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Thymol is a phenolic compound that affects physiology in different cell models. However, whether thymol affects Ca²⁺ homeostasis in prostate cancer cells is unknown. The action of this compound on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and viability in PC3 human prostate cancer cells was explored. The results show that thymol at concentrations of 100-1500 μM caused [Ca²⁺]i rises in a concentration-dependent manner. Removal of extracellular Ca²⁺ reduced thymol’s effect by approximately 80%. Thymol-induced Ca²⁺ entry was confirmed by Mn²⁺ entry-induced quench of fura-2 fluorescence, and was inhibited by approximately 30% by Ca²⁺ entry modulators (nifedipine, econazole, SKF96365), and the protein kinase C (PKC) inhibitor GF109203X. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished thymol-induced [Ca²⁺]i rises. Treatment with thymol also abolished thapsigargin-induced [Ca²⁺]i rises. Thymol-induced Ca²⁺ release from the endoplasmic reticulum was abolished by the phospholipase C (PLC) inhibitor U73122. Thymol at 100-900 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, thymol induced [Ca²⁺]i rises by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels and other unknown channels. Thymol also induced Ca²⁺-dissociated cell death.

The effect of the anti-inflammatory compound NPC-14686 on intracellular Ca²⁺ concentration ([Ca²⁺](i)) and viability in OC2 human oral cancer cells was investigated. The Ca²⁺-sensitive fluorescent probe fura-2 was used to examine [Ca²⁺](i). NPC-14686 induced [Ca²⁺](i) rises in a concentration-dependent fashion. The effect was reduced approximately by 10% by removing extracellular Ca²⁺. NPC-14686- elicited Ca²⁺ signal was decreased by nifedipine, econazole, SKF96365, and GF109203X. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished NPC-14686-induced [Ca²⁺](i) rises. Conversely, pretreatment with NPC-14686 abolished thapsigargin or BHQ-induced [Ca²⁺](i) rises. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²⁺](i) rises. At 20-100 μM, NPC-14686 inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid-acetoxymethyl ester (BAPTA/AM). NPC-14686 between 20 μM and 40 μM also induced apoptosis. Collectively, in OC2 cells, NPC-14686 induced [Ca²⁺](i) rises by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-regulated store-operated Ca²⁺ channels. NPC-14686 also caused Ca²⁺-independent apoptosis.

Safrole is a carcinogen found in plants. The effect of safrole on cytosolic free Ca²⁺ concentrations ([Ca²⁺](i)) and viability in SCM1 human gastric cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺](i). Safrole at concentrations of 150-450 μM induced a [Ca²⁺](i) rise in a concentration-dependent manner. The response was reduced by 60% by removing extracellular Ca²⁺. Safrole-evoked Ca²⁺ entry was not altered by nifedipine, econazole, SKF96365, and protein kinase C activator or inhibitor. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished safrole-evoked [Ca²⁺](i) rises. Conversely, treatment with safrole abolished thapsigargin or BHQ-evoked [Ca²⁺](i) rises. Inhibition of phospholipase C (PLC) with U73122 abolished safrole-induced [Ca²⁺](i) rises. At 250-550 μM, safrole decreased cell viability concentration-dependently, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that safrole (350-550 μM) induced apoptosis concentration-dependently. These studies suggest that in SCM1 human gastric cancer cells, safrole induced [Ca²⁺](i) rises by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via non-store-operated Ca²⁺ entry pathways. Safrole-induced cell death may involve apoptosis.

The effect of the antidepressant doxepin on cytosolic Ca²⁺ concentrations ([Ca²⁺](i)) and viability in PC3 human prostate cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺](i). Doxepin at concentrations of 500-1000 μM induced a [Ca²⁺](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Doxepin-evoked Ca²⁺ entry was suppressed by Ca²⁺ entry blockers (nifedipine, econazole, SK&F96365), and protein kinase C (PKC) modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibit doxepin-induced [Ca²⁺](i) rise. Incubation with doxepin nearly inhibited thapsigargin or BHQ-induced [Ca²⁺](i) rise. Inhibition of phospholipase C (PLC) with U73122 failed to alter doxepin-induced [Ca²⁺](i) rise. At concentrations of 200-250 μM, doxepin killed cells in a concentration-dependent manner. This cytotoxic effect was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/PI staining data implied that doxepin (200 and 250 μM) did not induce apoptosis. Collectively, in PC3 cells, doxepin induced a [Ca²⁺](i) rise by evoking PLC-independent Ca²⁺ release from stores including the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels. Doxepin caused cell death that was independent of [Ca²⁺](i) rises.

Deoxycholic acid (DOA) is one of the secondary bile acids used as a mild detergent for the isolation of membrane associated proteins. This study examined whether the secondary bile acid, DOA, altered Ca(2+) movement, cell viability and apoptosis in SCM1 human gastric cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)]i. DOA-evoked [Ca(2+)]i rises concentration dependently. The response was reduced by removing extracellular Ca(2+). DOA-evoked Ca(2+) entry was inhibited by store-operated Ca(2+) channel inhibitors (nifedipine, econazole and SKF96365), the protein kinase C (PKC) activator phorbol 12-myristate 13 acetate (PMA) and the PKC inhibitor GF109203X. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (TG) abolished DOA-evoked [Ca(2+)]i rises. Conversely, treatment with DOA abolished TG-evoked [Ca(2+)]i rises. Inhibition of phospholipase C with U73122 abolished DOA-evoked [Ca(2+)]i rises. At 100-500 μM, DOA decreased cell viability, which was not changed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). DOA between 100 and 300 μM also induced apoptosis. Collectively, in SCM1 cells, DOA-induced [Ca(2+)]i rises by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via store-operated Ca(2+) channels. DOA also caused Ca(2+)-independent apoptosis.

Methoxychlor, an organochlorine pesticide, is thought to be an endocrine disrupter that affects Ca²⁺ homeostasis and cell viability in different cell models. This study explored the action of methoxychlor on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) and apoptosis in HA59T human hepatoma cells. Fura-2, a Ca²⁺-sensitive fluorescent dye, was applied to measure [Ca²⁺]i. Methoxychlor at concentrations of 0.1-1 μM caused a [Ca²⁺]i rise in a concentration-dependent manner. Removal of external Ca²⁺ abolished methoxychlor's effect. Methoxychlor-induced Ca²⁺ influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Methoxychlor-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SK&F96365, and protein kinase C modulators. Methoxychlor killed cells at concentrations of 10-130 μM in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent methoxychlor's cytotoxicity. Methoxychlor (10 and 50 μM) induced apoptosis concentration-dependently as determined by using Annexin V/propidium iodide staining. Together, in HA59T cells, methoxychlor induced a [Ca²⁺]i rise by inducing Ca²⁺ entry via protein kinase C-sensitive Ca²⁺-permeable channels, without causing Ca²⁺ release from stores. Methoxychlor also induced apoptosis that was independent of [Ca²⁺]i rises.

