Thermosensitive transient receptor potential channels in vagal afferent neurons of the mouse
Lei Zhang,
Sarahlouise Jones,
Kate Brody,
Marcello Costa, and
Simon J. H. Brookes
Department of Human Physiology and Centre for Neuroscience, Flinders University, Adelaide, South Australia, 5001, Australia
Submitted 8 October 2003
; accepted in final form 5 January 2004
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ABSTRACT
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A number of transient receptor potential (TRP) channels has recently been shown to mediate cutaneous thermosensitivity. Sensitivity to warm and cool stimuli has been demonstrated in both human and animal gastrointestinal tract; however, the molecular mechanisms that underlie this have not been determined. Vagal afferent neurons with cell bodies in the nodose ganglion are known to mediate nonnociceptive sensation from the upper gut. In this study, isolated cultured nodose ganglion from the mouse neurons showed changes in cytoplasmic-free Ca2+ concentrations over a range of temperatures, as well as to icilin (a TRPM8 and TRPN1 agonist) and capsaicin (a TRPV1 agonist). RT-PCR was used to show the presence of six temperature-sensitive TRP channel transcripts (TRPV14, TRPN1, and TRPM8) in whole nodose ganglia. In addition, RT-PCR of single nodose cell bodies, which had been retrogradely labeled from the upper gut, detected transcripts for TRPV1, TRPV2, TRPV4, TRPN1, and TRPM8 in a proportion of cells. Immunohistochemical labeling detected TRPV1 and TRPV2 proteins in nodose ganglia. The presence of TRP channel transcripts and proteins was also detected in cells within several regions of the gastrointestinal tract. Our results reveal that TRP channels are present in subsets of vagal afferent neurons that project to the stomach and may confer temperature sensitivity on these cells.
primary afferent neurons; nodose ganglion; stomach; retrograde labeling
ACCURATE DETECTION OF TEMPERATURE is required for thermal homeostasis and for avoiding damage by excessively high or low temperatures. Recent studies (13, 20, 24, 25) have identified key molecular components of neuronal temperature sensitivity in brain slices from the preoptic area/anterior hypothalamus (POAH) and in cultured dorsal root ganglion neurons. These studies have shown that several temperature-activated, nonselective cation channels cause generator potentials and firing behavior in thermally sensitive neurons. In cultured dorsal root ganglion cells, warming and cooling can evoke significant changes in cytoplasmic-free Ca2+ concentrations ([Ca2+]cyt) (7, 31). The cloning and functional reconstitution in heterologous cell types of transient receptor potentials (TRP)V1 (VR1), TRPV2 (VRL-1), TRPV3 and TRPV4 (TRPO4, VR-OAC), TRPN1 (ANKTM1), and TRPM8 (CMR1) have suggested that the increase in [Ca2+]cyt in response to thermal stimuli may be mediated by this group of Ca2+ permeable TRP channels (1, 2, 9, 17, 23, 29, 30, 35).
Thermosensitive afferent nerve fibers have been identified projecting to the esophagus, stomach, duodenum, and rectum both in animals and humans (5, 6, 34, 42). In the feline vagus nerves, three types of thermosensitive unmyelinated fibers can be distinguished by cold (1036°C), warm (3950°C), and mixed (1035°C and 4050°C) temperatures (5, 6), suggesting that the vagus nerve may mediate some thermosensitivity. Although spinal afferent temperature sensitivity has been extensively studied, the molecular basis of thermosensitivity in vagal afferent neurons has not been examined. In the present study, we investigated the responses of isolated nodose ganglion neurons to thermal stimuli by measuring [Ca2+]cyt using fura-2 AM. By using RT-PCR and immunohistochemistry, we further examined the expressions of TRPV1, TRPV2, TRPV3, TRPV4, TRPN1, and TRPM8 in nodose ganglia and in the gut wall. With the use of single-cell RT-PCR from neurons retrogradely labeled from the stomach, we also assayed the expression of these TRP channels in individual nodose ganglion neurons projecting to gut.
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MATERIAL AND METHODS
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Animals.
Adult 129SV mice of either sex (2040 g) and adult guinea pigs (250400 g) were housed in groups, maintained on a 12:12-h light-dark cycle at 23°C and given ad libitum access to tap water and food. Animals were housed in accordance with the Australian Code of Practice for the Care and Use of Animals for Scientific Purposes (1977) published by the National Health and Medical Research Council of Australia. Animals used for immunohistochemistry (n = 4) and RT-PCR (n = 4) were killed using halothane inhalation (mice) and by stunning and exsanguination (guinea pigs) in a manner approved by the institutional animal ethics committee.
Preparation of isolated nodose ganglia neurons.
Freshly dissociated nodose ganglia were enzymatically dissociated by incubation in serum-free DMEM (GIBCO-BRL) containing collagenase (type VII, 1 mg/ml; Sigma), DNase I (0.1 mg/ml; Ambion), and protease (2.4 mg/ml; Sigma) in 5% CO2 for 30 min at 37°C. Nodose ganglia were then mechanically dissociated by passing through a fire-polished glass Pasteur pipette 1620 times, before separation along a Percoll (Sigma) gradient. Individual cells were harvested by centrifugation (700 g for 3 min) before resuspension in DMEM with 10% (vol/vol) fetal bovine serum (GIBCO-BRL), 100 units/ml penicillin (GIBCO-BRL), 100 µg/ml streptomycin (GIBCO-BRL), and 20 ng/ml NGF (Invitrogen). Isolated nodose ganglia cells were used for [Ca2+]cyt measurement or picked up for single-cell RT-PCR.
Measurement of the [Ca2+]cyt.
