SEARCH

SEARCH BY CITATION

Keywords:

  • natural products;
  • (–)-carvone;
  • DRG neurons;
  • calcium imaging;
  • TRPV1 channels

Abstract

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

(–)-Carvone is an antinociceptive monoterpene found as the main active constituent of essential oils obtained from plants of the genus Mentha. Here, we have investigated the pharmacology of this monoterpene in dorsal root ganglia (DRG) neurons and TRPV1-expressing HEK293 cells. (–)-carvone at pharmacological active concentrations did not reveal significant cytotoxicity to the cells used in this study, as investigated by neutral red and propidium iodide flow cytometry assays. In calcium imaging experiments 1 mM (–)-carvone increased the cytosolic calcium levels in DRG neurons from 120.6 ± 5.0 nM (basal) to 310.7 ± 23.1 nM (P < 0.05). These effects were completely abolished when neurons were preincubated with calcium-free bath solution or ruthenium-red (5 µM) and capsazepine (10 µM), suggesting the possibility of TRPV1 channel-activation by (–)-carvone. Activity of (–)-carvone on TRPV1 channels was further investigated in HEK293 cells expressing recombinant human TRPV1 channels revealing dose-dependent calcium transients with an EC50 of 1.3 ± 0.2 mM (Hill coefficient = 2.5). In conclusion, we show for the first time the ability of (–)-carvone to induce increases in cytosolic calcium concentration through TRPV1 activation. © 2013 International Society for Advancement of Cytometry


INTRODUCTION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Monoterpenes are the main components of essential oils (EO) extracted from aromatic plants and possess distinct biological activities, already described elsewhere (1). Carvone is a monoterpene representing the main active constituent of the EO of Mentha sp. and commonly extracted from its leaves (2). R-(–)-carvone (abbreviated as (–)-Cv) is an enantiomer of carvone being the major constituent of peppermint (Mentha spicata) EO and displaying antinociceptive and anticonvulsant activities in addition to other pharmacological effects (3, 4). However, few studies have been proposed to elucidate the mechanisms involved with these pharmacological profiles of (–)-Cv at the cellular level. Regarding nociception, channels of the transient receptor potential (TRP) family have become increasingly important as novel sources of action for effective analgesic drugs (5). Among these channels, the TRP vanilloid type 1 (TRPV1) is well-known due to its role on nociception (5, 6). This channel is sensitive to protons, heat and chemicals like capsaicin, vanilloids, cannabinoids, and various inflammatory mediators (7). In a different context, Calixto et al. (8) highlighted the great contribution of plant-derived compounds such as capsaicin, menthol, or resinferatoxin as chemical tools to better understand the roles of TRP channels and their functions. Similar to menthol, camphor is another example for an antinociceptive monoterpene acting via TRP channels of theTRPV1 and TRPV3 types (9). Our hypothesis has been that (–)-Cv also affects nociceptors. In fact, it was described that (–)-Cv presents some activity on TRPV3 types (10). Therefore, we have nowstudied whether (–)-carvone directly activates TRPV1 channels.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Materials

All salts used in physiological solutions, glucose and NaOH, were purchased from Vetec (Duque de Caxias, Rio de Janeiro, Brazil) or Sigma-Aldrich, Canada. Ethylene glycol tetra-acetic acid (EGTA), acid-N-[2-hydroxyethyl] piperazine-N'-[2-ethanesulfonic acid] (HEPES), capsaicin (Cap), capsazepine (CPZ), dimethylsulfoxide (DMSO), caffeine (Caff), propidium iodide (PI), ionomycin (Iono), poly-L-lysine, thapsigargin (Thaps), and ruthenium red (Ru-red) were purchased from Sigma-Aldrich (USA or Canada). The enzymes papain and collagenase type-IA were purchased from Worthington Biochem. Corp. (USA) and Invitrogen (USA), respectively.

