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Expression of Cannabinoid Type 1 Receptors in Human Odontoblast Cells
Basic Research—Biology
Expression of Cannabinoid Type 1 Receptors in Human Odontoblast Cells Kehua Que, PhD,* Dan He, MD,* Ying Jin, MD,† Ligeng Wu, MD,* Fang Wang, MD,* Zhiying Zhao, MD,* Jing Yang, MD,‡ and Jiayin Deng, PhD§ Abstract Introduction: The aim of this study was to investigate the functional expression of cannabinoid type 1 (CB1) receptors in human odontoblasts (HODs) and the possible internal mechanism. Methods: In the present study, we examined the molecular and functional expression of the CB1 receptors in cultured HOD-like cells and native HODs obtained from healthy wisdom teeth. Results: Immunohistochemistry and immunofluorescence revealed that CB1 receptors localize to native HODs and HOD-like cells, respectively. Both reversetranscription polymerase chain reaction and Western blot analysis confirmed gene and protein expression of CB1 receptors. The ultrastructural distribution by immunoelectron microscopy also found that CB1 receptors labeled by colloidal gold particles distribute sparsely in the cytoplasm and odontoblastic processes. In functional assays, 2-arachidonyl glycerol, as an agonist of CB receptors, elicited the increase of intracellular fluorescence intensity that could be inhibited by a CB1specific receptor antagonist rather than a selective CB2 receptor antagonist with fluo-3AM Ca2+ fluorescence. The source of the increase of intracellular fluorescence intensity elicited by CB1 receptors was from extracellular Ca2+ but not intracellular Ca2+ stores. The process of 2-arachidonyl glycerol activating CB1 receptors modulated transient receptor potential vanilloid 1–mediated Ca2+ entry via the cyclic adenosine monophosphate signaling pathway. Conclusions: We conclude that HODs can express functional CB1 receptors that may play an important role in mediating the physiological function in tooth pulp. (J Endod 2016;:1–6)
Key Words Cannabinoid type 1 receptors, functional expression, human odontoblasts, transient receptor potential vanilloid 1 channels
T
he endocannabinoid Significance system is comprised of In dental pulp, CB1 receptors could play an imporendogenous ligands of tant role in dental sensory transduction, biominercannabinoid receptors 1 alization, tissue repair, and so on. Related studies and 2 (CB1 and CB2, of CB receptors could provide a new way for drug respectively) in addition treatment of pulp diseases in the future. to other receptors activated by endogenous cannabinoids (anandamine [AEA] and 2-arachidonyl glycerol [2-AG]) such as certain ionotropic channels, which are widely distributed in various mammalian tissues and are potential pharmaceutical targets for treating diseases (1). The endocannabinoid system is involved in multifarious physiologically and pathologically regulated effects in humans, including analgesia, anti-inflammatory, nerve protection, and immune and endocrine regulation (1). CB2 receptors are mainly expressed in immune cells, particularly B cells and natural killer cells (2, 3). CB1 receptors are distributed mainly in the nervous system, mediating pain control, memory, cognitive functions, and homeostasis in the central nervous system (4). In some peripheral tissues, the activation of CB1 receptors is involved in different complex physiological functions, such as analgesia and anti-inflammatory and immune regulation (1). However, studies on the endocannabinoid system in dental pulp tissues are lacking, and only a few studies have reported on the expression and possible modulation of physiological functions of CB1 receptors in human dental pulp (5–8). Several studies reported that the expression of CB1 receptors in nerve fibers of human and rat dental pulp, respectively, possibly modulated dental hypersensitivity and pain (5, 6). A study also reported that CB1 receptors in human dental pulp cells (HDPCs) may be involved in the regulation of matrix metalloproteinase-2, implicating that CB1 receptors may be associated with dental pulp tissue repair (7). Meanwhile, Tsumura et al (8) reported that the activation of the CB1 receptors can modulate extracellular Ca2+ entry to form ‘‘reparative dentin’’ in rat odontoblasts (ODs). Human odontoblasts (HODs) are very special and highly differentiated postmitotic cells that play an important role in immune defense (9), inflammation regulation (9), sensory transduction (10), biomineralization, and tissue repair in human pulp (8, 9, 11). However, evidence for the functional expression of CB1 receptors in HODs is still insufficient because of species differences. The expression of transient receptor potential vanilloid 1 (TRPV1) channels has been reported in rat (12), mouse (13), and human (14) ODs. TRPV1 channels are molecular sensors suggested to detect temperature-related chemical or mechanical stimuli in tooth pulp (12, 13). A ‘‘cross talk’’ mechanism between CB1 receptors and TRPV1 channels has been found in some cells (8, 15–18), including rat ODs (8).
From the *Department of Endodontics, College of Stomatology, Tianjin Medical University, Tianjin; †Department of Endodontics, Wuxi Stomatology Hospital, Jiangsu; Department of Implant, Stomatology College of Nan Kai University; and § Department of Periodontics, College of Stomatology, Tianjin Medical University, Tianjin, China. Address requests for reprints to Dr Jiayin Deng, Department of Periodontics, College of Stomatology, Tianjin Medical University, Num12, Road Qixiangtai, Heping District, Tianjin 300070, China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2016.10.004 ‡
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Cannabinoid Type 1 Receptors in Human Odontoblast Cells
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Basic Research—Biology The ‘‘cross talk’’ mechanism refers to the interaction among different intracellular substances or signal transduction pathways without affecting others. In fact, all sorts of intracellular information tend to be mixed together to form intracellular information networks and play a collaborative effect. Hence, a similar ‘‘cross talk’’ mechanism between CB1 receptors and TRPV1 channels in HODs was investigated in the present study. Meanwhile, the molecular and subcellular expression characteristics of CB1 receptors in HODs were also studied.
coverslips were incubated for 30 minutes at 37 C with their respective secondary antibodies (goat polyclonal antirabbit IgG Alexa 594 [Thermo Fisher Scientific, Waltham, MA] and goat polyclonal antimouse IgG-fluorescein isothiocyanate [ZSGB-BIO, Beijing, China]). Nuclei were counterstained with 40 ,6-diamidino-2-phenylindole for 8 minutes at room temperature. Coverslips were mounted on glass slides with mounting medium, and IF was observed by upright fluorescence microscopy. The control groups were incubated with PBS in place of primary antibody, and the rest of the steps were consistent with the experimental groups.
Materials and Methods Cell Culture All healthy immature and intact third molars extracted for orthodontic and impacted reasons were collected from adults between 16 and 18 years old following a protocol approved by these patients. The HODs and HDPCs were cultured as described by About et al (19). In brief, HODs and HDPCs derived from dental pulp explants were grown in an incubator with a constant temperature of 37 C and a humidified atmosphere of 5% CO2 and 95% air. These conditions were present in a minimum essential medium supplemented with 2 mmol/L L-glutamine, 100 UI/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL amphotericin B, and 10% fetal bovine serum with or without 2 mmol/L b-glycerophosphate. The cells were collected by trypsinization (0.25% trypsin-EDTA digestive juices) and subcultured when the density reached approximately 80% confluence. Cells from passage 4 to 6 were used in all subsequent experiments. Immunohistochemistry Staining Healthy third molar teeth were fixed in 4% paraformaldehyde for 30 hours at 4 C and demineralized in a decalcifying solution of 10% EDTA until fully decalcified. Decalcified teeth were cut into paraffin sections after being routinely dehydrated, cleared, and immersed in wax. The expression of CB1 receptors in dental pulp was detected using the Elivison 2-step method according to the manufacturer’s instruction. Briefly, the sections were deparaffinized, retrieved through citric acid buffer microwave antigen retrieval, and blocked using endogenous peroxidase by 3% solution of hydrogen peroxide in deionized water. The sections were incubated overnight at 4 C with the rabbit polyclonal anti-CB1R antibody diluted 1:100 in phosphate-buffered saline (PBS). Next, the specimens were incubated with polymer helper and polyperoxidase–anti-rabbit immunoglobulin (Ig)G, respectively, for 20 minutes at 37 C. The localization of CB1 receptors was displayed by the DAB Kit (ZSGB-BIO) and counterstained with hematoxylin. The PBS buffer solution was used to replace the anti-CB1R antibody as the negative control in these experiments.