NPC-14686 has been shown to have anti-inflammatory effect in previous studies, but the mechanisms are unclear. The effect of NPC-14686 on cytosolic Ca^(2+) concentrations ([Ca^(2+)]_i) and viability in MG63 human osteosarcoma cells was explored. The Ca^(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca^(2+)]_i. NPC-14686 at concentrations of 100-500 μM induced a [Ca^(2+)]_i rise in a concentrationdependent manner. The response was reduced by 80% by removing Ca^(2+). NPC-14686 induced Mn2+ influx leading to quenching of fura-2 fluorescence. NPC-14686-evoked Ca^(2+) entry was suppressed by nifedipine, econazole, SK&F96365, and protein kinase C inhibitor. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca^(2+)]_i rise. At 20-50 μM, NPC-14686 decreased cell viability, which was not reversed by chelating cytosolic Ca^(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that NPC-14686 (30-50 μM) induced apoptosis in a concentration-dependent manner. NPC-14686 also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, NPC-14686 induced a [Ca^(2+)]_i rise by inducing phospholipase C-dependent Ca^(2+) release from the endoplasmic reticulum and Ca^(2+) entry via protein kinase C-sensitive store-operated Ca^(2+) channels. NPC-14686 induced cell death that might involve apoptosis via mitochondrial pathways.

Thymol is a natural essential oil present in many plants and has many different effects in various cell types. However, the effect of thymol on the physiology of Madin-Darby canine kidney (MDCK) renal tubular cells is unknown. The action of the phytochemical thymol on cytosolic Ca^(2+) concentrations ([Ca^(2+)]i) and apoptosis in MDCK renal tubular cells was explored. Fura-2, a Ca^(2+)-sensitive fluorescent dye, was used to assess [Ca^(2+)]i. Thymol at concentrations of 200-500 μM caused a [Ca^(2+)]; rise in a concentration-dependent manner. Removal of extracellular Ca^(2+) partially reduced the effects of thymol. Thymol-induced Ca^(2+) entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In a Ca^(2+)-free medium, treatment with the endoplasmic reticulum Ca^(2+) pump inhibitor thapsigargin inhibited thymol-induced [Ca^(2+)]i increases. Treatment with thymol also inhibited thapsigargin-induced [Ca^(2+)]i rise. Thymol killed cells at concentrations of 300-500 μM in a concentration-dependent fashion. Chelating cytosolic Ca^(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent thymol cytotoxicity. Thymol (400 and 500 μM) induced apoptosis detected by using Annexin V/propidium iodide staining. At 400 or 500 μM, thymol increased levels of reactive oxygen species (ROS). Together, in MDCK cells, thymol induced a [Ca^(2+)]i rise by inducing Ca^(2+) release from the endoplasmic reticulum and Ca^(2+) entry via protein kinase C-sensitive store-operated Ca^(2+) channels. Our data suggest that thymol-induced apoptosis might involve ROS production.

Thymol is a phenolic compound that affects physiology in different cell models. However, whether thymol affects Ca²⁺ homeostasis in prostate cancer cells is unknown. The action of this compound on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and viability in PC3 human prostate cancer cells was explored. The results show that thymol at concentrations of 100-1500 μM caused [Ca²⁺]i rises in a concentration-dependent manner. Removal of extracellular Ca²⁺ reduced thymol’s effect by approximately 80%. Thymol-induced Ca²⁺ entry was confirmed by Mn²⁺ entry-induced quench of fura-2 fluorescence, and was inhibited by approximately 30% by Ca²⁺ entry modulators (nifedipine, econazole, SKF96365), and the protein kinase C (PKC) inhibitor GF109203X. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin abolished thymol-induced [Ca²⁺]i rises. Treatment with thymol also abolished thapsigargin-induced [Ca²⁺]i rises. Thymol-induced Ca²⁺ release from the endoplasmic reticulum was abolished by the phospholipase C (PLC) inhibitor U73122. Thymol at 100-900 μM decreased cell viability, which was not reversed by pretreatment with the Ca²⁺ chelator 1,2-bis(2-aminophenoxy) ethane-N,N,N’,N’-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Together, in PC3 cells, thymol induced [Ca²⁺]i rises by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels and other unknown channels. Thymol also induced Ca²⁺-dissociated cell death.

The effect of the natural product 3,3'-diindolylmethane (DIM) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MG63 human osteosarcoma cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). DIM at concentrations of 40-80 μM induced a [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). DIM-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SK&F96365 and protein kinase C modulators. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca(2+)](i) rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca(2+)](i) rise. At concentrations of 10-50 μM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered 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 implicate that DIM (20 and 40 μM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca(2+)](i) rise by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. DIM caused cell death that may involve apoptosis.

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.

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca2+]i levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca2+-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca2+]i in a concentration-dependent fashion. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. This Ca2+ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca2+ channels or by modulation of protein kinase C activity. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca2+ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca2+]i rise, suggesting that thapsigargin released Ca2+ from the endoplasmic reticulum. Inhibition of p...

Effect of sertraline, an antidepressant, on cytosolic free Ca2+ levels ([Ca2+]i) in human cancer cells is unclear. This study examined if sertraline altered basal [Ca2+]i levels in suspended OC2 human oral cancer by using fura-2 as a Ca2+-sensitive fluorescent probe. At concentrations of 10−100 μM, sertraline induced a [Ca2+]i rise in a concentration-dependent fashion. The Ca2+ signal was reduced partly by removing extracellular Ca2+ indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. Sertraline induced Mn2+ influx, leading to quench of fura-2 fluorescence suggesting Ca2+ influx. This Ca2+ influx was inhibited by suppression of phospholipase A2, inhibition of store-operated Ca2+ channels or by modulation of protein kinase C activity. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin or 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished sertraline-induced Ca2+ release. Conversely, pretreatment with sertraline greatly reduced the inhibitor-induced [Ca2+]i rise, suggesting that sertraline released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change sertraline-induced [Ca2+]i rise. Together, in human oral cancer cells, sertraline induced [Ca2+]i rises by causing phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels.

