The differential effects of superoxide anion, hydrogen peroxide and hydroxyl radical on cardiac mitochondrial oxidative phosphorylation

The involvement of reactive oxygen species (ROS) in cardiac ischemia-reperfusion injuries is well-established, but the deleterious effects of hydrogen peroxide (H(2)O(2)), hydroxyl radical (HO*) or superoxide anion (O(2)*(-) ) on mitochondrial function are poorly understood. Here, we report that incubation of rat heart mitochondria with each of these three species resulted in a decline of the ADP-stimulated respiratory rate but not substrate-dependent respiration. These three species reduced oxygen consumption induced by an uncoupler without alteration of the respiratory chain complexes, but did not modify mitochondrial membrane permeability. HO* slightly decreased F1F0-ATPase activity and HO* and O(2)*(-) partially inhibited the activity of adenine nucleotide translocase; H(2)O(2) failed to alter these targets. They inhibited NADH production by acting specifically on aconitase for O(2)*(-) and alpha-ketoglutarate dehydrogenase for H(2)O(2) and HO*. Our results show that O(2)*(-), H(2)O(2) and HO* act on different mitochondrial targets to alter ATP synthesis, mostly through inhibition of NADH production.     

Bioavailability and spatial distribution of fatty acids in the rat retina after dietary omega-3 supplementation

Spatial changes of FAs in the retina in response to different dietary n-3 formulations have never been explored, although a diet rich in EPA and DHA is recommended to protect the retina against the effects of aging. In this study, Wistar rats were fed for 8 weeks with balanced diet including either EPA-containing phospholipids (PLs), EPA-containing TGs, DHA-containing PLs, or DHA-containing TGs. Qualitative changes in FA composition of plasma, erythrocytes, and retina were evaluated by gas chromatography-flame ionization detector. Following the different dietary intakes, changes to the quantity and spatial organization of PC and PE species in retina were determined by LC coupled to MS/MS and MALDI coupled to MS imaging. The omega-3 content in the lipids of plasma and erythrocytes suggests that PLs as well as TGs are good omega-3 carriers for retina. However, a significant increase in DHA content in retina was observed, especially molecular species as di-DHA-containing PC and PE, as well as an increase in very long chain PUFAs (more than 28 carbons) following PL-EPA and TG-DHA diets only. All supplemented diets triggered spatial organization changes of DHA in the photoreceptor layer around the optic nerve. Taken together, these findings suggest that dietary omega-3 supplementation can modify the content of FAs in the rat retina.     

Myocardial ischemic postconditioning against ischemia-reperfusion is impaired in ob/ob mice

Ischemic postconditioning (IPCD) significantly reduces infarct size in healthy animals and protects the human heart. Because obesity is a major risk factor of cardiovascular diseases, the effects of IPCD were investigated in 8- to 10-wk-old leptin-deficient obese (ob/ob) mice and compared with wild-type C57BL/6J (WT) mice. All animals underwent 30 min of coronary artery occlusion followed by 24 h of reperfusion associated or not with IPCD (6 cycles of 10-s occlusion, 10-s reperfusion). Additional mice were killed at 10 min of reperfusion for Western blotting. IPCD reduced infarct size by 58% in WT mice (33+/-1% vs. 14+/-3% for control and IPCD, respectively, P<0.05) but failed to induce cardioprotection in ob/ob mice (53+/-4% vs. 56+/-5% for control and IPCD, respectively). In WT mice, IPCD significantly increased the phosphorylation of Akt (+77%), ERK1/2 (+41%), and their common target p70S6K1 (+153% at Thr389 and +57% at Thr421/Ser424). In addition, the phosphorylated AMP-activated protein kinase (AMPK)-to-total AMPK ratio was also increased by IPCD in WT mice (+64%, P<0.05). This was accompanied by decreases in phosphatase and tensin homolog deleted on chromosome 10 (PTEN), MAP kinase phosphatase (MKP)-3, and protein phosphatase (PP)2C levels. In contrast, IPCD failed to increase the phosphorylation state of all these kinases in ob/ob mice, and the level of the three phosphatases was significantly increased. Thus, although IPCD reduces myocardial infarct size in healthy animals, its cardioprotective effect vanishes with obesity. The lack of enhanced phosphorylation by IPCD of Akt, ERK1/2, p70S6K1, and AMPK might partly explain the loss of cardioprotection in this experimental model of obese mice.     

