Involvement of TRPA1 activation in acute pain induced by cadmium in mice
© Miura et al; licensee BioMed Central Ltd. 2013
Received: 30 November 2012
Accepted: 26 February 2013
Published: 28 February 2013
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© Miura et al; licensee BioMed Central Ltd. 2013
Received: 30 November 2012
Accepted: 26 February 2013
Published: 28 February 2013
Cadmium (Cd) is an environmental pollutant and acute exposure to it causes symptoms related to pain and inflammation in the airway and gastrointestinal tract, but the underlying mechanisms are still unclear. TRPA1 is a nonselective cation channel expressed in sensory neurons and acts as a nociceptive receptor. Some metal ions such as Ca, Mg, Ba and Zn are reported to modulate TRPA1 channel activity. In the present study, we investigated the effect of Cd on cultured mouse dorsal root ganglion neurons and a heterologous expression system to analyze the effect of Cd at the molecular level. In addition, we examined whether Cd caused acute pain in vivo.
In wild-type mouse sensory neurons, Cd evoked an elevation of the intracellular Ca concentration ([Ca2+]i) that was inhibited by external Ca removal and TRPA1 blockers. Most of the Cd-sensitive neurons were also sensitive to cinnamaldehyde (a TRPA1 agonist) and [Ca2+]i responses to Cd were absent in TRPA1(−/−) mouse neurons. Heterologous expression of TRPA1 mutant channels that were less sensitive to Zn showed attenuation of Cd sensitivity. Intracellular Cd imaging revealed that Cd entered sensory neurons through TRPA1. The stimulatory effects of Cd were confirmed in TRPA1-expressing rat pancreatic cancer cells (RIN-14B). Intraplantar injection of Cd induced pain-related behaviors that were largely attenuated in TRPA1(−/−) mice.
Cd excites sensory neurons via activation of TRPA1 and causes acute pain, the mechanism of which may be similar to that of Zn. The present results indicate that TRPA1 is involved in the nociceptive or inflammatory effects of Cd.
Cadmium (Cd) is a ubiquitous environmental pollutant distributed in rocks, soil, the atmosphere and water . Human exposure occurs mainly from consumption of contaminated food, smoking and inhalation by workers in metal industries (reviewed by [2, 3]). Cd is more efficiently absorbed by the lungs (25-50%) than the gastrointestinal tract (5%) . Thus, Cd inhalation is often a problem in metal workers and cigarette smokers.
Chronic Cd toxicity has been well studied and the main target organs are the kidney, liver, lung cardiovascular, immune and reproductive systems (reviewed by ), with resultant, renal tubular dysfunction and subsequent induction of osteomalacia known as “itai-itai disease” (reviewed by ). In acute exposure, on the other hand, Cd causes symptoms related to pain and inflammation in the airway or gastrointestinal tract. After exposure to a high level of metal dust or fumes containing Cd causes irritation of the upper respiratory tract and induces coughing in the early stage, various clinical manifestations known as metal fume fever occur, including airway inflammation, pulmonary edema, chest pain and flu-like symptoms. Cd ingestion causes abdominal pain, severe nausea and diarrhea (reviewed by ). These reports suggest that Cd stimulates sensory processing involved in pain and inflammation, but the underlying mechanisms are unclear.
TRPA1 and TRPV1 are nonselective cation channels expressed in nociceptive neurons and act as polymodal receptor . For example, TRPA1 channel is activated by a range of natural products (allyl isothiocyanate, cinnamaldehyde and allicin found in mustard oil, cinnamon, and garlic, respectively) [8, 9], environmental irritants (acrolein and formalin) [10, 11], α,β-unsaturated aldehyde (cigarette smoke) , reactive oxygen species  and cold temperature , and contributes to the perception of noxious stimuli. TRPV1 is also activated by various stimuli such as capsaicin, protons, and noxious heat, and plays an important role in sensory transduction [15, 16].
It has been reported that some metal ions regulate TRP channel activities. For example, nickel activates TRPV1 in the mM range, which is associated with nickel-induced contact dermatitis . It has also been reported that Na, Mg and Ca are able to activate TRPV1, which contributes to the nociceptive responses to elevated ionic strength . Moreover, Na negatively regulates TRPV1, since removal of external Na activates TRPV1 .
Some metal ions such as Ca, Mg, Ba and Zn modulate TRPA1 channel activity [20–23]. Among them, Zn, in the same metal ionic group as Cd, stimulates sensory nerves and causes nociception in mice through direct activation of TRPA1 [21, 22].
