Effect of p-chloroamphetamine on calcium movement and viability in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, the effect of p-chloroamphetamine, a neurotoxin that depletes intracellular serotonin, on intracellular Ca2+ concentration ([Ca2+]i) and viability was measured by using the Ca2+-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium. p-Chloroamphetamine (> or = 10 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. p-Chloroamphetamine-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. p-Chloroamphetamine-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which p-chloroamphetamine failed to increase [Ca2+]i; also, pretreatment with p-chloroamphetamine reduced 50% of thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not p-chloroamphetamine)-induced [Ca2+]i rise. Overnight incubation with 1-500 microM p-chloroamphetamine decreased cell viability. These findings suggest that p-chloroamphetamine evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release, and is cytotoxic.     

Nordihydroguaiaretic acid-induced Ca2+ handling and cytotoxicity in human prostate cancer cells

The effect of nordihydroguaiaretic acid (NDGA), a compound commonly used as a lipoxygenases inhibitor, on intracellular free Ca2+ levels ([Ca2+]i) in PC3 human prostate cancer cells was investigated. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. NDGA increased [Ca2+]i in a concentration-dependent manner with an EC50 of 30 microM. The Ca2+ signal comprised a gradual and sustained increase. Removal of extracellular Ca2+ partly decreased the NDGA-induced [Ca2+]i increase, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and intracellular Ca2+ release. NDGA-induced Ca2+ influx was independently confirmed by measuring NDGA-induced Mn2+ -coupled quench of fura-2 fluorescence. The NDGA-induced Ca2+ influx was not affected by L-type Ca2+ channel blockers. In Ca2+ -free medium, the NDGA-induced [Ca2+]i increase was abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with NDGA abolished thapsigargin-induced [Ca2+]i increase. NDGA-induced intracellular Ca2+ release was not altered by inhibition of phospholipase C. Overnight treatment with 20-50 microM NDGA inhibited cell proliferation rate in a concentration-dependent manner. Several other lipoxygenases inhibitors did not alter [Ca2+]i. Collectively, this study shows that in prostate cells, NDGA induced a [Ca2+]i increase via releasing stored Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity, and by causing Ca2+ influx. NDGA also caused cytotoxicity at higher concentrations.     

Effect of NPC-15199 on Ca2+ levels in renal tubular cells

In Madin-Darby canine kidney (MDCK) cells, effect of NPC-15199 on intracellular Ca2+ concentration ([Ca2+]i) was investigated by using fura-2. NPC-15199 (100-1000 microM) caused a rapid and sustained increase of [Ca2+]i in a concentration-dependent manner (EC50=500 microM). NPC-15199-induced [Ca2+]i rise was prevented by 70% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. In Ca2+-free medium, carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 microM), a mitochondrial uncoupler, and thapsigargin (1 microM), an inhibitor of the endoplasmic reticulum (ER) Ca2(+)-ATPase, caused a monophasic [Ca2+]i rise, respectively, after which the increasing effect of NPC-15199 (1 mM) on [Ca2+]i was substantially attenuated; also, pretreatment with NPC-15199 abolished CCCP- and thapsigargin-induced [Ca2+]i rises. U73122, an inhibitor of phospholipase C, [corrected] abolished 10 microM ATP (but not 1 mM NPC-15199)-induced [Ca2+]i rise. These results suggest that NPC-15199 rapidly increases [Ca2+]i by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via as yet unidentified mechanism(s).     

Effect of maprotiline on Ca(2+) movement in human neuroblastoma cells

In human neuroblastoma IMR32 cells, the effect of the anti-depressant maprotiline on baseline intracellular Ca2+ concentrations ([Ca2+]i) was explored by using the Ca2+-sensitive probe fura-2. Maprotiline at concentrations greater than 100 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50 = 200 microM). Maprotiline-induced [Ca2+]i rise was reduced by 50% by removal of extracellular Ca2+. Maprotiline-induced [Ca2+]i rises were inhibited by half by nifedipine, but was unaffected by verapamil or diiltiazem. 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 maprotiline on [Ca2+]i was abolished. U73122, an inhibitor of phospholipase C, did not affect maprotiline-induced [Ca2+]i rises. These findings suggest that in human neuroblastoma cells, maprotiline increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum via a phospholiase C-independent manner.     

