Cytotoxicity of Auranofin was evaluated using MTS assay and the Trypan blue dye exclusion method. With fluorescent dyes SR-FLICA and 7-AAD apoptotic death and necrotic death were differentiated by Flow cytometry. A concentration dependent decrease in the viability occurred and cells were shifted to the apoptotic phase. Intracellular calcium ([Ca2+]i) was recorded using florescence microscopy and a calcium sensitive dye (Fluo-4 AM) with a strong negative correlation (r = −0.713) to viability. Pharmacological modulators 2-APB (50 μM), Nimodipine (10 μM), Caffeine (10 mM), SKF 96365(20 μM) were used to modify calcium entry and release. Auranofin induced a sustained increase of [Ca2+]i in a concentration and time dependent manner. The use of different blockers of calcium channels did not reveal the source for the rise of [Ca2+]i. Overall, elevation of [Ca2+]i by Auranofin might be crucial for triggering Ca2+-dependent apoptotic pathways. Therefore, in anti-cancer therapy, modulating [Ca2+]i should be considered as a crucial factor for the induction of cell death in cancer cells.Auranofin, a transition metal complex is used for the treatment of rheumatoid arthritis but is also an effective anti-cancer drug. We investigate the effects of Auranofin in inducing cell death by apoptosis and whether these changes are correlated to changes of intracellular calcium concentration ([Ca Cancer is a result of uncontrolled cell-proliferation, invasion and destruction of healthy tissue. A defective apoptotic program is characteristic of all cancers. One of the main anticancer treatment strategies is to induce apoptosis in cancer cells. To achieve this goal, metal-based anti-cancer drugs have been developed  . Today, The mechanism of antitumor activity of gold compound is “DNA-independent”, and different from those of platinum drugs . Marzano2+]i) and there are considerable amounts of evidence on the central role of [Ca2+]i in proliferation and cell death. An increase in [Ca2+]i was shown to induce cell death in HeLa-S3 treated with cisplatin [2+]i, which could occur through an influx of Ca2+ through Ca2+-selective channels at the plasma membrane or by release from intracellular stores, is a key factor to trigger apoptosis. Boehning and coworkers emphasized the role of InsP3R-mediated calcium release from the endoplasmic reticulum in inducing apoptosis in response to specific stimuli [2+-response by specifically activating TRPA1 channels expressed in human differentiated neuroblastoma cell lines (nIMR-32). This mechanism is independent of the inhibition of the thioredoxin reductase [2+]i by mobilizing calcium from multiple storage sites, in a study done in neutrophils [2+]i in the induction of apoptosis by Auranofin in the breast cancer cell line MCF-7.Apoptosis is also regulated by intracellular calcium signaling , the control value and the values for low concentrations  were significantly different (p < 0.001) from the data taken with higher concentrations . In addition, a significant difference was found between concentrations of 3.1 μΜ and 12.5 μΜ or 25 μΜ (p = 0.002).Viability of MCF-7 cells were significantly impaired with increasing concentrations of Auranofin . Concentp > 0.05). At concentrations of 12.5 μM and 25 μM, the “live” population was found to be almost eliminated. The live population dropped significantly with concentrations higher than 6.25 μM (53.5 ± 31.6%). As it was not possible to do a morphological analysis of the cells by FACS, a clear distinction could not be made between debris and necrotic population which is a limitation of this method, nevertheless considering the total cytotoxicity (apoptosis + necrosis) in the two groups, untreated versus e.g., 12.5 μM of Auranofin, the total cytotoxicity was 17% and 97% respectively. A comparison of different concentrations for the different groups is illustrated in Increasing concentrations of Auranofin (in a single experiment) resulted in more apoptotic cells A. Under 50 of Auranofin calculated from FACS analysis was 5.1 μM. Pearson’s correlation was tested for the live and apoptotic population and showed a strong reciprocal (r = −0.964) link with respect to concentration, which means, the apoptotic population increased proportionally as viability decreased. Hence a concentration dependent increase in apoptotic population was detected.The IC2+]i  of Auranofin showed a significant increase in [Ca2+]i for the last three concentration within two hours as compared to initial [Ca2+]i .Auranofin triggered increase of [Ca2+]i A. The up [Ca2+]i B in more2+]i imaging experiments and the viability (MTS) assay, showed that the degree with which the viability was decreasing is closely related to increase in [Ca2+]i , caffeine (10 mM), nimodipine (10 μM) caffeine (10 mM) and SKF 96365 (20 μM)) did not change [Ca2+]i significantly . Thapsigargin did not change the effect caused by Auranofin significantly. The rise of [Ca2+]i was significantly different compared to the drug free condition (Control with thapsigargin only). p < 0.05 for all time points. SERCA was found to play no major role in Auranofin induced [Ca2+]i rise  based anti-arthritic drug. Here we examine its ability to induce cell death in a human breast cancer cell-line (MCF-7) and whether its cytotoxicity is related to a drug induced modulation of i, also plays a vital role in modulating pathways involving any of the above mentioned targets. Multiple studies support the central role for [Ca2+]i in the initiation of apoptosis in cancer cells. Splettstoesser and coworkers have reported that cisplatin induces concentration dependent [Ca2+]i by IP3-receptor mediated pathway in HeLa-S3 [2+]i elevation resulted in an increase of cytotoxicity and apoptosis in neuroblastoma cells treated with cisplatin and/or arsenic trioxide [2+ shift was regarded as an earlier marker of cytotoxicity in a study conducted with a panel of toxins tested in different mammalian cell lines [2+]i is modulated by Auranofin, as the strong reciprocal relation of “[Ca2+]i” versus “viability” indicates an important role of [Ca2+]i in inducing apoptosis.As calcium is an important signaling ion, which exerts effects on many enzyme and protein functions, it is likely that [Ca HeLa-S3 . A studytrioxide . Cytosolll lines . Our stu2+-conductance at the cell membrane [2+ from intracellular stores [2+]i and cell death [2+]i but the additional release of calcium from TG-sensitive calcium stores of the endoplasmic reticulum that triggers apoptosis [2+]i i .2+]i elevation directly caused an activation of caspases. Our studies with the blockers did not reveal the exact mechanism of [Ca2+]i elevation. Taking into consideration the complexity of Ca2+ signaling, at this juncture it remains difficult to elucidate the exact mechanism behind the apoptosis induced by elevation of [Ca2+]i. In addition the amount of calcium released from the ER or the mitochondria and the calcium entry through the plasma membrane for the observed elevations in [Ca2+]i needs further investigation in order to distinguish whether their functions are interconnected.With our analysis, we could not conclusively answer the question whether Auranofin induced apoptosis was a direct effect of the rise of intracellular calcium or an indirect effect and whether the i level for at least 30 min (control). The baseline for [Ca2+]i was obtained by averaging 20–25 frames at the beginning of the experiment (before drug application) and was considered as 100%. For each experiment at least 15–25 cells were recorded. Results were analyzed offline. For each experiment the Regions of Interest (ROI) were selected and only cells which were intact over the entire experiment were selected for analysis. Data (averaging the intensity of the fluorescent in the ROI) were exported to excel. Background values corresponding to each image were subtracted. The values obtained from the control conditions were averaged and set as control value (100%) to calculate the percentage change in intensity over time by the equation = (F/F0) × 100. Where, F is the intensity at a given time and F0 is the average intensity of the control window. The results were represented as 3D graph with time and concentration plotted against percentage increase in [Ca2+]i. Images were represented in “rainbow” scale  to highlight the difference in fluorescent intensities. Blue color represents lowest [Ca2+]i and yellow-red shows regions of high [Ca2+]i).Cells were plated in a 35 mm “eazygrip” BD Falcon tissue culture plates . The plates were washed with Tyrode’s buffer  for emptying the ER stores, 2-APB (50 μM) for inhibiting store-operated calcium (SOCE) release and a IP3-receptor blocker i and viable cells to apoptotic cells. Differences were considered significant when p-values were smaller than 0.05.Post hoc analysis using Bonferroni correction (2+]i in MCF-7 cell line. Prolonged elevation of calcium might be a key factor for triggering a Ca2+-dependent apoptotic pathway. To the best of our knowledge, this is the first study which analyzes the modulation of [Ca2+]i by Auranofin in a breast cancer cell line.Results indicate that the Auranofin induced an elevation of [Ca2+]i is a crucial factor to induce cell death in cancer cells. An answer to the mechanism of calcium modulation by Auranofin, by studying the calcium channels and pumps will help to improve the efficiency of drug action. To understand the exact mechanisms of [Ca2+]i modulation, further experiments are needed.We conclude that modulating [Ca


