alpha-keto-beta-methyl-n-valeric acid diminishes reactive oxygen species and alters endoplasmic reticulum Ca(2+) stores

Mitochondrial dysfunction and oxidative stress occur in neurodegenerative diseases. Other results show that bombesin-releasable calcium stores (BRCS) from the endoplasmic reticulum (ER) are exaggerated in fibroblasts from patients with Alzheimer's disease (AD) compared with controls and in fibroblasts from a young control treated with H(2)O(2). We hypothesize that alterations in oxidative stress underlie the exaggeration in BRCS in AD, and that appropriate antioxidants may be useful in treating this abnormality. Two indicators of different oxidant species were used to determine the effects of select oxidants on cellular oxidation status: carboxydichlorofluorescein (c-DCF) to detect reactive oxygen species (ROS), and 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF) to detect nitric oxide (NO(.-)). Various conditions that induce ROS, including H(2)O(2), oxygen/glucose deprivation, and 3-morpholinosyndnonimine (SIN-1), were used to test the ability of alpha-keto-ss-methyl-n-valeric acid (KMV) to scavenge ROS. KMV diminished c-DCF-detectable ROS that were induced by H(2)O(2), oxygen/glucose deprivation, or SIN-1 in PC12 cells, primary neuronal cultures, or fibroblasts. Furthermore, KMV reduced the H(2)O(2)-induced increase in BRCS and diminished the elevation in BRCS in cells from AD patients to control levels. On the other hand, DAF-detectable NO(.-) induced by SIN-1 was not scavenged by KMV and did not exaggerate BRCS. The results indicate that KMV is an effective antioxidant of c-DCF-detectable ROS. The effects of KMV are not cell type specific, but are ROS specific. The same H(2)O(2)-induced ROS that reacts with KMV may also underlie the changes in BRCS related to AD. Thus, KMV ameliorates the effects of ROS on calcium homeostasis related to oxidative stress and to AD.     

Oxidative stress increases internal calcium stores and reduces a key mitochondrial enzyme

Fibroblasts from patients with genetic and non-genetic forms of Alzheimer's disease (AD) show many abnormalities including increased bombesin-releasable calcium stores (BRCS), diminished activities of the mitochondrial alpha-ketoglutarate dehydrogenase complex (KGDHC), and an altered ability to handle oxidative stress. The link between genetic mutations (and the unknown primary event in non-genetic forms) and these other cellular abnormalities is unknown. To determine whether oxidative stress could be a convergence point that produces the other AD-related changes, these experiments tested in fibroblasts the effects of H(2)O(2), in the presence or absence of select antioxidants, on BRCS and KGDHC. H(2)O(2) concentrations that elevated carboxy-dichlorofluorescein (c-H(2)DCF)-detectable ROS increased BRCS and decreased KGDHC activity. These changes are in the same direction as those in fibroblasts from AD patients. Acute treatments with the antioxidants Trolox, or DMSO decreased c-H(2)DCF-detectable ROS by about 90%, but exaggerated the H(2)O(2)-induced increases in BRCS by about 4-fold and did not alter the reduction in KGDHC. Chronic pretreatments with Trolox more than doubled the BRCS, tripled KGDHC activities, and reduced the effects of H(2)O(2). Pretreatment with DMSO or N-acetyl cysteine diminished the BRCS and either had no effect, or exaggerated the H(2)O(2)-induced changes in these variables. The results demonstrate that BRCS and KGDHC are more sensitive to H(2)O(2) derived species than c-H(2)DCF, and that oxidized derivatives of the antioxidants exaggerate the actions of H(2)O(2). The findings support the hypothesis that select abnormalities in oxidative processes are a critical part of a cascade that leads to the cellular abnormalities in cells from AD patients.     

