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.     

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 diindolylmethane on Ca(2+) movement and viability in HA59T human hepatoma cells

The effect of diindolylmethane, a natural compound derived from indole-3-carbinol in cruciferous vegetables, on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in HA59T human hepatoma cells is unclear. This study explored whether diindolylmethane changed [Ca²⁺]_i in HA59T cells. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Diindolylmethane at concentrations of 1?C50???M evoked a [Ca²⁺]_i rise in a concentration-dependent manner. The signal was reduced by removing Ca²⁺. Diindolylmethane-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, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca²⁺]_i rise. Incubation with diindolylmethane inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca²⁺]_i rise. At concentrations of 10?C75???M, diindolylmethane killed cells in a concentration-dependent manner. The cytotoxic effect of diindolylmethane was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N??,N??-tetraacetic acid. Propidium iodide staining data suggest that diindolylmethane (25?C50???M) induced apoptosis in a concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane induced a [Ca²⁺]_i rise by causing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A₂-sensitive channels. Diindolylmethane induced cell death that may involve apoptosis.     

Effects of Antrodia camphorata on viability,apoptosis, [Ca2+]i,and MAPKs phosphorylation in MG63 human osteosarcoma cells

The present study explored the effect of Antrodia camphorata (AC) on viability, apoptosis, mitogen‐activated protein kinases (MAPKs) phosphorylation, and Ca²⁺ regulation in MG63 human osteosarcoma cells. AC (25–50 µg/ml) did not affect cell viability, but at 100–200 µg/ml decreased viability and induced apoptosis in a concentration‐dependent manner. AC at concentrations of 25–200 µg/ml did not alter basal [Ca²⁺]_i, but at 25 µg/ml decreased [Ca²⁺]_i increases induced by ATP, bradykinin, histamine, and thapsigargin. ATP, bradykinin, and histamine increased cell viability while thapsigargin decreased it. AC (25 µg/ml) pretreatment failed to alter bradykinin‐ and thapsigargin‐induced effects on viability, but potentiated ATP‐ and histamine‐induced increases in viability. Immunoblotting showed that MG63 cells did not have background phospho‐JNK and phospho‐p38 mitogen‐activated protein kinases (MAPKs); and AC did not induce the phosphorylation of these two MAPKs. Conversely, the cells had significant background phospho‐ERK MAPK that was inhibited by 200 µg/ml AC. The ERK‐specific inhibitor PD98059 also induced cell death. Collectively, in MG63 cells, AC exerted multiple effects on viability and [Ca²⁺]_i, caused apoptosis probably via inhibition of ERK MAPK phosphorylation. Drug Dev Res 68:71–78, 2007. © 2007 Wiley‐Liss, Inc.     

Octyl Gallate Inhibits ATP-induced Intracellular Calcium Increase in PC12 Cells by Inhibiting Multiple Pathways

Phenolic compounds affect intracellular free Ca²⁺ concentration ([Ca²⁺]_i) signaling. The study examined whether the simple phenolic compound octyl gallate affects ATP-induced Ca²⁺ signaling in PC12 cells using fura-2-based digital Ca²⁺ imaging and whole-cell patch clamping. Treatment with ATP (100 µM) for 90 s induced increases in [Ca²⁺]_i in PC12 cells. Pretreatment with octyl gallate (100 nM to 20 µM) for 10 min inhibited the ATP-induced [Ca²⁺]_i response in a concentration-dependent manner (IC₅₀=2.84 µM). Treatment with octyl gallate (3 µM) for 10 min significantly inhibited the ATP-induced response following the removal of extracellular Ca²⁺ with nominally Ca²⁺-free HEPES HBSS or depletion of intracellular Ca²⁺ stores with thapsigargin (1 µM). Treatment for 10 min with the L-type Ca²⁺ channel antagonist nimodipine (1 µM) significantly inhibited the ATP-induced [Ca²⁺]_i increase, and treatment with octyl gallate further inhibited the ATP-induced response. Treatment with octyl gallate significantly inhibited the [Ca²⁺]_i increase induced by 50 mM KCl. Pretreatment with protein kinase C inhibitors staurosporin (100 nM) and GF109203X (300 nM), or the tyrosine kinase inhibitor genistein (50 µM) did not significantly affect the inhibitory effects of octyl gallate on the ATP-induced response. Treatment with octyl gallate markedly inhibited the ATP-induced currents. Therefore, we conclude that octyl gallate inhibits ATP-induced [Ca²⁺]_i increase in PC12 cells by inhibiting both non-selective P2X receptor-mediated influx of Ca²⁺ from extracellular space and P2Y receptor-induced release of Ca²⁺ from intracellular stores in protein kinase-independent manner. In addition, octyl gallate inhibits the ATP-induced Ca²⁺ responses by inhibiting the secondary activation of voltage-gated Ca²⁺ channels.     