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.

The effect of nortriptyline, a tricyclic antidepressant, on Ca2+ regulation and viability in human prostate cancer cells (PC3) is unclear. The present study examined whether nortriptyline altered basal [Ca2+]i levels in suspended PC3 cells using fura-2 as a Ca2+-sensitive fluorescent probe. Nortriptyline (50–500 µM) increased [Ca2+]i in a concentration-dependent fashion. The Ca2+ signal was partially reduced by removing extracellular Ca2+, indicating that Ca2+ entry and release both contributed to the [Ca2+]i rise. Nortriptyline induced Mn2+ influx, leading to quench of fura-2 fluorescence, suggesting Ca2+ influx. This Ca2+ influx was inhibited by activation of protein kinase C, but not by inhibition of L-type Ca2+ channels. In Ca2+-free medium, pretreatment with the endoplasmic reticulum Ca2+ pump inhibitor, thapsigargin nearly abolished nortriptyline-induced Ca2+ release. Conversely, pretreatment with nortriptyline greatly reduced the inhibitor-induced [Ca2+]i rise, suggesting that nortriptyline released Ca2+ from the endoplasmic reticulum. Inhibition of phospholipase C did not change the nortriptyline-induced [Ca2+]i rise. Nortriptyline at a concentration of 10 µM increased viability in a Ca2+-independent manner. At 50 µM, nortriptyline killed 45% of cells. Nortriptyline at 10 µM did not induce apoptosis, but at 50 µM induced significant apoptosis measured by propidium iodide staining. Together, in PC3 cells, nortriptyline induced [Ca2+]i rises by causing the phospholipase C-independent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via the protein kinase C-sensitive pathway. Nortriptyline also induced both cell proliferation and death in a concentration-dependent manner. Apoptosis was involved in the cell death. Drug Dev Res 71:323–330, 2010. © 2010 Wiley-Liss, Inc.

The effect of maprotiline on cytosolic free Ca2+ concentrations ([Ca2+]i) and cell viability was explored in human osteosarcoma cells (MG63), using the fluorescent dyes fura-2 and WST-1, respectively. Maprotiline at concentrations of ≥20 µM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. The maprotiline-induced Ca2+ influx was sensitive to inhibition by aristolochic acid (a phospholipase A2 inhibitor). In Ca2+-free medium, after treatment with 1 µM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 200 µM maprotiline failed to induce a [Ca2+]i rise. At concentrations of 50–100 µM maprotiline killed cells in a concentration-dependent manner. The cytotoxic effect of 60 µM maprotiline was slightly enhanced by prechelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Propidium iodide staining data suggested that maprotiline induced apoptosis between concentrations of 60–70 µM, which was enhanced by BAPTA. Collectively, in MG63 cells, maprotiline induced [Ca2+]i rises by causing Ca2+ release from the endoplasmic reticulum and Ca2+ influx from phospholipase A2-regulated Ca2+ channels. Furthermore, maprotiline caused apoptosis that was regulated by Ca2+. Drug Dev Res 71: 268–274, 2010. © 2010 Wiley-Liss, Inc.

The effect of Antrodia camphorata (AC) on human oral cancer cells has not been explored. This study examined the effect of AC on the viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca(superscript 2+) regulation of OC2 human oral cancer cells. AC at a concentration of 25μM induced an increase in cell viability, but AC at concentrations ≥50μg/ml decreased viability in a concentration-dependent manner. AC at concentrations of 100-200μg/ml induced apoptosis in a concentration-dependent manner as demonstrated by propidium iodide staining. AC (25μg/ml) did not alter basal [Ca(superscript 2+)](subscript i), but decreased the [Ca2(superscript +)](subscript i) increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin, and histamine increased cell viability whereas thapsigargin decreased it. AC (25μg/ml) pretreatment failed to alter ATP-induced increase in viability, potentiated bradykinininduced increase in viability, decreased histamine-induced increase in viability and reversed thapsigargininduced decrease in viability. Immunoblotting suggested that AC induced phosphorylation of ERK and JNK MAPKs, but not p38 MAPK. Collectively, for OC2 cells, AC exerted multiple effects on their viability and [Ca(superscript 2+)](subscript i), induced their ERK and JNK MAPK phosphorylation, and probably evoked their apoptosis.

1. Short-wave diathermy (SWD) is a form of radiofrequency radiation that is used therapeutically by physiotherapists. The cellular mechanisms of SWD are unclear. The present study was performed to explore the effect of different conditions of short-wave exposure on the proliferation of cultured human chondrocytes. 2. Cells exposed to short waves once per day for seven consecutive days exhibited a significant increase in proliferation by 42% compared with the control cells. In cells that were treated with short waves twice per day for seven consecutive days, or only once on Day 1 and then examined for proliferation on Day 7, cell proliferation was greater than the control cells by 40% and 30%, respectively. 3. Given the importance of mitogen-activated protein kinases (MAPK) in the proliferation of different cell types, efforts were extended to explore the role of three major types of MAPK; that is, extracellular signal-regulated kinase (ERK), c-Jun NH(2)-terminal protein kinase (JNK) and p38. 4. It was found that the level of phosphorylated ERK (phospho-ERK 1 and ERK 2) increased significantly within 5-120 min following consecutive exposure to short waves for 7 days. Exposure to short waves failed to alter the intensity of phosphorylated JNK and p38 within 0-240 min. 5. Cells were exposed to short waves once for seven consecutive days in the presence of 0, 10 micromol/L, 20 micromol/L or 50 micromol/L PD98059 (an ERK inhibitor). PD98059 totally inhibited short waves-induced enhancement of proliferation without altering normal control viability. In the presence of short waves and PD98059, the cell viability was lower than the normal control. Together, the data suggest that short waves could increase proliferation in human chondrocytes through activation of the ERK pathway, which is also involved in maintaining normal cell proliferation under physiological conditions.

