NADPH oxidase is required for NMDA receptor-dependent activation of ERK in hippocampal area CA1

Previous studies have shown that N-methyl-D-aspartate (NMDA) receptor activation results in production of reactive oxygen species (ROS) and activation of extracellular signal-regulated kinase (ERK) in hippocampal area CA1. In addition, application of ROS to hippocampal slices has been shown to result in activation of ERK in area CA1. To determine whether these events were linked causally, we investigated whether ROS are required for NMDA receptor-dependent activation of ERK. In agreement with previous studies, we found that treatment of hippocampal slices with NMDA resulted in activation of ERK in area CA1. The NMDA receptor-dependent activation of ERK was either blocked or attenuated by a number of antioxidants, including the general antioxidant N-acetyl-L-cysteine (L-NAC), the superoxide-scavenging enzyme superoxide dismutase (SOD), the membrane-permeable SOD mimetic Mn(III) tetrakis (4-benzoic acid) porphyrin (MnTBAP), the hydrogen peroxide-scavenging enzyme catalase, and the catalase mimetic ebselen. The NMDA receptor-dependent activation of ERK also was blocked by the NADPH oxidase inhibitor diphenylene iodonium (DPI) and was absent in mice that lacked p47(phox), one of the required protein components of NADPH oxidase. Taken together, our results suggest that ROS production, especially superoxide production via NADPH oxidase, is required for NMDA receptor-dependent activation of ERK in hippocampal area CA1.     

The X+ chronic granulomatous disease as a fabulous model to study the NADPH oxidase complex activation

Chronic granulomatous disease (CGD) is a rare inherited disorder in which phagocytes lack NADPH oxidase activity. Patients with CGD suffer from recurrent bacterial and fungal infections because of the absence of superoxide anions (O2- degrees ) generatingsystem. The NADPH oxidase complex is composed of a membranous cytochrome b558, cytosolic proteins p67phox, p47phox, p40phox and two small GTPases Rac2 and Rap1A. Cytochrome b558 consists of two sub-units gp91phox and p22phox. The most common form of CGD is due to mutations in CYBB gene encoding gp91phox. In some rare cases, the mutated gp91phox is normally expressed but is devoided of oxidase activity. These variants called X+ CGD, have provided interesting informations about oxidase activation mechanisms. However modelization of such variants is necessary to obtain enough biological material for studies at the molecular level. A cellular model (knock-out PLB-985 cells) has been developed for expressing recombinant mutated gp91phox for functional analysis of the oxidase complex. Recent works demonstrated that this cell line genetically deficient in gp91phox is a powerful tool for functional analysis of the NADPH oxidase complex activation.     

Thapsigargin and flavin adenine dinucleotide ex vivo treatment rescues trafficking-defective gp91phox in chronic granulomatous disease leukocytes

Mutations in leukocyte NADPH oxidase genes lead to defective respiratory burst in leukocytes and cause chronic granulomatous diseases (CGD) in humans. The most common form of CGD is caused by mutations in the membrane-bound oxidase component gp91phox, which is encoded by the CYBB gene on the X chromosome. We previously reported on a patient with a CYBB mutation (H338Y) that prevents the intracellular trafficking and expression of gp91phox on leukocytes. The capacity of the leukocytes to produce reactive oxygen species (ROS) was rescued by treatment with thapsigargin and flavin adenine dinucleotide (FAD). The increase in ROS production was not due to the increase in cytoplasmic calcium induced by thapsigargin because the treatment of calcium ionophore did not have the same effect. Protein and cellular analyses on leukocytes and cells transfected with GFP-tagged gp91phox mutant showed that treated cells expressed more Endo H-resistant gp91phox protein on the cell surface and are more effective in killing bacteria. Thapsigargin- and FAD-treated CGD leukocytes had enhanced activity in protecting mice from Staphylococcus-induced peritoneal abscess formation in a mouse model of CGD. These results indicate that thapsigargin–FAD ex vivo treatment is effective in rescuing the ROS-producing activity of leukocytes in selected CGD patients.     

Role of components of the phagocytic NADPH oxidase in oxygen sensing.