Cyanidin-3-glucoside Inhibits ATP-induced Intracellular Free Ca2+ Concentration,ROS Formation and Mitochondrial Depolarization in PC12 Cells

Flavonoids have an ability to suppress various ion channels. We determined whether one of flavonoids, cyanidin-3-glucoside, affects adenosine 5''-triphosphate (ATP)-induced calcium signaling using digital imaging methods for intracellular free Ca²⁺ concentration ([Ca²⁺]_i), reactive oxygen species (ROS) and mitochondrial membrane potential in PC12 cells. Treatment with ATP (100µM) for 90 sec induced [Ca²⁺]_i increases in PC12 cells. Pretreatment with cyanidin-3-glucoside (1µ g/ml to 100µg/ml) for 30 min inhibited the ATP-induced [Ca²⁺]_i increases in a concentration-dependent manner (IC₅₀=15.3µg/ml). Pretreatment with cyanidin-3-glucoside (15µg/ml) for 30 min significantly inhibited the ATP-induced [Ca²⁺]_i responses following removal of extracellular Ca²⁺ or depletion of intracellular [Ca²⁺]_i stores. Cyanidin-3-glucoside also significantly inhibited the relatively specific P2X2 receptor agonist 2-MeSATP-induced [Ca²⁺]_i responses. Cyanidin-3-glucoside significantly inhibited the thapsigargin or ATP-induced store-operated calcium entry. Cyanidin-3-glucoside significantly inhibited the ATP-induced [Ca²⁺]_i responses in the presence of nimodipine and ω-conotoxin. Cyanidin-3-glucoside also significantly inhibited KCl (50 mM)-induced [Ca²⁺]_i increases. Cyanidin-3-glucoside significantly inhibited ATP-induced mitochondrial depolarization. The intracellular Ca²⁺ chelator BAPTA-AM or the mitochondrial Ca²⁺ uniporter inhibitor RU360 blocked the ATP-induced mitochondrial depolarization in the presence of cyanidin-3-glucoside. Cyanidin-3-glucoside blocked ATP-induced formation of ROS. BAPTA-AM further decreased the formation of ROS in the presence of cyanidin-3-glucoside. All these results suggest that cyanidin-3-glucoside inhibits ATP-induced calcium signaling in PC12 cells by inhibiting multiple pathways which are the influx of extracellular Ca²⁺ through the nimodipine and ω-conotoxin-sensitive and -insensitive pathways and the release of Ca²⁺ from intracellular stores. In addition, cyanidin-3-glucoside inhibits ATP-induced formation of ROS by inhibiting Ca²⁺-induced mitochondrial depolarization.     

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.     

