Calcium influx through TRP channels induced by short-lived reactive species in plasma-irradiated solution

Non-equilibrium helium atmospheric-pressure plasma (He-APP), which allows for a strong non-equilibrium chemical reaction of O2 and N2 in ambient air, uniquely produces multiple extremely reactive products, such as reactive oxygen species (ROS), in plasma-irradiated solution. We herein show that relatively short-lived unclassified reactive species (i.e., deactivated within approximately 10 min) generated by the He-APP irradiation can trigger physiologically relevant Ca2+ influx through ruthenium red- and SKF 96365-sensitive Ca2+-permeable channel(s), possibly transient receptor potential channel family member(s). Our results provide novel insight into understanding of the interactions between cells and plasmas and the mechanism by which cells detect plasma-induced chemically reactive species, in addition to facilitating development of plasma applications in medicine.

Scientific RepoRts | 6:25728 | DOI: 10.1038/srep25728 Experimental Helium atmospheric-pressure plasma (He-APP) irradiation and calcium live-imaging system. Non-equilibrium APP can produce a variety of chemically reactive species due to its high reactivity. In particular, if helium (He) is used as the working gas, the presence of metastable helium (He * ) in the plasma can greatly enhance the generation of reactive species such as ROS due to its high internal energy (~20 eV) [11][12][13][14][15][16][17]22 . Figure 1 schematically illustrates the experimental He-APP irradiation system and set-up for live-imaging of [Ca 2+ ] i after injection of the He-APP irradiated solution. In this system, He gas serves as the source gas, with its flow rate (f) through the dielectric tube regulated by a mass flow controller (MFC), and typically, f = 3 L/min. When the high-voltage (V p-p ) power supply (with a frequency of 10 kHz) of this system is turned on, dielectric barrier discharge plasma is generated and flows out from the nozzle of the quartz glass tube (6-mm inner diameter), irradiating a 3-mL volume of live-imaging HEPES-buffered saline (HBS; containing 150 mM NaCl, 5 mM KCl, 2 mM CaCl 2 , 1 mM MgCl 2 , and 10 mM HEPES [pH adjusted to 7.4 with NaOH]), with or without 5.6 mM D-glucose. Typically, V p-p = 5.0 kV, L g = 23 mm, and d ele = 38 mm. The parameter t i is defined as the duration of plasma irradiation, and t r is defined as the time until injection of plasma-irradiated HBS after completion of the plasma irradiation process. During and after the addition of plasma-irradiated solution (indirect plasma irradiation), real-time changes in [Ca 2+ ] i are measured using a confocal microscope (FV1000, Olympus) with a fluo-4 AM calcium indicator (F-14201, Invitrogen).

Production of hydrogen peroxide (H 2 O 2 ) and hydroxyl (OH) radicals in solution by He-APP irradiation.
A wide variety of plasma-produced chemically reactive species are expected, and the specific species produced and their reactivity are expected to vary over time. In terms of their life span in the solution, these species can be classified as long-lived (life span on the order of hours or more), short-lived (life span on the order of minutes), or extremely short-lived (life span on the order of seconds or less). One of the long-lived products is H 2 O 2 , which can significantly impact biological responses. The H 2 O 2 concentration (C H2O2 ) in the solution after plasma irradiation for t i was estimated using a colorimetric staining probe (WAK-H2O2, Kyoritsu Chemical-Check Laboratory). As shown in Fig. 2a, C H2O2 increased linearly with t i . Only 2.9 μ M H 2 O 2 was generated in 3 mL of HBS after plasma irradiation for 10 s. This level of H 2 O 2 reportedly causes minimal cytotoxicity and does not appreciably affect cellular proliferation 23 .