It has been hypothesized that O(2) sensing in type I cells of the carotid body and erythropoietin (EPO)-producing cells of the kidney involves protein components identical to the NADPH oxidase system responsible for the respiratory burst of phagocytes. In the present study, we evaluated O(2) sensing in mice with null mutant genotypes for two components of the phagocytic oxidase. Whole body plethysmography was used to study unanesthetized, unrestrained mice. When exposed to an acute hypoxic stimulus, gp91(phox)-null mutant and wild-type mice increased their minute ventilation by similar amounts. In contrast, p47(phox)-null mutant mice demonstrated increases in minute ventilation in response to hypoxia that exceeded that of their wild-type counterparts: 98.0 +/- 18.0 vs. 20.0 +/- 13.0% (n = 11, P = 0.003). In vitro recordings of carotid sinus nerve (CSN) activity demonstrated that resting (basal) neural activity was marginally elevated in p47(phox)-null mutant mice. With hypoxic challenge, mean CSN discharge was 1.5-fold greater in p47(phox)-null mutant than in wild-type mice: 109.61 +/- 13.29 vs. 72.54 +/- 7.65 impulses/s (n = 8 and 7, respectively, P = 0.026). Consequently, the hypoxia-evoked CSN discharge (stimulus-basal) was approximately 58% larger in p47(phox)-null mutant mice. Quantities of EPO mRNA in kidney were similar in gp91(phox)- and p47(phox)-null mutant mice and their respective wild-type controls exposed to hypobaric hypoxia for 72 h. These findings confirm the previous observation that absence of the gp91(phox) component of the phagocytic NADPH oxidase does not alter the O(2)-sensing mechanism of the carotid body. However, absence of the p47(phox) component significantly potentiates ventilatory and chemoreceptor responses to hypoxia. O(2) sensing in EPO-producing cells of the kidney appears to be independent of the gp91(phox) and p47(phox) components of the phagocytic NADPH oxidase.     

Genetic deficiency of NADPH oxidase does not diminish,but rather enhances,LPS-induced acute inflammatory responses in vivo

Reactive oxygen species (ROS) and oxidative stress are thought to play a central role in the etiology of cell dysfunction and tissue damage in sepsis. However, there is limited and controversial evidence from in vivo studies that ROS mediate cell signaling processes that elicit acute inflammatory responses during sepsis. Because NADPH oxidase is one of the main cellular sources of ROS, we investigated the role of this enzyme in lipopolysaccharide (LPS)-induced acute inflammation in vivo, utilizing mice deficient in the gp91^phox or p47^phox subunits of NADPH oxidase. Age-and body weight-matched C57BL/6J wild-type (WT) and gp91^phox?/? and p47^phox?/? mice were injected ip with 50 μg LPS or saline vehicle and sacrificed at various time points up to 24 h. We found that LPS-induced acute inflammatory responses in serum and tissues were not significantly diminished in gp91^phox?/? and p47^phox?/? mice compared to WT mice. Rather, genetic deficiency of NADPH oxidase was associated with enhanced gene expression of inflammatory mediators and increased neutrophil recruitment to lung and heart. Furthermore, no protection from LPS-induced septic death was observed in either knockout strain. Our findings suggest that NADPH oxidase-mediated ROS production and cellular redox signaling do not promote, but instead limit, LPS-induced acute inflammatory responses in vivo.     

Mutagenesis of an arginine- and lysine-rich domain in the gp91(phox) subunit of the phagocyte NADPH-oxidase flavocytochrome b558

