Kam-Chung Lee

Tamoxifen-induced increases in cytoplasmic free Ca2+ levels in human breast cancer cells.

Tamoxifen has been shown to increase cytoplasmic free Ca2+ levels [Ca2+]i in renal tubular cells and bladder cancer cells, and to alter Ca2+ signaling in MCF-7 breast cancer cells. The present study examined the effect of tamoxifen on [Ca2+]i in ZR-75-1 human breast cancer cells using fura-2 as an indicator. Tamoxifen increased [Ca2+]i at a concentration above 2 μM with an EC50 of 5 μM. Removing extracellular Ca2+ reduced the response by 48 ± 2%. In Ca2+-free medium, after tamoxifen-induced [Ca2+]i increased had returned to baseline, adding 3 mM Ca2+ increased [Ca2+]i in a concentration-dependent manner. Further, pretreatment with 10 μM tamoxifen abolished the [Ca2+]i increase induced by 1 μM thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor); and conversely, pretreatment with thapsigargin prevented tamoxifen from releasing more Ca2+. Tamoxifen (10 μM)-induced Ca2+ release was not changed by inhibiting phospholipase C activity with 2 μM U73122. Trypan blue exclusion assay revealed that tamoxifen (1–10 μM) did not alter viability after 1 min of incubation, but killed 10% of cells after 3–10 min of incubation. Together, this study shows that tamoxifen (>2 μM) induced a significant, immediate increase in [Ca2+]i in ZR-75-1 breast cancer cells. Tamoxifen acted by releasing Ca2+ from the endoplasmic reticulum Ca2+ stores in a manner independent of phospholipase C activity, and by inducing Ca2+ entry from extracellular medium. Tamoxifen may be of mild cytotoxicity after acute exposure.

Effect of timosaponin A-III, from Anemarrhenae asphodeloides Bunge (Liliaceae), on calcium mobilization in vascular endothelial and smooth muscle cells and on vascular tension

The effects of timosaponin A-III (TA-III), from Rhizoma Anemarrhenae, on Ca(2+) mobilization in vascular endothelial cells and smooth muscle cells and on vascular tension have been explored. TA-III increased intracellular Ca(2+) concentrations ([Ca(2+)](i)) in endothelials cells at a concentration larger than 5 microM with an EC(50) of 15 microM, and increased [Ca(2+)](i) in smooth muscle cells at a concentration larger than 1 microM with an EC(50) of 8 microM. Within 5 min, the [Ca(2+)](i) signal was composed of a gradual rise, and the speed of rising depended on the concentration of TA-III. The [Ca(2+)](i) signal was abolished by removing extracellular Ca(2+) and was recovered after reintroduction of Ca(2+). The TA-III-induced [Ca(2+)](i) increases in smooth muscle cells were partly inhibited by 10 microM nifedipine or 50 microM La(3+), but was insensitive to 10 microM verapamil and diltiazem. TA-III (10-100 microM) inhibited 0.3 microM phenylephrine-induced vascular contraction, which was abolished by pretreatment with 100 microM N(omega)-nitro-L-arginine (L-NNA) or by denuding the aorta. TA-III also increased [Ca(2+)](i) in renal tubular cells with an EC(50) of 8 microM. Collectively, the results show for the first time that TA-III causes [Ca(2+)](i) increases in the vascular system. TA-III acted by causing Ca(2+) influx without releasing intracellular Ca(2+). TA-III induced relaxation of phenylephrine-induced vascular contraction via inducing release of nitric oxide from endothelial cells.

Effect of betulinic acid on intracellular-free Ca2+ levels in Madin Darby canine kidney cells

The effect of betulinic acid, an anti-tumor and apoptosis-inducing natural product, on intracellular-free levels of Ca(2+) ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was examined by using fura-2 as a Ca(2+) dye. Betulinic acid caused significant increases in [Ca(2+)](i) concentration dependently between 25 and 500 nM with an EC(50) of 100 nM. The [Ca(2+)](i) signal was composed of an initial gradual rise and a plateau. The response was decreased by removal of extracellular Ca(2+) by 45+/-10%. In Ca(2+)-free medium, pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) abolished 250 microM betulinic acid-induced [Ca(2+)](i) increases. Conversely, pretreatment with betulinic acid only partly inhibited thapsigargin-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) increase after pretreatment with 250 nM betulinic acid in Ca(2+)-free medium for 5 min. This [Ca(2+)](i) increase was not altered by the addition of 20 microM SKF96365 and 10 microM econazole. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) abolished 250 nM betulinic acid-induced Ca(2+) release. Pretreatment with 10 microM La(3+) inhibited 250 nM betulinic acid-induced [Ca(2+)](i) increases by 85+/-3%; whereas 10 microM of verapamil, nifedipine and diltiazem had no effect. In Ca(2+) medium, pretreatment with 2.5 nM betulinic aid for 260 s potentiated 10 microM ATP and 1 microM thapsigargin-induced [Ca(2+)](i) increases by 33+/-3% and 45+/-3%, respectively. Trypan blue exclusion revealed that acute exposure of 250 nM betulinic acid for 2-30 min decreased cell viability by 6+/-2%, which could be prevented by pretreatment with 2 microM U731222. Together, the results suggest that betulinic acid induced significant [Ca(2+)](i) increases in MDCK cells in a concentration-dependent manner, and also induced mild cell death. The [Ca(2+)](i) signal was contributed by an inositol 1,4, 5-trisphosphate-dependent release of intracellular Ca(2+) from thapsigargin-sensitive stores, and by inducing Ca(2+) entry from extracellular medium in a La(3+)-sensitive manner.

Effect of diethylstilbestrol (DES) on intracellular Ca2+ levels in renal tubular cells

The effect of the estrogen diethylstilbestrol (DES) on intracellular Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) cells was investigated, using the fluorescent dye fura-2 as a Ca(2+) indicator. DES (10-50 microM) evoked [Ca(2+)](i) increases in a concentration-dependent manner. Extracellular Ca(2+) removal inhibited 45 +/- 5% of the Ca(2+) response. In Ca(2+)-free medium, pretreatment with 50 microM DES abolished the [Ca(2+)](i) increases induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor); and pretreatment with CCCP and thapsigargin partly inhibited DES-induced [Ca(2+)](i) signals. Adding 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 50 microM DES in Ca(2+)-free medium, suggesting that DES may induce capacitative Ca(2+) entry. 17beta-Estradiol (2-20 microM) increased [Ca(2+)](i), but 100 microM diethylstilbestrol dipropionate had no effect. Pretreatment with the phospholipase C inhibitor U73122 (1 microM) to abolish inositol 1,4,5-trisphosphate formation inhibited 30% of DES-induced Ca(2+) release. DES (20 microM) also increased [Ca(2+)](i) in human normal hepatocytes and osteosarcoma cells. Cumulatively, this study shows that DES induced rapid and sustained [Ca(2+)](i) increases by releasing intracellular Ca(2+) and triggering extracellular Ca(2+) entry in renal tubular cells.

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 CP55,940s action appears to be dissociated from stimulation of cannabinoid receptors.

NPC-14686, a novel anti-inflammatory agent, increased intracellular Ca2+ concentrations in MDCK renal tubular cells

The effect of NPC-14686 (Fmoc-L-homophenylalanine), a novel anti-inflammatory agent on intracellular free Ca(2+) concentrations ([Ca(2+)](i)) in Madin Darby canine kidney (MDCK) renal tubular cells, was investigated, using fura-2 as a Ca(2+) dye. At concentrations between 10 and 200 microM NPC-14686 increased [Ca(2+)](i) concentration dependently. The [Ca(2+)](i) signal comprised an initial rise and a sustained phase. Ca(2+) removal inhibited the Ca(2+) signals by 90%. In Ca(2+)-free medium, pretreatment with 100 microM NPC-14686 nearly abolished the [Ca(2+)](i) increase induced by 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) and abolished the [Ca(2+)](i) increase induced by 2 microM carbonylcyanide m-chlorophenylhydrazone (CCCP) (a mitochondrial uncoupler). NPC-14686 (100 microM) induced a slight [Ca(2+)](i) increase after pretreatment with 2 microM CCCP and 1 microM thapsigargin. Addition of 3 mM Ca(2+) elicited a [Ca(2+)](i) increase in cells pretreated with 100 microM NPC-14686 in Ca(2+)-free medium. Inhibition of inositol-1,4,5-trisphosphate (IP(3)) production by suppressing phospholipase C with 2 microM U73122 did not alter NPC-14686-induced Ca(2+) release. Trypan blue exclusion revealed that incubation with 10 or 200 microM NPC-14686 for 1-30 min decreased cell viability by 10-20% concentration dependently. Collectively, the results demonstrate that, in MDCK tubular cells, NPC-14686 induced Ca(2+) release followed by Ca(2+) entry, with the latter playing a major role. NPC-14686 appears to release intracellular Ca(2+) in an IP(3)-uncoupled manner. NPC-14686 may be of mild cytotoxicity.

