A critical investigation of NADPH oxidase activity in human spermatozoa

It has been suggested that human spermatozoa contain an NADPH oxidase that could generate reactive oxygen species involved in signalling pathways to promote fertility. The proposal depends on observations that the addition of NADPH to purified human spermatozoa stimulates chemiluminescence by the superoxide (O2-) probe, lucigenin. We confirmed these observations, but demonstrated that lucigenin increases NADPH consumption by spermatozoa and stimulates artefactual O2- production via a diphenyleneiodonium (DPI) sensitive flavoprotein. In the absence of cytochrome c, DPI-inhibitable NADPH oxidation by permeabilized spermatozoa was 8 times too small to account for the rate of NADPH-stimulated cytochrome c reduction. Thus NADPH can directly reduce cytochrome c by a flavoprotein dependent mechanism making this O2- assay also unreliable in sperm suspensions. We were unable to observe O2- production by 40 x 10(6) spermatozoa/ml using electron paramagnetic resonance spectroscopy but could identify O(2)(-) generation from 2000 4beta-phorbol-12-myristate-13-actetate (PMA)-stimulated leukocytes. Using spectrophotometry, we did not detect the reduced cytochrome b(558) component of the neutrophil NADPH oxidase in human spermatozoa. No hydrogen peroxide generation was observed using a sensitive Amplex Red assay. We conclude that human spermatozoa do not possess significant NADPH oxidase activity and that the mechanism by which NADPH promotes capacitation must be re-evaluated.     

Prostasomes inhibit the NADPH oxidase activity of human neutrophils

Prostasomes are particular lipid vesicles secreted by the prostate in human semen and involved in several physiological functions such as the improvement of sperm motility or immunomodulation. We have previously shown that they reduced the overall reactive oxygen species (ROS) production of seminal polymorphonuclear neutrophils (PMN). The present study was conducted to define the mechanism by which prostasomes inhibit the ROS production of blood and seminal PMN. The luminol chemiluminescence measuring total ROS production of blood PMN stimulated by either a phorbol ester (PMA) or a chemoattractant peptide, formyl-Met-Leu-Phe (fMLP) was significantly inhibited by prostasomes. The NADPH oxidase activity of the PMN was measured by 2-methyl-6-(p-methoxyphenyl)-3,7-dihydroimidazo[1, 2-a]pyrazin-3-one (MCLA) chemiluminescence. Prostasomes inhibited the NADPH oxidase activity of blood or seminal PMN and increased the lag-phase of the enzyme after PMA stimulation. Prostasomes also inhibited significantly the NADPH oxidase activity of fMLP stimulated blood PMN, but the inhibition was not significant for seminal PMN. The lipid composition of blood PMN was analysed and compared to the lipid composition of prostasomes. This showed that prostasomes had a high cholesterol:phospholipid molar ratio and a high proportion of sphingomyelin. Together with the fact that prostasomes can rigidify the plasma membrane of blood PMN, these results led us to postulate that prostasomes inhibit the NADPH oxidase activity of PMN by lipid transfer from the prostasomes to the plasma membrane of the PMN.     

The pH dependence of NADPH oxidase in human eosinophils

NADPH oxidase generates reactive oxygen species that are essential to innate immunity against microbes. Like most enzymes, it is sensitive to pH, although the relative importance of pH_o and pH_i has not been clearly distinguished. We have taken advantage of the electrogenic nature of NADPH oxidase to determine its pH dependence in patch-clamped individual human eosinophils using the electron current to indicate enzyme activity. Electron current stimulated by PMA (phorbol myristate acetate) was recorded in both perforated-patch configuration, using an NH₄₊ gradient to control pH_i, and in excised, inside-out patches of membrane. No electron current was detected in cells or excised patches from eosinophils from a patient with chronic granulomatous disease. When the pH was varied symmetrically (pH_o= pH_i) in cells in perforated-patch configuration, NADPH oxidase-generated electron current was maximal at pH 7.5, decreasing drastically at higher or lower values. Varying pH_o and pH_i independently revealed that this pH dependence was entirely due to effects of pH_i and that the oxidase is insensitive to pH_o. Surprisingly, the electron current in inside-out patches of membrane was only weakly sensitive to pH_i, indicating that the enzyme turnover rate per se is not strongly pH dependent. The most likely interpretation is that assembly or deactivation of the NADPH oxidase complex has one or more pH-sensitive steps, and that pH-dependent changes in electron current in intact cells mainly reflect different numbers of active complexes at different pH.     

