Functional Expression of IP, 5-HT4, D1, A2A, and VIP Receptors in Human Odontoblast Cell Line

Odontoblasts are involved in sensory generation as sensory receptor cells and in dentin formation. We previously reported that an increase in intracellular cAMP levels by cannabinoid 1 receptor activation induces Ca2+ influx via transient receptor potential vanilloid subfamily member 1 channels in odontoblasts, indicating that intracellular cAMP/Ca2+ signal coupling is involved in dentinal pain generation and reactionary dentin formation. Here, intracellular cAMP dynamics in cultured human odontoblasts were investigated to understand the detailed expression patterns of the intracellular cAMP signaling pathway activated by the Gs protein-coupled receptor and to clarify its role in cellular functions. The presence of plasma membrane Gαs as well as prostaglandin I2 (IP), 5-hydroxytryptamine 5-HT4 (5-HT4), dopamine D1 (D1), adenosine A2A (A2A), and vasoactive intestinal polypeptide (VIP) receptor immunoreactivity was observed in human odontoblasts. In the presence of extracellular Ca2+, the application of agonists for the IP (beraprost), 5-HT4 (BIMU8), D1 (SKF83959), A2A (PSB0777), and VIP (VIP) receptors increased intracellular cAMP levels. This increase in cAMP levels was inhibited by the application of the adenylyl cyclase (AC) inhibitor SQ22536 and each receptor antagonist, dose-dependently. These results suggested that odontoblasts express Gs protein-coupled IP, 5-HT4, D1, A2A, and VIP receptors. In addition, activation of these receptors increased intracellular cAMP levels by activating AC in odontoblasts.


Introduction
Plasma membrane 'multistimulatory receptor proteins', which are capable of receiving various external and internal stimuli, can be categorized as follows: G protein-coupled (metabotropic) receptors (GPCRs) or ligand-gated ion channels (ionotropic receptors). GPCRs activate the intracellular second messenger pathway to transmit cellular information via G protein activation. These G proteins are composed of α, β, and γ subunits and are identified by their Gα subunits. They are typically classified into four families; Gα s , Gα i , Gα q , and Gα 12/13 [1,2], which are expressed in most cell types. There are two principal intracellular signaling pathways induced by GPCR activation: the cAMP and phosphatidylinositol signaling pathways. Both Gα s and Gα i affect cAMP-generating enzyme adenylyl cyclase (AC), whereas Gα q activates phospholipase Cβ, which divides phosphatidylinositol 4,5-bisphosphate into diacylglycerol and inositol 1,4,5-trisphosphate. Ligand binding to G s protein-coupled receptors activates AC to catalyze the conversion of ATP to cAMP. cAMP regulates the actions of the four following proteins: protein kinase A, cAMP-dependent exchange protein, cyclic nucleotide-gated channels, and Popeye domaincontaining proteins, and these specific downstream cAMP effectors play important roles in the regulation of various physiological functions [3,4], such as the regulation of hormone 2 of 17 synthesis, thyroid cell mitogenesis, bone resorption, and cardiac excitation/contraction coupling by the sympathetic nervous system [2]. Ca 2+ influx induced by activation of transient receptor potential (TRP) channels, such as TRP vanilloid subfamily member 1 (TRPV1), and by activation of mechanosensitive Piezo1 channels in odontoblasts plays a critical role in producing and transmitting sensations to dentin ('odontoblast mechanosensory/hydrodynamic receptor model') as well as in developmental, physiological, and pathological dentin formation [5][6][7]. The application of various stimuli, including thermal, osmotic, and chemical stimuli, induces membrane deformation via dentinal fluid movement. Membrane deformation is detected as a mechanical stimulus via TRPV1, TRPV2, TRPV4, TRP ankyrin subfamily member-1 (TRPA1), and Piezo channels in odontoblasts [5][6][7][8]. Ca 2+ influx via the TRP and Piezo1 channels acts as the primary intracellular signal for ATP release from pannexin-1 (PANX-1) and glutamate from glutamate-permeable anion channels. The released ATP and glutamate act as neurotransmitters and activate the P2X receptor subtype 3 and metabotropic glutamate receptors in pulpal neurons, respectively. The activation of these receptors is involved in generating dentinal pain [6,7,9,10]. Functional crosstalk between cannabinoid 1 (CB1) receptors and TRPV1 channels in odontoblasts has also been demonstrated. CB1 receptors functionally couple primarily with G i -and G s -mediated pathways to regulate intracellular cAMP levels [11]. The application of 2-arachidonyl glycerol (2-AG) (a nonselective CB1 and CB2 receptor agonist, but not a TRPV1 agonist) increases the intracellular free Ca 2+ concentration ([Ca 2+ ] i ) in rat odontoblasts. The increase in 2-AG-induced [Ca 2+ ] i is significantly inhibited by a TRPV1 and TRP melastatin subfamily member 8 (TRPM8) channel antagonist, whereas a specific TRPM8 channel antagonist does not affect the increase in [Ca 2+ ] i . In addition, this increase is significantly suppressed by a selective CB1 receptor antagonist and AC inhibitor, whereas no effect has been observed with a selective CB2 receptor antagonist. These results suggest that cAMP signals are produced by AC, which is activated by the CB1 receptor, resulting in enhanced Ca 2+ influx via TRPV1 channel activation in odontoblasts [8]. These reports imply that intracellular cAMP levels are capable of mediating Ca 2+ signaling and may participate in dentin formation and dentinal pain.
