Are Free Radicals Involved in the Pathobiology of Human Essential Hypertension?

Possible involvement of reactive oxygen species and nitric oxide in the pathogenesis of human essential hypertension was investigated. It was observed that both superoxide anion and hydrogen peroxide production by polymorphonuclear leukocytes and the plasma levels of lipid peroxides are higher in uncontrolled essential hypertension compared with normal controls. Nitric oxide levels measured as its stable metabolite nitrite, as an index of nitric oxide synthesis, revealed its levels to be low in hypertensive patients. Superoxide anion, hydrogen peroxide, lipid peroxides and nitric oxide levels reverted to normal values after the control of hypertension by drugs. The concentrations of anti-oxidants such as vitamin E and superoxide dismutase were found to be decreased in patients with uncontrolled hypertension. Several anti-hypertensive drugs inhibited lipid peroxidation in vitro. Angiotensin-II, a potent vasoconstrictor, stimulated free radical generation in normal leukocytes which could be blocked by calmodulin antagonists. These results suggest that an increase in free radical generation and a simultaneous decrease in the production of nitric oxide and anti-oxidants such as SOD and vitamin E occurs in essential hypertension. This increase in free radical generation can inactivate prostacyclin and nitric oxide and decrease their half life which can lead to an increase in peripheral vascular resistance and hypertension.     

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes.

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

ESR studies on active oxygen radicals produced in the respiratory burst of human polymorphonuclear leukocytes

The mechanism and process of production of active oxygen radicals in the respiratory burst of polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate) was studied in this paper. The experimental results indicate that when the PMA was dilute enough or at the beginning of stimulation even when the PMA concentration was high, the spectrum of hydroxyl radical spin adducts, DMPO-OH, was dominant in the ESR spectra. However, at the maximum level of the respiratory burst, the spectrum of superoxide anion spin adducts, DMPO-OOH, was dominant.     

Dipyridamole inhibits hydroxylamine augmented nitric oxide (NO) production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism

Polymorphonuclear neutrophils (PMN) are thought to play a role in reperfusion injury and ischemia. These effects are partly mediated by toxic oxygen species (superoxide anion, hydrogen peroxide and hydroxyl radical) acting at the level of the endothelium. It was demonstrated recently that the superoxide anion reacts with nitric oxide (NO) and that interaction leads to the generation of highly toxic peroxynitrite. Several drugs were tested so far in order to affect PMN function. It was demonstrated that dipyridamole (2,6-bis-diethanolamino-4,8-dipiperidinopyrimido-(5,4-d)-pyrimidine) can influence neutrophil function by inhibiting adenosine uptake. However, this action can not fully explain all of the observed effects of dipyridamole action on PMN metabolism. The aim of our study was to evaluate the influence of dipyridamole on nitric oxide production by activated polymorphonuclear neutrophils. Incubation of PMNs with hydroxylamine (HA) and phorbol myristate acetate (PMA) generated nitrite (36.4+/-4.2 nmol/h 2x10(6) PMN), dipyridamole at 100 micromol/l, 50 micromol/l and 10 micromol/l caused a considerable drop in nitrite production (11.8+/-1.8, 19.7+/-2.7 and 27.4+/-3.2 nmol/h, respectively). Neither adenosine nor the adenosine analogue could mimic the dipyridamole effect. Moreover theophylline, an adenosine inhibitor could not reverse the dipirydamole action on PMN metabolism. We also found that dipyridamole inhibited hydrogen peroxide release from neutrophils. Catalase that scavenges hydrogen peroxide also largely abolished nitric oxide release from PMN. It is evident that dipyridamole inhibits hydroxylamine-augmented nitric oxide production by activated polymorphonuclear neutrophils through an adenosine-independent mechanism.     

The superoxide anion and singlet molecular oxygen: their role in the microbicidal activity of the polymorphonuclear leukocyte

Superoxide dismutase was found to partially inhibit both chemiluminescence and nitroblue tetrazolium (NBT) reduction from intact human polymorphonuclear leukocytes. This capacity to reduce NBT was lost when the polymorphonuclear leukocytes were sonicated, but could be regained if exogenous NADPH (or NADH) was added to the system. Superoxide dismutase was found to inhibit this NADPH- and NADH-dependent NBT reduction. A mechanism is proposed that relates superoxide anion generation to the univalent reduction of O₂ by the activated NADPH (and NADH) oxidase. The relationship of superoxide anion production to NBT reduction, singlet molecular oxygen generation, and chemiluminescence is discussed.     

