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 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.     

Effect of the Anti‐Breast Cancer Drug Tamoxifen on Ca2+ Movement in Human Osteosarcoma Cells

Abstract:The anti‐breast cancer drug tamoxifen has recently been shown to cause an increase in [Ca²⁺]_i in renal tubular cells, breast cells and bladder cells. Because tamoxifen is known to interact with oestrogens leading to modulation of bone metabolism, the present study was aimed at exploring whether tamoxifen could alter Ca²⁺ signaling in human osteoblast‐like MG63 cells. Cytosolic free Ca²⁺ levels were recorded by using the Ca²⁺‐sensitive dye fura‐2. Tamoxifen induced a sustained [Ca²⁺]_i increase at concentrations above 1 μM with an EC₅₀ of 8 μM. Removal of extracellular Ca²⁺ reduced the response by 40%, suggesting that tamoxifen induced both Ca²⁺ influx and store Ca²⁺ release. Tamoxifen‐induced Ca²⁺ influx was confirmed as tamoxifen caused Mn²⁺ influx‐induced quench of fura‐2 fluorescence. In Ca²⁺‐free medium, pretreatment with 10 μM tamoxifen abolished the [Ca²⁺]_i increase induced by 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), and by 2 μM carbonylcyanide m‐chlorophenylhydrazone (a mitochondrial uncoupler). Conversely, pretreatment with thapsigargin and carbonylcyanide m‐chlorophenylhydrazone only reduced 64% of tamoxifen‐induced [Ca²⁺]_i increases. Addition of 2 μM U73122 to inhibit phospholipase C activity abolished the [Ca²⁺]_i increase induced by 1 μM histamine, a phospholipase C‐dependent Ca²⁺ mobilizer, without affecting 10 μM tamoxifen‐induced Ca²⁺ release. The [Ca²⁺]_i increase induced by 10 μM tamoxifen was not altered by 10 μM of nifedipine, verapamil and diltiazem. Together, the data show that tamoxifen induced a lasting increase in [Ca²⁺]_i in human osteoblast‐like cells by causing Ca²⁺ influx and releasing Ca²⁺ from multiple stores in a phospholipase C‐independent manner.     

Effect of MK‐886 on Ca2+ Level and Viability in PC3 Human Prostate Cancer Cells

Abstract: 3‐[1‐(p‐chlorobenzyl)‐5‐(isopropyl)‐3‐tert‐butylthioindol‐2‐yl]‐2, 2‐dimethylpropanoic acid (MK‐886) is widely used for inhibition of leucotriene synthesis in in vitro studies, however, many of its other effects have been reported. The present study investigated the effect of MK‐886 on cytosolic‐free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in human PC3 prostate cancer cells. [Ca²⁺]_i in suspended cells was measured by using fura‐2. MK‐886 at concentrations of 1 µM and above increased [Ca²⁺]_i in a concentration‐dependent manner with an EC₅₀ value of 20 µM. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. MK‐886 evoked Mn²⁺ quenching of fura‐2 fluorescence, implicating Ca²⁺ entry. MK‐886‐induced Ca²⁺ influx was inhibited by store‐operated Ca²⁺ entry inhibitors nifedipine, econazole and SKF96365. In Ca²⁺‐free medium, after pre‐treatment with 10 µM MK‐886, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pre‐treatment abolished MK‐886‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter MK‐886‐induced [Ca²⁺]_i rises. MK‐886 at concentrations of 1–100 µM concentration‐dependently decreased cell viability with an IC₅₀ value of 60 µM. The cytotoxic effect of MK‐886 was not inhibited by pre‐chelating cytosolic Ca²⁺ with BAPTA/AM. Together, in PC3 cells, MK‐886 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via store‐operated Ca²⁺ channels. Independently, MK‐886 was cytotoxic to cells in a Ca²⁺‐independent manner.     

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.     

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 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.     

