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.     

Fendiline mobilizes intracellular Ca2+ in Chang liver cells

1. The effects of the antianginal drug fendiline (N-[3,3-diphenylpropyl]-alpha-methyl-benzylamine) on intracellular free Ca2+ levels ([Ca2+](i)) in Chang liver cells were evaluated using fura-2 as a fluorescent Ca2+ indicator. 2. Fendiline (1-100 micromol/L) increased [Ca2+](i) in a concentration-dependent manner, with an EC50 of 25 micromol/L. 3. The [Ca2+](i) response was composed of an initial rise and a slow decay to a sustained phase. Removal of extracellular Ca2+ partly reduced the [Ca2+](i) signals. 4. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was reduced by 65% following pretreatment with 1 micromol/L thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) to deplete Ca2+ stored in the endoplasmic reticulum. 5. After pretreatment with 10 micromol/L fendiline in Ca2+-free medium for several minutes, addition of 3 mmol/L Ca2+ induced an increase in [Ca2+](i) of a magnitude four-fold greater than control. This increase in [Ca2+](i) was not reduced by 10 micromol/L SKF96365, econazole, nifedipine or verapamil. 6. Fendiline (10 micromol/L)-induced release of intracellular Ca2+ was not altered by inhibition of phospholipase C with 2 micromol/L 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino) hexyl)-1H-pyrrole-2,5-dione (U73122). 7. The results of the present study show that fendiline induces an increase in [Ca2+](i) in Chang liver cells by releasing stored Ca2+ in an inositol 1,4,5-trisphosphate-independent manner and by causing extracellular Ca2+ influx.     

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.     

Mechanism underlying histamine-induced intracellular Ca2+ movement in PC3 human prostate cancer cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells and the underlying mechanism were evaluated using fura-2 as a Ca2+ dye. Histamine at concentrations between 0.1 and 50 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 1 microM. The [Ca2+]i response comprised an initial rise and a slow decay, which returned to baseline within 3 min. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In the absence of extracellular Ca2+, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 10 microM histamine did not increase [Ca2+]i. After pretreatment with 10 microM histamine in a Ca2+-free medium for several minutes, addition of 3 mM Ca2+ induced [Ca2+]i increases. Histamine (10 microM)-induced intracellular Ca2+ release was abolished 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), and by 10 microM pyrilamine but was not altered by 50 microM cimetidine. Collectively, the present study shows that histamine induced [Ca2+]i transients in PC3 human prostate cancer cells by stimulating H1 histamine receptors leading to Ca2+ release from the endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, and by inducing Ca2+ entry.     

Novel effects of 5,8,11-eicosatriynoic acid, a lipoxygenase inhibitor, on Ca2+ mobilization in Madin Darby canine kidney cells

