Molecular basis of dental sensitivity: The odontoblasts are multisensory cells and express multifunctional ion channels

Accepted Manuscript Title: MOLECULAR BASIS OF DENTAL SENSITIVITY: THE ODONTOBLASTS ARE MULTISENSORY CELLS AND EXPRESS MULTIFUNCTIONAL ION CHANNELS Authors: A. Sol´e-Magdalena, M. Mart´ınez-Alonso, C.A. Coronado, L.M. Junquera, J. Cobo, J.A. Vega PII: DOI: Reference:

S0940-9602(17)30126-7 https://doi.org/10.1016/j.aanat.2017.09.006 AANAT 51187

To appear in: Received date: Revised date: Accepted date:

17-4-2017 22-8-2017 10-9-2017

Please cite this article as: Sol´e-Magdalena, A., Mart´ınez-Alonso, M., Coronado, C.A., Junquera, L.M., Cobo, J., Vega, J.A., MOLECULAR BASIS OF DENTAL SENSITIVITY: THE ODONTOBLASTS ARE MULTISENSORY CELLS AND EXPRESS MULTIFUNCTIONAL ION CHANNELS.Annals of Anatomy https://doi.org/10.1016/j.aanat.2017.09.006 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

MOLECULAR BASIS OF DENTAL SENSITIVITY: THE ODONTOBLASTS ARE MULTISENSORY CELLS AND EXPRESS MULTIFUNCTIONAL ION CHANNELS

A. Solé-Magdalena1*, M. Martínez-Alonso1*, C.A. Coronado2, L.M. Junquera3,4, J. Cobo3,5, J.A. Vega1,2

1Departamento

de Morfología y Biología Celular Universidad de Oviedo, Spain de Ciencias Médicas, Universidad Autónoma de Chile, Chile 3Departamento de Especialidades Médico-Quirúrgicas, Universidad de Oviedo, Spain 4Servicio de Cirugía Maxilofacial, Hospital Universitario Central de Asturias, Oviedo, Spain 5Instituto Asturiano de Odontología, Oviedo, Spain 2Facultad

*These authors contributed equally to this paper *To whom all correspondence should be addressed José A. Vega, M.D., Ph.D. Departamento de Morfología y Biología Celular Facultad de Medicina y Ciencias de la Salud C/ Julián Clavería, 6 33006 Oviedo, Spain Email: [email protected] Abstract.- Odontoblasts are the dental pulp cells responsible for the formation of dentin. In addition, accumulating data strongly suggest that they can also function as sensory cells that mediate the early steps of mechanical, thermic, and chemical dental sensitivity. This assumption is based on the expression of different families of ion channels involved in various modalities of sensitivity and the release of putative neurotransmitters in response to odontoblast stimulation which are able to act on pulp sensory nerve fibers. This review updates the current knowledge on the expression of transient-potential receptor ion channel and acid-sensing ion channels in odontoblasts, nerve fibers innervating them and trigeminal sensory neurons, as well as 1

in pulp cells. Moreover, the innervation of the odontoblasts and the interrelationship been odontoblasts and nerve fibers mediated by neurotransmitters was also revisited. These data might provide the basis for novel therapeutic approaches for the treatment of dentine sensibility and/or dental pain.

Keywords: odontoblasts, dentine sensitivity, transient-receptor potential ion channels, acid-sensing ion channels, ATP, ATP receptors

Introduction Odontoblasts are specialized cells of the dental pulp originating from the neural crest (Mayor and Theveneau, 2013) which migrate beneath the oral epithelium and finally differentiate into cells that will organize and regulate the synthesis of the mineralized dentin matrix (Arana-Chavez and Massa, 2004; Bleicher, 2014; Kawashima and Okjii, 2016). During maturation, odontoblasts exhibit change in gene expression, acquiring a typical morphology with a body and a long process (Simon et al., 2009; Byers and Westenbroek, 2011). The cell bodies of the odontoblasts are located at the dentin-pulp interface complex where they are organized in a palisade between the mineralized tissues (enamel and dentine) and the living tissue (the pulp) of the tooth. In fact, odontoblasts are connected at their apical pole (i.e. the zones connecting the bodies with the processes) by numerous junctional complexes (tight junctions and desmosome-like) forming a selective barrier controlling the relationship and trafficking between dentin and pulp and vice versa under physiological and pathological conditions. The odontoblast processes are contained in dentinal tubules bathing in the dentinal fluid (Fig. 1; see Murray et al., 2003; Bleicher, 2014). 2

In addition to the formation of the dentin, the odontoblasts presumably mediate the early stage of sensory processes, playing a key role in mechanical, thermal, and chemical sensation, thus, in dental sensitivity and pain (Gillam, 1995; Maurin et al., 2003). Because of their location in the tooth, odontoblasts are the first targets of external stimuli (chemicals changes in dentinal fluid, thermal variations or mechanical forces), and can therefore act as sensory cells for the detection of these sensory signals. Dentin sensitivity is due to the direct exposure of dentin to mechanical, chemical and/or thermal stimuli. In this way, the

odontoblasts express

mechanosensitive, chemosensitive and thermosensitive transient receptor potential (TRP; Magloire et al., 2010) and acid-sensing (ASIC; Solé-Magdalena et al., 2012) ion channels, which are regarded as necessary to initiate those sensory processes (Delmas and Coste, 2013; Nilius and Szallasi, 2014; Ranade et al., 2015; Sharif-Naeini, 2015). Furthermore, it has been demonstrated that gating ion channels present in odontoblasts induce release of ATP, which is the primary transmitter between odontoblasts and nerve fibers (Egbuniwe et al., 2014; Liu et al., 2015; Lee et al., 2017) thus initiating the transmission of a given sensory modality to the central nervous system. This review is an update of the molecular basis of mechano-, chemical- and thermosensitivity in odontoblasts, based on the presence of ion channels, primarily belonging to the TRP and ASICs superfamilies, in these cells. The blockade of these channels would provide a novel therapeutic intervention for the treatment of dentin sensitivity and tooth pain (see El Karim et al., 2011; Alexander et al., 2013; Kweon and Shu, 2013;

3

Holzer and Izzo, 2014; Kaneko and Szallasi, 2014; Nilius and Szallasi, 2014; SousaValente et al., 2014; Baron and Lingueglia, 2015).

Innervation of the odontoblasts Nerves entering the dental pulp consist of sensory and postganglionic sympathetic fibres arising from the trigeminal and the superior sympathetic ganglia neurons, respectively. Within the dental pulp they innervate the blood vessels, the pulp cells, and the odontoblasts through fibers evolved from the so-called subodontoblastic plexus (Raschkow’s plexus; Fig. 2). Odontoblasts, both at the dentine-pulp border and within the dentinal tubules, are innervated by a dense network of myelinated Aδ and unmyelinated C sensory nerve fibers. However, the definitive structure of the contacts between the odontoblasts and the subodontoblastic plexus remains to be elucidated (see Allard et al., 2006). The Aδ fibers are principally located at the pulp-dentin border and reach the basal odontoblast layer, while the C fibers enter the dentin tubules (Byers and Närhi, 1999; Struys et al., 2007). Byers (1984) observed that more than 40% of the dentinal tubules contain nerve fibres and many of them more than one. An elegant and detailed study carried out by Cardá and Peydró (2006) in human teeth demonstrated that about 30-70% of odontoblast processes are in contact with nerve endings that enter the innermost segments of the dentinal tubules, no further than 200 µm from the pulp. They also observed that each dentinal tubule contains a single nerve fiber which varies in relationship to the odontoblast process from simple adjacency to encircling. Nevertheless, no synapse-like structures have been observed (Byers et al., 1987; Ibuki et al., 1996; Cardá and Peydró, 2006). 4

The pulp is a highly vascular tissue with a dense capillary plexus under the odontoblast layer that is innervated by postganglionic sympathetic nerve fibers (Yoshida and Ohshima, 1996). The pattern of innervation of odontoblasts is directly regulated by local nerve attractive and nerve repulsive molecules (such as nerve growth factor, glial-cell line derived neutrophic factor, sema7A, or reelin) released from the pulp cells and the odontoblasts themselves; these molecules direct the nerve terminals to appropriate sites (Mitsiadis and Luukko, 1995; Luukko et al., 1997; Maurin et al., 2004, 2005). For example, nerve growth factor and its high affinity signaling receptor TrkA regulate the development, density and maintenance of both nociceptive and postganglionic nerve fibers (Kirstein and Fariñas, 2002). Consistently, the dental pulp of TrkA-deficient mice lack nerve fibers in tooth pulp, including sympathetic fibers, calcitonin gene-related peptide and substance P nociceptive fibers (Matsuo et al., 2001; Ichikawa et al., 2004).