The present study explored the effect of Antrodia camphorata (AC) on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation, and Ca2+ regulation in MG63 human osteosarcoma cells. AC (25–50 µg/ml) did not affect cell viability, but at 100–200 µg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25–200 µg/ml did not alter basal [Ca2+]i, but at 25 µg/ml decreased [Ca2+]i increases induced by ATP, bradykinin, histamine, and thapsigargin. ATP, bradykinin, and histamine increased cell viability while thapsigargin decreased it. AC (25 µg/ml) pretreatment failed to alter bradykinin- and thapsigargin-induced effects on viability, but potentiated ATP- and histamine-induced increases in viability. Immunoblotting showed that MG63 cells did not have background phospho-JNK and phospho-p38 mitogen-activated protein kinases (MAPKs); and AC did not induce the phosphorylation of these two MAPKs. Conversely, the cells had significant background phospho-ERK MAPK that was inhibited by 200 µg/ml AC. The ERK-specific inhibitor PD98059 also induced cell death. Collectively, in MG63 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis probably via inhibition of ERK MAPK phosphorylation. Drug Dev Res 68:71–78, 2007. © 2007 Wiley-Liss, Inc.

Safrole is a carcinogen found in plants. The effect of safrole on cytosolic free Ca²⁺ concentrations ([Ca²⁺](i)) and viability in SCM1 human gastric cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺](i). Safrole at concentrations of 150-450 μM induced a [Ca²⁺](i) rise in a concentration-dependent manner. The response was reduced by 60% by removing extracellular Ca²⁺. Safrole-evoked Ca²⁺ entry was not altered by nifedipine, econazole, SKF96365, and protein kinase C activator or inhibitor. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished safrole-evoked [Ca²⁺](i) rises. Conversely, treatment with safrole abolished thapsigargin or BHQ-evoked [Ca²⁺](i) rises. Inhibition of phospholipase C (PLC) with U73122 abolished safrole-induced [Ca²⁺](i) rises. At 250-550 μM, safrole decreased cell viability concentration-dependently, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/propidium iodide staining data suggest that safrole (350-550 μM) induced apoptosis concentration-dependently. These studies suggest that in SCM1 human gastric cancer cells, safrole induced [Ca²⁺](i) rises by inducing PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via non-store-operated Ca²⁺ entry pathways. Safrole-induced cell death may involve apoptosis.

The effect of the anti-inflammatory compound NPC-14686 on intracellular Ca²⁺ concentration ([Ca²⁺](i)) and viability in OC2 human oral cancer cells was investigated. The Ca²⁺-sensitive fluorescent probe fura-2 was used to examine [Ca²⁺](i). NPC-14686 induced [Ca²⁺](i) rises in a concentration-dependent fashion. The effect was reduced approximately by 10% by removing extracellular Ca²⁺. NPC-14686- elicited Ca²⁺ signal was decreased by nifedipine, econazole, SKF96365, and GF109203X. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished NPC-14686-induced [Ca²⁺](i) rises. Conversely, pretreatment with NPC-14686 abolished thapsigargin or BHQ-induced [Ca²⁺](i) rises. Inhibition of phospholipase C with U73122 abolished NPC-14686-induced [Ca²⁺](i) rises. At 20-100 μM, NPC-14686 inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid-acetoxymethyl ester (BAPTA/AM). NPC-14686 between 20 μM and 40 μM also induced apoptosis. Collectively, in OC2 cells, NPC-14686 induced [Ca²⁺](i) rises by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-regulated store-operated Ca²⁺ channels. NPC-14686 also caused Ca²⁺-independent apoptosis.

Protriptyline, a tricyclic anti-depressant, is used primarily to treat the combination of symptoms of anxiety and depression. However, the effect of protriptyline on prostate caner is unknown. This study examined whether the anti-depressant protriptyline altered Ca(2+) movement and cell viability in PC3 human prostate cancer cells. The Ca(2+)-sensitive fluorescent dye fura-2 was used to measure [Ca(2+)](i). Protriptyline evoked [Ca(2+)](i) rises concentration-dependently. The response was reduced by removing extracellular Ca(2+). Protriptyline-evoked Ca(2+) entry was inhibited by store-operated channel inhibitors (nifedipine, econazole and SKF96365), protein kinase C activator (phorbol 12-myristate 13 acetate, PMA) and protein kinase C inhibitor (GF109203X). Treatment with the endoplasmic reticulum Ca(2+) pump inhibitor 2,5-di-tert-butylhydr-oquinone (BHQ) in Ca(2+)-free medium inhibited 60% of protriptyline-evoked [Ca(2+)](i) rises. Conversely, treatment with protriptyline abolished BHQ-evoked [Ca(2+)](i) rises. Inhibition of phospholipase C with U73122 suppressed 50% of protriptyline-evoked [Ca(2+)](i) rises. At concentrations of 50-70 µM, protriptyline decreased cell viability in a concentration-dependent manner; which were not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in PC3 cells, protriptyline evoked [Ca(2+)](i) rises by inducing phospholipase C-associated Ca(2+) release from the endoplasmic reticulum and other stores, and Ca(2+) influx via protein kinase C-sensitive store-operated Ca(2+) channels. Protriptyline caused cell death that was independent of [Ca(2+)](i) rises.

The effect of the antidepressant doxepin on cytosolic Ca²⁺ concentrations ([Ca²⁺](i)) and viability in PC3 human prostate cancer cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺](i). Doxepin at concentrations of 500-1000 μM induced a [Ca²⁺](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Doxepin-evoked Ca²⁺ entry was suppressed by Ca²⁺ entry blockers (nifedipine, econazole, SKF96365), and protein kinase C (PKC) modulators. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) partly inhibit doxepin-induced [Ca²⁺](i) rise. Incubation with doxepin nearly inhibited thapsigargin or BHQ-induced [Ca²⁺](i) rise. Inhibition of phospholipase C (PLC) with U73122 failed to alter doxepin-induced [Ca²⁺](i) rise. At concentrations of 200-250 μM, doxepin killed cells in a concentration-dependent manner. This cytotoxic effect was not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'- tetraacetic acid/acetoxy methyl (BAPTA/AM). Annexin V/PI staining data implied that doxepin (200 and 250 μM) did not induce apoptosis. Collectively, in PC3 cells, doxepin induced a [Ca²⁺](i) rise by evoking PLC-independent Ca²⁺ release from stores including the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ channels. Doxepin caused cell death that was independent of [Ca²⁺](i) rises.