Single neurons were seeded on poly-l-ornithine-coated glass coverslips (22 mm diameter) and incubated in DMEM with 10% (vol/vol) fetal bovine serum, 100 IU/ml penicillin, 100 µg/ml streptomycin, and 20 ng/ml NGF overnight (5% CO2-37C). Cells were washed twice with DMEM and then loaded for 30 min at 37°C with 5 µM fura-2 AM and 0.0075% (wt/vol) pluronic F-127 (Molecular Probes) in DMEM medium followed by a 10-min incubation in DMEM to allow desertification of fura-2 AM. Coverslips were placed in a recording chamber and continuously superfused with modified Hanks' solution plus 1.8 mM Ca2+. Temperature was controlled by a cell micro control system (Norfolk, VA) consisting of a Peltier unit and a resistive heater with feedback control, and was monitored by a thermistor placed within 160 µm of the field of view. Thermal stimuli {cold (2812°C), warm (2838°C), moderate heating (3845°C), noxious heating (4556°C) and chemical stimuli [capsaicin, icilin, and tetrodotoxin (Sigma)]} were applied by superfusion to stimulate neuronal cell bodies. Fura-2 fluorescence was measured on a Nikon inverted microscope (Image Systems) using a charge-coupled device camera. [Ca2+]cyt in nodose ganglion neurons was estimated by analyzing plots of fluorescence ratio (excitation wavelengths, 340/380 nm; emission wavelength, 510 nm) as a function of time.
Retrograde labeling and isolation of labeled nodose ganglion cells.
Mice used for retrograde labeling experiments (n = 6) received injections of the fluorescent tracer 1,1'-didodecyl-3,3,3',3',-tetramethyl indocarbocyanine (DiI) [D-383, (Molecular Probes) 2 mg/ml dissolved in dimethyl formamide, (Sigma) D-4551] under halothane anesthesia (25% in oxygen at a rate of 12 l/min). A total volume of 34 µl of DiI was injected over several sites in the forestomach. The tracer was pressure injected through a glass micropipette with a tip diameter of
10 µm. After each injection, the glass pipette was held in position for
20 s, and the site was swabbed with sterile saline to minimize tracer leakage. Animals were housed individually after surgery and were killed 6 days after surgery by a lethal dose of sodium pentobarbitone and the nodose ganglia was immediately removed. Single-cell suspensions were prepared as described (see Preparation of isolated nodose ganglia neurons) using enzymatic and mechanical methods. Individual labeled neurons were identified by using a Nikon inverted light/fluorescence microscope. With the use of an Ultra Micro Pump-II (World Precision Instruments, Sarasota, FL), retrogradely labeled neurons were drawn into a glass micropipette with a tip diameter of
80 µm mounted on a micromanipulator and collected individually into 0.2-ml PCR tubes. Tubes were immediately placed in liquid nitrogen and stored at 80°C until used.
RT-PCR and single-cell RT-PCR.
Freshly isolated mouse tissue was homogenized, and total RNA was isolated by using the RNeasy kit (Qiagen) according to the manufacturer's protocol. This was followed by 1 unit of DNase I (Ambion) treatment for 15 min at 37°C before PCR, to remove residual DNA contamination. Reverse transcription was then performed by using the Ominiscript reverse transcriptase kit (Qiagen) with oligo(dT) (17) primer in a volume of 20 µl for 60 min at 37°C according to the manufacturer's instructions. The primers used for PCR amplification are summarized in Table 1. PCR was conducted with a RoboCycler Gradient 96 (Stratagene) using HotStart Taq Master Mix kit (Qiagen) for 30 cycles (GAPDH) and 40 cycles (TRP channels) under the following conditions: initial denaturation was 15 min at 95°C, then 1 min at 94°C, followed by a 1-min annealing step at 5056°C and 1-min elongation at 72°C, and a final elongation of 10 min at 72°C. Single-cell RT-PCR was performed by using a one-step RT-PCR kit (Qiagen) and gene-specific primers, for 50 cycles under the following conditions: reverse transcription at 52°C for 30 min, initial denaturation; 15 min at 95°C and then 1 min at 94°C, followed by a 1-min annealing step at 5056°C and 1 min elongation at 72°C. To detect multiple transcripts in the same DiI-labeled cell, single-cell aliquots were divided into six parts, each of which was subjected to one-step RT-PCR as describe above. The RT-PCR products were separated on 1.5% (wt/vol) agarose gel in Tris-acetate EDTA buffer, stained with 0.5% (wt/vol) ethidium bromide (Sigma) and analyzed by a FluoroImager 875 (Molecular Dynamics). Sequencing of PCR products to verify amplification specificity was carried out following the manufacturer's protocol (ABI PRISM sequencing protocol) using Ampli-Taq FS Big Dye Terminator III.
Immunohistochemistry.
Mouse (n = 4) and guinea pig tissues (n = 4) were removed from a freshly killed animal and fixed overnight in modified Zamboni's fixative (2% formaldehyde and 15% saturated picric acid in 0.1 M sodium phosphate buffer). Tissue for whole mounts was then cleared by using three 10-min washes in DMSO and three 10-min washes in PBS (0.15 M NaCl in 0.01 M sodium phosphate buffer pH 7.2). Tissue was prepared for polyethylene glycol (PEG) (Sigma) sections by clearing through four 10-min incubations in 80% ethanol, and then two 15-min incubations in 100% ethanol, three 10-min washes in DMSO, and finally two 10-min washes in 100% ethanol. Tissue was then infiltrated in mol wt 1,000 PEG for 30 min, embedded in mol wt 1,450 PEG, and 7-µm sections were cut. Sections were incubated in primary antisera raised against TRPV1 (ABR; cat. no. Pa1747) and anti-sheep polyclonal antibody against TRPV2 (gift from Dr. Xin-Fu Zhou, Department of Human Physiology, Flinders University, Adelaide, Australia) at room temperature for 1 h. Sections were rinsed with phosphate buffer and incubated in anti-rabbit IgG secondary antibody conjugated with Cy3 (Jackson ImmunoResearch Laboratories) (1:2,000 dilution) for 2 h at room temperature. Sections were examined and photographed by using an Olympus AX-70 fluorescence microscope. The cross-sectional area of nerve cell bodies was determined by using NIH image (National Institutes of Health, Bethesda, MD).
