Increase in IP3 and intracellular Ca2+ induced by phosphate depletion in LLC-PK 1 cells

The mechanisms by which Pi depletion rapidly regulates gene expression and cellular function have not been clarified. Here, we found a rapid increase in intracellular ionized calcium [Ca(2+)](i) by phosphate depletion in LLC-PK(1) cells using confocal microscopy with the green-fluorescence protein based calcium indicator "yellow cameleon 2.1." The increase of [Ca(2+)](i) was observed in the presence or absence of extracellular Ca(2+). At the same time, an approximately twofold increase in intracellular inositol 1,4,5-triphosphate (IP(3)) occurred in response to the acute Pi depletion in the medium. Furthermore, 2-aminoethoxydiphenyl borate completely blocked the [Ca(2+)](i) increase induced by Pi depletion. These results suggest that Pi depletion causes IP(3)-mediated release of Ca(2+) from intracellular Ca(2+) pools and rapidly increases [Ca(2+)](i) in LLC-PK(1) cells.     

Interleukin-8 primes oxidative burst in neutrophil-like HL-60 through changes in cytosolic calcium

In response to a variety of stimuli, neutrophils release large amount of reactive oxygen species (ROS) generated by NADPH oxidase. This process known as the respiratory burst is dependent on cytosolic free calcium concentration ([Ca(2+)](i)). Proinflammatory cytokines such as interleukin-8 (IL-8) may modulate ROS generation through a priming phenomenon. The aim of this study was to determine the effect of human IL-8 on ROS production in neutrophil-like dimethylsulfoxide-differentiated HL-60 cells (not equalHL-60 cells) and further to examine the role of Ca(2+) mobilization during the priming. IL-8 at 10 nM induced no ROS production but a [Ca(2+)](i) rise (254 +/- 36 nM). IL-8 induced a strongly enhanced (2 fold) ROS release during stimulation with 1 microM of N-formyl-L-methionyl-L-leucyl-L-phenylalanine (fMLF). This potentiation of ROS production is dependent of extracellular Ca(2+) (17.0+/-4.5 arbitrary units (A.U.) in the absence of Ca(2+) versus 56.6 +/- 3.9 A.U. in the presence of 1.25 mM of Ca(2+)). Also, IL-8 enhanced fMLF-stimulated increase in [Ca(2+)](i) (375 +/- 35 versus 245 +/- 21 nM, 0.1 microM of fMLF). IL-8 had no effect on not equalHL-60 cells in response to 1 microM of thapsigargin (472 +/- 66 versus 470 +/- 60 nM). In conclusion, Ca(2+) influx is necessary for a full induction of neutrophil priming by IL-8.     

The anti-anginal drug fendiline elevates cytosolic Ca(2+) in rabbit corneal epithelial cells

The effect of the anti-anginal drug fendiline on intracellular free Ca(2+) levels ([Ca(2+)](i)) in a rabbit corneal epithelial cell line (SIRC) was explored using fura-2 as a fluorescent Ca(2+) indicator. At a concentration above 1 microM, fendiline increased [Ca(2+)](i) in a concentration-dependent manner with an EC(50) value of 7 microM. The [Ca(2+)](i) response consisted of an immediate rise and an elevated phase. Extracellular Ca(2+) removal decreased half of the [Ca(2+)](i )signal. Fendiline induced quench of fura-2 fluorescence by Mn(2+) (50 microM), suggesting the presence of Ca(2+) influx across the plasma membrane. This Ca(2+) influx was abolished by La(3+) (50 microM), but was insensitive to dihydropyridines, verapamil and diltiazem. Fendiline (10 microM)-induced store Ca(2+) release was largely reduced by pretreatment with thapsigargin (1 microM) (an endoplasmic reticulum Ca(2+) pump inhibitor) to deplete the endoplasmic reticulum Ca(2+). Conversely, pretreatment with 10 microM fendiline abolished thapsigargin-induced Ca(2+) release. Fendiline (10 microM)-induced Ca(2+) release was not altered 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). Cumulatively, this study shows that fendiline induced concentration-dependent [Ca(2+)](i )increases in corneal epithelial cells by releasing the endoplasmic reticulum Ca(2+) in a phospholipase C-independent manner, and by causing Ca(2+) influx.     

