Comparison of proton channel, phagocyte oxidase, and respiratory burst levels between human eosinophil and neutrophil granulocytes.

Robust production of reactive oxygen species (ROS) by phagocyte NADPH oxidase (phox) during the respiratory burst (RB) is a characteristic feature of eosinophil and neutrophil granulocytes. In these cells the voltage-gated proton channel (Hv1) is now considered as an ancillary subunit of the phox needed for intense ROS production. Multiple sources reported that the expression of phox subunits and RB is more intensive in eosinophils than in neutrophils. In most of these studies the eosinophils were not isolated from healthy individuals, and a comparative analysis of Hv1 expression had never been carried out. We performed a systematic comparison of the levels of essential phox subunits, Hv1 expression and ROS producing capacity between eosinophils and neutrophils of healthy individuals. The expression of phox components was similar, whereas the amount of Hv1 was approximately 10-fold greater in eosinophils. Furthermore, Hv1 expression correlated with Nox2 expression only in eosinophils. Additionally, in confocal microscopy experiments co-accumulation of Hv1 and Nox2 at the cell periphery was observed in resting eosinophils but not in neutrophils. While phorbol-12-myristate-13-acetate-induced peak extracellular ROS release was approximately 1.7-fold greater in eosinophils, oxygen consumption studies indicated that the maximal intensity of the RB is only approximately 1.4-fold greater in eosinophils. Our data reinforce that eosinophils, unlike neutrophils, generate ROS predominantly extracellularly. In contrast to previous works we have found that the two granulocyte types display very similar phox subunit expression and RB capacity. The large difference in Hv1 expression suggests that its support to intense ROS production is more important at the cell surface

Naloxone inhibits immune cell function by suppressing superoxide production through a direct interaction with gp91phox subunit of NADPH oxidase

<p>Abstract</p> <p>Background</p> <p>Both (-) and (+)-naloxone attenuate inflammation-mediated neurodegeneration by inhibition of microglial activation through superoxide reduction in an opioid receptor-independent manner. Multiple lines of evidence have documented a pivotal role of overactivated NADPH oxidase (NOX2) in inflammation-mediated neurodegeneration. We hypothesized that NOX2 might be a novel action site of naloxone to mediate its anti-inflammatory actions.</p> <p>Methods</p> <p>Inhibition of NOX-2-derived superoxide by (-) and (+)-naloxone was measured in lipopolysaccharide (LPS)-treated midbrain neuron-glia cultures and phorbol myristate acetate (PMA)-stimulated neutrophil membranes by measuring the superoxide dismutase (SOD)-inhibitable reduction of tetrazolium salt (WST-1) or ferricytochrome c. Further, various ligand (³H-naloxone) binding assays were performed in wild type and gp91<it>^phox-/-</it>neutrophils and transfected COS-7 and HEK293 cells. The translocation of cytosolic subunit p47<it>^phox</it>to plasma membrane was assessed by western blot.</p> <p>Results</p> <p>Both (-) and (+)-naloxone equally inhibited LPS- and PMA-induced superoxide production with an IC50 of 1.96 and 2.52 μM, respectively. Competitive binding of ³H-naloxone with cold (-) and (+)-naloxone in microglia showed equal potency with an IC50 of 2.73 and 1.57 μM, respectively. ³H-Naloxone binding was elevated in COS-7 and HEK293 cells transfected with gp91^{<it>phox</it>}; in contrast, reduced ³H-naloxone binding was found in neutrophils deficient in gp91^{<it>phox </it>}or in the presence of a NOX2 inhibitor. The specificity and an increase in binding capacity of ³H-naloxone were further demonstrated by 1) an immunoprecipitation study using gp91^{<it>phox </it>}antibody, and 2) activation of NOX2 by PMA. Finally, western blot studies showed that naloxone suppressed translocation of the cytosolic subunit p47^{<it>phox </it>}to the membrane, leading to NOX2 inactivation.</p> <p>Conclusions</p> <p>Strong evidence is provided indicating that NOX2 is a non-opioid novel binding site for naloxone, which is critical in mediating its inhibitory effect on microglia overactivation and superoxide production.</p

Ginkgo Biloba Extract Ameliorates Oxidative Phosphorylation Performance and Rescues Aβ-Induced Failure