Taxol induces oxidative neuronal cell death by enhancing the activity of NADPH oxidase in mouse cortical cultures

We examined the involvement of oxidative stress in neuronal cell death induced by taxol, a microtubule-stabilizing anti-cancer drug and investigated whether NADPH oxidase plays a role in taxol-induced neuronal cell death in mouse cortical cultures. Cell death was assessed by measuring lactate dehydrogenase in the bathing media after 24-h exposure to taxol. Taxol (30-1000nM) induced the concentration-dependent neuronal death with apoptotic features. The neuronal death induced by taxol was significantly attenuated not only by anti-apoptotic drugs such as z-VAD-fmk and cycloheximide but also by antioxidants such as trolox, ascorbic acid and tempol. Vinblastine, a microtubule-depolymerizing anti-cancer drug, also induced neuronal death. The neuronal cell death induced by vinblastine was also attenuated by z-VAD-fmk, but not by antioxidants and NADPH oxidase inhibitors. Exposure the cortical cultures to taxol for 80min formed neurite beadings visualized by fluorescence immunocytochemistry for tubulin. Treatment with either trolox or apocynin, an NADPH oxidase inhibitor, did not affect formation of the neurite beadings. RT-PCR and Western blot analysis revealed that exposure to taxol increased the expression of p47(phox) and gp91(phox) and induced translocation of the p47(phox) to the membrane in cortical cultures. Exposure to taxol markedly increased cellular 2,7-dichlorofluorescin diacetate fluorescence, an indicator for reactive oxygen species. Apocynin and trolox markedly inhibited the taxol-induced increase of the fluorescence. Moreover, treatment with NADPH oxidase inhibitors or suppression of gp91(phox) by siRNA significantly attenuated the taxol-induced neuronal death. These results indicate that taxol induces oxidative neuronal apoptosis by enhancing the activity of NADPH oxidase.     

The role of the NADPH oxidase complex, p38 MAPK, and Akt in regulating human monocyte/macrophage survival

M-CSF induces PI 3-kinase activation, resulting in reactive oxygen species (ROS) production. Previously, we reported that ROS mediate macrophage colony-stimulating factor (M-CSF)-induced extracellular regulated kinase (Erk) activation and monocyte survival. In this work, we hypothesized that M-CSF-stimulated ROS products modulated Akt1 and p38 activation. Furthermore, we sought to clarify the source of these ROS and the role of ROS and Akt in monocyte/macrophage survival. Macrophages from p47(phox-/-) mice, lacking a key component of the NADPH oxidase complex required for ROS generation, had reduced cell survival and Akt1 and p38 mitogen-activated protein kinase (MAPK) phosphorylation compared with wild-type macrophages in response to M-CSF stimulation, but had no difference in M-CSF-stimulated Erk. To understand how ROS affected monocyte survival and signaling, we observed that NAC and DPI decreased cell survival and Akt1 and p38 MAPK phosphorylation. Using bone marrow-derived macrophages from mice expressing constitutively activated Akt1 (Myr-Akt1) or transfecting Myr-Akt1 constructs into human peripheral monocytes, we concluded that Akt is a positive regulator of monocyte survival. Moreover, the p38 MAPK inhibitor, SB203580, inhibited p38 activity and M-CSF-induced monocyte survival. These findings demonstrate that ROS generated from the NADPH oxidase complex contribute to monocyte/macrophage survival induced by M-CSF via regulation of Akt and p38 MAPK.     

Chemoattractant-induced NADPH oxidase activity in human monocytes is terminated without any association of receptor-ligand complex to cytoskeleton

When the chemotactic peptide formylmethionyl-leucyl-phenylalanine binds to its cell surface receptor, a transmembrane signal is generated that activates the superoxide-producing NADPH oxidase of human phagocytes. Comparing monocytes and neutrophils with regard to the production of superoxide anion induced by the peptide, we found a similar time-course for both types of cells. In neutrophils, ligand binding induced a conversion of the receptor to a high-affinity form, a change suggested to be due to an association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton. This event has been hypothesized to terminate the signal that activates the NADPH oxidase and thereby results in cessation of the cellular production of superoxide anion. Neutrophils preincubated with the cytoskeleton-disrupting drug cytochalasin B showed an increased and prolonged superoxide anion production after activation with the peptide, thus indicating that the cytoskeleton is involved in terminating this response. Formylmethionyl-leucyl-phenylalanine was also found to induce polymerization of actin in monocytes; however, cytochalasin B had no effect on the peptide-induced generation of superoxide anion in these cells. Furthermore, also in monocytes, ligand binding induced a conversion of the receptor to a high-affinity form; however, the receptor-ligand complex did not coisolate with the Triton X-100-insoluble cytoskeleton. These results indicate that, in monocytes, the NADPH oxidase activating pathway is terminated without any association of the receptor-ligand complex to the Triton X-100-insoluble cytoskeleton.     

