Plasma Membrane Oxidoreductases Research Articles

The effect of inhibitors of plasma membrane H+ - ATPase and oxidoreductases on NH4+ uptake by Pisum arvense roots

The effect of inhibitors of plasma membrane oxidoreductases (quinacrine and dicumarol) and H<sup>+</sup>-ATPase (dicyclohexylcarbodiimide and orthovanadate) on ammonium uptake by <i>Pisum arvense</i> seedlings and the activities of H<sup>+</sup>-ATPase and NADH-ferricyanide oxidoreductase was investigated. The uptake solution contained 50 µM NH<sub>4</sub><sup>+</sup>. In I h experiments, quinacrine and dicumarol depressed strongly and irreversibly the rate of NH<sub>4</sub><sup>+</sup> uptake and markedly inhibited the activity of NADH-ferri-cyanide oxidoreductase in the plasma membrane vesicles prepared from root cells. Simultaneously, sodium orthovanadate inhibited the activity of plasma membrane H<sup>+</sup>-ATPase increased the rate of NH<sub>4</sub><sup>+</sup> uptake. Dicyclohexylcarbodiimide inhibited H<sup>+</sup>-ATPase activity and increased efflux of NH<sub>4</sub><sup>+</sup> from roots to ambient solution. The results indicate on the lack of direct connection between uptake rate of 50 µM NH<sub>4</sub><sup>+</sup> and H<sup>+</sup>-ATPase activity, and suggest that membrane redox systems play a predominant role in this process.

Protein Disulfide Isomerase Regulates Sickle Erythrocyte Volume Via ET-1 Dependent Casein Kinase II Mechanism

Abstract 2114Disordered K+ efflux and osmotically induced water loss leads to red blood cell (RBC) dehydration and plays a role in the pathophysiology of Sickle Cell Disease. We previously reported that activation of endothelin-1 (ET-1) receptors in sickle erythrocyte was partially responsible for dense sickle cell formation. However, the mechanism by which ET-1 regulates RBC volume remains unclear. Serine/threonine kinases have been shown to regulate K+ transport in RBC. Casein Kinase II (CK2), a serine/threonine kinase, phosphorylates acidic proteins, regulates calmodulin activity and cytoskeletal proteins and is present in RBC. CK2 activity is blocked by apigenin, emodin, heparin, and ornithine decarboxylase. Previous reports have shown a role for flavonoids such as apigenin as substrates for erythrocyte plasma membrane oxidoreductases. We recently observed a role for Protein Disulfide Isomerase (PDI) in regulating cellular hydration and K+ efflux in human RBC. PDI catalyzes disulfide interchange reactions in the plasma membrane, mediates redox modifications and is up-regulated under hypoxic conditions. However the relationship between CK2 and PDI in the setting of cellular hydration status is un-explored. Our results indicate that erythrocyte membrane CK2 activity increases when sickle cells are incubated with 500 nM ET-1 for 30 min (2.8 ± 0.1 to 4.9 ± 0.01 nmol/min/mL * 106 cell) an event that is blunted by pre-incubation with the ET-1 B receptor blocker, BQ788 (2.5 ± 0.1 nmol/min/mL * 106 cell, n=3, p<0.04) and 20 μM apigenin (2.7 ± 0.4 nmol/min/mL * 106 cell, n=3, p<0.04). We examined the role of CK2 activation on cellular dehydration. We incubated sickle erythrocytes for 3 hours in deoxygenation-oxygenation cycles in the presence or absence of 20μM apigenin or 2μM 4,5,6,7-tetrabromobenzotriazole (TBB), a specific CK2 inhibitor, and measured the changes in erythrocyte density by phthalate oil density analysis. We observed that inhibition of CK2 led to reduced deoxygenation-stimulated cellular dehydration in sickle erythrocytes by apigenin (D50= 1.106 to 1.100 g/mL) or TBB (D50 =1.097 g/mL). We then studied the role of CK2 inhibitors on PDI activity by Insulin Turbidity Assay and observed that apigenin and TBB led to significant reductions in PDI activity in vitro (64% and 42% respectively). We also studied the effects of the flavonoids: naringenin, naringin, apigenin and rutin on PDI activity and observed reductions in PDI activity that were greater with apigenin>rutin>TBB>naringin>naringenin (n=2, P<0.05). Furthermore, we observed that K+ flux via Gardos channel activation is correlated with PDI activity in vitro in sickle erythrocytes. Taken together our results implicate CK2 and PDI as intermediate regulators of ET-1 stimulated cellular volume systems in red blood cells. Supported by NIH R01-HL09632 to AR. Disclosures:No relevant conflicts of interest to declare.

