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.     

Novel effect of Y-24180, a presumed specific platelet-activating factor receptor antagonist, on Ca2+ levels and growth of human osteosarcoma cells.

In human osteosarcoma MG63 cells, the effect of Y-24180, a presumed specific platelet-activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) and proliferation was measured by using fura-2 and tetrazolium as fluorescent dyes, respectively. Y-24180 (1-5 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was inhibited by 35% by dihydropyridines or removal of extracellular Ca(2+), but was not altered by verapamil and diltiazem. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which 5 microM Y-24180 failed to increase [Ca(2+)](i); conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited histamine-induced, but not 5 microM Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with 0.1-5 microM Y-24180 inhibited cell proliferation in a concentration-dependent manner. Together, these findings suggest that Y-24180 acts as a potent and cytotoxic Ca(2+) mobilizer in human osteosarcoma cells, by inducing both extracellular Ca(2+) influx and intracellular Ca(2+) release. Alterations in cytosolic Ca(2+) regulation may lead to interferences of various cellular functions; thus, attention should be exercised in using Y-24180 as a selective PAF receptor antagonist.     

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.     

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.     

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.     

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.     

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.     

Desipramine-induced Ca2+ movement and cytotoxicity in PC3 human prostate cancer cells.

The effect of the antidepressant desipramine on intracellular Ca(2+) movement and viability in prostate cancer cells has not been explored previously. The present study examined whether desipramine could alter Ca(2+) handling and viability in human prostate PC3 cancer cells. Cytosolic free Ca(2+) levels ([Ca(2+)](i)) in populations of cells were measured using fura-2 as a probe. Desipramine at concentrations above 10 microM increased [Ca(2+)](i) in a concentration-dependent manner. The responses saturated at 300 microM desipramine. The Ca(2+) signal was reduced by half by removing extracellular Ca(2+), but was unaffected by nifedipine, nicardipine, nimodipine, diltiazem or verapamil. In Ca(2+)-free medium, after treatment with 300 microM desipramine, 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) failed to release Ca(2+) from endoplasmic reticulum. Conversely, desipramine failed to release more Ca(2+) after thapsigargin treatment. Inhibition of phospholipase C with U73122 did not affect desipramine-induced Ca(2+) release. Overnight incubation with 10-800 microM desipramine decreased viability in a concentration-dependent manner. Chelation of cytosolic Ca(2+) with BAPTA did not reverse the decreased cell viability. Collectively, the data suggest that in PC3 cells, desipramine induced a [Ca(2+)](i) increase by causing Ca(2+) release from endoplasmic reticulum in a phospholipase C-independent fashion and by inducing Ca(2+) influx. Desipramine decreased cell viability in a concentration-dependent, Ca(2+)-independent manner.     

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.     

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 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.     

Thimerosal-induced cytosolic Ca2+ elevation and subsequent cell death in human osteosarcoma cells.

The effect of the oxidizing agent thimerosal on cytosolic free Ca(2+) concentration ([Ca(2+)]i) and proliferation has not been explored in human osteoblast-like cells. This study examined whether thimerosal alters Ca(2+) levels and causes cell death in MG63 human osteosarcoma cells. [Ca(2+)]i and cell death were measured using the fluorescent dyes fura-2 and WST-1, respectively. Thimerosal at concentrations above 5 microM increased [Ca(2+)]i in a concentration-dependent manner. The Ca(2+) signal was reduced by 80% by removing extracellular Ca(2+). The thimerosal-induced Ca(2+) influx was sensitive to blockade of La(3+), and dithiothreitol (50 microM) but was insensitive to nickel and several L-type Ca(2+) channel blockers. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), thimerosal failed to induce [Ca(2+)]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change thimerosal-induced [Ca(2+)]i rises. At concentrations of 5, 10 and 20 microM thimerosal killed 33, 55 and 100% cells, respectively. The cytotoxic effect of 5 microM thimerosal was reversed by 54% by prechelating cytosolic Ca(2+) with BAPTA. Collectively, in MG63 cells, thimerosal induced a [Ca(2+)]i rise by causing Ca(2+) release from endoplasmic reticulum stores and Ca(2+) influx from extracellular space. Furthermore, thimerosal can cause Ca(2+)-related cytotoxicity in a concentration-dependent manner.     

