Human heart mitochondria do not produce physiologically relevant quantities of nitric oxide

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.     

Mitochondria produce reactive nitrogen species via an arginine-independent pathway

We measured the contribution of mitochondrial nitric oxide synthase (mtNOS) and respiratory chain enzymes to reactive nitrogen species (RNS) production. Diaminofluorescein (DAF) was applied for the assessment of RNS production in isolated mouse brain, heart and liver mitochondria and also in a cultured neuroblastoma cell line by confocal microscopy and flow cytometry. Mitochondria produced RNS, which was inhibited by catalysts of peroxynitrite decomposition but not by nitric oxide (NO) synthase inhibitors. Disrupting the organelles or withdrawing respiratory substrates markedly reduced RNS production. Inhibition of complex I abolished the DAF signal, which was restored by complex II substrates. Inhibition of the respiratory complexes downstream from the ubiquinone/ubiquinol cycle or dissipating the proton gradient had no effect on DAF fluorescence. We conclude that mitochondria from brain, heart and liver are capable of significant RNS production via the respiratory chain rather than through an arginine-dependent mtNOS.     

Mitochondrial nitric oxide synthase is not eNOS, nNOS or iNOS

Recent studies indicated that there is a distinct mitochondrial nitric oxide synthase (mtNOS) enzyme, which may be identical to the other known NOS isoforms. We investigated the possible involvement of the endothelial, the neuronal, and the inducible NOS isoforms (eNOS, nNOS, iNOS, respectively) in mitochondrial NO production. Mouse liver mitochondria were prepared by Percoll gradient purification from wild-type and NOS knockout animals. NOS activity was measured by the arginine conversion assay, NO production of live mitochondria was visualized by the fluorescent probe DAF-FM with confocal microscopy and measured with flow cytometry. Western blotting or immunoprecipitation was performed with 12 different anti-NOS antibodies. Mitochondrial NOS was purified by arginine, 2,5 ADP and calmodulin affinity columns. We observed NO production and NOS activity in mitochondria, which was not attenuated by classic NOS inhibitors. We also detected low amounts of eNOS protein in the mitochondria, however, NO production and NOS activity were intact in eNOS knockout animals. Neither nNOS nor iNOS were present in the mitochondria. Furthermore, we could not find mitochondrial targeting signals in the sequences of either NOS proteins. Taken together, the presented data do not support the hypothesis that any of the known NOS enzymes are present in the mitochondria in physiologically relevant levels.     

Nitric oxide synthase in porcine heart mitochondria: evidence for low physiological activity

The capacity of isolated porcine heart mitochondria to produce nitric oxide (NO) via mitochondrial NO synthase (NOS) was evaluated. The mitochondrial NOS content and activity (0.2 nmol NO x mg mitochondrial protein(-1) x min(-1)) were approximately 10 times lower than previously reported for the rat liver. No evidence for mitochondrial NOS-generated NO was found in mitochondrial suspensions based on the lack of NO production and the lack of effect of either L-arginine or NOS inhibitors on the rate of respiration. The reason that even the low mitochondrial NOS activity did not result in net NO production and metabolic effects is because the mitochondrial metabolic breakdown of NO (1-4 nmol NO x mg mitochondrial protein(-1) x min(-1)) was greater than the maximum rate of NO production measured in homogenates. These data suggest that NO production at the mitochondria via NOS is not a significant source of NO in the intact heart and does not regulate cardiac oxidative phosphorylation.     

Mitochondrial function and nitric oxide metabolism are modified by enalapril treatment in rat kidney

