In many cells, the activation of phospholipase C-coupled receptors hydrolyzes membrane phosphoinositides and produces the depletion of endoplasmic reticulum Ca2+ stores, followed by the sustained elevation of [Ca2+]i from Ca2+ entry across the plasma membrane via store-operated Ca2+ entry (SOCE). Ca2+ entry is also increased in a store-independent manner by arachidonate-regulated Ca2+ (ARC) channels. Using rat parotid salivary gland cells, we examined multiple pathways of Ca2+ entry/elevation to determine if they activated cell signaling proteins and whether this occurred in a pathway-dependent manner. We observed that SOCE activates extracellular signal-related kinases 1 and 2 (ERK1/2) to ∼3-times basal levels via a receptor-independent mechanism when SOCE was initiated by depleting Ca2+ stores using the endoplasmic reticulum Ca2+-ATPase inhibitor thapsigargin (TG). TG-initiated ERK1/2 phosphorylation increased as rapidly as that initiated by the muscarinic receptor agonist carbachol, which promoted an increase to ∼5-times basal levels. Notably, ERK1/2 phosphorylation was not increased by the global elevation of [Ca2+]i by Ca2+ ionophore or by Ca2+ entry via ARC channels in native cells, although ERK1/2 phosphorylation was increased by Ca2+ ionophore in Par-C10 and HSY salivary cell lines. Agents and conditions that blocked SOCE in native cells, including 2-aminoethyldiphenyl borate (2-APB), SKF96363, and removal of extracellular Ca2+, also reduced TG- and carbachol-stimulated ERK1/2 phosphorylation. TG-promoted ERK1/2 phosphorylation was blocked when SRC and Protein Kinases C (PKC) were inhibited, and it was blocked in cells pretreated with β-adrenergic agonist isoproterenol. These observations demonstrate that ERK1/2 is activated by a selective mechanism of Ca2+ entry (SOCE) in these cells, and suggest that ERK1/2 may contribute to events downstream of SOCE.The regulation of intracellular Ca In various non-excitable cells, the increases in [Ca2+]i involve extracellular Ca2+ entry into the cell via SOCE, which is initiated by the release of Ca2+ stores from the endoplasmic reticulum via inositol 1,4,5-trisphosphate receptors (IP3R)/Ca2+ channels when the activation of G-protein-coupled receptors produces IP3 and diacylglycerol from phosphatidylinositol-4,5-bisphosphate (PIP2) hydrolysis by phospholipase C 2+ signals may be initiated by ARC channels, which produce oscillatory increases in [Ca2+]i rather than the sustained increases in [Ca2+]i that are produced by SOCE. ARC channels rely on the generation of arachidonic acid and are store-independent, since their activation does not depend on the loss of Ca2+ from the endoplasmic reticulum 2+-sensitive ion movements that occur in response to Ca2+ entry via the SOCE pathway in salivary gland cells, the entry of extracellular Ca2+ via the P2X7 receptor/ion channel also activates Ca2+-sensitive ion channels and initiates fluid secretion and saliva formation 2+-ATPase on the endoplasmic reticulum membrane, which depletes endoplasmic reticulum Ca2+ stores and thereby promotes Ca2+ entry.Receptor-mediated increases in [Ca2+-sensitive K+ and Cl− channels which are opened downstream of receptor-mediated [Ca2+]i elevation via SOCE, and these channels also are opened when [Ca2+]i is increased more directly via Ca2+ ionophores 2+]i can also initiate signaling cascades, in some cases by transactivating receptors or by increasing the phosphorylation of signaling proteins. The signaling and physiological events downstream of an increase in [Ca2+]i may be activated uniquely by a specific mechanism of Ca2+ entry and elevation. For example, Ca2+-sensitive Adenylyl Cyclase 8 (AC8) is stimulated by the entry of extracellular Ca2+ into cells via SOCE but not by ARC channels, Ca2+ release from intracellular stores, or global increases in [Ca2+]i2+-dependent changes in the phosphorylation of cell signaling proteins can be promoted in similar fashion by both receptor ligands and Ca2+ ionophores The stimulation of fluid secretion in salivary gland cells involves Ca2+ from the endoplasmic reticulum has long been known to promote Ca2+ influx across the plasma membrane via SOCE , which serve as Ca2+ sensors that links the plasma membrane Ca2+ channels to the endoplasmic reticulum Ca2+ stores CRAC current (Ca-release-activated current) and STIM1, Orai1, and TRPC proteins form different Ca2+ influx pathways that involve different complexes of these proteins 2+ channel/STIM1 complex have been found, including Golli-BG21, IP3 receptors, and other proteins The depletion of Ca2+ homeostasis, the activation of cell signaling proteins, and the stimulation of epithelial ion transport mechanisms and fluid secretion 2+ elevation might initiate signaling effects downstream of specific Ca2+ entry pathways into native parotid cells. In particular, having observed in early studies that SOCE produced an increase in ERK1/2 phosphorylation, we sought to examine whether similar effects were produced by the elevation of [Ca2+]i by other mechanisms of Ca2+ entry/elevation, including ARC channels and Ca2+ ionophore. We also examined the dependence of ERK1/2 activation on various signaling proteins. To gain a more complete understanding of the contributions SOCE to ERK1/2 activation, we compared the effects of receptor-independent activation of SOCE to that of muscarinic receptor activation in a variety of conditions.Rat parotid acinar cells have been used as a model system to study receptor-initiated CaThe investigation conforms to the “Guide for the Care and Use of Laboratory Animals” published by the National Institutes of Health . Our protocol was submitted to and approved by the Institutional Animal Care and Use Committee .2 (PGE2)  were dissolved in cell culture grade dimethyl sulfoxide (D2650) from Sigma-Aldrich. 2-APB (100065) and phorbol 12-myristate 13-acetate  were obtained from EMD Millipore. Fura-2 AM was purchased from Invitrogen/Molecular Probes. Polyclonal ERK2 (SC-154) was purchased from Santa Cruz Biotechnology, Inc. Phospho-Thr202/Tyr204-ERK1/2 (9101) and Phospho-(Ser) PKC substrate (2261) antibodies were purchased from Cell Signaling Technology. Anti-rabbit IgG (AP307P) secondary antibody used for Western blotting was obtained from Chemicon. All other chemicals were reagent grade or better.Carbamyl choline (carbachol) (C4382), isoproterenol (I5627), and 2′(3′)-O-(4-Benzoylbenzoyl) adenosine 5′-triphosphate triethylammonium salt  were purchased from Sigma-Aldrich. Thapsigargin (1138), ionomycin (1704), PP2 (1407), GF109203X (0741), Go6976 (2293), and SKF96363 (1147) were purchased from Tocris Bioscience. Arachidonic acid  and prostaglandin E2PO4, 25 mM Na HEPES, 1.8 mM CaCl2, 0.8 mM MgCl2, 5 mM Na butyrate, 5.6 mM glucose, pH 7.4. In experiments using BzATP, this solution was modified to contain 1 mM CaCl2 without MgCl2. Aliquots (1.5 ml) of cells were equilibrated for ∼5–10 min prior to treatments with various agents or vehicles.Parotid acinar cells were prepared from male Sprague-Dawley rats  as described previously 2 at 37°C.Par-C10 cells were grown to near confluence in DMEM-F12 (1∶1) medium containing 2.5% fetal bovine serum and supplements as previously described 2O) for similar periods of time. At the end of the treatment period, the suspended native parotid cells were collected by rapid sedimentation. Cells were lysed in ice-cold lysis buffer  glycerol, 1% v/v Igepal) containing protease and phosphatase inhibitors oC. For experiments conducted using cell lines, the cells were treated as indicated in a 37°C incubator for the appropriate times. The cells were removed from the incubator, washed 3 times in phosphate-buffered saline solution, lysed, and sedimented at 15,000×g. The cleared supernatants were diluted with 5× Laemmli sample buffer, boiled for 5 min, and stored at −20°C prior to electrophoresis. Samples were separated using SDS-polyacrylamide gel electrophoresis with an 8% separating gel and a 3% stacking gel. Proteins were transferred to nitrocellulose. Immunoblots were probed overnight with various antibodies according to the supplier’s specifications. Proteins were visualized on X-ray film using enhanced chemiluminescence reagents, and proteins were quantified by densitometry using the Image J software program from the National Institutes of Health as described previously Native rat salivary gland cells were treated at 37°C with various agents as indicated in the figure legends, and control cells were exposed to vehicle .Alterations in i can initiate or modulate cell signaling events in a variety of cells, we compared the effects of [Ca2+]i elevation by SOCE and other mechanisms on ERK1/2 signaling in rat parotid acinar cells. TG, which inhibits Ca2+ uptake into the endoplasmic reticulum Ca2+ stores and thereby initiates SOCE, produced an increase in ERK1/2 activation (phosphorylation) within 2 min , which increases Ca2+ entry into salivary gland and other cells via ARC channels 2, a product of arachidonic acid metabolism, also did not produce significant changes in ERK1/2 phosphorylation , we reexamined the contribution of PKC to ERK1/2 activation .Rat parotid acinar cells exposed to arachidonic acid exhibit a slow increase in [Cavia SOCE . If adde2+]i in rat parotid acinar cells. The main physiological function of these salivary gland cells is to initiate fluid secretion and saliva formation in response to neurotransmitters that bind to and activate phospholipase C-linked G-protein-coupled receptors. This results in the generation of the second messenger IP3 and the promotion of Ca2+ release from intracellular stores and the subsequent Ca2+ entry across the plasma membrane via SOCE. The elevation of [Ca2+]i opens Ca2+-sensitive K+ and Cl− channels that contribute to net fluid secretion into the acinar lumen 2+-sensitive ion channels and subsequent ionic changes and secondary ionic movements via other ion transport proteins can be initiated by [Ca2+]i elevation via multiple pathways, including increases initiated by G-protein-coupled receptors, P2X7Rs/ion channels, and Ca2+ ionophores These studies were conducted to examine the selectivity of cell signaling downstream of various mechanisms that increase [Ca2+]i by SOCE in the absence of G-protein-coupled receptor activation. In the absence of extracellular Ca2+, the increase in [Ca2+]i is modest and also is transient. In contrast, a large increase in ERK1/2 phosphorylation upon the addition of extracellular Ca2+ to store-depleted cells accompanies the large increase in [Ca2+]i via SOCE under these conditions.The data demonstrate that ERK1/2 phosphorylation is increased by the TG-initiated elevation of i via SOCE.2-APB and SKF96365 inhibit SOCE in salivary gland cells in vitro, and a triple mutation of serine to alanine mutation in all three ERK1/2 target sites on STIM1  resulted in a reduction in SOCE 2+ via SOCE 2+ into cells in which Ca2+ stores were first depleted by TG , and these oscillations were probably SOCE-independent 2+ entry via receptor-independent SOCE, but not by Ca2+ entry via ARC channels and Ca2+ ionophore. TG and carbachol shared multiple characteristics as agents that increased ERK1/2 phosphorylation, including the kinetics and significant increases above basal levels, the dependences on similar signaling and regulatory proteins, and the reductions by agents and conditions that block SOCE. The data suggest that part of the receptor-initiated stimulation of ERK1/2 is dependent on SOCE; in addition, we suggest that the receptor-independent stimulation of ERK1/2 may rely on phospholipase C activation. Calcium ionophore and the activation of ARC channels were insufficient to activate ERK1/2; thus our findings indicate that the activation of cell signaling proteins by Ca2+ entry into native rat cells is much more discriminatory than the activation of ion transport proteins by [Ca2+]i. These studies demonstrate the selective activation of cellular responses downstream of a specific mechanisms of Ca2+ entry into these cells, and how SOCE may contribute to cellular processes beyond to its role in elevating [Ca2+]i.In summary, we observed that ERK1/2 was activated in rat parotid acinar cells by Ca


