scholarly journals Absence of Proton Channels in COS-7 Cells Expressing Functional NADPH Oxidase Components

2002 ◽  
Vol 119 (6) ◽  
pp. 571-580 ◽  
Author(s):  
Deri Morgan ◽  
Vladimir V. Cherny ◽  
Marianne O. Price ◽  
Mary C. Dinauer ◽  
Thomas E. DeCoursey
Keyword(s):  
Nadph Oxidase ◽  
Superoxide Anion ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Proton Channel ◽  
Charge Movement ◽  
Wild Type ◽  
Electrophysiological Properties ◽  
Proton Channels ◽  
Proton Currents ◽  
Gp91phox Subunit

Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase is an enzyme of phagocytes that produces bactericidal superoxide anion (O2−) via an electrogenic process. Proton efflux compensates for the charge movement across the cell membrane. The proton channel responsible for the H+ efflux was thought to be contained within the gp91phox subunit of NADPH oxidase, but recent data do not support this idea (DeCoursey, T.E., V.V. Cherny, D. Morgan, B.Z. Katz, and M.C. Dinauer. 2001. J. Biol. Chem. 276:36063–36066). In this study, we investigated electrophysiological properties and superoxide production of COS-7 cells transfected with all NADPH oxidase components required for enzyme function (COSphox). The 7D5 antibody, which detects an extracellular epitope of the gp91phox protein, labeled 96–98% of COSphox cells. NADPH oxidase was functional because COSphox (but not COSWT) cells stimulated by phorbol myristate acetate (PMA) or arachidonic acid (AA) produced superoxide anion. No proton currents were detected in either wild-type COS-7 cells (COSWT) or COSphox cells studied at pHo 7.0 and pHi 5.5 or 7.0. Anion currents that decayed at voltages positive to 40 mV were the only currents observed. PMA or AA did not elicit detectable H+ current in COSWT or COSphox cells. Therefore, gp91phox does not function as a proton channel in unstimulated cells or in activated cells with a demonstrably functional oxidase.

scholarly journals Role of nucleotides and phosphoinositides in the stability of electron and proton currents associated with the phagocytic NADPH oxidase

2006 ◽  
Vol 400 (3) ◽  
pp. 431-438 ◽  
Author(s):  
Gábor L. Petheő ◽  
Nathalie C. Girardin ◽  
Nicolas Goossens ◽  
Gergely Z. Molnár ◽  
Nicolas Demaurex
Keyword(s):  
Nadph Oxidase ◽  
Proton Channel ◽  
Soluble Factors ◽  
Phosphatase Inhibitors ◽  
Stabilizing Effect ◽  
Cytosolic Side ◽  
The Stability ◽  
Inside Out ◽  
Proton Currents

The phagocytic NADPH oxidase (phox) moves electrons across cell membranes to kill microbes. The activity of this lethal enzyme is tightly regulated, but the mechanisms that control phox inactivation are poorly understood for lack of appropriate assays. The phox generates measurable electron currents, Ie, that are associated with inward proton currents, IH. To study the inactivation of the phox and of its associated proton channel, we determined which soluble factors can stabilize Ie (induced by the addition of NADPH) and IH (initiated by small depolarizing voltage steps) in inside-out patches from PMA-activated human eosinophils. Ie decayed rapidly in the absence of nucleotides (τ≈6 min) and was maximally stabilized by the combined addition of 5 mM ATP and 50 μM of the non-hydrolysable GTP analogue GTP[S] (guanosine 5′-[γ-thio]triphosphate) (τ≈57 min), but not by either ATP or GTP[S] alone. IH also decayed rapidly and was stabilized by the ATP/GTP[S] mixture, but maximal stabilization of IH required further addition of 25 μM PI(3,4)P2 (phosphoinositide 3,4-bisphosphate) to the cytosolic side of the patch. PI(3,4)P2 had no effect on Ie and its stabilizing effect on IH could not be mimicked by other phosphoinositides. Reducing the ATP concentration below millimolar levels decreased IH stability, an effect that was not prevented by phosphatase inhibitors but by the non-hydrolysable ATP analogue ATP[S] (adenosine 5′-[γ-thio]triphosphate). Our data indicate that the assembled phox complex is very stable in eosinophil membranes if both ATP and GTP[S] are present, but inactivates within minutes if one of the nucleotides is removed. Stabilization of the phox-associated proton channel in a highly voltage-sensitive conformation does not appear to involve phosphorylation but ATP binding, and requires not only ATP and GTP[S] but also PI(3,4)P2, a protein known to anchor the cytosolic phox subunit p47phox to the plasma membrane.

