scholarly journals High rates of extracellular superoxide generation by cultured human fibroblasts: involvement of a lipid-metabolizing enzyme

1996 ◽  
Vol 318 (3) ◽  
pp. 805-812 ◽  
Author(s):  
Valerie B. O'DONNELL ◽  
Angelo AZZI
Keyword(s):  
Nadph Oxidase ◽  
Human Fibroblasts ◽  
Extracellular Enzyme ◽  
Competitive Inhibitor ◽  
Superoxide Generation ◽  
Diphenylene Iodonium ◽  
Cultured Human Fibroblasts ◽  
Prostaglandin H ◽  
Metabolizing Enzyme ◽  
Hydroperoxyeicosatetraenoic Acid

Expression of NADPH oxidase and low superoxide generation (approx. 0.06 nmol/min per 106 cells) by cytokine- or ionophore-stimulated human fibroblasts is known. However, we here show that these cells also contain an ectoplasmic enzyme, distinct from NADPH oxidase, which can generate superoxide (2.19±0.14 nmol/min per 106 cells) at levels similar to phorbol ester-stimulated monocytes on exogenous NADH addition. Superoxide generation was temperature-dependent, insensitive to chelation (desferal), and had a Km(app)(NADH) of 11.5 µM. Inhibitor studies showed that there was no involvement of NADPH oxidase (diphenylene iodonium, diphenyl iodonium), prostaglandin H synthase (indomethacin), xanthine oxidase (allopurinol), cytochrome P-450 (metyrapone) or mitochondrial respiration (rotenone, antimycin A). NAD+ was a competitive inhibitor, whereas NADPH supported 40% of the rate seen with NADH. No luminescence was observed after the addition of lactate, malate, pyruvate, GSH or l-cysteine. NADH-stimulated superoxide generation was enhanced by the addition of (3–30 µM) arachidonic acid, linoleic acid or (5S)-hydroxyeicosatetraenoic acid [(5S)-HETE] but not palmitic acid, (15S)-hydroperoxyeicosatetraenoic acid [(15S)-HPETE], (15S)-HETE or (12S)-HETE. Several features suggest involvement of an enzyme related to 15-lipoxygenase, and, in support of this, we show superoxide generation and NADH oxidation by recombinant rabbit reticulocyte 15-lipoxygenase. The large amounts of superoxide measured suggest that the fibroblast extracellular enzyme could be a major source of reactive oxygen species after tissue damage.

scholarly journals The superoxide-generating NADPH oxidase of human neutrophils is electrogenic and associated with an H+ channel

1987 ◽  
Vol 246 (2) ◽  
pp. 325-329 ◽  
Author(s):  
L M Henderson ◽  
J B Chappell ◽  
O T G Jones
Keyword(s):  
Membrane Potential ◽  
Nadph Oxidase ◽  
Human Neutrophils ◽  
Superoxide Generation ◽  
Compensatory Movement ◽  
Diphenylene Iodonium

The membrane potential of cytoplasts, derived from human neutrophils, was depolarized by the activation of the superoxide-generating NADPH-dependent oxidase. The extent of the depolarization was inhibited by diphenylene iodonium and was therefore due directly to the activity of the oxidase, which must be electrogenic. The extent of the depolarization was influenced by alteration of the delta pH across the cytoplast membrane, indicating that the outward translocation of H+ eventually compensates for superoxide generation. The depolarization of the potential is enhanced by Cd2+, a blocker of H+ currents, suggesting that the compensatory movement is via an H+ channel.

scholarly journals Identification of a superoxide-generating NADPH oxidase system in human fibroblasts

1991 ◽  
Vol 275 (1) ◽  
pp. 241-245 ◽  
Author(s):  
B Meier ◽  
A R Cross ◽  
J T Hancock ◽  
F J Kaup ◽  
O T G Jones
Keyword(s):  
Nadph Oxidase ◽  
Human Fibroblasts ◽  
Light And Electron Microscopy ◽  
Specific Inhibitor ◽  
Outer Cell ◽  
Oxidase System ◽  
Diphenylene Iodonium ◽  
Outer Cell Membrane ◽  
System 1 ◽  
Generating System

Human fibroblasts have the capacity to release superoxide radicals upon stimulation of an electron transport system similar to the NADPH oxidase of leukocytes. Two components of the NADPH oxidase system, (1) a flavoprotein of 45 kDa which binds diphenylene iodonium (a compound described as a specific inhibitor of the leukocyte NADPH oxidase), and (2) a low-potential cytochrome b, are present in fibroblast membranes. Fibroblasts exhibit these compounds at lower concentrations than do polymorphonuclear leukocytes or B-lymphocytes. The superoxide-generating system is rather uniformly associated with the outer cell membrane, as shown by light and electron microscopy. Superoxide release upon stimulation with various agents was prevented by the addition of micromolar concentrations of diphenylene iodonium, making an NADPH oxidase a likely source.

scholarly journals Influence of chlorate on proteoglycan biosynthesis by cultured human fibroblasts.

1988 ◽  
Vol 263 (26) ◽  
pp. 12886-12892 ◽  
Author(s):  
H Greve ◽  
Z Cully ◽  
P Blumberg ◽  
H Kresse
Keyword(s):  
Human Fibroblasts ◽  
Cultured Human Fibroblasts

scholarly journals Fatty alcohol metabolism in cultured human fibroblasts. Evidence for a fatty alcohol cycle.

1987 ◽  
Vol 262 (36) ◽  
pp. 17412-17419 ◽  
Author(s):  
W B Rizzo ◽  
D A Craft ◽  
A L Dammann ◽  
M W Phillips
Keyword(s):  
Fatty Alcohol ◽  
Human Fibroblasts ◽  
Alcohol Metabolism ◽  
Cultured Human Fibroblasts

L-iduronidase in cultured human fibroblasts and liver

1971 ◽  
Vol 42 (2) ◽  
pp. 340-345 ◽  
Author(s):  
Reuben Matalon ◽  
J.A. Cifonelli ◽  
Albert Dorfman
Keyword(s):  
Human Fibroblasts ◽  
Cultured Human Fibroblasts

scholarly journals The transport of neutral amino acids in cultured human fibroblasts.

1980 ◽  
Vol 255 (3) ◽  
pp. 929-936 ◽  
Author(s):  
G.C. Gazzola ◽  
V. Dall'Asta ◽  
G.G. Guidotti
Keyword(s):  
Amino Acids ◽  
Human Fibroblasts ◽  
Neutral Amino Acids ◽  
Cultured Human Fibroblasts

scholarly journals Transport of l-Isoleucine by Cultured Human Fibroblasts

1974 ◽  
Vol 249 (11) ◽  
pp. 3430-3435
Author(s):  
Richard E. Hillman ◽  
Elaine F. Otto
Keyword(s):  
Human Fibroblasts ◽  
Cultured Human Fibroblasts

scholarly journals Na/K/Cl cotransport in cultured human fibroblasts.

1985 ◽  
Vol 260 (3) ◽  
pp. 1445-1451
Author(s):  
N E Owen ◽  
M L Prastein
Keyword(s):  
Human Fibroblasts ◽  
Cultured Human Fibroblasts
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