Dentine sensitivity and ion channels The dentine sensitivity is caused by its exposition to mechanical, chemical or thermal stimuli. When enamel is removed or injured, and the dentine tubules opened, the liquid content of the dentinal tubules, the odontoblasts, the trigeminal nerve entering the tooth, and probably also the dental pulp cells, are exposed to the sensitivizing agents. Thus, dentin sensitivity results from the activation of dental (i.e., trigeminal) sensory neurons for various mechanical, thermic or chemical stimuli which affect the dentinal fluid, the dentinal tubule content (nerves and odontoblast processes) or the

5

dental pulp cells. Nevertheless, the molecular mechanisms underlying tooth-dentine sensitivity have not been fully elucidated. Three hypotheses are currently considered to explain dentinal sensitivity. The first hypothesis (nerve theory; Fig. 3a) is based on the direct stimulation of dental nerves and the properties of trigeminal primary afferents innervating the tooth, especially the functional expression of mechanosensitive, chemosensitive or thermosensitive ion channels. The second hypothesis (hydrodynamic theory; Fig. 3b) attributes dentinal sensitivity to nerve stimulation by fluid movement within dentinal tubules: cold stimulation causes an outward fluid flow while a hot stimulation induces an inward fluid flow in dentinal tubules (Linsuwanont et al., 2007). Moreover, the pulp cells and nerves can also detect the temperature and chemical composition of the fluid (Andrew and Matthews, 2002). The third hypothesis (odontoblastic theory; Fig. 3c) is consistent with the capability of the odontoblasts to sense diverse stimuli. The presence in these cells of specific ion channels strongly suggest that they are able to detect mechanical, chemical, and thermal signals (Chung and Oh, 2013; Chung et al., 2013). These three theories are not mutually exclusive and cannot be considered separately because of the presence of nerves and odontoblast processes within the dentinal tubules, bathing in the dentinal fluid, and the close apposition of the odontoblasts to the dentinal or basal nerves terminals. Thus, external stimuli modifying the dentinal fluid induce responses in the odontoblasts, in the nerves, and in the odontoblast-nerve complex, and this may represent a unique polymodal sensory system responsible for dentine sensibility. In this context the odontoblasts play a pivotal role in signal

6

transduction, but how they sense signals and how these signals are transmitted to axons are questions which have been resolved in part only recently. Various studies in non-vertebrates and vertebrates have identified several ion channels that are responsible, or are required, for detecting a range of thermal, chemical or mechanical stimuli, and stimuli-transduction ion channels are known for most sensory modalities (Belmonte and Viana, 2008; Damann et al., 2008; Nilius and Szallasi, 2014; Jardín et al., 2017). The identification of ion channels selectively activated by different stimuli, supported the concept that the specificity of a sensory cell is determined by their expression of a particular ion channel conferring its selectivity to respond to a unique stimulus. Nevertheless, it is currently accepted that the ion channels proposed as specific transducers are not as neatly and selectively associated with the distinct types of sensibility. In fact, ion channels originally associated with the transduction of one particular form of energy are also activated by different stimuli. In addition, some ion channels associated with a specific type of stimulus are expressed in sensory cells functionally defined as specific for other sensitivities. In other words, a specific ion channel can be expressed in more than a sensory cell type, and each cell type may express more than one type of ion channel. Thus, the capacity exhibited by the different functional types of sensory cells, to preferentially detect a specific stimulus is the result of a characteristic combinatorial expression of different ion channels (Liedtke, 2007; Belmonte and Viana, 2008; Ezak et al., 2010); and this is the case for the trigeminal neurons (Vandewauw et al., 2013) and the odontoblasts (Magloire et al., 2010). 7

Several classes of ion channels have been identified in odontoblasts which are involved in nociception and other types of sensitivity, such as L-type Ca2+ channels, mechanosensitive K+ channels and voltage-gated Na+ channels (Guo and Davidson, 1998; Magloire et al., 2003, 2009, 2010; Allard et al., 2000, 2006; Solé-Magdalena et al., 2011; Ichikawa et al., 2012). Here we focused on two superfamilies of ion channels: TRP ion channels and degenerin/epithelial Na+ channels (DEG/ENa+C), particularly ASICs.

The superfamily of transient receptor potential ion channels (TRP) TRPs are integral membrane proteins that function as ion channels. They are nonselective cation channels, a few are highly Ca2+ selective and some are permeable for highly hydrated Mg2+. The TRP superfamily is subdivided into seven subfamilies: TRPC (canonical), TRPV (vanilloid), TRPM (melastatin), TRPP (polycystin), TRPML (mucolipin), TRPA (ankyrin) and TRPN (NOMPC-like); the latter being found only in invertebrates and fishes. At least 28 different TRP proteins have been identified in mammals. Structurally, a typical TRP protein contains six putative transmembrane domains (S1 to S6) with a pore-forming reentrant loop between S5 and S6. Intracellular N- and Ctermini are variable in length and consist of a variety of domains (Clapham et al., 2005; Hellmich and Gaudet, 2014; Nilius and Szallasi, 2014). This ion channel superfamily shows a variety of gating mechanisms with modes of activation ranging from ligand binding, voltage and changes in temperature to covalent modifications of nucleophilic residues (see for a review Eid and Cortright, 2009; Nilius and Owsianik, 2011; Nilius and Szallasi, 2014). 8

The superfamily of acid-sensing ion channels ASICs are Na+-selective cation channels which are voltage-insensitive and amiloridesensitive and monitor moderate deviations from the physiological values of extracellular pH (Waldmann et al., 1997; Lingueglia, 2007; Lumpkin and Caterina, 2007; Baron and Lingueglia, 2015). Six ASIC proteins encoded by four genes have been identified: ASIC1a, ASIC1b, ASIC2a, ASIC2b, ASIC3, and ASIC4 which differ in their kinetics, external pH sensitivity, tissue distribution and pharmacological properties (Krishtal, 2003). The pH values required for half-maximal activation are 6.2-6.8 for ASIC1a, 5.9-6.2 for ASIC1b, 4.9 for ASIC2a and 6.5-6.7 for ASIC3 (Kress and Waldmann et al., 2006; Hanukoglu, 2017). In addition, some ASICs may work as mechanosensors (or are required for mechanosensation) and nociception (Wemmie et al., 2006; Holzer, 2009, 2011; Sherwood et al., 2012; Zha, 2013; Holzer and Izzo, 2014; Omerbašić et al., 2015). Structurally, ASICs consist of two transmembrane domains and a large extracellular loop (Sherwood et al., 2012).

The members of these two families of ion channels exhibiting mechanosensitivity, thermosensitivity and chemosensitivity are summarized in table 1. In the following pages the occurrence of these ion channels in the odontoblast and the nerve fibres supplying them will be detailed. Regarding the trigeminal neurons, almost all the ion channels involved in all sensory modalities have been detected in these cells. In fact, in the murine trigeminal neurons 17 of the 28 TRP channel genes were detectable (TRPA1, TRPC1, TRPC3, TRPC4, TRPC5, TRPM2, TRPM3, TRPM4, TRPM5, TRPM6, 9

TRPM7, TRPM8, TRPV1, TRPV2, TRPV4, TRPML1 and TRPP2; Vandewauw et al., 2013), while in the human trigeminal ganglion 10 TRPs (TRPC1, TRPM2, TRPM3, TRPM7, TRPM8, TRPV1, TRPV2, TRPV3 , TRPV4 and TRPML1) and ASIC1-3 have been identified (Flegel et al., 2015; Fu et al., 2016).

