Mechanisms of diethylstilbestrol-induced calcium movement in MG63 human osteosarcoma cells

The effect of the estrogen diethylstilbestrol (DES) on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteoblasts was explored by using fura-2 as a Ca(2+) indicator. DES at concentrations between 5--20 microM induced an immediate increase in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 70%. Pretreatment with 50 microM La(3+) or 10 microM of nifedipine, verapamil and diltiazem did not change 20 microM DES-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 20 microM DES in Ca(2+)-free medium. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store partly inhibited 20 microM DES-induced Ca(2+) release, but addition of carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and thapsigargin together abolished DES-induced Ca(2+) release. Conversely, pretreatment with 20 microM DES abrogated CCCP- and thapsigargin-induced Ca(2+) release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 20 microM DES-induced Ca2+ release. Another estrogen 17beta-estradiol also increased [Ca(2+)](i) in a concentration-dependent manner with an EC50 of 7 microM. Together, the data indicate that in human osteoblasts, DES increased [Ca(2+)](i) via causing Ca(2+) release from both mitochondria and the endoplasmic reticulum in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

Effect of the antidepressant desipramine on cytosolic Ca(2+) movement and proliferation in human osteosarcoma cells

In human osteosarcoma MG63 cells, the effect of desipramine, an antidepressant, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2. Desipramine (>10 micromol/l) caused a rapid and sustained rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 200 micromol/l). Desipramine-induced [Ca(2+)](i) rise was prevented by 80% by removal of extracellular Ca(2+) but was not altered by voltage-gated Ca(2+) channel blockers. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of desipramine on [Ca(2+)](i) was abolished; also, pretreatment with desipramine partly reduced thapsigargin-induced [Ca(2+)](i) increase. U73122, an inhibitor of phospholipase C, did not affect desipramine-induced [Ca(2+)](i) rise. Overnight incubation with 10 micromol/l desipramine did not alter cell proliferation, but killed 32 and 89% of cells at concentrations of 100 and 200 micromol/l, respectively. These findings suggest that desipramine rapidly increases [Ca(2+)](i) in osteoblasts by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release, and is cytotoxic at high concentrations.     

Dual effect of tamoxifen, an anti-breast-cancer drug, on intracellular Ca(2+) and cytotoxicity in intact cells

The effect of tamoxifen on Ca(2+) signaling and viability in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Tamoxifen evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 1 and 50 microM with an EC50 of 10 microM. The response was decreased by extracellular Ca(2+) removal. In Ca(2+)-free medium, pretreatment with 5 microM tamoxifen abolished the [Ca(2+)](i) increase induced by the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM), but pretreatment with brefeldin A (50 microM; a Ca(2+) mobilizer of the Golgi complex), thapsigargin (an inhibitor of the endoplasmic reticulum Ca(2+) pump), and carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler), only partly inhibited tamoxifen-induced [Ca(2+)](i) increases. This suggests that tamoxifen released Ca(2+) from multiple pools. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 5 microM tamoxifen in Ca(2+)-free medium. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) did not alter 5 microM tamoxifen-induced Ca(2+) release. The [Ca(2+)](i) increase induced by 5 microM tamoxifen was not altered by La(3+), nifedipine, verapamil, or diltiazem. Tamoxifen (1-10 microM) decreased cell viability in a concentration- and time-dependent manner. Tamoxifen (5 microM) also increased [Ca(2+)](i) in neutrophils, bladder cancer cells, and prostate cancer cells from humans and glioma cells from rats. Collectively, it was found that tamoxifen increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol 1,4, 5-trisphosphate and also by triggering Ca(2+) influx from extracellular space. The [Ca(2+)](i) increase was accompanied by cytotoxicity.     

Effect of capsazepine on cytosolic Ca(2+) levels and proliferation of human prostate cancer cells.

Capsazepine has been widely used as a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on most cell types is unknown. In human PC3 prostate cancer cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was investigated 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 75 microM. Capsazepine-induced [Ca(2+)](i) rise was reduced by 60% by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was contributed by extracellular Ca(2+) influx and intracellular Ca(2+). Consistently, the capsazepine (200 microM)-induced [Ca(2+)](i) rise was decreased by La(3+) by half. 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 effect of capsazepine on [Ca(2+)](i) was inhibited by 80%. Conversely, pretreatment with capsazepine partly reduced thapsigargin-induced [Ca(2+)](i) rise. 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. These findings suggest that in human PC3 prostate cancer cells, capsazepine increases [Ca(2+)](i) by evoking Ca(2+) influx and releasing Ca(2+) from the endoplasmic reticulum via a phospholiase C-independent manner. Overnight incubation with capsazepine (200 microM) killed 37% of cells, which could not be prevented by chelating intracellular Ca(2+) with BAPTA.     