RT-PCR Cultured cells were grown to confluence, and total RNA was extracted from the cells using Trizol reagent (Cwbio, Beijing, China) according to the guidelines provided by the manufacturer. The purified total RNA was reverse transcribed into first-strand complementary DNA using M-MLV reverse transcriptase (Promega, Madison, WI). PCR amplification was performed for an initial denaturation of 3 minutes at 95 C followed by 30 cycles of 20 seconds at 95 C, 20 seconds at 58 C, 20 seconds at 72 C, and a final step of 10 minutes at 72 C. PCR products were displayed on ethidium bromide–stained 1.7% agarose gels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize the relative expression levels. The primer sequences set for PCR were as follows: DSPP (product size = 293 bp): forward 50 -TTAATAGAAGTAAAAGAAATCC-30 and reverse 50 -CATCCTCCCTTCTCAAAAAGAT-30 , nestin (product size = 718 bp): forward 50 -AGGTTGGAGACAAGGTTGCGA CGG-30 and reverse 50 -TCGGGACTGGTGAGGTCAAATCTC-30 , CB1 receptors (product size = 115 bp): forward 50 -GCTGCCTAAATCCACTCTGC-30 and reverse 50 TGGACATGAAATGGCAGAAA-30 , and GAPDH (product size = 226 bp): forward 50 -GGAGGTGAAGGTCGGAGTC-30 and reverse 50 -GAAGATGGTGATGGGATTTC-30 . WB Confluent cells were washed with ice-cold PBS and lysed in a buffer containing 50 mmol/L Tris-HCL (pH = 7.4), 150 mmol/L NaCl, 1% NP-40 (Beyotime, Nantong, China), 0.1% sodium dodecyl sulfate, and 1 mmol/L phenylmethane sulfonyl fluoride. The total protein concentration was determined using bicinchoninic acid protein assay. Proteins were fractionated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane. Nonspecific antigens were blocked with 5% nonfat milk for 1 hour at room temperature, sequentially probed with corresponding primary antibodies and horseradish peroxidase–conjugated secondary antibodies, and visualized with the eclectrochemiluminescence (Solarbio) system.
IEM Immunofluorescence Staining Cells from the same batch were used in the following experiments including immunofluorescence (IF) staining, reverse-transcription polymerase chain reaction (RT-PCR), Western blotting (WB), immunoelectron microscopy (IEM), and measurement of intracellular Ca2+. Cells were seeded onto coverslips and fixed in 4% paraformaldehyde for 15 minutes at room temperature. After permeating for 5 minutes with 1% Triton X-100 (Solarbio, Beijing, China) in PBS, nonspecific binding sites were blocked with 10% complete serum for 2 hours at 37 C. Slides were incubated overnight at 4 C with the following primary antibodies: mouse polyclonal antibody against human dentin sialophosphoprotein (DSPP; Santa Cruz Biotechnology, Santa Cruz, CA; dilution: 1:100), rabbit polyclonal antibody against human nestin (Proteintech Group, Chicago, IL; dilution: 1:50), and rabbit polyclonal antibody against human CB1 (Santa Cruz Biotechnology, dilution: 1:100). After washing, the 2
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Culture HOD-like cell tissue samples were taken out from 4% paraformaldehyde fixation fluid with 0.1% glutaraldehyde. The samples were washed twice in a sucrose flushing fluid for 30 minutes and sealed with 0.5 mol/L NH4Cl for 1 hour. Then, the tissues were dehydrated at 0 and 20 C for 30 and 60 minutes, respectively, and at 35 C 4 times for 60 minutes. At 35 C, the tissues were orderly saturated with 1:1, 1:2, 0:1, and 0:1 ethanol/Lowicryl K4M (Seebio, Shanghai, China) embedding medium for 60 minutes. Precool capsules, labels, tweezers, toothpicks, and embedding medium were stored at 35 C. The samples were placed at the bottom of capsules, slowly mixed with the embedding medium, and covered with lids. Then, the samples were placed in brackets and polymerized at 35 C for 24 hours in an ultraviolet polymerization box. Ultrathin sections were made and mounted on nickel grids. The grids were incubated in Tris-buffered saline solution (TBS) (pH = 7.4) for 5 minutes, sealed with TBS (pH = 7.4) supplemented JOE — Volume -, Number -, - 2016
Basic Research—Biology with 2% BSA for 10 minutes, and incubated in rabbit antihuman antibody (Santa Cruz Biotechnology, dilution = 1:100) at 4 C overnight. The grids were washed in TBS (pH = 7.4) 6 times for 2 minutes and TBS (pH = 8.2) twice for 2 minutes. Afterward, the grids were sealed with TBS (pH = 8.2) supplemented with 2% BSA for 10 minutes and subsequently floated on drops of goat antirabbit antibody (Laboratory Aurion, Wageningen, Netherlands; dilution = 1:40) conjugated with gold particles of 10 nm for 2 hours at room temperature. The grids were washed orderly in TBS (pH = 8.2), TBS (pH = 7.4), and distilled water 5 times for 2 minutes. The sections on nickel grids were dried and stained with uranium acetate, air dried, and viewed under a transmission electron microscope (TEM).
Measurement of Intracellular Ca2+ The HODs were cultured in glass culture bottles for at least 24 hours to ensure adequate adhesion, and experiments were performed at 70% confluence. The HODs were loaded with 1 mmol/L fluo-3 acetoxymethyl ester (fluo-3AM; Beyotime, Nanjing, Jiangsu, China) for 30 minutes at 37 C in darkness and in Ca2+-free standard solution with (in mmol/L) 136 NaCl, 5 KCl, 0.5 MgCl2, 10 N0 -a-hydroxythylpiperazine-N0 -ethanesulfanic acid, 10 glucose, and 12 NaHCO3 (pH = 7.4 by Tris). The samples were further incubated in fresh cell culture media without fluo-3AM. The cells were stimulated with the nonselective CB receptor agonist 2-AG. Then, the selective CB1 receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl -N-1-piperidinyl1H-pyrazole-3-carboxamide (AM251; Tocris, Bristol, UK), the selective CB2 receptor antagonist 6-iodopravadoline (AM630; Tocris), nonselective TRPV1 and transient receptor potential melastatin 8 (TRPM8) channel antagonist capsazepine (CPZ), selective TRPM8 channel antagonist N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N(2-thienylmethyl) benzamide hydrochloride salt (AMTB), and the selective cyclic adenosine monophosphate (cAMP) antagonist 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536; Tocris) were used to block the agonist responses. Ca2+-dependent fluorescence intensity was measured and analyzed with a confocal laser scanning microscope. By confocal laser scanning microscopy, we dynamically observed the changes in intracellular fluorescence intensity, collected different periods of fluorescence images, and analyzed the changes in the
concentration of intracellular Ca2+ that were proportional to the fluorescence intensity.
