High pH–Sensitive TRPA1 Activation in Odontoblasts Regulates Mineralization

Abstract

Calcium hydroxide and mineral trioxide aggregate are widely used for indirect and direct pulp capping and root canal filling. Their dissociation into Ca2+ and OH- in dental pulp creates an alkaline environment, which activates reparative/reactionary dentinogenesis. However, the mechanisms by which odontoblasts detect the pH of the extracellular environment remain unclear. We examined the alkali-sensitive intracellular Ca2+ signaling pathway in rat odontoblasts. In the presence or absence of extracellular Ca2+, application of alkaline solution increased intracellular Ca2+ concentration, or [Ca2+]i. Alkaline solution–induced [Ca2+]i increases depended on extracellular pH (8.5 to 10.5) in both the absence and the presence of extracellular Ca2+. The amplitude was smaller in the absence than in the presence of extracellular Ca2+. Each increase in [Ca2+]i, activated by pH 7.5, 8.5, or 9.5, depended on extracellular Ca2+ concentration; the equilibrium binding constant for extracellular Ca2+ concentration decreased as extracellular pH increased (1.04 mM at pH 7.5 to 0.11 mM at pH 9.5). Repeated applications of alkaline solution did not have a desensitizing effect on alkali-induced [Ca2+]i increases and inward currents. In the presence of extracellular Ca2+, alkaline solution–induced [Ca2+]i increases were suppressed by application of an antagonist of transient receptor potential ankyrin subfamily member 1 (TRPA1) channels. Ca2+ exclusion efficiency during alkaline solution–induced [Ca2+]i increases was reduced by a Na+-Ca2+ exchanger antagonist. Alizarin red and von Kossa staining revealed increased mineralization levels under repeated high pH stimulation, whereas the TRPA1 antagonist strongly reduced this effect. These findings indicate that alkaline stimuli—such as the alkaline environment inside dental pulp treated with calcium hydroxide or mineral trioxide aggregate—activate Ca2+ mobilization via Ca2+ influx mediated by TRPA1 channels and intracellular Ca2+ release in odontoblasts. High pH–sensing mechanisms in odontoblasts are important for activating dentinogenesis induced by an alkaline environment.