15d-PGJ2 inhibits oxidized LDL-induced macrophage proliferation by inhibition of GM-CSF production via inactivation of NF-kappaB

Macrophage-derived foam cells play an important role in atherosclerotic lesions. Oxidized low-density lipoprotein (Ox-LDL) induces macrophage proliferation via production of GM-CSF in vitro. This study investigated the effects of 15-deoxy-Delta(12,14)-prostaglandin J(2) (15d-PGJ(2)), a natural ligand for peroxisome proliferator-activated receptor gamma, on macrophage proliferation. Mouse peritoneal macrophages and RAW264.7 cells were used for proliferation study and reporter gene assay, respectively. Twenty microgram per milliliter of Ox-LDL induced [3H]thymidine incorporation in mouse peritoneal macrophages, and 15d-PGJ(2) inhibited Ox-LDL-induced [3H]thymidine incorporation in a dose-dependent manner. Ox-LDL increased GM-CSF release and GM-CSF mRNA expression, and activated GM-CSF gene promoter, all of which were prevented by 15d-PGJ(2) or 2-cyclopenten-1-one, a cyclopentenone ring of 15d-PGJ(2). The suppression of GM-CSF promoter activity by 15d-PGJ(2) and 2-cyclopenten-1-one was mediated through reduction of NF-kappaB binding to GM-CSF promoter. These results suggest that 15d-PGJ(2) inhibits Ox-LDL-induced macrophage proliferation through suppression of GM-CSF production via NF-kappaB inactivation.     

Effect of Y-24180 on Ca2+ movement and proliferation in renal tubular cells

In canine renal tubular cells, the effect of Y-24180, a presumed specific platelet activating factor (PAF) receptor antagonist, on intracellular Ca(2+) concentration ([Ca(2+)](i)) was measured by using fura-2 as a Ca(2+)-sensitive fluorescent probe. Y-24180 (0.1-10 microM) caused a rapid and sustained [Ca(2+)](i) rise in a concentration-dependent manner. The [Ca(2+)](i) rise was prevented by 30% by removal of extracellular Ca(2+), but was not changed by dihydropyridines, verapamil and diltiazem. Y-24180-induced Ca(2+) influx was confirmed by Mn(2+)-influx induced quench of fura-2 fluorescence. 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 5 microM Y-24180 on [Ca(2+)](i) was abolished; conversely, depletion of Ca(2+) stores with 5 microM Y-24180 abolished thapsigargin-induced [Ca(2+)](i) rise. U73122, an inhibitor of phoispholipase C, inhibited ATP-, but not Y-24180-induced [Ca(2+)](i) rise. Overnight treatment with Y-24180 did not alter cell proliferation rate. Collectively, these results suggest that Y-24180 acts as a potent, but not cytotoxic, Ca(2+) mobilizer in renal tubular cells, by stimulating both extracellular Ca(2+) influx and intracellular Ca(2+) release. Since alterations in Ca(2+) movement may interfere many cellular signaling processes unrelated to modulation of PAF receptors, caution must be applied in using this chemical as a selective PAF receptor antagonist.     

Modulation of intracellular Ca(2+) via alpha(1B)-adrenoreceptor signaling molecules,G alpha(h) (transglutaminase II) and phospholipase C-delta 1

We characterized the alpha(1B)-adrenoreceptor (alpha(1B)-AR)-mediated intracellular Ca(2+) signaling involving G alpha(h) (transglutaminase II, TGII) and phospholipase C (PLC)-delta 1 using DDT1-MF2 cell. Expression of wild-type TGII and a TGII mutant lacking transglutaminase activity resulted in significant increases in a rapid peak and a sustained level of intracellular Ca(2+) concentration ([Ca(2+)](i)) in response to activation of the alpha(1B)-AR. Expression of a TGII mutant lacking the interaction with the receptor or PLC-delta 1 substantially reduced both the peak and sustained levels of [Ca(2+)](i). Expression of TGII mutants lacking the interaction with PLC-delta 1 resulted in a reduced capacitative Ca(2+) entry. Reduced expression of PLC-delta 1 displayed a transient elevation of [Ca(2+)](i) and a reduction in capacitative Ca(2+) entry. Expression of the C2-domain of PLC-delta 1, which contains the TGII interaction site, resulted in reduction of the alpha(1B)-AR-evoked peak increase in [Ca(2+)](i), while the sustained elevation in [Ca(2+)](i) and capacitative Ca(2+) entry remained unchanged. These findings demonstrate that stimulation of PLC-delta 1 via coupling of the alpha(1B)-AR with TGII evokes both Ca(2+) release and capacitative Ca(2+) entry and that capacitative Ca(2+) entry is mediated by the interaction of TGII with PLC-delta 1.     