Before all experiments, R-(–)-carvone (Sigma-Aldrich, USA or Canada) was vigorously dissolved in a Krebs-Ringer-HEPES (KRH) solution (containing in mM: NaCl 140, KCl 4.5, CaCl2 2, MgCl2 1, HEPES 10, glucose 10, pH = 7.4 adjusted with NaOH) supplemented with 0.1% DMSO. All lipophilic compounds used in this study were diluted to form a final volume ≤0.1% of DMSO.

TRPV1-cDNA from Rattus norvegicus (rTRPV1) was kindly donated by Dr. David Julius (UCSF, USA) which had been cloned as described by Caterina et al. (11). The plasmid pmCherry-N1 was purchased from Clontech (USA).

Animals

In this study, we used 4-week old Wistar rats (R. norvegicus) weighing around 100 g, randomly housed in appropriate cages at 22°C ± 2°C on a 12-h light cycle with free access to food (Purina, Brazil) and water. They were killed by cervical dislocation, and all procedures were carried out in accordance with the guide lines of the Institutional Animal Care and Use Committee of the Federal University of Paraíba, Brazil (CEPA, process number #306/08).

Primary Culture of Dorsal Root Ganglion Neurons

Dorsal root ganglion (DRG) neurons were surgically dissected from thoracic and lumbar levels and immediately washed three times by centrifugation (150× g/1 min) with cold phophate-buffered saline (PBS) (low Ca2+), consisting of (in mM): NaCl 137, KCl 5.6, MgCl2 3.5, NaH2PO4 0.4, Na2HPO4 0.4, NaHCO3 4.2, CaCl2 0.05, HEPES 10, pH = 7.4 (adjusted with NaOH). Then, DRG neurons were exposed for 20 min to 0.1% papain at 37°C and then for an additional 30 min to 0.25% collagenase type-I. Thereafter, ganglia were mechanically dissociated using fired-polished glass Pasteur pipettes and resuspended in DMEM (Invitrogen, USA) supplemented with 10% FBS, 100 IU/ml penicillin and 100 μg/ml streptomycin. Neurons were then plated onto coverslips pre-treated with poly-L-lysine (10 μg/ml) and cultured at 37°C and 5% CO2 atmosphere.

HEK293 Cell Culture

Human embryonic kidney (HEK) 293 cells were cultured in MEM medium (Invitrogen, Canada) supplemented with 10% fetal bovine serum (FBS), 100 IU/ml penicillin, and 100 μg/ml streptomycin. The cells were handled under sterile environment and cultured at 37°C and 5% CO2 atmosphere.

Neutral Red Assay for Determination of Cell Viability

The neutral red assay is an indicator of intact cell membrane integrity. This test is based on the uptake of neutral red, a supravital dye, by uninjured cells accumulating it in the lysosome. The neutral red assay was performed in a modified form as described by Borenfreund and Puerner (12). Briefly, freshly isolated DRG neurons from Wistar Rats were plated at density of 5 × 105 per mL in 96-well plates using DMEM, supplemented with 10% FBS, penicillin (100 UI/ml) and streptomycin (100 µg/ml). The culture medium was removed 24 h after cell seeding and replaced with medium containing (–)-Cv at 0.5, 1, 2, or 4 mM concentrations. Doxorubicin was used at 1, 10, and 100 µM, as a positive control to induce cell death. After 24 h of treatment with the drugs, neutral red solution (50 µg/mL) was added followed by incubation for 3 h at 37°C. The cells were then washed rapidly with PBS containing calcium and following addition of a solution containing 1% glacial acetic acid and 50% ethanol. The reaction was agitated for 20 min on a plate shaker, and the absorbance was then measured at 540 nm in a spectrophotometer (Biotek Instruments EL800, USA).

