However, downstream signaling pathways for the induced emesis remain unknown. The 5-HT3R channel has high permeability to extracellular calcium (Ca2+) and upon stimulation allows increased Ca2+ influx. We examined the contribution of Ca2+/calmodulin-dependent protein kinase IIα (Ca2+/CaMKIIα), interaction of 5-HT3R with calmodulin, and extracellular signal-regulated kinase 1/2 (ERK1/2) signaling to 2-Me-5-HT-induced emesis in the least shrew. Using fluo-4 AM dye, we found that 2-Me-5-HT augments intracellular Ca2+ levels in brainstem slices and that the selective 5-HT3R antagonist palonosetron, can abolish the induced Ca2+ signaling. Pre-treatment of shrews with either: i) amlodipine, an antagonist of L-type Ca2+ channels present on the cell membrane; ii) dantrolene, an inhibitor of ryanodine receptors (RyRs) Ca2+-release channels located on the endoplasmic reticulum (ER); iii) a combination of their less-effective doses; or iv) inhibitors of CaMKII (KN93) and ERK1/2 (PD98059); dose-dependently suppressed emesis caused by 2-Me-5-HT. Administration of 2-Me-5-HT also significantly: i) enhanced the interaction of 5-HT3R with calmodulin in the brainstem as revealed by immunoprecipitation, as well as their colocalization in the area postrema (brainstem) and small intestine by immunohistochemistry; and ii) activated CaMKIIα in brainstem and in isolated enterochromaffin cells of the small intestine as shown by Western blot and immunocytochemistry. These effects were suppressed by palonosetron. 2-Me-5-HT also activated ERK1/2 in brainstem, which was abrogated by palonosetron, KN93, PD98059, amlodipine, dantrolene, or a combination of amlodipine plus dantrolene. However, blockade of ER inositol-1, 4, 5-triphosphate receptors by 2-APB, had no significant effect on the discussed behavioral and biochemical parameters. This study demonstrates that Ca2+ mobilization via extracellular Ca2+ influx through 5-HT3Rs/L-type Ca2+ channels, and intracellular Ca2+ release via RyRs on ER, initiate Ca2+-dependent sequential activation of CaMKIIα and ERK1/2, which contribute to the 5-HT3R-mediated, 2-Me-5-HT-evoked emesis.Stimulation of 5-HT Recently, it has been demonstrated that increased luminal glucose levels result in 5-HT release from EC cells, which subsequently activates vagal afferent 5-HT3Rs, leading to activation of the Ca2+/calmodulin-dependent kinase II (CaMKII) signaling pathway in the brainstem DVC-gut circuit in rats 3Rs including pain The central/peripheral-acting agent 2-Methyl serotonin (2-Me-5-HT) is considered a “more selective” 5-HT3Rs in the process of vomiting via the use of in vivo pharmacology, ex-vivo and/or in vitro immunoprecipitation, immunohistochemistry, immunocytochemistry and Western blot on isolated EC cells and/or tissues of both small intestine and brainstem in the least shrew.In the present study we sought to evaluate the potential involvement of the above-discussed transduction signals downstream of 5-HTad libitum as described previously Adult least shrews were bred in the animal facility of Western University of Health Sciences. Previous studies had demonstrated no gender differences, so both males and females were used. Shrews were housed in groups of 5–10 on a 14∶10 light:dark cycle, fed with food and water 2A receptor antagonist SR46349B was purchased from Sanofi . The 5-HT6 receptor antagonists Ro-046790 and Ro-4368554 were purchased from Sigma/RBI  and Tocris , respectively. The CaMKII inhibitor KN93 and its inactive analog KN92 as well as ERK1/2 inhibitor PD98059 were obtained from Calbiochem . The L-type Ca2+ channel antagonist amlodipine besylate was purchased from Tocris . The ryanodine receptor antagonist dantrolene  and the inositol-1, 4, 5-triphosphate receptor antagonist 2-APB, were obtained from Santa Cruz Biotechnology . Unless otherwise stated, the above drugs were dissolved in water. KN92, dantrolene sodium and 2-APB were dissolved in 25% DMSO in water. PD98059 was dissolved in 0.5% ethanol, 0.5% Tween-80 in saline. All drugs were administered at a volume of 0.1 ml/10 g of body weight.2-Methylserotonin maleate salt (2-Me-5-HT) was purchased from Sigma/RBI . Palonosetron was a generous gift from Helsinn Health Care . The 5-HT2; 2 CaCl2; 10 glucose; and 26.2 NaHCO3, and aerated with 95% O2/5% CO2. Transverse brainstem slices (200 µm-thick) containing the DVC emetic nuclei identified as previously reported in our lab 2+ indicator fluo-4 AM  for 30 min in dark at room temperature. The fluo-4 AM loaded slices were pinned to sylgard blocks  and pre-treated with either the selective 5-HT3R antagonist palonosetron (1 µM) or its vehicle (control) for 30 min. The pretreated slices were simultaneously placed in an open bath imaging chamber  containing aCSF and mounted on the confocal imaging stage assembled with model 710 NLO  laser scanning confocal imaging workstation with inverted microscope (Olympus IX81 or Zeiss Axio Observer Z1). Since only 1 section (200 µm-thick) containing the emetic nuclei could be prepared from each shrew brainstem, one slice from 4 different shrews were used to investigate the effect of palonosetron on 2-Me-5-HT-elicited Ca2+ increase in the AP region among the brainstem DVC emetic nuclei. 2-Me-5-HT (1 µM) was added to aCSF containing palonosetron or vehicle at the end of pretreatment using a hand pipette, exactly at the 400th sec during the whole 1200-sec Ca2+ image-acquisition period.Adult least shrews were anesthetized in lethal isoflurane chamber and subsequently decapitated. Brainstems were quickly removed and transferred to ice-cold artificial cerebrospinal fluid  buffer, containing (in mM): 124 NaCl; 5 KCl; 1.3 MgCl2+ responses were acquired at roughly one image per second with an imaging depth of 10 µm, which is equivalent to two or three cells thick. The sampling depth was 16-bit (Zeiss 710). Ca2+ spark recordings were made using Zeiss C-Apochromat 63x/1.20 water immersion objective. ROIs were examined post hoc and analyzed with ImageJ. Analysis of time series recordings was achieved by hand using the time series analyzer plugin for ImageJ. For presentation purposes, the fractional fluorescence intensity was calculated as F/F0. After Ca2+ image acquisition, the data was analyzed by NIH-approved Fiji ImageJ software using the time series analyzer plugin for ImageJ. The captured images were visualized and cells with different level (500–60000) of fluorescence intensities were identified. Regions of interest were selected from the initial frame captured at 0 sec with cells showing initial fluorescence intensities between 5000–25000 and the values of fluorescence intensities at different time points were identified by time series analyzer to plot the graphs of selected regions of interest. To show the changes in Ca2+ levels before and after 2-Me-5-HT treatment, the average fluorescence intensities were calculated for at least 12 regions of interest in each acquisition for all time points. The data is represented in a graph as the ratio (F/F0) of final fluorescence intensity (F) for each time point to the initial fluorescence intensities (F0) at 0 sec for ROIs and is the mean value of 4 individual experiments.Slices were illuminated at 488 nm with a krypton argon laser and the emitted light was collected using a photomultiplier tube. Line scans were imaged at rates from 422 to 822 lines generated every 1 s, depending on line length. To ensure that sparks within the region of interest (ROI), the AP region of the brainstem, were imaged, global CaOn the day of the experiment shrews were brought from the animal facility, separated into individual cages and allowed to adapt for at least two hours (h). Daily food was withheld 2 h prior to the start of the experiment but shrews were given 4 mealworms each prior to emetogen injection, to aid in identifying wet vomits as described previously 2+ channel antagonist amlodipine ; ii) ryanodine receptor (RyRs) antagonist dantrolene ; iii) inositol-1,4,5 triphosphate (IP3) receptor antagonist 2-APB ; iv) serotonin 5-HT2A receptor antagonist SR46349B ; v) serotonin 5-HT6 receptor antagonists Ro-046970 or Ro-4368554 , vi) active inhibitor of CaMKII KN93 ; vii) inactive analog of KN93, KN92 ; or viii) ERK1/2 inhibitor PD98059 . Thirty minutes later, each treated shrew received a 5 mg/kg emetic dose of 2-Me-5-HT (i.p.). The number of animals vomiting within groups and the frequency of vomits for the next 30 min were recorded. The antiemetic effects of a combination of semi-active doses of amlodipine (s.c.) with dantrolene (i.p.) were further investigated. Thus, different groups of shrews (n = 8–11 per group) were pre-treated either with their corresponding vehicles (Aml 0 + Dan 0), amlodipine 5 mg/kg + dantrolene vehicle (Aml 5 + Dan 0), amlodipine vehicle + dantrolene 10 mg/kg (Aml 0 + Dan 10), or amlodipine 5 mg/kg + dantrolene 10 mg/kg (Aml 5 + Dan 10), 30 min prior to 2-Me-5-HT administration. The indices of induced emesis were recorded as described above. Each shrew was used once and then euthanized with an overdose of pentobarbital  following the termination of each experiment.We have previously demonstrated that a 5 mg/kg intraperitoneal (i.p.) injection of 2-Me-5-HT produces a robust frequency of vomits in all tested animals Adult least shrews treated with 2-Me-5-HT  were rapidly anesthetized with isoflurane and decapitated at the indicated time points post-treatment . Brainstem and small intestine were quickly removed. Further division of the small intestine to recognize the jejunal segment was performed according to Ray et al The optimal-cutting-temperature compound-embedded brainstems  were cut into 20 µm sections using a cryostat and mounted onto slides. Sections from brainstem were observed with a light microscope and those containing the whole DVC subjected to immunohistochemistry. Slides were washed in PBS three times, fixed with 4% PF for 2 h at 4°C, then washed 3 times with PBS, permeabilized with 0.1% Triton X-100 for 30 min at 4°C, and washed again 3 times with PBS. After blockade for 1 h with the blocking buffer containing 5% bovine serum albumin (BSA) in PBS, histological sections of brainstem and intestine were co-incubated overnight at 4°C with goat anti-CaMKIIα  and rabbit anti-phospho-CaMKIIα (Thr286) antibodies  to analyze CaMKIIα phosphorylation at Thr286 site. Sections were then washed 3 times (10 min each) in PBS and incubated in Alexa Fluor 594 donkey anti-goat IgG and Alexa Fluor 488 donkey anti-rabbbit IgG  for 2 h at room temperature. Images for the whole DVC region and for the individual areas (AP/NTS/DMNX) were acquired under a confocal microscope (Nikon) with Metamorph software using 20× and 100× objectives, respectively. Nuclei of cells were stained with DAPI. DAPI is excited at 345 nm and emits at 458 nm, producing blue fluorescence.3R-CaM colocalization on brainstem slices (20 µm) and jejunum slices (10 µm). Sections were co-incubated overnight at 4°C with rabbit anti-5-HT3R  and mouse