Intracellular cAMP signaling-induced Ca²⁺ influx mediated by calcium homeostasis modulator 1 (CALHM1) in human...

In odontoblasts, intracellular Ca²⁺ signaling plays key roles in reactionary dentin formation and generation of dentinal pain. Odontoblasts also express several G_s protein-coupled receptors that promote production of cyclic AMP (cAMP). However, the crosstalk between intracellular cAMP and Ca²⁺ signaling, as well as the role of cAMP in the cellular functions of odontoblasts, remains unclear. In this study, we measured intracellular cAMP levels and intracellular free Ca²⁺ concentration ([Ca²⁺]_i). We also investigated the effect of intracellular cAMP on mineralization by the odontoblasts. In the presence of extracellular Ca²⁺, the application of forskolin (adenylyl cyclase activator) or isoproterenol (G_s protein-coupled beta-2 adrenergic receptor agonist) increased intracellular cAMP levels and [Ca²⁺]_i in odontoblasts. The [Ca²⁺]_i increases could not be observed by removing extracellular Ca²⁺, indicating that cAMP is capable to activate Ca²⁺ entry. Forskolin-induced [Ca²⁺]_i increase was inhibited by a protein kinase A inhibitor in odontoblasts. The [Ca²⁺]_i increase was sensitive to Gd³⁺, 2APB, or Zn²⁺ but not verapamil, ML218, or La³⁺. In immunofluorescence analyses, odontoblasts were immunopositive for calcium homeostasis modulator 1 (CALHM1), which was found close to ionotropic ATP receptor subtype, P2X₃ receptors. When CALHM1 was knocked down, forskolin-induced [Ca²⁺]_i increase was suppressed. Alizarin red and von Kossa staining showed that forskolin decreased mineralization. These findings suggest that activation of adenylyl cyclase elicited increases in the intracellular cAMP level and Ca²⁺ influx via protein kinase A activation in odontoblasts. Subsequent cAMP-dependent Ca²⁺ influx was mediated by CALHM1 in odontoblasts. In addition, the intracellular cAMP signaling pathway in odontoblasts negatively mediated dentinogenesis.