Endoplasmic Ca2 Research Articles

Investigation of 2,6-diisopropylphenol (propofol)-evoked Ca2+ movement and cell death in human glioblastoma cells

This study examined whether propofol altered [Ca2+]i and caused cell death in DBTRG-05MG cells. Propofol at 400–1000μM increased [Ca2+]i in a concentration-dependent manner. The signal was decreased partially by removal of extracellular Ca2+. Propofol-induced Ca2+ influx was not altered by nifedipine, econazole, SK&F96365, and protein kinase C (PKC) activators; but was inhibited by PKC inhibitor. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished propofol-induced [Ca2+]i rise. Incubation with propofol inhibited thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished propofol-induced [Ca2+]i rise. At 300–700μM, propofol killed cells in a concentration-dependent manner. The cytotoxic effect of propofol was partly reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/PI staining further showed that 300–500μM propofol evoked apoptosis. Propofol also increased reactive oxygen species (ROS) production. Overall, propofol induced a [Ca2+]i rise by inducing PLC- and PKC-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via non store-operated Ca2+ channels. Propofol induced cell death that might involve ROS-mediated apoptosis.

Store‐operated calcium entry regulate mesenchymal stem cell proliferation

Mesenchymal stem cells (MSC) are a unique class of stromal cells which like other stem cells have the special ability to self‐renew and differentiate. MSCs have multi‐lineage differentiation potential, and can be coaxed to differentiate into adipocytes, chondrocytes, osteocytes or neurons in vitro. It is this remarkable plasticity which makes the MSCs important for cell‐based therapy and regenerative applications. Thus, understanding the molecular mechanisms that control growth and differentiation of MSCs is of clinical importance. Calcium (Ca2+) is an essential signaling messenger that modulates a variety of cellular functions ranging from cell growth to differentiation to cell death. One of the prime physiological modalities that regulate cellular Ca2+homeostasis is store‐operated calcium entry (SOCE). SOCE represents a universal phenomenon of Ca2+influx across the plasma membrane which is triggered, following activation of PM‐GPCRs, by IP3‐mediated depletion of endoplasmic reticulum Ca2+stores. Studies from this laboratory and others have suggested the non‐selective cation channel TRPC1 as an integral component of store‐operated Ca2+channel (SOC). Further, TRPC1 forms a complex with the recently identified calcium‐selective channel Orai1 and following store‐depletion is activated by the endoplasmic reticulum‐associated Ca2+sensor STIM1. The objective of this study was to identify the ‘TRPC1‐STIM1‐Orai1’ component of Ca2+signaling and to test the significance of SOCE in MSC proliferation. Using RT‐PCR we identified the expression of TRPC1, STIM1 and Orai1, and co‐immunoprecipitation analysis identified their functional interaction. Further, pharmacological inhibition of SOCE in proliferating MSCs resulted in growth attenuation. In conclusion, our study suggests the ‘TRPC1‐STIM1‐Orai1’ complex as the core SOCE machinery that is critical for MSC proliferation

Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells

AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca2+ level ([Ca2+]i) and viability in human glioblastoma cells. Main methodsThe Ca2+-sensitive fluorescent dye fura-2 was applied to measure [Ca2+]i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry. Key findingsCarvacrol at concentrations of 400–1000μM induced a [Ca2+]i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca2+. Carvacrol-induced Ca2+ signal was not altered by nifedipine, econazole, SK&F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca2+ was removed, incubation with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca2+]i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca2+]i rise. At concentrations of 200–800μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N–-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that carvacrol (200, 400 and 600μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600μM, carvacrol induced production of ROS. SignificanceIn human glioblastoma cells, carvacrol induced a [Ca2+]i rise by inducing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ entry via protein kinase C-sensitive, non store-operated Ca2+ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis.

Effect ofm-3m3FBS on Ca2+handling and viability in OC2 human oral cancer cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-60 μM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 μM m-3M3FBS pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. At concentrations between 5 and 100 μM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 μM) induced apoptosis in a Ca2+-independent manner. Collectively, in OC2 cells, m-3M3FBS induced [Ca2+]i rise by causing inositol 1,4,5-trisphosphate-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive store-operated Ca2+ channels. M-3M3FBS also induced Ca2+-independent cell death and apoptosis.

