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

The 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.     

Effects of econazole on Ca2+ levels in and the growth of human prostate cancer PC3 cells

1. Econazole is used clinically as an antifungal drug with many different in vitro effects. However, the effects of econazole on prostate cancer cells are unknown. The effects of econazole on intracellular Ca2+ concentrations ([Ca2+]i) in and the proliferation of human PC3 prostate cancer cells was explored in the present study using fura-2 and tetrazolium as fluorescent dyes. 2. At a concentration of 0.1 micromol/L, econazole started to increase [Ca2+]i in a concentration-dependent manner. The econazole-induced increase in [Ca2+]i was reduced by 48% by removal of extracellular Ca2+, suggesting that the econazole-induced increase in [Ca2+]i was composed of extracellular Ca2+ influx and intracellular Ca2+. 3. This econazole-induced Ca2+ influx was via an L-type Ca2+ channel-like pathway. In Ca2+-free medium, 1 micromol/L thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic increase in [Ca2+]i, after which the effect of econazole to increase [Ca2+]i was substantially inhibited. Conversely, pretreatment with 5 micromol/L econazole to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. 4. The phospholipase C (PLC) inhibitor U73122 (2 micromol/L) abolished the increase in [Ca2+]i induced by 10 micromol/L ATP (a Ca2+ mobilizer that needs inositol 1,4,5-trisphosphate). 5. Overnight incubation with 1-30 micromol/L econazole inhibited proliferation of PC3 cells in a concentration-dependent manner. 6. These findings suggest that, in PC3 cells, econazole increases [Ca2+]i by stimulating Ca2+ influx into cells and Ca2+ release from the endoplasmic reticulum via a PLC-independent mechanism. Econazole is cytotoxic at submicromolar concentrations.     

Effect of calmidazolium on Ca(+2) movement and proliferation in human osteosarcoma cells

In human MG63 osteosarcoma cells, the effect of calmidazolium on [Ca(2+)](i) and proliferation was explored using fura-2 and ELISA, respectively. Calmidazolium, at concentrations greater than 0.1 micromol/L, caused a rapid increase in [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 0.5 micromol/L). The calmidazolium-induced [Ca(2+)](i) increase was reduced by 66% by removal of extracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic increase in [Ca(2+)](i), after which the effect of calmidazolium to increase [Ca(2+)](i) was completely inhibited. U73122, an inhibitor of phospholipase C (PLC), abolished histamine (but not calmidazolium)-induced increases in [Ca(2+)](i). Pretreatment with phorbol 12-myristate 13-acetate to activate protein kinase C inhibited the calmidazolium-induced increase in [Ca(2+)](i) in Ca(2+)-containing medium by 47%. Separately, it was found that overnight treatment with 2-10 micromol/L calmidazolium inhibited cell proliferation in a concentration-dependent manner. These results suggest that calmidazolium increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing release of intracellular Ca(2+) from the endoplasmic reticulum in a PLC-independent manner. Calmidazolium may be cytotoxic to osteosarcoma cells.     

Mechanism underlying histamine-induced intracellular Ca movement in PC3 human prostate cancer cells

The effect of histamine on intracellular free Ca ²⁺levels ([Ca ²⁺] _i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca ²⁺dye. Histamine at concentrations between 0.1 and 50 μM increased [Ca ²⁺] _iin a concentration-dependent manner with an EC ₅₀value of 1 μM. The [Ca ²⁺] _iresponse comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca ²⁺removal inhibited 50% of the [Ca ²⁺] _isignal. In the absence of extracellular Ca ²⁺, after cells were treated with 1 μM thapsigargin (an endoplasmic reticulum Ca ²⁺pump inhibitor), 10 μM histamine did not increase [Ca ²⁺] _i. After pretreatment with 10 μM histamine in a Ca ²⁺-free medium for several minutes, addition of 3 mM Ca ²⁺induced [Ca ²⁺] _iincreases. Histamine (10 μM)-induced intracellular Ca ²⁺release was abolished by inhibiting phospholipase C with 2 μM 1-(6-((17 β-3- methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 10 μM pyrilamine but was not altered by 50 μM cimetidine. Collectively, the present study shows that histamine induced [Ca ²⁺] _itransients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca ²⁺release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca ²⁺entry.     

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

The 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.     

