) for serotonin (5-HT) type 3 (5-HT3) receptor (R) in rat brain (positive control). a: Low-magnification view of section of rat brain incubated with antisense probe for 5-HT3-R. Strong signal (dark purple) is present in the cortex and pyramidal cell layer. Magnification, ×100. b: Higher magnification of rat brain cortex shown in a. Strong signal for 5-HT3-R mRNA (dark purple) in the cytoplasm of neurons contrasts with negative nuclei. Magnification, ×400. c: Low-magnification view a rat brain section in a incubated with sense probe for 5-HT3-R (sense probe negative control). No signal is present, confirming specificity of NISH reaction. Magnification, ×100.

To localize 5-HT3-R mRNA in NEB cells in lungs of different animal species (rabbit, hamster, mouse) and humans, we combined immunostaining with NISH. First, the lung sections were immunostained for amine or peptide to identify NEB within the airway epithelium, followed by NISH to localize the 5-HT3-R mRNA signal. In rabbit fetal lungs, NEB cells immunoreactive for 5-HT were typically located at airway branch points (Fig. 2a). NISH with antisense 5-HT3-R RNA probe showed a strong signal for 5-HT3-R mRNA within the same NEB identified by 5-HT immunostaining (Fig. 2b). An adjacent section immunostained for 5-HT but incubated with a sense 5-HT3-R RNA probe showed no signal, indicating specificity of NISH (Fig. 2c). In the lungs of fetal hamster and neonatal mouse, NEB were identified by immunostaining for CGRP followed by NISH with antisense RNA probe for 5-HT3-R. Strong specific signal for 5-HT3-R mRNA was identified in NEB cells from both species (Fig. 3, a-c). Similarly, a strong signal for 5-HT3-R mRNA was detected in NEB cells of human neonatal lung previously immunostained for bombesin/GRP, a well-characterized marker of NEB in human lung (Fig. 4, a and b). Our immunocytochemical colocalization studies for the detection of 5-HT and 5-HT3-R have shown coexpression of both epitopes in the same NEB cells. As expected, 5-HT was localized in the cytoplasm of NEB cells, whereas 5-HT3-R showed punctuate and linear plasma membrane staining as demonstrated in composite image obtained by means of confocal microscopy (Fig. 5).


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Fig. 2.   Immunolocalization of 5-HT in rabbit fetal lung (27 days gestation) combined with NISH for 5-HT3-R. a: Clusters of 5-HT-immunoreactive neuroepithelial body (NEB) cells located at airway branch points (arrowheads). Immunoperoxidase method for 5-HT; magnification, ×250. b: NISH using antisense 5-HT3-R probe on same section as in a. Specific signal for 5-HT3-R mRNA (dark purple) is localized in cytoplasm of same NEB cells (arrowheads) shown in a. Magnification, ×250. c: NISH using sense 5-HT3-R probe (sense probe negative control) applied on lung section first immunostained for 5-HT to localize NEB cells. No dark purple signal is present in NEB cells (arrowheads) immunoreactive for 5-HT (brown). Magnification, ×400.



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Fig. 3.   Immunostaining for calcitonin gene-related peptide (CGRP) combined with NISH for 5-HT3R in NEB cells of hamster and mouse lungs. a: Section of hamster fetal lung immunostained for CGRP showing NEB cell positivity (arrowheads; immunoperoxidase method for CGRP). Magnification, ×400. b: NISH using antisense 5-HT3-R probe on same section as in a. Strong signal (dark purple) in NEB cells (arrowheads) is depicted. Magnification, ×400. c: Section of neonatal mouse lung showing colocalization of CGRP and 5-HT3-R mRNA in NEB cells (arrowheads; immunoperoxidase method for CGRP followed by NISH for 5-HT3-R). Magnification, ×400. Focal membrane staining of airway epithelial cells (b and c) is nonspecific and possibly represents an edge artifact.



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Fig. 4.   Immunolocalization for bombesin combined with NISH for 5-HT3-R in NEB cells of human neonatal lung. a: NEB cells positive for bombesin (arrowheads) within airway epithelium (immunoperoxidase method for bombesin). Magnification, ×400. b: NISH using antisense 5-HT3-R probe on the same section as in a. Strong signal (dark purple) localized in cytoplasm of same NEB cells (arrowheads) as in a. Magnification, ×400.



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Fig. 5.   Composite confocal microscopy image of immunostaining for 5-HT and 5-HT3-R in rabbit fetal NEB cells in culture. Typical NEB cell cluster with expression of 5-HT in cytoplasm (green signal) and 5-HT3-R on plasma membrane (red signal; double immunofluorescence with confocal microscopy). Magnification, ×800.

