Time-lapse fluorescence microscopy revealed that mdivi-1 blocked NMDA-induced mitochondrial fission but not that triggered by sustained AMPA receptor activation, showing that mdivi-1 inhibits excitotoxic mitochondrial fragmentation in a source specific manner. Similarly, mdivi-1 strongly reduced NMDA-triggered necrotic-like neuronal death and, to a lesser extent, AMPA-induced toxicity. Interestingly, neuroprotection provided by mdivi-1 against NMDA, but not AMPA, correlated with a reduction in cytosolic Ca2+ ([Ca2+]cyt) overload and calpain activation indicating additional cytoprotective mechanisms. Indeed, mdivi-1 depolarized mitochondrial membrane and depleted ER Ca2+ content, leading to attenuation of mitochondrial [Ca2+] increase and enhancement of the integrated stress response (ISR) during NMDA receptor activation. Finally, lentiviral knockdown of Drp1 did not rescue NMDA-induced mitochondrial fission and toxicity, indicating that neuroprotective activity of mdivi-1 is Drp1-independent. Together, these results suggest that mdivi-1 induces a Drp1-independent protective phenotype that prevents predominantly NMDA receptor-mediated excitotoxicity through the modulation of mitochondrial function and intracellular Ca2+ signaling.Excessive dynamin related protein 1 (Drp1)-triggered mitochondrial fission contributes to apoptosis under pathological conditions and therefore it has emerged as a promising therapeutic target. Mitochondrial division inhibitor 1 (mdivi-1) inhibits Drp1-dependent mitochondrial fission and is neuroprotective in several models of brain ischemia and neurodegeneration. However, mdivi-1 also modulates mitochondrial function and oxidative stress independently of Drp1, and consequently the mechanisms through which it protects against neuronal injury are more complex than previously foreseen. In this study, we have analyzed the effects of mdivi-1 on mitochondrial dynamics, Ca All experiments were conducted in accordance with the Directives of the European Union on animal ethics and welfare. All possible efforts were made to minimize animal suffering and the number of animals used.® medium, B-27 supplement, antibiotic-antimycotic, calcein acetoxymethyl ester , JC-1 and rhodamine 123 were purchased from Invitrogen . N-Methyl-D-aspartic acid (NMDA), mdivi-1, HBSS, glycine, poly-L-ornithine, glutamine, thapsigargin, tunycamicin, EGTA and FCCP were obtained from Sigma . α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA), kainate and cyclothiazide (CTZ) were obtained from Tocris Biosciences . Cytotox 96® for LDH release quantification was purchased from Promega . The plasmid expressing mitochondria-targeted Ca2+ indicator (2mtD4cpv) was kindly provided by Roger Tsien . Lentiviral particles carrying a Drp1-shRNA vector were obtained from Santa Cruz Biotechnology .Neurobasal® medium supplemented with B27, glutamine (2 mM) and antibiotic-antimycotic mixture, and seeded onto poly-L-ornithine-coated 48 well plates or glass coverslips (7 mm in diameter) at 1.5 × 105 cells per well. For confocal single cell imaging experiments, cells were plated onto glass-bottom μ-dishes . The medium was replaced by serum-free, supplemented Neurobasal® medium 24 h later. The cultures were essentially free of astrocytes and microglia and were maintained at 37°C and 5% CO2. Cultures were used at 8–10 days in vitro (DIV).Cortical neurons were obtained from the cortical lobes of E18 Sprague-Dawley rat embryos according to previously described procedures  according to the manufacturer’s instructions and plated and maintained as described above.For transfection of cells, 4 × 10Drp1 knockdown was carried out by lentiviral delivery of expression constructs encoding target-specific shRNA (Santa Cruz Biotechnology). Neurons were infected at 2 DIV following standard procedures and treated with puromycin (1 μg/ml) from 4 DIV to 7 DIV for selection of cells expressing shRNA. For imaging experiments infected neurons were plated onto 7 mm glass coverslips in 48-well plates. Cultures were used at 9 DIV. All the procedures with lentiviral particles were performed in a biosafety level 2 (BSL-2) laboratory.2+ and Mg2+-free HBSS containing 20 mM HEPES, 10 mM glucose, 10 μM glycine and 2.6 mM CaCl2 (incubation buffer) and z-stacks of the yellow fluorescent protein (YFP) were acquired through a 63× objective by inverted LCS SP2 or TCS SP8X confocal microscopes  at an acquisition rate of 1 stack/5 min during the indicated time period. To evaluate mitochondrial fission in neurons expressing 2mtD4cpv and lentiviral shRNA, neurons were fixed after treatment and YFP fluorescence was acquired through a Plan-Apochromat 20X/0.8 NA objective in an inverted widefield Zeiss Axio Observer microscope , equipped with an AxioCam MRm camera. After the time-lapse or cell fixation, number of cells with tubular and fragmented mitochondrial network was counted for data analysis.Neurons expressing mitochondria-targeted 2mtD4cpv were exposed to agonists in Ca2+]cyt were carried out as previously described  in which F represents the fluorescence value for a given time point and F0 represents the mean of the resting fluorescence level.Measurements of cyt, [Ca2+]mit and ∆Ψm, basal line-extracted area under curve was calculated from single cell imaging time-lapse curves. Normality tests were carried out using GraphPad Prism software, and Student’s t-test or Mann-Whitney’s U test were applied for parametric and nonparametric tests, respectively. Statistical significance was determined at p < 0.05.All data are expressed as mean ± SEM  before and during application of NMDA or AMPA/CTZ and cell viability was assessed 24 h later by calcein fluorescence analysis. NMDA at 30 μM and 100 μM induced a reduction in neuronal viability of 15.4 ± 1.82% and 29.1 ± 1.36% compared to control , that was attenuated by mdivi-1 to 3.9 ± 1.26% and 15.6 ± 2.1%, respectively Figure . On the Since calpains are major mediators of excitotoxic necrosis Wang,  we next 2+-activated proteases and mitochondrial dynamics influence Ca2+ signaling  were assessed by time-lapse fluorescence microscopy. Application of NMDA (30 μM), KA (100 μM) and AMPA (25 μM) induced a fast [Ca2+]cyt increase with a peak amplitude of 367.7 ± 8.4%, 304.2 ± 10.2% and 282.1 ± 9% compared with resting levels (100%) that was reduced by 50 μM mdivi-1 to 306.0 ± 8.2%, 164.5 ± 9.3% and 123 ± 3%, respectively  contribute to excitotoxicity  induced a fast [Ca2+]cyt peak of 336.5 ± 9.7% over basal levels (100%) that was attenuated to 247.3 ± 12.2% by 50 μM of mdivi-1 , exposed to NMDA or AMPA/CTZ and mitochondrial Ca2+ levels ([Ca2+]mit) assessed by time-lapse confocal microscopy. NMDA  induced a [Ca2+]mit peak increase of 216.9 ± 6.7% compared to resting levels (100%) that was attenuated to 179.4 ± 4.7% by 50 μM of mdivi-1  we compared thapsigargin-induced [Ca2+]cyt increase in control and mdivi-1-treated neurons. We observed that inhibition of SERCA pumps triggered a rise in [Ca2+]cyt of 162.2 ± 5.8% of resting levels (100%) that was diminished by 10 μM and 50 μM of mdivi-1 to 142.4 ± 4.2% and 115.7 ± 1.6%, respectively  from the ER. In summary, these results indicate that mdivi-1 regulates the [Ca2+]cyt rise after activation of ionotropic glutamate receptors and VGCCs while reducing mitochondrial Ca2+ overload and basal ER Ca2+ content.On the other hand, we previously reported that endoplasmic reticulum Cay Figure . Alternal Figure  but redue Figure , showing2+ homeostasis leads to the induction of the UPR , a fundamental component of the unfolded protein response (UPR) and the core of the ISR  mdivi-1 does not act as a calpain inhibitor; and (ii) that it may additionally protect against excitotoxicity independently of its effects on the early [Ca2+]cyt rise. Finally, since mdivi-1 failed at inhibiting AMPA/CTZ-induced mitochondrial fission, these results strongly suggest as well that the inhibition of NMDA-induced mitochondrial fission provided by mdivi-1 depends on its effects on [Ca2+]cyt overload during excitotoxicity, rather than to its direct effect on Drp1.Inhibition of calpain activation also correlated with the reduction of the NMDA-induced [Ca2+]ER (Foyouzi-Youssefi et al., 2+ crosstalk between ER and cytosol and its downregulation protects neurons against excitotoxicity (D’Orsi et al., 2+ levels are crucial for ER correct function and rapid and complete depletion of ER Ca2+ stores leads to ER stress and apoptosis (Verkhratsky and Petersen, 2+ was partially depleted by mdivi-1, with no significant evidence of ER stress induction and neuronal death. However, we found that mdivi-1 enhanced eIF2α phosphorylation in both basal and excitotoxic conditions. Phosphorylation of eIF2α inhibits global protein synthesis and triggers the ISR, a cytoprotective cellular mechanism that has been previously linked to neuroprotection during excitotoxicity (Sokka et al., Anti-apoptotic Bcl-2 protein reduces [Ca2+]mit and respiration. We observed that mdivi-1 reduced resting [Ca2+]mit, indicating that the lowered [Ca2+]ER was unlikely a consequence of an enhanced Ca2+ export into mitochondria. Importantly, mdivi-1 depolarized the mitochondrial membrane, which could in part explain the reduced matrix free [Ca2+], since Ca2+ accumulation inside the mitochondria is membrane-potential dependent (Drago et al., 2+ overload is reduced (Castilho et al., 2+]cyt signals triggered by NMDA, AMPA and KA. On the other hand, regardless of the extent of the [Ca2+]cyt increase, mdivi-1 attenuated the [Ca2+]mit rise triggered by both NMDA and AMPA/CTZ, an effect that can be reproduced by uncouplers and electron transport chain inhibitors and that attenuates excitotoxicity (Stout et al., 2+ overload during excitotoxicity.Another key finding of this study is that mdivi-1 lowers mitochondrial membrane potential, [Ca2+]cyt increase results in Drp1 activation that leads to mitochondrial fragmentation (Cribbs and Strack, 2+] and mitochondrial function during excitotoxicity are independent of Drp1 inhibition. This assumption was further supported by the lack of reduction of NMDA-induced mitochondrial fission and toxicity after lentiviral Drp1 knockdown. In agreement with these findings, previous studies reported that dominant mutant Drp1K38A was unable to inhibit mitochondrial fission and neuronal death triggered by necrotic glutamate stimuli (Young et al., [Ca2+ homeostasis disruption in neurons.Thus, further research is needed to reveal the molecular mechanisms involved in excitotoxic early mitochondrial fission, and exhaustive controls should be carried out when using mdivi-1 as a Drp1 inhibitor in conditions of intracellular Ca2+ signaling through Drp1-independent mechanisms (Figure 2+ overload and necrotic cell death, a feature that may expand its therapeutic potential for the treatment of brain diseases in which glutamate receptor overactivation is involved, such as ischemic brain injury and Alzheimer’s disease.In summary, we describe here that mdivi-1 strongly protects against NMDA-induced excitotoxicity by modulating mitochondrial function and intracellular Cas Figure . To our AR: designed and performed the experiments and drafted the manuscript. EA and CM: designed the project and experiments, helped with the interpretation of the results and critically revised the manuscript. All authors have read and approved of the manuscript.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.


