Mitochondrial Nitric Oxide Synthase Research Articles

Nicotine Exposure Causes Nitrosative and Oxidative Stress in Glomerular Podocytes, Resulting in Renal Damage

Introduction: Nicotine consumption is the leading cause of preventable disease and death. Patients with hypertension or kidney disease are more likely to exhibit renal pathologies associated with nicotine, such as glomerulopathy. In endothelial cells, nicotine was linked to mitochondrial oxidative stress and nitric oxide synthase (NOS) remodeling. However, there is a gap in knowledge regarding the effects of nicotine in glomerular epithelial cells, podocytes. We hypothesized that nicotine promotes nitrosative stress in the podocytes by producing peroxynitrite (ONOO-) and mitochondrial damage, thereby reducing glomerular function. Methods: We performed confocal imaging on human cultured podocytes to detect ONOO− (HPF), Ca2+ (Fluo-8) and nitric oxide NO (DAF-FM) in response to nicotine. Seahorse assay (Agilent XFe24) was used to test mitochondrial respiration. Then, nicotine was chronically infused in Dahl SS rats (0.2 mg/kg/day s.c., 0.4% NaCl, 21 days), and blood pressure was recorded with telemetry. Glomerular damage was evaluated, and electron microscopy was employed to assess mitochondrial ultrastructure. Electron paramagnetic resonance (EPR) spectroscopy was used to access the NO levels in cultured podocytes and in vivo. OriginPro was used for statistical analysis. Results: Acute application of nicotine promoted intracellular Ca2+ and ONOO− transients in podocytes. The ONOO− response was blocked in the presence of SOD, indicating that ONOO− production requires superoxide. The application of specific α7 or α4β2, α2β4, α4β4, and α3β4 nicotinic acetylcholine receptor (nAChR) agonists elicited Ca2+ transients but did not produce ONOO−, suggesting that nitrosative stress occurs independently from Ca2+ influx or nAChR activation. Incubation with nicotine (12 hrs) resulted in a decrease in podocytes’ mitochondrial respiration (two-sample t-test, p<0.05). In vivo, nicotine infusion did not affect blood pressure but promoted mitochondrial damage in podocytes, which exhibited swelling, loss of cristae, and mitophagy (t-test, p<0.05). Histopathological assessment showed higher glomerular damage in nicotine-exposed rats (t-test, p<0.05). Both EPR and confocal approaches demonstrated nicotine-mediated changes in NO bioavailability and an increase in NOS2 activity (t-test, p<0.05) Conclusion: Nicotine elicits superoxide-mediated nitrosative stress and peroxynitrite formation in podocytes, promoting mitochondrial damage and glomerular dysfunction. Revealing the mechanisms of nicotine-mediated damage in the podocytes impacts our progress towards new treatments for smoking and vaping-associated renal pathologies. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Bisphenol A aggravate selenium deficiency-induced apoptosis via miR-215-3p/Dio1 to activate ROS/PI3K/AKT pathway in chicken arterial.

Both bisphenol A (BPA) and selenium (Se) deficiency can affect the expression of microRNAs (miRNAs), which can specifically regulate its target mRNA and induce apoptosis, and play a significant role in cardiovascular injury diseases. To explore the mechanism of apoptosis induced by BPA and Se deficiency in chicken arterial endothelial tissue and the role of miRNAs in this process, the model of BPA exposure/Se deficiency in chicken and PAEC cells have been employed. The targeting relationship between miR-215-3p and iodothyronine deiodinase 1 (Dio1) in PAEC was verified by double luciferase gene report. The level of miR-215-3p was detected by qRT-PCR. The oxidative stress level of arterial endothelial cells was detected by oxidative stress kit and DCFH-DA probe method. The PI3K/AKT pathway, mitochondrial dynamics, and apoptosis-related genes were detected by qRT-PCR and western blot. The mitochondrial ATP level and nitric oxide synthases (NOSs) level were detected with the kit. TUNEL, acridine orange/ethidium bromide,and flow cytometry were used to detect the level of apoptosis. The results showed that BPA exposure and Se deficiency led to overexpression of miR-215-3p, aggravated oxidative stress, inhibited activation of PI3K/AKT pathway, promoted mitochondrial division, increased expression of apoptosis related genes, and finally led to apoptosis of chicken arterial endothelial cells. We also established knockdown/overexpression models of miR-215-3p and Dio1 in vitro, and found that overexpression of miR-215-3p and knockout of Dio1 can induce apoptosis. Interestingly, miR-215-3p-Inhibitor and N-acetyl- l-cysteine (NAC) partially prevented apoptosis caused by BPA exposure and Se deficiency, and LY294002 aggravated apoptosis. These results suggest that BPA exposure aggravates the apoptosis of Se deficient arterial endothelial cells in chickens by regulating the ROS/PI3K/AKT pathway activated by miR-215-3p/Dio1. The miR-215-3p/Dio1 axis provides a new way to understand the toxic mechanism of BPA exposure and Se deficiency, and reveals a new regulatory model of apoptosis damage in vascular diseases.

