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
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ABSTRACT |
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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
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INTRODUCTION |
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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 -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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MATERIALS AND METHODS |
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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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RESULTS |
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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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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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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
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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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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
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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)
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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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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This work was supported by grants from the Nicole Fealdman Sudden Infant Death Syndrome Research Fund and Canadian Institutes of Health Research (MOP-12742).
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FOOTNOTES |
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* 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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REFERENCES |
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1.
Cho, T,
Chan W,
and
Cutz E.
Distribution and frequency of neuroepithelial bodies in postnatal rabbit lung. Quantitative study with monoclonal antibody against serotonin.
Cell Tissue Res
255:
353-362,
1989[ISI][Medline].
2.
Cutz, E.
Structure, molecular markers, ontogeny and distribution of pulmonary neuroepithelial bodies.
In: Cellular and Molecular Biology of Airway Chemoreceptors, edited by Cutz E.. Austin, TX: Landes, 1997, p. 1-33.
3.
Cutz, E,
Spiers V,
and
Yeger H.
Cell biology of pulmonary neuroepithelial bodiesvalidation of an vitro model. Effects of hypoxia and Ca++ ionophore on serotonin content and exocytosis of dense core vesicles.
Anat Rec
236:
41-52,
1993[ISI][Medline].
4.
Davies, PA,
Pistis M,
Hanna MC,
Peter JA,
Lambert JJ,
Hales TG,
and
Kirkness EF.
The 5-HT3B subunit is a major determinant of serotonin-receptor function.
Nature
392:
359-363,
1999[ISI].
5.
Derkach, V,
Surprenant A,
and
North RA.
5-HT3 receptors are membrane ion channels.
Nature
339:
706-709,
1989[ISI][Medline].
6.
Fozard, YR.
The Principal Actions of 5-HT. Oxford, UK: Oxford University Press, 1989.
7.
Fu, XW,
Nurse CA,
Wang YT,
and
Cutz E.
Selective modulation of membrane currents by hypoxia in intact airway chemoreceptors from neonatal rabbit.
J Physiol (Lond)
514.1:
139-150,
1999
8.
Gonzalez, C,
Almarez L,
Obeso A,
and
Rigual R.
Carotid body chemoreceptor: from natural stimuli to sensory discharges.
Physiol Rev
74:
829-898,
1994
9.
Gu, J,
Agrawal N,
Wang P,
Cohen M,
and
Downey J.
A primary-secondary antibody complex method of immunocytochemistry using rabbit polyclonal antibodies to detect antigens in rabbit tissue.
Cell Vision
2:
52-58,
1995.
10.
Hamill, OP,
Marty A,
Neher E,
Sakmann B,
and
Sigworth FJ.
Improved patch-clamp techniques for high resolution current from cells and cell-free membrane patches.
Pfluegers Arch
391:
85-100,
1981[ISI][Medline].
11.
Higashi, H,
and
Nishi S.
5-Hydroxytryptamine receptors of visceral primary afferent neurones on rabbit nodose ganglia.
J Physiol (Lond)
323:
543-567,
1992[ISI][Medline].
12.
Huganir, RL,
Delcour AH,
Greengard P,
and
Hess GP.
Phosphorylation of the nicotinic acetylcholine receptor regulates its rate of desensitization.
Nature
321:
774-776,
1986[ISI][Medline].
13.
Jackson, MB.
The 5-HT3 receptor channel.
Annu Rev Physiol
57:
447-468,
1995[ISI][Medline].
14.
Kazuyoshi, K.
Distribution and functional properties of 5-HT3 receptors in the rat hippocampal dentate gyrus: a patch-clamp study.
J Neurophysiol
21:
1935-1947,
1994.
15.
Kilpatrick, GJ,
Jones BJ,
and
Tyers MB.
Identification and distribution of 5-HT3 receptors in rat brain using radioligand binding.
Nature
330:
746-748,
1987[ISI][Medline].
16.
Lambert, JJ,
Peters JA,
Hales TO,
and
Dempster J.