5-HT-Induced Current Mediated by 5-HT3-R

Application of 5-HT (5-150 µM) elicited an inward membrane current in 80% of NEB cells (n = 55) in lung slices from neonatal hamsters. At a holding potential of -60 mV, rapidly superfused 5-HT (5-150 µM) induced concentration-dependent currents that displayed desensitization in the continued presence of the agonist (Fig. 6Aa). Peak currents ranged from -6 to -196 pA at -60-mV holding membrane potential. When NEB cells were depolarized with perfusion of 50 µM 5-HT, the inward current response become smaller (Fig. 6Ba). The current-voltage relationship of the 5-HT inward current is shown in Fig. 6Bb, reversing at -2.8 ± 0.5 mV (n = 4).


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Fig. 6.   5-HT- and 5-HT agonist-induced inward currents, and current-voltage (I-V) relationships in NEB cells. Aa: 5-HT-induced currents at various concentrations. The inward current reached its peak amplitude within 30 s and then declined, with a half-time (t1/2) of ~31 s at 50 µM 5-HT concentration. Ab: 2-methyl-5-HT (2-Me-5-HT)-induced current. Drugs (50 µM) were applied as follows: 5-HT; 180-s recovery period; 2-methyl-5-HT; 180-s recovery period; and 5-HT. Note that the peak amplitude of the 2-methyl-5-HT-induced current was smaller than that of the 5-HT-induced current. Holding potential of all recordings was -60 mV. Ba: 5-HT (50 µM)-induced currents at various holding potentials. Bb: I-V relationship for the same cell as in Ba. The current reversed in -3 mV.

Responses to 5-HT were mimicked by the selective 5-HT3-R agonist 2-methyl-5-HT (50 µM; Fig. 6Ab). The amplitude range of the inducing current by 2-methyl-5-HT (5-150 µM) was -6 to -132 pA, ~80-50% of the inducing currents by 5-HT (Figs. 6 and 7). In most cells, the time course of the half-time of desensitization (t1/2, the time required for the response to decay by 50% with continuous 50 µM 5-HT application) could be fitted with a single exponential with a time constant of 33.4 ± 3.2 s (n = 16). The desensitization time course of 2-methyl-5-HT was 29.04 ± 5.4 s (n = 6).


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Fig. 7.   Dose-response curves for various 5-HT agonists. Dose-response curves for 5-HT () and 2-methyl-5-HT (open circle ) at different concentrations (5, 10, 25, 50, 100, and 150 µM). Recordings were performed as in Fig. 6. The membrane potential was at -60 mV. All responses were normalized to the peak current induced by 100 µM 5-HT and plotted against the log 5-HT or 2-methyl-5-HT. Each point represents the mean ± SE between 4 and 21 NEB cells.

Dose-Response Curves of 5-HT and 2-Methyl-5-HT Inward Current

The relationship between the mean peak current and 5-HT and 2-methyl-5-HT concentrations (Fig. 7) was fitted with the Hill equation
I/I<SUB>max</SUB><IT>=</IT>1<IT>/</IT>{1<IT>+</IT>(EC<SUB>50</SUB><IT>/</IT>[5-HT])<SUP><IT>n</IT></SUP>}
where I is the measured peak current for a given 5-HT concentration, Imax is the maximal response, n is the Hill coefficient, and EC50 is the concentration of 5-HT or 2-methyl-5-HT required for half-maximal activation (43, 47). As shown in Fig. 6, EC50 for the receptor was 25.3 ± 2.0 µM, and the Hill coefficient was 1.01 ± 0.1 (n = 6). The EC50 for 2-methyl-5-HT was 55.2 ± 9.2 µM, and the Hill coefficient was 0.99 ± 0.1 (n = 6). These data suggest that two (or more) agonist binding sites are required for maximal activation of the receptor (13). Errors in these estimates may arise from the method of 5-HT application and/or receptor desensitization (13, 43, 47), although our results are comparable to other studies, for example, guinea pig myenteric neurons (EC50 = 38.7 µM, Hill coefficient = 1.02; see Ref. 46).