Molecular mechanisms of anthracycline cardiovascular toxicity.

Anthracyclines are effective chemotherapeutic agents, commonly used in the treatment of a variety of hematologic malignancies and solid tumors. However, their use is associated with a significant risk of cardiovascular toxicities and may result in cardiomyopathy and heart failure. Cardiomyocyte toxicity occurs via multiple molecular mechanisms, including topoisomerase II-mediated DNA double-strand breaks and reactive oxygen species (ROS) formation via effects on the mitochondrial electron transport chain, NADPH oxidases (NOXs), and nitric oxide synthases (NOSs). Excess ROS may cause mitochondrial dysfunction, endoplasmic reticulum stress, calcium release, and DNA damage, which may result in cardiomyocyte dysfunction or cell death. These pathophysiologic mechanisms cause tissue-level manifestations, including characteristic histopathologic changes (myocyte vacuolization, myofibrillar loss, and cell death), atrophy and fibrosis, and organ-level manifestations including cardiac contractile dysfunction and vascular dysfunction. In addition, these mechanisms are relevant to current and emerging strategies to diagnose, prevent, and treat anthracycline-induced cardiomyopathy. This review details the established and emerging data regarding the molecular mechanisms of anthracycline-induced cardiovascular toxicity.

Modulations of Cardiac Functions and Pathogenesis by Reactive Oxygen Species and Natural Antioxidants

Homeostasis in the level of reactive oxygen species (ROS) in cardiac myocytes plays a critical role in regulating their physiological functions. Disturbance of balance between generation and removal of ROS is a major cause of cardiac myocyte remodeling, dysfunction, and failure. Cardiac myocytes possess several ROS-producing pathways, such as mitochondrial electron transport chain, NADPH oxidases, and nitric oxide synthases, and have endogenous antioxidation mechanisms. Cardiac Ca2+-signaling toolkit proteins, as well as mitochondrial functions, are largely modulated by ROS under physiological and pathological conditions, thereby producing alterations in contraction, membrane conductivity, cell metabolism and cell growth and death. Mechanical stresses under hypertension, post-myocardial infarction, heart failure, and valve diseases are the main causes for stress-induced cardiac remodeling and functional failure, which are associated with ROS-induced pathogenesis. Experimental evidence demonstrates that many cardioprotective natural antioxidants, enriched in foods or herbs, exert beneficial effects on cardiac functions (Ca2+ signal, contractility and rhythm), myocytes remodeling, inflammation and death in pathological hearts. The review may provide knowledge and insight into the modulation of cardiac pathogenesis by ROS and natural antioxidants.

Acute hypobaric hypoxia and cardiac energetic response in prepubertal rats: Role of nitric oxide.

What is the central question of this study? In adult rat hearts, exposure to hypobaric hypoxia increases tolerance to hypoxia-reoxygenation, termed endogenous cardioprotection. The mechanism involves the nitric oxide system and modulation of mitochondrial oxygen consumption. What is the cardiac energetic response in prepubertal rats exposed to hypobaric hypoxia? What is the main finding and its importance? Prepubertal rats, unlike adult rats, did not increase tolerance to hypoxia-reoxygenation in response acute exposure to hypobaric hypoxia, which impaired cardiac contractile economy. This finding could be related to a failure to increase nitric oxide synthase expression, hence modulation of mitochondrial oxygen consumption and ATP production. Studies in our laboratory showed that exposure of rats to hypobaric hypoxia (HH) increased the tolerance of the heart to hypoxia-reoxygenation (H/R), involving mitochondrial and cytosolic nitric oxide synthase (NOS) systems. The objective of the present study was to evaluate how the degree of somatic maturation could alter this healthy response. Prepubertal male rats were exposed for 48h to a simulated altitude of 4400m in a hypobaric chamber. The mechanical energetic activity in perfused hearts and the contractile functional capacity of NOS in isolated left ventricular papillary muscles were evaluated during H/R. Cytosolic nitric oxide (NO), production of nitrites/nitrates (Nx), expression of NOS isoforms, mitochondrial O2 consumption and ATP production were also evaluated. The left ventricular pressure during H/R was not improved by HH. However, the energetic activity was increased. Thus, the contractile economy (left ventricular pressure/energetic activity) decreased in HH. Nitric oxide did not modify papillary muscle contractility after H/R. Cytosolic p-eNOS-Ser1177 and inducible NOS expression were decreased by HH, but no changes were observed in NO production. Interestingly, HH increased Nx levels, but O2 consumption and ATP production in mitochondria were not affectedby HH. Prepubertal rats exposed to HH preserved cardiac contractile function, but with a high energetic cost, modifying contractile economy. Although thiscould be related to the decreased NOS expression detected, cytosolic NO production was preserved, maybe through the Nx metabolic pathway, without modification of mitochondrial ATP production and O2 consumption. In this scenario, the treatment was unable to increase tolerance to H/R as observed in adult animals.

