Effect of fluoxetine on [Ca²âº]i and cell viability in OC2 human oral cancer cells.

Fluoxetine is a serotonin-specific reuptake inhibitor that has been used as an antidepressant. This study examined the effect of fluoxetine on cytosolic free Ca²âº concentrations ([Ca2⁺]i) and viability in OC2 human oral cancer cells. The Ca²âº-sensitive fluorescent dye fura-2 was used to measure [Ca²âº]i, and the water soluble tetrazolium (WST-1) regent was used to measure viability. Fluoxetine induced [Ca²âº]i rises concentration-dependently. The response was reduced by half by removing extracellular Ca²âº. Fluoxetine-induced Ca²âº entry was enhanced by activation of protein kinase C (PKC) with phorbol 12-myristate 13 acetate (PMA) but was inhibited by inhibition of the enzyme with GF109203X. In Ca²âº-free medium, treatment with the endoplasmic reticulum Ca²âº pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) or thapsigargin abolished fluoxetine-evoked [Ca²âº]i rise. Conversely, treatment with fluoxetine inhibited BHQ/thapsigargin-evoked [Ca²âº]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished fluoxetine-induced [Ca²âº]i rise. At 20-80 µM, fluoxetine decreased cell viability concentration-dependently, which was not altered by chelating cytosolic Ca²âº with 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). At 20-60 µM, fluoxetine induced apoptosis as detected by annexin V/propidium iodide (PI) staining. Together, in OC2 cells, fluoxetine induced [Ca²âº]i rises by evoking PLC-dependent Ca²âº release from the endoplasmic reticulum and Ca²âº entry via PKC-regulated mechanisms. Fluoxetine also caused Ca²âº-independent apoptosis.

Effect of sertraline on [Ca2+](i) and viability of human MG63 osteosarcoma cells.

The antidepressant, sertraline, has been shown to have diverse in vitro effects. This study examined whether sertraline altered [Ca(2+)](i) in MG63 human osteosarcoma cells by using fura-2 as a Ca(2+)-sensitive fluorescent dye. At 50-200 µM, sertraline induced a [Ca(2+)](i) rise in a concentration-dependent manner. Ca(2+) response was decreased by removing extracellular Ca(2+), suggesting that Ca(2+) entry and release contributed to the [Ca(2+)](i) signal. Sertraline-induced Ca(2+) entry was inhibited by nifedipine, La(3+), Gd(3+), and SK&F96365. When extracellular Ca(2+) was removed, pretreatment with the endoplasmic reticulum (ER) Ca(2+) pump inhibitor, thapsigargin, or 2,5-di-tert-butylhydroquinone (BHQ) abolished the sertraline-evoked [Ca(2+)](i) rise. Incubation with sertraline also abolished the thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C (PLC) with U73122 abolished the sertraline-induced [Ca(2+)](i) rise. At 20-30 µM, overnight treatment with sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of sertraline was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data demonstrate that sertraline (30 µM) evoked apoptosis. Sertraline (20 and 30 µM) also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, sertraline evoked a [Ca(2+)](i) rise by inducing PLC-dependent Ca(2+) release from the ER and Ca(2+) entry by L-type Ca(2+) channels and store-operated Ca(2+) channels. Sertraline induced cell death that may involve apoptosis by mitochondrial pathways.

M-3M3FBS-induced Ca² ⁺ movement and apoptosis in HA59T human hepatoma cells.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca² ⁺ concentrations ([Ca² ⁺ ]i ) in HA59T human hepatoma cells is unclear. This study explored whether m-3M3FBS elevated basal [Ca² ⁺ ]i levels in suspended cells by using fura-2 as a Ca² ⁺ -sensitive fluorescent dye. M-3M3FBS at concentrations of 10- 50 µM increased [Ca² ⁺ ]i in a concentration-dependent fashion. The Ca² ⁺ signal was reduced partly by removing extracellular Ca² ⁺ . M-3M3FBS-induced Ca² ⁺ influx was inhibited by nifedipine, econazole, SK&F96365, aristolochic acid, and GF109203X. In Ca² ⁺ -free medium, 50 µM m-3M3FBS pretreatment inhibited the [Ca² ⁺ ]i rise induced by the endoplasmic reticulum Ca² ⁺ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin partly reduced m-3M3FBS-induced [Ca² ⁺ ]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca² ⁺ ]i rise. At concentrations between 10 and 40 µM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca² ⁺ with 1,2-bis(2- aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data suggest that m-3M3FBS induced apoptosis in a concentration-dependent manner. M-3M3FBS also increased levels of reactive oxygen species. Together, in human hepatoma cells, m-3M3FBS induced a [Ca² ⁺ ]i rise by inducing phospholipase C-independent Ca² ⁺ release from the endoplasmic reticulum and Ca² ⁺ entry via protein kinase C-sensitive store-operated Ca² ⁺ channels. M-3M3FBS induced cell death that might involve apoptosis via mitochondrial pathways.