The renal and cardiac benefits of renin-angiotensin system (RAS) inhibition in hypertension exceed those attributable to blood pressure reduction, and seem to involve mitochondrial function changes. To investigate whether mitochondrial changes associated with RAS inhibition are related to changes in nitric oxide (NO) metabolism, four groups of male Wistar rats were treated during 2 wk with a RAS inhibitor, enalapril (10 mg x kg(-1) x day(-1); Enal), or a NO synthase (NOS) inhibitor, N(omega)-nitro-L-arginine methyl ester (L-NAME) (1 mg x kg(-1) x day(-1)), or both (Enal+L-NAME), or were untreated (control). Blood pressure and body weight were lower in Enal than in control. Electron transfer through complexes I to III and cytochrome oxidase activity were significantly lower, and uncoupling protein-2 content was significantly higher in kidney mitochondria isolated from Enal than in those from control. All of these changes were prevented by L-NAME cotreatment and were accompanied by a higher production/bioavailability of kidney NO. L-NAME abolished mitochondrial NOS activity but failed to inhibit extra-mitochondrial kidney NOS, underscoring the relevance of mitochondrial NO in those effects of enalapril that were suppressed by L-NAME cotreatment. In Enal, kidney mitochondria H(2)O(2) production rate and MnSOD activity were significantly lower than in control, and these effects were not prevented by L-NAME cotreatment. These findings may clarify the role of NO in the interactions between RAS and mitochondrial metabolism and can help to unravel the mechanisms involved in renal protection by RAS inhibitors.     

Nitric oxide ameliorates hydrophobic bile acid-induced apoptosis in isolated rat hepatocytes by non-mitochondrial pathways

Hydrophobic bile acids are toxic to isolated rat hepatocytes by mechanisms involving mitochondrial dysfunction and oxidative stress. In the current study we examined the role of nitric oxide (NO), a potential mediator of apoptosis, during bile acid-induced apoptosis. Freshly isolated rat hepatocytes and hepatic mitochondria generated NO and peroxynitrite (ONOO(-)) in a concentration- and time-dependent manner when exposed to the toxic bile salt glycochenodeoxycholate (GCDC) (25-500 microm), which was prevented by the nitric-oxide synthase (NOS) inhibitors N(G)-monomethyl-N-arginine monoacetate (l-NMMA) and 1400W. Relationships between hepatocyte NO production and apoptosis were examined by comparing the effects of NOS inhibitors with other inhibitors of GCDC-induced apoptosis. Inhibitors of caspases 8 and 9, the mitochondrial permeability transition blocker cyclosporin A, and the antioxidant idebenone reduced NO generation and apoptosis in GCDC-treated hepatocytes. In contrast, NOS inhibitors had no effect on GCDC-induced apoptosis despite marked reduction of NO and ONOO(-). However, treatment with the NO donors S-nitroso-N-acetylpenicillamine and spermine NONOate [N-(-aminoethyl)N-(2-hydroxy-2-nitrohydrazino)-1,2-ethylenediamine) inhibited apoptosis and caspase 3 activity while significantly elevating NO levels above GCDC-stimulated levels. Neither NO donors nor NOS inhibitors affected GCDC-induced mitochondrial permeability transition or cytochrome c release from liver mitochondria or GCDC-induced mitochondrial depolarization from isolated hepatocytes, suggesting that NO inhibits bile acid-induced hepatocyte apoptosis by a non-mitochondrial-dependent pathway. In conclusion, whereas NO produced from GCDC-treated hepatocytes neither mediates nor protects against bile acid-induced apoptosis, higher levels of NO inhibit GCDC-induced hepatocyte apoptosis by caspase-dependent pathways.     

Mitochondrial nitric oxide synthase is constitutively active and is functionally upregulated in hypoxia

Nitric oxide is a potent modulator of mitochondrial respiration, ATP synthesis, and K_ATP channel activity. Recent studies show the presence of a potentionally new isoform of the nitric oxide synthase (NOS) enzyme in mitochondria, although doubts have emerged regarding the physiological relevance of mitochondrial NOS (mtNOS). The aim of the present study were to: (i) examine the existence and distribution of mtNOS in mouse tissues using three independent methods, (ii) characterize the cross-reaction of mtNOS with antibodies against the known isoforms of NOS, and (iii) investigate the effect of hypoxia on mtNOS activity. Nitric oxide synthase activity was measured in isolated brain and liver mitochondria using the arginine to citrulline conversion assay. Mitochondrial NOS activity in the brain was significantly higher than in the liver. The calmodulin inhibitor calmidazolium completely inhibited mtNOS activity. In animals previously subjected to hypoxia, mtNOS activity was significantly higher than in the normoxic controls. Antibodies against the endothelial (eNOS), but not the neuronal or inducible isoform of NOS, showed positive immunoblotting. Immunogold labeling of eNOS located the enzyme in the matrix and the inner membrane using electron microscopy. We conclude that mtNOS is a constitutively active eNOS-like isoform and is involved in altered mitochondrial regulation during hypoxia.     

