Pharmacological regulation of mitochondrial nitric oxide synthase

Alcohol hangover is the combination of negative mental and physical symptoms which can be experienced after a single episode of alcohol consumption, starting when blood alcohol concentration approaches zero. We previously demonstrated that hangover provokes mitochondrial dysfunction, oxidative stress, imbalance in antioxidant defenses, and impairment in cellular bioenergetics. Chronic and acute ethanol intake induces neuroapoptosis but there are no studies which evaluated apoptosis at alcohol hangover. The aim of the present work was to study alcohol residual effects on intrinsic and extrinsic apoptotic signaling pathways in mice brain cortex. Male Swiss mice received i.p. injection of ethanol (3.8 g/kg) or saline. Six hours after injection, at alcohol hangover onset, mitochondria and tissue lysates were obtained from brain cortex. Results indicated that during alcohol hangover a loss of granularity of mitochondria and a strong increment in mitochondrial permeability were observed, indicating the occurrence of swelling.

Alcohol-treated mice showed a significant 35% increase in Bax/Bcl-2 ratio and a 5-fold increase in the ratio level of cytochrome c between mitochondria and cytosol. Caspase 3, 8 and 9 protein expressions were 32%, 33% and 20% respectively enhanced and the activity of caspase 3 and 6 was 30% and 20% increased also due to the hangover condition. Moreover, 38% and 32% increments were found in PARP1 and p53 protein expression respectively and on the contrary, SIRT-1 was almost 50% lower than controls due to the hangover condition. The present work demonstrates that alcohol after-effects could result in the activation of mitochondrial and non-mitochondrial apoptosis pathways.

Hydrogen peroxide is the main metabolite effective in redox regulation and it is considered an insulinomimetic agent, with insulin signalling being essential for normal mitochondrial function in cardiomyocytes. Therefore, the aim of this work was to deeply analyse the heart mitochondrial H₂O₂ metabolism, in the early stage of type 1 diabetes. Diabetes was induced by Streptozotocin (STZ, single dose, 60 mg × kg⁻¹, ip.) in male Wistar rats and the animals were sacrificed 10 days after injection. Mitochondrial membrane potential and ATP production, using malate-glutamate as substrates, in the heart of diabetic animals were like the ones observed in control group. Mn-SOD activity was lower (15%) in the heart of diabetic rats even though its expression was increased (29%). The increment in heart mitochondrial H₂O₂ production (117%) in diabetic animals was accompanied by an enhancement in the activities and expressions of glutathione peroxidase (26% and 42%) and of catalase (200% and 133%), with no changes in the peroxiredoxin activity, leading to [H₂O₂]_ss ∼40 nM. Heart mitochondrial lipid peroxidation and protein nitration were higher in STZ-injected animals (45% and 42%) than in control group. The mitochondrial membrane potential and ATP production preservation suggest the absence of irreversible damage at this early stage of diabetes 1. The increase in mitochondrial [H₂O₂]_ss above the physiological range, but still below supraphysiological concentration (∼100 nM) seems to be part of the adaptive response triggered in cardiomyocytes due to the absence of insulin. The signs of mitochondrial dysfunction observed in this very early stage of diabetes are consistent with the mitochondrial entity called ″complex I syndrome″.

Rat brain and heart display different endogenous protective responses against hypobaric hypoxia in an age-dependent way. The aim of the present work was to evaluate the effects of acute hypobaric hypoxia (HH, 48 h) on brain and heart mitochondrial function as well as the participation of nitric oxide (NO) in old rats (22-month old).

Cortical mitochondria from rats exposed to HH decreased respiratory rates (37 %, state 3) and membrane potential (20 %), but NO and H₂O₂ production increased by 48 %, and 23 %, respectively. Hippocampal mitochondria preserved O₂ consumption and H₂O₂ production, decreased membrane potential (18 %) and increased NO production (46 %). By contrast, HH decreased NO production (53 %) in mitochondria from left heart ventricles associated with increased cytochrome oxidase activity (39 %) and decreased NADPH oxidase activity (31 %). Also, a tendency to increase complex I-III (24 %) and complex II-III (65 %) activity was observed.

In conclusion, after HH hippocampal and cortical mitochondria showed mild uncoupling and increased NO production. However, only the hippocampus preserved O₂ consumption and H₂O₂ levels. Interestingly, heart mitochondria showed a decreased ROS production through increased cytochrome oxidase activity associated with a decrease in NO production. This may be interpreted as a self-protective mechanism against hypoxia.