Propidium Iodide Staining and Flow Cytometry for Quantification of Dead Cells

HEK293 cells (5 × 105 cells/ml) were treated with (–)-Cv at 0.5, 1, 5, or 10 mM concentrations and compared to cells treated with doxorubicin (25 µM). After 6 h of exposure, the samples were collected by centrifugation for 5 min at 150×g, resuspended and stained for 5 min with propidium iodide (2 µg/ml) in the dark, at room temperature. The cells were analyzed using a Becton Dickinson FACS Calibur flow cytometer (Becton Dickinson, Mountain View, CA) equipped with a 488-nm argon ion laser. Samples were detected with photomultiplier tubes (PMTs) at 585 nm ± 20 nm (channel FL-2) and acquired as list mode files (FCS files). The PMT signal was amplified logarithmically to distinguish populations of permeable to propidium iodide, which are presumably dead cells, from viable one. To exclude cell debris from the analysis, a polygon gate was set according to their light scattering properties (in a forward vs. side scatter plot), based on the acquisition of data for 10,000 cells. Fluorescence signals were collected in logarithmic mode, and the cytofluorimetric data were analyzed by the Summit software (Dako, USA) and plotted as a histogram of FL2 staining. Further details are in Supporting Information Table 1 (Author Checklist: Location of MIFlowCyt-Compliant Items).

Heterologous Expression of rTRPV1

In this study, HEK293 cells were transiently transfected with rTRPV1 plasmids by the modified calcium phosphate co-precipitation method (13, 14). In brief, 0.5 μg of rTRPV1 was mixed into 300 μl Hank's Balanced Salt Solution (HBSS, Sigma-Aldrich, USA) followed by dropwise addition of 15 μl CaCl2 (2.5 M). The mix was carefully added to a 30 mm Petri dish containing HEK293 cells in 80% confluence. After 10 h of incubation (5% CO2/37°C), the medium was replaced by complete MEM, and the cells were assayed after 12–24 h. Fifty ng pmCherry-N1 was co-transfected with rTRPV1 to indicate cells positive for TRPV1 (mCh+). Mock-transfected cells were transfected only with pmCherry-N1 (mCh-), and the control group was submitted to the same protocol, however in the absence of DNA.

Calcium Imaging

DRG neurons (3 × 104/well) were loaded with 5 μM Fluo-3/AM in the presence of 0.1% pluronic acid (both reagents from Invitrogen, USA) for 20 min in DMEM medium containing 10% FBS. Then, cells were gently washed with Mg2+- or Ca2+-free Krebs-Ringer-HEPES (KRH) buffer, incubated in this solution for further 30 min, then subjected to calcium imaging with the inverted Microscope ECLIPSE -TiS (Nikon, Melville, NY) coupled to a 14-bit high-resolution CCD camera CoolSNAP HQ2 (Photometrics, Tucson, AZ). Data were analyzed with NIS-Element for advanced research software (Nikon) using image acquisition rates of one frame per second collected by an automatic shutter system. Fluorescence excitation and emission wavelengths were 488 and 515 nm, respectively (15).

TRPV1 activity was further evaluated by labeling transfected HEK293 cells with Fura-2/AM (Invitrogen, USA) followed by calcium imaging using a fluorescence microscope (Leica, Germany) equipped with Ex340/380; Em510 nm filters. In these experiments cells were incubated with Fura-2/AM (5 μM) for 20 min and then gently washed with KRH for additional 30 min. Fluorescence ratio (R) was later analyzed with the Leica AF6000 software (Leica, Germany). Transfected cells were selected using mCherryFP filters (Ex587; Em610 nm).

The cytosolic calcium concentration ([Ca2+]i) was estimated by applying the equation [Ca2+]i = Kd * (FFmin)/(FmaxF) where Kd is the dissociation constants of Fluo-3 (450 nM) or Fura-2 (224 nM), F is the variable fluorescence intensity, Fmax and Fmin are, respectively, the maximum and minimum fluorescence obtained in the presence of ionomycin (5 μM) or EGTA (20 mM) (16). F was substituted for R in the formula above when Fura-2 was used as fluorophore. Calcium imaging data, using DRG neurons or HEK293 transfected cells were representative for three independent experiments of single cell analysis of at least 10 cells each.

