Due to its small size (<10 μm) measurement of intracellular Ca2+ in Plasmodium is technically challenging, and thus Ca2+ regulation in this human pathogen is not well understood. Here we analyze Ca2+ homeostasis via a new approach using transgenic P. falciparum expressing the Ca2+ sensor yellow cameleon (YC)-Nano. We found that cytosolic Ca2+ concentration is maintained at low levels only during the intraerythrocytic trophozoite stage (30 nM), and is increased in the other blood stages (>300 nM). We determined that the mammalian SERCA inhibitor thapsigargin and antimalarial dihydroartemisinin did not perturb SERCA activity. The change of the cytosolic Ca2+ level in P. falciparum was additionally detectable by flow cytometry. Thus, we propose that the developed YC-Nano-based system is useful to study Ca2+ signaling in P. falciparum and is applicable for drug screening.Calcium (Ca Plasmodium, and remains a major global public health problemMalaria is caused by the intracellular protozoan parasite 2+).Malaria parasite infection begins with the inoculation of sporozoite stage parasites by the mosquito, followed by a period of parasite replication in the liver, and then release of merozoite stages into the blood to begin the intraerythrocytic cycle. It is the asexual repetition of red blood cell (RBC) invasion, development, multiplication via schizogony, and RBC rupture to release new merozoites which amplifies the blood stream parasite burden and provokes malarial symptoms and pathology. Some of the intra-erythrocytic parasites transform to non-replicative macro- and microgametocytes, which are the only stages transmissible to mosquitoes following the taking of a blood meal and which mediate sexual stage development in the mosquito midgut. The life cycle completes by the formation of sporozoites with midgut oocysts, and accumulation of these invasive sporozoites in the mosquito salivary gland. Herein we focus on the intraerythrocytic asexual cycle and gametocytes to characterize the storage and flow of calcium  pathway in both Plasmodium falciparum and the distantly related apicomplexan parasite, Toxoplasma gondii82+ storage compartments in P. falciparum2+ through the sarco/endoplasmic reticulum Ca2+-ATPase (PfSERCA or PfATP6) pump11P. falciparum genome contains only one SERCA gene2+ homeostasis in P. falciparum is an appealing approach to antimalarial drug development.Calcium is a universal secondary messenger for intracellular signaling in eukaryotic cells and regulates a variety of cellular functions through fluctuation of cytosolic free Ca2+ has been studied using synthetic chemical fluorescent Ca2+ indicators; however, these types of fluorochromes have drawbacks2+ concentration in a specific compartmentalized space in the eukaryotic cells including malaria parasitesP. falciparum, and the degree of accumulation appears to differ among parasite strains depending on the gene copy number encoding the transporter located on this membrane2+ concentration of less than 100 nM which is proposed for P. falciparum2+ concentration using chemical Ca2+ indicators must be approached with caution. To overcome these limitations, we consider here a recently designed genetically encoded Ca2+ indicator, yellow cameleon-Nano (YC-Nano) which is ultrasensitive and able to detect nanomolar changes of Ca2+ concentration in mammalian cell lines2+ biosensors were based on cyan fluorescent protein (CFP) fused with the Ca2+ binding protein, calmodulin (CaM), and yellow fluorescent protein (YFP) fused with M13 peptide. The CaM domain binds to M13 peptide in the presence of Ca2+, which in turn shortens the distance and thereby increases fluorescence resonance energy transfer (FRET) efficiency between the two fluorescent proteins. The advantage of the biosensor compared with fluorochromes is the nature of the output, which is the ratio of the emitted YFP signal to the emitted CFP signal. This ratio metric output reduces the artifacts introduced by unstable focusing due to parasite movement during live cell imaging process, which may occur with non-ratiometric indicators such as fluorochromes.The cell biology of intracellular CaP. falciparum the application of YC-Nano and the successful measurement of changes of cytosolic Ca2+. Live cell confocal microscopy revealed that cytosolic Ca2+ was maintained at a low concentration only at the trophozoite stage, and increased as intraerythrocytic development progressed. We show that the mammalian SERCA pump inhibitor thapsigargin (TG) and dihydroartemisinin (dART), a current first-line antimalarial, did not change the cytosolic Ca2+ concentration, indicating that these compounds do not inhibit P. falciparum SERCA pump activity. Docking analysis supported the insensitivity of the parasite to TG and dART. We also demonstrate detection of the FRET signal by a flow cytometry method, indicating that the transgenic reporter parasite is applicable for the high-throughput screening of compounds targeting P. falciparum Ca2+ homeostasis.Here we report for 2+ in P. falciparum, we generated transgenic lines expressing fluorescent protein-based Ca2+ biosensors YC-Nano15 or YC-Nano50 driven by the P. falciparum heat shock protein 86 (PfHSP86) constitutive promoter  surrounding the parasite. The ratio of YFP and CFP was determined by confocal microscopy with Tyrode’s buffer containing different concentrations of Ca2+ (0–500 nM). The obtained calibration curves revealed a dissociation constant value of 15.5 nM and 45.8 nM for YC-Nano15 and YC-Nano50, respectively ; minimum between 0–30 sec) to 2.66  in trophozoite stage parasites, confirming that YC-Nano50 has a large dynamic range in P. falciparum. The calculated median cytosolic Ca2+ concentration in the trophozoite stage was 30.0 (interquartile range: 5.6–55.0) nM. We found calculated Ca2+ concentrations in other stages of the parasite were much higher; 372.5 (253.0–483.0) nM at ring, 310.0 (256.2–514.9) nM at schizont, 949.6 (785.1–995.2) nM at merozoite, 131.3 (70.1–185.1) nM at gametocyte (stage III) and 521.8 (387.2–942.2) nM at gametocyte (stage IV–V) stages  and gametocytes, at higher FRET signals than in the trophozoite stage . To esti) stages .2+ storage compartment to maintain and regulate the cytosolic Ca2+ concentration in eukaryotic cells, and uptake of Ca2+ from cytosol to ER is regulated by SERCA. In Plasmodium conflicting reports describe the responses of malaria parasites against SERCA inhibitors, specifically thapsigargin (TG)212+ homeostasis, and found that 15 μM CPA, a SERCA specific inhibitor consistently reported to inhibit P. falciparum SERCA (PfSERCA)2+ 2+ ; whereasntration . The eff applied . The poso effect .2+ rich environments in the human body and in the culture - for example, 45–86 nM in the RBC cytosol, ~40 μM in the PV space, and ~1 mM in the human plasma252+-free medium after selective membrane permeabilization. Firstly, iRBCs were treated with streptolysin O (SLO) to selectively permeabilize the RBC membrane, but not the PVM and parasite plasma membrane (PPM). When TG was added to SLO-treated iRBC, no effect was observed, but subsequent addition of CPA increased the cytosolic Ca2+  for TG and PfSERCA of −9.58 kcal/mol and 95.62 nM, respectively; whereas those for TG and human SERCA were −10.82 kcal/mol and 11.77 nM, respectively. A more negative free energy value indicates a stronger molecular interaction, and thus the results suggest a 8.1-fold weaker interaction between PfSERCA and TG than that between human SERCA and TG. Next, to optimize the interaction between protein and TG in detail, we performed energy minimizations from homology modeling, then we made an energy minimized structure of complex of human SERCA and PfSERCA with TG  at the same position in the SERCA structure indicated a difference of TG binding pocket and binding energy. For example, L1046 of PfSERCA occupied the location where F834 of human SERCA exists, which results in more open space in the TG-binding pocket of PfSERCA. The total value between PfSERCA and TG was −99.0, a value higher than that between human SERCA and TG (−100.8). Because a more negative value of the estimated van der Waals force indicates more stability, these results also suggest that the interaction between PfSERCA and TG is less stable than that between human SERCA and TG. These homology modeling and binding energy calculations between PfSERCA and TG support the hypothesis of P. falciparum insensitivity against TG.To gain insights into the difference between human SERCA and  with TG . Interac with TG . Five amP. falciparum has not developed resistance against these drugs outside of Southeast Asia2+ homeostasis as implicated from the latter hypothesis. We exposed parasites to different concentration of dART  and found that dART had no effect on the parasite cytosolic Ca2+ concentration even at 100 μM  derivatives are currently the most commonly used anti-malarial drugs, due to their low cost and because t 100 μM . SequentPfSERCA or human SERCA were performed with 200 individual genetic algorithm from homology modeling resulted that the estimated free energy and KI for PfSERCA and dART were −6.96 kcal/mol and 7.85 μM, respectively. For human SERCA and dART the values for estimated free energy and KI were −7.47 kcal/mol and 3.37 μM, respectively; suggesting, firstly, a 2.3-fold weaker interaction between PfSERCA and dART than that between human SERCA and dART; and, secondly, a much weaker interaction of dART with both human and PfSERCA than the case for TG. These modeling calculations support the observed inactivity of dART against cytosolic Ca2+ homeostasis in P. falciparum.Docking simulation of dART with 2+ level in the Ca2+-containing medium with the calcium ionophore A23187, but not CPA, even after SERCA was inhibited with CPA as a SERCA specific inhibitor , suggesting that this reduction reflect Ca2+ influx from PV into parasite cytosol in SLO-treated iRBC. These results indicate that our system is able to detect the existence of parasite Ca2+ in the PV space and intracellular Ca2+ storage compartments in addition to the ER.In the present study we detected an increase of cytosolic Canhibitor , therebylic Ca2+ . Althougncreased , suggestarasites , indicatP. falciparum (tert-butylhydroquinone (BHQ), in addition to CPA and TG, to compare the FRET signals obtained by confocal microscopy ), 1.13 ± 0.03, 0.99 ± 0.02, and 0.98 ± 0.02, for CPA, BHQ, TG, and DMSO, respectively , 0.26 ± 0.01, and 32.8 ± 4.8 μM, respectively; thus validating the pharmacological activity of all compounds (2 = 0.9956) existed between FRET signals obtained from confocal microscopy and flow cytometry  power of 457 nm laser beams for excitation. Introduction of the Perfect Focus System and galvano scanner enabled stable capture images every 1 second at a 512 × 512 pixel resolution, which is critical to monitor changes in organisms of sizes less than 10 μm diameter, such as the malaria parasite. With these optimizations the FRET signals from this organism became stable for 10 minutes or more.In this study we generated transgenic 2+ concentrations throughout the blood stages of the malaria parasite. The cytosolic Ca2+ concentration is high for all stages , with the exception of the trophozoite (30 nM) , a non-SERCA-type Ca2+-transporting P-ATPase which is located on the parasite plasma membrane, may participate to this process2+ level significantly increased in the schizont stage, both mechanisms appear to be less active in this mature parasite form. The estimated cytosolic Ca2+ concentration was highest at the merozoite stage, significantly higher than the schizont stage . Ca2+ signaling is known to be involved in the egress of the merozoites from the RBC, as well as the invasion into new RBC by triggering the secretion of microorganelles such as exonemes and micronemes in the merozoite stage parasite72+ level at released merozoites indicates that the Ca2+ secretion signals have been initiated. Glushakova et al. reported that cytosolic Ca2+ level increased at the schizont stage, reaching