Effect of 2,5-dimethylphenol on Ca2+ movement and viability in PC3 human prostate cancer cells

The phenolic compound 2,5-dimethylphenol is a natural product. 2,5-Dimethylphenol has been shown to affect rat hepatic and pulmonary microsomal metabolism. However, the effect of 2,5-dimethylphenol on Ca^2+?signaling and cyotoxicity has never been explored in any culture cells. This study explored the effect of 2,5-dimethylphenol on cytosolic free Ca^2+?levels ([Ca²⁺]_i) and cell viability in PC3 human prostate cancer cells. 2,5-Dimethylphenol at concentrations between 500?μM and 1000?μM evoked [Ca²⁺]_i rises in a concentration-dependent manner. This Ca^2+?signal was inhibited by approximately half by the removal of extracellular Ca²⁺. 2,5-Dimethylphenol-induced Ca^2+?influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Pretreatment with the protein kinase C (PKC) inhibitor GF109203X, nifedipine or the store-operated Ca^2+?entry inhibitors (econazole or SKF96365) inhibited 2,5-dimethylphenol-induced Ca^2+?signal in Ca²⁺-containing medium by ～30%. Treatment with the endoplasmic reticulum Ca^2+?pump inhibitor thapsigargin in Ca²⁺-free medium abolished 2,5-dimethylphenol-induced [Ca²⁺]_i rises. Conversely, treatment with 2,5-dimethylphenol abolished thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 reduced 2,5-dimethylphenol-evoked [Ca²⁺]_i rises by ～80%. 2,5-Dimethylphenol killed cells at concentrations of 350–1000?μM in a concentration-dependent fashion. Chelation of cytosolic Ca^2+?with 1,2-bis(2-aminophenoxy)ethane-N, N, N′, N′-tetraacetic acid/AM (BAPTA/AM) did not prevent 2,5-dimethylphenol’s cytotoxicity. Together, in PC3 cells, 2,5-dimethylphenol induced [Ca²⁺]_i rises that involved Ca^2+?entry through PKC-regulated store-operated Ca^2+?channels and PLC-dependent Ca^2+?release from the endoplasmic reticulum. 2,5-Dimethylphenol induced cytotoxicity in a Ca²⁺-independent manner.     

Effect of celecoxib on Ca2+ handling and viability in human prostate cancer cells (PC3)

Celecoxib has been shown to have an antitumor effect in previous studies, but the mechanisms are unclear. Ca²⁺ is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human suspended PC3 prostate cancer cells was explored by using fura-2 as a fluorescent dye. Celecoxib at concentrations between 5 and 30 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. Celecoxib-induced Ca²⁺ influx was not blocked by L-type Ca²⁺ entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺-free medium, 30 μM of celecoxib failed to induce a [Ca²⁺]_i rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca²⁺ pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca²⁺ release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca²⁺]_i rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca²⁺ with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca²⁺]_i rises by causing phospholipase C–independent Ca²⁺ release from the ER and Ca²⁺ influx via non-L-type, phospholipase A₂-regulated Ca²⁺ channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.     

Exploring the impact of a naturally occurring sapogenin diosgenin on underlying mechanisms of Ca2+ movement and cytotoxicity in human prostate cancer cells

Literature has shown that diosgenin, a naturally occurring sapogenin, inducedcytotoxic effects in many cancer models. This study investigated the effect of diosgenin on intracellular Ca²⁺ concentration ([Ca²⁺]i) and cytotoxicity in PC3 human prostate cancer cells. Diosgenin (250‐1000 μM) caused [Ca²⁺]i rises which was reduced by Ca²⁺ removal. Treatment with thapsigargin eliminated diosgenin‐induced [Ca²⁺]i increases. In contrast, incubation with diosgeninabolished thapsigargin‐caused [Ca²⁺]i increases. Suppression of phospholipase C with U73122 eliminated diosgenin‐caused [Ca²⁺]i increases. Diosgenin evoked Mn²⁺ influx suggesting that diosgenin induced Ca²⁺ entry. Diosgenin‐induced Ca²⁺influx was suppressed by PMA, GF109203X, and nifedipine, econazole, or SKF96365. Diosgenin (250‐600 μM) concentration‐dependently decreased cell viability. However, diosgenin‐induced cytotoxicity was not reversed by chelation of cytosolic Ca²⁺ with BAPTA/AM. Together, diosgenin evoked [Ca²⁺]i increases via Ca²⁺ release and Ca²⁺ influx, and caused Ca²⁺‐non‐associated deathin PC3 cells. These findings reveal a newtherapeutic potential of diosgenin for human prostate cancer.     

