Unusual Pungency from Extra-Virgin Olive Oil Is Attributable to Restricted Spatial Expression of the Receptor of Oleocanthal

Oleocanthal exclusively activates TRPA1 in HEK 293 cells expressing human TRP channels. a, OC activates hTRPA1-expressing HEK 293 cells. Representative whole-cell current trace activated by OC (5 μm) and AITC (100 μm) in the presence of extracellular Ca²⁺. V_h = −60 mV. The horizontal bars indicate the duration of compound application. The current–voltage (I–V) curves were expanded from the current response indicated (a) in the traces. b, HEK 293 cells expressing hTRPV1, hTRPV2, and hTRPV4 are not excited by OC. Fifty micromolar OC does not evoke calcium influx into hTRPV1 (n = 100), 2 (n = 77), and 4 (n = 39) as assessed by fura-2 ratiometric imaging (F₃₄₀/F₃₈₀). The channel-specific ligands, capsaicin, lysophosphatidylcholine (LPC), and GSK1016790A, were applied as controls for activity in HEK cells expressing TRPV1, TRPV2, and TRPV4, respectively. The curves in b were generated using mean values of peak amplitude. Error bars indicate SEM.

Ibuprofen excites trigeminal neurons through TRPA1 channel activation. a, IBU (10 mm) evokes calcium influx into cultured rat trigeminal neurons as assessed by fura-2 ratiometric imaging (F₃₄₀/F₃₈₀). In a, the representative trace of one neuron shows that cells sensitive to IBU are also activated by OC (5 μm) and AITC (50 μm). The horizontal bars indicate the duration of compound application. The 40 mm KCl solutions were applied as a control for neuronal activity. b, Dose–response curves for IBU-evoked responses in rat trigeminal neurons. The high IBU concentrations (>30 mm) required to complete the dose–response curve did not permit to reach saturation, as the increase of the ionic strength and osmolarity of the medium affected cell viability. The curve was generated by averaging the peak amplitude of the responses (fura-2 ratio) of all the cells (n > 60 per data point). IBU chemical structure is indicated on the graph. c, Sensitivity to OC and IBU is correlated in sensory neurons. The scatterplot displays relationship between 10 mm IBU and 5 μm OC-evoked responses in trigeminal neurons. Each dot represents intracellular calcium increase in a single cell. Small calcium influxes induced by IBU (gray circle) were observed in all the examined cells (n = 56) insensitive to OC. d, Representative whole-cell current traces generated in DRG neurons, in the presence of extracellular Ca²⁺. V_h = −60 mV. Ten millimolar IBU elicited current responses in sensory neurons expressing TRPA1 (AITC sensitive) (top) but not in sensory neurons lacking the TRPA1 channel (AITC insensitive) (bottom). e, TRPA1 is required for trigeminal neuron response to IBU. HC-030031 (HC) (20 μm) decreased 10 mm IBU-evoked responses down to the calcium influx measured in all the OC-insensitive cells (examined cells, n = 55) (left) and TRPA1-deficient trigeminal neurons display only small nonspecific IBU-evoked calcium influx (n = 128 from 5 mice) (right) similar to those observed in the OC-insensitive cells (left). *p < 0.05 versus control [vehicle for left panel; wild-type (WT) for right panel], °p < 0.05 versus OC-sensitive cells in vehicle solution, t test. Error bars indicate SEM.

Ibuprofen exclusively activates TRPA1 in HEK 293 cells expressing human TRP channels. a, IBU activates hTRPA1-expressing HEK 293 cells. Representative whole-cell current trace activated by IBU (10 mm) and AITC (100 μm) in the presence of extracellular Ca²⁺. V_h = −60 mV. b, HEK 293 cells expressing hTRPV1, hTRPV2, and hTRPV4 are not excited by IBU. Ten millimolar IBU does not evoke calcium influx into hTRPV1 (n = 95), 2 (n = 82), and 4 (n = 38) as assessed by fura-2 ratiometric imaging (F₃₄₀/F₃₈₀). The channel-specific ligands, capsaicin, lysophosphatidylcholine (LPC), and GSK1016790A, were applied as controls for activity in HEK cells expressing TRPV1, TRPV2, and TRPV4, respectively. The curves in b were generated using mean values of peak amplitude. Error bars indicate SEM.

The human throat and nose are more sensitive than the anterior tongue to OC. a, Irritation perceived on the anterior tongue after its immersion in either 25 ml of an extra-virgin olive oil (EVOO) (OC source) or a horseradish solution (AITC source), and irritation perceived in the throat after swallowing 3.5 ml of the same solutions (subjects = 12). *p < 0.05 versus olive oil, t test. b, Irritation perceived either on the anterior tongue after its immersion in 25 ml of solution, or in the throat after swallowing 3.5 ml of the same solutions (subjects = 13). The four solutions tested were as follows: water plus 0.25% ethanol, water plus 0.25% ethanol plus 660 μm OC, corn oil, and corn oil plus 660 μm OC. The addition of 0.25% ethanol was required to dissolve OC in water. *p < 0.05 versus control, °p < 0.05 versus water, t test. c, Irritation perceived in the nares after application of OC, IBU, trans-cinnamaldehyde, and sucrose. Each stimulus dissolved in saline solution plus 0.1% ethanol was applied in the subject nares with a nasal sprayer device (volume, 0.3 ml). The four concentrations used for each stimulus were tested in independent sessions and in duplicate (subjects = 11). Mean intensity rating of saline solution plus 0.1% ethanol alone was similar to the sucrose solution (=5.14). All perceived irritation ratings were performed on a gLMS scale. Values represent mean of gLMS ratings ± SEM. d, OC activation curves in cultured rat trigeminal neurons (green) and current density curves of heterologously expressed hTRPA1 channels in HEK 293 cells (red) compared with the perceived intensity curve of the compound in the nares (black). Error bars indicate SEM.

TRPA1 is expressed in a subset of nerve fibers in pharyngeal epithelium, but not in nerve fibers of taste buds within fungiform papillae. Histochemistry and immunohistochemistry of human fungiform papilla containing a taste bud (a–f) and upper pharyngeal epithelium (g–l) are shown. a and g show the bright-field view of the tissue samples, b and h show the DNA stain DAPI (blue) to reveal the nuclei, c and i are the overlay of bright-field and DAPI to reveal tissue structure, d and j show the neuronal marker protein PGP9.5 (green), e and k show the TRPA1 receptor (red), and f and l show the overlay of PGP9.5 and TRPA1; yellow depicts the superposition of TRPA1 reactivity (red) and neuronal PGP9.5 reactivity (green). Eight to 10 slides were examined for the pharyngeal tissue (from 1 subject) and the fungiform papilla tissue (from 4 subjects). Scale bars: a–f, 20 μm; g–l, 40 μm.