Two such fundamental calcium pumps are plasma membrane calcium ATPase (PMCA) and Sarco/endoplasmic reticulum calcium ATPase (SERCA) which play a pivotal role in maintaining intracellular calcium homeostasis. This intracellular Ca2+ homeostasis is often disturbed by the protozoan parasite Leishmania donovani, the causative organism of visceral leishmaniasis. In the present study we have dileneated the involvement of PMCA4 and SERCA3 during leishmaniasis. We have observed that during leishmaniasis, intracellular Ca2+ concentration was up-regulated and was further controlled by both PMCA4 and SERCA3. Inhibition of these two Ca2+-ATPases resulted in decreased parasite burden within the host macrophages due to enhanced intracellular Ca2+. Contrastingly, on the other hand, activation of PMCA4 was found to enhance the parasite burden. Our findings also highlighted the importance of Ca2+ in the modulation of cytokine balance during leishmaniasis. These results thus cumulatively suggests that these two Ca2+-ATPases play prominent roles during visceral leishmaniasis.Calcium is an ubiquitous cellular signaling molecule that controls a variety of cellular processes and is strictly maintained in the cellular compartments by the coordination of various Ca Leishmania donovani, an obligatory intracellular protozoan parasite which resides and multiplies within the host macrophages Leishmania responses, the parasites induce various immune-silencing mechanisms for its intracellular survival inside the hostile environment of macrophages. Impairment of the classical protein kinase C (PKC) signaling within the host macrophages is one of the major manipulative strategies through which the parasite exerts its immunosuppressive effects.2+ along with DAG and phosphatidylserine for their activation.PKC is structurally related to serine-threonine family. On the basis of structural and regulatory properties, PKC has been grouped into three subfamilies 2+-ATPase resides in the sarcoplasmic reticulum (SR) which is involved in the transport of Ca2+ from the cytosol to the lumen of the SR at the expense of ATP hydrolysis during muscle relaxation. The Plasma membrane Ca2+-ATPase (PMCA4) also obtains energy to pump calcium out of the cell by hydrolysing adenosine triphosphate (ATP).Calcium plays crucial roles in different physiological, biochemical and signaling pathways in both animal and plant cells. Moreover, calcium is one of the most widespread second messengers involved in several signal transductions. Intracellular organelles like mitochondria and endoplasmic reticulum, are the store house of calcium; they constantly accumulate and release calcium during certain cellular events to maintain the calcium homeostasis Leishmania parasite has achieved a great deal of attention because it impairs Ca2+-dependent PKC signaling in the infected macrophages Leishmania donovani derived glycolipid, leads to the down-regulation of classical PKC activity along with upregulation of Ca2+-independent atypical PKC-ζ expression Leishmania donovani, within the host macrophages Several studies have implicated the role of PKCs in the control of intracellular microbial replication. In this context, the 2+-ATPases were induced in Leishmania infected macrophages. Moreover, the enhanced PMCA4 expression was found to increase the parasite burden. Similar result was obtained upon thapsigargin treatment, a SERCA3 inhibitor. In contrast, enhancement of intracellular calcium by ionomycin treatment induced the expression of calcium dependent PKCs. This in turn was found to regulate the profile of TH1 and TH2 cytokines differentially. Altogether, these results clearly suggested that the enhanced intracellular calcium was regulated by both PMCA4 and SERCA3 during visceral leishmaniasis.In the present communication, we were prompted to study the role of PMCA4 and SERCA3 in visceral leishmaniasis and found that both CaDulbecco’s Modified Eagle’s Medium (DMEM), M-199 medium (M199), penicillin, streptomycin, EGTA and ionomycin were purchased from Sigma Chemicals . Fetal calf serum (FCS) was obtained from Gibco BRL Grand Island, NY, USA and ELISA Assay Kit (Quantikine M) for tumor necrosis factor-α (TNF-α), Interferon-γ (IFN-γ), IL-12, IL-10 were from R&D Systems .L. donovani organisms (strain MHOM/IN/1983/AG-83) were maintained in Medium 199 (Sigma) plus 10% fetal calf serum (Gibco).RAW 264.7 macrophages were maintained in complete Dulbecco's modified Eagle's medium (SIGMA) containing 10% FCS.2+-ATPase (PMCA4) (10 µM trifluoperazine). These were then infected with L. donovani in a 1∶10 ratio. Unbound parasites were washed off after 4 hr. Cells were then incubated for 24 hr, fixed with chilled methanol, and stained with Giemsa following of which amastigotes were microscopically counted.Macrophages were plated on eight well chamber slides  and were treated for 30 min with inhibitors of Sarco-endoplasmic Reticulum Calcium ATPase (SERCA3) (1 µM thapsigargin) and Plasma Membrane CaCells were scraped using chilled PBS and centrifuged at 2000 rpm for 10 min at 4°C. The pellet was resuspended in 100 µl of extraction buffer containing 50 mM Tris-HCl, 50 mM EDTA, 1 mM PMSF, 50 mM β-ME and protease inhibitors and kept on ice for 30 min. Cells were sonicated and spun at 100000 g for 35 min at 4°C. The pellet was collected and resuspended in 100 µl of extraction buffer containing 50 mM Tris-HCl, 50 mM EDTA, 1 mM PMSF, 50 mM β-ME, protease inhibitors including 0.2% NP-40. The cell suspension was vortexed at 4°C for 30 min and centrifuged at 12,000 rpm for 10 min. Supernatant was collected and used for the measurement of PMCA4 activity.et.al2, 1 mM MgCl2, and 1 mM ATP for 30 min. The reaction was arrested at 45 min by the addition of 8%  cold tri-chloro-acetic-acid (TCA). Tubes were vortexed and 40 mM of ammonium molybdate together with 2% ascorbic acid was added and incubated at room temperature for 10 min and the absorbance was measured at 820 nm.The method described in this protocol is a modified one from the procedure reported by Sarkadi Control and infected macrophages were scrapped from the plate using chilled PBS. Cells were centrifuged and pellet dissolved in calcium free buffer . Cells were counted by hemocytometer. 3 µl of Fura-2/AM  was added in 1 ml cell solution at 37°C for 30 min. Cells were centrifuged and the pellets were dissolved in calcium free buffer and transferred in quartz cuvette. After 60 sec., one set of cells were stimulated with 1 µM thapsigargin followed by 10 µM trifluoperazine. In another set of cells, after 90 sec., ionomycin (1 µM) stimulation was given followed by 50 µM EGTA. At indicated time points, samples were analyzed by flow cytometry (F = 340/380).The conditioned medium of macrophage culture was assayed for mouse cytokines by the sandwich ELISA kit (BD Biosciences). The assay was performed according to the manufacturer’s instructions.3VO4. The protease inhibitor mixture consisted of 0.33 mM leupeptin, 0.2 mM phenyl-methyl-sulfonyl-fluoride (PMSF), 0.35 mM antipain, 0.24 mg of chymostatin per ml, 0.35 mM pepstatin, and 4.8 TIU of aprotinin per ml. The suspension was sonicated at 4°C and centrifuged at 7000 RPM for 10 min at 4°C, and the supernatant was used for the experiments. An amount of 40 µg protein was subjected to 10% sodium dodecyl sulfate polyacrylamide gel electrophoresis, and was subsequently transferred to a nitrocellulose membrane. The membrane was blocked overnight with 3% bovine serum albumin in Tris-saline buffer , and immunoblotting was performed to examine the expression levels of SERCA3, PMCA4, PKC-β and -ζ.The adherent cell population was scraped and centrifuged at 3000 RPM for 15 min at 4°C. The cells were then resuspended in ice-cold extraction buffer containing 10 mM Tris-HCl (pH 7.5), 4.5 mM EGTA, 2.5 mM EDTA, protease inhibitor mixture, and 1 mM NaRNA was isolated according to the standard protocol Immunoblots and PCR products were analyzed using a model GS-700 Imaging Densitometer and Molecular Analyst .t test was used to assess the significance of differences between the mean values for the control and experimental groups. A difference with P<0.05 was considered significant, a difference with P<0.001 was considered highly significant and ns implied non-significant.In vitro cultures were done in triplicate. Data, shown as means ± standard deviations, from one experiment performed at least three times. The Student Leishmania donovani infection is associated with abrogated calcium dependent PKC activation in macrophages Leishmania donovani infection. The addition of a calcium ionophore (1 µM), ionomycin, 2+ using EGTA (50 µM), the fast initial increase in response to ionomycin disappeared but slower sustained elevation in cytosolic Ca2+ was observed  was observed. Therefore, our results indicated that the intracellular calcium ion release is relatively high in Leishmania donovani infected macrophages.observed . The addobserved . Interes2+ level, whereas, blocking of PMCA4 with TFP resulted in a sharp and sustained elevation in intracellular Ca2+ concentration.Moreover, SERCA3 blocking resulted in a transient decrease in intracellular Ca2+-ATPases, PMCA4 and SERCA3. Therefore, we analyzed the protein and mRNA expression status of these two ATPases in Leishmania donovani infected macrophages. PMCA4 and SERCA3 both have several isoforms. PMCA4 activation relates to PKC isotypes, where as SERCA3 is ubiquitous. We therefore have selected both of them in our study. The expression of PMCA4 and SERCA3 was examined in Leishmania donovani infected macrophages at different time points of infection. The results showed that both the SERCA3 and PMCA4 expression gradually increased from 3 to 12 hr of post infection  protein expression in infected macrophages compared to that of the uninfected control macrophages. To study the role of Ca2+, an elevator, ionomycin and a chelator, EGTA of Ca2+ were selected. Interestingly, ionomycin (1 µM) treatment resulted in significant up-regulation of PKC-β expression in the infected macrophages compared to that of the uninfected one. However, treatment with a calcium ion chelator, EGTA (2 mM), abrogated the ionomycin induced enhancement of PKC-β expression in the infected macrophages. On the other hand, when both EGTA and ionomycin were used, no significant enhancement of the PKC-β expression in the infected macrophages . When ionomycin (1 µM) was used, it was found that the mRNA expression of both the PMCA4 and SERCA3 significantly augmented in the infected macrophages compared to that of the untreated one. Interestingly, treatment with EGTA or EGTA+ionomycin resulted in a sharp decline in the PMCA4 and SERCA3 mRNA expression in the infected macrophages . The result presented here revealed that the PMCA activity was indeed increased under thapsigargin induced SERCA inhibition . This wa2+ acts as a second messenger controlling many different aspects of cellular function 2+ homeostasis by regulating certain Ca2+ channels and pumps 2+-ATPases (SERCA). Since intracellular organelles are limited by their storage capacity, long-term calcium homeostasis is regulated solely by the plasma membrane Ca2+-ATPases (PMCA) Cytoplasmic Ca2+ concentration is found to be enhanced 2+-dependent PKC activation is impaired during leishmaniasis 2+ and Ca2+-ATPases during Leishmania donovani infection. Consistent with the previous reports, we observed an upregulation in the intracellular Ca2+ concentration in Leishmania infected macrophages, which was further increased by trifluoperazine (TFP), a PMCA4 inhibitor. Interestingly, SERCA3 inhibition by thapsigargin (Tg) resulted in a transient decline in intracellular Ca2+ level. It was also observed that both ATPases (SERCA3 and PMCA4) expressions were augmented during the course of infection. These expressions were further enhanced by ionomycin treatment. However, EGTA abrogated their expression, implying that excess Ca2+ from the infected cells might be pumped out via the activation of Ca2+-ATPases.During leishmaniasis, intracellular Ca2+ concentration significantly affect the activation of classical PKCs. During visceral leishmaniasis, Ca2+-dependent PKC activation is impaired, whereas PKC-ζ, , is induced Leishmania infected macrophages, the impaired PKC-β  expression was induced by ionomycin treatment, whereas PKC-ζ expression was decreased. This may suggest that the differential function of cellular Ca2+ may be involved in the impairment of Ca2+ -dependent PKCs during pathogenesis Intracellular Ca2+ signaling effectively modulate the pro- and anti-inflammatory cytokine production during pathogenesis 2+ signaling in macrophages seems to be one of the major strategies employed by the Leishmania parasite for its survival within the macrophages. In the present study, ionomycin treatment did not change the enhanced release of IL-10 and decreased release of IL-12 and TNF-α in L. donovani infected macrophages, may be that ionomycin play different role in the regulation of pro- and anti-inflammatory cytokines 2+ generated during Leishmania infection, induces Th2 type immune response in favour of parasitemia.It has been reported that Ca2+ signaling is involved in the transformation of promastigotes to axenic amastigotes of Leishmania donovani2+ is responsible for apoptosis of intracellular Leishmania donovani amastigotes 2+-ATPases helps in pumping out the extra Ca2+ from the intracellular milieu of Leishmania infected cells, which in-turn helps the survivility of Leishmania parasites within the host cells.From the foregoing study it has been demonstrated that PMCA4 activity is significantly elevated in thapsigargin treated cells along with the enhancement of parasite burden, while the inhibition of PMCA4 activity showed significant reduction under this condition. It is reported that Ca