Increasing Nrf2 Activity as a Treatment Approach in Neuropsychiatry.

Nuclear factor erythroid 2-related factor 2 (Nrf2) is a transcription factor encoded by NFE2L2. Under oxidative stress, Nrf2 does not undergo its normal cytoplasmic degradation but instead travels to the nucleus, where it binds to a DNA promoter and initiates transcription of anti-oxidative genes. Nrf2 upregulation is associated with increased cellular levels of glutathione disulfide, glutathione peroxidase, glutathione transferases, thioredoxin and thioredoxin reductase. Given its key role in governing the cellular antioxidant response, upregulation of Nrf2 has been suggested as a common therapeutic target in neuropsychiatric illnesses such as major depressive disorder, bipolar disorder and schizophrenia, which are associated with chronic oxidative and nitrosative stress, characterised by elevated levels of reactive oxygen species, nitric oxide and peroxynitrite. These processes lead to extensive lipid peroxidation, protein oxidation and carbonylation, and oxidative damage to nuclear and mitochondrial DNA. Intake of N-acetylcysteine, coenzyme Q10 and melatonin is accompanied by increased Nrf2 activity. N-acetylcysteine intake is associated with improved cerebral mitochondrial function, decreased central oxidative and nitrosative stress, reduced neuroinflammation, alleviation of endoplasmic reticular stress and suppression of the unfolded protein response. Coenzyme Q10, which acts as a superoxide scavenger in neuroglial mitochondria, instigates mitohormesis, ameliorates lipid peroxidation in the inner mitochondrial membrane, activates uncoupling proteins, promotes mitochondrial biogenesis and has positive effects on the plasma membrane redox system. Melatonin, which scavenges mitochondrial free radicals, inhibits mitochondrial nitric oxide synthase, restores mitochondrial calcium homeostasis, deacetylates and activates mitochondrial SIRT3, ameliorates increased permeability of the blood-brain barrier and intestine and counters neuroinflammation and glutamate excitotoxicity.

The Effect of Chalcones on the Main Sources of Reactive Species Production: Possible Therapeutic Implications in Diabetes Mellitus.

Diabetes mellitus (DM) is characterized by hyperglycaemia, resulting from defects in insulin secretion, insulin action or both. There are several factors such as hyperlipidemia and oxidative stress (OS), namely the production of reactive oxygen/nitrogen species (ROS/RNS), that actively contribute to the development and worsening of DM. Chalcones, also termed as benzalacetophenone or benzylidene acetophenone, present a 1,3-diaryl-2-propen-1-one scaffold that has been shown to be highly promising in the development of new antioxidant compounds. Considering the potential interest of antioxidant therapy, the present review scrutinizes the role of the main sources of ROS/RNS production during DM. The modulatory effect of chalcones against nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, xanthine oxidase, mitochondrial respiratory chain and nitric oxide synthase, is also thoroughly discussed, establishing, whenever possible, a structure-activity relationship (SAR). From the SAR analysis, it can be stated that the presence of catechol groups, hydroxyl and methoxyl substituents in the chalcones scaffold improves their modulatory activity against the main sources of ROS/RNS production in DM.

The mitochondrial NO-synthase/guanylate cyclase/protein kinase G signaling system underpins the dual effects of nitric oxide on mitochondrial respiration and opening of the permeability transition pore.