The effect of 5,8,11-eicosatriynoic acid, a widely used lipoxygenase inhibitor, on Ca2+ fate in Madin Darby canine kidney cells was examined by using fura-2 as a Ca2+ probe. At concentrations between 2-100 microM 5,8,11-eicosatriynoic acid increased [Ca2+]i concentration-dependently with an EC50 of 20 microM . Extracellular Ca2+ removal decreased the Ca2+ signals, indicating that 5,8,11-eicosatriynoic acid triggered Ca2+ release and Ca2+ influx. 5,8,11 -Eicosatriynoic acid (30 microM) induced a [Ca2+]i increase in Ca2+-free medium after pretreatment with carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor for 20 min. Conversely, 5,8,11-eicosatriynoic acid pretreatment almost abolished the Ca2+ release induced by carbonylcyanide m-chlorophenylhydrazone and thapsigargin. These results suggest that 30 microM 5,8,11-eicosatriynoic acid released Ca2+ from the endoplasmic reticulum, mitochondria and other stores. Addition of 3 mM Ca2+ increased [Ca2+]i after preincubation with 2-50 microM 5,8,11-eicosatriynoic acid for 10 min. in Ca2+-free medium concentration-dependently. Pretreatment with 10 microM La3+ abolished 30 microM 5,8,11-eicosatriynoic acid -induced [Ca2+]i increases, but adding La3+ during the decay phase had no effect. 5,8,11-Eicosatriynoic acid-induced Ca2+ release was not altered by inhibiting 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), but was decreased by 60% by 40 microM aristolochic acid. Several other lipoxygenase inhibitors such as baicalein (50 microM), 5.8.11.14-eicosatetraynoic acid (ETYA; 0.1-0.2 mM), caffeic acid (5-50 microM), esculetin (5-50 microM), alpha-pentyl-3-(2-quinolinylmethoxy)-benzenemethanol (REV-5901; 0.1-0.2 mM) and alpha-pentyl-4-(2-quinolinylmethoxy)-benzenemethanol (L-655238; 80-100 microM) had no effect on [Ca2+]i. Collectively, the data suggest that the lipoxygenase inhibitor 5,8,11-eicosatriynoic acid induced a [Ca2+]i increase in renal tubular cells concentration-dependently, by releasing intracellular Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing extracellular Ca2+ influx in a La3+-sensitive manner.     

Effect of the antianginal drug bepridil on intracellular Ca2+ release and extracellular Ca2+ influx in human neutrophils

To understand more fully the effects of bepridil, an antiarrhythmic and antianginal drug, on myocardial ischemia-reperfusion injury and systemic immune responses, its effect on intracellular Ca2+ levels ([Ca2+]i) in human neutrophils was investigated by using fura-2 as a fluorescent probe. Bepridil (10-200 microM) increased [Ca2+]i in a concentration-dependent fashion. This signal was partly inhibited by removal of extracellular Ca2+. In a Ca(2+)-free medium, pretreatment with bepridil (100 microM) abolished the Ca2+ release induced by thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, and by carbonylcyanide m-chlorophenylhydrazone (2 microM), a mitochondrial uncoupler. Pretreatment with carbonylcyanide m-chlorophenylhydrazone and thapsigargin, respectively, partly inhibited bepridil-induced Ca2+ release. Addition of Ca2+ (3 mM) increased [Ca2+]i after pretreatment with bepridil (100 microM) in a Ca(2+)-free medium. Bepridil (100 microM)-induced Ca2+ release was not altered when phospholipase C was inhibited by U73122 (2 microM). Both Ca2+ release and Ca2+ entry induced by bepridil (100 microM) were augmented by activating protein kinase C with phorbol 12-myristate 13-acetate (10 nM), and were suppressed by inhibiting protein kinase C with GF 109203X (2 microM). Treatment with bepridil (10-20 microM) for 30 min increased the production of reactive oxygen intermediates (ROI) by more than 50%. Collectively, it was found that bepridil increased [Ca2+]i concentration-dependently in human neutrophils by releasing Ca2+ from the endoplasmic reticulum, mitochondria and, possibly, other compartments in a phospholipase C-independent manner. Bepridil also activated Ca2+ influx. The activity of protein kinase C may regulate bepridil-induced Ca2+ release and Ca2+ entry.     

Zn2+ increases resting cytosolic Ca2+ levels and abolishes capacitative Ca2+ entry induced by ATP in MDCK cells

