Role of LOX-1 in Ang II-induced oxidative functional damage in renal tubular epithelial cells

The lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1), plays an important role in angiotension II (Ang II)-induced hypertensive renal injury associated with pro-inflammatory responses, tubular functional damage and cellular apoptosis. In this study, we report on the role of LOX-1 in Ang II-induced oxidative functional damage and underlying signaling in human renal proximal tubular epithelial cells (HRPTEpiCs). The exposure to Ang II enhanced the expression of the NADPH oxidases (the p22phox, p47phox and Nox4 subunits), LOX-1 and the adhesion molecule, ICAM-1. It also promoted monocytic U937 cell adherences to HRPTEpiCs, increased reactive oxygen species formation and stimulated apoptosis, which was concomitant with an increase in the activation of p38 and p44/42 mitogen-activated protein kinases (MAPK). Furthermore, the Ang II treatment disturbed the balance of the Bcl-2 family proteins, destabilized mitochondrial membrane potential, and subsequently triggered the release of cytochrome c into the cytosol, causing the activation of caspase-3. The NADPH oxidase inhibitors and LOX-1 small interfering RNA markedly ameliorated these detrimental effects by reducing LOX-1 expression and MAPK activation. The p38 and p44/42MAPK inhibitors also inhibited the Ang II-induced functional damage without affecting LOX-1 expression in the HRPTEpiCs. These observations suggest that LOX-1 mediates Ang II-induced renal tubular epithelial dysfunction. In addition, MAPK pathway activation occurs downstream of the Ang II/reactive oxygen species/LOX-1 cascade.     

Role of NADPH oxidases in the control of vascular gene expression

All vascular cells, including endothelial cells and smooth muscle cells, express components of the leukocyte NADPH oxidase such as p22phox, p47phox, and Rac. Endothelial cells and fibroblasts also express the leukocyte NADPH oxidase subunit gp91phox/nox2, whereas in smooth muscle cells nox1 and nox4 are found. The different vascular NADPH oxidases represent important sources for the basal as well as the agonist-induced superoxide anion (O(2) .-) generation in the vasculature. In vascular smooth muscle cells, activation of the NADPH oxidases and the subsequent formation of O(2) .- has been demonstrated for various agents including angiotensin II, thrombin, lysophosphatidylcholine, and tumor necrosis factor alpha. By influencing the activity of p38 mitogen-activated protein kinase and AKT, NADPH oxidase-derived O(2) .- increases the expression of several pro-arteriosclerotic genes, such as monocyte chemoattractant protein-1, tissue factor, and vascular endothelial growth factor. Thus, the vascular NADPH oxidases play an important role in mediating the signal transduction cascade of pro-arteriosclerotic stimuli.     

NADPH oxidase-dependent formation of reactive oxygen species contributes to angiotensin II-induced epithelial-mesenchymal transition in rat peritoneal mesothelial cells

The objective of the present study was to investigate the role of NADPH oxidase-dependent formation of reactive oxygen species (ROS) in the angiotensin II (Ang II)-induced epithelial-mesenchymal transition (EMT) and in the accumulation of extracellular matrix (ECM) in rat peritoneal mesothelial cells (RPMCs). Primary cultured RPMCs were incubated with serum-free media for 24 h in order to arrest and synchronize cell growth. The cells were treated with Ang II (10?? M) up to 48 h. Cells were pretreated with an Ang II type I receptor antagonist (losartan, 10?? M), or an inhibitor of NADPH, oxidase diphenyleneiodonium (DPI) (10?? M), for 1 h before addition of Ang II. The dichlorofluorescein (DCF)-sensitive cellular ROS were measured by fluorometric assay and confocal microscopy. RT-PCR was employed to detect the mRNA expression for the NADPH oxidase subunit p47phox, plasminogen activator inhibitor-1 (PAI-1), α-smooth muscle actin (α-SMA) and E-cadherin. PAI-1, α-SMA and p47phox protein expression were examined by Western blot analysis. Ang II significantly induced the production of intracellular ROS. DPI and losartan inhibited Ang II-induced ROS generation by 86.8% and 77.4% (p<0.05), respectively. Ang II significantly stimulated NADPH oxidase subunit p47phox mRNA and protein expression in RPMCs. Both losartan and DPI inhibited Ang II-induced up-regulation of p47phox mRNA by 37.3% and 67.8% (p<0.05), respectively. Ang II also stimulated α-SMA mRNA and protein expression and down-regulated E-cadherin mRNA expression in RPMCs. This effect was suppressed by both losartan and DPI. Ang II significantly up-regulated PAI-1 mRNA and protein expression and these were significantly suppressed by both losartan and DPI. In conclusion, NADPH oxidase-dependent formation of ROS mediates Ang II dependent EMT and accumulation of ECM in rat peritoneal mesothelial cells. NADPH oxidase may represent a potential therapeutic target in the management of peritoneal fibrosis.     

