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. 2019 Apr;95(4):433-441.
doi: 10.1124/mol.118.114561. Epub 2019 Jan 24.

trans-Anethole of Fennel Oil Is a Selective and Nonelectrophilic Agonist of the TRPA1 Ion Channel

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Free PMC article

trans-Anethole of Fennel Oil Is a Selective and Nonelectrophilic Agonist of the TRPA1 Ion Channel

Tosifa Memon et al. Mol Pharmacol. .
Free PMC article

Abstract

Transient receptor potential (TRP) cation channels are molecular targets of various natural products. TRPA1, a member of TRP channel family, is specifically activated by natural products such as allyl isothiocyanate (mustard oil), cinnamaldehyde (cinnamon), and allicin (garlic). In this study, we demonstrated that TRPA1 is also a target of trans-anethole in fennel oil (FO) and fennel seed extract. Similar to FO, trans-anethole selectively elicited calcium influx in TRPA1-expressing mouse sensory neurons of the dorsal root and trigeminal ganglia. These FO- and anethole-induced calcium responses were blocked by a selective TRPA1 channel antagonist, HC-030031. Moreover, both FO and trans-anethole induced calcium influx and transmembrane currents in HEK293 cells stably overexpressing human TRPA1 channels, but not in regular HEK293 cells. Mutation of the amino acids S873 and T874 binding site of human TRPA1 significantly attenuated channel activation by trans-anethole, whereas pretreating with glutathione, a nucleophile, did not. Conversely, activation of TRPA1 by the electrophile allyl isothiocyanate was abolished by glutathione, but was ostensibly unaffected by mutation of the ST binding site. Finally, it was found that trans-anethole was capable of desensitizing TRPA1, and unlike allyl isothiocyanate, it failed to induce nocifensive behaviors in mice. We conclude that trans-anethole is a selective, nonelectrophilic, and seemingly less-irritating agonist of TRPA1.