Putative mechanosensors in odontoblasts and trigeminal afferent fibers and neurons To understand what mechanoproteins are, it is necessary to have in mind that they are not receptors and that mechanical forces are not ligands. The force is rather an “antiligand” because it can disrupt intramolecular or intermolecular interactions to trigger a conformational transmission (see Sukharev and Anishkin, 2004). Most of the ion channels candidates for mechanosensors belong to the superfamilies of DEG/ENaC and TRP ion channels (see for a review Gillespie and Walker, 2001; Lumpkin and Caterina, 2007; Arnadottir and Chalfie, 2010; Delmas and Coste, 2013; Ranade et al., 2015). Moreover, members of the two potassium pore (K2P) channels and the product of Piezo1 and Piezo2 genes have been also identified as components of mechanically activated cation channels (Coste et al., 2010; Delmas and Coste, 2013; Ranade et al., 2015). The putative mechanosensors TRPV4, TRPM3, TRPP1 and TRPP2 are expressed in cultured mouse odontoblasts (Son et al., 2009; Sato et al., 2013), whereas the adult rat odontoblasts express TRPM7, TRPC1, TRPC6, and TRPV4 (Kwon et al., 2014), and human odontoblasts display immunoreactivity for TRPV4, ASIC2 and the β- and ϒENaC, but not α-ENaC, subunits (Solé-Magdalena et al., 2011). TRPP1 and TRPP2, which

10

act together as a mechanical receptor, are present at the surface of odontoblasts, and appear to be located at the base of the primary cilium (Thivichon-Prince et al., 2009). TRPM3, TRPV4, TRPA1, ASIC3, ENaC-α and ENaC-ϒ ion channels have been detected in primary afferent dental neurons (Hermanstyne et al., 2008; Vandewauw et al., 2013).

Putative thermosensors in odontoblasts and trigeminal afferent fibers/neurons Six members of the TRP superfamily, TRPA1, TRPM8, TRPV1, TRPV2, TRPV3, and TRPV4, proposed to participate in thermosensation have been detected in sensory nerves, but only some of them have been detected in the odontoblasts or trigeminal afferent neurons. TRPV1, TRPV2, TRPV3, and TRPV4 have incompletely overlapping functions over a broad thermal range from warm to hot. While TRPA1 and TRPM8 respond to cool and cold, TRPV1 and TRPV2 are activated by painful levels of heat (>43°C and >52°C, respectively), TRPV3 and TRPV4 respond to non-painful warmth (3339°C), TRPM 8 is activated by non-painful cool temperatures (<25°C), and TRPA 1 is activated by painful cold (<18°C; Palkar et al., 2015). TRPV1 is also responsive to noxious stimuli and various chemical agents (Reid, 2005; Nieto-Posadas et al., 2011; Wetsel, 2011; Vay et al., 2012; Voets, 2012). Heat- and cold-sensing TRP channels, TRPV1, TRPV2, and TRPV3, TRPM8 and TRPA1 are functionally expressed in odontoblasts (Son et al., 2009; El Karim et al., 2011; Kim et al., 2012; Sato et al., 2013), but the results vary widely among species. Cultured mouse odontoblastic cells express TRPV1, TRPV2, TRPV3, TRPV4, and TRPM3, but not TRPM8 and TRPA1 (Son et al., 2009), while odontoblasts from adults rats express TRPA1 (Byers and Westenbroek, 2011) and lack TRPV1 or TRPV2 (Yeon et al., 2009). In 11

partial disagreement with these findings, Tsumura el al. (2012, 2013) have detected functional expression of TRPV1, TRPM8 and TRPA1 channels in rat odontoblasts. The human odontoblasts contain TRPM8, TRPA1 along with TRPV1 (Okumura et al., 2005; El Karim et al., 2011, Tazawa et al., 2017). Finally, in human immortalized dental pulp cells derived from an odontoblast phenotype, Egbuniwe et al. (2014) demonstrated expression of mRNA for TRPA1, TRPV1, and TRPV4 but not TRPM8. Nevertheless, although all these putative thermal sensors are present in odontoblasts, it is unlikely that these cells serve as thermal sensors (Yeon et al., 2009). TRPV1, TRPV2 and TRPA1 are also expressed in dental afferents whereas primary afferent trigeminal neurons express TRPV1, TRPV4, TRPA1, TRPM8 and TRPM3 (Story et al., 2003; Park et al., 2006; Chung et al., 2013). In the human dental pulp, TRPA1 was expressed in a large number of axons branching in the peripheral pulp as well as in the cell body of odontoblasts (Kim et al., 2012).

Putative chemosensors in odontoblasts and trigeminal afferent fibers and neurons The chemosensory capacity of the sensory systems relies on the appropriate expression of chemoreceptors, which detect chemical stimuli and transduce sensory information into cellular signals. The molecular mechanism of chemosensation in the trigeminal system has been revised recently by Viana (2010), but without any direct reference to the dental sensitivity. Most of the authors suggest that chemosensation is determined primarily by the chemical activation of nociceptors and thermoreceptors, and that activation by chemicals involves the direct activity of an ion channel by chemical stimuli: the so12

called ionotrophic transduction (Wood and Docherty, 1997; Lee et al., 2005). The ionotropic channels include some TRPs, ASICs and two-pore K2P channels in trigeminal neurons (Caterina et al., 1997; Tominaga and Tominaga, 2005; Bautista and Julius, 2008; Viana, 2010; Flegel et al., 2015), and at least TRPM5 in the odontoblasts (Khatibi Shahidi et al., 2015). The data about the presence of primary TRP and ASIC ion channels involved in mechanosensitivity and thermosensitivity in odontoblasts, pulpar nerve fibres and trigeminal neurons are summarized in the figure 4. Moreover, figure 5 show pictures of human odontoblasts showing immunoreactivity for some ion channels mentioned in this review.

Other mechano- and thermos-sensitive proteins in odontoblasts In addition to the above mentioned TRP and ASIC ion channels, members of the twopore domain potassium (K2p) channels family, and the product of Piezo1 and Piezo2 genes are also considered as putative mechanotransducer channels (Lumpkin and Caterina, 2007; Coste et al., 2010; Lumpkin et al., 2010; Ranade et al., 2015; SharifNaeini, 2015). K2P ion channels consist of six subfamilies within the superfamily of K+-selective channel subunits (Yost, 2003; Sabbadinia and Yost, 2009), and two members of this superfamily, TREK1 and TRAAK, are among the few channels for which a direct mechanical gating has been demonstrated (Maingret et al., 2000). TREK/TRAAK channels are co-expressed with TRP-thermo channels, and can also work as nociceptors, thermosensors and controlling pain produced by mechanical stimulation 13

(Maingret et al, 1999a, 1999b, 2000; Chemin et al, 2005; Alloui et al, 2006; Noël et al., 2009). Interestingly, TREK1 and TREK2 are present in neurons innervating the dental pulp (Hermanstyne et al., 2008), and the transcripts of TREK1 are expressed in the human odontoblasts (Magloire et al., 2003). On the other hand, it has been demonstrated that the products of Piezo1 and Piezo2 genes are rapidly adapting, mechanically activated ion channels (Ranade et al., 2014; Volkers et al., 2015). Piezo2 has been reported in trigeminal neurons innervating tissues other than dental pulp (Bron et al., 2014) and, recently, Khatibi Shahidi et al. (2015) found occurrence of Piezo2 in murine odontoblasts.

The primary cilium of the odontoblasts and its relation to TRP and ASIC ion channels Typically, the odontoblasts contain a primary cilium than is involved in odontogenesis (Thivichon-Prince et al., 2009), but also in detection of fluid movement in dentinal tissue (elicited by high pressure, osmotic, chemical or thermal stimuli) that may cause a cilium deformation thus initiating a signal transduction pathway (Magloire et al., 2004). The primary cilium is a cell system which senses and transduces mechanical and chemical stimuli (Pazour and Witman, 2003; Praetorius and Spring, 2005; Muhammad et al., 2012; Prasad et al., 2014). Both processes involve Ca2+ entry and require the presence of stretch-activated Ca2+ channels (polycystins) localized in the cilia (Delmas, 2004; Delmas et al., 2004; Praetorius and Spring, 2005). It is also noteworthy that the membrane of the cilium contains multiple sensory and channel proteins, including a variety of Ca2+-ion permeable channels that play a role in Ca2+-mediated fluid-flow mechanosensation (Anishkin and Kung, 2013; Delling et al., 2013). Very probably, 14

ASICs, which have been localized in the cilia of various tissues, are among these ion channels (Kikuchi et al., 2008, 2010; Viña et al., 2015), as well TRP channels, since the cilium is a unique Ca2+ compartment regulated by a heteromeric TRP channel (Giamarchi et al., 2006; Delling et al., 2013). On the other hand, the mechanosensory and chemosensory functions of the cilium are those of ASIC2 (see Sherwood et al., 2012), thus it can be speculated that ASIC2 participates in the ciliary function.