The NADPH oxidase family and its inhibitors

The classical nicotinamide adenine dinucleotide phosphate (NADPH) oxidase was originally detected in neutrophils as a multicomponent enzyme that catalyzes the generation of superoxide from oxygen and the reduced form of NADPH. This enzyme is composed of two membrane-bound subunits (p22phox and gp91phox), three cytosolic subunits (p67phox, p47phox, and p40phox) and a small G-protein Rac (Rac1 and Rac2). Recently, it has been demonstrated that there are several isoforms of nonphagocytic NADPH oxidase. Endothelial cells, vascular smooth muscle cells or adventitial fibroblasts possess multiple isoforms of this enzyme. The new homologs, along with gp91phox are now designated the Nox family of NADPH oxidases and are key sources of reactive oxygen species in the vasculature. Reactive oxygen species play a significant role in regulating endothelial function and vascular tone. However, besides the participation in the processes of physiological cell, these enzymes can also be the perpetrator of oxidative stress that causes endothelial dysfunction. This review summarizes the current state of knowledge of the structure and functions of NADPH oxidase and NADPH oxidase inhibitors in the treatment of disorders with endothelial damage.     

NADPH oxidase-dependent signaling in endothelial cells: role in physiology and pathophysiology

Reactive oxygen species (ROS) including superoxide (O(2)(.-)) and hydrogen peroxide (H(2)O(2)) are produced endogenously in response to cytokines, growth factors; G-protein coupled receptors, and shear stress in endothelial cells (ECs). ROS function as signaling molecules to mediate various biological responses such as gene expression, cell proliferation, migration, angiogenesis, apoptosis, and senescence in ECs. Signal transduction activated by ROS, "oxidant signaling," has received intense investigation. Excess amount of ROS contribute to various pathophysiologies, including endothelial dysfunction, atherosclerosis, hypertension, diabetes, and acute respiratory distress syndrome (ARDS). The major source of ROS in EC is a NADPH oxidase. The prototype phagaocytic NADPH oxidase is composed of membrane-bound gp91phox and p22hox, as well as cytosolic subunits such as p47(phox), p67(phox) and small GTPase Rac. In ECs, in addition to all the components of phagocytic NADPH oxidases, homologues of gp91(phox) (Nox2) including Nox1, Nox4, and Nox5 are expressed. The aim of this review is to provide an overview of the emerging area of ROS derived from NADPH oxidase and oxidant signaling in ECs linked to physiological and pathophysiological functions. Understanding these mechanisms may provide insight into the NADPH oxidase and oxidant signaling components as potential therapeutic targets.     

血管紧张素Ⅱ刺激高草酸尿症大鼠肾脏NADPH氧化酶的表达

目的： 观察肾素-血管紧张素系统（RAS）和NADPH氧化酶在高草酸尿症大鼠肾脏氧化应激（OS）形成中的相互作用。方法: 采用0.8%乙二醇饮水法诱导建立高草酸尿症大鼠模型。动物分6个组（n=8）,A组:空白组;B组：高草酸尿症组;C组：高草酸尿症+apocynin治疗组;D组：单纯apocynin治疗组;E组：高草酸尿症+losartan治疗组;F组：单纯losartan治疗组。后4组分别灌胃给予apocynin（0.2 g·kg-1·d-1）或losartan（30 mg·kg-1·d-1）。4周后检测大鼠尿液、肾组织中的OS指标（尿8-IP和肾组织SOD活性）,放免法检测肾组织血管紧张素Ⅱ（AngⅡ）的含量,免疫组化法观察NADPH氧化酶亚单位P47phox蛋白在肾脏中的表达位置,RT-PCR法检测肾组织p47phox mRNA的表达水平。结果: p47phox在各组大鼠肾脏中都有广泛表达,表达部位包括肾皮质、内髓、外髓。与A组比较,B组尿液8-IP明显增多,肾组织SOD活性降低,肾组织AngⅡ含量增多,p47phox mRNA在肾组织中的表达水平也明显增多。使用apocynin（C组）和losartan（E组）均可抑制肾组织p47phox mRNA的表达,同时肾脏的OS程度减轻。结论: 在高草酸尿症大鼠模型中,肾脏p47phox mRNA表达增多,导致肾脏OS;同时肾脏RAS也被激活,后者可通过刺激p47phox mRNA的表达而促进肾脏OS程度增加。     