Single-Channel Conductance of 5-HT3-R Estimated From Fluctuation Analysis

The apparent single-channel conductance of the 5-HT3-R channel was estimated using fluctuation analysis (43, 47). To obtain a steady, nondesensitizing response suitable for noise analysis, a low concentration (3 µM) of 5-HT was applied by bath perfusion (Fig. 8, inset). The rising phase of the response was accompanied by a small increase in current noise, as revealed by the high gain. The plot of current variance vs. mean current was fitted with linear regression (Fig. 8), and the average single-channel conductance was estimated from the relationship (47)
&ggr;=&dgr;<SUP>2</SUP>/&Dgr;I(V<SUB>h</SUB><IT>−V</IT><SUB>eq</SUB>)
where delta 2 is the 5-HT-induced current variance, Delta I is the 5-HT-induced mean change in membrane current, Vh is the holding potential, and Veq is the reversal potential of the 5-HT response. The mean single-channel conductance obtained from the above relationship was 8.7 ± 1.8 pS at -60 mV (n = 4), a value comparable to that reported for guinea pig myenteric neurons (9 pS; see Ref. 5) but higher than rat petrosal neurons (2.7 pS; see Ref. 47).


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Fig. 8.   Estimation of 5-HT3-R single-channel conductance from fluctuation analysis of the 5-HT-induced whole cell current in NEB cells. Inset: section of the record used for fluctuation analysis; inward current produced by perfusion of 3 µM 5-HT. The variance of current noise was plotted vs. mean current, and single-channel conductance, estimated from the least squares fit of the data, was 6.5 pS. Membrane noise was increased during the rising phase of the current evoked at a holding potential of -60 mV.

Pharmacological Characterization of the 5-HT3-R

5-HT-induced current blocked by antagonist. The NEB cells were perfused with external solution containing each 5-HT antagonist for 30 s before simultaneous application with 50 µM 5-HT. Application 50 µM ICS-205-930, a selective 5-HT3 antagonist, reversibly inhibited the 5-HT inward current ~76.5 ± 5.9% (n = 9, P < 0.01; Figs. 9A and 10). In contrast, the selective 5-HT2 receptor antagonist ketanserin and the 5-HT1A receptor antagonist p-MPPI reduced the 5-HT inward currents by 18.8 ± 7.7% (n = 5) and 13.02 ± 2.2% (n = 5), respectively (Fig. 9, B and C). We also assessed the combined effects of ICS-205-930 with ketanserin or ICS-205-930 with p-MPPI, since ICS-205-930 did not completely block 5-HT inward currents. Application of 50 µM ICS-205-930 with 50 µM ketanserin or with 50 µM p-MPPI reduced 5-HT inward currents by 73.1 ± 3.2% (n = 5) and 72.0 ± 2.7% (n = 5), respectively (Fig. 10). This result indicates that ICS-205-930 is the most effective antagonist of the 5-HT-induced current response. Ketanserin and p-MPPI had minor effects on the 5-HT-induced current response.


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Fig. 9.   Pharmacological properties of the 5-HT3-R in NEB cells. The cells were perfused by each 5-HT antagonist during simultaneous application of 50 µM 5-HT. A: the response was reversibly blocked (~76%) by the specific 5-HT3-R antagonist ICS-205-930 (50 µM). B: the response was reversibly blocked (~18%) by the 5-HT2 receptor antagonist ketanserin (50 µM). C: the response was reversibly blocked (~13%) by the 5-HT1A receptor antagonist p-4-iodo-N-{2-[4-(methoxyphenyl)-1-piperazinyl]ethyl}-N-2-pyridinylbenzamide (p-MPPI hydrochloride; 50 µM).



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Fig. 10.   Summary graph of the blocking effects of selective antagonists on 5-HT-induced current in NEB cells. Effects of antagonists individually and in combination are shown as percent of block of 5-HT-induced current (50 µM). Application of ICS-205-930 had the most significant inhibitory effect on 5-HT-induced current in NEB cells (**P < 0.01). Ketanserin and p-MPPI alone or in combination with ICS-205-930 showed <20% inhibitory effect. n, No. of cells.