Mechanisms of 5-hydroxytryptamine-induced inhibition in the porcine myometrium

1 The present experiments were designed to clarify the mechanisms of the inhibitory response of 5-hydroxytryptamine (5-HT) in the porcine uterine circular muscle. 2 Inhibitory responses induced by 5-HT (1 n m –1 μm ) were not affected by apamin (1 μm ), charybdotoxin (100 n m ) or glibenclamide (20 μm ) but were significantly attenuated by 4-aminopyridine (3 m m ) and tetraethylammonium (3 m m ). 3 Imidazole (100 μm ) decreased but 3-isobutyl-1-methylxanthine (30 μm ), milrinone (30 μm ) and Ro 20–1724 (10 and 30 μm ) potentiated the 5-HT-induced inhibition. On the other hand, zaprinast (3–30 μm ) had no significant effect on the inhibitory response of 5-HT. 4 5-HT caused a time (0–5 min)-and concentration (1 n m –1 μm )-dependent increase in the tissue cyclic AMP level, but had no effect on the tissue cyclic GMP level. A significant correlation (P < 0.05) was observed between the inhibition of contraction and tissue cyclic AMP level. 5 The effect of 5-HT on contractile force and cytosolic Ca²⁺ level ([Ca²⁺]_i) was investigated using fura-PE3-loaded myometrial strips. A low concentration of 5-HT (≤ 10 n m ) inhibited the spontaneous contraction without changing the amplitude of the spontaneous [Ca²⁺]_i increase, but a higher concentration of 5-HT (≥ 100 n m ) decreased the resting [Ca²⁺]_i and inhibited both the spontaneous [Ca²⁺]_i increase and spontaneous contraction. 6 High-K⁺ (50 m m ) caused increases in muscle contractile force and [Ca²⁺]_i. 5-HT concentration-dependently inhibited the high-K⁺-induced contraction (EC₅₀, 45 n m ) with only a small decrease in [Ca²⁺]_i increase. 7 Carbachol also caused increases in muscle contractile force and [Ca²⁺]_i. 5-HT significantly decreased both the carbachol-induced contraction and [Ca²⁺]_i increase, but was more potent at inhibition of contractile force than [Ca²⁺]_i. 8 In Ca²⁺ -loaded myometrial strips, carbachol, but not caffeine, caused a transient increase in [Ca²⁺]_i and contraction in the absence of external Ca²⁺ (EGTA, 1 m m ). 5-HT inhibited both the carbachol-induced increases in [Ca²⁺]_i release and contractile force. 9 In the β-escin permeabilized myometrium, 5-HT significantly inhibited the Ca²⁺-induced contraction. 10 The present results indicate that 5-HT stimulates tissue cyclic AMP production, and inhibits the porcine uterine muscle contractility by a reduction in [Ca²⁺]_i and in Ca²⁺ sensitivity of the contractile elements. Activation of K⁺ channels might be partially involved in 5-HT-induced inhibition of the myometrial contractility.     

Nonylphenol‐induced cytosolic Ca2+ elevation and death in renal tubular cells

Nonylphenol is an environmental endocrine disrupter. The effect of nonylphenol on intracellular free Ca²⁺ levels ([Ca²⁺]_i) and viability in Madin‐Darby canine kidney (MDCK) cells was explored. Nonylphenol increased [Ca²⁺]_i in a concentration‐dependent manner (EC₅₀～0.8 μM). Nonylphenol‐induced Mn²⁺ entry demonstrated Ca²⁺ influx and removal of extracellular Ca²⁺ partly decreased the [Ca²⁺]_i rise. The [Ca²⁺]_i rise was inhibited by the protein kinase C activator, phorbol 13‐myristate acetate (PMA) but not by L‐type Ca²⁺ channel blockers. In Ca²⁺‐free medium, nonylphenol‐induced [Ca²⁺]_i rise was partly inhibited by pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor). Conversely, nonylphenol pretreatment abolished thapsigargin‐induced Ca²⁺ release. Nonylphenol‐induced Ca²⁺ release was unaltered by inhibition of phospholipase C. At concentrations of 5–100 μM, nonylphenol killed cells in a concentration‐dependent manner. The cytotoxic effect of 100 μM nonylphenol was not affected by preventing [Ca²⁺]_i rises with BAPTA/AM. Collectively, this study shows that nonylphenol induced [Ca²⁺]_i increase in MDCK cells via evoking Ca²⁺ entry through protein kinase C‐regulated Ca²⁺ channels, and releasing Ca²⁺ from endoplasmic reticulum and other stores in a phospholipase C‐independent manner. Nonylphenol also killed cells in a Ca²⁺‐independent fashion. Drug Dev Res, 2009. © 2009 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).     

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 Methoxychlor on Ca(2+) Movement and Viability in MDCK Renal Tubular Cells

The effect of the insecticide methoxychlor on the physiology of renal tubular cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in MDCK renal tubular cells using the Ca²⁺‐sensitive fluorescent dye fura‐2. Methoxychlor at 5–20 μM increased [Ca²⁺]_i in a concentration‐dependent manner. The signal was reduced by 80% by removing extracellular Ca²⁺. Methoxychlor‐induced Ca²⁺ entry was not affected by nifedipine and SK&F96365 but was inhibited by econazole and protein kinase C modulators. In Ca²⁺‐free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5‐di‐tert‐butylhydroquinone (BHQ) partly inhibited methoxychlor‐induced [Ca²⁺]_i rise. Incubation with methoxychlor also inhibited thapsigargin‐ or BHQ‐induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 nearly abolished methoxychlor‐induced [Ca²⁺]_i rise. At 5–15 μM, methoxychlor slightly increased cell viability, whereas at 20 μM, it decreased viability. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca²⁺ with 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N,N‐tetraacetic acid/AM (BAPTA/AM). Annexin V‐FITC data suggest that 10 μM methoxychlor inhibited apoptosis, while 20 μM methoxychlor enhanced apoptosis. Methoxychlor (10 and 20 μM) increased the production of reactive oxygen species. Together, in renal tubular cells, methoxychlor induced [Ca²⁺]_i rise by inducing phospholipase C‐dependent Ca²⁺ release from multiple stores and Ca²⁺ entry via protein kinase C‐ and econazole‐sensitive channels. Methoxychlor slightly enhanced or inhibited cell viability in a concentration‐dependent, Ca²⁺‐independent manner. Methoxychlor induced cell death that may involve apoptosis via mitochondrial pathways.     

