Peroxynitrite Produced in Isolated Guinea Pig Cardiac Mitochondria: Role of Mitochondrial NOS

The presence of an endogenous nitric oxide synthase (NOS) isoform in cardiac mitochondria remains controversial. NOS catalyzes production of the gaseous signaling molecule nitric oxide (NO • ) from arginine. During cardiac ischemia/reperfusion (IR) injury, NO • can react with superoxide (O 2 •− ), derived from a free electron and O 2 , to form the strong oxidant peroxynitrite (ONOO − ), which initiates deleterious post‐translational modifications of cellular proteins and can cause tissue damage. We hypothesized that isolated mitochondria contain endogenous NOS. Our aim was to assess production of ONOO − in isolated cardiac mitochondria under conditions of oxidative/nitrosative stress, and to determine if ONOO − production is dependent on the presence of endogenous mitochondrial NOS. To investigate first if isolated guinea pig cardiac mitochondria contain NOS, Western blot analyses were performed using whole‐heart tissue and percoll‐purified cardiac mitochondria samples with anti‐eNOS and anti‐nNOS antibodies. The bands showed that the reacted eNOS antibody (140 kDa), but not the nNOS, antibody, was detected in both the whole‐heart and isolated mitochondria samples, where band density was much higher. Additionally, mitochondria were suspended in respiratory buffer (pH 7.15), and treated with succinate, a complex II substrate, and CaCl 2 followed by menadione, a redox cycler that generates large amounts of O 2 •− . In physiological buffer, added L‐tyrosine is oxidized by ONOO − to form the fluorescent dimer dityrosine (diTyr). We found that exposing isolated mitochondria energized with succinate to CaCl 2 and menadione resulted in an increase in the diTyr fluorescence signal, indicating that the simulated IR injury conditions enhanced mitochondrial ONOO − production. To verify that mitochondrial ONOO − production was dependent on NOS, mitochondria were incubated with the NOS inhibitors L‐NAME/L‐NNA, PTIO, a potent global NO • scavenger, and TEMPOL, a superoxide dismutase (SOD) mimetic. L‐NAME, L‐NNA and PTIO significantly decreased succinate, Ca 2+ and menadione‐mediated ONOO − production. TEMPOL also decreased ONOO − levels by converting O 2 •− to H 2 O 2 , as assessed by the HRP/amplex red assay. Some reports furnish evidence that NOS migrates from the cytosol to locate in mitochondrial outer membrane. These results suggest that mitochondrial NOS is responsible for the production of ONOO − during simulated oxidative/ nitrosative stress. Support or Funding Information National Heart, Lung, and Blood Institute Training Grant T35 HL072483