Results These cells exhibited positive immunohistochemistry (IHC) staining (shown in brown) for DSPP (Fig. 1A) and nestin (Fig. 1B), indicating that these cells were ODs. IHC staining for CB1 receptors was mainly observed in native OD cellular processes, and weak positive staining was observed in cell bodies (Fig. 1C). Conversely, IHC staining for TRPV1 channels was mainly observed in cell processes of ODs with more intense staining than CB1 receptors (Fig. 1D). Moreover, the blank control showed no IHC staining (Fig. 1E), in contrast to that of protein. IF staining showed a positive expression of DSPP (green) and nestin (red) on tall, columnar, and polarized cells from cultured human dental pulp tissues (Fig. 2A–F), indicating that the morphologies and functions of these cells were similar to those of HOD cells. These cells are known as HOD-like cells and are used to study OD cells in vitro. Meanwhile, cells were positive for CB1 immunoreaction (red), and the nuclei were shown in blue (Fig. 2G–I). Gene and protein expression levels of CB1 in HOD-like cells were shown with RT-PCR and WB (Fig. 2K and L). Electrophoresis of RT-PCR products revealed strong intensity bands corresponding to predicted sizes of GAPDH (226 bp), DSPP (293 bp), nestin (718 bp), and CB1 (115 bp) (Fig. 2J and K). Using WB, the odontoblastic protein expression in cultured cells was confirmed by the expression of the odontogenic markers DSPP and nestin (Fig. 2L). Further WB assays identified the expression of CB1 receptors and TRPV1 channels in HOD-like cells (Fig. 2L). Using IEM, CB1 receptors and TRPV1 channels labeled with tiny immune colloidal gold particles were observed. CB1 receptors distribute sparsely in the cytoplasm (Fig. 3A) and processes (Fig. 3E) in the ultrathin sections of HOD-like cells. No gold particles were observed clearly on the mitochondria (Fig. 3B), endoplasmic reticulum (ER) (Fig. 3C), and primary cilium (Fig. 3D). In the contrast, TRPV1 channels were labeled with colloidal gold particles and distributed densely in the cytoplasm (Fig. 3F–H), mitochondria (Fig. 3F), ER (Fig. 3G), primary cilium (Fig. 3H), and odontoblastic processes (Fig. 3I).
Figure 1. IHC localization of (C) CB1 and (D) TRPV1 in human dental pulp ex vivo. These ODs cells were positive for (A) DSPP, (B) nestin, (C) CB1, and (D) TRPV1 immunoreaction (brown) compared with (E) the blank control (200, scale bars = 20 mm). D, dentin; ODs, odontoblasts; P, pulp. JOE — Volume -, Number -, - 2016
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Figure 2. The expression of CB1 in HOD-like cells was evaluated by IF, RT-PCR, and WB. (A–I) Observation of HOD-like cells by IF staining. There cells were positive for (B and C) DSPP (green), (E and F) nestin (red), and (H and I) CB1 (red) immunoreaction, and nuclei are shown in blue. Scale bars = 50 mm. (J–L) Molecular expression of CB1 receptors in HOD-like cells using RT-PCR and WB. Representative results from at least 3 independent experiments are shown.
To only activate CB1 receptors in ODs, we used the nonselective CB receptor agonist 2-AG, which cannot activate TRPV1 channels (Fig. 4). With extracellular Ca2+ (2.5 mmol/L), the application of 1 mmol/L 2-AG alone rapidly increased the fluorescence intensity to peak values followed by a rapid decline to baseline levels. With the addition of 1 mmol/L 2-AG and 10 nmol/L AM251 (a selective CB1 receptor antagonist), the increase of fluorescence intensity was significantly reduced (Fig. 4A). However, the addition of 1 mmol/L 2-AG and 10 nmol/L AM630 (a selective CB2 receptors antagonist) did not cause significant changes in the fluorescence intensity (Fig. 4B). Moreover, 2-AG was administered in the absence of extracellular Ca2+, but no fluorescence
intensity increase was observed; the fluorescence intensity immediately increased after the addition of 2.5 mmol/L extracellular Ca2+ (Fig. 4C). In contrast, when 1 mmol/L 2-AG and 2.5 mmol/L extracellular Ca2+ were immediately administered together on preapplication Ca2+-free standard solution, the fluorescence intensity also immediately increased (Fig. 4D). However, the 1 mmol/L 2-AG–induced increase of fluorescence intensity in the presence of extracellular Ca2+ was inhibited by 1 mmol/L CPZ (nonselective TRPV1 and TRPM8 channel antagonists) (Fig. 4E) but was not affected by the administration of 1 mmol/L AMTB (a selective TRPM8 channel antagonist) (Fig. 4F). In addition, the 2-AG–induced increase of fluorescence intensity was inhibited
Figure 3. Immunoelectron micrographs of HOD-like cells; 10 nm colloidal gold particles labeled (A–E) CB1 and (F–I) TRPV1 in HOD-like cells (60,000; scale bars = 200 nm). (A) Cytoplasm. (B) and (F) Mitochondria. (C) and (G) Endoplasmic reticulum. (D) and (H) Primary cilium. (E) and (I) Odontoblastic processes.
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Figure 4. Intracellular calcium levels in HOD-like cells were evaluated after being treated with different agonists and antagonists against CB1 and CB2, TRPV1 and TRPM8, and cAMP. Examples of traces of changes of intracellular fluorescence intensity by 1 mmol/L 2-AG with or without (A) 10 nmol/L AM251, (B) 10 nmol/L AM630, (E) 1 mmol/L CPZ, (F) 1 mmol/L AMTB, or (G) 1 mmol/L SQ22536 in the presence of extracellular Ca2+ (2.5 mmol/L) (white boxes at bottom). (C and D) Changes of intracellular fluorescence intensity by the administration of extracellular Ca2+ (2.5 mmol/L) (gray lines at top) after 1 mmol/L 2-AG application or with 1 mmol/L 2-AG simultaneously in the absence of extracellular Ca2+ (white boxes at bottom). Gray lines and black lines at the top indicate times of addition of these antagonists and CB receptors agonist 2-AG to external solution, respectively. (H–L) Summary bar graphs of changes of intracellular fluorescence intensity by 1 mmol/L 2-AG with (gray columns) or without (H) 10 nmol/L AM251, (I) 10 nmol/L AM630, (J) 1 mmol/L CPZ, (K) 1 mmol/L AMTB, or (L) 1 mmol/L SQ22536 (open columns). Each bar denotes the mean standard error of several experiments. Statistically significant differences between columns (shown by solid lines) are indicated by asterisks: *P < .05.
significantly by 1 mmol/L SQ22536 (a selective cAMP antagonist) in the presence of extracellular Ca2+ (2.5 mmol/L) (Fig. 4G). Meanwhile, the statistical analysis of the fluorescence intensity peak in 1 mmol/L 2-AG with or without 10 nmol/L AM251 (Fig. 4H), 1 mmol/L CPZ (Fig. 4J), or 1 mmol/L SQ22536 (Fig. 4L) showed significant differences (P < .05). But, the statistical analysis of the fluorescence intensity peak in 1-mmol/ L 2-AG with or without 10-nmol/L AM630 (Fig. 4I) or 1-mmol/L AMTB (Fig. 4K) showed no significant differences (P > .05).