Investigation of carvedilol-evoked Ca²+ movement and death in human oral cancer cells.

The effect of carvedilol on cytosolic free Ca²âº concentrations ([Ca²âº](i)) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²âº](i) levels in suspended OC2 cells by using fura-2 as a Ca²âº-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²âº](i) in a concentration-dependent fashion. The Ca²âº signal was decreased by 50% by removing extracellular Ca²âº. Carvedilol-induced Ca²âº entry was not affected by the store-operated Ca²âº channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²âº-free medium, incubation with the endoplasmic reticulum Ca²âº pump inhibitor thapsigargin did not change carvedilol-induced [Ca²âº](i) rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²âº release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²âº](i) release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²âº](i) rise. Carvedilol at 5-50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²âº was chelated with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acetoxymethyl ester (BAPTA/AM). Annexin V/propidium iodide staining assay suggests that apoptosis played a role in the death. Collectively, in OC2 cells, carvedilol induced [Ca²âº](i) rise by causing phospholipase C-independent Ca²âº release from mitochondria and non-endoplasmic reticulum stores, and Ca²âº influx via protein kinase C-regulated channels. Carvedilol (up to 50 µM) induced cell death in a Ca²âº-independent manner that involved apoptosis.

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

Capsazepine has been widely used as a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on most cell types is unknown. In human PC3 prostate cancer cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was investigated by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 75 microM. Capsazepine-induced [Ca(2+)](i) rise was reduced by 60% by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was contributed by extracellular Ca(2+) influx and intracellular Ca(2+). Consistently, the capsazepine (200 microM)-induced [Ca(2+)](i) rise was decreased by La(3+) by half. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the effect of capsazepine on [Ca(2+)](i) was inhibited by 80%. Conversely, pretreatment with capsazepine partly reduced thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. These findings suggest that in human PC3 prostate cancer cells, capsazepine increases [Ca(2+)](i) by evoking Ca(2+) influx and releasing Ca(2+) from the endoplasmic reticulum via a phospholiase C-independent manner. Overnight incubation with capsazepine (200 microM) killed 37% of cells, which could not be prevented by chelating intracellular Ca(2+) with BAPTA.

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.

Independent [Ca2+]i increases and cell proliferation induced by the carcinogen safrole in human oral cancer cells.

The effect of the carcinogen safrole on intracellular Ca2+ movement and cell proliferation has not been explored previously. The present study examined whether safrole could alter Ca2+ handling and growth in human oral cancer OC2 cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at a concentration of 325 microM started to increase [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 40% by removing extracellular Ca2+, and was decreased by 39% by nifedipine but not by verapamil or diltiazem. In Ca2+-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) barely induced a [Ca2+]i rise; in contrast, addition of safrole after thapsigargin treatment induced a small [Ca2+]i rise. Neither inhibition of phospholipase C with 2 microM U73122 nor modulation of protein kinase C activity affected safrole-induced Ca2+ release. Overnight incubation with 1 microM safrole did not alter cell proliferation, but incubation with 10-1000 microM safrole increased cell proliferation by 60+/-10%. This increase was not reversed by pre-chelating Ca2+ with 10 microM of the Ca2+ chelator BAPTA. Collectively, the data suggest that in human oral cancer cells, safrole induced a [Ca2+]i rise by causing release of stored Ca2+ from the endoplasmic reticulum in a phospholipase C- and protein kinase C-independent fashion and by inducing Ca2+ influx via nifedipine-sensitive Ca2+ entry. Furthermore, safrole can enhance cell growth in a Ca2+-independent manner.

Effect of nordihydroguaiaretic acid on intracellular Ca(2+) concentrations in C6 glioma cells.

The effect of nordihydroguaiaretic acid (NDGA) on Ca(2+) signaling in C6 glioma cells has been investigated. NDGA (5-100 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 NDGA-induced [Ca(2+)]i signals by 52+/-2%. After incubation of cells with NDGA in Ca(2+)-free medium for 4 min, addition of 3 mM CaCl2 induced a concentration-dependent increase in [Ca(2+)]i. NDGA (100 microM)-induced [Ca(2+)]i increases in Ca(2+)-containing medium was not changed by pretreatment with 10 microM nifedipine or verapamil. In Ca(2+)-free medium, pretreatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM) abolished 100 microM NDGA-induced [Ca(2+)]i increases. Inhibition of phospholipase C with 2 microM U73122 had little effect on 100 microM NDGA-induced Ca(2+) release. Several other lipoxygenase inhibitors had no effect on basal [Ca(2+)]i. Collectively, the results suggest that NDGA increased [Ca(2+)]i in glioma cells in a lipoxygenase-independent manner, by releasing Ca(2+) from the endoplasmic reticulum in a manner independent of phospholipase C activity and by causing Ca(2+) influx.