Selective enrichment of NADPH oxidase activity in phagosomes from guinea pig polymorphonuclear leukocytes

Recent studies have demonstrated that the activated NADPH oxidase, the enzyme responsible for the stimulation of O₂ consumption with O ₂ ^– formation during phagocytosis, is located in the plasma membrane of leukocytes. The present work deals with whether the activation induced by phagocytosis involves the enzyme of the entire membrane or only that of the portion of the membrane that interacts with the phagocytosable particle and forms the phagosome. The results presented show that the activity of the NADPH oxidase of phagosomal membrane, isolated by centrifugation of homogenates on discontinuous sucrose gradients, is increased 12.6-fold with respect that of homogenate. In contrast, the activities of 5-nucleotidase and of acidp-nitrophenyl phosphatase, enzyme markers of the plasma membrane not activated during phagocytosis and uniformly distributed on the entire membrane, are increased only about threefold with respect to that of homogenate. These results indicate that during phagocytosis the activation of NADPH oxidase is a segmentary response that involves only the enzyme that forms the phagocytic vacuole. This fact is relevant for the function of toxic intermediates of oxygen reduction that are discharged in direct contact with the engulfed agent.     

NADPH氧化酶NOX家族与疾病的关系

还原型烟酰胺腺嘌呤二核苷酸磷酸(nicotinamide adenine dinucleotide phosphate, NADPH)氧化酶的非吞噬细胞氧化酶(non-phagocytic cell oxidase, NOX)家族是许多非吞噬细胞中活性氧(reactive oxygen species, ROS)的主要来源.正常状态下,通过该途径产生的ROS作为信号分子参与了细胞分化、增殖、凋亡等的调节,但在环境胁迫下,NOX蛋白家族在感受细胞外信息刺激时,能够迅速活化产生过量的ROS,引起的氧化压力会诱导机体多种疾病的发生、发展.本文主要从NADPH氧化酶NOX家族蛋白的结构、活化、功能及与疾病发生、发展的关系等方面进行简述.     

Regulation of the NADPH oxidase activity and anti-microbial function of neutrophils by arachidonic acid

Arachidonic acid (AA), a second-messenger molecule released from membrane phospholipids by phospholipase A₂ in activated cells, is a stimulator of neutrophil responses, including the oxygen-dependent respiratory burst. The polyunsaturated fatty acid is also the precursor of biologically active eicosanoids. There are several mechanisms by which AA stimulates the respiratory burst. These include the direct binding of AA to S100 proteins which regulate the assembly of the NADPH oxidase as well as the activation of key signaling molecules which control the respiratory burst. Arachidonic acid also stimulates it own release from membrane phospholipids and this contributes to optimal respiratory burst activity. Thus, increased levels of AA at sites of inflammation will influence the magnitude and course of the inflammatory response, not only by directly affecting the function of infiltrating neutrophils and other leukocytes, but also through its metabolites generated by lipoxygenases and cyclooxygenases.     

NADPH氧化酶家族与高血压血管重构

高血压及其并发症的发生、发展与血管重构密切相关。高血压过程中血管重构的类型、特点以及发生机制已成为高血压研究领域的热点之一。高血压血管重构的机制尚未完全明了,研究表明活性氧诱导的氧化应激参与了高血压血管重构的各个环节。NADPH氧化酶是体内生成活性氧的主要酶类,大量研究证实NADPH氧化酶来源的ROS在高血压血管重构的发生、发展中起重要作用。本文就NADPH氧化酶介导高血压血管重构的作用及机制作一综述。     

NADPH oxidase activity is necessary for acute intermittent hypoxia-induced phrenic long-term facilitation

