Mechanism of [Ca2+]i rise induced by angiotensin 1–7 in MDCK renal tubular cells

The effect of angiotensin 1–7 (Ang 1–7) on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) in MDCK renal tubular cells was explored. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Ang 1–7 at concentrations of 10–50 µM induced a [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Ang 1–7 evoked store operated Ca²⁺ entry that was inhibited by La³⁺ and aristolochic acid. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin prevented Ang 1–7 from releasing more Ca²⁺. Inhibition of phospholipase C with U73122 abolished Ang 1–7-induced [Ca²⁺]_i rise. Ang 1–7-induced [Ca²⁺]_i rise was abolished by the angiotensin type 1 receptor antagonist losartan, but was not affected by the angiotensin type 2 receptor antagonist PD 123,319. In sum, in MDCK cells, Ang 1–7 stimulated angiotensin type 1 receptors leading to a [Ca²⁺]_i rise that was composed of phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A2-sensitive store-operated Ca²⁺ channels.     

Effect of clotrimazole on cytosolic Ca2+ rise and viability in HA59T human hepatoma cells

Abstract

Clotrimazole is an antimycotic imidazole derivative that interferes with cellular Ca²⁺ homeostasis. This study examined the effect of clotrimazole on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in HA59T human hepatoma cells. The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Clotrimazole induced [Ca²⁺]_i rises in a concentration-dependent manner. The response was reduced by removing extracellular Ca²⁺. Clotrimazole-evoked Ca²⁺ entry was suppressed by store-operated channel inhibitors (nifedipine, econazole and SK&F96365) and protein kinase C modulators (GF109203X and phorbol, 12-myristate, 13-acetate). In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone abolished clotrimazole-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished clotrimazole-induced [Ca²⁺]_i rise. At 10–40?µM, clotrimazole inhibited cell viability, which was not reversed by chelating cytosolic Ca²⁺. Clotrimazole at 10 and 30?µM also induced apoptosis. Collectively, in HA59T cells, clotrimazole-induced [Ca²⁺]_i rises by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via store-operated Ca²⁺ channels. Clotrimazole also caused apoptosis.     

The mechanism of protriptyline-induced Ca2+ movement and non-Ca2+-triggered cell death in PC3 human prostate cancer cells

Abstract

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 Ca²⁺ movement and cell viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Protriptyline evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Protriptyline-evoked Ca²⁺ 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 Ca²⁺ pump inhibitor 2,5-di-tert-butylhydr-oquinone (BHQ) in Ca²⁺-free medium inhibited 60% of protriptyline-evoked [Ca²⁺]_i rises. Conversely, treatment with protriptyline abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 suppressed 50% of protriptyline-evoked [Ca²⁺]_i rises. At concentrations of 50–70?µM, protriptyline decreased cell viability in a concentration-dependent manner; which were not reversed by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-acetoxymethyl ester (BAPTA/AM). Collectively, in PC3 cells, protriptyline evoked [Ca²⁺]_i rises by inducing phospholipase C-associated Ca²⁺ release from the endoplasmic reticulum and other stores, and Ca²⁺ influx via protein kinase C-sensitive store-operated Ca²⁺ channels. Protriptyline caused cell death that was independent of [Ca²⁺]_i rises.     

Effect of diindolylmethane on Ca2+ homeostasis and viability in PC3 human prostate cancer cells

The effect of the natural product diindolylmethane 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. Diindolylmethane at concentrations of 20–50 µM induced [Ca²⁺]_i rise in a concentration-dependent manner. The response was reduced partly by removing Ca²⁺. Diindolylmethane-evoked Ca²⁺ entry was suppressed by nifedipine, econazole, SK&F96365, protein kinase C modulators and aristolochic acid. In the absence of extracellular Ca²⁺, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished diindolylmethane-induced [Ca²⁺]_i rise. Incubation with diindolylmethane also inhibited thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 reduced diindolylmethane-induced [Ca²⁺]_i rise. At concentrations of 50–100 µM, diindolylmethane killed cells in a concentration-dependent manner. This cytotoxic effect was not altered by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA). Annexin V/PI staining data implicate that diindolylmethane (50 and 100 µM) induced apoptosis in a concentration-dependent manner. In conclusion, diindolylmethane induced a [Ca²⁺]_i rise in PC3 cells by evoking phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via phospholipase A₂-sensitive store-operated Ca²⁺ channels. Diindolylmethane caused cell death in which apoptosis may participate.     