Site-directed mutagenesis was used to generate a series of mutants harboring point or multiple substitutions within the hydrophilic, polybasic domain of gp91(phox) encompassed by residues 86-102, which was previously identified as a site of interaction with p47(phox) during phagocyte NADPH oxidase assembly. Recombinant wild-type or mutant gp91(phox) was expressed in a human myeloid leukemia cell line in which the endogenous gp91(phox) gene was disrupted by gene targeting. NADPH oxidase activity was measured in a cytochrome c reduction assay following granulocytic differentiation of cells that expressed recombinant gp91(phox). Expression of a gp91(phox) mutant in which amino acids 89-97 were replaced with nine alternate amino acids abolished NADPH oxidase activity. Expression of gp91(phox) mutants R89T, D95A, D95R, R96A, R96E, or K102T did not significantly affect NADPH oxidase activity. However, mutations of individual or paired arginine residues at positions 91 and 92 had substantial effects on superoxide generation. The R91E/R92E mutation completely abolished both NADPH oxidase activity and membrane-translocation of the cytosolic oxidase proteins p47(phox), p67(phox), Rac1, and Rac2. The phorbol 12-myristate 13-acetate-induced rate of superoxide production was reduced by approximately 75% in cells expressing R91T/R92A, R91E, or R92E gp91(phox) along with an increased lag time to the maximal rates of superoxide production relative to cells expressing wild-type gp91(phox). Taken together, these results demonstrate that Arg91 and Arg92 of gp91(phox) are essential for flavocytochrome b558 function in granulocytes and suggest that these residues participate in the interaction of gp91(phox) with the cytosolic oxidase proteins.     

Altered NMDA receptor trafficking contributes to sleep deprivation-induced hippocampal synaptic and cognitive impairments

Recent evidence indicates that continuous wakefulness (sleep deprivation, SD) causes impairments in behavioral performance and hippocampal long-term potentiation (LTP) in animals. However, the mechanisms by which SD impairs long-term synaptic plasticity and cognitive function are not clear. Here, we report that 24-h SD in mice results in impaired hippocampus-dependent contextual memory and LTP and, unexpectedly, in reductions of the surface expression of NMDA receptor (NMDAR) subunit NR1 and NMDAR-mediated excitatory post-synaptic currents at hippocampal perforant path-dentate granule cell synapses. The results suggest that the reduction of functional NMDAR in hippocampal neurons may underlie the SD-induced deficits in hippocampus-dependent contextual memory and long-term synaptic plasticity.     

Gene therapy for inherited diseases using heamatopoietic stem cells--gene therapy for patients with chronic granulomatous disease

The possibility of gene therapy for inherited diseases with a single gene mutation in Figure 1 had been verified by the successful treatment with bone marrow transplantation. As the gene therapy method and theory has been progressing rapidly, it is expected that gene therapy will overcome the complications of bone marrow transplantation. Of these inherited diseases, chronic granulomatous disease (CGD) is the one of the most expected disease for gene therapy. CGD is an inherited immune deficiency caused by mutations in any of the following four phox genes encoding subunits of the superoxide generating phagocyte NADPH oxidase. It consists of membranous cytochrom b558 composed of gp91 phox and p22 phox, and four cytosolic components, p47 phox, p67 phox, rac p21 and p40 phox, which translocate to the membrane upon activation. In our group study, more than 220 CGD patients has been enrolled. The incidence of CGD patients was estimated as 1 out of 250,000 births. The expected life span of the CGD patients is 25 to 30 years old by the Kaplan Meier analysis. Comparing with the ratio of CGD subtype in US and Europe, that with p47phox deficiency is lower (less than 10%/o vs. 23%) and that of gp91 phox deficiency is higher (more than 75% vs. 60%). Prophylactic administration of ST antibiotics and IFN-gamma and bone marrow transplantation have been successfully employed in our therapeutic strategy. However, it is necessary to develop the gene therapy technology for CGD patients as more promising treatment. In the current study we constructed two retrovirus vectors; MFGS-gp91/293 SPA which contains only the therapeutic gp91 phox gene, a bicistronic retrovirus pHa-MDR-IRES-gp91/PA317 which carries a multi drug resistant gene (MDR1) and the gp91phox gene connected with an internal ribosome entry site (IRES). We demonstrate high efficiency transduction of gp 91 phox to CGD EB virus established cell line with high levels of functional correction of the oxidase by MFGS-gp91 and by pHa-MDR-IRES-gp91, respectively. We also demonstrate sufficient transduction of gp91 phox to CD34+ hematopoietic stem cell from the patients with gp91 phox deficiency by MFGS-gp91/293 SPA. Our current studies suggest that the combination of the 293-SPA packaging system and the bicistronic retrovirus system inserted MDR1 gene make our CGD gene therapy more feasible for clinical application.     

