Regulation of intracellular Mg2+ by superoxide in amnion cells.

Changes of intracellular free Mg2+ concentration ([Mg2+]i) in human amnion cells induced by superoxide anion were determined using a highly Mg(2+)-sensitive fluorescent dye Mg(2+)-fura2 or Mg(2+)-indol. Superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, induced decrease of [Mg2+]i. The decrease was significantly inhibited by an anion channel blocker, 4,4'diisothiocyano-2,2' disulfonic acid stilbene (DIDS). Superoxide dismutase (SOD), injected into cells by cell fusion, also inhibited the change of [Mg2+]i, but catalase did not. Superoxide anion induced prompt increase of intracellular pH (pHi) as well as decrease of [Mg2+]i and subsequently activated the increase of intracellular free Ca2+ ([Ca2+]i) and the release of arachidonate. In contrast to superoxide anion, NH4Cl which induces increase of pHi in amnion cells increased [Mg2+]i. The elevation of basal level of [Mg2+]i by Mg(2+)-ionophore inhibited the change of [Ca2+]i and the release of arachidonate induced by superoxide anion. These results suggest that superoxide anion, transported through anion channels into cells, decreases [Mg2+]i directly, not due to a pH-effect and that the decrease of [Mg2+]i may regulate biological functions of the cells via increase of [Ca2+]i.     

Superoxide anion increases intracellular pH, intracellular free calcium, and arachidonate release in human amnion cells.

We determined the effects of superoxide anion, produced by addition of xanthine oxidase to hypoxanthine, on the intracellular pH (pHi) and intracellular free calcium concentration ([Ca2+]i) and release of arachidonate in human cultured amnion cells. Superoxide anion induced a prompt increase of pHi and subsequent increase of [Ca2+]i. The evoked pHi was inhibited by pretreatment with anion channel blockers but not affected by omission of extracellular Na+ or addition of amiloride. The increase of [Ca2+]i was inhibited significantly by the absence of extracellular calcium or by the addition of a calcium channel blocker, cobalt. NH4Cl, which can generally increase pHi, also increased [Ca2+]i of amnion cells. But the increase of [Ca2+]i induced by the NH4Cl was significantly less than that induced by the amount of superoxide anion causing a similar increase in pHi. These results show that superoxide anion, crossed through anion channel in membrane, increased [Ca2+]i at least partially via increase of pHi and that the calcium mobilization was dependent on both extracellular and intracellular sources. Superoxide anion induced the release of arachidonate in a dose-dependent manner and this induction was inhibited by omission of extracellular calcium. These data suggest that the release of arachidonate was dependent on the increase of [Ca2+]i. We also determined the viability of cells in the presence of superoxide anion by flow cytometry. Superoxide anion at the levels used in these experiments did not change the percentage of viable cells. These findings suggested that superoxide anion may regulate biological functions in amnion cells via pHi, [Ca2+]i mobilization, and the release of arachidonate without damaging the cells.     

Ca2+ is mobilized by hydroxyl radical but not by superoxide in RTH-149 cells: the oxidative switching-on of Ca2+ signaling

Differential effects of superoxide and hydroxyl radical on intracellular calcium were investigated in trout hepatoma cells (RTH-149). [Ca2+]i variations were recorded using confocal imaging, fluo-3 loading, and exposure to various mixtures consisting of hypoxanthine/xanthine oxidase (HX/XO), and of sub-stimulatory concentrations of H2O2 and Cu2+ . No [Ca2+]i variation was found with HX/XO, a slight [Ca2+]i rise with a mixture of Cu2+ and HX/XO, a sustained rise with Cu2+ and H2O2, and the highest rise with Cu2+, H2O2 and HX/XO. Fluorimetric assay using dihydrorhodamine 123 revealed a correlation between the oxidizing power of a mixture and its effect on [Ca2+]i. The [Ca2+]i rise induced by Cu2+, H2O2 and HX/XO, was partially reduced in Ca2+ free medium or in the presence of SOD, converted into Ca2+ transient by verapamil, and almost abolished by the PLC inhibitor U73122 or in the presence of the hydroxyl radical quencher TEMPOL. Data indicate that Ca2+ is mobilized by hydroxyl radical but not by superoxide. The mechanism consists of PLC activation causing intracellular Ca2+ release, while Ca2+ entry potentiates Ca2+ release thus leading to sustained [Ca2+]i rise. A role of hydroxyl radicals in the oxidative switching-on of Ca2+ signaling is discussed.     