In the present study, we examined the effect of Cd on sensory neurons in vitro and on nociceptive behavior in vivo using wild-type and TRPA1(−/−) mice. To examine the neuronal activity, we used fura-2-based Ca-imaging techniques since some TRP channels are highly Ca permeable . We investigated the effect of Cd on cultured mouse dorsal root ganglion (DRG) neurons, which are a useful model of nociception in vitro. We also used a heterologous expression system to analyze the effect of Cd at the molecular level using Ca-imaging and patch-clamp techniques. In addition, we examined whether Cd induced acute pain in vivo.
The present study indicates that Cd induced [Ca2+]i increases in mouse primary sensory neurons, which were also sensitive to the TRPA1 agonist cinnamaldehyde and selectively inhibited by TRPA1 blockers. The Cd-induced [Ca2+]i responses were absent in TRPA1 (−/−) mouse DRG neurons. [Ca2+]i responses to Cd were confirmed in RIN-14B cells expressing TRPA1 endogenously. Cd evoked current responses in heterologously expressed TRPA1. In wild-type mice, intraplantar injection of Cd induced pain-related behaviors, which were largely attenuated in TRPA1 (−/−) mice. These results suggested that Cd elicited acute pain through the activation of TRPA1.
It is known that Cd and Zn bind cysteine and histidine residues in metallothionein, a cysteine-rich metal-binding protein , and in some metal ion transporters . Zn directly activates heterologously expressed TRPA1 [21, 22] and specific intracellular cysteine and histidine residues of TRPA1 bind Zn . Our findings indicated that two cysteine (C641, C1021) residues and one histidine (H983) residue affected Cd sensitivity, since mutant TRPA1 channels (C641S/C1021S, H983A) showed attenuation of Cd sensitivity like Zn. These properties were confirmed by the patch-clamp experiments using heterologously expressed mutant TRPA1 channels. These amino acid residues were located in the intracellular domain and the N-terminal C641 has been identified as a residue involved in some reactive chemicals . Thus, it seems likely that Cd activates TRPA1 through recognition of the same specific amino acid residues for its recognition sites as those for Zn.
Furthermore, Cd imaging using Leadmium Green showed that Cd entered mouse sensory neurons. The increases of fluorescent intensity of Leadmium Green in TRPA1(−/−) mouse DRG neurons were significantly lower than in wild-type ones. These results suggested that Cd may be able to permeate into neurons though TRPA1. It was confirmed in human TRPA1-expressing HEK293 cells. It has been reported that Zn permeates through TRPA1 and acts on the inner domain of this channel . In TRPA1(−/−) mouse DRG neurons, however, Cd influx remained at higher concentrations, suggesting that Cd might enter through other pathways. It has been reported that Cd permeates through voltage-dependent Ca channels (L-type; , T-type; ), an Fe transporter (DMT1; ), Zn transporters (ZIP8; , ZIP14; ) and TRPV6 . These channels or transporters are reported to be expressed in neurons [33, 35–37]. Thus, in wild-type mouse DRG neurons, Cd entering through these pathways might also activate TRPA1.
In this study, we used 30–300 μM Cd, concentrations that were much higher than reported blood or urine Cd levels in exposed humans (blood-Cd:10 μg/L, urine-Cd:7 μg/L creatinine, ). It is reported that workers exposed to Cd fumes at 8.63 mg/m3 for 5 h exhibited symptoms of coughing and slight irritation of the throat and mucosa . In the case of acute Cd exposure such as airway instillation, local concentrations would be higher than blood or urine concentrations. Similarly, in research on acute toxicity to Zn in vitro, submillimolar concentrations were used (300 μM; , 30 μM; , 30 μM, ). In the present study, intraplantar injection of Cd (2 nmol/paw), the amount of which was somewhat higher than reports for Zn in in vivo experiments (0.6 nmol, intraplantarly; , 0.05 nmol, intratracheally; ), elicited nociceptive behaviors in wild-type mice. On the other hand, Cd induced significantly fewer behavioral changes in TRPA1(−/−) mice, suggesting the involvement of TRPA1 in Cd-induced acute pain.
Recently, functional TRPA1 expression has been reported in non-neuronal cells such as lung fibroblast cells, epithelial cells and smooth muscle cells, which release IL-8 in response to TRPA1 agonists and contribute to lung inflammation . It is also reported that Cd promotes secretion of IL-8 and IL-6 from airway epithelial cells . For lung inflammation, therefore, not only neuronal but also non-neuronal TRPA1 may be involved in Cd toxicity.