The elevated PVR causes an increase in afterload in the right ventricle, leading to right ventricular hypertrophy, right heart failure, and eventually death. Understanding the pathogenic mechanisms of PAH is important for developing more effective therapeutic approach for the disease. An increase in cytosolic free Ca2+ concentration ([Ca2+]cyt) in pulmonary arterial smooth muscle cells (PASMC) is a major trigger for pulmonary vasoconstriction and an important stimulus for PASMC migration and proliferation which lead to pulmonary vascular wall thickening and remodeling. It is thus pertinent to define the pathogenic role of Ca2+ signaling in pulmonary vasoconstriction and PASMC proliferation to develop new therapies for PAH. [Ca2+]cyt in PASMC is increased by Ca2+ influx through Ca2+ channels in the plasma membrane and by Ca2+ release or mobilization from the intracellular stores, such as sarcoplasmic reticulum (SR) or endoplasmic reticulum (ER). There are two Ca2+ entry pathways, voltage-dependent Ca2+ influx through voltage-dependent Ca2+ channels (VDCC) and voltage-independent Ca2+ influx through store-operated Ca2+ channels (SOC) and receptor-operated Ca2+ channels (ROC). This paper will focus on the potential role of VDCC, SOC, and ROC in the development and progression of sustained pulmonary vasoconstriction and excessive pulmonary vascular remodeling in PAH.Pulmonary circulation is an important circulatory system in which the body brings in oxygen. Pulmonary arterial hypertension (PAH) is a progressive and fatal disease that predominantly affects women. Sustained pulmonary vasoconstriction, excessive pulmonary vascular remodeling,  The only organ in the body to receive the entire cardiac output (CO) at one time is the lung. To receive a high flow of volume from the entire CO, the pulmonary circulatory system must maintain a low-resistance and low-pressure system to carry blood to the pulmonary capillaries. Deoxygenated venous blood flows through the pulmonary artery to the pulmonary capillaries where oxygen and carbon dioxide gas exchange occurs. Pulmonary hypertension (PH) is a severe chronic disorder that affects the pulmonary circulatory system. This disorder is often a deadly hemodynamic irregularity that may be idiopathic, heritable, or secondary to other diseases such as chronic obstructive pulmonary disease (COPD). Pulmonary arteries are thin and have low myogenic tone compared to systemic arteries. Therefore, pulmonary arteries rely on arterial distension and recruitment for decreasing pulmonary vascular resistance (PVR) after increased blood flow or increased CO . In the lungs, increased PVR is the major cause for the development of PH. An increased PVR results in increased RV afterload which leads to right heart failure and eventually death . arteries + PVRcapillaries + PVRveins) . , 5. 4, 5There are 15 orders of branching in the pulmonary arteries between the main pulmonary artery and the capillaries in the human lung . The pulin situ thrombosis, and arterial wall stiffening, together these attributes increase PVR leading to right heart failure cyt in PASMC, which leads to smooth muscle contraction by both Ca2+-dependent and Ca2+-independent mechanisms. Ca2+ directly activates myosin light chain kinase (MLCK) leading to contraction and migration. Additionally, an increase in [Ca2+]cyt stimulates Ca2+-dependent signal transduction proteins. In a healthy individual, the pulmonary circulatory system has low resistance and easily dilates to accommodate the entire CO with a marginal change in pressure. In contrast to the systemic circulatory system, which has higher resistance and pressure, hypoxia causes the pulmonary arteries to constrict while the systemic arteries to dilate [2+]cyt in PASMC, increased contractile elements in lung tissue, and enhanced sensitivity of contractile proteins. Several studies in the past have demonstrated that the amplitude of contraction can be affected by any change in the ratio of myosin light chain kinase (MLCK): MLCP activity. MLCK activity is dependent on Ca2+ calmodulin; hence, the rise in [Ca2+]cyt is the primary determinant of smooth muscle contraction. The increase in sensitivity of smooth muscle contractility to [Ca2+]cyt  may be caused by inhibition of MLCP activity. It has been found that the predominant pathological cause in idiopathic pulmonary arterial hypertension (IPAH) in patients is a loss of vascular compliance and an increase in PVR due to pulmonary vascular remodeling and vasoconstriction cyt in PASMC. Vascular smooth muscle contraction decreases the radius of pulmonary blood vessels, leading to sustained vasoconstriction and to increased PVR. The mechanism of contraction in pulmonary vascular smooth muscle is different than that of striated cardiac or skeletal muscles. Skeletal muscles undergo fast contractions, which involve Ca2+ binding to troponin. However, in PASMC slow, tonic contractions are initiated through changes in [Ca2+]cyt that cause a Ca2+-activated phosphorylation of myosin. When [Ca2+]cyt rises, Ca2+ binds to calmodulin (CaM) and activates myosin light chain kinase (MLCK) which then phosphorylates the myosin light chain. Phosphorylation of MLC increases myosin ATPase activity that hydrolyzes ATP to release energy. The subsequent cycling of the myosin cross-bridges produces a sliding motion of myosin-actin filaments and results in contraction of the smooth muscles (2+ blocks the high K+-induced and phenylephrine- (PE-) induced contraction in isolated rat pulmonary arterial rings, illustrating that Ca2+ influx is necessary for smooth muscle contraction  and transcription factors  can stimulate cell proliferation (2+]cyt is an important stimulus for cellular proliferation (2+ in the sarcoplasmic reticulum/endoplasmic reticulum (SR/ER) is vital for cell growth cyt is essential for cell proliferation.In addition to causing PASMC contraction, increased .–30.2+]cyferation  31].In .In 2+]cyl growth . It has d growth . Ca2+ incle (G1) . Thus, t2+-dependent pathways play an important role in the regulation of numerous physiological and pathophysiological processes in PASMC such as contraction, proliferation, and migration cyt, and contractile Ca2+ sensitivity, altogether contributing to vasoconstriction, and thus leading to PH. Ca2+ influx in PASMC is mainly regulated by voltage-dependent Ca2+ channels and voltage-independent nonselective cation channels. Intracellular Caigration . Studies2+ channels (VDCC)  have established roles in the regulation of blood pressure; and dihydropyridine Ca2+ channel blockers have been clinically used for the treatment of hypertension. The Ca2+ influx mechanisms in PASMC via VDCC are influenced by changes in the membrane potential, an electrochemical driving force for Ca2+ entry. PASMCs maintain a resting membrane potential (EM) at about −40 to −60 mV, which is significantly less negative than the equilibrium potential for K+ (EK is about −85 mV), suggesting that resting EM is regulated not only by K+ currents, but also background cation  currents through voltage-independent cation channels and/or nonselective cation channels . At rest, the membrane is more permeable to K+, so background K+ currents through voltage-dependent and voltage-independent K+ channels are the major determinants for the resting EM, although Na+, Cl−, and Ca2+ currents also contribute to the regulation of the resting EM. The activity of various K+ channels in the plasma membrane contributes to maintaining the EM of PASMC. Inhibition of K+ channels causes membrane depolarization, whereas activation of K+ channels causes membrane repolarization and hyperpolarization. Among more than 50 different types of K+ channels, voltage-gated K+ (KV) channels are shown to be ubiquitously expressed in smooth muscle cells cyt , or capacitative Ca2+ entry (CCE), is identified as one of the important mechanisms that regulate [Ca2+]cyt, as addressed by Casteels and Droogmans in early 80s. The groups of channels that mediate SOCE are termed as store-operated Ca2+ channels (SOC) cyt and thus produces vasoconstriction, promotes cell cycle progression, and ultimately leads to PASMC proliferation and vascular remodeling cyt in a membrane-delimited fashion. TRPC6 is insensitive to PLC-IP3 activation at the cellular level cyt may presumably induce BKCa (like TRPV4) and regulate membrane potential depolarization. Thus, TRPM4-mediated depolarization may support Ca2+ influx. These findings were further substantiated by diminishing the expression of TRPM4 in the cerebral vasculature, which resulted in a loss of autoregulation of cerebral blood flow in response to changes in perfusion pressure cyt. Variations in the expression and function of the ion channels necessary for Ca2+ influx are key features in the development and pathogenesis of PAH. In PAH, a rise in [Ca2+]cyt in PASMC due to enhanced SOCE/ROCE (due to upregulated SOC/SOC components) and voltage-dependent Ca2+ entry in PASMC from IPAH patients also activates many signal transduction proteins  and transcription factors  thus influencing gene expression and promoting cell proliferation. Experiments have therefore shown that Ca2+ channels and significant signaling pathways are possible sites for therapeutic targets. In order to understand the pathogenic mechanisms of PAH, it is important to consider CaFuture experiments detailing the specific downstream effects of enhanced SOCE and ROCE will shed light on important therapeutic strategies for PAH. More specifically, understanding the role of other proteins such as transcription factors, signal transduction proteins, and nuclear transporters and their effect on vasoconstriction and proliferation could provide future pharmacological blockades of SOC/ROC. Additionally, downregulation of these proteins could reveal therapeutic effects for PH. Application of new experimental methods along with further use of well-established methods is crucial to the advancement of the field of pulmonary hypertension.