Effects of forskolin and Ca2+ on 5-HT-induced-current. Desensitization of the nicotinic receptor is known to be accelerated by the adenylate cyclase activator forskolin (28), presumably because of elevated cytoplasmic cAMP concentrations (22) and cAMP-dependent phosphorylation (12). Forskolin (30 µM) decreased the desensitization half-time in NG 108-15 cells and in hippocampal neurons (42). For this reason, we examined the effects of desensitization by forskolin in NEB cells. The amplitude of the 5-HT (50 µM)-induced current was -141.2 ± 9.1 pA, and t1/2 was 31.8 ± 9.2 s (n = 7). After application of 30 µM forskolin with 50 µM 5-HT, the inward current was -153.0 ± 17.2 pA (Fig. 11); t1/2 was 31.2 ± 3.7 s (n = 5). Application of forskolin did not induce significant changes in either the amplitude or desensitization t1/2 of inward currents in response to 5-HT. Thus the rate of desensitization and amplitude of current in NEB cells were not affected by the adenylate cyclase activator forskolin. An additional observation suggesting that the second messengers or G proteins were not involved was that the amplitude of the 5-HT-induced currents remained relatively constant for over 1 h in cells with internal solution lacking ATP and GTP (data not shown). During the continued application of 5-HT (50 µM) at a holding potential of -60 mV, the amplitude was -141.2 ± 9 (n = 3; Fig. 11); t1/2 was 29.3 ± 3.0 s (n = 6) when the perfusing solution contained Ca2+ (2.4 mM) and Mg2+ (1 mM). However, in the solution with reduced Ca2+ (0.1 mM Ca2+ and 0.1 mM Mg2+), the rate of desensitization was much slower; t1/2 was 50.9 ± 4.0 s (n = 9, P < 0.05) at -60 mV, and amplitude was increased to -214.3 ± 18.3 (n = 9, P < 0.01; Fig. 11). This suggests that in NEB cells the enhanced amplitude of the inward current and acceleration of desensitization by the decreased extracellular concentration of divalent cation are similar to that seen with 5-HT responses in NCB-20 cells (a mouse neuroblastoma), a Chinese hamster brain cell hybrid line (16), and in Xenopus oocytes (21, 40).


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Fig. 11.   Effects of forskolin and Ca2+ on 5-HT-induced currents (50 µM). Forskolin (30 µM) had no significant effect on 5-HT-induced current in NEB cells. Ca2+ and Mg2+ removal significantly increased (**P < 0.01) the amplitude of the 5-HT-induced current. n, No. of cells.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

The present study is the first to report expression of functional 5-HT3-R in pulmonary NEB cells. We used NISH to localize 5-HT3-R mRNA transcripts in NEB cells and observed a strong positive signal for 5-HT3-R localized in NEB cells in lungs of different animal species and humans, whereas other lung cell types showed no reactivity. To demonstrate expression of 5-HT3-R in NEB cells at a protein level, we used the double-labeling immunohistochemical method. In agreement with previous studies on 5-HT3-R immunolocalization (46), we have used NEB cell cultures to enhance the immunodetection of this low-abundance epitope. Our immunohistochemical colocalization studies have confirmed that 5-HT-immunoreactive NEB cells also expressed 5-HT3-R immunoreactivity. As demonstrated by confocal microscopy, 5-HT3-R immunoreactivity was predominantly localized to the plasma membrane of NEB cells. Such localization is consistent with a function as a ligand-gated ion channel. We have shown previously that another 5-HT receptor subtype (5-HT2c-R), modulated via intracellular signaling, is expressed in alveolar type 2 cells of the rat lung, with no signal observed in NEB cells (38). These findings suggest that 5-HT3-R expression in the lung may be restricted to NEB cells.

To characterize the electrophysiological properties of 5-HT3-R in NEB cells, we used a fresh lung slice preparation from neonatal hamster, a species with well-defined NEB cell morphology (31). The advantage of the fresh lung slice preparation is that it allows assessment of ionic currents in intact NEB in their "natural" environment, avoiding possible secondary effects of isolation and culture procedures (7). The present study demonstrated that, in the majority of NEB cells tested (~80%), application of 5-HT elicited a fast inward current. This response was mimicked by application of 2-methyl-5-HT, a 5-HT3-R agonist. Furthermore, ionic currents induced by 5-HT were significantly blocked by the specific 5-HT3-R antagonist ICS-205-930 but not by p-MPPI, a 5-HT1A receptor antagonist, or ketanserin, a 5-HT2 receptor antagonist. The general pharmacological profile of 5-HT3-R in NEB cells is that of a ligand-gated cation channel similar to that described in native neuronal cells or experimental expression systems (21, 23, 42). However, there are subtle differences in the pharmacological and functional properties of 5-HT3-R in different cell types. For example, the reversal potential of 5-HT3-R currents in NEB cells was -2.8 mV, which is close to that reported for the hippocampal dentate gyrus in rat brain slices (14). For rabbit ciliary ganglia, the reversal potential was reported as -12 mV (32), but, in the nodose ganglion cells of the rabbit and in a mouse neuroblastoma cell line (NIE-115), the reversal potentials measured under similar conditions were +7 and +20 mV, respectively (11, 23). These variations between experimental preparations suggest that subtypes or structural variations in 5-HT3-R may exist either in their subunits and/or in their combination that makes up a functional molecular unit (14, 34).