Plasma membrane electron transport in pancreatic β-cells is mediated in part by NQO1

Plasma membrane electron transport (PMET), a cytosolic/plasma membrane analog of mitochondrial electron transport, is a ubiquitous system of cytosolic and plasma membrane oxidoreductases that oxidizes cytosolic NADH and NADPH and passes electrons to extracellular targets. While PMET has been shown to play an important role in a variety of cell types, no studies exist to evaluate its function in insulin-secreting cells. Here we demonstrate the presence of robust PMET activity in primary islets and clonal β-cells, as assessed by the reduction of the plasma membrane-impermeable dyes WST-1 and ferricyanide. Because the degree of metabolic function of β-cells (reflected by the level of insulin output) increases in a glucose-dependent manner between 4 and 10 mM glucose, PMET was evaluated under these conditions. PMET activity was present at 4 mM glucose and was further stimulated at 10 mM glucose. PMET activity at 10 mM glucose was inhibited by the application of the flavoprotein inhibitor diphenylene iodonium and various antioxidants. Overexpression of cytosolic NAD(P)H-quinone oxidoreductase (NQO1) increased PMET activity in the presence of 10 mM glucose while inhibition of NQO1 by its inhibitor dicoumarol abolished this activity. Mitochondrial inhibitors rotenone, antimycin A, and potassium cyanide elevated PMET activity. Regardless of glucose levels, PMET activity was greatly enhanced by the application of aminooxyacetate, an inhibitor of the malate-aspartate shuttle. We propose a model for the role of PMET as a regulator of glycolytic flux and an important component of the metabolic machinery in β-cells.

Transplasma membrane electron transport comes in two flavors.

All tested cells possess transplasma membrane electron transfer (tPMET) systems that are capable of reducing extracellular electron acceptors at the cost of cytosolic electron donors. In mammals, classically NAD(P)H- and NADH-dependent systems have been distinguished. The NADH-dependent system has been suggested to be involved in non-transferrin-bound iron (NTBI) reduction and uptake. Recently we reported that transplasma membrane ascorbate/dehydroascorbate cycling can promote NTBI reduction and uptake by human erythroleukemia (K562) cells (D.J.R. Lane and A. Lawen, J Biol Chem 28 (2008), 12701-12708). This system, involves i) cellular import of dehydroascorbate, ii) intracellular reduction of dehydroascorbate to ascorbate using metabolically-derived reducing equivalents, iii) export of ascorbate down its concentration gradient, iv) direct reduction of low molecular weight iron chelates by ascorbate, and v) uptake of iron (II) into the cell. We here propose the consideration of this system as a novel form of tPMET which shares with classical enzyme-mediated tPMET systems the net transfer of reducing equivalents from the cytoplasmic compartment to the extracellular space, but lacks the involvement of the plasma membrane oxidoreductases responsible for the latter. Thus, transplasma membrane electron transfer can and does occur at two mechanistically distinct levels: i) enzyme-mediated transmembrane electron transfer and ii) transmembrane metabolite shuttling/cycling.

