Mitochondrial Nitric Oxide Synthase: A Ubiquitous Regulator of Oxidative Phosphorylation? T. E. Bates, A. Loesch,* G. Burnstock,* and J. B. Clark Department of Neurochemistry, Institute of Neurology, University of London, Queen Square, London, WC1N 3BG, England; and *Department of Anatomy and Developmental Biology, University College London, Gower Street, London, WC1E 6BT, England Received November 13, 1995 In this article we demonstrate the immunocytochemical localization of nitric oxide synthase in mitochondria isolated from heart, skeletal muscle, and kidney, using a monoclonal antibody directed against the endothelial form of nitric oxide synthase. The possibility that mitochondrially located nitric oxide synthase is a ubiquitous regulator of mitochondrial oxidative phosphorylation in mammalian cells is discussed. © 1996 Academic Press, Inc. In a recent paper we presented immunocytochemical evidence for the presence of nitric oxide synthase (NOS) in non-synaptic brain mitochondria and liver mitochondria (1). We also suggested that mitochondrially located NOS (mtNOS) might be involved in the regulation of oxidative phosphorylation, because of the ability of nitric oxide (NO) (a product of NOS activity) to bind to cytochrome oxidase and inhibit electron transport (2). If mtNOS is an intrinsic regulator of mito- chondrial electron transport, then one would predict that it would be present in mitochondria from other mammalian tissues. Therefore in this study we have investigated the mitochondrial localiza- tion of NOS within mitochondria isolated from heart, skeletal muscle and kidney utilizing a postembedding immunogold procedure with silver enhancement. MATERIALS AND METHODS Isolation of mitochondria. Preparations of heart, skeletal muscle and kidney mitochondria were isolated from adult male Wistar rats using minor modifications of established (non-enzymatic) methods (3-5). Isolated mitochondria were incubated in respiration medium with 2.5 mM malate and 10 mM glutamate at 37°C prior to pelleting in a centrifuge at 16,000g. Immunocytochemistry. Isolated mitochondria were fixed for 1h at 4°C with 2% paraformaldehyde and 0.05% glutaral- dehyde in 100 mM phosphate buffer pH 7.4, dehydrated through graded ethanol and embedded in Unicryl resin (Agar, U.K.). Ultrathin sections (on uncoated nickel grids) were processed for post-embedding colloidal gold immunocytochem- istry as previously reported (1,6). Mouse anti-eNOS monoclonal antibody (Affiniti, isotype: IgG1, specificity: human and rat) was used at a dilution of 1:25-1:50; gold-labelled goat anti-mouse IgG: 10nm (BioCell, U.K.) was used at a dilution of 1:25. Signal was intensified with silver using a silver enhancing kit (Biocell, U.K.). After the immunoprocedure, the ultrathin sections were stained with uranyl acetate and lead citrate and subsequently examined with a JEM-1010 electron microscope. The specificity of the immunoprocedure was investigated routinely, including the omission of the primary antibody step, resulting in elimination of positive labelling. The monoclonal anti-eNOS antibody used was raised in mouse using a 20.4 kDa protein fragment corresponding to amino acids 1030 to 1209 of human eNOS as an antigen. In order to establish the percentage of mitochondria positive (and negative) for eNOS, mitochondria were counted upon examination of electron micrographs. Data is expressed as mean ± S.E.M. (n  number of microscopic fields studied). RESULTS Silver enhanced gold immunolabelling for eNOS was seen within mitochondrial preparations from heart, skeletal muscle and kidney (Fig 1a–c). A high percentage of mitochondria from all three tissues were positive for eNOS. However, the labelling signal varied from mitochondrion to mitochondrion. Approximately 86.4% ± 3.3% of mitochondria in heart (154 mitochondria out of a total of 176 examined; n  6), 70.3% ± 3.8% in skeletal muscle (132 mitochondria out of a total of 184 examined; n  6) and 63.7% ± 4.4% in kidney (234 mitochondria out of a total of 351 examined; n  8) displayed the silver-gold signal/immunodeposit. The immunodeposit was seen BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS 218, 40–44 (1996) ARTICLE NO. 0008 40 0006-291X/96 $18.00 Copyright © 1996 by Academic Press, Inc. All rights of reproduction in any form reserved.