to 1–10 μM range just prior egress using a chemical indicator Fura Red, which is consistent to our estimated cytosolic Ca2+ concentration of 949 nM at the merozoite stageP. falciparum calcium-dependent protein kinase 5 (PfCDPK5) and PfCDPK1 are plant-like protein kinase family members and have been proposed to act during these stepsPfCDPKs are activated at Ca2+ concentrations as high as the 310 nM estimated at the schizont stage, one report proposed that the Kd value for Ca2+ of a purified beetroot CDPK is 770 nMet al. successfully observed Ca2+ oscillation during P. berghei sporozoite movement using another calcium biosensor, TN-XXL, which has a Kd of 830 nM2+ level in the released merozoites was noted by Biagini et al. using Fluo 4-AM, but the concentration was not determined due to the limited resolution of the system2+ level less than 100 nM during ring stage development. Because CDPK1 is also expressed in both male and female gametocytes2+ concentration estimated at the gametocyte stage is consistent with possible CDPK1 activity in the high Ca2+ level environment.To our knowledge this is the first report to estimate cytosolic Ca (30 nM) . The trohibitors , indicat2+ concentration in P. falciparum, we conducted four experiments to evaluate: 1) the effect of TG on the P. falciparum cytosolic Ca2+ homeostasis, 2) the effect of dART on the P. falciparum cytosolic Ca2+ homeostasis, 3) the feasibility to detect Ca2+ storage(s) beside ER, and 4) the feasibility to establish a high-throughput method to detect the change of the Ca2+ level by flow-cytometry. Uptake of Ca2+ from cytosol to ER is largely regulated by SERCA; however, there are conflicting observations regarding malaria parasite responses against the SERCA inhibitor TG. One report concluded that PfSERCA was TG-insensitive2+ with synthetic chemical indicators Fura 2-AM or Fluo 3-AM. Our analysis using a parasite line expressing a biosensor revealed that the Ca2+ concentration at the trophozoite stage was 30 nM, which was much lower than the dissociation constant of Fura 2- or Fluo 3-based indicators . Using the YC-Nano50 biosensor with a Kd value of 45.8 nM, which is superior than the chemical indicators to evaluate Ca2+ concentration at the trophozoite stage of P. falciparum, we were able to clarify that TG had no effect on the Ca2+ homeostasis of P. falciparum. SERCA of apicomplexan parasites, including Plasmodium, is evolutionally more closely related to one of the two types of plant SERCA than to mammalian SERCA2+-ATPases have developed insensitivity to TGPfSERCA is TG-insensitive. Docking models of TG with PfSERCA and mammalian SERCA also suggest a clear difference in the shape of the TG binding pocket between the two SERCA structures. Based on these differences in the sensitivity against TG and the structure of the TG binding pocket, TG may serve as a seed compound for a structure-based drug design to develop selective anti-malarial compounds.To validate the robustness of our established system to monitor the Ca2+-ATPase activity using PfSERCA expressed on Xenopus oocyte membrane proposed that ART had effect on PfSERCA42PfSERCA was a target of ART442+ concentration in the parasite cytosol. Docking models of dART and both SERCA showed that the affinity of dART to both SERCA is at the micromolar level, suggesting that dART may not be effective against both SERCA. Together, our data indicate that dART plays at most a minor role to modulate P. falciparum Ca2+ homeostasis.Because both TG and ART are composed of sesquiterpene lactone, and since TG is a highly selective inhibitor for mammalian SERCA2+ in the malaria parasite122+ storage sites  and PfATP4, have been annotated among the 13 P-type ATPases. PfATP4 is localized on the PPM and is considered to transport not only Na+ but also Ca2+322+ increase after calcium ionophore stimulation in SLO-treated iRBC than that in saponin-treated iRBC. The DV was reported to contain only moderate amounts of Ca2+ and no dynamic changes of the Ca2+ concentration were observed in DV following induced cytosolic Ca2+ bursts2+ storages, active participation of these compartments to maintain cytosolic Ca2+ homeostasis of malaria parasites is still unclearThe ER is the most important organelle storing Cage sites . In thisP. falciparum expressing YC-Nano50 biosensor and showed that this parasite is a suitable and powerful tool in which to study Ca2+ homeostasis in the trophozoite stage of P. falciparum. We determined, for the first time, that the resting Ca2+ concentrations at schizont, merozoite, ring, and late gametocyte stages are higher than 300 nM. We also showed that TG and dART did not affect the cytosolic Ca2+ level of trophozoite stage of this parasite. FRET signals are detectable by flow cytometry and correlate with a microscope-based assay, indicating that the developed flow cytometry-based system is applicable for drug screens targeting mechanisms which maintain P. falciparum Ca2+ homeostasis.In conclusion, we generated a transgenic Thapsigargin (TG), cyclopiazonic acid (CPA), calcium ionophore A23187, 2,5-Di-t-butyl-1,4-butylhydroquinone (BHQ), and dimethyl sulfoxide (DMSO) were purchased from Sigma Aldrich Chemical Co . Dihydroartemisinin (dART) was purchased from Tokyo chemical industry Co . Stock solutions of all drugs were dissolved in DMSO.P. falciparum transfection were constructed based on the Invitrogen Multisite Gateway® system . DNA fragments encoding YC-Nano15 and -50 were amplified from corresponding plasmid templates by PCR amplification and recombined with pDONR™P2R-P3 to generate pENT23-YC-Nano15 and -50, respectively. Expression vectors, pLN-YC-Nano15 and 50  containing a blasticidin-s deaminase (BSD) selectable markerPlasmids for 5 and 50 , were geP. falciparum Dd2 parasite line was originally obtained from National Institute of Health, USA. The parasites were maintained with O+ human RBC at 2% hematocrit in fibrinogen-free human plasma-containing complete RPMI medium and transfection was performed as describedThe P. falciparum parasites (3–6% parasitemia) were used for live cell imaging experiments. Ca2+ measurements were performed using trophozoite parasites which were obtained by 5% sorbitol synchronization before 18–24 hr experimentationTM TC treated hydrophilic coated dish . After keeping the iRBC in the dish for 30 min, ICM were replaced with phenol red- and plasma-free RPMI medium containing 0.5% AlbuMAX® I. Time-lapse imaging was performed at 37 °C using an A1R confocal microscope system configured with an inverted microscope  with 60× or 100× oil objective lens . The inverted microscope configuration acts as a stable system with the Perfect Focus System . The water chamber stage and the objective lens were kept at 37 °C with a temperature controller . The fluorescence resonance energy transfer (FRET) image analysis between cyan fluorescent protein (CFP) and yellow fluorescent protein (YFP) was performed by confocal microscopy. YC-Nano was excited at 457 nm for both CFP and YFP, and emissions were detected for CFP (482/35 nm) and YFP (525/50 nm). Time-lapse images were captured every 1 sec at a 512 × 512 pixel resolution by confocal microscopy. For the first 30 sec, time-lapse images were taken without chemical compounds. Chemical compounds  were added directly to the edge of the chamber containing transgenic parasites. The parasite cytosolic region was used for the analysis as a region of interest (ROI) and background fluorescence was subtracted. The imaging analysis was carried out using NIS-Element Advanced Research imaging software (Nikon). The R/R0 value was calculated for each parasite, where R is the YFP/CFP ratio and R0 is the mean YFP/CFP ratio before adding the drug as baseline (time between 0–30 s).YC-Nano-expressing 2+ free Tyrode’s buffer  to remove Ca2+ from the extracellular medium. Finally, Ca2+ free Tyrode’s buffer was used for time lapse imaging.To selectively permeabilize the RBC membrane only, iRBC were treated with 20 U/ml streptolysin O  in PBS for 6 min at room temperature, washed three times with ICM, and kept in ICM2+  were prepared with calcium chloride. Parasites were re-suspended in the different concentrations of Ca2+-containing buffer, kept for 10 min, and observed under the confocal microscope. Images were obtained from 10 independent parasites for each Ca2+ concentration and the fractional change of the YFP to CFP ratio (ΔR/R0 where ΔR = R − R0) was calculated. The ΔR/R0 values were normalized by dividing by the ΔR/R0 value with 500 nM Ca2+ buffer and plotted using GraphPad Prism6 software .To generate the calibration curve, iRBCs were permeabilized with saponin and prepared for analysis as described above. Tyrode’s buffer containing different concentration of Ca2+ concentration in the different stages of P. falciparum, we employed an in situ Ca2+ calibration method with time course change of Ca2+ concentration in the individual intact iRBC, which is more precise than single images of the parasite2+Kd is a dissociation constant  or with 10 mM CaCl2 and 10 μM A23187 (Saturated Ca2+ condition), respectively. Fmin and Fmax are the fluorescence intensities at 458 nm excitation at the moment of Rmin or Rmax. Obtained Rmin, Rmax, Fmin, and Fmax values were 1.36, 1233, 2.66, and 503, respectively. The different stages of P. falciparum were assayed in plasma-free RPMI medium containing 0.5% AlbuMAX® I. The acquisition of FRET images and the calculation of the resting cytosolic Ca2+ concentration were performed using NIS-Element Advanced Research imaging software (Nikon) with the above formula. For example, Ca2+ concentration of one schizont was calculated as 345.6 nM using above equation from YFP/CFP value of 2.34. Obtained Ca2+ concentration using Grynkiewicz equation with above condition was limited to 1000 nM to avoid unacceptably large fluctuations by NIS-Element Advanced Research imaging software.To estimate the resting cytosolic CaTM, Beckman Coulter, Inc., Brea, CA). Before assay parasite-iRBCs were washed twice in phenol red- and plasma-free RPMI medium containing 0.5% AlbuMAX® I. To measure CFP and FRET signals, iRBC were excited with a 405 nm laser and fluorescence was collected in the CFP channel with a standard 450/50 filter, while the FRET signal was measured with a 525/20 filter. To measure YFP signal, parasite-iRBCs were excited with a 488 nm laser and emission was taken with 525/20 filter. For each sample a minimum of five thousand YFP positive iRBCs were evaluated and non-infected RBCs were used as a baseline for signal detection. To measure the changes of cytosolic Ca2+, baseline Ca2+ levels were first measured for 60 sec for each sample, followed by addition of different Ca2+ inhibitors  for 3 min. FlowJo software  was used to analyze the obtained data. The mean fluorescence intensities of the iRBC before and after adding inhibitors were obtained and the Rpost ratio of iRBC after adding inhibitors were normalized by Rpre of iRBC before adding inhibitors to obtain the FRET signal changes of each inhibitor.The FRET signal of parasites was measured by flow cytometry , assay to determine IC50 using a protocol available at WorldWide Antimalarial Resistance Network .http://www.rcsb.org; 2AGV). A model structure of human SERCA and PfSERCA were generated by a homology modeling based on the rabbit SERCA structure using Modeller9.14  http://www.pymol.org). Binding free energies of TG (PubChem CID: 446378) and dART (PubChem CID: 71939-50-9) with human/PfSERCA were estimated by docking simulations using AutoDock4.26 energy evaluations were performed. Other model structures of TG and selected 144 amino acid residues located near the binding region were constructed by 2000 steps energy minimizations using AMBER99SB force fieldThe coordinates of the crystal structure of complex between rabbit SERCA and TG was downloaded from the Protein Data Bank .How to cite this article: Pandey, K. et al. Ca2+ monitoring in Plasmodium falciparum using the yellow cameleon-Nano biosensor. Sci. Rep.6, 23454; doi: 10.1038/srep23454 (2016).


