Calcium hydroxide and mineral trioxide aggregate are widely used for indirect and direct pulp capping and root canal filling. Their dissociation into Ca(2+) and OH(-) in dental pulp creates an alkaline environment, which activates reparative/reactionary dentinogenesis. However, the mechanisms by which odontoblasts detect the pH of the extracellular environment remain unclear. We examined the alkali-sensitive intracellular Ca(2+) signaling pathway in rat odontoblasts. In the presence or absence of extracellular Ca(2+), application of alkaline solution increased intracellular Ca(2+) concentration, or [Ca(2+)]i Alkaline solution-induced [Ca(2+)]i increases depended on extracellular pH (8.5 to 10.5) in both the absence and the presence of extracellular Ca(2+) The amplitude was smaller in the absence than in the presence of extracellular Ca(2+) Each increase in [Ca(2+)]i, activated by pH 7.5, 8.5, or 9.5, depended on extracellular Ca(2+) concentration; the equilibrium binding constant for extracellular Ca(2+) concentration decreased as extracellular pH increased (1.04 mM at pH 7.5 to 0.11 mM at pH 9.5). Repeated applications of alkaline solution did not have a desensitizing effect on alkali-induced [Ca(2+)]i increases and inward currents. In the presence of extracellular Ca(2+), alkaline solution-induced [Ca(2+)]i increases were suppressed by application of an antagonist of transient receptor potential ankyrin subfamily member 1 (TRPA1) channels. Ca(2+) exclusion efficiency during alkaline solution-induced [Ca(2+)]i increases was reduced by a Na(+)-Ca(2+) exchanger antagonist. Alizarin red and von Kossa staining revealed increased mineralization levels under repeated high pH stimulation, whereas the TRPA1 antagonist strongly reduced this effect. These findings indicate that alkaline stimuli-such as the alkaline environment inside dental pulp treated with calcium hydroxide or mineral trioxide aggregate-activate Ca(2+) mobilization via Ca(2+) influx mediated by TRPA1 channels and intracellular Ca(2+) release in odontoblasts. High pH-sensing mechanisms in odontoblasts are important for activating dentinogenesis induced by an alkaline environment.

Merkel cells (MCs) have been proposed to form a part of the MC-neurite complex with sensory neurons through synaptic contact. However, the detailed mechanisms for intercellular communication between MCs and neurons have yet to be clarified. The present study examined the increases in intracellular free Ca 2+ concentration ([Ca 2+ ] i ) induced by direct mechanical stimulation of MCs. We also measured [Ca 2+ ] i in the trigeminal ganglion neurons (TGs) following direct mechanical stimulation to the MCs in an MC-TGs coculture. The MCs were isolated from hamster buccal mucosa, while TGs were isolated from neonatal Wistar rats. Both cell populations showed depolarization-induced [Ca 2+ ] i . Direct mechanical stimulation to MCs increased [Ca 2+ ] i , showing stimulation strength dependence. In the MC-TGs coculture, the application of direct mechanical stimulation to MCs resulted in increased [Ca 2+ ] i in the TGs. These changes were significantly suppressed by antagonists of glutamate-permeable anion channels (4,4′-diisothiocyanato-2,2′-stilbenedisulfonic acid; DIDS), and non-competitive antagonist of the N -methyl- D -aspartate (NMDA) receptors (MK801). Apyrase, an ATP-degrading enzyme, and suramin, a non-selective P2 purinergic receptor antagonist, did not exert inhibitory effects on these [Ca 2+ ] i increases in the TGs following MC stimulation. These results indicated that MCs are capable of releasing glutamate, but not ATP, in response to cellular deformation by direct mechanical stimulation. The released glutamate activates the NMDA receptors on TGs. We suggest that MCs act as mechanoelectrical transducers and establish synaptic transmission with neurons, through the MC-neurite complex, to mediate mechanosensory transduction.

