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Trans-activation of TRPV1 by D1R in mouse dorsal root ganglion neurons
Biochemical and Biophysical Research Communications 465 (2015) 832e837
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Trans-activation of TRPV1 by D1R in mouse dorsal root ganglion neurons Dong Woo Lee a, 1, Pyung Sun Cho b, 1, Han Kyu Lee b, Sang Hoon Lee b, Sung Jun Jung b, *, Seog Bae Oh a, c, ** a
Pain Cognitive Function Research Center, Dental Research Institute and Department of Neurobiology and Physiology, School of Dentistry, Seoul National University, Seoul, Republic of Korea Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea c Department of Brain and Cognitive Sciences, College of Natural Sciences, Seoul National University, Seoul, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2015 Accepted 21 August 2015 Available online 28 August 2015
TRPV1, a ligand-gated ion channel expressed in nociceptive sensory neurons is modulated by a variety of intracellular signaling pathways. Dopamine is a neurotransmitter that plays important roles in motor control, cognition, and pain modulation in the CNS, and acts via a variety of dopamine receptors (D1R eD5R), a class of GPCRs. Although nociceptive sensory neurons express D1-like receptors, very little is known about the effect of dopamine on TRPV1 in the peripheral nervous system. Therefore, in this study, we examined the effects of D1R activation on TRPV1 in mouse DRG neurons using Ca2þ imaging and immunohistochemical analysis. The D1R agonist SKF-38393 induced reproducible Ca2þ responses via Ca2þ influx through TRPV1 rather than Ca2þ mobilization from intracellular Ca2þ stores. Immunohistochemical analysis revealed co-expression of D1R and TRPV1 in mouse DRG neurons. The PLC-specific inhibitor blocked the SKF-38393-induced Ca2þ response, whereas the PKC, DAG lipase, AC, and PKA inhibitors had no effect on the SKF-38393-induced Ca2þ response. Taken together, our results suggest that the SKF-38393-induced Ca2þ response results from the direct activation of TRPV1 by a PLC/DAGmediated membrane-delimited pathway. These results provide evidence that the trans-activation of TRPV1 following D1R activation may contribute to the modulation of pain signaling in nociceptive sensory neurons. © 2015 Elsevier Inc. All rights reserved.
Keywords: Dopamine Dopamine receptor SKF-38393 TRPV1 Diacylglycerol Nociceptor
1. Introduction Transient receptor potential vanilloid-1 (TRPV1), a representative transducer of nociceptive sensory neurons, plays an important role in nociception. TRPV1 is activated by noxious heat, protons, and chemicals such as capsaicin [1]. Notably, the activation of TRPV1 is modulated by a variety of intracellular signaling pathways. cAMP-dependent PKA regulates TRPV1 desensitization by direct phosphorylation [2]. Furthermore, protein kinase C (PKC) is
* Corresponding author. Department of Biomedical Science, Graduate School of Biomedical and Engineering, Hanyang University, 222 Wangsimni-ro, Seongdonggu, Seoul, 133-791, Republic of Korea. ** Corresponding author. Department of Neurobiology and Physiology, School of Dentistry, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Republic of Korea. E-mail addresses: [email protected] (S.J. Jung), [email protected] (S.B. Oh). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.08.096 0006-291X/© 2015 Elsevier Inc. All rights reserved.
involved in the activation of TRPV1 [3]. In our previous studies, we demonstrated a direct activation of TRPV1 by diacylglycerol (DAG) [4,5]. Dopamine is a neurotransmitter of the catecholamine family that plays numerous important roles in the central nervous system (CNS) [6]. Dopamine exerts cellular effects by binding with a specific membrane receptor. Dopamine receptors are a class of G protein-coupled receptors (GPCRs) that affects cells through various intracellular signaling pathways. Dopamine receptors are classified into five subtypes, D1ReD5R, which are categorized into D1-like (D1R, D5R) and D2-like (D2ReD4R) subfamilies based on their molecular structures and functional properties [6]. Receptors in the D1-like subfamilies activate adenylyl cyclase (AC) and increase intracellular cAMP concentrations, whereas those in the D2like subfamilies result in an opposite response [7,8]. Previous studies revealed another D1-like receptor elicits the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into DAG and inositol triphosphate (IP3) through the activation of phospholipase C (PLC)
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[8e10]. The distribution and contribution of dopamine receptors in the CNS are well understood [11,12]. In addition, dopaminergic fibers that originate from the area A11 in the diencephalon project to the dorsal horn of the spinal cord and represent a major source of dopamine in the spinal cord [13,14], which exerts anti-nociceptive action by modulating nociceptive transmission through D2-like receptors [15,16]. A recent study showed that dopamine receptors were expressed not only in the CNS but also in DRG neurons in the peripheral nervous system. A subpopulation of DRG neurons expresses tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine [17]. mRNAs and alternatively spliced transcripts of dopamine receptors have been detected in peripheral sensory neurons [18], and the expression of dopamine receptors in DRG neurons was also confirmed by immunofluorescence imaging [19]. Interestingly, the expression of dopamine receptors was limited to small diameter DRG neurons, which mainly represent nociceptive sensory neurons, and although D1R and D5R were primarily expressed, D2R was not observed [20]. It has been demonstrated that dopamine influences the properties of various ion channels in DRG neurons through diverse intracellular signaling pathways [20,21]. Moreover, it was reported that spontaneous glutamate release was enhanced by the activation of D1-like receptors via PLC and TRPV1 in rat somatosensory cortical neurons [22]. However, to date, the effect of dopamine on TRPV1 in nociceptive sensory neurons remains poorly understood. In this study, we examined whether D1-like receptors and TRPV1 are co-expressed in DRG neurons, and if so, whether the activation of D1-like receptors recruits an intracellular signaling pathway to modulate TRPV1 activity in nociceptive sensory neurons. 2. Materials and methods All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the School of Dentistry, Seoul National University (Seoul, Korea). Adult C57BL/6J wild-type male mice (OrientBio, Korea) were used. Animals were housed in a conventional facility with a 12:12 h light cycle (lights on 8.00am) and ad libitum access to water and chow. Mice were acclimatized at least one week prior to experiments. 2.1. Preparation of DRG neurons DRG neurons were prepared as previously described [5]. Briefly, DRG neurons were obtained from 6-week-old mice and incubated in 1 mg/ml collagenase A (Roche) and 2.4 U/ml dispase-II (Roche) in HBSS for 40 min at 37 C. Subsequently, these neurons were digested with 0.25% trypsin (Sigma) for 5 min at 37 C, followed by 0.25% trypsin inhibitor (Sigma). Furthermore, the neurons were mechanically dissociated with a flame-polished Pasteur pipette in the presence of 0.05% DNase I (Sigma). Moreover, the neurons were plated on Poly-D-Lysine (PDL)-coated 12 mm glass coverslip and grown in a neurobasal defined medium (with 5% B-27 supplement, Invitrogen) in the presence of 5% CO2 at 37 C. DRG neurons were then maintained for 24 h before use. 2.2. Ca2þ imaging The Ca2þ response of DRG neurons was measured using the fluorescent Ca2þ probe (indicator) Fluo-3 AM (Enzo Life Science, US). DRG neurons were loaded with Fluo-3 AM (5 mM) mixed with 1 ml pluronic acid (20% solution in DMSO, Life Technologies) in
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DMEM medium (with 10% FBS) for 40 min at 37 C. Loaded cells, which were previously placed on coverslips, were then mounted onto the chamber and placed under an inverted microscope (Olympus IX70, Japan). We used an external bath of the following composition (mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, with pH adjusted to 7.3e7.4. Intracellular calcium concentrations ([Ca2þ]i) were measured by microfluorometry with an intensified CCD camera (QIClick, QImaging, Canada) coupled to the microscope and a computer with software (MetaMorph® NX, Molecular Devices, US). Ca2þ responses were presented as a pseudoratio (⊿F/F) to estimate comparative fluorescence intensity [23].