The Ca2+ entering from outside into the cell triggers Ca2+ release from the sarcoplasmic reticulum (SR) stores. To refill the depleted Ca2+ stores in the SR, another type of Ca2+ channels in the cell membrane, known as store‐operated Ca2+ channels (SOCC), are activated. These SOCCs are verapamil and nifedipine resistant, but are SKF 96465 (SK) and gadolinium (Gd3+) sensitive. Both SK and Gd3+ have been shown to reduce [Ca2+]i in the smooth muscle, but their effects on blood pressure have not been reported. Our results demonstrated that both SK and Gd3+ produced a dose‐dependent reduction in blood pressure in rat. The combination of SK and verapamil produced an additive action in lowering the blood pressure. Furthermore, SK, but not Gd3+ suppressed proliferation of vascular smooth muscle cells in the absence or presence of lysophosphatidic acid (LPA). SK decreased the elevation of [Ca2+]i induced by LPA, endothelin‐1 (ET‐1) and angiotensin II (Ang II), but did not affect the norepinephrine (NE)‐evoked increase in [Ca2+]i. On the other hand, Gd3+ inhibited the LPA and Ang II induced change in [Ca2+]i, but had no effect on the ET‐1 and NE induced increase in [Ca2+]i. The combination of verapamil and SK abolished the LPA‐ or adenosine‐5′‐triphosphate (ATP)‐induced [Ca2+]i augmentation. These results suggest that SOCC inhibitors, like VOCC blocker, may serve as promising drugs for the treatment of hypertension.The voltage‐operated Ca In view of the fact that hypertension is associated with an increase in smooth muscle cell proliferation and [Ca2+]i3+ on blood pressure, cell proliferation and [Ca2+]i. The specificity of the effect of SK was also examined by using various agents such as Ang II, ET‐1, NE and LPA, which are known to increase the [Ca2+]i and blood pressure. Some experiments were carried out to investigate the effect of SOCC blocker, SK in combination with VOCC antagonist, verapamil on blood pressure and [Ca2+]i.In view of the effect of different vasoactive agents such as Ang II, NE, ET‐1 and LPA on blood pressure 3H] ‐ thymidine was purchased from Amersham . DMEM and foetal bovine serum (FBS) were obtained from Invitrogen , whereas the A10 VSMC line was from the American Type Culture Collection .The use of animals and experimental protocol were according to the guidelines of the Canadian Council of Animal Care as approved by the Animal Care Committee of the University of Manitoba. SKF 96365 was purchased from Biomol Research Laboratory . Gadolinium, L‐α‐LPA and verapamil were obtained from Sigma Chemicals ; Fura‐2 acetoxymenthyl/ester (Fura 2‐AM) was purchased from Molecular Probes ; ‐thymidine incorporation into the DNA of the cells 3H]‐thymidine was added. The reaction was terminated after 22 hrs by keeping the cell culture plates on the ice and removing the culture medium. The cells were washed three times with 1 ml HEPES buffer . These cells were then incubated for 1 hr in cold 5% trichloroacetic acid on the ice, washed two more times with 0.5 ml HEPES buffer, and incubated with 0.2 ml NaOH (0.5 N) for 1 hr. The aliquots were transferred to scintillation vial. The radioactivity was counted in a Beckman LS 6500 scintillation counter after the addition of 10 ml Cytoscint‐ES .A10 VSMC were cultured in DMEM containing 10% FBS and 0.01 mg/ml gentamicin  at 37°C with 95% air and 5% CO6 cells/ml by adding HEPES buffer. The fluorescence intensity of Fura‐2 was determined by a SLM DMX–1100 dual‐wavelength spectrofluorometer ; the ratio (R) of fluorescence signal at 340/380 (nM) was calculated automatically. The Rmax and Rmin values were determined by the addition of 40 μl Triton X‐100 (10%) and 20 μl EGTA (40 mM) to a cuvette with 2 ml cell suspension respectively. The [Ca2+]i was calculated according to the following formula: [Ca2+]i = 224 × [(R − Rmin)/(Rmax − R)] × Sf2/Sb2, where Sf2 and Sb2 are the fluorescence proportionality coefficients obtained at 380 nm under Rmin and Rmax conditions respectively The cultured A10 VSMC were incubated with 0.25% trypsin‐1 mM EDTA for 2 min. and then the cells were harvested and centrifuged at 240 × g for 5 min. at room temperature. The supernatant was discarded and the cells were incubated with 10 μM Fura 2‐AM in HEPES buffer for 40 min. at 37°C. The cells were then washed twice with HEPES buffer and the cell number was adjusted to 0.3 × 10anova analysis, the comparison of mean values of the two groups was performed by Student's t‐test. P values less than 0.05 were considered to be significantly different.The data are expressed as mean ± S.E.M. Statistical analysis was performed with the Microcal Origin Version 6 . The data analysis was carried out by one‐way 3+ intravenously. The blood pressure was monitored before and after treatment. In preliminary experiments, both SK (45–450 μg/100 g b.w.) and Gd3+ (16–160 μg/100 g b.w.) were found to lower blood pressure in a dose‐dependent manner. As shown in Figure 3+, Figures 3+, Figures 3+ treatment.The effects of SOCC blockers on blood pressure in rats were tested by injecting different doses of SK or GdIn view of the importance of LPA in the development of hypertension 3+ treatment had no significant effect on cell number. To confirm this finding, [3H] thymidine incorporation, an index of DNA synthesis and cell proliferation, was examined in cultured A10 VSMC. As shown in Figure 3H] thymidine incorporation with or without LPA was significantly inhibited by SK treatment, meanwhile Gd3+ had no significant effect on [3H] thymidine incorporation.The ratio of arterial lumen and wall thickness is an essential factor for the regulation of blood pressure 2+ concentration is a key factor for the control of vascular tone as well as cell proliferation 3+ on the various vasoactive agonists ‐evoked changes in [Ca2+]i were examined in cultured A10 VSMC. Figure 3+ had no significant effect on basal [Ca2+]i; however, LPA‐induced increase in [Ca2+]i was inhibited by both SK and Gd3+ in a concentration‐dependent manner. But these antagonists had no significant effect on NE‐induced increase in [Ca2+]i  and Ca2+ release from intracellular store 2+]i , a pore forming protein  and a transient receptor (which regulates the function of SOCC) 2+ influx in VSMC 3+ on cell proliferation and [Ca2+]i observed in this study may be as a result of their effects on different targeting sites in SOCC. Because the expression of the transient receptor component of SOCC is up‐regulated in the vasculature of hypertensive rats It has been shown that SOCCs play an important role in mitogenic response to growth factor in VSMC 3+ have been shown to block Ca2+ influx through SOCC in VSMC and their inhibitory effects are agonist and cell‐type dependent. For instance, in arteriolar smooth muscle cells 3+ produced a concentration‐dependent inhibition of SOCC‐mediated Ca2+ entry; however, the effects of SK on cyclopiazonic acid and thapsigargin depletion‐induced SOCC activation were different. In our study, both SK and Gd3+ inhibited LPA‐induced alteration of [Ca2+]i as observed previously 2+]i, induced by NE. This is because NE evoked the increase in [Ca2+]i mainly by cell membrane depolarization 2+]i through SOCC which was blocked by SK 3+ produced no effect on ET‐1‐induced [Ca2+]i change in our experiments, indicating the distinct effect of these two blockers on SOCC in VSMC. Taken together, LPA, Ang II, NE and ET‐1, were all observed to increase in [Ca2+]i, but the mechanisms seemed to be different.It is noteworthy that both SK and Gd2+]i increase in VSMC. Other investigators have reported an additive inhibitory effect on ET‐1‐induced vasoconstriction in cerebral arteries by using SK and nifedipine (VOCC blocker) 3+ on SOCC. In this regard, it has been reported that these two agents in higher concentrations may also inhibit ROCC and other Ca2+ channels We have shown that both SK and verapamil, when used in combination, produced a synergistic effect on LPA‐ and ATP‐induced [CaThe authors declare no conflict of interest with any grant funding agency.