CORM-3, a water soluble CO-releasing molecule,uncouples mitochondrial respiration via interaction with the phosphate carrier

Carbon monoxide is continuously produced in small quantities in tissues and is an important signaling mediator in mammalian cells. We previously demonstrated that CO delivered to isolated rat heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) is able to uncouple mitochondrial respiration. The aim of this study was to explore more in depth the mechanism(s) of this uncoupling effect. We found that acceleration of mitochondrial O₂ consumption and decrease in membrane potential induced by CORM-3 were associated with an increase in mitochondrial swelling. This effect was independent of the opening of the mitochondrial transition pore as cyclosporine A was unable to prevent it. Interestingly, removal of phosphate from the incubation medium suppressed the effects mediated by CORM-3. Blockade of the dicarboxylate carrier, which exchanges dicarboxylate for phosphate, decreased the effects induced by CORM-3 while direct inhibition of the phosphate carrier with N-ethylmaleimide completely abolished the effects of CORM-3. In addition, CORM-3 was able to enhance the transport of phosphate into mitochondria as evidenced by changes in mitochondrial phosphate concentration and mitochondrial swelling that evaluates the activity of the phosphate carrier in de-energized conditions. These results indicate that CORM-3 activates the phosphate carrier leading to an increase in phosphate and proton transport inside mitochondria, both of which could contribute to the non-classical uncoupling effect mediated by CORM-3. The dicarboxylate carrier amplifies this effect by increasing intra-mitochondrial phosphate concentration.     

A carbon monoxide-releasing molecule (CORM-3) uncouples mitochondrial respiration and modulates the production of reactive oxygen species

Carbon monoxide (CO), produced during the degradation of heme by the enzyme heme oxygenase, is an important signaling mediator in mammalian cells. Here we show that precise delivery of CO to isolated heart mitochondria using a water-soluble CO-releasing molecule (CORM-3) uncouples respiration. Addition of low-micromolar concentrations of CORM-3 (1–20 μM), but not an inactive compound that does not release CO, significantly increased mitochondrial oxygen consumption rate (State 2 respiration) in a concentration-dependent manner. In contrast, higher concentrations of CORM-3 (100 μM) suppressed ADP-dependent respiration through inhibition of cytochrome c oxidase. The uncoupling effect mediated by CORM-3 was inhibited in the presence of the CO scavenger myoglobin. Moreover, this effect was associated with a gradual decrease in membrane potential (ψ) over time and was partially reversed by malonate, an inhibitor of complex II activity. Similarly, inhibition of uncoupling proteins or blockade of adenine nucleotide transporter attenuated the effect of CORM-3 on both State 2 respiration and Δψ. Hydrogen peroxide (H₂O₂) produced by mitochondria respiring from complex I-linked substrates (pyruvate/malate) was increased by CORM-3. However, respiration initiated via complex II using succinate resulted in a fivefold increase in H₂O₂ production and this effect was significantly inhibited by CORM-3. These findings disclose a counterintuitive action of CORM-3 suggesting that CO at low levels acts as an important regulator of mitochondrial respiration.     

Lipid Composition of the Human Eye: Are Red Blood Cells a Good Mirror of Retinal and Optic Nerve Fatty Acids?

Background

The assessment of blood lipids is very frequent in clinical research as it is assumed to reflect the lipid composition of peripheral tissues. Even well accepted such relationships have never been clearly established. This is particularly true in ophthalmology where the use of blood lipids has become very common following recent data linking lipid intake to ocular health and disease. In the present study, we wanted to determine in humans whether a lipidomic approach based on red blood cells could reveal associations between circulating and tissue lipid profiles. To check if the analytical sensitivity may be of importance in such analyses, we have used a double approach for lipidomics.

Methodology and Principal Findings

Red blood cells, retinas and optic nerves were collected from 9 human donors. The lipidomic analyses on tissues consisted in gas chromatography and liquid chromatography coupled to an electrospray ionization source-mass spectrometer (LC-ESI-MS). Gas chromatography did not reveal any relevant association between circulating and ocular fatty acids except for arachidonic acid whose circulating amounts were positively associated with its levels in the retina and in the optic nerve. In contrast, several significant associations emerged from LC-ESI-MS analyses. Particularly, lipid entities in red blood cells were positively or negatively associated with representative pools of retinal docosahexaenoic acid (DHA), retinal very-long chain polyunsaturated fatty acids (VLC-PUFA) or optic nerve plasmalogens.