Deletion mutagenesis of p22phox subunit of flavocytochrome b558: identification of regions critical for gp91phox maturation and NADPH oxidase activity

The heterodimeric flavocytochrome b558, comprised of the two integral membrane proteins p22phox and gp91phox, mediates the transfer of electrons from NADPH to molecular oxygen in the phagocyte NADPH oxidase to generate the superoxide precursor of microbicidal oxidants. This study uses deletion mutagenesis to identify regions of p22phox required for maturation of gp91phox and for NADPH oxidase activity. N-terminal, C-terminal, or internal deletions of human p22phox were generated and expressed in Chinese hamster ovary cells with transgenes for gp91phox and two other NADPH oxidase subunits, p47phox, and p67phox. The results demonstrate that p22phox-dependent maturation of gp91phox carbohydrate, cell surface expression of gp91phox, and the enzymatic function of flavocytochrome b558 are closely correlated. Whereas the 5 N-terminal and 25 C-terminal amino acids are dispensable for these functions, the N-terminal 11 amino acids of p22phox are required, as is a hydrophilic region between amino acids 65 and 90. Upon deletion of 54 residues at the C terminus of p22phox (amino acids 142-195), maturation and cell surface expression of gp91phox was still preserved, although NADPH oxidase activity was absent, as expected, due to removal of a proline-rich domain between amino acids 151-160 that is required for recruitment of p47phox. Antibody binding studies indicate that the extreme N terminus of p22phox is inaccessible in the absence of cell permeabilization, supporting a model in which both the N- and C-terminal domains of p22phox extend into the cytoplasm, anchored by two membrane-embedded regions.     

Gp91phox contributes to NADPH oxidase activity in aortic fibroblasts but not smooth muscle cells

Reactive oxygen species (ROS) derived from vascular NADPH oxidase are important in normal and pathological regulation of vessel growth and function. Cell-specific differences in expression and function of the catalytic subunit of NADPH oxidase may contribute to differences in vascular cell response to NADPH oxidase activation. We examined the functional expression of gp91phox on NADPH oxidase activity in vascular smooth muscle cells (SMC) and fibroblasts (FB). As measured by dihydroethidium fluorescence in situ, superoxide (O2-*) levels were greater in adventitial cells compared with medial SMC in wild-type aorta. In contrast, there was no difference in O2-* levels between adventitial cells and medial SMC in aorta from gp91phox-deficient (gp91phox KO) mice. Adventitial-derived FB and medial SMC were isolated from the aorta of wild-type and gp91phox KO mice and grown in culture. Consistent with the observations in situ, basal and stimulated ROS levels were reduced in FB isolated from aorta of gp91phox KO compared with FB from wild-type aorta, whereas ROS levels were similar in SMC derived from gp91phox KO and wild-type aorta. There were no differences in expression of superoxide dismutase between gp91phox KO and wild-type FB to account for these observations. Because gp91phox is associated with membranes, we examined NADPH-stimulated O2-. production in membrane-enriched fractions of cell lysate. As measured by chemiluminescence, NADPH oxidase activity was markedly greater in wild-type FB compared with gp91phox KO FB but did not differ among the SMCs. Confirming functional expression of gp91phox in FB, antisense to gp91phox decreased ROS levels in wild-type FB. Finally, deficiency of gp91phox did not alter expression of the gp91phox homolog NOX4 in isolated FB. We conclude that the neutrophil subunit gp91phox contributes to NADPH oxidase function in vascular FB, but not SMC.     