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 17beta-estradiol on intracellular Ca(2+) levels in renal tubular cells.

The effect of 17beta-estradiol on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney cells was investigated by using the fluorescent dye fura-2. 17Beta-estradiol (5-100 micromol/l) induced instantaneous increases in [Ca(2+)](i) in a concentration-dependent manner. Ca(2+) removal inhibited 45 +/- 15% of the Ca(2+) signal. In Ca(2+)-free medium, pretreatment with 50 micromol/l 17beta-estradiol abolished the [Ca(2+)](i) increases induced by 2 micromol/l carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler), 1 micromol/l thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) and 50 micromol/l brefeldin A (an antibiotic which disperses the Golgi complex), but pretreatment with brefeldin A, CCCP and thapsigargin only partly inhibited the 17beta-estradiol-induced [Ca(2+)](i) signal. Adding 3 mmol/l Ca(2+) increased [Ca(2+)](i) in cells pretreated with 5-100 micromol/l 17beta-estradiol in Ca(2+)-free medium. Pretreatment with 1 micromol/l U73122 to abolish the formation of inositol-1,4,5-trisphosphate inhibited 50% of the Ca(2+) release induced by 50 micromol/l 17beta-estradiol. 17Beta-estradiol (20 micromol/l) also increased [Ca(2+)](i) in human bladder cancer cells and prostate cancer cells. Collectively, this study shows that 17beta-estradiol evoked a significant internal Ca(2+) release and external Ca(2+) entry possibly in a nongenomic manner.     

Xestospongin C, a novel blocker of IP3 receptor, attenuates the increase in cytosolic calcium level and degranulation that is induced by antigen in RBL-2H3 mast cells

1. We evaluated the role of the cross-linking of Fc epsilon RI-mediated inositol 1,4,5-triphosphate (IP(3)) in the increase in cytosolic Ca(2+) level ([Ca(2+)](i)) using xestospongin C, a selective membrane permeable blocker of IP(3) receptor, in RBL-2H3 mast cells. 2. In the cells sensitized with anti-dinitrophenol (DNP) IgE, DNP-human serum albumin (DNP-HSA) and thapsigargin induced degranulation of beta-hexosaminidase and a sustained increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited both of these changes that were induced by DNP-HSA without changing those induced by thapsigargin. 3. In the absence of external Ca(2+), DNP-HSA induced a transient increase in [Ca(2+)](i). Xestospongin C (3 - 10 microM) inhibited this increase in [Ca(2+)](i). 4. In the cells permeabilized with beta-escin, the application of IP(3) decreased Ca(2+) in the endoplasmic reticulum (ER) as evaluated by mag-fura-2. Xestospongin C (3 - 10 microM) inhibited the effect of IP(3). 5. After the depletion of Ca(2+) stores due to stimulation with DNP-HSA or thapsigargin, the addition of Ca(2+) induced capacitative calcium entry (CCE). Xestospongin C (3 - 10 microM) inhibited the DNP-HSA-induced CCE, whereas it did not affect the thapsigargin-induced CCE. 6. These results suggest that Fc epsilon RI-mediated generation of IP(3) contributes to Ca(2+) release not only in the initial phase but also in the sustained phase of the increase in [Ca(2+)](i), resulting in prolonged Ca(2+) depletion in the ER. The ER Ca(2+) depletion may subsequently activate CCE to achieve a continuous [Ca(2+)](i) increase, which is necessary for degranulation in the RBL-2H3 mast cells. Xestospongin C may inhibit Ca(2+) release and consequently may attenuate degranulation.     

