The peripheral sensory nerve must be maintained to perceive environmental changes. Daily physiological mechanical stimulations, like gravity, floor reaction force and occlusal force, influence the nerve homeostasis directly or indirectly. Although the direct axonal membrane stretch enhances axon outgrowth via mechanosensitive channel activation, the indirect mechanisms remain to be elucidated. In this study, we identified the indirect pathways where Wnt5a was a molecular cue released by mechanically stimulated rat periodontal ligament (rPDL) cells. qRT-PCR and ELISA showed that mechanically stimulated rPDL cells enhanced Wnt5a expression level and Wnt5a protein in a Ca²⁺-dependent manner. The inhibitors of PI3K (LY294002) and MEK1/2 (U0126) suppressed the Akt/PKB and ERK1/2 phosphorylation, respectively, in western blotting analysis and consequently abolished the increase in Wnt5a expression. Similarly, PF573228, a focal adhesion kinase inhibitor, attenuated Akt- and ERK1/2-phosphorylation and Wnt5a expression. Importantly, the culture medium of stretched PDL cells enhanced neurite elongation, sprouting, and branching in trigeminal ganglion neurons which project to PDL. Moreover, treatment with an anti-Wnt5a antibody (to neutralize Wnt5a activity), AP7677a (anti-Ryk antibody, to block Ryk receptor activity), or strictinin (Ror1 inhibitor) suppressed the morphological changes. These findings reveal the indirect mechanisms that Wnt5a, released from the connective tissues in response to mechanical stimulation, enhances the outgrowth of the peripheral nerves. Our study suggests that the peripheral connective tissues regulate peripheral nerve homeostasis and that Wnt5a signaling could be targeted for the treatment of peripheral nerve disorders.

The transient receptor potential vanilloid 1 (TRPV1) channel is a polymodal receptor in sensory nerves and involved in pain sensation. TRPV1 has at least three distinct activation modes that are selectively induced by different stimuli capsaicin, noxious heat, and protons. Although many mode-selective TRPV1 antagonists have been developed for their anticipated analgesic effects, there have been few successful reports because of adverse effects due to burn injuries and hyperthermia. Eugenol is a vanilloid that has been used as an analgesic in the dental treatment, and its TRPV1 activation ability has been reported. However, our knowledge about the underlying mechanisms of the antagonistic effects of eugenol on TRPV1 activation induced by three different modes is limited. Here, we show that eugenol dose-dependently inhibited the capsaicin-activated inward currents of mouse TRPV1 expressed in human embryonic kidney 293 (HEK293) cells. Under low pH conditions, low concentrations of eugenol only enhanced the proton-induced TRPV1 currents, whereas high eugenol concentrations initially potentiated but then immediately abrogated TRPV1 currents. Finally, eugenol had no modulatory effects on heat-activated TRPV1 in electrophysiological and Fura-2-based Ca2+ imaging experiments. Our results demonstrate that eugenol is a mode-selective antagonist of TRPV1 and can be evaluated as a lead compound of analgesics targeting TRPV1 without serious side effects.

The transient receptor potential vanilloid type 1 (TRPV1) channel is a non-selective cation channel expressed with transient receptor potential ankyrin type 1 (TRPA1) in small and medial size neurons of the dorsal root ganglions and trigeminal ganglions. TRPV1 is activated by capsaicin, thermal stimuli higher than 43°C, mechanical stress, and protons (H+). Although the TRPV1 channel does not have positively charged residues at regular intervals on its transmembrane segments, alterations in membrane potential also affect the state of TRPV1 channel. In the presence of capsaicin, voltage-dependent probability of opening of the TRPV1 channel and its kinetics have been examined, but the characteristics in the low pH remain unclear. To understand the voltage-dependency of the TRPV1 channel activation, we recorded capsaicin- and proton-induced mouse TRPV1 channel currents in a heterologous expression system. Outward current evoked by depolarizing square pulses in the presence of capsaicin or protons was fitted to a two-exponential function with a time-independent component. The voltage-dependent changes in amplitude of the three components displayed shallow curves and the changes in their ratio to the total current display similar tendencies in the presence of capsaicin and under the low pH. However, the fast and slow time constants in the presence of capsaicin were respectively 5- and 8-fold lower than those obtained under low pH conditions. These results suggest that the TRPV1 channel slowly drives the feed-forward cycle of pain sensation, and capsaicin and protons differently modulate the voltage-dependent TRPV1 channel gating.