Nitric oxide and mitochondrial status in noise-induced hearing loss

The study investigated the distribution of nitric oxide (NO) within isolated outer hair cells (OHCs) from the cochlea, its relationship to mitochondria and its modulation of mitochondrial function. Using two fluorescent dyes--4,5-diamino-fluorescein diacetate (DAF-2DA), which detects NO, and tetramethyl rhodamine methyl ester (TMRM+), a mitochondrial membrane potential dye--it was found that a relatively greater amount of the DAF fluorescence in OHCs co-localized with mitochondria in comparison to DAF fluorescence in the cytosole. This study also observed reduced mitochondrial membrane potential of OHCs and increased DAF fluorescence following exposure of the cells to noise (120 dB SPL for 4 h) and to an exogenous NO donor, NOC-7 (>350 mm). Antibody label for nitrotyrosine was also increased, indicating NO-related formation of peroxynitrite in both mitochondria and the cytosol. The results suggest that NO may play an important physiological role in regulating OHC energy status and act as a potential agent in OHC pathology.     

Do mitochondria make nitric oxide? no?

Several papers have claimed that mitochondria contain nitric oxide synthase (NOS) and make nitric oxide (NO*) in amounts sufficient to affect mitochondrial respiration. However, we found that the addition of L-arginine or the NOS inhibitor L-NMMA to intact rat liver mitochondria did not have any effect on the respiratory rate in both State 3 and State 4. We did not detect mitochondrial NO* production by the oxymyoglobin oxidation assay, or electrochemically using an NO* electrode. An apparent NO* production detected by the Griess assay was identified as an artifact. NO* generated by eNOS added to the mitochondria could easily be detected, although succinate-supplemented mitochondria appeared to consume NO*. Our data show that NO* production by normal rat liver mitochondria cannot be detected in our laboratory, even though the levels of production claimed in the literature should easily have been measured by the techniques used. The implications for the putative mitochondrial NOS are discussed.     

Nitric oxide synthase activity in brain tissues from llama fetuses submitted to hypoxemia.

The fetal llama (Lama glama; a species adapted to live in chronic hypoxia in the highlands of the Andes) did not increase cerebral blood flow and reduce the brain oxygen uptake during hypoxemia. Although nitric oxide (NO) is a normal mediator in the regulation of vascular tone and synaptic transmission, NO overproduction by hypoxemia could produce neuronal damage. We hypothesized that nitric oxide synthase (NOS) activity is either maintained or reduced in the central nervous system of the llama fetuses submitted to chronic hypoxemia. Approximately 85% of the Ca(2+)-dependent NOS activity was soluble, at least 12% was associated with the mitochondrial fraction, and less than 5% remains associated with microsomes. To understand the role of NO in chronic hypoxemia, we determined the effect of 24-h hypoxemia on NOS activity in the central nervous system. No changes in activity or the subcellular distribution of NOS activity in brain tissues after hypoxemia were found. We proposed that the lack of changes in NOS activity in the llama under hypoxemia could be a cytoprotective mechanism inherent to the llama, against possible toxic effects of NO.     

Functional evidence for nitric oxide production by skeletal-muscle mitochondria from lipopolysaccharide-treated mice

The possible existence of a mitochondrially localized nitric oxide (NO) synthase (mtNOS) is controversial. To clarify this, we studied the ability of intact mitochondria to generate NO and the effect of mitochondrial NO on respiration. Respiratory rates and oxygen kinetics (P(50) values) were determined by high-resolution respirometry in skeletal-muscle mitochondria from control mice and mice injected with Escherichia coli lipopolysaccharide (LPS). In the presence of the NOS substrate L-arginine, mitochondria from LPS-treated mice had lower respiration rates and higher P(50) values than control animals. These effects were prevented by the NOS inhibitor L-NMMA. Our results suggest that mitochondrially derived NO is generated by an LPS-inducible NOS protein other than iNOS and modulates oxygen consumption in mouse skeletal muscle.     

