Single Cell Analysis of Rat Odontoblasts for Functional Expression of P2X Purinoceptors

Objectives: Odontoblasts form dentin at the outermost surface of tooth pulp. Increasing number of evidences in recent years along with the locational advantage support the secondary role of odontoblasts as sensory or immune cells. Extracellular ATP is well characterized signaling molecule in both neuronal and immune systems, and its potential involvement in inter-odontoblast communications was demonstrated recently.
Methods: In an effort to elaborate the purinergic signaling mechanism in odontoblasts, current study investigated whether and which P2X subtypes are expressed in individual odontoblasts isolated from incisal teeth of 8-10 week old rats through single cell RT-PCR, and their response to ATP application in vitro by calcium imaging expreriment.
Results: While RT-PCR analysis of tooth pulp detected all seven P2X1- P2X7 subtypes, single cell RT-PCR analysis of acutely isolated rat odontoblasts revealed P2X2, P2X4, P2X6 and P2X7 expression in 40-60% of cells tested, with no evidence for P2X1, P2X3 and P2X5 expression. Increase of intracellular Ca2+ concentration in response to 100μM ATP, which was repeated after pretreatment of thapsigargin but abolished under 0 Ca2+ condition, suggested function of ionotropic P2X receptors in odontoblasts. Positive calcium response to 2’,3’-O-(benzoyl-4-benzoyl)-ATP (BzATP) and negative response to α,β-methylene ATP confirmed P2X2, P2X4 and P2X7 as functional subunits in rat 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) further confirmed functional P2X4 subtype in odontoblasts. The functional expression of P2X4 and P2X7 was corroborated with scRT-PCR analysis of the cells harvested after positive response from the calcium imaging experiment.
Conclusions: Overall, this study demonstrates heterogeneous expression of P2X purinergic receptor subtypes in subsets of individual odontoblasts, with potential role of P2X2, P2X4 and P2X7 receptors in odontoblastic functions as signal mediators for extracellular ATP.