Gene/Protein Disease Symptom Drug Enzyme Compound
Pivot Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound   Target Concepts: Gene/Protein Disease Symptom Drug Enzyme Compound

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Query: EC:1.6.3.1 (NADPH oxidase)
11,281 document(s) hit in 31,850,051 MEDLINE articles (0.00 seconds)

The potential role of cytosolic phospholipase A2 (cPLA2) in the regulation of the electrogenic arachidonic acid (AA)-activatable H+ translocator of neutrophils was investigated. (1) The trifluoromethyl ketone analogue of arachidonate (AACOCF3), a newly developed selective blocker of cPLA2, inhibited both the N-formylmethionyl-leucylphenylalanine (fMLP)- and the phorbol-ester-induced rheogenic H+ efflux (K0.5 approximately 5 microM) and abrogated the stimulus-triggered release of AA from these cells. The drug failed to reduce the fMLP-evoked Ca2+ signal or protein tyrosine phosphorylation and did not affect the activity of protein kinase C. By using the patch-clamp technique we verified that the agent did not interfere with the voltage- and the pH-dependent activation of the H+ conductance of the peritoneal macrophages and therefore is not a direct blocker of the H+ channel itself. AACOCF3, however, slightly decreased the AA-induced stimulation of the H+ currents. We conclude that AA, liberated by the agonist-induced stimulation of cPLA2, is a direct activator of H+ conductance. (2) AACOCF3 did not inhibit superoxide generation, indicating that activation of cPLA2 may not be a prerequisite for turning on NADPH oxidase. (3) Since neither acid generation by the oxidase, nor the basal or stimulated Na+/H+ exchange (the predominant acid-eliminating mechanism) were influenced by the drug, we could use AACOCF3 to address whether the H+ channel in fact opens and plays any physiological role during activation of neutrophils. Stimulus-induced cytosolic alkalinization was smaller, whereas depolarization became larger, in the presence of AACOCF3. Stimulated H+ conductance therefore does contribute to intracellular pH (pHi) homoeostasis and membrane potential changes of intact neutrophils.
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PMID:Electrogenic H+ pathway contributes to stimulus-induced changes of internal pH and membrane potential in intact neutrophils: role of cytoplasmic phospholipase A2. 923 Jan 34

This investigation was undertaken to clarify the mechanisms of superoxide anion (O2-) generation in rat peritoneal mast cells. Compound 48/80, a typical histamine liberator mediated by calcium influx, elicited O2- generation from the mast cells in a dose-dependent fashion. It was demonstrated by immunohistochemical study and Western blot analysis that the mast cells contained the 47-kDa phagocyte oxidase (p47phox) protein, which was one cytosolic component of the NADPH oxidase system. Arachidonic acid stimulated O2- generation in the mast cells, but other unsaturated fatty acids had no effect. On the other hand, 48/80-induced O2- generation was inhibited by phospholipase A2 inhibitors, such as arachidonyl trifluoromethyl ketone and manoalide. Forskolin, isoprenaline, and dibutyryl cyclic AMP inhibited the O2- generation, and KT-5720, a cyclic AMP-dependent protein kinase (A-kinase) inhibitor, markedly enhanced the O2- generation. These findings suggest that O2- is generated by a NADPH oxidase-like enzyme system in mast cells and that this enzyme system is activated by arachidonic acid released by cytosolic phospholipase A2. Thus, it is regulated by the cyclic AMP-A kinase system.
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PMID:The mechanisms of compound 48/80-induced superoxide generation mediated by A-kinase in rat peritoneal mast cells. 923 5