In addition, recent studies have shown that rat odontoblasts are immunoreactive to G s protein-coupled β 2 and calcitonin gene-related peptide (CGRP) receptor antibodies [12,13], and mouse odontoblasts express the parathyroid hormone receptor as observed in in situ hybridization assays [14]. The immunofluorescent expression of prostaglandin (PG) I 2 (IP) receptors [15] and the mRNA expression of dopamine (DA) D 1 (D 1 ) receptors in rat odontoblasts have also been reported [16]. Human dental pulp cells express mRNAs of all four adenosine receptor subtypes (A 1 , A 2A , A 2B , and A 3 receptors) in RT-PCR analysis. The expression levels of G s protein-coupled A 2A and G s/q protein-coupled A 2B receptor mRNA are higher than those of G i/o protein-coupled A 1 and A 3 receptors [17]. Furthermore, the existence of nerve endings that are located near odontoblasts and that contain vasoactive intestinal polypeptide (VIP) implies the expression of VPAC 1 and VPAC 2 as subtypes of VIP receptors in odontoblasts [18]. Moreover, the 5-hydroxytryptamine (5-HT) receptors are involved in enamel morphogenesis and maturation in mice [19]. Among them, 5-HT 4 is a ubiquitously expressed G s protein-coupled receptor. However, the functional expression of G s protein-coupled (IP, D 1 , A 2A , VPAC 1/2 , and 5-HT 4 ) receptors and the detailed intracellular cAMP signaling pathway following their receptor activation in odontoblasts remain unclear.
In the present study, we selected specific G s protein-coupled receptors for screening based on their known morphological and mRNA expression patterns in odontoblasts and their anatomical/functional relationships; the intracellular cAMP dynamics involving these receptors were assessed in single, living odontoblasts.

Cell Culture
A human odontoblast cell line was obtained from a healthy third molar and immortalized by transfection with the human telomerase transcriptase gene [20][21][22]. This cell line represents a pure population of cells with odontoblast properties and exhibits the mRNA expression of dentin sialophosphoprotein (DSPP), type 1 collagen, and alkaline phosphatase [20]. Human odontoblasts were cultured in basal medium (pH 7.4) (alphaminimum essential medium containing 10% fetal bovine serum, 100 U/mL penicillinstreptomycin (Thermo Fisher Scientific Inc., Waltham, MA, USA), and amphotericin B (Sigma-Aldrich, St. Louis, MO, USA)) at 37 • C in a 5% CO 2 incubator for 48 h. The odontoblast suspension was adjusted to a density of 5 × 10 4 cells/mL.