The exploration of effect of terfenadine on Ca2+ signaling in renal tubular cells

Terfenadine, an antihistamine used for the treatment of allergic conditions, affected Ca²⁺-related physiological responses in various models. However, the effect of terfenadine on cytosolic free Ca²⁺ levels ([Ca²⁺]_i) and its related physiology in renal tubular cells is unknown. This study examined whether terfenadine altered Ca²⁺ signaling and caused cytotoxicity in Madin–Darby canine kidney (MDCK) renal tubular cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i. Cell viability was measured by the fluorescent reagent 4-[3-[4-lodophenyl]-2-4(4-nitrophenyl)-2H-5-tetrazolio-1,3-benzene disulfonate] water soluble tetrazolium-1 (WST-1) assay. Terfenadine at concentrations of 100–1000?μM induced [Ca²⁺]_i rises concentration dependently. The response was reduced by approximately 35% by removing extracellular Ca²⁺. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone (BHQ) partly inhibited terfenadine-evoked [Ca²⁺]_i rises. Conversely, treatment with terfenadine abolished BHQ-evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 inhibited 95% of terfenadine-induced Ca²⁺ release. Terfenadine-induced Ca²⁺ entry was supported by Mn²⁺-caused quenching of fura-2 fluorescence. Terfenadine-induced Ca²⁺ entry was partly inhibited by an activator of protein kinase C (PKC), phorbol 12-myristate 13 acetate (PMA) and by three modulators of store-operated Ca²⁺ channels (nifedipine, econazole, and SKF96365). Terfenadine at 200–300?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 MDCK cells, terfenadine induced [Ca²⁺]_i rises by evoking PLC-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via PKC-sensitive store-operated Ca²⁺ entry. Furthermore, terfenadine caused cell death that was not triggered by preceding [Ca²⁺]_i rises.     

Effect of thapsigargin on Ca2+ fluxes and viability in human prostate cancer cells

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.     

Mechanisms of resveratrol-induced changes in [Ca2+]i and cell viability in PC3 human prostate cancer cells

Abstract

Resveratrol is a natural compound that affects cellular Ca²⁺ homeostasis and viability in different cells. This study examined the effect of resveratrol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and viability in PC3 human prostate cancer cells. The Ca²⁺-sensitive fluorescent dye fura-2 was used to measure [Ca²⁺]_i and WST-1 was used to measure viability. Resveratrol-evoked [Ca²⁺]_i rises concentration-dependently. The response was reduced by removing extracellular Ca²⁺. Resveratrol-evoked Ca²⁺ entry was not inhibited by nifedipine, econazole, SKF96365 and the protein kinase C inhibitor GF109203X, but was nearly abolished by the protein kinase C activator phorbol 12-myristate 13 acetate. In Ca²⁺-free medium, treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-di-tert-butylhydroquinone decreased resveratrol-evoked rise in [Ca²⁺]_i. Conversely, treatment with resveratrol inhibited BHQ-evoked rise in [Ca²⁺]_i. Inhibition of phospholipase C with U73122 did not alter resveratrol-evoked rise in [Ca²⁺]_i. Previous studies showed that resveratrol between 10 and 100?µM induced cell death in various cancer cell types including PC3 cells. However, in this study, resveratrol (1–10?μM) increased cell viability, which was abolished by chelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetra-acetic acid-acetoxymethyl ester (BAPTA/AM). Therefore, it is suggested that in PC3 cells, resveratrol had a dual effect on viability: at low concentrations (1–10?µM) it induced proliferation, whereas at higher concentrations it caused cell death. Collectively, our data suggest that in PC3 cells, resveratrol-induced rise in [Ca²⁺]_i by evoking phospholipase C-independent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry, via protein kinase C-regulated mechanisms. Resveratrol at 1–10?µM also caused Ca²⁺-dependent cell proliferation.     

Caffeine-induced Ca2+ oscillations in type I horizontal cell of carp retina: A mathematical model

Oscillations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) have been observed in a variety of cell types. In the present study, we constructed a mathematical model to simulate the caffeine-induced [Ca²⁺]_i oscillations based on experimental data obtained from isolated type I horizontal cell of carp retina. The results of model analysis confirm the notion that the caffeine-induced [Ca²⁺]_i oscillations involve a number of cytoplasmic and endoplasmic Ca²⁺ processes that interact with each other. Using this model, we evaluated the importance of store-operated channel (SOC) in caffeine-induced [Ca²⁺]_i oscillations. The model suggests that store-operated Ca²⁺ entry (SOCE) is elicited upon depletion of the endoplasmic reticulum (ER). When the SOC conductance is set to 0, caffeine-induced [Ca²⁺]_i oscillations are abolished, which agrees with the experimental observation that [Ca²⁺]_i oscillations were abolished when SOC was blocked pharmacologically, verifying that SOC is necessary for sustained [Ca²⁺]_i oscillations.     