Statistical analysis.
All data are expressed as percent means ± SE. Statistical evaluation of the data was performed by unpaired Student's t-test. A value of P < 0.05 was taken as significant.
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RESULTS
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Increases in [Ca2+]cyt in response to thermal stimuli in subpopulations of isolated nodose ganglia neurons.
To investigate the molecular mechanisms underlying vagus nerve-mediated thermosensitivity, we examined responses of isolated vagal afferent neurons to different thermal stimuli by measuring [Ca2+]cyt with fura-2 AM. In the presence of 1.8 mM Ca2+, subsets of mouse nodose neurons exhibited substantial increases in fura-2 fluorescence ratios in response to cooling (from 28°C to 12°C), warming (from 2838°C), moderate heating (3845°C), and noxious heating (4556°C) as shown in Fig. 1. The majority of neurons tested responded to warming from 2838°C (89.58 ± 39.5%; N = 289 neurons), whereas fewer responded to cooling (11.05 ± 3.5%; n = 305 neurons), moderate heating (20.55 ± 6.8%; n = 286 neurons), or noxious heating (10.66 ± 6.9%; n = 301 neurons). A small percentage (4.24 ± 3.2%; n = 126 neurons) of neurons exhibited significant increases in [Ca2+]cyt in response to each stimulus; cooling, warming, moderate heating, and noxious heating. The amplitude of increase in [Ca2+]cyt in response to each thermal stimulus (cooling, warming, heating, and noxious heat) were not significantly influenced by the addition of 600 nM tetrodotoxin, which blocks voltage-gated sodium channels and thus prevents action-potential-mediated depolarization (4050 neurons in each group, n = 2).

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Fig. 1. Increase in cytoplasmic-free Ca2+ concentration ([Ca2+]cyt) in isolated nodose neurons in response to thermal stimuli. Isolated neurons were loaded with fura-2 AM and fluorescence at 340 and 380 nm was recorded. Subsets of neurons show a rapid increase in [Ca2+]cyt. when cooling (A), warming (B), moderate heating (C), noxious heating (D), and mixed thermal stimuli (E) were applied (solid traces). The change in bath temperature is represented schematically below the traces although it was not directly recorded during the study. Removal of external Ca2+ completely suppressed the response to thermal stimuli (dashed traces). Experiments were performed in triplicate. The average responses of 7090 neurons and 20 neurons from different experiments are presented in AD and E, respectively.
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When Ca2+ was omitted from the bathing medium, thermally induced [Ca2+]cyt increases in neurons were abolished (dashed traces in Fig. 1), indicating that the transient increase in [Ca2+]cyt may have been due to Ca2+ influx through Ca2+ permeable channels. To test whether the increases in [Ca2+]cyt observed in nodose ganglia neurons caused by cooling (2812°C) or moderate heating (3845°C) were due to Ca2+ influx through TRP channels, we examined the effects of icilin (a known opener of TRPN1 and TRPM8) and capsaicin (an opener of TRPV1). No detectable responses were observed in cold-responsive cells to 1 or 10 µM icilin. However, at 100 µm, icilin evoked changes in [Ca2+]cyt in many cells. Of the cells that had responded to cooling from 28°C to 12°C, the majority (80.3 ± 25.2%) exhibited a significant increase in [Ca2+]cyt. In contrast, in cells that had not responded to cooling, only 5.1 ± 4.9% showed a similar response (P < 0.05, n = 3). Likewise, of the cells that had responded to an increase from 3845°C, 90.2 ± 34.1% had a significant increase in [Ca2+]cyt after exposure to capsaicin (10 µM), whereas few (7.0 ± 4.0%) of the unresponsive cells exhibited a detectable response (P < 0.05, n = 3). Changes in [Ca2+]cyt evoked by icilin and capsaicin were abolished when calcium was removed from the extracellular medium (Fig. 2), similar to the situation with temperature-evoked changes. Our results demonstrate that nodose ganglion neurons that respond to cooling are also activated by icilin, suggesting the presence of TRPN1 and/or TRPM8 and that neurons that respond to medium heat are also activated by capsaicin, suggesting the presence of TRPV1.

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Fig. 2. Increase in [Ca2+]cyt in cooling- and moderate heating-responsive neurons in response to icilin and capsaicin. The majority of cooling- and moderate heating-responsive neurons show a rapid increase in [Ca2+]cyt when 100 µM icilin (A) or 10 µM capsaicin (B) were applied, respectively (solid traces). Removal of external Ca2+ completely suppressed the response to thermal stimuli (dashed trace). Experiments were performed in triplicate. The average response of 3040 neurons from different experiments are presented.
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TRP channels are present in nodose ganglia, stomach, and small intestine.
