Human heart mitochondria do not produce physiologically relevant quantities of nitric oxide Attila Csordás a , Eszter Pankotai a , James A. Snipes b , Attila Cselenyák a , Zsolt Sárszegi c , Attila Cziráki c , Balázs Gaszner c , Lajos Papp c , Rita Benkő a , Levente Kiss a , Endre Kovács a , Márk Kollai a , Csaba Szabó a , David W. Busija b , Zsombor Lacza a,b, ⁎ a Department of Human Physiology and Clinical Experimental Research, Semmelweis University, Budapest, Hungary b Department of Physiology/Pharmacology, Wake Forest University Medical Center, Winston-Salem, NC, USA c Heart Institute, Faculty of Medicine, University of Pécs, Hungary Received 14 April 2006; accepted 17 October 2006 Abstract Previous studies raised the possibility that nitric oxide synthase is present in heart mitochondria (mtNOS) and the existence of such an enzyme became generally accepted. However, original experimental evidence is rather scarce and positive identification of the enzyme is lacking. We aimed to detect an NOS protein in human and mouse heart mitochondria and to measure the level of NO released from the organelles. Western blotting with 7 different anti-NOS antibodies failed to detect a NOS-like protein in mitochondria. Immunoprecipitation or substrate-affinity purification of the samples concentrated NOS in control preparations but not in mitochondria. Release of NO from live respiring human mitochondria was below 2 ppb after 45 min of incubation. In a bioassay system, mitochondrial suspension failed to cause vasodilation of human mammary artery segments. These results indicate that mitochondria do not produce physiologically relevant quantities of NO in the heart and are unlikely to have any physiological importance as NO donors, nor do they contain a recognizable mtNOS enzyme. © 2006 Elsevier Inc. All rights reserved. Keywords: Mitochondrion; Nitric oxide; Circulation; Vasodilatation Introduction Nitric oxide (NO) is a key signaling molecule in the mito- chondrial environment. Due to its affinity to metal complexes NO can react with the active center of several electron transport chain enzymes and in doing so it inhibits mitochondrial res- piration (Brown, 1999). The metabolism of NO is also closely associated with mitochondria: the bioactivation of nitroglycerin is the function of mitochondrial enzymes, and on the other hand, NO is inactivated by cytochrome c oxidase (Chen et al., 2002; Pearce et al., 2002), although in hypoxic conditions, yeast and rat liver mitochondria produce NO from nitrite through the activity of cytochrome c oxidase (Kozlov et al., 1999; Castello et al., 2006). Therefore, NO production by mitochondria can be an effective means of self-regulation and it may serve as a specific and novel drug target for the induction of ischemic preconditioning, preservation of tissues during transplantation, and even against degenerative diseases. Several studies raised the possibility that a distinct mitochondrial NO synthase enzyme (mtNOS) exists, although this theory was challenged recently (for contrasting reviews see Giulivi, 2003; Brookes, 2004; Lacza et al., 2006). The majority of these earlier experiments were conducted on mitochondria from liver or brain. In the porcine heart it was shown that mtNOS is insignificant and cannot produce enough NO to have any effect on the respiratory chain enzymes (French et al., 2001). In contrast, several other studies described the participation of heart mtNOS in various pathological mechan- isms and the existence of such an enzyme became generally accepted (Kanai et al., 2001; Costa et al., 2002; Manzo-Avalos et al., 2002; Boveris et al., 2002; Valdez et al., 2004). To the best Life Sciences 80 (2007) 633 – 637 www.elsevier.com/locate/lifescie ⁎ Corresponding author. H-1082 Budapest, Üllői út 78/a, Hungary. Tel.: +36 1 2100306; fax: +36 1 3343162. E-mail address: zlacza@mac.com (Z. Lacza). 0024-3205/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2006.10.009