Warren Su

Mechanism of Bradykinin-Induced Ca2+ Mobilization in MG63 Human Osteosarcoma Cells

Background: The effect of bradykinin on intracellular free Ca²⁺ levels ([Ca²⁺]_i) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca²⁺ dye. Methods/Results: Bradykinin (0.1 nM–1 µM) increased [Ca²⁺]_i in a concentration-dependent manner with an EC₅₀ value of 0.5 nM. The [Ca²⁺]_i signal comprised an initial peak and a fast decay which returned to baseline in 2 min. Extracellular Ca²⁺ removal inhibited the peak [Ca²⁺]_isignals by 35 ± 3%. Bradykinin (1 nM) failed to increase [Ca²⁺]_i in the absence of extracellular Ca²⁺after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca²⁺ pump inhibitor; 1 µM). Bradykinin (1 nM)-induced intracellular Ca²⁺ release was nearly abolished by inhibiting phospholipase C with 2 µM 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The [Ca²⁺]_iincrease induced by 1 nM bradykinin in Ca²⁺- free medium was abolished by 1 nM HOE 140 (a B2 bradykinin receptor antagonist) but was not altered by 100 nM Des-Arg-HOE 140 (a B1 bradykinin receptor antagonist). Pretreatment with 1 pM pertussis toxin for 5 h in Ca²⁺ medium inhibited 30 ± 3% of 1 nM bradykinin-induced peak [Ca²⁺]_i increase. Conclusions: Together, this study shows that bradykinin induced [Ca²⁺]_i increases in a concentration-dependent manner, by stimulating B2 bradykinin receptors leading to mobilization of Ca²⁺ from the thapsigargin-sensitive stores in a manner dependent on inositol-1,4,5-trisphosphate, and also by inducing extracellular Ca²⁺ influx. The bradykinin response was partly coupled to a pertussis toxin-sensitive G protein pathway.

Effect of the organotin compound triethyltin on Ca2+ handling in human prostate cancer cells.

The effects of triethyltin on Ca2+ mobilization in human PC3 prostate cancer cells have been explored. Triethyltin increased [Ca2+]i at concentrations larger than 3 microM with an EC50 of 30 microM. Within 5 min, the [Ca2+]i signal was composed of a gradual rise and a sustained phase. The [Ca2+]i signal was reduced by half by removing extracellular Ca2+. The triethyltin-induced [Ca2+]i increases were inhibited by 40% by 10 microM nifedipine, nimodipine and nicardipine, but were not affected by 10 microM of verapamil or diltiazem. In Ca2+-free medium, pretreatment with thapsigargin (1 microM), an endoplasmic reticulum Ca+ pump inhibitor, reduced 200 microM triethyltin-induced Ca+ increases by 50%. Pretreatment with U73122 (2 microM) to inhibit phospholipase C did not alter 200 microM triethyltin-induced [Ca2+]i increases. Incubation with triethyltin at a concentration that did not increase [Ca2+]i (1 microM) in Ca2+-containing medium for 3 min potentiated ATP (10 microM)- or bradykinin (1 microLM)-induced [Ca2+]i increases by 41 +/- 3% and 51 +/- 2%, respectively. Collectively, this study shows that the environmental toxicant triethyltin altered Ca2+ handling in PC3 prostate cancer cells in a concentration-dependent manner: at higher concentrations it increased basal [Ca2+]i; and at lower concentrations it potentiated agonists-induced [Ca2+]i increases.

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.

Mechanisms of diethylstilbestrol-induced calcium movement in MG63 human osteosarcoma cells.

The effect of the estrogen diethylstilbestrol (DES) on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteoblasts was explored by using fura-2 as a Ca(2+) indicator. DES at concentrations between 5--20 microM induced an immediate increase in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) of 10 microM. Removing extracellular Ca(2+) reduced the Ca(2+) signal by 70%. Pretreatment with 50 microM La(3+) or 10 microM of nifedipine, verapamil and diltiazem did not change 20 microM DES-induced [Ca(2+)](i) increases. Addition of 3 mM Ca(2+) increased [Ca(2+)](i) in cells pretreated with 20 microM DES in Ca(2+)-free medium. Pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store partly inhibited 20 microM DES-induced Ca(2+) release, but addition of carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler) and thapsigargin together abolished DES-induced Ca(2+) release. Conversely, pretreatment with 20 microM DES abrogated CCCP- and thapsigargin-induced Ca(2+) release. Inhibition of phospholipase C activity with 2 microM U73122 did not alter 20 microM DES-induced Ca2+ release. Another estrogen 17beta-estradiol also increased [Ca(2+)](i) in a concentration-dependent manner with an EC50 of 7 microM. Together, the data indicate that in human osteoblasts, DES increased [Ca(2+)](i) via causing Ca(2+) release from both mitochondria and the endoplasmic reticulum in a phospholipase C-independent manner, and by causing Ca(2+) influx.