scholarly journals Voltage-Gated Proton Channels Find Their Dream Job Managing the Respiratory Burst in Phagocytes

Physiology ◽  
2010 ◽  
Vol 25 (1) ◽  
pp. 27-40 ◽  
Author(s):  
Thomas E. DeCoursey
Keyword(s):  
Reactive Oxygen Species ◽  
Nadph Oxidase ◽  
Respiratory Burst ◽  
Proton Channel ◽  
Oxygen Species ◽  
Proton Extrusion ◽  
Proton Channels ◽  
Voltage Gated ◽  
Voltage Gated Ion Channels ◽  
Voltage Sensing Domain

The voltage-gated proton channel bears surprising resemblance to the voltage-sensing domain (S1–S4) of other voltage-gated ion channels but is a dimer with two conduction pathways. The proton channel seems designed for efficient proton extrusion from cells. In phagocytes, it facilitates the production of reactive oxygen species by NADPH oxidase.

scholarly journals Hu-Lu-Ba-Wan Attenuates Diabetic Nephropathy in Type 2 Diabetic Rats through PKC-α/NADPH Oxidase Signaling Pathway

2013 ◽  
Vol 2013 ◽  
pp. 1-10 ◽  
Author(s):  
Lishan Zhou ◽  
Hui Dong ◽  
Yi Huang ◽  
Lijun Xu ◽  
Xin Zou ◽  
...  
Keyword(s):  
Diabetic Nephropathy ◽  
Nadph Oxidase ◽  
Signaling Pathway ◽  
Superoxide Anion ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Renal Tissue ◽  
Diabetic Rats ◽  
Control Group ◽  
Type 2 Diabetic

Hu-Lu-Ba-Wan (HLBW) is a Chinese herbal prescription used to treat kidney deficiency. The aim of this study was to explore the effect and mechanism of HLBW on diabetic nephropathy (DN) in type 2 diabetic rats. The rat model of DN was established by being fed a high-fat diet and intravenous injection of streptozotocin. Then, HLBW decoction was administered for 16 weeks. Blood glucose level, lipid profile, renal function, 24-hour total urinary protein, and albumin content were examined. Renal morphology and superoxide anion levels were evaluated. The activity of nicotinamide-adenine dinucleotide phosphate (NADPH) and protein kinase C-alpha (PKC-α) related genes expression in renal tissue were also determined. Our data demonstrated that HLBW significantly improved hyperglycemia, hyperlipidemia, and proteinuria in diabetic rats compared with those of control group. HLBW also alleviated glomerular expansion and fibrosis, extracellular matrix accumulation and effacement of the foot processes. Additionally, HLBW reduced superoxide anion level, NADPH oxidase activity, the protein and mRNA expressions of p47phox, and the protein expression of phosphorylated PKC-αin renal tissue. These results suggest that HLBW is effective in the treatment of DN in rats. The underlying mechanism may be related to the attenuation of renal oxidative stress via PKC-α/NADPH oxidase signaling pathway.

scholarly journals Hypoxic induction of gene expression in chronic granulomatous disease- derived B-cell lines: oxygen sensing is independent of the cytochrome b558-containing nicotinamide adenine dinucleotide phosphate oxidase

Blood ◽  
1996 ◽  
Vol 87 (2) ◽  
pp. 756-761 ◽  
Author(s):  
RH Wenger ◽  
HH Marti ◽  
CC Schuerer-Maly ◽  
I Kvietikova ◽  
C Bauer ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
B Cell ◽  
Cell Lines ◽  
Nicotinamide Adenine Dinucleotide ◽  
Oxygen Sensing ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Wild Type ◽  
Superoxide Generation ◽  
Cytochrome B558 ◽  
Nadph Oxidase Complex