Discussion The present study shows for the first time that HODs express functional CB1 receptors. Except for molecular expression characteristics of CB1 in HODs, the subcellular characteristics of CB1 receptors observed by IEM was also studied and compared with that of TRPV1 channels for the first time. Moreover, this study showed the physiological properties not only of CB1 receptors but also of TRPV1 channels. In addition, we further clarified the possible functional coupling of CB1 receptors with TRPV1 channels. The cultured HOD-like cells used in this study derived from pulp tissue explants using a previously validated method (14). The odontoblastic phenotype of the cells was confirmed using the gene expression of odontogenic markers DSPP and nestin. In addition to ODs, the results of our IHC revealed the expression of CB1 receptors in some of the cells of the dental pulp ex vivo. The dental pulp is populated by different cell types, including fibroblasts, undifferentiated mesenchymal cells, and defense cells. The result of the present study was similar to that of a previous work, which also showed the expression of CB1 receptors in dental pulp cells (7). In the brain, the 2-AG content is at least 50 times higher than AEA (20). 2-AG acts as the main endocannabinoid for CB1 receptors in the central nervous system and plays an important physiological role with JOE — Volume -, Number -, - 2016
specific binding sites on the CB1 receptors. However, 2-AG could not activate the TRPV1 channels compared with AEA, which will interfere with the study of CB1 receptors. Hence, 2-AG was used as the specific agonist of CB1 receptors in this present study. Only 1 study reported that rat tooth pulp ODs express CB1 receptors (8). In the present study, our results showed that CB1 receptors were positively but not strongly expressed in cell bodies and in some processes of IHC. Using IF staining, RT-PCR, and WB, we also showed the expression of CB1 receptors in HOD-like cells; the results were similar to those described by IHC in native HODs. We further studied the subcellular expression characteristics of CB1 receptors in HODs, and interesting molecules were mapped using IEM at high resolution by applying a set of affinity reagents to sections and locating them with suitable markers; thus, IEM plays an important role in cell biology by quantifying structures and assessing the spatial distributions of molecules (21). Under IEM, the number of CB1 receptors labeled with colloidal gold particles was considerably less than that of TRPV1 channels. In the present study, the expression characteristics of TRPV1 channels under a TEM were reported for the first time. Extensive and obvious expression of TRPV1 channels under a TEM suggested that HOD-like cells could sense and transmit nociceptive stimuli. The results showed that the cytoplasm and cell processes comprised the coexpression areas, which are the first areas to sense extracellular stimuli in HODs and may also be the sites that play the ‘‘cross talk’’ function. However, whether the expression and distribution of CB1 receptors and TRPV1 channels could vary under the effect of external stimuli or infectious substance still needs to be further studied. We showed that the 2-AG–induced fluorescence intensity increase was sensitive to CB1-specific receptor antagonist AM251 instead of CB2specific receptor antagonist AM630, indicating that HODs express CB1 instead of CB2 receptors (1, 2, 22, 23). In the present study, the fluorescence intensity did not increase after the application of 2-AG
Cannabinoid Type 1 Receptors in Human Odontoblast Cells
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Basic Research—Biology in the absence of extracellular Ca2+, indicating that the increase in intracellular Ca2+ by the activation of CB1 receptors was evoked by Ca2+ influx from extracellular medium instead of the Ca2+ release from the intracellular Ca2+ stores. Although 2-AG is a lipid that can easily penetrate across the cell membrane, the ER was not affected by 2-AG possibly because of the expression of CB1 receptors in the ER (main intracellular Ca2+ stores) of HODs as shown by IEM. In addition, the Ca2+ influx stimulated by 2-AG activating CB1 receptors was inhibited by CPZ. Although CPZ acts as an antagonist for TRPV1 and TRPM8 channels, the specific antagonist of TRPM8 channels was also applied but had no obvious effect on the 2-AG–induced increase in fluorescence intensity. 2-AG is an agonist of CB receptors instead of TRPV1 channels, and, thus, there could be a coupling mechanism between TRPV1 channels and CB1 receptors. Meanwhile, the increase of fluorescence intensity by the application of 1 mmol/L 2-AG was also inhibited by adenylyl cyclase antagonist SQ22536. This phenomenon indicates that the ‘‘cross talk’’ mechanism between CB1 receptor and TRPV1 channel activation of CB1 receptors modulates the TRPV1-mediated Ca2+ entry via the intracellular cAMP signaling pathway, which is catalyzed by ATP via adenylyl cyclase. Similar results and conclusions have been confirmed in the study from Tsumura et al (8). Moreover, 1 study also showed that in HEK-293 cells the coexpression of CB1 receptors and TRPV1 channels pretreated by CB1 agonists inhibited or stimulated TRPV1 gating using capsaicin, which was dependent on the concomitant activation of the cAMP signaling pathway (24). CB1 receptors have antihyperalgesic and antinociceptive effects for pain, whereas TRPV1 channels could act as molecular sensors for nociceptive stimuli (1, 25–28). Thus, a functional coexpression or cross talk pathway for CB1 receptors and TRPV1 channels could play an important role in pulp pain sensation and analgesia (15–18). Several studies have reported the effects of the coexpression of CB1 receptors and TRPV1 channels on pain. Hermann et al (24) reported that TRPV1 appears to be partly responsible for the transmission of pain during thermal and inflammatory hyperalgesia, whereas CB1 receptors were suggested to partly counteract hyperalgesia by inhibiting TRPV1-mediated nociception. The TRPV1 channels activated by potent synthetic agonists are immediately followed by desensitization in which the receptors lose reactivity to corresponding agonists to produce powerful analgesic effects in vivo (29). Several studies also reported agents capable of activating both CB1 receptors and TRPV1 channels at the same time; for instance, the combined application of AEA and capsaicin showed strong effects for analgesia (24). Controlling pain was always a difficult research topic, and studies on the coexpression of CB1 receptors and TRPV1 channels may provide new pain treatments in the future. In summary, we provided evidence that HODs express functional CB1 receptors suggesting that CB1 receptors could play roles in dental sensory transduction, biomineralization, and tissue repair.
Acknowledgments Kehua Que and Dan He contributed equally to this work. Supported by the Research Fund for the Doctoral Program of Higher Education of China (20131202120010), Tianjin Municipal Natural Science Foundation (14JCQNJC13500), and Science and Technology Development Project of Tianjin Educational Commission (20130132) in China. The authors deny any conflicts of interest related to this study.