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 CaCa2+ concentration ([Ca2+]i) and proliferation was examined by using the Ca(2 +)-sensitive fluorescent dye fura-2 and the viability detecting fluorescent dye tetrazolium, respectively. Celecoxib (> or =1 micro M) caused an increase of [CaCa2+]i in a concentration-dependent manner. Celecoxib-induced [CaCa2+]i increase was partly reduced by removal of extracellular CaCa2+. Celecoxib-induced CaCa2+ influx was independently suggested by MnCa2+ influx-induced fura-2 fluorescence quench. In Ca(2 +)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2 +)-ATPase, caused a monophasic [CaCa2+]i increase, after which celecoxib only induced a tiny [CaCa2+]i increase; conversely, pretreatment with celecoxib completely inhibited thapsigargin-induced [CaCa2+]i increases. U73122, an inhibitor of phospholipase C, abolished ATP (but not celecoxib)-induced [CaCa2+]i increases. Overnight incubation with 1 or 10 micro M celecoxib decreased cell viability by 80% and 100%, respectively. These data indicate that celecoxib evokes a [CaCa2+]i increase in renal tubular cells by stimulating both extracellular CaCa2+ influx and intracellular CaCa2+ release and is highly toxic to renal tubular cells in vitro.     

Effect of nortriptyline on Ca2+ handling in SIRC rabbit corneal epithelial cells

To explore the effect of nortriptyline, a tricyclic antidepressant, on cytosolic free Ca2+ concentrations ([Ca2+]i) in corneal epithelial cells, [Ca2+]i levels in suspended SIRC rabbit corneal epithelial cells were measured by using fura-2 as a Ca2+-sensitive fluorescent dye. Nortriptyline at concentrations between 20-200 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced partly by removing extracellular Ca2+. Nortriptyline-induced Ca2+ influx was inhibited by the store-operated Ca2+ channel blockers econazole and SK&F96365, the phospholipase A2 inhibitor aristolochic acid, and alteration of activity of protein kinase C. In Ca2+-free medium, 200 microM nortriptyline pretreatment greatly inhibited the rise of [Ca2+]i induced by the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin. Conversely, pretreatment with thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ; another endoplasmic reticulum Ca2+ pump inhibitor) nearly abolished nortriptyline-induced [Ca2+]i rise. Inhibition of phospholipase C with U73122 decreased nortriptyline-induced [Ca2+]i rise by 75%. Taken together, nortriptyline induced [Ca2+]i rises in SIRC cells by causing phospholipase C-dependent Ca2+ release from the endoplasmic reticulum and Ca2+ influx via store-operated Ca2+ channels.     

The antidepressant mirtazapine-induced cytosolic Ca2+ elevation and cytotoxicity in human osteosarcoma cells

The effect of the antidepressant mirtazapine on cytosolic free Ca2+ concentration ([Ca2+]i) and viability has not been explored in any cell type. This study examined whether mirtazapine alters Ca2+ levels and causes cell death in osteoblast-like cells using MG63 human osteosarcoma cells as a model. [Ca2+]i and cell viability were measured using the fluorescent dyes fura-2 and WST-1, respectively. Mirtazapine at concentrations above 250 microM increased [Ca2+]i in a concentration-dependent manner. The Ca2+ signal was reduced by 60% by removing extracellular Ca2+. The mirtazapine-induced Ca2+ influx was sensitive to blockade of nifedipine and verapamil. In Ca(2+)-free medium, after pretreatment with 1.5 mM mirtazapine, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 2 microM CCCP (a mitochondrial uncoupler), and 1 microM ionomycin failed to release more stored Ca2+; conversely, pretreatment with thapsigargin, CCCP and ionomycin abolished mirtazapine-induced Ca2+ release. Inhibition of phospholipase C with 2 microM U73122 did not change mirtazapine-induced [Ca2+]i, increase. Seal of Ca2+ movement across the plasma membrane with 50 microM extracellular La3+ enhanced 1 microM thapsigargin-induced [Ca2+]i increase, suggesting that Ca2+ efflux played a role in lowering thapsigargin-induced [Ca2+]i increase; however, the same La3+ treatment did not alter mirtazapine-induced [Ca2+]i increase. At concentrations of 500 microM and 1000 microM, mirtazapine killed 30% and 60% cells, respectively. The cytotoxicity was not reversed by chelating cytosolic Ca2+ with BAPTA. Collectively, in MG63 cells, mirtazapine induced a [Ca2+]i increase by causing Ca2+ release from stores and Ca2+ influx from extracellular space. Furthermore, mirtazapine caused cytotoxicity at higher concentrations in a Ca(2+)-dissociated manner.     

