Overexpression of metallothionein decreases sensitivity of pulmonary endothelial cells to oxidant injury

1997 ◽  
Vol 273 (4) ◽  
pp. L856-L865 ◽  
Author(s):  
Bruce R. Pitt ◽  
Margaret Schwarz ◽  
Elizabeth S. Woo ◽  
Emily Yee ◽  
Karla Wasserloos ◽  
...  
Keyword(s):  
Lipid Peroxidation ◽  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Metal Binding ◽  
High Performance ◽  
Binding Capacity ◽  
Peroxyl Radicals ◽  
Intact Cells ◽  
Tert Butyl Hydroperoxide ◽  
Liquid Chromatography Separation

Metallothionein (MT) is a low-molecular-weight cysteine-rich protein with extensive metal binding capacity and potential nonenzymatic antioxidant activity. Despite the sensitivity of vascular endothelium to either heavy metal toxicity or oxidative stress, little is known regarding the role of MT in endothelial cells. Accordingly, we determined the sensitivity of cultured sheep pulmonary artery endothelial cells (SPAEC) that overexpressed MT to tert-butyl hydroperoxide ( t-BOOH), hyperoxia, or 2,2′-azobis(2,4-dimethylvaleronitrile) (AMVN; peroxyl radical generator). Nontoxic doses of 10 μM Cd increased MT levels from 0.21 ± 0.03 to 2.07 ± 0.24 μg/mg and resulted in resistance to t-BOOH and hyperoxia as determined by reduction of Alamar blue or [3H]serotonin transport, respectively. SPAEC stably transfected with plasmids containing either mouse or human cDNA for MT were resistant to both t-BOOH and hyperoxia. In addition, we examined transition metal-independent, noncytotoxic AMVN-induced lipid peroxidation after metabolic incorporation of the oxidant-sensitive fluorescent fatty acid cis-parinaric acid into phospholipids and high-performance liquid chromatography separation. SPAEC that overexpressed MT after gene transfer completely inhibited peroxyl oxidation of phosphatidylserine, phosphatidylcholine, and sphingomyelin (but not phosphatidylethanolamine) noted in wild-type SPAEC. These data show for the first time that MT can 1) protect pulmonary artery endothelium against a diverse array of prooxidant stimuli and 2) directly intercept peroxyl radicals in a metal-independent fashion, thereby preventing lipid peroxidation in intact cells.

Endopeptidases 3.4.24.15 and 24.16 in endothelial cells: potential role in vasoactive peptide metabolism

2003 ◽  
Vol 284 (6) ◽  
pp. H1978-H1984 ◽  
Author(s):  
M. Ursula Norman ◽  
Shane B. Reeve ◽  
Vincent Dive ◽  
A. Ian Smith ◽  
Rebecca A. Lew
Keyword(s):  
Endothelial Cells ◽  
High Performance ◽  
Vascular Endothelial Cells ◽  
Umbilical Vein ◽  
Hybrid Cell ◽  
Fluorescent Substrate ◽  
Intact Cells ◽  
Aortic Endothelial Cells

The closely related metalloendopeptidases EC 3.4.24.15 (EP24.15; thimet oligopeptidase) and 24.16 (EP24.16; neurolysin) cleave a number of vasoactive peptides such as bradykinin and neurotensin in vitro. We have previously shown that hypotensive responses to bradykinin are potentiated by an inhibitor of EP24.15 and EP24.16 (26), suggesting a role for one or both enzymes in bradykinin metabolism in vivo. In this study, we have used selective inhibitors that can distinguish between EP24.15 and EP24.16 to determine their activity in cultured endothelial cells (the transformed human umbilical vein endothelial hybrid cell line EA.hy926 or ovine aortic endothelial cells). Endopeptidase activity was assessed using a specific quenched fluorescent substrate [7-methoxycoumarin-4-acetyl-Pro-Leu-Gly-d-Lys(2,4-dinitrophenyl)], as well as the peptide substrates bradykinin and neurotensin (assessed by high-performance liquid chromatography with mass spectroscopic detection). Our results indicate that both peptidases are present in endothelial cells; however, EP24.16 contributes significantly more to substrate cleavage by both cytosolic and membrane preparations, as well as intact cells, than EP24.15. These findings, when coupled with previous observations in vivo, suggest that EP24.16 activity in vascular endothelial cells may play an important role in the degradation of bradykinin and/or other peptides in the circulation.