Rac-1-mediated O2- secretion requires Ca2+ influx in neutrophil-like HL-60 cells

Neutrophil-like HL-60 cells reacted to N -formyl- l -Methionyl- l -Leucyl- l -P henylalanine (f MLP) with a rise in the intracellular calcium concentration ([Ca2]i), NADPH oxidase activation, and increased superoxide anion (O2-) production. [Ca2+]i mobilization and superoxide production were largely dependent on extracellular calcium (Ca2+]e) and a capacitative calcium entry. The monomeric G-protein, Rac-1, regulates NADPH oxidase activity. We tested the effect of removal of Ca2+]e on Rac-1 plasma membrane sequestration and activation of NADPH oxidase using immunodetection and a double labelling fluorescent method. Results showed that Rac-1 activation is mediated via a pertussis toxin (PTX)-sensitive heteromeric G-protein pathway, and that Rac-1 membrane sequestration was preceded by [Ca2+]i mobilization following entry of Ca2+ e. Therefore, we propose that O2- production is dependent on activation of PTX-sensitive G-proteins and sequestration of Rac-1 in the plasma membrane, following entry of Ca2+ e.     

Wine polyphenols stimulate superoxide anion production to promote calcium signaling and endothelial-dependent vasodilatation

The present study was aimed to evaluate the mechanisms involved in the vasorelaxant effects of red wine polyphenol compounds (RWPC) in small mesenteric rat arteries. RWPC produce relaxation in small mesenteric arteries. This relaxant effect was abolished by endothelial denudation, NO-synthase blockade with L-NAME and partial depolarization with KCl or L-NAME plus KCl. Incubation with the reactive oxygen species scavenger, superoxide dismutase (SOD) plus catalase, or inhibition of NAD(P)H-dependent oxidoreductases with diphenyleneiodonium also inhibited RWPC induced vascular relaxation. Application of RWPC elicited a transient increase in intracellular calcium concentration ([Ca2+]i) in bovine aortic endothelial cells (BAEC), which was attenuated by a mixture of SOD and catalase. Incubation of BAEC with RWPC increased the SOD inhibitable production of O2-. These results suggest the involvement of O2- in the [Ca2+]i increase evoked by RWPC, leading to the activation of enzymes involved in the release of endothelial relaxant factors and subsequent vasodilatation of resistance arteries.     

Evidence for a platelet-activating factor receptor on human alveolar macrophages.

In this report we demonstrate evidence which strongly suggests that human alveolar macrophages possess receptor for the platelet activating factor (PAF). We investigated the effects of PAF by measuring (a) the intracellular free calcium concentration [Ca2+]i, using the fura-2 method in single isolated cells and (b) the production of superoxide anion. PAF increased [Ca2+]i in a dose-dependent manner (EC50 = 1 x 10(-8) M), whereas lyso-PAF had no effect. The initial increase of [Ca2+]i was followed by a slow decrease to a sustained elevation of [Ca2+]i significantly above basal values. While the initial rise in [Ca2+]i was only slightly reduced in Ca(2+)-free medium (1 mM EGTA), the sustained phase was totally abolished. The sustained calcium increase was also blocked after preincubation of AM with the calcium-channel blocker nitrendipine. PAF increased the production of superoxide anion (O2-) by human alveolar macrophages in a dose- dependent manner. The effects of PAF on [Ca2+]i and (O2-) could be blocked by the PAF-specific antagonist WEB 2086 dose dependently, indicating a receptor-mediated event.     

