Mitochondria produce reactive nitrogen species via an arginine-independent pathway.
We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.
Lacza
Zsombor
Z
Department of Physiology/Pharmacology, Wake Forest University Health Sciences, Winston-Salem, NC 27157, USA. zlacza@mac.com
Kozlov
Andrey V
AV
Pankotai
Eszter
E
Csordás
Attila
A
Wolf
Gerald
G
Redl
Heinz
H
Kollai
Márk
M
Szabó
Csaba
C
Busija
David W
DW
Horn
Thomas F W
TF
D000818:Animals; D001120:Arginine / Q000378:metabolism*; D002478:Cells, Cultured; D004579:Electron Transport / Q000502:physiology*; D005434:Flow Cytometry; D006801:Humans; D051379:Mice; D018613:Microscopy, Confocal; D008928:Mitochondria / Q000378:metabolism*; D026361:Reactive Nitrogen Species / Q000096:biosynthesis*
D026361:Reactive Nitrogen Species; D001120:Arginine