Mitochondrial Enzymes and Endoplasmic Reticulum Calcium Stores as Targets of Oxidative Stress in Neurodegenerative Diseases

Considerable evidence indicates that oxidative stress accompanies age-related neurodegenerative diseases. Specific mechanisms by which oxidative stress leads to neurodegeneration are unknown. Two targets of oxidative stress that are known to change in neurodegenerative diseases are the mitochondrial enzyme alpha-ketoglutarate dehydrogenase complex (KGDHC) and endoplasmic reticulum calcium stores. KGDHC activities are diminished in all common neurodegenerative diseases and the changes are particularly well documented in Alzheimer's disease (AD). A second change that occurs in cells from AD patients is an exaggerated endoplasmic reticulum calcium store [i.e., bombesin-releasable calcium stores (BRCS)]. H(2)O(2), a general oxidant, changes both variables in the same direction as occurs in disease. Other oxidants selectively alter these variables. Various antioxidants were used to help define the critical oxidant species that modifies these responses. All of the antioxidants diminish the oxidant-induced carboxy-dichlorofluorescein (cDCF) detectable reactive oxygen species (ROS), but have diverse actions on these cellular processes. For example, alpha-keto-beta-methyl-n-valeric acid (KMV) diminishes the H(2)O(2) effects on BRCS, while trolox and DMSO exaggerate the response. Acute trolox treatment does not alter H(2)O(2)-induced changes in KGDHC, whereas chronic treatment with trolox increases KGDHC almost threefold. The results suggest that KGDHC and BRCS provide targets by which oxidative stress may induce neurodegeneration and a useful tool for selecting antioxidants for reversing age-related neurodegeneration.     

Differential alterations in antioxidant capacity in cells from Alzheimer patients

Oxidative stress occurs in brains of Alzheimer's disease (AD) patients. A major question in AD research is whether the oxidative stress is just secondary to neurodegeneration. To test whether oxidative stress is an inherent property of AD tissues, the ability of cultured fibroblasts bearing the AD Presenilin-1 246 Ala-->Glu mutation to handle reactive oxygen species (ROS) was compared to controls. Although ROS in cells from AD subjects were only slightly less than cells from controls under basal conditions (-10%) or after exposure to H(2)O(2) (-16%), treatment with antioxidants revealed clear differences. Pretreatment with DMSO, a hydroxyl radical scavenger, reduced basal and H(2)O(2)-induced ROS levels significantly more in cells from controls (-22%, -22%) than in those from AD subjects (-4%, +14%). On the other hand, pretreatment with Trolox diminished H(2)O(2)-induced ROS significantly more in cells from AD (-60%) than control subjects (-39%). In summary, cells from AD patients have greater Trolox sensitive ROS and less DMSO sensitive ROS than controls. The results demonstrate that fibroblasts bearing this PS-1 mutation have altered means of handling oxidative stress and appear useful for determining the mechanism underlying the altered redox metabolism.     

Macelignan protects HepG2 cells against tert-butylhydroperoxide-induced oxidative damage

In this study, we investigated the protective effect of macelignan, isolated from Myristica fragrans Houtt. (nutmeg) against tert-butylhydroperoxide (t-BHP)-induced cytotoxicity in a human hepatoma cell line, HepG2. The tetrazolium dye colorimetric test (MTT test) and lactate dehydrogenase (LDH) assay were used to monitor cell viability and necrosis, respectively. Lipid peroxidation [malondialdehyde (MDA) formation] was estimated by the fluorometric method. Intracellular reactive oxygen species (ROS) formation was measured using a fluorescent probe 2',7'-dichlorofluorescein diacetate (DCFH-DA), and DNA damage was detected using single cell gel electrophoresis (comet assay). The results showed that macelignan significantly reduced the cell growth inhibition and necrosis caused by t-BHP. Furthermore, macelignan ameliorated lipid peroxidation as demonstrated by a reduction in MDA formation in a dose-dependent manner. It was also found that macelignan reduced intracellular ROS formation and DNA damaging effect caused by t-BHP. These results strongly suggest that macelignan has significant protective ability against oxidative damage caused by reactive intermediates.     