Apart from TRPV1, temperature-sensitive TRP channels have not been identified in either vagal sensory neurons that project to the gut nor in the gut itself. We used RT-PCR and immunohistochemistry to determine whether TRPV1, TRPV2, TRPV3, TRPV4, TRPN1, and TRPM8 are expressed in nodose ganglia, stomach, and small intestine. Because all of the six TRP channel transcripts have been previously reported in dorsal root ganglia (DRG), we used DRGs as positive controls. Electrophoresis of PCR amplification products revealed intense single bands, corresponding to the predicted sizes of TRPV1, TRPV3, TRPV4, and TRPN1 sequences in dorsal root ganglia, nodose ganglia, superior cervical ganglia, stomach, and small intestine (Fig. 3). TRPV2 was detected in the stomach and small intestinal muscle and TRPM8 in stomach and small intestinal mucosa, whereas both channels were also detected in dorsal root and nodose ganglia. GAPDH, a housekeeping gene was used as a control transcript and was equally detected in all tested tissues. In every case, nucleotide sequence analysis of PCR products confirmed their identity with published sequences.

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Fig. 3. Detection of thermosensitive transient receptor potential (TRP) channel transcripts in whole mouse nodose ganglion and gut. RT-PCR for detection of TRPV1, TRPV2, TRPV3, TRPV4, TRPM8, TRPN1, and GAPDH was carried out by using their specific primers (Table 1) as described in the materials and methods. PCR fragments were separated on a 1.5% agarose gel and bands were analyzed by size and sequence. The results are representatives of those obtained in 3 independent experiments. Molecular weight marker (MWM) used for TRPV3 and TRPM8 PCR was spp-1/HindIII DNA; for TRPN1, it was 100-bp DNA ladder, and for all others, pUC19/Hpall DNA was used.
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To confirm PCR results and to further study the distribution of TRPV channels in nodose ganglion neurons, immunohistochemistry was performed by using antisera raised against TRPV1 and TRPV2. Immunoreactivity was detected in 37.4 ± 9.7 and 6.7 ± 3.7% (n = 800 neurons) of all nodose ganglion neurons in mouse, respectively (Fig. 4, A and D). Antisera preabsorbed with an excess amount of the cognate peptide exhibited no staining (data not shown). The majority of TRPV1 nerve cell bodies were of small and medium diameter (290 ± 98 µm2; Fig. 5A, n = 50 neurons from 4 animals), whereas the majority of TRPV2-immunoreactive nerve cell bodies was of medium and large diameter (502 ± 187 µm2; Fig. 5A, n = 50 neurons from 4 animals). TRPV1 and TRPV2 immunoreactivity was also detected in 19.5 ± 3.8 and 5.2 ± 2.0% (n = 1,000 neurons) of all nodose ganglion neurons in guinea pig, respectively (Fig. 4, B and E); however, the majority of TRPV1 (454 ± 133 µm2; Fig. 5B, n = 50 neurons from 4 animals) and TRPV2 (587 ± 251 µm2, Fig. 5B, n = 50 neurons from 4 animals nerve cells bodies) were of medium and large diameter. When TRPV1 and TRPV2 antisera were applied to mouse gastrointestinal preparations, no convincing immunoreactivity was detected (data not shown). In guinea pig small intestine, however, TRPV1 immunoreactivity was detected in subpopulations of mucosal epithelium cells (Fig. 4C) and TRPV2-immunoreactivity was present in fibers and nerve cell bodies in the myenteric plexus (Fig. 4F).

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Fig. 4. Detection by immunohistochemistry of expression of TRPV1 and TRPV2 in mouse and guinea pig nodose ganglia and gut. Whole mounts or sectioned tissues were fixed and subject to immunohistochemistry for TRPV1 and TRPV2 channels. TRPV1-immunoreactive cells are shown in mouse (A) and guinea pig (B) nodose ganglia and guinea pig small intestine mucosa (C). TRPV2 immunoreactive cells are shown in mouse (D) and guinea pig (E) nodose ganglia and guinea pig small intestine myenteric plexus (F). The results are representative of those obtained in at least 3 independent experiments.
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Fig. 5. A frequency distribution of cell body sizes of neurons immunoreactive for TRPV1 and TRPV2 in the mouse nodose ganglion and guinea pig nodose ganglion. Filled bars represent TRPV1-immunoreactive cells, open bars represent TRPV2 immunoreactive cells. In mouse nodose ganglion, the average size of TRPV2 neurons was slightly larger than the TRPV1 neurons, although there is an overlap between the 2 populations. In guinea pig nodose ganglion, TRPV2-immunoreactive cells were on average larger than the TRPV1 neurons. A range of 4250 neurons from 4 animals for each species was measured.
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Expression of TRP channels in vagal afferent neuronal cell bodies projecting to the gut.
Neurons of the nodose ganglia provide afferent innervation to a number of thoracic and abdominal organs including the esophagus, stomach, and small intestine as well as heart and respiratory tract. To examine whether temperature-sensitive TRP channels are expressed in vagal afferent neurons projecting to gut, the tracer DiI was injected into the ventral forestomach. After 6 days for transport, the left and right nodose ganglia of four animals contained 27 ± 9 and 14 ± 2 DiI-labeled cell bodies, respectively (Fig. 6A). A cell suspension was obtained by enzymatic digestion (N = 4 animals) and individual DiI-labeled neurons were collected into PCR tubes and one-step RT-PCR was carried out. TRPV1, TRPV2, TRPV4, TRPN1, and TRPM8 transcripts were individually detected in a 20, 12.5, 80, 20, and 12.5% (n = 20 neurons for each TRP channel) of DiI-labeled vagal afferent neurons (Fig. 6, BF); however, TRPV3 was not detected (0 of 20 cells tested). To examine multiple TRP channel expression in the same neuron, mRNA from single DiI-labeled cells was aliquoted into six equal parts and one-step RT-PCR was carried out. Coexpression of temperature-sensitive TRP channel mRNAs was found in a proportion of DiI-labeled cells (TRPV1 and TRPV2: 30%; TRPV1, TRPV2, TRPV4, and TRPN1: 10%; TRPV1 and TRPN1: 50%; n = 20 neurons) (Fig. 6G). Control experiments were carried out in which some of the Krebs solution, in which single cells had been suspended, was collected into the micropipette and subjected to RT-PCR. On each occasion, no PCR product was detected (n = 20 samples).