Arabidopsis nitric oxide synthase1 is targeted to mitochondria and protects against oxidative damage and dark-induced senescence

The Arabidopsis thaliana protein nitric oxide synthase1 (NOS1) is needed for nitric oxide (NO) synthesis and signaling during defense responses, hormonal signaling, and flowering. The cellular localization of NOS1 was examined because it is predicted to be a mitochondrial protein. NOS1-green fluorescent protein fusions were localized by confocal microscopy to mitochondria in roots. Isolated mitochondria from leaves of wild-type plants supported Arg-stimulated NO synthesis that could be inhibited by NOS inhibitors and quenched by a NO scavenger; this NOS activity is absent in mitochondria isolated from nos1 mutant plants. Because mitochondria are a source of reactive oxygen species (ROS), which participate in senescence and programmed cell death, these parameters were examined in the nos1 mutant. Dark-induced senescence of detached leaves and intact plants progressed more rapidly in the mutant compared with the wild type. Hydrogen peroxide, superoxide anion, oxidized lipid, and oxidized protein levels were all higher in the mutant. These results demonstrate that NOS1 is a mitochondrial NOS that reduces ROS levels, mitigates oxidative damage, and acts as an antisenescence agent.     

New insights into the mitochondrial nitric oxide production pathways

Considerable evidence has appeared over the past few years that nitric oxide (NO) is an important anoxic metabolite and a potent signal molecule in plants. Several pathways operative in different cell compartments, lead to NO production. Mitochondria, being a major NO producing compartment, can generate it by either nitrite reduction occurring at nearly anoxic conditions or by the oxidative route via nitric oxide synthase (NOS). Recently we compared both pathways by ozone collision chemiluminescence and by DAF fluorescence. We found that nitrite reduction to NO is associated with the mitochondrial membrane fraction but not with the matrix. In case of the nitric oxide synthase pathway, an L-arginine dependent fluorescence was detected but its response to NOS inhibitors and substrates was untypical. Therefore the existence of NOS or NOS-like activity in barley root mitochondria is very doubtful. We also found that mitochondria scavenge NO. In addition, we found indirect evidence that mitochondria are able to convert NO to gaseous intermediates like NO₂, N₂O and N₂O₃.Key words: nitrate reductase, nitric oxide synthase, nitric oxide, mitochondria, DAF fluorescenceMitochondria are known as powerhouses of the cell. These organelles harbour the citric acid cycle and electron transport chain. Almost all the eukaryotic mitochondria share these basic functions. In addition to the energy generation, mitochondria are one of the major producers of reactive oxygen species¹ and involved in retrograde signalling.² Recent evidence suggests that mitochondria are one of the major producers of nitric oxide (NO) in plants.^3,4 Since nitric oxide has gained high importance, this novel property of mitochondria stimulated interest in NO signalling research.Eukaryotic mitochondria may produce NO by two distinct pathways. One is an oxidative pathway which uses L-arginine as a substrate and produces NO and citrulline⁷ and the other is a reductive pathway which uses nitrite as a substrate and produces NO at low oxygen conditions.^5,6     

Nitric oxide and mitochondrial status in noise-induced hearing loss

This study investigated the distribution of nitric oxide (NO) within isolated outer hair cells (OHCs) from the cochlea, its relationship to mitochondria and its modulation of mitochondrial function. Using two fluorescent dyes—4,5-diaminofluorescein diacetate (DAF-2DA), which detects NO, and tetramethyl rhodamine methyl ester (TMRM⁺), a mitochondrial membrane potential dye—it was found that a relatively greater amount of the DAF fluorescence in OHCs co-localized with mitochondria in comparison to DAF fluorescence in the cytosole. This study also observed reduced mitochondrial membrane potential of OHCs and increased DAF fluorescence following exposure of the cells to noise (120 dB SPL for 4 h) and to an exogenous NO donor, NOC-7 (>350 nm). Antibody label for nitrotyrosine was also increased, indicating NO-related formation of peroxynitrite in both mitochrondria and the cytosol. The results suggest that NO may play an important physiological role in regulating OHC energy status and act as a potential agent in OHC pathology.     

Mitochondrial nitric-oxide synthase stimulation causes cytochrome c release from isolated mitochondria. Evidence for intramitochondrial peroxynitrite formation.

