TRPA1 and TRPV1 channels participate in atmospheric-pressure plasma-induced [Ca2+]i response

Masayoshi Kawase,Weijian Chen,Mazvita R Nyasha,Toshiro Kaneko,Shota Sasaki,Makoto Kanzaki,Hiroyasu Hatakeyama,Kota Kawaguchi

doi:10.1038/s41598-020-66510-y

Masayoshi Kawase, Weijian Chen + Show 6 more

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https://doi.org/10.1038/s41598-020-66510-y

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Journal: Scientific Reports	Publication Date: Jun 16, 2020
Citations: 5	License type: open-access

Affiliation: Tohoku University, Kitasato University

Abstract

Despite successful clinical application of non-equilibrium atmospheric pressure plasma (APP), the details of the molecular mechanisms underlying APP-inducible biological responses remain ill-defined. We previously reported that exposure of 3T3L1 cells to APP-irradiated buffer raised the cytoplasmic free Ca2+ ([Ca2+]i) concentration by eliciting Ca2+ influx in a manner sensitive to transient receptor potential (TRP) channel inhibitors. However, the precise identity of the APP-responsive channel molecule(s) remains unclear. In the present study, we aimed to clarify channel molecule(s) responsible for indirect APP-responsive [Ca2+]i rises. siRNA-mediated silencing experiments revealed that TRPA1 and TRPV1 serve as the major APP-responsive Ca2+ channels in 3T3L1 cells. Conversely, ectopic expression of either TRPA1 or TRPV1 in APP-unresponsive C2C12 cells actually triggered [Ca2+]i elevation in response to indirect APP exposure. Desensitization experiments using 3T3L1 cells revealed APP responsiveness to be markedly suppressed after pretreatment with allyl isothiocyanate or capsaicin, TRPA1 and TRPV1 agonists, respectively. APP exposure also desensitized the cells to these chemical agonists, indicating the existence of a bi-directional heterologous desensitization property of APP-responsive [Ca2+]i transients mediated through these TRP channels. Mutational analyses of key cysteine residues in TRPA1 (Cys421, Cys621, Cys641, and Cys665) and in TRPV1 (Cys258, Cys363, and Cys742) have suggested that multiple reactive oxygen and nitrogen species are intricately involved in activation of the channels via a broad range of modifications involving these cysteine residues. Taken together, these observations allow us to conclude that both TRPA1 and TRPV1 channels play a pivotal role in evoking indirect APP-dependent [Ca2+]i responses.

Highlights

Despite successful clinical application of non-equilibrium atmospheric pressure plasma (APP), the details of the molecular mechanisms underlying APP-inducible biological responses remain ill-defined
By employing cell-based [Ca2+]i analyses of both siRNA-mediated silencing (Figs. 1 and 2) and exogenous expression strategies (Figs. 3 and 4), we demonstrated both TRPA1 and TRPV1 channels to be molecules www.nature.com/scientificreports was applied at approximately 50~100 sec., and ionomycin was added. (B) Quantification of the indirect APP-responsive [Ca2+]i transients in C2C12 myoblasts expressing either TRPA1 or TRPV1, alone or in combination, with or without 100 μM ruthenium red (RR) from 3 independent experiments
The effects of exogenous expression of TRPA1 and TRPV1, alone or in combination, are indicated by *(P < 0.05), and the effects of RR are indicated by #(P < 0.05). (C) Quantification of agonist-dependent [Ca2+]i transients in C2C12 myoblasts exogenously expressing either TRPA1 or TRPV1 channels from 3 independent experiments

Summary

Introduction

Despite successful clinical application of non-equilibrium atmospheric pressure plasma (APP), the details of the molecular mechanisms underlying APP-inducible biological responses remain ill-defined. Mutational analyses of key cysteine residues in TRPA1 (Cys[421], Cys[621], Cys[641], and Cys665) and in TRPV1 (Cys[258], Cys[363], and Cys742) have suggested that multiple reactive oxygen and nitrogen species are intricately involved in activation of the channels via a broad range of modifications involving these cysteine residues. These TRP channel inhibitors suppressed APP-inducible enhancement of YOYO-1 uptake into 3T3L1 fibroblasts[12] These pharmacological results strongly suggest TRP channel(s) to be intimately involved in the observed APP-inducible biological responses especially in Ca2+ influx, though the molecular identity and the activation mechanisms of the APP-responsive channel(s) remain unclear. We found that TRPA1 and TRPV1 both function as indirect APP-responsive Ca2+-permeable channels

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