Capsazepine elevates intracellular Ca2+ in human osteosarcoma cells, questioning its selectivity as a vanilloid receptor antagonist

Capsazepine is thought to be a selective antagonist of vanilloid type 1 receptors; however, its other in vitro effect on different cell types is unclear. In human MG63 osteosarcoma cells, the effect of capsazepine on intracellular Ca(2+) concentrations ([Ca(2+)](i)) and cytotoxicity was explored 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 100 microM. Capsazepine-induced [Ca(2+)](i) rise was partly reduced by removal of extracellular Ca(2+), suggesting that the capsazepine-induced [Ca(2+)](i) rise was composed of extracellular Ca(2+) influx and intracellular Ca(2+). 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 capsazepine on [Ca(2+)](i) was inhibited by 75%. Conversely, pretreatment with capsazepine to deplete intracellular Ca(2+) stores totally prevented thapsigargin from releasing more Ca(2+). 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. Overnight treatment with 1-100 microM capsazepine inhibited cell proliferation in a concentration-dependent manner. These findings suggest that in human MG63 osteosarcoma cells, capsazepine 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. Capsazepine may be mildly cytotoxic.     

Calcium elevation elicited by reverse mode Na+/Ca2+ exchange activity is facilitated by intracellular calcium stores in bovine chromaffin cells

The Na(+)/Ca(2+) exchanger (NCX) in plasma membranes either moves Ca(2+) out of (forward mode) or into (reverse mode) cells depending on the electrochemical gradient of these ions across the membrane. In this report, we characterize the sources responsible for the elevation in [Ca(2+)](i) elicited by reverse mode NCX activity. The elevation in [Ca(2+)](i) elicited by reverse mode NCX activity was significantly diminished by thapsigargin. KB-R7943 could only partially suppress the [Ca(2+)](i) change. Measurement of the [Ca(2+)](i) concurrent with reverse mode NCX current by perforated whole-cell patch showed that elevation in [Ca(2+)](i), but not the current, was inhibited by thapsigargin. The change in [Ca(2+)](i) response elicited by nicotinic acetylcholine receptor agonist was inhibited by thapsigargin. These suggest the importance of intracellular Ca(2+) stores in facilitating the [Ca(2+)](i) elevation elicited by reverse mode NCX activity under physiological condition.     

Effect of crocin on experimental atherosclerosis in quails and its mechanisms

In the present study, we examined the prophylaxis effect of crocin on experimental atherosclerosis and its possible mechanisms. The atherosclerosis formation was induced by hyperlipidamic diet in quails. At the 9th week, serum lipid, MDA and NO were measured, and HE staining was used to investigate the histopathological changes of aorta. Bovine aortic endothelial cells (EC) were obtained from the thoracic aorta of newborn calves. After incubation of the cells with Ox-LDL (50 mg x L(-1)) for 24 h, the activities of LDH, NO in culture media and activity of NOS in endothelial cells were measured, flow cytometer was used to determine the rate of endothelial cells apoptosis. Peritoneal macrophages were obtained from thioglycolate-injected mice. Cholesterol and free cholesterol in cells were assayed after incubation of the cells with Ox-LDL. Bovine aortic smooth muscle cells (SMC) were obtained from the thoracic aorta of newborn calf. Proliferation was induced by 100 microg x L(-1) Ox-LDL and antiproliferative effect of crocin on SMCs were observed. SMCs cycle phases were measured by flow cytometry. SMCs were loaded with Fluo-3/AM and [Ca2+]i was measured by Laser Scanning Confocal Microscope (LSCM). Crocin could reduce the level of serum TC, TG, LDL-C and inhibit the formation of aortic plaque. Crocin could reduce MDA and inhibit the descending of NO in serum. Compared with control, Ox-LDL group could increase the activity of LDH and decrease activity of NO in culture media and activity of NOS in endothelial cells, preincubated with crocin, the effects of Ox-LDL were inhibited. Crocin could decrease the EC apoptosis induced by Ox-LDL. Crocin concentration-dependently inhibited the TC and CE elevation induced by Ox-LDL in macrophages. Crocin could inhibit the proliferation of SMCs induced by Ox-LDL. In the presence or absence of extracellular Ca2+, crocin concentration-dependently inhibited the [Ca2+]i elevation induced by 120 mg x L(-1)Ox-LDL, In the absence of extracellular Ca2+, crocin could inhibit the [Ca2+]i elevation induced by CHCl3 in a concentration-dependent manner. The results indicated that crocin could inhibit the formation of atherosclerosis in quails. Crocin had protective effects on endothelial cells. Crocin could decrease CE in macrophages and uptake of Ox-LDL, inhibiting the formation of foam cell, which would promote the initiation and progression of atherosclerosis. Crocin could inhibit the [Ca2+]i elevation in smooth muscle cell, Ca2+ is an important second messenger that regulates a variety of cellular processes, including smooth muscle cell proliferation and gene expression . Crocin exerted antiatherosclerotic effects through decreasing the level of Ox-LDL that plays an important role in the initiation and progression of atherosclerosis.     

