Modulation of peroxynitrite produced via mitochondrial nitric oxide synthesis during Ca2+ and succinate-induced oxidative stress in cardiac isolated mitochondria

Biochim Biophys Acta Bioenerg. 2020 Dec 1;1861(12):148290. doi: 10.1016/j.bbabio.2020.148290. Epub 2020 Aug 20.


We hypothesized that NO is generated in isolated cardiac mitochondria as the source for ONOO- production during oxidative stress. We monitored generation of ONOO- from guinea pig isolated cardiac mitochondria subjected to excess Ca2+ uptake before adding succinate and determined if ONOO- production was dependent on a nitric oxide synthase (NOS) located in cardiac mitochondria (mtNOS). Mitochondria were suspended in experimental buffer at pH 7.15, and treated with CaCl2 and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O2•-. L-tyrosine was added to the mitochondrial suspension where it is oxidized by ONOO- to form dityrosine (diTyr) in proportion to the ONOO- present. We found that exposing mitochondria to excess CaCl2 before succinate resulted in an increase in diTyr and amplex red fluorescence (H2O2) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa2+ and succinate, increased mitochondrial ONOO- production via NO and O2•-. Changes in mitochondrial ONOO- production dependent on NOS were evidenced by using NOS inhibitors L-NAME/L-NNA, TEMPOL, a superoxide dismutase (SOD) mimetic, and PTIO, a potent global NO scavenger. L-NAME and L-NNA decreased succinate and menadione-mediated ONOO- production, PTIO decreased production of ONOO-, and TEMPOL decreased ONOO- levels by converting more O2•- to H2O2. Electron microscopy showed immuno-gold labeled iNOS and nNOS in mitochondria isolated from cardiomyocytes and heart tissue. Western blots demonstrated iNOS and nNOS bands in total heart tissue, bands for both iNOS and nNOS in β-tubulin-free non-purified (crude) mitochondrial preparations, and a prominent iNOS band, but no nNOS band, in purified (Golgi and ER-free) mitochondria. Prior treatment of guinea pigs with lipopolysacharride (LPS) enhanced expression of iNOS in liver mitochondria but not in heart mitochondria. Our results indicate that release of ONOO- into the buffer is dependent both on O2•- released from mitochondria and NO derived from a mtCa2+-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.

Keywords: Heart; Liver mitochondria; Mitochondrial oxidant stress; Nitric oxide; Nitric oxide synthase; Peroxynitrite; Superoxide.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Animals
  • Calcium / pharmacology*
  • Electron Transport / drug effects
  • Free Radical Scavengers / metabolism
  • Guinea Pigs
  • Hydrogen Peroxide / metabolism
  • Isoenzymes / metabolism
  • Isoenzymes / ultrastructure
  • Membrane Potential, Mitochondrial / drug effects
  • Mitochondria, Heart / drug effects
  • Mitochondria, Heart / metabolism*
  • Mitochondria, Heart / ultrastructure
  • Nitric Oxide / biosynthesis*
  • Nitric Oxide Synthase / antagonists & inhibitors
  • Nitric Oxide Synthase / metabolism
  • Nitric Oxide Synthase / ultrastructure
  • Oxidative Stress / drug effects*
  • Peroxynitrous Acid / metabolism*
  • Spectrometry, Fluorescence
  • Stress, Physiological / drug effects
  • Succinic Acid / pharmacology*
  • Superoxide Dismutase / metabolism
  • Time Factors


  • Free Radical Scavengers
  • Isoenzymes
  • Peroxynitrous Acid
  • Nitric Oxide
  • Succinic Acid
  • Hydrogen Peroxide
  • Nitric Oxide Synthase
  • Superoxide Dismutase
  • Calcium