Physicochemical roles of soluble metal cations in the outer membrane of Escherichia coli K-12

1986 ◽  
Vol 32 (7) ◽  
pp. 594-601 ◽  
Author(s):  
F. G. Ferris ◽  
T. J. Beveridge
Keyword(s):  
Cell Surface ◽  
Outer Membrane ◽  
Metal Binding ◽  
Metal Salt ◽  
Binding Capacity ◽  
Cell Surface Hydrophobicity ◽  
Surface Hydrophobicity ◽  
Metallic Ions ◽  
Intact Cells ◽  
K 12

Atomic absorption spectroscopy of isolated native and EDTA-modified (lipopolysaccharide-depleted) outer membrane revealed trace amounts of potassium, manganese, and iron (1.0–7.0 nmol/mg dry weight outer membrane). Sodium, magnesium, and calcium were approximately one order of magnitude more plentiful, but EDTA-modified outer membrane was deficient in calcium. When metal-binding assays were conducted to find the binding capacity of native and EDTA-modified outer membrane, potassium bound poorly compared with sodium. However, there was no difference in the binding of these ions between the OM preparations. In contrast, reduced amounts of magnesium, calcium, manganese, and iron III bound to the EDTA-modified OM. Partitioning of intact cells in a biphasic dextran–polyethyleneglycol system indicated that the reduced lipopolysaccharide content of the EDTA-modified outer membrane increased the hydrophobicity of the cell surface. Exposure of control and EDTA-treated cells to divalent metal salt solutions before phase partitioning also increased cell surface hydrophobicity. Freeze-etching showed that sodium ions had no effect on the membrane fractures observed in control cells, but with EDTA-treated cells, this cation increased the occurrence of small outer membrane fractures (plateaus) which are characteristic of EDTA treatment. Both magnesium and manganese increased the frequency of outer membrane cleavage in control cells, whereas calcium did not. In contrast, all three divalent metallic ions increased the frequency and extent of cleavage in the outer membrane of EDTA-treated cells.

scholarly journals Oxidized glutathione decreases luminal Ca2+ content of the endothelial cell ins(1,4,5)P3-sensitive Ca2+ store

1995 ◽  
Vol 312 (2) ◽  
pp. 485-489 ◽  
Author(s):  
P N Henschke ◽  
S J Elliott
Keyword(s):  
Signal Transduction ◽  
Endothelial Cells ◽  
Plasma Membrane ◽  
Endothelial Cell ◽  
Pulmonary Artery ◽  
Vascular Tissue ◽  
Oxidant Stress ◽  
Inhibitory Effect ◽  
Oxidized Glutathione ◽  
Intact Cells

The model oxidant, t-butyl hydroperoxide (t-buOOH), inhibits Ins(1,4,5)P3-dependent Ca2+ signalling in calf pulmonary artery endothelial cells. Metabolism of t-buOOH within the cytosol is coupled to the oxidation of glutathione. In this study, we investigated whether oxidized glutathione (GSSG) is the intracellular moiety responsible for mediating the effects of t-buOOH on Ca2+ signalling. The increase in cytosolic [Ca2+] stimulated by application of 2,5-di-t-butylhydroquinone (BHQ) was used to estimate the luminal Ca2+ content of the Ins(1,4,5)P3-sensitive store in intact cells. Luminal Ca2+ content was unaffected by t-buOOH (0.4 mM, 0-3 h) unless intracellular GSSG content was concomitantly elevated. The effect was specific for increased GSSG and was not replicated by depletion of GSH. These results suggest that cytosolic GSSG, produced endogenously within the endothelial cell, decreases the luminal Ca2+ content of Ins(1,4,5)P3-sensitive Ca2+ stores. Depletion of internal Ca2+ stores by GSSG may represent a key mechanism by which some forms of oxidant stress inhibit signal transduction in vascular tissue. At the plasma membrane, t-buOOH is known to inhibit the capacitative Ca2+ influx pathway. Increased intracellular GSSG potentiated the inhibitory effect of t-buOOH on Ca2+ influx, thereby providing the first evidence that activity of the capacitative Ca2+ influx channel is sensitive to thiol reagents formed endogenously within the cell.