D5 dopamine receptor regulation of reactive oxygen species production, NADPH oxidase, and blood pressure

Activation of D1-like receptors (D1 and/or D5) induces antioxidant responses; however, the mechanism(s) involved in their antioxidant actions are not known. We hypothesized that stimulation of the D5 receptor inhibits NADPH oxidase activity, and thus the production of reactive oxygen species (ROS). We investigated this issue in D5 receptor-deficient (D5-/-) and wild-type (D5+/+) mice. NADPH oxidase protein expression (gp91(phox), p47(phox), and Nox 4) and activity in kidney and brain, as well as plasma thiobarbituric acid-reactive substances (TBARS) were higher in D5-/- than in D5+/+ mice. Furthermore, apocynin, an NADPH oxidase inhibitor, normalized blood pressure, renal NADPH oxidase activity, and plasma TBARS in D5-/- mice. In HEK-293 cells that heterologously expressed human D5 receptor, its agonist fenoldopam decreased NADPH oxidase activity, expression of one of its subunits (gp91(phox)), and ROS production. The inhibitory effect of the D5 receptor activation on NADPH oxidase activity was independent of cAMP/PKA but was partially dependent on phospholipase D2. The ability of D5 receptor stimulation to decrease ROS production may explain, in part, the antihypertensive action of D5 receptor activation.     

Impaired long-term memory and long-term potentiation in N-type Ca2+ channel-deficient mice

Voltage-dependent N-type Ca(2+) channels, along with the P/Q-type, have a crucial role in controlling the release of neurotransmitters or neuromodulators at presynaptic terminals. However, their role in hippocampus-dependent learning and memory has never been examined. Here, we investigated hippocampus-dependent learning and memory and synaptic plasticity at hippocampal CA3-CA1 synapses in mice deficient for the alpha(1B) subunit of N-type Ca(2+) channels. The mutant mice exhibited impaired learning and memory in the Morris water maze and the social transmission of food preference tasks. In particular, long-term memory was impaired in the mutant mice. Interestingly, among activity-dependent long-lasting synaptic changes, theta burst- or 200-Hz-stimulation-induced long-term potentiation (LTP) was decreased in the mutant, compared with the wild-type mice. This type of LTP is known to require brain-derived neurotrophic factor (BDNF). It was found that both BDNF-induced potentiation of field excitatory postsynaptic potentials and facilitation of the frequency of miniature excitatory postsynaptic currents (mEPSCs) were reduced in the mutant. Taken together, these results demonstrate that N-type Ca(2+) channels are required for hippocampus-dependent learning and memory, and certain forms of LTP.     

P2X7 receptor-NADPH oxidase axis mediates protein radical formation and Kupffer cell activation in carbon tetrachloride-mediated steatohepatitis in obese mice

While some studies show that carbon tetrachloride-mediated metabolic oxidative stress exacerbates steatohepatitic-like lesions in obese mice, the redox mechanisms that trigger the innate immune system and accentuate the inflammatory cascade remain unclear. Here we have explored the role of the purinergic receptor P2X7-NADPH oxidase axis as a primary event in recognizing the heightened release of extracellular ATP from CCl(4)-treated hepatocytes and generating redox-mediated Kupffer cell activation in obese mice. We found that an underlying condition of obesity led to the formation of protein radicals and posttranslational nitration, primarily in Kupffer cells, at 24h post-CCl(4) administration. The free radical-mediated oxidation of cellular macromolecules, which was NADPH oxidase and P2X7 receptor-dependent, correlated well with the release of TNF-α and MCP-2 from Kupffer cells. The Kupffer cells in CCl(4)-treated mice exhibited increased expression of MHC Class II proteins and showed an activated phenotype. Increased expression of MHC Class II was inhibited by the NADPH oxidase inhibitor apocynin , P2X7 receptor antagonist A438709 hydrochloride, and genetic deletions of the NADPH oxidase p47 phox subunit or the P2X7 receptor. The P2X7 receptor acted upstream of NADPH oxidase activation by up-regulating the expression of the p47 phox subunit and p47 phox binding to the membrane subunit, gp91 phox. We conclude that the P2X7 receptor is a primary mediator of oxidative stress-induced exacerbation of inflammatory liver injury in obese mice via NADPH oxidase-dependent mechanisms.     