anti-CaM antibodies  to analyze co-localization of 5-HT3R and CaM. After washing in PBS, the immunoreactivities were visualized by incubation with Alexa Fluor 594 donkey anti-rabbit IgG and Alexa Fluor 488 donkey anti-mouse IgG  or rhodamine red anti-rabbit and FITC-conjugated anti-mouse secondary antibodies . Images for the whole DVC region and for the individual nuclei (AP/NTS/DMNX) were acquired under a confocal microscope (Nikon) with Metamorph software using 200× objective.Immunohistochemistry was also performed to analyze 5-HT3R and CaM in the brainstem of least shrews, the animals (n = 3 per group) were treated either with vehicle, the 5-HT3R agonist 2-Me-5-HT , the 5-HT3R antagonist palonosetron , or a combination of both agents. The time to first vomit was generally within 15 minutes of 2-Me-5-HT injection. Thus, each shrew brainstem was isolated 20 min after 2-Me-5-HT treatment, homogenized in cold lysis buffer  containing protease- and phosphatase-inhibitors cocktail , and centrifuged at 10000×g for 20 min at 4°C. Total protein concentrations in supernatants were confirmed using BCA protein assay kit . A 1 mg protein extract from each brain lysate was immunoprecipitated overnight at 4°C with 10 µg rabbit anti-5-HT3R antibody (sc-28958) or rabbit IgG  and then incubated with 50% Protein A/G agarose slurry  for 1 h with occasional mixing at 4°C. After washing 3 times with lysis buffer by centrifuging at 700×g for 1 min at 4°C, supernatant was discarded, and 50 µl of 1.5× SDS-PAGE sample buffer was added to the saved pellets, heated at 100°C for 10 min, and centrifuged at 700×g for 1 min. Supernatants containing 5-HT3R immunoprecipitates were subjected to Western blot for the detection of 5-HT3R and CaM. Inputs from various groups were used to confirm the expression of 5-HT3R and CaM. GAPDH served as an internal standard. All samples were subjected to 12% SDS-polyacrylamide gel electrophoresis. Proteins were transferred to a polyvinylidene difluoride membrane for 90 min at 90 V. After blocking with TBST solution  containing 5% BSA for 1 h at room temperature, membranes were incubated overnight at 4°C with mouse anti-CaM antibody , goat anti-5-HT3R antibody  or mouse anti-GAPDH antibody . Infrared fluorescent-labeled anti-goat and anti-mouse secondary antibodies  were then used. Bound antibodies were visualized using Odyssey imaging system and analyzed semi-quantitatively based on densitometric values using Quantity-One 1D software (Bio-Rad). The ratios of CaM (∼17 kD) to 5-HT3R (∼55 kD) precipitated by 5-HT3R antibody were calculated and are shown as fold change of control.To assess the 2-Me-5-HT-induced interaction of 5-HTTo determine the time-dependent profile of CaMKIIα and ERK activation, different groups of animals (n = 3 per group) were sacrificed at 5, 10, 20, 30, 60 min following 2-Me-5-HT administration . In addition, different groups of least shrews (n = 3 per treatment group) were pretreated with either palonosetron , SR46349B , 2-APB , amlodipine , dantrolene , amlodipine  + dantrolene , KN93 , PD98059 , or corresponding vehicles, 30 min before 2-Me-5-HT injection . Shrew brainstems were then removed at specific intervals after 2-Me-5-HT treatment and homogenized in lysis buffer. Protein extracts from brainstem lysates were subjected to Western blot. Primary antibodies included rabbit anti-phospho-CaMKII (Thr286) , mouse anti-CaMKII , rabbit anti-phospho-ERK1/2  and mouse anti-ERK1/2  antibodies. Infrared fluorescent-labeled anti-rabbit or anti-mouse secondary antibodies  were used. Bound antibodies were visualized correspondingly using Odyssey imaging system and analyzed semi-quantitatively based on densitometric values using Quantity-One 1D software (Bio-Rad). The ratios of pCaMKIIα (∼50 kD) to CaMKIIα and pERK1/2 (42/44 kD) to ERK1/2 were calculated and presented as fold change of control.The enterochromaffin (EC) cell isolation from naïve shrews was performed via a slight modification of the method described by Schäfermeyer and co-workers 3R stimulation with 1 µM 2-Me-5-HT (or its vehicle as control) for 20 min at 37°C. After treatment, the collected cell pellets were re-suspended in lysis buffer, sonicated and centrifuged . Supernatants containing total protein were quantified and used to analyze CaMKIIα phosphorylation at Thr286 by Western blot, as described above. For immunocytochemistry, control and treated EC cells (n = 3 experiments per group) were fixed with 4% PF in PBS for 10 min followed by treatment with cold methanol for 10 min at 4°C. The cells were also co-stained with rabbit anti-CaMKIIα  and mouse anti-phospho-CaMKIIα (Thr286) . The immunoreactivities were visualized by incubation with rhodamine red anti-rabbit and FITC-conjugated anti-mouse antibodies.The EC cells (n = 3 expeiments per group) were pre-incubated for 30 min with either 1 µM palonosetron or its vehicle at 37°C, followed by 5-HTpost hoc test. The percentage of animals vomiting across groups at different doses was compared using the chi square test. Statistical significance for differences between two groups was tested by unpaired t-test, among groups (≥3) was tested by one-way ANOVA followed by Tukey’s multiple comparison tests. For time-course analyses of CaMKIIα and ERK activation, one-way ANOVA followed by Dunn’s post hoc test was used. All results are presented as mean ± SEM. P<0.05 was considered significant.The vomit frequency data were analyzed using the Kruskal-Wallis non-parametric one-way analysis of variance (ANOVA) and followed by Dunn’s 3Rs regulates neuronal function by directly gating its corresponding ion channel to produce an increase in Ca2+ influx which rapidly induces neuronal depolarization 2+ signal can be partly due to subsequent extracellular Ca2+ influx via enhancement of voltage-operated Ca2+ channels 2+ from ER stores through the process of Ca2+-induced Ca2+ release (CICR) Activation of 5-HT3R-mediated emesis, changes in intracellular Ca2+ signaling were first examined. Thus, incubation of isolated least shrew brainstem slices containing the DVC emetic loci with the selective 5-HT3R agonist 2-Me-5-HT (1 µM) resulted in a rapid increase in intracellular Ca2+ concentration monitored via an increase in fluo-4 AM fluorescence intensity, as shown by the increased F/F0 ratio  slightly reduced the baseline Ca2+ levels and fully suppressed the 2-Me-5-HT-induced enhancement of intracellular Ca2+ signaling  elicited vomiting in all the tested shrews  dose-dependently suppressed both the vomit frequency  = 14.77, P<0.01)  = 11.98; P<0.01) in response to 2-Me-5-HT (2+ release from the ER via ryanodine receptors (RyRs) and/or inositol-1, 4, 5-triphosphate receptors (IP3Rs), were involved in 2-Me-5-HT-induced vomiting. Administration of dantrolene , a blocker of RyRs, dose-dependently suppressed both the 2-Me-5-HT-induced vomit frequency  = 23.35, P<0.001) and the percentage  = 15.42, P<0.01) of shrews vomiting with significant reductions occurring at 5, 10 and 20 mg/kg doses  = 15.49, P<0.01, respectively) in shrews pretreated with lower but combined doses of amlodipine (5 mg/kg) plus dantrolene (10 mg/kg)  had no effect on 2-Me-5-HT-evoked vomiting responses   and the -Me-5-HT . In fact0 mg/kg) . In contesponses . These b3R stimulation induces extracellular Ca2+ influx which may secondarily affect the cytosolic Ca2+ sensor protein, calmodulin (CaM), since an increase in free cytoplasmic Ca2+ concentration can lead to activation of CaM and CaMKIIα 1A-, 5-HT2A- and 5-HT2C-recptors 2+ channels, voltage-gated sodium channels and voltage-gated potassium channels 5-HT3R activation, we investigated the interaction of CaM with 5-HT3R in the least shrew brainstem via co-immunoprecipitation. We have previously demonstrated that the 5-HT3R antagonist, palonosetron, dose-dependently suppresses vomiting evoked by 2-Me-5-HT with approximately 70% maximal protection at 5 mg/kg 3R antibody. The resulting 5-HT3R immunoprecipitates were used to detect 5-HT3R and CaM. As shown in 3Rs and CaM (P<0.05 vs. control), whereas following 5-HT3R blockade with palonosetron, 2-Me-5-HT failed to increase the interaction of 5-HT3R with CaM (P>0.05 vs. control). As with palonosetron, amlodipine suppressed the interaction of 5-HT3R-CaM (data not shown).To determine the regulation of CaM following 5-HT3R with CaM in brainstem in response to 2-Me-5-HT treatment by immunohistochemistry. Brainstems from the above-discussed experimental shrews were isolated, sections were prepared and immunolabeled for 5-HT3R and CaM. The colocalization between 5-HT3R and CaM in different DVC emetic loci in the brainstem  were then evaluated. Brainstem sections obtained from the 2-Me-5-HT-treated shrews exhibited significantly enhanced 5-HT3R-CaM colocalization in AP area relative to vehicle control, whereas the brainstem sections obtained from least shrews pretreated with palonosetron followed by 2-Me-5-HT  did not show significant alteration in 5-HT3R-CaM colocalization, which was similar to control  can inhibit the 2-Me-5-HT -induced CaMKIIα activation, the tested animals were sacrificed 20 min after 2-Me-5-HT administration. The results revealed that palonosetron pretreatment abolished the 2-Me-5-HT-evoked increase in pCaMKIIα  (CaMKIIα is a downstream kinase which is activated by CaMe-5-HT) .We further confirmed our Western blot results with immunohistochemistry . Brainst3Rs present on EC cells to promote release of endogenous serotonin from these cells and the induced release is sensitive to selective corresponding antagonists 3Rs.The 5-HT-releasing EC cells present in the GIT are involved in the induction of emesis (see introduction). Furthermore, 2-Me-5-HT can activate 5-HT3R-mediated CaMKIIα activation, we performed immunoblots on protein extracts of brainstems obtained from 2-Me-5-HT-treated shrews respectively pretreated with either amlodipine , dantrolene , or a combination of amlodpine  and dantrolene   = 13.76, P<0.01) in a dose-dependent and potent manner  . In the g, i.p.)   . These ror, KN93 . Thus, Kt manner . In factt manner . These o2+-mobilizing stimuli 2+/CaMKII regulates ERK1/2 signaling in response to 2-Me-5-HT administration . Our attained time profile indicates that following 2-Me-5-HT administration, both pERK1 and pERK2 levels (pERK1/2) increased markedly in the least shrew brainstem at the 5  and 10 min  exposure intervals, but returned towards baseline levels at 20 and 30 min   ; ii) intracellular Ca2+ release from ER stores through RyRs by dantrolene ; iii) of both of these channels by lower but combined doses of amlodipine (5 mg/kg) and dantrolene (10 mg/kg) (P<0.05); or iv) CaMKII activity by its inhibitor KN93 (10 mg/kg) (P<0.05) (3R-mediated ERK1/2 activation is a Ca2+/CaMKII-dependent process.It has been reported that CaMKII mediates ERK1/2 activation in response to Cad 30 min 7A. Therecontrol) 7B. This n of ERK ; P<0.05 (P<0.05) 7D. On th(P<0.05) . Therefo2  = 10.48, P<0.01) in response to 2-Me-5-HT injection in a dose-dependent manner with ∼90% protection at 5 mg/kg (P<0.01)  30 min prior to 2-Me-5-HT (5 mg/kg) injection. PD98059 reduced both the frequency  = 12.18, P<0.001) and percentage of shrews vomiting (χ(P<0.01) . A 5 mg/(P<0.01) . Thus, 52ARs and 5-HT3Rs 2ARs may be involved in emetic response evoked by 2-Me-5-HT, we evaluated the effect of 5-HT2A/C R antagonist, SR46349B 3R, and not the 5-HT2AR subtype, is specifically involved in 2-Me-5-HT-induced emesis-related responses.It has been suggested that functional interaction exists between 5-HT6Rs 6Rs are also not involved in vomiting.In addition, 2-Me-5-HT has affinity for 5-HT3R antagonists against CINV began in the early 1980s. To date, understanding of emetic signals downstream of 5-HT3R has remained elusive. Since chemotherapeutics such as cisplatin induce vomiting via concomitant release of several different emetogenic neurotransmitters 3R “preferring” agonist 2-Me-5-HT in the least shrew. The advantage of this model over the long-standing and well-established ferret model is that unlike ferrets, shrews vomit consistently and in a dose-dependent manner in response to systemic administration of serotonin 3R activation in the induction of 2-Me-5-HT-evoked vomiting. Our findings support the hypothesis that, following 5-HT3R activation, 2-Me-5-HT causes an influx of extracellular Ca2+ through 5-HT3Rs/L-type Ca2+ channels, which subsequently evokes Ca2+-induced Ca2+ release (CICR) from intracellular ER Ca2+ stores via activation of RyRs Ca2+ channels present on the ER membrane. The enhanced Ca2+ mobilization is also sequentially linked to the intracellular activation of the CaMKIIα-ERK pathway in the brainstem, which plays an important role in 2-Me-5-HT-induced vomiting.  and L-type Ca2+ channels (e.g. nifedipine) 2+ channels, causes vomiting in the least shrew in a dose-dependent manner. Not only palonosetron and nifedipine on their own can suppress FPL 64176-induced vomiting in a dose-dependent and potent manner, their ineffective but combined doses demonstrate significantly greater antiemetic efficacy against vomiting caused by several emetogens including FPL64176, 2-Me-5-HT and cisplatin in vivo findings support the proposed cross-talk that occurs between 5-HT3Rs and L-type Ca2+ channels in vitro2+ channel blocker, amlodipine. Furthermore, both nifedipine and amlodipine are effective antiemetics against vomiting caused by diverse emetogens Stimulation of 5-HT2+ release from the endoplasmic reticulum (ER) can occur via at least two classes of receptors present in ER membrane termed IP3Rs and RyRs 2+ channels and RyRs appear to exist which plays an important role in intracellular Ca2+ release following voltage-dependent Ca2+ entry through L-type Ca2+channels 3Rs and RyRs. We found that the 5-HT3R-mediated vomiting was insensitive to the IP3R antagonist, 2-APB, but in contrast, was dose-dependently suppressed by the RyR antagonist, dantrolene. Furthermore, a combination of the semi-effective doses of amlodipine and dantrolene, was more potent than each antagonist being tested alone. These behavioral findings suggest that 5-HT3R stimulation drives extracellular Ca2+ through both 5-HT3Rs and L-type Ca2+ channels, which subsequently trigger Ca2+ release via RyRs from intracellular ER stores (i.e. CICR), which greatly amplifies free Ca2+ levels in the cytoplasm. Our in vivo findings are consistent with a previous in vitro cellular study which demonstrated that 5-HT3R activation evokes extracellular Ca2+ entry which then triggers such Ca2+ release from intracellular stores in a RyRs-sensitive manner (i.e. CICR) Intracellular Ca2+ concentration can lead to activation of CaM and subsequent CaMKIIα 2+ sensor CaM can regulate diverse functions by binding to hundreds of target proteins 3R and CaM in both the shrew brainstem and their colocalization in the jejunum following 2-Me-5-HT administration, since the observed association is sensitive to the 5-HT3R antagonist palonosetron. Indeed, it is already known that CaM can interact with several G-protein-coupled receptors including serotonergic 5-HT1A2A2C1-receptors 3R activation by 2-Me-5-HT, CaM might influence the localization, clustering, and trafficking of 5HT3R as well as 5HT3R-mediated signal transduction via direct or indirect binding to 5-HT3R. Moreover, not only does CaM bind to L-type Ca2+ channels (LTCC) 3R interaction can be also suppressed by the L-type Ca2+ channel antagonist, amlodipine. Thus, our findings suggest that 5-HT3R-CaM interaction appears to be regulated by 5-HT3R and LTCC activities which support the proposed crosstalk between 5-HT3R and L-type Ca2+ channels 3R signaling in general and in emesis in particular remains to be fully characterized, and more systematic experiments remain to be conducted, especially, (1) investigation of the consequences of 5-HT3R-CaM interaction on 5-HT3R function as an ion channel; and (2) cellular studies investigating the specific interruption of the 5-HT3R-CaM interaction with specific small molecules or peptides directly targeting the protein complexes as well as the influence of this specific blockade on 5-HT3R-mediated signaling pathway and emesis.An increase in free cytoplasmic Ca2+ and functions as an intracellular signaling protein. Phosphorylated CaMKII (pCaMKII) has a relatively unique property that allows prolonged phosphorylation in response to transient Ca2+ signals making it an excellent marker for cellular activation. Furthermore, enhanced current through L-type voltage-gated Ca2+ channels can stimulate CaMKII activity which is required for various effects including induction of long-term potentiation 2+/CaMKII signaling is involved in the 5-HT3R-mediated 2-Me-5-HT-induced vomiting. In fact both vomit frequency and the degree of CaMKIIα activation appear to have a temporal relationship, since within 20 min of systemic injection, 2-Me-5-HT not only caused maximal number of vomits, but also induced maximal increase in CaMKIIα phosphorylation at Thr286 in brainstem as revealed by Western blots and immunohistochemistry. Similar to the reported differential increases in c-Fos immunoreactivity in the AP, NTS and DMNX of the least shrew in response to 2-Me-5-HT administration in vitro. Both Western blots of total protein extracted from least shrew EC cells and immunocytochemistry of EC cells exhibited substantial increases in pCaMKIIα levels. Moreover, pretreatment with the 5-HT3R antagonist palonosetron reversed the 2-Me-5-HT-induced increases in pCaMKIIα in the above-discussed in vivo and in vitro experiments. Since 5-HT3Rs are expressed in distinct cells in the GIT including functionally discrete classes of neurons as well as EC cells, 5-HT3R stimulation may involve the activation of both neuronal and nonneuronal pathways 3Rs present on the surface of EC cells by 2-Me-5-HT can induce release of endogenous serotonin which can be prevented by prior exposure to selective 5-HT3R antagonists 3Rs on vagal nerve endings to initiate the vomiting reflex 3Rs 2+ channel antagonist amlodipine, the RyR antagonist dantrolene, or a combination of their less effective doses, but not by the IP3R antagonist 2-APB, which is consistent with the earlier discussed effects of these Ca2+ modulators on 2-Me-5-HT-induced vomiting presented in this study. In addition, the CaMKII inhibitor KN93  3R-mediated vomiting and is under regulation of extracellular Ca2+ influx through 5-HT3R/L-type Ca2+ channels as well as intracellular Ca2+ release from the ER stores via the RyRs.CaMKII is a protein kinase that is widely expressed in a variety of tissues 3R stimulation is directly coupled to ERK1/2 phosphorylation. This upregulation of ERK1/2 was abolished by prior treatment with either palonosetron, amlodipine, dantrolene, KN93, or the ERK inhibitor PD98059, suggesting that extracellular Ca2+ influx, CICR from ER stores via RyRs, and CaMKII activation are sequential prior components of the ERK1/2 cascade involved in 5-HT3R-mediated signaling pathway. Our behavioral evidence that inhibition of ERK1/2 activation with PD98059 attenuated 2-Me-5-HT-induced emesis provides further credibility for the involvement of ERK1/2 in the induction of 5-HT3R-mediated emesis.We have recently demonstrated that significant activation of ERK1/2 is associated with peak vomit frequency during both the immediate and delayed phases of emesis caused by cisplatin in the least shrew 3R selective agonist, it does possess affinity for 5-HT2ARs and 5-HT6Rs 2A receptor-mediated head-twitch behavior 2AR stimulation can increase intracellular Ca2+ levels and affect L-type Ca2+ currents 2AR potentiates 5-HT3R function 2AR antagonist SR46349B, does not reduce the ability of 2-Me-5-HT to either induce vomiting or activate CaMKIIα in the shrew brainstem. Moreover, i.p. administration of the selective 5-HT2AR agonist, DOI, produces the head-twitch response in the least shrew 6R antagonists (Ro-046790 and Ro-04368554). Both antagonists failed to suppress 2-Me-5-HT-evoked vomiting in the least shrew. Since we have recently demonstrated cAMP/PKA signaling is involved in mediation of cyclophosphamide-induced emesis 6Rs can activate the cAMP/PKA cascade 6R nor its downstream signaling is involved in the induced vomiting (data not shown). Thus, the discussed findings strongly demonstrate that 5-HT3Rs (but not 5-HT2ARs or 5-HT6Rs) are specifically involved in the mediation of 2-Me-5-HT-induced emesis and related downstream signaling.Although 2-Me-5-HT is generally considered a 5-HT3R-mediated emesis: 2-Me-5-HT acts in both the brainstem DVC and the GIT emetic loci to increase extracellular influx of Ca2+ through both 5-HT3Rs and the L-type Ca2+ channels, which leads to CICR from intracellular ER calcium stores via RyRs. This 5-HT3R activation-induced increase in intracellular Ca2+ concentration initiates attachment of CaM to the 5-HT3R, and causes Ca2+-dependent activation of CaMKIIα which further results in ERK1/2 activation and vomiting  colocalization in the least shrew brainstem nucleus tractus solitaries (NTS) and dorsal motor nucleus of the vagus (DMNX).Effects of 2-Me-5-HT treatment on 5-HT Shrews were treated with 2-Me-5-HT  or vehicle for 20 min. 5-HT3R-CaM colocalization was determined through co-stained brainstem slices with 5-HT3R (red) and CaM (green). Graphs A and B are representative images (200×) of NTS (A) and DMNX (B). Nuclei were shown with DAPI stains. Scale bar, 10 µm.(TIF)Click here for additional data file.Figure S2Effects of 2-Me-5-HT treatment on pCaMKIIα in the least shrew brainstem nucleus tractus solitaries (NTS) and dorsal motor nucleus of the vagus (DMNX). Shrews were treated with 2-Me-5-HT  or vehicle for 20 min. CaMKIIα activation was determined through co-stained brainstem slices with CaMKIIα (red) and pCaMKIIα (green). Graphs A and B are representative images (100×) of NTS (A) and DMNX (B). Nuclei were shown with DAPI stains. Scale bar, 10 µm.(TIF)Click here for additional data file.