Effect of celecoxib on Ca(2+) 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. Ca(2+) is a key second messenger in most cells. The effect of celecoxib on cytosolic free Ca(2+) concentrations ([Ca(2+)](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 [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+). Celecoxib-induced Ca(2+) influx was not blocked by L-type Ca(2+) entry inhibitors or protein kinase C/A modulators [phorbol 12-myristate 13-acetate (PMA), GF109203X, H-89], but was inhibited by the phospholipase A(2) inhibitor, aristolochic acid. In Ca(2+)-free medium, 30 μM of celecoxib failed to induce a [Ca(2+)](i) rise after pretreatment with thapsigargin (an endoplasmic reticulum [ER] Ca(2+) pump inhibitor). Conversely, pretreatment with celecoxib inhibited thapsigargin-induced Ca(2+) release. Inhibition of phospholipase C with U73122 did not change celecoxib-induced [Ca(2+)](i) rises. Celecoxib induced slight cell death in a concentration-dependent manner, which was enhanced by chelating cytosolic Ca(2+) with BAPTA. Collectively, in PC3 cells, celecoxib induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the ER and Ca(2+) influx via non-L-type, phospholipase A(2)-regulated Ca(2+) channels. These data may contribute to the understanding of the effect of celecoxib on prostate cancer cells.     

The anti-anginal drug fendiline increases intracellular Ca(2+) levels in MG63 human osteosarcoma cells

The effect of fendiline, an anti-anginal drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored by using fura-2 as a Ca(2+) indicator. Fendiline at concentrations between 1 and 200 microM increased [Ca(2+)](i) in a concentration-dependent manner and the signal saturated at 100 microM. The Ca(2+) signal was inhibited by 65+/-5% by Ca(2+) removal and by 38+/-5% by 10 microM nifedipine, but was unchanged by 10 microM La(3+) or verapamil. In Ca(2+)-free medium, pre-treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store inhibited fendiline-induced intracellular Ca(2+) release. The Ca(2+) release induced by 50 microM fendiline appeared to be independent of IP(3) because the [Ca(2+)](i) increase was unaltered by inhibiting phospholipase C with 2 microM U73122. Collectively, the results suggest that in MG63 cells fendiline caused an increase in [Ca(2+)](i) by inducing Ca(2+) influx and Ca(2+) release in an IP(3)-independent manner.     

Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells

The effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability is largely unknown. This study examined whether melittin alters Ca(2+) levels and causes 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. Melittin at concentrations above 0.5 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 75% by removing extracellular Ca(2+). The melittin-induced Ca(2+) influx was also implicated by melittin-caused Mn(2+) influx. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), melittin-induced Ca(2+) release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca(2+) release. At concentrations of 0.5-20 microM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 microM melittin was nearly completely reversed by prechelating cytosolic Ca(2+) with BAPTA. Melittin at 0.5-2 microM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca(2+)](i) rise by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, melittin can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.     

Novel effect of carvedilol on Ca2+ movement in renal tubular cells

The effect of carvedilol on intracellular free Ca(2+) levels ([Ca(2+)](i)) has not been explored previously. This study was aimed to examine the effect of carvedilol on Ca(2+) handling in renal tubular cells. Madin-Darby canine kidney cells were used as a model for renal tubular cells and fura-2 was used as a fluorescent Ca(2+) probe. Carvedilol increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 5 microM. Extracellular Ca(2+) removal partly inhibited the [Ca(2+)](i) signals. Carvedilol-induced Ca(2+) influx was verified by measuring Mn(2+)-induced quench of fura-2 fluorescence. Carvedilol-induced store Ca(2+) release was reduced by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) but not with 5 microM ryanodine or 2 microM carbonylcyanide m-chlorophenylhydrazone (a mitochondrial uncoupler). Carvedilol (30 microM)-induced Ca(2+) release was not affected by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-l)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; 2 microM), but was potentiated by increasing cAMP levels or inhibiting protein kinase C. The carvedilol-induced Ca(2+) mobilization was not significantly sequestered by the endoplasmic reticulum or mitochondria. This study shows that carvedilol increased [Ca(2+)](i) in renal tubular cells by causing Ca(2+) release from the endoplasmic reticulum and other unknown stores in an inositol-1,4,5-trisphosphate-independent manner, and by inducing Ca(2+) influx. The Ca(2+) release was modulated by cAMP and protein kinase C.     

