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1 J Physiol 595. (7) pp Store-operated calcium entry is required for sustained contraction and Ca + oscillations of airway smooth muscle Jun Chen and Michael J. Sanderson Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, M, US The Journal of Physiology Key points irway hyper-responsiveness in asthma is driven by excessive contraction of airway smooth muscle cells (SMCs). gonist-induced Ca + oscillations underlie this contraction of SMCs and the magnitude of this contraction is proportional to the Ca + oscillation frequency. Sustained contraction and Ca + oscillations require an influx of extracellular Ca +,although the mechanisms and pathways mediating this Ca + influx during agonist-induced SMC contraction are not well defined. y inhibiting store-operated calcium entry (SOCE) or voltage-gated Ca + channels (VGCCs), we show that SOCE, rather than Ca + influx via VGCCs, provides the major Ca + entry pathway into SMCs to sustain SMCs contraction and Ca + oscillations. SOCE may therefore serve as a potential target for new bronchodilators to reduce airway hyper-responsiveness in asthma. bstract sthma is characterized by airway hyper-responsiveness: the excessive contraction of airway smooth muscle. The extent of this airway contraction is proportional to the frequency of Ca + oscillations within airway smooth muscle cells (SMCs). Sustained Ca + oscillations require aca + influx to replenish Ca + losses across the plasma membrane. Our previous studies implied store-operated calcium entry (SOCE) as the major pathway for this Ca + influx. In the present study, we explore this hypothesis, by examining the effects of SOCE inhibitors (GSK7975 and GSK5498) as well as L-type voltage-gated Ca + channel inhibitors (nifedipine and nimodipine) on airway contraction and Ca + oscillations and SOCE-mediated Ca + influx in SMCs within mouse precision-cut lung slices. We found that both GSK7975 and GSK5498 were able to fully relax methacholine-induced airway contraction by abolishing the Ca + oscillations, in a manner similar to that observed in zero extracellular Ca + ([Ca + ] e ). In addition, GSK7975 and GSK5498 inhibited increases in intracellular Ca + ([Ca + ] i ) in SMCs with depleted Ca + -stores in response to increased [Ca + ] e, demonstrating a response consistent with the inhibition of SOCE. However, GSK7975 and GSK5498 did not reduce Ca + release via IP 3 receptors stimulated with IP 3 released from caged-ip 3. y contrast, nifedipine and nimodipine only partially reduced airway contraction, Ca + oscillation frequency and SOCE-mediated Ca + influx. These data suggest that SOCE is the major Ca + influx pathway for SMCs with respect to sustaining agonist-induced airway contraction and the underlying Ca + oscillations. The mechanisms of SOCE may therefore form novel targets for new bronchodilators. (Received pril 6; accepted after revision 6 July 6; first published online July 6) Corresponding author J. Chen: Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, 55 Lake venue North, Worcester, M 655, US. jun.chen@umassmed.edu DOI:.3/JP7694

2 34 J. Chen and M. J. Sanderson J Physiol 595. bbreviations HR, airway hyper-responsiveness; SM, airway smooth muscle; SMCs, airway smooth muscle cells; [Ca + ] e, extracellular Ca + concentration; [Ca + ] i, intracellular Ca + concentration; [Ca + ] SR,Ca + concentration within the SR; Caged-IP 3, caged-iso-ins(,4,5)p 3 /propionoxymethyl ester; G5, GSK5498; G7, GSK7975; HSS, Hanks balanced salt solution; I CRC,Ca + release-activated Ca + current; IP 3, inositol-,4,5-trisphosphate; IP 3 R, inositol-,4,5-trisphosphate receptor Ca + channels;, methacholine; OG, Oregon Green; PCLS, precision-cut lung slices; PM, plasma membrane; RyRs, ryanodine receptors; S, sulfobromophthalein; SERC, sarcoplasmic reticulum Ca + transport TPases; shss, Hanks balanced salt solution supplemented with mm Hepes; SOCE, store-operated calcium entry; SR, sarcoplasmic reticulum; STIM, stromal interaction molecule; VGCCs, voltage-gated Ca + channels. Introduction sthma is characterized by excessive airway smooth muscle (SM) contraction (i.e. airway hyperresponsiveness; HR). lthough HR has been clinically managed with bronchodilators and corticosteroids, these approaches are not always adequate for severe asthma and the development of new or alternative therapies would be beneficial. Unfortunately, efforts aiming towards this goal are hindered by a poor understanding of the aetiology of HR (n et al. 7; Gauthier et al. 5). Consequently, in the present study, we focus on the fundamental Ca + -dependent mechanisms of SM contraction and how these may serve as novel targets for new bronchodilators. SM contraction is dependent on the phosphorylation of the regulatory myosin light-chain mediated by myosin light-chain kinase, which is activated by the binding of calmodulin and calcium (Pfitzer, ). Hence, an increase of intracellular calcium concentration ([Ca + ] i ) induces the contraction of SM. Our previous studies have demonstrated that the pattern of increased [Ca + ] i in SM cells (SMCs) induced by agonists consists of repetitive Ca + waves propagating along the length of the SMC, a phenomenon called Ca + oscillations. Most importantly, the frequency of these Ca + oscillations regulates the extent of SM contraction (Perez & Sanderson, 5; ai & Sanderson, 6a). The mechanism underlying these Ca + oscillations relies on the cyclic release of Ca + from (and re-uptake of Ca + to) the sarcoplasmic reticulum (SR). Ca + release mainly occurs via inositol-,4,5-trisphosphate (IP 3 ) receptor Ca + channels (IP 3 R) activated by IP 3 produced in response to agonist activation of phospholipase Cβ via G-protein coupled receptors. Ca + re-uptake to SR occurs via SR Ca + transport TPases (SERC) (Sathish et al. 8; Prakash et al. 9). Some Ca + efflux also occurs via ryanodine receptors (RyRs) during the initial Ca + oscillations (Kannan et al. 997; Croisier et al. 5). Importantly, although the initiation of agonist-induced Ca + oscillations does not require an influx of extracellular Ca +, the maintenance of Ca + oscillations, and thus sustained SM contraction, does require Ca + influx to replenish Ca + lost across the plasma membrane (PM) mediated by PM Ca + TPases (erridge et al. 3). variety of Ca + entry pathways are proposed to contribute to Ca + homeostasis in SMCs including, but not limited to, voltage-gated Ca + channels (VGCCs), receptor-operated Ca + channels and reverse-mode Na + /Ca + exchangers (Dai et al. 6; Dai et al. 7; Flores-Soto et al. 3). However, the most important Ca + influx pathway coupled with Ca + loss from SR is probably the store-operated calcium entry (SOCE). SOCE