Effect of thymol on Ca²âº homeostasis and viability in PC3 human prostate cancer cells.

Thymol is a phenolic compound that affects physiology in different cell models. However, whether thymol affects Ca²âº homeostasis in prostate cancer cells is unknown. The action of this compound on cytosolic Ca²âº concentrations ([Ca²âº]i) and viability in PC3 human prostate cancer cells was explored. The results show that thymol at concentrations of 100-1500 µM caused [Ca²âº]i rises in a concentration-dependent manner. Removal of extracellular Ca²âº reduced thymol's effect by approximately 80%. Thymol-induced Ca²âº entry was confirmed by Mn²âº entry-induced quench of fura-2 fluorescence, and was inhibited by approximately 30% by Ca²âº entry modulators (nifedipine, econazole, SKF96365), and the protein kinase C (PKC) inhibitor GF109203X. In Ca²âº-free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin abolished thymol-induced [Ca²âº]i rises. Treatment with thymol also abolished thapsigargin-induced [Ca²âº]i rises. Thymol-induced Ca²âº release from the endoplasmic reticulum was abolished by the phospholipase C (PLC) inhibitor U73122. Thymol at 100-900 µM decreased cell viability, which was not reversed by pretreatment with the Ca²âº chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, thymol induced [Ca²âº]i rises by inducing PLC-dependent Ca²âº release from the endoplasmic reticulum and Ca²âº entry via PKC-sensitive store-operated Ca²âº channels and other unknown channels. Thymol also induced Ca²âº-dissociated cell death.

Ca²âº Movement Induced by Deltamethrin in PC3 Human Prostate Cancer Cells.

This study explored the effect of deltamethrin, a pesticide, on intracellular free Ca²âº concentration ([Ca²âº]i) in PC3 human prostate cancer cells. Deltamethrin at concentrations between 5 µM and 20 µM evoked [Ca²âº]i rises in a concentration-dependent manner. This Ca²âº signal was inhibited by 22% by removal of extracellular Ca²âº. Nifedipine, econazole, and SKF96365 also inhibited the Ca²âº signal. Treatment with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) in Ca²âº-free medium nearly abolished deltamethrin-induced [Ca²âº]i rises. Treatment with deltamethrin also inhibited most of BHQ-induced [Ca²âº]i rises. Inhibition of phospholipase C (PLC) with U73122 failed to alter deltamethrin-evoked [Ca²âº]i rises. Deltamethrin killed cells at concentrations of 20-100 µM in a concentration-dependent fashion. Chelation of cytosolic Ca²âº with 1,2-bis (2-aminophenoxy) ethane-N, N, N', N'-tetraacetic acid/acetoxymethyl ester (BAPTA/AM) did not prevent deltamethrin's cytotoxicity. Together, in PC3 human prostate cancer cells, deltamethrin induced [Ca²âº]i rises that involved Ca²âº entry through store-operated Ca²âº channels and PLC-independent Ca²âº release from the endoplasmic reticulum. Deltamethrin induced cytotoxicity in a Ca²âº-independent manner.

Effect of 2,5-dimethylphenol on Ca(2+) 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 ([Ca(2+)]i) and cell viability in PC3 human prostate cancer cells. 2,5-Dimethylphenol at concentrations between 500 µM and 1000 µM evoked [Ca(2+)]i rises in a concentration-dependent manner. This Ca(2+ )signal was inhibited by approximately half by the removal of extracellular Ca(2+). 2,5-Dimethylphenol-induced Ca(2+ )influx was confirmed by Mn(2+)-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 Ca(2+)-containing medium by ∼30%. Treatment with the endoplasmic reticulum Ca(2+ )pump inhibitor thapsigargin in Ca(2+)-free medium abolished 2,5-dimethylphenol-induced [Ca(2+)]i rises. Conversely, treatment with 2,5-dimethylphenol abolished thapsigargin-induced [Ca(2+)]i rises. Inhibition of phospholipase C (PLC) with U73122 reduced 2,5-dimethylphenol-evoked [Ca(2+)]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 [Ca(2+)]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 Ca(2+)-independent manner.

Effect of sertraline on Ca²âº fluxes in rabbit corneal epithelial cells.