Effect of thymol on Ca homeostasis and viability in human glioblastoma cells

The effect of the natural essential oil thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in human glioblastoma cells was examined. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Thymol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Thymol-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 thymol-induced [Ca²⁺]_i rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, thymol 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 thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca²⁺]_i rise by inducing phospholipase C- and protein kinase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via non store-operated Ca²⁺ channels. Thymol induced cell death that may involve apoptosis.     

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.     

2-Benzyloxybenzaldehyde inhibits formyl peptide-stimulated increase in intracellular Ca2+ in neutrophils mainly by blocking Ca2+ entry

2-Benzyloxybenzaldehyde (CCY1a) inhibited the formyl-Met-Leu-Phe (fMLP)-induced elevation of cytosolic [Ca²⁺] ([Ca²⁺]_i) in rat neutrophils. The late plateau phase, but not the initial Ca²⁺ spike, of the fMLP-induced [Ca²⁺]_i change was inhibited by CCY1a. In the absence of external Ca²⁺, CCY1a had no appreciable effect on either the fMLP- or cyclopiazonic acid (CPA)-induced [Ca²⁺]_i elevation. CCY1a failed to inhibit [Ca²⁺]_i changes induced by N-ethylmaleimide, GEA3162, ionomycin or sphingosine, but slightly inhibited the Ca²⁺ signals elicited by ATP or interleukin-8 (IL-8). In a classical Ca²⁺ readdition protocol, addition of CCY1a after cell activation strongly inhibited the [Ca²⁺]_i response to fMLP, whilst that to CPA was only slightly reduced. CCY1a nearly abrogated the fMLP-stimulated Mn²⁺ influx but was less effective on the CPA-induced response. CCY1a attenuated the levels of tyrosine-phosphorylated bands in the 70–85 kDa molecular mass range. CCY1a had no effect on the basal [Ca²⁺]_i level, the pharmacologically isolated plasma membrane Ca²⁺-ATPase activity or on the mitochondrial membrane potential. Thus, CCY1a blocks fMLP-induced Ca²⁺ entry into neutrophils probably by blocking the relevant Ca²⁺ channel directly or, alternatively, indirectly through the attenuation of tyrosine phosphorylation of some cellular proteins.     

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.     

Effect of protriptyline on [Ca2+]i and viability in MG63 human osteosarcoma cells

Tricyclic antidepressants (TCA) have been clinically prescribed in the auxiliary treatment of cancer patients. Although protriptyline, a type of TCA, was used primarily in the clinical treatment of mood disorders in cancer patients, the effect of protriptyline on physiology in human osteosarcoma is unknown. This study examined the effect of protriptyline on cytosolic free Ca^2+?concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells. Protriptyline between 50 and 250?μM evoked [Ca²⁺]_i rises concentration-dependently. Protriptyline induced influx of Mn²⁺, indirectly implicating Ca^2+?influx. Protriptyline-evoked Ca^2+?entry was inhibited by nifedipine by 20% but was not altered by econazole, SKF96365, GF109203X, and phorbol-12-myristate-13-acetate (PMA). In Ca²⁺-free medium, treatment with protriptyline inhibited the endoplasmic reticulum Ca^2+?pump inhibitor thapsigargin-evoked [Ca²⁺]_i rises. Conversely, treatment with thapsigargin inhibited 45% of protriptyline-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 failed to alter protriptyline-evoked [Ca²⁺]_i rises. Protriptyline at 50–250?μM decreased cell viability, which was not reversed by pretreatment with the Ca^2+?chelator 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, our data suggest that in MG63 cells, protriptyline induced [Ca²⁺]_i rises by evoking Ca^2+?release from the endoplasmic reticulum and other stores in a PLC-independent manner, and Ca^2+?entry via a nifedipine-sensitive Ca^2+?pathway. Protriptyline also caused Ca²⁺-independent cell death.     