The mechanism of carvacrol-evoked [Ca2+]i rises and non-Ca2+-triggered cell death in OC2 human oral cancer cells.

Carvacrol is one of the main substances of essential oil which triggers intracellular Ca(2+) mobilization and causes cytotoxicity in diverse cell models. However, the mechanism of carvacrol-induced Ca(2+) movement and cytotoxicity is not fully understood. This study examined the effect of carvacrol on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)), cell viability and apoptosis in OC2 human oral cancer cells. Carvacrol induced a [Ca(2+)](i) rise and the signal was reduced by removal of extracellular Ca(2+). Carvacrol-induced Ca(2+) entry was not altered by store-operated Ca(2+) channel inhibitors and protein kinase C (PKC) activator, but was inhibited by a PKC inhibitor. In Ca(2+) -free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) or 2,5-di-tert-butylhydroquinone (BHQ) inhibited carvacrol-induced [Ca(2+)](i) rise. Conversely, incubation with carvacrol inhibited TG or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C (PLC) with U73122 abolished carvacrol-induced [Ca(2+)](i) rise. Carvacrol decreased cell viability, which was not reversed when cytosolic Ca(2+) was chelated with BAPTA-AM (1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester). Carvacrol-induced apoptosis and activation of reactive oxygen species (ROS) and caspase-3. Together, carvacrol induced a [Ca(2+)](i) rise by inducing PLC-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via PKC-sensitive, non store-operated Ca(2+) channels. Carvacrol-induced ROS- and caspase-3-associated apoptosis.

Effect of the environmental pollutant bisphenol A dimethacylate (BAD) on Ca2+ movement and viability in OC2 human oral cancer cells.

The environmental pollutant bisphenol A dimethacylate (BAD) has been used as a dental composite. The effect of BAD on cytosolic Ca(2+) concentrations ([Ca(2+)]i) and viability in OC2 human oral cancer cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)]i. BAD induced [Ca(2+)]i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca(2+). BAD-evoked Ca(2+) entry was suppressed by nifedipine, econazole, and SK&F96365. In Ca(2+)-free medium, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin abolished BAD-induced [Ca(2+)]i rise. Inhibition of phospholipase C with U73122 did not alter BAD-induced [Ca(2+)]i rise. At 10-30µM, BAD inhibited cell viability, which was not reversed by chelating cytosolic Ca(2+). BAD (20-30µM) also induced apoptosis. Collectively, in OC2 cells, BAD induced a [Ca(2+)]i rise by evoking phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. BAD also caused apoptosis.

Effect of m-3m3FBS on Ca2+ handling and viability in OC2 human oral cancer cells.

The effect of 2,4,6-trimethyl-N-(meta-3-trifluoromethyl-phenyl)-benzenesulfonamide (m-3M3FBS), a presumed phospholipase C activator, on cytosolic free Ca2+ concentrations ([Ca2+]i) in OC2 human oral cancer cells is unclear. This study explored whether m-3M3FBS changed basal [Ca2+]i levels in suspended OC2 cells by using fura-2 as a Ca2+-sensitive fluorescent dye. M-3M3FBS at concentrations between 10-60 µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. M-3M3FBS-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers nifedipine, econazole and SK&F96365, and by the phospholipase A2 inhibitor aristolochic acid. In Ca2+-free medium, 30 µM m-3M3FBS pretreatment inhibited the [Ca2+]i rise induced by the endoplasmic reticulum Ca2+ pump inhibitors thapsigargin and 2,5-di-tert-butylhydroquinone (BHQ). Conversely, pretreatment with thapsigargin, BHQ or cyclopiazonic acid partly reduced m-3M3FBS-induced [Ca2+]i rise. Inhibition of inositol 1,4,5-trisphosphate formation with U73122 did not alter m-3M3FBS-induced [Ca2+]i rise. At concentrations between 5 and 100 µM m-3M3FBS killed cells in a concentration-dependent manner. The cytotoxic effect of m-3M3FBS was not reversed by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Propidium iodide staining data suggest that m-3M3FBS (20 or 50 µM) induced apoptosis in a Ca2+-independent manner. Collectively, in OC2 cells, m-3M3FBS induced [Ca2+]i rise by causing inositol 1,4,5-trisphosphate-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via phospholipase A2-sensitive store-operated Ca2+ channels. M-3M3FBS also induced Ca2+-independent cell death and apoptosis.

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

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

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

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

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.