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Abstract
Eugenol is widely used in dentistry to relieve pain. We have recently demonstrated voltage-gated Na+ and Ca2+ channels as molecular targets for its analgesic effects, and hypothesized that eugenol acts on P2X3, another pain receptor expressed in trigeminal ganglion (TG), and tested the effects of eugenol by whole-cell patch clamp and Ca2+ imaging techniques. In the present study, we investigated whether eugenol would modulate 5'-triphosphate (ATP)-induced currents in rat TG neurons and P2×3-expressing human embryonic kidney (HEK) 293 cells. ATP-induced currents in TG neurons exhibited electrophysiological properties similar to those in HEK293 cells, and both ATP- and α, β-meATP-induced currents in TG neurons were effectively blocked by TNP-ATP, suggesting that P2×3 mediates the majority of ATP-induced currents in TG neurons. Eugenol inhibited ATP-induced currents in both capsaicin-sensitive and capsaicin-insensitive TG neurons with similar extent, and most ATP-responsive neurons were IB4-positive. Eugenol inhibited not only Ca2+ transients evoked by α, β-meATP, the selective P2×3 agonist, in capsaicin-insensitive TG neurons, but also ATP-induced currents in P2×3-expressing HEK293 cells without co-expression of transient receptor potential vanilloid 1 (TRPV1). We suggest, therefore, that eugenol inhibits P2×3 currents in a TRPV1-independent manner, which contributes to its analgesic effect.
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Fig. 1.
Comparison of current profiles of 10 μM ATP-induced currents in P2X3-expressing HEK293 cells and rat TG neurons. (A) Representative current traces activated by 10 μM ATP in P2X3-expressing HEK293 cells (left, n=60) and rat TG neurons (right, n=56). (B) Records of 10 μM ATP-induced currents activated by twice applications of 10 μM ATP with the interval of 90s in a P2X3-expressing HEK293 cell (a) and rat TG neuron (b). The normalized amplitude ratio (C2/C1) in P2X3-expressing HEK293 cells and that in TG neurons (c). (C) Inhibition of 10 μM ATP (a)- and 100 μM α, β-meATP (b)-induced currents by 1 μM TNP-ATP in small TG neurons. The summary of the inhibition of TNP-ATP on ATP (n=5)- and α, β-meATP (n=5)-induced currents (mean±SEM, p<0.05) (c). Black points indicate the time point of ATP or α,β-meATP application.
Fig. 2.
Eugenol inhibited ATP-induced P2X currents in both capsaicin-insensitive and capsaicin-sensitive rat TG neurons. (A) Representative current traces of 10 μM ATP-induced P2X currents under control (Aa and Ab, left), and eugenol (1 mM) (Aa and Ab, right) in both capsaicin-insensitive (Aa) and capsaicin-sensitive (Ab) rat TG neurons. (B) The summary of the inhibition of ATP-induced P2X currents in both capsaicin-insensitive (Ba) and capsaicin-sensitive (Bb) rat TG neurons. The amplitude of second (C2) and third currents (C3) was normalized compared to the first one (C1). Eugenol-induced inhibition in capsaicin-insensitive neurons (n=15) was similar to that obtained in capsaicin-sensitive neurons (n=10) (mean±SEM, p>0.05). Black points indicate the time point of ATP application.
Fig. 3.
Eugenol inhibited α,β-meATP-induced Ca transients in rat TG neurons. (A) α,β-meATP (100 βM) induced Ca2+ transients in small-sized TG neurons. Eugenol (1 mM) abolished α,β-meATP-induced Ca2+ transients in capsaicin (0.5 μM)-insensitive TG neurons. (B) The summary of the inhibition of α,β-meATP-induced Ca2+ transients by eugenol in capsaicin-insensitive TG neurons (mean±SEM, n=9, p<0.05). The amplitude changes of Ca2+ transients induced by second (combined application with eugenol) and third (3rd) α,β-meATP applications were normalized compared to the first one (1st).
Fig. 4.
Eugenol inhibited ATP-induced P2X3 currents in both P2X3-expressing and P2X3/TRPV1-coexpressing HEK293 cells. (A) Representative current traces of 10 μM ATP-induced P2X3 currents under control (Aa and Ab, left), and eugenol (1 mM) (Aa and Ab, right) in both P2X3-expressing (Aa) and P2X3/TRPV1-coexpressing (Ab) HEK293 cells. (B) The summary of the inhibition of ATP-induced P2X3 currents in both P2X3-expressing (Aa) and P2X3/TRPV1-coexpressing (Ab) HEK293 cells. The amplitude of second (C2) and third currents (C3) was normalized compared to the first one (C1). Eugenol-induced inhibition in P2X3-expressing cells (n=13) was similar to that obtained in P2X3/TRPV1-coexpressing cells (n=12) (mean±SEM, p>0.05). Black points indicate the time point of ATP application.
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