Hypotonic-induced Stretching of Plasma Membrane Activates Transient Receptor Potential Vanilloid Channels and Sodium-Calcium Exchangers in Mouse Odontoblasts

Introduction: A number of transient receptor potential (TRP) channels have been identified as membranebound sensory proteins in odontoblasts. However, the activation properties of these channels remain to be clarified. The purpose of this study was to investigate hypotonic stimulation induced Ca2+ entry via TRP vanilloid subfamily member (TRPV) 1, TRPV2, and TRPV4 channels, which are sensitive to osmotic and mechanical stimuli, and their functional coupling with Na+-Ca2+ exchangers (NCXs) in mouse odontoblast lineage cells. Methods: We examined TRP channel activity by measuring intracellular-free Ca2+ concentration by using fura-2 fluorescence and ionic current recordings with whole-cell patch-clamp methods. Protein localization and messenger RNA expression were characterized using immunofluorescence and reverse-transcription polymerase chain reaction analyses. Results: Extracellular hypotonic solution induced stretching of plasma membrane resulted in the. activation of. Ca2+ influx and inward currents. TRPV1, TRPV2, and TRPV4 channel antagonists inhibited the hypotonic stimulation induced Ca2+ entry and currents. Their respective agonists activated Ca2+ entry. Although the increase in the intracellular free Ca2+ concentration decayed rapidly after the applications of these TRPV channel agonists, NCX inhibitors significantly prolonged the decay time constant. The messenger RNA expression of TRPV1, TRPV2, and TRPV4 channels; NCX isoforms 2 and 3; and dentin sialophosphoprotein were upregulated after 24 hours of exposure to the hypotonic culture medium. Conclusions: These results indicate that stretching of the odontoblast membrane activates TRPV1-, TRPV2-, and TRPV4-mediated Ca2+ entry, and increased intracellular-free Ca2+ concentration is extruded via NCXs. These results suggest that odontoblasts can act as sensors that detect stimuli applied to exposed dentin and drive a number of cellular functions including dentinogenesis and/or sensory transduction.