⊿F=F ¼ ðF1 Fbase Þ=Fbase F1 ¼ measured intensity of the cell after stimulation. Fbase ¼ measured intensity of the cell before stimulation. 2.3. Immunohistochemistry Immunohistochemical analysis was performed as previously described [24]. Briefly, Mice were perfused with physiological saline and subsequently with 4% paraformaldehyde. The DRG neurons from the lumbar segments were obtained and immersed in a post fixative at 4 C overnight and then transferred to 30% sucrose in PBS for 48 h. Serial frozen transverse sections (10 mm thick) were mounted on slides. All procedures were performed at room temperature (RT) unless otherwise stated. Tissue sections were washed in PBS and then incubated in a blocking solution containing 5% NGS, 2% BSA, 2% FBS, and 0.1% Triton X-100 for 1 h. The sections were incubated overnight at 4 C with the primary guinea pig anti-TRPV1 antibody (1:1000; Chemicon AB5566) and primary mouse anti-D1R antibody (1:200; Millipore MAB5290), then washed 3 times with PBS. Next, sections were incubated with a CY3-conjugated donkey anti-guinea pig IgG antibody (1:1000; Jackson ImmunoResearch, West Grove, PA, US) and an FITC-conjugated donkey anti-mouse IgG antibody (1:500; Jackson ImmunoResearch, West Grove, PA, US) for 1 h. After being washed with PBS, sections were mounted in Vectashield, mounting media (Vector Laboratories, Inc., Burlingame, CA, US), which contained DAPI to detect nuclei and visualized using a confocal microscope (LSM 700, Zeiss, Germany). Colocalization of TRPV1 and D1R was examined by using Zen 2012 software (Zeiss, Germany). Images of double-stained sections are submitted to background correction. Then, diverse coefficients are counted. The coefficients and interpretation of their results showing either absence or presence of co-localization. 2.4. Drugs SKF-38393, SCH-23390, capsaicin, capsazepine, SQ 22536, H-89, RHC 80267, bisindolylmaleimide (BIM), and thapsigargin were obtained from Sigma (Saint Louis, MO). U73122 and U73343 were purchased from Tocris Bioscience (Bristol, UK). 2.5. Statistical analyses Statistical analysis of the normalized Ca2þ responses (2nd SKF38393 response relative to 1st SKF-38393 response) was performed using SPSS software (SPSS Statistics 17.0, IBM). Doseeresponse analysis was performed using Origin 6.1 software (MicroCal, Northampton, MA). Data were compared by one-way ANOVA followed by Bonferroni's post-hoc test. Differences were considered to be significant when the P value was less than 0.05. All data are presented as mean ± SEM; the number of cells tested is indicated in parentheses where applicable.
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3. Results 3.1. D1R agonist SKF-38393 induces Ca2þ response in DRG neurons in a dose-dependent manner The effects of D1R activation in DRG neurons were determined in small sized cells. We examined whether D1R agonist SKF-38393 induces Ca2þ responses in DRG neurons using Fluo-3 AM-based Ca2þ imaging. Application of 1 mM SKF-38393 (10 s) induced reproducible Ca2þ responses during sequential application of SKF38393 (83.74 ± 4.53%; n ¼ 8; p > 0.05) (Fig. 1A, C). SCH-23390 (10 mM), a D1R selective antagonist, inhibited Ca2þ responses elicited by SKF-38393, indicating that the SKF-38393-induced Ca2þ response was mediated through the activation of D1R (6.31 ± 1.04%; n ¼ 8; p < 0.005) (Fig. 1B, C). SKF-38393-sensitive DRG neurons exhibited a concentration-dependent increase in [Ca2þ]i, with the half-maximal concentration (EC50) of SKF-38393, 547 nM (Fig. 1D). Taken together, our results indicate that the activation of D1R causes a transient increase in [Ca2þ]i with a dosedependent manner in a subpopulation of DRG neurons. 3.2. SKF-38393-induced Ca2þ response results from influx of extracellular Ca2þ We examined the source of increase in [Ca2þ]i by SKF-38393. To exclude extracellular Ca2þ influx, 0 mM Ca2þ solution was applied for 100 s before the 2nd SKF-38393 application. The 2nd SKF38393-induced Ca2þ response was abolished by pretreatment with 0 mM Ca2þ in the bath solution compared with the Ca2þ response to 1st SKF-38393 application (4.70 ± 1.12%; n ¼ 8; p < 0.005) (Fig. 2). Next, we used thapsigargin, a selective inhibitor of the sarcoplasmic/endoplasmic Ca2þ-ATPase, to exclude Ca2þ mobilization from intracellular Ca2þ stores [25]. The SKF-38393-induced Ca2þ response did not change in the presence of 1 mM thapsigargin (71.53 ± 6.55%; n ¼ 8; p > 0.05), and the depletion of
intracellular Ca2þ stores was confirmed by a following slight Ca2þ response to application of thapsigargin (Fig. 2). These results suggest that SKF-38393-induced Ca2þ response is a consequence of an influx of extracellular Ca2þ rather than Ca2þ released from intracellular stores. 