The effect of sertraline, a selective serotonin reuptake inhibitor (SSRI), on cytosolic free Ca²âº concentrations ([Ca²âº](i)) in a rabbit corneal epithelial cell line (SIRC) is unclear. This study explored whether sertraline changed basal [Ca²âº](i) levels in suspended SIRC cells by using fura-2 as a Ca²âº-sensitive fluorescent dye. Sertraline at concentrations between 10-100 µM increased [Ca²âº](i) in a concentration-dependent manner. The Ca²âº signal was reduced by 23% by removing extracellular Ca²âº. Sertraline induced Mn²âº influx, leading to quench of fura-2 fluorescence, suggesting Ca²âº influx. This Ca²âº influx was inhibited by phospholipase A2 inhibitor aristolochic acid, but not by store-operated Ca²âº channel blockers and protein kinase C/A modulators. In Ca²âº-free medium, pretreatment with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin, cyclopiazonic acid or 2,5-di-tert-butylhydroquinone greatly inhibited sertraline-induced Ca²âº release. Inhibition of phospholipase C with U73122 abolished sertraline-induced [Ca²âº](i) rise. At concentrations of 5-50 µM, sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of 25 µM sertraline was not reversed by prechelating cytosolic Ca²âº with BAPTA/AM. Collectively, in SIRC cells, sertraline induced [Ca²âº](i) rises by causing phospholipase C-dependent Ca²âº release from the endoplasmic reticulum and Ca²âº influx via phospholipase A2-sensitive Ca²âº channels. Sertraline-caused cytotoxicity was mediated by Ca²âº-independent pathways.

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 Ca(2+) concentrations ([Ca(2+)](i)) and viability in HA59T human hepatoma cells is unclear. This study explored whether diindolylmethane changed [Ca(2+)](i) in HA59T cells. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Diindolylmethane at concentrations of 1-50 µM evoked a [Ca(2+)](i) rise in a concentration-dependent manner. The signal was reduced by removing Ca(2+). Diindolylmethane-induced Ca(2+) influx was not inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators but was inhibited by aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca(2+)](i) rise. Incubation with diindolylmethane inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca(2+)](i) rise. At concentrations of 10-75 µM, diindolylmethane killed cells in a concentration-dependent manner. The cytotoxic effect of diindolylmethane was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid. Propidium iodide staining data suggest that diindolylmethane (25-50 µM) induced apoptosis in a concentration-dependent manner. Collectively, in HA59T cells, diindolylmethane induced a [Ca(2+)](i) rise by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via phospholipase A(2)-sensitive channels. Diindolylmethane induced cell death that may involve apoptosis.

Safflower extract: a novel renal fibrosis antagonist that functions by suppressing autocrine TGF-beta.

Progressive renal disease is characterized by the accumulation of extracellular matrix proteins in the renal interstitium. Hence, developing agents that antagonize fibrogenic signals is a critical issue facing researchers. The present study investigated the blood-circulation-promoting Chinese herb, safflower, on fibrosis status in NRK-49F cells, a normal rat kidney interstitial fibroblast, to evaluate the underlying signal transduction mechanism of transforming growth factor-beta (TGF-beta), a potent fibrogenic growth factor. Safflower was characterized and extracted using water. Renal fibrosis model was established both in vitro with fibroblast cells treated with beta-hydroxybutyrate and in vivo using rats undergone unilateral ureteral obstruction (UUO). Western blotting was used to examine protein expression in TGF-beta-related signal proteins such as type I and type II TGF-beta receptor, Smads2/3, pSmad2/3, Smads4, and Smads7. ELISA was used to analyze bioactive TGF-beta1 and fibronectin levels in the culture media. Safflower extract (SE) significantly inhibited beta-HB-induced fibrosis in NRK cells concomitantly with dose-dependent inhibition of the type I TGF-beta1 receptor and its down-stream signals (i.e., Smad). Moreover, SE dose-dependently enhanced inhibitory Smad7. Thus, SE can suppress renal cellular fibrosis by inhibiting the TGF-beta autocrine loop. Moreover, remarkably lower levels of tissue collagen were noted in the nephron and serum TGF-beta1 of UUO rats receiving oral SE (0.15 g/3 ml/0.25 kg/day) compared with the untreated controls. Hence, SE is a potential inhibitor of renal fibrosis. We suggest that safflower is a novel renal fibrosis antagonist that functions by down-regulating TGF-beta signals.

Effect of anandamide on cytosolic Ca(2+) levels and proliferation in canine renal tubular cells.