Effects of estradiol, progesterone and testosterone on the function of carp, Cyprinus carpio, phagocytes in vitro

The modulation of phagocytic cells by beta-estradiol, 11-ketotestosterone and progesterone was analyzed in common carp Cyprinus carpio. Carp kidney leukocytes were cultured in RPMI 1640 medium containing 0.1, 1, 10, 100 or 1000 nM concentration of each hormone. The production of superoxide anion, nitric oxide (NO) and phagocytosis were measured in vitro. Similar concentrations of cortisol were used as control. Phagocytic activities of carp macrophages was suppressed by treatment with beta-estradiol, progesterone and 11-ketotestosterone. The production of NO in carp macrophages was suppressed by progesterone and 11-ketotestosterone. However, carp macrophages incubated with beta-estradiol, progesterone and 11-ketotestosterone did not show any difference in the production of superoxide anion in comparison with control macrophages in the absence of hormones. Carp macrophages treated with cortisol suppressed phagocytosis and the production of nitric oxide and superoxide anion.     

Oxygen-derived radicals from Trypanosoma cruzi-stimulated human neutrophils

This study provides biochemical and electron spin resonance spectroscopic evidence that contract of human polymorphonuclear leukocytes with antibody-coated Trypanosoma cruzi triggers the respiratory burst. Oxygen consumption, superoxide anion and hydrogen peroxide release were stimulated under conditions of polymorphonuclear leukocyte-mediated killing. This stimulation did not occur under non-killing conditions when antibody was omitted. A common mechanism of cytotoxicity of human polymorphonuclear leukocytes against different T. cruzi forms is suggested by the triggering of the respiratory burst by antibody-coated epimastigotes and trypomastigotes.     

Effect of resveratrol on some activities of isolated and in whole blood human neutrophils

Resveratrol, which is a polyphenol present in red wines and vegetables included in human diets, exerts many biological effects. The aim of the present study was to investigate its effect on some activities of polymorphonuclear leukocytes, particularly the generation of superoxide anion ((O2)(-)) in whole blood, hypochlorous acid (HOCl) and nitric oxide (NO) production by isolated cells, and chemotaxis. Resveratrol showed significant dose-dependent inhibitory effect on all these activities. In particular, it inhibited O2(-) generation in stimulated but not in resting neutrophils, decreased HOCl much more than O2(-) production indicating an effect on myeloperoxidase secretion since HOCl production is directly and proportionally dependent on O2(-) generation and reduced cell motility. The small dose of resveratrol (4.38 nM) used is attainable with a diet including red wine and vegetables confirming its protective role against some pathological processes such as inflammation, coronary heart disease, and cancer.     

Scavenging effect of schizandrins on active oxygen radicals

The reactive oxygen radicals produced from human polymorphonuclear leukocytes (PMN) stimulated with PMA (phorbol myristate acetate), hydroxyl radicals generated by a Fenton reaction, and superoxide anion radicals produced by irradiating solutions of riboflavin in the presence of EDTA have been taken as the models for production of oxygen radicals. With the use of the electron spin resonance spin trapping method, the scavenging effects of schizandrol A (solA) (5 x 10(-4) M) and schizandrin B (sinB) (5 x 10(-4) M) have been studied and compared with the effects of vitamin E (5 x 10(-4) M) and vitamin C (5 x 10(-4) M). It has been found that in cell system the scavenging effects of sinB and solA, as judged by ESR spin trappings, on hydrpxyl radicals (.OH) are greater than vitamin E and vitamin C and the scavenging effects on superoxide anion (O2) are greater than vitamin E but lower than vitamin C. With respect to the Fenton reaction, sinB has the strogest scavenging effect on .OH (77%) and solA has strong scavenging effect on .OH (63%), both of them larger than that of vitamin E (35%) and vitamin C (56%). In the riboflavin/EDTA system, the scavenging effect of sinB (46%) is smaller than that of vitamin C (96%) but larger than that of vitamin E (23%); the scavenging effect of solA is not obvious (14%). With the use of spin probe oximetry, the oxygen consumption during the respiratory burst of stimulated PMN has been measured when exposed to schizandrins. The experiment results demonstrated that they do not affect the activity of production of active oxygen radicals in the respiratory burst of PMN stimulated with PMA.     