Effect of melamine on [Ca2+]i and viability in PC3 human prostate cancer cells

Melamine is thought to be an endocrine disrupter that affects physiology in cells. This study examined the effect of melamine on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. Melamine evoked [Ca²⁺]_i rises concentration-dependently. Melamine-evoked Ca²⁺ entry was inhibited by nifedipine, econazole, SKF96365, GF109203X and phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin inhibited melamine-evoked [Ca²⁺]_i rise. Conversely, treatment with melamine abolished thapsigargin-evoked [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter melamine-evoked [Ca²⁺]_i rise. Melamine at 500–800 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). Collectively, our data suggest that in PC3 cells, melamine induced [Ca²⁺]_i rises by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ entry via protein kinase C-regulated store-operated Ca²⁺ entry. Melamine also caused Ca²⁺-independent cell death.     

Effect of clomiphene on [Ca2+]i rises and cell viability in rabbit corneal epithelial cells

The effect of clomiphene a first‐line therapy for WHO group II (eu‐estrogenic) infertility on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability has not been explored in rabbit corneal epithelial cells (SIRC). This study examined whether clomiphene altered [Ca²⁺]_i levels and caused cell death in SIRC cells. [Ca²⁺]_i and cell viability were measured using the fluorescent dyes fura‐2 and WST‐1, respectively. Clomiphene at concentrations ≥5 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The clomiphene‐induced Ca²⁺ influx was insensitive to blockade of L‐type Ca²⁺ channel blockers. In Ca²⁺‐free medium, after pretreatment with 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), clomiphene failed to increase [Ca²⁺]_i. Inhibition of phospholipase C with 2 µM U73122 did not change clomiphene‐induced [Ca²⁺]_i rises. At concentrations of 0.5–20 µM, clomiphene killed cells in a concentration‐dependent manner. The cytotoxic effect of 15 µM clomiphene was not reversed by prechelating cytosolic Ca²⁺ with BAPTA/AM. Collectively, in SIRC cells, clomiphene‐induced [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from non‐L‐type Ca²⁺ channels. Clomiphene‐caused cytotoxicity was not mediated by a preceding [Ca²⁺]_i rise. Drug Dev Res 69:272–278, 2008 ©2008 Wiley‐Liss, Inc.     

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.     

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 capsaicin on Ca2+ fluxes in Madin‐Darby canine renal tubular cells

The effect of capsaicin, a transient receptor potential vanniloid‐1 (TRPV1) receptor agonist, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin Darby canine kidney (MDCK) cells is unclear. This study explored whether capsaicin changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura‐2 as a Ca²⁺‐sensitive fluorescent dye. Capsaicin at concentrations between 10–100 µM increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced by 80% by removing extracellular Ca²⁺. Capsacin induced Mn²⁺ influx, leading to quench of fura‐2 fluorescence suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid and the non‐selective Ca²⁺ entry blocker La³⁺, but not by store‐operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, and protein kinase C/A modulators. In Ca²⁺‐free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished capsaicin‐induced Ca²⁺ release. Conversely, pretreatment with capsaicin partly reduced thapsigargin‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 did not alter capsaicin‐induced [Ca²⁺]_i rise. The TRPV1 receptor antagonist capsazepine also induced significant Ca²⁺ entry and Ca²⁺ release. Collectively, in MDCK cells, capsaicin induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2‐regulated, La³⁺‐sensitive Ca²⁺ channels in a manner dissociated from stimulation of TRPV1 receptors. Drug Dev Res, 2009. © 2009 Wiley‐Liss, Inc.     

L‐type Ca2+ channel opener BayK 8644‐induced Ca2+ influx and Ca2+ release in human oral cancer cells (OC2)

The effect of BayK 8644, a chemical widely used to activate L‐type Ca²⁺ channels, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in human oral cancer cells (OC2) has not been explored to date. The present study examined whether BayK 8644 altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura‐2. BayK 8644 (10 pM–10 µM) increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. BayK 8644‐induced Ca²⁺ influx was blocked by nifedipine, but was not altered by the store‐operated Ca²⁺ entry inhibitors, econazole and SKF96365; protein kinase C modulators phorbol 12‐myristate 13‐acetate (PMA) and GF109203X; the protein kinase A inhibitor H89; and the phospholipase A₂ inhibitor, aristolochic acid. In Ca²⁺‐free medium, after pretreatment with 1 µM BayK 8644, 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; and conversely, thapsigargin pretreatment abolished BayK 8644‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change BayK 8644‐induced [Ca²⁺]_i rises. Collectively, in OC2 cells, BayK 8644 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum; and Ca²⁺ influx via L‐type Ca²⁺ channels. Drug Dev Res 69: 2008. © 2008 Wiley‐Liss, Inc.     