Statistical Analysis

All data were expressed as the mean ± SEM. Levels for statistical significance were set at P < 0.05 using ANOVA followed by Dunnet or Tukey tests. EC50 values were acquired by plotting normalized data to the Hill equation: f = Min + (Max – Min)/(1 + EC50/[CvOL]n), where Max and Min represent the maximum and minimum values, respectively; EC50 is the half-maximal effective concentration of the tested drug, and n is the Hill coefficient.

RESULTS

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

To determine the non-toxic concentration range of (–)-Cv, we have performed cytotoxicity assays using the same cell types used subsequent calcium imaging. During these tests doxorubicin was used as positive control. Neutral red assays in DRG neurons indicated that (–)-Cv did not show any cytotoxic effect on these cells after 24 h of incubation when used at concentrations lower than 2 mM (Fig. 1). However, 4 mM of the monoterpene reduced cell viability by about 40.0% ± 2.1% (P < 0.05) (Fig. 1A). The vehicle (0.1% DMSO) did not reveal any cytotoxic effects under the same experimental conditions (Fig. 1A). Flow cytometry experiments of HEK293 cells stained with propidium iodide demonstrated that concentrations higher than 5 mM of (–)-Cv resulted in a reduction of cell viability (55.0 ± 6.7% compared to control experiments) with an EC50 of 4.9 ± 0.1 mM (Figs. 1B and 1C).

thumbnail image

Figure 1. Determination of (–)-carvone cytotoxicity on DRG neurons and HEK293 cells. (A) Neutral red assay in DRG neurons (5 × 105 cells/ml), after 24 h of incubation with (–)-Cv (0.5–4 mM), doxorubicin (Doxo, 10 μM) or vehicle. Absorbance levels were normalized to the control group which had not been exposed to any drug. (B) Cytotoxicity of (–)-carvone, (–)-Cv, on HEK293 cells determined by flow cytometry using propidium iodide staining. HEK293 cells (1 × 105 cells/ml) were submitted to 6 h-treatment with (–)-Cv (0.5–10 mM), Doxo (50 μM) or vehicle. (C) Bar plot showing statistical analysis of data shown in (B). Data are reported as mean values ± S.E. n = three independent experiments with each of them carried out in duplicate, **P < 0.01, ***P < 0.001, as determined by ANOVA followed by the Dunnett test. [Color figure can be viewed in the online issue which is available at wileyonlinelibrary.com]

Download figure to PowerPoint

Small-diameter DRG neurons (15–30 µm) responded with transient increases of cytosolic calcium concentration ([Ca2+]i) upon stimulation with 1 mM (–)-Cv. In calcium imaging experiments, the relative fluorescence intensity increased from 1.0 ± 0.0 (basal) to 2.0 ± 0.6 arbitrary units a few seconds after drug application (Fig. 2A); however, no changes in [Ca2+]i were observed when the vehicle alone was incubated under the same conditions (Fig. 2A). Ionomycin (5 μM) was used to induce the maximum fluorescence in these cells and to verify the population of vital cells. To investigate whether the [Ca2+]i transients induced by (–)-Cv resulted from calcium release of intracellular stores, DRG neurons were bathed in calcium-free KRH. A caffeine (3 mM) and thapsigargin (1 μM) solution was used to induce internal calcium release (17, 18). While (–)-Cv (1 mM) in the absence of extracellular calcium was not able to cause any fluorescence change in these conditions, [Ca2+]i transients were observed following application of caffeine plus thapsigargin solution (Fig. 2B).