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.     

Effects of reactive oxygen species on brain synaptic plasma membrane Ca(2+)-ATPase.

The regulation of free intracellular calcium [Ca2+]i is altered in neurons from the aged brain, possibly due to reductions in the activity of Ca2+ transporters. The plasma membrane Ca(2+)-ATPase (PMCA) plays a critical role in Ca2+ homeostasis, and its kinetic properties change in aged rat brain. These changes could be due to oxidative modification of PMCA as a result of age-related chronic oxidative stresses. The present studies were undertaken to determine the sensitivity of the neuronal PMCA to in vitro exposure of synaptic plasma membranes (SPMs) to reactive oxygen species (ROS). We examined the effects of three oxidants including peroxyl radicals generated by azo-initiators, 2,2'-Azobis 2-amidinopropane dihydrochloride (AAPH) and 4,4'-Azobis 14-cyanovaleric acid (ACVA), hydrogen peroxide (H2O2), and peroxynitrite (ONOO-). Synaptic plasma membranes briefly exposed to these oxidants were analyzed for functional and structural alterations in PMCA. Although all three oxidants led to significant loss of PMCA activity, the effect of ONOO- was the most potent, followed by peroxyl radicals and H2O2. Kinetic analysis of PMCA activity after oxidant treatment showed decreases in Vmax without significant changes in K(act). Immunoblots revealed oxidant-induced cross-linking of PMCA molecules that were partially reversed under reducing conditions and completely reversed with addition of urea. The PMCA appears to be very sensitive to inhibition by ROS and hence may be a target of oxidative stress in the aging brain. Reduction in its activity may contribute to age-related alterations in neuronal [Ca2+]i regulation.     

Role of mitochondrial reactive oxygen species in hypoxia-dependent increase in intracellular calcium in pulmonary artery myocytes

Previous studies examining the role of mitochondria-derived reactive oxygen species (ROS) in hypoxic responses have been mainly conducted in isolated lungs and cultured pulmonary artery smooth muscle cells (PASMCs) using mitochondrial inhibitors, and yielded largely conflicting results. Here we report that in freshly isolated mouse PASMCs, which are devoid of the mixed responses from multi-types of cells in lungs and significant changes in gene expression in cultured cells, the mitochondrial electron transport chain (ETC) complex I, II, or III inhibitors blocked hypoxia-induced increases in intracellular ROS and Ca2+ concentration ([ROS]i and [Ca2+]i) without effects on their resting levels. Inhibition of the complex I plus II and/or III did not produce an additive effect. Glutathione peroxidase-1 (Gpx1) or catalase gene overexpression to enhance H2O2 removal remarkably reduced hypoxic increases in [ROS]i and [Ca2+]i, whereas Gpx1 gene deletion had the opposite effect. None of these genetic modifications changed the resting [ROS]i and [Ca2+]i. H2O2 at 51 microM caused a similar increase in DCF fluorescence ([ROS]i) as that by hypoxia, but only induced 33% of hypoxic increase in [Ca2+]i. Moreover, H2O2 (5.1 microM) reversed the inhibition of the hypoxia-induced increase in [Ca2+]i by rotenone. Collectively, our study using various mitochondrial inhibitors and genetic approaches demonstrates that in response to acute hypoxia, the mitochondrial ETC molecules prior to the complex III ubisemiquinone site act as a functional unit to increase the generation of ROS, particularly H2O2, which is important for, but may not fully cause, the hypoxic increase in [Ca2+]i in freshly isolated PASMCs.     

Priming effect of calcium ionophores on phorbol ester-induced respiratory burst in mouse peritoneal neutrophils.

