Mitochondrial Nitric Oxide Production Research Articles

Combined Inhibition of MCL1 By AZD5991 and BCL2/Bclxl By AZD4320 As Promising Strategies to Overcome Acute Leukemia Tumor Burden and Niche Chemoresistance

Background. Abnormal regulation of anti-apoptotic proteins belonging to the BCL2 family contributes to therapeutic resistance of many hematological neoplasms. Although Venetoclax, a selective inhibitor of the BCL2 protein, has proven to exert beneficial effects in acute leukemias, the activation of another anti-apoptotic protein, MCL1, a key factor to Venetoclax acquired resistance, frequently reduces its benefits. Additionally, it is known that BCL-xL plays an important pro-survival role in cancer cells by interacting with BCL-2 Hence, we hypothesize that the co-inhibition of MCL1, Bcl-2 and BCL-xL would represent an improved strategy for the treatment of patients with acute leukemia. Thus, in this study, we assessed the efficacy of specific inhibitors of MCL1, BCL2 and BCLxL antiapoptotic proteins. Aims. To investigate the in vitro, ex vivo and in vivo effects of AZD5991 (AstraZeneca), a novel MCL1 inhibitor compound, alone or in combination with AZD4320 (BCL-2/BCL-XL inhibitor compound), venetoclax, cytarabine and doxorubicin. Methods.MCL1 mRNA expression data were obtained from TCGA AML study, performed in mononuclear cells isolated from 163 AML patients (median age 57.5 years [range: 18-88]). Bone marrow (BM) aspirates (25) from healthy donors (HD), Myelodysplastic syndrome (MDS), de novo AML, and acute lymphoblastic leukemia (ALL) patients at diagnosis were submitted to analysis of MCL1 protein levels by flow cytometry and immunofluorescence (IF). A panel of myeloid (OCI-AML3, MOLM13, U937, HL60, KG1a, K562) and lymphoblastic (RS4;11, Jurkat) leukemia cell lines were treated with increasing concentrations of AZD5991 for 48h and the 50% inhibiting concentration and apoptosis (annexin-V stain) rates were determined. Mononuclear and hematopoietic stem (CD34+) cells from HD were submitted to cytotoxic assays. Leukemia cell lines were also tested for reactive oxygen species (ROS) and nitric oxide (NO) production (DAF dye), mitochondrial membrane potential (DilC1 probe) and protein expression (flow cytometry, Western blot) in a time course of 0 to 48h, alone or in combination. Moreover, we tested the AZD5991/AZD4320 action in malignant cells cultured in a 3D-system (in the presence of the mesenchymal cells) and in a monocyte monolayer (2D) in order to evaluate tumor microenvironment (TME) participation. We also developed a xenograft mouse model using U937 leukemia cell line. Statistical analyzes were performed using Kruskal Wallis, ANOVA or Mann-Whitney tests (p<.05). Results. The TCGA study (CBIO Portal) analysis showed high MCL1 gene expression in AML patients was observed (p<.0001). Immunofluorescence and flow cytometry showed that MCL1, BCL-2 and BCL-xL protein levels higher in MDS, AML and ALL BM cells compared to normal cells. All leukemia cell lines were dose-dependent sensitive to AZD5991. Growth inhibiting concentrations were OCI-AML3/0.04μM, U937/4.3μM, RS4;11/3.5μM, Jurkat/0.3μM. Apoptosis was increased in a time and concentration-dependent manner, with greater intensity in OCI-AML3 cells. AZD5991 was able to impair mitochondrial membrane potential with reduced mitochondrial ROS and NO production (p<.01) and decreased MCL1, Bax and Bak but increased Bim protein levels. Coculture in a 3D-system showed that AZD5991 treatment reduced patients’ cell growth, without modulating HD cells. In the 2D-coculture, programmed monocytes intensified apoptosis with synergistic effect between AZD5991 and AZD4320. In vitro experiments demonstrated an additive effect between AZD5991 and AZD4320, venetoclax, cytarabine or doxorubicin. In addition, xenotransplanted mice confirmed the efficacy of AZD5991 compound alone or in combination with AZD4320 due to reduction of tumor weight (fold decrease (FD): 3.8; 2.8) and tumor volume (FD: 6.5; 2.8) compared to vehicle-treated group, respectively. Conclusion. MCL1 overexpression in mononuclear cells from patients with acute leukemia and that present or not resistance to venetoclax treatment, is associated with poor prognosis and relapse disease. Our results demonstrated that co-targeting MCL-1, BCL-2 and BCL-xL, through the AZD5991 and AZD4320 compounds in the treatment of acute leukemias, may enhance therapeutic specificity, overcome chemoresistance and contribute with the cure of these aggressive malignant disorders. Funding: FAPESP grants #2017/21801-2, #2019/25247-5, #2021/05320-0.