Mitochondria accumulate large amounts of quercetin: prevention of mitochondrial damage and release upon oxidation of the extramitochondrial fraction of the flavonoid

Quercetin uptake in Jurkat cells is extremely rapid and associated with a remarkable accumulation of the flavonoid, dependent on its binding to intracellular components. Cell-associated quercetin is biologically active, quantitatively consumed to promote survival in the presence of reactive species, such as peroxynitrite (ONOO −), or reduction of extracellular oxidants via activation of plasma membrane oxidoreductases. In alternative, quercetin is very slowly released upon post-incubation in drug-free medium, an event significantly accelerated by extracellular albumin. Quercetin uptake is also observed in isolated mitochondria, resulting in an enormous accumulation of the flavonoid, consumed under conditions associated with prevention of lipid peroxidation induced by ONOO −. Interestingly, remarkable quercetin accumulation is also detected in the mitochondria isolated from quercetin-pre-loaded cells, and exposure to either ONOO − or extracellular oxidants caused the parallel loss of both the mitochondrial and cytosolic fractions of the flavonoid. In conclusion, Jurkat cells accumulate large amounts of quercetin and even larger amounts of the flavonoid further accumulate in their mitochondria. Intramitochondrial quercetin appears to be functional for prevention of mitochondrial damage as well as for redistribution to the cytosol, when the fraction of the flavonoid therein retained is progressively consumed either by cell-permeant oxidants or by activation of plasma membrane oxidoreductases.

Transplasma membrane electron transport comes in two flavors

AbstractAll tested cells possess transplasma membrane electron transfer (tPMET) systems that are capable of reducing extracellular electron acceptors at the cost of cytosolic electron donors. In mammals, classically NAD(P)H‐ and NADH‐dependent systems have been distinguished. The NADH‐dependent system has been suggested to be involved in non‐transferrin‐bound iron (NTBI) reduction and uptake. Recently we reported that transplasma membrane ascorbate/dehydroascorbate cycling can promote NTBI reduction and uptake by human erythroleukemia (K562) cells (D.J.R. Lane and A. Lawen, J Biol Chem 283 (2008), 12701‐12708). This system, involves i) cellular import of dehydroascorbate, ii) intracellular reduction of dehydroascorbate to ascorbate using metabolically‐derived reducing equivalents, iii) export of ascorbate down its concentration gradient, iv) direct reduction of low molecular weight iron chelates by ascorbate, and v) uptake of iron (II) into the cell. We here propose the consideration of this system as a novel form of tPMET which shares with classical enzyme‐mediated tPMET systems the net transfer of reducing equivalents from the cytoplasmic compartment to the extracellular space, but lacks the involvement of the plasma membrane oxidoreductases responsible for the latter. Thus, transplasma membrane electron transfer can and does occur at two mechanistically distinct levels: i) enzyme‐mediated transmembrane electron transfer and ii) transmembrane metabolite shuttling/cycling.

Electron Pathways through Erythrocyte Plasma Membrane in Human Physiology and Pathology: Potential Redox Biomarker?

Erythrocytes are involved in the transport of oxygen and carbon dioxide in the body. Since pH is the influential factor in the Bohr-Haldane effect, pHi is actively maintained via secondary active transports Na+/H+exchange and HC3–/Cl–anion exchanger. Because of the redox properties of the iron, hemoglobin generates reactive oxygen species and thus, the human erythrocyte is constantly exposed to oxidative damage. Although the adult erythrocyte lacks protein synthesis and cannot restore damaged proteins, it is equipped with high activity of protective enzymes. Redox changes in the cell initiate various signalling pathways. Plasma membrane oxido-reductases (PMORs) are trans-membrane electron transport systems that have been found in the membranes of all cells and have been extensively characterized in the human erythrocyte. Erythrocyte PMORs transfer reducing equivalents from intracellular reductants to extracellular oxidants, thus their most important role seems to be to enable the cell respond to changes in intra- and extra-cellular redox environments.So far the activity of erythrocyte PMORs in disease states has not been systematically investigated. This review summarizes present knowledge on erythrocyte electron transfer activity in humans (health, type 1 diabetes, diabetic nephropathy, and chronic uremia) and hypothesizes an integrated model of the functional organization of erythrocyte plasma membrane where electron pathways work in parallel with transport metabolons to maintain redox homeostasis.

