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

Abstract

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.