Functional expression of TRPM8 and TRPA1 channels in rat odontoblasts.

Odontoblasts produce dentin during development, throughout life, and in response to pathological conditions by sensing stimulation of exposed dentin. The functional properties and localization patterns of transient receptor potential (TRP) melastatin subfamily member 8 (TRPM8) and ankyrin subfamily member 1 (TRPA1) channels in odontoblasts remain to be clarified. We investigated the localization and the pharmacological, biophysical, and mechano-sensitive properties of TRPM8 and TRPA1 channels in rat odontoblasts. Menthol and icilin increased the intracellular free Ca(2+) concentration ([Ca(2+)]i). Icilin-, WS3-, or WS12-induced [Ca(2+)]i increases were inhibited by capsazepine or 5-benzyloxytriptamine. The increase in [Ca(2+)]i elicited by allyl isothiocyanate (AITC) was inhibited by HC030031. WS12 and AITC exerted a desensitizing effect on [Ca(2+)]i increase. Low-temperature stimuli elicited [Ca(2+)]i increases that are sensitive to both 5-benzyloxytriptamine and HC030031. Hypotonic stimulation-induced membrane stretch increased [Ca(2+)]i; HC030031 but not 5-benzyloxytriptamine inhibited the effect. The results suggest that TRPM8 channels in rat odontoblasts play a role in detecting low-temperature stimulation of the dentin surface and that TRPA1 channels are involved in sensing membrane stretching and low-temperature stimulation. The results also indicate that odontoblasts act as mechanical and thermal receptor cells, detecting the stimulation of exposed dentin to drive multiple cellular functions, such as sensory transduction

Thermal and membrane stretch sensitivity of TRPM8 and TRPA1 channels.

<p>(<b>A and C</b>) Representative traces of [Ca²⁺]_i increase in response to cool-stimulation from 35°C to 22±1°C or cold-stimulation from 26°C to 13±1°C, with or without 10 µM 5-BOT (<b>A</b>) or 100 µM HC030031 (<b>C</b>), in presence of extracellular Ca²⁺ (white boxes at bottom). Black bars indicate when cool- or cold-stimulation was applied, and white bars show application of 5-BOT (<b>A</b>) or HC030031 (<b>C</b>) to external solution. (<b>B</b>) Summary bar graphs of increase in [Ca²⁺]_i upon cool-stimulation with (gray column) or without (open columns) 10 µM 5-BOT. (<b>D</b>) Summary bar graphs of increase in [Ca²⁺]_i upon cold-stimulation with (gray column) or without (open columns) 100 µM HC030031. Each bar indicates mean ± SE of 5 (<b>B</b>) and 8 (<b>D</b>) experiments. Statistically significant differences between columns (shown by solid lines) are denoted by asterisks: *<i>P</i><0.05. (<b>E and G</b>) Example traces of transient [Ca²⁺]_i increase in response to 200 mOsm/L hypotonic solution with or without 10 µM 5-BOT (<b>E</b>) or 100 µM HC030031 (<b>G</b>) in presence of extracellular Ca²⁺ (white boxes at bottom). Bars denote when hypotonic solution (black) and 5-BOT (<b>E</b>) or HC030031 (<b>G</b>) (white) were added to external solution. (<b>F and H</b>) Summary bar graphs of increase in [Ca²⁺]_i upon addition of 200 mOsm/L hypotonic solution without (open columns in <b>F</b> and <b>H</b>) or with (gray column in <b>F </b><b>and </b><b>H</b>) 5-BOT or HC030031. Each bar indicates mean ± SE of several experiments (see text). Statistically significant differences between columns (shown by solid lines) are denoted by asterisks: *<i>P</i><0.05.</p

Localization and distribution of TRPM8 channels in odontoblasts.

<p>(<b>A–D</b>) Tall, columnar, and polarized secretory odontoblasts positive for TRPM8 channel immunoreactivity (brown and green) were observed at dental pulp section in the mandibular incisor (<b>A and B</b>) and molar (<b>C and D</b>) by immunohistochemical (<b>A and </b><b>C</b>) and immunofluorescence (<b>B </b><b>and D</b>) analysis. Immunoreactivity was also observed in mature odontoblasts and those obtained from incisors and molars of 16-week-old rats (not shown). TRPM8 channels were localized across entire cell membrane. Arrowheads indicate distal portion of membrane, and asterisks show cellular processes inside dentinal tubules of odontoblasts. Nuclei are shown in blue. Arrows: dentopulpal junction. Scale bars: 20 µm. No fluorescence was detected in negative controls (insets of <b>A–D</b>).</p

[Ca²⁺]_i increase induced by a TRPM8 channel agonists.

<p>(<b>A–D</b>) Representative traces of transient [Ca²⁺]_i increase in response to 100 µM menthol (<b>A</b>), 1 µM icilin (<b>B</b>), 10 µM WS3 (<b>C</b>), or 500 nM WS12 (<b>D</b>) in presence of extracellular Ca²⁺ (white boxes at bottom). Addition of 10 µM CPZ (<b>B </b><b>and C</b>) or 5-BOT (<b>D</b>) inhibited [Ca²⁺]_i increase. Bars in each figure indicate when agonists (black) or antagonists (white) were added to external solution. (<b>E</b>) Summary bar graphs show increase in [Ca²⁺]_i by agonists with (gray columns) or without (open columns) antagonists. Agonists and antagonists used are denoted on left side of graph. (<b>F and G</b>) Example trace of transient [Ca²⁺]_i increase in response to 10 µM WS3 with (white box at bottom) or without extracellular Ca²⁺ (<b>F</b>). Black bars denote when WS3 was added to external solution. Summary bar graphs of [Ca²⁺]_i increase without (gray column) or with (open column) extracellular Ca²⁺. Resting value is shown as <i>F/F₀</i> = 1.0. Each bar (in <b>E </b><b>and </b><b>G</b>) indicates mean ± SE of several experiments (see text). Statistically significant differences between columns (shown by solid lines) are denoted by asterisks: *<i>P</i><0.05.</p

Localization and distribution of TRPA1 channels.

<p>(<b>A–D</b>) Tall, columnar, and polarized secretory odontoblasts were positive for TRPA1 channel immunoreactivity. Construction of <b>A–D</b> is same as in Fig. 1. Although TRPA1 channels were localized on entire cell membrane, intense immunoreactivity was observed on distal membrane of odontoblasts and cellular processes inside dentinal tubules.</p