Arachidonic acid-dependent activation of a p22(phox)-based NAD(P)H oxidase mediates angiotensin II-induced mesangial cell protein synthesis and fibronectin expression via Akt/PKB

Angiotensin II (Ang II) induces protein synthesis and hypertrophy through arachidonic acid (AA)- and redoxdependent activation of the serine-threonine kinase Akt/PKB in mesangial cells (MCs). The role of NAD(P)H oxidase component p22( phox ) was explored in this signaling pathway and in Ang II-induced expression of the extracellular matrix protein fibronectin. Ang II causes activation of Akt/PKB and induces fibronectin protein expression, effects abrogated by phospholipase A(2) inhibition and mimicked by AA. Ang II and AAalso elicited an increase in fibronectin expression that was reduced with a dominant negative mutant of Akt/PKB. Exposure of the cells to hydrogen peroxide stimulates Akt/PKB activity and fibronectin synthesis. The antioxidant N-acetylcysteine abolished Ang II- and AA-induced Akt/PKB activation and fibronectin expression. Western blot analysis revealed high levels of p22( phox ) in MCs. Antisense (AS) but not sense oligonucleotides for p22( phox ) prevented ROS generation in response to Ang II and AA. AS p22( phox ) inhibited Ang II- or AA-induced Akt/PKB as well as protein synthesis and fibronectin expression. These data provide the first evidence, in MCs, of activation by AAof a p22( phox )-based NAD(P)H oxidase and subsequent generation of ROS. Moreover, this pathway mediates the effect of Ang II on Akt/PKB-induced protein synthesis and fibronectin expression.     

Evaluation of the expression of NADPH oxidase components during maturation of HL-60 cells to neutrophil lineage

To understand the expression of NADPH oxidase components during neutrophil maturation, we examined the expression of mRNAs and proteins for NADPH oxidase components, and the superoxide-producing activity using HL-60 cells incubated with dimethyl sulfoxide (DMSO). Northern blot and Western blot analyses revealed that gp91(phox), p67(phox), and p47(phox) were expressed after myelocyte stages, whereas p22(phox), p40(phox), and rac-2 were expressed from the promyelocyte stage. Furthermore, immunocytochemical staining of DMSO-induced HL-60 cells indicated that gp91(phox), p67(phox), and p47(phox) were detected only after myelocyte stages (myelocytes, metamyelocytes, band cells, and segmented cells), whereas p22(phox), p40(phox), and rac-2 were detected from the promyelocyte stage. In addition, nitro blue tetrazolium (NBT) assay showed that superoxide could be produced after myelocyte stages but not produced before promyelocyte stages. Moreover, almost the same results as those with DMSO-induced HL-60 cells were obtained using human bone-marrow cells by immunocytochemical staining and NBT assay, except that p22(phox) was detected by immunocytochemical staining after myelocyte stages in bone-marrow cells. Together, these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during neutrophil maturation.     

Mechanism and characteristics of stimuli-dependent ROS generation in undifferentiated HL-60 cells

