Translocation of proteins homologous to human neutrophil p47phox and p67phox to the cell membrane in activated hemocytes of Galleria mellonella

Activation of the superoxide forming respiratory burst oxidase of human neutrophils, crucial in host defence, requires the cytosolic proteins p47phox and p67phox which translocate to the plasma membrane upon cell stimulation and activate flavocytochrome b558, the redox centre of this enzyme system. We have previously demonstrated the presence of proteins (67 and 47kDa) in hemocytes of the insect Galleria mellonella homologous to proteins of the superoxide-forming NADPH oxidase complex of neutrophils. The work presented here illustrates for the first time translocation of homologous hemocyte proteins, 67 and 47kDa from the cytosol to the plasma membrane upon phorbol 12-myristate 13 acetate (PMA) activation. In hemocytes, gliotoxin (GT), the fungal secondary metabolite significantly suppressed PMA-induced superoxide generation in a concentration dependent manner and reduced translocation to basel nonstimulated levels. Primarily these results correlate translocation of hemocyte 47 and 67kDa proteins with PMA induced oxidase activity. Collectively results presented here, demonstrate further cellular and functional similarities between phagocytes of insects and mammals and further justify the use of insects in place of mammals for modelling the innate immune response to microbial pathogens.     

Assembly of NADPH Oxidase in Human Neutrophils Is Modulated by the Opacity-Associated Protein Expression State of Neisseria gonorrhoeae

Neisseria gonorrhoeae (the gonococcus, Gc) triggers a potent inflammatory response and recruitment of neutrophils to the site of infection. Gc survives exposure to neutrophils despite these cells'' antimicrobial products, such as reactive oxygen species (ROS). ROS production in neutrophils is initiated by NADPH oxidase, which converts oxygen into superoxide. The subunits of NADPH oxidase are spatially separated between granules (gp91^phox/p22^phox) and the cytoplasm (p47^phox, p67^phox, and p40^phox). Activation of neutrophils promotes the coassembly of NADPH oxidase subunits at phagosome and/or plasma membranes. While Gc-expressing opacity-associated (Opa) proteins can induce neutrophils to produce ROS, Opa-negative (Opa⁻) Gc does not stimulate neutrophil ROS production. Using constitutively Opa⁻ and OpaD-positive (OpaD⁺) Gc bacteria in strain FA1090, we now show that the difference in ROS production levels in primary human neutrophils between these backgrounds can be attributed to differential assembly of NADPH oxidase. Neutrophils infected with Opa⁻ Gc showed limited translocation of NADPH oxidase cytoplasmic subunits to cellular membranes, including the bacterial phagosome. In contrast, these subunits rapidly translocated to neutrophil membranes following infection with OpaD⁺ Gc. gp91^phox and p22^phox were recruited to Gc phagosomes regardless of bacterial Opa expression. These results suggest that Opa⁻ Gc interferes with the recruitment of neutrophil NADPH oxidase cytoplasmic subunits to membranes, in particular, the p47^phox “organizing” subunit, to prevent assembly of the holoenzyme, resulting in an absence of the oxidative burst.     

NADPH oxidases and MMP-9/TIMP-1 balance in human glomeruli: putative links to diabetic nephropathy

Study aim Glomerular basement membrane thickening, the hallmark of diabetic nephropathy, is thought to be related to an enhanced oxidative stress and reduced matrix proteolysis. Our study concerned the mRNA and protein expression of NADPH oxidase (NOX) components, MMP‐2, MMP‐9 and TIMP‐1 in freshly isolated human glomeruli as well as enzymatic activities and their modulation by glucose, H₂O₂ and angiotensin‐2. Material and methods NOX, cytosolic and membrane‐bound associated proteins and mRNA were analysed by RT‐PCR and Western blotting after glomerular extraction. Oxidase activity was identified by cytochrome c reduction and chemiluminescence. Gelatinases and inhibitors were semiquantitatively assessed by RT‐PCR, gelatin zymography and ELISA in a model of glomerular conditioned survival. Results NOX‐2, NOX‐4 and membrane‐bound and cytosolic factors could be observed in freshly extracted glomeruli (RNA + protein). p40^phox, p67^phox and p47^phox molecular weights were increased compared to their phagocytic counterparts advocating for specific glomerular analogues, and a slight specific oxidase activity was retrieved in isolated glomeruli. Also, mRNA coding for MMP‐2, ‐9 and TIMP‐1, ‐2 were detected. High glucose concentrations (25 mm) reduced TIMP‐1 release in glomerular survival media and MMP‐2 activity in glomerular extracts. On the opposite, angiotensin‐2 significantly induced MMP‐2 and ‐9 activities in the survival media as well as H₂O₂ in glomerular extracts, while addition of 25 mm glucose blunted these findings. Conclusion Glomerular matrix remodelling, the backbone of renal fibrosis in diabetic patients, could be induced by H₂O₂ from specific glomerular NADPH oxidases under the influence of extra‐cellular glucose and angiotensin‐2 and could participate in the control of MMP activities.     