Mercury-induced Ca2+ increase and cytotoxicity in renal tubular cells

The effect of mercury (Hg2+), a known nephrotoxicant, on intracellular free Ca2+ levels ([Ca2+]i) in Madin Darby canine kidney (MDCK) cells was explored. [Ca2+]i was measured by using the Ca2+ -sensitive dye fura-2. Hg2+ increased [Ca2+]i in a concentration-dependent manner with an EC50 of 6 microM. The Ca2+ signal comprised a gradual increase. Removal of extracellular Ca2+ decreased the Hg2+ -induced [Ca2+]i increase by 27%, suggesting that the Ca2+ signal was due to both extracellular Ca2+ influx and store Ca2+ release. In Ca2+ -free medium, the Hg2+ -induced [Ca2+]i increase was nearly abolished by pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), and conversely, pretreatment with Hg2+ abolished thapsigargin-induced Ca2+ increase. Hg2+ -induced Ca2+ release was not altered by inhibition of phospholipase C but was potentiated by activation of protein kinase C. Overnight treatment with 1 microM Hg2+ did not alter cell proliferation rate and mitochondrial activity, but 10 microM Hg2+ killed all cells. Collectively, this study shows that Hg2+ induced protein kinase C-regulated [Ca2+]i increases in renal tubular cells via releasing store Ca2+ from the endoplasmic reticulum in a manner independent of phospholipase C activity. Hg2+ also caused cytotoxicity at higher concentrations.     

2-O-methyl PAF as a Ca2+ mobilizer in Madin Darby canine kidney cells

In Madin-Darby canine kidney (MDCK) cells, the effect of 2-O-methyl PAF, an inactive analogue of platelet activating factor (PAF), on intracellular Ca2+ concentration ([Ca2+]i) was measured by using the Ca2+-sensitive fluorescent dye fura-2. 2-O-methyl PAF (> or = 15 microM) caused a rapid rise of [Ca2+]i in a concentration-dependent manner. 2-O-methyl PAF-induced [Ca2+]i rise was partly reduced by removal of extracellular Ca2+. 2-O-methyl PAF-induced extracellular Ca2+ influx was also suggested by Mn2+ influx-induced fura-2 fluorescence quench. The 2-O-methyl PAF-induced Ca2+ influx was blocked by nifedipine, verapamil and diltiazem. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which 2-O-methyl PAF failed to increase [Ca2+]i; also, pretreatment with 2-O-methyl PAF depleted thapsigargin-sensitive Ca2+ stores. U73122, an inhibitor of phospholipase C, abolished ATP (but not 2-O-methyl PAF)-induced [Ca2+]i rise. These findings suggest that 2-O-methyl PAF evokes a rapid increase in [Ca2+]i in renal tubular cells by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release.     

Effect of t-butyl hydroperoxide on Ca2+ movement in PC12 pheochromocytoma cells

The effect of the oxidant t-butyl hydroperoxide on intracellular free levels of Ca2+ ([Ca2+]i) in PC12 pheochromocytoma cells was examined by using fura-2 as a fluorescent dye. t-Butyl hydroperoxide induced an increase in [Ca2+]i in a concentration-dependent fashion between 50-250 microM with an EC50 of 100 microM. The [Ca2+]i signal consisted of a slow rise and a sustained phase. The response was decreased by 65% by removal of extracellular Ca2+. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) abolished 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase, and conversely, pretreatment with t-butyl hydroperoxide abrogated thapsigargin-induced [Ca2+]i increase. The 150 microM t-butyl hydroperoxide-induced [Ca2+]i increase in Ca2+ medium was reduced by 42 +/- 5% by pretreatment with 0.1 microM nicardipine but not by 10 microM verapamil, nifedipine, nimodipine or diltiazem, or by 50 microM La3+ or Ni2+. Pretreatment with 10 microM t-butyl hydroperoxide for 40 min did not affect 10 microM ATP-induced [Ca2+]i increase. Together, the results show that t-butyl hydroperoxide induced significant [Ca2+]i increase in PC12 cells by causing store Ca2+ release from the thapsigargin-sensitive endoplasmic reticulum pool in an inositol 1,4,5-trisphosphate-independent manner and by inducing Ca2+ influx via a nicardipine-sensitive pathway.     

