Abstract WP274: Inhibition of Na+/H+ Exchanger Isoform 1 and/or Voltage Gated Proton Channel Improves Outcomes in an Embolic Stroke Model

While Na+/H+ exchanger isoform 1 inhibitor (NHEi) has been reported to be neuro- and cardio-protective in experimental stroke and myocardial infarction, respectively, clinical use of NHEi for cardioprotection has stalled due to increased cerebrovascular events. NHEi has been demonstrated to increase Hv1 activity and we recently showed that NHEi activates NADPH oxidase and results in amplified superoxide formation in a voltage gated proton channel Hv1-dependent manner. In the CNS, Hv1 is localized primarily to microglia and deletion of Hv1 is neuroprotective after permanent and transient middle cerebral artery occlusion (MCAO). In the current study, we hypothesized that beneficial effect of NHEi after MCAO will be greater in a rat model lacking Hv1 due to loss of a potentially deleterious increase in Hv1 activation and NADPH oxidase activity. The wild type (WT) and Hv1 knockout (KO) rats (n=4-6) were treated with vehicle or NHEi (KR-32568, 2 mg/kg, i.v.) at 30 min after embolic MCAO. The neurological deficiency, infarct size, HT index, and edema ratio were assessed 3 days after surgery (Table). Compare to WT rats, KO rats had smaller infarct, less edema, and better neurological outcomes as previously found in the suture model. NHEi decreased infarct size and edema in both strains. While there was no significant difference in HT between WT and KO rats, the HT was less in WT rats with NHEi. Functional outcomes were significantly improved with NHEi in WT group, while the KO groups had a trend for a better outcomes with NHEi. These data indicate that NHE inhibition in the acute stroke period is similarly effective in both WT and Hv1 KO animals in providing neurovascular protection. Our data do not support the hypothesis that a deleterious increase in Hv1 dependent NADPH oxidase activity limits the beneficial actions of NHEi in embolic stroke. Further studies are needed to explore the underlying mechanism of the interaction between NHEi and the Hv1 channel in ischemic stroke.

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DJC Suppresses Advanced Glycation End Products-Induced JAK-STAT Signaling and ROS in Mesangial Cells

Evidence-based Complementary and Alternative Medicine ◽

10.1155/2017/2942830 ◽

2017 ◽

Vol 2017 ◽

pp. 1-11 ◽

Cited By ~ 3

Author(s):

Min Sun ◽

Yan Li ◽

Wenjie Bu ◽

Jindong Zhao ◽

Jianliang Zhu ◽

...

Keyword(s):

Nadph Oxidase ◽

Advanced Glycation End Products ◽

Oxidase Activity ◽

Mesangial Cells ◽

Dependent Manner ◽

Advanced Glycation ◽

Nadph Oxidase Activity ◽

Glycation End Products ◽

End Products ◽

Anti Inflammatory

The antidiabetic properties and anti-inflammatory effects of Danzhi Jiangtang Capsules (DJC) have been demonstrated in clinical and laboratory experiments. In this study, we explored whether DJC can ameliorate advanced glycation end products- (AGEs-) mediated cell injury and the precise mechanisms of DJC in treating diabetic nephropathy (DN). Western blot analysis was employed to assess the expressions of iNOS, COX2, and SOCS and the phosphorylation of JAK2, STAT1, and STAT3 in glomerular mesangial cells (GMCs) after treatment with DJC. TNF-α, IL-6, and MCP-1 were determined using double-antibody sandwich ELISA. ROS and NADPH oxidase activity were measured by DCFH-DA assay and lucigenin-enhanced chemiluminescence, respectively. DJC significantly reversed the AGEs-induced expression of COX2 and iNOS. Moreover, DJC inhibited the AGEs-induced JAK2-STAT1/STAT3 activation, resulting in the inhibition of inflammatory cytokines such as IL-6, MCP-1, and TNF-αin a concentration-dependent manner. The ability of DJC to suppress STAT activation was also verified by the observation that DJC significantly increased the SOCS3 protein level. DJC reversed the AGEs-induced accumulation of ROS and NADPH oxidase activity, thus confirming that DJC possesses antioxidant activity. The results suggest that the anti-inflammatory effects of DJC in GMCs may be due to its ability to suppress the JAK2-STAT1/STAT3 cascades and reduce ROS production.