Measurement of Intracellular cAMP Level
For the live-cell cAMP sensor assay, the green upward cAMP BacMam sensor (green upward cAMP difference detector in situ; Montana Molecular, Bozeman, MT, USA) was used. The green upward cAMP BacMam sensor is a vector supplied as a nonreplicating baculovirus expressed by infecting the cells. The vector contains a gene encoding a fluorescent protein that functions as a transient cAMP-sensitive fluorescent biosensor. The fluorescence intensity of the protein increases when the vector specifically binds to cAMP in live mammalian cells. The cAMP sensor was transfected in human odontoblasts by incubation in basal medium containing 16.7% BacMam sensor and 0.4% Na-butyrate at 37 • C for 36 h. BacMam-sensor-transfected human odontoblasts were rinsed with fresh standard solution and then mounted on a dish on the stage of a microscope (IX73, Olympus, Tokyo, Japan) incorporated with HCImage software, an excited wavelength selector, and an intensified charge-coupled device camera system (Hamamatsu Photonics, Shizuoka, Japan). The green fluorescence emission was measured at 517 nm at an excitation wavelength of 506 nm. The intracellular cAMP levels were expressed as the fluorescence intensity ratio (F/F 0 unit) of the fluorescence intensity (F) to the resting value (F 0 ). The F/F 0 baseline (F/F 0 baseline ) was denoted as the mean value for 30 s before the first application of each receptor agonist and was set at 1.0. To evaluate the pharmacological effect of the G s protein-coupled receptor antagonist on its agonist-induced increase in cAMP levels, we first calculated the ∆F value by the following equation, where F/F 0 peak is a peak F/F 0 value and F/F 0 baseline is F/F 0 baseline by G s proteincoupled receptor agonist application with or without its antagonist. We then normalized the ∆F value during agonist application with (∆F antagonist ) or without (∆F agonist as 100%) antagonist application. A standard solution with or without each receptor agonist, antagonist, or SQ22536 was applied via superfusion using a rapid gravity-fed perfusion system (ValueLink8.2 Controller; AutoMate Scientific, Barkeley, CA, USA). A series of repeated applications (2 min) of each receptor agonist with or without the antagonist or SQ22536 was applied to the cells, and they were washed with standard solution until the F/F 0 value returned to baseline. All experiments were performed at 28 ± 1 • C.

Statistical Analysis
Data are expressed as the mean ± standard error of the mean of N observations, where N represents the number of independent experiments. One-way ANOVA with Tukey's post-hoc test was used to determine the parametric statistical significance. Statistical significance was set at p < 0.05. All statistical analyses were performed using GraphPad Prism 7.0 software (GraphPad Software, La Jolla, CA, USA).

Immunofluorescence Analysis of Human Odontoblast Markers
Immunoreactivity for DSPP ( Figure 1A

Human Odontoblasts Were Immunopositive for Gαs Protein and IP, 5-HT4, D1, A2A, Receptor Antibodies
Immunofluorescence analysis revealed that the human odontoblasts were im positive for Gαs protein ( Figure 2A

The IP Receptor Agonist Increased Intracellular cAMP Levels in Odontoblasts
The intracellular cAMP levels in human odontoblasts were measured using an mNeon Green-based cAMP sensor. The application of 10 nM beraprost (a potent IP receptor agonist) to human odontoblasts transiently increased intracellular cAMP levels, which reached peak values of 1.82 ± 0.11 F/F 0 units (N = 9; Figure 3A,B), 1.43 ± 0.03 F/F 0 units (N = 7; Figure 3C), or 1.30 ± 0.16 F/F 0 units (N = 4; Figure 3E). Beraprost-induced intracellular cAMP level increases were significantly and reversibly inhibited by an AC inhibitor (1 µM SQ22536). In addition, these heightened cAMP levels were significantly suppressed by a selective IP receptor antagonist (Ro1138452) to 51.8 ± 4.39% (N = 7; Figure 3C,D) with a 1 µM application and to 42.0 ± 5.55% (N = 4; Figure 3D,E) with a 10 µM application. Intracellular cAMP levels were recovered by removing SQ22536 and the IP receptor antagonist.
Biomolecules 2023, 13, x FOR PEER REVIEW 7 of 18 µM application and to 42.0 ± 5.55% (N = 4; Figure 3D,E) with a 10 µM application. Intracellular cAMP levels were recovered by removing SQ22536 and the IP receptor antagonist.
Biomolecules 2023, 13, x FOR PEER REVIEW 10 of 18 (D) denotes the mean ± SE across the number of experiments shown in parenthesis. Statistically significant differences between columns (solid lines) are indicated with asterisks: * p < 0.05.
shows the reversible effect of SQ22536. (D) Bar graphs of inhibitory effects of 50 nM ZM241385 (middle column) or 500 nM ZM241385 (lower column) on the normalized value of 100 nM PSB0777induced increases in the intracellular cAMP in the presence of ZM241385 to that in the absence of ZM241385. Each bar in (B) and (D) denotes the mean ± SE across the number of experiments shown in parenthesis. Statistically significant differences between columns (solid lines) are indicated with asterisks: * p < 0.05.