Effect of olvanil (N-vanillyl-cis-9-octadecenoamide) on cytosolic Ca2+ increase in renal tubular cells

In canine renal tubular cells, effect of olvanil, a presumed cannabinoid and vanilloid receptor modulator, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Olvanil (5-100 microM) caused a rapid and sustained [Ca2+]i rise in a concentration-dependent manner. Olvanil-induced [Ca2+]i rise was prevented by 70 and 90% by removal of extracellular Ca2+ and La3+, respectively, but was not changed by dihydropyridines, verapamil and diltiazem. 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 olvanil on [Ca2+]i was abolished; also, pretreatment with olvanil partly reduced thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phoispholipase C, abrogated ATP-, but partly inhibited olvanil-, induced [Ca2+]i rise. Two cannabinoid receptor antagonists (AM251 and AM281; 5 microM) and a vanilloid receptor antagonist (capsazepine; 100 microM) did not alter olvanil (50 microM)-induced [Ca2+]i rise. These results suggest that olvanil rapidly increases [Ca2+]i in renal tubular cells, by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release via mechanism(s) independent of stimulation of cannabinoid and vanilloid receptors.     

Effect of diethylstilbestrol on Ca2+ handling and cell viability in human breast cancer cells

In human breast cancer cells, the effect of the widely prescribed estrogen diethylstilbestrol (DES) on intracellular Ca2+ concentrations ([Ca2+]i) and cell viability was explored by using fura-2 and trypan blue exclusion, respectively. DES caused a rise in [Ca2+]i in a concentration-dependent manner (EC50 = 15 microM). DES-induced [Ca2+]i rise was reduced by 80 % by removal of extracellular Ca2+. DES-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that DES induced extracellular Ca2+ influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca2+]i rise, after which the increasing effect of DES on [Ca2+]i was greatly inhibited. Conversely, pretreatment with DES to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+, whereas ionomycin added afterward still released some Ca2+. These findings suggest that in human breast cancer cells, DES increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum. Acute trypan blue exclusion studies suggest that 10-20 NM DES killed cells in a time-dependent manner.     

Effect of desipramine on Ca2+ levels and growth in renal tubular cells

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 Ca2+ concentration ([Ca2+]i) was measured by using fura-2. Desipramine (>25 microM) caused a rapid and sustained rise of [Ca2+]i in a concentration-dependent manner (EC50=50 microM). Desipramine-induced [Ca2+]i rise was prevented by 40% by removal of extracellular Ca2+ but was not altered by L-type Ca2+ channel blockers. In Ca2+-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca2+-ATPase, caused a monophasic [Ca2+]i rise, after which desipramine failed to release more Ca2+; in addition, pretreatment with desipramine partly decreased thapsigargin-induced [Ca2+]i increase. U73122, an inhibitor of phospholipase C, did not change desipramine-induced [Ca2+]i rise. Incubation with 10-100 microM desipramine enhances or inhibits cell proliferation in a concentration- and time-dependent manner. The inhibitory effect of desipramine on proliferation was not extracellular Ca2+-dependent. Apoptosis appears to contribute to desipramine-induced cell death. Together, these findings suggest that desipramine increases baseline [Ca2+]i in renal tubular cells by evoking both extracellular Ca2+ influx and intracellular Ca2+ release, and can cause apoptosis.     

Novel effect of Y-24180, a presumed specific platelet activation factor receptor antagonist, on Ca2+ levels and growth of human prostate cancer cells

In human prostate cancer PC3 cells, the effect of Y-24180, a presumed specific platelet activation factor (PAF) receptor antagonist, on intracellular Ca2+ concentration ([Ca2+]i) was measured by using fura-2 as a Ca2+-sensitive fluorescent probe. Y-24180 (1-10 microM) caused a rapid and sustained [Ca2+]i rise in a concentration-dependent manner. The [Ca2+]i rise was prevented by 40% by removal of extracellular Ca2+, but was not changed by dihydropyridines, verapamil and diltiazem. 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 10 microM Y-24180 on [Ca2+]i was reduced by 67%; conversely, depletion of Ca2+ stores with 10 microM Y-24180 abolished thapsigargin-induced [Ca2+]i rise. U73122, an inhibitor of phospholipase C, inhibited ATP-, but not Y-24180-induced [Ca2+]i rise. Activation of protein kinase C with phorbol-12-myristate-13-acetate (PMA) enhanced Y-24180-induced [Ca2+]i rise by 70%. Overnight treatment with 0.1-10 microM Y-24180 inhibited cell proliferation in a concentration-dependent manner. Collectively, these results suggest that Y-24180 acts as a potent and cytotoxic Ca2+ mobilizer in prostate cancer cells, by stimulating both extracellular Ca2+ influx and intracellular Ca2+ release. Since alterations in Ca2+ movement may interfere with many cellular signalling processes unrelated to modulation of PAF receptors, caution must be applied in using this reagent as a selective PAF receptor antagonist.     