We therefore postulated that plasma membrane cation channels might be a common site of inhibition by structurally distinct cationic neurotoxicants that could modulate ATP-induced Ca2+ signals in astrocytes. To test this, we examined the capacity of two dopaminergic neurotoxicants to alter ATP-dependent Ca2+ waves and transients in primary murine striatal astrocytes: MPP+, the active metabolite of 1-methyl 4-phenyl-1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA). Both compounds acutely decreased ATP-induced Ca2+ transients and waves in astrocytes and blocked OAG-induced Ca2+ influx at micromolar concentrations, suggesting the transient receptor potential channel, TRPC3, as an acute target. MPP+ inhibited 1-oleoyl-2-acetyl-sn-glycerol (OAG)-induced Ca2+ transients similarly to the TRPC3 antagonist, pyrazole-3, whereas 6-OHDA only partly suppressed OAG-induced transients. RNAi directed against TRPC3 inhibited the ATP-induced transient as well as entry of extracellular Ca2+, which was augmented by MPP+. Whole-cell patch clamp experiments in primary astrocytes and TRPC3-overexpressing cells demonstrated that acute application of MPP+ completely blocked OAG-induced TRPC3 currents, whereas 6-OHDA only partially inhibited OAG currents. These findings indicate that MPP+ and 6-OHDA inhibit ATP-induced Ca2+ signals in astrocytes in part by interfering with purinergic receptor mediated activation of TRPC3, suggesting a novel pathway in glia that could contribute to neurotoxic injury.Dopaminergic nuclei in the basal ganglia are highly sensitive to damage from oxidative stress, inflammation, and environmental neurotoxins. Disruption of adenosine triphosphate (ATP)-dependent calcium (Ca However, disruptions in astrocyte Ca2+ signaling could have serious implications for neuronal function and survival during states of neurological injury and disease. Transient increases in intracellular Ca2+ are essential for astrocytic regulation of neuronal excitability in both the healthy and diseased brain 2+-dependent release of ATP, deprecations in Ca2+ signaling in astrocytes could predispose regional populations of neurons to excitotoxic injury. Ca2+ signaling in astrocytes could also be dysregulated during neuroinflammation, as indicated by recent studies which demonstrated that ATP release from activated microglia evokes P2Y purinergic receptor-dependent release of neuroactive compounds from astrocytes, including ATP, D-serine and glutamate 2+ signaling could contribute to neuronal dysfunction following exposure to oxidative or neurotoxic insults or during conditions of chronic neuroinflammation.Calcium (Ca2+. P2 receptors are divided into two subfamilies: ionotropic (P2X) receptors and metabotropic (P2Y) receptors. P2Y receptors are activated by low physiological ATP concentrations (<10 µM), whereas P2X receptors are activated by high concentrations of ATP that are released during pathological events such as ischemia and oxidative stress 3) that induces release of Ca2+ from the endoplasmic reticulum. Additionally, PLC activation generates diacylglycerol (DAG) that stimulates a more prolonged influx of extracellular Ca2+, resulting in a biphasic Ca2+ response that causes opening of receptor operated channels including TRPC3 2+ influx pathways are affected by neurotoxic exposures is not well understood. This is important because sustained increases in [Ca2+]i are required for exocytosis of ATP in astrocytes following purinergic stimulation Extracellular ATP activates purinergic receptors (P2 receptors) on astrocytes 2+ responses to purinergic signals is the transient receptor potential (TRP) channel, a family of selective and non-selective cation channels that are widely expressed in diverse cell types, including astrocytes 2+ signaling through activation of P2Y Gq GPCR receptors that activate the PLC-IP3-DAG pathway 2+-permeable, non-selective cation channel is associated with vulnerability to the dopaminergic neurotoxicants, MPP+, the active metabolite of 1-methyl 4-phenyl 1,2,3,6-tetrahydropyridine (MPTP), and 6-hydroxydopamine (6-OHDA), an endogenous dopamine oxidation product that is generated at higher levels during aging. We propose a novel mechanism in astrocytes in which ATP-induced Ca2+ transients are acutely suppressed by neurotoxic cationic molecules through inhibition of purinergic signaling and through blockade of TRPC3 channel function, thereby altering the global amplitude of the intracellular Ca2+ response. To examine mechanisms by which these neurotoxicants could dysregulate ATP-induced Ca2+ signaling in astrocytes, we acutely exposed cultured striatal astrocytes to each compound and determined the effect on ATP- or OAG-induced Ca2+ transients using real-time fluorescence imaging, as well as the capacity of each compound to inhibit electrophysiologic Ca2+ currents in cells over-expressing TRPC3 and in primary astrocytes. The results of these studies indicated that acute application of each compound prior to stimulation with ATP or OAG decreased the amplitude of evoked transients, in addition to diminishing TRPC3-dependent electrophysiological currents induced by OAG. These data suggest that TRPC3 is a receptor-operated channel in astrocytes linked to the activation of metabotropic purinergic receptors and that selected cationic neurotoxicants suppress Ca2+ transients in astrocytes in part by inhibiting this pathway, implicating this channel as a target that may be relevant to neurotoxic injury from structurally diverse cationic molecules.A possible target of neurotoxic compounds that could decrease CaAll chemical reagents were from Sigma-Aldrich  unless otherwise stated. C57BL/6J mice were obtained from Jackson Laboratories . Cell culture media and fluorescent dyes were obtained from Invitrogen .2 in a humidified incubator for approximately three weeks until reaching a mature phenotype. Confluent astrocytes were sub-cultured onto 4-well poly-D-lysine-coated chambered glass slides  and allowed to grow to approximately 75% confluence. In our laboratory, cultures consistently yield>98% astrocytes as determined by immunofluorescence staining for glial fibrillary acidic protein Primary astrocytes were isolated from 1–3 day old C57BL/6J mice from either cortex or striatum, as previously described All procedures involving animals were conducted under a protocol approved by the Animal Care and Use Committee at Colorado State University (IACUC Protocol No. 12-3646A). According to the guidelines of the National Institutes of Health, every effort was made to minimize pain and discomfort and all terminal procedures for isolation of primary cells were conducted under deep isofluorane anesthesia.+, 6-OHDA, or 3,4-dihydroxyphenylacetic acid/DOPAC) were added 30 seconds prior to each agonist. Images of Fluo-4 fluorescence were collected every 500 milliseconds for 120 seconds with camera binning set at 4×4 pixels and an exposure time of 20 milliseconds. Images were collected on a Zeiss Axiovert 200 M microscope equipped with a Hammatsu ORCA-ER cooled charge-coupled device camera. Fluorescent intensity was expressed relative to the baseline image (F/F0), where F0 is the fluorescence level prior to stimulation. Datasets were analyzed using Slidebook software .Astrocytes were grown to approximately 75% confluence on 4-well chamber slides and incubated with Fluo-4 AM (2 µM)  for 15 minutes at 37°C prior to imaging. Cells were imaged in media  supplemented with 10 mM HEPES (pH 7.4) at 25°C. Groups of approximately 15–30 contiguous cells per field of view were identified for imaging. Cells were stimulated with ATP (1 µM) to selectively activate metabotropic purinergic receptors, rather than ionotropic receptors 2+ wave propagation studies, astrocytes were sub-cultured onto poly-D-lysine coated 30 mm round glass coverslips and placed in a flow chamber . After a 10 second collection of baseline Fluo-4 intracellular Ca2+ intensity, wave studies were mechanically-induced with a 5 µm diameter glass micropipet using a micromanipulator, as described previously +, 6-OHDA or DOPAC (100 µM) were added 30 seconds prior to stimulation and images were acquired every 500 milliseconds for 60 seconds. Fluorescent intensity was expressed relative to the baseline image (F/F0) in all cells within the wave activation site. Wave amplitude and distance were determined using Slidebook software .For Ca2+ transients were determined by real-time fluorescence imaging following stimulation with ATP (1 µM) or OAG (100 µM) in the presence of the phospholipase C (PLC) inhibitor, U73122 (10 µM), or its inactive analog, U73433 (10 µM). After a 10 min preincubation period with the inhibitor or analog, the cells were stimulated with 1 µM ATP or 100 µM OAG and images of Fluo-4 fluorescence collected every 500 milliseconds for 60 seconds. Fluorescent intensity was expressed relative to the baseline image (F/F0) in all cells. Pyrazole 3 , a selective extracellular inhibitor of TRPC3, was incubated with astrocytes for 30 minutes prior to imaging at 37°C. Following incubation with Pyr3, cells were stimulated with ATP or OAG as described. For experiments without extracellular Ca2+, Ca2+-free imaging medium supplemented with 2 mM EGTA (pH 7.4) was used in replacement of imaging medium, with all other experimental conditions held constant. Luciferase measurements were made using a 96-well luminescence plate reader .Astrocytic Ca2 and sub-cultured when confluent using 0.25% trypsin-EDTA (Gibco). HEK 293 cells were transfected by electroporation with a plasmid that encodes TRPC3 channels tagged with yellow fluorescent protein (TRPC3-YFP)2 