MiP2005: Session 3

Mitochondrial Physiology Network 10.9: 39-40 (2005) - download pdf


Mitochondrial nitrogen radical synthesis by a NOS-independent mechanism.

Zsombor Lacza1,2, E Pankotai2, A Csordás2, G Wolf3, M Kollai2, C Szabó2, DW Busija1, TFW Horn3

1Dept. Physiology/Pharmacology, Wake Forest University School of Medicine, Medical Center Blvd., Winston-Salem, NC 27157, USA; 2Inst. Human Physiology Clinical Experimental Research, Semmelweis University, Üllöi út 78/a, 1082 Budapest, Hungary; 3Inst. Medical Neurobiology, Otto-von-Guericke University, Leipziger Str.44, D-39120 Magdeburg, Germany. - zlacza@mac.com

    Mitochondria are suspected to produce nitric oxide (NO) and reactive nitrogen species (RNS), which regulate the action of the respiratory chain. However, the existence of a distinct mitochondrial NO synthase enzyme (mtNOS) is debated and the mechanism by which mitochondria produce RNS is unclear [1]. We hypothesized that not mtNOS, but the respiratory chain enzymes are responsible for 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. Fluorescence was detected by confocal microscopy and flow cytometry. Respiring mitochondria produced reactive nitrogen species, which were inhibited by catalysts of peroxynitrite decomposition. Mitochondria from different regions had varying morphology, but their DAF fluorescence was similar. Withdrawal of arginine and calcium or the application of nitric oxide synthase inhibitors failed to decrease DAF fluorescence. In contrast, disrupting the integrity of the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of Complex I abolished the DAF signal, which was restored in the presence of Complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone cycle or even dissipating the proton gradient had no effect on DAF fluorescence indicating that the redox state of ubiquinone significantly affects the reaction. 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 mitochondrial nitric oxide synthase.

1.  Lacza Z, Pankotai E, Csordás A, Gerő D, Kiss L, Horváth EM, Kollai M, Busija DW, Szabó C (2005) Mitochondrial NO and reactive nitrogen species production: does mtNOS exist? (2005) Nitric Oxide - Biology and Chemistry (in press).

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