Oral administration of 12 µmol PEITC resulted in drastically suppressing ovarian tumor growth in a preclinical mouse model. Our in vitro studies demonstrated that PEITC suppress the growth of SKOV-3, OVCAR-3 and TOV-21G human ovarian cancer cells by inducing apoptosis in a concentration-dependent manner. Growth inhibitory effects of PEITC were mediated by inhibition of EGFR and AKT, which are known to be overexpressed in ovarian tumors. PEITC treatment caused significant down regulation of constitutive protein levels as well as phosphorylation of EGFR at Tyr1068 in various ovarian cancer cells. In addition, PEITC treatment drastically reduced the phosphorylation of AKT which is downstream to EGFR and disrupted mTOR signaling. PEITC treatment also inhibited the kinase activity of AKT as observed by the down regulation of p-GSK in OVCAR-3 and TOV-21G cells. AKT overexpression or TGF treatment blocked PEITC induced apoptosis in ovarian cancer cells. These results suggest that PEITC targets EGFR/AKT pathway in our model. In conclusion, our study suggests that PEITC could be used alone or in combination with other therapeutic agents to treat ovarian cancer.Ovarian cancer is the leading cause of cancer related deaths in women. Genetic alterations including overexpression of EGFR play a crucial role in ovarian carcinogenesis. Here we evaluated the effect of phenethyl isothiocyanate (PEITC) in ovarian tumor cells  Ovarian cancer is one of the leading causes of gynecologic cancer-related deaths among women in western countries Activation of various tyrosine kinases including EGFR is important in ovarian cancer pathogenesis. Approximately 70% of ovarian tumors express activated EGFR One of the major downstream pathways that are regulated by EGFR is AKT. Activation of EGFR leads to the activation of AKT by its phosphorylation at Ser-473 in vivo in a tumor xenograft model.PEITC is a major isothiocyanate present in cruciferous vegetables Antibodies against Cl-caspase3, Cl-PARP, p-EGFR (Tyr-1068), EGFR, p-AKT (Ser-473), p- mTOR (Ser 2481), Raptor and AKT antibodies were obtained from Cell Signaling Technology . Rictor antibody was obtained from Novusbio . PEITC, actin antibody, TGF, SRB, MCDB105 and Medium 199 were procured from Sigma Aldrich . RPMI and McCoy 5A were purchased from Mediatech . AnnexinV apoptosis kit was procured from BD biosciences .6 SKOV-3 cells were injected subcutaneously into both right and left flanks. Ten mice were assigned randomly to each group. Since each mouse was implanted two xenografts, each group had twenty tumors. Mice in the control group received PBS, whereas, mice in the treatment group received 12 µmol PEITC suspended in PBS by oral gavage every day. Tumor growth was monitored until day 42 as described by us previously Four to six week old female athymic nude mice were purchased from Charles River Laboratories . Institutional Animal Care and Use Committee (IACUC), Texas Tech University Health Sciences Center approved the use of mice and their treatment, and all the experiments were carried out in strict compliance with regulations. Mice were fed with antioxidant-free AIN-76A special diet for a week before starting the experiment. About 5×102/95% air.SKOV-3, OVCAR-3, TOV-21G cells lines were procured from American Type Culture Collection . SKOV-3 cells were maintained in McCoy’s 5A medium supplemented with 10% Fetal Bovine Serum (FBS). OVCAR-3 cells were maintained in RPMI medium supplemented with 20% FBS, 10 mM sodium pyruvate, 10 mM HEPES, 10 mg/L bovine insulin and 4.5 g/L glucose. TOV21G cells were maintained in 1∶1 mixture of MCDB105 and Medium 199 supplemented with 15% FBS. A 1% antibiotic mixture was used in all the above media. All the cell lines were maintained at 37°C in a humidified incubator circulated with 5% COAbout 5,000 cells in 0.1 ml medium were plated in 96 well plates and allowed to attach overnight. Desired concentrations of PEITC were added to the cells and incubated at 37°C for 24 h. The cells were then processed and stained with 0.4% SRB solution and the absorbance was read at 570 nm using a Biotek plate reader as described by us previously SKOV-3, OVCAR-3 and TOV-21G cells were exposed to varying concentrations of PEITC alone or in presence of TGF. In another experiment, cells were treated with 15 µM PEITC for different time intervals. For concentration dependent study SKOV3 cells were treated with 7.5, 15 and 30 µM PEITC for 24 hours. Cells were collected, lysed, and 20–80 µg of protein was subjected to SDS gel electrophoresis followed by immunoblotting as described by us previously 6 cells per well in a six-well plate and allowed to attach overnight. Cells were then treated with or without PEITC. After 24 hours, cells were washed, suspended in binding buffer and incubated for 15 minutes with Annexin V-FITC. Fluorescence was measured using C6 Accuri flow cytometer  with a minimum of 10,000 events per sample as described by us previously SKOV-3, OVCAR-3 or TOV-21G cells were plated at a density of 0.3×10Immunoprecipitation assay was performed to examine the effect of PEITC on the complex of mTOR with Rictor and Raptor. Briefly, SKOV3 cells were treated with 15 µM PEITC for 24 hours. The cell lysates from control and PEITC treated cells were immunoprecipitated with the mTOR antibody , as described by us earlier SKOV-3 or OVCAR-3 cells were treated with 50 ng/ml TGF for 1 hour followed by incubation with different concentrations of PEITC for 24 hours. Cells were then processed for western blotting as described above.5 cells were transfected with 0.5 µg of the AKT plasmid diluted in Opti-MEM serum-free medium to which Fugene reagent was added before the mixture was added to cells. Cells were incubated with plasmid-Fugene mixture for 6 hours and then media was replaced with fresh growth medium and incubated for another 24 hours. Transfected cells were treated with 10 µM PEITC for 24 hours. Whole cell lysates were subjected to Western blot analysis and actin was used as loading control.SKOV-3 cells were transiently transfected with plasmid containing wil-type AKT  by using Fugene (Roche). Briefly, 0.3×10t-test was used to compare the control and treated groups. In experiments involving more than three groups, non-parametric analysis of variance followed by Bonferroni post hoc multiple comparison test was used. All statistical tests were two sided. Differences were considered statistically significant when the p value was less than 0.05.All the statistical analysis was performed using Prism 5.0 . The data represents mean ± S.D. Student’s 6 cells in both right and left flanks. Mice were randomly divided into control and treatment group. Tumor bearing mice were fed 12 µmol PEITC every day and tumor growth was recorded periodically. Our results demonstrate that PEITC substantially suppressed the growth of ovarian tumors in athymic nude mice. For example, at day 42, the average tumor volume in mice from treatment group was approximately 60 mm3, whereas average tumor volume in mice from control group was approximately 220 mm3  of EGFR and AKT . HoweverTo further strengthen our observation that PEITC-induced apoptosis was mediated by AKT inhibition in ovarian cancer cells, we transiently transfected SKOV-3 cells with AKT encoding plasmid for 24 hours, resulting in almost 7 fold increase in the expression of AKT. Correspondingly, phosphorylation of AKT at Ser-473 also increased substantially. AKT overexpression completely prevented PEITC induced suppression of AKT phosphorylation. Furthermore, AKT overexpression abrogated PEITC induced apoptosis as evaluated by cleavage of PARP . Taken tRecent literature suggests that EGFR-AKT activation regulates mTOR activity which is important for cell proliferation in vivo by suppressing EGFR-AKT pathway and identifies EGFR-Akt axis as a target of PEITC.In the present study, we showed the mechanism by which PEITC suppressed the growth of ovarian cancer cells in culture as well as in animal model. Our results show that oral administration of 12 µmol PEITC to athymic nude mice significantly suppressed the growth of SKOV-3 ovarian tumor xenografts. Tumor growth suppression by PEITC treatment was associated with increased apoptosis in the tumor cells, which in turn was linked with the inhibition of EGFR and AKT activation. Inhibition of phosphorylation of EGFR and AKT were confirmed in SKOV-3 and OVCAR-3 ovarian cancer cells by western blotting. PEITC also blocked TGF-mediated activation of both EGFR and AKT. Overexpression of AKT by transient transfection substantially blocked the apoptosis inducing effects of PEITC. Our results showed that PEITC inhibits ovarian tumor growth Constitutive activation of EGFR has been reported in various cancers including breast, prostate and ovarian AKT is pivotal to EGFR activation. Activated EGFR further activates AKT by phosphorylating it at Ser 473 It is important to note that oral administration of PEITC has reasonable bioavailability. Oral administration of 10 µmol PEITC in rats resulted in approximately 9.2±0.6 µM PEITC in the plasma after 0.44 hour of treatment in vitro and in vivo by inhibiting EGFR signaling. Our results also provide evidence that PEITC suppress the phosphorylation of AKT, which is regulated by EGFR. Taken together, our study provides support for the use of PEITC in pre-clinical and clinical settings for the management of ovarian cancer.In conclusion, our results established that PEITC suppresses the growth of ovarian cancer