Altered vascular activation due to deficiency of the NADPH oxidase component p22phox

BackgroundReactive oxygen species generated by nicotinamide adenine dinucleotide phosphate (NADPH) oxidase play important roles in vascular activation. The p22^phox subunit is necessary for the activity of NADPH oxidase complexes utilizing Nox1, Nox2, Nox3, and Nox4 catalytic subunits.MethodsWe assessed p22^phox-deficient mice and human tissue for altered vascular activation.ResultsMice deficient in p22^phox were smaller than their wild-type littermates but showed no alteration in basal blood pressure. The wild-type littermates were relatively resistant to forming intimal hyperplasia following carotid ligation, and the intimal hyperplasia that developed was not altered by p22^phox deficiency. However, at the site of carotid artery ligation, the p22^phox-deficient mice showed significantly less vascular elastic fiber loss compared with their wild-type littermates. This preservation of elastic fibers was associated with a reduced matrix metallopeptidase (MMP) 12/tissue inhibitor of metalloproteinase (TIMP) 1 expression ratio. A similar decrease in the relative MMP12/TIMP1 expression ratio occurred in human coronary artery smooth muscle cells upon knockdown of the hydrogen peroxide responsive kinase CK1αLS. In the ligated carotid arteries, the p22^phox-deficient mice showed reduced expression of heterogeneous nuclear ribonucleoprotein C (hnRNP-C), suggesting reduced activity of CK1αLS. In a lung biopsy from a human patient with p22^phox deficiency, there was also reduced vascular hnRNP-C expression.ConclusionsThese findings indicate that NADPH oxidase complexes modulate aspects of vascular activation including vascular elastic fiber loss, the MMP12/TIMP1 expression ratio, and the expression of hnRNP-C. Furthermore, these findings suggest that the effects of NADPH oxidase on vascular activation are mediated in part by protein kinase CK1αLS.     

Vasoactive intestinal peptide dampens formyl-peptide-induced ROS production and inflammation by targeting a MAPK-p47phox phosphorylation pathway in monocytes

Reactive oxygen species (ROS) produced by the phagocyte NADPH oxidase (NOX2) are required for microbial clearance; however, when produced in excess they exacerbate inflammatory response and injure surrounding tissues. NOX2 is a multicomponent enzyme composed of membrane-associated cytochrome b588 and cytosolic components p47^phox, p67^phox, p40^phox, and rac1/2. We investigated whether vasoactive intestinal peptide (VIP), an endogenous immune-modulatory peptide, could affect ROS production by NOX2 in primary human phagocytes. VIP did not modulate basal ROS production by phagocytes, but it inhibited monocyte and not neutrophil ROS production in response to the bacterial peptide N-formyl-methionyl-leucyl-phenylalanine (fMLF). The action of VIP was essentially mediated by high-affinity G-protein coupled receptors VPAC1 as its specific agonist, [ALA^11,22,28]VIP, mimicked VIP-inhibitory effect, whereas the specific VPAC1 antagonist, PG97-269, blunted VIP action. Further, we showed that VIP inhibited fMLF-induced phosphorylation of ERK1/2 (extracellular signal–regulated kinase 1/2), p38MAPK (p38 mitogen-activated protein kinase) pathways, and phosphorylation of p47^phox on Ser345 residue. Also, VIP exerted an anti-inflammatory effect in a model of carrageenan-induced inflammation in rats. We thus found that VIP exerts anti-inflammatory effects by inhibiting the “MAPK–p47^phox phosphorylation–NOX2 activation” axis. These data suggest that VIP acts as a natural anti-inflammatory agent of the mucosal system and its analogs could be novel anti-inflammatory molecules.     

