Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts

ATP modulates various functions in the dental pulp cells, such as intercellular communication and neurotransmission between odontoblasts and neurons, proliferation of dental pulp cells, and odontoblast differentiation. However, functional expression patterns and their biophysical properties of ionotropic ATP (P2X) receptors (P2X1–P2X7) in odontoblasts were still unclear. We examined these properties of P2X receptors in mouse odontoblasts by patch-clamp recordings. K+-ATP, nonselective P2X receptor agonist, induced inward currents in odontoblasts in a concentration-dependent manner. K+-ATP-induced currents were inhibited by P2X4 and P2X7 selective inhibitors (5-BDBD and KN62, respectively), while P2X1 and P2X3 inhibitors had no effects. P2X7 selective agonist (BzATP) induced inward currents dose-dependently. We could not observe P2X1, 2/3, 3 selective agonist (αβ-MeATP) induced currents. Amplitudes of K+-ATP-induced current were increased in solution without extracellular Ca2+, but decreased in Na+-free extracellular solution. In the absence of both of extracellular Na+ and Ca2+, K+-ATP-induced currents were completely abolished. K+-ATP-induced Na+ currents were inhibited by P2X7 inhibitor, while the Ca2+ currents were sensitive to P2X4 inhibitor. These results indicated that odontoblasts functionally expressed P2X4 and P2X7 receptors, which might play an important role in detecting extracellular ATP following local dental pulp injury.

Odontoblasts originate from the neural crest and are located at the interface between the dentin and dental pulp. The primary function of odontoblasts is dentin formation known as dentinogenesis, during developmental, physiological, and pathological processes. In addition, recent studies have indicated that odontoblasts are sensory receptor cells for dentin sensitivity, known as the "hydrodynamic odontoblast receptor theory" Shibukawa et al., 2015;Nishiyama et al., 2016) by communicating intercellularly with neurons via neurotransmitter, ATP and glutamate. Membrane deformation caused by dentinal fluid movement activates the mechanosensitive-transient receptor potential (TRP) channel; ATP is released to the extracellular space through pannexin-1, which are plasma membrane ATPpermeable channels, and activates P2X 3 receptors on the neuron to establish neurotransmission between odontoblast and neurons (Shibukawa et al., 2015). Glutamate mediates neurotransmission between odontoblasts and metabotropic glutamate (mGlu) receptors in trigeminal ganglion neurons through glutamate-permeable anion channels (Nishiyama et al., 2016). Both ATP and glutamate also mediate intercellular odontoblast-odontoblast communication by activation of P2Y and mGlu receptors, respectively Shibukawa et al., 2015;Nishiyama et al., 2016). However, these previous studies suggested that odontoblasts did not express P2X 3 receptors, and P2X receptors could not mediate intercellular odontoblast-odontoblast communication . Therefore, the functional expression and the expression patterns of P2X receptors in odontoblasts have remained unclear.
To elucidate the functional expression and biophysical/pharmacological properties of P2X receptors, we measured the plasma membrane currents induced by P2X receptor activation in mouse odontoblasts.

Whole-Cell Patch-Clamp Recording Technique
Whole-cell recordings were performed using a conventional patch-clamp recording configuration under voltage-clamp conditions. Patch pipettes (4-9 M ) were pulled from capillary tubes by using a DMZ universal puller (Zeitz Instruments, Martinsried, Germany), which were filled with an intracellular solution. The intracellular solution contained 140 mM KCl, 10 mM NaCl, and 10 mM HEPES (pH was adjusted to 7.2 by Tris). Whole-cell currents were measured using a patchclamp amplifier (L/M-EPC-7+; Heka Elektronik, Lambrecht, Germany). The current traces were monitored and stored using pCLAMP (Molecular Device, Foster City, CA, USA) after digitizing the analog signals at 10 kHz (DigiData 1440A, Molecular Device) and filtering the signals digitally at 3 kHz using pCLAMP. The data were analyzed offline by using pCLAMP and the technical graphics/analysis program ORIGIN (MicroCal Software, Northampton, MA, USA). The solution temperature when measuring the whole-cell currents was maintained at 30 • C.