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Abstract 211: Inhibition of Heme Oxygenase-1 (HO-1) Induces Mild Hypertension in Pregnant Rats

Hypertension ◽

10.1161/hyp.60.suppl_1.a211 ◽

2012 ◽

Vol 60 (suppl_1) ◽

Author(s):

Eric M George ◽

Frank Spradley ◽

Joey P Granger

Keyword(s):

Oxidative Stress ◽

Blood Pressure ◽

Nadph Oxidase ◽

Oxidase Activity ◽

Late Gestation ◽

Heme Oxygenase 1 ◽

Maternal Circulation ◽

Pregnant Rats ◽

Nadph Oxidase Activity ◽

Significant Difference

In the preeclamptic patient, inadequate remodeling of the maternal vasculature exacerbates this effect, causing dramatically increased oxidative stress in the placenta, which has been shown to be an important component of the maternal hypertension. There is also increasing awareness that HO-1 may act as an important regulator of placental function during normal pregnancy and decreases in HO-1 activity have been implicated in the pathogenesis of preeclampsia. While previous work in pregnant mice demonstrated that pharmacological inhibition of HO-1 leads to elevations in blood pressure, the mechanisms involved in the hypertension are unclear. The purpose of this study was to test the hypothesis that HO inhibition in late gestation leads to increases in maternal blood pressure by altering angiogenic balance and increasing placental oxidative stress in pregnant rats. HO activity was inhibited with tin mesoporphyrin (SnMP) was administered on gestational day 14, and blood pressure was measured on gestational day 19 by indwelling carotid catheter before sacrifice. In response to SnMP treatment, maternal MAP was significantly increased (99±1 vs 113±2 mmHg, p<0.05, n=15 per group). Placental sFlt-1 (631±47 vs 648±26 pg/mg, p=0.76) levels in the placenta were not affected by HO inhibition. Additionally, there was no significant difference in free VEGF in the maternal circulation (287±22 vs 329±14 pg/ml, p=0.11). There was, however, a significant increase in placental NADPH oxidase activity in SnMP treated rats (2021±238 vs 3005±301 RLU/min/mg, p<0.05) as determined by NADPH dependent lucigenin luminescence. This is likely due to decreased production of bilirubin, which is known to inhibit NADPH oxidase activity, and suggests an important role for HO-1 as an antioxidant in the developing placenta.

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The Phosphoinositide-Binding Protein p40phox Regulates NADPH Oxidase Activation Rather Than Assembly during FcγIIA Receptor-Induced Phagocytosis.

Blood ◽

10.1182/blood.v108.11.678.678 ◽

2006 ◽

Vol 108 (11) ◽

pp. 678-678 ◽

Cited By ~ 1

Author(s):

Wei Tian ◽

Xing Jun Li ◽

Natalie D. Stull ◽

Chang-Il Suh ◽

Sergio Grinstein ◽

...

Keyword(s):