Nitric oxide (NO) is synthesized by members of the NO synthase (NOS) family. Recently the existence of a mitochondrial NOS (mtNOS), its Ca(2+) dependence, and its relevance for mitochondrial bioenergetics was reported (Ghafourifar, P., and Richter, C. (1997) FEBS Lett. 418, 291-296; Giulivi, C., Poderoso, J. J., and Boveris, A. (1998) J. Biol. Chem. 273, 11038-11043). Here we report on the possible involvement of mtNOS in apoptosis. We show that uptake of Ca(2+) by mitochondria triggers mtNOS activity and causes the release of cytochrome c from isolated mitochondria in a Bcl-2-sensitive manner. mtNOS-induced cytochrome c release was paralleled by increased lipid peroxidation. The release of cytochrome c as well as increase in lipid peroxidation were prevented by NOS inhibitors, a superoxide dismutase mimic, and a peroxynitrite scavenger. We show that mtNOS-induced cytochrome c release is not mediated via the mitochondrial permeability transition pore because the release was aggravated by cyclosporin A and abolished by blockade of mitochondrial calcium uptake by ruthenium red. We conclude that, upon Ca(2+)-induced mtNOS activation, peroxynitrite is formed within mitochondria, which causes the release of cytochrome c from isolated mitochondria, and we propose a mechanism by which elevated Ca(2+) levels induce apoptosis.     

4,5‐diaminofluoroscein imaging of nitric oxide synthesis in crayfish terminal ganglia

We have analyzed the synthesis of nitric oxide in the terminal abdominal ganglion of the crayfish using the fluorescent probe 4,5‐Diaminofluoroscein diacetate, DAF‐2 DA. Following DAF‐2 loading, ganglia showed cell‐specific patterns of fluorescence in which the occurrence of strongly fluorescent cell bodies was highest in specific anterior, central, and posterior regions. We found that preincubation with the nitric oxide synthase (NOS) inhibitor L ‐NAME prevented much of the initial development of DAF‐2 fluorescence, whereas the inactive isomer D ‐NAME had no effect. Washout of preincubated L ‐NAME caused increased cell‐specific fluorescence due to endogenous NOS activity. Application of the NOS substrate L ‐arginine also resulted in an increase of DAF‐2 fluorescence in a cell‐specific manner. We bath applied the NO donor SNAP to increase exogenous NO levels which resulted in DAF‐2 fluorescence increases in most cells. We therefore presume that the cell‐specific pattern of DAF‐2 fluorescence indicates the distribution of neurones actively synthesizing NO. The similarity between the DAF‐2 staining pattern and previously published studies of NOS activity are discussed. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 361–369, 2002     

An internally positioned signal can direct attachment of a glycophospholipid membrane anchor

All known glycophosphatidylinositol (GPI)-anchored membrane proteins contain a COOH-terminal hydrophobic domain necessary for signalling anchor attachment. To examine the requirement that this signal be at the COOH terminus of the protein, we constructed a chimeric protein, DAFhGH, in which human growth hormone (hGH) was fused to the COOH terminus of decay accelerating factor (DAF) (a GPI-anchored protein), thereby placing the GPI signal in the middle of the chimeric protein. We show that the fusion protein appears to be processed at the normal DAF processing site in COS cells, producing GPI-anchored DAF on the cell surface. This result indicates that the GPI signal does not have to be at the COOH terminus to direct anchor addition, suggesting that the absence of a hydrophilic COOH-terminal extension (beyond the hydrophobic domain) is not a necessary requirement for GPI anchoring. A similar DAFhGH fusion, containing an internal GPI signal in which the DAF hydrophobic domain was replaced with the signal peptide of hGH, also produced GPI-anchored cell surface DAF. The signal for GPI attachment thus exhibits neither position specificity nor sequence specificity. In addition, mutant DAF or DAFhGH constructs lacking an NH2-terminal signal peptide failed to produce GPI-anchored protein, suggesting that membrane translocation is necessary for anchor addition.     