Phrenic long-term facilitation (pLTF) following acute intermittent hypoxia (AIH) is a form of spinal, serotonin-dependent synaptic plasticity that requires reactive oxygen species (ROS) formation. We tested the hypothesis that spinal NADPH oxidase activity is a necessary source of ROS for pLTF. Sixty minutes post-AIH (three 5-min episodes of 11% O₂, 5 min intervals), integrated phrenic and hypoglossal (XII) nerve burst amplitudes were increased from baseline, indicative of phrenic and XII LTF. Intrathecal injections (∼C₄) of apocynin or diphenyleneiodonium chloride (DPI), two structurally and functionally distinct inhibitors of the NADPH oxidase complex, attenuated phrenic, but not XII, LTF. Immunoblots from soluble (cytosolic) and particulate (membrane) fractions of ventral C₄ spinal segments revealed predominantly membrane localization of the NADPH oxidase catalytic subunit, gp91^phox, whereas membrane and cytosolic expression were both observed for the regulatory subunits, p47^phox and RAC1. Immunohistochemical analysis of fixed tissues revealed these same subunits in presumptive phrenic motoneurons of the C₄ ventral horn, but not in neighbouring astrocytes or microglia. Collectively, these data demonstrate that NADPH oxidase subunits localized within presumptive phrenic motoneurons are a major source of ROS necessary for AIH-induced pLTF. Thus, NADPH oxidase activity is a key regulator of spinal synaptic plasticity, and may be a useful pharmaceutical target in developing therapeutic strategies for respiratory insufficiency in patients with, for example, cervical spinal injury.     

Fungal metabolite gliotoxin inhibits assembly of the human respiratory burst NADPH oxidase

Reactive oxygen species are a critical weapon in the killing of Aspergillus fumigatus by polymorphonuclear leukocytes (PMN), as demonstrated by severe aspergillosis in chronic granulomatous disease. In the present study, A. fumigatus-produced mycotoxins (fumagillin, gliotoxin [GT], and helvolic acid) are examined for their effects on the NADPH oxidase activity in human PMN. Of these mycotoxins, only GT significantly and stoichiometrically inhibits phorbol myristate acetate (PMA)-stimulated O2- generation, while the other two toxins are ineffective. The inhibition is dependent on the disulfide bridge of GT, which interferes with oxidase activation but not catalysis of the activated oxidase. Specifically, GT inhibits PMA-stimulated events: p47phox phosphorylation, its incorporation into the cytoskeleton, and the membrane translocation of p67phox, p47phox, and p40phox, which are crucial steps in the assembly of the active NADPH oxidase. Thus, damage to p47phox phosphorylation is likely a key to inhibiting NADPH oxidase activation. GT does not inhibit the membrane translocation of Rac2. The inhibition of p47phox phosphorylation is due to the defective membrane translocation of protein kinase C (PKC) betaII rather than an effect of GT on PKC betaII activity, suggesting a failure of PKC betaII to associate with the substrate, p47phox, on the membrane. These results suggest that A. fumigatus may confront PMN by inhibiting the assembly of the NADPH oxidase with its hyphal product, GT.     

Neuronal expression of the NADPH oxidase NOX4, and its regulation in mouse experimental brain ischemia

Ischemia-induced neuronal damage has been linked to elevated production of reactive oxygen species (ROS) both in animal models and in humans. NADPH oxidase enzymes (NOX-es) are a major enzymatic source of ROS, but their role in brain ischemia has not yet been investigated. The present study was carried out to examine the expression of NOX4, one of the new NADPH oxidase isoforms in a mouse model of focal permanent brain ischemia. We demonstrate that NOX4 is expressed in neurons using in situ hybridization and immunohistochemistry. Ischemia, induced by middle cerebral artery occlusion resulted in a dramatic increase in cortical NOX4 expression. Elevated NOX4 mRNA levels were detectable as early as 24 h after the onset of ischemia and persisted throughout the 30 days of follow-up period, reaching a maximum between days 7 and 15. The early onset suggests neuronal reaction, while the peak period corresponds to the time of neoangiogenesis occurring mainly in the peri-infarct region. The occurrence of NOX4 in the new capillaries was confirmed by immunohistochemical staining. In summary, our paper reports the presence of the ROS producing NADPH oxidase NOX4 in neurons and demonstrates an upregulation of its expression under ischemic conditions. Moreover, a role for NOX4 in ischemia/hypoxia-induced angiogenesis is suggested by its prominent expression in newly formed capillaries.     