Superoxide anion impairs contractility in cultured aortic smooth muscle cells

We examined the effects of superoxide anion (O) generated by xanthine plus xanthine oxidase (X/XO) on the intracellular Ca(2+) concentration ([Ca(2+)](i)) and muscle contractility in cultured bovine aortic smooth muscle cells (BASMC). Cells were grown on collagen-coated dish for the measurement of [Ca(2+)](i). Pretreatment with X/XO inhibited ATP-induced Ca(2+) transient and Ca(2+) release-activated Ca(2+) entry (CRAC) after thapsigargin-induced store depletion, both of which were reversed by superoxide dismutase (SOD). In contrast, Ca(2+) transients induced by high-K(+) solution and Ca(2+) ionophore A-23187 were not affected by X/XO. BASMC-embedded collagen gel lattice, which was pretreated with xanthine alone, showed contraction in response to ATP, thapsigargin, high-K(+) solution, and A-23187. Pretreatment of the gel with X/XO impaired gel contraction not only by ATP and thapsigargin, but also by high-K(+) solution and A-23187. The X/XO-treated gel showed normal contraction; however, when SOD was present during the pretreatment period. These results indicate that O(2)(-) attenuates smooth muscle contraction by impairing CRAC, ATP-induced Ca(2+) transient, and Ca(2+) sensitivity in BASMC.     

Transient Ca2+ changes in endothelial cells induced by low doses of reactive oxygen species: Role of hydrogen peroxide

Cultured human and rat endothelial cells were used to study cellular toxicity and Ca2+ signalling upon exposure to reactive oxygen species. Superoxide and hydrogen peroxide (O2·–/H2O2) were produced by the hypoxanthine/xanthine oxidase system (HX/XO) and caused intracellular Ca2+ concentration ([Ca2+]i) to rise steadily when activities above 2 mU/ml were used. These Ca2+ increases were also measured when the glucose/glucose oxidase (G/GO) system above 5 mU/ml was used to produce hydrogen peroxide (H2O2). Gross morphological changes appeared to parallel elevated [Ca2+]i levels preceding cell death. However, when HX/XO or G/GO were used at non toxic doses rapid and transient changes in [Ca2+]i were measured. These treatments did not alter subsequent receptor mediated Ca2+ signalling induced by ATP (10 M) or histamine (100 M). Superoxide dismutase (50 U/ml), which dismutates O2·minus; into H2O2 al ient [Ca2+]i responses. H2O2 added directly was able to induce similar Ca2+ transients when concentrations of at least 500 M were used. Buffering trace amounts of iron (o-phenanthroline; 200 M) in order to inhibit úOH radical formation was not effective to alter Ca2+ changes. Experiments performed in Ca2+-free buffer showed a similar rise in [Ca2+]i and readdition of Ca2+ to the extracellular medium indicated the activation of store operated Ca2+ entry. Blocking Ca2+-ATPases of the endoplasmatic reticulum with thapsigargin (1 M) inhibited ROS induced transient increases and cells preincubated with pertussis toxin (200 nM) showed unchanged Ca2+ transients after exposure to both enzyme systems. Phospholipase C inhibitor U73122 (2 M) effectively reduced hydrogen peroxide induced emptying of intracellular stores. Taken together, we demonstrate that enzymatically produced non-toxic H2O2 rather than O· ndash; or · OH causes calcium signalling from thapsigargin sensitive stores, and activates store operated Ca2+ entry at least partially by activating phospholipase C. These changes clearly differ from pathological oxidative stress associated with a progressive increase in [Ca2+]i.     

Selenium prevents diabetes-induced alterations in [Zn2+]i and metallothionein level of rat heart via restoration of cell redox cycle

Intracellular free zinc concentration ([Zn2+]i) is very important for cell functions, and its excessive accumulation is cytotoxic. [Zn2+]i can increase rapidly in cardiomyocytes because of mobilization of Zn2+ from intracellular stores by reactive oxygen species (ROS). Moreover, ROS have been proposed to contribute to direct and/or indirect damage to cardiomyocytes in diabetes. To address these hypotheses, we investigated how elevated [Zn2+]i in cardiomyocytes could contribute to diabetes-induced alterations in intracellular free calcium concentration ([Ca2+]i). We also investigated its relationship to the changes of metallothionein (MT) level of the heart. Cardiomyocytes from normal rats loaded with fura-2 were used to fluorometrically measure resting [Zn2+]i (0.52 +/- 0.06 nM) and [Ca2+]i (26.53 +/- 3.67 nM). Fluorescence quenching by the heavy metal chelator N,N,N',N'-tetrakis(2-pyridylmethyl)ethylenediamine was used to quantify [Zn2+]i. Our data showed that diabetic cardiomyocytes exhibited significantly increased [Zn2+]i (0.87 +/- 0.05 nM ) and [Ca2+]i (49.66 +/- 9.03 nM), decreased levels of MT and reduced glutathione, increased levels of lipid peroxidation and nitric oxide products, and decreased activities of superoxide dismutase, glutathione reductase, and glutathione peroxidase. Treatment (4 wk) of diabetic rats with sodium selenite (5 micromol.kg body wt(-1).day(-1)) prevented these defects induced by diabetes. A comparison of present data with previously observed beneficial effects of selenium treatment on diabetes-induced contractile dysfunction of the heart can suggest that an increase in [Zn2+]i may contribute to oxidant-induced alterations of excitation-contraction coupling in diabetes. In addition, we showed that oxidative stress is involved in the etiology of diabetes-induced downregulation of heart function via depressed endogenous antioxidant defense mechanisms.     

