Aziz Elimadi

Lipoic acid prevents hypertension, hyperglycemia, and the increase in heart mitochondrial superoxide production

BACKGROUND The present study was designed to investigate whether the effects of dietary supplementation with alpha-lipoic acid could prevent the increase in mitochondrial superoxide production in the heart as well as the enhanced formation of advanced glycation end-products (AGE) that are associated with the development of hypertension and insulin resistance in chronically glucose-fed rats. METHODS Sprague Dawley rats were either given or not given a 10% D-glucose solution to drink during 4 weeks, combined either with a normal chow diet or with alpha-lipoic acid supplemented diet. The oxidative stress was evaluated by measuring the heart mitochondrial superoxide production using the lucigenin chemiluminescence method. The formation of AGE was also assessed in plasma and aorta. RESULTS Chronic administration of glucose resulted in a 29% increase in blood pressure, 30% increase in glycemia, 286% increase in insulinemia, and 408% increase in insulin resistance index. Chronic glucose feeding also resulted in a 22% greater mitochondrial superoxide anion production in heart and in an increase of 63% in AGE content in aorta. Increases in blood pressure, aorta AGE content and heart mitochondrial superoxide production were prevented in the rats fed glucose supplemented with lipoic acid. The simultaneous treatment with lipoic acid also attenuated the rise in insulin levels as well as in insulin resistance in the glucose fed rats. CONCLUSIONS These findings demonstrate that alpha-lipoic acid supplementation prevents development of hypertension and hyperglycemia, presumably through its antioxidative properties, as reflected by prevention of an increase in heart mitochondrial superoxide anion production and in AGE formation in the aorta of chronically glucose treated rats.

Attenuation of liver normothermic ischemia-reperfusion injury by preservation of mitochondrial functions with S-15176, a potent trimetazidine derivative

We investigated the antiischemic properties of a new compound, S-15176, in an experimental model of rat liver subjected to 120-min normothermic ischemia followed by 30-min reperfusion. Rats were divided into groups, pretreated with different doses of S-15176 (1.25, 2.5, 5 and 10 mg/kg/day by intramuscular injection) or solvent alone, and subjected to the ischemia--reperfusion process. Another group served as the sham-operated controls. Ischemia--reperfusion induced huge alterations of hepatocyte functions, namely, a decrease in ATP content and bile flow, and membrane leakage of alanine aminotransferase (ALAT) and aspartate aminotransferase (ASAT). These effects were associated with alterations in mitochondrial functions characterized by (1) a decrease in ATP synthesis, (2) a decrease in NAD(P)H levels and mitochondrial membrane potential, and (3) an increase in mitochondrial swelling reflecting the generation of permeability transition. Pretreatment of rats with S-15176 alleviated these deleterious ischemia--reperfusion effects at both the cellular and mitochondrial levels in a dose-dependent manner. The protection of mitochondrial functions was almost complete at a dosage of 10 mg/kg/day. In addition, in vitro, S-15176 totally abolished the swelling of isolated mitochondria induced by a calcium overload with an IC(50) value of 10 microM. These data demonstrate that S-15176 protects mitochondria against the deleterious effects of ischemia-reperfusion and suggest that this protective effect could be related to the inhibition of the mitochondrial permeability transition.

Evidence for the existence of [3H]-trimetazidine binding sites involved in the regulation of the mitochondrial permeability transition pore