Reduced oxygenation of a variety of cells results in transcriptional upregulation of several genes, including the hematopoietic hormone erythropoietin, the angiogenic vascular endothelial growth factor (VEGF), and glycolytic enzymes such as aldolase. Recently, the heme protein cytochrome b558 of the nicotinamide adenine dinucleotide phosphate (NADPH) oxidase complex has been proposed as a key component of the oxygen-sensing mechanism. Cytochrome b558 consists of the p22phox and gp91phox subunits and is essential for superoxide generation in phagocytes and B lymphocytes. Mutations in these subunits result in cytochrome b558-negative chronic granulomatous disease (cytb- CGD), an inherited disorder in humans characterized by reduced microbicidal activity due to deficient superoxide generation. To test whether NADPH oxidase is involved in oxygen sensing, we exposed wild- type B-cell lines as well as cytb- CGD-derived B cell lines, deficient in either p22phox or gp91phox, to hypoxia (1% oxygen) or CoCl2 (100 mumol/L) and compared the mRNA levels of VEGF and aldolase with the untreated controls. Northern blot analysis revealed unimpaired basal and inducible expression of VEGF and aldolase mRNA in all four cytb- CGD-derived B-cell lines compared with wild-type cells. Furthermore, reconstitution of cytochrome b558 expression in cytb- CGD-derived B cells by transfection with p22phox or gp91phox expression vectors did not modify VEGF and aldolase mRNA expression. Thus, cytochrome b558 of the NADPH oxidase complex appears not to be essential for hypoxia- activated gene expression and can be excluded as a candidate for the putative universal oxygen sensor.

scholarly journals Voltage-gated proton channels exist in the plasma membrane of human oocytes

2019 ◽  
Vol 34 (10) ◽  
pp. 1974-1983 ◽  
Author(s):  
R Ya Smith ◽  
D Morgan ◽  
L Sharma ◽  
V V Cherny ◽  
N Tidswell ◽  
...  
Keyword(s):  
Large Scale ◽  
Human Sperm ◽  
Proton Channel ◽  
Human Oocytes ◽  
Proton Channels ◽  
Voltage Gated ◽  
Registration Number ◽  
Heterologous Expression Systems ◽  
Competing Interest ◽  
Proton Currents

Abstract STUDY QUESTION Do human oocytes express voltage-gated proton channels? SUMMARY ANSWER Human oocytes exhibit voltage-gated proton currents. WHAT IS KNOWN ALREADY Voltage-gated proton currents have been reported in human sperm, where they contribute to capacitation and motility. No such studies of human oocytes exist. STUDY DESIGN, SIZE, DURATION Voltage-clamp studies were undertaken using entire oocytes and vesicles derived from oocytes and in excised patches of membrane from oocytes. PARTICIPANTS/MATERIALS, SETTING, METHODS Frozen, thawed human metaphase II oocytes were obtained from material donated to the gamete repository at the Rush Center for Advanced Reproductive Care. Prior to patch clamping, oocytes were warmed and equilibrated. Formation of an electrically tight seal requires exposing bare oolemma. Sections of the zona pellucida (ZP) were removed using a laser, followed by repeated pipetting, to further separate the oocyte from the ZP. Patch-clamp studies were performed using the whole-cell configuration on oocytes or vesicles derived from oocytes, and using inside-out patches of membrane, under conditions optimized to detect voltage-gated proton currents. MAIN RESULTS AND THE ROLE OF CHANCE Proton currents are present at significant levels in human oocytes where they exhibit properties similar to those reported in other human cells, as well as those in heterologous expression systems transfected with the HVCN1 gene that codes for the voltage-gated proton channel. LARGE SCALE DATA N/A LIMITATIONS, REASONS FOR CAUTION Human oocytes are large cells, which limits our ability to control the intracellular solution. Subtle effects of cryopreservation by vitrification and subsequent warming on properties of HVCN1, the HVCN1 gene product, cannot be ruled out. WIDER IMPLICATIONS OF THE FINDINGS Possible functions for voltage-gated proton channels in human oocytes may now be contemplated. STUDY FUNDING/COMPETING INTEREST(S) NIH R35GM126902 (TED), Bears Care (DM). No competing interests. TRIAL REGISTRATION NUMBER N/A.

scholarly journals Endothelin-1-Induced Microvascular ROS and Contractility in Angiotensin-II-Infused Mice Depend on COX and TP Receptors

