TGF-ß Signaling Pathways in the Development of Diabetic Retinopathy.

Diabetic retinopathy (DR), a prevalent complication of diabetes mellitus affecting a significant portion of the global population, has long been viewed primarily as a microvascular disorder. However, emerging evidence suggests that it should be redefined as a neurovascular disease with multifaceted pathogenesis rooted in oxidative stress and advanced glycation end products. The transforming growth factor-ß (TGF-ß) signaling family has emerged as a major contributor to DR pathogenesis due to its pivotal role in retinal vascular homeostasis, endothelial cell barrier function, and pericyte differentiation. However, the precise roles of TGF-ß signaling in DR remain incompletely understood, with conflicting reports on its impact in different stages of the disease. Additionally, the BMP subfamily within the TGF-ß superfamily introduces further complexity, with BMPs exhibiting both pro- and anti-angiogenic properties. Furthermore, TGF-ß signaling extends beyond the vascular realm, encompassing immune regulation, neuronal survival, and maintenance. The intricate interactions between TGF-ß and reactive oxygen species (ROS), non-coding RNAs, and inflammatory mediators have been implicated in the pathogenesis of DR. This review delves into the complex web of signaling pathways orchestrated by the TGF-ß superfamily and their involvement in DR. A comprehensive understanding of these pathways may hold the key to developing targeted therapies to halt or mitigate the progression of DR and its devastating consequences.

Naringin prevents diabetic nephropathy in rats through blockage of oxidative stress and attenuation of the mitochondrial dysfunction.

We have studied the effects of naringin (NAR), a flavonoid from citric fruits, on morphology, ultrastructure and function of the kidney in streptozotocin (STZ)-induced diabetic rats. Two groups of animals were used: (1) control rats and (2) STZ rats (60 mg STZ/kg b.w.). At 3 days after induction, one group of STZ-treated rats received 40 mg NAR/kg b.w. daily. NAR blocked completely alterations in the biochemical renal markers in STZ rats except the increase in serum urea that was partially avoided by the flavonoid. NAR ameliorated the kidney morphological lesions from STZ rats. STZ treatment induced round and smaller mitochondria, which was avoided by NAR. Citrate synthase, isocitrate and malate dehydrogenases, enzyme activities of the Krebs cycle, were decreased in STZ rats. NAR abolished this decrease in the latter proteins. NAR also prevented a decrease in the ATP synthase activity of the mitochondria from renal cortex by about 49% in STZ rats, returning the enzyme activity to control values. The nephroprotection caused by NAR is mediated through counteraction of oxidative stress in mitochondria of proximal tubules. NAR might be a therapeutic strategy to reduce the complication of diabetic nephropathy in type 1 diabetic patients.

Loss of dystrophin is associated with increased myocardial stiffness in a model of left ventricular hypertrophy.

Transition from compensated to decompensated left ventricular hypertrophy (LVH) is accompanied by functional and structural changes. Here, the aim was to evaluate dystrophin expression in murine models and human subjects with LVH by transverse aortic constriction (TAC) and aortic stenosis (AS), respectively. We determined whether doxycycline (Doxy) prevented dystrophin expression and myocardial stiffness in mice. Additionally, ventricular function recovery was evaluated in patients 1 year after surgery. Mice were subjected to TAC and monitored for 3 weeks. A second group received Doxy treatment after TAC. Patients with AS were stratified by normal left ventricular end-diastolic wall stress (LVEDWS) and high LVEDWS, and groups were compared. In mice, LVH decreased inotropism and increased myocardial stiffness associated with a dystrophin breakdown and a decreased mitochondrial O2 uptake (MitoMVO2). These alterations were attenuated by Doxy. Patients with high LVEDWS showed similar results to those observed in mice. A correlation between dystrophin and myocardial stiffness was observed in both mice and humans. Systolic function at 1 year post-surgery was only recovered in the normal-LVEDWS group. In summary, mice and humans present diastolic dysfunction associated with dystrophin degradation. The recovery of ventricular function was observed only in patients with normal LVEDWS and without dystrophin degradation. In mice, Doxy improved MitoMVO2. Based on our results it is concluded that the LVH with high LVEDWS is associated to a degradation of dystrophin and increase of myocardial stiffness. At least in a murine model these alterations were attenuated after the administration of a matrix metalloprotease inhibitor.