Effect of thymol on Ca2+ homeostasis and viability in human glioblastoma cells

The effect of the natural essential oil thymol on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in human glioblastoma cells was examined. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Thymol at concentrations of 400-1000 μM induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca(2+). Thymol-induced Ca(2+) 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 Ca(2+) was removed, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca(2+)](i) rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca(2+)](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 Ca(2+) 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 [Ca(2+)](i) rise by inducing phospholipase C- and protein kinase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via non store-operated Ca(2+) channels. Thymol induced cell death that may involve apoptosis.     

Effect of nordihydroguaiaretic acid on intracellular Ca(2+) concentrations in hepatocytes.

The effect of nordihydroguaiaretic acid (NDGA) on Ca(2+) signaling in human hepatoma cells (HA22/VGH) has been investigated. NDGA (5-50 microM) increased [Ca(2+)](i) concentration-dependently. The [Ca(2+)](i) increase comprised an initial rise and an elevated phase over a time period of 4 min. Removal of extracellular Ca(2+) reduced 10-50 microM NDGA induced [Ca(2+)](i) signals by 45+/-5%. Consistently, the 50 microM NDGA-induced [Ca(2+)](i) increase in Ca(2+)-containing medium was reduced by 41+/-2% by 10 microM of La(3+), nifedipine or verapamil. In Ca(2+)-free medium, pretreatment with 20 microM NDGA for 6 min abolished the [Ca(2+)](i) increase induced by the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM). Conversely, 20 microM NDGA failed to increase [Ca(2+)](i) after 1 microM thapsigargin had depleted the endoplasmic reticulum Ca(2+) store. Inhibition of phospholipase C with 2 microM U73122 had little effect on 20 microM NDGA-induced Ca(2+) release. Several other lipoxygenase inhibitors had no effect on basal [Ca(2+)](i). Together, the data suggest that NDGA increased [Ca(2+)](i) in hepatocytes in a lipoxygenase-independent manner, by releasing Ca(2+) from the endoplasmic reticulum and causing Ca(2+) influx.     

Calmidazolium-induced rises in cytosolic calcium concentrations in Madin Darby canine kidney cells

The effect of calmidazolium on Ca(2+) signaling in Madin Darby canine kidney (MDCK) cells was investigated using fura-2 as a Ca(2+) probe. Calmidazolium at 2-5 microM increased [Ca(2+)](i) concentration dependently. The [Ca(2+)](i) rise induced by 2-5 microM calmidazolium comprised an immediate rise and a slow decay. External Ca(2+) removal partly inhibited the Ca(2+) signals, suggesting that calmidazolium activated external Ca(2+) influx and internal Ca(2+) release. In Ca(2+)-free medium, pretreatment with 3 microM calmidazolium abolished the Ca(2+) release induced by 1 microM thapsigargin, an endoplasmic reticulum Ca(2+) pump inhibitor, and, vice versa, pretreatment with thapsigargin inhibited calmidazolium-induced Ca(2+) release. This indicates that thapsigargin-sensitive Ca(2+) store was the source of calmidazolium-induced Ca(2+) release. Calmidazolium (3 microM) induced Mn(2+) quench of fura-2 fluorescence at 360 nm excitation wavelength, which was suppressed by 0.1 mM La(3+). Addition of 3 mM Ca(2+) increased [Ca(2+)](i) after pretreatment with 3-5 microM calmidazolium in Ca(2+)-free medium. This implies that calmidazolium activated concentration-dependent capacitative Ca(2+) entry. Calmidazolium (3 microM) augmented the capacitative Ca(2+) entry induced by 1 microM thapsigargin or 0.1 mM ATP by 38%. Calmidazolium (3 microM)-induced Ca(2+) release was blocked by pretreatment with 40 microM aristolochic acid and 2 microM U73122 (2 microM) to inhibit phospholipase A(2) and phospholipase, respectively, but pretreatment with 0.1 mM propranolol to inhibit phospholipase D had no effect. This suggests that calmidazolium induced internal Ca(2+) release in a manner dependent on phospholipases C- and A(2)-coupled events.     