Nitric oxide attenuates hydrogen peroxide-mediated injury to porcine pulmonary artery endothelial cells

1997 ◽  
Vol 272 (6) ◽  
pp. L1133-L1141 ◽  
Author(s):  
M. P. Gupta ◽  
V. Evanoff ◽  
C. M. Hart
Keyword(s):  
Nitric Oxide ◽  
Lipid Peroxidation ◽  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Cell Injury ◽  
Vascular Endothelial Cell ◽  
H2o2 Treatment ◽  
No Donors ◽  
Synthase Inhibitor ◽  
Porcine Pulmonary Artery

To examine the role of nitric oxide (.NO) in vascular endothelial cell injury, cultured porcine pulmonary artery endothelial cells (PAEC) were treated with H2O2 (100-500 microM) for 30 min in the presence or absence of the .NO donors (+/-)S-nitroso-N-acetylpenicillamine (SNAP) or diethylamine nitric oxide (DEANO). H2O2 caused dose-dependent PAEC cytotoxicity detected 2 h after H2O2 treatment as the release of lactate dehydrogenase. SNAP (100 microM) and DEANO (100 microM) attenuated H2O2-induced cytotoxicity if present during H2O2 treatment. In contrast, restricting treatment with .NO donors to periods before (30 min) or after (2 h) incubation with H2O2 did not prevent PAEC injury. Furthermore, the .NO synthase inhibitor NG-nitro-L-arginine methyl ester (1 mM) sensitized PAEC to H2O2-induced injury. SNAP also attenuated H2O2-induced PAEC lipid peroxidation even if restricted to periods before or after exposure to H2O2. Thus, although .NO effectively attenuated H2O2-mediated PAEC lipid peroxidation and cytotoxicity, these effects were clearly dissociated, suggesting that the antiperoxidative effects of .NO are not sufficient to account for its cytoprotective properties.

Localization of carbonic anhydrase on pulmonary artery endothelial cells in culture

1982 ◽  
Vol 53 (4) ◽  
pp. 914-919 ◽  
Author(s):  
U. S. Ryan ◽  
P. L. Whitney ◽  
J. W. Ryan
Keyword(s):  
Endothelial Cells ◽  
Plasma Membrane ◽  
Carbonic Anhydrase ◽  
Pulmonary Artery ◽  
Venous Blood ◽  
Carbonic Anhydrase Activity ◽  
Intact Cells ◽  
Cells In Culture ◽  
Carbonic Anhydrase B ◽  
Arterial Blood

Bovine pulmonary artery endothelial cells in culture possess carbonic anhydrase activity and immunoreactivity. The intact cells and cell homogenates lower the pH of 25 mM triethanolamine sulfate buffer saturated with CO2 (starting pH 8.1). The intact cells are more reactive than the cell homogenates, and the enzymic activity is enriched in association with the plasma membrane fraction. Specific immunofluorescence is obtained when the cells are incubated with rabbit antibovine erythrocyte carbonic anhydrase B and then with goat antirabbit immunoglobulin G coupled to fluorescein. At the level of electron microscopy, antibodies to carbonic anhydrase B are reactive with sites along the plasma membrane and associated caveolae. Multivesicular bodies are the only intracellular sites labeled and appear to correspond to the globular sites of intracellular immunofluorescence. Cells maintained and propagated in culture in the absence of an exogenous source of carbonic anhydrase nonetheless possess carbonic anhydrase activity, suggesting that the cells are capable of synthesizing the enzyme. Taken together, our results indicate that pulmonary artery endothelial cells possess carbonic anhydrase situated so that the enzyme could readily catalyze the dehydration of plasma HCO-3 to facilitate CO2 excretion and participate in the regulation of blood pH as central venous blood is converted into systemic arterial blood.

Hypoxia inhibits L-arginine synthesis from L-citrulline in porcine pulmonary artery endothelial cells

1995 ◽  
Vol 269 (5) ◽  
pp. L581-L587 ◽  
Author(s):  
Y. Su ◽  
E. R. Block
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Biosynthetic Pathway ◽  
Argininosuccinate Synthetase ◽  
Argininosuccinate Lyase ◽  
Arginine Biosynthesis ◽  
Intact Cells ◽  
Porcine Pulmonary Artery