Baculovirus mediated expression of human phagocytic cell oxidase cytochrome b558 in sf9 insect cells

Chronic granulomatous disease (CGD) results from deficient production of components of the phagocyte NADPH oxidase. Most commonly affected is cytochrome b₅₅₈, a heterodimer composed of a 22-kDa protein (p22^phox noncovalently bound to a 91-kDa transmembrane glycoprotein (gp91^phox). CGD phagocytes lack both p22^phox and gp91^phox peptides when either gene is affected, suggesting that both peptides must be produced for individual subunit stability. Both genes have been cloned, but eukaryotic expression of recombinant gp91^phox has not been reported. To investigate the stability and interaction of cytochrome b₅₅₈ subunits, we introduced p22^phox and gp91^phox cDNA into recombinant baculoviruses. Recombinant gp91^phox (rgp91^phox) and p22^phox (rp22^phox) were detected individually and together in the same cells by in situ immunofluorescence and by SDS-PAGE immunoblotting of membranes from sf9 cells infected with baculovirus constructs. Formation of rp22^phox/ rgp91^phox complexes was demonstrated by coprecipitation using subunit-specific antibodies. This study demonstrates for the first time that cDNA encoding either subunit is capable of initiating production of stable recombinant cytochrome b₅₅₈ subunits in eukaryotic cells.     

Leu505 of Nox2 is crucial for optimal p67phox-dependent activation of the flavocytochrome b558 during phagocytic NADPH oxidase assembly

The role of Leu505 of Nox2 on the NADPH oxidase activation process was investigated. An X-CGD PLB-985 cell line expressing the Leu505Arg Nox2 mutant was obtained, exactly mimicking the phenotype of a previously published X91+-CGD case. In a reconstituted cell-free system (CFS), NADPH oxidase and iodonitrotetrazolium (INT) reductase activities were partially maintained concomitantly with a partial cytosolic factors translocation to the plasma membrane. This suggests that assembly and electron transfer from NADPH occurred partially in the Leu505Arg Nox2 mutant. Moreover, in a simplified CFS using purified mutant cytochrome b558 and recombinant p67phox, p47phox, and Rac1proteins, we found that the Km for NADPH and for NADH was about three times higher than those of purified WT cytochrome b558, indicating that the Leu505Arg mutation induces a slight decrease of the affinity for NADPH and NADH. In addition, oxidase activity can be extended by increasing the amount of p67phox in the simplified CFS assay. However, the maximal reconstituted oxidase activity using WT purified cytochrome b558 could not be reached using mutant cytochrome b558. In a three-dimensional model of the C-terminal tail of Nox2, Leu505 appears to have a strategic position just at the entry of the NADPH binding site and at the end of the alpha-helical loop (residues 484-504), a potential cytosolic factor binding region. The Leu505Arg mutation seems to affect the oxidase complex activation process through alteration of cytosolic factors binding and more particularly the p67phox interaction with cytochrome b558, thus affecting NADPH access to its binding site.     

The influence of calcium on morphology and histamine content of isolated intact rat mast cell granules

