Induced Superoxide Production Research Articles

Mechanism of Angiotensin II-induced Superoxide Production in Cells Reconstituted with Angiotensin Type 1 Receptor and the Components of NADPH Oxidase

The mechanism of angiotensin II (Ang II)-induced superoxide production was investigated with HEK293 or Chinese hamster ovary cells reconstituted with the angiotensin type 1 receptor (AT(1)R) and NADPH oxidase (either Nox1 or Nox2) along with a pair of adaptor subunits (either NOXO1 with NOXA1 or p47(phox) with p67(phox)). Ang II enhanced the activity of both Nox1 and Nox2 supported by either adaptor pair, with more effective activation of Nox1 in the presence of NOXO1 and NOXA1 and of Nox2 in the presence of p47(phox) and p67(phox). Expression of several AT(1)R mutants showed that interaction of the receptor with G proteins but not that with beta-arrestin or with other proteins (Jak2, phospholipase C-gamma1, SH2 domain-containing phosphatase 2) that bind to the COOH-terminal region of AT(1)R, was necessary for Ang II-induced superoxide production. The effects of constitutively active alpha subunits of G proteins and of various pharmacological agents implicated signaling by a pathway comprising AT(1)R, Galpha(q/11), phospholipase C-beta, and protein kinase C as largely, but not exclusively, responsible for Ang II-induced activation of Nox1 and Nox2 in the reconstituted cells. A contribution of Galpha(12/13), phospholipase D, and phosphatidyl-inositol 3-kinase to Ang II-induced superoxide generation was also suggested, whereas Src and the epidermal growth factor receptor did not appear to participate in this effect of Ang II. In reconstituted cells stimulated with Ang II, Nox2 exhibited a more sensitive response than Nox1 to the perturbation of protein kinase C, phosphatidylinositol 3-kinase, or the small GTPase Rac1.

Mechanisms and Consequences of Neutrophil Interaction with the Endothelium

Leukocyte recruitment into inflamed tissue proceeds in a cascade-like fashion. The first contact of neutrophils with the endothelium is mediated by selectins and their counterreceptors, followed by rolling of neutrophils along the endothelial wall of postcapillary venules and integrin-mediated arrest. While rolling, neutrophils collect different inflammatory signals that can activate several pathways. In addition to activation of neutrophils by ligation of G-protein-coupled receptors with chemokines and other chemoattractants, integrins and selectin ligands are also able to signal into the cell, where they initiate neutrophil extravasation, promote cytoskeletal rearrangement, and ultimately induce superoxide production and degranulation. These signaling pathways may be targeted by therapeutic interventions to inhibit specific functions of neutrophils without affecting others. This Review is focused on the signaling events during the interaction of neutrophils with the endothelium.

Olmesartan Ameliorates Progressive Glomerular Injury in Subtotal Nephrectomized Rats through Suppression of Superoxide Production

Angiotensin type 1 receptor blockers are more effective than other antihypertensive agents in slowing the progression of renal disease. Angiotensin II (Ang II) induces production of NAD(P)H oxidase-dependent superoxide in vascular and mesangial cells, but the direct role of Ang II in glomerular superoxide production remains unknown. Here we examined the effect of Ang II on superoxide production both ex vivo and in vivo. Ang II increased superoxide generation in isolated normal glomeruli in a dose-dependent manner, and co-incubation with olmesartan, an angiotensin type 1 receptor blocker, suppressed such increase. Subtotal nephrectomized rats (Nx, n=8) showed impaired renal function, increased glomerular sclerosis, and significantly high superoxide production in glomeruli. These changes were inhibited in olmesartan-treated (n=8), but not hydralazine-treated (n=8) Nx rats. Oxidative stress and nitrosative stress were observed in Nx glomeruli, as evidenced by increased levels of carbonyl protein and nitrotyrosine formation, respectively. These changes were inhibited by 8-week treatment with olmesartan. The apoptosis observed in Nx glomeruli was also suppressed by olmesartan. Superoxide generation in Nx glomeruli was blocked by an NAD(P)H oxidase inhibitor, diphenylene iodinium. The mRNA expression levels of two NAD(P)H oxidase subunits were increased in Nx, and olmesartan significantly reduced the mRNA expression levels. These results indicate that Ang II directly induced superoxide production through activation of NAD(P)H oxidase, and olmesartan would inhibit superoxide production and oxidative stress independent of its blood pressure-lowering effect. These findings support the notion that superoxide plays a primary role in glomerular injury in chronic kidney disease.