Differences in intracellular calcium mobilization by interferon-beta and interferon-gamma in RPMI-4788 cells

Human interferon (IFN) stimulates a 1.5- to 1.7-fold transient increase in the concentration of cytoplasmic-free calcium ion ([Ca2+]i) within 10-20 s upon exposure of RPMI-4788 cells to IFN. This early event of IFN-induced [Ca2+]i mobilization was measurable by loading the cells with Fura-2AM, a fluorescent Ca2+ indicator. The mobilization induced by IFN-beta or IFN-gamma was dependent on the concentration of each IFN. The increased [Ca2+]i gradually returned to its resting level within 60 s. The addition of EGTA (0.5-10 mM) to medium induced a marked decrease in the amount of [Ca2+]i mobilized by IFN-beta and a partial decrease by IFN-gamma. This finding suggests that the mechanisms of [Ca2+]i mobilization by IFN-beta and IFN-gamma might be different. While IFN-beta-induced mobilization may be mainly from an influx of the extracellular calcium ion ([Ca2+]o), IFN-gamma-induced mobilization may be a summation of an influx of [Ca2+]o and a release from intracellular Ca2+ stores.     

内皮素对培养心肌细胞内游离钙浓度的作用

实验用培养新生ＳＤ大鼠心室肌细胞,以Ｆｕｒａ－２／ＡＭ荧光指示剂负载检测收肌细胞内游离钙浓度（「Ｃａ＾２＋」）的变化,探讨内皮素－１（ＥＴ－１）对「Ｃａ＾２＋」ｉ的作用及其机制。结果显示：ＥＴ－１引起心肌细胞「Ｃａ＾２＋」ｉ升高有两个时相,瞬时相持续相。ＥＴ－１诱导的瞬时相「Ｃａ＾２＋」ｉ升高呈浓度依赖性,预先用ＥＴＡ特异性受阻断剂ＢＱ１２３处理,可阻断ＥＴ－１引起的「Ｃａ＾２＋」ｉ升高,揭示上述     

Extracellular ATP itself elicits superoxide generation in guinea pig peritoneal macrophages.

Extracellular ATP itself elicited the generation of superoxide (O2-) in guinea pig peritoneal macrophages associated with an increase in cytosolic calcium ([Ca2+]i). The ATP-induced O2- generation was completely inhibited by pretreatment with pertussis toxin (PT) accompanied by the suppression of [Ca2+]i mobilization. Pre-exposure to a small amount of phorbol myristate acetate (PMA) primed the ATP-induced generation of O2- without a change of [Ca2+]i. The results suggest that ATP-induced O2- generation is mediated by [Ca2+]i mobilization and by PT-sensitive G protein.     

Na(+)-ionophore, monensin-induced rise in cytoplasmic free calcium depends on the presence of extracellular calcium in FRTL-5 rat thyroid cells