humidified incubator. Currents were recorded using an AxoPatch 200B amplifier equipped with an Axon CV 203BU headstage (Molecular Devices). Recording electrodes (1–3 MΩ) were pulled, polished, and coated with wax to reduce capacitance. GΩ seals were obtained in Mg-PSS. Currents were filtered at 1 kHz, digitized at 40 kHz, and stored for subsequent analysis. Clampex and Clampfit versions 10.2 (Molecular Devices) were used for data acquisition and analysis, respectively. All patch-clamp experiments were performed at 25°C. The extracellular bathing solution contained (mM) 140 NaCl, 5 CsCl, 0.1 MgCl2, 0.05 CaCl2, 10 Glucose, 10 HEPES (pH 7.4). Pipette solution contained (mM) 130 CsOH, 110 aspartic acid, 15 CsCl, 1 MgCl2, 3.6 CaCl2, 10 EGTA, 10 HEPES (pH 7.4). The calculated reversal potentials for monovalent and divalent cations are 0 mV and −24 mV, respectively. Reversal potentials for monovalent anions are 1.6 mV for these solutions. Cells were initially voltage clamped at 0 mV, and voltage ramps from −100 mV to +100 mV were applied every 4 seconds. After baseline currents were recorded, OAG (100 µM) or ATP (1 µM) was administered to activate TRPC3 currents. When the evoked current reached a steady state MPP+ (100 µM), 6-hydroxydopamine, 6-OHDA (100 µM), or Pyr3 (10 µM) were administered to examine the effect of these compounds on OAG- or ATP-induced TRPC3 currents.HEK 293 cells were cultured in Dulbecco's 1× high glucose modified Eagle's medium supplemented with 10% fetal bovine serum and 0.5% penicillin-streptomycin . Cells were incubated at 37°C with 6% COTotal RNA from cultured astrocytes were prepared using the RNeasy Mini Kit  with on-column DNase digestion. RNA was quantified using a Nanodrop system , with the average yield of RNA from 75–100 ng/μL. Whole brain total RNA was prepared with the Qiagen RNeasy Mini Kit and used for a positive control. cDNA was synthesized using reverse transcriptase  and reverse transcriptase-PCR was conducted. PCR products were visualized with a 1.5% agarose gel with ethidium bromide staining on a BioRad Chemidoc XRS imager. Primers used are listed in Primary striatal astrocytes (70% confluency) were transfected for 72 hrs with 25–50 pmol of siRNA oligonucleotides  using the Transit-X2 reagent  per the manufacturer's protocol. Knockdown was confirmed by reverse transcriptase PCR.Differences between three or more means with one independent variable was performed using one-way analysis of variance (ANOVA) followed by the Tukey's multiple comparison post hoc test using Prism software . Two-component variant analysis was performed using two-way ANOVA and Sidak's multiple comparison test. Comparisons of two groups were analyzed using the Student's t-test. Results are expressed as the mean ± SEM from a minimum of 3 independent studies and for all experiments, p<0.05 was considered significant.2+ waves between astrocytes depends heavily upon release of ATP by SNARE proteins  and connexin 43 hemi-channels, which is stimulated by a transient rise in the intracellular Ca2+ concentration 2+ transients was investigated in 2+ in primary striatal astrocytes, as determined by real-time fluorescence microscopy (2+ release from intracellular stores was followed by a sustained increase in Ca2+ associated with entry from the extracellular space. The acute effects of 1-methyl-4-phenylpyridinium ion (MPP+) and 6-hydroxydopamine (6-OHDA) on ATP-induced Ca2+ transients were determined by adding increasing concentrations of each neurotoxicant to the medium 30 sec prior to stimulation with ATP. This was done to test the direct effect of each cationic neurotoxicant on Ca2+ response independent of longer term alterations such as changes in gene expression or channel translocation. Increasing concentrations of MPP+ from 0.01 to 10 µM caused a concentration-dependent decrease in the maximum amplitude of ATP-induced Ca2+ transients in striatal astrocytes , had no inhibitory effect on ATP-induced transients  30 sec prior to stimulation of Ca2+ waves resulted in a dramatic decrease in the amplitude of the wavefront throughout the field of astrocytes. Quantification of the maximum intensity of the intracellular Ca2+ transient at the wavefront in each group is shown in + was more effective than 6-OHDA at suppressing mechanically-induced Ca2+ waves in striatal astrocytes, although both compounds resulted in greater than 50% inhibition of the maximum amplitude of the wavefront. 100 µM MPP+ and 6-OHDA were equally effective in diminishing the distance of propagation of Ca2+ waves, whereas acute administration of 100 µM DOPAC resulted in only a slight decrease in the extent of wave propagation  had no inhibitory effect. Stimulation with 1 µM ATP in Ca2+-free imaging medium reduced the peak amplitude of the Ca2+ transient but abolished the sustained phase of Ca2+ entry from the extracellular space  that can trigger influx of extracellular Ca2+ through plasma membrane TRP channels 2+ entry in astrocytes to the overall ATP-induced transient, we stimulated astrocytes with the DAG analog, 1-oleoyl-2-acetyl-sn-glycerol (OAG), a membrane permeable analog of DAG, that directly activates TRPC 3, 6 and 7 2+ that was not prevented by pretreatment with U73122  and caffeine  were suppressed, although not completely abolished, by pretreatment (60 sec) with Pyr3  and MPP+ (10 µM) further reduced ATP-induced Ca2+ transients, although the initial response to ATP was still present. Pyr3 completely blocked the intracellular response to OAG (100 µM). . . To detedenosine . Neithery 10 min p<0.00012+ entry but did not affect IP3-dependent transients following stimulation with ATP  were measured after 30 sec pre-incubation with each compound (2+ transients were significantly attenuated by MPP+ (10 µM) compared to control , and were blocked with the TRPC3 inhibitor, Pyr3  currents that were blocked by MPP+  (+ (+ or Pyr3 (+-mediated inhibition.To directly measure the effects of MPPechnique . Using t(100 µM) . OAG-dep(100 µM) , indicat0 µM)  signaling in astrocytes is essential for modulating diverse physiological processes in the CNS, including synaptic transmission, neurovascular coupling and metabolic activity in glial cells 2+ signaling during disease states and neurotoxic stress are not well understood. We therefore examined whether selected toxicants of the dopaminergic system could alter ATP-dependent puringergic Ca2+ signaling in astrocytes in part by acutely inhibiting TRPC3 channels, which could provide insight into novel glial-specific signaling mechanisms underlying the sensitivity of dopaminergic nuclei to oxidative and neurotoxic injury. Although these toxins are structurally distinct and represent endogenous (6-OHDA) and exogenous (MPP+) insults, they may share a common mechanism of action in their capacity to acutely disrupt astrocytic Ca2+ responses to purinergic stimuli that are critical for maintaining neuronal homeostatic and trophic functions.Calcium  could influence certain long term intracellular effects of MPP+ or 6-OHDA. Although these channels are expressed in astrocytes + and 6-OHDA + likely precludes ROS formation as a primary mechanism. Furthermore, acute addition of DOPAC had no effect on ATP-induced Ca2+ transients  or PMA (phorbol myristate acetate) was unable to completely abolish ATP-induced Ca2+ responses 2+ only partially decreased the amplitude of the initial ATP-induced transient but completely abolished the sustained phase of the Ca2+ response associated with entry of extracellular Ca2+ , which directly activates TRPC3 channels + and 6-OHDA. Expression of DAG-sensitive TRPC6/7 was not detected in primary striatal astrocytes, further supporting TRPC3 as the likely site of inhibition of extracellular Ca2+ entry.To identify possible sites of inhibition of MPP1 µM ATP , indicatlar Ca2+ , indicat2+ influx in striatal astrocytes independent of PLC, because U73122 failed to inhibit OAG-dependent increases in intracellular Ca2+  channels + efflux via K2P channels could oppose any potential Na+/Ca2+ influx via TRPC3 in native cells, yielding little or no net charge movement and could explain the lack of inward current at hyperpolarizing potentials in our patch clamp recordings. In contrast, ATP evoked large inward and outward currents in astrocytes that were suppressed by both Pyr3 and MPP+ . This receptor is inhibited by toxicants such as polybrominated diphenyl ethers (PBDE's) and non-coplanar polychlorinated biphenyl compounds (PCB's), which alter Ca2+ homeostasis in neural cells + is non-coplanar + and 6-OHDA more strongly suggest that they function as channel blockers at the plasma membrane, as demonstrated in recent studies with the divalent metal, Mn2+, which reversibly inhibited OAG-induced Ca2+ transients in astrocytes by competitively decreasing Ca2+ influx through TRPC3 + and, to a lesser extent 6-OHDA, acutely decreases ATP-dependent Ca2+ transients in astrocytes by partly by inhibiting the channel activity of TRPC3. These data do not preclude the possibility of longer term changes in Ca2+ signaling due to altered gene expression or translocation of TRPC3 2+ responses of astrocytes to ATP.Ca2+ from intracellular stores but have deprecations in mGluR-mediated inward currents, indicating an important role for TRPC3 in stimulus-dependent Ca2+ entry + or 6-OHDA decreases ATP-dependent Ca2+ transients partly by blocking entry of extracellular Ca2+, thereby suppressing downstream signaling responses to critical gliotransmitters such as ATP that are associated with Ca2+ wave activity and intercellular communication between astrocytes  Expression of ionotropic P2X receptors in primary cortical and striatal astrocytes was determined by reverse transcriptase-PCR (rtPCR) and indicated that all P2X receptor subtypes are expressed in both cortical and striatal astrocytes. (b) Metabotropic P2Y receptors were broadly expressed in cortical astrocytes except P2Y4 and P2Y13, whereas striatal astrocytes expressed all P2Y receptors. (c) Expression of TRPC subfamily receptors was determined by rtPCR. TRPC receptors 1–4 were detected in primary astrocytes. Results are representative of 2 independent experiments in separate cultures of primary astrocytes.(DOCX)Click here for additional data file.Table S1Expression of P2X/Y and TRPC receptors in primary astrocytes. (a) Expression of ionotropic P2X receptors in primary cortical and striatal astrocytes was determined by reverse transcriptase-PCR (rtPCR) and indicated that all P2X receptor subtypes are expressed in both cortical and striatal astrocytes. (b) Metabotropic P2Y receptors were broadly expressed in cortical astrocytes except P2Y4 and P2Y13, whereas striatal astrocytes expressed all P2Y receptors. (c) Expression of TRPC subfamily receptors was determined by rtPCR. TRPC receptors 1–4 were detected in primary striatal astrocytes. Results are representative of 2 independent experiments in separate cultures of primary astrocytes.(DOCX)Click here for additional data file.