However, it is unclear whether a toxic cytosolic Ca2+elevation mediates the fungicidal activity of eugenol. In the present study, no significant difference in yeast survival was observed following transient eugenol treatment in the presence or absence of extracellular Ca2+. Furthermore, using yeast expressing apoaequorin to report cytosolic Ca2+ and a range of eugenol derivatives, antifungal activity did not appear to be coupled to Ca2+ influx or cytosolic Ca2+ elevation. Taken together, these results suggest that eugenol toxicity is not dependent on a toxic influx of Ca2+. In contrast, careful control of extracellular Ca2+ (using EGTA or BAPTA) revealed that tolerance of yeast to eugenol depended on Ca2+ influx via Cch1p. These findings expose significant differences between the antifungal activity of eugenol and that of azoles, amiodarone and carvacrol. This study highlights the potential to use eugenol in combination with other antifungal agents that exhibit differing modes of action as antifungal agents to combat drug resistant infections.Eugenol is a plant-derived phenolic compound which has recognised therapeutical potential as an antifungal agent. However little is known of either its fungicidal activity or the mechanisms employed by fungi to tolerate eugenol toxicity. A better exploitation of eugenol as a therapeutic agent will therefore depend on addressing this knowledge gap. Eugenol initiates increases in cytosolic Ca There is increasing interest in the use of plant-derived antimicrobial compounds as both natural preservatives and in the treatment of fungal infections. A driver for this interest is that antifungal therapies are limited to a few classes of compounds which are undermined by the emergence of resistance and tolerance coupled with innate toxicity of these compounds to the host organism Eugenol is the major constituent of essential oils from clove, cinnamon and bay leaves and is a member of a wider group of diverse amphipathic phenolic compounds which display antifungal activity 2+ in Saccharomyces cerevisiae and that their toxicity correlates with the amplitude and duration of cytosolic Ca2+ elevation; consequently, it has been proposed that the Ca2+ elevation represents a toxic shock responsible, at least in part, for the antifungal activity of these compounds 2+ elevation in yeast the cch1Δ mutant (which is deficient in a subunit of the yeast high affinity plasma membrane Ca2+ channel) exhibits reduced Ca2+ influx in response to eugenol but is hypersensitive to eugenol. This raises the exciting possibility that the mechanisms by which yeast respond to eugenol and the related compounds amiodarone and carvacrol differ and that a Cch1p-mediated Ca2+ influx forms part of a cell signalling response to enable yeast to survive eugenol stress.Previous studies have shown that amiodarone and carvacrol, two amphipathic phenolic compounds related to eugenol, induce increases in cytosolic Ca2+ in eugenol toxicity in detail. We show that the eugenol-induced cytosolic Ca2+ elevation in yeast is unlikely to represent a toxic burst of Ca2+. Importantly, Ca2+ influx appears to be limited to a signalling role that is crucial for protecting yeast against eugenol stress.The present study investigates the role of Camid1Δ and cch1Δ mutants were derived from the parental S. cerevisiae strain JK9-3da  by replacing the MID1 and CCH1 genes by a KanMX cassette 2Single and double vcx1Δ and pmc1Δ mutants were derived from the parental strain S. cerevisiae strain BY4742  by gene replacement with a KanMX cassette (EUROSCARF) and cultured in YPD contained 1% yeast extract and 2% peptone. All growth media contained 2% (w/v) glucose (and 2% (w/v) agar for solid media), and where indicated, supplemented with BAPTA ethane-N,N,N′,N′-tetraacetic acid), EGTA (ethylene glycol tetraacetic acid) or CaCl2 using stock solutions of BAPTA , EGTA  or 2 M CaCl2. pH was approximately 6.5 for YPD-based growth media and between 5.0 and 5.5 for SCM-based growth media. Eugenol, isoeugenol, estragole, o-eugenol, acetyl eugenol and methyl eugenol (Sigma Aldrich) were in liquid form and made to 100x stocks in ethanol and stored at 4°C.8 cells/ml). OD600 was determined after 8x dilution of culture in water. To obtain cells in mid-log growth phase, 0.5 ml of overnight cultures were subcultured into 10 ml of fresh SCM-leu to give an OD600 of 0.8 and incubated shaking at 150 rpm for up to 3 hours until an OD600 between 1.6 and 2.4 was reached. Cells were pelleted (at room temperature using 200 g for 5 minutes) and resuspended in fresh SCM-leu to an OD600 of 2.4. 4 µl of 0.5 mM coelentrazine  in absolute methanol was added to 1 ml of cells and incubated in the dark for 2 hours at 30 C shaking at 150 rpm. Coelentrazine loaded cells were pelleted in a microcentrifuge (5000 rpm for 20 seconds) and resuspended in fresh SCM-leu to an OD600 of 8. Luminescence from 20 µl samples of mid-log growth phase cells was recorded as previously reported 2, 10 mM HEPES, 2% glucose, pH 7.5 with Tris base), BAPTA buffer  or SCM-leu. Luminescence (expressed in arbitrary units (AU) per 0.2 seconds) was measured for up to 10 minutes after which cells were lysed with 1.6 M CaCl2 in 20% (v/v) ethanol to determine total (summed) luminescence. Total luminescence was in much greater excess over luminescence induced by eugenol (or eugenol derivatives) indicating that the availability of aequorin-coelentrazine complex was sufficient for the reporting induced Ca2+cyt elevations.Cells expressing apoaequorin were grown overnight in SCM-leu in a shaking (150 rpm) incubator to an optical density at 600 nm (OD600) of 8 (approximately 1×101 ml of overnight cultures of JK9-3da (and derived mutants) was added to 9 ml of fresh SCM-leu to an OD600 of 1.6 and incubated for approximately 4 hours shaking at 150 rpm until an OD600 between 3 and 4 was reached and cells were in mid-logarithmic growth phase. Culture was pelleted (200 g for 5 minutes) and resuspended in 5 ml of 2% glucose, repelleted and resuspended in 1 ml of 2% glucose. 0.5 ml cell samples were pelleted in a microcentrifuge (5000 rpm for 20 seconds) and resuspended in 0.5 ml of Ca buffer or BAPTA buffer and adjusted to an OD600 of 20. 100 µl of cells were pelleted and resuspended in 0.5 ml of corresponding buffer containing eugenol and incubated at room temperature for 10 minutes (unless otherwise stated). Following incubation, cells were pelleted in a microcentrifuge and resuspended in 1 ml of SCM-leu, repelleted and resuspended in 100 µl of SCM-leu. 10-fold serial dilution was performed in sterile water and 5 µl of each dilution was placed onto SCM-leu containing 2% agar. Images of plates were taken after two days growth.8 cells/ml. Following 10-fold serial dilution of each yeast strain using sterile water, 5 µl drops were spotted onto YPD  plates containing varying concentrations of eugenol. Images of plates were taken after two days growth at 30 C.JK9-3da (and derived mutants) were cultured overnight in 10 ml SCM-leu to an OD600 of 8, pelleted (200 g for 5 minutes) and resuspended in 10 ml of sterile water and resuspended to 0.5×102+ or BAPTA to 10−4 cells/ml and 1 ml aliquoted to wells except the initial well in which 2 ml was aliquoted. Eugenol, eugenol derivatives or BAPTA were added to the first well and a two-fold dilution series achieved by mixing, removing 1 ml and adding this to the second well; this was repeated for subsequent wells except the final well which served as a eugenol (or BAPTA)-free control. Plates were sealed and incubated shaking at 150 rpm for 48 hours after which OD600 was determined . For BY4742 strains, sensitivities to eugenol were determined as above except culture media was YPD. Student' t tests were performed on the IC50 values to determine p values and whether mean IC50 values were significantly differently.Sensitivities of yeast growth to eugenol (and derivatives) were assayed by a dilution method in 24 well plates as described by Edlind et al. 2+ in eugenol toxicity, 10 minute transient exposure of wild type (Jk9-3da) yeast to varying concentrations of eugenol were performed in the presence  the eugenol-induced cytosolic Camembrane  followednd Mid1p  revealin2+ elevation further, we compared the toxicity of a range of eugenol derivatives with their ability to induce cytosolic Ca2+ elevations (50) against the cytosolic Ca2+ elevation induced following addition of 3.2 mM eugenol (or derivative). Notably, both estragole (hydroxyl group replaced with methoxy group) and o-eugenol  exhibited significantly greater toxicity towards yeast (1.017±0.0435 mM and 0.949±0.0285 mM respectively) than eugenol (p<0.02 and <0.01 respectively) but with reduced elevation of cytosolic Ca2+. Isoeugenol however  was significantly more toxic to yeast than eugenol  and induced greater cytosolic Ca2+ elevation compared to that for eugenol. Taken together, these results show no correlation between Ca2+ elevation and toxicity and do not support a mode of action based on a toxic elevation in cytosolic Ca2+. Consistent with this, estragole and methyl eugenol both failed to evoke measurable Ca2+ elevation using a concentration (i.e. 3.2 mM) which is 2 to 3 times greater than the determined IC50. It is noteworthy that the IC50 values determined for the eugenol derivatives are unlikely to simply reflect differences in hydrophobicity because acetyl eugenol, isoeugenol and eugenol  have virtually identical hydrophobicity (log P) values of approximately 2.5 To investigate the relationship between toxicity and cytosolic Caevations . Figure 2+ elevation in response to amiodarone and carvacrol correlates with drug toxicity 2+ influx across the plasma membrane; reducing extracellular Ca2+ with EGTA blocks cytosolic Ca2+ elevation and rescuses growth inhibition by amiodarone 2+ influx 2+ mediating amiodarone and carvacrol toxicity, vma2Δ mutants which lack vacuolar H+ pumping activity (and as a consequence have impaired Ca2+ sequestration into the vacuole via Ca2+/H+ antiport) exhibit prolonged cytosolic Ca2+ elevations and have increased sensitivity to amiodarone and carvacrol 2+ from the cytosol and to negate eugenol toxicity using the pmc1Δ  indicating that a reduced capacity to remove Ca2+ from the cytosol across the vacuolar membrane did not affect eugenol toxicity. Taken together, these data are consistent with eugenol toxicity being independent of a general cytotoxic Ca2+ elevation.In contrast to the present study, the amplitude and duration of cytosolic CaS. cerevisiae to eugenol; the hyper-sensitivity of cch1Δ mutants suggested that a Cch1p-mediated Ca2+ influx may be necessary to protect yeast against the toxic effects of eugenol 2 or 10 mM EGTA (to increase or reduce extracellular Ca2+ respectively) were conducted. Reducing extracellular Ca2+ reduced the tolerance of the wild type (Jk9-3da) and mid1Δ strains to levels similar to that observed for the cch1Δ mutants  was a crucial factor in determining the tolerance of  mutants . Furtheriae e.g.  Cch1p act 3.2 mM , A & G bt 3.2 mM  indicate2+ more precisely, growth experiments were conducted using a Ca2+-free synthetic complete medim (SCM-leu-Ca); this permitted a more robust yet subtle control of extracellular Ca2+ levels using the Ca2+ chelator, BAPTA (which exhibits higher specificity for Ca2+ and is less pH sensitive than EGTA). 50 of eugenol (the concentration of eugenol resulting in 50% inhibition of growth when compared to growth in the absence of eugenol) for the wild type strain was strongly dependent on extracellular Ca2+. In the presence of 100 µM extracellular Ca2+, the IC50 of eugenol for wild type yeast was 1.62±0.133 mM and this decreased to 1.27±0.031 and 0.96±0.043 mM in the presence of 0.05 and 0.2 mM BAPTA, respectively. Importantly, the growth of wild type yeast in the absence of eugenol was unaffected by the presence of 0.05 and 0.2 mM BAPTA  was similar to that for wild type yeast in the presence of 0.2 mM BAPTA (0.96±0.043 mM) indicating that the Ca2+-dependent tolerance to eugenol exhibited by wild type yeast is dependent on Ca2+ influx via Cch1p. Furthermore, the cch1Δ mutant was more sensitive to reductions in extracellular Ca2+ than wild type yeast as it was unable to grow in media containing more than 0.0625 mM BAPTA  is also enhanced by extracellular Ca2+ sequestration (using 1 mM EGTA); however, in contrast to that observed with eugenol, the addition of 3 mM Ca2+ to the growth media reduced azole activity by 3-fold 2+ influx and calcineurin activation in the azole tolerance mechanisms employed by yeast. Interestingly, eugenol tolerance is independent of calcineurin activation 2+-dependent tolerance mechanisms employed by yeast in response to the antifungal properties of eugenol and the commercially available azoles.Taken together, the data show that Ca eugenol  is in ag eugenol  and is c2+ elevation is unlikely to be responsible for eugenol toxicity in yeast and that the role of Ca2+ in eugenol toxicity appears confined to a Cch1p-dependent Ca2+ influx which is necessary to enhance eugenol tolerance in yeast. Although the downstream targets of the resulting Ca2+ signal remain unknown, our data suggest that the signalling pathways employed by yeast to tolerate eugenol toxicity will be distinct to those employed in azole and amiodarone tolerance. This is supported further by the discovery that eugenol and amiodarone employ different modes of action with respect to antifungal activity. These differences highlight the potential to use eugenol in combination therapies which aim to augment the efficacy of commercially available azoles and other promising antifungal drugs.In conclusion, our data show that a toxic elevation in cytosolic CaFigure S1Viability of cch1Δ (A and G), mid1Δ (B and H) and cch1Δmid1Δ (C and I) cells after 10 minutes exposure to varying concentrations of eugenol suspended in Ca buffer  and BAPTA buffer . Yeast cultures are spotted on to SCM-leu media containing 2% agar; left most spots are growth after 2 days following inoculation with 5 µl of culture. Serial 10-fold dilution of the left most inoculum is shown to the right. Ca2+-dependent aequorin luminescence from cch1Δ (D and J), mid1Δ (E and K) and cch1Δmid1Δ (F and L) cells in response to 3.2 and 9.6 mM eugenol in Ca buffer  and BAPTA buffer . Eugenol was added at 40 seconds (indicated by arrow). Traces represent mean (± SEM) from at least 5 independent experiments. SEM vales are illustrated using grey shading. Luminescence was recorded every 0.2 seconds and is expressed in arbitrary units (AU). Inset is data from the main figure on an expanded y axis.(TIF)Click here for additional data file.