Molecular composition and regulation of the Nox family NAD(P)H oxidases

Reactive oxygen species (ROS) are conventionally regarded as inevitable deleterious by-products in aerobic metabolism with a few exceptions such as their significant role in host defense. The phagocyte NADPH oxidase, dormant in resting cells, becomes activated during phagocytosis to deliberately produce superoxide, a precursor of other microbicidal ROS, thereby playing a crucial role in killing pathogens. The catalytic center of this oxidase is the membrane-integrated protein gp91(phox), tightly complexed with p22(phox), and its activation requires the association with p47(phox), p67(phox), and the small GTPase Rac, which normally reside in the cytoplasm. Since recent discovery of non-phagocytic gp91(phox)-related enzymes of the NAD(P)H oxidase (Nox) family--seven homologues identified in humans--deliberate ROS production has been increasingly recognized as important components of various cellular events. Here, we describe a current view on the molecular composition and post-translational regulation of Nox-family oxidases in animals.     

The Nox/Duox family of ROS-generating NADPH oxidases

Reactive oxygen species (ROS) generated by the NADPH oxidases are conventionally thought to be cytotoxic and mutagenic and at high levels induce an oxidative stress response. The phagocyte NADPH oxidase catalyzes the NADPH-dependent reduction of molecular oxygen to generate superoxide O2-., which can dismute to generate ROS species. Together, these ROS participate in host defence by killing or damaging invading microbes. Flavocytochrome b558 is the catalytic core of the phagocyte NADPH oxidase and consists of a large glycoprotein gp91phox or Nox-2 and a small protein p22phox. The other components of the NADPH oxidase are cytosolic proteins, namely p67phox, p47phox, p40phox and Rac. A defect in any of the genes encoding gp91phox, p22phox, p67phox or p47phox results in chronic granulomatous disease, a genetic disorder characterized by severe and recurrent infections. Evidence is rapidly accumulating that low level of ROS were produced by NADPH oxidase homologs in non-phagocytic cells. To date, six human homologs (Nox-1, Nox-3, Nox-4, Nox-5, Duox-1 and Duox-2) have been recently identified in a variety of non-phagocytic cells. The identification of Nox-1 was quickly followed by the cloning of Nox-3, Nox-4, and Nox-5. In parallel, two very large members of the Nox family were discovered, namely Duox-1 and Duox-2, initially also referred to as thyroid oxidases. The physiological functions of Nox-dependent ROS generation are in progress and still require detailed characterization. Activation mechanisms and tissue distribution of the different members of the Nox family are very different, suggesting distinct physiological functions. Nox family enzymes are likely to be involved in a variety of physiological events including cell proliferation, host defence, differentiation, apoptosis, senescence and activation of growth-related signaling pathways. An increase and a decrease in the function of Nox enzymes can contribute to a wide range of pathological processes.     

Role of NADPH oxidase in the mechanism of lung neutrophil sequestration and microvessel injury induced by Gram-negative sepsis: studies in p47phox-/- and gp91phox-/- mice

We addressed the role of O(2) generated by the NADPH oxidase complex in the mechanism of polymorphonuclear leukocyte (PMN) accumulation and transalveolar migration and lung microvascular injury. Studies were made in mice lacking the p47(phox) and gp91(phox) subunits of NADPH oxidase (p47(phox-/-) and gp91(phox-/-)) in which PMN are incapable of the respiratory burst. The mice were challenged i.p. with live Escherichia coli to induce sepsis. We observed time-dependent increases in PMN sequestration and migration from 1 to 6 h after challenge with 2 x 10(8) E. coli. The responses in knockout mice were greater post-E. coli challenge compared with control mice; i.e., transalveolar PMN migration post-E. coli challenge increased by approximately 50% in the null mice above values in wild type. The increased PMN infiltration was associated with decreased lung bacterial clearance. The generation of the chemoattractant macrophage-inflammatory protein-2 in lung tissue was greater in NADPH oxidase-defective mice after E. coli challenge than control mice; moreover, macrophage-inflammatory protein-2 Ab pretreatment prevented the PMN infiltration. We also observed that E. coli failed to increase lung microvascular permeability in p47(phox-/-) and gp91(phox-/-) mice despite the greater lung PMN sequestration. Thus, O(2) production is required for the induction of sepsis-induced lung microvascular injury. We conclude that NADPH oxidase-derived O(2) generation has an important bactericidal role, such that an impairment in bacterial clearance in NADPH oxidase-defective mice results in increased chemokine generation and lung tissue PMN infiltration.     