These Ca2+-signals were completely inhibited by removal of extracellular Ca2+, confirming their dependence on influx of extracellular Ca2+. The 4-αPDD Ca2+-response recorded in the presence of cyclopiazonic acid (CPA), which depletes the intracellular stores preventing any signal amplification through store release, was used as a measure of Ca2+-influx across the cell membrane. This response was blocked by HC067047, a TRPV4 antagonist. Under voltage clamp conditions, the TRPV4 agonist GSK1016790A stimulated a membrane current, which was again inhibited by HC067047. Following incubation with 25mM D-glucose TRPV4 expression was reduced in comparison with RMECs cultured under control conditions, as were 4αPDD-induced Ca2+-responses in the presence of CPA and ion currents evoked by GSK1016790A. Molecular expression of TRPV4 in the retinal vascular endothelium of 3 months’ streptozotocin-induced diabetic rats was also reduced in comparison with that in age-matched controls. We conclude that hyperglycaemia and diabetes reduce the molecular and functional expression of TRPV4 channels in retinal microvascular endothelial cells. These changes may contribute to diabetes induced endothelial dysfunction and retinopathy.Retinal endothelial cell dysfunction is believed to play a key role in the etiology and pathogenesis of diabetic retinopathy. Numerous studies have shown that TRPV4 channels are critically involved in maintaining normal endothelial cell function. In the current paper, we demonstrate that TRPV4 is functionally expressed in the endothelium of the retinal microcirculation and that both channel expression and activity is downregulated by hyperglycaemia. Quantitative PCR and immunostaining demonstrated molecular expression of TRPV4 in cultured bovine retinal microvascular endothelial cells (RMECs). Functional TRPV4 activity was assessed in cultured RMECs from endothelial Ca2+-responses recorded using fura-2 microfluorimetry and electrophysiological recordings of membrane currents. The TRPV4 agonist 4α-phorbol 12,13-didecanoate (4-αPDD) increased [Ca Drugs were dissolved in dimethyl sulfoxide (DMSO). Superfusion with DMSO alone at the maximum bath concentration used had no effect. Summary data for each protocol represents at least 5 separate cells from at least 3 isolations.RMECs, plated on 1% gelatin-coated coverslips (zero thickness), were incubated with the membrane permeable CaRMECs were seeded onto 35mm culture dishes for 72 hours before performing recordings. These were placed on the stage of an inverted microscope and perfused with the relevant solution at 33°C. Membrane currents were recorded using the whole cell voltage-clamp technique, with an Axopatch 200B amplifier , and digitized with an analog-to-digital converter . Data were collected, digitized and analysed using pCLAMP software . Pipette resistances were 3-5MΩ and mean access resistance was 14.9±0.8MΩ. Currents were normalized to cell capacitance (mean cell capacitance was 38.1±1.7pF). The junction potential for the bath and pipette solutions used was measured at 11mV and corrected offline. Drugs were dissolved in DMSO, which had no effect on membrane currents at -80mV and +80mV at the maximum concentration used.2+-microfluorimetry experiments was (mM): NaCl, 140; KCl, 6; D-glucose, 5; CaCl2, 2; MgCl2, 1.3; HEPES, 10; pH set to 7.4 with NaOH. Low Ca2+ Hanks’ solution contained no added Ca2+ and 1 mM EGTA. External bath solution for electrophysiological recordings was (mM): NaCl, 150; CsCl, 6; CaCl2, 1.5; MgCl2, 1; D-glucose, 5; HEPES, 10; adjusted to pH 7.4 with Tris. The pipette solution was (mM) Cs-aspartate, 100; CsCl, 20; BAPTA, 10; CaCl2, 0.08; Na2ATP, 4; MgCl2, 1; HEPES, 10; adjusted to pH 7.2 with Tris. Stock solutions of 4α-phorbol 12,13-didecanoate (4α-PDD) and GSK1016790A (Sigma-Aldrich), and HC067047  were made up in DMSO and dissolved in bath solution to give the required final drug concentration.The composition of the external Hanks’ solution used for CaSummary data have been presented as mean±standard error of the mean (SEM) throughout. The statistical significance of apparent differences was tested using paired or unpaired t-tests or one-way Analysis of Variance with a Bonferroni post-hoc test corrected for multiple comparisons, with significance set at P<0.05.We began by confirming the expression of TRPV4 at the mRNA  and prot2+] ([Ca2+]i). In 8 similar experiments the mean value of R340/380 was increased by 0.20±0.03, from a baseline of 1.17±0.02 to 1.37±0.02 . The specificity of this response was tested by transfecting RMECs with siRNA targeted against TRPV4. This reduced mRNA by approximately 60% when compared with untransfected cells and with those transfected with a negative control siRNA sequence i rise in the presence of CPA and the GSK1016790A/HC067047 sensitive currents were used as measures of functional expression of TRPV4 in the endothelial cell membrane in subsequent experiments.The amplitudes of the 4-αPDD evoked i evoked by 4-αPDD reflected activation of TRPV4 channels functionally expressed in the endothelial cell membrane. Cells grown in 25mM D-glucose produced no significant increase in [Ca2+]i during superfusion with 4-αPDD (Culturing RMECs for 72 hours in a solution containing 25mM of D-glucose reduced TRPV4 expression at both mRNA  and prot release , which mh 4-αPDD . ResponsTRPV4 mediated whole-cell currents were used as an alternative measure of TRPV4 activity in the endothelial cell membrane. This was dramatically down-regulated following 72 hours incubation with 25mM D-glucose, with no statistically significant change in current following addition of GSK1016790A at either -80mV or +80mV . In contA final series of experiments was carried out to investigate the effects of diabetes on retinovascular TRPV4 expression in vivo. We made use of the STZ-rat model, with which we have considerable previous experience. Retinas2+-responses to 4-αPDD were almost completely abolished following siRNA knock-down of TRPV4, providing strong evidence of agonist specificity (2+, indicating that Ca2+-entry through channels in the cell membrane was a necessary step (2+, indicating that the TRPV4 Ca2+-signal was substantially dependent on store release. Although this protocol could not distinguish between direct activation of intracellular TRPV4 channels by 4-αPDD and signal amplification through Ca2+-induced Ca2+-releasei (2+ alone had little or no effect on [Ca2+]i (Functional TRPV4 expression in the cell membrane was also assessed directly by recording membrane ion currents . Consistactivator,40, stimactivator,28,41,42activator. TRPV4 hactivator,42. Sinc7°C[2+]i  and only7°C[2+]i . Limited [Ca2+]i . Most im2+]i-responses to 4-αPDD and GSK1016790A/HC067047 sensitive membrane currents (Endothelial expression of TRPV4 mRNA and protein were downregulated in RMECs grown under high glucose conditions  and in rcurrents . HyperosGiven their key role in endothelial signalling, downregulation of TRPV4 function might be expected to have significant vascular consequences. TRPV4 channels have been implicated in endothelium dependent vasodilatory responses to a wide range of stimuli, including increased flow and shear stress–19, agonIn summary, we have provided direct evidence of functional TRPV4 expression in the cell membrane of the retinal microvascular endothelium and demonstrated that this expression is downregulated both by hyperglycaemic culture conditions in vitro and by diabetes in vivo. Given that TRPV4 signalling has been demonstrated to play a role in several types of endothelium-dependent vasodilatation, it is tempting to speculate that loss of TRPV4 function may contribute to endothelial dysfunction in diabetes. The contribution of TRPV4 channel downregulation to the loss of endothelium-dependent vasodilatation and the pathogenesis of diabetic retinopathy now warrants further investigation.


