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

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 Ca 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 CaCl and then the complex II substrate Na-succinate, followed by menadione, a quinone redox cycler, to generate O. 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 CaCl before succinate resulted in an increase in diTyr and amplex red fluorescence (HO) signals, indicating that mitochondrial oxidant stress, induced by elevated mtCa and succinate, increased mitochondrial ONOO production via NO and O. 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 O to HO. 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 O released from mitochondria and NO derived from a mtCa-inducible nNOS isoform, possibly attached to mitochondria, and a mtNOS isoform like iNOS that is non-inducible.