It has been widely believed that undifferentiated human promyelocytic leukemia cells (HL-60) have no ability to generate reactive oxygen species (ROS) responding to stimuli. We report here that undifferentiated HL-60 cells possess NADPH oxidase and that generation of superoxide can be measured using a highly sensitive chemiluminescence dye, L-012. Five subunits of NADPH oxidase, namely, gp91(phox), p22(phox), p67(phox), p47(phox), and Rac 2, were detected in undifferentiated HL-60 cells by immunoblotting analysis. The contents of these NADPH oxidase components in the cells were increased with the differentiation induced by phorbol myristate acetate (PMA), except for p22(phox). Messenger RNAs of these subunits were also detected by the RT-PCR method, and their expressions increased except that of p22(phox) with the differentiation induced by PMA. Kinetic analysis using L-012 revealed that HL-60 cells generated substantial amounts of ROS by various stimulants, including formylmethionyl-leucyl-phenylalanine, PMA, myristic acid, and a Ca2+ ionophore, A23187. Both diphenyleneiodonium (an inhibitor of FAD-dependent oxidase) and apocynin (a specific inhibitor of NADPH oxidase) suppressed this stimuli-dependent ROS generation. Genistein, staurosporine, uric acid, and sodium azide inhibited the ROS generation in undifferentiated HL-60 cells in a similar way to that in undifferentiated neutrophils. These results suggested that the mechanism of ROS generation in undifferentiated HL-60 cells is the same as that in primed neutrophils.     

Regulation of NADPH oxidase in vascular endothelium: the role of phospholipases, protein kinases, and cytoskeletal proteins

The generation of reactive oxygen species (ROS) in the vasculature plays a major role in the genesis of endothelial cell (EC) activation and barrier function. Of the several potential sources of ROS in the vasculature, the endothelial NADPH oxidase family of proteins is a major contributor of ROS associated with lung inflammation, ischemia/reperfusion injury, sepsis, hyperoxia, and ventilator-associated lung injury. The NADPH oxidase in lung ECs has most of the components found in phagocytic oxidase, and recent studies show the expression of several homologues of Nox proteins in vascular cells. Activation of NADPH oxidase of nonphagocytic vascular cells is complex and involves assembly of the cytosolic (p47(phox), p67(phox), and Rac1) and membrane-associated components (Noxes and p22(phox)). Signaling pathways leading to NADPH oxidase activation are not completely defined; however, they do appear to involve the cytoskeleton and posttranslation modification of the components regulated by protein kinases, protein phosphatases, and phospholipases. Furthermore, several key components regulating NADPH oxidase recruitment, assembly, and activation are enriched in lipid microdomains to form a functional signaling platform. Future studies on temporal and spatial localization of Nox isoforms will provide new insights into the role of NADPH oxidase-derived ROS in the pathobiology of lung diseases.     

Regulation of proliferation of skeletal muscle precursor cells by NADPH oxidase

Skeletal muscle precursor cells are adult stem cells located among muscle fibers. Proliferation, migration, and subsequent differentiation of these cells are critical steps in the repair of muscle injury. We document in this study the roles and mechanisms through which the NAPDH oxidase complex regulates muscle precursor cell proliferation. The NADPH oxidase subunits Nox2, Nox4, p22(phox), p47(phox), and p67(phox) were detected in primary human and murine skeletal muscle precursor cells. In human muscle precursor cells, NADPH oxidase-fusion proteins were localized in the cytosolic and membrane compartments of the cell, except for p47(phox), which was detected in the nucleus. In proliferating subconfluent precursor cells, both Nox2 and Nox4 contributed to O(2)(-) production. However, Nox4 expression was significantly attenuated in differentiated myotubes. Proliferation of precursor cells was significantly reduced by antioxidants (N-acetylcysteine and apocynin), inhibition of p22(phox) expression by using siRNA oligonucleotides, and reduction of Nox4 and p47(phox) activities with dominant-negative vectors and siRNA oligonucleotides resulted in attenuation of activities of the Erk1/2, PI-3 kinase/AKT and NFkappaB pathways and significant reduction in cyclin D1 levels. We conclude that NADPH oxidase is expressed in skeletal muscle precursor cells and that its activity plays an important role in promoting proliferation of these cells.     

Evaluation of the expression of NADPH oxidase components during maturation of HL-60 clone 15 cells to eosinophilic lineage