We herein show that relatively short-lived unclassified reactive species  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.Non-equilibrium helium atmospheric-pressure plasma (He-APP), which allows for a strong non-equilibrium chemical reaction of O Plasma medicine is a rapidly emerging field, and a number of researchers have reported innovative applications of non-equilibrium atmospheric-pressure plasma (APP)123781112131415161232+), which play key roles in many cellular processes, such as endocytosis, exocytosis, intermediate metabolism, neurotransmission, muscle contraction, cell motility, and cell division2+]i) in mammalian cells is normally maintained at extremely low levels (~100 nM) compared with the extracellular calcium concentration ([Ca2+]i ~1 mM) and calcium concentration in the endoplasmic reticulum (ER) as an intracellular Ca2+ store. Temporary opening of calcium ion channels in the cell membrane or the ER membrane leads to a sharp rise in [Ca2+]i, resulting in the activation of various signal transduction pathways that regulate cell function. Some of these ion channels can open and close in response to extracellular mechanical, electrical, and chemical stimuli1920Our research focuses on cytoplasmic calcium ions  channels triggered by the plasma-produced reactive species. Numerous previous reports suggest that biological responses in cells exposed to plasma-irradiated solution are mediated through calcium signaling cascades. The results of the present study therefore enhance 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.*) in the plasma can greatly enhance the generation of reactive species such as ROS due to its high internal energy (~20 eV)111213141516172+]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 (Vp-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 , with or without 5.6 mM D-glucose. Typically, Vp-p = 5.0 kV, Lg = 23 mm, and dele = 38 mm. The parameter ti is defined as the duration of plasma irradiation, and tr 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 [Ca2+]i are measured using a confocal microscope  with a fluo-4 AM calcium indicator .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  in the solution after plasma irradiation for ti was estimated using a colorimetric staining probe . As shown in H2O2 increased linearly with ti. Only 2.9 μM H2O2 was generated in 3 mL of HBS after plasma irradiation for 10 s. This level of H2O2 reportedly causes minimal cytotoxicity and does not appreciably affect cellular proliferationA 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 Hti  in the HBS, direct addition of this level of H2O2 to HBS (H2O2 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 potential1234252O2 administration. As time proceeds, the extremely short-lived chemically reactive species apparently rapidly break down into short-lived and long-lived species such as H2O2.Total production of the extremely short-lived OH radical species in the solution after plasma irradiation for d [TA]ti . Althougti = 10 s) to cells in culture resulted in gradual and sometimes oscillatory increases in [Ca2+]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 [Ca2+]i  failed to induce any increase in [Ca2+]i (black line). These results suggest that chemically reactive species other than H2O2 in the plasma-irradiated HBS induced the increase in [Ca2+]i, possibly ROS, which could have been generated in the HBS as one of the initial reaction products , as shown in Administration of plasma-irradiated HBS  in the HBS , plays the predominant role in triggering changes in [Ca2+]i, although higher concentrations of H2O2 (30 μM) have been shown to induce increases in [Ca2+]i mediated through TRPA1 channels in other cell types2+]i responses induced by plasma-irradiated HBS were not observed in the present study in MCF-7 human breast adenocarcinoma cells (data not shown).Based upon these observations, we conclude that ROS in plasma-irradiated HBS, rather than H2+]i spike after addition of calcium-containing plasma-irradiated HBS , the characteristic [Ca2+]i spike was suppressed in cells treated with nominally calcium-free plasma-irradiated HBS ([Ca2+] = 0 mM) (black line). In addition, the [Ca2+]i responses evoked by plasma-irradiated HBS were completely blunted in the presence of ruthenium red (RR) and SKF-96365 (SKF), noncompetitive pan inhibitors of multiple TRP channels28292+]i responses even in the presence of RR (blue line) and SKF (green line). Thus, these results indicate that the observed [Ca2+]i increase following administration of plasma-irradiated HBS was mediated via the influx of extracellular Ca2+ and could not be attributed to calcium release from the ER. These results also strongly suggest that TRP channel(s) are involved in [Ca2+]i responses elicited by chemically reactive species produced in plasma-irradiated HBS.Although we observed a sharp [Ca2+]i responses elicited by plasma-irradiated HBS, HBS plasma irradiated for 1, 3, or 30 s was administrated to cells, and [Ca2+]i responses of individual cells were then carefully analyzed. Whereas administration of plasma-irradiated HBS (ti = 10 s) resulted in a significant increase in [Ca2+]i after a 70-s lag period, as shown in ti = 30 s) induced large, sharp spikes in [Ca2+]i (lasting for ~100 s), followed by prolonged periods (at least 10 min) of lower [Ca2+]i, although these lower levels were still significantly higher than the control (red line). As expected, (ti = 3 s) plasma-irradiated HBS required a much longer lag period (~100 s) before commencement of the significant increase in [Ca2+]i, although in this case, the [Ca2+]i peak was not as sharp as that observed with administration of (ti = 30 s) plasma-irradiated HBS (blue line).To explore the underlying mechanism of the [Ca2+]i responses elicited by administration of plasma-irradiated HBS were highly complex and appeared to involve a variety of ROS  generated in the HBS by differing intensities (periods) of plasma irradiation, we examined the [Ca2+]i responses at the single-cell level based on analyses of four parameters , as shown [Ca2+]i represents the maximum value of [Ca2+]i after addition of plasma-irradiated HBS; tdelay represents the time required to reach a level of 10% of the increase in [Ca2+]i after exposure to plasma-irradiated HBS; trise represents the interval between the time the 10% level in the increase in [Ca2+]i is attained and the time the 90% level in the increase in [Ca2+]i is attained; and tfall represents the interval between the time the 90% level in the increase in [Ca2+]i is attained and the time [Ca2+]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 ti in tiincreased, more cells exhibited a higher MAX[Ca2+]i, and the mean/median values for MAX[Ca2+]i increased , and the [Ca2+]i response was then carefully analyzed. As expected, the [Ca2+]i responses changed dramatically as the retention time after plasma-irradiation (tr) increased. As tr increased, the increase in [Ca2+]i was lower, and the time required to reach the maximal value increased . In addition, single-cell analyses of [Ca2+]i responses in the same manner as described above showed that most of cells exhibited lower MAX[Ca2+]i values with increasing tr, although some cells maintained a high MAX[Ca2+]i. Similarly, although both tdelay and trise tended to be longer for most of the cells, some cells maintained short tdelay and trise. Taken together, these observations demonstrate that the chemically reactive species in plasma-irradiated HBS that exhibit the highest potency in evoking [Ca2+]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 tr-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 [Ca2+]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 trise is strongly correlated with tfall   responsible for these [Ca2+]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.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 waves123+ influx  and 4 thannel(s) , possiblroblasts , 6, 7, w+ influx . The potadiation . Althoug2 and N2 in ambient air, resulting in the efficient generation of chemically reactive species including ozone/O332+]i responses evoked by plasma-irradiated HBS involve RR- and SKF-sensitive Ca2+-permeable channel(s) (212O2 (~30 μM), S-nitroso-N-acetyl-DL-penicillamine (SNAP), which is an NO donor, as well as by other inflammatory mediators. These [Ca2+]i responses are blunted by substitution of the redox-sensitive cysteine residues in TRPA1, which can be modified by oxidative covalent reaction 2+ mobilization from an oscillatory to a sustained elevated pattern via calcium release–activated calcium (CRAC)–mediated capacitive Ca2+ entry through Orai channel activated by stromal interaction molecule 1 (STIM1) on ER, which is also possibly involving TRP channel(s)352+ influx is clearly triggered through RR- and SKF-sensitive Ca2+-permeable channels, possibly TRP channel(s), secondary calcium dynamics (e.g. oscillatory increases in 372+]i responses in 3T3L1 cells are mediated via TRP channel(s)2O2) reactive species. As research indicates that TRP channels are promising drug targetsIn this study, we utilized non-equilibrium APP, which allows for a strong non-equilibrium chemical reaction of Oannel(s) , presumaPlasma medicine is a rapidly emerging field that combines plasma physics, life sciences, and clinical medicine with the goal of developing therapeutic applications for physical plasma1232 atmosphere. For observation, cells were re-plated onto glass-bottomed recording chambers and cultured for 1–3 additional days.Mouse 3T3-L1 fibroblasts (ATCC CL-173) and human breast adenocarcinoma MCF-7 cells (RBRC-RCB1904) were obtained from ATCC and RIKEN BioResource Center, respectively. 3T3-L1 fibroblasts were maintained in DMEM supplemented with 10% calf serum (CS), 100 U/mL penicillin, and 100 μg/mL streptomycin (growth medium) at 37 °C in an 8% COtr) after plasma irradiation, the plasma-irradiated HBS was added to cells.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 (2+]i were expressed as (F − F0)/F0, where F and F0 represent the fluorescence intensity of fluo 4 and the averaged fluorescence intensity of the dye before stimulation with plasma-irradiated HBS, respectively.The acetoxymethyl (AM) ester form of fluo-4  was dissolved in dimethyl sulfoxide (DMSO) at 5 mM. 3T3-L1 cells in a glass-bottomed recording chamber  were incubated in serum-free DMEM-HG containing 5 μM fluo 4-AM and 0.03% Cremophor-EL  for 30 min at 37 °C. The cells were then washed with HBS containing 5.6 mM D-glucose . Images How to cite this article: Sasaki, S. et al. Calcium influx through TRP channels induced by short-lived reactive species in plasma-irradiated solution. Sci. Rep.6, 25728; doi: 10.1038/srep25728 (2016).


