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 membrane-bound 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 Ca(2+) 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(+)-Ca(2+) exchangers (NCXs) in mouse odontoblast lineage cells. METHODS: We examined TRP channel activity by measuring intracellular-free Ca(2+) 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 Ca(2+) influx and inward currents. TRPV1, TRPV2, and TRPV4 channel antagonists inhibited the hypotonic stimulation-induced Ca(2+) entry and currents. Their respective agonists activated Ca(2+) entry. Although the increase in the intracellular free Ca(2+) 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 up-regulated 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 Ca(2+) entry, and increased intracellular-free Ca(2+) 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.

Expression of P2X1 and P2X4 receptors in rat trigeminal ganglion neurons.

Extracellular ATP, an essential pain mediator, is received by cell-surface ionotropic P2X and/or metabotropic P2Y receptors. Although the contribution of P2X3 and/or P2X(2/3) receptors toward the pain mechanism is well described in trigeminal ganglion neurons, the expression of other subtypes of P2X receptor remains to be clarified. We examined expression of P2X receptor mRNA and measured intracellular free Ca²âº concentration ([Ca²âº]i) by the activation of these receptors by fura-2 fluorescence in primary cultured rat trigeminal ganglion neurons. Real-time reverse transcription-PCR analysis revealed mRNA expression of P2X receptor subtype P2X1, P2X3, and P2X4 in trigeminal ganglion neurons. In the presence of extracellular Ca²âº, the application of P2X receptors agonists, ATP, α,ß-methylene ATP or ß,γ-methylene ATP induced Ca²âº influx significantly. The ATP-induced increase in [Ca²âº]i was inhibited by a series of selective antagonists for P2X1, P2X3, or P2X4 receptors. These results indicate that trigeminal ganglion neurons functionally express P2X1, P2X3, and P2X4 receptors and that these receptors are involved in the mediation of not only nociceptive but also neuropathic pain in the orofacial area.