Effect of methoxychlor on Ca2+ handling and viability in OC2 human oral cancer cells

The effect of the insecticide methoxychlor on the physiology of oral cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) in human oral cancer cells (OC2) by using the Ca(2+)-sensitive fluorescent dye fura-2. Methoxychlor at 5-20 μM increased [Ca(2+)](i) in a concentration-dependent manner. The signal was reduced by 70% by removing extracellular Ca(2+). Methoxychlor-induced Ca(2+) entry was not affected by nifedipine, econazole, SK&F96365 and protein kinase C modulators but was inhibited by the phospholipase A2 inhibitor aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished methoxychlor-induced [Ca(2+)](i) rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 did not alter methoxychlor-induced [Ca(2+)](i) rise. At 5-20 μM, methoxychlor killed cells in a concentration-dependent manner. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM). Annexin V-FITC data suggest that methoxychlor (10 and 20 μM) evoked apoptosis in a concentration-dependent manner. Together, in human OC2, methoxychlor induced a [Ca(2+)](i) rise probably by inducing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive channels. Methoxychlor induced cell death that may involve apoptosis.     

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.     

Effect of betulinic acid on intracellular-free Ca(2+) levels in Madin Darby canine kidney cells

The effect of betulinic acid, an anti-tumor and apoptosis-inducing natural product, on intracellular-free levels of Ca(2+) ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was examined by using fura-2 as a Ca(2+) dye. Betulinic acid caused significant increases in [Ca(2+)](i) concentration dependently between 25 and 500 nM with an EC(50) of 100 nM. The [Ca(2+)](i) signal was composed of an initial gradual rise and a plateau. The response was decreased by removal of extracellular Ca(2+) by 45+/-10%. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) abolished 250 microM betulinic acid-induced [Ca(2+)](i) increases. Conversely, pretreatment with betulinic acid only partly inhibited thapsigargin-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 250 nM betulinic acid in Ca(2+)-free medium for 5 min. This [Ca(2+)](i) increase was not altered by the addition of 20 microM SKF96365 and 10 microM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) abolished 250 nM betulinic acid-induced Ca(2+) release. Pretreatment with 10 microM La(3+) inhibited 250 nM betulinic acid-induced [Ca(2+)](i) increases by 85+/-3%; whereas 10 microM of verapamil, nifedipine and diltiazem had no effect. In Ca(2+) medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 microM ATP and 1 microM thapsigargin-induced [Ca(2+)](i) increases by 33+/-3% and 45+/-3%, respectively. Trypan blue exclusion revealed that acute exposure of 250 nM betulinic acid for 2-30 min decreased cell viability by 6+/-2%, which could be prevented by pretreatment with 2 microM U731222. Together, the results suggest that betulinic acid induced significant [Ca(2+)](i) increases in MDCK cells in a concentration-dependent manner, and also induced mild cell death. The [Ca(2+)](i) signal was contributed by an inositol 1,4, 5-trisphosphate-dependent release of intracellular Ca(2+) from thapsigargin-sensitive stores, and by inducing Ca(2+) entry from extracellular medium in a La(3+)-sensitive manner.     

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

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

AA-861-induced Ca(2+) mobilization in Madin Darby canine kidney cells

The effect of 2,3,5-trimethyl-6-(12-hydroxy-5,10-dodecadiynyl)-1, 4-benzoquinone (AA-861), a 5-lipoxygenase inhibitor, on Ca(2+) mobilization in Madin Darby canine kidney (MDCK) cells has been examined by fluorimetry using fura-2 as a Ca(2+) indicator. AA-861 at 10-200 microM increased [Ca(2+)](i) concentration dependently. The signal comprised an initial rise and a sustained phase. Ca(2+) removal inhibited the Ca(2+) signals by reducing both the initial rise and the sustained phase. In Ca(2+)-free medium, pretreatment with 50 microM AA-861 abolished the Ca(2+) release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, and carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler. Pretreatment with CCCP, thapsigargin and gly-phe-beta-naphthylamide to deplete the Ca(2+) stores in mitochondria, the endoplasmic reticulum, and lysosomes, respectively, only partly inhibited AA-861-induced Ca(2+) release. This suggests AA-861 released Ca(2+) from multiple internal pools. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 50 microM AA-861 in Ca(2+)-free medium. AA-861 (50 microM)-induced internal Ca(2+) release was not altered by inhibition of phospholipase C with U73122 (2 microM) but was inhibited by 40% by inhibition of phospholipase A(2) with aristolochic acid (40 microM). Collectively, we found that AA-861 increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple internal stores in a manner independent of the formation of inositol-1,4,5-trisphosphate, followed by Ca(2+) entry from external medium.     

Effect of oleamide on Ca(2+) signaling in human bladder cancer cells.