In this review we summarize the concepts regarding the application of the “leukemia stem cell” theory to the development of CBF AML; we will analyze the studies investigating the leukemogenetic role of t and inv(16)/t, the proposed theories of its clonal evolution, and the role played by the hematopoietic niches in preserving the disease. Finally, we will discuss the clinical implications of stem cell modeling of CBF AML for the therapy of the disease.Even though clonally originated from a single cell, acute leukemia loses its homogeneity soon and presents at clinical diagnosis as a hierarchy of cells endowed with different functions, of which only a minority possesses the ability to recapitulate the disease. Due to their analogy to hematopoietic stem cells, these cells have been named “leukemia stem cells,” and are thought to be chiefly responsible for disease relapse and ultimate survival after chemotherapy. Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) is cytogenetically characterized by either the t or the inv(16)/t chromosomal abnormalities, which, although being pathognomonic, are not sufficient  This ultimately results in impaired hematopoietic differentiation and clonal expansion [ per se to determine overt leukemia, additional mutations act on these preleukemic clones, leading eventually to the emergence of the clinical disease.Core Binding Factor (CBF) Acute Myeloid Leukemia (AML) is cytogenetically defined by the presence of the t8;21) or the inv(16)(p13q22)/t [In fact, even though originated by the evolution of a single genetically alterated cell, CBF AML, as most acute leukemias and cancers, loses its original homogeneity soon after its initial clonal expansion  Figure . This isBased on their analogy to hematopoietic stem cells (HSC), that is, their role at the top of a hierarchical architecture, their immature characteristics, and their ability to recapitulate the disease with the morphological, immunophenotypic, and functional characteristics of the origin, these cells have been named “leukemia stem cells” (LSC) –5. Despi5 cells. Later studies with more immunodepressed models, that is, NOD/SCID/IL2rg−/− mice, have challenged these measures and proved that also CD34+CD38+ [Given their definition based on a function, these cells are best identified, retrospectively, by means of a model of serial xenotransplantation of the human disease in immunodepressed mice. This model was first established in 1994  by trans34+CD38+  and CD3434+CD38+ ; (2) the34+CD38+ ; (4) int34+CD38+ ; (5) L-IResearch studies following Lapidot et al. , howeverSubclonal genetic and functional heterogeneity is currently thought as a universal feature of cancer , even ifThe recent advances in high-throughput sequencing methodologies have enabled scientists to fully map the genome of leukemic cells and to serially track the development of the subclones constituting the disease. This allowed confirming the “LSC theory” and demonstrating how L-IC share a common gene expression profile (GEP) with HSC ; the pre per patient, with 5 of them in genes recurrently mutated in AML [These data also established AML as characterized by an average of 13 different mutationsd in AML . Computad in AML , 17, 18.In fact, the existence of a preleukemic phase of AML was suggested by a series of historical works on asymmetric X chromosome inactivation, highlighting the existence of a stage of clonal hematopoiesis in most cases of AML . This phThese observations are in line with the historical model of leukemogenesis proposed by Gilliland & Griffin as their “two-hit” theory : the fir per se to confer self-renewal ability and L-IC properties to committed hematopoietic progenitors (HPP), as evidenced by the capacity of experimentally engineered MLL+ committed granulocyte/macrophage progenitors (GMP) to serially transfer leukemia to secondary recipient mice [At the same time, the acquisition of L-IC function by the cells is not determined only by genetics, but it is rather the result of complex and still poorly clarified interactions between genetics, epigenetics, and interactions with the microenvironment. For instance, the fact that L-IC showed a CD34+/CD38− immature immunophenotype, similar to that of normal HSC, initially led to hypothesize that driver mutations leading to AML could only happen in HSC. Later evidence, though, showed how mixed lineage leukemia (MLL) fusion proteins, produced by translocations involving chromosome 11q23, are sufficientent mice . In factThus, at present at least three scenarios have been hypothesized to describe the early phases of leukemogenesis , 31 Fig. In the This model of leukemogenesis hypothesizes possibly different effects by the same mutation when occurring in HSC or in early committed HPP. According to this model, quiescence and multipotency of HSC would endow their leukemic counterparts with an intrinsic better resistance to chemotherapy and allow the persistence of a larger number of L-IC after induction therapy. This could explain why a “stemness-related” GEP is prognostic for most cases of AML . On the α subunit  or β subunit (CBFB) die during embryogenesis without the development of HSC in the aorta-gonad-mesonephros (AGM) region [ Nematodes to humans [CBF as an heterodimer is essential for the development and homeostasis of definitive hematopoiesis , 34; emb) region , 36. RUNo humans , 37, whio humans . In a reo humans .RUNX1-RUNX1T1 (formerly AML-ETO) lacks the RUNX1 transcription activation domain and as such acts as a dominant repressor for many RUNX1-responsive hematopoietic genes , 37. CBFRecently, an experimental mouse model has been developed  in whichOn the opposite, an alternatively spliced isoform of the RUNX1-RUNX1T1/AML1-ETO transcript, AML1-ETO9a, which includes an extra exon (9a) of the ETO gene, leads to the rapid development of acute leukemia in a mouse retroviral transduction-transplantation model . DiffereIn any case, the preleukemic clone originating from CBF translocations is primed for a second hit by the direct effects of both RUNX1-RUNX1T1  and CBFBCancer is a complex microenvironment, in which intrinsic  as well as extrinsic determinants  cooperate to determine overall clinical malignancy .In vivo, the hematopoietic niches  are involved in HSC physiology as primary extrinsic determinants  expression by leukemic blasts [ in vivo and probably the chance to evolve as granulocytic sarcoma.It is highly likely that these physiological properties of the hematopoietic niches are used by AML L-IC in their favour. In fact, AML L-IC, identified by their immunophenotype, have been maintained long-termC niches . MoreoveC niches , 57, whiC niches . At the C niches . This abC niches . At the C niches , thus imC niches ; a fractC niches . APP is c blasts . We beliBesides their role in influencing stem cell properties, there are other ways by which hematopoietic niches might reduce chemosensitivity of leukemic blasts: for example, by inducing quiescence, thus preserving leukemic cells from cycle-dependent drugs ; by chemCBF AML patients have consistently showed enhanced survival after intensive chemotherapy, with long-term OS approaching 60% . Even thAs such, the biological basis explaining the intrinsic chemosensitivity of CBF AML, especially during consolidation therapy, remains still unclarified. As L-IC are thought to ultimately be responsible for leukemia relapse , it is p2 in both induction and consolidation therapy, has increased the OS chance in a randomized setting, without significantly increasing treatment mortality risk [Monoclonal antibodies are currently widely used in various fields of medicine to exert precise targeting of pivotal molecular pathways according to therapeutic needs. In the case of AML, CD33 has been chosen as a potential target based on its diffuse expression by myeloid progenitors and AML blasts but not by HSC. Gemtuzumab ozogamicin (GO), composed by a humanized monoclonal anti-CD33 antibody complexed with an antimitotic drug (Mitomycin), is the latest drug approved for clinical use and recently challenged in 3 wide European studies and one American trial . Differeity risk –85; on tity risk –85. Agaiity risk . GO is city risk . As advoity risk , 87, we Autologous hematopoietic stem cell transplantation (ASCT) has been tested intensively in AML treatment as a tool to enhance consolidation therapy; in most cases, these attempts have proven unsuccessful with regard to long-term OS. A notable exception is CBF AML, where ASCT used after consolidation as part of first-line therapy improves DFS and OS in several studies , 88–90;  momentum with Plerixafor.Once the bulk of disease is cleared by chemotherapy, a more specific treatment is needed to eradicate the disease by acting on residual L-IC surviving in protective hematopoietic niches. Monoclonal antibodies (mAb) may represent a potential approach to minimal residual disease eradication (reviewed in ). Among NCT01238211) and the German Acute Myeloid Leukemia Study Group (AMLSG) , while its use in the maintenance of patients achieving clinical remission with persistent molecular transcripts has recently failed to improve OS in another trial [Modern technologies are enabling at lower cost the complete genetic definition of AML patients. It is tantalizing to try and guess how this level of complexity will translate into the clinical practice: we can imagine a future where patients, following a debulking based on chemotherapy, will be treated individually on the basis of molecularly defined leukemia blueprints by a combination of molecular drugs. The results obtained by combining tyrosine-kinase inhibitors and chemotherapy in the treatment of Philadelphia-positive ALL provide an example of how the chance of cure can be ameliorated following a deeper molecular characterization of this disease . In the er trial .Often in science true progress comes from the contamination of ideas obtained from different fields of interest: the application of concepts derived from stem cell biology has already helped us to understand how leukemia originates and propagates; in the future, stem cell modeling of acute leukemia will lead us to a better way to treat the disease.