Hydralazine inhibits compression and acrolein‐mediated injuries in ex vivo spinal cord

We have previously shown that acrolein, a lipid peroxidation byproduct, is significantly increased following spinal cord injury in vivo, and that exposure to neuronal cells results in oxidative stress, mitochondrial dysfunction, increased membrane permeability, impaired axonal conductivity, and eventually cell death. Acrolein thus may be a key player in the pathogenesis of spinal cord injury, where lipid peroxidation is known to be involved. The current study demonstrates that the acrolein scavenger hydralazine protects against not only acrolein-mediated injury, but also compression in guinea pig spinal cord ex vivo. Specifically, hydralazine (500 mumol/L to 1 mmol/L) can significantly alleviate acrolein (100-500 mumol/L)-induced superoxide production, glutathione depletion, mitochondrial dysfunction, loss of membrane integrity, and reduced compound action potential conduction. Additionally, 500 mumol/L hydralazine significantly attenuated compression-mediated membrane disruptions at 2 and 3 h following injury. This was consistent with our findings that acrolein-lys adducts were increased following compression injury ex vivo, an effect that was prevented by hydralazine treatment. These findings provide further evidence for the role of acrolein in spinal cord injury, and suggest that acrolein-scavenging drugs such as hydralazine may represent a novel therapy to effectively reduce oxidative stress in disorders such as spinal cord injury and neurodegenerative diseases, where oxidative stress is known to play a role.

Abstract 777: Caveolin-1 Regulates NADPH Oxidase Subcellular Location And Activity In Endothelial Cells.

Background: The biological setting or location where reactive oxygen species (ROS) are produced is a new concept which may provide a mechanistic explanation for redox signaling specificity. In this regard, we recently reported that NADPH oxidase, a major superoxide producing enzyme system, is localized and functional in endothelial cell caveolae. Here, we investigate the role of caveolin-1, the main structural protein of caveolae, in the organization and regulation of NADPH oxidase activity and ROS mediated responses to cytokine challenge. Methods and Results: Superoxide production was measured by DHE assay in endothelial cells derived from wild type (wt) and caveolin-1 knockout (Cav−/ −) mouse lungs (MLEC). TNFα failed to induced superoxide production in Cav−/ − endothelial cell cultures (MOI = 0). However, infection of knockout cells with full-length caveolin-1 containing Adenovirus at MOI of 10 and 50 progressively restored TNFα effect on superoxide production with MOI 50 nearly reaching those observed in wt cells. The distribution of NADPH oxidase within the plasma membrane showed localization to membrane rafts fractions in wt cells which shifted to the non-raft membrane fractions in Cav-1−/ − MLEC. Re-expression of caveolin-1 (MOI 50) restored raft targeting of NADPH oxidase and both gp91 and p22phox co-precipitated with newly expressed caveolin-1 immunoprecipitated from raft fractions. To evaluate the cellular consequences of a potential redox signaling relay involving caveolin-1, we examined an NADPH oxidase dependent pathway involving activation of JNK1/2 MAP kinases and downstream upregulation of ICAM. We found that phosphorylation of JNK1/2 observed in wt MLEC in response to TNFα was significantly attenuated in Cav−/ − MLEC. In addition, TNFα did not induce upregulation of ICAM expression in caveolin-1 knockout MLEC or wt cells pretreated with DPI. Conclusions: Collectively, these results indicate that caveolin-1 is a necessary component of the molecular machinery for proper subcellular positioning required for cytokine activation of NADPH oxidase and superoxide production in endothelial cells. Thus, caveolin-1 may constitute a potential therapeutic target for altering ROS associated with cardiovascular pathology.