Intercellular signal transmission between odontoblasts and nerves How is the firing of odontoblasts transmitted to nerve endings supplying them? As previously mentioned, no specific synaptic-like structures have been observed between nerve fibers and odontoblasts even when they are in close proximity (Byers, 1984; Cardá and Peydró, 2006). Nevertheless, release of mediators from stimulated odontoblasts into the gap-space between odontoblasts and nerves, followed by signaling to dental afferents, are sine qua non conditions supporting the hypothesis that odontoblasts are sensory cells, and, thus, the odontogenic theory of dentine sensitivity. Among the proposed mediators are nitric oxide (Korkmaz et al., 2005), galanin (Suzuki et al., 2002), glutamate (Nishiyama et al., 2016) and, especially, extracellular purines such as ATP and adenosine (see for a review Lim and Mitchel, 2012; Lee et al., 2017); the process of signaling throughout purines is known as purinergic transmission. Thus, purinergic signaling refers to the process by which purines and pyrimidines mediate cellular responses following stimulation of specific receptors. Every cell has a supply of cytoplasmic ATP that can be released under physiological conditions through fusion of 15

ATP-containing vesicles with the plasma membrane in a classic release pathway. Also, ATP can passively efflux out of the cell through permeant membrane pores or channels (anion channels, connexin and pannexin hemichannels), or via a hybrid mechanism throughout Ca2+-dependent fusion of vesicles containing permeant channels with the plasma membrane (Reigada and Mitchell, 2005). Odontoblasts expressed the gap junction protein connexin43, or pannexin-1 which can form transmembrane hemichannels for ATP release (Liu et al., 2012; Fig. 6). Multiple release mechanisms may exist within the same cell able to be activated by different stimuli, although ATP-release is frequently triggered by mechanical stimuli (Xia et al., 2012). Released ATP acts over specific receptors and is then rapidly dephosphorylated by a series of extracellular enzymes converting ATP directly into AMP and pyrophosphate (Goding et al., 2003; Zimmermann, 2006). The purinergic receptors fall into two main categories termed P1 and P2 receptors (Burnstock, 1978). P1 are receptors for adenosine, and P2 receptors for ATP. P1 receptors are G protein-coupled receptors. P2 receptors are subclassified into ionotropic cation channels P2X receptors and G-protein-coupled P2Y receptors. To date, seven P2X receptors (P2X1R-7R) and eight P2Y receptors (P2Y1R, P2Y2R, P2Y4R, P2Y6R, P2Y11R, P2Y12R, P2Y13R and P2Y14R) have been identified in mammals (Burnstock, 2006, 2006; Coddou et al., 2011; von Kugelgen and Harden, 2011). The P2X receptors, differ in their rates of inactivation and sensitivity to ATP, the P2X7R requiring the highest concentrations of ATP for activation and inactivating most slowly (North, 2002). P2XRs are expressed in the nociceptive trigeminal ganglion cells (Staikopoulos et al., 2007; Kim et al., 2008) as well as in the dental pulp cells (Alavi et 16

al., 2001; Renton et al., 2003; Wang et al., 2016). Moreover, myelinated and unmyelinated P2X positive nerve fibers were detected in the subodontoblastic plexus in close association with odontoblasts (Cook et al., 1997; Alavi et al., 2001; Sharma and Pradeep, 2006). In rats, P2XR2 and P2XR3 receptors were found in both pulp nerves and a subpopulation of trigeminal ganglion neurons (Matsuka et al., 2001; Jiang and Gu, 2002; Staikopoulos et al., 2007; Chung et al., 2008; Kuroda et al., 2012). Furthermore, odontoblast themselves express several P2XRs of different subtypes (Lee et al., 2017; Shiozaki et al., 2017) thus suggesting that ATP might regulate the physiology of the odontoblasts throughout autocrine-paracrine mechanisms, since inhibition of interodontoblastic communication can be reached blocking of ATP release (Lee et al., 2017). Furthermore, P2Y receptor subtypes are present in pulp cells (Wang et al., 2016), trigeminal neurons (Li et al., 2014; Kawaguchi et al., 2015) and trigeminal satellite glial cells (Magni et al, 2015), as well as in odontoblasts (Sato et al., 2015; Wang et al., 2016). It has been traditionally accepted that mechanical distortion or direct stimulation of the odontoblasts lead to ATP release, presumably acting via ion channels. The activation of TRPA1 and TRPV4, but not TRPV1, in human odontoblast-like cells causes an increase in ATP release than can be abolished by selective TRP antagonists (Egbuniwe et al., 2014). Also, following mechanical stimuli activity of TRPV1, TRPV3, TRPV4 and TRPA1, but not TRPM8, there is a release of ATP via pannexina-1 that transmits a signal to P2X3 receptors of trigeminal neurons (Shibukawa et al., 2013, 2015; Sato et al., 2015; Fig. 6).

17

Future perspectives: TRPs, ASICs, and the ATP signaling system as targets for the treatment of dentin sensitivity The expression of mechanosensing, thermosensing and chemosensing ion channels by odontoblasts; the close relationships between odontoblasts and nerve fibers; the release of ATP by the odontoblasts in response to, at least, mechanical stimulation; the existence of an enzymatic apparatus for ATP degradation in dental pulp; and the expression of ATP receptors in dental afferents, strongly suggests that ATP might act as a signaling molecule between odontoblasts and neurons in the sensory transduction sequence for dentinal sensibility (Lim and Mitchell, 2012; Shibukawa et al., 2015). Taken together, these steps in the transmission of signals demonstrate the key role of odontoblasts as sensory cells, lending support to the odontoblastic theory of dentine sensitivity (Fig. 6). Consistenty, the disruption of these sequential events using appropriate pharmacological modulators for TRP and ASIC ion channels (El Karim et al., 2011; Alexander et al., 2013) or prurinergic transmission (Burnstock, 2006,2009) would provide a novel therapeutic approach in the treatment of dentine sensitivity.

18

References Alavi AM, Dubyak GR, Burnstock G. Immunohistochemical evidence for ATP receptors in human dental pulp. J Dent Res. 2001; 80: 476-83. Alexander SP, Benson HE, Faccenda E, Pawson AJ, Sharman JL, Catterall WA, Spedding M, Peters JA, Harmar AJ; CGTP Collaborators. The Concise Guide to PHARMACOLOGY 2013/14: ion channels. Br J Pharmacol. 2013; 170:1607-51. Allard B, Couble ML, Magloire H, Bleicher F. Characterization and gene expression of high

conductance

calcium-activated

potassium

channels

displaying

mechanosensitivity in human odontoblasts. J Biol Chem. 2000; 275:25556-61. Allard B, Magloire H, Couble ML, Maurin JC, Bleicher F. Voltage-gated sodium channels confer excitability to human odontoblasts: possible role in tooth pain transmission. J Biol Chem. 2006; 281: 29002-10. Alloui A, Zimmermann K, Mamet J, Duprat F, Noël J, Chemin J, Guy N, Blondeau N, Voilley N, Rubat-Coudert C, Borsotto M, Romey G, Heurteaux C, Reeh P, Eschalier A, Lazdunski M. TREK-1, a K+ channel involved in polymodal pain perception. EMBO J. 2006; 25: 2368-76. Andrew D, Matthews B. Properties of single nerve fibres that evoke blood flow changes in cat dental pulp. J Physiol. 2002. 542:921-8. Anishkin A, Kung C. Stiffened lipid platforms at molecular force foci. Proc Natl Acad Sci USA 2013; 110:4886-92. Arana-Chavez VE, Massa LF. Odontoblasts: the cells forming and maintaining dentine. Int J Biochem Cell Biol. 2004; 36:1367-73.