Caffeine-induced Ca2+ oscillations in type I horizontal cell of carp retina: A mathematical model

Oscillations in intracellular free Ca²⁺ concentration ([Ca²⁺]_i) have been observed in a variety of cell types. In the present study, we constructed a mathematical model to simulate the caffeine-induced [Ca²⁺]_i oscillations based on experimental data obtained from isolated type I horizontal cell of carp retina. The results of model analysis confirm the notion that the caffeine-induced [Ca²⁺]_i oscillations involve a number of cytoplasmic and endoplasmic Ca²⁺ processes that interact with each other. Using this model, we evaluated the importance of store-operated channel (SOC) in caffeine-induced [Ca²⁺]_i oscillations. The model suggests that store-operated Ca²⁺ entry (SOCE) is elicited upon depletion of the endoplasmic reticulum (ER). When the SOC conductance is set to 0, caffeine-induced [Ca²⁺]_i oscillations are abolished, which agrees with the experimental observation that [Ca²⁺]_i oscillations were abolished when SOC was blocked pharmacologically, verifying that SOC is necessary for sustained [Ca²⁺]_i oscillations.     

The investigation of minoxidil-induced [Ca2+]i rises and non-Ca2+-triggered cell death in PC3 human prostate cancer cells

Minoxidil is clinically used to prevent hair loss. However, its effect on Ca²⁺ homeostasis in prostate cancer cells is unclear. This study explored the effect of minoxidil on cytosolic-free Ca²⁺ levels ([Ca²⁺]_i) and cell viability in PC3 human prostate cancer cells. Minoxidil at concentrations between 200 and 800?μM evoked [Ca²⁺]_i rises in a concentration-dependent manner. This Ca²⁺ signal was inhibited by 60% by removal of extracellular Ca²⁺. Minoxidil-induced Ca²⁺ influx was confirmed by Mn²⁺-induced quench of fura-2 fluorescence. Pre-treatment with the protein kinase C (PKC) inhibitor GF109203X, PKC activator phorbol 12-myristate 13 acetate (PMA), nifedipine and SKF96365 inhibited minoxidil-induced Ca²⁺ signal in Ca²⁺ containing medium by 60%. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5-ditert-butylhydroquinone (BHQ) in Ca²⁺-free medium abolished minoxidil-induced [Ca²⁺]_i rises. Conversely, treatment with minoxidil abolished BHQ-induced [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) with U73122 abolished minoxidil-evoked [Ca²⁺]_i rises. Overnight treatment with minoxidil killed cells at concentrations of 200–600?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 minoxidil’s cytotoxicity. Together, in PC3 cells, minoxidil induced [Ca²⁺]_i rises that involved Ca²⁺ entry through PKC-regulated store-operated Ca²⁺ channels and PLC-dependent Ca²⁺ release from the endoplasmic reticulum. Minoxidil-induced cytotoxicity in a Ca²⁺-independent manner.     

Carvacrol-induced [Ca2+]i rise and apoptosis in human glioblastoma cells

AimsThis study examined whether the essential oil component carvacrol altered cytosolic free Ca²⁺ level ([Ca²⁺]_i) and viability in human glioblastoma cells.Main methodsThe Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Cell viability was measured by detecting reagent WST-1. Apoptosis and reactive oxygen species (ROS) were detected by flow cytometry.Key findingsCarvacrol at concentrations of 400–1000 μM induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Carvacrol-induced Ca²⁺ signal was not altered by nifedipine, econazole, SK&;F96365, and protein kinase C activator phorbol myristate acetate (PMA), but was inhibited by the protein kinase C inhibitor GF109203X. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished carvacrol-induced [Ca²⁺]_i rise. Incubation with carvacrol also abolished thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished carvacrol-induced [Ca²⁺]_i rise. At concentrations of 200–800 μM, carvacrol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed 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 carvacrol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. At concentrations of 200, 400 and 600 μM, carvacrol induced production of ROS.SignificanceIn human glioblastoma cells, carvacrol induced a [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive, non store-operated Ca²⁺ channels. Carvacrol induced cell death that might involve ROS-mediated apoptosis.     