It is reported that Cd produces reactive oxygen species (ROS)  that mediates Ca signaling involved in Cd-induced cell death . Since ROS are also known to activate TRPA1 [13, 45], we examined whether Cd elicited ROS production in mouse DRG neurons. However, Cd failed to produce ROS under our experimental conditions (30 or 300 μM, 2 min), using CM-H2DCFDA, a fluorescent ROS indicator (data not shown).
The present study demonstrates that Cd excites sensory neurons via activation of TRPA1 and causes acute pain, the mechanism of which may be similar to that of Zn. Our present data show that TRPA1 contributes to the nociceptive or inflammatory effects of Cd. However, further studies are necessary to completely understand the pathological conditions of acute Cd toxicity.
All protocols for experiments on animals were approved by the Committee on Animal Experimentation of Tottori University (♯11–T–2). All efforts were made to minimize the number of animals used.
We used adult mice of either sex (4–16 weeks old). C57BL/6 J mice, TRPA1-null mice (kindly provided by Dr. D. Julius, University of California) were euthanized by inhalation of CO2 gas. Mouse DRG cells were isolated and cultured as described previously . In brief, DRG cells were removed, dissected and freed from connective tissue under a dissecting microscope in phosphate-buffered saline (PBS: in mM, 137 NaCl, 10 Na2HPO4, 1.8 KH2PO4, 2.7 KCl) supplemented with 100 U/ml penicillin G and 100 μg/ml streptomycin. Then isolated ganglia were cut into small pieces and enzymatically digested for 30 min at 37°C in PBS containing collagenase (1 mg/ml, type II, Worthington, USA) and DNase I (1 mg/ml, Roche Molecular Biochemicals, USA). Subsequently, the ganglia were immersed in PBS-containing trypsin (10 mg/ml, Sigma, USA) and DNase I (1 mg/ml) for 15 min at 37°C. After enzyme digestion, the enzyme-containing solution was aspirated and the ganglia were washed with culture medium (Dulbecco’s-modified Eagle’s medium [DMEM, Sigma] supplemented with 10% fetal bovine serum [Sigma]), penicillin G (100 U/ml) and streptomycin (100 μg/ml). DRG cells were obtained by gentle trituration with a fine-polished Pasteur pipette. Then the cell suspension was centrifuged (800 rpm, 2 min, 4°C) and the pellet-containing cells were resuspended with the culture medium. Aliquots were placed on glass cover slips coated with poly-D-lysine (Sigma) and cultured in a humidified atmosphere of 95% air and 5% CO2 at 37°C. In the present experiment, cells cultured within 24 h were used.
The RIN-14B cells were purchased from DS Pharma Biomedical Co., Ltd. (Osaka, Japan). Cells were cultured in RPMI1640 medium (Wako) supplemented with 10% FBS, 100 U/ml penicillin G and 100 μg/ml streptomycin.
Cells were transfected using 1 μg of human TRPA1 (hTRPA1, gift from Ardem Patapoutian) and mutants of hTRPA1 (C641S/C1021S, H983A ), which were made using a modified QuickChange Site-Directed Mutagenesis method (Stratagene, La Jolla, CA, USA). Human embryonic kidney (HEK) 293 cells were cultured in DMEM supplemented with 10% FBS, 100 U/ml penicillin G and 100 μg/ml streptomycin. Cells were transfected with the expression vectors using a transfection reagent (Lipofectamine 2000 or Lipofectamine Reagent together with Plus Reagent, Invitrogen) and used 24 h after transfection.