Recently, a new type of intercellular Ca2+ wave was observed in vitro in a linear arrangement of smooth muscle cells. The intercellular wave was denominated ultrafast Ca2+ wave and it was suggested to be the result of the interplay between membrane potential and Ca2+ dynamics which depended on influx of extracellular Ca2+, cell membrane depolarization and its intercel- lular propagation. In the present study we measured experimentally the conduction velocity of the membrane depolarization and performed simulations of the ultrafast Ca2+ wave along coupled smooth muscle cells. Numerical results reproduced a wide spectrum of experimental observations, including Ca2+ wave velocity, electrotonic membrane depolarization along the network, effects of inhibitors and independence of the Ca2+ wave speed on the intracellular stores. The numerical data also provided new physiological insights suggesting ranges of crucial model parameters that may be altered experimentally and that could significantly affect wave kinetics allowing the modulation of the wave characteristics experimentally. Numerical and experimental results supported the hypothesis that the propagation of membrane depolarization acts as an intercellular messenger mediating intercellular ultrafast Ca2+ waves in smooth muscle cells.Vascular smooth muscle cells exhibit intercellular Ca Nevertheless, Ca2+ and IP3 fluxes through gap junctions are small and thus, their passive diffusion should have a limited effect on Ca2+ mobilization at distant sites2+ waves, the propagation of an increase in intracellular Ca2+ concentration. Such intercellular Ca2+ waves have been induced in vitro by mechanical, electrical or chemical stimuli22+ wave as an electrically propagated wave5789102+ waves observed in arterial SMCs3 diffusion, and Ca2+ diffusion these models reproduced experimental observations like asynchronous Ca2+ flashings, recruitment of cells and vasomotion in absence of endothelium131415162+ wave and to investigate the particular conditions for intercellular ultrafast Ca2+ wave to occur as well as the properties of the membrane depolarization. Our study showed the direct interplay between the Ca2+ wave and the spreading of the membrane depolarization. We tested, discussed and demonstrated that an intercellular ultrafast Ca2+ wave is driven by the propagation of cell membrane depolarization and its speed is not dependent on the intracellular Ca2+ stores. Simulations predicted novel results and opened the field for further experimental studies to investigate the effect of electrical coupling and whole-cell conductance on Ca2+ wave velocity and on the propagation speed of membrane depolarization.Communication between vascular smooth muscle cells (SMCs) plays an important role in coordinating vascular function and compromised intercellular signaling may underlie pathological conditions. Continuous electrical and ionic movements take place between coupled cells which affect resting states and enable conduction of signals. Electrical current, inositol 1,4,5-trisphosphate IP and Ca2+2+], normalized by the steady state concentration before activation ([Ca2+]0), is depicted in 2+ increase and each cell reached a new steady state with an asymptotic [Ca2+] that decreased exponentially with the distance from the stimulated site. We measured a typical scale of 4,16 cells   cell.s−1.For the set of parameters corresponding to the numerical control case (see Methods), the time evolution of the  wave was not a trivial consequence of the absence of [Ca2+] increase of the stimulated cell, we designed a numerical experiment in which we imposed a [Ca2+] profile to the first cell , through the RyRs, did not substantially contribute to the mechanism of ultrafast Ca2+ wave propagation, nor to the rate and amplitude of the [Ca2+] increase. Nevertheless, in the case of a minimal CICR, this event would be subsequent to the onset of the [Ca2+] increase and then would not affect drastically the ultrafast Ca2+ wave velocity. We simulated experiments inhibiting each of the two intracellular Ca2+ sources by modifying F for the Ca2+ release from IP3 sensitive stores and C for the CICR mechanism , but did not suppress the propagation . This new experimental result supported the postulated hypothesis that the Ca2+ release through IP3Rs was not involved in the ultrafast Ca2+ wave mechanism. Altogether the experimental and theoretical results obtained by blocking the intracellular stores, demonstrated the hypothesis that the CICR did not contribute significantly to the mechanism of the ultrafast Ca2+ wave propagation nor did it to the fast Ca2+ increase process along the SMCs network.The other source of Cation see  nor the −1 (n = 3) for pSMCs and 94,1 ± 24 cell.s−1 (n = 4) for A7r5 and were not statistically different . Numerically, we also measured the conduction velocity by extracting the onset times at which the depolarization reached 50% of its maximal level. For the values of the coupling constant g and whole-cell conductance GCa under study, we confirmed the linear relationship between the speed of the ultrafast Ca2+ wave and the conduction speed of the membrane depolarization  or experimental conditions  in a region of the parameter space that may correspond to a situation closer to in vitro conditions.Our findings indicated that the propagation of membrane depolarization mediated by the electrical coupling between cells was at the origin of the observed ultrafast Ca2+ -activated chloride channels −1)2829302+ wave was most probably a consequence of the membrane depolarization spreading. Nonetheless, if a delay between the spreading of the depolarization and the initial entry of the Ca2+ through VOCCs was expected, it would be conserved for the whole network without affecting the velocities. Comparison between the velocities of the ultrafast Ca2+ wave and the membrane depolarization propagation revealed no statistical difference for both types of vascular SMCs. Altogether these results suggest that it is likely that the velocity of the ultrafast Ca2+ wave was determined by the propagation speed of the membrane depolarization followed by Ca2+ entry through VOCCs. Similarly to gastrointestinal electrical slow waves propagation2+ wave in cultured vascular SMCs was not limited by the diffusion of Ca2+ in relation to conduction by CICR. On the contrary, it was mediated by current spread that had more spatial influence compared to the diffusion of second messengers of Ca2+ or IP3. Moreover, the model predicted that the direct relation between those two phenomena held for a wide range of electrical coupling coefficient and whole cell conductance for VOCCs. This fact, may endorse new experimental approaches to explore the physiological consequences in the vasculature.In a tissue made of electrically unexcitable cells, a change in the membrane potential could electrotonically propagate intercellularly with an exponential decrease in amplitude over distance. This coupling can only extend a few millimeters but can be very rapid (a few milliseconds)2+ wave under various physiological conditions, including extreme physiological conditions where wave properties could only be extrapolated from experiments. Conceptually, the present approach represented a significant advance by combining new theoretical and new experimental data for both Ca2+ and membrane potential dynamics. Numerically, we found that the ultrafast Ca2+ was not regenerated, it had a velocity and a spatial range in agreement with experiments. We validated the electrotonic behavior of the membrane depolarization spread and measured both in experiments and simulations the propagation speed. The model served also as a support for the experimental findings in which the intracellular Ca2+ sources were not involved in the generation of ultrafast Ca2+ wave. In our model, VOCCs were necessary for Ca2+ entry after depolarization. This Ca2+ influx event progressively propagated along the SMCs network with an intensity depending on the amplitude of the membrane depolarization. Importantly, the model predicted that a membrane depolarization was still present even when the VOCCs were inactive. It also allowed us to estimate valid ranges of critical parameters that are challenging to measure experimentally, such as electrical coupling coefficient and whole-cell conductance for VOCCs. Finally, the model reproduced, validated and allowed a better understanding of recently reported experimental data. In the context of the vascular system, analysis and modeling of SMCs mechanisms should ultimately provide new insights into the dynamic coordination of vasculature and thereby contribute to the regulation of tissue perfusion.In this work we provided an accurate framework to investigate the behavior of ultrafast Ca2+ fluoroscence and membrane depolarization measurements with microelectrode technics (The cell culture of primary SMCs (pSMCs) and rat aortic SM cell line (A7r5), pattern microfabrication, mechanical stimulation protocols , Ca2+ fltechnics , were pr3 receptors. 2-APB was first dissolved at 100 mM in DMSO, and then diluted to 30 μM in buffer solution. Cells were allowed to preincubate in 2-APB for 10 minutes.We used the drug 2-Aminoethoxy-diphenylborate (2-APB)  that blocks IPμM), PA (50 μM), Nifedipine (10 μM).Ryanodine . A custom Matlab program  controlled simultaneously the micromanipulator via the serial interface and the acquisition card, handling the synchronization of the mechanical stimulation and the recording of the running depolarization. Thus, performing twice the mechanical stimulation in the same cell with the same conditions and registering the changes in the membrane potential for two different cells within the cell network, it was possible to estimate the spreading velocity of the membrane depolarization. The spreading velocity is calculated by dividing the difference in the onset times of membrane depolarization for the two different membrane potential registrations (two different cells in the same network) by the separation distance between them. The crucial part that allowed the conduction velocity calculation was the development of a program that allowed us to have a recording system that controls both the moment of stimulation and the registration of electrical events.The experimental data presented here are expressed as means ± SEM (n). Statistical significance was tested using Student’s t-test on paired data; P < 0.05 was considered significant; n represents the number of experiments.2+] and the membrane potential dynamics in SMCs were taken from2+ increases2+ and the membrane potential dynamics are described by + and Cl− (vK and vCl) in + and Cl−, depolarizing the cell membrane of the stimulated SMC−1 to cells.s−1 assuming a typical length for a single SMC of 100–150 μm, estimated from the cells spatial configuration used in ref. The equations describing the cytosol Ca+ and the2+ dynamics in each SMC (i), is described by five variables: the Ca2+ concentration in the cytosol (ci); the Ca2+ concentration in the sarcoplasmic reticulum (si); the cell membrane potential (vi); the open-state probability of Ca2+ activated potassium channels (wi) and the IP3 concentration (Ii). Cells are connected to their nearest neighbors via gap-junctional electrical coupling.The Ca2+ dynamics and electrophysiology in a SMC is given by the following equations:The mathematical approach for modeling the CaThe JVOCC models the Ca2+ influx through VOCCs:+/Ca2+ exchange:2+ induced Ca2+ release (CICR):2+ extrusion from the SMCs by the Ca2+ -ATPase pumps:2+ release from IP3-sensitive stores:+-K+-ATPase:2+ -activated;+ efflux:2+ and the voltage activation of the K+ channels:3 degradation:back and SR uptake constant B values to avoid spontaneous oscillations and ensure the stability of the system during the stimulation.Model parameters were taken fromCa = 0,036195 μ.M.mV−1.s−1 and the value of the electrical coupling coefficient g = 1000 s−1. The latter corresponds to the gap-junctional conductance measured experimentally363738g is related to the gap junctional conductance G by g = G/Cm, where Cm is the cell membrane capacitance.For the control case we fixed the whole-cell conductance for VOCCs Gg from 10 to 5000 s−1 and GCa from 0,026 to 0,0525 μM.mV−1.s−1.To further investigate the effect these two crucial parameters on the wave, we used How to cite this article: Quijano, J. C. et al. Intercellular ultrafast Ca2+ wave in vascular smooth muscle cells: numerical and experimental study. Sci. Rep. 6, 31271; doi: 10.1038/srep31271 (2016).


