Lack of mitochondrial nitric oxide production in the mouse brain

Zsombor Lacza, Thomas F.W. Horn, James A. Snipes, Jie Zhang, Sanjoy Roychowdhury, Eszter M. Horváth, Jorge P. Figueroa, Márk Kollai, Csaba Szabó, David W. Busija

Research output: Contribution to journalArticlepeer-review

37 Scopus citations

Abstract

Based on our initial finding that the nitric oxide (NO) sensitive fluorochrome diaminofluorescein (DAF) was localized to mitochondria in cultured primary neurons, we investigated whether brain mitochondria produce NO through a mitochondrial NO synthase (mtNOS) enzyme. Isolated brain mitochondria were loaded with DAF and subjected to flow cytometry analysis. Neither the application of NOS inhibitors nor the genetic disruption of either NOS gene diminished the DAF-fluorescence. However, peroxynitrite scavengers reduced the mitochondrial DAF fluorescence, indicating that the DAF signal is not specific to NO. Chemiluminescence detection in the head space gas and a Clark-type NO-sensitive electrode in the solution failed to detect NO release in brain mitochondria. NOS activity in mitochondria was only 1% of the whole brain NOS activity level, which may be attributed to extramitochondrial contamination. Extensive immunoblotting and immunoprecipitation experiments failed to show the presence of endothelial, neuronal, or inducible NOS in mouse brain mitochondria using a variety of primary antibodies. Arginine, calmodulin or 2,5-ADP affinity purification protocols successfully concentrated eNOS and nNOS from full brain tissue but failed to show any signal in mitochondria. We conclude that mouse brain mitochondria do not contain NOS isoforms, nor do they produce NO through a NOS-dependent mechanism.

Original languageEnglish (US)
Pages (from-to)942-951
Number of pages10
JournalJournal of neurochemistry
Volume90
Issue number4
DOIs
StatePublished - Aug 2004
Externally publishedYes

Keywords

  • Diaminofluorescein
  • FP15
  • L-NAME
  • Mitochondrial nitric oxide synthase
  • Mitochondrion
  • Peroxynitrite

ASJC Scopus subject areas

  • Biochemistry
  • Cellular and Molecular Neuroscience

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