Role of the endothelium and nitric oxide synthases in modulating superoxide formation induced by endotoxin and cytokines in porcine pulmonary arteries

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Abstract
Background: The interactive roles of cytokines, endotoxins, superoxide (O2• − ) and nitric oxide (NO) in the pathogenesis of adult respiratory distress syndrome (ARDS) have not been fully elucidated. The effects of tumour necrosis factor-α (TNF-α), interleukin 1α (IL-1α), and lipopolysaccharide (LPS) and the role of NO and the endothelium in mediating O2• − formation were therefore investigated in intact porcine pulmonary arteries in vitro. Methods: Intrapulmonary artery (PA) segments were obtained from White Landrace pigs (25–35 kg) and incubated with LPS, IL-1α, and TNF-α and O2• − release was measured by the superoxide dismutase (SOD) inhibitable reduction of ferricytochrome c. The source of O2• − formation was determined using a number of enzyme inhibitors. The role of NO was explored using NO synthase (NOS) inhibitors and the distribution of NOS isoforms and peroxynitrite (ONOO−, an index of NO–O2• − interactions) assessed by immunocytochemistry. Results: LPS, IL-1α, and TNF-α promoted the formation of O2• − from PA compared with untreated controls in a time and dose dependent manner, an effect markedly enhanced by removal of the endothelium but completely inhibited by the NADPH oxidase inhibitor diphenylene iodonium chloride (DPI). l-NAME and the eNOS inhibitor N5-(1-iminoethyl)-ornithine (l-NIO) enhanced O2• − formation from PA (with endothelium) in response to IL-1α and TNF-α but had no effect on LPS mediated O2• − formation, whereas l-NAME and the iNOS inhibitor l-N6-(1-iminoethyl)-lysine-HCl (l-NIL) enhanced O2• − formation only in response to LPS. Conclusions: LPS, IL-1α, and TNF-α promote O2• − formation through an upregulation of NADPH oxidase activity which is augmented by removal of the endothelium, as well as the inhibition of eNOS (in the case of cytokines) and iNOS (in the case of LPS). The concomitant expression of NOS isoforms (and NO formation) with that of NADPH oxidase may therefore constitute a protective system designed to remove O2• − through the formation of ONOO−. If this is so, the integrity of the endothelium may be axiomatic in the progression and severity of ARDS.