Both non-arginine-depleted and arginine-depleted pulmonary artery endothelial cells (PAEC) actively convert citrulline into arginine. Exposure to hypoxia for 4-24 h inhibited arginine synthesis from citrulline in intact cells and in cell homogenates. The conversion of L-citrulline to L-argininosuccinate by argininosuccinate synthetase (AS) was inhibited by exposure to hypoxia for 4, 12, or 24 h. The conversion of argininosuccinate to arginine by argininosuccinate lyase was inhibited by exposure to hypoxia for 24 h but not for 4-12 h. The decrease of L-arginine biosynthesis during hypoxia coincided with the increase of intracellular glutamine content and was abrogated by preventing an increase in intracellular glutamine. In addition, AS activity was inversely related to glutamine content in the medium. These results indicate that hypoxia inhibited the L-arginine biosynthetic pathway via decreased activity of AS. The latter is related to increased glutamine content. Hypoxic inhibition of arginine synthesis from citrulline did not result in a decrease of arginine content, suggesting that PAEC are able to maintain intracellular arginine for up to 24 h despite reduction in the L-arginine biosynthetic pathway.

Oxidant stress decreases Na+/H+ antiport activity in bovine pulmonary artery endothelial cells

1994 ◽  
Vol 267 (6) ◽  
pp. L649-L659 ◽  
Author(s):  
M. Cutaia ◽  
N. Parks
Keyword(s):  
Endothelial Cells ◽  
Pulmonary Artery ◽  
Oxidant Stress ◽  
Buthionine Sulfoximine ◽  
Redox System ◽  
Membrane Function ◽  
Acid Recovery ◽  
Tert Butyl Hydroperoxide ◽  
Protein Sulfhydryl ◽  
Protein Sulfhydryl Groups

We determined the effects of oxidant stress by the use of tert-butyl hydroperoxide (t-BOOH) on Na+/H+ exchange in pulmonary artery endothelial cells. Monolayers were exposed to the hydroperoxide, followed by measurement of intracellular pH and the rate of recovery from acidosis by utilizing the pH-sensitive probe 2',7'-bis(carboxyethyl)-5(6)- carboxyfluorescein. t-BOOH (0.4 mM) decreased the rate of acid recovery after a 2-h exposure without evidence of overt cytotoxicity (51Cr-release assay). Glutathione repletion (N-acetyl-L-cysteine) abolished the effect of the hydroperoxide. Lowering intracellular glutathione with buthionine sulfoximine decreased the acid recovery rate at a dose of t-BOOH (0.04 mM) that was not normally associated with a change in this parameter. Preincubation with vitamin E had no protective effect. Dithiothreitol abolished the effect of the hydroperoxide, suggesting oxidation of protein sulfhydryl groups as a mechanism for the altered kinetics of acid recovery. There was no difference in cell buffering capacity between control and treated monolayers. The findings suggest that the decrease in Na+/H+ antiport activity in this model of oxidant stress represents an early perturbation of membrane function and illustrate the role of the glutathione redox system in maintaining the functional integrity of the Na+/H+ antiport in these cells.

scholarly journals Chromatography-Independent Fractionation and Newly Identified Molecular Features of the Adzuki Bean (Vigna angularis Willd.) β-vignin Protein

2021 ◽  
Vol 22 (6) ◽  
pp. 3018
Author(s):  
Biane Philadelpho ◽  
Victória Souza ◽  
Fabiani Souza ◽  
Johnnie Santos ◽  
Fabiana Batista ◽  
...  
Keyword(s):  
Metal Binding ◽  
Cardiovascular Health ◽  
Binding Capacity ◽  
Biological Activities ◽  
Electrophoretic Analysis ◽  
Adzuki Bean ◽  
Molecular Features ◽  
Health Promoting ◽  
High Degree

Adzuki seed β-vignin, a vicilin-like globulin, has proven to exert various health-promoting biological activities, notably in cardiovascular health. A simple scalable enrichment procedure of this protein for further nutritional and functional studies is crucial. In this study, a simplified chromatography-independent protein fractionation procedure has been optimized and described. The electrophoretic analysis showed a high degree of homogeneity of β-vignin isolate. Furthermore, the molecular features of the purified protein were investigated. The adzuki bean β-vignin was found to have a native size of 146 kDa, and the molecular weight determined was consistent with a trimeric structure. These were identified in two main polypeptide chains (masses of 56–54 kDa) that are glycosylated polypeptides with metal binding capacity, and one minor polypeptide chain with a mass 37 kDa, wherein these features are absent. The in vitro analysis showed a high degree of digestibility of the protein (92%) and potential anti-inflammatory capacity. The results lay the basis not only for further investigation of the health-promoting properties of the adzuki bean β-vignin protein, but also for a possible application as nutraceutical molecule.

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