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Fig. 6. Identification of vagal afferent neurons with gut projection by retrograde labeling in mouse nodose ganglia and detection by single-cell RT-PCR of expression of thermosensitive TRP channels in traced afferent neurons. A: 1,1'-dodecyl-3,3,3',3',-tetramethyl indocarbocyanine (DiI)-containing dispersed nodose ganglion nerve cell bodies retrogradely labeled from the stomach. Individual DiI-labeled vagal afferent neurons were collected into single PCR tubes. One-step RT-PCR was carried out to detect the presence of TRPV1, TRPV2, TRPV3, TRPV4, TRPN1, and TRPM8 (BF). Multiple TRP transcripts were also detected in the same DiI-labeled neurons (G). Results are representatives of those obtained in three independent experiments.
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DISCUSSION
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In this study, we have detected mRNAs of six temperature-sensitive TRP channels in nodose ganglia, in various tissue layers from several regions of gut, and in sympathetic ganglia. Five of the six TRP channel transcripts were also detected in single vagal afferent neurons projecting to stomach and upper gut. Interestingly, several TRP channel transcripts could be detected in the same single neurons. We further found that many neurons in the nodose ganglia show changes in [Ca2+]cyt in response to thermal stimuli. Similar [Ca2+]cyt responses could be evoked by icilin and capsaicin, which are known openers of TRPM8 /TRPN1 and TRPV1, respectively. Both temperature and icilin/capsaicin-induced [Ca2+]cyt responses required extracellular Ca2+.
Technical interpretation of results.
Our study detected six temperature-sensitive TRP channel transcripts in a variety of tissues. All PCR products were sequenced and their authenticity confirmed against published sequences. To our knowledge, this is the first systematic study of these channels in the nodose ganglion. However, although RT-PCR has extremely high sensitivity, the presence of mRNA transcripts does not necessarily reflect the abundance or even presence of the associated protein. Where immunohistochemical studies were carried out, they supported the findings of this study. TRPV1 has been detected immunohistochemically in both the nodose ganglion and in gut tissues. Likewise, TRPV2 has also been detected immunohistochemically in the nodose ganglion (1, 16, 18, 19, 22). The present study confirmed these findings and extended them by showing immunoreactivity in both guinea pig and mouse ganglia. It has been reported that 40% of nodose ganglion neurons in the rat, retrogradely labeled from the stomach are immunoreactive for TRPV1(22). Our finding that 20% of retrogradely labeled neurons in the mouse express TRPV1 transcripts is comparable with this. The same report also demonstrated VR1-immunoreactive nerve fibers in the stomach wall but few of these were of vagal origin (22). This led to the suggestion that vagal afferent neurons may express very low levels of TRPV1 in their peripheral axons, below the level of detectability of immunohistochemistry. Ward et al. (36) further showed that gut VR1-like fibers appear to be predominantly spinal in origin. Currently, antisera to the other temperature-sensitive TRP channels studied here (TRPV3, TRPV4, TRPN1, and TRPM8) are not commercially available, thus detection of the channel proteins is not currently possible.
Results of Ca2+ imaging demonstrated that temperature-responsive neurons are present in nodose ganglia. Because temperature-induced changes in [Ca2+]cyt were rapidly abolished when extracellular Ca2+ was excluded, it is likely that they were caused by an influx of Ca2+ through membrane-located ion channels rather than from intracellular stores. TRP channels are the best candidates for mediating temperature-induced Ca2+ influx. Studies of cells in the POAH and in cultured dorsal root ganglion neurons have revealed that warming and cold-activated nonselective cation channels underlie the thermoreceptor potential and spike generation. Suto and colleagues in 1998 (7) and Gotoh et al. in 1999 (8) further showed that warming and cooling raises [Ca2+]cyt in cultured dorsal root ganglionic cells. Overexpression of thermosensitive Ca2+-permeable TRP channels suggests that they could account for much of the increase in [Ca2+]cyt evoked by thermal stimuli. However, activation of TRP channels is also likely to depolarize neurons, possibly leading to trains of action potentials that could increase Ca2+ entry via voltage-gated Ca2+ channels. Our observation that increases in [Ca2+]cyt in response to thermal stimuli were not inhibited by tetrodotoxin suggests that Ca2+ influx via an indirect, action potential-mediated mechanism probably does not contribute to the overall change in [Ca2+]cyt evoked by thermal stimuli.
TRPV1 is expressed in small- and medium-diameter spinal afferent neurons that respond to both noxious thermal stimuli (>42°C) and capsaicin (2). TRPV2 is expressed in medium- and large-diameter sensory neurons and is activated by higher temperatures (>52°C) (1). TRPV3 has been localized in dorsal root ganglion neurons of all sizes, as well as in the superior cervical and trigeminal ganglion (28, 29, 35, 39) and responds close to core body temperature (3638°C). TRPV4 is expressed in anterior hypothalamic structures and is activated by warm temperature (>34°C) (9, 10, 37, 38). TRPN1 is expressed in small- and medium-sized dorsal root ganglia neurons and in fibroblasts and is activated by cold temperature (<18°C) and icilin (30). TRPM8 is expressed in small-diameter sensory neurons in dorsal root ganglia and trigeminal ganglia and is activated by cool/cold temperature (<25°C) as well as by menthol and icilin (17, 23). In our experiments, significant increases in [Ca2+]cyt were detected in subsets of nodose ganglion neurons in response to changes in temperature that corresponded to the activation threshold for TRPM8, TRPN1, TRPV3, TRPV4, TRPV1, and TRPV2. The requirement for extracellular Ca2+ in temperature-evoked changes of [Ca2+]cyt in the present study is also compatible with TRP channel involvement.
