2026 Volume 49 Issue 1 Pages 149-153
Matching transformation system (MA-T), an on-demand aqueous chlorine dioxide production solution is utilized in various applications, including antibacterial and cancer therapies. Herein, we focused on the ability of MA-T in oxidizing human transient receptor potential cation channel subfamily V member 4 (hTRPV4), which functions as a redox sensor, using Ca2+ imaging and patch-clamp experiments. We found that 10 ppm MA-T activated hTRPV4 in HEK293T cells. Moreover, MA-T induced increase in cytosolic calcium concentration through hTRPV4 activation in human non-pigmented ciliary epithelial cells. Our study findings suggest MA-T as a potential agent for TRPV4-related diseases.
Transient receptor potential (TRP) channels are widely expressed ion channels in mammalian tissues that are involved in sensing external environmental changes and transducing signals.1,2) TRP vanilloid 4 (TRPV4) is a non-selective ion channel activated by osmotic, mechanical, and thermal stimuli,3,4) and it has been implicated in systemic water homeostasis.5) Previously, TRPV4 activation in the eyes of mice has been reported to facilitate aqueous humor outflow and reduce intraocular pressure through the trabecular meshwork.6) Moreover, TRPV4 is expressed in human non-pigmented ciliary epithelial cells, and its activation may be involved in intraocular pressure reduction through melatonin increase in aqueous humor.7)
Aqueous chlorine dioxide, which is formed from sodium chlorite solutions, has been used to disinfect tap water in Western countries. However, their association with several toxic reactions on cells has been reported.8,9) Matching transformation system (MA-T), an on-demand aqueous chlorine dioxide solution is a stable radical which is formed under chemical equilibrium condition in aqueous solution and is reported to have a strong microbicidal property10) against various microorganisms such as bacteria, fungi, and viruses, and can be applied in the medical field.11,12) In addition, a stable radical in MA-T exhibits a high stability and safety with rapid chlorine dioxide generation only upon contact with biological targets through a catalytic process.10) Due to this selectivity, it demonstrates greater safety compared with conventional radical species that attack non-selectively. In this catalytic process, MA-T causes generation of aqueous chlorine dioxide radicals, which participate in reversible reactions with water molecules and chlorite ions, thereby sustaining a dynamic equilibrium that continuously produces reactive species capable of inducing oxidation reactions.13)
In this study, we evaluated the effect of MA-T on human TRPV4 (hTRPV4) activity using both Ca2+ imaging and electrophysiological approaches.
High concentrations of MA-T (40000 ppm) were provided by Earth Chemical Co., Ltd. (Tokyo, Japan). A TRPV4 activator GSK1016790A, TRPV4 inhibitors HC067047 and SK2193874 were obtained from FUJIFILM Wako Pure Chemical Corporation (Osaka, Japan) and Tocris Bioscience (Bristol, U.K.), respectively.
Molecular CloningFull-length human TRPV4 cDNAs were obtained from Life Technologies (Carlsbad, CA, U.S.A.) cloned into pcDNA3.1 vector.
Cell CultureHEK293T cells were maintained in Dulbecco’s Modified Eagle Medium (FUJIFILM Wako Pure Chemical Corporation) supplemented with 10% fetal bovine serum (Biowest SAS), 100 U/mL penicillin and 100 μg/mL streptomycin (Thermo Fisher Scientific, Waltham, MA, U.S.A.), and 2 mM l-glutamine (GlutaMAX; Thermo Fisher Scientific), at 37°C in 5% CO2 atmosphere. For Ca2+ imaging, 1 μg of plasmid DNA containing hTRPV4 in pcDNA3.1 was transfected into HEK293T cells using Lipofectamine Plus Reagent (Thermo Fisher Scientific) in OPTI-MEM medium (Thermo Fisher Scientific). After a 3- to 4-h incubation, the cells were reseeded onto coverslips and incubated further at 37°C in a 5% CO2 atmosphere. Human non-pigmented ciliary epithelial cells (HNPCEpiCs, ScienCell Research Laboratories, Carlsbad, CA, U.S.A.) were cultured in Epithelial Cell Medium (EpiCM, ScienCell Research Laboratories) in an incubator set at 37°C in a 5% CO2 atmosphere. After 7- to 8 d of incubation, the cells were reseeded onto coverslips and incubated further at 37°C in 5% CO2 atmosphere.
siRNA TransfectionSmall interfering RNAs (siRNAs) targeting human TRPV4 (Thermo Fisher Scientific) and a negative control siRNA (Thermo Fisher Scientific) were purchased from Invitrogen. Cells were transfected with 100 nM siRNA using LipofectamineTM RNAiMAX (Thermo Fisher Scientific) 24 h after passaging, according to the manufacturer’s instructions; 6 h after transfection, the medium was replaced with fresh EpiCM. Cells were used for subsequent assays 48 h after transfection.
