Electron spin resonance characterization of the NAD(P)H oxidase in vascular smooth muscle cells

Endogenously produced reactive oxygen species are important for intracellular signaling mechanisms leading to vascular smooth muscle cell (VSMC) growth. It is therefore critical to define the potential enzymatic sources of ROS and their regulation by agonists in VSMCs. Previous studies have investigated O-2(.-) production using lucigenin-enhanced chemiluminescence. However, lucigenin has been recently criticized for its ability to redox cycle and its propensity to measure cellular reductase activity independent from O-2(.-). To perform a definitive characterization of VSMC oxidase activity, we used electron spin resonance trapping of O-2(.-) with DEPMPO. We confirmed that the main source of O-2(.-) from VSMC membranes is an NAD(P)H oxidase and that the O-2(.-) formation from mitochondria, xanthine oxidase, arachidonate-derived enzymes, and nitric oxide synthases in VSMC membranes was minor. The VSMC NAD(P)H oxidase(s) are able to produce more O-2(.-) when NADPH is used as the substrate compared to NADH (the maximal NADPH signal is 2.4- +/- 0.4-fold higher than the NADH signal). The two substrates had similar EC50's (similar to 10-50 muM). Stimulation with angiotensin II and platelet-derived growth factor also predominantly increased the NADPH-driven signal (101 +/- 8% and 83 +/- 1% increase above control, respectively), with less of an effect on NADH-dependent O-2(.-) (17 +/- 3% and 36 +/- 5% increase, respectively). Moreover, incubation of the cells with diphenylene iodonium inhibited predominantly NADPH-stimulated O-2(.-). In conclusion, electron spin resonance characterization of VSMC oxidase activity supports a major role for an NAD(P)H oxidase in O-2(.-) production in VSMCs, and provides new evidence concerning the substrate dependency and agonist-stimulated activity of this key enzyme. (C) 2001 Elsevier Science Inc.