The Nonselective VIP Receptor Agonist Increased Intracellular cAMP Levels in Odontoblasts
The application of 1 nM VIP transiently increased intracellular cAMP levels to peak values of 1.25 ± 0.02 F/F0 units (N = 7; Figure 7A,B), 1.30 ± 0.01 F/F0 units (N = 11; Figure  7C), or 1.45 ± 0.06 F/F0 units (N = 3; Figure 7E). VIP-induced intracellular cAMP level increases were significantly and reversibly inhibited by an AC inhibitor (1 µM SQ22536). In addition, a nonselective VIP receptor antagonist (VIP(6-28)) significantly suppressed VIPinduced increases in intracellular cAMP level to 66.4 ± 5.48% (N = 11; Figure 7C,D) with a 10 nM application and to 55.8 ± 14.2% (N = 3; Figure 7D,E) with a 100 nM application. Intracellular cAMP levels were recovered by removing SQ22536 and the VIP receptor antagonist.    to that in the absence of VIP . Each bar in (B,D) denotes the mean ± SE across the number of experiments shown in parenthesis. Statistically significant differences between columns (shown by solid lines) are indicated with asterisks: * p < 0.05.

Discussion
Here, the functional expression of the Gα s protein and the G s protein-coupled IP, 5-HT 4 , D 1 , A 2A , and VIP receptors in human odontoblasts was demonstrated. The activation of these Gα s protein-coupled receptors regulates the AC signal transduction pathway to induce cAMP production.
Prostanoids are cyclooxygenase metabolites of arachidonic acid and include PGs, PGD 2 , PGE 2 , PGF 2α , PGI 2 , and thromboxane A 2 . These are synthesized and released upon cell stimulation and act on cells in the vicinity of their synthesis to exert their actions [26]. They also belong to a subclass of eicosanoids consisting of the prostaglandins, thromboxanes, and prostacyclins. Prostaglandins have a broad range of pathophysiological effects, including a contribution to inflammation and pain perception. The specific receptors for PGD 2 , PGE 2 , PGF 2 , PGI 2 , and TXA 2 are termed the DP, EP, FP, IP, and TP receptors, respectively [26]. In human embryonic kidney 293 (HEK293) cells expressing IP receptors, beraprost dose-dependently increased cAMP levels with a half-maximal (50%) effective concentration (EC 50 ) of 10.4 nM [27]. SQ22536 dose-dependently blocked cAMP level increase during platelet aggregation with a half-maximal (50%) inhibitory concentration (IC 50 ) of 1 µM [28]. SQ22536 (1 µM) also suppressed the 2-AG-induced increase in [Ca 2+ ] i in rat odontoblasts [8]. Ro1138452 is a potent and selective antagonist that has a high affinity for human IP receptors, but showed no significant potency for non-IP prostanoid receptors (EP 1 , EP 3 , FP, and TP receptors) [29]. The IC 50 of Ro1138452 was 100 nM in IP-receptor-expressing CHO-K1 cells [29]. However, Ro1138452 has been used at higher concentrations, such as 1-10 µM, than the IC 50 [30,31]. In this study, 10 nM beraprost, 1 µM SQ22536, and 1 µM Ro1138452 were used. Each concentration of these reagents was appropriate for activating or inhibiting the IP receptors or AC. PGI 2 synthase (PGIS) immunoreactivity has been detected in odontoblasts and has been reported to increase with mechanical stimulation and maturation [32]. An experimental force was previously applied to the maxillary first and second molars by inserting an elastic band between them for 6-72 h; thereafter, the mRNA levels of PGIS, IP receptor, and TRPV1 were shown to be upregulated in rat molar pulp. In addition, rat odontoblasts showed PGIS, IP receptor, and TRPV1 immunoreactivities in that study, indicating the contribution of the PGIS-IPreceptor-TRPV1 axis to functional cellular processes in odontoblasts [15]. Extracellular PGI 2 also potentiates/sensitizes TRPV1 channel activity via IP receptor activation and cAMP-dependent protein kinase A activity [33]. In our previous research, we found that TRPV1 channel activity (which is sensitive to mechanical stimulation) is closely involved in the generation of dentinal pain. Additionally, cAMP signals enhance Ca 2+ influx via TRPV1 channel activation in odontoblasts [8]. Although further studies are required in this regard, IP receptor activation by extracellular PGI 2 during inflammatory responses in dental pulp may modulate dentinal pain intensity by modulating mechanosensitive ion channels via cAMP signaling. Furthermore, Iloprost-a stable, long-acting PGI 2 analog-enhances the mineralization of human dental pulp cells and reactionary dentin formation [34]. Our ongoing experiments aim to clarify the contribution of the IP receptor to odontoblast cellular functions, including dentin formation and sensory transduction in dentinal sensitivity.