Our observation that responses to icilin were largely restricted to cells that responded to cooling/cold and that cells that responded to capsaicin also tended to respond to moderate heating points to involvement of the TRPN1/TRPM8 and TRPV1, respectively. However, it should be noted that in the present study, icilin induced an increase in [Ca2+]cyt in nodose ganglion neurons only at 100 µM, compared with 0.110 µM when used to activate TRPM8 overexpressed in Xenopus oocytes (17). Nevertheless this is compatible with the concentration of icilin that activate TRPN1 channel. In Xenopus oocytes, overexpressed ANKTM1 was activated by 25100 µM icilin (30). It is possible that the Ca2+ influx induced by cooling/cold in nodose ganglionic neurons is mediated primarily by ANKTM1 and not TRPM8. Further experiments would be required to determine whether this is the case.
It has to be borne in mind that the procedures involved in isolation and culture of nodose ganglion neurons may have affected their responsiveness. Ji et al. (15) showed that TRPV1 protein is upregulated by NGF, via activation of the p38 MAPK pathway in primary sensory neurons after inflammation. In the present study, we added NGF in cultured nodose ganglion neurons to avoid downregulation of TRPV1 before carrying out Ca2+ imaging studies. Whether TRPV1 levels or levels of other TRP channels in nodose ganglion neurons are altered by these culture procedures remains to be determined.
Functional significance.
Although thermosensitive afferent fibers have been identified in the esophagus, stomach, duodenum, and rectum in animals and humans (5, 6, 34, 42), the specific pathways involved in gastrointestinal temperature sensation have not been fully defined. In animal experiments, both vagal and splanchnic fibers appear to be activated by thermal stimuli in gut (46, 11, 27). Zamyatina in 1954 (42) reported that instillation of water at a temperature of 45 or 50°C into the stomach of the cat increased afferent impulses in the splanchnic nerve. Riedel in 1976 (26) also recorded increased afferent discharge in the splanchnic nerve after warming the abdomen, and Gupta and Nier in 1979 (11) recorded activity in the splanchnic nerve after cooling the stomach and duodenum (11). On the other hand, three types of thermosensitive unmyelinated fibers were reported by EI Ouazzani and Mei in 1979 and 1981 (5, 6) in the vagus nerve of the cat with responses to cold (1036°C), warm (3950°C), and mixed (1035 and 4050°C) temperatures. Together, this suggests that thermoreceptors in the esophagus, stomach, small intestine, and proximal colon relay information to the central nervous system via both splanchnic and vagal afferent fibers. It is likely that under normal physiological conditions, ingested material would provide only transient thermal stimuli to the esophagus and perhaps upper stomach, which are densely innervated by vagal afferent neurons.
The widespread distribution of TRP channels in tissues in the present study suggest that they may be involved in functions other than thermal sensitivity. TRPV1 is also activated by H+ (2), which is associated with tissue damage and inflammation. It has been shown that ablation of TRPV1-bearing afferent fibers, increased damage by a variety of stimuli that damage the gastric mucosa (12). It is likely that TRPV1 on spinal afferents plays an important role in triggering protective hyperemia if the gastric mucosal barrier is breached. TRPV1 is also activated by anandamide, an endogenous cannabinoid found in the central nervous system. It is possible that TRPV1 could function as a ligand-gated ion channel that modulates sensory input to the central nervous system in the presence of endogenous anandamide. To date, other endogenous ligands are yet to be identified for the other four TRP channels although the ability of menthol and icilin to open TRPM8 and TRPN1 may be suggestive (3, 17, 23, 30). Our observation that all of the temperature-sensitive TRP channel transcripts were also detected in sympathetic ganglion neurons, which are not believed to play an afferent role, strongly suggests that TRP channels do have other functions in neurons, apart from sensory transduction. We found that TRPV4 was expressed in 80% of nerve cells labeled by DiI applied to the stomach. This was a considerably higher proportion than for any of the other temperature-sensitive TRP channels. It is possible that TRPV4 is involved in detection of mechanical stimuli, because it can be activated by mechanical and osmotic stimuli in the skin and in inner ear hair cells (32, 38). Many vagal afferent fibers to the stomach and esophagus are mechanosensitive, including those with intraganglionic laminar endings (40, 41) and those projecting to the mucosa (21). Temperature-sensitive TRP channels, in particular TRPV1, were also located in nonneuronal cells in the present study. TRPN1 is expressed in human fibroblasts, whereas TRPM8 is also expressed in a variety nonneuronal tissues and is upregulated in prostate cancer (14, 17, 23, 33). In both cases, expression of these temperature-sensitive TRP channels appear to be affected by the mechanisms of carcinogenesis.
Our study also demonstrated the presence of multiple TRP channel transcripts in single vagal neurons. Consistent with this, we found that subsets of nodose ganglion neurons responded to a wider range of thermal stimuli than could be explained by any single channel. Other studies have also shown the presence of more than one temperature-sensitive TRP channel in neurons. It has been shown that TRPV1 coexists with TRPN1 (23, 30) or with TRPV3 (28, 29) in some dorsal root ganglion neurons. EI Ouazzani and Mei (5, 6) demonstrated electrophysiologically that single vagal afferents can detect a broad spectrum of thermal stimuli. Whether this wide dynamic range of response is due to the presence of different homomeric channel complexes in single neurons or formation of heteromeric channel complexes is unclear. With the use of a heterologous expression system, Smith et al. (28) showed that TRPV1 and TRPV3 could form heterologous ion channels with properties that differed from the homologous constituents.
