PIEZO1 promotes ATP release from periodontal ligament cells following compression force.

OBJECTIVES: Orthodontic mechanical force on the periodontal ligament induces extracellular adenosine triphosphate (ATP) release. However, mechanosensitive molecules have not been confirmed functionally in periodontal ligament cells. In the present study, we examined the roles of mechanosensitive PIEZO channels in the mechanically stimulated release of ATP in human periodontal ligament fibroblasts (HPdLFs). MATERIALS AND METHODS: To examine PIEZO expression in HPdLFs, we performed reverse transcription-quantitative polymerase chain reaction, fluorescent immunostaining, and Ca2+ imaging. ATP concentrations were measured in culture medium after applications of the PIEZO1 agonist Yoda1 and compression force in a newly developed in vitro weight-loaded cell model (IVWLC) using balance weights and a 48-well plate. The mechanosensitive channel inhibitor GsMTx4 and the ATP-releasing route inhibitors clodronic acid, meclofenamic acid, and probenecid were used. To suppress PIEZO1 expression, short interference RNA (siRNA) treatment of the PIEZO1 gene was performed. RESULTS: PIEZO1 mRNA was expressed more abundantly than PIEZO2 mRNA in HPdLFs. HPdLF cell bodies were immunoreactive to anti-PIEZO1 antibody. Yoda1 increased intracellular Ca2+ and extracellular ATP concentrations in a dose-dependent manner. ATP release was inhibited by GsMTx4 and inhibitors of ATP release routes. In the IVWLC, HPdLFs released ATP in response to compression force but not in response to hypoxic stimulation that was simultaneously applied to cells. Mechanically stimulated ATP release was inhibited by GsMTx4, inhibitors of ATP-releasing routes and siRNA treatment of PIEZO1. CONCLUSIONS: PIEZO1 on the cell membranes of HPdLFs is activated by compression force and then induces ATP release via intracellular Ca2+-dependent exocytosis and ATP-permeable channels.

Novel approaches to the study of viscosity discrimination in rodents.

Texture has enormous effects on food preferences. The materials used to study texture discrimination also have tastes that experimental animal can detect; therefore, such studies must be designed to exclude taste differences. In this study, to minimize the effects of material tastes, we utilized high- and low-viscosity forms of carboxymethyl cellulose (CMC-H and CMC-L, respectively) at the same concentrations (0.1-3%) for viscosity discrimination tests in rats. In two-bottle preference tests of water and CMC, rats avoided CMC-H solutions above 1% (63 mPa·s) but did not avoid less viscous CMC-L solutions with equivalent taste magnitudes, suggesting that rats spontaneously avoided high viscosity. To evaluate low-viscosity discrimination, we performed conditioned aversion tests to 0.1% CMC, which initially showed a comparable preference ratio to water in the two-bottle preference tests. Conditioning with 0.1% CMC-L (1.5 mPa·s) did not induce aversion to 0.1% CMC-L or CMC-H. However, rats acquired a conditioned aversion to 0.1% CMC-H (3.6 mPa·s) even after latent inhibition to CMC taste by pre-exposure to 0.1% CMC-L. These results suggest that rats can discriminate considerably low viscosity independent of CMC taste. This novel approach for viscosity discrimination can be used to investigate the mechanisms of texture perception in mammals.