We also know that the mitochondrial Ca2+ transporters and the release of catecholamine are faster at 37°C with respect to room temperature. Therefore, we planned this investigation to gain further insight into the contribution of mitochondrial Ca2+ buffering to the shaping of [Ca2+]c and catecholamine release transients, using some novel experimental conditions that have not been yet explored namely: (1) perifusion of bovine chromaffin cells (BCCs) with saline at 37°C and their repeated challenging with the physiological neurotransmitter acetylcholine (ACh); (2) separate blockade of mitochondrial Ca2+ uniporter (mCUP) with Ru360 or the mitochondrial Na+/Ca2+ exchanger (mNCX) with CGP37157; (3) full blockade of the mitochondrial Ca2+ cycling (mCC) by the simultaneous inhibition of the mCUP and the mNCX. Ru360 caused a pronounced delay of [Ca2+]c clearance and augmented secretion. In contrast, CGP37157 only caused a tiny delay of [Ca2+]c clearance and a mild decrease in secretion. The mCC resulting in continued Ca2+ uptake and its release back into the cytosol was interrupted by combined Ru360 + CGP37157 (Ru/CGP), the protonophore carbonyl cyanide‐p‐trifluoromethoxyphenylhydrazone, or combined oligomycin + rotenone (O/R); these three treatments caused a mild but sustained elevation of basal [Ca2+]c that, however, was not accompanied by a parallel increase in basal secretion. Nevertheless, all treatments caused a pronounced augmentation of ACh‐induced secretion, with minor changes of the ACh‐induced [Ca2+]c transients. Combined Ru/CGP did not alter the resting membrane potential in current‐clamped cells. Additionally, Ru/CGP did not increase basal [Ca2+]c near subplasmalemmal sites and caused a mild decrease in the size of the readily releasable vesicle pool. Our results provide new functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh‐elicited exocytotic response through its efficient Ca2+ removal by the mCUP, M2 shapes the lower [Ca2+]c elevations required for new vesicle supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh responses, in comparison with the mNCX.From experiments performed at room temperature, we know that the buffering of Ca Regulation by mitochondria of exocytosis at 37°C. To measure ACh‐stimulated [Ca2+]c elevations in BCCs loaded with fura‐2 or FFP18, cells were plated at a density of 5 × 105 cells/coverslip in 25‐mm 6‐well plates. Cells were kept for 2–4 days in a water‐saturated incubator at 37°C, in a 5% CO2‐95% air atmosphere, and used 1–4 days thereafter. After 24 h, the medium was replaced by serum‐free fresh medium and subsequently changed every 2 days.BCCs were isolated from adrenal glands of calves . Cells were introduced in a 100 μL‐microchamber for their superfusion at the constant rate of 2 mL/min. The liquid flowing from the superfusion chamber reached an electrochemical detector model 641 VA , placed just at the outlet of the microchamber, which monitors online the amount of catecholamines secreted under the amperometric mode. Cells were stimulated to secrete with short pulses (5 sec) of a Krebs–HEPES solution containing 30 μmol/L ACh (concentration corresponding to EC50 of ACh to produce a secretory response in BCCs). Solutions were rapidly exchanged through electrovalves commanded by a computer. This amperometric strategy permits the online recording of reproducible catecholamine release responses during long time periods of 30–60 min. In this study, all experiments were performed at the constant temperature of 37 ± 1°C. The maximal amperometric signal stimulated with ACh corresponding to secretion peak (SP) was evaluated. To estimate the quantity of catecholamine secreted per pulse, the signal expressed in nano ampere (nA) corresponding to SP was compared to peak of amperometric signal obtained with different concentrations of adrenaline (1–20 μmol/L). An analysis of amperometric charge of SP was calculated by integrating the amperometric current over time during the stimulus duration by means of Origin Pro 8 SR2 software, version 8.0891 .An online amperometric method was used to measure at real time the rate of ACh‐stimulated catecholamine release from BCCs populations  or the near‐membrane Ca2+ indicator FFP18  equipped with an oil immersion objective . Once the cells were placed under the microscope, they were continuously superfused by means of a five‐way superfusion system at 1 mL/min with a common outlet 0.28 mm‐tube driven by electrically controlled valves with Tyrode's solution, at 37°C. Fura‐2 was excited alternatively at 340 ± 10 nm and 387 ± 10 nm using a Küber CODIX xenon 8 lamp . Emitted fluorescence was collected through a 540 ± 20 nm emission filter and measured with an intensified charge coupled device camera . Fluorescence images were generated at 1‐sec intervals. Images were digitally stored and analyzed using LAS AF software . A Tyrode external solution was used of the same composition as the one described above, where drugs were added to this solution. The rates of exponential decay of ACh‐induced [Ca2+]c responses were compared between BCCs treated and untreated with agents, which interfere with Ca2+ transport by mitochondria.At the moment of Ca2, 2 CaCl2, 5 KCl, 10 HEPES, and 10 glucose, pH 7.35 with NaOH.Resting membrane potential (Em), capacitance increments (ΔCm), and calcium currents were recorded using standard patch‐clamp techniques with an EPC‐10 amplifier and PULSE software  on the stage of a Nikon Diaphot microscope equipped with Narishige MWH‐3 micromanipulators , as previously described : 145 glutamic acid, 1 MgCl2, 8 NaCl, and 10 HEPES, pH 7.2 with CsOH. Leak currents in voltage‐clamp were automatically subtracted by the PULSE program by applying a Pn/P4 hyperpolarizing protocol.We used two intracellular solutions; solution 1 for current clamp recordings to measure membrane potential variations was composed of (in mmol/L): 135 KCl, 9 NaCl, 10 HEPES, 1 MgCl2+. Depolarizations were adjusted to match calcium current sizes of both pulses . The probes Fura‐2 AM and FFP18 were supplied by Invitrogen . FCCP, oligomycin, and rotenone were obtained from Sigma . CGP37157 was obtained from Tocris  and Ru‐360 from Calbiochem .The following chemicals were used for the bovine cell culture: DMEM, gentamicin and penicillin‐streptomycin were from GIBCO , l‐glutamine was from Sigma , fetal bovine serum was from PAA laboratories , and collagenase‐A from 2+ concentrations data analysis was carried out on a personal computer; data obtained from LAS AF Lite software, version 2.6.0  were exported to Excel tables . Graphs and the mathematical analyses were performed using the Graphpad Prism software, version 5.01 . Areas or peaks heights were calculated by integrating the calcium transient over time during the stimulus duration by means of Origin Pro 8 SR2 software, version 8.0891 . Results shown in the text and figures are expressed as mean ± SEM. Unless otherwise stated, statistical analyses were carried out with analysis of variance (ANOVA) one‐way test, and Tukey post hoc analyses; *, **, or *** show a statistical significance of P < 0.05, P < 0.01 or P < 0.001, respectively. Kinetic parameters of [Ca2+]c transients were determined using Origin Pro 8 SR2 software, version 8.0891 . The [Ca2+]c transients were fitted to the exponential decay equationx is the time, y starts at y0 and decays down or goes up to plateau, and t is the time or rate constant equal to the reciprocal of the x axis. The kinetic data of catecholamine release transients were calculated using a similar approach.Regarding cytosolic Ca2+ transporters are highly sensitive to temperature; for instance in BCCs the plasmalemmal Na+‐dependent Ca2+ efflux works fivefold faster at physiological compared with room temperature ; only at the final pulses (P9–P12) a mild decay in peak height was observed  the rate of secretion reached baseline in all pulses P1–P12) , are given in Reproducible bulk responses were further illustrated by monitoring their kinetic parameters, calculated as shown in the insets to μmol/L Ru360; this was double than the concentration of 1 μmol/L usually used to target the mCUP in permeabilized cells . τcl was augmented by 340%, indicating that Ru360 considerably delayed the clearance of the [Ca2+]c transient elicited by ACh.If subplasmalemmal mitochondria take up Caτcl; because the width did not change, the enhanced response seemed to be essentially due to an augmented rate of initial secretion.Secretion responses were drastically augmented in the presence of Ru360, as shown in the original record from an example cell batch Fig. C and in μmol/L in neuronal cultures . The first interesting observation was that cell exposure to Ru/CGP immediately enhanced the basal [Ca2+]c, as shown in the original trace of the example cell of 2+]c is the [Ca2+]c transient elicited by ACh P5, that if measured with respect to the new baseline was 40% smaller than P4. The second interesting finding was that basal [Ca2+]c remained elevated along the time period Ru/CGP was present (8 min); at ACh pulses P6, P7, and P8, the transients had smaller amplitudes than controls P1–P4. Upon Ru/CGP washout, baseline [Ca2+]c recovered its initial level; in spite of this, the transient remained around 40% lower than the initial responses. The inset in 2+]c in the presence of Ru/CGP. Thus, in resting conditions it seems that the mCC is effectively working to maintain low the