Increasing NaCl Induces Superoxide Production in Macula Densa Cell Line

To study whether the cultured macula densa cell line, MMDD1 shares the same characteristics of superoxide (O2−) production as the isolated, perfused macula densa, we measured the O2− in the cell line. We also used laser capture microdissection (LCM) and PCR to examine which isoforms of NAD(P)H oxidase are expressed in the rat macula densa and compared with the cell line. The cultured cells were loaded with lucigenin (5 μM) and placed in a luminometer for O2− measurement. When NaCl was increased from 10 to 80 mM, O2− production rose from 0.27 ± 0.05 to 0.63 ± 0.08 units/105 cells (p < 0.01 vs 10 mM NaCl). When the NAD(P)H oxidase inhibitor apocynin (10−5 M) was added for 30 min, O2− production was 0.31 ± 0.07 units/105 cells (p < 0.01 vs 10 mM NaCl). With RT-PCR, we found NOX2 and NOX4 are expressed in the cell line. To determine which NOX isoforms are expressed in the macula densa, we performed LCM followed by single-cell-PCR on frozen sections of rat kidney. The macula densa cells were identified by their anatomical location and morphology and dissected with the laser. We confirmed that the captured cells are macula densa by nNOS expression. We found that NOX2 and NOX4 are expressed by the macula densa. In summary, we found that increasing luminal NaCl induces O2− production by the cultured macula densa cells, primarily from NAD(P)H oxidase. Both rat macula densa and the cell line express NOX2 and NOX4 isoforms.

Depolarization of the macula densa induces superoxide production via NAD(P)H oxidase

Superoxide (O(2)(-)) enhances tubuloglomerular feedback by scavenging nitric oxide at the macula densa. However, the singling pathway of O(2)(-) production in the macula densa is not known. We hypothesized that the increase in tubular NaCl concentration that initiates tubuloglomerular feedback induces O(2)(-) production by the macula densa via NAD(P)H oxidase, which is activated by macula densa depolarization. We isolated and microperfused the thick ascending limb of the loop of Henle and attached macula densa in rabbits. A fluorescent dye, dihydroethidium, was used to detect O(2)(-) production at the macula densa. When luminal NaCl was switched from 10 to 80 mM, a situation of initiating maximum tubuloglomerular feedback response, O(2)(-) production significantly increased. To make sure that the shifts in the oxyethidium/dihydroethidium ratio were due to changes in O(2)(-), we used tempol (10(-4) M), a stable membrane-permeant superoxide dismutase mimetic. With tempol present, when we switched from 10 to 80 mM NaCl, the increase in oxyethidium/dihydroethidium ratio was blocked. To determine the source of O(2)(-), we used the NAD(P)H oxidase inhibitor apocynin. When luminal NaCl was switched from 10 to 80 mM in the presence of apocynin, O(2)(-) production was inhibited by 80%. To see whether the effect of increasing luminal NaCl involves Na-K-2Cl cotransporters, we inhibited them with furosemide. When luminal NaCl was switched from 10 to 80 mM in the presence of furosemide, O(2)(-) production was blocked. To test whether depolarization of the macula densa induces O(2)(-) production, we artificially induced depolarization by adding valinomycin (10(-6) M) and 25 mM KCl to the luminal perfusate. Depolarization alone significantly increases O(2)(-) production. We conclude that increasing luminal NaCl induces O(2)(-) production during tubuloglomerular feedback. O(2)(-) generated by the macula densa is primarily derived from NAD(P)H oxidase and is induced by depolarization.

Various Molecular Species of Diacylglycerol Hydroperoxide Activate Human Neutrophils via PKC Activation.