3.3. SKF-38393 induces Ca2þ response through TRPV1 in nociceptive sensory neurons We next examined whether the SKF-38393-induced Ca2þ response was associated with TRPV1. The Ca2þ response was elicited by the 1st SKF-38393 application, and the 2nd SKF-38393induced Ca2þ response was blocked by a pretreatment with a TRPV1 blocker, 10 mM capsazepine (6.59 ± 1.24%; n ¼ 8; p < 0.005) (Fig. 3A, C). To identify TRPV1-expressing nociceptive neurons, a TRPV1 agonist, 300 nM capsaicin, was applied for 5 s at the end of each experiment. In 86 out of 134 DRG neurons that had capsaicinsensitivity (64%; n ¼ 86/134), we found that 57 neurons responded to SKF-38393 (66%; n ¼ 57/86). Twenty nine out of 86 neurons (34%; n ¼ 29/86) responded only to capsaicin. No neurons were found to respond to SKF-38393 alone. In small DRG neurons of TRPV1 knock-out mice, no Ca2þ response was induced by SKF38393 (2.21 ± 1.24%; n ¼ 7; p < 0.005) (Fig. 3B). To test whether voltage-activated Ca2þ channels (VACCs) contributed to the SKF38393-induced Ca2þ response, 100 mM CdCl2 was added to the bath solution to non-selectively block all VACCs without influencing TRPV1 [26]. SKF-38393 evoked Ca2þ responses even in the presence of Cd2þ (79.61 ± 7.27%; n ¼ 8; p > 0.05) (Fig. 3B, C). Taken together, these results indicate that SKF-38393 activates TRPV1 channels in nociceptive sensory neurons. 3.4. D1R and TRPV1 are co-expressed in DRG neurons Our results above suggest D1R and TRPV1 might be expressed together in the same DRG neurons. Thus, we examined whether
Fig. 1. SKF-38393 induces Ca2þ response in DRG neurons. A, Sequential application of 1 mM SKF-38393 (SKF) evokes reproducible Ca2þ responses (n ¼ 8). B, The SKF-38393-induced Ca2þ response was blocked by a 100 s pretreatment of 10 mM SCH-23390, a D1R selective antagonist (SCH, n ¼ 8). C, Summary of normalized Ca2þ response (2nd SKF-38393 response compared to 1st SKF-38393 response) (*p < 0.005; non-paired t-test). D, Doseeresponse curve of SKF-38393-induced Ca2þ response in mouse DRG neurons showed that the EC50 is 547 nM (n ¼ 10 in respective concentration). Results are presented as the mean ± SEM.
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Fig. 2. The SKF-38393-induced Ca2þ response results from extracellular Ca2þ influx. The SKF-38393-induced Ca2þ response was abolished by pretreatment of 0 mM Ca2þ in the bath solution (n ¼ 8), but not 1 mM thapsigargin (Thap, n ¼ 8). Summary of normalized Ca2þ response (2nd SKF-38393 response compared to 1st SKF-38393 response; *p < 0.005; oneway ANOVA). Results are presented as the mean ± SEM.
Fig. 3. SKF-38393 induces Ca2þ response through activation of TRPV1. A, SKF-38393-induced Ca2þ response was blocked by the pretreatment of capsazepine (CPZ, 10 mM, n ¼ 8). However, the SKF-38393-induced Ca2þ response was maintained in the Cd2þ condition (1 mM, n ¼ 8). B, In DRG neurons of TRPV1 (/) mice, SKF-38393 did not induce Ca2þ responses (n ¼ 7). C, Summary of normalized Ca2þ response (2nd SKF-38393 response compared to 1st SKF-38393 response; *p < 0.005; one-way ANOVA). Results are presented as the mean ± SEM. D, Co-expression of D1R and TRPV1 in DRG neurons. Nuclei of DRG neurons were stained with DAPI (blue). DRG sections were stained with monoclonal antibodies to D1R (green) and TRPV1 (red). Merged images indicate co-expression of D1R and TRPV1. Scale bars shown are 50 mm.
D1R and TRPV1 were co-expressed in DRG neurons using immunohistochemical analysis (Fig. 3D). Consistent with previous reports [27], TRPV1-positive fluorescence was expressed in 60 DRG neurons (60/142, 42.3%). Also, D1R-positive fluorescence were found in 33 DRG neurons (33/142, 23.2%), which is in accordance with a previous study that D1R and D5R were expressed in DRG neurons [20]. Double immunofluorescence revealed that DRG neurons that were immunoreactive for TRPV1 and D1R (23/142, 16.2%) and majority of D1R-immunoreactive neurons were colocalized with TRPV1 (69.7%). 3.5. SKF-38393-induced Ca2þ response in DRG neuron results from direct activation of TRPV1 by DAG Activation of D1R increases intracellular cAMP levels via adenylyl cyclase (AC) [7,8], and D1R stimulates PLC pathway and
hydrolyzes PIP2 into DAG and inositol IP3 [8e10,22]. Both AC and PLC pathways can affect the function of TRPV1 either directly or by phosphorylation [2,28]. We identified the signaling molecules that mediate SKF-38393-induced TRPV1 activation (Fig. 4). Pretreatment with U73122 (2 mM), a specific PLC inhibitor, abolished SKF38393-induced Ca2þ response (3.79 ± 1.25%; n ¼ 8; p < 0.005), whereas U73343, an inactive-form of U73122, did not influence the Ca2þ response elicited by SKF-38393 (74.74 ± 5.36%; n ¼ 8; p > 0.05). The SKF-38393-induced Ca2þ response remained in the presence of 1 mM BIM, a specific PKC inhibitor, (77.20 ± 3.73%; n ¼ 8; p > 0.05) 1 mM RHC 80267, a DAG lipase inhibitor, (78.38 ± 10.54%; n ¼ 8; p > 0.05), or their cocktail (77.92 ± 4.58%; n ¼ 8; p > 0.05), indicating that the PLC pathway mediated SKF38393-induced Ca2þ response, but without PKC- and DAG lipasedependent activation of TRPV1. Next, we tested whether activation of the AC/PKA signaling pathway by D1R was involved in the
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Fig. 4. SKF-38393 induces a Ca2þ response through direct activation of TRPV1 by the PLC/DAG pathway. A, The SKF-38393-induced Ca2þ response was abolished in the presence of U73122 (2 mM, n ¼ 8), but not U73343 (2 mM, n ¼ 8). Also, 1 mM BIM (n ¼ 8), 1 mM RHC 80267 (n ¼ 8), and a mixture of both (n ¼ 8) had no effect on the SKF-38393-induced Ca2þ response. The SKF-38393-induced Ca2þ response was not blocked by the pretreatment with SQ 22,536 (10 mM, n ¼ 8) or H-89 (10 mM, n ¼ 8). B, Summary of normalized Ca2þ response (2nd SKF-38393 response compared to 1st SKF-38393 response; *p < 0.005; one-way ANOVA). Results are presented as the mean ± SEM.