Adoptive immunotherapy, including transfer of activated NK cells, is currently under active investigation for patients with refractory and relapsed acute myeloid leukemia (AML). However, a highly immunosuppressive microenvironment sustained in part by tumor-derived exosomes, creates a major hurdle for adoptive immunotherapy. We recently reported that exosomes, virus-size (30-100nm) membrane-bound vesicles, represent one immunosuppressive mechanism operating in AML. We found high levels of exosomes in plasma of newly-diagnosed AML patients prior to any therapy. These exosomes carried an immunosuppressive cargo, including membrane-associated TGF-β1, PD-1, MICA/MICB and markers of myeloid blasts. Infusion of activated NK cells in patients with AML did not result in the expected recovery of NK cell activity. We hypothesize that the presence of immunosuppressive plasma exosomes in refractory/relapsed AML patients may impair anti-tumor activity of adoptive cell therapies.Venous blood (20-50 mL) was obtained from patients with refractory/relapsed AML (n=7). Exosome fractions were isolated from the patients' plasma and plasma of normal controls by using mini-size exclusion chromatography with Sepharose 2A. Protein levels, numbers and the size of exosomes (qNano) and their morphology (transmission electron microscopy) were determined. Exosomes were characterized by Western blots for expression of exosome markers, Tsg101 and CD81, and myeloid cell-surface markers associated with AML, interleukin-3 receptor alpha chain (CD123) and C-type lectin-like molecule-1 (CLL-1), CD44, CD96 and TGF-β1. Isolated normal human NK cells were co-incubated with AML exosomes and multiparameter flow cytometry was used to monitor changes in expression levels (mean fluorescence intensity) of NKG2D on NK cells.Exosome fractions isolated from AML patients' plasma with refractory/relapsed AML had mean protein content of 36 µg protein/mL plasma. AML exosomes contained blast markers, CD123, CLL-1, CD44, CD96 suggesting their leukemia origin. They were also enriched in TGF-β1, which is known to interfere with NK cell activity and promote Treg expansion. Co-incubation of AML exosomes with activated NK cells resulted in down-regulation of NKG2D expression with a concomitant reduction of NK-cell cytotoxicity. Antibody neutralization of TGF-β1 carried by AML exosomes significantly abrogated their immunosuppressive activity.The persistently elevated levels of biologically-active TGF-β1+ exosomes carrying leukemia blast markers in plasma of refractory/relapsed AML patients indicate that existing immune suppression will interfere with anti-leukemia effects of adoptive cell therapies.


Transient elevations of cytosolic Ca2+ may boost ATP synthesis, but ATP is also consumed by ion pumps to maintain a low Ca2+ in cytosol. In differentiation process plasma membrane Ca2+ ATPase (PMCA) is considered as one of the major players for Ca2+ homeostasis. From four PMCA isoforms, the fastest PMCA2 and PMCA3 are expressed predominantly in excitable cells. In the present study we assessed whether PMCA isoform composition may affect energy balance in differentiating PC12 cells. We found that PMCA2-downregulated cells showed higher basal O2 consumption, lower NAD(P)H level, and increased activity of ETC. These changes associated with higher [Ca2+]c resulted in elevated ATP level. Since PMCA2-reduced cells demonstrated greatest sensitivity to ETC inhibition, we suppose that the main source of energy for PMCA isoforms 1, 3, and 4 was oxidative phosphorylation. Contrary, cells with unchanged PMCA2 expression exhibited prevalence of glycolysis in ATP generation. Our results with PMCA2- or PMCA3-downregulated lines provide an evidence of a novel role of PMCA isoforms in regulation of bioenergetic pathways, and mitochondrial activity and maintenance of ATP level during PC12 cells differentiation.A close link between Ca Neuronal development is highly organized sequence of events eventually leading to the formation of functional nerve cells. Neurogenesis occurs globally during development and, to some extent, is also active in adult nervous system . The for2+, as a link between ATP handling and neurogenesis is suggested by numerous studies [2+ concentration ([Ca2+]c) may on the one hand boost ATP synthesis but, on the other, ATP is required for homeostatic maintenance of a low resting [Ca2+]c in neuronal cells c according to the equation [Ca2+]free = Kd((F − Fmin)/(Fmax − F)), where Kd = 345 nM. Maximal signal (Fmax) was obtained with 10 μM ionomycin, while the minimal signal (Fmin) was obtained with 10 mM EGTA. In a separate set of experiments, cells were preincubated for 30 min with 10 μM BAPTA-AM and [Ca2+]c were monitored as above.Approximately 1 × 10μM) added in presence of glucose and pyruvate  2 h before measurement (+glucose + 2-DG + pyruvate). Short (3 h treatment) and long (48 h treatment) time effects of 6 μM oligomycin action were determined in glucose-containing buffer.If not stated otherwise, the given parameters were assessed in a buffer containing 5 mM D-glucose (+glucose). 1 mM KCN was added alone 20 min before measurement to glucose-containing buffer (+glucose + KCN). The contribution of glycolysis to ATP synthesis was determined with 2-DG (20 μM oligomycin (for 3 h or 48 h) or with 20 μM 2-deoxyglucose (for 2 h) in the presence of glucose. ~1 × 106 cells were double stained with Annexin V/Propidium iodide using Annexin V-FITC Apoptosis Detection Kit I according to the manufacturer's protocol and analyzed with FACScan Becton Dickinson. The fluorescence recorded from 104 cells was measured in each experiment. The data were plotted using CellQuest Becton Dickinson software. The basal level of mortality was determined with or without 0.1% DMSO (solvent for oligomycin) in the presence of glucose. Due to lack of differences, the data obtained without DMSO were chosen for further comparisons.Cells were incubated with either 6 ATP was measured in nontreated cells and upon treatment with inhibitors, as described in Drug Treatment section. ATP concentration was determined using adenosine 5′-triphosphate (ATP) Bioluminescent Assay Kit on GloMax 20/20 luminometer (Promega) and normalized to the protein content. In experiments with oligomycin, cells incubated with 0.1% DMSO (oligomycin solvent) were used as a negative control and ATP values from these cells were subtracted from ATP values obtained in oligomycin (+) experiments. For each set of measurements a second negative control (no cells) was included, and a background fluorescence was further subtracted from all other values.Medium of a 48 h culture of differentiated cells was used to quantify basal glucose consumption and lactate release using Lactate Assay Kit and Glucose Assay Kit. The same method was used to assessed glucose and lactate concentration following treatments with inhibitors (see Results and Drug Treatment sections).6 cells were suspended in a buffer containing 50 mM TRIS-HCl, pH 8.0, 50 mM acetyl-CoA, 0.1% Triton X-100, and 100 mM dithionitrobenzoic acid (DTNB) and the reaction was initiated by the addition of oxaloacetate to a final concentration of 250 mM. After 5 min of incubation the absorbance of a resulting product thionitrobenzoic acid (TNB) was determined spectrophotometrically at 412 nm c and these changes were associated with reduced protein level of examined PMCA isoforms. Thereby, we next clarified the possible relationship between [Ca2+]c and ATP content H profile suggested more effective energy conversion into ATP through OxPh.Further we assessed the activity of all four ETC complexes and normalized the results to citrate synthase activity. We observed higher activity of complexes I, III, and IV in _2 line Figures , which eβIII-tubulin, a protein found almost exclusively in neurons, was present at higher amounts in _2 and _3 lines in comparison to control cells. After incubation with oligomycin its level decreased to comparable values in all lines H level, all accompanied by higher consumption of NADH by ETC due to increased activity of proton-pumping complexes. Therefore, the rate of oxidative phosphorylation when PMCA2 level is reduced may be controlled at the activity of ETC proteins which is supported by the metabolic study demonstrated in isolated mitochondria [2+ produced by PMCA2 downregulation may transiently activate TCA cycle dehydrogenases. This would be expected because the elevations in [Ca2+]c are thought to increase ATP consumption by Ca2+ clearance mechanisms c are, however, able to modify mitochondrial metabolism suggesting a tight relationship between PMCA2 and ATP production. Because even small changes in cellular energy state may initiate cellular pathology and death, fast reacting PMCA2 and PMCA3 isoforms may be considered as cellular bodyguards protecting against Ca2+ overload and subsequent mitochondrial failure.In summary, our findings demonstrate that changes in ATP pool during differentiation are tightly coupled to Ca


Rhododendron dauricum L., has been proven to be an important multifunctional physiologically active component, but its vasoactive mechanism is not clear. The present study was performed to observe the vasoactive effects of farrerol on rat aorta and to investigate the possible underlying mechanisms. Isolated aortic rings of rat were mounted in an organ bath system and the myogenic effects stimulated by farrerol were studied. Intracellular Ca2+ ([Ca2+]in) was measured by molecular probe fluo-4-AM and the activities of L-type voltage-gated Ca2+ channels (LVGC) were studied with whole-cell patch clamp in cultured vascular smooth muscle cells (VSMCs). The results showed that farrerol significantly induced dose-dependent relaxation on aortic rings, while this vasorelaxation was not affected by NG-nitro-l-arginine methylester ester or endothelium denudation. In endothelium-denuded aortas, farrerol also reduced Ca2+-induced contraction on the basis of the stable contraction induced by KCl or phenylephrine (PE) in Ca2+-free solution. Moreover, after incubation with verapamil, farrerol can induce relaxation in endothelium-denuded aortas precontracted by PE, and this effect can be enhanced by ruthenium red, but not by heparin. With laser scanning confocal microscopy method, the farrerol-induced decline of [Ca2+]in in cultured VSMCs was observed. Furthermore, we found that farrerol could suppress Ca2+ influx via LVGC by patch clamp technology. These findings suggested that farrerol can regulate the vascular tension and could be developed as a practicable vasorelaxation drug.Farrerol, isolated from  HyperAlthough there are many drugs currently approved to treat hypertension, such as indapamide, metoprolol, enalapril, losartan, nifedipine , most ofRhododendron dauricum L., and has been extensively used to alleviate symptoms associated with bronchial asthma in China in, gradually restoring the calcium levels to the basal level measured before the application of farrerol. The above results indicate that farrerol could decrease [Ca2+]in in cultured