Econazole-evoked [Ca2+]i Rise and Non-Ca2+-triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effects of econazole, an antifungal drug applied for treatment of keratitis and mycotic corneal ulcer, on cytosolic-free Ca²⁺ concentrations ([Ca²⁺]_i) and viability of corneal cells was examined by using SIRC rabbit corneal epithelial cells as model. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Econazole at concentrations ≥ 1 µM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The econazole-induced Ca(2+) influx was insensitive to L-type Ca²⁺ channel blockers and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 20 µM econazole, [Ca²⁺]_i rises induced by 1 µM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) were abolished. Conversely, thapsigargin pretreatment also abolished econazole-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 µM U73122 did not change econazole-induced [Ca²⁺]_i rises. At concentrations between 10 and 80 µM, econazole killed cells in a concentration-dependent manner. The cytotoxic effect of 20 µM econazole was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. This shows that in SIRC cells econazole induces [Ca²⁺]_i rises by causing Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ influx from unknown pathways. Econazole-caused cytotoxicity was independent from a preceding [Ca²⁺]_i rise.     

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

The effect of carvedilol on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) in OC2 human oral cancer cells is unknown. This study examined if carvedilol altered basal [Ca²⁺]_i levels in suspended OC2 cells by using fura-2 as a Ca²⁺-sensitive fluorescent probe. Carvedilol at concentrations between 10 and 40 µM increased [Ca²⁺]_i in a concentration-dependent fashion. The Ca²⁺ signal was decreased by 50% by removing extracellular Ca²⁺. Carvedilol-induced Ca²⁺ entry was not affected by the store-operated Ca²⁺ channel blockers nifedipine, econazole, and SK&F96365, but was enhanced by activation or inhibition of protein kinase C. In Ca²⁺-free medium, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin did not change carvedilol-induced [Ca²⁺]_i rise; conversely, incubation with carvedilol did not reduce thapsigargin-induced Ca²⁺ release. Pretreatment with the mitochondrial uncoupler carbonylcyanide m-chlorophenylhydrazone (CCCP) inhibited carvedilol-induced [Ca²⁺]_i release. Inhibition of phospholipase C with U73122 did not alter carvedilol-induced [Ca²⁺]_i rise. Carvedilol at 5–50 µM induced cell death in a concentration-dependent manner. The death was not reversed when cytosolic Ca²⁺ 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 [Ca²⁺]_i rise by causing phospholipase C-independent Ca²⁺ release from mitochondria and non-endoplasmic reticulum stores, and Ca²⁺ influx via protein kinase C-regulated channels. Carvedilol (up to 50 μM) induced cell death in a Ca²⁺-independent manner that involved apoptosis.     

Diethylstilbestrol-Induced Estrogen Receptor-Dependent [Ca2+]i Rises and Apoptosis in Chinese Hamster Ovary (CHO) Cells

The effect of the synthetic estrogen diethylstilbestrol (DES) on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability was explored in Chinese hamster ovary (CHO-K1). [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. DES at concentrations ≥ 1∝ increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. In Ca²⁺-free medium, after pretreatment with 50∝ DES, 1∝ thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor)-induced [Ca²⁺]_i rises were abolished. Conversely, thapsigargin pretreatment abolished DES-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with U73122 did not alter DES-induced [Ca²⁺]_i rises. At a concentration of 5∝, DES increased cell viability. At concentrations of 100–200 μ M, DES decreased viability in a concentration-dependent manner. The effect of 5 and 100 μM DES on viability was partly reversed by prechelating cytosolic Ca²⁺ with 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′ -tetraacetic acid (BAPTA). DES-induced cell death was induced via apoptosis as demonstrated by propidium iodide staining. DES (100 μ M)-induced [Ca²⁺]_i rises were largely inhibited by pretreatment with the estrogen receptor antagonist ICI-182,780 (100 μ M). ICI-182,780 did not affect 5 μ M DES-induced increase in viability but partly reversed 100 μ M DES-induced cell death. Collectively, in CHO-K1 cells, DES induced [Ca²⁺]_i rises by stimulating estrogen receptors leading to Ca²⁺ release from the endoplasmic reticulum in a phospholipase C-independent manner, and Ca²⁺ influx. DES-caused cytotoxicity was mediated by an estrogen receptor- and Ca²⁺-dependent pathway.     

