The aminosteroid U73122 is frequently used as a phospholipase C (PLC) inhibitor and as such was used to investigate PLC-dependent activation and modulation of the transient receptor potential ankyrin type 1 (TRPA1) receptor channel. However, U73122 was recently shown to activate recombinant TRPA1 directly, albeit this interaction was not further explored. Our aim was to perform a detailed characterization of this agonistic action of U73122 on TRPA1. We used Fura-2 calcium microfluorimetry and the patch clamp technique to investigate the effect of U73122 on human and mouse wild type and mutant (C621S/C641S/C665S) TRPA1 expressed in HEK293t cells, as well as native TRPA1 in primary afferent neurons from wild type and TRPV1 and TRPA1 null mutant mice. In addition, we measured calcitonin gene-related peptide (CGRP) release from skin isolated from wild-type and TRPA1 null mutant mice. Human and mouse TRPA1 channels were activated by U73122 in the low nanomolar range. This activation was only partially dependent upon modification of the N-terminal cysteines 621, 641, and 665. U73122 also activated a subpopulation of neurons from wild-type and TRPV1 null mutant mice, but this effect was absent in mice deficient of TRPA1. In addition, U73122 evoked marked calcitonin gene-related peptide (CGRP) release from skin preparations of wild type but not TRPA1 null mutant mice. Our results indicate that U73122 is a potent and selective TRPA1 agonist. This effect should be taken into account when U73122 is used to inhibit PLC in TRPA1-expressing cells, such as primary nociceptors. In addition, U73122 may present a novel lead compound for the development of TRPA1-targeting drugs.
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Averbeck B, Reeh PW (2001) Interactions of inflammatory mediators stimulating release of calcitonin gene-related peptide, substance P and prostaglandin E(2) from isolated rat skin. Neuropharmacology 40:416–423
Babes A, Fischer MJ, Reid G, Sauer SK, Zimmermann K, Reeh PW (2010) Electrophysiological and neurochemical techniques to investigate sensory neurons in analgesia research. Methods Mol Biol 617:237–259. https://doi.org/10.1007/978-1-60327-323-7_19
Babes A et al (2016) Photosensitization in Porphyrias and photodynamic therapy involves TRPA1 and TRPV1. J Neurosci 36:5264–5278. https://doi.org/10.1523/JNEUROSCI.4268-15.2016
Bandell M et al (2004) Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41:849–857
Bautista DM et al (2005) Pungent products from garlic activate the sensory ion channel TRPA1. Proc Natl Acad Sci U S A 102:12248–12252. https://doi.org/10.1073/pnas.0505356102
Bellono NW, Kammel LG, Zimmerman AL, Oancea E (2013) UV light phototransduction activates transient receptor potential A1 ion channels in human melanocytes. Proc Natl Acad Sci U S A 110:2383–2388. https://doi.org/10.1073/pnas.1215555110
Bleasdale JE, Thakur NR, Gremban RS, Bundy GL, Fitzpatrick FA, Smith RJ, Bunting S (1990) Selective inhibition of receptor-coupled phospholipase C-dependent processes in human platelets and polymorphonuclear neutrophils. J Pharmacol Exp Ther 255:756–768
Bretag AH (1969) Synthetic interstitial fluid for isolated mammalian tissue. Life Sci 8:319–329
Caterina MJ, Schumacher MA, Tominaga M, Rosen TA, Levine JD, Julius D (1997) The capsaicin receptor: a heat-activated ion channel in the pain pathway. Nature 389:816–824. https://doi.org/10.1038/39807
Cho H, Youm JB, Ryu SY, Earm YE, Ho WK (2001) Inhibition of acetylcholine-activated K(+) currents by U73122 is mediated by the inhibition of PIP(2)-channel interaction. Br J Pharmacol 134:1066–1072. https://doi.org/10.1038/sj.bjp.0704347
Chuang HH et al (2001) Bradykinin and nerve growth factor release the capsaicin receptor from PtdIns(4,5)P2-mediated inhibition. Nature 411:957–962. https://doi.org/10.1038/35082088