has been recently identified as a Ca + influx pathway in SMCs (y et al. 4; Peel et al. 6; Prakash et al. 6; Peel et al. 8; Sieck et al. 8; Liu et al. ). The mechanism mediating SOCE is multifaceted, involving a PM resident Ca + channel called Orai and an SR membrane-spanning, Ca + sensing protein called stromal interaction molecule (STIM) (Liou et al. 5; Roos et al. 5; Feske et al. 6; Soboloff et al. ; Jairaman & Prakriya, 3; aba et al. 4; Prakriya & Lewis, 5; Steinckwich et al. 5). STIM detects decreases in Ca + concentration within the SR ([Ca + ] SR ), which induces the oligomerization and translocation of STIM to a region near the PM, where it interacts with Orai to generate alocalca + influx current (Lewis, 7; Soboloff et al. ). Therefore, SOCE appears to be an appropriate Ca + influx pathway that can be readily coupled to the decrease of [Ca + ] SR occurring during agonist-induced Ca + oscillations in SMCs (Croisier et al. 3). Importantly, our mathematical studies predict that the Ca + oscillation frequency of SMCs is dependent on the magnitude of SOCE, rather than other Ca + influx pathways (Croisier et al. 3), emphasizing SOCE as a key parameter for airway contraction. Furthermore, SOCE may be regulated by caveolin- (Sathish et al. ), with linkage to CD38 and cyclic DP ribose, indicating that the extent of SOCE may be enhanced with the upregulation of CD38 and caveolar protein during airway inflammation (Sathish et al. 4). Similarly, STIM aggregation in SMCs has been shown to increase with inflammatory stimulation, again, suggesting a link between increased SOCE and HR (Croisier et al. 3; Wylam et al. 5). The obvious inverse corollary of these ideas is that a reduction in SOCE would reduce the Ca + oscillation frequency of SMCs to induce airway relaxation. Consequently, to characterize the significance of SOCE in airway contraction and the underlying Ca + oscillations

3 J Physiol 595. Store-operated Ca + entry in airway smooth muscle Ca + oscillations 35 of SMCs, we tested the effects of two newly developed SOCE inhibitors, GSK7975 (G7) and GSK5498 (G5) (shmole et al. ; Derler et al. 3; Rice et al. 3), as well as two classic L-type VGCC inhibitors, nifedipine and nimodipine, on agonist-induced airway contraction and Ca + oscillations of SMCs in mouse precision cut lung slices (PCLS). The effect of these drugs on SOCE mediated Ca + influx was further confirmed in SOCE-activated PCLS. Our results show that SOCE, rather than VGCCs, is required for Ca + influx to maintain agonist-induced Ca + oscillations and the contraction of SM. Thus, SOCE provides a potential novel target for new bronchodilators to reduce HR. Methods Ethical approval nimal maintenance and experimental procedures complied with the requirements of the nimal Welfare ct, US Public Health Service Policy and NIH guidelines and were approved by the Institutional nimal Care and Use Committee of the University of Massachusetts Medical School. ll experiments were performed on tissue collected immediately after death of animals not subject to any other treatments. ll authors understand the ethical principles under which The Journal of Physiology operates and our work complied with the animal ethics policy and checklist as reported by Grundy (Grundy, 5). Chemicals and reagents GSK-7975 was obtained from obious Inc. (Gloucester, M, US); GSK-5498 was obtained from MedChem Express (Monmouth Junction, NJ, US); Oregon Green 488 PT- M was obtained from Life Technologies (Grand Island, NY, US); caged-iso-ins(,4,5)p 3 /propionoxymethyl ester (caged-ip 3 ) was obtained from Enzo Life Sciences (Farmingdale, NY, US); ryanodine was obtained from bcam Inc. (Cambridge, M, US); all other reagents were obtained from either Sigma-ldrich (St Louis, MO, US) or Thermo Fisher Scientific (Pittsburgh, P, US). Hanks balanced salt solution (HSS) was supplemented with mm Hepes buffer (shss) and adjusted to ph 7.4. Hanks -Ca + solution (-Ca + shss) was prepared by supplementing HSS without Ca + with mm Hepes,.9 mm MgSO 4 and mm Na H -EGT. The stock solutions for all compounds were prepared in DMSO with final working solutions containing.5% (or less) DMSO. Corresponding concentrations of DMSO were included in all control solutions. Preparation of PCLS The detailed protocol has been described previously (Perez & Sanderson, 5). riefly, female alb/c mice (8 weeks), purchased from Charles River reeding Laboratories (Needham, M, US), were killed by cervical dislocation. fter opening the thoracic cavity, the lungs were inflated with ml of.8% agarose in shss at 37 C via an intratracheal catheter. Subsequently,.3 ml of air was injected to flush the agarose within the airway into the distal alveoli. fter the agarose was gelled by cooling the lungs with 4 C shss, the lung lobes were removed and sectioned into 8 μm thick slices with a vibratome (VF-; Precisionary Instruments, Greenville, NC, US). The PCLS were maintained in Dulbecco s modified Eagle s medium with antibiotics at 37 C and % CO for up to 3 days. ll experiments were performed at 37 C with constant perfusion using a custom-made, temperature-controlled microscope enclosure as described previously (ai & Sanderson, 6b). Measurement of airway contraction and relaxation The detailed protocol has been described previously (ai & Sanderson, 9). riefly, PCLS were placed on a cover-glass that was mounted in a custom-made Plexiglas support, and held down by a μm nylon mesh with a hole aligned over a selected airway. perfusion chamber was created by placing another smaller cover-glass on the top of the nylon mesh and sealing the edges with silicone grease. irway contraction and relaxation in response to different compounds was monitored with an inverted microscope (Diaphot; Nikon, Tokyo, Japan; or IX7; Olympus, Tokyo, Japan) with a objective. Phase-contrast images were collected at a rate of one image per s with a CCD camera, which was controlled by custom-programed software based on Video Savant 4 (IO Industries, Montreal, Canada). The change in lumen area of a selected airway was analysed using ImageJ (NIH, ethesda, MD, US) as described previously (Tan & Sanderson, 4). To summarize drug-induced relaxation (%), we initially normalized the contractile state of an airway (measured at the mid-time point at 5 min in Figs 3) to its own initial contraction (measured at 5 min) by the formulae: % relaxation (R) = (area 5min area 5min )/( area 5min ). We then normalized the drug induced relaxation to the amount of relaxation that occurred in the control response by the formulae: % normalized relaxation = (R drug R control )/( R control ). To characterize and compare rates of airway relaxation under various conditions, we calculated the time constant (t ) for each response: each experimental airway contraction (from 5.5 to 35 min in Figs,, C and 3) was normalized to the initial control contractile response (at 5.5 min) and then subtracted from control contraction at each time point. This isolates the experimental-induced relaxation from the control