Increased protein S-nitrosylation in mitochondria: a key mechanism of exercise-induced cardioprotection.

Endothelial nitric oxide synthase (eNOS) activation in the heart plays a key role in exercise-induced cardioprotection during ischemia-reperfusion, but the underlying mechanisms remain unknown. We hypothesized that the cardioprotective effect of exercise training could be explained by the re-localization of eNOS-dependent nitric oxide (NO)/S-nitrosylation signaling to mitochondria. By comparing exercised (5days/week for 5weeks) and sedentary Wistar rats, we found that exercise training increased eNOS level and activation by phosphorylation (at serine 1177) in mitochondria, but not in the cytosolic subfraction of cardiomyocytes. Using confocal microscopy, we confirmed that NO production in mitochondria was increased in response to H2O2 exposure in cardiomyocytes from exercised but not sedentary rats. Moreover, by S-nitrosoproteomic analysis, we identified several key S-nitrosylated proteins involved in mitochondrial function and cardioprotection. In agreement, we also observed that the increase in Ca2+ retention capacity by mitochondria isolated from the heart of exercised rats was abolished by exposure to the NOS inhibitor L-NAME or to the reducing agent ascorbate, known to denitrosylate proteins. Pre-incubation with ascorbate or L-NAME also increased mitochondrial reactive oxygen species production in cardiomyocytes from exercised but not from sedentary animals. We confirmed these results using isolated hearts perfused with L-NAME before ischemia-reperfusion. Altogether, these results strongly support the hypothesis that exercise training increases eNOS/NO/S-nitrosylation signaling in mitochondria, which might represent a key mechanism of exercise-induced cardioprotection.

Interaction of nitric oxide with the components of the plant mitochondrial electron transport chain.

Mitochondria are not only major sites for energy production but also participate in several alternative functions, among these generation of nitric oxide (NO), and its different impacts on this organelle, is receiving increasing attention. The inner mitochondrial membrane contains the chain of protein complexes, and electron transfer via oxidation of various organic acids and reducing equivalents leads to generation of a proton gradient that results in energy production. Recent evidence suggests that these complexes are sources and targets for NO. Complex I and rotenone-insensitive NAD(P)H dehydrogenases regulate hypoxic NO production, while complex I also participates in the formation of a supercomplex with complex III under hypoxia. Complex II is a target for NO which, by inhibiting Fe-S centres, regulates reactive oxygen species (ROS) generation. Complex III is one of the major sites for NO production, and the produced NO participates in the phytoglobin-NO cycle that leads to the maintenance of the redox level and limited energy production under hypoxia. Expression of the alternative oxidase (AOX) is induced by NO under various stress conditions, and evidence exists that AOX can regulate mitochondrial NO production. Complex IV is another major site for NO production, which can also be linked to ATP generation via the phytoglobin-NO cycle. Inhibition of complex IV by NO can prevent oxygen depletion at the frontier of anoxia. The NO production and action on various complexes play a major role in NO signalling and energy metabolism.

The Effect of Mitochondrial Complex I-Linked Respiration by Isoflurane Is Independent of Mitochondrial Nitric Oxide Production