VSMCs.Laser scanning confocal microscopy method was employed to investigate ex influx currents were measured using whole-cell patch clamp technique. In order to obtain currents of a bigger magnitude, Ba2+ was used as the charge carrier. Currents appeared at a voltage of about −40 mV, and peaked at around 0 mV. The maximal current density was −10.72 ± 0.45 pA/pF (n = 8). At a test potential of 0 mV, 14.02 μM farrerol reduced the Ca2+ current by 59.50% (The inhibitory potency of farrerol on Cay 59.50% .2.2.Rhododendron dauricum L. (known as man-shan-hong in Chinese), has been extensively used for the treatment of bronchitis and asthma in China. The present study revealed that farrerol has vasorelaxation properties. This is the first study of the myogenic and electric effects of farrerol on rat aorta.Farrerol, a flavonone, isolated from 2+ availability, which was confirmed by three different experiments in the present study. Myogenic experiments showed that farrerol mainly inhibited the contraction component induced by [Ca2+]ex influx. The calcium assays demonstrated that farrerol inhibited the [Ca2+]in elevation induced by depolarization. Moreover, the patch clamp experiments indicated that farrerol reduced [Ca2+]ex influx currents through LVGC, which is also consistent with previous studies showing that inhibiting the entry of [Ca2+]ex is a contributing mechanism to the vasorelaxation induced by farrerol. Ca2+ plays essential roles in regulating the vascular tone ex may be another critical mechanism involved in the relaxation. The fact that farrerol can reduce the increase in [Ca2+]in elicited by KCl suggests that farrerol may inhibit voltage-dependent Ca2+ channels activity, resulting in decreased Ca2+ entry and [Ca2+]in. The measurements of [Ca2+]in confirmed this observation.It is well known that a high concentration of extracellular KCl leads to the depolarization of the cell membrane, which induces the increase in transmembrane Catraction . Farrero2+ influx through ROC and by release of Ca2+ from the sarcoplasmic reticulum [2+ release from sarcoplasmic reticulum stores to decrease [Ca2+]in and relax the aorta. At the same time, farrerol (35.94 μM) was observed to inhibit contraction when PE produced a steady contraction followed by gradual Ca2+ input in a Ca2+-free Krebs’ solution, which indicated that farrerol blockade of ROC to decrease the influx of [Ca2+]ex may be a critical mechanism in relaxing the aorta.Previous studies showed that PE, an α-adrenoreceptor agonist, causes aortic contraction by Caeticulum –42. In t−5 M ruthenium red, but not by 50 mg/L heparin, suggested that the contraction action of farrerol on aorta may be mediated by blocking ryanodine receptors to release Ca2+ from the sarcoplasmic reticulum.We found that farrerol (35.94 μM ) induced relaxation both in the endothelium-denuded and -intact aortas pretreated for 30 min with 1 μM verapamil and subsequently contracted by PE. This effect of farrerol, enhanced by 102+ from the sarcoplasmic reticulum. These results suggest that farrerol may be a possible therapeutic agent for the treatment of hypertension diseases.Taken together, our results suggest that farrerol relaxes contracted rat aorta through the involvement of LVGC as well as ROC. Furthermore, the relaxation effect of farrerol may be mediated by blocking ryanodine receptors to release Ca3.3.1.N,N,N′,N′-tetraacetic acid (EGTA), acetylcholine chloride, and phenylephrine (PE) were purchased from Sigma . 4-(2-Hydroxyethyl) piperazine-1-ethanesulfonic acid (HEPES) was purchased from AMRESCO Company . Verapamil, ruthenium red (RR), and heparin (HP) were from Sigma Company. Potassium chloride (KCl), sodium chloride (NaCl), magnesium chloride (MgCl2), glucose, calcium chloride (CaCl2) and all the other reagents were of analytical purity. Farrerol (v/v), which has been shown to be devoid of any observable effects on the vascular muscle tone and rat aorta VSMCs.Ethylene glycol-bis (2-aminoethylether)-Farrerol  was prep3.2.Male Sprague Dawley rats  were fed a diet containing 11% fat, 60% vegetable starch, and 29% protein, supplied by the Animal Center of Shanxi Medical University. Rats were housed in cages, had free access to food and water, and were maintained in a 12 h light/dark cycle. All protocols and procedures of our experiments described were approved by the Animal Care and Use Committee of the Shanxi Medical University , and all efforts were made to minimize the number of animals used and their suffering, in accordance with the ethical guidelines for animal research at Shanxi Medical University.3.3.4·7H2O, 1.2; KH2PO4, 1.2; CaCl2, 2.5; NaHCO3, 25.0; and Glucose, 11.0, bubbled with 95% O2 + 5% CO2 (pH 7.4). The aortas were cleaned of adherent connective tissues and cut into rings of 4 mm length. The rings were suspended on two wire hooks in water-jacketed tissue baths containing 5 mL of the Krebs’ solution kept at 37 °C and gassed with 95% O2 + 5% CO2. The upper hook was connected to a force transducer and changes in isometric force were recorded using Chart 5.4  and saved on a computer hard disk. Passive tension was adjusted to 2 g and all subsequent measurements representing the force were generated above this baseline in release. Endothelium-denuded aortic rings were exposed to 10−6 M verapamil in measurements were carried out using an OLYMPUS FV1000  confocal microscope at room temperature. Fluo-4-AM was excited at 494 nm, and the emission was recorded at 516 nm; the measured fluorescence was subtracted from the background and autofluorescence. The fluorescent intensity was used to represent the changes in intracellular Ca2+ levels.VSMCs were plated on dishes and loaded with 2 μM Fluo-4-AM for 30 min at 37 °C in a physiological salt solution . Patch pipettes were filled with a solution containing 130 mM CsCl, 2 mM MgCl2, 5 mM Mg-ATP (Adenosine triphosphate), 5 mM HEPES, 10 mM Glucose and 10 mM EGTA at pH 7.2. CsCl was included in the pipettes solution to eliminate K+ currents. The voltage protocol was designed according to the characteristics of IBa in rat aortic VSMCs. IBa was measured at holding potentials of −60 mV and subjected to step depolarizations of 200 ms to +60 mV in 10 mV increments every 1 s. Voltage-activated calcium currents were recorded under a flowing bath solution containing 80 mM BaCl2, 30 mM TEA (Tetraethylammonium chloride), 2 mM MgCl2, 5 mM HEPES and 10 mM Glucose at pH 7.4. Data were collected after stabilization of the current amplitude, usually 1–2 min after the whole-cell configuration had been obtained. The input resistance of the cells must exceed 1 GΩ.Whole cell L-type Ca3.5.n” represents the number of rats. The EC50 was defined as the concentration of farrerol that induced 50% of the maximum relaxation from the contraction elicited by PE (1 μM) or KCl (60 mM) and was calculated from the concentration–response curve resulting from a nonlinear regression (curve fit) performed using GraphPad Prism . Statistical analysis was done using the Student’s t-test and one-way analysis of variance (ANOVA). In all cases, statistical significance was defined when a p value of <0.05 was obtained. Laser confocal data were analyzed with the FV10-ASW 3.0 software . Patch clamp data were analyzed by the softwares IGOR Pro 6.1  and PULSEFIT .All data are presented as means ± SD. The letter “4.2+]in in VSMCs. These data provide strong evidence that farrerol inhibits both entry [Ca2+]ex and release of intracellular stored Ca2+ in VSMCs, and suggest that farrerol has potential effects on the regulation of the hypertension and could be developed as a practicable vasorelaxation drug.In conclusion, farrerol could markedly induce relaxation of rat aortic rings. The involvement of LVGC as well as ROC contributes to the relaxant effects of farrerol in rat aorta. Furthermore, this activity correlated well with its potency to reduce [Ca


With a prevalence of 1–2%, epilepsies belong to the most frequent neurological diseases worldwide. Although antiepileptic drugs are available since several decades, the incidence of patients that are refractory to medication is still over 30%. Antiepileptic effects of κ opioid receptor (κ receptor) agonists have been proposed since the 1980s. However, their clinical use was hampered by dysphoric side effects. Recently, G‐protein biased κ receptor agonists were developed, suggesting reduced aversive effects.We investigated the effects of the κ receptor agonist U‐50488H and the G‐protein biased partial κ receptor agonist 6′‐GNTI in models of acute seizures and drug‐resistant temporal lobe epilepsy and in the conditioned place avoidance (CPA) test. Moreover, we performed slice electrophysiology to understand the functional mechanisms of 6′‐GNTI.−1) or 6′‐GNTI (10–30 nmoles) treatment in the mouse model of intra‐hippocampal injection of kainic acid. Single cell recordings on hippocampal pyramidal cells revealed enhanced inhibitory signalling as potential mechanisms causing the reduction of paroxysmal activity. Effects of 6′‐GNTI were blocked in both seizure models by the κ receptor antagonist 5′‐GNTI. Moreover, 6′‐GNTI did not induce CPA, a measure of aversive effects, while U‐50488H did.As previously shown for U‐50488H, 6′‐GNTI markedly increased the threshold for pentylenetetrazole‐induced seizures. All treated mice displayed reduced paroxysmal activity in response to U‐50488H (20 mg·kgin vivo.Our data provide the proof of principle that anticonvulsant/antiseizure and aversive effects of κ receptor activation can be pharmacologically separated  Intracisternally saline infused animals, and animals treated with 5′‐GNTI alone, were included as control groups. Infusion was stopped at the first appearance of tonic–clonic seizures and animals were immediately killed by cervical displacement. Threshold was calculated from volume injected and body weight  and then deeply anaesthetized with sevoflurane through a precise vaporizer . Mice were injected with 50 nL of a 20 mM kainic acid solution into the left hippocampus as previously described  male mice were sedated with ketamine (160 mg·kg−1) 20 min before and after surgery as an analgesic treatment. The locally injected kainic acid model was chosen because it is a model of trauma‐induced epilepsy that allows the investigation of self‐sustained paroxysmal events, and it was characterized as a model for drug‐resistant epilepsy  instead of the contralateral electrode. All animals received meloxicam . EEGs were filtered for epileptiform spikes defined as high amplitude discharges (>3 × baseline) lasting less than 70 ms. This definition allows the exclusion of other EEG events induced by hippocampal kainic acid administration, such as high‐voltage sharp waves that last considerably longer  and automatically analysed using et al., Twenty‐eight C57BL/6 N wild‐type young adult  male mice were used for the conditioned place avoidance (CPA) paradigm. Tests were conducted in a custom‐made, three‐chamber apparatus . To analyse the influence of drug treatment on motor activity, the distance travelled in the