4 36 J. Chen and M. J. Sanderson J Physiol 595. relaxation. The time constant (t ) of these relaxation data was calculated from the formula: y = y + e x/t,where x represents time, and y represents the normalized contraction at each time point. Measurement of intracellular Ca + signalling PCLS were loaded with shss containing μm Oregon Green (OG) 488 PT--M,.% Pluronic F-7 and μm sulfobromophthalein (S) in the dark at C for h, followed by shss containing μm S for an additional min at room temperature to allow the de-esterification of OG. The intracellular Ca + signalling of SMCs in PCLS, mounted in the perfusion chamber (described above), was examined with either a custom-built, video-rate scanning confocal microscope or two-photon laser microscope (Perez & Sanderson, 5; ai & Sanderson, 6b). For real-time or time-lapse experiments, fluorescence images were recorded at a rate of 5 images per second, or at a rate of image per s, respectively, using custom-written image acquisition software based on Video Savant. The fluorescence intensity irway rea (% Initial rea) µm t = min shss t = 5 min nm nm.3 mm Ca + / -Ca + t = 5 min nm in -Ca free calcium control Figure. Response of airways to in the presence and absence of extracellular Ca +, representative phase-contrast images of an airway in a PCLS under resting conditions (left, scale bar = µm), exposed to nm (middle) and in the absence of extracellular Ca + (right)., the response of airways to nm in the presence ( control) and absence (free calcium) of extracellular Ca +. Each line in represents the mean and each point represents the mean ± SEM at selected time points of the airway lumen area normalized to the initial size at t = s.datain control represent six airways from five mice, data in free calcium represent three airways from three mice. [Colour figure can be viewed at wileyonlinelibrary.com] in a region of interest (8 pixels) within an SMC, with respect to time, was determined using custom-written software. Relative fluorescence intensity was expressed as F t /F, which is a ratio of the fluorescence intensity at a particular time (F t ) normalized to the initial time (F ). Flash photolysis of caged-ip 3 PCLS were initially incubated with OG as described above and subsequently incubated with shss containing 4 μm irway rea (% Initial rea) C irway rea (% Initial rea) µm t = min shss t = 5 min nm nm + GSK-7975 nm + GSK-5498 t = 5 min nm + GSK-7975 µm 9 8 µm 5 µm 7 µm µm 5 control µm 5 µm 7 µm µm 5 control Figure. Effects of SOCE blockers, GSK-7975 and GSK-5498, on -induced airway contraction, representative phase-contrast images of an airway in a PCLS under resting conditions (left, scale bar = µm), treated with nm (middle) and with µm GSK-7975 (right)., the effects of GSK-7975 and C, GSK-5498 (concentrations ranging from µm to µm) on airways contracted with nm. Controls were treated with vehicle of.5% DMSO in shss. Each line in and C represents the mean and each point represents the mean ± SEM at selected time points of the airway lumen area normalized to the initial size at t = s.datain represent eight to airways for each line from eight mice. Data in C represent four to six airways for each line from five mice. [Colour figure can be viewed at wileyonlinelibrary.com]

5 J Physiol 595. Store-operated Ca + entry in airway smooth muscle Ca + oscillations 37 caged-ip 3,.% Pluronic F-7 and μm S for h, followed by de-esterification in shss containing μm S for min. Details of the flash photolysis set-up and protocols have been described previously (Leybaert & Sanderson, ; Tan & Sanderson, 4). riefly, a pulse of UV light was generated from a mercury arc lamp with a band-pass filter (3 nm) and focused to a point into our custom-built confocal microscope. The intensity and duration of the flash, regulated by exposure time ( ms) and neutral density filters (with an optical density of.7.9), determined the amount of IP 3 released by photolysis of caged-ip 3,andthereby the amount of Ca + released from internal store via IP 3 R of SR. UV exposure with the same intensity and duration was repeated three times in the same region of interest of an SMC. Only the SMCs that showed similar Ca + signalling in response to the first and third exposures were used for analysis, suggesting irway rea (% Initial rea) irway rea (% Initial rea) nm + nifedipine nm + nimodipine 9 8 µm 5 µm 7 µm µm 5 control µm 5 µm 7 µm µm 5 control Figure 3. Effects of VGCC inhibitors, nifedipine and nimodipine, on -induced airway contraction, the effects of nifedipine and, nimodipine (concentrations ranging from to µm) on airways contracted with nm. Controls were treated with the vehicle of.5% DMSO in shss. Each line represents the mean and each point represents the mean ± SEM at selected time points of the airway lumen area normalized to the initial size at t = s.datain represent four to seven airways for each line from four mice. Data in represent four to eight airways for each line from four mice. [Colour figure can be viewed at wileyonlinelibrary.com] a capacity to release similar small amounts of IP 3 for at least three times and the submaximal Ca + signal response to each exposure. Different compounds were administrated to PCLS for 5 min before the second exposure, then followed by washout after exposure; the response of SMC to the second exposure represents the effect of a specific compound on the IP 3 Rpathway.The Ca + -dependent fluorescence changes in SMCs treated with different compounds were recorded and analysed as described above. Preparation of SOCE-activated PCLS This protocol and its validation have been demonstrated previously (ai & Sanderson, 6b). riefly, PCLS were simultaneously exposed to mm caffeine and 5 μm ryanodine for 5 min, followed by a thorough washout with shss prior to the experiments. This treatment gates RyRs of the SR in the open state, and thereby induces the depletion of the SR Ca + store. This, in turn, leads to a persistent Ca + influx via SOCE to elevate the [Ca + ] i. s a result, the [Ca + ] i is determined by the extracellular Ca + concentration ([Ca + ] e ). The PCLS treated with the caffeine and ryanodine remain viable for >5 h,during which the treatment was irreversible. Statistical analysis ll data are reported as the mean ± SEM; ll differences between experimental groups were tested by NOV followed Tukey s honestly significant difference analysis; the sample number for statistical tests