Dario L. Santos

Diabetes and mitochondrial oxidative stress: A study using heart mitochondria from the diabetic Goto-Kakizaki rat

Increasing evidence shows that the overproduction of reactive oxygen species, induced by diabetic hyperglycemia, contributes to the development of several cardiopathologies. The susceptibility of diabetic hearts to oxidative stress, induced in vitro by ADP-Fe2+ in mitochondria, was studied in 12-month-old Goto-Kakizaki rats, a model of non-insulin dependent diabetes mellitus, and normal (non-diabetic) Wistar rats. In terms of lipid peroxidation the oxidative damage was evaluated on heart mitochondria by measuring both the O2 consumption and the concentrations of thiobarbituric acid reactive substances. Diabetic rats display a more intense formation of thiobarbituric acid reactive substances and a higher O2 consumption than non-diabetic rats. The oxidative damage, assessed by electron microscopy, was followed by an extensive effect on the volume of diabetic heart mitochondria, as compared with control heart mitochondria. An increase in the susceptibility of diabetic heart mitochondria to oxidative stress can be explained by reduced levels of endogenous antioxidants, so we proceeded in determinating α-tocopherol, GSH and coenzyme Q content. Although no difference of α-tocopherol levels was found in diabetic rats as compared with control rat mitochondria, a significant reduction in GSH (21.5% reduction in diabetic rats) and coenzyme Q levels of diabetic rats was observed. The data suggest that a significant decrease of coenzyme Q9, a potent antioxidant involved in the elimination of mitochondria-generated reactive oxygen species, may be responsible for an increased susceptibility of diabetic heart mitochondria to oxidative damage.

Brain and liver mitochondria isolated from diabeticGoto-Kakizaki rats show different susceptibility to induced oxidative stress

Increased oxidative stress and changes in antioxidant capacity observed in both clinical and experimental diabetes mellitus have been implicated in the etiology of chronic diabetic complications. Many authors have shown that hyperglycemia leads to an increase in lipid peroxidation in diabetic patients and animals reflecting a rise in reactive oxygen species production. The aim of the study was to compare the susceptibility of mitochondria from brain and liver of Goto‐Kakizaki (12‐month‐old diabetic) rats (GK rats), a model of non‐insulin dependent diabetes mellitus, to oxidative stress and antioxidant defenses.

Inhibitory effect of carvedilol in the high-conductance state of the mitochondrial permeability transition pore

The mitochondrial permeability transition is a widely studied, but poorly understood, phenomenon in mitochondrial bioenergetics. It has been recognised that this phenomenon is related to the opening of a protein pore in the inner mitochondrial membrane, and that opening of this pore is the cause of some forms of mitochondrial dysfunction. In this work, we propose that carvedilol, a multi-role cardioprotective compound, may act as an inhibitor of the high-conductance state of the mitochondrial permeability transition pore, a conclusion supported by the finding that carvedilol provides differential protection against mitochondrial swelling in sucrose and KCl-based media, and that it is unable to protect against calcium-induced depolarisation of the mitochondrial membrane. We also show that carvedilol inhibits the oxidation of mitochondrial thiol groups and that, beyond causing a slight depression of the membrane potential, it has no inhibitory effect on mitochondrial calcium uptake.A decrease in the number of oxidised protein thiol groups may be the main mechanism responsible for this selective inhibition of the permeability transition pore in heart mitochondria. These effects may be important for the role of carvedilol in some cardiac pathologies.

Cationic solid lipid nanoparticles interfere with the activity of antioxidant enzymes in hepatocellular carcinoma cells.

Solid lipid nanoparticles (SLN) are colloidal drug and/or gene carriers developed from solid lipids and surfactants that are considered safe. Cationic SLN, usually used for formulating poorly water-soluble drugs and for gene delivery purposes, as positively charged particles may attach to cellular surfaces and be internalized more easily than negatively charged SLN, but they can also cause damage. The main aim of this work was to test a set of cationic SLN and investigate its influence on the amount of reactive oxygen species (ROS), on antioxidant enzymes activities and on possible oxidative damage to membrane lipids in HepG2 cells. The Dichlorofluorescein assay revealed great increase in ROS presence after cell exposure to SLN. While the exposure to SLN increased the activities of superoxide dismutase and glutathione peroxidase it decreased glutathione reductase activity. Although no significant increase in thiobarbituric reactive species was found, a decrease in sulfhydryl groups was detected. These results indicate that cationic SLN caused oxidative stress in HepG2 cells, but under reported exposure conditions HepG2 cells could attenuate the stress and thus the damage to cellular components was minimal.

