Identification of a neuronal nitric oxide synthase in isolated cardiac mitochondria using electrochemical detection

Abstract

Mitochondrial nitric oxide synthase (mtNOS), its cellular NOS isoform, and the effects of mitochondrially produced NO on bioenergetics have been controversial since mtNOS was first proposed in 1995. Here we functionally demonstrate the presence of a NOS in cardiac mitochondria. This was accomplished by direct porphyrinic microsensor measurement of Ca 2؉ -dependent NO production in individual mitochondria isolated from wild-type mouse hearts. This NO production could be inhibited by NOS antagonists or protonophore collapse of the mitochondrial membrane potential. The similarity of mtNOS to the neuronal isoform was deduced by the absence of NO production in the mitochondria of knockout mice for the neuronal, but not the endothelial or inducible, isoforms. The effects of mitochondrially produced NO on bioenergetics were studied in intact cardiomyocytes isolated from dystrophindeficient (mdx) mice. mdx cardiomyocytes are also deficient in cellular endothelial NOS, but overexpress mtNOS, which allowed us to study the mitochondrial enzyme in intact cells free of its cytosolic counterpart. In these cardiomyocytes, which produce NO beat-to-beat, inhibition of mtNOS increased myocyte shortening by approximately one-fourth. Beat-to-beat NO production and altered shortening by NOS inhibition were not observed in wildtype cells. A plausible mechanism for the reversible NO inhibition of contractility in these cells involves the reaction of NO with cytochrome c oxidase. This suggests a modulatory role for NO in oxidative phosphorylation and, in turn, myocardial contractility.cardiomyocytes ͉ cytochrome oxidase ͉ oxidative phosphorylation ͉ respiration ͉ chronoamperometry T he existence of a nitric oxide synthase (NOS) that is localized in the mitochondria (mtNOS) was originally described in a series of immunohistochemical studies published between 1995 (1, 2) and 1996 (3). Because all of the known NOS isoforms are encoded by nuclear DNA and NOS is not encoded by mitochondrial DNA, this finding implied that one of the recognized NOS isoforms was targeted to the mitochondria after protein synthesis in the cytosol. In these early studies, it was reported that the endothelial NOS (eNOS) isoform was localized to the inner mitochondrial membrane in all tissues that were tested, which included brain, kidney, liver, and skeletal and cardiac muscle. Between 1997 and 1998, more in-depth studies using a variety of NO detection techniques with isolated rat liver mitochondria (4), submitochondrial particles (SMPs; ref. 4), and purified NOS enzyme (5-7) added further support for the existence of a mtNOS. However, these studies were unable to determine whether the enzyme was novel or related to the neuronal (nNOS), inducible (iNOS), or eNOS isoforms. In the last three years, some laboratories have extended their studies to the investigation of the functional implications of mtNOS (8-11), while others have used a NO-sensitive dye to stain the mitochondria in intact cells and demonstrate the presence of NO within these organelles (12). Howeve...

Biological chemistry of reactive oxygen and nitrogen and radiation-induced signal transduction mechanisms

“…Kanai et al, 2001;Paxinou et al, 2001;Sarkela et al, 2001;Elfering et al, 2002). However, recent careful measurements of concentration-response curves for mitochondrial respiration in brain slices bring into question the role of cytochrome c oxidase.…”

“…Thus, endogenously generated NO protects cells against H 2 O 2 exposure only when cells are actively respiring (Paxinou et al, 2001). The importance of NO in regulating cellular ROS levels is underlined by the recent identification of a Ca 2 þ -activated NOS isoform in the mitochondria of cells (Kanai et al, 2001;Elfering et al, 2002). Pharmacological inhibition of cellular NOS stimulates O 2 À production, and NO scavengers induce cellular oxidative stress (Niu et al, 1996;Goda et al, 1997;Janssen et al, 1998a, b).…”

“…The NOS-1 gene is made up of 27 exons and encodes for several different isoforms with molecular sizes ranging from 150 to 165 kDa. A specific isoform of NOS-1 has been identified in the mitochondria of liver and cardiomyocytes (Kanai et al, 2001;Elfering et al, 2002), and its mRNA transcript has been identified in most if not all tissues examined (Elfering et al, 2002). The mitochondrial NOS-1 is myristoylated in contrast to other NOS-1 isoforms.…”

A novel method of measuring nitric-oxide-dependent fluorescence using 4,5-diaminofluorescein (DAF-2) in the isolated Langendorff-perfused rabbit heart

“…It is likely that the basal fluorescence signal could originate from a number of sources since NO synthases can be found in endothelial cells (Miethke et al, 2003), neurones and nerve fibres of cardiac ganglia (Sosunov et al, 1996;Sosunov et al, 1995) and subcellularly located within cardiac myocytes e.g. sarcoplasmic reticulum and mitochondria (Xu et , 1999;Kanai et al, 2001). We have performed preliminary experiments applying this novel NO measurement technique to the innervated isolated heart preparation which we developed (Ng et al, 2001).…”

Docking of Endothelial Nitric Oxide Synthase (eNOS) to the Mitochondrial Outer Membrane

“…There is an ongoing debate as to the identity of mtNOS; immunologic cross-reaction of mtNOS with antibodies against endothelial NOS (eNOS) (1,3,7), neuronal NOS (4), and inducible NOS (6) have been reported. A recent investigation (8) has identified a neuronal NOS in cardiomyocyte mitochondria. It has been suggested that mtNOS plays important roles in oxidative stress and apoptosis (9,10), regulation of mitochondrial respiration (11,12), and modulation of intracellular Ca 2ϩ homeostasis (13).…”