The effect of Zn2+ on Ca2+ signaling in Madin Darby canine kidney (MDCK) cells was investigated by measuring the changes in the fluorescence of the Ca2+-sensitive dye fura-2. Zn2+ significantly increased cytoplasmic free Ca2+ levels ([Ca2+]i) at concentrations of 2-100 microM. The maximum response was obtained at concentrations of 25-100 microM. The [Ca2+]i rise induced by 100 microM Zn2+ consisted of a gradual rise and a plateau phase, and was primarily mediated by La3+-sensitive extracellular Ca2+ influx because the [Ca2+]i rise was abolished by pretreatment with 100 microM La3+ or removal of extracellular Ca2+, and that Zn2+ induced Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength which was prevented by pretreatment with 100 microM La3+. Pretreatment with 100 microM Zn2+ for 220 s did not reduce the [Ca2+]i rise induced by the endoplasmic reticulum (ER) Ca2+ pump inhibitor, thapsigargin, suggesting that Ca2+ release from the ER played a minor role in the Zn2+-induced [Ca2+]i rise. Zn2+ (100 microM) nearly abolished the capacitative Ca2+ entry induced by ATP (100 microM). We also investigated the effect of Zn2+ pretreatment on the [Ca2+]i rise induced by ATP. Zn2+ (100 microM) affected ATP-induced [Ca2+]i rise by abolishing capacitative Ca2+ entry and increasing [Ca2+]i on its own without altering Ca2+ release from intracellular sources. The effect of Zn2+ on [Ca2+]i was dissociated from changes in membrane potential.     

Fluoxetine-induced Ca2+ signals in Madin-Darby canine kidney cells

The effect of fluoxetine on Ca2+ signaling in Madin-Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca2+]i concentration-dependently between 5 microM and 200 microM with an EC50 value of 40 microM. The response was reduced by external Ca2+ removal by 30%40%. In Ca2+-free medium pretreatment with 1 microM thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+ pump, abolished 100 microM fluoxetine-induced Ca2+ release. Addition of 3 mM Ca2+ to Ca2+-free medium increased [Ca2+]i when cells were pretreated with 100 microM fluoxetine. Suppression of 1,4,5-trisphosphate (IP3) formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 100 microM fluoxetine-induced Ca2+ release. Fluoxetine (5-100 microM) also increased [Ca2+]i in neutrophils, prostate cancer cells and bladder cancer cells from human and rat glioma cells.     

Tamoxifen-induced Ca2+ mobilization in bladder female transitional carcinoma cells

This study examined the effect of tamoxifen, an anti-breast cancer drug, on Ca2+ handling in bladder female transitional cancer cells. Changes in cytosolic free Ca2+ levels were recorded by using the Ca2+-sensitive dye fura-2. In a dose-dependent manner, tamoxifen induced intracellular free Ca2+ concentrations ([Ca2+]i) increases between 5 and 20 microM with an EC50 of 10 microM. External Ca2+ removal reduced the response by 60+/-6%. Addition of 3 mM Ca2+ caused a [Ca2+]i increase after pretreatment with 10 microM tamoxifen in Ca2+-free medium. In Ca2+-free medium, pretreatment with 10 microM tamoxifen abolished the [Ca2+]i increase induced by 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 1 microM thapsigargin prevented tamoxifen from releasing more Ca2+. Inhibition of phospholipase C-dependent inositol 1,4,5-tris-phosphate formation with 2 microM U73122 did not alter 10 microM tamoxifen-induced Ca2+ release. The [Ca2+]i increase induced by 5 microM tamoxifen was not altered by 10 microM La3+, nifedipine, verapamil, and diltiazem. Collectively, it was found that tamoxifen increased [Ca2+]i in bladder cancer cells by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from external medium.     

Effect of fluoxetine on intracellular Ca2+ levels in bladder female transitional carcinoma (BFTC) cells.