Calculation of the Change in Ionic Permeability Induced by P2X Receptor Activation
We calculated the relative change in the total ionic permeability induced by the activation of P2X receptor by using the following Equation 1: P P2X /P control = 10 ErevF/2. 303RT (1) where P P2X is the relative total ionic permeability after the activation of P2X receptor by the agonist (BzATP or K + -ATP), P control is 1.0 for the reversal potentials (E rev s) measured without any P2X receptor agonist in the ECS, E rev is the change in E rev by P2X receptor agonist, F is Faraday's constant, R is gas constant, and T is absolute temperature. The temperature was maintained at 30 • C while measuring the ramp currents.

Statistical Analysis
All data are presented as mean ± standard deviation (SD) of N observations, where N represents the number of cells tested or the number of experiments. Steel-Dwass multiple comparisons were used to determine nonparametric statistical significance. P < 0.05 were considered significant.

Outwardly Rectifying Current in Odontoblasts
Mouse odontoblast lineage cells have a cell capacitance of 32.7 pF ± 6.2 (N = 6). Current amplitudes were normalized to these single cell capacitance values and expressed as current densities (pA/pF). Depolarized voltage steps from −100 to +80 mV at a holding potential (Vh) of −70 mV (lower in Figure 1A) elicited outward currents (upper in Figure 1A) with a reversal potential of −61 mV (−60.3 ± 1.8; Figure 1B) in the standard ECS. These outward currents showed slow activation and noninactivation during 400 ms depolarization pulses. The currentvoltage relationship of the currents showed outward rectification with increasing membrane potentials ( Figure 1B).