Background: Anesthetic preconditioning (APC) of the myocardium is mediated in part by reversible alteration of mitochondrial function. Nitric oxide (NO) inhibits mitochondrial respiration and may mediate APC-induced cardioprotection. In this study, the effects of isoflurane on different states of mitochondrial respiration during the oxidation of complex I-linked substrates and the role of NO were investigated. Methods: Mitochondria were isolated from Sprague-Dawley rat hearts. Respiration rates were measured polarographically at 28ºC with a computer-controlled Clark-type O₂ electrode in the mitochondria (0.5 mg/mL) with complex I substrates glutamate/malate (5 mM). Isoflurane (0.25 mM) was administered before or after adenosine diphosphate (ADP)-initiated state 3 respiration. The NO synthase (NOS) inhibitor L-N⁵-(1-iminoethyl)-ornithine (L-NIO, 10 μM) and the NO donor S-nitroso-N-acetylpenicillamine (SNAP, 1 μM) were added before or after the addition of ADP. Results: Isoflurane administered in state 2 increased state 2 respiration and decreased state 3 respiration. This attenuation of state 3 respiration by isoflurane was similar when it was given during state 3. L-NIO did not alter mitochondrial respiration or the effect of isoflurane. SNAP only, added in state 3, decreased state 3 respiration and enhanced the isoflurane-induced attenuation of state 3 respiration. Conclusion: Isoflurane has clearly distinguishable effects on different states of mitochondrial respiration during the oxidation of complex I substrates. The uncoupling effect during state 2 respiration and the attenuation of state 3 respiration may contribute to the mechanism of APC-induced cardioprotection. These effects of isoflurane do not depend on endogenous mitochondrial NO, as the NOS inhibitor L-NIO did not alter the effects of isoflurane on mitochondrial respiration.

Mitochondrial nitric oxide production supported by reverse electron transfer

Heart phosphorylating electron transfer particles (ETPH) produced NO at 1.2 ± 0.1 nmol NO. min−1 mg protein−1 by the mtNOS catalyzed reaction. These particles showed a NAD+ reductase activity of 64 ± 3 nmol min−1 mg protein−1 sustained by reverse electron transfer (RET) at expenses of ATP and succinate. The same particles, without NADPH and in conditions of RET produced 0.97 ± 0.07 nmol NO. min−1 mg protein−1. Rotenone inhibited NO production supported by RET measured in ETPH and in coupled mitochondria, but did not reduce the activity of recombinant nNOS, indicating that the inhibitory effect of rotenone on NO production is due to an electron flow inhibition and not to a direct action on mtNOS structure. NO production sustained by RET corresponds to 20% of the total amount of NO released from heart coupled mitochondria. A mitochondrial fraction enriched in complex I produced 1.7 ± 0.2 nmol NO. min−1 mg protein−1 and reacted with anti-75 kDa complex I subunit and anti-nNOS antibodies, suggesting that complex I and mtNOS are located contiguously. These data show that mitochondrial NO production can be supported by RET, and suggest that mtNOS is next to complex I, reaffirming the idea of a functional association between these proteins.

Ampelopsin sodium induces mitochondrial-mediated apoptosis in human lung adenocarcinoma SPC-A-1 cell line.

Ampelopsin is a well-known flavonoid which has variety of biological and pharmacological actions including anticancer effects and induction of apoptosis on the several cancer cell lines. The present study aimed to evaluate the role of ampelopsin sodium (Amp-Na) in the mitochondrial-mediated apoptosis of human lung adenocarcionma SPC-A-1 cells. The analysis of cell proliferation and ultrastructure were performed. Furthermore, to clarify its action mechanism by determining the mitochondrial membrane potential (Δψm), intracellular calcium (Ca2+) concentration, mitochondrial nitric oxide (NO) level and total ATPase activity. The results showed that Amp-Na markedly inhibited the SPC-A-1 cell proliferation and caused ultrastructural apoptosis feature in SPC-A-1 cells in a dose-dependent manner. Amp-Na led to a rapid and sustained Ca2+ elevation and Δψm reduction, and induced the mitochondrial NO production and decreased the total ATPase activity in SPC-A-1 cells. The results enhance the potential of Amp-Na as a therapeutic drug for treating lung cancer, and provide new information for mechanism of Amp-Na which induces mitochondrial-mediated apoptosis in tumor cells.

