Regulation of brain mitochondrial H₂O₂ production by membrane potential and NAD(P)H redox state

TLDR

Mitochondrial ROS production at Complex I is linked to neural cell death, yet how NADH‑linked substrates regulate ROS without inhibitors remains poorly understood. The study tested whether isolated brain mitochondria generate H₂O₂ in a manner controlled by membrane potential (ΔΨ) and the NAD(P)H redox state. H₂O₂ production was quantified with Amplex Red fluorescence while ΔΨ was tracked via a tetraphenylphosphonium electrode and NAD(P)H redox state monitored by autofluorescence. H₂O₂ release correlated with ΔΨ and NAD(P)H reduction at high potentials, but 30 % of maximal production persisted even with uncoupler‑induced depolarization, demonstrating that ROS generation is modulated by ΔΨ and NAD(P)H redox state across physiological energy demands.

Abstract

Abstract Mitochondrial production of reactive oxygen species (ROS) at Complex I of the electron transport chain is implicated in the etiology of neural cell death in acute and chronic neurodegenerative disorders. However, little is known regarding the regulation of mitochondrial ROS production by NADH‐linked respiratory substrates under physiologically realistic conditions in the absence of respiratory chain inhibitors. This study used Amplex Red fluorescence measurements of H 2 O 2 to test the hypothesis that ROS production by isolated brain mitochondria is regulated by membrane potential (ΔΨ) and NAD(P)H redox state. ΔΨ was monitored by following the medium concentration of the lipophilic cation tetraphenylphosphonium with a selective electrode. NAD(P)H autofluorescence was used to monitor NAD(P)H redox state. While the rate of H 2 O 2 production was closely related to ΔΨ and the level of NAD(P)H reduction at high values of ΔΨ, 30% of the maximal rate of H 2 O 2 formation was still observed in the presence of uncoupler ( p ‐trifluoromethoxycarbonylcyanide phenylhydrazone) concentrations that provided for maximum depolarization of ΔΨ and oxidation of NAD(P)H. Our findings indicate that ROS production by mitochondria oxidizing physiological NADH‐dependent substrates is regulated by ΔΨ and by the NAD(P)H redox state over ranges consistent with those that exist at different levels of cellular energy demand.