Trimetazidine is an anti‐ischaemic drug effective in different experimental models but its mechanism of action is not fully understood. Data indicate that mitochondria could be the main target of this drug. The aim of this work was to investigate the binding of [3H]‐trimetazidine on a purified preparation of rat liver mitochondria. [3H]‐trimetazidine binds to two populations of mitochondrial binding sites with Kd values of 0.96 and 84 μm. The total concentration of binding sites is 113 pmol mg−1 protein. Trimetazidine binding sites are differently distributed. The high‐affinity ones are located on the outer membranes and represent only a small part (4%) of total binding sites, whereas the low‐affinity ones are located on the inner membranes and are more abundant (96%) with a Bmax=108 pmol mg−1 protein. Drug displacement studies with pharmacological markers for different mitochondrial targets showed that [3H]‐trimetazidine binding sites are different from previously described mitochondrial sites. The possible involvement of [3H]‐trimetazidine binding sites in the regulation of the mitochondrial permeability transition pore (MTP), a voltage‐dependent channel sensitive to cyclosporin A, was investigated with mitochondrial swelling experiments. Trimetazidine inhibited the mitochondrial swelling induced by Ca2+ plus tert‐butylhydroperoxide (t‐BH). This effect was concentration‐dependent with an IC50 value of 200 μm. Assuming that trimetazidine effectiveness may be related to its structure as an amphiphilic cation, we compared it with other compounds exhibiting the same chemical characteristic both for their ability to inhibit MTP opening and to displace [3H]‐trimetazidine bound to mitochondria. Selected compounds were drugs known to interact with various biological membranes. A strong correlation between swelling inhibition potency and low‐affinity [3H]‐trimetazidine binding sites was observed: r=0.907 (n=24; P<0.001). These data suggest that mitochondrial sites labelled with [3H]‐trimetazidine may be involved in the MTP inhibiton.

Dose-related inversion of cinnarizine and flunarizine effects on mitochondrial permeability transition

We investigated the effects of cinnarizine and flunarizine on mitochondrial permeability transition, ATP synthesis, membrane potential and NAD(P)H oxidation. Both drugs were effective in inhibiting the mitochondrial permeability transition induced either by Ca2+ alone or in the presence of tert-butylhydroperoxide. This protective effect occurred at low concentrations (< 50 microM) of these drugs and was accompanied by the inhibition of NAD(P)H oxidation and the restoration of the mitochondrial membrane potential decreased by a high concentration of Ca2+ (25 microM). However, at higher concentrations (> 50 microM) of cinnarizine and flunarizine and in the absence of both tert-butylhydroperoxide and Ca2+, their effects on the mitochondria were reversed as follows: mitochondrial permeability transition was generated, mitochondrial NAD(P)H was oxidized and membrane potential collapsed. These deleterious effects were not antagonized by cyclosporine A, the most potent inhibitor of the mitochondrial permeability transition, but by 2,6-di-tert-butyl-4-methylphenol, a known antioxidant agent. This mitochondrial effect was neither accompanied by an increase in malondialdehyde production nor by an increase in H2O2 generation, which attested that the effect of both drugs was not due to an increase in reactive oxygen species production. The dual effects of both cinnarizine and flunarizine on mitochondrial functions is discussed with regard to both the protective effect afforded by these drugs against ischemia-reperfusion injury and their side effect observed in some therapeutic situations where an overdosage seems likely.

[3H]-Trimetazidine mitochondrial binding sites: regulation by cations, effect of trimetazidine derivatives and other agents and interaction with an endogenous substance

Trimetazidine, an antiischaemic drug, has been shown to restore impaired mitochondrial functions. Specific binding sites for [3H]‐trimetazidine have been previously detected in liver mitochondria. In the present study we confirm this observation and provide additional evidence for the involvement of these sites in the pharmacological effects of the drug. Inhibition experiments using a series of trimetazidine derivatives revealed the presence of three classes of binding sites. An N‐benzyl substituted analogue of trimetazidine exhibited a very high affinity (Ki=7 nM) for one of these classes of sites. Compounds from different pharmacological classes were evaluated for their ability to inhibit [3H]‐trimetazidine binding. Among the drugs tested pentazocine, ifenprodil, opipramol, perphenazine, haloperidol, and to a lower extent prenylamine, carbetapentane and dextromethorphan competed with high affinity, suggesting a similarity of high affinity [3H]‐trimetazidine sites with sigma receptors. [3H]‐Trimetazidine binding was modulated by pH. Neutral trimetazidine had about 10 fold higher affinity than protonated trimetazidine for its mitochondrial binding sites. Various cations also affected [3H]‐trimetazidine binding. Ca2+ was the most potent inhibitor and totally suppressed the binding of [3H]‐trimetazidine to the sites of medium affinity. An endogenous cytosolic ligand was able to displace [3H]‐trimetazidine from its binding sites. Its activity was not affected by boiling for 15 min, suggesting a non‐protein compound. These data suggest that mitochondrial [3H]‐trimetazidine binding sites could have a physiological relevance and be involved in the antiischaemic effects of the drug.

