Jue-Long Wang

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.

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.

Mechanisms of histamine-induced intracellular Ca2+ release and extracellular Ca2+ entry in MG63 human osteosarcoma cells

The effect of histamine on intracellular free Ca2+ levels ([Ca2+](i)) in MG63 human osteosarcoma cells was explored using fura-2 as a Ca2+ dye. Histamine increased ([Ca2+](i)) in a concentration-dependent fashion with an EC(50) value of 0.5 microM. Extracellular Ca2+ removal inhibited the ([Ca2+](i)) signals. Histamine failed to increase ([Ca2+](i)) in Ca2+-free medium after cells were pretreated with thapsigargin (an endoplasmic reticulum Ca2+ pump inhibitor). Addition of Ca2+ induced concentration-dependent ([Ca2+](i)) increases after preincubation with histamine in Ca2+-free medium. Histamine-induced intracellular Ca2+ release was abolished by inhibiting phospholipase C with 1-(6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione (U73122). The ([Ca2+](i)) increase induced by histamine in Ca2+ medium was abolished by cimetidine, but was not altered by pyrilamine, nifedipine, verapamil, and La(3+). Together, this study shows that histamine increased in ([Ca2+](i)) in osteosarcoma cells by stimulating H2 histamine receptors. The Ca2+ signal was caused by Ca2+ release from the endoplasmic reticulum in a phospholipase C-dependent manner. The Ca2+ release was accompanied by Ca(2+) influx.

Effect of Thymol on Ca2+ Homeostasis and Viability in MG63 Human Osteosarcoma Cells

Aims: The effect of the natural product thymol on cytosolic Ca²⁺ concentrations ([Ca²⁺]_i) and viability in MG63 human osteosarcoma cells was examined. Methods: The Ca²⁺-sensitive fluorescent dye fura-2 was applied to measure [Ca²⁺]_i. Results: Thymol at concentrations of 200–1,000 µmol/l induced a [Ca²⁺]_i rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca²⁺. Thymol-induced Ca²⁺ entry was inhibited by nifedipine, econazole, SK&F96365 and protein kinase C modulators. When extracellular Ca²⁺ was removed, incubation with the endoplasmic reticulum Ca²⁺ pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited the thymol-induced [Ca²⁺]_i rise. Incubation with thymol also inhibited the thapsigargin or BHQ-induced [Ca²⁺]_i rise. Inhibition of phospholipase C with U73122 abolished the thymol-induced [Ca²⁺]_i rise. At concentrations of 100–600 µmol/l, thymol 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/AM. Annexin V/propidium iodide staining data suggest that thymol (200 and 400 µmol/l) induced apoptosis in a concentration-dependent manner. Thymol (200 and 400 µmol/l) also increased levels of reactive oxygen species. Conclusions: In MG63 cells, thymol induced a [Ca²⁺]_i rise by inducing phospholipase C-dependent Ca²⁺ release from the endoplasmic reticulum and Ca²⁺ entry via protein kinase C-sensitive store-operated Ca²⁺ channels. Thymol induced cell death that may involve apoptosis via mitochondrial pathways.

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.

Effect of the Antidepressant Desipramine on Cytosolic Ca2+ Movement and Proliferation in Human Osteosarcoma Cells

In human osteosarcoma MG63 cells, the effect of desipramine, an antidepressant, on intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured by using fura-2. Desipramine (>10 µmol/l) caused a rapid and sustained rise of [Ca²⁺]_i in a concentration-dependent manner (EC₅₀ = 200 µmol/l). Desipramine-induced [Ca²⁺]_i rise was prevented by 80% 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 (ER) Ca²⁺-ATPase, caused a monophasic [Ca²⁺]_i rise, after which the increasing effect of desipramine on [Ca²⁺]_i was abolished; also, pretreatment with desipramine partly reduced thapsigargin-induced [Ca²⁺]_i increase. U73122, an inhibitor of phospholipase C, did not affect desipramine-induced [Ca²⁺]_i rise. Overnight incubation with 10 µmol/l desipramine did not alter cell proliferation, but killed 32 and 89% of cells at concentrations of 100 and 200 µmol/l, respectively. These findings suggest that desipramine rapidly increases [Ca²⁺]_i in osteoblasts by stimulating both extracellular Ca²⁺ influx and intracellular Ca²⁺ release, and is cytotoxic at high concentrations.

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 sertraline on [Ca2+]i and viability of human MG63 osteosarcoma cells

The antidepressant, sertraline, has been shown to have diverse in vitro effects. This study examined whether sertraline altered [Ca2+]i in MG63 human osteosarcoma cells by using fura-2 as a Ca2+-sensitive fluorescent dye. At 50–200 µM, sertraline induced a [Ca2+]i rise in a concentration-dependent manner. Ca2+ response was decreased by removing extracellular Ca2+, suggesting that Ca2+ entry and release contributed to the [Ca2+]i signal. Sertraline-induced Ca2+ entry was inhibited by nifedipine, La3+, Gd3+, and SK&F96365. When extracellular Ca2+ was removed, pretreatment with the endoplasmic reticulum (ER) Ca2+ pump inhibitor, thapsigargin, or 2,5-di-tert-butylhydroquinone (BHQ) abolished the sertraline-evoked [Ca2+]i rise. Incubation with sertraline also abolished the thapsigargin or BHQ-induced [Ca2+]i rise. Inhibition of phospholipase C (PLC) with U73122 abolished the sertraline-induced [Ca2+]i rise. At 20–30 µM, overnight treatment with sertraline killed cells in a concentration-dependent manner. The cytotoxic effect of sertraline was not reversed by chelating cytosolic Ca2+ with 1,2-bis(2-aminophenoxy)ethane-N,N,N’,N’-tetraacetic acid (BAPTA). Annexin V/propidium iodide staining data demonstrate that sertraline (30 µM) evoked apoptosis. Sertraline (20 and 30 µM) also increased levels of reactive oxygen species. Together, in human osteosarcoma cells, sertraline evoked a [Ca2+]i rise by inducing PLC-dependent Ca2+ release from the ER and Ca2+ entry by L-type Ca2+ channels and store-operated Ca2+ channels. Sertraline induced cell death that may involve apoptosis by mitochondrial pathways.

Effects of Antrodia camphorata extracts on the viability, apoptosis, [Ca2+]i, and MAPKs phosphorylation of OC2 human oral cancer cells.

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.