The abilities of three calcium ionophores (A23187, 4-bromo-A23187, and ionomycin) to modulate the respiratory burst of neutrophils induced by phorbol ester and to increase the concentration of free intracellular Ca2+ ([Ca2+]i) were compared. The production of reactive oxygen species (ROS) was determined by luminol-dependent chemiluminescence and [Ca2+]i was determined with the Fura-2 fluorescent probe. A23187 (0.05-2 microM) and ionomycin (0.001-0.5 microM) but not 4-bromo-A23187 amplified 3-4-fold the respiratory burst induced by phorbol ester. The integral response (total production of ROS over 6 min) had a bell-shaped dependence on the concentration of ionomycin and A23187 with increase and decrease at low and high concentrations of the ionophores, respectively. The maximal effect was found at 0.5 microM ionomycin and 2 microM A23187, these concentrations resulting in transient increases in [Ca2+]i to 1776 +/- 197 and 955 +/- 27 nM, respectively. The ionophores had no effect in calcium-free media, though they increased [Ca2+]i to approximately 400 nM through the mobilization of intracellular Ca2+. In cells with exhausted stores of Ca2+, the addition of 1.5 mM Ca2+ combined with phorbol ester amplified twofold the production of ROS. The inhibition of phospholipase A2 with 4-bromophenacyl bromide significantly decreased the production of ROS. Thus, the entrance of Ca2+ and generation of arachidonic acid under the influence of phospholipase A2 are necessary for the ionophore-induced priming of production of ROS during cell activation with phorbol esters.     

Detachment of cultured cells from the substratum induced by the neutrophil-derived oxidant NH2Cl: synergistic role of phosphotyrosine and intracellular Ca2+ concentration

The neutrophil-derived, membrane-permeating oxidant, NH2Cl, (but not the non-membrane-permeating chloramine, taurine-NHCl) induced detachment of fetal mouse cardiac myocytes and other cell types (fibroblasts, epithelial cells, and endothelial cells) from the culture dish, concomitant with cell shrinkage ("peeling off"). Stimulated human neutrophils also induced peeling off of cultured mouse cardiac myocytes when the latter were pretreated with inhibitors of .OH and elastase. Immunofluorescence microscopy revealed that the NH2Cl-induced peeling off of WI-38 fibroblasts is accompanied by disorganization of integrin alpha 5 beta 1, vinculin, stress fibers, and phosphotyrosine (p-Tyr)- containing proteins. Decrease in the content of the p-Tyr-containing proteins of the NH2Cl-treated cells was analyzed by immunoblotting techniques. Coating of fibronectin on the culture dish prevented both NH2Cl-induced peeling off and a decrease in p-Tyr content. Preincubation with a protein-tyrosine phosphatase inhibitor, sodium orthovanadate (Na3VO4), also prevented NH2Cl-induced peeling off, suggesting that dephosphorylation of p-Tyr is necessary for peeling off. NH2Cl-induced peeling off was accompanied by an increase in intracellular Ca2+ concentration ([Ca2+]i) in mouse cardiac myocytes and WI-38 fibroblasts. The absence of extracellular Ca2+ prevented both NH2Cl-induced peeling off and increased [Ca2+]i, both of which did occur on subsequent incubation of the cells in Ca2+-containing medium. These observations suggest that an increase in [Ca2+]i is also necessary for peeling off. Depletion of microsomal and cytosolic Ca2+ by incubation with the microsomal Ca2+-ATPase inhibitor 2',5'-di(tert- butyl)-1,4-benzohydroquinone (BHQ) plus EGTA prevented both NH2Cl- induced increases in [Ca2+]i and peeling off. Direct inhibition of microsomal Ca2+ pump activity by NH2Cl may participate in the NH2Cl- induced [Ca2+]i increment. A combination of p-Tyr dephosphorylation by genistein (an inhibitor of tyrosine kinase) and an increase in [Ca2+]i by BHQ could also induce peeling off. All these observations suggest a synergism between p-Tyr dephosphorylation and increased [Ca2+]i in NH2Cl-induced peeling off.     