The present study aimed to investigate whether scopolin exhibits beneficial effects on high-fat diet (HFD)-induced hepatic steatosis in mice. The involvement of sirtuin 1 (SIRT1) as a molecular target for scopolin was also explored. Scopolin decreased the Km of SIRT1 for p53 and nicotinamide adenine dinucleotide without altering Vmax in a cell-free system. Scopolin alleviated oleic acid-induced lipid accumulation and downregulation of SIRT1 activity in HepG2 cells, and these beneficial effects of scopolin were abolished in the presence of SIRT1 inhibitor. Mice administered 0.02% scopolin for 8 weeks exhibited improved phenotypes of HFD-induced hepatic steatosis along with increased hepatic SIRT1 activity and protein expression. Scopolin resulted in increased deacetylation of sterol regulatory element-binding protein 1c with subsequent downregulation of lipogenic genes, and enhanced deacetylation of protein peroxisome proliferator-activated receptor-γ coactivator 1α with upregulation of fatty acid oxidation genes in livers. Scopolin also enhanced deacetylation of nuclear factor-kappa enhancer binding protein and liver kinase B1 (LKB1), facilitating LKB1/AMP-activated protein kinase signaling cascades. Scopolin attenuated hepatic steatosis through activation of SIRT1-mediated signaling cascades, a potent regulator of lipid homeostasis. Increased hepatic SIRT1 activity and protein expression appeared to be associated with these beneficial effects of scopolin. SIRT1 has been shown to deacetylate many non-histone proteins in hepatocytes, including sterol regulatory element-binding protein 1c (SREBP1c)4, peroxisome proliferator-activated receptor-γ coactivator 1α (PGC-1α)5, liver kinase B1 (LKB1)6, and NF-κB8. Deacetylated SREBP1c transcription factor results in the reduced expression of lipogenic genes such as acetyl-CoA carboxylases (ACC) and fatty acid synthase (FAS)10, whereas deacetylation of PGC-1α enhances its function upregulating the transcription of carnitine palmitoyltransferase 1 (CPT1) needed for mitochondrial fatty acid oxidation13. Once LKB1 is deacetylated, it is translocated to the cytoplasm and then engaged in the phosphorylation of AMP-activated protein kinase (AMPK) which leads to both decreased lipogenesis via the mammalian target of rapamycin (mTOR)/ liver X receptor α (LXRα) signaling cascade and increased fatty acid oxidation via ACC phosphorylation14.Hepatic steatosis, defined as the accumulation of triglycerides (TGs) in hepatocytes, results from an imbalance between pathways leading to TG synthesis and catabolism. In hepatocytes with prolonged TG accretion, nuclear factor-kappa enhancer binding protein (NF-κB) that functions as a proinflammatory master switch is activated, leading to a subacute inflammatory response. These pathways involved in the regulation of hepatic lipid metabolism and inflammation share a common regulator, sirtuin 1 (SIRT1) that is a nicotinamide adenine dinucleotide 20. Furthermore, Rollinger et al.18 showed that scopolin isolated from S. carniolica Jaqc increased the extracellular acetylcholine concentration, which is responsible for regulating learning and memory, in rat brains up to 300% compared to the basal release, potentially reducing the risk of Alzheimer’s disease.Scopolin  along with other compounds such as scopoletin (0.38% w/w) and acetophenone glycoside (0.26% w/w) reversed high-fat diet (HFD)-induced abnormal increments in visceral adiposity, hepatic steatosis, and hyperlipidemia in mice15. A more recent study has demonstrated that scopoletin, a molecule lacking the glucopyranoside chain of scopolin, exhibited protective effects against ethanol-induced hepatic lipid accumulation in HFD-fed mice25. Nevertheless, the protective role of scopolin against adiposity and metabolic diseases has not yet been reported in in vitro or in vivo systems. The present study investigated whether scopolin isolated from A. iwayomogi exerts beneficial effects on HFD-induced hepatic steatosis in mice. We explored the potential involvement of SIRT1 as a molecular target for scopolin in the process of reducing hepatic lipid accumulation.Dietary intervention with several bioactive phytochemicals has been proven to be an effective approach to reducing the risk of hepatic steatosis1H-NMR (600 MHz) and 13C-NMR (150 MHz) on a Varian Gemini 2000 spectrometer . 1H-NMR (DMSO-d6), δ 7.97 , 7.29 , 7.15 , 5.09 , 5.06 , 3.79 , 3.47 , 3.43 , 3.33 , 3.18 , 2.52 ; 13C-NMR (DMSO-d6), δ 162.2 (C-2), 151.4 (C-7), 150.5 (C-9), 147.6 (C-6), 145.9 (C-4), 114.9 (C-3), 113.9 (C-10), 111.3 (C-5), 101.1 (C-1′), 78.6 (C-3′), 78.1 (C-5′), 74.5 (C-2′), 71.1 (C-4′), 62.1 (C-6′), 57.7 (OCH3). The structure of scopolin (C16H18O9) is shown in Fig. Isolated white amorphous scopolin powder was measured by A. iwayomogi ethanol extracts revealed that the scopolin yield was 0.97 ± 0.12% (w/w)  Fig. .Scopolin (100 μM) effectively reversed oleic acid-induced lipid accumulation in HepG2 cells, as shown by reduced Oil-Red O stained lipid accumulation, and this beneficial effect of scopolin was blocked in the presence of the SIRT1 inhibitor EX-527 Fig. . Next, tp < 0.05) without affecting food intake  of either acetylated peptide substrate or NAD+ was not affected by scopolin. Whereas, scopolin significantly lowered the Km of both acetylated peptide substrate (62% reduction) and NAD+ (68% reduction)  Fig. . These rSIRT1 deacetylase activity was measured in the nuclear extracts of liver tissues from animals fed experimental diets. HFD feeding for 8 weeks significantly decreased hepatic SIRT1 activity compared with that in ND-fed mice, while scopolin significantly restored HFD-induced downregulation of SIRT1 activity Fig. . ImmunobScopolin administration significantly reversed the HFD-induced downregulation of hepatic adiponectin receptor 2 (AdipoR2) and SIRT1 genes. mRNA expression of lipogenic genes, such as LXRα, ACC, LPL, and FAS, was significantly downregulated, whereas the expression of peroxisome proliferator-activated receptor alpha (PPARα) and CPT1, regulators of fatty acid oxidation, was significantly upregulated in the livers of SPD mice than in the HFD mice. Hepatic mRNA expression of the proinflammatory cytokines, MCP-1, TNFα, and IL-6 was all significantly downregulated by scopolin administration Fig. . Westernet al.26 and the US Food and Drug Administration . On the basis of our findings that the yield of scopolin isolated from dried A. iwayomogi extract was 0.97% in the present study, a daily human dose of 97 mg scopolin would correspond to approximately 9.9 g extract of A. iwayomogi. The toxicity of scopolin has yet to be reported. In rats, the no observed adverse effect level (NOAEL) value of orally administered A. iwayomogi Kitamura extract was reported as exceeding 2,000 mg/kg/day27, which is equivalent to 19.5 g/60-kg human/day. The absorption, pharmacokinetics, bioavailability, biodistribution, and metabolism of scopolin are unknown. A scopolin pharmacokinetics study in rats revealed that after a single oral administration of scopolin (100 mg/kg), the mean maximum plasma concentration of scopolin was 3.33 μM/ mL, the mean Tmax was 20 min, and the mean elimination half-life was 33 min16.In this study, the mice were fed the 0.02% scopolin-supplemented diet  which are corresponding to an intake of approximately 1.6 mg/kg body weight (97 mg/60-kg person), when calculated on the basis of normalization to body surface area as recommended by Reagan-Shaw 30. The latter is considered as an early event associating hepatic lipogenesis and inflammatory stress. Although NASH did not develop in our 8-week HFD-feeding protocol, the hepatic expression of pro-inflammatory cytokines could have facilitated the deterioration of steatosis to NASH if the experiment had been conducted for a longer duration.The present study found that scopolin significantly alleviated hepatic steatosis induced by HFD in mice, as demonstrated by the improved parameters of lipid droplets in liver tissues, hepatic lipid levels, and plasma AST and ALT activities, and adiponectin levels as well. These beneficial effects of scopolin on hepatic steatosis appear to be associated with increased deacetylation of the SREBP1c protein with subsequent downregulation of lipogenic genes , along with increased deacetylation of the PGC-1α protein with upregulation of genes involved in fatty acid oxidation . Furthermore, scopolin increased the deacetylation of a proinflammatory transcription factor, NF-κB, with subsequent downregulation of its target genes , in the absence of inflammatory phenotypes. HFD-induced fatty liver diseases can progress from simple steatosis to nonalcoholic steatohepatitis . It has repeatedly been observed by us, and by other investigators that mice fed the HFD for 10 weeks showed simple steatosis with the absence of necrosis or signs of inflammation. Moreover, HFD feeding for 10 weeks significantly elevated levels of serum ALT and AST, as well as the expression of hepatic pro-inflammatory cytokines such as TNF-α and MCP-1A. iwayomogi extract which is majorly composed of scopolin, reduced body weight gain and hepatic lipid levels in mice fed a high-fat diet15. At the same time, our in vitro observation that scopolin (100 µM) effectively reversed oleic acid-induced lipid accumulation in HepG2 cells raised the possibility that scopolin could reduce hepatic lipids independent of alterations in visceral adiposity  develop obesity and insulin resistance33 and exhibit increased lipid accumulation and inflammation in the liver and adipose tissues34. Complementary to these findings, transgenic mice with a 2–4-fold overexpression of SIRT1 show lower lipid-induced inflammation, better glucose tolerance, and protection from hepatic steatosis8. Recently, SIRT1 