We have proposed that diacylglycerol hydroperoxide-induced unregulated signal transduction causes oxidative stress-related diseases. In this study, we investigated which molecular species of diacylglycerol hydroperoxide activated human peripheral neutrophils. All diacylglycerol hydroperoxides, diacylglycerol hydroxides, and diacyglycerols tested in the present study induced superoxide production by neutrophils. The ability to activate neutrophils among molecular species containing the same fatty acid composition was as follows; diacylglycerol hydroperoxide>diacylglycerol hydroxide≥diacylglycerol. The diacylglycerol hydroperoxide composed of linoleate was a stronger activator for neutrophils than that composed of arachidonate. 1-Palmitoyl-2-linoleoylglycerol hydroperoxide (PLG-OOH) was the strongest stimulator for neutrophils. We reconfirmed that PLG-OOH activated protein kinase C (PKC) in neutrophils. PLG-OOH induced the phosphorylation of p47phox, a substrate of PKC and a cytosolic component of NADPH oxidase, in neutrophils, as did N-formyl-methionyl-leucyl-phenylalanine or 4β-phorbol-12β-myristate-13α-acetate. Moreover, the time course of p47phox phosphorylation was comparable to that of superoxide production. These results suggest that PLG-OOH activated intracellular protein kinase C. PLG-OOH, produced via an uncontrolled process, can act as a biological second messenger to cause inflammatory disease from oxidative stress.

Vasculoprotective Effects of Anti-Tumor Necrosis Factor-α Treatment in Aging

Vascular aging is associated with dysregulation of tumor necrosis factor (TNF)-alpha expression. TNF-alpha is a master regulator of vascular proatherogenic phenotypic changes, and it has been linked to endothelial dysfunction and apoptosis. To test the hypothesis that anti-TNF-alpha treatment exerts vasculoprotective effects in aging, aged (29 months old) F344 rats were treated with etanercept (1 mg/kg/week for 4 weeks), which binds and inactivates TNF-alpha. In aged carotid arteries, relaxations to acetylcholine were decreased, and endothelial O2* production was increased (as shown by dihydroethidine fluorescence measurements). Etanercept treatment significantly improved responses to acetylcholine and decreased vascular NAD(P)H oxidase activity and expression. In aged carotid and coronary arteries, there were increases in DNA fragmentation rate and caspase 3/7 activity (indicating an increased rate of apoptotic cell death), which were attenuated by etanercept treatment. In aged vessels, there was an up-regulation of inflammatory markers, including inducible nitric-oxide synthase and intercellular adhesion molecule-1, which was decreased by etanercept treatment. In carotid arteries of young animals, recombinant TNF-alpha elicited endothelial dysfunction, oxidative stress, and increased apoptosis and proinflammatory gene expression, mimicking many of the symptoms of vascular aging. Thus, we propose that anti-TNF-alpha treatment exerts anti-aging vasculoprotective effects.

Involvement of p38 MAP Kinase in Not Only Activation of the Phagocyte NADPH Oxidase Induced by Formyl-methionyl-leucyl-phenylalanine but Also Determination of the Extent of the Activity

Activated NADPH oxidase in neutrophils produces superoxide. We investigated the role of p38 MAP kinase in activating NADPH oxidase stimulated by the bacteria-derived peptide fMLP. fMLP-stimulated superoxide production was completely abolished by SB203580, a p38 MAP kinase inhibitor, whereas anisomycin, a p38 MAP kinase activator, did not induce superoxide production, indicating that p38 MAP kinase was essential, but not sufficient, for NADPH oxidase activation. Anisomycin pretreatment strongly activated p38 MAP kinase in fMLP-stimulated cells, accompanied by greatly increased superoxide production, suggesting that p38 MAP kinase determines the extent of the fMLP-stimulated NADPH oxidase activity. Furthermore, superoxide production was remarkably reactivated by cytochalasin B addition after fMLP-stimulated production had disappeared, and this was correlated with highly activated p38 MAP kinase. These results suggest that p38 MAP kinase is involved not only in activating NADPH oxidase stimulated by fMLP but also in determining the extent of its activity.