is provided where appropriate. P <.5 was considered statistically significant. Statistical analysis was performed with Origin 6 software (OriginLab Corporation, Northampton, M, US). Results Response of airways to methacholine () was used as the agonist of choice throughout the present study to investigate the role of SOCE and L-type VGCCs in sustained agonist-induced contraction of airways; a concentration of nm was used to induce a non-saturated response of airway contraction and Ca + oscillations in SMCs as described previously (Perez & Sanderson, 5; ai & Sanderson, 6a). ctivation of muscarinic receptors via on other cell types, apart from SMCs, in PCLS appeared to have no direct effect on airway contraction (Meurs et al. 3). Prior to the experiments, airway viability was confirmed by an initial exposure to. The exposure of the PCLS to nm induced airway contraction with a lumen reduction of 5 % of the initial luminal area within min. Removal of resulted in an immediate airway relaxation that was completed within 5 min

6 38 J. Chen and M. J. Sanderson J Physiol 595. (Fig. ). The subsequent longer-duration exposure to led to a similar airway contraction but one that slowly relaxed (t = 9.9 ± 55. s) until a more stable lumen size of % (of the initial size) was established. This state persisted for another min when the agonist was present. This time-dependent control response was used to compare both the rate and magnitude of relaxation induced by various compounds and also the reversibility of action (Fig., control). To confirm airway viability at the end of each experiment, and tested drugs were fully washed out and the airway was exposed for a third time to (Fig. ). Effect of an absence of extracellular Ca + When extracellular Ca + was removed (-[Ca + ] e ) to mimic a complete absence of Ca + influx in the presence of nm, the contracted airway immediately underwent a rapid full relaxation (t = 8.8 ± s) and remained relaxed until extracellular Ca + was restored (Fig., free calcium). Under control conditions with nm, the airway displayed an absolute relaxation of.7 ± 3.7%(at5min);in-Ca +,theairway displayed a normalized relaxation of 86.9 ± 8.6% (at 5 min). These data indicate that Ca + influx was required to sustain airway contraction. Effects of SOCE inhibitors on airway contraction Treatment of -contracted airways with SOCE blockers, G7 and G5 relaxed airway contraction in a concentration-dependent manner (Fig. ; see also Supporting information, Video S). Specifically, the largest relaxation was induced by μm G7 or G5, which relaxed the airway lumen by 84.5 ± 4.3% and 86.9 ±.%, respectively. The magnitude of this relaxation was very similar to that induced by -Ca + shss, although the rate of relaxation was slower. G5 initially relaxed the airway at a faster rate (t = 8.8 ±.7 s for μm) compared to G7 (t = 85. ±.3 s for μm)(fig.). Lower concentrations of G7 or G5 induced less relaxation (Fig. and C) with an IC 5 of 4.5 and μm, respectively (Fig. 4). Upon removal of G7 and G5, the airway re-contracted and an almost full re-contraction was achieved within min (Fig. and C). These data indicate that a near complete relaxation of airway contraction is induced when SOCE is inhibited and that these drug effects are reversible. Effects of VGCC inhibitors on airway contraction y contrast to SOCE inhibitors, treatment of -contracted airways with the L-type VGCC inhibitors, nifedipine and nimodipine, only induced a partial relaxation of the airways (Figs 3 and 4), even at concentrations that were considered high relative to their normal efficacy ( 5 μm). Nifedipine, at concentrations ranging from to μm, relaxed the airway lumen up to 5.4 ± 7.6% (at μm). However, although there was a significant difference compared to the control response, there was no significant difference between the responses induced by each concentration of nifedipine (Figs 3 and 4). Similarly, nimodipine relaxed the airway up to 33.8 ± 9.% (at μm) but, again, although there was a significant difference from the control response, there was no significant difference Normalized irway Relaxation (%) Time Constant (second) control -Ca + shss 5 Concentration (µm) + G7 G5 nife nimo + GSK-5498 GSK Nifedipine Nimodipine Figure 4. Summary of relaxation effects and relaxation time of SOCE inhibitor and VGCC inhibitor on -induced airway contraction, concentration-dependent relaxation effects and, relaxation time (time constant, t ) of SOCE inhibitors, GSK-7975 (G7) and GSK-5498 (G5), and VGCC inhibitors, nifedipine (nife) and nimodipine (nimo), on airways contracted with nm. The percentage relaxation induced by each compound (measured at 5 min as shown in the traces of Figs and 3) is normalized to its own control contraction (measured at 5 min). The time constant was calculated based on the relaxation induced by the maximal concentration ( µm) of each drug; control indicates the base-line relaxation that occurred during sustained exposure to nm. Each point in represents the mean ± SEM, data represent four to airways for each point from at least mice. P <.5, significant difference from nifedipine; # P <.5, significant difference from nimodipine; + P <.5, significant difference from control. [Colour figure can be viewed at wileyonlinelibrary.com]

7 J Physiol 595. Store-operated Ca + entry in airway smooth muscle Ca + oscillations 39 between the responses induced by each nimodipine concentration (Figs 3 and 4). In addition, the rate of this relaxation was slow compared to G5 and G7 (t for μm nifedipine and μm nimodipine were 47. ± 3.9 s and 97.7 ± 5.8 s, respectively, as summarized in Fig. 4). These non-significant results with increasing concentrations of nifedipine and nimodipine indicate that their efficacy rapidly reached saturation ( μm) and suggest that the effects associated with higher concentrations are probably related to indirect mechanisms. Overall, as shown in Fig. 4, SOCE inhibitors G5 and G7 induced greater, as well as faster, airway relaxation at all concentrations compared to L-type VGCC inhibitors nifedipine and nimodipine. Effects of an absence of extracellular Ca + on -induced Ca + oscillations To understand the mechanisms underlying airway relaxation induced by SOCE blockers, we first examined how an absence of Ca + influx (zero extracellular Ca + ) would affect Ca + oscillations in SMCs. In response to nm, SMCs displayed Ca + oscillations with a mean frequency of 5 ± 3min and a mean fluorescence intensity ratio of. ±.6 (Fig. 5; see also Supporting information, Video S). In accordance with the contractile responses, these Ca + oscillations only stopped following removal of. To mimic an absence of Ca + influx, extracellular Ca + was removed with -Ca + shss