Antioxidants ◽  
2019 ◽  
Vol 8 (6) ◽  
pp. 193 ◽  
Author(s):  
Christopher S. Wilcox ◽  
Cheng Wang ◽  
Dan Wang
Keyword(s):  
Angiotensin Ii ◽  
Nadph Oxidase ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Ros Generation ◽  
Oxygen Species ◽  
Ang Ii ◽  
Wild Type ◽  
Endothelin 1 ◽  
Ros Metabolism ◽  
Tp Receptors

(1) Background: Angiotensin II (Ang II) and endothelin 1 (ET-1) generate reactive oxygen species (ROS) that can activate cyclooxygenase (COX). However, thromboxane prostanoid receptors (TPRs) are required to increase systemic markers of ROS during Ang II infusion in mice. We hypothesized that COX and TPRs are upstream requirements for the generation of vascular ROS by ET-1. (2) Methods: ET-1-induced vascular contractions and ROS were assessed in mesenteric arterioles from wild type (+/+) and knockout (−/−) of COX1 or TPR mice infused with Ang II (400 ng/kg/min × 14 days) or a vehicle. (3) Results: Ang II infusion appeared to increase microvascular protein expression of endothelin type A receptors (ETARs), TPRs, and COX1 and 2 in COX1 and TPR +/+ mice but not in −/− mice. Ang II infusion increased ET-1-induced vascular contractions and ROS, which were prevented by a blockade of COX1 and 2 in TPR +/+ mice. ET-1 increased the activity of aortic nicotinamide adenine dinucleotide phosphate (NADPH) oxidase and decreased superoxide dismutase (SOD) 1, 2, and 3 in Ang-II-infused mice, which were prevented by a blockade of TPRs. (4) Conclusion: Activation of vascular TPRs by COX products are required for ET-1 to increase vascular contractions and ROS generation from NADPH oxidase and reduce ROS metabolism by SOD. These effects require an increase in these systems by prior infusion of Ang II.

scholarly journals Electron and proton transport by NADPH oxidases

2005 ◽  
Vol 360 (1464) ◽  
pp. 2315-2325 ◽  
Author(s):  
Nicolas Demaurex ◽  
Gábor L Petheö
Keyword(s):  
Patch Clamp ◽  
Nadph Oxidase ◽  
White Blood Cells ◽  
Proton Channel ◽  
Redox Activity ◽  
Patch Clamp Technique ◽  
Proton Channels ◽  
Clamp Technique ◽  
Electron And Proton Transport ◽  
Excised Patches

The NADPH oxidase is the main weapon of phagocytic white blood cells that are the first line of defence of our body against invading pathogens, and patients lacking a functional oxidase suffer from severe and recurrent infections. The oxidase is a multisubunit enzyme complex that transports electrons from cytoplasmic NADPH to molecular oxygen in order to generate superoxide free radicals. Electron transport across the plasma membrane is electrogenic and is associated with the flux of protons through voltage-activated proton channels. Both proton and electron currents can be recorded with the patch-clamp technique, but whether the oxidase is a proton channel or a proton channel modulator remains controversial. Recently, we have used the inside–out configuration of the patch-clamp technique to record proton and electron currents in excised patches. This approach allows us to measure the oxidase activity under very controlled conditions, and has provided new information about the enzymatic activity of the oxidase and its coupling to proton channels. In this chapter I will discuss how the unique characteristics of the electron and proton currents associated with the redox activity of the NADPH oxidase have extended our knowledge about the thermodynamics and the physiological regulation of this remarkable enzyme.

Cadmium decreased superoxide anion derived from NADPH oxidase through overload of calcium in wheat seedling

2020 ◽  
Vol 52 (5) ◽  
Author(s):  
Hongjuan Jiang ◽  
Yi Fu ◽  
Cuixiang Li ◽  
Mengying Chen ◽  
Zewei Gu ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Superoxide Anion ◽  
Wheat Seedling

scholarly journals Sex-dependent dysregulation of human neutrophil responses by bisphenol A

2021 ◽  
Vol 20 (1) ◽  
Author(s):  
Wioletta Ratajczak-Wrona ◽  
Marzena Garley ◽  
Malgorzata Rusak ◽  
Karolina Nowak ◽  
Jan Czerniecki ◽  
...  
Keyword(s):  
Nadph Oxidase ◽  
Bisphenol A ◽  
Total Concentration ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Human Neutrophils ◽  
Boyden Chamber ◽  
No Production ◽  
Pathogen Elimination ◽  
Latex Beads