Modulation by cADPr of Ca2+ mobilization and oxidative response in dimethylsulfoxide- or retinoic acid-differentiated HL-60 cells

In human phagocytic cells, reactive oxygen species (ROS) generation in response to N-formyl-L-Methionyl-L-Leucyl-L-Phenylalanine (fMLF) is largely dependent on cytosolic free calcium concentration ([Ca2+]i). Cyclic ADP-ribose (cADPr) is able to regulate Ca2+ release from intracellular stores through the ryanodine receptor but its potential role in biological responses has so far not been determined. In this study, we examined whether extracellular and intracellular cADPr is required in fMLF-induced [Ca2+]i rise and consequently in the oxidative response in human neutrophil-like HL-60 cells differentiated with dimethylsulfoxide or all-trans-retinoic acid (ATRA). We establish that extracellular cADPr cannot elicit [Ca2+]i elevation. Furthermore, we demonstrate that 8-Br-cADPr, a functional antagonist of cADPr, inhibits Ca2+ entry into HL-60 cells differentiated with ATRA and stimulated with fMLF (95+/-4 and 148+/-5 nM respectively, n=3). Finally, we show that this partial inhibition of Ca2+ mobilization is unrelated to ROS production (10.0+/-0.3 vs. 9.6+/-0.5 A.U., n=3). In conclusion, we showed that cADPr can control fMLF-induced Ca2+ influx but is unable to regulate a Ca2+-dependent biological response, i.e. H2O2 production.     

Human round spermatids from azoospermic men exhibit oocyte-activation and Ca2+ oscillation-inducing activities

During mammalian fertilization, intracellular Ca2+ oscillations are important for both oocyte activation and embryonic development. As the ability of round spermatids (ROS) to induce Ca2+ oscillations and oocyte activation is different between species, we examined Ca2+ oscillation- and oocyte activation-inducing abilities of human ROS originating from patients with non-obstructive azoospermia. Human ROS from 11 non-obstructive azoospermic patients were collected during their TESE-ICSI cycles. Following injection into mature unfertilized mouse oocytes, we examined the oocyte-activating and Ca2+ oscillation-inducing activities of ROS by using Ca2+ imaging and confocal laser scanning microscopy (mouse test). In these 11 cases, clinical TESE-ICSI using mature testicular spermatozoa was successful, with the exception of one case in which only one sperm-injected oocyte was not fertilized. The mean fertilization rate was 70.1% (40-100%); the mean cleavage rate was 97.9% (46/47). Two pregnancies were established from 10 transfer cycles (PR; 20%). When the ROS from these patients were injected into mouse oocytes, the ROS from all patients induced at least some intracellular Ca2+ oscillations (25-100%). In all patients, 40 out of 82 oocytes injected with ROS exhibited normal oscillation patterns of [Ca2+]i.Human spermatogenetic cells acquired oocyte-activating and Ca2+ oscillation-inducing abilities at the round spermatid stage, an earlier stage than found for rodent cells. These data indicate that human ROS might be useful for clinical treatments of non-obstructive azoospermic patients exhibiting mature spermatozoa in biopsied specimens.     

Molecular Profiling Reveals Diversity of Stress Signal Transduction Cascades in Highly Penetrant Alzheimer's Disease Human Skin Fibroblasts

