Abstract
Primary sensory afferent neurons detect environmental and painful stimuli at their peripheral termini. A group of transient receptor potential ion channels (TRPs) are expressed in these neurons and constitute sensor molecules for the stimuli such as thermal, mechanical, and chemical insults. We examined whether a mouse sensory neuronal line, N18D3, shows the sensory TRP expressions and their functionality. In Ca2+ imaging and electrophysiology with these cells, putative TRPV4-mediated responses were observed. TRPV4-specific sensory modalities including sensitivity to a specific agonist, hypotonicity, or an elevated temperature were reproduced in N18D3 cells. Electrophysiological and pharmacological profiles conformed to those from native TRPV4 of primarily cultured neurons. The TRPV4 expression in N18D3 was also confirmed by RT-PCR and Western blot analyses. Thus, N18D3 cells may represent TRPV4-expressing sensory neurons. Further, using this cell lines, we discovered a novel synthetic TRPV4-specific agonist, MLV-0901. These results suggest that N18D3 is a reliable cell line for functional and pharmacological TRPV4 assays. The chemical information from the novel agonist will contribute to TRPV4-targeting drug design.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Alessandri-Haber N, Joseph E, Dina OA, Liedtke W, Levine JD (2005) TRPV4 mediates pain-related behavior induced by mild hypertonic stimuli in the presence of inflammatory mediator. Pain 118(1–2):70–79
Alessandri-Haber N, Dina OA, Joseph EK, Reichling D, Levine JD (2006) A transient receptor potential vanilloid 4-dependent mechanism of hyperalgesia is engaged by concerted action of inflammatory mediators. J Neurosci 26(14):3864–3874
Alexander R, Kerby A, Aubdool AA, Power AR, Grover S, Gentry C, Grant AD (2013) 4α-phorbol 12,13-didecanoate activates cultured mouse dorsal root ganglia neurons independently of TRPV4. Br J Pharmacol 168:761–772
Bang S, Hwang SW (2009) Polymodal ligand sensitivity of TRPA1 and its modes of interactions. J Gen Physiol 133(3):257–262
Bang S, Kim KY, Yoo S, Kim YG, Hwang SW (2007a) Transient receptor potential A1 mediates acetaldehyde-evoked pain sensation. Eur J Neurosci 26(9):2516–2523
Bang S, Kim KY, Yoo S, Lee SH, Hwang SW (2007b) Transient receptor potential V2 expressed in sensory neurons is activated by probenecid. Neurosci Lett 425(2):120–125
Bang S, Yoo S, Yang TJ, Cho H, Kim YG, Hwang (2010a) Resolvin D1 attenuates activation of sensory transient receptor potential channels leading to multiple anti-nociception. Br J Pharmacol 161(3):707–720
Bang S, Yoo S, Yang TJ, Cho H, Hwang SW (2010b) Farnesyl pyrophosphate is a novel pain-producing molecule via specific activation of TRPV3. J Biol Chem 285(25):19,362–19,371
Bang S, Yoo S, Yang T, Cho H, Hwang S (2011a) 17(R)-resolvin D1 specifically inhibits TRPV3 leading to peripheral antinociception. Br J Pharmacol 165(3):683–692
Bang S, Yoo S, Yang TJ, Cho H, Hwang SW (2011b) Isopentenyl pyrophosphate is a novel antinociceptive substance that inhibits TRPV3 and TRPA1 ion channels. Pain 152(5):1156–1164
Bang S, Yoo S, Yang TJ, Cho H, Hwang SW (2012) Nociceptive and pro-inflammatory effects of dimethylallyl pyrophosphate via TRPV4 activation. Br J Pharmacol 166(4):1433–1443