Abstract Background In the present study, we aimed to investigate selected functions of human neutrophils exposed to bisphenol A (BPA) under in vitro conditions. As BPA is classified among xenoestrogens, we compared its action and effects with those of 17β-estradiol (E2). Methods Chemotaxis of neutrophils was examined using the Boyden chamber. Their phagocytosis and nicotinamide adenine dinucleotide phosphate hydrogen (NADPH) oxidase activity were assessed via Park’s method with latex beads and Park’s test with nitroblue tetrazolium. To assess the total concentration of nitric oxide (NO), the Griess reaction was utilized. Flow cytometry was used to assess the expression of cluster of differentiation (CD) antigens. The formation of neutrophil extracellular traps (NETs) was analyzed using a microscope (IN Cell Analyzer 2200 system). Expression of the investigated proteins was determined using Western blot. Results The analysis of results obtained for both sexes demonstrated that after exposure to BPA, the chemotactic capacity of neutrophils was reduced. In the presence of BPA, the phagocytic activity was found to be elevated in the cells obtained from women and reduced in the cells from men. Following exposure to BPA, the percentage of neutrophils with CD14 and CD284 (TLR4) expression, as well as the percentage of cells forming NETs, was increased in the cells from both sexes. The stimulatory role of BPA and E2 in the activation of NADPH oxidase was observed only in female cells. On the other hand, no influence of E2 on the expression of CD14 and CD284, chemotaxis, phagocytosis, and the amount of NET-positive neutrophils was found for both sexes. The study further showed that BPA intensified NO production and iNOS expression in the cells of both sexes. In addition, intensified expression of all tested PI3K-Akt pathway proteins was observed in male neutrophils. Conclusions The study demonstrated the influence of BPA on neutrophil functions associated with locomotion and pathogen elimination, which in turn may disturb the immune response of these cells in both women and men. Analysis of the obtained data showed that the effect of this xenoestrogen on the human neutrophils was more pronounced than E2.

scholarly journals Akt isoforms differentially regulate neutrophil functions

Blood ◽  
2010 ◽  
Vol 115 (21) ◽  
pp. 4237-4246 ◽  
Author(s):  
Jia Chen ◽  
Haiyang Tang ◽  
Nissim Hay ◽  
Jingsong Xu ◽  
Richard D. Ye
Keyword(s):  
Kinase Inhibitor ◽  
Nicotinamide Adenine Dinucleotide Phosphate ◽  
Leading Edge ◽  
Neutrophil Activation ◽  
Enzyme Release ◽  
Wild Type ◽  
Akt Isoforms ◽  
Phosphoinositide 3 Kinase ◽  
O2 Production

In neutrophils, the phosphoinositide 3-kinase/Akt signaling cascade is involved in migration, degranulation, and O2− production. However, it is unclear whether the Akt kinase isoforms have distinct functions in neutrophil activation. Here we report functional differences between the 2 major Akt isoforms in neutrophil activation on the basis of studies in which we used individual Akt1 and Akt2 knockout mice. Akt2−/− neutrophils exhibited decreased cell migration, granule enzyme release, and O2− production compared with wild-type and Akt1−/− neutrophils. Surprisingly, Akt2 deficiency and pharmacologic inhibition of Akt also abrogated phorbol ester-induced O2− production, which was unaffected by treatment with the phosphoinositide 3-kinase inhibitor LY294002. The decreased O2− production in Akt2−/− neutrophils was accompanied by reduced p47phox phosphorylation and its membrane translocation, suggesting that Akt2 is important for the assembly of phagocyte nicotinamide adenine dinucleotide phosphate oxidase. In wild-type neutrophils, Akt2 but not Akt1 translocated to plasma membrane upon chemoattractant stimulation and to the leading edge in polarized neutrophils. In the absence of Akt2, chemoattractant-induced Akt protein phosphorylation was significantly reduced. These results demonstrate a predominant role of Akt2 in regulating neutrophil functions and provide evidence for differential activation of the 2 Akt isoforms in neutrophils.

Sign in / Sign up

Export Citation Format

Share Document