K + -ATP-Induced Inward Current in Odontoblasts
In the standard ECS, the addition of four different concentration of extracellular K + -ATP (10, 50, 100, and 200 µM) evoked inward currents at Vh of −70 mV, in a concentration-dependent manner (Figures 2A-E). A semilogarithmic plot ( Figure 2F) illustrates membrane current densities (pA/pF) as a function of the applied concentration of extracellular K + -ATP, with an equilibrium binding constant (EC 50 ) of 52.9 µM (N = 6). A series of three times of repeated applications of 100 µM K + -ATP (10 s in duration at 40 s intervals) elicited a significant desensitizing effect of current ( Figure 2G), showing that the current amplitudes decreased with increasing times of repeated application. The amplitudes of K + -ATP induced current at the second and third application were significantly decreased by 78.6 ± 3.2% (N = 6, P < 0.05) and 48.9 ± 8.0% (N = 6, P < 0.05), respectively, over that at the first application ( Figure 2H).
To further analyze the changes in the total ionic permeability induced by P2X receptor activation, we recorded ramp currents to determine the current-voltage (I-V) relationship and analyze E rev s during application of P2X receptor agonists. When we applied a voltage-ramp protocol from −100 to +100 mV (0.2 mV/ms) at a Vh of −70 mV (upper, Figures 6L,M), outwardly rectifying currents were observed in standard ECS (Figures 6L-N). Applications of 300 µM BzATP ( Figure 6L) and 100 µM K + -ATP ( Figure 6M) increased both inward and outward current components with changes in E rev s. E rev s of the ramp currents were shifted +57 mV toward depolarizing potentials by 100 µM K + -ATP and +51 mV to them by 300 , and with 20 µM NF449 (most right). Each bar denotes the mean ± SD of three separate experiments. Statistically significant differences between bars (shown by solid lines) are indicated by asterisks, *P < 0.05. Significant differences were found in the K + -ATP-induced currents between in the presence and absence of KN62 or 5-BDBD, while we could not observe any significant differences in the peak current density in K + -ATP-induced currents between in the absence or presence of NF110 or NF449.  µM BzATP, on comparing Erevs obtained without K + -ATP and BzATP in standard ECS (N = 3, Figure 6N). We calculated the relative changes in the total ionic permeability induced by P2X receptor activations using Equation (1). The relative P P2X was 7.1 for BzATP-induced current and 8.8 for K + -ATP-induced one.
In our previous study, we have shown that ATP, as intercellular-/neuro-transmitter, which was released from mechanically stimulated odontoblasts increased the intracellular Ca 2+ concentration in odontoblasts as well as neurons located nearby the stimulated odontoblasts Shibukawa et al., 2015;Nishiyama et al., 2016). An application of P2X 3 receptor antagonist did not elicit mechanical stimulationinduced response in nearby odontoblasts, but did in the neurons (Shibukawa et al., 2015). These results were in line with the present results showing the implication of the lack of P2X 3 receptor in odontoblasts, while the P2X 3 receptor in the TG neurons plays an important role in the sensory transduction sequence by receiving ATP from mechanically stimulated odontoblasts (Shibukawa et al., 2015). We could not obtain the results showing whether the P2X 2 , P2X 5 , and P2X 6 are expressed functionally in odontoblasts or not in this study, since there are no commercially available selective pharmacological ligands for these P2X receptor subtypes to date. Although, we showed that odontoblasts exhibit functional expression of P2X 4 and P2X 7 receptors but not P2X 1 , P2X 2/3 , P2X 3 , and P2X 4/6 , further study is warranted to throw light on the expression of P2X 2 , P2X 5 , and P2X 6 (homomer) receptors.
The peak amplitudes of K + -ATP-induced currents were increased by the removal of Ca 2+ , whereas they were decreased by the removal of Na + from the extracellular medium. In addition, K + -ATP-induced currents were almost completely abolished in the Na + and Ca 2+ -free ECS. Thus, the K + -ATP induced current was composed by Na + and Ca 2+ conductance. It has been well-known that P2X receptors show relative high Ca 2+ permeability (Samways et al., 2014). In this study, K + -ATPinduced Ca 2+ currents in the Na + -Free ECS were significantly inhibited by P2X 4 receptor antagonist (5-BDBD), while not by P2X 7 receptor antagonist (KN62), indicating that P2X 4 receptor in odontoblast has high Ca 2+ permeability. These results are also in line with the results showing that P2X 4 receptor showed highest relative Ca 2+ permeability among the P2X FIGURE 6 | Continued graph shows the peak current densities evoked by 100 µM K + -ATP in Ca 2+ free ECS without or with 20 nM KN62 and 10 nM 5-BDBD. (K) Summary bar graph shows the peak current densities evoked by 100 µM K + -ATP in Na + free ECS without or with 20 nM KN62 and 10 nM 5-BDBD. Each bar denotes the mean ± SD of three separate experiments. Statistically significant differences between bars (shown by solid lines) are indicated by asterisks, *P < 0.05. (L,M) I-V relationships of the currents induced by P2X receptor agonists. Voltage-ramp protocol from −100 to +100 mV (0.2 mV/ms) at a Vh of −70 mV was applied to the cells (bottoms in L,M). Traces show the example currents, which were recorded in standard ECS and in standard ECS with 300 µM BzATP (L) or 100 µM K + -ATP (M). (N) I-V relationships for ramp current without (closed squares) or with 100 µM K + -ATP (closed circles) and 300 µM BzATP (closed triangles). The data points show the mean ± SD of three separate experiments. Reversal potential (E rev ) for the currents were −72.3 ± 4.1 mV in standard ECS, −15.0 ± 6.0 mV in the ECS with K + -ATP, or −21.3 ± 9.4 mV in standard ECS with BzATP.
