The phospholipase C inhibitor U73122 is a potent agonist of the polymodal transient receptor potential ankyrin type 1 (TRPA1) receptor channel
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.
KeywordsNociceptor Dorsal root ganglion TRP channel Pain Signal transduction
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.
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).
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