The role of cytosolic phospholipase A2 (cPLA2) and its mode of activation by opsonized zymosan (OZ) was studied in human neutrophils in comparison with activation by PMA. The activation of cPLA2 by 1 mg/ml OZ or 50 ng/ml PMA is evidenced by its translocation to the membrane fractions on stimulation. This translocation is consistent with dithiothreitol (DTT)-resistant phospholipase A2 (PLA2) activity detected in the membranes of activated cells. Neutrophils stimulated by either OZ or PMA exhibited an immediate stimulation of extracellular-signal-regulated kinases (ERKs). The inhibition of ERKs, DTT-resistant PLA2 and NADPH oxidase activities by the MAP kinase kinase inhibitor PD-98059 indicates that ERKs mediate the activation of cPLA2 and NADPH oxidase stimulated by either OZ or PMA. The protein kinase C (PKC) inhibitor GF-109203X inhibited epidermal growth factor receptor peptide kinase activity, the release of [3H]arachidonic acid, DTT-resistant PLA2 activity and superoxide generation induced by PMA, but did not inhibit any of these activities induced by OZ. PKC activity was similarly inhibited by GF-109203X in membrane fractions separated from neutrophils stimulated by either PMA or OZ. In the presence of the tyrosine kinase inhibit orgenistein, ERKs, PLA2 and NADPH oxidase activities were inhibited in cells stimulated by OZ, whereas they were hardly affected in cells stimulated by PMA. The results suggest that the activation of cPLA2 by PMA or OZ is mediated by ERKs. Whereas PMA stimulates ERKs activity through a PKC-dependent pathway, signal transduction stimulated by OZ involves tyrosine kinase activity leading to activation of ERKs via a PKC-independent pathway.
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PMID:Cytosolic phospholipase A2 and its mode of activation in human neutrophils by opsonized zymosan. Correlation between 42/44 kDa mitogen-activated protein kinase, cytosolic phospholipase A2 and NADPH oxidase. 930 39

Arachidonic acid (AA) can trigger activation of the phagocyte NADPH oxidase in a cell-free assay. However, a role for AA in activation of the oxidase in intact cells has not been established, nor has the AA generating enzyme critical to this process been identified. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected that lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid or 1,25-dihydroxyvitamin D3, indicating that cPLA2 is not involved in the differentiation process. Neither cPLA2 nor stimulated [3H]AA release were detectable in differentiated cPLA2-deficient PLB-985 cells, demonstrating that cPLA2 is the major type of PLA2 activated in phagocytic-like cells. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but the addition of exogenous AA fully restores oxidase activity. This establishes an essential requirement of cPLA2-generated AA for activation of phagocyte NADPH oxidase.
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PMID:Essential requirement of cytosolic phospholipase A2 for activation of the phagocyte NADPH oxidase. 941 1

The NADPH oxidase producing-superoxide is the major mechanism by which phagocytes kill invading pathogens. The human myeloid cell line PLB-985 was transfected to express p85 cytosolic phospholipase A2 (cPLA2) antisense mRNA and stable clones were selected which lack detectable cPLA2. cPLA2-deficient PLB-985 cells differentiate similarly to control PLB-985 cells in response to retinoic acid, DMSO or 1,25 dihydroxyvitamin D3 indicating that cPLA2 is not involved in the differentiation process. Despite the normal synthesis of NADPH oxidase subunits during differentiation of cPLA2-deficient PLB-985 cells, these cells fail to activate NADPH oxidase in response to a variety of soluble and particulate stimuli, but addition of exogenous arachidonic acid (AA) fully restores oxidase activity. This establishes an essential requirement of cPLA2 generated AA for activation of phagocyte NADPH oxidase. In order to elucidate the mechanism by which cPLA2 regulates the oxidase, the role of cPLA2 in NADPH oxidase associated H+ channel was studied. Activation of differentiated PLB cells resulted in a Zn+2 sensitive alkalization, indicating H+ channel activity. In contrast, differentiated PLB-D cells failed to activate the H+ channel, but addition of exogenous AA fully restored this activity, indicating an essential and specific physiological requirement of cPLA2-generated AA for activation of the H+ channel. The presence of the H+ channel inhibitor, Zn+2, caused significant inhibition of NADPH oxidase activity, suggesting a role of the NADPH oxidase associated H+ channel in regulating oxidase activity.
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PMID:Cytosolic phospholipase A2 is required for the activation of the NADPH oxidase associated H+ channel in phagocyte-like cells. 1089 15