The available data on the involvement of nitric oxide (NO) and mitochondrial calcium-dependent NO synthase (mtNOS) in the control of mitochondrial respiration and the permeability transition pore (mPTP) are contradictory. We have proposed that the mitochondrial mtNOS/guanylate cyclase/protein kinase G signaling system (mtNOS-SS) is also implicated in the control of respiration and mPTP, providing the interplay between NO and mtNOS-SS, which, in turn, may result in inconsistent effects of NO. Therefore, using rat liver mitochondria, we applied specific inhibitors of the enzymes of this signaling system to evaluate its role in the control of respiration and mPTP opening. Steady-state respiration was supported by pyruvate, glutamate, or succinate in the presence of hexokinase, glucose, and ADP. When applied at low concentrations, l-arginine (to 500µm) and NO donors (to 50µm) activated the respiration and increased the threshold concentrations of calcium and d,l-palmitoylcarnitine required for the dissipation of the mitochondrial membrane potential and pore opening. Both effects were eliminated by the inhibitors of NO synthase, guanylate cyclase, and kinase G, which denotes the involvement of mtNOS-SS in the activation of respiration and deceleration of mPTP opening. At high concentrations, l-arginine and NO donors inhibited the respiration and promoted pore opening, indicating that adverse effects induced by an NO excess dominate over the protection provided by mtNOS-SS. Thus, these results demonstrate the opposite impact of NO and mtNOS-SS on the respiration and mPTP control, which can explain the dual effects of NO.

Arginase exhibits negative regulation of respiration in isolated murine cardiac mitochondria independent of mitochondrial nitric oxide synthase

ObjectiveMitochondrial dysfunction underlies the development of various cardiovascular diseases. Previously, we reported the presence of mitochondrial nitric oxide synthase (mtNOS) in cardiomyocytes and for the first time observed that NOS inhibitors decreased the state IIIu respiration in isolated cardiac mitochondria but had no significant effect on basal/state II or ATP production. These effects of NOS inhibitors on mitochondrial respiration in isolated mitochondria were significantly different than the effects on mitochondrial respiration in intact cells suggesting novel functional role of mtNOS. Arginase regulates the levels of arginine available for the local nitric oxide synthases, thereby regulating the NO production. Cardiomyocytes specifically express arginase II localized in mitochondria and has been shown to regulate cardiomyocyte contractility. We hypothesized that inhibition of arginase II increases respiration in isolated murine cardiac mitochondria in vitro.MethodsCardiac mitochondria were isolated from three‐month‐old C57bl6 (n= 4–5) mice and treated with specific arginase inhibitor, S‐(2‐boronoethyl)‐L‐cysteine hydrochloride, S‐(2‐boronoethyl)‐L‐cysteine, BEC (100μM) for 30 minutes in assay buffer containing 10 mM pyruvate and 2 mM malate. Mitochondria (5 μg/well) were plated into cell plates and oxygen consumption rate (OCR) was measured using Agilent Seahorse XFe24 analyzer after serial injections of ADP (5 mM), oligomycin (5 μM), FCCP (5 μM) and antimycin and rotenone (10 μM and 2 μM). Respiratory parameters were reported as OCR (pico moles of oxygen/minute).ResultsArginase inhibition did not significantly alter the basal/state II respiration (106.2 ± 6.2 vs 98.8 ± 1.6) or state IVo (87.1 ± 8.0 vs 80.9 ± 7.7) or proton leak (53.2 ± 11.6 vs 41.7 ± 5.7). Interestingly, arginase inhibition increased the ADP‐induced or state III respiration by 25% (329.9 ± 18.7 vs 263.5 ± 20.2, p=0.05) and ATP production by 30% (237.5 ± 13.5 vs 182.7 ± 15.5, p=0.03) compared to untreated mitochondria. Uncoupler‐induced or state IIIu respiration (state IIIu) showed strong trend towards increase by 33% (237.5 ± 13.6 vs 182.7 ± 15.5, p=0.059) in isolated mitochondria treated with arginase inhibitor.ConclusionsArginase II negatively regulates the mitochondrial respiration in the heart and thus along with mtNOS it may play critical role in cardiovascular physiology and pathophysiology.Support or Funding InformationSupport: National Institute of Health: National Institute of General Medical Sciences and National Institute of Neurological Disorders and Stroke (Katakam: R01NS094834).This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Differential Respiratory Response of Isolated Cardiac Mitochondria from Young and Aged Mice towards Nitric Oxide Synthase Inhibitors