The effect of the antidepressant fluoxetine on Ca2+ signaling in cultured cells was largely unknown. The effect of various concentrations of fluoxetine on [Ca 2+] i in populations of bladder female transitional cancer (BFTC) cells was evaluated by using fura-2 as a Ca2+ probe. Fluoxetine increased [Ca 2+] i concentration dependently (20-100 microM) with an EC50 value of 30 microM. The response was inhibited by 50-60% on extracellular Ca2+ removal. In Ca2+ -free medium, pretreatment with 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca2+ pump) abolished 50 microM fluoxetine-induced Ca2+ release; whereas pretreatment with fluoxetine did not alter the thapsigargin-induced Ca2+ response. Addition of 3 mM Ca2+ increased [Ca 2+] i after pretreatment with 50 microM fluoxetine in Ca2+ -free medium, suggestive of fluoxetine-induced capacitative Ca2+ entry. Suppression of inositol 1,4,5-trisphosphate formation by 2 microM U73122 (a phospholipase C inhibitor) did not affect 50 microM fluoxetine-induced Ca2+ release. Collectively, this study shows that fluoxetine increased [Ca 2+] i in bladder cancer cells in a concentration-dependent fashion, by releasing Ca2+ from thapsigargin-sensitive Ca2+ stores in an IP3-independent manner, and by inducing Ca2+ influx from extracellular medium.     

Tamoxifen-induced [Ca2+]i rise and apoptosis in corneal epithelial cells

The effect of tamoxifen on cytosolic free Ca2+ concentrations ([Ca2+]i) and viability has not been explored in corneal epithelial cells. This study examined whether tamoxifen altered [Ca2+]i and viability in SIRC corneal epithelial cells. Tamoxifen at concentrations > or = 1 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 6 microM. The Ca2+ signal was reduced substantially by removing extracellular Ca2+. Tamoxifen induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was insensitive to Ca2+ entry inhibitors and protein kinase C modulators. After pretreatment with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), tamoxifen-induced [Ca2+]i rises were abolished; conversely, tamoxifen pretreatment abolished thapsigargin-induced [Ca2+]i rises. Inhibition of phospholipase C with U73122 did not change the [Ca2+]i rises. At concentrations of 5-30 microM, tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 15 microM tamoxifen was not reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Apoptosis was induced by 5-30 microM tamoxifen. Tamoxifen (30 microM did not induce production of reactive oxygen species (ROS). Collectively, in SIRC cells, tamoxifen induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via unknown pathways. Tamoxifen-caused cytotoxicity was partly mediated by a Ca2+-independent apoptotic pathway.     

Effect of the neuroprotective agent riluzole on intracellular Ca2+ levels in IMR32 neuroblastoma cells

Riluzole is an effective neuroprotective drug. Its effect on intracellular free Ca2+ levels ([Ca2+]i) has not been explored. This study examined the effect of riluzole on [Ca2+]i in IMR32 neuroblastoma cells using fura-2 as a Ca2+ probe. Riluzole 0.1-1 mM increased [Ca2+]i in a concentration-dependent manner. Removal of extracellular Ca2+ inhibited the response by 52 +/- 5%. The [Ca2+]i increase induced by 0.2 mM riluzole was unaltered by 0.1 mM La3+ or 10 microM verapamil, but was inhibited by 51 +/- 4% by 10 microM nifedipine. In Ca2+-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) reduced the 0.2 mM riluzole-induced Ca2+ release by 44 +/- 3%; this reduction was augmented to 66 +/- 5% by additionally depleting the Ca2+ stores in the Golgi complex with 50 microM brefeldin A. Inhibition of inositol 1,4,5-trisphosphate formation by 2 microM U73122, a phospholipase C inhibitor, did not affect Ca2+ release induced by 0.2 microM riluzole. It was concluded that the neuroprotective agent riluzole increased [Ca2+]i in IMR32 neuroblastoma cells concentration-dependently by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner and also by inducing nifedipine-sensitive Ca2+ influx.     