Deltamethrin-Induced [Ca2+ ]i Rise and Death in HGB Human Glioblastoma Cells

The effect of the pesticide deltamethrin on cytosolic free Ca ²⁺ concentrations ([Ca²⁺ ]i) and viability in human glioblastoma DBTRG-05MG cells is explored. [Ca²⁺ ]i was measured in suspended cells using fura-2 as a Ca²⁺ -sensitive fluorescent dye. Deltamethrin at concentrations of 5-60 μM increased [Ca²⁺]i in a concentration-dependent fashion. The Ca²⁺ signal was reduced partly by removing extracellular Ca ²⁺ . Deltamethrin induced Mn ²⁺ entry leading to quenching of fura-2 fluorescence. Deltamethrin-induced [Ca²⁺ ]i rise was not inhibited by nifedipine, econazole, SKF96365, and phorbol 12-myristate 13 acetate, but was inhibited by the protein kinase C (PKC) inhibitor GF109203X via blocking Ca²⁺ release. In Ca²⁺ -free medium, 50 μM deltamethrin pretreatment abolished the [Ca²⁺]i rise induced by the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) or 2,5-di-tertbutylhydroquinone (BHQ). Conversely, pretreatment with TG/BHQ abolished deltamethrin-induced [Ca²⁺]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 suppressed 50% of deltamethrin-induced [Ca²⁺]i rise. At concentrations between 10 and 80 μM deltamethrin killed cells in a concentration-dependent manner. The cytotoxic effect of deltamethrin was not reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/ propidium iodide staining data suggest that deltamethrin (10-40 μM) induced apoptosis in a concentrationdependent manner. Deltamethrin also increased levels of reactive oxygen species. Together, in human glioblastoma cells, deltamethrin induced a [Ca²⁺]i rise by inducing phospholipase C- and PKC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via non-store-operated Ca²⁺ channels. Deltamethrin induced cell death that might involve apoptosis via mitochondrial pathways.

Signaling by intracellular Ca2+ and H+ in larval mosquito (Aedes aegypti) midgut epithelium in response to serosal serotonin and lumen pH

The midgut of larval mosquitoes (Aedes aegypti) mediates a cycle of alkali secretion in the anterior segment (AMG) followed by partial reacidification in the posterior segment (PMG); both processes are serotonin-dependent. Here we report that intracellular Ca2+(Cai2+) as indicated by Fura-2 fluorescence, is elevated in both tissues in response to serotonin, but the time courses differ characteristically in the two gut segments, and Ca2+-free solution abolishes the serotonin response in AMG, but not in PMG, whereas Thapsigargin, an inhibitor of endoplasmic Ca2+ transport, abolished responsiveness to 5-HT in PMG. These results suggest the origins for the Ca2+ signal differ between the two tissues. Quantitative real-time RT-PCR revealed expression of 5 putative 5-HT receptor types in AMG, including 5-HT2-like receptors which would be expected to initiate a Ca2+ signal. None of these receptors were highly expressed in PMG. Cyclic AMP (cAMP) is a secretagogue for both tissues, but H89, an inhibitor of Protein Kinase A (PKA), is also a secretagogue, suggesting that the stimulatory effect of cAMP involves a non-PKA pathway. Cytochalasins B and D block the effect of 5-HT in AMG, suggesting a vesicle-fusion mechanism of activation of the basal V-ATPase in this tissue. Finally, in PMG, elevation of luminal pH increases (Cai2+) and decreases intracellular pH as measured by BCECF fluorescence. These responses suggest that the rate of acid secretion by PMG might be responsive to local demand for luminal reacidification as well as to serosal serotonin.

Effect of thapsigargin on Ca2+ fluxes and viability in human prostate cancer cells

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2+]i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2+-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca2+]i in a concentration-dependent fashion. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. This Ca2+ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2+ channels or by modulation of protein kinase C activity. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2+ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2+]i rise, suggesting that thapsigargin released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca2+]i rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca2+ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive Ca2+ channels. Thapsigargin also induced cell death via Ca2+-dependent pathways and Ca2+-independent apoptotic pathways.