Endosomal acidification and activation of NADPH oxidase isoforms are upstream events in hyperosmolarity-induced hepatocyte apoptosis

Hyperosmotic exposure of rat hepatocytes induced a rapid oxidative-stress(ROS) response as an upstream signal for proapoptotic CD95 activation. This study shows that hyperosmotic ROS formation involves a rapid ceramide- and protein kinase Czeta (PKCzeta)-dependent serine phosphorylation of p47phox and subsequent activation of NADPH oxidase isoforms. Hyperosmotic p47phox phosphorylation and ROS formation were sensitive to inhibition of sphingomyelinases and were strongly blunted after knockdown of acidic sphingomyelinase (ASM) or of p47phox protein. Hyperosmolarity induced a rapid bafilomycin- and 4,4 '-diisothiocyanostilbene-2,2 '-disulfonic acid disodium salt (DIDS)-sensitive acidification of a vesicular compartment, which was accessible to endocytosed fluorescein isothiocyanate-dextran and colocalized with ASM, PKCzeta, and the NADPH oxidase isoform Nox 2 (gp91phox). Bafilomycin and DIDS prevented the hyperosmolarity-induced increase in ceramide formation, p47phox phosphorylation, and ROS formation. As shown recently (Reinehr, R., Becker, S., H?ngen, A., and H?ussinger, D. (2004) J. Biol. Chem. 279, 23977-23987), hyperosmolarity induced a Yes-dependent activation of JNK and the epidermal growth factor receptor (EGFR), followed by EGFR-CD95 association, EGFR-catalyzed CD95-tyrosine phosphorylation, and translocation of the EGFR-CD95 complex to the plasma membrane, where formation of the deathinducing signaling complex occurs. These proapoptotic responses were not only sensitive to inhibitors of sphingomyelinase, PKCzeta, or NADPH oxidases but also to ASM knockdown, bafilomycin, and DIDS, i.e. maneuvers largely preventing hyperosmolarity-induced endosomal acidification and/or ceramide formation. In hepatocytes from p47phox knock-out mice, hyperosmolarity failed to activate the CD95 system. The data suggest that hyperosmolarity induces endosomal acidification as an important upstream event for CD95 activation through stimulation of ASM-dependent ceramide formation and activation of NADPH oxidase isoforms.     

Hypoxic pulmonary hypertension: role of superoxide and NADPH oxidase (gp91phox)

Chronic exposure to low-O2 tension induces pulmonary arterial hypertension (PAH), which is characterized by vascular remodeling and enhanced vasoreactivity. Recent evidence suggests that reactive oxygen species (ROS) may be involved in both processes. In this study, we critically examine the role superoxide and NADPH oxidase plays in the development of chronic hypoxic PAH. Chronic hypoxia (CH; 10% O2 for 3 wk) caused a significant increase in superoxide production in intrapulmonary arteries (IPA) of wild-type (WT) mice as measured by lucigenin-enhanced chemiluminescence. The CH-induced increase in the generation of ROS was obliterated in NADPH oxidase (gp91phox) knockout (KO) mice, suggesting that NADPH oxidase was the major source of ROS. Importantly, pathological changes associated with CH-induced PAH (mean right ventricular pressure, medial wall thickening of small pulmonary arteries, and right heart hypertrophy) were completely abolished in NADPH oxidase (gp91phox) KO mice. CH potentiated vasoconstrictor responses of isolated IPAs to both 5-hydroxytryptamine (5-HT) and the thromboxane mimetic U-46619. Administration of CuZn superoxide dismutase to isolated IPA significantly reduced CH-enhanced superoxide levels and reduced the CH-enhanced vasoconstriction to 5-HT and U-46619. Additionally, CH-enhanced superoxide production and vasoconstrictor activity seen in WT IPAs were markedly reduced in IPAs isolated from NADPH oxidase (gp91phox) KO mice. These results demonstrate a pivotal role for gp91phox-dependent superoxide production in the pathogenesis of CH-induced PAH.