Preservation of mitochondrial functional integrity in mitochondria isolated from small cryopreserved mouse brain areas

Studies of mitochondrial bioenergetics in brain pathophysiology are often precluded by the need to isolate mitochondria immediately after tissue dissection from a large number of brain biopsies for comparative studies. Here we present a procedure of cryopreservation of small brain areas from which mitochondrial enriched fractions (crude mitochondria) with high oxidative phosphorylation efficiency can be isolated. Small mouse brain areas were frozen and stored in a solution containing glycerol as cryoprotectant. Crude mitochondria were isolated by differential centrifugation from both cryopreserved and freshly explanted brain samples and were compared with respect to their ability to generate membrane potential and produce ATP. Intactness of outer and inner mitochondrial membranes was verified by polarographic ascorbate and cytochrome c tests and spectrophotometric assay of citrate synthase activity. Preservation of structural integrity and oxidative phosphorylation efficiency was successfully obtained in crude mitochondria isolated from different areas of cryopreserved mouse brain samples. Long-term cryopreservation of small brain areas from which intact and phosphorylating mitochondria can be isolated for the study of mitochondrial bioenergetics will significantly expand the study of mitochondrial defects in neurological pathologies, allowing large comparative studies and favoring interlaboratory and interdisciplinary analyses.     

Brain mitochondrial nitric oxide synthase: in vitro and in vivo inhibition by chlorpromazine

Mouse brain mitochondria have a nitric oxide synthase (mtNOS) of 147 kDa that reacts with anti-nNOS antibodies and that shows an enzymatic activity of 0.31-0.48 nmol NO/min mg protein. Addition of chlorpromazine to brain submitochondrial membranes inhibited mtNOS activity (IC50 = 2.0 +/- 0.1 microM). Brain mitochondria isolated from chlorpromazine-treated mice (10 mg/kg, i.p.) show a marked (48%) inhibition of mtNOS activity and a markedly increased state 3 respiration (40 and 29% with malate-glutamate and succinate as substrates, respectively). Respiration of mitochondria isolated from control mice was 16% decreased by arginine and 56% increased by NNA (Nomega-nitro-L-arginine) indicating a regulatory activity of mtNOS and NO on mitochondrial respiration. Similarly, mitochondrial H2O2 production was 55% decreased by NNA. The effect of NNA on mitochondrial respiration and H2O2 production was significantly lower in chlorpromazine-added mitochondria and absent in mitochondria isolated from chlorpromazine-treated mice. Results indicate that chlorpromazine inhibits brain mtNOS activity in vitro and can exert the same action in vivo.     

Mitochondrial Dysfunction in Brain Cortex Mitochondria of STZ-Diabetic Rats: Effect of l-Arginine

Mitochondrial dysfunction has been implicated in many diseases, including diabetes. It is well known that oxygen free radical species are produced endogenously by mitochondria, and also nitric oxide (NO) by nitric oxide synthases (NOS) associated to mitochondrial membranes, in consequence these organelles constitute main targets for oxidative damage. The aim of this study was to analyze mitochondrial physiology and NO production in brain cortex mitochondria of streptozotocin (STZ) diabetic rats in an early stage of diabetes and the potential effect of l-arginine administration. The diabetic condition was characterized by a clear hyperglycaemic state with loose of body weight after 4 days of STZ injection. This hyperglycaemic state was associated with mitochondrial dysfunction that was evident by an impairment of the respiratory activity, increased production of superoxide anion and a clear mitochondrial depolarization. In addition, the alteration in mitochondrial physiology was associated with a significant decrease in both NO production and nitric oxide synthase type I (NOS I) expression associated to the mitochondrial membranes. An increased level of thiobarbituric acid-reactive substances (TBARS) in brain cortex homogenates from STZ-diabetic rats indicated the presence of lipid peroxidation. l-arginine treatment to diabetic rats did not change blood glucose levels but significantly ameliorated the oxidative stress evidenced by lower TBARS and a lower level of superoxide anion. This effect was paralleled by improvement of mitochondrial respiratory function and a partial mitochondrial repolarization.In addition, the administration of l-arginine to diabetic rats prevented the decrease in NO production and NOSI expression. These results could indicate that exogenously administered l-arginine may have beneficial effects on mitochondrial function, oxidative stress and NO production in brain cortex mitochondria of STZ-diabetic rats.