Calcium is an important regulator of cell function, and may be influenced by the intracellular sodium content. In the present study, the Na(+)-ionophore, monensin, was used to investigate the interrelationship between changes in intracellular Na+ concentration ([Na+]i) and elevation of cytosolic Ca2+ concentration ([Ca2+]i) in FRTL-5 thyroid cells. Cytoplasmic Ca2+ levels were measured using the fluorescent dye, indo-1. Monensin induced a dose-dependent increase in [Ca2+]i in FRTL-5 cells. Inhibitors of intracellular Ca2+ release, TMB-8 and ryanodine, were unable to prevent the monensin effect on [Ca2+]i. The alpha 1-receptor antagonist, prazosin, did not block the monensin-stimulated increase in [Ca2+]i. In the absence of extracellular calcium there was a marked diminution in the monensin effect on [Ca2+]i, yet calcium channel antagonists (nifedipine, diltiazem and verapamil) did not inhibit the response. Replacement of Na+ by choline chloride in the medium depressed the monensin-evoked rise in [Ca2+]i by up to 84%. Furthermore, addition of the Na(+)-channel agonist, veratridine, elicited an increase in [Ca2+]i, even though less dramatic than that caused by monensin. Ouabain increased the resting cytosolic Ca2+ concentration as well as the magnitude of the monensin effect on [Ca2+]i. The absence of any effect on the Na(+)-ionophore evoked increase in [Ca2+]i upon addition of tetrodotoxin (TTX) excluded a possible involvement of TTX-sensitive Na+ channels. These data show that the rise in [Ca2+]i induced by increasing [Na+]i is largely dependent on both external Na+ and Ca2+. Calcium entry appears not to involve voltage-dependent or alpha 1-receptor sensitive Ca2+ channels, but may result from activation of an Na(+)-Ca2+ exchange system.     

Voltage-regulated calcium channels involved in the regulation of enkephalin synthesis are blocked by phorbol ester treatment

Treatment of bovine chromaffin cells with 40 mM KCl stimulates a 3-fold increase in total methionine enkephalin immunoreactivity (medium plus cells) and a 4-fold increase in proenkephalin mRNA (mRNAenk). These effects of KCl, which are dependent on extracellular calcium, can be blocked by treatment with 12-O-tetradecanoylphorbol-13-acetate (TPA), although release of methionine enkephalin appears less affected. Using fura-2-loaded chromaffin cells and a dual-excitation wavelength spectrofluorometer, we have examined whether the actions of KCl and TPA on methionine enkephalin synthesis and release can be explained by changes in intracellular free calcium ([Ca2+]i). KCl produced a rapid 600 nM increase in [Ca2+]i from resting levels of approximately 170 nM. Subsequently, [Ca2+]i declined to a new steady-state plateau which was approximately 275 nM higher than the original resting levels. The postdepolarization plateau of [Ca2+]i was reduced by TPA, (-)-(R)-202,791 (a dihydropyridine calcium channel antagonist), and LaCl3 (a nonselective calcium channel blocker). TPA also inhibited potentiation of the KCl-stimulated plateau of [Ca2+]i due to (+)-(S)-202,791, a calcium channel agonist. In contrast, TPA had no effect on resting [Ca2+]i and only slightly inhibited the initial rapid KCl-stimulated increase in [Ca2+]i. The inhibitory effects were maintained for 24 h in the continuous presence of TPA. We conclude 1) that TPA inhibits enkephalin synthesis by inactivating dihydropyridine-sensitive voltage-dependent calcium channels, 2) that these channels alone maintain elevated [Ca2+]i following KCl depolarization, and 3) that sustained elevation in [Ca2+]i is necessary in order to increase enkephalin synthesis in KCl-treated chromaffin cells.     

Immunoglobulin E receptor cross-linking induces oscillations in intracellular free ionized calcium in individual tumor mast cells

Fura-2 fluorescence in single rat basophilic leukemia cells was monitored to study the rise in intracellular free ionized calcium ([Ca2+]i) produced by aggregation of immunoglobulin E receptors. Repetitive transient increases in [Ca2+]i were induced by antigen stimulation and were measured using digital video imaging microscopy at high time resolution. The [Ca2+]i oscillations were not dependent upon changes in the membrane potential of the cells and were observed in cells stimulated with antigen either with or without extracellular Ca2+. Transient oscillations in [Ca2+]i were also observed when calcium influx was blocked with La3+. These results suggested that during antigen stimulation of cells under normal physiological conditions, release of Ca2+ from intracellular stores makes an important contribution to the initial increase in [Ca2+]i. Oscillations in [Ca2+]i are not induced by elevating [Ca2+]i with the calcium ionophore ionomycin. Mitochondrial calcium buffering is not required for [Ca2+]i oscillations to occur. The results show that rat basophilic leukemia cells have significant stores of calcium and that release of calcium from these stores can participate in both the initial rise and the oscillations in [Ca2+]i.     