Dai Y et al (2007) Sensitization of TRPA1 by PAR2 contributes to the sensation of inflammatory pain. J Clin Invest 117:1979–1987. https://doi.org/10.1172/jci30951
DelloStritto DJ et al (2016) Differential regulation of TRPV1 channels by H2O2: implications for diabetic microvascular dysfunction. 111:21. https://doi.org/10.1007/s00395-016-0539-4
Dittert I, Benedikt J, Vyklicky L, Zimmermann K, Reeh PW, Vlachova V (2006) Improved superfusion technique for rapid cooling or heating of cultured cells under patch-clamp conditions. J Neurosci Methods 151:178–185. https://doi.org/10.1016/j.jneumeth.2005.07.005
Earley S, Gonzales AL, Garcia ZI (2010) A dietary agonist of transient receptor potential cation channel V3 elicits endothelium-dependent vasodilation. Mol Pharmacol 77:612–620. https://doi.org/10.1124/mol.109.060715
Eberhardt MJ et al (2012) Methylglyoxal activates nociceptors through transient receptor potential channel A1 (TRPA1): a possible mechanism of metabolic neuropathies. J Biol Chem 287:28291–28306. https://doi.org/10.1074/jbc.M111.328674
Gees M et al (2013) Mechanisms of transient receptor potential vanilloid 1 activation and sensitization by allyl isothiocyanate. Mol Pharmacol 84:325–334. https://doi.org/10.1124/mol.113.085548
Gijsen HJ, Berthelot D, Zaja M, Brone B, Geuens I, Mercken M (2010) Analogues of morphanthridine and the tear gas dibenz[b,f][1,4]oxazepine (CR) as extremely potent activators of the human transient receptor potential ankyrin 1 (TRPA1) channel. J Med Chem 53:7011–7020. https://doi.org/10.1021/jm100477n
Hinman A, Chuang HH, Bautista DM, Julius D (2006) TRP channel activation by reversible covalent modification. Proc Natl Acad Sci U S A 103:19564–19568. https://doi.org/10.1073/pnas.0609598103
Horowitz LF, Hirdes W, Suh BC, Hilgemann DW, Mackie K, Hille B (2005) Phospholipase C in living cells: activation, inhibition, Ca2+ requirement, and regulation of M current. J Gen Physiol 126:243–262. https://doi.org/10.1085/jgp.200509309
Ibarra Y, Blair NT (2013) Benzoquinone reveals a cysteine-dependent desensitization mechanism of TRPA1. Mol Pharmacol 83:1120–1132. https://doi.org/10.1124/mol.112.084194
Jin W, Lo TM, Loh HH, Thayer SA (1994) U73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells. Brain Res 642:237–243
Jordt SE et al (2004) Mustard oils and cannabinoids excite sensory nerve fibres through the TRP channel ANKTM1. Nature 427:260–265. https://doi.org/10.1038/nature02282
Kadkova A, Synytsya V, Krusek J, Zimova L, Vlachova V (2017) Molecular basis of TRPA1 regulation in nociceptive neurons. A review. Physiol Res 66:425–439
Karashima Y, Damann N, Prenen J, Talavera K, Segal A, Voets T, Nilius B (2007) Bimodal action of menthol on the transient receptor potential channel TRPA1. J Neurosci 27:9874–9884. https://doi.org/10.1523/JNEUROSCI.2221-07.2007
Karashima Y, Prenen J, Meseguer V, Owsianik G, Voets T, Nilius B (2008) Modulation of the transient receptor potential channel TRPA1 by phosphatidylinositol 4,5-biphosphate manipulators. Pflugers Arch - Eur J Physiol 457:77–89. https://doi.org/10.1007/s00424-008-0493-6
Kistner K et al (2016) Systemic desensitization through TRPA1 channels by capsazepine and mustard oil - a novel strategy against inflammation and pain. Sci Rep 6:28621. https://doi.org/10.1038/srep28621
Klein RR et al (2011) Direct activation of human phospholipase C by its well known inhibitor u73122. J Biol Chem 286:12407–12416. https://doi.org/10.1074/jbc.M110.191783
Klose A, Huth T, Alzheimer C (2008) 1-[6-[[(17beta)-3-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5- dione (U73122) selectively inhibits Kir3 and BK channels in a phospholipase C-independent fashion. Mol Pharmacol 74:1203–1214. https://doi.org/10.1124/mol.108.047837
Leitner MG et al (2016) Direct modulation of TRPM4 and TRPM3 channels by the phospholipase C inhibitor U73122. Br J Pharmacol 173:2555–2569. https://doi.org/10.1111/bph.13538
