Faculty Collaboration Database

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 01;1861(12):148290

Date

08/24/2020

Pubmed ID

32828729

Pubmed Central ID

PMC7560995

DOI

10.1016/j.bbabio.2020.148290

Scopus ID

2-s2.0-85089838074 (requires institutional sign-in at Scopus site)   9 Citations

Abstract

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.

Author List

Gerdes HJ, Yang M, Heisner JS, Camara AKS, Stowe DF

Authors

Amadou K. Camara PhD Professor in the Anesthesiology department at Medical College of Wisconsin
David F. Stowe MD, PhD Professor in the Anesthesiology department at Medical College of Wisconsin

MESH terms used to index this publication - Major topics in bold

Animals
Calcium
Electron Transport
Free Radical Scavengers
Guinea Pigs
Hydrogen Peroxide
Isoenzymes
Membrane Potential, Mitochondrial
Mitochondria, Heart
Nitric Oxide
Nitric Oxide Synthase
Oxidative Stress
Peroxynitrous Acid
Spectrometry, Fluorescence
Stress, Physiological
Succinic Acid
Superoxide Dismutase
Time Factors