scholarly journals Insight into the nature and site of oxygen-centered free radical generation by endothelial cell monolayers using a novel spin trapping technique

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 699-707 ◽  
Author(s):  
BE Britigan ◽  
TL Roeder ◽  
DM Shasby
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Free Radical ◽  
Hydroxyl Radical ◽  
Spin Trap ◽  
Spin Trapping ◽  
Cathepsin G ◽  
Cell Monolayers ◽  
Radical Production ◽  
Radical Generation

Abstract Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA- stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy- radical generation.

scholarly journals Insight into the nature and site of oxygen-centered free radical generation by endothelial cell monolayers using a novel spin trapping technique

Blood ◽  
1992 ◽  
Vol 79 (3) ◽  
pp. 699-707
Author(s):  
BE Britigan ◽  
TL Roeder ◽  
DM Shasby
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Free Radical ◽  
Hydroxyl Radical ◽  
Spin Trap ◽  
Spin Trapping ◽  
Cathepsin G ◽  
Cell Monolayers ◽  
Radical Production ◽  
Radical Generation

Spin trapping, a sensitive and specific means of detecting free radicals, is optimally performed on cell suspensions. This makes it unsuitable for the study of adherent endothelial cell monolayers because disrupting the monolayer to induce a cell suspension could introduce confounding factors. This problem was eliminated through the use of endothelial cells that were grown to confluence on microcarrier beads. Using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), the nature of free radical species generated by suspensions of microcarrier bead adherent porcine pulmonary endothelial cells under various forms of oxidant stress was examined. Exposure of these endothelial cells to paraquat resulted in the spin trapping of superoxide (.O2-). Endothelial cell incubation in the presence of either bolus or continuous fluxes of hydrogen peroxide (H2O2) yielded spin trap evidence of hydroxyl radical formation, which was preventable by pretreating the cells with deferoxamine. Chromium oxalate which eliminates extracellular electron paramagnetic resonance spectrometry (EPR) signals, prevented the detection of DMPO spin adducts generated by paraquat but not H2O2-treated endothelial cells. When endothelial cells were coincubated with PMA-stimulated monocytes evidence of both .O2- and hydroxyl radical production was detected, whereas with PMA- stimulated neutrophils only .O2- production could be confirmed. Neutrophil elastase, cathepsin G, and the combination of PMA and A23187 have previously been suggested to induce endothelial cell oxy-radical generation. However, exposure of endothelial cells to each of these agents did not yield DMPO spin adducts or cyanide-insensitive endothelial cell O2 consumption. These data indicate that endothelial cell exposure: to paraquat induces extracellular .O2- formation; to H2O2 leads to intracellular hydroxyl radical production; and to elastase, cathepsin G, or A23187/PMA does not appear to cause oxy- radical generation.

scholarly journals The oxidation of oxyhaemoglobin by glyceraldehyde and other simple monosaccharides

1984 ◽  
Vol 217 (3) ◽  
pp. 615-622 ◽  
Author(s):  
P J Thornalley ◽  
S P Wolff ◽  
M J C Crabbe ◽  
A Stern
Keyword(s):  
Free Radicals ◽  
Free Radical ◽  
Phosphate Buffer ◽  
Spin Trap ◽  
Spin Trapping ◽  
Free Radical Production ◽  
Radical Production ◽  
Hydroxyalkyl Radical ◽  
Aldehyde Derivative

Glyceraldehyde and other simple monosaccharides oxidize oxyhaemoglobin to methaemoglobin in phosphate buffer at pH 7.4 and 37 degrees C, with the concomitant production of H2O2 and an alpha-oxo aldehyde derivative of the monosaccharide. Simple monosaccharides also reduce methaemoglobin to ferrohaemichromes (non-intact haemoglobin) at pH 7.4 and 37 degrees C. Carbonmonoxyhaemoglobin is unreactive towards oxidation by autoxidizing glyceraldehyde. Free-radical production from autoxidizing monosaccharides with haemoglobins was observed by the e.s.r. technique of spin trapping with the spin trap 5,5-dimethyl-l-pyrroline N-oxide. Hydroxyl and l-hydroxyalkyl radical production observed from monosaccharide autoxidation was quenched in the presence of oxyhaemoglobin and methaemoglobin. The haemoglobins appear to quench the free radicals by reaction with the free radicals and/or the ene-diol precursor of the free radical.

scholarly journals Mitochondrial metabolism of a hydroperoxide to free radicals in human endothelial cells: an electron spin resonance spin-trapping investigation