Figures

Fig. 1.
Fig. 1.
FO activates mouse and human TRPA1. (A) Representative calcium imaging traces of neuronal phenotypes observed among 1328 neurons analyzed from DRG cultures of three adult wild-type C57BL/6 mice. The y-axis is the 340/380-nm ratio, a measure of intracellular calcium, and x-axis is the duration of the experiment in minutes. Arrows on x-axis indicate 15-second treatments with each stimulus. AITC, 100 μM allyl isothiocyanate; Cap, 300 nM capsaicin; FO, 1:10,000-fold diluted fennel oil; [K+]o, 30 mM extracellular K+; Me, 400 μM menthol. (B) Response frequency, measured as percentage of cells activated, of FO (1:10,000-fold diluted) responses in presence and absence of 30 μM HC-030031 (HC), a selective TRPA1 antagonist. (C) Representative calcium imaging traces for FO dilutions (x-axis) in normal HEK293 cells (top) and HEK293 cells stably overexpressing human TRPA1 (bottom). (D) Response frequency of FO dilutions in normal (n = 676) and human TRPA1-expressing HEK293 cells (n = 1695).
Fig. 2.
Fig. 2.
Anethole elicits calcium response in TRPA1-expressing mouse DRG neurons. (A) Representative calcium imaging traces of neuronal phenotypes observed among 1375 neurons analyzed from DRG cultures of three adult wild-type C57BL/6 mice. The y-axis is the 340/380-nm ratio, a measure of intracellular calcium, and x-axis is the duration of the experiment in minutes. Arrows on x-axis indicate 15-second treatment with given stimuli. AITC, 100 μM allyl isothiocyanate; anethole, 130 μM trans-anethole; Cap, 300 nM capsaicin; FO, 1:10,000-fold diluted fennel oil; [K+]o, 30 mM extracellular K+; Me, 400 μM menthol. (B) Response frequency (n = 964) of 130 μM anethole in presence and absence of 30 μM HC-030031 (HC), a selective TRPA1 antagonist.
Fig. 3.
Fig. 3.
Distribution of peptidergic and nonpeptidergic neuronal subtype neurons activated by anethole, AITC, and capsaicin. (Top) Representative images show CGRP+, CGRP+ IB4+ (overlaid images of CGRP+ and IB4+), and IB4+ neurons (from left to right). White arrows point at examples of CGRP+ and IB4+ neurons. (Middle) Representative traces for each neuronal subtype. (Bottom) Pie charts show average distribution of neuronal cell types, i.e., CGRP+ (green), CGRP+ IB4+ (yellow), IB4+ (red), and CGRP− IB4− or none (gray) activated by anethole (n = 504), AITC (n = 731), and capsaicin (n = 945) from DRG cultures of three adult CD-1 mice.
Fig. 4.
Fig. 4.
Anethole elicits calcium response in hTRPA1-HEK293 cells. (A) Representative calcium imaging traces for anethole dose response over time (x-axis) in normal HEK293 cells (top) and HEK293 cells stably overexpressing human TRPA1 (bottom). The y-axis is the 340/380-nm ratio, a measure of intracellular calcium, and x-axis is the duration of the experiment in minutes. Arrows on x-axis indicate 15-second treatment with given stimuli. (B) Response frequency, measured as percentage of cells activated, of anethole dose response in normal human TRPA1–expressing HEK293 cells (n = 1195).
Fig. 5.
Fig. 5.
Anethole induces transmembrane currents in hTRPA1-HEK293 cells. (A) A representative time course of current measured at the indicated holding potentials (Vh) from HEK293 cells stably overexpressing human TRPA1. Anethole was applied at a concentration of 66 μM. (B) I-V relationship of transmembrane current measured before (control) and during administration of anethole (maximum current, Imax). (C) Dose-response relationship. Circles represent mean values of Imax recorded at Vh = 100 mV. Error bars indicate S.E.M. The solid line is the best fit with logistic function. EC50 = 80.8 μM. Shown in parentheses are the number of cells.
Fig. 6.
Fig. 6.
Anethole is a nonelectrophilic agonist of TRPA1. HEK293-GCaMP6–overexpressing cells transiently transfected with wild-type hTRPA1 (A1 WT) and hTRPA1-ST (A1 ST) mutant plasmid robustly respond to AITC and anethole. Pretreatment of AITC with 20 mM GSH for 10 minutes abolished AITC responses in both A1 WT and A1 ST cells (***P < 0.001), while the response to anethole when pretreated with GSH was not affected. In cells transfected with (A1 ST) plasmid, the response to AITC was not significantly affected, while response to anethole was significantly diminished (*P < 0.05; **P < 0.01). Data are the average response ± S.E.M. of treatments (x-axis) normalized to the ionomycin response (y-axis) for the respective plasmid (n = 3). ns, not significant.
Fig. 7.
Fig. 7.
Desensitization of anethole-elicited calcium responses. Representative calcium imaging trace illustrating desensitization of calcium responses, indicated by the declining magnitude of responses upon the second and third applications of anethole, each elicited by a 15-second application of 1.3 mM anethole on mouse DRG neurons (A) and HEK293 cells overexpressing human TRPA1 (C). (B and D) The average percentage response magnitude of second and third responses in mouse DRG neurons (n = 372) and hTRPA1-HEK293 cells (n = 833) as a percentage of the first response to 1.3 mM anethole, indicating significant desensitization of anethole-induced calcium responses over time (***P < 0.001). The second and third responses to anethole (x-axis) were normalized to the first response of each neuron or HEK293 cell. Average percentage response ± S.E.M. values are presented.
Fig. 8.
Fig. 8.
Repetitive stimulation of TRPA1 by anethole reveals a desensitization of the TRPA1-mediated whole-cell current to the agonist. (A) A representative trace of the whole-cell current recorded from an hTRPA1-HEK293 cell with the plasma membrane potential clamped at −70 mV. Application of anethole (100 µM) is indicated by bars above the trace. (B) Statistical analysis of results illustrated in (A). Magnitude of the second and third responses was normalized to that of the first response. Shown are the mean ± S.E.M. values. *P < 0.05; N.S., not significant (P > 0.05); paired-sample t test, n = 4 cells. Anethole was applied for 1.5 minutes.
Fig. 9.
Fig. 9.
Nocifensive response induced by subcutaneous injection of anethole. Paw-licking behavior of adult wild-type C57BL/6 mice was recorded as response duration in seconds ±S.E.M. (y-axis) observed over 15-minute duration after subcutaneous injection of given treatments in left hind paw. (A) Response duration of paw-licking behavior every 5 minutes. (B) Sum of paw-licking response duration for given treatments (x-axis) during 15-minute observation period. DRG Obs. was used as vehicle. Significant difference (*P < 0.05) in paw-licking response duration was observed between vehicle-treated (n = 6) and 10 mM AITC– treated (n = 7) mice, while the difference between vehicle- and 6.6 mM anethole– treated (n = 6) or 66 mM anethole– treated (n = 5) mice was not significant.

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