The intracellular Ca imaging in individual cells were performed with the fluorescent Ca indicator fura-2 by dual excitation using a fluorescent-imaging system controlling illumination and acquisition (Aqua Cosmos, Hamamatsu Photonics, Hamamatsu, Japan) as described previously . Briefly, to load fura-2, cells were incubated for 40 min at 37°C with 10 μM fura-2 AM (Molecular Probes, Eugene, Oregon, USA) in HEPES-buffered solution (in mM: 134 NaCl, 6 KCl, 1.2 MgCl2, 2.5 CaCl2, and 10 HEPES, pH 7.4). A coverslip with fura-2-loaded cells was placed in an experimental chamber mounted on the stage of an inverted microscope (Olympus IX71) equipped with an image acquisition and analysis system. Cells were illuminated every 5 s with lights at 340 and 380 nm, and the respective fluorescence signals of 500 nm were detected. The fluorescence emitted was projected onto a charge-coupled device camera (ORCA-ER, Hamamatsu Photonics) and the ratios of fluorescent signals (F340/F380) for [Ca2+]i were stored on the hard disk of a computer (Endeavor pro 2500, Epson). Cells were continuously superfused with HEPES-buffered solution at a flow rate of ~2 ml/min through a Y-tube pipette. The composition of high-K solution was (in mM) 80 KCl, 1.2 MgCl2, 2.5 CaCl2, and 10 HEPES, pH 7.4). For Ca-free external solution, Ca was omitted. All experiments were carried out at room temperature (22-25°C).
HEK293 cells expressing hTRPA1 and mutants of hTRPA1 on coverslip were mounted in an experimental chamber and superfused with HEPES-buffered solution as for Ca imaging experiments. The pipette solution contained (in mM: 140 KCl, 1.2 MgCl2, 2 ATPNa2, 0.2 GTPNa3, 10 HEPES, 10 EGTA, pH 7.2 with KOH). The resistance of patch electrodes ranged from 4 to 5 MΩ. The whole-cell currents were sampled at 5 kHz and filtered at 1 kHz using a patch-clamp amplifier (Axopatch 200B; Molecular Devices, Sunnyvale, CA) in conjunction with an A/D converter (Digidata 1322A; Molecular Devices). Membrane potential was clamped at −60 mV and voltage ramp pulses from −100 mV to +80 mV for 100 ms were applied every 5 s.
For single-cell Cd imaging, we used Leadmium Green (Molecular Probes), a specific indicator for lead and cadmium. To load Leadmium Green, cells were incubated for 40 min at 37°C with 50 ng/μl Leadmium Green-AM in HEPES-buffered solution. Cd imaging was performed using the same apparatus as for Ca imaging. Cells were illuminated with light at 490 nm and fluorescence signals of 520 nm were collected. For Cd imaging, cells were superfused with Ca-free external solution. Signals were expressed as the relative change in fluorescence, F/F0, where F and F0 indicate fluorescence at any given period and the initial period, respectively.
Mice were placed in cages for 30 min before experiments. Twenty microliters of the HEPES-buffered solution (vehicle), which was similar in composition to that used in in vitro experiments, was first injected intraplantarly into the right hind paw as a control. The numbers of times each mouse licked, bit and flicked the injected paw were counted for 15 min after the injection. Subsequently, the same amount of Cd (2 nmol/paw) was injected into the left hind paw and the number of pain-related behaviors were counted for 15 min.
The following drugs were used (vehicle and concentration for stock solution). Capsaicin (ethanol, 0.01 M), HC-030031 (dimethyl sulfoxide: DMSO, 0.01 M), AP18 (DMSO, 0.1 M) and Cremophor EL (distilled water: DW, 1%) were from Sigma. Ruthenium red (DW, 0.01 M), cadmium chloride (DW, 1 M) and zinc chloride (DMSO, 0.1 M) were from Wako (Osaka, Japan). N-(4-t-butylphenyl)-4-(3-chloropyridin-2-yl)tetrahydropyrazine-1(2H)-carboxamide (BCTC, DMSO, 0.01 M) was from BIOMOL Research Laboratories, Inc., (Pennsylvania, USA). A967079 (DMSO, 0.01 M) was from Focus Biomolecules, Llc (Pennsylvania, USA). N,N,N',N'-tetrakis-(2-pyridylmethyl) ethylenediamine (TPEN, 0.05 M) was from Dojindo Molecular Technologies, Inc.,(Kumamoto, Japan). Cinnamaldehyde (DMSO, 1 M) was from Nacalai Tesque, Inc. (Kyoto, Japan). All other drugs used were of analytical grade.
The data are presented as the mean ± SEM (n=number of cells). For comparison of two groups, data were analyzed by the unpaired Student’s t test, and for multiple comparisons, one-way ANOVA following by Turkey-Kramer test was used. Differences with a P-value of less than 0.05 were considered significant. Values of the 50% effective concentrations (EC50) were determined using Origin version 9.0 J (Origin-Lab).
Dorsal root ganglion
Intracellular Ca2+ concentration
Transient receptor potential ankyrin 1
Transient receptor potential vanilloid 1.
This work was supported by KAKENHI, Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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