The properties of the 5-HT3 receptors in clonal cell lines studied by patch-clamp techniques.
Br J Pharmacol
97:
27-40,
1989[Abstract].
17.
Lauweryns, JM,
Cokelaere M,
and
Deleersynder M.
Intrapulmonary neuroepithelial bodies in newborn rabbits. Influence of hypoxia, hyperoxia, hypercapnia, nicotine, reserpine, L-DOPA, and 5-HTP.
Cell Tissue Res
182:
425-440,
1977[ISI][Medline].
18.
Lauweryns, JM,
Cokelaere M,
and
Lerut T.
Cross-circulation studies on the influence of hypoxia and hypoxaemia on neuroepithelial bodies in young rabbits.
Cell Tissue Res
193:
373-386,
1978[ISI][Medline].
19.
Lauweryns, JM,
and
VanLommel A.
Effects of various vagotomy procedures on the reaction to hypoxia of rabbit neuroepithelial bodies: modulation by intrapulmonary axon reflexes?
Exp Lung Res
11:
319-339,
1986[ISI][Medline].
20.
Lopez-Barneo, J.
Oxygen-sensing by ion channels and the regulation of cellular functions.
Trends Neurosci
19:
435-440,
1996[ISI][Medline].
21.
Maricq, A,
Peterson VA,
Brake A,
Myers RM,
and
Julius D.
Primary structure and functional expression of the 5HT3 receptor, a serotonin-gated ion channel.
Nature
254:
432-437,
1993.
22.
Middleton, P,
Jaramillo F,
and
Schuetze SM.
Forskolin increases the rate of acetylcholine receptor desensitization at rat soleus endplates.
Proc Natl Acad Sci USA
83:
4967-4971,
1986[Abstract].
23.
Neijt, HC,
Dutts LJ,
and
Vijverberg HPM
Pharmacological characterization of serotonin 5-HT3 receptor-mediated electrical response in cultures of mouse neuroblastoma cells.
Neuropharmacology
27:
301-307,
1988[ISI][Medline].
24.
Nurse, CA,
and
Zhang M.
Acetylcholine contributes to hypoxic chemotransmission in co-cultures of rat type 1 cells and petrosal neurons.
Respir Physiol
115:
189-199,
1999[ISI][Medline].
25.
Peters, JA,
Hales TG,
and
Lambert JJ.
Divalent-cations modulate 5-HT3 receptor-inducd currents in N1E-115 neuroblastoma cells.
Eur J Pharmacol
151:
491-495,
1988[ISI][Medline].
26.
Polak, JM,
Becker KL,
Cutz E,
Gail DB,
Goniakowska-Witalinska L,
Gosney JR,
Lauweyns JM,
Linnoila L,
McDowell EM,
and
Miller YE.
Lung endocrine cell markers, peptides, and amines.
Anat Rec
236:
169-171,
1993[ISI][Medline].
27.
Robertson, B,
and
Bevan S.
Properties of 5-hydroxytryptamine3 receptor-gated currents in adult rat dosal root ganglion neurones.
Br J Pharmacol
102:
272-276,
1991[Abstract].
28.
Seamon, KB,
Padgett W,
and
Daly JW.
Forskolin: unique diterpene activator of adenylate in membranes and in intact cells.
Proc Natl Acad Sci USA
78:
3363-3367,
1981[Abstract].
29.
Seuwen, K,
and
Pouyssegur J.
Serotonin as a growth factor.
Biochem Pharmacol
39:
985-990,
1990[ISI][Medline].
30.
Shirahta, M,
and
Sham SK.
Roles of ion channels in carotid body chemotransmission of acute hypoxia.
Jpn J Physiol
49:
213-228,
1999[ISI][Medline].
31.
Sorokin, SP,
and
Hoyt RF.
Neuroepithelial bodies and solitary small-granule cells.
In: Lung Cell Biology, edited by Massaro D.. New York: Dekker, 1989, p. 191-344.
32.