Histamine is released by “sequential exocytosis” in mast cells. The exocytosis involves fusion of the plasma membrane with the perigranular membrane and further fusions of adjacent perigranular membranes. To study a possible direct effect of Ca²⁺ on granule membrane fusions, mast cell granule suspensions were prepared from sonicated rat mast cells. With the sonication method used, more than 60% of the granules obtained were found to be homogeneous, electron dense and surrounded by a perigranular membrane, when observed in the electron microscope. These granules correspond to normal, histamine-containing granules found in untreated mast cells and are therefore named “intact” granules. The other granules were swollen, less electron dense and without a perigranular membrane. These “changed” granules are formed during the histamine release process. Aliquots of the granule suspension were incubated in 0.34 M sucrose buffered with 10 mM HEPES, pH 7.0, containing different concentrations of CaCl₂, MgCl₂ (10 mM, 1 mM, 100 μM, 10 μM) or NaCl (10 mM). Only with the highest concentration (10 mM) of Ca²⁺ or Mg²⁺ was it possible to visualize an apposition of the perigranular membranes of “intact”, normal granules. No elimination of the individual membrane structures could be observed at the place of membrane contact. Thus, we found no signs of membrane fusions. The histamine content was lower in the suspensions incubated with lower concentrations of these ions or with 10 mM NaCl. Ca²⁺ and Mg²⁺ in high concentrations seemed to stabilize the perigranular membranes instead of initiating histamine release. Therefore, changes in the Ca²⁺-ion concentration per se do not explain the membrane fusions seen in mast cells during “sequential exocytosis”.     

P40phox associates with the neutrophil Triton X-100-insoluble cytoskeletal fraction and PMA-activated membrane skeleton: a comparative study with P67phox and P47phox

NADPH oxidase is an O2*- -generating enzyme found in phagocytes such as neutrophils. It is composed of a membrane-bound cytochrome b, the cytosolic proteins p67phox, p47phox, p40phox, and the G-protein p21rac. The system is dormant in resting cells but acquires catalytic activity on exposure to appropriate stimuli. Cytochrome b, p67phox, p47phox, and rac2 associate with the cytoskeleton and membrane skeleton of activated neutrophils. It is not known whether p40phox associates with the cytoskeleton. The purpose of this study was to analyze the subcellular distribution of p40phox. When resting neutrophils were lysed in Triton X-100 or octyl glucoside buffer and separated into detergent-soluble and detergent-insoluble fractions, p40phox and p67phox were mainly associated with the detergent-insoluble fraction (defined as the cytoskeleton), whereas p47phox was mainly found in the soluble fraction. Neutrophil activation by phorbol myristate acetate (PMA) induced p47phox translocation to the cytoskeleton but did not affect the distribution of p40phox or p67phox. Using immunofluorescence confocal microscopy, we found that p40phox colocalized with filamentous actin. In neutrophils from a p67phox-deficient patient with detectable p40phox, p40phox associated with the cytoskeleton only after activation by PMA. A complex containing the three proteins was isolated from the cytoskeleton of activated neutrophils. When activated membranes were treated with Triton X-100 buffer, p40phox, p47phox, and p67phox were found in the membrane skeleton enriched in NADPH-oxidase activity; some p40phox and p47phox was found in the soluble membrane fraction, but no p67phox was detected. These findings show that p40phox, like p67phox and p47phox, binds to the cytoskeleton and membrane skeleton. In addition, p40phox can dissociate from p67phox in activated membranes.     

Inhibition of the neutrophil NADPH oxidase by adenosine is associated with increased movement of flavocytochrome b between subcellular fractions

Adenosine is a potent inhibitor of reactive oxygen species (ROS) production by the NADPH oxidase in fMLF-stimulated neutrophils. Although much is known about the pharamacology and signal transduction of this effect, it is not known how adenosine affects assembly and localization of the NADPH oxidase components within the neutrophil. We report here that adenosine pretreatment of fMLF-stimulated neutrophils results in decreased plasma membrane/secretory granule content of the flavocytochrome b components (p22^phox and gp91^phox) of the NADPH oxidase, which correlates with inhibition of ROS production. Adenosine treatment did not affect upregulation of secretory and specific granule surface markers, confirming that degranulation was not impaired by adenosine. However, adenosine treatment did result in increased movement of cell-surface flavocytochrome b to heavy granule fractions in fMLF-stimulated neutrophils. These data suggest that adenosine-mediated effects on neutrophil ROS production are due, in part to endocytosis and/or redistribution of flavocytochrome b between various subcellular compartments.     