Mitochondrial respiratory dysfunction is proposed to be an important underlying mechanism in the aging‐induced cardiac abnormalities. Identifying the mechanisms involved in the development of aging‐induced respiratory dysfunction in cardiac mitochondria could be useful to develop therapeutic strategies to reduce the risk of developing cardiovascular diseases. Role of mitochondrial nitric oxide synthase (NOS) to aging‐induced respiratory changes in cardiac mitochondria are not studied previously. Here we treated the isolated cardiac mitochondria from young (3 months) and old mice (18 months) hearts with neuronal NOS inhibitor (nNOS, ARL 17477 dihydrochloride or ARL) and endothelial NOS inhibitor (eNOS, N5‐(1‐Iminoethyl)‐L‐ornithine or NIO) and studied the respiratory characteristics using Seahorse Xfe24 analyzer.ResultsMitochondria from the old mice hearts exhibited significantly increased basal respiration (24.7%, 169.0±14.0 vs 135.5±5.1, p≤0.05) with unchanged ATP production. They also showed a significant decrease in state IIIu respiration (23%, 635.4±40.2 vs 826.4±49.2, p≤0.05) and also the membrane potential. Both ARL and NIO treatments significantly decreased state IIIu respiration (22.1%, 643.7±34.4 vs 826.4±49.2, p≤0.05 & 23.9%, 448±18.5 vs 587.1±34.8, p≤0.05 respectively) in mitochondria from young mice hearts without affecting membrane potential and oxidative stress parameters. Interestingly, ARL and NIO neither induced similar responses in the mitochondria from the old mice hearts nor they corrected the aging‐induced decline in state IIIu respiration and membrane potential. Mitochondria from the old mice hearts have increased nNOS protein levels (421%, 1.98±0.80 vs 0.38±0.20, p≤0.05) with decreased protein S‐nitrosylation (46.25%, 8.3±1.5 vs 15.44±3.2, p≤0.07). The observed differential respiratory responses of cardiac mitochondria from young and old mice following nNOS and eNOS inhibitors were associated with reduced protein S‐nitrosylation in mitochondria from young mice hearts (64.2%, 5.53±0.35 vs 15.44±3.2, p≤0.05 & 65.7%, 5.3±0.84 vs 15.44±3.2, p≤0.05 respectively) with unaltered nitrosylation in mitochondria from old mice hearts.ConclusionsThus the functional loss of mitochondrial nNOS and eNOS in the aged hearts accompanied by the reduced NO‐mediated regulation of mitochondrial proteins contributes to the aging‐induced mitochondrial dysfunction.Support or Funding InformationAmerican Heart Association (PVG: 14SDG20490359 and VNS: 16PRE31450006) and National Institute of Health: National Institute of Neurological Disorders and Stroke and National Institute of General Medical Sciences (PVK: R01NS094834).This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Interaction between Mitochondrial Reactive Oxygen Species, Heme Oxygenase, and Nitric Oxide Synthase Stimulates Phagocytosis in Macrophages.

Macrophages are cells of the innate immune system that populate every organ. They are required not only for defense against invading pathogens and tissue repair but also for maintenance of tissue homeostasis and iron homeostasis. The aim of this study is to understand whether heme oxygenase (HO) and nitric oxide synthase (NOS) contribute to the regulation of nicotinamide adenine dinucleotide phosphate oxidase (NOX) activity and phagocytosis, two key components of macrophage function. This study was carried out using resting J774A.1 macrophages treated with hemin or vehicle. Activity of NOS, HO, or NOX was inhibited using specific inhibitors. Reactive oxygen species (ROS) formation was determined by Amplex® red assay, and phagocytosis was measured using fluorescein isothiocyanate-labeled bacteria. In addition, we analyzed the fate of the intracellular heme by using electron spin resonance. We show that both enzymes NOS and HO are essential for phagocytic activity of macrophages. NOS does not directly affect phagocytosis, but stimulates NOX activity via nitric oxide-triggered ROS production of mitochondria. Treatment of macrophages with hemin results in intracellular accumulation of ferrous heme and an inhibition of phagocytosis. In contrast to NOS, HO products, including carbon monoxide, neither clearly affect NOX activity nor clearly affect phagocytosis, but phagocytosis is accelerated by HO-mediated degradation of heme. Both enzymes contribute to the bactericidal activity of macrophages independently, by controlling different pathways.

NO – синтаза митохондрий – в физиологических и патологических условиях при сердечно-сосудистых заболеваниях

NO – синтаза митохондрий – в физиологических и патологических условиях при сердечно-сосудистых заболеваниях

Protective Effect of Tempol against Cisplatin-Induced Ototoxicity.