Odontoblasts play a crucial role in dentin formation and sensory transduction following the application of stimuli to the dentin surface. Various exogenous and endogenous stimuli elicit an increase in the intracellular free calcium concentration ([Ca2+]i) in odontoblasts, which is mediated by Ca2+ release from intracellular Ca2+ stores and/or Ca2+ influx from the extracellular medium. In a previous study, we demonstrated that the depletion of Ca2+ stores in odontoblasts activated store-operated Ca2+ entry (SOCE), a Ca2+ influx pathway. However, the precise biophysical and pharmacological properties of SOCE in odontoblasts have remained unclear. In the present study, we examined the functional expression and pharmacological properties of Ca2+ release-activated Ca2+ (CRAC) channels that mediate SOCE and evaluated the alkali sensitivity of SOCE in rat odontoblasts. In the absence of extracellular Ca2+, treatment with thapsigargin (TG), a sarco/endoplasmic reticulum Ca2+-ATPase inhibitor, induced an increase in [Ca2+]i. After [Ca2+]i returned to near-resting levels, the subsequent application of 2.5 mM extracellular Ca2+ resulted in an increase in [Ca2+]i which is a typical of SOCE activation. Additionally, application of 2-methylthioadenosine diphosphate trisodium salt (2-MeSADP), a P2Y1,12,13 receptor agonist, or carbachol (CCh), a muscarinic cholinergic receptor agonist, in the absence of extracellular Ca2+, induced a transient increase in [Ca2+]i. The subsequent addition of extracellular Ca2+ resulted in significantly higher [Ca2+]i in 2-MeSADP- or CCh-treated odontoblasts than in untreated cells. SOCE, that is activated by addition of extracellular Ca2+ in the TG pretreated odontoblasts was then suppressed by Synta66, BTP2, or lanthanum, which are CRAC channel inhibitors. Treatment with an alkaline solution enhanced SOCE, while treatment with HC030031, a TRPA1 channel antagonist, inhibited it. The amplitude of SOCE at pH 9 in the presence of HC030031 was higher than that at pH 7.4 in the absence of HC030031. These findings indicate that CRAC channel-mediated alkali-sensitive SOCE occurs in odontoblasts. SOCE is mediated by P2Y and muscarinic-cholinergic receptors, which are activated by endogenous ligands in odontoblasts.

Increased intracellular free Ca2+ concentrations elicit plasma membrane depolarization, which leads to the activation of K+ currents. However, the precise properties of K+ currents activated by depolarization in odontoblasts remain to be elucidated. The present study identified biophysical and pharmacological characteristics of time-dependent and voltage-activated K+ currents in freshly dissociated rat odontoblasts using patch-clamp recordings in a whole-cell configuration. Using a holding potential of −70 mV, outwardly rectifying time- and voltage-dependent currents were activated by depolarizing voltage. To record pure K+ conductance, we substituted Cl− in both the extracellular and intracellular solutions with gluconate−. Under these conditions, observation of K+ concentration changes in the extracellular solution showed that reversal potentials of tail currents shifted according to the K+ equilibrium potential. The activation kinetics of outward K+ currents were relatively slow and depended on the membrane potential. Kinetics of steady-state inactivation were fitted by a Boltzmann function. The half-maximal inactivation potential was −38 mV. Tetraethylammonium chloride, 4-aminopyridine, and α-dendrotoxin inhibited outward currents in odontoblasts in a concentration-dependent manner, suggesting that rat odontoblasts express the α-subunit of the time- and voltage-dependent K+ channel (Kv) subtypes Kv1.1, 1.2, and/or 1.6. We further examined the effects of Kv activity on mineralization by alizarin red and von Kossa staining. Continuous application of tetraethylammonium chloride to human odontoblasts grown in a mineralization medium over a 21-day period exhibited a dose-dependent decrease in mineralization efficiency compared to cells without tetraethylammonium chloride. This suggests that odontoblasts functionally express voltage-dependent K+ channels that play important roles in dentin formation.

Bradykinin (BK) and its receptors, B and B, in trigeminal ganglion (TG) neurons are involved in the regulation of pain. Recent studies have revealed that B receptors are expressed in neonatal rat TG neurons; however, the intracellular signaling pathway following B receptor activation remains to be elucidated. To investigate the mechanism by which B receptor activation leads to intracellular Ca mobilization, we measured the intracellular free Ca concentration ([Ca]) in primary-cultured TG neurons. The application of Lys-[Des-Arg]BK (B receptor agonist) increased the [Ca] in these TG neurons even in the absence of extracellular Ca. Pretreatment with inhibitors of ryanodine receptors or sarco/endoplasmic reticulum Ca-ATPase suppressed the increase in Lys-[Des-Arg]BK-induced [Ca]. The Lys-[Des-Arg]BK-induced [Ca] increase was unaffected by phospholipase-C inhibitor. B receptor activation-induced [Ca] increase was suppressed by phosphodiesterase inhibitor and enhanced by adenylyl cyclase inhibitor. These results suggest that B receptor activation suppresses intracellular cAMP production via adenylyl cyclase inhibition and mobilizes intracellular Ca via ryanodine receptors that access intracellular Ca stores.