has also been shown to be a potential therapeutic target for treatment of fatty liver disease36. Liver-specific deletion of SIRT1 in mice markedly promotes the development of hepatic steatosis, independent of the presence of obesity, insulin resistance and inflammation37.SIRT1 is one of seven mammalian homologs of Sir2 that catalyzes NAD+ in a cell-free system. Scopolin decreased the Km of SIRT1 for p53 and NAD+ with no effect on the Vmax  was extracted using 50% (v/v) ethanol to obtain a dried ethanol extract of 0.7 kg (yield: 14%). This dried extract was suspended in water (1.5 L) and partitioned with chloroform (3 × 1.5 L) to obtain water-soluble (456.7 g) and water-insoluble (97.6 g) subfractions. The water-soluble fraction was further adsorbed onto a non-ionic porous resin  and desorbed with 30%, 60%, and 100% methanol. The 30% methanol fraction (92.2 g) was chromatographed on a Sephadex LH-20 gel (Sigma-Aldrich) column by using 40% methanol to yield five fractions (Fr 1–Fr 5). Fr 2 (24.1 g) was subjected to separation on an MCI CHP20P gel (Sigma-Aldrich) column by using 5% methanol to yield six fractions (Fr 1–Fr 6). Fr 2 was concentrated, yielding scopolin (4.15 g).The powder of A. iwayomogi extract, scopolin compound (0.1 mg) and dried 50% ethanol extract of A. iwayomogi (1 mg) were immersed in 1 mL aqueous 50% ethanol, and then filtered through a 0.2 µm membrane filter. HPLC analyses were performed using a Waters HPLC system  with an auto sampler. Chromatographic separation was achieved using a Sunfire C18 column  at 30 °C with a flow rate of 0.9 mL/min using a gradient mobile phase composed of 0.1% (v/v) aqueous formic acid (A) and acetonitrile (B) with a gradient elution condition of 85–84% A at 0–5 min, 84–72% A at 5–10 min, 72–69% A at 10–15 min, and 69–40% A at 15–20 min. The injection volume was 10 μL, and the detection wavelength was set at 330 nm.To identify the scopolin peak from the high-performance liquid chromatography (HPLC) chromatogram of 2-humidified incubator and grown in minimum essential medium supplemented with 10% fetal bovine serum, penicillin (100 U/mL) and streptomycin (100 μg/mL). Scopolin was dissolved in dimethyl sulfoxide (DMSO) at concentrations of 100 mM, and then added to the reaction mixture for a final concentration of 100 μM. To deposit lipids in HepG2 cells, the cells were incubated for 24 h with serum-free medium containing 1 mM oleic acid with fatty acid-free bovine serum albumin. Lipid accumulation in HepG2 cells was evaluated by Oil-Red O staining in the presence of the SIRT1 inhibitor EX527 (Sigma-Aldrich). Briefly, the cells were washed twice with phosphate-buffered saline , and then fixed with 2 mL 10% formalin solution in PBS for 1 h at room temperature. After two rinses with PBS, the cells were stained with 0.5% Oil-Red O (Sigma-Aldrich) in isopropanol for 30 min with gentle agitation. Cells were washed twice with distilled water and photographed under a light microscope. Lipids of the cells were extracted in 2 mL 100% isopropanol and their optical density values were measured in a spectrophotometer at 600 nm.HepG2 human hepatic carcinoma cells  were cultured at 37 °C in a 5% CO+ (500 µM) in PBS at 37 °C on a horizontal shaker for 45 min. The reaction was stopped by adding 2 mM nicotinamide and a developing solution that binds to deacetylated lysine to form a fluorophore. Following a 10-min incubation at 37 °C, fluorescence was read in a fluorescence microplate reader . For endogenous SIRT1 activity measurement, the assay was modified using nuclear extracts containing approximately 10 µg protein. The nuclear extracts from liver tissues or HepG2 cells were prepared by using the Nuclear Extract kit  according to the manufacturer’s instructions. Data for endogenous SIRT1 activation were normalized to protein concentrations in the nuclear extracts measured using Bradford assay reagents .SIRT1 activity was measured by using a SIRT1 Fluorometric Drug Discovery Kit . In this assay, SIRT1 activity is assessed by the degree of deacetylation of a standardized substrate containing an acetylated lysine side chain. The substrate utilized is a peptide containing amino acids 379–382 of human p53 (Arg-His-Lys-Lys [Ac]), an established target of SIRT1 activity; SIRT1 activity is directly proportional to the degree of deacetylation of Lys-382. For SIRT1 activator screening, scopolin (100 µM) was added to a human recombinant SIRT1 enzyme and incubated with the peptide substrate (25 µM) and NADad libitum. Thereafter, the mice were divided into three groups (n = 8/group) and fed one of three experimental diets: Normal diet (ND), HFD, and HFD supplemented with scopolin (SPD). The ND was a purified diet based on the AIN-76 rodent diet. The HFD was identical to the ND, except for the addition of 200 g fat/kg (170 g lard and 30 g corn oil) and 0.1% (w/w) cholesterol. The SPD was identical to the HFD, but also contained 0.02% (w/w) scopolin. All animals had access to diet and water ad libitum. Mice were kept on the treatment for 8 weeks. Weekly body weight and daily food intake were measured throughout the experimental period. At the end of the experiment, the mice were anaesthetized with 0.15 mL avertin  per 10 g body weight after a 12-hr fast. Blood was drawn from the abdominal aorta into an EDTA-coated tube, and plasma was obtained by centrifuging the blood at 2,000 × g for 15 min at 4 °C. The livers were excised, rinsed with PBS, and weighed, and a portion of each liver was fixed in 10% formalin for further analysis. Plasma and liver samples were stored at −80 °C until analysis.Five-week-old male C57BL/6 N mice weighing 18–20 g were obtained from Orient Bio . Mice were housed in a pathogen-free facility at Yonsei University  at room temperature with a 12-h light/dark cycle. All animal experiments were performed in accordance with the Korea Food and Drug Administration guidelines. All experimental protocols were reviewed and approved by the Institutional Animal Care and Use Committee of the Yonsei Laboratory Animal Research Center. During one week of acclimatization, animals were fed a commercial diet  and tap water et al.43 and re-dissolved in 2 mL ethanol. Hepatic TG, cholesterol, and fatty acid contents were determined by using commercial kits . Plasma activities of aspartate aminotransferase (AST) and alanine aminotransferase (ALT) were measured by using commercial kits . Plasma levels of adiponectin, monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor-alpha (TNFα), and IL-6 were determined by using ELISA kits .Liver tissues were homogenized, and total hepatic lipids were extracted as described by Folch Liver tissue specimens fixed in 10% buffered formalin were embedded in paraffin, cut into 5-μm-thick slices, and stained with hematoxylin and eosin (H&E) for histological examination of fat droplets. Steatosis and inflammation were numerically scored according to semi-quantitative pathological standards. Briefly, the pathological degree of steatosis was scored as follows: No steatosis = 0; minimal steatosis = 1; slight steatosis = 2; moderate steatosis = 3; marked steatosis = 4; severe steatosis = 5. In addition, the degree of lobular inflammation was scored as follows: No inflammation foci = 0; 1–2 inflammation foci = 1; 3–4 inflammation foci = 2; >4 inflammation foci = 3.Total mRNA from the liver samples or HepG2 cells was isolated using TRIzol reagent . RT-PCR was performed using a Superscript II kit (Invitrogen). Forward and reverse primer sets for target and internal marker genes are listed in Supplementary Table Liver tissues were lysed in Western lysis buffer consisting of 100 mM Tris-HCl (pH 7.4), 5 mM EDTA, 50 mM NaCl, 50 mM sodium pyrophosphate, 50 mM NaF, 100 mM orthovanadate, 1% Triton X-100, 1 mM phenylmethylsulfonyl fluoride, 2 μg/mL aprotinin, 1 μg/mL pepstatin A, and 1 μg/mL leupeptin. Samples were incubated on ice with frequent vortexing for 10 min and centrifuged for 20 min at 1,300 × g. The protein concentration of each supernatant was quantified using a protein assay reagent from the Bradford assay (Bio-Rad) in accordance with the manufacturer’s instructions.Proteins were loaded onto an 8% SDS-PAGE and transferred to a nitrocellulose membrane . After transfer, membranes were blocked with 5% bovine serum albumin (BSA) in Tris-buffered saline with 0.05% Tween 20 and probed with the specified primary antibodies  overnight at 4 °C. Primary antibodies were sourced as follows: SIRT1, β-actin, and NF-κB p65 were obtained from Santa Cruz Biotechnology ; Ac-NF-κB p65, AMPK, phospho (p)-AMPK (Thr172), mTOR, p-mTOR (Thr2448), S6 Kinase 1 (S6K1), p-S6K1 (Thr389), ACC, and p-ACC (Ser79) were obtained from Cell Signaling Technology . After washing, the membranes were incubated with secondary antibodies in Tris-buffered saline with 0.05% Tween 20 for 1 hr. The blots were then developed using an ECL detection kit (Amersham) according to the manufacturer’s instructions.2, 8.5% glycerol, 1 mM dithiothreitol, 0.1% Triton X-100, 2% BSA, and 1 mg/mL nonfat milk for 6 hr at 4 °C, and incubated with 2 μg of SREBP1c, PGC-1α, or LKB1 antibody (Santa Cruz Biotechnology). The immunocomplex was captured by incubating the samples with protein A-agarose (Santa Cruz Biotechnology) suspension overnight at 4 °C on a rocking platform. Agarose beads were collected by centrifugation and washed three times with PBS containing protease inhibitors. After microcentrifugation, the pellet was washed with 30 μL SDS-PAGE sample buffer and boiled for 5 min at 100 °C. An aliquot of the supernatant was subjected to electrophoresis on 10% SDS-PAGE and immunoblotted with an antibody against acetyl-lysine .Nuclear extracts from liver tissues were brought to a final volume of 1 mL with buffer containing 10 mM PBS, 50 mM KCl, 0.05 mM EDTA, 2.5 mM MgClp < 0.05.All data are expressed as means ± SEM. Statistical analysis was performed using one-way ANOVA and analyzed further by Duncan’s multiple range test for statistical difference. Differences among experimental groups were considered to be statistically significant at Supplementary Information