Ca2+ ImagingCalcium ion (Ca2+) imaging was performed 1 d after transfection of HEK293T cells expressing hTRPV4, 7 or 8 d after initiating culture from a frozen stock of HNPCEpiCs, and 2 or 3 d after siRNA transfection of HNPCEpiCs. Each cell line was mounted onto coverslips in an open chamber and superfused with a standard bath solution (140 mM NaCl, 5 mM KCl, 2 mM MgCl2, 2 mM CaCl2, 10 mM HEPES, and 10 mM glucose; pH 7.4). Free cytosolic Ca2+ concentrations in HEK293T cells and HNPCEpiCs were measured by dual-wavelength microfluorometry using a Fura-2 radiometric indicator (Thermo Fisher Scientific) with excitation at 340/380 nm and emission at 510 nm. The Fura-2 ratio was calculated using the MetaFluor software (Molecular Devices).
ElectrophysiologyWhole-cell patch-clamp recordings were performed 1 d after transfection of HEK293T cells expressing hTRPV4, 9 or 10 d after initiating culture from frozen HNPCEpiC stock. The standard bath solution was identical to that used in the Ca2+ imaging experiments. The pipette solution contained 140 mM KCl, 5 mM EGTA, and 10 mM HEPES, pH 7.4 (adjusted with KOH). Data from the whole-cell voltage-clamp recordings were sampled at 10 kHz and filtered at 5 kHz for analysis using an Axon 1550B amplifier with pCLAMP software (Axon Instruments). The membrane potential was clamped at −60 mV for all conditions. All the experiments were performed at room temperature (24–26°C).
To evaluate the effect of MA-T on hTRPV4, we first performed Ca2+ imaging on HEK293T cells overexpressing hTRPV4 at room temperature. We observed an increase in the Fura-2 ratio (intracellular calcium concentrations, [Ca2+]i) induced by low concentrations of MA-T (10 or 20 ppm) in hTRPV4 overexpressing cells, but not in the Mock-transfected cells (Figs. 1A, 1B). To confirm this activation of hTRPV4, we performed whole-cell patch-clamp experiments using hTRPV4 overexpressing HEK293T cells. MA-T (10 ppm) induced activation currents in the cells treated with 100 nM GSK1016790A, a specific TRPV4 agonist (n = 6, Fig. 1C), but not in the cells that underwent Mock transfection (n = 4, Fig. 1E). Moreover, the currents evoked by 10 ppm MA-T were significantly inhibited by 20 μM HC067047, a specific TRPV4 antagonist (n = 9, Figs. 1D, 1F). To confirm that MA-T-induced hTRPV4 activation depends on oxidative effects, we treated cells with 20 ppm MA-T in the presence of 5 mM dithiothreitol (DTT), a strong reducing agent. The [Ca2+]i increases induced by 20 ppm MA-T were completely inhibited (Fig. 1G).
(A) Fura-2 ratio changes due to MA-T (5, 10, 20 ppm) and GSK1016790A (100 nM) treatments (n = 49). (B) Comparison of Fura-2 ratio changes due to MA-T (0, 5, 10, 20 ppm) treatment between hTRPV4 overexpressing (n = 49) and Mock-transfected (n = 192) HEK293T cells. Statistical significance was evaluated using Dunnett’s multiple comparison test. Data are presented as the mean ± standard error of the mean (S.E.M.). (C) Representative MA-T (10 ppm)- and GSK1016790A (100 nM)-induced current. (D) MA-T (10 ppm)-induced hTRPV4 current inhibited by HC067047 (20 μM). (E) Representative current trace induced by MA-T (10 ppm) in Mock-transfected cells. (F) Comparison of changes in current density between MA-T (10 ppm) + HC067047 (20 μM) and MA-T (10 ppm) (n = 9). (G) Comparison of normalized Fura-2 ratio changes following MA-T (20 ppm, n = 8) inhibition by dithiothreitol (DTT, 5 mM, n = 19). Statistical significance was evaluated using Student’s t test. Data are presented as the mean ± S.E.M.
To elucidate the effect of MA-T on ocular cells, HNPCEpiCs, which control intraocular pressure, we exposed to Ca2+ imaging experiments. MA-T (10 and 20 ppm)-induced [Ca2+]i increased in response to treatment with 100 nM GSK1016790A, a TRPV4 agonist (Fig. 2A). Furthermore, treatment with 10 ppm MA-T induced inward currents in HNPCEpiCs (Fig. 2B). In addition, to confirm the involvement of hTRPV4 in the MA-T response, HNPCEpiCs were transfected with siRNA targeting human TRPV4 (siTRPV4) or a negative control (siControl). GSK1016790A-induced increases in [Ca2+]i were significantly lower in siTRPV4-transfected cells compared with siControl cells (Fig. 2C). In particular, 20 ppm MA-T-induced increases in [Ca2+]i were significantly attenuated in siTRPV4 HNPCEpiCs, indicating that the response to MA-T depends on hTRPV4 expression (Figs. 2D, 2E).