1994 ◽  
Vol 304 (3) ◽  
pp. 707-713 ◽  
Author(s):  
V O'Donnell ◽  
M J Burkitt
Keyword(s):  
Endothelial Cells ◽  
Free Radicals ◽  
Endothelial Cell ◽  
Free Radical ◽  
Spin Trapping ◽  
Cell Killing ◽  
Lipid Hydroperoxides ◽  
Cell Integrity ◽  
Human Endothelial Cells ◽  
Tert Butylhydroperoxide

Oxidative damage to the vascular endothelium may be an important event in the promotion of atherosclerosis. Several lines of evidence suggest that lipid hydroperoxides may be responsible for the induction of such damage. Hydroperoxides cause loss of endothelial cell integrity, increase the permeability of the endothelium to macromolecules, and compromise its ability to control vascular tone via the secretion of vasoactive molecules in response to receptor stimulation. The molecular mechanisms responsible for these effects are, however, poorly understood. In this paper, we describe an e.s.r. spin-trapping investigation into the metabolism of the model hydroperoxide compound tert-butylhydroperoxide to reactive free radicals in intact human endothelial cells. The hydroperoxide is shown to undergo a single electron reduction to form free radicals. Experiments with metabolic poisons indicate that the mitochondrial electron-transport chain is the source of electrons for this reduction. The metal-ion-chelating agent desferrioxamine was found to prevent cell killing by tert-butylhydroperoxide, but did not affect free radical formation, suggesting that free metal ions may serve to promote free-radical chain reactions involved in cell killing following the initial conversion of the hydroperoxide to free radicals by mitochondria. These processes may well be responsible for many of the reported effects of hydroperoxides on endothelial cell integrity and function.

Involvement of Ca2+ in the H2O2-induced increase in endothelial permeability

1996 ◽  
Vol 270 (6) ◽  
pp. L973-L978 ◽  
Author(s):  
A. Siflinger-Birnboim ◽  
H. Lum ◽  
P. J. Del Vecchio ◽  
A. B. Malik
Keyword(s):  
Hydrogen Peroxide ◽  
Endothelial Cells ◽  
Endothelial Cell ◽  
Binding Sites ◽  
Endothelial Permeability ◽  
Extracellular Medium ◽  
Critical Determinant ◽  
Cell Monolayers ◽  
Sensitive Membrane

We studied the role of Ca2+ in mediating the hydrogen peroxide (H2O2)-induced increase in endothelial permeability to 125I-labeled albumin using bovine pulmonary microvessel endothelial cells (BMVEC). Changes in cytosolic-free Ca2+ ([Ca2+]i) were monitored in BMVEC monolayers loaded with the Ca(2+)-sensitive membrane permeant fluorescent dye fura 2-AM. H2O2 (100 microM) produced a rise in [Ca2+]i within 10 s that was reduced by the addition of EGTA to the medium. Uptake of 45Ca2+ from the extracellular medium increased in the presence of H2O2 (100 microM) compared with control monolayers, suggesting that the H2O2-induced rise in [Ca2+]i is partly the result of extracellular Ca2+ influx. The effects of [Ca2+]i on endothelial permeability were addressed by pretreatment of BMVEC monolayers with BAPTA-AM (3-5 microM), a membrane permeant Ca2+ chelator, before the H2O2 exposure. BAPTA-AM produced an approximately 50% decrease in the H2O2-induced increase in endothelial permeability compared with endothelial cell monolayers exposed to H2O2 alone. The increase in endothelial permeability was independent of Ca2+ influx, since LaCl3 (0-100 microM), which displaces Ca2+ from binding sites on the cell surface, did not modify the permeability response. These results indicate that the rise in [Ca2+]i produced by H2O2 is a critical determinant of the increase in endothelial permeability.

scholarly journals Thrombin-induced gap formation in confluent endothelial cell monolayers in vitro

Blood ◽  
1983 ◽  
Vol 62 (3) ◽  
pp. 549-556 ◽  
Author(s):  
M Laposata ◽  
DK Dovnarsky ◽  
HS Shin
Keyword(s):  
Endothelial Cells ◽  
Endothelial Cell ◽  
Umbilical Vein ◽  
Cyclic Adenosine Monophosphate ◽  
Adenosine Monophosphate ◽  
Gap Formation ◽  
Culture Dish ◽  
Cell Monolayers ◽  
Umbilical Vein Endothelial Cells