Mitochondria and chromaffin cell function

Chromaffin cells are an excellent model for stimulus?Csecretion coupling. Ca²⁺ entry through plasma membrane voltage-operated Ca²⁺ channels (VOCC) is the trigger for secretion, but the intracellular organelles contribute subtle nuances to the Ca²⁺ signal. The endoplasmic reticulum amplifies the cytosolic Ca²⁺ ([Ca²⁺]_C) signal by Ca²⁺-induced Ca²⁺ release (CICR) and helps generation of microdomains with high [Ca²⁺]_C (HCMD) at the subplasmalemmal region. These HCMD induce exocytosis of the docked secretory vesicles. Mitochondria close to VOCC take up large amounts of Ca²⁺ from HCMD and stop progression of the Ca²⁺ wave towards the cell core. On the other hand, the increase of [Ca²⁺] at the mitochondrial matrix stimulates respiration and tunes energy production to the increased needs of the exocytic activity. At the end of stimulation, [Ca²⁺]_C decreases rapidly and mitochondria release the Ca²⁺ accumulated in the matrix through the Na⁺/Ca²⁺ exchanger. VOCC, CICR sites and nearby mitochondria form functional triads that co-localize at the subplasmalemmal area, where secretory vesicles wait ready for exocytosis. These triads optimize stimulus?Csecretion coupling while avoiding propagation of the Ca²⁺ signal to the cell core. Perturbation of their functioning in neurons may contribute to the genesis of excitotoxicity, ageing mental retardation and/or neurodegenerative disorders.     

The molecular basis of chronic granulomatous disease

Summary and conclusions CGD is a rare inherited immunodeficiency syndrome, caused by the phagocytes' inability to produce (sufficient) reactive oxygen metabolites. This dysfunction is due to a defect in the NADPH oxidase, the enzyme responsible for the production of superoxide. It is composed of several subunits, two of which, gp9l^phox and p22^phox, form the membrane-bound cytochrome b558, while its three cytosolic components, p47^phox p67^phox and p40^phox, have to translocate to the membrane upon activation. This is a tightly and intricately controlled process that involves, among others, several low-molecular weight GTP-binding proteins. Gp91^phox is encoded on the X-chromosome and p22^phox, p47^phox and p67^phox on different autosomal chromosomes, and a defect in one of these components leads to CGD. This explains the variable mode of inheritance seen in this syndrome.Clinically CGD manifests itself typically already at a very young age with recurrent and serious infections, most often caused by catalase-positive pathogens.Modern treatment options, including prophylaxis with trimethoprim-sulfamethoxazole and rIFN- as well as early and aggressive anti-infection therapy, have improved the prognosis of this disease dramatically.CGD, as a very well-characterized inherited affection of the hematopoietic stem cells, is predestined to be among the first diseases to profit from the advances in cutting-edge therapeutics, such as gene therapy and in utero stem cell transplantation.     

Biochemical changes contributing to functional quiescence in lacrimal gland acinar cells after chronic ex vivo exposure to a muscarinic agonist

Profound secretory dysfunction can be associated with relatively modest lymphocytic infiltration of the lacrimal and salivary glands of Sjögren's syndrome (SjS) patients. SjS patients' sera contain autoantibodies to M3 muscarinic acetylcholine receptors (MAChR) that have variously been reported to have agonistic and antagonistic effects. We sought to identify consequences of chronic agonist stimulation by maintaining acinar cells from rabbit lacrimal glands for 20 h in the presence or absence of 10 µm carbachol (CCh). Exposure to CCh diminished the cells' ability to elevate cytosolic Ca²⁺ and secrete β‐hexosaminidase in response to acute stimulation with 100 µm CCh, but it enhanced their secretory responses to phenylephrine and ionomycin. Secretory vesicles appeared normal by electron microscopy, but confocal fluorescence microscopy revealed depletion of the secretory vesicle membrane marker, rab3D, and decreased ability to recruit secretory transport vesicles in response to acute 100 µm CCh. Additionally, the apical cortical actin cytoskeleton was disrupted and diminished compared to the basal–lateral cortical network. Subcellular fractionation analyses revealed that total membrane phase protein content was increased. The contents of β‐hexosaminidase and MAChR relative to total protein were not significantly altered, and MAChR abundance in the plasma membrane fraction was increased as the result of redistribution from endomembrane pools. However, relative cellular contents of the heterotrimeric guanosine triphosphate (GTP)‐binding proteins, G_q and G₁₁, were decreased. Additional biochemical changes included decreased contents of 47 kDa G_s and G_i3, protein kinase Cα and rab3D and polymeric immunoglobulin (Ig) receptors; internalization of Na,K‐ATPase from the plasma membranes to endomembrane compartments and decreased content of β‐hexosaminidase in the lysosomes. The observations demonstrate that chronic exposure to a MAChR agonist induces refractoriness to optimal stimulation, without causing receptor downregulation, by downregulating postreceptor‐signalling mediators and effectors. The cells' secretory mechanisms for IgA and electrolytes also appear to be impaired, as does their ability to properly sort proteins to the lysosomes.     