Abstract 4328: Resveratrol inhibits NO-mediated oxidative stress in murine prostate cancer cells

Abstract Background: Nitric oxide (NO) is an important signaling molecule in the immune, nervous and cardiovascular systems. NO involves in the regulation of cellular behavior as well as cytotoxic events. Studies demonstrated that resveratrol (RES) (a polyphenolic compound in the skin of red fruits) at high concentration (≥50μM) induces NO production in endothelial F-2 cells and in human umbilical vein endothelial cells. However, the impact of RES on the biological functions of NO are still poorly understood. Therefore, the goal of this study was to investigate the impact of RES on NO-mediated oxidative stress at mitochondria level in mouse prostate cancer (PCa) cells. Experimental Procedures: The transgenic adenocarcinoma of the mouse prostate (TRAMP) cells (mirrors the pathogenesis of human prostate cancer) were cultured in Dulbecco's Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine serum (FBS), penicillin 100 IU/ml, streptomycin 100 μg/ml, 0.005mg/ml bovine insulin, and 10nM Dehydroisoandrosterone in 5% CO2 at 370C. Cells were harvested by trypsinization after 80% confluency (at 4-5 pass), counted (105/ml), and incubated with RES (50uM), RES (50uM) + Deta-NONOate (750uM), Deta-NONOate (750uM), RES (50uM) +L-NMMA (750uM), and L-NMMA (750uM). Deta-NONOate was used as nitric oxide donor while L-NMMA was used as nitric oxide inhibitor. Thereafter, cell viability, NO-estimation in culture supernatant, mitochondrial membrane potential (MMP), total ROS-production in mitochondria and cytoplasm, and apoptotic assay (Annexin-V and caspase-3/7) were carried out. Results and Conclusion: PCa cells incubated with RES showed decreased cell viability, decreased NO production in culture supernatant, decreased MMP, and decreased level of ROS in mitochondria. However, PS-externalization, and total ROS in cytoplasm were found high after normalization with control cells. Further, treatment with Deta-NONOate results in decreased cell viability, decreased total ROS in the cytoplasm, and decreased MMP, while mitochondrial ROS and NO production in culture supernatant were observed highly significant as compared control cells. Furthermore, cells were also exposed to the combination of RES and NO which suggest that RES reduced NO-mediated cell killing and reactive oxygen species generation in TRAMP cells. However, no significant changes were observed after L-NMMA exposure to TRAMP cells. The result demonstrates that RES protect cells from NO-mediated cell killing and induced NO-independent apoptosis in PCa cells. This suggests that role of RES as a potential therapeutic agent to control cancer. However, further investigation is needed to explore potential mechanism of action of RES in controlling cancer proliferation. Citation Format: Sanjay Kumar, James Stokes, Karyn Scissum Gunn, Selvarangan Ponnazhagan, Upender Manne, Manoj K. Mishra. Resveratrol inhibits NO-mediated oxidative stress in murine prostate cancer cells. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4328.

Nitric oxide metabolism in heart mitochondria

Normal cardiac function is accomplished through a continuous energy supply provided by mitochondria. Heart mitochondria are the major source of reactive oxygen and nitrogen species: superoxide anion (O2-) and nitric oxide (NO). NO production by mitochondrial NOS (mtNOS) is modified by metabolic state and shows an exponential dependence on Δψ. The interaction between mtNOS and complexes I and IV might be a mechanism involved in the regulation of mitochondrial NO production. NO exerts a high affinity, reversible and physiological inhibition of cytochrome c oxidase activity. A second effect of NO on the respiratory chain is accomplished through its interaction with ubiquinol-cytochrome c oxidoreductase. The ability of mtNOS to regulate mitochondrial O2 uptake and O2- and H2O2 productions through the interaction of NO with the respiratory chain is named mtNOS functional activity. Together, heart mtNOS allows NO to optimize the balance between cardiac energy production and utilization, and to regulate the steady-state concentrations of other oxygen and nitrogen species.

Plant mitochondria: Source and target for nitric oxide

Plant mitochondria generate nitric oxide (NO) under anoxia through the action of cytochrome c oxidase and other electron transport chain components on nitrite. This reductive mechanism operates under aerobic conditions at high electron transport rates. Indirect evidence also indicates that the oxidative pathway of NO production may be associated with mitochondria. We review the consequences of mitochondrial NO production, including the inhibition of oxygen uptake by cytochrome c oxidase, the inhibition of aconitase and succinate dehydrogenase, the induction of alternative oxidase, and the nitrosylation of several proteins, including glycine decarboxylase. The importance of these events in adaptation to abiotic and biotic stresses is discussed.

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 4days 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 (NOSI) 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.