in the presence of nm and on-going Ca + oscillations (Fig. 5). With the loss of Ca + influx, these Ca + oscillations slowed and stopped within s, a response consistent with airway relaxation in -Ca + shss. To further confirm that Ca + oscillation require Ca + influx for sustained activity, rather than just for the initiation of activity, PCLS were initially perfused with -Ca + shss (Fig. 5C). Upon exposure to nm in -[Ca + ] e, Ca + oscillations were initiated but rapidly decreased in frequency and stopped within s. This transient Ca + signalling is consistent with an initial release of Ca + from SR stores and a high dependence on Ca + influx for sustained Ca + oscillations. Effects of SOCE inhibitors on -induced Ca + oscillations We next examined the effect of SOCE inhibitors on agonist-induced Ca + oscillations in SMCs. Similar to the experiments with -[Ca + ] e, pre-treatment of PCLS with μm G7 only allowed a transient period of -induced Ca + oscillations that lasted for s (Fig. 5D). The addition of SOCE inhibitors G7 or G5 (G5 data not shown) after the initiation of -induced Ca + oscillations also inhibited the Ca + oscillations (Fig. 5E H; summarized in Fig. 6); (Fig. 5E) and 5 μm (Fig. 5F) G7 completely inhibited the Ca + oscillations within 9 s and s, respectively (see Supporting information, Video S3). However, lower concentrations of G7 ( and μm)onlyslowedtheca + oscillation frequency from 5 ± min to 9 ± 4min and 38 ± 3min, respectively, after 5 min (Fig. 5G and H). summary of the concentration-dependent inhibition of Ca + oscillation frequency by G5 and G7 is provided in Fig. 6. These data imply that a reduced Ca + influx, as a result of SOCE inhibition, leads to slowing or cessation of Ca + oscillations. Effects of VGCC inhibitors on -induced Ca + oscillations y contrast to the effects of -[Ca + ] e,g7org5,the L-type VGCC inhibitors, nifedipine and nimodipine, did not induce the cessation of -induced Ca + oscillations at any concentration tested (up to μm) (Fig. 6 E, nifedipine data not shown), even though these concentrations are relatively high with respect to their reported normal efficacy ( 5 μm). However, nifedipine and nimodipine induced a slowing of the Ca + oscillation frequency from 54 ± 3min (control) to 36 ± 8 min and ± 4 min, respectively, after 5 min of exposure at μm (Fig. 6 and ). However, this decrease in frequency was similar to that induced by μm nifedipine and nimodipine, again indicating that the response to VGCC inhibitors is not concentration-dependent above μm (i.e. saturated at low concentrations). This partial effect on Ca + oscillation frequency is consistent with the concentration independent relaxation effect (above μm) of VGCC inhibitors on airway contraction (Figs 3 and 4). possible explanation for the limited effect of nifedipine and nimodipine on airway contraction and Ca + oscillation frequency is that these compounds are inactive in SMCs of PCLS. To confirm that nifedipine and nimodipine were indeed capable of inhibiting VGCCs in PCLS, we tested their effect on KCl-induced Ca + oscillations. Our previous studies have shown that these slow, depolarization-induced Ca + oscillations are mediated by a constant Ca + influx through VGCCs associated with a periodic overfilling of the SR with Ca + and its release via RyRs by Ca + -induced Ca + release (Perez & Sanderson, 5). ccordingly, as shown in Fig. 6F, both nifedipine and nimodipine, at a -fold lower concentration ( μm), quickly inhibited 5 mm KCl-induced Ca + oscillations (within s). These results suggest a high efficiency and specificity of these compounds for L-type VGCCs. y contrast, μm G7 did not stop the KCl-induced oscillations within min of exposure (Fig. 6G), although some slowing in frequency was evident with longer exposure. This result suggests that KCl-induced oscillations do not utilize SOCE-mediated

8 3 J. Chen and M. J. Sanderson J Physiol 595. influx. Collectively, these results with nifedipine and nimodipine indicate that substantial Ca + influx still occurs during agonist-induced Ca + oscillations when L-type VGCCs are completely blocked. Ca + Effects of SOCE inhibitors on Ca + influx in SOCE-activated PCLS To further characterize the action of SOCE and VGCC inhibitors on SOCE-mediated Ca + influx, we utilized SOCE-activated PCLS. These PCLS were prepared by simultaneous exposure to mm caffeine and 5 μm ryanodine: the caffeine stimulates the opening of RyRs on SR, whereas ryanodine irreversibly locks the RyRs in a submaximal open state. The open RyRs results in a constitutive Ca + efflux from the SR and leads to the Ca + depletion of SR, which, in turn, activates and maintains the opening of store-operated Ca + channels onthecellmembranetoenablesoce(yet al. 4). Caffeine and ryanodine treatment results in an immediate transient increase in [Ca + ] i (Ca + mainly from SR) followed by a sustained elevation in [Ca + ] i (Fig. 7), which results from the balance between persistent Ca + efflux from the SR and Ca + influx through store-operated Ca + channels. In this scenario, the [Ca + ] i in SMCs is determined by the [Ca + ] e. This is confirmed (Fig. 7) by the repetitive decrease of [Ca + ] i in response to treatment with -[Ca + ] e and the increase of [Ca + ] i when Intensity (F t /F ) Intensity (F t /F ) C Intensity (F t /F ) D Intensity (F t /F ) nm nm GSK7975 µm nm E Intensity (F t /F ) F Intensity (F t /F ) G Intensity (F t /F ) H Intensity (F t /F ).5 -Ca + shss nm + GSK-7975 µm nm + GSK µm -Ca + nm nm + GSK7975 µm nm + GSK7975 µm Figure 5. Effect of SOCE inhibitor, GSK-7975, on -induced Ca + oscillations in SMCs, a representative trace of the Ca + oscillations shows that nm induced Ca + oscillations (sustained for more than min, representative of 93 from at least mice);, nm -induced Ca + oscillations were stopped by zero extracellular Ca + (-[Ca + ] e ) in s (representative of four from three mice); C, nm only induced transient Ca + oscillations in SMCs pre-treated with -[Ca + ] e (representative of three from two mice); D, nm only induced transient Ca + oscillations in an SMC pre-treated with µm GSK-7975 (representative of three from three mice); E, nm -induced Ca + oscillations were stopped by µm GSK-7975 in 9 s (representative of four from three mice); F, nm -induced Ca + oscillations were stopped by 5 µm GSK-7975 in s (representative of five from four mice); G, andh, nm -induced Ca + oscillations were slowed down in a concentration-dependent manner by µm and µm GSK-7975, respectively (representative of nine to from five mice). Representative traces are expressed as intensity (F t ) normalized to the initial intensity at t = s(f ), measured from a region of interest with size of 8 pixels within a single SMCs.