The effect of the endogenous cannabinoid anandamide on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and proliferation is largely unknown. This study examined whether anandamide altered Ca(2+) levels and caused Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Anandamide at concentrations above 5 muM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 78% by removing extracellular Ca(2+). The anandamide-induced Ca(2+) influx was insensitive to L-type Ca(2+) channel blockers and the cannabinoid receptor antagonist AM 251, but was inhibited differently by aristolochic acid, WIN 55,212-2 (a cannabinoid receptor agonist), phorbol ester, GF 109203X and forskolin. After pretreatment with thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), anandamide-induced Ca(2+) release was inhibited. Inhibition of phospholipase C with U73122 did not change anandamide-induced Ca(2+) release. At concentrations of 100 muM and 200 muM, anandamide killed 50% and 95% cells, respectively. The cytotoxic effect of 100 muM anandamide was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Collectively, in MDCK cells, anandamide induced [Ca(2+)](i) rises by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, anandamide can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.

Further evidence of the usefulness of Acute Physiology and Chronic Health Evaluation II scoring system in acute paraquat poisoning.

OBJECTIVE AND METHOD: We have previously successfully applied the Acute Physiology and Chronic Health Evaluation (APACHE) II system to assess the severity of patients with acute paraquat poisoning, and this article investigates further evidence of the usefulness of APACHE II system in predicting the in-hospital mortality of 64 patients with acute paraquat poisoning over a period of 12 years. The predictive factors including APACHE II score, plasma paraquat concentration, severity index of paraquat poisoning (SIPP), and estimated ingestion dosage of paraquat for evaluating the outcome in paraquat-poisoned patients were assessed. RESULTS: Overall mortality was 71.9%: 46 out of 64 patients died. Non-survivors (n = 46) had a higher APACHE II score (23.3 +/- 12.7) than survivors (n = 18) (6.1 +/- 4.2) (p < 0.001). The plasma paraquat concentration, SIPP, and estimated ingestion dosage of paraquat were significantly higher in non-survivors than in survivors (p < 0.05, in all comparisons). By multiple logistic regression analysis, only the APACHE II score and peak data of blood sugar in 24 h after admission were capable of predicting in-hospital mortality. By using the area under receiver operating characteristic curves (AURC), the APACHE II system yielded better discriminative power (AURC = 0.893) than SIPP (AURC = 0.674), plasma paraquat concentration (AURC = 0.676), and estimated ingestion dosage of paraquat (AURC = 0.673). An APACHE II score greater than 13 predicted in-hospital mortality with 67% sensitivity and 94% specificity. CONCLUSIONS: The APACHE II score is a simple, reproducible, and practical tool for evaluating the severity of acute paraquat poisoning.

The effect of the phenol compound ellagic acid on Ca(2+) homeostasis and cytotoxicity in liver cells.

Ellagic acid, a natural phenol compound found in numerous fruits and vegetables, causes various physiological effects in different cell models. However, the effect of this compound on Ca(2+) homeostasis in liver cells is unknown. This study examined the effect of ellagic acid on intracellular Ca(2+) concentration ([Ca(2+)]i) and established the relationship between Ca(2+) signaling and cytotoxicity in liver cells. The data show that ellagic acid induced concentration-dependent [Ca(2+)]i rises in HepG2 human hepatoma cells, but not in HA22T, HA59T human hepatoma cells or AML12 mouse hepatocytes. In HepG2 cells, this Ca(2+) signal response was reduced by removing extracellular Ca(2+) and was inhibited by store-operated Ca(2+) channel blockers (2-APB, econazole or SKF96365) and the protein kinase C (PKC) inhibitor GF109203X. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin abolished ellagic acid-induced [Ca(2+)]i rises. Conversely, incubation with ellagic acid abolished thapsigargin-induced [Ca(2+)]i rises. Inhibition of phospholipase C (PLC) with U73122 also abolished ellagic acid-induced [Ca(2+)]i rises. Ellagic acid (25-100µM) concentration-dependently caused cytotoxicity in HepG2, HA22T or HA59T cells, but not in AML12 cells. Furthermore, this cytotoxic effect was partially prevented by prechelating cytosolic Ca(2+) with BAPTA-AM only in HepG2 cells. Together, in HepG2 cells, ellagic acid induced [Ca(2+)]i rises by inducing PLC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via PKC-sensitive store-operated Ca(2+) channels. Moreover, ellagic acid induced Ca(2+)-associated cytotoxicity.

2-O-methyl PAF as a Ca2+ mobilizer in Madin Darby canine kidney cells.

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura-2. 2-O-methyl PAF (> or = 15 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. 2-O-methyl PAF-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca2+ influx was blocked by nifedipine, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which 2-O-methyl PAF failed to increase [Ca2+]i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca2+]i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release.

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells.

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.

Effect of maprotiline on Ca(2+) movement in human neuroblastoma cells.