Orai1 may associate with other proteins to form a signaling complex, which is essential for regulating a variety of physiological functions. In this study, we studied the possible interaction between Orai1 and large conductance Ca2+‐activated potassium channel (CBKa). Using RNA interference technique, we demonstrated that the SOCE and its associated membrane hyperpolarization were markedly suppressed after knockdown of Orai1 with a specific Orai1 siRNA in rat mesenteric artery smooth muscle. Moreover, isometric tension measurements showed that agonist‐induced vasocontraction was increased after Orai1 was knocked down or the tissue was incubated with CBKa blocker iberiotoxin. Coimmunoprecipitation data revealed that CBKa and Orai1 could reciprocally pull down each other. In situ proximity ligation assay further demonstrated that Orai1 and CBKa are in close proximity. Taken together, these results indicate that Orai1 physically associates with CBKa to form a signaling complex in the rat mesenteric artery smooth muscle. Ca2+ influx via Orai1 stimulates CBKa, leading to membrane hyperpolarization. This hyperpolarizing effect of Orai1‐CBKa coupling could contribute to reduce agonist‐induced membrane depolarization, therefore preventing excessive contraction of the rat mesenteric artery smooth muscle in response to contractile agonists.Orai1, a specific nonvoltage‐gated Ca Orai1 is widely expressed in many cell types, including vascular smooth muscle cells (VSMCs) and endothelial cells Beech . Functio2+‐activated potassium (KCa) channels in modulating SOCE  primary antibody was from Alomone Lab. Orai1 specific siRNA for rat was obtained from Invitrogen. The sequence is as follows: CAACAGCAAUCCGGAGCUU  and iberiotoxin (IbTX) were purchased from Sigma‐Aldrich. Thapsigargin (TG) and endothelin 1 (ET‐1) were obtained from Calbiochem. Anti‐Orai1 (sc‐74778) primary antibody was purchased from Santa Cruz. Anti‐BKμg/mL streptomycin for 5 to 7 days before experimental use. Cells were grown at 37°C in a 5% CO2 humidified incubator. The first passage was used in all experiments.All animal experiments were conducted in accordance with NIH publication no. 8523 and were approved by the Animal Experimentation Ethics Committee of Anhui Medical University. Primary cultured VSMCs were isolated from Sprague–Dawley rats. Briefly, mesenteric artery was dissected. After rubbing off endothelial layer, smooth muscle layer was peeled off and then digested with 0.2% collagenase type IA and 0.9% papain for 1 h. The dispersed VSMCs were cultured in Dulbecco modified Eagle's medium (DMEM) containing 10% fetal bovine serum (FBS), 100 U/mL penicillin and 100 4(3)] at 37°C for 10 min. Cells were incubated with/without IbTX (50 nmol/L) for 10 min, or with scrambled siRNA or Orai1 siRNA for 24 h. Cells were treated with 4 μmol/L TG in the dark for 8 to 15 min in a Ca2+‐free physiological saline solution (0Ca2+‐PSS), which contained (in mmol/L): 140 NaCl, 5 KCl, 1 MgCl2, 10 glucose, 0.2 EGTA, 5 Hepes, pH 7.4. SOCE was then initiated by applying 1 mmol/L extracellular Ca2+, resulting in a marked membrane hyperpolarization. Changes in fluorescence were measured by Nikon Diaphot inverted microscope.Membrane potential was measured as previously described . The membrane carrying the transferred proteins was incubated at 4°C overnight with the primary antibody at 1:250 dilution in PBST buffer containing 0.1% Tween 20 and 5% nonfat dry milk. Immunodetection was accomplished using horseradish peroxidase‐conjugated secondary antibody. Antibody binding was detected by the ECL system.Immunoprecipitation and immunoblots were performed as previously described , 25.2 NaHCO3, and 11.1 glucose) oxygenated with a gas mixture of 95% O2 and 5% CO2. The segments were mounted in a DMT myograph  under a normalized tension as previously described . After the washout, the rings were challenged with 1 μmol/L Phe to test their contractile responses and subsequently exposed to 1 μmol/L acetylcholine to verify endothelial integrity. The contractile response to Phe (10−7.5–10−5 mol/L) or ET‐1 (10‐10‐10‐8) were obtained by cumulatively adding agonists into the bath with or without the pretreatment of IbTX (50 μmol/L) for 10 min. For the Orai1 knockdown experiment, mesenteric artery was incubated with Orai1 siRNA or scrambled siRNA for 24 h before experiments.Mesenteric artery tension measurement was performed as previously reported  was measured as previously described  for 40 min in the dark at 37°C. Ca2+ stores were depleted by treating cells with 4 μmol/L TG for 10 min in 0Ca2+‐PSS. Ca2+ influx was initiated by applying 1 mmol/L extracellular Ca2+. Cells were pretreated with scrambled siRNA or Orai1 siRNA for 24 h before experiments. Fluorescence signal was recorded by Leica TCS SP5 confocal laser system. Changes in [Ca2+]i were displayed as the ratio of fluorescence relative to the intensity before applying extracellular Ca2+ (F1/F0).Cytosolic CaCa was detected by using in situ PLA kit Duolink , following the manufacturer's instructions. Briefly, VSMCs were freshly isolated from mesenteric arteries and attached on coverslips. The cells were fixed and permeabilized. After blocked with Duolink blocking solution, VSMCs were incubated with anti‐Orai1 and anti‐BKCa  antibodies overnight at 4°C in Duolink antibody diluent. Negative control slides were incubated with anti‐Orai1 antibody alone. Then cells were washed with physiological saline solution and incubated with Duolink secondary antibodies conjugated with oligonucleotides  in a preheated humidity chamber for 1 h at 37°C. Subsequently, cells were incubated with a ligation solution containing two oligonucleotides and one ligase. The oligonucleotides hybridize to the two PLA probes only if they are in close proximity (<40 nm separation), whereas the ligase joins the two hybridized oligonucleotides to form a close circle. Ligation of the oligonucleotides was followed by a rolling‐circle amplification reaction using the ligated circle as a template, resulting in a repeated sequence product. The amplification products were then detected by a fluorescence (Texas Red channel)‐labeled complementary oligonucleotide detection probes. Slides were mounted with Duolink mounting medium containing DAPI nuclear stain. PLA signals (red fluorescent dots) were visualized and imaged using a Leica TCS SP5 confocal microscope.Interaction of Orai1 with BKt test or two‐way analysis of variance (ANOVA) followed by the Bonferroni post hoc test when more than two treatments were compared. A value of P < 0.05 was considered statistically significant.Collected data were presented as means ± SE. The significance was analyzed using two‐tailed Student's μmol/L TG for 8–10 min in 0Ca2+‐PSS resulted in a rise in [Ca2+]i, which indicated the Ca2+ release from intracellular Ca2+ stores. Subsequent addition of extracellular Ca2+ (1 mmol/L) initiated SOCE   fluorescence . We subsequently used Orai1 siRNA to test the role of Orai1 in [CaThe effect of Orai1 siRNA on SOCE‐induced membrane hyperpolarization was then examined. Incubation of VSMCs with Orai1 siRNA 1:250) for 24 h significantly inhibited this hyperpolarization compared with the treatment of scrambled siRNA  this Orai1‐mediated membrane hyperpolarization was decreased by BKCa blocker; (3) inhibition of Orai1 activity by transfection with Orai1‐specific siRNA or preincubation of BKCa blocker markedly enhanced vasocontraction of rat mesenteric arteries in response to contractile agonists; (4) coimmunoprecipitation data revealed that anti‐Orai1 antibody could pull down BKCa, and anti‐BKCa antibody could inversely pull down Orai1;(5) PLA analysis showed that Orai1 and BKCa were physically interacted in VSMCs. Taken together, these results indicate that Orai1 physically interacts with BKCa to form a signaling complex in rat mesenteric artery VSMCs, and that Ca2+ influx via Orai1 activates BKCa, causing membrane hyperpolarization. Furthermore, this hyperpolarizing effect of Orai1‐BKCa coupling could contribute to suppress contractile agonist‐induced membrane depolarization, preventing excessive contraction of smooth muscle in response to contractile agonists.This study demonstrated that in rat mesenteric arteries, (1) SOCE was mediated by Orai1, and Ca2+ influx through Orai1 should result in a decrease in smooth muscle contraction, instead of contraction increase. However, in isometric tension experiments, our data showed that smooth muscle contractility was significantly enhanced after Orai1 proteins were knocked down. Therefore, we hypothesized that Ca2+ influx via Orai1 may activate BKCa, leading to a decrease of smooth muscle contraction. BKCa blocker was used to test this hypothesis. Our data showed that Orai1‐mediated membrane hyperpolarization was decreased by BKCa blocker, indicating that Orai1‐BKCa coupling plays an important functional role in regulating SOCE and its associated membrane hyperpolarization in VSMCs of rat mesenteric arteries. Our coimmunoprecipitation and PLA results further demonstrated that Orai1 and BKCa indeed have physical interaction, which would allow an efficient signal transduction between Orai1 and BKCa.Orai1 channel plays an essential role in SOCE. To elucidate the role of Orai1 in smooth muscle of rat mesenteric artery, Orai1 expression was knocked down using specific siRNA. Interestingly, Orai1 siRNA not only effectively suppressed Orai1 protein expression, but also reduced SOCE as well as membrane hyperpolarization. Presumably, suppressing CaCa. are abundantly expressed in many types of smooth muscle cells, we hypothesized that the membrane hyperpolarization initiated by Orai1‐BKCa coupling may inactivate voltage‐gated Ca2+ channels (VGCCs), which dominantly regulate Ca2+ influx in VSMCs in response to contractile agonists, thereby contribute to reduce vascular contraction. To test this hypothesis, endogenous contractile agonists Phe and ET‐1 were used, which bind to α1‐adrenoceptor and endothelin receptor and induce Ca2+ influx via VGCCs, resulting in membrane depolarization and vascular contraction of VSMCs. Additionally, the store Ca2+ release was induced by both agonists, which could initiate SOCE. According to the Orai1‐BKCa coupling model of this study, we suggest that this SOCE may exert its effect to hyperpolarize the plasma membrane and thereby reduce agonist‐mediated membrane depolarization and vasocontraction. Our isometric tension data showed that treatment of the smooth muscle with Orai1 siRNA or BKCa blocker increased vasocontraction to Phe, indicating that Orai1‐BKCa coupling is functionally involved in agonist‐induced contraction in VSMCs of rat mesenteric arteries.Since both Orai1 and BKCa channels could interact with nonvoltage‐gated Ca2+ channels to produce a signal transduction between these proteins. TRPC1‐BKCa coupling contributes to reduce membrane depolarization in response to agonist‐induced vascular contraction, thereby preventing excessive contraction of aortic smooth muscle cells (Kwan et al. Ca physically associates with Orai1 to mediate Ca2+ signaling, store refilling and migration in microglia (Ferreira and Schlichter Ca in VSMCs of rat mesenteric arteries to regulate muscle contraction. It is possible that live cell may use different signaling complexes such as TRPC1‐BKCa and Orai1‐BKCa to response to different agonists. However, even we demonstrated that Orai1 and BKCa form a signal complex to regulate vascular tone, the role of Orai1‐BKCa coupling in human disease still remained. The functional change in Orai1‐BKCa interaction may be linked with some vascular diseases, such as hypertension, diabetes, and atherosclerosis. Therefore, the future study will benefit the understanding of pathological relevance of Orai1‐BKCa coupling.Some evidence suggests that KCa to form a signaling complex in VSMCs of rat mesenteric arteries. Ca2+ influx via Orai1 activates BKCa, causing membrane hyperpolarization. This hyperpolarizing effect of Orai1‐BKCa could contribute to prevent excessive contraction of smooth muscle in response to contractile agonists.In conclusion, we verified that Orai1 physically interacted with BKNone declared.