Abstract When thrombin is incubated with confluent monolayers of human umbilical vein endothelial cells in vitro, there is a change in the shape of the endothelial cells that results in gaps in the monolayer, disrupting the integrity of the endothelium and exposing the subendothelium. Using a grid assay to measure this phenomenon, we observed that up to 80% of the surface area once covered by cells was uncovered after a 15-min incubation with 10(-2) U/ml (10(-10)M) thrombin. The effect was apparent within 2 min and did not remove cells from the surface of the culture dish. The gaps in the monolayer completely disappeared within 2 hr after exposure to thrombin. The effect of thrombin was inhibited by preincubation of thrombin with hirudin or antithrombin III plus heparin or by preincubation of the monolayers with dibutyryl cyclic adenosine monophosphate (dbcAMP). Histamine also induced gap formation in endothelial cell monolayers. Both pyrilamine and cimetidine prevented the histamine-induced effect, but they had no effect on thrombin- induced gap formation. Intact monolayers were not disrupted by bradykinin, serotonin, C5a, or C3a. Our results suggest that small amounts of thrombin can induce repeated and transient exposure of the subendothelium, a situation believed to be conducive to atherogenesis and thrombosis.

Myocardial alterations due to free-radical generation

1984 ◽  
Vol 246 (6) ◽  
pp. H776-H783 ◽  
Author(s):  
K. P. Burton ◽  
J. M. McCord ◽  
G. Ghai
Keyword(s):  
Endothelial Cells ◽  
Free Radicals ◽  
Hydroxyl Radical ◽  
Cardiac Myocytes ◽  
Hydroxyl Radicals ◽  
Vascular Endothelial Cells ◽  
Tissue Injury ◽  
Myocardial Damage ◽  
Vascular Endothelial ◽  
Radical Generation

Oxygen-derived free radicals have been proposed as general mediators of tissue injury in a variety of disease states. Recent interest has focused on the possibility that free radicals may be involved in ischemic myocardial damage. However, the exact types of damage that result from myocardial exposure to free radicals remains to be established. The purpose of this study was to evaluate the effects of superoxide and hydroxyl radicals on myocardial structure and function in an isolated perfused rabbit interventricular septal preparation. Superoxide was generated by adding purine (2.3 mM) and xanthine oxidase (0.01 U/ml) to the physiological solutions perfusing the septa. Hydroxyl radical generation was catalyzed by the addition of 2.4 microM Fe3+-loaded transferrin to the system. Exposure of normal septa to superoxide-generating solutions resulted in the development of structural alterations in the vascular endothelium including the development of vacuoles. Membranous cellular debris was evident in the extracellular space and within the vessels. Cardiac myocytes showed evidence of mild alterations. Exposure of septa to solutions capable of generating hydroxyl radicals resulted in more extensive and severe damage. Vascular endothelial cells showed evidence of vacuoles or blebs and edema. Severe swelling of mitochondria was evident in cardiac myocytes and vascular endothelial cells. In addition, myocytes often showed blebbing of the basement membrane. Normal septa exposed to superoxide showed no significant decrease in developed tension, whereas hydroxyl radical exposure resulted in a significant decrease in myocardial function.(ABSTRACT TRUNCATED AT 250 WORDS)

scholarly journals The spin trapping of pyrimidine nucleotide free radicals in a Fenton system

1989 ◽  
Vol 261 (3) ◽  
pp. 831-839 ◽  
Author(s):  
W D Flitter ◽  
R P Mason
Keyword(s):  
Free Radicals ◽  
Hydroxyl Radical ◽  
Spin Trap ◽  
Gamma Ray ◽  
Thiobarbituric Acid ◽  
Spin Trapping ◽  
Pyrimidine Nucleotide ◽  
Gamma Ray Irradiation ◽  
Radical Attack ◽  
Fenton System

The reaction of the hydroxyl radical, generated by a Fenton system, with pyrimidine deoxyribonucleotides was investigated by using the e.s.r. technique of spin trapping. The spin trap t-nitrosobutane was employed to trap secondary radicals formed by the reaction of the hydroxyl radical with these nucleotides. The results presented here show that hydroxyl-radical attack on thymidine, 2-deoxycytidine 5-monophosphate and 2-deoxyuridine 5-monophosphate produced nucleotide-derived free radicals. The results indicate that .OH radical attack occurs predominantly at the carbon-carbon double bond of the pyrimidine base. The e.s.r. studies showed a good correlation with previous results obtained by authors who used x- or gamma-ray irradiation to generate the hydroxyl radical. A thiobarbituric acid assay was also used to monitor the damage produced to the nucleotides by the Fenton system. These results showed qualitative agreement with the spin-trapping studies.

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