DA, which is synthesized by tyrosine hydroxylase (TH), is the predominant catecholamine neurotransmitter in the brain, and it affects many physiological functions, such as the control of coordinated movements and hormone secretion as well as motivational and emotional behaviors. DA binds to DA receptors (D 1-5 receptors), which are classified as either D 1 -like receptors (D 1 and D 5 receptors) or D 2 -like receptors (D 2-4 receptors) [16]. SKF83959 is a D 1 -like receptor partial agonist that has shown very high D 1 and D 5 receptor affinity (K i = 1.18 and 7.56 nM) compared to D 2 (K i = 920 nM) and D 3 (K i = 399 nM) [35]. LE300 is a potent D 1 and D 5 receptor antagonist with a K i of 1.9 and 7.5 nM, respectively, and demonstrated 20-fold selectivity for human D 1 -like receptors compared to D 2 -like receptors [36,37]. LE300 (150-5000 nM) also suppressed SKF38393-induced cAMP accumulation in HEK293 cells expressing D 1 receptors [36]. In this study, higher concentrations of SKF38393 (1 µM) and LE300 (10 µM and 100 µM) were used than those used in previous studies. These concentrations were sufficient to activate or antagonize both the D 1 -like and/or D 2 -like receptors. We showed D 1 receptor immunoreactivity in human odontoblasts in the present study. Not only D 5 receptor, but also D 2 -like receptors, might also be expressed in odontoblasts; however, further study will be needed. It has been reported that, in line with the present results, rat odontogenic stem cells express D 1 and D 3 receptors. These receptors appear to be functionally involved in tooth repair by transducing DA signals from pulp-injury-activated platelets [38]. Odontoblasts in both rat incisors and molars reportedly express DA and TH, and the DA promotes the odontoblastic differentiation of pre-odontoblasts through intracellular cAMP/protein kinase A (PKA) signaling via D 1 -like receptor activation on the odontoblast [16]. In previous studies, we have shown that Ca 2+ signals following the mechanical stimulation of odontoblasts elicit the release of ATP via pannexin-1 and glutamate via glutamate-permeable anion channels, extracellularly [6,9,39]. The released ATP, glutamate, and ADP (which is subsequently hydrolyzed from ATP) establish a paracrine signaling network between odontoblasts through the activation of ionotropic ATP (P2X) receptors, metabotropic glutamate receptors, and metabotropic ADP (P2Y) receptors, respectively; an intercellular odontoblast paracrine/autocrine communication is thereby established. The expression of both DA and TH as well as the DA receptor in odontoblasts [16], taken together with the present results, suggests that DA-induced cAMP signaling following G s protein-coupled DA receptor activation may establish intercellular odontoblast paracrine and/or autocrine communication to promote odontoblastic differentiation and dentinogenesis. However, further studies are required to confirm this hypothesis.
Four adenosine receptors (A 1 , A 2A , A 2B , and A 3 ) have been cloned in a variety of species [40]. All adenosine receptors are seven-transmembrane GPCRs linked to a variety of intercellular, intracellular, and transmembrane signal transduction pathways [40]. PSB0777 exhibited high affinity for human A 1 (Ki = 541 nM) and A 2A (Ki = 360 nM) receptors, and no or negligible affinity for A 2B and A 3 receptors, indicating high selectivity for human A 1 and A 2A receptors [41]. In addition, PSB0777 dose-dependently accumulated cAMP with an EC 50 of 117 nM in CHO cells stably expressing human A 2A receptors. The pEC 50 for cAMP production in HEK 293T cells expressing wild-type A 2A receptors was 8.1 [42]. ZM241385 had a high affinity for A 2A receptors in guinea pigs, with a pA 2 of 8.5. ZM241385 had a low affinity for A 1 and A 2B receptors in guinea pigs with a pA 2 of 7.06 and 5.95, respectively. ZM241385 has also shown a very low affinity for cloned rat A 3 receptors in CHO cells, with a pIC 50 of 3.82. Moreover, 250 nM ZM241385 reduced basolateral amygdala pyramidal cell intrinsic excitability, which is mediated by A 2A receptor activation [43]. In addition, 100 nM ZM241385 significantly prevented CA1 pyramidal neuronal damage caused by oxygen glucose deprivation in a model of cerebral ischemia [44]. Thus, 100 nM PSB0777 as well as 50 and 500 nM ZM241385, which were used in the present study, were sufficient to activate or antagonize A 2A receptors, respectively. Odontoblasts are mechanosensory receptor cells that can detect mechanical stimulation through dentinal fluid movement caused by stimuli applied to the dentin surface, and this process occurs via