basal [Ca2+]c. The simple blockade of one of the two Ca2+ transporters is insufficient to elevate basal [Ca2+]c; blockade of both transporters is therefore needed to suppress the mCC and to enhance basal [Ca2+]c. This is surprising given the reported low Ca2+ affinity of the mCUP in BCCs  in P5 with respect to P4  were used to abort mCC. 2+]c transients elicited by repeated ACh pulsing of an example fura‐2‐loaded BCC. As in the case of Ru/CGP and FCCP, O/R also caused an elevation of basal [Ca2+]c; however, such elevation was milder than that elicited by FCCP, as shown in the inset of 2+]c transient was slightly reduced in the presence of O/R, as shown in pulses P5 and P10 in Protonophores might disturb not only the Ca2+]c, combined O/R did not affect baseline secretion  with the low‐Ca2+ affinity probe FFP18 ; alteration of cell excitability by the compounds could contribute to the effects on [Ca2+]c and secretion transients triggered by ACh. Given separately or in combination, CGP37157 and Ru360 did not alter the resting Em (panels A–C). However, cell perifusion of the cells with a saline solution containing 75 mmol/L K+ caused the expected immediate depolarization to 0 mV of BCCs. Mean pooled data from 6 to 11 cells corroborated the lack of effects of Ru360 and CGP37157, either given alone or in combination, on the resting Em of BCCs.Current‐clamped BCCs had a resting Em of around −60 mV, and they seldom fired an isolated action potential; these data are consistent with those from previous reports  and 339 ± 43 fF (Bmax). Bmin decreased by 31% in the presence of Ru/CGP; however, Bmax was not significantly altered  with aequorin with different affinities for Ca2+, we suggested the existence of two different mitochondrial pools, M1 and M2, which take up Ca2+ at very different rates in response to activation of VACCs of BCCs. Pool M1 takes up Ca2+ at a rate of about 50 μmol/L/s, 30% of the mCUP Vmax, whereas uptake by pool M2 is about 150 times slower  takes about a minute after the 5‐sec ACh pulse to recover baseline levels. During this period, which overlays the ACh pulse, the long delay of the [Ca2+]c clearance could explain the higher total secretion.Repeated pulsing with ACh at 37°C produced reproducible catecholamine release responses for a long time period Fig. . This inransient . It is p2+]c and secretion transients was an expected outcome; inhibition of the mNCX by the compound would mitigate the rate of release of mitochondrial Ca2+ into the cytosol, leading to lower rate of vesicle transport and decreased secretion. Combined Ru/CGP, however, decreased the ACh [Ca2+]c transient but the potentiation of secretion remained. Again we can implicate the Ca2+‐dependent inactivation of VACCs to explain this apparent paradox.That CGP37157 diminished mildly the ACh‐elicited [Ca2+]c. However, if given together the basal [Ca2+]c underwent a mild elevation that was sustained along the 6‐min time period of cell perifusion with Ru/CGP  in order to release the Ca2+ stored in the mitochondrial matrix. In support of this were the experiments with FCCP and O/R that also blocks the mCC and hence, caused an elevated basal [Ca2+]c. The fact Ru/CGP did not elevate [Ca2+]nm suggests that the elevation of the bulk basal [Ca2+]c is unrelated to mitochondrial pool M1; rather, it suggests that the basal [Ca2+]c elevation is associated with the mCC of the M2 mitochondrial pool. It seems, therefore, that this low‐rate of Ca2+ cycling across the M2 pool is contributing to Ca2+ redistribution to the cell core, to activate vesicle transport toward exocytotic sites. Thus, the drastic potentiation of ACh secretory responses by Ru/CGP  and 170 vesicles (Bmax). The maximum number of vesicles of the RRVP, in cells exposed to Ru/CGP was similar, indicating that the pool was unchanged. It seems therefore that enhanced secretion elicited by the blockade of the mCC is due to the continued supply of new vesicles to a stable docked pool that is undergoing exocytosis during repeated ACh stimulation at 37°C, a kind of facilitation of exocytosis occurring during mild elevations of [Ca2+]c before stimulus application.One last point deserves attention. The double‐pulse experiments with measurement of ΔCm suggests that Ru/CGP did not augment the readily releasable pool Neher . These e2+]c signals and the exocytotic response in CCs ; this triad shapes the c elevations required for new vesicles supply to the exocytotic machinery, from the large reserve vesicle pool at the cell core. The mCUP of the M1 pool seems to play a more prominent role in controlling the ACh response, in comparison with the mNCX.In conclusion, our results provide the first functional features in support of the view that in BCCs there are two subpopulations of mitochondria, M1 underneath the plasmalemma nearby exocytotic sites and M2 at the core cell nearby vesicle transport sites. While M1 serves to shape the ACh‐elicited exocytotic response through its efficient CaNone declared.


The present study explored, whether thioridazine elicits eryptosis. Methods: [Ca2+]i has been estimated from Fluo3-fluorescence, cell volume from forward scatter, phosphatidylserine exposure from annexin-V-binding, and hemolysis from hemoglobin release. Results: A 48 hours exposure to thioridazine was followed by a significant increase of [Ca2+]i (30 µM), decrease of forward scatter (30 µM), and increase of annexin-V-binding (≥12 µM). Nominal absence of extracellular Ca2+ and p38 kinase inhibitor SB203580 (2 µM) significantly blunted but did not abolish annexin-V-binding following thioridazine exposure. Conclusions: Thioridazine stimulates eryptosis, an effect in part due to entry of extracellular Ca2+ and activation of p38 kinase.Background: Thioridazine, a neuroleptic phenothiazine with antimicrobial efficacy is known to trigger anemia. At least in theory, the anemia could result from stimulation of suicidal erythrocyte death or eryptosis, which is characterized by cell shrinkage and by phospholipid scrambling of the cell membrane with phosphatidylserine exposure at the erythrocyte surface. Triggers of eryptosis include increase of cytosolic Ca Thioridazine, a phenothiazine drug, has both antipsychotic efficacy ,2 and an2+ concentration ([Ca2+]i) due to Ca2+ entry through Ca2+-permeable cation channels [2+]i leads to cell shrinkage due to activation of Ca2+-sensitive K+ channels i further leads to translocation of phosphatidylserine from the inner leaflet of the cell membrane to the erythrocyte surface i, cell volume and phosphatidylserine abundance at the erythrocyte surface has been determined.The present study explored, whether thioridazine triggers eryptosis. To this end, the effect of thioridazine on i). Accordingly, [Ca2+]i was determined utilizing Fluo3 fluorescence. To this end, erythrocytes were loaded with Fluo3-AM and Fluo3 fluorescence determined in flow cytometry following prior incubation of the erythrocytes in Ringer solution without or with thioridazine. As illustrated in Both, cell membrane scrambling and cell shrinkage could have resulted from increase of cytosolic Ca2+ entry was required for the effect of thioridazine on cell membrane scrambling, erythrocytes were exposed to 30 µM of thioridazine for 48 hours, either in the presence of 1 mM Ca2+ or in the absence of Ca2+ and presence of Ca2+ chelator EGTA (1 mM). As shown in 2+. However, even in the absence of extracellular Ca2+, thioridazine still significantly increased the percentage of annexin-V-bindiung erythrocytes pointing to additional mechanisms involved.In order to explore whether extracellular CaIn order to explore whether the additional mechanisms could include p38 kinase, erythrocytes were exposed in further experiments to 6–30 µM of thioridazine for 48 h in either the presence or absence of the p38 kinase inhibitor SB203580 (2 µM). As shown in N-acetyl-cysteine (1 mM). As a result, a 48 h exposure to thioridazine (30 µM) increased the percentage of annexin V binding erythrocytes again to a similar value in the presence and absence of N-acetyl-cysteine.Additional experiments were performed in the absence and presence of pancaspase inhibitor zVAD (10 µM). As illustrated in i.e., the stimulation of eryptosis, the suicidal erythrocyte death. Exposure of human erythrocytes to thioridazine decreased cell volume and triggered erythrocyte of eryptosis. The concentrations required to trigger eryptosis were within the range of concentrations (6 µg/mL ≈ 15 µM) encountered in vivo i), activation of Ca2+ sensitive K+ channels i further contributed to the triggering of cell membrane scrambling by thioridazine. However, even in the absence of extracellular Ca2+ thioridazine still significantly triggered cell membrane scrambling, an observation pointing to the involvement of additional mechanisms. Mechanisms mediating the stimulation of eryptosis following osmotic shock include p38 kinase [Thioridazine influenced erythrocyte cell volume most likely by stimulating entry and/or impairing extrusion of Cachannels ,77, K+ eed water . The Ca2channels , which achannels . Ca2+ isridazine . Stimula8 kinase , which i8 kinase . As show8 kinase , p38 kinWhatever mechanisms involved in the triggering of phosphatidylserine translocation by thioridazine, phosphatidylserine exposed at the surface of eryptotic cells fosters the binding of the affected erythrocytes to phagocytosing cells with subsequent engulfment and thus clearance of those cells . The clePhosphatidylserine exposing erythrocytes may further adhere to endothelial CXCL16/SR-PSO of the vascular wall  and, thuBesides causing anemia ,8, thiorOn the other hand, triggering of eryptosis may result in the clearance of defective erythrocytes prior to rupture of the cell membrane and thus prevent release of cellular hemoglobin, which is filtered in renal glomerula and subsequently occludes renal tubules .in vitro 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, 1 CaCl2; pH 7.4 at 37 °C for 48 h. Where indicated, erythrocytes were exposed to thioridazine  at the indicated concentrations (dissolved in 0.001% DMSO). In Ca2+-free Ringer solution, 1 mM CaCl2 was substituted by 1 mM glycol-bis(2-aminoethylether)-N,N,N',N'-tetraacetic acid (EGTA) and/or p38 kinase inhibited by addition of SB203580 .Leukocyte-depleted erythrocytes were kindly provided by the blood bank of the University of Tuebingen. The study is approved by the ethics committee of the University of Tuebingen (184/2003V). 