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References 1. Pertwee RG, Howlett AC, Abood ME, et al. International Union of Basic and Clinical Pharmacology. LXXIX. Cannabinoid receptors and their ligands: beyond CB1 and CB2. Pharmacol Rev 2010;62:588–631. 2. Pertwee RG, Ross RA. Cannabinoid receptors and their ligands. Prostaglandins Leukot Essent Fatty Acids 2002;66:101–21. 3. Pertwee RG. Pharmacology of cannabinoid CB1 and CB2 receptors. Pharmacol Ther 1997;74:129–80. 4. Mechoulam R, Parker LA. The endocannabinoid system and the brain. Annu Rev Psychol 2013;64:21–47. 5. Beneng K, Renton T, Yilmaz Z, et al. Cannabinoid receptor CB1-immunoreactive nerve fibres in painful and non-painful human tooth pulp. J Clin Neurosci 2010; 17:1476–9. 6. Mitrirattanakul S, Poomsawat S, Fuangtharnthip P. Cannabinoid receptor 1 (CB1R) expression in rat dental pulp. Oral Sci Int 2012;9:17–20. 7. Miyashita K, Oyama T, Sakuta T, et al. Anandamide induces matrix metalloproteinase-2 production through cannabinoid-1 receptor and transient receptor potential vanilloid-1 in human dental pulp cells in culture. J Endod 2012;38: 786–90. 8. Tsumura M, Sobhan U, Muramatsu T, et al. TRPV1-mediated calcium signal couples with cannabinoid receptors and sodium-calcium exchangers in rat odontoblasts. Cell Calcium 2012;52:124–36. 9. Goldberg M, Farges JC, Lacerda-Pinheiro S, et al. Inflammatory and immunological aspects of dental pulp repair. Pharmacol Res 2008;58:137–47. 10. Magloire H, Couble ML, Thivichon-Prince B, et al. Odontoblast: a mechano-sensory cell. J Exp Zool B Mol Dev Evol 2009;312B:416–24. 11. Goldberg M, Smith AJ. Cells and extracellular matrices of dentin and pulp: a biological basis for repair and tissue engineering. Crit Rev Oral Biol Med 2004;15:13–27. 12. Okumura R, Shima K, Muramatsu T, et al. The odontoblast as a sensory receptor cell? The expression of TRPV1 (VR-1) channels. Arch Histol Cytol 2005;68:251–7. 13. Son AR, Yang YM, Hong JH, et al. Odontoblast TRP channels and thermo/mechanical transmission. J Dent Res 2009;88:1014–9. 14. El Karim IA, Linden GJ, Curtis TM, et al. Human odontoblasts express functional thermo-sensitive TRP channels: implications for dentin sensitivity. Pain 2011;152: 2211–23. 15. Ahluwalia J, Urban L, Capogna M, et al. Cannabinoid 1 receptors are expressed in nociceptive primary sensory neurons. Neuroscience 2000;100:685–8. 16. Kulkarni-Narla A, Brown DR. Opioid, cannabinoid and vanilloid receptor localization on porcine cultured myenteric neurons. Neurosci Lett 2001;308:153–6. 17. Coutts AA, Irving AJ, Mackie K, et al. Localisation of cannabinoid CB(1) receptor immunoreactivity in the guinea pig and rat myenteric plexus. J Comp Neurol 2002;448:410–22. 18. Cristino L, De Petrocellis L, Pryce G, et al. Immunohistochemical localization of cannabinoid type 1 and vanilloid transient receptor potential vanilloid type 1 receptors in the mouse brain. Neuroscience 2006;139:1405–15. 19. About I, Bottero MJ, de Denato P, et al. Human dentin production in vitro. Exp Cell Res 2000;258:33–41. 20. Iversen L. Cannabis and the brain. Brain 2003;126:1252–70. 21. Mayhew TM, Lucocq JM. Developments in cell biology for quantitative immunoelectron microscopy based on thin sections: a review. Histochem Cell Biol 2008;130: 299–313. 22. Matsuda LA, Lolait SJ, Brownstein MJ, et al. Structure of a cannabinoid receptor and functional expression of the cloned cDNA. Nature 1990;346:561–4. 23. Munro S, Thomas KL, Abu-Shaar M. Molecular characterization of a peripheral receptor for cannabinoids. Nature 1993;365:61–5. 24. Hermann H, De Petrocellis L, Bisogno T, et al. Dual effect of cannabinoid CB1 receptor stimulation on a vanilloid VR1 receptor-mediated response. Cell Mol Life Sci 2003;60:607–16. 25. Pedersen SF, Owsianik G, Nilius B. TRP channels: an overview. Cell Calcium 2005; 38:233–52. 26. Gees M, Colsoul B, Nilius B. The role of transient receptor potential cation channels in Ca2+ signaling. Cold Spring Harb Perspect Biol 2010;2:a003962. 27. Caterina MJ, Leffler A, Malmberg AB, et al. Impaired nociception and pain sensation in mice lacking the capsaicin receptor. Science 2000;288:306–13. 28. Akopian AN, Ruparel NB, Jeske NA, et al. Role of ionotropic cannabinoid receptors in peripheral antinociception and antihyperalgesia. Trends Pharmacol Sci 2009;30: 79–84. 29. Szallasi A, Blumberg PM. Vanilloid (Capsaicin) receptors and mechanisms. Pharmacol Rev 1999;51:159–212.
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Expression of Cannabinoid Type 1 Receptors in Human Odontoblast Cells Kehua Que, PhD,* Dan He, MD,* Ying Jin, MD,† Ligeng Wu, MD,* Fang Wang, MD,* Zhiying Zhao, MD,* Jing Yang, MD,‡ and Jiayin Deng, PhD§ Abstract Introduction: The aim of this study was to investigate the functional expression of cannabinoid type 1 (CB1) receptors in human odontoblasts (HODs) and the possible internal mechanism. Methods: In the present study, we examined the molecular and functional expression of the CB1 receptors in cultured HOD-like cells and native HODs obtained from healthy wisdom teeth. Results: Immunohistochemistry and immunofluorescence revealed that CB1 receptors localize to native HODs and HOD-like cells, respectively. Both reversetranscription polymerase chain reaction and Western blot analysis confirmed gene and protein expression of CB1 receptors. The ultrastructural distribution by immunoelectron microscopy also found that CB1 receptors labeled by colloidal gold particles distribute sparsely in the cytoplasm and odontoblastic processes. In functional assays, 2-arachidonyl glycerol, as an agonist of CB receptors, elicited the increase of intracellular fluorescence intensity that could be inhibited by a CB1specific receptor antagonist rather than a selective CB2 receptor antagonist with fluo-3AM Ca2+ fluorescence. The source of the increase of intracellular fluorescence intensity elicited by CB1 receptors was from extracellular Ca2+ but not intracellular Ca2+ stores. The process of 2-arachidonyl glycerol activating CB1 receptors modulated transient receptor potential vanilloid 1–mediated Ca2+ entry via the cyclic adenosine monophosphate signaling pathway. Conclusions: We conclude that HODs can express functional CB1 receptors that may play an important role in mediating the physiological function in tooth pulp. (J Endod 2016;:1–6)
Key Words Cannabinoid type 1 receptors, functional expression, human odontoblasts, transient receptor potential vanilloid 1 channels
T
he endocannabinoid Significance system is comprised of In dental pulp, CB1 receptors could play an imporendogenous ligands of tant role in dental sensory transduction, biominercannabinoid receptors 1 alization, tissue repair, and so on. Related studies and 2 (CB1 and CB2, of CB receptors could provide a new way for drug respectively) in addition treatment of pulp diseases in the future. to other receptors activated by endogenous cannabinoids (anandamine [AEA] and 2-arachidonyl glycerol [2-AG]) such as certain ionotropic channels, which are widely distributed in various mammalian tissues and are potential pharmaceutical targets for treating diseases (1). The endocannabinoid system is involved in multifarious physiologically and pathologically regulated effects in humans, including analgesia, anti-inflammatory, nerve protection, and immune and endocrine regulation (1). CB2 receptors are mainly expressed in immune cells, particularly B cells and natural killer cells (2, 3). CB1 receptors are distributed mainly in the nervous system, mediating pain control, memory, cognitive functions, and homeostasis in the central nervous system (4). In some peripheral tissues, the activation of CB1 receptors is involved in different complex physiological functions, such as analgesia and anti-inflammatory and immune regulation (1). However, studies on the endocannabinoid system in dental pulp tissues are lacking, and only a few studies have reported on the expression and possible modulation of physiological functions of CB1 receptors in human dental pulp (5–8). Several studies reported that the expression of CB1 receptors in nerve fibers of human and rat dental pulp, respectively, possibly modulated dental hypersensitivity and pain (5, 6). A study also reported that CB1 receptors in human dental pulp cells (HDPCs) may be involved in the regulation of matrix metalloproteinase-2, implicating that CB1 receptors may be associated with dental pulp tissue repair (7). Meanwhile, Tsumura et al (8) reported that the activation of the CB1 receptors can modulate extracellular Ca2+ entry to form ‘‘reparative dentin’’ in rat odontoblasts (ODs). Human odontoblasts (HODs) are very special and highly differentiated postmitotic cells that play an important role in immune defense (9), inflammation regulation (9), sensory transduction (10), biomineralization, and tissue repair in human pulp (8, 9, 11). However, evidence for the functional expression of CB1 receptors in HODs is still insufficient because of species differences. The expression of transient receptor potential vanilloid 1 (TRPV1) channels has been reported in rat (12), mouse (13), and human (14) ODs. TRPV1 channels are molecular sensors suggested to detect temperature-related chemical or mechanical stimuli in tooth pulp (12, 13). A ‘‘cross talk’’ mechanism between CB1 receptors and TRPV1 channels has been found in some cells (8, 15–18), including rat ODs (8).