Ruthenium red,inhibitor of mitochondrial Ca2+ uniporter,inhibits curcumin-induced apoptosis via the prevention of intracellular Ca2+ depletion and cytochrome c release

Curcumin, a natural, biologically active compound extracted from rhizomes of Curcuma species, has been shown to possess potent anti-inflammatory, anti-tumor, and anti-oxidative properties. The mechanism by which curcumin initiates apoptosis remains poorly understood. In the present report we investigated the effect of curcumin on the activation of the apoptotic pathway in human leukemia U937 cells. Curcumin induces apoptosis in U937 cells via a mechanism that appears to involve down-regulation of the anti-apoptotic Bcl-xL, and IAP proteins, release of cytochrome c, and activation of caspase 3. Ruthenium red, an inhibitor of mitochondrial uniporter, specifically inhibits curcumin-induced apoptosis in U937 cells. Cotreatment with ruthenium red markedly prevented the activation of caspase 3, cytochrome c release, and cell death, suggesting a role for intracellular Ca(2+) in this process. Curcumin induced a marked depletion of [Ca(2+)](i) in Caki cells bathed with both Ca(2+)-containing and -free solutions. Thapsigargin (TG), cyclopiazonic acid (CPA), and dantolene (DAN) had no effect. Ruthenium red, an inhibitor of mitochondrial uniporter, only attenuated the curcumin-induced [Ca(2+)](i) depletion in a dose-dependent manner. These data indicate that curcumin acts as a stimulator of intracellular Ca(2+) uptake into mitochondria via uniporter pathway and may involve in the execution of apoptosis.     

Ca(2+)-independent F-actin assembly and disassembly during Fc receptor- mediated phagocytosis in mouse macrophages

Phagocytosis of IgG-coated particles by macrophages is presumed to involve the actin-based cytoskeleton since F-actin accumulates beneath forming phagosomes, and particle engulfment is blocked by cytochalasins, drugs that inhibit actin filament assembly. However, it is unknown whether Fc receptor ligation affects the rate or extent of F-actin assembly during phagocytosis of IgG-coated particles. To examine this question we have used a quantitative spectrofluorometric method to examine F-actin dynamics during a synchronous wave of phagocytosis of IgG-coated red blood cells by inflammatory mouse macrophages. We observed a biphasic rise in macrophage F-actin content during particle engulfment, with maxima at 1 and 5 min after the initiation of phagocytosis. F-actin declined to resting levels by 30 min, by which time particle engulfment was completed. These quantitative increases in macrophage F-actin were reflected in localized changes in F-actin distribution. Previous work showed that the number of IgG-coated particles engulfed by macrophages is unaffected by buffering extracellular calcium or by clamping cytosolic free calcium concentration ([Ca2+]i) to very low levels (Di Virgilio, F., B. C. Meyer, S. Greenberg, and S. C. Silverstein. 1988. J. Cell Biol. 106: 657-666). To determine whether clamping [Ca2+]i in macrophages affects the rate of particle engulfment, or the assembly or disassembly of F-actin during phagocytosis, we examined these parameters in macrophages whose [Ca2+]i had been clamped to approximately less than 3 nM with fura 2/AM and acetoxymethyl ester of EGTA. We found that the initial rate of phagocytosis, and the quantities of F-actin assembled and disassembled were similar in Ca(2+)-replete and Ca(2+)-depleted macrophages. We conclude that Fc receptor-mediated phagocytosis in mouse macrophages is accompanied by an ordered sequence of assembly and disassembly of F-actin that is insensitive to [Ca2+]i.     

