Advertisement

The phospholipase C inhibitor U73122 is a potent agonist of the polymodal transient receptor potential ankyrin type 1 (TRPA1) receptor channel

  • Cristian Neacsu
  • Susanne K. Sauer
  • Peter W. Reeh
  • Alexandru BabesEmail author
Original Article

Abstract

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.

Keywords

Nociceptor Dorsal root ganglion TRP channel Pain Signal transduction 

Notes

Acknowledgements

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.

Authors’ contribution

C.N. and S.K.S conducted experiments; C.N., S.K.S., and A.B. analyzed data; A.B., C.N., S.K.S., and P.W.R. wrote the manuscript. All authors read and approved the manuscript.

Compliance with ethical standards

Conflict of interest

The authors declare that they have no conflict of interest.

Research involving animals

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).

Supplementary material

210_2019_1722_MOESM1_ESM.docx (12 kb)
ESM 1 (DOCX 12 kb)
210_2019_1722_Fig8_ESM.png (23 kb)
Figure S1

(PNG 23 kb)

210_2019_1722_MOESM2_ESM.tif (48 kb)
High resolution image (TIF 48 kb)
210_2019_1722_Fig9_ESM.png (1.1 mb)
Figure S2

(PNG 1123 kb)

210_2019_1722_MOESM3_ESM.tif (6.4 mb)
High resolution image (TIF 6592 kb)

References

  1. 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 40:416–423CrossRefGoogle Scholar
  2. 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 CrossRefGoogle Scholar
  3. 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 CrossRefGoogle Scholar
  4. Bandell M et al (2004) Noxious cold ion channel TRPA1 is activated by pungent compounds and bradykinin. Neuron 41:849–857CrossRefGoogle Scholar
  5. 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 CrossRefGoogle Scholar
  6. 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 CrossRefGoogle Scholar
  7. 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 255:756–768Google Scholar
  8. Bretag AH (1969) Synthetic interstitial fluid for isolated mammalian tissue. Life Sci 8:319–329CrossRefGoogle Scholar
  9. 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 CrossRefGoogle Scholar
  10. 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 CrossRefGoogle Scholar
  11. 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 CrossRefGoogle Scholar
  12. 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 CrossRefGoogle Scholar
  13. 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
  14. 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 CrossRefGoogle Scholar
  15. 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 CrossRefGoogle Scholar
  16. 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 CrossRefGoogle Scholar
  17. 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 CrossRefGoogle Scholar
  18. 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 CrossRefGoogle Scholar
  19. 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 CrossRefGoogle Scholar
  20. 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 CrossRefGoogle Scholar
  21. 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 CrossRefGoogle Scholar
  22. Jin W, Lo TM, Loh HH, Thayer SA (1994) U73122 inhibits phospholipase C-dependent calcium mobilization in neuronal cells. Brain Res 642:237–243CrossRefGoogle Scholar
  23. 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 CrossRefGoogle Scholar
  24. Kadkova A, Synytsya V, Krusek J, Zimova L, Vlachova V (2017) Molecular basis of TRPA1 regulation in nociceptive neurons. A review. Physiol Res 66:425–439Google Scholar
  25. 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 CrossRefGoogle Scholar
  26. 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 CrossRefGoogle Scholar
  27. 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 CrossRefGoogle Scholar
  28. 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 CrossRefGoogle Scholar
  29. 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 CrossRefGoogle Scholar
  30. 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 CrossRefGoogle Scholar
  31. 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 CrossRefGoogle Scholar
  32. 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 CrossRefGoogle Scholar
  33. 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 CrossRefGoogle Scholar
  34. 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
  35. 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 253:688–697Google Scholar
  36. 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 CrossRefGoogle Scholar
  37. Tominaga M et al (1998) The cloned capsaicin receptor integrates multiple pain-producing stimuli. neuron 21:531–543CrossRefGoogle Scholar
  38. 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 CrossRefGoogle Scholar
  39. 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 CrossRefGoogle Scholar
  40. 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 CrossRefGoogle Scholar
  41. 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 CrossRefGoogle Scholar

Copyright information

© Springer-Verlag GmbH Germany, part of Springer Nature 2019

Authors and Affiliations

  1. 1.Institute for Physiology and PathophysiologyFriedrich-Alexander University of Erlangen-NurembergErlangenGermany
  2. 2.Department of Anatomy, Physiology and Biophysics, Faculty of BiologyUniversity of BucharestBucharestRomania

Personalised recommendations