Mathematical treatment of chemiluminescence data allowing an optimised kinetic analysis of vascular NAD(P)H-dependent superoxide anion production

Regulation of vascular redox homeostasis plays a central role in the contro l of vascular tone (e.g.: redox modulation of endothelial NO synthase and s oluble guanylate cyclase activities). Superoxide anion is one of the main r eactive oxygen species involved in the modulation of the redox status of th e vessel. It is crucial to know if pathological situations or drug treatmen ts are able to modify NAD(P)H oxidase-dependent production of O-2(-). As a steady-state approach is the only way for a valid assessment of these param eters, a mathematical treatment allowing visualisation of the linear portio n of the initial velocity of O-2(-) production was developed. Using two vas cular preparations (rat aortic rings and mouse thoracic aortae), the chemil uminescence (CL) in the presence of lucigenin was recorded every second in the 15-30 minute period after successive injections of NADPH and superoxide dismutase (SOD). Because, both O-2(-) and photons are labile and evanescen t products, visualisation of the steady-state requires calculation of their cumulative production by integral calculus using a first order integral eq uation. The cumulative production of CL and/or O-2(-) was plotted as a func tion of the recording time. The initial velocities, as a function of the NA DPH concentrations, were determined from the linear portion of these plots. For a precise molar quantification of O-2(-) production, a calibration cur ve of initial velocities using xanthine oxidase + xanthine as a source of O -2(-) was plotted by comparing CL and cytochrome C reduction. The NADPH-dep endent CL production exhibited Michaelian behavior and SOD acted as a nonco mpetitive inhibitor. For example, the values for NADPH oxidase in rat aorta were: Km = 237 +/- 2 muM; Vmax 500-600 pmol O-2(-)/min/ring.