6‐Methylsulfinylhexyl isothiocyanate activates carbonic anhydrase‐dependent HCO ₃ ⁻ /H ⁺ /Na ⁺ /Ca ²⁺ transport via SLC4As–NHE–NCX–PMCA axis in odontoblasts

Ca²⁺-permeable transient receptor potential channels, which play an important role in the developmental/reactionary dentinogenesis by odontoblasts, are sensitive to wasabi sulfinyls. We investigated the effects of the wasabi sulfinyls 6-methylsulfinylhexyl isothiocyanate (6-MSITC) and its eight derivatives on ion transport mechanisms which promote mineralisation by odontoblasts. 6-MSITC significantly increased the mineralisation efficiency in cultured odontoblasts, and we also observed a significant increase in medium pH. Inhibitors of carbonic anhydrase (CA) and plasma membrane Ca²⁺-ATPase (PMCA) significantly reduced 6-MSITC-induced mineralisation. Odontoblasts expressed the HCO₃ ^--transporting solute carrier family 4 (SLC4A) members SLC4A1, SLC4A2, SLC4A3, SLC4A4, SLC4A8 and SLC4A9, as well as CA I and CA II. 6-MSITC enhanced reactionary dentinogenesis beneath the cavities prepared on rat mandibular first molars. We recorded 6-MSITC-induced outward currents, which were suppressed by inhibitors of CA, Na⁺-H⁺ exchanger (NHE), and Na⁺-Ca² ⁺ exchanger (NCX). These results indicated that 6-MSITC has a strong ability to form reactionary dentin by activating or upregulating intracellular CA and electrically neutral HCO₃ ^-/H⁺ transport via SLC4As/NHE. Exchanging Na⁺ with H⁺ using NHE resulted in the reversal of the transmembrane Na⁺ gradient. This activated the Ca²⁺ influx mode of NCX, and the subsequent accumulation of intracellular Ca²⁺ was then extruded by PMCA activity to produce reactionary dentin. Thus, 6-MSITC activates CA-mediated SLC4As-NHE-NCX-PMCA coupling and is useful in dentin regenerative medicine. KEY POINTS: Ca²⁺ signalling in odontoblasts plays an important role not only in developmental/reactionary dentinogenesis, but also in the generation of dentinal (tooth) pain. The wasabi sulfinyls including 6-methylsulfinylhexyl isothiocyanate (6-MSITC) promote mineralisation by odontoblasts and significantly increase pH in medium with cultured odontoblasts. We showed that 6-MSITC has a strong ability to form reactionary dentin through the upregulation and activation of intracellular carbonic anhydrase (CA) and electrically neutral HCO₃ ^-/H⁺ transport by members of the HCO₃ ^--transporting solute carrier family 4 (SLC4As) and Na⁺-H⁺ exchanger (NHE). Na⁺ accumulation by NHE activity resulted in the reversal of the transmembrane Na⁺ gradient. This activated the Ca²⁺ influx mode of the Na⁺-Ca² ⁺ exchanger (NCX), resulting in the accumulation of intracellular Ca²⁺. It was then extruded using plasma membrane Ca²⁺-ATPase (PMCA) to produce reactionary dentin. 6-MSITC activates CA-mediated SLC4As-NHE-NCX-PMCA coupling and is useful in dentin regenerative medicine.