Here we demonstrate that increase in intracellular Ca2+ via transient receptor potential canonical channel-1 (TRPC1) regulates autophagy, thereby preventing cell death in two morphologically distinct cells lines. The addition of DMOG or DFO, a cell permeable hypoxia-mimetic agents, or serum starvation, induces autophagy in both epithelial and neuronal cells. The induction of autophagy increases Ca2+ entry via the TRPC1 channel, which was inhibited by the addition of 2APB and SKF96365. Importantly, TRPC1-mediated Ca2+ entry resulted in increased expression of autophagic markers that prevented cell death. Furthermore, hypoxia-mediated autophagy also increased TRPC1, but not STIM1 or Orai1, expression. Silencing of TRPC1 or inhibition of autophagy by 3-methyladenine, but not TRPC3, attenuated hypoxia-induced increase in intracellular Ca2+ influx, decreased autophagy, and increased cell death. Furthermore, the primary salivary gland cells isolated from mice exposed to hypoxic conditions also showed increased expression of TRPC1 as well as increase in Ca2+ entry along with increased expression of autophagic markers. Altogether, we provide evidence for the involvement of Ca2+ influx via TRPC1 in regulating autophagy to protect against cell death.Autophagy is a cellular catabolic process needed for the degradation and recycling of protein aggregates and damaged organelles. Although Ca In addition, since autophagy delivers a fresh pool of amino acids and other essential molecules to the cell, initiation of autophagy is highly beneficial particularly during nutritional stress situations or tissue remodeling during development and embryogenesis.6 Consequently, impaired or altered autophagy is often implicated in several pathologies, like neurodegenerative disorders and cancer,9 which again highlight its importance.Autophagy is a cellular process responsible for the delivery of proteins or organelles to lysosomes for its degradation. Autophagy participates not only in maintaining cellular homeostasis, but also promotes cell survival during cellular stress situations.2+ has a vital role in the regulation of a large number of cellular processes such as cell proliferation, survival, migration, invasion, motility, and apoptosis.11 To perform functions on such a broad spectrum, the cells have evolved multiple mechanisms regulating cellular Ca2+ levels, mainly by regulating the function of various Ca2+ channels present in different locations. Mitochondrial, ER, lysosomal, and cytosolic Ca2+ levels are regulated by Ca2+ permeable ion channels localized either on the membranes of the intracellular organelles or on the plasma membrane.10 The Ca2+ permeable channels, including families of TRPCs, Orais, voltage-gated, two-pore, mitochondrial Ca2+ uniporter, IP3, and ryanodine receptors have all been identified to contribute towards changes in intracellular Ca2+ ([Ca2+]i).14 Channels of the TRPCs and Orai families have been related to several Ca2+-dependent physiological processes in various cell types, ranging from cell proliferation to contractility, to apoptosis under both physiological and pathological conditions.12 Moreover, it has been suggested that intracellular Ca2+ is one of the key regulators of autophagy;15 however, the possible role of Ca2+ in autophagy is still inconclusive. Many reports also suggest that Ca2+ inhibits autophagy,18 whereas others have indicated a stimulatory role for Ca2+ towards autophagy.21 Furthermore, the identity of the major Ca2+ channel(s) involved in autophagy is not known. Members of the TRPC family have been suggested as mediators of Ca2+ entry into cells. Activation of the G-protein (Gq/11–PLC pathway) leads to the generation of second messenger IP3.22 IP3 binds to the IP3R, which initiates Ca2+ release from the ER stores, thereby facilitating stromal interacting molecule-1 (STIM1) to rearrange and activate Ca2+ entry via the store-operated channels.22 Two families of proteins (TRPCs and Orais) have been identified as potential candidates for SOC-mediated Ca2+ entry.22 However, their role in autophagy has not yet been determined. Thus, here we investigated the role of Ca2+ entry channels (TRPCs and Orais) in autophagy and show that both hypoxia-mimetic and nutrient depression induces autophagy in two different cell lines. Furthermore, our data indicates that autophagy was dependent on TRPC1-mediated increase in intracellular Ca2+ levels, suggesting that TRPC1 has an important role in regulating autophagy and inhibiting cell death.Ca23 Thus, we also evaluated the expression of p62, which was significantly decreased in DMOG, DFO, and serum-deprived conditions  cells and neuroblastic SHSY-5Y cells were treated with 1 mM DMOG or DFO or were subjected to serum-free media. After treatment, the cells were lysed and expressions of the autophagic marker proteins were analyzed using western blotting. Both autophagic markers Beclin-1 and LC3A were upregulated in both cells treated with DMOG or DFO or in serum-deprived conditions . Similarnditions . To haveutophagy . AlthougTo confirm that hypoxia-mimetic agents, DMOG and DFO, induce autophagy that inhibits apoptosis, cell viability assays were performed. Treatment with 1 mM DMOG or 1 mM DFO showed no significant decrease in cell viability in both SHSY-5Y and HSG cells . Moreove2+ entry has been suggested to have an essential role in regulating cell survival.26 Furthermore, alterations in intracellular Ca2+ levels has been shown to regulate autophagy.15 Thus, we next evaluated Ca2+ levels in both epithelial and neuronal cells. Cells were pretreated with 1 mM DMOG or 1 mM DFO or in serum-free media for 6 h and were then incubated with Fura-2 to measure intracellular Ca2+ levels in both SHSY-5Y and HSG cells. To evaluate Ca2+ entry, ER Ca2+ stores were depleted by the addition of thapsigargin . Importantly, in the absence of extracellular Ca2+, the increase in [Ca2+]i evoked by Tg (first peak) was not significantly different in DMOG-treated cells, when compared with untreated HSG cells (2+ (1 mM), which initiates store-mediated Ca2+ entry, was significantly increased in DMOG-treated HSG cells (2+ entry was observed in HSG cells that were serum starved (2+ entry (data not shown). We next compared if these effects are same in SHSY-5Y cells, and all hypoxic-inducing treatments again resulted in an increase in [Ca2+]i activated by store depletion in SHSY-5Y . The cell viability was also affected when cells were pretreated with DMOG or DFO in the presence of TRPC channel inhibitor SKF, where a significant decrease in cell survival was observed (2-Aminoethoxydiphenyl borate (2APB) and SKF96365 hydrochloride (SKF) are potent TRPC channel inhibitors; of 2APB  and SKF  of 2APB . Similarobserved . Pretreaobserved . Moreoveobserved . Similar2+ entry channel, electrophysiological recordings were performed in hypoxia conditions. The addition of Tg caused the appearance of an inward current, which reversed between 0 and −5 mV in SHSY-5Y cells (28 which was induced by store deletion (baseline currents without Tg are shown in I–V curves), reversal potential around 0 mV, slightly inward rectifying and nonselective, suggesting that TRPC1 could contribute to the endogenous Ca2+ entry channel in these cells. Importantly, DMOG and DFO treatment significantly facilitated TRPC1-mediated Ca2+ currents without altering the current–voltage (I–V) relationship  involved in this process, cell lysates were obtained under these conditions and were used to evaluate the expression of various proteins involved in Ca2+ entry. Importantly, cells pretreated with 1 mM DMOG, or 1 mM DFO, or in serum-free media showed a significant increase in TRPC1 levels . Hereby, our data indicate that autophagy was dependent on TRPC1, suggesting that TRPC1 has an important role in regulating hypoxia inducing autophagy and inhibiting cell death.To establish the identity of the Ca5Y cells  and in H5Y cells . The chationship . Moreovecurrents . To furt1 levels . In cont1 levels . TRPC1 e1 levels . To furt2+ entry and could modulate autophagy. Thus, to study the importance of TRPC1 channels in hypoxia and serum depletion-induced autophagy, we knocked down TRPC1 in both SHSY-5Y and HSG cells using siRNA  a known autophagy inhibitor,4 was used. The cells were pretreated with 1 mM 3-MA, along with DMOG, or DFO, or serum-free media and protein expression of STIM1, Orai1, TRPC1, and autophagy markers were analyzed. Importantly, cells treated with 3-MA showed no increase in DMOG or serum-deprived increase in TRPC1 expression  receptor (IP3R).16 In Huntington's disease cell model, lithium ions have been shown to stimulate autophagy in a novel mTOR-independent manner.40 Lithium acted through inhibition of the inositol monophosphatases, thereby reducing the IP3 levels, which reduced the stimulatory effect of lithium on autophagy. This is consistent with the IP3R triple knockout chicken DT40 cells, which showed higher autophagy levels than their wild-type counterparts, verifying the IP3R-mediated inhibition of autophagy.18 Autophagy has also been shown to be initiated by chemical inhibition of IP3Rs or suppressing its expression thereby suppressing the Ca2+ signaling.41 This is consistent with our data as loss of IP3R will have a decrease in TRPC1-mediated Ca2+ entry. Although we have not fully identified the mechanism as to how TRPC1 regulates autophagy, it has been shown that IP3Rs facilitate the formation of anti-autophagic Bcl-2–Beclin-1 complex thereby decreasing the amount of free Beclin-1 available to induce autophagy. We suggest that perhaps similar mechanism might be present where binding of IP3R with TRPC1 will inhibit the formation of anti-autophagic complex and more research is needed to understand the mechanism via which TRPC1 regulates autophagy. Binding of xestospongin-B to the IP3R also induced the dissociation of the complex, which was prevented by Bcl-2 overexpression, whereas siRNA against Bcl-2 abolished the IP3R–Beclin-1 interaction.17The notion that Ca2+ mobilizing agents, like ionomycin, ATP, vitamin D3, sarco/endoplasmic reticulum Ca2+ ATPase (SERCA) inhibitors thapsigargin and alisol have been reported to act as an activator of autophagy by elevating [Ca2+]i.43 Our data not only support these studies, but also identify that Ca2+ entry via TRPC1 is essential for autophagy. Of course, prolonged treatment with these agents will lead to ER Ca2+ depletion and subsequent ER stress, which itself might also be a trigger for autophagy. The addition of BAPTA-AM, a fast and potent intracellular Ca2+ buffer, however, prevented the induction of autophagy, indicating the importance of cytosolic Ca2+.44 The thapsigargin-induced autophagy was still present in unfolded protein response (UPR)-deficient cells, suggesting again a direct involvement of Ca2+ in the induction of autophagy.45 The nutrient deprivation-induced autophagy was also inhibited by BAPTA-AM,46 suggesting that other autophagy inducers  might also lead via enhanced Ca2+ signaling to autophagy induction,15 which is again consistent with the data presented here. Although our data suggest that TRPC1 was important for autophagy , we cannot completely rule out the possible role of other Ca2+ channels in this process. Gordon et al.31 already