Energy deficiency and mitochondrial failure have been recognized as a prominent, early event in Alzheimer's disease (AD). Recently, we demonstrated that chronic exposure to amyloid-beta (Abeta) in human neuroblastoma cells over-expressing human wild-type amyloid precursor protein (APP) resulted in (i) activity changes of complexes III and IV of the oxidative phosphorylation system (OXPHOS) and in (ii) a drop of ATP levels which may finally instigate loss of synapses and neuronal cell death in AD. Therefore, the aim of the present study was to investigate whether standardized Ginkgo biloba extract LI 1370 (GBE) is able to rescue Abeta-induced defects in energy metabolism

The O2.- generating oxidase activation of bovine neutrophils. Evidence for synergism of multiple cytosolic factors in a cell-free system.

International audienceActivation of the O2.- generating oxidase in neutrophils can be achieved with a cell-free oxidase-activating system, which consists of a high speed supernatant (cytosol), a particulate fraction enriched in plasma membrane, GTP-gamma-S, arachidonic acid and Mg ions. Cytosolic proteins from bovine neutrophils were fractionated by chromatography on Mono Q and Mono S columns into two fractions, neither of which was able to activate efficiently the membrane-bound oxidase. However, when combined and added to the cell-free system under optimized conditions, they acted synergistically, activating the oxidase to virtually the same extent as crude cytosol. This synergism demonstrates that more than one cytosolic factor is required for oxidase activation, and can be used to trace the cytosolic factors during the course of their purification

Activation of bovine neutrophil oxidase in a cell free system. GTP-dependent formation of a complex between a cytosolic factor and a membrane protein.

International audienceThe effect of guanine nucleotides on activation of the O2-. generating oxidase in a cell free system consisting of bovine neutrophils membranes, cytosol and arachidonic acid has been studied. In a complete system, GTP-gamma-S was stimulatory and GDP-beta-S inhibitory. When cytosol was omitted, both nucleotides acted as inhibitors. Activation parameters have been explored in a preincubation step prior to the oxidase assay. Stimulation was found to be maximal at 7 to 100 microM GTP-gamma-S. Whereas the time course of activation was monophasic when activation was performed at room temperature, it became biphasic at 2 degrees C, with a first plateau of activation attained after 1 min, followed by a slow rise lasting for more than 30 min. The following lines of evidence demonstrated that oxidase activation resulted from the formation of a complex between cytosolic factor(s) and a target protein in the plasma membrane. 1/ When activated membranes, in a suspension containing cytosol, arachidonic acid and GTP-gamma-S, were separated from soluble components by centrifugation and washed, their oxidase remained fully active. 2/ The activity of the washed membranes was lost upon addition of GDP-beta-S, urea and deoxycholate, but was preserved by addition of glutaraldehyde, a cross-linking reagent. The results of experiments in which cytosol and membrane fractions were incubated separately with GTP-gamma-S, suggested that GTP-gamma-S first interacts with a factor present in the cytosol, before reacting with a target protein in the plasma membrane

Parameters of activation of the membrane-bound O2- generating oxidase from bovine neutrophils in a cell-free system.

International audienceParameters governing the extent of activation of the O2- generating oxidase in a cell-free system derived from bovine neutrophils were examined. The reconstituted system consisted of the following: a particulate fraction enriched in plasma membrane and containing the oxidase, a soluble fraction containing cytosolic factor(s) required for oxidase a soluble fraction containing cytosolic factor(s) required for oxidase activation, a non hydrolyzable analog of GTP, and either arachidonic acid or sodium dodecyl sulfate. When the amount of arachidonic acid or sodium dodecyl sulfate was maintained at a fixed value with respect to the amount of membrane used, a sigmoidal response of oxidase activity to increasing amounts of cytosol added was observed. In contrast, when the concentration of arachidonic acid or sodium dodecyl sulfate was properly adjusted with respect to that of membrane and cytosol, the curve relating oxidase activity to cytosol was hyperbolic, pointing to a simple michaelian relationship for the dependence of oxidase activation on the activating factor(s) of cytosol. Another parameter affecting oxidase activation was the ionic strength of the reconstitution medium, the extent of activation being lower at high ionic strength