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2+ concentrations ([Ca2+]i) was explored by using the Ca2+-sensitive probe fura-2. Maprotiline at concentrations greater than 100 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50 = 200 microM). Maprotiline-induced [Ca2+]i rise was reduced by 50% by removal of extracellular Ca2+. Maprotiline-induced [Ca2+]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. 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 abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2+]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.

Capsazepine elevates intracellular Ca2+ in human osteosarcoma cells, questioning its selectivity as a vanilloid receptor antagonist.

Capsazepine is thought to be a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on different cell types is unclear. In human MG63 osteosarcoma cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 100 microM. Capsazepine-induced [Ca(2+)](i) rise was partly reduced by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was composed of extracellular Ca(2+) influx and intracellular Ca(2+). 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 capsazepine on [Ca(2+)](i) was inhibited by 75%. Conversely, pretreatment with capsazepine to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. Overnight treatment with 1-100 microM capsazepine inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, capsazepine 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. Capsazepine may be mildly cytotoxic.

Neurotoxic effects of carambola in rats: the role of oxalate.

BACKGROUND AND PURPOSE: Carambola (star fruit) has been reported to contain neurotoxins that cause convulsions, hiccups, or death in uremic patients, and prolong barbiturate-induced sleeping time in rats. The constituent responsible for these effects remains uncertain. Carambola contains a large quantity of oxalate, which can induce depression of cerebral function and seizures. This study was conducted to investigate the role of oxalate in carambola toxicity in rats. MATERIALS AND METHODS: The effects on barbiturate-induced sleeping time and death caused by intraperitoneal administration of carambola juice were observed in Sprague-Dawley rats. To obtain a dose-dependent response curve and evaluate the lethal dose, rats were treated with serial amounts of pure carambola juice diluted with normal saline in a volume of 1:1. To test the role of oxalate in the neurotoxic effect of carambola, either 5.33 g/kg carambola after oxalate removal or 5.33 g/kg of pure carambola juice diluted with normal saline were administered intraperitoneally, while the control group was given normal saline before pentobarbital injection. The effects of carambola and oxalate-removed carambola on barbiturate-induced sleeping time were compared with those of saline. To assess the lethal effect of oxalate in carambola, we gave rats chemical oxalate at comparable concentrations to the oxalate content of carambola. RESULTS: Carambola juice administration prolonged barbiturate-induced sleeping time in a dose-dependent manner. The sleeping time of rats that received normal saline and 1.33 g/kg, 2.67 g/kg, 5.33 g/kg, and 10.67 g/kg of carambola juice were 66 +/- 16.6, 93.7 +/- 13.4, 113.3 +/- 11.4, 117.5 +/- 29.0, and 172.5 +/- 38.8 minutes, respectively. The three higher-dose groups had longer sleeping times than controls (p < 0.05 or 0.005). This effect was eliminated after the removal of oxalate from carambola juice. Four of eight rats in the 10.67-g/kg group and all rats in the 21.33 g/kg and chemical oxalate groups died after seizure. Lethal doses of carambola juice were rendered harmless by the oxalate removal procedure. CONCLUSIONS: Oxalate is a main constituent of carambola neurotoxicity. This finding suggests that patients with carambola intoxication should be treated for oxalate toxicosis.

Effect of gossypol on intracellular Ca2+ regulation in human hepatoma cells.

Gossypol is a natural toxicant present in cottonseeds, and is hepatotoxic to animals and human. The effect of gossypol on cytosolic free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatocytes was explored using fura-2 as a fluorescent Ca2+ indicator. Gossypol increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 2 microM. The Ca2+ signal was reduced by removing extracellular Ca2+ or by 10 microM La3+, but was not affected by nifedipine, verapamil or diltiazem. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ partly reduced 10 microM gossypol-induced Ca2+ release; and conversely pretreatment with gossypol abolished thapsigargin-induced Ca2+ release. The Ca2+ release induced by 10 microM gossypol was not changed by inhibiting phospholipase C with 2 microM U73122 or by depleting ryanodine-sensitive Ca2+ stores with 50 microM ryanodine. Together, the results suggest that in human hepatocytes, gossypol induced a [Ca2+]i increase by causing store Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and by inducing Ca2+ influx.

Effect of NPC-15199 on Ca2+ levels in renal tubular cells.