SKF-38393-induced Ca2þ response. Pretreatment with 10 mM SQ 22,536, an AC inhibitor (74.49 ± 8.45%; n ¼ 8; p > 0.05) and 10 mM H-89, a PKA inhibitor (84.10 ± 5.62%; n ¼ 8; p > 0.05) had no effect to SKF-38393-induced Ca2þ response. Taken together, these results indicate that D1R agonist SKF-38393 activates TRPV1 through PLC signaling pathway, and imply that DAG is a strong candidate for downstream signaling molecules in PLC pathway. 4. Discussion In the present study, we found that stimulation of D1R transactivates TRPV1 in mouse DRG neurons, and DAG endogenously produced by the PLC/DAG pathway following D1R activation may mediate the direct activation of TRPV1. We focused on the excitatory effect of dopamine in DRG neurons using a specific D1R agonist, SKF-38393, because the biphasic effects of dopamine depend on the subclass of dopamine, D1 or D2like, receptors. The excitatory effect of dopamine was mediated by D1-like receptor-linked intracellular signaling pathways. In contrast, the inhibitory effect was mediated by D2-like receptors [29,30]. A recent study characterized the expression of D1R and D5R, but not D2R, in DRG neurons by Western blot and immunohistochemical analysis [20]. Based on these reports, we inferred that the excitatory effect of dopamine would mainly function in nociceptive sensory neurons. In this study, we demonstrated the excitatory effect of dopamine and trans-activation of TRPV1 by D1R stimulation. However, further experiments will be required to determine whether dopamine induces an identical excitatory effect via D1-like receptors.
Our results show that the effect of D1R activation was mediated by TRPV1. In the presence of thapsigargin, the SKF-38393 response was not changed. This result was consistent with a previous study that showed activation of TRPV1 by D1R in rat somatosensory cortical neurons [22]. Given that VACCs may contribute to another portion of Ca2þ response, we verified that VACCs were not involved in the SKF-38393-induced Ca2þ response. In the experiments with capsazepine or TRPV1 knock-out mice, moreover, we did not observe SKF-38393-induced Ca2þ response. Previous studies suggested the existence of a phosphatidylinositol-linked D1-like receptor [8e10]. However, it is unclear which downstream signaling molecule links D1R to TRPV1. In the present study, we found that the PLC/DAG pathway mediates D1R activation in nociceptive DRG neurons. Previously, we reported that endogenous DAG directly activates TRPV1 produced by GPCR activation [4]. Moreover, we have recently shown that group 1 mGluR5 activates the TRPV1 channel in a membrane-delimited manner; this process is mediated by DAG [5]. In another studies, it was suggested that modulation of TRPV1 activity was determined by PLC isoforms, such as PLCb and PLCd [31] and that TRPV1 activation via PLC was mediated by 2-arachidonoylglycerol and 1arachidonoylglycerol as endogenous TRPV1 activators [32]. In the present study, however, DAG lipase was not involved in a link between D1R and TRPV1. Taken together, these data suggest that DAG, produced by D1R activation, is the most probable endogenous ligand for direct activation of TRPV1. In addition, we found that the effect of SKF-38393 persisted in the presence of an AC inhibitor or PKA inhibitor. This result is in contrast with a previous study that capsaicin-induced currents resulted from TRPV1 activation were
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abolished by AC and PKA inhibitors in mouse DRG neurons [33]. Although the experimental conditions were different, it seems that more studies are needed to characterize the intracellular signaling pathways for TRPV1 modulation; an indirect trans-activation through intracellular signaling pathways or direct activation produced by a specific agonist. The role of dopamine receptors in the pain transmission can be considered at spinal cord or at peripheral site. However, descending pain modulation of dopamine in spinal cord is related to the dopaminergic nerve and D2-like receptors [15,16]. Recently, it has been reported that epidermal cells such as keratinocytes or melanocytes express TH, the rate-limiting enzyme in the dopamine metabolic pathway [17,34,35]. If dopamine receptors are expressed in the epidermal nerve fibers, particularly unmyelinated afferent fibers, dopamine released from epidermal cells may excite nociceptive primary afferent through TRPV1 activation. Another source of dopamine may be in the form of it being released by itself in DRG neurons [17,19,36,37]. To understand the physiological implications of trans-activation of TRPV1 by D1R activation in DRG neurons, in vivo studies are needed to explore the role of dopamine in peripheral pain modulation. In conclusion, our present study demonstrates that activation of D1R trans-activates TRPV1 in mouse nociceptive DRG neurons. The D1R agonist SKF-38393 induces Ca2þ responses through direct activation of TRPV1 by a PLC/DAG intracellular signaling pathway without the involvement of the AC/PKA cascade. Our finding regarding the effect of a D1R agonist on nociceptive sensory neurons provides an insight into the role of dopamine in pain modulation. Acknowledgments This work was supported by the research fund of Hanyang University (HY-2012-N) and the Pioneer research center through National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning Korea government (MSIP) (2012R1A3A2048834 and 2011-0027921). Transparency document Transparency document related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2015.08.096. References [1] D.E. Clapham, TRP channels as cellular sensors, Nature 426 (2003) 517e524. [2] G. Bhave, W. Zhu, H. Wang, et al., cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation, Neuron 35 (2002) 721e731. [3] L.S. Premkumar, G.P. Ahern, Induction of vanilloid receptor channel activity by protein kinase C, Nature 408 (2000) 985e990. [4] D.H. Woo, S.J. Jung, M.H. Zhu, et al., Direct activation of transient receptor potential vanilloid 1(TRPV1) by diacylglycerol (DAG), Mol. Pain 4 (2008) 42. [5] Y.H. Kim, C.K. Park, S.K. Back, et al., Membrane-delimited coupling of TRPV1 and mGluR5 on presynaptic terminals of nociceptive neurons, J. Neurosci. 29 (2009) 10000e10009. [6] J.N. Joyce, Multiple dopamine receptors and behavior, Neurosci. Biobehav. Rev. 7 (1983) 227e256. [7] R.M. Huff, Signaling pathways modulated by dopamine receptors, in: The Dopamine Receptors, Springer, 1997, pp. 167e192. [8] C. Missale, S.R. Nash, S.W. Robinson, et al., Dopamine receptors: from structure to function, Physiol. Rev. 78 (1998) 189e225. [9] L.Q. Jin, S. Goswami, G. Cai, et al., SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain, J. Neurochem. 85 (2003) 378e386.
