Tumour necrosis factor α activates a p22^phox-based NADH oxidase in vascular smooth muscle

Increasing experimental evidence suggests that non-phagocytic cells express a potent superoxide (O₂^-.)-producing NADH oxidase that might be related to the phagocytic NADPH oxidase. Here we show that the cytokine tumour necrosis factor α(TNF-α) activates, in a time- and dose-dependent manner, a O₂^-.-producing NADH oxidase in cultured rat aortic smooth-muscle cells. Dose-response experiments for NADH showed an upward shift of the curve for TNF-α-treated cells, suggesting that TNF-α increased the amount of available enzyme. Using the anti-sense transfection technique, we further demonstrate that the molecular identity of this oxidase includes p22^phox (the α subunit of cytochrome b₅₅₈ and part of the electron transfer component of the phagocytic NADPH oxidase), which we recently cloned from a rat vascular smooth-muscle cell cDNA library. In addition, prolonged treatment with TNF-α increased p22^phox mRNA expression without affecting p22^phox mRNA stability, and only when transcriptional activity was intact. These findings identify a p22^phox-containing NADH oxidase as a source for cytokine-induced free radical production in vascular smooth-muscle cells and clarify some of the mechanisms involved in the regulation of vascular oxidase activity.