Nadph Oxidase ◽

Oxidase Activity ◽

Superoxide Production ◽

Null Mouse ◽

Target Sequence ◽

Resting Cells ◽

Nadph Oxidase Activity ◽

Px Domain ◽

Significant Difference ◽

Nadph Oxidase Activation

Abstract Many critical features of the organization and regulation of the phagocyte NADPH oxidase, a complex multi-subunit enzyme that generates superoxide for microbial killing, remain poorly defined. The active enzyme includes a membrane-bound flavocytochrome b along with p47phox, p67phox, p40phox, and Rac-GTP that are present in the cytosol of resting cells. p67phox is linked by high affinity interactions with both p47phox and p40phox, which appear to translocate as a trimeric complex upon cellular activation. The p47phox subunit acts as an adaptor to promote translocation by docking at a proline-rich target sequence on the flavocytochrome, and p67phox is a Rac-GTP effector containing a domain that activates electron transport. In contrast, the function of p40phox, which is not required for high level oxidase activity in cell free systems, is poorly understood. Recently, our group showed that p40phox plays key role in the activation of superoxide production during phagocytosis of IgG-opsonized targets in COSphoxFcγR cells. This model cell line contains stable transgenes for the flavocytochrome, p47phox, p67phox, and the FcγIIA receptor, without or with an additional transgene for p40phox. p40phox-dependent coupling of FcγR-mediated phagocytosis to superoxide production required an intact p40phox PX domain, which binds to phosphatidylinositol-3-phosphate (PI3P), a phosphoinositide generated by class III PI3 kinases in phagosome membranes (Suh et al J Exp Med 203, 1915Suh et al J Exp Med 203, 2006). Furthermore, a newly developed p40phox-null mouse exhibits reduced neutrophil NADPH oxidase activity in response to selected agonists, including IgG-opsonized targets (Ellson et al J Exp Med 203, 1927Ellson et al J Exp Med 203, 2006). In the current study, we investigated whether p40phox is required for translocation of p67phox during phagocytosis. We generated COSphoxFcγR cells expressing YFP-tagged p67phox from a stable transgene instead of untagged p67phox. Following incubation with IgG-opsonized sheep red blood cells (IgG-RBC), p67phox was detected on phagosome membranes at both early stages of phagosome cup formation and after closure, independent of whether or not p40phox was also co-expressed. However, NADPH oxidase activity was not detected in IgG-RBC phagosomes in COSphoxFcγR-p67phox-YFP cells unless p40phox was present. PMA-activated superoxide production was independent of p40phox, and Western blotting indicated there was no significant difference in expression of the other oxidase subunits in COSphoxFcγR-p67phox-YFP cells without or with the p40phox transgene. Further studies in PLB-985 granulocytes expressing stable transgenes for either YFP-tagged p67phox or p40phox showed that the PI3K inhibitor wortmannin inhibited phagosome NADPH oxidase activity and translocation of p40phox, but localization of p67phox to phagosomes was unaffected. These results indicate that although p40phox positively regulates NADPH oxidase activation during phagocytosis, recruitment of p67phox to the phagosome is independent of p40phox. Taken together, these data suggest that the PX domain of p40phox acts as a PI3P-dependent switch to activate the membrane-assembled NADPH oxidase complex.

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Polydatin prevents angiotensin II-induced cardiac hypertrophy and myocardial superoxide generation

Experimental Biology and Medicine ◽

10.1177/1535370214561958 ◽

2014 ◽

Vol 240 (10) ◽

pp. 1352-1361 ◽

Cited By ~ 6

Author(s):

Qi Zhang ◽

Yingying Tan ◽

Nan Zhang ◽

Fanrong Yao

Keyword(s):

Nadph Oxidase ◽

Cardiac Hypertrophy ◽

Oxidase Activity ◽

Weight Ratio ◽

Heart Weight ◽

Leucine Incorporation ◽

Dependent Manner ◽

Ang Ii ◽

Nadph Oxidase Activity ◽

Cultured Cardiomyocytes

Our studies and others recently demonstrate that polydatin, a resveratrol glucoside, has antioxidative and cardioprotective effects. This study aims to investigate the direct effects of polydatin on Ang II-induced cardiac hypertrophy to explore the potential role of polydatin in cardioprotection. Our results showed that in primary cultured cardiomyocytes, polydatin blocked Ang II-induced cardiac hypertrophy in a dose-dependent manner, which were associated with reduction in the cell surface area and [3H]leucine incorporation, as well as attenuation of the mRNA expressions of atrial natriuretic factor and β-myosin heavy chain. Furthermore, polydatin prevented rat cardiac hypertrophy induced by Ang II infusion, as assessed by heart weight-to-body weight ratio, cross-sectional area of cardiomyocyte, and gene expression of hypertrophic markers. Further investigation demonstrated that polydatin attenuated the Ang II-induced increase in the reactive oxygen species levels and NADPH oxidase activity in vivo and in vitro. Polydatin also blocked the Ang II-stimulated increases of Nox4 and Nox2 expression in cultured cardiomyocytes and the hearts of Ang II-infused rats. Our results indicate that polydatin has the potential to protect against Ang II-mediated cardiac hypertrophy through suppression of NADPH oxidase activity and superoxide production. These observations may shed new light on the understanding of the cardioprotective effect of polydatin.

