Respiratory uncoupling by UCP1 and UCP2 and superoxide generation in endothelial cell mitochondria

Abstract

Mitochondria represent a major source of reactive oxygen species (ROS), particularly during resting or state 4 respiration wherein ATP is not generated. One proposed role for respiratory mitochondrial uncoupling proteins (UCPs) is to decrease mitochondrial membrane potential and thereby protect cells from damage due to ROS. This work was designed to examine superoxide production during state 4 (no ATP production) and state 3 (active ATP synthesis) respiration and to determine whether uncoupling reduced the specific production of this radical species, whether this occurred in endothelial mitochondria per se, and whether this could be modulated by UCPs. Superoxide formation by isolated bovine aortic endothelial cell (BAE) mitochondria, determined using electron paramagnetic resonance spectroscopy, was approximately fourfold greater during state 4 compared with state 3 respiration. UCP1 and UCP2 overexpression both increased the proton conductance of endothelial cell mitochondria, as rigorously determined by the kinetic relationship of respiration to inner membrane potential. However, despite uncoupling, neither UCP1 nor UCP2 altered superoxide formation. Antimycin, known to increase mitochondrial superoxide, was studied as a positive control and markedly enhanced the superoxide spin adduct in our mitochondrial preparations, whereas the signal was markedly impaired by the powerful chemical uncoupler p-(trifluoromethoxyl)-phenyl-hydrazone. In summary, we show that UCPs do have uncoupling properties when expressed in BAE mitochondria but that uncoupling by UCP1 or UCP2 does not prevent acute substrate-driven endothelial cell superoxide as effluxed from mitochondria respiring in vitro.

Reactive Oxygen and Targeted Antioxidant Administration in Endothelial Cell Mitochondria

“…To determine the concentration of TPP ϩ inside the mitochondrial matrix, it is necessary to know the mitochondrial matrix volume and the extent to which TPP ϩ is bound to mitochondrial protein as opposed to freely present within the matrix. Mitochondrial matrix volumes and the TPPϩ binding correction, under the conditions of incubation, were determined as we have previously reported (20).…”

“…This was accomplished through simultaneous recording of oxygen consumption and potential as we have previously carried out using electrodes sensitive to oxygen and to the distribution of the tetraphenylphosphonium ion (TPP ϩ ) (19,20). Mitochondria were incubated in respiratory buffer plus 5-M rotenone to inhibit electron entry at Complex-I, and 0.1-M nigericin to abolish the ⌬pH (34) across the mitochondrial membrane.…”

“…One approach to this involves the synthesis of compounds linking agents such as redox forms of Q compounds (ubiquinol and ubiquinone) or vitamin E to compounds like triphenylphosphonium, a lipophilic cation avidly taken up into the relatively negative mitochondrial matrix (17,18). In fact, we and several others have used the tetraphenylphosphonium ion to measure mitochondrial membrane potential (19,20).…”

“…One approach to this involves the synthesis of compounds linking agents such as redox forms of Q compounds (ubiquinol and ubiquinone) or vitamin E to compounds like triphenylphosphonium, a lipophilic cation avidly taken up into the relatively negative mitochondrial matrix (17,18). In fact, we and several others have used the tetraphenylphosphonium ion to measure mitochondrial membrane potential (19,20).One such compound, an alkyltriphenylphosphonium cation, termed mitoQ (mitoquinol or mitoquinone or a mixture of these redox forms), consists of compound Q covalently bound to the cation triphenylphosphonium. By virtue of its delocalized positive charge, mitoQ accumulates several hundredfold in mitochondria (18,21) and has been used to modulate ROS in the mitochondrial matrix (22).…”

Investigation on the Mechanism of Qi-Invigoration from a Perspective of Effects of Sijunzi Decoction on Mitochondrial Energy Metabolism

“…Mitochondria were isolated by differential centrifugation using a modified protocol of Fink et al [72]. Protein determinations were carried out by Bradford method using BSA as a standard [73].…”