Bender FL, Mederos YSM, Li Y, Ji A, Weihe E, Gudermann T, Schafer MK (2005) The temperature-sensitive ion channel TRPV2 is endogenously expressed and functional in the primary sensory cell line F-11. Cell Physiol Biochem 15(1–4):183–194
Brierley SM, Page AJ, Hughes PA, Adam B, Liebregts T, Cooper NJ, Holtmann G, Liedtke W, Blackshaw LA (2008) Selective role for TRPV4 ion channels in visceral sensory pathways. Gastroenterology 134(7):2059–2069
Chen W, Mi R, Haughey N, Oz M, Hoke A (2007) Immortalization and characterization of a nociceptive dorsal root ganglion sensory neuronal line. J Peripher Nerv Syst 12(2):121–130
Dhaka A, Viswanath V, Patapoutian A (2006) Trp ion channels and temperature sensation. Annu Rev Neurosci 29:135–161
Guler AD, Lee H, Iida T, Shimizu I, Tominaga M, Caterina M (2002) Heat-evoked activation of the ion channel, TRPV4. J Neurosci 22(15):6408–6414
Hwang SW, Oh U (2007) Current concepts of nociception: nociceptive molecular sensors in sensory neurons. Curr Opin Anaesthesiol 20(5):427–434
Jahnel R, Bender O, Munter LM, Dreger M, Gillen C, Hucho F (2003) Dual expression of mouse and rat VRL-1 in the dorsal root ganglion derived cell line F-11 and biochemical analysis of VRL-1 after heterologous expression. Eur J Biochem 270(21):4264–4271
Kim JG, Koh SH, Lee YJ, Lee KY, Kim Y, Kim S, Lee MK, Kim SH (2005) Differential effects of diallyl disulfide on neuronal cells depend on its concentration. Toxicology 211(1–2):86–96
Kim KY, Bang S, Han S, Nguyen YH, Kang TM, Kang KW, Hwang SW (2008) TRP-independent inhibition of the phospholipase C pathway by natural sensory ligands. Biochem Biophys Res Commun 370(2):295–300
Koh SH, Kim SH, Kwon H, Kim JG, Kim JH, Yang KH, Kim J, Kim SU, HJ Y, Do BR, Kim KS, Jung HK (2004) Phosphatidylinositol-3 kinase/Akt and GSK-3 mediated cytoprotective effect of epigallocatechin gallate on oxidative stress-injured neuronal-differentiated N18D3 cells. Neurotoxicology 25(5):793–802
Liedtke W, Choe Y, Marti-Renom MA, Bell AM, Denis CS, Sali A, Hudspeth AJ, Friedman JM, Heller S (2000) Vanilloid receptor-related osmotically activated channel (VR-OAC), a candidate vertebrate osmoreceptor. Cell 103(3):525–535
Nilius B, Voets T (2013) The puzzle of TRPV4 channelopathies. EMBO Rep 14(2):152–163
Park SA, Choi KS, Bang JH, Huh K, Kim SU (2000) Cisplatin-induced apoptotic cell death in mouse hybrid neurons is blocked by antioxidants through suppression of cisplatin-mediated accumulation of p53 but not of Fas/Fas ligand. J Neurochem 75(3):946–953
Park IH, Kim MK, Kim SU (2008) Ursodeoxycholic acid prevents apoptosis of mouse sensory neurons induced by cisplatin by reducing P53 accumulation. Biochem Biophys Res Commun 377(4):1025–1030
Raymon HK, Thode S, Zhou J, Friedman GC, Pardinas JR, Barrere C, Johnson RM, Sah DW (1999) Immortalized human dorsal root ganglion cells differentiate into neurons with nociceptive properties. J Neurosci 19(13):5420–5428
Rimmerman N, Bradshaw HB, Hughes HV, Chen JS, SS H, McHugh D, Vefring E, Jahnsen JA, Thompson EL, Masuda K, Cravatt BF, Burstein S, Vasko MR, Prieto AL, O'Dell DK, Walker JM (2008) N-palmitoyl glycine, a novel endogenous lipid that acts as a modulator of calcium influx and nitric oxide production in sensory neurons. Mol Pharmacol 74(1):213–224