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.     

Expression of a functional extracellular calcium-sensing receptor in human aortic endothelial cells

Extracellular Ca(2+) concentration ([Ca(2+)](o)) regulates the functions of many cell types through a G protein-coupled [Ca(2+)](o)-sensing receptor (CaR). Whether the receptor is functionally expressed in vascular endothelial cells is largely unknown. In cultured human aortic endothelial cells (HAEC), RT-PCR yielded the expected 555-bp product corresponding to the CaR, and CaR protein was demonstrated by fluorescence immunostaining and Western blot. RT-PCR also demonstrated the expression in HAEC of alternatively spliced variants of the CaR lacking exon 5. Although stimulation of fura 2-loaded HAEC by several CaR agonists (high [Ca(2+)](o), neomycin, and gadolinium) failed to increase intracellular Ca(2+) concentration ([Ca(2+)](i)), the CaR agonist spermine stimulated an increase in [Ca(2+)](i) that was diminished in buffer without Ca(2+) and was abolished after depletion of an intracellular Ca(2+) pool with thapsigargin or after blocking IP(3)- and ryanodine receptor-mediated Ca(2+) release with xestospongin C and with high concentration ryanodine, respectively. Spermine stimulated an increase in DAF-FM fluorescence in HAEC, consistent with NO production. Both the increase in [Ca(2+)](i) and in NO production were reduced or absent in HAEC transfected with siRNA specifically targeted to the CaR. HAEC express a functional CaR that responds to the endogenous polyamine spermine with an increase in [Ca(2+)](i), primarily due to release of IP(3)- and ryanodine-sensitive intracellular Ca(2+) stores, leading to the production of NO. Expression of alternatively spliced variants of the CaR may result in the absence of a functional response to other known CaR agonists in HAEC.     

Losartan inhibits cellular uptake of oxidized LDL by monocyte-macrophages from hypercholesterolemic patients

Angiotensin II (Ang II) and oxidized LDL (Ox-LDL) are risk factors for atherosclerosis, and both of them contribute to macrophage cholesterol accumulation, the hallmark of early atherosclerosis. As Ang II was shown to increase macrophage uptake of Ox-LDL, we investigated the effect of losartan, an Ang II receptor antagonist with antiatherogenic properties, on the cellular uptake of Ox-LDL by human monocyte-derived macrophages (HMDM) from hypercholesterolemic patients. Eight normotensive hypercholesterolemic patients were treated with losartan (50 mg/day) for a period of 4 weeks. Losartan therapy did not significantly affect the degradation of native LDL by the patients' HMDM. However, losartan therapy significantly reduced HMDM uptake of Ox-LDL as shown by a 78% reduction in Ox-LDL cell-association and a 21% reduction in Ox-LDL degradation. CD36 (an Ox-LDL receptor) mRNA expression in HMDM obtained after losartan treatment was decreased by 54% compared to HMDM obtained before treatment. The ability of losartan to inhibit HMDM CD36 mRNA expression and, hence, Ox-LDL uptake and macrophage foam cell formation is probably related to the blockage of Ang II binding to the cell surface and thus to the prevention of Ang II atherogenic effects.     

Ca(2+) homeostasis, Ca(2+) signalling and somatodendritic vasopressin release in adult rat supraoptic nucleus neurones