In conclusion, the present study provides evidence that temperature-sensitive TRP channels are present in vagal afferent neurons in the nodose ganglion. These TRP channels may play a role in the sensory innervation of the upper gastrointestinal tract and are likely to confer temperature sensitivity on some vagal afferent neurons. Our studies showed that thermal stimuli induced increase in [Ca2+]cyt in vagal afferent neurons, probably by opening temperature-sensitive TRP channels. Whether this also occurs in the corresponding sensory nerve endings in the gut wall is yet to be established. Our results do not preclude other roles for TRP channels apart from temperature sensitivity.
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GRANTS
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This work was supported by a grant from AstraZeneca of Sweden. S. J. H. Brookes is a senior research fellow of the National Health and Medical Research Council, Canberra, Australia.
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ACKNOWLEDGMENTS
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We thank Greg J. Barritt (Department of Biochemistry, Flinders University) for helpful advice and discussions for calcium image experiments.
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FOOTNOTES
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Address for reprint requests and other correspondence: S. J. H. Brookes, Dept. of Human Physiology, and Centre for Neuroscience, Flinders Univ., GPO Box 2100, Adelaide, South Australia, 5001, Australia (E-mail: simon.brookes{at}flinders.edu.au).
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.
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REFERENCES
|
---|
- Caterina M, Rosen TA, Tominaga M, Brake A, and Julius D. A capsaicin-receptor homologue with a high threshold for noxious heat. Nature 398: 436441, 1999.[CrossRef][ISI][Medline]
- Caterina M, Schumacher MA, Tominaga M, Rosen TA, Levine JD, and Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389: 816824, 1997.[CrossRef][ISI][Medline]
- Chung M, Lee H, and Caterina M. Warm temperatures activate TRPV4 in mouse 308 keratinocytes. J Biol Chem 278: 3203732046, 2003.[Abstract/Free Full Text]
- Delbro D, Lisander B, and Andersson SA. Atropine-sensitive gastric excitation by local heating- the possibility of visceral axon reflex arrangement. Acta Physiol Scand 114: 319320, 1982.[ISI][Medline]
- El Ouazzani T and Mei N. Vagal thermoreceptors in the gastro-intestinal area. Their role in the regulation of the digestive motility. Exp Brain Res 34: 419434, 1979.[ISI][Medline]
- El Ouazzani T and Mei N. Electrophysiologic properties and role of the vagal thermoreceptors of lower esophagus and stomach of cat. Gastroenterology 83: 9951001, 1982.[ISI][Medline]
- Gotoh H, Akatsuka H, and Suto K. Warm cells revealed by microfluorimetry of Ca2+ in cultured dorsal root ganglion neurons. Brain Res 796: 319322, 1998.[CrossRef][ISI][Medline]
- Gotoh H, Kajikawa M, Kato H, and Suto K. Intracellular Mg2+ surge follows Ca2+ increase during depolarization in cultured neurons. Brain Res 828: 163168, 1999.[CrossRef][ISI][Medline]
- Guler AD, Lee H, Iida T, Shimizu I, Tominaga M, and Caterina M. Heat-evoked activation of the ion channel, TRPV4. J Neurosci 22: 64086414, 2002.[Abstract/Free Full Text]
- Voets T, Prenen J, Vriens J, Watanabe H, Janssens A, Wissenbach U, Bodding M, Droogmans G, and Nilius B. Molecular determinants of permeation through the cation channel TRPV4. J Biol Chem 277: 3370433710, 2002.[Abstract/Free Full Text]
- Gupta B, Nier K, and Hensel H. Cold-sensitive afferents from the abdomen. Pflügers Arch 380: 203204, 1979.[ISI][Medline]
- Holzer P. Sensory neurone responses to mucosal noxae in the upper gut: relevance to mucosal integrity and gastrointestinal pain. Neurogastroenterol Motil 14: 459475, 2002.[CrossRef][ISI][Medline]
- Hori A, Minato K, and Kobayashi S. Warming-activated channels of warm-sensitive neurons in rat hypothalamic slices. Neurosci Lett 275: 9396, 1999.[CrossRef][ISI][Medline]
- Jaquemar D, Schenker T, and Trueb B. An ankyrin-like protein with transmembrane domains is specifically lost after oncogenic transformation of human fibroblasts. J Biol Chem 274: 73257333, 1999.[Abstract/Free Full Text]
- Ji R, Samad T, Jin S, Schmoll R, and Woolf C. p38 MAPK activation by NGF in primary sensory neurons after inflammation increases TRPV1 levels and maintains heat hyperalgesia. Neuron 36: 5758, 2002.[ISI][Medline]
- Jia Y, McLeod R, Wang X, Parra L, Egan R, and Hey J. Anandamide induces cough in conscious guinea-pigs through VR1 receptors. Br J Pharmacol 137: 831836, 2002.[Abstract/Free Full Text]
- McKemy DD, Neuhausser WM, and Julius D. Identification of a cold receptor reveals a general role for TRP channels in thermosensation. Nature 416: 5258, 2002.[CrossRef][ISI][Medline]