Higher efficiency of the liver phosphorylative system in diabetic Goto‐Kakizaki (GK) rats

Liver mitochondrial bioenergetics of Goto‐Kakizaki (GK) rats (a model of non‐insulin dependent diabetes mellitus) reveals a ΔΨ upon energization with succinate significantly increased relatively to control animals. The repolarization rate following ADP phosphorylation is also significantly increased in GK mitochondria in parallel with increased ATPase activity. The increase in the repolarization rate and ATPase activity is presumably related to an improved efficiency of F0F1‐ATPase, either from a better phosphorylative energy coupling or as a consequence of an enlarged number of catalytic units. Titrations with oligomycin indicate that diabetic GK liver mitochondria require excess oligomycin pulses to completely abolish phosphorylation, relative to control mitochondria. Therefore, accepting that the number of operational ATP synthase units is inversely proportional to the amount of added oligomycin, it is concluded that liver mitochondria of diabetic GK rats are provided with extra catalytic units relative to control mitochondria of normal rats. Other tissues (kidney, brain and skeletal muscle) were evaluated for the same bioenergetic parameters, confirming that this feature is exclusive to liver from diabetic GK rats.

Nerolidol effects on mitochondrial and cellular energetics

In the present work, we evaluated the potential toxic effects of nerolidol, a sesquiterpenoid common in plants essential oils, both on mitochondrial and cellular energetics. Samples of enriched natural extracts of nerolidol (a racemic mixture of cis and trans isomers) were tested on rat liver mitochondria and a decrease in phosphorylative system was observed but not in the mitochondrial respiratory chain activity, which reflects a direct effect on F1-ATPase. Hence, respiratory control ratio was also decreased. Cellular ATP/ADP levels were significantly decreased in a concentration-dependent manner, possibly due to the direct effect of nerolidol on F(0)F(1)-ATPsynthase. Nerolidol stimulates respiratory activity probably due to an unspecific effect, since it does not show any protonophoric effect. Furthermore, we observed that mitochondrial permeability transition was delayed in the presence of nerolidol, possibly due to its antioxidant activity and because this compound decreases mitochondrial transmembrane electric potential. Our results also show that, in human hepatocellular liver carcinoma cell line (HepG2), nerolidol both induces cell death and arrests cell growth, probably related with the observed lower bioenergetic efficiency.

Are fentanyl and remifentanil safe opioids for rat brain mitochondrial bioenergetics

Fentanyl and remifentanil are potent opioid widely used in routine anesthesia procedures. This study evaluates and compares the effects of fentanyl/remifentanil in isolated brain mitochondria bioenergetic status. Fentanyl and remifentanil in clinical concentrations does not interfere with rat brain isolated mitochondria. Do not withstand, fentanyl concentrations >4 microg/mL, induces an impairment of the respiratory chain characterized by a decrease in respiratory control ratio, state 3 and uncoupled respiration. Additionally, membrane potential collapses and ADP/O were reduced. Remifentanil follows the same profile but with effects at higher concentrations (>10 microg/mL). High concentrations of fentanyl and remifentanil interfere with mitochondrial electron chain (complexes III, IV) and on mitochondrial phosphorylation unit (complex V). Mitochondrial permeability transition pore was not induced by both fentanyl and remifentanil in tested concentrations. These data provide the first indication that fentanyl and remifentanil (microg/mL range) alters mitochondrial metabolism. Fentanyl showed a stronger inhibitory effect on mitochondrial bioenergetics.