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.

Cellular and Molecular Mechanisms of Neuronal Degeneration in Early-Stage Diabetic Retinopathy.

BACKGROUND: Studies on the early retinal changes in Diabetic Retinopathy (DR) have demonstrated that neurodegeneration precedes vascular abnormalities like microaneurysms or intraretinal hemorrhages. Therefore, there is a growing field of study to analyze the cellular and molecular pathways involved to allow for the development of novel therapeutics to prevent the onset or delay the progression of DR. Molecular Mechanisms: Oxidative stress and mitochondrial dysfunction contribute to neurodegeneration through pathways involving polyol, hexosamine, advanced glycation end products, and protein kinase C. Potential interventions targeting these pathways include aldose reductase inhibitors and protein kinase C inhibitors. Neurotrophic factor imbalances, notably brain-derived neurotrophic factor and nerve growth factor, also play a role in early neurodegeneration, and supplementation of these neurotrophic factors show promise in mitigating neurodegeneration. Cellular Mechanisms: Major cellular mechanisms of neurodegeneration include caspase-mediated apoptosis, glial cell reactivity, and glutamate excitotoxicity. Therefore, inhibitors of these pathways are potential therapeutic avenues. Vascular Component: The nitric oxide pathway, critical for neurovascular coupling, is disrupted in DR due to increased reactive oxygen species. Vascular Endothelial Growth Factor (VEGF), a long-known angiogenic factor, has demonstrated both damaging and neuroprotective effects, prompting a careful consideration of long-term anti-VEGF therapy. CONCLUSION: Current DR treatments primarily address vascular symptoms but fall short of preventing or halting the disease. Insights into the mechanisms of retinal neurodegeneration in the setting of diabetes mellitus not only enhance our understanding of DR but also pave the way for future therapeutic interventions aimed at preventing disease progression and preserving vision.

Mitochondrial H2O2 metabolism as central event of heart complex I syndrome in early diabetes.

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″.

Endotoxemia impairs heart mitochondrial function by decreasing electron transfer, ATP synthesis and ATP content without affecting membrane potential.

Acute endotoxemia (LPS, 10 mg/kg ip, Sprague Dawley rats, 45 days old, 180 g) decreased the O2 consumption of rat heart (1 mm³ tissue cubes) by 33% (from 4.69 to 3.11 µmol O2/min. g tissue). Mitochondrial O2 consumption and complex I activity were also decreased by 27% and 29%, respectively. Impaired respiration was associated to decreased ATP synthesis (from 417 to 168 nmol/min. mg protein) and ATP content (from 5.40 to 4.18 nmol ATP/mg protein), without affecting mitochondrial membrane potential. This scenario is accompanied by an increased production of O2·â» and H2O2 due to complex I inhibition. The increased NO production, as shown by 38% increased mtNOS biochemical activity and 31% increased mtNOS functional activity, is expected to fuel an increased ONOO⁻ generation that is considered relevant in terms of the biochemical mechanism. Heart mitochondrial bioenergetic dysfunction with decreased O2 uptake, ATP production and contents may indicate that preservation of mitochondrial function will prevent heart failure in endotoxemia.

Diastolic function during hemorrhagic shock in rabbits.