Lymphocytes can produce respiratory burst and oxygen radicals as polymorphonuclear leukocytes

The respiratory burst and production of oxygen radicals by lymphocytes stimulated with phorbol myristate acetate (PMA) was studied and compared with that of polymorphonuclear leukocytes (PMN) by electron paramagnetic resonance (EPR) and spin trapping technique. Superoxide anion and hydroxyl radicals spin adducts of DMPO were detected in the stimulated PMN system, but only hydroxyl radical spin adducts of DMPO were detected in the stimulated lymphocyte system. It was proved by Superoxide dismutase (SOD) and catalase that the hydroxyl radicals produced in the stimulated lymphocyte system came from Superoxide anions, just like the hydroxyl radicals produced in the stimulated PMN.     

Effect of stobadine on oxygen free radical generation in stimulated human polymorphonuclear leukocytes

The generation both superoxide and a mixture of reactive oxygen species was recorded in a suspension of human polymorphonuclear leukocytes stimulated with phorbol myristate acetate. While stobadine dose-dependently decreased chemiluminescence, only its highest concentration used reduced significantly superoxide generation. The results suggest that stobadine is a more effective scavenger of free radicals rather than a quencher of superoxide anion.     

Free radical scavenging action of the medicinal herb Limonium wrightii from the Okinawa islands

Free radical scavenging action of Limonium wrightii O. kunthe was examined in vitro and in vivo by using electron spin resonance spectrometer and chemiluminescence analyzer. A water extract of L. wrightii showed a strong scavenging action for the 1,1-diphenyl-2-picrylhydrazyl, or superoxide anion and moderate for hydroxyl radical. The extract also depressed production of reactive oxygen species from polymorphonuclear leukocytes stimulated by phorbor-12-mysistate acetate and inhibited lipid peroxidation in rat liver microsomes. When the extract was given intraperitoneally to mice prior to carbon tetrachloride (CCl4) treatment, CCl4-induced liver toxicity, as seen by an elevation of serum aspartate aminotransferase and alanine aminotransferase activities, was significantly reduced. Gallic acid was identified as the active component of L. wrightii with a strong free radical scavenging action. Our results demonstrate the free radical scavenging action of L. wrightii and that gallic acid contributes to these actions.     

Changes in protein phosphorylation in guinea pig polymorphonuclear leukocytes by treatment with membrane-perturbing agents which stimulate superoxide anion production

Phosphorylation of proteins was examined in guinea pig polymorphonuclear leukocytes in relation to the effects of membrane-perturbing agents, which stimulate superoxide anion production, and their inhibitors. The phosphorylation was detected by 32P autoradiography after separation by two-dimensional electrophoresis of proteins phosphorylated in 32P-preloaded cells. Though phosphorylation of various proteins was stimulated by each of the membrane-perturbing agents, the stimulation was especially marked in six proteins. Phorbol myristate acetate and digitonin enhanced the phosphorylation of the six proteins, while myristate and concanavalin A increased the phosphorylation of five and three proteins, respectively, out of the six proteins. p-Bromophenacyl bromide, an inhibitor of phospholipase A2, inhibited the stimulatory effect of phorbol myristate acetate on both superoxide anion production and protein phosphorylation. Trifluoperazine, a calmodulin inhibitor, also inhibited the effect of phorbol myristate acetate on both, except for an increase in the phosphorylation of one out of the six proteins. alpha-Methylmannoside, an inhibitor of concanavalin A binding, inhibited the stimulation of the phosphorylation of the three proteins by concanavalin A. The results indicate that the activation of superoxide anion production by the membrane-perturbing agents in guinea pig polymorphonuclear leukocytes is accompanied by the phosphorylation of, at least some of, these six proteins.     

Inhibition of superoxide generation from fMLP-stimulated leukocytes by high concentrations of nitric oxide or peroxynitrite: characterization by electron spin resonance spectroscopy

This present study examined the effects of high concentrations of nitric oxide (NO*) and peroxynitrite (ONOO-) on superoxide (O2*-) production from formyl-methionyl-leucyl-phenylalanine (fMLP)-stimulated polymorphonuclear leukocytes (PMNs) by using electron spin resonance (ESR) and spin trapping with 5-(diethoxyphosphoryl)-5-methyl-1-pyrroline-N-oxide (DEPMPO). We demonstrated that ONOO- (100 microM) decreased the ESR signal of DEPMPO-OOH from fMLP-activated PMNs, indicating the inhibition of O2*- generation, while it enhanced the signal of DEPMPO-OH. Inhibition of the respiratory burst was also observed when PMNs were pre-exposed to high concentrations of NO* (100 microM), generated by the NO* donor NOR-1, 30 min prior to stimulation with fMLP. NOR-1 inhibited O2*- generation more effectively under conditions in which ONOO-was formed concurrently. The ability of high concentrations of either ONOO- or NO* to inhibit O2*-generation from fMLP-stimulated PMNs is relevant to pathophysiological conditions, such as severe inflammation, in which NO* or ONOO- production can be significantly elevated.     