Total production of the extremely short-lived OH radical species in the solution after plasma irradiation for t i (also determined using chemical dosimetry based on terephthalic acid [TA] 24 ) also increased linearly with t i (Fig. 2b). Although plasma irradiation resulted in a significant increase in the generation of OH radicals along with the generation of H 2 O 2 (e.g., 10 μ M H 2 O 2 generated at t i = 30s) in the HBS, direct addition of this level of H 2 O 2 to HBS (H 2 O 2 control) failed to generate any detectable level of OH radicals. Therefore, the observed OH radicals in HBS were generated by He-APP irradiation. OH radicals are believed to play an important role in plasma-mediated biological responses targeted in plasma medicine due to their high reactivity and oxidation potential [1][2][3][4]25,26 . The presence of OH radicals in the solution therefore indicates that many different chemically reactive species are actually produced, with reactions involving OH radicals serving as potential triggers for inducing various biological responses after plasma irradiation of HBS. The production of OH radicals could not The parameter t i is defined as the duration of plasma irradiation; t r is defined as the time between completion of the plasma irradiation process and injection of plasma-irradiated HBS; V p-p represents the applied peak-topeak voltage; d ele is defined as the distance between the electrodes; and L g is defined as the distance between the powered electrode and the edge of the glass tube. Typically, V p-p = 5.0 kV; L g = 23 mm; and d ele = 38 mm. The flow rate of He gas (f) is regulated by a mass flow controller (MFC), and typically, f = 3 L/min. Scientific RepoRts | 6:25728 | DOI: 10.1038/srep25728 be mimicked by direct H 2 O 2 administration. As time proceeds, the extremely short-lived chemically reactive species apparently rapidly break down into short-lived and long-lived species such as H 2 O 2 . 2+ ] i in 3T3L1 fibroblasts. Administration of plasma-irradiated HBS (t i = 10 s) to cells in culture resulted in gradual and sometimes oscillatory increases in [Ca 2+ ] i after a relatively long lag period (~70 s), whereas administration of naive HBS containing 10% calf serum (10% CS/HBS) as a positive control induced a rapid increase in [Ca 2+ ] i (Fig. 3a, (Fig. 3b,c, black line). These results suggest that chemically reactive species other than H 2 O 2 in the plasma-irradiated HBS induced the increase in [Ca 2+ ] i , possibly ROS, which could have been generated in the HBS as one of the initial reaction products (e.g., OH radicals), as shown in Fig. 2b.

Plasma-irradiated HBS elicited an increase in [Ca
To clarify the possible involvement of OH radicals in the increase in [Ca 2+ ] i induced by plasma-irradiated HBS, we compared [Ca 2+ ] i responses in the absence and presence of 5.6 mM D-glucose (an OH radical scavenger) in the HBS (Fig. 4b,c). In the case of glucose-free HBS (Fig. 4a), [Ca 2+ ] i was significantly higher compared with HBS containing 5.6 mM glucose. On the other hand, because D-glucose serves not only as an OH radical scavenger but also as the major energy source for living cells, the additional verification is necessary. We therefore examined effect of D-mannitol as another OH radical scavenger 26 , displaying less-permeable/less-metabolizable property, and found that 5.6 mM D-mannitol similarly suppressed the plasma-induced [Ca 2+ ] i increase (Fig. 4d). These results suggest that the effect of glucose metabolism would be minimal for at least a few minutes of this live-cell imaging. In the presence of 5.6 mM D-glucose, further addition of 5.6 mM D-mannitol additively suppressed the [Ca 2+ ] i level, strongly suggesting that ROS produced by an OH radical-initiated reaction are responsible for the increase in [Ca 2+ ] i elicited by the exposure of cells to plasma-irradiated HBS.
Based upon these observations, we conclude that ROS in plasma-irradiated HBS, rather than H 2 O 2 (~2.9 μ M), plays the predominant role in triggering changes in [Ca 2+ ] i , although higher concentrations of H 2 O 2 (30 μ M) have been shown to induce increases in [Ca 2+ ] i mediated through TRPA1 channels in other cell types 27 . It should be noted that the [Ca 2+ ] i responses induced by plasma-irradiated HBS were not observed in the present study in MCF-7 human breast adenocarcinoma cells (data not shown). Thus, these results indicate that the observed [Ca 2+ ] i increase following administration of plasma-irradiated HBS was mediated via the influx of extracellular Ca 2+ and could not be attributed to calcium release from the ER. These results also strongly suggest that TRP channel(s) are involved in [Ca 2+ ] i responses elicited by chemically reactive species produced in plasma-irradiated HBS.

Single-cell analyses of [Ca 2+ ] i responses induced by short-lived products in plasma-irradiated HBS.
To explore the underlying mechanism of the [Ca 2+ ] i responses elicited by plasma-irradiated HBS, HBS plasma irradiated for 1, 3, or 30 s was administrated to cells, and [Ca 2+ ] i responses of individual cells were then carefully analyzed. Whereas administration of plasma-irradiated HBS (t i = 10 s) resulted in a significant increase in [Ca 2+ ] i after a 70-s lag period, as shown in Figs 3 and 4, plasma-irradiated HBS (t i = 30 s) induced large, sharp spikes in [Ca 2+ ] i (lasting for ~100 s), followed by prolonged periods (at least 10 min) of lower [Ca 2+ ] i , although these lower levels were still significantly higher than the control (Fig. 6a, red line). As expected, (t i = 3 s) plasma-irradiated HBS required a much longer lag period (~100 s) before commencement of the significant increase in [Ca 2+ ] i , although in this case, the [Ca 2+ ] i peak was not as sharp as that observed with administration of (t i = 30 s) plasma-irradiated HBS (Fig. 6a, blue line).