Hemorrhage (H) is associated with a left ventricular (LV) dysfunction. However, the diastolic function has not been studied in detail. The main goal was to assess the diastolic function both during and 120 min after bleeding, in the absence and in the presence of L-NAME. Also, the changes in mRNA and protein expression of nitric oxide synthase (NOS) isoforms were determined. New Zealand rabbits were divided into three groups: Sham group, H group (hemorrhage 20% blood volume), and H L-NAME group (hemorrhage treated with L-NAME). We evaluated systolic and diastolic ventricular functions in vivo and in vitro (Langendorff technique). Hemodynamic parameters and LV function were measured before, during, and at 120 min after bleeding. We analyzed the isovolumic relaxation using t ½ in vivo (closed chest). After that, hearts were excised and perfused in vitro to measure myocardial stiffness. Samples were frozen to measure NOS mRNA and protein expression. The t½ increased during bleeding and returned to basal values 120 min after bleeding. L-NAME blunted this effect. Data from the H group revealed a shift to the left in the LV end diastolic pressure-volume curve at 120 min after bleeding, which was blocked by L-NAME. iNOS and nNOS protein expression and mRNA levels increased at 120 min after the hemorrhage. Acute hemorrhage induces early and transient isovolumic relaxation impairment and an increase in myocardial stiffness 120 min after bleeding. L-NAME blunted the LV dysfunction, suggesting that NO modulates ventricular function through iNOS and nNOS isoforms.

Combined treatment with niclosamide and camptothecin enhances anticancer effect in U87 MG human glioblastoma cells.

Glioblastoma multiforme (GBM) is one of the deadliest cancers of the brain. Its ability to infiltrate healthy brain tissues renders it difficult to remove surgically. Furthermore, it exhibits high rates of radio- and chemoresistance, making the survival rates of patients with GBM poor. Therefore, novel effective therapies for GBM remain urgently in demand. Niclosamide is an anti-helminthic drug and recently it has been receiving attention due to its reported anticancer effects in cancer models, including GBM. Furthermore, camptothecin (CPT) is a naturally-occurring alkaloid and has been previously reported to be a potential chemotherapeutic agent by targeting the nuclear topoisomerase I. In the present study, the possible combined chemotherapeutic effects of niclosamide and CPT on the human glioblastoma cell line U87 MG was investigated by MTT assay and western blot analysis. Niclosamide exhibited synergistic activities with CPT to suppress the proliferation of U87 MG cells. Additionally, niclosamide suppressed cell proliferation and induced cell death mainly by triggering ER stress and autophagy, whilst CPT induced cell apoptosis mainly through p53-mediated mitochondrial dysfunction and activation of the MAPK (ERK/JNK) pathways. Overall, these findings suggest that co-administration of niclosamide and CPT may provide a novel therapeutic treatment strategy for GBM.

Temporal evolution of cardiac mitochondrial dysfunction in a type 1 diabetes model. Mitochondrial complex I impairment, and H2O2 and NO productions as early subcellular events.

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.

Decrease lysyl oxidase activity in hearts of copper-deficient bovines.

BACKGROUND: Lysyl oxidase (LOX) is a metalloenzyme that requires Cu as a cofactor and it is responsible for the formation of collagen and elastin cross-linking. The objective of this work was to measure the LOX enzyme activity in the heart of bovines with Cu deficiency induced by high molybdenum and sulfur levels in the diet. METHODS: Eighteen myocardial samples were obtained from Cu-deficient (n = 9) and control (n = 9) Holstein bovines during two similar assays. The samples were frozen in liquid nitrogen and stored at -70 °C to measure enzymatic activity. A commercial kit was used, following producer instructions. RESULTS: The results showed that LOX activity from the hearts of Cu-deficient bovines is 29 % lower than the ones of control bovines, being this difference statistically significant (p = 0.03). CONCLUSION: To our knowledge, this is the first report that determined LOX enzymatic activity in bovine heart of Cu-deficient animals. The microscopic alterations found in these animals in our previous work, could be explained by a diminished LOX activity. The results are in agreement with other authors, who found a relationship between LOX activity and dietary Cu intake. The information provided by this work could help to clarify the pathogenesis of cardiac lesions in cattle with dietary Cu deficiency.

Role of matrix metalloproteinase-2 in the cardioprotective effect of ischaemic postconditioning.