Strategic localization of heart mitochondrial NOS: a review of the evidence

Heart mitochondria play a central role in cell energy provision and in signaling. Nitric oxide (NO) is a free radical with primary regulatory functions in the heart and involved in a broad array of key processes in cardiac metabolism. Specific NO synthase (NOS) isoforms are confined to distinct locations in cardiomyocytes. The present article reviews the chemical reactions through which NO interacts with biomolecules and exerts some of its crucial roles. Specifically, the article discusses the reactions of NO with mitochondrial targets and the subcellular localization of NOS within the myocardium and analyzes the available data about heart mitochondrial NOS activity and identity. The article also describes the regulation of heart mtNOS by the distinctive mitochondrial environment by showing the effects of Ca(2+), O(2), l-arginine, mitochondrial transmembrane potential, and the metabolic states on heart mitochondrial NO production. The article depicts the effects of NO on heart function and highlights the relevance of NO production within mitochondria. Finally, the evidence on the functional implications of heart mitochondrial NOS is delineated with emphasis on chronic hypoxia and ischemia-reperfusion studies.

Identification and characterization of a functional mitochondrial angiotensin system

The renin-angiotensin (Ang) system regulates multiple physiological functions through Ang II type 1 and type 2 receptors. Prior studies suggest an intracellular pool of Ang II that may be released in an autocrine manner upon stretch to activate surface membrane Ang receptors. Alternatively, an intracellular renin-Ang system has been proposed, with a primary focus on nuclear Ang receptors. A mitochondrial Ang system has not been previously described. Here we report that functional Ang II type 2 receptors are present on mitochondrial inner membranes and are colocalized with endogenous Ang. We demonstrate that activation of the mitochondrial Ang system is coupled to mitochondrial nitric oxide production and can modulate respiration. In addition, we present evidence of age-related changes in mitochondrial Ang receptor expression, i.e., increased mitochondrial Ang II type 1 receptor and decreased type 2 receptor density that is reversed by chronic treatment with the Ang II type 1 receptor blocker losartan. The presence of a functional Ang system in human mitochondria provides a foundation for understanding the interaction between mitochondria and chronic disease states and reveals potential therapeutic targets for optimizing mitochondrial function and decreasing chronic disease burden with aging.

The emerging roles of nitric oxide (NO) in plant mitochondria

In recent years nitric oxide (NO) has been recognized as an important signal molecule in plants. Both, reductive and oxidative pathways and different subcellular compartments appear involved in NO production. The reductive pathway uses nitrite as substrate, which is exclusively generated by cytosolic nitrate reductase (NR) and can be converted to NO by the same enzyme. The mitochondrial electron transport chain is another site for nitrite to NO reduction, operating specifically when the normal electron acceptor, O 2, is low or absent. Under these conditions, the mitochondrial NO production contributes to hypoxic survival by maintaining a minimal ATP formation. In contrast, excessive NO production and concomitant nitrosative stress may be prevented by the operation of NO-scavenging mechanisms in mitochondria and cytosol. During pathogen attacks, mitochondrial NO serves as a nitrosylating agent promoting cell death; whereas in symbiotic interactions as in root nodules, the turnover of mitochondrial NO helps in improving the energy status similarly as under hypoxia/anoxia. The contribution of NO turnover during pathogen defense, symbiosis and hypoxic stress is discussed in detail.

Reciprocal regulation of cellular nitric oxide formation by nitric oxide synthase and nitrite reductases

SummaryOur mini-review focuses on dual regulation of cellular nitric oxide (NO) signaling pathways by traditionally characterized enzymatic formation from L-arginine via the actions of NO synthases (NOS) and by enzymatic reduction of available cellular nitrite pools by a diverse class of cytosolic and mitochondrial nitrite reductases. Nitrite is a major metabolic product of NO and is found in all cell and tissue types that utilize NO signaling processes. Xanthine oxidoreductase (XOR) has been previously characterized as a housekeeping enzyme responsible for cellular uric acid formation via enzymatic conversion of hypoxanthine and xanthine. It has become apparent that XOR possesses multi-functional enzymatic activities outside the realm of xanthine metabolism and a small but significant literature also established a compelling functional association between administered sodium nitrite, XOR activation, and pharmacologically characterized NO transductive effects in positive cardiovascular function enhanced pulmonary perfusion, and protection against ischemia/reperfusion injury and hypoxic damage and oxidative stress. Similar positive vascular and cellular effects were observed to be functionally associated with mitochondrial aldehyde dehydrogenase and cytochrome c/cytochrome c oxidase. The profound implications of a reciprocal regulatory mechanism responsible for cytosolic and mitochondrial NO production are discussed below.