9 J Physiol 595. Store-operated Ca + entry in airway smooth muscle Ca + oscillations 3 shss containing.3 mm Ca + was replaced. Thus, these SMCs are considered SOCE-activated, a condition that is irreversible and persists after the washout of caffeine and ryanodine (ai & Sanderson, 6b), making these PCLS an ideal tool for assessing the effects of compounds on SOCE-mediated Ca + influx. To evaluate the effect of G5 and G7 on SOCE, these inhibitors were applied when changing the [Ca + ] e to zero and remained in place when the [Ca + ] e was subsequently increased to.3 mm. n inhibition of SOCE is indicated by a reduced increase in F t /F intensity upon Ca + add-back (Fig. 7 E). The percentage Ca + increase induced by shss in the presence of each drug (drug average intensity F t /F,I drug, over a period from 9 to min in Fig. 7D and E and 8 and C) is normalized to the Ca + increase induced by shss in the absence of drug (own control I) by the formulae: % Ca + increase (CI) = [I drug (9 min) I zero-calcium (4 7 min) ]/ [I shss (8 min) I zero-calcium (4 7 min) ], then further normalized to the amount of Ca + increase that occurred in the control response by the formulae: % normalized Ca + increase = (CI drug CI control )/( CI control ). Intensity (F t /F ) Intensity (F t /F ) C Ca + Oscillations frequency (min ) 5 * # * # * # * # * # * # 5 Concentrarion (µm) Nimodipine Nifedipine GSK-7975 GSK-5498 nm + nimodipine µm nm + nimodipine 5 µm D Intensity (F t /F ) E Intensity (F t /F ) F Intensity (F t /F ) G Intensity (F t /F ) nm + nimodipine µm nm + nimodipine µm KCI 5 mm + nifedipine µm KCI 5 mm GSK-7975 µm Figure 6. Summary effects of SOCE inhibitor and VGCC inhibitor on or KCl induced Ca + oscillations in SMCs, the concentration-dependent effect of SOCE inhibitors, GSK-7975 and GSK-5498, and VGCC inhibitors, nifedipine and nimodipine, on the frequency of Ca + oscillations induced by nm insmcs. E, representative Ca + oscillation traces show that nm -induced Ca + oscillations were partially slowed by nimodipine with concentrations ranging from µm to µm (representative of four to six from five mice). Note that, even at µm, nimodipine did not stop the -induced Ca + oscillations. F, a representative Ca + oscillation traces show that µm nifedipine stopped 5 mm KCl-induced Ca + oscillations within min (representative of five from four mice); G, µm GSK-7975 did not stop 5 mm KCl-induced Ca + oscillations after treatment for min (representative of three from three mice). Representative traces are expressed as intensity (F t ) normalized to the initial intensity at t = s(f ), measured from a region of interest with size of 8 pixels within a single SMCs. Each point in () represents the mean ± SEM, data represent four to SMCs for each point from at least mice. P <.5, significant difference from nifedipine; # P <.5, significant difference from nimodipine. [Colour figure can be viewed at wileyonlinelibrary.com]

10 3 J. Chen and M. J. Sanderson J Physiol 595. s shown in Fig. 7D and E (note the F t /F intensity in last 5 min), G7 and G5 inhibited an increase in [Ca + ] i of SMCs in a concentration-dependent manner. Specifically, μm G5 and G7 both fully prevented SOCE-mediated Ca + increases upon the addition of.3 mm [Ca + ] e (see Supporting information, Video S3). Lower concentrations of G7 and G5 partially inhibited SOCE-mediated increases with an IC 5 of 4. and 3.7 μm, respectively (Fig. 8). y contrast, the L-type VGCC inhibitors had only a small effect on SOCE-mediated increases in [Ca + ] i in response to.3 mm [Ca + ] e at all concentrations up to μm (Fig. 8). Effects of SOCE blockers on the activation of the IP 3 R n inhibition of Ca + release by the IP 3 R could contribute to the decreased frequency of agonist-induced Ca + oscillations as described previously (Tan & Sanderson, 4). To exclude this possibility, the action of G7 and G5 on IP 3 R sensitivity to IP 3 was evaluated by UV photolysis of caged-ip 3 in SMCs. Under control conditions (in shss), SMCs displayed a transient Ca + spike in response to a single UV flash. similar Ca + signal in the same SMC was generated by a second and third UV flash, indicating that this approach was reproducible for at least three time trials (Fig. 9). s a positive control, the effect of μm chloroquine, a type bitter-taste receptor (TSR) agonist that attenuates the Ca + release from SR via inhibition of IP 3 R (Tan & Sanderson, 4), was administrated to the PCLS for 5 min prior to the second flash. s shown in Fig. 9 and E, μm chloroquine significantly reduced the amount of Ca + released from SR by 43.5 ± 8.%. However, treatment with either G7 or G5 (at μm) had no effect on the IP 3 -induced Ca + transient (Fig. 9C E). These results support the notion Intensity (F t /F ) C Ryanodine 5 µm + Caffeine mm t = min SMC epithelium.3 mm Ca + shss -Ca + shss t = min t = 5 min t = min epithelium µm SMC t = 5 min t = min µm.3 mm Ca + shss.3 mm Ca + shss -Ca + shss.3 mm Ca+ shss + GSK-7975 µm D Intensity (F t /F ) E Intensity (F t /F ) Ca +.3 mm Ca + -Ca +.3 mm Ca + GSK-7975 in -Ca + GSK-5498 in -Ca + GSK-7975 in.3 mm Ca GSK-5498 in.3 mm Ca Control µm µm 5 µm µm Control µm µm 5 µm µm Figure 7. Effects of SOCE inhibitors, GSK-7975 and GSK-5498, on SOCE-mediated Ca + influx in SOCE-activated PCLS, a representative Ca + signal trace shows that treatment with caffeine and ryanodine resulted in an immediate transient increase in intracellular Ca + concentration ([Ca + ] i ) followed by a sustained elevation in [Ca + ] i ( min); after wash off, the [Ca + ] i in SMC can be manipulated by the [Ca + ] e (8 4 min)., andc, representative two-photon fluorescence microscope images (scale bar = µm) showing SMCs adjacent to airway epithelium in the same airway of a SOCE-activated PCLS with exposure to.3 mm Ca + shss ( and C, left), -Ca + shss ( and C, middle) and.3 mm Ca + shss again (, right, as control) or.3 mm Ca + shss with µm GSK-7975 (C, right); the time points in () and(c) correspond to the time trace in D and E. Note that, in the presence of µm GSK-7975, the [Ca + ] i of SMCs did not increase as [Ca + ] e increased (C, right compared to, right). The effects of (D) GSK-7975 and (E) GSK-5498, with concentrations ranging from µm to µm, on SOCE-mediated Ca + influx. Note that the level of decreased [Ca + ] i after 9 min indicates the inhibitory level of SOCE. Controls were treated with the vehicle of.5% DMSO in shss. Traces are expressed as intensity (F t ) normalized to the initial intensity at t = s(f ), measured from a region of interest with size of 8 pixels within a single SMC. Each line in D and E represents the mean and each point represents the mean ± SEM at selected time points. Data in D represent three to six SMCs for each line from five mice. Data in E represent three to four SMCs for each line from four mice. [Colour figure can be viewed at wileyonlinelibrary.com]