Mercuric chloride alters the membrane potential and intracellular calcium level in mouse pancreatic islet cells

In this study, mercuric chloride was applied to the primary cultures of mouse pancreatic islet cells for studying its effects on resting membrane potential and the intracellular free calcium ion concentration ([Ca 2+), using the techniques of electrophysiology and fluorometry. It was observed that mercuric chloride (1-100 microM) caused a rapid and sustained depolarization, and induced a rapid first phase and a large sustained second phase of elevation in fura-2 fluorescence ratio in islet cells. The depolarization and increased lCa2+]i induced by mercuric chloride could be inhibited by dithiothreitol (a sulfhydryl-containing reducing agent). Removing Ca2+ from the external medium inhibited the mercuric chloride-induced elevation of [Ca2+]i. The increased [Ca2+]i may also originate from the endoplasmic reticulum of pancreatic islet cells, since caffeine (an activator of Ca2+ release from endoplasmic reticulum) and thapsigargin (an inhibitor of endoplasmic reticulum Ca2+-ATPase) could antagonize the effect of mercuric chloride. Moreover, in the absence of glucose in the medium, the response of islet cells to mercuric chloride was a rapid first phase of increased [Ca2+]i followed by a small sustained second phase. Readministration of 5 mM glucose was sufficient but transient to restore sustained phase of increased [Ca2+]i. The increase of [Ca2+]i in islet cells induced by a lower concentration of mercuric chloride (5 microM) was potentiated in higher glucose (7.5 mM) medium. Tolbutamide, an inhibitor of the ATP-sensitive K+-channel, could also inhibit the effect of mercuric chloride. These findings suggest that mercuric chloride initially interacts with the sulfhydryl groups of membrane-bound proteins, which may be an ATP-sensitive K+ channel, to cause depolarization of the islet cells. This depolarization triggers Ca2+ influx and then the release of Ca2+ from the endoplasmic reticulum.     

Novel effect of N-palmitoyl-L-serine phosphoric acid on cytosolic Ca2+ levels in human osteoblasts

The effect of N-palmitoyl-L-serine phosphoric acid (L-NASPA), which has been used as an inhibitor of lysophosphatidic acid receptors, on intracellular Ca2+ concentration ([Ca2+]i) in human osteosarcoma MG63 cells was measured by using fura-2. L-NASPA (0.1-10 microM) caused a rapid and transient plateau [Ca2+]i rise in a concentration-dependent manner (EC50=0.5 microM). The L-NASPA-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+ but was not altered by L-type voltage-gated Ca2+ channel blockers. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, induced a [Ca2+]i rise, after which the increasing effect of L-NASPA on [Ca2+]i was completely inhibited; also, pretreatment with L-NASPA partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phospholipase C, abolished histamine (but not L-NASPA)-induced [Ca2+]i rise. Overnight incubation with 1 microM L-NASPA did not affect cell proliferation, but 10-20 microM L-NASPA exerted 4% and 15% inhibition, respectively. Collectively, L-NASPA rapidly increased [Ca2+]i in MG63 cells by evoking both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at higher concentrations.     

Mechanisms of AM404-induced [Ca(2+)](i) rise and death in human osteosarcoma cells

The effect of N-(4-hydroxyphenyl) arachidonoyl-ethanolamide (AM404), a drug commonly used to inhibit the anandamide transporter, on intracellular free Ca2+ levels ([Ca2+]i) and viability was studied in human MG63 osteosarcoma cells using the fluorescent dyes fura-2 and WST-1, respectively. AM404 at concentrations > or = 5 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 60 microM. The Ca2+ signal was reduced partly by removing extracellular Ca2+. AM404 induced Mn2+ quench of fura-2 fluorescence implicating Ca2+ influx. The Ca2+ influx was sensitive to La3+, Ni2+, nifedipine and verapamil. In Ca2+-free medium, after pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), AM404-induced [Ca2+]i rise was abolished; and conversely, AM404 pretreatment totally inhibited thapsigargin-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 did not change AM404-induced [Ca2+]i rise. At concentrations between 10 and 200 microM, AM404 killed cells in a concentration-dependent manner presumably by inducing apoptotic cell death. The cytotoxic effect of 50 microM AM404 was partly reversed by prechelating cytosolic Ca2+ with BAPTA/AM. Collectively, in MG63 cells, AM404 induced [Ca2+]i rise by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca2+ influx via L-type Ca2+ channels. AM404 caused cytotoxicity which was possibly mediated by apoptosis.     