A study of ERECTA cytoplasmic domain predicts a primary phosphorylation site in its activation segment and indicates that signal transduction by ERECTA differs in specific developmental processes. erecta and erecta erl1 erl2 mutants. These experiments indicated that ERECTA’s ability to phosphorylate is functionally significant, and that while the cytoplasmic juxtamembrane domain is important for ERECTA function, the C-terminal tail is not. An analysis of multiple putative phosphorylation sites identified four amino acids in the activation segment of the kinase domain as functionally important. Homology of those residues to functionally significant amino acids in multiple other plant RLKs emphasizes similarities in RLK function. Specifically, our data predicts Thr812 as a primary site of phosphor-activation and potential inhibitory phosphorylation of Tyr815 and Tyr820. In addition, our experiments suggest that there are differences in the molecular mechanism of ERECTA function during regulation of stomata development and in elongation of above-ground organs.In plants, extracellular signals are primarily sensed by plasma membrane-localized receptor-like kinases (RLKs). ERECTA is a leucine-rich repeat RLK that together with its paralogs ERECTA-like 1 (ERL1) and ERL2 regulates multiple aspects of plant development. ERECTA forms complexes with a range of co-receptors and senses secreted cysteine-rich small proteins from the EPF/EPFL family. Currently the mechanism of the cytoplasmic domain activation and transmission of the signal by ERECTA is unclear. To gain a better understanding we performed a structure–function analysis by introducing altered ERECTA genes into  Intercellular communications are essential for development of multicellular organisms where cell proliferation and differentiation must be cooperative and structured to attain a desired shape and function. Plants especially rely on intercellular communications as cell behavior is often position-dependent. To detect extracellular signals, plant cells have a large group of receptor-like kinases (RLKs). These receptors possess a structurally diverse extracellular ligand-sensing domain, a single-pass transmembrane domain, and a cytoplasmic serine/threonine/tyrosine kinase domain.ERECTA (ER), ERECTA-like 1 (ERL1) and ERECTA-like 2 (ERL2)  RLKs appeared early during land plant evolution and are involved in the regulation of multiple developmental processes . During henotype . An analERECTA, ERL1, and ERL2 receptors form homo- and heterodimers (ERf protein structure consists of an extracellular leucine-rich domain (LRR), a single-span transmembrane domain, and a cytoplasmic Ser/Thr kinase domain flanked by a juxtamembrane domain (JMD) and a C-terminal tail . Previouer-105 and er-105 erl1-2 erl2-1 mutants have been described previously (–3 of Osmocoat 15-9-12 (Scotts). All plants were grown at 20 °C under long-day conditions (18 h light/6 h dark).The Arabidopsis ecotype Columbia (Col) was used as the wild-type (WT). The ERECTA-RLUC sequence by overlap extension PCR using pESH427 as a template . The er-105 erl1-2 erl2-1 mutants were selected based on kanamycin resistance and the homozygous status of the erl1-2 mutation was confirmed by PCR with the primers erl1g3659 (GAGCTTGGACATATAATC), erl1g4411.rc (CCGGAGAGATTGTTGAAGG), and JL202 (CATTTTATAATAACGCTGCGGACATCTAC). In addition, for transgenic lines transformed with pPZK102, pPZK110, and pPZK111 constructs, the homozygous status of the erl1-2 mutation was confirmed by analysis of kanamycin resistance in the progeny. The quantitative phenotypic analysis of er erl1 erl2 plants transformed with the described constructs has been done in T3 generation once their genetic status was established.In all plasmids except pPZK111 the substitutions/deletions were introduced into the genomic Renilla luciferase activity with a 20/20n single-tube luminometer in T1 inflorescences or in T2 8-d-old seedlings using the Renilla Luciferase Reporter Assay (Promega). The protein concentration in each sample was determined using the Bradford assay.ERECTA-RLUC protein expression was measured by monitoring Measurements of stomata index and clustering were done on the abaxial side of cotyledons from 17-d-old seedlings using differential interference contrast (DIC) microscopy. For DIC, seedlings were incubated in a solution of 9:1 ethanol:acetic acid overnight, rehydrated with an ethanol series to 50% (v/v) ethanol, and then cleared in a mixture of 8:1:1 chloral hydrate:distilled water:glycerol.The crude microsomal proteins were isolated from 11-d-old WT and T2 T807D seedlings (~0.4g per sample) using a method described by http://www.ebi.ac.uk/Tools/msa/clustalw2/).Full-length amino acid sequences of ERECTA family proteins from different species were retrieved from the NCBI database and aligned using ClustalW2 (ER (At2g26330), ERL1 (At5g62230), and ERL2 (At5g07180).Arabidopsis Genome Initiative numbers for the genes discussed here are as follows: The activity of a RLK’s kinase domain is often modulated by the flanking regions: the JMD and the C-terminal tail. In some receptors those regions inhibit kinase function, in others they are essential for the enzymatic activity . PhosphoRenilla Luciferase (RLUC) at the C-terminus of the receptor to monitor the level of protein expression. The luciferase assay is a fast, reliable, and relatively cheap method to measure protein levels. Most significantly, it reflects the protein concentration in Arabidopsis extracts . In our earlier experiments, the genomic ERECTA (pKUT196) rescued 100% of transgenic er-105 plants in the T1 generation (er-105 plants while ΔE921-E976 ERECTA-RLUC (pPZK110) rescued only 16% (Supplementary Table S1). As a result, the frequency of complementation in the T1 generation has not been used as a measure of a construct functionality. Instead, for each construct we analyzed multiple T1 plants with the goal of finding three to four independent transgenic lines with relatively similar protein expression. While analysis of protein expression detected a variation in the amount of ERECTA produced in different transgenic lines, the general ability of a construct to rescue the ERECTA phenotype did not correlate with the level of protein expression in selected lines  was used as a positive control. The constructs were transformed into ed lines . For exaer-105 and er erl1/+ erl2, similar to the positive control pESH427 . Interestingly, these two substitutions had a very similar negative impact on ERECTA function, and the phenotype of plants expressing T812A and T812D did not differ statistically. Therefore, phosphorylation of Thr812 is unlikely to play a major role in the activation of ERECTA.Next, we analyzed multiple Ser/Thr/Tyr in the activation segment by substituting them to Ala or as a phosphomimic to Asp. Alanine and aspartate substitution of Ser801, Ser803, Ser806, and Tyr808 in the activation loop did not have any effect on ERECTA function in control of organ elongation . SubstitAnother residue that is important for ERECTA function is Thr807. The T807A substitution substantially reduced functionality of ERECTA, while organ elongation in plants expressing ERECTA with T807D substitution was similar to the wild-type or even greater . Based oer-105, suggesting that with those substitutions ERECTA retained a very low level of functionality  or disruption of the kinase activity by the K676E substitution (pPZK 101) destroyed ERECTA’s ability to regulate stomata development and fertility in 2 mutant . In Fig.Considerable progress has been made recently in understanding the composition of receptor–ligand complexes formed by ERf in the plasma membrane . Howeverin vitro  immediately preceding an aspartate (D) in the catalytic loop. Previous research has established that ERECTA is a weak kinase in vitro . In ArabThr/Ser residues preceding the GlyThr motif are the primary phosphorylation sites that are essential for the activation of mammalian Ser/Thr kinases . S856 inin vivo to play an important role in BR signaling and their phosphorylation is predicted to have a negative impact on the kinase activity (Plant receptor-like kinases have dual specificity, phosphorylating both Ser/Thr and Tyr residues. Two tyrosine residues in the BRI1 P+1 loop, Tyr1052 and Tyr1057, have been shown activity b. ResiduPhosphorylation events in the JMD and the C-terminal regions often alter activity of receptor-like kinases . HoweverA majority of the RLK/Pelle kinases have an N-terminal extension in front of the N-terminal lobe of the kinase domain (er erl1 erl2 background. These results suggest that there are distinct signaling requirements for ERECTA in different developmental processes and imply that ERECTA might transmit the signal to downstream targets in different ways. The receptor-like kinases BAK1 and SCRAMBLED have also been shown to control multiple pathways using distinct signaling mechanisms with different requirements for their kinase domain function (Our structure–function analysis indicates that ERECTA function differs in the specific developmental processes in which it participates. The kinase function is absolutely essential for ERECTA’s ability to regulate stomata formation and flower structure. Simultaneously, the kinase-dead ERECTA is able to partially rescue stem and pedicel elongation defects in the Supplementary DataClick here for additional data file.