Hydrophobic bile acids impair gallbladder emptying in vivo and inhibit gallbladder muscle contraction in response to CCK-8 in vitro. This study was aimed at determining the mechanisms of muscle cell dysfunction caused by bile acids in guinea pig gallbladders. Muscle cells were obtained by enzymatic digestion. Taurochenodeoxycholic acid (TCDC), a hydrophobic bile acid, caused a contraction of up to 15% and blocked CCK-induced contraction. Indomethacin abolished the TCDC-induced contraction. Hydrophilic bile acid tauroursodeoxycholic acid (TUDC) had no effect on muscle contraction but prevented the TCDC-induced contraction and its inhibition on CCK-induced contraction. Pretreatment with NADPH oxidase inhibitor PH2I, xanthine oxidase inhibitor allopurinol, and free-radical scavenger catalase also prevented TCDC-induced contraction and its inhibition of the CCK-induced contraction. TCDC caused H2O2 production, lipid peroxidation, and increased PGE2 synthesis and activities of catalase and SOD. These changes were significantly inhibited by pretreatment of PH2I or allopurinol. Inhibitors of cytosolic phospholipase A2 (cPLA2), protein kinase C (PKC), and mitogen-activating protein kinase (MAPK) also blocked the TCDC-induced contraction. It is concluded that hydrophobic bile acids cause muscle cell dysfunction by stimulating the formation of H2O2 via activation of NADPH and xanthine oxidase. H2O2 causes lipid peroxidation and activates cPLA2 to increase PGE2 production, which, in turn, stimulates the synthesis of free-radical scavengers through the PKC-MAPK pathway.
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PMID:Effects of bile acids on the muscle functions of guinea pig gallbladder. 1206 95

The antioxidants butylated hydroxytoluene (BHT, 1 mM) and D-alpha-tocopherol (10 microM) completely attenuated protein degradation in murine myotubes in response to both proteolysis-inducing factor (PIF) and angiotensin II (Ang II), suggesting that the formation of reactive oxygen species (ROS) plays an important role in this process. Both PIF and Ang II induced a rapid and transient increase in ROS formation in myotubes, which followed a parabolic dose-response curve, similar to that for total protein degradation. Antioxidant treatment attenuated the increase in expression and activity of the ubiquitin-proteasome proteolytic pathway by PIF and Ang II, by preventing the activation of the transcription factor nuclear factor-kappaB (NF-kappaB), through inhibition of phosphorylation of the NF-kappaB inhibitor protein (I-kappaB) and its subsequent degradation. ROS formation by both PIF and Ang II was attenuated by diphenyleneiodonium (10 microM), suggesting that it was mediated through the NADPH oxidase system. ROS formation was also attenuated by trifluoroacetyl arachidonic acid (10 microM), a specific inhibitor of cytosolic phospholipase A2, U-73122 (5 microM) and D609 (200 microM), inhibitors of phospholipase C and calphostin C (300 nM), a highly specific inhibitor of protein kinase C (PKC), all known activators of NADPH oxidase. Myotubes containing a dominant-negative mutant of PKC did not show an increase in ROS formation in response to either PIF or Ang II. The two Rac1 inhibitors W56 (200 microM) and NSC23766 (10 microM) also attenuated both ROS formation and protein degradation induced by both PIF and Ang II. Rac1 is known to mediate signalling between the phosphatidylinositol-3 kinase (PI-3K) product and NADPH oxidase, and treatment with LY24002 (10 microM), a highly selective inhibitor of PI-3K, completely attenuated ROS production in response to both PIF and Ang II, and inhibited total protein degradation, while the inactive analogue LY303511 (100 microM) had no effect. ROS formation appears to be important in muscle atrophy in cancer cachexia, since treatment of weight losing mice bearing the MAC16 tumour with D-alpha-tocopherol (1 mg kg(-1)) attenuated protein degradation and increased protein synthesis in skeletal muscle.
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PMID:Role of reactive oxygen species in protein degradation in murine myotubes induced by proteolysis-inducing factor and angiotensin II. 1753 11