Plasma Membrane Oxidoreductases: Effects on Erythrocyte Metabolism and Redox Homeostasis

Plasma membrane oxidoreductases (PMORs) have been found in the membranes of all cells. These systems have been studied extensively in the human erythrocyte, so much is known about their activity and effect on erythrocyte cellular functioning. PMORs have been shown to be involved in a number of events associated with cell growth and function in other cell lines, but perhaps their most important role, especially in the nucleus- free mature erythrocyte, is as a redox sensor. The PMOR reduces extracellular oxidants by using the reducing power of intracellular antioxidants, making the cell metabolism respond to changes in the local redox environment. Thus, the activity of the PMOR is closely linked to the metabolic status of the erythrocyte. The main intracellular reductant for this system is ascorbic acid; however, the cell must also have the ability to supply NADH for full activity. Nuclear magnetic resonance studies on the effects of extracellular oxidants on intracellular metabolism have increased our knowledge of the intimate link between PMOR activity and metabolism, and these studies are reviewed here in detail.

Developmental regulation of the H 2O 2-producing system and of a basic peroxidase isoenzyme in the Zinnia elegans lignifying xylem

The lignin content, H 2O 2 production and peroxidase (EC 1.11.1.7) isoenzyme patterns of the lignifying xylem of the first internode from Zinnia elegans L. stems were analyzed at three different developmental stages. The results showed that both H 2O 2 production and the catalytic activity of a basic peroxidase isoenzyme were strongly regulated developmental processes, which coincided temporally with the onset of lignification. H 2O 2 production by the lignifying xylem was stimulated by the sulfhydryl blockers, N-ethyl-maleimide (NEM) and N-pyrenyl-maleimide (NPM), and this sulfhydryl blocker-stimulated H 2O 2 production was inhibited by imidazole, suggesting that it was performed by an NADPH oxidase-like enzyme. These distinctive features of H 2O 2 production by the lignifying Z. elegans xylem open up the possibility of unambiguously identifying this enzymatic system responsible for the delivery of H 2O 2 to xylem peroxidases from the vast array of plasma membrane oxidoreductases present in plant cells.

Reduction of trans-4,5-Dihydroxy-1,2-dithiane by Cellular Oxidoreductases Activates gadd153/chop andgrp78 Transcription and Induces Cellular Tolerance in Kidney Epithelial Cells

trans-4,5-Dihydroxy-1,2-dithiane, the intramolecular disulfide form of dithiothreitol (DTTox) transcriptionally activates the stress-responsive genes gadd153(chop) and grp78. Herein, we used a renal epithelial cell line, LLC-PK1, to investigate the mechanism(s) whereby DTTox activates a molecular stress response. DTTox activated both grp78 and gadd153 transcriptionally, but gadd153 mRNA stability also increased suggesting that both transcriptional and posttranscriptional mechanisms are involved. DTTox did not activate hsp70 transcription indicating that a heat shock response was not induced. Structure-activity studies showed that DTTox analogues lacking the intramolecular disulfide were inactive. Furthermore, the ring-open intermolecular disulfide form of DTTox, 2-hydroxyethyl disulfide, was only a weak inducer of grp78 and gadd153 but was a strong inducer of hsp70 mRNA and a potent oxidant that lowered the NADPH/NADP+ ratio and depleted reduced glutathione (GSH). DTTox had little effect on the overall GSH and NADPH levels; thus cells were not undergoing oxidative stress; however, the NADPH/NADP+ ratio decreased slightly indicating that reducing equivalents were consumed. LLC-PK1 cells reduced DTTox to DTT, and the kinetics as well as the concentration dependence for reduction correlated with induction of both grp78 and gadd153 mRNA. Prior treatment with DTTox rendered cells tolerant to the potent nephrotoxicant S-(1,1,2, 2-tetrafluoroethyl)-L-cysteine. Bacitracin, an inhibitor of plasma membrane oxidoreductases, blocked DTTox reduction and gene activation as well as DTTox-induced tolerance. Thus, activation of stress genes and induction of cellular tolerance by DTTox is mediated by a novel mechanism involving cellular oxidoreductases.