The serious and growing impact of the neurodegenerative disorder Alzheimer''s disease (AD) as an individual and societal burden raises a number of key questions: Can a blanket test for Alzheimer''s disease be devised forecasting long-term risk for acquiring this disorder? Can a unified therapy be devised to forestall the development of AD as well as improve the lot of present sufferers? Inflammatory and oxidative stresses are associated with enhanced risk for AD. Can an AD molecular signature be identified in signaling pathways for communication within and among cells during inflammatory and oxidative stress, suggesting possible biomarkers and therapeutic avenues? We postulated a unique molecular signature of dysfunctional activity profiles in AD-relevant signaling pathways in peripheral tissues, based on a gain of function in G-protein-coupled bradykinin B2 receptor (BKB2R) inflammatory stress signaling in skin fibroblasts from AD patients that results in tau protein Ser hyperphosphorylation. Such a signaling profile, routed through both phosphorylation and proteolytic cascades activated by inflammatory and oxidative stresses in highly penetrant familial monogenic forms of AD, could be informative for pathogenesis of the complex multigenic sporadic form of AD. Comparing stimulus-specific cascades of signal transduction revealed a striking diversity of molecular signaling profiles in AD human skin fibroblasts that express endogenous levels of mutant presenilins PS-1 or PS-2 or the Trisomy 21 proteome. AD fibroblasts bearing the PS-1 M146L mutation associated with highly aggressive AD displayed persistent BKB2R signaling plus decreased ERK activation by BK, correctible by gamma-secretase inhibitor Compound E. Lack of these effects in the homologous PS-2 mutant cells indicates specificity of presenilin gamma-secretase catalytic components in BK signaling biology directed toward MAPK activation. Oxidative stress revealed a JNK-dependent survival pathway in normal fibroblasts lost in PS-1 M146L fibroblasts. Complex molecular profiles of signaling dysfunction in the most putatively straightforward human cellular models of AD suggest that risk ascertainment and therapeutic interventions in AD as a whole will likely demand complex solutions.     

Hypoxic remodelling of Ca2+ mobilization in type I cortical astrocytes: involvement of ROS and pro-amyloidogenic APP processing

Chronic hypoxia (CH) alters Ca2+ homeostasis in various cells and may contribute to disturbed Ca2+ homeostasis of Alzheimer's disease. Here, we have employed microfluorimetric measurements of [Ca2+]i to investigate the mechanism underlying augmentation of Ca2+ signalling by chronic hypoxia in type I cortical astrocytes. Application of bradykinin evoked significantly larger rises of [Ca2+]i in hypoxic cells as compared with control cells. This augmentation was prevented fully by either melatonin (150 micro m) or ascorbic acid (200 micro m), indicating the involvement of reactive oxygen species. Given the association between hypoxia and increased production of amyloid beta peptides (AbetaPs) of Alzheimer's disease, we performed immunofluorescence studies to show that hypoxia caused a marked and consistent increased staining for AbetaPs and presenilin-1 (PS-1). Western blot experiments also confirmed that hypoxia increased PS-1 protein levels. Hypoxic increases of AbetaP production was prevented with inhibitors of either gamma- or beta-secretase. These inhibitors also partially prevented the augmentation of Ca2+ signalling in astrocytes. Our results indicate that chronic hypoxia enhances agonist-evoked rises of [Ca2+]i in cortical astrocytes, and that this can be prevented by antioxidants and appears to be associated with increased AbetaP formation.     

Aromatic monoamine-induced immediate oxidative burst leading to an increase in cytosolic Ca2+ concentration in tobacco suspension culture

Aromatic monoamines may contribute to both chemical and physical protection of plants. Addition of phenylethylamine (PEA) and benzylamine to tobacco suspension culture (cell line BY-2) induced a very rapid and transient generation of two active oxygen species (AOS), H2O2 and superoxide anion, both detected with chemiluminescence. Electron spin resonance spectroscopy revealed that hydroxy radicals are also produced. With laser-scanning confocal microscopy, fluorescence spectroscopy and microplate fluorescence reading, intracellular H2O2 production was detected using dichlorofluorescin diacetate as a fluorescent probe. Following AOS production, cytosolic Ca2+ concentration ([Ca2+]c) of the tobacco cells, monitored with luminescence of transgenic aequorin, increased and attained to a peak level 12 s after PEA addition. The PEA-induced increase in [Ca2+]c was inhibited by a Ca2+ chelator, Ca2+ antagonists and AOS scavengers, suggesting that PEA-induced AOS triggered a Ca2+ influx across the plasma membrane.     