Production and interaction of oxygen and nitric oxide free radicals in PMA stimulated macrophages during the respiratory burst

The activity of nitric oxide synthase (NOS) during the respiratory burst in phorbol-1,2-myristate-1,3-acetate (PMA) stimulated macrophages has been the topic of much debate in the literature. To help clarify the role of NOS, we have examined the chemiluminescence arising from peroxynitrite production, nitrite/nitrate and nitric oxide production, and oxygen consumption during the respiratory burst in PMA-stimulated macrophages. The Griess reaction was used to measure nitrite/nitrate, spin trapping with N-methyl D-glucamine dithiocarbamate (MGD)2-Fe2+ was used to quantify nitric oxide, and the spin probe 2,2,6,6-tetramethylpiperidine-N-oxyl-4-ol (TEMPOL) was used to measure oxygen consumption. Oxygen free radical production (hydroxyl and superoxide free radicals) was also investigated using the spin trap 5,5-dimethyl-1-pyroline-1-oxide (DMPO). The chemiluminescence emitted by the PMA-stimulated macrophages and nitrite/nitrate in the culture system were both found to increase. However, the rate of nitric oxide release remained constant, indicating that the activity of NOS is not enhanced during the respiratory burst in PMA stimulated macrophages.     

Increased peroxynitrite activity in AIDS dementia complex: implications for the neuropathogenesis of HIV-1 infection

Oxidative stress is suggested to be involved in several neurodegenerative diseases. One mechanism of oxidative damage is mediated by peroxynitrite, a neurotoxic reaction product of superoxide anion and nitric oxide. Expression of two cytokines and two key enzymes that are indicative of the presence of reactive oxygen intermediates and peroxynitrite was investigated in brain tissue of AIDS patients with and without AIDS dementia complex and HIV-seronegative controls. RNA expression of IL-1beta, IL-10, inducible nitric oxide synthase, and superoxide dismutase (SOD) was found to be significantly higher in demented compared with nondemented patients. Immunohistochemical analysis showed that SOD was expressed in CD68-positive microglial cells while inducible nitric oxide synthase was detected in glial fibrillary acidic protein (GFAP)-positive astrocytes and in equal amounts in microglial cells. Approximately 70% of the HIV p24-Ag-positive macrophages did express SOD, suggesting a direct HIV-induced intracellular event. HIV-1 infection of macrophages resulted in both increased superoxide anion production and elevated SOD mRNA levels, compared with uninfected macrophages. Finally, we show that nitrotyrosine, the footprint of peroxynitrite, was found more intense and frequent in brain sections of demented patients compared with nondemented patients. These results indicate that, as a result of simultaneous production of superoxide anion and nitric oxide, peroxynitrite may contribute to the neuropathogenesis of HIV-1 infection.     

Nucleotide receptor signalling and the generation of reactive oxygen species

Elevated levels of extracellular nucleotides are present at sites of inflammation, platelet degranulation and cellular damage or lysis. These extracellular nucleotides can lead to the activation of purinergic (nucleotide) receptors on various leukocytes, including monocytes, macrophages, eosinophils, and neutrophils. In turn, nucleotide receptor activation has been linked to increased cellular production and release of multiple inflammatory mediators, including superoxide anion, nitric oxide and other reactive oxygen species (ROS). In the present review, we will summarize the evidence that extracellular nucleotides can facilitate the generation of multiple ROS by leukocytes. In addition, we will discuss several potential mechanisms by which nucleotide-enhanced ROS production may occur. Delineation of these mechanisms is important for understanding the processes associated with nucleotide-induced antimicrobial activities, cell signalling, apoptosis, and pathology. This work was supported by National Institutes of Health Grants HL56396 and AI50500. The first author was supported by the Hematology Training Program NIH 5 T32 HL07899 at the University of Wisconsin.     