thumbnail image

Figure 2. (–)-Carvone-induced [Ca2+]i transients in DRG neurons. Calcium responses were measured as increases in fluorescence emission (Ex488, Em515 nm) following exposure of DRG neurons to agonists/antagonists in KRH solution (A). Calcium-free KRH solution (B). TRPV1 blockers Ru-red (5 μM) and 10 μM CPZ (C). After treatment with vehicle (DMSO 0.1%). Cells were stimulated with 1 mM (–)-Cv followed by ionomycin (Iono, 5 µM). (D) The plot shows the peak values of [Ca2+]i levels along the measured time kinetics (see methods). Data are expressed as mean values ± S.E. of three independent experiments in which the response of each 10 cells were measured in duplicate. PS means physiological solution. ***P < 0.001, ANOVA followed by the Tukey test.

Download figure to PowerPoint

To assess the activation of TRPV1 channels as part of (–)-Cv-mediated effects, DRG neurons were pre-incubated with the TRPV1 blockers Ru-red (5 μM) and CPZ (10 μM) (7), separately, for 5 min before application of the vehicle and 1 mM (–)-Cv. Capsaicin (Cap; 10 μM) was used as TRPV1-activator (11) which increased, as expected, the relative fluorescence from 1.0 ± 0.0 (basal) to 2.4 ± 0.2 arbitrary units (F340/F380) (P < 0.05). The Cap-induced effect was reduced to fluorescence levels of unstimulated cells (1.0 ± 0.04 (P < 0.05) and 1.1 ± 0.02 (P < 0.05)) by Ru-red and CPZ, respectively (data not shown). Interestingly, a similar effect was also observed with (–)-Cv, since the fluorescence increase induced by this monoterpene (2.0 ± 0.9 arbitrary units, P < 0.05) was completely blocked (0.9 ± 0.02 and 0.0 ± 0.03 arbitrary units) in the presence of 5 μM Ru-red and 10 μM CPZ, respectively (Fig. 2C). Quantification of measured fluorescence emission values showed that basal [Ca2+]i levels of 120.6 ± 5.0 nM increased to 310.7 ± 23.1 nM (P < 0.001) after addition of 1 mM (–)-Cv. Such effect was diminished to 168.9 ± 13.0 nM (P < 0.001), 136.1 ± 12.2 nM (P < 0.001) and 127.2 ± 14.1 nM (P < 0.001), when the neurons had been pre-incubated with calcium-free KRH, Ru-red (5 μM) or CPZ (10 μM), respectively (Fig. 2D).

For further confirmation of interaction between (–)-Cv and the TRPV1 channels, calcium imaging was performed with HEK293 cells expressing recombinant rat TRPV1 channels. As observed for DRG neurons, 1 mM (–)-Cv increased Fura-2 fluorescence in HEK cells co-transfected with rTRPV1 and mCherry (TRPV1+) but not in mock-transfected cells (TRPV1-) (Figs. 3A and 3B). As expected, 5 μM Cap increased [Ca2+]i levels in these cells when applied after (–)-Cv (Fig. 3B). It is important to mention that neither (–)-Cv (1mM) nor Cap (10 μM) induced any changes in [Ca2+]i levels of untransfected HEK293 cells (MCh-) (Fig. 3B). We further quantified (–)-Cv-induced [Ca2+]i transients in TRPV1+ cells and observed that (–)-Cv increased the calcium levels in these cells in a concentration-dependent manner, exhibiting an EC50 of 1.3 ± 0.2 mM (R2 = 0.95; Hill coefficient = 2.5) (Fig. 3B). At 1 mM (-)-Cv concentration we observed a [Ca2+]i increase from 96.6 ± 3.7 nM (basal) to 729.0 ± 55.2 nM (P <0.05) in cells bathed with physiological solution alone. Such effect was completely blocked (04.7 ± 3.9 nM) when cells had been preincubated for 5 min with 10 μM CPZ (Fig. 3C). As expected, [Ca2+]i transients induced by 5 μM Cap were also blocked by CPZ under the same experimental conditions (data not shown).