Ruan B, Pong K, Jow F, Bowlby M, Crozier RA, Liu D, Liang S, Chen Y, Mercado ML, Feng X, Bennett F, von Schack D, McDonald L, Zaleska MM, Wood A, Reinhart PH, Magolda RL, Skotnicki J, Pangalos MN, Koehn FE, Carter GT, Abou-Gharbia M, Graziani EI (2008) Binding of rapamycin analogs to calcium channels and FKBP52 contributes to their neuroprotective activities. Proc Natl Acad Sci U S A 105(1):33–38
Ryu JJ, Yoo S, Kim KY, Park JS, Bang S, Lee SH, Yang TJ, Cho H, Hwang SW (2010) Laser modulation of heat and capsaicin receptor TRPV1 leads to thermal antinociception. J Dent Res 89(12):1455–1460
Sanfeliu C, Wright JM, Kim SU (1999) Neurotoxicity of isoniazid and its metabolites in cultures of mouse dorsal root ganglion neurons and hybrid neuronal cell line. Neurotoxicology 20(6):935–944
Stein AT, Ufret-Vincenty CA, Hua L, Santana LF, Gordon SE (2006) Phosphoinositide 3-kinase binds to TRPV1 and mediates NGF-stimulated TRPV1 trafficking to the plasma membrane. J Gen Physiol 128:509–522
Story GM, Peier AM, Reeve AJ, Eid SR, Mosbacher J, Hricik TR, Earley TJ, Hergarden AC, Andersson DA, Hwang SW, McIntyre P, Jegla T, Bevan S, Patapoutian A (2003) ANKTM1, a TRP-like channel expressed in nociceptive neurons, is activated by cold temperatures. Cell 112(6):819–829
Strotmann R, Harteneck C, Nunnenmacher K, Schultz G, Plant TD (2000) OTRPC4, a nonselective cation channel that confers sensitivity to extracellular osmolarity. Nat Cell Biol 2(10):695–702
Suzuki M, Mizuno A, Kodaira K, Imai M (2003) Impaired pressure sensation in mice lacking TRPV4. J Biol Chem 278(25):22,664–22,668
Vincent F, Acevedo A, Nguyen MT, Dourado M, DeFalco J, Gustafson A, Spiro P, Emerling DE, Kelly MG, Duncton MA (2009) Identification and characterization of novel TRPV4 modulators. Biochem Biophys Res Commun 389(3):490–494
Vriens J, Owsianik G, Janssens A, Voets T, Nilius B (2007) Determinants of 4 alpha-phorbol sensitivity in transmembrane domains 3 and 4 of the cation channel TRPV4. J Biol Chem 282(17):12,796–12,803
Watanabe H, Vriens J, Suh SH, Benham CD, Droogmans G, Nilius B (2002) Heat-evoked activation of TRPV4 channels in a HEK293 cell expression system and in native mouse aorta endothelial cells. J Biol Chem 277(49):47,044–47,051
Wood JN, Bevan SJ, Coote PR, Dunn PM, Harmar A, Hogan P, Latchman DS, Morrison C, Rougon G, Theveniau M et al (1990) Novel cell lines display properties of nociceptive sensory neurons. Proc Biol Sci 241(1302):187–194
Zimmermann K, Hein A, Hager U, Kaczmarek JS, Turnquist BP, Clapham DE, Reeh PW (2009) Phenotyping sensory nerve endings in vitro in the mouse. Nat Protoc 4(2):174–196
Acknowledgements
This work was supported by grants from the National Research Foundation of Korea (NRF-2017R1A2B2001817, NRF-2017M3C7A1025600) and Korea Health Technology R&D Project of Ministry of Health & Welfare (HI15C2099).
Author information
Authors and Affiliations
Corresponding authors
Ethics declarations
Conflict of interest
The authors declare that they have no conflict of interest.
Rights and permissions
About this article
Cite this article
Yoo, S., Choi, SI., Lee, S. et al. Endogenous TRPV4 Expression of a Hybrid Neuronal Cell Line N18D3 and Its Utilization to Find a Novel Synthetic Ligand. J Mol Neurosci 63, 422–430 (2017). https://doi.org/10.1007/s12031-017-0993-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12031-017-0993-y