19

Arnadottir J, Chalfie M. Eukaryotic mechanosensitive channels. Annu Rev Biophys. 2010; 39: 111-37. Baron A, Lingueglia E. Pharmacology of acid-sensing ion channels - Physiological and therapeutical perspectives. Neuropharmacology 2015; 94:19-35. Bautista D, Julius D. Fire in the hole: pore dilation of the capsaicin receptor TRPV1. Nat Neurosci. 2008; 11:528-9. Belmonte C, Viana F. Molecular and cellular limits to somatosensory specificity. 2008; 4:14. Bleicher F. Odontoblast physiology. Exp Cell Res. 2014; 325:65-71. Bron R, Wood RJ, Brock JA, Ivanusic JJ. Piezo2 expression in corneal afferent neurons. J Comp Neurol. 2014; 522:2967-79. Burnstock G. A basis for distinguishing two types of purinergic receptor. In: Cell membrane receptors for drugs and hormones: a multidisciplinary approach. Straub RW, Bolis L, editors. New York, NY: Raven Press, pp. 107-118, 1978. Burnstock G. Purinergic P2 receptors as targets for novel analgesics. Pharmacol Ther. 2006; 110:433-54. Burnstock G. Purines and sensory nerves. Handb Exp Pharmacol. 2009; 194:333-92. Byers MR. Dental sensory receptors. Int Rev Neurobiol. 1984; 25:39-94. Byers MR, Närhi MV. Dental injury models: experimental tools for understanding neuroinflammatory interactions and polymodal nociceptor functions Crit Rev Oral Biol Med. 1999; 10: 4-39. Byers MR, Närhi MV, Dong WK. Sensory innervation of pulp and dentin in adult dog teeth as demonstrated by autoradiography. Anat Rec. 1987; 218: 207-15. 20

Byers MR, Westenbroek RE. Odontoblasts in developing, mature and ageing rat teeth have multiple phenotypes that variably express all nine voltage-gated sodium channels. Arch Oral Biol. 2011; 56:1199-220. Cardá C, Peydró A. Ultrastructural patterns of human dentinal tubules, odontoblasts processes and nerve fibres. Tissue Cell. 2006; 38: 141-50. Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D. The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 1997; 389:816-24. Chemin J, Patel AJ, Duprat F, Lauritzen I, Lazdunski M, Honoré E. A phospholipid sensor controls mechanogating of the K+ channel TREK-1. EMBO J 2005; 24: 44-53. Chung MK, Güler AD, Caterina MJ. TRPV1 shows dynamic ionic selectivity during agonist stimulation. Nat Neurosci. 2008; 11:555-64. Chung G, Jung SJ, Oh SB. Cellular and molecular mechanisms of dental nociception. J Dent Res. 2013; 92: 948-55. Chung G, Oh BS. TRP channels in dental pain. Open Pain J. Supp 1 2013; 6: 31-6. Clapham DE, Julius D, Montell C, Schultz G. International Union of Pharmacology. XLIX. Nomenclature and structure-function relationships of transient receptor potential channels. Pharmacol Rev. 2005; 57: 427-50. Coddou C, Yan Z, Obsil T, Huidobro-Toro JP, Stojilkovic SS. Activation and regulation of purinergic P2X receptor channels. Pharmacol Rev. 2011; 63:641-83. Cook SP, Vulchanova L, Hargreaves KM, Elde R, McCleskey EW. Distinct ATP receptors on pain-sensing and stretch-sensing neurons. Nature 1997; 387:505-8.

21

Coste B, Mathur J, Schmidt M, Earley TJ, Ranade S, Petrus MJ, Dubin AE, Patapoutian A. Piezo1 and Piezo2 are essential components of distinct mechanically activated cation channels. Science 2010; 330: 55-60. Damann N, Voets T, Nilius B. TRPs in our senses. Curr Biol 2008; 18:R880-R889. Delling M, DeCaen PG, Doerner JF, Febvay S, Clapham DE. Primary cilia are specialized calcium signalling organelles. Nature 2013; 504: 311-4. Delmas P. Polycystins: from mechanosensation to gene regulation. Cell 2004; 118:1458. Delmas P, Coste B.

Mechano-gated ion channels in sensory systems. Cell 2013;

155:278-84. Delmas P Padilla F, Osorio N, Coste B, Raoux M, Crest M. Polycystins, calcium signaling, and human diseases. Biochem Biophys Res Commun. 2004; 322:1374-83. Eid SR, Cortright DN. Transient receptor potential channels on sensory nerves. Handb Exp Pharmacol. 2009; 194, 261-81. Egbuniwe O, Grover S, Duggal AK, Mavroudis A, Yazdi M, Renton T, Di Silvio L, Grant AD. TRPA1 and TRPV4 activation in human odontoblasts stimulates ATP release. J Dent Res. 2014; 93: 911-7. El Karim IA, Linden GJ, Curtis TM, About I, McGahon MK, Irwin CR, Lundy FT. Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity. Pain 2011; 152: 2211-23. Ezak MJ, Hong E, Chaparro-Garcia A, Ferkey DM. Caenorhabditis elegans TRPV channels function in a modality-specific pathway to regulate response to aberrant sensory signaling. Genetics 2010; 185:233-44. 22

Flegel C, Schöbel N, Altmüller J, Becker C, Tannapfel A, Hatt H, Gisselmann G. RNA-Seq Analysis of Human Trigeminal and Dorsal Root Ganglia with a Focus on Chemoreceptors. PLoS One 2015; 10:e0128951. Fu H, Fang P, Zhou HY, Zhou J, Yu XW, Ni M, Zheng JY, Jin Y, Chen JG, Wang F, Hu ZL. Acid-sensing ion channels in trigeminal ganglion neurons innervating the orofacial region contribute to orofacial inflammatory pain. Clin Exp Pharmacol Physiol. 2016; 43:193-202. Giamarchi A, Padilla F, Coste B, Raoux M, Crest M, Honoré E, Delmas P. The versatile nature of the calcium-permeable cation channel TRPP2. EMBO Rep 2006; 7:787-93. Gillam DG. Mechanisms of stimulus transmission across dentin- a review. J West Soc Periodontol Periodontal Abstr 1995; 43: 53-65. Gillespie PG, Walker RG. Molecular basis of mechanosensory transduction. Nature 2001; 413: 194-202. Goding JW, Grobben B, Slegers H. Physiological and pathophysiological functions of the ecto-nucleotide pyrophosphatase/phosphodiesterase family. Biochim Biophys Acta 2003; 1638:1-19 Guo L, Davidson RM. Potassium and chloride channels in freshly isolated rat odontoblasts. J Dent Res. 1998; 77: 341-50. Hanukoglu I. ASIC and ENaC type sodium channels: conformational states and the structures of the ion selectivity filters. FEBS J. 2017; 284:525-45 Hellmich UA, Gaudet R. Structural biology of TRP channels. Handb Exp Pharmacol. 2014; 23:963-90.

23

Hermanstyne TO, Markowitz K, Fan L, Gold MS. Mechanotransducers in rat pulpal afferents. J Dent Res. 2008; 87: 834-8. Holzer P. Acid-sensitive ion channels and receptors. Handb Exp Pharmacol. 2009; 194: 283-332. Holzer P. Acid sensing by visceral afferent neurones. Acta Physiol. 2011; 201: 63-75. Holzer P, Izzo AA. The pharmacology of TRP channels. Br J Pharmacol. 2014; 171: 246973. Ibuki T, Kido MA, Kiyoshima T, Terada Y, Tanaka T. An ultrastructural study of the relationship between sensory trigeminal nerves and odontoblasts in rat dentin/pulp as demonstrated by the anterograde transport of wheat germ agglutininhorseradish peroxidase (WGA-HRP). J Dent Res. 1996; 75:1963-70. Ichikawa H, Matsuo S, Silos-Santiago I, Jacquin MF, Sugimoto T. The development of myelinated nociceptors is dependent upon trks in the trigeminal ganglion. Acta Histochem. 2004; 106: 337-43. Ichikawa H, Kim HJ, Shuprisha A, Shikano T, Tsumura M, Shibukawa Y, Tazaki M. Voltage-dependent sodium channels and calcium-activated potassium channels in human odontoblasts in vitro. J Endod. 2012; 38: 1355-62. Jardín I, López JJ, Diez R, Sánchez-Collado J, Cantonero C, Albarrán L, Woodard GE, Redondo PC, Salido GM, Smani T, Rosado JA. TRPs in Pain Sensation. Front Physiol. 2017; 8:392. Jiang J, Gu J. Expression of adenosine triphosphate P2X3 receptors in rat molar pulp and trigeminal ganglia. Oral Surg Oral Med Oral Pathol Oral Radiol Endod. 2002; 94:622-6. 24