Effect of thimerosal on Ca2+ movement and apoptosis in PC3 prostate cancer cells

AbstractThe present study evaluated the effects of thimerosal, a vaccine preservative, on cytosolic free Ca2+ concentrations ([Ca2+]i) in human prostate cancer cells (PC3). Thimerosal (10–200 µM) increased [Ca2+]i in a concentration‐dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Thimerosal‐induced Ca2+ influx was inhibited by econazole, SKF963656, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators [phorbol 12‐myristate 13‐acetate (PMA) and GF109203X]. In Ca2+‐free medium, a 200‐µM thimerosal‐induced [Ca2+]i rise was partly inhibited after pretreatment with 2,5‐di‐tert‐butylhydroquinone (BHQ) (an endoplasmic reticulum Ca2+ pump inhibitor). Thimerosal at 1–7 µM induced cell death in a concentration‐dependent manner that was not reversed when cytosolic Ca2+ was chelated with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Propidium iodide staining suggests that apoptosis played a role in the death. Collectively, in PC3 cells, thimerosal induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store‐operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Thimerosal also induced cell death in a Ca2+‐independent apoptotic manner. Drug Dev Res 72: 330–336, 2011. © 2011 Wiley‐Liss, Inc.

P02-214 - Possible involvement of the calreticulin gene in the evolution of cognition in humans

The chromosomal region, 19p 13.2, has been suggested to be linked with schizoaffective disorder (Hamshere et al. 2005). This region harbors the calreticulin gene, protein encoded by which is an endoplasmic Ca+2-binding molecular chaperone. Development-dependent, tissue-specific expression of this gene in the gray matter coincides with the expression of psychoses phenotypes. We have recently reported instances of mutations within the core promoter (−48 G>C, −205 C>T) and coding sequence [exon 5 (c: 682 C>T, pro228ser)] of the gene in schizoaffective disorder. In view of the mounting evidence on the genetic overlap in the psychiatric spectrum, we investigated this gene in 386 patients afflicted with schizophrenia, schizoaffective disorder, major affective disorder and 600 controls by PCR/SSCA. We found that a unique mutation within the core promoter of the gene, located at a conserved genomic block, co-occurring with four cases of psychoses including schizophrenia, schizoaffective disorder and bipolar disorder type I. This unique mutation reverts the human promoter sequence to the ancestral types observed in chimpanzee, rhesus Macaque and mouse, implying that the genomic block harboring this nucleotide may be involved in the evolution of human-specific higher-order functions of the brain (i.e. cognition), that are ubiquitously impaired in psychoses. Our findings propose that calreticulin is not only a promising candidate in the spectrum of psychoses, but also, a gene that may be important in the process of human-unique higher-level functions of the brain.

The mechanism of sertraline-induced [Ca2+]i rise in human OC2 oral cancer cells

Effect of sertraline, an antidepressant, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in human cancer cells is unclear. This study examined if sertraline altered basal [Ca(2+)](i) levels in suspended OC2 human oral cancer by using fura-2 as a Ca(2+)-sensitive fluorescent probe. At concentrations of 10-100 μM, sertraline induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+) indicating that Ca(2+) entry and release both contributed to the [Ca(2+)](i) rise. Sertraline induced Mn(2+) influx, leading to quench of fura-2 fluorescence suggesting Ca(2+) influx. This Ca(2+) influx was inhibited by suppression of phospholipase A2, inhibition of store-operated Ca(2+) channels or by modulation of protein kinase C activity. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished sertraline-induced Ca(2+) release. Conversely, pretreatment with sertraline greatly reduced the inhibitor-induced [Ca(2+)](i) rise, suggesting that sertraline released Ca(2+) from the endoplasmic reticulum. Inhibition of phospholipase C did not change sertraline-induced [Ca(2+)](i) rise. Together, in human oral cancer cells, sertraline induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via store-operated Ca(2+) channels.