Diacylglycerol increases cytosolic free Ca2+ concentration in rat pituitary cells. Relationship to thyrotropin-releasing hormone action

To elucidate possible functions of elevation of endogenous diacylglycerol induced by thyrotropin-releasing hormone in pituitary cells, we have studied the actions of two synthetic diacylglycerols, sn-1-oleoyl-2-acetylglycerol (OAG) and sn-1,2-dioctanoylglycerol (DiC8), on cytosolic free calcium concentration ([Ca2+]i) in GH4C1 cells. OAG induced an immediate increase in [Ca2+]i which gradually reached a peak that was twice the basal level after the first min; [Ca2+]i then returned to remain at basal level after 3 min. The increase in [Ca2+]i was dependent on the concentration of OAG added with two apparent potencies; half-maximal actions on [Ca2+]i were observed at 70 nM and greater than 20 microM. The increase in [Ca2+]i induced by OAG was blocked completely by chelating extracellular calcium, or by pretreatment with calcium channel blockers. The phorbol ester 12-O-tetradecanoylphorbol-13-acetate, which itself induces a rise in [Ca2+]i in these cells that is similar in time course, magnitude, and drug sensitivity to that of OAG, blocked completely the actions of subsequent exposure to OAG. Analogous results were obtained using DiC8, although DiC8 induced a transient inhibition to 75% of basal levels of [Ca2+]i after the initial increase in [Ca2+]i, and DiC8 was less potent than OAG. These data indicated that diacylglycerols induce influx of extracellular calcium in these cells, possibly by activation of voltage-dependent Ca2+ channels. Furthermore, diacylglycerols and phorbol esters appear to utilize a common pathway in eliciting these actions on [Ca2+]i, possibly involving activation of a protein kinase C. These actions of diacylglycerol provide a pathway by which thyrotropin-releasing hormone may act to enhance calcium channel activity.     

Thimerosal increases the responsiveness of the calcium receptor in human parathyroid and rMTC6-23 cells

Parathyroid cells express a plasma membrane calcium receptor (CaR), which is stimulated by a rise in extracellular calcium concentration ([Ca2+]ext). A decreased sensitivity to [Ca2+]ext occurs in adenomatous parathyroid cells in patients with primary hyperparathyroidism, but the underlying functional mechanism is not yet fully understood. This study explored whether CaR responsiveness is influenced by increasing the affinity of IP3 receptors--a major signalling component of other G-protein-coupled receptors. The sulphydryl reagent thimerosal was used to increase the responsiveness of IP3-receptors. Quantitative fluorescence microscopy in Fura-2-loaded cells was used to investigate the effects of thimerosal on the cytoplasmic calcium concentrations ([Ca2+]i) in human parathyroid cells and to compare its effects in a rat medullary thyroid carcinoma cell line (rMTC6-23) also expressing CaR. During incubation in Ca(2+)-free medium, thimerosal 5 microM induced a rapid sustained rise in [Ca2+]i in human parathyroid cells and no further [Ca2+]i increase appeared in response to the CaR agonist Gd3+ (100 microM). Thimerosal 1 microM induced only slow and minimal changes of basal [Ca2+]i and allowed a rapid response to Gd3+ 20 nM (a concentration without effect in control cells). The slope of the thimerosal-induced [Ca2+]i responses was steeper following exposure to CaR agonists. In the presence of 1 mM [Ca2+]ext, thimerosal (0.5 microM) induced a sharp increase in [Ca2+]i to a peak (within 60 s), followed either by return to basal [Ca2+]i or by a plateau of slightly higher amplitude. Similar results were obtained using rMTC6-23 cells. Thimerosal increases the responsiveness to CaR agonists through modulation of the sensitivity of the IP3 receptor in both parathyroid and rMTC6-23 cells.     

Calcium-dependent PAF-stimulated generation of reactive oxygen species in a human keratinocyte cell line.