Effects of Anaplasma phagocytophila on NADPH oxidase components in human neutrophils and HL-60 cells

The human granulocytic ehrlichiosis agent, Anaplasma phagocytophila, resides and multiplies exclusively in cytoplasmic vacuoles of granulocytes. A. phagocytophila rapidly inhibits the superoxide anion (O(2)(-)) generation by human neutrophils in response to various stimuli. To determine the inhibitory mechanism, the influence of A. phagocytophila on protein levels and localization of components of the NADPH oxidase were examined. A. phagocytophila decreased levels of p22(phox), but not gp91(phox), p47(phox), p67(phox), or P40(phox) reactive with each component-specific antibody in human peripheral blood neutrophils and HL-60 cells. Double immunofluorescence labeling revealed that p47(phox), p67(phox), Rac2, and p22(phox) did not colocalize with A. phagocytophila inclusions in neutrophils or HL-60 cells, and p22(phox) levels were also reduced. A. phagocytophila did not prevent either membrane translocation of cytoplasmic p47(phox) and p67(phox) or phosphorylation of p47(phox) upon stimulation by phorbol myristate acetate. The inhibitory signals for O(2)(-) generation was independent of several signals required for A. phagocytophila internalization. These results suggest that rapid alteration in p22(phox) induced by binding of A. phagocytophila to neutrophils is involved in the inhibition of O(2)(-) generation. Absence of colocalization of NADPH oxidase components with the inclusion further protects A. phagocytophila from oxidative damage.     

Activation of the granule pool of the NADPH oxidase accelerates apoptosis in human neutrophils

Oxidative stress induces apoptosis in many types of cells, including human neutrophils. Our objective was to determine whether reactive oxygen species (ROS) produced by activated neutrophils are associated with accelerated apoptosis. Exposing neutrophils to ionomycin or phorbol myristate acetate (PMA) induced intracellular H2O2 production and rapid onset of apoptosis, measured as condensed chromatin, cellular shrinkage, and DNA fragmentation. Neutrophils activated with formyl-methionyl-leucyl-phenylalanine (fMLP) generated mainly extracellular H2O2 and did not undergo apoptosis. Exogenously added H2O2, together with the catalase blocker sodium azide, induced apoptosis to the same extent and with similar kinetics as PMA and ionomycin. Adenosine inhibited ionomycin-induced intracellular H2O2 production and apoptosis. Neither PMA nor ionomycin caused apoptosis in dimethyl sulfoxide-differentiated HL-60 cells, which are incapable of intracellular H2O2 production, whereas H2O2 induced apoptosis more efficiently in these cells than in neutrophils. We propose that activated neutrophils use intracellularly formed H2O2 to commit suicide.     

Regulation of phagolysosome pH in bovine and human neutrophils: the role of NADPH oxidase activity and an Na+/H+ antiporter

Fluorescein-labelled Staphylococcus aureus were used to follow changes in phagolysosome (PL) pH of bovine and human neutrophils following phagocytosis. Under aerobic conditions there was an alkalinisation of the PL followed by a slow decline. Under anaerobic conditions no alkalinisation of the PL was seen, and pharmacological inhibition of the NADPH oxidase with diphenyleneiodonium (DPI) resulted in a rapid acidification of the PL following phagocytosis. The inclusion of amiloride, an inhibitor of Na+/H+ antiporter activity, produced a more rapid alkalinisation phase following phagocytosis under aerobic conditions and reduced, but did not abolish, the acidification phases seen under anaerobic conditions or following treatment of neutrophils with DPI. The results suggest that PL pH is influenced by NADPH oxidase activity and to a lesser extent by a Na+/H+ antiporter. The antibacterial effectiveness of neutrophil granule proteins may be affected under conditions that influence the functioning of these two systems.     

Protein-A activates membrane bound multicomponent enzyme complex, NADPH oxidase in human neutrophils

Protein-A, 42KD cell wall glycoprotein of S. aureus Cowan I enhance mononuclear and polymorphonuclear cell counts in vivo and possesses antitoxic, antitumor, properties. In order to explain the mechanism of its function, the respiratory burst phenomenon in cell and cell free system was studied using lucigenin-dependent chemiluminescence technique. A dose dependent increase in protein A-mediated generation of superoxide radical was observed in resting and PMA stimulated neutrophils. Superoxide dismutase (SOD) was used to confirm the production of superoxide radicals (O^-₂). To understand the mechanism of protein-A induced O^-₂ generation; NADPH oxidase activity was measured in cell free system using NADPH as a substrate. A significant increase in NADPH oxidase activity was observed in the membrane and post-nuclear supernatant fraction of activated human neutrophils. Cytosolic fraction showed slight enzyme activation. Protein A (SpA)-induced NADPH oxidase activation in the membrane fraction was observed even in the absence of the substrate NADPH. These data indicate that protein A attenuate the NADPH oxidase system to produce O^-₂ radicals.