OBJECTIVE: Superoxide-generating NADPH oxidase consists of the membrane-bound cytochrome b558 (gp91phox and p22Phox) and the cytosolic components (p67phox, p47phox, p40phox and rac). In this study, we evaluated the superoxide-generating activity and the expression of NADPH oxidase components during eosinophilic maturation using HL-60 clone 15 cell line. MATERIALS AND METHODS: HL-60 clone 15 cells were matured to eosinophils by incubation with 0.5 mM butyrate for 7 days, and NADPH oxidase components were detected by Northern blot, Western blot analyses and immunocytochemical staining. Moreover, superoxide-generating activity was examined by nitro blue tetrazolium (NBT) assay. RESULTS: Northern blot and Western blot analyses revealed that mRNAs and proteins for gp91phox, p67phox and p47phox were expressed after eosinophilic myelocyte stages, whereas mRNAs and proteins for p40phox and rac-2 were expressed from the promyelocyte stage. Interestingly, p22phox mRNA was expressed from the promyelocyte stage, but its protein was expressed after eosinophilic myelocyte stages. Consistent with the results of Western blotting, immunocytochemical staining of butyrate-induced HL-60 clone 15 cells indicated that gp91phox, p22phox, p67phox and p47phox were detected after eosinophilic myelocyte stages (eosinophilic myelocytes, eosinophilic metamyelocytes, eosinophilic band cells and eosinophilic-segmented cells), whereas p40phox and rac-2 were expressed from the promyelocyte stage. Moreover, almost the same results as those with butyrate-treated HL-60 clone 15 cells were obtained using human bone marrow cells by immunocytochemical staining. Furthermore, nitro blue tetrazolium (NBT) assay indicated that superoxide could be produced after eosinophilic myelocyte stages but not produced before the promyelocyte stage. CONCLUSIONS: Together these observations indicate that all the components for NADPH oxidase are expressed, and the superoxide-producing activity is obtained after myelocyte stages during eosinophilic maturation.     

血管紧张素Ⅱ通过NADPH氧化酶源性氧化应激促进肾小球系膜细胞单核细胞趋化蛋白1表达

目的 探讨氧化应激在血管紧张素(Ang)Ⅱ诱导的系膜细胞单核细胞趋化蛋白1(MCP-1)表达中的作用.方法 体外培养人肾小球系膜细胞,应用即时定量PCR检测MCP-1表达;荧光探针2,7-二氯二氢荧光素乙酰乙酸(DCFDA)检测细胞内活性氧的产生;化学发光法检测尼克酰胺腺嘌呤二核苷酸磷酸(NADPH)氧化酶活性;Western blot检测p47phox和p67phox膜转位.24只雄性C57BL/6小鼠随机分为三组:正常对照组、模型组和夹竹桃麻素治疗组.采用酶联免疫吸附试验(ELISA)检测尿中8-isoprostane和白蛋白水平.结果 Ang Ⅱ呈剂量依赖性诱导MCP-1表达,100nmol/L AngⅡ诱导的MCP-1表达是对照组的3.56倍.Ang Ⅱ呈时间依赖性和剂量依赖性促进系膜细胞活性氧产生.AngⅡ刺激3 min,系膜细胞内活性氧产生明显增加,至60 min达到高峰.1、10和100 nmol/L AngⅡ刺激60 min,活性氧产生分别是对照组的1.82、2.92和4.08倍.AT1R拮抗剂氯沙坦完全阻断AngⅡ诱导的活性氧产生,而AT2R拮抗剂PDl23319无抑制作用.NADPH氧化酶抑制剂夹竹桃麻素和二联苯碘几乎完全阻断AngⅡ诱导的活性氧产生,而线粒体复合体Ⅰ抑制剂鱼藤酮、黄嘌呤氧化酶抑制剂别嘌醇、环氧化酶抑制剂吲哚美辛、脂氧化酶抑制剂去甲二氢化愈创木酸、细胞色素P450氧化酶抑制剂酮康唑以及一氧化氮合成酶抑制剂N-硝基-L-精氨酸甲酯(L-NAME)对Ang Ⅱ诱导的活性氧产生均无明显影响.Ang Ⅱ显著刺激NADPH氧化酶活化及p47 phox和p67phox膜转位.氯沙坦、乙酰半胱氨酸(NAC)、夹竹桃麻素和二联苯碘阻断AngⅡ诱导的MCP-1表达.AngⅡ灌注后尿中8-isoprostane、白蛋白排泄及肾组织中MCP-1表达分别是对照组的5.45、16.65和2.50倍;肾组织中NADPH氧化酶活性以及p47phox和p67phox膜转位分别是对照组的2.69、2.97和2.67倍.NADPH氧化酶抑制剂夹竹桃麻素显著减轻AngⅡ诱导的蛋白尿和氧化应激,并抑制MCP-1表达.结论 NADPH氧化酶来源的活性氧调控AngⅡ诱导的MCP-1表达,抑制NADPH氧化酶活性可减轻Ang Ⅱ诱导的肾脏损害.     