Effect of celecoxib on Ca2+ movement and cell proliferation in human osteoblasts

In human osteoblasts, the effect of the widely prescribed cyclooxygenase-2 inhibitor celecoxib on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cell proliferation was explored by using fura-2 and the tetrazolium assay, respectively. Celecoxib at concentrations greater than 1microM caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner ( EC 50= 10 microM). Celecoxib-induced [Ca(2+)](i) rise was reduced by 90% by removal of extracellular Ca(2+), and by 30% by l-type Ca(2+) channel blockers. Celecoxib-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that celecoxib-induced extracellular Ca(2+) influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of celecoxib on [Ca(2+)](i) was greatly inhibited. Conversely, pretreatment with celecoxib to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phoispholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not celecoxib-induced, [Ca(2+)](i) rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, partly inhibited celecoxib-induced [Ca(2+)](i) rise in Ca(2+)-containing medium. Separately, overnight treatment with 1-100microM celecoxib inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human osteoblasts, celecoxib increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Celecoxib may be cytotoxic at higher concentrations.     

Effect of diallyl disulfide on Ca 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 50-1000 μM 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 500-1000 μM, 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 (500 μM) induced apoptosis in a Ca²⁺-independent manner. Annexin V/PI staining further shows that 10 μM and 500 μM 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 nortriptyline on cytosolic Ca2+ regulation and viability in PC3 human prostate cancer cells

The 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 the antidepressant maprotiline on calcium movement and the viability of renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of maprotiline, an antidepressant, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fura-2. Maprotiline (>2.5 µM) caused a rapid rise of [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ 200 µM). Maprotiline-induced [Ca²⁺]_i rise was reduced by removal of extracellular Ca²⁺ or by addition of La³⁺, but was not altered by voltage-gated Ca²⁺-channel blockers. Maprotiline-induced Mn²⁺ influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused Ca²⁺ influx. In Ca²⁺-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca²⁺-ATPase, caused a monophasic [Ca²⁺]_i rise, after which the increasing effect of maprotiline on [Ca²⁺]_i was nearly abolished; also, pretreatment with maprotiline reduced a portion of thapsigargin-induced [Ca²⁺]_i rise. U73122, an inhibitor of phospholipase C, abolished [Ca²⁺]_i rise induced by ATP (but not by maprotiline). Overnight incubation with 1–10 µM maprotiline enhanced cell viability, but 20–50 µM maprotiline decreased it. These findings suggest that maprotiline rapidly increases [Ca²⁺]_i in renal tubular cells by stimulating both extracellular Ca²⁺ influx and intracellular Ca²⁺ release, and may modulate cell proliferation in a concentration-dependent manner.     

Mechanisms underlying the effect of an oral antihyperglycaemic agent glyburide on calcium ion (Ca2+) movement and its related cytotoxicity in prostate cancer cells

Glyburide is an agent commonly used to treat type 2 diabetes and also affects various physiological responses in different models. However, the effect of glyburide on Ca²⁺ movement and its related cytotoxicity in prostate cancer cells is unclear. This study examined whether glyburide altered Ca²⁺ signalling and viability in PC3 human prostate cancer cells and investigated those underlying mechanisms. Intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in suspended cells were measured by using the fluorescent Ca²⁺-sensitive dye fura-2. Cell viability was examined by WST-1 assay. Glyburide at concentrations of 100–1000 μM induced [Ca²⁺]_i rises. Ca²⁺ removal reduced the signal by approximately 60%. In Ca²⁺-containing medium, glyburide-induced Ca²⁺ entry was inhibited by 60% by protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate, PMA) and inhibitor (GF109203X), and modulators of store-operated Ca²⁺ channels (nifedipine, econazole and SKF96365). Furthermore, glyburide induced Mn²⁺ influx suggesting of Ca²⁺ entry. In Ca²⁺-free medium, inhibition of phospholipase C (PLC) with U73122 significantly inhibited glyburide-induced [Ca²⁺]_i rises. Treatment with the endoplasmic reticulum (ER) Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) abolished glyburide-evoked [Ca²⁺]_i rises. Conversely, treatment with glyburide abolished BHQ-evoked [Ca²⁺]_i rises. Glyburide at 100–500 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, glyburide induced [Ca²⁺]_i rises by Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels and Ca²⁺ release from the ER in a PLC-dependent manner. Glyburide also caused Ca²⁺-independent cell death. This study suggests that glyburide could serve as a potential agent for treatment of prostate cancer.     