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 forward scatter (FSC) of the cells was determined, and annexin-V fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm utilizing a FACS Calibur .After incubation under the respective experimental condition, 50 µL cell suspension were washed in Ringer solution containing 5 mM CaCl2 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 150 µL Ringer. Following this, Ca2+-dependent fluorescence intensity was measured with an excitation wavelength of 488 nm and an emission wavelength of 530 nm utilizing a FACS Calibur .After incubation, erythrocytes were washed in Ringer solution and then loaded with Fluo-3/AM  in Ringer solution containing 5 mM CaClFor the determination of hemolysis the samples were centrifuged  after incubation, 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.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 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.2+ concentration.In conclusion, thioridazine is shown to stimulate eryptosis, which is characterized by cell membrane scrambling and cell shrinkage. The substance is partially effective by activation of p38 kinase and by increase of cytosolic Ca


The dysregulation of Ca2+ homeostasis has been suggested as an important event in driving the expression of the malignant phenotypes, such as proliferation, migration, invasion, and metastasis. Cell migration is an early prerequisite for tumor metastasis that has a significant impact on patient prognosis. During cell migration, the exquisite spatial and temporal organization of intracellular Ca2+ provides a rapid and robust way for the selective activation of signaling components that play a central role in cytoskeletal reorganization, traction force generation, and focal adhesion dynamics. A number of known molecular components involved in Ca2+ influx pathways, including stromal interaction molecule (STIM)/Orai-mediated store-operated Ca2+ entry (SOCE) and the Ca2+-permeable transient receptor potential (TRP) channels, have been implicated in cancer cell migration and tumor metastasis. The clinical significance of these molecules, such as STIM proteins and the TRPM7 channel, in tumor progression and their diagnostic and prognostic potentials have also been demonstrated in specific cancer types. In this review, we summarize the recent advances in understanding the important roles and regulatory mechanisms of these Ca2+ influx pathways on malignant behaviors of tumor cells. The clinical implications in facilitating current diagnostic and therapeutic procedures are also discussed.Intracellular Ca Surprisingly, SPCA2 induced Ca2+ influx independently of its ATPase function, as a SPCA2 mutant with impaired Ca2+-ATPase activity increased basal [Ca2+]i and anchorage-independent growth to a similar extent to that of wild-type SPCA2. The results from immunofluorescent staining and surface biotinylation in breast cancer cells showed that SPCA2 is partially localized to the plasma membrane where it interacts with the N-terminus of SOC channel Orai1 to elicit the constitutive STIM1-independent Ca2+ influx. As a result, the Ca2+ dependent nuclear translocation of nuclear factor of activated T cells (NFAT) was upregulated in breast cancer cells. The SPCA2-Orai1 complex thus elicits a novel type of constitutive store-independent Ca2+ signalling that promotes breast tumorigenesis.In addition to STIM1-mediated activation of Orai1, an entirely different signalling mechanism, in which the Ca isoform ,76, are ocessing . The limactation . Howevert cancer , SPCA2 wt cancer . Additio2+ entry pathways in various non-excitable and excitable cells i: Intracellular CaThe authors have no conflicts of interest to declare.Y-F C and M-R S collected information, conceived the concept, prepared figures, and drafted the manuscript. Y-T C and W-T C were involved in drafting part of the manuscript. All of the authors read and approved the final manuscript.


Hydatid cyst is still endemic in various regions of the world. It is the most frequent cause of liver cysts worldwide. Urticaria is sometimes the first manifestation of the disease. However anaphylactic reaction and urticaria have been very rarely reported in the literature. Traditionally, surgery has been the only accepted mode of treatment; however, percutaneous treatment has recently been proposed as an alternative. Cases of anaphylaxis have been reported after percutaneous drainage of hydatid cyst. However, anaphylaxis usually develops within a few hours. Herein, we describe the case of a patient who presented with hydatid cyst causing chronic urticaria and late anaphylactic reaction following percutaneous aspiration of a liver hydatid cyst. We emphasize that physicians should be aware of hydatid cyst as a possible etiology for seemingly chronic spontaneous urticaria, especially in endemic regions. Patients should be kept under observation for at least one day due to the risk of early and late anaphylaxis after percutaneous aspiration treatment. Echinococcus granulosus and resides in the liver (65%) and lungs (25%). As anaphylactic reactions and allergic symptoms generally involve cases of HC rupture the treatment regimens for spontaneous cases have also been reported [Urticaria is a heterogeneous group of diseases. All types and subtypes of urticaria share a common distinctive skin reaction pattern. Chronic urticaria (CU) is defined as urticaria that persists for more than six weeks and is further classified into subgroups such as physiologic, spontaneous, and other ones. Chronic persistent bacterial, viral, parasitic, or fungal infections have been suspected of triggering urticarial symptoms in patients with chronic spontaneous urticaria (CSU) . One of reported . In this3, sedimentation rate: 45 mm/hour, and C-Reactive Protein: 14.3 mg/dL. On chest X-ray a cavity with irregular borders and nonhomogenous infiltration was seen in the upper lobes (Helicobacter pylori antigens, and hepatitis markers were found to be normal. The patient was referred to a Tuberculosis Clinic. He was called for a control checkup six months after treatment and presented with a gain in weight and restoration of appetite but urticarial lesions continued in the patient whenever he did not take the antihistamine drugs. Chest X-ray showed a nearly resolved lesion (A 21-year-old male patient presented to our polyclinic with a 3-month history of urticaria. Urticaria treatment was started with pheniramine maleate of 22.7 mg at a twice daily dose in an outside clinic setting. A decrease in symptoms with this treatment was not seen and urticaria reoccurred on days when the patient did not take the antihistamines. The patient was therefore referred to our department for further investigations into the etiology of the urticaria. The patient, who appeared cachectic on presentation, had a complaint of a 4-5 kg weight loss in the last 6 months. Physical examination findings were blood pressure of 110/70, heart rate of 76/min, respiratory rate of 16/min, and body temperature of 37°C. Other physical examination findings were normal. Laboratory findings were as follows: leukocyte: 14800/mmer lobes . A sputud lesion . An abdoEchinococcus specific IgE to support a HC mediated Type 1 hypersensitivity reaction causing urticaria in our patient were not conducted.Urticaria is a dermatologic reaction that can be caused by several different factors. Chronic urticaria is defined as urticaria that persists for more than six weeks . In our There are 3 different types of treatment for HC, medical treatment, surgical treatment, and percutaneous drainage. Traditionally, surgical treatment has been the most accepted method of treatment. However surgical treatment carries a high morbidity rate of 14%–60% and a mortality rate of about 7% . In receChronic spontaneous urticaria cases should be carefully investigated for chronic bacterial, viral, fungal, and parasitic infections. Particularly in regions where HC is endemic, HC as an etiology should be kept in mind. Due to the high risk of anaphylaxis that can develop during procedures in the treatment of HC, procedures of this sort should be conducted in settings that may possibly require emergency interventions with adrenaline and have intubation sets available. Patients should be kept under observation in a clinical setting for at least a day before being discharged due to the possibility of the development of late anaphylactic reactions.