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 and N2O.

Reactive oxygen species and nitric oxide in plant mitochondria: origin and redundant regulatory systems

Plant mitochondria differ from their mammalian counterparts in many respects, which are due to the unique and variable surroundings of plant mitochondria. In green leaves, plant mitochondria are surrounded by ample respiratory substrates and abundant molecular oxygen, both resulting from active photosynthesis, while in roots and bulky rhizomes and fruit carbohydrates may be plenty, whereas oxygen levels are falling. Several enzymatic complexes in mitochondrial electron transport chain (ETC) are capable of reactive oxygen species (ROS) formation under physiological and pathological conditions. Inherently connected parameters such as the redox state of electron carriers in the ETC, ATP synthase activity and inner mitochondrial membrane potential, when affected by external stimuli, can give rise to ROS formation via complexes I and III, and by reverse electron transport (RET) from complex II. Superoxide radicals produced are quickly scavenged by superoxide dismutase (MnSOD), and the resulting H(2)O(2) is detoxified by peroxiredoxin-thioredoxin system or by the enzymes of ascorbate-glutathione cycle, found in the mitochondrial matrix. Arginine-dependent nitric oxide (NO)-releasing activity of enzymatic origin has been detected in plant mitochondria. The molecular identity of the enzyme is not clear but the involvement of mitochondria-localized enzymes responsible for arginine catabolism, arginase and ornithine aminotransferase has been shown in the regulation of NO efflux. Besides direct control by antioxidants, mitochondrial ROS production is tightly controlled by multiple redundant systems affecting inner membrane potential: NAD(P)H-dependent dehydrogenases, alternative oxidase (AOX), uncoupling proteins, ATP-sensitive K(+) channel and a number of matrix and intermembrane enzymes capable of direct electron donation to ETC. NO removal, on the other hand, takes place either by reactions with molecular oxygen or superoxide resulting in peroxynitrite, nitrite or nitrate ions or through interaction with non-symbiotic hemoglobins or glutathione. Mitochondrial ROS and NO production is tightly controlled by multiple redundant systems providing the regulatory mechanism for redox homeostasis and specific ROS/NO signaling.

Neuronal nitric oxide synthase in heart mitochondria: a matter of life or death

Neuronal nitric oxide synthase in heart mitochondria: a matter of life or death

Large-conductance K + channel opener CGS7184 as a regulator of endothelial cell function

Large-conductance Ca 2+-activated potassium (BK Ca) channels are present in endothelium, but their regulatory role remains uncharacterized. The aim of the present study was to investigate the pharmacological effects of the BK Ca channel opener ethyl-1-[[(4-chlorophenyl)amino]oxo]-2-hydroxy-6-trifluoromethyl-1H-indole-3-carboxylate (CGS7184) on endothelium in the aorta and coronary circulation, particularly with regard to nitric oxide (NO)-dependent regulation of vascular tone, as well as effects of CGS7184 on NO production, calcium homeostasis, and mitochondrial function in cultured endothelial cells. The vasorelaxant action of CGS7184 was studied in coronary circulation and in the aorta using isolated perfused guinea pig heart and rat aortic rings, respectively. The effects of CGS7184 on calcium homeostasis, mitochondrial membrane potential, NO production, and mitochondrial respiration were tested in cultures of EA.hy 926 endothelial cells. The BK Ca channel opener CGS7184 caused a concentration-dependent (0.03–3 µM) relaxation of the rat aorta and coronary vasodilatation in the isolated guinea pig heart. Both responses were profoundly inhibited by the nitric oxide (NO) synthase (NOS) inhibitor N G-nitro- l-arginine methyl ester ( l-NAME) (100 µM). CGS7184 (5 µM) also increased basal NO production in EA.hy 926 cells by approximately two-fold. Moreover, CGS7184 induced a concentration-dependent (0.1–10 µM) elevation in intracellular calcium concentration. Interestingly, CGS7184 affected mitochondrial function by causing mitochondrial potential depolarization and an increase in oxygen consumption in EA.hy 926 endothelial cells. The BK Ca channel opener CGS7184 activates NOS pathways and modulates mitochondrial function in the endothelium. Both effects may be triggered by the CGS7184-induced modulation of intracellular Ca 2+ homeostasis in EA.hy 926 endothelial cells.