Liang WZ, Lu CH (2012) Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells. Life Sci 90:703–711. https://doi.org/10.1016/j.lfs.2012.03.027
Macmillan D, McCarron JG (2010) The phospholipase C inhibitor U-73122 inhibits ca(2+) release from the intracellular sarcoplasmic reticulum ca(2+) store by inhibiting ca(2+) pumps in smooth muscle. Br J Pharmacol 160:1295–1301. https://doi.org/10.1111/j.1476-5381.2010.00771.x
Macpherson LJ, Dubin AE, Evans MJ, Marr F, Schultz PG, Cravatt BF, Patapoutian A (2007) Noxious compounds activate TRPA1 ion channels through covalent modification of cysteines. nature 445:541–545. https://doi.org/10.1038/nature05544
Mogami H, Lloyd Mills C, Gallacher DV (1997) Phospholipase C inhibitor, U73122, releases intracellular Ca2+, potentiates Ins(1,4,5)P3-mediated Ca2+ release and directly activates ion channels in mouse pancreatic acinar cells. Biochem J 324(Pt 2):645-51. https://doi.org/10.1042/bj3240645
Smith RJ, Sam LM, Justen JM, Bundy GL, Bala GA, Bleasdale JE (1990) Receptor-coupled signal transduction in human polymorphonuclear neutrophils: effects of a novel inhibitor of phospholipase C-dependent processes on cell responsiveness. J Pharmacol Exp Ther 253:688–697
Stenger B et al (2015) Activation of the chemosensing transient receptor potential channel A1 (TRPA1) by alkylating agents. Arch Toxicol 89:1631–1643. https://doi.org/10.1007/s00204-014-1414-4
Tominaga M et al (1998) The cloned capsaicin receptor integrates multiple pain-producing stimuli. neuron 21:531–543
Wang S et al (2008a) Phospholipase C and protein kinase a mediate bradykinin sensitization of TRPA1: a molecular mechanism of inflammatory pain. Brain J Neurol 131:1241–1251. https://doi.org/10.1093/brain/awn060
Wang YY, Chang RB, Waters HN, McKemy DD, Liman ER (2008b) The nociceptor ion channel TRPA1 is potentiated and inactivated by permeating calcium ions. J Biol Chem 283:32691–32703. https://doi.org/10.1074/jbc.M803568200
Xu H, Delling M, Jun JC, Clapham DE (2006) Oregano, thyme and clove-derived flavors and skin sensitizers activate specific TRP channels. Nat Neurosci 9:628–635. https://doi.org/10.1038/nn1692
Zurborg S, Yurgionas B, Jira JA, Caspani O, Heppenstall PA (2007) Direct activation of the ion channel TRPA1 by Ca2+. Nat Neurosci 10:277–279. https://doi.org/10.1038/nn1843
C.N. and A.B. acknowledge support from the UEFISCDI-CNCS grant PNIII-P4-ID-PCE-2016-0475 from the Romanian Ministry of Research and Innovation. A.B. received generous support from the Alexander von Humboldt Foundation. C.N. and P.W.R. received intramural support from the ‘Emerging Fields Initiative-Redox Medicinal Chemistry’ of the Erlangen University. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
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All applicable international, national, and/or institutional guidelines for the care and use of animals were followed. This article does not contain any studies with human participants performed by any of the authors.
Breeding and euthanasia and all procedures of animal handling were prospectively approved by the Animal Welfare Authority of the District Government of Unterfranken in Würzburg (Germany) and the Institutional Animal Care Department (University of Erlangen, Germany) in accordance with the German regulations of animal care and welfare (Tierschutzgesetz). Experiments were performed in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC).
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Neacsu, C., Sauer, S.K., Reeh, P.W. et al. The phospholipase C inhibitor U73122 is a potent agonist of the polymodal transient receptor potential ankyrin type 1 (TRPA1) receptor channel. Naunyn-Schmiedeberg's Arch Pharmacol 393, 177–189 (2020). https://doi.org/10.1007/s00210-019-01722-2
- Dorsal root ganglion
- TRP channel
- Signal transduction