However, it is still unclear if mitochondrial Ca2+ flux can regulate the generation of Ca2+ waves (CaWs) and triggered activities in cardiac myocytes. In the present study, intracellular/cytosolic Ca2+ (Cai2+) was imaged in Fluo-4-AM loaded mouse ventricular myocytes. Spontaneous sarcoplasmic reticulum (SR) Ca2+ release and CaWs were induced in the presence of high (4 mM) external Ca2+ (Cao2+). The protonophore carbonyl cyanide p-(trifluoromethoxy)phenylhydrazone (FCCP) reversibly raised basal Cai2+ levels even after depletion of SR Ca2+ in the absence of Cao2+ , suggesting Ca2+ release from mitochondria. FCCP at 0.01 - 0.1 µM partially depolarized the mitochondrial membrane potential (Δψm) and increased the frequency and amplitude of CaWs in a dose-dependent manner. Simultaneous recording of cell membrane potentials showed the augmentation of delayed afterdepolarization amplitudes and frequencies, and induction of triggered action potentials. The effect of FCCP on CaWs was mimicked by antimycin A (an electron transport chain inhibitor disrupting Δψm) or Ru360 , but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor), excluding the contribution of intracellular ATP levels. The effects of FCCP on CaWs were counteracted by the mitochondrial permeability transition pore blocker cyclosporine A, or the mitochondrial Ca2+ uniporter activator kaempferol. Our results suggest that mitochondrial Ca2+ release and uptake exquisitely control the local Ca2+ level in the micro-domain near SR ryanodine receptors and play an important role in regulation of intracellular CaWs and arrhythmogenesis. Recent studies have suggested that mitochondria may play important roles in the Ca In particular, the regulation of intracellular CaWs by mitochondrial Ca2+ flux under high intracellular Ca2+ conditions was investigated. We found that the protonophore carbonyl cyanide p-(trifluoromethoxy) phenylhydrazone (FCCP) depolarizes Δψm and subsequently causes Ca2+ release from mPTPs, which promotes SR spontaneous Ca2+ release and CaWs. The exacerbation of CaWs by mitochondrial Ca2+ release is able to cause triggered activities, manifesting increased arrhythmogenesis during mitochondrial dysfunction.In the present study, we aim to test whether mitochondrial CaGuide for the Care and Use of Laboratory Animals, published by the National Institutes of Health . Mice of 2-4 months old (either sex) were used in this study. All experiments were performed at 35-37°C. All animal experimental procedures were reviewed and approved by the Institutional Animal Care and Use Committee (IACUC) at the Rutgers-New Jersey Medical School, and in accordance with the 2+-free Tyrode’s solution containing 0.5 mg/ml collagenase  and 0.1 mg/ml protease  for 10 to 15 minutes. The enzyme solution was then washed out and the hearts were removed from the perfusion apparatus. The left ventricles were placed in petri dishes, and were then gently teased apart with forceps. Finally, the myocytes were filtered through nylon mesh (200 µm). The Ca2+ concentration was gradually increased to 1.0 mM, and the cells were stored at room temperature and used within 8 hours. Ventricular myocytes were enzymatically isolated from mouse hearts as previously described . BrieflyΔψm was assessed by the decline in TMRM fluorescence intensity. Ventricular myocytes were loaded with tetramethylrhodamine methylester , a lipophilic cation, for 40 min at room temperature. TMRM fluorescence was monitored with confocal fluorescence microscopy (Ex/Em: 540/580nm) and recorded using an Ixon Charge-Coupled Device (CCD) camera (Andor Technology). FCCP-induced depolarization of 2 in normal Tyrode's solution at room temperature for 30 min. Loaded myocytes were washed free of calcein (CoCl2 was continuously perfused) just before imagining, and excited at 484 nm. Emitted fluorescence was acquired at 520 nm every 2 min. The percentage of calcein leak, estimated from the fluorescence decrease, was used as the index of mPTP opening.Measurement of the mPTP opening was conducted as previously described ,25. IsolF/F0 values. The F/F0 value was expressed as 0 during the periods when the cell was not exposed to the excitation light. The average values measured at 3 consecutive 200 ms-exposures were used to evaluate F/F0 values every 2 min. The baseline value (i.e. before perfusion of FCCP) was normalized to 1. The F/F0 level at 6 min after FCCP treatment was compared between different groups. Changes in mitochondrial superoxide production were monitored using MitoSOX Red (Invitrogen/Molecular Probes). Isolated cardiac myocytes were loaded with MitoSOX Red (5 μM) for 30 min at 37°C followed by washout. MitoSOX Red fluorescence (EX/EM: 485/585 nm) was monitored using a Nikon Eclipse TE200 inverted microscope and recorded using an Ixon Charge-Coupled Device camera (Andor Technology) operating at ~10 fps with a spatial resolution of 500×400 pixels. MitoSOX Red fluorescence is presented as background-subtracted 2+ indicator (Mag-fluo-4) [2+. Thus the free intracellular Mg2+ concentration ([Mg2+]i) changes reflect reciprocal changes of [ATP]i. Ventricular myocytes were loaded with Mag-fluo-4 AM  for 20 min in Tyrode’s solution. Mag-fluo-4 fluorescence (EX/EM: 485/530 nm) was monitored using a Nikon Eclipse TE200 inverted microscope and recorded using an Ixon CCD camera (Andor Technology) operating at ~10 fps with a spatial resolution of 500×400 pixels. The fluorescence images (3 consecutive 200 ms-exposures) were obtained every 1 or 2 min in the absence and presence of 100 nM and 1 µM FCCP, respectively. The changes of intracellular ATP levels were estimated indirectly by using a fluorescent Mg-fluo-4) , based oi2+ fluorescence (EX/EM: 485/530 nm) was monitored using a Nikon Eclipse TE200 inverted microscope and recorded using an Ixon Charge-Coupled Device camera (Andor Technology) operating at ~50 fps with a spatial resolution of 500×400 pixels, as described in previous studies o functions as a promoting factor. This high [Ca2+]o-induced CaW model was used for the subsequent experiments. To investigate the potential regulatory effect of mitochondrial Ca2+ level ([Cai2+]) ( i2+] were counteracted by the mPTP blocker cyclosporine A (CsA) , and was subsequently reduced to 27.9 ± 3.06 min-1 by 1 µM CsA  (i2+] levels were elevated from F/F0 =1 to 1.26 ± 0.07 by 100 nM FCCP (p < 0.01), and were subsequently reversed by 1 µM CsA  : 17.3 ± 3.1 min-1 . These results suggest that depolarization of Δψm (by FCCP) may affect CaW behavior via mitochondrial Ca2+ release through mPTP opening. We then examined the effect of FCCP (10 nM - 1 µM) on CaWs. A representative recording is shown in ai2+])   in a dos A (CsA) . The fre01, n=8) . The bas05, n=8) . In orde2+ transients were caused, secondarily, by triggered action potentials. This phenomenon is well demonstrated in 2+ fluorescence (2+ fluorescence intensity (0), and membrane potential . Since TAs are a major mechanism for arrhythmias, these results strongly imply that mitochondria play a role in arrhythmogenesis via regulation of CaWs. The exacerbation of CaWs by mitochondrial Ca release is sufficient for triggering action potentials and thus increases arrhythmogenicity.During perfusion with 100 and 500 nM FCCP , high-amrescence , whole-cntensity F/F0, aotential , MP were2+ level was dramatically elevated . As shown in 2+ content  was significantly decreased in the presence of 1 µM FCCP compared to the control. Blocking sNCX by a rapid application of lithium (Li+) raised basal Cai2+ levels extremely high and restored CaWs  completely eliminated spontaneous CaWs, while the basal Cared CaWs , while C to 8 mM . 1F0-ATP synthase, and ATP may be consumed as the ATP synthase operates in the reverse mode. Therefore, one can assume that the decrease of SR Ca2+ content may be caused by the reduction of SERCA2a activity due to the lack of ATP. To test this assumption, we used oligomycin, an inhibitor of F1F0-ATP synthase, to attenuate ATP depletion in the present of FCCP. As shown in 1/F0-ATP synthase. It is likely the decrease of SR Ca2+ content at 1 µM FCCP  by 1 µM FCCP, but not by 100nM FCCP during the same time course .It has been shown, that in the presence of high concentration of FCCP, a protonophoric uncoupler, there is no driving force for Fi2+ levels  the entry of Ca2+ through the sarcolemmal membrane, and 3) SR Ca2+ release. In the following experiments, we dissected the component of the Cai2+ elevation caused by mitochondrial Ca2+ efflux in the presence of 1 µM FCCP , suggesting the elevated basal [Cai2+] in the absence of extracellular Ca2+ can be attributed to intracellular sources, i.e. the mitochondria and SR. To further analyze the contribution from mitochondrial release, we depleted the SR Ca2+ by fast exposure to caffeine (10mM) before treatment with FCCP in Ca2+-free Tyrode’s solution. A representative recording is shown in i2+] was still elevated  by FCCP when both extracellular and SR sources were eliminated, suggesting that mitochondrial Ca2+ release contributes partially to the basal [Cai2+]. We postulate that this elevation of [Cai2+] may locally regulate RyR Ca release function at cytosolic face of RyRs and effectively facilitate CaW and TA generation. We have observed FCCP-induced elevation of basal Ca+ levels . This el µM FCCP . The tot n = 11;  by 1 µM i2+] plays an important role in influencing the RyRs, and subsequently modulating CaWs, Ca2+ flux via other mitochondrial Ca2+ transporters should also be effective . This assumption was tested by modifying the function of mCUs, which mediate Ca2+ uptake into mitochondria , however its effect was strongly potentiated by the addition of 2 µM Ru360. A direct potentiating effect of Ru360 on CaWs was also observed in the absence of FCCP  on CaWs (data not shown).In addition, we also tested the effects of the mCU blocker Ru360 on CaWs. As shown in  of FCCP . Based oi2+] were mimicked by antimycin A (10 µM), an electron transport chain (complex III) inhibitor, which also depolarizes Δψm   . 