Extracellular ATP Induces Calcium Signaling in Odontoblasts

Odontoblasts form dentin at the outermost surface of tooth pulp. An increasing level of evidence in recent years, along with their locational advantage, implicates odontoblasts as a secondary role as sensory or immune cells. Extracellular adenosine triphosphate (ATP) is a well-characterized signaling molecule in the neuronal and immune systems, and its potential involvement in interodontoblast communications was recently demonstrated. In an effort to elaborate the ATP-mediated signaling pathway in odontoblasts, the current study performed single-cell reverse transcription polymerase chain reaction (RT-PCR) and immunofluorescent detection to investigate the expression of ATP receptors related to calcium signal in odontoblasts from incisal teeth of 8- to 10-wk-old rats, and demonstrated an in vitro response to ATP application via calcium imaging experiments. While whole tissue RT-PCR analysis detected P2Y 2 , P2Y 4 , and all 7 subtypes (P2X 1 to P2X 7 ) in tooth pulp, single-cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2Y 2 , P2Y 4 , P2X 2 , P2X 4 , P2X 6 , and P2X 7 expression in only a subset (23% to 47%) of cells tested, with no evidence for P2X 1 , P2X 3 , and P2X 5 expression. An increase of intracellular Ca 2+ concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin or under the Ca 2+ -free condition, suggested function of both ionotropic and metabotropic ATP receptors in odontoblasts. The enhancement of ATP-induced calcium response by ivermectin and inhibition by 5-(3-bromophenyl)-1,3-dihydro-2H-benzofuro[3,2-e]-1,4-diazepin-2-one (5-BDBD) confirmed a functional P2X 4 subtype in odontoblasts. Positive calcium response to 2′,3′-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP suggested P2X 2 , P2X 4 , and P2X 7 as functional subunits in rat odontoblasts. Single-cell RT-PCR analysis of the cells with confirmed calcium response and immunofluorescent detection further corroborated the expression of P2X 4 and P2X 7 in odontoblasts. Overall, this study demonstrated heterogeneous expression of calcium-related ATP receptor subtypes in subsets of individual odontoblasts, suggesting extracellular ATP as a potential signal mediator for odontoblastic functions.