The nitric oxide oxidative pathway in mitochondria.

PODEROSO JJ; LISDERO CL; SCHÖPFER F; CARRERAS MC; CADENAS E; BOVERIS A

Los Angeles, EEUU

Congreso; Third International Conference Biochemistry and Molecular Biology of Nitric Oxide.; 1998

We have previously reported that NO increases superoxide anion (O2-) and hydrogen peroxide (H2O2) production by mitohcondria by reacting with ubiquinol (QH2); the sequence of oxidative pathways involve the following reactions [1] NO + QH2 ® Q- + NO-, [2] Q- + O2 ® O2- + Q, [3] O2- + O2- ® H2O2 + O2, and [4] NO + O2- ® ONOO- (peroxynitrite). The aim of this study was to analyze the influence of NO oxidative pathway on NO decay (-d[NO]/dt) and its effects on liver mitochondria and submitochondrial particles (SMP) To that purpose mitochondria isolated from Sprague-Dawley rats were exposed to a single 0.2-2 µM NO pulse and NO kinetic changes were recorded simultaneously with O2 mitochondrial uptake, membrane potential and H2O2 production rate (d[H2O2]/dt). Oxygen uptake was already inhibited at 0.2 µM NO during a lapse dependent on the initial NO levels. In the presence of succinate 8 mM as substrate, the spontaneous -d[NO]/dt increased 75% with addition of SMP (0.1 mg protein/ ml) and moreover, increased 20%, by addition of  2 µM QH2-0 or decreased 30% either with the addition of 2 µM SOD or in the absence of substrate, indicating that NO concentration in mitochondria is partially regulated by reactions 1-4. The d[H2O2]/dt was dependent both on NO and mitochondrial protein concentration. At 0.5 µM NO, d[H2O2]/dt remained stable and stopped after a lapse inversely related ro protein concentration r=0.99, p<0.05 but without changes in H2O2 total production, concomitantly -d[NO]/dt was directly related to SMP concentration. In agreement wuth reaction [1], the supplementation of SMP with QH2 (QH2-0 or QH2-2, 20-120 nmol. mg protein-1) markedly increased d[H2O2]/dt. Since d[H2O2]/dt increased after a lag time and was sustained after NO was extinguished; sources of O2- other than reaction [1] may be expected. In this way, we observed that ONOO- oxidized ubiquinol according to reaction [5]: ONOO- + QH2 ® .NO2 + Q- and subsequently reaction [2]; likewise, SMP supplemented with 0.25-2 µM ONOO- released 0.015-0.1 µM min-1 O2-. It can be assumed that, at least, part of NO-induced O2- release depends on the ONOO- formed in mitochondria by reactions [1-3]. The data suggest that, a cohort of oxidative reactions that take place in mitochondria may modulate NO concentration and its effects; also, oxidative reactions and the reversibility of NO effects should depend on QH2 concentration in mitochondrial membranes.