Cannabinoids desensitize capsaicin and mustard oil responses in sensory neurons via TRPA1 activation

Abstract

Although the cannabinoid agonists R-(+)-(2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrol[1,2,3-de]-1,4-benzoxazin-6-yl)-(1-naphthalenyl) methanone mesylate [WIN 55,212-2 (WIN)] and (R,S)-3-(2-iodo-5-nitrobenzoyl)-1-(1-methyl-2-piperidinylmethyl)-1H-indole (AM1241) exert peripheral antihyperalgesia in inflammatory pain models, the mechanism for cannabinoid-induced inhibition of nociceptive sensory neurons has not been fully studied. Because TRPV1 and TRPA1 channels play important roles in controlling hyperalgesia in inflammatory pain models, we investigated their modulation by WIN and AM1241. The applications of WIN (>5 microM) and AM1241 (>30 microM) inhibit responses of sensory neurons to capsaicin and mustard oil. To determine potential mechanisms for the inhibition, we evaluated cannabinoid effects on nociceptors. WIN and AM1241 excite sensory neurons in a concentration-dependent manner via a nonselective Ca2+-permeable channel. The expression of TRP channels in CHO cells demonstrates that both WIN and AM1241 activate TRPA1 and, by doing so, attenuate capsaicin and mustard oil responses. Using TRPA1-specific small interfering RNA or TRPA1-deficient mice, we show that the TRPA1 channel is a sole target through which WIN and mustard oil activate sensory neurons. In contrast, AM1241 activation of sensory neurons is mediated by TRPA1 and an unknown channel. The knockdown of TRPA1 activity in neurons completely eliminates the desensitizing effects of WIN and AM1241 on capsaicin-activated currents. Furthermore, the WIN- or AM1241-induced inhibition of capsaicin-evoked nocifensive behavior via peripheral actions is reversed in TRPA1 null-mutant mice. Together, this study demonstrates that certain cannabinoids exert their peripheral antinocifensive actions via activation of the TRPA1 channel on sensory neurons.

Figure 1.

ACEA, WIN, and AM1241 inhibit I_CAP in TG neurons. A, TG neurons were pretreated with ACEA (25 μm), WIN (25 μm), or AM1241 (30 μm), as well as CAP (0.5 μm), and evaluated for desensitizing I_CAP (0.5 μm applied for 40 s). Drugs were applied at an interval of 3 min with CAP pretreatments for 30 s, and ACEA, WIN, and AM1241 pretreatments for 2 min. Numbers of recorded neurons are indicated within bars. B, ACEA, WIN, and AM1241 inhibit I_CAP in a concentration-dependent manner. I_CAP was plotted as a function of cannabinoid concentrations applied to neurons for 2 min. Vehicle (0.1% DMSO)-treated I_CAP is at log [drug] = −10 point. n = 8–13. C, Typical I_WIN, I_AM1241, and I_CAP traces recorded during experiments; results of experiments are summarized on A. Durations of particular drug applications are marked with horizontal bars. Data were generated from TG neurons cultured for 24–48 h. Error bars are SEM. *p < 0.05; **p < 0.01.

Figure 2.

ACEA, WIN, and AM1241 inhibit I_MO in TG neurons. A, Pretreatment of TG neurons with ACEA (25 μm), WIN (25 μm), or AM1241 (30 μm), as well as MO (50 μm), desensitize I_MO (50 μm applied for 2 min). Drugs were applied at an interval of 3 min with MO, ACEA, WIN, and AM1241 pretreatments for 2 min. Numbers of recorded neurons are indicated within bars. B, Typical I_WIN, I_AM1241, and I_MO traces recorded during experiments represented on A. Durations of particular drug applications are marked with horizontal bars. Data were generated from TG neurons cultured for 24–48 h. Error bars are SEM. **p < 0.01.

Figure 3.