Formation, stabilisation and fusion of the readily releasable pool of secretory vesicles

Calcium-triggered exocytosis of neurotransmitter or hormone-filled vesicles has developed as the main mechanism for cell-to-cell communication in animals. Consequently, in the course of evolution this form of exocytosis has been optimized for speed. Since many of the maturation processes of vesicles are intrinsically slow, the solution has been to develop a pool of vesicles that are fully matured and can be fused very rapidly upon stimulation. Vesicles in this readily releasable pool are characterized by very low release rate constants at the resting cytosolic [Ca²⁺] ([Ca²⁺]_i) and very high release rate constants at stimulated [Ca²⁺]_i. Here I review the kinetic and molecular requirements for the existence of such a pool of vesicles, focusing on chromaffin cells of the adrenal medulla. I discuss how the use of assay methods with different time resolution may lead to fundamentally different conclusions about the role of proteins in exocytosis. Finally, I review recent evidence that the soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) complex, formed between proteins residing in the vesicle and the plasma membrane, is involved in formation and stabilization of the readily releasable vesicle pool, whereas synaptotagmin, a Ca²⁺- and phospholipid-binding vesicular protein, is involved in setting the Ca²⁺ dependence of the fusion process itself. Future studies are likely to focus on the interaction between these two classes of proteins.     

From pharmacomechanical coupling to G-proteins and myosin phosphatase

A brief summary of recent studies of pharmacomechanical coupling is presented, with emphasis on the role of GTP-binding proteins and Ca²⁺-independent regulation of contraction (Ca²⁺-sensitization/desensitization) through regulatory myosin light chain (MLC₂₀) phosphorylation and dephosphorylation. Pharmacomechanical regulation of cytosolic [Ca²⁺] is largely, though not solely, controlled by the phosphatidylinositol cascade and Ca²⁺-pumps of the plasma membrane and the sarcoplasmic reticulum. The monomeric GTPase, RhoA, is a major upstream component of Ca²⁺-sensitization. Its crystal structure and apparently obligatory translocation to the plasma membrane for activation of its downstream effectors are described. Inhibition of RhoA activity by a membrane-permeant ADP-ribosylating bacterial exoenzyme, DC3B, causes severe depression of the tonic component of agonist-induced contraction, suggesting that this component is largely due to Ca²⁺-sensitization. A relatively specific inhibitor (Y27632) of Rho-kinase, a downstream effector of Ca²⁺-sensitization (Uehata et al 1997), also inhibits oxytocin-induced Ca²⁺-sensitization of myometrium. The major mechanism of physiological, G-protein-coupled Ca²⁺-sensitization is through inhibition of smooth muscle myosin phosphatase (SMPP-1M), whereas conventional or novel protein kinase Cs play very little or no role in this process. Mechanisms of Ca²⁺-desensitization include inhibition of myosin light chain kinase and activation of SMPP-1M. Activation of SMPP-1M in phasic smooth muscle can be attributed, at least in part, to the synergistic phosphatase activating activities of a cyclic nucleotide-dependent kinase and its major substrate, telokin.     

