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.