One of the major adverse effects of cisplatin chemotherapy is hearing loss. Cisplatin-induced ototoxicity hampers treatment because it often necessitates dose reduction, which decreases cisplatin efficacy. This study was performed to investigate the effect of Tempol on cisplatin-induced ototoxicity in an auditory cell line, House Ear Institute-Organ of Corti 1 (HEI-OC1). Cultured HEI-OC1 cells were exposed to 30 μM cisplatin for 24 h with or without a 2 h pre-treatment with Tempol. Cell viability was determined using 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) assay and apoptotic cells were identified using terminal deoxynucleotidyl transferase dUTP nick end labeling of nuclei (TUNEL) assay and flow cytometry. The effects of Tempol on cisplatin-induced cleaved poly(ADP-ribose) polymerase, cleaved caspase, and mitochondrial inducible nitric oxide synthase expression were evaluated using western blot analysis. Levels of intracellular reactive oxygen species (ROS) were measured to assess the effects of Tempol on cisplatin-induced ROS accumulation. Mitochondria were evaluated by confocal microscopy, and the mitochondrial membrane potential was measured to investigate whether Tempol protected against cisplatin-induced mitochondrial dysfunction. Cisplatin treatment decreased cell viability, and increased apoptotic features and markers, ROS accumulation, and mitochondrial dysfunction. Tempol pre-treatment before cisplatin exposure significantly inhibited all these cisplatin-induced effects. These results demonstrate that Tempol inhibits cisplatin-induced cytotoxicity in HEI-OC1, and could play a preventive role against cisplatin-induced ototoxicity.

Mitochondrial nitric oxide synthase and its role in the mechanisms of cell adaptation to hypoxia

Mitochondrial nitric oxide synthase (mtNOS) actively participates in mechanisms of regulation of cell adaptation to extreme factors. It is closely interacted with other mitochondrial regulatory factors and is involved in adaptation reactions of cells to hypoxia, ischemia and other pathogenic action. Analysis of the role of mtNOS in cell functioning in hypoxia condition and its influence on apoptosis is presented in the article. Complex mechanism of cell adaptation with participation of mitochondrial nitric oxide (NO) is considered. Mitochondrial NO is a modulator of cell respiration, synthesis of ATP, activity of mitochondrial ATP-sensitive potassium channels, mitochondrial megapore and the specific protein factor to hypoxia adaptation. The possibility of pharmacological regulation of mtNOS activity is discussed. This approach seems to be promising for searching of new drugs for the pharmacotherapy of diseases with hypoxia and ischemia in their pathogenesis. There is possible to regulate cell homeostasis, for example, resistance to hypoxia by modulating the activity of mtNOS and synthesis of mitochondrial NO.

Melatonin: A Potential Anti-Oxidant Therapeutic Agent for Mitochondrial Dysfunctions and Related Disorders

Mitochondria play a central role in cellular physiology. Besides their classic function of energy metabolism, mitochondria are involved in multiple cell functions, including energy distribution through the cell, energy/heat modulation, regulation of reactive oxygen species (ROS), calcium homeostasis, and control of apoptosis. Simultaneously, mitochondria are the main producer and target of ROS with the result that multiple mitochondrial diseases are related to ROS-induced mitochondrial injuries. Increased free radical generation, enhanced mitochondrial inducible nitric oxide synthase (iNOS) activity, enhanced nitric oxide (NO) production, decreased respiratory complex activity, impaired electron transport system, and opening of mitochondrial permeability transition pores have all been suggested as factors responsible for impaired mitochondrial function. Because of these, neurodegenerative diseases, such as Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), and aging, are caused by ROS-induced mitochondrial dysfunctions. Melatonin, the major hormone of the pineal gland, also acts as an anti-oxidant and as a regulator of mitochondrial bioenergetic function. Melatonin is selectively taken up by mitochondrial membranes, a function not shared by other anti-oxidants, and thus has emerged as a major potential therapeutic tool for treating neurodegenerative disorders. Multiple in vitro and in vivo experiments have shown the protective role of melatonin for preventing oxidative stress-induced mitochondrial dysfunction seen in experimental models of PD, AD, and HD. With these functions in mind, this article reviews the protective role of melatonin with mechanistic insights against mitochondrial diseases and suggests new avenues for safe and effective treatment modalities against these devastating neurodegenerative diseases. Future insights are also discussed.