From the *Department of Endodontics, College of Stomatology, Tianjin Medical University, Tianjin; †Department of Endodontics, Wuxi Stomatology Hospital, Jiangsu; Department of Implant, Stomatology College of Nan Kai University; and § Department of Periodontics, College of Stomatology, Tianjin Medical University, Tianjin, China. Address requests for reprints to Dr Jiayin Deng, Department of Periodontics, College of Stomatology, Tianjin Medical University, Num12, Road Qixiangtai, Heping District, Tianjin 300070, China. E-mail address: [email protected] 0099-2399/$ - see front matter Copyright ª 2016 American Association of Endodontists. http://dx.doi.org/10.1016/j.joen.2016.10.004 ‡
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Cannabinoid Type 1 Receptors in Human Odontoblast Cells
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Basic Research—Biology The ‘‘cross talk’’ mechanism refers to the interaction among different intracellular substances or signal transduction pathways without affecting others. In fact, all sorts of intracellular information tend to be mixed together to form intracellular information networks and play a collaborative effect. Hence, a similar ‘‘cross talk’’ mechanism between CB1 receptors and TRPV1 channels in HODs was investigated in the present study. Meanwhile, the molecular and subcellular expression characteristics of CB1 receptors in HODs were also studied.
coverslips were incubated for 30 minutes at 37 C with their respective secondary antibodies (goat polyclonal antirabbit IgG Alexa 594 [Thermo Fisher Scientific, Waltham, MA] and goat polyclonal antimouse IgG-fluorescein isothiocyanate [ZSGB-BIO, Beijing, China]). Nuclei were counterstained with 40 ,6-diamidino-2-phenylindole for 8 minutes at room temperature. Coverslips were mounted on glass slides with mounting medium, and IF was observed by upright fluorescence microscopy. The control groups were incubated with PBS in place of primary antibody, and the rest of the steps were consistent with the experimental groups.
Materials and Methods Cell Culture All healthy immature and intact third molars extracted for orthodontic and impacted reasons were collected from adults between 16 and 18 years old following a protocol approved by these patients. The HODs and HDPCs were cultured as described by About et al (19). In brief, HODs and HDPCs derived from dental pulp explants were grown in an incubator with a constant temperature of 37 C and a humidified atmosphere of 5% CO2 and 95% air. These conditions were present in a minimum essential medium supplemented with 2 mmol/L L-glutamine, 100 UI/mL penicillin, 100 mg/mL streptomycin, 0.25 mg/mL amphotericin B, and 10% fetal bovine serum with or without 2 mmol/L b-glycerophosphate. The cells were collected by trypsinization (0.25% trypsin-EDTA digestive juices) and subcultured when the density reached approximately 80% confluence. Cells from passage 4 to 6 were used in all subsequent experiments. Immunohistochemistry Staining Healthy third molar teeth were fixed in 4% paraformaldehyde for 30 hours at 4 C and demineralized in a decalcifying solution of 10% EDTA until fully decalcified. Decalcified teeth were cut into paraffin sections after being routinely dehydrated, cleared, and immersed in wax. The expression of CB1 receptors in dental pulp was detected using the Elivison 2-step method according to the manufacturer’s instruction. Briefly, the sections were deparaffinized, retrieved through citric acid buffer microwave antigen retrieval, and blocked using endogenous peroxidase by 3% solution of hydrogen peroxide in deionized water. The sections were incubated overnight at 4 C with the rabbit polyclonal anti-CB1R antibody diluted 1:100 in phosphate-buffered saline (PBS). Next, the specimens were incubated with polymer helper and polyperoxidase–anti-rabbit immunoglobulin (Ig)G, respectively, for 20 minutes at 37 C. The localization of CB1 receptors was displayed by the DAB Kit (ZSGB-BIO) and counterstained with hematoxylin. The PBS buffer solution was used to replace the anti-CB1R antibody as the negative control in these experiments.
RT-PCR Cultured cells were grown to confluence, and total RNA was extracted from the cells using Trizol reagent (Cwbio, Beijing, China) according to the guidelines provided by the manufacturer. The purified total RNA was reverse transcribed into first-strand complementary DNA using M-MLV reverse transcriptase (Promega, Madison, WI). PCR amplification was performed for an initial denaturation of 3 minutes at 95 C followed by 30 cycles of 20 seconds at 95 C, 20 seconds at 58 C, 20 seconds at 72 C, and a final step of 10 minutes at 72 C. PCR products were displayed on ethidium bromide–stained 1.7% agarose gels. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a housekeeping gene to normalize the relative expression levels. The primer sequences set for PCR were as follows: DSPP (product size = 293 bp): forward 50 -TTAATAGAAGTAAAAGAAATCC-30 and reverse 50 -CATCCTCCCTTCTCAAAAAGAT-30 , nestin (product size = 718 bp): forward 50 -AGGTTGGAGACAAGGTTGCGA CGG-30 and reverse 50 -TCGGGACTGGTGAGGTCAAATCTC-30 , CB1 receptors (product size = 115 bp): forward 50 -GCTGCCTAAATCCACTCTGC-30 and reverse 50 TGGACATGAAATGGCAGAAA-30 , and GAPDH (product size = 226 bp): forward 50 -GGAGGTGAAGGTCGGAGTC-30 and reverse 50 -GAAGATGGTGATGGGATTTC-30 . WB Confluent cells were washed with ice-cold PBS and lysed in a buffer containing 50 mmol/L Tris-HCL (pH = 7.4), 150 mmol/L NaCl, 1% NP-40 (Beyotime, Nantong, China), 0.1% sodium dodecyl sulfate, and 1 mmol/L phenylmethane sulfonyl fluoride. The total protein concentration was determined using bicinchoninic acid protein assay. Proteins were fractionated by 12% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and electrotransferred to a polyvinylidene difluoride membrane. Nonspecific antigens were blocked with 5% nonfat milk for 1 hour at room temperature, sequentially probed with corresponding primary antibodies and horseradish peroxidase–conjugated secondary antibodies, and visualized with the eclectrochemiluminescence (Solarbio) system.
IEM Immunofluorescence Staining Cells from the same batch were used in the following experiments including immunofluorescence (IF) staining, reverse-transcription polymerase chain reaction (RT-PCR), Western blotting (WB), immunoelectron microscopy (IEM), and measurement of intracellular Ca2+. Cells were seeded onto coverslips and fixed in 4% paraformaldehyde for 15 minutes at room temperature. After permeating for 5 minutes with 1% Triton X-100 (Solarbio, Beijing, China) in PBS, nonspecific binding sites were blocked with 10% complete serum for 2 hours at 37 C. Slides were incubated overnight at 4 C with the following primary antibodies: mouse polyclonal antibody against human dentin sialophosphoprotein (DSPP; Santa Cruz Biotechnology, Santa Cruz, CA; dilution: 1:100), rabbit polyclonal antibody against human nestin (Proteintech Group, Chicago, IL; dilution: 1:50), and rabbit polyclonal antibody against human CB1 (Santa Cruz Biotechnology, dilution: 1:100). After washing, the 2
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Culture HOD-like cell tissue samples were taken out from 4% paraformaldehyde fixation fluid with 0.1% glutaraldehyde. The samples were washed twice in a sucrose flushing fluid for 30 minutes and sealed with 0.5 mol/L NH4Cl for 1 hour. Then, the tissues were dehydrated at 0 and 20 C for 30 and 60 minutes, respectively, and at 35 C 4 times for 60 minutes. At 35 C, the tissues were orderly saturated with 1:1, 1:2, 0:1, and 0:1 ethanol/Lowicryl K4M (Seebio, Shanghai, China) embedding medium for 60 minutes. Precool capsules, labels, tweezers, toothpicks, and embedding medium were stored at 35 C. The samples were placed at the bottom of capsules, slowly mixed with the embedding medium, and covered with lids. Then, the samples were placed in brackets and polymerized at 35 C for 24 hours in an ultraviolet polymerization box. Ultrathin sections were made and mounted on nickel grids. The grids were incubated in Tris-buffered saline solution (TBS) (pH = 7.4) for 5 minutes, sealed with TBS (pH = 7.4) supplemented JOE — Volume -, Number -, - 2016
Basic Research—Biology with 2% BSA for 10 minutes, and incubated in rabbit antihuman antibody (Santa Cruz Biotechnology, dilution = 1:100) at 4 C overnight. The grids were washed in TBS (pH = 7.4) 6 times for 2 minutes and TBS (pH = 8.2) twice for 2 minutes. Afterward, the grids were sealed with TBS (pH = 8.2) supplemented with 2% BSA for 10 minutes and subsequently floated on drops of goat antirabbit antibody (Laboratory Aurion, Wageningen, Netherlands; dilution = 1:40) conjugated with gold particles of 10 nm for 2 hours at room temperature. The grids were washed orderly in TBS (pH = 8.2), TBS (pH = 7.4), and distilled water 5 times for 2 minutes. The sections on nickel grids were dried and stained with uranium acetate, air dried, and viewed under a transmission electron microscope (TEM).