The activation of matrix metalloproteinases (MMPs) contributes to myocardial injury at the onset of reperfusion; however, their role in ischaemic postconditioning is unknown. The aim of the present study was to examine the effects of ischaemic postconditioning on MMP activity in isolated rabbit hearts. The isolated rabbit hearts were subjected to 30 min of global ischaemia followed by 180 min of reperfusion (I/R group; n = 8). In the ischaemic postconditioning group (n = 8), a postconditioning protocol was performed (2 cycles of 30 s reperfusion-ischaemia). In other experiments, we added doxycycline, an MMP inhibitor, at 25 (n = 7) or 50 micromol l(1) (n = 8) during the first 2 min of reperfusion. Coronary effluent and left ventricular tissue were collected during pre-ischaemic conditions and at different times during the reperfusion period to measure MMP-2 activity and cardiac protein nitration. We evaluated ventricular function and infarct size. In the I/R group, infarct size was 32.1 +/- 5.2%; Postcon reduced infarct size to 9.5 +/- 3.8% (P < 0.05) and inhibited MMP-2 activity during reperfusion. The administration of doxycycline at 50 micromol l(1) inhibited MMP-2 activity and cardiac protein nitration and reduced the infarct size to 9.7 +/- 2.8% (P < 0.05). A lower dose of doxycycline (25 micromol l(1)) failed to inhibit MMP-2 activity and did not modify the infarct size. Our results strongly suggest that ischaemic postconditioning may exert part of its cardioprotective effects through the inhibition of MMP-2 activity.

Effectiveness of two rehabilitation strategies provided by nurses for stroke patients in Mexico.

OBJECTIVE: Rehabilitation strategies have been developed to improve functional state in stroke patients. The main objective of this study was to evaluate the effectiveness of the early rehabilitation at hospital and its continuity at home provided by nurses, on the functional recovery of basic and social activities in stroke patients compared with conventional care. DESIGN: A randomised clinical trial was carried out in three general hospitals of the Mexican Institute of Social Security (IMSS) in Mexico City between April 2003-May 2004. PARTICIPANTS: Stroke patients. METHODS: Two rehabilitation strategies provided by nurses for stroke patients were compared: physiotherapy plus caregiver education in rehabilitation (strategy 1, S1) vs. education alone (strategy 2, S2). The main outcome variables were the basic (Barthel index) and social (Frenchay activities index) activities of daily living, of each patient. Age, sex, morbidity, stroke symptoms, complications, neurological damage (Canadian Scale), cognitive state (mini-mental state examination questionnaire) and duration of hospitalisation were defined as the control variables. Patients were evaluated at baseline and months one, three and six thereafter. RESULTS: One hundred and ten patients with ischaemic stroke were enrolled and randomised; 59 were assigned to S1 and 51 to S2. Comparison of the outcome variables showed that patients improved significantly over time, but no differences were observed between groups. We observed no significant difference in strategy performance with regard to the basic and instrumental activities of daily living. RELEVANCE TO CLINICAL PRACTICE: Participants who received physiotherapy with additional caregiver education benefit no more than those whose caregivers received education alone. Those countries that do not have integral rehabilitation programmes for stroke patients should understand their importance and budget resources for them. Meanwhile, both caregiver education and nurses trained in specific care and physiotherapy are alternatives that benefit these patients.

(+)-Catechin inhibits heart mitochondrial complex I and nitric oxide synthase: functional consequences on membrane potential and hydrogen peroxide production.

In order to study the in vitro effect of flavan-3-ol (+)-catechin on the enzymatic activities of mitochondrial complex I and nitric oxide synthase (mtNOS), as well as the consequences on the membrane potential and H2O2 production rate, isolated mitochondria from rat heart were exposed to 3 nM to 100 µM (+)-catechin. NADH-Q1 reductase (complex I) and mtNOS activities were inhibited 25% and 50%, respectively, by the addition of 10 nM (+)-catechin to the reaction medium. Moreover, in the nM range, (+)-catechin decreased state 4 mitochondrial membrane potential by about 10 mV, but failed to change the membrane potential measured in the presence of ADP. (+)-Catechin (10 nM) inhibited not only complex I activity, but also the H2O2 production rate (35%) sustained by malate-glutamate, in accordance with the decrease observed in mitochondrial membrane potential. Considering (+)-catechin concentrations lower than 10 nM, linear and positive correlations were obtained between mitochondrial complex I activity and either NO (r2 = 0.973) or H2O2 production rates (r2 = 0.958), suggesting a functional association among these parameters. Altogether, the results indicate that (+)-catechin, at nM concentrations, inhibits mitochondrial complex I activity, leading to membrane potential decline and consequently to reduction in H2O2 and NO production rates. The decrease in mtNOS activity could also be a consequence of the direct action of (+)-catechin on the NOS structure, this effect being in accordance with the functional interaction between complex I and mtNOS, as previously reported.