A growing body of evidence suggests oxidative stress involvement in neurodegenerative diseases; however, it remains to be determined whether oxidative stress is a cause, result, or epiphenomenon of the pathological processes. This review concerns the current issue, focusing on Alzheimer disease (AD), Parkinson disease (PD), and amyotrophic lateral sclerosis (ALS). Several studies have indicated that oxidative stress initially occurs in the disease-specific, site-restricted sources such as amyloid-beta in the cerebral cortex of AD brain, alpha-synuclein in the brain stem of PD brain, and glutamate receptor-coupled Ca2+ channel in the motor system of ALS spinal cord. Subsequent events in the neurons common to these diseases are glutamate-induced neurotoxicity and increased cytosolic Ca2+ levels, resulting in activation of Ca2+ -dependent enzymes including NADPH oxidase, cytosolic phospholipase A2, xanthine oxidase, and neuronal nitric oxide synthase (NOS). These enzymes produce reactive oxygen and nitrogen species (ROS/RNS), which oxidatively modify nucleic acid, lipid, sugar, and protein, leading to nuclear damage, mitochondrial damage, proteasome inhibition, and endoplasmic reticulum (ER) stress. Mitochondrial damage results in both ROS leakage from the electron transport system and Ca2+ release. Nuclear damage induces p53 activation, and proteasome inhibition reduces p53 degradation. The resultant increased p53 levels in the nucleus induce Bax activation and Bcl-2 inhibition, followed by a release of cytochrome c into the cytosol that truncates procaspase-9. ER stress triggers activation of caspase-12 as well as caspase-9 via the tumor necrosis factor (TNF) receptor-associated factor-2 / apoptosis-signaling kinase-1 / c-Jun N-terminal kinase pathway. Oxidative stress also stimulates astrocytes and microglia to yield and secrete cytokines such as TNFa and FasL that cause not only neuronal caspase-8 activation but also glial inflammatory response through induction of nuclear factor-kappaB-mediated, proinflammatory gene products including cytokines, chemokines, growth factors, cell adhesion molecules, and ROS/RNS-producing enzymes. The activated caspases truncate procaspase-3 to exert classical apoptosis. Moreover, oxidative DNA damage leads to the release and nuclear truncation of mitochondrial apoptosis-inducing kinase, which triggers apoptosis-like programmed cell death via cyclophilin A. These observations could indicate crucial implications for oxidative stress in several steps of the pathomechanisms of neurodegenerative diseases.
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PMID:[The role for oxidative stress in neurodegenerative diseases]. 1830 64

Up-regulation of cytosolic phospholipase A2 (cPLA2) by cigarette smoke extract (CSE) may play a critical role in airway inflammatory diseases. However, the mechanisms underlying CSE-induced cPLA2 expression in human tracheal smooth muscle cells (HTSMCs) remain unknown. CSE induced cPLA2 protein and mRNA expression, and ROS generation was attenuated by pretreatment with a reactive oxygen species (ROS) scavenger (N-acetylcysteine), or inhibitors of NADPH oxidase (diphenyleneiodonium chloride, apocynin) and transfection with p47phox siRNA, suggesting that CSE-induced cPLA2 expression was mediated through NADPH oxidase activation and ROS production in HTSMCs. Furthermore, CSE-induced cPLA2 expression was attenuated by pretreatment with the inhibitors of MEK1/2 (U0126), p38 MAPK (SB202190), and JNK (SP600125), which were further confirmed by transfection with siRNAs of JNK1, p42, and p38 to down-regulate the expression of respective proteins and reduce cPLA2 expression. Induction of cPLA2 by CSE was attenuated by selective inhibitors of NF-kappaB (helenalin) and AP-1 (curcumin). Moreover, promoter assays revealed that increases of cPLA2, NF-kappaB, and AP-1 luciferase activities stimulated by CSE were attenuated by these inhibitors. These results suggest that in HTSMCs, CSE induced NADPH oxidase activation leading to phosphorylation of p42/p44 MAPK, p38 MAPK, and JNK. These reactions induced nuclear transcription NF-kappaB and AP-1 activities which were essential for CSE-induced cPLA2 gene expression.
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PMID:Cigarette smoke extract induces cytosolic phospholipase A2 expression via NADPH oxidase, MAPKs, AP-1, and NF-kappaB in human tracheal smooth muscle cells. 1928 Jul 14

ROS (reactive oxygen species) overproduction is an important underlying factor for the activation of astrocytes in various neuropathological conditions. In the present study, we examined ROS production in astrocytes and downstream effects leading to changes in the signalling cascade, morphology and membrane dynamics using menadione, a redox-active compound capable of inducing intracellular ROS. NAD(P)H oxidase-mediated menadione-induced ROS production, which then stimulated phosphorylation of p38 MAPK (mitogen-activated protein kinase) and ERK1/2 (extracellular-signal-regulated kinase 1/2), and increased actin polymerization and cytoskeletal protrusions. We also showed that astrocyte plasma membranes became more molecularly ordered under oxidative stress, which was abrogated by down-regulating cPLA2 (cytosolic phospholipase A2) either with a pharmacological inhibitor or by RNA interference. In addition, mild disruption of F-actin with cytochalasin D suppressed menadione-enhanced phosphorylation of cPLA2 and membrane alterations. Taken together, these results suggest an important role for ROS derived from NAD(P)H oxidase in activation of astrocytes to elicit biochemical, morphological and biophysical changes reminiscent of reactive astrocytes in pathological conditions.
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PMID:NAD(P)H oxidase-mediated reactive oxygen species production alters astrocyte membrane molecular order via phospholipase A2. 1939 62


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