Nonylphenol-induced Ca2+ elevation and Ca2+-independent cell death in human osteosarcoma cells

The effect of the environmental toxicant nonylphenol on cytosolic free Ca2+ concentration ([Ca2+]i) and proliferation has not been explored in human osteoblast-like cells. This study examined whether nonylphenol alters Ca2+ levels and causes cell death in MG63 human osteosarcoma cells. [Ca2+]i and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Nonylphenol at concentrations above 3 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+. The nonylphenol-induced Ca2+ influx was insensitive to blockade of L-type Ca2+ channel blockers. After pretreatment with 10 microM nonylphenol, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to induce [Ca2+]i rises. Inhibition of phospholipase C with 2 microM U73122 did not change nonylphenol-induced [Ca2+]i rises. The nonylphenol-induced [Ca2+]i rises were enhanced or inhibited by phorbol myristate acetate or GF 109203X, respectively. At concentrations of 10 and 20 microM nonylphenol killed 55% and 100% cells, respectively. The cytotoxic effect of 10 microM nonylphenol was unaltered by pre-chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, nonylphenol induced [Ca2+]i rises by causing Ca2+ release from intracellular stores and Ca2+ influx from extracellular space. Furthermore, nonylphenol can cause Ca2+-unrelated cytotoxicity in a concentration-dependent manner.     

Effect of nortriptyline on intracellular Ca2+ handling and proliferation in human osteosarcoma cells

The effect of the antidepressant nortriptyline, on bone cells is unknown. In human osteosarcoma MG63 cells, the effect of nortriptyline on intracellular Ca2+ concentration ([Ca2+]i) and proliferation was measured by using fura-2 and tetrazolium, respectively. Nortriptyline (> or = 10 microM) caused a [Ca2+]i rise in a concentration-dependent manner (EC50 = 200 microM). Nortriptyline-induced [Ca2+]i rise was prevented by 60% by removal of extracellular Ca2+ but was not altered by voltage-gated Ca2+ channel blockers. 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 nortriptyline on [Ca2+]i was abolished; also, pretreatment with nortriptyline abolished thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, did not affect nortriptyline-induced [Ca2+]i rise; however, activation of protein kinase C decrease nortriptyline-induced [Ca2+]i rise by 32%. Overnight incubation with 50 and 100 microM nortriptyline killed 78% and 97% of cells, respectively; while 10 microM nortriptyline had no effect. These data suggest that nortriptyline rapidly increases [Ca2+]i in human osteosarcoma cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic at high concentrations.     

Effect of triethyltin on Ca2+ movement in Madin-Darby canine kidney cells

The effects of the environmental toxicant, triethyltin, on Ca2+ mobilization in Madin-Darby canine kidney (MDCK) cells have been examined. Triethyltin induced an increase in cytosolic free Ca2+ levels ([Ca2+]i) at concentrations larger than 2 microM in a concentration-dependent manner. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was partly reduced by removing extracellular Ca2+. In Ca(2+)-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca2+ pump inhibitor, reduced 50 microM triethyltin-induced [Ca2+]i increase by 80%. Conversely, pretreatment with triethyltin abolished thapsigargin-induced Ca2+ release. Pretreatment with U73122 (2 microM) to inhibit phospholipase C-coupled inositol 1,4,5-trisphosphate formations failed to alter 50 microM triethyltin-induced Ca2+ release. Incubation with triethyltin at a concentration (1 microM) that did not increase basal [Ca2+]i for 3 min did not alter ATP (10 microM)- and bradykinin (1 microM)-induced [Ca2+]i increases. Collectively, this study shows that triethyltin altered Ca2+ movement in renal tubular cells by releasing Ca2+ from multiple stores in an inositol 1,4,5-trisphosphate-independent manner, and by inducing Ca2+ influx.     

The ether lipid ET-18-OCH3 increases cytosolic Ca2+ concentrations in Madin Darby canine kidney cells.