mechanosensitive TRP (TRPV1, TRPV2, TRPV4, and TRPA1) and Piezo1 channel activations. Ca 2+ entry via TRP/Piezo1 channels activates the release of ATP from pannexin-1 channels into the extracellular space. The released ATP then activates the P2X receptor subtype 3 on the neurons that innervate the dental pulp, establishing intercellular synaptic-like communication between odontoblasts and neurons. This communication mediates the sensory signal transduction pathway in the generation of dentinal sensitivities, known as the 'odontoblast mechanosensory/hydrodynamic receptor model' [6,7,10]. The released ATP is also hydrolyzed to ADP by nucleoside triphosphate diphosphohydrolase-2 in Schwann cells or the odontoblast membrane [45]. ADP subsequently activates P2Y receptors in trigeminal ganglion (TG) neurons that innervate the dental pulp and neighboring odontoblasts to form paracrine signals. The activation of P2Y receptors establishes not only odontoblast-odontoblast but also odontoblast-TG neuron chemical communication, which may drive reactionary dentin formation and further sensory signal transduction [5][6][7]22]. Odontoblasts are known to express alkaline phosphatase (ALP) activity, which hydrolyzes ATP to adenosine; therefore, further studies should investigate whether ALP activity in odontoblasts contributes to the hydrolysis of ATP to adenosine, which then activates odontoblast A 2A receptors to establish inter-odontoblast paracrine communication.
VIP is a neuropeptide that is widely distributed in the central and peripheral nervous systems. VIP triggers biological responses through interactions with two receptor subtypes, VPAC 1 and VPAC 2 , which are mainly coupled to the Gα s protein and stimulate cellular AC activity [46]. VIP promotes cAMP production with an EC 50 of 0.5-2 nM in COS cells (fibroblast-like cell lines derived from monkey kidney tissue) expressing chimeric VPAC 1 receptors between humans and rats [47]. Applications of VIP (1-100 nM) caused reversible increases in [Ca 2+ ] i in gonadotropin-releasing hormone (GnRH) neurons, and increases in [Ca 2+ ] i did not show dose dependency [48]. VIP , has been used as a VPAC 1 and VPAC 2 receptor antagonist in various studies [48][49][50]. Moreover, 100 nM VIP(6-28) inhibits VIP-induced increases in GnRH neuronal firing [48]. In the present study, VIP (1 nM) and VIP(6-28) (10 nM and 100 nM) were used, and the concentrations of these reagents were appropriate for activating or inhibiting the VIP receptors. Neuropeptide VIP immunoreactivity is present in human dental pulp [51] and in TG neurons. VIP-positive nerve fibers project into the odontoblastic region and in the vicinity of blood vessels. Additionally, the levels of VIP in dental pulp tissue are higher in moderately carious teeth than in non-carious and grossly carious teeth [52]. Although further studies are needed to confirm this hypothesis, the distribution of VIP in dental pulp tissue of carious teeth and/or VIP released from nerve endings near the odontoblastic region may affect odontoblasts via the VIP-VPAC 1 receptor axis, enhancing tertiary dentin formation and/or modulating the tooth pain threshold. We have previously shown that the mechanical stimulation of peptidergic C neurons of TG, which mimics increasing tissue pressure due to dental pulp inflammation, elicits CGRP release [13]. The released CGRP increases intracellular cAMP levels in rat odontoblasts via the CGRP-CGRP receptor axis, which may establish axon reflex in dental pulp. Additionally, TG neurons are capable of releasing adrenomedullin to establish intercellular neuron-endothelial cell communication as an axon reflex (our personal communication from YS). Therefore, our ongoing research will focus on the possible modification of odontoblast and/or dental pulp function(s) mediated by intercellular 'retrograde' communication from neurons to the odontoblasts via the VIP-VPAC 1 receptor axis.
Overall, further studies need to clarify the cellular function(s) mediated by intracellular cAMP signaling following G s protein-coupled receptor activation in odontoblasts, as well as to explore the origin of ligand synthesis in dental pulp, such as endothelial cells, fibroblasts, nerve terminals, or blood cells, for odontoblasts expressing the GPCR-regulated AC signal transduction pathway. Clarification of the intercellular communication pathways between ligand-releasing cells and odontoblasts is also of immense interest.
In conclusion, here, functional IP, 5-HT 4 , D 1 , A 2A , and VIP receptor expression in human odontoblasts was demonstrated, which activates the intracellular cAMP signaling pathway.