The plasma membrane NADH-diaphorase is active during selective phases of the cell cycle in mouse neuroblastoma cell line NB41A3. Its relation to cell growth and differentiation.

Plasma membrane oxidoreductases have been described in all cells and use extracellular impermeant electron acceptors (DCIP, Ferricyanide) that are reduced by NADH. They appear to regulate the overall cell activity in response to oxidative stress from the cellular environment. An NADH-DCIP reductase has been described at the plasma membrane of NB41A3, a neuroblastoma cell line (Zurbriggen and Dryer (1993) Biochim. Biophys. Acta 1183, 513-520) whose activation with extracellular impermeant substrates promotes cell growth. Elutriation was performed to separate cells and the various fractions were analysed for enzyme activity on intact cells combined with flow cytometry. These studies showed that the enzyme is mostly induced and activated during the G1 and during the G2/M-phases. These observations were further corroborated with specific inhibitors of the cell cycle. A three-fold increase in enzyme activity was observed in the presence of alpha-amanitin, a specific cell cycle inhibitor of the G1-phase. Taxol, a specific inhibitor of the M-phase, also induces a significant increase in enzyme activity. FACS analysis of taxol -treated and alpha-amanitin-treated cells corroborated these data. The cells have been synchronized and the enzyme activity was measured at different time intervals. An activity increase was observed after ca. 2-3 h, that corresponds to a raise in the M-phase, according to FACS data. Furthermore, NTera-2 cells - a human neuroblastoma cell line that differentiates into fully mature neurones in the presence of retinoic acid - exhibit a 50% decrease in the enzyme activity during the G0-phase upon differentiation, compared to undifferentiated cells. Together the data presented in this paper show that this plasma membrane NADH-diaphorase affects cell growth and differentiation and is strongly modulated at various phases of the cell cycle.

Effects of plasma membrane oxidoreductases on Ca2+ mobilization and protein phosphorylation in rat brain synaptosomes

We have investigated the possible role of plasma membrane oxidoreductases in the Ca2+ export mechanisms in rat brain synaptic membranes. Ca2+ efflux in nerve terminals is controlled both by a high-affinity/low capacity Mg-dependent ATP-stimulated Ca2+ pump and by a low affinity/high capacity ATP-independent Na(+)-Ca2+ exchanger. Both Ca2+ efflux mechanisms were strongly inhibited by pyridine nucleotides, in the order NADP greater than NAD greater than NADPH greater than NADH with IC50 values of ca. 10 mM for NADP and ca. 3 mM for the other agents in the case of the ATP-driven Ca2+ pump and with IC50 values between 8 and 10 mM for the Na(+)-Ca2+ exchanger. Oxidizing agents such as DCIP3 and ferricyanide inhibited the ATP-driven Ca2+ efflux mechanism but not the Na(+)-Ca2+ exchanger. In addition, full activation of plasma membrane oxidoreductases requires both an acceptor and an electron donor; therefore the combined effects of both substrates added together were also studied. When plasma membrane oxidoreductases of the synaptic plasma membrane were activated in the presence of both NADH (or NADPH) and DCIP or ferricyanide, the inhibition of the ATP-driven Ca2+ pump was optimal; by contrast, the pyridine nucleotide-mediated inhibition of the Na(+)-Ca2+ exchanger was partially released when both substrates of the plasma membrane oxidoreductases were present together. Furthermore, the activation of plasma membrane oxidoreductases also strongly inhibited intracellular protein phosphorylation in intact synaptosomes, mediated by either cAMP-dependent protein kinase, Ca2+ calmodulin-dependent protein kinases, or protein kinase C.