Effect of oleamide on Ca2+ signaling in human bladder cancer cells

Abstract The effect of oleamide, a sleep-inducing endogenous lipid in animal models, on intracellular free levels of Ca 2+ ([Ca 2+ ] i ) in non-excitable and excitable cells was examined by using fura-2 as a fluorescent dye. [Ca 2+ ] i in pheochromocytoma cells, renal tubular cells, osteoblast-like cells, and bladder cancer cells were increased on stimulation of 50 μM oleamide. The response in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10–100 μM) increased [Ca 2+ ] i in a concentration-dependent fashion with an EC 50 of 50 μM. The [Ca 2+ ] i signal comprised an initial rise and a sustained plateau and was reduced by removing extracellular Ca 2+ by 85 ± 5%. After pre-treatment with 10–100 μM oleamide in Ca 2+ -free medium, addition of 3 mM Ca 2+ increased [Ca 2+ ] i in a manner dependent on the concentration of oleamide. The [Ca 2+ ] i increase induced by 50 μM oleamide was reduced by 100 μM La 3+ by 40%, but was not altered by 10 μM nifedipine, 10 μM verapamil, and 50 μM Ni 2+ . In Ca 2+ -free medium, pre-treatment with thapsigargin (1 μM), an endoplasmic reticulum Ca 2+ pump inhibitor, abolished 50 μM oleamide-induced [Ca 2+ ] i increases; conversely, pretreatment with 50 μM oleamide reduced 1 μM thapsigargin-induced [Ca 2+ ] i increases by 50 ± 3%. Suppression of the activity of phospholipase C with 2 μM U73122 failed to alter 50 μM oleamide-induced Ca 2+ release. Linoleamide (10–100 μM), another sleep-inducing lipid with a structure similar to that of oleamide, also induced an increase in [Ca 2+ ] i . Together, it was shown that oleamide induced significant [Ca 2+ ] i increases in cells by a phospholipase C-independent release of Ca 2+ from thapsigargin-sensitive stores and by inducing Ca 2+ entry.

Effect of 17β-Estradiol on Intracellular Ca2+ Levels in Renal Tubular Cells

The effect of 17β-estradiol on intracellular Ca²⁺ concentrations ([Ca²⁺]_i) in Madin Darby canine kidney cells was investigated by using the fluorescent dye fura-2. 17β-Estradiol (5–100 µmol/l) induced instantaneous increases in [Ca²⁺]_i in a concentration-dependent manner. Ca²⁺ removal inhibited 45 ± 15% of the Ca²⁺ signal. In Ca²⁺-free medium, pretreatment with 50 µmol/l 17β-estradiol abolished the [Ca²⁺]_i increases induced by 2 µmol/l carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler), 1 µmol/l thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor) and 50 µmol/l brefeldin A (an antibiotic which disperses the Golgi complex), but pretreatment with brefeldin A, CCCP and thapsigargin only partly inhibited the 17β-estradiol-induced [Ca²⁺]_i signal. Adding 3 mmol/lCa²⁺ increased [Ca²⁺]_i in cells pretreated with 5–100 µmol/l 17β-estradiol in Ca²⁺-free medium. Pretreatment with 1 µmol/l U73122 to abolish the formation of inositol-1,4,5-trisphosphate inhibited 50% of the Ca²⁺ release induced by 50 µmol/l 17β-estradiol. 17β-Estradiol (20 µmol/l) also increased [Ca²⁺]_i in human bladder cancer cells and prostate cancer cells. Collectively, this study shows that 17β-estradiol evoked a significant internal Ca²⁺ release and external Ca²⁺ entry possibly in a nongenomic manner.

Effect of nordihydroguaiaretic acid on intracellular Ca2+ concentrations in C6 glioma cells

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