A novel action of the antianginal drug bepredil: induction of internal Ca(2+) release and external Ca(2+) influx in Madin-Darby canine kidney (MDCK) epithelial cells

The effect of the antianginal drug bepridil on Ca(2+) signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Bepridil at 10-50 microM evoked a significant rise in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in a dose-dependent manner. The [Ca(2+)](i) rise consisted of an immediate initial rise and a slow decay. Removal of external Ca(2+) partly inhibited the Ca(2+) signals by reducing both the initial rise and the decay phase, suggesting that bepridil activated both external Ca(2+) influx and internal Ca(2+) release. In Ca(2+)-free medium, pretreatment with 50 microM bepridil nearly abolished the Ca(2+) release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, and vice versa, pretreatment with thapsigargin inhibited most of the bepridil-induced Ca(2+) release, suggesting that the thapsigargin-sensitive Ca(2+) store was the main source of bepridil-induced Ca(2+) release. Bepridil (50 microM) induced considerable Mn(2+) quench of fura-2 fluorescence at an excitation wavelength of 360 nm, which was partly inhibited by La(3+) (0.1 mM). Consistently, La(3+) (0.1 mM) pretreatment significantly inhibited the bepridil-induced [Ca(2+)](i) rise. Addition of 3 mM Ca(2+) induced a significant [Ca(2+)](i) rise after prior incubation with 10-50 microM bepridil in Ca(2+)-free medium, suggesting that bepridil induced dose-dependent capacitative Ca(2+) entry. However, 50 microM bepridil inhibited 1 microM thapsigargin-induced capacitative Ca(2+) entry by 38%. Pretreatment with aristolochic acid (40 microM) so as to inhibit phospholipase A(2) inhibited 50 microM bepridil-induced internal Ca(2+) release by 42%, but inhibition of phospholipase C with U73122 (2 microM) or inhibition of phospholipase D with propranolol (0.1 mM) had little effect, suggesting that bepridil induced internal Ca(2+) release in an inositol 1,4,5-trisphosphate-independent manner that could be modulated by phospholipase A(2)-coupled events. This is the first report providing evidence that bepridil, currently used as an antianginal drug, induced a rise in [Ca(2+)](i) in a non-excitable cell line.     

Ca(2+) mobilization induced by W-7 in MG63 human osteosarcoma cells.

The effect of N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide hydrochloride (W-7), a widely used calmodulin inhibitor, on intracellular free Ca(2+)levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca(2+)probe. W-7 (20-1000 micro m) induced an increase in [Ca(2+)](i)in a dose-dependent manner, with an EC(50)of 100 microm. The [Ca(2+)](i)signal comprised an initial rise and a sustained plateau without significant decay within 5 min. External Ca(2+)removal decreased the Ca(2+)signals by reducing the peak and sustained phase, indicating W-7-activated intracellular Ca(2+)release and extracellular Ca(2+)influx. W-7 (500 microm) failed to induce a [Ca(2+)](i)increase in a Ca(2+)-free medium after pre-treatment with thapsigargin (1 microm), an endoplasmic reticulum Ca(2+)pump inhibitor. Conversely, W-7 pre-treatment abolished the Ca(2+)release induced by thapsigargin. This suggests that W-7 (500 microm ) released internal Ca(2+)mainly from the endoplasmic reticulum. The addition of 3 mm Ca(2+)increased [Ca(2+)](i)dose-dependently after preincubation with 20-1000 microm W-7 in a Ca(2+)-free medium, implying that W-7 induced capacitative Ca(2+)entry. W-7-induced Ca(2+)release was not altered by inhibiting phospholipase C with 2 microm 1-(6-((17 beta - 3-methoxyestra-1,3, 5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) (U73122). Tryphan blue assay demonstrated that W-7 (200 microm) caused gradual cell death within 30 min of the initial drug exposure. Together, it was found that W-7 induced [Ca(2+)](i)increases in human osteosarcoma cells by releasing internal Ca(2+)from the endoplasmic reticulum, and also by triggering Ca(2+)influx. W-7 may be cytotoxic to osteosarcoma cells.     

Effects of the antianginal drug fendiline on Ca2+ movement in hepatoma cells.