Detection of superoxide anion generation by equine spermatozoa

To identify the generation of the superoxide anion by equine spermatozoa. Multiple ejaculates collected from 3 Thoroughbred stallions. Induced superoxide production by reduced nicotinamide adenine dinucleotides (NAD[P]H; ie, reduced nicotinamide adenine dinucleotide [NADH] and reduced nicotinamide adenine dinucleotide phosphate [NADPH]) was measured by use of a nitroblue tetrazolium (NBT) reduction assay on whole spermatozoa and a cytochrome c reduction assay on isolated membrane fractions of spermatozoa. Localization of superoxide generation was determined by use of NBT cytochemistry. A dose-dependent increase in NBT reduction was found in the presence of NADPH, which was inhibited by superoxide dismutase (SOD). The flavoprotein inhibitor, diphenyleneiodonium (DPI; 5 or 15 microM), significantly decreased NBT reduction. Cytochrome c reduction by plasma membranes of spermatozoa was significantly higher in the presence of NADPH than in its absence. Cytochemical staining of equine spermatozoa in the presence of NADPH and NADH revealed diaphorase labeling in the spermatozoon midpiece and head. This staining was inhibited by DPI and SOD. Results of our study indicate that superoxide generation is associated with a membrane-associated NAD(P)H oxidase present in equine spermatozoa, although mitochondrial generation of superoxide is also detected. This oxidase may play a role in cell signaling or may also contribute to cytopathic effects associated with oxidative stress in equine spermatozoa.

Tocopheryl phosphate, a novel natural form of vitamin E: in vitro and in vivo studies

Tocopheryl phosphate (TP) has been found to be present in a number of tissues as well as foodstuffs. When added to smooth muscle cells in vitro, TP exhibits higher growth inhibition potency relatively to alpha tocopherol (T). The same stronger inhibition is observed in CD36 expression. TP inhibits PMA induced superoxide production in macrophages at lower concentrations than AT. If AT is incubated with digitonin permeabilized smooth muscle cells or macrophages, addition of [32P]ATP results in the synthesis of [32P]-tocopherol. Tocopheryl phosphate has been shown to be hydrolyzed by alkaline phosphatase at a rate of 1/1000 relative to p-nitro-phenyl-phosphate. In a rabbit model of atherosclerosis (2% cholesterol in the diet) TP (0.3–1.33 mg/kg diet) diminishes the amount of the atherosclerotic plaque of more than 60%, while tocopheryl acetate was only about 20% protective. In plasma of animals supplemented with tocopheryl acetate, tocopherol concentration was highly. In animals supplemented with tocopheryl phosphate plasma concentration of tocopherol was low while that of tocopheryl phosphate was high. The hypothesis will be discussed that tocopherol acts at the cellular level after phosphorylation to tocopheryl-phosphate. Such a reaction implies the existence of a kinase and of a phosphatase to dephosphorylate it. A possible interference of T phosphorylation with the phosphorylation of inositol derivatives and a possible molecular mechanism of TP similar to that of phosphatidylinositol phosphates will be also considered.

Synthetic smooth muscle cell phenotype is associated with increased nicotinamide adenine dinucleotide phosphate oxidase activity: Effect on collagen secretion

Smooth muscle cells (SMCs) from prosthetic vascular grafts secrete higher levels of collagen than aortic SMCs under basal conditions and during incubation with oxidized low-density lipoprotein. We postulated that reactive oxygen species (ROS) contributed to the observed difference. The objective of this study was to assess the effect of ROS on collagen secretion by aortic and graft SMCs and explore the mechanism involved. SMCs isolated from canine aorta or Dacron thoracoabdominal grafts were incubated with 6-anilinoquinoline-5,8-quinone (LY83583), an agent that induces superoxide production. Type I collagen in the conditioned medium was measured by enzyme-linked immunosorbent assay, and superoxide anion production was measured by lucigenin assay. LY83583 stimulated a rapid increase in collagen production by graft SMCs that paralleled the LY83583-induced increase in superoxide production. The increase in both collagen and superoxide was greater in graft SMCs than aortic SMCs. Collagen and superoxide production were inhibited by superoxide scavengers. Nicotinamide adenine dinucleotide phosphate (NADPH) induced significantly more superoxide production by graft SMCs than aortic SMCs, suggesting that the NADPH oxidase system was more active in graft SMCs. NADPH oxidase inhibitors blocked the superoxide and collagen production induced by LY83583. In SMCs, the synthetic phenotype is associated with increased NADPH oxidase activity and elevated superoxide production in response to an oxidative stress. Superoxide, in turn, leads to increased collagen production. The inflammatory process after prosthetic vascular graft implantation causes oxidative stress that can stimulate collagen production by graft SMCs, contributing to the progression of intimal hyperplasia. The exaggerated response of graft SMCs to oxidative stress offers a potential target for therapeutic interventions.