Ketoconazole-Evoked [Ca2+]i Rises and Non-Ca2+-Triggered Cell Death in Rabbit Corneal Epithelial Cells (SIRC)

The effect of ketoconazole on cytosolic free Ca^{2 +} concentrations ([Ca^{2 +}]_i) and proliferation has not been explored in corneal cells. This study examined whether ketoconazole alters Ca^{2 +} levels and causes cell death in SIRC rabbit corneal epithelial cells. [Ca^{2 +}]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Ketoconazole at concentrations of 5 μ M and above increased [Ca^{2 +}]_i in a concentration-dependent manner. The Ca^{2 +} signal was reduced partly by removing extracellular Ca^{2 +}. The ketoconazole-induced Ca^{2 +} influx was insensitive to L-type Ca^{2 +} channel blockers and protein kinase C modulators. In Ca^{2 +}-free medium, after pretreatment with 50 μ M ketoconazole, thapsigargin-(1 μ M)-induced [Ca^{2 +}]_i rises were abolished; conversely, thapsigargin pretreatment nearly abolished ketoconazole-induced [Ca^{2 +}]_i rises. Inhibition of phospholipase C with 2 μ M U73122 did not change ketoconazole-induced [Ca^{2 +}]_i rises. At concentrations between 5 and 100 μ M, ketoconazole killed cells in a concentration-dependent manner. The cytotoxic effect of 50 μ M ketoconazole was not reversed by prechelating cytosolic Ca^{2 +} with BAPTA. In summary, in corneal cells, ketoconazole-induced [Ca^{2 +}]_i rises by causing Ca^{2 +} release from the endoplasmic reticulum and Ca^{2 +} influx from unknown pathways. Furthermore, the cytotoxicity induced by ketoconazole was not caused via a preceding [Ca^{2 +}]_i rise.     

Tamoxifen-Induced [Ca2+]i Rises and Ca2+-Independent Cell Death in Human Oral Cancer Cells

The purpose of this study was to explore the effect of tamoxifen on cytosolic free Ca²⁺ concentrations ([Ca²⁺]_i) and cell viability in OC2 human oral cancer cells. [Ca²⁺]_i and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Tamoxifen at concentrations above 2 μM increased [Ca²⁺]_i in a concentration-dependent manner. The Ca²⁺ signal was reduced partly by removing extracellular Ca²⁺. The tamoxifen-induced Ca²⁺ influx was sensitive to blockade of L-type Ca²⁺ channel blockers but insensitive to the estrogen receptor antagonist ICI 182,780 and protein kinase C modulators. In Ca²⁺-free medium, after pretreatment with 1 μM thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor), tamoxifen-induced [Ca²⁺]_i rises were substantially inhibited; and conversely, tamoxifen pretreatment inhibited a part of thapsigargin-induced [Ca²⁺]_i rises. Inhibition of phospholipase C with 2 μM U73122 did not change tamoxifen-induced [Ca²⁺]_i rises. At concentrations between 10 and 50 μM tamoxifen killed cells in a concentration-dependent manner. The cytotoxic effect of 23 μM tamoxifen was not reversed by prechelating cytosolic Ca²⁺ with BAPTA. Collectively, in OC2 cells, tamoxifen induced [Ca²⁺]_i rises, in a nongenomic manner, by causing Ca²⁺ release from the endoplasmic reticulum, and Ca²⁺ influx from L-type Ca²⁺ channels. Furthermore, tamoxifen-caused cytotoxicity was not via a preceding [Ca²⁺]_i rise.     

Effect of tetramethylpyrazine (TMP) on Ca2+ signal transduction and cell viability in a model of renal tubular cells

Tetramethylpyrazine (TMP) is a compound purified from herb. Its effect on Ca²⁺ concentrations ([Ca²⁺]_i) in renal cells is unclear. This study examined whether TMP altered Ca²⁺ signaling in Madin‐Darby canine kidney (MDCK) cells. TMP at 100–800 μM induced [Ca²⁺]_i rises, which were reduced by Ca²⁺ removal. TMP induced Mn²⁺ influx implicating Ca²⁺ entry. TMP‐induced Ca²⁺ entry was inhibited by 30% by modulators of protein kinase C (PKC) and store‐operated Ca²⁺ channels. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 93% of TMP‐evoked [Ca²⁺]_i rises. Treatment with TMP abolished BHQ‐evoked [Ca²⁺]_i rises. Inhibition of phospholipase C (PLC) abolished TMP‐induced responses. TMP at 200–1000 μM decreased 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. Together, in MDCK cells, TMP induced [Ca²⁺]_i rises by evoking PLC‐dependent Ca²⁺ release from endoplasmic reticulum and Ca²⁺ entry via PKC‐sensitive store‐operated Ca²⁺ entry. TMP also caused Ca²⁺‐independent cell death.     