This study investigated the effect of the anti-anginal drug, fendiline, on intracellular free Ca2+ levels ([Ca2+]i) in HA/ 22 human hepatoma cells by using fura-2 as a fluorescent Ca2+ dye. Fendiline (1-100 microM) increased [Ca2+]i with an EC50 of 25 microM. Removal of extracellular Ca2+ reduced the [Ca2+]i signals by 51 +/- 5%. Fendiline (10 microM)-induced Ca2+ release was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Inhibition of phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122) did not alter 10 microM fendiline-induced Ca2+ release. Several other calmodulin antagonists, such as phenoxybenzamine (100-200 microM), trifluoperazine (5-50 microM), and fluphenazine-N-chloroethane (2-100 microM), had no effect on [Ca2+]i. Together, it was found that fendiline increased [Ca2+]i in human hepatoma cells by discharging Ca2+ from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ entry. This effect of fendiline does not appear to be via antagonism of calmodulin.     

Dual effect of flurbiprofen on cell proliferation and agonist-induced Ca(2+) movement in human osteosarcoma cells

In human MG63 osteosarcoma cells, the effect of flurbiprofen on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and proliferation was explored. The proliferation was enhanced by 20-120 microM flurbiprofen, and was decreased by 140-200 microM flurbiprofen. The effect of flurbiprofen on the increases in cytosolic free Ca(2+) levels ([Ca(2+)](i)) induced by ATP, bradykinin, histamine and thapsigargin (an inhibitor of the endoplasmic reticulum Ca(2+) ATPase), was examined. In cell preincubated with 20 or 80 microM flurbiprofen, the [Ca(2+)](i) increases induced by all agonists were attenuated. In the presence of 20 microM flurbiprofen, the decreased [Ca(2+)](i) responses with the agonists were attributed to a defective Ca(2+) influx because this decrease was unobserved in agonists-induced [Ca(2+)](i) increases in the absence of extracellular Ca(2+). In the presence of 80 microM flurbiprofen, both the Ca(2+) influx component and the Ca(2+) releasing (from organelles) component were defective. These results suggest that flurbiprofen could alter proliferation and inhibit [Ca(2+)](i) increases.     

Dual effect of the antianginal drug fendiline on bladder female transitional carcinoma cells: mobilization of intracellular CA2+ and induction of cell death

The effect of fendiline, an antianginal drug, on cytosolic free Ca2+ levels ([Ca2+]i) in populations of bladder female transitional carcinoma (BFTC) cells was explored using fura-2 as a Ca2+ indicator. Fendiline at concentrations between 3 and 200 micromol/l increased [Ca2+]i in a concentration-dependent manner and the signal saturated at 100 micromol/l. The [Ca2+]i signal was biphasic, with an initial rise and a slow decay. Ca2+ removal inhibited the Ca2+ signal by about half in peak amplitude. Adding 3 mmol/l Ca2+ increased [Ca2+]i in cells pretreated with 100 micromol/l fendiline in Ca2+ -free medium, suggesting that fendiline induced Ca2+ influx via capacitative Ca2+ entry. In Ca2+ -free medium, pretreatment with 1 micromol/l thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete the endoplasmic reticulum Ca2+ store inhibited most of the 100 micromol/l fendiline-induced internal Ca2+ release; and conversely, pretreatment with 100 micromol/l fendiline partly inhibited 1 micromol/l thapsigargin-induced Ca2+ release. This indicates that the major internal Ca2+ store of fendiline-induced [Ca2+]i increases is located in the endoplasmic reticulum. The Ca2+ release induced by 100 micromol/l fendiline may be partly mediated by inositol 1,4,5-trisphosphate, because the [Ca2+]i increase was inhibited by 50% by inhibiting phospholipase C with 2 micromol/l U73122. Fendiline (100 micromol/l) decreased cell viability by 12-44% after being added to cells for 2- 30 min. Together, the findings indicate that in BFTC cells, fendiline exerts a dual effect: mobilization of intracellular Ca2+ and induction of cell death.