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Trans-activation of TRPV1 by D1R in mouse dorsal root ganglion neurons Dong Woo Lee a, 1, Pyung Sun Cho b, 1, Han Kyu Lee b, Sang Hoon Lee b, Sung Jun Jung b, *, Seog Bae Oh a, c, ** a
Pain Cognitive Function Research Center, Dental Research Institute and Department of Neurobiology and Physiology, School of Dentistry, Seoul National University, Seoul, Republic of Korea Department of Biomedical Science, Graduate School of Biomedical Science and Engineering, Hanyang University, Seoul, Republic of Korea c Department of Brain and Cognitive Sciences, College of Natural Sciences, Seoul National University, Seoul, Republic of Korea b
a r t i c l e i n f o
a b s t r a c t
Article history: Received 19 August 2015 Accepted 21 August 2015 Available online 28 August 2015
TRPV1, a ligand-gated ion channel expressed in nociceptive sensory neurons is modulated by a variety of intracellular signaling pathways. Dopamine is a neurotransmitter that plays important roles in motor control, cognition, and pain modulation in the CNS, and acts via a variety of dopamine receptors (D1R eD5R), a class of GPCRs. Although nociceptive sensory neurons express D1-like receptors, very little is known about the effect of dopamine on TRPV1 in the peripheral nervous system. Therefore, in this study, we examined the effects of D1R activation on TRPV1 in mouse DRG neurons using Ca2þ imaging and immunohistochemical analysis. The D1R agonist SKF-38393 induced reproducible Ca2þ responses via Ca2þ influx through TRPV1 rather than Ca2þ mobilization from intracellular Ca2þ stores. Immunohistochemical analysis revealed co-expression of D1R and TRPV1 in mouse DRG neurons. The PLC-specific inhibitor blocked the SKF-38393-induced Ca2þ response, whereas the PKC, DAG lipase, AC, and PKA inhibitors had no effect on the SKF-38393-induced Ca2þ response. Taken together, our results suggest that the SKF-38393-induced Ca2þ response results from the direct activation of TRPV1 by a PLC/DAGmediated membrane-delimited pathway. These results provide evidence that the trans-activation of TRPV1 following D1R activation may contribute to the modulation of pain signaling in nociceptive sensory neurons. © 2015 Elsevier Inc. All rights reserved.
Keywords: Dopamine Dopamine receptor SKF-38393 TRPV1 Diacylglycerol Nociceptor
1. Introduction Transient receptor potential vanilloid-1 (TRPV1), a representative transducer of nociceptive sensory neurons, plays an important role in nociception. TRPV1 is activated by noxious heat, protons, and chemicals such as capsaicin [1]. Notably, the activation of TRPV1 is modulated by a variety of intracellular signaling pathways. cAMP-dependent PKA regulates TRPV1 desensitization by direct phosphorylation [2]. Furthermore, protein kinase C (PKC) is
* Corresponding author. Department of Biomedical Science, Graduate School of Biomedical and Engineering, Hanyang University, 222 Wangsimni-ro, Seongdonggu, Seoul, 133-791, Republic of Korea. ** Corresponding author. Department of Neurobiology and Physiology, School of Dentistry, Seoul National University, 101 Daehak-ro, Jongno-gu, Seoul, 110-744, Republic of Korea. E-mail addresses: [email protected] (S.J. Jung), [email protected] (S.B. Oh). 1 These authors contributed equally to this work. http://dx.doi.org/10.1016/j.bbrc.2015.08.096 0006-291X/© 2015 Elsevier Inc. All rights reserved.
involved in the activation of TRPV1 [3]. In our previous studies, we demonstrated a direct activation of TRPV1 by diacylglycerol (DAG) [4,5]. Dopamine is a neurotransmitter of the catecholamine family that plays numerous important roles in the central nervous system (CNS) [6]. Dopamine exerts cellular effects by binding with a specific membrane receptor. Dopamine receptors are a class of G protein-coupled receptors (GPCRs) that affects cells through various intracellular signaling pathways. Dopamine receptors are classified into five subtypes, D1ReD5R, which are categorized into D1-like (D1R, D5R) and D2-like (D2ReD4R) subfamilies based on their molecular structures and functional properties [6]. Receptors in the D1-like subfamilies activate adenylyl cyclase (AC) and increase intracellular cAMP concentrations, whereas those in the D2like subfamilies result in an opposite response [7,8]. Previous studies revealed another D1-like receptor elicits the hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) into DAG and inositol triphosphate (IP3) through the activation of phospholipase C (PLC)
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[8e10]. The distribution and contribution of dopamine receptors in the CNS are well understood [11,12]. In addition, dopaminergic fibers that originate from the area A11 in the diencephalon project to the dorsal horn of the spinal cord and represent a major source of dopamine in the spinal cord [13,14], which exerts anti-nociceptive action by modulating nociceptive transmission through D2-like receptors [15,16]. A recent study showed that dopamine receptors were expressed not only in the CNS but also in DRG neurons in the peripheral nervous system. A subpopulation of DRG neurons expresses tyrosine hydroxylase (TH), the rate-limiting enzyme for the synthesis of dopamine [17]. mRNAs and alternatively spliced transcripts of dopamine receptors have been detected in peripheral sensory neurons [18], and the expression of dopamine receptors in DRG neurons was also confirmed by immunofluorescence imaging [19]. Interestingly, the expression of dopamine receptors was limited to small diameter DRG neurons, which mainly represent nociceptive sensory neurons, and although D1R and D5R were primarily expressed, D2R was not observed [20]. It has been demonstrated that dopamine influences the properties of various ion channels in DRG neurons through diverse intracellular signaling pathways [20,21]. Moreover, it was reported that spontaneous glutamate release was enhanced by the activation of D1-like receptors via PLC and TRPV1 in rat somatosensory cortical neurons [22]. However, to date, the effect of dopamine on TRPV1 in nociceptive sensory neurons remains poorly understood. In this study, we examined whether D1-like receptors and TRPV1 are co-expressed in DRG neurons, and if so, whether the activation of D1-like receptors recruits an intracellular signaling pathway to modulate TRPV1 activity in nociceptive sensory neurons. 2. Materials and methods All surgical and experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the School of Dentistry, Seoul National University (Seoul, Korea). Adult C57BL/6J wild-type male mice (OrientBio, Korea) were used. Animals were housed in a conventional facility with a 12:12 h light cycle (lights on 8.00am) and ad libitum access to water and chow. Mice were acclimatized at least one week prior to experiments. 2.1. Preparation of DRG neurons DRG neurons were prepared as previously described [5]. Briefly, DRG neurons were obtained from 6-week-old mice and incubated in 1 mg/ml collagenase A (Roche) and 2.4 U/ml dispase-II (Roche) in HBSS for 40 min at 37 C. Subsequently, these neurons were digested with 0.25% trypsin (Sigma) for 5 min at 37 C, followed by 0.25% trypsin inhibitor (Sigma). Furthermore, the neurons were mechanically dissociated with a flame-polished Pasteur pipette in the presence of 0.05% DNase I (Sigma). Moreover, the neurons were plated on Poly-D-Lysine (PDL)-coated 12 mm glass coverslip and grown in a neurobasal defined medium (with 5% B-27 supplement, Invitrogen) in the presence of 5% CO2 at 37 C. DRG neurons were then maintained for 24 h before use. 2.2. Ca2þ imaging The Ca2þ response of DRG neurons was measured using the fluorescent Ca2þ probe (indicator) Fluo-3 AM (Enzo Life Science, US). DRG neurons were loaded with Fluo-3 AM (5 mM) mixed with 1 ml pluronic acid (20% solution in DMSO, Life Technologies) in
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DMEM medium (with 10% FBS) for 40 min at 37 C. Loaded cells, which were previously placed on coverslips, were then mounted onto the chamber and placed under an inverted microscope (Olympus IX70, Japan). We used an external bath of the following composition (mM): 140 NaCl, 5 KCl, 2 CaCl2, 1 MgCl2, 10 glucose, and 10 HEPES, with pH adjusted to 7.3e7.4. Intracellular calcium concentrations ([Ca2þ]i) were measured by microfluorometry with an intensified CCD camera (QIClick, QImaging, Canada) coupled to the microscope and a computer with software (MetaMorph® NX, Molecular Devices, US). Ca2þ responses were presented as a pseudoratio (⊿F/F) to estimate comparative fluorescence intensity [23].