Effects of timolol on Ca2+ handling and viability in human prostate cancer cells

Timolol is a medication used widely to treat glaucoma. Regarding Ca²⁺ signaling, timolol was shown to modulate Ca²⁺-related physiology in various cell types, however, the effect of timolol on Ca²⁺ homeostasis and cell viability has not been explored in human prostate cancer cells. The aim of this study was to explore the effect of timolol on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Timolol at concentrations of 100–1000?μM induced [Ca²⁺]_i rises. The Ca²⁺ signal in Ca²⁺-containing medium was reduced by removal of extracellular Ca²⁺ by approximately 75%. Timolol (1000?μM) induced Mn²⁺ influx suggesting of Ca²⁺ entry. Timolol-induced Ca²⁺ entry was partially inhibited by three inhibitors of store-operated Ca²⁺ channels: nifedipine, econoazole and SKF96365, and by a protein kinase C (PKC) activator (phorbol 12-myristate 13 acetate [PMA]) or an inhibitor (GF109203X). In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished timolol-evoked [Ca²⁺]_i rises. Conversely, treatment with timolol abolished thapsigargin-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 abolished timolol-induced [Ca²⁺]_i rises. Timolol at concentrations between 200 and 600?μM killed cells in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not reverse cytotoxicity of timolol. Together, in PC3 cells, timolol induced [Ca²⁺]_i rises by evoking Ca²⁺release from the endoplasmic reticulum in a PLC-dependent manner, and Ca²⁺ influx via PKC-regulated store-operated Ca²⁺ entry. Timolol also caused cell death that was not linked to preceding [Ca²⁺]_i rises.     

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

The 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.     

Effects of sphingosine-1-phosphate and sphingosylphosphorylcholine on intracellular Ca2+ and cell death in prostate cancer cell lines

The sphingolipid metabolites sphingosine-1-phosphate (S1P) and sphingosylphosphorylcholine (SPC) can be involved in cellular growth and apoptosis, by both receptor-dependent and -independent mechanisms. We investigated the role of S1P and SPC in intracellular Ca2+ elevation, cell proliferation and cell death in DU 145 and PC3 hormone-refractory prostate cancer cell lines. S1P and SPC increased intracellular Ca2+ levels, most likely in a receptor-independent manner. Surprisingly, both S1P and SPC did not stimulate but rather reduced cell growth through induction of apoptosis. Therefore, antagonists targeted against S1P, SPC and their receptors do not appear to be promising new approaches in the treatment of hormone-refractory prostate cancer.     

组织和细胞中Ca~(2+)浓度测定方法的研究进展

组织和细胞内的Ca2+与信号传导以及生理病理应答反应具有重要的联系,因此测定其含量及动态变化具有重要的研究意义。近年来Ca2+的测定技术和相关机制研究在国内外发展迅速,包括45Ca跨膜流动测定方法、X射线微区分析方法、离子选择微电极方法、核磁共振波谱方法、原子吸收分光光度法测定方法、荧光探针方法等,该文就相关方面介绍这些方法的特点和主要应用情况。     

人参皂苷Rg_2对培养心肌细胞内游离Ca~(2+)含量的影响

众所周知 ,Ca2 + 在心肌的舒缩过程中具有调控作用 ,文献[1] 曾研究了人参皂苷Rg2 对休克动物心功能的改善作用 ,该作用是否与Ca2 + 有关 ,故观察了Rg2 对体外培养心肌细胞内游离Ca2 + 含量的影响。1　材料与方法　　药物 :人参皂苷Rg2 (Rg2 )由吉林省中医中药研究院制备 ,纯度为 98 5 % ,Fura 2 /AM和DME/F12培养基购自Sigma公司。动物 :新生 4d的Wistar大鼠乳鼠 ,由中山医科大学实验动物中心提供。心肌细胞培养参照文献[2 ] 进行 ,取乳鼠心肌 ,用胰蛋白酶反复消化 ,制成单细胞悬液。用DME/F12培养液调细胞浓度 ,接种培养皿 ,形成…     

Hydroxylated PCB induces Ca2+ oscillations and alterations of membrane potential in cultured cortical cells