The in vitro effect of desipramine on renal tubular cell is unknown. In Madin–Darby canine kidney (MDCK) cells, the effect of desipramine on intracellular Ca 2+ concentration ([Ca 2+ ] i ) was measured by using fura-2. Desipramine (>25 μM) caused a rapid and sustained rise of [Ca 2+ ] i in a concentration-dependent manner (EC 50 =50 μM). Desipramine-induced [Ca 2+ ] i rise was prevented by 40% by removal of extracellular Ca 2+ but was not altered by L-type Ca 2+ channel blockers. In Ca 2+ -free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca 2+ -ATPase, caused a monophasic [Ca 2+ ] i rise, after which desipramine failed to release more Ca 2+ ; in addition, pretreatment with desipramine partly decreased thapsigargin-induced [Ca 2+ ] i increase. U73122, an inhibitor of phospholipase C, did not change desipramine-induced [Ca 2+ ] i rise. Incubation with 10–100 μM desipramine enhances or inhibits cell proliferation in a concentration- and time-dependent manner. The inhibitory effect of desipramine on proliferation was not extracellular Ca 2+ -dependent. Apoptosis appears to contribute to desipramine-induced cell death. Together, these findings suggest that desipramine increases baseline [Ca 2+ ] i in renal tubular cells by evoking both extracellular Ca 2+ influx and intracellular Ca 2+ release, and can cause apoptosis.

The differences in the intracellular Ca2+ responses to hormones in platelets from systemic lupus erythematosus (SLE) patients compared to normal humans have not been explored. This study examined the Ca2+ signaling and density of platelets in normal, inactive and active SLE patients. The platelet number per µl in inactive and normal groups did not differ, whereas the number in active SLE patients was smaller than the other two groups by 60%. The intracellular free Ca2+ levels ([Ca2+]i) in response to stimulation of four endogenous Ca2+ mobilizing hormones, 100 µM arachidonic acid (AA), 10 µM ADP, 10 nM platelet activation factor (PAF) and 1 µM thrombin, were investigated using the Ca2+-sensitive fluorescent dye, fura-2. The AA-induced [Ca2+]i rises in normal and inactive groups were similar. In contrast, the AA-induced [Ca2+]i rises in the active SLE group were significantly smaller than in the normal and inactive groups. The defect in the AA-induced [Ca2+]i rises in active SLE groups appears to be caused by defective Ca2+ influx and Ca2+ releasing pathways because the AA-induced responses were not altered by removal of extracellular Ca2+, whereas the AA-induced responses in normal and inactive SLE groups were reduced by removal of extracellular Ca2+, and the AA-induced Ca2+ release was smaller in the active SLE group. PAF, ADP and thrombin all induced [Ca2+]i rises in the three groups, but no significant differences were found among the three groups. Together, the results indicate that cell density and Ca2+ signaling in platelets from active SLE patients are altered in response to particular stimulators. In these regards, platelets from inactive SLE patients appear to be similar to those from normal humans.

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.

Methoxychlor, an organochlorine pesticide, is thought to be an endocrine disrupter that affects Ca²⁺ homeostasis and cell viability in different cell models. This study explored the action of methoxychlor on cytosolic free Ca²⁺ concentrations ([Ca²⁺]i) and apoptosis in HA59T human hepatoma cells. Fura-2, a Ca²⁺-sensitive fluorescent dye, was applied to measure [Ca²⁺]i. Methoxychlor at concentrations of 0.1-1 μM caused a [Ca²⁺]i rise in a concentration-dependent manner. Removal of external Ca²⁺ abolished methoxychlor's effect. Methoxychlor-induced Ca²⁺ influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Methoxychlor-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SKF96365, and protein kinase C modulators. Methoxychlor killed cells at concentrations of 10-130 μM in a concentration-dependent fashion. Chelation of cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy) ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent methoxychlor's cytotoxicity. Methoxychlor (10 and 50 μM) induced apoptosis concentration-dependently as determined by using Annexin V/propidium iodide staining. Together, in HA59T cells, methoxychlor induced a [Ca²⁺]i rise by inducing Ca²⁺ entry via protein kinase C-sensitive Ca²⁺-permeable channels, without causing Ca²⁺ release from stores. Methoxychlor also induced apoptosis that was independent of [Ca²⁺]i rises.

Thymol is a natural essential oil present in many plants and has many different effects in various cell types. However, the effect of thymol on the physiology of MDCK renal tubular cells is unknown. The action of the phytochemical thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]i) and apoptosis in Madin-Darby canine kidney (MDCK) renal tubular cells was explored. Fura-2, a Ca²⁺-sensitive fluorescent dye, was used to assess [Ca²⁺]i. Thymol at concentrations of 200-500 μM caused a [Ca²⁺]i rise in a concentration-dependent manner. Removal of extracellular Ca²⁺ partially reduced the effects of thymol. Thymol-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SKF96365 and protein kinase C modulators. In a Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin inhibited thymol-induced [Ca²⁺]i increases. Treatment with thymol also inhibited thapsigargin-induced [Ca²⁺]i rise. Thymol killed cells at concentrations of 300-500 μM in a concentrationdependent fashion. Chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM) did not prevent thymol cytotoxicity. Thymol (400 and 500 μM) induced apoptosis detected by using Annexin V/propidium iodide staining. At 400 or 500 μM, thymol increased levels of reactive oxygen species. Together, in MDCK cells, thymol induced a [Ca²⁺]i rise by inducing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels. Our data suggest that thymol-induced apoptosis might involve reactive oxygen species (ROS) production.

We examined the effect of 2,5-di-tert-butylhydroquinone (BHQ) on intracellular Ca2+ concentrations ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. BHQ increased [Ca2+]i in a dose-dependent manner with an EC50 of 40 microM. The Ca2+ signal showed a slow onset, a gradual decay and a sustained plateau in normal Ca2+ medium. Depletion of the endoplasmic reticulum Ca2+ store by incubation with 0.1 mM BHQ for 6 min abolished the [Ca2+]i increase evoked by bradykinin or ATP, suggesting that BHQ depleted the inositol 1,4,5-trisphosphate (IP3)-sensitive Ca2+ store. Removal of extracellular Ca2+ reduced the BHQ response by 50%. The Ca2+ signal was initiated by Ca2+ release from the internal store, followed by capacitative Ca2+ entry which was abolished by 100 microM La3+ or 50 microM Gd3+ and was partly inhibited by 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF 96365). After depletion of the endoplasmic reticulum Ca2+ store, by incubation with another inhibitor of the endoplasmic reticulum Ca2+ pump, thapsigargin for 30 min, BHQ did not elevate [Ca2+]i, suggesting that the BHQ-induced Ca2+ influx was largely due to capacitative Ca2+ entry, and that BHQ released Ca2+ from the thapsigargin-sensitive endoplasmic reticulum store. We investigated the mechanism of decay of the BHQ response. Pretreatemt with La3+ (or Gd3+) or alkalization of the extracellular medium to pH 8 significantly potentiated the Ca2+ signal, whereas pretreatment with carbonylcyanide m-chlorophenylhydrazone (CCCP) or oligomycin, or removal of extracellular Na+, had no effect. Collectively, our results suggest that BHQ increased [Ca2+]i in MDCK cells by depleting the endoplasmic reticulum Ca2+ store followed by capacitative Ca2+ entry, with both pathways contributing equally. The decay of the BHQ response is effected by Ca2+ efflux via the plasma membrane Ca2+ pump, but not by efflux via Na+/Ca2+ exchange or sequestration by the mitochondria.