Complex I syndrome in striatum and frontal cortex in a rat model of Parkinson disease.

Mitochondrial dysfunction named complex I syndrome was observed in striatum mitochondria of rotenone treated rats (2 mg rotenone/kg, i. p., for 30 or 60 days) in an animal model of Parkinson disease. After 60 days of rotenone treatment, the animals showed: (a) 6-fold increased bradykinesia and 60% decreased locomotor activity; (b) 35-34% decreases in striatum O2 uptake and in state 3 mitochondrial respiration with malate-glutamate as substrate; (c) 43-57% diminished striatum complex I activity with 60-71% decreased striatum mitochondrial NOS activity, determined both as biochemical activity and as functional activity (by the NO inhibition of active respiration); (d) 34-40% increased rates of mitochondrial O2•- and H2O2 productions and 36-46% increased contents of the products of phospholipid peroxidation and of protein oxidation; and (e) 24% decreased striatum mitochondrial content, likely associated to decreased NO-dependent mitochondrial biogenesis. Intermediate values were observed after 30 days of rotenone treatment. Frontal cortex tissue and mitochondria showed similar but less marked changes. Rotenone-treated rats showed mitochondrial complex I syndrome associated with cellular oxidative stress in the dopaminergic brain areas of striatum and frontal cortex, a fact that describes the high sensitivity of mitochondrial complex I to inactivation by oxidative reactions.

Biochemical and Morphological Alterations in Hearts of Copper-Deficient Bovines.

Copper deficiency is an important disease of cattle that produces several clinical signs and lesions, due to alterations in copper-dependent enzymes. One of the organs affected by this deficiency is the heart (falling disease), but nevertheless, these cardiac lesions have not been extensively studied in bovines. The aim of this work was to propose a possible pathogenic mechanism for cardiac lesions in cattle affected by copper deficiency. Because of the possible existence of oxidative distress caused by low levels of copper-zinc-superoxide dismutase and cytochrome oxidase, ultrastructural and histological lesions have been evaluated in the heart of bovines in which a Cu deficiency had been induced using high molybdenum and sulfur levels in the diet. Our results indicated that copper deficiency produces significant damage in myocardium with high levels of lipid oxidation and a significant reduction in copper-zinc-superoxide dismutase activity leading to an oxidative distress situation. However, cytochrome oxidase activity was not significantly reduced. Histological observation revealed a significant increase in the amount of connective tissue, enlarged basement membranes of myocytes, and numerous Anichkov cells, in the hearts of deficient animals. Ultrastructural observation showed a significant enhancement in the mitochondrial volume density, with presence of lesions such as swelling and cristae disruption. We conclude that copper deficiency in bovines causes morphological lesions in the heart due to an oxidative damage produced by copper-dependent enzyme alterations.

Mitochondrial metabolic states and membrane potential modulate mtNOS activity.

The mitochondrial metabolic state regulates the rate of NO release from coupled mitochondria: NO release by heart, liver and kidney mitochondria was about 40-45% lower in state 3 (1.2, 0.7 and 0.4 nmol/min mg protein) than in state 4 (2.2, 1.3 and 0.7 nmol/min mg protein). The activity of mtNOS, responsible for NO release, appears driven by the membrane potential component and not by intramitochondrial pH of the proton motive force. The intramitochondrial concentrations of the NOS substrates, L-arginine (about 310 microM) and NADPH (1.04-1.78 mM) are 60-1000 times higher than their KM values. Moreover, the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. Nitric oxide release was exponentially dependent on membrane potential as reported for mitochondrial H2O2 production [S.S. Korshunov, V.P. Skulachev, A.A. Satarkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15-18.]. Agents that decrease or abolish membrane potential minimize NO release while the addition of oligomycin that produces mitochondrial hyperpolarization generates the maximal NO release. The regulation of mtNOS activity, an apparently voltage-dependent enzyme, by membrane potential is marked at the physiological range of membrane potentials.

