Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS.
Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.
Lacza
Zsombor
Z
Department of Physiology/Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC, USA. zlacza@mac.com
Snipes
James A
JA
Zhang
Jie
J
Horváth
Eszter M
EM
Figueroa
Jorge P
JP
Szabó
Csaba
C
Busija
David W
DW
D000818:Animals; D001120:Arginine / Q000378:metabolism; D015153:Blotting, Western; D005434:Flow Cytometry; D005456:Fluorescent Dyes; D051379:Mice; D008810:Mice, Inbred C57BL; D018345:Mice, Knockout; D018613:Microscopy, Confocal; D008930:Mitochondria, Liver / Q000201:enzymology*; D009569:Nitric Oxide / Q000378:metabolism; D019001:Nitric Oxide Synthase / Q000235:genetics / Q000502:physiology*; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; D011233:Precipitin Tests
D005456:Fluorescent Dyes; D009569:Nitric Oxide; D001120:Arginine; D019001:Nitric Oxide Synthase; D052248:Nitric Oxide Synthase Type I; D052247:Nitric Oxide Synthase Type II; D052250:Nitric Oxide Synthase Type III; C496321:Nos1 protein, mouse; C496317:Nos2 protein, mouse; C496315:Nos3 protein, mouse