Another well-defined property of ligand-gated ion channels is desensitization, whereby the current reaches a maximum amplitude and then declines during the continued presence of the agonist. Desensitization of the 5-HT3-R has been characterized previously in different preparations, including mouse hippocampal neurons (42), rat superior cervical ganglion neurons (43), and a variety of clonal cell lines (42, 43). In NEB cells, forskolin had no significant effects on either the amplitude or desensitization time of inward currents induced by 5-HT. Thus the rate of desensitization and the amplitude of current in NEB cells do not appear to be affected by forskolin, a well-known adenylate cyclase activator (28). Other studies also suggest that the second messengers or G proteins are not involved in modulation of the amplitude and desensitization of the 5-HT3-R, as demonstrated in rat superior cervical ganglion neurons, NIE-115 cells, and rat dorsal root ganglion neurons (25, 27, 43). Initial reports indicated that the 5-HT3 channel was impermeable to Ca2+; however, other studies have shown significant Ca2+ permeability (10, 41, 43) and that removal of Ca2+ slows desensitization (25). In NEB cells, desensitization was also slower after the reduction of Ca2+ in the extracellular solution. One interpretation of these results is that Ca2+ enter the cell through opened 5-HT3 channels and that this contributes to the rapid desensitization by acting at an intracellular site of the channel. It is likely that Ca2+ exerts, at least in part, its effect on desensitization of the 5-HT3 channel at an intracellular site, because of the finding that intracellular 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid slows the rate of desensitization (40).

The physiological significance of 5-HT3-R in pulmonary NEB is at present speculative. NEB cells are a known source of endogenous 5-HT in the lung, and acute hypoxia appears to be the main "physiological" stimulus for 5-HT release. In addition, a variety of stimuli, including chronic hypoxia, chemicals, and carcinogens, have been shown to alter the amine and/or peptide content in NEB cells of human and experimental animals, but the precise mechanisms are unknown (2). Because NEB are postulated to function as airway O2 sensors, the release of 5-HT during hypoxia may modulate synaptic output via autoreceptors on NEB cells and excitation of opposed vagal afferents, the cell bodies of which reside in the nodose ganglion (19, 34). In support of the potential role of 5-HT3-R as presynaptic (auto)receptors are studies on amine, particularly dopamine modulation of chemoreceptor function in CB glomus cells (8). Although the role of dopamine in glomus cell hypoxia chemotransduction appears to be mostly inhibitory, application of 5-HT in cultures of petrosal neurons produces a rapid excitatory response (24). In the case of NEB, hypoxia-stimulated 5-HT release may provide positive feedback via 5-HT3-R and thus increase 5-HT secretion, augmenting the hypoxia signaling. Such signal amplification may be a compensatory mechanism to increase the amplitude of the hypoxia signal generated from discrete cell clusters widely distributed over a large surface area of the lung. The plausibility of this scenario is supported by recent studies on the role of nicotinic ACh receptor (another ion-gated cation channel) in hypoxic chemotransduction in CB glomus cells (30). In this model, a small decease in O2 tension augments the activity of nicotinic ACh receptors, causing influx of Ca2+ and Na2+, which in turn leads to membrane depolarization and activation of voltage-gated K+ and Ca2+ channels, causing a further increase in intracellular Ca2+ and neurotransmitter release. According to this model, the hypoxia inhibition of oxygen-sensitive K+ channels would participate to further depolarize the glomus cells. Hypoxia-induced 5-HT release from NEB cells may also act as an excitatory neurotransmitter activating postsynaptic receptors on vagal afferents. Previous studies in rabbits have demonstrated that activation of 5-HT3-R results in depolarization of both nodose neurons and isolated vagus nerve (10). Further studies are required to define the precise role of 5-HT3-R in NEB cell function and in the chemotransduction of the hypoxic stimulus.


    ACKNOWLEDGEMENTS

This work was supported by grants from the Nicole Fealdman Sudden Infant Death Syndrome Research Fund and Canadian Institutes of Health Research (MOP-12742).


    FOOTNOTES

* X. W. Fu and D. Wang contributed equally to this work.

Address for reprint requests and other correspondence: E. Cutz, Div. of Pathology, Dept. of Pediatric Laboratory Medicine, The Hospital for Sick Children, 555 University Ave., Toronto, Ontario, Canada M5G 1X8 (E-mail: ernest.cutz{at}sickkids.on.ca).

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.

Received 8 January 2001; accepted in final form 18 May 2001.


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