Effect of diallyl disulfide on Ca2+ movement and viability in PC3 human prostate cancer cells

The effect of diallyl disulfide (DADS) on cytosolic Ca2+ concentrations ([Ca2+]i) and viability in PC3 human prostate cancer cells is unclear. This study explored whether DADS changed [Ca2+]i in PC3 cells by using fura-2. DADS at 50–1000μM increased [Ca2+]i in a concentration-dependent manner. The signal was reduced by removing Ca2+. DADS-induced Ca2+ influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators; but was inhibited by aristolochic acid. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) nearly abolished DADS-induced [Ca2+]i rise. Incubation with DADS inhibited thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not alter DADS-induced [Ca2+]i rise. At 500–1000μM, DADS killed cells in a concentration-dependent manner. The cytotoxic effect of DADS was partly reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Propidium iodide staining suggests that DADS (500μM) induced apoptosis in a Ca2+-independent manner. Annexin V/PI staining further shows that 10μM and 500μM DADS both evoked apoptosis. DADS also increased reactive oxygen species (ROS) production. Collectively, in PC3 cells, DADS induced [Ca2+]i rise probably by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive channels. DADS induced Ca2+-dependent cell death, ROS production, and Ca2+-independent apoptosis.

Effect of capsazepine on [Ca2+]i in MDCK renal tubular cells

AbstractThe current study explored whether capsazepine changed basal cytosolic free Ca2+ concentrations ([Ca2+]i) levels in suspended Madin Darby canine kidney (MDCK) cells cells by using fura‐2 as a Ca2+‐selective fluorescent dye. At concentrations of 10–200 µM, capsazepine increased [Ca2+]i in a concentration‐dependent manner. The Ca2+ signal was partially reduced by 40% by removing extracellular Ca2+. Capsazepine induced Mn2+ quench of fura‐2 fluorescence, indirectly implicating Ca2+ entry. Capsazepine‐induced Ca2+ influx was unchanged by L‐type Ca2+ entry inhibitors and protein kinase C modulators [phorbol 12‐myristate 13‐acetate (PMA) and GF109203X]. In Ca2+‐free medium, 100 µM capsazepine‐induced Ca2+ release was substantially suppressed by pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Pretreatment with capsazepine nearly abolished thapsigargin‐induced Ca2+ release. Inhibition of phospholipase C with U73122 did not change capsazepine‐induced [Ca2+]i rises. Collectively, in MDCK cells, capsazepine induced [Ca2+]i rises by causing phospholipase C‐independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via non‐L‐type Ca2+ channels. Drug Dev Res 72: 323–329, 2011. © 2010 Wiley‐Liss, Inc.

The pharmacophore of thapsigargin

A derivative of thapsigargin (1) is currently in clinical phase 1 trials for treatment of prostate, bladder, and breast cancer. Thapsigargin has been targeted towards solid tumours by coupling the natural product to a peptide, which selectively is cleaved by enzymes present in excess in the cell walls of the blood vessels nourishing solid tumours. Human kallikrein 2 is an example of such a proteolytic enzyme [1]. The cytoxicity of thapsigargin relates to the ability to inhibit the sarco- or endoplasmic Ca2+ ATPase (SERCA). A pharmacophore model suggest that lipophilic interactions between the acyl groups at O-3, O-8, O-10 and CH3–15 are of major importance for the affinity to the binding cavity. Analysis of the X-ray structure of the complex of thapsigargin and SERCA [2] reveals that the segment F(834)FRY(837) andthe segment A(306)IPEGL(311) form the sides of the cavity.

Effect of nortriptyline on cytosolic Ca2+ regulation and viability in PC3 human prostate cancer cells

AbstractThe effect of nortriptyline, a tricyclic antidepressant, on Ca2+ regulation and viability in human prostate cancer cells (PC3) is unclear. The present study examined whether nortriptyline altered basal [Ca2+]i levels in suspended PC3 cells using fura‐2 as a Ca2+‐sensitive fluorescent probe. Nortriptyline (50–500 µM) increased [Ca2+]i in a concentration‐dependent fashion. The Ca2+ signal was partially reduced by removing extracellular Ca2+, indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. Nortriptyline induced Mn2+ influx, leading to quench of fura‐2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by activation of protein kinase C, but not by inhibition of L‐type Ca2+ channels. In Ca2+‐free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin nearly abolished nortriptyline‐induced Ca2+ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor‐induced [Ca2+]i rise, suggesting that nortriptyline released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change the nortriptyline‐induced [Ca2+]i rise. Nortriptyline at a concentration of 10 µM increased viability in a Ca2+‐independent manner. At 50 µM, nortriptyline killed 45% of cells. Nortriptyline at 10 µM did not induce apoptosis, but at 50 µM induced significant apoptosis measured by propidium iodide staining. Together, in PC3 cells, nortriptyline induced [Ca2+]i rises by causing the phospholipase C‐independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via the protein kinase C‐sensitive pathway. Nortriptyline also induced both cell proliferation and death in a concentration‐dependent manner. Apoptosis was involved in the cell death. Drug Dev Res 71:323–330, 2010. © 2010 Wiley‐Liss, Inc.