Inhibition of thrombin-induced Ca²⁺ influx in platelets by R59949, an inhibitor of diacylglycerol kinase

Objectives The aim of this study was to determine whether diacylglycerol kinase (DGK) is involved in transplasmalemmal Ca²⁺ influx of platelets. Methods Effects of R59949, an inhibitor of diacylglycerol kinase, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) and mRNA expression of DGK isozymes were investigated using washed human platelet suspensions. Key findings Thrombin‐induced increase in [Ca²⁺]_i was significantly inhibited by pretreatment of platelets with R59949, while thapsigargin‐induced increase in [Ca²⁺]_i was comparable in platelets with and without R59949 pretreatment. Thapsigargin‐induced increase in [Ca²⁺]_i was markedly attenuated in the presence of SKF‐96365. In the presence of SKF‐96365, thrombin‐induced increase in [Ca²⁺]_i was significantly attenuated, and additional treatment with R59949 caused a further decrease in [Ca²⁺]_i. Pretreatment of platelets with 1‐butanol significantly attenuated thrombin‐induced increase in [Ca²⁺]_i, while thrombin‐induced increase in [Ca²⁺]_i was augmented in the presence of propranolol. mRNA expression of DGK‐α and DGK‐γ, which are known to be inhibited by R59949, in platelets was confirmed by RT‐PCR analysis. Conclusions R59949 inhibited a store‐depletion‐insensitive component of transplasmalemmal Ca²⁺ entry induced by thrombin, while store‐operated Ca²⁺ entry was not affected by R59949. The results of this study suggest that phosphatidic acid is involved in thrombin‐induced Ca²⁺ influx of platelets.     

3,3'-Diindolylmethane alters Ca2+ homeostasis and viability in MG63 human osteosarcoma cells

Abstract: The effect of the natural product 3,3′‐diindolylmethane (DIM) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells was explored. The Ca²⁺‐sensitive fluorescent dye fura‐2 was applied to measure [Ca²⁺]_i. DIM at concentrations of 40–80 μM induced a [Ca²⁺]_i rise in a concentration‐dependent manner. The response was reduced partly by removing Ca²⁺. DIM‐evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited or abolished DIM‐induced [Ca²⁺]_i rise. Incubation with DIM also inhibited thapsigargin or BHQ‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished DIM‐induced [Ca²⁺]_i rise. At concentrations of 10–50 μM, DIM killed cells in a concentration‐dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data implicate that DIM (20 and 40 μM) induced apoptosis in a concentration‐dependent manner. In sum, in MG63 cells, DIM induced a [Ca²⁺]_i rise by evoking phospholipase C‐dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C‐sensitive store‐operated Ca²⁺ channels. DIM caused cell death that may involve apoptosis.     

Effects of MK‐886, a leukotriene synthesis inhibitor,on [Ca2+]i and apoptosis in MG63 human osteosarcoma cells

The effect of MK‐886 (3‐[1‐(p‐chlorobenzyl)‐5‐(isopropyl)‐3‐tert‐butylthioindol‐2‐yl]‐2, 2‐dimethylpropanoic acid), a compound widely used to inhibit leukotriene synthesis, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in osteosarcoma cells has not been explored. This study examined whether MK‐886 altered [Ca²⁺]_i levels in suspended MG63 human osteosarcoma cells using fura‐2. MK‐886 at 0.1 μM and above increased [Ca²⁺]_i in a concentration‐dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. MK‐886 induced Mn²⁺ quenching of fura‐2 fluorescence, implicating Ca²⁺ entry. MK‐886‐induced Ca²⁺ influx was inhibited by store‐operated Ca²⁺ entry inhibitors, nifedipine, econazole, and SKF96365; and by the protein kinase C modulators, phorbol 12‐myristate 13‐acetate (PMA) and GF109203X. In Ca²⁺‐free medium, after pretreatment with 5 μM MK‐886, 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)‐induced [Ca²⁺]_i rises were abolished; conversely, thapsigargin pretreatment nearly abolished MK‐886‐induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not change MK‐886‐induced [Ca²⁺]_i rises. Collectively, in MG63 osteosarcoma cells, MK‐886 induced [Ca²⁺]_i rises by causing phospholipase C‐independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via protein kinase C‐regulated store‐operated Ca²⁺ entry. Drug Dev Res 69: 49–57, 2008. © 2008 Wiley‐Liss, Inc.     