Calcium channel blockers inhibit the (Ca2+ + Mg2+)-ATPase activity and the 125I-calmodulin binding in brain membranes

Ca2+ channel blockers belonging to three distinct chemical groups (dihydropyridines, phenylalkylamines and diphenylalkylamines) differentially inhibit the (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes (Santos et al., J. Neurochem. 52, S49D, 1989). We now report that (-)-desmethoxyverapamil and flunarizine are the most potent inhibitors of the Ca(2+)-activated ATPase activity of synaptic plasma membranes, decreasing the Vmax by 41% and 37%, respectively, with no significant effects on the Km for Ca2+ (162.7 +/- 14.9 nM free [Ca2+]), while nitrendipine did not affect these parameters. Trifluoperazine was the most potent inhibitor of the Ca(2+)-activated ATPase of synaptic plasma membranes with an IC50 of 8-10 microM. To clarify whether the inhibitory effects of Ca2+ channel blockers and of trifluoperazine on the (Ca2+ + Mg2+)-ATPase occur through the inhibition of the interaction of calmodulin with the enzyme, we studied their effects on the binding of 125I-calmodulin to the membrane proteins separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), followed by electrotransfer to nitrocellulose and autoradiography. The autoradiograms revealed Ca(2+)-dependent CaM binding proteins of about 140, 70 and 55 kDa. Trifluoperazine (30-40 microM) inhibited by 50-60% the binding of 125I-calmodulin to the 140 kDa band, which probably includes the (Ca2+ + Mg2+)-ATPase protein. Flunarizine and (-)-desmethoxyverapamil (100 microM) inhibited the 125I-calmodulin binding to the 140 kDa peptides by 100 and 90%, respectively, and they inhibited by 55 and 40%, respectively, the binding of 125I-calmodulin to the peptides in the 70-55 kDa range, whereas nitrendipine did not show any effect. The results suggest that the inhibitory effects of (-)-desmethoxyverapamil and flunarizine, as well as trifluoperazine, on the (Ca2+ + Mg2+)-ATPase activity of synaptic plasma membranes are mediated by inhibition of the calmodulin interaction with the enzyme.

Mitochondrial function is not affected by renal morphological changes in diabetic goto-kakizaki rat.

Renal disease is a common complication of diabetes mellitus. The pathogenesis of diabetic nephropathy is not well understood, but hyperglycemia seems to be a crucial factor. Recent evidence indicates that the overproduction of reactive oxygen species, observed in both clinical and experimental diabetes, and mitochondrial dysfunction are key factors in pathogenic process. The objective of this investigation was to test the hypothesis of whether hyperglycemia could affect kidney morphology and mitochondrial bioenergetics as well as susceptibility to oxidative stress in 12-month-old diabetic Goto-Kakizaki (GK) rats, a model of type 2 diabetes mellitus. We observed that there were no significant differences in the kidney respiratory function and phosphorylation capacity between GK and age-matched control Wistar rats. Mitochondria from kidneys of diabetic rats were equally susceptible to in vitro oxidative damage as those from normal rats, while coenzyme Q and α -tocopherol concentrations were similar in both types of preparations. However, the kidney of GK rats presented in most glomerulus a capillary basement membrane thickening with mesangial widening, in evolution to segmental glomerular sclerosis, and, in some interlobular arteries, excessive deposition of PAS-positive material at the tunica intima. The results show that the mild prolonged hyperglycemia and the kidney structural changes observed in GK rats are not sufficient to cause renal dysfunction and were not associated with functional and biochemical alterations in mitochondria.

Measuring Mitochondrial Membrane Potential with a Tetraphenylphosphonium-Selective Electrode.

Mitochondrial bioenergetics is based on the generation of the protonmotive force by the electron transport chain. The protonmotive force is used by mitochondria for different critical aspects of its normal function, ranging from calcium accumulation to the synthesis of ATP. The transmembrane electric potential (ΔΨ) is the major component of the protonmotive force and is also the main responsible for ATP synthesis by mitochondrial ATP synthase. Although several methods can be used to measure the ΔΨ, the use of the tetraphenylphosphonium cation (TPP+)‐selective electrode is still a method of election due to its sensitivity. The method is based on the accumulation of TPP+ by energized mitochondria, which develop a negative charge in the matrix due to the ejection of protons. This unit describes how to build a custom‐made TPP+‐selective electrode and how to establish the necessary set‐up to follow ΔΨ fluctuations in isolated mitochondrial fractions.