WIN and AM1241 activate Ca²⁺-permeable, nonselective currents in subsets of TG neurons. A, Application of indicated concentrations of AM1241 (AM), WIN, MO, and CAP trigger [Ca²⁺]_i accumulation in subset of TG neurons. Numbers of analyzed (numerator) and total recorded (denominator) neurons are indicated within bars. CAP was applied for 1 min, whereas MO, WIN, and AM1241 were applied for 3 min. B, Characteristic Ca²⁺-imaging traces from n = 10 TG neurons that responded to both AM1241 and WIN. Application durations are noted by horizontal bars. C, Typical whole-cell fast and slow inward currents in TG neurons generated by AM1241 (30 μm), WIN (25 μm), and ACEA (25 μm). The currents were acquired from separate neurons bathed in SES (2 mm Ca²⁺) (see Materials and Methods for buffer compositions). D, Concentration–response curves for I_WIN, I_ACEA, and I_AM1241. Data for each point were generated from separate neurons by application of cannabinoids for 2 min. n = 6–18. Whole-cell recordings were performed with SES and SIS. E, I–V relationships (averaged from 4–8 traces) for I_AM1241, I_WIN, and I_ACEA were obtained by recording from neurons maintained in a Mg²⁺-free SES physiological solution with 2 mm Ca²⁺. The electrodes were filled with Cs-containing SIS without Mg²⁺. I–V curves were recorded from separate neurons. Depolarizing voltage ramp protocol is presented in the inset. TG neurons were cultured for 24–48 h. Error bars are SEM. **p < 0.01.

Figure 4.

WIN and AM1241 activate the TRPA1 channel expressed in CHO cells. Representative traces show activation of TRPV1 and TRPA1 by ACEA and WIN or AM1241, respectively. CAP and MO were used as control stimuli for TRPV1 and TRPA1, respectively. Agonists were applied sequentially to CHO cells expressing pcDNA3 (marked as CHO cells), GFP, TRPV1, TRPA1, or the combination of TRPV1 and TRPA1. The horizontal bars mark duration of agonist application. Types and concentrations of agonists are denoted above horizontal bars. Recordings were done in whole-cell patch voltage-clamp configuration with SES and SIS as external and internal solutions.

Figure 5.

WIN and AM1241 desensitize I_CAP and I_MO in TRPV1- and/or TRPA1-expressing CHO cells. A, WIN (25 μm), ACEA (25 μm), and AM1241 (30 μm) as well as CAP (0.3 μm) inhibit I_CAP (0.3 μm) in TRPV1/TRPA1-expressing CHO cells. B, TRPV1-gating ACEA, but not TRPA1-gating WIN and AM1241 attenuates I_CAP (0.3 μm) in TRPV1-expressing CHO cells. C, ACEA and WIN (25 μm each), as well as MO (50 μm), inhibit I_MO (50 μm) in TRPA1–TRPV1-expressing CHO cells, whereas only TRPA1-gating agonists WIN and MO still show inhibition of I_MO in TRPA1-expressing CHO cells. CHO cells were treated 2 min with MO, ACEA, WIN, and AM1241, and 30 s with CAP. I_CAP were recorded by CAP application during 40 s, whereas MO was applied for 2 min to record I_MO. The interval between treatment and I_MO or I_CAP registration was 3 min. The numbers of responsive CHO cells are shown inside the bars. Error bars are SEM. *p < 0.05; **p < 0.01.

Figure 6.