We have determined effects of caffeine and its xanthine metabolites on pancreatic acinar IP3R-mediated Ca2+ signalling and experimental AP.Caffeine reduces toxic Ca3 or toxins that induce AP and effects of xanthines, non-xanthine phosphodiesterase (PDE) inhibitors and cyclic adenosine monophosphate and cyclic guanosine monophosphate (cAMP/cGMP) determined. The intracellular cytosolic calcium concentration ([Ca2+]C), mitochondrial depolarisation and necrosis were assessed by confocal microscopy. Effects of xanthines were evaluated in caerulein-induced AP (CER-AP), taurolithocholic acid 3-sulfate-induced AP (TLCS-AP) or palmitoleic acid plus ethanol-induced AP (fatty acid ethyl ester AP (FAEE-AP)). Serum xanthines were measured by liquid chromatography-mass spectrometry.Isolated pancreatic acinar cells were exposed to secretagogues, uncaged IP3-mediated Ca2+ oscillations, while monomethylxanthines had little effect. Caffeine and dimethylxanthines inhibited uncaged IP3-induced Ca2+ rises, toxin-induced Ca2+ release, mitochondrial depolarisation and necrotic cell death pathway activation; cAMP/cGMP did not inhibit toxin-induced Ca2+ rises. Caffeine significantly ameliorated CER-AP with most effect at 25 mg/kg (seven injections hourly); paraxanthine or theophylline did not. Caffeine at 25 mg/kg significantly ameliorated TLCS-AP and FAEE-AP. Mean total serum levels of dimethylxanthines and trimethylxanthines peaked at >2 mM with 25 mg/kg caffeine but at <100 µM with 25 mg/kg paraxanthine or theophylline.Caffeine, dimethylxanthines and non-xanthine PDE inhibitors blocked IP3R-mediated pancreatic acinar Ca2+ signals but only caffeine ameliorated experimental AP. Caffeine is a suitable starting point for medicinal chemistry.Caffeine and its dimethylxanthine metabolites reduced pathological IP Acute pancreatitis is a major health problem without specific drug therapy.Coffee consumption reduces the incidence of acute alcoholic pancreatitis.2+ oscillations by inhibition of inositol 1,4,5-trisphosphate receptor-(IP3R)-mediated signalling.Caffeine blocks physiological intracellular Ca2+ overload from abnormal Ca2+ signalling is implicated as a critical trigger in the pathogenesis of acute pancreatitis.Sustained cytosolic Ca3R-mediated, sustained cytosolic Ca2+ elevations, loss of mitochondrial membrane potential and necrotic cell death pathway activation in pancreatic acinar cells.Caffeine and its dimethylxanthine metabolites inhibit IP2+ elevations in pancreatic acinar cells.Neither specific phosphodiesterase inhibitors nor cyclic adenosine monophosphate and cyclic guanosine monophosphate inhibit sustained Ca3R-mediated Ca2+ overload in experimental acute pancreatitis.Serum levels of xanthines after 25 mg/kg caffeine administration are sufficient to inhibit IP3R-mediated signalling inhibition.Caffeine but not theophylline or paraxanthine administered at 25 mg/kg significantly ameliorated pancreatic injury in experimental acute pancreatitis through IP2+ overload and of its consequences as novel potential therapy for acute pancreatitis.These findings support an approach of inhibition of CaMethylxanthine-based structures are suitable starting points for drug discovery and development to treat acute pancreatitis.Acute pancreatitis (AP) has an incidence of 30 per 100 000 per annum in the UK, commonly caused by gallstones or alcohol excess.2+ signals control normal secretion from pancreatic acinar cells but can become a critical trigger in pathogenesis. Physiological concentrations of acetylcholine (ACh) and cholecystokinin (CCK) generate repetitive elevations in the cytosolic Ca2+ concentration ([Ca2+]C) within the cellular apical pole that elicit stimulus metabolism coupling to generate ATP from mitochondria and stimulus-secretion coupling to initiate exocytosis.2+]C that are global and sustained. These abnormal elevations induce premature activation of intracellular enzymes, mitochondrial dysfunction, impaired autophagy, vacuolisation and necrosis, all of which contribute to the pathogenesis of AP.2+ chelation prevents zymogen activation and vacuolisation through attenuation of Ca2+ overload in acinar cells in vitro2+ release-activated Ca2+ channel, also known as the store-operated Ca2+ entry (SOCE) channel, by Orai1 inhibitor GSK-7975A, reduces Ca2+ overload and necrosis in both mouse17Intracellular Ca2+ release from intracellular stores occurs predominantly via inositol 1,4,5-trisphosphate receptor (IP3R) Ca2+ channels.3R in the apical region, close to the luminal membrane,3R types 2 and 3 are predominantly responsible for physiological Ca2+ signalling and enzyme secretion.2+ release in pancreatic acinar cells primarily via IP3Rs, an effect inhibited by double knockout of IP3R types 2 and 318Excessive Ca2+ signalling.2+ release from IP3Rs by inhibition of phospholipase C-mediated production of IP33Rs3Rs.2+ release from ryanodine receptors (RyRs) by increasing the sensitivity of RyRs to Ca2+ itself as observed in multiple cells,3Rs predominate.29Caffeine  belongs to the methylxanthine class of small, purine-based planar molecules and has several pharmacological actions,3-mediated Ca2+ signalling may be protective in AP since the incidence of AP is inversely proportional to the amount of coffee consumed.3R-mediated signalling have not been clarified. As data suggest caffeine and/or related methylxanthines may be protective in AP, we sought to determine their actions on toxin-induced, IP3R-mediated [Ca2+]C changes and cell death in vitro, and in three models of AP in vivo.The effects of caffeine on IPAdult male CD1 mice (8–12 weeks old) were housed at 23±2°C under a 12 h light/dark cycle with ad libitum access to standard laboratory chow and water. For in vivo experiments, animals were deprived of food but were allowed access to water from 12 h before the start of the experiments.3/PM (2 µM) and/or tetramethyl rhodamine methyl ester  were loaded for 30 min at room temperature. Confocal images were acquired on a Zeiss LSM510 system  with a 63× C-Apochromat water immersion objective (NA 1.2). ΔΨM was recorded in the perigranular mitochondrial cell region. IP3 was uncaged by UV excitation of whole cells  every three seconds where indicated. All fluorescence measurements were expressed as changes from basal fluorescence (F/F0 ratio), where F0 represents initial fluorescence at the start of each experiment.Fresh pancreatic acinar cells were isolated as described.For CCK-induced cell death, a time-course propidium iodide (50 µM) necrosis assay was run at 37°C using a POLARstar Omega Plate Reader . Isolated murine pancreatic acinar cells (75 µL) were added to a caffeine solution (75 µL) at selected concentrations or the same volume of physiological saline (for controls) prior to CCK (50 nM) addition.g for 2 min), resuspended and transferred to a microplate. Data were calculated as background-subtracted (cell-free blanks) percentage of total death (in 0.02% TritonX). Data were normalised to minimum and maximum fluorescence using the formula (F-Fmax)/(Fmax − Fmin)+1. All experiments were in triplicate.In TLCS-induced cell injury, an end-point propidium iodide (100 µg/mL) necrosis assay was employed. Cells were incubated with respective test solutions and agitated by rotary inversion for 30 min at 37°C, centrifuged /1% and tetrahydrofuran v/v, Eluent B 100% acetonitrile/0.1% FA v/v. The QTRAP5500 was operated in positive electrospray ionisation (ESI) mode and two MRM transitions were monitored for caffeine (195.3/138.0 and 195.3/110.0), theobromine (181.1/124.0 and 181.1/96.0), paraxanthine (181.2/124.0 and 181.2/142.0), theophylline (181.7/96.0 and 181.7/124.0) and internal standard (paracetamol—152.064/110.0 and 152.064/65.0) with a 100 ms dwell time. Also, 1 µL of 100 µM internal standard was added to 50 µL of each mouse serum sample and subjected to acetone precipitation (8:1 v/v) at −20°C for 1 h. Samples were centrifuged at 14 000g for 10 min at 4°C, then supernatant vacuum centrifuged to a volume of 50 µL. A 10 µL aliquot was injected into the liquid chromatography-mass spectrometry system. All xanthine serum concentrations were determined using a calibration curve of 1–100 µM for each analyte, spiked in mouse serum.Serum was analysed on a QTRAP5500 hybrid triple-quadrupole/linear ion trap instrument with TurboIon V Ion source , with inline LC ) and Gemini C18, 3 µm, 2.1×100 mm column . Eluent A comprised HHyperstimulation AP was induced by either 7 or 12 intraperitoneal injections of 50 µg/kg caerulein hourly (CER-AP), with saline controls. Bile acid AP was induced by retrograde infusion of 50 µL taurolithocholate acid sulfate  into the pancreatic duct as described, with saline injection (sham) controls.Details of caffeine dose optimisation and administration of other methylxanthines are described in supplementary materials and methods. In CER-AP, mice received seven intraperitoneal injections of 1, 5, 10 or 25 mg/kg of caffeine  hourly, beginning 2 h after the first caerulein injection, and were humanely killed at 12 h for determination of severity. The effect of caffeine was also assessed in both 7-injection and 12-injection CER-AP models at 24 h. In TLCS-AP, caffeine (25 mg/kg regimen) was begun 1 h after TLCS infusion and severity determined after humane killing at 24 h. In FAEE-AP, two intraperitoneal injections of caffeine  were administered from an hour after the second POA/ethanol injection.Results are presented as means±SEM from three or more independent experiments. In all figures, vertical bars denote mean±SE values. Statistical analysis was performed using Student's t test or analysis of variance in Origin 8.5  and a value of p<0.05 considered significant.3/PM from SiChem GmbH . Unless otherwise stated, all other chemicals were from Sigma  of the highest grade available.Fluo 4-AM, TMRM and Hoechst 33342 were from Thermo Fisher Scientific ; ci-IP2+]C oscillations in pancreatic acinar cells that were concentration-dependently inhibited by caffeine at 500 µM to 2 mM  caused C that were inhibited in a concentration-dependent manner by caffeine (3 and 5 mM)  Bi, ii. Tnd 5 mM) Biii. The2+]C signals were investigated. An elevated Ca2+ plateau followed hyperstimulation with 10 nM CCK  did not affect the CCK-induced [Ca2+]C plateau, whereas 10 mM caffeine caused complete inhibition (2+]C oscillations (see online supplementary figure S3A–D).Methylxanthines are PDE inhibitors and simultaneous increases in cAMP and cGMP may synergistically inhibit C signals in vitro, consistent with an effect of caffeine on this signalling pathway.In contrast of the dramatic effects of caffeine on caerulein-induced pancreatic injury, theophylline and paraxanthine did not exert significant protective effects in CER-AP with both 10 and 25 mg/kg regimens (see online supplementary figure S5A–E). To further explore these unexpected findings, the serum levels of theophylline and paraxanthine were measured from both dose regimens. Serum levels of theophylline and paraxanthine 10 min after the last xanthine injection were each <100 µM in the 25 mg/kg regimen and <50 µM in the 10 mg/kg regimen (see online supplementary figure S6). These dimethylxanthine concentrations were previously shown not to alter IPSince caffeine treatment was markedly protective in CER-AP at 12 h after induction by seven caerulein injections, its effects on more severe disease at a later time point were compared . CER-AP TLCS-AP caused dramatic increases of pancreatic and systemic injury markers compared with the sham group at 24 h A–E, with2+ signals in vitro,Since caffeine inhibits FAEE-induced Ca3R-mediated Ca2+ release from the pancreatic acinar endoplasmic reticulum store into the cytosol and their potential application in AP. It has been shown that caffeine inhibits IP3Rs3 production in a concentration-dependent manner.3R-mediated Ca2+ release is attributable at least in part to an action on the IP3R, since xanthines inhibited IP3R-mediated Ca2+ release elicited by uncaged IP3. Caffeine, theophylline and paraxanthine prevented physiological Ca2+ signalling and toxic elevations of [Ca2+]C induced by agents (CCK and TLCS) that cause AP, in a concentration-dependent manner (500 µM to 10 mM), also inhibiting falls in ΔΨM and necrotic cell death pathway activation. An inhibitory action on PDE preventing cAMP/cGMP degradation could not account for the effects on toxic [Ca2+]C overload since additional cAMP/cGMP did not prevent these. Extending these findings in vivo, caffeine significantly reduced the severity of multiple, diverse models of AP. The combined concentrations of dimethylxanthine and trimethylxanthine after the 25 mg/kg caffeine protocol were within the range over which effects on both IP3R-mediated Ca2+ release and toxic elevations of [Ca2+]C were identified. Despite the half-life of caffeine in mice of ∼60 min,3Rs but within the effective range on PDE (approaching 100 µM 10 min after the last dimethylxanthine injection),3R-mediated Ca2+ release.This study defines the inhibitory effects of methylxanthines on IP2+]C overload induced by CCK hyperstimulation was similar to that seen here with quasi-physiological ACh-elicited Ca2+ oscillations, as previously in pancreatic acinar cells2+ oscillations since both xanthine-based