Kaneko Y, Szallasi A. Transient receptor potential (TRP) channels: a clinical perspective. Br J Pharmacol. 2014; 171: 2474-507. Kawaguchi A, Sato M, Kimura M, Ichinohe T, Tazaki M, Shibukawa Y. Expression and function of purinergic P2Y12 receptors in rat trigeminal ganglion neurons. Neurosci Res. 2015; 98:17-27. Kawashima N, Okiji T. Odontoblasts: Specialized hard-tissue-forming cells in the dentinpulp complex. Congenit Anom (Kyoto). 2016; 56:144-53. Khatibi Shahidi M, Krivanek J, Kaukua N, Ernfors P, Hladik L, Kostal V, Masich S, Hampl A, Chubanov V, Gudermann T, Romanov RA, Harkany T, Adameyko I, Fried K. Threedimensional Imaging Reveals New Compartments and Structural Adaptations in Odontoblasts. J Dent Res. 2015; 94:945-54. Kikuchi S, Ninomiya T, Kawamata T, Ogasawara N, Kojima T, Tachi N, Tatsumi H. The acid-sensing ion channel 2 (ASIC2) of ciliated cells in the developing rat nasal septum. Arch Histol Cytol. 2010; 73:81-9. Kikuchi S, Ninomiya T, Kawamata T, Tatsumi H. Expression of ASIC2 in ciliated cells and stereociliated cells. Cell Tissue Res. 2008; 333:217-24. Kim YS, Jung HK, Kwon TK, Kim CS, Cho JH, Ahn DK, Bae YC. Expression of transient receptor potential ankyrin 1 in human dental pulp. J Endod. 2012; 38: 1087-92. Kim YS, Paik SK, Cho YS, Shin HS, Bae JY, Moritani M, Yoshida A, Ahn DK, Valtschanoff J, Hwang SJ, Moon C, Bae YC. Expression of P2X3 receptor in the trigeminal sensory nuclei of the rat. J Comp Neurol, 2008; 506:627-39. Kirstein M, Fariñas I. Sensing life: regulation of sensory neuron survival by neurotrophins. Cell Mol Life Sci. 2002; 59: 1787-802. 25

Korkmaz Y, Baumann MA, Steinritz D, Schröder H, Behrends S, Addicks K, Schneider K, Raab WH, Bloch W (2005) NO-cGMP signaling molecules in cells of the rat molar dentin-pulp complex. J Dent Res 84:618-623. Kress M, Waldmann R. Acid sensing ionic channels. Curr Top Membr. 2006; 57:241-76. Krishtal O. The ASICs: signaling molecules? Modulators? Trends Neurosci. 2003; 26: 477-83. Kuroda H, Shibukawa Y, Soya M, Masamura A, Kasahara M, Tazaki M, Ichinohe T. Expression of P2X₁ and P2X₄ receptors in rat trigeminal ganglion neurons. Neuroreport. 2012; 23:752-6. Kweon HJ, Suh BC. Acid-sensing ion channels (ASICs): therapeutic targets for neurological diseases and their regulation. BMB Rep. 2013; 46:295-304. Kwon M, Baek SH, Park CK, Chung G, Oh SB. Single-cell RT-PCR and immunocytochemical detection of mechanosensitive transient receptor potential channels in acutely isolated rat odontoblasts. Arch Oral Biol. 2014; 59:1266-71. Lee BM, Jo H, Park G, Kim YH, Park CK, Jung SJ, Chung G, Oh SB. Extracellular ATP Induces Calcium Signaling in Odontoblasts. J Dent Res. 2017; 96:200-7. Lee Y, Lee CH, Oh U. Painful channels in sensory neurons. Mol Cells. 2005; 20:315-24. Li N, Lu ZY, Yu LH, Burnstock G, Deng XM1, Ma B. Inhibition of G protein-coupled P2Y2 receptor induced analgesia in a rat model of trigeminal neuropathic pain. Mol Pain. 2014; 10:21. Liedtke

WB.

Chapter

22:

TRPV

Channels’

Function

in

Osmo-

and

Mechanotransduction. In: Liedtke WB, Heller S, editors. TRP Ion Channel

26

Function in Sensory Transduction and Cellular Signaling Cascades. Boca Raton (FL): CRC Press, 2007. Lim JC, Mitchell CH. Inflammation, Pain, and Pressure—Purinergic Signaling in Oral Tissues. J Dent Res. 2012; 91: 1103-9. Lingueglia E. Acid-sensing ion channels in sensory perception. J Biol Chem. 2007; 282: 17325-9. Linsuwanont P, Palamara JEA, Messer HH. An investigation of thermal stimulation in intact teeth. Arch Oral Biol. 2007; 52: 218-27. Liu X, Yu L, Wang Q, Pelletier J, Fausther M, Sévigny J, Malmström HS, Dirksen RT, Ren YF. Expression of ecto-ATPase NTPDase2 in human dental pulp. J Dent Res. 2012; 91:261-7. Liu X, Wang C, Fujita T, Malmstrom HS, Nedergaard M, Ren YF, Dirksen RT. External Dentin Stimulation Induces ATP Release in Human Teeth. J Dent Res. 2015; 94:1259-66. Lumpkin EA, Caterina MJ. Mechanisms of sensory transduction in the skin. Nature 2007; 445: 858-65. Lumpkin EA, Marshall KL, Nelson AM. The cell biology of touch. J Cell Biol. 2010; 191:237-48. Luukko K, Suvanto P, Saarma M, Thesleff I. Expression of GDNF and its receptors in developing tooth is developmentally regulated and suggests multiple roles in innervation and organogenesis. Dev Dyn. 1997; 210: 463-71. Magloire H, Couble ML, Romeas A, Bleicher F. Odontoblast primary cilia: facts nd hypotheses. Cell Biol Int. 2004; 28: 93-9. 27

Magloire H, Couble ML, Thivichon-Prince B, Maurin JC, Bleicher F. Odontoblast: a mechano-sensory cell. J Exp Zool B Mol Dev Evol. 2009; 312B:416-24. Magloire H, Lesage F, Couble ML, Lazdunski M, Bleicher F. Expression and localization of TREK-1 K+ channels in human odontoblasts. J Dent Res. 2003; 82:542-5. Magloire H, Maurin JC, Couble ML, Shibukawa Y, Tsumura M, Thivichon-Prince B, Bleicher F. Topical review. Dental pain and odontoblasts: facts and hypotheses. J Orofac Pain 2010; 24: 335-49. Magni G, Merli D, Verderio C, Abbracchio MP, Ceruti S. P2Y2 receptor antagonists as anti-allodynic agents in acute and sub-chronic trigeminal sensitization: role of satellite glial cells. Glia. 2015; 63:1256-69. Maingret F, Fosset M, Lesage F, Lazdunski M, Honoré E. TRAAK is a mammalian neuronal mechano-gated K+ channel. J Biol Chem. 1999a; 274: 1381-7. Maingret F, Lauritzen I, Patel AJ, Heurteaux C, Reyes R, Lesage F, Lazdunski M, Honoré E. TREK-1 is a heat-activated background K(+) channel. EMBO J. 2000; 19: 2483-91. Maingret F, Patel AJ, Lesage F, Lazdunski M, Honoré E. Mechano- or acid stimulation, two interactive modes of activation of the TREK-1 potassium channel. J Biol Chem. 1999b; 274: 26691-6. Matsuka Y, Neubert JK, Maidment NT, Spigelman I. Concurrent release of ATP and substance P within guinea pig trigeminal ganglia in vivo. Brain Res. 2001; 915:24855 Matsuo S, Ichikawa H, Henderson TA, Silos-Santiago I, Barbacid M, Arends JJ, Jacquin MF. trkA modulation of developing somatosensory neurons in oro-facial tissues:

28

tooth pulp fibers are absent in trkA knockout mice. Neuroscience 2001; 105: 74760. Maurin JC, Couble ML, Didier-Bazes M, Brisson C, Magloire H, Bleicher H. Expression and localization of reelin in human odontoblasts. Matrix Biol. 2004; 23: 277-85. Maurin JC, Couble ML, Thivichon-Prince B, Magloire H. Odontoblast: a key cell involved in the perception of dentinal pain. Med Sci (Paris) 2003; 29: 293-9. Article in French Maurin JC, Delorme G, Machuca-Gayet I, Couble ML, Magloire H, Jurdic P, Bleicher F. Odontoblast expression of semaphorin 7A during innervation of human dentin. Matrix Biol. 2005; 24: 232-8. Mayor R, Theveneau E. The neural crest. Development 2013; 140:2247-51. Mitsiadis T, Luukko K. Neurotrophins in odontogenesis. Int J Dev Biol. 1995; 39: 195202. Muhammad H, Rais Y, Miosge N, Ornan EM. The primary cilium as a dual sensor of mechanochemical signals in chondrocytes. Cell Mol Life Sci. 2012; 69:2101-7. Murray PE, About I, Lumley PJ, Franquin JC, Windsor LJ, Smith AJ. Odontoblast morphology and dental repair. J Dent. 2003; 31: 75-82. Nieto-Posadas A, Jara-Oseguera A, Rosenbaum T. TRP channel gating physiology. Curr Top Med Chem. 2011; 11: 2131-50. Nilius B, Owsianik G. The transient receptor potential family of ion channels. Genome Biol. 2011; 12, 218. Nilius B, Szallasi A. Transient receptor potential channels as drug targets: from the science of basic research to the art of medicine. Pharmacol Rev. 2014; 66: 676-814.

29

Nishiyama A, Sato M, Kimura M, Katakura A, Tazaki M, Shibukawa Y. Intercellular signal communication among odontoblasts and trigeminal ganglion neurons via glutamate. Cell Calcium. 2016; 60:341-55. Noël J, Zimmermann K, Busserolles J, Deval E, Alloui A, Diochot S, Guy N, Borsotto M, Reeh P, Eschalier A, Lazdunski M. The mechano-activated K+ channels TRAAK and TREK-1 control both warm and cold perception. EMBO J. 2009; 28:1308-18. North RA. Molecular physiology of P2X receptors. Physiol Rev. 2002; 82:1013-67. Okumura R, Shima K, Muramatsu T, Nakagawa K, Shimono M, Suzuki T, Magloire H, Shibukawa Y. The odontoblast as a sensory receptor cell? The expression of TRPV1 (VR-1) channels. Arch Histol Cytol. 2005; 68: 251-7. Omerbašić D, Schuhmacher LN, Bernal Sierra YA, Smith ES, Lewin GR. ASICs and mammalian mechanoreceptor function. Neuropharmacology 2015; 94:80-6. Palkar R, Lippoldt EK, McKemy DD. The molecular and cellular basis of thermosensation in mammals. Curr Opin Neurobiol. 2015; 34:14-9. Park CK, Kim MS, Fang Z, Li HY, Jung SJ, Choi SY, Lee SJ, Park K, Kim JS, Oh SB. Functional expression of thermo-transient receptor potential channels in dental primary afferent neurons: implication for tooth pain. J Biol Chem. 2006; 281:1730411. Pazour GJ, Witman GB. The vertebrate primary cilium is a sensory organelle. Curr Opin Cell Biol. 2003; 15: 105-10. Praetorius HA, Spring KR. A physiological view of the primary cilium. Annu Rev Physiol. 2005; 67: 515-29.

30

Prasad RM, Jin X, Nauli SM. Sensing a sensor: identifiying the mechanosensory function of primary cilia. Biosensors 2014; 2014; 4: 47-62. Ranade SS, Syeda R, Patapoutian A. Mechanically Activated Ion Channels. Neuron 2015; 87:1162-79. Ranade SS, Woo SH, Dubin AE, Moshourab RA, Wetzel C, Petrus M, Mathur J, Bégay V, Coste B, Mainquist J, Wilson AJ, Francisco AG, Reddy K, Qiu Z, Wood JN, Lewin GR, Patapoutian A. Piezo2 is the major transducer of mechanical forces for touch sensation in mice. Nature 2014; 516:121-5. Reid G. ThermoTRP channels and cold sensing: what are they really up to? Pflugers Arch. 2005; 451: 250-63. Reigada D, Mitchell CH. (2005). Release of ATP from RPE cells involves both CFTR and vesicular transport. Am J Physiol Cell Physiol. 2005; 288:C132-C140. Renton T, Yiangou Y, Baecker PA, Ford AP, Anand P. Capsaicin receptor VR1 and ATP purinoceptor P2X3 in painful and nonpainful human tooth pulp. J Orofac Pain. 2003; 17:245-50. Sabbadinia M, Yost C. Molecular Biology of Background K Channels: Insights from K2P Knockout Mice. J Mol Biol. 2009; 385: 1331-44. Sato M, Furuya T, Kimura M, Kojima Y, Tazaki M, Sato T, Shibukawa Y. Intercellular Odontoblast Communication via ATP Mediated by Pannexin-1 Channel and Phospholipase C-coupled Receptor Activation. Front Physiol. 2015; 6:326. Sato M, Sobhan U, Tsumura U, Kuroda H, Soya M, Masamura A, Nishiyama A, Katakura A, Ichinohe T, Tazaki M, Shibukawa Y. Hypotonic-induced stretching of plasma

31

membrane activates transient receptor potential vanilloid channels and sodium– calcium exchangers in mouse odontoblasts. J Endod. 2013; 39: 779-87. Sharif-Naeini R. Contribution of mechanosensitive ion channels to somatosensation. Prog Mol Biol Transl Sci. 2015; 131:53-71 Sharma CG, Pradeep AR. Gingival crevicular fluid osteopontin levels in periodontal health and disease. J Periodontol. 2006; 77:1674-80 Sherwood TW, Frey EN, Askwith CC. Structure and activity of the acid-sensing ion channels. Am J Physiol Cell Physiol. 2012; 303:C699-C710. Shibukawa Y, Sato M, Kimura M, Sobhan U, Shimada M, Nishiyama A, Kawaguchi A, Soya M, Kuroda H, Katakura A, Ichinohe T, Tazaki M. Odontoblasts as sensory receptors: transient receptor potential channels, pannexin-1, and ionotropic ATP receptors mediate intercellular odontoblast-neuron signal transduction. Pflugers Arch. 2015; 467: 843-63. Shibukawa Y, Sato M, Tsumura M, Kuroda H, Sobhan U, Tazaki M. Odontoblast as sensory receptor cell: TRP channels, pannexin 1 and P2×3 receptor coupling mediate sensory transduction in dentin, in: Proceedings of the Tooth, Morphogenesis and Differentiation Meeting, La Londe Les Maures, France, May 21– 26, 2013. Shiozaki Y, Sato M, Kimura M, Sato T, Tazaki M, Shibukawa Y. Ionotropic P2X ATP Receptor Channels Mediate Purinergic Signaling in Mouse Odontoblasts. Front Physiol. 2017; 8:3. Simon S, Smith AJ, Lumley PJ, Berdal A, Smith G, Finney S, Cooper PR. Molecular characterization of young and mature odontoblasts. Bone 2009; 45: 693-703. 32

Solé-Magdalena A, Revuelta EG, Menénez-Díaz I, Calavia MG, Cobo T, García-Suárez O, Pérez-Piñera P, De Carlos F, Cobo J, Vega JA. Human odontoblasts express transient receptor protein and acid-sensing ion channel mechanosensor proteins. Microsc Res Tech. 2011; 74: 457-63. Son AR, Yang YM, Hong JH, Lee SI, Shibukawa Y, Shin DM. Odontoblast TRP channels and thermo/mechanical transmission. J Dent Res. 2009; 88: 1014-9. Sousa-Valente J, Andreou AP, L Urban L, Nagy. Transient receptor potential ion channels in primary sensory neurons as targets for novel analgesics. Br J Pharmacol. 2014; 171: 2508-27. Staikopoulos V, Sessle BJ, Furness JB, Jennings EA. Localization of P2X2 and P2X3 receptors in rat trigeminal ganglion neurons. Neuroscience 2007; 144:208-16 Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A. ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 2003; 112: 819-29. Struys T, De Peralta T, Vandenabeele F, Raab WHM, Politis C, Schepers S, Vrienlinck L, Lambrichts I. Neuro-odontonlast relationships. Chin J Dent Res. 2007; 10: 7-13. Sukharev S, Anishkin A. Mechanosensitive channels: what can we learn from 'simple' model systems? Trends Neurosci. 2004; 27: 345-51. Suzuki H, Iwanaga T, Yoshie H, Li J, Yamabe K, Yanaihara N, Langel U, Maeda T. Expression of galanin receptor-1 (GALR1) in the rat trigeminal ganglia and molar teeth. Neurosci Res. 2002; 42:197-207.