thumbnail image

Figure 3. Induction of [Ca2+]i transients by (–)-carvone in HEK293 cells expressing rat recombinant TRPV1 channels. (A) Calcium imaging experiments with Fura-2 (340/380 nm) in transfected cells (TRPV1+) or mock cells (TRPV1−). Images in the left panel represent the merging between transmitted light (TL) and mCherry fluorescence (mCh) at 587 nm for identification of transfected cells (red). The other images represents the ratio of fluorescence emission following excitation (340/380 nm) during calcium imaging experiment without any drug (basal) (upper panels), followed by incubation with 1 mM (−)-Cv and 5 μM ionomycin (Iono) (lower panels). (B) Left panel: Representative traces reflecting ratios of fluorescence emission at F340/F380 excitation after incubation with 1mM (–)-Cv, 5 μM capsaicin (Cap) and 5 μM ionomycin (Iono) in TRPV1+ and TRPV1− cells. Right panel: Concentration-dependent induction of [Ca2+]i levels of TRPV1+ cells (for equation see Method Section). (C) Effects of 1 mM (–)-Cv on TRPV1+ cells pre-incubated with capsazepine (CPZ, 10 μM) or physiological solution (PS) alone. Data are expressed as mean values ± S.E. of 15 individually analyzed cells (n = 3). ***P < 0.001, ANOVA followed by the Tukey test.

Download figure to PowerPoint

DISCUSSION

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Compounds derived from natural products are potential targets for the development of new analgesic drugs (1). Recent studies have demonstrated that the monoterpene (–)-Cv is a neuroactive molecule with promising therapeutic applications, since this substance exerts distinct effects on both central and peripheral nervous systems (3, 4). Nevertheless, more data are still required for better understanding of possible therapeutic applications for (–)-Cv.

Cellular cytotoxicity tests belong to the most commonly used tools for efficient screening in drug discovery and development. These assays usually evaluate the integrity of the cell membrane, lysosomes, or mitochondria, since interruptions of these structures are indicative of cell death and can provide information about the susceptibility of cellular organelles and compartments (19, 20). The neutral red assay, for example, is a valuable cytotoxicity assay based on the cell ability to incorporate the neutral red, a supravital dye, within the lysosomes of viable cells (21, 22). According to our data, (–)-Cv induced minor cytotoxic effects on DRG neurons with concentrations less than 4 mM, and the drug did not cause any membrane damage (Fig. 1A).

This was further confirmed by flow cytometry assays using the DNA-sensitive dye propidium iodide (23, 24) in HEK293 cells pre-incubated with (–)-Cv (0.5–10 mM). The data showed that (–)-Cv exert low cytotoxicity on HEK293 cells, presenting an EC50 of about 5 mM for cell death (Fig. 1B). These results indicate that pharmacological activities of lower concentrations of (–)-Cv would not be related to changes in cell death.

It is well known that Ca2+ is an essential ion involved directly or indirectly in several intracellular events; thus monitoring its concentrations experimentally can be an appropriate choice for initial evaluation of the pharmacological activity of many bioactive molecules (25, 26). (–)-Cv promoted a rapid increase in [Ca2+]i of DRG neurons (Fig. 2A). The rapid increase of [Ca2+]i levels in eukaryotic cells may result from ion-channel activation for example, or can be originated internally from intracellular stores (27, 28). To evaluate which of these pathways were activated by (–)-Cv in DRG neurons, the cells were pre-incubated in calcium-free bath solution prior to application of the monoterpene. Changes in [Ca2+]i levels were no longer induced by 1mM (–)-Cv under these conditions, but persisted when caffeine and thapsigargin solution was added to the cell culture, indicating that (–)-Cv effects in DRG neurons depended on the presence of extracellular Ca2+ (Fig. 2B).