Characterization of diabetic osteoarthritic cartilage and role of high glucose environment on chondrocyte activation: toward pathophysiological delineation of diabetes mellitus-related osteoarthritis

To examine the relationship between osteoarthritis (OA) and type 2 diabetes mellitus (DM). OA cartilage from DM and non-DM patients undergoing knee replacement were stimulated by IL-1β for 24 h and release of interleukin-6 (IL-6) and prostaglandin E2 (PGE2) was measured. Primary cultured murine chondrocytes were stimulated for 24 and 72 h with or without IL-1β (5 ng/mL) under normal-glucose (5.5 mM) or high-glucose (25 mM) conditions. The expression and release of pro-inflammatory mediators (IL-6, cyclooxygenase 2 [COX2]/PGE2) were analyzed by quantitative RT-PCR and ELISA/EIA. Glucose uptake was assessed with ((14)C)-2-deoxyglucose. Reactive oxygen species (ROS) and nitric oxide (NO) production were measured. To analyze the mechanism of IL-1β-induced inflammation, cells were pretreated or treated with inhibitors of glucose transport (cytochalasin B), the polyol pathway (epalrestat), mitochondrial oxidative stress (MitoTEMPO) or nitric oxide synthase (l-NAME). With IL-1β stimulation, IL-6 and PGE2 release was greater in human DM than non-DM OA cartilage (2.7- and 3-fold, respectively) (P < 0.05). In vitro, with IL-1β stimulation, IL-6 and COX2 mRNA expression, IL-6 and PGE2 release, and ROS and NO production were greater under high-than normal-glucose conditions in cultured chondrocytes. IL-1β-increased IL-6 release was reduced with cytochalasin B, epalrestat, L-NAME or MitoTEMPO treatment (-45%, -62%, -38% and -40%, respectively). OA cartilages from DM patients showed increased responsiveness to IL-1β-induced inflammation. Accordingly, high glucose enhanced IL-1β-induced inflammation in cultured chondrocytes via oxidative stress and the polyol pathway. High glucose and diabetes may thus participate in the increased inflammation in OA.

Acute ketamine impairs mitochondrial function and promotes superoxide dismutase activity in the rat brain.

Ketamine is often associated with altered mitochondrial function and oxidative stress. Nevertheless, limited data are still available regarding the in vivo action of ketamine in mitochondrial bioenergetics and redox state. Accumulating evidence supports a role for nitric oxide (NO) as a possible modulator of ketamine's side effects. In the present study, we investigated the role of NO modulation on ketamine anesthesia at the level of brain mitochondrial function and redox status. Adult male rats received a single dose of ketamine (50, 100, or 150 mg/kg IP) or a combination of ketamine and N-nitro-L-arginine (3 mg/kg IP). Animals were killed 6 hours after treatment. Brain and blood samples were collected for plasma NO determination and mitochondria isolation. Several variables of brain mitochondrial function were evaluated. Ketamine interfered with complex I function, revealing increased oxygen consumption in state 4, impaired oxidative phosphorylation efficiency of glutamate-malate substrate, and decreased NADH-ubiquinone oxidoreductase activity. In addition, mitochondrial NO synthase (mtNOS) activity and NO plasma levels were increased for the 50 and 100 mg/kg doses. Ketamine administration increased hydrogen peroxide generation and triggered superoxide dismutase activity. All these effects could totally or partially be prevented by mtNOS inhibition through N-nitro-L-arginine. Acute ketamine administration impaired the function of mitochondrial complex I leading to increased mtNOS activity, increased generation of hydrogen peroxide and NO, resulting in superoxide dismutase triggering, and improved antioxidant activity. The present findings clarify the role of NO modulation in ketamine anesthesia, providing new data on a relevant clinical mechanism.

Tempol, a Superoxide Dismutase Mimetic Agent, Ameliorates Cisplatin-Induced Nephrotoxicity through Alleviation of Mitochondrial Dysfunction in Mice

BackgroundMitochondrial dysfunction is a crucial mechanism by which cisplatin, a potent chemotherapeutic agent, causes nephrotoxicity where mitochondrial electron transport complexes are shifted mostly toward imbalanced reactive oxygen species versus energy production. In the present study, the protective role of tempol, a membrane-permeable superoxide dismutase mimetic agent, was evaluated on mitochondrial dysfunction and the subsequent damage induced by cisplatin nephrotoxicity in mice.Methods and FindingsNephrotoxicity was assessed 72 h after a single i.p. injection of cisplatin (25 mg/kg) with or without oral administration of tempol (100 mg/kg/day). Serum creatinine and urea as well as glucosuria and proteinuria were evaluated. Both kidneys were isolated for estimation of oxidative stress markers, adenosine triphosphate (ATP) content and caspase-3 activity. Moreover, mitochondrial oxidative phosphorylation capacity, complexes I–IV activities and mitochondrial nitric oxide synthase (mNOS) protein expression were measured along with histological examinations of renal tubular damage and mitochondrial ultrastructural changes. Tempol was effective against cisplatin-induced elevation of serum creatinine and urea as well as glucosuria and proteinuria. Moreover, pretreatment with tempol notably inhibited cisplatin-induced oxidative stress and disruption of mitochondrial function by restoring mitochondrial oxidative phosphorylation, complexes I and III activities, mNOS protein expression and ATP content. Tempol also provided significant protection against apoptosis, tubular damage and mitochondrial ultrastructural changes. Interestingly, tempol did not interfere with the cytotoxic effect of cisplatin against the growth of solid Ehrlich carcinoma.ConclusionThis study highlights the potential role of tempol in inhibiting cisplatin-induced nephrotoxicity without affecting its antitumor activity via amelioration of oxidative stress and mitochondrial dysfunction.