Multiple mechanisms that maintain Ca(2+) homeostasis and provide for Ca(2+) signalling operate in the somatas and neurohypophysial nerve terminals of supraoptic nucleus (SON) neurones. Here, we examined the Ca(2+) clearance mechanisms of SON neurones from adult rats by monitoring the effects of the selective inhibition of different Ca(2+) homeostatic molecules on cytosolic Ca(2+) ([Ca(2+)](i)) transients in isolated SON neurones. In addition, we measured somatodendritic vasopressin (AVP) release from intact SON tissue in an attempt to correlate it with [Ca(2+)](i) dynamics. When bathing the cells in a Na(+)-free extracellular solution, thapsigargin, cyclopiazonic acid (CPA), carbonyl cyanide 3-chlorophenylhydrazone (CCCP), and the inhibitor of plasma membrane Ca(2+)-ATPase (PMCA), La(3+), all significantly slowed down the recovery of depolarisation (50 mM KCl)-induced [Ca(2+)](i) transients. The release of AVP was stimulated by 50 mM KCl, and the decline in the peptide release was slowed by Ca(2+) transport inhibitors. In contrast to previous reports, our results show that in the fully mature adult rats: (i) all four Ca(2+) homeostatic pathways, the Na(+)/Ca(2+) exchanger, the endoplasmic reticulum Ca(2+) pump, the plasmalemmal Ca(2+) pump and mitochondria, are complementary in actively clearing Ca(2+) from SON neurones; (ii) somatodendritic AVP release closely correlates with intracellular [Ca(2+)](i) dynamics; (iii) there is (are) Ca(2+) clearance mechanism(s) distinct from the four outlined above; and (iv) Ca(2+) homeostatic systems in the somatas of SON neurones differ from those expressed in their terminals.     

Phospholipase A2-independent Ca2+ entry and subsequent apoptosis induced by melittin in human MG63 osteosarcoma cells

Melittin, a peptide from bee venom, is thought to be a phospholipase A(2) activator and Ca(2+) influx inducer that can evoke cell death in different cell types. However, the effect of melittin on cytosolic free Ca(2+) concentration ([Ca(2+)](i)) and viability has not been explored in human osteoblast-like cells. This study examined whether melittin altered [Ca(2+)](i) and killed cells in MG63 human osteosarcoma cells. [Ca(2+)](i) changes and cell viability were measured by using the fluorescent dyes fura-2 and WST-1, respectively. Melittin at concentrations above 0.075 microM increased [Ca(2+)](i) in a concentration-dependent manner. The Ca(2+) signal was abolished by removing extracellular Ca(2+). Melittin-induced Ca(2+) entry was confirmed by Mn(2+) quenching of fura-2 fluorescence at 360 nm excitation wavelength which was Ca(2+)-insensitive. The melittin-induced Ca(2+) influx was unchanged by modulation of protein kinase-C activity with phorbol 12-myristate 13-acetate (PMA) and GF 109203X, or inhibition of phospholipase A(2) with AACOCF(3) and aristolochic acid; but was substantially inhibited by blocking L-type Ca(2+) channels. At concentrations of 0.5 microM and 1 microM, melittin killed 33% and 45% of cells, respectively, via inducing apoptosis. Lower concentrations of melittin failed to kill cells. The cytotoxic effect of 1 microM melittin was completely reversed by pre-chelating cytosolic Ca(2+) with BAPTA. Taken together, these data showed that in MG63 cells, melittin induced a [Ca(2+)](i) increase by causing Ca(2+) entry through L-type Ca(2+) channels in a manner independent of protein kinase-C and phospholipase A(2) activity; and this [Ca(2+)](i) increase subsequently caused apoptosis.     

Rotavirus infection induces an increase in intracellular calcium concentration in human intestinal epithelial cells: role in microvillar actin alteration

Rotaviruses, which infect mature enterocytes of the small intestine, are recognized as the most important cause of viral gastroenteritis in young children. We have previously reported that rotavirus infection induces microvillar F-actin disassembly in human intestinal epithelial Caco-2 cells (N. Jourdan, J. P. Brunet, C. Sapin, A. Blais, J. Cotte-Laffitte, F. Forestier, A. M. Quero, G. Trugnan, and A. L. Servin, J. Virol. 72:7228-7236, 1998). In this study, to determine the mechanism responsible for rotavirus-induced F-actin alteration, we investigated the effect of infection on intracellular calcium concentration ([Ca(2+)](i)) in Caco-2 cells, since Ca(2+) is known to be a determinant factor for actin cytoskeleton regulation. As measured by quin2 fluorescence, viral replication induced a progressive increase in [Ca(2+)](i) from 7 h postinfection, which was shown to be necessary and sufficient for microvillar F-actin disassembly. During the first hours of infection, the increase in [Ca(2+)](i) was related only to an increase in Ca(2+) permeability of plasmalemma. At a late stage of infection, [Ca(2+)](i) elevation was due to both extracellular Ca(2+) influx and Ca(2+) release from the intracellular organelles, mainly the endoplasmic reticulum (ER). We noted that at this time the [Ca(2+)](i) increase was partially related to a phospholipase C (PLC)-dependent mechanism, which probably explains the Ca(2+) release from the ER. We also demonstrated for the first time that viral proteins or peptides, released into culture supernatants of rotavirus-infected Caco-2 cells, induced a transient increase in [Ca(2+)](i) of uninfected Caco-2 cells, by a PLC-dependent efflux of Ca(2+) from the ER and by extracellular Ca(2+) influx. These supernatants induced a Ca(2+)-dependent microvillar F-actin alteration in uninfected Caco-2 cells, thus participating in rotavirus pathogenesis.     