We studied the mechanisms underlying the bradykinin-evoked changes in intracellular calcium concentration ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells. Bradykinin evoked a [Ca2+]i transient in a dose-dependent manner, measured by fura-2 fluorimetry and digital video imaging. The transient consisted of a rise and a decay and [Ca2+]i returned to baseline without oscillations. External Ca2+ influx occurred, as demonstrated by Mn2+ quench and external Ca2+ removal measurements. Bradykinin acted by stimulating bradykinin B2 receptors as evidenced by blockade by D-arginyl-L-arginlyl-L-prolyl-trans-4-hydroxy-L-prolylglycyl -3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolineca rbonyl-L-(2alpha,3beta,7alphabeta)-octahydro-1 H-indole-2-carbonyl-L-arginine (HOE 140) but not by D-arginyl-L-arginlyl-L-prolyl-trans-4-hydroxy-L-proylglycyl- 3-(2-thienyl)-L-alanyl-L-seryl-D-1,2,3,4-tetrahydro-3-isoquinolinecar bonyl-L-(2alpha,3beta,7alphabeta)-octahydro-1 H-indole-2-carbonyl ([Des-Arg]HOE 140). The [Ca2+]i signal was abolished by 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1 H-pyrrole-2,5-dione (U73122) and partially inhibited by neomycin, implying mediation by phospholipase C. The transient was initiated by a release of Ca2+ from internal stores since it was abolished by pretreatment with thapsigargin or cyclopiazonic acid. The mobilization of the internal Ca2+ store subsequently triggered a 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1 H-imidazole hydrochloride (SKF 96365)-insensitive Ca2+ entry. Pretreatment with carbonylcyanide m-chlorophynylhydrozone and gly-phe-beta-naphthylamide did not alter the transient, thus excluding the participation of mitochondria and lysosomes. Efflux via Ca2+ pumps contributed to the decay of the transient. Efflux via Na+/Ca2+ exchange or sequestration by mitochondria and lysosomes was insignificant. The transient was blunted by the protein kinase C activator phorbol 12-myristate 13-acetate, and was enhanced by the protein kinase C inhibitors sphingosine and chelerythrine, the protein kinase A inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone, N-[2-(p-bromocinnamylamino)ethyl]5-isoquinolinesulfonamide (H-89), the agent 8-(diethylamino)octyl 3,4,5-trimethoxybenzoate (TMB-8), and agents that elevated levels of 3',5'-cyclic guanosine monophosphate. The transient did not heterologously desensitize with that evoked by ATP, ADP or UTP.

The effect of the natural product 3,3'-diindolylmethane (DIM) on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in MG63 human osteosarcoma cells was explored. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). DIM at concentrations of 40-80 μM induced a [Ca(2+)](i) rise in a concentration-dependent manner. The response was reduced partly by removing Ca(2+). DIM-evoked Ca(2+) entry was suppressed by nifedipine, econazole, SKF96365 and protein kinase C modulators. In the absence of extracellular Ca(2+), incubation with the endoplasmic reticulum Ca(2+) pump inhibitors thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished DIM-induced [Ca(2+)](i) rise. Incubation with DIM also inhibited thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished DIM-induced [Ca(2+)](i) rise. At concentrations of 10-50 μM, DIM killed cells in a concentration-dependent manner. This cytotoxic effect was not altered 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 implicate that DIM (20 and 40 μM) induced apoptosis in a concentration-dependent manner. In sum, in MG63 cells, DIM induced a [Ca(2+)](i) rise by evoking phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via protein kinase C-sensitive store-operated Ca(2+) channels. DIM caused cell death that may involve apoptosis.

The effect of the environmental contaminant, bisphenol A, on cytosolic free Ca(2+) concentrations ([Ca(2+)](i)) in Madin-Darby canine kidney (MDCK) cells is unclear. This study explored whether bisphenol A changed basal [Ca(2+)](i) levels in suspended MDCK cells by using fura-2 as a Ca(2+)-sensitive fluorescent dye. Bisphenol A, at concentrations between 50 and 300 µM, increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced, partly, by removing extracellular Ca(2+). Bisphenol A induced Mn(2+) influx, leading to quenching of fura-2 fluorescence, suggesting Ca(2+) influx. This Ca(2+) influx was inhibited by phospholipase A2 inhibitor aristolochic acid, store-operated Ca(2+) channel blockers nifedipine and SKF96365, and protein kinase C inhibitor GF109203X. In Ca(2+)-free medium, pretreatment with the mitochondrial uncoupler, carbonylcyanide m-chlorophenylhydrazone (CCCP), and the endoplasmic reticulum Ca(2+) pump inhibitors, thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ), inhibited bisphenol A-induced Ca(2+) release. Conversely, pretreatment with bisphenol A abolished thapsigargin (or BHQ)- and CCCP-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished bisphenol-induced [Ca(2+)](i) rise. Bisphenol A caused a concentration-dependent decrease in cell viability via apoptosis in a Ca(2+)-independent manner. Collectively, in MDCK cells, bisphenol A induced [Ca(2+)](i) rises by causing phospholipase C-dependent Ca(2+) release from the endoplasmic reticulum and mitochondria and Ca(2+) influx via phospholipase A2-, protein kinase C-sensitive, store-operated Ca(2+) channels.