(A) Fura-2 ratio changes due to MA-T (2, 10, 20 ppm), GSK1016790A (100 nM) and ionomycin (2 μM) treatments (n = 42). (B) Representative MA-T (10 ppm)-induced current. (C) Comparison of normalized Fura-2 ratio changes due to GSK1016790A (100 nM) in siControl (n = 601) and siTRPV4 (n = 375). Fura-2 ratio changes (D) and their normalized comparison (E) due to MA-T (20 ppm) in siControl (n = 303) and siTRPV4 (n = 999). Statistical significance was evaluated using Student’s t test. Data are presented as the mean ± S.E.M.
To further elucidate the effect of MA-T on hTRPV4, we examined the effects of TRPV4 antagonists on MA-T-induced [Ca2+]i increases. The elevation of [Ca2+]i induced by 100 nM GSK1016790A was significantly inhibited by pretreatment with 20 μM HC067047, confirming hTRPV4-mediated activation. Similarly, 10 ppm MA-T-induced increases in [Ca2+]i were completely inhibited by 20 μM HC067047, whereas the 20 ppm MA-T responses were partially but significantly inhibited by both 20 μM HC067047 and 300 nM GSK2193874, a potent and selective TRPV4 blocker, suggesting that MA-T-induced [Ca2+]i increases in HNPCEpiC are primarily mediated by hTRPV4 activation (Figs. 3A, 3B).
Fura-2 ratio changes (A) and their normalized comparison (B) following GSK1016790A (100 nM, n = 21) or MA-T (10 ppm, n = 22) inhibition by HC067047 (HC, 20 μM), and MA-T (20 ppm) inhibition by HC067047 (20 μM, n = 777) or GSK2193874 (GSK2 300 nM, n = 520). Data are presented as the mean ± S.E.M. Statistical significance was evaluated using Student’s t test between the addition of TRPV4 antagonists (HC067047 or GSK2193874) versus GSK1016790A or MA-T (10 or 20 ppm).
In this study, Ca2+ imaging and whole-cell patch-clamp experiments suggested that low concentrations of MA-T activate hTRPV4 in both HEK293T cells and HNPCEpiC.
MA-T has been investigated not only as an antibacterial agent but also as a cancer drug because of its safety, which is based on the minimal generation of chlorine dioxide caused by the catalytic action of Lewis acids.13) MA-T (100 or 150 ppm) is currently available on the market as a sanitizer and deodorant in airplanes and hotels in Japan, without any health hazards. Therefore, we evaluated the potential of MA-T as eye drops by focusing on hTRPV4, which is believed to play a role in reducing intraocular pressure. Among the TRP channels, TRPV4 is reported as an oxidative stress sensor, shows high oxidation sensitivity.14) Therefore, transient production of minimum chlorine dioxide from MA-T might directly induce TRPV4 activation, similar to hydrogen peroxide, inducing calcium influx through TRPV4 in lung microvascular endothelial cells.15)
Although our experiments focused only on TRPV4, the influence of MA-T on other proteins including additional TRP channels, should be further investigated. The partial inhibitory effects of HC067047 and GSK2193874 on 20 ppm MA-T-induced increases in [Ca2+]i in HNPCEpiCs suggest that higher concentrations of MA-T might activate additional ion channels (Figs. 3A, 3B). Furthermore, the inhibitory effects of siTRPV4 on 20 ppm MA-T-induced [Ca2+]i increases in HNPCEpiCs were greater than those observed for 100 nM GSK1016790A-induced responses and for the inhibition produced by TRPV4 antagonists under the same conditions (Figs. 2C–2E, 3A, 3B). In addition, the complete inhibition of 20 ppm MA-T-induced [Ca2+]i increases by 5 mM DTT in hTRPV4 overexpressing HEK293T cells (Fig. 1G) further supports the involvement of oxidative stress-sensitive mechanisms. These findings indicate that MA-T might also activate downstream pathways or secondary oxidative stress-sensitive channels modulated by hTRPV4.
The molecular mechanisms underlying hTRPV4 activation require detailed elucidation. TRPV4 agonists have been suggested as potential agents for glaucoma.6) However, the function of TRPV4 in controlling interocular pressure remains controversial. Treatment with GSK1016790A lowered interocular pressure in rats, with the baseline of interocular pressure being higher in TRPV4⁻/⁻ mice.16) However, intraocular injection of HC067047, a TRPV4 antagonist, lowered interocular pressure in glaucomatous mouse.17) Since TRPV4 responds to osmotic and mechano stimuli,18,19) controlling interocular pressure through TRPV4 is desirable in glaucoma treatment. Further experiments are necessary to confirm whether MA-T or TRPV4 agonists reduce intraocular pressure in patients with glaucoma.
In this study, we demonstrated that MA-T can induce hTRPV4 activation. In the future, we hope that MA-T can be developed as a mild drug for TRPV4-related diseases.
The authors thank Mr. Kazuyoshi Nishikawa, as a Professional Engineer (Chemistry), of the MA-T Business Center of Earth Chemical Co., Ltd. This study was supported by the Earth Chemical Co., Ltd.
Fumitaka Fujita was employed by Mandom Corporation. The other authors declare that this research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