NADPH oxidase activity of neutrophil specific granules: requirements for cytosolic components and evidence of assembly during cell activation

Neutrophils and other phagocytic cells support host defense by ingesting microbes and destroying them with reactive oxygen species or oxygen independent mechanisms. Production of ROS is initiated by the phagocyte NADPH oxidase (phox), an enzyme system composed of several constituents. During activation of the cell cytosolic phox proteins (p47phox, p67phox, p40phox, and Rac2) translocate to the plasma membrane and specific granules fuse with the plasma membrane increasing the amount of flavocytochrome b(558). The resultant assembly of phox components results in formation of a complete complex and expression of activity. In this study, we evaluated the oxidase activity of specific granules. In the SDS cell-free system, specific granules expressed oxidase activity in the presence of cytosol in a manner similar to plasma membrane. In contrast to plasma membrane, activity of specific granules was latent, diminishing rapidly over time. In addition, this subcellular fraction contained an inhibitor, possibly related to contamination with azurophilic granules explaining previously published discrepant results. Experiments with recombinant p47phox, p67phox, and dilute cytosol or fractionated cytosol as a source of Rac demonstrated that specific granules have requirements identical to specific granules for oxidase activity. Finally, analysis of neutrophils stimulated with PMA demonstrated translocation of p47phox and to p67phox to specific granules as well as plasma membrane. Both plasma membrane and specific granules from PMA stimulated cells expressed oxidase activity with addition of NADPH demonstrating an assembled oxidase complex. These studies establish a critical role for specific granules as a site for assembly and activation of the oxidase enzyme system and an important constituent for the microbicidal activity of the neutrophil.     

Priming of the neutrophil NADPH oxidase activation: role of p47phox phosphorylation and NOX2 mobilization to the plasma membrane

Neutrophils play an essential role in host defense against microbial pathogens and in the inflammatory reaction. Upon activation, neutrophils produce superoxide anion (O*2), which generates other reactive oxygen species (ROS) such as hydrogen peroxide (H2O2), hydroxyl radical (OH*) and hypochlorous acid (HOCl), together with microbicidal peptides and proteases. The enzyme responsible for O2* production is called the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase or respiratory burst oxidase. This multicomponent enzyme system is composed of two trans-membrane proteins (p22phox and gp91phox/NOX2, which form the cytochrome b558), three cytosolic proteins (p47phox, p67phox, p40phox) and a GTPase (Rac1 or Rac2), which assemble at membrane sites upon cell activation. NADPH oxidase activation in phagocytes can be induced by a large number of soluble and particulate factors. Three major events accompany NAPDH oxidase activation: (1) protein phosphorylation, (2) GTPase activation, and (3) translocation of cytosolic components to the plasma membrane to form the active enzyme. Actually, the neutrophil NADPH oxidase exists in different states: resting, primed, activated, or inactivated. The resting state is found in circulating blood neutrophils. The primed state can be induced by neutrophil adhesion, pro-inflammatory cytokines, lipopolysaccharide, and other agents and has been characterized as a "ready to go" state, which results in a faster and higher response upon exposure to a second stimulus. The active state is found at the inflammatory or infection site. Activation is induced by the pathogen itself or by pathogen-derived formylated peptides and other agents. Finally, inactivation of NADPH oxidase is induced by anti-inflammatory agents to limit inflammation. Priming is a "double-edged sword" process as it contributes to a rapid and efficient elimination of the pathogens but can also induce the generation of large quantities of toxic ROS by hyperactivation of the NADPH oxidase, which can damage surrounding tissues and participate to inflammation. In order to avoid extensive damage to host tissues, NADPH oxidase priming and activation must be tightly regulated. In this review, we will discuss some of the mechanisms of NADPH oxidase priming in neutrophils and the relevance of this process to physiology and pathology.     

Ketamine reduces inducible superoxide generation in human neutrophils in vitro by modulating the p38 mitogen-activated protein kinase (MAPK)-mediated pathway