Tatsumi, H,
and
Katayama Y.
The action of 5-hydroxytryptamine in the rabbit ciliary ganalion.
J Auton Nerv Syst
20:
137-145,
1987[ISI][Medline].
33.
Tecott, LH,
Maricq AV,
and
Julius D.
Nervous system distribution of serotonin 5-HT3 receptor mRNA.
Proc Natl Acad Sci USA
90:
1430-1434,
1993[Abstract].
34.
Van Lommel, A,
Lauweryns JM,
and
Berthoud HR.
Pulmonary neuroepithelial bodies are innervated by vagal afferent nerves: an investigation with in vivo anterograde DiI tracing and confocal microscopy.
Anat Embryol (Berl)
197:
325-330,
1998[ISI][Medline].
35.
Verdoorn, TA,
Burnashev N,
Monyer H,
Seeburo PH,
and
Sakmann B.
Structural determinants for ion flow through recombinant glutamate receptor channels.
Science
252:
1715-1718,
1991[ISI][Medline].
36.
Waeber, C,
Heyer D,
and
Palacio JM.
5-Hydroxytryptamine 3 receptors in the human brain: autoradiographic visualization using.
Neuroscience
31:
393-410,
1989[ISI][Medline].
37.
Wang, D,
and
Cutz E.
Simultaneous detection of messenger ribonucleic acids for bombesin/gastrin-relasing peptide and its receptor in rat brain by nonradiolabeled double in situ hybridization.
Lab Invest
70:
775-780,
1994[ISI][Medline].
38.
Wang, D,
Post M,
and
Cutz E.
Expression of serotonin receptors2c in rat type II pneumocytes.
Am J Respir Cell Mol Biol
20:
1175-1180,
1999
39.
Wang, D,
Youngson C,
Wong V,
Yeger H,
Dinauer M,
Miera EVD,
Rudy B,
and
Cutz E.
NADPH-oxidase and a hydrogen peroxide-sensitive K+ channel may function as an oxygen sensor complex in airway chemoreceptors and small cell lung carcinoma cell lines.
Proc Natl Acad Sci USA
93:
13182-13187,
1996
40.
Yakel, JL,
Lagrutta A,
Adelman JP,
and
North RA.
Single amino acid substitution affects desensitization of the 5-hydroxytryptamine type 3 receptor expressed in Xenopus oocytes.
Proc Natl Acad Sci USA
90:
5030-5033,
1993[Abstract].
41.
Yakel, JL,
Shao XM,
and
Jackson MB.
The selectivity of the channel coupled to the 5-HT3 receptor.
Brain Res
533:
46-52,
1990[ISI][Medline].
42.
Yakel, JL,
Trussell LO,
and
Jackson MB.
Three serotonin responses in cultured mouse hippocampal and striatal neurons.
J Neurosci
8:
1273-1285,
1988[Abstract].
43.
Yang, J,
Mathie A,
and
Hille B.
5-HT3 receptor channels in dissociated rat superior cervical-ganglion neurons.
J Physiol (Lond)
448:
237-256,
1992[Abstract].
44.
Youngson, C,
Nurse CA,
Yeger H,
and
Cutz E.
Oxygen sensing in airway chemoreceptors.
Nature
365:
153-155,
1993[ISI][Medline].
45.
Zaccone, G,
Fasulo S,
Ainis L,
and
Licata A.
Paraneurons in the gills and airways of fishes.
Microsc Res Tech
37:
4-12,
1997[ISI][Medline].
46.
Zhai, J,
Gershon MD,
Walsh JH,
Wong HC,
and
Kirchgessner AL.
Inward currents in neurons from newborn guinea pig intestine: mediation by 5-hydroxytryptamine type 3 receptors.
J Pharmacol Exp Ther
291:
374-382,
1999
47.
Zhong, H,
Zhang M,
and
Nurse CA.
Electrophysiological characterization of 5-HT receptors on rat petrosal neurons in dissociated cell culture.
Brain Res
816:
544-553,
1999[ISI][Medline].