As the profiles of [Ca 2+ ] i responses elicited by administration of plasma-irradiated HBS were highly complex and appeared to involve a variety of ROS (which then partially/gradually degenerated) generated in the HBS by differing intensities (periods) of plasma irradiation, we examined the [Ca 2+ ] i responses at the single-cell level based on analyses of four parameters (MAX [Ca2+]i , t delay , t rise , t fall ), as shown  attained; and t fall represents the interval between the time the 90% level in the increase in [Ca 2+ ] i is attained and the time [Ca 2+ ] i returns to the 10% level. Each of these parameters was determined for each cell examined, and the results are depicted as a function of t i in Fig. 6c, d, e. As t i increased, more cells exhibited a higher MAX [Ca2+]i , and the mean/median values for MAX [Ca2+]i increased (Fig. 6c). In addition, both t delay and t rise declined with increasing t i (Fig. 6d,e). Thus, the nature of the [Ca 2+ ] i response induced by administration of plasma-irradiated HBS is apparently defined by the concentrations of chemically reactive species that are generated by plasma irradiation, in a manner dependent on t i .
Most of the chemically reactive species generated by plasma irradiation are assumed to be short-lived and to dissipate rapidly over time. Therefore, we next investigated the amount of time plasma-irradiated HBS retains its potency for evoking [Ca 2+ ] i responses (Fig. 7). Plasma-irradiated HBS was administrated to cells at 30, 300, or 600 s after irradiation (t i = 30 s), and the [Ca 2+ ] i response was then carefully analyzed. As expected, the [Ca 2+ ] i responses changed dramatically as the retention time after plasma-irradiation (t r ) increased. As t r increased, the increase in [Ca 2+ ] i was lower, and the time required to reach the maximal value increased (i.e., the response was slower). In addition, single-cell analyses of [Ca 2+ ] i responses in the same manner as described above showed that most of cells exhibited lower MAX [Ca2+]i values with increasing t r , although some cells maintained a high MAX [Ca2+]i . Similarly, although both t delay and t rise tended to be longer for most of the cells, some cells maintained short t delay and t rise . Taken together, these observations demonstrate that the chemically reactive species in plasma-irradiated HBS that exhibit the highest potency in evoking [Ca 2+ ] i responses have a life span of the order of several minutes. Furthermore, the results of single-cell analyses suggest that differences in results between retention experiments is due to t r -dependent deactivation-associated changes in the composition of the chemically reactive species in plasma-irradiated HBS retained for different periods before administration. The above results indicate that the nature of the [Ca 2+ ] i response is strongly influenced by the concentration and composition of chemically reactive species in the plasma-irradiated HBS, which apparently have life spans on the order of several minutes. In addition, these results indicate that t rise is strongly correlated with t fall (Fig. 8a); that is, a sharper rise in [Ca 2+ ] i tends to cause a sharper fall in [Ca 2+ ] i .

Discussion
Despite the promising potential of plasma medicine based on non-equilibrium APP technology, a crucial issue remains unresolved. That is, how do cells decipher and respond to the wide array of highly complex and interrelated stimuli evoked by APP irradiation, including irradiation with UV rays, ROS, RNS, electric fields, and shock waves [1][2][3][4] ? In an attempt to dissect these complex stimuli that directly and/or indirectly affect cellular functions, we focused on the biologically active elements generated in the medium (a biological buffer) after non-equilibrium He-APP irradiation by monitoring intracellular Ca 2+ dynamics. A key finding of the present study is that plasma-irradiated HBS contains chemically reactive species that can induce physiologically relevant increases    (Figs 3 and 4) through RR-and SKF-sensitive Ca 2+ -permeable channel(s) (Fig. 5), possibly a member(s) of the TRP channel family [28][29][30] . Based upon our detailed analyses of the [Ca 2+ ] i responses in 3T3-L1 fibroblasts (Figs 3-7), we can conclude that plasma irradiation creates very potent, but relatively short-lived, chemically reactive species that trigger Ca 2+ influx (Fig. 8b). The potency of these species is not attributable to only H 2 O 2 , which is one of the end products of plasma irradiation (Fig. 3). Although elucidating the nature of the plasma-generated bioactive species and clarifying the identity of Ca 2+ -permeable channel(s) responsible for these [Ca 2+ ] i responses will be challenging, our novel findings provide important insights that will enhance our understanding of plasma-mediated biological responses, at least with respect to the associated constituents, consisting presumably of multiple reactive species uniquely created in the medium by non-equilibrium He-APP irradiation.