DRG neurons express a wide variety of calcium-permeable ion channels. Some of these channels are part of the transient receptor potential (TRP) family, which has been widely studied in the recent years and provide confirmed targets for several natural compounds like monoterpenes (8–10). In particular, the TRP vanilloid type 1 (TRPV1) is certainly the most investigated channel in this regard, and several studies have demonstrated its importance in nociception mediated by DRG neurons. TRPV1 modulators are being investigated as potential analgesic candidates for a variety of pain complications (5). To investigate whether (–)-Cv acts on TRP channels and specifically on TRPV1, DRG neurons were preincubated with TRP and TRPV1 antagonists such as Ru-red or CPZ. In these experiments, we demonstrated that Ca2+ influx in these cells induced by 1 mM (–)-Cv was completely blocked by Ru-red or CPZ (Figs. 2C and 2D) suggesting for the first time TRPV1 channel-activation by (–)-Cv. Regarding the concentration range in which the monoterpenes have shown their effects, our work is consistent with the literature, since previous studies point at mM activity ranges of monoterpenes with similar structure to (–)-carvone. For instance, camphor activated TRPV1 at 10 mM concentration. Other monoterpenes with similar structure to carvone, have also demonstrated effects on TRPV3 only in the mM range (29, 30) and TRPA1 (31).

For further confirmation of the effects of (–)-Cv on TRPV1 channels, we heterologously expressed this channel in HEK293 cells (see methods). In these experiments, capsaicin was used as positive control to confirm the funcionality of hTRPV1 channels in HEK293 cells. (–)-Cv increased cytosolic calcium levels only in cells transfected with hTRPV1 (TRPV1+), while mock transfected cells (TRPV1-) or non-transfected cells (mCh-) remained inertly during the same conditions (Figs. 3A and 3B). Additionally, we demonstrated that (–)-Cv acts in a concentration-dependent manner exhibiting an EC50 of 1.3 ± 0.2 mM (Hill = 2.5) for the increase in [Ca2+]i (Fig. 3B). Subsequently, by using the TRPV1-specific antagonist CPZ, we confirmed that (–)-Cv is a TRPV1-channel activator (Fig. 3C) supporting the conclusions of our experiments with DRG neurons. Recent studies suggested that activation of TRPV1 channels, followed by their rapid desensitization, is a plausible explanation for the analgesic effects of other monoterpenes such as camphor for example (26). The same hypothesis has been used to explain the paradoxical analgesic effect of capsaicin (32). From these findings, we suggest that the antinociceptive effect of (–)-Cv is related to the desensitization of TRPV1 channels. However, additional experiments are needed to confirm this hypothesis.

In conclusion, this study demonstrates for the first time, that (–)-Cv, an antinociceptive monoterpene present in distinct aromatic plants, exhibited low cytotoxicity in bothneural and epithelial cells. We have also shown the ability of (–)-Cv to induce increases of [Ca2+]i in DRG neurons through TRPV1 activation, which was further confirmed in recombinant TRPV1-expressing HEK293 cells.

Acknowledgements

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

We are thankful for Dr. David Julius for kindly providing us the rTRPV1 construct.. J.C.R.G.'s work at the University of Western Ontario was partially supported by internal funds to M.A.M.P. and to V.F.P. H.D.N.S's master thesis and A.N.N.'s Ph.D. thesis research were funded by CNPq and FAPESP, respectively. H.U. and D.A.M.A. are CNPq fellows and J.C.R.G. is CAPES fellow.

LITERATURE CITED

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. INTRODUCTION
  4. MATERIALS AND METHODS
  5. RESULTS
  6. DISCUSSION
  7. Acknowledgements
  8. LITERATURE CITED
  9. Supporting Information

Additional Supporting Information may be found in the online version of this article.

FilenameFormatSizeDescription
CYTO_22236_sm_SuppTable1.doc54KSupporting Information: MIFlowCyt checklist.

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.