11 J Physiol 595. Store-operated Ca + entry in airway smooth muscle Ca + oscillations 33 Intensity (F t /F ) C Intensity (F t /F ) Normalized mount of Ca + influx (%) Concentration (µm) -Ca +.3 mm Ca + nifedipine in -Ca Ca +.3 mm Ca + nimodipine in -Ca Nifedipine Nimodipine GSK-5498 GSK-7975 nifedipine in.3 mm Ca + nimodipine in.3 mm Ca + Control µm µm 5 µm µm Control µm µm 5 µm µm Figure 8. Summary effects of SOCE inhibitors and VGCC inhibitors on SOCE-mediated Ca + influx in SOCE-activated PCLS, the concentration-dependent inhibitory effects of SOCE inhibitors, GSK-7975 and GSK-5498, and VGCC inhibitors, nifedipine and nimodipine, on SOCE-mediated Ca + influx in SOCE-activated PCLS. The % Ca + influx for each compound is calculated by the average intensity F t /F (I) over a period from 9 min to min normalized to its own control (the increased I in response to the change of [Ca + ] e from to.3 mm). The effects of VGCC inhibitors, nifedipine and C nimodipine, with concentrations ranging from µm to µm, on SOCE-mediated Ca + influx in SOCE-activated PCLS. Note that the level of decreased [Ca + ] i after 9 min indicates the inhibitory level of SOCE. Controls were treated with the vehicle of.5% DMSO in shss. Traces are expressed as intensity (F t ) normalized to the initial intensity at t = s(f ), measured from a region of interest with size of 8 pixels within a single SMCs. Each point in represents the mean ± SEM, data represent three to six SMCs for each point from at least mice. Each line in and C represents the mean and each point represents the mean ± SEM at select time points. Data in represent three to five SMCs for each line from four mice. Data in C represent three to four SMCs for each line from four mice. P <.5, significant difference from nifedipine; # P <.5, significant difference from nimodipine; + P <.5, significant difference from control. [Colour figure can be viewed at wileyonlinelibrary.com] that the cessation of Ca + oscillations induced by G7 and G5 probably resulted from their inhibitory effect on SOCE-mediated Ca + influx rather than decreased IP 3 R function. Discussion The molecular basis for SOCE, based on STIM and Orai, was discovered almost a decade ago (Lewis, 7), although direct evidence for the role of SOCE in SM contraction is limited. Ohga et al. (8) showed that a SOCE inhibitor YM-58483/TP- attenuated ovalbumin-induced bronchoconstriction in guinea pigs, implying an important role of SOCE in bronchoconstriction. However, the present study only provided indirect evidence in vivo or from tracheal muscle strips that might not represent the physiology of SM from smaller airways surrounded by lung parenchyma. On the other hand, with a variety of Ca + channels in the PM of SM, the identity of the major pathway accounting for the SR refilling during agonist-induced SM contraction remains controversial. Our previous mathematical modelling predicts that SOCE might be the major pathway (Croisier et al. 3). Therefore, we used mouse PCLS, which reflect the in situ physiology of SM of relatively smaller airways, to investigate the effects of two specific SOCE inhibitors, G7 and G5, and two classical L-type VGCC inhibitors, nifedipine and nimodipine, on -induced airway contraction and the underlying Ca + oscillations within SMCs. We first confirmed the significance of Ca + influx for sustained airway contraction with -[Ca + ] e that mimics condition where Ca + influx is totally absent. s predicted, -induced airway contraction was quickly and almost completely relaxed when Ca + influx could not occur. Next, we further examined the Ca + oscillations in SMCs, which mediate airway contraction by activating myosin light-chain kinase to induce the phosphorylation of regulatory myosin light-chain. In accordance with the contraction data, -induced Ca + oscillations in SMCs also stopped quickly when there was no Ca + influx. To address which channel pathway accounts for this Ca + influx, we first examined the effects of G7 and G5; these compounds are novel pyrazol derivatives that inhibit SOCE via both Orai and Orai3 with an IC 5 of approximately 4 μm in cell studies (Derler et al. 3). We found that both G7 and G5 (at μm) almost fully relaxed the pre-contracted airway and completely stopped -induced Ca + oscillations. These responses were extremely similar to those observed in -[Ca + ] e,interms of the extent and speed of relaxation. We also examined the role of SOCE on the initiation of Ca + oscillations by treating PCLS with -[Ca + ] e or G7 prior to exposure. Under either of these conditions, only

12 34 J. Chen and M. J. Sanderson J Physiol 595. Intensity (F t /F ) Intensity (F t /F ) C shss shss shss shss Choloroquine µm shss shss GSK-7975 µm shss Intensity (F t /F ) D Intensity (F t /F ) E shss GSK-5498 µm shss Intensity (F t /F ) st flash nd flash 3 rd flash *.6.4. shss Cn Ch G7 G5 shss Figure 9. Effects of SOCE inhibitors, GSK-7975 and GSK-5498, on Ca + signalling induced by IP 3 released from UV photolysis of caged-ip 3 in SMCs Representative Ca + signal traces show that a single SMC was exposed to a pulse of UV illumination (5 ms) during resting conditions (first flash), after 5 min of incubation (second flash) with either shss (Cn,, as negative control), µm choloroquine (Ch,, as positive control), µm GSK-7975 (G7, C) and µm GSK-5498 (G5, D), and after washout of the each compound (third flash). Note that only chloroquine inhibited the IP 3 -induced Ca + signalling as shown in the summary of results in E. Traces are expressed as intensity (F t ) normalized to the initial intensity at t = s (F ), measured from a region of interest with asizeof8 pixels within a single SMC. Each bar in E represents the mean ± SEM with four to nine SMCs from three mice. P <.5 indicates a significant difference from its own control in the first and third flash.

