Depolarization-induced Intracellular Free Calcium Concentration Increases Show No Desensitizing Effect in Rat Odontoblasts.

Odontoblasts play an important role in the transduction of the sensory signals underlying dentinal pain. Transmembrane voltage-independent Ca(2+) influx in odontoblasts has been well described. Voltage-dependent Ca(2+) influx has also been reported, but its biophysical properties remain unclear. The aim of the present study was to investigate the desensitizing effect of voltage-dependent Ca(2+) influx in rat odontoblasts by measuring depolarization-induced intracellular free Ca(2+) concentrations ([Ca(2+) ]i ). Odontoblasts on dental pulp slices from newborn rats were acutely isolated and [Ca(2+) ]i measured by using fura-2 fluorescence. Repeated application of extracellular high-K(+) solution (50 mM), which induces membrane depolarization-elicited repeated and transient increases in [Ca(2+) ]i in the presence of extracellular Ca(2+). Increases in depolarization-induced [Ca(2+) ]i showed no significant desensitizing effect (p >0.05; Friedman test). These results suggest that odontoblasts express a voltage-dependent Ca(2+) influx pathway with no desensitizing properties.

Transient Receptor Potential Cation Channel Subfamily Vanilloid Member 3 is not Involved in Plasma Membrane Stretch-induced Intracellular Calcium Signaling in Merkel Cells.

Merkel cells (MCs), which form part of the MC-neurite complex, making contact with sensory afferents to drive mechanosensory transduction mechanisms, express transient receptor potential (TRP) cation channel subfamily vanilloid (V) members 1, 2, and 4, as well as ankyrin subfamily member 1. While these proteins are involved in sensing plasma membrane stretch, less is known about the functional properties of TRPV subfamily member 3 (TRPV3) during membrane stretch in MCs. The aim of this study was to determine whether TRPV3 channels were involved in mechanosensory activity by measuring intracellular free Ca(2+) concentrations ([Ca(2+)]i) in MCs isolated from hamster buccal mucosa. Application of a hypotonic extracellular solution to quinacrine-positive MCs elicited a transient increase in [Ca(2+)]i. When TRPV3 channel antagonist 2,2-diphenyltetrahydrofuran was added to the hypotonic extracellular solution, however, no effect was observed on hypotonic stimulation-induced increase in [Ca(2+)]i. These results suggest that TRPV3 channels are not involved in the mechanosensory mechanism during membrane stretch in MCs.

Use of barometric pressure and electromyography measurement techniques to elucidate the mechanisms by which bolus passes from the oral cavity to the oropharynx during swallowing.

OBJECTIVES: The exact timings associated with swallowing are difficult to measure with the currently available methods. In this study, we aimed to elucidate the timings of action of the swallowing organs in the oral and pharyngeal stages of swallowing by recording the barometric pressure (BP) and tongue muscle activities. METHODS: Dry and water swallows were studied in 10 adults using electromyography and small barometers. BPs were recorded during swallowing in the oral cavity (BP-o) and pharynx (BP-p), associated with muscle activities of the anterior (TA) and posterior (TP) parts of the tongue and of the suprahyoid (SHy) muscle. To analyze the temporal pattern of each activity, times of onset, cessation, and peak were measured. RESULTS: Two characteristic waveforms were obtained. BP-o peaked immediately after onset and decreased to atmospheric pressure following a short plateau. However, BP-p gradually increased, reached a peak, and returned to the atmospheric pressure immediately before the end of BP-o. Since pressure increments indicated that the sensor was compressed in a closed space, onset and cessation of BP-p could correspond to the duration of nasopharyngeal closure. The onset of BP-p and the peak time of BP-o occurred in close succession. Thus, nasopharyngeal closure could be evaluated from BP-o. The sensor and EMG measured durations of oral and pharyngeal stages as 0.4 and 0.6 sec, respectively. TA activation began earlier than the TP. TA and TP peaks appeared before the BP-o peak, suggesting that the tongue begins the activity for swallowing before nasopharyngeal closure. CONCLUSIONS: This study revealed movements of swallowing organs in the two stages with high temporal resolution. BP-o detected the duration of nasopharyngeal closure.

Intracellular Ca2+ mobilization pathway via bradykinin B1 receptor activation in rat trigeminal ganglion neurons.

Bradykinin (BK) and its receptors, B1 and B2, in trigeminal ganglion (TG) neurons are involved in the regulation of pain. Recent studies have revealed that B1 receptors are expressed in neonatal rat TG neurons; however, the intracellular signaling pathway following B1 receptor activation remains to be elucidated. To investigate the mechanism by which B1 receptor activation leads to intracellular Ca2+ mobilization, we measured the intracellular free Ca2+ concentration ([Ca2+]i) in primary-cultured TG neurons. The application of Lys-[Des-Arg9]BK (B1 receptor agonist) increased the [Ca2+]i in these TG neurons even in the absence of extracellular Ca2+. Pretreatment with inhibitors of ryanodine receptors or sarco/endoplasmic reticulum Ca2+-ATPase suppressed the increase in Lys-[Des-Arg9]BK-induced [Ca2+]i. The Lys-[Des-Arg9]BK-induced [Ca2+]i increase was unaffected by phospholipase-C inhibitor. B1 receptor activation-induced [Ca2+]i increase was suppressed by phosphodiesterase inhibitor and enhanced by adenylyl cyclase inhibitor. These results suggest that B1 receptor activation suppresses intracellular cAMP production via adenylyl cyclase inhibition and mobilizes intracellular Ca2+ via ryanodine receptors that access intracellular Ca2+ stores.

Merkel Cells Release Glutamate Following Mechanical Stimulation: Implication of Glutamate in the Merkel Cell-Neurite Complex.

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 Ca2+ concentration ([Ca2+]i) induced by direct mechanical stimulation of MCs. We also measured [Ca2+]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 [Ca2+]i. Direct mechanical stimulation to MCs increased [Ca2+]i, showing stimulation strength dependence. In the MC-TGs coculture, the application of direct mechanical stimulation to MCs resulted in increased [Ca2+]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 [Ca2+]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.

High pH-Sensitive Store-Operated Ca2+ Entry Mediated by Ca2+ Release-Activated Ca2+ Channels in Rat Odontoblasts.

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.

Potassium Currents Activated by Depolarization 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.