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.     

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.     

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.     

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.     

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.     

The mechanism of sertraline-induced [Ca2+]i rise in human OC2 oral cancer cells

Effect of sertraline, an antidepressant, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in human cancer cells is unclear. This study examined if sertraline altered basal [Ca(2+)](i) levels in suspended OC2 human oral cancer by using fura-2 as a Ca(2+)-sensitive fluorescent probe. At concentrations of 10-100 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 suppression of phospholipase A2, inhibition of store-operated Ca(2+) channels or by modulation of protein kinase C activity. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished sertraline-induced Ca(2+) release. Conversely, pretreatment 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 did not change sertraline-induced [Ca(2+)](i) rise. Together, in human oral cancer cells, sertraline induced [Ca(2+)](i) rises by causing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) influx via store-operated Ca(2+) channels.     

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.     

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.     

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.     

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.     

Effect of riluzole on cytosolic Ca2+ increase in human osteosarcoma cells

In human osteosarcoma MG63 cells, the effect of the neuroprotective drug riluzole on the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2. Riluzole (50-500 micromol/l) caused a rapid and sustained plateau increase in [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 150 micromol/l). The riluzole-induced rise in [Ca(2+)](i) was prevented by 58 and 20% by extracellular Ca(2+) removal and nifedipine, respectively, but was not changed by La(3+) and verapamil. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, caused a monophasic increase in [Ca(2+)](i), after which the increasing effect of riluzole on [Ca(2+)](i) was attenuated by 84%; also, pretreatment with riluzole abolished the thapsigargin-induced [Ca(2+)](i) increase. U73122, an inhibitor of phospholipase C, abrogated the ATP (but not riluzole)-induced rise in [Ca(2+)](i). A low concentration (6 micromol/l) of riluzole selectively potentiated the bradykinin (but not ATP and histamine)-induced increase in [Ca(2+)](i). These results suggest that riluzole rapidly increases [Ca(2+)](i) by stimulating both the extracellular Ca(2+) influx via a nifedipine-sensitive pathway and intracellular Ca(2+) release from the ER via an as yet unidentified mechanism(s).     

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.     

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.     

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.     

Triethyltin increases cytosolic Ca(2+) levels in human osteoblasts

In human osteosarcoma MG63 cells, effect of triethyltin, an environmental toxicant, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2. Triethyltin (1-50 μM) caused a rapid and sustained plateau rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50)=10 μM). Triethyltin-induced [Ca(2+)](i) rise was prevented by 50% 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 triethyltin on [Ca(2+)](i) was attenuated by 60%; also, pretreatment with triethyltin abolished thapsigargin-induced [Ca(2+)](i) increase. Depletion of mitochondrial Ca(2+) with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 μM) did not affect triethyltin-induced Ca(2+) release. U73122, an inhibitor of phoispholipase C, abolished ATP (but not triethyltin)-induced [Ca(2+)](i) rise. A low concentration (1 μM) of triethyltin failed to alter ATP and bradykinin-induced [Ca(2+)](i) rises. These findings suggest that triethyltin rapidly increases [Ca(2+)](i) in osteoblasts by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release via as yet unidentified mechanism(s).     

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.     

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.     

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.     

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.