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca2+ concentration ([Ca2+]i) was investigated by using fura-2. NPC-15199 (100-1000 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50=500 microM). NPC-15199-induced [Ca2+]i rise was prevented by 70% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an inhibitor of the endoplasmic reticulum (ER) Ca2(+)-ATPase, caused a monophasic [Ca2+]i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca2+]i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca2+]i rises. U73122, an inhibitor of phospholipase C, [corrected] abolished 10 microM ATP (but not 1 mM NPC-15199)-induced [Ca2+]i rise. These results suggest that NPC-15199 rapidly increases [Ca2+]i by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via as yet unidentified mechanism(s).

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells.

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.

Cadmium-induced cytosolic Ca2+ elevation and subsequent apoptosis in renal tubular cells.

Cadmium (Cd2+) is an industrial and environmental metal. The effect of Cd2+ on intracellular free-Ca2+ levels ([Ca2+](i)) and viability in Madin Darby canine kidney cells was explored. Cd2+increased [Ca2+](i) in a concentration-dependent manner with an EC50 of 85 microM. Cd2+-induced Mn2+ entry demonstrated Ca2+ influx. Removal of extracellular Ca2+ decreased the [Ca2+](i) signal by 60%. The [Ca2+](i) signal was inhibited by La3+ but not by L-type Ca2+ channel blockers. In Ca2+-free medium, Cd2+-induced [Ca2+]i signal was abolished by pre-treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+pump inhibitor) and 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler). Cd2+-induced Ca2+ release was not altered by inhibition of phospholipase C. At concentrations between 10 and 100 microM, Cd2+killed cells in a concentration-dependent manner. The cytotoxic effect of 100 microM Cd2+was reversed by pre-chelating cytosolic Ca2+with BAPTA. Cd2+-induced apoptosis was demonstrated by propidium iodide. Collectively, this study shows that Cd2+ induced a [Ca2+](i) increase in Madin Darby canine kidney cells via evoking Ca2+ entry through non-selective Ca2+ channels, and releasing stored Ca2+ from endoplasmic reticulum and mitochondria in a phospholipase C-independent manner.

Defect in regulation of Ca2+ movement in platelets from patients with systemic lupus erythematosus.

The differences in the intracellular Ca(2+) responses to hormones in platelets from systemic lupus erythematosus (SLE) patients compared to normal humans have not been explored. This study examined the Ca(2+) signaling and density of platelets in normal, inactive and active SLE patients. The platelet number per mul in inactive and normal groups did not differ, whereas the number in active SLE patients was smaller than the other two groups by 60%. The intracellular free Ca(2+) levels ([Ca(2+)](i)) in response to stimulation of four endogenous Ca(2+) mobilizing hormones, 100 microM arachidonic acid (AA), 10 microM ADP, 10 nM platelet activation factor (PAF) and 1 microM thrombin, were investigated using the Ca(2+)-sensitive fluorescent dye, fura-2. The AA-induced [Ca(2+)](i) rises in normal and inactive groups were similar. In contrast, the AA-induced [Ca(2+)](i) rises in the active SLE group were significantly smaller than in the normal and inactive groups. The defect in the AA-induced [Ca(2+)](i) rises in active SLE groups appears to be caused by defective Ca(2+) influx and Ca(2+) releasing pathways because the AA-induced responses were not altered by removal of extracellular Ca(2+), whereas the AA-induced responses in normal and inactive SLE groups were reduced by removal of extracellular Ca(2+), and the AA-induced Ca(2+) release was smaller in the active SLE group. PAF, ADP and thrombin all induced [Ca(2+)](i) rises in the three groups, but no significant differences were found among the three groups. Together, the results indicate that cell density and Ca(2+) signaling in platelets from active SLE patients are altered in response to particular stimulators. In these regards, platelets from inactive SLE patients appear to be similar to those from normal humans.

Chitosan enhances platelet adhesion and aggregation.

In this study, chitosan (MW=50,000) was tested for its enhancing platelet activity in rabbit platelet suspensions and the possible mechanisms involved were further investigated. Our results showed that after initial (5 min) and long-term (30 min) contact of platelets with chitosan, the platelet adhesion to chitosan-coated microtiter plates was dose-dependently increased compared to that of solvent control. Similarly, chitosan also dose-dependently increased the platelet aggregation and the intracellular free Ca(2+) rise of Fura-2-AM loaded platelets. Additionally, in the presence of FITC-labeled anti-CD41/CD61, chitosan significantly enhanced the expression of platelet glycoprotein IIb/IIIa complex assayed by a flow cytometer. It is concluded that chitosan is an effective inducer for platelet adhesion and aggregation and the mechanisms of action of chitosan may be associated, at least partly, with the increasing [Ca(2+)](i) mobilization and enhancing expression of GPIIb/IIIa complex on platelet membrane surfaces.