Effect of Nortriptyline on Ca(superscript 2+) Handling in SIRC Rabbit Corneal Epithelial Cells

To explore the effect of nortriptyline, a tricyclic antidepressant, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) in corneal epithelial cells, [Ca²⁺]i levels in suspended SIRC rabbit corneal epithelial cells were measured by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Nortriptyline at concentrations between 20-200 microM increased [Ca²⁺]i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Nortriptyline-induced Ca²⁺ influx was inhibited by the store-operated Ca²⁺ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and alteration of activity of protein kinase C. In Ca²⁺-free medium, 200 microM nortriptyline pretreatment greatly inhibited the rise of [Ca²⁺]i induced by the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ; another endoplasmic reticulum Ca²⁺ pump inhibitor) nearly abolished nortriptyline-induced [Ca²⁺]i rise. Inhibition of phospholipase C with U73122 decreased nortriptyline-induced [Ca²⁺]i rise by 75%. Taken together, nortriptyline induced [Ca²⁺]i rises in SIRC cells by causing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via store-operated Ca²⁺ channels.

Econazole‐induced Ca2+ fluxes and apoptosis in human oral cancer cells

AbstractThe effect of econazole on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability was explored in human oral cancer cells (OC2), using the fluorescent dyes fura‐2 and WST‐1, respectively. Econazole at concentrations of >1 µM increased [Ca2+]i in a concentration‐dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The econazole‐induced Ca2+ influx was sensitive to blockade of aristolochic acid (phospholipase A2 inhibitor) and GF109203X (PKC inhibitor). In Ca2+‐free medium, after treatment with 1 µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 30 µM econazole failed to induce a [Ca2+]i rise. Inhibition of phospholipase C with 2 µM U73122 substantially suppressed econazole‐induced [Ca2+]i rise. At concentrations of 5–70 µM econazole killed cells in a concentration‐dependent manner. The cytotoxic effect of 50 µM econazole was enhanced by prechelating cytosolic Ca2+ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). The ERK MAPK inhibitor, PD98059 (10 µM), also enhanced 20 µM econazole‐induced cell death. Propidium iodide staining data suggest that econazole induced apoptosis between concentrations of 10–70 µM. Collectively, in OC2 cells, econazole induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2/PKC‐regulated Ca2+ channels. Furthermore, econazole caused cell death appeared to be regulated by ERK MAPK. Drug Dev Res 71: 240–248, 2010. © 2010 Wiley‐Liss, Inc.

Maprotiline‐induced Ca2+ fluxes and apoptosis in human osteosarcoma cells

AbstractThe effect of maprotiline on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability was explored in human osteosarcoma cells (MG63), using the fluorescent dyes fura‐2 and WST‐1, respectively. Maprotiline at concentrations of ≥20 µM increased [Ca2+]i in a concentration‐dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The maprotiline‐induced Ca2+ influx was sensitive to inhibition by aristolochic acid (a phospholipase A2 inhibitor). In Ca2+‐free medium, after treatment with 1 µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 200 µM maprotiline failed to induce a [Ca2+]i rise. At concentrations of 50–100 µM maprotiline killed cells in a concentration‐dependent manner. The cytotoxic effect of 60 µM maprotiline was slightly enhanced by prechelating cytosolic Ca2+ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Propidium iodide staining data suggested that maprotiline induced apoptosis between concentrations of 60–70 µM, which was enhanced by BAPTA. Collectively, in MG63 cells, maprotiline induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2‐regulated Ca2+ channels. Furthermore, maprotiline caused apoptosis that was regulated by Ca2+. Drug Dev Res 71: 268–274, 2010. © 2010 Wiley‐Liss, Inc.