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Large-conductance calcium-activated potassium channel activity is absent in human and mouse neutrophils and is not required for innate immunity

AJP Cell Physiology ◽

10.1152/ajpcell.00450.2006 ◽

2007 ◽

Vol 293 (1) ◽

pp. C45-C54 ◽

Cited By ~ 42

Author(s):

Kirill Essin ◽

Birgit Salanova ◽

Ralph Kettritz ◽

Matthias Sausbier ◽

Friedrich C. Luft ◽

...

Keyword(s):

Innate Immunity ◽

Patch Clamp ◽

Nadph Oxidase ◽

Oxidase Activity ◽

Bk Channels ◽

Bk Channel ◽

Proton Channel ◽

Nadph Oxidase Activity ◽

Intracellular Ca ◽

Channel Currents

Large-conductance Ca2+-activated K+(BK) channels are reported to be essential for NADPH oxidase-dependent microbial killing and innate immunity in leukocytes. Using human peripheral blood and mouse bone marrow neutrophils, pharmacological targeting, and BK channel gene-deficient (BK−/−) mice, we stimulated NADPH oxidase activity with 12- O-tetradecanoylphorbol-13-acetate (PMA) and performed patch-clamp recordings on isolated neutrophils. Although PMA stimulated NADPH oxidase activity as assessed by O2−and H2O2production, our patch-clamp experiments failed to show PMA-activated BK channel currents in neutrophils. In our studies, PMA induced slowly activating currents, which were insensitive to the BK channel inhibitor iberiotoxin. Instead, the currents were blocked by Zn2+, which indicates activation of proton channel currents. BK channels are gated by elevated intracellular Ca2+and membrane depolarization. We did not observe BK channel currents, even during extreme depolarization to +140 mV and after elevation of intracellular Ca2+by N-formyl-l-methionyl-l-leucyl-phenylalanine. As a control, we examined BK channel currents in cerebral and tibial artery smooth muscle cells, which showed characteristic BK channel current pharmacology. Iberiotoxin did not block killing of Staphylococcus aureus or Candida albicans. Moreover, we addressed the role of BK channels in a systemic S. aureus and Yersinia enterocolitica mouse infection model. After 3 and 5 days of infection, we found no differences in the number of bacteria in spleen and kidney between BK−/−and BK+/+mice. In conclusion, our experiments failed to identify functional BK channels in neutrophils. We therefore conclude that BK channels are not essential for innate immunity.

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Apocynin Attenuates Cerebral Infarction after Transient Focal Ischaemia in Rats

Journal of International Medical Research ◽

10.1177/147323000703500411 ◽

2007 ◽

Vol 35 (4) ◽

pp. 517-522 ◽

Cited By ~ 63

Author(s):

LL Tang ◽

K Ye ◽

XF Yang ◽

JS Zheng

Keyword(s):

Nadph Oxidase ◽

Cerebral Infarction ◽

Infarct Size ◽

Oxidase Activity ◽

Nicotinamide Adenine Dinucleotide Phosphate ◽

Treated Group ◽

Oxidase Inhibitor ◽

Sprague Dawley ◽

Nadph Oxidase Activity ◽

Focal Ischaemia

This study investigated whether inhibition of reduced nicotinamide adenine dinucleotide phosphate (NADPH) oxidase attenuates cerebral infarction after transient focal ischaemia in rats. Focal ischaemia (1.5 h) was produced in male Sprague-Dawley rats (250 − 280 g) by middle cerebral artery occlusion. Some rats also received treatment with 50 mg/kg apocynin, a NADPH oxidase inhibitor, by intraperitoneal injection 30 min prior to reperfusion. Two hours after reperfusion, brains were harvested to measure NADPH oxidase activity and superoxide levels. After 24 h, the remaining brains were harvested to investigate infarct size. NADPH oxidase activity and superoxide level were all augmented 2 h after reperfusion compared with controls. Apocynin treatment significantly reduced NADPH oxidase activity and superoxide levels. Cerebral infarct size was significantly smaller in the apocynin-treated group compared with those undergoing ischaemia/reperfusion alone. These results indicate that inhibition of NADPH oxidase attenuates cerebral infarction after transient focal ischaemia in rats, suggesting that inhibition of NADPH oxidase may provide a therapeutic strategy for ischaemic stroke.

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