0F1-ATPase) inhibitor (n=5) nor iodoacetic acid  in CaW activation under our present experimental conditions.On the contrary, neither oligomycin , an ATP synthase  in mitochondria may lead to aberrant calcium homeostasis –33. In o2+ flux in modulation of CaWs and TAs in mouse ventricular myocytes. The major findings of this present study include: 1) The protonophore FCCP, which can depolarize Δψm and open mPTPs, increase basal [Cai2+] and potentiate CaWs and TAs in a dose-dependent manner in mouse ventricular myocytes; 2) the effect of FCCP on CaWs can be antagonized by CsA, an mPTP blocker; 3) Ca2+ flux through mCU also affects CaW behaviors; 4) the effect of FCCP on SCWs can be mimicked by antimycin A , but not by oligomycin (an ATP synthase inhibitor) or iodoacetic acid (a glycolytic inhibitor). We postulate that mitochondrial Ca2+ flux controls the local Ca2+ homeostasis in the micro-domain at the cytosolic face of RyR. It is well known that the majority of RyRs are located in the dyadic regions of the junctional SR membrane and face the transverse tubule in ventricular myocytes  (4 mM). This model indicates that Ca2+ influx from sarcolemma can elevate cytosolic levels and trigger CaWs. This sarcolemmal Ca2+ influx may be caused by non-selective cationic channels, such as TRPCs [2+ fluxes via mitochondrial Ca2+ transporters dynamically change cytosolic Ca2+ levels and consequently regulate CaW behaviors. Although only a mild (but significant) elevation of basal [Cai2+] induced by mitochondrial efflux was detected by measuring global Ca2+  when basal [Cai2+] was elevated. Based on the results shown in 2+ content below the CaW threshold level. It should be noted that FCCP has biphasic effects on CaWs . FCCP pri2+] . Early s2+]  and/or reduced mitochondrial Ca2+ uptake (via mCU) followed by increased diastolic [Ca2+]i and enhanced SR Ca2+ release. On the contrary, the inhibition of CaWs at higher concentrations (e.g. 1 µM) could be caused by reduced SR Ca2+ content due to consumption of ATP and consequent reduction of SERCA activity, as well as excessive extrusion of Ca2+ through the sarcolemma. In addition, we cannot exclude the possibility of reduced RyR activity since ATP may act as a direct activator of SR RyR o-induced CaW model in this study. We believe that this model is still relevant since Ca overload may occur under various pathological conditions, such as ischemia-reperfusion, hypertrophy, and heart failure.In order to induce CaWs consistently and study the regulatory effects of mitochondrial dysfunction, we have exclusively used a high (4 mM) [Ca


This study aims to clarify the relationship amongst absolute and relative concentrations of K+, K+, Ca2+, Mg2+, Na+/K+, Ca2+/Mg2+ together with IL6, TNFα as proinflammatory cytokines and caspase3 as proapoptotic biomarker were determined in plasma of 25 Saudi autistic male patients and compared to 16 age and gender matching control samples.Na2+ and a significantly higher K+ compared to age and gender matching controls. On the other hand both Mg2+ and Na+ were non-significantly altered in autistic patients. Pearson correlations revealed that plasma concentrations of the measured cytokines and caspase-3 were positively correlated with Ca2+ and Ca2+/K+ ratio. Reciever Operating Characteristics (ROC) analysis proved that the measured parameters recorded satisfactory levels of specificity and sensitivity.The obtained data recorded that Saudi autistic patients have a remarkable lower plasma caspase3, IL6, TNFα, CaAlteration of the selected measured ions confirms that oxidative stress and defective mitochondrial energy production could be contributed in the pathogenesis of autism. Moreover, it highlights the relationship between the measured ions, IL6, TNFα and caspase3 as a set of signalling pathways that might have a role in generating this increasingly prevalent disorder. The role of ions in the possible proinflammation and proapoptic mechanisms of autistics' brains were hypothesized and explained. Children with Autism Spectrum Disorders (ASD) have impairments in three core domains: socialization, communication, and restricted interests and repetitive behaviors -4. ReseaAlthough the etiology of the disorder is unknown, recent studies have suggested that the susceptibility to autism is clearly attributable to genetic factors ,8. In ad2+ have long been associated with apoptotic neuronal cell death. Ca2+ ionophores have been shown to induce ultrastructural changes, such as cell shrinkage, chromatin condensation, and DNA fragmentation, consistent with apoptosis , providing evidence that brain cells cytosolic [Mg2+] is regulated to equilibrate any changes in rapidly available free energy. Moreover, it has also been shown that the measurement of brain Mg2+ can help in the differential diagnosis of neurodegenerative diseases sharing common clinical features.Magnesium . All subjects enrolled in the study  had written informed consent provided by their parents and assented to participate if developmentally able. They were enrolled through the ART Center (Autism Research & Treatment Center) clinic . The ART Center clinic sample population consisted of children diagnosed on the ASD. The diagnosis of ASD was confirmed in all subjects using the Autism Diagnostic Interview-Revised (ADI-R) and the Autism Diagnostic Observation Schedule (ADOS) and 3DI . The ages of all autistic children who participated were between the ages of 4 and 12 years old. All were simplex cases. All are negative for fragile × gene study. The control group recruited from Well baby Clinic at King Khaled University hospital with mean age 4-11 year old. Subjects were excluded from the investigation if they had organic aciduria, dysmorphic features, or diagnosis of Fragile × or other serious neurological , psychiatric  or known medical conditions. All participants were screened via parental interview for current and past physical illness. Children with known endocrine, cardiovascular, pulmonary, liver, kidney or other medical disease were excluded from the study. None of the recruited autistic patients were on special diets or alternative treatments.A written consent was obtained from the parents of each individual case, according to the guidelines of the ethical committee of King Khalid Hospital, King Saud University.After overnight fast, 10 ml blood samples were collected from both groups in test tubes containing sodium heparin as anticoagulant. Tubes were centrifuged at 3500 rpm at room temperature for 15 minutes, plasma was obtained and deep freezed (at -80°C) until analysis time.2+ procedure is based on the reaction of Ocresolphthalein complexone (O-CPC) with Ca2+ to form a chromogenic complex that absorbs light which is measured photometrically at 575 nm. Mg2+ interference is prevented by sequestration with 8-hydroxyquinoline. 2-Ethylaminoethanol is used to establish the reaction pH at 12. Dimethyl sulfoxide is used to lower the dielectric constant of the reaction mixture and to repress the ionization of cresolphthalein complexone . Vitamin E which is known to attenuate A β-induced apoptosis despite Ca2+ accumulation in brain cells is significantly lower in Saudi autistic patients [2+- induced apoptosis in brain cells of Saudi autistics.This suggested increased influx of blood-to-brain Capatients . This co+ concentrations in plasma of autistic and control subjects. It could be easily noticed that autistic patients recorded raised concentrations of K+ compared to controls. This could be attributed to the altered Na+/K+ ATPase activity previously reported by El-Ansary et al. [Table y et al. , which m+ which reflect the remarkable higher rate of k+ efflux from brain to blood in autistic patients could be easily related to the significant lower Ca2+, the unchanged Na+, lower Ca2+/Na+ ratios and to the speculated higher brain caspase3 activity. Xiao et al. [2 and Na+ condition induced apoptotic neuronal death, and that the K+ efflux via NMDA receptor channels was likely a key event in NMDA-induced apoptosis. This postulation could be supported by Pigozzi et al. [2+ into neuron cells can accelerate apoptosis by accelerating the expression of growth arrest and DNA Damage inducible gene 153 (GADD153) and by inducing a prolonged efflux of K+ out of the cell. This is in good agreement with the elevated K+ and the reduced Ca2+ concentrations in plasma of autistic patients compared to controls as a report of the present study. Moreover, the significantly impaired Ca2+ and K+ concentrations in plasma of autistic patients could be easily related to the postulated increase of brain cytokines (TNFα and IL6) after infiltration from plasma to brain. Experimental evidence demonstrates that ion channels are targeted by cytokines, which can specifically modulate their function [2+ influx from blood to brain [The recorded higher plasma concentrations of Ko et al.  showed pi et al.  who provfunction  and TNFαto brain . These sROC analysis presented in Figure The authors declare that they have no competing interests.AE designed the study and drafted the manuscript. ABB helped to draft the manuscript and performed the statistical analysis. LA provided samples and participated in the design of the study. All authors have read and approved the final manuscript.