Isolated rat heart perfused with 1.5–7.5 μM NO solutions or bradykinin, which activates endothelial NO synthase, showed a dose-dependent decrease in myocardial O2uptake from 3.2 ± 0.3 to 1.6 ± 0.1 (7.5 μM NO, n = 18, P < 0.05) and to 1.2 ± 0.1 μM O2 ⋅ min−1 ⋅ g tissue−1 (10 μM bradykinin, n = 10, P < 0.05). Perfused NO concentrations correlated with an induced release of hydrogen peroxide (H2O2) in the effluent ( r = 0.99, P < 0.01). NO markedly decreased the O2 uptake of isolated rat heart mitochondria (50% inhibition at 0.4 μM NO, r = 0.99, P < 0.001). Cytochrome spectra in NO-treated submitochondrial particles showed a double inhibition of electron transfer at cytochrome oxidase and between cytochrome b and cytochrome c, which accounts for the effects in O2uptake and H2O2 release. Most NO was bound to myoglobin; this fact is consistent with NO steady-state concentrations of 0.1–0.3 μM, which affect mitochondria. In the intact heart, finely adjusted NO concentrations regulate mitochondrial O2uptake and superoxide anion production (reflected by H2O2), which in turn contributes to the physiological clearance of NO through peroxynitrite formation.

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Although transcriptional effects of thyroid hormones have substantial influence on oxidative metabolism, how thyroid sets basal metabolic rate remains obscure. Compartmental localization of nitric-oxide synthases is important for nitric oxide signaling. We therefore examined liver neuronal nitric-oxide synthase-␣ (nNOS) subcellular distribution as a putative mechanism for thyroid effects on rat metabolic rate. At low 3,3 ,5-triiodo-L-thyronine levels, nNOS mRNA increased by 3-fold, protein expression by one-fold, and nNOS was selectively translocated to mitochondria without changes in other isoforms. In contrast, under thyroid hormone administration, mRNA level did not change and nNOS remained predominantly localized in cytosol. In hypothyroidism, nNOS translocation resulted in enhanced mitochondrial nitric-oxide synthase activity with low O 2 uptake. In this context, NO utilization increased active O 2 species and peroxynitrite yields and tyrosine nitration of complex I proteins that reduced complex activity. Hypothyroidism was also associated to high phospho-p38 mitogenactivated protein kinase and decreased phospho-extracellular signal-regulated kinase 1/2 and cyclin D1 levels. Similarly to thyroid hormones, but without changing thyroid status, nitric-oxide synthase inhibitor N -nitro-L-arginine methyl ester increased basal metabolic rate, prevented mitochondrial nitration and complex I derangement, and turned mitogen-activated protein kinase signaling and cyclin D1 expression back to control pattern. We surmise that nNOS spatial confinement in mitochondria is a significant downstream effector of thyroid hormone and hypothyroid phenotype.Hypothyroidism is a prevalent disorder associated to low oxygen utilization and low tissue proliferation rate (1). In addition to non-genomic effects (2), thyroid hormones influence transcription of a number of nuclear and mitochondrial-encoded respiratory genes (3). Although direct or transcriptional effects have considerable impact on oxidative metabolism and hemodynamic function, much is still unknown about how thyroid hormones set the metabolic rate of the body (4); consonant with slowness of transcriptional mechanisms, treatment of hypothyroidism may require weeks of hormone administration to normalize the altered functions (5). In the last decade, the effects of nitric oxide (NO) 2 expanded from the vascular system to the intracellular milieu. In this context, subcellular localization of nitric oxide-synthases (NOS) with effector molecules is an important regulatory mechanism for NO signaling (6, 7). Accordingly, we are interested in the traffic of a posttranslationally modified variant of neuronal nitric-oxide synthase-␣ (nNOS) to mitochondria (formerly named mitochondrial nitric-oxide synthase or mtNOS), which vectorially directs NO to the matrix compartment (8, 9). mtNOS could be low in adult rodents, but a modulated increase has been associated to thyroid status (10), release of cytochrome c (11), mitochondrial protein nitration (12), liver and brain development (13,...

Sepsis-associated multiple organ failure is a major cause of mortality characterized by a massive increase of reactive oxygen and nitrogen species (ROS/RNS) and mitochondrial dysfunction. Despite intensive research, determining events in the progression or reversal of the disease are incompletely understood. Herein, we studied two prototype sepsis models: endotoxemia and cecal ligation and puncture (CLP)-which showed very different lethality rates (2.5% and 67%, respectively)-, evaluated iNOS, ROS and respiratory chain activity, and investigated mitochondrial biogenesis and dynamics, as possible processes involved in sepsis outcome. Endotoxemia and CLP showed different iNOS, ROS/RNS, and complex activities time-courses. Moreover, these alterations reverted after 24-h endotoxemia but not after CLP. Mitochondrial biogenesis was not elicited during the first 24 h in either model but instead, 50% mtDNA depletion was observed. Mitochondrial fusion and fission were evaluated using real-time PCR of mitofusin-2 (Mfn2), dynamin-related protein-1 (Drp1), and using electron microscopy. During endotoxemia, we observed a decrease of Mfn2-mRNA levels at 4-6 h, and an increase of mitochondrial fragmentation at 6 h. These parameters reverted at 24 h. In contrast, CLP showed not only decreased Mfn2-mRNA levels at 12-18 h but also increased Drp1-mRNA levels at 4 h, and enhanced and sustained mitochondrial fragmentation. The in vivo pretreatment with mdivi-1 (Drp1 inhibitor) significantly attenuated mitochondrial dysfunction and apoptosis in CLP. Therefore, abnormal fusion-to-fission balance, probably evoked by ROS/RNS secondary to iNOS induction, contributes to the progression of sepsis. Pharmacological targeting of Drp1 may be a potential novel therapeutic tool for sepsis.