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

The 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.     

Novel effects of a sleep‐inducing lipid,oleamide, on Ca2+ signaling in renal tubular cells

The effect of oleamide, a sleep‐inducing endogenous lipid in animal models, on intracellular free levels of Ca²⁺ ([Ca²⁺]_i) in Madin‐Darby renal tubular cells was examined using fura‐2 as a fluorescent dye. Oleamide (5–50 μM) increased [Ca²⁺]_i in a concentration‐dependent fashion with an EC₅₀ value of 20 μM. The [Ca²⁺]_i signal comprised an initial rise and an elevated phase and was reduced by removing extracellular Ca²⁺ by 50%. After pretreatment with 5–50 μM oleamide in Ca²⁺‐free medium, addition of 3 mM Ca²⁺ increased [Ca²⁺]_i in a manner dependent on the concentration of oleamide. In Ca²⁺‐free medium, pretreatment with thapsigargin (1 μM), an endoplasmic reticulum Ca²⁺ pump inhibitor, abolished [Ca²⁺]_i increases induced by 20 μM oleamide; conversely, pretreatment with 20 μM oleamide reduced 1 μM thapsigargin‐induced [Ca²⁺]_i increases by 50%. Suppression of the activity of phospholipase C with 2 μM U73122 abolished 20 μM oleamide‐induced Ca²⁺ release. Collectively, these data demonstrate that oleamide induced significant [Ca²⁺]_i increases in renal tubular cells by a phospholipase C‐dependent release of Ca²⁺ from thapsigargin‐sensitive stores and by inducing Ca²⁺ entry via store‐operated Ca²⁺ entry. Drug Dev. Res. 54:40–44, 2001. © 2001 Wiley‐Liss, Inc.     

Lindane (γ‐Hexachlorocyclohexane) Induces Internal Ca2+ Release and Capacitative Ca2+ Entry in Madin‐Darby Canine Kidney Cells

The effect of lindane (γ‐hexachlorocyclohexane), an organochlorine pesticide, on Ca²⁺ mobilization in Madin‐Darby canine kidney cells was examined by fluorimetry using fura‐2 as a Ca²⁺ indicator. Lindane (5–200 μM) increased [Ca²⁺]_i concentration‐dependently. The [Ca²⁺]_i signal comprised an immediate initial rise followed by a persistent phase. Ca²⁺ removal inhibited the [Ca²⁺]_i signal by reducing both the initial rise and the sustained phase. This implies lindane‐triggered Ca²⁺ influx and Ca²⁺ release. In Ca²⁺‐free medium, 0.15 mM lindane increased [Ca²⁺]_i after pretreatment with carbonylcyanide m‐chlorophenylhydrazone (CCCP; 2 μM), a mitochondrial uncoupler, and two endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin and cyclopiazonic acid. Conversely, pretreatment with lindane abolished CCCP‐ and thapsigargin‐induced Ca²⁺ release. This suggests that 0.15 mM lindane released Ca²⁺ from the endoplasmic reticulum, mitochondria and other stores. La³⁺ (1 mM) partly inhibited 0.1 mM lindane‐induced [Ca²⁺]_i increase, confirming that lindane induced Ca²⁺ influx. Addition of 3 mM Ca²⁺ increased [Ca²⁺]_i after pretreatment with 0.15 mM lindane for 750 sec. in Ca²⁺‐free medium, which indicates lindane‐induced capacitative Ca²⁺ entry. Lindane (0.15 mM)‐induced Ca²⁺ release was not reduced by inhibiting phospholipase C with 2 μM U73122, but was inhibited by 70% by the phospholipase A₂ inhibitor aristolochic acid (40 μM).