In this study, we utilized non-equilibrium APP, which allows for a strong non-equilibrium chemical reaction of O 2 and N 2 in ambient air, resulting in the efficient generation of chemically reactive species including ozone/O 3 14 , nitric oxide/NO 14 , atomic oxygen/O 15 , and OH radicals 15 as well as their unclassified derivatives at a specific constituent ratio. Some of these chemically reactive species reportedly exert both beneficial and detrimental biological effects on cells, mediated by various ROS-sensing mechanisms 3,31 . Our results provide compelling evidence that the [Ca 2+ ] i responses evoked by plasma-irradiated HBS involve RR-and SKF-sensitive Ca 2+ -permeable channel(s) (Fig. 5), presumably members of TRP channel. Indeed, it is increasingly apparent that a subclass of TRP channels function as chemosensors for detecting various reactive species, such as ROS and RNS 21,32 . For example, TRPA1 is reportedly activated by H 2 O 2 (~30 μ M), S-nitroso-N-acetyl-DL-penicillamine (SNAP), which is an NO donor, as well as by other inflammatory mediators. These [Ca 2+ ] i responses are blunted by substitution of the redox-sensitive cysteine residues in TRPA1, which can be modified by oxidative covalent reaction (e.g., S-nitrosylation) 33 . In addition to TRPA1, the channel activity of many other TRPs (TPRV1, V2, V3, V4, and C5) has been shown to be regulated in a redox-sensitive manner with different electron acceptor (oxidation) capacities 34 . Furthermore, oxidative stress reportedly led to a phenotypic shift in Ca 2+ mobilization from an oscillatory to a sustained elevated pattern via calcium release-activated calcium (CRAC)-mediated capacitive Ca 2+ entry through Orai channel activated by stromal interaction molecule 1 (STIM1) on ER, which is also possibly involving TRP channel(s) 35,36 . While observed Ca 2+ influx is clearly triggered through RR-and SKF-sensitive Ca 2+ -permeable channels, possibly TRP channel(s), secondary calcium dynamics (e.g. oscillatory increases in Fig. 3) might be related to STIM1 activation. In addition, it has been reported that plasma-produced species can directly peroxidate lipids 37,38 and that TRP channels are activated by oxidized lipids 39 , and thereby, it is also possible that plasma-produced species may activate TRP channels through lipid peroxidation. Considering these highly sensitive redox-responsive characteristics of TRP channels in light of our results (Figs 3-7), it is highly likely that the [Ca 2+ ] i responses in 3T3L1 cells are mediated via TRP channel(s) 40 , which are activated by relatively short-lived ROS (deactivated within approximately 10 min) rather than extremely short-lived (OH radicals) or long-lived (H 2 O 2 ) reactive species. As research indicates that TRP channels are promising drug targets 41 , plasma-irradiated solutions could be utilized in medical applications, though the interactions between TRP channels and plasma-induced chemically reactive species need to be further elucidated.
Plasma medicine is a rapidly emerging field that combines plasma physics, life sciences, and clinical medicine with the goal of developing therapeutic applications for physical plasma [1][2][3][4] . The biological responses to direct cold plasma and plasma-irradiated solutions have been the subject of considerable recent research. The sensitivity of cells to chemically reactive species is thought to depend on the unique properties of each cell type. However, many previous reports on the biological responses to plasma-irradiated solutions may actually involve calcium influx through TRP channels, as reported in this paper. We anticipate that our results will contribute to further progress in the field of plasma medicine.
Indirect plasma irradiation. HBS without cells was directly irradiated with APP. The applied voltage and frequency were 5.0 kV and 10 kHz, respectively. The helium gas flow rate was 3 L/min. After a prescribed time (t r ) after plasma irradiation, the plasma-irradiated HBS was added to cells.  (Fig. 4). Images were acquired every 2 s using a confocal microscope (FV1000, Olympus). Changes in [Ca 2+ ] i were expressed as (F − F 0 )/F 0 , where F and F 0 represent the fluorescence intensity of fluo 4 and the averaged fluorescence intensity of the dye before stimulation with plasma-irradiated HBS, respectively.