Polychlorinated biphenyls (PCBs) are known as environmental pollutants that may cause adverse health effects. Although some congeners have been shown to affect brain development or function, the molecular mechanisms mediating their toxicity are not yet fully understood. Since signal transduction via intracellular Ca²⁺ is crucial for neuronal development and plasticity, we investigated the effect of PCBs on Ca²⁺ homeostasis and membrane potential in cultured mouse cortical cells. Acute exposure to hydroxylated PCB 106 [4(OH)‐2′,3,3′,4′,5′‐pentachlorobiphenyl, OH‐PCB 106, 0.1 μm ] caused recurring Ca²⁺ oscillations that were classified into three prototypes. Although extracellular Ca²⁺ deprivation significantly reduced the oscillations, 54% of the cells still showed different patterns of oscillations or gradual increase in the intracellular Ca²⁺ concentration, indicating possible involvement of multiple Ca²⁺ channels in a cell‐specific manner. Such a possibility was further confirmed by differential responses to several channel/receptor blockers, including nifedipine, ryanodine, xestospongine and tetrodotoxin. Although all chemicals had partial inhibition action in different subsets of neurons, nifedipine blocked the OH‐PCB 106 action in the largest subpopulation of cells and with the greatest magnitude. Ryanodine also blocked the action with a similar magnitude, but in a smaller subpopulation of cells. Moreover, OH‐PCB 106 induced depolarization of the plasma membrane in all the recorded cells. Taken together, our results indicate that OH‐PCB 106 alters membrane potential as well as Ca²⁺ dynamics in part by inducing extracellular influx and/or intracellular release of Ca²⁺. These mechanisms may be responsible for their neurotoxicity. Copyright © 2009 John Wiley & Sons, Ltd.     

Capacitative Ca2+ entry associated with α1-adrenoceptors in rat aorta

In rat aorta, depletion of internal Ca²⁺ stores by addition of noradrenaline (1μM) induces a biphasic response (an initial phasic response and a tonic one) mediated by two different intracellular Ca²⁺ pools. This response cannot be repeated, suggesting a depletion of internal Ca²⁺ stores sensitive to noradrenaline. In absence of the agonist, this depletion is the signal for the entry of extracellular Ca²⁺, not only to refill the stores but also, under our experimental conditions, to activate the contractile proteins thus inducing an increase in the resting tone (IRT) that constitutes functional evidence of this Ca²⁺ entry. The ionic channels involved in the mechanism of the IRT have been studied in the present work. The fact that the addition of nimodipine (10^–15– 10^–11M) selectively inhibits the IRT suggests that this mechanical response is mediated by Ca²⁺ influx through dihydropyridine-sensitive Ca²⁺ channels. Moreover, the inhibitory action of nimodipine is attenuated by glibenclamide (10μM). Cromakalim (10^–10–10^–6M) also inhibits the IRT concentration dependently, and this inhibition is antagonized by glibenclamide (10μM). These results relate the ATP-dependent K⁺ channels to the mechanism of the IRT. The refilling of the two internal Ca²⁺ compartments sensitive to noradrenaline was, like the IRT, altered in presence of the compounds tested, since the subsequent contractile response to noradrenaline was decreased. The present results suggest that nimodipine treatment inhibits the refilling of the Ca²⁺ compartment responsible for the tonic contraction induced by noradrenaline in Ca²⁺-free medium, whereas the refilling of the Ca²⁺ pool responsible for the phasic response to noradrenaline remained unaltered. Both the phasic and tonic responses to noradrenaline in Ca²⁺-free medium decreased after treatment with cromakalim. We can therefore assume that the refilling of both Ca²⁺ compartments sensitive to noradrenaline was inhibited. In conclusion, these results are consistent with the contraction of the rat aorta in response to noradrenaline in Ca²⁺-free medium consisting of an initial phasic response and a tonic one. The former is due to the release of internal Ca²⁺ from a compartment refilled through a special channel that is cromakalim but not dihydropyridine sensitive. The tonic response is due to Ca²⁺ release from another compartment refilled through a cromakalim- and dihydropyridine-sensitive Ca²⁺ channel. The Ca²⁺ entry through this latter channel intervenes in the IRT observed during the refilling of these stores previously depleted by noradrenaline, and the opening state of this channel is also modulated by ATP-dependent K²⁺ channels. Received: 7 May 1996 / Accepted: 30 January 1997