The Na+/Ca2+ exchanger is active and working in the reverse mode in human umbilical artery smooth muscle cells

The data presented in this work suggest that in human umbilical artery (HUA) smooth muscle cells, the Na(+)/Ca(2+) exchanger (NCX) is active and working in the reverse mode. This supposition is based on the following results: (i) microfluorimetry in HUA smooth muscle cells in situ showed that a Ca(2+)-free extracellular solution diminished intracellular Ca(2+) ([Ca(2+)](i)), and KB-R7943 (5microM), a specific inhibitor of the Ca(2+) entry mode of the exchanger, also decreased [Ca(2+)](i) (40.6+/-4.5% of Ca(2+)-free effect); (ii) KB-R7943 produced the relaxation of HUA rings (-24.7+/-7.3gF/gW, n=8, p<0.05); (iii) stimulation of the NCX by lowering extracellular Na(+) increases basal [Ca(2+)](i) proportionally to Na(+) reduction (Delta fluorescence ratio=0.593+/-0.141 for Na(+)-free solution, n=8) and HUA rings' contraction (peak force=181.5+/-39.7 for 130mM reduction, n=8), both inhibited by KB-R7943 and a Ca(2+)-free extracellular solution. In conclusion, the NCX represents an important Ca(2+) entry route in HUA smooth muscle cells.     

Biophysical re-equilibration of Ca2+ fluxes as a simple biologically plausible explanation for complex intracellular Ca2+ release patterns

Physiological regulation of Ca(2+) release from the endoplasmic reticulum (ER) is critical for cell function. Recent direct measurements of free [Ca(2+)] inside the ER ([Ca(2+)](ER)) revealed that [Ca(2+)](ER) itself is a key regulator of ER Ca(2+) handling. However, the role of this new regulatory process in generating various patterns of Ca(2+) release remains to be elucidated in detail. Here, we incorporate the recently quantified experimental correlations between [Ca(2+)](ER) and Ca(2+) movements across the ER membrane into a mathematical model ER Ca(2+) handling. The model reproduces basic experimental dynamics of [Ca(2+)](ER). Although this was not goal in model design, the model also exhibits mechanistically unclear experimental phenomena such as "quantal" Ca(2+) release, and "store charging" by increasing resting cytosolic [Ca(2+)]. While more complex explanations cannot be ruled out, on the basis of our data we propose that "quantal release" and "store charging" could be simple re-equilibration phenomena, predicted by the recently quantified biophysical dynamics of Ca(2+) movements across the ER membrane.     

E-LDL upregulates TOSO expression and enhances the survival of human macrophages

Uptake of modified lipoproteins by macrophages causes foam cell formation and promotes atherosclerosis. Atherogenic lipoproteins are cytotoxic and induce cell death under certain conditions but may also enhance macrophage survival. Macrophages treated with enzymatically modified LDL (E-LDL) were subjected to GeneChip analysis and the antiapoptotic gene TOSO was found induced. TOSO mRNA is upregulated and apoptosis is reduced in E-LDL but not in oxidized LDL (Ox-LDL) loaded macrophages. FLIP(L) abundance was suggested to mediate the antiapoptotic properties of TOSO; however, FLIP(L) was not changed. Ox-LDL is internalized predominantly by scavenger receptors such as CD36 while E-LDL particles are preferentially internalized by Fc- and complement-receptor dependent phagocytosis and internalization of phagobeads by macrophages upregulates TOSO. In COS-7 cells however, phagocytotic activity was not affected by TOSO. These data indicate that E-LDL-generated foam cells are protected from cell death most likely through the expression of TOSO by a FLIP(L) independent mechanism.