and non-xanthine-based PDE inhibitors reduced ACh-elicited Ca2+ oscillations. Nevertheless, PDE inhibition is unlikely to have contributed to the reduction of toxic [Ca2+]C overload as this was not affected by application of cell-permeable cAMP/cGMP analogues, but was immediately reversed upon caffeine administration. It is also unlikely that any increase in SERCA activity occurred in response to caffeine and downstream rises in cyclic nucleotide levels since no decrease in [Ca2+]C was induced by analogues of cAMP and cGMP, which have been shown to upregulate SERCA via phospholamban.2+]C overload are consistent with a primary effect on IP3R-mediated Ca2+ release.The concentration range over which caffeine inhibited toxic [Ca2+]C plateaus, rather SOCE will have been inhibited secondarily to reduction of store depletion, the principal driver of SOCE in non-excitable cells.21SOCE in pancreatic acinar and ductal cells occurs predominantly via Orai channels and is regulated in part by TRP channels.2+ release via RyR ameliorates both caerulein2+ release from RyRs in excitatory cells,2+ signalling in pancreatic acinar cells,2+ overload observed here might appear contradictory. However, in contrast to the situation in muscle cells, caffeine can only release Ca2+ in pancreatic acinar cells under quite exceptional circumstances and then only when present at a low concentration (1 mM); indeed, this effect is abolished by stepping up the caffeine concentration.2+ signalling is blocked by inhibiting IP3Rs pharmacologically3R2 and IP3R3) results in a failure of Ca2+ signal generation and secretion.2+ release in fundamental investigations of pancreatic acinar and other electrically non-excitable cells.27Inhibition of second messenger-mediated CaLittle, if any, protective effect of caffeine on experimental AP can be attributed to actions on adenosine receptors, which have both inhibitory  and excitatory  actions mediated in part through changes in cAMP.52High doses of caffeine were required to reduce the severity of experimental AP, with the most effective 25 mg/kg regimen extending into toxicity, indicative of a very narrow therapeutic index. At this dose, the number of hourly injections had to be reduced from seven to two in FAEE-AP to avoid mortality; in CER-AP, 50 mg/kg resulted in caffeine intoxication syndrome, although at 25 mg/kg no visible side effects were observed. In humans, even 10 mg/kg caffeine would be likely to induce caffeine intoxication, with florid neuro-excitotoxic and other undesirable side effects.3R-mediated signalling. Medicinal chemistry starting with the template of caffeine and/or other compounds that inhibit IP3R-mediated signalling could lead to more potent, selective and safer drug candidates for AP.Our study has demonstrated proof of principle that caffeine causes marked amelioration of experimental AP, largely through inhibition of IP


Interestingly, skeletal myotubes over-expressing MG53 or PRY-SPRY display a reduced intracellular Ca2+ release in response to K+-membrane depolarization or caffeine stimulation, suggesting a reduction in RyR1 channel activity. Expressions of TRPC3, TRPC4, and calmodulin 1 are increased in the myotubes, and MG53 directly binds to TRPC3, which suggests a possibility that TRPC3 also participates in the enhanced extracellular Ca2+ entry. Thus, MG53 could participate in regulating extracellular Ca2+ entry via Orai1 during SOCE and also intracellular Ca2+ release via RyR1 during skeletal muscle contraction.Mitsugumin 53 (MG53) participates in the membrane repair of various cells, and skeletal muscle is the major tissue that expresses MG53. Except for the regulatory effects of MG53 on SERCA1a, the role(s) of MG53 in the unique functions of skeletal muscle such as muscle contraction have not been well examined. Here, a new MG53-interacting protein, Orai1, is identified in skeletal muscle. To examine the functional relevance of the MG53-Orai1 interaction, MG53 was over-expressed in mouse primary or C2C12 skeletal myotubes and the functional properties of the myotubes were examined using cell physiological and biochemical approaches. The PRY-SPRY region of MG53 binds to Orai1, and MG53 and Orai1 are co-localized in the plasma membrane of skeletal myotubes. MG53-Orai1 interaction enhances extracellular Ca Membrane depolarization induces muscle action potential in skeletal muscle cells, and the action potential spreads along the surface of the plasma membrane and to the interior of skeletal muscle cells via transverse (t)-tubule invaginations. The spreading of the action potential activates dihydropyridine receptors , which, in turn, activates ryanodine receptor 1  membrane) via physical interactions between DHPR and RyR1. This results in the release of Ca2+ ions from the SR to the cytosol via RyR1 and skeletal muscle contraction by the binding of the released Ca2+ ions to contractile proteins. Ca2+ ions also activate RyR1 by binding to RyR1, which is called Ca2+-induced Ca2+ release (CICR), and CICR in skeletal myotubes plays a role in maximizing and maintaining the Ca2+ supply for skeletal muscle contraction12+ entry, such as store-operated Ca2+ entry (SOCE) via Orai1 or canonical-type transient receptor potential cation channels (TRPC), partially contributes to the Ca2+ supply for skeletal muscle contraction6782+ channel responsible for SOCE in skeletal muscle32+-ATPase 1a (SERCA1a) uptakes Ca2+ from the cytosol to the SR in order to reduce cytosolic Ca2+ to its resting level and to replenish the SR with Ca2+ ions3412Skeletal muscle contraction is accomplished by the operation of excitation-contraction (EC) coupling1234Mitsugumin 53  is expressed in skeletal and cardiac muscle, in lungs, and in kidneys, but skeletal muscle is the major site for MG53 expression14151617181917161921221619162514152+ movements in skeletal muscle using mouse primary skeletal myotubes, rabbit skeletal muscle tissue, a C2C12 mouse skeletal muscle cell line, and biochemical and cell physiological approaches.Apart from the roles of MG53 in the membrane repair system, there have been a few reports on the roles of MG53 in the unique functions of skeletal muscle, such as contraction and relaxation. MG53 facilitates the differentiation of C2C12 skeletal myoblasts to myotubes by enhancing vesicle trafficking and membrane fusion162327‘bona fide’ tissue to express MG53, Orai1, and STIM1, and with anti-Orai1  in MG53, cDNAs for four GST-fused MG53 domains were constructed : GST-TRIant Blue . All GST2+-quenching of intracellular fura-2 fluorescence33342+ is known to permeate cells via a SOCE mechanism but is insensitive to both the surface membrane extrusion processes and SR uptake by Ca2+ pumps. When Mn2+ binds to intracellular fura-2, it quenches the fura-2 fluorescence measured at excitation wavelength of 360 nm. Thus, the rate of Mn2+ fluorescence quenching  represents a measurement of the unidirectional Ca2+ entry. To examine the functional relevance of MG53 in skeletal SOCE, MG53 was over-expressed in C2C12 skeletal myotubes. The SR Ca2+ storage of the myotubes loaded with fura-2 was depleted with the addition of thapsigargin (TG) in the absence of extracellular Ca2+, and the addition of extracellular Mn2+ led to a quenching of intracellular fura-2 fluorescence   and extracellular Ca2+ application (SOCE) were applied to the myotubes  . C2C12 shemistry . To indumyotubes . MG53 inmyotubes . To exammyotubes , and themyotubes . Enhancemyotubes . As Oraimyotubes . Enhance2+ entry  were evaluated via immunoblot assays using the lysate of myotubes over-expressing MG53 or PRY-SPRY , JP2, calsequestrin, and mitsugumin 29. However, the expression level of calmodulin 1 (CaM1), a Ca2+-dependent protein, was significantly up-regulated by the over-expression of full-length MG53 or PRY-SPRY  exerts an inhibitory effect on RyR1 channel activity362+-CaM1 interaction with RyR1 may contribute to the reduced Ca2+ release via RyR1 in myotubes with the over-expressions of either MG53 or PRY-SPRY. In addition, it is not likely that CaM1 itself directly affects either MG53 or the enhanced SOCE via the MG53-Orai1 interaction, because there was no direct interaction between MG53 and CaM1  have been revealed (GenBank accession number: NM_001008274.3): PRY-SPRY domain forms a binding pocket that is significantly broader and deeper2+ levels (in the present study), while STIM1 over-expression does not2+ levels of skeletal muscle2+ by the MG53-Orai1 interaction, suggesting that TRPC3 could serve not only as proteins mediating SOCE3482+ movements for skeletal EC coupling.The knock-down of MG53 in skeletal muscle increases SERCA1a activity2+-CaM1 inhibits Ca2+ release via RyR1 at micromolar cytosolic Ca2+ concentrations in skeletal muscle, and the inhibition is independent of the kinase activity of CaM1362+-CaM1 that can inhibit RyR1 channel activity. Therefore, it is possible that, as a compensatory mechanism against extreme SOCE by the MG53-Orai1 interaction, Ca2+-CaM1 down-regulates RyR1 activity in order to regulate cytosolic Ca2+ levels. This is well supported by a previous report that CaM1 participates in regulating cytosolic Ca2+ levels by regulating Ca2+ release from the SR in skeletal muscle at elevated cytosolic Ca2+ levels2+ levels in skeletal muscle, and CaM1 could be a functional competitor of MG53 in regulating Ca2+ homeostasis in skeletal muscle.CaAll surgical interventions, including pre- and post-surgical animal care, were carried out in accordance with the Laboratory Animals Welfare Act, the Guide for the Care and Use of Laboratory Animals, and the Guidelines and Policies for Rodent Survival Surgery approved by the Institutional Animal Care and Use Committee of the College of Medicine at The Catholic University of Korea.E. coli (DH5α), as previously described30Using full-length mouse MG53 DNA (GenBank accession number: NM_001079932) as a template, GST-fused full-length MG53 or MG53 domains were constructed and were expressed in 2+ movement in skeletal muscle, which included Orai134323930The triad vesicles enriched with triad proteins that mediate intra and extracellular Ca2+ imaging experiment) coated with Matrigel . For the expression of GFP-tagged full-length MG53 or PRY-SPRY, immature primary skeletal myotubes on differentiation day 3 were transfected with each DNA construct using FuGENE6 transfection reagent . The C2C12 mouse skeletal myoblast cell line  was grown and differentiated to myotubes, as previously described192 incubator in high DMEM with 10% FEB, 100 units/ml of penicillin, and 100 μg/ml of streptomycinMouse primary skeletal myoblasts were derived from mouse skeletal muscle using a single-cell cloning method, and then were proliferated and differentiated to myotubes, as previously described12323946472+-quenching assay are described elsewhere  after TG-induced SR Ca2+ storage depletion without extracellular Ca2+ (by 50 μM BAPTA-AM), and the Mn2+-quenching of intracellular fura-2 fluorescence was measured at 360 nm (fura-2 excitation wavelength)2+ was expressed as the percent decrease in the intracellular fura-2 fluorescence per unit time.Details of the principles and the procedure involved in the Mnlsewhere 333435. BFully differentiated mouse primary skeletal myotubes on differentiation day 5 were solubilized, and the solubilized lysate  was subjected to SDS-PAGE  and immunoblot assay, as previously described1232394619394630322+ imaging experiments were performed, as previously described123239462+ levels) or with 5 μM fluo-4-AM (for other measurements) in an imaging buffer  at 37 °C for 45 min. The myotubes were transferred to an inverted stage microscope  equipped with a 40X oil-immersion objective (NA 1.30). During single-myotube Ca2+ imaging, images of the myotubes were captured using a high-speed monochromator with a 75 W xenon lamp  and a 12-bit charge-coupled device camera . The data were displayed and analyzed using image acquisition and analysis software . Either caffeine or KCl was dissolved in the imaging buffer and applied via an auto-perfusion system . To measure the amount of releasable Ca2+ from the SR to cytosol, TG  was manually applied to the myotubes in the absence of extracellular Ca2+ in order to avoid extracellular Ca2+ entry. DMSO (0.05%) alone had no effect on the release of Ca2+. For the measurement of SOCE, the SR Ca2+ storage was depleted with TG (2.5 μM) in the absence of extracellular Ca2+, and once the cytosolic Ca2+ level returned to the baseline, 2 mM Ca2+ was added to the myotubes to measure SOCE. To analyze the Ca2+ release obtained from the Ca2+ imaging experiments, the peak amplitude, which exhibited similar increases or decreases in peak areas, was considered. For long-term Ca2+ releases such as SOCE and TG responses, the areas under the curves were analyzed. All reagents for Ca2+ imaging experiments were obtained from Sigma-Aldrich .Single-myotube Cap < 0.05.The results are presented as the mean ± S.E. for the number of myotubes shown in the parenthesis in How to cite this article: Ahn, M. K. et al. Mitsugumin 53 regulates extracellular Ca2+ entry and intracellular Ca2+ release via Orai1 and RyR1 in skeletal muscle. Sci. Rep.6, 36909; doi: 10.1038/srep36909 (2016).Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.