Some mitogens cause rapid increases in free calcium in fibroblasts

Quiescent 3T3 fibroblasts grown on microcarrier beads and loaded with the [Ca2+] indicator quin2 had a cytosolic free Ca2+ concentration ( [Ca2+]i) of 154 +/- 11 nM (SE; n = 32). Stimulation with the mitogens vasopressin, epidermal growth factor (EGF) or prostaglandin F2 alpha (PGF2 alpha) caused a very rapid increase in [Ca2+]i to a maximum of 200-500 nM after 60-90 s. [Ca2+]i declined thereafter to a level above that in quiescent cells which was maintained for at least 15 min. In contrast no immediate effects on [Ca2+]i were detected after the addition of the mitogens insulin or 12-O-tetradecanoylphorbol 13-acetate (TPA). These studies indicate that early changes in [Ca2+]i may be involved in the action on fibroblasts of some, but not all, mitogens.     

Involvement of prostaglandins in histamine H1 receptor-operated Ca2+ entry in human gingival fibroblasts

In the absence of external Ca2+, 100 microM histamine evoked a transient increase in intracellular Ca2+ ([Ca2+]i), and subsequent addition of Ca2+ to the medium resulted in a sustained increase in [Ca2+]i in fura-2-loaded human gingival fibroblasts. These Ca2+ mobilizations are attributed to Ca2+ release from intracellular stores and Ca2+ entry, respectively. When the histamine H1 antagonist chlorpheniramine was added after the histamine-induced transient increase in [Ca2+]i, the Ca2+ entry induced by the addition of Ca2+ was inhibited. In the fibroblasts pretreated with cyclooxygenase inhibitors, indomethacin (1 microM) or aspirin (100 microM), histamine-induced Ca2+ entry was significantly inhibited, but not the transient [Ca2+]i increase. These results suggest that the histamine-induced Ca2+ entry requires the continuous binding of histamine to the H1 receptors and is regulated by prostaglandins, which are probably produced due to the H1 receptor activation.     

Volume-sensitive NADPH oxidase activity and taurine efflux in NIH3T3 mouse fibroblasts

Reactive oxygen species (ROS) are produced in NIH3T3 fibroblasts during hypotonic stress, and H(2)O(2) potentiates the concomitant release of the organic osmolyte taurine (Lambert IH. J Membr Biol 192: 19-32, 2003). The increase in ROS production [5-(and-6)-carboxy-2', 7'-dichlorodihydrofluorescein diacetate fluorescence] is detectable after a reduction in the extracellular osmolarity from 335 mosM (isotonic) to 300 mosM and reaches a maximal value after a reduction to 260 mosM. The swelling-induced ROS production is reduced by the flavoprotein inhibitor diphenylene iodonium chloride (25 microM) but is unaffected by the nitric oxide synthase inhibitor N omega-nitro-l-arginine methyl ester, indicating that the volume-sensitive ROS production is NADPH oxidase dependent. NIH3T3 cells express the NADPH oxidase components: p22 phox, a NOX4 isotype; p47 phox; and p67 phox (real-time PCR). Exposure to the Ca2+-mobilizing agonist ATP (10 microM) potentiates the release of taurine but has no effect on ROS production under hypotonic conditions. On the other hand, addition of the protein kinase C (PKC) activator phorbol 12-myristate 13-acetate (PMA, 100 nM) or the lipid messenger lysophosphatidic acid (LPA, 10 nM) potentiates the swelling-induced taurine release as well as the ROS production. Overexpression of Rac1 or p47 phox or p47 phox knockdown [small interfering (si)RNA] had no effect on the swelling-induced ROS production or taurine release. NOX4 knockdown (siRNA) impairs the increase in the ROS production and the concomitant taurine release following osmotic exposure. It is suggested that a NOX4 isotype plus p22 phox account for the swelling-induced increase in the ROS production in NIH3T3 cells and that the oxidase activity is potentiated by PKC and LPA but not by Ca2+.     

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.