The role of mitochondria in oxidative and nitrosative stress during ischemia/reperfusion in the rat kidney

Reoxygenation following ischemia causes tissue oxidative stress. We studied the role of oxidative stress caused by kidney ischemia/reperfusion (I/R) on the mitochondria of renal tissue slices. I/R caused the mitochondria to be swollen, fragmented, and have lower membrane potential. The mitochondria generated more reactive oxygen species (ROS) and nitric oxide (NO) in situ as measured by fluorescence of ROS- and NO-sensitive probes. Infusion of lithium ion, an inhibitor of glycogen kinase synthase-3, caused phosphorylation of its Ser-9 and restored the membrane potential and decreased ROS production of the mitochondrial fraction. Ischemic kidney and hypoxic rat preconditioning improved mitochondrial membrane potential and lowered ROS production caused by subsequent I/R similar to lithium ion infusion. Preconditioning normalized NO production in mitochondria as well. The drop in the mitochondrial membrane potential was prevented by NO synthase inhibition, demonstrating a strong contribution of NO to changes in mitochondrial energy metabolism during the I/R transition. Mitochondria in the I/R-stressed kidney contained less cytochrome c and more pro-apoptotic Bax, consistent with apoptotic degradation.

Nitric oxide differentially regulates T-cell function in rheumatoid arthritis and systemic lupus erythematosus

Experimental and clinical evidence for T-cell involvement in the pathogenesis of rheumatoid arthritis (RA) is compelling, and points to a local dysregulation of T-cell function in the inflamed joint. Nitric oxide (NO) has been shown to regulate T-cell function under physiological conditions, but overproduction of NO may contribute to lymphocyte dysfunction in RA. NO has recently been recognized as a key signaling intermediate for T-cell activation and mitochondrial biogenesis. NO is synthesized from L-arginine by NO synthetases (NOS). Three distinct isoforms of NOS are known, including neuronal NOS (nNOS), inducible NOS (iNOS), and endothelial NOS (eNOS) enzymes. We previously detected the expression of eNOS and nNOS and the absence of iNOS in human PBL, while during inflammation macrophages and monocytes express iNOS. Systemic lupus erythematosus (SLE) is a systemic autoimmune disease of unknown origin characterized by the involvement of multiple organs. Several studies carried out on patients with both RA and SLE have documented increased endogenous NO synthesis, but its contribution to T-lymphocyte mitochondrial biogenesis and T-cell dysregulation is not known. We investigated the role of NO in T-cell mitochondrial biogenesis in RA and SLE. The mitochondrial mass, NO production and cytoplasmic Ca2+ levels were measured by flow cytometry. Mitochondria were visualized using transmission electron microscope. T cells from RA patients produce >2.5 times more NO than T cells from healthy donors (P < 0.001). Unexpectedly, the mitochondrial mass was found to be similar in RA and control T cells (P = 0.65), whilst increased NO production was associated with increased cytoplasmic Ca2+ concentrations in RA T cells (P < 0.001). We observed that T-cell NO production decreased in most RA patients following anti-TNF treatment. Although lupus T cells produced comparable amounts of NO to normal T cells, lupus monocytes produced twice as much NO as normal monocytes (P = 0.015). We also observed increased mitochondrial mass (47.7 ± 2.8%; P = 0.00017) and increased cytoplasmic (38 ± 6.4%; P = 0.0023) Ca2+ content in T cells from SLE patients when compared with control donors. Electron microscopy revealed that T cells of lupus patients contained 8.76 ± 1 mitochondria, while control donors contained 3.18 ± 0.28 mitochondria per cell (P = 0.0009). In addition, lupus lymphocytes harbor several-fold enlarged mega-mitochondria. These data suggest that monocytes are the primary source of NO in SLE, while T lymphocytes are the primary source of NO in RA. Although the iNOS pathway is not as rapid as eNOS or nNOS, it is thought to be capable of generating much larger quantities of NO (nanomolar range) than the constitutive NOS isoforms (picomolar range), explaining the differences in T-cell mitochondrial biogenesis in SLE and RA. Since mitochondria can take up, store and release Ca2+, increased mitochondrial mass may account for altered Ca2+ handling in SLE. Furthermore increased NO production may contribute to T-cell dysfunction in both SLE and RA.