13 J Physiol 595. Store-operated Ca + entry in airway smooth muscle Ca + oscillations 35 induced transient Ca + oscillations. These data suggest that the initial stage of agonist-induced Ca + oscillations mainly relies on the release of Ca + from internal stores and that sustained Ca + oscillations requires Ca + influx via a SOCE pathway to replenish the Ca + lost through PM Ca + TPases. It is worth noting that, although G7 and G5 induced airway relaxation with an IC 5 of 4.5 and μm, respectively (i.e. similar to the IC 5 tested in the cell studies), it appears that a concentration up to μm is required for either G7 or G5 to induce a maximal effect on the PCLS. This dose is much higher than a dose expected in a cell study ( μm) and probably is a result of the considerable thickness and more complex structure in the PCLS compared to the single cells. The possibility that VGCC is an important contributor to Ca + influx in SM is supported by observations that VGCC inhibitors relaxed KCl-induced airway contraction, where it implies that SM contraction results from membrane depolarization and an associated Ca + influx via VGCCs (Prakash et al. 997;Du et al. 6). However, the KCl-induced Ca + oscillations are fundamentally different from the agonist-induced Ca + oscillations. With KCl, membrane depolarization increases [Ca + ] i via VGCCs, which results in a progressive overfilling of the SR with Ca +. This, in turn, results in a sensitization of the RyRs, with the consequence of unloading the overfilled SR Ca + store via Ca + -induced Ca + release (Perez & Sanderson, 5; ai & Sanderson, 6a). y contrast, agonist-induced Ca + oscillations utilize internal Ca + release via an IP 3 R. Hence, although VGCCs are important for slow KCl-induced Ca + oscillations, they do not appear to be important for fast agonist-induced Ca + oscillations. This non-essential role of VGCCs in SM contraction has been suggested previously (Janssen, ) and our data obtained in the present study, as well as those from previous studies (Perez & Sanderson, 5), are consistent with this hypothesis in that neither airway contraction, nor Ca + oscillations induced by can be completely inhibited by nifedipine or nimodipine (up to μm). The possibility that nifedipine or nimodipine were inactive is ruled out by their clear ability, at low concentrations, to inhibit KCl-induced Ca + oscillations. Nevertheless, a similar partial airway relaxation and limited reduction in Ca + oscillation frequency was observed with all concentrations of VGCC inhibitors. These results imply that the activity of VGCCs accounts for a relatively small amount of Ca + influx during agonist-induced Ca + oscillations. Given that the agonist-induced Ca + oscillations might only result in a gradual decrease in [Ca + ] SR rather than a full depletion of the SR Ca + (Croisier et al. 3), the maintenance of Ca + oscillations probably only requires a small Ca + influx, mediated by slow responding of SOCE together with VGCCs, rather than a large Ca + influx that requires full activation of SOCE. Under this scenario, an absence of Ca + influx from VGCCs, although proportionally small, would be sufficient to demonstrate an effect on the Ca + oscillation frequency. However, with the loss of this Ca + influx, the SR Ca + content might be reduced further. This, in turn, will result in an elevation in the SOCE current. Thus, the cell compensates for the loss of Ca + influx via VGCCs with an increase in SOCE, with the consequence of a limited effect on the Ca + oscillation frequency. This scenario is addressed in our accompanying mathematical model. y contrast, the inhibition of SOCE by G5 or G7 induced the rapid cessation of the agonist-induced Ca + oscillations and eventually full airway relaxation. This implies that, although VGCCs contributes to Ca + influx, not only are they unable to compensate for the loss of Ca + influx via SOCE, but also they are unable to provide sufficient Ca + influx to generate and sustain agonist-induced Ca + oscillations, as SOCE does. major reason for this is that, unlike SOCE, there is no linkage between SR Ca + depletion and the Ca + influx via VGCCs. This lack of dependence of VGCC-mediated Ca + influx on SR Ca + content is also supported by the success of nifedipine, but the failure of G7, to stop KCl-induced Ca + oscillations, suggesting that KCl-induced Ca + oscillations are mainly driven by VGCC-mediated Ca + influx and, more importantly, might not require the involvement of SOCE as a result of decreased SR Ca + content. The slowing of KCl-induced Ca + oscillations with longer exposure of G7 may result from the limited non-specific action of G7 on VGCCs (Derler et al. 3), even though this inhibitory effect appeared to be much weaker than that provided by nifedipine. Therefore, the relatively specific inhibition of SOCE via G7 or G5, together with the inability of VGCCs to either compensate or match for the inhibited SOCE, resulted in almost complete cessation of the agonist-induced Ca + oscillations and thus full airway relaxation. It is important to note that, although the full airway relaxation takes time to develop, the cessation of Ca + oscillations was, to some extent, more rapid. This observation can be explained by the fact that the generation of Ca + oscillations require a threshold level of Ca + influx (Croisier et al. 3), which could not be provided when SOCE was blocked via G7 or G5. However, a small amount of Ca + influx via VGCCs and other Ca + influx pathway might still exist to account for a weak but persistant airway contraction until the intracellular Ca + is totally run down. To further corroborate that G7 and G5 were acting onsoce,weutilizedsoce-activatedpclsinwhicha persistent Ca + influx was generated by full Ca + depletion of the SR (ai & Sanderson, 6b). Compared to the previous techniques that stimulate SOCE by inhibiting

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