Intracellular cAMP increase evokes Ca²⁺ influx in odontoblasts

Objectives: Odontoblasts are sensory receptor cells that play key roles in dentin formation and sensory generation of dentinal pain, following the application of various stimuli to the dentin surface. In our previous study, we reported that odontoblasts expressed cannabinoid 1 (CB1) receptors and transient receptor potential vanilloid subfamily member 1 (TRPV1) channels. We also revealed cyclic AMP (cAMP)-mediated crosstalk between CB1 receptors and TRPV1 channels in odontoblasts. The activation of CB1 receptors enhanced cAMP production by stimulating adenylyl cyclase following activation of the Gs alpha subunit of G proteins (Gs protein). The intracellular cAMP increases facilitated Ca²⁺ influx via TRPV1 channels induced by stimuli to dentin surface in odontoblasts. These results suggested that cAMP might play important roles in dentin formation and/or sensory transduction mechanisms for tooth pain. However, the detailed intracellular cAMP signaling pathway and the participation of cAMP in Ca²⁺ signaling in odontoblasts remain unclear. In the present study, we therefore investigated the crosstalk between intracellular cAMP and Ca²⁺ signaling in odontoblasts.
Methods: We simultaneously measured intracellular cAMP levels and Ca²⁺ concentrations using mNeonGreen-based cAMP sensor and fura-2 in odontoblasts.
Results: In the presence of extracellular Ca²⁺, the application of forskolin (an adenylyl cyclase activator) or isoproterenol (an agonist of the Gs protein-coupled beta-2 adrenergic receptors) increased intracellular cAMP levels in a dose-dependent manner. The increases were inhibited by application of SQ22536, an adenylyl cyclase inhibitor. In the presence of extracellular Ca²⁺, the application of forskolin or isoproterenol increased both intracellular cAMP levels and Ca²⁺ concentrations. In the absence of extracellular Ca²⁺, application of forskolin or isoproterenol increased intracellular cAMP levels, but not intracellular Ca²⁺ concentrations.
Conclusions: These results suggested that activation of Gs protein-coupled receptors increased intracellular cAMP by stimulation of adenylyl cyclase. The increase in intracellular cAMP levels has an ability to elicit Ca²⁺ influx from extracellular medium in odontoblasts.