Mitochondrial metabolic states regulate nitric oxide and hydrogen peroxide diffusion to the cytosol.

Mitochondria isolated from rat heart, liver, kidney and brain (respiratory control 4.0-6.5) release NO and H2O2 at rates that depend on the mitochondrial metabolic state: releases are higher in state 4, about 1.7-2.0 times for NO and 4-16 times for H2O2, than in state 3. NO release in rat liver mitochondria showed an exponential dependence on membrane potential in the range 55 to 180 mV, as determined by Rh-123 fluorescence. A similar behavior was reported for mitochondrial H2O2 production by [S.S. Korshunov, V.P. Skulachev, A.A. Starkov, High protonic potential actuates a mechanism of production of reactive oxygen species in mitochondria. FEBS Lett. 416 (1997) 15_18.]. Transition from state 4 to state 3 of brain cortex mitochondria was associated to a decrease in NO release (50%) and in membrane potential (24-53%), this latter determined by flow cytometry and DiOC6 and JC-1 fluorescence. The fraction of cytosolic NO provided by diffusion from mitochondria was 61% in heart, 47% in liver, 30% in kidney, and 18% in brain. The data supports the speculation that NO and H2O2 report a high mitochondrial energy charge to the cytosol. Regulation of mtNOS activity by membrane potential makes mtNOS a regulable enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.

Mitochondrial nitric oxide synthase, a voltage-dependent enzyme, is responsible for nitric oxide diffusion to cytosol.

The mitochondrial metabolic state regulates the rate of mitochondrial NO production and release to the cytosol. Nitric oxide release of rat heart mitochondria decreased markedly from 2.2 to 1.2 nmol NO/min. mg protein in the state 4 to state 3 transition. The activity of mtNOS, responsible for NO release, is driven by the membrane potential and not by intramitochondrial pH changes. The release of NO by rat liver mitochondria showed an exponential dependence on membrane potential. A similar behavior was reported for heart mitochondrial H2O2 production. The fraction of heart cytosolic NO provided by diffusion from mitochondria is 90%. The intramitochondrial concentrations of L-arginine and NADPH are higher than their KM values, and the changes in their concentrations in the state 4-state 3 transition are not enough to explain the changes in NO release. These data indicate that the redox state of the respiratory chain components regulates H2O2 production and mitochondrial membrane potential modulates NO release, and support the speculation that NO and H2O2 are a biological signal that reports a high mitochondrial energy charge to the cytosol. The marked regulation of mtNOS activity, as a voltage-dependent enzyme and at the physiological range of membrane potentials, makes mtNOS a highly sensitive enzyme that in turn regulates mitochondrial O2 uptake and H2O2 production.

Mitochondrial contribution to the molecular mechanism of heart acclimatization to chronic hypoxia: role of nitric oxide.

A remarkable number of adaptive responses; including changes in the cardiovascular, respiratory and hematologic systems; takes place during acclimatization to natural or simulated high altitude. This adaptation to chronic hypoxia confers the heart an improved tolerance to all major deleterious consequences of acute O2 deprivation, not only reducing infarct size but also alleviating post-ischemic contractile dysfunction and ventricular arrhythmias. There is growing evidence about the involvement of mitochondria and NO in the establishment of cardioprotection. This review focuses on evidence about the putative role of different effectors of heart acclimatization to chronic hypoxia. Along with classical parameters, we consider NO, specially that generated by mtNOS, mitochondrial respiratory chain, mitoK(ATP) channels, reactive oxygen species and control of gene expression by HIF-1.