Measurement of Intracellular Ca2+ The HODs were cultured in glass culture bottles for at least 24 hours to ensure adequate adhesion, and experiments were performed at 70% confluence. The HODs were loaded with 1 mmol/L fluo-3 acetoxymethyl ester (fluo-3AM; Beyotime, Nanjing, Jiangsu, China) for 30 minutes at 37 C in darkness and in Ca2+-free standard solution with (in mmol/L) 136 NaCl, 5 KCl, 0.5 MgCl2, 10 N0 -a-hydroxythylpiperazine-N0 -ethanesulfanic acid, 10 glucose, and 12 NaHCO3 (pH = 7.4 by Tris). The samples were further incubated in fresh cell culture media without fluo-3AM. The cells were stimulated with the nonselective CB receptor agonist 2-AG. Then, the selective CB1 receptor antagonist 1-(2,4-dichlorophenyl)-5-(4-iodophenyl)-4-methyl -N-1-piperidinyl1H-pyrazole-3-carboxamide (AM251; Tocris, Bristol, UK), the selective CB2 receptor antagonist 6-iodopravadoline (AM630; Tocris), nonselective TRPV1 and transient receptor potential melastatin 8 (TRPM8) channel antagonist capsazepine (CPZ), selective TRPM8 channel antagonist N-(3-aminopropyl)-2-{[(3-methylphenyl)methyl]oxy}-N(2-thienylmethyl) benzamide hydrochloride salt (AMTB), and the selective cyclic adenosine monophosphate (cAMP) antagonist 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ22536; Tocris) were used to block the agonist responses. Ca2+-dependent fluorescence intensity was measured and analyzed with a confocal laser scanning microscope. By confocal laser scanning microscopy, we dynamically observed the changes in intracellular fluorescence intensity, collected different periods of fluorescence images, and analyzed the changes in the
concentration of intracellular Ca2+ that were proportional to the fluorescence intensity.
Results These cells exhibited positive immunohistochemistry (IHC) staining (shown in brown) for DSPP (Fig. 1A) and nestin (Fig. 1B), indicating that these cells were ODs. IHC staining for CB1 receptors was mainly observed in native OD cellular processes, and weak positive staining was observed in cell bodies (Fig. 1C). Conversely, IHC staining for TRPV1 channels was mainly observed in cell processes of ODs with more intense staining than CB1 receptors (Fig. 1D). Moreover, the blank control showed no IHC staining (Fig. 1E), in contrast to that of protein. IF staining showed a positive expression of DSPP (green) and nestin (red) on tall, columnar, and polarized cells from cultured human dental pulp tissues (Fig. 2A–F), indicating that the morphologies and functions of these cells were similar to those of HOD cells. These cells are known as HOD-like cells and are used to study OD cells in vitro. Meanwhile, cells were positive for CB1 immunoreaction (red), and the nuclei were shown in blue (Fig. 2G–I). Gene and protein expression levels of CB1 in HOD-like cells were shown with RT-PCR and WB (Fig. 2K and L). Electrophoresis of RT-PCR products revealed strong intensity bands corresponding to predicted sizes of GAPDH (226 bp), DSPP (293 bp), nestin (718 bp), and CB1 (115 bp) (Fig. 2J and K). Using WB, the odontoblastic protein expression in cultured cells was confirmed by the expression of the odontogenic markers DSPP and nestin (Fig. 2L). Further WB assays identified the expression of CB1 receptors and TRPV1 channels in HOD-like cells (Fig. 2L). Using IEM, CB1 receptors and TRPV1 channels labeled with tiny immune colloidal gold particles were observed. CB1 receptors distribute sparsely in the cytoplasm (Fig. 3A) and processes (Fig. 3E) in the ultrathin sections of HOD-like cells. No gold particles were observed clearly on the mitochondria (Fig. 3B), endoplasmic reticulum (ER) (Fig. 3C), and primary cilium (Fig. 3D). In the contrast, TRPV1 channels were labeled with colloidal gold particles and distributed densely in the cytoplasm (Fig. 3F–H), mitochondria (Fig. 3F), ER (Fig. 3G), primary cilium (Fig. 3H), and odontoblastic processes (Fig. 3I).
Figure 1. IHC localization of (C) CB1 and (D) TRPV1 in human dental pulp ex vivo. These ODs cells were positive for (A) DSPP, (B) nestin, (C) CB1, and (D) TRPV1 immunoreaction (brown) compared with (E) the blank control (200, scale bars = 20 mm). D, dentin; ODs, odontoblasts; P, pulp. JOE — Volume -, Number -, - 2016
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Figure 2. The expression of CB1 in HOD-like cells was evaluated by IF, RT-PCR, and WB. (A–I) Observation of HOD-like cells by IF staining. There cells were positive for (B and C) DSPP (green), (E and F) nestin (red), and (H and I) CB1 (red) immunoreaction, and nuclei are shown in blue. Scale bars = 50 mm. (J–L) Molecular expression of CB1 receptors in HOD-like cells using RT-PCR and WB. Representative results from at least 3 independent experiments are shown.
To only activate CB1 receptors in ODs, we used the nonselective CB receptor agonist 2-AG, which cannot activate TRPV1 channels (Fig. 4). With extracellular Ca2+ (2.5 mmol/L), the application of 1 mmol/L 2-AG alone rapidly increased the fluorescence intensity to peak values followed by a rapid decline to baseline levels. With the addition of 1 mmol/L 2-AG and 10 nmol/L AM251 (a selective CB1 receptor antagonist), the increase of fluorescence intensity was significantly reduced (Fig. 4A). However, the addition of 1 mmol/L 2-AG and 10 nmol/L AM630 (a selective CB2 receptors antagonist) did not cause significant changes in the fluorescence intensity (Fig. 4B). Moreover, 2-AG was administered in the absence of extracellular Ca2+, but no fluorescence
intensity increase was observed; the fluorescence intensity immediately increased after the addition of 2.5 mmol/L extracellular Ca2+ (Fig. 4C). In contrast, when 1 mmol/L 2-AG and 2.5 mmol/L extracellular Ca2+ were immediately administered together on preapplication Ca2+-free standard solution, the fluorescence intensity also immediately increased (Fig. 4D). However, the 1 mmol/L 2-AG–induced increase of fluorescence intensity in the presence of extracellular Ca2+ was inhibited by 1 mmol/L CPZ (nonselective TRPV1 and TRPM8 channel antagonists) (Fig. 4E) but was not affected by the administration of 1 mmol/L AMTB (a selective TRPM8 channel antagonist) (Fig. 4F). In addition, the 2-AG–induced increase of fluorescence intensity was inhibited
Figure 3. Immunoelectron micrographs of HOD-like cells; 10 nm colloidal gold particles labeled (A–E) CB1 and (F–I) TRPV1 in HOD-like cells (60,000; scale bars = 200 nm). (A) Cytoplasm. (B) and (F) Mitochondria. (C) and (G) Endoplasmic reticulum. (D) and (H) Primary cilium. (E) and (I) Odontoblastic processes.