Effect of the carcinogen thapsigargin on human prostate cancer cells is unclear. This study examined if thapsigargin altered basal [Ca²⁺](i) levels in suspended PC3 human prostate cancer cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Thapsigargin at concentrations between 10 nM and 10 µM increased [Ca²⁺](i) in a concentration-dependent fashion. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺ indicating that Ca²⁺ entry and release both contributed to the [Ca²⁺](i) rise. This Ca²⁺ influx was inhibited by suppression of phospholipase A2, but not by inhibition of store-operated Ca²⁺ channels or by modulation of protein kinase C activity. In Ca²⁺-free medium, pretreatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-(t-butyl)-1,4-hydroquinone (BHQ) nearly abolished thapsigargin-induced Ca²⁺ release. Conversely, pretreatment with thapsigargin greatly reduced BHQ-induced [Ca²⁺](i) rise, suggesting that thapsigargin released Ca²⁺ from the endoplasmic reticulum. Inhibition of phospholipase C did not change thapsigargin-induced [Ca²⁺](i) rise. At concentrations of 1-10 µM, thapsigargin induced cell death that was partly reversed by chelation of Ca²⁺ with BAPTA/AM. Annexin V/propidium iodide staining data suggest that apoptosis was partly responsible for thapsigargin-induced cell death. Together, in PC3 human prostate cancer cells, thapsigargin induced [Ca²⁺](i) rises by causing phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx via phospholipase A2-sensitive Ca²⁺ channels. Thapsigargin also induced cell death via Ca²⁺-dependent pathways and Ca²⁺-independent apoptotic pathways.

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.

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, SKF96365 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.

The effect of Antrodia camphorata (AC) on human oral cancer cells has not been explored. This study examined the effect of AC on the viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ regulation of OC2 human oral cancer cells. AC at a concentration of 25 microM induced an increase in cell viability, but AC at concentrationsor = 50 microg/ml decreased viability in a concentration-dependent manner. AC at concentrations of 100-200 microg/ml induced apoptosis in a concentration-dependent manner as demonstrated by propidium iodide staining. AC (25 microg/ml) did not alter basal [Ca2+]i, but decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin, and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment failed to alter ATP-induced increase in viability, potentiated bradykinin-induced increase in viability, decreased histamine-induced increase in viability and reversed thapsigargin-induced decrease in viability. Immunoblotting suggested that AC induced phosphorylation of ERK and JNK MAPKs, but not p38 MAPK. Collectively, for OC2 cells, AC exerted multiple effects on their viability and [Ca2+]i, induced their ERK and JNK MAPK phosphorylation, and probably evoked their apoptosis.

Antrodia camphorata (AC) has been used as a health supplement in Asia to control different cancers; however, the cellular mechanisms of its effects are unclear. The effect of AC on cultured human prostate cancer cells (PC3) has not been explored. This study examined the effect of AC on viability, apoptosis, mitogen-activated protein kinases (MAPKs) phosphorylation and Ca2+ handling in PC3 cells. AC at concentrations of 5-50 microg/ml did not affect cell viability, but at 100-200 microg/ml decreased viability and induced apoptosis in a concentration-dependent manner. AC at concentrations of 25-200 microg/ml did not alter basal [Ca2+]i, but at a concentration of 25 microg/ml decreased the [Ca2+]i increases induced by ATP, bradykinin, histamine and thapsigargin. ATP, bradykinin and histamine increased cell viability whereas thapsigargin decreased it. AC (25 microg/ml) pretreatment inhibited ATP-, bradykinin-, and histamine-induced enhancement on viability, but reversed thapsigargin-induced cytotoxicity. Immunoblotting showed that AC (200 microg/ml) did not induce the phosphorylation of ERK, JNK, and p38 MAPKs. Collectively, in PC3 cells, AC exerted multiple effects on viability and [Ca2+]i, caused apoptosis via pathways unrelated to [Ca2+]i signal and phosphorylation of ERK, JNK and p38 MAPKs.

Melittin, a peptide from bee venom, is thought to be a phospholipase A(2) activator and Ca(2+) influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca(2+)](i) and killed cells in MG63 human osteosarcoma cells. [Ca(2+)](i) changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was abolished by removing extracellular Ca(2+). Melittin-induced Ca(2+) entry was confirmed by Mn(2+) quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca(2+)-insensitive. The melittin-induced Ca(2+) influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A(2) with AACOCF(3) and aristolochic acid; but was substantially inhibited by blocking L-type Ca(2+) channels. At concentrations of 0.5 microM and 1 microM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 microM melittin was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca(2+)](i) increase by causing Ca(2+) entry through L-type Ca(2+) channels in a manner independent of protein kinase-C and phospholipase A(2) activity; and this [Ca(2+)](i) increase subsequently caused apoptosis.

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.

The differences in the intracellular Ca(2+) responses to hormones in platelets from systemic lupus erythematosus (SLE) patients compared to normal humans have not been explored. This study examined the Ca(2+) signaling and density of platelets in normal, inactive and active SLE patients. The platelet number per mul in inactive and normal groups did not differ, whereas the number in active SLE patients was smaller than the other two groups by 60%. The intracellular free Ca(2+) levels ([Ca(2+)](i)) in response to stimulation of four endogenous Ca(2+) mobilizing hormones, 100 microM arachidonic acid (AA), 10 microM ADP, 10 nM platelet activation factor (PAF) and 1 microM thrombin, were investigated using the Ca(2+)-sensitive fluorescent dye, fura-2. The AA-induced [Ca(2+)](i) rises in normal and inactive groups were similar. In contrast, the AA-induced [Ca(2+)](i) rises in the active SLE group were significantly smaller than in the normal and inactive groups. The defect in the AA-induced [Ca(2+)](i) rises in active SLE groups appears to be caused by defective Ca(2+) influx and Ca(2+) releasing pathways because the AA-induced responses were not altered by removal of extracellular Ca(2+), whereas the AA-induced responses in normal and inactive SLE groups were reduced by removal of extracellular Ca(2+), and the AA-induced Ca(2+) release was smaller in the active SLE group. PAF, ADP and thrombin all induced [Ca(2+)](i) rises in the three groups, but no significant differences were found among the three groups. Together, the results indicate that cell density and Ca(2+) signaling in platelets from active SLE patients are altered in response to particular stimulators. In these regards, platelets from inactive SLE patients appear to be similar to those from normal humans.

In Madin-Darby canine kidney (MDCK) cells, the effect of maprotiline, an antidepressant, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2. Maprotiline (2.5 microM) caused a rapid rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50) 200 microM). Maprotiline-induced [Ca(2+)](i) rise was reduced by removal of extracellular Ca(2+) or by addition of La(3+), but was not altered by voltage-gated Ca(2+)-channel blockers. Maprotiline-induced Mn(2+) influx-associated fura-2 fluorescence quench directly suggests that maprotiline caused 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 maprotiline on [Ca(2+)](i) was nearly abolished; also, pretreatment with maprotiline reduced a portion of thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phospholipase C, abolished [Ca(2+)](i) rise induced by ATP (but not by maprotiline). Overnight incubation with 1-10 microM maprotiline enhanced cell viability, but 20-50 microM maprotiline decreased it. These findings suggest that maprotiline rapidly increases [Ca(2+)](i) in renal tubular cells by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release, and may modulate cell proliferation in a concentration-dependent manner.