Alcohol hangover is defined as the combination of mental and physical symptoms experienced the day after a single episode of heavy drinking, starting when blood alcohol concentration approaches zero. We previously evidenced increments in free radical generation and an imbalance in antioxidant defences in non-synaptic mitochondria and synaptosomes during hangover. It is widely known that acute alcohol exposure induces changes in nitric oxide (NO) production and blocks the binding of glutamate to NMDAR in central nervous system. Our aim was to evaluate the residual effect of acute ethanol exposure (hangover) on NO metabolism and the role of NMDA receptor-PSD95-nNOS pathway in non-synaptic mitochondria and synaptosomes from mouse brain cortex. Results obtained for the synaptosomes fraction showed a 37% decrease in NO total content, a 36% decrease in NOS activity and a 19% decrease in nNOS protein expression. The in vitro addition of glutamate to synaptosomes produced a concentration-dependent enhancement of NO production which was significantly lower in samples from hangover mice than in controls for all the glutamate concentrations tested. A similar patter of response was observed for nNOS activity being decreased both in basal conditions and after glutamate addition. In addition, synaptosomes exhibited a 64% and 15% reduction in NMDA receptor subunit GluN2B and PSD-95 protein expression, respectively. Together with this, glutamate-induced calcium entry was significant decreased in synaptosomes from alcohol-treated mice. On the other hand, in non-synaptic mitochondria, no significant differences were observed in NO content, NOS activity or nNOS protein expression. The expression of iNOS remained unaltered in synaptosomes and non-synaptic mitochondria. Here we demonstrated that hangover effects on NO metabolism are strongly evidenced in synaptosomes probably due to a disruption in NMDAR/PSD-95/nNOS pathway.

Glaucoma is a neurodegenerative disease that affects eye structures and brain areas related to the visual system. Oxidative stress plays a key role in the development and progression of the disease. The aims of the present study were to evaluate the mitochondrial function and its participation in the brain redox metabolism in an experimental glaucoma model.

3-month-old female Wistar rats were subjected to cauterization of two episcleral veins of the left eye to elevate the intraocular pressure. Seven days after surgery, animals were sacrificed, the brain was carefully removed and the primary visual cortex was dissected. Mitochondrial bioenergetics and ROS production, and the antioxidant enzyme defenses from both mitochondrial and cytosolic fractions were evaluated.

When compared to control, glaucoma decreased mitochondrial ATP production (23%, p < 0.05), with an increase in superoxide and hydrogen peroxide production (30%, p < 0.01 and 28%, p < 0.05, respectively), whereas no changes were observed in membrane potential and oxygen consumption rate. In addition, the glaucoma group displayed a decrease in complex II activity (34%, p < 0.01). Moreover, NOX4 expression was increased in glaucoma compared to the control group (27%, p < 0.05). Regarding the activity of enzymes associated with the regulation of the redox status, glaucoma showed an increase in mitochondrial SOD activity (34%, p < 0.05), mostly due to an increase in Mn-SOD (50%, p < 0.05). A decrease in mitochondrial GST activity was observed (11%, p < 0.05). GR and TrxR activity were decreased in both mitochondrial (16%, p < 0.05 and 20%, p < 0.05 respectively) and cytosolic (21%, p < 0.01 and 50%, p < 0.01 respectively) fractions in the glaucoma group. Additionally, glaucoma showed an increase in cytoplasmatic GPx (50%, p < 0.01). In this scenario, redox imbalance took place resulting in damage to mitochondrial lipids (39%, p < 0.01) and proteins (70%, p < 0.05).

These results suggest that glaucoma leads to mitochondrial function impairment in brain visual targets, that is accompanied by an alteration in both mitochondrial and cytoplasmatic enzymatic defenses. As a consequence of redox imbalance, oxidative damage to macromolecules takes place and can further affect vital cellular functions. Understanding the role of the mitochondria in the development and progression of the disease could bring up new neuroprotective therapies.

The aim of this work was to study the early events that occur in heart mitochondria and to analyse the temporal evolution of cardiac mitochondrial dysfunction in a type 1 diabetes model. Male Wistar rats were injected with Streptozotocin (STZ, single dose, 60 mg × kg⁻¹, i.p.) and hyperglycemic state was confirmed 72 h later. The animals were sacrificed 10 or 14 days after STZ-injection. Heart mitochondrial state 3 O₂ consumption sustained by malate-glutamate (21%) or by succinate (16%), and complexes I-III (27%), II-III (24%) and IV (22%) activities were lower in STZ group, when animals were sacrificed at day 14, i.e. ~11 days of hyperglycemia. In contrast, after 10 days of STZ-injection (~7 days of hyperglycemia), only the state 3 O₂ consumption sustained by malate-glutamate (23%) and its corresponding respiratory control (30%) were lower in diabetic rats, in accordance with complex I-III activity reduction (17%). Therefore, this time (~7 days of hyperglycemia) has been considered as an “early stage” of cardiac mitochondrial dysfunction. At this point, mitochondrial production rates of H₂O₂ (117%), NO (30%) and ONOO⁻ (~225%), and mtNOS expression (29%) were higher; and mitochondrial SOD activity (15%) and [GSH + GSSG] (28%) were lower in diabetic rats. Linear correlations between the modified mitochondrial parameters and glycemias were observed. PGC-1α expression was similar between groups, suggesting that mitochondrial biogenesis was not triggered in this initial phase of mitochondrial dysfunction. Consequently, complex I, H₂O₂ and NO could be considered early subcellular signals of cardiac mitochondrial dysfunction, with NO and H₂O₂ being located upstream de novo synthesis of mitochondria.