⊿F=F ¼ ðF1 Fbase Þ=Fbase F1 ¼ measured intensity of the cell after stimulation. Fbase ¼ measured intensity of the cell before stimulation. 2.3. Immunohistochemistry Immunohistochemical analysis was performed as previously described [24]. Briefly, Mice were perfused with physiological saline and subsequently with 4% paraformaldehyde. The DRG neurons from the lumbar segments were obtained and immersed in a post fixative at 4 C overnight and then transferred to 30% sucrose in PBS for 48 h. Serial frozen transverse sections (10 mm thick) were mounted on slides. All procedures were performed at room temperature (RT) unless otherwise stated. Tissue sections were washed in PBS and then incubated in a blocking solution containing 5% NGS, 2% BSA, 2% FBS, and 0.1% Triton X-100 for 1 h. The sections were incubated overnight at 4 C with the primary guinea pig anti-TRPV1 antibody (1:1000; Chemicon AB5566) and primary mouse anti-D1R antibody (1:200; Millipore MAB5290), then washed 3 times with PBS. Next, sections were incubated with a CY3-conjugated donkey anti-guinea pig IgG antibody (1:1000; Jackson ImmunoResearch, West Grove, PA, US) and an FITC-conjugated donkey anti-mouse IgG antibody (1:500; Jackson ImmunoResearch, West Grove, PA, US) for 1 h. After being washed with PBS, sections were mounted in Vectashield, mounting media (Vector Laboratories, Inc., Burlingame, CA, US), which contained DAPI to detect nuclei and visualized using a confocal microscope (LSM 700, Zeiss, Germany). Colocalization of TRPV1 and D1R was examined by using Zen 2012 software (Zeiss, Germany). Images of double-stained sections are submitted to background correction. Then, diverse coefficients are counted. The coefficients and interpretation of their results showing either absence or presence of co-localization. 2.4. Drugs SKF-38393, SCH-23390, capsaicin, capsazepine, SQ 22536, H-89, RHC 80267, bisindolylmaleimide (BIM), and thapsigargin were obtained from Sigma (Saint Louis, MO). U73122 and U73343 were purchased from Tocris Bioscience (Bristol, UK). 2.5. Statistical analyses Statistical analysis of the normalized Ca2þ responses (2nd SKF38393 response relative to 1st SKF-38393 response) was performed using SPSS software (SPSS Statistics 17.0, IBM). Doseeresponse analysis was performed using Origin 6.1 software (MicroCal, Northampton, MA). Data were compared by one-way ANOVA followed by Bonferroni's post-hoc test. Differences were considered to be significant when the P value was less than 0.05. All data are presented as mean ± SEM; the number of cells tested is indicated in parentheses where applicable.
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3. Results 3.1. D1R agonist SKF-38393 induces Ca2þ response in DRG neurons in a dose-dependent manner The effects of D1R activation in DRG neurons were determined in small sized cells. We examined whether D1R agonist SKF-38393 induces Ca2þ responses in DRG neurons using Fluo-3 AM-based Ca2þ imaging. Application of 1 mM SKF-38393 (10 s) induced reproducible Ca2þ responses during sequential application of SKF38393 (83.74 ± 4.53%; n ¼ 8; p > 0.05) (Fig. 1A, C). SCH-23390 (10 mM), a D1R selective antagonist, inhibited Ca2þ responses elicited by SKF-38393, indicating that the SKF-38393-induced Ca2þ response was mediated through the activation of D1R (6.31 ± 1.04%; n ¼ 8; p < 0.005) (Fig. 1B, C). SKF-38393-sensitive DRG neurons exhibited a concentration-dependent increase in [Ca2þ]i, with the half-maximal concentration (EC50) of SKF-38393, 547 nM (Fig. 1D). Taken together, our results indicate that the activation of D1R causes a transient increase in [Ca2þ]i with a dosedependent manner in a subpopulation of DRG neurons. 3.2. SKF-38393-induced Ca2þ response results from influx of extracellular Ca2þ We examined the source of increase in [Ca2þ]i by SKF-38393. To exclude extracellular Ca2þ influx, 0 mM Ca2þ solution was applied for 100 s before the 2nd SKF-38393 application. The 2nd SKF38393-induced Ca2þ response was abolished by pretreatment with 0 mM Ca2þ in the bath solution compared with the Ca2þ response to 1st SKF-38393 application (4.70 ± 1.12%; n ¼ 8; p < 0.005) (Fig. 2). Next, we used thapsigargin, a selective inhibitor of the sarcoplasmic/endoplasmic Ca2þ-ATPase, to exclude Ca2þ mobilization from intracellular Ca2þ stores [25]. The SKF-38393-induced Ca2þ response did not change in the presence of 1 mM thapsigargin (71.53 ± 6.55%; n ¼ 8; p > 0.05), and the depletion of
intracellular Ca2þ stores was confirmed by a following slight Ca2þ response to application of thapsigargin (Fig. 2). These results suggest that SKF-38393-induced Ca2þ response is a consequence of an influx of extracellular Ca2þ rather than Ca2þ released from intracellular stores. 