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

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

VEGF signaling through NADPH oxidase-derived ROS

Angiogenesis is a key process involved in normal development and wound repair, as well as ischemic heart and limb diseases, and atherosclerosis. Vascular endothelial growth factor (VEGF), a potent angiogenesis factor, stimulates proliferation, migration, and tube formation of endothelial cells (ECs), primarily through the VEGF receptor type2 (VEGFR2). Reactive oxygen species (ROS) function as signaling molecules to mediate biological responses. In ECs, NADPH oxidase is one of the major sources of ROS and consists of catalytic subunits (Nox1, Nox2, and Nox4), p22phox, p47phox, p67phox, and the small GTPase Rac1. VEGF stimulates ROS production via activation of gp91phox (Nox2)-based NADPH oxidase, and ROS are involved in VEGFR2-mediated signaling linked to EC migration and proliferation. Moreover, ROS derived from NADPH oxidase are involved in postnatal angiogenesis. Localizing NADPH oxidase and its regulators at the specific subcellular compartment is an important mechanism for activating specific redox signaling events. This review focuses on a role of NADPH oxidase-derived ROS in angiogenesis and critical regulators involved in generation of spatially and temporally restricted ROS-dependent VEGF signaling at leading edge, focal adhesions/complexes, caveolae/lipid rafts, and cell-cell junctions in ECs. Understanding these mechanisms should facilitate the development of new therapeutic strategies to modulate new blood vessel formation.     

Priming of neutrophil oxidative burst in diabetes requires preassembly of the NADPH oxidase

Hyperglycemia associated with diabetes mellitus results in the priming of neutrophils leading to oxidative stress that is, in part, responsible for diabetic complications. p47phox, a NADPH oxidase cytosolic subunit, is a key protein in the assembly of the NADPH oxidase leading to superoxide generation. Little is known about the priming mechanism of oxidative pathways in neutrophils of people with diabetes. In this study, the kinetics of p47phox activation was investigated by comparing neutrophils from diabetic and healthy subjects, and the mechanism of hyperglycemia-induced changes was studied by using neutrophil-like HL-60 cells as a model. In resting neutrophils from diabetic subjects, p47phox prematurely translocates to the cell membrane and preassembles with p22phox, a NADPH oxidase membrane subunit. This premature p47phox translocation and preassembly with p22phox were also observed in HL-60 cells cultured with high glucose (HG; 25 mM) and with the specific ligand for the receptor for advanced glycation end products (RAGE), S100B. Phosphorylation of ERK1/2, but not p38 MAPK, was the primary signaling pathway, as evidenced by PD98059 suppressing the translocation of p47phox in HL-60 cells incubated with HG and S100B. HL-60 cells cultured in HG and S100B exhibited a 1.8-fold increase in fMLP-induced superoxide generation compared with those cultured in normal glucose (5.5 mM). These data suggest that HG and increased AGE prime neutrophils and increase oxidative stress inducing the translocation of p47phox to the cell membrane and preassembly with p22phox by stimulating a RAGE-ERK1/2 pathway.     

Oxygen radical generation and expression of NADPH oxidase genes in bottlenose dolphin (Tursiops truncatus) neutrophils

Oxygen radical generation by stimulation with phorbol myristate acetate (PMA) was evaluated in bottlenose dolphin neutrophils. A Cypridina luciferin analog-dependent chemiluminescent assay demonstrated that dolphin neutrophils generate superoxide by the addition of PMA, and that its superoxide-forming activity is completely suppressed by diphenylene iodonium, a specific inhibitor of NADPH oxidase. These results indicate that dolphin neutrophils possess NADPH oxidase activity. Furthermore, the NADPH oxidase activity (hydrogen peroxide production) in dolphin neutrophils, as well as in human neutrophils, was greater at 37 degrees C than at a lower temperature. RT-PCR with specific primers revealed that dolphin neutrophils expressed the mRNAs of the major NADPH oxidase components, which included membrane-associated flavocytochrome b (gp91(phox) and p22(phox)) and cytosolic factors (p40(phox), p47(phox), and p67(phox)), implying the existence of these protein homologues in dolphin neutrophils.