The effect of oleamide, a sleep-inducing endogenous lipid in animal models, on intracellular free levels of Ca(2+) ([Ca(2+)](i)) in non-excitable and excitable cells was examined by using fura-2 as a fluorescent dye. [Ca(2+)](i) in pheochromocytoma cells, renal tubular cells, osteoblast-like cells, and bladder cancer cells were increased on stimulation of 50 microM oleamide. The response in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10-100 microM) increased [Ca(2+)](i) in a concentration-dependent fashion with an EC(50) of 50 microM. The [Ca(2+)](i) signal comprised an initial rise and a sustained plateau and was reduced by removing extracellular Ca(2+) by 85 +/- 5%. After pre-treatment with 10-100 microM oleamide in Ca(2+)-free medium, addition of 3 mM Ca(2+) increased [Ca(2+)](i) in a manner dependent on the concentration of oleamide. The [Ca(2+)](i) increase induced by 50 microM oleamide was reduced by 100 microM La(3+) by 40%, but was not altered by 10 microM nifedipine, 10 microM verapamil, and 50 microM Ni(2+). In Ca(2+)-free medium, pre-treatment with thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, abolished 50 microM oleamide-induced [Ca(2+)](i) increases; conversely, pretreatment with 50 microM oleamide reduced 1 microM thapsigargin-induced [Ca(2+)](i) increases by 50 +/- 3%. Suppression of the activity of phospholipase C with 2 microM U73122 failed to alter 50 microM oleamide-induced Ca(2+) release. Linoleamide (10-100 microM), another sleep-inducing lipid with a structure similar to that of oleamide, also induced an increase in [Ca(2+)](i). Together, it was shown that oleamide induced significant [Ca(2+)](i) increases in cells by a phospholipase C-independent release of Ca(2+) from thapsigargin-sensitive stores and by inducing Ca(2+) entry.     

Mechanisms underlying ketoconazole-induced Ca(2+) mobilization in Madin-Darby canine kidney cells

The effect of ketoconazole on Ca(2+) signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Ketoconazole evoked increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) concentration dependently. The response was decreased by external Ca(2+) removal. In Ca(2+)-free medium, pretreatment with ketoconazole abolished the [Ca(2+)](i) rise induced by thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+) pump. Addition of 3 mM Ca(2+) induced a significant [Ca(2+)](i) rise after preincubation with 150 microM ketoconazole in Ca(2+)-free medium. Pretreatment with aristolochic acid (40 microM) to inhibit phospholipase A(2) inhibited the 150-microM-ketoconazole-induced internal Ca(2+) release by 37%, but inhibition of phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) (2 microM) had no effect. Collectively, we found that ketoconazole increases [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from thapsigargin-sensitive pools in a manner independent of the production of inositol-1,4,5-trisphosphate, followed by Ca(2+) influx from the external space.     

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

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

The mechanism of sertraline-induced [Ca(2+) ](i) rise in human PC3 prostate cancer cells

The effect of sertraline, an antidepressant, on cytosolic-free Ca(2+) levels ([Ca(2+) ](i) ) in human cancer cells is unclear. This study examined whether sertraline altered basal [Ca(2+) ](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca(2+) -sensitive fluorescent probe. At concentrations of 10-150 μM, sertraline induced a [Ca(2+) ](i) rise in a concentration-dependent fashion. The Ca(2+) signal was reduced partly by removing extracellular Ca(2+) indicating that Ca(2+) entry and release both contributed to the [Ca(2+) ](i) rise. Sertraline induced Mn(2+) influx, leading to quench of fura-2 fluorescence suggesting Ca(2+) influx. This Ca(2+) influx was inhibited by the suppression of store-operated Ca(2+) channels or by the modulation of protein kinase C activity. In Ca(2+) -free medium, pre-treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone nearly abolished sertraline-induced Ca(2+) release. Conversely, pre-treatment with sertraline greatly reduced the inhibitor-induced [Ca(2+) ](i) rise, suggesting that sertraline released Ca(2+) from the endoplasmic reticulum. Inhibition of phospholipase C inhibited sertraline-induced [Ca(2+) ](i) rise. At 20-30 μM, sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of sertraline was enhanced by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM. Annexin V-FITC data suggest that sertraline (20 and 30 μM) evoked apoptosis in a concentration-dependent manner. Together, in PC3 human prostate cancer cells, sertraline induced [Ca(2+) ](i) rises by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and via multiple Ca(2+) influx pathways that involve store-operated Ca(2+) channels. Sertraline also induced apoptosis that was not triggered by [Ca(2+) ](i) rise.