Histamine-Induced increases in intracellular free Ca2+ levels in hepatoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in HA22/VGH human hepatoma cells were evaluated using fura-2 as a fluorescent Ca2+ dye. Histamine (0.2-5 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of about 1 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the [Ca2+]i signal. In Ca2+-free medium, after cells were treated with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase of a magnitude 7-fold greater than control. Histamine (5 microM)-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 2 microM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122), and by 5 microM pyrilamine but was not altered by 50 microM cimetidine. Together, this study shows that histamine induced [Ca2+]i increases in human hepatoma cells by stimulating H1, but not H2, histamine receptors. The [Ca2+]i signal was caused by Ca2+ release from thapsigargin-sensitive endoplasmic reticulum in an inositol 1,4,5-trisphosphate-dependent manner, accompanied by Ca2+ entry.     

CP55,940 increases intracellular Ca2+ levels in Madin-Darby canine kidney cells

The effect of CP55,940, a presumed CB1/CB2 cannabinoid receptor agonist, on intracellular free Ca2+ levels ([Ca2+]i) in Madin-Darby canine kidney cells was examined by using the fluorescent dye fura-2 as a Ca2+ indicator. CP55,940 (2-50 microM) increased [Ca2+]i concentration-dependently with an EC50 of 8 microM. The [Ca2+]i signal comprised an initial rise and a sustained phase. Extracellular Ca2+ removal decreased the maximum [Ca2+]i signals by 32+/-12%. CP55,940 (20 microM)-induced [Ca2+]i signal was not altered by 5 microM of two cannabinoid receptor antagonists, AM-251 and AM-281. CP55,940 (20 microM)-induced [Ca2+]i increase in Ca2+-free medium was inhibited by 86+/-3% by pretreatment with 1 microM thapsigargin, an endoplasmic reticulum Ca2+ pump inhibitor. Conversely, pretreatment with 20 microM CP55,940 in Ca2+-free medium for 6 min abolished thapsigargin-induced [Ca2+]i increases. CP55,940 (20 microM)-induced intracellular Ca2+ release was not inhibited when inositol 1,4,5-trisphosphate formation was abolished by suppressing phospholipase C with 2 microM U73122. Collectively, this study shows that CP,55940 induced significant [Ca2+]i increases in canine renal tubular cells by releasing stored Ca2+ from the thapsigargin-sensitive pools in an inositol 1,4,5-trisphosphate-independent manner, and also by causing extracellular Ca2+ entry. The CP55,940's action appears to be dissociated from stimulation of cannabinoid receptors.     

Characterization of histamine-induced increases in intracellular free Ca2+ concentrations in Chang liver cells.

The effect of histamine on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were investigated by using fura-2 as a Ca2+ dye. Histamine (0.2-50 microM) increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 0.8 microM. The [Ca2+]i response comprised an initial rise, a slow decay, and a sustained phase. Extracellular Ca2+ removal inhibited 50% of the maximum [Ca2+]i signal and abolished the sustained phase. After pretreatment with 5 microM histamine in Ca2+-free medium for 4 min, addition of 3 mM Ca2+ induced a [Ca2+]i increase with a magnitude 7-fold greater than control. In Ca2+-free medium, after treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor), 5 microM histamine failed to increase [Ca2+]i. Histamine (5 microM)-induced intracellular Ca2+ release was abolished     

Clomiphene, an ovulation-inducing agent, mobilizes intracellular Ca2+ and causes extracellular Ca2+ influx in bladder female transitional carcinoma cells