- Michael G and Priestley JV. Differential expression of the mRNA for the vanilloid receptor subtype 1 in cells of the adult rat dorsal root and nodose ganglia and its downregulation by axotomy. J Neurosci 19: 18441854, 1999.[Abstract/Free Full Text]
- Nozawa Y, Nishihara K, Yamamoto A, Nakano M, Ajioka H, and Matsuura N. Distribution and characterization of vanilloid receptors in the rat stomach. Neurosci Lett 309: 3336, 2001.[CrossRef][ISI][Medline]
- Okazawa M, Takao K, Hori A, Shiraki T, Matsumura K, and Kobayashi S. Ionic basis of cold receptors acting as thermostats. J Neurosci 22: 39944001, 2002.[Abstract/Free Full Text]
- Page A and Blackshaw LA. An in vitro study of the properties of vagal afferent fibres innervating the ferret oesophagus and stomach. J Physiol 512: 907916, 1998.[Abstract/Free Full Text]
- Patterson L, Zheng H, Ward S, and Berthoud H. Vanilloid receptor (VR1) expression in vagal afferent neurons innervating the gastrointestinal tract. Cell Tissue Res 311: 277287, 2003.[ISI][Medline]
- Peier AM, Moqrich A, Hergarden AC, Reeve AJ, Andersson DA, Story GM, Earley TJ, Dragoni I, McIntyre P, Bevan S, and Patapoutian A. A TRP channel that senses cold stimuli and menthol. Cell 108: 705715, 2002.[ISI][Medline]
- Reid G, Babes A, and Pluteanu F. A cold- and menthol-activated current in rat dorsal root ganglion neurones: properties and role in cold transduction. J Physiol 545: 595614, 2002.[Abstract/Free Full Text]
- Reid G and Flonta ML. Physiology: cold current in thermoreceptive neurons. Nature 413: 480, 2001.[CrossRef][ISI][Medline]
- Riedel W. Warm receptors in the dorsal abdominal wall of the rabbit. Pflügers Arch 361: 205206, 1976.[ISI][Medline]
- Rozsa Z, Mattila J, and Jacobson ED. Substance P mediates a gastrointestinal thermoreflex in rats. Gastroenterology 95: 265276, 1988.[ISI][Medline]
- Smith GD, Gunthorpe MJ, Kelsell RE, Hayes PD, Reilly P, Facer P, Wright JE, Jerman JC, Walhin JP, Ooi L, Egerton J, Charles KJ, Smart D, Randall AD, Anand P, and Davis JB. TRPV3 is a calcium-permeable temperature-sensitive cation channel. Nature 418: 181186, 2002.[CrossRef][ISI][Medline]
- Smith GD, Gunthorpe MJ, Kelsell RE, Hayes PD, Reilly P, Facer P, Wright JE, Jerman JC, Walhin JP, Ooi L, Egerton J, Charles KJ, Smart D, Randall AD, Anand P, and Davis JB. TRPV3 is a temperature-sensitive vanilloid receptor-like protein. Nature 418: 186190, 2002.[CrossRef][ISI][Medline]
- Story G, Peier A, Reeve A, Eid S, Mosbacher J, Hricik T, Earley T, Hergarden A, Andersson D, Hwang S, McIntyre P, Jegla T, Bevan S, and Patapoutian A. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112: 819829, 2003.[ISI][Medline]
- Suto K and Gotoh H. Calcium signaling in cold cells studied in cultured dorsal root ganglion neurons. Neuroscience 92: 11311135, 1999.[CrossRef][ISI][Medline]
- Suzuki M, Mizuno A, Kodaira K, and Imai M. Impaired pressure sensation with mice lacking TRPV4. J Biol Chem 278: 2266422668, 2003.[Abstract/Free Full Text]
- Tsavaler L, Shapero M, Morkowski S, and Laus R. Trp-p8, a novel prostate-specific gene, is up-regulated in prostate cancer and other malignancies and shares high homology with transient receptor potential calcium channel proteins. Cancer Res 61: 37603769, 2001.[Abstract/Free Full Text]
- Villanova N, Azpiroz F, and Malagelada JR. Perception and gut reflexes induced by stimulation of gastrointestinal thermoreceptors in humans. J Physiol 502: 215222, 1997.[Abstract]
- Voets T, Prenen J, Vriens J, Watanabe H, Janssens A, Wissenbach U, Bodding M, Droogmans G, and Nilius B. TRPV3 is a temperature-sensitive vanilloid receptor-like protein. J Biol Chem 418: 186190, 2002.
- Ward S, Bayguinov J, Won K, Grundy D, and Berthoud H. Distribution of the vanilloid receptor (VR1) in the gastrointestinal tract. J Comp Neurol 465: 121135, 2003.[CrossRef][ISI][Medline]
- Watanabe H, Vriens J, Suh S, Benham C, Droogmans G, and Nilius B. Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells. J Biol Chem 277: 4704447051, 2002.[Abstract/Free Full Text]
- Wissenbach U, Bodding M, Freichel M, and Flockerzi V. Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. FEBS Lett 103: 525535, 2000.
- Xu H, Ramsey IS, Kotecha SA, Moran MM, Chong JA, Lawson D, Ge P, Lilly J, Silos-Santiago I, Xie Y, DiStefano PS, Curtis R, and Clapham DE. A heat-sensitive TRP channel expressed in keratinocytes. Nature 296: 20462049, 2002.
- Zagorodnyuk V and Brookes SJ. Transduction sites of vagal mechanoreceptors in the guinea pig esophagus. J Neurosci 20: 62496255, 2000.[Abstract/Free Full Text]
- Zagorodnyuk V, Chen BN, and Brookes SJ. Intraganglionic laminar endings are mechano-transduction sites of vagal tension receptors in the guinea-pig stomach. J Physiol 534: 255268, 2001.[Abstract/Free Full Text]
- Zamyatina O. Electrophysiological characteristics and functional significance of afferent impulses originating in intestinal wall. Trans IP Pavlov Inst Physiol 3: 193198, 1954.