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

Abstract

Regulation of vascular redox homeostasis plays a central role in the control of vascular tone (e.g.: redox modulation of endothelial NO synthase and soluble guanylate cyclase activities). Superoxide anion is one of the main reactive oxygen species involved in the modulation of the redox status of the vessel. It is crucial to know if pathological situations or drug treatments are able to modify NAD(P)H oxidase-dependent production of O2-. As a steady-state approach is the only way for a valid assessment of these parameters, a mathematical treatment allowing visualisation of the linear portion of the initial velocity of O2- production was developed. Using two vascular preparations (rat aortic rings and mouse thoracic aortae), the chemiluminescence (CL) in the presence of lucigenin was recorded avery second in the 15-30 minute period after successive injections of NADPH and superoxide dismutase (SOD). Because, both O2- and photons are labile and evanescent products, visualisation of the steady-state requires calculation of their cumulative production by integral calculus using a first order integral equation. The cumulative production of CL and/or O2- was plotted as a function of the recording time. The initial velocities, as a function of the NADPH concentrations, were determined from the linear portion of these plots. For e precise molar quantification of O2- production, a calibration curve of initial velocities using xanthine oxidase + xanthine as a source of O2- was plotted by comparing CL and cytochrome C reduction. The NADPH-dependent CL production exhibited Michaelian behavior and SOD acted as a noncompetitive inhibitor. For example, the values for NADPH oxidase in rat aorta were: Km = 237 ± 2 μM; Vmax = 500-600 pmol O2-/min/ring.