Effect of bisphenol A on Ca2+ fluxes and viability in Madin-Darby canine renal tubular cells

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca²⁺]_i levels in suspended MDCK cells by using fura-2 as a Ca²⁺-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced, partly, by removing extracellular Ca²⁺. Bisphenol A induced Mn²⁺ influx, leading to quenching of fura-2 fluorescence, suggesting Ca²⁺ influx. This Ca²⁺ influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca²⁺ channel blockers nifedipine and SK&F96365, and protein kinase C inhibitor GF109203X. In Ca²⁺-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca²⁺ pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A–induced Ca²⁺ release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca²⁺]_i rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca²⁺-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca²⁺]_i rises by causing phospholipase C–dependent Ca²⁺ release from the endoplasmic reticulum and mitochondria and Ca²⁺ influx via phospholipase A2–, protein kinase C–sensitive, store-operated Ca²⁺ channels.     

Intracellular calcium concentrations in human bladder tumor cells could be increased by NPC‐14686, a novel antiinflammatory agent

NPC‐14686 (Fmoc‐L‐homophenylalanine), a novel antiinflammatory agent, increases intracellular Ca²⁺ concentrations ([Ca²⁺]_i] in T24 bladder tumor cells. Using fura‐2 as a Ca²⁺ probe, NPC‐14686 (10–200 μM) increased [Ca²⁺]_i in a concentration‐dependent manner. The [Ca²⁺]_i increase comprised an initial slow rise and a plateau over a time period of 5 min. Ca²⁺ removal partly inhibited the Ca²⁺ signals. In Ca²⁺‐free medium, pretreatment with 100 μM NPC‐14686 abolished the [Ca²⁺]_i increases induced by 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor] and 2 μM carbonylcyanide m‐chlorophenylhydrazone (a mitochondrial uncoupler). However, 100 μM NPC‐14686 still slightly increased [Ca²⁺]_i after Ca²⁺ stores had been depleted by pretreating with 2 μM CCCP and 1 μM thapsigargin. These results suggest that NPC‐14686 released Ca²⁺ from multiple pools. Adding 3 mM Ca²⁺ increased [Ca²⁺]_i in cells pretreated with 100 μM NPC‐14686 in Ca²⁺‐free medium, indicating that NPC‐14686 activated capacitative Ca²⁺ entry. Inhibiting formation of inositol‐1,4,5‐trisphosphate (IP₃] by blocking phospholipase C with 2 μM U73122 had little effect on NPC‐14686‐induced Ca²⁺ release. Activating protein kinase C with phorbol 12‐myristate 13‐acetate (PMA) significantly potentiated NPC‐14686‐induced [Ca²⁺]_i increase. NPC‐14686 (100 μM) also increased [Ca²⁺]_i in MDCK renal cells, BFTC bladder tumor cells, and MS‐1 endothelial cells. Together, the findings suggest that in T24 bladder tumor cells NPC‐14686 induced Ca²⁺ release followed by Ca²⁺ entry. The Ca²⁺ release was unlinked to IP₃ and the [Ca²⁺]_i signal could be modulated by protein kinase C. Drug Dev. Res. 50:147–152, 2000. © 2000 Wiley‐Liss, Inc.     

The mechanism of carvacrol-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in OC2 human oral cancer cells

Carvacrol is one of the main substances of essential oil which triggers intracellular Ca²⁺ mobilization and causes cytotoxicity in diverse cell models. However, the mechanism of carvacrol-induced Ca²⁺ movement and cytotoxicity is not fully understood. This study examined the effect of carvacrol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i), cell viability and apoptosis in OC2 human oral cancer cells. Carvacrol induced a [Ca²⁺]_i rise and the signal was reduced by removal of extracellular Ca²⁺. Carvacrol-induced Ca²⁺ entry was not altered by store-operated Ca²⁺ channel inhibitors and protein kinase C (PKC) activator, but was inhibited by a PKC inhibitor. In Ca²⁺ -free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited carvacrol-induced [Ca²⁺]_i rise. Conversely, incubation with carvacrol inhibited TG or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol-induced [Ca²⁺]_i rise. Carvacrol decreased cell viability, which was not reversed when cytosolic Ca²⁺ was chelated with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid-acetoxymethyl ester). Carvacrol-induced apoptosis and activation of reactive oxygen species (ROS) and caspase-3. Together, carvacrol induced a [Ca²⁺]_i rise by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive, non store-operated Ca²⁺ channels. Carvacrol-induced ROS- and caspase-3-associated apoptosis.