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Figure 4. Intracellular calcium levels in HOD-like cells were evaluated after being treated with different agonists and antagonists against CB1 and CB2, TRPV1 and TRPM8, and cAMP. Examples of traces of changes of intracellular fluorescence intensity by 1 mmol/L 2-AG with or without (A) 10 nmol/L AM251, (B) 10 nmol/L AM630, (E) 1 mmol/L CPZ, (F) 1 mmol/L AMTB, or (G) 1 mmol/L SQ22536 in the presence of extracellular Ca2+ (2.5 mmol/L) (white boxes at bottom). (C and D) Changes of intracellular fluorescence intensity by the administration of extracellular Ca2+ (2.5 mmol/L) (gray lines at top) after 1 mmol/L 2-AG application or with 1 mmol/L 2-AG simultaneously in the absence of extracellular Ca2+ (white boxes at bottom). Gray lines and black lines at the top indicate times of addition of these antagonists and CB receptors agonist 2-AG to external solution, respectively. (H–L) Summary bar graphs of changes of intracellular fluorescence intensity by 1 mmol/L 2-AG with (gray columns) or without (H) 10 nmol/L AM251, (I) 10 nmol/L AM630, (J) 1 mmol/L CPZ, (K) 1 mmol/L AMTB, or (L) 1 mmol/L SQ22536 (open columns). Each bar denotes the mean standard error of several experiments. Statistically significant differences between columns (shown by solid lines) are indicated by asterisks: *P < .05.
significantly by 1 mmol/L SQ22536 (a selective cAMP antagonist) in the presence of extracellular Ca2+ (2.5 mmol/L) (Fig. 4G). Meanwhile, the statistical analysis of the fluorescence intensity peak in 1 mmol/L 2-AG with or without 10 nmol/L AM251 (Fig. 4H), 1 mmol/L CPZ (Fig. 4J), or 1 mmol/L SQ22536 (Fig. 4L) showed significant differences (P < .05). But, the statistical analysis of the fluorescence intensity peak in 1-mmol/ L 2-AG with or without 10-nmol/L AM630 (Fig. 4I) or 1-mmol/L AMTB (Fig. 4K) showed no significant differences (P > .05).
Discussion The present study shows for the first time that HODs express functional CB1 receptors. Except for molecular expression characteristics of CB1 in HODs, the subcellular characteristics of CB1 receptors observed by IEM was also studied and compared with that of TRPV1 channels for the first time. Moreover, this study showed the physiological properties not only of CB1 receptors but also of TRPV1 channels. In addition, we further clarified the possible functional coupling of CB1 receptors with TRPV1 channels. The cultured HOD-like cells used in this study derived from pulp tissue explants using a previously validated method (14). The odontoblastic phenotype of the cells was confirmed using the gene expression of odontogenic markers DSPP and nestin. In addition to ODs, the results of our IHC revealed the expression of CB1 receptors in some of the cells of the dental pulp ex vivo. The dental pulp is populated by different cell types, including fibroblasts, undifferentiated mesenchymal cells, and defense cells. The result of the present study was similar to that of a previous work, which also showed the expression of CB1 receptors in dental pulp cells (7). In the brain, the 2-AG content is at least 50 times higher than AEA (20). 2-AG acts as the main endocannabinoid for CB1 receptors in the central nervous system and plays an important physiological role with JOE — Volume -, Number -, - 2016
specific binding sites on the CB1 receptors. However, 2-AG could not activate the TRPV1 channels compared with AEA, which will interfere with the study of CB1 receptors. Hence, 2-AG was used as the specific agonist of CB1 receptors in this present study. Only 1 study reported that rat tooth pulp ODs express CB1 receptors (8). In the present study, our results showed that CB1 receptors were positively but not strongly expressed in cell bodies and in some processes of IHC. Using IF staining, RT-PCR, and WB, we also showed the expression of CB1 receptors in HOD-like cells; the results were similar to those described by IHC in native HODs. We further studied the subcellular expression characteristics of CB1 receptors in HODs, and interesting molecules were mapped using IEM at high resolution by applying a set of affinity reagents to sections and locating them with suitable markers; thus, IEM plays an important role in cell biology by quantifying structures and assessing the spatial distributions of molecules (21). Under IEM, the number of CB1 receptors labeled with colloidal gold particles was considerably less than that of TRPV1 channels. In the present study, the expression characteristics of TRPV1 channels under a TEM were reported for the first time. Extensive and obvious expression of TRPV1 channels under a TEM suggested that HOD-like cells could sense and transmit nociceptive stimuli. The results showed that the cytoplasm and cell processes comprised the coexpression areas, which are the first areas to sense extracellular stimuli in HODs and may also be the sites that play the ‘‘cross talk’’ function. However, whether the expression and distribution of CB1 receptors and TRPV1 channels could vary under the effect of external stimuli or infectious substance still needs to be further studied. We showed that the 2-AG–induced fluorescence intensity increase was sensitive to CB1-specific receptor antagonist AM251 instead of CB2specific receptor antagonist AM630, indicating that HODs express CB1 instead of CB2 receptors (1, 2, 22, 23). In the present study, the fluorescence intensity did not increase after the application of 2-AG
Cannabinoid Type 1 Receptors in Human Odontoblast Cells
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Basic Research—Biology in the absence of extracellular Ca2+, indicating that the increase in intracellular Ca2+ by the activation of CB1 receptors was evoked by Ca2+ influx from extracellular medium instead of the Ca2+ release from the intracellular Ca2+ stores. Although 2-AG is a lipid that can easily penetrate across the cell membrane, the ER was not affected by 2-AG possibly because of the expression of CB1 receptors in the ER (main intracellular Ca2+ stores) of HODs as shown by IEM. In addition, the Ca2+ influx stimulated by 2-AG activating CB1 receptors was inhibited by CPZ. Although CPZ acts as an antagonist for TRPV1 and TRPM8 channels, the specific antagonist of TRPM8 channels was also applied but had no obvious effect on the 2-AG–induced increase in fluorescence intensity. 2-AG is an agonist of CB receptors instead of TRPV1 channels, and, thus, there could be a coupling mechanism between TRPV1 channels and CB1 receptors. Meanwhile, the increase of fluorescence intensity by the application of 1 mmol/L 2-AG was also inhibited by adenylyl cyclase antagonist SQ22536. This phenomenon indicates that the ‘‘cross talk’’ mechanism between CB1 receptor and TRPV1 channel activation of CB1 receptors modulates the TRPV1-mediated Ca2+ entry via the intracellular cAMP signaling pathway, which is catalyzed by ATP via adenylyl cyclase. Similar results and conclusions have been confirmed in the study from Tsumura et al (8). Moreover, 1 study also showed that in HEK-293 cells the coexpression of CB1 receptors and TRPV1 channels pretreated by CB1 agonists inhibited or stimulated TRPV1 gating using capsaicin, which was dependent on the concomitant activation of the cAMP signaling pathway (24). CB1 receptors have antihyperalgesic and antinociceptive effects for pain, whereas TRPV1 channels could act as molecular sensors for nociceptive stimuli (1, 25–28). Thus, a functional coexpression or cross talk pathway for CB1 receptors and TRPV1 channels could play an important role in pulp pain sensation and analgesia (15–18). Several studies have reported the effects of the coexpression of CB1 receptors and TRPV1 channels on pain. Hermann et al (24) reported that TRPV1 appears to be partly responsible for the transmission of pain during thermal and inflammatory hyperalgesia, whereas CB1 receptors were suggested to partly counteract hyperalgesia by inhibiting TRPV1-mediated nociception. The TRPV1 channels activated by potent synthetic agonists are immediately followed by desensitization in which the receptors lose reactivity to corresponding agonists to produce powerful analgesic effects in vivo (29). Several studies also reported agents capable of activating both CB1 receptors and TRPV1 channels at the same time; for instance, the combined application of AEA and capsaicin showed strong effects for analgesia (24). Controlling pain was always a difficult research topic, and studies on the coexpression of CB1 receptors and TRPV1 channels may provide new pain treatments in the future. In summary, we provided evidence that HODs express functional CB1 receptors suggesting that CB1 receptors could play roles in dental sensory transduction, biomineralization, and tissue repair.
Acknowledgments Kehua Que and Dan He contributed equally to this work. Supported by the Research Fund for the Doctoral Program of Higher Education of China (20131202120010), Tianjin Municipal Natural Science Foundation (14JCQNJC13500), and Science and Technology Development Project of Tianjin Educational Commission (20130132) in China. The authors deny any conflicts of interest related to this study.
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