family (Egan and Khakh, 2004). The cation permeability for P2X 7 receptor remains debatable, which also shows high Ca 2+ permeability; however, the Ca 2+ curries ∼5% of the total inward current through P2X 7 (Jarvis and Khakh, 2009;Samways et al., 2014). It has been known that the cation permeability by P2X receptor activation were modulated by divalent cations as "Ca 2+ dependent block" (Jarvis and Khakh, 2009;Kasuya et al., 2016), in which the ion permeability of P2X receptors are inhibited by the extracellular Ca 2+ in a concentration-dependent manner. The Ca 2+ dependent block for P2X receptors was also welldescribed in P2X 7 receptors (Yan et al., 2011;Liang et al., 2015). When we removed extracellular Ca 2+ from the standard ECS (Ca 2+ -free ECS), the amplitude of K + -ATP-induced currents augmented compared to those in the standard ECS. These K + -ATP-induced Na + currents were strongly inhibited by an antagonist of P2X 7 receptors, but not of P2X 4 receptors. Thus, the results indicated that the ionic permeability of P2X 7 receptors in odontoblasts were also blocked by extracellular Ca 2+ , showing Ca 2+ dependent block. In addition, the main ionic component of cation permeability for P2X 4 receptor was Ca 2+ , whereas that for P2X 7 was Na + . We could observe each residual K + -ATPinduced Ca 2 and Na + current component after P2X 4 and P2X 7 receptor inhibition. These were carried by other P2X receptor activation, including P2X 2 , P2X 5 , and P2X 6 receptors; however, further study will be needed.
In previous studies, we showed that P2X receptors, as well as ionotropic glutamate receptors, could not mediate intercellular odontoblast-odontoblast communication Nishiyama et al., 2016). We could hardly record evoked-inward currents [that were activated by intercellular mediator(s)] in the odontoblast located 5 µm away from the mechanically stimulated odontoblast . These previous results had implied that the ionotropic receptor [such as ionotropic ATP (P2Xs) and/or glutamate receptors] activation by released intercellular transmitters-not only of ATP but also of glutamate-following mechanical stimulation of the odontoblasts, are hardly involved in the inter-odontoblast communication. In the present study, the EC 50 of K + -ATP on the inward currents in odontoblasts was 52.9 µM. Comparing the EC 50 of ATP for P2X 1 -P2X 6 receptor activation, this value was of 5-50 times higher concentration than those reported by other studies, in which they have reported EC 50 ranging from 1.0 to 10 µM (Jarvis and Khakh, 2009). The value of EC 50 of K + -ATP in this study was in line with that for EC 50 of ATP for P2X 7 receptor activation (100 µM : Khakh et al., 2001). Recently, the concentration of released ATP by external dentin cold-stimulation has been reported to be "nM" range in an in vitro human tooth perfusion model (Egbuniwe et al., 2014;Liu et al., 2015). Based on our results, P2X receptor subtypes expressed in odontoblasts need ∼1000 times as high of a concentration of extracellular ATP to be activated (ca. 50-100 µM range), as compared to the ATP concentration by dentin stimulation induced releases. Therefore, by the existence of the differences in the affinity of ATP, P2X receptors in odontoblasts seem not to mediate intercellular odontoblast-odontoblast communication Shibukawa et al., 2015), while P2X receptors in neurons mediate neurotransmission between odontoblasts and neurons for sensory transduction sequence for the dentinal pain (Kuroda et al., 2012;Shibukawa et al., 2015).
On the other hand, ATP that leaked from injured cells in dental pulp could activate P2X receptors in odontoblasts. The intracellular concentration of ATP is in the mM range (Imamura et al., 2009). Following tissue injury, the increase in the local concentration of ATP at the injured site reached the sub-mM range, which such high enough concentration of ATP might be capable to activate P2X receptors in odontoblasts. Thus, we imply that P2X receptors in odontoblasts play an important role in the biophylaxis function for the dental pulp to detect local tissue injury, rather than intercellular odontoblast communication.

AUTHOR CONTRIBUTIONS
Yuta S carried out the measurement membrane currents. Yoshiyuki S, TS, and MT participated with design of the study. Yuta S and Yoshiyuki S performed the statistical analysis. Yoshiyuki S conceived of the study, and participated in its design and coordination and helped to draft the manuscript. All authors read and approved the final manuscript.