Differential effects of zidovudine and zidovudine triphosphate on mitochondrial permeability transition and oxidative phosphorylation

The effects of zidovudine (ZDV) and zidovudine triphosphate (ZDV‐3P) on Ca2+‐induced mitochondrial permeability transition (MPT), respiratory control ratio (RCR) and ATP synthesis have been investigated on isolated rat liver mitochondria. ZDV slightly but significantly decreased RCR and ATP synthesis but was ineffective in inhibiting MPT. In contrast, ZDV‐3P did not alter RCR and ATP synthesis but strongly inhibited MPT (IC50=3.0±0.9 μM). The effect of ZDV‐3P on mitochondrial swelling required a preincubation time. When incubated 10 min with mitochondria, ZDV‐3P (8 μM) totally inhibited the rate of swelling. ADP, ATP and atractyloside, which are agents known to interact with the mitochondrial adenine nucleotide carrier (ANC), antagonized the effect of ZDV‐3P on mitochondrial swelling. Indeed, the IC50 value of ZDV‐3P increased from 3.0 to 17.4, 93.6 and 66.5 μM, in the presence of 20 μM, ADP, ATP or atractyloside, respectively. ZDV‐3P did not displace [3H]‐ATP from its mitochondrial binding site(s) whereas ADP and atractyloside did, suggesting that ZDV‐3P and [3H]‐ATP do not share the same binding sites. ZDV‐3P did not affect either mitochondrial respiration or ATP synthesis but inhibited Ca2+‐dependent mitochondrial swelling. It was concluded that mitochondrial toxic effects observed during the chronic administration of ZDV cannot be related to its active metabolite (ZDV‐3P).

Combining ursodeoxycholic acid or its NO- releasing derivative NCX-1000 with lipophilic antioxidants better protects mouse hepatocytes against amiodarone toxicity

Nonalcoholic steatohepatitis (NASH) is a common and potentially severe form of liver disease. This study aimed to determine the effect of ursodeoxycholic acid and its NO-releasing derivative NCX-1000 alone or in combination with antioxidants on cultured mouse hepatocytes treated with amiodarone to mimic certain aspects of hepatocyte injury found in NASH. Isolated mouse hepatocytes were incubated with ursodeoxycholic acid or NCX-1000 (0-100 micromol/L) combined or not combined with the hydrophilic antioxidants butylated hydroxytoluene and ascorbic acid (0-100 micromol/L) or with the lipophilic antioxidant alpha-tocopherol (0-100 micromol/L) 15 min before adding amiodarone (50 micromol/L) to the culture medium. Twenty hours later, necrosis, apoptosis, superoxide anion production, and malondialdehyde levels were assessed in cultured cells. Amiodarone led to a dose-dependent decrease in cell viability with an LD50 of 50 micromol/L and increased production of superoxide anion and lipid peroxidation. NCX-1000 showed a better protective potential than ursodeoxycholic acid against the toxic effects of amiodarone. The hydrophilic antioxidants had no effect on the toxicity of amiodarone, whereas alpha-tocopherol at a concentration >100 micromol/L almost completely suppressed it. Ursodeoxycholic acid and NCX-1000 protection was additive only when they were combined with alpha-tocopherol, not with butylated hydroxytoluene or ascorbic acid. In addition, all the antioxidants tested reduced the superoxide anion detected, but only alpha-tocopherol prevented lipid peroxidation induced by amiodarone. The combination of lipophilic antioxidants with ursodeoxycholic acid or NCX-1000 enhances their protective potential and could represent an interesting therapeutic approach to explore for the treatment of NASH.