33

Tazawa K, Ikeda H, Kawashima N, Okiji T. Transient receptor potential melastatin (TRPM) 8 is expressed in freshly isolated native human odontoblasts. Arch Oral Biol. 2017; 75:55-61. Thivichon-Prince B, Couble ML, Giamarchi A, Delmas P, Franco B, Romio L, Struys T, Lambrichts I, Ressnikoff D, Magloire H, Bleicher F. Primary cilia of odontoblasts: possible role in molar morphogenesis. J Dent Res. 2009; 88: 910-5. Tominaga M, Tominaga T. Structure and function of TRPV1. Pflugers Arch. 2005; 451:143-50. Tsumura M, Sobhan U, Muramatsu T, Sato M, Ichikawa H, Sahara Y, Tazaki M, Shibukawa Y. TRPV1-mediated calcium signal couples with cannabinoid receptors and sodium-calcium exchangers in rat odontoblasts. Cell Calcium 2012; 52: 124-36. Tsumura M, Sobhan U, Sato M, Shimada M, Nishiyama A, Kawaguchi A, Soya M, Kuroda H, Tazaki M, Shibukawa Y. Functional expression of TRPM8 and TRPA1 channels in rat odontoblasts. PLoS One 2013; 8: e82233. Vandewauw I, Owsianik G, Voets T. Systematic and quantitative mRNA expression analysis of TRP channel genes at the single trigeminal and dorsal root ganglion level in mouse. BMC Neurosci. 2013; 14:21. Vay L, Gu C, McNaughton PA. The thermo-TRP ion channel family: properties and therapeutic implications. Br J Pharmacol. 2012; 165:787-801. Viana F. Chemosensory properties of the trigeminal system. ACS Chem Neurosci 2010; 2: 38-50. Viña E, Parisi V, Abbate F, Cabo R, Guerrera MC, Laurà R, Quirós LM, Pérez-Varela JC, Cobo T, Germanà A, Vega JA, García-Suárez O. Acid-sensing ion channel 2 (ASIC2) is 34

selectively localized in the cilia of the non-sensory olfactory epithelium of adult zebrafish. Histochem Cell Biol. 2015 143: 59-68. Voets T. Quantifying and modeling the temperature-dependent gating of TRP channels. Rev Physiol Biochem Pharmacol. 2012; 162: 91-119. Volkers L, Mechioukhi Y, Coste B. Piezo channels: from structure to function. Pflugers Arch. 2015; 467:95-9. von Kugelgen I, Harden TK. Molecular pharmacology, physiology, and structure of the P2Y receptors. Adv Pharmacol. 2011; 61:373-415 Waldmann R, Champigny G, Bassilana F, Heurteaux C, Lazdunski M. A proton-gated cation channel involved in acid-sensing. Nature 1997; 386: 173-7. Wang W, Yi X, Ren Y, Xie Q. Effects of Adenosine Triphosphate on Proliferation and Odontoblastic Differentiation of Human Dental Pulp Cells. J Endod. 2016; 42:1483-9. Wemmie JA, Price MP, Welsh MJ. Acid-sensing ion channels: advances, questions and therapeutic opportunities. Trends Neurosci. 2006; 29:578-86. Wetsel WC. Sensing hot and cold with TRP channels. Int J Hyperthermia 2011; 27:38898. Wood JN, Docherty R. Chemical activators of sensory neurons. Annu Rev Physiol. 1997; 59:457-82. Xia J, Lim JC, Lu W, Beckel JM, Macarak EJ, Laties AM, Mitchell CH. Neurons respond directly to mechanical deformation with pannexin-mediated ATP release and autostimulation of P2X7 receptors. J Physiol. 2012; 590: 2285-304 Yeon KY, Chung G, Shin MS, Jung SJ, Kim JS, Oh SB. Adult rat odontoblasts lack noxious thermal sensitivity. J Dent Res. 2009; 88: 328-32. 35

Yoshida S, Ohshima H. Distribution and organization of peripheral capillaries in dental pulp and their relationship to odontoblasts. Anat Rec. 1996; 245: 313-26. Yost CS. Update on tandem pore (2P) domain K+ channels. Curr Drug Targets 2003; 4: 347-51. Zha XM. Acid-sensing ion channels: trafficking and synaptic function. Mol Brain 2013; 6:1. Zimmermann H. Ectonucleotidases in the nervous system. Novartis Found Symp. 2006; 276:113-28

36

Legends for figures Figure 1.- A: Schematic representation of the odontoblasts and their relations to the dentin and nerve fibers. Pictures show dontoblasts immunolabelled with anti-vimentin (B), anti-TRPV4 (C) and anti-PGP 9.5 antibodies (D). Vimentin and PGP 9.5 are an intermediate cytoskeletal protein and a cytosolic protein, respectively, present in the odontoblasts and their processes. TRPV4 is an ion channel detected in the odontoblasts and related to different modalities of sensitivity. d: dentin; O: odontoblasts; p: pulp.

37

Figure 2.- Double immunofluorecence showing the innervation (green fluorescence) of human odontoblasts (red fluorescence). The odontoblasts were immunolabelled for detection of vimentin which was found in both the cell bodies and processes. Spots of green fluorescence (arrows) indicate axons immunolabelled for detection of neurofilaments proteins (NFP). NFP-positive nerve profiles contact odontoblast cell bodies and also enter the deep segment of dentinal canals. Objective 60x/1.25 Oil; pinhole airy 1, XY resolution 156 nm and Z resolution 334 nm.

38

Figure 3.- Schematic representation of the three main theories explaining the dentin sensitivity. The nerve theory postulates the direct stimulation of dentinal tubules and pulpar nerve terminals; the hydrodynamic theory assumes stimulation of dental nerves by dentinal fluid; and the odontoblastic theory postulates direct stimulation of odontoblast, and is based on the expression of several ion channels by these cells.

39

Figure 4.- Schematic representation of the distribution of TRP and ASIC ion channels in odontoblasts, dental nerve fibres and trigeminal ganglia neurons.

40

Figure 5.- Immunohistochemical localization of some TRP and ASIC1 ion channels in human odontoblasts. Positive immunostaining was detected in both the cell bodies and processes. d: dentin; o: odontoblasts; p: pulp.

41

Figure 6.- Schematic representation of the molecular mechanisms coupling direct odontoblast stimulation or activation of odontoblastic TRP/ASIC ion channels that results in ATP outflow. The extracellular ATP binds to specific receptors present in nerve fibers, odontoblasts and other pulp cells acting via synaptic-like (grey lines), autocrine (red line) or paracrine (blue line) mechanisms.

42

Table 1.- Members of the TRP and Deg/ENaC superfamilies exhibiting mechano-, thermo- and chemo-sensitivity (based on Belmonte and Viana, 2008) Mechanosensing TRPC1 PRPC6 TRPV1 TRPV2 TRPV4 TRPM3 TRPM4 TRPM8 TRPA1 TRPP2 ASIC 1 ASIC2 ASIC3

Thermosensing

Chemiosensing

TRPV1 TRPV2 TRPV3 TRPV4

TRPV1

TRPM8 TRPA1

ASIC1 ASIC2 ASIC3

ASIC1 ASIC2 ASIC3

43