The effect of the ether lipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphorylcholine (ET-18-OCH3) on the intracellular free Ca2+ concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was studied using fura-2 as the Ca2+ probe. In Ca2+ medium, ET-18-OCH3 induced a significant rise in [Ca2+]i at concentrations between 10-100 microM with a concentration-dependent delay of 45-175 s. The [Ca2+]i signal was composed of a gradual rise and a sustained plateau. In Ca2+-free medium, ET-18-OCH3 (10-100 microM) induced a Ca2+ release from internal Ca2+ stores with a concentration-dependent delay of 45-175 s. This discharge of internal Ca2+ triggered capacitative Ca2+ entry in a concentration-dependent manner. This capacitative Ca2+ entry was not inhibited by econazole (25 microM), 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF96365; 50 microM), nifedipine (10 microM), verapamil (10 microM), diltiazem (10 microM) and cadmium (0.5 microM). Methyl 2-(phenylthio)ethyl-1,4-dihydro-2,4,6-trimethylpyridine-3,5-dicarboxylat e (PCA-4248), a platelet-activating factor (PAF) receptor antagonist, inhibited 25 microM ET-18-OCH3-induced [Ca2+]i rise in a concentration-dependent manner between 1-20 microM, with 20 microM exerting a complete block. The [Ca2+]i rise induced by ET-18-OCH3 (25 microM) was not altered when the production of inositol 1,4,5-trisphosphate (IP3) was suppressed by the phospholipase C inhibitor U73122 (2 microM), but was partly inhibited by the phospholipase D inhibitor propranolol (0.1 mM) or the phospholipase A2 inhibitor aristolochic acid (20-40 microM). In Ca2+-free medium, pretreatment with 25 microM ET-18-OCH3 completely depleted the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-sensitive Ca2+ store. In contrast, pretreatment with thapsigargin abolished 0.1 mM ATP-induced [Ca2+]i rise without altering the ET-18-OCH3-induced [Ca2+]i rise. This suggests that ET-18-OCH3 depleted thapsigargin-sensitive Ca2+ stores and also released Ca2+ from thapsigargin-insensitive stores. The thapsigargin-insensitive stores involve mitochondria because the mitochondria uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM) induced a release of mitochondrial Ca2+ which was abolished by pretreatment with 25 microM ET-18-OCH3. ET-18-OCH3 (25 microM) induced a significant Mn2+ quench of fura-2 fluorescence at 360 nm excitation wavelength confirming that ET-18-OCH3 induced capacitative Ca2+ entry. La3+ (0.1 mM) or Gd3+ (50 microM) abolished the ET-18-OCH3-induced Mn2+ quench and [Ca2+]i rise. Our data imply that ET-18-OCH3 induced a [Ca2+]i rise in MDCK cells by activating PAF receptors leading to an internal Ca2+ release followed by capacitative Ca2+ entry. Phospholipase D and phospholipase A2, but not phospholipase C, might be involved in mediating the capacitative Ca2+ entry. La3+ abolished the ET-18-OCH3-induced [Ca2+]i rise presumably by inhibiting PAF receptors.     

Oxidation by thimerosal increases calcium levelsin renal tubular cells

The effect of thimerosal, a reactive oxidant, on cytoplasmic free Ca2+ concentrations ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored by using the Ca2+-sensitive dye fura-2. Thimerosal acted in a concentration-dependent manner with an EC50 of 0.5 microM. The Ca2+ signal comprised a gradual rise and a sustained elevation. Removal of extracellular Ca2+ reduced 80% of the signal. In Ca2+-free medium, the [Ca2+]i rise induced by 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) was completely inhibited by pretreatment with 5 microM thimerosal. The thimerosal (5 microM)-induced Ca2+ release was not changed by inhibition of phospholipase C with 2 microM U73122. Collectively, this study shows that thimerosal induced [Ca2+]i rises in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity.