Effect of celecoxib on Ca2+ movement and cell proliferation in human osteoblasts

In human osteoblasts, the effect of the widely prescribed cyclooxygenase-2 inhibitor celecoxib on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cell proliferation was explored by using fura-2 and the tetrazolium assay, respectively. Celecoxib at concentrations greater than 1microM caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner ( EC 50= 10 microM). Celecoxib-induced [Ca(2+)](i) rise was reduced by 90% by removal of extracellular Ca(2+), and by 30% by l-type Ca(2+) channel blockers. Celecoxib-induced Mn(2+)-associated quench of intracellular fura-2 fluorescence also suggests that celecoxib-induced extracellular Ca(2+) influx. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of celecoxib on [Ca(2+)](i) was greatly inhibited. Conversely, pretreatment with celecoxib to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). U73122, an inhibitor of phoispholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not celecoxib-induced, [Ca(2+)](i) rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, partly inhibited celecoxib-induced [Ca(2+)](i) rise in Ca(2+)-containing medium. Separately, overnight treatment with 1-100microM celecoxib inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human osteoblasts, celecoxib increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing intracellular Ca(2+) release from the endoplasmic reticulum via a phospholiase C-independent manner. Celecoxib may be cytotoxic at higher concentrations.     

Effect of capsazepine on cytosolic Ca(2+) levels and proliferation of human prostate cancer cells.

Capsazepine has been widely used as a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on most cell types is unknown. In human PC3 prostate cancer cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was investigated by using fura-2 and tetrazolium, respectively. Capsazepine caused a rapid rise in [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 75 microM. Capsazepine-induced [Ca(2+)](i) rise was reduced by 60% by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was contributed by extracellular Ca(2+) influx and intracellular Ca(2+). Consistently, the capsazepine (200 microM)-induced [Ca(2+)](i) rise was decreased by La(3+) by half. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the effect of capsazepine on [Ca(2+)](i) was inhibited by 80%. Conversely, pretreatment with capsazepine partly reduced thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca(2+) mobilizer)-induced, but not capsazepine-induced, [Ca(2+)](i) rise. These findings suggest that in human PC3 prostate cancer cells, capsazepine increases [Ca(2+)](i) by evoking Ca(2+) influx and releasing Ca(2+) from the endoplasmic reticulum via a phospholiase C-independent manner. Overnight incubation with capsazepine (200 microM) killed 37% of cells, which could not be prevented by chelating intracellular Ca(2+) with BAPTA.     

Fluoxetine inhibits ATP-induced [Ca(2+)](i) increase in PC12 cells by inhibiting both extracellular Ca(2+) influx and Ca(2+) release from intracellular stores

Fluoxetine, a widely used antidepressant, has additional effects, including the blocking of voltage-gated ion channels. We examined whether fluoxetine affects ATP-induced calcium signaling in PC12 cells using fura-2-based digital calcium imaging, an assay for [3H]-inositol phosphates (IPs) and whole-cell patch clamping. Treatment with ATP (100 microM) for 2 min induced increases in intracellular free Ca(2+) concentrations ([Ca(2+)](i)). Treatment with fluoxetine (100 nM to 30 microM) for 5 min inhibited the ATP-induced [Ca(2+)](i) increases in a concentration-dependent manner (IC(50) = 1.85 microM). Treatment with fluoxetine (1.85 microM) for 5 min significantly inhibited the ATP-induced responses following the removal of extracellular Ca(2+) or depletion of intracellular Ca(2+) stores. Whereas treatment for 10 min with nimodipine (1 microM) significantly inhibited the ATP-induced [Ca(2+)](i) increase, treatment with fluoxetine further inhibited the ATP-induced response. Treatment with fluoxetine significantly inhibited [Ca(2+)](i) increases induced by 50 mM K(+). In addition, treatment with fluoxetine markedly inhibited ATP-induced inward currents in a concentration-dependent manner. However, treatment with fluoxetine did not inhibit ATP-induced [3H]-IPs formation. Therefore, we conclude that fluoxetine inhibits ATP-induced [Ca(2+)](i) increases in PC12 cells by inhibiting both the influx of extracellular Ca(2+) and the release of Ca(2+) from intracellular stores without affecting IPs formation.     

Effect of calmidazolium on Ca(+2) movement and proliferation in human osteosarcoma cells

In human MG63 osteosarcoma cells, the effect of calmidazolium on [Ca(2+)](i) and proliferation was explored using fura-2 and ELISA, respectively. Calmidazolium, at concentrations greater than 0.1 micromol/L, caused a rapid increase in [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 0.5 micromol/L). The calmidazolium-induced [Ca(2+)](i) increase was reduced by 66% by removal of extracellular Ca(2+). In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum Ca(2+)-ATPase, caused a monophasic increase in [Ca(2+)](i), after which the effect of calmidazolium to increase [Ca(2+)](i) was completely inhibited. U73122, an inhibitor of phospholipase C (PLC), abolished histamine (but not calmidazolium)-induced increases in [Ca(2+)](i). Pretreatment with phorbol 12-myristate 13-acetate to activate protein kinase C inhibited the calmidazolium-induced increase in [Ca(2+)](i) in Ca(2+)-containing medium by 47%. Separately, it was found that overnight treatment with 2-10 micromol/L calmidazolium inhibited cell proliferation in a concentration-dependent manner. These results suggest that calmidazolium increases [Ca(2+)](i) by stimulating extracellular Ca(2+) influx and also by causing release of intracellular Ca(2+) from the endoplasmic reticulum in a PLC-independent manner. Calmidazolium may be cytotoxic to osteosarcoma cells.     

The anti-anginal drug fendiline increases intracellular Ca(2+) levels in MG63 human osteosarcoma cells

The effect of fendiline, an anti-anginal drug, on cytosolic free Ca(2+) levels ([Ca(2+)](i)) in MG63 human osteosarcoma cells was explored by using fura-2 as a Ca(2+) indicator. Fendiline at concentrations between 1 and 200 microM increased [Ca(2+)](i) in a concentration-dependent manner and the signal saturated at 100 microM. The Ca(2+) signal was inhibited by 65+/-5% by Ca(2+) removal and by 38+/-5% by 10 microM nifedipine, but was unchanged by 10 microM La(3+) or verapamil. In Ca(2+)-free medium, pre-treatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+) store inhibited fendiline-induced intracellular Ca(2+) release. The Ca(2+) release induced by 50 microM fendiline appeared to be independent of IP(3) because the [Ca(2+)](i) increase was unaltered by inhibiting phospholipase C with 2 microM U73122. Collectively, the results suggest that in MG63 cells fendiline caused an increase in [Ca(2+)](i) by inducing Ca(2+) influx and Ca(2+) release in an IP(3)-independent manner.     

Triethyltin increases cytosolic Ca(2+) levels in human osteoblasts

In human osteosarcoma MG63 cells, effect of triethyltin, an environmental toxicant, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2. Triethyltin (1-50 μM) caused a rapid and sustained plateau rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50)=10 μM). Triethyltin-induced [Ca(2+)](i) rise was prevented by 50% by removal of extracellular Ca(2+) but was not altered by voltage-gated Ca(2+) channel blockers. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of triethyltin on [Ca(2+)](i) was attenuated by 60%; also, pretreatment with triethyltin abolished thapsigargin-induced [Ca(2+)](i) increase. Depletion of mitochondrial Ca(2+) with carbonylcyanide m-chlorophenylhydrazone (CCCP; 2 μM) did not affect triethyltin-induced Ca(2+) release. U73122, an inhibitor of phoispholipase C, abolished ATP (but not triethyltin)-induced [Ca(2+)](i) rise. A low concentration (1 μM) of triethyltin failed to alter ATP and bradykinin-induced [Ca(2+)](i) rises. These findings suggest that triethyltin rapidly increases [Ca(2+)](i) in osteoblasts by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release via as yet unidentified mechanism(s).     

Dual effect of tamoxifen, an anti-breast-cancer drug, on intracellular Ca(2+) and cytotoxicity in intact cells

The effect of tamoxifen on Ca(2+) signaling and viability in Madin Darby canine kidney (MDCK) cells was investigated by using fura-2 as a Ca(2+) probe. Tamoxifen evoked a rise in cytosolic free Ca(2+) levels ([Ca(2+)](i)) concentration-dependently between 1 and 50 microM with an EC50 of 10 microM. The response was decreased by extracellular Ca(2+) removal. In Ca(2+)-free medium, pretreatment with 5 microM tamoxifen abolished the [Ca(2+)](i) increase induced by the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin (1 microM), but pretreatment with brefeldin A (50 microM; a Ca(2+) mobilizer of the Golgi complex), thapsigargin (an inhibitor of the endoplasmic reticulum Ca(2+) pump), and carbonylcyanide m-chlorophenylhydrazone (CCCP; a mitochondrial uncoupler), only partly inhibited tamoxifen-induced [Ca(2+)](i) increases. This suggests that tamoxifen released Ca(2+) from multiple pools. Addition of 3 mM Ca(2+) induced a [Ca(2+)](i) rise after pretreatment with 5 microM tamoxifen in Ca(2+)-free medium. Inhibiting inositol 1,4,5-trisphosphate formation with the phospholipase C inhibitor U73122 (2 microM) did not alter 5 microM tamoxifen-induced Ca(2+) release. The [Ca(2+)](i) increase induced by 5 microM tamoxifen was not altered by La(3+), nifedipine, verapamil, or diltiazem. Tamoxifen (1-10 microM) decreased cell viability in a concentration- and time-dependent manner. Tamoxifen (5 microM) also increased [Ca(2+)](i) in neutrophils, bladder cancer cells, and prostate cancer cells from humans and glioma cells from rats. Collectively, it was found that tamoxifen increased [Ca(2+)](i) in MDCK cells by releasing Ca(2+) from multiple Ca(2+) stores in a manner independent of the production of inositol 1,4, 5-trisphosphate and also by triggering Ca(2+) influx from extracellular space. The [Ca(2+)](i) increase was accompanied by cytotoxicity.     

Effect of betulinic acid on intracellular-free Ca(2+) 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 the antidepressant desipramine on cytosolic Ca(2+) movement and proliferation in human osteosarcoma cells

In human osteosarcoma MG63 cells, the effect of desipramine, an antidepressant, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2. Desipramine (>10 micromol/l) caused a rapid and sustained rise of [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 200 micromol/l). Desipramine-induced [Ca(2+)](i) rise was prevented by 80% by removal of extracellular Ca(2+) but was not altered by voltage-gated Ca(2+) channel blockers. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, caused a monophasic [Ca(2+)](i) rise, after which the increasing effect of desipramine on [Ca(2+)](i) was abolished; also, pretreatment with desipramine partly reduced thapsigargin-induced [Ca(2+)](i) increase. U73122, an inhibitor of phospholipase C, did not affect desipramine-induced [Ca(2+)](i) rise. Overnight incubation with 10 micromol/l desipramine did not alter cell proliferation, but killed 32 and 89% of cells at concentrations of 100 and 200 micromol/l, respectively. These findings suggest that desipramine rapidly increases [Ca(2+)](i) in osteoblasts by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release, and is cytotoxic at high concentrations.     

Effects of (1R,9S)-beta-hydrastine on intracellular calcium concentration in PC12 cells

(1R,9S)-beta-hydrastine (BHS) decreases the basal intracellular Ca(2+) concentration ([Ca(2+)](i)) in PC12 cells.(5) This study examined the effects of (1R,9S)-BHS on [Ca(2+)](i) in PC12 cells. (1R,9S)-BHS at 10-100 microM in combination with a high extracellular K+ level (56 mM) inhibited the release of dopamine in a concentration-dependent manner with an IC(50) value of 66.5 microM. BHS at 100 microM inhibited the sustained increase in [Ca(2+)](i) induced by a high K+ level (56 mM), and had an inhibitory effect on the 2 microM nifedipine-induced blockage of the K+ -stimulated sustained increase in [Ca(2+)](i). In addition, (1R,9S)-BHS at 100 microM prevented the rapid and sustained increase in [Ca(2+)](i) elicited by 20 mM caffeine, but did not have an effect on the increase induced by 1 microM thapsigargin, in the presence of external Ca(2+). These results suggest that the active sites of (1R,9S)-BHS are mainly L-type Ca(2+) channels and caffeine-sensitive Ca(2+)-permeable channels in PC12 cells.     

Melittin-induced [Ca2+]i increases and subsequent death in canine renal tubular cells

The effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability is largely unknown. This study examined whether melittin alters Ca(2+) levels and causes Ca(2+)-dependent cell death in Madin-Darby canine kidney (MDCK) cells. [Ca(2+)](i) and cell death were measured using the fluorescent dyes fura-2 and WST-1 respectively. Melittin at concentrations above 0.5 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was reduced by 75% by removing extracellular Ca(2+). The melittin-induced Ca(2+) influx was also implicated by melittin-caused Mn(2+) influx. After pretreatment with 1 microM thapsigargin (an endoplasmic reticulum Ca(2+) pump inhibitor), melittin-induced Ca(2+) release was inhibited; and conversely, melittin pretreatment abolished thapsigargin-induced Ca(2+) release. At concentrations of 0.5-20 microM, melittin killed cells in a concentration-dependent manner. The cytotoxic effect of 0.5 microM melittin was nearly completely reversed by prechelating cytosolic Ca(2+) with BAPTA. Melittin at 0.5-2 microM caused apoptosis as assessed by flow cytometry of propidium iodide staining. Collectively, in MDCK cells, melittin induced a [Ca(2+)](i) rise by causing Ca(2+) release from endoplasmic reticulum and Ca(2+) influx from extracellular space. Furthermore, melittin can cause Ca(2+)-dependent cytotoxicity in a concentration-dependent manner.     

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 methoxychlor on Ca2+ handling and viability in OC2 human oral cancer cells

The effect of the insecticide methoxychlor on the physiology of oral cells is unknown. This study aimed to explore the effect of methoxychlor on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) in human oral cancer cells (OC2) by using the Ca(2+)-sensitive fluorescent dye fura-2. Methoxychlor at 5-20 μM increased [Ca(2+)](i) in a concentration-dependent manner. The signal was reduced by 70% by removing extracellular Ca(2+). Methoxychlor-induced Ca(2+) entry was not affected by nifedipine, econazole, SK&F96365 and protein kinase C modulators but was inhibited by the phospholipase A2 inhibitor aristolochic acid. In Ca(2+)-free medium, treatment with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) inhibited or abolished methoxychlor-induced [Ca(2+)](i) rise. Incubation with methoxychlor also inhibited thapsigargin- or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 did not alter methoxychlor-induced [Ca(2+)](i) rise. At 5-20 μM, methoxychlor killed cells in a concentration-dependent manner. The cytotoxic effect of methoxychlor was not reversed by chelating cytosolic Ca(2+) with 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/AM (BAPTA/AM). Annexin V-FITC data suggest that methoxychlor (10 and 20 μM) evoked apoptosis in a concentration-dependent manner. Together, in human OC2, methoxychlor induced a [Ca(2+)](i) rise probably by inducing phospholipase C-independent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via phospholipase A(2)-sensitive channels. Methoxychlor induced cell death that may involve apoptosis.     

Effect of riluzole on cytosolic Ca2+ increase in human osteosarcoma cells

In human osteosarcoma MG63 cells, the effect of the neuroprotective drug riluzole on the intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured using fura-2. Riluzole (50-500 micromol/l) caused a rapid and sustained plateau increase in [Ca(2+)](i) in a concentration-dependent manner (EC(50) = 150 micromol/l). The riluzole-induced rise in [Ca(2+)](i) was prevented by 58 and 20% by extracellular Ca(2+) removal and nifedipine, respectively, but was not changed by La(3+) and verapamil. In Ca(2+)-free medium, thapsigargin, an inhibitor of the endoplasmic reticulum (ER) Ca(2+)-ATPase, caused a monophasic increase in [Ca(2+)](i), after which the increasing effect of riluzole on [Ca(2+)](i) was attenuated by 84%; also, pretreatment with riluzole abolished the thapsigargin-induced [Ca(2+)](i) increase. U73122, an inhibitor of phospholipase C, abrogated the ATP (but not riluzole)-induced rise in [Ca(2+)](i). A low concentration (6 micromol/l) of riluzole selectively potentiated the bradykinin (but not ATP and histamine)-induced increase in [Ca(2+)](i). These results suggest that riluzole rapidly increases [Ca(2+)](i) by stimulating both the extracellular Ca(2+) influx via a nifedipine-sensitive pathway and intracellular Ca(2+) release from the ER via an as yet unidentified mechanism(s).     

Monensin causes transient calcium ion influx into mouse splenic lymphocytes in a sodium ion-independent fashion

Monensin, a Na(+) ionophore, can increase cytosolic free Ca(2+) concentration ([Ca(2+)](i)) in many cell types, but no studies have investigated the mechanism underlying a monensin-induced increase in [Ca(2+)](i) in immune cells. In view of this, we investigated the effect of monensin on [Ca(2+)](i) and cytosolic free Na(+) concentration ([Na(+)](i)) in mouse splenic lymphocytes using a fluorescence Ca(2+) indicator, fura-2, and a fluorescence Na(+) indicator, sodium-binding benzofuran isophthalate (SBFI), respectively. Monensin (1-100 microM) caused transient and sustained increases in [Ca(2+)](i) and [Na(+)](i), respectively, in a concentration-dependent manner. The monensin-induced increase in [Ca(2+)](i) was abolished by the omission of extracellular Ca(2+) or 1-[beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl]-1H-imidazole hydrochloride (SKF-96365, 100-150 microM), and was largely inhibited by Ni(2+) (2-5 mM). The omission of extracellular Na(+) failed to inhibit the monensin-induced increases in [Ca(2+)](i). Furthermore, tetrodotoxin (1-10 microM), 5-(N,N-dimethyl)-amiloride (DMA, 10-20 microM), 2-[4-[(2,5-difluorophenyl)methoxy]phenoxy]-5-ethoxyaniline (SEA0400, 3-10 microM), verapamil (10-200 microM), nifedipine (10-200 microM), omega-agatoxin IVA (0.2-10 microM), omega-conotoxin GVIA (1-10 microM), omega-conotoxin MVIIC (0.5-10 microM), and nordihydroguaiaretic acid (NDGA, 1-10 microM) had no effect on the increases in [Ca(2+)](i). Monensin-induced Mn(2+) influx into splenic lymphocytes. The Mn(2+) influx was completely inhibited by SKF-96365. These results suggest that monensin transiently increases [Ca(2+)](i) in mouse splenic lymphocytes by stimulating Ca(2+) entry via non-selective cation channels in a Na(+)-independent manner.     

Effect of carvedilol on Ca2+ movement and cytotoxicity in human MG63 osteosarcoma cells

Carvedilol is a useful cardiovascular drug for treating heart failure, however, the in vitro effect on many cell types is unclear. In human MG63 osteosarcoma cells, the effect of carvedilol on intracellular Ca2+ concentrations ([Ca2+]i) and cytotoxicity was explored by using fura-2 and tetrazolium, respectively. Carvedilol at concentrations greater than 1 microM caused a rapid rise in [Ca2+]i in a concentration-dependent manner (EC50=15 microM). Carvedilol-induced [Ca2+]i rise was reduced by 60% by removal of extracellular Ca2+. Carvedilol-induced Mn2+-associated quench of intracellular fura-2 fluorescence also suggests that carvedilol induced extracellular Ca2+ influx. 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 carvedilol on [Ca2+]i was inhibited by 50%. Conversely, pretreatment with carvedilol to deplete intracellular Ca2+ stores totally prevented thapsigargin from releasing more Ca2+. U73122, an inhibitor of phospholipase C, abolished histamine (an inositol 1,4,5-trisphosphate-dependent Ca2+ mobilizer)-induced, but not carvedilol-induced, [Ca2+]i rise. Pretreatment with phorbol 12-myristate 13-acetate and forskolin to activate protein kinase C and adenylate cyclase, respectively, did not alter carvedilol-induced [Ca2+]i rise. Separately, overnight treatment with 0.1-30 microM carvedilol inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, carvedilol increases [Ca2+]i by stimulating extracellular Ca2+ influx and also by causing intracellular Ca2+ release from the endoplasmic reticulum and other stores via a phospholipase C-independent manner. Carvedilol may be cytotoxic to osteoblasts.     

Effect of oleamide on Ca(2+) signaling in human bladder cancer cells.

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 microM oleamide. The response in human bladder cancer cells (T24) was the greatest and was further explored. Oleamide (10-100 microM) increased [Ca(2+)](i) in a concentration-dependent fashion with an EC(50) of 50 microM. 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 microM 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 microM oleamide was reduced by 100 microM La(3+) by 40%, but was not altered by 10 microM nifedipine, 10 microM verapamil, and 50 microM Ni(2+). In Ca(2+)-free medium, pre-treatment with thapsigargin (1 microM), an endoplasmic reticulum Ca(2+) pump inhibitor, abolished 50 microM oleamide-induced [Ca(2+)](i) increases; conversely, pretreatment with 50 microM oleamide reduced 1 microM thapsigargin-induced [Ca(2+)](i) increases by 50 +/- 3%. Suppression of the activity of phospholipase C with 2 microM U73122 failed to alter 50 microM oleamide-induced Ca(2+) release. Linoleamide (10-100 microM), 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.     

Direct effects of hydrogen peroxide on airway smooth muscle tone: roles of Ca2+ influx and Rho-kinase

Reactive oxidant species are implicated in the chronic airway inflammation related to asthma and chronic obstructive pulmonary disease. This study was designed to determine mechanisms underlying contraction induced by hydrogen peroxide (H(2)O(2)), a clinical marker of oxidative stress, in airway smooth muscle. Isometric tension and fluorescent intensities of fura-2, an index of intracellular Ca(2+) concentrations ([Ca(2+)](i)), were measured in epithelium-denuded tracheal smooth muscle tissues isolated from guinea pigs. H(2)O(2) (0.01-1 mM) caused contraction with an augmentation of [Ca(2+)](i) in a concentration-dependent manner in the normal physiological solution containing 2.4 mM of extracellular Ca(2+) concentrations. The contractile force and [Ca(2+)](i) by H(2)O(2) (1 mM) were approximately half of those in response to 1 microM methacholine. However, contraction by H(2)O(2) was not generated under the condition that extracellular Ca(2+) concentrations were less than 0.15 mM. Verapamil (10 microM), an inhibitor of voltage-operated Ca(2+) channels, partially but significantly inhibited the H(2)O(2)-induced contraction. In contrast, SKF-96365 (1-{beta-[3-(4-methoxyphenyl)propoxy]-4-methoxyphenethyl}-1H-imidazole hydrochloride) (100 microM), a non-selective inhibitor of Ca(2+) channels, completely abolished both the contraction and the increase in [Ca(2+)](i) elicited by H(2)O(2). Moreover, Y-27632 ((R)-(+)-trans-N-(4-Pyridyl)-4-(1-aminoethyl)-cyclohexanecarboxamide) (0.03-10 microM), an inhibitor of Rho-kinase, caused a concentration-dependent inhibition of the H(2)O(2)-induced contraction. In conclusion, both the Ca(2+) influx from the extracellular side and the Ca(2+) sensitization by Rho-kinase are involved in the regulation of airway smooth muscle tone induced by H(2)O(2). An inhibition of the Rho/Rho-kinase pathway may be beneficial for the treatment of airflow limitation mediated by oxidative stress.     

Effect of thymol on Ca2+ homeostasis and viability in human glioblastoma cells

The effect of the natural essential oil thymol on cytosolic Ca(2+) concentrations ([Ca(2+)](i)) and viability in human glioblastoma cells was examined. The Ca(2+)-sensitive fluorescent dye fura-2 was applied to measure [Ca(2+)](i). Thymol at concentrations of 400-1000 μM induced a [Ca(2+)](i) rise in a concentration-dependent fashion. The response was decreased partially by removal of extracellular Ca(2+). Thymol-induced Ca(2+) 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 Ca(2+) was removed, incubation with the endoplasmic reticulum Ca(2+) pump inhibitor thapsigargin or 2,5-di-tert-butylhydroquinone (BHQ) abolished thymol-induced [Ca(2+)](i) rise. Incubation with thymol also abolished thapsigargin or BHQ-induced [Ca(2+)](i) rise. Inhibition of phospholipase C with U73122 abolished thymol-induced [Ca(2+)](i) rise. At concentrations of 200-800 μM, thymol killed cells in a concentration-dependent manner. This cytotoxic effect was not changed by chelating cytosolic Ca(2+) 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 thymol (200, 400 and 600 μM) induced apoptosis in a concentration-dependent manner. Collectively, in human glioblastoma cells, thymol induced a [Ca(2+)](i) rise by inducing phospholipase C- and protein kinase C-dependent Ca(2+) release from the endoplasmic reticulum and Ca(2+) entry via non store-operated Ca(2+) channels. Thymol induced cell death that may involve apoptosis.     

Mechanism of action of angiotensin II in human isolated subcutaneous resistance arteries

1. Human isolated subcutaneous arteries were mounted in a myograph and isometric tension measured. In some experiments, intracellular calcium [Ca(2+)]i was also measured using fura-2. 2. Angiotensin II (100 pM - 1 microM) increased [Ca(2+)]i and tone in a concentration-dependent manner. The effects of angiotensin II (100 nM) were inhibited by an AT1-receptor antagonist, candesartan (100 pM). 3. Ryanodine (10 microM), had no effect on angiotensin II-induced responses, but removal of extracellular Ca(2+) abolished angiotensin II-induced rise in [Ca(2+)]i and tone. Inhibition of Ca(2+) entry by Ni(2+) (2 mM), also inhibited angiotensin II responses. The dihydropyridine, L-type calcium channel antagonist, amlodipine (10 microM), only partially attenuated angiotensin II responses. 4. Inhibition of protein kinase C (PKC) by chelerythrine (1 microM), or by overnight exposure to a phorbol ester (PDBu; 500 nM) had no effect on angiotensin II-induced contraction. 5. Genistein (10 microM), a tyrosine kinase inhibitor, inhibited angiotensin II-induced contraction, but did not inhibit the rise in [Ca(2+)]i, suggesting that at this concentration it affected the calcium sensitivity of the contractile apparatus. Genistein did not affect responses to norepinephrine (NE) or high potassium (KPSS). 6. A selective MEK inhibitor, PD98059 (30 microM), inhibited both the angiotensin II-induced contraction and rise in [Ca(2+)]i, but had no effect on responses to NE or KPSS. 7. AT1 activation causes Ca(2+) influx via L-type calcium channels and a dihydropyridine-insensitive route, but does not release Ca(2+) from intracellular sites. Activation of tyrosine kinase(s) and the ERK 1/2 pathway, but not classical or novel PKC, also play a role in angiotensin II-induced contraction in human subcutaneous resistance arteries.