During inflammation and other pathological states, the lipid mediator platelet-activating factor (PAF) and reactive oxygen species (ROS) are both generated. We have been investigating the effect of exogenous PAF on ROS formation in the human keratinocyte cell line (HaCaT). ROS production, measured using luminol-enhanced chemiluminescence (CL), proved to be rapid, transient, PAF receptor-mediated, and totally dependent on an increase in intracellular Ca2+ ([Ca2+]i) and on the presence of extracellular Ca2+. Repeated administration of PAF resulted in refractoriness to the agonist in terms of both capacities to increase [Ca2+]i and generate ROS. The cells, however, continued to respond fully to other stimulants (bradykinin, epidermal growth factor, thapsigargin). The PAF-induced increases in [Ca2+]i (monitored using the fluorescent probe Fluo-3) were also rapid and transient and paralleled those of ROS generation. Relatively specific inhibitors of potential ROS-producing systems were administered in an attempt to characterize the ROS producing system(s). Inhibitors of xanthine oxidase, phospholipase A2, lipoxygenase, cyclooxygenase and NO synthase did not interfere with PAF evoked ROS. The flavoprotein inhibitor diphenyleneiodonium and the mitochondrial cytochrome oxidase inhibitor KCN, prevented generation of ROS, making NAD(P)H a candidate for the electron source of the ROS and the mitochondria a potential major site of formation.     

Selective role for superoxide in InsP3 receptor-mediated mitochondrial dysfunction and endothelial apoptosis

Reactive oxygen species (ROS) play a divergent role in both cell survival and cell death during ischemia/reperfusion (I/R) injury and associated inflammation. In this study, ROS generation by activated macrophages evoked an intracellular Ca2+ ([Ca2+]i) transient in endothelial cells that was ablated by a combination of superoxide dismutase and an anion channel blocker. [Ca2+]i store depletion, but not extracellular Ca2+ chelation, prevented [Ca2+]i elevation in response to O2*- that was inositol 1,4,5-trisphosphate (InsP3) dependent, and cells lacking the three InsP3 receptor (InsP3R) isoforms failed to display the [Ca2+]i transient. Importantly, the O2*--triggered Ca2+ mobilization preceded a loss in mitochondrial membrane potential that was independent of other oxidants and mitochondrially derived ROS. Activation of apoptosis occurred selectively in response to O2*- and could be prevented by [Ca2+]i buffering. This study provides evidence that O2*- facilitates an InsP3R-linked apoptotic cascade and may serve a critical function in I/R injury and inflammation.     

Alteration of cytosolic free calcium homeostasis by SIN-1: high sensitivity of L-type Ca2+ channels to extracellular oxidative/nitrosative stress in cerebellar granule cells

Exposure of cerebellar granule neurones in 25 mm KCl HEPES-containing Locke's buffer (pH 7.4) to 50-100 microm SIN-1 during 2 h decreased the steady-state free cytosolic Ca2+ concentration ([Ca2+]i) from 168 +/- 33 nm to 60 +/- 10 nm, whereas exposure to > or = 0.3 mm SIN-1 produced biphasic kinetics: (i) decrease of [Ca2+]i during the first 30 min, reaching a limiting value of 75 +/- 10 nm (due to inactivation of L-type Ca2+ channels) and (ii) a delayed increase of [Ca2+]i at longer exposures, which correlated with SIN-1-induced necrotic cell death. Both effects of SIN-1 on [Ca2+]i are blocked by superoxide dismutase plus catalase and by Mn(III)tetrakis(4-benzoic acid)porphyrin chloride. Supplementation of Locke's buffer with catalase before addition of 0.5-1 mm SIN-1 had no effect on the decrease of [Ca2+]i but further delayed and attenuated the increase of [Ca2+]i observed after 60-120 min exposure to SIN-1 and also protected against SIN-1-induced necrotic cell death. alpha-Tocopherol, the potent NMDA receptor antagonist (+)-MK-801 and the N- and P-type Ca2+ channels blocker omega-conotoxin MVIIC had no effect on the alterations of [Ca2+]i upon exposure to SIN-1. However, inhibition of the plasma membrane Ca2+ ATPase can account for the increase of [Ca2+]i observed after 60-120 min exposure to 0.5-1 mm SIN-1. It is concluded that L-type Ca2+ channels are a primary target of SIN-1-induced extracellular nitrosative/oxidative stress, being inactivated by chronic exposure to fluxes of peroxynitrite of 0.5-1 microm/min, while higher concentrations of peroxynitrite and hydrogen peroxide are required for the inhibition of the plasma membrane Ca2+ ATPase and induction of necrotic cell death, respectively.