Effect of Celecoxib on Ca2+ Fluxes and Proliferation in MDCK Renal Tubular Cells

The effect of celecoxib on renal tubular cells is largely unexplored. In Madin Darby canine kidney (MDCK) cells, the effect of celecoxib on intracellular Ca^{2 +} concentration ([Ca^{2 +}]_i) and proliferation was examined by using the Ca^{2 +}-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (≥1 μ M) caused an increase of [Ca^{2 +}]_i in a concentration-dependent manner. Celecoxib-induced [Ca^{2 +}]_i increase was partly reduced by removal of extracellular Ca^{2 +}. Celecoxib-induced Ca^{2 +} influx was independently suggested by Mn^{2 +} influx-induced fura-2 fluorescence quench. In Ca^{2 +}-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca^{2 +}-ATPase, caused a monophasic [Ca^{2 +}]_i increase, after which celecoxib only induced a tiny [Ca^{2 +}]_iincrease; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [Ca^{2 +}]_i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [Ca^{2 +}]_i increases. Overnight incubation with 1 or 10 μ M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [Ca^{2 +}]_i increase in renal tubular cells by stimulating both extracellular Ca^{2 +} influx and intracellular Ca^{2 +} release and is highly toxic to renal tubular cells in vitro.     

Regulation of intracellular calcium concentration by nanosecond pulsed electric fields

Changes in [Ca²⁺]_i response of individual Jurkat cells to nanosecond pulsed electric fields (nsPEFs) of 60 ns and field strengths of 25, 50, and 100 kV/cm were investigated. The magnitude of the nsPEF-induced rise in [Ca²⁺]_i was dependent on the electric field strength. With 25 and 50 kV/cm, the [Ca²⁺]_i response was due to the release of Ca²⁺ from intracellular stores and occurred in less than 18 ms. With 100 kV/cm, the increase in [Ca²⁺]_i was due to both internal release and to influx across the plasma membrane. Spontaneous changes in [Ca²⁺]_i exhibited a more gradual increase over several seconds. The initial, pulse-induced [Ca²⁺]_i response initiates at the poles of the cell with respect to electrode placement and co-localizes with the endoplasmic reticulum. The results suggest that nsPEFs target both the plasma membrane and subcellular membranes and that one of the mechanisms for Ca²⁺ release may be due to nanopore formation in the endoplasmic reticulum.     

Ca2+ Signaling and Cell Death Induced by Protriptyline in HepG2 Human Hepatoma Cells

The effect of protriptyline on Ca²⁺ physiology in human hepatoma is unclear. This study explored the effect of protriptyline on [Ca²⁺]_i and cytotoxicity in HepG2 human hepatoma cells. Protriptyline (50–150 μM) evoked [Ca²⁺]_i rises. The Ca²⁺ entry was inhibited by removal of Ca²⁺. Protriptyline‐induced Ca²⁺ entry was confirmed by Mn²⁺‐induced quench of fura‐2 fluorescence. Except nifedipine, econazole, SKF96365, GF109203X, and phorbol 12‐myristate 13 acetate did not inhibit Ca²⁺ entry. Treatment with the endoplasmic reticulum Ca²⁺ pump inhibitor 2,5‐di‐tert‐butylhydroquinone (BHQ) inhibited 40% of protriptyline‐induced response. Treatment with protriptyline abolished BHQ‐induced response. Inhibition of phospholipase C (PLC) suppressed protriptyline‐evoked response by 70%. At 20–40 μM, protriptyline killed cells which was not reversed by the Ca²⁺ chelator 1,2‐bis(2‐aminophenoxy)ethane‐N,N,N′,N′‐tetraacetic acid‐acetoxymethyl ester (BAPTA/AM). Together, in HepG2 cells, protriptyline induced [Ca²⁺]_i rises that involved Ca²⁺ entry through nifedipine‐sensitive Ca²⁺ channels and PLC‐dependent Ca²⁺ release from endoplasmic reticulum. Protriptyline induced Ca²⁺‐independent cell death.