BACKGROUND/METHODS: The effect of clomiphene, an ovulation-inducing agent, on cytosolic free Ca2+ levels ([Ca2+]i) in populations of BFTC human bladder cancer cells was explored by using fura-2 as a Ca2+ indicator. RESULTS: Clomiphene at concentrations between 10 and 75 microM increased [Ca2+]i in a concentration-dependent manner and the signal saturated at 50 microM. The [Ca2+]i signal was biphasic with an initial rise and a slow decay. Ca2+ removal inhibited the Ca2+ signal by about 40-50% in maximum [Ca2+]i. Adding 3 mM Ca2+ increased [Ca2+]i in cells pretreated with 50 microM clomiphene in Ca2+-free medium, suggesting that clomiphene induced capacitative Ca2+ entry. In Ca2+-free medium, pretreatment with 50 microM brefeldin A (to disrupt the Golgi complex Ca2+ store), 1 microM thapsigargin (to inhibit the endoplasmic reticulum Ca2+ pump), and CCCP (to uncouple mitochondria) inhibited 85% of clomiphene-induced intracellular Ca2+ release. Conversely, pretreatment with 50 microM clomiphene in Ca2+-free medium abolished the [Ca2+]i increase induced by brefeldin, thapsigargin or CCCP. The intracellular Ca2+ release was unaltered by inhibiting formation of inositol-1,4,5-trisphosphate (IP3) with 2 mM 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122; a phospholipase C inhibitor). CONCLUSION: The [Ca2+]i increase induced by 50 microM clomiphene was not affected by 10 microM of nifedipine, verapamil or diltiazem. Collectively, the results suggest that clomiphene releases intracellular Ca2+ in an IP3-independent manner and also activates extracellular Ca2+ influx.     

The carcinogen safrole increases intracellular free Ca2+ levels and causes death in MDCK cells

The effect of the carcinogen safrole on intracellular Ca2+ movement in renal tubular cells has not been explored previously. The present study examined whether safrole could alter Ca2+ handling in Madin-Darby canine kidney (MDCK) cells. Cytosolic free Ca2+ levels ([Ca2+]i) in populations of cells were measured using fura-2 as a fluorescent Ca2+ probe. Safrole at concentrations above 33 microM increased [Ca2+]i in a concentration-dependent manner with an EC50 value of 400 microM. The Ca2+ signal was reduced by 90% by removing extracellular Ca2+, but was not affected by nifedipine, verapamil, or diltiazem. Addition of Ca2+ after safrole had depleted intracellular Ca(2+)-induced dramatic Ca2+ influx, suggesting that safrole caused store-operated Ca2+ entry. In Ca(2+)-free medium, after pretreatment with 650 microM safrole, 1 microM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor) failed to release more Ca 2+. Inhibition of phospholipase C with 2 microM U73122 did not affect safrole-induced Ca2+ release. Trypan blue exclusion assays revealed that incubation with 650 microM safrole for 30 min did not kill cells, but killed 70% of cells after incubation for 60 min. Collectively, the data suggest that in MDCK cells, safrole induced a [Ca2+] increase by causing Ca2+ release from the endoplasmic reticulum in a phospholipase C-independent fashion, and by inducing Ca2+ influx via store-operated Ca2+ entry. Furthermore, safrole can cause acute toxicity to MDCK cells.     

Independent effects of the broccoli-derived compound sulforaphane on Ca2+ influx and apoptosis in Madin-Darby canine renal tubular cells

This study explored whether sulforaphane changed basal [Ca2+]i levels in suspended Madin-Darby canine kidney (MDCK) cells by using fura-2 as a Ca(2+)-sensitive fluorescent dye. Sulforaphane at concentrations between 2.5-10 microM increased [Ca2+]i in a concentration-dependent manner. This Ca2+ influx was inhibited by phospholipase A2 inhibitor aristolochic acid but not by Ca2+ channel blockers such as nifedipine, nimodipine, nicardipine, diltiazem, verapamil, econazole and SK&F96365. The Ca2+ signal was abolished by removing extracellular Ca2+. In Ca(2+)-free medium, pretreatment with sulforaphane did not alter the endoplasmic reticulum Ca2+ pump inhibitor thapsigargin-induced Ca2+ release suggesting sulforaphane did not induce slow Ca2+ release from endoplasmic reticulum. At concentrations between 1 and 20 microM, sulforaphane induced concentration-dependent decrease in cell viability which was not affected by pre-chelation of cytosolic Ca2+ with BAPTA/AM. Flow cytometry data suggest that 20 (but not 5 and 10) microM sulforaphane induced significant increase in sub G1 phase indicating involvement of apoptosis. Collectively, in MDCK cells, sulforaphane induced [Ca2+]i rises by causing Ca2+ entry through phospholipase A2-sensitive pathways without inducing Ca2+ release from the endoplasmic reticulum. Sulforaphane also induced Ca(2+)-independent cell death that might involve apoptosis.