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.

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 lignans isolated from Hernandia nymphaeifolia on estrogenic compounds-induced calcium mobilization in human neutrophils.

The effect of five lignans isolated from Hernandia nymphaeifolia on estrogenic compounds (17beta-estradiol, tamoxifen and clomiphene)-induced Ca(2+) mobilization in human neutrophils was investigated. The five lignans were epi-yangambin, epi-magnolin, epi-aschantin, deoxypodophyllotoxin and yatein. In Ca(2+)-containing medium, the lignans (50-100 microM) inhibited 10 microM 17beta-estradiol- and 5 microM tamoxifen-induced increases in intracellular free Ca(2+) levels ([Ca(2+)](i)) without changing 25 microM clomiphene-induced [Ca(2+)](i) increase. 17beta-estradiol and tamoxifen increased [Ca(2+)](i) by causing Ca(2+) influx and Ca(2+) release because their responses were partly reduced by removing extracellular Ca(2+). In contrast, clomiphene solely induced Ca(2+) release. The effect of the lignans on these two Ca(2+) movement pathways underlying 17beta-estradiol- and tamoxifen-induced [Ca(2+)](i) increases was explored. All the lignans (50-100 microM) inhibited 10 microM 17beta-estradiol-and 5 microM tamoxifen-induced Ca(2+) release, and 17beta-estradiol-induced Ca(2+) influx. However, only 100 microM epi-aschantin was able to reduce tamoxifen-induced Ca(2+) influx while the other lignans had no effect. Collectively, this study shows that the lignans altered estrogenic compounds-induced Ca(2+) signaling in human neutrophils in a multiple manner.

Novel action of lignans isolated from Hernandia nymphaeifolia on Ca2+ signaling in renal tubular cells

The effect of five lignans, epi-aschantin, epi-magnolin, epi-yangambin, deoxypodophyllotoxin and yatein, isolated from Hernandia nymphaeifolia on Ca(2+) signaling in Madin-Darby canine kidney cells was examined using fura-2 as a Ca(2+) indicator. These lignans at concentrations between 10 and 100 microM increased [Ca(2+)](i) in a concentration-dependent manner. Removal of extracellular Ca(2+) abolished the Ca(2+) signals evoked by 50 microM of the lignans. La(3+)(50 microM) abolished the Ca(2+) signals induced by 100 microM of epi-aschantin, epi-magnolin and epi-yangambin, and 20 microM deoxypodophyllotoxin, but inhibited by 60% 50 microM yatein-induced responses. All five lignans (50-100 microM) inhibited by 42-65% thapsigargin-induced capacitative Ca(2+) entry, and inhibited by 23-61% thapsigargin-induced intracellular Ca(2+) release. Epi-yangambin (100 microM), epi-magnolin (100 microM), and epi-aschantin (100 microM) inhibited by 8-38% 10 microM ATP-induced Ca(2+) release. Trypan blue exclusion revealed that incubation with deoxypodophyllotoxin or yatein (but not the other lignans) decreased cell viability in a concentration-dependent manner. Together, the results suggest that, in renal tubular cells, these lignans exert multiple actions on Ca(2+) signaling. They caused Ca(2+) influx but reduced thapsigargin-induced capacitative Ca(2+) entry and also thapsigargin- and ATP-induced Ca(2+) release. Additionally, deoxypodophyllotoxin and yatein may be cytotoxic.

Fendiline mobilizes intracellular Ca2+ in Chang liver cells.

1. The effects of the antianginal drug fendiline (N‐[3,3‐diphenylpropyl]‐α‐methyl‐benzylamine) on intracellular free Ca2+ levels ([Ca2+]i) in Chang liver cells were evaluated using fura‐2 as a fluorescent Ca2+ indicator.