drug‐paired or saline‐paired chamber was recorded on the first treatment day using the VideoMot 2 System.et al., n = 4 per group) by an overdose of thiopental (150 mg·kg−1), and brains were fixed by transcardial perfusion with 4% paraformaldehyde in PBS . Immunohistochemistry was performed on coronal 40 μm vibratome sections incubated free‐floating in blocking solution  for 90 min. Primary antibodies against Zif268  or pERK  were applied overnight at room temperature followed by horseradish peroxidase conjugated secondary antibodies  and 3,3′‐diaminobenzidine for detection. Specificity of antibodies was controlled by avoiding primary antibodies or by pre‐absorbing the primary antibody with the respective blocking peptide  overnight at +4°C before the application on the section.Immunohistochemistry was performed as described elsewhere  optimized for slice preparation (slice solution), containing (in mM) 118 NaCl, 3 KCl, 1.3 MgSO4, 1.4 NaH2PO4, 10 glucose, 26 NaHCO3 and 2.5 CaCl2, which was bubbled continuously with carbogen . Kynurenic acid (1 mM) was also added to the slice solution to reduce glutamate‐mediated excitotoxicity. Mid‐ventral, transverse sections of the hippocampus (300 μm) were cut using a vibrating slicer . Slices were then transferred to a carbogenated ACSF (bath) solution, which contained the following (in mM): 124 NaCl, 3 KCl, 1.3 MgSO4, 1.4 NaH2PO4, 10 glucose, 26 NaHCO3 and 2.5 CaCl2 (300–305 mOsm·L−1). Slices were stored at room temperature (22°C) in the bath solution for at least 30 min following slicing; the bath solution was also used to perfuse slices for all experiments. Slices were placed into a recording chamber attached to a fixed stage of a moveable upright microscope  and held submerged by a platinum and polyester fibre ‘harp’. Slices were continuously perfused with a warmed (34 ± 0.5°C), carbogenated bath ACSF at a rate of 2–3 mL·min−1 for at least 20 min prior to recording.Five C57BL/6 N male wild‐type mice (25 g) between 8–16 weeks of age were used for 2, to which 0.05–0.1% neurobiotin was added, and the pH was adjusted to 7.27–7.30 with KOH, (275–285 mOsm·L−1). Recordings were made using a Multiclamp 700B amplifier data and were acquired using a pCLAMP 10.3 via a Digidata 1322 interface .Pipettes were pulled from thin‐walled borosilicate glass  with a two‐stage puller  to a tip resistance of 5–7 MΩ when backfilled with an internal solution containing (in mM) 126 K‐gluconate, 10 HEPES, 4 KCl, 5 MgATP, 0.3 NaGTP, 1 EGTA and 0.3 CaClCA3 and CA1 pyramidal neurons were visually identified using infrared differential interference contrast optics and selected for recordings based on characteristic pyramidal morphology and the presence of a large apical dendrite. After obtaining the whole cell patch clamp configuration, neurons were held in a voltage clamp at −60 mV for 5–10 min before beginning experiments and between experimental measurements. Only neurons that showed stable resting membrane potential (RMP), holding a current (in a voltage clamp) in a series of control measurements, and which showed stable access resistance throughout the entire experiment, were selected for analysis.A receptor‐mediated. Spontaneous IPSCs and EPSCs were also measured during 2 min continuous voltage clamp recordings at −40 mV. The RMP of neurons was measured by averaging the potential over a 30 s period of passive current clamp recording. Drug effects on postsynaptic conductances were measured using a series of hyperpolarizing voltage steps conducted from a holding potential of −40 mV. Eight successive hyperpolarizing steps were used; the initial step hyperpolarized the membrane 10 mV, and each successive step increased by a −10 mV increment such that the final step moved the membrane from −40 to −120 mV. Each successive hyperpolarizing step was shortened by Xms to limit voltage‐mediated damage to the cell membrane. Several (>2) sets of recordings of neuronal and synaptic properties  were taken at 5 min intervals before drug application.A stimulation isolation unit  was used to elicit synaptic responses onto recorded neurons. Stimulating electrodes (bath ACSF‐filled patch pipettes) were placed in either the mossy fibre layer for CA3 recordings or the Shaffer collaterals for CA1 recordings, exciting axons from the dentate gyrus and CA3 respectively. Stimulation intensities were adjusted to evoke responses of approximately half‐maximal amplitude. Synaptic responses that were evoked as neurons were held at −40 mV, which allowed inhibitory (likely GABAergic) and excitatory responses to be differentiated as outward and inward currents respectively. At −40 mV, most neurons showed both evoked EPSCs and inhibitory postsynaptic current (IPSCs). Holding neurons at −60 mV eliminated the IPSC component of evoked responses, suggesting the IPSC was largely GABA6′‐GNTI, dissolved in 10 mL ACSF to a final concentration of 1 μM, was applied via bath perfusion over a period of 2–3 min and then was washed out with ACSF. Recordings were taken immediately prior to drug application, during application, 1 min after application and successively every 5 min until drug effects washed out. The electrophysiology was performed unblinded due to practical reasons.et al., in vivo experiments and to generate figures. For the statistical analysis, a one‐way ANOVA with a Dunnett post hoc test was applied to in vivo experiments. For electrophysiology, the two‐tailed, paired t‐tests were applied for RMP analysis, and a one‐way ANOVA was used to compare drug effects on IPSC. CPA data were analysed using paired t‐test and two‐way ANOVA with a Bonferroni post hoc test. A P value lower than 0.05 was considered significant.The data and statistical analysis comply with the recommendations on experimental design and analysis in pharmacology . The κ receptor agonist U‐50488H and antagonist 5′‐GNTI were purchased from Tocris Bioscience,  while the G‐protein biased partial κ receptor agonist 6′‐GNTI was provided by the drug supply programme of National Institute on Drug Abuse (NIDA). pentylenetetrazole, U‐50488H, 5′‐GNTI and 6′‐GNTI were dissolved in saline, and the pH was adjusted to 7.2. Diazepam was dissolved in DMSO (10 mg·mL) and further diluted with saline. Diazepam and U‐50488H were applied i.p. For EEG recordings and CPA experiments, 5′‐GNTI (3 or 10 nmoles per 3 μL) and 6′‐GNTI (1–30 nmoles per 3 μL) were given i.c.v., while for the pentylenetetrazole experiments, they were administered intracisternally.F8,43 = 11.37). This effect reached a plateau at about 3 nmoles (P < 0.05). Pretreatment of animals with 3 or 10 nmoles of the specific κ receptor antagonist 5′‐GNTI reversed this effect.Based on our previous finding that the full κ receptor agonist U‐50488H was able to restore the reduced pentylenetetrazole‐seizure threshold in pDyn‐KO mice, we investigated the effects of 6′‐GNTI in this model of acute seizures. As shown in Figure et al., n = 21). During hpds, mice displayed behavioural arrest and stereotypies. To investigate the potentials of κ receptor agonists in this model, we first tested whether the full κ receptor agonist U‐50488H affected these EEG abnormalities. As shown in Figure P < 0.05 for 20 mg·kg−1; F4,29 = 5.081) resulted in a significant reduction of total time spent in hpds . In contrast to diazepam‐treated mice, which were not moving (most probably due to the highly sedative effects of diazepam), U‐50488H‐treated animals were actively exploring the cage during this time. The onset of suppression of spike trains is very rapid for both diazepam and U‐50488H, as shown in Figure Unilateral injection of kainic acid into the dorsal hippocampus of mice was characterized as a model for drug‐resistant epilepsy based on the non‐responsiveness of paroxysmal discharges to acute carbamazepine, phenytoin or valproate treatment  as well as the number of events  were reduced, resulting in a significant reduction of total time of hpds . Although 6′‐GNTI is only a partial κ receptor agonist, animals displayed a strong reduction of spike trains after the treatment with 10 or 30 nmoles  induced strong place avoidance . In contrast, 6′‐GNTI induced neither avoidance nor preference for the drug‐conditioned chamber at any tested dose  with saline, 2.5 mg·kget al., n = 7; one‐way ANOVA, P < 0.05 for 6′‐GNTI vs. control; F2,20 = 23.28), the effect was reversed upon washout of the drug. In a separate experiment, we tested whether the specific κ receptor antagonist 5′‐GNTI was able to block the increased eIPSC amplitude. After 6′‐GNTI application (demonstrating the responsiveness of cells to 6′‐GNTI), slices were bathed in ACSF containing 5′‐GNTI but not 6′‐GNTI. Under these conditions, eIPSC amplitudes were on control level , and reversed on washout. No effect on pyramidal cell spontaneous IPSCs amplitudes was observed , as these were suggested to have higher informative values in terms of human seizures. Irrespective of the settings, 6′‐GNTI induced reduction of inter‐ictal spikes and seizure‐like events, at least at the highest dose tested. In contrast, the modern antiepileptic drugs showed significant reduction of spike trains, but were mostly ineffective on hippocampal paroxysmal discharges.in vitro.In conclusion, our data provide proof for the principle that anticonvulsant and antiseizure effects can be achieved without inducing place‐aversion by applying a partial κ receptor agonist, which preferentially activates the G‐protein over the β‐arrestin pathway, at least The authors declare no conflicts of interest.Declaration acknowledges that this paper adheres to the principles for transparent reporting and scientific rigour of preclinical research recommended by funding agencies, publishers and other organisations engaged with supporting research.This Figure S1 Treatment design for U‐50488H and 6′‐GNTI applied on kainic acidinjected mice . Ratios of total recorded events 60 min post/60 min pre‐treatment are given. * P < 0.05, significant difference; one‐way ANOVA with Dunnett's post hoc test..Figure S3 Immunohistochemistry for Zif268  and pERK1  was performed 30 minutes after i.c.v. application of saline , 2.5 mg/kg U‐ 50488H  or 30 nmoles 6′‐GNTI . Note the strong labelling for Zif268 in the nucleus accumbens and the moderate labeling for pERK1 in the dorsal raphe after 6′‐GNTI injection. In d and h the primary antibodies, Zif 268 (n = 4) and pERK 1 (n = 4) respectively, were pre‐absorbed with the blocking peptide, resulting in a lack of labeling as compared to c and g (sections obtained from the same brains). Scale bars in c for a‐d = 50 μm, in g for e‐h = 25 μm.Figure S4 Effects of 6′‐GNTI  and 5′‐GNTI treatments  on time of hpds on kainic acid injected mice compared t saline (n = 7). 6′‐GNTI effect was entirely reversed upon coadministration with 5′‐GNTI (n = 4). Instead, 5′‐GNTI alone (n = 5) had no effect on the time spent in hpds. * P <0.05, significant difference; one‐way ANOVA with Bonferroni's post hoc test.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.Supporting info itemClick here for additional data file.