Oxidative-Nitrosative Stress and Poly(ADP-Ribose) Polymerase (PARP) Activation in Experimental Diabetic Neuropathy

Poly(ADP-ribose) polymerase (PARP) activation, an important factor in the pathogenesis of diabetes complications, is considered a downstream effector of oxidative-nitrosative stress. However, some recent findings suggest that it is not necessarily the case and that PARP activation may precede and contribute to free radical and oxidant-induced injury. This study evaluated the effect of PARP inhibition on oxidative-nitrosative stress in diabetic peripheral nerve, vasa nervorum, aorta, and high glucose-exposed human Schwann cells. In vivo experiments were performed in control rats and streptozocin (STZ)-induced diabetic rats treated with and without the PARP inhibitor 3-aminobenzamide (ABA) (30 mg . kg(-1) . day(-1) i.p. for 2 weeks after 2 weeks of untreated diabetes). Human Schwann cells (HSC) (passages 7-10; ScienCell Research Labs) were cultured in 5.5 or 30 mmol/l glucose with and without 5 mmol/l ABA. Diabetes-induced increase in peripheral nerve nitrotyrosine immunoreactivity, epineurial vessel superoxide and nitrotyrosine immunoreactivities, and aortic superoxide production was reduced by ABA. PARP-1 (Western blot analysis) was abundantly expressed in HSC, and its expression was not affected by high glucose or ABA treatment. High-glucose-induced superoxide production and overexpression of nitrosylated and poly(ADP-ribosyl)ated protein, chemically reduced amino acid-(4)-hydroxynonenal adducts, and inducible nitric oxide synthase were decreased by ABA. We concluded that PARP activation contributes to superoxide anion radical and peroxynitrite formation in peripheral nerve, vasa nervorum, and aorta of STZ-induced diabetic rats and high- glucose-exposed HSC. The relations between oxidative-nitrosative stress and PARP activation in diabetes are bi- rather than unidirectional, and PARP activation cannot only result from but also lead to free radical and oxidant generation.

Leishmania pifanoiAmastigotes Avoid Macrophage Production of Superoxide by Inducing Heme Degradation

Whereas infections of macrophages by promastigote forms of Leishmania mexicana pifanoi induce the production of superoxide, infections by amastigotes barely induce superoxide production. Several approaches were employed to gain insight into the mechanism by which amastigotes avoid eliciting superoxide production. First, in experiments with nitroblue tetrazolium, we found that 25% of parasitophorous vacuoles (PVs) that harbor promastigotes are positive for the NADPH oxidase complex, in contrast to only 2% of PVs that harbor amastigotes. Second, confocal microscope analyses of infected cells labeled with antibodies to gp91phox revealed that this enzyme subunit is found in PVs that harbor amastigotes. Third, in immunoblots of subcellular fractions enriched with PVs from amastigote-infected cells and probed with antibodies to gp91phox, only the 65-kDa premature form of gp91phox was found. In contrast, subcellular fractions from macrophages that ingested zymosan particles contained both the 91- and 65-kDa forms of gp91phox. This suggested that only the immature form of gp91phox is recruited to PVs that harbor amastigotes. Given that gp91phox maturation is dependent on the availability of heme, we found that infections by Leishmania parasites induce an increase in heme oxygenase 1 (HO-1), the rate-limiting enzyme in heme degradation. Infections by amastigotes performed in the presence of metalloporphyrins, which are inhibitors of HO-1, resulted in superoxide production by infected macrophages. Taken together, we propose that Leishmania amastigotes avoid superoxide production by inducing an increase in heme degradation, which results in blockage of the maturation of gp91phox, which prevents assembly of the NADPH oxidase enzyme complex.