Conclusions and Significance

LC-ESI-MS is more appropriate than gas chromatography for lipidomics on red blood cells, and further extrapolation to ocular lipids. The several individual lipid species we have identified are good candidates to represent circulating biomarkers of ocular lipids. However, further investigation is needed before considering them as indexes of disease risk and before using them in clinical studies on optic nerve neuropathies or retinal diseases displaying photoreceptors degeneration.     

A detailed identification study on high-temperature degradation products of oleic and linoleic acid methyl esters by GC-MS and GC-FTIR

GC-MS and GC-FTIR were complementarily applied to identify oxidation compounds formed under frying conditions in methyl oleate and linoleate heated at 180°C. The study was focused on the compounds that originated through hydroperoxide scission that remain attached to the glyceridic backbone in fats and oils and form part of non-volatile molecules. Twenty-one short-chain esterified compounds, consisting of 8 aldehydes, 3 methyl ketones, 4 primary alcohols, 5 alkanes and 1 furan, were identified. In addition, twenty non-esterified volatile compounds, consisting of alcohols, aldehydes and acids, were also identified as major non-esterified components. Furanoid compounds of 18 carbon atoms formed by a different route were also identified in this study. Overall, the composition of the small fraction originated from hydroperoxide scission provides a clear idea of the complexity of the new compounds formed during thermoxidation and frying.     

Adenosine A(1)-receptor induced late preconditioning and myocardial infarction: reperfusion duration is critical

We investigated the influence of coronary artery reperfusion (CAR) duration on the infarct-limiting properties of adenosine A(1)-receptor stimulation-induced delayed preconditioning (A(1)-DPC) compared with ischemia-induced delayed preconditioning (I-DPC). Sixty-one chronically instrumented conscious rabbits successfully underwent the following protocol. On day 1, rabbits were randomly divided into four groups: control (saline, iv), I-DPC (six 4-min coronary artery occlusion/4-min reperfusion cycles), A(1)-DPC(100) (N(6)-cyclopentyladenosine, 100 microg/kg iv), and A(1)-DPC(400) (N(6)-cyclopentyladenosine, 400 microg/kg iv). On day 2 (i.e., 24 h later), rabbits underwent a 30-min coronary artery occlusion after which CAR was started and maintained for either 3 or 72 h. Infarct size (percentage of the area at risk) was determined by triphenyltetrazolium chloride staining. After 3 h of CAR, I-DPC, A(1)-DPC(100), and A(1)-DPC(400) significantly decreased infarct size (36 +/- 5, 41 +/- 4, 38 +/- 5%, respectively) compared with control (55 +/- 3%). After 72 h of CAR, infarct sizes were not significantly different among the four groups. This result was confirmed by histologic analysis. Thus A(1)-DPC at the two investigated doses, as well as I-DPC, decreased infarct size after 3 h but not 72 h of CAR.     

Active Rho is localized to podosomes induced by oncogenic Src and is required for their assembly and function

Transformation of fibroblasts by oncogenic Src causes disruption of actin stress fibers and formation of invasive adhesions called podosomes. Because the small GTPase Rho stimulates stress fiber formation, Rho inactivation by Src has been thought to be necessary for stress fiber disruption. However, we show here that Rho[GTP] levels do not decrease after transformation by activated Src. Inactivation of Rho in Src-transformed fibroblasts by dominant negative RhoA or the Rho-specific inhibitor C3 exoenzyme disrupted podosome structure as judged by localization of podosome components F-actin, cortactin, and Fish. Inhibition of Rho strongly inhibited Src-induced proteolytic degradation of the extracellular matrix. Furthermore, development of an in situ Rho[GTP] affinity assay allowed us to detect endogenous Rho[GTP] at podosomes, where it colocalized with F-actin, cortactin, and Fish. Therefore, Rho is not globally inactivated in Src-transformed fibroblasts, but is necessary for the assembly and function of structures implicated in tumor cell invasion.