However, for our own species the Homo sapiens, they are not. A major question is why are there only two types of gametes (sperm- and egg cells), two types of sex steroids, , while the reproduction-related behaviour of the gamete producers displays a much greater variability than just two prominent forms, namely heterosexual males and heterosexual females? It indicates that in addition to a few sex-determining genes ( = the first pillar), other factors play a role. A second possible pillar is the still poorly understood cognitive memory system in which electrical phenomena and its association with the plasma membrane membrane-cytoskeletal complex of cells play a major role . This paper advances a third pillar, that hitherto has been almost completely ignored, namely the cellular Ca2+-homeostasis system, more specifically its sex-specific differences. Differential male-female genetics- and hormone-based Ca2+-homeostasis with effects on gender-related processes has been named Calcigender before. It will be argued that it follows from the principles of Ca2+- physiology and homeostasis that all individuals of a sexually reproducing animal population have a personalized gender behaviour. Thus, subdividing gender-behaviours in hetero-, homo-, bi-, trans- etc. which all result from a differential use of the very same basic physiological principles, is too primitive a system that may yield false sociological interpretations.Are  Some obvious examples of animals in which females extrude much more calcium from their body related to reproduction than males are birds  and mammals that produce milk, with its extraordinarily huge Ca2+-concentration of about 50 millimolar (mM) compared to the very low Ca2+-concentration of about 100 nanomolar (nM) in the cytoplasm of resting cells  and egg cells. There are no intermediate types of sex cells between sperm- and egg cells. By convention the larger type (egg cells) is called the female gamete, and the individual that produces them is the female. The smaller sperm cells are called male gametes, and the producers are called males. These definitions of sexual forms, which date from before the term gender was introduced, do not include any reference to reproduction-related behaviour. Thus with respect to biological sex, one is either male or female. Individuals that have the two types of gonads, either occurring together or alternating, are called hermaphrodites.Heterosexual: a male and a female feel attracted to members and the opposite (second) sex/gender form, and usually cooperate to produce an offspring involving the fusion of a sperm- and an egg cell.Homosexual: individuals of the same sex form are more attracted to each other than to members of the other sex. Homosexuality occurs in many animal species.Gender: as will be outlined later, Gender can have different meanings. Males and females behave differently, and furthermore, not all genetically male or female organisms display the typical reproduction-oriented behaviour typical for their genetic sex. Sex and gender are invariably properties of the whole body, meaning that all cells participate.Calcigender: Males and females differ in their Ca2+ physiology  a progeny and for being evolutionarily successful. Yet, this answer implies that testes and ovaries are aware of the reproduction-oriented drive the whole organism might have. This cannot possibly be the case: ovaries and testes do not plan for the future by producing gametes. According to the current status of evolutionary theory i, e.g. by activation of a GPCR that uses Ca2+ as secondary messenger, may cause the sudden release of Ca2+ from internal storage sites. The SERCA pump will quickly restore equilibrium. In their analysis of the mode of action of another type of lipophilic molecule, namely the farnesol ester juvenile hormone, De Loof et al i in such cells. Hence my statement that all cells of a differentiated organism differ from each other, is not based upon exact measurements. It is deduced from a theoretical statistics-based argument, namely far too many causal agents play a role.From the study of comparative physiology of reproduction in vertebrates and invertebrates, it has become clear that many aspects of reproduction involving (hetero) gametes came into existence long ago in evolution, and that they have been very well conserved since . For bioSex kills is one of the numerous theories explaining aging phenomena [poisoned by Ca2+ than the male one [2+ undoubtedly has in the body result from the fact that short-lived rising Ca2+ concentrations ( = toxic puffs) are causal to them, e.g. in biochemical signalling (Ca2+ as secondary messenger). As an example, in many species, females live longer than males, despite, or better thanks to the toxicity of Ca2+. Females have better means to remove excess Ca2+ from their body, and can therefore better cope with the toxicity of Ca2+. Yet, decalcification of the skeleton resulting in osteoporosis that affects woman more than man is a negative aspect.I can imagine that without a knowledgeable insight into the fundamentals of animal physiology and development, the hypothesis that sexual reproduction results from an evolutionarily ancient sort of prokaryotic  infection of a proto-eukaryotic cell that eventually became the common ancestor of all eukaryotes, is at first encounter perceived as both counterintuitive and far-sought. Yet there are sound scientific arguments to support this view . Not onlhenomena . In addi2+-homeostasis, the social environment and other aspects of Nurture enables a huge variability in gender variants. In this context, a major gender-related topic of discussion is the place of homosexuality in an animal population, and the way this is perceived in human society in particular. It is known since long that homosexuality, one of the many possible gender variants, occurs in several animal species. For biologists this illustrates that this gender form must have a physiological basis that occurs widely in the animal kingdom, and that can generate variability in reproduction-related behaviour. The key actors in Ca2+-homeostasis are proteins that reside in membranes: Ca2+ channels and Ca2+-pumps. Bao and Swaab (2013) attribute little weight to the role of post-partum Nurture in steering the coming into existence of gender orientation, e.g. hetero- versus homosexuality. I support this view: Nature, thus genetics, is much more important than Nurture in defining gender and gender orientation. Differential sex steroid production and differential Ca2+-homeostasis are already effective long before birth.The interplay between genetics, sex hormones, Cathere are probably as many different gender variants as there are sexually reproducing individuals, and this not only in humans. Hence, there is no need to make subcategories in the gender variants with the purpose to install a moral hierarchy as they are all physiologically equal. This does not mean that all gender variants contribute equally well to the well-being of the whole population. I hope that growing awareness that the Ca2+- homeostasis system which is a key signalling system in all eukaryotes, acts as one of the corner stones on which gender is based, may contribute to a better acceptance of the variability of in gender forms as a natural, largely genetic (inborn) system.In conclusion: In this paper I advanced the physiology-based view that


By using TRPV1-transfected HeLa cells, we investigated the underlying mechanisms involved in CAP-induced TRPV1-mediated cell death, the dependence of CAP dose, and the participation of mitochondrial dysfunction and cytosolic Ca2+ increase. Together, our results contribute to elucidate the pathophysiological steps that follow after TRPV1 stimulation with CAP. Low concentrations of CAP (1 μM) induce cell death by a mechanism involving a TRPV1-mediated rapid and transient intracellular Ca2+ increase that stimulates plasma membrane depolarization, thereby compromising plasma membrane integrity and ultimately leading to cell death. Meanwhile, higher doses of CAP induce cell death via a TRPV1-independent mechanism, involving a slow and persistent intracellular Ca2+ increase that induces mitochondrial dysfunction, plasma membrane depolarization, plasma membrane loss of integrity, and ultimately, cell death.The transient receptor potential (TRP) ion channel family consists of a broad variety of non-selective cation channels that integrate environmental physicochemical signals for dynamic homeostatic control. Involved in a variety of cellular physiological processes, TRP channels are fundamental to the control of the cell life cycle. TRP channels from the vanilloid (TRPV) family have been directly implicated in cell death. TRPV1 is activated by pain-inducing stimuli, including inflammatory endovanilloids and pungent exovanilloids, such as capsaicin (CAP). TRPV1 activation by high doses of CAP (>10 μM) leads to necrosis, but also exhibits apoptotic characteristics. However, CAP dose–response studies are lacking in order to determine whether CAP-induced cell death occurs preferentially via necrosis or apoptosis. In addition, it is not known whether cytosolic Ca Transient receptor potential (TRP) channels belong to a polymodal family of ion channels that act as molecular transducers and integrators of a variety of environmental physicochemical stimuli, such as temperature, osmotic pressure, mechanical stress, and exogenous and endogenous ligands , 2009. TTRPV1 is activated by pain-inducing stimuli, including inflammatory endovanilloids, TNF-α, TGF-β, heat (37–42°C), acids (pH < 6.3), and pungent exovanilloids, such as capsaicin (CAP) or resiniferatoxin . TRPV1 a2+, generating mitochondrial membrane depolarization and decreased cell viability  induce a fast and transient increase in intracellular Ca2 atmosphere.HeLa cells were obtained from ATCC . The culture medium used was Dulbecco’s Modified Eagle Medium/F12 supplemented with 10% fetal bovine serum and 50 U/ml penicillin–streptomycin. Cells were incubated in a conventional incubator at 37°C and a 95% air/5% COHeLa cells were cultured at 70–80% confluence and then were transfected with pcDNA3.1 containing the full length of rat TRPV1 (GenBankTM accession no. NM031982) using Lipofectamine (ThermoFisher). Transfected cells were selected using Geneticin  to generate a stable cell line encoding TRPV1. Stable TRPV1 expression was checked by RT-PCR and flow cytometry weekly  ↔ Vulnerable (V) ↔ Dead (D). To determine probability for the A state (PA), data were normalized with respect to internal controls using the following equation: AP = X – DC/AC -DC, where X corresponds to living cells in each experimental condition, DC corresponds to living cells in the presence of 10% ethanol to induce cell death, and AC corresponds to living cells without treatment.HeLa cells were exposed to different experimental conditions in Dulbecco’s Modified Eagle Medium/F12 supplemented with 1% bovine serum albumin instead of fetal bovine serum. Cellular death was determined as described in the literature  and anal2, 0.8 mM MgCl2, 5.6 mM glucose, 10 mM HEPES, pH 7.4 (adjusted with Tris base)], rinsed, and allowed to equilibrate for 5–10 min. Next, HeLa cells were cultured on 12-mm glass cover slips in a recording chamber mounted on an epifluorescence Olympus IX81 microscope  equipped with a multiple-excitation filter wheel. CAP-induced activity was recorded for a minimum recording time of 2 s. Maximum resolution was obtained with a Plan Apo 40X 1.3 NA oil objective lens.Cell cultures were loaded with Fura-2 AM  for 30 min at room temperature in buffer solution  as described previously (4(3) (200 nM) was applied extracellularly for approximately 20 min to ensure dye distribution across the cell membrane. Changes in fluorescence intensity were monitored by sampling every 10 s for 30 min with a 515 nm excitation filter and an emission filter of at least 600 nm. Fluorescence data were transformed to mV using a calibration curve from HeLa cells as described previously , and reverse transcription was performed to create a cDNA library using reverse transcriptase M-MLV . An equal amount of RNA was used as template in each reaction. The PCR reactions were performed using GoTaq Master Mix  following the manufacturer’s instructions.HeLa cells were collected by trypsinization and fixed with 4% paraformaldehyde for 30 min. Next, the cells were blocked and permeabilized using a PBS solution with 5% bovine serum albumin  and 2% Tween-20  for 1 h and then incubated with an anti-TRPV1 antibody  in blocking solution overnight at 4°C. After washing with PBS, the cells were incubated with anti-goat biotinylated secondary antibody  for 1 h at 37°C. The cells were washed with PBS and incubated in the dark with streptavidin-Alexa Fluor 488  for 1 h at 37°C. The Alexa Fluor signal was measured with a 530/30 bandpass filter using an argon laser at 488 nm integrated into a FACScalibur flow cytometer . Debris and duplets were excluded from the analysis, and a minimum of 10,000 cells were acquired in each experiment. Data were analyzed with FlowJo software .Cyanide-4-(trifluoromethoxy)phenylhydrazone, FCCP, ionomycin, rodamine123, and PI were obtained from Sigma-Aldrich . CAP and CPZ were obtained from Tocris Bioscience . Buffers, ethanol, and salts were purchased from Merck .post hoc test were used to compare multiple groups using Statgraphics Plus 5.0 . A p-value < 0.05 was used to indicate statistical significance.All results are presented as the mean ± SD from at least three independent assays for each experimental condition. Fisher’s least significant difference test and an ANOVA test followed by the Bonferroni A), vulnerable (V), and dead (D)] was established by means of flow cytometry dot plot analysis to determine cell death with or without mitochondrial involvement. Wild-type HeLa (wt-HeLa) cells were exposed to 10 μM FCCP  for 0, 30, and 120 min, and 10% ethanol  for 0, 1, and 12 h. The three-state cell death model displayed a pronounced progression from A (PIlowRho123hi), to V , to D . The induction of an intermediate PIlowRho123lowV-phenotype indicates loss of mitochondrial membrane potential without plasma membrane disruption , while PIhiRho123hiV-phenotype indicates plasma membrane disruption, but without loss of mitochondrial membrane potential . Cell phenotype did not remain constant, and the proportion of cells in the three states varied over time. Cells achieved a full phenotype shift toward the D state after 3 h of exposure to FCCP  and after 9 h of exposure to 10% ethanol . Cells reached the intermediate V state at approximately 1 h of exposure to either FCCP or 10% ethanol, and PA was greater for cells exposed to FCCP than to 10% ethanol. Therefore, HeLa cell physiology appeared more sensitive to mitochondrial dysfunction than to plasma membrane disruption, as shown with PA progression curves.The participation of TRPV1 expression and activity to sensitize cells to CAP-induced death was tested using a three-state cell death model. The three-state model I after 2 min of exposure to CAP reaching basal levels shortly before 10 min . However, st-TRPV1 HeLa cells treated with 10 μM CAP showed a fast and persistent increase in [Ca2+]I, without return to basal levels after 10 min. Interestingly, when the latter cells were simultaneously exposed to 10 μM CPZ, the fast increase in [Ca2+]I was prevented, showing a slow and constant increase in [Ca2+]I reaching levels similar to those observed in the absence of CPZ . Thus, 10 μM CAP elicits two Ca2+ signals combined, a TRPV1-dependent fast and transient increase and another slow and persistent Ca2+ increase, without participation of TRPV1. As a positive control, cells were responsive to ionomycin .To investigate the intracellular effect of CAP-mediated TRPV1 stimulation, we measured Ca2+ levels was mediated by TRPV1, we were prompted to determine the participation of extracellular Ca2+ on the CAP-induced increase on intracellular Ca2+ levels. Figure 4C depicts both the transient and the persistent increases in [Ca2+]I from experiments as shown in Figure 4B. St-TRPV1 HeLa cells exposed to 1 μM CAP showed a transient, but not persistent increase in [Ca2+]I, while cells exposed to 10 μM CAP showed a persistent increase in [Ca2+]I. The addition of 10 μM CPZ prevented both the transient increase in [Ca2+]I independent of CAP concentration, but CPZ failed into inhibit the persistent increase in [Ca2+]I when 10 μM CAP was present. Interestingly, after removal of extracellular Ca2+, transient Ca2+ increases were prevented for both 1 and 10 μM CAP treatments, suggesting that CAP-induced transient increases in [Ca2+]I are dependent on extracellular Ca2+ and considering that it is CPZ-sensitive, this influx is mediated TRPV1. However, after extracellular Ca2+ depletion, 10 μM CAP was still able to stimulate an increase in [Ca2+]I, potentially due to a secondary mechanism involving Ca2+ mobilization from an intracellular storage compartment .Whether the increase in intracellular Ca2+]I  and that CAP induced a disruption in membrane integrity but not mitochondrial function , we determined whether CAP challenge could depolarize plasma membrane potential. St-TRPV1 HeLa cells exposed to 10 μM CAP for 30 min increased the fluorescence of the membrane potential fluorescent indicator DIBAC4(3), suggesting that exposure to 10 μM CAP induced a significant depolarization of the plasma membrane from approximately –80 mV in non-treated st-TRPV1 HeLa cells to –35 mV . Interestingly, selective inactivation of TRPV1 with 10 μM CPZ prevented the CAP-induced plasma membrane depolarization, suggesting that TRPV1 participates in plasma membrane depolarization induced by CAP.Considering that stimulation of st-TRPV1 with CAP generated a fast increase in [Ca2+, st-TRPV1 HeLa cells were exposed to 10 μM CAP in the absence of extracellular Ca2+. These results showed that in the absence of external Ca2+, CAP-induced plasma membrane depolarization was significantly decreased. Often, intracellular Ca2+ increases are followed by increases in intracellular Na+, which severely contribute to plasma membrane depolarization . Thus, CAP-induced plasma membrane depolarization appears to be an additive effect of Ca2+ and Na+ influxes. DIBAC4(3) efficiency to measure plasma membrane potential was validated using valinomycin and gramicidin , which depend on intracellular Ca2+ mobilization (2+ concentration through mitochondrial refilling and/or ATP-dependent Ca2+ compartmentalization processes (2+ increases, likely via the release of Ca2+ stores caused by mitochondrial failure or mitochondrial fission.TRPV1 expression exhibits dose-dependent cytotoxic effects, including mitochondrial store-dependent Caoverload , membranoverload , pyknotitivation , and celtivation . Indeed,tivation , therebytoxicity . Furtherlization . Optimalrocesses . Thus, C2+. Although transient increases in Ca2+ did not induce a change in cell state, heavy stimulation of TRPV1 with 100 μM CAP was able to trigger a toxic Ca2+ overload, likely due to intracellular mitochondrial Ca2+ release. Mitochondrial Ca2+ exchange with the cytoplasm has been previously reported (2+ uptake has been proposed to generate microdomains of low Ca2+ concentrations across the cytoplasm (2+-dependent ion channels, capable of regulating the electrochemical gradients required to mobilize Ca2+ into intracellular stores (2+ influx, leading to CAP-induced cell death.Stable TRPV1 expression and stimulation induces membrane depolarization through an increase in intracellular Careported  and Ca2+ytoplasm . In factr stores . Thus, tin toto experiments (2+ overload (Endogenous TRPV1 expression in many tissues, such as the brain, heart, skin, and testis, has been reported to play a role in cell death . For exaeriments . In facteriments . Moreoveeriments . These feriments , mitochoeriments , and intoverload . Neverth2+ signaling modifications that lead to a plasma membrane potential depolarization contributing to cell death.Taken together, the results shown in this study suggest that the expression and specific activation of TRPV1 can induce TRPV1-dependent CaRR-B, CC, SG, BR, CL, IM, CE, FS, and PO critically revised and edited this manuscript. RR-B, FS, and PO participated in the research design. RR-B, CC, BR, CL, IM, and PO conducted the experiments and performed data analyses. R-RB, SG, FS, and PO contributed to the figure design. RR-B, SG, IM, CE, FS, and PO wrote the paper.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.