3.3. SKF-38393 induces Ca2þ response through TRPV1 in nociceptive sensory neurons We next examined whether the SKF-38393-induced Ca2þ response was associated with TRPV1. The Ca2þ response was elicited by the 1st SKF-38393 application, and the 2nd SKF-38393induced Ca2þ response was blocked by a pretreatment with a TRPV1 blocker, 10 mM capsazepine (6.59 ± 1.24%; n ¼ 8; p < 0.005) (Fig. 3A, C). To identify TRPV1-expressing nociceptive neurons, a TRPV1 agonist, 300 nM capsaicin, was applied for 5 s at the end of each experiment. In 86 out of 134 DRG neurons that had capsaicinsensitivity (64%; n ¼ 86/134), we found that 57 neurons responded to SKF-38393 (66%; n ¼ 57/86). Twenty nine out of 86 neurons (34%; n ¼ 29/86) responded only to capsaicin. No neurons were found to respond to SKF-38393 alone. In small DRG neurons of TRPV1 knock-out mice, no Ca2þ response was induced by SKF38393 (2.21 ± 1.24%; n ¼ 7; p < 0.005) (Fig. 3B). To test whether voltage-activated Ca2þ channels (VACCs) contributed to the SKF38393-induced Ca2þ response, 100 mM CdCl2 was added to the bath solution to non-selectively block all VACCs without influencing TRPV1 [26]. SKF-38393 evoked Ca2þ responses even in the presence of Cd2þ (79.61 ± 7.27%; n ¼ 8; p > 0.05) (Fig. 3B, C). Taken together, these results indicate that SKF-38393 activates TRPV1 channels in nociceptive sensory neurons. 3.4. D1R and TRPV1 are co-expressed in DRG neurons Our results above suggest D1R and TRPV1 might be expressed together in the same DRG neurons. Thus, we examined whether
Fig. 1. SKF-38393 induces Ca2þ response in DRG neurons. A, Sequential application of 1 mM SKF-38393 (SKF) evokes reproducible Ca2þ responses (n ¼ 8). B, The SKF-38393-induced Ca2þ response was blocked by a 100 s pretreatment of 10 mM SCH-23390, a D1R selective antagonist (SCH, n ¼ 8). C, Summary of normalized Ca2þ response (2nd SKF-38393 response compared to 1st SKF-38393 response) (*p < 0.005; non-paired t-test). D, Doseeresponse curve of SKF-38393-induced Ca2þ response in mouse DRG neurons showed that the EC50 is 547 nM (n ¼ 10 in respective concentration). Results are presented as the mean ± SEM.
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Fig. 2. The SKF-38393-induced Ca2þ response results from extracellular Ca2þ influx. The SKF-38393-induced Ca2þ response was abolished by pretreatment of 0 mM Ca2þ in the bath solution (n ¼ 8), but not 1 mM thapsigargin (Thap, n ¼ 8). Summary of normalized Ca2þ response (2nd SKF-38393 response compared to 1st SKF-38393 response; *p < 0.005; oneway ANOVA). Results are presented as the mean ± SEM.
Fig. 3. SKF-38393 induces Ca2þ response through activation of TRPV1. A, SKF-38393-induced Ca2þ response was blocked by the pretreatment of capsazepine (CPZ, 10 mM, n ¼ 8). However, the SKF-38393-induced Ca2þ response was maintained in the Cd2þ condition (1 mM, n ¼ 8). B, In DRG neurons of TRPV1 (/) mice, SKF-38393 did not induce Ca2þ responses (n ¼ 7). C, Summary of normalized Ca2þ response (2nd SKF-38393 response compared to 1st SKF-38393 response; *p < 0.005; one-way ANOVA). Results are presented as the mean ± SEM. D, Co-expression of D1R and TRPV1 in DRG neurons. Nuclei of DRG neurons were stained with DAPI (blue). DRG sections were stained with monoclonal antibodies to D1R (green) and TRPV1 (red). Merged images indicate co-expression of D1R and TRPV1. Scale bars shown are 50 mm.
D1R and TRPV1 were co-expressed in DRG neurons using immunohistochemical analysis (Fig. 3D). Consistent with previous reports [27], TRPV1-positive fluorescence was expressed in 60 DRG neurons (60/142, 42.3%). Also, D1R-positive fluorescence were found in 33 DRG neurons (33/142, 23.2%), which is in accordance with a previous study that D1R and D5R were expressed in DRG neurons [20]. Double immunofluorescence revealed that DRG neurons that were immunoreactive for TRPV1 and D1R (23/142, 16.2%) and majority of D1R-immunoreactive neurons were colocalized with TRPV1 (69.7%). 3.5. SKF-38393-induced Ca2þ response in DRG neuron results from direct activation of TRPV1 by DAG Activation of D1R increases intracellular cAMP levels via adenylyl cyclase (AC) [7,8], and D1R stimulates PLC pathway and
hydrolyzes PIP2 into DAG and inositol IP3 [8e10,22]. Both AC and PLC pathways can affect the function of TRPV1 either directly or by phosphorylation [2,28]. We identified the signaling molecules that mediate SKF-38393-induced TRPV1 activation (Fig. 4). Pretreatment with U73122 (2 mM), a specific PLC inhibitor, abolished SKF38393-induced Ca2þ response (3.79 ± 1.25%; n ¼ 8; p < 0.005), whereas U73343, an inactive-form of U73122, did not influence the Ca2þ response elicited by SKF-38393 (74.74 ± 5.36%; n ¼ 8; p > 0.05). The SKF-38393-induced Ca2þ response remained in the presence of 1 mM BIM, a specific PKC inhibitor, (77.20 ± 3.73%; n ¼ 8; p > 0.05) 1 mM RHC 80267, a DAG lipase inhibitor, (78.38 ± 10.54%; n ¼ 8; p > 0.05), or their cocktail (77.92 ± 4.58%; n ¼ 8; p > 0.05), indicating that the PLC pathway mediated SKF38393-induced Ca2þ response, but without PKC- and DAG lipasedependent activation of TRPV1. Next, we tested whether activation of the AC/PKA signaling pathway by D1R was involved in the
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Fig. 4. SKF-38393 induces a Ca2þ response through direct activation of TRPV1 by the PLC/DAG pathway. A, The SKF-38393-induced Ca2þ response was abolished in the presence of U73122 (2 mM, n ¼ 8), but not U73343 (2 mM, n ¼ 8). Also, 1 mM BIM (n ¼ 8), 1 mM RHC 80267 (n ¼ 8), and a mixture of both (n ¼ 8) had no effect on the SKF-38393-induced Ca2þ response. The SKF-38393-induced Ca2þ response was not blocked by the pretreatment with SQ 22,536 (10 mM, n ¼ 8) or H-89 (10 mM, n ¼ 8). B, Summary of normalized Ca2þ response (2nd SKF-38393 response compared to 1st SKF-38393 response; *p < 0.005; one-way ANOVA). Results are presented as the mean ± SEM.
