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Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy

^a Department of Biochemical Sciences `A. Rossi Fanelli' and CNR Center of Molecular Biology, University of Rome `La Sapienza', I-00185 Roma, Italy
^b CNR–Institute of Biophysics, Pisa, Italy

With the electro-driven import of rhodamine 123, we used single cell fluorescence microscopy to single out the contribution of nitric oxide (NO) in controlling mitochondrial membrane potential expressed by (stationary growing) rhabdomyosarcoma and neuroblastoma cells in culture. The experimental design and the computer-aided image analysis detected and quantitated variations of fluorescence signals specific to mitochondria. We observed that 1) the two cell lines display changes of fluorescence dependent on mitochondrial energization states; 2) mitochondrial fluorescence decreases after exposure of the cells to a NO releaser; 4) the different fluorescence intensity measured under stationary growing conditions, or after activation and inhibition of constitutive NO synthase, is consistent with a steady-state production of NO. Direct comparison of single cell fluorescence with bulk cytofluorimetry proved that the results obtained by the latter method may be misleading because of the intrinsic-to-measure lack of information about distribution of fluorescence within different cell compartments. The kinetic parameters describing the reactions between cytochrome oxidase, NO, and O₂ may account for the puzzling (20-fold) increase of the K_M for O₂ reported for cells and tissues as compared to purified cytochrome c oxidase, allowing an estimate of in vivo NO flux.—Sarti, P., Lendaro, E., Ippoliti, R., Bellelli, A., Benedetti, P. A., Brunori, M. Modulation of mitochondrial respiration by nitric oxide: investigation by single cell fluorescence microscopy. FASEB J. 13, 191–197 (1999)

Key Words: NO • cell respiration • mitochondria • cytochrome oxidase • NOR₁ • neuroblastoma

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