The TRPA1 channel mediates WIN and AM1241 responses in TG neurons. A–D, Knockdown of TRPA1 in TG neurons with rat TRPA1-specific siRNAs ALab, AA, or AB oblates positive control (MO 20 μm) responses (A), as well as AM1241 (30 μm; C) and WIN (25 μm; D), but not CAP (0.3 μm; B) responses measured as internal Ca²⁺ accumulation (Δ[Ca²⁺]_i). Treatments of TG neurons with Drosophila TRPA1-specific (ADr) and scrambled (#1) siRNAs have no effects on MO, CAP, WIN, and AM1241 responses (A–D). All data were compared with mock (None) transfected TG neurons. CAP application was for 1 min, MO for 2 min, and WIN and AM1241 for 4 min each. Types of siRNA transfection are indicated on x-axis. Data were collected from all neurons (i.e., both transfected and untransfected; marked “All”) or from only transfected neurons (marked “transf”) that were identified by ALab conjugated with Alexa Fluor 488. Together, one round of siRNA transfection was performed and TG neurons were cultured for 3 d. The numbers of responsive cells over the total number of studied cells are indicated within the columns. E, F, MO (25 μm; E)-, WIN (25 μm; F)-, and CAP (50 nm; E)-activated accumulation of [Ca²⁺]_i in TG neurons from WT and TRPA1 KO mice. Indicated drugs were separately delivered to at least 60 neurons. The numbers of responsive neurons are indicated within bars. Mouse lines are also noted. G, This panel shows CAP and WIN treatments of two groups of TG neurons (BrF) transfected with Alexa Fluor 488-labeled ALab (ALab-siRNA). The yellow arrows show ALab containing neurons that are responsive to CAP. The red arrow indicates a neuron that is untransfected and activated by WIN, whereas transfected neurons are unresponsive to WIN application. Error bars are SEM. **p < 0.01; ***p < 0.001.

Figure 7.

The TRPA1 channel mediates WIN- and AM1241-induced inhibitions of I_CAP in TG neurons. A, Oblation of TRPA1 function with ALab-siRNA, but not mock and ADr-siRNA, transfections reversed inhibitory effects of WIN (25 μm) and AM1241 (30 μm), but not CAP (0.3 μm), on I_CAP (0.3 μm) in TG neurons. TG neurons were treated 2 min with WIN and AM1241, and 30 s with CAP. I_CAP were generated by 40 s application of CAP. Interval between treatments and I_CAP recording was 3 min. The numbers of analyzed TG neurons are shown inside the bars. One round of siRNA transfection was used. TG neurons were cultured for 3 d. B–D, I_WIN, I_AM1241, and I_CAP traces recorded during experiments on mock (C), ADr (D), and ALab (E) siRNA-transfected TG neurons. Durations of particular drug applications are marked with horizontal bars.

Figure 8.

Effect of WIN 55,212 and AM1241 on CAP-induced nocifensive behavior in WT and TRPA1 KO mice. All behavioral tests were performed on male mice. Behavior was measured by observers blinded to treatment allocation, and outcome of nocifensive behavior was measured as a grooming or flinching. Numbers of animals (n) used for each bar of the bar graphs are indicated. A, B, Evaluation of the effect of injection of vehicle or 5 μg (A) or 2.5 μg (B) in the contralateral paw (Contra) to desensitize CAP-induced nocifensive behavior in ipsilateral (Ipsi) hindpaw of WT mice. n = 6–8 (for A and B). C, Evaluation of the effect of preinjection of vehicle or AM1241 (40 μg) in the contralateral paw (Contra) to desensitize CAP-induced nocifensive behavior in ipsilateral (Ipsi) hindpaw of WT mice. n = 8–12. D, Role of the TRPA1 channel in central mechanisms of WIN-induced antinociception. Evaluation of the effect of injection of vehicle and CAP (0.5 μg) or WIN (5 μg) and CAP (0.5 μg) in the ipsilateral paw (Ipsi) to desensitize CAP-induced nocifensive behavior in WT and TRPA1 KO mice. n = 6. E, Role of the TRPA1 channel in peripheral mechanisms of WIN-induced antinociception Evaluation of the effect of injection of vehicle and CAP (0.5 μg) or WIN (2.5 μg) and CAP (0.5 μg) in the ipsilateral paw (Ipsi) to desensitize CAP-induced nocifensive behavior in WT and TRPA1 KO mice. n = 8–10. F, Role of the TRPA1 channel in peripheral mechanisms of AM1241 (AM)-induced antinociception. Evaluation of the effect of preinjection of vehicle or AM1241 (40 μg) in the ipsilateral paw (Ipsi) to desensitize CAP-induced nocifensive behavior in WT as well as TRPA1KO mice. n = 7–8.