SKF-38393-induced Ca2þ response. Pretreatment with 10 mM SQ 22,536, an AC inhibitor (74.49 ± 8.45%; n ¼ 8; p > 0.05) and 10 mM H-89, a PKA inhibitor (84.10 ± 5.62%; n ¼ 8; p > 0.05) had no effect to SKF-38393-induced Ca2þ response. Taken together, these results indicate that D1R agonist SKF-38393 activates TRPV1 through PLC signaling pathway, and imply that DAG is a strong candidate for downstream signaling molecules in PLC pathway. 4. Discussion In the present study, we found that stimulation of D1R transactivates TRPV1 in mouse DRG neurons, and DAG endogenously produced by the PLC/DAG pathway following D1R activation may mediate the direct activation of TRPV1. We focused on the excitatory effect of dopamine in DRG neurons using a specific D1R agonist, SKF-38393, because the biphasic effects of dopamine depend on the subclass of dopamine, D1 or D2like, receptors. The excitatory effect of dopamine was mediated by D1-like receptor-linked intracellular signaling pathways. In contrast, the inhibitory effect was mediated by D2-like receptors [29,30]. A recent study characterized the expression of D1R and D5R, but not D2R, in DRG neurons by Western blot and immunohistochemical analysis [20]. Based on these reports, we inferred that the excitatory effect of dopamine would mainly function in nociceptive sensory neurons. In this study, we demonstrated the excitatory effect of dopamine and trans-activation of TRPV1 by D1R stimulation. However, further experiments will be required to determine whether dopamine induces an identical excitatory effect via D1-like receptors.
Our results show that the effect of D1R activation was mediated by TRPV1. In the presence of thapsigargin, the SKF-38393 response was not changed. This result was consistent with a previous study that showed activation of TRPV1 by D1R in rat somatosensory cortical neurons [22]. Given that VACCs may contribute to another portion of Ca2þ response, we verified that VACCs were not involved in the SKF-38393-induced Ca2þ response. In the experiments with capsazepine or TRPV1 knock-out mice, moreover, we did not observe SKF-38393-induced Ca2þ response. Previous studies suggested the existence of a phosphatidylinositol-linked D1-like receptor [8e10]. However, it is unclear which downstream signaling molecule links D1R to TRPV1. In the present study, we found that the PLC/DAG pathway mediates D1R activation in nociceptive DRG neurons. Previously, we reported that endogenous DAG directly activates TRPV1 produced by GPCR activation [4]. Moreover, we have recently shown that group 1 mGluR5 activates the TRPV1 channel in a membrane-delimited manner; this process is mediated by DAG [5]. In another studies, it was suggested that modulation of TRPV1 activity was determined by PLC isoforms, such as PLCb and PLCd [31] and that TRPV1 activation via PLC was mediated by 2-arachidonoylglycerol and 1arachidonoylglycerol as endogenous TRPV1 activators [32]. In the present study, however, DAG lipase was not involved in a link between D1R and TRPV1. Taken together, these data suggest that DAG, produced by D1R activation, is the most probable endogenous ligand for direct activation of TRPV1. In addition, we found that the effect of SKF-38393 persisted in the presence of an AC inhibitor or PKA inhibitor. This result is in contrast with a previous study that capsaicin-induced currents resulted from TRPV1 activation were
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abolished by AC and PKA inhibitors in mouse DRG neurons [33]. Although the experimental conditions were different, it seems that more studies are needed to characterize the intracellular signaling pathways for TRPV1 modulation; an indirect trans-activation through intracellular signaling pathways or direct activation produced by a specific agonist. The role of dopamine receptors in the pain transmission can be considered at spinal cord or at peripheral site. However, descending pain modulation of dopamine in spinal cord is related to the dopaminergic nerve and D2-like receptors [15,16]. Recently, it has been reported that epidermal cells such as keratinocytes or melanocytes express TH, the rate-limiting enzyme in the dopamine metabolic pathway [17,34,35]. If dopamine receptors are expressed in the epidermal nerve fibers, particularly unmyelinated afferent fibers, dopamine released from epidermal cells may excite nociceptive primary afferent through TRPV1 activation. Another source of dopamine may be in the form of it being released by itself in DRG neurons [17,19,36,37]. To understand the physiological implications of trans-activation of TRPV1 by D1R activation in DRG neurons, in vivo studies are needed to explore the role of dopamine in peripheral pain modulation. In conclusion, our present study demonstrates that activation of D1R trans-activates TRPV1 in mouse nociceptive DRG neurons. The D1R agonist SKF-38393 induces Ca2þ responses through direct activation of TRPV1 by a PLC/DAG intracellular signaling pathway without the involvement of the AC/PKA cascade. Our finding regarding the effect of a D1R agonist on nociceptive sensory neurons provides an insight into the role of dopamine in pain modulation. Acknowledgments This work was supported by the research fund of Hanyang University (HY-2012-N) and the Pioneer research center through National Research Foundation of Korea (NRF) funded by the Ministry of Science, ICT and Future Planning Korea government (MSIP) (2012R1A3A2048834 and 2011-0027921). Transparency document Transparency document related to this article can be found at http://dx.doi.org/10.1016/j.bbrc.2015.08.096. References [1] D.E. Clapham, TRP channels as cellular sensors, Nature 426 (2003) 517e524. [2] G. Bhave, W. Zhu, H. Wang, et al., cAMP-dependent protein kinase regulates desensitization of the capsaicin receptor (VR1) by direct phosphorylation, Neuron 35 (2002) 721e731. [3] L.S. Premkumar, G.P. Ahern, Induction of vanilloid receptor channel activity by protein kinase C, Nature 408 (2000) 985e990. [4] D.H. Woo, S.J. Jung, M.H. Zhu, et al., Direct activation of transient receptor potential vanilloid 1(TRPV1) by diacylglycerol (DAG), Mol. Pain 4 (2008) 42. [5] Y.H. Kim, C.K. Park, S.K. Back, et al., Membrane-delimited coupling of TRPV1 and mGluR5 on presynaptic terminals of nociceptive neurons, J. Neurosci. 29 (2009) 10000e10009. [6] J.N. Joyce, Multiple dopamine receptors and behavior, Neurosci. Biobehav. Rev. 7 (1983) 227e256. [7] R.M. Huff, Signaling pathways modulated by dopamine receptors, in: The Dopamine Receptors, Springer, 1997, pp. 167e192. [8] C. Missale, S.R. Nash, S.W. Robinson, et al., Dopamine receptors: from structure to function, Physiol. Rev. 78 (1998) 189e225. [9] L.Q. Jin, S. Goswami, G. Cai, et al., SKF83959 selectively regulates phosphatidylinositol-linked D1 dopamine receptors in rat brain, J. Neurochem. 85 (2003) 378e386.
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