Many cellular stresses and inflammatory stimuli can activate p38 mitogen-activated protein kinase (MAPK), a serine/threonine kinase in the MAPK family. The different stimuli act via different receptors or signalling pathways to induce phosphorylation of the cytosolic protein p47^phox, one subunit of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase. Formyl–methionyl–leucyl–phenylalanine (fMLP) has been shown to induce the p38 MAPK phosphorylation during the respiratory burst in human neutrophils. Here, we show that treatment with S(+)-ketamine or R(-)-ketamine at different concentrations (50, 100, 200, 400 µM) reduced fMLP-induced superoxide anion generation and p47^phox phosphorylation in neutrophils in a concentration-dependent manner (y = −0·093x + 93·35 for S(+)-ketamine and y = −0·0982x + 95·603 for R(-)-ketamine, respectively). While treatment with 50 µM ketamine inhibited fMLP-induced superoxide generation by 10%, treatment with 400 µM S(+)-ketamine and R(-)-ketamine reduced fMLP-induced superoxide generation to 60·5 ± 8·3% and 60·0 ± 8·5%, respectively, compared with that in neutrophils treated with fMLP alone. Furthermore, treatment with ketamine down-regulated both fMLP-induced p47^phox and isoproterenol-induced p38 MAPK phosphorylation and superoxide production. Interestingly, treatment with SB203580, the p38 MAPK inhibitor, also mitigated fMLP-induced superoxide anion generation and p38 MAPK and p47^phox phosphorylation as well as apoptosis in a concentration-dependent fashion in neutrophils. Therefore, ketamine racemes inhibited fMLP-induced superoxide anion generation and p47^phox phosphorylation by modulating fMLP-mediated p38 MAPK activation in neutrophils.     

S100A10/p11: family, friends and functions

S100A10, also known as p11 or annexin 2 light chain, is a member of the S100 family of small, dimeric EF hand-type Ca²⁺-binding proteins that generally modulate cellular target proteins in response to intracellular Ca²⁺ signals. In contrast to all other S100 proteins, S100A10 is Ca²⁺ insensitive because of amino acid replacements in its Ca²⁺-binding loops that lock the protein in a permanently active state. Within cells, the majority of S100A10 resides in a tight heterotetrameric complex with the peripheral membrane-binding protein annexin A2 that directs the complex to specific target membranes, in particular the plasma membrane and the membrane of early endosomes. Several other Ca²⁺-independent interaction partners of S100A10 have been described in the recent past. Many of these interactions, which have been shown to be of functional significance for the respective partner, involve plasma membrane-resident proteins. In most of these cases, S100A10, probably residing in a complex with annexin A2, appears to regulate the intracellular trafficking of the respective target protein and thus its functional expression at the cell surface. In this paper, we review the current information on S100A10 protein interactions placing a particular emphasis on data that contribute to an understanding of the mechanistic basis of the S100A10 action. Based on these data, we propose that S100A10 functions as a linker tethering certain transmembrane proteins to annexin A2 thereby assisting their traffic to the plasma membrane and/or their firm anchorage at certain membrane sites.     

A comparison of synaptic protein localization in hippocampal mossy fiber terminals and neurosecretory endings of the neurohypophysis using the cryo-immunogold technique

In central synapses synaptic vesicle docking and exocytosis occurs at morphologically specialized sites (active zones) and requires the interaction of specific proteins in the formation of a SNARE complex. In contrast, neurosecretory terminals lack active zones. Using the cryo-immunogold technique we analyzed the localization of synaptic vesicle proteins and of proteins of the docking complex at active zones. This was compared to the localization of the identical proteins in neurosecretory terminals. In addition we compared the vesicular and granular localization of the proteins investigated. Synaptic vesicles in rat hippocampal mossy fiber synapses and microvesicles in the neurosecretory terminals of the neurohypophysis contained in common the proteins VAMP II (a v-SNARE), SV2, rab3A, and N-type Ca²⁺ channels. Only minor immunolabeling for these proteins was observed at neurosecretory granules. These results support the notion of a close functional identity of microvesicles from neurosecretory endings of the neurohypophysis and of synaptic vesicles. The vesicular pool of N-type Ca²⁺ channels may serve their stimulation-induced translocation into the plasma membrane. We find increased labeling for VAMP II, SNAP-25, N-type Ca²⁺ channels and of rab3A at the active zones of mossy fiber synapses. Labeling at release sites is by far highest for Bassoon, a high molecular weight protein of the active zone. The labeling pattern implies an association of Bassoon with presynaptic dense projections. Bassoon is absent from neurosecretory terminals and VAMP II, SNAP-25, rab3A, and N-type Ca²⁺ channels reveal a scattered distribution over the plasma membrane. The competence of the presynaptic active zone for selective vesicle docking may not primarily result from its contents in SNARE proteins but rather from the preformation of presynaptic dense projections as structural guides for vesicle exocytosis.