Retinoic acid induces expression of SLP-76: Expression with c-FMS enhances ERK activation and retinoic acid-induced differentiation/G0 arrest of HL-60 cells

Retinoic acid (RA) is known to cause MAPK signaling which propels G0 arrest and myeloid differentiation of HL-60 human myeloblastic leukemia cells. The present studies show that RA up-regulated expression of SLP-76 (Src-homology 2 domain-containing leukocyte-specific phospho-protein of 76 kDa), which became a prominent tyrosine-phosphorylated protein in RA-treated cells. SLP-76 is a known adaptor molecule associated with T-cell receptor and MAPK signaling. To characterize functional effects of SLP-76 expression in RA-induced differentiation and G0 arrest, HL-60 cells were stably transfected with SLP-76. Expression of SLP-76 had no discernable effect on RA-induced ERK activation, subsequent functional differentiation, or the rate of RA-induced G0 arrest. To determine the effects of SLP-76 in the presence of a RA-regulated receptor, SLP-76 was stably transfected into HL-60 cells already overexpressing the colony stimulating factor-1 (CSF-1) receptor, c-FMS, from a previous stable transfection. SLP-76 now enhanced RA-induced ERK activation, compared to parental c-FMS transfectants. It also enhanced RA-induced differentiation, evidenced by enhanced paxillin expression, inducible oxidative metabolism and superoxide production. RA-induced RB tumor suppressor protein hypophosphorylation was also enhanced, as was RA-induced G0 cell cycle arrest. A triple Y to F mutant SLP-76 known to be a dominant negative in T-cell receptor signaling failed to enhance RA-induced paxillin expression, but enhanced RA-induced ERK activation, differentiation and G0 arrest essentially as well as wild-type SLP-76. Thus, SLP-76 overexpression in the presence of c-FMS, a RA-induced receptor, had the effect of enhancing RA-induced cell differentiation. This is the first indication to our knowledge that RA induces the expression of an adapter molecule to facilitate induced differentiation via co-operation between c-FMS and SLP-76.

Role of oxidative stress in angiotensin-II mediated contraction of human conduit arteries in patients with cardiovascular disease

Background Angiotensin II is a powerful vasoconstrictor involved in the development of high blood pressure and in the regulation of cardiovascular growth. Recent reports have suggested that in addition to the classical pathways involved in transducing responses to receptor activation, formation of reactive oxygen species by angiotensin II may also be involved. We investigated the importance of oxidative stress in angiotensin II induced contraction in human conduit arteries from patients with cardiovascular disease. Methods and results Isometric contraction studies using human radial arteries entailed probes modulating the redox-dependent reactions to define the oxidative pathways involved in angiotensin II contraction. In situ oxidative fluorescence was employed to detect immediate superoxide tissue production in radial and internal mammary arteries. Treatment with TEMPOL, human superoxide dismutase, diphenyleneiodonium, oxypurinol, NG-monomethyl l-arginine considerably decreased contractile response to angiotensin II in radial arteries. Similarly, angiotensin II-stimulated arterial superoxide production was reduced in the presence of the above inhibitors. On the contrary, used as controls, norepinephrine vasoconstriction was not associated with increase of superoxide and neither ciprofloxacin nor aminophylline altered basal or angiotensin II induced superoxide generation. Conclusions Our findings provide evidence for the role of oxidative pathways in contractile response of human conduit arteries to angiotensin II. Angiotensin II induced superoxide anion production may be mediated by multiple inter-dependent rate-limiting enzymes in both types of artery. Our studies may have important implication for future therapeutic approaches involving inhibition of angiotensin II mediated superoxide generation in hypertension and prevention of cardiovascular disease. Condensed abstract We studied the role of oxidant species in contraction responses to angiotensin II in human conduit arteries. Treating radial artery segments with the anti-oxidants with a range of inhibitors, affecting the redox dependent pathways, markedly reduced contraction to angiotensin II. In parallel experiments, oxidative fluorescence was assessed and compared in human radial and internal mammary artery. Angiotensin II induced superoxide anion production may be mediated by multiple inter-dependent rate-limiting enzymes in both types of artery.