suggested the dependence of autophagy on intracellular stored Ca2+ and most Ca2+-mobilizing agents promote Ca2+ release from the ER.15 Ca2+-dependent autophagy is also enhanced by the pharmacological agent PK11195.47 Similarly, it has been shown that inhibition of plasma membrane Ca2+ channels prevented the autophagy induction caused by the increase of intra-axonal Ca2+ in the rat optic nerve after crush lesion;48 however, the source as well as the Ca2+ channels responsible for this has not been identified. These results along with the data presented here clearly show that the ER is probably the main, but not the only origin for Ca2+ during Ca2+-induced autophagy. Lowering of ER [Ca2+] also prevents apoptosis driven by mitochondrial Ca2+ overload, underscoring that both autophagy and apoptosis are closely related.15 Our data also suggest that Ca2+ entry via the TRPC1 channel was initially protective. However, most of the compounds used for Ca2+-induced autophagy have also been shown to promote apoptosis.39 Therefore, one might propose that autophagy is indirectly activated by Ca2+ that tries to counter apoptosis20 to protect these cells and loss of these functions could perhaps lead to apoptosis. Here, thus we propose a model where increase in intracellular Ca2+ via TRPC1 channel promotes autophagy before the activation of apoptosis and increases cell survival.Ca50 Cells were maintained at 37 °C with 95% humidified air and 5% CO2 and were passaged as needed. Culture medium was changed twice weekly and cells were maintained in complete media, until reaching 90% confluence. For RNAi experiments, siRNA that targets the coding sequence of human TRPC1 or TRPC3 were obtained from Ambion  and a FITC-conjugated non targeting siRNA was used as control. Cells were transfected with individual siRNA (50 nM) using Lipofectamine 2000 in Opti-MEM medium as per supplier's instructions  and assayed after 48 h. Antibodies that were used in this study are described in the figures. All other reagents used were of molecular biology grade obtained from Sigma  chemicals unless mentioned otherwise. To subject mice to chronic hypoxia, mice were placed in a plexiglass chamber maintained at 21%  or 10% O2 (hypoxic) by controlling the inflow rates of air and nitrogen. The O2 concentration was monitored continuously and after 3 days, mice were anesthetized and salivary glands were obtained and used for the study.Human salivary gland (HSG) cells and SHSY-5Y neuroblastoma cells were cultured in their respective medium along with various supplements, as previously described.5 cells/well. The cultures were grown for 24 h followed by the addition of fresh medium before the experiment. Cell viability was measured by using the MTT method. Twenty microliters of MTT reagent (5 mg/ml MTT in PBS) was added to each well and incubated in a CO2 incubator for 4 h. The resulting formazan dye was extracted with 100 μl of 0.01 N HCl in isopropanol and the absorbance was measured in a micro plate reader  at 570 and 650 nm. Cell viability was expressed as a percentage of the control culture.Cells were seeded on 96-well plates at a density of 0.5 × 10et al.51 and Sukumarn et al.52 Protein concentrations were determined using the Bradford reagent , and 25–50 μg of proteins were resolved on 4–12% SDS-Tris-acetate gels, transferred to PVDF membranes and probed with respective antibodies Peroxidase conjugated respective secondary antibodies were used to label the proteins. The proteins were detected by enhanced chemiluminescence detection kit . Densitometric analysis was performed using imageJ analysis and results were corrected for protein loading by normalization for β-actin expression as described in Singh et al.55 For cell surface biotinylation, cells were treated as required and incubated for 20 min with 1.5 mg/ml Sulfo-NHS-LC-Biotin (Pierce) in PBS (pH 8.0) on ice and followed the protocol as mentioned before.56 Following biotin labeling, cells were washed and solubilized. Biotinylated proteins were pulled down with NeutrAvidin-linked beads (Pierce), resolved on 4–12% SDS-Tris-acetate gels, and individual proteins were detected by western blotting.Cells were collected and stored at −80 °C. Crude lysates were prepared from SH-SY5Y, HSG cells, and salivary tissues, as described previously in Singh For the immunofluorescence assays, the SH-SY5Y and HSG cells were grown overnight on coverslips. Cells were transfected with fluorescent-tagged LC3A plasmid using Lipofectamine 2000 in Opti-MEM medium as per supplier's instructions (Invitrogen) and treated with the desired chemicals. After 24 h, the cells were washed twice with phosphate-buffered saline, and fixed for 30 min using 3% paraformaldehyde. Confocal images were collected using a MRC1024-krypton/argon laser scanning confocal equipped with a Zeiss LSM 510 Meta photomicroscope .μM fura-2  for 45 min, washed twice with Ca2+-free SES  buffer. For fluorescence measurements, the fluorescence intensity of Fura-2-loaded control cells was monitored with a CCD camera-based imaging system  mounted on an Olympus  XL70 inverted microscope equipped with an Olympus × 40 (1.3 NA) objective. A monochrometer dual wavelength enabled alternative excitation at 340 and 380 nm, whereas the emission fluorescence was monitored at 510 nm with an Okra Imaging camera . The images of multiple cells collected at each excitation wavelength were processed using the C imaging, PCI software (Compix), to provide ratios of Fura-2 fluorescence from excitation at 340 nm to that from excitation at 380 nm (F340/F380). Dispersed salivary gland cells were placed on glass-bottom poly-D lysine plates and used for the study. Fluorescence traces shown represent [Ca2+]i values that are averages from at least 30–40 cells and are a representative of results obtained in at least three to four individual experiments. [Ca2+]i in individual cells was estimated on the basis of the Grynkiewicz equation formula: [Ca2+]i=KD × B × (R−Rmin)/(Rmax−R), where KD is the indicator's dissociation constant for Ca2+(0.22 μM); R is ratio of fluorescence intensity at two different wavelengths (340/380 nm); Rmax and Rmin are the ratios of Ca2+-free and Ca2+-bound Fura-2, respectively; and B is the ratio of the fluorescence intensity of the second excitation wavelength at zero and saturating Ca2+ concentrations.57 The bar diagram shown in the figures represents the [Ca2+]i in nanomolar concentrations.Cells were incubated with 2 2, 1; CaCl2, 1; Hepes, 10; Glucose, 10; pH 7.3 (NaOH). For Na-based divalent ion-free (DVF), the external solutions contained (mM) 165 NaCl, 5 CsCl, 10 EDTA, 10 HEPES and 10 glucose, pH 7.4. Whole-cell currents were recorded using an Axopatch 200B . The patch pipette had resistances between 3 and 5 M after filling with the standard intracellular solution that contained the following (mM): cesium methane sulfonate, 150; NaCl, 8; Hepes, 10; EGTA, 10; pH 7.2 (CsOH). With a holding potential 0 mV, voltage ramps ranging from −100 mV to +100 mV and 100 ms duration were delivered at 2-s intervals after whole-cell configuration was formed. Currents were recorded at 2 kHz and digitized at 5–8 kHz. pClamp 10.1 software was used for data acquisition and analysis. Basal leaks were subtracted from the final currents and average currents are shown. All experiments were carried out under room temperature.For patch clamp experiments, coverslips with cells were transferred to the recording chamber and perfused with an external Ringer's solution of the following composition (mM): NaCl, 145; CsCl, 5; MgCl*P<0.05 or **P<0.01, respectively.Data analysis was performed using MicroSoft Excel or Origin 7.0 (OriginLab) and Graphpad prism 6.0. Statistical comparisons were made using one-way ANOVA. Experimental values are expressed as means±S.E.M. Differences in the mean values were considered to be significant at


Oscillatory spikes rely on repetitive exchanges of Ca2+ between the endoplasmic reticulum (ER) and the cytosol, due to the regulation of inositol 1,4,5-trisphosphate receptors. Mitochondria also sequester and release Ca2+, thus affecting Ca2+ signalling. Mitochondrial Ca2+ activates key enzymes involved in ATP synthesis. We propose a new integrative model for Ca2+ signalling and mitochondrial metabolism in electrically non-excitable cells. The model accounts for (1) the phase relationship of the Ca2+ changes in the cytosol, the ER and mitochondria, (2) the dynamics of mitochondrial metabolites in response to cytosolic Ca2+ changes, and (3) the impacts of cytosol/mitochondria Ca2+ exchanges and of mitochondrial metabolism on Ca2+ oscillations. Simulations predict that as expected, oscillations are slowed down by decreasing the rate of Ca2+ efflux from mitochondria, but also by decreasing the rate of Ca2+ influx through the mitochondrial Ca2+ uniporter (MCU). These predictions were experimentally validated by inhibiting MCU expression. Despite the highly non-linear character of Ca2+ dynamics and mitochondrial metabolism, bioenergetics were found to be robust with respect to changes in frequency and amplitude of Ca2+ oscillations.Oscillations of cytosolic Ca Mitochondria sequester and release Ca2+, thereby affecting the shape, the frequency and the amplitude of the Ca2+ spikes in the cytosol122+ ([Ca2+]m) linked to the transfer of Ca2+ from the cytosol to mitochondria during [Ca2+]c signals stimulates mitochondrial metabolism and allows the coupling of ATP supply with energy demand456In most organisms, mitochondria play an important role in ATP production and act as Ca2+]m and [Ca2+]c are similar, in the 100 nM range3) is produced and triggers cytosolic Ca2+ oscillations2+ between the cytosol and the endoplasmic reticulum (ER), through the biphasic regulation of the IP3 receptor (IP3R) by cytosolic Ca2+. Fast activation and slow inhibition of the opening of the IP3R by Ca2+ indeed suffice to generate either Ca2+ oscillations in classical deterministic models or repetitive spiking, if noise is considered to play a predominant role in cellular Ca2+ dynamics10112+ entry into mitochondria occurs through a multistep mechanism. By extruding protons out of mitochondria, the respiratory chain creates a large inside-negative potential difference across the inner mitochondrial membrane. This ΔΨ, which is harnessed by the ATP synthase for the production of ATP, allows the Mitochondrial Calcium Uniporter (MCU) to transport Ca2+ inside mitochondria132+ entry then depolarizes the mitochondria, thus reducing its own driving force. When [Ca2+]c returns to its basal value, extrusion of Ca2+ out of mitochondria occurs through both a Na+-Ca2+ exchanger (NCX) and a H+-Ca2+ exchanger, possibly identified as LETM1, although the contribution of this channel to mitochondrial Ca2+ transport is not yet firmly established1516At rest, [Ca1Fo-ATPase to phosphorylate ADP into ATP. An increase in [Ca2+]m activates metabolism as pyruvate dehydrogenase and two rate-limiting enzymes of the TCA cycle, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, are up-regulated by Ca2+2+ from the cytosol into mitochondria allows for the enhancement of mitochondrial ATP production by the F1Fo-ATPase. Cytosolic Ca2+ also directly influences mitochondrial metabolism as a component of the MAS, the aspartate-glutamate