Abnormal mitochondrial L-arginine transport contributes to the pathogenesis of heart failure and rexoygenation injury.

BackgroundImpaired mitochondrial function is fundamental feature of heart failure (HF) and myocardial ischemia. In addition to the effects of heightened oxidative stress, altered nitric oxide (NO) metabolism, generated by a mitochondrial NO synthase, has also been proposed to impact upon mitochondrial function. However, the mechanism responsible for arginine transport into mitochondria and the effect of HF on such a process is unknown. We therefore aimed to characterize mitochondrial L-arginine transport and to investigate the hypothesis that impaired mitochondrial L-arginine transport plays a key role in the pathogenesis of heart failure and myocardial injury.Methods and ResultsIn mitochondria isolated from failing hearts (sheep rapid pacing model and mouse Mst1 transgenic model) we demonstrated a marked reduction in L-arginine uptake (p<0.05 and p<0.01 respectively) and expression of the principal L-arginine transporter, CAT-1 (p<0.001, p<0.01) compared to controls. This was accompanied by significantly lower NO production and higher 3-nitrotyrosine levels (both p<0.05). The role of mitochondrial L-arginine transport in modulating cardiac stress responses was examined in cardiomyocytes with mitochondrial specific overexpression of CAT-1 (mtCAT1) exposed to hypoxia-reoxygenation stress. mtCAT1 cardiomyocytes had significantly improved mitochondrial membrane potential, respiration and ATP turnover together with significantly decreased reactive oxygen species production and cell death following mitochondrial stress.ConclusionThese data provide new insights into the role of L-arginine transport in mitochondrial biology and cardiovascular disease. Augmentation of mitochondrial L-arginine availability may be a novel therapeutic strategy for myocardial disorders involving mitochondrial stress such as heart failure and reperfusion injury.

Human IGF1 regulates midgut oxidative stress and epithelial homeostasis to balance lifespan and Plasmodium falciparum resistance in Anopheles stephensi.

Insulin and insulin-like growth factor signaling (IIS) regulates cell death, repair, autophagy, and renewal in response to stress, damage, and pathogen challenge. Therefore, IIS is fundamental to lifespan and disease resistance. Previously, we showed that insulin-like growth factor 1 (IGF1) within a physiologically relevant range (0.013–0.13 µM) in human blood reduced development of the human parasite Plasmodium falciparum in the Indian malaria mosquito Anopheles stephensi. Low IGF1 (0.013 µM) induced FOXO and p70S6K activation in the midgut and extended mosquito lifespan, whereas high IGF1 (0.13 µM) did not. In this study the physiological effects of low and high IGF1 were examined in detail to infer mechanisms for their dichotomous effects on mosquito resistance and lifespan. Following ingestion, low IGF1 induced phosphorylation of midgut c-Jun-N-terminal kinase (JNK), a critical regulator of epithelial homeostasis, but high IGF1 did not. Low and high IGF1 induced midgut mitochondrial reactive oxygen species (ROS) synthesis and nitric oxide (NO) synthase gene expression, responses which were necessary and sufficient to mediate IGF1 inhibition of P. falciparum development. However, increased ROS and apoptosis-associated caspase-3 activity returned to baseline levels following low IGF1 treatment, but were sustained with high IGF1 treatment and accompanied by aberrant expression of biomarkers for mitophagy, stem cell division and proliferation. Low IGF1-induced ROS are likely moderated by JNK-induced epithelial cytoprotection as well as p70S6K-mediated growth and inhibition of apoptosis over the lifetime of A. stephensi to facilitate midgut homeostasis and enhanced survivorship. Hence, mitochondrial integrity and homeostasis in the midgut, a key signaling center for IIS, can be targeted to coordinately optimize mosquito fitness and anti-pathogen resistance for improved control strategies for malaria and other vector-borne diseases.