Rosmarinic acid inhibits the formation of reactive oxygen and nitrogen species in RAW264.7 macrophages

Antioxidant action of Rosmarinic acid (Ros A), a natural phenolic ingredient in many Lamiaceae herbs such as Perilla frutescens, sage, basil and mint, was analyzed in relation to the Iκ-B activation in RAW264.7 macrophages. Ros A inhibited nitric oxide (NO) production and inducible nitric oxide synthase (iNOS) protein synthesis induced by lipopolysaccharide (LPS), and also effectively suppressed phorbol 12-myristate 13-acetate (PMA)-induced superoxide production in RAW264.7 macrophages in a dose-dependent manner. Peroxynitrite-induced formation of 3-nitrotyrosine in bovine serum albumin and RAW264.7 macrophages were also inhibited by Ros A. Moreover, Western blot analysis demonstrated that LPS-induced phosphorylation of Iκ-Bα was abolished by Ros A. Ros A can act as an effective protector against peroxynitrite-mediated damage, and as a potent inhibitor of superoxide and NO synthesis; the inhibition of the formation of reactive oxygen and nitrogen species are partly based on its ability to inhibit the serine phosphorylation of Iκ-Bα.

Evolution of anaphylatoxins, their diversity and novel roles in innate immunity: Insights from the study of fish complement

Anaphylatoxins are small molecules (∼9 kDa) that are generated as a result of the activation of the complement system. These molecules play an important role in inflammation, and they are responsible for the activation of various innate and adaptive immune processes. The study of these important inflammatory molecules has been restricted to mammalian species so far. Recent studies have shown that teleost fish, unlike any other known animal species, contain multiple forms of the C3a anaphylatoxin, all of which are functionally active and play a prominent role in inducing superoxide production in fish leukocytes. The C5a anaphylatoxin has also been characterized in these animals, and like in mammals, it plays an important role in leukocyte chemotaxis and in triggering the respiratory burst of leukocytes. Interestingly, it has been shown that rainbow trout anaphylatoxins play an unexpected role in enhancing phagocytosis of particles. C5a and C3a receptors have recently been cloned and characterized in rainbow trout, suggesting that the duplication of these receptors from a common ancestor occurred before the emergence of teleosts. The studies derived from these molecules in teleost fish indicate that the basic structure and function of anaphylatoxins and their receptors, have been conserved for more than 300 million years.

The Synthetic Chemoattractant Peptide WKYMVm Induces Superoxide Production by Human Eosinophils via the Phosphoinositide 3-Kinase-Mediated Activation of ERK1/2

Background: Eosinophils play a key role in allergic inflammation and parasitic infections. The synthetic peptide, Trp-Lys-Tyr-Met-Val-D-Met (WKYMVm), has been previously shown to activate eosinophils and thus to enhance respiratory burst through the formyl peptide receptors. Objective: This study was undertaken to determine the intracellular signaling pathway involved in WKYMVm-stimulated superoxide production by human eosinophils. Methods: Purified eosinophils from peripheral blood were stimulated with various concentrations (10^–3 to 10 µM) of WKYMVm and the involvement of PI3-kinase and MAP kinases in WKYMVm-triggered superoxide production was investigated using pharmacological inhibitors. Results: WKYMVm-induced superoxide production by eosinophils was strongly inhibited by pretreatment with the PI3-kinase inhibitor LY294002. In addition, pretreatment with the ERK1/2 kinase inhibitor PD98059 resulted in marked inhibition of superoxide production induced by WKYMVm. Indeed, WKYMVm strongly induced phosphorylation of ERK1/2. The ERK1/2 activation by the peptide was transient and peaked after 2 min of stimulation. Furthermore, ERK1/2 activation by WKYMVm was completely inhibited by pretreatment with the PI3-kinase inhibitor LY294002, but not by the PKC inhibitor Ro-31-8220. Conclusion: These results suggest that WKYMVm stimulates human eosinophils to induce superoxide production via a PI3-kinase-mediated ERK1/2 pathway.