Activation of CGRP receptors increased intracellular cAMP level in odontoblasts

Abstract: Objectives : Our previous studies suggested that Ca ²⁺ signaling in odontoblasts is involved in dentin formation and generation of dentinal pain. In the studies, we have reported the expression of transient receptor potential vanilloid subfamily member 1 (TRPV1) channels and cannabinoid 1 (CB1) receptors in odontoblasts, and functional crosstalk between them. In odontoblasts, increase in intracellular Ca ²⁺ concentration induced by TRPV1 channel activation secretes endocannabinoids. We also demonstrated that cyclic AMP (cAMP) regulates CB1 receptor-TRPV1 channel crosstalk. These results suggest that cAMP may participate in dentin formation and dentinal pain generation. However, the detailed intracellular cAMP signaling pathway in odontoblasts and the role of cAMP in odontoblast function remain unclear. In this study, we examined the cAMP signaling elicited by activation of adenylate cyclase or Gs protein-coupled receptors in odontoblasts.Methods : Odontoblasts were acutely isolated from the incisors of newborn Wistar rats. Odontoblasts were cultured in α-MEM containing FBS for 24-36 hours, and then we added mNeon Green-based cAMP sensor. After culture for 24 hours, intracellular cAMP level was measured from odontoblasts.Results : In the presence of extracellular Ca ²⁺ , application of forskolin, an adenylate cyclase activator, dose-dependently increased intracellular cAMP level in odontoblasts. The increases were decreased by repeated application of forskolin. In the presence of extracellular Ca ²⁺ , application of CGRP, a CGRP receptor agonist, increased intracellular cAMP level in odontoblasts. The increases were significantly inhibited by application of a selective CGRP receptor antagonist or an adenylate cyclase inhibitor in odontoblasts.Conclusion : These results suggested that activation of adenylate cyclase increased intracellular cAMP level in odontoblasts. We also showed expression of CGRP receptors in odontoblasts. In addition, these results indicate that CGRP receptor activation increased intracellular cAMP level by activation of adenylate cyclase.