carrier (AGC), is stimulated by modest increases in cytosolic Ca2+2+ and mitochondrial dynamics, it is useful to resort to computational modelling to clarify their interplay. Several models have been proposed. Many of them focus on mitochondrial metabolism, but less on the effect of this metabolism on Ca2+ signalling20212223242+ exchanges to incorporate Ca2+ handling by mitochondria. These models shed new light on important specific questions, such as the effect of mitochondria on the amplitude of the Ca2+ spikes2+ oscillationsβ-cells2+ wave propagation in mitochondrial suspensionsUptake by mitochondria of pyruvate, the end product of cytosolic glycolysis, is at the onset of the oxidative phosphorylation cascade. A pyruvate dehydrogenase transforms substrates into acetyl-CoA which enters the Krebs cycle, also called the acid citric cycle or tricarboxylic acid cycle (TCA). This 9-step cycle converts the chemical energy of pyruvate into the reducing power of NADH. In addition, the activity of the malate-aspartate shuttle (MAS) also increases mitochondrial NADH2+ in non-excitable cells. These observations concern the effect of Ca2+ on mitochondrial metabolism, as well as changes in cytosolic Ca2+ dynamics occurring when the kinetic properties of the mitochondria are modified. The model is mainly built from the combination of previously published kinetic expressions. One of our aims is to clarify this complex issue by using simple expressions when possible and by motivating the resort to complex mathematical expressions when necessary. This is for example the case for the kinetics of the MCU that we analysed in details. We also found that a reversible Ca2+ leak between the cytosol and the mitochondria, which could correspond to the low conductance mode of the mitochondrial permeability transition pore, was necessary to account for a number of experimental observations. The phase relationship between Ca2+ peaks in the cytosol, the ER, and the mitochondria32Here we extend these previous studies to propose a model accounting for a variety of already published experimental observations centred on Ca2+ oscillations. We tested this counter-intuitive prediction by silencing the MCU in HeLa cells. We then switched to the validation of the model concerning mitochondrial metabolism by comparing computationally obtained time series with the evolution of NADH2+ spike2+ oscillations42+ spikes. Besides a strong robustness of mitochondrial metabolism with respect to the characteristics of the cytosolic Ca2+ changes, the model predicted that ATP synthesis by mitochondria is most efficient for frequencies and amplitudes of Ca2+ spikes usually observed experimentally.We found that increasing the activity of the MCU first increases and then decreases the frequency of Ca2+ microdomains. More specifically, we do not explicitly incorporate MAM (mitochondria-associated ER membranes) in the modelThe processes considered in the model are schematized in 2+ dynamics272+ and H+. The model is defined by 7 evolution equations and 4 conservation relations given here below ). The fluxes appearing in these equations are defined by Eqs. We adopt a fully deterministic approach, which is appropriate to obtain predictions about the average effect of the different individual fluxes on Ca2+ concentrationCytosolic Cac is the Ca2+ buffering capacity of the cytosol. The different volumes of the compartments are taken into account via α = VER/Vc and δ = Vm/Vc. Jx is a bidirectional Ca2+ leak . Ca2+ exchanges with the extracellular medium are not taken into account since they do not play a major role in the interplay between cytosolic and mitochondrial Ca2+ during the early spikes following stimulation2+ fluxes through the mitochondrial Ca2+/H+ exchanger. Although this is not firmly established2+/H+ exchanger is thought to correspond to the LETM1 protein2+ export during physiological stimulation of HeLa cellsUsing the framework of rapid buffering approximation2+ concentrationm is the Ca2+ buffering capacity of mitochondria.Mitochondrial Ca3 receptors3 receptors by Ca2+10Fraction of inactivated IPMitochondrial NADH concentrationMitochondrial ADP concentrationCytosolic ADP concentrationp is a constant that includes both the membrane capacitance and the Faraday constant.Voltage difference across the inner mitochondrial membrane (positive corresponding to an excess of positive charges in the cytosol)2+ER is the Ca2+ buffering capacity of the ER.Conservation of intracellular CaConservation of total (oxidized and reduced) NADHConservation of di- and trisphosphorylated adenine nucleotides in mitochondriaConservation of di- and trisphosphorylated adenine nucleotides in the cytosol2+ transporters, known as SCaMCs, mediate net transfer of ADP and/or ATP to the mitochondrial matrix. However, on the short time scales investigated in this study, we expect this transporter to have a minor role since SCaMCs are much slower than the adenine nucleotide translocases (ANT) and have a low affinity for cytoplasmic Ca2+The conservation relations described by Eqs. (10) and  do not awhere 2+ flux through the IP3 receptor that is rapidly activated at low cytosolic Ca2+ and more slowly inhibited at high cytosolic Ca2+3 that is taken as a parameter whose value directly reflects the level of cell stimulation.This is a classical expression for the Ca2+ flux through an unidirectional SERCA ATPase, that transports Ca2+ from the cytosol into the ER, against the concentration gradient, using the energy provided by the hydrolysis of ATPIt represents the Ca2+ fluxes and metabolism. Most of these processes have been already individually described by complex models quantitatively accounting for energetic-redox mechanisms under various (patho) physiological conditions. Assembling these models would lead to an extremely complex network. Our aim here is to build an intelligible model, centred around Ca2+ signalling and its interplay with mitochondrial metabolism. The kinetic expressions used are thus a trade-off between complexity and appropriate conceptualization.We now describe the kinetic expressions for mitochondrial CaMCU stands for the flux of Ca2+ from the cytosol into mitochondria that occurs through the MCU, defined with respect to the mitochondrial volume. This flux is cooperatively stimulated by cytosolic Ca2+ through the MICU1 subunit422+ uniporter), it is in agreement with the observed properties of this channel. 2+ concentration (black curve). This curve, entirely based on the expression and the parameter values proposed by Magnus and Keizeret al. (2013) on the MCU2+ is removed (K2 → ∞), the curve (in red) becomes less sigmoidal and is shifted to the left, as observed for the MICU1 knock-down. As shown by the blue curve, the simplified expression proposed by Bertram et al.2+] > 1 μM. Eq. 2+ cannot bind to the transporter when subunits are in the so-called “tense” form. Concerning the dependence of this rate on the mitochondrial potential, we have simplified the expression used by Magnus and Keizer and simply used Eyring’s theory. Thus, the rate constant of Ca2+ transport increases with the potential difference across the mitochondrial membrane in an exponential manner. As explained in the ‘Results’ section, this change was moreover required to account for the observation that energising mitochondria leads to a decreased frequency of Ca2+ oscillations1Jet al.2+ extrusion out of mitochondria mediated by the Na+/Ca2+ exchanger, defined with respect to the mitochondrial volume. This channel exchanges 1 Ca2+ for 3 Na+ and is thus electrogenic. It is assumed that the cytosolic Na+ concentration remains constant and that channel activity is favoured by a large ratio of Ca2+ concentrations between the cytosol and mitochondria. As reported previouslyc towards Cm). The dependency on the potential has the same form as for the MCU.This is the expression proposed by Bertram m does not drop in MCU-knocked down cells2+ from the cytosol at nanomolar concentrations, at which the MCU is inactivec oscillations when the NCX is totally inhibited2+ accumulation in mitochondria and arrest of oscillations. As explained in the Results section, we hypothesize that this flux may reflect the low conductance state of the mitochondrial Permeability Transition Pore (mPTP)This flux, defined with respect to the mitochondrial volume, was not considered in previous models. Its molecular nature remains to be fully identified (see ‘Results’). We found necessary to consider this flux to account for many experimental results and, in particular, for the fact that Cet al.2+-sensitive reversible phosphorylation. This rate not only accounts for the Pyruvate DeHydrogenase(PDH)-catalysed reaction but gathers the glycolytic pathway (kGLY) and the Krebs cycle. The Krebs cycle reduces NAD+ into NADH, hence the dependency on the adenine nicotinamide ratio. [NAD+] is computed from the conservation relationship (Eq. (9)). We have modified the value of et al.2+, the latter one occurring at the levels of both the isocitrate- and the α-ketoglutarate dehydrogenases. In agreement with experimental dataD value for activation by Ca2+ (q2) is taken equal to 0.1 μM.This expression is taken from Bertram 2+ increases (100 nM < S0.5 < 350 nM)182+ levels at which the MCU becomes active, although the cross-talk between the Ca2+ activation of MAS and the mitochondrial dehydrogenases may vary from tissue to tissue. By activating the Krebs cycle, mitochondrial Ca2+ increase would lead to a decrease in the amount of α-ketoglutarate, a key-metabolite of the MAS182+ with a threshold value q2 that is the same as the threshold for activation of the Krebs cycle by mitochondrial Ca2+. As the AGC is electrogenic, JAGC also appears in Eq. (7).The aspartate-glutamate carrier is part of the MAS NADH shuttle. The two mammalian carriers, aralar and citrin, are activated by moderate cytosolic CaO thus represents both the rate at which NADH is oxidized (Eq. (4)) and the rate at which H+ are extruded. In the rate expression for JO O is multiplied by a1 to scale the NADH production into a change in voltage due to the proton flux. Eq. et al.Downstream the Krebs cycle, NADH is then oxidized in the Electron Transport Chain (ETC) to extrude protons from mitochondria. Jr JO Eq. , the cha. et al. 062028. Ac-ATPm; ATPc-ADPm; ADPc-ATPm; ADPc-ADPm). The α’s stand for the fact that only a fraction of nucleotides has access to the transporter. ADP and ATP are negatively charged , which explains the potential dependence of this flux. It is defined with respect to the mitochondrial volume. This equation has been erroneously transcribed and simplified in previous publicationsThis rather complex expression1Fo-ATPase. This expression, proposed by Bertram et al.m (i.e. with decreasing ADPm), but for a steep sigmoidal dependency on the mitochondrial potential. ATP synthesis is driven by the proton flux from the cytosol into the mitochondria, which depolarizes the membrane. Thus JF1FO also enters the evolution equation for ΔΨ (Eq. (7)), with the scaling factor a2.This is the rate of ATP synthesis by the F2+ activity and ATP consumption in the cytoplasm. SERCA pumps  or with two pooled siRNA sequences against the MCUHeLa cells were from ATCC and were grown in RPMI