Effect of m-3M3FBS on Ca(superscript 2+) Movement in PC3 Human Prostate Cancer Cells

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) in PC3 human prostate cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca²⁺]i levels in suspended PC3 cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-50 microM increased [Ca²⁺]i in a concentration-dependent manner. The Ca²⁺ signal was reduced by 60% by removing extracellular Ca²⁺. M-3M3FBS-induced Ca²⁺ influx was inhibited by the store-operated Ca²⁺ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca²⁺-free medium, 30 microM m-3M3FBS pretreatment greatly inhibited the [Ca²⁺]i rise induced by the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or BHQ. Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid reduced the major part of m-3M3FBS-induced [Ca²⁺]i rise. Inhibition of phospholipase C with U73122 did not much alter m-3M3FBS-induced [Ca²⁺]i rise. Collectively, in PC3 cells, m-3M3FBS induced [Ca²⁺]i rises by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via store-operated Ca²⁺ channels.

Intracellular Ca2+ release‐dependent inactivation of Ca2+ currents in thalamocortical relay neurons

Neuronal Ca(2+) channels are rapidly inactivated by a mechanism that is termed Ca(2+)-dependent inactivation (CDI). In this study we investigated the influence of intracellular Ca(2+) release on CDI of high-voltage-activated Ca(2+) channels in rat thalamocortical relay neurons by combining voltage-clamp, Ca(2+) imaging and immunological techniques. Double-pulse protocols revealed CDI, which depended on the length of the conditioning pulses. Caffeine caused a concentration-dependent increase in CDI that was accompanied by an increase in the duration of Ca(2+) transients. Inhibition of ryanodine receptors and endoplasmic Ca(2+) pumps (by thapsigargin or cyclopiazonic acid) resulted in a reduction of CDI. In contrast, inhibition of inositol 1,4,5-tris-phosphate receptors by intracellular application of 2-aminoethoxy diphenyl borate or heparin did not influence CDI. The block of transient receptor potential channels by extracellular application of 2-aminoethoxy diphenyl borate, however, resulted in a significant reduction of CDI. The central role of L-type Ca(2+) channels was emphasized by the near-complete block of CDI by nifedipine, an effect only surpassed when Ca(2+) was replaced by Ba(2+) and chelated by 1,2-bis(o-aminophenoxy)ethane-N,N,N',N',-tetraacetic acid (BAPTA). Trains of action potential-like stimuli induced a strong reduction in high-voltage-activated Ca(2+) current amplitude, which was significantly reduced when intracellular Ca(2+) stores were made inoperative by thapsigargin or Ba(2+)/BAPTA. Western blotting revealed expression of L-type Ca(2+) channels in thalamic and hippocampal tissue but not liver tissue. In summary, these results suggest a cross-signalling between L-type Ca(2+) channels and ryanodine receptors that controls the amount of Ca(2+) influx during neuronal activity.

Effect of the antidepressant paroxetine on Ca2+ movement in PC3 human prostate cancer cells

AbstractThe effect of the antidepressant paroxetine on cytosolic free Ca2+ concentrations ([Ca2+]i) in PC3 human prostate cancer cells is unclear. This study explored whether paroxetine changed basal [Ca2+]i levels in suspended PC3 cells by using fura‐2 as a Ca2+‐sensitive fluorescent dye. Paroxetine at concentrations between 10–150 µM increased [Ca2+]i in a concentration‐dependent manner. The Ca2+ signal was reduced by 55% by removing extracellular Ca2+. Paroxetine‐induced Ca2+ influx was inhibited by the store‐operated Ca2+ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and protein kinase C modulators. In Ca2+‐free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin, 2,5‐di‐tert‐butylhydroquinone (BHQ), or cyclopiazonic acid (CPA) all abolished paroxetine‐induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 inhibited paroxetine‐induced [Ca2+]i rise by 80%. Collectively, in PC3 cells, paroxetine induced [Ca2+]i rise by causing phospholipase C‐dependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store‐operated Ca2+ channels in a manner regulated by protein kinase C and phospholipase A2. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.