The effect of oxidants on Ca(2+) movement in osteoblasts is unclear. In this study, we show that 4,4'-dithiodipyridine (4,4'-DTDP), a reactive disulphide that mobilizes Ca(2+) in muscle, induces an increase in cytoplasmic free-Ca(2+) concentrations ([Ca(2+)](i)) in MG63 human osteosarcoma cells loaded with the Ca(2+)-sensitive dye fura-2. 4,4'-DTDP acted in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 35%. In Ca(2+)-free medium, the 4,4'-DTDP-induced [Ca(2+)](i) increase was not changed by depleting store Ca(2+) with 50 microM brefeldin A (a Golgi apparatus permeabilizer), by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP, a mitochondrial uncoupler), by 1 microM thapsigargin (an inhibitor of the endoplasmic reticulum Ca(2+) pump) or by 5 microM ryanodine. Ca(2+) signals induced by 4,4'-DTDP in Ca(2+)-containing medium were not affected by modulation of protein kinase C activity or suppression of phospholipase C activity. However, 4,4'-DTDP-induced Ca(2+) release was inhibited by a thiol-selective reducing reagent, dithiothreitol (0.05-2.5 mM), in a concentration-dependent manner. Collectively, this study shows that 4,4'-DTDP induced [Ca(2+)](i) increases in human osteosarcoma cells via releasing store Ca(2+) from multiple stores in a manner independent of protein kinase C or phospholipase C activity. The store Ca(2+) release induced by 4,4'-DTDP appears to be associated with thiol oxidation. Furthermore, overnight incubation with 4,4'-DTDP inhibited cell activity in a concentration-dependent manner.

The anti-breast cancer drug tamoxifen has recently been shown to cause an increase in intracellular free-Ca(2+) concentrations ([Ca(2+)](i)) in renal tubular cells, breast cells and bladder cells. Because tamoxifen is known to alter ovary function in human patients and in rats, the present study was aimed at exploring whether tamoxifen could alter Ca(2+) movement in Chinese hamster ovary (CHO-K1) cells. Cytosolic free-Ca(2+) levels in populations of cells have been explored by using fura-2 as a fluorescent Ca(2+) indicator. Tamoxifen at concentrations above 1 micro M increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 8 micro M. The Ca(2+) signal was reduced by removing extracellular Ca(2+), but was not affected by nifedipine, verapamil, diltiazem or ICI 182,780 (an estrogen receptor antagonist). Pretreatment with 1 micro M thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) abolished 10 micro M tamoxifen-induced Ca(2+) release. Neither inhibition of phospholipase C with 2 micro M U73122 nor depletion of ryanodine-sensitive Ca(2+) stores with 50 micro M ryanodine affected tamoxifen-induced Ca(2+) release. Cell proliferation assays using ELISA revealed that overnight incubation with 5-10 micro M tamoxifen inhibited cell proliferation by 20%, and 20 micro M tamoxifen killed all cells. Together, the results suggest that, in CHO-K1 cells, tamoxifen induced a [Ca(2+)](i) increase by causing store-Ca(2+) release from the endoplasmic reticulum in an phospholipase C-independent manner, and by inducing Ca(2+) influx. The action of tamoxifen appears to be dissociated from estrogen receptor activation. Longer incubation with tamoxifen (5 micro M) was cytotoxic.

We studied the effect of thapsigargin on intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. Thapsigargin elevated [Ca2+]i dose dependently with an EC50 of approximately 0.15 microM. The Ca2+ signal consisted of a slow rise, a gradual decay and a plateau. Depletion of the endoplasmic reticulum Ca2+ store with thapsigargin for 7 min abolished the [Ca2+]i increases evoked by bradykinin. Removal of extracellular Ca2+ reduced the thapsigargin response by approximately 50%. The Ca2+ signal was initiated by Ca2+ release from the internal store followed by capacitative Ca2+ entry (CCE). The thapsigargin-evoked CCE was abolished by La3 and Gd3+, and was partly inhibited by SKF 96365 and econazole. After depletion of the internal Ca2+ store for 30 min with another inhibitor of the internal Ca2+ pump, cyclopiazonic acid, thapsigargin failed to increase [Ca2+]i, thus suggesting that the thapsigargin-evoked Ca2+ influx was solely due to CCE. We investigated the mechanism of decay of the thapsigargin response. Pretreatment with La3+ (or Gd3+) or alkalization of extracellular medium to pH 8 significantly potentiated the Ca2+ signal; whereas pretreatment with carbonylcyanide m-chlorophynylhydrozone (CCCP) or removal of extracellular Na+ had no effect. Collectively, our results imply that thapsigargin increased [Ca2+]i in MDCK cells by depleting the internal Ca2+ store followed by CCE, with both pathways contributing equally. The decay of the thapsigargin response might be significantly governed by efflux via the plasmalemmal Ca2+ pump.

We have studied the effects of La3+ on ATP-evoked rises in intracellular calcium levels ([Ca2+]i) measured by fura-2 fluorimetry in Madin Darby canine kidney (MDCK) cells. ATP evoked [Ca2+]i rises dose-dependently with an EC50 of 2.5 microM. The trigger for the Ca2+ signal was a release of Ca2+ from the inositol-1,4,5-trisphosphate (IP3)-sensitive stores because the signal was completely blocked by pretreatment with the endoplasmic reticulum (ER) Ca2+ pump inhibitor thapsigargin (TG) or the phospholipase C (PLC) inhibitor U73122. Both the peak height and area under the curve of 10 microM ATP-evoked Ca2+ signal was reduced by approximately 50% by extracellular Ca2+ removal, suggesting that ATP induced capacitative Ca2+ entry. La3+ inhibited the ATP-evoked Ca2+ signal dose-dependently when added before or after ATP. Pretreatment of 0.1 mM La3+ inhibited approximately 90% of the Ca2+ signal induced by 10 microM ATP. The mechanisms underlying the La3+ inhibition appear to involve not only block of capacitative Ca2+ entry but also interference with ATP binding to the ATP receptors.