High rates of extracellular superoxide generation by cultured human fibroblasts: involvement of a lipid-metabolizing enzyme.
Expression of NADPH oxidase and low superoxide generation (approx. 0.06 nmol/min per 10(6) 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 10(6) 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 K(m) (app)(NADH) of 11.5 microM. 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 microM) 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.
O'Donnell
V B
VB
Institute of Biochemistry and Molecular Biology, Bern, Switzerland.
Azzi
A
A
D000818:Animals; D001093:Arachidonate 15-Lipoxygenase / Q000378:metabolism; D002460:Cell Line; D003574:Cytochrome c Group / Q000378:metabolism; D004579:Electron Transport; D005110:Extracellular Space / Q000378:metabolism; D005227:Fatty Acids / Q000378:metabolism; D005347:Fibroblasts / Q000378:metabolism; D006801:Humans; D007700:Kinetics; D050356:Lipid Metabolism; D008163:Luminescent Measurements; D009243:NAD / Q000378:metabolism / Q000494:pharmacology; D009249:NADP / Q000378:metabolism / Q000494:pharmacology; D019255:NADPH Oxidases / Q000378:metabolism; D010084:Oxidation-Reduction; D011817:Rabbits; D017382:Reactive Oxygen Species / Q000378:metabolism; D011994:Recombinant Proteins / Q000378:metabolism; D013481:Superoxides / Q000378:metabolism*
D003574:Cytochrome c Group; D005227:Fatty Acids; D017382:Reactive Oxygen Species; D011994:Recombinant Proteins; D009243:NAD; D013481:Superoxides; D009249:NADP; D001093:Arachidonate 15-Lipoxygenase; D019255:NADPH Oxidases