medium containing 5% fetal calf serum in a 5% COThe equivalent of 4 × 105 cells were scraped in Laemmli-loading buffer. Samples were subjected to Western-blot analysis using the indicated primary antibody followed by anti-rabbit HRP-conjugated antibody. Detection was performed using ECL plus reagent .2+ variations was performed by loading cells with 3 μM Fluo-4-AM (Invitrogen) in EM buffer containing 120 mM NaCl, 7 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, 5 mM glucose, 25 mM HEPES pH 7.3, as described previouslyAnalysis of Ca2+ oscillations occur in the cytosol, the ER and the mitochondria with well-defined phase relationships2+ concentrations in the cytosol (black), the ER (red) and the mitochondria (blue) in the presence of first a relatively high (from 0 to 200 s), followed by a low IP3 concentration. The period of oscillations is of the order of 30 s, while their half-width is ~1/5 of the period. A detailed view of one peak is shown in 2+ in the cytosol, during sustained oscillations. From this starting point, Ca2+ slowly increases both in the cytosol and in the ER, while Cm is still decreasing. Thus, release of mitochondrial Ca2+ is responsible for the increase in Cc, which itself allows the replenishment of the ER. The rise in Cc then stimulates the IP3R and CER starts decreasing fast. Cm only starts increasing during the fast rising phase of the cytosolic Ca2+ peak. Then, Cc reaches its maximum, slightly before CER gets to its minimum value , the frequency of cytosolic Ca2+ oscillations is lower, but the amplitude is higher. In addition to these modifications, mitochondria also alter the shape of the cytosolic Ca2+ peak. Indeed, when JMCU = JNCX = Jx = 0, the rate of cytosolic Ca2+ decrease is significantly lower  has a very strong influence on the values of Cm associated with changes in Cc  stimulate and inhibit the oscillatory behaviour in HeLa cells and in fibroblastsWe next modified the rate constant of the MCU, which is the mitochondrial Ca2+ entry into mitochondria through the MCU slows down Ca2+ oscillations appeared as a rather counter-intuitive prediction of the model. We tested this prediction experimentally using HeLa cells transiently transfected with scrambled siRNA or siRNA against MCU  to 97 s  in the cells that do not express the MCU. Experiments thus confirmed that MCU inhibition leads to a decrease in the frequency of Ca2+ oscillations in HeLa cells. Our interpretation of this observation is that, in the absence of MCU, the very low level of mitochondrial Ca2+ does not allow the release of mitochondrial Ca2+ between two ER-generated spikes, release that is responsible for the ‘pacemaker-like’ Ca2+ trigger of the IP3 receptor occurring in control cells.That decreasing the rate of Ca MCU see . Indeed,μM histamine, only 3% of MCU siRNA treated cells displayed a Ca2+ response while 39% of control cells oscillated. However, this shift is not the only reason for the observed decrease in period in the cells inhibited for the MCU. Indeed, the period of Ca2+ oscillations at the highest histamine dose in anti-MCU siRNA treated cells  is still larger than that seen at the lowest histamine dose in Scrbl siRNA treated cells . Thus, oscillations in the absence of MCU at maximal stimulation, which corresponds to the smallest possible period, are still slower than those obtained at low levels of stimulation in the presence of MCU, pointing to a specific role of the MCU in the control of the frequency of oscillations.The increase in period observed in the experiments is, however, much larger than that predicted by the model. We thus investigated if the MCU siRNA treatment shifts the effective dose response to histamine. 2+ exchange between the mitochondria and the cytosol  and 2+ oscillations. As this flux is bidirectional, it could either boost or slow down Ca2+ oscillations. 2+ oscillations, as for the NCX. Interestingly, in hepatocytes, an inhibition of the PTP by cyclosporin A (CSA) results in an increase in the interspike intervalIn the model, there is another flux allowing a Ca (2) and ). This f2+ oscillations in HeLa cells, we stimulated CSA-treated cells with histamine. As shown in 2+ oscillations in response to both 0.3 and 1 μM histamine. It should be noted that CSA has been reported to stimulate SERCA pumps in addition to its effect on the mPTP2+ pumping back to ER might thus also participate in the effect shown in 2+ oscillations was eliminated in the presence of mitochondrial inhibitionx.To further challenge the prediction that the low conductance mode of the mPTP could be involved during cytosolic Ca2+ peak in the cytosol. The Ca2+ peak simulated in μM amplitude. We chose this type of stimulation to optimise the comparison with experimental data. 2+ spike. It is in agreement with observations in phenylephrine-stimulated hepatocytes2+ from the cytosol into mitochondria .We next examined how mitochondrial metabolism affects CasGLY Eq.  that rep2+ rises in mitochondria have been suggested as essential for physiological cell bioenergetics622+ regulation of mitochondrial metabolism to be both versatile and robust. Given the complexity and the highly non-linear character of the regulation of mitochondrial metabolism, this question is best addressed by modelling. Thus, we simulated Ca2+ spikes of different frequencies and amplitudes and computed the resulting average values of mitochondrial NADH and ATP concentrations. The results shown in 2+ pulses lasting 10 s, with a 100 nM basal level of Ca2+. We found that maximal average values of NADHm and ATPm were obtained for frequencies and amplitudes of Ca2+ spiking that are typically observed in HeLa cells (~0.04 s−1 and ~800 nM). Interestingly, optimised metabolism is naturally obtained in the model for the parameter values listed in 2+ and mitochondrial metabolism.Stimulus-induced Cam is an increasing function of both the frequency and the amplitude of the Ca2+ spikes. As shown in m does not regain its basal level between the Ca2+ spikes, the minimal value reached being fixed by the oscillations’ period. Moreover, AGC also influences this minimal value, as it is activated by low levels of cytosolic Ca2+. In consequence, one observes a frequency modulation of the average [NADH]m, which is more pronounced with a lower amplitude of the Ca2+ spikes. There is a similar amplitude modulation of the average [ATP]m, more pronounced with a lower frequency of the Ca2+ spikes  the effect of mitochondrial metabolism on Ca2+ cycling between ER, cytoplasm and mitochondria during oscillations induced by IP3-generating agonists, and (2) the effect of changes in cytosolic Ca2+ on mitochondrial metabolism. The model is built from a selection of previously proposed kinetic expressions for the various fluxes and reaction rates, except for the increase of mitochondrial NADH mediated by the malate-aspartate shuttle, which, to the best of our knowledge, has not been considered in previous models. We have modified the expressions for the MCU concerning its dependence with respect to the voltage, and for the SERCA pump, where the ATP requirement was included in the rate equation  of yet poorly identified molecular nature has been reported in mitochondria isolated from liver and cardiac cells15Other modes of Ca2+ exchanges on the characteristics of Ca2+ oscillations. A surprising result of the simulations is that the frequency of oscillations can decrease when inhibiting the activity either of the NCX .


As a first step toward this goal, we therefore analyzed, using quantitative real-time PCR, the gene expression of 65 potential LAAs  was calculated as the C(t) of target gene ‘X' minus the geometric mean C(t) of reference genes HPRT1, PPIH and TFRC in a sample. ΔC(t) for each target gene ‘Xn healthy donor samples was also computed in this manner. To calculate ΔΔC(t), median ΔC(t) of gene ‘X' in healthy donor blood or BM (depending on the source of the AML sample) was subtracted from the ΔC(t) of X in the AML sample (ΔC(t) of X in AML sample−median ΔC(t) of X in healthy donor samples). Statistical analysis was performed using GraphPad Prism .A total of 52 samples  and 22 bone marrow aspirate (BM) samples) from 48 AML patients were analyzed, which included 4 patients for whom both PB and BM samples were available. The average age of the patients was 52 years (range 24–86); 52% of the patients were women. A total of 7 patients had favorable cytogenetics, whereas 11 were classified as adverse, 13 patients had FLT3 mutations (including 8 patients with FLT3-ITD) and 9 patients had mutations in NPM1 . RNA andHBG2, ordinarily expressed in the fetal liver, spleen and BM but not usually in adulthood, which was recently identified as a leukemia antigen in a study of induced immune responses to GVAX/K562 vaccination in chronic myeloid leukemia (CML),11 was found to be frequently overexpressed in AML, often to a high level  of putative LAAs compared with healthy donor tissues . Every Agh level  and 2. S samples . We wereE of WT1  but not d MAGEC1  that werd MAGEC1 .2 We therefore also quantified tissue expression of these putative LAAs in a range of normal tissues that included samples (10 PB and 7 BM) collected under institutional review board-approved protocols at the NIH clinical center from 17 healthy donors with an average age of 41, 70% of whom were male, with ficoll purification and RNA extraction as described above. In addition, purified total RNA from a wide panel of human organs was obtained from Clontech   . ExpressCA, USA) .Several technical features are worthy of note. We found that samples with RIN scores of less than 7.0 resulted in higher than expected C(t) outputs, which correspond to lower gene expression when compared with samples with higher RIN scores  and were14This work has several obvious limitations. We performed this work on ‘real world' first-presentation primary samples from three different leukemia centers in an attempt to limit bias introduced by presentation and referral patterns. Nevertheless, all institutions are highly specialized tertiary academic medical centers located in the northeastern and southeastern United States. Although we do have patient demographics on age and gender  and all 15The ideal AML LAA would be expressed in most or all cases of AML but not in healthy tissues. Using a novel, highly sensitive and reproducible, real-time reverse transcription–PCR array testing only high-quality RNA we show in this work that the majority of proposed ‘LAAs' are expressed in the leukemia cell line K562 but often not in primary samples from AML patients. Although we identified no antigen that was universally overexpressed in all AML samples, every patient did have at least one potentially targetable antigen overexpressed. We also noted significant healthy organ-specific tissue expression of many LAAs, highlighting the possibility of ‘off-target' effects, a finding not evident from the study of expression levels in PB and BM alone. This list of genes overexpressed in AML, together with information regarding expression in a wide range of healthy tissue types, may be of use in AML as a reference for the selection of antigenic targets in adoptive T-cell therapy and may also have use in the PCR-based detection of minimal residual disease.