Phosphatidylserine abundance at the erythrocyte surface was quantified utilizing annexin-V-binding, cell volume from forward scatter, [Ca2+]i from Fluo3-fluorescence, ceramide from antibody binding, and reactive oxidant species (ROS) from 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. A 48 h treatment of human erythrocytes with cantharidin significantly increased the percentage of annexin-V-binding cells (≥10 μg/mL), significantly decreased forward scatter (≥25 μg/mL), significantly increased [Ca2+]i (≥25 μg/mL), but did not significantly modify ceramide abundance or ROS. The up-regulation of annexin-V-binding following cantharidin treatment was not significantly blunted by removal of extracellular Ca2+ but was abolished by kinase inhibitor staurosporine (1 μM) and slightly decreased by p38 inhibitor skepinone (2 μM). Exposure of erythrocytes to cantharidin triggers suicidal erythrocyte death with erythrocyte shrinkage and erythrocyte membrane scrambling, an effect sensitive to kinase inhibitors staurosporine and skepinone.The natural phosphoprotein phosphatase inhibitor cantharidin, primarily used for topical treatment of warts, has later been shown to trigger tumor cell apoptosis and is thus considered for the treatment of malignancy. Similar to apoptosis of tumor cells, erythrocytes may undergo eryptosis, a suicidal cell death characterized by cell shrinkage and translocation of cell membrane phosphatidylserine to the erythrocyte surface. Signaling of eryptosis includes increase of cytosolic Ca Cantharidin, a traditional Chinese natural product, has been successfully used for the treatment of warts, molluscum contagiosum, and callus removal . Canthar2+ activity ([Ca2+]i), ceramide i was estimated utilizing Fluo3-fluorescence, ceramide abundance utilizing specific antibodies, and abundance of reactive oxidant species utilizing 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA) fluorescence. The involvement of phosphorylation was tested utilizing kinase inhibitors staurosporine and skepinone.The present study explored whether cantharidin stimulates eryptosis. To this end, erythrocytes from healthy volunteers were exposed to cantharidin, phosphatidylserine abundance at the erythrocyte surface determined using annexin-V-binding and cell volume estimated from forward scatter in flow cytometry. Moreover, i). As illustrated in 2 in the presence and absence of 1 mM sodium pyruvate.Both phospholipid scrambling of the erythrocyte membrane and cell shrinkage could be triggered by activation of Ca2, was similar in the presence or absence of pyruvate. A further series of experiments explored whether cantharidin-induced translocation of phosphatidylserine to the cell surface required entry of extracellular Ca2+. To this end, erythrocytes were incubated for 48 h in the absence or presence of 25 or 50 μg/mL cantharidin, both in the presence or nominal absence of extracellular Ca2+. As illustrated in 2+ did not significantly blunt the effect of cantharidin on annexin-V-binding. Instead, cantharidin significantly increased the percentage of annexin-V-binding erythrocytes to similarly high levels in the absence and in the presence of extracellular Ca2+. Thus, triggering of eryptosis did not require entry of extracellular Ca2+.As illustrated in 2+]i by ceramide. Thus, specific antibodies were utilized to quantify ceramide abundance at the erythrocyte surface. As a result, the ceramide abundance was similar following a 48 h incubation in the absence of cantharidin , presence of 10 μg/mL cantharidin  and presence of 50 μg/mL cantharidin . Thus, cantharidin did not enhance ceramide abundance.Eryptosis could be stimulated independently from increased i), which may contribute to the triggering of cell shrinkage by activation of Ca2+ sensitive K+ channels, K+ exit, cell membrane hyperpolarization, Cl− exit and thus cellular loss of KCl with osmotically obliged water i, an effect presumably due to stimulation of Ca2+ entry. Moreover, the increase of [Ca2+]i following cantharidin treatment could have contributed to stimulation of eryptosis. However, cantharidin treatment was able to trigger eryptosis even in the absence of Ca2+ entry, an observation pointing to the operation of a Ca2+ insensitive mechanism. Such a mechanism could have been ceramide. However, the cantharidin-induced eryptosis does apparently not involve ceramide formation or translocation.Removal of extracellular CaCantharidin further did not trigger oxidative stress, a known stimulator of eryptosis . In nucl+,K+,2Cl− cotransport on the other, carriers under the control of several kinases [+ in turn fosters cell membrane scrambling [Instead, the effect of cantharidin on both, cell membrane scrambling and cell volume was apparently related to its inhibitory effect on phosphoprotein phosphatases ,5. The e kinases ,48,49. Trambling . The senThe stimulation of eryptosis by cantharidin may add to the toxicity of the substance. Phosphatidylserine exposing erythrocytes are engulfed by macrophages and thus rapidly cleared from circulating blood and stimulation of eryptosis may thus lead to anemia . Moreovein vitro for 48 h at a hematocrit of 0.4% in Ringer solution containing (in mM) 125 NaCl, 5 KCl, 1 MgSO4, 32 N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), 5 glucose, and 1 CaCl2; the pH was adjusted to 7.4 and the temperature kept at 37 °C. Where indicated, erythrocytes were exposed to cantharidin .Fresh Lithium-Heparin-anticoagulated blood samples were kindly provided by the blood bank of the University of Tübingen. The study was approved by the ethics committee of the University of Tübingen (184/2003 V). The blood was centrifuged at 120 g for 20 min at 23 °C and the platelets and leukocytes-containing supernatant was disposed. Erythrocytes were incubated 2 and then stained with Annexin-V-FITC  in this solution at 37 °C for 20 min under protection from light. In the following, the geometric mean of the forward scatter (FSC) was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur . In some experiments erythrocytes were preincubated in Ca2+ free solution. For determination of annexin-V-binding, addition of Ca2+ was required during the 15 min incubation with FITC-annexin-V. Immediately thereafter measurements were done so that the exposure to Ca2+ was too short to trigger significant phosphatidylserine translocation.After incubation under the respective experimental condition, a 150 μL cell suspension was washed in Ringer solution containing 5 mM CaClFor the determination of hemolysis, the samples were centrifuged  after incubation under the respective experimental conditions and the supernatants were harvested. As a measure of hemolysis, the hemoglobin (Hb) concentration of the supernatant was determined photometrically at 405 nm. The absorption of the supernatant of erythrocytes lysed in distilled water was defined as 100% hemolysis.2 and 5 μM Fluo-3/AM. The cells were incubated at 37 °C for 30 min and washed twice in Ringer solution containing 5 mM CaCl2. The Fluo-3/AM-loaded erythrocytes were resuspended in 200 μL Ringer. Then, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur.After incubation, a 150 μL cell suspension was washed in Ringer solution and then loaded with Fluo-3/AM  in Ringer solution containing 5 mM CaClOxidative stress was determined utilizing 2′,7′-dichlorodihydrofluorescein diacetate (DCFDA). After incubation, a 150 μL suspension of erythrocytes was washed in Ringer solution and then stained with DCFDA  in Ringer solution containing DCFDA at a final concentration of 10 μM. Erythrocytes were incubated at 37 °C for 30 min in the dark and then washed three times in Ringer solution. The DCFDA-loaded erythrocytes were resuspended in 200 μL Ringer solution, and ROS-dependent fluorescence intensity was measured at an excitation wavelength of 488 nm and an emission wavelength of 530 nm on a FACS Calibur (BD).For the determination of ceramide, a monoclonal antibody-based assay was used. After incubation, cells were stained for 1 h at 37 °C with 1 μg/mL anti ceramide antibody  in PBS containing 0.1% bovine serum albumin (BSA) at a dilution of 1:10. The samples were washed twice with PBS-BSA. Subsequently, the cells were stained for 30 min with polyclonal fluorescein isothiocyanate (FITC) conjugated goat anti-mouse IgG and IgM specific antibody  diluted 1:50 in PBS-BSA. Unbound secondary antibody was removed by repeated washing with PBS-BSA. The samples were then analyzed by flow cytometric analysis with an excitation wavelength of 488 nm and an emission wavelength of 530 nm.t test as appropriate. n denotes the number of different erythrocyte specimens studied. Since different erythrocyte specimens used in distinct experiments are differently susceptible to triggers of eryptosis, the same erythrocyte specimens have been used for control and experimental conditions.Data are expressed as arithmetic means ± SEM. As indicated in the figure legends, statistical analysis was made using ANOVA with Tukey’s test as post-test and Cantharidin stimulates erythrocyte cell membrane scrambling and cell shrinkage, both hallmarks of eryptosis, the suicidal erythrocyte death. The effect of cantharidin on cell membrane scrambling and cell shrinkage is abrogated by kinase inhibitor staurosporine and may thus be due to the known inhibitory effect of cantharidin on protein phosphatases.