Fc-receptor-mediated intracellular delivery of Cu/Zn-superoxide dismutase (SOD1) protects against redox-induced apoptosis through a nitric oxide dependent mechanism

BACKGROUND: Using specific antibodies against bovine Cu/Zn-superoxide dismutase (EC 1.15.1.1, SOD1) we demonstrated that anti-SOD antibodies (IgG1) are able to promote the intracellular translocation of the antioxidant enzyme. The transduction signalling mediated by IgG1 immune complexes are known to promote a concomitant production of superoxide and nitric oxide leading to the production of peroxynitrites and cell death by apoptosis. The Fc-mediated intracellular delivery of SOD1 thus limited the endogenous production of superoxide. It was thus of interest to confirm that in the absence of superoxide anion, the production of nitric oxide protected cells against apoptosis. Study in greater detail clearly stated that under superoxide anion-free conditions, nitric oxide promoted the cell antioxidant armature and thus protected cells against redox-induced apoptosis. MATERIALS AND METHODS: The murine macrophage cell-lines J774 A1 were preactivated or not with interferon-gamma and were then stimulated by IgG1 immune complexes (IC), free SOD1 or SOD1 IC and superoxide anion, nitric oxide, peroxynitrite, and tumor necrosis factor-alpha (TNF-alpha) production was evaluated. The redox consequences of these activation processes were also evaluated on mitochondrial respiration and apoptosis as well as on the controlled expression of the cellular antioxidant armature. RESULTS: We demonstrated that SOD1 IC induced a Fcgamma receptor (FcgammaR)-dependent intracellular delivery of the antioxidant enzyme in IFN-gamma activated murine macrophages (the J774 AI cell line). The concomitant stimulation of the FcyR and the translocation of the SOD1 in the cytoplasm of IFN-gamma-activated macrophages not only reduced the production of superoxide anion but also induced the expression of the inducible form of nitric oxide synthase (iNOS) and the related NO production. This inducing effect in the absence of superoxide anion production reduced mitochondrial damages and cell death by apoptosis and promoted the intracellular antioxidant armature. CONCLUSIONS: To define the pharmacologic mechanism of action of bovine SOD1, we attempted to identify the second messengers that are induced by SOD1 IC. In this work, we propose that Fc-mediated intracellular delivery of the SOD1 that reduced the production of superoxide anion and of peroxynitrite, promoted a NO-induced protective effect in inducing the antioxidant armature of the cells. Taken together, these data suggested that specific immune responses against antigenic SOD1 could promote the pharmacological properties of the antioxidant enzyme likely via a NO-dependent mechanism.     

Oxidation of tetrahydrobiopterin by peroxynitrite: implications for vascular endothelial function.

Subsaturating levels of tetrahydrobiopterin (BH(4)), an essential cofactor for nitric oxide synthase (NOS), can lead to endothelial dysfunction as a result of decreased production of nitric oxide. Furthermore, insufficient BH(4) can also result in NOS-uncoupled production of reactive oxygen intermediates, such as superoxide anion and hydrogen peroxide. Nitric oxide and superoxide react rapidly to form peroxynitrite, which may be the reactive species responsible for many of the toxic effects of nitric oxide. Here we show that BH(4) is a primary target for peroxynitrite-catalyzed oxidation because at pH 7.4, physiologically relevant concentrations of BH(4) are oxidized rapidly by low concentrations of peroxynitrite. Peroxynitrite oxidizes BH(4) to quinonoid 5,6-dihydrobiopterin and a large proportion of the quinonoid isomer readily loses its side chain to form 7,8-dihydropterin which is not a cofactor for nitric oxide synthase. Thus, abnormally low levels of BH(4) can promote a cycle of its own destruction mediated by nitric oxide synthase-dependent formation of peroxynitrite. This mechanism might contribute to vascular endothelial dysfunction induced by oxidative stress.     

Analysis of the pathways of nitric oxide utilization in mitochondria

The regulatory role that mitochondria play in cell dysfunction and cell-death pathways involves the concept of a complex and multisite regulation of cellular respiration and energy production signaled by cellular and intercellular messengers. Hence, the role of nitric oxide, as a physiological regulator acting directly on the mitochondrial respiratory chain acquires further relevance. This article provides a survey of the major regulatory roles of nitric oxide on mitochondrial functions as an expression of two major metabolic pathways for nitric oxide consumption: a reductive pathway, involving mitochondrial ubiquinol and yielding nitroxyl anion and an oxidative pathway involving superoxide anion and yielding peroxynitrite. The modulation of the decay pathways for nitrogen-and oxygen-centered radicals is further analyzed as a function of the redox transitions of mitochondrial ubiquinol. The interplay among these redox processes and its implications for mitochondrial function is discussed in terms of the mitochondrial steady-state levels (and gradients) of nitric oxide and superoxide anion.