Resolving Contributions of Oxygen-Consuming and ROS-Generating Enzymes at the Synapse

Figure 1

NADPH oxidases are actively consuming O₂ and producing H₂O₂ in freshly isolated synaptosomes. Successive injections of 200 μM deoxygenated NADPH batches on synaptosomes triggered reproducible oxygen consumption (a, b) and H₂O₂ production as detected simultaneously by HRP/Amplex Red fluorescence (c, d) or using electrochemical sensor (e, f). The effects of inclusion of the NOX inhibitors Vas-2870 (10 μM) (a, c, e) or ebselen (10 μM) (b, d, f) are shown. (g) Quantifications of the overall O₂ and H₂O₂ fluxes induced by the three added doses of NADPH. The two methods of H₂O₂ detection yielded similar results. Both of the added NOX inhibitors, VAS-2870 (10 μM) and Ebselen (10 μM), caused significant inhibition of NADPH-triggered oxygen flux (h) and H₂O₂ flux (i). Values are given as mean ± SEM, paired Student -test was used for paired groups to determine statistical significance in comparison with NADPH-induced activity, , and .